WO2005046575A2 - Amino acid prodrugs - Google Patents

Abstract

The present invention is directed to a prodrug comprised of an amino acid bonded to a medicament or drug having a hydroxy, amino, carboxy or acylating derivative thereon. The prodrug has the same utility as the drug from which it is made, but it has enhanced therapeutic properties. In fact, the prodrugs of the present invention enhance at least two therapeutic qualities, as defined herein. The present invention is also directed to pharmaceutical compositions containing same.

Description

AMINO ACID PRODRUGS

SCOPE OF THE INVENTION

The invention relates to amino acid derivatives of pharmaceutical compounds, methods of treating particular ailments, which are ameliorated by the administration of these drugs and pharmaceutical compositions containing these drugs. The current invention involves improving many physicochemical, biopharmaceutical, and clinical efficacy of various drugs using amino acids as covalently bonded carriers for these drugs.

The development of chemical compounds for the treatment of disorders, maladies and diseases has become increasingly difficult and costly. The probability of success for such development is often discouragingly low. Further, the time for development can approach or exceed ten years, leaving large numbers of patients without remedy for an extended period of time.

Even in cases in which effective pharmaceutical compounds have been developed, there are often disadvantages associated with their administration. These disadvantages can include aesthetic, and pharmacokinetic bafflers affecting the effectiveness of some existing pharmaceutical compounds, For example, unpleasant taste or smell of a pharmaceutical compound or composition can be a significant barrier to patient compliance with respect to the administration regimen. The undesirable solubility characteristics of a pharmaceutical compound can lead to difficulty in formulation of a homogeneous composition. Other disadvantages associated with known pharmaceutical compounds include: poor absorption of orally administered formulations; poor bioavailability of the pharmaceutical compounds in oral formulations; lack of dose proportionality; low stability of pharmaceutical compounds in vitro and in vivo; poor penetration of the blood/brain barrier; excessive first-pass metabolism of pharmaceutical compounds as they pass through the liver; excessive enterohepatic recirculation; low absorption rates; ineffective compound release at the site of action; excessive irritation, 1 H:\work\1652\17690\SPEC\1769Q.spec without claimsll.doc for example, gastro-intestinal irritability and/or ulceration; painful injection of parenterally administered pharmaceutical compounds and compositions; excessively high dosages required for some pharmaceutical compounds and compositions, and other undesirable characteristics. Some pharmaceutical compounds are processed by the body to produce toxic by-products with harmful effects.

The art is continually seeking new chemical compounds for the treatment of a wide variety of disorders, with improved properties to overcome the disadvantages of known pharmaceutical compounds mentioned above.

The present invention has overcome many problems associated with currently marketed drugs by making prodrugs. The concept of prodrugs is well known, and there are a number of examples of such prodrugs enumerated in the literature and there are a number of prodrugs available in the market, including such diverse groups as statin drugs, ACE inhibitors, antiviral drugs such as Acyclovir and the like.

The present invention, however, uses amino acids as the moiety to make the prodrugs. The prodrugs of the present invention have a number of advantages. For example, when amino acid prodrugs are administered by a number of routes such as oral, IV, rectal or other such methods, these prodrugs are converted into active drug molecules. A significant advantage of the amino acid prodrug is that it is non-toxic, and hence either assimilated into the body or safely excreted. This is quite unlike the characteristics exhibited by a number of prodrugs available in the market, where the promoiety itself is toxic, as is the case with statin drugs, Enalapril, Benazapril and the like group of ace inhibitors, and a number of antibiotics such as pivoxil, isopropyl, Axetil, Cilexetil and the like groups, which are highly toxic, thereby reducing the therapeutic index of the active drug.

On the other hand, the amino acid prodrugs of the present invention also impart a number of advantages as shown herein below.

2 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc SUMMARY OF THE INVENTION

The present invention is directed to a pharmaceutically active prodrug, having amino acid covalently bonded to a pharmaceutical compound to form said acid prodrug, which is administered in this form to the subject, such as a mammal.

The amino acid is an ideal model to be used as a prodrug, because it is capable of forming various types of linkages between itself and the drug. By definition, an amino acid has at least two functionalities thereon, an amino group (NH₂) and a carboxy group (COOH). For example, the α- amino acids have the well known structure H

H₂N- -COOH

Ro H wherever _n Ro i .s t ,he si .de group or c ,hai •n oft,he ami •no aci .d... Th, e H₂N — C COOH as defined herein, is the main chain ofthe amino acid. Thus, for example, beside the amino group and the carboxyl group on the main chain, the side chain may have functional groups thereon. It is the functional group on the amino acid moiety that permits the covalent linkage to occur between the amino acid and the drug.

The drug or medicament useful in the present invention contains functional groups thereon that permit the drug to react with and form a covalent bond with the amino acid. Examples of functional groups present on the drugs include NH₂, OH, COOH or acid derivatives thereof, such as esters, amides and the like.

The mode of attachment between the pharmaceutical compound and the amino acid can be via: 1) An ester bond (-CO-O-) arising from condensation of a carboxylic acid and an alcohol or phenolic hydroxyl group, or through transesterification, for example:

3 H:\work\l₆52\17690\SPEC\17690.spec without claimsll.doc a) Where the pharmaceutical compound has an aliphatic or aromatic hydroxyl group an ester bond can be formed with the backbone carboxylic acid group of the amino group under esterification conditions; or b) Where the pharmaceutical compound has an aliphatic or aromatic hydroxyl group and the amino acid has a side chain carboxylic acid group, an ester bond can be formed therebetween under esterification conditions; or c) Where the pharmaceutical compound has a carboxylic acid group and the amino acid has a side chain aliphatic or aromatic hydroxyl group, an ester bond can be formed therebetween under esterification condition; or d) Where the pharmaceutical compound has an ester group with a substituted or unsubstituted acyloxy (e.g., alkoxy- or arylalkoxy-, aryloxy carbonyl) substituent (compound-O-CO-substituent) and the amino acid has a backbone carboxylic acid group, an ester bond can be formed therebetween through transesterification; or e) Where the pharmaceutical compound has an ester group with a substituted or unsubstituted acyloxy (e.g., alkoxy- or arylalkoxy-, aryloxy carbonyl) substituent (compound-O-CO-substituent) and the amino acid has a side chain carboxylic acid group, an ester bond can be formed therebetween through transesterification; or f) Where the pharmaceutical compound has an ester group with a substituted or unsubstituted alkoxy- or arylalkoxy- or aryloxy carbonyl substituent

(compound-CO-O-Substituent) and the amino acid has a side chain aliphatic or aromatic hydroxyl group, an ester bond can form therebetween though transesterification; or g) The alcohol and carboxylic acid moieties may be on the same molecule such they can form an internal ester. For example, certain compounds like Gabapentin can form an internal ester under ester forming conditions, is also included with the scope ofthe present invention. 2) An amide bond (-CO-NH -) arising from condensation of a carboxylic acid and an amine, for example:

H:\work\1652\I7690\SPEC\17690.spec without claimsll.doc a) Where the pharmaceutical compound has an amino group and the amino acid has a backbone carboxylic acid group, an amide can be formed under amide forming conditions; or b) Where the pharmaceutical compound has an amino group and the amino acid has a side chain carboxylic acid group, an amide bond can form therebetween under amide forming conditions; or c) Where the pharmaceutical compound has a carboxylic acid group and the amino acid has a backbone amino group, an amide bond can form therebetween under amide forming conditions; or d) Where the pharmaceutical compound has a carboxylic acid group and the amino acid has a side chain amino group, an amide bond can be formed therebetween under amide forming conditions.

Thus, the present invention is directed to the prodrugs thus formed. As shown hereinbelow the prodrug thus formed has advantages not realized relative to the drug without the amino acid attached thereto. For example, it can improve bioavailability, efficacy, be less toxic, exhibit greater solubility in water and/or improve the ability of the drug to pass into the cell membrane or through blood brain barrier, exhibit less side effects, such as gastro-intestinal irritability, enhanced therapeutic index and the like.

Thus, the present invention is directed to a method of improving the therapeutic quality of a drug wherein the improvement in the therapeutic quality is selected from the group consisting of improved efficacy, enhanced therapeutic index, increased solubility in the mammal's internal fluid, improved passage through the cell membrane, improved passage through the blood brain barrier, decreased side effects, such as significantly decreased irritation and/or ulcerations, less toxicity, enhanced absorption ratio and the like relative to the corresponding drug administered to the patient in the non-prodrug form, said method comprising reacting the drug with an amino acid to form a covalent bond therebetween and administering the product thereof (hereinafter "prodrug") to a patient. The prodrugs of the present invention have at least one improved quality. In 5 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc fact, they exhibit preferably at least two ofthe improved qualities cited hereinabove. Other advantages ofthe prodrug include the wide availability ofthe amino acids and the ease in which the reactions take place. The reaction to form the amide is generally efficient and yield are very high, presumably above about 70% and more preferably above about 80% and most preferably above about 90%.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 graphically compares the efficacy of L- serine ester of (±) Ibuprofen (FI), L- threonine ester (+) Ibuprofen (F2) and L-hydroxyproline ester of (+) Ibuprofen (F3), (+) Ibuprofen (i.e., the racemic mixture) and Ibuprofen (S)(+), after one hour dosing, based on the antagonizing property of Acetylcholine induced writhe in Albino mice.

Figure 2 graphically compares the efficacy of L- serine ester of (+) Ibuprofen, (FI), L- threonine ester of, (I) Ibuprofen (F2), L-hydroxyproline ester of (+) Ibuprofen (F3), ± Ibuprofen and S(+) Ibuprofen after 3 hour dosing, based on the antagonizing property of Acetylcholine induced writhes in albino mice.

Figure 3 depicts graphically the dose response relationship to mean clotting time (MCT) in minutes for the L-serine ester of acetylsalicylic acid (Formulation 1).

Figure 4 depicts graphically the dose response relationship to mean clotting time (MCT) minutes for the L-hydroxyproline ester of acetylsalicylic acid (Formulation 2).

Figure 5 depicts the dose response relationship to mean clotting time (MCT) in minutes for the L-threonine ester of acetylsalicylic acid (Formulation 3)

Figure 6 depicts the dose response relationship to mean clotting time (MCT) in minutes for control (acetylsalicylic acid).

H:\work\16₅2\17690\SPEC\17690.spec without claimsILdoc Figure 7 graphically compares the relative efficacy of L-serine (ester of acetyl salicyclic acid (F.l), L-threonine ester of acetyl salicylic acid (F.2), L-hydroxyproline ester of acetylsalicylic acid (F.3), and acetylsalicylic acid (PC) as a function of mean clotting time in minutes.

DETAD ED DESCRIPTION OF THE PRESENT INVENTION As used here, the term "drug", "medicament ", and "pharmaceutical" are being used interchangeably and refer to the active compound that is administered to the patient without attachment ofthe amino acid thereto. Moreover, as used herein, the drug contains a functional group thereon capable of reacting with the amino acid, such as

NH₂, OH, COOH or acylating derivatives thereof (e.g., ester, anhydride, amide, and the like) and the like. When the drug is linked to an amino acid, the term "amino acid prodrug" or "prodrug ofthe present invention" or synonym thereto is utilized.

Among the amino acids useful as promoieties (i.e., reacting with the drugs) are those containing the free amino and/or carboxylic acid groups of all conventional amino acids. Of those, some preferred embodiments include those amino acids having relatively high solubility in aqueous media, for example, in deionized water at unbuffered aqueous solution at 25°C, of at least 100 g L, and more preferably, at least 250 g/L, and even more preferably at least 500 g/L. For example, glycine and proline have solubilities in deionized water at 25°C of approximately 250 g/L and 1620 g/L, respectively.

Other amino acids useful as promoieties are those containing basic amino side chains, such as lysine. For example, lysine has solubility in deionized water at 25 °C of approximately 700 g/L.

Among other amino acids useful as promoieties are those containing hydroxyl side chains, such as hydroxyproline, serine, and threonine. For example, threonine, hydroxyproline and serine have solubilities in deionized water at 25°C of approximately 100 g/L, 369 g/L and 420 g/L, respectively. 7 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc Other preferred embodiments include those amino acids with relatively low solubility in aqueous media, for example, in deionized water at 25 °C of at most 10 g/L, or for example, at most 2 g/L, or for example at most 0.6 g/L. For example, the solubility of tyrosine in deionized water at 25°C is approximately 0.5 g/L. Such prodrugs could be used to produce formulations with extended release characteristics, due to the limited solubility ofthe prodrugs.

Among other amino acids useful as promoieties are those containing carboxylic acid side chains, such as glutamic acid and aspartic acid. Other amino acids useful as promoieties are the non-essential amino acids, and the non-naturally occurring amino acids.

The following reaction schemes depict the reactions discussed hereinabove with respect to reaction of hydroxyl, carboxyl and amine containing drugs with various amino acids. In the schemes below, R is the drug less the functional OH, COOH or NH₂ group H C- R_n whichever is present, and Ri is , wherein R₀ is the side chain ofthe amino acid listed hereinbelow:

Reaction Scheme A: Where the hydroxyl group ofthe drug is reacted with the carboxyl group of an amino acid to from the ester prodrug

R-OH + HOOC-R1- NH2 -» R-O-(C=O)-Rι- NH₂

Drug Amino Acid Amino acid Ester Prodrug

Reaction Scheme B: Where the carboxyl group ofthe drug is reacted with the hydroxyl group of a hydroxy amino acid wherein the hydroxy group is on the side chain to form the ester prodrug.

H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

Drug Hydroxy Amino Acid Amino Acid Ester Prodrug

Reaction Scheme C: Where the amine group ofthe drug is reacted with the carboxyl group ofthe amino acid to from the amide prodrug

R- NH₂ + HOOC-R1- NH2 -» R-NH(C=O)-R NH₂ Drug Amino Acid Amino Acid Amide Prodrug

Reaction Scheme D: Where the carboxyl group ofthe drug is reacted with the carboxyl group ofthe amino acid to form the anhydride prodrug.

R-COOH + HOOC-R1- NH2 ^■> R-(C=O)-O-(C=O)-Rι- NH₂

Drug Amino Acid Amino Acid Anhydride Prodrug

Reaction Scheme E: Where the amine group ofthe drug is reacted with the amine group ofthe amino acid to form the azo prodrug derivative.

R- NH₂ + NH2-R1- COOH ^■» R-N=N-Rι-COOH

Drug Amino Acid Amino Acid Azo Prodrug

Reaction Scheme F: Where the carboxyl group ofthe drug is reacted with amine group ofthe amino acid to form the amide prodrug.

R-COOH + NH₂-R₁- COOH ^• R-(C=O)NH-Rι- COOH

Drug Amino Acid Amino Acid Amide Prodrug

In the above schemes A-F, the preferred amino acids used are shown hereinbelow:

H:\work\1652\17δ90\SPEC\17690.spec without claimsILdoc

glycine (gly) sarcosine (sar) alanine (ala) valine (val) isoleucine (lie) leuciπe (leu)

setine (ser) homoserine threonine (thr) carnitiπe choline aspartate (asp) glutamate (glu)

ornithine

proline (pro) asparagine (asn) glutamine (gln) histidine (his)

phenylalanine (phe) tyrosine (fyi) tryptophan (trp) kyurenine yurenio acid hypaphorine

As used herein the term "amino acid" refers to an organic compound having therein a carboxyl group (COOH) and an amino group (NH₂) or salts thereof. In solution, these two terminal groups ionize to form a double ionized, through overall neutral entity identified as zwitterions. The amine donates an electron to the carboxyl group and the ionic ends are stabilized in aqueous solution by polar water molecules. 10 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc It is the side groups that distinguish the amino acids from each other. Some amino acids, such as lysine, have amino groups on the side chain; other amino acids have side chains containing hydroxy groups, such as threonine, serine, hydroxyproline, and tyrosine; some amino acids have carboxy groups on the side chain, such as glutamic acid or aspartic acid. These functional groups on the side chain also can form a covalent bond with the drug, such as esters, amides, and the like. When these side groups become involved in these linkages, such as hydroxy group, the bond may be depicted as OAA, wherein AA is an amino acid residue having a side chain with a hydroxy group, but without the hydroxy group. Thus, AA by this definition, refers to the amino acid without the hydroxy side group since it took part in the reaction in forming the ester. Moreover, when an ester is formed between the hydroxy group ofthe amino acid and the OH group ofthe drug, the hydroxy group on the carboxy group forms a byproduct with the hydrogen ofthe hydroxy group, thus, the resulting product does not have the OH group on the carboxy group, but just the acyl moiety. When the bond is depicted as C(=O)-NHAA, this means that the amino acid forms as an amide bond between the carboxy group on the drug and the amino group ofthe amino acid. However, as written, since the NH from the amide bond comes from the amino acid, AA is the amino acid without the amino group.

The preferred amino acids are the naturally occurring amino acids. It is more preferred that the amino acids are the α-amino acids. It is also preferred that the amino acids are in the L-configuration. The preferred amino acids include the twenty essential amino acids. The preferred amino acids are Lysine (Lys), Leucine (Leu), Isoleucine (He), Glycine (Gly), Aspartic Acid (Asp), Glutamic Acid (Glu), Methionine (Met), Alanine (Ala), Valine (Val), Proline (Pro), Histidine (His), Tyrosine (Tyr), Serine (Ser), Norleucine (Nor), Arginine (Arg), Phenylalanine (Phe), Tryptophan (Trp), Hydroxyproline (Hyp), Homoserine (Hsr), Carnitine (Car), Ornithine (Ort), Canavanine (Cav), Asparagine (Asn), Glutamine (Gin), Carnosine (Can), Taurine (Tau), djenkolic Acid (Djk), γ-aminobutyric Acid (GABA), Cysteine (Cys) Cystine (Dcy), Sarcosine , 11 H:\work\l 6₅2\17690\SPEC\17690.spec without claimsll.doc (Sar), Threonine (Thr) and the like. The even more preferred amino acids are the twenty essential amino acids, Lys, Leu, lie, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Thr, Arg, Phe, Trp, Gin, Asn, Cys and Ser.

The prodrugs are prepared from a drug having a group thereon which can react with the amino acid.

The preferred drugs that are reacted with amino acids in accordance with various schemes are as follows: Reaction Schemes

Drugs A B C D E F

Cyclosporins YES

Lopinavir YES YES YES

Ritonavir YES YES YES Cefdinir YES YES YES YES YES

Zileuton YES YES YES

Nelfmavir YES YES YES

Flavoxate YES YES YES

Candesarten YES YES YES YES YES Propofol YES

Nisoldipine YES YES YES YES YES

Amlodipine YES YES YES YES YES

Ciprofloxacin YES YES YES YES

Ofloxacin YES YES YES YES Fosinopril YES YES YES

Enalapril YES ^' YES YES

Ramipril YES YES YES

Benazepril YES YES YES

Moexipril YES YES YES Trandolapril YES YES YES 12 H:\work\16₅2\1769Q\SPEC\17690.spec without claimsll.doc Cromolyn YES YES YES YES Amoxicillin YES YES YES YES YES YES Cefuroxime YES YES YES YES YES YES Ceftazimide YES YES YES YES YES YES

> Ce odoxime YES YES YES YES YES YES Atovaquone YES Gancyclovir YES YES YES Penciclovir YES YES YES Famciclovir YES YES YES

) Acyclovir YES YES YES Niacin YES YES YES Bexarotene YES YES YES Propoxyphene YES Salsalate YES YES YES YES

» Acetaminophen YES ibuprofen YES YES YES Lovastatin YES YES YES YES Simavastatin YES YES YES YES Atorvastatin YES YES YES YES

) Pravastatin YES YES YES YES Fluvastatin YES YES YES YES Nadolol YES Valsartan YES YES YES Methylphenidate YES YES YES YES

> Sulfa Drugs YES YES Sulfasalazine YES Methylprednisolone YES Medroxyprogesterone YES Estramustine YES

) Miglitol YES 13 H:\work\1652\1769Q\SPEC\17690.spec without claimsll.doc Mefloquine YES YES

Capacitabine YES

Danazol YES

Eprosartan YES YES YES

Divalproex YES YES YES

Fenofibrate YES YES YES

Gabapentin* YES YES YES YES YES

Omeprazole YES

Lansoprazole YES

Megestrol YES

Metformin YES

Tazorotene YES YES YES

Sumitriptan YES

Naratriptan YES

Zolmitriptan YES

Aspirin YES YES YES

Olmesartan YES YES YES

Sirolimus YES

Tacrolimus YES

Clopidogrel YES YES YES

Amphotericin B YES YES YES YES

Tenofovir YES

Unoprostone YES YES YES

Fulvestrant YES

Cefditoren YES YES YES

Efavirenz YES

Eplerenone YES YES YES

Treprostinil YES YES YES YES

Adefovir YES

14 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc The prodrug ofthe present invention contains amino groups and as such are basic in nature. They are capable of forming a wide variety of pharmaceutically acceptable salts with various inorganic and organic acids. These acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmaceutically acceptable anions, such as the hydrochloride, hydrobromide, hydroiodide, nitride, sulfate, bisulfate, phosphate, formate, acetate, citrate, tartate, lactate, and the like.

As indicated herein, in one embodiment, the present invention is directed to a prodrug wherein the prodrug comprises a drug, e.g., cyclosporine and an amino acid esterified to the MeBmt (x-y=CH=CH) or dihydro MeBmt moiety, (x-y=CH₂CH₂). The amino acid is attached to the cyclosporine and to the other other drugs by a covalent bond.

The compounds ofthe present invention are prepared by art recognized techniques. For examples, if the drug contains an OH group, said as cyclosporin, then an amino acid or an acylating derivatives thereof, such as the acid halide, e.g., amino acid fluoride, amino acid chloride, or an amino acid alkyl ester wherein alkyl group contains 1-6 carbon atoms is reacted with the carboxy group ofthe drug, e.g., cyclosporine under esterification condition. Preferably, the reaction is conducted in the presence of an acid, such as hydrochloric acid, hydrobromic acid, p-toluenesulfonic acid and the like.

Alternatively, as described hereinabove, if the drug has an amino group thereon, then the amino acid may be reacted with the drug under amide forming conditions to form an amide as the covalent bond. Or if the drug has a carboxy group or acylating derivative thereon, it may be reacted with the amino group ofthe amino acid to form an amide under amide forming conditions to form an amide bond between the amino acid and the drug. Additionally if the drug has a carboxy group therein, the hydroxy group ofthe side chain ofthe amino acid may be reacted with the carboxy group or acylating derivative, therein under esterification conditions to form the ester linkage between the amino acid and the drug, as described hereinabove.

15 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc If the amino acid has a group thereon which is reactive under the reaction conditions it is protected by a protecting group known in the art. After the completion ofthe reaction, the protecting group is removed. Examples of protecting groups that could be used are described in the book entitled, "Protective Group in Organic Synthesis" by Theodora W. Greene, John Wiley & Sons, 1981 , the contents of which are incorporated by reference.

For example, if amino acids with carboxylic groups in their side chains, for example, aspartic acid and glutamic acid, are used in the aforementioned synthesis, these will generally require protection ofthe side chain carboxylic acid. Suitable protecting groups can be esters, such as cyclohexyl esters, t-butyl esters, benzyl esters, allyl esters, 9- fluorophenyl-methyl groups or adamantyl groups, such as 1-or 2-adamantyl which can be protected after the esterfication reaction is completed using techniques known to one of ordinary skill in the art.

If amino acids with hydroxyl groups in their side chains, for example, serine, threonine, hydroxyproline, and the like and amino acids with phenolic groups in their side chains, for example, tyrosine, and the like are used in the aforementioned esterification, reaction, they will desirably require protection ofthe chain hydroxyl or phenolic group. Suitable for protecting groups for the hydroxyl side chain groups can be ethers, such as benzyl ether or t-butyl ether. Removal ofthe benzyl ether can be effected by liquid hydrogen fluoride, while the t-butyl ether can be removed by treatment with trifluoroacetic acid. Suitable protecting groups for phenolic side chain groups can be ethers, as above, including benzyl or t-butyl ether or 2,6-dichlorobenzyl, 2- bromobenzyloxycarbonyl, 2,4-dintrophenyl and the like.

Moreover, the products can be purified to be made substantially pure by techniques known to one of ordinary skill in the art, such as by chromatography, e.g., HPLC, crystallization and the like. By substantially "pure" it is meant that the product contains no more than about 10% impurity therein.

16 H:\work\l 6₅2\17690\SPEC\17690.spec without claimsll.doc The prodrugs can be made into pharmaceutical compositions including prodrugs of, or pharmaceutical acceptable salts, pharmaceutical acceptable solvates, esters, enantiomers, diastereomers, N-Oxides, polymorphs, thereof, as described herein, along with a pharmaceutical acceptable carrier, and optionally but desirably pharmaceutically acceptable excipients using techniques known to one of ordinary skill in the art.

The prodrugs utilized in the present method are used in therapeutically effective amounts.

The physician will determine the dosage of the prodrugs of the present invention which will be most suitable and it will vary with the form of administration and the particular compound chosen, and furthermore, it will vary depending upon various factors, including but not limited to the patient under treatment and the age ofthe patient, the severity ofthe condition being treated and the like and the identify ofthe prodrug administered. He will generally wish to initiate treatment with small dosages substantially less than the optimum dose ofthe compound and increase the dosage by small increments until the optimum effect under the circumstances is reached. It will generally be found that when the composition is administered orally, larger quantities of the active agent will be required to produce the same effect as a smaller quantity given parenterally. The compounds are useful in the same manner as the corresponding drug in the non-prolong form and the dosage level is ofthe same order of magnitude as is generally employed with these other therapeutic agents. When given parenterally, the compounds are administered generally in dosages of, for example, about 0.001 to about 10,000 mg/kg/day, also depending upon the host and the severity ofthe condition being treated and the compound utilized.

In a preferred embodiment, the compounds utilized are orally administered in amounts ranging from about 0.01 mg to about 1000 mg per kilogram of body weight per day, depending upon the particular mammalian host or the disease to be treated, more preferably from about 0.1 to about 500 mg/kg body weight per day. This dosage 17 H:\work\16₅2\1769₀\SPEC\1769Q.spec without claimsll.doc regimen may be adjusted by the physician to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies ofthe therapeutic situation.

The prodrug may be administered in any convenient manner, such as by oral, intravenous, intramuscular or subcutaneous routes.

The prodrug may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly into the food ofthe diet. For oral therapeutic administration, the prodrug may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% ofthe prodrug. The percentage ofthe compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% ofthe weight ofthe unit. The amount ofthe prodrug used in such therapeutic compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention contain between about 200 mg and about 4000 mg of prodrug. The tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials ofthe above type, a liquid carrier.

Various other materials may be present as coatings or otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such 18 H:\work\1652\17690\SPEC\l 7690.spec without claimsll.doc as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations. For example, sustained release dosage forms are contemplated wherein the active ingredient is bound to an ion exchange resin which, optionally, can be coated with a diffusion barrier coating to modify the release properties ofthe resin or wherein the prodrug ofthe present invention is associated with a sustained release polymer known in the art, such as hydroxypropylmethylcellulose and the like.

The prodrug may also be administered parenterally or intraperitoneally. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, e.g., PEG 100, PEG 200, PEG 300, PEG 400, and the like, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form is usually sterile and must be fluid to the extent that syringability exists. It must be stable under the conditions of manufacture and storage and usually must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and one or more liquid polyethylene glycol, e.g. as disclosed herein and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersions and by the use of surfactants. The prevention ofthe action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, tbimerosal, and the like. In many cases, it will be preferable to 19 H:\work\16₅2\17690\SPEC\17690.spec without claimsll.doc include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the prodrug in the required amount in the appropriate solvent with various ofthe other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders, the above solutions are vacuum dried or freeze-dried, as necessary.

The prodrug can also be applied topically, as e.g., through a patch using techniques known to one of ordinary skill in the art. The prodrug can be administered buccally by preparing a suitable formulation of the prodrug of the present invention and utilizing procedures well known to those skilled in the art. These formulations are prepared with suitable non-toxic pharmaceutically acceptable ingredients. These ingredients are known to those skilled in the preparation of buccal dosage forms. Some of these ingredients can be found in Remington's Pharmaceutical Sciences, 17^th edition, 1985, a standard reference in the field. The choice of suitable carriers is highly dependent upon the exact nature ofthe buccal dosage form desired, e.g., tablets, lozenges, gels, patches and the like. All of these buccal dosage forms are contemplated to be within the scope ofthe present invention and they are formulated in a conventional manner.

The formulation ofthe pharmaceutical compositions may be prepared using conventional methods using one or more physiologically and/or pharmaceutically acceptable carriers or excipients. Thus, the compounds and their pharmaceutically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, or rectal administration. For oral administration, the pharmaceutical compositions may take the 20 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (for example, pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropylmethyl cellulose); fillers (for example, lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (for example, magnesium stearate, talc, or silica); disintegrants (for example, potato starch, or sodium starch glycolate); or wetting agents (for example, sodium lauryl sulfate). The tablets may be coated by methods well known in the art.

Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicles before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives, such as suspending agents (for example, sorbitol syrup, co syrup, cellulose derivatives or hydrogenated edible oils and fats); emulsifying agents (for example, lecithin or acacia); non-aqueous vehicles (for example, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (for example, methyl or propyl p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated to give controlled release ofthe active prodrug.

The prodrug ofthe present invention may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, for example, in ampoules, or in multi- dose containers, with an added preservative. The compositions may take such forms as suspension, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the prodrug may be in the powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

21 H:\work\1652\17690\SPEC\l 7690.spec without claimsll.doc The prodrugs ofthe present invention may also be formulated in rectal compositions such as suppositories or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the prodrug ofthe present invention may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the prodrugs may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions containing the prodrugs ofthe present invention may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredients. The pack may for example comprise metal or plastic foil, such as blister pack. The pack or dispenser device may be accompanied by instructions for administration.

In tablet form, it is desirable to include a lubricant which facilitates the process of manufacturing the dosage units; lubricants may also optimize erosion rate and drag flux. If a lubricant is present, it will be present on the order of 0.01 wt. % to about 2 wt. %, preferably about 0.01 wt. % to 0.5 wt, %, ofthe dosage unit. Suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, sodium stearylfumarate, talc, hydrogenated vegetable oils and polyethylene glycol. As will be appreciated by those skilled in the art, however, modulating the particle size of the components in the dosage unit and/or the density ofthe unit can provide a similar effect- -i.e., improved manufacturability and optimization of erosion rate and drug flux—without addition of a lubricant.

22 H:\work\16₅2\17690\SPEC\17690.spec without claimsll.doc Other components may also optionally be incorporated into the dosage unit. Such additional optional components include, for example, one or more disintegrants, diluents, binders, enhancers, or the like. Examples of disintegrants that may be used include, but are not limited to, crosslinked polyvinylpyrrolidones, such as crospovidone (e.g., Polyplasdone® XL, which may be obtained from GAF), cross-linked carboxylic methylcelluloses, such as croscanmelose (e.g., Ac-di-sol®, which maybe obtained from FMC), alginic acid, and sodium carboxymethyl starches (e.g., Explotab®, which may be obtained from Edward Medell Co., Inc.), agar bentonite and alginic acid. Suitable diluents are those which are generally useful in pharmaceutical formulations prepared using compression techniques, e.g., dicalcium phosphate dihydrate (e.g., Di-Tab®, which may be obtained from Stauffer), sugars that have been processed by crystallization with dextrin (e.g., co-crystallized sucrose and dextrin such as Di-Pak®, which may be obtained from Amstar), calcium phosphate, cellulose, kaolin, mannitol, sodium chloride, dry starch, powdered sugar and the like. Binders, if used, are those that enhance adhesion. Examples of such binders include, but are not limited to, starch, gelatin and sugars such as sucrose, dextrose, molasses, and lactose. Permeation enhancers may also be present in the novel dosage units in order to increase the rate at which the active agents pass through the buccal mucosa. Examples of permeation enhancers include, but are not limited to, dimethylsulfoxide ("DMSO"), dimethyl formamide ("DMF"), N,N-dimethylacetamide ("DMA"), decylmefhylsulfoxide ("CioMSO"), polyethylene glycol monolaurate ("PEGML"), glycerol monolaurate, lecithin, the 1 -substituted azacycloheptan-2-ones, particularly 1-n- dodecylcyclazacycloheptan-2-one (available under the trademark Azone.RTM. from Nelson Research & Development Co., Irvine, Calif.), lower alkanols (e.g., ethanol), SEP A® (available from Macrochem Co., Lexington, Mass.), cholic acid, taurocholic acid, bile salt type enhancers, and surfactants such as Tergitol®, Nonoxynol-9® and TWEEN-80®.

Flavorings may be optionally included in the various pharmaceutical formulations. Any suitable flavoring may be used, e.g., mannitol, lactose or artificial sweeteners such as 23 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc aspartame. Coloring agents may be added, although again, such agents are not required. Examples of coloring agents include any ofthe water soluble FD&C dyes, mixtures of the same, or their corresponding lakes.

In addition, if desired, the present dosage units may be formulated with one or more preservatives or bacteriostatic agents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chloride, or the like.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents for pharmaceutical active substances well known in the art. Except insofar as any conventional media or agent is incompatible with the prodrug, their use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of prodrug calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

The prodrug is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore described. A unit dosage, for example, contains the principal active compound in amounts ranging from about 10 mg e.g. in humans, or as low as 1 mg (for small animals) to about 2000 mg. If placed in solution, the concentration ofthe prodrug preferably ranges from about 10 mg/mL to about 250 mg/mL. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration ofthe said ingredients. In the case of buccal administration, the prodrugs are preferably in the buccal unit dosage form present in an amount ranging from about 10 to about 50 mg. 24 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc The prodrugs ofthe present invention are effective in treating disease or conditions in which the corresponding drug (without the amino acid prodrug ofthe present invention) normally are used.

As used herein the term "treating" refers to reversing, alleviating or inhibiting the progress of a disease, disorder or condition, or one or more symptoms of such disease, disorder or condition, to which such term applies. As used herein, "treating" may also refer to decreasing the probability or incidence ofthe occurrence of a disease, disorder or condition in a mammal as compared to an untreated control population, or as compared to the same mammal prior to treatment. For example, as used herein, "treating" may refer to preventing a disease, disorder or condition, and may include delaying or preventing the onset of a disease, disorder or condition, or delaying or preventing the symptoms associated with a disease, disorder or condition. As used herein, "treating" may also refer to reducing the severity of a disease, disorder or condition or symptoms associated with such disease, disorder or condition prior to a mammal's affliction with the disease, disorder or condition. Such prevention or reduction ofthe severity of a disease, disorder or condition prior to affliction relates to the administration ofthe composition ofthe present invention, as described herein, to a subject that is not at the time of administration afflicted with the disease, disorder or condition. As used herein "treating" may also refer to preventing the recurrence of a disease, disorder or condition or of one or more symptoms associated with such disease, disorder or condition. The terms "treatment" and "therapeutically," as used herein, refer to the act of treating, as "treating" is defined above.

As used herein the term "patient" or "subject" refers to a warm blooded animal, and preferably mammals, such as, for example, cats, dogs, horses, cows, pigs, mice, rats and primates, including humans. The preferred patient is humans.

25 H:\work\1652\1769Q\SPEC\17690.spec without claimsll.doc The prodrugs ofthe present invention exhibit the same utility as the corresponding drug without the amino acid linkage. The prodrug exhibits an enhanced therapeutic quality. That is, they exhibit at least one and more preferably at least two enhanced therapeutic qualities relative to the drug which has not been transformed to the prodrug ofthe present invention prior to administration. These include, but are not limited to a. improved taste, smell b. Desired octanol/water partition coefficient (i.e., solubility in water/fat)

The various amino acids have different solubility in aqueous solutions. By selecting a particular amino acid, the octanol water partition coefficient can be affected. For example, many drugs in the following list are highly hydrophobic. The amino acids are highly hydrophilic. For example, assume propofol is the drug and lysine is the amino acid. Propofol is completely insoluble in water, while lysine is soluble to the extent of 700 mg/ml. When these two diverse molecules are esterified via an ester bond, the resulting lysine ester of propfol has a solubility in water in excess of 250 mg/ml.

On the other hand, cromolyn sodium is highly water soluble. For all practical purpose, it is not absorbed when administered orally. By affecting its water solubility one could improve absorption. In this case, one would look for conditions opposite to that of propofol, i.e., the goal is to decrease water solubility. By choosing apporoprieate low water soluble amino acids, such as tyrosine, one can achieve proper hydrophilic/lipophilic balance. c. Improved stability in- vitro and in- vivo d. Enhanced penetration of blood-brain barrier e. Elimination of first-pass effect in liver, i.e., the drug not metabolized in liver and therefore more drug in system circulation f. Reduction of entero-hepatic recirculation (this improves bio-availability) g. Painless injections with parenteral formulations h. Improved bio-availability i. Improved changes in the rate of absorption (increase vs lack thereof) 26 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc j. Reduced side effects k. Dose proportionality

A dose proportionality claim requires that when the drug is administered in escalating doses, proportionally escalating amounts of active drag is delivered into the blood stream. This is measured by determining the area under the plasma concentration vs. time curve obtained after administering a drug via any route other than IV route and measuring the same in plasma/blood. A simple mathematical procedure is as follows: For example, a drug is administered at e.g., 3 different doses, 10, 100 and 1000 mg, orally to a patient, the area under the plasma concentration time curve (AUC) is measured. Then each total AUC is divided by the dose, and the result should be the same for all three doses. If it is the case, then there is dose proportionality. Lack of dose proportionality indicates any one or more ofthe pharmacokinetic/pharmacological mechanisms are saturable, including absorption, metabolism or the number of receptor sites available for pharmacological response.

For example in the above study, assume the AUC values of 100, 1000 and 10,000 are obtained, in this case the dose proportionality is inappropriate. When there is lack of dose proportionality, there is either more or less amount of drug in the plasma, depending upon which mechanism is saturable. The following are the possibilities:

Saturable Absorption. If this is the case, as the dose is increased, proportionally less and less ofthe drug is absorbed, hence overall AUC will decrease as the dose is increased.

Saturable metabolism of elimination. If thus is the case, then more and more ofthe drag will be circulating in the blood, and the AUC will increase with increasing dose.

Saturable pharmacological receptor sites: In this case, since all the receptor sites will eventually be occupied by the drug, any additional drag will not increase the response. Thus, increasing dose will not result in increasing response.

27 H:\work\1652\17690\SPEC\l 7690.spec without claimsILdoc Dose proportionality is an excellent response profile, since one can predict accurately the pharmacological response and curative power at all doses. Thus dose proportionality is a desirable quality for any drug. Furthermore, achievement of dose proportionality is also dependent upon the formulation, and fed/fasted differences.

1. Selective hydrolysis of the prodrug at site of action m. Controlled release properties n. Targeted drug delivery o. Reduction in toxicity, hence, improved therapeutic ratio p. Reduced dose q. Alteration of metabolic pathway to deliver more drag at the site of action r. Increased solubility in aqueous solution s. Enhanced efficacy

Thus, various dosage forms available with amino acid pro-drugs and they are prepared by conventional methods: i. Oral liquid dosage (Controlled release and immediate release liquids containing sugar and sugar free, dye and dye free, alcohol and alcohol free formulations, including chewable tablets) ii. Oral solid dosage (Controlled release and immediate release tablets, capsules and caplets iii. Intravenous (Injections, both ready to use and lyophilized powders) iv. Intramuscular (Injections, both ready to use and lyophilized powders) v. Subcutaneous (injections, both ready to use and lyophilized powders) vi. Transdermal (Mainly patches) vii. Nasal (Sprays, formulations for nebulizer treatments) viii. Topical (Creams, ointments) ix. Rectal (Creams, ointments and suppositories) x. Vaginal (Creams, ointments and pessaries) xi. Ocular (Drops and ointments) 28 H:\work\lf552\1769Q\SPEC\1 690.spec without claimsll.doc xii. Buccal (Chewable and now chewable tables)

Many drugs discussed herein, especially in the table hereinbelow are characteristically highly hydrophobic and readily precipitate in the presence of even very minor amounts of water, e.g., on contact with the body (e.g., stomach fluids). It is accordingly extremely difficult to provide e.g., oral formulations which are acceptable to the patient in terms of form and taste, which are stable on storage and which can be administered on a regular basis to provide suitable and controlling patient dosing.

Proposed liquid formulations, e.g., for oral administration of a number of drugs shown herein in the table have heretofore been based primarily on the use of ethanol and oils or similar excipient as carrier media. Thus, the commercially available drink-solutions of a number of drags employ ethanol and olive oil or com oil as carrier medium in conjunction with solvent systems comprising e.g., ethanol and LABRIFIL and equivalent excipient as carrier media. For example, the commercially available

Cyclosporin drink solution employs ethanol and olive oil or com oil as carrier medium in conjunctions with a Labroid as a surfactant. See e.g., U.S. Patent NO. 4,388,307. Use ofthe drink solution and similar composition as proposed in the art is however accompanied by a variety of difficulties.

Further, the palatability ofthe known oil based system has proved problematic. The taste ofthe known drink-solution of several drags is, in particular, unpleasant. Admixture with an appropriate flavored drink, for example, chocolate drink preparation, at high dilution immediately prior to ingestion has generally been practiced in order to make regular therapy at all acceptable. Adoption of oil-based systems has also required the use of high ethanol concentrations which is itself inherently undesirable, in particular where administration to children is foreseen. In addition, evaporation ofthe ethanol, e.g., from capsules (adopted in large part, to meet problems of palatability, as discussed or other forms (e.g., when opened)) results in the development of a drug precipitate. Where such compositions are presented in, for example, soft gelatin encapsulated form, 29 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc this particular difficulty necessitates packaging ofthe encapsulated product in an airtight component, for example, an air-tight blister or aluminum-foil blister package. This in turn renders the product both bulky and more expensive to produce. The storage characteristics ofthe aforesaid formulations are, in addition, far from ideal.

Bioavailability levels achieved using existing oral dosage system for a number of drugs described herein are also low and exhibit wide variation between individuals, individual patient types and even for single individuals at different times during the course of therapy. Reports in the literature indicates that currently available therapy employing the commercially available drag drink solution provides an average absolute bioavailability of approximately 10-30% only, with the marked variation between individual groups, e.g., between liver (relatively low bioavailability) and bone-marrow (relatively high bioavailability) transplant recipients. Reported variation in bioavailability between subjects has varied from one or a few percent for some patients, to as much as 90% or more for others. And as already noted, marked change in bioavailability for individuals with time is frequently observed. Thus, there is a need for a more uniform and high bioavailability of a number drags shown herein in patients.

Use of such dosage forms is also characterized by extreme variation in required patient dosing. To achieve effective therapy, drag blood or blood serum levels have to be maintained within a specified range. This required range can in turn, vary, depending on the particular condition being treated, e.g., whether therapy is to prevent one or more pharmacological actions of a specific drug and when alternative therapy is employed concomitantly with principal therapy. Because ofthe wide variations in bioavailability levels achieved with conventional dosage forms, daily dosages needed to achieve required blood serum levels will also vary considerably from individual to individual and even for a single individual. For this reason it may be necessary to monitor blood/blood-serum levels of patients receiving drug therapy at regular and frequent intervals. Monitoring of blood/blood-serum levels has to be carried out on a regular

30 H:\work\16₅2\1769₀\SPEC\17690.spec without claimsll.doc basis. This is inevitably time consuming and inconvenient and adds substantially to the overall cost of therapy.

It is also the case that blood/blood serum levels of a number of drags described herein achieved using available dosage systems exhibit extreme variation between peak and trough levels. That is for each patient, effective drag levels in the blood vary widely between administrations of individual dosages.

There is also a need for providing a number of drugs described herein, especially the beta-lactum antibiotics, Cyclosporin, cephalosporins, steroids, quinolone antibiotics and Cyclosporin, in a water-soluble form for injection. It is well known that Cremaphore L® (CreL) used in cairrent formulations of a number of drags described hereinbelow is a polyoxyethylated derivative of castor oil and is a toxic vehicle. There have been a number of incidences of anaphylaxis due to the castor oil component. At present there is no formulation that would allow many of these drags to be in aqueous solution at the concentrations needed due to poor water solubility ofthe drag.

Beyond all these very evident practical difficulties lies the occurrence of undesirable side reactions already alluded to, observed employing available oral dosage forms.

Several proposals to meet these various problems have been suggested in the art, including both solid and liquid oral dosage forms. An overriding difficulty which has however remained is the inherent insolubility ofthe several ofthe drags shown in the table hereinbelow in aqueous media, hence preventing the use of a dosage form which can contain the drugs in sufficiently high concentration to permit convenient use and yet meet the required criteria in terms of bioavailability, e.g. enabling effective absorption from the stomach or gut lumen and achievement of consistent and appropriately high blood/blood-serum levels.

31 H:\work\l 6₅2\17690\SPEC\17690.spec without claimsILdoc The particular difficulties encountered in relation to oral dosing with these drags have inevitably led to restrictions in the use of specific drug therapy for the treatment of relatively less severe or endangering disease conditions. For example, taking Cyclosporin as a test drug, a particular area of difficulty in this respect has been the adoption of Cyclosporin therapy in the treatment of autoimmune diseases and other conditions affecting the skin, for example for the treatment of atopic dermatitis and psoriasis and, as also widely proposed in the art, for hair growth stimulation, e.g. in the treatment of alopecia due to ageing or disease.

Thus while oral Cyclosporin therapy has shown that the drag is of considerable potential benefit to patients suffering e.g. from psoriasis, the risk of side-reaction following oral therapy has prevented common use. Various proposals have been made in the art for application of Cyclosporins, e.g. Cyclosporin, in topical form and a number of topical delivery systems have been described. Attempts at topical application have however failed to pro ide any demonstrably effective therapy.

However, the present invention overcomes the problems described hereinabove. More specifically, the prodrug ofthe present invention significantly enhances its solubility in aqueous solutions relative to the non-prodrag form ofthe pharmaceutical, thereby avoiding the need to utilize a carrier, such as ethanol or castor oil when administered as a solution. Moreover, the prodrugs of these drugs, in accordance with the present invention, do not exhibit the side effects ofthe prior art formulations. Further, it has been found that when many ofthe drags in the table hereinbelow is administered in its prodrug form in accordance with the present invention, there is enhanced oral absorption, thereby enhancing significantly its bioavailability and its efficacy.

The preferred drugs used in combination with the amino acids are forming prodrugs are listed hereinbelow in the following table and the benefits found are as listed in the penultimate column ofthe table. In the table, the key is as follows: a) Improved taste smell 32 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc b) Desired Octanol/water partition coefficient (i.e. solubility in water) c) improved stability in vitro and in vivo d) Penetration of blood-brain barrier e) Elimination of first pass effect in liver f) Reduction of enterohepatic recirculation g) Painless injections with parenteral formulations h) Improved bioavailability i) Increased rate of absorption j) Reduced side effects k) Dose proportionability 1) Selective hydrolysis ofthe prodrag at site of actions m) Controlled release properties n) Targeted drug delivery o) Reduction in toxicity, hence improved therapeutic ratio p) Reduced dose q) Alteration of metabolic pathway to deliver more drug at site of action.

Moreover, the table indicates the utility ofthe prodrag. The utility ofthe prodrag is the same as the corresponding drug (without the amino acid moiety attached). The utiluity is described in the literature such as the Physicians Desk Reference, 2004 edition, the contenets of which are incorporated by reference.

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Ceftazidime All doses expressed as drug base Lys, Leu, He, Gly, Asp, Lys, Leu, He, Gly, Hyp, Ser, Thr, a, b, c, g, antibiotic, treatmtent off

Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Gly, & Lys h, i,j, k, l, lower respiratory tract fl Powder for IV 0.1-5 gm 0.25-4 gm 0.5-2 gm His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, °, P, q infections, including ~ Oral Tab/Cap Phe, Tφ, Hyp, Hsr, Car, Thr, Arg, Phe, Tφ, 0.1-1 gm 0.25-1 gm 0.5-1 gm pneumonia caused by \ Oral Liquid Ort, Cav, Asn, Gin, Can, Gin, Asn, Cys and 0.1-2.5 0.25-2 0.5-1 pseudomonas, H. r; jk, GABA, Cys, Ser, Hyp, Sar gm 5ml gm/5ml gm/5ml Tau, D influenzae, Klebsiella, jϋ Dcy, Thr, and Sar Enterbacter, E. coli, prgtgus rnirabilis, streptococcus^ staphylococcus; skin anp skin structure infections,^";: caused by p^eudomona⁵sE aeruginosa, Klebsiella, Hfe coli, Proteus enterbactesg staphylococcus, O streptococcus, urinary feact infections casued by pseudomonas aeruginosa, enterbacter, proteus, Klebsiella, E. coli; bone and joint infections caused by pseudomonas, eruginosa, Klebsiella, Enterbacter, or staphylococcus; gynecologic infections including endometritis, pelvic cellulits and infections ofthe female genital tract caused by E. coli, intra-abdominal infections and central nervous system infections, including meningitis

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The following non-limiting examples further illustrate the invention:

Synthesis of Various Amino Acid Derivatives of Selected Drugs I. Propofol Derivatives

Propofol (2,6-diisopropylphenol) is a low molecular weight phenol which widely used as a central nervous system anesthetic, and posses sedative and hypnotic activities. It is administered intravenously in the induction and maintenance of anesthesia and/or sedation in mammals. The major advantages of Propofol are that it can induce anesthesia rapidly, minimal side effects and upon withdrawal, the patient recovers quickly without prolonged sedation. PROPOFOL

Propofol has been shown to have a large number of therapeutic applications, which are quite varying and somewhat surprising. For example, it has been shown to be an effective antioxidant, anti-emetic, anti-pruritic, anti-epileptic, anti-inflammatory, and even seems to possess anti-cancer properties.

Mechanism of Action:

The mechanism of action of Propofol has been extensively studied. Its central nervous system anesthetic activity has been shown to be related its high affinity for a specific subclass of GABA receptors (Collins G.G.S., 1988, Br. J. Pharmacology. 542, 225-232). However, there are a number of different receptors in the brain which are substrates for propofol, hence its varied activities.

57 H:\work\16₅2\17690\SPEC\1769Q.spec without claimsll.doc Propofol also has significant biological effect as an antioxidant. Because of this generalized activity of propofol, it is theoretically useful in the treatment of a number of inflammatory processes where oxidation is an important factor. For example, cyclooxygenase mediated prostaglandin synthesis results in inflammation. By inhibiting oxidation in the respiratory tract, one could use propofol in the treatment of acid aspiration, adult/infant respiratory distress syndrome, airway obstructive diseases, asthma, cancer and a number of other similar pathological conditions.

Since oxidative tissue damage is a very common occurrence, it has been suggested that propofol could be useful in the treatment of Parkinson's disease, Alzheimer disease, Friedrich's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, spinal chord injuries, and various other neurodegenerative diseases.

Propofol is currently available in the US market as an intravenous emulsion marketed by Astra Zenaca under the brand name Diprivan®. It is one the most widely used short acting central nervous system anesthetics in the market. The concentration of propofol is 10 mg/mL in non-pyrogenic sterile emulsion and the formula contains soybean oil, glycerol, egg lecithin, disodium edetate and sodium hydroxide.

A significant disadvantage of Propofol is that it is completely insoluble in water. Even at very low concentrations of 10 mg/mL, the drug precipitates out of an aqueous solution in room temperature. Therefore, manufacturers of this formulation use heroic methods to emulsify this product in water using extraordinarily complex and toxic emulsifying agents. For example, manufacturers ofthe IV formulations use egg lecithin, Cremaphor L®, castor oil, and other similar emulsifiers.

However, use of such emulsifiers is associated with number of problems. It is well now that various types of Cremaphor L® emulsifiers can precipitate allergic reactions. Egg lecithin and castor oil have been shown to produce anaphylactic shock in some patients. Furthermore, maintenance of stability of propofol in these emulsions is short lived and 58 H:\wori \1652\17690\SPEC\17690.spec without claimsll.doc more expensive. Moreover, the presence of egg lecithin and castor oil make the emulsion prone to microbial growth. It may be possible to dissolve propofol in water by complexing it with cyclodextrin, but cyclodextrin has not been approved by the FDA for use in intravenous therapy.

Heretofore, no one has made a safe prodrug of propofol. The British patents 1,102,011, and 1,160,468 and US Patent 3,389,138 describe the various phenol esters of amino acids, wherein the propofol is attached to a number of side chains which when released in the body produce toxic effects.

US Patent 6,451,854 describe a number of substituted alpha amino acetic acid esters of propofol, wherein propofol and the side chain were substituted with a number of different chemical groups. All the N,N-disubstituted glycine esters of propofol have not shown to be non-toxic and there many ofthe compounds described are derivative of propofol. Thus when released in the body after the cleavage of ester by the enzymes, many ofthe active drugs released are not propofol, and hence they do not possesses any toxicity data and are entirely new molecules with unknown therapeutic efficacy in man.

In yet another published paper on the water soluble salts of amino acid esters of anesthetic agent propofol, (Int. J. Pharmaceutics, 175[2]: 195-204, 1998) authors have synthesized a number of water soluble derivatives of propofol. However, when these prodrugs are cleaved by esterase enzymes, substituted non-natural amino acids with unknown toxicity profile are released in the body.

Until now there has been no pharmaceutical preparation has been available in the market that can deliver propofol without harmful side effects. The present invention however, has produced a number of water soluble, non-toxic derivatives of propofol which are suitable for delivering propofol in the body without any harmful side effects and without the needs for toxic and expensive additives, solubilizers and emulsifiers.

59 H:\work\1652\17690\SPEC\1769Q.spec without claimsll.doc Accordingly, in one aspect, the present invention is directed to a class of prodrugs of Propofol. The prodrug consists ofthe carboxyl group of an amino acid esterified to the free hydroxyl group present on the propofol molecules.

More specifically, one aspect ofthe present invention is directed to, the compounds of the formulae POFOL

or pharmaceutically acceptable salts thereof; wherein AA is an amino acid, in which the carboxyl group of AA is reacted with the hydroxyl group ofthe Propofol.

In antoher aspect, the present invention is also directed to a pharmaceutical composition comprising a therapeutically effective amount ofthe various Propofol prodrugs above and a pharmaceutical carrier therefor.

In another embodiment, the present invention is directed to a method of treating a patient in need of propofol therapy, which method comprises administering to said patient an effective amount ofthe Propofol.

In a further embodiment, the present invention is directed to a method of enhancing the solubility of propofol in an aqueous solution comprising reacting the hydroxyl functionality ofthe Propofol and isolating the products thereof.

In a still further embodiment, the present invention is directed to a method of substantially and in a therapeutically efficacious manner, reducing or eliminating the potential toxic side effects of current formulations containing toxic exepients when

60 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc administered to a patient which comprises reacting the hydroxyl functionality ofthe propofol molecule with carboxyl function of selected amino acids to form an ester covalent bond respectively and isolating the product thereof and administering said product to the patient.

The current invention shows that when unsubstituted naturally occurring amino acids are esterified to propofol, the resulting prodrugs are highly water soluble, (>200 mg/L in water), release non-toxic amino acids upon cleavage in the body and require none ofthe toxic emulsifier, additives and other exepients.

Furthermore, it has been shown that the current invention also produced drugs, while they are prodrugs of propofol ofthe present invention are highly effective central nervous system anesthetics. Thus the current amino acid prodrugs are effective central nervous system anesthetics, with or without releasing the active parent drug.

The amino acid esters ofthe present invention are at least 10 times more soluble that propofol in water in room temperature. Especially the glycine, proline and lysine esters of propofol are soluble at the range of more than 100 mg/ml, and in case of lysine it is greater than 250 mg/mL.

While the prodrugs ofthe present invention are not expected to possess any antioxidant activity due to blockage ofthe phenolic group responsible for such; however the present inventor has found that the prodrugs of propofol are effective anesthetics with or without releasing propofol. The propofol prodrugs described release the propofol when administered in vivo and the resulting drug maintains its pharmacological and anti- oxidant properties.

The prodrug of propofol ofthe present invention clearly provides a number of advantages over propofol, for example, all ofthe side chains cleaved from these prodrugs are naturally occurring essential amino acids and hence are non-toxic. This 61 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc results in high therapeutic index. Secondly all the prodrugs are readily cleaved in the body to release propofol. Furthermore, due their high water solubility, they can be easily administered by either forming an in-situ solution just before IV administration using lyophilized sterile powder or providing the drug in solution in prefilled syringe or bottles for infusion. The aminoacid esters are more stable than propofol since OH group in propofol is blocked to oxidation. Thus the propofol prodrugs ofthe present invention are more effective then propofol itself without the toxicity and other pharmaceutical problems associated with current marketed formulations.

The prodrugs of propofol ofthe present invention possess anti-inflammatory, anti- oxidant, anti-cancer, anti-convulsive, anti-emetic and anti-pruritic properties.

These prodrugs of propofol ofthe present invention are effective in treating diseases or conditions in which Propofol normally are used. The prodrugs disclosed herein are transformed within the body to release the active compound and enhances the therapeutic benefits ofthe Propofol by reducing or eliminating biopharmaceutical and pharmacokenetic barriers associated with each of them. However it should be noted that these prodrugs themselves will have sufficient activity without releasing any active drug in the mammals. Since the prodrugs are more soluble in water then Propofol, it does not need to be associated with a carrier vehicle, such as alcohol or castor oil which may be toxic or produce unwanted side reactions. Moreover, oral formulations containing the prodrugs of Propofol are absorbed into the blood and are quite effective.

Thus, the prodrug ofthe present invention enhances the therapeutic benefits by removing biopharmaceutical and pharmacokenetic barriers of existing drugs.

Furthermore, the prodrugs are easily synthesized in high yields using reagents which are readily and commercially available.

62 H:\work\16₅2\17690\SPEC\1769₀.spec without claimsILdoc Overview:

The procedure for the synthesis ofthe glycine, L-proline, and L-lysine esters of Propofol is depicted hereinbelow. However, these are exemplary and any amino acid produrugs thereof can be prepared using the following methodology. The complete procedure and analytical data is given in the Experimental Section. In general, as shown in the following scheme Propofol (10 g) was coupled with the N-Boc protected amino acid (1 equivalent) with l-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC) in the presence of a catalytic amount of 4-(N,N-dimethyamino)-pyridine (DMAP). The EDC was removed by extraction with water. After drying over sodium sulfate, filtration, and concentration the crude protected amino acid ester of Propofol was purified by flash chromatography to generate the protected esters in 50-60% yield. The protecting groups were then removed by stirring the protected esters in diethyl ether saturated with hydrochloric acid (gas) at room temperature. Yields for the deprotection step were generally 60-95%. After filtration and drying the hydrochloride salts ofthe glycine and L-proline esters of Propofol did not require additional purification. The hydrochloride salt ofthe L-lysine-Propofol ester was crystallized once from ethanol to remove a trace of mono-protected L-lysine-Propofol ester.

Synthetic Sequence:

63 H:\work\l 6₅2\17690\SPEC\17690.spec without claimsll.doc 1. SPIO010

Propofol Boc-glyciπe SP10010

2. SPIO011

SCHEME

Synthesis of the glycine, L-proline, and L-lysine esters of Propofol: a) EDC, DMAP, CH₂C1₂; b) HCl (g), Et₂O.

Experimental Section:

The synthesis of SPIOOIO, SPIOOll and SPI0013 were conducted in batches. Generally a small-scale experiment was performed first followed by a larger batch. Reagents mentioned in the experimental section were purchased at the highest obtainable purity from Aldrich, Acros, or Bachem, except for solvents, which were purchased from either Fisher Scientific or Mallinkrodt.

64 H:\work\16₅2\17690\SPEC\17690.spec without claimsll.doc 1) SPIOOIO

Propofol (9.98 g, 55.97 mmole) was dissolved in dichloromethane (200 mL) at room temperature, under an argon atmosphere. N-t-Butyloxocarbonyl-glycine (11.2 g, 63.91 mmole) was added along with l-(3-dimemylaminoρropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 11.1 g, 57.9 mmole) and 4-( ,N-dimethylamino)-pyridine (DMAP, 1.5 g, 12.27 mmole). After stirring for 21 hours under an argon atmosphere at room temperature, water (200 mL) was added and the layers were separated. The dichloromethane layer was washed again with water (200 mL) and dried for 1 hour over sodium sulfate (5 g). After filtration and concentration under reduced pressure, the remaining oil was purified by flash chromatography on silica gel (250 g), eluting with hexanes/ethyl acetate (10:1). The procedure generated the protected N-BOC protected glycine ester of Propofol as a white solid (11.34g, 60% yield).

tert-Butoxycarbonylamino-acetic acid 2,6-diisopropylphenyl ester:

1H NMR (300 MHz, CDC1₃): δ = 7.25-7.13 (m, 3H), 5.18 (br s, IH), 4.22 (d, 2H, J= 5.7 Hz), 2.89 (m, 2H), 1.46 (s, 9H), 1.18 (d, 12H, J= 6.9 Hz).

¹³C NMR (75 MHz, CDC1₃): δ = 169.35, 155.75, 145.22, 140.35, 126.90, 124.14, 80.32, 42.66, 28.54, 27.79, 23.57.

The Propofol-Boc-gycine ester (11.28 g, 33.6 mmole) was dissolved in anhydrous diethyl ether (200 mL) at room temperature. Hydrochloric acid (gas) was passed through the solution for 45 minutes while stirring. The mixture was allowed to stir at room temperature for 48 hours under an argon atmosphere. After 48 hours hexanes (200 mL) were added and the precipitate was filtered. The white solid was dried under high

65 H:\work\16₅2\17690\SPEC\17690.spec without claimsll.doc vacuum for 5 hours at 88 °C. The experiment produced SPIOOIO (8.73 g, 95% yield, purity 99.9% by HPLC) as a white solid.

SPIO010

Amino-acetic acid 2,6-diisopropyl-phenyl ester, hydrochloride:

1H NMR (300 MHz, CDC1₃): δ = 8.77 (br s, 3H), 7.20-7.08 (m, 3H), 4.14 (m, 2H), 2.87 (m, 2H), 1.11 (d, 12H, J= 7 Hz).

¹³C NMR (75 MHz, CDCI₃): δ =166.42, 144.84, 140.42, 127.10, 124.06, 40.47, 27.61, 23.55.

CHN analysis: calc: C 61.87, H 8.16, N 5.15; found: C 61.14, H 8.20, N 5.14.

2) SPIOOll

Propofol (10.03 g, 56.23 mmole) was dissolved in dichloromethane (100 mL) at room temperature, under an argon atmosphere. N-t-Butyloxocarbonyl-L-proline (14.04 g, 65.22 mmole) was added along with l-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 11.95 g, 62.33 mmole) and 4-(N,N-dimethylamino)-pyridine (DMAP, 1.1 g, 9.0 mmole). After stirring for 3 hours under an argon atmosphere at room temperature, water (100 mL) was added and the layers were separated. The dichloromethane layer was washed again with water (100 mL) and dried for 1 hour over sodium sulfate (5 g). After filtration and concentration under reduced pressure, the remaining oil was purified by flash chromatography on silica gel (250 g), eluting with hexanes/ethyl acetate (10:1). The procedure generated the protected N-BOC protected

66 H:\work\16₅2\17690\SPEC\17690.spec without claimsILdoc L-proline ester of Propofol as a clear oil (11.34g, 66% yield) that solidified on standing in the freezer.

Pyrrolidine-l,2-dicarboxylic acid 1-tert-butyl ester 2-(2,6-diisopropyl-phenyl) ester:

^lH NMR (300 MHz, CDC1₃): δ = 7.31-7.20 (m, 3H), 4.73 (m, IH), 3.70-3.50 (m, 2H), 3.20-2.94 (m, 2H), 2.46-2.20 (m, 2H), 2.20-2.0 (m, 2H), 1.55 (m, 9H), 1.25 (m, 12H).

¹³C NMR (75 MHz, CDC1₃): δ = 171.87, 171.01, 154.34, 153.93, 145.35 145.23, 140.06, 140.21, 126.69, 126.53, 123.95, 80.28, 79.89, 59.14, 46.67, 46.42, 31.10, 30.17, 28.61, 28.56, 28.56, 27.44, 27.18, 23.47.

The Propofol-Boc-L-proline ester (13.95 g, 37.14 mmole) was dissolved in anhydrous diethyl ether (100 mL) at room temperature. Hydrochloric acid (gas) was passed through the solution for 60 minutes while stirring. The mixture was allowed to stir at room temperature for 22 hours under an argon atmosphere. After 22 hours hexanes (50 mL) were added and the precipitate was filtered. The white solid was dried under high vacuum for 5 hours at 88 °C. The experiment produced SPIOOll (9.1 g, 81% yield, purity 99.1% by HPLC) as a white solid.

SPI0011 Pyrrolidine-2(S)-carboxylic acid 2,6-diisopropyl-phenyl ester, hydrochloride:

67 H:\work\1652\l 7690\SPEC\17690.spec without claimsll.doc ^!H NMR (300 MHz, CDC1₃): δ = 10.15 (hr s, 2H), 7.27-7.14 (m, 3H), 4.78 (t, IH, J= 7.8 Hz), 3.56 (m, 2H), 2.85 (m, 2H), 2.64 (m, IH), 2.40 (m, IH), 2.20 (m, IH), 2.05 (m, IH), 1.18 (m, 12H).

¹³C NMR (75 MHz, CDC1₃): δ = 168.30, 144.23, 139.74, 126.98, 123.96, 51.58, 38.21, 29.32, 26.64, 26.18, 23.71, 23.02, 21.67.

CHN analysis: calc: C 65.48, H 8.40, N 4.49; found: C 65.50, H 8.43, N 4.50.

3) SPI0013

The dicyclohexylamine salt of di-N-boc-L-lysine (23.62 g, 0.0447 mole) was added to diethyl ether (200 mL) and potassium hydrogen sulfate (9.14 g) in water (200 mL) that was cooled in an ice/water bath. After strirring for 20 minutes, the layers were separated. The ether layer was extracted three times with cold water (100 mL). The ether layer was then dried over sodium sulfate (15 g) for one hour, filtered, and concentrated under reduced pressure. The procedure generated the free acid of N,N'- di- boc-L-lysine (15.5 g, 100% recovery).

Propofol (8.0 g, 45 mmole) was dissolved in dichloromethane (100 mL) at room temperature, under an argon atmosphere. N, N'-di-t-Butyloxocarbonyl-L-lysine (15.5 g, 44.7 mmole) was added along with l-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 8.62 g, 45 mmole) and 4-(N,N-dimethylarnino)-pyridine (DMAP, 0.55 g, 4.5 mmole). After stirring for 3 hours under an argon atmosphere at room temperature, water (100 mL) was added and the layers were separated. The dichloromethane layer was washed again with water (100 mL) and dried for 1 hour over sodium sulfate (5 g). After filtration and concentration under reduced pressure, the remaining oil was purified by flash chromatography on silica gel (250 g), eluting with hexanes/ethyl acetate (9:1). The procedure generated the protected N-BOC protected L- lysine ester of Propofol as a white foam (12.42 g, 55% yield). 68 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

2(S),6-Bis-t-butoxycarbonylamino-hexanoic acid 2,6-diisopropyl-phenyl ester:

Η NMR (300 MHz, CDC1₃): 5 = 7.28-7.15 (m, 3H), 5.22 (d, IH, J= 8.4 Hz), 4.70 (m, IH), 4.59 (m, IH), 3.17 (m, 2H), 2.93 (m, 2H), 2.09 (m, IH), 1.86 (m, IH), 1.67-1.54 (m, 4H), 1.48 (s, 9H), 1.46 (s, 9H), 1.20 (m, 12H).

¹³C MR (75 MHz, CDC1₃): δ = 171.82, 156.10, 155.65, 145.25, 140.30, 126.80, 124.03, 80.14, 79.28, 53.76, 40.29, 32.09, 28.66, 28.54, 27.48, 23.91, 23.10.

The Propofol-di-Boc-L-lysine ester (12.34 g, 24.37 mmole) was dissolved in anhydrous diethyl ether (250 mL) at room, temperature. Hydrochloric acid (gas) was passed through the solution for 60 minutes while stirring and cooling in an ice/water bath. The mixture was allowed to stir at room temperature for 48 hours under an argon atmosphere. After 48 hours the precipitate was filtered and crystallized from ethanol (100 mL). The white solid was dried under high vacuum for 4 hours at 90 °C. The experiment produced SPI0013 (5.5 g, 60% yield, purity 98.6% by HPLC) as a white solid.

2(S),6-Diamino-hexanoic acid 2,6-diisopropyl-phenyl ester, dihydrochloride:

69 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc ^lH NMR (300 MHz, CDC1₃): δ = 9.05 (br s, 3H), 8.35 (br s, 3H), 7.26-7.13 (m, 3H), 4.43 (t, IH, J= 6 Hz), 3.0-2.6 (m, 4H), 2.09 (m, 2H), 1.80-1.50 (m, 4H), 1.10 (d, 12H, J= 7 Hz).

¹³C NMR (75 MHz, CDC1₃): δ = 168.30, 144.23, 139.74, 126.98, 123.96, 51.58, 38.21, 29.32, 26.64, 23.71, 23.02, 21.67.

CHN analysis: calc: C 56.99, H 8.50, N 7.38; found: C 56.48, H 8.56, N 7.30.

II. PRO DRUGS OF NON-STEROIDAL ANTI-INFLAMMATORY DRUGS (NSAIDs)

The NSAIDs comprise a class of structurally distinctive, carboxylic acid moiety attached to a planar aromatic functionality, Examples include: acetyl salicyclic acid, salicyclic acid, diflunisal, ibuprofen, fenoprofen, carprofen, flurbiprofen, ketoprofen, naproxen, sulindac, indomethacin, etodolac, tolmetin, ketorolac, diclofenac, and meclofenamate. The NSADIs posess anti-inflammatory, analgesic, antipyretic and anti-clotting activity.

Examples ofthe chemical structures of this uniques class of compounds showing wide variety of pharmacological activities are shown below.

70 H:\work\1652\l 7690\SPEC\17690.spec without claimsll.doc

Salicylic Acid

cety sa cy c cid Diflunisal

Fenoprofen

Ketoprofen Naproxen Etodolac

Tolmetin Ketorolac

NSAIDs are widely used for the treatment of acute and chronic pain, management of edema, tissue damage resulting from inflammatory joint diseases and also, effective anti- clotting agents in the treatment of myocardial infraction. A number ofthe agents also possess antipyretic activity in addition to analgesic and anti-inflammatory action, thus useful in reducing fever.

Some drugs in the above group have also been prescribed for Rheumatoid Arthritis, Osteoarthritis, acute gout, ankolysing spondylitis, and dysmenorrhea.

71 H:\work\l 6₅2\17690\SPEC\17690.spec without claimsll.doc Mechanism of Action:

The major mechanism by which the NSAIDs produce their therapeutic effect is via inhibition of prostaglandin synthesis. Specifically NSAIDs inhibit cyclooxygenases, such as COX-1 and COX-2 enzymes, where these two enzymes are responsible for synthesis of prostaglandins. While COX-1 enzyme is important for the regulation of platelet aggregation, regulation of blood flow in kidney and stomach, and regulation of gastric acid secretion, COX-2 enzyme plays an important role in the pain and inflammatory processes. NSAIDs significantly increase clotting time and can be used for prophylaxis of thromboembolism and myocardial infarction.

All NSAIDs are relatively medium to strong organic acids with pKa's in the 3-6 range. Most of them are carboxylic acid derivatives. Acidic group is essential for COX inhibitory activity and in physiological pH, all the NSAIDs are ionized. All of them have quite varying hydrophilic lipophilic balance, and these are functions of their aryl, aromatic and aliphatic side chains and other heterocyclic variations in their structures. Most ofthe NSAIDs are highly bound to plasma proteins and often competitively replace other drugs which have similar affinity for plasma proteins. Hence concomitant administration of NSAIDs with other therapeutic class must be carefully evaluated to prevent drug interactions. Most ofthe drugs, due to acidic carboxyl group are metabolized by the mammals via conjugation. The major pathway of metabolic clearance of a number of NSAIDs is glucuronidation followed by renal elimination.

Use of acetylsalicylic acid (aspirin) in the prophylaxis of coronary heart diseases is now well known, and this drug has proved to be a lifesaver for a number of patients with myocardial infarction. Several additional uses have already been documents for aspirin, for example, it was recently reported in the medical journal Lancet (Vol 349, p 1641) that aspirin reduces the risk of stroke in patients with early warning signs of transient ischemic heart attacks. Pre-eclampsia and fetal growth retardation, both caused by blockages ofthe blood vessels ofthe placenta, are two ofthe commonest complications of pregnancy - there are millions of cases of pre-eclampsia in the world each year. In a 72 H:\work\l 652\17690\SPEC\17690.spec without claimsll.doc trial involving more than 9000 women in 16 countries, a daily dose of 60mg aspirin reduced the risk of pre-eclampsia by 13 per cent. (Aspirin Foundation website). Aspirin has also been shown to be effective in some studies to prevent colon cancer, lung cancer and pancreatic cancer in post-menopausal women. Since aspirin can improve blood flow, its usefulness in the treatment of diabetes certain forms of dementia such as Alzheimer' s disease are becoming increasingly clear.

Because of their unique pharmaceutical potential, the NSAIDs have attracted considerable attention in the press. The primary area of clinical investigation for above drugs has been as non-steroidal anti-inflammatory agents, in particular in relation to their application to patients suffering from pain, arthritis, (Rheumatoid and Osteo) other inflammatory reactions, fever and for the prophylaxis of coronary heart diseases. These drugs are also used in the treatment of migraine headache, menstrual syndromes, back pain and gout.

Despite the very major contribution which NSAIDs have made, difficulties have been encountered in providing more effective and convenient means of administration (e.g., galenic formulations, for example, oral dosage form, which are both convenient and for the patient as well as providing appropriate bioavailability and allowing dosaging at an appropriate and controlled dosage rate) as well as the reported occurrence of undesirable side reactions; in particular severe gastric and duodenal ulcers, mucosal erythema, and edema, erosions, perforations, blood in stool, ulcerative colitis have been obvious serious impediments to their wider use or application. The dual injury theory involves NSAID- mediated direct damage, followed by a systemic effect in which prostaglandin synthesis is inhibited. Topical injury may also occur as a result ofthe biliary excretion of active hepatic metabolites and subsequent duodenogastric reflux. (Arthritis and Rheumatism 1995; 38(1):5-18) The effects are additive; either topical or systemic mechanisms alone are sufficient to produce gastro duodenal mucosal damage.

73 H:\work\1652\1769Q\SPEC\17690.spec without claimsll.doc Moreover, the above mentioned NSAIDs are characteristically highly hydrophobic and readily precipitate in the presence of even very minor amounts of water, e.g., on contact with the body (e.g., stomach fluids). It is accordingly extremely difficult to provide e.g., oral formulations which are acceptable to the patient in terms of form and taste, which are stable on storage and which can be administered on a regular basis to provide suitable and controlling patient dosaging.

Proposed liquid formulations, e.g., for oral administration of NSAIDs, have heretofore been based primarily on the use of natural gums, like Xanthan, cellulose, citric acid, and lime flavor etc. See e.g., U. S . Patent NO. 5,780,046. Commercially available NSAIDs drink-solution employs incompatible orange color and berry flavor, citric acid, Xanthan Gum, polysorbate 80, pregelatinized starch, glycerin, sodium benzoate, and additional artificial colors and flavors. Use ofthe drink solution and similar composition as proposed in the art is however accompanied by a variety of difficulties.

Further, the palatability ofthe known oil based system has proved problematic. The taste of he known drink-solution is, in particular, unpleasant. Admixture with an appropriate flavored drink, for example, chocolate drink preparation, at high dilution immediately prior to ingestion has generally been practiced in order to make regular therapy at all acceptable. Adoption of oil based systems has also required the use of high ethanol concentrations to itself inherently undesirable, in particular where administration to children is forseen. In addition, evaporation ofthe ethanol, e.g., from capsules (adopted in large part, to meet problems of palatability, as discussed or other forms (e.g., when opened) results in the development of a NSAID precipitate. Where such compositions are presented in, for example, soft gelatin encapsulated form; this particular difficulty necessitates packaging ofthe encapsulated product in an air-tight component, for example, an air-tight blister or aluminum-foil blister package. This in turn renders the product both bulky and more expensive to produce. The storage characteristics ofthe aforesaid formulations are, in addition, far from ideal.

74 H:\ ork\1652\17690\SPEC\17690.spec without claimsILdoc Gastric irritability ofthe NSAIDs has been a topic of great concern to the practicing physicians and as well as patients. Acute uses of aspirin, fenoprofen, flurbiprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, and piroxicam produce serious GI side effects. Even Ibuprofen is shown to cause severe gastric lesions upon long term use. Gastrointestinal toxicity is the most frequently encountered side effect associated with NSAIDs and presents considerable concern. Approximately one half of all hospital admissions for a bleeding ulcer are attributed to the use of NSAIDs, aspirin, or the two taken in combination during the week prior to admission. (Faulkner G, Prichard P, Somerville K, et al. Aspirin and bleeding peptic ulcers in the elderly. Br Med J. 1988; 297:1311-1313). A survey of Tennessee Medicaid patients who were hospitalized with GI complications showed that patients who used NSAIDs had approximately a fourfold greater risk for developing GI hemorrhage or peptic ulcer disease than patients not taking NSAIDs. (Griffin MR, Piper JM, Daugherty JR, et al. Nonsteroidal anti-inflammatory drug use and increased risk for peptic ulcer disease in elderly persons. Ann Intern Med. 1991; 114:257-263). Serious GI events, according to the FDA, occur in as many as 2% to 4% of patients per year who are taking continuous NSAiD therapy for rheumatoid arthritis. The relative risk of gastric ulcer (4.725), duodenal ulcer (1.1 to 1.6), bleeding (3.8), perforation, and death are all increased by NSAID use when such patients are compared to those who do not take these products. In 1989, patients with rheumatoid arthritis had approximately 20,000 hospitalizations per year with an estimated cost of $10,000 per stay. (Fries JF, Miller SR, Spitz PW, et al. Toward an epidemiology of gastropathy associated with nonsteroidal anti-inflammatory drug use. J Gastroenterology. 1989; 96:647-655).

There is also a need for providing some ofthe NSAIDs in a water soluble form for injection. It is well known that high concentrations of alcohol and tromethamine used to form a salt in the current formulations of Ketorolac are toxic. At present there is no formulation that would allow the NSAIDs to be in aqueous solution at the concentrations needed due to poor water solubility ofthe drug.

75 H:\work\I652\17690\SPEC\17690.spec without claimsll.doc Beyond all these very evident practical difficulties lies the occurrence of undesirable side reactions already alluded to, observed employing available oral dosage forms.

Several proposals to meet these various problems have been suggested in the art, including both solid and liquid oral dosage forms. An overriding difficulty which has however remained is the inherent insolubility ofthe NSAIDs in aqueous media, hence preventing the use of a dosage form which can contain NSAIDs in sufficiently high concentration to permit convenient use and yet meet the required criteria in terms of bioavailability, e.g. enabling effective resorption from the stomach or gut lumen and achievement of consistent and appropriately high blood/blood-serum levels.

The present prodrugs of NSAIDs overcome the problems described hereinabove. More specifically, an embodiment ofthe present invention is directed to a prodrug of NSAID which significantly enhances its solubility in aqueous solutions, thereby avoiding the need to utilize a carrier, such as ethanol or castor oil when administered as a solution. Moreover, the prodrugs of NSAID, in accordance with the present invention, do not exhibit the side effects ofthe prior art formulations. Further, the prodrugs ofthe present invention are almost completely devoid of gastric irritability upon oral administration, thereby enhancing significantly the therapeutic index ofthe prodrugs tested and their efficacy.

Accordingly, in one aspect, the present invention is directed to a prodrug of NSAIDs. The preferred prodrugs ofthe NSAIDs have the formula

76 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

Ketoprofen Naproxen Etodolac

Indomethacin Tolmetin Ketorolac

or pharmaceutically acceptable salts thereof; wherein Y is either NH-AA or O-AA and AA is an amino acid, in which either an amine group or the hydroxyl group of AA is reacted with the carboxylic acid group ofthe NSAIDs.

The present invention is also directed to a pharmaceutical composition comprising a therapeutically effective amount ofthe various NSAIDs above and a pharmaceutical carrier therefor.

77 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc In another embodiment, the present invention is directed to a method of treating a patient in need of NSAID therapy, which method comprises administering to said patient an effective amount ofthe NSAIDs.

In a further embodiment, the present invention is directed to a method of enhancing the solubility of NSAID in an aqueous solution comprising reacting the carboxyl functionality of each ofthe NSAIDs and isolating the products thereof.

In a still further embodiment, the present invention is directed to a method of substantially and in a therapeutically efficacious manner, reducing or eliminating the gastric mucosal damage of NSAIDs when administered to a patient which comprises reacting the carboxyl functionality of each ofthe NSAID molecule with either amine or hydroxyl function of selected amino acids to form either an amide or ester covalent bond respectively and isolating the product thereof and administering said product to the patient.

A. Synthesis of Ibuprofen Amino Acid Derivaties Overview:

The procedure for the synthesis ofthe L-serine, L-threonine, and L-hydroxyproline esters of Ibuprofen is outlined in Synthetic Sequence section. The complete procedure and analytical data is given in the Experimental Section. Again, these synthetic schemes are exemplary. The scheme is applicable for other amino acids in the preparation ofthe NSAID prodrugs ofthe present invention. In general, (+)-lbuprofen (4-10 g, in batches) was coupled with the N-benzyloxy/benzyl ester protected a ino acids (1 equivalent) with l-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 1 equivalent) in the presence of a catalytic amount of 4-(N,N- dimethyamino)-pyridine (DMAP). Once the reactions were complete, any excess EDC was removed by extraction with water, DMAP was removed by extraction with dilute acid, and Ibuprofen was removed by extraction with sodium bicarbonate. After drying over sodium sulfate, filtration, and concentration the crude protected amino acid esters 78 H:\work\1652\l 7690\SPEC\17690.spec without claimsll.doc of (±)-Ibuprofen were either used directly or purified by flash chromatography on silica gel to generate the protected esters in good yield (85-95%). The column chro iatography was generally not necessary if a slight excess of Ibuprofen and coupling agent were used, and a thorough extraction procedure was conducted. The protecting groups were removed by hydrogenation (25-35 psi H₂) in the presence of 10% palladium on carbon and hydrochloric acid. Yields for the deprotection step ranged from 70-90%. After filtration and drying the hydrochloride salts ofthe serine and threonine esters of (±)- Ibuprofen were purified by crystallization. The hydrochloride salt ofthe L- hydroxyproline-Ibuprofen ester was a gel that would not solidify/crystallize. In this case the hydrogenation was repeated without the use of acid and the neutral compound was purified.

Because the ibuprofen started as a mixture of enantiomers, the final products were delivered as a mixture of diastereomers except for the threonine ester. In the case ofthe threonine ester of Ibuprofen, washing with water, acetone or acetonitrile could readily separate the final diastereomeric salts. The insoluble isomer (SPI0016A) was determined to be the active isomer by comparison with an authentic standard prepared from S-(+)-Ibuprofen. The serine and hydroxyproline esters of (±)-lbuproferx could not be readily separated in this fashion.

79 H:\work\l 6₅2\17690\SPEC\17690.spec without claimsll.doc Synthetic Sequence:

1. SPI0015

(±)-lbuprofen Z-Ser-OBz SPI001501

2. SP!0016A and SPI0016B

(±)-lbuprofen Z-Thr-OBz

C)

SPI0016A SPI0016B 3. SPI0017

(±)-lbuprofen Z-Hyp-OBz SPI001701 SPI0017

Synthesis of the L-serine, L-threonine, and L-hydroxyproline esters of (±)-Ibuprofen: a) EDC, DMAP, CH₂C1₂; b) HCl, 10% Pd/C, EtOH c) acetone, d) 10% Pd/C, EtOH.

80 H:\work\16₅2\17690\SPEC\17690.spec without claimsll.doc Experimental Section:

The synthesis of SPI0015, SPI0016 and SPI0017 were conducted in two or three batches. Reagents mentioned in the experimental section were purchased at the highest obtainable purity from Sigma- Aldrich, Acros, or Bachem, except for solvents, which were purchased from either Fisher Scientific or Mallinkrodt.

1) Preparation of (±)-Ibuprofen-L-serine ester, hydrochloride (SPI0015).

(±)-Ibuprofen (5.04 g, 24.4 mmole), N-carbobenzyloxy-L-serine benzyl ester (8.11 g, 24.6 mmole), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC,

4.87g, 25.4 mmole), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.40 g, 3.27 mmole) were dissolved in dichloromethane (150 mL) at room temperature, under an argon atmosphere. After stirring for 22 hours under an argon atmosphere at room temperature, water (100 mL) was added and the layers were separated. The dichloromethane layer was washed again with water (100 mL) and dried for 1 hour over sodium sulfate (5 g). After filtration and concentration under reduced pressure, the remaining oil was purified by flash chromatography on silica gel (250 g), eluting with hexanes/ethyl acetate (3:1). The procedure generated the protected L-serine-(±)- Ibuprofen ester (SPI001501) as a colorless solid (11.4 g, 90% yield).

SPI001501

2(S)-Benzyloxycarbonylamino-3-[2(R,S)-(4-isobutyl-phenyl)-propionyloxy]-propioπic acid benzyl ester:

1H NMR (300 MHz, CDC1₃): δ = 7.40-7.20 (m, 10H), 7.14-7.01 (m, 4H), 5.50 (d, HH, J= 8.4 Hz), 5.29 (d, HH, J= 8.4 Hz), 5.11-5.02 (m, 2.5H), 4.90 (d, fcH, J= 12 Hz), 4.62

81 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc (m, IH), 4.49-4.43 (m, IH), 4.36-4.32 (m, IH), 3.59 (m, IH), 2.39-2.35 (m, 2H), 1.78 (m, IH), 1.42-1.39 (m, 3H), 0.85 (d, 6H, J= 6.6 Hz).

¹³C NMR (75 MHz, CDC1₃): δ = 174.05, 169.19, 169.07, 155.68, 140.73, 137.20, 136.12, 135.05, 134.91, 129.44, 128.67, 128.65, 128.60, 128.41, 128.33, 128.30, 128.19, 127.19, 127.16, 67.75, 67.32, 64.51, 64.32, 53.71, 45.16, 45.02, 30.35, 22.60, 18.27.

The protected Ibuprofen-L-serine ester (22.50 g, 43.4 mmole) was dissolved in ethanol (200 mL) at room temperature and added to a Parr bottle that contained 10% palladium on carbon (3.86 g, 50% wet) under a nitrogen atmosphere. Hydrochloric acid (10 mL 37% HCl in 30 mL water) was added and the nitrogen atmosphere was replaced with hydrogen gas (25 psi). After 4 hours of shaking, the palladium catalyst was removed by filtration through celite. The ethanol/water was removed under reduced pressure. The remaining white solids were washed with water (25 mL), acetone (20 mL) and dried under high vacuum (4 hours at 88 °C). The experiment produced (±)-lbuprofen-L-serine ester, hydrochloride SPI0015 (11.3 g, 80% yield) as a colorless solid.

SPI0015

2(S)-Amino-3-[2(R,S)-(4-isobutylphenyl)-propionyloxy]-propionic acid, hydrochloride; ((R,S)-Ibuprofen-L-Serine ester, hydrochloride):

^!H NMR (300 MHz, DMSO): δ = 8.92 (br s, 3H), 7.22 (t, 2H, J= 7.5 Hz), 7.10 (d, 2H, J= 7.5 Hz), 4.56 (m, IH), 4.37-4.20 (m, 2H), 3.83 (q, IH, J= 6.9 Hz), 2.41 (d, 2H, J= 6.9 Hz), 1.80 (m, IH), 1.41 (d, 3H, J= 6.9 Hz), 0.85 (d, 6H, J= 6.9 Hz).

82 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc ¹³C NMR (75 MHz, DMSO): δ = 173.36, 173.32, 168.08, 168.04, 139.70, 128.96, 129.92, 127.20, 127.05, 62.47, 51.59, 51.49, 44.28, 44.00, 43.90, 29.68, 22.28, 18.70, 18.42.

HPLC analysis:

99.13% purity; rt = 3.133 min; Luna C18 5u column (sn 167917-13); 4.6x250 mm; 254 nm; 50% ACN/50% TFA buffer (0.1%); 35 C; 20 ul inj.; lml/min; 1 mg/mL sample size; sample dissolved in mobile phase.

CHN analysis: calc: C 58.27, H 7.33, N 4.25; found: C 58.44, H 7.46, N 4.25.

Melting point: 169.5 - 170.5 °C

2a) Preparation and Separation of (±)-Ibuprofen-L-threonine ester, hydrochloride (SPI0016A and SPI0016B).

(±)-Ibuprofen (4.15 g, 20.11 mmole), N-carbobenzyloxy-L-threonine benzyl ester (6.90 g, 20.11 mmole), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 3.95 g, 20.6 mmole), and 4-(N,N-dimethylamino)-ρyridine (DMAP, 0.25 g, 2.0 mmole) were dissolved in dichloromethane (50 mL) at room temperature, under an argon atmosphere. After stirring for 19 hours, the dichloromethane layer was washed with water (50 mL), 5% hydrochloric acid (2x25 mL), water (25 mL), saturated sodium bicarbonate (2x25 mL), and water (50 mL). After drying for one hour over sodium sulfate (5 g), filtration, and concentration under reduced pressure, the remaining oil was used without further purification. The procedure generated the protected L-threonine- (±)-lbuprofen ester (SPI001601) as a light yellow oil (10.2 g, 95.3% yield), which solidified on standing.

83 H:\work\1652\l 7690\SPEC\1769Q.spec without claimsll.doc

2(S)-Benzyloxycarbonylamino-3-[2(R,S)-(4-isobutyl-phenyl)-propionyloxy]-butyric acid benzyl ester:

Η NMR (300 MHz, CDC1₃): δ = 7.40-7.15 (m, 10H), 7.14-7.01 (m, 4H), 5.48-5.25 (m, 2H), 5.11-5.01 (m, 3H), 4.90 (d, %H, J= 12 Hz), 4.68 (d, HH, J= 12 Hz), 4.48 (m, IH), 3.60-3.48 (m, IH), 2.39(m, 2H), 1.79 (m, IH), 1.42-1.35 (m, 3H), 1.27 (d, 1.5 H, J= 6.6 Hz), 1.17 (d, 1.5 H, J= 6.6 Hz), 0.85 (m, 6 H).

¹³C NMR (75 MHz, CDC1₃): δ = 173.32, 169.70, 169.30, 156.55, 140.75, 137.38,

137.22, 136.14, 135.07, 134.99, 129.45, 129.41, 128.65, 128.39, 128.22, 127.21, 127.14, 70.97, 70.70, 67.81, 67.66, 67.53, 57.83, 45.19, 30.39, 22.61, 18.57, 18.30, 17.18, 16.87.

The protected Ibuprofen-L-threonine ester (10.15 g, 19.0 mmole) was dissolved in warm ethanol (150 mL) and added to a Parr bottle that contained 10% palladium on carbon (3.4 g, 50% wet) under a nitrogen atmosphere. Hydrochloric acid (6 mL 37% HCl in 20 mL water) was added and the nitrogen atmosphere was replaced with hydrogen gas (30 psi). After 3 hours of shaking, the palladium catalyst was removed by filtration tlirough celite (30 g). The ethanol/water was removed under reduced pressure. The experiment produced (±)-Ibuprofen-L-threonine ester, hydrochloride (SPI0016A and SPI0016B, 6.4 g, 97% crude yield) as a colorless solid. The crude mixture of diastereomers was stirred in acetone (200 mL) for 2 hours at room temperature under an argon atmosphere. After 2 hours the solids (2.84 g, SPI0016A) were filtered. The filtrate (SPI0016B, 3.0 g) was concentrated under reduced pressure.

84 H:\work\l₆52\17690\SPEC\17690.spec without claimsILdoc 1.) Purification of SPI0016A (active isomer):

After 3 batches ofthe S-Ibuprofen-L-threonine ester (SPI0016A) had been completed, the batches were combined (8.78 g total) and crystallized three times from DIUF water (100 mL). Each time a small amount of zwitterion was generated. In order to regenerate the salt, the solid generated (from each crystallization) was dissolved in 1% hydrochloric acid in ethanol (3 mL 37% hydrochloric acid in 100 mL ethanol). The ethanol solution was then concentrated under reduced pressure at room temperature. After the third crystallization and regeneration procedure, the salt (5.6 g) was stirred in acetonitrile (100 mL) for 44 hours at room temperature, under an argon atmosphere. The salt was then filtered and dried under high vacuum at 50-55 ° until the weight was constant (5.5 g).

Cl SPI0016A

2(S)- Amino-3 (R)-[2(S)-(4-isobutyl-phenyl)-propionyloxy] -butyric acid; (S-Ibuprofen-L-threonine ester, hydrochloride, active isomer):

1H NMR (300 MHz, DMSO): δ = 8.76 (br s, 3H), 7.19 (d, 2H, J= 8.1 Hz), 7.11 (d, 2H, J= 8.1 Hz), 5.28 (dq, IH, J= 6.3, 3.6 Hz), 4.14 (q, IH, J= 3.6 Hz), 3.80 (q, IH, J- 7.2 Hz), 2.41 (d, 2H, J= 7.2 Hz), 1.80 (m, IH), 1.37 (d, 3H, J- 7.2 Hz), 1.21 (d, 3H, J= 6.3 Hz), 0.85 (d, 6H, J= 6.6 Hz).

¹³C NMR (75 MHz, DMSO): δ = 172.66, 168.24, 139.68, 137.24, 128.95, 126.97, 67.98, 55.35, 44.23, 43.83, 29.66, 22.24, 18.52, 16.47.

CHN analysis: calc: C 59.38, H 7.62, N 4.07; found: C 59.17, H 7.63, N 4.04. 85 H:\work\16₅2\17690\SPEC\17690.spec without claimsILdoc HPLC analysis:

98.28% purity; r.t.= 6.951 min.; 60% TFA (0.1%)/40% acetonitrile; 1 mL/min; 37.5 C;

Luna C18, 3u column (SN 167917-13), 4.6x250 mm; 22 ul injection.

Optical rotation: + 24.5 °

Melting Point: 189-190 °C

2) Purification of SPI0016B (inactive isomer): After 3 batches of the R-Ibuprofen-L-threonine ester (SPI0016B) had been completed, the batches were combined (9.02 g total) and crystallized from DIUF water (50 mL). A small amount of zwitterion was generated during the crystallization. In order to regenerate the salt, the solid generated was dissolved in 1% hydrochloric acid in ethanol (3 mL 37% hydrochloric acid in 100 mL ethanol). The ethanol solution was then concentrated under reduced pressure at room temperature. The remaining salt (5.93 g) was crystallized three times from hot toluene (100 mL) with the addition of a small amount on acetone (1 mL). The salt was then filtered and dried under high vacuum at room temperature until the weight was constant (5.1 g).

2(S)- Amino-3 (R)- [2(R)-(4-isobutyl-phenyl)-propionyloxy] -butyric acid; (R-Ibuprofen-L-threonine ester, hydrochloride, inactive isomer):

86 H:\work\1052\17690\SPEC\17690.spec without claimsll.doc 1H NMR (300 MHz, DMSO): δ = 8.82 (br s, 3H), 7.23 (d, 2H, J= 7.8 Hz), 7.10 (d, 2H, J= 7.8 Hz), 5.27 (m, IH), 4.18 (m, IH), 3.80 (q, IH, J= 7.2 Hz), 2.41 (d, 2H, J= 7.2 Hz), 1.81 (m, IH), 1.41 (d, 3H, J= 6.9 Hz), 1.34 (d, 3H, J= 6.3 Hz), 0.85 (d, 6H, J= 6.3 Hz).

¹³C NMR (75 MHz, DMSO): δ = 72.56, 168.08, 139.64, 136.98, 128.84, 127.14, 68.8, 55.29, 44.28, 29.69, 22.28, 18.24, 16.41.

CHN analysis: calc: C 59.38, H 7.62, N 4.07; found: C 59.30, H 7.60, N 4.05.

HPLC analysis:

98.43% purity; r.t .= 6.19 min.; 60% TFA (0.1%)/40% acetonitrile; 1 mL/min; 37.5 C;

Luna C18, 3u column (SN 167917-13), 4.6x250 mm; 22 ul injection.

Optical Rotation: + 10.4 °

Melting Point: 176-177 °C

2b) Preparation of the S-(+)-Ibuprofen-L-threonine ester, hydrochloride standard (SPI0016S).

S-(+)-lbuprofen (2.0 g, 9.69 mmole), N-carbobenzyloxy-L-threonine benzyl ester (3.25 g, 9.91 mmole), l-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 1.90 g, 9.91 mmole), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.12 g, 1.0 mmole) were dissolved in dichloromethane (25 mL) at room temperature, under an argon atmosphere. After stirring for 4 hours, the dichloromethane layer was washed with water (25 mL), 5% hydrochloric acid (25 mL), saturated sodium bicarbonate (2x25 mL), and water (25 mL). After drying for one hour over sodium sulfate (5 g), filtration, and concentration under reduced pressure, the remaining oil was used without further purification. The procedure generated the protected S-(+)-Ibuprofen-L-threonine ester (SPI001601S) as a light yellow oil (5.01 g, 98 % yield), which solidified on standing. 87 H:\work\1652\17690\SPEC\I 7690.spec without claimsll.doc

2(S)-Berrzyloxycarbonylamino-3-[2(R,S)-(4-isobutyl-phenyl)-propionyloxy]-butyric acid benzyl ester:

1H NMR (300 MHz, CDC1₃): δ = 7.35-7.23 ( , 10H), 7.10 (d, 2H, J= 7.8 Hz), 7.05 (d, 2H, J= 7.8 Hz), 5.48-5.25 (m, 2H), 5.17-5.01 (m, 4H), 4.50 (dd, IH, J= 9.6, 1.8 Hz), 3.50 (q, IH, J= 7.2 Hz), 2.40 (d, 2H, J= 1.1 Hz), 1.80 (m, IH), 1.37 (d, 3H, J= 7.2 Hz), 1.17 (d, 3 H, J= 6.3 Hz), 0.86 (d, 6 H, J= 6.6 Hz).

¹³C NMR (75 MHz, CDC1₃): δ = 173.29, 169.69, 156.51, 140.68, 137.21, 136.08, 135.06, 129.40, 128.70, 128.66, 128.57, 128.38, 128.24, 127.14, 70.70, 67.80, 67.53, 57.87, 45.19, 45.11, 30.39, 22.61, 18.57, 16.87.

The protected S-(+)-Ibuρrofen-L-threonine ester (5.0 g, 9.40 mmole) was dissolved in warm ethanol (100 mL) and added to a Parr bottle that contained 10% palladium on carbon (1.0 g, 50% wet) under a nitrogen atmosphere. Hydrochloric acid (1 mL 37% HCl in 10 mL water) was added and the nitrogen atmosphere was replaced with hydrogen gas (32 psi). After 2 hours of shaking, the palladium catalyst was removed by filtration through celite (30 g). The ethanol/water was removed under reduced pressure. The experiment produced S-(+)-Ibuprofen-L-threonine ester, hydrochloride (SPI0016S, 2.8 g, 85% crude yield) as a colorless solid. The salt was stirred in acetone (50 mL) for 3 hours at room temperature under an argon atmosphere. After 3 hours the solids (2.24 g, 69% yield) were filtered and dried under high vacuum at room temperature, until the weight was constant.

88 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

Ci SPI0016S

2(S)-Amino-3(R)-[2(S)-(4-isobutyl-phenyl)-propionyloxy]-butyric acid; (S-Ibuprofen-L-threonine ester, hydrochloride, active isomer):

1H NMR (300 MHz, DMSO): δ = 8.76 (br s, 3H), 7.19 (d, 2H, J= 8.1 Hz), 7.11 (d, 2H, J= 8.1 Hz), 5.28 (dq, IH, J= 6.3, 3.6 Hz), 4.14 (q, IH, J= 3.6 Hz), 3.80 (q, IH, J= 7.2 Hz), 2.41 (d, 2H, J= 7.2 Hz), 1.80 (m, IH), 1.37 (d, 3H, J= 7.2 Hz), 1.21 (d, 3H, J= 6.3 Hz), 0.85 (d, 6H, J= 6.6 Hz).

¹³C NMR (75 MHz, DMSO): δ = 172.66, 168.24, 139.68, 137.24, 128.95, 126.97, 67.98, 55.35, 44.23, 43.83, 29.66, 22.24, 18.52, 16.47.

HPLC analysis:

98.28% purity; r.t.= 6.951 min.; 60% TFA (0.1%)/40% acetonitrile; 1 mL/min; 37.5 C; Luna C18, 3u column (SN 167917-13), 4.6x250 mm; 22 ul injection.

Optical rotation: + 26.5 °

Melting Point: 189-190 °C

3) Preparation of the (±)-Ibuprofen-L-hydroxyproline ester (SPI0017).

(±)-lbuprofen (5.10 g, 24.7 mmole), N-carbobenzyloxy-L-hydroxyproline benzyl ester (8.80 g, 24.7 mmole), l-(3-dimemylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 5.10g, 26.0 mmole), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.30 g, 2.40 mmole) were dissolved in dichloromethane (100 mL) at room temperature, under an 89 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc argon atmosphere. After stirring for 24 hours under an argon atmosphere at room temperature, water (100 mL) was added and the layers were separated. The dichloromethane layer was washed again with water (100 mL), 5% sodium bicarbonate (2x50 mL) and dried for 1 hour over sodium sulfate (5 g). After filtration and concentration under reduced pressure, the remaining oil was used without further purification. The procedure generated the protected (±)-lbuprofen-L-hydroxyproline ester (SPI001701) as a light yellow oil (11.5 g, 85% yield).

SPI001701

4(R)-[2-(4-Isobutyl-phenyl)-propionyloxy]-pyrrolidine-2(S)-carboxylic acid; ((R, S)-lbuprofen-L-hydroxyproline ester) :

^XH NMR (300 MHz, CDC1₃): δ = 7.33-7.02 (m, 14H), 5.25-4.95 (m, 5H), 4.51-4.19 (m, IH), 3.75-3.50 (m, 3H), 2.40 (d, 2H, J= 6.9 Hz), 2.15 (m, IH), 1.81 (m, IH), 1.44 (d, 3H, J= 7.0 Hz), 0.87 (d, 6H, J= 6.6 Hz).

¹³C NMR (75 MHz, CDC1₃): δ = 173.99, 171.93, 171.72, 154.68, 154.15, 140.70, 137.23, 137.04, 136.23, 135.44, 135.23, 129.41, 128.59, 128.47, 128.35, 128.19, 128.08, 127.89, 127.02, 72.86, 72.16, 67.40, 67.18, 67.09, 58.12, 57.83, 52.66, 52.49, 52.13, 45.15, 36.63, 35.67, 32.07, 30.33, 29.23, 22.90, 22.58, 18.36.

The protected Ibuprofen-L-hydroxyproline ester (11.40 g, 43.4 mmole) was dissolved in ethanol (150 mL) at room temperature and added to a Parr bottle that contained 10% palladium on carbon (2.73 g, 50% wet) under a nitrogen atmosphere. The nitrogen atmosphere was replaced with hydrogen gas (34 psi). After 5 hours of shaking, the palladium catalyst was removed by filtration through celite. The ethanol was removed

90 H:\work\1652\17690\SPEC\1769Q.spec without claimsILdoc under reduced pressure. The remaining white solids (6.60 g) were washed with DIUF water (50 mL), diethyl ether (50 mL) and dried under high vacuum until the weight was constant. The experiment produced (±)-Ibuprofen-L-hydroxyproline ester SPI0017 (5.64 g, 84% yield) as a colorless solid.

SPI0017

4(R)-[2-(4-Isobutyl-phenyl)-propionyloxy]-pyrrolidine-2(S)-carboxylic acid; ((R,S)-Ibuprofen-L-hydroxyproline ester):

1H NMR (300 MHz, CDC1₃): δ = 7.22 (d, 2H, J= 7.2 Hz), 7.09 (d, 2H, J= 7.2 Hz), 5.27 (m, IH), 4.40 (t, 0.5H, J= 7 Hz), 4.24 (t, 0.5 H, J= 9 Hz), 3.75 (m, IH), 3.61(m, IH), 3.28 (d, 0.5H, J= 13 Hz), 3.15 (d, 0.5H, J= 13 Hz), 2.42-2.10 (m, 4H), 1.78 (m, IH), 1.40 (br t, 3H, J= 6 Hz), 0.82 (d, 6H, J= 6 Hz), (mixture of diastereomers)

¹³C NMR (75 MHz, CDC1₃): δ = 173.28, 173.23, 168.98, 139.88, 137.33, 137.23, 129.12, 127.26, 127.17, 72.58, 57.60, 57.50, 50.24, 50.12, 44.34, 44.15, 34.31, 34.16, 29.77, 22.34, 18.43, 18.23. (mixture of diastereomers)

HPLC analysis:

100% purity; r.t.= 5.35, 5.22 min.; 55% TFA (0.1%), 45% ACN; 1 mL/min; 32.3 C, Luna C18, serial # 188255-37; 20 ul inj..

CHN analysis: calc: C 67.69, H 7.89, N 4.39; found: C 67.47, H 7.87, N 4.30.

Melting Point: 198-199 °C 91 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc Efficacy (anti nociceptive potential) of Synthesis ofthe L-serine, L-threonine, and L-hydroxyproline esters of (±)-Ibuprofen by employing acetylcholine induced abdominal constriction method in male albino mice:

The present study was conducted to evaluate the efficacy of L-serine, L-threonine, and L-hydroxyproline esters of (±)-lbuprofen taking into account the antagonizing property on acetylcholine induced writhe as an index in albino mice. Ibuprofen (racemic mixture) and ibuprofen (S)-(+) served as reference controls.

Different new formulations of ibuprofen and reference controls viz., ibuprofen (racemic mixture) and ibuprofen (S)-(+) were administered by gavage to male albino mice (Swiss strain), using 5% (v/v) Tween 80 in milli Q water as the vehicle. The study was conducted at two dose levels viz. 50mg and lOOmg/kg body weight along with a vehicle control group. At each dose level 10 animals were used. All the doses were expressed as ibuprofen molar equivalents. The doses used as well as the molar equivalents are presented below.

92 H:\work\1652\l 7690\SPEC\17690.spec without claimsll.doc Table 2: Formulation: Molar Equivalent:

Table 3: Test Item: Group: Dose(mg/kg): Equivalent wt. Ofthe test item:

The efficacy in terms of antagonizing effect on acetylcholine induced single writhe at two dose levels - 50.0 and 100.0 mg/kg for the three formulations and reference controls are presented below.

93 H:\work\l 652\17₆90\SPEC\17690.spec without claimsll.doc Table 4: Test Item: Group: Dose (mg/kg): Number of animals showing absence of single writhe (out of 10)

Statistical analysis employing Chi - square test procedure did not show any statistically significant difference among the formulations in comparison to reference control, while comparing the number of animals not showing writhe in each groups, as the respective "p" was found to be greater than 0.05, the level of significance.

From clinical observation based on the number of animals not showing writhes due to administration of acetylcholine, (±)-lbuprofen-L-hydroxyproline ester was found to be 94 H:\work\l₆S2\l 769Q\SPEC\17690.spec without claimsll.doc more effective in antagonizing the acetylcholine induced writhe when compared to other formulations and Ibuprofen (racemic) and Ibuprofen (S)-(+).

Table 5: Summary of Efficacy of L-serine, L-threonine, and L-hydroxyproline esters of (±)-Ibuprofen, Ibuprofen (racemic mixture) and Ibuprofen (S)-(+) - Based on Antagonizing Property of Acetylcholine Induced Writhe in Albino Mice

Table 6:

95 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc The data were subjected to statistical analysis employing Chi - square test procedure for evaluating the efficacy ofthe new formulations in comparison to the reference controls. The test did not show any statistically significant difference among the formulations in comparison to reference control, while comparing the number of animals not showing writhe in each groups, as the respective "p" was found to be greater than 0.05, the level of significance.

The data is also summarized in FIGS. 1 and 2. From clinical observations and bar diagram for comparative efficacy (FIGS. 1 and 2) based on the number of animals not showing writhes due to administration of acetylcholine, (±)-Ibuprofen-L-hydroxyproline ester was found to be more effective in antagonizing the acetylcholine induced writhe when compared to other formulations and Ibuprofen (racemic) and Ibuprofen (S)-(+).

CONCLUSION The present study was conducted to evaluate the relative efficacy of new formulations of ibuprofen. For this the antagonizing property of new formulations on acetylcholine writhes was taken as an index to determine the relative efficacy ofthe formulations, ibuprofen (racemic mixture and ibuprofen (S)-(+) served as reference controls. The study was conducted at two dose levels (50.0 and 100.0 mg/kg) along with a vehicle control group.

The efficacy in terms of antagonizing effect of acetylcholine induced single writhe at two dose levels - 50.0 and 100.0 mg/kg for the three formulations and reference controls are presented below.

96 H:\work\1052\17690\SPEC\17690.spec without claimsll.doc Table 7: Test Item: Group: Dose (mg/kg): No. of animals showing absence of single writhe (out of 10)

Statistical analysis employing Chi - square test procedure did not show any statistically significant difference among the formulations in comparison to reference control, while comparing the number of animals not showing writhe in each groups, as the respective "p" was found to be greater than 0.05, the level of significance.

However from clinical observation based on the number of animals not showing writhes due to administration of acetylcholine (±)-Ibuprofen-L-hydroxyproline ester was found

97 H:\work\16₅2\17690\SPEC\17690.spec without claimsILdoc to be more effective in antagonizing the acetylcholine induced writhe when compared to other formulations and Ibuprofen (racemic) and Ibuprofen (S)-(+).

Gastric mucosal irritation potential of L-serine, L-threonine, and L-hydroxyproline esters of (±)-Ibuprofen in fasted male albino rats

SUMMARY

The present study was conducted to determine the relative potential of new formulations of ibuprofen (L-serine, L-threonine, and L-hydroxyproline esters of (±)-lbuprofen) to cause gastric mucosal irritation lesions in fasted male albino rats. Ibuprofen (racemic mixture) and Ibuprofen(S)-(+) served as reference controls.

Different new formulations of ibuprofen and ibuprofen (racemic mixture) and ibuprofen(S)-(+) were administered by gavage to fasted male albino rats (Wistar strain), using 5% solution of Tween 80 in milli Q water as the vehicle. The study was conducted at two dose levels viz. 200mg and 300mg/kg body weight along with a vehicle control group. At each dose level 5 animals were used. All the doses were expressed as ibuprofen (racemic mixture) molar equivalents. The doses used as well as the molar equivalents were presented below.

Table 8: Formulation: Molar Equivalent

98 H:\work\1652\1769Q\SPEC\17690.spec without claimsll.doc The various groups used are tabulated hereinbelow:

Table 9: Test item: group: Dose (mg kg) Equivalent wt.

The rats were fasted for a period of 18 to 22 hours before dosing. The test item was administered as a single dose by gavage. Three hours after drug administration, the animals were killed humanely by CO₂ gas inhalation. The stomach was dissected out and observed for

• the quantity of mucous exudate,

• degree of hyperemia and thickening of stomach wall,

• hemorrhagic spots (focal or diffuse), nature of hemorrhages (petechial or ecchymotic) along with the size and

• perforations or any other lesions

99 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc The observations on gastric mucosal irritation of animals of various groups were summarized below

Table 10: Test item: Group: Dose (mg/kg): Observation

100 H:\work\1652\l 7690\SPEC\17690.spec without claimsll.doc

The results ofthe present study showed that none ofthe formulations of ibuprofen had caused any evidence of irritation of gastric mucosa in fasted male albino rats of male sex at the two dose levels tested (200mg and 300mg/kg body weight). In contrast both ibuprofen (racemic mixture) and ibuprofen (S) - (+) had caused irritation of gastric mucosa at the two dose levels tested. Further ibuprofen(S ) - (+) was found to be more gastric mucosal irritant than ibuprofen (racemic mixture).

Overview Ketoprofen S(+) Threonine Ester Synthesis: The procedure for the synthesis ofthe L-threonine esters of Ketoprofen is outlined in Synthetic Sequence section. This synthesis is exemplary and is equally applicable for the other amino acids. The complete procedure and analytical data is given in the Experimental Section. In general, (±)-Ketoprofen (5 g) was coupled with N-boc-L- threonine t-butyl ester¹ (1 equivalent) with l-(3-dimethylaminopropyl)-3- ethylcarbodiimide, hydrochloride (EDC, 1 equivalent) in the presence of a catalytic amount of 4-(N,N-dimethyamino)-pyridine (DMAP). Once the reaction was complete, any excess EDC was removed by extraction with water, DMAP was removed by extraction with dilute acid, and Ketoprofen was removed by extraction with sodium bicarbonate. After drying over sodium sulfate, filtration, and concentration the crude

101 H:\work\16₅2\17690\SPEC\I7690.spec without claimsll.doc protected L-threonine-(+)-Ketoprofen was purified by flash chromatography on silica gel to generate the protected L-threonine ester in good yield (98%). The protecting groups were removed by treatment with 2M hydrochloric acid in diethyl ether to cleave the boc group, followed by treatment with trifluoroacetic acid to remove the t-butyl ester. After drying, the mixture of L-threonine-R,S(±)-Ketoprofen esters was separated by crystallization from acetonitrile. The hydrochloride salt ofthe L-threonine-S(+)- Ketoprofen ester preferentially precipitated from acetonitrile. A sample of an optically pure standard was prepared starting with S(+)-ketoprofen for comparison. After drying and analysis, a sample of L-threonine-S(+)-Ketoprofen ester, hydrochloride (1.75 g) was separated from the mixture.

Synthetic Sequence: SPI0018A

(±)-Ketoprofen Boc-T r-θ-tBu b) c)

SPI0018A SPI0018A SPI0018B

Synthesis of the L-threonine esters of (±)-Ketoprofen: a) EDC, DMAP, CH₂C1₂; b) HCl (2M); c) TFA; d) ACN (crystallization).

Experimental Section:

The synthesis of SPI0018A was conducted in a single batch. Reagents mentioned in the experimental section were purchased at the highest obtainable purity from Sigma- 102 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc Aldrich, Acros, or Bachem, except for solvents, which were purchased from either Fisher Scientific or Mallinkrodt.

Preparation and Separation of S(+)-Ketoprofen-L-threonine ester, hydrochloride (SPI0018A).

(±)-Ketoprofen (5.32 g, 20.92 mmol), N-t-butylcarbonyl-L-threonine t-butyl ester (Boc- Thr-OtBu, 5.17 g, 18.72 mmol, (prepared in accordance with the literature), l-(3- dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 4.0 g, 20.9 mmol), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.22 g) were dissolved in dichloromethane (50 mL) at room temperature, under an argon atmosphere. After stirring for 5 hours, the dichloromethane layer was washed with water (50 mL), 5% hydrochloric acid (2x25 mL), water (25 mL), saturated sodium bicarbonate (2x25 mL), and water (50 mL). After drying for one hour over sodium sulfate (5 g), filtration, and concentration under reduced pressure, the remaining oil (10.3 g) was purified by column chromatography on silica gel (150 g), eluting with hexanes/ethyl acetate (2:1). After combining the product containing fractions, concentration and drying under high vacuum, the procedure generated the protected L-threonine-(±)-Ketoprofen ester (SPI001801) as a clear oil (9.42 g, 98% yield),

3-[2(R,S)-(3-Benzoyl-phenyl)-propionyloxy]-2(S)-tert-butoxycarbonylamino-butyric acid tert-butyl ester: (mix of diastereomers)

1H NMR (300 MHz, CDC1₃): δ = 7.83-7.42 (m, 9H), 5.43 (dd, IH, J= 13.2, 6.9 Hz), 5.10 (dd, IH, J= 20.7, 9.3), 4.29 (t, IH, J= 11.7 Hz), 3.75 (q, IH, J= 7.2 Hz), 1.50-1.42 (m, 19.5H), 1.30-1.18 (m, 4.5H).

103 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc ¹³C NMR (75 MHz, CDC1₃): δ =. 196.18, 172.62, 172.55, 168.85, 168.58, 155.81, 140.33, 140.23, 137.86, 137.39, 132.46, 132.42, 131.54, 131.38, 130.00, 129.31, 129.13, 129.02, 128.54, 128.27, 82.50, 82.37, 80.05, 71.38, 71.22, 57.59, 57.52, 45.46, 45.31, 28.40, 27.98, 27.84, 18.54, 18.48, 17.19, 16.84.

The protected (R,S)-Ketoprofen-L-threonine ester (9.42 g, 18.41 mmol) was dissolved in dichloromethane (25 mL) under an argon atmosphere, at room temperature. Anhydrous hydrochloric acid in diethyl ether (2M, 25 mL) was added to the solution and the mixture was allowed to stir for 17 hours at room temperature. The mixture was concentrated under reduced pressure. The remaining foam (8.2 g) was dissolved in a mixture of dichloromethane (10 mL) and trifluoroacetic acid (20 mL). After stirring at room temperature for 6.5 hours the solution was concentrated under reduced pressure. Toluene (25 mL) was added to the remaining oil and the mixture was concenfrated a second time. A mixture of ethanol (20 mL) and anhydrous hydrochloric acid in diethyl ether (2M, 20 mL) was added and the solution was concentrated a third time. After drying under high vacuum for 2 hours at room temperature, the experiment produced (±)-Ketoprofen-L-threonine ester, hydrochloride (mix of diastereomers, 7.11 g, 98% crude yield) as an off-white solid. The crude mixture of diastereomers (7.0 g) was crystallized 3 times from acetonitrile (200 mL). After the third crystallization, the remaining white solid was dried under high vacuum at 50 °C until the weight was constant (4 hours). The experiment produced L-threonine-S(+)-Ketoprofen ester, hydrochloride SPI0018A (2.2 g, 30% yield from SPI001801).

2(S)-Amino-3(R)-[2(S)-(3-benzoyl-phenyl)-propionyloxy]-butyric acid, hydrochloride (L-threonine-S(+)-Ketoprofen ester, hydrochloride):

104 H:\work\l 652\17690\SPEC\17690.spec without claimsll.doc 1H NMR (300 MHz, DMSO): δ = 14.08 (br s, IH), 8.72 (br s, 3H), 7.74-7.51 (m, 9H), 5.29 (t, IH, J= 4.5 Hz), 4.16 (m, IH), 3.97 (q, IH, J= 6.3 Hz), 1.42 (d, 3H, J= 6.9 Hz), 1.23 (d, 3H, J= 6.3 Hz).

¹³C NMR (75 MHz, DMSO): δ = 195.34, 172.26, 168.21, 140.42, 137.05, 136.74, 132.66, 131.66, 129.48, 128.73, 128.49, 128.30, 68.23, 55.31, 44.00, 18.44, 16.45.

CHN analysis: calc: C 61.30, H 5.66, N 3.57; found: C 61.02, H 5.58, N 3.58.

HPLC analysis:

98.28% purity; r.t.= 25.14min.; 55% DIUF water (0.1% TFA)/45% methanol; 1 mL/min;

36.4 C; Luna Cl 8, 5u column (serial # 211739-42), 4.6x250 mm; 20 ul injection.

Optical rotation: + 27.0 ° (20 C, 174.4 mg/10 mL ethanol, 589 nm); Melting Point: 166-167 °C

Preparation of the S-(+)-Ketoprofen-L-threonine ester, hydrochloride standard.

(+)-Ketoprofen (1.87 g, 7.74 mmol), N-t-butylcarbonyl-L-threonine t-butyl ester (Boc- Thr-OtBu, 2.25 g, 8.14 mmol, prepared in accordance with the literature method), l-(3- dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 1.65 g, 8.60 mmol), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.1 g) were dissolved in dichloromethane (25 mL) at room temperature, under an argon atmosphere. After stirring for 4 hours, the dichloromethane layer was washed with water (25 mL). After drying for one hour over sodium sulfate 5 g), filtration, and concenfration under reduced pressure, the remaining oil was used without purification. The procedure generated the protected L-threonine- (+)-Ketoprofen ester as a clear oil (4.01 g, ~100% yield).

105 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

'HNMR (300 MHz, CDC1₃): δ = 7.81-7.42 (m, 9H), 5.43 (m, IH), 5.10 (d, IH, J= 9.3), 4.29 (d, IH, J= 9.6 Hz), 3.75 (q, IH, J= 7.2 Hz), 1.50-1.42 (m, 21H), 1.18 (d, 3H, J= 6.3 Hz).

¹³C NMR (75 MHz, CDC1₃): δ = 196.4, 172.79, 168.99, 155.94, 140.44, 137.99, 137.51, 132.59, 131.50, 130.13, 129.31, 129.25, 129.15, 128.66, 128.40, 82.68, 80.24, 71.37, 57.71, 45.43, 28.53, 28.10, 18.99, 16.96.

The protected (S)-Ketoprofen-L-threonine ester (3.92 g, 7.66 mmol) was dissolved in anhydrous hydrochloric acid in diethyl ether (2M, 50 mL) and stirred for 17 hours at room temperature. The mixture was concentrated under reduced pressure. The remaining foam (3.4 g) was dissolved in a mixture of dichloromethane (20 mL) and trifluoroacetic acid (20 mL). After stirring at room temperature for 6.5 hours the solution was concentrated under reduced pressure. Toluene (25 mL) was added to the remaining oil and the mixture was concentrated a second time. A mixture of ethanol (20 mL) and anhydrous hydrochloric acid in diethyl ether (2M, 20 mL) was added and the solution was concentrated a third time. After drying under high vacuum for 2 hours at room temperature, the experiment produced S(+)-Ketoprofen-L-threonine ester, hydrochloride (3.05g crude) as an off-white solid. The crude material was stirred with acetone (50 mL) for 2 hours at room temperature under an argon atmosphere. The remaining white solid was filtered and dried under high vacuum at 50 °C until the weight was constant (4 hours). The experiment produced L-threonine-S(+)-Ketoprofen ester, hydrochloride (2.04 g, 67 % yield).

106 H:\work\16₅2\1769₀\SPEC\17690.spec without claimsILdoc 1H NMR (300 MHz, DMSO): δ = 14.08 (br s, IH), 8.72 (br s, 3H), 7.74-7.51 (m, 9H), 5.29 (t, IH, J= 4.5 Hz), 4.16 (m, IH), 3.97 (q, IH, J= 6.3 Hz), 1.42 (d, 3H, J= 6.9 Hz), 1.23 (d, 3H, J= 6.3 Hz).

¹³C NMR (75 MHz, DMSO): δ = 195.34, 172.26, 168.21, 140.42, 137.05, 136.74, 132.66, 131.66, 129.48, 128.73, 128.49, 128.30, 68.23, 55.31, 44.00, 18.44, 16.45.

HPLC analysis:

99.43% purity; r.t.= 25.14min.; 55% DIUF water (0.1% TFA)/45% methanol; 1 mL/min; 36.4 C; Luna Cl 8, 5u column (serial # 211739-42), 4.6x250 mm; 20 ul injection.

Optical rotation: + 27.1 ° (20 C, 177.8 mg/10 mL ethanol, 589 nm); Melting Point: 166-167 °C

C. Amino Acid Derivaties of Aspirin

Overview:

The procedure for the synthesis ofthe L-serine, L-threonine, and L-hydroxyproline esters of acetylsalicylic acid is outlined in Synthetic Sequence section and is exemplary for other amino acids. The complete procedure and analytical data is given in the Experimental Section. In general, acetylsalicyloyl chloride (10 g-25 g, in batches) was coupled with the N-benzyloxy/benzyl ester protected amino acids in the presence of pyridine. Once the reactions were complete (24 to 48 hours at room temperature), the mixture was poured into ice-cold 2N hydrochloric acid. The dichloromethane fraction was then washed with sodium bicarbonate, water and brine. After drying over sodium sulfate, filtration, and concentration the crude protected amino acid esters of acetylsalicylic acid were purified by flash chromatography on silica gel. The procedure generated the protected amino acid esters of acetylsalicylic acid in yields ranging from 68% to 95%. The protecting groups were removed by hydrogenation (20 psi H₂) in the presence of 10% palladium on carbon. The amino acid esters of acetylsalicylic acid were extracted away from the palladium catalyst with water, concentrated, and dried. 107 HΛwork\1652\17690\SPEC\17690.spec without claimsll.doc The final compounds were washed with solvent (water, dioxane, acetonitrile, and/or dichloromethane) until pure and dried under high vacuum until a constant weight was achieved.

Synthetic Sequence:

1. SPIB00102

Acetylsalicylic acid Z-Thr-OBzl SPIB0010201 SPIB00102

2. SPIB00101

Acetylsalicylic acid Z-Ser-OBzl SPIB0010101 SPIB00101 3. SPIB00103

Acetylsalicylic acid

SPIB00103

Synthesis of the L-serine, L-threonine, and L-hydroxyproline esters of acetylsalicylic acid: a) pyridine, CH₂C1₂; b) 10% Pd/C, EtOH, EtOAc.

Experimental Section: The synthesis of SPIBOOIOI, SPD300102 and SPIB00103 was conducted in one or two batches. Reagents mentioned in the experimental section were purchased at the highest 108 H:\work\l 652\17690\SPEC\17690.spec without claimsll.doc obtainable purity from Lancaster, Sigma- Aldrich, Acros, or Bachem, except for solvents, which were purchased from either Fisher Scientific or Mallinkrodt.

1) SPD300102: 2-O-Acetylsalicylic acid (2S, 3R)-(-)-threonine ester A mixture of N-carbobenzyloxy-L-threonine benzyl ester (Z-Thr-OBzl, 21.77 g, 63.40 mmole) and pyridine (25 mL) in anhydrous dichloromethane (500 mL) was cooled in an ice bath while under a nitrogen atmosphere. Acetylsalicyloyl chloride (17.63 g, 88.76 mmole) was added and the mixture was allowed to warm to room temperature and stir overnight. After 24 hours, the mixture was poured into ice-cold 2N hydrochloric acid (400 mL). After mixing, the layers were separated and the dichloromethane fraction was washed with water (500 mL), saturated sodium bicarbonate solution (500 mL), water (500 mL), brine (500 mL) and dried over sodium sulfate (25 g). After filtration, concentration under reduced pressure, and drying under high vacuum, the remaining yellow oil (35. 43 g) was purified by flash chromatography on silica gel (300 g, 0.035- 0.070 mm, 6 nm pore diameter), eluting with hexanes/ethyl acetate (3:1). After concentration ofthe product containing fractions under reduced pressure and drying under high vacuum until the weight was constant, the experiment produced the protected acetylsalicylic-L-threonine ester SPD30010201 (28.1 g, 88% yield) as a colorless oil.

1H NMR (300 MHz, CDC1₃): δ = 7.74 (IH, d, J= 7.5 Hz), 7.51 (IH, dt, J= 7.5, 1.5 Hz), 7.34-7.17 (11H, m), 7.06 (IH, d, J= 7.2 Hz), 5.62 (2H, m), 5.13 (4H, m), 4.65 (IH, dd, J= 9.6, 2.4 Hz), 2.29 (3H, s), 1.38 (3H, d, J= 6.6 Hz).

109 H:\work\16₅2\1769₀\SPEC\17690.spec without claimsll.doc ¹³C NMR (75 MHz, CDC1₃): δ = 169.35, 169.22, 162.73, 156.26, 150.41, 135.79, 134.67, 133.77, 131.24, 128.35, 128.24, 128.08, 127.95, 125.78, 123.51, 122.61, 71.22, 67.72, 67.26, 57.64, 20.98, 16.88.

The protected acetylsalicylic-L-threonine ester SPD30010201 (14.50 g, 28.68 mmole) was dissolved in ethanol (100 mL) and ethyl acetate (100 mL) at room temperature and added to a Parr bottle that contained 10% palladium on carbon (3.0 g, 50% wet) under a nitrogen atmosphere. The nitrogen atmosphere was replaced with hydrogen gas (20 psi). After 20 hours of shaking, the palladium catalyst was removed by filtration through celite. The remaining solids (palladium/celite and product) were washed with water (600x4 mL) until the product was removed. The ethanol and water fractions were concentrated under reduced pressure at room temperature. The remaining solids were washed with water (20 mL) and dioxane (20 mL) for 48 hours. After filtration, the remaining white solid was dried at room temperature under high vacuum until the product weight was constant (16 hours). The experiment produced acetylsalicylic-L- threonine ester, SPD300102 (4.40 g, 55% yield) as a white solid.

SPIB00102

1H NMR (300 MHz, D₂O-DCl): δ = 8.00 (IH, dd, J= 7.8, 1.5 Hz), 7.74 (IH, dt, J= 7.8, 1.5 Hz), 7.47 (IH, dt, J= 7.8, 1.5 Hz), 7.27 (IH, dd, J= 7.8, 1.5 Hz), 5.76 (IH, dq, J= 6.9, 3.0 Hz), 4.49 (IH, d, J= 3.0 Hz), 2.39 (3H, s), 1.55 (3H, d, J= 6.9 Hz).

¹³C NMR (75 MHz, D₂O-DCl): δ = 173.03, 168.84, 163.97, 149.56, 135.32, 131.26,

126.85, 123.48, 121.49, 69.16, 56.36, 20.45, 15.86.

110 H:\work\16S2\17690\SPEC\17690.spec without claimsILdoc HPLC analysis:

98.7% purity; rt= 6.233 min; Luna C18 5u column (sn 167917-13); 4.6x250 mm; 254 nm; 35% MeOH/65% TFA (0.1%) pH= 1.95; 35 C; 20 ul inj.; Iml/min; sample dissolved in mobile phase with 1 drop phosphoric acid.

CHN analysis: calc: C 55.51, H 5.38, N 4.98; found: C 55.37, H 5.40, N 5.03.

Melting point: 153.5 °C (dec.)

2) SPIBOOIOI : 2-O-Acetylsalicylic acid (2S)-(+)-serine ester A mixture of N-carbobenzyloxy-L-serine benzyl ester (Z-Ser-OBzl, 23.17 g, 70.34 mmole) and pyridine (30 mL) in anhydrous dichloromethane (500 mL) was cooled in an ice bath while under a nitrogen atmosphere. Acetylsalicyloyl chloride (21.07 g, 106.1 mmole) was added and the mixture was allowed to warm to room temperature and stir over two days. After 48 hours, the mixture was poured into ice-cold 2N hydrochloric acid (400 mL). After mixing, the layers were separated and the dichloromethane fraction was washed water (500 mL), saturated sodium bicarbonate solution (500 mL), water (500 mL), brine (500 mL) and dried over sodium sulfate (25 g). After filtration, concentration under reduced pressure, and drying under high vacuum, the remaining brown solid (47.19 g) was purified by flash chromatography on silica gel (200 g, 0.035-0.070 mm, 6 nm pore diameter), eluting with hexanes/ethyl acetate (3:1). After concenfration ofthe product containing fractions under reduced pressure and drying under high vacuum until the weight was constant, the experiment produced the protected acetylsalicylic-L-serine ester SPD30010101 (32.97 g, 95% yield) as a white solid.

Ill H:\work\16₅2\17690\SPEC\17690.spec without claimsll.doc

^lK NMR (300 MHz, CDC1₃): δ = 7.74 (IH, d, J= 7.8 Hz), 7.55 (IH, dt, J= 7.8, 1.5 Hz), 7.33-7.21 (11H, m), 7.08 (IH, d, J= 7.5 Hz), 5.68 (IH, d, J= 8.4 Hz), 5.20 (2H, s), 5.12 (2H, s), 4.77 (IH, m), 4.66 (IH, dd, J= 11.4, 3.3 Hz), 4.57 (IH, dd, J- 11.4, 3.3 Hz), 2.30 (3H, s).

¹³C NMR (75 MHz, CDC1₃): δ = 169.45, 169.09, 163.68, 163.35, 155.57, 150.77, 135.87, 134.75, 134.07, 131.44, 128.50, 128.43, 128.27, 128.14, 128.04, 125.92, 123.71, 122.18, 67.83, 67.27, 64.63, 53.55, 21.03.

The protected acetylsalicylic-L-serine ester SPIB0010101 (21.0 g, 42.7 mmole) was dissolved in ethanol (100 mL) and ethyl acetate (100 mL) at room temperature and added to a Parr bottle that contained 10% palladium on carbon (4.20 g, 50% wet) under a nitrogen atmosphere. The nifrogen atmosphere was replaced with hydrogen gas (20 psi). After 5 hours additional 10% palladium catalyst (4.26 g) was added and the hydrogen atmospere was returned (20 psi). After an additional 20 hours of shaking at room temperature, the palladium catalyst was removed by filtration through celite. The remaining solids (palladium/celite and product) were washed with water (1500x2 mL) until the product was removed. The ethanol and water fractions were concentrated under reduced pressure at room temperature. The remaining solid (7.17 g) was dissolved in DIUF water (4.3 L), filtered tlirough celite to remove insoluble material, and concentrated under high vacuum at room temperature. The white solid was then washed with 1,4-dioxane (100 mL) and DIUF water (50 mL) overnight. After 24 hours the solid was filtered and dried under high vacuum until the weight was constant (24 hours).

112 H:\work\16₅2\17690\SPEC\17690.spec without claimsll.doc The experiment produced the acetylsalicylic-L-serine ester SPIBOOIOI (6.17 g, 54% yield) as a white solid.

SPIB00101

1H NMR (300 MHz, D₂O-DCl): δ = 8.05 (IH, dd, J= 7.8, 1.5 Hz), 7.75 (IH, dt, J= 7.8, 1.5 Hz), 7.47 (IH, dt, J= 7.8, 0.9 Hz), 7.27 (IH, dd, J= 7.8, 0.9 Hz), 4.87 (IH, dd, J= 12.6, 4.2 Hz), 4.79 (IH, dd, J= 12.6, 3.0 Hz), 4.62 (IH, dd, J- 4.2, 3.0 Hz), 2.39 (3H, s).

¹³C NMR (75 MHz, D₂O-DCl): δ = 173.01, 168.58, 164.54, 149.72, 135.39, 131.59, 126.87, 123.62, 121.15, 62.38, 52.05, 20.44.

HPLC analysis:

98.1% purity; r.t.= 5.839 min.; 65% TFA (0.1%)/35% methanol; 1 mL/min; 35 C; Luna C18, 3u column (SN 184225-37), 4.6x250 mm; 22 ul injection; DAD1B, Sig= 240, 4 Ref= 550,100.

CHN analysis: calc: C 53.93, H 4.90, N 5.24; found: C 54.02, H 5.00, N 5.23.

Melting point: 147.0 °C (dec.)

3) SPIB00103: 2-O- Acetylsalicylic acid (2S, 4R)-4-hydroxyproline ester A mixture of N-carbobenzyloxy-L-hydroxyproline benzyl ester (Z-Ser-OBzl, 21.5 g, 60.5 mmole)¹ and pyridine (25 mL) in anhydrous dichloromethane (500 mL) was cooled in an ice bath while under a nitrogen atmosphere. Acetylsalicyloyl chloride (13.2 113 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc g, 66.6 mmole) was added and the mixture was allowed to warm to room temperature and stir overnight. After 24 hours, additional acetylsalicyloyl chloride (5.0 g, 25.2 mmole) was added and the mixture was allowed to stir overnight. After 48 hours, the mixture was poured into ice-cold IN hydrochloric acid (500 mL). After mixing, the layers were separated and the dichloromethane fraction was washed with water (500 mL), saturated sodium bicarbonate solution (500 mL), water (500 mL), brine (500 mL) and dried over sodium sulfate (25 g). After filtration, concenfration under reduced pressure, and drying under high vacuum, the remaining yellow oil (40.7 g) was purified by flash chromatography on silica gel (460 g, 0.035-0.070 mm, 6 nm pore diameter), eluting with heptane/ethyl acetate (3:1). After concenfration of the product containing fractions under reduced pressure and drying under high vacuum until the weight was constant, the experiment produced the protected acetylsalicylic-L-hydroxyproline ester SPIB0010301 (21.31 g, 68% yield) as a colorless oil.

1H NMR (300 MHz, CDC1₃): δ = 7.92 (IH, d, J= 7.8 Hz), 7.56 (IH, t, J= 7.8 Hz), 7.34- 7.21 (10H, m), 7.09 (IH, d, J= 7.8 Hz), 5.48 (IH, s), 5.21 (2H, m), 5.03 (2H, d, J=15 Hz), 4.57 (IH, m), 3.85 (2H, m), 2.53 (IH, m), 2.28 (4H, m).

¹³C NMR (75 MHz, CDC1₃): δ = 171.72, 171.49, 169.25, 163.47, 163.30, 154.52, 153.93, 150.54, 136.05, 135.94, 135.21, 135.00, 134.17, 134.12, 128.43, 128.32, 128.28, 128.20, 128.05, 127.98, 127.94, 127.79, 125.89, 123.70, 122.46, 122.38, 73.24, 72.59, 67.33, 67.11, 66.97, 58.02, 57.69, 52.47, 52.15, 36.74, 35.65, 20.90.

114 H:\work\1652\17690\SPEC\l 7690.spec without claimsll.doc The protected acetylsalicylic-L-hydroxyproline ester SPE80010301 (10.6 g, 20.5 mmole) was dissolved in ethanol (75 mL) and ethyl acetate (75 mL) at room temperature and added to a Parr bottle that contained 10% palladium on carbon (3.0 g, 50% wet) under a nitrogen atmosphere. The nifrogen atmosphere was replaced with hydrogen gas (20 psi). After 17 hours of shaking at room temperature, the reaction mixture was washed with water (500 mL) for two hours. The organic layer (top) was removed via pipette and the aqueous layer was filtered through celite. The water fraction was concentrated under reduced pressure at room temperature. The remaining solid (6.71 g) was then washed with anhydrous dichloromethane (35 mL) overnight. After 24 hours the solid was filtered and dried under high vacuum until the weight was constant (24 hours). The experiment produced acetylsalicylic-L-hydroxyproline ester, SPIB00301

(2.87 g, 47.7 % yield) as a white solid.

SPIB00103

1H NMR (300 MHz, D₂O-DCl): δ = 8.09 (IH, d, J= 7.5 Hz), 7.75 (IH, t, J= 7.5 Hz), 7.48 (IH, t, J= 7.5 Hz), 7.28 (IH, d, J= 7.5 Hz), 5.69 (IH, m), 4.76 (IH, t, J=7.5 Hz), 3.86 (IH, dd, J= 13.5, 3.9 Hz), 3.74 (IH, d, J= 13.5 Hz), 2.81 (IH, dd, J= 15.0, 7.5 Hz), 2.60 (IH, m), 2.40 (3H, s).

¹³C NMR (75 MHz, D₂O-DCl): δ = 173.13, 170.25, 164.31, 149.65, 135.36, 131.54, 126.87, 123.54, 121.37, 73.86, 58.34, 50.95, 34.38, 20.48.

HPLC analysis:

98.3% purity; r.t.= 7.201 min.; 65% TFA (0.1%)/35% methanol; 1 mL/min; 35 C; Luna C18, 3u column (SN 184225-37), 4.6x250 mm; 22 ul injection; DADIB, Sig= 240, 4

Ref= 550,100. 115 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc CHN analysis: calc: C 57.34, H 5.16, N 4.78; found: C 57.09, H 5.23, N 4.91.

Melting point: 162 °C (dec.)

Gastric Mucosa irritation potential ofthe L-serine, L-threonine, and L- Hydroxyproline esters of acetylsalicylic acid compared to acetylsalicylic acid:-

The present study was conducted to determine the relative potential of new formulations of aspirin (L-serine, L-threonine, and L-Hydroxyproline esters of acetylsalicylic acid) to cause gastric mucosal irritation/lesions in fasted male albino rats. Aspirin served as reference control.

Different new formulations of aspirin and aspirin were administered by gavage to fasted male albino rats (Wistar strain), using 0.5% (w/v) Carboxymethylcellulose (CMC) in Phosphate Buffer (pH 2.6) solution as the vehicle. The study was conducted at two dose levels viz. lOOmg and 200mg/kg body weight along with a vehicle confrol group. At each dose level 5 animals were used. All the doses were expressed as aspirin molar equivalents. The doses used as well as the molar equivalents were presented below.

Table 11 :Formulation: Molar equivalent

116 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc Table 12: Test Item: Group: Dose (mgper kg) [in terms of acetylsalicylic acid] Equivalent weight ofthe Test item [mg]

The rats were fasted for a period of 18 to 22 hours before dosing. The test item was administered as a single dose by gavage. Three hours after drug administration, the animals were killed humanely by CO₂ gas inhalation. The stomach was dissected out and observed for

• the quantity of mucous exudate,

• degree of hyperemia and thickening of stomach wall,

• hemorrhagic spots (focal or diffuse), nature of hemorrhages (petechial or ecchymotic) along with the size and

• perforations

The observations on gastric mucosal irritation of animals of various groups were summarized below

117 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc Table 13: Test Item: Group: Dose mg/kg (as acetylsalicylic acid): Observation

In conclusion it was observed that none ofthe L-serine, L-threonine, and L- Hydroxyproline esters of acetylsalicylic acid induced any evidence of irritation of gastric mucosa at the two doses tested viz., 100 and 200mg/kg body weight In contrast, aspirin (acetylsalicylic acid) has caused irritation ofthe gastric mucosal in all the fasted male albino rats at the dose level of 200mg/kg. However at the dose level of lOOmg/kg aspirin failed to cause any evidence of gastric mucosal irritation in the male rats.

118 H:\work\16₅2\17690\SPEC\17690.spec without claimsll.doc Further none ofthe animals of different test groups showed any clinical symptoms of toxicity through out the observation period of three hours.

Efficacy of L-serine, L-threonine, and L-Hydroxyproline esters of acetylsalicylic acid compared to acetylsalicylic acid on Clotting Time in rats estimated one hour after dosing

Observations of blood clotting time

The data on the mean clotting time (MCT) ofthe animals of low, intermediate and high dose groups of different formulations, vehicle control and positive control groups estimated one hour after dosing were presented below (Table 14):

Table 14: Summary of Mean Clotting Time (± S.D.) in Minutes - L-serine, L-threonine, and L-Hydroxyproline esters of acetylsalicylic acid and Aspirin (Positive control): Low dose: Intermediate dose: High dose

FIG. 3-6 depict the group mean data of animals regarding the dose relationship + mean clotting time in minutes for the L-series ester of aspirin and for the control.

The statistical analysis showed a significant improvement at 5% significance level in the efficacy for the high dose and mid dose when compared to the vehicle control group (Figure 7).

119 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc FIG. 4 shows the group mean data of animals. It provides the dose response relationship to mean clotting time (MCT) in minutes with respect to L-hydroxyproline ester of asperin. The statistical analysis of FIG. 4 showed a significant improvement at 5% significance level in the efficacy for the high dose and low dose when compared to the vehicle control group (Figure 6)

FIG. 5 depicts the dose response relationship to mean clotting time (MCT) in minutes of L-threonine ester of acetylsalicylic acid. The statistical analysis showed a significant improvement at 5% significance level in the efficacy for the high dose when compared to the vehicle control.

FIG. 6 depicts the dose response relationship to mean clotting time for acetylsalicylic acid. The statistical analysis showed a significant improvement at 5% significance level in the efficacy for the intermediate and high dose when compared to the vehicle confrol. The dose response effect were statistically significant and clearly evident (Figure 7).

CONCLUSION

The present study was conducted to evaluate the efficacy of new formulations of aspirin using blood clotting time as an index in albino rats. Aspirin served as positive control. The study was conducted at three dose levels with the new formulations and positive control along with a vehicle control group.

Doses

The doses for the main study were selected based on the dose range finding experiments with acetylsalicylic acid. All the doses were expressed as aspirin molar equivalents. The doses used for the main experiment for different formulations and positive control were same and presented below.

120 H:\work\1652\17690\SPEC\17690.spec without claimsn.doc Table 15: Test Item: Low Dose (mg/kg): Intermediate dose 9mg/kg): High dose (mg/kg)

Efficacy (Blood clotting time)

The efficacy in terms of time required for the blood clotting time at different dose levels - low, intermediate and high dose for different formulations and acetylsalicylic acid are presented below.

Table 16: Low dose: Intermediate dose: High dose

L-serine, L-threonine, and L-Hydroxyproline esters of acetylsalicylic acid are as significant as acetylsalicylic acid with respect to clotting time observed after one hour but are far superior in terms ofthe absence of gastric irritation at all levels compared to acetylsalicylic acid.

Efficacy of L-serine, L-threonine, and L-Hydroxyproline esters of acetylsalicylic acid compared to acetylsalicylic acid on Clotting Time in rats estimated two hours after dosing

121 H:\work\16₅2\17₆90\SPEC\17690.spec without claimsll.doc The present study was conducted to evaluate the efficacy of L-serine, L-threonine, and L-Hydroxyproline esters of acetylsalicylic acid compared to acetylsalicylic acid using blood clotting time, estimated 2 hours (± 10 minutes) after dosing, as an index in albino rats. Aspirin served as positive control. Male albino rats were exposed to aspirin and to 3 new formulations of aspirin at one dose level of 20mg/kg body weight. No vehicle control group was used. The doses were expressed as aspirin molar equivalents. The doses used for the main experiment for different formulations and positive control was presented below.

Table 17: Test Item: Dose in terms of Acetylsalicylic acid 9mg/kg)

Efficacy (Blood clotting time)

The efficacy in terms of time required for the blood clotting time at the dose level of 20 mg/kg body weight for different formulations and aspirin (positive confrol) are presented below.

Observations of Blood clotting time

The data on the mean clotting time (MCT) ofthe animals, estimated 2 hours (± 10 minutes) after dosing, at the dose level of 20mg/kg body weight for different formulations, vehicle control and positive control are presented below

122 H:\work\1652\1769Q\SPEC\17690.spec without claimsll.doc Table 18: Summary of Mean Clotting Time (± S.D.) in Minutes of L-serine, L-threonine, and L-Hydroxyproline esters of acetylsalicylic acid compared to acetylsalicylic acid (Positive confrol)

L-serine, L-threonine, and L-Hydroxyproline esters of acetylsalicylic acid were found to be effective on clotting time.

In conclusion, it was observed that based on the time required for the blood to clot (clotting time), when estimated 2 hours after dosing, the amino acid prodrugs were efficacous. However, the L-threonine ester of acetylsalicyclic acid was found to have relatively better efficacy than the other two formulations.

As shown by FIG. 7 the statistical analysis showed that L-threonine, and L- Hydroxyproline esters of acetylsalicylic acid are as effective as acetylsalicylic acid there is no significant difference at 5% significance level for L-Hydroxyproline ester of acetylsalicylic acid and L-threonine ester of acetylsalicylic with respect to positive control for the mean blood clotting time observed after two hours. However, combined with the gastric irritation potential, the L-serine, L-threonine, and L-Hydroxyproline esters of acetylsalicylic acid are far superior.

123 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc There are a number of screening tests to determine the utility ofthe prodrugs created according to the disclosed methods. These include both in vitro and in vivo screening methods.

The in vitro methods include acid/base hydrolysis ofthe prodrugs, hydrolysis in pig pancreas hydrolysis in rat intestinal fluid, hydrolysis in human gastric fluid, hydrolysis in human intestinal fluid, and hydrolysis in human blood plasma. These assays are described in Simmons, DM, Chandran, VR and Portmann, GA, Danazol Amino Acid Prodrugs: In Vitro and In Situ Biopharmaceutical Evaluation, Drug Development and Industrial Pharmacy, Vol 21, Issue 6, Page 687, 1995, the contents of all of which are incorporated by reference.

The compounds ofthe present invention are effective in treating diseases or conditions in which NSAIDs normally are used. The prodrugs disclosed herein are transformed within the body to release the active compound and enhances the therapeutic benefits of the NSAIDs by reducing or eliminating biopharmaceutical and pharmacokenetic barriers associated with each of them. However it should be noted that these prodrugs themselves will have sufficient activity without releasing any active drug in the mammals. Since the prodrugs is more soluble in water then Ibuprofen or other NSAIDs, it does not need to be associated with a carrier vehicle, such as alcohol or castor oil which may be toxic or produce unwanted side reactions. Moreover, oral formulations containing the NSAID prodrugs are absorbed into the blood and are quite effective.

Thus, the prodrug ofthe present invention enhances the therapeutic benefits by removing biopharmaceutical and pharmacokenetic barriers of existing drugs.

Furthermore, the prodrugs are easily synthesized in high yields using reagents which are readily and commercially available.

124 H:\work\16S2\17690\SPEC\17690.spec without claimsILdoc IV. Proline Derivative of Acetaminophen Overview:

The procedure for the synthesis ofthe L-proline ester of acetaminophen is outlined in Synthetic Sequence section. The synthesis is exemplary. The complete procedure and analytical data is given in the Experimental Section. Acetaminophen (10 g) was coupled with Boc-L-proline with EDC in the presence of a catalytic amount of DMAP. Once the reaction was complete (3 hours at room temperature), the solution was washed with water. After drying over sodium sulfate, filtration, and concentration the crude protected amino acid ester of acetaminophen was purified by flash chromatography on silica gel. The procedure generated the protected L-proline ester of acetaminophen in 72%. The protecting group was removed by dissolving the ester in dichloromethane and passing hydrogen chloride through the solution at room temperature. After filtration, the final salt was stirred in tefrahydrofuran until pure. The yield for the deprotection step was 91.4% after filfration and drying under high vacuum at 90 °C for 4 hours.

Synthetic Sequence:

125 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

Acetaminophen boc-L-proline SPI001401 b)

Synthesis ofthe L-proline ester of acetaminophen: a) EDC, DMAP, CH₂C1₂; b) HCl (g), CH₂C1₂.

Experimental Section:

The synthesis of SPI0014 was conducted in one batch. Reagents mentioned in the experimental section were purchased at the highest obtainable purity from Lancaster, Sigma- Aldrich, or Acros, except for solvents, which were purchased from either Fisher Scientific or Mallinkrodt.

SPI0014: Pyrrolidine-2(S)-carboxylic acid 4-acetylamino-phenyl ester, hydrochloride

A mixture of Boc-L-proline (14.39 g, 68.80 mmole), acetaminophen (10.02 g, 66.28 mmole), EDC (12.9 g, 67.29 mmole) and DMAP (1.10 g, 9.0 mmole) in anhydrous dichloromethane (100 mL) was stirred for 3 hours at room temperature under an argon 126 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc atmosphere. After 3 hours, water (120 mL) was added. After mixing for 5 minutes, the layers were separated and the dichloromethane fraction was washed with water (120 mL) and dried over sodium sulfate (5 g). After filtration, concentration under reduced pressure, and drying under high vacuum, the remaining oil (24.10 g) was purified by flash chromatography on silica gel (100 g, 0.035-0.070 mm, 6 nm pore diameter), eluting with hexanes/ethyl acetate (1 :2). After concentration ofthe product containing fractions under reduced pressure and drying at high vacuum until the weight was constant, the experiment produced the protected acetaminophen-L-proline ester SPI001401 (16.71 g, 72.3% yield) as a white solid (foam).

SPI001401

1H NMR (300 MHz, CDC1₃): δ = 8.83 (1/2 H, s), 8.70 (1/2 H, s), 7.58 (1/2 H, d, J= 7.5 Hz), 7.46 (1/2 H, d, J= 7.5 Hz), 6.96 (2 H, m), 4.47 (1 H, ), 3.59-3.45 (2H, m), 2.36 (1 H, m), 2.17-1.90 (6 H, m), 1.46 (9 H, m).

¹³C NMR (75 MHz, CDC1₃): δ = 171.91, 171.75, 169.02, 154.44, 153.78, 146.36, 146.21, 121.44, 121.23, 120.82, 80.41, 80.17, 59.16, 46.78, 46.55, 31.06, 30.11, 28.50,

24.57, 24.28, 23.78.

The protected acetaminophen-L-proline ester SPI001401 (16.60 g, 47.64 mmole) was dissolved in dichloromethane (400 mL) and hydrogen chloride gas was passed through the solution for 2 hours at room temperature. The remaining solids were allowed to settle (for 1 hour). The dichloromethane was carefully decanted away from the white precipitate. Tetrahydrofuran (200 mL) was added to the precipitate and the mixture stirred for 2 hours under an argon atmosphere. After filtration, the remaining white solid 127 H:\work\1652\17690\SPECU7690.sρec without claimsll.doc was dried under high vacuum at 90 °C until the product weight was constant (4 hours). The experiment produced acetaminophen-L-proline ester, hydrochloride SPI0014 (12.4 g, 91.4% yield) as a white solid.

1H NMR (300 MHz, CDCL₃-DMSO): δ = 10.41 (IH, br s), 10.26 (IH, s), 9.55 (IH, br s), 7.70 (2H, d, J- 9 Hz), 7.12 (2H, d, J- 9 Hz), 4.66 (t, IH, J= 8.4 Hz), 3.33 (2H, m), 2.43 (IH, m), 2.28 (IH, m), 2.08 (s, 3H), 2.04 (2H, m).

¹³C NMR (75 MHz, CDCL₃-DMSO): δ = 168.08, 167.25, 144.55, 137.40, 121.12, 119.64, 58.53, 45.33, 27.74, 23.86, 23.08.

HPLC analysis:

99.45% purity; rt= 5.733 min; Luna C18 5u column (sn 167917-13); 4.6x250 mm; 254 nm; 15% MeOH/85% hexane sulfonate buffer (1 lOmMol, pH= 6); 35 C; 20 ul inj.; Iml/min; 5 mg/mL sample size.

CHN analysis: calc: C 54.84, H 6.02, N 9.84; found: C 54.66, H 5.98, N 9.65.

Melting point: 221-222 °C

V. Amino Acid Derivative of Cyclosporine A

The macrocyclic immunosuppresants comprise a class of structurally distinctive, cyclic, poly, N-methylated undecaptides, and similar semi-synthetic macrolide structures commonly possessing pharmacological, in particular immunosuppressive, anti- inflammatory and/or anti-parasitic activity. The first ofthe cyclosporine to be isolated was the naturally occurring fungal metabolite Ciclosporin or Cyclosporine also known as cyclosporine A, which has the formula:

128 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc

wherein MeBmt represents N-methyl-(4R)-4-but-2E-en-l-yl-4-methyl-(L) threonyl residue ofthe formula CH,

CH₂

HO (R) ^CH. ^CH^ ( )^ CH,

N CH- CO (S) CH, in which -x-y- is CH=CH -(trans). Other similar products include, sirolimus (b), tacrolimus (c), and pimecrolimus (d), having the following structures:

129 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc ( )

(c)

Tacrolimus

130 H:\work\16₅2\17690\SPEC\17690.spec without claimsll.doc (d)

Pimecrolimus

The class comprised by the cyclosporines is thus now very large indeed and includes, for example, [Thr]²-, [Val]²-, [Nva]²- and [Nva]²-[Nva]⁵-Ciclosporin (also known as cyclosporines C, D, G and M respectively), [Dihydrop-MeBmt] ¹-[Val]²-ciclosρorin (also known as dihydro-cyclosporine D), [(D)Ser]⁸-Ciclosporin, [Melle]¹¹ -Ciclosporin, [(D)MeVal]¹¹ -Ciclosporin (also known as cyclosporine H), [MeAla]⁶-Ciclosρorin, [(D)Pro]³-Ciclosporin and so on.

In accordance with conventional nomenclature for cyclosporines, these are defined throughout the present specification and claims by reference to the structure of cyclosporine (i.e., Cyclosporine A). This is done by first indicating the amino acid residues present which differ from those present ion Ciclosporin (e.g., "[(D)Pro]³" to indicate that the cyclosporine in question has a -(D)Pro- rather than -Sar- residue at the 3 -position) and then applying the term Cyclosporine to characterize remaining residues which are identical to those present in Cyclosporine A.

131 H:\work\16₅2\1769₀\SPEC\17690.spec without claimsll.doc As used herein, the term "cyclosporines" refers to the various types of cyclosporines, in which x-y in the MeBmt residue has a cis or trans CH=CH or in which x-y therein is also included in those derivatives in which one or more of those amino acids in positions 2-11 of Cyclosporine A is replaced by a different amino acid. It is preferred; however, that not more than two ofthe amino acids are replaced in the formula of cyclosporine A and more preferentially not more than one ofthe amino acids is replaced by an amino acid.

In addition, amino acid residues referred to by abbreviation, e.g., -Ala-, -Me Val- and - Abu-, are, in accordance with conventional practice, to be understood as having the (L)-configuration unless otherwise indicated, e.g. as in the case of "-(D)Ala-". Residue abbreviations preceded by "Me" as in the case of "-MeLeu-", represent α-N-methylated residues. Individual residues ofthe cyclosporine molecule are numbered, as in the art, clockwise and starting with the residue -MeBmt-, dihydro-MeBmt- etc...in position 1. The same numerical sequence is employed throughout the present specification and claims.

Because of their unique pharmaceutical potential, the macrocyclic immunosuppressants have attracted considerable attention in the press. The term "macrocyclic immuno- suppressants" includes various natural and semi-synthetic derivatives of cyclosporine, and other macrolides such as sirolimus, tacrolimus and pimecrolimus. The primary area of clinical investigation for above drugs has been as immunosuppressive agents, in particular in relation to its application to recipients of organ transplants, e.g., heart, lung, combined heart-lung, liver, kidney, pancreatic, bone-marrow, skin and corneal transplants, and in particular allogenic organ fransplants. These drugs are also used in the treatment of psoriasis, atompic dermatitis, rheumatoid arthritis and nephritic syndrome.

Macrocyclic immunosuppressants are also useful for treating various autoimmune diseases and inflammatory conditions and especially inflammatory conditions with an 132 H:\work\16S2\17690\SPEC\17690.spec without claimsll.doc aetiology, including an autoimmune component, such as arthritis (for example, rheumatoid arthritis, arthritis chronica progredient and arthritis deformons) and rheumatic diseases. Specific autoimmune diseases for which cyclosporine therapy has been proposed or applied include, autoimmune hematological disorder (including, e.g., hemolytic anemia, aplastic anemia, pure red cell anemia, and idiopathic thrombocytopaenia), systemic lupus erythematosus, polychondritis, sclerodoma, Wegener granulamatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, Steven- Johnson syndrome, idiopathic sprue, autoimmune inflammatory bowel disease, including, e.g., ulcerative colitis and Crohn's disease), endocrine opthalmopathy Graves disease, sarcoidosis, multiple sclerosis, primary billiary cirrhosis, juvenile diabetes (diabetes mellitus type I), uvetis (anterior and posterior), keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstial lung fibrosis, psoriatic arthritis, atopic dermatitis and glomerulonephritis (with and without nephrotic syndrome, e.g., including idiopathic nephritic syndrome or minimal change nephropathy).

Furthermore, macrocyclic immunosuppressants also have applicability as an anti- parasitic, in particular anti-protozoal agent, and are suggested to be useful for treating malaria, coccidiomycosis and schistomsomiasis. More recently, they have been taught to be useful as an agent for reversing or abrogating anti-neoplastic agent resistance contumors, and the like.

Despite the very major contribution which macrocyclic immunosuppressants have made, difficulties have been encountered in providing more effective and convenient means of administration (e.g., galenic formulations, for example, oral dosage form, which are both convenient and for the patient as well as providing appropriate bioavailability and allowing dosaging at an appropriate and controlled dosage rate) as well as the reported occurrence of undesirable side reactions; in particular nephrotoxic reactions have been obvious serious impediments to its wider use or application.

133 H:\work\16₅2\17690\SPEC\1769₀.spec without claimsll.doc Moreover, the above mentioned macrocyclic immunosuppressants are characteristically highly hydrophobic and readily precipitate in the presence of even very minor amounts of water, e.g., on contact with the body (e.g., stomach fluids). It is accordingly extremely difficult to provide e.g., oral formulations which are acceptable to the patient in terms of form and taste, which are stable on storage and which can be administered on a regular basis to provide suitable and controlling patient dosaging.

Proposed liquid formulations, e.g., for oral administration of macrocyclic immunosuppressants, have heretofore been based primarily on the use of ethanol and oils or similar excipients as carrier media. Thus, the commercially available macrocyclic immunosupressant drink-solution employs ethanol and olive oil or corn-oil as carrier medium in conjunction with solvent systems comprising e.g., ethanol and LABRIFIL and equivalent excipients as carrier media. Thus, the commercially available macrocyclic immunosupressant drink solution employs ethanol and olive oil or corn-oil as carrier medium in conjunctions with a Labrifil as a surfactant. See e.g., U.S. Patent NO. 4,388,307. Use ofthe drink solution and similar composition as proposed in the art is however accompanied by a variety of difficulties.

Further, the palatability ofthe known oil based system has proved problematic. The taste ofthe known drink-solution is, in particular, unpleasant. Admixture with an appropriate flavored drink, for example, chocolate drink preparation, at high dilution immediately prior to ingestion has generally been practiced in order to make regular therapy at all acceptable. Adoption of oil based systems has also required the use of high ethanol concentrations to itself inherently undesirable, in particular where administration to children is forseen. In addition, evaporation ofthe ethanol, e.g., from capsules (adopted in large part, to meet problems of palatability, as discussed or other forms (e.g., when opened) results in the development of a macrocyclic immunosupressant precipitate. When such compositions are presented in, for example, soft gelatin encapsulated form an additional problem arises. This particular difficulty necessitates packaging of the encapsulated product in an air-tight component, for 134 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc example, an air-tight blister or aluminum-foil blister package. This in turn renders the product both bulky and more expensive to produce. The storage characteristics ofthe aforesaid formulations are, in addition, far from ideal.

Bioavailability levels achieved using existing oral macrocyclic immunosupressant dosage system are also low and exhibit wide variation between individuals, individual patient types and even for single individuals at different times during the course of therapy. Reports in the literature indicates that currently available therapy employing the commercially available macrocyclic immunosupressant drink solution provides an average absolute bioavailability of approximately 30% only, with the marked variation between individual groups, e.g., between liver (relatively low bioavailability) and bone- marrow (relatively high bioavailability) transplant recipients. Reported variation in bioavailability between subjects has varied from one or a few percent for some patients, to as much as 90% or more for others. And as already noted, marked change in bioavailability for individuals with time is frequently observed. Thus, there is a need for a more uniform and high bioavailability of macrocyclic immunosupressant in patients.

Use of such dosage forms is also characterized by extreme variation in required patient dosaging. To achieve effective immunosuppressive therapy, blood or blood serum levels compounds ofthe cyclosporin have to be maintained within a specified range. This required range can in turn, vary, depending on the particular condition being treated, e.g., whether therapy is to prevent transplant rejection or for the control of an autoimmune disease, or condition and on whether or not alternative immunosuppressive therapy is employed concomitantly with any ofthe immunosuppressants ofthe formula a-d therapy. Because ofthe wide variations in bioavailability levels achieved with conventional dosage forms, daily dosages needed to achieve required blood serum levels will also vary considerably from individual to individual and even for a single individual. For this reason it is necessary to monitor blood/blood-serum levels of patients receiving macrocyclic immunosuppressant therapy at regular and frequent intervals. Monitoring of blood/blood-serum levels, which is generally performed by 135 H:\work\l 652\17690\SPEC\17690.spec without claimsll.doc RIA or equivalent immunoassay technique, e.g. employing monoclonal antibody based technology, has to be carried out on a regular basis. This is inevitably time consuming and inconvenient and adds substantially to the overall cost of therapy.

It is also the case that blood/blood serum macrocyclic immunosuppressant levels achieved using available dosage systems exhibit extreme variation between peak and trough levels. That is for each patient, effective macrocyclic immunosuppressant levels in the blood vary widely between administrations of individual dosages.

There is also a need for providing macrocyclic immunosuppressant in a water soluble form for injection. It is well known that Cremephore L used in a current formulations of macrocyclic immunosuppressants is a polyoxyethylated derivative of castor oil and is a toxic vehicle. There have been a number of incidences of anaphylaxis due to the castor oil component. At present there is no formulation that would allow the macrocyclic immunosuppressants to be in aqueous solution at the concenfrations needed due to poor water solubility ofthe drug.

Beyond all these very evident practical difficulties lies the occurrence of undesirable side reactions already alluded to, observed employing available oral dosage forms.

Several proposals to meet these various problems have been suggested in the art, including both solid and liquid oral dosage forms. An overriding difficulty which has however remained is the inherent insolubility ofthe macrocyclic immunosuppressants in aqueous media, hence preventing the use of a dosage form which can contain macrocyclic immunosuppressants in sufficiently high concenfration to permit convenient use and yet meet the required criteria in terms of bioavailability, e.g. enabling effective resorption from the stomach or gut lumen and achievement of consistent and appropriately high blood/blood-serum levels.

136 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc The particular difficulties encountered in relation to oral dosaging with macrocyclic immunosuppressants have inevitably led to restrictions in the use of macrocyclic immunosuppressant therapy for the freatment of relatively less severe or endangering disease conditions. A particular area of difficulty in this respect has been the adoption of macrocyclic immunosuppressant therapy in the freatment of autoimmune diseases and other conditions affecting the skin, for example for the treatment of atopic dermatitis and psoriasis and, as also widely proposed in the art, for hair growth stimulation, e.g. in the treatment of alopecia due to ageing or disease.

Thus while oral macrocyclic immunosuppressant therapy has shown that the drug is of considerable potential benefit to patients suffering e.g. from psoriasis, the risk of side- reaction following oral therapy has prevented common use. Various proposals have been made in the art for application of macrocyclic immunosuppressants, e.g. cyclosporine, in topical form and a number of topical delivery systems have been described. Attempts at topical application have however failed to provide any demonstrably effective therapy.

However, the present invention overcomes the problems described hereinabove. More specifically, an embodiment ofthe present invention is a prodrug of macrocyclic immunosuppressant which significantly enhances its solubility in aqueous solutions, thereby avoiding the need to utilize a carrier, such as ethanol or castor oil when administered as a solution. Moreover, the prodrugs of macrocyclic immunosuppressant, in accordance with the present invention, do not exhibit the side effects ofthe prior art formulations. Further, the inventor has found that the macrocyclic immunosuppressant prodrugs ofthe present invention enhance its absorption when administered in the prodrug form to a patient, thereby enhancing significantly its bioavailability and its efficacy.

Accordingly, in one aspect, the present invention is directed to a prodrug of macrocyclic immunosuppressants. The prodrug consists of an amino acid esterified to the free 137 H:\work\l 6₅2\17690\SPEC\17690.spec without claimsll.doc hydroxy group present on the side chain of cyclosporine, sirolimus, tacrolimus and either one ofthe hydroxyl groups ofthe pimecrolimus molecule.

For example, an aspect ofthe present invention is directed to, the compounds ofthe formulas

(a) CH,

^"CH,

N CH CO CYCLO

CH,

138 H:\work\16₅2\17690\SPEC\17690.spec without claimsll.doc (b)

(c)

139 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc (d)

or pharmaceutically acceptable salts thereof; wherein CYCLO represents the residues at positions 2-11 ofthe cyclosporine molecule; x-y is CH=CH or CH₂CH₂ and AA is an amino acid or a dipeptide ofthe formula GLY- AA. In the latter case, GLY is glycine and AA is any α-amino acid. In the dipeptide structure, an AA is attached to the drug via OH group using glycine as the spacer. Glycine is esterified to cyclosporine and then glycine is bonded to any AA via amide linkage using amino group of glycine and carboxylic acid group of AA.

The present invention is also directed to a pharmaceutical composition comprising a therapeutically effective amount ofthe compounds ofthe Formulas a-d above and a pharmaceutical carrier therefor.

In another embodiment, the present invention is directed to a method of treating a patient in need of macrocyclic immunosuppressant therapy, which method comprises administering to said patient an effective amount ofthe compounds of Formulas a-d.

140 H:\work\l 652\17690\SPEC\17690.spec without claimsILdoc In a further embodiment, the present invention is directed to a method of enhancing the solubility of macrocyclic immunosuppressant in an aqueous solution comprising reacting the hydroxy functionality in the MeBmt moiety at position 1 ofthe cyclosporine molecule as well as the specified hydroxyl functions in formulas b-d with an amino acid or acylating derivative thereof under ester forming conditions or by using a simple amino acid or a dipeptide structure wherein the AA is attached to drug using glycine as the spacer and isolating and isolating the product thereof.

In a still further embodiment, the present invention is directed to a method of enhancing the bioavailability of macrocyclic immunosuppressants when administered to a patient which comprises reacting the hydroxy functionality in the MeBmt moiety in position of the cyclosporine molecule with an amino acid or acylating derivative under ester forming conditions and as well as the specified hydroxyl functions in formulas b-d with an amino acid or acylating derivative thereof under ester forming conditions or by using a simple amino acid or a dipeptide structure wherein the AA is attached to drug using glycine as the spacer and isolating the product thereof and administering said product to the patient.

Overview: The procedure for the synthesis of the N-(L-proline)-glycine and N-(L-lysine)-glycine esters of Cyclosporine A. is outlined in Synthetic Sequence section. These examples are exemplary ofthe synthetic scheme using amino acids. The complete procedure and analytical data is given in the Experimental Section. Cyclosporine A (15 g) was coupled with chloroacetic anhydride (4 equivalent) in anhydrous pyridine. The experiment produced the chloroacetate ester of Cyclosporine A (SPI001201, 14 g, 88% yield) in good yield. The chloroacetate ester (10.1 g) was then treated with sodium azide in DMF to generate the azidoacetate ester of Cyclosporine A (SPI001202, 9.9 g, 97% yield). The azidoacetate (9.8 g) was then reduced with tin chloride (9 g) to prepare the glycine ester of Cyclosporine A (8.54 g, 89% yield). The glycine ester of Cyclosporine A (SPI001203) was then coupled with a two-fold excess of either boc-L-proline or Boc- 141 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc L-lysine using EDC as the coupling agent. After purification by column chromatography, the boc protecting groups were removed from the dipeptide esters of Cyclosporine A at low temperature (5 °C) by treatment with 2M hydrochloric acid in diethyl ether. The L-lysine-glycine ester salt of Cyclosporine A did not require additional purification and was dried. The L-proline-glycine ester salt of Cyclosporine A required purification. The salt was converted to the free-base with sodium bicarbonate and purified by filtration through silica gel (eluting with acetone). The salt was then formed at low temperature with dilute anhydrous hydrochloric acid and dried.

142 H:\work\16₅2\17690\SPEC\17690.spec without claimsll.doc Synthetic Sequence:

Cyclosporin A SPI001201

SPI0022

Synthesis of the N-(L-proline)-glycine and N-(L-Lysine)-glycine esters of Cyclosporine A: a) pyridine; b) NaN₃, DMF; c) SnCl₂, methanol; d) boc-L-lysine, EDC; e) boc-L-proline, EDC; f) HCl, Et₂O.

143 H:\work\l 652\17690\SPEC\17690.spec without claimsILdoc Experimental Section:

The synthesis of SPI0022 and SPI0023 was conducted in batches. Generally a small- scale experiment was performed first followed by a larger batch. Reagents mentioned in the experimental section were purchased at the highest obtainable purity from Aldrich, Acros, or Bachem, except for solvents, which were purchased from either Fisher

Scientific or Mallinckrodt. The Cyclosporine A (USP grade) used in these procedures was provided by Signature Pharmaceuticals, Inc.

1) SPI001201

Cyclosporine A (15.01 g, 0.0124 moles) was dissolved in anhydrous pyridine (35 mL) at room temperature, under an argon atmosphere. The solution was cooled to 5 °C in an ice/water batch and chloroacetic anhydride (9.10 g, 0.053 moles) was added. After stirring for 10 minutes, the ice bath was removed and the solution was allowed to stir under an argon atmosphere at room temperature for 17 hours. After 17 hours, diethyl ether (200 mL) was added. The ether was washed with water (2x100 mL) and dried for 1 hour over sodium sulfate (10 g). After filfration and concentration under reduced pressure, the remaining yellow foam was dried under high vacuum (1 hour at room temperature) and purified by flash chromatography on silica gel (200 g), eluting with heptane/acetone (2:1). After combining and concentrating the product containing fractions, the remaining light yellow foam (14.8 g) was purified a final time by crystallization from hot diethyl ether (140 mL). After cooling (-10 °C, 2 hours), filtration, and drying under high vacuum, the procedure generated the chloroacetate ester of Cyclosporine A SPI001201 as a white solid (14.0 g, 88.3% yield). 144 H:\work\16S2\17690\SPEC\17690.spec without claimsll.doc Cyclosporine A chloroacetate ester:

^XE NMR (300 MHz, CDC1₃): δ = 8.50 (d, IH, J= 9.6 Hz), 7.95 (d, IH, J= 6.6 Hz), 7.46 (d, IH, J= 9.0 Hz), 7.40 (d, IH, J= 7.8 Hz), 5.35-4.52 (m, 15 H), 4.37 (t, IH, J= 7.2 Hz), 4.12 (d, IH, J= 14.7 Hz), 3.89 (d, IH, J= 14.7 Hz), 3.45-3.0 (m, 15 H), 2.8-2.5 (m, 6H), 2.5-1.5 (m, 16H), 1.5-0.7 (m, 53 H).

¹³C NMR (75 MHz, CDC1₃): δ = 173.78, 173.37, 172.86, 172.61, 171.28, 171.18, 170.91, 170.79, 168.78, 167.64, 167.18, 128.77, 126.68, 75.46, 65.95, 58.89, 57.47, 55.80, 55.31, 54.86, 54.34, 50.19, 48.91, 48.35, 48.02, 44.80, 40.96, 39.44, 37.07, 35.93, 33.85, 33.25, 32.40, 31.74, 31.50, 30.38, 30.12, 29.82, 29.53, 25.13, 24.92, 24.78, 24.40, 23.99, 23.75, 22.85, 21.94, 21.41, 21.25, 20.84, 19.85, 18.79, 18.32, 17.89, 17.82, 15.46, 15.24, 10.08.

2) SPI001202

The chloroacetate ester of Cyclosporine A SPI001201 (10.10 g, 7.89 mmole) was dissolved in anhydrous N,N-dimethlformamide (30 mL) at room temperature. Sodium azide (2.15 g, 33.0 mmole) was added. The mixture was allowed to stir at room temperature for 24 hours in the dark, under an argon atmosphere. After 24 hours, diethyl ether (150 mL) was added and the precipitate was filtered. The ether was washed with water (2x100 mL), dried over sodium sulfate (15 g) for 30 minutes, filtered, and 145 H:\work\l₆₅2\1769₀\SPEC\1769₀.spec without claimsll.doc concentrated under reduced pressure. The remaining white solid was dried under high vacuum for 1 hour at room temperature. The experiment produced the azidoacetate ester of Cyclosporine A SPI001202 (9.90 g, 97% yield) as a white solid, which was used without further purification.

Cyclosporine A azidoacetate ester:

1H NMR (300 MHz, CDC1₃): δ = 8.48 (d, IH, J= 9.3 Hz), 7.95 (d, IH, J= 6.9 Hz), 7.45 (d, IH, J= 9.0 Hz), 7.39 (d, IH, J= 7.8 Hz), 5.5-4.5 (m, 15 H), 4.31 (t, IH, J= 6.6 Hz), 4.04 (d, IH, J= 17.3 Hz), 3.53 (d, IH, J= 17.3 Hz), 3.45-3.0 (m, 15 H), 2.8-2.5 (m, 6H), 2.5-1.5 (m, 16H), 1.5-0.7 (m, 53 H).

¹³C NMR (75 MHz, CDC1₃): δ = 173.76, 173.32, 172.82, 172.53, 171.13, 170.89, 170.76, 170.69, 169.70, 168.20, 167.49, 128.63, 126.61, 74.96, 58.91, 57.39, 55.56, 55.21, 54.80, 54.23, 50.14, 48.99, 48.23, 48.24, 47.93, 44.71, 40.89, 39.33, 39.22, 37.02, 35.83, 33.81, 32.96, 32.31, 31.67, 31.42, 30.31, 30.09, 29.76, 29.47, 25.08, 24.92, 24.84, 24.67, 24.51, 24.40, 23.94, 23.82, 23.71, 21.85, 21.33, 21.25, 20.82, 19.79, 18.71, 18.25, 17.92, 17.81, 15.17, 10.03.

3) SPI001203

The azidoacetate ester of Cyclosporine A SPI001202 (9.80 g, 7.62 mmole) was dissolved in methanol (250 mL) at room temperature. Water (40 mL) was added 146 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc followed by tin (II) chloride (5 g, 26.3 mmole). The solution was allowed to stir for 1 hour at room temperature when an additional quantity of tin (II) chloride (4 g, 21.0 mmole) was added. The solution was allowed to stir for an additional 2 hours at room temperature. Water (200 mL) containing ammonium hydroxide (40 mL, 29%) was added. After filtration, the solution was concentrated (to 200 mL) under reduced pressure. The remaining aqueous solution was extracted with ethyl acetate (2x200 mL). The ethyl acetate fractions were combined, dried over sodium sulfate (20 g), filtered and concentrated under reduced pressure. The remaining clear foam was purified by filfration through silica gel (150 g), eluting with dichloromethane/methanol (20:1). The procedure generated the glycine ester of Cyclosporine A as a clear, solid foam (8.54g, 89% yield).

Glycine ester of Cyclosporine A:

1H NMR (300 MHz, CDC1₃): δ = 8.60 (d, IH, J= 9.6 Hz), 8.06 (d, IH, J= 6.9 Hz), 7.53 (d, IH, J= 8.4 Hz), 7.51 (d, IH, J= 6.6 Hz), 5.7-4.52 (m, 15 H), 4.41 (t, IH, J= 6.9 Hz), 3.5-3.0 (m, 17 H), 2.82-2.5 (m, 8H), 2.5-1.5 (m, 16H), 1.5-0.7 (m, 53 H).

¹³C NMR (75 MHz, CDC1₃): δ = 174.10, 173.67, 173.23, 172.72, 172.55, 171.18, 171.10, 170.73, 170.61, 169.68, 167.77, 128.82, 126.42, 73.83, 58.57, 57.32, 55.99, 55.20, 54.74, 54.31, 50.08, 48.82, 48.28, 47.90, 44.70, 43.81, 40.74, 39.33, 39.24, 37.02, 35.84, 33.72, 33.07, 32.39, 31.72, 31.41, 30.25, 29.98, 29.74, 29.51, 25.05, 24.81, 24.73, 24.54, 24.31, 23.91, 23.78, 23.68, 21.86, 21.33, 21.25, 20.68, 19.76, 18.74, 18.24, 17.94, 17.79, 15.18, 10.03.

4) SPI0022

147 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

The glycine ester of Cyclosporine A (SPI001203, 2.0 g, 1.59 mmole) was dissolved in anhydrous dichloromethane (25 mL) with boc-L-lysine (1.31 g, 3.78 mmole) and EDC (0.75 g, 3.9 mmole), under an argon atmosphere at room temperature. The boc-L-lysine was prepared from the dicyclohexylamine salt (2.0 g in 50 mL ether) by extraction with cold potassium hydrogen sulfate solution (1 g in 50 mL water) followed by cold water (2x50 mL). The ether containing the boc-L-lysine was dried over sodium sulfate (5 g), filtered, concentrated and dried under high vacuum for one hour at room temperature. A few crystals of DMAP were added to the mixture of EDC, boc-L-lysine, and the glycine ester of Cyclosporine A and the solution was allowed to stir for 4 hours at room temperature. The dichloromethane solution was extracted with DIUF water (50 mL), 5% sodium bicarbonate solution (50 mL), and with DIUF water (50 mL). After drying over sodium sulfate (10 g), the dichloromethane solution was filtered and concenfrated under reduced pressure. The remaining white foam (3.O1 g) was purified by flash column chromatography on silica gel (50 g), eluting with heptane/acetone (2:1). The product containing fractions were combined, concentrated under reduced pressure, and dried under high vacuum. The purified protected intermediate (2.34 g white solid, 92.8% yield) was placed in a flask under an argon atmosphere, which was cooled in an ice-water bath. Cold anhydrous 2 M hydrochloric acid in diethyl ether (20 mL) was added and the solution stirred for 8 hours (at 5 °C). The mixture was slowly allowed to warm to room temperature overnight. After stirring for a total of 20 hours, the flask was cooled again in an ice- water bath for 30 minutes. The product was filtered and dried under high vacuum for 1 hour at room temperature and then at 50 °C for 4 hours. The

148 H:\work\l 652\17690\SPEC\17690.spec without claimsILdoc experiment produced Cyclosporine A N-(L-lysine)-glycine ester, dihydrochloride trihydrate (SPI0022, 1.59 g, 73.9% yield) as a white solid.

1H NMR (300 MHz, CDC1₃, NMR data is for the free base): δ = 8.58 (d, IH, J= 9.3 Hz), 8.04 (d, IH, J= 6 Hz), 7.80 (d, IH, J= 6 Hz), 7.49 (d, 2H, J= 8.4 Hz), 5.70-4.6 (m, 17 H), 4.41 (m, IH), 4.28 (dd, IH, J= 17, 7.2 Hz), 3.67 (d, IH, J= 17 Hz), 3.46 (s 3H), 3.4-2.8 (m, 16 H), 2.8-2.5 (m, 8H), 2.5-1.35 (m, 24H), 1.5-0.7 (m, 50 H). ¹³C NMR (75 MHz, CDC1₃, NMR data is for the free base): δ = 175.23, 173.77, 173.34, 172.75, 172.63, 171.34, 171.22, 170.94, 170.84, 170.91, 169.89, 169.70, 128.74, 126.67, 74.41, 58.82, 57.43, 55.91, 55.21, 54.81, 54.42, 50.17, 48.89, 48.31, 47.98, 44.78, 41.92, 40.82, 40.69, 39.44, 39.32, 27.19, 35.91, 34.88, 33.71, 33.25, 33.12, 32.44, 31.83, 31.50, 30.38, 30.06, 29.81, 29.55, 25.14, 24.90, 24.52, 24.43, 24.00, 23.76, 21.93, 21.42, 21.29, 20.81, 19.84, 18.82, 18.32, 17.96, 17.86, 15.21, 10.10.

^• CHN analysis: Calculated for C₇₀H_12gCl₂N₁₄O₁₅-3H₂O: C 55.50, H 8.92, and N 12.74; found: C 58.28, H 8.98, and N 13.16.

HPLC analysis: 99.60%o purity; r.t.= 14.763 min.; 80% acetonitrile/20% Tris base in DIUF water; 1 mL/min; 60C; Synergi Hydro RP, 4u column (serial # 163383-7), 4.6x250 mm; 20 ul; UV= 210 nm.

Melting point: 196.0-198 °C (unconected)

149 H:\work\1652\I7690\SPEC\17690.spec without claimsILdoc 5) SPI0023

The glycine ester of Cyclosporine A (SPIO01203, 7.50 g, 5.95 mmole) was dissolved in anhydrous dichloromethane (50 mL) with boc-L-proline (2.56 g, 11.90 mmole) and EDC (2.28 g, 11.9 mmole), under an argon atmosphere at room temperature. A few crystals of DMAP were added to the mixture of EDC, boc-L-proline, and the glycine ester of Cyclosporine A and the solution was allowed to stir for 3 hours at room temperature. The dichloromethane solution was extracted with DIUF water (50 mL), 5% sodium bicarbonate solution (2x50 mL), and with DIUF water (50 mL). After drying over sodium sulfate (10 g), the dichloromethane was filtered and concenfrated under reduced pressure. The remaining white foam (9.50 g) was purified by flash column chromatography on silica gel (150 g), eluting with heptane/acetone (2:1 followed by 1:1). The product containing fractions were combined, concentrated under reduced pressure, and dried under high vacuum (7.94 g white solid, 91.7% yield) for 10 minutes at room temperature.

The purified protected intermediate (6.46 g) was placed in a flask under an argon atmosphere, which was cooled in an ice-water bath. Cold anhydrous 2 M hydrochloric acid in diethyl ether (150 mL) was added and the solution stirred for 8 hours (at 5 °C). The mixture was slowly allowed to warm to room temperature overnight. After stirring for a total of 20 hours, the flask was cooled again in an ice-water bath for 30 minutes. The product was filtered and dried under high vacuum for 30 minutes at room temperature. The Cyclosporine A N-(L-proline)-glycine ester, hydrochloride (5.17 g, 84.6% yield, and 90% purity by HPLC) was converted to the free base by dissolving the 150 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc salt in DIUF water (25 mL) that contained sodium bicarbonate (1 g). The free base was extracted with dichloromethane (3x25 mL), which was dried over sodium sulfate (5 g), filtered and concenfrated. The remaining off-white solid (5 g) was purified by filfration through silica gel (100 g), eluting with acetone. The product containing fractions were combined, concentrated under reduced pressure, and dried under high vacuum for 30 minutes at room temperature. The hydrochloride salt was regenerated by dissolving the free base (3.8 g) in diethyl ether (25 mL) and adding it to anhydrous 2M hydrochloric acid (5 mL) in heptane (50 mL), while cooling in an ice- ater bath. After 20 minutes at 5 °C, the white solid was filtered and dried under high vacuum for 6 hours at room temperature. The experiment produced Cyclosporine A N-(L-proline)-glycine ester, hydrochloride (SPI0023, 3.8 g) as a white solid.

1H NMR (300 MHz, CDC1₃): δ = 14.20 (br s, 2H), 8.62 (d, IH, J= 10 Hz), 8.06 (d, 1H₉ J= 6.9 Hz), 7.61 (d, IH, J= 8.1 Hz), 7.48 (d, IH, J= 9 Hz), 5.70-5.50 (m, 3H), 5.40-4.6O ( , 12H), 4.37 (m, IH), 4.20 (d, IH, J= 18 Hz), 3.97 (d, IH, J= 18 Hz), 3.70 (m, IH), 3.45 (s, 3H), 3.23-3.08 (m, 12H), 2.66 (s, 3H), 2.60 (s, 3H), 2.50-1.80 ( , 15H), 1.78-1.20 (m, 15H), 1.15-0.66 (m, 46H).

¹³C NMR (75 MHz, CDC ): δ = 174.15, 173.49, 172.67, 172.59, 171.86, 171.20, 171.13, 171.02, 170.83, 169.68, 168.77, 167.55, 128.30, 127.10, 80.09, 75.58, 62.65, 59.35, 57.36, 55.53, 55.30, 54.78, 54.35, 53.60, 50.25, 50.09, 48.92, 48.18, 48.12, 44.62, 40.59, 40.02, 39.43, 39.30, 37.13, 35.88, 33.74, 33.07, 32.19, 32.01, 31.86, 31.50, 31.43, 30.43, 29.93, 29.72, 29.30, 29.16, 27.56, 26.04, 25.00, 24.86, 24.74, 24.39, 20.96, 19.81, 18.71, 18.26, 18.09, 17.85, 17.79, 15.09, 14.30, 10.00.

CHN analysis:

Calculated for C₆₉H₁₂₂ClN₁₃Oι₄: C 59.48, H 8.83, and N 13.07; found: C 59.84, H 9.02, and N 12.65. 151 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc HPLC analysis:

99.59% purity; r.t.= 10.613 min.; 85% acetonitrile/15% Tris base in DIUF water; 1.2 mL/min; 60C; Synergi Hydro RP, 4u column (serial # 163383-7), 4.6x250 mm; 20 ul; UV= 210 nm.

Melting point: 197.0-199 °C (uncorrected)

These prodrugs of cyclosporin ofthe present invention are effective in treating diseases or conditions in which macrocyclic immunosuppressants normally are used. These prodrugs are transformed within the body to release the active compound and enhances the therapeutic benefits ofthe macrocyclic immunosuppressants by reducing or eliminating biopharmaceutical and pharmacokenetic barriers associated with each of them. However it should be noted that these prodrugs themselves will have sufficient activity without releasing any active drug in the mammals. Since the prodrugs are more soluble in water then cyclosporine or other macrocyclic immunosuppressants, it does not need to be associated with a carrier vehicle, such as alcohol or castor oil which may be toxic or produce unwanted side reactions. Moreover, oral formulations containing the prodrugs ofthe prodrugs are absorbed into the blood and are quite effective.

Thus, the prodrug of cyclosporin of the present invention enhances the therapeutic benefits by removing biopharmaceutical and pharmacokenetic barriers of existing drugs.

Furthermore, the prodrugs are easily synthesized in high yields using reagents which are readily and commercially available.

VI. Valproic Acid Esters

Valproic acid (2-Propylpentanoic acid) is low molecular weight carboxylic acid derivative which is widely used as an anti-convulsive agent, useful in the treatment of epilepsy and also possess vasodilatation activity in the brain to relieve migraine

152 H:\work\1652\17690\SPEC\l 7690.spec without claimsll.doc headaches. It is administered orally to confrol epileptic episodes in humans and also alleviate severe pain associated with migraine headaches.

VALPROIC ACID

Valproic acid has been shown to have a large number of therapeutic applications, which are quite varying and somewhat surprising. For example, in addition to its efficacy in the treatment of epilepsy and migraine headaches, it has been shown to be effective in the treatment of certain psychiatric illnesses, such as bipolar disorder, mood stabilization, control of aggression, impulsivity in personality disorder, agitation in dementia, and has also been of use as adjunct therapy in the treatment of post traumatic stress disorder (PTSD).

Mechanism of Action:

In spite of being used in the treatment of epilepsy for a number of years, the exact mechanism of action of Valproic acid is still unknown. It has been postulated that it exerts its action by increasing concentration of gamma-amino butyric acid (GABA) in the brain. Gamma-amino butyric acid is a neurotransmitter, a chemical that nerves use to communicate with one another.

Valproate is the drug of choice in myoclonic epilepsy, with or without generalized tonic- clonic seizures, including juvenile myoclonic epilepsy of Janz that begins in adolescence or early adulthood. Photosensitive myoclonus is usually easily controlled. Valproate also is effective in the treatment of benign myoclonic epilepsy, postanoxic myoclonus, and, with clonazepam, in severe progressive myoclonic epilepsy that is characterized by tonic-clonic seizures as well. It also may be prefened in certain stimulus-sensitive (reflex, startle) epilepsies.

153 H:\work\16₅2\17690\SPEC\17690.spec without claimsll.doc Although Valproate may be effective for infantile spasms; it is relatively contraindicated in children whose spasms are due to hyperglycinemia or other underlying metabolic (mitochondria!) abnormalities. In general, atonic and akinetic seizures in patients with Lennox-Gastaut syndrome are difficult to control, but Valproate is the drug of choice for treatment of mixed seizure types. Since this drug has been useful in some patients who are refractory to all other antiepileptic drugs, it may warrant a trial iα nearly all nonresponsive patients regardless of seizure type.

In spite of it usefulness, hepatotoxicity may be fatal, but is idiosyncratic and not preventable by routinely monitoring liver enzymes. Hepatotoxicity occurs in very young children, most often those on multiple anticonvulsants. Valproate-induced cytopenias may be dose-related and warrant monitoring of complete blood counts during therapy. Encephalopathy with hyperammonemia without liver function test abnormalities may occur. Pregnant women in first month are at risk for neural tube defects.

Valproic acid is a low molecular weight liquid with characteristic odor. Taken orally it has unpleasant taste and can severely irritate mouth and throat. In order to convert Valproic acid into a solid dosage form convenient for oral administration, a number of derivatives with covalent and ionic bond with the carboxylic acid have been made. A simple sodium salt of Valproic acid, resulting in Valproate sodium is available as a solid. However a stable coordination complex, know as Divalproex sodium was formed by partial neutralization of two molecules of Valproic acid with one atom of sodium. This product is the most widely available Valproic acid hemi salt marketed by Abbott Laboratories in the USA under the brand name Depakote®. Depakote® is also available in extended release formulation for oral administration.

A significant disadvantage of Valproic acid is that it in liquid form it is difficult to administer. Furthermore, administration of Valproic acid in different forms does not uniformly produce desired bioavailability. For example, the overall bioavailability of Valproate from Valproic acid, its sodium salt, Divalproex®, and their, extended release 154 H:\work\1652\17690\SPEC\l 7690.spec without claimsll.doc formulations are not quite interchangeable. Since continuous monitoring of plasma profile of Valproic acid is essential, any change in plasma concentration due to changes in the formulation adversely affect overall therapeutic outcome.

In order to improve the therapeutic effectiveness, uniform blood profile, develop pharmaceutically elegant formulation and reduce first pass metabolism, present invention discusses prodrugs of Valproic acid which overcome some ofthe difficulties stated above.

Until now there has been no pharmaceutical preparation has been available in the market that can deliver Valproic acid with out harmful side effects. The present invention however, has produced a number of water soluble, non-toxic derivatives of Valproic acid which are suitable for delivering Valproic acid consistently in the body without any harmful side effects and without the needs for expensive additives, and exepients.

Accordingly, in one aspect, the present invention is directed to a class of prodrugs of Valproic acid. The prodrug consists ofthe hydroxyl group of an amino acid esterified to the free carboxyl group present on the Valproic acid molecules. In another embodiment, the amine group ofthe amino acid is reacted with COOH group to form an amide linkage.

More specifically, an embodiment ofthe present invention is directed to, the compounds ofthe formula

VALPROIC ACID

155 H:\work\16S2\1769Q\SPEC\17690.spec without claimsll.doc or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AA and AA is an amino acid, in which either an amine group or the hydroxyl group is reacted with the carboxylic acid group of Valproic Acid.

The present invention is also directed to a pharmaceutical composition comprising a therapeutically effective amount ofthe various Valproic acid prodrugs above and a pharmaceutical carrier therefor.

In another embodiment, the present invention is directed to a method of treating a patient in need of Valproic acid therapy, which method comprises administering to said patient an effective amount ofthe Valproic acid.

In a further embodiment, the present invention is directed to a method of converting liquid Valproic acid into a solid powder by reacting the carboxyl functionality ofthe Valproic acid with either amine or hydroxyl functionality of an amino acid and isolating the products thereof.

In a still further embodiment, the present invention is directed to a method of substantially and in a therapeutically efficacious manner, reducing or eliminating the potential first pass metabolism thereby improving the consistent therapeutic effect by administering to a patient a prodrug which comprises reacting the COOH functionality ofthe Valproic acid molecule with either NH2 or OH functionality of selected amino acids to form an ester or amide covalent bond respectively and isolating the product thereof and administering said product to the patient.

It has been found that when unsubstituted naturally occurring amino acids are esterified to Valproic acid, the resulting prodrugs are pharmaceutically elegant free flowing powders, and are rapidly absorbed into the body and release non-toxic amino acids upon cleavage in the body and require none ofthe emulsifiers, additives and other exepients.

156 H:\work\1652\17690\SPEC\l 769Q.spec without claimsll.doc Furthermore, it has been shown *-;Jat the current invention also produced drugs, while they are prodrugs of Valproic acid; they were highly effective anti-epileptics and were exhibiting such effect intact. Thus the current amino acid prodrugs are effective anti- epileptics and useful in the freatment of a number of psychiatric illnesses and exhibit such potential with or without releasing the active parent drug.

The Valproic acid prodrug bulk density is much higher than the corresponding sodium salts, and they are suitable for compacting large weight tablets and capsules. Furthermore, their do not exhibit bitter taste and unusual odor ofthe Valproic acid.

While the prodrugs my invention are not supposed to possess any acidic activity due to blockage ofthe carboxylic acid group responsible for such, however it has been shown that the prodrugs are effective anti-epileptics with or without releasing Valproic acid. However, all ofthe Valproic acid prodrugs described are released in vivo the active drug with all its pharmacological and psychoactive properties.

The prodrug of Valproic acid clearly provides a number of advantages over Valproic acid, for example, all ofthe side chains cleaved from these prodrugs are naturally occurring essential amino acids hence are non-toxic. This results in high therapeutic index. Secondly all the prodrugs are readily cleaved in the body to release Valproic acid. Furthermore, due their high water solubility, they can be easily administered by either forming an in-situ solution just before TV administration using lyophilized sterile powder or providing the drug in solution in prefilled syringe or bottles for infusion. The aminoacid esters are more stable than Valproic acid since COOH group in Valproic acid is blocked to reaction with bases. Thus the Valproic acid prodrags invented here are more effective then Valproic acid itself without the toxicity and other pharmaceutical problems associated with current marketed formulations.

The procedure for the synthesis ofthe L-serine, L-threonine, and L-hydroxyproline esters of valproic acid (2-propylpentanoic acid) is outlined in Synthetic Sequence 157 H:\work\16S2\17690\SPEC\17690.spec without claimsll.doc section and is exemplary for tM preparation ofthe various prodrugs ofthe present invention. The complete procedure and analytical data is given in the Experimental Section. In general, valproic acid (2-8 g, in batches) was coupled with the N- benzyloxy/benzyl ester protected amino acids using EDC in the presence of a catalytic amount of DMAP. Once the reactions were complete (20 hours at room temperature), the mixture was extracted with DIUF water, dried over sodium sulfate, and concentrated under reduced pressure. The crude material was either used directly for the deprotection step or purified by column chromatography. The procedure generated the protected amino acid esters of valproic acid in yields ranging from 72% to 92%. The protecting groups were removed by hydrogenation (30 psi H₂) in the presence of 10% palladium on carbon. The amino acid esters of valproic acid were exfracted away from the palladium catalyst with ethanol, concentrated, and dried. The final salts were formed by acidification with hydrochloric acid. The crude salts (yields ranging from 57% to 92%) were then purified by the methods described in the Experimental Section.

158 H:\work\1652\17690\SPEC\l 769Q.spec without claimsll.doc Synthetic Sequence: 1. SPIC001

Valproic acid Z-Ser-OBzl SPIC00101 SPIC001

2. SPIC002

Valproic acid Z-Hyp-OBzl SPIC00201 SPIC002 3. SPIC003

Valproic acid Z-Thr-OBzl SPIC00301 SPIC003

Synthesis of the L-serine, L-threonine, and L-hydroxyproline esters of valproicacid: a) EDC, DMAP, CH₂C1₂; b) H₂, 10% Pd/C, EtOH, EtOAc; c) HCl.

159 H:\work\l 6₅2\17690\SPEC\17690.spec without claimsILdoc Experimental Section:

The synthesis of SPIC001, SPIC002 and SPIC003 was conducted in one or two batches. Reagents mentioned in the experimental section were purchased at the highest obtainable purity from Lancaster, Sigma- Aldrich, Acros, or Bachem, except for solvents, which were purchased from either Fisher Scientific or Mallinkrodt.

1) SPIC001: 2-Propylpentanoic acid 2(S)-amino-2-carboxy-ethyl ester, hydrochloride

(L-Serine-valproic acid ester, hydrochloride)

A mixture of 2-propylpentanoic acid (valproic acid, 6.48 g, 44.93 mmole), N- carbobenzyloxy-L-serine benzyl ester (Z-Ser-OBzl, 14.80 g, 44.93 mmole), EDC (8.61 g, 44.91mmole), and DMAP (549 mg, 4.49 mmole) in anhydrous dichloromethane (50 mL) was stirred under an argon atmosphere at room temperature for 20 hours. After 20 hours, the dichloromethane was washed with water (3 x50 mL), dried over magnesium sulfate (5 g), filtered and concentrated under reduced pressure. The remaining colorless oil (20.87 g) was purified by column chromatography on silica gel (150 g, 0.035-0.070 mm, 6 nm pore diameter), eluting with hexanes/ethyl acetate (3:1). After concentration of the product containing fractions under reduced pressure and drying under high vacuum until the weight was constant, the experiment produced the protected L-serine- valproate ester SPIC00101 (18.9 g, 92% yield) as a colorless oil.

160 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc 1H NMR (300 MHz, DMSO): δ = 7.96 (IH, d, J= 8.1 Hz), 7.35 (10H, m), 5.14 (2H, s), 5.05 (2H, s), 4.51 (IH, m), 4.29 (2H, m), 2.29 (IH, m), 1.50-1.25 (4H, m), 1.25-1.10 (4H, m), 0.80 (6H, t, j= 6.6 Hz).

¹³C NMR (75 MHz, DMSO): δ = 174.88, 169.15, 155.85, 136.58, 135.45, 128.26,

128.18, 127.47, 127.71, 127.57, 66.32, 65.66, 62.47, 53.09, 44.20, 33.86, 33.79, 19.95, 13.85.

The protected L-serine-valproate ester SPIC00101 (18.9 g, 41.48 mmole) was dissolved in ethanol (60 mL) and ethyl acetate (60 mL) at room temperature and added to a Parr bottle (500 mL) that contained 10% palladium on carbon (3.0 g, 50% wet) under a nitrogen atmosphere. The nitrogen atmosphere was replaced with hydrogen gas (30 psi). After 4 hours of shaking, additional palladium catalyst (1.0 g) in ethanol\ethyl acetate (1:1, 100 L) was added and the reaction mixture shook overnight under hydrogen gas (30 psi) at room temperature. After 24 hours the catalyst was removed by filtration through a thin layer of activated carbon. The ethanol and ethyl acetate were concenfrated under reduced pressure at room temperature. After drying under high vacuum, the remaining solids were acidified with hydrochloric acid in diethyl ether (2M, 24.6 L). The mixture was stored in a refrigerator for two hours before filtration and washing with additional cold diethyl ether (10 mL). After filtration, the remaining white solid was dried at room temperature under high vacuum until the product weight was constant (24 hours). The experiment produced L-serine-valproic acid ester, hydrochloride SPIC001 (6.34 g, 57% yield) as a white solid.

SPIC001

161 H:\work\16₅2\17690\SPEC\17690.spec without claimsILdoc 1H NMR (300 MHz, DMSO): δ = 8.73 (br s, 3H), 4.47 (dd, IH, J= 12.9, 4.5 Hz), 4.31 (dd, 2H, J= 12.9, 3.6 Hz), 2.36 (m, IH), 1.50 (m, 2H), 1.39 (m, 2H), 1.20 (m, 4H), 0.84 (t, 6H, J= 7 Hz).

¹³C NMR (75 MHz, DMSO): δ = 174.67, 168.19, 61.84, 51.16, 44.12, 33.76, 33.58, 20.07, 19.92, 13.97, 13.89.

HPLC analysis:

98.49% purity; rt= 4.767 min; Luna C18 5u column (sn 167917-13); 4.6x250 mm; 254 nm; 33% ACN/66% DIUF water; 35 C; 20 ul inj.; Iml/min; 20 mg/mL sample size; sample dissolved in mobile phase.

CHN analysis: calc: C 49.34, H 8.28, N 5.23; found: C 49.22, H 8.35, N 5.24.

Melting point: 159-160 °C

2) SPIC002: 4(R)-(2-Propyl-pentanoyloxy)-pyrrolidine-2(S)-carboxylic acid

(L-Hydroxyproline-valproic acid ester)

A mixture of 2-propylpentanoic acid (valproic acid, 4.32 g, 30 mmole), N- carbobenzyloxy-L-hydroxyproline benzyl ester (Z-Hyp-OBzl, 10.66 g, 30 mmole)¹, EDC (5.74 g, 30 mmole), and DMAP (366 mg, 3 mmole) in anhydrous dichloromethane (30 mL) was stirred under an argon atmosphere at room temperature for 20 hours. After 20 hours, the dichloromethane was washed with water (3x30 mL), dried over magnesium sulfate (5 g), filtered and concentrated under reduced pressure. The remaining colorless oil SPIC00201 (11.95 g, 24.7 mmole, 82.4% yield) was used without purification.

162 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

1H NMR (300 MHz, CDC1₃): δ = 7.29 (10H, m), 5.28-5.00 (5H, m), 4.55 (1/2H, t, J= 8 Hz), 4.46 (1/2H, t, J= 8 Hz), 3.80-3.60 (2H, m), 2.43-2.16 (3H, m), 1.60-1.45 (2H, m), 1.40-1.32 (2H, ), 1.28-1.20 (4H, m), 0.86 (6H, m).

¹³C NMR (75 MHz, DMSO): δ = 174.74, 171.40, 171.05, 153.79, 153.31, 136.34, 136.20, 135.57, 135.38, 128.24, 128.13, 127.95, 127.87, 127.67, 127.52, 127.28, 127.10, 72.29, 71.53, 66.34, 66.10, 57.66, 57.19, 52.27, 51.89, 44.13, 40.33, 35.78, 34.79, 34.04, 33.92, 33.35, 20.00, 19.91, 13.79, 13.73.

The protected L-hydroxyproline- valproate ester SPIC00201 (17.24 g, 35.79 mmole) was dissolved in ethanol (50 mL) and ethyl acetate (100 mL) at room temperature and added to a Parr bottle (500 mL) that contained 10% palladium on carbon (3.5 g, 50% wet) under a nitrogen atmosphere. The nitrogen atmosphere was replaced with hydrogen gas (30 psi). After 15 hours of shaking, the catalyst was removed by filfration through a thin layer of celite and activated carbon. The ethanol and ethyl acetate mixture was concentrated under reduced pressure at room temperature. After drying overnight under high vacuum at room temperature, the experiment produced L-hydroxyproline- valproic acid ester SPIC002 (9.2 g, 99.8% yield) as a white solid. In order to remove trace impurities, the zwitterion was purified by reverse-phase column chromatography (50 g ODS silica gel) in two batches. The zwitterion was placed on the column in DIUF water and eluted with mixture of DIUF water/methanol (2:1, 1:1, 1:2, 100% methanol).

163 H:\work\l 6₅2\17690\SPEC\17690.spec without claimsILdoc The product containing fractions were combined, concentrated under reduced pressure at 20 °C (or less), and dried under high vacuum at room temperature until the weight was constant (24 hours, 6.4 g white solid recovered).

SPIC002

1H NMR (300 MHz, CDC1₃): δ = 12.40 (br s, IH), 8.32 (br s, IH), 5.28 (m, IH), 4.11 (t, IH, J= 7.2 Hz), 3.59 (m, IH), 3.34 (br d, IH, J= 10.5 Hz), 2.50-2.22 (m, 3H), 1.62-1.50 (m, 2H), 1.50-1.32 (m, 2H), 1.32-1.19 (m, 4H), 0.88 (t, 6H, J= 7.2 Hz).

¹³C NMR (75 MHz, CDC1₃): δ = 175.99, 173.35, 71.83, 59.56, 49.77, 45.08, 36.19, 34.51, 20.87, 14.31.

HPLC analysis: 99.20% purity; r.t.= 7.228 min.; 70% DIUF water/30% acetonitrile; 1 mL/min; 36.8C; Luna C18, 5u column (serial # 167917-13), 4.6x250 mm; 22 ul injection; sample dissolved in mobile phase.

CHN analysis: calc: C 60.68, H 9.01, N 5.44; found: C 60.58, H 9.12, N 5.48.

Melting point: 179.0-180.0 °C

3) SPIC003: 2-Propyl-pentanoic acid2(S)-amino-2-carboxy-l(R)-methyl-ethyl ester, hydrochloride

164 H:\work\16₅2\17690\SPEC\17690.spec without claimsll.doc (L-Threonine-valproic acid ester, hydrochloride)

A mixture of 2-propylpentanoic acid (valproic acid, 4.32 g, 30 mmole), N- carbobenzyloxy-L-threonine benzyl ester (Z-Thr-OBzl, 10.30 g, 30 mmole), EDC (5.74 g, 30 mmole), and DMAP (366 mg, 3.0 mmole) in anhydrous dichloromethane (30 mL) was stirred under an argon atmosphere at room temperature for 20 hours. After 20 hours, the dichloromethane was washed with water (3x30 mL), dried over magnesium sulfate (5 g), filtered and concentrated under reduced pressure. The remaining colorless oil (13.44 g) was purified by column chromatography on silica gel (100 g, 0.035-0.070 mm, 6 nm pore diameter), eluting with hexanes/ethyl acetate (4:1). After concentration ofthe product containing fractions under reduced pressure and drying under high vacuum until the weight was constant, the experiment produced the protected L-threonine-valproate ester SPIC00301 (12.65 g, 89.8% yield) as a colorless oil.

1H NMR (300 MHz, CDC1₃): δ = 7.40-7.05 (1 IH, m), 5.45 (IH, m), 5.17-5.02 (4H, m), 4.53 (IH, d, J= 9.6 Hz), 2.24 (IH, m), 1.58-1.40 (2H, m), 1.40-1.15 (9H, m), 0.86 (6H, m).

13 C, NMR (75 MHz, DMSO): δ = 174.24, 169.29, 156.48, 136.61, 135.34, 128.26, 128.20, 127.74, 127.67, 127.58, 69.04, 66.33, 65.78, 57.62, 44.50, 33.89, 33.80, 20.03, 19.91, 16.40, 13.87.

165 H:\work\1652\17690\SPEC\1769Q.spec without claimsILdoc The protected L-threonine- valproate ester SPIC00301 (12.65 g, 26.9 mmole) was dissolved in ethanol (50 mL) and ethyl acetate (50 mL) at room temperature and added to a Parr bottle (500 mL) that contained 10% palladium on carbon (2.53 g, 50% wet) under a nitrogen atmosphere. The nifrogen atmosphere was replaced with hydrogen gas (30 psi). After 20 hours the catalyst was removed by filtration tlirough a thin layer of activated carbon, washing with ethanol (25 mL). The ethanol and ethyl acetate were concenfrated under reduced pressure at room temperature. After drying under high vacuum, the remaining solids (6.13 g) were acidified with hydrochloric acid (3.1 mL cone.) in DIUF water (50 mL). The solution was filtered a second time through activated carbon and dried overnight in a freeze-dryer. The experiment produced L- threonine-valproic acid ester, hydrochloride SPIC003 (6.52 g, 86.0 % yield) as a white solid.

The combined batches ofthe L-threonine- valproic acid ester, hydrochloride SPIC003 (8.8 g) were purified by crystallization form acetonitrile. After the salt was dissolved in hot acetonitrile (225 mL), the material was treated activated acrbon, filtered, and placed in a 5 °C refrigerator overnight. The white solids were filtered after 18 hours, washed with cold acetonitrile (10 mL), and dried under high vacuum at room temperature until the product weight was constant (24 hours). The process recovered L-threonine-valproic acid ester, hydrochloride SPIC003 (6.82 g, 77.5 % recovery) as a white solid.

166 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc

SPIC003

1H NMR (300 MHz, DMSO): δ = 8.71 (br s, 3H), 5.28 (m, IH), 4.16 (d, IH, J= 2.7Hz), 2.33 (m, IH), 1.56-1.40 (m, 2 H), 1.37-1.27 (m, 5H), 1.21-1.13 (m, 4H), 0.84 (t, 6H, J= 6.6 Hz).

¹³C NMR (75 MHz, DMSO): δ = 173.97, 168.19, 67.69, 55.42, 44.43, 33.95, 33.78, 20.07, 19.95, 16.54, 13.94.

HPLC analysis:

98.88% purity; r.t.= 4.864 min.; 70% DIUF water/30% acetonitrile; 1 mL/min; 40C; Luna C18, 5u column (serial # 211739-42), 4.6x250 mm; 20 ul injection; sample dissolved in mobile phase.

CHN analysis: calc: C 51.15, H 8.59, N 4.97; found: C 51.29, H 8.59, N 4.98.

Melting point: 144 °C

Solubility ofthe above esters wee determined in water at room temperature by dissolving excess of each ofthe drag and allowing them to settle for a few hours. The resulting solutions were centrifuged at 1500rpm for 3 min and the supernatant liquid was analyzed. It was shown that these esters possess solubility in water in excess of 50 mg/mL.

167 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc There are a number of screening tests to determine the utility ofthe prodrugs created according to the disclosed methods. These include both in vitro and in vivo screening methods.

The in vitro methods include acid/base hydrolysis of the prodrags, hydrolysis in pig pancreas, hydrolysis in rat intesetinal fluid, hydrolysis in human gastric fluid, hydrolysis in human intestinal fluid, and hydrolysis in human blood plasma. These assays are described in Simmons, DM, Chandran, VR and Portmann, GA, Danazol, Amino Acid Prodrugs: In Vitro and In Situ Biopharmaceutical Evaluation, Drag Development and Industrial Pharmacy, Vol. 21, Issue 6, Page 687, 1995, the contents of all of which are incorporated by reference.

Prodrags of Valproic acid ofthe present invention are effective in treating diseases or condidiotns in which Valproic acid normally are used. The prodrags disclosed herein are transformed within the body to release the active compond and enhances the therapeutic benefits ofthe Valproic acid by reducing or eliminating biopharmaceutical and pharmacokinetic barriers associated with each of them. However it should be noted that these prodrags themselves will have sufficient activity without releasing any active drug in the mammals.

Thus, the prodrag ofthe present invention enhances the therapeutic benefits by removing biopharmaceutical and pharmacokenetic barriers of existing drags. Furthermore, the prodrugs are easily synthesized in high yields using reagents which are readily and commercially available.

VII WATER SOLUBLE PRODRUGS OF FIBRIC ACID DERIVATIVES

Fibric acid derivatives are useful anti-hyperlipidemic drugs useful in the treatment of hyperlipidemia in mammals where the symptoms are elevated triglycerides, low HDL (High density lipoproteins or "good" cholesterol, and elevated cholesterol. Fibric Acid derivatives are also useful in reducing LDL (Low density lipoproteins, or "bad" 168 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc cholesterol). The general structure ofthe fibric acid analogs is represented below, where X is various mixed aliphatic and aromatic functionalities. Specific derivatives included in this formula are clofibric acid, fenofibric acid, ciprfibrate and gemfibrozil and the like.

FIBRIC ACID ANALOGS

Typical examples ofthe chemical moiety X in the above structure are shown below.

Ciprofi ric Acid

Clofibric Acid

Fenofibric Acid

Gemfibrozil

169 H:\work\I 6S2\17690\SPEC\17690.spec without claimsll.doc Fibric acid analogs shown in the structure above have been shown to have a large number of therapeutic applications, which are quite varying and somewhat surprising. Broadly, these derivatives are useful in the freatment dyslipidemia and dyslipoproteinemia. Dyslipidemia and dyslipoproteinemia are herein defined to include the group selected from hypercholesterolemia, abnormal and elevated levels of cholesterol, abnormal and elevated levels of LDL cholesterol, abnormal and elevated levels of total cholesterol, abnormal and elevated levels of plasma cholesterol, abnormal and elevated levels of triglycerides, hypertrigylceridaemia, abnormal levels of lipoproteins, abnormal and elevated levels of low density lipoproteins (LDLs), abnormal and elevated levels of very low density lipoproteins, abnormal and elevated levels of very low intermediate density lipoproteins, abnormal levels of high density lipoproteins, hyperlipidemia, hyperchylomicronemia, abnormal levels of chylomicrons, related disorders, and combinations thereof such as those described in The ILIB Lipid Handbook for Clinical Practice, Blood Lipids and Coronary Heart Disease, Second Edition, A. M. Gotto et al, International Lipid Information Bureau, New York, N.Y., 2000, which is hereby incorporated by reference.

Mechanism of Action:

The mechanism of action of Fibric acid derivatives seen in clinical practice have been explained in-vivo in transgenic mice and in vitro in human hepatocyte cultures by the activation of peroxisome proliferator activated receptor alpha (PPAR-alpha). Through this mechanism, Fibric acid derivatives increase lipolysis and elimination of triglyceride- rich particles from plasma by activating lipoprotein lipase and reducing production of apoprotein C-III (an inhibitor of lipoprotein lipase activity).

The resulting fall in triglycerides produces an alteration in the size and composition of LDL from small, dense particles (which are thought to be atherogenic due their susceptibility to oxidation), to large buoyant particles. These larger particles have greater affinity for cholesterol receptors and are catabolized rapidly. Activation of PPAR-alpha also induces an increase in the synthesis of apoproteins A-I, A-II, and HDL cholesterol. 170 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc Fibric Acid derivatives are also useful in the treatment of gout, as they reduce serum uric acid levels in hyperurecemic patients.

Hyperlipidemia types include type I, type Ila, type lib, type III, type IV, and type V. These types can be characterized according to the levels relative to normal of lipids

(cholesterol and triglycerides) and lipoproteins described above. Different classifications are derived from Drug Facts and Comparisons, 52nd Edition (1998) page 1066 which is hereby incorporated by reference.

Many of the fibric acid derivatives when administered orally do not have sufficient bioavilability and absorption are variable, enatic and depended upon food. In fact absolute bioavialbility of many ofthe fibric acid derivatives is not possible since the prodrags of fibric acids currently marketed as insoluble in water, hence a parenteral formuation is difficult or not available. Furthermore, since these drugs usually administered as esters, they are in fact prodrags. These prodrugs have to be metabolized in the body to release active drug, which are the fibric acids. However, due to the ester formation of these drugs, they are quite insoluble in water, hence are difficult to formulate, and are not easily broken down in the body to release active drugs.

Many ofthe Fibric acid derivatives are low to medium molecular weight solids with characteristic odor. Taken orally it has unpleasant taste and can severely irritate mouth and throat. Taken with food provides more blood concentration compared to fasting. This fed/fast difference in bioavailability is more pronounced when Fibric acid derivatives are compared against their corresponding prodrag derivatives. Overall bioavailability has been reported anywhere between 40-60 and quite variable among patients.

One ofthe significant problems associated with currently marketed fibric acid derivatives being that when these prodrags are cleaved in the body, they release the prodrug moiety, which themselves are highly toxic. For example, in the case of 171 H:\work\16₅2\17690\SPEC\1769₀.spec without claimsll.doc fenofibrate and gemfibrozil isopropyl alcohol is released as the esterase enzyme cleave the pro-moiety from the fenofibric acid. It is well know isopropanol is highly toxic when released into any ofthe mammalian tissues.

In order to improve the therapeutic effectiveness, uniform blood profile, develop pharmaceutically elegant formulation and improve the solubility ofthe drug in water, present invention discusses alternative prodrags of Fibric acid derivatives which overcome many ofthe difficulties stated above.

Accordingly, in one aspect, the present invention is directed to alternate class of prodrags of Fibric acid derivatives. The prodrag consists ofthe hydroxyl group of an amino acid esterified to the free carboxyl group present on the Fibric acid derivatives molecules. In another embodiment, the amine group ofthe amino acid is reacted with COOH ofthe fibric acids to form an amide linkage.

More specifically, in one aspect ofthe present invention is directed to, the compounds of the formulas

FIBRIC ACID ANALOGS

where x is as defined hereinabove

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AA and AA is an amino acid, in which either an amine group or the hydroxyl group is reacted with the carboxylic acid group of Fibric acid derivatives.

172 H:\work\16S2\17690\SPEC\17690.spec without claimsll.doc The present invention is also directed in an embodiment to a pharmaceutical composition comprising a therapeutically effective amount ofthe various Fibric acid derivatives prodrags above and a pharmaceutical carrier therefor.

In another embodiment, the present invention is directed to a method of freating a patient in need of Fibric acid derivatives therapy, which method comprises administering to said patient an effective amount ofthe Fibric acid derivatives.

In a further embodiment, the present invention is directed to a method of converting liquid Fibric acid derivatives into a solid powder by reacting the carboxyl functionality ofthe Fibric acid derivatives with either amine or hydroxyl functionality of an amino acid and isolating the products thereof.

In a still further embodiment, the present invention is directed to a method of substantially and in a therapeutically efficacious manner, make the derivatives absorbed easily upon oral administration thereby improving the consistent therapeutic effect by administering to a patient a prodrag which comprises reacting the COOH functionality ofthe Fibric acid derivatives molecule with either NH₂ or OH functionality of selected amino acids to form an ester or amide covalent bond respectively and isolating the product thereof and administering said product to the patient.

It was determined that when unsubstituted naturally occurring amino acids are esterified to Fibric acid derivatives, the resulting prodrags are pharmaceutically elegant free flowing powders, and are rapidly absorbed into the body and release non-toxic amino acids upon cleavage in the body and require none ofthe emulsifiers, additives and other exepients.

Furthermore, it has been found that the current invention also produced drugs, while they are prodrugs of Fibric acid derivatives; they were highly effective anti-hyperlipidemics and were exhibiting such effect intact. Thus the current amino acid prodrugs are 173 H:\work\l 6₅2\17690\SPEC\17690.spec without claimsll.doc effective anti-hyperlipidemics and useful in the treatment of a number of high cholesterol related illnesses and exhibit such potential with or without releasing the active parent drug.

While the prodrags of fibric acidn ofthe present invention are not expected to possess any acidic activity due to blockage ofthe carboxylic acid group responsible for such, however it has been shown that the prodrugs of fibric acid are effective anti- hyperlipidemics with or without releasing Fibric acid derivatives. However, all ofthe Fibric acid derivatives prodrags described are released in vivo the active drag with all its pharmacological and cholesterol lowering properties.

The present invention clearly provides a number of advantages over Fibric acid derivatives, for example, all ofthe side chains cleaved from these prodrugs are naturally occurring essential amino acids hence are non-toxic. This results in high therapeutic index. Secondly all the prodrags are readily cleaved in the body to release Fibric acid derivatives. Furthermore, due their high water solubility, they can be easily administered by either forming an in-situ solution just before IV administration using lyophilized sterile powder or providing the drag in solution in prefilled syringe or bottles for infusion. The aminoacid esters are more stable than Fibric acid derivatives since COOH group in Fibric acid derivatives is blocked to reaction with bases. Thus the Fibric acid derivatives prodrugs described here are more effective then Fibric acid derivatives itself without the toxicity and other pharmaceutical problems associated with current marketed formulations.

The prodrags of this invention are anti-hyperlipidemic drags useful in the treatment of hyperlipidemia in mammals where the symptoms are elevated triglycerides, low HDL (High density lipoproteins or "good" cholesterol, and elevated cholesterol. Fibric Acid derivatives are also useful in reducing LDL (Low density lipoproteins, or "bad" cholesterol).

174 H:\work\16₅2\17690\SPEC\17690.spec without claimsπ.doc Typical examples of synthesis of L-threonine, L-hydroxyproline and L-serine esters of Fibric acid derivatives are shown in the synthetic processes outlined below. These procedures are applicable to all other compounds ofthe Fibric acid derivatives class as well.

Synthesis of Fibric acid derivatives Prodrugs

The procedure for the synthesis ofthe L-serine, L-threonine, and L-hydroxyproline esters of fenofibric acid is outlined in Synthetic Sequence section and is exemplary. The complete procedure and analytical data is given in the Experimental Section. In general, fenofibric acid ( 100 g batches) was prepared from 4-chloro-4 ' - hydroxybezophenone in accordane with the procedures in the literature. Fenofibric acid was coupled with the t-butyl esters of N-Boc protected amino acid (L-serine, L- threonine, and L-hydroxyproline) using EDC as the coupling agents and a catalytic amount of DMAP. The protecting groups were removed at low temperature (5 °C, 3-6 days) with a mixture of hydrochloric acid in acetic acid (IM) with dichloromethane. The amino acid ester salts of fenofibric acid were purified by crystallization from ethyl acetate, and dried under high vacuum.

Synthetic Sequence:

175 H:\work\16₅2\17690\SPEC\17690.spec without claimsll.doc

SPIBDD202

Synthesis ofthe L-serine, L-threonine, and L-hydroxyproline esters of fenofibric acid: a) Boc-Ser-OtBu, EDC, DMAP, CH₂C1₂; b) Boc-Thr-OtBu, EDC, DMAP, CH₂C1₂; c) Boc-Hyp-OtBu, EDC, DMAP, CH₂C1₂; d) HCl, AcOH, CH₂C1₂.

176 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc Experimental Section: The synthesis of SPIB00201, SPIB00202 and SPIB00203 was conducted in one or two batches. Reagents mentioned in the experimental section were purchased at the highest obtainable purity from Lancaster, Sigma- Aldrich, Acros, or Bachem, except for solvents, i which were purchased from either Fisher Scientific or Mallinkrodt.

1) Synthesis of fenofibric acid:

A mixture of 4-chloro-4'-hydroxybezophenone (116 g, 0.500 mole) and sodium hydroxide (120 g, 3.00 mole) in acetone (1 L) was heated to reflux for 2 hours. The heating was stopped and the heating source was removed. A mixture of chloroform (179 g, 1.50 mole) in acetone (300 mL) was added drop- wise. The reaction mixture was stirred overnight without heating. The mixture was heated to reflux for 8 hours and then allowed to cool to room temperature. The precipitate was removed by filtration and washed with acetone (100 mL). The filfrate was concentrated under reduced pressure to give a brown oil. Water (200 mL) was added to the brown oil and was acidified (to ρH=l) with IN hydrochloric acid. The precipitate, which formed was filtered and dried under high vacuum. The remaining yellow solid (268 g) was recrystallized from toluene in 4 batches (400 mL toluene each). After filfration and drying under high vacuum, the experiment produced fenofibric acid (116 g, 73% yield) as a light yellow solid.

1H NMR (300 MHz, DMSO-d₆): δ = 13.22 (IH, s, br), 7.72 (4H, d, J= 8.4 Hz), 7.61 (2H, d, J= 7.8 Hz), 6.93 (2H, d, J= 7.8 Hz), 1.60 (6H, s). ¹³C NMR (75 MHz, DMSO-d₆): δ = 192.96, 174.18, 159.35, 136.84, 136.12, 131.67, 131.02, 129.12, 128.43, 116.91, 78.87, 25.13.

177 H:\work\I6₅2\17690\SPEC\17690.spec without claimsILdoc 2) SPD300201: L-serine-fenofibric acid ester

To a mixture of fenofibric acid (11.6 g, 36.3 mmol), N-carbobenzyloxy-L-serine t-butyl ester (Boc-Ser-OtBu, 8.62 g, 33.0 mmol), EDC (7.59 g, 39.6 mmol), and DMAP (484 mg, 3.96 mmol) cooled in an ice-water bath was added anhydrous dichloromethane (150 mL) dropwise. After the addition was complete, the ice bath was removed and the reaction mixture was stirred under an argon atmosphere at room temperature for 20 hours. After 20 hours, the additional dichloromethane (200 mL) was added and the solution was washed with water (2x200 mL) and brine (200 mL). After drying over sodium sulfate and filtration, the solution was concenfrated under reduced pressure. The remaining yellow oil (21.2 g) was purified by column chromatography on silica gel (400 g, 0.035-0.070 mm, 6 nm pore diameter), eluting with heptane/ethyl acetate (3:1). After concentration ofthe product-containing fractions under reduced pressure and drying under high vacuum until the weight was constant, the experiment produced the protected L-serine-fenofibric acid ester SPIB0020101 (16.2 g, 87% yield) as a light yellow oil.

1H NMR (300 MHz, CDC1₃): δ = 7.75 (2H, d, J= 9.0 Hz), 7.72 (2H, d, J= 9.0 Hz), 7.45 (2H, d, J= 8.7 Hz), 6.86 (2H, d, J= 8.7 Hz), 5.04 (IH, d, J= 6.9 Hz), 4.55-4.42 (3H, m), 1.66 (3H, s), 1.65 (3H, s), 1.43 (9H, s), 1.39 (9H, s).

¹³C NMR (75 MHz, CDC1₃): δ = 193.92, 172.99, 168.07, 159.24, 154.87, 138.24, 136.19, 131.94, 131.06, 130.40, 128.41, 117.26, 82.88, 80.13, 79.24, 65.44, 53.44, 28.27, 27.92, 25.70, 25.30.

To a stirred solution ofthe protected L-serine-fenofibric acid ester SPD30020101 (16.2 g, 28.8 mmol) in anhydrous dichloromethane (100 mL) cooled to 5 °C, under an argon atmosphere was added a solution of hydrogen chloride in acetic acid (400 mL, IM, 400

178 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc mmol) drop-wise. The reaction mixture stirred for 3 days at 5 °C. After three days the mixture was concentrated under reduced pressure and dried under high vacuum to remove acetic acid. To the remaining light yellow oil (24.7 g) was added ethyl acetate (lOO mL). The solution was concenfrated and dried a second time. To the remaining i light yellow oil (17.0 g) was added ethyl acetate (65 mL). The mixture was heated to reflux for 5 minutes and cooled to room temperature. The precipitate was removed by filfration and dried under high vacuum overnight at room temperature, then at 43 °C for one hour. The experiment produced the L-serine-fenofibric acid ester, hydrochloride SPEB00201 (7.66 g, 60% yield) as a white solid,

'HNMR (300 MHz, DMSO-de): δ = 14.12 (IH, s, br), 8.77 (3H, s, br), 7.72 (4H, m), 7.62 (2H, d, J= 8.4 Hz), 6.92 (2H, d, J= 9.0 Hz), 4.62 (IH, dd, J= 12.0, 4.2 Hz), 4.50 (IH, dd, J= 12.0, 2.4 Hz), 4.41 (IH, m), 1.64 (3H, s), 1.63 (3H, s). ¹³C NMR (75 MHz, DMSO-d₆): δ = 193.06, 171.70, 168.06, 158.72, 136.93, 136.06, 131 .73, 131.09, 129.62, 128.49, 117.64, 79.02, 62.99, 51.11, 25.04, 24.94.

HPLC analysis: 100% purity; r.t.= 4.361 min.; 55% TFA (0.1%), 45% ACN; 1 mL/min; 32.3 C, Luna C18, serial # 167917-13; 20 ul inj., NB275-49.

CH M analysis: calc: C 54.31, H 4.79, N 3.17; found: C 54.37, H 4.78, N 3.12. Melting point: 151°C (dec.)

179 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc 3) SPIB00202: L-threonine-fenofibric acid ester

To a mixture of fenofibric acid (25.5 g, 79.9 mmol), N-carbobenzyloxy-L-threonine t- butyl ester (Boc-Thr-OtBu, 20.0 g, 72.6 mmol, prepared by the literature method), EDC (16.7 g, 87.1 mmol), and DMAP (1.06 g, 8.71 mmol) cooled in an ice-water bath was added anhydrous dichloromethane (200 mL), dropwise. After the addition was complete, the ice bath was removed and the reaction mixture was stirred under an argon atmosphere at room temperature for 20 hours. After 20 hours, additional EDC (1.39 g, 7.26 mmol) was added and the experiment was allowed to stir over the weekend at room temperature under an argon atmosphere. After 4 days, additional dichloromethane (300 mL) was added and the solution was washed with water (300 mL) and brine (300 mL). After drying over sodium sulfate and filtration, the solution was concentrated under reduced pressure. The remaining yellow oil (53.5 g) was purified by column chromatography on silica gel (500 g, 0.035-0.070 mm, 6 nm pore diameter), eluting with heptane/ethyl acetate (3:1). After concentration ofthe product-containing fractions under reduced pressure and drying under high vacuum until the weight was constant, the experiment produced the protected L-threonine-fenofibric acid ester SPIB0020201 (34.1 g, 82% yield) as a white foam,

1H NMR (300 MHz, CDC1₃): δ = 7.74 (2H, d, J= 8.4 Hz), 7.72 (2H, d, J= 8.4 Hz), 7.45 (2H, d, J= 8.4 Hz), 6.87 (2H, d, J= 8.4 Hz), 5.47 (IH, m), 4.98 (IH, d, J= 9.9 Hz), 4,31 (IH, d, J= 9.9 Hz), 1.65 (3H, s), 1.64 (3H, s), 1.45 (9H, s), 1.42 (9H, s), 1.22 (3H, d, J= 6.3 Hz).

¹³C NMR (75 MHz, CDC1₃): δ = 193.94, 172.14, 168.70, 159.26, 155.62, 138.28, 136.18, 131.90, 131.08, 130.37, 128.43, 117.40, 82.70, 80.17, 79.38, 72.02, 57.46, 28.30, 27.99, 26.44, 24.79, 16.90.

180 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc To a stirred solution ofthe protected L-threonine-fenofibric acid ester SPIB0020201 (34.1 g, 59.2 mmol) in anhydrous dichloromethane (100 mL) cooled to 5 °C, under an argon atmosphere was added a solution of hydrogen chloride in acetic acid (600 mL, IM, 600 mmol) drop-wise. The reaction mixture was kept for 6 days at 5 °C. The mixture was concentrated under reduced pressure and dried under high vacuum to remove acetic acid. To the remaining white solid (45.8 g) was added ethyl acetate (500 mL). The mixture was heated to reflux for 10 minutes and cooled to room temperature. The precipitate was removed by filtration and dried under high vacuum overnight at room temperature. The experiment produced the L-threonine-fenofibric acid ester, hydrochloride SPD300202 (26.3 g, 97% yield) as a white solid.

1H NMR (300 MHz, DMSO-d₆): δ = 14.10 (IH, s, br), 8.84 (3H, s, br), 7.73 (4H, m), 7.63 (2H, d, J= 8.1 Hz), 6.89 (2H, d, J= 8.7 Hz), 5.44 (IH, m), 4.31 (IH, s), 1.64 (3H, s), 1.62 (3H, s), 1.38 (3H, d, J= 6.3 Hz).

¹³C NMR (75 MHz, DMSO-de): δ = 193.04, 171.00, 168.13, 158.76, 136.90, 136.08, 131.70, 131.06, 129.49, 128.48, 117.41, 78.99, 69.40, 55.21, 25.59, 24.22, 16.06.

HPLC analysis: 98.59% purity; r.t.= 4.687 min.; 55% TFA (0.1%), 45% ACN; 1 mL/min; 32.3 C, Luna C18, serial # 167917-13; 20 ul inj., NB275-49, DAD1 B, Sig=210.4, Ref=550,100.

CHN analysis: calc: C 55.27, H 5.08, N 3.07; found: C 54.98, H 5.13, N 3.03.

Melting point: 160.5 °C (dec.)

181 H:\work\1652\I7690\SPEC\17690.spec without claimsll.doc 4) SP1B00203: L-hydroxyproline-fenofibric acid ester

To a mixture of fenofibric acid (24.9 g, 78.1 mmol), N-carbobenzyloxy-L- hydroxyproline t-butyl ester (Boc-Hyp-OtBu, 20.4 g, 71.0 mmole, prepared in accordance with the procedure in the literature), EDC (16.3 g, 85.2 mmol), and DMAP (1.04 g, 8.52 mmol) cooled in an ice-water bath was added anhydrous dichloromethane (200 mL) dropwise. After the addition was complete, the ice bath was removed and the reaction mixture was stirred under an argon atmosphere at room temperature for 20 hours. After 20 hours, additional EDC (1.63 g, 8.52 mmol) was added and the experiment was allowed to stir over the weekend at room temperature under an argon atmosphere. After 4 days the solution was washed with water (200 mL) and brine (200 mL). After drying over sodium sulfate and filtration, the solution was concentrated under reduced pressure. The remaining yellow oil (49.4 g) was purified by column chromatography on silica gel (500 g, 0.035-0.070 mm, 6 nm pore diameter), eluting with heptane/ethyl acetate (2:1). After concenfration ofthe product containing fractions under reduced pressure and drying under high vacuum until the weight was constant, the experiment produced the protected L-hydroxyproline-fenofibric acid ester SPIB0020301 (26.4 g, 63% yield) as a colorless oil.

1H NMR (300 MHz, CDC1₃): δ = 7.76 (2H, d, J= 8.1 Hz), 7.73 (2H, d, J= 8.1 Hz), 7.46 (2H, d, J- 8.1 Hz), 6.84 (2H, d, J= 8.1 Hz), 5.32 (IH, m), 4.13 (0.38H, t, J= 7.8 Hz), 4.00 (0.62H, t, J= 7.8 Hz), 3.67 (1.62H, m), 3.46 (0.38H, d, J=12.6 Hz), 2.29 (IH, ), 2.15 (IH, m), 1.68 (3H, s), 1.66 (3H, s), 1.44-1.38 (18H, m).

182 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc ¹³C NMR (75 MHz, CDC1₃): δ = 193.88, 172.98, 171.14, 159.25, 153.48, 138.23, 136.16, 131.99, 131.08, 130.36, 128.44, 117.03, 116.91, 81.48, 80.32, 80.20, 79.19, 74.03, 73.26, 58.23, 51.88, 51.58, 36.33, 35.31, 31.92, 28.29, 28.00, 25.89, 24.95.

To a stfrred solution ofthe protected L-hydroxyproline-fenofibric acid ester

SPIB0020301 (26.0 g, 44.2 mmol) in anhydrous dichloromethane (100 mL) cooled to 5 °C, under an argon atmosphere was added a solution of hydrogen chloride in acetic acid (450 mL, IM, 450 mmol) drop-wise. The reaction mixture stirred for 4 days at 5 °C. After four days the mixture was concenfrated under reduced pressure and dried under high vacuum to remove acetic acid. To the remaining yellow oil (31.5 g) was added ethyl acetate (200 mL). The mixture was sonicated and then concenfrated under reduced pressure and dried under high vacuum. To the remaining white solid (23.2 g) was added ethyl acetate (300 mL). The ethyl acetate mixture was heated to reflux for 10 minutes and cooled to room temperature. The precipitate was removed by filtration and dried under high vacuum overnight at room temperature. The experiment produced the L- hydroxyproline-fenofϊbric acid ester, hydrochloride SPIB00203 (15.8 g, 76% yield) as a white solid.

1H NMR (300 MHz, DMSO-d₆): δ = 14.07 (IH, s, br), 10.75 (IH, s, br), 9.40 (IH, s, br), 7.71 (4H, d, J= 8.1 Hz), 7.60 (2H, d, J= 8.1 Hz), 6.96 (2H, d, J= 8.1 Hz), 5.42 (IH, m), 4.24 (IH, t, J= 9.0 Hz), 3.61 (IH, dd, J= 13.2, 4.2 Hz), 3.28 (IH, d, J= 13.2 Hz), 2.35 (2H, m), 1.66 (3H, s), 1.64 (3H, s).

¹³C NMR (75 MHz, DMSO-d₆): δ = 193.00, 171.52, 169.14, 158.81, 136.87, 136.09, 131.81, 131.05, 129.48, 128.46, 117.28, 78.99, 73.79, 57.54, 50.23, 34.13, 25.69, 24.49.

HPLC analysis: 183 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc 100% purity; r.t.= 8.369 min.; 60% DIUF water (0.1% TFA)/40% acetonitrile; 1 mL/min; 36.4 C; Luna Cl 8, 5u column (serial # 191070-3), 4.6x250 mm; 20 ul injection; DAD1 A, Sig = 210.4, Ref = 550,100.

HPLC-MS (ESI): calculated: M⁺ = 431 ; found M+H= 432.3

Melting point: 187.5 °C (dec.)

Solubility ofthe above esters were determined in water at room temperature by dissolving excess of each ofthe drug and let them settle for few hours. The resulting solutions were centrifuged at 1500rpm for 3 min and the supernatant liquid was analyzed. It was shown that these esters possess solubility in water in excess of 50 mg/mL.

EXPERIMENTAL

Rats were checked for time zero triglyceride level in blood. Then the rats were set on high sugar diet, such as 30% surcorse in water for 1 week. Then at the end of 1 week, rats were tested for triglycerides, and were put on normal diet. From day 7-14 the rats were administered either test or control drag. Triglycerides were again tested on the 14^th day in rat blood.

In the Fenofibrate (control) vs L-Serine Ester of Fenofibric acid (test drag), 3 rats each for each ofthe drug and control at equivalent doses of 50, 100 and 200 mg/kg were tested.

The results are shown below.

184 H:\work\It552\17690\SPEC\17690.spec without claimsILdoc SUMMARY - DOSE RANGE FINDING STUDY - HYPOLIPIDEMIC PROPERTY - FENOFIBRATE AND ITS FORMULATION Test Substance: L-Serine Ester of Fenofibric Acid Vehicle: 1% Tween 80 in milli Q -water

From the above results, it can be concluded the highly water soluble serine ester was effectively performed. There are a number of screening tests to determine the utility of the prodrags created according tio the disclosed methods. These include both in vitro and in vivo screening methods.

185 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc The in vitro methods include acid/base hydrolysis ofthe prodrags, hydrolysis in pig pancreas hydrolysis in rat intestinal fluid, hydrolysis in human gastric fluid, hydrolysis in human intestinal fluid, and hydrolysis in human blood plasma. These assays are dscribed in Simmons, DM, Chandran, VR and Portmann, GA, Danazol Amino Acid Prodrugs: In Vitro and In Situ Biopharmaaceutical Evaluation, Drug Development and Industrial Pharmacy, Vol 21, Issue 6, Page 687, 1995, the contents of allof which are incorporated by reference.

The prodrags of Fibric Acid ofthe present invention are effective in treating diseases or conditions in which Fibric acid derivatives normally are used. The prodrugs disclosed herein are transformed within the body to release the active compound and enhances the therapeutic benefits ofthe Fibric acid derivatices by reducing or eliminating biopharmaceutical and pharmacokinetic barriers associated with each of them. However it should be noted that these prodrags themselves will have sufficient activity without releasing any active drag in the mammals.

Thus, the prodrug ofthe present invention enhances the therapeutic benefits by removing biopharmaceutical and pharmacokenetic barriers of existing drugs.

Furtheremore, the prodrugs are easily synthesized in high yields using reagents which are readily and commercially available.

The prodrags of Fibric acid ofthe present invention are effective in treating diseases or conditions in which Fibric acid derivatives normally are used. The prodrags disclosed herein are transformed within the body to release the active compound and enhances the therapeutic benefits ofthe Fibric acid derivatives by reducing or eliminating biopharmaceutical and pharmacokenetic barriers associated with each of them. However it should be noted that these prodrugs themselves will have sufficient activity without releasing any active drug in the mammals.

186 H:\work\l 652\17690\SPECU7690.spec without claimsll.doc Thus, the prodrug of fibric acid ofthe present invention enhances the therapeutic benefits by removing biopharmaceutical and pharmacokenetic barriers of existing drags. Furthermore, the prodrugs are easily synthesized in high yields using reagents which are readily and commercially available.

In the formula hereinabove and in the claims it is to be understood that the AA has the following definition in the following contents

O II C — NH — AA -

1)

AA in this definition refers to the amino acid residue without an amino group either on the main chain or the side chain.

AA in this definition is an amino acid residue less the hydroxy group on the side chain.

NH -AA

O

3)

AA refers to an amino acid group without the carboxy group, either on the main chain or side group.

187 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc 4) OAA- This is a ester bond between the hydroxy group ofthe drug and the carboxy group ofthe amino acid either on the main chain or side chain. Thus, as written OAA is

wherein Ro is the side chain amino acid as defined hereinabove.

Alternatively, it may refer to an ester bond between the carboxy group ofthe drug and the hydroxy group on the side chain of those amino acids which have a hydroxy group thereon such as threonine, serine, hydroxyproline, tyrosine and the like. The hydroxy group forms part ofthe ester linkage which is depicted hereinabove with O. Thus, as written, the AA refers to an amino acid with a hydroxy group on the side chain, but as depicted OAA, the AA is without the hydroxy group since the oxygen atom is depicted in the formula.

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope ofthe invention, which is defined by the scope ofthe appended claims. Other aspects, advantages, and modifications are within the scope ofthe following claims.

188 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

Claims

IN THE CLAIMS:

1. A method for enhancing at least two of the therapeutic qualities of a drug having a functionality group selected form the group consisting of hydroxy, amino, carboxy or acylating derivatives of said carboxy group, said improved therapeutic quality being selected from the group consisting of: (a) improved taste or smell (b) desired octanol/water partition coefficient (c) improved stability (d) enhanced penetration of blood-brain barrier (e) elimination of first pass effect in the liver (f) reduction of intero-hepatic recirculation (g) painless injection with parental formulation (h) improved bioavailability (i) improved changes in the rate of absorption (j) reduced side effects (k) dose proportionality (1) selective hydrolysis of the prodrag at site of action (m) controlled release properties (n) targeted drag delivery (o) reduction in toxicity (p) reduced dose (q) alteration of metabolic pathway to deliver more drag at site of action (r) increased solutility in aqueous solution and (s) enhanced efficacy, the method comprising (a) reacting the drag with an amino acid under conditions effective to form a covalent bond between the drag and the amino acid and (b) administering the product of (a) to a patient in need thereof.

2. The method according to Claim 1 wherein the amino acid is a naturally occurring amino acid. 189 H:\work\1652\1769Q\SPEC\17690.spec without claimsll.doc

3. The method according to Claim 1 whrein the amino acid is a α-L-amino acid.

4. The method according to Claim 1 whreinn the amino acid is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Tφ, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

5. The method according to Claim 1 wherein AA is proline, glycine, lysine, hydroxy proline or alanine.

6. The method according to Claim 5 wherein AA is Lys or hydroxyproline.

7. The method according to Claim 6 wherein AA is Lys.

8. The method according to Claim 1 wherein the drag is Cyclosporin, Lopinavir, Ritonavir, Cefdinir, Zileuton, Nelfinavir, Flavoxate, Candesarten, Propofol, Nisoldipine, Amlodipine, Ciprofloxacin, Ofloxacin, Fosinopril, Enalapril, Ramipril, Benazepril, Moexipril, Trandolapril, Cromolyn, Amoxicillin, Cefuroxime, Ceftazimide, Cefpodoxime, Atovaquone, Gancyclovir, Penciclovir, Famciclovir, Acyclovir, Niacin, Bexarotene, Propoxyphene, Salsalate, Acetaminophen, Ibuprofen, Lovastatin, Simavastatin, Atorvastatin, Pravastatin, Fluvastatin, Nadolol, Valsartan, Methylphenidate, Sulfa Drags, Sulfasalazine, Methylprednisolone, Medroxyprogesterone, Estramustine, Miglitol, Mefloquine, Capacitabine, Danazol, Eprosartan, Divalproex, Fenofibrate, Gabapentin, Omeprazole, Lansoprazole, Megesfrol, Metformin, Tazorotene, Sumitriptan, Naratriptan, Zolmitriptan, Aspirin, Olmesartan, Sirolimus, Tacrolimus, Clopidogrel, Amphotericin, Tenofovir, Unoprostone, Fulvesfrant, Cefditoren, Efavirenz, Eplerenone, Treprostinil, or Adefovir.

9. A compound ofthe formula:

190 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc CH,

CH,

N CH CO CYCLO -

CH, or

191 H:\work\l 652\17690\SPEC\1769G.spec without claimsILdoc

or pharmaceutically acceptable salts thereof; wherein CYCLO represents the residues at positions 2-11 of a cyclosporin molecule x-y is CH=CH or CH₂CH₂ and R² is OAA or OGly-AA, AA is an amino acid residue wittiout the OH groups ofthe carboxy group and where Gly is a glycyl residue and is a

192 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc spacer group attached to the oxygen atom ofthe cyclosporin, sirolimus, tacrolimus or pimecrolimus.

10. The compound according to Claim 9 wherein AA is naturally occurring α L- amino acid.

11. The compound according to Claim 9 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

12. The compound according to Claim 9 wherein AA is proline, glycine, lysine or ornithine.

13. The compound according to Claim 9 wherein AA is Lys or ornithine.

14. The compound according to Claim 9 wherein the compound has the formula HC CH

CH I CH₂ \ H CH-CH₃ AAt I /

MeVal — N — CH — C — Abu — MeGly

MΘLΘU ^' MeLeu

MθLeu DAIa Ala MeLeu — Val

193 H:\work\1652\1769G\SPEC\1769G.spec without claimsll.doc

15. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 9-14 and a pharmaceutically acceptable carrier therefor.

16. A method of treating a patient in need of cyclosporin therapy, which method comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 9-14.

17. A method of enhancing the dose proportionality of cyclosporin comprising reacting the hydroxy functionality in the MeBmt moiety at position 1 ofthe cyclosporin molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof and administering the product to a patient.

18. A compound of the formula:

or pharmaceutically acceptable salts thereof; wherein AA is an amino acid residue ofthe hydroxy group ofthe carboxy group.

19. The compound according to Claim 18 wherein AA is a naturally occuring L- cc-amino acid.

20. The compound according to Claim 18 wherein AA is Lys, Leu, He, Gly, Asp,

Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys,

Sar or Dcy. 194 H:\work\1652\17690\SPEC\1769G.spec without claimsll.doc

21. The compound according to Claim 18 wherein AA is proline, glycine, lysine, hydroxyproline or alanine

22. The compound according to Claim 18 wherein AA is Lys or hydroxy proline.

23. The compound according to Claim 18 wherein AA is Lys.

24. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 18-23 or a pharmaceutically acceptable carrier therefor.

25. A method of treating a patient in need of lopinavir therapy, which method comprises administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 18-23.

26. A method of enhancing the solubility of lopinavir in an aqueous solution comprising reacting the hydroxy functionality ofthe lopinavir molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

27. A method of enhancing the bioavailability of lopinavir when administered to a patient which comprises reacting the hydroxy functionality ofthe lopinavir molecule with an amino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

28. A method of treating AIDS in a mammal comprising administering to said mammal a compound according to any one of Claims 18-23 in an amount therapeutically effective to inhibit HIV protease.

29. A compound ofthe formula: 195 H:\work\16S2\1769Q\SPEC\17690.spec without claimsll.doc

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AAi; AA_t is an amino acid with a OH on the side chain thereof without the OH group; and AA is an amino acid less the NH₂ group.

30. The compound according to Claim 29 wherein AA and AAi are L-α-naturally occurring amino acid.

31. The compound according to Claim 29 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy and AA] is Ser, Thr, Tyr, or hydroxyproline.

32. The compound according to Claim 29 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine or AAi_. is Thr, hydroxyproline or serine.

33. The compound according to Claim 29 wherein AA or AAj_. is Serine or hydroxyproline.

34. The compound according to Claim 29 wherein AA or AAj is hydroxyproline.

35. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 29-34 or a pharmaceutically acceptable carrier therefor.

36. A method of treating a patient in need of cefdinir therapy, which method 196 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc comprises administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 29-34.

37. A method of enhancing the solubility of cefdinir in an aqueous solution comprising reacting the carboxylic acid functionality ofthe cefdinir molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

38. A method of enhancing the bioavailability of cefdinir when administered to a patient which comprises reacting the carboxylic acid functionality ofthe cefdinir molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

39. A method of treating infectious diseases in mammals caused by pathogenic micro organisms, said method comprising administering to said mammal in need of treatment a therapeutically effective amount of a compound according to any one of Claims 29-34.

40. A compound ofthe formula:

ZILEUTON or pharmaceutically acceptable salts thereof; wherem AA is an amino acid less the OH on the carboxy group.

41. The compound according to Claim 40 wherein AA is an L-α-naturally occurring amino acid.

197 H:\work\16S2\17690\SPEC\17690.spec without claimsILdoc

42. The compound according to Claim 40 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

43. The compound according to Claim 40 wherein AA is proline, glycine, lysine, hydroxyproline or Alanine

44. The compound according to Claim 40 wherein AA is Lys or hydroxy proline.

45. The compound according to Claim 40 wherein AA is Lys.

46. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 40-45 or a pharmaceutically acceptable carrier therefor.

47. A method of treating a patient in need of zileuton therapy, which method comprises administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 40-45.

48. A method of enhancing the solubility of zileuton in an aqueous solution comprising reacting the hydroxy functionality ofthe zileuton molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

49. A method of treating asthma in mammals comprising administering a pharmaceutically effective amount of a compound according to any one of Claims 40-45 to said mammal.

50. A compound ofthe formula:

198 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc

or pharmaceutically acceptable salts thereof; wherein AA is an amino acid less the hydroxy group ofthe carboxy group thereof.

51. The compound according to Claim 50 wherein AA is an L-naturally occurring α- amino acid.

52. The compound according to Claim 50 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, ISfor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

53. The compound according to Claim 50 wherein AA is proline, glycine, lysine, hydroxyproline or alanine

54. The compound according to Claim 50 wherein AA is Lys or hydroxy proline.

55. The compound according to Claim 50 wherein AA is Lys.

56. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 50-55 and a pharmaceutically acceptable carrier therefor.

199 H:\work\l 652\17690\SPEC\17690.spec without claimsll.doc

57. A method of freating a patient in need of nelfinavir therapy, which method comprises administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 50-55.

58. A method of enhancing the solubility of nelfinavir in an aqueous solution comprising reacting the hydroxy functionality ofthe nelfinavir molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

59. A method of enhancing the bioavailability of nelfinavir when administered to a patient which comprises reacting the hydroxy functionality ofthe nelfinavir molecule with an amino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

60. A method of treating AIDS caused by HIV in a mammal which comprises administering to said mammal in need of such freatment a therapeutically effective amount of a compound according to any one of Claims 50-55.

61. A compound of the formula:

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AAi and AA_Ϊ is attached via an ester bond and is an amino acid residue with a hydroxy group on the side chain without the hydroxy group, and AA is attached via an amide bond and is an amino acid residue less the amino group.

200 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

62. The compound according to Claim 61 -wherein AA and AAj_. are independently naturally occurring L-α-amino acid.

63. The compound according to Claim 61 wherein AA is of Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAi is Thr, Ser, Tyr, or hydroxyproline.

64. The compound according to Claim 61 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine and AA], is Thr, Ser or hydroxyproline.

65. The compound according to Claim 61 wherein AA or AAj_. is Serine or hydroxyproline.

66. The compound according to Claim 61 wherein AA or AAi is hydroxyproline.

67. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 61-66 and a pharmaceutically acceptable carrier therefor.

68. A method of freating a patient in need of flavoxate therapy, which method comprises administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 61 -66.

69. A method of enhancing the solubility of flavoxate in an aqueous solution comprising reacting the carboxylic acid functionality ofthe flavoxate molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

70. A method of enhancing the bioavailability of flavoxate when administered to

201 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc a patient which comprises reacting the carboxylic acid functionality ofthe flavoxate molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

71. A method of treating urinary spasms in mammals comprising administering to said mammal in need of treatment or pharmaceutically effective of a compound according to any one of Claims 61-66.

72. A compound ofthe formula:

CANDESARTAN CILEXETIL

or pharmaceutically acceptable salts thereof; wherein the R is either NH-AA! or O-AA and AA is attached via an amide bond an amino acid residue less the amino group ofthe main chain or side chain thereof and AAi is an amino acid residue having a hydroxy group on the side chain thereof without the hydroxy group.

73. The compound according to Claim 72 wherein AA_Ϊ is Ser, Try or Tyr o rhydroxyproline.

74. The compound according to Claim 72 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

202 H:\work\16S2\17690\SPEC\17690.spec without claimsll.doc

75. The compound according to Claim 72 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine or AAj is serine, threonine or hydroxyproline.

76. The compound according to Claim 72 wherein AA or AA_! is Serine or hydroxyproline.

77. The compound according to Claim 72 wherein AA or AAj. is hydroxyproline.

78. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 72-77 and a pharmaceutically acceptable carrier therefor.

79. A method of treating a patient in need of candesartan therapy, which method comprises administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 72-77.

80. A method of enhancing the solubility of candesartan in an aqueous solution comprising reacting the carboxylic acid functionality ofthe candesartan molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

81. A method of enhancing the bioavailability of candesartan when administered to a patient which comprises reacting the carboxylic acid functionality ofthe candesartan molecule with an amino acid or acylating derivative under ester or amide fonriing conditions, isolating the product thereof and administering said product to the patient.

82. A method of treating hypertension in mammals comprising administering to said mammal in need thereof a pharmaceutically effective amount of a compound according to any of Claims 72-77. 203 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

83. A compound ofthe formula:

or pharmaceutically acceptable salts thereof; wherein AA is an amino acid less a hydroxy group on the carboxy group.

84. The compound according to Claim 83 wherein AA is a naturally occurring L- amino acid.

85. The compound according to Claim 83 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

86. The compound according to Claim 83 wherein AA is proline, glycine, lysine, hydroxyproline or alanine

87. The compound according to Claim 83 wherein AA is Lys, Gly or proline.

88. The compound according to Claim 83 wherein AA is proline.

89. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 83-88 and a pharmaceutically acceptable carrier therefor.

204 H:\work\l 6₅2\17690\SPEC\17690.spec without claimsll.doc

90. A method of treating a patient in need of propofol therapy, which method comprises administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 83-88.

91. A method of enhancing the safety profile with longer anesthetic effect of propofol in an aqueous solution comprising reacting the hydroxy functionality ofthe propofol molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

92. A method of enhancing the bioavailability of propofol when administered to a patient which comprises reacting the hydroxy functionality ofthe propofol molecule with an amino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

93. A method of providing central nervous system anesthesia to a patient comprising administering to a patient in need thereof a therapeutically effective amount of a compound according to any one of Claims 83-88.

94. Compounds ofthe formula: AMLODIPINE NISOLDIPINE

205 H:\work\16₅2\17690\SPEC\17690.spec without claimsILdoc

or pharmaceutically acceptable salts thereof; R2 1S

II O ₄ is NH-AA_! or O-AA,

R₅ is AA₂, wherein AA is attached via an ester bond and is an amino acid residue having a hydroxy group on the side chain without the hydroxy group,

AAi is an amino acid residue without the amino group,

206 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc AA₂ is an amino acid residue less the hydroxy group on the carboxy group, AA₃ is an amino acid residue less the hydrogen atom on the amino group.

95. The compound according to Claim 94 wherein AA, AA_1? AA₂ and AA₃ are independently naturally occurring Lα-amino acid.

96. The compound according to Claim 94 wherein AAi, AA₂ and AA₃ are independently residues of Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Tip, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy and AA is Ser, Thr, hydroxyproline or Tyr.

97. The compound according to Claim 94 wherein AAi, AA₂ and AA₃ are independently residues of proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

98. The compound according to Claim 94 wherein AA, AA_ls AA₂ and AA₃ are independently amino acid residues of Serine or hydroxyproline.

99. The compound according to Claim 94 wherein AA, AAi, AA and AA₃ are independently amino acid residues of hydroxyproline.

100. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 94-99 and a pharmaceutically acceptable carrier therefor.

101. A method of treating a patient in need of calcium channel blocker therapy, said method comprising administering to said patient a therapeutically effective amount of the compound according to any one of Claims 94-100.

207 H:\work\1652\17690\SPECU7690.spec without claimsll.doc

102. A method of enhancing the solubility of a calcium channel blocker selected from the group consisting of amlodipine or nisoldipine in an aqueous solution comprising reacting the carboxylic acid functionality ofthe calcium channel blockers molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

103. A method of enhancing the bioavailability of calcium channel blocker selected from the group consisting of amlodipine or nisoldipine when administered to a patient which comprises reacting the carboxylic acid functionality ofthe calcium channel blockers molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

104. A method of treating hypertension in mammals which comprises administering to a mammal in need thereof a therapeutically effective amount of a compound according to any one of Claims 94-100.

105. Compounds ofthe formula:

OFLOXACIN CIPROFLOXACIN

or pharmaceutically acceptable salts thereof; wherein the R is either NH-AA or O-AAi[ wherein AA is an amino acid residue less an amino group and AAi is an amino acid residue having a hydroxy group on the side chain thereof without said hydroxy group.

208 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

106. The compound according to Claim 105 wherein AA and AAi are a naturally occurring Lα-amino acid.

107. The compound according to Claim 105 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAi is Tyr, Ser, Thr or hydroxyproline.

108. The compound according to Claim 105 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

109. The compound according to Claim 105 wherein AA or AAi is Serine or hydroxyproline.

110. The compound according to Claim 105 wherein AA or AAi is hydroxyproline.

111. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 105-109 and a pharmaceutically acceptable carrier therefor.

112. A method of treating a patient infected with an infection by a microorganism to which of oxacin and ciprofloxacin is toxic therapy, which method comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 105-109.

113. A method of enhancing the solubility of quinolone antibiotic having a carboxy group or acylating group in an aqueous solution comprising reacting the carboxylic acid functionality ofthe quinolone antibiotics molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

209 H:\work\16₅2\17690\SPEC\1769₀.spec without claimsll.doc

114. A method of enhancing the bioavailability of quinolone antibiotic having a carboxyl group or acylating group when administered to a patient which comprises reacting the carboxylic acid functionality ofthe quinolone antibiotics molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

115. A method of treating infectious diseases in mammals caused by of pathogenic micro organisms to which of loxacin and ciprofloxacin is toxic comprising administering to said mammal an antibiotic effective amount of a compound according to any one of Claims 105-109 using various formulations ofthe quinolone antibiotic compounds.

116. Compounds of the formulas:

ENALAPRIL

RA IPRIL

210 H:\work\16₅2\17690\SPEC\17690.spec without claimsll.doc FOSINOPRIL Na

BENA2EPRIL

PERINDOPRIL

MOEXIPRIL

211 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc TRANDOLAPRIL

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AAi and AA is an amino acid residue without an amino group and AAi is an amino acid residue having an OH group on the side chain thereof, without said OH group.

117. The compound according to Claim 116 wherein AA and AAj_. are independently naturally occurring L-α-amino acid.

118. The compound according to Claim 116 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAi is hydroxyproline, Tyr, or Ser or Thr.

119. The compound according to Claim 116 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine or AA_Ϊ is Serine, hydroxyproline or Thr.

120. The compound according to Claim 116 wherein AA or AA_! is independently Serine or hydroxyproline.

121. The compound according to Claim 116 wherein AA or AAi is hydroxyproline.

122. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 116-121 and a pharmaceutically acceptable carrier therefor.

212 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc

123. A method of treating a patient having hypertension, which method comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 116-121.

124. A method of enhancing the solubility of ace inhibitors having a carboxy acylating group in an aqueous solution comprising reacting the carboxylic acid functionality ofthe ace inhibitor molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

125. A method of enhancing the bioavailability of ace inhibitors having a carboxy or acylating group when administered to a patient which comprises reacting the carboxylic acid functionality ofthe ace inhibitors molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

126. A compound of the formula: CROMOLYN

or pharmaceutically acceptable salts thereof; wherein AA is attached via an ester bond and is an amino acid residue less a hydroxy group in the carboxy group, and R is an ethyl group.

127. The compound according to Claim 126 wherein AA is a naturally occurring L-α- amino acid.

213 H:\work\16₅2\1769₀\SPEC\17690.spec without claimsll.doc

128. The compound according to Claim 126 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Tφ, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

129. The compound according to Claim 126 wherein AA is proline, glycine, lysine, hydroxyproline or alanine

130. The compound according to Claim 126 wherein AA is Lys or hydroxy proline.

131. The compound according to Claim 126 wherein AA is Lys.

132. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 126-131 and a pharmaceutically acceptable carrier therefor.

133. A method of treating a patient in need of cromolyn sodium therapy, which method comprises administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 126-131.

134. A method of enhancing the solubility of cromolyn sodium in an aqueous solution comprising reacting the hydroxy functionality ofthe cromolyn sodium molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

135. A method of enhancing the bioavailability of cromolyn sodium when administered to a patient which comprises reacting the hydroxy functionality ofthe cromolyn sodium molecule with an amino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

214 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

136. Compounds of the formula:

XICILLIN

CEFUROXIME

CEFTAZIDIME

CEFPODOXIME

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AAi and AA is an amino acid residue less an amino a group and AAj is an amino acid residue with a hydroxy group on the side chain thereof, without said OH group.

215 H:\work\l 652\17690\SPEC\17690.spec without claimsll.doc

137. The compound according to Claim 136 wherein AA and AAi are independently naturally occurring L-oc-amino acid.

138. The compound according to Claim 136 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAi is Ser, Thr, Tyr or hydroxyproline.

139. The compound according to Claim 136 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

140. The compound according to Claim 136 wherein AA or AAi is Serine or hydroxyproline.

141. The compound according to Claim 136 wherein AA or AA₁ is hydroxyproline.

142. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 136-141 and a pharmaceutically acceptable carrier therefor.

143. A method of treating a patient infected with a microorganism to which a amoxicillin, cefuroxime, ceftazidime and cexpodoxine is toxic, said method comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 13 -141.

144. A method of enhancing the solubility of cephalosporin antibiotics having a carboxylic acid functionality thereon in an aqueous solution comprising reacting the carboxylic acid functionality ofthe cephalosporin antibiotics molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

216 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc

145. A method of enhancing the bioavailability of cephalosporin antibiotics having a carboxylic acid functionality thereon when administered to a patient which comprises reacting the carboxylic acid functionality ofthe cephalosporin antibiotics molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

146. A compound ofthe formula:

or pharmaceutically acceptable salts thereof; wherein AA is attached via an ester bond and is an amino acid residue without the hydroxy group on the carboxy group.

147. The compound according to Claim 146 wherein AA is a naturally occurring L- - amino acid.

148. The compound according to Claim 146 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

149. The compound according to Claim 146 wherein AA is proline, glycine, lysine, hydroxyproline or alanine

150. The compound according to Claim 146 wherein AA is Lys or hydroxy proline.

151. The compound according to Claim 146 wherein AA is Lys. 217 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

152. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 146-151 and a pharmaceutically acceptable carrier therefor.

153. A method of treating malaria in mammals caused by plasmodium parasite, which method comprising administering to said patient a therapeutically effective amount of the compound according to any one of Claims 146-151.

154. A method of enhancing the solubility of atovaquone in an aqueous solution comprising reacting the hydroxy functionality ofthe atovaquone molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

155. A method of enhancing the bioavailability of atovaquone when administered to a patient which comprises reacting the hydroxy functionality ofthe atovaquone molecule with an amino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

156. Compounds of the formula:

218 H:\work\l 652\17690\SPEC\17690.spec without claimsll.doc

or pharmaceutically acceptable salts thereof; wherein AA is attached via an ester bond and is an amino acid residue less the hydroxy group ofthe carboxy group.

157. The compound according to Claim 156 wherein AA is a L-narurally occuring - amino acid.

158. The compound according to Claim 156 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy, with the proviso that when the drug is Acyclovir then AA is not Ala or Gly.

159. The compound according to Claim 156 wherein AA is proline, lysine, hydroxyproline or alanine

160. The compound according to Claim 156 wherein AA is Lys or proline.

161. The compound according to Claim 156 wherein AA is Lys.

162. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 156-161 and a pharmaceutically acceptable carrier therefor.

163. A method of treating a patient suffering from human cytomegalovirus, which method comprising administering to said patient a therapeutically effective amount of the compound according to any one of Claims 156-161. 219 H:\work\l 652\17690\SPEC\17690.spec without claimsll.doc

164. A method of enhancing the solubility of nucleoside analogs having an OH functionality in an aqueous solution comprising reacting the hydroxy functionality ofthe nucleoside analogs molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

165. A method of enhancing the bioavailability of nucleoside analogs having an oct group therein when administered to a patient which comprises reacting the hydroxy functionality ofthe nucleoside analogs molecule with an amino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

166. A compound of the formula:

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AAj. and AA is an amino acid less an amino group, AA]_. is an amino acid residue having a hydroxy group on the side chain without said hydroxy group thereon.

167. The compound according to Claim 166 wherein AA and AAi are independently naturally occurring L-α-amino acid.

168. The compound according to Claim 166 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AA] is Tyr, Thr, Ser or hydroxyproline.

220 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

169. The compound according to Claim 166 wherein AA is Alanine, proline, glycine, lysine, serine, threonine, or hydroxyproline.

170. The compound according to Claim 166 wherein AA or AA] is Serine or hydroxyproline.

171. The compound according to Claim 166 wherein AA or AAi is hydroxyproline.

172. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 166-171 and a pharmaceutically acceptable carrier therefor.

173. A method of reducing the lipid concentration in the body of a mammal, which method comprising administering to said patient a therapeutically effective amount of the compound according to any one of Claims 166-171.

174. A method of enhancing the solubility of niacin in an aqueous solution comprising reacting the carboxylic acid functionality ofthe niacin molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

175. A method of enhancing or reducing the bioavailability of niacin when administered to a patient which comprises reacting the carboxylic acid functionality of the niacin molecule with an amino acid or acylating derivative under ester or amide for ing conditions, isolating the product thereof and administering said product to the patient.

176. A compound of the formula:

221 H:\work\l 652\17690\SPEC\17690.spec without claimsILdoc

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AA_Ϊ and AA is an amino acid residue without the amino group and AAi is an amino acid residue having a hydroxy group on the side chain thereof without said hydroxy group.

177. The compound according to Claim 176 wherein AA and AAi are a naturally occurring L-α-amino acid.

178. The compound according to Claim 176 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, ' Sar or Dcy or AA] is Tyr, Ser, Thr or hydroxyproline.

179. The compound according to Claim 176 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

180. The compound according to Claim 176 wherein AA or AAj is Serine or hydroxyproline.

181. The compound according to Claim 176 wherein AA or AAi is hydroxyproline.

182. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 176-181 and a pharmaceutically acceptable carrier therefor.

222 H:\work\1652\17690\SPEC\1769Q.spec without claimsll.doc

183. A method of treating a skin condition in a patient requiring activation of retinoid X receptors, which method comprises administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 176-181.

184. A method of enhancing the solubility of bexarotene in an aqueous solution comprising reacting the carboxylic acid functionality ofthe bexarotene molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

185. A method of enhancing the bioavailability of bexarotene when administered to a patient which comprises reacting the carboxylic acid functionality ofthe bexarotene molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

186. A compound of the formula: H₅

PROPOXYPHENE

or pharmaceutically acceptable salts thereof; wherein AA is attached via an ester bond and is an amino acid residue less the hydroxy group on the carboxy group.

187. The compound according to Claim 186 wherein AA is a naturally occurring L-α- amino acid.

223 H:\work\16₅2\17690\SPEC\17690.spec without claimsll.doc

188. The compound according to Claim 186 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

189. The compound according to Claim 186 wherein AA is proline, glycine, lysine, hydroxyproline or alanine.

190. The compound according to Claim 186 wherein AA is Lys or hydroxy proline.

191. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 186-190 and a pharmaceutically acceptable carrier therefor.

192. A method of treating pain in a mammal, which method comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 186-190.

193. A method of enhancing the solubility of propoxyphene in an aqueous solution comprising reacting the hydroxy functionality ofthe propoxyphene molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

194. A method of enhancing the bioavailability of propoxyphene when administered to a patient which comprises reacting the hydroxy functionality ofthe propoxyphene molecule with an amino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

195. A compound of the formula:

224 H:\work\l 652\1 690\SPEC\17690.spec without claimsll.doc

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AA_! and AA is an amino acid residue without an amino group and AA_Ϊ is an amino acid residue with a hydroxy group on the side chain less said hydroxy group.

196. The compound according to Claim 195 wherein AA and AA_\ are independently naturally occurring L-α-amino acid.

197. The compound according to Claim 195 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAi is Tyr, Ser, Thr or hydroxyproline.

198. The compound according to Claim 195 wherein AA is proline, glycine, lysine, hydroxyproline or alanine.

199. The compound according to Claim 195 wherein AA is Lys or hydroxy proline.

200. The compound according to Claim 195 wherein AA is Lys.

201. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 195-200 or a pharmaceutically acceptable carrier therefor.

202. A method of treating inflammation in a patient, said method comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 195-200.

225 H:\work\16₅2\17690\SPEC\1769₀.spec without claimsll.doc

203. A method of enhancing the solubility of salsalate in an aqueous solution comprising reacting the hydroxy functionality ofthe salsalate molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

204. A method of enhancing the bioavailability of salsalate when administered to a patient which comprises reacting the hydroxy functionality ofthe salsalate molecule with an amino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

205. A compound ofthe formula:

or pharmaceutically acceptable salts thereof; wherein AA is an amino acid residue less the hydroxy group of a carboxy group thereon.

206. The compound according to Claim 205 wherein AA is a naturally occurring L-α- amino acid.

207. The compound according to Claim 205 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

208. The compound according to Claim 205 wherein AA is proline, glycine, lysine, hydroxyproline or alanine.

209. The compound according to Claim 205 wherein AA is Lys or proline. 226 H:\work\l 652\17690\SPEC\17690.spec without claimsll.doc

210. The compound according to Claim 205 wherein AA is proline.

211. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 205-209 and a pharmaceutically acceptable carrier therefor.

212. A method of treating a patient suffering from pain or who has a fever comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 205-209.

213. A method of enhancing the solubility of acetaminophen in an aqueous solution comprising reacting the hydroxy functionality ofthe acetaminophen molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

214. A method of enhancing the bioavailability of acetaminophen when administered to a patient which comprises reacting the hydroxy functionality ofthe acetaminophen molecule with an amino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

215. A compound of the formula:

IBUPROFEN

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AAi and AA is an amino acid residue without an amino group thereon and AAi is an amino acid residue with a hydroxy group on the side chain thereof without said hydroxy group.

227 H:\work\16S2\17690\SPEC\17690.spec without claimsILdoc

216. The compound according to Claim 215 wherein AA and AAj_. are naturally occurring L-α-amino acid.

217. The compound according to Claim 215 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAi is Thr, Ser, Tyr or hydroxyproline.

218. The compound according to Claim 215 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

219. The compound according to Claim 215 wherein AA or AA] is Serine or hydroxyproline.

220. The compound according to Claim 215 wherein AA or AAi is hydroxyproline.

221. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 215-220 and a pharmaceutically acceptable carrier therefor.

222. A method of treating pain, fever or inflammation in a patient comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 215-220.

223. A method of enhancing the solubility of ibuprofen in an aqueous solution comprising reacting the carboxylic acid functionality ofthe ibuprofen molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

224. A method of enhancing the bioavailability of ibuprofen when administered to a patient which comprises reacting the carboxylic acid functionality ofthe ibuprofen 228 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

225. A method of reducing the gastric irritation of ibuprofen comprising reacting the carboxylic acid functionalitiy ofthe ibuprofen molecule with an amino acid or acylating derivative under either ester or amide forming conditions and administering the product to a patient in need thereof, said product resulting less gastric irritability then ibuprofen.

226. Compounds ofthe formula

SIMAVASTATIN

229 H:\work\1652\1769Q\SPEC\17690.spec without claimsll.doc

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O- AAi and AA is an amino acid residue without an amino group and AAi is an amino acid residue having a side chain containing a hydroxy group without said hydroxy group thereon.

227. The compound according to Claim 226 wherein AA and AAj_. are naturally occurring L-α-amino acid.

228. The compound according to Claim 226 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAj. is Tyr, Ser, Thr or hydroxyproline.

230 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc

229. The compound according to Claim 226 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

230. The compound according to Claim 226 wherein AA or AAi is Serine or hydroxyproline.

231. The compound according to Claim 226 wherein AA or AAi is hydroxyproline.

232. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 226-231 and a pharmaceutically acceptable carrier therefor.

233. A method of lowering the cholesterol concentration in a mammal in need of such treatment a patient in need of statin drug therapy, which method comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 226-231.

234. A compound ofthe formula:

or pharmaceutically acceptable salts thereof; wherein AA is attached via an ester bond and is an amino acid residue without the hydroxy group ofthe carboxy group thereon.

235. The compound according to Claim 234 wherein AA is a naturally occurring L-α- a ino acid.

231 H:Work\16S2\17690\SPEC\1769Q.spec without claimsll.doc

236. The compound according to Claim 234 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

237. The compound according to Claim 234 wherein AA is proline, glycine, lysine, hydroxyproline or alanine.

238. The compound according to Claim 234 wherein AA is Lys or hydroxy proline.

239. The compound according to Claim 234 wherein AA is Lys.

240. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 234-239 or a pharmaceutically acceptable carrier therefor.

241. A method of treating a patient suffering from conditions selected from the group consisting of angina, pectoris and hypertension, which method comprising administering to said patient in need of treatment of a therapeutically effective amount of the compound according to any one of Claims 234-239.

242. A compound ofthe formula:

or pharmaceutically acceptable salts thereof; 232 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc wherein R is either NH-AA or O-AAi and AA is an amino acid residue without the amino group and AAj. is an amino acid residue having a hydroxy group on the side chain without said hydroxy group.

243. The compound according to Claim 242 wherein AA and AAi are a naturally occurring L-α-amino acid.

244. The compound according to Claim 242 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAi is Ser, Thr, Tyr or hydroxyproline.

245. The compound according to Claim 242 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

246. The compound according to Claim 242 wherein AA or AAi_. is Serine or hydroxyproline.

247. The compound according to Claim 242 wherein AA or AA] is hydroxyproline.

248. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 242-247 and a pharmaceutically acceptable carrier therefor.

249. A method of freating a patient with hypertension comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 242-247.

250. A method of enhancing the solubility of valsartan in an aqueous solution comprising reacting the carboxylic acid functionality ofthe valsartan molecule with an

233 H:\work\16₅2\1769₀\SPEC\17690.spec without claimsILdoc amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

251. A method of enhancing the bioavailability of valsartan when administered to a patient which comprises reacting the carboxylic acid functionality ofthe valsartan molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

252. A compound ofthe formula:

METHYLPHENIDATE

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AA and AA is an amino acid residue without the amino group and AAi is an amino acid residue having a hydroxy group on the side chain without said hydroxy group.

253. The compound according to Claim 252 wherein AA and AAi are a naturally occurring L-α-amino acid.

254. The compound according to Claim 252 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAi is Tyr, Ser, Thr, or hydroxyproline.

255. The compound according to Claim 252 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

234 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

256. The compound according to Claim 252 wherein AA or AAi is Serine or hydroxyproline.

257. The compound according to Claim 252 wherein AA or AA] is hydroxyproline.

258. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 252-257 and a pharmaceutically acceptable carrier therefor.

259. A method of treating a patient suffering from attention deficit disorder and narcolepsy comprising administering to a patient in need of such treatment a therapeutically effective amount ofthe compound according to any one of Claims 252- 257.

260. A method of enhancing the solubility of methylphenidate in an aqueous solution comprising reacting the carboxylic acid functionality ofthe methylphenidate molecule with an amino acid or acylating derivative thereof under either ester or amide foπning conditions and isolating the product thereof.

261. A method of enhancing the bioavailability of methylphenidate when administered to a patient which comprises reacting the carboxylic acid functionality of the methylphenidate molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

262. A compound ofthe formula:

235 H:\work\l 652\17690\SPEC\17690.spec without claimsll.doc

or pharmaceutically acceptable salts thereof; wherein AA is attached via an ester bond and is an amino acid residue without the hydroxy group ofthe carboxy group thereon.

263. The compound according to Claim 262 wherein AA is a naturally occurring L-α- amino acid.

264. The compound according to Claim 262 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

265. The compound according to Claim 262 wherein AA is proline, glycine, lysine, hydroxyproline or alanine.

266. The compound according to Claim 262 wherein AA is Lys or hydroxy proline.

267. The compound according to Claim 262 wherein AA is Lys.

268. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 262-267 and a pharmaceutically acceptable carrier therefor.

269. A method of freating inflammation resulting from tissue damage, infection, allergy or auto-immune disease, which method comprising administering to said patient 236 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc in need of treatment a therapeutically effective amount ofthe compound according to any one of Claims 262-267.

270. A method of enhancing the solubility of methylprednisolone in an aqueous solution comprising reacting the hydroxy functionality ofthe methylprednisolone molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

271. A method of enhancing the bioavailability of methylprednisolone when administered to a patient which comprises reacting the hydroxy functionality ofthe methylprednisolone molecule with an amino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

272. A compound of the formula:

or pharmaceutically acceptable salts thereof, where AA is attached via an ester bond and is an amino acid residue less the hydroxy group ofthe carboxy group thereon.

273. The compound according to Claim 272 wherein AA is a naturally occurring L-α- amino acid.

237 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc

274. The compound according to Claim 272 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

275. The compound according to Claim 272 wherein AA is proline, glycine, lysine, hydroxyproline or alanine

276. The compound according to Claim 272 wherein AA is Lys or hydroxy proline.

277. The compound according to Claim 272 wherein AA is Lys.

278. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 272-277 and a pharmaceutically acceptable carrier therefor.

279. A method of producing contraception to a patient, which method comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 272-277.

280. A method of enhancing the solubility of medroxyprogesterone in an aqueous solution comprising reacting the hydroxy functionality ofthe medroxyprogesterone molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

281. A method of enhancing the bioavailability of medroxyprogesterone when administered to a patient which comprises reacting the hydroxy functionality ofthe medroxyprogesterone molecule with an amino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

238 H:\work\l₆52\17690\SPEC\17690.spec without claimsll.doc

282. A compound ofthe formula:

or pharmaceutically acceptable salts thereof; wherein AA is attached via an ester bond and is an amino acid residue less the OH group on the carboxy group.

283. The compound according to Claim 282 wherein AA is an α-amino acid.

284. The compound according to Claim 282 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

285. The compound according to Claim 282 wherein AA is proline, glycine, lysine, hydroxyproline or alanine.

286. The compound according to Claim 282 wherein AA is Lys or hydroxy proline.

287. The compound according to Claim 282 wherein AA is Lys.

288. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 282-287 and a pharmaceutically acceptable carrier therefor.

239 H:\work\16S2\17690\SPECΛ17690.spec without claimsll.doc

289. A method of treating a patient in need of estramustine therapy, which method comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 282-287.

290. A method of enhancing the solubility of estramustine in an aqueous solution comprising reacting the hydroxy functionality ofthe estramustine molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

291. A compound of the formula:

or pharmaceutically acceptable salts thereof; wherein AA is attached via an ester bond and an amino acid residue less the OH group on a carboxy group thereon.

292. The compound according to Claim 291 wherein AA is a naturally occurring L-α- amino acid.

293. The compound according to Claim 291 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

294. The compound according to Claim 291 wherein AA is proline, glycine, lysine, hydroxyproline or alanine.

295. The compound according to Claim 291 wherein A A is Lys or hydroxy proline.

296. The compound according to Claim 291 wherein AA is Lys. 240 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc

297. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 291-296 and a pharmaceutically acceptable carrier therefor.

298. A method of treating type II diabetes in a patient comprising administering to a patient in need thereof a therapeutically effective amount ofthe compound according to any one of Claims 291-296.

299. A method of enhancing the solubility of miglitol in an aqueous solution comprising reacting the hydroxy functionality ofthe miglitol molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

300. A method of enhancing the bioavailability of miglitol when administered to a patient which comprises reacting the hydroxy functionality ofthe miglitol molecule with an amino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

301. A compound of the formula:

or pharmaceutically acceptable salts thereof; wherein AA is attached via an ester bond and is an amino acid residue without the OH group on the carboxy group thereon.

241 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

302. The compound according to Claim 301 wherein AA is a naturally occurring L-α- amino acid.

303. The compound according to Claim 301 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

304. The compound according to Claim 301 wherein AA is proline, glycine, lysine, hydroxyproline or alanine

305. The compound according to Claim 301 wherein AA is Lys or hydroxy proline.

306. The compound according to Claim 301 wherein AA is Lys.

307. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 301-306 and a pharmaceutically acceptable carrier therefor.

308. A method of treating malaria in a patient, which method comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 301 -306.

309. A method of enhancing the solubility of mefloquine in an aqueous solution comprising reacting the hydroxy functionality ofthe mefloquine molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

310. A method of enhancing the bioavailability of mefloquine when administered to a patient which comprises reacting the hydroxy functionality ofthe mefloquine molecule

242 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc with an amino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

311. A compound of the formula:

or pharmaceutically acceptable salts thereof; wherein AA forms an ester bond and is an amino acid residue less a OH group on a carboxy group thereon.

312. The compound according to Claim 311 wherein AA is a naturally occurring L-α- amino acid.

313. The compound according to Claim 311 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

314. The compound according to Claim 311 wherein AA is proline, glycine, lysine, hydroxyproline or alanine

315. The compound according to Claim 311 wherein A A is Lys or hydroxy proline.

316. The compound according to Claim 311 wherein AA is Lys.

243 H:\work\l 6S2\17δ90\SPEC\17690.spec without claimsll.doc

317. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 311-315 and a pharmaceutically acceptable carrier therefor.

318. A method of treating a patient suffering from endometriosis, which method comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 311-315.

319. A method of enhancing the solubility of danazol in an aqueous solution comprising reacting the hydroxy functionality ofthe danazol molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

320. A method of enhancing the bioavailability of danazol when administered to a patient which comprises reacting the hydroxy functionality ofthe danazol molecule with an amino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

321. A compound of the formula:

EPROSARTAN

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AAi and AA is an amino acid residue without an amino group and AAi an amino acid residue having an hydroxyl group on the side chain thereon without said hydroxy group..

244 H:\work\1652\1769Q\SPEC\17690.spec without claimsll.doc

322. The compound according to Claim 321 wherein AA and AAi are a naturally occurring L-α-amino acid.

323. The compound according to Claim 321 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAi is Tyr, Ser, Thr, or hydroxyproline.

324. The compound according to Claim 321 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Aanine.

325. The compound according to Claim 321 wherein AA or AAi is Serine or hydroxyproline.

326. The compound according to Claim 321 wherein AA or AAi hydroxyproline.

327. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 321-326 and a pharmaceutically acceptable carrier therefor.

328. A method of freating a patient in need of eprosartan therapy, which method comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 321 -326.

329. A method of enhancing the solubility of eprosartan in an aqueous solution comprising reacting the carboxylic acid functionality ofthe eprosartan molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

330. A method of enhancing the bioavailability of eprosartan when administered to a patient which comprises reacting the carboxylic acid functionality ofthe eprosartan 245 H:\work\1652\17690\SPEC\1769Q.spec without claimsILdoc molecule with an amino acid or acylating derivative under ester or amide fonning conditions, isolating the product thereof and administering said product to the patient.

331. A compound ofthe formula: VALPROICACID

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AAi AA is an amino acid residue less the amino group and AAj_. is an amino acid residue having a hydroxy group on the side chain without said hydroxy group.

332. The compound according to Claim 331 wherein AA and AAiare a naturally occurring L-α-amino acid.

333. The compound according to Claim 331 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAi is Thr, Ser, Tyr or hydroxyproline.

334. The compound according to Claim 331 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

335. The compound according to Claim 331 wherein AA or AA] is Serine or hydroxyproline.

336. The compound according to Claim 331 wherein AA or AAi is hydroxyproline.

337. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 331-336 and a pharmaceutically acceptable carrier therefor. 246 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

338. A method of freating a patient suffering from epilepsy comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 331-336.

339. A method of enhancing the solubility of divalproex. in an aqueous solution comprising reacting the carboxylic acid functionality ofthe divalproex molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

340. A method of enhancing the bioavailability of divalproex when administered to a patient which comprises reacting the carboxylic acid functionality ofthe divalproex molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

341. A compound of the formula:

FENOFIBRATE

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AAi and AA an amino acid residue without the amino group and AA^ is an amino acid residue having a hydroxy group on the side chain without the hydroxy group thereon.

342. The compound according to Claim 341 wherein AA and AAj are a naturally occurring L-α-amino acid.

247 H:\work\1652\17690\SPEC\17690.spec withoutclaimsII.doc

343. The compound according to Claim 341 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAi is Thr, Ser, Tyr or hydroxyproline.

344. The compound according to Claim 341 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

345. The compound according to Claim 341 wherein AA or AAi is Serine or hydroxyproline.

346. The compound according to Claim 341 wherein AA or AAj is hydroxyproline.

347. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 341-346 and a pharmaceutically acceptable carrier therefor.

348. A method of treating a patient in need of fenofibrate therapy, said method comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 341-346.

349. A method of enhancing the solubility of fenofibrate in an aqueous solution comprising reacting the carboxylic acid functionality ofthe fenofibrate molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

350. A method of enhancing the bioavailability of fenofibrate when administered to a patient which comprises reacting the carboxylic acid functionality ofthe fenofibrate molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

248 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

351. Compounds of the formula:

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AAi and AA is an amino acid residue without the amino group and AAi is an amino acid residue having a hydroxy group on the side chain without said hydroxy group.

352. The compound according to Claim 351 wherein AA and AAj are a naturally occurring L-α-amino acid.

353. The compound according to Claim 351 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AA] is Thr, Ser, Tyr or hydroxyproline.

354. The compound according to Claim 351 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

355. The compound according to Claim 351 wherein AA or AAi is Serine or hydroxyproline.

356. The compound according to Claim 351 wherein AA or AA_t is hydroxproline or tyrosine.

357. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 351-356 and a pharmaceutically acceptable carrier therefor.

249 H:\work\l 652\17690\SPEC\17690.spec without claimsll.doc

358. A method of freating a patient in need of gabapentin therapy, which method comprises administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 351-356.

359. A method of enhancing the solubility of gabapentin in an aqueous solution comprising reacting the carboxylic acid functionality ofthe gabapentin molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

360. A method of enhancing the bioavailability of gabapentin when administered to a patient which comprises reacting the carboxylic acid functionality ofthe gabapentin molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

361. Compounds of the formula:

OMEPRAZOLE

LANSOPRAZOLE

or pharmaceutically acceptable salts thereof;

250 H:\work\16S2\17690\SPEC\17690.spec without claimsILdoc wherein AA is attached via an amino bond and is an amino acid residue without the hydroxy group on the carboxy group.

362. The compound according to Claim 361 wherein AA is a naturally occurring L-α- amino acid.

363. The compound according to Claim 361 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

364. The compound according to Claim 361 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

365. The compound according to Claim 361 wherein AA is Serine or hydroxyproline.

366. The compound according to Claim 361 wherein AA is hydroxyproline.

367. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 361-366 and a pharmaceutically acceptable carrier therefor.

368. A method of treating gastric hyperacidity in a patient, said method comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 361-366.

369. A method of enhancing the solubility of proton pump inhibitors in an aqueous solution comprising reacting the amine functionality ofthe proton pump inhibitors molecule with the carboxylic acid moiety ofthe amino acid thereof under amide forming conditions and isolating the product thereof.

251 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

370. A method of enhancing the bioavailability of proton pump inhibitors when administered to a patient which comprises reacting the amine functionality ofthe proton pump inhibitors molecule with the carboxylic acid moiety ofthe amino acid under amide forming conditions, isolating the product thereof and administering said product to the patient.

371. A compound ofthe formula:

or pharmaceutically acceptable salts thereof; wherein AA is attached via an ester bond and is an amino acid residue without a hydroxy group on the carboxy group.

372. The compound according to Claim 371 wherein AA is a naturally occurring L-oc- amino acid.

373. The compound according to Claim 371 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

374. The compound according to Claim 371 wherein AA is proline, glycine, lysine, hydroxyproline or alanine.

375. The compound according to Claim 371 wherein AA is Lys or hydroxy proline.

376. The compound according to Claim 371 wherein AA is Lys. 252 H:\work\16₅2\17690\SPEC\1769₀.spec without claimsll.doc

377. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 371-376 and a pharmaceutically acceptable carrier therefor.

378. A method of freating anorexia in a patient in need of megestrol therapy, comprising administering to a patient in need thereof a therapeutically effective amount ofthe compound according to any one of Claims 371-376.

379. A method of enhancing the solubility of megestrol in an aqueous solution comprising reacting the hydroxy functionality ofthe megestrol molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

380. A method of enhancing the bioavailability of megestrol when administered to a patient which comprises reacting the hydroxy functionality ofthe megesfrol molecule with an a ino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

381. A compound of the formula:

METFORMIN or pharmaceutically acceptable salts thereof, wherein R is CO-AA, where AA is attached via an amide bond and is an amino acid residue without the carboxy group.

382. The compound according to Claim 381 wherein AA is a naturally occurring L-α- amino acid.

253 H:\work\ie52\1769Q\SPEC\17690.spec without claimsILdoc

383. The compound according to Claim 381 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

384. The compound according to Claim 381 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

385. The compound according to Claim 381 wherein AA is Serine or hydroxyproline.

386. The compound according to Claim 381 wherein AA is hydroxyproline.

387. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 381-386 and a pharmaceutically acceptable carrier therefor.

388. A method of treating hyperglycemia in a patient comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 381-386.

389. A method of enhancing the solubility of metformin in an aqueous solution ι comprising reacting the amine functionality ofthe metformin molecule with the carboxylic acid moiety ofthe amino acid thereof under amide forming conditions and isolating the product thereof.

390. A method of enhancing the bioavailability of metformin when administered to a patient which comprises reacting the amine functionality ofthe metformin molecule with the carboxylic acid moiety ofthe amino acid under amide forming conditions, isolating the product thereof and administering said product to the patient.

391. A compound of the formula: 254 H:\work\l 652\17690\SPEC\17690.spec without claimsll.doc

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AAi and AA is an amino acid residue without the amino group and AAi is an amino acid residue having a hydroxy group on the residue chain thereon and without said hydroxy group.

392. The compound according to Claim 391 wherein AA and AA_! are a naturally occurring L-α-amino acid.

393. The compound according to Claim 391 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAi is Tyr, Ser, Thr or hydroxyproline.

394. The compound according to Claim 391 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

395. The compound according to Claim 391 wherein AA or AAi is Serine or hydroxyproline.

396. The compound according to Claim 391 wherein AA or AAi is hydroxyproline.

397. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 391-396 and a pharmaceutically acceptable carrier therefor.

255 H:\work\l₆52\17690\SPEC\17690.spec without claimsll.doc

398. A method of treating psoriasis and acne in a patient in comprising administering to patient affected therewith a therapeutically effective amount ofthe compound according to any one of Claims 391-396.

399. A method of enhancing the solubility of tazarotene in an aqueous solution comprising reacting the carboxylic acid functionality ofthe tazarotene molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

400. A method of enhancing the bioavailability of tazarotene when administered to a patient which comprises reacting the carboxylic acid functionality ofthe tazarotene molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

401. Compounds of the formula:

256 H:\work\1652\l 7690\SPEC\17690.spec without claimsll.doc ZOLMITRIPTAN

or pharmaceutically acceptable salts thereof; wherein R is CO-AA and AA is attached via an amide bond and is an amino acid residue without the carboxy group thereon.

402. The compound according to Claim 397 wherein AA is a naturally occurring L-α- amino acid.

403. The compound according to Claim 401 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

404. The compound according to Claim 401 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

405. The compound according to Claim 401 wherein AA is Serine or hydroxyproline.

406. The compound according to Claim 401 wherein AA is hydroxyproline.

407. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 401-406 and a pharmaceutically acceptable carrier therefor.

257 H:\work\16₅2\17690\SPEC\17690.spec without claimsILdoc

408. A method of freating migraine headaches in a patient in need of said treatment, which method comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 401-406.

409. A method of enhancing the solubility of Selective 5-HT receptor agonists in an aqueous solution comprising reacting the amine functionality ofthe Selective 5-HT receptor agonists molecule with the carboxylic acid moiety ofthe amino acid thereof under amide forming conditions and isolating the product thereof.

410. A method of enhancing the bioavailability of Selective 5-HT receptor agonists when administered to a patient which comprises reacting the amine functionality ofthe Selective 5-HT receptor agonists molecule with the carboxylic acid moiety ofthe amino acid under amide forming conditions, isolating the product thereof and administering said product to the patient.

411. A compound of the formula: ^ASPIRIN or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AAi and AA is an amino acid residue without the amino group and AAi is an amino acid residue with a hydroxy group on the side chain without said hydroxy group.

412. The compound according to Claim 411 wherein AA and AAi are a naturally occurring L-α-amino acid.

413. The compound according to Claim 411 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAi is Tyr, Ser, Thr or hydroxyproline. 258 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

414. The compound according to Claim 411 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

415. The compound according to Claim 411 wherein AA or AAi is Serine or hydroxyproline.

416. The compound according to Claim 411 wherein AA or AAi is hydroxyproline.

417. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 411-416 and a pharmaceutically acceptable carrier therefor.

418. A method of enhancing the solubility of aspirin in an aqueous solution comprising reacting the carboxylic acid functionality ofthe aspirin molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

419. A method of enhancing the bioavailability of aspirin when administered to a patient which comprises reacting the carboxylic acid functionality ofthe aspirin molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

420. A method of reducing the gastric irritability in the stomach resulting from aspirin administration to a mammal which method comprises reacting the carboxylic acid functionality ofthe aspirin molecule with an amino acid or acylating derivative thereof under either amide or ester forming conditions, and administering the formed product to said mammal, wherein the gastric irritability is reduced relative to aspirin.

421. A Compound of the formula:

259 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc OLMESARTAN

or pharmaceutically acceptable salts thereof; wherein the R is either NH-AA or O-AAi and AA is an amino acid residue without the amino group and AAi is an amino acid residue without the hydroxy group on the carboxy group thereon.

422. The compound according to Claim 421 wherein AA and AAi are a naturally occurring L-α-amino acid.

423. The compound according to Claim 421 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAi is Tyr, Ser, Thr, or hydroxyproline.

424. The compound according to Claim 421 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

425. The compound according to Claim 421 wherein AA or AAi is Serine or hydroxyproline.

426. The compound according to Claim 421 wherein AA or AAi is hydroxyproline.

260 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc

427. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 421-426 and a pharmaceutically acceptable carrier therefor.

428. A method of freating hypertension in a patient in need of said therapy, comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 421-426.

429. A method of enhancing the solubility of olmesartan in an aqueous solution comprising reacting the carboxylic acid functionality ofthe olmesartan molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

430. A method of enhancing the bioavailability of olmesartan when administered to a patient which comprises reacting the carboxylic acid functionality ofthe olmesartan molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

431. Compounds of the formula:

261 H:\work\16₅2\1769₀\SPEC\17690.spec without claimsll.doc

262 H:\work\16₅2\17690\SPEC\17690.spec without claimsll.doc

or pharmaceutically acceptable salts thereof; wherein AA is attached via an ester bond and is an amino acid residue without the hydroxy group on the carboxy group thereon.

432. The compound according to Claim 431 wherein AA is a naturally occurring L-α- amino acid.

433. The compound according to Claim 431 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

434. The compound according to Claim 431 wherein AA is proline, glycine, lysine, hydroxyproline or alanine

435. The compound according to Claim 431 wherein AA is Lys or hydroxy proline.

436. The compound according to Claim 431 wherein AA is Lys.

263 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

437. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 431 and a pharmaceutically acceptable carrier therefor.

438. A method of treating a patient in need of immunosuppresant therapy, which method comprising administering to said patient a therapeutically effective amount of the compound according to any one of Claims 431-436.

439. A method of enhancing the solubility of immunosuppresants in an aqueous solution comprising reacting the hydroxy functionality ofthe immunosuppresants molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

440. A method of enhancing the bioavailability of immunosuppresants when administered to a patient which comprises reacting the hydroxy functionality ofthe immunosuppresants molecule with an amino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

441. A compound of the formula:

CLOPIDOGREL

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AAi and AA is an amino acid residue without an amino group and AAi is an amino acid residue having a hydroxy group on the side chain thereof, without said hydroxy group.

264 H:\work\l 652\17690\SPEC\17690.spec without claimsILdoc

442. The compound according to Claim 441 wherein AA and AAi are a naturally occurring L-α-amino acid.

443. The compound according to Claim 441 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAi is Tyr, Ser, Thr or hydroxyproline.

444. The compound according to Claim 441 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Aanine.

445. The compound according to Claim 441 wherein AA or AAi is Serine or hydroxyproline.

446. The compound according to Claim 441 wherein AA or AAi is hydroxyproline.

447. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 441-446 and a pharmaceutically acceptable carrier therefor.

448. A method of freating myocardial infarction in a mammal, said method comprising administering to said a mammal in need thereof a therapeutically effective amount ofthe compound according to any one of Claims 441-446.

449. A method of enhancing the solubility of clopidogrel in an aqueous solution comprising reacting the carboxylic acid functionality ofthe clopidogrel molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

450. A method of enhancing the bioavailability of clopidogrel when administered to a patient which comprises reacting the carboxylic acid functionality ofthe clopidogrel 265 H:\work\16₅2\17690\SPEC\17690.spec without claimsILdoc molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

451. A compound of the formula:

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AAi, AA is an amino acid residue without the amino group and AAi is an amino acid residue having a hydroxy group on the side chain without the hydroxy group.

452. The compound according to Claim 451 wherein AA and AAi are a naturally occurring L-α-amino acid.

453. The compound according to Claim 451 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Aa, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or A i is Tyr, Ser, Thr, or hydroxyproline.

454. The compound according to Claim 451 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

455. The compound according to Claim 451 wherein AA or AAj_. is Serine or hydroxyproline.

456. The compound according to Claim 451 wherein AA or AAi is hydroxyproline. 266 H;\work\1652\17690\SPEC\17690.spec without claimsll.doc

457. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 451-456 and a pharmaceutically acceptable carrier therefor.

458. A method of treating a patient in need of amphotericin B therapy, which method comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 451-456.

459. A method of enhancing the solubility of amphotericin B in an aqueous solution comprising reacting the carboxylic acid functionality ofthe amphotericin B molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

460. A method of enhancing the bioavailability of amphotericin B when administered to a patient which comprises reacting the carboxylic acid functionality ofthe amphotericin B molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

461. Compound of the formula:

and acceptable pharmaceutical salts thereof; wherein R is O-AA, where AA is attached via an ester bond and is an amino acid without the OH group on the carboxy group.

267 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

462. The compound according to Claim 461 wherein AA is a naturally occurring L-α- amino acid.

463. The compound according to Claim 461 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Aa, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

464. The compound according to Claim 461 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

465. The compound according to Claim 461 wherein AA is Serine or hydroxyproline.

466. The compound according to Claim 461 wherein AA is hydroxyproline.

467. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 461-466 and a pharmaceutically acceptable carrier therefor.

468. A method of treating a patient in need of tenofovir therapy, which method comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 461-466.

469. A method of enhancing the solubility of tenofovir in an aqueous solution comprising reacting the phosphoric acid functionality ofthe tenofovir molecule with an amino acid or acylating derivative thereof under either ester foπning conditions and isolating the product thereof.

470. A method of enhancing the bioavailability of tenofovir when administered to a patient which comprises reacting the phosphoric acid functionality ofthe tenofovir

268 H:\work\l 6₅2\17690\SPEC\17690.spec without claimsll.doc molecule with an amino acid or acylating derivative under ester foraiing conditions, isolating the product thereof and administering said product to the patient. ,

471. Compound of the formula:

UNOPROSTONE

or pharmaceutically acceptable salts thereof; wherein R is either NH-AA or O-AA and

AA is an amino acid residue less an amino group and AAi is an amino acid residue having a hydroxy group on the side chain thereof, without said hydroxy group.

472. The compound according to Claim 471 wherein AA, and AAi are independently a naturally occurring L-α-amino acid.

473. The compound according to Claim 471 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAi is Tyr, Ser, Thr or hydroxyproline.

474. The compound according to Claim 471 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

475. The compound according to Claim 471 wherein AA or AAi is Serine or hydroxyproline.

476. The compound according to Claim 471 wherein AA or AAi is hydroxyproline.

477. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 471-476 and a pharmaceutically acceptable carrier therefor.

269 H:\work\16₅2\17690\SPEC\17690.spec without claimsll.doc

478. A method of freating glaucoma in a patient comprising administering to a patient a therapeutically effective amount ofthe compound according to any one of Claims 471- 476.

479. A method of enhancing the solubility of unoprostone in an aqueous solution comprising reacting the carboxylic acid functionality ofthe unoprostone molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

480. A method of enhancing the bioavailability of unoprostone when administered to a patient which comprises reacting the carboxylic acid functionality ofthe unoprostone molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

481. Compound of the formula:

or pharmaceutically acceptable salts thereof, wherein AA is attached via an ester bond and is an amino acid without the OH on the carboxy group.

482. The compound according to Claim 481 wherein AA is a naturally occurring L-α- amino acid.

483. The compound according to Claim 481 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Aa, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

270 H:\work\16₅2\1769₀\SPEC\17690.spec without claimsll.doc

484. The compound according to Claim 481 wherein AA is proline, glycine, lysine, hydroxyproline or alanine

485. The compound according to Claim 481 wherein AA is Lys or hydroxy proline.

486. The compound according to Claim 481 wherein AA is Lys.

487. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 481-486 and a pharmaceutically acceptable carrier therefor.

488. A method of freating breast cancer, comprising administering to a patient in need thereof, which a therapeutically effective amount ofthe compound according to any one of Claims 481-486.

489. A method of enhancing the solubility of fulvesfrant in an aqueous solution comprising reacting the hydroxy functionality ofthe fulvesfrant molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

490. A method of enhancing the bioavailability of fulvesfrant when administered to a patient which comprises reacting the hydroxy functionality ofthe fulvesfrant molecule with an amino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

491. Compound of the formula:

271 H:\work\l 652\17690\SPEC\17690.spec without claimsILdoc

and pharmaceutically acceptable salts thereof, wherein R is either NH-AA or O-AAi, AA is an amino acid residue without an amino group and AAi is an amino acid residue having a hydroxy group on the side chain thereof without the hydroxy group.

492. The compound according to Claim 491 wherein AA and AAi are a naturally occurring L-α-amino acid.

493. The compound according to Claim 491 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAi is Tyr, Ser, Thr or hydroxyproline.

494. The compound according to Claim 491 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

495. The compound according to Claim 491 wherein AA or AAi is Serine or hydroxyproline.

496. The compound according to Claim 491 wherein AA or AAi is hydroxyproline.

497. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 491-496 and a pharmaceutically acceptable carrier therefor. 272 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

498. A method of freating a patient infected with microorganism comprising administering to said patient in need of such treatment a therapeutically effective amount ofthe compound according to any one of Claims 491-496.

499. A method of enhancing the solubility of cefditoren in an aqueous solution comprising reacting the carboxylic acid functionality ofthe cefditoren molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

500. A method of enhancing the bioavailability of cefditoren when administered to a patient which comprises reacting the carboxylic acid functionality ofthe cefditoren molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

501. Compounds of the formula: R

or pharmaceutically acceptable salts thereof; wherein R is CO-AA and AA is attached via an amide bond and is an amino acid residue without the OH group on the carboxy group.

502. The compound according to Claim 501 wherein AA is a naturally occurring L-α- amino acid.

273 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc

503. The compound according to Claim 501 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

504. The compound according to Claim 501 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

505. The compound according to Claim 501 wherein AA is Serine or hydroxyproline.

506. The compound according to Claim 501 wherein AA is hydroxyproline.

507. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 501-505 and a pharmaceutically acceptable carrier therefor.

508. A method of freating a patient with AIDS infection, which method comprising administering to a patient in need of treatment a therapeutically effective amount ofthe compound according to any one of Claims 501-505.

509. A method of enhancing the solubility of Efavirenz in an aqueous solution comprising reacting the amine functionality ofthe Efavirenz molecule with the carboxylic acid moiety ofthe amino acid thereof under amide forming conditions and isolating the product thereof.

510. A method of enhancing the bioavailability of Efavirenz when administered to a patient which comprises reacting the amine functionality ofthe Efavirenz molecule with the carboxylic acid moiety ofthe amino acid under amide forming conditions, isolating the product thereof and administering said product to the patient.

511. Compound of the formula:

274 H:\work\16₅2\17690\SPEC\I7690.spec without claimsll.doc

or pharmaceutically acceptable salts thereof; R is either NH-AA or O-AAi and AA is an amino acid group without the NH₂ group and AAi is an amino acid residue having a hydroxy group on the side chain thereof without the hydroxy group.

512. The compound according to Claim 511 wherein AA and AAi are naturally occurring L-α-amino acid.

513. The compound according to Claim 511 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy or AAi is Tyr, Thr, Ser or hydroxyproline.

514. The compound according to Claim 511 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

515. The compound according to Claim 511 wherein AA or AAi is Serine or hydroxyproline.

516. The compound according to Claim 511 wherein AA or AAi is hydroxyproline.

517. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 511-516 and a pharmaceutically acceptable carrier therefor.

275 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc

518. A method of freating hypertension, which method comprising administering to a patient in need of freatment of a therapeutically effective amount ofthe compound according to any one of Claims 511-516.

519. A method of enhancing the solubility of eplerenone in an aqueous solution comprising reacting the carboxylic acid functionality ofthe eplerenone molecule with an amino acid or acylating derivative thereof under either ester or amide forming conditions and isolating the product thereof.

520. A method of enhancing the bioavailability of eplerenone when administered to a patient which comprises reacting the carboxylic acid functionality ofthe eplerenone molecule with an amino acid or acylating derivative under ester or amide forming conditions, isolating the product thereof and administering said product to the patient.

521. Compound of the formula

He

or pharmaceutically acceptable salts thereof; wherein AA is attached via an ester bonded and is an amino acid residue.

522. The compound according to Claim 521 wherein AA is a naturally occurring L-α- amino acid.

276 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

523. The compound according to Claim 521 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

524. The compound according to Claim 521 wherein AA is proline, glycine, lysine, hydroxyproline or alanine

525. The compound according to Claim 521 wherein AA is Lys or hydroxy proline.

526. The compound according to Claim 521 wherein AA is Lys.

527. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 521-526 and a pharmaceutically acceptable carrier therefor.

528. A method of treating a cardiovascular related condition in a patient in need of freprostinil therapy, which method comprising administering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 521- 526.

529. A method of enhancing the solubility of freprostinil in an aqueous solution comprising reacting the hydroxy functionality ofthe freprostinil molecule with an amino acid or acylating derivative thereof under ester forming conditions and isolating the product thereof.

530. A method of enhancing the bioavailability of freprostinil when administered to a patient which comprises reacting the hydroxy functionality ofthe freprostinil molecule with an amino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

277 H:\work\16S2\17690\SPEC\17690.spec without claimsll.doc

531. Compound of the formula:

and acceptable pharmaceutical salts thereof; wherein R is O-AA, where AA is attached via an ester bond and is an amino acid residue without the hydroxy group on the carboxy group.

532. The compound according to Claim 531 wherein AA is a naturally occurring L-α- amino acid.

533. The compound according to Claim 531 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

534. The compound according to Claim 531 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Alanine.

535. The compound according to Claim 531 wherein AA is Serine or hydroxyproline.

536. The compound according to Claim 531 wherein AA is hydroxyproline.

537. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 531-536 or a pharmaceutically acceptable carrier therefor.

278 H:\work\16S2\17690\SPEC\17690.spec without claimsll.doc

538. A method of treating AIDS in a patient comprising admiriistering to said patient a therapeutically effective amount ofthe compound according to any one of Claims 531- 536.

539. A method of enhancing the solubility of adefovir in an aqueous solution comprising reacting the phosphoric acid functionality ofthe adefovir molecule with an amino acid or acylating derivative thereof under either ester forming conditions and isolating the product thereof.

540. A method of enhancing the bioavailability of adefovir when administered to a patient which comprises reacting the phosphoric acid functionality ofthe adefovir molecule with an amino acid or acylating derivative under ester forming conditions, isolating the product thereof and administering said product to the patient.

541. Compounds of the formula R-N=N-AAι-COOH

SULFO DRUG AZO DERIVATIVE or

R-NH CO-AA-NH₂

SULFA DRUG AMIDE DERIVATIVE or pharmaceutically acceptable salts thereof wherein R is the sulfanilamide moiety ofthe general class of sulfa drugs and AAi is an amino acid residue without the amino group and without the carboxy group.

542. The compound according to Claim 541 wherein AA is a naturally occurring L-α- amino acid.

543. The compound according to Claim 541 wherien AA is in the L-isomer.

279 H:\work\1652\17690\SPEC\17690.spec without claimsll.doc

544. The compound according to Claim 541 wehrein AA. is Lys, Leu, He, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr, Ag, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

545. The compound according to Claim 541 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline, or Alanine.

546. The compound according to Claim 541 wherein AA is Serine or hydroxyproline.

547. The compound according to Claim 541 wherein AA is hydroxyproline.

548. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 541-547 and a pharmaceutically acceptable carrier therefor.

549. A method of enhancing the solubility of sulfa drugs in an aqueous solution comprising reacting the amine ofthe sulfa drugs molecule with an amino acid or acylating derivative thereof under either azo or amide forming conditions and isolating the product thereof.

550. A method of enhancing the bioavailability of sulfa drugs when administered to a patient which comprises reacting the amine functionality ofthe sulfa drugs molecule with an amino acid or acylating derivative under azo or amide forming conditions, isolating the product thereof and administering said product to the patient.

551. A method of providing clear aqueous intravenous formulation of Sulfa drugs when administered to a patient which comprises reacting the arriine ofthe Sulfa drugs molecule with an amino acid or acylating derivative under azo or amide forming conditions, isolating the product thereof and administering said product to the patient parenterally. 280 H:\work\1652\17690\SPEC\17690.spec without claimsILdoc

552. A method of providing an appropriate formulation of Sulfa drugs when administered to a patient which comprises reacting the amine ofthe Sulfa drugs molecule with an amino acid or acylating derivative under azo or amide forming conditions, isolating the product thereof and administering said product to the patient either rectally, transdermally, buccally, vaginally, or nasally.

553. A compound of the formula: GLUTAMIC ACID AZO SALICYLIC ACID DERIVATIVE

or pharmaceutically acceptable salts thereof; wherein the chemical structure of salazine is 5-amino salicylic acid and Ri is the amino acid residue without the carboxy and amino groups.

554. The compound according to Claim 503 wherein AA is an α-amino acid.

555. The compound according to Claim 503 wherein AA is Lys, Leu, He, Gly, Asp, Glu, Met, Aa, Val, Pro, His, Tyr, Ser, Nor, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.

556. The compound according to Claim 553 wherein AA is proline, glycine, lysine, serine, threonine, hydroxyproline or Aanine.

557. The compound according to Claim 553 wherein AA is Serine or hydroxyproline.

558. The compound according to Claim 553 wherein AA is hydroxyproline.

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559. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of Claims 553-558 and a pharmaceutically acceptable carrier therefor.

560. A method of enhancing the solubility of salazine in an aqueous solution comprising reacting the amine functionality of trie salazine molecule with an amino acid or acylating derivative thereof under either azo bond forming conditions and isolating the product thereof.

561. A method of enhancing the bioavailability of salazine when administered to a patient which comprises reacting the amine functionality ofthe salazine molecule with an amino acid or acylating derivative under azo ^"bond forming conditions, isolating the product thereof and administering said product to the patient.

562. A method of providing clear aqueous intravenous formulation of Salazine when administered to a patient which comprises reacting the amine ofthe Salazine molecule with an amino acid or acylating derivative under azo bond forming conditions, isolating the product thereof and administering said product to the patient parenterally.

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563. A method of treating ulcerative colitis, crohn's disease, and other inflammatory infectious conditions ofthe colon and rectum in mammals, which method comprises administering to said mammal in need thereof a therapeutically effective amount of a compound according to any one of Claims 553-558.

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