US20050059606A1 - Novel peptides as NS3-serine protease inhibitors of hepatitis C virus - Google Patents

Novel peptides as NS3-serine protease inhibitors of hepatitis C virus Download PDF

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Publication number
US20050059606A1
US20050059606A1 US10/934,141 US93414104A US2005059606A1 US 20050059606 A1 US20050059606 A1 US 20050059606A1 US 93414104 A US93414104 A US 93414104A US 2005059606 A1 US2005059606 A1 US 2005059606A1
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United States
Prior art keywords
compound
alkyl
mmol
cycloalkyl
aryl
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Abandoned
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US10/934,141
Inventor
Anil Saksena
Viyyoor Girijavallabhan
Raymond Lovey
Edwin Jao
Ashok Arasappan
Tejal Parekh
Patrick Pinto
Frank Bennett
Jinping McCormick
Haiyan Wang
Russell Pike
Stephane Bogen
Yi-Tsung Liu
F. Njoroge
Ashit Ganguly
Terence Brunck
Scott Kemp
Odile Levy
Marguerita Lim-Wilby
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Merck Sharp and Dohme Corp
Dendreon Pharmaceuticals LLC
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Dendreon Corp
Schering Corp
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Priority to US10/934,141 priority Critical patent/US20050059606A1/en
Publication of US20050059606A1 publication Critical patent/US20050059606A1/en
Assigned to SCHERING CORPORATION reassignment SCHERING CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WANG, HAIYAN, PAREKH, TEJAL, MCCORMICK, JINPING L., LIU, YI-TSUNG, NJOROGE, F. GEORGE, PIKE, RUSSELL E., PINTO, PATRICK A., ARASAPPAN, ASHOK, BENNETT, FRANK, BOGEN, STEPHANE L., GIRIJAVALLABHAN, VIYYOOR M., JAO, EDWIN, LOVEY, RAYMOND G., SAKSENA, ANIL K., GANGULY, ASHIT K.
Assigned to CORVAS INTERNATIONAL, INC. reassignment CORVAS INTERNATIONAL, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KEMP, SCOTT JEFFREY, LEVY, ODILE ESTHER, BRUNCK, TERENCE K., LIM-WILBY, MARGUERITA
Assigned to DENDREON SAN DIEGO LLC reassignment DENDREON SAN DIEGO LLC MERGER (SEE DOCUMENT FOR DETAILS). Assignors: CORVAS INTERNATIONAL, INC.
Assigned to DENDREON CORPORATION reassignment DENDREON CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DENDREON SAN DIEGO LLC
Assigned to DRONE ACQUISITION SUB INC. reassignment DRONE ACQUISITION SUB INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DENDREON CORPORATION, AND ITS WHOLLY OWNED SUBSIDIARIES, DENDREON HOLDINGS, LLC, DENDREON DISTRIBUTION, LLC, AND DENDREON MANUFACTORING, LLC
Assigned to DENDREON PHARMACEUTICALS, INC. reassignment DENDREON PHARMACEUTICALS, INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: DRONE ACQUISITION SUB INC.
Abandoned legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel hepatitis C virus (“HGV”) protease inhibitors, pharmaceutical compositions containing one or more such inhibitors, methods of preparing such inhibitors and methods of using such inhibitors to treat hepatitis C and related disorders.
  • HCV hepatitis C virus
  • This invention specifically discloses novel peptide compounds as inhibitors of the HCV NS3/NS4a serine protease.
  • Hepatitis C virus is a (+)-sense single-stranded RNA virus that has been implicated as the major causative agent in non-A, non-B hepatitis (NANBH), particularly in blood-associated NANBH (BB-NANBH)(see, International Patent Application Publication No. WO 89/04669 and European Patent Application Publication No. EP 381 216).
  • NANBH is to be distinguished from other types of viral-induced liver disease, such as hepatitis A virus (HAV), hepatitis B virus (HBV), delta hepatitis virus (HDV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV), as well as from other forms of liver disease such as alcoholism and primary biliar cirrhosis.
  • HAV hepatitis A virus
  • HBV hepatitis B virus
  • HDV delta hepatitis virus
  • CMV cytomegalovirus
  • EBV Epstein-Barr virus
  • This approximately 3000 amino acid polyprotein contains, from the amino terminus to the carboxy terminus, a nucleocapsid protein (C), envelope proteins (E1 and E2) and several non-structural proteins (NS1, 2, 3, 4a, 5a and 5b).
  • NS3 is an approximately 68 kda protein, encoded by approximately 1893 nucleotides of the HCV genome, and has two distinct domains: (a) a serine protease domain consisting of approximately 200 of the N-terminal amino acids; and (b) an RNA-dependent ATPase domain at the C-terminus of the protein.
  • the NS3 protease is considered a member of the chymotrypsin family because of similarities in protein sequence, overall three-dimensional structure and mechanism of catalysis.
  • Other chymotrypsin-like enzymes are elastase, factor Xa, thrombin, trypsin, plasmin, urokinase, tPA and PSA.
  • the HCV NS3 serine protease is responsible for proteolysis of the polypeptide (polyprotein) at the NS3/NS4a, NS4a/NS4b, NS4b/NS5a and NS5a/NS5b junctions and is thus responsible for generating four viral proteins during viral replication. This has made the HCV NS3 serine protease an attractive target for antiviral chemotherapy.
  • NS4a protein an approximately 6 kda polypeptide
  • NS3/NS4a serine protease activity of NS3 It has been determined that the NS4a protein, an approximately 6 kda polypeptide, is a co-factor for the serine protease activity of NS3.
  • Autocleavage of the NS3/NS4a junction by the NS3/NS4a serine protease occurs intramolecularly (i.e., cis) while the other cleavage sites are processed intermolecularly (i.e., trans).
  • NS3/NS4a junction contains a threonine at P1 and a serine at P1′.
  • the Cys ⁇ Thr substitution at NS3/NS4a is postulated to account for the requirement of cis rather than trans processing at this junction. See, e.g., Pizzi et al. (1994) Proc. Natl. Acad. Sci ( USA ) 91:888-892, Failla et al.
  • NS3/NS4a cleavage site is also more tolerant of mutagenesis than the other sites. See, e.g., Kollykhalov et al. (1994) J. Virol. 68:7525-7533. It has also been found that acidic residues in the region upstream of the cleavage site are required for efficient cleavage. See, e.g., Komoda et al. (1994) J. Virol. 68:7351-7357.
  • Inhibitors of HCV protease include antioxidants (see, International Patent Application Publication No. WO 98/14181), certain peptides and peptide analogs (see, International Patent Application Publication No. WO 98/17679, Landro et al. (1997) Biochem. 36:9340-9348, ingallinella et al. (1998) Biochem. 37:8906-8914, Llinàs-Brunet et al. (1998) Bioorg. Med. Chem. Lett. 8:1713.-1718), inhibitors based on the 70-amino acid polypeptide eglin c (Martin et al. (1998) Biochem.
  • HCV has been implicated in cirrhosis of the liver and in induction of hepatocellular carcinoma.
  • the prognosis for patients suffering from HCV infection is currently poor.
  • HCV infection is more difficult to treat than other forms of hepatitis due to the lack of immunity or remission associated with HCV infection.
  • Current data indicates a less than 50% survival rate at four years post cirrhosis diagnosis.
  • Patients diagnosed with localized resectable hepatocellular carcinoma have a five-year survival rate of 10-30%, whereas those with localized unresectable hepatocellular carcinoma have a five-year survival rate of less than 1%.
  • a still further object of the present invention is to provide methods for modulating the activity of serine proteases, particularly the HCV NS3/NS4a serine protease, using the compounds provided herein.
  • Another object herein is to provide methods of modulating the processing of the HCV polypeptide using the compounds provided herein.
  • the present invention provides a novel class of inhibitors of the HCV protease, pharmaceutical compositions containing one or more of the compounds, methods of preparing pharmaceutical formulations comprising one or more such compounds, and methods of treatment, prevention or amelioration or one or more of the symptoms of hepatitis C. Also provided are methods of modulating the interaction of an HCV polypeptide with HCV protease.
  • compounds that inhibit HCV NS3/NS4a serine protease activity are preferred.
  • the presently disclosed compounds generally contain about four or more amino acid residues and less than about twelve amino acid residues.
  • the present application discloses peptide compounds, defined further below.
  • the present invention provides a compound of Formula I: or a pharmaceutically acceptable salt, solvate or derivative thereof, wherein:
  • the present invention provides a compound of Formula II: wherein X, P6, P5, P4, P3, P2, P1a, P1b and P1′ are as defined above.
  • Z is O, NH or NR 12 where R 12 has been defined before.
  • P2 when connected with the N atom adjacent to C atom it is attached to, forms a cyclic ring, with the proviso that said cyclic ring does not contain a carbonyl group as part of the cyclic ring structure.
  • the cyclic ring moiety comprised of P2, the carbon atom to which P2 is attached, and the nitrogen atom adjacent to that carbon atom is noted above as: which may denote a five-membered ring or a six-membered ring structure.
  • Preferred representatives for that cyclic ring structure are selected from the following: wherein:
  • the present invention discloses compounds of Formula III: where the various elements are as defined above.
  • alkyl refers to a monovalent group derived from a straight or branched chain saturated hydrocarbon by the removal of a single atom having from 1 to 8 carbon atoms, preferably from 1 to 6;
  • Also included in the invention are tautomers, rotamers, enantiomers and other optical isomers of compounds of Formula I, Formula II and Formula II, as well as pharmaceutically acceptable salts, solvates and derivatives thereof.
  • a further feature of the invention is pharmaceutical compositions containing as active ingredient a compound of Formula I, Formula II or Formula III (or its salt, solvate or isomers) together with a pharmaceutically acceptable carrier or excipient.
  • the invention also provides methods for preparing compounds of Formulas I, II and III, as well as methods for treating diseases such as, for example, HCV and related disorders.
  • the methods for treating comprise administering to a patient suffering from said disease or diseases a therapeutically effective amount of a compound of Formula I, II or III, or pharmaceutical compositions comprising a compound of Formula I, II or III.
  • the present invention discloses compounds of Formula I as inhibitors of HCV protease, especially the HCV NS3/NS4a serine protease, or a pharmaceutically acceptable derivative thereof, where the various definitions are given above.
  • the present invention discloses compounds of Formula II as inhibitors of HCV protease, especially the HCV NS3/NS4a serine protease, or a pharmaceutically acceptable derivative thereof, where the various definitions are given above.
  • the present invention discloses compounds of Formula III as inhibitors of HCV protease, especially the HCV NS3/NS4a serine protease, or a pharmaceutically acceptable derivative thereof, where the various definitions are given above:
  • the compounds of the invention may form pharmaceutically acceptable salts with organic or inorganic acids, or organic or inorganic bases.
  • suitable acids for such salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, malonic, salicylic, malic, fumaric, succinic, ascorbic, maleic, methanesulfonic and other mineral and carboxylic acids well known to those skilled in the art.
  • suitable bases are, for example, NaOH, KOH, NH 4 OH, tetraalkylammonium hydroxide, and the like.
  • this invention provides pharmaceutical compositions comprising the inventive peptides as an active ingredient.
  • the pharmaceutical compositions generally additionally comprise a pharmaceutically acceptable carrier diluent, excipient or carrier (collectively referred to herein as carrier materials). Because of their HCV inhibitory activity, such pharmaceutical compositions possess utility in treating hepatitis C and related disorders.
  • the present invention discloses methods for preparing pharmaceutical compositions comprising the inventive compounds as an active ingredient.
  • the active ingredients will typically be administered in admixture with suitable carrier materials suitably selected with respect to the intended form of administration, i.e. oral tablets, capsules (either solid-filled, semi-solid filled or liquid filled), powders for constitution, oral gels, elixirs, dispersible granules, syrups, suspensions, and the like, and consistent with conventional pharmaceutical practices.
  • the active drug component may be combined with any oral non-toxic pharmaceutically acceptable inert carrier, such as lactose, starch, sucrose, cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, talc, mannitol, ethyl alcohol (liquid forms) and the like.
  • suitable binders, lubricants, disintegrating agents and coloring agents may also be incorporated in the mixture.
  • Powders and tablets may be comprised of from about 5 to about 95 percent inventive composition.
  • Suitable binders include starch, gelatin, natural sugars, corn sweeteners, natural and synthetic gums such as acacia, sodium alginate, carboxymethylcellulose, polyethylene is glycol and waxes.
  • lubricants there may be mentioned for use in these dosage forms, boric acid, sodium benzoate, sodium acetate, sodium chloride, and the like.
  • Disintegrants include starch, methylcellulose, guar gum and the like.
  • Sweetening and flavoring agents and preservatives may also be included where appropriate.
  • disintegrants namely disintegrants, diluents, lubricants, binders and the like, are discussed in more detail below.
  • compositions of the present invention may be formulated in sustained release form to provide the rate controlled release of any one or more of the components or active ingredients to optimize the therapeutic effects, i.e.
  • Suitable dosage forms for sustained release include layered tablets containing layers of varying disintegration rates or controlled release polymeric matrices impregnated with the active components and shaped in tablet form or capsules containing such impregnated or encapsulated porous polymeric matrices.
  • Liquid form preparations include solutions, suspensions and emulsions. As an example may be mentioned water or water-propylene glycol solutions for parenteral injections or addition of sweeteners and pacifiers for oral solutions, suspensions and emulsions. Liquid form preparations may also include solutions for intranasal administration.
  • Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier such as inert compressed gas, e.g. nitrogen.
  • a pharmaceutically acceptable carrier such as inert compressed gas, e.g. nitrogen.
  • a low melting wax such as a mixture of fatty acid glycerides such as cocoa butter is first melted, and the active ingredient is dispersed homogeneously therein by stirring or similar mixing. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool and thereby solidify.
  • solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration.
  • liquid forms include solutions, suspensions and emulsions.
  • the compounds of the invention may also be deliverable transdermally.
  • the transdermal compositions may take the form of creams, lotions, aerosols and/or emulsions and can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.
  • the compound is administered orally, intravenously or subcutaneously.
  • the pharmaceutical preparation is in a unit dosage form.
  • the preparation is subdivided into suitably sized unit doses containing appropriate quantities of the active components, e.g., an effective amount to achieve the desired purpose.
  • the quantity of the inventive active composition in a unit dose of preparation may be generally varied or adjusted from about 1.0 milligram to about 1,000 milligrams, preferably from about 1.0 to about 950 milligrams, more preferably from about 1.0 to about 500 milligrams, and typically from about 1 to about 250 milligrams, according to the particular application.
  • the actual dosage employed may be varied depending upon the patient's age, sex, weight and severity of the condition being treated. Such techniques are well known to those skilled in the art.
  • the human oral dosage form containing the active ingredients can be administered 1 or 2 times per day.
  • the amount and frequency of the administration will be regulated according to the judgment of the attending clinician.
  • a generally recommended daily dosage regimen for oral administration may range from about 1.0 milligram to about 1,000 milligrams per day, in single or divided doses.
  • Capsule refers to a special container or enclosure made of methyl cellulose, polyvinyl alcohols, or denatured gelatins or starch for holding or containing compositions comprising the active ingredients.
  • Hard shell capsules are typically made of blends of relatively high gel strength bone and pork skin gelatins. The capsule itself may contain small amounts of dyes, opaquing agents, plasticizers and preservatives.
  • Tablet refers to a compressed or molded solid dosage form containing the active ingredients with suitable diluents.
  • the tablet can be prepared by compression of mixtures or granulations obtained by wet granulation, dry granulation or by compaction.
  • Oral gel refers to the active ingredients dispersed or solubilized in a hydrophillic semi-solid matrix.
  • Powder for constitution refers to powder blends containing the active ingredients and suitable diluents which can be suspended in water or juices.
  • Diluent refers to substances that usually make up the major portion of the composition or dosage form. Suitable diluents include sugars such as lactose, sucrose, mannitol and sorbitol; starches derived from wheat, corn, rice and potato; and celluloses such as microcrystalline cellulose.
  • the amount of diluent in the composition can range from about 10 to about 90% by weight of the total composition, preferably from about 25 to about 75%, more preferably from about 30 to about 60% by weight, even more preferably from about 12 to about 60%.
  • Disintegrant refers to materials added to the composition to help it break apart (disintegrate) and release the medicaments.
  • Suitable disintegrants include starches; “cold water soluble” modified starches such as sodium carboxymethyl starch; natural and synthetic gums such as locust bean, karaya, guar, tragacanth and agar; cellulose derivatives such as methylcellulose and sodium carboxymethylcellulose; microcrystalline celluloses and cross-linked microcrystalline celluloses such as sodium croscarmellose; alginates such as alginic acid and sodium alginate; clays such as bentonites; and effervescent mixtures.
  • the amount of disintegrant in the composition can range from about 2 to about 15% by weight of the composition, more preferably from about 4 to about 10% by weight.
  • Binder refers to substances that bind or “glue” powders together and make them cohesive by forming granules, thus serving as the “adhesive” in the formulation. Binders add cohesive strength already available in the diluent or bulking agent. Suitable binders include sugars such as sucrose; starches derived from wheat, corn rice and potato; natural gums such as acacia, gelatin and tragacanth; derivatives of seaweed such as alginic acid, sodium alginate and ammonium calcium alginate; cellulosic materials such as methylcellulose and sodium carboxymethylcellulose and hydroxypropylmethylcellulose; polyvinylpyrrolidone; and inorganics such as magnesium aluminum silicate.
  • the amount of binder in the composition can range from about 2 to about 20% by weight of the composition, more preferably from about 3 to about 10% by weight, even more preferably from about 3 to about 6% by weight.
  • Lubricant refers to a substance added to the dosage form to enable the tablet, granules, etc. after it has been compressed, to release from the mold or die by reducing friction or wear.
  • Suitable lubricants include metallic stearates such as magnesium stearate, calcium stearate or potassium stearate; stearic acid; high melting point waxes; and water soluble lubricants such as sodium chloride, sodium benzoate, sodium acetate, sodium oleate, polyethylene glycols and d'l-leucine. Lubricants are usually added at the very last step before compression, since they must be present on the surfaces of the granules and in between them and the parts of the tablet press.
  • the amount of lubricant in the composition can range from about 0.2 to about 5% by weight of the composition, preferably from about 0.5 to about 2%, more preferably from about 0.3 to about 1.5% by weight.
  • Glident material that prevents caking and improve the flow characteristics of granulations, so that flow is smooth and uniform.
  • Suitable glidents include silicon dioxide and talc.
  • the amount of glident in the composition can range from about 0.1% to about 5% by weight of the total composition, preferably from about 0.5 to about 2% by weight.
  • Coloring agents that provide coloration to the composition or the dosage form.
  • excipients can include food grade dyes and food grade dyes adsorbed onto a suitable adsorbent such as clay or aluminum oxide.
  • the amount of the coloring agent can vary from about 0.1 to about 5% by weight of the composition, preferably from about 0.1 to about 1%.
  • Bioavailability refers to the rate and extent to which the active drug ingredient or therapeutic moiety is absorbed into the systemic circulation from an administered dosage form as compared to a standard or control.
  • Conventional methods for preparing tablets are known. Such methods include dry methods such as direct compression and compression of granulation produced by compaction, or wet methods or other special procedures. Conventional methods for making other forms for administration such as, for example, capsules, suppositories and the like are also well known.
  • Another embodiment of the invention discloses the use of the pharmaceutical compositions disclosed above for treatment of diseases such as, for example, hepatitis C and the like.
  • the method comprises administering a therapeutically effective amount of the inventive pharmaceutical composition to a patient having such a disease or diseases and in need of such a treatment.
  • the compounds of the invention may be used for the treatment of HCV in humans in monotherapy mode or in a combination therapy mode such as, for example, in combination with antiviral agents such as, for example, ribavirin and/or interferon such as, for example, ⁇ -interferon and the like.
  • antiviral agents such as, for example, ribavirin and/or interferon such as, for example, ⁇ -interferon and the like.
  • the invention includes tautomers, rotamers, enantiomers and other stereoisomers of the compounds also.
  • inventive compounds may exist in suitable isomeric forms. Such variations are contemplated to be within the scope of the invention.
  • Another embodiment of the invention discloses a method of making the compounds disclosed herein.
  • the compounds may be prepared by several techniques known in the art. Representative illustrative procedures are outlined in the following reaction schemes. It is to be understood that while the following illustrative schemes describe the preparation of a few representative inventive compounds, suitable substitution of any of both the natural and unnatural amino acids will result in the formation of the desired compounds based on such substitution. Such variations are contemplated to be within the scope of the invention.
  • N-Cbz-hydroxyproline methyl ester available from Bachem Biosciences, Incorporated, King of Prussia, Pa.
  • compound (2.1) 3.0 g
  • toluene 30 mL
  • ethyl acetate 30 mL
  • the mixture was stirred vigorously, and then a solution of NaBr/water (1.28 g/5 mL) was added.
  • TEMPO 2,2,6,6-tetramethyl-1-piperidinyloxy free radical
  • Ethyl acetate 1000 mL was added to the lower acetonitrile layer, and then the layer was washed with 10% aqueous KH 2 PO 4 (2 ⁇ 700 mL), and brine.
  • the filtrate was evaporated under vacuum in a 25° C. water bath, taken up in fresh ethyl acetate (1000 mL), and washed successively with 0.1 N HCl, 0.1 N NaOH, 10% aqueous KH 2 PO 4 , and brine.
  • the organic solution was dried over anhydrous MgSO 4 , filtered, and evaporated under vacuum.
  • Step B Compound (3.2).
  • reactors for the solid-phase synthesis of peptidyl argininals are comprised of a reactor vessel with at least one surface permeable to solvent and dissolved reagents, but not permeable to synthesis resin of the selected mesh size.
  • Such reactors include glass solid phase reaction vessels with a sintered glass frit, polypropylene tubes or columns with frits, or reactor KansTM made by Irori Inc., San Diego Calif. The type of reactor chosen depends on volume of solid-phase resin needed, and different reactor types might be used at different stages of a synthesis.
  • the tert-butyl L-pyroglutamate was dissolved in acetonitrile (1.5 L) with 4-dimethylaminopyridine (6.0 g, 49.1 mmol) and cooled to 0° C.
  • a solution of di-tert-butyl-dicarbonate (154 g, 705 mmol) in acetonitrile (150 mL) was added over 30 min and after an additional 30 min the cooling bath was removed. After stirring at room temperature for 48 hours, the reaction mixture was concentrated. The residue was dissolved in ether (1 L) and hexanes (1 L), then washed with saturated aqueous sodium bicarbonate (2 ⁇ 100 mL) and saturated aqueous sodium chloride.
  • the reaction mixture was immersed in an ice/water bath and 30% aqueous hydrogen peroxide (10 drops) was added. The solution was stirred for 20 minutes at 0° C., then the reaction mixture was concentrated in vacuo to remove the tetrahydrofuran.
  • the aqueous solution was diluted with water (10 mL) and extracted with dichloromethane (3 ⁇ 40 mL). The organic layers were dried (sodium sulfate), filtered and concentrated. The residue was dissolved in dichloromethane (20 mL) and triethylsilane (310 ⁇ L, 2.0 mmol), then cooled to ⁇ 78° C. and boron trifluoride etherate (270 ⁇ L, 2.13 mmol) was added dropwise.
  • the solid was dissolved in a minimal amount of hot methanol, then crystallized by the addition of isopropyl ether, followed by 10% ethyl acetate in hexanes. The solid (658.6 mg) was collected and recrystallized again to give the title compound (520 mg, 1.335 mmol, 53%).
  • the reaction mixture was stirred at room temperature for 17 h, then concentrated to remove the dioxane and diluted with water (200 mL). The solution was washed with ether (3 ⁇ 100 mL). The pH of the aqueous solution was adjusted to 2 by the addition of citric acid (caution! foaming! and water (100 mL). The mixture was extracted with dichloromethane (400 mL, 100 mL, 100 mL) and the combined organic layers were dried (sodium sulfate), filtered and concentrated to give the title compound.
  • the solution was washed with ether (3 ⁇ 75 mL).
  • the pH of the aqueous solution was adjusted to 2 by the addition of citric acid (approx. 20 g, caution! foaming! and water (100 mL).
  • the mixture was extracted with dichloromethane (4 ⁇ 100 mL), and the combined organic layers were dried (sodium sulfate), filtered and concentrated.
  • the crude product contained a major impurity which necessitated a three step purification.
  • the crude product was dissolved in dichloromethane (50 mL) and trifluoroacetic acid (50 mL) and stirred for 5 h before being concentrated.
  • the residue was purified by preparatory reverse-phase HPLC.
  • the mixture was extracted with ethyl acetate (3 ⁇ 150 mL) and the combined organic layers were washed with saturated aqueous sodium chloride (100 mL), dried (sodium sulfate), filtered and concentrated.
  • the crude product was dissolved in saturated aqueous sodium bicarbonate(100 mL) and washed with ether (3 ⁇ 75 mL).
  • the pH of the aqueous layer was adjusted to 3 by the addition of citric acid, then extracted with dichloromethane (4 ⁇ 100 mL).
  • the combined organic layers were dried (sodium sulfate), filtered and concentrated to the title compound (1.373 g, 2.94 mmol, 42%).
  • Procedure A Coupling reaction: To the resin suspended in DMF (10-15 mL/gram resin) was added Fmoc-amino acid (1 eq), HOBt (1 eq), TBTU (1 eq) and DIEA (1 eq). The mixture was let to react for 448 hours. The reactants were drained and the resin was washed successively with dimethylformamide, dichloromethane, methanol, dichloromethane and diethylether (use 10-15 mL solvent/gram resin). The resin was then dried in vacuo.
  • Procedure B Coupling reaction: To the resin suspended in N-methylpyrrolidine (NMP) (10-15 mL/gram resin) was added Fmoc-amino acid (2 eq), HOAt (2 eq), HATU (2 eq) and diisopropylethylamine (4 eq). The mixture was let to react for 4-48 hours. The reactants were drained and the resin was washed successively with dimethylformamide, dichloromethane, methanol, dichloromethane and diethylether (use 10-15 mL solvent/gram resin). The resin was then dried in vacuo.
  • NMP N-methylpyrrolidine
  • Fmoc deprotection The Fmoc-protected resin was treated with 20% piperidine in dimethylformamide (10 mL reagent/g resin) for 30 minutes. The reagents were drained and the resin was washed successively with dimethylformamide, dichloromethane, methanol, dichloromethane and diethyl ether (10 mL solvent/gram resin).
  • Procedure D Boc deprotection: The Boc-protected resin was treated with a 1:1 mixture of dichloromethane and trifluoroacetic acid for 20-60 minutes (10 mL solvent/gram resin). The reagents were drained and the resin was washed successively with dichloromethane, dimethylformamide, 5% diisopropylethylamine in dimethylformamide, dimethylformamide, dichloromethane and dimethylformamide (10 mL solvent/gram resin).
  • Procedure E Acetylation with acetic anhydride: The resin was suspended in dimethylformamide.
  • the acetylating reagent prepared by adding 5 mmol (0.47 mL) acetic anhydride and 5 mmol (0.70 mL) triethylamine to 15 mL Dimethylformamide, was added to the resin and the resin was agitated for 30 minutes. The resin was washed successively with dimethylformamide, dichloromethane, methanol, dichloromethane and diethyl ether (10 mL solvent/gram resin).
  • Procedure F Semicarbazone hydrolysis: The resin was suspended in the cleavage cocktail (10 mL/g resin) consisting of trifluoroacetic acid: pyruvic acid: dichloromethane: water 9:2:2:1 for 2 hours. The reactants were drained and the procedure was repeated three more times. The resin was washed successively with dichloromethane, water and dichloromethane and dried under vacuum.
  • Procedure G HF cleavage: The dried peptide-nVal(CO)-G-O-PAM resin (50 mg) was placed in an HF vessel containing a small stir bar. Anisole (10% of total volume) was added as a scavenger. In the presence of glutamic acid and cysteine amino acids, thioanisole (10%) and 1,2-ethanedithiol (0.2%) were also added. The HF vessel was then hooked up to the HF apparatus (from Immuno Dynamics, Incorporated) and the system was flushed with nitrogen for five minutes. It was then cooled down to ⁇ 70° C. with a dry ice/isopropanol bath.
  • HF was distilled to the desired volume (10 mL HF/g resin). The reaction was let to proceed for one and a half hour at 0° C. Work up consisted of removing all the HF using nitrogen. Dichloromethane was then added to the resin and the mixture was stirred for five minutes. This was followed by the addition of 20% acetic acid in water (4 mL). After stirring for 20 minutes, the resin was filtered using a fritted funnel and the dichloromethane was removed under reduced pressure. Hexane was added to the remaining residue and the mixture was agitated, and the layers separated (this was repeated twice to remove scavengers). Meanwhile, the resin was soaked in 1 mL methanol.
  • the aqueous layer (20% HOAC) was added back to the resin and the mixture was agitated for five minutes and then filtered. The methanol was removed under reduced pressure and the aqueous layer was lyophilized. The peptide was then dissolved in 10-25% methanol (containing 0.1% trifluoroacetic acid) and purified by reverse phase HPLC.
  • Procedure H HF pyridine cleavage: To dried peptide-nVal(CO)-G-O-PAM resin (50 mg) in a HDPE 20 mL scintillation vial containing a 1 ⁇ 2 inch football stir bar was added thioanisole (100 ⁇ L) using a pipetman. A commercially available solution of 70% HF/Pyridine (1 mL) was added. The vial was immediately sealed tight with a polypropylene lined cap and placed in an ice bath with gentle stirring. After stirring in a 0° C. bath for one hour, the bath was removed and the mixture was stirred at room temperature for one hour.
  • the vial was inspected periodically and if necessary the resin which built up on the sides of the flask was dispersed back into the HF solution. The vial was cooled back to 0° C. and its cap was carefully removed. The vial were then cooled to ⁇ 15° C. using a salt/ice bath. A 10 mL B-D syringe body equipped with a 23 gauge needle was held above the vial. To this syringe body was added methoxytrimethylsilane (2 mL) using a repetitive autopipette.
  • step Ib above (22.7 g, 75 mmol) was refluxed in 6N HCl (480 mL) for 4 h.
  • the reaction mixture was allowed to cool to room temperature, then washed with dichloromethane (3 ⁇ 100 mL).
  • the aqueous layer was concentrated to dryness.
  • the residue was triturated with toluene/n-heptane (3 ⁇ ), dried in vacuo to give crude 3-amino-2-hydroxy-hexanoic acid.
  • the crude 3-amino-2-hydroxy-hexanoic acid was dissolved in hydrogen chloride saturated ethanol (40 mL). After 4 h, the solution was concentrated under reduced pressure.
  • step Id above To the compound of step Id above (4.02 g, 8.1 mmol) in ethanol (40.5 mL) was slowly added 1N lithium hydroxide (64.8 mL, 64.8 mmol). After 3 h, Dowex 50WX8400 ion exchange resin was added until the pH of the solution reached 4, and the mixture was stirred for 5 min. The mixture was filtered, washed with methanol, and concentrated. The solution was diluted with water, washed with ether. The aqueous solution was concentrated in vacuo. The resulting solid was triturated with toluene, then dried in vacuo to yield 1.44 g of the title compound (38%) as a white solid.
  • 1N lithium hydroxide 64.8 mL, 64.8 mmol
  • Boc-Gly-PAM resin 5 g, 3.35 mmol was deprotected according to Procedure D in a 250 mL fritted solid phase reaction vessel equipped with a nitrogen inlet. It was then coupled to Boc-nVal(dpsc)-OH (step Ie above) (2.81 g, 6 mmol)according to Procedure A. The resin was then subjected to Boc deprotection according to procedure D.
  • step 1 The resin obtained in step 1 (3.5 9, 1.80 mmol) was reacted with Fmoc-Pro-OH (4.5 mmol, 1.52 g) according to Procedure A. After 18 hours, qualitative ninhydrin analysis showed colorless beads and solution indicating a high yield of coupling.
  • step II above 100 mg was transferred to a fritted polypropylene tube and was deprotected according to Procedure C.
  • a ninhydrin assay on a small aliquot gave dark blue resin and solution, indicating a high yield for the deprotection.
  • the resin was resuspended in DMF (1 mL) and coupled to Fmoc-Val-OH (51 mg, 0.15 mmol) according to Procedure A.
  • a small aliquot was taken for qualitative ninhydrin analysis which showed colorless beads and a dark red solution indicating a high yield of coupling.
  • the compound of the previous step (100 mg) was deprotected according to Procedure C.
  • a ninhydrin assay on a small aliquot gave dark blue resin and solution showing a high yield for the deprotection.
  • the resin was resuspended in DMF (1 mL) and was coupled to Fmoc-Val-OH (51 mg, 0.15 mmol), according to Procedure A for 20 hours.
  • a small aliquot was taken for qualitative ninhydrin analysis which showed colorless beads and solution indicating a high yield of coupling.
  • the compound of the previous step (100 mg) was deprotected according to Procedure C.
  • a ninhydrin assay on a small aliquot gave dark blue resin and solution showing a high yield for the deprotection.
  • the resin was resuspended in DMF (1 mL) and was coupled to Fmoc-Glu(OtBu)-OH (64 mg, 0.15 mmol), according to Procedure A for 5 hours.
  • a small aliquot was taken for qualitative ninhydrin analysis which showed colorless beads and solution indicating a high yield of coupling.
  • the compound of the previous step (100 mg) was deprotected according to Procedure C.
  • a ninhydrin assay on a small aliquot gave dark blue resin and solution showing a high yield for the deprotection.
  • the resin was resuspended in DMF (1 ml) and was coupled to Fmoc-Glu(OtBu)-OH (64 mg, 0.15 mmol), according to Procedure A for 5 hours.
  • a small aliquot was taken for qualitative ninhydrin analysis which showed colorless beads and solution indicating a high yield of coupling.
  • the resin of the previous step (100 mg) was subjected to HF cleavage condition (Procedure G) to yield the desired crude product.
  • the material was purified by HPLC using a 2.2 ⁇ 25 cm reverse phase column, containing a C-18 resin comprised of 10 micron size gel particles with a 300 angstrom pore size, eluting with a gradient using 5-25% acetonitrile in water.
  • Analytical HPLC using a 4.6 ⁇ 250 mm reverse phase column, containing a C-18 resin comprised of 5 micron size gel particles with a 300 angstrom pore size, eluting with 5-25% acetonitrile (containing 0.1% trifluoroacetic acid) showed one peak at 17.5 minutes.
  • Low resolution mass spectrum confirmed the desired mass (MH + 798.5).
  • Trifluoroacetic acid (4.15 mL, 41.54 mmol) was added dropwise to a cooled solution (0° C.) of Boc-nVal-aldehyde (4.18 9, 20.77 mmol) (obtained in example I, step Ib(part I)), ethylisocyanoacetate (2.72 mL, 24.93 mmol), and pyridine (6.72 mL, 83.09 mmol) in dichloromethane (83 mL). After two hours, the reaction was brought to room temperature and stirred uncapped for 48 hours. The reaction mixture was concentrated and dissolved in ethylacetate (80 mL).
  • step I above The product obtained in step I above (1.12 g, 3.38 mmol) was treated for one hour with 10 mL saturated solution of anhydrous HCl in ethanol. It was then concentrated and triturated with n-heptane to a solid (0.9 g, 99.2%).
  • Step III Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-OH (Steps a-f Below)
  • the resin from the previous step was transferred to a 1 L plastic bottle and cleaved in the presence of 525 ml solution of acetic acid: trifluoroethanol: dichloromethane (1:1:3) with vigorous shaking for two hours.
  • the resin was filtered using a fritted funnel and washed 3 ⁇ 50 mL with dichloromethane.
  • the brownish red filtrate was concentrated to an oil which was then treated three times with 50 ml of a 1:1 mixture of dichloromethane: n-heptane.
  • the crude off-white powder (13 g) was then dissolved in minimum amount of methanol and purified by HPLC using a 2.2 ⁇ 25 cm reverse phase column, containing a C-18 resin comprised of 10 micron size gel particles with a 300 angstrom pore size, eluting with a gradient ranging from 15-55% acetonitrile in water.
  • the pure fractions were pulled and concentrated to a fluffy, white product (7.5 g, 65%).
  • step IV To the compound obtained in step IV above (0.94 g, 1 mmol) in ethanol (15 mL) was added 1N lithium hydroxide (4 mL, 4 mmol) and the reaction was stirred at room temperature for two hours. The reaction was stopped by the addition of enough Dowex ion exchange resin (50 ⁇ 8-400) to obtain an acidic solution, pH ⁇ 3. After stirring for 15 minutes, the reaction mixture was filtered and concentrated. The crude product was subjected to HPLC purification using a 5.5 ⁇ 30 cm reverse phase column, containing a C-18 resin comprised of 5 micron size gel particles with a 300 angstrom pore size, eluting with a 30 minute gradient using 5-30% acetonitrile in water. The desired fractions were pulled and concentrated to a white solid (238 mg, 26%).
  • step V above 129 mg, 0.14 mmol was coupled to allylamine (13 l, 0.17 mmol) in the presence of HOBt (58.5 mg, 0.38 mmol), EDC (54.3 mg, 0.28 mmol) and diisopropylethylamine (124 l, 0.71 mmol) in dimethylformamide (10 ml). After 18 hours, the reaction mixture was concentrated and the remaining residue was picked-up in ethylacetate and washed three times each with 5 mL portions of 1N sodium bisulfate, saturated sodium bicarbonate and brine. After drying over sodium sulfate, the organic layer was concentrated to give a white precipitate which was taken to the next step without further purification (115 mg, 85%).
  • step VI Under a stream of nitrogen gas, the product of step VI above (115.2 mg, 0.12 mmol) was dissolved in dimethylsulfoxide (5 mL) and toluene (5 mL). Water soluble carbodiimide (EDC, 232.2 mg, 1.21 mmol) was then added in one batch. The reaction mixture was cooled to 0° C. and dichloroacetic acid (52 l, 0.60 mmol) was added dropwise. Stirring at 0° C. continued for 15 minutes. The ice bath was removed and the reaction continued for two hours at room temperature. The toluene was removed under reduced pressure. The remaining solution was diluted with ethylacetate and washed three times each with 5 mL portions of 1N sodium bisulfate, saturated sodium bicarbonate and brine. It was then concentrated to a yellowish foam (85.5 mg, 74.4%).
  • EDC Water soluble carbodiimide
  • step VII above (0.86 g, 0.91 mmol) was treated with a 1:1 mixture of dichloromethane: trifluoroacetic acid (20 ml) for one hour.
  • the reaction mixture was then concentrated and the remaining residue was purified using a 2.2 ⁇ 25 cm reverse phase HPLC column, containing a C-18 resin comprised of 10 micron size gel particles with a 300 angstrom pore size, eluting with a 30 minutes gradient using 10-25% acetonitrile in water.
  • the purified fractions were pulled and lyophilized to a white powder (21.5 mg, 28.5%).
  • Step I Synthesis of Fmoc-nVal-Gly-Oallyl (Steps a-c Below) a) Synthesis of allyl isocyanoacetate
  • step al (99.92 g, 0.81 mol) dissolved in acetonitrile (810 mL) was added allyl bromide (92 mL, 1.05 mol). After refluxing for four hours, a dark brown solution was obtained. The reaction mixture was concentrated and the remaining residue was picked-up in ether (1.5 L) and washed three times with water (500 ml). The organic layer was dried and concentrated to a dark brown syrup. The crude was purified by vacuum distillation at 7 mm Hg (98° C.) to a clear oil (78.92 g, 77.7%). NMR ⁇ ppm (CDCI3): 5.9 (m, 1 H), 5.3 (m, 2H), 4.7 (d, 2H), 4.25 (s, 2H).
  • step b2 To the product of step b2 (21.70 g, 66.77 mmol) in dichloromethane (668 mL) was added triethylamine (37.23 mL, 267.08 mmol) and the solution was cooled to 0° C. A suspension of pyridine sulfur trioxide complex (42.51 g, 267.08 mmol) in dimethylsulfoxide (96 mL) was added to the chilled solution. After one hour, thin layer chromatography in 2:3 ethylacetate: hexane confirmed the completion of the reaction.
  • step I a2 To a solution of 9-fluorenylmethoxycarbonyl-norvalinal obtained from step b3 (5.47 g, 16.90 mmol) in dichloromethane (170 mL) was added allyl isocyanoacetate (step I a2 above) (2.46 mL, 20.28 mmol) and pyridine (5.47 mL, 67.61 mmol). The reaction mixture was cooled to 0° C. and trifluoroacetic acid (3.38 mL, 33.80 mmol) was added dropwise. The reaction was stirred at 0 C for 1 h, and then at room temperature for 48 hours. Thin layer chromatography taken in ethylacetate confirmed the completion of the reaction.
  • step II The product obtained from step II above (28.2 mg, 0.11 mmol) was coupled to Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-OH (example II, step III) (84 mg, 0.11 mmol) in the presence of HOAt (23.6 mg, 0.17 mmol), HATU (48.4 mg, 0.13 mmol), diisopropylethylamine (100.8 ⁇ l, 0.58 mmol) in dimethylformamide (20 mL) for 4 hours at room temperature. The DMF was removed under reduced pressure and the remaining residue was picked up in ethylacetate and washed with 1N sodium bisulfate, saturated sodium bicarbonate and brine. After drying over sodium sulfate, the organic layer was concentrated to give a white solid (100.3 mg, 91 %) which was taken to the next step without further purification. Low resolution mass spectrum confirmed the desired mass (M+Na + 974.5).
  • the product of the previous step (51.5 mg, 0.054 mmol) was dissolved in dimethylsulfoxide (1.2 mL) and toluene (1.2 mL).
  • the product of the previous step (40.4 mg, 0.042 mmol) was treated with a 1:1 mixture of dichloromethane: trifluoroacetic acid (4 mL) for two hours.
  • the reaction mixture was then concentrated and purified on a 1 ⁇ 25 cm reverse phase HPLC column, containing a C-18 resin comprised of 10 micron size gel particles with a 300 angstrom pore size, eluting with a 30 minute gradient using 10-30% acetonitrile in water.
  • the desired fractions were pulled and concentrated to a white powder (8.5 mg, 24%).
  • the product of the previous step (0.12 g, 0.06 mmol) was treated with Boc-Orn-OtBu.AcOH (0.86 g, 2.47 mmol) in the presence of diisopropylethylamine (0.05 mL, 0.31 mmol) and dimethylsulfoxide (1.2 mL) for 18 hours.
  • the reagents were drained and the procedure was repeated for 5 hours using fresh reagents. All reagents were drained and the resin was washed thoroughly with 2 mL portions of dimethylformamide, methanol and dichloromethane.
  • step II The resin obtained in example IV (step I) (0.24 g, 0.12 mmol) was treated with t-butyl N-(2-aminoethyl)-carbamate (0.78 mL, 4.94 mmol) in the presence of diisopropylethylamine (0.11 mL, 0.62 mmol) and dimethylsulfoxide (2.4 mL) for 18 hours. The reagents were drained and the procedure was repeated for 5 hours using fresh reagents. All reagents were drained and the resin was washed thoroughly with 2 mL portions of dimethylformamide, methanol and dichloromethane. Step II. Synthesis of Fmoc-Val-Gly(N-Et(NH-Boc))-nVal(dpsc)Gly-PAM Resin
  • step II above (0.12 g, 0.06 mmol) was treated with a 1:1 mixture of trifluoroacetic acid: dichloromethane according to Procedure D.
  • the free amine generated was then capped with the addition of benzoyl chloride (0.02 mL, 0.19 mmol) in the presence of diisopropylethylamine (0.06 mL, 0.37 mmol) in N-methylpyrrolidine (1.24 mL).
  • step II The product obtained from step II above (19 mg, 0.103 mmol) was coupled to Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-OH (example II, step IIIf) (50 mg, 0.069 mmol) in the presence of HOAt (14.1 mg, 0.103 mmol), HATu (28.8 mg, 0.076 mmol), diisopropylethylamine (60 ⁇ l, 0.345 mmol) in dimethylformamide (10 mL) for 4 hours at room temperature. The DMF was removed under reduced pressure and the remaining residue was picked up in ethylacetate and washed with 1N sodium bisulfate, saturated sodium bicarbonate and brine. After drying over sodium sulfate it was concentrated to give a white solid (40 mg, 68%) which was taken to the next step without further purification. Low resolution mass spectrum confirmed the desired mass (M+Na + 876.5).
  • the product of the previous step (40 mg, 0.047 mmol) was dissolved in dimethylsulfoxide (4 mL) and toluene (4 mL).
  • Water soluble carbodiimide (EDC, 89.8 mg, 0.47 mmol) was then added in one batch.
  • the reaction mixture was cooled to 0° C. and dichloroacetic acid (20 l, 0.23 mmol) was added dropwise. Stirring at 0° C. continued for 15 minutes. The ice bath was removed and the reaction was slowly brought to room temperature. The reaction was stopped after 90 minutes. The toluene was removed under reduced pressure.
  • the product of the previous step (39.9 mg, 0.047 mmol) was treated with a 1:1 mixture of dichloromethane: trifluoroacetic acid (10 mL) for two hours.
  • the reaction mixture was concentrated and the remaining residue was subjected to HPLC purification using a 1 ⁇ 25 cm reverse phase column, containing a C-18 resin comprised of 10 micron size gel particles with a 300 angstrom pore size, eluting with a 30 minute gradient using 5-25% acetonitrile in water.
  • the purified fractions were pulled and lyophilized to a white powder (3.6 mg, 10%). Low resolution mass spectrum confirmed the desired mass (MH + 740.0).
  • step I The resin obtained from example I (step I) (0.70 g, 0.36 mmol) was coupled with Boc-Pro(4t-MeNHFmoc)-OH according to procedure B for 18 hours, with 99.98% efficiency. The resin was then subjected to deprotection according to procedure D to obtain the title product.
  • Step II Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val -Pro(4t-MeNHFmoc)-nVal-(dpsc)-Gly-PAM Resin
  • the Fmoc side chain protecting group of the product obtained from the previous step was removed according to procedure C to afford the title compound.
  • Step IV Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro(4t-MeNHBzl(3-OPh))-nVal-(dpsc)-Gly-PAM Resin
  • the resin obtained from the previous step (0.15 g, 0.05 mmol) was coupled to 3-phenoxy benzoic acid (0.02 g, 0.10 mmol) according to procedure B with 99.97% efficiency.
  • Step V Synthesis of Ac-Glu-Glu-Val-Val-Pro(4t-MeNHBzl(3-OPh))-nVal-(CO)-Gly-OH
  • the resin obtained from the previous step was subjected to semicarbazone hydrolysis according to procedure F, followed by HF cleavage according to procedure H to yield the crude product.
  • the material was subjected to HPLC purification using a 1 ⁇ 25 cm reverse phase column, containing a C-18 resin comprised of 10 micron size gel particles with a 300 angstrom pore size, eluting with a 30 minute gradient ranging from 10-35% acetonitrile in water.
  • step I The resin obtained from example I (step I) (1.3 g, 0.67 mmol) was coupled with Boc-Pro(4t-NHFmoc)-QH (0.61 g, 1.34 mmol) according to procedure B for 18 hours, at which time qualitative ninhydrin test confirmed completion of the reaction. The resin was then subjected to deprotection according to Procedure C to obtain the title product.
  • Step II Synthesis of Boc-Pro(4t-NHBzl)-nVal-(dpsc)-Gly-PAM Resin
  • the resin obtained from the previous step (0.13 g, 0.05 mmol) was transferred to a fritted polypropylene tube and was suspended in N-methylpyrrolidine (1.5 mL). It was then capped with benzoyl chloride (0.02 mL, 0.16 mmol) in the presence of diisopropylethylamine (0.05 mL, 0.32 mmol) for four hours to yield the title product.
  • the compound of the previous step (100 mg, 0.05 mmol) was deprotected according to Procedure D.
  • a ninhydrin assay on a small aliquot gave dark blue resin and solution showing a high yield for the deprotection.
  • the resin was resuspended in N-methylpyrrolidine (1.47 mL) and coupled to Fmoc-Val-OH (0.03 g, 0.10 mmol) according to Procedure B.
  • a small aliquot was taken for qualitative ninhydrin analysis which showed colorless beads and a dark red solution indicating a high yield of coupling.
  • the compound of the previous step (100 mg, 0.03 mmol) was deprotected according to Procedure D.
  • a ninhydrin assay on a small aliquot gave dark blue resin and solution showing a high yield for the deprotection.
  • the resin was resuspended in N-methylpyrrolidine (1.47 mL) and was coupled to Fmoc-Glu(OtBu)-OH (0.04 g, 0.10 mmol), according to Procedure B for 5 hours.
  • a small aliquot was taken for qualitative ninhydrin analysis which showed colorless beads and solution indicating a high yield of coupling.
  • Step VI Synthesis of Fmoc-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro(4t-NHBzl)-nVal-(dpsc)-Gly-PAM Resin
  • the compound of previous step (100 mg) was deprotected according to Procedure C and acylated according to Procedure E.
  • the resin was vacuum dried and a small aliquot was taken for qualitative ninhydrin analysis which showed colorless beads and solution indicating a high yield of coupling.
  • the resin was then subjected to semicarbazone hydrolysis followed by HF cleavage reactions according to Procedures F and H, respectively.
  • the crude product was subjected to HPLC purification using a 1 ⁇ 25 cm reverse phase column, containing a C-18 resin comprised of 10 micron size gel particles with a 300 angstrom pore size, eluting with a 30 minute gradient using 10-40% acetonitrile in water.
  • Step I Synthesis of ethyl (R,S)-2-hydroxy-3-amino hexanoate hydrochloride
  • step Ib The product obtained in example I(step Ib) (1.99 g, 6.59 mmol) was refluxed in 6N HCl (42 mL) for four hours. After cooling to room temperature, the reaction mixture was extracted with dichloromethane (3 ⁇ 30 mL) and the aqueous layer was concentrated to dryness (1.65 g crude). Some of the resulting product (0.88 g, 4.8 mmol) was stirred in 20 mL saturated solution of anhydrous HCl in ethanol for 90 minutes. The mixture was concentrated to a white solid (0.95 g, 93%).
  • Spectrophotometric assay for the HCV serine protease was performed on the inventive compounds by following the procedure described by R. Zhang et al, Analytical Biochemistry, 270 (1999) 268-275, the disclosure of which is incorporated herein by reference.
  • the assay based on the proteolysis of chromogenic ester substrates is suitable for the continuous monitoring of HCV NS3 protease activity.
  • X A or P
  • chromophoric alcohols 3- or 4-nitrophenol, 7-hydroxy4-methyl-coumarin, or 4-phenylazophenol
  • the prewarming block was from USA Scientific (Ocala, Fla.) and the 96-well plate vortexer was from Labline Instruments (Melrose Park, Ill.).
  • a Spectramax Plus microtiter plate reader with monochrometer was obtained from Molecular Devices (Sunnyvale, Calif.).
  • HCV NS3/NS4A protease (strain 1a) was prepared by using the procedures published previously (D. L. Sali et al, Biochemistry, 37 (1998) 3392-3401). Protein concentrations were determined by the Biorad dye method using recombinant HCV protease standards previously quantified by amino acid analysis.
  • the enzyme storage buffer 50 mM sodium phosphate pH 8.0, 300 mM NaCl, 10% glycerol, 0.05% lauryl maltoside and 10 mM DTT
  • the assay buffer 25 mM MOPS pH 6.5, 300 mM NaCl, 10% glycerol, 0.05% lauryl maltoside, 5 ⁇ M EDTA and 5 pM DTT
  • Substrate Synthesis and Purification The synthesis of the substrates was done as reported by R. Zhang et al, (ibid.) and was initiated by anchoring Fmoc-Nva-OH to 2-chlorotrityl chloride resin using a standard protocol (K. Barlos et al, Int. J. Pept. Protein Res., 37 (1991), 513-520). The peptides were subsequently assembled, using Fmoc chemistry, either manually or on an automatic ABI model 431 peptide synthesizer.
  • N-acetylated and fully protected peptide fragments were cleaved from the resin either by 10% acetic acid (HOAc) and 10% trifluoroethanol (TFE) in dichloromethane (DCM) for 30 min, or by 2% trifluoroacetic acid (TFA) in DCM for 10 min.
  • HOAc acetic acid
  • TFE trifluoroethanol
  • TFE trifluoroacetic acid
  • ester substrates were assembled using standard acid-alcohol coupling procedures (K. Holmber et al, Acta Chem. Scand., B33 (1979) 410-412). Peptide fragments were dissolved in anhydrous pyridine (30-60 mg/ml) to which 10 molar equivalents of chromophore and a catalytic amount (0.1 eq.) of para-toluenesulfonic acid (pTSA) were added. Dicyclohexylcarbodiimide (DCC, 3 eq.) was added to initiate the coupling reactions. Product formation was monitored by HPLC and found to be complete following 12-72 hour reaction at room temperature.
  • DCC dicyclohexylcarbodiimide
  • Spectra of Substrates and Products Spectra of substrates and the corresponding chromophore products were obtained in the pH 6.5 assay buffer. Extinction coefficients were determined at the optimal off-peak wavelength in 1-cm cuvettes (340 nm for 3-Np and HMC, 370 nm for PAP and 400 nm for 4-Np) using multiple dilutions. The optimal off-peak wavelength was defined as that wavelength yielding the maximum fractional difference in absorbance between substrate and product (product OD—substrate OD)/substrate OD).
  • HCV protease assays were performed at 30° C. using a 200 ⁇ l reaction mix in a 96-well microtiter plate. Assay buffer conditions (25 mM MOPS pH 6.5, 300 mM NaCl, 10% glycerol, 0.05% lauryl maltoside, 5 ⁇ M EDTA and 5 pM DTT) were optimized for the NS3/NS4A heterodimer (D. L. Sali et al, ibid.)).
  • 150 ⁇ l mixtures of buffer, substrate and inhibitor were placed in wells (final concentration of DMSO 4 % v/v) and allowed to preincubate at 30° C. for approximately 3 minutes.
  • the plates were monitored over the length of the assay (60 minutes) for change in absorbance at the appropriate wavelength (340 nm for 3-Np and HMC, 370 nm for PAP, and 400 nm for 4-Np) using a Spectromax Plus microtiter plate reader equipped with a monochrometer (acceptable results can be obtained with plate readers that utilize cutoff filters).
  • the resulting data were fitted using linear regression and the resulting slope, 1/(K i (1+[S] o /K m ), was used to calculate the K i * value.

Abstract

The present invention discloses novel peptide compounds which have HCV protease inhibitory activity as well as methods for preparing such compounds. In another embodiment, the invention discloses pharmaceutical compositions comprising such peptides as well as methods of using them to treat disorders associated with the HCV protease.

Description

    FIELD OF THE INVENTION
  • The present invention relates to novel hepatitis C virus (“HGV”) protease inhibitors, pharmaceutical compositions containing one or more such inhibitors, methods of preparing such inhibitors and methods of using such inhibitors to treat hepatitis C and related disorders. This invention specifically discloses novel peptide compounds as inhibitors of the HCV NS3/NS4a serine protease.
    • BACKGROUND OF THE INVENTION
  • Hepatitis C virus (HCV) is a (+)-sense single-stranded RNA virus that has been implicated as the major causative agent in non-A, non-B hepatitis (NANBH), particularly in blood-associated NANBH (BB-NANBH)(see, International Patent Application Publication No. WO 89/04669 and European Patent Application Publication No. EP 381 216). NANBH is to be distinguished from other types of viral-induced liver disease, such as hepatitis A virus (HAV), hepatitis B virus (HBV), delta hepatitis virus (HDV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV), as well as from other forms of liver disease such as alcoholism and primary biliar cirrhosis.
  • Recently, an HCV protease necessary for polypeptide processing and viral replication has been identified, cloned and expressed; (see, e.g., U.S. Pat. No. 5,712,145). This approximately 3000 amino acid polyprotein contains, from the amino terminus to the carboxy terminus, a nucleocapsid protein (C), envelope proteins (E1 and E2) and several non-structural proteins (NS1, 2, 3, 4a, 5a and 5b). NS3 is an approximately 68 kda protein, encoded by approximately 1893 nucleotides of the HCV genome, and has two distinct domains: (a) a serine protease domain consisting of approximately 200 of the N-terminal amino acids; and (b) an RNA-dependent ATPase domain at the C-terminus of the protein. The NS3 protease is considered a member of the chymotrypsin family because of similarities in protein sequence, overall three-dimensional structure and mechanism of catalysis. Other chymotrypsin-like enzymes are elastase, factor Xa, thrombin, trypsin, plasmin, urokinase, tPA and PSA. The HCV NS3 serine protease is responsible for proteolysis of the polypeptide (polyprotein) at the NS3/NS4a, NS4a/NS4b, NS4b/NS5a and NS5a/NS5b junctions and is thus responsible for generating four viral proteins during viral replication. This has made the HCV NS3 serine protease an attractive target for antiviral chemotherapy.
  • It has been determined that the NS4a protein, an approximately 6 kda polypeptide, is a co-factor for the serine protease activity of NS3. Autocleavage of the NS3/NS4a junction by the NS3/NS4a serine protease occurs intramolecularly (i.e., cis) while the other cleavage sites are processed intermolecularly (i.e., trans).
  • Analysis of the natural cleavage sites for HCV protease revealed the presence of cysteine at P1 and serine at P1′ and that these residues are strictly conserved in the NS4a/NS4b, NS4b/NS5a and NS5a/NS5b junctions. The NS3/NS4a junction contains a threonine at P1 and a serine at P1′. The Cys→Thr substitution at NS3/NS4a is postulated to account for the requirement of cis rather than trans processing at this junction. See, e.g., Pizzi et al. (1994) Proc. Natl. Acad. Sci (USA) 91:888-892, Failla et al. (1996) Folding & Design 1:35-42. The NS3/NS4a cleavage site is also more tolerant of mutagenesis than the other sites. See, e.g., Kollykhalov et al. (1994) J. Virol. 68:7525-7533. It has also been found that acidic residues in the region upstream of the cleavage site are required for efficient cleavage. See, e.g., Komoda et al. (1994) J. Virol. 68:7351-7357.
  • Inhibitors of HCV protease that have been reported include antioxidants (see, International Patent Application Publication No. WO 98/14181), certain peptides and peptide analogs (see, International Patent Application Publication No. WO 98/17679, Landro et al. (1997) Biochem. 36:9340-9348, ingallinella et al. (1998) Biochem. 37:8906-8914, Llinàs-Brunet et al. (1998) Bioorg. Med. Chem. Lett. 8:1713.-1718), inhibitors based on the 70-amino acid polypeptide eglin c (Martin et al. (1998) Biochem. 37:11459-11468, inhibitors affinity selected from human pancreatic secretory trypsin inhibitor (hPSTI-C3) and minibody repertoires (MBip) (Dimasi et al. (1997) J. Virol. 71:7461-7469), cVHE2 (a “camelized” variable domain antibody fragment) (Martin et al.(1997) Protein Eng. 10:607-614), and α1-antichymotrypsin (ACT) (Elzouki et al.) (1997) J. Hepat. 27:42-28). A ribozyme designed to selectively destroy hepatitis C virus RNA has recently been disclosed (see, BioWorld Today 9(217): 4 (Nov. 10, 1998)).
  • Reference is also made to the PCT Publications, No. WO 98/17679, published Apr. 30, 1998 (Vertex Pharmaceuticals Incorporated); WO 98/22496, published May 28, 1998 (F. Hoffmann-La Roche AG); and WO 99/07734, published Feb. 18, 1999 (Boehringer Ingelheim Canada Ltd.).
  • HCV has been implicated in cirrhosis of the liver and in induction of hepatocellular carcinoma. The prognosis for patients suffering from HCV infection is currently poor. HCV infection is more difficult to treat than other forms of hepatitis due to the lack of immunity or remission associated with HCV infection. Current data indicates a less than 50% survival rate at four years post cirrhosis diagnosis. Patients diagnosed with localized resectable hepatocellular carcinoma have a five-year survival rate of 10-30%, whereas those with localized unresectable hepatocellular carcinoma have a five-year survival rate of less than 1%.
  • Reference is made to A. Marchetti et al, Synlett, S1, 1000-1002 (1999) describing the synthesis of bicylic analogs of an inhibitor of HCV NS3 protease. A compound disclosed therein has the formula:
    Figure US20050059606A1-20050317-C00001
  • Reference is also made to WO 00/09558 (Assignee: Boehringer Ingelheim Limited; Published Feb. 24, 2000) which discloses peptide derivatives of the formula:
    Figure US20050059606A1-20050317-C00002
    where the various elements are defined therein. An illustrative compound of that series is:
    Figure US20050059606A1-20050317-C00003
  • Reference is also made to WO 00/09543 (Assignee: Boehringer Ingelheim Limited; Published Feb. 24, 2000) which discloses peptide derivatives of the formula:
    Figure US20050059606A1-20050317-C00004
    where the various elements are defined therein. An illustrative compound of that series is:
    Figure US20050059606A1-20050317-C00005
  • Current therapies for hepatitis C include interferon-α (INFα) and combination therapy with ribavirin and interferon. See, e.g., Beremguer et al. (1998) Proc. Assoc. Am. Physicians 110(2):98-112. These therapies suffer from a low sustained response rate and frequent side effects. See, eg. Hoofnagle et al. (1997) N. Engl. J. Med. 336:347. Currently, no vaccine is available for HCV infection.
  • Pending and copending U.S. patent applications, Ser. No. 60/220,110, filed Jul. 21, 2000, Ser. No. 60/220,107, filed Jul. 21, 2000, Ser. No. 60/220,108, filed Jul. 21, 2000, Ser. No. 60/220,101, filed Jul. 21, 2000, Ser. No. 60/254,869, filed Dec. 12, 2000, Ser. No. 60/194,607, filed Apr. 5, 2000, and Ser. No. 60/198,204, filed Apr. 19, 2000 disclose various types of peptides as NS-3 serine protease inhibitors of hepatitis C virus.
  • There is a need for new treatments and therapies for HCV infection. It is, therefore, an object of this invention to provide compounds useful in the treatment or prevention or amelioration of one or more symptoms of hepatitis C.
  • It is a further object herein to provide methods of treatment or prevention or amelioration of one or more symptoms of hepatitis C.
  • A still further object of the present invention is to provide methods for modulating the activity of serine proteases, particularly the HCV NS3/NS4a serine protease, using the compounds provided herein.
  • Another object herein is to provide methods of modulating the processing of the HCV polypeptide using the compounds provided herein.
    • SUMMARY OF THE INVENTION
  • In its many embodiments, the present invention provides a novel class of inhibitors of the HCV protease, pharmaceutical compositions containing one or more of the compounds, methods of preparing pharmaceutical formulations comprising one or more such compounds, and methods of treatment, prevention or amelioration or one or more of the symptoms of hepatitis C. Also provided are methods of modulating the interaction of an HCV polypeptide with HCV protease. Among the compounds provided herein, compounds that inhibit HCV NS3/NS4a serine protease activity are preferred. The presently disclosed compounds generally contain about four or more amino acid residues and less than about twelve amino acid residues. Specifically, the present application discloses peptide compounds, defined further below.
  • In its first embodiment, the present invention provides a compound of Formula I:
    Figure US20050059606A1-20050317-C00006
    or a pharmaceutically acceptable salt, solvate or derivative thereof, wherein:
      • Z is O, NH or NR12;
      • X is alkylsulfonyl, heterocyclylsulfonyl, heterocyclylalkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylcarbonyl, heterocyclylcarbonyl, heterocyclylalkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, heterocyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkyaminocarbonyl, heterocyclylaminocarbonyl, arylaminocarbonyl, or heteroarylaminocarbonyl moiety, with the proviso that X may be additionally optionally substituted with R12 or R13;
      • X1 is H; C1-C4 straight chain alkyl; C1-C4 branched alkyl or; CH2-aryl (substituted or unsubstituted);
      • R12 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocyclyl, heterocyclylalkyl, aryl, alkylaryl, arylalkyl, heteroaryl, alkylheteroaryl, or heteroarylalkyl moiety, with the proviso that R12 may be additionally optionally substituted with R13.
      • R13 is hydroxy, alkoxy, aryloxy, thio, alkylthio, arylthio, amino, alkylamino, arylamino, alkylsulfonyl, arylsulfonyl, alkylsulfonamido, arylsulfonamido, carboxy, carbalkoxy, carboxamido, alkoxycarbonylamino, alkoxycarbonyloxy, alkylureido, arylureido, halogen, cyano, or nitro moiety, with the proviso that the alkyl, alkoxy, and aryl may be additionally optionally substituted with moieties independently selected from R13.
      • P1a, P1b, P2, P3, P4, P5, and P6 are independently:
        • H; C1-C10 straight or branched chain alkyl; C2-C10 straight or branched chain alkenyl;
        • C3-C8 cycloalkyl, C3-C8 heterocyclic; (cycloalkyl)alkyl or (heterocyclyl)alkyl, wherein said cycloalkyl is made up of 3 to 8 carbon atoms, and zero to 6 oxygen, nitrogen, sulfur, or phosphorus atoms, and said alkyl is of 1 to 6 carbon atoms;
        • aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein said alkyl is of 1 to 6 carbon atoms;
        • wherein all aforesaid alkyl, alkenyl, cycloalkyl, heterocyclyl; (cycloalkyl)alkyl and (heterocyclyl)alkyl moieties may be optionally substituted with R13. Additionally, the atoms of P1a and P1b may be joined to each other in such a fashion to form a spirocyclic or spiroheterocyclic ring, with said spirocyclic or spiroheterocyclic ring containing zero to 6 oxygen, nitrogen, sulfur, or phosphorus atoms, and may be optionally substituted with R13.
      • P1′ is H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocyclyl, heterocyclyl-alkyl, aryl, aryl-alkyl, heteroaryl, or heteroaryl-alkyl; with the proviso that said P1′ may be additionally optionally substituted with R13.
        Among the above-stated definitions for X, R12, R13, P1a, P1b, P2, P3, P4, P5, and P1′, the preferred groups for the various moieties are as follows: Preferred moieties for X are:
        Figure US20050059606A1-20050317-C00007
        wherein Alkyl is C1 to C4 straight or branched chain, and wherein Aryl is phenyl or substituted phenyl.
        Preferred moieties for P6 are:
        Figure US20050059606A1-20050317-C00008
        with n being 1-4.
        Preferred moieties for P5 are:
        Figure US20050059606A1-20050317-C00009
        with n being 1-4; additionally, P6 and P5 may be the same or different. Preferred moieties for R1 are OH, O-t-Bu, OR3, NHR3, NH-phenyl or NH-trityl.
        Preferred moieties for R3 are H, Cl to C4 straight or branched chain alkyl. P3 and P4 may be the same or different and preferred moieties for P3 and P4 are:
        Figure US20050059606A1-20050317-C00010
        Preferred moieties for P2 are:
        Figure US20050059606A1-20050317-C00011
        wherein n=0, 1, 2 or 3;
        Preferred moieties for P1a and P1b are:
        Figure US20050059606A1-20050317-C00012
        Preferred moieties for P1′ are:
        Figure US20050059606A1-20050317-C00013
  • In another embodiment, the present invention provides a compound of Formula II:
    Figure US20050059606A1-20050317-C00014
    wherein X, P6, P5, P4, P3, P2, P1a, P1b and P1′ are as defined above. Z is O, NH or NR12 where R12 has been defined before. In Formula II, P2, when connected with the N atom adjacent to C atom it is attached to, forms a cyclic ring, with the proviso that said cyclic ring does not contain a carbonyl group as part of the cyclic ring structure. The cyclic ring moiety comprised of P2, the carbon atom to which P2 is attached, and the nitrogen atom adjacent to that carbon atom is noted above as:
    Figure US20050059606A1-20050317-C00015
    which may denote a five-membered ring or a six-membered ring structure. Preferred representatives for that cyclic ring structure are selected from the following:
    Figure US20050059606A1-20050317-C00016
    wherein:
      • R2 and R3 may be the same or different and are selected from H; C1-C6 straight chain alkyl; C1-C6 branched alkyl or cycloalkyl;
      • R4is CO-Alkyl (alkyl being C1-C6 straight chain or branched or cycloalkyl); CO-aryl; COO-alkyl or COO-aryl;
      • R5 and R6 may be the same or different and are selected from H; C1-C3 straight chain alkyl; or C1-C3 branched alkyl;
      • R7 and R8 may be the same or different and are selected from H; C1-C3 straight chain alkyl; C1-C3 branched alkyl or CH2OH;
      • R9 and R10 may be the same or different and are selected from H; C1-C3 straight chain alkyl; C1-C3 branched alkyl; COOMe; COOH or CH2OH;
      • R11 is C1-C6 straight chain alkyl; C1-C6 branched alkyl; cyclocalkyl; or CH2-aryl (substituted or unsubstituted);
      • Z1 and Z2 may be the same or different and are selected from S; O; or CH2;
      • Z3 is CH2; S, SO2; NH or NR4;
      • Z4 and Z5 may be the same or different and are selected from S, O or CH2.
  • In yet another embodiment, the present invention discloses compounds of Formula III:
    Figure US20050059606A1-20050317-C00017
    where the various elements are as defined above.
  • Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. Thus, for example, the term alkyl (including the alkyl portions of alkoxy) refers to a monovalent group derived from a straight or branched chain saturated hydrocarbon by the removal of a single atom having from 1 to 8 carbon atoms, preferably from 1 to 6;
      • aryl—represents a carbocyclic group having from 6 to 14 carbon atoms and having at least one benzenoid ring, with all available substitutable aromatic carbon atoms of the carbocyclic group being intended as possible points of attachment. Preferred aryl groups include phenyl, 1-naphthyl, 2-naphthyl and indanyl, and especially phenyl and substituted phenyl;
      • aralkyl—represents a moiety containing an aryl group linked vial a lower alkyl;
      • alkylaryl—represents a moiety containing a lower alkyl linked via an aryl group;
      • cycloalkyl—represents a saturated carbocyclic ring having from 3 to 8 carbon atoms, preferably 5 or 6, optionally substituted.
      • heterocyclic—represents, in addition to the heteroaryl groups defined below, saturated and unsaturated cyclic organic groups having at least one O, S and/or N atom interrupting a carbocyclic ring structure that consists of one ring or two fused rings, wherein each ring is 5-, 6- or 7-membered and may or may not have double bonds that lack delocalized pi electrons, which ring structure has from 2 to 8, preferably from 3 to 6 carbon atoms, e.g., 2- or 3-piperidinyl, 2- or 3-piperazinyl, 2- or 3-morpholinyl, or 2- or 3-thiomorpholinyl;
      • halogen—represents fluorine, chlorine, bromine and iodine;
      • heteroaryl—represents a cyclic organic group having at least one O, S and/or N atom interrupting a carbocyclic ring structure and having a sufficient number of delocalized pi electrons to provide aromatic character, with the aromatic heterocyclyl group having from 2 to 14, preferably 4 or 5 carbon atoms, e.g., 2-, 3- or 4-pyridyl, 2- or 3-furyl, 2- or 3-thienyl, 2-, 4- or 5-thiazolyl, 2- or 4-imidazolyl, 2-, 4- or 5-pyrimidinyl, 2-pyrazinyl, or 3- or 4-pyridazinyl, etc. Preferred heteroaryl groups are 2-, 3- and 4-pyridyl; such heteroaryl groups may also be optionally substituted.
  • Also included in the invention are tautomers, rotamers, enantiomers and other optical isomers of compounds of Formula I, Formula II and Formula II, as well as pharmaceutically acceptable salts, solvates and derivatives thereof.
  • A further feature of the invention is pharmaceutical compositions containing as active ingredient a compound of Formula I, Formula II or Formula III (or its salt, solvate or isomers) together with a pharmaceutically acceptable carrier or excipient.
  • The invention also provides methods for preparing compounds of Formulas I, II and III, as well as methods for treating diseases such as, for example, HCV and related disorders. The methods for treating comprise administering to a patient suffering from said disease or diseases a therapeutically effective amount of a compound of Formula I, II or III, or pharmaceutical compositions comprising a compound of Formula I, II or III.
  • Also disclosed is the use of a compound of Formulas I, II or III for the manufacture of a medicament for treating HCV and related disorders.
    • DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
  • In one embodiment, the present invention discloses compounds of Formula I as inhibitors of HCV protease, especially the HCV NS3/NS4a serine protease, or a pharmaceutically acceptable derivative thereof, where the various definitions are given above.
  • In another embodiment, the present invention discloses compounds of Formula II as inhibitors of HCV protease, especially the HCV NS3/NS4a serine protease, or a pharmaceutically acceptable derivative thereof, where the various definitions are given above.
  • In another embodiment, the present invention discloses compounds of Formula III as inhibitors of HCV protease, especially the HCV NS3/NS4a serine protease, or a pharmaceutically acceptable derivative thereof, where the various definitions are given above:
  • Representative compounds of the invention which exhibit excellent HCV protease inhibitory activity are listed below in Table 1 along with their activity (ranges of Ki* values in nanomolar, nM).
    TABLE 1
    Compounds and HCV protease continuous assay results
    Compound from Ki*
    Example No. Range
    1 b
    2 b
    3 c
    4 b
    5 b
    6 c
    7 c
    8 b
    9 b
    10 c
    11 c
    12 b
    13 c
    14 b
    15 b
    16 c
    17 c
    18 c
    19 c
    20 c
    21 c
    22 c
    23 c
    24 c
    25 a
    26 b
    27 a
    28 c
    29 c
    30 c
    31 b
    32 b
    33 b
    34 c
    35 c
    36 a
    37 b
    38 a
    39 a
    40 a
    41 c
    42 a
    43 a
    44 a
    45 a
    46 c
    47 c
    48 b
    49 a
    50 c
    51 b
    52 b
    53 b
    54 b
    55 a
    56 b
    57 b
    58 c
    59 b
    60 b
    61 a
    62 a
    63 a
    64 b
    65 b
    66 b
    67 b
    68 c
    69 c
    70 c
    71 b
    72 b
    73 c
    74 c
    75 c
    76 c
    77 c
    78 b
    79 a
    80 a
    81 a
    82 a
    83 a
    84 a
    85 a
    86 a
    87 a
    88 b

    HCV continous assay Ki* range:
  • Category a=1-100 nM; Category b=101-999 nM; Category c=1000-10,000.
  • Some of the types of the inventive compounds and methods of synthesizing the various types of the inventive compounds of both Formula I and Formula II are listed below, then schematically described, followed by the illustrative Examples.
    Figure US20050059606A1-20050317-C00018
    Figure US20050059606A1-20050317-C00019
    Figure US20050059606A1-20050317-C00020
    Figure US20050059606A1-20050317-C00021
  • Depending upon their structure, the compounds of the invention may form pharmaceutically acceptable salts with organic or inorganic acids, or organic or inorganic bases. Examples of suitable acids for such salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, malonic, salicylic, malic, fumaric, succinic, ascorbic, maleic, methanesulfonic and other mineral and carboxylic acids well known to those skilled in the art. For formation of salts with bases, suitable bases are, for example, NaOH, KOH, NH4OH, tetraalkylammonium hydroxide, and the like.
  • In another embodiment, this invention provides pharmaceutical compositions comprising the inventive peptides as an active ingredient. The pharmaceutical compositions generally additionally comprise a pharmaceutically acceptable carrier diluent, excipient or carrier (collectively referred to herein as carrier materials). Because of their HCV inhibitory activity, such pharmaceutical compositions possess utility in treating hepatitis C and related disorders.
  • In yet another embodiment, the present invention discloses methods for preparing pharmaceutical compositions comprising the inventive compounds as an active ingredient. In the pharmaceutical compositions and methods of the present invention, the active ingredients will typically be administered in admixture with suitable carrier materials suitably selected with respect to the intended form of administration, i.e. oral tablets, capsules (either solid-filled, semi-solid filled or liquid filled), powders for constitution, oral gels, elixirs, dispersible granules, syrups, suspensions, and the like, and consistent with conventional pharmaceutical practices. For example, for oral administration in the form of tablets or capsules, the active drug component may be combined with any oral non-toxic pharmaceutically acceptable inert carrier, such as lactose, starch, sucrose, cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, talc, mannitol, ethyl alcohol (liquid forms) and the like. Moreover, when desired or lo needed, suitable binders, lubricants, disintegrating agents and coloring agents may also be incorporated in the mixture. Powders and tablets may be comprised of from about 5 to about 95 percent inventive composition. Suitable binders include starch, gelatin, natural sugars, corn sweeteners, natural and synthetic gums such as acacia, sodium alginate, carboxymethylcellulose, polyethylene is glycol and waxes. Among the lubricants there may be mentioned for use in these dosage forms, boric acid, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include starch, methylcellulose, guar gum and the like.
  • Sweetening and flavoring agents and preservatives may also be included where appropriate. Some of the terms noted above, namely disintegrants, diluents, lubricants, binders and the like, are discussed in more detail below.
  • Additionally, the compositions of the present invention may be formulated in sustained release form to provide the rate controlled release of any one or more of the components or active ingredients to optimize the therapeutic effects, i.e.
  • HCV inhibitory activity and the like. Suitable dosage forms for sustained release include layered tablets containing layers of varying disintegration rates or controlled release polymeric matrices impregnated with the active components and shaped in tablet form or capsules containing such impregnated or encapsulated porous polymeric matrices.
  • Liquid form preparations include solutions, suspensions and emulsions. As an example may be mentioned water or water-propylene glycol solutions for parenteral injections or addition of sweeteners and pacifiers for oral solutions, suspensions and emulsions. Liquid form preparations may also include solutions for intranasal administration.
  • Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier such as inert compressed gas, e.g. nitrogen.
  • For preparing suppositories, a low melting wax such as a mixture of fatty acid glycerides such as cocoa butter is first melted, and the active ingredient is dispersed homogeneously therein by stirring or similar mixing. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool and thereby solidify.
  • Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions and emulsions.
  • The compounds of the invention may also be deliverable transdermally. The transdermal compositions may take the form of creams, lotions, aerosols and/or emulsions and can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.
  • Preferably the compound is administered orally, intravenously or subcutaneously.
  • Preferably, the pharmaceutical preparation is in a unit dosage form. In such form, the preparation is subdivided into suitably sized unit doses containing appropriate quantities of the active components, e.g., an effective amount to achieve the desired purpose.
  • The quantity of the inventive active composition in a unit dose of preparation may be generally varied or adjusted from about 1.0 milligram to about 1,000 milligrams, preferably from about 1.0 to about 950 milligrams, more preferably from about 1.0 to about 500 milligrams, and typically from about 1 to about 250 milligrams, according to the particular application. The actual dosage employed may be varied depending upon the patient's age, sex, weight and severity of the condition being treated. Such techniques are well known to those skilled in the art.
  • Generally, the human oral dosage form containing the active ingredients can be administered 1 or 2 times per day. The amount and frequency of the administration will be regulated according to the judgment of the attending clinician. A generally recommended daily dosage regimen for oral administration may range from about 1.0 milligram to about 1,000 milligrams per day, in single or divided doses.
  • Some useful terms are described below:
  • Capsule—refers to a special container or enclosure made of methyl cellulose, polyvinyl alcohols, or denatured gelatins or starch for holding or containing compositions comprising the active ingredients. Hard shell capsules are typically made of blends of relatively high gel strength bone and pork skin gelatins. The capsule itself may contain small amounts of dyes, opaquing agents, plasticizers and preservatives.
  • Tablet—refers to a compressed or molded solid dosage form containing the active ingredients with suitable diluents. The tablet can be prepared by compression of mixtures or granulations obtained by wet granulation, dry granulation or by compaction.
  • Oral gel—refers to the active ingredients dispersed or solubilized in a hydrophillic semi-solid matrix.
  • Powder for constitution refers to powder blends containing the active ingredients and suitable diluents which can be suspended in water or juices.
  • Diluent—refers to substances that usually make up the major portion of the composition or dosage form. Suitable diluents include sugars such as lactose, sucrose, mannitol and sorbitol; starches derived from wheat, corn, rice and potato; and celluloses such as microcrystalline cellulose. The amount of diluent in the composition can range from about 10 to about 90% by weight of the total composition, preferably from about 25 to about 75%, more preferably from about 30 to about 60% by weight, even more preferably from about 12 to about 60%.
  • Disintegrant—refers to materials added to the composition to help it break apart (disintegrate) and release the medicaments. Suitable disintegrants include starches; “cold water soluble” modified starches such as sodium carboxymethyl starch; natural and synthetic gums such as locust bean, karaya, guar, tragacanth and agar; cellulose derivatives such as methylcellulose and sodium carboxymethylcellulose; microcrystalline celluloses and cross-linked microcrystalline celluloses such as sodium croscarmellose; alginates such as alginic acid and sodium alginate; clays such as bentonites; and effervescent mixtures. The amount of disintegrant in the composition can range from about 2 to about 15% by weight of the composition, more preferably from about 4 to about 10% by weight.
  • Binder—refers to substances that bind or “glue” powders together and make them cohesive by forming granules, thus serving as the “adhesive” in the formulation. Binders add cohesive strength already available in the diluent or bulking agent. Suitable binders include sugars such as sucrose; starches derived from wheat, corn rice and potato; natural gums such as acacia, gelatin and tragacanth; derivatives of seaweed such as alginic acid, sodium alginate and ammonium calcium alginate; cellulosic materials such as methylcellulose and sodium carboxymethylcellulose and hydroxypropylmethylcellulose; polyvinylpyrrolidone; and inorganics such as magnesium aluminum silicate. The amount of binder in the composition can range from about 2 to about 20% by weight of the composition, more preferably from about 3 to about 10% by weight, even more preferably from about 3 to about 6% by weight.
  • Lubricant—refers to a substance added to the dosage form to enable the tablet, granules, etc. after it has been compressed, to release from the mold or die by reducing friction or wear. Suitable lubricants include metallic stearates such as magnesium stearate, calcium stearate or potassium stearate; stearic acid; high melting point waxes; and water soluble lubricants such as sodium chloride, sodium benzoate, sodium acetate, sodium oleate, polyethylene glycols and d'l-leucine. Lubricants are usually added at the very last step before compression, since they must be present on the surfaces of the granules and in between them and the parts of the tablet press. The amount of lubricant in the composition can range from about 0.2 to about 5% by weight of the composition, preferably from about 0.5 to about 2%, more preferably from about 0.3 to about 1.5% by weight.
  • Glident—material that prevents caking and improve the flow characteristics of granulations, so that flow is smooth and uniform. Suitable glidents include silicon dioxide and talc. The amount of glident in the composition can range from about 0.1% to about 5% by weight of the total composition, preferably from about 0.5 to about 2% by weight.
  • Coloring agents—excipients that provide coloration to the composition or the dosage form. Such excipients can include food grade dyes and food grade dyes adsorbed onto a suitable adsorbent such as clay or aluminum oxide. The amount of the coloring agent can vary from about 0.1 to about 5% by weight of the composition, preferably from about 0.1 to about 1%.
  • Bioavailability—refers to the rate and extent to which the active drug ingredient or therapeutic moiety is absorbed into the systemic circulation from an administered dosage form as compared to a standard or control.
  • Conventional methods for preparing tablets are known. Such methods include dry methods such as direct compression and compression of granulation produced by compaction, or wet methods or other special procedures. Conventional methods for making other forms for administration such as, for example, capsules, suppositories and the like are also well known.
  • Another embodiment of the invention discloses the use of the pharmaceutical compositions disclosed above for treatment of diseases such as, for example, hepatitis C and the like. The method comprises administering a therapeutically effective amount of the inventive pharmaceutical composition to a patient having such a disease or diseases and in need of such a treatment.
  • In yet another embodiment, the compounds of the invention may be used for the treatment of HCV in humans in monotherapy mode or in a combination therapy mode such as, for example, in combination with antiviral agents such as, for example, ribavirin and/or interferon such as, for example, α-interferon and the like.
  • As stated earlier, the invention includes tautomers, rotamers, enantiomers and other stereoisomers of the compounds also. Thus, as one skilled in the art appreciates, some of the inventive compounds may exist in suitable isomeric forms. Such variations are contemplated to be within the scope of the invention.
  • Another embodiment of the invention discloses a method of making the compounds disclosed herein. The compounds may be prepared by several techniques known in the art. Representative illustrative procedures are outlined in the following reaction schemes. It is to be understood that while the following illustrative schemes describe the preparation of a few representative inventive compounds, suitable substitution of any of both the natural and unnatural amino acids will result in the formation of the desired compounds based on such substitution. Such variations are contemplated to be within the scope of the invention.
  • Abbreviations which are used in the descriptions of the schemes, preparations and the examples that follow are:
      • THF: Tetrahydrofuran
      • DMF: N,N-Dimethylformamide
      • EtOAc: Ethyl acetate
      • AcOH: Acetic acid
      • HOOBt: 3-Hydroxy-1,2,3-benzotriazin-4(3H)-one
      • EDCI:1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
      • NMM: N-Methylmorpholine
      • ADDP: 1,1′-(Azodicarbobyl)dipiperidine
      • DEAD: Diethylazodicarboxylate
      • DCC: Dicyclohexylcarbodiimide
      • HOBt: Hydroxybezotriazole
      • HATU: O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
      • hexafluorophosphate
      • TEMPO: 2,2,6,6-tetramethyl-1-piperidinyloxy free radical
      • TBTU: 2-(1H-Benzotriazole-1-yl)-1,1,3,3,-tetramethyluronium tetrafluoroborate
      • PAM: 4-Hydroxymethylphenylacetamidomethyl
      • DTT: Dithiothreitol
      • Hünigs base (DIPEA or DIEA): Diisopropylethyl amine
      • DCM: Dichloromethane
      • MeOH: Methanol
      • EtOH: Ethanol
      • Et2O: Diethyl ether
      • PyBrOP: Bromo-tris-pyrrolidinophosphonium hexafluorophosphate
      • Trt: Trityl
      • PMB: Para-methoxybenzyl
      • Bzl=Bn: Benzyl
      • Boc: tert-Butyloxycarbonyl
      • Cbz: Benzyloxycarbonyl
      • Cp: Cylcopentyldienyl
      • Ts: p-toluenesulfonyl
      • Me: Methyl
        General Preparative Schemes:
  • The following schemes describe the methods of synthesis of intermediate building blocks:
    Figure US20050059606A1-20050317-C00022
    Figure US20050059606A1-20050317-C00023
    Figure US20050059606A1-20050317-C00024
    Figure US20050059606A1-20050317-C00025
    Figure US20050059606A1-20050317-C00026
    Preparation of Intermediates:
  • The procedures to modify an amino acid with N-Boc, N-Cbz, COOBzl, COOBut, Obzl, Obut, COOMe, both putting them on or taking them off in the presence of each other in various combinations, are generally well known to those skilled in the art. Any modifications from the known procedures are, noted herein.
  • Commercially Available Intermediates:
  • The following amino acids, used as P2 units in the preparation of the various inventive compounds, are commercially available, and were converted to their N-Boc derivatives with di-tert-butyldicarbonate, using known procedures.
    Figure US20050059606A1-20050317-C00027
    The following N-Boc-amino acids, used as P2 units, are commercially available.
    Figure US20050059606A1-20050317-C00028
    The following N-Boc-amino acid, used as P2 unit, is commercially available. After coupling the carboxylic acid, the Fmoc is removed by known treatment with piperidine before subsequent coupling.
    Figure US20050059606A1-20050317-C00029
    Certain intermediates which were not commercially available were synthesized, as needed, by following the procedures given below:
    Figure US20050059606A1-20050317-C00030
    A. Mesylate:
  • A mixture of triphenylphosphine (8.7 g), toluene (200 mL), and methanesulfonic acid (2.07 mL) was stirred at 15° C. while slowly adding diethylazidodicarboxylate (7.18 g) to maintain the temperature below 35° C. The mixture was cooled to 20° C., and the N-Boc amino acid (7.4 g, Bachem Biosciences, Inc.), and Et3N (1.45 mL) were added, and then the mixture was stirred at 70° C. for 5 hr. The mixture was cooled to 5° C., the organic supernate decanted, and solvent was removed from it in vacuo. The residue was stirred with Et2O (200 mL) until a precipitate deposits, the mixture was filtered, and the ethereal solution was chromatographed on silica gel (5:95 to 20:80 EtOAc-Et2O) to obtain the product (9.3 g), which was carried into the next step.
  • B. Azide
  • Sodium azide (1.98 g) was added to a solution of the product of the step above (9.3 g) in DMF (100 mL), and the mixture stirred at 70° C. for 8 hr. The mixture was cooled, and poured into 5% aqueous NaHCO3, and extracted with EtOAc. The organic layer was washed with brine, then dried over anhydrous MgSO4. The mixture was filtered, and the filtrate evaporated in vacuo, to obtain the product (6.2 g), which was carried into the next step.
  • C. N-Cbz(4-N-Boc)-PrO—OMe
  • A solution of the product of the step above (0.6 g) in dioxane (40 mL) was treated with di-tert-butyldicarbonate (0.8 g), 10% Pd-C (0.03 g), and hydrogen at one atmosphere for 18 hr. The mixture was filtered, the filtrate evaporated in vacuo, and the residue chromatographed on silica gel (1:1 to 2:1 Et2O-hexane) to obtain the product.
    D. N-Cbz(4-N-Boc)-PrO-OH was prepared using known ester hydrolysis using LiOH.
    Figure US20050059606A1-20050317-C00031
  • These were prepared by following the procedure of U. Larsson, et al., Acta Chem. Scan., (1994), 48(6), 517-525. A solution of oxone® (20.2 g, from Aldrich Chemical Co.) in water (110 mL) was added slowly to a 0° C. solution of the sulfide (7.2 g, from Bachem Biosciences, Inc.) in MeOH (100 mL). The cold bath was removed and the mixture stirred for 4 hr. The mixture was concentrated to ½ volume on a rotary evaporator, cold water (100 mL) added, extracted with EtOAc, the extract washed with brine, and then it was dried over anhydrous MgSO4. The mixture was filtered, and the filtrate evaporated in vacuo, to obtain the product as a white solid (7.7 g). A portion was crystallized from (i-Pr)2O to obtain an analytical sample, [α]D+8.6 (c 0.8, CHCl3). Using the same procedure, the other sulfides shown were oxidized to sulfones to lead to the subject targets.
    • PREPARATIVE EXAMPLE 1 Preparation of Ac-Glu(OBut)Glu(OBut)-Val-Val-OH(1.6) (Scheme 1)
  • Step A Compound (1.1).
  • To a mixture of Ac-Glu(OBzl)-OH (2.0 g), H-Glu(OBzl)-OMe.HCl (2.0 g), HOOBt (1.35 g), N-methylmorpholine (0.87 mL), and dimethylformamide (70 mL) at −20° C. was added EDCl (2.50 g) and stirred for 48 hr. The reaction mixture was poured into 5% aqueous KH2PO4 (500 mL) and extracted with ethyl acetate (2×150 mL). The combined organic layer was washed with cold 5% aqueous K2CO3, then 5% aqueous KH2PO4, then brine, and dried over anhydrous MgSO4. The mixture was filtered and evaporated under vacuum. The residue was triturated with hexane (200 mL), and filtered to leave the title compound (3.4 g, 96% yield), TLC (EtOAc) Rf=0.7.
  • Step B Compound (1.2)
  • A solution of Compound (1.1) from Step A (3.4 g), MeOH (125 mL), and 1 M aqueous LiOH (7.25 mL) was stirred at room temperature for 18 hr. The mixture was concentrated, treated with cold 0.5 N HCl (250 mL), and extracted with ethyl acetate (2×150 mL). The combined organic layer was washed with cold water, then brine. The mixture was evaporated under vacuum, triturated with Et2O-hexane, and filtered to leave the title compound (2.8 g, 85% yield).
  • Step C Compound (1.3)
  • In essentially the same manner as in Step A, but substituting proportional amounts of compound (1.2) the product of Step B for Ac-Glu(OBzl)-OH, and H-Val-OMe-HCl for H-Glu(OBzl)-Ome, the title compound was prepared (80% yield).
  • Step D Compound (1.4)
  • In essentially the same manner as in Step B, but substituting a proportional amount of compound (1.3) the product of Step C for compound (1.1) the product of Step A, the title compound was prepared (41% yield); C25H43N3O9 (529.64), mass spec. (FAB) M+1=530.3; [a]D−24.6 (c=0.7, MeOH).
  • Step E Compound (1.5)
  • In essentially the same manner as in Step A, but substituting proportional amounts of compound (1.4), the product of Step D for Ac-Glu(OBzl)-OH, and H-Val-OBzl.HCl for H-Glu(OBzl)-Ome, the title compound was prepared (86% yield), C37H58N4O10 (718.90) mass spec. (FAB) M+1=719.3.
  • Step F Compound (1.6)
  • A mixture of compound (1.5) the product of Step E (2.6 g), 10% Pd/C (0.2 g), and EtOH-dioxane (1:1, 240 mL) was stirred under 1 atm. H2 for 18 hr. The mixture was filtered and evaporated to dryness under vacuum to afford the title compound (2.1 g, 93% yield), C30H52N4O10 (628.77) mass spec. (FAB) M+1=629.5.
    • PREPARATIVE EXAMPLE 2
  • Step A Compound (2.2)
    Figure US20050059606A1-20050317-C00032
  • In a pot were combined N-Cbz-hydroxyproline methyl ester (available from Bachem Biosciences, Incorporated, King of Prussia, Pa.), compound (2.1) (3.0 g), toluene (30 mL), and ethyl acetate (30 mL). The mixture was stirred vigorously, and then a solution of NaBr/water (1.28 g/5 mL) was added. To this was added 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (TEMPO, 17 mg, from Aldrich Chemicals, Milwaukee, Wis.). The stirred mixture was cooled to 5° C. and then was added a prepared solution of oxidant [commercially available bleach, Clorox® (18 mL), NaHCO3 (2.75 g) and water to make up 40 mL] dropwise over 0.5 hr. To this was added 2-propanol (0.2 mL). The organic layer was separated, and the aqueous layer extracted with ethyl acetate. The organic extracts were combined, washed with 2% sodium thiosulfate, then saturated brine. The organic solution was dried over anhydrous MgSO4, filtered, and evaporated the filtrate under vacuum to leave a pale yellow gum suitable for subsequent reactions (2.9 g, 97% yield), C14H15NO5 (277.28), mass spec. (FAB) M+1=278.1.
    Step B Compound (2.3).
    Figure US20050059606A1-20050317-C00033
  • Compound (2.2) from Step A above (7.8 g) was dissolved in dichloromethane (100 mL), and cooled to 15° C. To this mixture was first added 1,3-propanedithiol (3.1 mL), followed by freshly distilled boron trifluoride etherate (3.7 mL). The mixture was stirred at room temperature for 18 h. While stirring vigorously, a solution of K2CO3/water (2 g/30 mL)was carefully added, followed by saturated NaHCO3 (10 mL). The organic layer was separated from the aqueous layer (pH −7.4), washed with water (10 mL), then brine. The organic solution was dried over anhydrous MgSO4, filtered, and evaporated under vacuum. The residue was chromatographed on silica gel, eluting with toluene, then a with a gradient of hexane-Et2O (2:3 to 0:1) to afford a brown oil (7.0 g, 68% yield), C17H21NO4S2 (367.48), mass spec. (FAB) M+1=368.1.
    Steg C Compound (2.4)
    Figure US20050059606A1-20050317-C00034
  • A solution of compound (2.3) from Step B above (45 g) in acetonitrile (800 mL) at 20° C. was treated with freshly distilled iodotrimethylsilane (53 mL) at once. The reaction was stirred for 30 min., then poured into a freshly prepared solution of di-t-butyldicarbonate (107 g), ethyl ether (150 mL), and diisopropylethylamine (66.5 mL). The mixture stirred for 30 min. more then was washed with hexane (2×500 mL). Ethyl acetate (1000 mL) was added to the lower acetonitrile layer, and then the layer was washed with 10% aqueous KH2PO4 (2×700 mL), and brine. The filtrate was evaporated under vacuum in a 25° C. water bath, taken up in fresh ethyl acetate (1000 mL), and washed successively with 0.1 N HCl, 0.1 N NaOH, 10% aqueous KH2PO4, and brine. The organic solution was dried over anhydrous MgSO4, filtered, and evaporated under vacuum. The residue (66 g) was chromatographed on silica gel (2 kg), eluting with hexane (2 L), then Et2O/hexane (55:45, 2 L), then Et2O (2 L) to afford an orange gum which slowly crystallized on standing (28 g, 69% yield), C14H23NO4S2 (333.46), mass spec. (FAB) M+1=334.1.
    Step D Compound (2.5)
    Figure US20050059606A1-20050317-C00035
  • A solution of compound (2.4) from Step C above (11 g) in dioxane (150 mL) at 20° C. was treated with 1 N aqueous LiOH (47 mL) and stirred for 30 h. The mixture was concentrated under vacuum in a 30° C. water bath to half volume. The remainder was diluted with water (300 mL), extracted with Et2O (2×200 mL). The aqueous layer was acidified to pH ˜4 with 12 N HCl (34 mL), extracted with ethyl acetate, and washed with brine. The organic solution was dried over anhydrous MgSO4, filtered, and evaporated under vacuum to leave the title compound (8.1 g, 78%), C13H21NO4S2 (319.44), mass spec. (FAB) M+1=320.1.
    Step E Compound (2.6).
    Figure US20050059606A1-20050317-C00036
  • To a solution of compound (2.4) from Step C above (1 g) in dioxane (5 mL), was added 4 N HCl-dioxane solution (50 mL). The mixture was stirred vigorously for 1 hr. The mixture was evaporated under vacuum in a 25° C. water bath. The residue was triturated with Et2O, and filtered to leave the title compound (0.76 g, 93% yield), C9H15NO2S2.HCl (269.81), mass spec. (FAB) M+1=234.0.
    Step F Compound (2.7).
    Figure US20050059606A1-20050317-C00037
  • A mixture of compound (2.6) from Step E above (1.12 g), N-Boc-cyclohexylglycine (1.0 g, from Sigma Chemicals, St. Louis, Mo.), dimethylformamide (20 mL), and PyBrOP coupling reagent (2.1 g) was cooled to 5° C. To this was added diisopropylethylamine (DIEA or DIPEA, 2.8 mL). The mixture was stirred cold for 1 min., then stirred at room temperature for 6 hr. The reaction mixture was poured into cold 5% aqueous H3PO4 (150 mL) and extracted with ethyl acetate (2×150 mL). The combined organic layer was washed with cold 5% aqueous K2CO3, then 5% aqueous KH2PO4, then brine. The organic solution was dried over anhydrous MgSO4, filtered, and evaporated under vacuum. The residue was chromatographed on silica gel, eluting with EtOAc-CH2Cl2 to afford a white solid (0.8 g, 50% yield), C22H36N2O5S2 (472.66), mass spec. (FAB) M+1=473.2.
    Step G Compound (2.8).
    Figure US20050059606A1-20050317-C00038
  • Following essentially the same procedure of Step D above, but substituting compound (2.7) (0.8 g) as starting material, compound (2.8) (0.7 g) was obtained C21H34N2O5S2 (458.64), mass spec. (FAB) M+1=459.2.
    Step H Compound (2.9).
    Figure US20050059606A1-20050317-C00039
  • Following essentially the same procedure of Step E above, but substituting compound (2.8) as starting material, compound (2.9) was obtained. C17H28N2O3S2.HCl (409.01), mass spec. (FAB) M+1=373.2.
    • PREPARATIVE EXAMPLE 3
  • Step A Compound (3.1)
    Figure US20050059606A1-20050317-C00040
  • Following essentially the same procedure of Preparative Example 2, Step B, substituting ethane dithiol for propane dithiol, compound (3.1) was obtained.
    Step B Compound (3.2).
    Figure US20050059606A1-20050317-C00041
  • Following essentially the same procedure of Preparative Example 2, Step C, substituting compound (3.1) for compound (2.3), the product compound (3.2) was obtained.
    Step C Compound (3.3)
    Figure US20050059606A1-20050317-C00042
  • Following essentially the same procedure of Preparative Example 2, Step D, substituting compound (3.2) for compound (2.4) the product compound (3.3) was obtained.
    Step D Compound (3.4)
    Figure US20050059606A1-20050317-C00043
  • Following essentially the same procedure of Preparative Example 2, Step E, substituting compound (3.2) for compound (2.4) the product compound (3.4) was obtained.
    Step E Compound (3.5)
    Figure US20050059606A1-20050317-C00044
  • Following essentially the same procedure of Preparative Example 2, Step F, substituting compound (3.4) for compound (2.6) the product compound (3.5) was obtained.
    Step F Compound (3.6)
    Figure US20050059606A1-20050317-C00045
  • Following essentially the same procedure of Preparative Example 2, Step G, substituting compound (3.5) for compound (2.7) the product compound (3.6) was obtained.
    Step G Compound (3.7)
    Figure US20050059606A1-20050317-C00046
  • Following essentially the same procedure of Preparative Example 2, Step H, substituting compound (3.5) for compound (2.7) the product compound (3.7) was obtained.
    • PREPARATIVE EXAMPLE 4
  • Step A Compound (4.1)
    Figure US20050059606A1-20050317-C00047
  • To a stirred solution of compound (4.01) (3.00 g, 12.0 mmol) prepared according to S. L. Harbeson et al., J.Med.Chem. 37 (18), 2918-2929 (1994), in DMF (15 mL) and CH2Cl2 (15 mL) at −20° C. was added HOOBt (1.97 g, 12.0 mmol), N-methyl morpholine (4.0 mL, 36.0 mmol) and EDCl (2.79 g, 14.5 mmol). The reaction mixture stirred for 10 minutes, followed by the addition of HCl.H2N-Gly-Oallyl (2.56 g, 13.0 mmol). The resulting solution was stirred at −20° C. for 2 hrs, then kept in the refrigerator overnight. The solution was concentrated to dryness, then diluted with EtOAc (150 mL). The EtOAc solution was then washed twice with saturated NaHCO3, H2O, 5% H3PO4, and brine, dried over Na2SO4, filtered and concentrated to dryness to give the product (4.1). LRMS m/z MH+=345.2.
    Step B Compound (4.2)
    Figure US20050059606A1-20050317-C00048
  • Following essentially the same procedure of Preparative Example 2, Step E, but substituting compound (4.1) from Step A above, as the starting material, compound (4.2) was obtained C11H20N2O4.HCl (280.75).
    Step C Compound (4.3)
    Figure US20050059606A1-20050317-C00049
  • Following essentially the same procedure of Preparative Example 1, Step A, reacting compound (4.2) from Step B above, with compound (3.3) from Preparative example 3, Step C, compound (4.3) was obtained. C28H37N3O7S2 (531.69).
    Step D Compound (4.4).
    Figure US20050059606A1-20050317-C00050
  • Following essentially the same procedure of Preparative Example 2, Step E, but substituting compound (4.3) from Step C above, as starting material, compound (4.4) was obtained. C18H30N4O5S2.HCl (483.05).
    Step E Compound (4.5)
    Figure US20050059606A1-20050317-C00051
  • Following essentially the same procedure of Preparative Example 1, Step A, reacting compound (4.4) from Step D above, with N-Boc-cyclohexylglycine, compound (4.5) was obtained. C31H50N4O8S2 (670.88).
    Step F Compound (4.6).
    Figure US20050059606A1-20050317-C00052
  • Following essentially the same procedure of Preparative Example 2, Step E, but substituting compound (4.5) from Step E above, as starting material, compound (4.6) was obtained. C26H42N4O6S2.HCl (607.23).
    Step G Compound (4.7).
    Figure US20050059606A1-20050317-C00053
  • Following essentially the same procedure of Preparative Example 1, Step A, reacting compound (4.6) from Step F above, with compound (1.4) from Preparative example 1, Step D, compound (4.7) was obtained. C51H83N7O14S2 (1082.38).
    Step H Compound (4.8).
    Figure US20050059606A1-20050317-C00054
  • A mixture of compound (4.7) from Step G above (0.47 g), dichloromethane (20 mL), methyl sulfoxide (0.9 mL), and 2,2-dichloroacetic acid (0.142 mL) was stirred at 5° C. To this was added a solution of 1 M dicyclohexylcarbodiimide in CH2Cl2 (9.6 mL), and the resulting mixture was stirred cold for 5 min., at room temperature for 3 h. A solution of oxalic acid (0.35 g) in methanol (3 mL) was added to destroy excess oxidant, stirred for 15 min., and then filtered to remove the precipitated urea. The filtrate was diluted with excess ethyl acetate, and washed with cold 0.1 N NaOH, then cold 0.2 N H3PO4, then brine, The organic solution was dried over anhydrous MgSO4, filtered, and evaporated under vacuum. The residue was chromatographed on silica gel, eluting with a gradient of MeOH—CH2Cl2 (1:99 to 5:95) to obtain the title compound (4.8) (0.25 g, 53% yield) C51H81N7O14S2 (1080.36), mass spec. (FAB) M+1=1080.6.
    Step I Compound (4.9).
    Figure US20050059606A1-20050317-C00055
  • Following essentially the same procedure of Preparative Example 2, Step D, but substituting compound (4.8) from Step H above, as the starting material, compound (4.9) was obtained C48H73N7O14S2 (1036.26), mass spec. (FAB) M+1=1036.6.
    Step J Compound (86).
    Figure US20050059606A1-20050317-C00056
  • Compound (4.9) from Step I above (0.10 g) was treated with a solution of anhydrous trifluoroacetic acid-dichloromethane (1:1, 10 mL) for 2 h. The solution was diluted with xylene (50 mL) and evaporated under vacuum. The residue was triturated with Et2O, and filtered to leave the title compound (86) (0.04 g), C40H61N7O14S2 (928.08), mass spec. (FAB) M+1=928.4.
    • EXAMPLES
  • Using the procedures of Preparative Example 1, Step A, and Preparative Example 2, Step F, for couplings; Preparative Example 1, Step B, Preparative Example 1, Step F, Preparative Example 2, Step D, and Preparative Example 4, Step J for ester deprotection; Preparative Example 2, Step E, and Preparative Example 4, Step J, for amine deprotection; and Preparative Example 4, Step H, for oxidation of hydroxyamides to ketoamides—together with the α-amino acids of the above examples or those commercially available or those described in the literature, in the necessary various combinations, the following compounds in Table 2 were prepared:
    TABLE 2
    Compound
    from Molecular
    Example No. STRUCTURE Weight
    1
    Figure US20050059606A1-20050317-C00057
         		950.149
    2
    Figure US20050059606A1-20050317-C00058
         		837.932
    3
    Figure US20050059606A1-20050317-C00059
         		849.944
    4
    Figure US20050059606A1-20050317-C00060
         		779.895
    5
    Figure US20050059606A1-20050317-C00061
         		892.112
    6
    Figure US20050059606A1-20050317-C00062
         		836.948
    7
    Figure US20050059606A1-20050317-C00063
         		949.164
    8
    Figure US20050059606A1-20050317-C00064
         		781.911
    9
    Figure US20050059606A1-20050317-C00065
         		894.128
    10
    Figure US20050059606A1-20050317-C00066
         		793.922
    11
    Figure US20050059606A1-20050317-C00067
         		1065.28
    12
    Figure US20050059606A1-20050317-C00068
         		777.879
    13
    Figure US20050059606A1-20050317-C00069
         		890.096
    14
    Figure US20050059606A1-20050317-C00070
         		948.133
    15
    Figure US20050059606A1-20050317-C00071
         		835.916
    16
    Figure US20050059606A1-20050317-C00072
         		825.921
    17
    Figure US20050059606A1-20050317-C00073
         		853.975
    18
    Figure US20050059606A1-20050317-C00074
         		966.192
    19
    Figure US20050059606A1-20050317-C00075
         		938.138
    20
    Figure US20050059606A1-20050317-C00076
         		915.166
    21
    Figure US20050059606A1-20050317-C00077
         		889.408
    22
    Figure US20050059606A1-20050317-C00078
         		1099.30
    23
    Figure US20050059606A1-20050317-C00079
         		931.062
    24
    Figure US20050059606A1-20050317-C00080
         		1043.28
    25
    Figure US20050059606A1-20050317-C00081
         		952.165
    26
    Figure US20050059606A1-20050317-C00082
         		811.894
    27
    Figure US20050059606A1-20050317-C00083
         		839.948
    28
    Figure US20050059606A1-20050317-C00084
         		827.894
    29
    Figure US20050059606A1-20050317-C00085
         		841.921
    30
    Figure US20050059606A1-20050317-C00086
         		1010.25
    31
    Figure US20050059606A1-20050317-C00087
         		996.219
    32
    Figure US20050059606A1-20050317-C00088
         		878.085
    33
    Figure US20050059606A1-20050317-C00089
         		765.868
    34
    Figure US20050059606A1-20050317-C00090
         		1025.22
    35
    Figure US20050059606A1-20050317-C00091
         		932.177
    36
    Figure US20050059606A1-20050317-C00092
         		876.069
    37
    Figure US20050059606A1-20050317-C00093
         		1043.19
    38
    Figure US20050059606A1-20050317-C00094
         		819.961
    39
    Figure US20050059606A1-20050317-C00095
         		987.083
    40
    Figure US20050059606A1-20050317-C00096
         		1022.26
    41
    Figure US20050059606A1-20050317-C00097
         		1080.41
    42
    Figure US20050059606A1-20050317-C00098
         		853.932
    43
    Figure US20050059606A1-20050317-C00099
         		982.278
    44
    Figure US20050059606A1-20050317-C00100
         		926.170
    45
    Figure US20050059606A1-20050317-C00101
         		870.062
    46
    Figure US20050059606A1-20050317-C00102
         		896.057
    47
    Figure US20050059606A1-20050317-C00103
         		823.905
    48
    Figure US20050059606A1-20050317-C00104
         		906.139
    49
    Figure US20050059606A1-20050317-C00105
         		793.922
    50
    Figure US20050059606A1-20050317-C00106
         		793.922
    51
    Figure US20050059606A1-20050317-C00107
         		910.149
    52
    Figure US20050059606A1-20050317-C00108
         		797.932
    53
    Figure US20050059606A1-20050317-C00109
         		829.931
    54
    Figure US20050059606A1-20050317-C00110
         		990.214
    55
    Figure US20050059606A1-20050317-C00111
         		877.998
    56
    Figure US20050059606A1-20050317-C00112
         		910.084
    57
    Figure US20050059606A1-20050317-C00113
         		797.867
    58
    Figure US20050059606A1-20050317-C00114
         		1044.31
    59
    Figure US20050059606A1-20050317-C00115
         		1004.24
    60
    Figure US20050059606A1-20050317-C00116
         		892.025
    61
    Figure US20050059606A1-20050317-C00117
         		932.09
    62
    Figure US20050059606A1-20050317-C00118
         		914.031
    63
    Figure US20050059606A1-20050317-C00119
         		1026.25
    64
    Figure US20050059606A1-20050317-C00120
         		954.097
    65
    Figure US20050059606A1-20050317-C00121
         		906.139
    66
    Figure US20050059606A1-20050317-C00122
         		793.922
    67
    Figure US20050059606A1-20050317-C00123
         		807.950
    68
    Figure US20050059606A1-20050317-C00124
         		920.166
    69
    Figure US20050059606A1-20050317-C00125
         		900.004
    70
    Figure US20050059606A1-20050317-C00126
         		1012.22
    71
    Figure US20050059606A1-20050317-C00127
         		940.069
    72
    Figure US20050059606A1-20050317-C00128
         		851.960
    73
    Figure US20050059606A1-20050317-C00129
         		964.176
    74
    Figure US20050059606A1-20050317-C00130
         		1004.24
    75
    Figure US20050059606A1-20050317-C00131
         		871.950
    76
    Figure US20050059606A1-20050317-C00132
         		966.192
    77
    Figure US20050059606A1-20050317-C00133
         		1006.26
    78
    Figure US20050059606A1-20050317-C00134
         		894.041
    79
    Figure US20050059606A1-20050317-C00135
         		851.960
    80
    Figure US20050059606A1-20050317-C00136
         		964.176
    81
    Figure US20050059606A1-20050317-C00137
         		934.106
    82
    Figure US20050059606A1-20050317-C00138
         		894.04
    83
    Figure US20050059606A1-20050317-C00139
         		956.112
    84
    Figure US20050059606A1-20050317-C00140
         		1026.25
    85
    Figure US20050059606A1-20050317-C00141
         		914.031
    86
    Figure US20050059606A1-20050317-C00142
         		928.099
    87
    Figure US20050059606A1-20050317-C00143
         		1060.27
    88
    Figure US20050059606A1-20050317-C00144
         		889.984
  • Additional compounds were prepared by the following procedures and examples. The compounds are listed in Tables immediately following the examples. The structures of many of the so-prepared compounds and their activity are given in the attached Table 3.
  • General Procedure for Preparation of the Compounds of Table 3:
  • Solid-phase synthesis is useful for the production of small amounts of certain compounds of the present invention. As with the conventional solid-phase synthesis of peptides, reactors for the solid-phase synthesis of peptidyl argininals are comprised of a reactor vessel with at least one surface permeable to solvent and dissolved reagents, but not permeable to synthesis resin of the selected mesh size. Such reactors include glass solid phase reaction vessels with a sintered glass frit, polypropylene tubes or columns with frits, or reactor Kans™ made by Irori Inc., San Diego Calif. The type of reactor chosen depends on volume of solid-phase resin needed, and different reactor types might be used at different stages of a synthesis.
    • General Procedure for the Synthesis of 4-alkylprolines (Intermediates Used in the Synthesis (Steps 14 Below):
  • Step 1
    • tert-Butyl N-α-t-butoxycarbonyl-L-pyroglutamate
  • Figure US20050059606A1-20050317-C00145
  • To a solution of L-pyroglutamic acid (100 g, 775 mmol) in tert-butyl acetate (1.3 L) was added 70% aqueous perchloric acid (25 mL). After stirring for 18 hours in a 3-liter round bottom flask sealed with a rubber septum, the reaction mixture was poured carefully into saturated aqueous sodium bicarbonate (800 mL) and extracted with ethyl acetate (1 L, then 300 mL). The combined organic layers were dried (sodium sulfate), filtered and concentrated to provide tert-Butyl L-pyroglutamate (100.5 g, 70% mass recovery). The tert-butyl L-pyroglutamate was dissolved in acetonitrile (1.5 L) with 4-dimethylaminopyridine (6.0 g, 49.1 mmol) and cooled to 0° C. A solution of di-tert-butyl-dicarbonate (154 g, 705 mmol) in acetonitrile (150 mL) was added over 30 min and after an additional 30 min the cooling bath was removed. After stirring at room temperature for 48 hours, the reaction mixture was concentrated. The residue was dissolved in ether (1 L) and hexanes (1 L), then washed with saturated aqueous sodium bicarbonate (2×100 mL) and saturated aqueous sodium chloride. The solution was dried (sodium sulfate), filtered and concentrated to give a yellow oil (140 g). The residue was purified by recrystallization from hexanes (800 ml) and ethyl acetate (25 mL), and the mother liquor was recrystallized from hexanes (300 mL) and ethyl acetate (10 mL) to give two crops of the title compound (122 g, 428 mmol, 55%) as a white solid. NMR data was consistent with previously reported material: R. A. August et al, J. Chem. Soc., Perkin Trans. 1 (1996) 507-514.
  • Step 2
    • tert-Butyl N-α-t-butoxycarbonyl-4-alkyl-L-pyroglutamate
  • Figure US20050059606A1-20050317-C00146
  • To a solution of the compound of Step 1 (1.15 g, 4.0 mmol) in tetrahydrofuran (20 mL) stirring at −78 ° C., was added a 1M solution of lithium hexamethyldisilazide in tetrahydrofuran (4.4 mL, 4.4 mmol) dropwise over 5 min. After 40 min, alkylbromide (4.8 mmol) in tetrahydrofuran (5 mL) was added. After 2 h at −78° C., the cooling bath was removed and saturated aqueous ammonium chloride (20 mL) was added. The solution was stirred for 20 minutes, then extracted with 50% ether/ethyl acetate (3×70 mL). The combined organic layers were washed with brine (30 mL), dried (sodium sulfate), filtered, and concentrated. The resulting residues were purified by flash chromatography and/or recrystallization to afford the title compounds.
    Compound Alkylbromide Yield (%)
    R═Bn benzyl bromide 63
    R═PMB p-methoxybenzyl bromide 57
    R═allyl allyl bromide 22
    R═CH2CO2Bn benzyl a-bromoacetate 51

    Step 3
    • 4-alkyl-proline
  • Figure US20050059606A1-20050317-C00147
  • (Modification of known procedure: C. Pedregal et al, Tetrahedron Letters 35 (13) (1994) 35(13) 2053-2056.) To a solution of tert-butyl N-tert-butoxycarbonyl-4-alkylpyroglutamate (2.0 mmol) in tetrahydrofuran (5 mL) stirring at −78 ° C., was added a 1 M solution of lithium triethylborohydride in tetrahydrofuran (2.4 mL, 2.4 mmol) dropwise over 5 min. After 30 min, the cooling bath was removed and saturated aqueous sodium bicarbonate (5 mL) was added. The reaction mixture was immersed in an ice/water bath and 30% aqueous hydrogen peroxide (10 drops) was added. The solution was stirred for 20 minutes at 0° C., then the reaction mixture was concentrated in vacuo to remove the tetrahydrofuran. The aqueous solution was diluted with water (10 mL) and extracted with dichloromethane (3×40 mL). The organic layers were dried (sodium sulfate), filtered and concentrated. The residue was dissolved in dichloromethane (20 mL) and triethylsilane (310 μL, 2.0 mmol), then cooled to −78° C. and boron trifluoride etherate (270 μL, 2.13 mmol) was added dropwise. Stirring was continued for 30 min, at which time additional triethylsilane (310 μL, 2.0 mmol) and boron trifluoride etherate (270 μL, 2.13 mmol) were added. After stirring at −78° C. for an additional 2 h, the cooling bath was removed and saturated aqueous sodium bicarbonate (4 mL) was added. After 5 min the mixture was extracted with dichloromethane (3×40 mL). The organic layers were dried (sodium sulfate), filtered and concentrated. The residue was dissolved in dichloromethane (5 mL) and trifluoroacetic (5 mL) and stirred at ambient temperature for 5 h. The solution was concentrated and then dried under high vacuum to afford the title compound.
    Compound Yield (%)
    R═Bn 72
    R═PMB 76
    R═allyl 65
    R═CH2CO2Bn 34

    Step 4
    • N-α-t-butoxycarbonyl-4-alkyl-proline
  • Figure US20050059606A1-20050317-C00148
  • To a solution of 4-alkylproline trifluoroacetate salt (1.5 mmol) in dioxane (7 mL), acetonitrile (12 mL) and diisopropylethylamine (700 μL, 4 mmol) was added di-tert-butyl-dicarbonate (475 mg, 2.18 mmol) in acetonitrile (5 mL). After stirring for 12 h at room temperature the solution was concentrated in vacuo, dissolved in saturated aqueous sodium bicarbonate(50 mL) and washed with ether (3×40 mL). The aqueous layer was acidified to pH 3 with citric acid, then extracted with dichloromethane (3×40 mL). The combined organic layers were dried (sodium sulfate), filtered and concentrated to afford the title compounds, which where pure enough to be used without further purification.
    Compound Yield (%)
    R═Bn 59
    R═PMB 69
    R═allyl 58
    R═CH2CO2Bn 67
    • Synthesis of Other Intermediates: 4-methylsulfinyl-2S-fluorenylmethyloxycarboaminobutyric Acid (Fmoc-Met(O2)-OH
  • Figure US20050059606A1-20050317-C00149
  • To a solution of N-α-fluorenylmethyloxycarbonyl-methionine (3.72 g, 10 mmol) in chloroform (225 mL) at 0° C., was added 56-87% m-chloroperbenzoic acid (20-31mmol) in portions over 10 min. The cold bath was removed and the reaction was stirred for 18 h. The solid (4.09 g) was collected by filtration (filtrate was discarded), dissolved in hot methanol (90 mL), allowed to cool, then filtered and concentrated. The crude product was recrystallized from ethyl acetate and hexanes to yield the title compound (2.577 g, 6.39 mmol, 64 %).
    • N-α-fluorenylmethyloxycarbonyl-3-methylsulfinylalanine (Fmoc-Cvs(O2,Me)—OH
  • Figure US20050059606A1-20050317-C00150
  • To a solution of N-α-fluorenylmethyloxycarbonyl-S-methylcysteine (893 mg, 2.5 mmol) in chloroform (50 mL) at 0° C., was added 56-87% m-chloroperbenzoic acid (1.555 g, 5.0-7.8 mmol, >2 eq) in portions over 10 min. The cold bath was removed and the reaction was stirred for 18 h. The solid was collected by filtration (filtrate was discarded), dried in vacuo, dissolved in hot ethyl acetate (150 mL), filtered and concentrated. The solid was dissolved in a minimal amount of hot methanol, then crystallized by the addition of isopropyl ether, followed by 10% ethyl acetate in hexanes. The solid (658.6 mg) was collected and recrystallized again to give the title compound (520 mg, 1.335 mmol, 53%).
    • Synthesis of N-tert-butoxycarbonyl-trans-4-(N-fluorenylmethyloxycarbonyl amino)-L-proline (Boc-Pro(4t-NHFmoc): (Steps 1-3 Below)
  • Step 1
    • N-tert-butoxycarbonyl-cis-4-chloro-L-proline benzyl ester
  • Figure US20050059606A1-20050317-C00151
  • A mixture of N- -t-butoxycarbonyl-trans-4-hydroxy-proline (8.79 g, 38 mmol), potassium carbonate (13.0 g, 94 mmol), benzyl bromide (4.5 ml, 38 mmol) and dimethylformamide (150 mL) was stirred for 18 h. Addition of ethyl acetate (100 mL) was followed by filtration. The white cloudy filtrate was clarified by the addition of 1M HCl (100 mL). The layers were separated and the aqueous layer was extracted with additional ethyl acetate (2×100 mL). The combined organic layers were washed with water (2×50 mL), dried (sodium sulfate), filtered and concentrated. Toluene was added to the crude benzyl ester, and the solution was filtered and reconcentrated. Dichloromethane (70 mL) and carbon tetrachloride (70 mL) was added, followed by triphenylphosphine (21.11 g, 80 mmol). The reaction mixture was stirred for 10 h, quenched with ethanol (7 mL) and stirred for 5 more h. The solution was concentrated to approx. 100 ml, then dichloromethane (40 mL) was added, followed by the addition of ether (200 mL) while stirring. The solution was cooled for 4 h, filtered and concentrated to give a yellow-brown oil which was purified by flash chromatography using ether/hexane/dichloromethane 2:2:1 to give the title compound (9.13 g, 26.9 mmol, 71 %) as a white solid.
  • Step 2
    • N-α-t-butoxycarbonyl-trans-4-azido-L-proline benzyl ester
  • Figure US20050059606A1-20050317-C00152
  • A solution of the compound of step 1 above (9.0 g, 26.5 mmol) and sodium azide (7.36 g, 113 mmol) in dimethylformamide (270 mL) was heated at 75° C. for 2 days. Water (100 mL) was added and the reaction mixture was extracted with ethyl acetate (3×100 mL). The combined organic layers were washed with water (3×50 mL), dried (sodium sulfate), filtered and concentrated. The oil was purified by flash chromatography using ethyl acetate/hexane 1:1 to give the title compound (8.59 g, 24.8 mmol, 94%).
  • Step 3
    • N-tert-butoxycarbonyl-trans-4-(N-fluorenylmethyloxycarbonyl_amino)-L-proline
  • Figure US20050059606A1-20050317-C00153
  • A mixture of the compound of step 2 above (8.59 g, 24.8 mmol) and 10% palladium on carbon (900 mg) in ethanol (500 mL) was hydrogenated at 50 psi for 14 h using a Parr hydrogenation apparatus. The mixture was filtered, concentrated, dissolved in methanol (60 mL), refiltered and concentrated to give a colorless oil. The oil was dissolved in water (53 mL) containing sodium carbonate (5.31 g, 50.1 mmol) and a solution of fluorenylmethyl succinyl carbonate (8.37 g, 29.8 mmol) in dioxane (60 mL) was added over 40 min. The reaction mixture was stirred at room temperature for 17 h, then concentrated to remove the dioxane and diluted with water (200 mL). The solution was washed with ether (3×100 mL). The pH of the aqueous solution was adjusted to 2 by the addition of citric acid (caution! foaming!) and water (100 mL). The mixture was extracted with dichloromethane (400 mL, 100 mL, 100 mL) and the combined organic layers were dried (sodium sulfate), filtered and concentrated to give the title compound.
    • N-α-t-butoxycarbonyl-4-trans-(N-fluorenylmethyloxycarbonyl aminomethyl)-L-proline (Boc-Pro(4t-MeNHFmoc)-OH) (Steps 1-4 Below)
  • Step 1
    • N-α-t-butoxycarbonyl cis-4-hydroxy-L-proline benzyl ester
  • Figure US20050059606A1-20050317-C00154
  • To a mixture of cis-hydroxy-L-proline (5 g, 38.1 mmol) in benzene (45 mL) and benzyl alcohol (45 mL) was added p-toluenesulfonic acid monohydrate (7.6 g, 40.0 mmol). The reaction mixture was heated at 125° C. for 20 h while water (2 ml) was removed using a Dean-Stark trap. The solution was filtered while still hot, and then ether (150 ml) was added. The solution was allowed to cool for three h at room temperature, then three h at 4° C. The resulting solid was collected, washed with ether (100 mL) and dried in vacuo for 1 h to give 13.5 grams of white solid. The solid was dissolved in dioxane (40 mL) and diisopropylethylamine (7.6 mL), and then di-tert-butyl-dicarbonate (10 g, 45.8 mmol) was added over 5 min while using an ice bath to maintain a constant reaction temperature. After 10 h at room temperature the reaction mixture was poured into cold water (200 mL) and extracted with ethyl acetate (3×200 mL). The combined organic layers were washed with water (3×100 mL) and saturated aqueous sodium chloride (50 mL), dried (sodium sulfate), filtered and concentrated. The crude product was purified by flash chromatography using 40-60% ethyl acetate in hexanes to give the title compound (10.04 g, 31.24 mmol, 82%).
  • Step 2
    • N-α-t-butoxycarbonyl cis-4-mesyloxy-L-proline benzyl ester
  • Figure US20050059606A1-20050317-C00155
  • To a solution of the compound of step 2 above (8.45 g, 26.3 mmol) in pyridine (65 mL) at 0° C., was added methanesulfonyl chloride (3.4 mL, 44 mmol) dropwise over 7 min. The reaction mixture was allowed to warm to room temperature over 2 h, then stirred overnight. A solution of 10% water in pyridine (20 mL) was added over 15 min and the reaction mixture was concentrated. The residue was dissolved in water and extracted with ethyl acetate (2×200 mL). The combined organic layers were washed with water (2×50 mL) saturated aqueous sodium bicarbonate (50 mL) and saturated aqueous sodium chloride (50 mL), dried (sodium sulfate), filtered and concentrated. The resulting residue was dissolved in toluene (100 mL) and concentrated to remove traces of pyridine. The residue was dried in vacuo for 30 min to afford the title compound (10.7 g, 102%), then used in the next step without purification.
  • Step 3
    • N-α-t-butoxycarbonyl-trans-4-(R)-cyano-L-proline benzyl ester
  • Figure US20050059606A1-20050317-C00156
  • A solution of the compound of Step 3 above (10.7 g, 26.3 mmol) and tetrabutylammonium cyanide (15.0 g, 56 mmol) in dimethylformamide (100 mL) was heated in an oil bath at 55° C. for 28 h. After cooling, water (150 mL) was added and the mixture was extracted with ethyl acetate (3×200 mL). The combined organic layers were washed with water (3×100 mL) and saturated aqueous sodium chloride (100 mL), dried (sodium sulfate), filtered and concentrated. The resulting residue was purified by flash chromatography (1:1 ether/hexanes) and then recrystallized from ethyl acetate/hexanes to provide the title compound (2.40 g, 7.26 mmol, 28%).
  • Step 4
    • N-α-t-butoxycarbonyl-4-trans-(N-fluorenyimethyloxycarbonyl aminomethyl)-L-proline
  • Figure US20050059606A1-20050317-C00157
  • A mixture the com pound of Step 3 above (2.31 g, 7 mmol), water (10 mL), methanol (85 mL) and 10% palladium on carbon (700 mg) was hydrogenated at 50 psi for 11 h using a Parr hydrogenation apparatus. The mixture was filtered and concentrated. Water (15 mL) and sodium carbonate (1.5 g, 14.2 mmol) was added to the residue. A solution of fluorenylmethyl succinyl carbonate (2.36 g, 7.0 mmol) in dioxane (17 mL) was added over 5 min and stirring was continued for 28 h at room temperature. The reaction was concentrated in vacuo to a 15 mL volume, and water (100 mL) was added. The solution was washed with ether (3×75 mL). The pH of the aqueous solution was adjusted to 2 by the addition of citric acid (approx. 20 g, caution! foaming!) and water (100 mL). The mixture was extracted with dichloromethane (4×100 mL), and the combined organic layers were dried (sodium sulfate), filtered and concentrated. The crude product contained a major impurity which necessitated a three step purification. The crude product was dissolved in dichloromethane (50 mL) and trifluoroacetic acid (50 mL) and stirred for 5 h before being concentrated. The residue was purified by preparatory reverse-phase HPLC. The pure 4-(N-fluorenylmethyloxycarbonyl aminomethyl)proline trifluoroacetate salt (1.887 g, 3.93 mmol) was dissolved in dioxane (10 mL), acetonitrile (20 mL) and diisopropylethylamine (1.4 mL, 8 mmol). To the reaction mixture was added a solution of di-tert-butyldicarbonate (1.1g, 5 mmol) in dioxane (5 mL). After stirring for 18 h, the pH of the solution was adjusted to 2 by the addition of citric acid (caution: foaming!) and water (100 mL). The mixture was extracted with ethyl acetate (3×150 mL) and the combined organic layers were washed with saturated aqueous sodium chloride (100 mL), dried (sodium sulfate), filtered and concentrated. The crude product was dissolved in saturated aqueous sodium bicarbonate(100 mL) and washed with ether (3×75 mL). The pH of the aqueous layer was adjusted to 3 by the addition of citric acid, then extracted with dichloromethane (4×100 mL). The combined organic layers were dried (sodium sulfate), filtered and concentrated to the title compound (1.373 g, 2.94 mmol, 42%).
  • Procedure to Synthesize Inventive Compounds:
  • Procedure A: Coupling reaction: To the resin suspended in DMF (10-15 mL/gram resin) was added Fmoc-amino acid (1 eq), HOBt (1 eq), TBTU (1 eq) and DIEA (1 eq). The mixture was let to react for 448 hours. The reactants were drained and the resin was washed successively with dimethylformamide, dichloromethane, methanol, dichloromethane and diethylether (use 10-15 mL solvent/gram resin). The resin was then dried in vacuo.
  • Procedure B: Coupling reaction: To the resin suspended in N-methylpyrrolidine (NMP) (10-15 mL/gram resin) was added Fmoc-amino acid (2 eq), HOAt (2 eq), HATU (2 eq) and diisopropylethylamine (4 eq). The mixture was let to react for 4-48 hours. The reactants were drained and the resin was washed successively with dimethylformamide, dichloromethane, methanol, dichloromethane and diethylether (use 10-15 mL solvent/gram resin). The resin was then dried in vacuo.
  • Procedure C: Fmoc deprotection: The Fmoc-protected resin was treated with 20% piperidine in dimethylformamide (10 mL reagent/g resin) for 30 minutes. The reagents were drained and the resin was washed successively with dimethylformamide, dichloromethane, methanol, dichloromethane and diethyl ether (10 mL solvent/gram resin).
  • Procedure D: Boc deprotection: The Boc-protected resin was treated with a 1:1 mixture of dichloromethane and trifluoroacetic acid for 20-60 minutes (10 mL solvent/gram resin). The reagents were drained and the resin was washed successively with dichloromethane, dimethylformamide, 5% diisopropylethylamine in dimethylformamide, dimethylformamide, dichloromethane and dimethylformamide (10 mL solvent/gram resin).
  • Procedure E: Acetylation with acetic anhydride: The resin was suspended in dimethylformamide. The acetylating reagent, prepared by adding 5 mmol (0.47 mL) acetic anhydride and 5 mmol (0.70 mL) triethylamine to 15 mL Dimethylformamide, was added to the resin and the resin was agitated for 30 minutes. The resin was washed successively with dimethylformamide, dichloromethane, methanol, dichloromethane and diethyl ether (10 mL solvent/gram resin).
  • Procedure F: Semicarbazone hydrolysis: The resin was suspended in the cleavage cocktail (10 mL/g resin) consisting of trifluoroacetic acid: pyruvic acid: dichloromethane: water 9:2:2:1 for 2 hours. The reactants were drained and the procedure was repeated three more times. The resin was washed successively with dichloromethane, water and dichloromethane and dried under vacuum.
  • Procedure G: HF cleavage: The dried peptide-nVal(CO)-G-O-PAM resin (50 mg) was placed in an HF vessel containing a small stir bar. Anisole (10% of total volume) was added as a scavenger. In the presence of glutamic acid and cysteine amino acids, thioanisole (10%) and 1,2-ethanedithiol (0.2%) were also added. The HF vessel was then hooked up to the HF apparatus (from Immuno Dynamics, Incorporated) and the system was flushed with nitrogen for five minutes. It was then cooled down to −70° C. with a dry ice/isopropanol bath. After 20 minutes, HF was distilled to the desired volume (10 mL HF/g resin). The reaction was let to proceed for one and a half hour at 0° C. Work up consisted of removing all the HF using nitrogen. Dichloromethane was then added to the resin and the mixture was stirred for five minutes. This was followed by the addition of 20% acetic acid in water (4 mL). After stirring for 20 minutes, the resin was filtered using a fritted funnel and the dichloromethane was removed under reduced pressure. Hexane was added to the remaining residue and the mixture was agitated, and the layers separated (this was repeated twice to remove scavengers). Meanwhile, the resin was soaked in 1 mL methanol. The aqueous layer (20% HOAC) was added back to the resin and the mixture was agitated for five minutes and then filtered. The methanol was removed under reduced pressure and the aqueous layer was lyophilized. The peptide was then dissolved in 10-25% methanol (containing 0.1% trifluoroacetic acid) and purified by reverse phase HPLC.
  • Procedure H: HF pyridine cleavage: To dried peptide-nVal(CO)-G-O-PAM resin (50 mg) in a HDPE 20 mL scintillation vial containing a ½ inch football stir bar was added thioanisole (100 μL) using a pipetman. A commercially available solution of 70% HF/Pyridine (1 mL) was added. The vial was immediately sealed tight with a polypropylene lined cap and placed in an ice bath with gentle stirring. After stirring in a 0° C. bath for one hour, the bath was removed and the mixture was stirred at room temperature for one hour. During the two hour cleavage process, the vial was inspected periodically and if necessary the resin which built up on the sides of the flask was dispersed back into the HF solution. The vial was cooled back to 0° C. and its cap was carefully removed. The vial were then cooled to −15° C. using a salt/ice bath. A 10 mL B-D syringe body equipped with a 23 gauge needle was held above the vial. To this syringe body was added methoxytrimethylsilane (2 mL) using a repetitive autopipette. After the syringe body dripped empty, additional portions of methoxytrimethylsilane (2×2 mL, 3 mL, (9 mL total)) were added until the HF was neutralized. The vial was stirred for 5 minutes and then removed from the bath and stirred for 20 min. The vial was loaded in the speed vac and concentrated. Methylene chloride (5 mL) was added and the resin was stirred for 10 minutes and filtered into a 16×100 mm test tube using a 5 mL disposable column. The methylene chloride filtrate was concentrated. The resin was additionally washed with 20% AcOH in water (2×3 mL) and diethyl ether (2 mL) into the before mentioned test tube. The mixture was agitated to dissolve the oil. The ether layer was then removed and the aqueous layer was lyophilized. The peptide was then dissolved in 10-25% methanol (containing 0.1% trifluoroacetic acid) and purified by reverse phase HPLC.
    • Example I Solid Phase Synthesis of Ac-EEWP-nV(CO)-G-OH
  • Figure US20050059606A1-20050317-C00158
    Step I. Synthesis of H-nVal(dpsc)-Gly-PAM Resin
    Figure US20050059606A1-20050317-C00159
    • a) Preparation of N-α-t-butoxycarbonyl-norvalinol
  • Figure US20050059606A1-20050317-C00160
  • To a solution of N-α-t-butoxycarbonyl-norvaline (25.0 g, 0.115 mol) in tetrahydrofuran (461 mL), cooled to 0° C., was added borane/tetrahydrofuran complex (461 mL of a 1.0M solution in THF) dropwise. After 1 h at 0° C., the solution was warmed to room temperature over a period of 1.5 h. TLC indicated that the reaction was complete. Methanol was added to quench the reaction. The solution was concentrated to yield the title compound (22.56 g, 96%) as a foamy syrup. TLC of the products indicated satisfactory purity. Rf=0.34 (40% ethyl acetate/hexanes).
    • b) Preparation of N-α-t-butoxycarbonyl-norvaline hydroxy t-butyl amide
  • Figure US20050059606A1-20050317-C00161
  • Part I: To a solution of the compound of step la (7.77 g, 38 mmol), in anhydrous dimethylsulfoxide (153 mL) and toluene (153 mL) was added EDC (73.32 g, 382 mmol). After the solution was cooled to 0° C., dichloroacetic acid (15.8 mL, 191 mmol) was added dropwise. After addition was complete, the reaction was stirred for 15 min. The solution was allowed to warm to room temperature over a period of 2 h. The reaction mixture was concentrated to remove the toluene, then dissolved in ethyl acetate. The solution was washed successively with 1N sodium bisulfate, saturated sodium bicarbonate and brine, dried over sodium sulfate, then concentrated to afford crude N-α-t-butoxycarbonyl-norvalinal which was used directly in the next step. Rf=0.84 (40% ethyl acetate/hexanes).
  • Part II: To a solution of the crude N- -t-butoxycarbonyl-norvalinal in dichloromethane (153 mL) was added t-butyl isocyanide (5.19 mL, 46 mmol) and 2,4,6-collidine (20.2mL, 153 mmol). After the solution was cooled to 0° C., trifluoroacetic acid (7.64 mL, 76 mmol) was added dropwise. After stirring for 1 h, the solution was stirred at room temperature for 3 days while allowing the solvent from the reaction mixture in an uncovered vessel to evaporate under ambient conditions. The reaction mixture was concentrated to remove the toluene, then dissolved in ethyl acetate. The solution was washed successively with 1N sodium bisulfate, saturated sodium bicarbonate and brine, dried over sodium sulfate, then concentrated. The residue was purified by flash silica gel chromatography eluting with 10-40% ethyl acetate/hexanes to afford 8.12 g of the title compound (70% yield) as a yellow syrup. Rf=0.44 (40% ethyl acetate/hexanes).
    • c) Preparation of Ethyl 3-(N-t-butoxycarbonyl)amino-2-hydroxy-hexanoate
  • Figure US20050059606A1-20050317-C00162
  • The compound of step Ib above (22.7 g, 75 mmol) was refluxed in 6N HCl (480 mL) for 4 h. The reaction mixture was allowed to cool to room temperature, then washed with dichloromethane (3×100 mL). The aqueous layer was concentrated to dryness. The residue was triturated with toluene/n-heptane (3×), dried in vacuo to give crude 3-amino-2-hydroxy-hexanoic acid. The crude 3-amino-2-hydroxy-hexanoic acid was dissolved in hydrogen chloride saturated ethanol (40 mL). After 4 h, the solution was concentrated under reduced pressure. The residue was triturated with n-heptane (3×), then dried in vacuo to give crude ethyl 3-amino-2-hydroxy-hexanoate. To the crude ethyl 3-amino-2-hydroxy-hexanoate in water (150 mL) was added potassium carbonate (37.32 g, 270 mmol). After dissolution was complete, dioxane was added (150 mL), followed by di-t-butyldicarbonate (17.7 g, 81.0 mmol). The reaction mixture was allowed to stir overnight, then concentrated under reduced pressure to remove the dioxane. The solution was extracted with ether (3×), then acidified to pH 2-3 with 1N sodium bisulfate. The solution was extracted with ethyl acetate (3×200 mL), dried over sodium sulfate, and the solvent was removed in vacuo to give 13.21 g of the title compound in 71 % yield.
    • d) Preparation of Ethyl N-α-t-butoxycarbonyl-norvalyl carboxylate diphenylmethylsemicarbazone (Part I-Part III Below)
  • Figure US20050059606A1-20050317-C00163
    • Part I: Synthesis of 1-t-Butoxycarbonyl-semicarbazid-4-yl diphenylmethane
  • Figure US20050059606A1-20050317-C00164
  • A solution of carbonyidiimidazole (16.2 g, 0.10 mole) in 225 mL of dimethylformamide was prepared at room temperature and allowed to stir under nitrogen. A solution of t-butyl carbazate (13.2 g, 0.100 mol) in 225 mL DMF was then added dropwise over a 30 min. period. Diphenylmethylamine (18.3 9, 0.10 mol) was added next over a 30min. period. The reaction was allowed to stir at room temperature under nitrogen for one hour. Water (10 mL) was added and the mixture was concentrated to about 150 mL under reduced pressure. This solution was poured into 500 mL water and extracted with 400 mL of ethyl acetate. The ethylacetate phase was extracted two times each with 75 mL 1 N HCl, H2O, saturated sodium bicarbonate solution and sodium chloride, and dried with magnesium sulfate. The mixture was filtered and the solution was concentrated to give 29.5 g (85% yield) of a white foam. This material could be purified by recrystallization from ethyl acetate/hexane, but was pure enough to use directly in the next step: mp 142-143° C. 1H NMR (CDCl3) d 1.45 (s, 9H), 6.10 (dd, 2H), 6.42 (s, 1H), 6.67 (bs, 1H), 7.21-7.31 (m, 1OH). Anal: Calcd. for C19H23N3O3: C, 66.84; H, 6.79; N, 12.31. Found: C, 66.46; H, 6.75; N; 12.90.
    • Part II): Synthesis of diphenylmethyl semicarbazide (dpsc) trifluoroacetate salt
  • Figure US20050059606A1-20050317-C00165
  • A solution of the product obtained in part I (above) (3.43 9,10 mmol) in 12.5 mL of dichloromethane was treated with 12.5 mL of trifluoroacetic acid at room temperature and allowed to stir for 30 min. The solution was added dropwise to 75 mL of ether and the resulting precipitate (2.7 g, 80%) was filtered on a glass funnel. mp 182-184° C. 1H NMR (CD3OD) d 6.05 (s, 1H), 7.21-7.35 (m, 10H). 13C NMR (CD3OD) d 57.6, 118.3 (q, CF3), 126.7, 127.9, 141.6, 156.9, 160.9 (q, CF3CO2H).
  • Part III): Preparation of Ethyl N-α-t-butoxycarbonyl-norvalyl carboxylate diphenylmethylsemicarbazone
  • To a cooled (0° C.) solution of the compound of step Ic above (13.21 g, 48 mmol) and EDC (92.1 g, 0.48 mol) in dimethylsulfoxide (20 mL) and toluene (20 mL), was added dichloroacetic acid (20.6 mL, 0.24 mol) dropwise. The reaction mixture was stirred for 15 min, then allowed to warm to room temperature over a 2 h period. The reaction mixture was concentrated under reduced pressure, then diluted with ethyl acetate, and washed successively with 1N sodium bisulfate, saturated sodium bicarbonate and brine. The organic layer was dried over sodium sulfate, and the solvent was removed in vacuo to afford crude ethyl N-α-t-butoxycarbonyl-norvalyl carboxylate. Ethyl N-α-t-butoxycarbonyl-norvalyl carboxylate was dissolved in ethanol (180 mL), and water (60 mL), diphenylmethylsemicarbazide (obtained in part 11 above)(34.12 g, 96 mmol) and sodium acetate (4.73 g, 57.6 mmol) was added. The reaction mixture was refluxed overnight, then allowed to cool to room temperature. The reaction mixture was concentrated under reduced solvent, then diluted with ethyl acetate. The organic layer was washed successively with 1N sodium bisulfate, saturated sodium bicarbonate and brine, dried over sodium sulfate, and the solvent was removed in vacuo. The residue was chromatographed over flash silica gel using 20-30% ethyl acetate/hexanes as eluent to afford 4.02g of the title compound in 17% yield as a white powder. Rf=0.60 (40% ethyl acetate/hexanes).
    • e) Preparation of N-α-t-butoxycarbonyl-norvalyl carboxylic acid diphenylmethylsemicarbazone (Boc-nVal(dpsc)-OH)
  • Figure US20050059606A1-20050317-C00166
  • To the compound of step Id above (4.02 g, 8.1 mmol) in ethanol (40.5 mL) was slowly added 1N lithium hydroxide (64.8 mL, 64.8 mmol). After 3 h, Dowex 50WX8400 ion exchange resin was added until the pH of the solution reached 4, and the mixture was stirred for 5 min. The mixture was filtered, washed with methanol, and concentrated. The solution was diluted with water, washed with ether. The aqueous solution was concentrated in vacuo. The resulting solid was triturated with toluene, then dried in vacuo to yield 1.44 g of the title compound (38%) as a white solid.
  • HPLC: Rt=16.2 and 17.7 minutes in a 30% to 90% acetonitrile gradient in 0.1% aqueous TFA buffer on a 4.6×250 mm, 5 mM particle, 100 Å pore, C18 column at a 1.0 mL/min flow rate.
    • f) Synthesis of H-nVal(dpsc)-Gly-PAM Resin
  • Figure US20050059606A1-20050317-C00167
  • The commercially available Boc-Gly-PAM resin (5 g, 3.35 mmol) was deprotected according to Procedure D in a 250 mL fritted solid phase reaction vessel equipped with a nitrogen inlet. It was then coupled to Boc-nVal(dpsc)-OH (step Ie above) (2.81 g, 6 mmol)according to Procedure A. The resin was then subjected to Boc deprotection according to procedure D.
  • Step II. Synthesis of Fmoc-Pro-nVal(dpsc)-Gly-PAM resin
  • The resin obtained in step 1 (3.5 9, 1.80 mmol) was reacted with Fmoc-Pro-OH (4.5 mmol, 1.52 g) according to Procedure A. After 18 hours, qualitative ninhydrin analysis showed colorless beads and solution indicating a high yield of coupling.
  • Step III. Synthesis of Fmoc-Val-Pro-nVal(dpsc)-Gly-PAM Resin
  • The compound of step II above (100 mg) was transferred to a fritted polypropylene tube and was deprotected according to Procedure C. A ninhydrin assay on a small aliquot gave dark blue resin and solution, indicating a high yield for the deprotection. The resin was resuspended in DMF (1 mL) and coupled to Fmoc-Val-OH (51 mg, 0.15 mmol) according to Procedure A. A small aliquot was taken for qualitative ninhydrin analysis which showed colorless beads and a dark red solution indicating a high yield of coupling.
  • Step IV. Synthesis of Fmoc-Val-Val-Pro-nVal(dpsc)-Gly-PAM Resin
  • The compound of the previous step (100 mg) was deprotected according to Procedure C. A ninhydrin assay on a small aliquot gave dark blue resin and solution showing a high yield for the deprotection. The resin was resuspended in DMF (1 mL) and was coupled to Fmoc-Val-OH (51 mg, 0.15 mmol), according to Procedure A for 20 hours. A small aliquot was taken for qualitative ninhydrin analysis which showed colorless beads and solution indicating a high yield of coupling.
  • Step V. Synthesis of Fmoc-Glu(OtBu)-Val-Val-Pro-nVal(dpsc)-Gly-PAM Resin
  • The compound of the previous step (100 mg) was deprotected according to Procedure C. A ninhydrin assay on a small aliquot gave dark blue resin and solution showing a high yield for the deprotection. The resin was resuspended in DMF (1 mL) and was coupled to Fmoc-Glu(OtBu)-OH (64 mg, 0.15 mmol), according to Procedure A for 5 hours. A small aliquot was taken for qualitative ninhydrin analysis which showed colorless beads and solution indicating a high yield of coupling.
  • Step VI. Synthesis of Fmoc-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(dpsc)-Gly-PAM Resin
  • The compound of the previous step (100 mg) was deprotected according to Procedure C. A ninhydrin assay on a small aliquot gave dark blue resin and solution showing a high yield for the deprotection. The resin was resuspended in DMF (1 ml) and was coupled to Fmoc-Glu(OtBu)-OH (64 mg, 0.15 mmol), according to Procedure A for 5 hours. A small aliquot was taken for qualitative ninhydrin analysis which showed colorless beads and solution indicating a high yield of coupling.
  • Step VII. Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(dpsc)-Gly-PAM Resin
  • The compound of the previous step (100 mg) was deprotected according to Procedure C and acylated according to Procedure E. The resin was vacuum dried and a small aliquot was taken for qualitative ninhydrin analysis which showed colorless beads and solution indicating a high yield of coupling.
  • Step VIII. Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal-(CO)-Gly-PAM Resin
  • The compound of the previous step (100 mg) was subjected to semicarbazone hydrolysis Procedure F.
  • Step IX. Synthesis of Ac-Glu-Glu-Val-Val-Pro-nVal-(CO)Gly-OH
  • The resin of the previous step (100 mg) was subjected to HF cleavage condition (Procedure G) to yield the desired crude product. The material was purified by HPLC using a 2.2×25 cm reverse phase column, containing a C-18 resin comprised of 10 micron size gel particles with a 300 angstrom pore size, eluting with a gradient using 5-25% acetonitrile in water. Analytical HPLC using a 4.6×250 mm reverse phase column, containing a C-18 resin comprised of 5 micron size gel particles with a 300 angstrom pore size, eluting with 5-25% acetonitrile (containing 0.1% trifluoroacetic acid) showed one peak at 17.5 minutes. Low resolution mass spectrum confirmed the desired mass (MH+ 798.5).
    • Table of Compounds Synthesized According to Example I
  • COMPOUND NAME SYNTHESIS
    Ac-EEVVP-nV-(CO)-G-OH example I
    Ac-EEVV-Sar-nV-(CO)-G-OH step II: used Fmoc-Sar-OH
    Ac-EEVV-Aze-nV-(CO)-G-OH step II: used Fmoc-azetidine-OH
    Ac-EEV-G(Chx)-P-nV-(CO)-G-OH step III: used Fmoc-
    Gly(CHx)—OH
    Ac-EEVFP-nV-(CO)-G-OH step III: used Fmoc-Phe-OH
    Ac-EEVIP-nV-(CO)-G-OH step III: used Fmoc-Ile-OH
    Ac-EEVV-dlPip-nV-(CO)-G-OH step II: used Boc-d,I-pipecolic
    acid
    Ac-EEVV-Tiq-nV-(CO)-G-OH step II: used Fmoc-Tiq-OH
    Ac-EEVV-C(Me)-nV-(CO)-G-OH step II: used Fmoc-
    Cys(Me)—OH
    Ac-EEVV-C(O2,Me)-nV-(CO)-G-OH step II: used Fmoc-
    Cys(O2,Me)—OH
    Ac-EEVV-C(2-AcOH)-nV-(CO)-G- step II: used Fmoc-
    OH Cys(2-AcOtBu)—OH
    Ac-EEVV-M(O2)-nV-(CO)-G-OH step II: used Fmoc-
    Met(O2)—OH
    Ac-EEVV-P(4t-Bn)-nV-(CO)-G-OH step II: used Boc-Pro(4t-Bn)-OH
    Ac-EEVV-P(4t-Bn(4-OMe))-nV- step II: used Boc-
    (CO)-G-OH Pro(4t-Bn(4-OMe))—OH
    Ac-EEVV-P(4t-allyl)-nV-(CO)-G-OH step II: used Boc-
    Pro(4t-allyl)-OH
    Ac-EEVVD-nV-(CO)-G-OH step II: used Fmoc-
    Asp(OtBu)—OH
    Ac-EEVVE-nV-(CO)-G-OH step II: used Boc-
    Glu(OtBu)—OH
    Ac-EEVVF-nV-(CO)-G-OH step II: used Fmoc-Phe-OH
    Ac-EEVV-P(4t-AcOH)-nV-(CO)-G- step II: used Boc-
    OH Pro(4t-AcOBn)-OH
    Ac-EESVP-nV-(CO)-G-OH step IV: used Fmoc-
    Ser(tBu)—OH
    Ac-EAVVP-nV-(CO)-G-OH Step V: used Fmoc-Ala-OH
    Ac-EEHVP-nV-(CO)-G-OH step IV: used Fmoc-His(Trt)-OH
    Ac-EENVP-nV-(CO)-G-OH step IV: used Fmoc-
    Asn(Trt)-OH
    Ac-EEVV-P(4t-Ph)-nV-(CO)-G-OH step II: used Boc-
    Pro(4t-Ph)—OH
    Ac-EEVV-P(3t-Me)-nV-(CO)-G-OH step II: used Boc-
    Pro(3t-Me)—OH
    Ac-EE-Orn-VP-nV-(CO)-G-OH step IV: used Fmoc-
    Orn(Boc)-OH
    Ac-EdEVVP-nV-(CO)-G-OH step V: used Fmoc-
    dGlu(OtBu)—OH
    Ac-EE-(s,s)alloT-VP-nV-(CO)-G- step IV: used Fmoc-(s,s)allo-
    OH Thr-OH
    Ac-EE-Dif-VP-nV-(CO)-G-OH step III: used Fmoc-Dif-OH
    Ac-EE-daba-VP-nV-(CO)-G-OH step III: used Fmoc-
    *Daba(Boc)-OH
    Ac-EEDVP-nV-(CO)-G-OH step IV: used Fmoc-
    Asp(OtBu)—OH
    Ac-EEEVP-nV-(CO)-G-OH step IV: used Fmoc-
    Glu(OtBu)—OH
    Ac-EETVP-nV-(CO)-G-OH step IV: used Fmoc-
    Thr(tBu)—OH
    Ac-AEVVP-nV-(CO)-G-OH step VI: used Fmoc-Ala-OH
    Ac-EELVP-nV-(CO)-G-OH step IV: used Fmoc-Leu-OH

    *Note: Daba denotes diaminobutyric acid
    • Example II Solution Phase Synthesis of Ac-EEWP-nV-(CO)-G-allylAm
  • Figure US20050059606A1-20050317-C00168
    Step I. Synthesis of Boc-nVal(CHOH)-Gly-OEt
    Figure US20050059606A1-20050317-C00169
  • Trifluoroacetic acid (4.15 mL, 41.54 mmol) was added dropwise to a cooled solution (0° C.) of Boc-nVal-aldehyde (4.18 9, 20.77 mmol) (obtained in example I, step Ib(part I)), ethylisocyanoacetate (2.72 mL, 24.93 mmol), and pyridine (6.72 mL, 83.09 mmol) in dichloromethane (83 mL). After two hours, the reaction was brought to room temperature and stirred uncapped for 48 hours. The reaction mixture was concentrated and dissolved in ethylacetate (80 mL). It was then extracted three times each with 10 mL portions of 1N sodium bisulfate, saturated sodium bicarbonate and brine. The organic layer was dried and concentrated and the remaining residue was subjected to purification using flash column chromatography in 1:4 ethylacetate: hexane followed by 2:3 ethylacetate: hexane. The desired fractions were pulled and concentrated to a yellowish flake (24.5 g, 78.6%). Thin layer chromatography in 2:3 ethylacetate: hexane showed one spot with an Rf of 0.16. Low resolution mass spectrum confirmed the desired mass (M+Na+ 489).
  • Step II. Synthesis of HCl.H-nVal(CHOH)-Gly-OEt
  • The product obtained in step I above (1.12 g, 3.38 mmol) was treated for one hour with 10 mL saturated solution of anhydrous HCl in ethanol. It was then concentrated and triturated with n-heptane to a solid (0.9 g, 99.2%).
  • Step III. Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-OH (Steps a-f Below)
  • a) Synthesis of Fmoc-Val-Pro-2ClTrt Resin
  • In a 1 L solid phase reaction vessel equipped with a nitrogen inlet, 25 g of Pro-2ClTrt resin (200-400 mesh, 0.64 mmol/g substitution) was suspended in dimethylformamide (213 mL). Fmoc-Val-OH (1.5 g, 32 mmol) was coupled for four hours according to Procedure A. A small aliquot was taken for colorimetric ninhydrin analysis which showed a 99.5% coupling efficiency in the production of the title compound.
  • b) Synthesis of Fmoc-Val-Val-Pro-2ClTrt Resin
  • The resin from the previous step (0.53 mmol/g) was deprotected according to Procedure C. It was then coupled to Fmoc-Val-OH (10.85 g, 32 mmol) according to Procedure A with 99.5% efficiency.
  • c) Synthesis of Fmoc-Glu(OtBu)-Val-Val-Pro-2ClTrt Resin
  • The resin from the previous step (0.504 mmol/g) was deprotected according to Procedure C. It was then coupled to Fmoc-Glu(OtBu)-OH (13.63 g, 32 mmol) according to Procedure A with 99.4% efficiency.
  • d) Synthesis of Fmoc-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-2ClTrt Resin
  • The resin from the previous step (0.461 mmol/g) was deprotected according to Procedure C. It was then coupled to Fmoc-Glu(OtBu)-OH (13.63 g, 32 mmol) according to procedure A with 99.2% efficiency to yield the titled compound.
  • e) Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-2ClTrt Resin
  • The resin from the previous step (0.42 mmol/g) was deprotected according to procedure C. The N-terminus was then capped according to Procedure D to yield the desired compound in 99.7% efficiency.
  • f) Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-OH
  • The resin from the previous step was transferred to a 1 L plastic bottle and cleaved in the presence of 525 ml solution of acetic acid: trifluoroethanol: dichloromethane (1:1:3) with vigorous shaking for two hours. The resin was filtered using a fritted funnel and washed 3×50 mL with dichloromethane. The brownish red filtrate was concentrated to an oil which was then treated three times with 50 ml of a 1:1 mixture of dichloromethane: n-heptane. The crude off-white powder (13 g) was then dissolved in minimum amount of methanol and purified by HPLC using a 2.2×25 cm reverse phase column, containing a C-18 resin comprised of 10 micron size gel particles with a 300 angstrom pore size, eluting with a gradient ranging from 15-55% acetonitrile in water. The pure fractions were pulled and concentrated to a fluffy, white product (7.5 g, 65%). Analytical HPLC using a 4.6×250 mm reverse phase column, containing a C-18 resin comprised of 5 micron size gel particles with a 300 angstrom pore size ran at 5-50% acetonitrile (containing 0.1 % trifluoroacetic acid) showed one peak with the retention time of 20.5 min. Low resolution mass spectrum confirmed the desired mass (MH+ 726.5).
  • Step IV. Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(CHOH)-Gly-OEt
  • The compound of step III above (Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-OH) (0.72 g, 1 mmol) was coupled to the compound of step II above (HCl.H-nVal(CHOH)-Gly-OEt) (0.27 g, 1 mmol) using HOAt (0.204 g, 1.5 mmol), HATU (0.418 g, 1.1 mmol) and diisopropylethylamine (0.87 mL, 5 mmol) in DMF at room temperature. After 18 hours, the reaction mixture was concentrated. The remaining residue was picked up in ethylacetate and washed three times each with 10 mL portions of 1N sodium bisulfate, saturated sodium bicarbonate and brine. It was then dried over sodium sulfate and concentrated to a crusty yellowish product which was taken to the next step without further purification (0.98 g). Analytical HPLC using a 4.6×250 mm reverse phase column, containing a C-18 resin comprised of 5 micron size gel particles with a 300 angstrom pore size, eluting with 5-50% acetonitrile (containing 0.1% trifluoroacetic acid) showed a 2:1 ratio of diastereomers with retention times of 21 minutes and 21.5 minutes, respectively.
  • Step V. Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(CHOH)-Gly-OH
  • To the compound obtained in step IV above (0.94 g, 1 mmol) in ethanol (15 mL) was added 1N lithium hydroxide (4 mL, 4 mmol) and the reaction was stirred at room temperature for two hours. The reaction was stopped by the addition of enough Dowex ion exchange resin (50×8-400) to obtain an acidic solution, pH ˜3. After stirring for 15 minutes, the reaction mixture was filtered and concentrated. The crude product was subjected to HPLC purification using a 5.5×30 cm reverse phase column, containing a C-18 resin comprised of 5 micron size gel particles with a 300 angstrom pore size, eluting with a 30 minute gradient using 5-30% acetonitrile in water. The desired fractions were pulled and concentrated to a white solid (238 mg, 26%).
  • Step VI. Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(CHOH)-Gly-allylamide
  • The compound of step V above (129 mg, 0.14 mmol) was coupled to allylamine (13 l, 0.17 mmol) in the presence of HOBt (58.5 mg, 0.38 mmol), EDC (54.3 mg, 0.28 mmol) and diisopropylethylamine (124 l, 0.71 mmol) in dimethylformamide (10 ml). After 18 hours, the reaction mixture was concentrated and the remaining residue was picked-up in ethylacetate and washed three times each with 5 mL portions of 1N sodium bisulfate, saturated sodium bicarbonate and brine. After drying over sodium sulfate, the organic layer was concentrated to give a white precipitate which was taken to the next step without further purification (115 mg, 85%). Analytical HPLC using a 4.6×250 mm reverse phase column, containing a C-18 resin comprised of 5 micron size gel particles with a 300 angstrom pore size, eluting with 5-50% acetonitrile (containing 0.1% trifluoroacetic acid) showed two diastereomeric peaks with retention times of 15.9 and 16.5 minutes, respectively. Low resolution mass spectrum confirmed the desired mass (M+Na+ 973.5).
  • Step VII. Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(CO)-YGly-allylamide
  • Under a stream of nitrogen gas, the product of step VI above (115.2 mg, 0.12 mmol) was dissolved in dimethylsulfoxide (5 mL) and toluene (5 mL). Water soluble carbodiimide (EDC, 232.2 mg, 1.21 mmol) was then added in one batch. The reaction mixture was cooled to 0° C. and dichloroacetic acid (52 l, 0.60 mmol) was added dropwise. Stirring at 0° C. continued for 15 minutes. The ice bath was removed and the reaction continued for two hours at room temperature. The toluene was removed under reduced pressure. The remaining solution was diluted with ethylacetate and washed three times each with 5 mL portions of 1N sodium bisulfate, saturated sodium bicarbonate and brine. It was then concentrated to a yellowish foam (85.5 mg, 74.4%).
  • Step VIII. Synthesis of Ac-Glu-Glu-Val-Val-Pro-nVal(CO)-Gly-allylamide
  • The product of step VII above (0.86 g, 0.91 mmol) was treated with a 1:1 mixture of dichloromethane: trifluoroacetic acid (20 ml) for one hour. The reaction mixture was then concentrated and the remaining residue was purified using a 2.2×25 cm reverse phase HPLC column, containing a C-18 resin comprised of 10 micron size gel particles with a 300 angstrom pore size, eluting with a 30 minutes gradient using 10-25% acetonitrile in water. The purified fractions were pulled and lyophilized to a white powder (21.5 mg, 28.5%). Analytical HPLC using a 4.6×250 mm reverse phase column, containing a C-18 resin comprised of 5 micron size gel particles with a 300 angstrom pore size, eluting with 5-75% acetonitrile (containing 0.1 % trifluoroacetic acid) showed one peak at 9.5 minutes. Low resolution mass spectrum confirmed the desired mass (MH+ 837.5).
    • Table of Compounds Synthesized According to Example II
  • COMPOUND NAME SYNTHESIS
    Ac-EEVVP-nV-(CO)-G-allylAm example II
    Ac-EEVVP-nV-(CO)-G-2PhEtAm step VI: used
    phenethylamine
    Ac-EEVVP-nV-(CO)-G-PropAm step VI: used
    propylamine
    Ac-EEVVP-nV-(CO)-G-propynylAm step VI: used
    propynylamine
    Ac-EEVVP-nV-(CO)-G-iPrAm step VI: used
    isopropylamine
    • Example III Solution Phase Synthesis of Ac-EEWP-nV-(CO)-G(Oallyl)
  • Figure US20050059606A1-20050317-C00170
    Step I. Synthesis of Fmoc-nVal-Gly-Oallyl (Steps a-c Below)
    Figure US20050059606A1-20050317-C00171
    a) Synthesis of allyl isocyanoacetate
  • a1) Ethyl isocyanoacetate (96.6 mL, 0.88 mol) was added dropwise to a chilled solution of ethanol (1.5 L) and potassium hydroxide (59.52 g, 1.0 mol). The reaction was slowly warmed to room temperature. After two hours, the precipitated product was filtered on a glass funnel and washed with several portions of chilled ethanol. The potassium salt of isocyanoacetic acid thus obtained was dried in vacuo to a golden-brown solid (99.92 g, 91.8%).
  • a2) To the product of step al (99.92 g, 0.81 mol) dissolved in acetonitrile (810 mL) was added allyl bromide (92 mL, 1.05 mol). After refluxing for four hours, a dark brown solution was obtained. The reaction mixture was concentrated and the remaining residue was picked-up in ether (1.5 L) and washed three times with water (500 ml). The organic layer was dried and concentrated to a dark brown syrup. The crude was purified by vacuum distillation at 7 mm Hg (98° C.) to a clear oil (78.92 g, 77.7%). NMR δ ppm (CDCI3): 5.9 (m, 1 H), 5.3 (m, 2H), 4.7 (d, 2H), 4.25 (s, 2H).
  • b) Synthesis of 9-fluorenylmethoxycarbonyl-norvalinal (Steps b1-b3 Below)
  • b1) Synthesis of 9-fluorenylmethoxycarbonyl-norvaline methyl ester:
  • To a chilled solution of Fmoc-norvaline (25 g, 73.75 mmol) in anhydrous methanol (469 mL) was added thionyl chloride (53.76 mL, 0.74 mol) over one hour. Thin layer chromatography in ethylacetate taken an hour later confirmed the completion of the reaction (Rf=0.85). The reaction mixture was concentrated and the remaining residue was picked-up in ethylacetate. The organic layer was washed with three 200 ml portions of saturated sodium bicarbonate followed by brine. The organic layer was dried and concentrated to afford the title product as a white solid (26.03 g) in quantitative yield. NMR δ ppm (CD3OD): 7.7 (m, 2H), 7.6 (m, 2H), 7.4 (m, 2H), 7.3 (m, 2H), 4.3 (m, 2H), 4.1 (m, 2H), 3.7 (s, 3H), 1.7 (m, 1H), 1.6 (m,1H), 1.4 (m, 2H), 0.95 (t, 3H).
  • b2) Synthesis of 9-fluorenylmethoxycarbonyl-norvalinol:
  • To the product of step b1 (26.03 g, 73.75 mmol) in tetrahydrofuran (123 mL) and methanol (246 mL) was added calcium chloride (16.37 g, 147.49 mmol). The reaction mixture was cooled to 0° C. and sodium borohydride (11.16 g, 0.3 mol) was added in several batches. Methanol (500 mL) was added to the thick paste obtained and the reaction was stirred at room temperature for 90 minutes. Thin layer chromatography in 2:3 ethylacetate: hexane confirmed the completion of the reaction (Rf=0.25). The reaction was quenched with the slow addition of 100 mL water at 0° C. The methanol was removed under reduced pressure and the remaining aqueous phase was diluted with ethylacetate (500 mL). The organic layer was washed three times each with 500 ml portions of water, saturated sodium bicarbonate and brine. The organic layer was dried over sodium sulfate and concentrated to a white solid (21.70 g, 90.5%). NMR δ ppm (CD3OD): 7.8 (m, 2H), 7.7 (m, 2H), 7.4 (m, 2H), 7.3 (m, 2H), 4.3-4.5 (m, 2H), 4.2 (m, 1H), 3.6 (s, 1H), 3.5 (s, 2H), 1.5 (m, 1H), 1.3-1.4 (m, 3H), 0.99 (m, 3H).
  • b3) Synthesis of 9-fluorenylmethoxycarbonyl-norvalinal:
  • To the product of step b2 (21.70 g, 66.77 mmol) in dichloromethane (668 mL) was added triethylamine (37.23 mL, 267.08 mmol) and the solution was cooled to 0° C. A suspension of pyridine sulfur trioxide complex (42.51 g, 267.08 mmol) in dimethylsulfoxide (96 mL) was added to the chilled solution. After one hour, thin layer chromatography in 2:3 ethylacetate: hexane confirmed the completion of the reaction. The dichloromethane was removed under reduced pressure and the remaining residue was picked-up in ethylacetate and washed with several 50 mL portions of water,1N saturated sodium bisulfate, saturated sodium bicarbonate and brine. The organic layer was concentrated to yield a white solid. Theoretical yield (21.57 g) was assumed and the reaction was taken to the next step without further purification.
    c) Synthesis of Fmoc-nVal(CHOH)-Gly-Oallyl
    Figure US20050059606A1-20050317-C00172
  • To a solution of 9-fluorenylmethoxycarbonyl-norvalinal obtained from step b3 (5.47 g, 16.90 mmol) in dichloromethane (170 mL) was added allyl isocyanoacetate (step I a2 above) (2.46 mL, 20.28 mmol) and pyridine (5.47 mL, 67.61 mmol). The reaction mixture was cooled to 0° C. and trifluoroacetic acid (3.38 mL, 33.80 mmol) was added dropwise. The reaction was stirred at 0 C for 1 h, and then at room temperature for 48 hours. Thin layer chromatography taken in ethylacetate confirmed the completion of the reaction. The reaction mixture was concentrated and subjected to flash column chromatography using a gradient composed of 20:80 ethylacetate: hexane to 70:30 ethylacetate: hexane. Fractions containing the desired product were pooled and concentrated to a white foam (6.88 g, 87.3%). TLC in-50:50 ethylacetate showed one spot (Rf=0.37). NMR δ ppm (CD3OD): 7.8 (m, 2H), 7.65 (m, 2H), 7.4 (m, 2H), 7.3 (m, 2H), 5.9 (m,1H), 5.1-5.4 (m, 2H), 4.55-4.65 (m, 2H), 4.3-4.4 (m, 2H), 4.15-4.25 (m, 1H), 4.01 (s, 1H), 3.9-4.0 (m, 3H),1.5-1.6 (m, 2H), 1.35-1.45 (m, 3H), 0.9 (m, 3H).
  • Step II. Synthesis of H-nVal(CHOH)-Gly-Oallyl
  • To Fmoc-nVal(CHOH)-Gly-Oallyl (300 mg, 0.64 mmol) (obtained in step Ic above) dissolved in tetrahydrofuran (5.8 mL) was added diethylamine (0.64 mL) and the resulting solution was stirred at room temperature for two hours. The reaction mixture was concentrated and the remaining solid was triturated with a 1:4 ether: hexane mixture. The product was collected on a glass funnel and washed several times with a 1:1 ether: hexane mixture (93.7 mg, 57%). Low resolution mass spectrum confirmed the desired mass (MH+ 245.0).
  • Step III. Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(CHOH)-Gly-Oallyl
  • The product obtained from step II above (28.2 mg, 0.11 mmol) was coupled to Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-OH (example II, step III) (84 mg, 0.11 mmol) in the presence of HOAt (23.6 mg, 0.17 mmol), HATU (48.4 mg, 0.13 mmol), diisopropylethylamine (100.8 μl, 0.58 mmol) in dimethylformamide (20 mL) for 4 hours at room temperature. The DMF was removed under reduced pressure and the remaining residue was picked up in ethylacetate and washed with 1N sodium bisulfate, saturated sodium bicarbonate and brine. After drying over sodium sulfate, the organic layer was concentrated to give a white solid (100.3 mg, 91 %) which was taken to the next step without further purification. Low resolution mass spectrum confirmed the desired mass (M+Na+ 974.5).
  • Step IV. Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(CO)-Gly-Oallyl
  • Under a stream of nitrogen gas, the product of the previous step (51.5 mg, 0.054 mmol) was dissolved in dimethylsulfoxide (1.2 mL) and toluene (1.2 mL).
  • Water soluble carbodiimide (EDC, 103.8 mg, 0.54 mmol) was then added in one batch. The reaction mixture was cooled to 0° C. and dichloroacetic acid (22.3 l, 0.27 mmol) was added dropwise. Stirring at 0° C. continued for 15 minutes. The ice bath was removed and the reaction was slowly brought to room temperature. The reaction was stopped after 90 minutes. The toluene was removed under reduced pressure. The reaction was diluted with ethylacetate and washed with 1N sodium bisulfate, saturated sodium bicarbonate and brine. It was then concentrated to a yellowish foam (40.4 mg, 79%) and taken to the next step without further purification.
  • Step V. Synthesis of Ac-Glu-Glu-Val-Val-Pro-nVal(CO)-Gly-Oallyl
  • The product of the previous step (40.4 mg, 0.042 mmol) was treated with a 1:1 mixture of dichloromethane: trifluoroacetic acid (4 mL) for two hours. The reaction mixture was then concentrated and purified on a 1×25 cm reverse phase HPLC column, containing a C-18 resin comprised of 10 micron size gel particles with a 300 angstrom pore size, eluting with a 30 minute gradient using 10-30% acetonitrile in water. The desired fractions were pulled and concentrated to a white powder (8.5 mg, 24%). Analytical HPLC using a 4.6×250 mm reverse phase column, containing a C-18 resin comprised of 5 micron size gel particles with a 300 angstrom pore size, ran at 5-50% acetonitrile (containing 0.1% trifluoroacetic acid) showed one peak at 15 minutes. Low resolution mass spectrum confirmed the desired mass (MH+ 838.0).
    • Table: Compounds Synthesized According to Example III
  • COMPOUND NAME SYNTHESIS
    Ac-EEVVPnV-(CO)-G-Oallyl example III
    Ac-EEVVP-nL-(CO)-G-Oallyl step I(b1): used Fmoc-nLeu-OH
    Ac-EEVVP-V-(CO)-G-Oallyl step I(b1): used Fmoc-Val-OH
    Ac-EEVVPL-(CO)-G-Oallyl step I(b1): used Fmoc-Leu-OH
    Ac-EEVVP-G(propynyl)-(CO)-G- step I(b1): used Fmoc-
    Oallyl Gly(propynyl)-OH
    Ac-EEVVPnV-(CO)-G-OEt step Ic: used ethyl isocyanoacetate
    Ac-EEVVP-G(allyl)-(CO)-G-Oallyl step I(b1): used Fmoc-
    Gly(allyl)-OH
    Ac-EEVVG-L-(CO)-G-Oallyl step I(b1): used Fmoc-Leu-OH,
    example II, step IIIa: used Gly-
    2ClTrt-resin
    Ac-EEVVPnV-(CO)-G-OtBu step Ic: used t-butyl
    isocyanoacetate
    Ac-EEVVP-G(allyl)-(CO)-G-OEt step Ic: used ethyl isocyanoacetate,
    step I(b1): used Fmoc-
    Gly(allyl)-OH
    Ac-EEVVP-C(Me)-(CO)-G-OMe step Ic: used methyl
    isocyanoacetate, step I(b1): used
    Boc-Cys(Me)—OH
    • Example IV Solid Phase Synthesis of Ac-EEW-G(N-Bu(4NH2,4-CO2H))-nV-(CO)-G-OH
  • Figure US20050059606A1-20050317-C00173
    Step I. Synthesis of bromoacetyl-nVal(dpsc)-Gly-PAM Resin
    Figure US20050059606A1-20050317-C00174
  • In a solid phase vessel, bromoacetic acid (1.29 g, 9.27 mmol) was coupled to the resin obtained in example I (step If) (1.5 g, 0.77 mmol) in the presence of diisopropylcarbodiimide (1.27 g, 10.04 mmol) and dimethylformamide (10 mL) for five hours. The reagents were filtered and the reaction was repeated once more for 18 hours, at which time qualitative ninhydrin test showed colorless beads and solution indicating that the reaction had gone to completion. All reagents were drained and the resin was washed thoroughly with 15 mL portions of dimethylformamide, methanol and dichloromethane.
    Step II. Synthesis of Gly(N-Bu(4NH-Boc,4-COOtBu)-nVal(dpsc)-Gly-PAM Resin
    Figure US20050059606A1-20050317-C00175
  • The product of the previous step (0.12 g, 0.06 mmol) was treated with Boc-Orn-OtBu.AcOH (0.86 g, 2.47 mmol) in the presence of diisopropylethylamine (0.05 mL, 0.31 mmol) and dimethylsulfoxide (1.2 mL) for 18 hours. The reagents were drained and the procedure was repeated for 5 hours using fresh reagents. All reagents were drained and the resin was washed thoroughly with 2 mL portions of dimethylformamide, methanol and dichloromethane.
  • Step III. Synthesis of Ac-Glu-Glu-Val-Val-Gly(N-Bu(4NH2,4-COOH)-nVal(CO)-Gly-OH
  • The following steps were carried out sequentially:
      • a) Fmoc-Val-OH (0.04 g, 0.10 mmol) was double coupled to the product of step II above (0.12 g, 0.05 mmol) according to Procedure B.
      • b) The resin was deprotected according to Procedure C and coupled to Fmoc-Val-OH (0.04 g, 0.10 mmol) according to Procedure B.
      • c) The resin was deprotected according to Procedure C and coupled to Fmoc-Glu(OtBu)-OH (0.04 g, 0.10 mmol) according to Procedure B.
      • d) The resin was deprotected according to Procedure C and coupled to Fmoc-Glu(OtBu)-OH (0.04 g, 0.10 mmol) according to Procedure B.
      • e) The resin was deprotected according to Procedure C and acylated at the N-terminus according to Procedure E.
      • f) The semicarbazone group of the product obtained in step e was hydrolyzed according to Procedure F, and the product was subjected to HF cleavage according to Procedure G. The crude material was subjected to HPLC purification using a 1×25 cm reverse phase column, containing a C-18 resin comprised of 10 micron size gel particles with a 300 angstrom pore size, eluting with a 30 minute gradient using 1040% acetonitrile in water. Analytical HPLC using a 4.6×250 mm reverse phase column, containing a C-18 resin comprised of 5 micron size gel particles with a 300 angstrom pore size, eluting with 5-75% acetonitrile (containing 0.1% trifluoroacetic acid) showed one peak at 8 minutes. Low resolution mass spectrum confirmed the presence of the desired product (MH+ 873.5).
    Table of Compounds Synthesized According to Example IV
  • COMPOUND NAME SYNTHESIS
    Ac-EEVV-G(N-Bu(4NH2,4-CO2H))-nV- example IV
    (CO)-G-OH
    Ac-EEVV-G(N-Et(CO2H))-nV-(CO)-G- step II: used -Ala(OtBu).HCl
    OH
    Ac-EEVV-G(N-EtPh(3,4diOMe))-nV- step II: used 3,4-
    (CO)-G-OH dimethoxyphenethylamine
    Ac-EEVV-G(N-Pe(5-NH2,5-CO2H))-nV- step II: used CBz-
    (CO)-G-OH Lys(OBzl).benzene sulfonate
    • Example V Solid Phase Synthesis of Ac-EEW-G(N-Et(NHBz))-nV-(CO)-G-OH
  • Figure US20050059606A1-20050317-C00176
    Step I. Synthesis of Gly(N-Et(NH-Boc))-nVal(dpsc)-Gly-PAM Resin
    Figure US20050059606A1-20050317-C00177
  • The resin obtained in example IV (step I) (0.24 g, 0.12 mmol) was treated with t-butyl N-(2-aminoethyl)-carbamate (0.78 mL, 4.94 mmol) in the presence of diisopropylethylamine (0.11 mL, 0.62 mmol) and dimethylsulfoxide (2.4 mL) for 18 hours. The reagents were drained and the procedure was repeated for 5 hours using fresh reagents. All reagents were drained and the resin was washed thoroughly with 2 mL portions of dimethylformamide, methanol and dichloromethane.
    Step II. Synthesis of Fmoc-Val-Gly(N-Et(NH-Boc))-nVal(dpsc)Gly-PAM Resin
    Figure US20050059606A1-20050317-C00178
  • Fmoc-Val-OH (0.07 g, 0.21 mmol) was double coupled to the product of step I above (0.24 g, 0.10 mmol) according to Procedure B.
    Step III. Synthesis of Fmoc-Val-Gly(N-Et(NHBz))-nVal(dpsc)-Gly-PAM Resin
    Figure US20050059606A1-20050317-C00179
  • The product obtained from step II above (0.12 g, 0.06 mmol) was treated with a 1:1 mixture of trifluoroacetic acid: dichloromethane according to Procedure D. The free amine generated was then capped with the addition of benzoyl chloride (0.02 mL, 0.19 mmol) in the presence of diisopropylethylamine (0.06 mL, 0.37 mmol) in N-methylpyrrolidine (1.24 mL).
  • Step IV. Synthesis of Ac-Glu-Glu-Val-Val-Gly(N-Et(NHBz))-nVal(CO)-Gly-OH
  • The following steps were carried out sequentially:
      • a) The resin obtained in step III above was deprotected according to Procedure C and coupled to Fmoc-Val-OH (0.04 g, 0.10 mmol) according to Procedure B.
      • b) The resin obtained in the previous step was deprotected according to Procedure C and coupled to Fmoc-Glu(OtBu)-OH (0.04 g, 0.10 mmol) according to Procedure B.
      • c) The resin obtained in the previous step was deprotected according to Procedure C and coupled to Fmoc-Glu(OtBu)-OH (0.04 g, 0.10 mmol) according to Procedure B.
      • d) The resin obtained in the previous step (0.12 g) was deprotected according to Procedure C and acylated at the N-terminus according to Procedure E.
      • e) The semicarbazone group of the product obtained in step d was hydrolyzed according to procedure F, and the product was subjected to HF cleavage according to procedure G. Analytical HPLC using a 4.6×250 mm reverse phase column, containing a C-18 resin comprised of 5 micron size gel particles with a 300 angstrom pore size, ran at 5-50% acetonitrile (containing 0.1% trifluoroacetic acid) showed one peak at 14 minutes. Low resolution mass spectrum confirmed the presence of the desired product (MH+ 905.5).
    Table of Compounds Synthesized According to Example V
  • COMPOUND NAME SYNTHESIS
    Ac-EEVV-G(N-Et(NHBz))-nV-(CO)-G- example V
    OH
    Ac-EEVV-G(N-Et(NHBzl(3-OPh)))-nV- step III: used 3-phenoxy-
    (CO)-G-OH benzoic acid as capping group
    Ac-EEVV-G(N-Prop(NHBz))-nV-(CO)-G- step I: used t-butyl N-(2-
    OH aminopropyl)-carbamate
    • Example VI
  • Solid Phase Synthesis of Ac-EEWP-nV(CO)-Am
    Figure US20050059606A1-20050317-C00180
    Step I. Formation of HOBt Ammonium Salt
  • Ammonium hydroxide (0.5 mL) was added dropwise to a slurry of HOBt (2 g, 13.07 mmol) in water (5 mL). The mixture was stirred at room temperature until a clear solution was obtained. The product was precipitated by the slow addition of acetone (50 mL). It was then filtered on a glass funnel and washed thoroughly with cold acetone (white powder, 2.23 g, 78%; mp 177-181° C.).
    Step II. Synthesis of HCl.H-nVal-(CHOH)-CONH2
    Figure US20050059606A1-20050317-C00181
  • Boc-nVal-(CHOH)—COOH (295 mg, 1.19 mmol) (example V, step II) was reacted with the product of the previous step (362 mg, 2.38 mmol) in the presence of EDC (342 mg, 1.78 mmol) in dimethylformamide (10 mL) at room temperature for 18 hours. The reaction mixture was concentrated and the remaining residue was picked up in ethylacetate (5 mL) and washed three times each with 5 mL portions of 1N sodium bisulfate, saturated sodium bicarbonate and brine. The organic layer was dried over sodium sulfate and concentrated to a white solid (170 mg, 58%). After drying in vacuo, the product was treated with SN anhydrous HCl in ethylacetate (5 mL) for 1 hour and concentrated (120 mg, 94%).
  • Step III. Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal-(CHOH)-CONH2
  • The product obtained from step II above (19 mg, 0.103 mmol) was coupled to Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-OH (example II, step IIIf) (50 mg, 0.069 mmol) in the presence of HOAt (14.1 mg, 0.103 mmol), HATu (28.8 mg, 0.076 mmol), diisopropylethylamine (60 μl, 0.345 mmol) in dimethylformamide (10 mL) for 4 hours at room temperature. The DMF was removed under reduced pressure and the remaining residue was picked up in ethylacetate and washed with 1N sodium bisulfate, saturated sodium bicarbonate and brine. After drying over sodium sulfate it was concentrated to give a white solid (40 mg, 68%) which was taken to the next step without further purification. Low resolution mass spectrum confirmed the desired mass (M+Na+ 876.5).
  • Step IV. Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal-(CO)-CONH2
  • Under a stream of nitrogen gas, the product of the previous step (40 mg, 0.047 mmol) was dissolved in dimethylsulfoxide (4 mL) and toluene (4 mL). Water soluble carbodiimide (EDC, 89.8 mg, 0.47 mmol) was then added in one batch. The reaction mixture was cooled to 0° C. and dichloroacetic acid (20 l, 0.23 mmol) was added dropwise. Stirring at 0° C. continued for 15 minutes. The ice bath was removed and the reaction was slowly brought to room temperature. The reaction was stopped after 90 minutes. The toluene was removed under reduced pressure. The reaction was diluted with ethylacetate and washed with 1N sodium bisulfate, saturated sodium bicarbonate and brine. It was then concentrated to a yellowish foam (40 mg, 53%) and taken to the next step without further purification. Low resolution mass spectrum confirmed the desired mass (M+Na+ 852.5).
  • Step V. Synthesis of Ac-Glu-Glu-Val-Val-Pro-nVal-(CO)-CONH2
  • The product of the previous step (39.9 mg, 0.047 mmol) was treated with a 1:1 mixture of dichloromethane: trifluoroacetic acid (10 mL) for two hours. The reaction mixture was concentrated and the remaining residue was subjected to HPLC purification using a 1×25 cm reverse phase column, containing a C-18 resin comprised of 10 micron size gel particles with a 300 angstrom pore size, eluting with a 30 minute gradient using 5-25% acetonitrile in water. The purified fractions were pulled and lyophilized to a white powder (3.6 mg, 10%). Low resolution mass spectrum confirmed the desired mass (MH+ 740.0).
    • Table of Compounds Synthesized According to Example VI
  • COMPOUND NAME SYNTHESIS
    Ac-EEVVP-nV-(CO)-Am example VI
    • Example VII Solid Phase Synthesis of Ac-EEW-P(4t-MeNHBzl(3-OPh))-nV-(CO)-G-OH
  • Figure US20050059606A1-20050317-C00182
    Step I. Synthesis of H-Pro(4t-MeNHFmoc)-nVal-(dpsc)-Gly-PAM Resin
    Figure US20050059606A1-20050317-C00183
  • The resin obtained from example I (step I) (0.70 g, 0.36 mmol) was coupled with Boc-Pro(4t-MeNHFmoc)-OH according to procedure B for 18 hours, with 99.98% efficiency. The resin was then subjected to deprotection according to procedure D to obtain the title product.
  • Step II. Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val -Pro(4t-MeNHFmoc)-nVal-(dpsc)-Gly-PAM Resin
  • The resin obtained from the previous step (0.7 g, 0.29 mmoJ) was double coupled to Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-OH (0.45 g, 0.72 mmol) (obtained similar to example II (step II) starting from H-Val-2ClTrt-resin) according to procedure B.
    Step III. Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro(4t-MeNH2)-nVal-(dpsc)-Gly-PAM Resin
    Figure US20050059606A1-20050317-C00184
  • The Fmoc side chain protecting group of the product obtained from the previous step was removed according to procedure C to afford the title compound.
  • Step IV. Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro(4t-MeNHBzl(3-OPh))-nVal-(dpsc)-Gly-PAM Resin
  • The resin obtained from the previous step (0.15 g, 0.05 mmol) was coupled to 3-phenoxy benzoic acid (0.02 g, 0.10 mmol) according to procedure B with 99.97% efficiency.
  • Step V. Synthesis of Ac-Glu-Glu-Val-Val-Pro(4t-MeNHBzl(3-OPh))-nVal-(CO)-Gly-OH
  • The resin obtained from the previous step was subjected to semicarbazone hydrolysis according to procedure F, followed by HF cleavage according to procedure H to yield the crude product. The material was subjected to HPLC purification using a 1×25 cm reverse phase column, containing a C-18 resin comprised of 10 micron size gel particles with a 300 angstrom pore size, eluting with a 30 minute gradient ranging from 10-35% acetonitrile in water. Analytical HPLC using a 4.6×250 mm reverse phase column, containing a C-18 resin comprised of 5 micron size gel particles with a 300 angstrom pore size, eluting with 5-75% acetonitrile (containing 0.1% trifluoroacetic acid) showed one peak at 14.5 minutes. Low resolution mass spectrum confirmed the presence of the desired product (MH+ 1049.5).
    • Table of Compounds Synthesized According to Example VII
  • COMPOUND NAME SYNTHESIS
    Ac-EEVV-P(4t-MeNHBzl(3-OPh))- example VII
    nV-(CO)-G-OH
    Ac-EEVV-P(4t-MeNHCO2Ph)-nV- step IV: used phenyl
    (CO)-G-OH chloroformate, DIEA, NMP
    Ac-EEVV-P(4t-MeNHCOPh)-nV- step IV: used benzoyl chloride,
    (CO)-G-OH DIEA, NMP
    Ac-EEVV-P(4t-MeNH-Fmoc)-nV- omitted steps III and IV
    (CO)-G-OH
    Ac-EEVV-P(4t-MeNHSO2Ph)-nV- step IV: used benzenesulfonyl
    (CO)-G-OH chloride, 2,4,6-collidine, NMP
    Ac-EEVV-P(4t-MeUreaPh)-nV- step IV: used phenyl isocyanate,
    (CO)-G-OH DIEA, NMP
    Ac-EEVV-P(4t-NH-Fmoc)-nV- step I: used Boc-Pro(4t-NH-Fmoc)-
    (CO)-G-OH OH
    • Example VIII Solid Phase Synthesis of Ac-EEW-P(4t-NHBZI)-nV-(CO)-G-OH
  • Figure US20050059606A1-20050317-C00185
    Step I. Synthesis of Boc-Pro(4t-NH2)-nVal-(dpsc)-Gly-PAM Resin
    Figure US20050059606A1-20050317-C00186
  • The resin obtained from example I (step I) (1.3 g, 0.67 mmol) was coupled with Boc-Pro(4t-NHFmoc)-QH (0.61 g, 1.34 mmol) according to procedure B for 18 hours, at which time qualitative ninhydrin test confirmed completion of the reaction. The resin was then subjected to deprotection according to Procedure C to obtain the title product.
    Step II. Synthesis of Boc-Pro(4t-NHBzl)-nVal-(dpsc)-Gly-PAM Resin
    Figure US20050059606A1-20050317-C00187
  • The resin obtained from the previous step (0.13 g, 0.05 mmol) was transferred to a fritted polypropylene tube and was suspended in N-methylpyrrolidine (1.5 mL). It was then capped with benzoyl chloride (0.02 mL, 0.16 mmol) in the presence of diisopropylethylamine (0.05 mL, 0.32 mmol) for four hours to yield the title product.
  • Step III. Synthesis of Fmoc-Val-Pro(4t-NHBzl)-nVal-(dpsc)-Gly-PAM resin
  • The compound of the previous step (100 mg, 0.05 mmol) was deprotected according to Procedure D. A ninhydrin assay on a small aliquot gave dark blue resin and solution showing a high yield for the deprotection. The resin was resuspended in N-methylpyrrolidine (1.47 mL) and coupled to Fmoc-Val-OH (0.03 g, 0.10 mmol) according to Procedure B. A small aliquot was taken for qualitative ninhydrin analysis which showed colorless beads and a dark red solution indicating a high yield of coupling.
  • Step IV. Synthesis of Fmoc-Val-Val-Pro(4t-NHBzl)-nVal-(dpsc)-Gly-PAM resin
  • The compound of the previous step (100 mg) was deprotected according to Procedure D. A ninhydrin assay on a small aliquot gave dark blue resin and solution showing a high yield for the deprotection. The resin was resuspended in N-methylpyrrolidine (1.47 mL) and was coupled to Fmoc-Val-OH (0.03, 0.10 mmol) as in step III.
  • Step V. Synthesis of Fmoc-Glu(OtBu)-Val-Val-Pro(4t-NHBzl)-nVal-(dpsc)-Gly-PAM Resin
  • The compound of the previous step (100 mg, 0.03 mmol) was deprotected according to Procedure D. A ninhydrin assay on a small aliquot gave dark blue resin and solution showing a high yield for the deprotection. The resin was resuspended in N-methylpyrrolidine (1.47 mL) and was coupled to Fmoc-Glu(OtBu)-OH (0.04 g, 0.10 mmol), according to Procedure B for 5 hours. A small aliquot was taken for qualitative ninhydrin analysis which showed colorless beads and solution indicating a high yield of coupling.
  • Step VI. Synthesis of Fmoc-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro(4t-NHBzl)-nVal-(dpsc)-Gly-PAM Resin
  • The compound of the previous step (100 mg) was deprotected according to Procedure D and coupled to Fmoc-Glu(OtBu)-OH (0.04 g, 0.10 mmol) in the same manner.
  • Step VII. Synthesis of Ac-Glu-Glu-Val-Val-Pro(4t-NHBzl)-nVal-(CO)-Gly-OH
  • The compound of previous step (100 mg) was deprotected according to Procedure C and acylated according to Procedure E. The resin was vacuum dried and a small aliquot was taken for qualitative ninhydrin analysis which showed colorless beads and solution indicating a high yield of coupling. The resin was then subjected to semicarbazone hydrolysis followed by HF cleavage reactions according to Procedures F and H, respectively. The crude product was subjected to HPLC purification using a 1×25 cm reverse phase column, containing a C-18 resin comprised of 10 micron size gel particles with a 300 angstrom pore size, eluting with a 30 minute gradient using 10-40% acetonitrile in water. Analytical HPLC using a 4.6×250 mm reverse phase column, containing a C-18 resin comprised of 5 micron size gel particles with a 300 angstrom pore size, ran at 5-50% acetonitrile (containing 0.1% trifluoroacetic acid) showed one peak at 13 minutes. Low resolution mass spectrum confirmed the presence of the desired product (MH+ 917.5).
    • Table of Compounds Synthesized According to Example VIII
  • COMPOUND NAME SYNTHESIS
    Ac-EEVV-P(4t-NHBzl)-nV-(CO)-G-OH example VIII
    Ac-EEVV-P(4t-NHBzl(4-OMe))-nV- step II: used 4-methoxy-
    (CO)-G-OH benzoyl chloride, DIEA, NMP
    Ac-EEVV-P(4t-NHBzl(4-OPh))-nV- step II: used 4-phenoxy-
    (CO)-G-OH benzoic acid
    Ac-EEVV-P(4t-NHBzl(3-OPh))-nV- step II: used 3-phenoxy-
    (CO)-G-OH benzoic acid
    Ac-EEVV-P(4t-NHBzl(3,4-OMeO))-nV- step II: used piperonyloyl
    (CO)-G-OH chloride, DIEA, NMP
    Ac-EEVV-P(4t-NHBzl(4F))-nV-(CO)-G- step II: used 4-fluoro-
    OH benzoyl chloride, DIEA, NMP
    Ac-EEVV-P(4t-NHiBoc)-nV-(CO)-G-OH step II: used isobutyl
    chloroformate, DIEA, NMP
    Ac-EEVV-P(4t-NHSO2Ph)-nV-(CO)-G- step II: used benzene sulfonyl
    OH chloride, 2,4,6-collidine,
    NMP
    Ac-EEVV-P(4t-NHSO2Ph(4-OMe))-nV- step II: used 4-methoxy-
    (CO)-G-OH benzene sulfonyl chloride,
    2,4,6-collidine, NMP
    Ac-EEVV-P(4t-UreaPh)-nV-(CO)-G-OH step II: used phenyl
    isocyanate, DIEA, NMP
    Ac-EEVV-P(4t-UreaPh(4-OMe))-nV- step II: used 4-methoxyphenyl
    (CO)-G-OH isocyanate, DIEA, NMP
    • Example IX: Solution Phase Synthesis of Ac-EEWP-nV-CO)—OH
  • Figure US20050059606A1-20050317-C00188
    Step I. Synthesis of ethyl (R,S)-2-hydroxy-3-amino hexanoate hydrochloride
    Figure US20050059606A1-20050317-C00189
  • The product obtained in example I(step Ib) (1.99 g, 6.59 mmol) was refluxed in 6N HCl (42 mL) for four hours. After cooling to room temperature, the reaction mixture was extracted with dichloromethane (3×30 mL) and the aqueous layer was concentrated to dryness (1.65 g crude). Some of the resulting product (0.88 g, 4.8 mmol) was stirred in 20 mL saturated solution of anhydrous HCl in ethanol for 90 minutes. The mixture was concentrated to a white solid (0.95 g, 93%).
  • Step II. Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(CHOH)-OEt
  • The product obtained from the previous step (88 mg, 0.41 mmol) was coupled to Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-OH (example II, step IIIf) (200 mg, 0.26 mmol) in the presence of HOAt (56.3 mg, 0.41 mmol), HATu (115.3 mg, 0.30 mmol), diisopropylethylamine (240 1, 1.37 mmol) in dimethylformamide (10 mL) for 18 hours at room temperature. The DMF was removed under reduced pressure and the remaining residue was subjected to HPLC purification using a 2.2×25 cm reverse phase column, containing a C-18 resin comprised of 10 micron size gel particles with a 300 angstrom pore size, eluting with a 30 minute gradient ranging from 15-50% acetonitrile in water. The desired fractions were pulled and concentrated to a solid (67 mg, 27.5%). Analytical HPLC using a 4.6×250 mm reverse phase column, containing a C-18 resin comprised of 5 micron size gel particles with a 300 angstrom pore size, eluting with 5-50% acetonitrile (containing 0.1 % trifluoroacetic acid) showed one peak at 23.5 minutes. Low resolution mass spectrum confirmed the desired mass (MH+ 883.5).
  • Step III. Synthesis of Ac-Glu(OtBu)-Glu(OtBu)-Val-Val-Pro-nVal(CHOH)-carboxylic acid
  • To the product obtained in the previous step (67 mg, 0.076 mmol) dissolved in ethanol (3.8 mL) was added 1N lithium hydroxide (3041, 0.304 mmol) and the reaction was stirred at room temperature for 90 minutes. The reaction was stopped by the addition of enough Dowex ion exchange resin (50×8-400) to obtain an acidic solution, pH ˜3. After stirring for 15 minutes, the reaction mixture was filtered and concentrated to a white solid (53.4 mg, 82.2%).
  • Step IV. Synthesis of Ac-Glu-Glu-Val-Val-Pro-nVal(CHOH)-carboxylic acid
  • The product obtained in the previous step (53.1 mg) stirred in a 1:1 mixture of trifluoroacetic acid: dichloromethane (10 mL) for 90 minutes. The reaction mixture was concentrated to a yellowish solid (50 mg) which was taken to the next step without further purification.
  • Step V. Synthesis of Ac-Glu-Glu-Val-Val-Pro-nVal(CO)-carboxylic acid
  • The product obtained in the previous step (55.7 mg, 0.075 mmol) was dissolved in dichloromethane (8 mL) and dimethylsulfoxide (2 mL). Triethylamine (125.5 l, 0.901 mmol) followed by pyridine sulfur trioxide (143.4 mg, 0.901 mmol) were added and the reaction was stirred at room temperature for two hours. Dichloromethane was removed under reduced pressure and the remaining residue was diluted with methanol (containing 0.1% TFA) and purified on a reverse phase HPLC column (1×25 cm) containing a C-18 resin comprised of 10 micron size gel particles with a 300 angstrom pore size, eluting with a 30 minute gradient using 5-15% acetonitrile in water. The desired fractions were pulled and concentrated to an oil (15.2 mg, 27.4%). Analytical HPLC using a 4.6×250 mm reverse phase column, containing a C-18 resin comprised of 5 micron size gel particles with a 300 angstrom pore size, ran at 5-50% acetonitrile (containing 0.1% trifluoroacetic acid) showed one peak at 11 minutes. Low resolution mass spectrum confirmed the desired mass (MH+ 741.0).
    • Table of Compound Synthesized According to Example IX
  • Ac-EEVVP-nV-(CO)-OH example IX

    Assay for HCV Protease Inhibitory Activity:
  • Spectrophotometric Assay: Spectrophotometric assay for the HCV serine protease was performed on the inventive compounds by following the procedure described by R. Zhang et al, Analytical Biochemistry, 270 (1999) 268-275, the disclosure of which is incorporated herein by reference. The assay based on the proteolysis of chromogenic ester substrates is suitable for the continuous monitoring of HCV NS3 protease activity. The substrates were derived from the P side of the NS5A-NS5B junction sequence (Ac-DTEDWX(Nva), where X=A or P) whose C-terminal carboxyl groups were esterified with one of four different chromophoric alcohols (3- or 4-nitrophenol, 7-hydroxy4-methyl-coumarin, or 4-phenylazophenol). Presented below are the synthesis, characterization and application of these novel spectrophotometric ester substrates to high throughput screening and detailed kinetic evaluation of HCV NS3 protease inhibitors.
  • Materials and Methods:
  • Materials: Chemical reagents for assay related buffers were obtained from Sigma Chemical Company (St. Louis, Mo.). Reagents for peptide synthesis were from Aldrich Chemicals, Novabiochem (San Diego, Calif.), Applied Biosystems (Foster City, Calif.) and Perseptive Biosystems (Framingham, Mass.). Peptides were synthesized manually or on an automated ABI model 431A synthesizer (from Applied Biosystems). UVNIS Spectrometer model LAMBDA 12 was from Perkin Elmer (Norwalk, Conn.) and 96-well UV plates were obtained from Coming (Coming, N.Y.). The prewarming block was from USA Scientific (Ocala, Fla.) and the 96-well plate vortexer was from Labline Instruments (Melrose Park, Ill.). A Spectramax Plus microtiter plate reader with monochrometer was obtained from Molecular Devices (Sunnyvale, Calif.).
  • Enzyme Preparation: Recombinant heterodimeric HCV NS3/NS4A protease (strain 1a) was prepared by using the procedures published previously (D. L. Sali et al, Biochemistry, 37 (1998) 3392-3401). Protein concentrations were determined by the Biorad dye method using recombinant HCV protease standards previously quantified by amino acid analysis. Prior to assay initiation, the enzyme storage buffer (50 mM sodium phosphate pH 8.0, 300 mM NaCl, 10% glycerol, 0.05% lauryl maltoside and 10 mM DTT) was exchanged for the assay buffer (25 mM MOPS pH 6.5, 300 mM NaCl, 10% glycerol, 0.05% lauryl maltoside, 5 μM EDTA and 5 pM DTT) utilizing a Biorad Bio-Spin P-6 prepacked column.
  • Substrate Synthesis and Purification: The synthesis of the substrates was done as reported by R. Zhang et al, (ibid.) and was initiated by anchoring Fmoc-Nva-OH to 2-chlorotrityl chloride resin using a standard protocol (K. Barlos et al, Int. J. Pept. Protein Res., 37 (1991), 513-520). The peptides were subsequently assembled, using Fmoc chemistry, either manually or on an automatic ABI model 431 peptide synthesizer. The N-acetylated and fully protected peptide fragments were cleaved from the resin either by 10% acetic acid (HOAc) and 10% trifluoroethanol (TFE) in dichloromethane (DCM) for 30 min, or by 2% trifluoroacetic acid (TFA) in DCM for 10 min. The combined filtrate and DCM wash was evaporated azeotropically (or repeatedly extracted by aqueous Na2CO3 solution) to remove the acid used in cleavage. The DCM phase was dried over Na2SO4 and evaporated.
  • The ester substrates were assembled using standard acid-alcohol coupling procedures (K. Holmber et al, Acta Chem. Scand., B33 (1979) 410-412). Peptide fragments were dissolved in anhydrous pyridine (30-60 mg/ml) to which 10 molar equivalents of chromophore and a catalytic amount (0.1 eq.) of para-toluenesulfonic acid (pTSA) were added. Dicyclohexylcarbodiimide (DCC, 3 eq.) was added to initiate the coupling reactions. Product formation was monitored by HPLC and found to be complete following 12-72 hour reaction at room temperature. Pyridine solvent was evaporated under vacuum and further removed by azeotropic evaporation with toluene. The peptide ester was deprotected with 95% TFA in DCM for two hours and extracted three times with anhydrous ethyl ether to remove excess chromophore. The deprotected substrate was purified by reversed phase HPLC on a C3 or C8 column with a 30% to 60% acetonitrile gradient (using six column volumes). The overall yield following HPLC purification was approximately 20-30%. The molecular mass was confirmed by electrospray ionization mass spectroscopy. The substrates were stored in dry powder form under desiccation.
  • Spectra of Substrates and Products: Spectra of substrates and the corresponding chromophore products were obtained in the pH 6.5 assay buffer. Extinction coefficients were determined at the optimal off-peak wavelength in 1-cm cuvettes (340 nm for 3-Np and HMC, 370 nm for PAP and 400 nm for 4-Np) using multiple dilutions. The optimal off-peak wavelength was defined as that wavelength yielding the maximum fractional difference in absorbance between substrate and product (product OD—substrate OD)/substrate OD).
  • Protease Assay: HCV protease assays were performed at 30° C. using a 200 μl reaction mix in a 96-well microtiter plate. Assay buffer conditions (25 mM MOPS pH 6.5, 300 mM NaCl, 10% glycerol, 0.05% lauryl maltoside, 5 μM EDTA and 5 pM DTT) were optimized for the NS3/NS4A heterodimer (D. L. Sali et al, ibid.)).
  • Typically, 150 μl mixtures of buffer, substrate and inhibitor were placed in wells (final concentration of DMSO 4 % v/v) and allowed to preincubate at 30° C. for approximately 3 minutes. Fifty pis of prewarmed protease (12 nM, 30° C.) in assay buffer, was then used to initiate the reaction (final volume 200 μl).The plates were monitored over the length of the assay (60 minutes) for change in absorbance at the appropriate wavelength (340 nm for 3-Np and HMC, 370 nm for PAP, and 400 nm for 4-Np) using a Spectromax Plus microtiter plate reader equipped with a monochrometer (acceptable results can be obtained with plate readers that utilize cutoff filters). Proteolytic cleavage of the ester linkage between the Nva and the chromophore was monitored at the appropriate wavelength against a no enzyme blank as a control for non-enzymatic hydrolysis. The evaluation of substrate kinetic parameters was performed over a 30-fold substrate concentration range (˜6-200 μM). Initial velocities were determined using linear regression and kinetic constants were obtained by fitting the data to the Michaelis-Menten equation using non-linear regression analysis (Mac Curve Fit 1.1, K. Raner). Tumover numbers (kcat) were calculated assuming the enzyme was fully active.
  • Evaluation of Inhibitors and Inactivators: The inhibition constants (Ki) for the competitive inhibitors Ac-D-(D-Gla)-L-I-(Cha)C-OH (27), Ac-DTEDWA(Nva)-OH and Ac-DTEDWP(Nva)-OH were determined experimentally at fixed concentrations of enzyme and substrate by plotting vo/vi vs. inhibitor concentration ([I]o) according to the rearranged Michaelis-Menten equation for competitive inhibition kinetics: vo/vi=1+[I]o/(Ki(1+[S]o/Km)), where vo is the uninhibited initial velocity, vi is the initial velocity in the presence of inhibitor at any given inhibitor concentration ([I]o) and [S]o is the substrate concentration used. The resulting data were fitted using linear regression and the resulting slope, 1/(Ki(1+[S]o/Km), was used to calculate the Ki* value.
  • The obtained Ki* values for the various compounds of the present invention are given in the Tables wherein the compounds have been arranged in the order of ranges of Ki* values. From these test results, it would be apparent to the skilled artisan that the compounds of the invention have excellent utility as NS3-serine protease inhibitors.
  • While the present invention has been described with in conjunction with the specific embodiments set forth above, many alternatives, modifications and other variations thereof will be apparent to those of ordinary skill in the art. All such alternatives, modifications and variations are intended to fall within the spirit and scope of the present invention.
    TABLE 3
    Ki*
    STRUCTURE NAME Range
    Figure US20050059606A1-20050317-C00190
         		Ac-EEVVP-L- (CO)-G-Oallyl
    Figure US20050059606A1-20050317-C00191
         		Ac-EEVVP-nL- (CO)-G-Oallyl a
    Figure US20050059606A1-20050317-C00192
         		Ac-EEVVP-nV- (CO)-G-Oallyl a
    Figure US20050059606A1-20050317-C00193
         		Ac-EEVVG-L- (CO)-G-Oallyl b
    Figure US20050059606A1-20050317-C00194
         		Ac-EEVVP-nV- (CO)-G-OEt c
    Figure US20050059606A1-20050317-C00195
         		Ac-EEVVP-nV- (CO)-G- 2PhEtAm c
    Figure US20050059606A1-20050317-C00196
         		Ac-EEVVP-nV- (CO)-Am b
    Figure US20050059606A1-20050317-C00197
         		Ac-EEVVP-nV- (CO)-OH c
    Figure US20050059606A1-20050317-C00198
         		Ac-EEVVP-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00199
         		Ac-EEVVP-nV- (CO)-G-OtBu b
    Figure US20050059606A1-20050317-C00200
         		Ac-EEVVP-nV- (CO)-iPrAm c
    Figure US20050059606A1-20050317-C00201
         		Ac-EEVVP- G(allyl)-(CO)-G- Oallyl b
    Figure US20050059606A1-20050317-C00202
         		Ac-EEVVP- G(allyl)-(CO)-G- OEt b
    Figure US20050059606A1-20050317-C00203
         		Ac-EEVVP-nV- (CO)-G-allylAm b
    Figure US20050059606A1-20050317-C00204
         		Ac-EEVVP-nV- (CO)-G- propynylamide c
    Figure US20050059606A1-20050317-C00205
         		Ac-EEVVP-nV- (CO)-G-Am c
    Figure US20050059606A1-20050317-C00206
         		Ac-EEVVP-nV- (CO)-G- Propylamide c
    Figure US20050059606A1-20050317-C00207
         		Ac-EEVVP- G(propynyl)- (CO)-G-Oallyl b
    Figure US20050059606A1-20050317-C00208
         		Ac-EEV-G(Ph)- P-nV-(CO)-G-OH b
    Figure US20050059606A1-20050317-C00209
         		Ac-EEVV-Sar- nV-(CO)-G-OH b
    Figure US20050059606A1-20050317-C00210
         		Ac-EEVV-Aze- nV-(CO)-G-OH a
    Figure US20050059606A1-20050317-C00211
         		Ac-EEVV-P(4t- O-2AcOH)-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00212
         		Ac-EEV-G(Chx)- P-nV-(CO)-G-OH a
    Figure US20050059606A1-20050317-C00213
         		Ac-EEVFP-nV- (CO)-G-OH b
    Figure US20050059606A1-20050317-C00214
         		Ac-EEVlP-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00215
         		Ac-EEVV-dlPip- nV-(CO)-G-OH a
    Figure US20050059606A1-20050317-C00216
         		Ac-EEVV-Tiq- nV-(CO)-G-OH a
    Figure US20050059606A1-20050317-C00217
         		Ac-EEVV-thioP- nV-(CO)-G-OH a
    Figure US20050059606A1-20050317-C00218
         		Ac-EEVV-C(Me) nV-(CO)-G-OH a
    Figure US20050059606A1-20050317-C00219
         		Ac-EEVV- C(O2,Me)-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00220
         		Ac-EEVV-C(2- AcOH)-nV-(CO)- G-OH a
    Figure US20050059606A1-20050317-C00221
         		Ac-EEVV- M(O2)-nV-(CO)- G-OH b
    Figure US20050059606A1-20050317-C00222
         		Ac-EEVVP- C(Me)-(CO)-G- OMe c
    Figure US20050059606A1-20050317-C00223
         		Ac-EEVV-P(4t- MeNHCO2Ph)- nV-(CO)-G-OH a
    Figure US20050059606A1-20050317-C00224
         		Ac-EEVV-P(4t- MeNHCOPh)- nV-(CO)-G-OH a
    Figure US20050059606A1-20050317-C00225
         		Ac-EEVV-P(4t- MeNH-Fmoc)- nV-(CO)-G-OH a
    Figure US20050059606A1-20050317-C00226
         		Ac-EEVV-P(4t- MeNHBzl(3- OPh))-nV-(CO)- G-OH a
    Figure US20050059606A1-20050317-C00227
         		Ac-EEVV-P(4t- MeNHSO2Ph)- nV-(CO)-G-OH a
    Figure US20050059606A1-20050317-C00228
         		Ac-EEVV-P(4t- NH-Fmoc)-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00229
         		Ac-EEVV-P(4t- MeUreaPh)-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00230
         		Ac-EEVV-P(4t- NHBzl)-nV-(CO) G-OH a
    Figure US20050059606A1-20050317-C00231
         		Ac-EEVV-P(4t- NHBzl(4-OMe))- nV-(CO)-G-OH a
    Figure US20050059606A1-20050317-C00232
         		Ac-EEVV-P(4t- NHBzl(4-OPh))- nV-(CO)-G-OH a
    Figure US20050059606A1-20050317-C00233
         		Ac-EEVV-P(4t- NHBzl(3-OPh))- nV-(CO)-G-OH a
    Figure US20050059606A1-20050317-C00234
         		Ac-EEVV-P(4t- Bn)-nV-(CO)- G-OH a
    Figure US20050059606A1-20050317-C00235
         		Ac-EEVV-P(4t- Bn(4-OMe))-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00236
         		Ac-EEVV-P(4t- allyl)-nV-(CO)- G-OH a
    Figure US20050059606A1-20050317-C00237
         		Ac-EEVV-P(4t- NHBzl(3,4- OMeO))-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00238
         		Ac-EEVV-P(4t- NHBzl(4F))-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00239
         		Ac-EEVV-P(4t- NHiBoc)-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00240
         		Ac-EEVV-P(4t- NHSO2Ph)-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00241
         		Ac-EEVV-P(4t- NHSO2Ph(4- OMe))-nV-(CO)- G-OH a
    Figure US20050059606A1-20050317-C00242
         		Ac-EEVV-P(4t- UreaPh)-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00243
         		Ac-EEVV-P(4t- UreaPh(4- OMe))-nV-(CO)- G-OH a
    Figure US20050059606A1-20050317-C00244
         		Ac-EEVVD-nV- (CO)-G-OH b
    Figure US20050059606A1-20050317-C00245
         		Ac-EEVVE-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00246
         		Ac-EEVVF-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00247
         		Ac-EEVV-P(4t- NH2)-nV-(CO)- G-OH b
    Figure US20050059606A1-20050317-C00248
         		Ac-EEVV-P(4t- AcOH)-nV-(CO)- G-OH a
    Figure US20050059606A1-20050317-C00249
         		Ac-EESVP-nV- (CO)-G-OH b
    Figure US20050059606A1-20050317-C00250
         		Ac-EAVVP-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00251
         		Ac-EEHVP-nV- (CO)-G-OH b
    Figure US20050059606A1-20050317-C00252
         		Ac-EENVP-nV- (CO)-G-OH b
    Figure US20050059606A1-20050317-C00253
         		Ac-EEVV-P(4t- Ph)-nV-(CO)-G- OH a
    Figure US20050059606A1-20050317-C00254
         		Ac-EEVV-P(3t- Me)-nV-(CO)-G- OH a
    Figure US20050059606A1-20050317-C00255
         		Ac-EEVV-G(N- Et(CO2H))-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00256
         		Ac-EEVV-G(N- EtPh(3,4diOMe))- nV-(CO)-G-OH b
    Figure US20050059606A1-20050317-C00257
         		Ac-EEVV-G(N- Bu(4NH2,4- CO2H))-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00258
         		Ac-EE-Orn-VP- nV-(CO)-G-OH b
    Figure US20050059606A1-20050317-C00259
         		Ac-EdEVVP-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00260
         		Ac-EE-(s,s)alloT VP-nV-(CO)-G- OH a
    Figure US20050059606A1-20050317-C00261
         		Ac-EE-Dif-VP- nV-(CO)-G-OH a
    Figure US20050059606A1-20050317-C00262
         		Ac-EE-daba-VP- nV-(CO)-G-OH b
    Figure US20050059606A1-20050317-C00263
         		Ac-EEDVP-nV- (CO)-G-OH c
    Figure US20050059606A1-20050317-C00264
         		Ac-EEEVP-nV- (CO)-G-OH b
    Figure US20050059606A1-20050317-C00265
         		Ac-EETVP-nV- (CO)-G-OH b
    Figure US20050059606A1-20050317-C00266
         		Ac-AEVVP-nV- (CO)-G-OH b
    Figure US20050059606A1-20050317-C00267
         		Ac-EELVP-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00268
         		Ac-EEVV-G(N- Et(NHBz))-nV- (CO)-G-OH b
    Figure US20050059606A1-20050317-C00269
         		Ac-EEVV-G(N- Et(NHBzl(3- OPh)))-nV-(CO)- G-OH a
    Figure US20050059606A1-20050317-C00270
         		Ac-EEVV-G(N- Prop(NHBz))-nV- (CO)-G-OH b
    Figure US20050059606A1-20050317-C00271
         		Ac-EEVV-G(N- Pe(5-NH2,5- CO2H))-nV- (CO)-G-OH a
    Figure US20050059606A1-20050317-C00272
         		Ac-EEA(1- Np)VP-nV-(CO)- G-OH b
    Figure US20050059606A1-20050317-C00273
         		Ac-EEA(2- Np)VP-nV-(CO)- G-OH b
    Figure US20050059606A1-20050317-C00274
         		Ac-EEhSVP-nV- (CO)-G-OH c
    Figure US20050059606A1-20050317-C00275
         		Ac-EEF(alpha- Me)VP-nV-(CO)- G-OH c
    Figure US20050059606A1-20050317-C00276
         		Ac-EEVLP-nV- (CO)-G-OH b
    Figure US20050059606A1-20050317-C00277
         		Ac-EEVG(t- Bu)P-nV-(CO)- G-OH a
    Figure US20050059606A1-20050317-C00278
         		Ac-EEVSP-nV- (CO)-G-OH c
    Figure US20050059606A1-20050317-C00279
         		A-EEVTP-nV- (CO)-G-OH c
    Figure US20050059606A1-20050317-C00280
         		Ac-EEV-nL-P- nV-(CO)-G-OH b
    Figure US20050059606A1-20050317-C00281
         		Ac-EEVDifP-nV- (CO)-G-OH b
    Figure US20050059606A1-20050317-C00282
         		Ac-EEVS(Me)P- nV-(CO)-G-OH c
    Figure US20050059606A1-20050317-C00283
         		Ac-EEVNP-nV- (CO)-G-OH c
    Figure US20050059606A1-20050317-C00284
         		Ac-EEVQP-nV- (CO)-G-OH c
    Figure US20050059606A1-20050317-C00285
         		Ac-EEFVP-nV- (CO)-G-OH b
    Figure US20050059606A1-20050317-C00286
         		Ac-EEVMP-nV- (CO)-G-OH b
    Figure US20050059606A1-20050317-C00287
         		Ac-EEVCP-nV- (CO)-G-OH b

Claims (54)

1. A compound, including enantiomers, stereoisomers, rotomers and tautomers of said compound, and pharmaceutically acceptable salts, solvates or derivatives thereof, with said compound having the general structure shown in Formula I:
Figure US20050059606A1-20050317-C00288
wherein:
Z is O, NH or NR12;
X is alkylsulfonyl, heterocyclylsulfonyl, heterocyclylalkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylcarbonyl, heterocyclylcarbonyl, heterocyclylalkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, heterocyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkyaminocarbonyl, heterocyclylaminocarbonyl, arylaminocarbonyl, or heteroarylaminocarbonyl moiety, with the proviso that X may be additionally optionally substituted with R12 or R13;
X1 is H; C1-C4 straight chain alkyl; C1-C4 branched alkyl or; CH2-aryl (substituted or unsubstituted);
R12 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocyclyl, heterocyclylalkyl, aryl, alkylaryl, arylalkyl, heteroaryl, alkylheteroaryl, or heteroarylalkyl moiety, with the proviso that R12 may be additionally optionally substituted with R13.
R13 is hydroxy, alkoxy, aryloxy, thio, alkylthio, arylthio, amino, alkylamino, arylamino, alkylsulfonyl, arylsulfonyl, alkylsulfonamido, arylsulfonamido, carboxy, carbalkoxy, carboxamido, alkoxycarbonylamino, alkoxycarbonyloxy, alkylureido, arylureido, halogen, cyano, or nitro moiety, with the proviso that the alkyl, alkoxy, and aryl may be additionally optionally substituted with moieties independently selected from R13.
P1a, P1b, P2, P3, P4, P5, and P6 are independently:
H; C1-C10 straight or branched chain alkyl; C2-C10 straight or branched chain alkenyl;
C3-C8 cycloalkyl, C3-C8 heterocyclic; (cycloalkyl)alkyl or (heterocyclyl)alkyl, wherein said cycloalkyl is made up of 3 to 8 carbon atoms, and zero to 6 oxygen, nitrogen, sulfur, or phosphorus atoms, and said alkyl is of 1 to 6 carbon atoms;
aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein said alkyl is of 1 to 6 carbon atoms;
wherein said alkyl, alkenyl, cycloalkyl, heterocyclyl; (cycloalkyl)alkyl and (heterocyclyl)alkyl moieties may be optionally substituted with R13, and further wherein said P1 a and P1 b may optionally be joined to each other to form a spirocyclic or,.spiroheterocyclic ring, with said spirocyclic or spiroheterocyclic ring containing zero to six oxygen, nitrogen, sulfur, or phosphorus atoms, and may be additionally optionally substituted with R13; and
P1′ is H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocyclyl, heterocyclyl-alkyl, aryl, aryl-alkyl, heteroaryl, or heteroaryl-alkyl; with the proviso that said P1′ may be additionally optionally substituted with R13.
2. The compound of claim 1, wherein X is selected from the group consisting of:
Figure US20050059606A1-20050317-C00289
wherein Alkyl is a C1 to C4 straight or branched chain, and Aryl is a phenyl or substituted phenyl.
3. The compound of claim 2, wherein X is —CO—CH3.
4. The compound of claim 2, wherein X is —CO-phenyl.
5. The compound of claim 1, wherein P5 and P6 are the same and are:
—(CH2)n—C(O)—R1, where n=14 and R1 is OH, O-t-Bu, OR3, NHR3, NH-phenyl or NH-trityl, with R3 being selected from H, C1-C4 straight or branched chain alkyl.
6. The compound of claim 1, wherein P5 and P6 are different and are:
—(CH2)n—C(O)—R1, where n=1-4 and R1 is OH, O-t-Bu, OR3, NHR3, NH-phenyl or NH-trityl, with R3 being selected from H, C1-C4 straight or branched chain alkyl.
7. The compound of claim 5, wherein P5 and P6 are —CH2—CH2—C(O)—O—C(CH3)3 or —CH2—CH2—C(O)—OH.
8. The compound of claim 6, wherein P5 and P6 are independently selected from —CH2—CH2—C(O)—O—C(CH3)3 or —CH2—CH2—C(O)—OH.
9. The compound of claim 1, wherein P3 and P4 are the same.
10. The compound of claim 1, wherein P3 and P4 are different.
11. The compound of claim 1, wherein P3 and P4 are independently selected from the group consisting of:
Figure US20050059606A1-20050317-C00290
12. The compound of claim 1, wherein P2 is selected from the group consisting of:
Figure US20050059606A1-20050317-C00291
wherein n is 0, 1, 2 or 3.
13. The compound of claim 1, wherein P1a and P1b are independently selected from the group consisting of:
Figure US20050059606A1-20050317-C00292
14. The compound of claim 1, wherein P1′ is selected from the group consisting of:
Figure US20050059606A1-20050317-C00293
15. The compound of claim 1, wherein Z is NH and X1 is H.
16. A compound, including enantiomers, stereoisomers, rotomers and tautomers of said compound, and pharmaceutically acceptable salts or solvates of said compound having the general structure shown in Formula II:
Figure US20050059606A1-20050317-C00294
wherein:
Z is O, NH or NR12;
X is alkylsulfonyl, heterocyclylsulfonyl, heterocyclylalkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylcarbonyl, heterocyclylcarbonyl, heterocyclylalkylcarbonyl, arylcarbonyl, heteroarylcarbonyl, alkoxycarbonyl, heterocyclyloxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, alkyaminocarbonyl, heterocyclylaminocarbonyl, arylaminocarbonyl, or heteroarylaminocarbonyl moiety, with the proviso that X may be additionally optionally substituted with R12 or R13;
R12 is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocyclyl, heterocyclylalkyl, aryl, alkylaryl, arylalkyl, heteroaryl, alkylheteroaryl, or heteroarylalkyl moiety, with the proviso that R12 may be additionally optionally substituted with R13;
R13 is hydroxy, alkoxy, aryloxy, thio, alkylthio, arylthio, amino, alkylamino, arylamino, alkylsulfonyl, arylsulfonyl, alkylsulfonamido, arylsulfonamido, carboxy, carbalkoxy, carboxamido, alkoxycarbonylamino, alkoxycarbonyloxy, alkylureido, arylureido, halogen, cyano, or nitro moiety, with the proviso that the alkyl, alkoxy, and aryl may be additionally optionally substituted with moieties independently selected from R13;
P1a, P1b, P2, P3, P4, P5, and P6 are independently:
H; C1-C10 straight or branched chain alkyl; C2-C10 straight or branched chain alkenyl;
C3-C8 cycloalkyl, C3-C8 heterocyclic; (cycloalkyl)alkyl or (heterocyclyl)alkyl, wherein said cycloalkyl is made up of 3 to 8 carbon atoms, and zero to six oxygen, nitrogen, sulfur, or phosphorus atoms, and said alkyl is of 1 to 6 carbon atoms; or
aryl, heteroaryl, arylalkyl, or heteroarylalkyl, wherein said alkyl is of 1 to 6 carbon atoms;
wherein said alkyl, alkenyl, cycloalkyl, heterocyclyi, (cycloalkyl)alkyl and (heterocyclyl)alkyl moieties may be optionally substituted with R13 and further wherein said P1 may optionally be a spirocyclic or spiroheterocyclic ring, with said spirocyclic or spiroheterocyclic ring containing zero to six oxygen, nitrogen, sulfur, or phosphorus atoms, and may be additionally optionally substituted with R13; and
P1′ is H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkyl-alkyl, heterocyclyl, heterocyclyl-alkyl, aryl, aryl-alkyl, heteroaryl, or heteroaryl-alkyl; with the proviso that said P1′ may be additionally optionally substituted with R13; and
Figure US20050059606A1-20050317-C00295
indicates a cyclic ring structure, with the proviso that said cyclic ring structure does not contain a carbonyl group as part of the cyclic ring.
17. The compound of claim 16, wherein said
Figure US20050059606A1-20050317-C00296
indicates a five-membered ring.
18. The compound of claim 16, wherein said
Figure US20050059606A1-20050317-C00297
indicates a six-membered ring.
19. The compound of claim 16, wherein X is selected from the group consisting of:
Figure US20050059606A1-20050317-C00298
wherein Alkyl is a C1 to C4 straight or branched chain, and Aryl is a phenyl or substituted phenyl.
20. The compound of claim 19, wherein X is —CO—CH3.
21. The compound of claim 19, wherein X is —CO-phenyl.
22. The compound of claim 16, wherein P5 and P6 are the same and are:
—(CH2)n—C(O)—R1, where n=14 and R1 is OH, O-t-Bu, OR3, NHR3, NH-phenyl or NH-trityl, with R3 being selected from H, C1-C4 straight or branched chain alkyl.
23. The compound of claim 16, wherein P5 and P6 are different and are:
—(CH2)n—C(O)—R1, where n=14 and R1 is OH, O-t-Bu, OR3, NHR3, NH-phenyl or NH-trityl, with R3 being selected from H, C1-C4 straight or branched chain alkyl.
24. The compound of claim 22, wherein P5 and P6 are —CH2CH2—C(O)—O—C(CH3)3 or —CH2—CH2—C(O)—OH.
25. The compound of claim 23, wherein P5 and P6 are independently selected from —CH2—CH2—C(O)—O—C(CH3)3 or —CH2—CH2C(O)—OH.
26. The compound of claim 16, wherein P3 and P4 are the same.
27. The compound of claim 16, wherein P3 and P4 are different.
28. The compound of claim 16, wherein P3 and P4 are independently selected from the group consisting of:
Figure US20050059606A1-20050317-C00299
29. The compound of claim 16, wherein P2 is selected from the group consisting of:
Figure US20050059606A1-20050317-C00300
wherein n=0, 1, 2, or 3; and
R2=R3=H; R2=C1 to C6 straight chainalkyl or cycloalkyl; R3=H
R4=COAlkyl (straight chain or cyclic, C1 to C6); COAryl; COOAlkyl; COOAryl
R5=H; R6=Alkyl (C1 to C3); R6=H; R5=Alkyl (C1 to C3)
R7=H; R8=Alkyl (C1 to C3), CH2OH; R8=H; R7=Alkyl (C1 to C3), CH2OH;
R7=R8=Alkyl (C1 to C3), CH2OH
R9=R10=Alkyl (C1 to C3); R9=H, R10=Alkyl (C1 to C3), COOMe, COOH, CH2OH;
R10=H, R9=Alkyl (C1 to C3), COOMe, COOH,CH2OH;
R11=Alkyl (C1 to C6 straight chain, branched or cyclic), CH2Aryl (may be substituted)
Z1=Z2=S, O; Z=S, Z2=O;Z =O,Z2=S;Z1=CH2,Z2=O;Z1=O,Z2=CH2;
Z1=S, Z2=CH2; Z1=CH2,Z2=S
Z3=CH2, S, SO2, NH, NR4
Z4=Z5=S, O
29. The compound of claim 16, wherein P1a and P1b are independently selected from the group consisting of:
Figure US20050059606A1-20050317-C00301
30. The compound of claim 16, wherein P1′ is selected from the group consisting of:
Figure US20050059606A1-20050317-C00302
31. The compound of claim 16, wherein Z is NH.
32. A pharmaceutical composition comprising as an active ingredient a compound of claim 1 or claim 16.
33. The pharmaceutical composition of claim 32 for use in treating disorders associated with Hepatitis C virus.
34. The pharmaceutical composition of claim 32 additionally comprising a pharmaceutically acceptable carrier.
35. A method of treating disorders associated with the HCV protease, said method comprising administering to a patient in need of such treatment a pharmaceutical composition which composition comprises therapeutically effective amounts of a compound of claim 1 or claim 16.
36. The method of claim 35, wherein said administration is via subcutaneous administration.
37. The use of a compound of claim 1 or claim 16 forthe manufacture of a medicament to treat disorders associated with the HCV protease.
38. A method of preparing a pharmaceutical composition for treating disorders associated with the HCV protease, said method comprising bringing into intimate contact a compound of claim 1 or claim 16 and a pharmaceutically acceptable carrier.
39. A compound exhibiting HCV protease inhibitory activity, including enantiomers, stereoisomers, rotamers and tautomers of said compound, and pharmaceutically acceptable salts or solvates of said compound, said compound being selected from the group of compounds with structures listed below:
Figure US20050059606A1-20050317-C00303
Figure US20050059606A1-20050317-C00304
Figure US20050059606A1-20050317-C00305
Figure US20050059606A1-20050317-C00306
Figure US20050059606A1-20050317-C00307
Figure US20050059606A1-20050317-C00308
Figure US20050059606A1-20050317-C00309
Figure US20050059606A1-20050317-C00310
Figure US20050059606A1-20050317-C00311
Figure US20050059606A1-20050317-C00312
Figure US20050059606A1-20050317-C00313
Figure US20050059606A1-20050317-C00314
Figure US20050059606A1-20050317-C00315
Figure US20050059606A1-20050317-C00316
Figure US20050059606A1-20050317-C00317
Figure US20050059606A1-20050317-C00318
Figure US20050059606A1-20050317-C00319
40. A pharmaceutical composition for treating disorders associated with the HCV protease, said composition comprising therapeutically effective amount of one or more compounds in claim 39 and a pharmaceutically acceptable carrier.
41. The pharmaceutical composition of claim 40, additionally containing an antiviral agent.
42. The pharmaceutical composition of claim 40 or claim 41, still additionally containing an interferon.
43. The pharmaceutical composition of claim 42, wherein said antiviral agent is ribavirin and said interferon is α-interferon.
44. A compound selected from the group consisting of:
Figure US20050059606A1-20050317-C00320
Figure US20050059606A1-20050317-C00321
Figure US20050059606A1-20050317-C00322
Figure US20050059606A1-20050317-C00323
Figure US20050059606A1-20050317-C00324
Figure US20050059606A1-20050317-C00325
Figure US20050059606A1-20050317-C00326
Figure US20050059606A1-20050317-C00327
Figure US20050059606A1-20050317-C00328
Figure US20050059606A1-20050317-C00329
Figure US20050059606A1-20050317-C00330
Figure US20050059606A1-20050317-C00331
Figure US20050059606A1-20050317-C00332
Figure US20050059606A1-20050317-C00333
Figure US20050059606A1-20050317-C00334
Figure US20050059606A1-20050317-C00335
Figure US20050059606A1-20050317-C00336
Figure US20050059606A1-20050317-C00337
Figure US20050059606A1-20050317-C00338
Figure US20050059606A1-20050317-C00339
Figure US20050059606A1-20050317-C00340
or an enantiomer, sterioisomer, rotamer or tautomer thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein the compound exhibits HCV inhibitory activity.
45. A pharmaceutical composition, comprising one or more compounds of claim 44.
47. A method of treatment of an hepatitis C virus associated disorder, comprising administering an effective amount of one or more compounds of claim 44.
47. A method of modulating the activity of hepatitis C virus (HCV) protease, comprising contacting HCV protease with one or more compounds of claim 44.
48. A method of treating, preventing, or ameliorating one or more symptoms of hepatitis C, comprising administering an effective amount of one or more compounds of claim 44.
49. The method of claim 47, wherein the HCV protease is the NS3/NS4a protease.
50. The method of claim 49, wherein the compound or compounds inhibit HCV NS3/NS4a protease.
51. A method of modulating the processing of hepatitis C virus (HCV) polypeptide, comprising contacting a composition containing the HCV polypeptide under conditions in which the polypeptide is processed with one or more compounds of claim 44.
52. The compound of claim 17, wherein P2 is selected from the group consisting of:
Figure US20050059606A1-20050317-C00341
wherein:
n is 0, 1, 2 or 3;
R20 is alkylene-COOH;
R21 is C(O)alkyl, CO2alkyl, C(O)aryl, CO2aryl, SO2alkyl, SO2aryl, CONHalkyl, or CONHaryl;
R22 is alkyl or alkylene-COOH; and
R23 is alkyl.
53. The compound of claim 52, wherein:
R20 is CH2COOH;
R21 is CO2Ph, COPh, CO2CH2-9-fluorenyl, CO-(3-phenoxyphenyl), SO2Ph or CONHPh;
R22 is methyl or CH2COOH; and
R23 is methyl.
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