CA2224211A1 - Syntheses of optically pure compounds useful as garft inhibitors and their intermediates - Google Patents

Abstract

The invention relates to methods of making compounds of formula (VII) and their enantiomers, where Ar is a substituted or unsubstituted five- or sixmembered aromatic group and B is either an amino acid linked through the amino portion to form an amide or a C1-C6 alcohol linked through the alcohol portion to form an ester. These compounds are advantageously employed as intermediates to prepare optically pure compounds that are active GARFT inhibitors. In one method, a compound of formula (III) or (XVII) is reacted with a compound of formula (IV), where X is a halogen.

Description

CA 02224211 1997-12-0~

WO 96/40674 PCT/US96/089~3 SYNY~ Rs OF OPTIC~ALLY P~RIZ COhrC)uNvo U:jh~lJh A~ GARFT T~T~TTORS AND TE~13IR INTFRI~IATl~S

Field of Invention The present invention relates to methods of making optically pure compounds that inhibit the enzyme glycinamide ribonucleotide formyl transferase (GARFT). The invention also relates to the optically pure intermediates useful for making the optically pure GARFT-inhibiting compounds and to the syntheses of these intermediates.
Backqround of Invention GARFT is a folate-dependent enzyme in the de novo purine biosynthesis pathway. This pathway is critical to cell division and proliferation. Shutting down this pathway is known to have an antiproliferative effect, in particular, an antitumor effect.
Compounds that inhibit GARFT consequently inhibit the growth and proliferation of the cells of higher organisms or microorganisms such as bacteria, yeast and fungi.
A number of folate analogs have been synthesized and studied for their ability to inhibit GARFT. A prototypical specific tight-binding inhibitor of GARFT, 5,10-dideazatetrahydrofolic acid, has been reported to show antitumor activity. See F.M.
Muggia, "Folate antimetabolites inhibitor to de novo purine synthesis," New Druqs, Concepts and Results in Cancer Chemothera~y, Kluwer Academic Publishers, Boston (1992), 65-87.

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W O 96/40674 rCTfUS96/08953 Antifolates or antifoles are a subclass of antiproliferative antimetabolites and are antagonists of the vitamin folic acid.
Typically, antifolates closely resemble the structure of folic acid and incorporate the characteristic P-benzoyl glutamate moiety of folic acid. The glutamate moiety of folic acid takes on a double negative charge at physiological pH. Thus, this compound and its analogs have an active energy-driven transport system to cross the cell membrane and exert a metabolic effect.
Compounds useful as antiproliferative agents or GARFT
inhibitors, such as certain glutamic acid derivatives, have been recently developed as described in U.S. Application Serial No. 08/
282,293 to Varney et al., filed July 28, 1994, and in International Application PCT/US94/00418 to Varney et al., filed on January 18, 1994, the disclosures of which are herein incorporated by reference. Such compounds also have antitumor, antiinflammatory, antipsoriatic and/or immunosuppressive activity.
The present invention relates to advantageous methods of making such compounds in optically pure forms in good yields. The invention also relates to optically pure intermediates useful for making the antiproliferative agents/GARFT inhibitors and to methods of making these intermediates.
Summarv of Invention In light of the pharmacological activity of GARFT-inhibiting or antiproliferative compounds, it would be desirable to develop a method for conveniently preparing such co...~ounds in optically pure forms. As generally understood by those skilled in the art, an CA 02224211 1997-12-0~

W096/40674 PCT~S96/08953 optically pure compound having one chiral center (i.e., one asyl~unetric carbon atom) i8 one that consist9 primarily of one of the two po9sible enantiomers (i.e., is enantiomerically pure), and an optically pure compound having more than one chiral center is one that is diastereomerically pure. As used herein, the term ~optically pure" is intended to mean a compound which consists of at least a sufficient amount of a single enantiomer (or diastereomer in the case of plural chiral centers) to yield a compound having the desired pharmacological activity. Preferably, "optically pure" is intended to mean a compound that consists of at least 90% of a single isomer (80~ enantiomeric or diastereomeric excess), preferably at least 95~ (90~ e.e. or d.e.), more preferably at least 97.5~ (95~ e.e. or d.e.), and most preferably at least 99~ (98~ e.e. or d.e.).
An object of the present invention is to develop methods for preparing in good yields GARFT- inhibiting, antiproliferative compounds in optically pure forms. More specifically, an object of the invention is to develop a method for synthesizing optically pure compounds of formula ~ ~Ar~ N~CC2R2 ~2N N H

where A is an oxygen, sulfur or selenium atom, Ar is a substituted or unsubstituted five- or six-membered aromatic group, and Rl and W O 96/40674 PCTAUS96/08953 ' R2 are ~ r~n~~~tly a hyd-oye., atom or a moiety that forms together with thc att~rhe~ C02 a readily hydrolyzable ester group.
Additional objects and advantages of the invention will be apparent from the detailed description, which follows.
The objccts of the invention have been achieved by a method of preparing optically pure compo~n~ of formula I

~ A ~ Ar ~ ~ ~ co,~, by reacting a cu...~ound of formula ~o ~ Ar 8 J ~ or its enantiomer -o ~~

where Ar is a substituted or un~ubstituted five- or six-membered aromatic group and B is either an amino acid linked through the amino portion to form an amide or a C1-C6 alcohol linked through the alcohol portion to form an ester, under conditions suitable to form a compound of formula I that is optically pure.
The diol compounds of formula VII and their enantiomers are optically purc intcrmediates uscful for making the optically pure GARFT inhibitors and antiproliferative agents. In formula VII, preferably Ar is a S-~emhered aromatic group having a sulfur ring atom. A preferrcd amino acid for B is dicthyl glutamatc, and a w096l40674 PCT~S96~8953 preferred Cl-C6 alcohol for B i9 methanol or ethanol. A preferred intermediate is 5-(3(S)-4-dihydroxy-butyl)-thiophene-2-carboxylic acid ethyl ester.
Intermediate compounds of formula VII and their enantiomers are advantageously prepared in accordance with the invention by a method comprising:
(a) reacting a compound of the formula III
o XJ m or its enantiomer o"",~/

~ J XVII

with a compound of the formula IV

~ Ar~B

O ~v where X is bromo, fluoro, chloro or iodo, and B and Ar are as defined above, to form a compound of the formula V

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W096l40674 PCT~S961089 ~ Ar~B

~0 v where Ar and B are as defined above, or its enantiomer;
(b) reacting the compound of formula V or its enantiomer with a reducing agent to obtain a compound of formula VI

~ o ~ Ar ~ B

where Ar and B are as defined above, or its enantiomer; and (c) reacting the compound of formula VI or its enantiomer with an acid under conditions suitable to form the compound of formula VII or its enantiomer.
Preferably, in step (a) the reaction is carried out in the presence of (i) a transition-metal catalyst containing one or more metals selected from palladium, copper and nickel, and (ii) a non-nucleophilic auxiliary base (iii) in a solvent in which at least one of the reactant compounds is at least partially soluble;
in step (b) the reducing agent is hydrogen gas in the presence of a metal catalyst containing palladium or platinum; and in step (c) the acid is p-toluenesulfonic acid.monohydrate and the reaction is carried out in methanol or ethanol.

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WO 96/40674 PCI'/US96/08953 ' other features and advantages of the invention will ~e apparent from the following detailed description, which illustrates preferred ~mho~iments of the invention.
Detailed Descr;Dtlon and Preferred ~mbo~iments of Invention A~ explained above, the invention relates generally to a method of making optically pure forms of antiproliferative co...pounds capable of inhibiting GARFT of formula I and their pharmaceutically acceptable salts:

~ ~ ~ Ar ~ ~ CO~

where:
A is an oxygen, sulfur or selenium atom;
Ar is a substituted or unsubstituted five- or six-membered aromatic group; and Rl and R2 are independently a hydrogen atom or a moiety that forms together with the attached C02 a readily hydrolyzable ester group. The carbon at position 6 shown in formula I is in either the pure R or the pure S configuration.
Preferably, Rl and R2 are independently selected from hydrogen, Cl-C6 alkyl, hydroxyalkyl, alkylaryl and arylalkyl.
More preferably, R1 and R2 are each hydrogen or a C1-C2 alkyl.
Preferably, A is sulfur.
Ar is preferably a five-membered aromatic group that i~
either substituted or unsubstituted. Illustrative example~ of CA 02224211 1997-12-0~

W096/40674 PCT~S96/08953' suitable substituents for the substituted species of Ar include alkyl groups such as methyl, ethyl and propyl, substituted alkyl groups, such as trifluoromethyl, and halogens, quch as bromo and chloro.
Preferred compounds of formula I are those of the subgenus defined by formula II:

where:
A' is sulfur or selenium;
Y is oxygen or sulfur;
C and D are independently hydrogen or a substituted or unsubstituted C1-C6 alkyl group; and Rl and R2 are independently hydrogen or a moiety that forms together with the attached CO2 a readily hydrolyzable ester group.
The C6 (asymmetric) carbon is in either the pure R or the pure S
configuration.
Preferably, R1 and R2 are independently selected from hydrogen, Cl-C6 alkyl, hydroxyalkyl, alkylaryl and arylalkyl.
More preferably, these substituents are independently hydrogen or a Cl-C2 alkyl. A i8 preferably sulfur. Preferably, Y is sulfur.
In preferred embodiments, C is hydrogen or methyl, and D is hydrogen or methyl. Illustrative examples of suitable substituents for the substituted species of C and D include alkyl groups such as methyl and ethyl.

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Although the compounds are depicted in formulae I and II in the 4-oxo form and are referred to as such throughout this description, the oxo group exists in tautomeric equilibrium with the corresponding 4-hydroxy group. It therefore will be understood that the tautomeric hydroxy forms are also intended to be covered by the formulae.
The compounds of formulae I and II in which R1 and R2 are hydrogen are active antitumor and antiproliferative compounds.
The compounds where Rl and R2 are each a moiety, preferably an ethyl group, that forms with the attached C02 a readily hydrolyzable ester group, are intermediates for forming the free glutamic acid forms of the compounds and can also be hydrolyzed in vi~o and thus act as prodrugs.
The pharmaceutically acceptable salts of the compounds of formulae I and II include, e.g., non-toxic metal salts, such as alkaline metal and alkaline earth metal salts, and substituted and unsubstit~ted ammonium salts of the glutamic acid compounds of the above formulae. Exemplary salts include sodium, potassium, lithium, calcium, magnesium, and substituted and unsubstituted pyridinium salts.
The invention relates to methods of making both chiral forms (C6 = asymmetric carbon atom) of compounds of formula I or II. In a preferred embodiment for making optically pure C6 (R) forms of the compounds of formula I or II, the following compound of formula III:

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W096/40674 PCT~Sg6/08953 o~/

XJ m is reacted under suitable conditions with a compound of formula IV:

o ~

where X is a halogen (bromo, fluoro, chloro or iodo), preferably bromo; B is either an amino acid, preferably diethyl glutamate, linked through the amino portion to form an amide, or a Cl-C6 alcohol, preferably methyl or ethyl alcohol, linked through the alcohol portion to form an ester; and Ar is as defined in formula I.
The compound of formula III may be prepared in accordance with methods known to the art, such as described in J. Am. Chem.
Soc., vol. 111 (1989), 7664-7665.
The reaction of the compounds of formulae III and IV is preferably carried out in the presence of (i) a suitable transition-metal catalyst, preferably palladium, copper, nickel or a mixture of two or more of these metals, and (ii) a non-nucleophilic auxiliary base, preferably a substituted amine (e.g., triethylamine or diethylamine), (iii) in a solvent in which at least one of the reactant compounds is at least partially CA 02224211 1997-12-0~

W O 96/40674 PCTrUS96/08953 ' soluble. The reaction i8 performed under conditions sufficient to obtain a ~ ound of formula V:

~ Ar~_"
X V

where Ar is as defined in formula I and B is as defined in formula IV.
The compound of formula V is then reduced under suitable conditions to obtain a compound of the formula VI:

vo~,~ Ar~B

/\ ~ o o ~

where Ar is as defined in formula I and B is as defined in formula IV. Preferably, the compound of formula V is reduced with hydrogen gas in the presence of a suitable metal catalyst, preferably palladium or platinum.
The compound of formula VI is reacted under conditions suitable to obtain an intermediate compound of formula VII:

Ho~~Ar~B

HO~ V~

CA 02224211 1997-12-0~

W096/40674 PCT~S96/08953' where Ar is a~ defined in formula I and B is as defined in formula IV. Preferably, the cG,.-~ound of formula VI i9 reacted with an acid, preferably p-toluenesulfonic acid.monohydrate, in an alcoholic solvent, preferably methanol or ethanol.
The compound of the formula VII is reacted with a sulfonylating agent, preferably p-toluenesulfonyl chloride or methanesulfonylchloride, in the presence of a non-nucleophilic base, preferably triethylamine or diisopropylethyl amine, to give an intermediate mono-sulfonylated compound. This intermediate is then reacted with a nitrogen nucleophile, preferably sodium azide, to obtain the compound of the formula VIII:

Ho~ Ar~B

N3 V~I

where Ar is as defined in formula I and B is as defined in formula IV.
The azide functionality of the compound of formula VIII is reduced under suitable conditions, preferably hydrogen gas in the presence of a metal catalyst, and subsequently protected with a suitable nitrogen-protecting group, preferably t-butoxycarbonyl, benzyloxycarbonyl or benzyl, to provide a compound of formula IX:

CA 02224211 1997-12-0~

W O 96/40674 PCTrUS96/08g53 Ho~~Ar~B

where Ar is as defined in formula I; B is as defined in formula IV; and R4 and R5 are independently hydrogen or a suitable nitrogen-protecting group that is readily removed in the presence of another sensitive functionality. Preferred nitrogen-protecting groups for R4 and R5 are t-butoxycarbonyl, benzyloxycarbonyl and benzyl.
The compound of formula IX is then reacted under conditions suitable to obtain a compound of the formula X:

AcA ~ ~Ar~B

RsR4N x where A and Ar are as defined in formula I; B is as defined in formula IV; R4 and R5 are as defined in formula IX; and Ac is an acyl group, preferably acetyl.
Preferably, the compound of formula IX is reacted with an acylating or sulfonylating agent, preferably methanesulfonyl chloride or p-toluenesulfonyl chloride, in the presence of a non-nucleophilic base, preferably triethylamine or diisopropylethylamine, in a suitable solvent in which at least one CA 02224211 1997-12-0~

w096/40674 PCT~S96/08953 of the reactants is at least partially soluble, to obtain an activated hydroxy group. The activated hydroxy group is subsequently di9placed with a suitable nucleophile, preferably a thioacid salt, more preferably potassium thioacetate, to obtain a cG~ ound of formula X. Alternatively, the co..-~ound of formula IX
is conveniently converted to the compound of formula X using (i) triphenylphosphine, (ii) diethyl, diisopropyl or dimethyl azadicarboxylate, and (iii) an acidic nucleophile, preferably thioacetic acid, (iv) in a suitable solvent.
The compound of the formula X is reacted under conditions suitable to obtain a compound of formula XI:
R5o~ y A ~ ~Ar ~ B

R,O,C o RsR4N~ ~1 where A and Ar are as defined in formula I; B is as defined in formula IV; R4 and R5 are as defined in formula IX; and R6 is hydrogen or a moiety that forms together with the attached C02 a readily hydrolyzable ester group, preferably C1-C6 alkyl, hydroxyalkyl, alkylaryl or aralkyl, more preferably a C1-C2 alkyl.
Preferably, the compound of formula X is treated with a nucleophilic base, preferably potassium carbonate, sodium carbonate, sodium hydroxide or potassium hydroxide, in an alcoholic solvent, preferably methanol, ethanol or isopropanol, in the presence of an alkylating agent, preferably dimethyl or diethyl chloromalonate, to obtain a compound of formula XI.

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Alternatively, the ~o~ ound of formula X i9 reacted with a nucleophilic base, preferably potassium carbonate, sodium carbonate, sodium hydroxide or potassium hydroxide, in an alcoholic solvent, preferably methanol, ethanol or isopropanol, to generate the oxidized dimer to obtain a compound of the formula XII:

~ ~ A ~ B

RsR4N XII

where A and Ar are as defined in formula I, B is as defined in formula IV, and R4 and R5 are as defined in formula IX. The compound of formula XII is then reacted with a reducing agent, preferably sodium borohydride, in an alcoholic solvent, preferably methanol or ethanol. The reduced product is then alkylated with a suitable alkylating agent, preferably diethyl or dimethyl chloromalonate, in the presence of a non-nucleophilic base, preferably sodium or potassium carbonate, to obtain the compound of formula XI.
The compound of the formula XI is then treated to remove either or both of the R4 and R5 protecting groups. For example, where a protecting group is t-butoxycarbonyl, the compound may be treated with trifluoroacetic acid and then neutralized to obtain a compound of the formula XIII:

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W O 96/406~4 ~ PCTAUS96108953 ' ~ ~Ar~B

~ / o O N xm where A and Ar are as defined in formula I, B i9 as defined in formula IV, and R6 is as defined in formula XI.
The compound of formula XIII is then converted under suitable conditions to a compound of formula XIV:

HN~ ~ ~ A ~ B

H2N N H XIV ~

where A and Ar are as defined in formula I, and B is as defined in formula IV.
Preferably, the compound of formula XIII is reacted with an alkylating agent, preferably trimethyl or triethyl oxonium tetrafluoroborate, in a suitable solvent, preferably dichloromethane, to form an intermediate lactim ether. The intermediate lactim ether is then reacted with guanidine in an alcoholic solvent, preferably methanol, ethanol or isopropanol, to form a compound of formula XIV. Alternatively, the compound of the formula XIII can be converted to the compound of the formula XIV by reacting the compound of the formula XIII with a thiolating agent, preferably P2S5 or (2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide), to form the thiolactam intermediate. The ether is then alkylated with an alkylating CA 02224211 1997-12-0~

W O 96/40674 PCTrUSg6/08953 agent, preferably methyl iodide or trimethyl or triethyl oxonium tetrafluoroborate, and then with guanidine in an alcoholic solvent, preferably methanol, ethanol or isopropanol, to obtain the compound of the formula XIV.
When B is an alcohol function so that the group attached with B forms an ester group, the co~ ound of the formula XIV may be hydrolyzed under basic conditions to form a compound of the formula XV: ~
HN ~ A ~ " ~ ~C~H

H2N N H ~ XV
where A and Ar are as defined in formula I.
The compound of the formula XV can be peptide-coupled by means well known to those skilled in the art with a glutamic acid diester hydrochloride to form a diester of the formula XVI:

H2~ )~Ar~N~cqR~

where A, Ar, R1, and R2 are as defined in formula I except that neither R1 nor R2 is hydrogen.
Finally, if desired, the compound of the formula XVI is hydrolyzed to the free glutamic acid form depicted in formula I
(i.e., both Rl and R2 are H).
Two methods may be used to prepare optically pure C6 (S) forms of the compounds depicted by formula I. The first method CA 02224211 1997-12-0~

w096/40674 PCT~S96/08953 begin~ with the compound of the formula XVII (J. Am. Chem. Soc., vol. 111 (1989), 7664-7665):
0"""~ .

X J XVII

This compound is the enantiomer of the compound of the formula III. The compound of the formula XVII i9 then used in a manner analogous to that described above employing the compound of the formula III and the remainder of the synthesis is as described abo~e, yielding an enantiomer of the formula XVI.
The second method to prepare the C6 (S) forms of the compounds of the formulae I and II employs the compound of formula VII. The compound of formula VII is reacted with a suitable protecting group, preferably a silylating agent, more preferably t-butyldimethylsilyl chloride, in the presence of a non-nucleophilic base, preferably triethylamine, in a suitable solvent, preferably dichloromethane, to obtain a compound of the formula XVIII:
Ho~~Ar~B

R~O XVI~

where Ar is as defined in formula I, B is as defined in formula IV, and R7 is a suitable hydroxy-protecting group, preferably t-butyldimethylsilyl.

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w096/40674 PCT~S96/08953 The compound of formula XVIII is reacted with an acylating or sulfonylating agent, preferably methanesulfonyl chloride, to obtain a compound of formula XIX:

R90 ~ Ar ~ B

~1~ ~

In formula XIX, Ar is as defined in formula I, B is as defined in formula IV, R7 is as defined in formula XVIII, and R8 i9 an acyl or sulfonyl group, preferably methanesulfonyl.
The compound of formula XIX is reacted with a reage~t suitable to remove the protecting group R7 and form an alcohol.
Where R7 is a silyl group, a preferred reagent is a fluoride salt, preferably tetrabutylammonium fluoride. The resulting alcohol is then treated with a strong non-nucleophilic base, preferably sodium or potassium hydride, to obtain a compound of formula XX:

~,~Ar ~B

xX
where Ar is as defined in formula I, and B is as defined in formula IV.

The compound of formula XX is reacted with a nitrogen-containing anion, preferably sodium azide, in the presence of a Lewis acid, preferably lithium or magnesium perchlorate, in a CA 02224211 1997-12-0~

w096/40674 PCT~S96/089~3 ' suitable ~olvent, preferably acetonitrile, to obtain a compound of formula XXI:

HO"" ~Ar~B

where Ar i8 as defined in formula I, and B is as defined in formula IV.
The compound of formula XXI is the enantiomer of the compound of formula VIII. The compound of formula XXI i9 processed in a manner analogous to that described above for the compound of formula VIII to prepare the compounds of the formula I having the C6 (S) configuration.
Exemplary compounds of the formula I made in accordance with the invention include: t2-(5-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-6][1,4]thiazin-6(R)-yl)-ethyl]-thiophene)-2-L-glutamic acid) diethyl ester; (2-(5-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-6][1,4]thiazin-6(S)-yl)-ethyl]-thiophene)-2-L-glutamic acid) diethyl ester;
2-(5-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-6][l~4]thiazin-6(R)-yl)-ethyl]-thiophene)-2-L-glutamic acid;
2-(5-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido [5,4-6][1,4]thiazin-6(S)-yl)-ethyl]-thiophene)-2-L-glutamic acid;
(2-(5-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido-[5,4-6][1,4]thiazin-6(R)-yl)-ethyl]-4-methylthiophene)-2-L-glutamic acid) diethyl ester; (2-(5-[2-(2-amino-4-oxo-CA 02224211 1997-12-0~

w096/40674 PCT~S96108953 4~6~7~8-tetrahydro-3H-pyrimido~5~4-6][l~4]thiazin-6(s)-yl)-ethyl]-4-methylthiophene)-2-L-glutamic acid) diethyl ester;
2-(5-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-6]-[l~4]thiazin-6(R)-yl)-ethyl]-4-methylthiophene)-2-L-glutamic acid; 2-(5-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-6][1,4]thiazin-6(S)-yl)-ethyl]-4-methylthiophene)-2-L-glutamic acid; (2-(5-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-6]tl,4]thiazin-6(R)-yl)-ethyl]-3-methylthiophene)-2-L-glutamic acid) diethyl ester;
(2-(5-[2-t2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido-[5~4-6][l~4]thiazin-6(s)-yl)-ethyl]-3-methylthiophenej-2-L-glutamic acid) diethyl ester; 2-(5-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-6][1,4]thiazin-6(R)-yl)-ethyl]-3-methylthiophene)-2-L-glutamic acid; and 2-(5-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-6]-[1,4]thiazin-6tS)-yl)-ethyl]-3-methylthiophene)-2-L-glutamic acid.
The compounds of the formulae I and II and their salts are particularly useful in the treatment of m~mT~1 ian hosts, such as human hosts, and in the treatment of avian hosts. These compounds produce any one or more of an antiproliferative, antibacterial, antiparasitic, antiviral, antipsoriatic, antiprotozoal, anticoccidial, antiinflammatory, immunosuppressive and antifungal effect. The compounds are especially useful in producing an antitumor effect in a vertebrate host harboring a tumor.

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W096/40674 PCT~S96/08953 The compounds of the formulae ~ and II, as well as their pharmaceutically acceptable salts, may be incorporated into convenient do9age forms, such as capsules, tablets and injectable preparations. Such pharmaceutical preparations may be prepared following conventional techniques of the pharmaceutical chemist involving steps such as mixing, granulation and compressing when necessary for tablet forms, or miY;n~, filling and dissolving the ingredients as appropriate, to give the desired products for oral, parenteral, topical, intravaginal, intranasal, intrabronchial, intraocular, intraaural and rectal ~min; stration.
Solid or liquid pharmaceutically acceptable carriers, diluents or excipients may be employed in the pharmaceutical compositions. Solid carriers include starch, lactose, calcium sulphate dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid. Liquid carriers include syrup, peanut oil, olive oil, saline solution and water. The carrier or diluent may include any prolonged-release material, such as glyceryl monostearate or glyceryl distearate, alone or with wax. When a liquid carrier is used, the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid (e.g. solution) or a nonaqueous or aqueous liquid suspension.
A dose of a composition contains at least an effective quantity of the active compound (i.e., a compound of the formula I
or a pharmaceutically acceptable salt thereof) and preferably is made up of one or more pharmaceutical dosage units. An effective CA 02224211 1997-12-0~

quantity i8 a quantity Rufficient to inhibit the folate metabolic pathway and derive the beneficial effects therefrom, e.g., through a~m;~;qtration of one or more of the pharmaceutical dosage units.
An exemplary daily dose for a vertebrate host contains an amount up to one gram of active compound per kilogram body weight of the host, preferably one-half of a gram, more preferably 100 milligrams, and even more preferably, about SO milligrams or less, per kilogram of the host weight. The selected dose may be administered to a warmblooded animal or mammal, for example, a human patient in need of treatment mediated by folate metabolic pathway inhibition, by any known method of administrating the dose including: topically, for example, as an ointment or cream;
orally; rectally, for example, as a suppository; parenterally by injection; or continuously by intravaginal, intranasal, intrabronchial, intraaural or intraocular infusion.
Preferred embodiments of the invention are described in the following examples, which are provided for illustrative purposes.
~xam~le 1: Pr-paration of 2-(5-~2-(2-Amino-4-oxo-4,6,7,8-t-trahydro-3~-pyr~mldo[5,4-61tl,4~thiazln-6(R)-yl)-ot_yl]-thiophene)-2-~-glutamic acid (lS) (a) ~ ation of 5-(2,2-Dimethyl-~1,3]dioxolan-4(S)-yl--t-_ynyl)-thioph~n~-2-carboxylic acid ethyl ~ster (1):
o X~~ ~ ~ I O~
o (1) CA 02224211 1997-12-0~

w096/40674 PcT~S9~089~

A solution cont~;n;n~ 75.61 g (0.60 mol) of crude 4-ethynyl-2,2-dimethyl-~1,3]dioxolane, 100.65 g (0.43 mol) of 5-bromo-thiophene-2-carboxylic acid ethyl ester (J. Am. Chem. Soc., vol.
111 (1989), 7664-7665), 14.84 g (0.01 mol) of tetrakis-(triphenylphosphine)-palladium(0), 4.B9 g (0.03 mol) of copper~I) iodide and 119 ml ~0.85 mol) of triethylamine in 700 ml of degassed acetonitrile was stirred under argon for 18 hours. The reaction mixture was concentrated under reduced pressure to a thick slurry, which was filtered through a sintered glass filter with washing of the filter cake with hexanes:EtOAc (20:1) and small amounts of CH2C12. The filtrate was concentrated under reduced pressure and the resulting residue was flash-chromatographed on silica gel, eluting 5-16~ EtOAc ~ethyl acetate) in hexanes. The yield of 5-(2,2-dimethyl-[1,3]dioxolan-4~S)-yl-ethynyl)-thiophene-2-carboxylic acid ethyl ester tl) was 74.61 g ~62~) as an amber oii.
[~]589 +36.6~ ~c = 0.88, MeOH).
IR ~neat) 2986, 2226, 1715, 1451, 1255, 1223, 1096, 1065 cm lH NMR ~CDC13) ~: 1.34 (t, 3H, J - 7.0 Hz), 1.42 ~s, 3H), 1.53 ~s, lH), 4.03 ~dd, lH, J - 6.2, 1.8 Hz), 4.34 (dd, lH, J =
6.4, 1.6 Hz), 4.34 (q, 2H, J ~ 7.1 Hz), 4.95 (dd, lH, J e 6.4, 0 Hz), 7.16 ~d, lH, J ~ 3.9 Hz), 7.63 ~d, lH, J - 3.9 Hz).
Anal. calc'd for C14H16O4S: C, 59.9B; H, 5.75; S, 11.44.
Found: C, 59.96; H, 5.71; S, 11.36.

CA 02224211 1997-12-0~

W O 96t40674 PCTnUS96/08g53 t~) Pr-p-r-tlon of S-t2-~2,2-Dlm thYl-11,3]-~ -4(8)-yl)-~thyl]-th~o~ o-2-c~rbo~yllc ~c~d ~thyl ~st-r (2):
- ~0 ><o~~S o~\~

~ (2) A Parr flask containing 75.10 g (268 mmol) of the acetylene compound (1) and 12.00 g of S~ Pd/C in 500 ml of EtOH ~ethanol) was shaken under 45 psi of hydrogen for 3 hours. The mixture was filtered through a pad of Celite (diatomaceous earth material) and the filtrate was concentrated under reduced pressure, yielding 73.25 g (96~-~ of 5-[2-t2,2-dimethyl-tl,3]-dioxolan-4(S)-yl)-ethyl]-thiophene-2-carboxylic acid ethyl ester (2) as a colorless oil. An analytical sample was obtained by flash chromatography on silica gel, eluting CH2Cl2.
[a]589 -12.1~ (c = 0.7B, MeOH).
IR (neat) 2984, 2938, 2874, 1709, 1462, 1263, 1094, 1069 cm lH NMR (CDC13) ~: 1.36 (t, 3H, J ~ 7.0 Hz), 1.37 (s, 3H), 1.43 (s, 3H), 1.94 (m, 2Hl, 2.96 (m, 2H), 3.55 (t, lH, J ~ 7.3 Hz), 4.04 (dd, lH, J = 6.1, l.S Hz), 4.12 (m, lH), 4.32 (q, 2H, J
= 7.0 Hz), 6.82 (d, lH, J = 3.7 Hz), 7.63 (d, lH, J = 4.0 Hz).
Anal. calc~d for C14H20O4S: C, 59.13; H, 7.09; S, 11.28.
Found: C, 59.23; H, 7.14; S, 11.31.

CA 02224211 1997-12-0~

W O g~40674 PCT~US96/08953 (c) Pr-p~r~tlon of 5-t3(S)-4-D~hydroxy-~utyl)-tb~oph-n--2-c-r~oYyl~c ac~d ethyl est~r (3):

H~ O (3) To a stirred solution of 65.98 g (232 mmoi) of the acetonide compound (2) in 300 ml of EtOH was added 17.65 g (93 mmol) of p-toluenesulfonic acid monohydrate. The reaction mixture was heated at 65~C for 3 hours, concentrated under reduced pressure and redissolved in EtOH, and heating was continued. This procedure was repeated until the starting material (2) had disappeared as determined by TLC (thin layer chromatography). The crude reaction was diluted with EtOAc, washed with saturated NaHCO3, washed with saturated NaCl, dried (MgSO4) and concentrated under reduced pressure. The residue was recrystallized from Et2O:hexanes, yielding 49.90 g of the diol (3). The mother liquors were flash-chromatographed on silica gel, eluting 30-100~
EtOAc in CH2Cl2. This yielded another 5.81 g of the diol (3) as a white, low-melting solid. The overall yield of the diol (3) was 55.71 g (98~).
[a]589 -28.7~ (c = 0.82, MeOH).
IR (neat) 3393 (broad), 2936, 1705, 1462, 1287, 1098, 1047 cm H NMR (CDCl3) ~: 1.36 (t, 3H, J = 7.2 Hz), 1.7 (br s, 2H), 1.76 (m, 2H), 2.99 (m, 2H), 3.48 (dd, lH, J = 7.4, 3.5 Hz), 3.68 CA 02224211 1997-12-0~

W09~40674 PCT~S96/08953 (dd, lH, J - 7.6, 3.2 Hz), 3.77 (m, lH), 4.32 (q, 2H, J = 7.2 Hz), 6.82 (d, lH, J = 3.9 Hz), 7.63 ~d, lH, J = 3.7 Hz).

Anal. calc'd for CllH1604S: C, 54.08; H, 6.60; S, 13.12.

Found: C, 53.81; H, 6.43; S, 13.37.

(d) Prep~rat~on of 5-(3(S)-~l~o~-4-p-tolylmethane-sulfonyloxy-butyl)- thior~-n--2-carboxyllc ac~d .",...~ ~ <0 To an ice-cold solution of 45.42 g ~186 mmol) of diol (3) and 31.1 ml (223 mmol) of triethylamine in 400 ml of CH2Cl2 was added 38.99 g (204 mmol) of p-toluenesulfonylchloride. The ice bath was removed and the reaction mixture was stirred at room temperature for 18 hours and poured into 0.SN HCl, and the resulting layers were separated. The aqueous layer was re-extracted with CH2C12.
The combined organic layers were washed with saturated NaCl, dried (MgSO4), and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel, first with 4-100% EtOAc in CH2C12 until the product (4) eluted and then with 0-10% MeOH in EtOAc to elute the starting diol (3). This produced 7.43 g of diol (3) and 49.23 g (66~) of the monotosylate (4) as a lightly colored oil, which solidified on standing.
[~]589 -8.2~ (c = 0.74, MeOH).

IR (neat) 3507 (broad), 2947, 1705, 1462, 1362, 1287, 1177, 1098 cm 1.

W O 96/40674 PCTrUS96/089S3 (CDCl3) ~: 1.36 (t, 3H, J = 6.9 Hz), 1.78 (m, 2H), 2.46 (8, 3H), 2.95 (m, 2H), 3.90 (m, 2H), 4.03 (m, lH), 4.32 (q, 2H, J = 7.0 Hz), 6.78 (d, lH, J = 3.7 Hz), 7.36 (d, 2H, J = 8.2 Hz), 7.61 (d, lH, J . 3.7 Hz), 7.79 (d, 2H, J = 8.2 Hz).
Anal. calc'd for C18H22O6S2: C, 54;25; N, 5.57; S, 16.09.
Found: C, 54.13; H, 5.57; S, 16.17.

(e) Preparation of 5-(4-Azido-3(S)-hydroxy-butyl)-t~or~ -2-carboxylic acid ethyl ester (5):

H~

N (5) To a stirred solution of 42.94 g (108 mmol) of the tosylate (4) in 250 ml of DMF (N,N-dimethylformamide) was added 31.52 g ~485 mmol) of sodium azide. The solution was heated under argon for 4 hours at 80~C. The reaction mixture was cooled and poured into 600 ml of saturated NaCl, and the resulting oil was separated. The aqueous solution was extracted three times with Et2O. The Et2O extracts were combined with the oil and washed once with saturated NaCl, dried (MgSO4), and concentrated, yielding 33.0 g of the azide (5) as a crude product that was sufficiently pure to use in the next step ~f~. An analytical sample of the azide (5) was obtained by flash chromatography on silica gel, eluting CH2Cl2-EtOAc (20:1) to produce a colorless oll .
~]5B9 -18.8~ (c = 0.84, MeOH).

CA 02224211 1997-12-0~

W096/40674 PCT~S96/08953 IR (neat) 3445 (broad), 2926, 2099, 1705, 1539, 1460, 1281, lH NMR (CDCl3) ~: 1.36 (t, 3H, J = 7.1 Hz), l.e5 (m, 2H), 2.99 tm, 2H), 3.35 (m, 2H), 3.79 (m, lH), 4.32 (q, 2H, J = 7.1 Hz), 6.82 (d, lH, J = 3.6 Hz), 7.63 (d, lH, J = 4.0 Hz).
Anal. calc'd for C11H15N3O3S2: C, 49.05; H, 5.61; N, 15.60;
S, 11.90. Found: C, 48.89; H, 5.70; N, 15.37; S, 12.02.
(f) Preparation of 5-(4-tert-Butoxycarbonylamino-3(S)-hydroxy-butyl)-thiophene-2-carboxyl~c acid ethyl ester (6):

J ~
~ (6) 0~~

A Parr flask containing 32.70 g (121 mmol) of the crude azide (5), 27.83 g (127 mmol) of di-tert-butyl dicarbonate, 4.5 g of 5 Pd/C, and 300 ml of THF was shaken under H2 at 30 psi. The . exothermic reaction was maintained at a pressure below 45 psi.
After 2 hours, the crude mixture was filtered through a pad of Celite and the filtrate was concentrated under reduced pressure.
The residue was purified by flash chromatography on silica gel,.
eluting 6-50~ EtOAc in CH2Cl2. There was thus obtained 35.04 g (84~) of the product (6) as a colorless oil.
~ [a]589 -24.8~ tc = 0.86, MeOH).

CA 02224211 1997-12-0~

IR ~neat) 3391 ~broad), 29ao, 1723, 1674, 1537, 1462, 1368;
1283, 1171, 1096 cm~l.
lH NMR (CDC13) ~: 1.36 (t, 3H, J ~ 7.0 Hz), 1.45 (s, 9H), 1.81 ~m, 2H), 2.94-3.07 (m, 3H), 3.26 (m, lH), 3.7S (m, lH), 4.32 (q, 2H, J e 7.1 Hz), 4.9 (br s, lH), 6.8i (d, lH, J = 3.7 Hz), 7.62 (d, lH, J = 4.0 Hz).
Anal. calc'd for C16H25NOSS: C, SS.9S; H, 7.34; N, 4.08;
S, 9.34. Found: C, S5.89; H, 7.42; N, 4.13; S, 9.4S.
(g) Pr-paration of 5-(4-tert-Butoxyc-r~o~ylamlno-3(S)-methane-ulfonyloxy-butyl)-thior~ ~--2-carboxylic acid ethyl ester (7):
~'\~
~7) o To an ice-cold solution of 34.34 g (100 mmol) of the alcohol (6) and 20.9 ml (lS0 mmol) of triethylamine in 250 ml of CH2C12 was added 9.3 ml (120 mmol) of methanesulfonyl chloride. After 30 minutes the reaction mixture was poured into 0.5N HCl and the layers were separated. The organic layer was washed with saturated NaHCO3 and then with saturated NaCl, dried (MgSO4), and concentrated under reduced pressure. This produced 38.36 g (91%) of the mesylate (7) as a yellow oil, which solidified on standing.

W O 96/40674 PCTAUS9~08953 This crude material (7) was used in the next step (h) without further purification.
lH NMR (CDC13) ~: 1.36 ~t, 3H, J . 7.0 Hz), 1.44 (s, 9HI, 2.09 (m, 2H), 3.00 (m, 2H), 3.0B (s, 3H), 3.34-3.56 (m, 2H), 4.32 (g, 2H, J - 7.0 Hz), 4.79 (m, lH), 4.95 (br t, lH), 6.~2 (d, lH, J . 3.7 Hz), 7.62 (d, lH, J - 3.8 Hz).
(h) Pr-p~ratiou of 5-~3(R)-Acetyl-ulfanyl-4-tert-~utoxyc~ ylamino-butyl)-thloF~~n--2-car~oxyl~c ac$d ethyl e~ter (8):

..~ 1-) _o To a stirred solution of 38.36 g (91.0 mmol) of the crude mesylate (7) in 650 ml of acetone was added 41.57 (364 mmol) of potassium thioacetate. After 14 hours, the starting mesylate (7) had disappeared as determined by TLC and the reaction mixture was a red-brown color and thick with precipitate. The crude reaction mixture was filtered, and the filtered precipitate was washed with acetone and Et2O until it was beige in color. The filtrate was concentrated under reduced pressures to a volume of 500 ml, diluted with EtOAc, washed with saturated NaCl, dried (MgSO4), and concentrated under reduced pressure. The crude oil was purified by flash chromatography on 250 g of silica gel, eluting h~ es:

CA 02224211 1997-12-0~

WOs6/4o674 PCT~S96/08953 EtOAc ~1:1) to remove baseline-colored impurities. The yield was 39.77 g (109~) of the thioacetate (8) as a red oil, which was used in step ~i) without further purification. An analytical sample was obtained by flash chromatography on silica gel, eluting hexanes:EtOAc (4:1), producing a colorless oil.
[a]589 +7.6~ (c = 0.66, MeOH).
IR (neat) 3376, 2978, 2932, 1712, 16B4, 1520, 1462, 1263, 1171, 1094 cm~1.
lH NMR (CDCl3) ~: 1.35 (t, 3H, J = 7.0 Hz), 1.44 (s, 9H), 1.90 (m, lH), 2.04 (m, lH), 2.36 (s, 3H), 2.95 (m, 2H), 3.36 (m, 2H), 3.60 (m, lH), 4.32 (q, 2H, J = 7.0 Hz), 4.78 (br t. lH), 6.7 (d, lH, J = 3.7 Hz), 7.61 (d, lH, J = 3.7 Hz).
Anal. calc'd for C18H27NO5S2: C, 53.84; H, 6.7B; N, 3.49; S, 15.97. Found: C, 53.79; H, 6.87; N, 3.38; S, 15.85.

(i) Pr~paration of 2-~1-(tert-Butoxycarbonylamino-m~thyl)-3(R)-(5-sthoxycarbonyl-thioph~ne-2-yl)-propyl~ulfanyl~-malonic acid dimsthyl sstsr (9):

~ . (9) ~o To a stirred, ice-cold solution of 39.77 g (99.0 mmol) of the crude thiolacetate.(8) and 13.91 ml (109.0 mmol) of dimethyl chloromalonate in 350 ml of MeOH was added 27.38 g (198.1 mmol) of CA 02224211 1997-12-0~

W O 96/40674 PCT~US96/08953 potassium carbonate. After 3 hours at 0~C the reaction mixture was stirred at room temperature for 2 hours until the starting ~8) material had disappeared (TLC). After pouring into H2O and extracting with EtOAc ~3x), the combined organic layers were washed with saturated NaCl, dried (MgSO4), and concentrated under reduced pressure, producing 49.80 g of a crude disulfide. To a stirred solution of 11.44 g (15.96 mmol) of this disulfide in EtOH
under argon was added 1.81 g (47.~5 mmol) of sodium borohydride.
After 4 hours, the reaction mixture was quenched with 0.5N HCl, diluted with EtOAc, and washed with more 0.5N HCl. The organic layer was washed two times with saturated NaCl, dried (MgSO4), and concentrated under reduced pressure to yield 10.88 g of a crude thiol/disulfide mixture, which was dissolved in degassed MeOH. To this solution was added, while stirring, 5.79 ml (45.36 mmol) of dimethyl chloromalonate and 8.36 g (60.49 mmol) of potassium carbonate. After stirring for 30 minutes under argon, the reaction mixture was diluted with EtOAc, washed twice with saturated NaCl, dried (MgSO4), and concentrated under reduced pressure. The resulting residue was purified by flash chromatography on silica gel, eluting hexanes:EtOAc (3 :l) . This yielded 4.20 g of the disulfide, which was recycled, and 6.96 g (45~) of the desired malonate (9) as a light-yellow oil.
[a] 589 +31.9~ (c = 0. 64, MeOH).

IR (neat) 3397, 2978, 2938, 1759, 1715, 1505, 1454, 1275, 1}65, 1093, 1020 cm~l.

lH NMR (CDCl3) ~: 1.36 (t, 3H, J = 7.0 Hz), 1.46 (s, 9H), 1.86 (m, lH), 2.03 (m, lH), 3.04 (m, 3H), 3.31 (m, 2H), 3.81 (s, 6H), 4.24 (8, lH), 4.34 (q, 2H, J ~ 7.0 Hz), 5.13 (br s, lH), 6.83 (d, lH, J - 3.7 Hz), 7.64 (d, lH, J . 3.7 Hz).

Anal. calc'd for C21H31NOaS2: C, 5~.51; H, 6.38; N, 2.36;

S, 13.10. Found: C, 51.29; H, 6.45; N, 2.78; S, 13.01.

(~) Preparation of 6-t2-(S-~thoxycarbonyl-thiophen-2-yl)-ethyl]-3(R)-oxo-th~omorpholine-2-carboxylic ac~d methyl ester (10):

o~~~\
~10) To an ice-cold solution of 29.13 g (59.60 mmol) of the malonate (9) in 225 ml of CH2Cl2 was added 35 ml of trifluoroacetic acid. After 1 hour at 0~C the reaction mixture showed no starting material by TLC. The reaction mixture was diluted with CH2Cl2 and continuously washed with saturated NaHCO3, making sure that the aqueous layer was alkaline. The organic layer was washed with saturated NaCl, dried (MgSO4), and concentrated under reduced pressure. The resulting crude amine was dissolved in MeOH and stirred for 2 hours at room temperature.
The volatiles were removed under reduced pressure. The resulting -crude lactam was flash-chromatographed on silica gel, eluting W096/40674 PCT~S96/08953 ' CH2C12:EtOAc (2:1). This produced 17.74 g (83~) of the desired product (10) as an amber oil.

[~l589 +30.9~ (c - 0.92, MeOH).

IR (neat) 3314, 3242, 2951, 1732, 1660, 1462, 1294, 1157, 1096, 1009 cm lH NMR (CDCl3) ~: 1.36 (t, 3H, J = 7.0 Hz), 1.94 (m, 2H), 2.95-3.25 (m, 2H), 3.42-3.67 (m, 3H), 3.79 and 3.82 (s, s, 3H), 4.14 and 4.25 (8, 9, lH), 4.32 (q, 2H, J = 7.0 Hz), 6.23 (m, lH), 6.82 (d, lH, J = 3.7 Hz), 7.63 (d, lH, J = 3.7 Hz).

Anal. calc'd for C15H1gNO5S2: C, 50.40; H, 5.36; N, 3.92; S, 17.94. Found: C, 50.33; H, 5.38; N, 3.82; S, 17.90.

(k) Preparation of 6-[2-(5-~thoxy~-r~Q~yl-th~ophen-2-yl)-ethyl]-3(R)-methoxy-5,6-dihydro-2H-[1,41thiazin~-2-carboxylic acid methyl ester (11):

o~ ~~~\

~O ~ ~ ) To a stirred solution of 11.799 g (33.01 mmol) of the lactam (10) in 70 ml of CH2Cl2 was added 6.835 g (46.21 mmol) of trimethyloxonium tetrafluoroborate. After stirring for 18 hours under argon at room temperature all the starting lactam was consumed. The reaction mixture was then cooled, and 50~ aqueous K2CO3 was added until the pH was alkaline. The KBF4 was filtered off and the layers were separated. The aqueous layer was CA 02224211 1997-12-0~

w096/40674 PCT~S96108953 ' re-extracted with CH2C12. The combined organic layers were washed with ~aturated NaCl, dried (Na2S04), and concentrated under reduced pressure, yielding 11.55 g (94~) of the crude lactim ether (11), which was used in the next step without further purification.

(1) Pr-p-ration of 5-t2-(2-Amlno-4-oxo-4~6~7~8-tetrahydro-3H-pyrimldo[5,4-b]thiaz~n-6(R)-yl)-ethyl~-th~o~-n--2-c~rboxyl~c acid ethyl ester (12):

1~ 1~o~\

A solution of sodium ethoxide was prepared by dissolving 0.845 g (36.75 mmol) of sodium metal in degassed absolute ethanol under argon. To this solution was added 3.63 g (38.00 mmol) of guanidine hydrochloride. After stirring for 15 minutes, a solution of 4.55 g ~12.25 mmol) of the lactim ether (11) in degassed absolute EtOH was added, and the mixture was heated at reflux for 1 hour. The cooled reaction was neutralized with 0.SN
HCl, diluted with EtOAc, and extracted repeatedly. The combined EtOAc layers were washed with saturated NaCl, dried (MgSO4), and concentrated under reduced pressure. The solid residue was slurried in hot EtOH, cooled, and filtered, producing 1.75 g ~33~) of the desired product (12) as a light-yellow solid ~m.p. 166~C, foams).

W096/40674 PCT~S96/089~3 la]589 +57.7~ ~c c 0.62, DMSO).
IR ~RBr) 3349 (broad), 2926, 1701, 1640, 1603, 1537, 1458, 1344, 1285, 1096 cm~l.
lH NMR (DMSO-d6) ~: 1.23 (t, 3H, J - 7.0 Hz), 1.80 (m, 2H), 2.B2-3.00 (m, 2H), 3.15-3.52 (m, 3H), 4.il (q, 2H, J . 7.0 Hz), 6.00 (s, 2H), 6.64 (8, lH), 6.95 (d, lH, J = 3.7 Hz), 7.59 (d, lH, J = 3.7 Hz), 10.04 (8, lH).
HRMS calc'd for C15H18N403S2: (M+ Na ) 389.0718.
Found: 389.0731.
(m) Preparation of s-t2-(2-Amlno-4-oxo-4~6~7~8-tetrahydro-3H-pyr~m~dot5,4-b~tl,4]thlaz~n-6(R)-yl)-ethyl~-thloph~e-2-carboxyllc acid (13):

~ ~~
~ (l3) A solution of 1.152 g (3.14 mmol) of the ester (12) in 10 ml of lN NaOH was stirred at room temperature for 2 hours. The reaction mixture was filtered to remove some yellowish material.
The filtrate was acidified with concentrated HC1 and then with 2N
HCl to a pH of 3, and the resultin~ precipitate was collected, washed with a small amount of H2O, and dried. There was thus obtained 894 mg (84~) of the desired acid (13) as an off-white solid (m.p. 283-285~C, with decomp.).
[~]589 +71.0~ (c = 0.60, lN NaOH).

CA 02224211 1997-12-0~

W O 96~40674 PCTrUS96/08953 IR (KBr) 3256 (broad), 2942, 1707, 1641, 1612, 1464, 1364, 1105 cm .
lH NMR (DMSO-d6) ~: 1.72 ~m, lH), 1.89 ~m, lH), 2.81-3.04 (m, 2H), 3.16-3.52 (m, 3H -- partially obscured by H2O), 6.08 (s, 2H), 6.68 (8, lH), 6.92 (d, lH, J - 4.0 Hz), 7.52 (d, lH, J = 3.7 Hz), 10.12 (s, lH), 12.80 (br s, lH).
Anal. calc'd for C13H14N4O3S2Ø60 H2O: C, 44.71; H, 4.39;
N, 16.04; S, 18.36. Found: C, 44.67; H, 4.37; N, 16.00; S, 18.25.
(n) Preparation of 2-{5-[2-(2-Amlno-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidotS,4-b~[1,4~th~az~n-6tR)-yl)-ethyl~-thiophen~}-2-L-glutam~c acid diethyl ester (14):

J ~4R~
a 'S R~H

To a stirred solution of 278 mg (0.82 mmol) of the acid (13), 117 mg (0.87 mmol) of l-hydroxybenzotriazole, 150 ml (0.87 mmol) of N,N-diisopropylethylamine and 207 mg (0.87 mmol) of L-glutamic acid diethyl ester hydrochloride in 8 ml of DMF, was added 165 mg (0.87 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. The reaction mixture was stirred under argon for 18 hours and then poured into an ice-cold saturated NaCl solution.
The gummy precipitate was collected and the aqueous filtrate was extracted twice with CH2C12. The combined precipitate and CH2C12 w096/40674 PCT~S96/08953' extract~ were ~ e~ twice with saturated NaCl and then dried (MgS04). The solvent was l~mo~ed under reduced pressure and the resulting residue was purified by flash chromatography on silica gel, eluting CH2Cl2:MeOH (10:1). This yielded 282 mg (65~) of the desired glutamate (1~) as a white solid ~m.p. 108-112~C).
[a]589 +35.0~ (c . 0.68, DMSO).
IR (~3r? 3343 (broad), 2930, 1732, 1634, 1545, 1450, 1344, 1207, 1024 cm lH NMR (DMSO-d6) ~: 1.13 (t, 3H, J = 7.0 Hz), 1.14 (t, 3H, J = 7.0 Hz), 1.91 (m, 4H), 2.37 (t, 2H, J = 7.3 Hz), 2.89 (m, 2H), 3.23 (m, 2H), 3.49 (m, lH), 4.00 (q, 2H, J = 7.0 Hz), 4.06 (q, 2H, J ~ 7.0 Hz), 4.34 (m, lH), 6.01 (s, 2H), 6.63 (s, lH), 6.90 (d, lH, J - 3.7 Hz), 7.66 (d, lH, J = 3.7 Hz), 8.60 (d, lH, J
= 7.3 Hz), 10.07 (s, lH).
Anal. calc'd for C22H29N5O6S2.1.0 H2O: C, 48.78; H, s.77;
N, 12.93; S, 11.84. Found: C, 48.77; H, 5.72; N, 12.81; S, 11.73.
(o) Pr-p~r~tlon of 2-(5-t2-(2-A~$no-~-oxo-4,6,7,8-t-trahydro-3~-pyr~dot5,4-b]~1,4~t~$~z$n-6(~)-yl)--thyl~ op~-n-)-2-L-glut~mlc ~c$d (15):

~ ~J~
~ a 1~ ~* ~

CA 02224211 1997-12-0~

w096/40674 PCT~S96/08953 A ~olution of 5.068 g (9.68 mmol) of the glutamate (14) in 35 ml of lN NaOH was stirred at room temperature for 3.5 hours. The reaction mixture was made acidic with concentrated HCl and then with 2N HCl until a pH of about 3. The resulting precipitate was collected by filtration, washed with H2O, and dried in vacuo, yielding 4.486 g (99~) of the desired acid (15) as an off-white solid (m.p. 191-194~C, foams).
[a]589 +61.9~ (c = 0.65, lN NaOH).
IR (KBr) 3389, 3235, 3086, 2924, 1701, 1624, 1545, 1340, 1148 cm 1H NMR (DMSO-d6) ~: 1.70-2.04 (m, 4H), 2.29 (t, 2~, J = 7.3 Hz), 2.90 (m, 2H), 3.13-3.53 (m, 3H -- partially obscured by H2O), 4.29 (m, lH), 6.30 ~s, 2H), 6.77 (s, lH), 6.89 (d, lH, J = 3.7 Hz), 7.66 (d, lH, J = 3.7 Hz), 8.50 (d, lH, J = 8.1 Hz), 10.30 (br s, lH).
Anal. calc'd for C18H21N5O6S2.1.80 H2O: C, 43.24; H, 4.96;
N, 14.01; S, 12.83. Found: C, 42.88; H, 4.61; N, 13.75; S, 12.60.
Biological and Biochomical E~aluation of Compound ~15):
The GAR-transformylase (GARFT) assay method of Young et al., Biochemistry, vol. 23 (1984), 3g79-3986, was modified and used as described below. Reactions mixtures were prepared containing the catalytic domain of the human GARFT, 0-250 nM test compound, 20 ~M glycinamide ribonucleotide (GAR), 10 or 20 ~M
N10-formyl-5,8-dideazafolate (FDDF), 50 mM HEPES-KOH (pH 7.5), and 50 mM KCl. The reaction was initiated with the addition of enzyme W096/40674 PCT~S96/08953 to a final concentration of 11 nM, followed by monitoring of the increa~- in a~sorbance at 294 nm at 20~C (~294 ~ 18.9 mM 1 cm 1).
The GARFT inhibition constant (~i) was determined from the depend~nce of the steady-state catalytic rate on the inhibitor and substrate concentration. The type of inhibition observed was determined to be competitive with r~spect to FDDF by the dependence of the apparent Ki (Ki app) on the concentration of FDDF and was shown to be described by Ki app ~ Ki + (Ki/Km)~FDDF].
The Michaelis constant for FDDF, Km, was determined independently by the dependence of the catalytic rate on the FDDF concentration.
Data for both the Km and Ki determinations were fitted by non-linear methods to the Michaelis equation or to the Michaelis equation for competitive inhibition, as appropriate. Data resulting from tight-bi n~i n~ inhibition was analyzed, and Ki was determined by fitting the data to the tight-binding equation of Morrison, Biochem. 8iophYs. Acta, vol. 185 (1969), 269-286, by non-linear methods. Compound ~15) was determined to have a GARFT
Ki of 3 nM.
~xam~l- 2: Pr-p~r-tlon of 5-(4-t-rt-Butoxyc~rbo~yl-mlno-3~R)-~y~.6a~-butyl)-thlsp~-ni--2-c-r~oxyl~c ~c~d ~thyl ~~t-r (21) (a) ~ r~tlou of 5-t4-(t-rt-Butyl-d~ t~yl-~ ~ylo y)-3(S)-hydroxy-butyl~-t~19p~- n-_ 2-c~bva~llc ~cld ~t~yl ~~t-r (16):

~O~ E:~o_\
~o /~

CA 02224211 1997-12-0~

w096/40674 PCT~S96/08953 To a stirred, ice-cold solution of 0.946 g ~3.87 mmol) of the diol (3) and 0.81 ml (5.81 mmol) of triethylamine in 10 ml of CH2C12 was added 0.701 g (4.65 mmol) of tert-butyldimethylsilyl chloride. The ice bath was removed and the reaction was stirred at room temperature. After stirring for 24 hours, an additional 117 mg (0.78 mmol) of tert-butyldimethylsilyl chloride and 0.16 ml (1.16 mmol) of triethylamine was added. After stirring for 5 days at room temperature, the reaction mixture was diluted with more CH2C12 and then washed sequentially with 0.5N HCl, saturated NaHCO3 and saturated NaCl. The organic layer was dried (MgSO4) and the solvent was removed under reduce'd pressure. The resulting crude residue was purified by flash chromatography on silica gel, eluting 0-20~ EtOAc in CH2C12. There was thus obtained 1.252 g (90~) of the desired product (16) as a colorless oil.
[a]589 -26.6~ (c = 0.82, MeOH).
IR (neat) 3486 (broad), 2953, 2930, 2859, 1709, 1462, 1259, 1096 cm lH NMR (CDC13) ~: 0.07 (s, 6H), 0.90 (s, 9H), 1.36 (t, 3H, J = 7.2 Hz), 1.78 (m, 2H), 2.96 (m, 2H), 3.43 (m, lH), 3.64 (m, 2H), 4.32 (q, 2H, J = 7.0 Hz), 6.82 (d, lH, J = 3.7 Hz), 7.62 (d, lH, J = 4.0 Hz).
Anal. calc'd'for C17H30O4SSi: C, 56.94; H, 8.43; S, 8.94.

Found: C, 56.74; H, 8.43; S, 8.89.

.

CA 02224211 1997-12-0~

(b) ~r-p r-tlon of 5-[4-(tert-Butyl-d~methyl-~yloxy)-3(S)-m than-sulfonyloxy-butyl]-~orh-n--2-~ Yylic ac~d ethyl ~oter (17):

~ O~S~ ~o_\

~0 ~ ) To a stirred, ice-cold solution of 23.19 g (64.68 mmol) of the alcohol ~16) and 13.5 ml (96.86 mmol) of triethylamine in 200 ml of CH2Cl2 was added 6.0 ml (77.52 mmol) of methanesulfonyl chloride. After 30 minutes at 0~C, the reaction mixture was warmed to room temperature. After another 30 minutes, the reaction mixture was diluted with CH2Cl2 and then washed sequentially with 0.5N HCl, saturated NaHCO3 and saturated NaCl.
The organic layer was dried (MgSO4) and the solvent was removed under reduced pressure, yielding 26.61 g (94%) of the desired mesylate (17) as a colorless oil, which was used in the next step without further purification.
lH NMR (CDC13) ~: 0.11 (s, 3H), 0.13 (s, 3H), 0.92 (s, 9H), 1.39 (t, 3H, J = 7.0 Hz), 2.08 (m, 2H), 3.03 (m, 2H), 3.10 (s, 3H), 3.80 (m, 2H), 4.36 (q, 2H, J = 7.0 Hz), 4.75 (m, lH), 6.88 (d, lH, J = 3.7 Hz), 7.66 (d lH, J = 3.7 Hz).

W 0 96/40674 rCT~US96108g53 (¢) Pr-~-r~tlo~ of 5-(4-~ -3(S)-meth~
~ulfo~yloxy-butyl)-thioph~ne-2-~ Yyl~c ~cld ~thyl e~t-r (18):

o~S4~o~0~

(1-) ~o To a stirred solution of 26.56 g (60.83 mmol) of protected alcohol (17) in 200 ml of THF (tetrahydrofuran) was added 67 ml (67.00 mmol) of a 1.0 M solution of tetrabutyl~onium fluoride in THF. After stirring for 1.5 hours at room temperature, the reaction mixture was diluted with EtOAc and washed with 0. SN HCl .
The aqueous layer was re-extracted with EtOAc. The combined organic layers were washed twice with saturated NaCl, dried ~MgSO4), and concentrated under reduced pressure. The residue was flash-chromatographed on silica gel, eluting 0-20~ EtOAc in CH2C12. This yielded 12.465 g (64~) of the desired product (18) as a colorless oil, which solidified on st~n~ing.
[~ 589 ~4 3~ (c = 0.78, MeOH) .
IR (neat) 3520, 2982, 2940, 1705, 1684, 1462, 1345, 1287, 1173, 1098 cm~l.
lH NMR (CDC13) ~: 1.36 ~t, 3H, J - 7.0 Hz), 2.09 ~m, 2H), 3.01 ~m, 2H), 3.12 (s, 3H), 3.80 (m, 2H), 4.32 (q, 2H, J = 7.0 Hz), 4.81 (m, lH), 6.86 (d, lH, J - 3.7 Hz), 7.63 ~d, lH, J = 3.7 Hz).

CA 02224211 1997-12-0~

W096l40674 PCT~S96/089~3 Anal. calc'd for C12H18O6S2: C, 44.70; H, 5.63; S, 19.89.

Found: C, 44.75; H, 5.70; S, 19.82.

(d) Pr-p~ratlon of 5-(2(R)-Ox~ranyl-ethyl)-thio~n~-2-carboxyllc acld ethyl ester (19):

~o 0~s 0_\
(l9) To a stirred, ice-cold solution of 559 mg ~1.73 mmol) of the mesylate (18) in 10 ml of THF was added 76 mg (1.90 mmol) of a 60 dispersion of sodium hydride (NaH) in mineral oil. After 1 hour, the ice bath was removed and the reaction was stirred at room temperature. After stirring for 18 hours, another 35 mg (0.88 mmol) of a 60% dispersion of NaH was added. After 4 days of stirring, the reaction mixture was diluted with EtOAc, washed with 0.5N HCl and then with saturated NaCl, dried (MgSO4), and concentrated under reduced pressure. The resulting crude residue was flash-chromatographed on silica gel, eluting CH2C12.
Consequently, 322 mg (82%) of the desired epoxide (19) was obtained as a colorless oil.
[~]589 +19.6~ (c = 1.14, MeOH).
IR (neat) 2984, 1707, 1460, 1283 1262, 1092 cm 1.
lH NMR (CDC13) ~: 1.36 (t, 3H, J = 7.0 Hz), 1.80-2.05 (m, 2H), 2.51 (dd, lH, J = 4.8, 2.6 Hz), 2.78 (dd, lH, J = 4.8, 4.1 CA 02224211 1997-12-0~

W096/40674 PCT~S96/08953 Hz), 2.99 tm, 3H), 4.32 (q, 2H, J - 7.0 Hz), 6.82 td, lH, J = 3.7 Hz), 7;63 (d, lH, J z 3.7 Hz).
Anal- calc'd for CllH14O3S: C, 58.38; H, 6.24; S, 14.17.
Found: C, 58.49; H, 6.29; S, 14.06.
~e) Preparation of 5-(4-Az~do-3(R)-hydroxy-butyl)-thlo~h~n~-2-Carh~Yyl~C ac~d ~thyl ~st~r (20):

~o ~~
~ (20) To a stirred solution of 200 mg (0.88 mmol) of the epoxide-(19) in 10 ml of CH3CN was added 296 mg (1.33 mmol) of magnesium perchlorate. After 10 minutes, the reaction mixture became homogeneous ana _-~i mg (2.21 mmol) of sodium azide was added. The reaction m--- re was heated to 75~C. After 18 hours, 25 mg (0.39 mmol) of soa;~m azide was added and heating was continued for another 18 hours. The reaction mixture was cooled and diluted with EtOAc, and was then washed with saturated NaCl. The aqueous layer was re-extracted with EtOAc. The combined organic layers were washed with saturated NaCl, dried (MgSO4), and concentrated under reduced pressure. The residue was flash-chromatographed on silica gel, eluting 20-25~ EtOAc in hexanes. This produced 187 mg (79~) of the azide (20) as a colorless oil.
[~]589 +18.9~ tc = 0.62, MeOH).

W096/40674 PCT~S96/08953 IR (neat) 3462 (broad), 2982, 2936, 2101, 1705, 1682, 1462, 1269, ~096 cm~1.

lH NMR (CDC13) ~: 1.36 (t, 3H, J = 7.0 Hz), 1.85 (m, 2H), 2.99 (m, 2H), 3.30 (m, 2H), 3.79 (m, lH), 4.32 (q, 2H, J = 7.0 Hz), 6.82 (d, lH, J - 3.7 Hz), 7.63 (d, lH, J ~ 3.7 Hz).

Anal. calc'd for CllH15O3S: C, 49.05; H, 5.61; N, 15.60; S, 11.90. Found: C, 48.97; H, 5.64; N, 15.51; S, 11.97.

(f) Preparat~on of 5-(4-t~rt-Butoxycarbonylamino-3(R)-hydlox~L~tyl)-t~iophen--2-carboxyllc acid ethyl estcr (21):

~~)~~~
~ (21) o/LO

A solution of 144 mg (0.53 mmol) of the azide (20), 14 mg of 5~ Pd/C and 128 mg (0.59 mmol) of di-tert-butyl dicarbonate in 10 ml of THF was stirred vigorously under 1 atmosphere of H2 for 18 hours. The reaction mixture was filtered through Celite and the solvent was removed under reduced pressure. The residue was flash-chromatographed on silica gel, eluting 9-20~ EtOAc in CH2C12. This yielded 166 mg (90~) of the desired product (21) as a colorless oil.
[~]589 +26.3~ (c = 0.76, MeOH).

Wos6/4o674 PCT~S96/08953 ' IR (neat) 3378 (broad), 2980, 2934, 1715, 1682, 1516, 1462, 1294, 1099 cm~1.

lH N ~ (CDCl3) ~: 1.33 (t, 3H, J = 7.0 Hz), 1.44 (9, 9H), 1.79 (m, 2H), 2.22 (br s, lH), 3.04 (m, 3H), 3.25 (m, lH), 3.73 (m, lH), 4.32 (q, 2H, J = 7.0 Hz), 6.81 (d, lH, J = 3.7 Hz), 7.62 (d, lH, J = 3.7 Hz).

Anal. calc'd for C16H25N05S: C, 55.95; H, 7.34; N, 4.08; S, 9.34. Found: C, 55.88; H, 7.35; N, 4.01; S, 9.40.

~g) Proparation of 5-(4-tert-Butoxycarbonyl~o-3(R)-moth-no_ulfonyloxy-butyl)-th~o~h~--2-carboxylic acld othyl ooter (22):
MSO~ . ~ O2Et HN
BOC

Starting with alcohol (21), compound (22) was prepared in 94%
crude yield according to the general procedure described for compound (7).
H NMR ICDC13) ~: 1.36 (t, 3H, J = 7.0 Hz), 1.45 (s, 9H), 2.07 (m, 2H), 3.00 (m, 2H), 3.06 (s, 3H), 3.37-3.50 (m, 2H), 4.32 (q, 2H, J = 7.0 Hz), 4.78 (m, lH), 4.91 (br t, lH), 6.84 (d, lH, J
= 4.0 Hz), 7.62 (d, lH, J = 4.0 Hz).

(h) ~p-~ation of 5-(3(S)-Acotyloulfanyl-4-tort-butoxycarbo~yl~ino-butyl)-thiophe~o-2-carboxylic ac~d othyl oot-r (23):

J~s ~02Et HN

CA 02224211 1997-12-0~

w096/40674 PCT~S96/08953 Starting with mesylate (22), compound (23) was prepared in 86S yield according to the general procedure described for compound (8).
[a]S89 -4.9~ (c = 0.61, MeOH).
IR ~neat) 3374, 2978, 2932, 1715, 1695, 1518, 1460, 1264, 1171, 1096 cm~l.
lH NMR (CDC13) ~: 1.35 (t, 3H, J = 7.0 Hz), 1.44 ~s, 9H), 1.90 (m, lH), 2.03 (m, lH), 2.36 ~s, 3H), 2.9S (m, 2H), 3.35 (m, 2H), 3.59 (m, lH), 4.31 (2H, q, J = 7.0 Hz), 4.74 (br t, lH), 6.79 (d, lH, J = 3.7 Hz), 7.62 (d, lH, J = 3.7 Hz).
Anal. calc'd for C18H27NO5S2: C, 53.84; H, 6.78; N, 3.49; S, lS.97. Found: C, 53.75; H, 6.86; N, 3.43; S, 16.07.

(i) Preparation of 2-[1-(tert-Butoxycarbonylamino-methyl)-3(S)-(5-ethoxycarbonyl-th~ophene-2-yl)-propyl~ulfanyl]-malonic acid dimethyl ester (24):

CO2Me MeO2C S~,02Et H~

To a stirred solution of 8.44 g (21.0 mmol) of thioacetate (23) and 3.22 ml (25.2 mmol) of dimethyl chloromalonate in 75 ml of degassed methanol at 0~C was added 5.81 g (42.0 mmol) of potassium carbonate. The reaction, under an Ar atmosphere, was allowed to stir for 1 hr at 0~C, then 1.5 hr at rt, poured into saturated NaCl and extracted twice with EtOAc. The combined w096/40674 PCT~S96/08953 organic layer was wa9hed twice more with saturated NaCl solution, dried (MgSO4), and concentrated under reduced pressure. The resulting residue was purified by flash chromatography on silica gel, eluting hexanes:EtOAc (3:1). This produced 9.678 g t94~) of the desired malonate (24) as a yellow oil.
[a]589 -32.8~ (c = 0.67, MeOH).
IR (neat) 3395 ~broad), 2978, 1755, 1715, I699, 1462, 1261 cm lH NMR (CDC13) ~: 1.36 (t, 3H, J = 7.0 Hz), 1.44 (s, 9H), 1.86 lm, lH), 2.05 (m, lH), 3.02 (m, 3H), 3.31 (m, 2H), 3.79 (s, 6H), 4.23 (s, lH), 4.33 (q, 2H, J = 7.0 Hz), 6.81 (d, lH, J = 3.7 Hz), 7.62 (d, lH, J = 3.7 Hz).
Anal. calc'd for C21H30NO8S2: C, 51.51; H, 6.38; N, 2.86; S, 13.10. Found: C, 51.58; H, 6.42; N, 2.79; S, 13.01.

(;) Preparation of 6-~2-(5-Buthoxycarbonyl-th~ophon-2-yl)-ethyl]-3(S)-oxo-thiomorpholine-2-carboxylic acid othyl estor (25):

o~ ) CO2Et N
H

Starting with malonate (24), compound (25) was prepared in 86~ yield according to the general procedure described for compound ~10).
[a]58g ~30-5~ (c = 1.01, MeOH).
IR (neat) 3320, 3229, 2951, 1738, 1703, 1669, 1460, 1281 W096l40674 PCT~S96/08953'-lH NMR (CDC13) ~: 1.36 (t, 3H, J . 7.0 Hz), 1.91-2.04 (m, 2H), 2.95-3.25 (m, 2H), 3.42-3.65 (m, 3H), 3.79 and 3.82 (s, s, 3H), 4.13 and 4.25 (s, s, lH), 4.32 (q, 2H, J = 7.0 Hz), 6.26 (m, lH), 6.82 (d, lH, J = 3.7 Hz), 7.64 (d, lH, J = 3.7 Hz).

Anal- calc'd for C15H19N~5S2 C~ 50;40; H~ 5-36; N~ 3-92; S~

17.94. Found: C, 50.47; H, 5.34; N, 3.95; S, 17.84.

(k) Preparation of 6-~2-(5-~uthoxyc-rh~yl-thiophen-2-yl)-othyl]-3(S)-mothoxy-5,6-dihydro-2H-tl,4]thiazinc-2-carboxylic acid ethyl e~ter (26):
MeO2CxS~_~ CO2Et Starting with lactam (25), compound (26) was prepared in quantitative crude yield according to the general procedure described for compound (11) and was used in the next step without purification.

(1) Preparation of 5-t2-(2-Amlno-4-oxo-4~6~7~8-tctrahydro-3~-pyr;m;do[5,4-b]thiazin-6(S)-yl)-cthyl]-th~oph~n~-2-carboxylic acid ~thyl ester (27):

HN~S~J I~CO2Et H Nl'N N

Starting with lactim ether (26), compound (27) was prepared in 45% yield according to the general procedure described for compound (12). (m.p. 181-lB4~C).

W O 96140674 PCTAUS96/08953 ' [~]589 ~59 7~ (c - 0.38, DMSO).

IR (~3r) 3335, 2926, 1705, 1632, 1593, 1456, 1335, 1260 cm 1.

lH NMR (DMSO-d6) ~: 1.23 (t, 3H, J = 7.0 Hz), 1.72 (m, lH), 1.88 (m, lH), 2.81-3.00 (m, 2H), 3.16-3.52 (m, 3H), 4.22 (q, 2H, J

_ 7.0 Hz), 6.00 (8, 2H), 6.65 (s, lH), 6:96 (d, lH, J ~ 3.7 Hz), 7.60 (d, lH, J z 3.7 Hz), 10.04 (s, lH).

Anal. calc'd for C15H18N4O3S2Ø4 H2OØ5 EtOH: C, 48.44;

H, 5.54; N, 14.12; S, 16.17. Found: C, 48.69; H, 5.52; N, 14.07;

S, 16.00.

~m) Preparat~on of 5-t2-(2-Amino-4-oxo-4~6~7~8-tetrahydro-3H
pyr~m~do[5,4-b~tl,4]thiaz~n-6(S)-yl)-ethyl] thi~h~~-2-c~r~yl~c ac~d (28):

~'$NJ CO2H

Starting with ester (27), compound (28) was prepared in 98 yield according to the general procedure described for compound (13). (m.p. 25B-261~C, with decomp.).
[a]589 -81.3~ (c = 0.63, lN NaOH).

IR (~3r) 3254 (broad), 2918, 1692, 1635, 1458, 1352, 1101 cm lH NMR (DMSO-d6) ~: 1.72 (m, lH), 1.89 (m, lH), 2.80-3.03 (m, 2H), 3.16-3.52 (m, 3H -- partially obscured by H2O), 6.09 (s, 2H), 6.80 (s, lH), 6.92 (d, lH, J = 3.7 Hz), 7.52 (d, lH, J = 3.7 Hz), 10.20 (s, lH), 12.80 (br s, lH).

HRMS calc'd for C13H14N403S2: (M+Na+) 361.0405. Found:
361.039Q.
(n) Propar-tlon of 2-({5-t2-(2-Am~no-4-oxo-4~6J7~8-tetrahydso-3R
pyr~do~5,4-b]~1,4]th~az~n-6(S)-yl)--thyl]-th~oF~-n~)-2-~-gIut~m~c ac~d d~othyl e~ter (29):

1~NJ NH~CO t Starting with acid (28), compound (29) was pr~,ed in 60%
yield according to the general procedure described for compound (14). (m.p. 105-110~C, with fo~m;ng).
[a]589 ~55-5~ (c - 0.53, DMSO).
IR (Br) 3345, 2930, 1734, 16S3, 1636, 1541, 1456, 1345 cm 1.
lH NMR (DMSO-d6) ~: 1.12 (t, 3H, J = 7.0 Hz), 1.14 (t, 3H, J = 7.0 Hz), 1.75-2.03 (m, 4H), 2.37 (t, 2H, J = 7.4 Hz), 2.B8 (m, 2H), 3.17 (m, 2H), 3.47 (m, lH), 4.01 (q, 2H, J = 7.0 Hz), 4.07 (q, 2H, J = 7.0 Hz), 4.34 (m, lH), 6.00 (s, 2H), 6.64 (s, lH), 6.89 (d, lH, J = 3.7 Hz), 7.65 (d, lH, J - 3.7Hz), 8.60 (d, lH, J
= 7.3 Hz), lO.OS (s, lH).
Anal. calc'd for C22H29N5O6S2.1.0 H2O: C, 48.78; H, 5.77; N, 12.93; S, 11.84. Found: C, 48.73; H, 5.74; N, 12.96; S, 11.92.

Wos6/40674 PCT~S96/08953 (o) Pr-parat~on of 2-(5-t2-(2-Amino-4-oXO-4,6,7,8-tetrahydro-3H-pyrl~ldol5~4-b~ 4~thiazin-6~s)-yl)-ethyll-th~or~e)-2-L
glut~ic acld ~30):

~NH~0ol1H

Starting with diester ~29), compound (30) was prepared in 97%
yield according to the procedure described for compound (15).
(m.p. 220~C with decomposition).
[~]589 -57.1~ (c=0.61, lN NaOH) IR(~3r) 3353, 3094, 2926, 1711, 1641, 1605, 1559, 1454, 1400, 1333, 1279, 1080 cm~1.
lH NMR ~DMSO-d6) ~: 1.70-2.05 (m, 4H), 2.29 (t, 2H, J = 7.4 Hz), 2.87 ~m, 2H), 3.15-3.48 (m, 3H -- partially obscured by H2O), 4.29 (m, lH), 6.03 (s, 2H), 6.66 (s, lH), 6.89 (d, lH, J = 3.7 Hz), 7.65 (d, lH, J = 3.7 Hz), 8.50 (d, lH, J = 7.7 Hz), 10.05 (s, lH), 12.50 (br s, 2H).
Anal. calc'd for Cl8H2lN5o6s2 ~ 2 14.21; S, 13.01. Found: C, 43.83; H, 4.78; N, 14.10; S, 12.90.

W096l40674 PCT~S96/08953 "

~3~Dl~ 3s ~r--at~on of 5-{2-l2-(2-Amlno-4-oxo-4~6~7~8-t-trahydro-3H-pyr~midotS,4-b]tl,4~thlazln-6~R)-yl)-ethyl]-4-methyl-th~rh~no}-2-~-glut-mlc acid (45) (a) ~- p-r~tion of 5-(2,2-Dimethyl-tl,3]dioxol~n-4(s)-yl-~thynyl)-4-methyl-th~orh~n~-2-r-r~QYyl~c acid ethyl ~ster (31): \

><C~3~o2Et o Starting with 4(S)-EthynyI-2,2-dimethyl-[1,3]dioxolane and 2-Bromo-3-methyl-thiophene-5-carboxyliC acid ethyl ester, prepared according to M. Nemec, Collection Czechoslov. Chem. Commun., compound (3i) was prepared in 67~ yield according to the general procedure described for compound (1).
[~]589 +36.7~ (c = 1.04, MeOH).
IR(neat) 2986, 2936, 2222, 1711, 1441, 1283, 1244, 1190 cm 1.
lH NMR (CDC13) ~: 1.36 (t, 3H, J = 7.0 Hz), 1.42 (s, 3H), 1.53 (s, 3H), 2.29 (s, 3H), 4.04 (dd, lH, J = 6.2, 1.8 Hz), 4.26 (dd, lH, J = 6.2, 1.8 Hz), 4.32 (q, 2H, J = 7.0 Hz), 4.99 (t, lH, J = 6.0 Hz), 7.50 (s, lH).
Anal. calc'd for C15H18O4S: C, 61.20; H, 6.16; S, 10.89.
Found: C, 61.33; H, 6.21; S, 10.78.

(b) ~pJ~ation of 5-[2-(2~2-Dimethyl~ 3~-dioxolan-4(S)-yl)-ethyl~-3-methyl-thiophene-2-carboxylic acid ethyl ester (32):

~02Et o CA 02224211 1997-12-0~

W O 96/40674 PCTAUS96/089~3 Starting with alkyne (31), co...~ound (32) was prepared in 98 yield according to the general procedure described for compound (2).
[a]589 -12.6~ (c = 0.94, MeOH).
IR (neat) 2984, 2936, 2872, 1707, 1456, 1371, 1246, 1069 cm lH NMR (CDCl3) ~: 1.35 (t, 3H, J = 7.0 Hz), 1.36 (s, 3H), 1.43 (s, 3H), 1.88 (m, 2H), 2.16 (9, 3H), 2.80-2.91 (m, 2H), 3.55 (dd, lH, J = 6.6, 0.7 Hz), 4.03 (dd, lH, J = 5.9, 1.8 Hz), 4.12 (m, lH), 4.31 (q, 2H, J = 7.0 Hz), 7.50 (s, lH).
Anal. calc'd for C15H22O4S: C, 60.37; H, 7.43; S, 10.75.
Found: C, 60.42; H, 7.48; S, 10.68.
(c) Preparat~on of 5-(3(S)-4-Dihydroxybutyl)-4-methyl-thlophene-2-carboxylic acid ethyI ester (33):

HO,~CO2Et HO

Starting with dioxolane (32), compound (33) was prepared in 92~ yield according to the general procedure described for compound (3).
[a]589 -24.3~ (c = 0.61, MeOH).
IR (~3r) 3264 (broad), 2924, 1707, 1458, 1447, 1260, 1179, 1074 cm~l.
lH NMR (CDCl3) ~: 1.35 (t, 3H, J = 7.0 Hz), 1.76 (m, 2H), 2.17 (s, 3H), 2.89 (m, 2H), 3.48 (dd, lH, J = 7.4, 3.3 Hz), 3.68 ~dd, lH, J - 7.7, 3.3 Hz), 3.75 (m, lH), 4.30 (q, 2H, J = 7.0 Hz), 7.50 (~, lH).

Anal. calc'd for C12H1804S: C, 55.79; H, 7.02; S, 12.41.

Found: C, 55.69; H, 6.99; S, 12.31.

(d) Preparation of 5-(3~s)-~dhv~r-4-p-toly~m thane~ulfonyloxy-butyl)-4-methyl-thl~r~~n~-2-carboxylic acld ethyl e8ter (34):

HO ~ CO2Et TsO

Starting with diol (33), compound (34) was prepared in 60~
yield according to the general procedure described for compound (4).
[a]589 ~5 7~ ~c = 0.74, MeOH).
IR (neat) 3491 (broad), 2982, 2928, 1703, 1449, 1360, 1250, 1177 cm 1.
H NMR (CDCl3) ~: 1.35 (t, 3H, J = 7.0 Hz), 1.71 (m, 2H), 2.13 (s, 3H), 2.46 (s, 3H), 2.85 (m, 2H), 3.B8-4.03 (m, 3H), 4.31 (q, 2H, J = 7.0 Hz), 7.36 (d, 2H, J = 8.1 Hz), 7.48 (s, lH), 7.79 (d, 2H, J = 8.4 Hz).
Anal. calc d for ClgH24O6S2: C, 55.32; H, 5.B6; S, 15.55.
Found: C, 55.37; H, 5.92; S, 15.48.

W096/40674 PCT~S96/08953 (-) Pr-p~r~tlo~ o~ 5-(4-Azido-3(S)-hr~Gxr-butyl)-4-methyl-thlo~h-~--2-r-rhQ~ylic acid ethyl ~ster (35):

HO~_~CO2Et N3~

Starting with tosylate (34), compound (35) was prepared in 96~ crude yield according to the general procedure described for compound (5) and used without purification.
lH NMR (CDCl3) ~: 1.36 ~t, 3H, J = 7.0 Hz), 1.80 (m, 2H), 2.16 (s, 3H), 2.83-2.96 (m, 2H), 3.29 (dd, lH, J = 12.1, 7.4 Hz), 3.40 (dd, lH, J = 12.1, 3.7 Hz), 3.78 (m, lH), 4.30 tq, 2H, J --7.0 Hz), 7.50 ts, lH).

(f) Preparation of 5-(4-tert-Butoxycarbonylam~no-3(S)-hydroxy-butyl)-4-methyl-thiophene-2-c-rboxyllc acid ethyl ester (36):

~ CO2Et HO S

HN
BOC

Starting with azide (35), compound (36) was prepared in 89 yield according to the general procedure described for compound (6).
t~]589 -21.7~ tc=0.71, MeOH).

WOs6/4o674 PCT~S96/08953 IR (neat) 3385 ~broad), 2978, 2932, 1715, 1682, 1520, 1454, 1254, ~177, 1073 cm~l.
lH N ~ (CDC13) ~: 1.35 ~t, 3H, J = 7.0 Hz), 1.44 (8, 9H), 1.75 (m, 2H), 2.16 (s, 3H), 2.80-2.94 (m, 2H), 3.08 (dd, lH, J s 14.3, 7.4 Hz), 3.28 (dd, lH, J ~ 14.3, 3;0 Hzj, 3.74 (m, lH), 4.30 (q, 2H, J 5 7.0 Hz), 7.49 (s, lH).
Anal. calc'd for C17H27N05S: C, 57.12; H, 7.61; N, 3.92; S, 8.97. Found: C, 57.05; H, 7.65; N, 3.96; S, 9.07.
(g) Preparation of 5-(4-t~rt-Butoxycarbonylam~no-3(S)-moth-nePulfonyloxy-butyl)-4-m~thyl-thioE~h~~--2-carboxyl~c acid ~thyl ~~t~r (37):

~CO2Et MSO S

HN
BOC

Starting with alcohol (36), compound (37) was prepared in 94 yield according to the general procedure described for compound (7) (~.p. 75-76~C).
~a]589 +7.8~ tc = 0.60, MeOH).
IR (~3r) 3362, 2982, 1699, 1680, 1530, 1350, 1278, 1175, 1071 cm lH NMR (CDC13) ~: 1.35 (t, 3H, J = 7.0 Hz), 1.45 ~s, 9H), 2.01 (m, 2H), 2.16 (s, 3H), 2.89 (m, 2H), 3.07 (s, 3H), 3.30-3.48 (m, 2H), 4.31 (q, 2H, J = 7.0Hz), 4.80 (m, lH), 4.95 (br t, lH), 7.50 (~, lH).

W O 96/40674 PCT~USg6tO8953 ' Anal. calc~d for C18H29N07S2: C, 49.63; H, 6.71; N, 3.22; S, 14.72. Found: C, 49.72; H, 6.76; N, 3.26; S, 14.82.

(h) Prqpar-tlon of 5-(3(R)-Acetylsulfanyl-4-tert-butoxyc~~h~ylam~no-butyl)-4-~thyl-th~orh~n~-2-c~h~yllc ac~d ethyl e~ter (38):

J~ - s~''''~co2Et HN

Starting with mesylate (37), compound (38) was prepared in 96~ yield according to the general procedure described for compound (8).
[~]589 -2.8~ (c = 0.78, MeOH).
IR (neat) 3376, 2978, 2932, 1699, 1516, 1454, 1250, 1173, 1073 cm~1.
1H NMR (CDC13) ~: 1.34 (t, 3H, J = 7.0Hz), 1.44 (9, 9H), 1.80-2.04 (m, 2H), 2.13 (s, 3H), 2.37 (s, 3H), 2.85 (m, 2H), 3.3B
(m, 2H), 3.60 (m, lH), 4.30 (q, 2H, J = 7.0Hz), 4.88 (br t, lH), 7.48 (s, lH).
Anal. calc'd for ClgH29N05S2 C, 54.91; H, 7.03; N, 3.37; S, 15.43. Found: C, 54.97; H, 7.05; N, 3.39; S, 15.32.

W O 96/40674 PCTrUS96/08953 r-p r-tlon of 2-tl-(tort-Butoxycarbo~yl~m~o-methyl)-3(R)-(S~ oxycar~onyI-3-methyl- thiorh~nc-2-yl)-propyl8ulfa~yl]-m~l~lc ~cld dim~thyl ~ster (39):

MeO2C 5~ CO2Et -HN

Starting with thioacetate (38), compound (39) was prepared in 90~ yield according to the general procedure described for compound (24).
[~]589 +22.7~ (c = 0.67, MeOH).
IR (neat) 3397, 2978, 1755, 1714, 1514, 1454, 1250, 1171, 1074 cm~l.
lH NMR (CDC13) ~: 1.34 (t, 3H, J = 7.0 Hz), 1.44 ls, 9H), 1.92 (m, 2H), 2.15 (s, 3H), 2.95-3.40 ~m, SH), 3.80 and 3.82 (s, s, 6H), 4.23 (s, lH), 4.31 (q, 2H, J = 7.0Hz), 5.18 (br s, lH), 7.49 (s, lH).
Anal. calc'd for C22H33NO8S: C, 52.46; H, 6.61; N, 2.78; S, 12.73. Found: C, 52.56; H, 6.62; N, 2.81; S, 12.71.
(;) Pr-paration of 6-[2-(5-8uthoxycarbonyl-3-methyl-thiopen-2-yl)--thyl}-3(R)-oxo-thiomorpholinc-2-carboxyl$c ac$d methyl oat-r (40):

o~S~ 02Et N
H

W096/40674 PCT~S96/08953 Starting with co~ ound (39), compound (40) was prepared in 87~ yield according to the general procedure described for compound (10). (m.p. 72-73~C).
[a] +19.1~ ~c=0.67, MeOH).
IR (thinfilm) 2954, 1738, 1694, 1651, 1464, 1296, 1196, 1074 cm lH NMR (CDCl3) ~: 1.35 (t, 3H, J = 7.0Hz), l.90 (m, 2H), 2.17 (s, 3H), 2.88 (m, 2H), 3.21-3.62 (m, 3H), 3.80 and 3.52 (s, s, 3H), 4.14 and 4.25 (s, s, lH), 4.31 (q, 2H, J = 7.0Hz), 6.22 (m, lH), 7.50 (s, lH).
Anal- calc d for C16H21N~5S2 C~ 51-73; ~ 5-70; N~ 3- 7; S~
17.26. Found: C, 51.65; H, 5.72; N, 3.73; S, 17.15.

(k) Preparation of 6-~2-(5-Ethoxycar~onyl-3-methyl-t~iopen-2-yl)-othyl]-3(R)-mothoxy-5,6-dihydro-2~-[1,4]thiazine-2-carboxylic acid methyl e~tsr (41):

X's) 02Et Starting with lactam (40), compound (41) was prepared in quantitative crude yield according to the general procedure described for compound (11) and was used without further purification.

Wos6/4o674 PCT~S96/08953 (1) Pr-p~r tlo~ of 5-~-(2-Amlno-4-oxo-4,6,7,8-t-tr~hydro-3H-r~ldol5~-blth~zl~-6(R)-y~ t~y~ -mothyl-thl~h~-2 c-~o-yl~c ~cld ~thyl est~r (42):

Starting with lactim ether ~41), compound (42) was prepared in 27~ yield according to the general procedure described for compound (12). (m.p. 120-140~C with decomp.).
[a]589 +57.1~ (c = 0.67, DMSO).
IR (~3r) 3337, 2926, 1701, 1653, 1635, 1599, 1449, 1250, 1072 cm lH NMR (DMSO-d6) ~: 1.22 (t, 3H, J = 7.0 Hz), 1.60-1.75 (m, lH~, 1.80-1.92 (m, lH), 2.10 (s, 3H), 2.86 (m, 2H), 3.12-3.52 (m, 3H~ 4.20 (q, 2H, J = 7.0 Hz), 6.02 (s, 2H), 6.65 (s, lH), 7.50 (s, lH), 10.08 (s, lH).
Anal. calc'd for C16H2oN4O3S2Ø8 H2O: C, 48.66; H, 5.51; N, 14.19; S, 16.24. Found: C, 48.77; H, 5.58; N, 14.07; S, 16.16.
HRMS calc'd for C16H20N4O3S2: ~M + H+) 381.1055. Found:

381.1067.

(~) Pr p r-tlo~ of 5-~2-(2-Ami~o-4-oxo-4,6,7,8-t-tr-hydro-3~-pyr~dot5,4-b]~1,4]thlazln-6(R)-yl)-ethyl~-4-methyl-thlqph-~--2-c--~o-yllc ac~d (43):

H2Nl~ 02H

Starting with ester (42), compound (43) was prepared in 92 yiel according to the general procedure described for compound (13). (m.p. 253~C, with decomp.) [~]589 +60.7~ ~c = 0.29, lN NaOH).
IR (KBr) 3339 (broad), 2922, 1641, 1539, 1451, 1346, 1269 cm lH NMR (DMSO-d6) ~: 1.64 (m, lH), 1.82 (m, lH), 2.09 (s, 3H), 2.78-2.94 (m, 2H), 3.10-3.55 (m, 3H -- partially obscured by H2O), 6.10 (s, 2H), 6.69 (s, lH), 7.42 (s, lH), 10.15 (s, lH), 12.75 (br s, lH).
HRMS calc'd for C14H16N4O3S2: (M + Na+) 375.0562. Found:
375.0575.
(n) Preparatlon of 5-{2-[2-(2-Amino-4-oxo-4,6,7,8-tetrahydro-3H-pyr~m~dot5,4-b~1,4]thiazin-6(R)-yl)-et~yl]-4-methyl-thlop~ }-2-~-glutamic acid diethyl e~ter (44):

HN~

W096/40674 PcT~Sg6/08953 Starting with acid (43), compound (44) was prepared in 71~
yield according to the general procedure described for compound (15). (m.p. 124~C with fo~
[a~589 +33.6~ ~c = 0.50, DMSO).
IR (KBr) 2996, 2860, 1734, 1653, 1636, 1559, 1456, 1206 cm 1 lH NMR (DMSO-d6) ~: 1.11 (t, 3H, J ~ 7.0 Hz), 1.15 (t, 3H, J = 7.0 Hz), 1.75-2.04 (m, 4H), 2.10 (s, 3H), 2.37 (t, 2H, J = 7.4 Hz), 2.85 (m, 2H), 3.12 (m, 2H), 3.50 (m, lH), 4.00 (q, 2H, J =
7.0 Hz), 4.06 (q, 2H, J = 7.0 Hz), 4.31 (m, lH), 6.01 (s, 2H), 6.65 (s, lH), 7.55 (s, lH), 8.53 (d, lH, J = 7.7 Hz), 10.08 (s, lH).
HRM 1 23 31 5 6 2 ( C ) ~ ou 670.0742.
(o) Preparat~on of 5-{2-~2-~2-Am~no-4-oxo-4,6,7,8-t~trahydro-3~-pyrimido[5,4-b~[1,4~thiaizin-6~R)-yl)-othyl~-4-mothyl-thioph~n~}-2-~-glutam~c acid (45):

H N J~S~
1~N N CO2H

Starting with diester (44), compound (45) was prepared in 77 yield according to the general procedure described for compound (15). (m.p. 210~C with decomp.).

W096/40674 PCT~S96/ 089~3 la]589 +64.4~ (c = 0.45, lN NaOH).

IR ~RBr) 3341 (broad), 2928, 1701, 1638, 1536, 1449, 1340 cm lH NMR (DMSO-d6) ~: 1.78-2.05 (m, 4H), 2.10 (s, 3H), 2.28 (t, 2H, J s 7.0Hz), 2.82 (m, 2H), 3.45 (m, 3H -- partially obscured by H2O), 4.25 (m, lH), 5.98 (s, 2H), 6.65 (s, lH), 7.54 (s, lH), 8.38 (d, lH, J = 7.7Hz), 10.05 (s, lH), 12.5 (br s, 2H).

Anal. calc'd for ClgH23N5O6SØ7 H2O: C, 46.18; H, 4.98; N, 14.17; S, 12.98. Found: C, 46.13; H, 4.99; N, 14.09; S, 13.02.

~xamDle 4: Preparation of N-(5-[2-~2-Aml~o-4(3H)-oxo-5,6,7,8-tetrahydropyr~mldo~5,6-b]~1,4~th~-zln-6(S)-yl)ethyl~-4-methylthono-2-yl)-L-glutam~c acld (64) (a) Preparat~on of 5-[2,2-Dimethyl-1,3-dloxolan-4(R)-yl-ethynyl]-4-methylthiophene-2-carboxylic acid ethyl ester (46):

S C02Et /~

Starting with 2,2-dimethyl-4(R)-ethynyl-1,3-dioxolane and 5-bromo-4-methylthiophene-2-carboxylic acid ethyl ester, compound (46) was prepared in 57~ yield according to the general procedure described for compound (1).
[~]589 -38.3~ (c 5 0.95, CH30H).
lH NMR (CDC13) ~: 1.36 (t, 3H, J = 7.0 Hz), 1.42 (s, 3H), 1.53 (s, 3H), 2.29 (s, 3H), 4.04 (dd, lH, J = 6.3, 8.1 Hz), 4.26 PC rluss6/osss3 (dd, 1~, J - 6.3, 8.1 Hz), 4.32 (q, 2H, J = 7.0 Hz), 4.99 ~t, lH, J , 6.3 Hz), 7.50 (~, lH).
Anal. calc'd for C15H1804S: C, 61.20; H, 6.16; S, 10.89.
Found: C, 61.33; H, 6.20; S, 10.80.
(b) P.~ ation of 5-(2-12,2-D~m thyl-1,3-dioxolan-4~R)-yl-~thyl)-4-m thylthiophen~-2-carboxylic acid ~thyl ~st~r ~47):
~' ~<q~ s CO2Et / OJ

Starting with alkyne (46), compound (47) was prepared in 96 yield according to the general procedure described for compound-(2).
[a]589 +13.4~ (c = 0.72, CH30H).
lH NMR (CDC13) ~ = 1.35 (t, 3H, J = 7.2 Hz), 1.36 (s, 3H), 1.43 (s, 3H), 1.79-1.99 ~m, 2H), 2.16 (s, 3H), 2.75-2.96 (m, 2H), 3.55 (dd, lH, J = 7.0, 7.7 Hz), 4.04 (dd, lH, J = 5.9, 7.7 Hz), 4.08-4.16 (m, lH), 4.33 ~q, 2H, J = 7.2 Hz), 7.50 (s, lH).
Anal. calc'd for C15H2204S: C, 60.38; H, 7.43; S, 10.74.
Found: C, 60.48; H, 7.40; ~, 10.69.
(c) ~. p--ation of S-t3(R),4-Dihyd~G~Lutyl~-4-m~thylthiophene-2-crr~o~ylic acld ethyl e~ter (48):

HO~."'~S~CO2R

- PCT~S96/08953 ' Starting with dioxolane (47), compound (48) was prepared in 89~ yiel~ according to the general procedure de~cribed for compound (3).
t~] 589 +25.4 ~ (c 5 0.82, CH30H) .
lH NMR (CDC13) ~ = 1.35 (t, 3H, J = 7.0 Hz), 1.73-1.81 ~m, 2H), 2.17 (s, 3H), 2.80-3.00 (m, 2H), 3.48 (dd, lH, J = 7.5, 10.8 Hz), 3.68 (dd, lH, J s 3.1, 10.8 Hz), 3.71-3.79 (m, lH), 4.31 (q, 2H, J = 7.0 Hz), 7.50 ~s, lH) .
Anal. calc'd for C12H1804S: C, 55.79; H, 7.02; S, 12.41 Found: C, 55.69; H, 7.03; S, 12.48.
td) Preparation of 5-[3(R)-Hydroxy-4-(p-toluenesulfonyloxy)-butyl~-4-methylth~ophene-2-~-rh~Yyl~c ac~d ethyl cster (49):
,~' HO~ ' S CO2Et TsO 49 Starting with diol ~48), compound (49) was prepared in 65~
yield according to the general procedure described for compound (4).
[a~ 589 +7.5~ (c = 0.59, CH30H) .
lH NMR ~CDC13) ~ 5 1.35 (t, 3H, J = 7.0 Hz), 1.68-1.79 (m, 2H), 2.13 (s, 3H), 2.46 (s, 3H), 2.78-2.95 (m, 2H), 3.82-3.94 (m, 2H), 4.03 (dd, lH, J = 1.8, 8.8 Hz), 4.31 (q, 2H, J = 7.0 Hz), 7.36 (d, 2H, J = 8.1 Hz), 7.48 (s, lH), 7.79 (d, 2H, J = 8.1 Hz).
Anal. calc'd for ClgH2406S2 C, 55.32; H, 5.86; S, 15.54.
Found: C, 55.26i H, 5.87; S, 15.64.

r-~-s-tlo~ of 5-~4-(tert-Butyld~methylsllyloxy)-3(S)-oa~tyll-4-methylthloph-ne-2-c~r~o~yllc acld ethyl ~st-r (50):

HOC~CO2R
~S- 50 Starting with diol (33), compound (50) was prepared in 95%
yield according to the general procedure described for compound (16).
[~]5~9 -23.5~ (c = 1.19, CH30H).
lH NMR (CDC13) ~: 0.07 (s, 6H), 0.90 (s, 9H), 1.35 (t, 3H, J = 7.1 Hz), 1.61-1.81 (m, 2H), 2.17 (s, 3H), 2.79-3.01 (m, 2H)-, 3.43 (dd, lH, J = 7.0,9.6 Hz), 3.63 (dd, lH, J = 3.3, 9.6 Hz), 3.66-3.71 (m, lH), 4.31 (q, 2H, J = 7.1.Hz), 7.50 (s, lH).
Anal. calc'd for C18H3204SSi: C, 58.03; H, 8.66; S, 8.60.
~ound: C, 57.71; H, 9.14; S, 8.47.
(f) Preparat~on of 5-~4-(tert-Butyldimethyl-llyloxy)-3(S)-(methanesulfonyloxy)butyl]-4-m~thylthlophono-2-carboxylic ac~d ethyl ecter (51):

MsOo~S CO2Et ~S,-Starting with alcohol (50), compound (51) was prepared in 84 yield according to the general procedure described for compound (17).

W O 96/40674 PCTnUS96/08953 ~a]589 ~3.9~ (c = 1.48, CH30H).

lH NMR ~CDC13) ~: 0.09 (s, 6H), 0.90 (s, 9H), 1.35 (t, 3H, J

= 7.0 Hz), 1.97-2.05 (m, 2H), 2.16 (s, 3H), 2.82-3.01 (m, 2H), 3.07 (8, 3H), 3.68-3.82 (m, 2H), 4.31 (q, 2H, J = 7.0 Hz), 4.69-4.77 (m, lH), 7.50 (s, lH).

Anal. calc'd for ClgH3406S2Si C, 50.64; H, 7.60; S, 14.23.

Found: C, 50.54; H, 7.58; S, 14.30.

(g) Prep~rat~on of 5-r4-Hydroxy-3(S)-(me~n~lfo~yloxy)butyl~-4-msthylthio~h~n~-2-carboxyl~c acid et~yl ester (52):

MsO~ ~CO2Et Starting with silyl ether (51), compound (52) was prepared in 39~ yield according to the general procedure described for compound (18).
[~]589 +14.2~ (c = 0.97, CH30H).
lH NMR (CDC13) ~: 1.35 (t, 3H, J = 7.0 Hz), 1.96-2.12 (m, 2H), 2.17 (s, 3H), 2.85-2.94 (m, 2H), 3.12 (s, 3H), 3.77 ~dd, lH, J = 6.6, 12.5 Hz), 3.86 (dd, lH, J = 2.9, 12.5 Hz), 4.31 (q, 2H, J

= 7.0 Hz), 4.7B-4.87 (m, lH), 7.50 (s, lH).
Anal. calc d for C13H2006S2: C, 46.41; H, 5.99; S, 19.06.
Found: C, 46.51; H, 6.04; S, 18.95.

W096/40674 PCT~S96/08953 (h) Pr-par~tlo~ of 5-t3(R),4-~Ga~L~tYl]-4-methylthi~rh~~-~-c~rboxyl~c acid ~t~yl ester (53):

S CO2Et 0~

Starting with mesylate (52), compound (53) was prepared in 70~ yield according to the general procedure described for compound (19).
[~]589 +23.5~ (c = 0.80, CH30H) .

H NMR (CDCl3) ~ = 1.35 (t, 3H, J = 7.1 Hz), 1.74-1.86 (m, lH), 1.89-2.01 (m, lH), 2.17 (s, 3H), 2.51 (dd, lH, J = 2.6, 14.8 Hz), 2.78 (dd, lH, J = 4.0, 14.8 Hz), 2.86-3.00 (m, 3H), 4.31 (q, 2H, J = 7.1 Hz), 7.50 (s, lH) .
Anal. calc'd for C12H16O3S: C, 59.98; H, 6.71; S, 13.34.
Found: C, 59.90; H, 6.73; S, 13.41.
(i) Preparation of 5-[4-Azido-3(R)-hydroxybutyl]-4-methylthiophene-2-carboxylic acid ethyl ester (54):
~' HO ~ S CO2Et Starting with tosylate (49), compound (54) was prepared in 91~ yield according to the general procedure described for compound (5).

W O 96140674 PCTrUSg6tO8953 Starting with epoxide (53), co~ ound (5~) was prepared in 79 yield according to the general procedure described for compound (20).

[a] 589 +11.0~ (c = 0.58, CH30H).

lH NMR ~CDCl3) ~: 1.35 ~t, 3H, J - 7.2 Hz), 1.75-1.84 (m, 2H), 2.17 (s, 3H), 2.80-3.00 (m, 2H), 3.29 ~dd, lH, J = 7.0, 12.3 Hz), 3.42 ~dd, lH, J = 3.1, 12.3 Hz), 3.75-3.83 (m, lH), 4.31 (q, 2H, J = 7.2 Hz), 7.50 (s, lH).

Anal. calc'd for C12H17N3O3S: C, 50.87; H, 6.05; N, 14.83; S, 11.31. Found: C, 50.94 H, 6.07; N, 14.75; S, 11.22.

(~) Prep~rat~on of 5-t4-(tort-Butoxycarbonylam~no)-3(R)-hydLo~yL~tyl]-4-m~thylth~ n --2-c~rboxylic ac$d ~thyl ~otor (55):

HO~ ~CO2Et HN

Starting with azide (54), compound (55) was prepared in 96 yield according to the general procedure described for compound (6).
[~]589 l22.6~ ~c = 0.66, CH30H).
lH NMR ~CDCl3) ~: 1.35 (t, 3H, J z 7.2 Hz), 1.45 ~s, 9H), 1.72-1.81 ~m, 2H), 2.16 ~s, 3H), 2.7B-2.99 ~m, 2H), 3.09 ~dd, lH, J = 7.4, 14.3 Hz), 3.29 (dd, lH, J = 2.9, 14.3 Hz), 3.70-3:78 (m, lH), 4.31 (q, 2H, J = 7.2 Hz), 7.50 (s, lH).
Anal. calc'd for C17H27N05S: C, 57.12; H, 7.61; N, 3.92; S, 8.97. Found: C, 57.05 H, 7.63; N, 3.84; S, 9.07.

PCT~S96/08953 (~) Pr-p-s-tlon of 5-~4-(t~rt-Butoxyc~r~onylam~no)-3(R)-~athan--ulfonyloxy)butyl]-4-methylthioph-n--2-carboxyl~c acid ~thyl ~-t-r (56):

MSO ~ S CO2Et HN ~

Starting with alcohol (55), compound (56) was prepared in 93 yield according to the general procedure described for compound (7).
[~]589 7.8~ (c = 0.98, CH30H) .
lH NMR (CDCl3) ~: 1.35 (t, 3H, J = 7.2 Hz), 1.45 (s, 9H), 1.98-2.06 ~m, 2H), 2.16 (s, 3H), 2.86-2.92 (m, 2H), 3.06 (s, 3~, 3.34-3.43 (m, 2H), 4.31 (q, 2H, J = 7.2 Hz), 4.77-4.84 (m, lH), 4.92 (broad, lH), 7.50 (s , lH) .
Anal. calc'd for C18H29NO7S-: C, 49.64; H, 6.71; N, 3.22; S, 14.72. Found: C, 49.57; H, 6.78; N, 3.28; S, 14.70.
~1) Preparatlon of 5-14-(tort-~utoxycarbonylamlno)-3(S)-(acetylthlo)butyl-4-methylthiophene-2-carboxylic acid sthyl ester (57):

Ac~CO2Et HN
BOC

Starting with mesylate (56), compound (57) was prepared in 92~ yield according to the general procedure described for compound (8).

W O 96/40674 PCTnUS96fO8953 ~a]589 l2.8~ (c . 0.93, CH30H).

1 (CDC13) ~: 1.34 (t, 3H, J = 7.0 Hz), 1.44 (9, 9H), 1.78-1.89 (m, lH), 1.93-2.04 (m, lH), 2.13 (8, 3H), 2.37 (9, 3H), 2.75-2.94 (m, 2H), 3.26-3.45 ~m, 2H), 3.56-3.64 (m, lH), 4.30 tq, 2H, J = 7.0 Hz), 4.75(broad, lH), 7.48 (8, lH).

Anal. calc'd for ClgH29NO5S2: C, 54.92; H, 7.03; N, 3.37; S, 15.43. Found: C, 54.81; H, 7.09; N, 3.43; S, 15.41.

(m) Preparat~on of 2-(~1-(tcrtBuoxycarbonylamlno)-4-)-5-ethoxycarbonyl-3-methylt~ien-2-yl)but-2(S)-yl]thlo) malonic acid diethyl e~ter (58):
~3' MeO2C~ ~/ S C~

MeO2C NH

Starting with thioacetate (57), compound ~58) was prepared in 88% yield according to the general procedure described for compound ~24).
[~]589 -23.7~ tc = 0.68, CH30H).
lH NMR (CDC13) ~: 1.34 (t, 3H, J = 1.1 Hz), 1.44 (s, 9H), 1.75-1.86 (m, lH), 1.90-1.99 (m, lH), 2.15 (s, 3H), 2.85-2.97 (m, 2H), 3.00-3.07-(m, lH), 3.23-3.39 (m, 2H), 3.80 ~8, 6H), 3.87 (s, lH), 4.31 (q, 2H, J = 7.1 Hz), 5.12 (broad, lH), 7.49 (s, lH).
Anal. calc'd for C22H33NO8S2: C, 52.47; H, 6.60; N, 2.78; S, 12.73. Found: C, 52.49; H, 6.64; N, 2.77; S, 12.64.

W O g6/40674 PCT~US96/08953 (n) Pr-p-r-t~o~ of 6(S)-[2-(5-ethoxyr~r~o~yl-3-m thylth~-n-2-yl)-~~hyl-3-oxo-1,4-thiazine-2-~-r~QYyl~c acld m-thyl ~st-r (59):

AleO2C~S~'CO2Et Starting with malonate (58), compound (59) was prepared in 88~ yield according to the general procedure described for compound (10).
[~]589 26.2~ (c = 0.81, CH30H).
lH NMR (CDC13) ~: 1.35 (t, 3H, J = 7.1 Hz), 1.83-2.03 (m, 2H), 2.16 (s, 3H), 2.83-2.97 (m, 2H), 3.39-3.67 (m, 3H), 3.60,3.82 (s, 3H), 4.14,4.25 (s, lH), 4.31 (q, 2H, J = 7.1 Hz), 6.29 (broad, lH), 7.50 (s, lH).
Anal. calc'd for C~ H21NO5S2: C, 51.73; H, 5.70; N, 3.79; S, 17.26. Found: C, 51.56; ~, 5.80; N, 3.70; S, 17.09.
(o) Preparation of 6(S)-~2-(5-Ethoxycarbonyl-3-m-thylth~en-2-yl)-thyl]-3-methoxy-1,4-thiazine-2-carboxyl~c acid methyl ester (60):

MeO2CX~CO2Et N

Starting with lactam (59), compound (60) was prepared in quantitative crude yield according to the general procedure W096l40674 PcT~ss6Jo89s3' described for compound (11) and was used without further purification.
(p) Pr-p-r-tion of 5-t2-(2-Am~no-4(3~)-oxo-5~6~7~8-t-trahydro-pyrimidot5,6-b~11,4~thiazin-6(S)-yl)-thyl~-4-m-thylthio~h~n~-2-c-rh~xylic acid ~thyl ~-tor (61):

H N ~ ~ ~ C02E~

Starting with imidoether (60), compound (61) was prepared in 27~ yield according to the general procedure described for compound (12).
[a] 589 41.50 (c = 0.66, DMSO) .
lH NMR (DMSO-d6) ~: 1.25 (t, 3H, J = 7.0 Hz), 1.62-1.73 (m, lH), 1.80-1.91 (m, lH), 2.13 (s, 3H), 2.79-2.97 (m, 3H), 3.19-3.26 (m, lH), 3.47-3.55 (m, lH), 4.22 tq, 2H, J = 7.0 Hz), 6.05 (s, 2H), 6.68 (s, lH), 7.53 (s, lH), 10.10 (s, lH).
(~) Propar~tion of 5-12-(2-Amlno-4(3H)-oxo-5,6,7,8-t-trahydro-pyrimidot5,6-b~[1,4]thiazin-6(S)-yl)cthyl~-4-m-thylth~oph~ne-2-carboxylic acid (62):

H N 1~ ~ N ~ C02H

WOs6/40674 PCT~S96/08953 -Starting with e~ter (61), co~ ound (62) was prepared in 77 yield according to the general procedure described for compound (13).
~ a]589 77-9~ (c ~ 0.58, lN NaOH).
lH NMR ~DMSO-d6) ~: 1.62-1.73 ~m, lH), 1.79-1.92 (m, lH), 2.12 ~s, 3H), 2.81-2.98 (m, 3H), 3.16-3.26 (m, lH), 3.50-3.58 (m, lH), 6.21(br s, 2H), 6.76 (br s, lH), 7.45 (s, lH), 10.24 (broad, lH), 12.76 (broad, lH).
Anal. calc'd for C14H16N4O3S2.1.4 H2O: C, 44.52; H, 5.02; N, 14.845,16.98. Found: C, 44.60; H, 4.86; N, 14.70; S, 16.92.

(r) Prepar~tion of N-(5-r2-(2-Am~no-4(3H)-oxo-5,6,7,8-totrahydl~y~ dot5,6-~]tl,4~thiaz~n-6(S)-yl)~thyl~-4-met~yltheno-2-yl)-L-glutamic acid diethyl e~ter ~63):

HN~ ~CO2Et H2N N H O CO2Et Starting with acid (62), compound (63) was prepared in 60~
yield according to the general procedure described for compound (14).

[a]589 -si.1~ (c = o . 61, DMSO).
lH NMR (DMSO-d6) ~: 1.15 (t, 3H, J = 7.1Hz), 1.17 ~t, 3H, J
= 7.1Hz), 1.61-1.72 ~m, lH), 1.77-2.07 ~m, 3H), 2.13 (s, 3H), 2.40 ~t, 2H, J = 7.5 Hz), 2.77-2.94 (m, 3H), 3.18-3.28 (m, lH), 3.50-3.56 (m, lH), 4.03 (q, 2H, J = 7.1 Hz), 4.08 (q, 2H, J = 7.1 PCT~S96/08953 W096t40674 Hz), 4.34 ~ddd, lH, J = 5.4, 7.7, 9.6 Hz), 6.02 ~8, 2H), 6.67 (s, lH), 7.58 (8, lH), 8.55 (d, lH, J 5 7.7 Hz), 10.06 (9, lH).

Anal. calc'd for C23H31N506S2. 2 12.81 S, 11.73. Found: C, 50.56; H, 5.92; N, 12.67; S, 11.54.
~s) Proparat~o~ of N-~5-[2-(2-Amino-4~3H)-oxo-5,6,7,8-tetrahyd~o~m~dot5,6-b1[1,4~ thlazin-6~S)-yl)~thyl~-4-m thyltheno-2-yl)-~-glutamlc acid t64):

HNJ~
H2N N H ~ CO2H

Starting with diester (63), compound ~64) was prepared in-85 yield according to the general procedure described for compound ~15).
[~]589 36.8~ (c = 0.57, lN NaOH).
lH NMR (DMSO-d6) ~: 1.61-1.72 (m, lH), 1.76-1.92 (m, 2H), 1.99- 2.08 (m, lH), 2.12 (s, 3H), 2.31 (t, 2H, J = 7.0 Hz), 2.79-2.94 (m, 3H), 3.17-3.28 (m, lH), 3.49-3.56 (m, lH), 4.30 (ddd, lH, J = 5.7, 7.7, g.8 Hz), 6.08 (s, 2H), 6.70 (s, lH), 7.58 (s, lH), 8.44 (d, lH, J = 7.7 Hz), 10.12 (br s, lH), 12.43 (broad, 2H).
Anal. calc'd for ClgH23N5O6S2Ø75 H2O: C, 46.09; H, 4.99;
N, 14.15; S, 12.95. Found: C, 46.09; H, 4.98; N, '4.01; S, 12.77.

Claims (19)

We Claim:

1. A compound of the formula or its enantiomer, wherein:
Ar is a substituted or unsubstituted five- or six-membered aromatic group; and B is an amino acid linked through the amino portion to form an amide, or a C1-C6 alcohol linked through the alcohol portion to form an ester.

2. A compound according to claim 1, wherein Ar is a 5-membered aromatic group having a sulfur ring atom.

3. A compound according to claim 1, wherein said amino acid is diethyl glutamate and said C1-C6 alcohol is methanol or ethanol.

4. A compound according to claim 1, which has the (S) configuration.

5. A compound according to claim 4, which is 5-(3(S)-4-dihydroxy-butyl)-thiophene-2-carboxylic acid ethyl ester.

6. A compound according to claim 1, which has the (R) configuration.

7. A method of making a compound of the formula or its enantiomer, wherein Ar is a substituted or unsubstituted five- or six-membered aromatic group and B is either an amino acid linked through the amino portion to form an amide or a C1-C6 alcohol linked through the alcohol portion to form an ester, comprising the steps of:
(a) reacting a compound of the formula or its enantiomer with a compound of the formula wherein X is bromo, fluoro, chloro or iodo, and B and Ar are as defined above, to form a compound of the formula or its enantiomer, wherein Ar and B are as defined above;
(b) reacting the compound of formula V or its enantiomer with a reducing agent to obtain a compound of the formula or its enantiomer, wherein Ar and B are as defined above; and (c) reacting the compound of the formula VI or its enantiomer with an acid to obtain the compound of the formula VII
or its enantiomer.

8. A method according to claim 7, wherein in step (a) the reacting is carried out in the presence of (i) a transition-metal catalyst containing one or more metals selected from the group consisting of palladium, copper and nickel, and (ii) a non-nucleophilic auxiliary base (iii) in a solvent in which at least one of the reactant compounds is at least partially soluble.

9. A method according to claim 7, wherein in step (b) the reducing agent is hydrogen gas in the presence of a metal catalyst containing palladium or platinum.

10. A method according to claim 7, wherein in step (c) the acid is p-toluenesulfonic acid.monohydrate and the reacting is carried out in methanol or ethanol.

11. A method according to claim 7, wherein in step (a), a compound of the formula III is reacted.

12. A method according to claim 7, wherein in step (a), a compound of the formula XVII is reacted.

13. A method of preparing a compound of the formula wherein A is an oxygen, sulfur or selenium atom, Ar is a substituted or unsubstituted five- or six-membered aromatic group, and R1 and R2 are independently a hydrogen atom or a moiety that forms together with the attached CO2 a readily hydrolyzable ester group, comprising:

reacting a compound of the formula or its enantiomer, wherein Ar is a substituted or unsubstituted five- or six-membered aromatic group and B is either an amino acid linked through the amino portion to form an amide or a C1-C6 alcohol linked through the alcohol portion to form an ester, to form the compound of the formula I that is optically pure.

14. A method according to claim 13, wherein the compound of the formula VII is prepared by steps comprising:
(a) reacting a compound of the formula or its enantiomer with a compound of the formula wherein X is bromo, fluoro, chloro or iodo, and B and Ar are as defined above, to form a compound of the formula or its enantiomer, wherein Ar and B are as defined above;
(b) reacting the compound of formula V or its enantiomer with hydrogen gas in the presence of a metal catalyst to obtain a compound of the formula or its enantiomer, wherein Ar and B are as defined above; and (c) reacting the compound of the formula VI or its enantiomer with an acid in an alcoholic solvent to obtain the compound of the formula VII or its enantiomer.

15. A method according to claim 14, wherein in step (a) the reacting is carried out in the presence of (i) a transition-metal catalyst containing one or more metals selected from the group consisting of palladium, copper and nickel, and (ii) a non-nucleophilic auxiliary base (iii) in a solvent in which at least one of the reactant compounds is at least partially soluble.

16. A method according to claim 13, wherein Ar is a 5-membered aromatic group having a sulfur ring atom.

17. A method according to claim 13, wherein the compound of the formula I has the (R) configuration.

18. A method according to claim 13, wherein the compound of the formula I has the (S) configuration.

19. A method according to claim 13, wherein the compound of the formula VII or its enantiomer is 5-(3(S)-4-dihydroxy-butyl)-thiophene-2-carboxylic acid ethyl ester.