CA2271147A1 - Combinatorial process for preparing thiazolidinone libraries - Google Patents

Abstract

This invention relates to a novel diverse combinatorial library of thiazolidinone compounds and to an apparatus providing a readily accessible source of individual members of the library. The apparatus can be used in assay kits and as a replaceable element in automated assay machines.

Description

WO 98l21584 PCT/US97/21002 COMBINATORIAL PROCESS FOR PREPARING THIAZOLIDINONE
LIBRARIES
This application claims the benefit of U.S.
Provisional Patent Application Serial No. 60/029,741, filed November 12, 1996.
Field of the Invention The present invention relates to diverse libraries of thiazolidinone compounds, methods of making such libraries, and an apparatus for storing and providing a readily accessible source of diverse thiazolidinone compounds. The apparatus harboring the present combinatorial libraries is a useful component of assay systems for identifying compounds for drug development.
Background of th.e Invention Research and development expenses account for a large outlay of capital in the ~oharmaceutical industry.
Synthesis of compounds is an expensive and time consuming phase of research and development. Historically, research chemists individually synthesized and analyzed high purity compounds for biological screening to develop pharmaceutical leads. Although such methods were successful in bringing new drugs to the market, the limitations of individual synthesis and complete compound characterization considerably slowed the discovery of new pharmaceutically active compounds. The need for more rapid and less expensive drug discovery methodology is increasingly important in today's competitive pharmaceutical industry.
Recently, modern drug discovery has utilized combinatorial chemistry to generate large numbers (102 -106) of compounds generically referred to as "libraries".
An important objective of combinatory chemistry is to generate a large number of novel compounds that can be screened to generate lead compounds for pharmaceutical research.
Theoretically the total number of compounds which may be produced for a given library is limited only by the number of reagents available to form substituents on the variable positions on the l~ubrary's molecular scaffold. The combinatorial process lends itself to automation, both in the generation of compounds and in their biological screening, thereby greatly enhancing the opportunity and efficiency of drug discovery.
Combinatorial chemistry may be performed in a manner where libraries of compounds are generated as mixtures with complete identification of the individual compounds postponed until after positive screening results are obtained. However, a preferred form of combinatorial chemistry is "parallel array synthesis", where individual reaction products are simultaneously synthesized, but are retained in separate vessels. For example, the individual library compounds can be prepared, stored, and assayed in separate wells of a rnicrotiter plate, each well containing one member of the parallel array. The use of standardized microtiter plates or equivalent apparatus, is advantageous because such an apparatus is readily accessed by programmed robotic machinery, both during library synthesis and during library sampling or assaying.
Typically, completion of the solution phase reactions in combinatorial chemistry schemes are ensured by selecting high yielding chemical reactions and/or by using one reagent in considerable excess. When one reagent is used in excess, comp~Letion of the reaction produces a mixture of the solub:Le product and at least one soluble unreacted reagent.
Combinatorial chemistry may be used at two distinct phases of drug development. In the discovery phase diverse libraries are created to find lead compounds. Tn a second optimization phase, strong lead compounds are more narrowly modified to find optimal molecular configurations.
The method of the present invention is based on the preparation of a novel diverse 7_ibrary of thiazolidinones useful in the identification of new lead compounds. The thiazolidinone library is prepax-ed by the cyclocondensation of alpha-mercaptocarboxylic acids, or esters thereof, with aryl imine:~. [See Y. Tanabe et al., Tetrahedron Letters, Vol. 32, No. 3, pp. 383-386 (1991).]
The library is created, stored, and used as an apparatus comprising a two-dimensional arx:ay of reservoirs, each reservoir containing a predetermined library reaction product differing from those in adjacent reservoirs.
Summary of the Invention The present invention provides combinatorial libraries of structurally related library compounds having a thiazolidinone scaffold with the general formula R~, S
~c O

R1 ~ R2:
wherein R1 is a substituent derived from an optionally substituted benzaldehyde of the formula CHO
/~

Ri R2 is hydrogen or an organic moiety derived from a primary amine of the formula R2DTH2, and R3 is an organic moiety derived from an alpha-mercaptocarboxylic acid of the formula SH
R3~ CO;rH
or ester thereof.
The invention further provides a method for preparing thiazolidinone libraries generally in accordance with Scheme 1 as set forth below.
Another embodiment of the present invention provides an assay kit for the identificas~ion of pharmaceutical lead thiazolidinone compounds, said kit comprising assay materials and a well plate apparatus or equivalent apparatus providing a two-dimensional array of defined reservoirs. The well plate apparatus provides a diverse combinatorial library, wherein Each well (reservoir) contains a unique reaction product of the thiazolidinone library. The well plate apparai~us is used to provide multiple reaction zones for mak_i.ng the library, to store and to provide a readily accessible source of library compounds.
Brief Description of the Drawinas Fig. 1 is a top view of a well plate in accordance with this invention.
Fig. 2 is a side view of a well plate apparatus for use in the process of this invention.
Detailed Description of the Invention The term "assay kit" as usE~d in accordance with the present invention refers to an <~ssemblage of two cooperative elements, namely (1) a well plate apparatus and (2) biological assay materials.

"Biological assay materials" are materials necessary to conduct a biological evaluation of the efficacy of any library compound in a screen relevant to a selected disease state.
A "library" is a collection of compounds created by a combinatorial chemical process, said compounds having a common scaffold with one or mor~= variable substituents.
The scaffold of the present invention is a thiazolidinone.
A "library compound" is an individual reaction product, a single compound or mixture of isomers, in a combinatorial library.
A "Lead compound" is a library compound in a selected combinatorial library for which the assay kit has revealed significant activity relevant to a selected disease state.
A "diverse library" means ~~ library where the substituents on the combinatorial library scaffold or core structure, are highly variable in constituent atoms, molecular weight, and structure, and the library, considered in its entirety, is :not a collection of closely related homologues or analogues (compare to "directed library").
A "directed library" is a collection of compounds created by a combinatorial chemical process, for the purpose of optimization of the activity of a lead compound, wherein each library compound has a common scaffold, and the library, considered in its entirety, is a collection of closely related homologues or analogues to the lead compound (compare with "diverse library").
The term "scaffold" as used in accordance with the present invention refers to the invariable region (a thiazolidinone core in the present invention) of the compounds which are members of the combinatorial library.
"Substituents" are chemical radicals which are bonded to or incorporated onto the thiazolidinone scaffold through the combinatorial synthesis process.
The different functional groups account for the diversity of the molecules throughout the library and are selected to impart diversity of biologics l activity to the scaffold in the case of diverse libraries, and optimization of a particular biological activity in the case of directed libraries.
"Reagent" means a reactant, any chemical compound used in the combinatorial synthesis to place substituents on the scaffold of a library.
"Parallel array synthesis" refers to the method of conducting combinatorial chemical synthesis of libraries wherein the individual combinatorial library compounds are separately prepared and stox-ed without prior and subsequent intentional mixing.
"Simultaneous synthesis" mE:ans making of library compounds within one production cycle of a combinatorial method (not making a11 library compounds at the same instant in time).
The "reaction zone" refers to the individual vessel location where the combinatorial. chemical library compound preparation process of the invention is carried out and where the individual library compounds are synthesized. Suitable reaction zones are the individual wells of a well plate apparatus.
"Well plate apparatus" refers to the structure capable of holding a plurality of library compounds in dimensionally fixed and defined positions.
"Non-interfering substituents" are those groups that do not significantly impede the process of the invention and yield stable thiazolidinone library compounds.
"Aryl" means one or more aromatic rings, each of 5 or 6 ring carbon atoms and includes substituted aryl having one or more non-interfering substituents.
Multiple aryl rings may be fused, as in naphthyl, or unfused, as in biphenyl.

-"Alkyl" means straight or branched chain or cyclic hydrocarbon having 1 to 20 carbon atoms.
"Substituted alkyl" is alkyl having one or more non-interfering substituents.
"Halo" means chloro, fluoro, iodo or bromo.
"Heterocycle" or "heterocyclic radical" means one or more rings of 5, 6 or 7 atoms with or without unsaturation or aromatic characi~er, optionally substituted, and at least one rang atom which is not carbon. Preferred heteroatoms :include sulfur, oxygen, and nitrogen. Multiple rings may be fused, as in quinoline or benzofuran, or unfused as in 4-phenylpyridine.
"Substituted heterocycle" or "Substituted heterocyclic radical" is heterocycle having one or more non-interfering substituents. Suitable radicals for substitution on the heterocyclic ring structure include, but are not limited to halo, C1~-C1p alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 alkoxy, C~-C12 aralkyl, C~-C12 alkaryl, C1-C10 alkylthio, arylthio, aryloxy, arylamino, C3-C10 cycloalkyl, C3-C1p cycloalkenyl, di(C1-C10)-alkylamino, C2-C12 alkoxya:Lkyl, C1-C6 alkylsulfinyl, C1-C10 alkylsulfonyl, arylsulfonyl, aryl, hydroxy, hydroxy(C1-C10)alkyl, aryloxy(C_L-C10)alkyl, C1-C10 alkoxycarbonyl, aryloxycarbonyl, C1-C10 alkanoyloxy, aryloyloxy, substituted alkoxy, fluoroalkyl, vitro, cyano, cyano(C1-C1p)alkyl, C1-C_Lp alkanamido, aryloylamido, arylaminosulfonyl, sulfonamido, heterocyclic radical, nitroalky:L, and -(CH2)m-Z-(C1-C10 alkyl), where m is 1 to 8 and Z is oxygen or sulfur.
"Organic moiety" means a substituent comprising a non-interfering substituent covalently bonded through at least one carbon atom. Suitably=_ radicals for substitution onto the connecting carbon atom include, but are not limited to hydrogen, ha:Lo, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 alkoxy, C~-C12 aralkyl, -g_ C~-C12 alkaryl, C1-C2p alkylthio, arylthio, aryloxy, arylamino, C3-C1p cycloalkyl, C3-Clp cycloalkenyl, di(C1-C1p)-alkylamino, C2-C12 alkoxyalkyl, C1-C6 alkylsulfinyl, C1-C1p alkylsulfonyl, arylsulfonyl, aryl, hydroxy, hydroxy(Cl-C1p)alkyl, aryloxy(Cl-Clp)alkyl, Cl-C10 alkoxycarbonyl, aryloxycarbonyl, C1-C1p alkanoyloxy, aryloyloxy, substituted alkoxy, fluoroalkyl, nitro, cyano, cyano(C1-C1p)alkyl, C1-C1p alkanamido, aryloylamido, arylaminosulfonyl, sulfonamido, heterocyclic radical, nitroalkyl, and -(CH2)m-Z-(C1-C10 alkyl), where m is 1 to 8 and Z is oxygen or sulfur.
"Optionally substituted benzaldehyde" means benzaldehyde or benzaldehyde having at least one non-interfering substituent covalently bound to the benzene 25 ring. Suitable radicals for substitution on the benzene ring include, but are not limited to halo, C1-Clp alkyl, C2-Clp alkenyl, C2-C1p alkynyl, C1-C1p alkoxy, C~-C12 aralkyl, C~-C12 alkaryl, C1-Clp alkylthio, arylthio, aryloxy, arylamino, C3-C1p cycloalkyl, C3-C10 cycloalkenyl, di(C1-C1p)-alkylamino, C2-C12 alkoxyalkyl, C1-C6 alkylsulfinyl, C1-Clp alkylsulfonyl, arylsulfonyl, aryl, hydroxy, hydroxy(C1-C1p)alkyl, aryloxy(C1-C1p)alkyl, C1-C1p alkoxycarbonyl, aryloxycarbonyl, C1-C10 alkanoyloxy, aryloyloxy, substituted alkoxy, fluoroalkyl, nitro, cyano, cyano(C1-Clp)alkyl, Cl-Clp alkanamido, aryloylamido, arylaminosulfonyl, sulfonamido, heterocyclic radical, nitroalkyl, and -(CH2)m-Z-(C1-C10 alkyl), where m is 1 to 8 and Z is oxygen or sulfur.
A diverse library of thiazolidinones is provided in accordance with the present invention. The thiazolidinone library embodied as an apparatus of this invention serves as a readily accessible source of diverse thiazolidinone compounds for use in identifying new biologically active thiazolidinone compounds through pharmaceutical and agricultural candidate screening assays, for use in studies defining structure/activity relationships, and/or for use in clinical investigation.
The library provided in accordance with the present invention includes thiazolidinone compounds of the formula F;3 S
,~ O
/ ; I
R 1 ~ Ft2 wherein R1 is hydrogen or a substituent derived from an optionally substituted benzaldehyde of the formula CHO
~R ~
R2 is hydrogen or an organic moiety derived from a primary amine of the formula R~;NH2, and R3 is an organic moiety derived from an alpha-mercaptocarboxylic acid of the formula SH
R3~ CO2H
or ester thereof.
In another embodiment of t:he present invention there is provided a library of compounds of Formula I above, wherein R1 is selected from non-interfering substituents, R2 is alkyl, substituted alkyl, or aryl, and R3 is hydrogen, alkyl, substituted alkyl, or aryl.
In another embodiment of this invention there is provided a library of compounds of Formula I above, wherein R1 is selected from non-interfering substituents, R2 is alkyl, substituted alkyl, or aryl, and R3 is hydrogen, C1-C10 alkyl or substituted (C1-C10 alkyl).

WO 98I21584 PCTlUS97121002 In still another embodiment of the present invention there is provided a library of compounds of Formula I
above, wherein R1 is a non-interfering substituent selected from the group consisting of halo, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 alkoxy, C12 aralkyl, C~-C12 alkaryl, C~_-C10 alkylthio, arylthio, aryloxy, arylamino, C3-C10 cyc7_oalkyl, C3-C10 cycloalkenyl, di(C1-C10)-alkylamino, C2-C12 alkoxyalkyl, C1-C6 alkylsulfinyl, C1-C10 all~:ylsulfonyl, arylsulfonyl, aryl, hydroxy, hydroxy(C1-C1p)alkyl, aryloxy(C1-C10)alkyl, C1-C10 alkoxycarbonyl, aryloxycarbonyl, C1-C10 alkanoyloxy, aryloyloxy, substituted alkoxy, fluoroalkyl, nitro, cyano, cyano(C1-C10)alkyl, C1-C10 alkanamido, aryloylamido, arylaminosulfony7_, sulfonamido) heterocyclic radical, nitroalkyl, or -(CH2)m-Z-(C1-C10 alkyl), where m is 1 to 8 and Z is oxygen or sulfur; R2 is C1-C10 alkyl, substituted (C1-C10 alkyl), or aryl; and R3 is hydrogen, C1-C10 alkyl, or substituted (C1-C10 alkyl).
The present invention also provides a method for preparing the library of thiazolidinone compounds of Formula I using combinatorial chemistry in a parallel array synthesis technique illu:~trated in the following reaction scheme:
Scheme 1.
R

/N
EtOH R 3~ C02H S O
-E- R2NH2 -s- N
\~~ RT ~ CH2C12) RT
R1 \~ R~ ~. ~ R2 The method comprises the ~>teps of preparing substituted imine intermediate:; by reacting series of optionally substituted benzaldehydes with series of primary amines, and then further reacting each intermediate with alpha-mercapt.ocarboxylic acids or esters thereof to prepare a library of thiazolidinone compounds with three sites of diversity, R1, R2 and R3, each derived respectively from the benzaldehyde reagent, the primary amine reagent, and the alpha-mercaptocarboxylic acid/ester reagent. Each compound is prepared in a separate reaction zone (i.e. parallel array synthesis), and the predetermined product compound is identified by the plate and reaction well number.
The benzaldehyde, primary amine and alpha-mercaptocarboxylic acid/ester reagents are either commercially available or prepared from commercially available starting materials. Benzaldehydes for use in accordance with this invention are compounds of the formula CHO

wherein R1 is a non-interferincr group, i.e., a substituent which does not interfere with formation of the imine intermediate or the reaction of that intermediate with an alpha-mercaptocarboxylic acid or ester thereof. Typically the benzaldehyde reactants have a molecular weight of 106 to about 600.
Illustrative of suitable benzaldehydes are compounds of the following formulas, wherein L is CHO:

L L L
/-' o L L
~i o' v 'o I I ~ ~ ,.
b °~ cH, L L
i i I I / _ Br I I
L L L
I \
c~ Br ° °~ F
L L L
/
_CI CH3 L L L
y \ F
V
F I
/ ~ C~ F
O /
~er F
L L L
\ \
/ /
'o CI

ci L l_ L
/ i \ \ /
/ /
o I \
~ c~, .i L HaC O L
I ' / ~ /
I
L
\ \~
I
~c~o F L L L
cl ~ i \
HaC~ O
F I
/~
O
~O
L L L
H C I oho H'c~o CHI
L O L L
\ w H3C ~ / H,c ~ o C~ ~ o HOC ' L o L L
HC O ~ \

L L L
~1 CHI /
\.
O~ HOC / 1 HOC O
~O
H~ ~- /C
L ~L L
J( ~~1 CI ~C.M/V'p i I O /

L
i O
O
The primary amines for use in the process for preparing the present library a.re represented by the general formula RNH2, wherein R. is an organic moiety.

Typically, amine reagents have a molecular weight ranging from about 30 to about 600.
Illustrative of suitable amines for use in preparation of the thiazolidinone library of this invention are the following:
dl-1-phenylethylamine (-)-norephedrine 1,2-dimethylpropylamine isopropylamine 2-methoxyisopropylamine dl-2-amino-1-propanol ethyl-3-aminobutyrate 1,3-dimethylbutylamine 3-amino-1-phenylbutane 2-amino-5-diethylaminopeni:ane 1,5-dimethylhexylamine sec-butylamine (+/-)-2-amino-1-butanol 3-aminopentane 2-aminopentane 3-aminoheptane 2-aminoheptane 2-aminooctane benzylamine 2-fluorobenzylamine 2-chlorobenzylamine 2,4-dichlorobenzylamine 2-methoxybenzylamine 2-ethoxybenzylamine 2-methylbenzylamine 3-fluorobenzylamine 3,4-dichlorobenzylamine 3,4-dimethoxybenzylamine 3-(trifluoromethyl)benzylamine 3-methylbenzylamine 4-fluorobenzylamine 4-chlorobenzylamine 4-methoxybenzylamine 4-methylbenzylamine 2,2,2-trifluoroethylamine 2-amino-1-phenylethanol 1-amino-2-propanol 3-amino-1,2-propanediol 2,2-diphenylethylamine beta-methylphenethylamine isobutylamine 2-methylbutylamine 2-ethylhexylamine n-decylamine n-undecylamine dodecylamine tridecylamine 1-tetradecylamine hexadecylamine octadecylamine ethylamine 2-(2-aminoethylamino)ethanol 2-methoxyethylamine 2-(2-aminoethoxy)ethanol ethanolamine phenethylamine 2-(2-chlorophenyl)ethylamine 2-(2-methoxyphenyl)ethylarnine 3-methoxyphenethylamine 2-(3,4-dimethoxyphenyl)ethylamine 4-bromophenethylamine 2-(4-chlorophenyl)ethylamine 2-(4-methoxyphenyl)ethylarnine tyramine 2-(4-aminophenyl)ethylamine 2-(p-tolyl)ethylamine taurine propargylamine allylamine 3,3-dimethylbutylamine 3,3-diphenylpropylamine isoamylamine propylamine 3-dimethylaminopropylaminE~

3-diethylaminopropylamine 3-(di-n-butylamino)propylamine 3-isopropoxypropylamine 3-ethoxypropylamine 3-amino-2-propanol 3-phenylpropylamine 4-amino-1-butanol 4-phenylbutylamine n-amylamine 5-amino-1-pentanol hexylamine 6-amino-1-hexanol n-heptylamine n-octylamine n-nonylamine dl-2-amino-1-pentanol dl-2-amino-1-hexanol 1-(3-aminopropyl)imidazol~=_ WO 98l21584 PCT/US97/21002 3,5-bis(trifluoromethyl)benzylamine 2,4-difluorobenzylamine 2,5-difluorobenzylamine 2,6-difluorobenzylamine 3,4-difluorobenzylamine 4-(trifluoromethyl)benzylamine 2-(trifluoromethyl)benzylamine 4-(2-aminoethyl)benzenesul.fonamide n-(4-aminobutyl)-n-ethyli~;oluminol n-butylamine 2-(1-cyclohexenyl)ethylami.ne 3-methoxypropylamine 3,4,5-trimethoxybenzylamine 3-butoxypropylamine aminomethylcyclopropane pentadecylamine 4-(2,4-di-tert-amylphenoxy)butylamine 3-chlorobenzylamine 4-fluoro-alpha-methylbenzylamine (r)-(+)-bornylamine n,n-di-n-butylethylenediamine (r)-(-)-1-cyclohexylethylamine n,n,2,2-tetramethyl-1,3-propanediamine 1-phenylalanine beta-napht.hyl-amide 2-(3-chlorophenyl)ethylami.ne 2-amino-1,3-propanediol 2-(2-thienyl)ethylamine 2,3-dimethoxybenzylamine 3,5-dimethoxybenzylamine 2,4-dichlorophenethylamine 2,5-dimethoxyphenethylamine 3-fluoro-5-(trifluoromethyl)benzylamine 4-(trifluoromethoxy)benzyl.amine 1-leucinol 1-leucine-4-nitroanilide (r)-(+)-1-(1-naphthyl)ethylamine (s)-(-)-1-(1-naphthyl)ethylamine 1-valinol d-valinol d-phenylalaninol 1-(+)-alpha-phenylglycinol.

d-(+)-alpha-methylbenzylamine 1(-)-alpha-methylbenzylami.ne (is,2r)-(+)-phenyl-propanclamine (s)-(+)-2-amino-1-propanol.

d-alaninol (r)-(-)-sec-butylamine (s)-(+)-sec-butylamine (s)-(+)-2-amino-1-butanol (r)-(-)-2-amino-1-butanol (r)-(-)-1-amino-2-propanol.

(s)-(+)-1-amino-2-propanol (s)-(-)-2-methylbutylamine (s)-(+)-1-cyclohexylethyla:mine oleylamine 1-adamantanemethylamine (ls,2r)-(+)-2-amino-1,2-diphenylethanol (lr,2s)-(-)-2-amino-1,2-diphenylethanol s-benzyl-1-cysteinol 2-(2-(aminomethyl)phenylthio)benzyl alcohol 3-fluorophenethylamine 2-aminobenzylamine 2-fluorophenethylamine 4-aminobenzylamine d-glucamine (+/-)-2,5-dihydro-2,5-dimethoxyfurfurylamine (s)-(+)-tetrahydrofurfurylamine 4-fluorophenethylamine (ls,2s)-(+)-thiomicamine (-)-3,4-dihydroxynorephedrine (r)-(+)-1-(p-tolyl)ethylamine (s)-(-)-1-(p-tolyl)ethylamine (s)-(-)-2-amino-1,1-diphen.yl-1-propanol (+/-)-exo-2-aminonorbornane (s)-(+)-2-(aminomethyl)pyrrolidine 3-amino-1-propanol vinyl ether geranylamine 4-(hexadecylamino)benzylamine (lr,2r,3r,5s)-(-)-isopinocampheylamine (ls,2s,3s,5r)-(+)-isopinocampheylamine n1-isopropyldiethylenetriamine (s)-tert-leucinol (r)-(-)-tetrahydrofurfurylamine dehydroabietylamine 2-bromo-4,5-dimethoxyphenethylamine (is,2r)-(-)-cis-1-amino-2-indanol (lr,2s)-(+)-cis-1-amino-2-indanol.

Suitable amines useful for forming the imine intermediates in preparation of the thiazolidinone libraries are further illustrated by the following formulas, wherein L is -NH2:

L L
L \
o /
L
L ~ \ L
O ' ~ /
F
F
\
/ \ L
/ F
CI /
F F
L I W _W L ~ L
F i / F
F
F
F F
F
\ L ~ / L CI \ L
CI CI CI
L
L ~ ~~L
/ C H3 \

O~CH3 L L
S
CI H3C ~
\ ~ S
o~
Q L ~ ~ _ L
ci i L , I . I
\ \

F
L L L
Br F
L ~L L
CI
i WO 98/21584 PCTlUS97/21002 L L L
HOC ~
HaC / HOC o L L
L

I L
CI
CI
Additional primary amines suitable for forming the imine intermediates in preparation of: the thiazolidinone libraries are further illustrated by the following formulas:

O
H 2N O~ O
H 2N ~ O'\
H2N O O'\ H2N
O
FRO ~ ~~
O O
H2N Oi H2N O
O
O O
O
H2NWp H O
zN ~J~o~
and NHCBz where CBz is benzyloxycarbonyl.
The alpha-mercaptocarboxylic acids are either commercially available, or they can be synthesized using standard techniques using commercially available starting materials. They are represented by the formula SH
R3~ C02H
wherein R3 is hydrogen or an organic moiety. More preferably R3 is hydrogen, alkyl, substituted alkyl or aryl. Most preferably R3 is hydrogen, Cl-Clp alkyl or substituted(C1-Clp)alkyl. The ~~orresponding esters can be prepared by reaction of the alpha-mercaptocarboxylic acids with ester-forming alcohols, most typically Cl-C10 WO 98l21584 PCTIUS9?/21002 primary alcohols. Exemplary of R3 groups in the above formula are methyl, ethyl, propyl, isopropyl, methoxymethyl, 2-chloroprop-1-yl, benzyl, 4-bromobenzyl, phenyl, and 3-methoxyphenyl. Preferably the alpha-s mercaptocarboxylic acids have a molecular weight of about 92 to about 700.
The preparation of the thiazolidinone library compounds of Formula I above comprises a two-step process wherein an imine intermediate is first formed and subsequently reacted with an alpha-mercaptocarboxylic acid or ester thereof, optionally in the presence of a catylst selected from A1C13, TiCl4 and Ti(OiPr)3. The progresslcompletion of the reactions can be determined by a number of conventional techniques including thin layer chromatography (TLC).
In the first step of the process for preparing the thiazolidinone libraries of the present invention, equivalent amounts of a primary amine and an optionally substituted benzaldehyde, each typically in solution in a suitable organic solvent, are combined in a reaction zone and reacted overnight, preferably at ambient temperature.
Suitable organic solvents are those in which each of the reactants are soluble and which do not interfere with the imine-forming condensation reaction. Suitable solvents include alcohols, such as methanol and ethanol; ethers, such as diethyl ether; esters, such as ethyl acetate; and halogenated hydrocarbon solvents, such as methylene chloride, chloroform, or 1,2-dichloroethane. The reaction temperature can optionally be elevated to about 30° to about 50° C. Following completion of the imine intermediate forming step, the reaction mixture is evaporated by vacuum to provide an imine intermediate in each reaction zone.
The imine intermediate in each reaction zone is dissolved in a halogenated hydrocarbon solvent, preferably methylene chloride, and reacted with an excess, typically a 2-4 fold excess on a molar basis, of an alpha-mercaptocarboxylic acid or an alpha-mercaptocarboxylic acid ester, optionally in the presence of a catalyst selected from AlCl3, TiCl4, and Ti(OiPr)3.
When a catalyst is added to the reaction mixture, it is typically added in less than or equal stoichiometric amounts relative to the amount of imine intermediate, for example, about 0.1 to about 1 molar equivalents per molar equivalent of imine intermediate. The reaction is typically carried out at ambient temperature; however, higher reaction temperatures up to about 60°C can be used. The progress of the reaction can be monitored, for example, by thin layer chromatography. Upon completion of the reaction, an aliquot of a slurry of a strong cation exchange (SCX) resin is added to the reaction mixture in the reaction zone to adsorb unreacted amine/imine reactants. The individual reation mixtures are then filtered and the SCX resin is washed two times with an organic solvent, for example, ethyl acetate, and the washings are combined with the original filtrate and evaporated to dryness. Alternatively, the amine/imine reactants can be extracted from. the reaction product mixture by washing a solution of the mixture in a non-water-miscible organic solvent with an aqueous acid, for example, 0.1 - 1.0 N hydrochloric acid. The resulting residue is dissolved in an organic solvent, preferably ethyl acetate, and washed with an aqueous base, for example, potassium hydroxide, to remove unreacted alpha-mercaptocarboxylic acid reactant from the library product. The organic solvent containing the library product is then dried, for example, by filtration through a pad comprising anhydrous sodium sulfate, into a clean well plate reservoir and evaporated to dryness under vacuum to provide a library product in each well plate reservoir.

The process of the present invention utilized in preparation of a library of thiazolidinones of Formula I
above may be carried out in any vessel capable of holding the liquid reaction medium. In one embodiment, the process of the invention is carried out in containers adaptable to parallel array synthesis. In particular, the thiazolidinone library of this invention can be formed in a 96-well plate as illustrated in Figures 1 and 2. The apparatus provides multiple reaction zones, most typically in a two-dimensional array of defined reservoirs, wherein a thiazolidinone library compound of this invention is prepared in each reservoir) Thus the diverse thiazolidinone library of the present invention comprises a plurality of reservoir arrays (e. g. well plates), each reservoir or well containing a library product of the thiazolidinone library. Accordingly the library compounds are typically identified by reference to their well plate number and their X column and Y row well plate coordinates.
Following simultaneous preparation of the library member compounds in the reservoir array, the compounds can be transferred in whole or in part to other reservoir arrays (e.g. well plates) to prepare multiple copies of the library apparatus or to subject the library to additional reaction conditions. Copies of the library apparatus (daughter well plates, each comprising a 2-dimensional array of defined reservoirs with each reservoir containing a predetermined reaction product of the library) are useful as replaceable elements in automated assay machines. The apparatus of this invention allows convenient access to a wide variety of structurally related thiazolidinone compounds. One preferred reservoir array for use in making and using this invention is a multi-well titer plate, typically a 96-well microtiter plate.

WO 98I21584 PCTlUS97/21002 Fig. 1 illustrates the top surface of a well plate apparatus of the present invention. The well plate (1) is a plastic plate with 96-wells (depressions) capable of holding liquids for parallel array synthesis. Individual reaction products are prepared :in each well and are labeled by the well plate coordinates. For example, the library compound at location (2;1, is identified by the alpha numeric coordinate, "A6".
Fig. 2 illustrates a side view of a modified well plate apparatus for use in prep<~ration of the library of the present invention. Well pl<~te (3) contains wells (4) with a filter (5) and a liquid reaction medium used in carrying out the process (6). 'rhe wells have an outlet at the bottom which is sealed by gasket (7) held in place by a top cover (8) and bottom cover (9) maintained in position by clamps t10).
Such well plates are typically prepared using standard 96-well plates. A hole is drilled in the bottom of each well in the plates and a porous frit is placed in the bottom of each well. The p_Late is then placed in the clamp assembly to seal the bottom of the wells.
Synthesis is initiated by <~dding reagents to their individual wells according to their assigned well coordinates. The plate is then capped and tumbled to mix the reagents. Following completion of the reaction to form the respective imine intermediates, an alpha-mercaptocarboxylic acid (or ester) is added followed by further mixing. Finally, a slurry of a strong ration exchange resin is added to each reaction zone after completion of the reaction to adsorb residual amine/imine reactants. The mixture is filtered and the filtrate in each reaction zone is washed wii~h aqueous base, dried, collected in a clean 96-well plate, and the solvent is evaporated to provide a library reaction product in each well. The reaction products are then analyzed, for example, by thin layer chromatography, mass spectrometry and/or nuclear magnetic resonance spectrometry.
In one embodiment of the present invention is an assay kit for the identification of pharmaceutical lead compounds. The assay kit comprises as essential parts, (1) a well plate apparatus (containing one of the thiazolidinone compounds in each of its individual wells), and (2) biological assay materials. The biological assay materials are generally known to be predictive of success for an associated disease state.
Illustrative of biological assay materials useful in the kit of this invention are those required to conduct the following assays:
In vitro assays:
Enzymatic inhibition Receptor-ligand binding Protein-Protein interaction Protein-DNA interaction Cell based, functional assays:
Transcriptional regulation Signal transduction/Second messenger Viral Infectivity Add, Incubate, & Read assays:
Scintillation Proximity Assays Angiotensin II IPA receptor binding assay Endothelia converting enzyme [125I] SPA assay HIV proteinase [125I] SPA enzyme assay Cholesteryl ester transfer (CETP) [3H] SPA assay Fluorescence Polarization Assays Fluorescence Correlation Spectroscopy Colorimeric biosensors Ca2+ -EGTA for Cell-based assays Receptor Gene Constructs for cell based assays WO 98I21584 PCTlUS97/21002 Luciferase, green fluorescent protein, Beta-lactamase Electrical cell impedance sensor assays Thiazolidinone Library Plates: Detailed Procedure.
A different amine reagent (100 pL of a 0.5 M_ solution in EtOH) was added to the wells of each row of a (several) 96-well glass titer plate (well volume of 1 mL), with care taken that a11 liquid was added to the bottom of the wells and with minimum splattering. A
different aldehyde (100 uL of a 0.5 M solution in EtOH) was then added to the wells of each column in the plate(s). The wells were capped and the plates shaken at ambient temperature overnight. The solvent was then evaporated by vacuum overnight (5-10 in/Hg, ambient temperature).
The residue in each well was then dissolved in dichloromethane (0.3 mL). An alpha-mercaptocarboxylic acid, for example, alpha-mercaptopropionic acid, (200 uL
of a 1.0 M solution in dichloromethane) was then added to all the wells of a plate(s). Different alpha-mercaptocarboxylic acids may be: added to the wells of different plates to create a library having diversity at R3 in Formula I. The plates) was capped and shaken for ten days at ambient temperature.
The plates) was then uncapped and a slurry of a strong cation exchange (SCX) resin (100 uL of a 0.4 g SCX
resin per 1.0 mL dichloromethan:e) was added to each well.
The plates) was capped and shaken at ambient temperature for twenty four hours. The pla.te(s) was then uncapped and the contents filtered into a clean titer plate(s).
The wells and the SCX resin were rinsed with ethylacetate and the washes added to the re~~pective filtrate (2 x 0.3 mL per well). The solvent was allowed to evaporate and the residue redissolved in ethylacetate (0.5 mL per well). Aqueous KOH was added to each well (200 uL of a 2.0 M_ solution) and the plates) was shaken at ambient temperature overnight. A saturated NaCl solution was added to each well (50uL per well) to effect a phase separation with the product in the organic phase. The organic phase was then transferred through a filter plate with a Na2S04/neutral almina pad (1:1) into clean titer plates (2.0 mL well volume). The wells of the reaction plates) were rinsed with ethylacetate (2 x 0.4 mL per well) and the washes were filtered and added to the filtrate. This process afforded plates containing about 25 ~.zmol of a library compound per well. Prior to final drying, samples of solution were taken from each well and submitted for thin layer chromatography and/or mass spectral analysis.

Claims (14)

We claim:

1. A library of thiazolidinone compounds wherein said library contains a plurality of diverse library compounds of the formula wherein R1 is a substituent derived from an optionally substituted benzaldehyde of the formula R2 is hydrogen or an organic moiety derived from a primary amine of the formula R2NH2, and R3 is an organic moiety derived from an alpha-mercaptocarboxylic acid of the formula or ester thereof.

2. The library of claim 1 wherein R1 is selected from non-interfering substituents, R2 is alkyl, substituted alkyl, or aryl, and R3 is hydrogen, alkyl, substituted alkyl, or aryl.

3. The library of claim 1 wherein the optionally substituted benzaldehyde has a molecular weight of about 106 to about 600.

4. The library of claim 1 wherein the amine has a molecular weight of about 31 to about 700.

5. A compound selected from the group consisting of the thiazolidinone library compounds of the library of claim 1.

6. A process for preparing a combinatorial library of thiazolidinone compounds of the formula having diversity in substituent groups R1, R2 and R3, wherein each library compound is made in a separate reaction zone, said process comprising the steps of reacting an amine of the formula R2NH2 with an aldehyde of the formula to form an intermediate imine compound of the formula in each reaction zone and reacting the intermediate imine in each zone with an alpha-mercaptocarboxylic acid of the formula or ester thereof, wherein in the above formulas R1 is a non-interfering substituent, R2 is alkyl, substituted alkyl, or aryl, and R3 is hydrogen, alkyl, substituted alkyl, or aryl.

7. The process of claim 6 wherein the reaction of the imine intermediate and the alpha-mercaptocarboxylic acid or ester thereof, is carried out in the presence of a catalyst selected from the group consisting of AlCl3, TiCl4 and Ti(OiPr)3.

8. The process of claim 6 further comprising the step of adding a strong cation exchange resin to the reaction mixture in each reaction zone after reaction of the intermediate imine and the alpha-mercaptocarboxylic acid, or ester thereof, and separating the resin from the product thiazolidinone.

9. The process of claim 8 further comprising the step of washing the product with an aqueous base.

10. An assay kit for identification of pharmaceutical lead compounds, said kit comprising biological assay materials and a well plate apparatus wherein each well in said apparatus contains a library compound of the library of claim 1.

11. The assay kit of claim 10 wherein the biological materials are selected for performing at least one assay test selected from the following group of assay tests:
In vitro assays:
Enzymatic inhibition Receptor-ligand binding Protein-Protein interaction Protein-DNA interaction Cell based, functional assays:
Transcriptional regulation Signal transduction/Second messenger Viral Infectivity Add, Incubate, & Read assays:
Scintillation Proximity Assays Angiotensin II IPA receptor binding assay Endothelia converting enzyme [125I] SPA assay HIV proteinase [125I] SPA enzyme assay Cholesteryl ester transfer (CETP) [3H] SPA assay Fluorescence Polarization Assays Fluorescence Correlation Spectroscopy Colorimeric biosensors Ca2+ -EGTA Yes for Cell-based assays Receptor Gene Constructs for cell based assays Luciferase, green fluorescent protein, betalactamase Electrical cell impedance sensor assays

12. An apparatus suitable as a replacement element in an automated assay machine as a source of individual members of a library of structurally related compounds, said apparatus comprising a 2-dimensional array of defined reservoirs, each reservoir containing a library compound, wherein said structurally related compounds are of the formula wherein R1 is a substituent derived from an optionally substituted benzaldehyde of the formula R2 is hydrogen or an organic moiety derived from a primary amine of the formula R2NH2, and R3 is an organic moiety derived from an alpha-mercaptocarboxylic acid of the formula or ester thereof.

13. The apparatus of claim 12 wherein the library compound in each reservoir is prepared in accordance with the process of claim 6 and wherein each reservoir provides one reaction zone.

14. The apparatus of claim 12 wherein the 2-dimensional array of defined reservoirs is a multi-well microtiter plate.