WO1998057173A1

WO1998057173A1 - Combinatorial process for preparing substituted phenylalanine libraries

Info

Publication number: WO1998057173A1
Application number: PCT/US1998/011909
Authority: WO
Inventors: Julia Marie Heerding; John William Lampe
Original assignee: Eli Lilly And Company
Priority date: 1997-06-10
Filing date: 1998-06-10
Publication date: 1998-12-17
Also published as: AU8063098A

Abstract

This invention relates to a novel diverse combinatorial library of substituted phenylalanine 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

COMBINATORIAL PROCESS FOR PREPARING SUBSTITUTED

PHENYLALANINE LIBRARIES

This Application claims the benefit of U.S. Provisional Application No. 60/049,054, filed June 10, 1997.

The present invention relates to diverse libraries of substituted phenylalanine compounds, methods of making such libraries, and an apparatus for storing and providing a readily accessible source of diverse substituted phenylalanine compounds . The apparatus harboring the present combinatorial libraries is a useful component of assay systems for identifying compounds for drug development.

Research and development expenses account for a large outlay of capital in the pharmaceutical 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 combinatorial 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 library'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 microtiter 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.

The reactions for production of the libraries can be conducted on reaction substrates (starting materials) where all reactants are in solution phase or where the substrate compounds are covalently coupled to an insoluble resin in particulate or bead form as a solid support. Typically, completion of 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 in solution phase reactions, completion of the reaction produces a mixture of a soluble product with at least one soluble unreacted reagent. The excess soluble reagent is separated from the product by using solid phase scavengers or by classical work-up procedures dependent on the chemical characteristics of the excess reagent and the product . Where the reaction substrate for the library is linked to a solid support for chemical modification, excess reagents can be separated by simple filtration and solid support washing techniques.

Combinatorial chemistry may be used at two distinct phases of drug development . In the discovery phase diverse libraries are created to find lead compounds. In 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 library of substituted phenylalanines useful in the identification of new lead compounds. The library is created, stored, and used as an apparatus comprising of a two-dimensional array of reservoirs, each reservoir containing a predetermined library reaction product differing from those in adjacent reservoirs.

The present invention provides combinatorial libraries of structurally related compounds of the general formula

wherein Ri and R₂ are each independently an organic moiety;

Y_l and Y2 are each independently selected from the group consisting of -CO-, -CO₂-, -C0NH-, -CSNH-, and -SO₂-; and Z is hydrogen or C1-C4 alkyl; or either of the groups R1Y1 or R2Y2 can be hydrogen.

The invention further provides a method for preparing substituted phenylalanine libraries generally in accordance with Scheme 1 as set forth below.

Another embodiment of the present invention provides an assay kit for the identification of pharmaceutical lead substituted phenylalanine 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 substituted phenylalanine library. The well plate apparatus is used to provide multiple reaction zones for making the library, to store the library and to provide a readily accessible source of library compounds.

Brief Description of the Drawings

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 used in accordance with the present invention refers to an assemblage 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 more variable substituents. The scaffold of the present invention is a substituted phenylalanine .

A "library compound" is an individual reaction product, a single compound or a 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 a 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 substituted phenylalanine core in the present invention) of the compounds which are members of the combinatorial library. "Solid support" is the solvent insoluble substrate to which the substituted phenylalanine scaffold is bound for subsequent synthesis of the library compound. It is represented by the symbol

and may be from organic or inorganic materials. "Substituents" are chemical radicals which are bonded to or incorporated onto the substituted phenylalanine 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 biological 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 stored without prior and subsequent intentional mixing.

"Simultaneous synthesis" means making of library compounds within one production cycle of a combinatorial method (not making all 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 substituted phenylalanine 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 character, optionally substituted, and at least one ring 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, Ci-Cio alkyl, C2-C10 alkenyl, C2-C10 alkynyl , C1-C10 alkoxy, C7-C12 aralkyl, C7-C12 alkaryl, C1-C10 alkylthio, arylthio, aryloxy, arylamino, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, di (C1-C10) -alkylamino, C2-C12 alkoxyalkyl, Ci- Cζ alkylsulfinyl, C1-C10 alkylsulfonyl, arylsulfonyl , aryl, hydroxy, hydroxy (C1-C10) alkyl, aryloxy (Cχ-Cιo) lkyl , C1-C10 alkoxycarbonyl , aryloxycarbonyl , C₁-C10 alkanoyloxy, aryloyloxy, substituted alkoxy, fluoroalkyl, nitro, cyano, cyano (C1-C10) alkyl, C1-C10 alkanamido, aryloylamido, arylaminosulfonyl, sulfonamido, heterocyclic .radical, nitroalkyl, and - (CH2)m-Z- (Cχ-Cιo 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. Suitable radicals for substitution onto the connecting carbon atom include, but are not limited to hydrogen, halo, C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 alkoxy, C7-C12 aralkyl, C7-C12 alkaryl, Cι_- C10 alkylthio, arylthio, aryloxy, arylamino, C3-C10 cycloalkyl, C3-C10 cycloalkenyl, di (C1-C10) -alkylamino, C2- C12 alkoxyalkyl, C3.-C6 alkylsulfinyl, Cχ-Cιo alkylsulfonyl, arylsulfonyl, aryl, hydroxy, hydroxy (Ci-Cio) alkyl, aryloxy (Ci-Cio) alkyl, Ci-Cio alkoxycarbonyl, aryloxycarbonyl, Cχ-Cιo alkanoyloxy, aryloyloxy, substituted alkoxy, fluoroalkyl, nitro, cyano, cyano (Ci-Cio) alkyl, Ci- Cio alkanamido, aryloylamido, arylaminosulfonyl, sulfonamido, heterocyclic radical, nitroalkyl, and - (CH2)πr Z- (Ci-Cio alkyl) , where m is 1 to 8 and Z is oxygen or sulfur.

A diverse library of substituted phenylalanines is provided in accordance with the present invention. The substituted phenylalanine library embodied as an apparatus of this invention serves as a readily accessible source of diverse substituted phenylalanine compounds for use in identifying new biologically active substituted phenylalanine 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 substituted phenylalanine compounds of the formula

wherein Ri and R₂ are each independently an organic moiety; Yi and Y2 are each independently selected from the group consisting of -CO-, -CO₂-, -C0NH-, -CSNH-, and -SO2-; and Z is hydrogen or C₁-C₄ alkyl; or either of the groups R₁Y₁ or R2Y₂ can be hydrogen.

In another embodiment of the present invention there is provided a library of compounds of Formula I above, wherein R_l and R₂ are each independently an organic moiety with radicals for substitution onto the connecting carbon selected from the group consisting of hydrogen, halo, C₁-C₁0 alkyl, C2-C₁0 alkenyl, C2-C₁₀ alkynyl, C₁-C₁₀ alkoxy, C7-C12 aralkyl, C7-C12 alkaryl, Cχ-Cιo alkylthio, arylthio, aryloxy, arylamino, C3-C₁0 cycloalkyl, C3-C10 cycloalkenyl, di (C1-C10) -alkylamino, C2-C12 alkoxyalkyl, Ci-Cδ alkylsulfinyl, C1-C10 alkylsulfonyl , arylsulfonyl, aryl, hydroxy, hydroxy (C1-C10) 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, arylaminosulfonyl, sulfonamido, heterocyclic radical, nitroalkyl, and - (CH2)m-Z- (C1-C10 alkyl), where m is 1 to 8 and Z is oxygen or sulfur.

In another embodiment of the present invention, there is provided a library of diverse substituted phenylalanine compounds of the formula

wherein Ri and R2 are each independently an organic moiety;

Yl and Y2 are each independently selected from the group consisting of -CO-, -CO2-, -C0NH-, -CSNH-, and -SO2-; and Z is hydrogen or C1-C4 alkyl; or either of the groups R₁Y1 or R2Y2 can be hydrogen.

In another embodiment of the present invention there is provided a library of compounds of Formula II above, wherein Rl and R2 are each independently an organic moiety with radicals for substitution onto the connecting carbon selected from the group consisting of hydrogen, halo, Cχ-Cιo alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 alkoxy, C7-C12 aralkyl, C7-C12 alkaryl, C1-C10 alkylthio, arylthio, aryloxy, arylamino, C3-C₁0 cycloalkyl, C3-C10 cycloalkenyl, di(Cι-Cιo) -alkylamino, C2-C12 alkoxyalkyl, Ci-Cβ alkylsulfinyl, Cχ-Cιo alkylsulfonyl , arylsulfonyl , aryl, hydroxy, hydroxy (C1-C10) alkyl, aryloxy (C1-C10) lkyl , C1-C10 alkoxycarbonyl, aryloxycarbonyl, C1-C10 alkanoyloxy, aryloyloxy, substituted alkoxy, fluoroalkyl, nitro, cyano, cyano (Ci-Cio) alkyl, Ci-Cio alkanamido, aryloylamido, arylaminosulfonyl, sulfonamido, heterocyclic radical, nitroalkyl, and - (CH2)m^_Z- (Ci-Cio alkyl), where m is 1 to 8 and Z is oxygen or sulfur.

The present invention also provides a method for preparing the library of substituted phenylalanine compounds of Formulas I and II using combinatorial chemistry in a parallel array synthesis technique illustrated in the following reaction scheme:

Scheme 1

The method is initiated by covalently coupling an amino-protected nitrophenylalanine with a solid support. Subsequent reaction steps comprise removing the amino- protecting group, acylating the deprotected amino group, reducing the nitro group, acylating the resulting amino group and finally cleaving the covalent bond to the solid support, optionally forming an ester of the resulting carboxylic acid. The resultant library of substituted phenylalanine compounds have three sites of diversity: Y3_.R1 and Y2R2_# derived from the acylating reagents, and Z derived from a lower alkanol . 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 numbers.

Acylating reagents suitable for use in preparing the library of the present invention are acyl halides, haloformates, sulfonylhalides, isocyantates or isothiocyanates . Such compounds are either commercially available or prepared from commercially available starting materials, including the corresponding acid derivatives of the acyl halides and sulfonylhalides. Typically the acylating reactants have a molecular weight of about 50 to about 600.

Acylating agents useful in the preparation of the libraries of the present invention include compounds of the formula RCOX, RSO2X, ROCOX, RN=C=0 or RN=C=S, wherein R is an organic moeity and X is halo.

Examples of suitable acyl halides of the formula RCOX, wherein R is an organic moeity and X is halo, are the following:

3 , 5-bis (trifluoromethyl) benzoyl chloride benzoyl chloride

2-bromobenzoyl chloride

2-fluorobenzoyl chloride pentafluorobenzoyl chloride

2, 4-difluorobenzoyl chloride 2, 6 -difluorobenzoyl chloride

2-chlorobenzoyl chloride

2,4-dichlorobenzoyl chloride 2,6-dichlorobenzoyl chloride o-acetylsalicyloyl chloride 2-methoxybenzoyl chloride 2,6-dimethoxybenzoyl chloride 2- (trifluoromethyl) benzoyl chloride o-toluoyl chloride 3-bromobenzoyl chloride 3 -fluorobenzoyl chloride 3-chlorobenzoyl chloride 3 ,4 -dichlorobenzoyl chloride m-anisoyl chloride 3,4-dimethoxybenzoyl chloride 3 ,4, 5-trimethoxybenzoyl chloride 3,5-dimethoxybenzoyl chloride 3-ethoxybenzoyl chloride isophthaloyl chloride trimesoyl chloride

3- (trifluoromethyl) enzoyl chloride m-toluoyl chloride 3- (chloromethyl) benzoyl chloride 4-bromobenzoyl chloride 4- fluorobenzoyl chloride 4-chlorobenzoyl chloride p-anisoyl chloride 4-ethoxybenzoyl chloride

4-n-butoxybenzoyl chloride 4-n-hexyloxybenzoyl chloride 4-heptyloxybenzoyl chloride 4-biphenylcarbonyl chloride terephthaloyl chloride

4- (trifluoromethyl) benzoyl chloride 4-tert-butylbenzoyl chloride p-toluoyl chloride 4 -ethylbenzoyl chloride 4-n-propylbenzoyl chloride 4-butylbenzoyl chloride 4-pentylbenzoyl chloride 4-hexylbenzoyl chloride 4-n-heptylbenzoyl chloride methyl oxalyl chloride ethyl oxalyl chloride heptafluorobutyryl chloride 2-acetoxyisobutyryl chloride pivaloyl chloride 3-chloropivaloyl chloride 2-bromopropionyl chloride 2 , 3-dibromopropionyl chloride 2,3-dichloropropionyl chloride o-acetylmandelic acid chloride itaconyl chloride methacryloyl chloride isobutyryl chloride

2 -ethylhexanoyl chloride acetyl chloride bromoacetyl chloride chloroacetyl chloride phenoxyacetyl chloride

4-chlorophenoxyacetyl chloride methoxyacetyl chloride phenylacetyl chloride 3,3-dimethylacryloyl chloride cinnamoyl chloride fumaryl chloride ethyl malonyl chloride tert-butylacetyl chloride isovaleryl chloride undecanoyl chloride lauroyl chloride myristoyl chloride palmitoyl chloride heptadecanoyl chloride stearoyl chloride propionyl chloride 3-bromopropionyl chloride 3-chloropropionyl chloride hydrocinnamoyl chloride succinyl chloride

3 - carbomethoxypropionyl chloride ethyl succinyl chloride butyryl chloride

4-bromobutyryl chloride

4-chlorobutyryl chloride valeryl chloride 5-chlorovaleryl chloride adipoyl chloride hexanoyl chloride

6-bromohexanoyl chloride pimeloyl chloride heptanoyl chloride suberoyl chloride octanoyl chloride

10-undecenoyl chloride

2 - chloro-2 , 2 -diphenylacetyl chloride dichloroacetyl chloride alpha- chlorophenylacetyl chloride

2-chloropropionyl chloride

2 - iodobenzoyl chloride

4 - iodobenzoyl chloride cyclopropanecarbonyl chloride trans-2 -phenyl- 1 - cyclopropanecarbonyl chloride cyclobutanecarbonyl chloride cyclopentanecarbonyl chloride

3-cyclopentylpropionyl chloride cyclohexanecarbonyl chloride

4- cyanobenzoyl chloride

2-furoyl chloride

1-naphthoyl chloride

2-naphthoyl chloride thiophene-2 -carbonyl chloride

2 - thiopheneacetyl chloride trimellitic anhydride chloride 2 , 6-pyridinedicarboxylic acid chloride

2-quinoxaloyl chloride

2-nitrobenzoyl chloride

3-nitrobenzoyl chloride 3, 5-dinitrobenzoyl chloride

4-nitrobenzoyl chloride

3,4- imethoxyphenylacetyl chloride

3-methyladipoyl chloride

3,5- ichlorobenzoyl chloride 2, 5 -difluorobenzoyl chloride

3, 4-difluorobenzoyl chloride

9-fluorenone-4-carbonyl chloride

3 , 5 -difluorobenzoyl chloride

(S) - (-) -N- (trifluoroacetyl)prolyl chloride benzyloxyacetyl chloride acetoxy acetyl chloride

3 - cyanobenzoyl chloride

2, 5 -dimethoxyphenylacetyl chloride

3-methoxyphenylacetyl chloride iminodibenzyl - 5- carbonyl chloride

2 ,4, 6-trimethylbenzoyl chloride tetrafluorosuccinyl chloride perfluorooctanoyl chloride diphenylacetyl chloride alpha-methyl valeroyl chloride methyl malonyl chloride ethyl glutaryl chloride

5-bromovaleryl chloride methyl adipyl chloride 3-cyclohexenecarbonyl chloride

3-isocyanato benzoyl chloride

2,4,6- triisopropylbenzoyl chloride fluoroacetyl chloride

2-ethoxybenzoyl chloride piperonyloyl chloride

2,4-dimethoxybenzoyl chloride

2,3,5,6- tetrachloroterephthaloyl chloride 5- (dimethylsulfamoyl) -2-methoxybenzoyl chloride 2- (4-chlorobenzoyl) benzoyl chloride 2 , 2 -bis (chloromethyl)propionyl chloride cinnamylidenemalonyl chloride 2-phenoxypropionyl chloride 2 -phenylbutyryl chloride 2-ethylbutyryl chloride p-tolylacetyl chloride gamma-methylvaleroyl chloride 3 , 3-dichloropivaloyl chloride

1-methyl-l-cyclohexanecarboxylic acid chloride 2- (2, , 5-trichlorophenoxy) acetyl chloride 4-chloro- 3 -nitrobenzoyl chloride 4-methyl-3-nitrobenzoyl chloride 2, 3 -dichlorobenzoyl chloride morpholine-4-carbonyl chloride p-chlorophenylacetyl chloride bicyclo [2.2.1] heptane-2 -carbonyl chloride d(-) -alpha- formyloxy-alpha-phenylacetyl chloride d(- ) -alpha-phenylglycine chloride hydrochloride trifluoroacetyl chloride pentafluoropropionyl chloride hexafluoroglutaryl chloride

2-chlorocinnamoyl chloride o-methoxycinnamyl chloride

5-nitro-2-furoyl chloride

2-chlorobutyryl chloride

4-phenylazobenzoyl chloride

4-n-amyloxybenzoyl chloride 4-decylbenzoyl chloride

4-octylbenzoyl chloride dl-2 -methylbutyryl chloride linolenoyl chloride linolelaidoyl chloride llh-eicosafluoroundecanoyl chloride

9h-hexadecafluorononanoy1 chloride

2, 3 -difluorobenzoyl chloride 2- (benzoyloxymethyl) benzoyl chloride

2,2-dimethylvaleroyl chloride

3, 5, 5-trimethylhexanoyl chloride phenothiazine-10-carbonyl chloride 3, 4-dimethyl benzoyl chloride

(+) -p- (2 -methylbutyl) benzoyl chloride

2,4-dichlorophenoxyacetic chloride pentadecanoyl chloride nonadecanoyl chloride neoheptanoyl chloride

9-anthracenecarbonyl chloride

2-ethoxy-l-naphthoyl chloride pyrrolidine carbonyl chloride m- (chlorosulfonyl) benzoyl chloride 2-n-propyl-n-valeroyl chloride

2-chloro-4-nitrobenzoyl chloride

2-phenoxybutyryl chloride

2-chloronicotinyl chloride

6-chloronicotinyl chloride 4- (trifluoromethoxy) benzoyl chloride

2- (trifluoromethoxy) benzoyl chloride

2, 6-dichloropyridine-4 -carbonyl chloride

3-chlorobenzo [b] thiophene- 2 -carbonyl chloride

4-chloromethylbenzoyl chloride neodecanoyl chloride

(phenylthio) acetyl chloride

4- carbethoxyhexafluorobutyryl chloride octafluoroadipoyl chloride

2-diazo-3 , 3, 3- trifluoropropionylchloride 2-bromobutyryl chloride arachidoyl chloride cis-vaccenoyl chloride

11-eicosenoyl chloride behenoyl chloride petroselinoyl chloride palmitoleoyl chloride tridecanoyl chloride 2-chloro-5-nitrobenzoyl chloride 3 -methylthiopropionyl chloride methyl 4 - chlorocarbonylbenzoate anthraquinone-2 -carbonyl chloride carbazole-n- carbonyl chloride 2-nitrophenoxyacetyl chloride 2-bromo-2-methylpropionyl chloride 2-fluoro-3- (trifluoromethyl) benzoyl chloride 2-fluoro-4- (trifluoromethyl) benzoyl chloride 2-fluoro-5- (trifluoromethyl) benzoyl chloride 3-fluoro-5- (trifluoromethyl) benzoyl chloride 4-fluoro-2- (trifluoromethyl) benzoyl chloride 4-fluoro-3- (trifluoromethyl) benzoyl chloride 2-fluoro-6- (trifluoromethyl) benzoyl chloride 2, 3 , 6- trifluorobenzoyl chloride 2, 4, 5 -trifluorobenzoyl chloride 2,4-di (trifluoromethyl) benzoyl chloride 2 , 6 -di (trifluoromethyl) benzoyl chloride 3- (trifluoromethoxy) benzoyl chloride m- (fluorosulfonyl) benzoyl chloride trans- 1, 2-cyclobutanedicarboxylic acid chloride 3-cyclohexylpropionyl chloride

4-ethyl -2,3-dioxo- 1 -piperazinecarbonylchloride isoxazole- 5-carbonyl chloride bromodifluoroacetyl chloride erucoyl chloride 2,4, 6-trifluorobenzoyl chloride dichlorochrysanthemic acid chloride isononanoyl chloride 1-adamantanecarbonyl chloride

2 , 5 -bis (trifluoromethyl) benzoyl chloride 2, 3, 4-trifluorobenzoyl chloride 2,3,4,5- tetrafluorobenzoyl chloride 2,4, 6-trichlorobenzoyl chloride 2, 4-dichloro-5-fluorobenzoyl chloride 4-methoxyphenylacetyl chloride trans-3- (trifluoromethyl) cinnamoyl chloride 3- (dichloromethyl) benzoyl chloride

4-isocyanato benzoyl chloride heneicosanoyl chloride

2-chloroisobutyryl chloride trans-4-nitrocinnamoyl chloride

3 , 4, 5 -trifluorobenzoyl chloride

5-fluoro-2- (trifluoromethyl) benzoyl chloride

2, 3 , 5 -trifluorobenzoyl chloride

2 - chloro-4 - fluorobenzoyl chloride (-) -alpha-chlorophenylacetyl chloride

2- (para-tolylsulfonyl) acetyl chloride

4 -methyl -4 -nitrohexanoyl chloride l-chloro-4-fluorosulfonyl-2-naphthoyl chloride

2 , 3-dibromo-3-phenylpropionyl chloride 2-menthoxyacetyl chloride

2 -phenyl -2- (phenylsulfonyl) acetyl chloride

4, 4,4-trifluorocrotonyl chloride

4, 4,4-trifluorobutyryl chloride

3,4-dichloro-2,5-thiophenedicarbonyl chloride pentachlorobenzoyl chloride

4,4,7,7- tetranitrosebacoyl chloride alpha, alpha' -dimethylsuccinyl chloride alpha-bromoisovaleryl chloride benzoyl chloride oleoyl chloride methyl suberyl chloride gamma- linolenoyl chloride (- ) -camphanic acid chloride

4,4'-stilbenedicarbonyl chloride chlorinated benzoyl chloride (lr) -(+) -camphanic chloride

2- (4-nitrophenoxy) tetradecanoyl chloride

7- [ (chlorocarbonyl)methoxy] -4-methylcoumarin

N,N-bis (2-chloroethyl) carbamoyl chloride (S- (-) -2-acetoxypropionyl chloride linoleoyl chloride 3 - chlorotetrafluoropropionyl chloride 3,4-dichloropentafluorobutyryl chloride

7H-dodecafluoroheptanoyl chloride

5H-octafluoropentanoyl chloride perfluorononanoyl chloride 3h-tetrafluoropropionyl chloride

2-bromo-2 ,3,3, 3- tetrafluoropropanoyl chloride arachidonoyl chloride pentachloropropionyl chloride

4-decenoyl chloride tridecafluoroheptanoyl chloride undecafluorocyclohexanecarbonyl chloride

4-n-nonylbenzoyl chloride

3 - (trichlorogermyl) propionylchloride

3 , 4, 5-triiodobenzoyl chloride 2- (phenylthio)propionyl chloride

2 ,2 ,2-triphenylacetyl chloride d( -) -alpha-azido-phenyl acetyl chloride

4-azido-benzoyl chloride difluoroacetyl chloride 5-chloropyrazine-2 -carbonyl chloride

N- (1-naphthalenesulfonyl) -1-phenylalanyl chloride

N- (4-nitrophenylsulfonyl) -1-phenylalanyl chloride

N- (p-toluenesulfonyl) -1-phenylalanyl chloride dimethylmalonyl chloride methyl sebacoyl chloride

2, 5 -dichloropyridine- 3 -carbonyl chloride

3- (2,5 xylyloxy) propionyl chloride.

Examples of suitable organohaloformates of the formula ROCOX, wherein R is an organic moeity and X is halo, are the following:

9-fluorenylmethyl chloroformate phenyl chloroformate

4-chlorophenyl chloroformate methyl chloroformate benzyl chloroformate vinyl chloroformate isobutyl chloroformate

2-ethylhexyl chloroformate ethyl chloroformate

2-bromoethyl chloroformate 2-chloroethyl chloroformate

1-chloroethyl chloroformate allyl chloroformate n-propyl chloroformate butyl chloroformate n-hexyl chloroformate octyl chloroformate

2,2,2-trichloro-l, 1-dimethylethyl chloroformate

2 , 2,2- trichloroethyl chloroformate cholesteryl chloroformate 4-nitrophenyl chloroformate

4-nitrobenzyl chloroformate

( - ) -menthyl chloroformate

4-t-butylcyclohexyl chloroformate cetyl chloroformate (+) -1- (9-fluorenyl) ethyl chloroformate isopropyl chloroformate

3-chlorocyclohexyl chloroformate decyl chloroformate oleyl chloroformate octadecyl chloroformate butenediol bischloroformate

2-chlorobenzyl chloroformate

4-chlorobutyl chloroformate

(+) -menthyl chloroformate 4, 5-dimethoxy-2-nitrobenzyl chloroformate cyclopentyl chloroformate t-butylcyclohexyl chloroformate menthylchloroformate p-tolyl chloroformate 4-bromophenyl chloroformate

4-fluorophenyl chloroformate

4-methoxyphenyl chloroformate 2-nitrophenyl chloroformate 4-methoxycarbonylphenyl chloroformate 1-chloro-2 -methylpropyl chloroformate (+/-) -1,2,2,2- tetrachloroethyl chloroformate 2,2-dichloroethyl chloroformate myristyl chloroformate cyclohexyl chloroformate chloromethyl chloroformate.

Examples of suitable organosulfonylhalides of the formula RSO2X, wherein R is an organic moeity and X is halo, are the following:

1-naphthalenesulfonyl chloride dansyl chloride 2-naphthalenesulfonyl chloride

2 -acetamido-4 -methyl - 5 - thiazolesulfonyl chloride

2-thiophenesulfonyl chloride

8-quinolinesulfonyl chloride benzenesulfonyl chloride pentafluorobenzenesulfonyl chloride

2,5-dichlorobenzenesulfonyl chloride

2-nitrobenzenesulfonyl chloride

2 , 4-dinitrobenzenesulfonyl chloride

3 , 5-dichloro-2-hydroxybenzenesulfonyl chloride 2,4, 6-triisopropylbenzenesulfonyl chloride

2-mesitylenesulfonyl chloride

3-nitrobenzenesulfonyl chloride p-bromobenzenesulfonyl chloride

4-fluorobenzenesulfonyl chloride 4-chlorobenzenesulfonyl chloride

4- chloro- 3 -nitrobenzenesulfonyl chloride pipsyl chloride

4-nitrobenzenesulfonyl chloride

4-methoxybenzenesulfonyl chloride 4-tert-butylbenzenesulfonyl chloride p-toluenesulfonyl chloride trifluoromethanesulfonyl chloride trichloromethanesulfonyl chloride isopropylsulfonyl chloride methanesulfonyl chloride alpha-toluenesulfonyl chloride trans-beta- styrenesulfonyl chloride

2,2,2-trifluoroethanesulfonyl chloride

1-hexadecanesulfonyl chloride ethanesulfonyl chloride

2-chloroethanesulfonyl chloride l-propanesulfonyl chloride

3-chloropropanesulfonyl chloride

1-butanesulfonyl chloride methyl 2- (chlorosulfonyl) benzoate

2 -nitro-4- (trifluoromethyl) benzenesulfonyl chloride 3- (trifluoromethyl) enzenesulfonyl chloride

1-octanesulfonyl chloride

4- (trifluoromethoxy) benzenesulphonyl chloride (1R) - (-) -10-camphorsulfonyl chloride d- (+) -10-camphorsulfonyl chloride (+/-) -10 -camphorsulfonyl chloride

2 -nitro-alpha- toluenesulfonyl chloride.

Examples of suitable isocyanate reagents of the formula RNCO, wherein R is an organic moeity, are the following: trans-2 -phenylcyclopropyl isocyanate phenyl isocyanate

2-bromophenyl isocyanate

2- fluorophenyl isocyanate

2,4-difluorophenyl isocyanate 2, 6-difluorophenyl isocyanate

2-chlorophenyl isocyanate

2, 3-dichloropheny1 isocyanate

2,4-dichlorophenyl isocyanate

2,5-dichlorophenyl isocyanate 2, 6 -dichlorophenyl isocyanate

2-methoxyphenyl isocyanate

2,4-dimethoxyphenyl isocyanate 2,5-dimethoxyphenyl isocyanate 2 -ethoxyphenyl isocyanate 2 - (trifluoromethyl) henyl isocyanate o-tolyl isocyanate 2 , 6 -dimethylphenyl isocyanate

2 -ethylphenyl isocyanate 3-bromophenyl isocyanate 3-fluorophenyl isocyanate

3 -chlorophenyl isocyanate 3, 4 -dichlorophenyl isocyanate

3-methoxyphenyl isocyanate

3- (trifluoromethyl) phenyl isocyanate m-tolyl isocyanate

4-bromophenyl isocyanate 4- fluorophenyl isocyanate

4-chlorophenyl isocyanate

4-methoxyphenyl isocyanate ethyl 4-isocyanatobenzoate

4- (trifluoromethyl) phenyl isocyanate p-tolyl isocyanate

N- (chlorocarbonyl) isocyanate benzoyl isocyanate tert-butyl isocyanate

(S) - ( - ) -alpha-methylbenzyl isocyanate isopropyl isocyanate methyl isocyanate ethyl isocyanatoacetate octadecyl isocyanate ethyl isocyanate 2-chloroethyl isocyanate allyl isocyanate n-propyl isocyanate butyl isocyanate cyclohexyl isocyanate 1-naphthyl isocyanate

(R) - (-) -1- (1-naphthyl) ethyl isocyanate

4-fluoro-3-nitrophenyl isocyanate 2-nitrophenyl isocyanate 3-nitrophenyl isocyanate

4 -nitrophenyl isocyanate

2 , 6 -diisopropylphenyl isocyanate benzyl isocyanate

3 - chloropropyl isocyanate ethoxycarbonyl isocyanate 3, 5 -bis (trifluoromethyl) phenyl isocyanate 2,4,6-tribromophenyl isocyanate 2 , 5-difluorophenyl isocyanate

2 , 4, 5-trichlorophenyl isocyanate 2 , 4, 6-trichlorophenyl isocyanate 2 -methoxycarbonylphenyl isocyanate

2 -ethoxycarbonylphenyl isocyanate 2 -isopropylphenyl isocyanate

2,3-dimethylphenyl isocyanate 4-methoxy-2-methylphenyl isocyanate 2,4-dimethylphenyl isocyanate 2,5-dimethylphenyl isocyanate 2-ethyl-6-methylphenyl isocyanate

3 - cyanophenyl isocyanate

5 - chloro-2,4-dimethoxyphenyl isocyanate 3 - chloro- 4 -methylphenyl isocyanate 3,5-dichlorophenyl isocyanate 5-chloro-2-methoxyphenyl isocyanate 3,4,5-trimethoxyphenyl isocyanate 3 , 5 -dimethoxypheny1 isocyanate 3- (methylthio) phenyl isocyanate 3 -ethoxycarbonylphenyl isocyanate 3-acetylphenyl isocyanate

3,4-dimethylphenyl isocyanate 3,5-dimethylphenyl isocyanate 2 -methoxy- 5 -methylphenyl isocyanate 3-ethylphenyl isocyanate 4-chloro-2-methoxyphenyl isocyanate

4 - chloro-2 -trifluoromethylphenyl isocyanate 4 - chloro- 3 -trifluoromethylphenyl isocyanate 4-iodophenyl isocyanate 4-phenoxypheny1 isocyanate 4-ethoxyphenyl isocyanate 4- (methylthio) phenyl isocyanate 4-acetylphenyl isocyanate

4-isopropylphenyl isocyanate 4-ethylphenyl isocyanate 4-n-butylphenyl isocyanate 3- (dichloromethylsilyl)propyl isocyanate octyl isocyanate

4-methyl-3-nitrophenyl isocyanate 4 - chloro- 2 -nitrophenyl isocyanate 2-methyl-4-nitrophenyl isocyanate 4-methyl-2 -nitrophenyl isocyanate 2- fluoro-5-nitrophenyl isocyanate 2 -methyl-5 -nitrophenyl isocyanate 3-bromopropyl isocyanate 2 , 4 , 6 - trimethylphenyl isocyanate 2- isopropyl- 6 -methylphenyl isocyanate 2, 6-diethylphenyl isocyanate

5-chloro- 2 -methylphenyl isocyanate 4 -chloro-2 -methylphenyl isocyanate 4- (trifluoromethoxy) phenyl isocyanate 4-trifluoromethylthiophenylisocyanate 2 , 4-dibromophenyl isocyanate

2,6-dibromo-4-ethylphenyl isocyanate 2,3,4,5- tetrachlorophenyl isocyanate 2 - chloro- 5 -trifluoromethylphenyl isocyanate 2- chloro- 6-methylphenyl isocyanate 2-n-carbobutoxyphenyl isocyanate 2,4,5-trimethylphenyl isocyanate 2-methyl-6- (t-butyl) phenyl isocyanate

2 -ethyl- 6 - isopropylphenyl isocyanate

3 - chloro-2 -methoxyphenyl isocyanate 3 -chloro-2 -methylphenyl isocyanate

3 -chloro-4-fluorophenyl isocyanate

4 - cyanophenyl isocyanate 4-bromo-2-methylphenyl isocyanate 4-bromo-2 , 6 -dimethylphenyl isocyanate 2, 6 -dibromo-4- fluorophenyl isocyanate 4-n-butoxyphenyl isocyanate 4-butoxycarbonylphenyl isocyanate phenethyl isocyanate 2 -methyl - 3 -nitrophenyl isocyanate hexyl isocyanate hexadecyl isocyanate methylene bis (o- chlorophenyl isocyanate) 4-chloro- 3 -nitrophenyl isocyanate 2 -chloro-4 -nitrophenyl isocyanate 4,5-dimethyl -2 -nitrophenyl isocyanate 2 - chloro- 5-nitrophenyl isocyanate 2 -methoxy-4 -nitrophenyl isocyanate 3-fluoro-4-methylphenyl isocyanate 5- fluoro-2 -methylphenyl isocyanate 3,5-dicarbomethoxyphenyl isocyanate 2,4-dichlorobenzyl isocyanate 2- (methylthio) phenyl isocyanate N- (methoxycarbonyl) isocyanate N- (phenox carbonyl) isocyanate 2-biphenylyl isocyanate 3 - iodopheny1 isocyanate 4-phenylphenyl isocyanate tetrahydro-2-pyranyl isocyanate 4- (tert-butyl) phenylisocyanate 1- (4-bromophenyl) ethyl isocyanate isocyanatoacetic acid n-butyl ester dodecyl isocyanate

6, 7-methylenedioxy-4-isocyanate-methylcoumarin (R) - (+) -alpha-methylbenzyl isocyanate (+/-) -1- (1-naphthyl) ethyl isocyanate (S) - (+) -1- (1-naphthyl) ethyl isocyanate 3, 4-difluorophenyl isocyanate

2 -methoxy- 5 -nitrophenyl isocyanate undecyl isocyanate ethyl 2-isocyanato-4-methyl valerate ethyl 6- isocyanatohexanoate ethyl 2-isocyanato-4-methylthiobutyrate ethyl 2 - isocyanatopropionate ethyl 3 - isocyanatopropionate ethyl 2 - isocyanato- 3 -methylbutyrate tert-butyl 3 - isothiocyanatopropionate ethyl 2 - isocyanato-3 -phenylpropionate

1, 3 -bis (isocyanatomethyl) cyclohexane 2- (trifluoromethoxy) phenyl isocyanate

4- (chloromethyl) phenyl isocyanate

1-adamanty1 isocyanate

1, 3 -bis (2 -isocyanato-2 -propyl) benzene n-amyl isocyanate n-heptyl isocyanate

2 - chloroethyl isocyanate, [ethyl -1,2 -14c]

1,1,3,3- tetramethylbutyl isocyanate

3,5-dinitrophenyl isocyanate

Examples of suitable isothiocyanates reagents of the formula RNCS, wherein R is an organic moeity, are the following: cyclohexyl isothiocyanate

1-naphthyl isothiocyanate trimethylsilyl isothiocyanate phenyl isothiocyanate

2-bromophenyl isothiocyanate

2-fluorophenyl isothiocyanate

2 -chlorophenyl isothiocyanate o-tolyl isothiocyanate

3-bromophenyl isothiocyanate

3 -fluorophenyl isothiocyanate

3 - chlorophenyl isothiocyanate m-tolyl isothiocyanate 4-bromophenyl isothiocyanate 4- fluorophenyl isothiocyanate

4- chlorophenyl isothiocyanate p-tolyl isothiocyanate ethoxycarbonyl isothiocyanate benzoyl isothiocyanate tert-butyl isothiocyanate tert-octyl isothiocyanate methyl isothiocyanate benyl isothiocyanate ethyl isothiocyanate phenethyl isothiocyanate allyl isothiocyanate

Other suitable acylating reagents for use in preparation of the substituted phenylalanine library of this invention are illustrated by the following formulas, wherein L is halo, PN is an amino-protecting group, and Po is a hydroxyl protecting group:

The preparation of the substituted phenylalanine library compounds of Formulas I and II above is initiated by the covalent attachment of an amino-protected nitrophenylalanine of the formula

to a solid support to give a solid support bound compound of the formula

wherein P_N is an amino-protecting group, and

is the solid support.

The solid support is preferably an organic polymer such as polyacrlyamide , cellulose, Wang resin, polystyrene or a polystyrene divinylbenzene copolymer such as Merrifield resin. Examples of inorganic solid supports are silica gel and alumina. The solid support is insoluble in typical organic solvents and water, and it has a particle size sufficient to allow its easy separation by filtration from residual soluble reagents and solvents. The solid support is selected to have functionality capable of reacting with the carboxy group of the nitrophenylalanine to covalently link the nitrophenylalanine to the solid support, typically through an ester or amide bond. The bond linking the nitrophenylalanine to the solid support should be stable under the reaction conditions used in preparation of the present library compounds, but readily cleavable to release the product library compound from the solid support without degradation of the library compound. One suitable solid support is Merrifield resin which includes a benzyl chloride functionality for reaction with the nitrophenylalanine carboxylate group to form a cleavable ester bond with the solid support. The ester bond is cleavable in the presence of base in protic solvents, but is stable under the reaction conditions used to form the present library compounds.

In one embodiment of the present invention, Merrifield resin having about 1.7 mmol chloride per gram of resin is reacted with approximately equimolar amounts of amino- protected nitrophenylalanine and K2CO3 (1:1:1; resin chloride: phenylalanine: K2CO3) in dimethylformamide and heated to about 55-60°C under nitrogen for 15-20 hours. Those reaction conditions are not critical; any of a wide variety of well known ester forming reaction conditions can be used. The progress of the coupling reaction can be monitored, for example, by taking aliquots of the resin from the reaction mixture, cleaving the linkage between the resin and the amino-protected nitrophenylalanine and quantifying the released amino-protected nitrophenylalanine using standard analytical techniques. Typical coupling ratios of nitrophenylalanine to the solid support range from about 0.5 to about 1.5 mmoles nitrophenylalanine per gram of solid support .

The preparation of the present library compounds is carried out on the support bound amino-protected nitrophenylalanine by a series of up to five reactions including deprotection of the protected alpha-amino group, acylation of the resulting alpha-amino group, reduction of the nitro group, acylation of the resulting amino group, and cleavage of the ester bond between the resulting acylated amino-phenylalanine to provide the corresponding acid or an ester thereof . The order in which the reactions are carried out is critical only in that the support bound phenylalanine is first reacted to have one free amino group before the first acylation reaction. Thus, in the first step either the amino protecting group can be removed or the nitro group can be reduced to produce a support-bound phenylalanine compound having one free amino group, and following acylation of that group, the resulting acylated intermediate is reacted to produce the second amino group for subsequent acylation. The support-bound product is then washed free of all unreacted reagents and thereafter cleaved from the solid support .

In one embodiment of this invention the amino- protecting group is removed from the solid support bound nitrophenylalanine to provide a compound of the formula

The nature of the amino-protecting group is not critical and those skilled in the art will readily appreciate which groups are acceptable and how they are removed to produce the corresponding amino compound. Examples of conventional protecting groups are t-butoxycarbonyl and benzyloxycarbony1. Thus, for example, t-butoxycarbonyl nitrophenylalanine is covalently bound to the solid support, and the t-butoxycarbonyl protecting group is subsequently removed by reaction with trifluoroacetic acid in a suitable organic solvent, for example, methylene chloride or dimethylformamide (DMF) to provide the corresponding support-bound nitrophenylalanine . Acylation of that product is carried out by reacting it with an acylating agent in an organic solvent, for example, methylene chloride, in the presence of about 0.1 equivalents of a strong tertiary amine base such as dimethylaminopyridine , and about 2 to about 4 equivalents, more preferably about 3 equivalents of a weaker tertiary amine base such as diisopropylethylamine, triethylamine, N- methylmorpholine or pyridine. The mixture is allowed to react for about 1 to 2 days preferably at a temperature of about 25°C to about 60°C, with continuous agitation. The support-bound acylated nitrophenylalanine is separated from the reaction mixture by filtration and washed free of residual reactants with a suitable organic solvent.

Conversion of the support bound nitrophenylalanine to the corresponding aminophenylalanine of the formula

is carried out under mild reducing conditions. For example, the reaction can be accomplished with stannous chloride in DMF at room temperature over a 1 to 3 -day reaction time. The product support-bound aminophenylalanine is washed several times with organic solvent and dried under vacuum. Acylation of the aminophenylalanine VI is accomplished under conditions similar to those used for acylating the phenylalanine alpha-amino group described. It is carried out with about 1 equivalent of an acylating agent in organic solvent in the presence of about 0.1 equivalent of a strong tertiary amine base and about 2 to about 4 equivalents of a weaker tertiary amine base with mixing for about 1 to 2 days at room temperature up to about 60°C. The reaction product represented by the formula

is separated from the reaction mixture by filtration and washed several times with an organic solvent .

The solid support bound library product is cleaved from the solid support, and the resulting solution of the library compound is separated from the solid support by filtration and evaporated to dryness. Thus, for example, the ester linkage between the solid support and the phenylalanine library compound is cleaved by contacting the product with a solution of a strong base, such as an alkali metal hydroxide, in a lower alkanol , for example 0.5 N NaOH in methanol, for 12 to 24 hours at room temperature to provide a solution of the library product. The solution is filtered to remove the solid support and the filtrate containing the product is neutralized to provide either a carboxylic acid or ester member of the library. To optimize the ester formation, the filtrate is neutralized with a strong acid, for example trifluoroacetic acid, and is allowed to dry by passive evaporation without vacuum or nitrogen flow. The ester is that corresponding to alcohol used in the cleavage reaction; for example, the methyl ester is formed when methanol is used as the solvent for the base catalyzed cleavage of the library product from that solid support. Similarly, the ethyl ester is formed when ethanol is used as the solvent for the cleavage reaction and the product solution is neutralized with a strong acid. The carboxylic acid library compounds are favored when weaker acids are used to neutralize the cleavage reaction mixture, for example, acetic acid, and when the product-containing filtrate is dried in vacuo after neutralization.

Optionally, library compounds of Formulas I and II above wherein the group RiYi is hydrogen can be synthesized by removing the amino-protecting group only after the reduction of the nitro group and acylation of the resulting amine. Similarly, compounds wherein the group ₂Y₂ is hydrogen can be synthesized by omitting the acylation step following reduction of the nitro group, and subsequent cleavage of the compound from the solid support, omitting the second acylation step. The absolute configuration of the phenylalanine library compounds corresponds to that of the starting nitrophenylalanine. Thus the library products can be prepared either as optically pure compounds or as racemic mixtures by selection of the desired starting compounds .

The process of the present invention utilized in preparation of a library of substituted phenylalanines 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 substituted phenylalanine library of this invention can be formed in a 96-well plate as illustrated in Figures 1 and 2. That apparatus provides multiple reaction zones most typically in a two-dimensional array of defined reservoirs, wherein one member of the substituted phenylalanine library of this invention is prepared in each reservoir. Thus the diverse substituted phenylalanine library of the present invention comprises a plurality of reservoir arrays (e.g. well plates) , each reservoir or well containing a library compound of the substituted phenylalanine 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 .

The initial steps of covalently bonding the nitrophenylalanine starting compound to the solid support and removal of the amino-protecting group can be carried out in the individual reaction zones, but it is preferably accomplished in a batch mode. The parallel array synthesis of library compounds is then continued by dispensing a slurry of the resulting support bound deprotected nitrophenylalanine into the individual reaction zones of apparatus in preparation for the first acylation step. 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 member 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 substituted phenylalanine 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.

Figure 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) , is identified by the alpha numeric coordinate, "A6".

Figure 2 illustrates a side view of a modified well plate apparatus for use in preparation of the library of the present invention. Well plate (3) contains wells (4) with a filter (5) , and a retaining frit (6) , and a liquid reaction medium used in carrying out the process (7) . The wells have an outlet at the bottom which is sealed by gasket (8) held in place by a top cover (9) and bottom cover (10) maintained in position by clamps (11) . 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 plate is then placed in the clamp assembly to seal the bottom of the wells. Synthesis proceeds by adding reagents to their individual wells according to their assigned plate coordinates. The plate is then capped and agitated to mix the reagents. Following completion of the reaction step, the solvent and residual reagents are removed by filtration and the resin bound product washed with appropriate solvents according to the procedure. The intermediate products can then be carried on to the next reaction step or a cleavage reaction. The intermediate products can be sampled and analyzed, for example, by cleavage from the solid support and then submitted to thin layer chromatography, mass spectrometry and/or nuclear magnetic resonance spectrometry. Upon completion of the desired reaction(s), the product is cleaved from the resin using a method known to those skilled in the art. The solid and solution are separated, the product optionally treated with neutralizing (acidic or basic) and/or purification conditions (for example, chromatography, solid phase extraction technology or solution phase extractions) and the volatile solvents and/or reagents optionally evaporated in a vacuum centrifuge.

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 substituted phenylalanine 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 Endothelin converting enzyme [¹²⁵I] SPA assay HIV proteinase [¹²⁵I] SPA enzyme assay Cholesteryl ester transfer (CETP) [³H] SPA assay Fluorescence Polarization Assays Fluorescence Correlation Spectroscopy Calorimetric biosensors Ca2⁺ - EGTA for Cell-based assays

Receptor Gene Constructs for cell based assays Luciferase, green fluorescent protein, Beta-lactamase Electrical cell impedance sensor assays

Example 1.

Substituted phenylalanine Library Plates: General Procedure with p-nitro-n-boc-phenylalanine and Merrifield resin.

Resin synthesis:

To a slurry of Merrifield resin (130 g, 1.7 mmol/g Cl, 0.22 mol Cl) in DMF (1.3 L) were added D-p-nitro-N-Boc- phenylalanine (35 g, 0.11 mmol), L-p-nitro-N-Boc- phenylalanine (35 g, 0.11 mmol) then anhydrous. K2CO3 (31 g, 0.22 mol) . The slurry was warmed at 55-60°C under 2 for 19 hours. Upon cooling to room temperature the solvent was removed via vacuum through a gas dispersion tube. The resin was then washed with CH2CI2 (0.5 L) , H2O (0.5 L), 1:1 THF:H2θ (1.0 L), THF (1.0 L), 10% aq HC1 (1.0 L), H2O (1.0 L), THF (1.0 L), then CH2CI2 (0.5 L total) was used to transfer the light yellow resin to a fritted funnel. After air drying under vacuum the solid was placed in a pyrex dish, covered with a large Kimwipe and dried in the vacuum oven (50°C, 20-25 in. Hg) for 48 hours. Upon drying the sample was pulverized and testing for loading via hydrolysis (0.5 N methanolic KOH, 18 h) . Analysis by quantitative HPLC methods provided an average loading (over three samples) of 1.01 mmol/g.

Removal of amino-protecting group: To a 0°C slurry of the resin bound p-nitro-N-Boc- phenylalanine (182 g) in CH2CI2 (1.4 L) was added trifluoroacetic acid (0.58 L) over 60 min. After 60 min more the ice bath was removed and the slurry allowed to stir 3 h longer. After removing the solvent via a gas dispersion tube, the resin was washed with CH2CI2 (1.0 L) , methanol (1.5 L) , CH2CI2 (1.5 L) , then CH2CI2 (1.0 L) was used to transfer the light yellow resin to a fritted funnel. After air drying under vacuum, the solid was placed in a pyrex dish, covered with a large Kimwipe and dried in the vacuum oven (50°C, 20-25 in. Hg) for 4 days. A total of 179 g of the title product was recovered.

Charging of plates : Plates were packed using an isopycnic slurry of CH2CI2 and DMF. For a one plate amount (2.36 g of a 0.91 mmol/g loaded resin in 114 mL total solvent, loaded with 1 mL of slurry/well) , 84 mL CH2CI2 and 30 mL DMF were required

(2.8:1) . After drying the plate in a vacuum oven, the plate contained 2.27 g of p-nitro-N-Boc-phenylalanine-resin, or 24 mg/well, or 22 μmol/well. Plates for the library were packed using the same ratio for the isopycnic slurry.

First acylation: Method A: To the loaded plate in a clamp were added to each well: the acylating agent R¹Y¹ (200 μL of a 1 M solution in CH2CI2. 200 μmol, 9.3 eq) then a solution of

DMAP (0.39 mg, 3.2 μmol, 0.15 eq) and pyridine (26 μL, 323 μmol, 15 eq) in CH2CI2 (500 μL total base solution). The wells were capped, the plate vortexed and agitated for 48 hours. The plate was drained, washed with CH2CI2, DMF, methanol, DMF, CH2CI2, and DMF (1 mL/well/washing solvent) and vacuumed dry on a teflon plenum.

Method B: To the loaded plate in a clamp were added a solution of DMAP (0.29 mg, 2.2 μmol, 0.10 eq) and diisopropylethyl amine (38 μL, 220 μmol, 10 eq) in CH2CI2 (500 μL total base solution) then a solution of the acylating agent (75 μL, 3.3 eq.). The wells were capped, the plate vortexed and agitated for 24 hours. The plate was drained, washed with CH2CI2, DMF, methanol, DMF, CH2CI2, and DMF (1 mL/well/washing solvent) .

Reduction of the nitro group:

To the plate in a clamp was added to each well SnCl2θ2H2θ (625 μL of a 1M solution in DMF, 625 μmol, 29 eq) . The wells were capped, the plate vortexed and agitated for 71 hours. The plate was drained, washed with DMF, methanol, DMF, methanol, DMF then CH2CI2 (l mL/well/washing solvent) and vacuumed dry on a teflon plenum.

Second acylation:

To the plate in a clamp were added to each well: the acylating agent R²Y² (200 μL of a 1 M solution in CH2CI2,

200 μmol, 9.3 eq) then a solution of DMAP (0.39 mg, 3.2 μmol, 0.15 eq) and pyridine (26 μL, 323 μmol, 15 eq) in CH2CI2 (500 μL total base solution) . The wells were capped, the plate vortexed and agitated for 52 hours. The plate was drained, washed with CH2CI2, DMF, methanol, DMF, methanol,

CH2CI2 and methanol (1 mL/well/washing solvent) and vacuumed dry.

Cleavage from the solid support:

To the plate in a clamp was added methanolic NaOH to each well (625 μL of a 0.5 N solution, 313 μmol, 14 eq) .

The wells were capped, the plate vortexed and agitated for 18 hours.

Neutralization:

Method A: The plate was drained into a 2 mL 96 well titer plate, then neutralized with 125-150 μL/well of a 2:1 v/v solution of methanol : cone HC1. Several wells were spotted onto pH paper to assure that all wells were pH 2. Method B: The plate was drained into a 2 mL 96 well titer plate, then neutralized with acetic acid (4 eq per well) . The pH was approximately 5-6.

Final Preparation:

The volatiles were evaporated ina vacuum centrifuge. Each well was reconstituted with 9:1 CH2CI2 :methanol (625 μL/well) and filtered through a fritted plate into a clean 1 mL titer plate. At this point, TLC's (60:40:2 toluene : ethyl acetate: acetic acid) were taken of each well; the solvents were then removed in a vacuum centrifuge as a final drying before submission.

Claims

We claim:

1. A library of substituted phenylalanine compounds wherein said library contains a plurality of diverse library compounds of the formula

wherein Ri and R2 are each independently an organic moiety;

Yl and Y2 are each independently selected from the group consisting of -CO-, -CO2-, -C0NH-, -CSNH-, and -SO2-; and Z is hydrogen or C₁-C4 alkyl; or either of the groups R1Y1 or

R2Y2 can be hydrogen.

2. The library of claim 1 wherein Ri and R2 are each independently derived from an acylating agent of the formula RCOX, RSO2X, ROCOX, RN=C=0 or RN=C=S, wherein R is an organic moeity and X is halo.

3. The library of claim 1 wherein the library contains a plurality of diverse library compounds of the formula

4. The library of claim 2 wherein the acylating agent has a molecular weight of about 50 to about 600.

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

6. A library of substituted phenylalanine compounds wherein said library contains a plurality of diverse library compounds of the formula

wherein Ri and R2 are each independ ^╬ÿently an org ^'a^"nic moiety; Yl and Y2 are each independently selected from the group consisting of -CO-, -CO2-, -CONH-, -CSNH-, and -SO2-; or either of the groups R1Y1 or R2Y2 can be hydrogen; and

is a covalently bound solid s ^╬ÿupport.

7. A compound selected from the group consisting of the library compounds of the library of claim 6.

8. A process for preparing a combinatorial library of substituted phenylalanine compounds of the formula

having diversity in substituent groups Ri, R2, Yl Y2 and Z wherein each library compound is made in a separate reaction zone, said process comprising the steps of a) covalently coupling a compound of the formula

to a solid support through the carboxy group to provide a compound of the formula

wherein PN an amine protecting group and

is the solid support;

b) removing the amine protecting group to provide the corresponding primary amine;

c) acylating the resulting primary amine with an acylating agent of the formula RiCOX, R1SO2X, RiOCOX,

R╧çN=C=0 or R╧çN=C=S , wherein i is an organic moeity and X is halo, to provide a compound of the formula

wherein Ri is an organic moeity and Yi is selected from the group consisting of -CO-, -CO2-, -CONH-, -CSNH-, and -SO2-;

d) reducing the nitro group to provide the corresponding primary amine of the formula

e) acylating the resulting primary amine with an acylating agent of the formula R2COX, R2SO2X, R2OCOX, R2N=C=0 or R╧çN=C=S, wherein R2 is an organic moeity and X is halo, to provide a compound of the formula

wherein RI and R2 are each independently an organic moeity and Yl and Y2 are independently selected from the group consisting of -CO-, -CO2-, -CONH-, -CSNH-, and -SO2-;

f) reacting the resulting solid support bound compound to cleave the covalent bond to the solid support.

9. A process for preparing a combinatorial library of substituted phenylalanine compounds of the formula

wherein R2Y2 is hydrogen and having diversity in substituent groups Ri, Yi, and Z wherein each library compound is made in a separate reaction zone, said process comprising the steps of a) covalently coupling a compound of the formula

wherein P_N is an amine protecting group and

is the solid support;

c) acylating the resulting primary amine with an acylating agent of the formula RiCOX, R1SO2X, RiOCOX, R__N=C=0 or R╧çN=C=S, wherein Ri is an organic moeity and X is halo, to provide a compound of the formula

wherein Ri is an organic moeity and Yi is selected from the group consisting of -CO-, -CO2-, -C0NH-, -CSNH-, and -SO2-;

e) acylating the resulting primary amine with an acylating agent of the formula R₂COX, R₂SO₂X, R₂OCOX, R2N=C=0 or R╬╣N=C=S, wherein R₂ is an organic moeity and X is halo, to provide a compound of the formula

wherein RI and R2 are each independently an organic moeity and Yl and Y2 are independently selected from the group consisting of -CO-, -CO₂-, -C0NH-, -CSNH-, and -SO2- ;

10. A process for preparing a combinatorial library of substituted phenylalanine compounds of the formula

wherein Y₁R₁ is hydrogen and having diversity in substituent groups R₂, Y₂ and Z wherein each library compound is made in a separate reaction zone, said process comprising the steps of a) covalently coupling a compound of the formula

wherein P is an amine protecting group and

is the solid support;

b) reducing the nitro group to provide the corresponding primary amine of the formula

c) acylating the resulting primary amine with an acylating agent of the formula R2COX, R2SO2X, R2OCOX, R2N=C=0 or R╧çN=C=S, wherein R2 is an organic moeity and X is halo, to provide a compound of the formula

wherein R2 is an organic moeity and Y2 is selected from the group consisting of -CO-, -CO2-, -C0NH-, -CSNH-, and -SO2 - ;

d) removing the amine protecting group to provide the corresponding primary amine of the formula

e) reacting the resulting solid support bound compound to cleave the covalent bond to the solid support.

11. The process of claim 8, 9, or 10 wherein the solid support is Merrifield resin of the formula

12. The process of claim 8, 9, or 10 wherein the coupling reaction with the solid support is effected in the presence of K2CO3.

13. The process of claim 8, 9, or 10 wherein the reduction of the nitro group is effected with SnCl2-

14. The process of claim 8 or 9 wherein the acylation step c) is carried out in an organic solvent with an acid chloride in the presence of diisopropylethylamine.

15. The process of claim 8 or 9 wherein the acylation of step c) is carried out with about ten equivalents of an acid chloride in methylene chloride in the presence of about 0.1 equivalents of dimethylaminopyridine and about three equivalents of diisopropylethylamine.

16. The process of claim 8, 9, or 10 wherein the product is a compound of formula I wherein Z is hydrogen.

17. The process of claim 8, 9, or 10 wherein the product is a compound of formula I wherein Z is C₁-C₄ alkyl.

18. 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, claim 3 or claim 6.

19. The assay kit of claim 18 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 Endothelin converting enzyme [¹²⁵I] SPA assay HIV proteinase [¹²⁵I] SPA enzyme assay

Cholesteryl ester transfer (CETP) [³H] SPA assay Fluorescence Polarization Assays Fluorescence Correlation Spectroscopy Calorimetric biosensors Ca2⁺ -EGTA Yes for Cell-based assays

20. 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 of said library, wherein said structurally related compounds are of the formula

wherein Ri and R2 are each independently an organic moiety; Yi and Y2 are each independently selected from the group consisting of -CO-, -CO2-, -C0NH-, -CSNH-, and -SO2-; and Z is hydrogen or C1-C4 alkyl or either of the groups R1Y1 or

R2Y2 can be hydrogen.

21. The apparatus of claim 20 wherein the library compound in each reservoir is prepared in accordance with the process of claim 8 , 9 , or 10 and wherein each reservoir provides one reaction zone.

22. The apparatus of claim 20 wherein the 2- dimensional array of defined reservoirs is a multi-well microtiter plate.