WO2003062230A1

WO2003062230A1 - Conbinatorial library of heterocycle compounds

Info

Publication number: WO2003062230A1
Application number: PCT/JP2002/000388
Authority: WO
Inventors: Naoki Kobayashi; Yasushi Okamoto; Kunizo Higurashi; Yumiko Kaku; Akira Ishibashi; Toshihiko Yamauchi
Original assignee: Eisai Co Ltd
Current assignee: Eisai Co Ltd
Priority date: 2002-01-21
Filing date: 2002-01-21
Publication date: 2003-07-31
Anticipated expiration: 2004-07-21

Abstract

This invention is directed to a combinatorial library of pyrrole compounds, process for preparing the library thereof. This library is useful for screening the biological activity of the pyrrole compounds.

Description

DESCRIPTION

COMBINATORIAL LIBRARY OF HETEROCYCLE COMPOUNDS

BACKGROUND OF THE INVENTION

This invention relates to a novel library containing a plurality of compounds, process for preparation thereof, and process for screening such a library for useful therapeutic compounds. Certain pyrrole compounds of the present invention have demonstrated activity as RAR (retinoic acid receptor) a agonists. Other therapeutic activities are expected, depending on the substituents attached to the compounds.

The traditional methods of drug discovery did eventually yield leads for new drugs. However, the time required for characterization of the compounds considerably limited the discovery of new pharmaceutically active compounds. In the present competitive environment of the pharmaceutical industry, the need has arisen for efficient, fast and less expensive methods for new drug discovery. Combinatorial chemistry offers a means of generating large numbers of related compounds generally referred as "library". See J. Hogan, Jr., Nature Biotechnology, 15, 328 (1997), "Combinatorial Chemistry and New Drugs," Scientific American, page69, April 1997, M. Pirrung, Chem. Rev., 97, 473 (1997), "A Hail of Silver Bullets, " Forbes, page 76, January 26, 1998. In simple terms, combinatorial chemistry is the parallel, simultaneous preparation of multiple derivatives of a central, fixed moiety often referred to as the "scaffold," "core" or "backbone". Theoretically, the number of compounds, which may be produced to establish a library, is limited only by the number of reagents available to form the derivatives.

Combinatorial chemistry may be used to generate libraries, which are mixtures of individual compounds and complete identification of the individual compounds are postponed until after positive screening. However, "parallel array synthesis" whereby individual reaction products are simultaneously synthesized but each reaction is carried out in a separate vessel is generally preferred. For example, a compound library may be prepared, stored and assayed in standard microtiter plates such as the plates often used in the biological sciences.

In combinatorial chemistry, the scaffold is typically attached to a solid support, e.g., a polymer, which is insoluble in the reaction solvent. When the entire reaction sequence has been completed, the library compounds are detached from the support. However, in some cases combinatorial libraries can be prepared in solution phase. It is also possible to tether the reactants to a support with the scaffold in solution. In all cases, the net effect is to produce a library of many derivatives of the scaffold each in its own small reaction vessel. Conceptually, preparation of a combinatorial library by parallel synthesis appears to be logical and straightforward. However, in practice, efficient preparation of such libraries has demanded development of new techniques and new equipment. Often synthetic sequences, which are routine in traditional organic chemistry, are difficult and unpredictable, if not impossible, when applied to combinatorial chemistry. In addition to the new technique and equipment developed in recent years, the application of robotics enables an organic chemist to build libraries of several thousand compounds in relatively short periods .

The need for large combinatorial libraries has been made acute by the advent of high throughput screening (HTS) technology (see W. Janzen, Laboratory Robotics and Automation, 8, 261, (1996)) . With this technology, thousands of compounds can be screened each day, thus creating a demand for large libraries to be introduced through the batteries of screens. Further HTS uses only microgram quantities of compound for each screen, so a library can be used for several screens before it is depleted.

Therefore the marriage of combinatorial chemistry and HTS has created a powerful tool for discovering new drug leads

(see "Combinatorial Chemistry", Chemical and Engineering News, page 43, February 24, 1997) .

BRIEF SUMMARY OF THE INVENTION

The present invention provides the combinatorial library containing a plurality of compounds of the Formula (1):

wherein X is 0, S, and NR⁷;

R^la, R^l and R⁷ are independently selected from the group consisting of hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, and optionally substituted aryl;

Z is optionally substituted aryl, a 5 to 14 membered aromatic monocyclic or fused polycyclic moiety that includes from 1 to 4 heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen;

Y is optionally substituted arylene, a 5 to 14 membered aromatic monocyclic or fused polycyclic moiety that includes from 1 to 4 heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen; or a pharmaceutically acceptable salt there of.

The present invention also provides a process for preparing the combinatorial library containing a plurality of heterocyclic compounds, comprising using a solid-supported intermediate of Formula (2) :

wherein g is a solid-support; b is a single bond, -0-, -OC(0)-, -NR⁶-, -S-, -S0₂NHC(0)-,

-NC(0)R⁶- or -NS(0)₂R⁶- where R⁶ is hydrogen, alkyl, aryl, alkenyl or alkynyl; f is optionally substituted arylene, a 5 to 14 membered aromatic monocyclic or fused polycyclic moiety that includes from 1 to 4 heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen; d is a single bond or optionally substituted alkylene;

R² is a stable moiety independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl or aryl;

R³ is a stable moiety independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl or aryl;

R⁵ is optionally substituted aryl, a 5 to 14 membered aromatic monocyclic or fused polycyclic moiety that includes from 1 to 4 heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used in this invention: The term "optionally substituted" refers that " the position being able to be substituted may have one or more substituents, which are independently selected from the group consisting of;

(1) halogen; (2) OH; (3) thiol; (4) nitro; (5) nitrilo; (6) oxo;

(7) azido; (8) guanidino; (9) hydrazino; (10) isocyano; (11) cyanato; (12) isocyanato; (13) thiocyanato; (14) isothiocyanato; (15) nitroso; (16) carbamide (ureido); (17) formyl; (18)

alkyl, C₂.₆ alkenyl, C₂.₆ alkynyl, C₃._B cycloalkyl, C_1-6 alkoxy, C₂.₆ alkenyloxy, C₂.₆ alkynyloxy, C₃.₆ cycloalkyloxy, alkylthio, C₂.₆ alkenylthio, C₂_g alkynylthio, C₃.₆ cycloalkylthio, C_1-6 alkylenedioxy, each of which may be independently substituted by 1 to 3 halogen atoms or 1 to 3 hydroxy groups; (20) C₆_₁₄ aryl; (21) 5 to 14 membered heterocyclic; (22) carboxyl; (23) trifluoromethyl; (24) C₆.₁₄ aryl

alkyl; (26) alkyl carbamoyl; (27)

alkoxy carbonyl; (28) C_1-6 alkyl carbonyl; (29)

alkyl carbonyloxy; (30)

alkyl sulfonyl . "Alcohols" include groups of the formula -OH. "Aldehydes" include groups of the formula -C(=0)H.

"Alkyl" refers to a substituted or unsubstituted, straight, branched or cyclic hydrocarbon chain containing of from 1 to 20 carbon atoms. Preferred alkyl groups are lower alkyl groups, i.e., alkyl groups containing from 1 to 6 carbon atoms. Preferred cycloalkyls have from 3 tolO, preferably 3-6, carbon atoms in their ring structure. Suitable examples of unsubstituted alkyl groups include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, iso-butyl, tert-butyl, sec- butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, cyclohexyl, and the like.

"Alkylene" is the two valences radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl group, and that groups may include one or more double or triple bonds. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone, and more preferably 20 or fewer and most preferred 10 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 3-6 carbons in the ring structure.

Particularly preferred alkyl substituents include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, iso-butyl, tert-butyl, sec-butyl, cyclobutyl, pentyl, hexyl, cyclohexyl, etc. Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. The aliphatic cyclic groups can be single or polycyclic containing between about 3 to 12 carbons per ring, but preferably between 3 and 9 carbons per ring.

"Alkenyl" refers to a substituted or unsubstituted, straight, branched or cyclic, unsaturated hydrocarbon chain that contains at least one double bond and 2 to 20, preferably 2 to 6, carbon atoms. Exemplary unsubstituted alkenyl groups include ethenyl (or vinyl) (-CH=CH₂) , 1-propenyl, 2-propenyl (or allyl) (- CH₂-CH=CH₂), 1,3-butadienyl (-CH=CHCH=CH₂) , 1-butenyl (CH=CHCH₂CH₃) , hexenyl, pentenyl, 1, 3, 5-hexatrienyl, and the like. Preferred cycloalkenyl groups contain having five to eight cabon atoms and at least one double bond. Examples of cycloalkenyl groups include cyclohexenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cycloheptadienyl, cyclooctatrienyl and the like. "Alkoxy" refers to a substituted or unsubstituted, -0-alkyl group. Exemplary unsubstituted alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy and the like . "Alkynyl" refers to a substituted or unsubstituted, straight, branched or cyclic unsaturated hydrocarbon chain containing at least one triple bond and 2 to 20, preferably 2 to 6, carbon atoms .

"Aprotic solvent" refers to polar solvents of moderately high dielectric constant which do not contain an acidic hydrogen. Examples of common aprotic solvents are dimethyl sulfoxide (DMSO) , dimethylformamide (DMF) , sulfolane, tetrahydrofuran (THF), diethyl ether (Et20) , methyl t-butyl ether or 1,2- dimethoxyethane . "Aryl" refers to any 5 to 14 membered monovalent aromatic monocyclic or fused polycyclic moieties . Preferred aryl groups include phenyl and naphthyl. The aryl group can be substituted at one or more positions with halo, alkyl, alkoxy, alkoxy carbonyl, haloalkyl, cyano, amino sulfonyl, aryl, sulfonyl, aminocarbonyl, carboxy, acylamino, alkyl sulfonyl, amino and substituted or unsubstituted substituents.

"Assay kit" as used in accordance with the present invention refers to an assemblage of at least 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. "Diverse library" means a library where the substituents on the combinatorial library scaffold 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") .

"Esters" include groups of the fomlula -C(=0)OR, where R is a substituent, preferably an alkyl group. Exemplary ester groups include methyl ester, ethyl ester, and the like. "Halogen" (or halo-) refers to fluorine, chlorine, bromine. The preferred halogen is fluorine or chlorine.

"Heterocyclic" (Het or heterocyclic) refers to a stable, saturated, partially unsaturated, or aromatic group containing 5 to 14, preferably 5 or 6, ring atoms. The ring can be substituted 1 or more times with a substituent. The ring can be mono-, bi- or polycyclic. The heterocyclic group consists of carbon atoms and from 1 to 4 heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. Examples of heterocyclic groups include acridinyl, benzothiazolidinyl, benzimidazolyl, benzofuranyl, benzothiazolyl, benzothiophenyl, carbazolyl, cinnolinyl, furanyl, imidazolyl, lH-indazolyl, indolyl, isoindolyl, isoquinolinyl, isothiazolyl, morpholinyl, oxazolyl, 1,2,3- oxadiazolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, piperazinyl, pteridinyl, purinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrole, quinazolinyl, quinolinyl, quinoxalinyl, thiazolyl, 1,3,4- thiadiazolyl, thiophenyl, 3, 5-triazinyl, triazolyl, thienyl, pyridyl, quinolyl and the like. It will be appreciated that many of the above heterocycles may exist in tautomeric forms. All such forms are included within the scope of this invention. "Inert atmosphere" refers to reaction conditions in which the mixture is covered with a layer of inert gas such as nitrogen or argon .

"Library" or "Combinatorial library" means a large number of chemical derivatives preferably used in screening for biological activity or other activity. "Library compound" is an individual reaction product (usually a single compound) in a combinatorial library. "Lead compound" means a compound in a selected combinatorial library for which the assay kit has revealed significant activity relevant to a selected disease state. "Nitriles" (or cyano) include groups of the formula -CN.

"Organic solvent" includes solvents containing carbon, such as halogenated hydrocarbons, ether, toluene, xylene, benzene, and tetrahydrofuran. "Pharmaceutically acceptable salt" and "salts thereof" means organic or inorganic salts of the pharmaceutically important molecule. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an ammonium ion, a sodium ion, an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically important organic molecule may have more than one charged atom in its structure. Situations where multiple charged atoms are part of the molecule may have multiple counterions. Hence, the molecule of a pharmaceutically acceptable salt may contain one or more than one charged atoms and may also contain, one or more than one counterion. The desired charge distribution is determined according to methods of drug administration. Examples of pharmaceutically acceptable salts are well known in the art but, without limiting the scope of the present invention, exemplary presentations can be found in the Physician's Desk Reference, The Merck Index, The Pharmacopoeia and Goodman & Gilman' s The Pharmacological Basis of Therapeutics.

"Protecting group" means a group used to protect a heteroatom such as oxygen, nitrogen, sulfur or phosphorus from chemical reaction. For example, an O-protecting group is used to protect an oxygen heteroatom, such as in a hydroxy group, from reaction. Examples of O-protecting groups include t-butoxycarbonyl (Boc) , t-butyl ether, benzyl ethers, and the like. Examples of amino-protecting (N-protecting) groups include acetyl (Ac) , 1-adamantanesulfonyl (AdS0₂) , 1-adamantaneacetyl (AdAc) , benzoyl (Bz) , t-butoxycarbonyl (Boc) , carbobenzoxy (Cbz) , 2-carboxybenzoyl (2-Cbz) , dansyl (DNS) , isovaleryl (Iva) , fluorenylmethoxycarbonyl (Fmoc) , methoxysuccinyl (MeOSuc) , nitropiperonyl, pyrenylethoxycarbonyl, nitroveratryl (NV) , nitrobenzyl, succinyl (Sue), tosyl (Ts) , and such aminoprotecting groups which are functionally equivalent thereto. Protecting groups are well known in the art, see for example Protective Groups in Organic Synthesis, Peter G. M. Wuts (Editor), Theodora W. Greene, 3^rd ed. (April 1999), Vch Pub; Protective groups in Organic Synthesis, Theodora W. Greene, Peter G. Wuts (Contributor), 2^nd ed., (May 1991) John Wiley & Sons. Preferred protecting groups include, but are not limited to, the "Boc" protecting group, trialkyl silyl groups such as TBS (tert-butyldimethylsilyl, Si (CH₃) ₂C (CH₃) ₃) , MEM (2- methoxyethoxymethyl) , MOM (methoxymethyl) , SEM (2- (trimethylsily) ethoxymethyl) , and THP (tetrahydropyranyl) .

"Protic solvent" refers to a solvent containing hydrogen that is attached to oxygen, and hence is appreciably acidic. Common protic solvents include such solvents as water, methanol (MeOH) , ethanol (EtOH) , 2-propanol (PrOH) , and 1-butanol (n-BuOH) . "Protic acid" refers to an acid having acidic hydrogen. Preferred protic acids include acetic acid, hydrochloric acid (HC1), formic acid, perchloric acid, sulfuric acid (H₂S0₄) , and phosphoric acid (H₃P0₄) in an aqueous medium. The most preferred protic acids are hydrochloric acid, sulfuric acid, and formic acid.

"Scaffold" as used in accordance with the present invention refers to the invariable region of the compounds that are members of the combinatorial library. That is, "scaffold" means a common chemical structure found within a library of organic compounds. Similarly, within a combinatorial chemical library the scaffold forms the basis for a diverse series of chemical derivatization, additions and subtractions. Importantly, regardless of the extent of the chemical derivatization performed on the scaffold, the product is within the scope of the combinatorial library.

"Solid support" broadly refers to any structure which is capable of supporting the chemical compound and is substantially inert to the chemical reactions conducted on the surface. Exemplary solid supports include, but are not limited to, metals, resins, polymers, gels, glass beads, silica gels, ceramic supports and other solid and semi-solid compositions. "Substantially pure" is intended to mean at least about 90 mole percent, more preferably at least about 95 mole percent, and most preferably at least about 98 mole percent of the desired enantiomer of stereoisomer is present compared to other possible configurations. "Substituted" means that the moiety contains at least one, preferably 1-3 substituent (s) . -Amino acids can be mono- substituted at the backbone nitrogen atom, substituted at the α -carbon (to form a a , -disubstituted -carbon) or substituted 1-3 times at one or more side chain atoms. Suitable substituents include hydrogen (H) and hydroxyl (-OH) , amino (-NR²) , oxy (-0-), carbonyl (-CO-), thiol, alkyl, alkenyl, alkynyl, alkoxy, halo, nitrile, nitro, aryl and heterocyclic groups. These substituents can optionally be further substituted with 1-3 substituents. Examples of substituted substituents include carboxamide, alkylmercapto, alkylsulphonyl, alkylamino, dialkylamino, carboxylate, alkoxycarbonyl, alkylaryl, aralkyl, alkylheterocyclic, (Cl- C4) fluoroalkyl groups (such as trifluoromethyl or 2,2,2- trifluoroethyl) and the like. "Thiols" include compounds of the formula -SH or -SR where R is a substituent, preferably alkyl. Exemplary thiols include methanthiol, ethanethiol, propanethiol, and the like. All other acronyms and abbreviations have the corresponding meaning as published in journals relative to the art of chemistry.

"Leaving group" means halo, oxo, thioxo radicals and activated alcohols such as a p-toluenesulfonyl activated alcohols and other groups that are susceptible to displacement and replacement by a nucleophile under selected conditions of temperature, solvent and time.

"TFA" means trifluoroacetic acid, "HC1" means hydrochloric acid, "THF" means tetrahydrofuran, "DMF" means dimethyl ormamide, "TEA" means triethylamine.

"Chemically derivatized" means the chemical manipulation such as addition to, oxidation of, substitution for, reduction of, or cyclization of the selected R group or R groups of the intermediate. Chemical derivatization also means the manipulation of two or more groups of the intermediate such that additional aryl or alkyl rings are formed and that rings may be fused or unfused to the intermediate ring, and that new ring may be substituted with further chemically derivatizable substituents .

A combinatorial library of compounds of Formula (1)

The present invention provides the combinatorial library containing a plurality of compounds of Formula (1):

wherein X is 0, S, and NR⁷;

The above library preferably contains compounds comprising from 2 to 1000000 compounds, and more preferably from 20 to 1000 compounds, of Formula (1) .

In the library mentioned above, Z is preferably optionally substituted 9-, 10- or 13- membered fused bicyclic or tricyclic aromatic heterocyclic group having 1 or 2 heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen;

Y is preferably optionally substituted phenyl, optionally substituted naphthyl, optionally substituted 5- or 6- membered aromatic heterocyclic ring having 1 or 2 heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen, or optionally substituted 9- or 10- membered fused bicyclic aromatic heterocyclic group having 1 or 2 heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen.

The library mentioned above is useful for screening the biological activity of the said compounds.

The optically active isomers and diastereomers of the compounds of Formula (1) are also considered part of this invention. Such optically active isomers may be prepared from their respective optically active precursors, or by resolving the racemic mixtures. This resolution can be carried out by derivatization with a chiral reagent followed by chromatography or by repeated crystallization. Removal of the chiral auxiliary by standard methods affords substantially optically pure isomers of the compounds of the present invention or their precursors . Further details regarding resolutions can be obtained in Jacques, et al . , Enantiomers, Racemates, and Resolutions, John Wiley & Sons, 1981.

In the library mentioned above, X is preferably NH.

The above library preferably contains compounds comprising from 2 to 1000000 pyrrole compounds, and more preferably from 20 to 1000 pyrrole compounds, of Formula (1) .

This library is particularly useful for screening the retinoic acid receptor agonizing activity of said compounds.

The pyrrole compounds of Formula (1) are useful whenever retinoic acid receptor agonis is efficacious, for example, as preventive and therapeutic agents for various cornification anomalies and skin diseases such as xeroderma pigmentosum, psoriasis, arthropathia psoriatica, acne or leukoplakia; various alopeciae such as alopeica areata, seborrheic alopecia or cachectic alopecia; various osteoporoses and osteopeniae such as postmenopausal osteoporosis, senile osteoporosis, steroidal osteoporosis, idiopathic osteoporosis, diabetic osteopenia, rheumatoid osteopenia or renal osteomalacia; diseases of bone and joint such as ectopic hyperostosis, osteoarthritis or shoulder periarthritis; automimmune diseases such as chronic rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, Behcet's disease, mycosis fungoides, systemic scleroderma, sudden thrombo- cytopenic purpura, yasthenia gravis, der atomyositis or nodular arteriosclerosis; various leukemiae such as acute promyelocytic leukemia, acute myelocytic leukemia or chronic leukemia; rejections of graft in organ transplantation; graft versus host diseases (GVHD) in born marrow transplantation or stem cell transplantation; nephropathies such as nephrotic syndrome; glomerulonephritis; malignant lymphomas such as mycosis fungoides; squamous cell carcinomas such as squamous cell carcinoma of head and neck; solid carcinomas such as bladder cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, prostatic cancer or pancreatic cancer; inflammations and allergic diseases such as atopic dermatitis or asthma; immune deficiencies and intractable infections such as immunodeficiency diseases, infections with cytomegalovirus due to lowered immune function or of fetus or opportunistic infection; hyperthyroidism; hypercalcemia; various fibroses such as pulmonary fibrosis, hepatic fibrosis or hepatic cirrhosis; atherosclerosis and restenosis after reconstructive operation of blood circulation; other nonmalignant hyperplastic diseases such as endometrial hyperplasia, benign prostatic hypertrophy, proliferative itreoretinopathy and dysplasia; diseases related to metabolism and transport of lipid such as hyperlipidemia; diabetes; wounds, dry eye syndrome; or solar skin injury; and as apoptosis induction accelerators.

The library of compounds of Formula (1) according to the present invention may be screened for biological activity. Generally, the library to be screened is exposed to a biological substance, usually a protein such as a receptor, enzyme, membrane binding protein or antibody; and the presence or absence of an interaction between the heterocycle derivative and the biological substance is determined. Typically this will comprise determining whether the biological substance is bound to one or more of the members of the library. Such binding may be determined by attaching a label to the biological substance. Commonly used labels include fluorescent labels. Other methods of labeling may be used, such as radioactive labels. It will be appreciated that certain compounds of Formula (1) may exist in, and be isolated in, isomeric forms, including tautomeric forms or cis- or trans-iso ers, as well as optically active, racemic or diastereomeric forms. The present invention encompasses library containing a plurality of compounds of Formula ( 1 ) in any of the tautomeric forms or as a mixture thereof . It is to be understood that the present invention encompasses library of compounds of Formula (1) as a mixture of diastereomers, as well as in the form of an individual diastereomer, and that the present invention encompasses library of compounds of Formula (1) as a mixture of enantiomers, as well as in the form of an individual enantiomer, any of which mixtures or form possesses agonism for retinoic acid receptors, it being well known in the art how to prepare or isolate particular forms and how to determine agonism for retinoic acid receptors by standard tests.

In addition, compounds of Formula (1) may exhibit polymorphism or may form a solvate with water or an organic solvent. The present invention also encompasses any such polymorphic form, any solvate or any mixture thereof.

Methods for acting as a retinoic acid receptor agonist

A pyrrole compound of Formula (1) is useful whenever retinoic acid receptor agonism is usuful, for example, as preventive and therapeutic agents for various cornification anomalies and skin diseases such as xeroderma pigmentosum, psoriasis, arthropathia psoriatica, acne or leukoplakia; various alopeciae such as alopeica areata, seborrheic alopecia or cachectic alopecia; various osteoporoses and osteopeniae such as postmenopausal osteoporosis, senile osteoporosis, steroidal osteoporosis, idiopathic osteoporosis, diabetic osteopenia, rheumatoid osteopenia or renal osteomalacia; diseases of bone and joint such as ectopic hyperostosis, osteoarthritis or shoulder periarthritis; automimmune diseases such as chronic rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, Behcet's disease, mycosis fungoides, systemic scleroderma, sudden thrombo- cytopenic purpura, myasthenia gravis, dermatomyositis or nodular arteriosclerosis; various leukemiae such as acute promyelocytic leukemia, acute myelocytic leukemia or chronic leukemia; rejections of graft in organ transplantation; graft versus host diseases (GVHD) in born marrow transplantation or stem cell transplantation; nephropathies such as nephrotic syndrome; glomerulonephritis; malignant lymphomas such as mycosis fungoides; squamous cell carcinomas such as squamous cell carcinoma of head and neck; solid carcinomas such as bladder cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, prostatic cancer or pancreatic cancer; inflammations and allergic diseases such as atopic dermatitis or asthma; immune deficiencies and intractable infections such as immunodeficiency diseases, infections with cytomegalovirus due to lowered immune function or of fetus or opportunistic infection; hyperthyroidism; hypercalcemia; various fibroses such as pulmonary fibrosis, hepatic fibrosis or hepatic cirrhosis; atherosclerosis and restenosis after reconstructive operation of blood circulation; other nonmalignant hyperplastic diseases such as endometrial hyperplasia, benign prostatic hypertrophy, proliferative itreoretinopathy and dysplasia; diseases related to metabolism and transport of lipid such as hyperlipidemia; diabetes; wounds, dry eye syndrome; or solar skin injury; and as apoptosis induction accelerators. The present invention further provides a method for the prevention and treatment of diseases against which the retinoic acid receptor agonism is efficacious by administering a pharmacologically effective amount of a compound of Formula

(1) .

The pyrrole compounds of Formula (1) are lowly toxic and highly safe, being useful also in this respect.

When the pyrrole compound of Formula (1) is to be administered for the above diseases, the route of administration may suitably be selected. Specifically, they may be orally administered as preventive or therapeutic agents in the form of tablets, powders, granules, capsules, syrups or the like, or may be parenterally administered in the form of suppositories, injections, external preparations or drops.

Although the dosage of the compound remarkably depends on the kind of diseases, the extent of symptom, the interval from sedation to the first administration, the age, sex and sensitivity of patient or the like, the compound may be administered generally in a dosage of about 0. 03 to 1000 mg, preferably 0.1 to 500 mg, still preferably of 0.1 to 100 mg per adult a day in several portions. When the compound is to be administered as an injection, the dosage of the compound is generally about 0.001 to 3 mg/kg, preferably about 3 to 1000 mg/kg.

Those pyrrole compounds may be isolated in the form of a salt. A salt of the compounds of formula (1) formed with a base or an acid such as one of those mentioned above are useful as a pharmaceutically acceptable salt for administration of the retinoic acid receptor agonistic agent and for preparation of a formulation of the agent. Other base addition salts or acid addition salts may be prepared and used in the isolation and purification of the compound.

Process for preparing the library

The process for preparing the library of compounds of the Formula (1) of the invention may be carried out in any reaction vessel capable of holding the liquid reaction medium and having, preferably, inlet and outlet means.

The present invention also provides a process for preparing the library of compounds of Formula (1) , by using a solid-supported intermediate of Formula (2) .

wherein g is a solid-support; b is a single bond, -0-, -0C(O)-, -NR⁶-, -S-, -S0₂NHC(0)-, -NC(0)R⁶- or -NS(0)₂R⁶- where R⁶ is hydrogen, alkyl, aryl, alkenyl or alkynyl; f is optionally substituted arylene, a 5 to 14 membered aromatic monocyclic or fused polycyclic moiety that includes from 1 to 4 heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen; d is a single bond or optionally substituted alkylene;

R² is a stable moiety independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl or aryl; R³ is a stable moiety independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl or aryl; R⁵ is optionally substituted aryl, a 5 to 14 membered aromatic monocyclic or fused polycyclic moiety that includes from 1 to 4 heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen.

The solid-supported intermediate of Formula (2) is prepared by reacting a solid-supported enone with aldehyde. As used herein and in the appended claims the term "solid supported-enone" means a solid support that at least has one enone moiety chemically attached thereto. For example, one embodiment of this compound may be represented by the Formula (3) :

wherein g is a solid-support, such as defined above; b is a single bond, -0-, -0C(0)-, -NR⁶-, -S-, -S0₂NHC(0)-, -NC(0)R⁶- or -NS(0)₂R⁶- where R⁶ is hydrogen, alkyl, aryl, alkenyl or alkynyl; f is optionally substituted arylene, a 5 to 14 membered aromatic monocyclic or fused polycyclic moiety that includes from 1 to 4 heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen; d is a single bond or optionally substituted alkylene;

R³ is a stable moiety independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl or aryl.

The preferred embodiment of reaction is comprising of, (a) reacting a compound of Formula (5) :

(wherein f is optionally substituted arylene, a 5 to 14 membered aromatic monocyclic or fused polycyclic moiety that includes from 1 to 4 heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen; R² is a stable moiety independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl or aryl; R³ is a stable moiety independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl or aryl) ;

with a compound of Formula (6) :

g^^"^ OH (6)

(wherein g is a solid-support) ; to form a corresponding intermediate of Formula (7) :

(wherein g, f, R² and R³ is as defined above) ;

(b) oxidizing and coupling the resulting solid-supported material (3-1) :

(wherein g, f, R² and R³ is as defined above) ;

with aldehyde of Formula (8) :

O

H-^R^S (8) (wherein R⁵ is aryl) ; to form a intermediate 1, 4-diketone (2-1) :

(c) cleaving the intermediate from the solid-support;

(d) cyclizing the 1, 4-diketone to get a compound of Formula (1) .

Alternatively, the solid-supported intermediate of Formula (2) is prepared by reacting a solid-supported aldehyde with enone. As used herein and in the appended claims the term "solid supported-aldehyde" means a solid support that at least has one aldehyde moiety chemically attached thereto. For example, one embodiment of this compound may be represented by the Formula (4) :

O

H (4)

wherein g is a solid-support, such as defined above; b is a single bond, -0-, -OC(O)-, -NR"-, -S-, -S0₂NHC(0)-, -NC(0)R⁶- or -NS(0)₂R⁶- where R⁶ is hydrogen, alkyl, aryl, alkenyl or alkynyl; f is optionally substituted arylene; d is a single bond or optionally substituted alkylene;

It is understood that f may be substituted one to three times with a substituent selected from the group consisting of alkyl, alkenyl, alkynyl, halo, hydroxy, alkoxy, alkylthio, carbonyl, amino, sulfonyl, aryl and where the substituents of the moieties substituted can themselves be substituted with one to three further substituents, if desired.

The preferred embodiment of reaction is comprising of, (a) reacting a compound of Formula (9) :

0 O

_HA_fA OH (9)

(wherein f is optionally substituted arylene) ;

with a compound of Formula (6) :

^g""^ OH (6)

(wherein g is a solid-support) ; to form a corresponding intermediate of Formula (4-1) :

O O s- 0 X.X H (4-1)

(wherein g and f are as defined above) ;

(b) coupling the resulting solid-supported material (4-1) with enone of Formula (10):

(wherein R² and R³ are as defined above ; R⁵ is aryl ) ; to form a intermediate 1 , 4-diketone ( 2-1 ) :

(c) cleaving the intermediate from the solid-support;

(d) cyclizing the 1, 4-diketone to get a compound of Formula (1) .

The preferred embodiment of reaction of preparing for formula (1) is as below;

route A

5 route B

route A and B

O "^•solid-support Two alternative routes for the synthesis of the pyrrole derivatives are shown in Scheme 1. The (1-hydroxyallyl) aryl carboxylic acids (1) were attached to Wang polystyrene resin

(2) using DIC. Oxidation of the resulting resin bound alcohol

(3) by Dess-Martin reagent gave the enone (4) . Stetter reaction of the enone (4) with the aldehydes (5) on solid support successfully gave the 1, 4-diketones (6) (route A). The formylaryl carboxylic acids (7) were attached to Wang polystyrene resin in the same manner (route B) . Stetter reaction of the solid-supported aldehydes (8) with the eonones (9) also successfully gave the 1, 4-diketones (6). Treatment of the resin with TFA in CH₂C1₂ resulted in the cleavage of 1, 4-diketones

(10) bearing carboxylic acid. Cyclization of the 1, 4-diketones

(10) with ammonium acetate gave the desired pyrrole compounds

(11) . The isolation of the pyrroles (11) was easily performed by the filtration after trituration with water.

The reagents for attaching the organic molecule to the solid support, as well as the reagents to displace the compound from the solid support are well known in the art. For example, see B. A. Bunin, The Combinatorial Index, Academic Press (1998); D. Obrecht and J. M. Villalgordo, Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries, Elsevier Science Ltd (1998) . Cyclization of the 1, 4-diketone to get the pyrrole compound is also well known in the art. For example, see H. Yoshimura, K. Kikuchi, S. Hibi, K. Tagami, T.Satoh, T. Yamauchi, A. Ishibashi, K. Tai, T. Hida, N. Tokuhara, and M. Nagai, J. Med. Chem. 43, 2929-2937 (2000). The oxidation proceeds smoothly with Dess-Martin reagent in DMF at room temperature for from several minutes to several hours.

The coupling reaction of aldehyde with enone in the presence of thiazolium salts and base to get 1, 4-diketones is well known as Stetter Reaction. For example, see H. Stetter, Angew. Chem. Int. Ed. Engl. (1976), 15, 639-712. However, the application of Stetter reaction to the solid phase synthesis has not been reported.

The temperature for the coupling procedure may be between about 50°C and 100°C but is preferably between about 60°C and 80°C and is most preferably at about 65°C.

The solvents suitable for the coupling procedure include protic or aprotic solvents. A preferred solvent is DMF. The thiazolium salts suitable for the coupling procedure include 5- (2-hydroxyethyl) -4-methylthiazonium halide that has alkyl or arylalkyl substituents at 3 position. A preferred thiazolium salt is 3-benzyl-5- (2-hydroxyethyl) -4- ethylthiazolium chloride. The amount of the thiazolium salt suitable for the coupling procedure may vary, but is between about 0.1 equivalent to 10 equivalent but is preferably btween about 1 equivalent to 3 equivalent and most preferably is 2 equivalent.

The bases suitable for the coupling procedure include organic or inorganic bases. A preferred base is triethylamine. The amount of triethylamine is between about 0.3 equivalent to 5 equivalent but is preferably between about 1 equivalent to 3 equivalent and most preferably is 2 equivalent. The time for the coupling reaction may vary, but generally is about 10 minutes and 3 days but preferably between about 2 hours and 18 hours .

Combinatorial Synthesis and Process for preparing libraries of compounds of the Formula (1)

The process for making libraries of compounds of the Formula (1) of the invention may be carried out in any reaction vessel capable of holding the liquid reaction medium and having, preferably, inlet and outlet means. For small scale synthesis of multiple products, the process of the invention is preferably carried out in containers adapted for parallel array synthesis. With parallel array synthesis, individual reaction products are prepared in each of multiple reaction zones. The reaction zones are physically separated from one another in a reaction vessel.

A preferred parallel synthesis embodiment of the present invention is a diverse compound library in the form of a plurality of wellplates, each wellplate having wells containing a separate reaction product (library compound) . In such cases, the library compounds are conveniently identified by their wellplate number and "x" column and "y" row coordinates. The process of making the library of phenyl ether compounds may be conveniently carried out in a conventional wellplate apparatus . It is particularly advantageous to carry out the method of the invention in a standard wellplate apparatus such as a plastic 96 well microtiter plate, or FLEXCHEM ™ 96 well Synthesis Assembly available from Robbins Scientific. Typically, the wellplate apparatus is in the form of a rigid or semi- rigid plate. Preferably the plate has a common surface containing openings of a plurality of reservoirs arranged in rows and columns. A standard form of wellplate apparatus is a rectangular plastic plate having 8 rows and 12 columns (total 96) of liquid retaining depressions, or reservoirs, on its surface. A wellplate apparatus may optionally have other elements or structure such as a top or cover (e.g., plastic or foil) , a bottom in a form such as a plate or reservoir, clamping means to secure the wellplate and prevent loss of its contained compounds .

The amount of solid bound compound introduced into such reaction zone will depend on the desired amount of each library compound that is needed for conducting biological assays, archival storage and other related needs. Typically desired amount of individual reaction product is from 1 microgram to 50 milligrams .

The amount of solid bound compound in each reaction zone is represented by the symbol "(n)", where (n) represents the equivalents of compound. Typically, from about 8 to about 800 diverse coupling reagents are employed serially to synthesize a library of compound. Combinatorial techniques are preferably very robust to work well for highly diverse groups of reactants. In the diverse compound library making process described herein the reactant is used in excess. The method of the invention contemplates solution phase reactions where a stoichiometric excess of the coupling reactant is used. The amount of coupling reactant used to ensure an excess is defined as at least 1.1 (n) and preferably a larger excess in the range of from 1.25 (n) to 5 (n) , where the variable (n) is as previously defined. The 1.1 multiplier is used to ensure at least a 10 % stoichiometric excess of coupling agent is present to drive the reaction to completion. The reaction zone is maintained at a temperature and for a time sufficient to permit reaction of solid bound compound with the coupling reactant, that is, to complete consumption of the solid bound compound and form an amount of compound necessary to conduct biological assays to determine the efficacy of the prepared library compounds.

The time, temperature, and pressure of the combinatorial reaction zones used for the creation of library compounds are not critical aspects of the invention. Reaction times for a single step of the reaction are generally from about 0.1 seconds to about 24 hours. The temperature of the reaction may be any temperature between the freezing point and the boiling point of the liquid reaction medium, but is generally between about -10°C and about 80°C, with 10°C to 40°C being preferred and ambient temperatures (about 20°C-30°C) being most preferred. The reactions may be conducted at subatmospheric pressure or superatmospheric pressure, but ambient atmospheric pressure (about 101325 Pa) is most often used.

Attaching the compounds to the solid support, cleaving the compounds from the solid support, and cyclization may be monitored by a number of conventional techniques, including, but not limited to, chromatography (preferably, thin layer chromatography) . Oxidation and coupling is described above. The library intermediate dissolved in the solvent phase of the cleavage reaction may be used for the next reaction after drying without purification.

The purification of the library compound dissolved in the solvent phase of the reaction may be done by any conventional chemical or physical method. Preferred are physical methods which are applicable to all members of a diverse library. Such methods include, for example: (i) ion exchange chromatography (ii) filtration, (iii) centrifugation, (iv) decantation, and (v) washing, and combination thereof. Filtration and ion exchange chromatography are particularly preferred forms of purification.

The last purification step of the process may optionally be supplemented by a solvent removal step in which the solute library compound is removed from its solvent by conventional processes known in the art; such as solvent evaporation, distillation, salting out, solvent extraction, and etc.

Screening of Libraries

The libraries of compounds of Formula (1) according to the present invention may be screened for biological activity. Generally, the library to be screened is exposed to a biological substance, usually a protein such as a receptor, enzyme, membrane binding protein or antibodies wherein the presence or absence of an interaction between the heterocycle derivative and the biological substance is determined. Typically this will comprise determining whether the biological substance is bound to one or more of the members of the library. Such binding may be determined by attaching a label to the biological substance. Commonly used labels include fluorescent labels. Other methods of labeling may be used, such as radio active labels. The degree of binding affinity may be determined by quantitating the amount or intensity of the bound label. Thus, various biologically active compounds may be selected by identifying which compounds bind the particular biological substance most effectively. Illustrative additional assays include, but are not limited to, in vitro assays such as enzymatic inhibition, receptor - ligand binding, protein - protein interaction, and protein - DNA interaction; cell based, functional assays such as transcriptional regulation, signal transduction / second messenger, and viral infectivity; add, incubate & read assays such as scintillation proximity assays (SPA) , fluorescence polarization assay, fluorescence correlation spectroscopy, colorimetric biosensors, cellular reporter assays using reporter genes such as luciferase, green fluorescent protein, 0 -lactamase, and the like; and electrical cell impedance sensor assays .

All of the above assays are known in the art to be predictive of success for an associated disease state.

Abbreviations used herein

ATRA = all trans retinoic acid CH₂C1₂ = dichloromethane DIC = diisopropylcarbodiimide

DMAP = Dimethylaminopyridine DMF = dimethylformamide DMSO = dimethylsulfoxide Et₃N = triethylamine

HTS = high throughout screening MeOH = methanol NH₄OAc = ammonium acetate NMR = Nuclear Magnetic Resonance TFA = trifluoroacetic acid

The following Examples are provided to further describe the invention and are not to be construed as limitations thereof.

EXAMPLES OF COMPOUNDS

The following compounds can be synthesized and included in the combinatorial library in accordance with the present invention.

37

38

39

Preparation of pyrrole compounds

The preparation of various pyrrole compounds in accordance with the present invention is exemplified below.

Stetter reaction successfully proceeded both for solid- supported aldehydes and for solid-supported enones . Aryl moieties are shown in Figure 1. Not all the combinations were prepared. The compounds actually prepared are listed in Table 1. The purity of final pyrrole compounds was confirmed by '^"H NMR spectra and was more than 80 %. We assumed that it was high enough for the biological assay. No further purification was done. The compounds were evaluated for their inhibition of LPS-induced mouse B-lymphocyte proliferation in the method reported by Apfel, and the results are also summarized in Table 1. All these compounds showed weaker inhibitory effect than compounds without any substituens on benzoic acid (data not shown) . The introduction of the small substituents at meta position on benzoic acid resulted in 10- to 200-fold decrease in the activity. The substitution of para-benzoic acid to thenoic acid still showed weak activity; however, the substitution of para-benzoic acid to meta-benzoic acid, naphthoic acid, and furoic acid lost the activity. 2,5-Di- substituted benzoic acid also lost the activity. In conclusion, we have developed the construction of 2,5- disubstituted-pyrrole library. We found Stetter reaction could be applied to the solid supported enones and/or aldehydes. Some compounds synthesized inhibited LPS-induced mouse B- lymphocyte proliferation. The present method opens an efficient way to develop SAR analysis of RAR agonists as well as the useful route of C-C bond formation on solid-phase synthesis.

Table 1. LPS-induced Mouse B-Cell Proliferation Inhibition.

a-p __c e-v 31 a-q _c e-w 45^d a-r _c f-r 23 a-s _c f-v 300 a-u _c f-w 240 a-aa _c g-r 15 b-o c g-v 95 b-p c g-w 64 b-r c h-r 2100 b-s c h-y __C b-t c i-o 230 b-x _c i-p _C b-z _c i-r 42 b-aa _c i-s c-m _^_c i-t c c-n __c i-w 62 c-o __c i-x 600 c-p _c i-z 480 c-q _c j-o c-r _c _c j-p c-s _c c j-r c-t c c j-s d-r 47 c j-t d-v 170 k-o^e 2.2 (± 0.5)^e d-w 210 k-r^e 0.76 (± 0.28)^e e-1 1500 k-w^e 1.3 (± 0.4)^e e-o 42 ATRA 1.0 e-p 140 ATRA^f O. i δ5±0.10^f e-r 18

^aSee Figure 1 for identities of partial structures.

""Relative IC₅₀ = IC₅₀/ATRA IC₅₀. ^c- = not detectable (relative IC₅₀ > 1000) .

"Values are means of two experiments. ^eReported in reference 12. (c) . ^fMeans of ATRA IC₅₀ (nM) ± SEM.

The preparation of various pyrrole compounds in accordance with the present invention is exemplified below for compounds 28 and 30.

Synthesis of compound 28

O ^{• • •}solid-support

To p-benzyloxybenzyl alcohol polystyrene (Wang) resin (600 mg, 0.63 mmol/g, 0.378 mmol) in DMF (12 ml) was added 3-bromo- 4- ( l-hydroxy-2-propenyl) benzoic acid (640 mg, 2.49 mmol), DIC (32 mg, 2.54 mmol) and DMAP (32 mg, 0.262 mmol) . After standing for 14 days at r.t., the resin was rinsed with 3 x 15 ml of DMF and 3 x 15 ml of CH₂C1₂. The resin was dried in vacuum overnight. The resin (307 mg) was then suspended in DMF (16 ml) followed by addition of Dess-Martin reagent (330 mg, 0.778 mmol) . After standing for 3.5 h at r.t., the resin was rinsed with 5 x 20 ml of DMF and 5 x 20 ml of CH₂C1₂. The resin was dried in vacuum overnight. The resin (100 mg) was then suspended in DMF (4 ml) followed by addition of 4, 5, 7-trimethylbenzofuran-2- carboxaldehyde (36 mg, 0.19 mmol), 3-benzyl-5- (2- hydroxyethyl) -4-methylthiazolium chloride (34 mg, 0.13 mmol), and Et₃N (0.017 ml, 0.12 mmol). After standing for 3 h at 65 °C, the reaction vessel was allowed to cool to r.t. And the resin was rinsed with 3 x 6 ml of DMF and 3 x 6 ml of CH₂C1₂. The resin was then suspended in 50% TFA/CH₂C1₂ (3 ml) for 2 h. The TFA solution was collected, concentrated by nitrogen stream and dried in vacuo . The residue was dissolved in MeOH (3 ml) followed by addition of NH₄OAc (100 mg, 1.3 mmol) . After stirring for 15 h at 70 °C, the reaction mixture was allowed to cool to r.t. Followed by addition of water (3 ml) . The precipitate was filtered, washed with water (3 x 5 ml) and dried in vacuo to give the substantially pure product 28 (6.3 mg) . 1 H-NMR (DMSO-d6., 400 MHz). Delta.; 2.25 (s, 3H) , 2.33 (s, 3H) , 2.42 (s, 3H) , 6.71 (d, IH, J = 4 Hz), 6.84 (d, IH, J = 4 Hz), 6.86 (s, IH) , 7.19 (s, IH) , 7.77 (d, IH, J=8 Hz), 7.96 (dd, IH, J=8 Hz, 2 Hz), 8.17 (d, IH, J = 2 Hz), 11.84 (br. s, IH) .

Synthesis of compound 30

O ^{• • •}solid-support

p-benzyloxybenzyl alcohol polystyrene (Wang) resin (75mg, 0.63 mmol/g, 0.047 mmol) was rinsed with 1.5 ml of DMF and filtered. To the resin was added DMF (0.5ml) and 3-bromo-4-formylbenzoic acid (0.5 ml, 0.28 M in DMF, 0.14 mmol), DIC (0.5 ml, 0.28 M in DMF, 0.14 mmol), and DMAP (0.5 l, 0.28 Min DMF, 0.14 mmol). After mixing for 16 h on ACT496 at 500 r.p.m.s at r.t., the resin was rinsed with 3 x 1.5 ml of DMF and 2 x 1.5 ml of CH₂C1₂. The resin was dried by nitrogen stream. The resin was rinsed with 2 x 1 ml of DMF, followed by addition of 3-benzyl-5- (2- hydroxyethyl) -4-methylthiazolium chloride (1ml, 0.093 Min DMF, 0.093 mmol), Et₃N (0.1 ml, 0.94 M in DMF, 0.094 mmol), and 4, 7-dimethylbenzofuran-2-yl vinyl ketone (0.5ml, 0.28MinDMF, 0.14 mmol) . After mixing for 4 h at 65 °C on ACT496 at 500 r.p.m.s, the reaction vessel was allowed to cool to r.t. and the resin was rinsed with 3 x 1.5 ml of DMF and 2 x 1.5 ml of CH₂C1₂. The resin was then suspended in 50% TFA/CH₂C1₂ (1 ml) and mixed for 1.5 h on ACT496 at 300 r.p.m.s at r.t. The TFA solution was collected and the resin was rinsed with 0.5 ml of CH₂C1₂. These washings were combined and concentrated in vacuo. The residue was dissolved in MeOH (2.5 ml) followed by addition of NH₄OAc (100 mg, 1.3 mmol) . After refluxing for 35 h, the reaction mixture was allowed to cool to r. t . followed by addition of water (4 ml) . The precipitate was filtered, washed with water (3 x 5 ml) and dried in vacuo to give the substantially pure product 30 (8.0 mg). 1 H-NMR (DMSO-d6., 400 MHz) . delta.; 2.43 (s, 3H) , 2.46 (s, 3H) , 6.73 (dd, IH, J = 2 Hz, J = 4 Hz) , 6.82 (dd, IH, J = 2 Hz, J = 4 Hz) , 6.92 (d, IH, J = 8 Hz), 6.96 (d, IH, J = 8 Hz), 7.20 (s, IH) , 7.75 (d, IH, J=8 Hz), 7.95 (dd, IH, J=8 Hz, 2 Hz), 8.17 (d, IH, J = 2 Hz), 11.86(br. s, IH) .

Methods for determining retinoic acid receptor agonist activity

The ability of a pyrrole compound of the present invention to be an effective retinoic acid receptor agonist is evaluated in the following assay.

Mouse splenocyte proliferation assay induced by E. coli lipopolysaccharide (LPS) , a well-known murine B-lymphocyte proliferation inducer, is performed according to the method previously reported with slight modifications. See C. Apfel, F. Bauer, M. Crettaz, L. Forni, M. Kamber, F. Kaufmann, P. LeMotte, W. Pirson, andM. A. Klaus, Proc. Natl. Acad. Sci. U.S.A.

1992, 89, 7129-7133. Compounds are tested in log dilutions of 1.0 x 10^"6 to 1.0 x 10^"12 M. BALB/c spleen cell suspensions are placed at 1.2 x 10⁵ cells/180 μL/well in 96-well culture plates, in RPMI-1640 medium containing antibiotics, 50 μM 2- mercaptoethanol, 10% FBS and 5 μg/mL LPS. Then 20 μL of each compound serially diluted is added to the wells and the plates are incubated for 3 days at 37 °C, 5% C0₂. Splenocytes are pulsed with 20 μL of 0.5 μCi of [³H] thymidine for further 6h. After incubation, cells are harvested on glass fiber filters and processed for ]3 -plate counting. The 50% inhibitory concentration (IC₅₀) values are calculated with nonlinear regression method. The inhibition of each compound is indicated as relative IC₅₀, where the IC₅₀ value of the compound is divided by that of ATRA.

It should be understood that a wide range of changes and modifications can be made to the embodiments described above. It is therefore intended that the foregoing description illustrates rather than limits this invention, and that it is the appended claims, including all equivalents, which define this invention.

CONDITIONS SUITABLE FOR THE COUPLING REACTION

Suitable conditions for this reaction include having a suitable solvent mixture with a base and a thiazolium salt, a suitable temperature and reacting for a suitable period of time.

A suitable solvent mixture is preferably of basic pH. Preferably the base is organic base, more preferably base is TEA or sodium acetate and most preferably the base is TEA. A suitable solvent is either protic or aprotic and preferably the solvent is DMF. A suitable thiazolium salt is 5- (2-hydroxyethyl) -4- methylthiazoniu halide that has alkyl or arylalkyl substituents at 3 position, preferably the thiazolium salt is 3-benzyl-5- (2-hydroxyethyl) -4-methyl-l, 3-thiazolium chloride or 3-ethyl-5- (2-hydroxyrthyl) -4-methyl-l, 3- thiazolium bromide and most preferably the thiazolium salt is 3-benzyl-5- (2-hydroxyethyl) -4-methyl-l, 3-thiazolium chloride . The reaction temperature is preferably between about 50°C and 100°C but more preferably between about 60°C and 80°C and most preferably at about 65°C.

The reaction time is preferably between about 10 minutes and 3 days, more preferably between about 2 hours and 18 hours. Under the above discussed preferred conditions the compounds (3) and (8), alternatively, compounds (4) and (10) react forming an intermediate (2) wherein g, b, f, d, R², R³ and R⁵ are as defined above .

0 O (2!

R⁵

R² R³

O O

R⁵

(2)

R² R³

This intermediate (2) is optionally chemically derivatized prior to the displacement and cyclization procedure to form corresponding intermediate (2'):

wherein the f' , R²', R³' and R⁵' substituents are each independently the same as substituents f, R², R³ and R⁵ respectively if not derivatized or are each independently the chemically derivatized substituents respectively. Examples of chemical derivatizationreactions include, but are not limited to, the following general derivatizations procedures .

Chemical reaction conditions suitable for BOC (t- butoxycarbonyl) removal/acylation, TMS (trimethylsilyl) or other silicone based alcohol-protecting group removal (F^~

/aqueous acid) , Mitsunobu reaction (0. Mitsunobu, Synthesis 1981, 1-28) , Suzuki coupling (N. Miyaura, A Suzuki, Chem. Commun. 1979, 866), Horner-Emmons type olefinations (W. S. Wadsworth, Jr., W. D. Emmons, J. Chem. Soc. 83, 1733 (1961)), reductive aminations (Klyeuv and Khidekel, Russ. Chem. Rev. 49, 14-27 (1980)), Sonogashira coupling (S. I. Kahn, M. W. Grinstaff, Tetrahedron Lett. 39, 8031 (1988)), and ester hydrolysis/amidation reaction condition.

After the optional derivatization of the intermediate, the displacement reaction whereby the intermediate (11) is formed.

o 0 0

R²' R^3*

In intermediate (11), f' , R² ' , R³ ' andR⁵' substituents are each independently the same as substituents f, R², R³ and R⁵ respectively if not derivatized or are each independently the chemically derivatized substituents respectively. Hence, each of f' , R²', R³' and R⁵' may be correspondingly the same as f, R², R³ and R⁵ or may represent the result of the optional chemical derivatization of the corresponding substituent, prior to displacement . It is understood from the synthetic routes that either R² ' or R³' is hydrogen or that both of R² ' and R³ ' are hydrogen.

CONDITIONS SUITABLE FOR THE DISPLACEMENT PROCEDURE The temperature for the displacement procedure may be between about 0°C and 60°C and is preferably at about 20°C. The solvents suitable for the displacement procedure include protic and aprotic solvent mixtures, aqueous and anhydrous solvent mixtures. A preferred solvent is TFA, a more preferred solvent mixture is TFA:CH₂C1₂, the most preferred solvent mixture is about 50 % mixture of TFA and CH₂C1₂. The time for the displacement reaction may vary, but generally is between about 5 minutes and 2 days but preferably between about 1 hour and 4 hours .

After the displacement reaction the intermediate (11) reacts with or without amine (12) or¹ sulfurization reagent in the presence of acid;

R⁷-NH2 (12)

where R⁷ is hydrogen, alkyl, alkenyl, alkynyl, carbonyl or aryl; to form the product (13) :

In product (13), the f' , R²', R³' andR⁵' are each independently the same as substituents f, R², R³ and R⁵ respectively if not derivatized or are each independently the chemically derivatized substituents respectively. Hence, each of f' , R ' , R³' and R⁵' may be correspondingly the same as f, R², R³ and R⁵ or may represent the result of the optional chemical derivatization of the corresponding substituent, prior to displacement and cyclization. It is understood from the synthetic routes that either R² or R³ is hydrogen or that both of R² and R³ are hydrogen.

CONDITIONS SUITABLE FOR THE CYCLIZATION PROCEDURE

The temperature for the cyclization procedure may be between about 60°C and 100°C and is preferably at about 65°C. The solvents suitable for the cyclization procedure include protic and aprotic solvent mixtures, aqueous and anhydrous solvent mixtures. A preferred solvent is alcohol, a more preferred solvent is methanol or ethanol, the most preferred solvent is methanol.

The time for the displacement reaction may vary, but generally is between about 10 minutes and 4 days but preferably between about 10 hour and 40 hours.

Optionally, the intermediate (2) can be derivatized before the displacement and cyclization procedure to thereby provide further options for the groups in the final product. The preferred, yet optional, derivatizations of the intermediate compound (2), and preferred conditions whereby optional derivatizations occur are as described above.

PARALLEL SYNTHESIS The solid supported mediated method of the invention may be carried out by way of parallel synthesis in any reaction vessel capable of holding the liquid reaction medium and having, preferably, inlet and outlet means.

For small-scale synthesis of multiple products, the solid support mediated method of the invention is preferably carried out in containers adaptable to parallel array syntheses. With parallel array synthesis, individual reaction products are prepared in each of multiple reaction zones. The reaction zones are physically separated from one another in a reaction vessel. Compounds can be added to the reaction vessel by multiple delivery apparatus, automated or robotic apparatus, any of that may be either manually or computer controlled. A preferred parallel synthesis embodiment of the present invention is a diverse heterocycle compound library in the form of a plurality of wellplates, each wellplate having wells containing a separate reaction product (library compound) . In such cases, their wellplate number and "x" column and "y" wellplate row coordinates conveniently identify the library compounds The process of making the library of heterocycle compounds may be conveniently carried out in a conventional wellplate apparatus. It is particularly advantageous to carry out the method of the invention in a standard wellplate apparatus such as a plastic 96 well microtiter plate.

Typically, the wellplate apparatus is in the form of a rigid or semi-rigid plate, the plate having a common surface containing openings of a plurality of reservoirs arranged in rows and columns. A standard form of wellplate apparatus is a rectangular plastic plate having 8 rows and 12 columns (total 96) of liquid retaining depressions, or reservoirs, on its surface. A wellplate apparatus may optionally have other elements of structure such as a top or cover (e.g., plastic or foil) , a bottom in a form such as a plate or reservoir, clamping means to secure the wellplate and prevent loss of its contained compounds .

The heterocycle library of compounds formed using the solid support mediated method aspects of the invention can be used to screen compounds for biological or other activity. Myriad biological assays are know in the art and can be used to screen the heterocycle library of compounds.

SCREENING OF HETEROCYCLE DERIVATIVE LIBRARIES

The libraries of plurality of compounds according to the solid support mediated method of the present invention (e.g., compounds (1)) may be screened for biological activity. Generally the library to be screened is exposed to a biological substance, usually a protein such as a receptor, enzyme, membrane binding protein or antibody, and the presence or absence of an interaction between the heterocycle derivative and the biological substance is determined. Typically this will comprise determining whether the biological substance is bound to one or more of the members of the library. Such binding may be determined by attaching a label to the biological substance. Commonly used labels include fluorescent labels. Other methods of labeling may be used, such as radioactive labels. The degree of binding affinity may be determined by quantitating the amount or intensity of the bound label. Thus, various biologically active compounds may be selected by identifying that compounds bind the particular biological substance most effectively. Illustrative additional assays include but are not limited to in vitro assays such as enzymatic inhibition, receptor - ligand binding, protein - protein interaction, and protein - DNA interaction; cell based, functional assays such as transcriptional regulation, signal transduction / second messenger, and viral infectivity; add, incubate and read assays such as scintillation proximity assays (SPA) , fluorescence polarization assay, fluorescence correlation spectroscopy, colorimetric biosensors, cellular reporter assays using reporter genes such as luciferase, green fluorescent protein, b-lactamase, and the like; and electrical cell impedance sensor assays. All of the above assays are known in the art to be predictive of success for an associated disease state.

Reagents obtained from commercial sources were used without further purification. Wang resin 1 was purchased from Novabiochem with a loading capacity of 0.63 mmol/g. All NMR spectra (400 MHz) were recorded on a Varian Unity 400 spectrometer.

Claims

1. A combinatorial library containing a plurality of compounds of Formula (1) :

wherein X is 0, S, or NR⁷;

2. The library according to claim 1, wherein the library contains 2 to 1000000 compounds of Formula (1) of claim 1.

3. The library according to claim 1, wherein the library contains 20 to 1000 compounds of Formula (1) of claim 1.

4. The library according to claim 1, wherein Z is optionally substituted 9-, 10- or 13- membered fused bicyclic or tricyclic aromatic heterocyclic group having 1 or 2 heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen;

Y is optionally substituted phenyl, optionally substituted naphthyl, optionally substituted 5- or 6- membered aromatic heterocyclic ring having 1 or 2 heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen, or optionally substituted 9- or 10- membered fused bicyclic aromatic heterocyclic group having 1 or 2 heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen.

5. The library according to claim 1, wherein X is NH.

6. The library according to claim 1, wherein the library contains 2 to 1000000 pyrrole compounds of Formula (1) of claim

1.

7. The library according to claim 1, wherein the library contains 20 to 1000 pyrrole compounds of Formula (1) of claim 1.

8. The library according to claim 1, wherein the library is a RAR (retinoic acid receptor) α agonizing library.

9. A process for preparing a combinatorial library containing a plurality of heterocyclic compounds; comprising using a solid-supported intermediate of Formula (2) :

R² is a stable moiety independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl or aryl; R³ is a stable moiety independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl or aryl;

10. The combinatorial library prepared by the process of claim 9.

11. A solid-supported intermediate of Formula (2):

wherein g is a solid-support; b is a single bond, -0-, -OC(O)-, -NR⁶-, -S-, -S0₂NHC(0)-,

12. A process for preparing the solid-supported intermediate of Formula (2) of claim 11, comprising reacting a solid- supported enone with aldehyde.

13. The process according to claim 12; wherein a solid- supported enone is represented by Formula (3) :

R² is a stable moiety independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl or aryl; R³ is a stable moiety independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl or aryl.

14. A process for preparing the solid-supported intermediate of Formula (2) of claim 11, comprising reacting a solid- supported aldehyde with enone.

15. The process according to claim 14; wherein a solid- supported aldehyde is represented by Formula (4) :

O g / H (4)

wherein g is a solid-support; b is a single bond, -0-, -OC(O)-, -NR⁶-, -S-, -S0₂NHC(0)-, -NC(0)R⁶- or -NS(0)₂R⁶- where R⁶ is hydrogen, alkyl, aryl, alkenyl or alkynyl; f is optionally substituted arylene, a 5 to 14 membered aromatic monocyclic or fused polycyclic moiety that includes from 1 to 4 heteroatoms which are the same or different and which are selected from sulfur, oxygen and nitrogen; d is a single bond or optionally substituted alkylene.

16. A reaction vessel, on which a plurality of compounds of Formula (2) of claim 11 are physically separated from each other.

17. An assay kit for identifying of pharmaceutical lead compounds, comprising biological assay materials and a wellplate apparatus; wherein the biological assay materials contain a plurality of compounds of Formula (1) of claim 1 which are separated from each other.

18. The assay kit according to claim 17, wherein said biological assay materials is for performing assay tests selected from the group consisting of in vitro assays, cell based functional assays and, incubate and read assays.

19. An assay kit for identifying of compounds which has RAR a agonizing activity, comprising biological assay materials and a wellplate apparatus; wherein the biological assay materials contain a plurality of compounds of Formula (1) of claim 1 which are separated from each other.

20. An apparatus suitable as a replacement element in an automated assay machine as a source of the combinatorial library of compounds, containing a 2-dimensional array of defined reservoirs; wherein each reservoir contains a plurality of compounds of Formula (1) of claim 1.

21. A process for agonizing RAR ; comprising administering a compound of formula (1) of claim 1 or a pharmaceutically acceptable salt thereof to a mammal in need of treatment.