COMBINATORIAL SYNTHESIS AND HIGH-THROUGHPUT SCREENING OF A REV-INHIBITING ARYLIDENEDIAMIDE ARRAY

Abstract: This invention relates to certain novel chemical compounds which inhibit the binding of a Rev protein to a Rev response element. These compounds reduce the binding activity of the protein to the response element by at least about 50 % and preferably 90 % or more. Another embodiment relates to a method for inhibiting the binding of a Rev protein to a Rev response element by administering one of these compounds to a subject in vitro.

Description: Combinatorial Synthesis and High-Throughput Screening of a Rev-Inhibiting Arylidenediamide Array Background of the Invention The discovery of new molecules has traditionally focused in two broad areas, biologically active molecules, which are used as drugs for the treatment of life-threatening diseases, and new materials, which are used in commercial, especially high technological application. In both areas, the strategy used to discover new molecules has involved two basic operations: (i) a more or less random choice of a molecular candidate, prepared either via chemical synthesis or isolated from natural sources, and (ii) the testing of the molecular candidate for the property or properties of interest. The discovery cycle is repeated indefinitely until a molecule possessing the desirable properties is located. In the majority of cases, the molecular types chosen for testing have belonged to rather narrowly defined chemical classes. For example, the discovery of new peptide hormones has involved work with peptides; the discovery of new therapeutic steroids has involved work with the steroid nucleus; the discovery of new surfaces to be used in the construction of computer chips or sensors has involved work with inorganic materials, etc. (for example, see R. Hirschmann, Angew. Chem., Int. Ed. in Engl. 1991, 30, 1278-1301). As a result, the discovery of new functional molecules, being ad hoc in nature and relying predominantly on. serendipity, has been an extremely time-consuming, laborious, unpredictable, and costly enterprise. A brief account of the strategies and tactics used in the discovery of new molecules is described below. The emphasis is on biologically interesting molecules. However, as discussed below, the technical problems encountered in the discovery of molecules and in the development of fabricated materials which can serve as new materials for high technological applications still limit the utility of most of these methods. Modern theories of biological activity state that biological activities, and therefore physiological states, are the result of molecular recognition events. For example, nucleotides can form complementary base pairs so that complementary single-stranded molecules hybridize resulting in double- or triple-helical structures that appear to be involved in regulation of gene expression. In another example, a biologically active molecule, referred to as a ligand, binds with another molecule, usually a macromolecule referred to as ligand-acceptor (e.g. a receptor or an enzyme), and this binding elicits a chain of molecular events which ultimately gives rise to a physiological state, e.g. normal cell growth and differentiation, abnormal cell growth leading to carcinogenesis, blood- pressure regulation, nerve-impulse-generation and propagation, etc. The biding between ligand and ligand-acceptor is geometrically characteristic and extraordinarily specific, involving appropriate three-dimensional structural arrangements and chemical interactions. Design and Synthesis of Mimetics of Biological Ligands A currently favored strategy for development of agents which can be used to treat diseases involves the discovery of forms of ligands of biological receptors, enzymes, or related macromolecules, which mimic such ligands and either boost (i.e., agonize) or suppress (i.e. , antagonize) the activity of the ligand. The discovery of such desirable ligan forms has traditionally been carried out either by random screening of molecules (produced through chemical synthesis or isolated from natural sources, for example, see K. Nakanishi, Ada Pharm. Nord. , 1992, 4, 319-328.), or by using so-called "rational" approach involving identification of lead-structure, usually the structure of the native ligand, and optimization of its properties through numerous cycles of structural redesign and biological testing (for example see Testa, B. & Kier, L.B. Med. Res. Rev. 1991, 11, 35-48 and Rotstein, S.H. & Mureko, M.A., J. Med. Chem. 1993, 36, 1700-1710.). Since most useful drugs have been discovered not through the "rational" approach but through the screening of randomly chosen compounds, a hybrid approach to drug discovery has recently emerged which is based on the use of combinatorial chemistry to construct huge libraries of randomly-built chemical structures which are screened for specific biological activities. (Brenner, S. & Lerner, R.A. Proc. Natl. Acad. Sci. USA 1992, 89, 5381). Most lead-structures which have been used in "rational" drug design are native polypeptide ligands of receptors or enzymes. The majority of polypeptide ligands, especially the small ones, are relatively unstable in physiological conditions, due to the tendency of the peptide bond to undergo facile hydrolysis in acidic media or in the presence of peptidases. Thus, such ligands are decisively inferior in a pharmacokinetic sense to nonpeptidic compounds, and are not favored "as drugs. An additional limitation of small peptides as drugs is their low affinity for ligan acceptors. This phenomenon is in sharp contract to the affmity demonstrated by large, folded polypeptides, e.g., proteins, for specific acceptors, e.g., receptors or enzymes, which can be in the subnanomolar range. For peptides to become effective drugs, they must be transformed into nonpeptide organic structures, i.e., peptide mimetics, which bind tightly, preferably in the nanomolar range, and can withstand the chemical and biochemical rigors of coexistence with biological fluids. Despite numerous incremental advances in the art of peptidornimetic design, no general solution to the problem of converting a polypeptide-ligand structure to a peptidornimetic has been defined. At present, "rational" peptidornimetic design is done on an ad hoc basis. Using numerous redesign-synmesis-screening cycles, peptide ligands belonging to a certain biochemical class have been converted by groups of organic chemists and pharmacologists to specific peptidomimetics; however, in the majority of cases the results in one biochemical area, e.g., peptidase inhibitor design using the enzyme substrate as a lead, cannot be transferred for use in another area, e.g. , tyrosine- kinase inhibitor design using die kinase substrate as a lead. In many cases, the peptidomimetics that result from a peptide structural lead using the "rational" approach comprise unnatural -amino acids. Many of these mimetics exhibit several of the troublesome features of native peptides (which also comprise alpha-amino acids) and are, thus, not favored for use as drugs. Recently, fundamental research on the use of nonpeptidic scaffolds, such as steroidal or sugar structures, to anchor specific receptor-binding groups in fixed geometric relationships have been described (see for example Hirschmann, R., et al. J. Am. Chem. Soc. 1992, 114, 9699-9701; Hirschmann, R. et al., J. Am. Chem. Soc, 1992, 114 9217-9218); however, the success of this approach remains to be seen. In an attempt to accelerate the identification of lead-structures, and also the identification of useful drug candidates through screening of randomly chosen compounds, researchers have developed automated methods for the generation of large combinatorial libraries of peptides and certain types of peptide mimetics, called "peptoids", which are screened for a desirable biological activity (see Gordon, E.M. et al. J. Med. Chem. 1994, 37, 1385-1401). For example, the method of H.M. Geysen, (Bioorg. Med. Chem. Letters, 1993, 3, 397-404; Proc. Natl. Acad. Sci. USA 1984, 81, 3998) employs a modification of Merrifield peptide synthesis, wherein the C-terminal amino acid residues of the peptides to be synthesized are linked to solid-support particles shaped as polyethylene pins; these pins are treated individually or collectively in sequence to introduce additional amino-acid residues forming me desired peptides. The peptides are - 3 - then screened for activity without removing them from the pins. Houghton, (Proc. Natl. Acad. Sci. USA 1985, 82, 5131; Eichler, J. & Houghten, R.A. Biochemistry, 1993, 32, 11035-11041, and US. Patent No. 4,631,211) utilizes individual polyethylene bags ("tea bags") containing C-terminal amino acids bound to a solid support. These are mixed and coupled with the requisite amino acids using solid phase synthesis techniques. The peptides produced are then recovered and tested individually. S.P.A. Fodor et al., (Science 1991, 251, 767) described light-directed, spatially addressable parallel-peptide synthesis on a silicon wafer to generate large arrays of addressable peptides that can be directly tested for binding to biological targets. These workers have also developed recombinant DNA genetic engineering methods for expressing huge peptide libraries on the surface of phages (Cwirla et al. Proc. Natl. Acad. Sci. USA 1990, 87, 6378; Barbas, et al. Proc. Natl. Acad. Sci. USA 1991, 88, 7979-7982). In another combinatorial approach, V.D. Huebner and D.V. Santi (US. Patent No. 5,182,366) utilized functio alized polystyrene beads divided into portions each of which was acylated with a desired amino acid; the bead portions were mixed together, then divided into portions each of which was re-subjected to acylation with a second desirable amino acid producing dipeptides, using me techniques of solid phase peptide synthesis. By using this synthetic scheme, exponentially increasing numbers of peptides were produced in uniform amounts which were then separately screened for a biological activity of interest. Zuckermann and co workers (For examples, see Zuckermann, et al. J. Med. Chem. 1994, 37, 2678-2685 & Zuckermann, et al. Int. J. Peptide Protein Res. 1992, 91, 1) also have developed similar methods for the synthesis of peptide libraries and applied these methods to the automation of a modular synthetic chemistry for the production of libraries of N-alkyl glycine peptide derivatives, called "peptoids", which are screened for activity against a variety of biochemical targets. (See also, Symon et al., 1992, Proc. Natl. Acad. Sic. USA 89:9367). Encoded combinatorial chemical syntheses have been described recently (Brenner, S. & Lerner, R.A. Proc. Natl. Acad. Sci. USA 1992, 89, 5381; Barbas, C.F. et al. Proc. Natl. Acad. Sci. USA 1992, 89, -4457-4461; see also Borchardt, A. & Still, W.C. J. Am. Chem. Soc. 1994, 116, 373-374; Kerr, J. et al. J. Am. Chem. Soc. 1993, 115, 2529-2531). M.J. Kurth and his group (Chen, C. et al. J. Am. Chem. Soc. 1994, 116, 2661- 2662.) have applied organic synthetic strategies to develop non-peptide libraries synthesized using multi-step processes on a polymer support. Although the method demonstrates the utility of standard organic synthesis in the application and development of chemical libraries, the synthetic conditions are limited by compatibility with the solid support. Although some means of quantification is needed to confirm this inhibitory action, conventional techniques include gel mobility shift assays and the like. These techniques are generally usable, but a need exists for improved assays and assaying methods for obtaining new compounds which possess this action. Summary of the Invention The present invention relates to certain novel chemical compounds which inhibit the binding of a Rev protein to a Rev response element. These compounds reduce the binding activity of the protein to the response element by at least about 50% and preferably 90% or more. Advantageously, these compounds do not precipitate at a concentrations of up to 6 μg/ml. Another embodiment relates to a method for inhibiting the binding of a Rev protein to a Rev response element through the use of one of these compounds, which can be aα^inistered to a subject or in vitro. These compounds of interest are selected from an n x m x p array of compounds which have a common scaffold structure and particular diversity elements, as explained herein. Preferred values for d e integers of n, m and p are n >_ 5, m >_ 2 and p >_ 1 or 2, with the product of n, m and p being greater than 25, with the most preferred array including 10,240 compounds with n = 8, m = 12 and p = 40. The arrays form yet another embodiment of the invention. Further details regarding these arrays and memods of making the same can also be found in copending application serial no. 08/375,838 filed January 20, 1995, the entire content of which is expressly incorporated herein by reference diereto. Definitions This invention discloses a system for the design, syn esis & use of logically arranged collections of syndietic product molecules called "molecular constructs" from structural elements in such a manner that the collection of molecular constructs possesses a constant structural element and a variable structural element. The definitions are shown below. - 5 - SUBSTΓΓUTE SHEET (RULE 26) A "construct" is a molecule which is a member of a collection of molecules containing a common constant structural and a common variable structural element. An "array" is a logical positional ordering of molecular constructs in Cartesian coordinates. A "bond" or "chemical bond" is used to describe a group of electrons that is shared between two atoms. This term also denotes an ionic, covalent or other attractive force between two atoms. A "building block" is any molecule useful in the assembly of a molecular construct. The terms "fragment" or "structural diversity element" refer to the common variable structural element of a molecular construct. The "molecular core" is the common constant structural element of a molecular construct. A "spatial address" is a position in the array defined by unique Cartesian coordinates. A "sub-array" is a set of spatial addresses within a given array contøixiing those molecular constructs having a common molecular core and differ from each other by 0 (zero) or 1 (one) change in a fragment. A "relative address" refers to a location within the array or sub array comparable to any selected address, and differing by 0 (zero) or only 1 (one) change in the common variable structural element. Inhibition and High-Throughput Screening of HIV Proteins The human immunodeficiency virus (HIV) has been implicated as the causative agent of acquired immune deficiency syndrome (AIDS) (Popovic; et al, Science, 198, 1984 497). The RNA genomic of the HIV retrovirus encodes an aspartic protease known as the HIV-1 protease (Kramer, R.A.; et al., Science, 1986, 231, 1580). This protease is required for maturation and proliferation of the infectious virion. The role of the HIV-1 protease is to cleave viral precursor proteins, in particular the Gag and Pol precursor proteins, into their active forms (Darke, P.L.; et al., Biochem. Biophys. Res. Comm, 1988, 156, 297). To date, numerous inhibitors of HIV-1 protease have been reported in the literature (Wlodawer, A.; Erickson, J.W., Annu. Rev. Biochem. 1993, 62, 543). Additionally, the RNA genome of the HIV retrovirus encodes for two viral regulatory proteins, Tat and Rev. Tat is a transcriptional activator, and Rev is a post- transcriptional protein that binds to a specific sequence of ribonucleic aid, the Rev Response Element (RRE). This action raises the intracellular concentrations of gag-pol and env messenger RNA (mRNA), thereby perpetuating the viral replication cycle, (for a review of this material see Green, M.R. AIDS Res. Rev. 1993, 3, 41-55.) Research in the field has shown that introduction of analogous sequences of nucleic acids to the mRNA within the RRE show a decrease in the concentration of Rev protein (Matsukura, M.; et al. Proc. Natl. Acad. Sci. USA 1989, 86, 4244-4248). Another valuable insight was the discovery that small molecules can provide this inhibitory action. Aminoglycoside antibiotics inhibit protein synthesis at what is believed to be at the level of ribosomal RNA (reviewed by Cundliffe, E. in The Ribosome: Structure, Function and Evolution Hill, W.F.; et al., eds. (American Society for Microbiology, Washington, D.C.) 1990, pp 479-490) Furthermore it was found that aminoglycoside antibiotics block the binding of Rev to RRE, inhibit Rev function and viral production in vivo (Zapp, M.L.; Stern, S.; Green, M.R. Cell 1993, 75, 969-978). An "operator" is a simultaneous and/or concurrent change in the condition of at least two spatial addresses in individual cells residing in an array or a sub-array that results in a structural change at least one molecular construct in the array. In particular, an operator in terms of this invention can be the reaction of at least one site on the molecular core capable of becoming or providing attachment for a structural diversity element, to add or change a structural motif thereon. Oilier operators which can be performed according to the patent include but are not limited to: addition of reagents or solvents; quality control protocols such as gas chromatography, high performance liquid chromatography, mass spectrometry, infrared spectroscopy, ultraviolet spectroscopy, nuclear magnetic resonance spectroscopy, fluorescence spectroscopy, melting pint, mass balance, combustion analysis and thin layer chromatography; biological and enzymological assays such as ELISA, spectroscopic inhibition assays, disc assays and binding affinity assays; mechanical motions or manipulations; passage of time which includes resting & evaporation; heating and cooling; iteration of previous steps in a synthesis; dilution and dispensation of products in a form suitable for the design purpose. The protein herein referred to as "Rev" or "the Rev Protein" means an HIV regulatory protein that is critical for key steps in the life cycle of the Human Immunodeficiency Virus (HIV). Detailed Description of the Invention This invention pertains to the logical layout, construction and testing of an array of chemical compounds blocking the binding of Rev Protein to the Rev Response Element, in which the desired properties of the compound can be measured and correlated to specific ordered changes in the fragments used to construct them. The array is ordered in such a fashion as to expedite assembly, to maximize the informational content derived from the testing and to facilitate the rapid extraction of that data from the testing process. This array and the coupled high-throughput screening has great utility in accelerating the development of compounds having the optimal properties for the inhibition of HIV replication. An application of this invention is the rapid determination and optimization of candidates for blocking the binding of Rev to RRE. The array is screened and the optimal candidates are chosen. Thus in one light the invention is the most powerful tool to date for the rapid screening and testing of compounds for IND candidacy against this target. This method is facilitated by virtue of selecting fragments based solely upon their ability to participate in the process of assembly into the arylidenediamide core and then the entirety of the library is screened for inhibition of the Rev-RRE binding. The logically arranged array of me present invention is fundamentally different from all known prior art. Testing of these arrays automatically results in the generation of complete relational structural information such that a positive result provides: (1) information on a compound within any given spatial address; (2) simultaneous juxtaposition of this information upon a set of systematically structural congeners; (3) the ability to extract relational structural information from negative results in the presence of positive results. Thus by analyzing the percent inhibition and correlating this information to the known structural components at each address, a valid Structure- Activity Relationship (SAR) is rapidly established. All known prior art is universally directed toward the maximization of structural diversity. By definition tiiis has excluded the acquisition of maximal data. In tiiese cases, the relationship between individual structural variations and any resulting changes in a measurable property of the compounds can not be directly obtained from these testing results. Methods in the prior art universally required extensive further experimentation to elucidate any relational information in a process which is costly, time consuming and one in which success is difficult to predict. This array is constructed from the arylidenediamide core shown below. The core candidate was selected so that the scaffold a) presents attachment points for at least two structural diversity elements; b) is able to present these structural diversity elements in controlled, varying spatial arrangements; c) can be constructed in a rapid concerted fashion. The array may be constructed using the following synthetic method: The substituents A, B, & C on the molecular core are structural diversity elements which may be the same or different and each represents straight or branched chain alkyl, carbocyclic aryl and substituted or heterocyclic derivatives thereof. As used herein, the phrase linear chain or branched chained alkyl groups means any substituted or unsubstituted acyclic carbon-containing compounds, including alkanes, alkenes and alkynes. Alkyl groups having up to 30 carbon atoms are preferred. Examples of alkyl groups include lower alkyl, for example, methyl, ethyl, n-propyl, iso- propyl, n-butyl, iso-butyl or tert-butyl; upper alkyl, for example, octyl, nonyl, decyl, and the like; lower alkylene, for example, ethylene, propylene, propyldiene, butylene, butyldiene; upper alkenyl such as 1-decene, 1-nonene, 2,6-dimethyl-5-octenyl, 6-ethyl-5- octenyl or heptenyl, and the like; alkynyl such as 1-ethynyl, 2-butynyl, 1-pentynyl and the like. The ordinary skilled artisan is familiar with numerous linear and branched alkyl groups, which are within the scope of the present invention. In addition, such alkyl group may also contain various substituents in which one or more hydrogen atoms has been replaced by a functional group. Functional groups include but are not limited to tertiary amine, amide, ester, ether, and halogen (fluorine, chlorine, bromine and iodine), to mention but a few. Specific substituted alkyl groups can be, for example, alkoxy such as methoxy, ethoxy, butoxy, pentoxy and d e like, dimethylamino, diethylamino, cyclopentylmethylamino, benzylmethylamino, dibenzylamino, and me like; formamido, acctamido, butanamido, and me like, methoxycarbonyl, ethoxycarbonyl or the like, or dimethyl or die yl etiier groups or the like. As used herein, substituted and unsubstituted carbocyclic groups of up to about 20 carbon atoms means cyclic carbon-containing compounds, including but not limited to cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and the like. Such cyclic groups may also contain various substituents in which one or more hydrogen atoms has been replaced by a functional group. Such functional groups include tiiose described above, and lower alkyl groups as described above. The cyclic groups of the invention may further comprise a hetero-atom. For example, in a specific embodiment, structural diversity element C is N,N-dimethyl aminoethyl. As used herein, substituted and unsubstituted aryl groups means a hydrocarbon ring bearing a system of conjugated double bonds, usually comprising (4p - 2) pi bond electrons, where p is an integer equal to or greater than 1. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anisyl, toluyl, xylenyl and the like. According to the present invention, aryl also includes aryloxy, aralkyl, aralkyloxy and heteroaryl groups, e.g., pyrimidine, morpholine, piperazine, piperidine, benzoic acid, toluene or thiophene and the like. These aryl groups may also be substimted with any number of a variety of functional groups. In addition to the functional groups described above in connection witii substimted alkyl groups and carbocyclic groups, functional groups on the aryl groups can be nitro groups. As mentioned above, structural diversity elements can also represent any combination of alkyl, carbocyclic or aryl groups; for example, 1-cyclohexylpropyl, benzylcyclohexylmethy, 2-cyclohexyl-propyl, 2,2-methylcyclohexylpropyl, 2,2- methylphenylpropyl, 2,2-methylphenylbutyl, and the like. The structural diversity element may also be a chemical bond or a connecting group that includes a terminal carbon atom for attachment to the quaternary nitrogen and may be different in adjacent n units. - 10 - In a preferred embodiment of the invention the structural diversity element "A" is derived from the set of 2-napthyl, α,c-,α-trifluoro p-toluyl,2,4-dichlorophenyl, and /?- toluyl. In another preferred embodiment of the invention, the structural diversity element "B" is derived from the set of c_,α,α,-tiifluoro-m-toluyl, α,α,α,-trifluoro-/7-toluyl, and 4-biphenylyl. In still anotiier preferred embodiment of the invention the structural diversity element "C" is derived from the set of 1,2,3,4-tetrahydronaphthyl, 2-(- /?-toluyl)ethyl, diphenylmethyl, 2,2-diphenyletiιyl, and (l-napthyl)methyl. In a highly preferred embodiment of the invention, these aforementioned sets are employed in combination such that the molecule of interest possesses structural diversity elements drawn from all three sets. The following experimental procedure is meant to exemplify but one embodiment of the present invention and are not intended to limit the invention thereto. Example A 10,240-component array is synthesized and assayed according to the teaching of the invention, from eight oxazolones (Building Block A), 32 aldehydes (Building Block B), and 40 amines (Building Block C). Tetrahydrofuran (THF) solutions of the building blocks are prepared to concentrations of 250 mM in "A" (oxazolone), 250 mM in "B" (aldehyde), and 500 mM in "C" (amine). Sufficient volumes of each solution are prepared to allow for the production of one row of reaction plates (Px, where x= 1-128 for AN 1001). A reaction plate contains 80 spatial addresses each (8X10) and a row contains 16 reaction plates. The entire array consists of 8 rows of those reaction plates which are recycled 16 at a time to complete production of the array. The initial cycle's first operator is spatial delivery of 200 μl (lcq., 50 μMoles) of the "A" building block solution starting at PI and ending at P16. The second operator is spatial delivery of 200 μl (leq., 50 μMoles) of the "B" Building Blocks to die same reaction plates. The third operator is addition to the same reaction plates of 50 μL of a 1 M (leq., 50 μMoles) solution of triethylamine in THF to all the spatial addresses that "A" and "B" building Blocks were added. The fourth operator is placement of the reaction blocks on *an agitator at 60 degrees centigrade for 1.5 hrs. The fifth operator is spatial addition of 100 μl (lcs., 50 μMoles) of the "C" building block solutions. The sixth operator is addition of 200 μL of THF to all die spatial addresses in the row or cycle. The seventh operator allows the reaction plates to stand at 25 degrees centigrade for 16 hrs. enabling evaporation of THF and completion of the synthesis of the molecular constructs. The following operators are then applied to distribute and reformat die molecular constructs for delivery and quality control. Heat the reaction plates to 60 degrees centigrade for 10 minutes and add 400 μl of dimediylsulfoxide (DMSO) to dissolve die molecular constructs (operator 8). Remove the solution from the reaction plates and place in a plastic microtiter plates in a spatial manner (operator 9). Spatially wash the reaction plates (each address) with 4 times 325 μL of DMSO and place in the same microtiter plates (operator 10). This affords 29.4 mM solutions of the molecular constructs in DMSO ready for further spatial distribution. Remove a 10 μL aliquot following a unique address pattern layout from each microtiter plate for quality control (operator 11). Spatially reformat these aliquots, dilute witii 300 μL of acetronitrile and subject these samples to analysis by High Performance Liquid Chromatography and Mass Spectrometry for quality control of the molecular constructs in each microtiter plate (operator 12). The above cycles and operators are repeated 7 more times to finish production and quality controlled validation of the array, AN 1001. The array was assayed in parallel at 6 μg/mL in reaction mixtures containing recombinant Rev protein, synthetic 32P-labelled RRE RNA, and a non-specific competitor RNA. The reaction is then filtered through nitrocellulose filters and washed with a low salt binding buffer. Rev-RRE complexes are retained in the filter while die unbound RRE filters through. Rev-RRE complexes retained in the filter are then quantified by scintillation counting. The percentage of inhibition is calculated according to die formula: %inh = (HCPM1/CPM0)) x 100 where CPM1 is the activity obtained in me presence of the drug and CPM0 is die average activity in the absence of the drug. Compounds of interest as potential Rev-RRE inhibitors are expected to cause at least 50% inhibition and not to precipitate at concentrations of 6 μg/mL or lower. Cursory examination of these assay results showed tiiat of 10,240 compounds, 752 of tiiese inhibited die binding of Rev to RRE at a value of greater than 50%. Raising the criterion of interest to 90% inhibition of complex formation afforded a set of 55 compounds which are examined for common functionality. These 55 compounds and tiieir inhibition values are listed in the following table, where A, B, and C are the structural diversity elements of the scaffold: - 12 - % inh A B C 98 2,4-dichlorophenyl phenyl cyclohexyl 97 2-napthyl α,α,α-trifluoro-m-toluyl 1 ,2,3 ,4-tetrahydronaphth- 1 -y 1 96 2-napthyl α,α,α-tτifluoro-m-toluyl diphenylmethyl 96 2,4-dichlorophenyl α,α,α-trifluoro-m-toluyl diphenylmethyl 96 2-napthyl α,α,α-trifluoro-m-toluyl 2-(ρ-toluyl)ethyl 96 2-napthyl α,α,α-trifluoro-p-toluyl diphenylmethyl 96 p-toluyl 2,4-difluorophenyl 2-hydroxyphenethyl 96 2-napthyl 3-quinolinyl ( 1 -napththyl)methyl 96 2-napthyl α,α,α-trifluoro-p-toluyl 4-phenylbut-2-yl 96 2,4-dichlorophenyl 4-biphenylyl diphenylmethyl 96 2-napthyl 4-(trifluoromethoxy)phenyl diphenylmethyl 95 2-napthyl α,α,α-tτifluoro-p-toluyl 2,2-diphenylethyl 95 2-napthyl α,α,α-trifluoro-p-toluyl 1 ,2,3 ,4-tetrahydronaphth- 1 -yl 95 2,4-dichlorophenyl 3-(phenoxy)phenyl 2-(p-toluyl)ethyl 95 p-toluyl 2-chlorophenyl 3-(4-morpholinyl)propyl 95 2-napthyl 2,4-dichlorophenyl 2-hydroxyphenethyl 95 2-napthyl α,α,α-trifluoro-m-toluyl 3-benzylpentamethylenyl 95 α,α,α-trifluoro- p-toluyl α,α,α-trifluoro-p-toluyl 2-(p-toluyl)ethyl 94 α,α,ct-trifluoro- ■p-toluyl α,α,α-trifluoro-p-toluyl 1 ,2,3 ,4-tetrahydronaphth- 1 -yl 94 α,α,α-trifluoro- p-toluyl 3-(phenoxy)phenyl diphenylmethyl 94 2,4-dichlorophenyl 4-biphenylyl 2-(p-toluyl)ethyl 94 2-napthyl m-anisyl diphenylmethyl 94 2-napthyl α,α,α-trifluoro-m-toluyl ( 1 -napththyl)methy 1 94 2,4-dichlorophenyl 4-biphenylyl benzyl 94 2,4-dichlorophenyl 3,5-difluorophenyl 2,2-diphenylethyl 94 2-napthyl α,α,α-trifluoro-m-toluyI 1 -hydroxy-4-methylpent-2-yl 94 2-napthyl 4-(trifluoromethoxy)phenyl 1 ,2,3 ,4-tetrahydronzphth- 1 -y 1 93 2-napthyl α,α,α-trifluoro-m-toluyl 2,2-diphenylethyl 93 2-napthyl α,α,α-trifluoro-m-toluyl pent-3-yl 93 2,4-dichlorophenyl α,α,α-tτifluoro-m-toluyl 2,2-diphenylethyl 93 2-napthyl α,α,α-trifluoro-m-toluyl diphenylmethyl 93 2-napthyl α,α,α-trifluoro-m-toluyl 2-furyl 93 2-napthyl 2,4-difluorophenyl (l-naphthyl)methyl 92 2-napthyl 3-chlorophenyl diphenylmethyl 13 - SUBSTΓΠJTE SHEET % inh A B C 92 2-napthyl α,α,α-trifluoro-p-toluyl 1-indanyl 92 2-napthyl α,α,α-trifluoro-m-toluyl 1-indanyl 92 α,α,α-trifluoro-p-toluyl 4-chlorophenyl 1 ,2,3 ,4-tetrahydronaphth- 1 -yl 92 2,4-dichlorophenyl 4-biphenylyl 1 ,2,3 ,4-tetrahydronaphth- 1 -yl 91 2-naphthyl m-anisyl phenethyl 91 phenyl 2-fluorophenyl 3-pyridinyl 91 2,4-dichlorophenyl 3,5-difluorophenyl 3-pyridinyl 91 2-napthyl 2,4-difluorophenyl 3-pyridinyl 91 2-napthyl α,α,α-trifluoro-p-toluyl 3-benzylpentamethylenyl 91 2-napthyl α,α,α-trifluoro-p-toluyl 3-phenylpropyl 91 α,α,α-trifluoro-p-toluyl α,α,α-trifluoro-p-toluyl N-phenyl-2,2'-aminodiethyl 91 2,4-dichlorophenyl 4-biphenylyl phenethyl 90 2,4-dichlorophenyl 4-biphenylyl 1-indanyl 90 2-napthyl 4-fluorophenyl 2,2-diphenylethyl 90 α,α,cc-trifluoro-p-toluyl 3-(phenoxy)phenyl 1 ,2,3 ,4-tetrahydronaphth- 1 -yl 90 2-napthyl α,α,α-trifluoro-m-toluyl 1 ,2,3 ,4-tetrahydronaphth- 1 -yl 90 2,4-dichloroρhenyl α,α,α-trifluoro-p-toluyl N-(α,α,α-trifluoro-m -toluyl)-2,2'-aminodiethyl 90 p-toluyl 2-chlorophenyl ( 1 -napththy l)methyl 90 α,α,α-tτifluoro-p-toluyl 3-(phenoxy)phenyl N-phenyl-2,2'-aminodiethyl 90 2,4-dichlorophenyl 4-biphenylyl ( 1 -napththyl)methyl 90 2,4-dichlorophenyl α,α,α-trifluoro-m-toluyl 1 ,2,3 ,4-tetrahydronaphth- 1 -y 1 Examination of these highly active molecules revealed that the first 25 molecules exhibit 94% ήihibition or better. Of these molecules, 12 draw all three structural diversity elements from the aforementioned sets, 9 draw two of tiiese structural diversity elements and die others draw one. - 14 -

Claims: The Claims What is claimed is: 1. An nxmxp array of chemical compounds, each of which has the following scaffold structure: wherein A, B, and C are diversity elements and at least one of A, B, or C is one of the following moieties: 2. The array of claim 1 wherein n, m and p are integers with n > 5, m > 2 and p > 1. 3. The array of claim 1 wherein the product of n, m and p is > 25. 4. The array of claim 1 wherein n, m and p are integers with n > 8, m > 12 and p > 2. 5. The array of claim 1 wherein n is 8, m is 32 and p is 40 and each compound has one of the following moieties: phenyl, m-methoxyphenyl, 2-napthyl, 2-thiophenyl, α,α,α-trifluoro-p- toluyl, 2,4-dichlorophenyl, p-toluyl, and m-toluyl for the element denoted as "A" 2,4 difluorophenyl, 2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, ,a,a- trifluoro-o-toluyl, α,α,α-trifluoro-p-toluyl, o-toluyl, m-toluyl, p-toluyl, 4- ethylphenyl, phenyl, 2-chlorophenyl, 3-chlorophenyl, 2,4-dichlorophenyl, m- anisyl, 4-(methylthio)phenyl, 4-biphenylyl, 1-naphtiιyl, 4-(trifluoromethoxy)phenyl, 3-(phenoxy)phenyl, 2-miopheneyl, 3-tiιiopheneyl, 3,5-difluorophenyl, 3-pyridinyl, 4-pyridinyl, 4-chlorophenyl, 3-quinolinyl, 4-quinolinyl, 2-furyl, 3-furyl, and 5- methylfur-2-yl for the element denoted as "B" and (2-tetrahydrofuryl)methyl, isobutyl, ( +)-hydroxypropyl,2-hydroxypenethyl, (-)-ephidryl, l-hydroxy-4-metiτylpent-2-yl, pentamethylenyl, 3- benzylpentamethylenyl, 2,2'-bis-ethylether, 4-phenylbut-2-yl, 3-phenylpropyl, benzyl, phenethyl, 1,2,3,4-tetrahydronaphthyl, 2-(p-toluyl)ethyl, diphenylmetiiyl, 2,2-diphenyletiιyl, 1-indanyl, 2-phenethyl, (l-naphthyl)methyl, (2-furyl)methyl, 3,4-dimedιoxyphenemyl, N-ethylcarboxy-2-2'-aminodiemyl, 2-(N- pyrrolidinyl)ethyl, N-phenyl-2,2'-aminodiemyl, l-benzyl-4-piperidinyl, N- piperonyl-2,2'-aminodietiιyl, 3-(4-morpholinyl)propyl, 3(l-imidazolyl)propyl, 3- (N,N-dimetiιylamino)propyl, N-(α,α,α-ttifluoro-m-toluyl)-2,2'-aminodiethyl, (2- pyridinyl)methyl, 2-(l-piperidinyl)ethyl, N-methyl-2,2'-aminodiedιyl, for the element denoted as "C". 6. The array of claim 1 wherein each compound is different. 7. A compound from the array of claim 1. 8. A compound from the array of claim 5. 9. A compound comprising the scaffold: wherein A, B, and C are diversity elements, and one of the following combinations of diversity elements: B C 2,4-dichlorophenyl phenyl cyclohexyl 2-napthyl α,α,α-trifluoro-m-toluyl 1 ,2,3,4-tetrahydronaphth- 1 -yl 2-napthyl α,α, -trifluoro-m-toluyl diphenylmethyl 2,4-dichlorophenyl α,α,cc-trifluoro-m-toluyl diphenylmethyl 2-napthyl α,α,α-trifluoro-m-toluyl 2-(p-toluyl)ethyl 2-napthyl α,α,α-trifluoro-p-toluyl diphenylmethyl p-toluyl 2,4-difluorophenyl 2-hydroxyphenethyl 2-napthyl 3-quinolinyl (l-napththyl)methyl 2-napthyl α,α,α-trifluoro-p-toluyl 4-phenylbut-2-yl 2,4-dichlorophenyl 4-biphenylyl diphenylmethyl 2-napthyl 4-(trifluoromethoxy)phenyl diphenylmethyl 2-napthyl α,α,α-trifluoro-p-toluyl 2,2-diphenylethyl 2-napthyl α,α,α-trifluoro-p-toluyl 1 ,2,3,4-tetrahydronaphth- 1 -yl 2,4-dichlorophenyl 3 -(phenoxy)pheny 1 2-(p-toIuyl)ethyl p-toluyl 2-chlorophenyl 3-(4-morpholinyl)propyl 2-napthyl 2,4-dichlorophenyl 2-hydroxyphenethyl 2-napthyl α,α,α-trifluoro-m-toluyl 3-benzylpentamethylenyl ,α,α-trifluoro-p-toluyl α,α,α-trifluoro-p-toluyl 2-(p-toluyl)ethyl α,α,α-trifluoro-p-toluyl α, ,α-trifluoro-p-toluyl 1 ,2,3,4-tetrahydronaphth- 1 -yl α,α,α-trifluoro-p-toluyl 3-(phenoxy)phenyl diphenylmethyl 2,4-dichlorophenyl 4-biphenylyl 2-(p-toluyl)ethyl 2-napthyl m-anisyl diphenylmethyl 2-napthyl ,α,α-trifluoro-m-toluyl ( 1 -napththy l)methy 1 2,4-dichlorophenyl 4-biphenylyl benzyl 2,4-dichlorophenyl 3 ,5-difluorophenyl 2,2-diphenylethyl 2-napthyl α,α,α-trifluoro-m-toluyl 1 -hydroxy-4-methylpent-2-yl 2-napthyl 4-(trifluoromethoxy)phenyl 1 ,2,3 ,4-tetrahydronzphth- 1 -y 1 A B 2-napthyl α,α,α-trifluoro-m-toluyl 2,2-diphenylethyl 2-napthyl α,α,α-trifluoro-m-toluyl pent-3-yl 2,4-dichlorophenyl α,α,α-trifluoro-m-toluyl 2,2-diphenylethyl 2-napthyl α,α,α-trifluoro-m-toluyl diphenylmethyl 2-napthyl α,α,α-trifluoro-m-toluyl 2-ruryl 2-napthyl 2,4-difluorophenyl ( 1 -naphthyl)methy 1 2-napthyl 3-chlorophenyl diphenylmethyl 2-napthyl α,α,α-trifluoro-p-toluyl 1-indanyl 2-napthyI α,α,α-trifluoro-m-toluyl 1-indanyl α,α,α-trifluoro-p-toluyl 4-chlorophenyl 1 ,2,3 ,4-tetrahydronaphth- 1 -y I 2,4-dichlorophenyl 4-biphenylyl 1 ,2,3 ,4-tetrahydronaphth- 1 -y 1 2-naphthyl m-anisyl phenethyl phenyl 2-fluorophenyl 3-pyridinyl 2,4-dichlorophenyl 3,5-difluorophenyl 3-pyridinyl 2-napthyl 2,4-difluorophenyl 3-pyridinyl 2-napthyl α,α,α-trifluoro-p-toluyl 3-benzylpentamethylenyl 2-napthyl α,α,α-trifluoro-p-toluyl 3-phenylpropyl α,α,α-trifluoro-p-toluyl α,α,α-trifluoro-p-toluyl N-phenyl-2,2'-aminodiethyl 2,4-dichlorophenyl 4-biphenylyl phenethyl 2,4-dichlorophenyl 4-biphenylyl 1-indanyl 2-napthyl 4-fluorophenyl 2,2-diphenylethyl α,α,α-trifluoro-p-toluyl 3-(phenoxy)phenyl 1 ,2,3 ,4-tetrahydronaphth- 1 -yl 2-napthyl α,α,α-trifluoro-m-toluyl 1 ,2,3,4-tetrahydronaphth- 1 -yl 2,4-dichlorophenyl α,α,α-trifluoro-p-toluyl N-(α,α,α-trifluoro-m -toluyl)-2,2'-aminodiethyl p-toluyl 2-chlorophenyl ( 1 -napththyl)methyl α,α,α-trifluoro-p-toluyl 3-(phenoxy)phenyl N-phenyl-2,2'-aminodiethyl 2,4-dichlorophenyl 4-biphenylyl ( 1 -napththyl)methyl 2,4-dichlorophenyl α,α,α-trifluoro-m-toluyl 1 ,2,3,4-tetrahydronaphth- 1 -yl 10. A method for inhibiting the binding of a Rev protein to a Rev response element which comprises administering one of the compounds of claim 1 to an subject or in vitro in an amount effective to at least partially inhibit the binding of the protein to die response element. 11. The method of claim 10 wherein the binding activity of die protein activity of die protein to the response element is reduced by at least about 50%. 12. The method of claim 10 wherein the compound does not precipitate at concentrations of up to 6 μg/ml. 13. A method for inhibiting the binding of a Rev protein to a Rev response element which comprises administering one of die compounds of claim 6 to an subject or in vitro in an amount effective to at least partially inhibit die binding of die protein to the response element. 14. The method of claim 13 wherein the binding activity of the protein to the response element is reduced by at least about 90%. 15. The method of claim 14 wherein the compound does not precipitate at concentrations of up to 6μg/ml. 16. A method of inhibiting the binding of a Rev protein to a Rev response element which comprises administering one of the compounds of claim 9 to an subject or in vitro in an amount effective to at least partially inhibit the binding of the protein to the response element.

Inventors: ARENAS JAIME E, BOLTON DAVID A, CASEBIER DAVID, FURTH PAUL, HOGAN JOSEPH C, TU CHENG, ZAMBIAS ROBERT A, ARENAS, JAIME, E., BOLTON, DAVID, A., CASEBIER, DAVID, FURTH, PAUL, HOGAN, JOSEPH, C., TU, CHENG, ZAMBIAS, ROBERT, A.
Applicants: ARQULE INC, ARQULE, INC.
Application year: 1996
Main classification: G01N33
IPC codes: A61K31, A61P31, C07B61, C07C237, C07D213, C07D215, C07D295, C07D307, C12Q1, G01N33, G01N37