WO1996022529A1 - A method of generating a plurality of chemical compounds in a spatially arranged array - Google Patents

Abstract

This invention is directed to an m x n array of different chemical compounds wherein each of said compounds has at least one structural diversity element selected from a group of amines and ketones and wherein the scaffold structure is selected from a group consisting of aminimide, imidazolone, sulfonylaminimide and phosphonylaminimide.

Description

A METHOD OF GENERATING A PLURALITY OF CHEMICAL COMPOUNDS IN A SPATIALLY ARRANGED 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 applications. 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. This 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, Angetv.

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, there are 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.

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 binding 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 ligand forms has traditionally been carried out either by random screening of molecules (produced through chemical synthesis or isolated from natural source's, for example, see K. Nakanishi, Acta Pharm. Nord., 1992, 4, 319-328.), or by using a so-called "rational" approach involving identification of a 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. & Murcko, 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 fluids, 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 ligand acceptors. This phenomenon is in sharp contrast to the affinity 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 nonpeptidic 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 peptidomimetic design, no general solution to the problem of converting a polypeptide-ligand structure to a peptidomimetic has been defined. At present, "rational" peptidomimetic design is done on an ad hoc basis. Using numerous redesign-synthesis-screening cycles, peptidic ligands belonging to a certain biochemical class have been converted by groups of organic chests 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 the 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 nonpeptide 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 the desired peptides. The peptides are then screened for activity without removing them from the pins. Houghton, (Proc. Natl. Acad. Sci. USA 1985, 82, 5131; Eichler, J. & Houghton, R. A. Biochemistry, 1993, 32, 11035-11041, and U.S. 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, 881, 7978-7982).

In another combinatorial approach, V. D. Huebner and D.V. Santi (U.S. Patent No. 5,182,366) utilized

functionalized 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 the

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 coworkers (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., Proc. Natl. Acad. Sci. USA, 1992, 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, 4,57-4461; see also Borchardt, A. & Still, W. C. J. Am. Chem. Soc. 1994, 226^", 373-374; Kerr, J. et al. J. Am. Chem. Soc. 1993, 225, 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.

The development of substrates or supports to be used in separations has involved either the

polymerization/crosslinking of monomeric molecules under various conditions to produce fabricated materials such as beads, gels, or films, or the chemical modification of various commercially available fabricated materials e.g., sulfonation of polystyrene beads, to produce the desired new materials. In the majority of cases, prior art support materials have been developed to perform specific separations or types of separations and are thus of limited utility.

Many of these materials are incompatible with biological macromolecules, e.g., reverse-phase silica frequently used to perform high pressure liquid chromatography can denature hydrophobic proteins and other polypeptides. Furthermore, many supports are used under conditions which are not

compatible with sensitive biomolecules, such as proteins, enzymes, glycoproteins, etc., which are readily denaturable and sensitive to extreme pH's. An additional difficulty with separations carried out using these supports is that the separation results are often support-batch dependent, i.e. they are irreproducible.

Recently a variety of coatings and composite-forming materials have been used to modify commercially available fabricated materials into articles with improved properties; however the success of this approach remains to be seen.

If a chromatographic support is equipped with molecules which bind specifically with a component of a complex

mixture, that component will be separated from the mixture and may be released subsequently by changing the experimental conditions (e.g., buffers, stringency, etc.) This type of separation is appropriately called "affinity chromatography" and remains an extremely effective and widely used separation technique (see Perry, E. S. in Techniques of Chemistry, Vol. 12 (J. Wiley) & May, S. W. in Separations and Purification 1978, 3rd ed.). It is certainly much more selective than traditional chromatographic techniques, e.g chromatography on silica, alumina, silica or alumina coated with long-chain hydrocarbons, polysaccharide and other types of beads or gels which in order to attain their maximum separating efficiency need to be used under conditions that are damaging to

biomolecules, e.g., conditions involving high pressure, use of organic solvents and other denaturing agents, etc. (for example see Stewart, D. J., et al. J. Biotechnology 1989, 22, 253-266; Brown, E., et al. Int. Symp. Affinity.

Chromatography & Molecular Interactions 1979, 86, 37-50).

The development of more powerful separation technologies depends significantly on breakthroughs in the field of materials science, specifically in the design and constructon of materials that have the power to recognize specific molecular shapes under experimental conditions resembling those found in physiological media, i.e. , these experimental conditions must involve an aqueous medium whose temperature and pH are close to the physiological levels and which contains none of the agents known to damage or denature biomolecules. The construction of these "intelligent" materials frequently involves the introduction of small molecules capable of specifically recognizing others into existing materials, e.g. surfaces, films, gels, beads, etc., by a wide variety of chemical modifications; alternatively molecules capable of recognition are converted to monomers and used to create the "intelligent" materials through polymerization reactions.

Advances in the ability to synthesize large numbers of peptides have made it possible to create a vast array of combinations of microenvironments within which different proteins may interact in equally. Kauvar (U.S. Patent

5,340,474) has developed a chromatographic method to obtain ligands which have the required affinity specific for a selected member of an array of analytes by providing maximal diversity in the choice of these ligands. A key to this technology is the use of a flow-through 96-well plate

compatible for assaying large numbers of parallel samples. Their short peptide-based ligands as paratope analogs (or "paralogs") contain an N-terminal amino acid spacer used for coupling to the sorbent. The C-terminal is capped with an amide group. Diversity is then created with the use of hydrophobic amino acids, enantiomeric amino acids, positively charged, negatively charged, and neutral (hydrophilic) residues, as well as intra-chain cyclization via the

formation of disulfide bonds between cysteine residues.

Protein is then loaded onto each column in the sorbent plate, and the proteins that are bound to the chromatographic sorbents are eluted, then collected into a second pretreated microplate (Benedek, K. et al. J. Chromatography 1992, 627, 51-61). Sets of paralogs are constructed by systematically varying five independent parameters drawn from protein structure literature: 1. a hydrophobic index; 2. an

isoelectric point derived from overall charge by averaging the pKa values of the ionizable side chains in solution at pH 7; 3. a hydrophobic moment; 4. an analogous lateral dipole moment; 5. a corrugation factor, defined as the measure of the scattering in the distribution of bulky side chains along the helical backbone (see Villar, H. O. & Kauvar, L. M. FEBS Letters 1994, 349, 125-130) and to defined reproducible patterns of cross-reaction which represent distinctive spectra of the primary antigen and its analogs using an immunoassay of molecular analogs against panels of antibodies (Cheung, P. Y. K., et al. Analytica Chimica Acta 1993, 283, 181-192)

Definitions

This invention discloses a system for the design, synthesis and use of logically arranged collections of synthetic 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.

A "construct" is a molecule which is a member of a collection of molecules containing a common constant

structural element 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 containing 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.

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 in 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. Other 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 point, 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.

SUMMARY OF THE INVENTION

This invention is directed to an m × n array of

different chemical compounds wherein each of said compounds has at least one structural diversity elements chosen from the group consisting of:

and wherein the scaffold structure is selected from the group consisting of:

This invention is still further directed to an m × n array of different chemical compounds wherein each of said compounds has at least one of the structural diversity elements defined herein and wherein the scaffold structure may be a chemical molecule having at least three carbon atoms and at least two sites on the molecule capable of undergoing a reaction to change the structure, usually by the addition of other- molecules to a site capable of reacting to form or attach a structural diversity element.

This invention is still yet further directed to an n × m array of chemical compounds called molecular constructs possessing a logical ordering of molecular constructs

comprising at least one k × l sub array within the array wherein each sub array is comprised of

a) at least k.l molecular constructs having a common molecular core and differing from the other k.l molecular constructs in the sub array by at least one change in the structural diversity

element attached to the molecular core; and b) each sub array within the array is related to all other sub arrays in that all corresponding molecular constructs within each sub array has at least one change in the structural diversity

elements.

Also, the array of chemical compounds above encompasses those circumstances wherein n, m, k and 1 are all integers greater than 1.

The above array of chemical compounds can also be directed to those circumstances wherein n > 5 and m > l, or n > 10 and m > 1, or even wherein n > 5 and m > 5. The specific integers used for m and n are not critical and any can be selected depending upon the desired form of the array.

The above defined array of chemical compounds is also directed to arrays wherein m multiplied by n is greater than 10, greater than 20, greater than 100, greater than 200, greater than 500, greater than 1000 or even greater than 5000. Again, the final number can be any multiple of the selected m and n values.

Still yet further the present invention is directed to an n × m array of chemical compounds called molecular

constructs possessing a logical ordering of molecular

constructs comprising at least one k × 1 sub array within the array the wherein each sub array is comprised of a) at least K.l molecular constructs having a common molecular core and differing from other k.l molecular constructs in the sub array by at least one change in the structural diversity element attached to the molecular core;

b) each sub array within the array is related to all other sub arrays in that all corresponding molecular constructs with each sub array has at least one change in the structural diversity elements; and

c) and wherein each molecular construct is equidistant from at least two of its neighboring molecular constructs.

A preferred array is that defined immediately above wherein when n and m are greater than 3 and the chemical compounds are surrounded on four sides by four equidistant neighboring other chemical compounds.

Also the present invention covers n × m arrays of chemical compounds called molecular constructs possessing a logical ordering of molecular constructs comprising at least one k × l sub array within the array wherein each sub array is comprised of

a) at least k.l molecular constructs having a common molecular core and differing from the other k.l molecular constructs in the sub array by at least one change in the structural diversity element attached to the molecular core; b) each sub array within the array is related to all other sub arrays in that all corresponding molecular constructs within each sub array has at least one change in the structural diversity elements; and

c) and wherein each molecular construct is separated from all other molecular constructs by a container material.

The contained materials for the above cited array may employ glass, polymers, silicon, or any other material known by those of ordinary skill in the art.

Further, the present invention is directed to an n × m × q array of chemical compounds called molecular constructs possessing a logical ordering of molecular constructs

comprising at least one k × l sub array within the array wherein each sub array is comprised of

a) at least k.l molecular constructs having a common molecular core and differing from the other k. l molecular constructs in the sub array by at least one change in the structural diversity element attached to the molecular core;

b) each sub array within the array is related to all other sub arrays in that all corresponding molecular constructs within each sub array has at least one change in the structural diversity elements; and

c) and wherein q is an integer > 1 and each array designated q₁...q_s where s is an integer > than 1, differs from the other q arrays by at least one function.

In addition, the present invention is directed to an n × m × q array wherein the function is the addition of an organic structure selected from the group consisting of an amine, an aldehyde, an alcohol, a ketone, a carboxylic acids, an ether and an epoxy, and wherein the function may or may not be an analytic technique.

The reactions which are the subject of this invention may be performed simultaneously by using a mechanical

apparatus such as multiple pipettes attached to an apparatus and other methods known to the skilled artisan. Brief Description of Drawings

Figure 1 is a graphical representation of an array vertex illustrating the relationship between the building blocks, their addresses and the various operators therefor; and

Figure 2 is a schematic diagram representing the

sequence of events for combining the building blocks to form the array.

Detailed Description of the Invention

This invention pertains to the logical layout,

construction and testing of arrays of chemical compound for one of a variety of applications, in which the desired properties of the compound can be measured and correlated to specific ordered changes in the fragments use 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 method has great utility in accelerating the development of compounds have the optimal properties for the desired application.

The arrays are constructed from logically ordered and arranged sub-arrays of compounds. Each sub-array consists of spatially addressable sets of structurally related individual chemical compounds, ranging in number from one to 10¹² and possessing the following properties: (1) a. common structural scaffold element referred to as a "molecular core" and (2) a variable structural diversity element referred to as a fragment, in such a manner that the variation between any two compounds within a given sub-array consists only of either zero (0) or one (l) change in a fragment. These arrays may in turn be arranged in such a manner to form higher order arrays consisting of sets of arrays and tested to provide information regarding the optimum structural features available for the application.

The sub-arrays are arranged in such a manner that the direct comparisons of compounds automatically yields

information regarding the effect known fragments have on a desired application, as well as on the effect on changes in physical and reactive properties. As provided by simple set theory for any number of independently variable structural diversity elements n, there exists n logical higher order array arrangements, such that relational information on the effect of variation of each of the a structural diversity elements can be obtained in a similar manner by comparison of testing data from the relative addresses in appropriately arranged sub-arrays.

An application of this invention is the rapid

determination and optimization of desired biological or physical activity. An array is screened and the optimum candidate is chosen. This process can be continued in n dimensions to provide an absolute structure activity

relationship ("SAR") picture of the candidate and selection in speeded by the rapid modular synthesis of arrays for use in testing. Thus in one light the invention is the most powerful tool to date for the rapid synthesis, screening and testing of compounds for IND candidacy. This method is facilitated by virtue of selecting fragments based solely upon their ability to react and participate in the process of assembly.

These arrays may be assembled to form a "super array" for exhaustive testing. This approach provides a large scale view over different structures, functionalities and spatial arrangements for exploring biological activity.

The physical construction of the array also permits the logical and rapid analysis of synthetic results for the assurance of purity and quality. By testing a series of loci within any given sub-array, it becomes possible to determine the efficacy of construction of that core, and eliminate those fragments (i.e., process development within the

assembly) which do not provide satisfactory results. This system, therefore possesses the ability to learn the utility of given reagents from previous results, and either delete them from further use or alter general conditions for their efficient incorporation into the array. Thus, both positive and negative results are of value in the ultimate

construction of the array, and there is no ambiguity in regards to the inclusion or exclusion of fragments.

A further application of this invention is the

facilitation of the optimal analyte or epitope binding ligand for attachment to a chromatographic support for separation or purification applications. A further application of this invention pertains to the ability to construct materials in a modular fashion, so as to facilitate their selection for such properties as strength, stability, reactivity or any other desired physical property. Whereas many methods rely upon logical choice for fragment candidates in such efforts, this method provides for the construction and testing of all candidates, thereby eliminating any compromises which

traditional methods make based on the limits of time,

manpower, and cost. By the screening of all possible

synthetic variations the selection of the optimal candidate is a matter of data and not chemical intuition. The desired affinity can be rapidly optimized and directly correlated and attributed to the singular change made within a given sub-array. Therefore the selection of a ligand is no longer a random, intuitive process, but one of complete confidence providing exhaustive data (cf. Kauvar, L.M. U.S. Patent

5,340,474).

Furthermore the invention provides for the development of seamless technology between planning, logistical

development, execution of assembly in either an arrayed or subarrayed manner, quality analysis, packaging, distribution, testing, interpretation and iteration. The invention

provides for the integrated design and delivery of a unified chemical discovery system, which by application of logic and implementation of information management, has been heretofore unknown. The invention provides for the occupation of all possible spatial addresses and therefore allows for complete analysis of desired properties. This concept can be extended toward the design and manufacture of appropriate hardware and software to support the integrated aspect of this modular construction.

The logically arranged arrays of the present invention are 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.

All known prior art is universally directed toward the maximization of structural diversity. By definition this has excluded the acquisition of maximal data. In these cases, the relationship between individual structural variations and any resulting changes in a measurable property of the

compounds can not be directly obtained from the testing results. The process of obtaining a compound having a desired physical property using methods of the prior art, while guided by intuition, is a random statistical process at best. Thus a positive result is not designed to give any additional information about the relationship between a specific structural modification and the corresponding change in the desired property, and a negative result can not provide any information at all. Methods in the prior art universally require 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.

These arrays may be constructed from a wide variety of molecular cores, several examples of whi :h are shown below. The criteria for core candidates are that the scaffold a) present 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.

In general the molecular cores are linear, branched or cyclic organic compounds. In particular, the molecular cores comprise a chemical molecule having at least three carbon atoms and at least two sites on the molecule capable of undergoing a reaction to change the structure, usually by the addition of other molecules to a site capable of reacting to form or attach a structural diversity element.

One example of a molecular core is an aminimide

molecule. This is a technology which has been previously described.

These compounds may be synthesized in a number of ways, from the reaction of an epoxide, an ester, and a hydrazine, as well as alkylation of a hydrazide, as shown below.

An example of a scaffold capable of forming a molecular core of an oxazolone molecule. Methylidene amides are formed from the sequential reaction of aldehydes, then amines with oxazolones. These compounds and their congeners may be in turn transformed into imidazolones:

These compounds and their methods of manufacture are

described in PCT Publications WO94/00509 and WO94/01102.

Sulfonylaminimides and phosphonylaminimides are still further examples of molecular cores which can be constructed in an analogous manner as their carbon-based counterparts, with the exception of sulfonate esters not participating in the reaction of an epoxide and hydrazine in the desired manner.

While the aminimide, oxazolone, sulphonylaminimide, and phosphonylaminimide are several examples of the concept of a molecular core, other molecular cores are possible according to the teachings of this invention. Further examples of possible molecular cores include, but are not limited to:

alkaloids, quinolines, isoquinolines, benzimidazoles,

benzothiazoles, purines, pyrimidines, thiazolidines,

imidazopyrazinones, oxazolopyridines, pyrroles, pyrrolidines, imidazolidones, quinolones, amino acids, macrolides, penems, saccharides, xanthins, benzothiadiazine, anthracyclines, dibenzocycloheptadienes, inositols, porphyrins, corrins, and carboskeletons presenting geometric solids (e.g.,

dodecahedrane).

Diels-Alder reactions, Darzens glycidic ester

condensations, Simmons-Smith cyclopropanations, rhodium catalyzed carbene additions, Ugi and Passerini reactions may all be done in such a manner, as to construct these arrays as described above. The application of this technology is facile and the format in which it is constructed is amenable to most organic transformations and reaction sequences.

The structural diversity elements may be the same or different, may be of a variety of structures and may differ markedly in their physical or functional properties, or may be the same; they may also be chiral or symmetric or from a compound which is chiral or symmetric. The structural diversity elements are preferably selected from:

1) amino acid derivatives of the form (AA)_n, which would include, for example, natural and synthetic amino acid residues (n = 1) including all of the naturally occurring alpha amino acids, especially alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine,

phenylalanine, proline, serine, threonine, tryptophan, tyrosine; the naturally occurring disubstituted amino acids, such as amino isobutyric acid, and isovaline, etc.; a variety of synthetic amino acid residues, including alpha- disubstituted variants, species with olefinic substitution at the alpha position, species having derivatives, variants or mimetics of the naturally occurring side chains; N-substituted glycine residues; natural and synthetic species known to functionally mimic amino acid residues, such as statine, bestatin, etc. Peptides (n = 2 - 30) constructed from the amino acids listed above, such as angiotensinogen and its family of physiologically important angiotensin hydrolysis products, as well as derivatives, variants and mimetics made from various combinations and permutations of all the natural and synthetic residues listed above.

Polypeptides (n = 31 - 70), such as big endothelin,

pancreastatin, human growth hormone releasing factor and human pancreatic polypeptide. Proteins (n > 70) including structural proteins such as collagen, functional proteins such as hemoglobin, regulatory proteins such as the dopamine and thrombin receptors.

2) a nucleotide derivative of the form (NUCL)_n, which includes natural and synthetic nucleotides (n = 1), such as adenosine, thymine, guanidine, uridine, cytosine, derivatives of these and a variety of variants and mimetics of the purine ring, the sugar ring, the phosphate linkage and combinations of some or all of these. Nucleotide probes (n = 2 - 25) and oligonucleotides (n > 25) including all of the various possible; homo and hetero-synthetic combinations and

permutations of the naturally occurring nucleotides;

derivatives and variants containing synthetic purine or pyrimidine species, or mimics of these; various sugar ring mimetics; and a wide variety of alternate backbone analogs, including but not limited to phosphodiester,

phosphorothionate, phosphorodithionate, phosphoramidate, alkyl phosphotriester, sulfamate, 3^,-thioformacetal,

methylene(methylimino), 3-N-carbamate, morpholino carbamate and peptide nucleic acid analogs.

3) a carbohydrate derivative of the form (CH)_n, which would include natural physiologically active carbohydrates; related compounds, such as glucose, galactose, sialic acids, iS-D-glucosylamine and nojorimycin, which are both inhibitors of glucosidase; pseudo sugars, such as 5a-carba-2-D- galactopyranose, which is known to inhibit the growth of Klebsiella pneumonia (n = 1); synthetic carbohydrate residues and derivatives of these (n = 1) and all of the complex oligomeric permutations of these as found in nature,

including high mannose oligosaccharides, the known antibiotic streptomycin (n > 1).

4) a naturally occurring or synthetic organic structural motif. The term "motif" is defined as an organic molecule having or containing a specific structure that has biological activity, such as a molecule having a

complementary structure to an enzyme active site, for

example. This term includes any of the well known basic structures of pharmaceutical compounds including

pharmacophores, or metabolites thereof. These basic

structures include beta-lactams, such as penicillin, known to inhibit bacterial cell wall biosynthesis; dibenzazepines, known to bind to CNS receptors and used as antidepressants; polyketide macrolides, known to bind to bacterial ribosymes, etc. These structural motifs are generally known to have specific desirable binding properties to ligand acceptors.

5) a reporter element, such as a natural or synthetic dye or a residue capable of photographic amplification which possesses reactive groups that may be synthetically

incorporated into the sulfaminimide structure or reaction scheme, and may be attached through the groups without adversely interfering or affecting with the reporting

functionality of the group. Preferred reactive groups are amino, thio, hydroxy, carboxylic acid, carboxylic acid ester, particularly methyl ester, acid chloride, isocyanate alkyl halides, aryl halides and oxirane groups.

6) an organic moiety containing a polymerizable group such as a double bond, or other functionalities capable of undergoing condensation polymerization or copolymerization. Suitable groups include vinyl groups, oxirane groups,

carboxylic acids, acid chlorides, esters, amides, azlactones, lactones and lactams. Other organic moiety such as those defined for R and R' may also be used.

7) a macromolecular component, such as a macromolecular surface or structures which may be attached to the

sulfaminimide modules via the various reactive groups

outlined above, in a manner where the binding of the attached species to a ligand-receptor molecule is not adversely affected and the interactive activity of the attached

functionality is determined or limited by the macromolecule. Examples of macromolecular components include porous and non-porous inorganic components, such as, for example, silica, alumina, zirconia, titania and the like, as commonly used for various applications, such as normal and reverse phase chromatographic separations, water purification, pigments for paints, etc.; porous and non-porous organic macromolecular components, including synthetic components such as

styrenedivinyl benzene beads, various methacrylate beads, PVA beads, and the like, commonly used for protein purification, water softening; and a variety of other applications, natural components such as native and functionalized celluloses, such as, for example, agarose and chitin, sheet and hollow fiber membranes made from nylon, polyether sulfone or any of the materials mentioned above. The molecular weight of these macromolecules may range from about 1000 Daltons to as high as possible. They may take the form of nano-particles (dp = 1000 - 5000 Angstroms), latex particles (dp = 1000 - 5000 Angstroms), porous or non-porous beads (dp = 0.5 - 1000 microns), membranes, gels, macroscopic surfaces or

functionalized or coated versions or composites.

Structural diversity elements may also be a chemical bond to a suitable organic moiety, a hydrogen atom, an organic moiety which contains a suitable electrophilic group, such as an aldehyde, ester, alkyl halide, ketone, nitrile, epoxide or the like; a suitable nucleophilic group, such as a hydroxyl, amino, carboxylate, amide, carbanion, urea or the like; or one of the other structural diversity elements defined below. In addition, structural diversity elements may join to form a ring, bi-cyclic or tri-cyclic ring system; or structure which connects to the ends of the repeating unit of the compound defined by the preceding formula; or may be separately connected to other moieties.

Structural diversity elements on a scaffold may be the same or different and each may be one or more atoms of carbon, nitrogen, sulfur, oxygen, any other inorganic

elements or combinations thereof. The structural diversity elements may be cyano, nitro, halogen, oxygen, hydroxy, alkoxy, thio, straight or branched chain alkyl, carbocyclic aryl and substituted or heterocyclic derivatives thereof.

Structural diversity elements may be different in adjacent molecular cores and have a selected stereochemical

arrangement about the carbon atom to which they are attached.

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, isopropyl, n-butyl, isobutyl 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 beptenyl, 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 hydroxyl, amino, carboxyl, 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 the like, polyhydroxy such as 1,2-dihydroxypropyl, 1,4-dihydroxy-1-butyl, and the like;

methylamino, ethylamino, dimethylamino, diethylamino,

triethylamino, cyclopentylamino, benzylamino, dibenzylamino, and the like; propionic, butanoic or pentanoic acid groups, and the like; formamido, acetamido, butanamido, and the like, methoxycarbonyl, ethoxycarbonyl or the like, chloroformyl, bromoformyl, 1, 1-chloroethyl, bromoethyl, and the like, or dimethyl or diethyl ether 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 those described above, and lower alkyl groups as described above. The cyclic groups of the invention may further comprise a heteroatom. For example, in a specific embodiment, structural diversity element A is cyclohexanol.

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 substituted with any number of a variety of

functional groups. In addition to the functional groups described above in connection with substituted 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,

benzylcyclohexylmethyl, 2-cyclohexyl-propyl,

2,2-methylcyclohexylpropyl, 2,2methylphenylpropyl,

2,2-methylphenylbutyl, and the like.

The structural diversity element may also be a

connecting group that includes a terminal carbon atom for attachment to the quaternary nitrogen and may be different in v djacent n units.

In one embodiment of the invention, at least one of the structural diversity elements represents an organic or inorganic macromolecular surface. Examples of preferred macromolecular surfaces include ceramics such as silica and alumina, porous and non-porous beads, polymers such as a latex in the form of beads, membranes, gels, macroscopic surfaces or coated versions or composites or hybrids thereof.

All publications, patents, and patent applications are herein specifically incorporated by reference to their relevant portions (cf. The Merck Index, 11th Ed., Budavari, S. Ed., Merck & Co., Rahway, NJ, 1989; Physicians Desk

Reference, 44th Ed., Barnhart, E. D. Publ., Medical Economics Company Inc., Oradell, NJ, 1990.

The following experimentals are meant to exemplify but one embodiment of the present invention and are not intended to limit the invention thereto.

Examples

A 10,240-component array is synthesized according to the teaching of the invention, from eight oxazolones (Building Block A), 32 aldehydes (Building Block B), and 40 amines (Building Block C).

AN 1001 Protocol: Tetrahydrofuran (THF) solutions of the building blocks are prepared according to the protocols generated on the spread sheets entitled "AN 1001 SOLUTION PROTOCOLS. CALCULATIONS, AND BUILDING BLOCK SELECTION". The Building Block solutions are 250 mM in "A", 250 mM in "B", and 500 mM in "C". 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 (8 X 10) and a row

contains 16 reaction plates. The entire array consists of 8 rows of these 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 (1 eq., 50 μmoles) of the "A" building block solution according to the spread sheet entitled "AN 1001 SPATIAL LAYOUT, "A" BUILDING

BLOCKS" starting at P1 and ending at P16. The second

operator is spatial delivery of 200 μl (1 eq., 50 μmoles) of the "B" Building Blocks to the same reaction plates according to the spread sheet entitled "AN 1001 SPATIAL LAYOUT, "B" BUILDING BLOCKS." The third operator is addition to the same reaction plates of 50 μL of a I M (1 eq., 50 μmoles) solution of triethylamine in THF to all the spatial addresses that "A" and "B" building Blocks were added. The forth 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 (1 eq., 50 μmoles) of the "C" building, block solutions according to the spread sheet entitled "AN 1001 SPATIAL LAYOUT, "C" BUILDING BLOCKS." The sixth

operator is addition of 200 μL of THF to all the spatial addresses in the row or cycle. The seventh operator allows the reaction plates to stand at 25 decrees 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 the molecular constructs for delivery and quality control. Heat the reaction plates to 60 degrees centigrade for 10 minutes and add 400 μl of dimethylsuIfoxide (DMSO) to dissolve the molecular constructs (operator 8). Remove the solution from the reaction plates and place in a plastic microtiter plates in a special manner (operator 9). Specially 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 spacial distribution. Remove a 10 μL aliquot following a unique address pattern layout from each microtiter plate for quality control (operator 11).

Specially reformat these aliquots, dilute with 300 μL of acetonitrile and subject these samples to analysis by High Performance Liquid Chromatography and Mass Spectrometry for quality control of the molecular constructs in the 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.

Claims

The Claims What is claimed is:

1. A method for constructing an array of synthetic molecular constructs, comprising the steps of:

a. forming a plurality of molecular constructs; b. laying out an array possessing a logical

ordering of sub-arrays of said molecular constructs;

c. providing each sub-array with molecular

constructs having a common molecular core and at least one structured diversity element which is different from the others; and

d. relating each sub-array within the array to all other sub arrays by said difference in said

structural diversity elements.

2. The method of Claim 1 where the molecular constructs are functionalized beads, plates, membranes, composites or combinations thereof.

3. A method of optimizing the ability of a first chemical compound to bind to a reaction site comprising the steps of:

a) ascertaining the three dimensional and

electrostatic configuration of the reaction site; b) selecting a scaffold backbone for attaching at least one structural diversity element that is complementary to the reaction site;

c) forming an n × mm array of different chemical compounds, wherein each chemical compound comprises said scaffold and at least one structural diversity element; and

d) simultaneously screening each of the compounds in the n × m array against the reaction site to determine the chemical compound having the most reactivity to the reaction site.

4. A method according to claim 3 which further - comprises (e) ascertaining which chemical compounds in the n × m array have any reactivity to the reaction site and assigning those compounds a location designated by (n,m).

5. A method according to claim 4 which further comprises (f) forming an o × p array comprising at least one of the compounds having reactivity to the reaction site and forming at least one homolog, isomer or analog of each of said at least one compound to form the o × p array.

6. A method according to claim 5 which further comprises (g) simultaneously screening each of the compounds in the o × p array to determine their reactivity to the reaction site.

7. A method according to claim 6 wherein steps (e) through (g) are repeated at least twice.

8. A method according to claim 1 which further comprises selecting the scaffold backbone to have the following structure:

9. A method according to claim 1 which further comprises selecting the scaffold backbone to have the following structure:

10. A method according to claim 1 which further comprises selecting the scaffold backbone to have the following structure:

11. A method according to claim 1 which further comprises selecting the scaffold backbone to have the following structure:

12. A method according to claim 1 which further comprises selecting the structural diversity elements is selected from the group consisting of but not limited to:

13. A method according to claim 3 wherein each compound having a binding ability is assigned a location in the array, which also provides structural and chemical data for that compound.

14. A method of ascertaining the spatial orientation of an n × m array of different chemical compounds comprising the steps of:

a) forming an n × m array of oriented wells;

b) adding reactive ingredients capable of forming chemical compounds into the wells;

c) determining the molecular weights of all ingredients added to each well and assigning each well a location (n,m);

d) allowing the reactive ingredients to combine to form a chemical compound in each well, and determining the molecular weight of the chemical compositions in any two wells;

e) matching the molecular weights of the chemical compounds in the any two wells with the molecular weights of the chemical ingredients added to each well (n,m) to determine the spatial orientation of the array.

15. A method according to claim 14 wherein the molecular weight is determined by mass spectroscopy.

16. A method of determining the structure of a chemical compound capable of binding to a reaction site comprising the steps of:

a) ascertaining the three dimensional structure and electrostatic configuration of the reaction site;

b) selecting a scaffold backbone for attaching structural diversity elements that is capable of being complementary to the reaction site; c) forming an n x m array of different chemical compounds, wherein each chemical compound comprises the scaffold backbone and at least one structural diversity element;

d) simultaneously screening each of the compounds in the n x m array against the reaction site to determine the chemical compound having the most reactivity to the molecular recognition site.

17. A method according to claim 16 wherein the scaffold has the structure:

18. A method according to claim 16 wherein the scaffold has the structure:

19. A method according to claim 16 wherein the scaffold has the structure:

20. A method according to claim 16 wherein the scaffold hps the structure:

21. A method according to claim 16 wherein at least one of the structural diversity elements is selected from the group consisting of but not limited to:

22. A method of determining the structure of at least a portion of a reaction site comprising the steps of a) selecting a scaffold backbone for attaching structural diversity elements;

b) forming an n × m array of different chemical compounds, wherein each chemical compound comprises the scaffold backbone and at least one structural diversity element;

c) simultaneously screening the n x m array against the reaction site to determine the chemical compound having the most reactivity to the reaction site;

d) determining the structure of the compound (n,m) in the array having the greatest reactivity; e) forming a second k × l array comprising the reactive compound having the greatest reactivity from the n × m array and its isomers, homologs and analogs;

f) simultaneously screening the k × l array of compounds against the reaction site to determine the chemical compound, having the greatest reactivity to the reaction site;

g) determining the structure and electrostatic configuration of the compound from the k × l array having the greatest reactivity;

f) forming at least one homolog, isomer or analog of each compound (n,m) having a reactivity to form the o × p array.

23. The rational development of a chemical

compound capable of reacting with a molecular recognition site comprising the steps of:

a) selecting a scaffold for attaching structural diversity elements;

b) simultaneously forming an n × m array of different chemical compounds, wherein each chemical compounds comprises a scaffold and at least one structural diversity element; and c) simultaneously screening the n × m array of compounds against a molecular recognition site to determine the chemical compound having the most reactivity to the molecular recognition site;

24. A method of forming an n × m array of chemical compounds comprising the steps of:

a) Forming an n × m array of the same molecular construct having a common molecular core and X, where X is greater than or equal to three, sites capable of undergoing chemical transformations;

b) simultaneously and/or concurrently performing an operation on each molecular construct in the n x m array to create a reaction on at least one site on each of the n × m molecules to form at least one different molecular construct;

c) performing either overlaid or orthogonal operations on the product of b. for sufficient cycles to produce an ordered, fully-addressable array of novel compounds.

25. A method of determining the structure of a chemical compound capable of binding to a reaction site comprising the steps of;

a) ascertaining the three dimensional structure and electrostatic configuration of the reaction site;

b) selecting a scaffold backbone for attaching structural diversity elements that is capable of being complementary to the reaction site; c) forming an n × m array of different chemical compounds, wherein each chemical compound comprises the scaffold backbone and at least one structural diversity element; and

d) simultaneously screening each of the compounds in the n × m array against the reaction site to determine the chemical compound having the most reactivity to the molecular recognition site.

26. A method according to claim 25 which further comprises the following steps:

e) simultaneously screening each of the compounds in the o × p array

f) determining the molecular weight of all ingredients added to each well and assigning each well a location (n,m);

g) allowing the reactive ingredients to combine to form a chemical compound in each well and determining the molecular weight of the chemical compositions in any two wells;

h) adding reactive ingredients capable of forming chemical compounds into the wells, is performed; and

i) simultaneously screening each of the compounds in the o × p array against a reactive site.

27. A method of determining the structure of at least a portion of a reaction site comprising the steps of:

a) selecting a scaffold backbone for attaching structural diversity elements;

b) forming an n × m array of different chemical compounds, wherein each chemical compound comprises the scaffold backbone and at least one structural diversity element;

c) simultaneously screening each compound in the n × m array against the reaction site to determine the chemical compound having the most reactivity to the reaction site;

d) determining the structure of the compound

(n,m) in the array having the greatest reactivity; e) forming a second k × l array comprising the most reactive compound from the n × m array and its isomers, homologs and analogs;

f) simultaneously screening the k × l array of compounds against the reaction site to determine the chemical compound having the greatest reactivity to the reaction site; and g) determining the structure and electrostatic configuration of the compound from the k × l array having the greatest reactivity.