A METHOD OF PREPARING CHEMICAL COMPOUNDS

Abstract: A method of preparing selected chemical compounds, the method comprising the steps of (a) generating a database of three-dimensional chemical structures by selecting appropriate organic molecules of a known structure as starting compounds and combining two or more of such organic molecules by one or more well-defined chemical reactions to form three-dimensional chemical structures, (b) defining search criteria for identifying potentially active compounds and conducting a search for hits, (c) selecting and synthesizing one or more chemical structures from the hits found in step (b) and (d) testing the selected chemical compounds synthesized in step (c) for activity.

Description: A METHOD OF PREPARING CHEMICAL COMPOUNDS FIELD OF INVENTION The present invention relates to a method of generating a database of chemical structures and searching the database, and the synthesis of selected chemical compounds based on the results of the search. BACKGROUND OF THE INVENTION In the last few years, docking or fitting small ligands into receptor or enzyme models, three-dimensional (3D) database searching, de novo design of compounds etc. have become very popular tools in the field of molecular modelling (cf. Trends in OSAR and Molecular Modeling. Proceedings of the 9th European Symposium on Structure-Activity Relationships: QSAR and Molecular Modeling, Ed. C.G. Wermuth, ESCOM, Leiden, 1993) . Some of the 3D molecular modelling tools such as 3D substructure searching depend on large structural databases containing small organic molecules in 3D coordinates (cf. Y.C. Martin, J. Med. Che . 35. 1992, pp. 2145-2154; and A. Rusinko III, Chemical Design Automation News j3, 1993, pp. 44-47). 3D databases of proprietary or commercially available compounds are often preferred in drug research because they provide a fast and relatively inexpensive way of getting physical access to selected compounds followed by biological testing in the new lead compound finding process. Alternatively, de novo design of bioactive molecules may be used to generate a large number of mostly novel structures. Computer programmes exist for generating such structures, e.g. the SPROUT programme for the design of structures with known pharmacophoric geometries (see V. Gillet et al., J. Computer-Aided Molecular Design 1_, 1993, pp. 127-153) . Once a database of novel structures has been created, another programme, CAESA, is used to rank the structures according to their ease of synthesis (see Z. Zsoldes et al. at page S6-5 in Molecular Graphics at the Frontier. 13th Annual Conference of the Molecular Graphics Society, July 9-13, 1994, at Northwestern University, Evanston IL, USA). Although 3D databases of de novo design compounds contain a large number of novel compounds, these are often difficult to obtain because complex chemical reactions are involved in their synthesis. A completely different approach is the creation of combinatorial chemical libraries in which a large number of compounds are synthesized to generate a physically existing library of chemical compounds which may then be screened for hits. For a review, see M. Gallop et al., J. Med. Chem. 37(9) . 1994, pp. 1233-1251. The generation of combinatorial chemical libraries requires considerable resources in terms of the time and cost required to establish such libraries. DESCRIPTION OF THE INVENTION The present invention relates to a method of synthesizing potential lead compounds based on a novel strategy of compiling a database including compounds for which known procedures of synthesis are available. Accordingly, the present invention relates to a method of preparing selected chemical compounds, the method comprising the steps of (a) generating a database of three-dimensional chemical structures by selecting appropriate organic molecules of a known structure as starting compounds and combining two or more of such organic molecules by one or more well-defined chemical reactions to form three-dimensional chemical structures, (b) defining search criteria for identifying potentially active compounds and conducting a search for hits, (c) selecting and synthesizing one or more chemical structures from the hits found in step (b) , and (d) testing the selected chemical compounds synthesized in step (c) for activity. In a further aspect the present invention relates to a method of preparing selected chemical compounds, the method comprising the steps of (a) generating a database of three-dimensional chemical structures by selecting appropriate organic molecules of a known structure as starting compounds and combining two or more of such organic molecules by one or more well-defined chemical reactions to form three-dimensional chemical structures, (b) defining search criteria for identifying potentially active compounds on the basis of known chemical structures exhibiting activity in appropriate assays, and conducting a search for hits, (c) selecting and synthesizing one or more chemical structures from the hits found in step (b) , and (d) testing the selected chemical compounds synthesized in step (c) for activity. In the present context, the term "selected chemical compounds" is intended to indicate compounds which are identified by the present method and, once they have been synthesized and tested for biological activity, are lead compounds for the development of pharmaceutically active products or compounds which are biologically active in other ways. The term "three-dimensional chemical structure" is intended to indicate compounds which may exist physically but are often hypothetical and exist as three-dimensional representations in a database. When the organic molecules used as the starting compounds are available as three-dimensional structures, the database is generated directly as a three-dimensional database. On the other hand, when the organic molecules are available as two-dimensional structures, the database is initially generated as a two-dimensional database. All or part of the two- dimensional chemical structures in the database are then converted to three-dimensional chemical structures. Conversion of two-dimensional chemical structures to three-dimensional structures may be done by means of commercially available software, e.g. CONCORD 3.01 (University of Texas at Austin, USA) . Other conversion programmes are discussed in R.S. Perlman, Chem. Design Automation News. August 1993, pp. 3-14. The term "appropriate organic molecules" is intended to indicate smaller compounds of a known structure which are either commercially available or readily synthesized by known reaction procedures. Representations of these compounds are selected as starting materials for inclusion in the database based on the ability of one such smaller compound to react with another by a well-known reaction mechanism to generate compounds, the two- or three-dimensional representations of which are included in the database. It is understood that not only two, but also three or four of such organic molecules may be combined to generate the three-dimensional chemical structures. Examples of such organic molecules are amines, carboxylic acids, alcohols, thiols, halides, ketones, ethers, nitriles or sulphonic acids. It is understood that these compounds may be both aliphatic, aromatic and heterocyclic. It is generally preferred to use organic molecules which contain only one reactive moiety (e.g. a functional group which takes part in the reaction) , while molecules containing more than one identical reactive moiety are typically eliminated as starting materials for the database in order to simplify the synthesis procedure. However, functional groups may be retained in the molecule if they are suspected to be important for the biological activity of the chemical compound resulting from the reaction. Commercially available software which may be used to generate the database is SYBYL/SPL 6.03 (SPL is the proprietary programming language of Tripos Associates Inc., USA, which may be used to write the source code) (it is understood that the programme may also be written in FORTRAN or another commercially available programming language) , UNITY 2.3 (Tripos Associates Inc. USA, which may be used for the database search) and CONCORD 3.01 (University of Texas at Austin, USA, which may be used for conversion of 2D to 3D chemical structures) . The software may be modified to streamline it for the present purpose. The principles employed to establish the database are shown in appended Fig. 1 illustrating the combination of a primary amine with a carboxylic acid to form an amide. In Fig. 1 green indicates Cl, pale blue indicates H, yellow indicates S, red indicates O, dark blue indicates N and grey indicates C. The term "well-defined chemical reactions" is intended to indicate reactions for which an established procedure is known. The advantage of the method of the invention over the previously described de novo design strategies is that, once a chemical structure has been selected after a search for hits in the database, it will be possible to synthesize it without major effort because the reaction required to synthesize the structure is an established one. This is in contrast to other database design programmes such as SPROUT (cf. V. Gillet et al., J. Computer-Aided Molecular Design 1_, 1993, pp. 127-153) which, lacking this selectivity at the outset, need to include some kind of scoring for "synthesizability". Another advantage of the method of the invention is that the number of three- dimensional chemical structures may be increased almost indefinitely by including an increasing number of starting organic molecules, and yet selected hits can often be prepared more easily than other de novo design structures or even proprietary compounds. On the other hand, hits obtained by searching the database are often original which is important for finding new lead compounds. Finally, the database can be custom-made to include certain essential structural elements which are thought to be essential for a certain biological function. It is generally preferred to include as many different types of reactions as possible in order to increase the diversity of the database. Examples of well-defined reactions are reactions for combining an amine with an acid resulting in an amide, an alcohol with an acid resulting in an ester, a thiol with an acid resulting in a thioester, an alcohol with an alkyl halide group resulting in an ether, etc. (e.g. as described in R.C. Larock, Comprehensive Organic Transformations; a Guide to Functional Group Preparations. VCH Publishers, 1989) . Representative reactions useful for generating entries into the database are: A. R'-NH j + R 2 -COOH → R 1 -NH-CO-R 2 B . R 1 -0H + R 2 -C00H → R^O-CO-R 2 C . R 1 -NH-R 2 + R 3 -COOH → R -N (R 2 ) -CO-R 3 D . R 1 -SH + R 2 -COOH → R 1 -S-C0-R 2 E . R 1 -NH + R 2 -Halo → R^N-R 2 F . R^NH + R 2 -0H → R 1 -N-R 2 Wherein R 1 , R 2 and R 3 may be the same or different and each represent an alkyl, aryl, aralkyl or heterocyclic group which may be substituted with any desired functional group. An example of generating a database according to step (a) of the method of the invention is shown in Table 1 below. Table 1 Database records Amine Acid Amide Starting organic molecules* 665 505 Theoretical amide combinations 335825 After sub-structure reduction 311 255 Theoretical amide combinations 79305 CONCORD 3D structures 78797 * Only one functional group is allowed in each starting organic molecule to reduce difficulties in synthesis The term "defining search criteria" is intended to indicate the parameters used to conduct the search in the generated database. Such parameters are often based on the structural features known to be required for the pharmaceutical activity of known pharmaceutically active compounds. Such structural features, e.g. specific groups or atoms, and their spatial interrelationships are also termed pharmacophores, e.g. as described in D.E. Clark and P. Willett, J. Molecular Graphics 10. December 1992, pp. 194-204. A pharmacophore mapping approach is described in Y.C. Martin et al., J. Computer-Aided Molecular Design 7, 1993, pp. 83-102. Another approach is termed the distance geometry method, e.g. described by A. Ghose and G. Crippen, J. Med. Chem. 28. 1985, p. 333 ff. A further example of an approach in which known software may be used to define search criteria is the so-called 3D-QSAR (three- dimensional quantitative structure activity relationship) , cf. for instance US 5,025,388. Selected compounds synthesized in step (c) of the present method are tested for biological activity by an established method, e.g. an established in vitro or in vivo assay such as receptor-binding assays, assays for activation of receptors on living cells by ligands, animal models for pharmacological activity etc. Once biological activity has been established, the compound is termed a "lead" compound, i.e. a candidate for further drug development. To limit the size of the three-dimensional database, only the most complex and diverse representatives of the starting organic molecules are retained in the database. This means that organic molecules which are merely substructures of more complex organic molecules are eliminated from the database (for the present purpose, this procedure is term "sub-structure reduction" abbreviated to SSR) . By this procedure, for instance, the number of amines in table 1 above is reduced from 665 to 311, and the number of acids from 505 to 255. In this way, the number of chemical structures making up the database may be substantially reduced without losing any significant structural information. In this embodiment, when the database is generated from two-dimensional representations of organic molecules, only those two-dimensional chemical structures which are retained in the database are converted to three-dimensional structures. In this embodiment, a hit found in the search of step (b) of the present method is the most complex representative of a certain class of compounds due to the nature of the SSR procedure. It may therefore be desirable to compare the hit structure with a database of two- or three-dimensional chemical structures generated from starting organic molecules which have not been subjected to SSR or with chemical structures found in a database of commercially available compounds. The purpose of this step is to establish a simpler structure fulfilling the search criteria of step (b) . This further step results in compounds which contain the required three-dimensional pharmacophores, the necessary reactive groups to make their synthesis comparatively simple, and as few unnecessary features as possible. EXAMPLE 1 Complexity of chemical structures in databases A database generated according to step (a) of the present method and including amide structures (cf. Table 1) was analysed for complexity (determined by the number of heteroatoms, rings, etc.) compared to commercially available 3D databases of chemical structures, that of the MayBridge Chemical Company, UK and the National Cancer Institute, USA. The results are shown in Table 2 below. Table 2 DATABASES MAYB. NCI STOCK EATS EATS TYPE 3D 3D 3D 2D full 3D REACTIONS - - - 1 1 Number of 35246 117650 4694 335825 78795 Structures Mean Atoms per 34.24 35.24 20.37 37.51 39.45 Structure Deviation from Mean 10.30 17.11 9.48 10.23 10.69 Mean Rings per 2.31 2.06 0.99 2.00 2.38 Structure Deviation from Mean 0.97 1.49 0.85 0.98 1.01 Mean Rotable 6.05 6.56 3.64 8.08 8.23 Bonds per Structure Deviation from Mean 3.05 5.03 3.25 3.41 3.53 Mean Halogen 1.35 0.46 0.50 0.70 0.88 Atoms per Structure Deviation from Mean 1.71 1.25 1.01 1.08 1.25 Mean Hetero 4.35 4.16 2.38 4.98 5.63 Atoms per Structure Deviation from Mean 1.98 2.84 1.78 2.06 2.66 In Table 2, "MAYB" refers to the MayBridge 3D database, "NCI" refers to the National Cancer Institute 3D database, "STOCK" refers to an internal chemical library of Novo Nordisk A/S used to provide starting materials for the present database, "EATS" refers to a database generated according to step (a) of the present method and is an abbreviation of easy-to-synthesize, "2D full" refers to the database generated from starting materials not subjected to SSR, and "3D" refers to the database generated from starting materials subjected to SSR and converted to 3D structures by means of the CONCORD 3.01 software. The results of the analysis show that the EATS 3D database is comparable to other large 3D databases which, in fact, contain the structures of physically existing compounds with potential biological activity. The compounds contained in the STOCK chemical library seem to contain half the preferred complexity and therefore appear to be a useful source for starting organic molecules. 3D search for pharmacophores 3D database searches were performed using published pharmacophores (cf. D.E. Clark and P. Willett, J. Molecular Graphics 10, December 1992, pp. 194-204) as search criteria and the Unity 2.3 software. The results are shown in Table 3 below. Table 3 DATABASES MAYB. NCI STOCK EATS TYPE 3D 3D 3D 3D REACTIONS AMIDE Number of Structures 35246 117650 4694 78795 Hits using query: 0 0 0 0 1) Anticholinergic Hits using query: 280 1337 4 2181 2) Antileukeamic Hits using query: 4 0 0 0 3) Adrenergic Hits using query: 25 370 2 150 4) Prostaglandin like Hits using query: 1 3 0 8 5) Antineoplastic Hits using query: 2254 12263 59 24472 6) Hallucinogenic Hits using query: 3 0 0 1 7) Serotoninic Hits using query: 131 105 15 1 8) Phorbol Identification of lead compounds The 3D amide database referred to in Table 1 was used to search for dopaminergic compounds using established structures of dopamine D2 antagonists as the search criteria. The search resulted in 374 hits, some of which are known to exhibit biological activity as dopamine D2 antagonists (cf. U. Norinder, J. Comp.-Aided Molecular Design 7. 1993, pp. 671-682) and therefore potential lead compounds for further development. RACLOPRI E pIC50 = 7.49 EATS HITS pIC50 = 6.59 EXAMPLE 2 Preparation of a novel glucagon receptor antagonist The known non-peptide glucagon receptor antagonist, CP-99,711, was reported to bind to the receptor with an IC 50 value of 4 +- 1 μM. Functionally it inhibited glucagon stimulated formation of cAMP with an IC 50 value of 7 +- 1 μM. By changing the flexible amino side chain to the more rigid 4- (dimethylamino)piperidino side chain as in Compound A the binding affinity was retained as was the functional activity. Scheme 1 Databases of three-dimensional chemical structures were generated by combining commercially available chemicals two and two by using simple well-defined chemical reactions (alkylation and acylation) . This provided three-dimensional databases on alkylated and acylated amines where starting materials were easily accessible. Based on the structure of Compound A, search criteria were defined using the intramolecular distances shown in Scheme 2. A search in the above generated three-dimensional databases was conducted resulting in approximately 240 hits comprising a number of novel structures. Variations of the tolerances for the parameters used when defining search criteria, e.g. molecular distances, will influence the number of hits. Scheme 2 Compound C Compound B Among the novel structures one, Compound B, was selected as potentially interesting. However, detailed knowledge of the desired type of compound resulted in a preference for the structure shown in Scheme 2 as Compound C. This compound was then prepared from commercially available N- ( ( 5- b enzy1oxy-lH-indol-3-yl)methyl) -N, -dimethylamine (5-benzyloxygramine) by a simple N-alkylation as described in S cheme 3. Compound C was found to be active in binding to the glucagon receptor. Scheme 3 Compound C

Claims: CLAIMS 1. A method of preparing selected chemical compounds, the method comprising the steps of (a) generating a database of three-dimensional chemical structures by selecting appropriate organic molecules of a known structure as starting compounds and combining two or more of such organic molecules by one or more well-defined chemical reactions to form three-dimensional chemical structures, (b) defining search criteria for identifying potentially active compounds and conducting a search for hits, (c) selecting and synthesizing one or more chemical structures from the hits found in step (b) , and (d) testing the selected chemical compounds synthesized in step (c) for activity. 2. A method according to claim 1, wherein the seach criteria in step (b) are based on known chemical structures exhibiting activity in appropriate assays. 3. A method according to claim 1, wherein only combinations wherein at least one of the organic molecules forms a part of the known structure is made for adoption in the database generated in step (a) . 4. A method according to claim 1, wherein the number of three-dimensional chemical structures generated in step (a) is limited by eliminating starting compounds which are substructures of more complex starting compounds. 5. A method according to claim 4, wherein hits found in step (b) of the method of claim 1 are compared to the full number of chemical structures generated in step (a) or found in a database of commercially available compounds with a view to establishing a simpler structure fulfilling the search criteria of step (b) . 6. A method of preparing selected chemical compounds, the method comprising the steps of (a) generating a database of three-dimensional chemical structures by selecting appropriate organic molecules of a known structure as starting compounds and combining two or more of such organic molecules by one or more well-defined chemical reactions to form three-dimensional chemical structures, (b) defining search criteria for identifying potentially active compounds on the basis of known chemical structures exhibiting activity in appropriate assays, and conducting a search for hits, (c) selecting and synthesizing one or more chemical structures from the hits found in step (b) , and (d) testing the selected chemical compounds synthesized in step (c) for activity. 7. A method according to claim 6, wherein the number of three-dimensional chemical structures generated in step (a) is limited by eliminating starting compounds which are substructures of more complex starting compounds. 8. A method according to claim 7, wherein hits found in step (b) of the method of claim 1 are compared to the full number of chemical structures generated in step (a) or found in a database of commercially available compounds with a view to establishing a simpler structure fulfilling the search criteria of step (b) .

Inventors: ANDERSEN KNUD ERIK, MADSEN PETER, NAERUM LARS, NOERSKOV-LAURITSEN LEIF, ANDERSEN, KNUD, ERIK, MADSEN, PETER, NAERUM, LARS, NOERSKOV-LAURITSEN, LEIF
Applicants: ANDERSEN KNUD ERIK, MADSEN PETER, NAERUM LARS, NOERSKOV LAURITSEN LEIF, NOVONORDISK AS, ANDERSEN, KNUD, ERIK, MADSEN, PETER, NAERUM, LARS, NOERSKOV-LAURITSEN, LEIF, NOVO NORDISK A/S
Application year: 1995
Main classification: G06F17
IPC codes: C07B61, C07D207, C07D209, C07D241, C07D401