Title of Invention	"METHOD OF DRUG DESIGN"
Abstract	The invention provides a method of identifying biologically active compounds comprising: (a) designing a first library of compounds of formula 1 to scan molecular diversity wherein each compound of the library has at least two pharmacophoric groups R1 to R5 as defined below and wherein compound of the library has same number of pharmacophoric groups; (b) assaying the first library of compounds in one or more biological assay(s); and (c) designing a second library wherein each compound of the second library contains one or more additional pharmacophoric group with respect to the first library; such that the/each component of the first and second library is a compound of formula 1.

Title of Invention

"METHOD OF DRUG DESIGN"

Abstract

The invention provides a method of identifying biologically active compounds comprising: (a) designing a first library of compounds of formula 1 to scan molecular diversity wherein each compound of the library has at least two pharmacophoric groups R1 to R5 as defined below and wherein compound of the library has same number of pharmacophoric groups; (b) assaying the first library of compounds in one or more biological assay(s); and (c) designing a second library wherein each compound of the second library contains one or more additional pharmacophoric group with respect to the first library; such that the/each component of the first and second library is a compound of formula 1.

Full Text	Method of Drug Design Field of the invention The invention relates to a method of identifying biologically active compounds, libraries of compounds. Background Small molecules involved in molecular interactions with a therapeutic target, be it enzyme or receptor, are often described in terms of binding elements or pharmacophoric groups which directly interact with the target, and non-binding components which form the framework of the bioactive molecule. In the case of peptide ligands or substrates for instance, usually a number of amino acid side chains form direct interactions with their receptor or enzyme, whereas specific folds of the peptide backbone (and other amino acid residues) provide the structure or scaffold that controls the relative positioning of these side chains. In other words, the three dimensional structure of the peptide serves to present specific side chains in the required fashion suitable for binding to a therapeutic target. The problem is that such models do not allowfor rapid identification of drug candidates owing to the necessity to synthesize an enormous amount of compounds to identify potenţial active compounds. A pharmacophoric group in the context of these libraries is an appended group or substituent, or part thereof, which imparts pharmacological activity to the molecule. Molecular diversity could be considered as consisting of diversity in pharmacophoric group combinations (diversity in substituents) and diversity in the way these pharmacophoric groups are presented (diversity in shape). Libraries of compounds in which either diversity of substituents, or diversity of shape, or both of these parameters are varied systematically are said to scan molecular diversity. Carbohydrate scaffoids provide a unique opportunity to create libraries of structurally diverse molecules, by varying the pharmacophoric groups, the scaffold and the positions of attachment of the pharmacophoric groups in a systematic manner. Such diversity libraries allow the rapid identification of minimal components or fragments containing at least two pharmacophoric groups required for an interaction with a biological target. These fragments can be further optimized to provide potent molecules for drug design. Therefore these types of carbohydrate libraries provide an excellent basis for scanning molecular diversity. In previous applications (W02004014929 and W02003082846) we demonstrated that arrays of novei compounds couid be synthesized in a combinatorial manner. The libraries of molecules described in these inventions were synthesized in a manner such that the position, orientation and Chemical characteristics of pharmacophoric groups around a range of chemical scaffoids, couid be modified and/or controlled. These applications demonstrate the synthesis and biological activity of a number of new chemical entities. Many drug discovery strategies fail owing to lack of knowledge of the bioactive conformation of, or the inability to successfully mimic the bioactive conformation of the natural ligand for a receptor. Libraries of compounds of the present invention allow for the systematic "scanning" of conformational space to identify the bioactive conformation of the target. Typically in the prior art, libraries based on molecular diversity are generated in a random rather than a systematic manner. This type of random approach requires large number of compounds to be included in the library to scan for molecular diversity. Further, this approach may also result in gaps in the model because of not effectively accessing all available molecular space. Therefore, one of the problems in the prior art is the necessity to synthesize an enormous amount of compounds to identify potenţial active compounds. Attempts nave been made to develop peptidomimetics using sugar scaffoids by Sofia et al. (Bioorganic & Medicinal Chemistry Letters (2003) 13, 2185-2189). Sofia describes the synthesis of monosaccharide scaffoids, specifically containing a carboxylic acid group, a masked amino group (N3) and a hydroxyl group as substitution points on the scaffold, with the two remaining hydroxyl groups being converted to their methyl ethers. Sofia teaches a specific subset of scaffoids not encompassed by the present invention and does not contemplate methods to simplify the optimization of phamnacophoric groups. Therefore there remains a need to provide a method of effectively scanning libraries designed from compounds with a wider range of different pharmacophoric groups. The present invention is directed to a method of drug design utilizing iterative scanning libraries, resulting in surprisingly efficient Identification of drug candidates, starting from a selected number of pharmacophores (e.g., two) in the first library and designing subsequent libraries with additional pharmacophores based on SAR information from the first library. The invention can provide a new method for the rapid identification of active molecules. In an embodiment, and to demonstrate the versatility of our invention, one of the G-protein coupled receptors (GPCR's) was chosen as a target: the somatostatin receptor (SST receptor). The tetradecapeptide somatostatin is widely distributed in the endocrine and exocrine system, where it has an essential role in regulating hormone secretion [1-3]. Five different subtypes have been identified to date (SST1-5), which are expressed in varying ratios throughout different tissues in the body. Somatostatin receptors are also expressed in tumours and peptide analogues of somatostatin affecting mainly SST5, such as octreotide, lanreotide, vapreotide and seglitide [4-7] have antiproliferative effects. They are used clinically for the treatment of hormone-producing pituitary, pancreatic, and intestinal tumours. SST5 is also implicated in angiogenesis, opening up the possibility of developing anti-angiogenic drugs that act on the SST5 receptor, for example for the use in oncology. The "core sequence" in somatostatin responsible for its biological activity is Phe-Trp-Lys (FWK), representing a motif of two aromatic groups and a positive charge, which is found in almost all SST receptor active compounds. It will be clearly understood that, if a prior art publication is referred to herein, this reference does not constitute an admission that the publication forms part of the common general knowledge in the art in Australia or in any other country. Summary of the invention In one form, the invention provides a method of identifying biologically active compounds comprising: (a) designing a first library of compounds of formula 1 to scan molecular diversity wherein each compound of the library has at least two pharmacophoric groups R1 to R5 as defined below and wherein compound of the library has same number of pharmacophoric groups; (b) assaying the first library of compounds in one or more biological assay(s); and (c) designing a second library wherein each compound of the second library contains one or more additional pharmacophoric group with respect to the first library; such that the/each component of the first and second library is a compound of formula 1: (Formula Removed) Formula l wherein the ring may be of any configuration; Z is sulphur, oxygen, CH2, C(0), C(0)NRA, NH, NRA or hydrogen, in the case where Z is hydrogen then R1 is not present, RA is selected from the set defined for R1 to R5 or wherein Z and R1 togetherform a heterocycle, X is oxygen or nitrogen providing that at least one X of Formula l is nitrogen, X may also combine independently with one of R1 to R5 to form an azide, RI to Rs are independently selected from the following non-pharmacophoric groups H, methyl and acetyl, and pharmacophoric groups, R1 to R5 are independently selected from the group which includes but is not limited to C2 to C20 alkyl or acyl excluding acetyl; C2 to C20 alkenyl, alkynyl, heteroalkyl; C5 to C20 aryl, heteroaryl, arylalkyl or heteroarylalkyl, which is optionally substituted, and can be branched or linear, or wherein X and the corresponding R moiety, R2 to R5 respectively, combine to form a heterocycle. In another form, the invention comprises biologically active compounds when identified by the method described above. In a preferred embodiment, the invention relates to said method wherein in the first library, three of the substituents R1-R5 are non-pharmacophoric groups and are selected from hydrogen or methyl or acetyl. In a preferred embodiment, the invention relates to said first method wherein in the first library, two of the substituents R1-R5 are non-pharmacophoric groups and are selected from hydrogen or methyl or acetyl. In a preferred embodiment, the invention relates to said first method wherein Z is sulphur or oxygen; In a preferred embodiment, the invention relates to said first method wherein at least one of the pharmacophoric groups is selected from aryl, arylalkyl, heteroaryl, heteroarylalkyl oracyl In a preferred embodiment, the invention relates to a library of compounds selected from compounds of formula 1 wherein in the first library, three of the non-pharmacophoric groups R1-R5 are hydrogen or methyl or acetyl when used according to said first method. In a preferred embodiment, the invention relates to a library of compounds selected from compounds of formula 1 wherein in the second library, two of the non-pharmacophoric groups R1-R5 are hydrogen or methyl or acetyl when used according to said first method. In a preferred embodiment, the invention relates to said first method wherein the/each component of the library is a compound selected from formula 2 or formula 3 or formula 4 (Formula Removed) In a preferred embodiment, the invention relates to said first method wherein the/each component of the library is a compound selected from formula 2 or formula 3 or formula 4 and wherein the/each compound is of the gluco- or galacto-or allo- configuration. In a preferred embodiment, the invention relates to said first method wherein the/each component of the library is a compound selected from formula 2 or formula 3 or formula 4 wherein the/each compound is of the galacto-configuration. In a preferred embodiment, the invention relates to said first method wherein the/each component of the library is a compound selected from formula 2 or formula 3 or formula 4 and wherein the/each compound is of the gluco-configuration. In a preferred embodiment, the invention relates to said first method wherein the/each component of the library is a compound selected from formula 2 or formula 3 or formula 4 and wherein the/each compound is of the allo-configuration. In a preferred embodiment, the invention relates to said first method wherein designing the library comprises molecular modeling to assess molecular diversity. In a preferred embodiment, the invention relates to said first method wherein R-i to R5 opţional substituents include OH, NO, NO2, NH2, N3, halogen, CF3, CHF2, CH2F, nitrile, alkoxy, aryloxy, amidine, guanidiniums, carboxylic acid, carboxylic acid ester, carboxylic acid amide, aryl, cycloalkyl, heteroalkyl, heteroaryl, aminoalkyl, aminodialkyl, aminotrialkyl, aminoacyl, carbonyl, substituted or unsubstituted imine, sulfate, sulfonamide, phosphate, phosphoramide, hydrazide, hydroxamate, hydroxamic acid, heteroaryloxy, aminoaryl, aminoheteroaryl, thioalkyl, thioaryl or thioheteroaryl, which may'optionally be further substituted. The term "halogen" denotes fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine. The term "alkyl" used either alone or in compound words such as "optionally substituted alkyl", "optionally substituted cycloalkyl", "arylalkyl" or "heteroarylalkyl", denotes straight chain, branched or cyclic alkyl, preferably C1-20 alkyl or cycloalkyl. Examples of straight chain and branched alkyl include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, amyl, isoamyl, sec-amyl, 1,2-dimethylpropyl,1, 1-dimethylpropyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl,1, 1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl,1, 2-dimethylbutyl, 1,3-dimethylbutyl, l^^trimethylpropyl.l, 1,2-trimethylpropyl, heptyl, 5-methylbexyl, 1-methylhexyl, 2,2-dimethypentyl, 3,3 dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3trimethylbutyl,1, 1,2-trimethylbutyl, 1,1,3-trimethylbutyl, octyl, 6-methylheptyl, 1-methylheptyl, 1,1,3,3tetramethylbutyl, nonyl,1-, 2-, 3-, 4-, 5-, 6-or 7methyloctyl,1-, 2-, 3-, 4-or 5-ethylheptyl, 1-, 2-or Spropylhexyl, decyl,1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-methylnonyl,1-, 2-, 3-, 4-, 5-or 6-ethyloctyl, 1-, 2-, 3or 4-propylheptyl, undecyM-, 2-, 3-, 4-, 5-, 6-, 7-, 8or 9-methyldecyl,1-, 2-, 3-, 4-, 5-, 6-or 7-ethylnonyl,1-, 2-, 3-, 4-or 5-propyloctyl,1-, 2-or 3-butylheptyl,1-pentylhexyl, dodecyl,1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-methylundecyl,1-, 2-, 3-, 4-, 5-, 6-, 7-or 8- ethyldecyl,1-, 2-, 3-, 4-, 5-or 6-propylnonyl, 1-, 2-, 3- or 4-butyloctyl, 1-2 pentylheptyl and the like. Examples of cyclic alkyl include mono-or polycyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and the like. The term "alkylene" used either alone or in compound words such as "optionally substituted alkylene" denotes the same groups as "alkyl" defined above except that an additional hydrogen has been removed to fomn a divalent radical. It will be understood that the opţional substituent may be attached to or form part of the alkylene chain. The term "alkenyl" used either alone or in compound words such as "optionally substituted alkenyl" denotes groups formed from straight chain, branched or cyclic alkenes including ethylenically mono-, di-or polyunsaturated alkyl or cycloalkyl groups as defined above, preferably C2-6 alkenyl. Examples of alkenyl include vinyl, allyl,1-methylvinyl, butenyl, iso-butenyl, 3-methyl-2butenyl, 1-pentenyl, cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl, cyclohexenyl,1-heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl,1-decenyl, 3-decenyl, 1,3-butadienyl, 1,4-pentadienyl, 1,3 cyclopentadienyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,3cyclohexadienyl, 1,4-cyclohexadienyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl and 1,3,5,7-cyclooctatetraenyl. The term "alkynyl" used either alone or in compound words, such as "optionally substituted alkynyl" denotes groups formed from straight chain, branched, or mono-or poly-or cyclic alkynes, preferably C 2-6 alkynyl. Examples of alkynyl include ethynyl,1-propynyl, 1-and 2butynyl, 2-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4pentynyl, 2-hexynyl, 3-hexylnyl, 4-hexynyl, 5-hexynyl, 10-undecynyl,4-ethyl-l-octyn-3-yl, 7-dodecynyl, 9-dodecynyl, 10-dodecynyl,3-methyl-1-dodecyn-3-yl, 2-tridecynyl, Utridecynyl, 3-tetradecynyl, 7-hexadecynyl, 3-octadecynyl and the like. The term "alkoxy" used either alone or in compound words such as "optionally substituted alkoxy" denotes straight chain or branched alkoxy, preferably C1-7 alkoxy. Examples of alkoxy include methoxy, ethoxy, npropyloxy, isopropyloxy and the different butoxy isomers. The term "aryloxy" used either alone or în compound words such as "optionally substituted aryloxy" denotes aromatic, heteroaromatic, arylalkoxy or heteroaryl alkoxy, preferably C6-13 aryloxy. Examples of aryloxy include phenoxy, benzyloxy,1-napthyloxy, and 2-napth'yloxy. The term "acyl" used either alone or in compound words such as "optionally substituted acy l "or "heteroarylacyl" denotes carbamoyl, aliphatic acyl group and acyl group containing an aromatic ring, which is referred to as aromatic acyl or a heterocyclic ring which is referred to as heterocyclic acyl. Examples of acyl include carbamoyl; straight chain or branched alkanoyl such as formyl, acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 2,2-dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl, and icosanoyl; alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl, t butoxycarbonyl, t-pentyloxycarbonyl and heptyloxycarbonyl; cycloalkylcarbonyl such as cyclopropylcarbonyl cyclobutylcarbonyl, cyclopentylcarbonyl and cyclohexylcarbonyl; alkylsulfonyl such as methylsulfonyl and ethylsulfonyl; alkoxysulfonyl such as methoxysulfonyl and ethoxysulfonyl; aroyl such as benzoyl, toluoyl and naphthoyl; aralkanoyl such as phenylalkanoyl (e. g. phenylacetyl, phenylpropanoyl, phenylbutanoyl, phenylisobutyl, phenylpentanoyl and phenylhexanoyl) and naphthylalkanoyl (e. g. naphthylacetyl, naphthlpropanoyl and naphthylbutanoyl); aralkenoyl such as phenylalkenoyl (e. g. phenylpropenoyl, phenylbutenoyl, phenylmethacrylyl, phenylpentenoyl and phenylhexenoyl and naphthylalkenoyl (e. g. naphthylpropenoyl, naphthylbutenoyl and naphthylpentenoyl); aralkoxycarbonyl such as phenylalkoxycarbonyl (e. g. benzyloxycarbonyl); aryloxycarbonyl such as phenoxycarbonyl and naphthyloxycarbonyl; aryloxyalkanoyl such as phenoxyacetyl and phenoxypropionyl; arylcarbamoyl such as phenylcarbamoyl; arylthiocarbamoyl such as phenylthiocarbamoyl; arylglyoxyloyl such as pnenylglyoxyloyl and naphthylglyoxyloyl; arylsulfonyl such as phenylsulfonyl and naphthylsulfonyl; heterocycliccarbonyl; heterocyclicalkanoyl such as thienylacetyl, thienylpropanoyl, thienylbutanoyl, thienylpentanoyl, thienylhexanoyl, thiazolylacetyl, thiadiazolylacetyl and tetrazolylacetyl; heterocyclicalkenoyl such as heterocyclicpropenoyl, heterocyclicbutenoyl, heterocyclicpentenoyl and heterocyclichexenoyl; and heterocyclicglyoxyloyl such as thiazolylglyoxyloyl and thienyglyoxyloyl. The term "aryl" used either alone or in compound words such as "optionally substituted aryl", "arylalkyl" or "heteroaryl" denotes single, polynuclear, conjugated and fused residues of aromatic hydrocarbons or aromatic heterocyclic ring systems. Examples of aryl include phenyl, biphenyl, terphenyl, quaterphenyl, phenoxyphenyl, naphthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl, benzanthracenyl, dibenzanthracenyl, phenanthrenyl, fluorenyl, pyrenyl, indenyl, azulenyl, chrysenyl, pyridyl, 4-phenylpyridyl, 3-phenylpyridyl, thienyl, furyl, pyrryl, pyrrolyl, furanyl, imadazo'lyl, pyrrolydinyl, pyridinyl, piperidinyl, indolyl, pyridazinyl, pyrazolyl, pyrazinyl, thiazolyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothienyl, purinyl, quinazolinyl, phenazinyl, acridinyl, benzoxazolyl, benzothiazolyl and the like. Preferably, the aromatic heterocyclic ring system contains 1 to 4 heteroatoms independently selected from N, O and S and containing up to 9 carbon atoms in the ring. The term "heterocycle" used ejther alone or in compound words as "optionally substituted heterocycle" denotes monocyclic or polycyclic heterocyclyl groups containing at least one heteroatom atom selected from nitrogen, sulphur and oxygen. Suitable heterocyclyl groups include N-containing heterocyclic groups, such as, unsaturated 3 to 6 membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl or tetrazolyl; saturated to 3 to 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, such as, pyrrolidinyl, imidazolidinyl, piperidin or piperazinyl ; unsaturated condensed heterocyclic groups containing 1 to 5 nitrogen atoms, such as, indolyl, isoindolyl, indolizinyl, benzimidazoyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl or tetrazolopyridazinyl; unsaturated 3 to 6-membered heteromonocyclic group containing an oxygen atom, such as, pyranyl or furyl; unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulphur atoms, such as, thienyl; unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as, oxazolyl, isoxazolyl or oxadiazolyl; saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as, morpholinyl; unsaturated condensed heterocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as, benzoxazolyl or benzoxadiazolyl; unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as, thiazolyl or thiadiazolyl; saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as thiazolidinyl; and unsaturated condensed heterocyclic group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as, benzothiazolyl or benzothiadiazolyl. In a preferred embodiment, the invention relates tq said first method wherein the compounds are synthesized. In a preferred embodiment, the invention relates to said first method wherein the biological assays are selected from peptide ligand class of GPCRs. In another aspect the invention provides a compound according to formula 1 in which at least one X is nitrogen, and said X is combined with the corresponding R2-R5 to form a heterocycle. The synthesis of the heterocyclic components of the present invention is disclosed in WO 2004/022572. In a preferred embodiment, the invention provides a compound according to formula 1 wherein X and R2 combine to form a heterocycle. In a preferred embodiment, the invention provides a compound according to formula 1 wherein the heterocycle is heteroaryl, includ ing triazoles, benzimidazoles, benzimidazolone, benzimidazolothione, imidazole, hydantoine, thiohydantoine and purine. Detailed Description of the invention The embodiments of the invention will be described with reference to the foilowing examples. Where appropriate, the foilowing abbreviations are used. Ac Acetyl DTPM 5-Acyl-1,3-dimethylbarbiturate Ph Phenyl TBDMS t-Butyldimethylsilyl TBDPS t-Butyldiphenylsilyl Bn benzyl Bz benzoyl Me methyl DCE 1,2-dichloroethane DCM dichloromethane, methylene chloride Tf trifluoromethanesulfonyl Ts 4-methylphenylsulfonyl, p-toluenesulfonyl DMF N,N-dimethylformamide DMAP N,N-dimethylaminopyridine α,α-DMT α,α-dimethoxytoluene, benzaldehyde dimethyl acetal DMSO dimethylsulfoxide DTT dithiothreitol DMTST Dimethyl(methylthio)sulphoniumtrifluoro-methanesulphonate TBAF tetra-n-butylammonium fluoride Part A: Preparation of building blocks: In order to fully enable the invention, there is described below methods for the preparation of certain building blocks used in the preparation of the compounds of the invention. The building blocks described are suitable for both solution and solid phase synthesis of the compounds of the invention. Example A: Synthesis of a 2.4 dinitroaen containina Galactopyranoside Buildina Block (Formula Removed) Conditions: (i) a,a-dimethoxytoluene (α,α-DMT), p-toluenesulphonic acid (TsOH), acetonitrile (MeCN), 76°C, 85%; (ii) Benzoylchloride (BzCI), triethylamine; DCM, 99%; (iii) methanol (MeOH)/MeCN/water, TsOH, 75°C, 98%; (iv) t-butyldiphenylsilylchloride (TBDPS-CI), imidazole, pyridine, 120°C, 99% ; (v) Tf2O, pyridine, DCM, 0°C, 100%;(b) NaN3, DMF, 16hr, RT, 99%. Example B: Synthesis of a 3-nitroaen containina Gulopyranoside Buildina Block(Formula Removed) Conditions: (i) (a) trifluoromethanesulfonic anhydride (Tf2O), pyridine, -20°C, dichloromethane (DCM), 1 hour, 100%, (b) sodium azide (NaN3), N,N-dimethylformamide (DMF), 50°C, 5 hours, quantitative; (ii) TsOH, MeCN/ MeOH/water (12:3:1), 90°C, 6 hours, 88%(iii) TBDPSCI, DMAP, pyridine, 120°C, 12 hours, 93% Examole C: Synthesis of a 2,6-dinitroaen substituted Glucopvranoside Building Block (Formula Removed) Conditions: (i) (a) Tosylchlodride, pyridine, RT, 24 hours, 33%(b) NaN3, DMF, RT, 168 hours. (Formula Removed) Example D: Synthesis of a 2-nitrogen containing Tallopyranoside Building Block Conditions: (i) TBDPSCI, imidazole, 1,2-DCE, reflux; (ii) NaOMe/MeOH; (iii) (a) Tf2O, pyridine, -20°C, DCM, 1 hour, (b) NaN3, DMF, 50°C, 5 hours; (iv) TsOH, MeCN/MeOH/water; (v) benzoylchloride, DMAP, 1,2-DCE, -20°C. Examole E: Synthesis of two 3-nitroaen containina Altropyranoside Buildina Block (Formula Removed) Conditions: (i) cyclohexanone dimethylacetal, TsOH, MeCN; (ii) p-methoxybenzaldehyde dimethylacetal, TsOH, MeCN; (iii) DIBAL, -78°C, diethyl ether; (iv) (a) Tf2O, pyridine, -20°C, DCM, 1 hour, (b) NaN3, DMF, 50°C, 5 hours; (v) TsOH, MeCN/MeOH/water; (vi) TBDPSCI, DMAP, 1,2-DCE; (vii) (a) CAN, (b) BzCI, DMAP, 1,2-DCE, (c) TsOH, MeCN/MeOH/water; (viii) TBDPSCI, DMAP, 1,2-DCE. Examole F: Svnthesis of a 2-nitroaen containina Glucopvranoside Buildina Block (Formula Removed) Conditions: (i) α,α-DMT, TsOH, MeCN; (ii) 1,2-DCE, BzCI, DMAP; (iii) TsOH, MeOH/MeCN; (iv) TBDPS-CI, DMAP, 1,2-DCE. (Formula Removed) Conditions: (i) TBDPSCI, DMAP, pyridine, 120°C, 0.5 hours, 81%; (ii) a. (Bu)2SnO, MeOH; b. Benzoylchloride, RT, 24 hour; Example G: Svnthesis of a 2-nitrogen containinq Allopvranoside Building Block (Formula Removed) Conditions: (i) DCM/pyridine, MsCI, DMAP, 0°C; (ii) sodium benzoate, dimethylsulphoxide (DMSO), 140°C; (iii) TsOH, MeOH/MeCN/water; (iv) TBDPS-Cl, imidazole, DCM, 1 hour, reflux. The Solid Phase Library Synthesis of Sugars is illustrated in Scheme 1. The reaction conditions are as follows: (A) 2P compound synthesis: R1=R2=OMe; i) 2-naphthalene methanol, DMTST, DCM; ii) TCA-Wang resin, BF3Et2O, DCM; iii) NaOMe, methanol; iv) a. KOtBu, DMF; b. Mel, DMF; v) HF 'proton sponge', AcOH, DMF, 65°C; vi) a. KOtBu, DMF; b. Mel, DMF; vii) 1,4-dithio-DL-threitol, KOtBu, DMF; viii) HBTU, Fmoc-ß-Ala-OH, DIPEA, DMF; ix) piperidine/DMF (1/4); x) TFA, Et3SiH, DCM (B) 3P compound synthesis: Ri=methyl-2-naphthyl, R2=OMe; i) 2-naphthalene methanol, DMTST, DCM; ii) TCA-Wang resin, BF3 Et2O, DCM; iii) NaOMe, methanol; iv) a. KOtBu, DMF; b. 2-bromomethyl-naphthalene, DMF; v) HF' proton sponge1, AcOH, DMF, 65°C; vi) a. KOtBu, DMF; b. Mei, DMF; vii) 1,4- dithio-DL-threitol, KOtBu, DMF; viii) HBTU, Fmoc-ß-Ala-OH, DlPEA, DMF; ix) piperidine/DMF (1/4); x) TFA, Et3SiH, DCM (C) 4P compound synthesis: Ri=methyl-2-naphthyl, R2=4-chlorobenzyl i) 2-naphthalene methanol, DMTST, DCM; ii) TCA-Wang resin, BF3Et2O, DCM; iii) NaOMe, methanol; iv) a. KOtBu, DMF; b. 2-bromomethyl-naphthalene, DMF; v) HF'proton sponge', AcOH, DMF, 65°C; vi) a. KOtBu, DMF; b. 4- chlorobenzylbromide, DMF; vii) 1,4-dithio-DL-threitol, KOtBu, DMF; viii) HBTU, Fmoc-p-Ala-OH, DIPEA, DMF; ix) piperidine/DMF (1/4); x) TFA, Et3SiH, DCM (Formula Removed) Scheme 1 Solid Phase library Synthesis ofsugars Scheme 2 Synthesis of allose 2,6 building block; an example of synthesis of 2P, 3P and 4P type compound The synthesis of the Allose 2,6N building block is illustrated in Scheme 2. The reaction conditions are as follows: i) p-methoxybenzaldehyde dimethylacetal, camphorsulfonic acid, N,N-dimethylformamide (DMF); ii) Tf20, pyridine, dichloromethane (DCM); iii) tetrabutylammonium benzoate, DMF, 55°C; iv) BH3THF, Bu2BOTf, DCM; v) methanesulfonylchloride, pyridine, DCM; vi) sodium azide, DMF, 85°C; vii) sodium methanolate (NaOMe), methanol; viii) n-butanol, ethylene diamine, reflux; ix) DTPM reagent, methanol; x) benzoic anhydride, pyridine; xi) trifluoroacetic acid, triethylsilane, DCM Designing libraries: The design of the libraries is based on the presentation of a positive charge and a varying number of aromatic substituents in different spaţial arrangements on a monosaccharide scaffold. Starting with a positive charge and one aromatic displayed on the core scaffold, actives from this first library were elaborated on by further variation and addition of more aromatic substituents to quickly identify highly active molecules. The first library of compounds comprises two pharmacophoric groups, known as a 2P library, in particular, one containing an aromatic and a positive charge. The library was designed such that each molecule presents two pharmacophoric groups in different relative orientation or presentation (e.g., distance, relative angle, i.e. relative position in space is different). Actives from this library were identified and SAR information from this library was used to design subsequent library of compounds wherein each compound may include three pharmacophoric groups, known as a 3P library. Subsequent libraries with four pharmacophoric groups are called 4P library, etc. Members of significantly improved activity were identified out of the second library and were selected for further drug development. The method of the invention includes real and virtual libraries. Thus, the molecules according to formula 1 are well suited for generating iterative scanning libraries, starting from a selected number of pharmacophores (eg, two) in the first library and designing subsequent libraries with additional pharmacophores based on SAR Information from the first library, thereby assisting in delineating pharmacophores. The 2P and 3P library of compounds were synthesized according to the building blocks as described in Examples A-G. The 2P library (Table 1) was designed to scan molecular diversity for 2P molecules, comprising an aromatic and a positive charge. The 2P library was screened for biological activity and the results are given in Table 1. Similarly, the 3P library was designed to scan molecular diversity for 3P molecules. Design of 3P library resulted from SAR obtained from 2P library in Table 1. The 3P library was screened for biological activity and the results are given in Table 2. A visual analysis of the results according to Table 1 (2P library) and Table 2 (3P Library) indicates that: 1.1,2 allose substitution according to formula 3 (and Scaffold C/D) presents the most active arrangement of molecules in the library wherein Z is oxygen, R-\ is naphthyl and R2 is propylamine or ethylamine. These compounds represent most actives at low nM range, and are suitable candidates for further drug development. 2. R-i as naphthyl is more active than the corresponding p-chlorobenzyl substituent. 3. 1, 2 allose according to formula 3 (Scaffold C/D) is more active than the corresponding 1, 2 glucose conformation (Scaffold A/B). 4. 1, 2 substitution according to formula 3 (Scaffold C/D) is more active than the corresponding 2, 6 substitution according to formula 4 (Scaffold G) 5. R2 as propylamine and ethylamine are more active than methylamine wherein Z, R1 and R2 are as described above. 6. 2, 3 allose substitution according to formula 3 (Scaffold C/D) presents the more actives wherein R2 is ethylamine, and R3 is p-chlorobenzyl compared to corresponding R2 as propylamine and ethylamine wherein R3 is p-chlorobenzyl substituent, and also wherein R2 is methylamine, ethylamine or propylamine and R3 is naphthyl. 7. 2, 3 glucose substitution according to formula 3 (scaffold A/B) presents the more actives wherein R2 is propylamine and R3 is naphthyl compared to corresponding R2 as methylamine or ethylamine, and also wherein R2 is methylamine, ethylamine or propylamineand R3 is p-chlorobenzyl. 8. 2, 4 and 3, 4 substitutions according to formula 3 (Scaffold G) present the least actives. Part B: Biological assays Example H. In vitro screening of compounds against somatostatin subtypes SSTR-1 to SSTR-5 General method Receptor membrane preparations containing the desired cloned receptor (for example cloned human somatostatin receptor subtype 5, SSTR5) and radio-labeled ligand were diluted at the concentration required for testing and according to the specific parameters associated with the selected receptor-ligand combination, including receptor Bmax, ligand Kd and any other parameters necessary to optimize the experimental conditions. When tested for competition activity to the reference ligand, "compound " was mixed with membrane suspension and the radiolabeled reference ligand (with or without an excess of unlabeled ligand to the receptor for determination of non-specific binding) and incubated at the temperature required by internai standard operating procedures. Following incubation, the binding reaction was stopped by the addition of ice-cold washing buffer and filtered on appropriate filters, which are then counted. Data analysis and curve-fitting was performed with XLfit (lDBS). Preparation ofcompounds 10mM solutions of test compounds in 100% DMSO were prepared. ~160 ni was used for each dilution (20 nl/well in triplicate). A 1.25 mM assay stock was prepared by making a 1:8 dilution of the 10 mM solution. To 30 ^L of the 10 mM solution was added 210 µL milli-Q H2O. A 1:5 dilution series in milli-Q H20 was then prepared. Final concentration Final concentration in SST4 assay in S ST 5 assay A. 240 µL of 1.25 mM 0.25 mM 0.125 mM B. 48 µL A+ 192 µLmQ 0.05 mM 0.025 mM C. 24 µL B+ 192 µLmQ 0.01 mM 0.005 mM etc Assays were performed in triplicate at each concentration within the 1:5 dilution series: 250µM, 50µM, 10µM, 2mM, 0.4µM, 0.08µM, 0.016µM, 0.0032 µM, etc. (for SST4 assay) and 125µM, 10µM, 2µM, 1uM, 0.5. µM, etc (for SST5 assay). Filter plate assay for SST5 receptor Human SST5 somatostatin receptor was transfected into HEK-293 EBNA cells. Membranes were suspended in assay buffer (50 mM Tris-HCI, 1 mM EGTA, 5 mM MgCI2, 10% sucrose, pH 7.5). The receptor concentration (Bmax) was 0.57 pmol/mg proteinKd for [125I]SST-14 Binding 0.31 nM, volume 0.4 ml per vial (400 microassays/vial), and protein concentration 1.03 mg/ml. After thawing the frozen receptor preparation rapidly, receptors were diluted with binding buffer, homogenized, and kept on ice. 1. Use Multiscreen glass fiber filter plates (Millipore, Cat No MAFCNOB10) precoated with o.5 % PEI for ~ 2hr at 4°C. Before use add 200 ul/well assay buffer and filter using Multiscreen Separation System. 2. Incubate 5.5 µg of membranes (40 ui of a 1:40 dilution), buffer and [125I]SST-14 (4 nM, -80 000 cpm, 2000 Ci/mmol) in a total volume of 200 ui for 60 min at 25°C. Calculate IC50 for SST-14 (a truncated version of the natural ligand SST-28) (Auspep, Cat No 2076) and SST-28 (Auspep, Cat No 1638). Prepare serial dilutions (1:5) of compounds, as descnbed above and instead of adding SST-14 in well, add 20 µl of compounds (Table 3). 3. Filter using Multiscreen Separation System with 5 x 0.2 ml ice-cold Assay buffer. 4. Remove the plastic underdrain and dry plate in oven for 1 hr at 40°C. 5. Seal tape to the bottom of the plate. 6. Add 50 ul/well scintillant (Supermix, Wallac, Cat No 1200-439). 7. Seal and count in the BJET, program 2. Table 3 (Table Removed) TB: total binding NSB: non-speciflc binding Part C: General Experimental Methods Example I: HPLC Method for compounds in Tables 1 and 2 The HPLC separation of compounds in Tables 1 and 2 was conducted under Method A or Method B as shown below. Method A Column: Agilent SB Zorbax C18 4.6x50mm (5um, 80 Â) LC mobile phase: 5% aqueous MeCN /1 min 100%MeCN/7-12min Method B Column: Agilent SB Zorbax C18 4.6x50mm (5um, 80 Â) LC mobile phase: 5% aq MeCN/1 min 30% aq MeCN/3min 40%aqMeCN/12 min 100%MeCN/13-15 min Key to Building Blocks for Tables 1 and 2 Table 1: * % SST5 radio- ligand binding displaced at conc (µM) for 2P library of compounds Table 2: * % SST5 radio- ligand binding displaced at conc (µM) for 3P library of compounds; ; R4 = X30; compounds 60-63, 119 and 156-159 are comparative compounds from 2P library "+ +": % SST5 radio- ligand binding displaced at conc (µM) >60% "+": % SST5 radio- ligand binding displaced at conc (µM) 60>+>40% "-": % SST5 radio- ligand binding displaced at conc (µM) - Table 1: Biological activity of example 2P library (Table Removed) Table 2: Biological activity of example 3P library (Table Removed) Throughout the specification and the claims (if present), unless the context requires otherwise, the term "comprise", or variations such as "comprises" or "comprising", will be understood to apply the inclusion of the stated integer or group of integers but not the exclusion of any other integer or group of integers. Throughout the specification and claims (if present), unless the context requires otherwise, the term "substantially" or "about" will be understood to not be limited to the value for the range qualified by the terms. It should be appreciated that various other changes and modifications can be made to any embodiment described without departing from the spirit and scope of the invention. References [1] Patel, Y.C. (1999) Somatostatin and its receptor family. Front. Neuroendocr. 20, 157-198 [2] Csaba, Z. and Dournaud, P. (2001) Cellular biology of somatostatin receptors. Neuropeptides 35, 1-23 [3] T Reisine, T. (1995) Somatostatin receptors; Am. J. Pysiol. (Gastrointest. Liver Physiol. 32) 269, G813-G820 [4] Bauer, W. et al. (1982) SMS201-995: A very potent and selective octapeptide analogue of somatostatin with prolonged action. Life Sci. 31, 1133-1140 [5] Lamberts, S.W.J. et al. (1996) Drug therapy: Octreotide. N. Eng. J. Med. 334, 246-254 [6] Robinson, C. and Castaner, J. (1994) Lanreotide acetate. Drugs Futune 19, 992-999 [7] Reisine, T. and Bell. G.l. (1995) Molecular biology of somatostatin receptors. Endocr. Rev. 16, 427-442 CLAIMS: 1. A method of identifying biologically active compounds comprising: (a)designing a first library of compounds of formula 1 to scan molecular diversity wherein each compound of the library has at least two pharmacophoric groups R1 to R5 as defined below and wherein compound of the library has same number of pharmacophoric groups; (b)assaying the first library of compounds in one or more biological assay(s); and (c)designing a second library wherein each compound of the second library contains one or more additional pharmacophoric groups with respect to the first library such that the/each component of the first and second library is a compound of formula 1: (Formula Removed) wherein the ring may be of any configuration; Z is sulphur, oxygen, CH2, C(0), C(0)NRA, NH, NRA or hydrogen, in the case where Z is hydrogen then R1 is not present, RA is selected from the set defined for R1 to R5, or wherein Z and R1 together form a heterocycle, X is oxygen or nitrogen providing that at least one X of Formula l is nitrogen, X may also combine independently with one of R1 to R5 to form an azide, R1 to R5 are independently selected from the foilowing non-pharmacophoric groups H, methyl and acetyl, and pharmacophoric groups R1 to R5 are independently selected from the group which includes but is not limited to C2 to C20 alkyl or acyl excluding acetyl; C2 to C20 alkenyl, alkynyl, heteroalkyl; C5 to C20 aryl, heteroaryl, arylalkyl or heteroarylalkyl, which is optionally substituted, and can be branched or linear, or wherein X and the corresponding R moiety, R2 to R5 respectively, combine to form a heterocycle. 2. A method according to claim 1 wherein in the first library, three of the substituents R1-R5 are non-pharmacophoric groups and are selected from hydrogen or methyl or acetyl. 3. A method according to claim 1 wherein in the first library, two of the substituents R1-R5 are non-pharmacophoric groups and are selected from hydrogen or methyl or acetyl. 4. A method according to claim 1 wherein Z is sulphur or oxygen. 5. A method according to any one of claims 1-4 wherein at least one of the pharmacophoric groups is selected from aryl, arylalkyl, heteroaryl, heteroarylalkyl or acyl. 6. A library of compounds selected from compounds of formula 1 when used according to claim 2. 7. A library of compounds selected from compounds of formula 1 when used according to claim 3. 8. A method according to any of claims 1 to 5 wherein the/each component of the library is a compound selected from formula 2 or formula 3 or formula 4. (Formula Removed) 9. A method according to claim 8 wherein the/each compound is of the gluco- or galacto- or allo- configuration. 10. A method according to claim 9 wherein the/each compound is of the gluco- configuration. 11. A method according to claim 9 wherein the/each compound is of the allo- configuration. 12. A method according to claim 9 wherein the/each compound is of the galacto- configuration. 13. A method according to claim 1 wherein designing the library comprises molecular modeling to assess molecular diversity. 14. A method according to claim 1 wherein R1 to R5 opţional substituents are selected from OH, NO, NO2, NH2, N3, halogen, CF3, CHF2, CH2F, nitrile, alkoxy, aryloxy, amidine, guanidiniums, carboxylic acid, carboxylic acid ester, carboxylic acid amide, aryl, cycloalkyl, heteroalkyl, heteroaryl, aminoalkyl, aminodialkyl, aminotrialkyl, aminoacyl, carbonyl, substituted or unsubstituted imine, sulfate, sulfonamide, phosphate, phosphoramide, hydrazide, hydroxamate, hydroxamic acid, heteroaryloxy, aminoaryl, aminoheteroaryl, thioalkyl, thioaryl or thioheteroaryl, which may optionally be further substituted. 15. A method according to any of claims 1 to 5 wherein the compounds are synthesized. 16. A method according to claim 1 wherein the biological assays involve peptide ligand class of GPCRs. 17. A compound according to formula 1 in which at least one X is nitrogen, and said X is combined with the corresponding R2-R5 to form a heterocycle. 18. A compound according to claim 17 wherein X and R2 combine to form a heterocycle. 19. A compound according to any one of claims 17 and 18 wherein the heterocycle is heteroaryl. 20. A compound according to any one of claims 17-19 wherein the heteroaryl is selected from triazoles, benzimidazoles, benzimidazolone, benzimidazolothione, imidazole, hydantoine, thiohydantoine and purine.

Full Text

Method of Drug Design
Field of the invention
The invention relates to a method of identifying biologically active compounds, libraries of compounds.
Background
Small molecules involved in molecular interactions with a therapeutic target, be it enzyme or receptor, are often described in terms of binding elements or pharmacophoric groups which directly interact with the target, and non-binding components which form the framework of the bioactive molecule. In the case of peptide ligands or substrates for instance, usually a number of amino acid side chains form direct interactions with their receptor or enzyme, whereas specific folds of the peptide backbone (and other amino acid residues) provide the structure or scaffold that controls the relative positioning of these side chains. In other words, the three dimensional structure of the peptide serves to present specific side chains in the required fashion suitable for binding to a therapeutic target. The problem is that such models do not allowfor rapid identification of drug candidates owing to the necessity to synthesize an enormous amount of compounds to identify potenţial active compounds.
A pharmacophoric group in the context of these libraries is an appended group or substituent, or part thereof, which imparts pharmacological activity to the molecule.
Molecular diversity could be considered as consisting of diversity in pharmacophoric group combinations (diversity in substituents) and diversity in the way these pharmacophoric groups are presented (diversity in shape). Libraries of compounds in which either diversity of substituents, or diversity of shape, or both of these parameters are varied systematically are said to scan molecular diversity.
Carbohydrate scaffoids provide a unique opportunity to create libraries of structurally diverse molecules, by varying the pharmacophoric groups, the scaffold and the positions of attachment of the pharmacophoric groups in a systematic manner. Such diversity libraries allow the rapid identification of minimal components or fragments containing at least two pharmacophoric groups required for an interaction with a biological target. These fragments can be further optimized to provide potent molecules for drug design. Therefore these types of carbohydrate libraries provide an excellent basis for scanning molecular diversity.
In previous applications (W02004014929 and W02003082846) we demonstrated that arrays of novei compounds couid be synthesized in a combinatorial manner. The libraries of molecules described in these inventions were synthesized in a manner such that the position, orientation and Chemical characteristics of pharmacophoric groups around a range of chemical scaffoids, couid be modified and/or controlled. These applications demonstrate the synthesis and biological activity of a number of new chemical entities.
Many drug discovery strategies fail owing to lack of knowledge of the bioactive conformation of, or the inability to successfully mimic the bioactive conformation of the natural ligand for a receptor. Libraries of compounds of the present invention allow for the systematic "scanning" of conformational space to identify the bioactive conformation of the target.
Typically in the prior art, libraries based on molecular diversity are generated in a random rather than a systematic manner. This type of random approach requires large number of compounds to be included in the library to scan for molecular diversity. Further, this approach may also result in gaps in the model because of not effectively accessing all available molecular space.
Therefore, one of the problems in the prior art is the necessity to synthesize an enormous amount of compounds to identify potenţial active compounds. Attempts
nave been made to develop peptidomimetics using sugar scaffoids by Sofia et al. (Bioorganic & Medicinal Chemistry Letters (2003) 13, 2185-2189). Sofia describes the synthesis of monosaccharide scaffoids, specifically containing a carboxylic acid group, a masked amino group (N3) and a hydroxyl group as substitution points on the scaffold, with the two remaining hydroxyl groups being converted to their methyl ethers. Sofia teaches a specific subset of scaffoids not encompassed by the present invention and does not contemplate methods to simplify the optimization of phamnacophoric groups.
Therefore there remains a need to provide a method of effectively scanning libraries designed from compounds with a wider range of different pharmacophoric groups.
The present invention is directed to a method of drug design utilizing iterative scanning libraries, resulting in surprisingly efficient Identification of drug candidates, starting from a selected number of pharmacophores (e.g., two) in the first library and designing subsequent libraries with additional pharmacophores based on SAR information from the first library.
The invention can provide a new method for the rapid identification of active molecules.
In an embodiment, and to demonstrate the versatility of our invention, one of the G-protein coupled receptors (GPCR's) was chosen as a target: the somatostatin receptor (SST receptor). The tetradecapeptide somatostatin is widely distributed in the endocrine and exocrine system, where it has an essential role in regulating hormone secretion [1-3]. Five different subtypes have been identified to date (SST1-5), which are expressed in varying ratios throughout different tissues in the body. Somatostatin receptors are also expressed in tumours and peptide analogues of somatostatin affecting mainly SST5, such as octreotide, lanreotide, vapreotide and seglitide [4-7] have antiproliferative effects. They are used
clinically for the treatment of hormone-producing pituitary, pancreatic, and intestinal tumours. SST5 is also implicated in angiogenesis, opening up the possibility of developing anti-angiogenic drugs that act on the SST5 receptor, for example for the use in oncology. The "core sequence" in somatostatin responsible for its biological activity is Phe-Trp-Lys (FWK), representing a motif of two aromatic groups and a positive charge, which is found in almost all SST receptor active compounds.
It will be clearly understood that, if a prior art publication is referred to herein, this reference does not constitute an admission that the publication forms part of the common general knowledge in the art in Australia or in any other country.
Summary of the invention
In one form, the invention provides a method of identifying biologically active compounds comprising:
(a) designing a first library of compounds of formula 1 to scan molecular
diversity wherein each compound of the library has at least two
pharmacophoric groups R1 to R5 as defined below and wherein compound
of the library has same number of pharmacophoric groups;
(b) assaying the first library of compounds in one or more biological assay(s);
and
(c) designing a second library wherein each compound of the second library
contains one or more additional pharmacophoric group with respect to the
first library;
such that the/each component of the first and second library is a compound of formula 1: (Formula Removed)
Formula l
wherein the ring may be of any configuration;
Z is sulphur, oxygen, CH2, C(0), C(0)NRA, NH, NRA or hydrogen, in the case where Z is hydrogen then R1 is not present, RA is selected from the set defined for R1 to R5 or wherein Z and R1 togetherform a heterocycle,
X is oxygen or nitrogen providing that at least one X of Formula l is nitrogen, X may also combine independently with one of R1 to R5 to form an azide,
RI to Rs are independently selected from the following non-pharmacophoric groups H, methyl and acetyl, and pharmacophoric groups, R1 to R5 are independently selected from the group which includes but is not limited to C2 to C20 alkyl or acyl excluding acetyl; C2 to C20 alkenyl, alkynyl, heteroalkyl; C5 to C20 aryl, heteroaryl, arylalkyl or heteroarylalkyl, which is optionally substituted, and can be branched or linear,
or wherein X and the corresponding R moiety, R2 to R5 respectively, combine to form a heterocycle.
In another form, the invention comprises biologically active compounds when identified by the method described above.
In a preferred embodiment, the invention relates to said method wherein in the first library, three of the substituents R1-R5 are non-pharmacophoric groups and are selected from hydrogen or methyl or acetyl.
In a preferred embodiment, the invention relates to said first method wherein in the first library, two of the substituents R1-R5 are non-pharmacophoric groups and are selected from hydrogen or methyl or acetyl.
In a preferred embodiment, the invention relates to said first method wherein Z is sulphur or oxygen;
In a preferred embodiment, the invention relates to said first method wherein at least one of the pharmacophoric groups is selected from aryl, arylalkyl, heteroaryl, heteroarylalkyl oracyl
In a preferred embodiment, the invention relates to a library of compounds selected from compounds of formula 1 wherein in the first library, three of the non-pharmacophoric groups R1-R5 are hydrogen or methyl or acetyl when used according to said first method.
In a preferred embodiment, the invention relates to a library of compounds selected from compounds of formula 1 wherein in the second library, two of the non-pharmacophoric groups R1-R5 are hydrogen or methyl or acetyl when used according to said first method.
In a preferred embodiment, the invention relates to said first method wherein the/each component of the library is a compound selected from formula 2 or formula 3 or formula 4 (Formula Removed)
In a preferred embodiment, the invention relates to said first method wherein the/each component of the library is a compound selected from formula 2 or formula 3 or formula 4 and wherein the/each compound is of the gluco- or galacto-or allo- configuration.
In a preferred embodiment, the invention relates to said first method wherein the/each component of the library is a compound selected from formula 2 or formula 3 or formula 4 wherein the/each compound is of the galacto-configuration.
In a preferred embodiment, the invention relates to said first method wherein the/each component of the library is a compound selected from formula 2 or formula 3 or formula 4 and wherein the/each compound is of the gluco-configuration.
In a preferred embodiment, the invention relates to said first method wherein the/each component of the library is a compound selected from formula 2 or formula 3 or formula 4 and wherein the/each compound is of the allo-configuration.
In a preferred embodiment, the invention relates to said first method wherein designing the library comprises molecular modeling to assess molecular diversity.
In a preferred embodiment, the invention relates to said first method wherein R-i to R5 opţional substituents include OH, NO, NO2, NH2, N3, halogen, CF3, CHF2, CH2F, nitrile, alkoxy, aryloxy, amidine, guanidiniums, carboxylic acid, carboxylic acid ester, carboxylic acid amide, aryl, cycloalkyl, heteroalkyl, heteroaryl, aminoalkyl, aminodialkyl, aminotrialkyl, aminoacyl, carbonyl, substituted or unsubstituted imine, sulfate, sulfonamide, phosphate, phosphoramide, hydrazide, hydroxamate, hydroxamic acid, heteroaryloxy, aminoaryl, aminoheteroaryl, thioalkyl, thioaryl or thioheteroaryl, which may'optionally be further substituted.
The term "halogen" denotes fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.
The term "alkyl" used either alone or in compound words such as "optionally

substituted alkyl", "optionally substituted cycloalkyl", "arylalkyl" or "heteroarylalkyl", denotes straight chain, branched or cyclic alkyl, preferably C1-20 alkyl or cycloalkyl. Examples of straight chain and branched alkyl include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, amyl, isoamyl, sec-amyl, 1,2-dimethylpropyl,1, 1-dimethylpropyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl,1, 1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl,1, 2-dimethylbutyl, 1,3-dimethylbutyl, l^^trimethylpropyl.l, 1,2-trimethylpropyl, heptyl, 5-methylbexyl, 1-methylhexyl, 2,2-dimethypentyl, 3,3 dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3trimethylbutyl,1, 1,2-trimethylbutyl, 1,1,3-trimethylbutyl, octyl, 6-methylheptyl, 1-methylheptyl, 1,1,3,3tetramethylbutyl, nonyl,1-, 2-, 3-, 4-, 5-, 6-or 7methyloctyl,1-, 2-, 3-, 4-or 5-ethylheptyl, 1-, 2-or Spropylhexyl, decyl,1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-methylnonyl,1-, 2-, 3-, 4-, 5-or 6-ethyloctyl, 1-, 2-, 3or 4-propylheptyl, undecyM-, 2-, 3-, 4-, 5-, 6-, 7-, 8or 9-methyldecyl,1-, 2-, 3-, 4-, 5-, 6-or 7-ethylnonyl,1-, 2-, 3-, 4-or 5-propyloctyl,1-, 2-or 3-butylheptyl,1-pentylhexyl, dodecyl,1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-methylundecyl,1-, 2-, 3-, 4-, 5-, 6-, 7-or 8- ethyldecyl,1-, 2-, 3-, 4-, 5-or 6-propylnonyl, 1-, 2-, 3- or 4-butyloctyl, 1-2 pentylheptyl and the like. Examples of cyclic alkyl include mono-or polycyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and the like.
The term "alkylene" used either alone or in compound words such as "optionally substituted alkylene" denotes the same groups as "alkyl" defined above except that an additional hydrogen has been removed to fomn a divalent radical. It will be understood that the opţional substituent may be attached to or form part of the alkylene chain.
The term "alkenyl" used either alone or in compound words such as "optionally substituted alkenyl" denotes groups formed from straight chain, branched or cyclic alkenes including ethylenically mono-, di-or polyunsaturated alkyl or cycloalkyl groups as defined above, preferably C2-6 alkenyl. Examples of alkenyl include
vinyl, allyl,1-methylvinyl, butenyl, iso-butenyl, 3-methyl-2butenyl, 1-pentenyl, cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl, cyclohexenyl,1-heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl,1-decenyl, 3-decenyl, 1,3-butadienyl, 1,4-pentadienyl, 1,3 cyclopentadienyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,3cyclohexadienyl, 1,4-cyclohexadienyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl and 1,3,5,7-cyclooctatetraenyl.
The term "alkynyl" used either alone or in compound words, such as "optionally substituted alkynyl" denotes groups formed from straight chain, branched, or mono-or poly-or cyclic alkynes, preferably C 2-6 alkynyl.
Examples of alkynyl include ethynyl,1-propynyl, 1-and 2butynyl, 2-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4pentynyl, 2-hexynyl, 3-hexylnyl, 4-hexynyl, 5-hexynyl, 10-undecynyl,4-ethyl-l-octyn-3-yl, 7-dodecynyl, 9-dodecynyl, 10-dodecynyl,3-methyl-1-dodecyn-3-yl, 2-tridecynyl, Utridecynyl, 3-tetradecynyl, 7-hexadecynyl, 3-octadecynyl and the like.
The term "alkoxy" used either alone or in compound words such as "optionally substituted alkoxy" denotes straight chain or branched alkoxy, preferably C1-7 alkoxy. Examples of alkoxy include methoxy, ethoxy, npropyloxy, isopropyloxy and the different butoxy isomers.
The term "aryloxy" used either alone or în compound words such as "optionally substituted aryloxy" denotes aromatic, heteroaromatic, arylalkoxy or heteroaryl alkoxy, preferably C6-13 aryloxy. Examples of aryloxy include phenoxy, benzyloxy,1-napthyloxy, and 2-napth'yloxy.
The term "acyl" used either alone or in compound words such as "optionally substituted acy l "or "heteroarylacyl" denotes carbamoyl, aliphatic acyl group and acyl group containing an aromatic ring, which is referred to as aromatic acyl or a heterocyclic ring which is referred to as heterocyclic acyl. Examples of acyl
include carbamoyl; straight chain or branched alkanoyl such as formyl, acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 2,2-dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl, and icosanoyl; alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl, t butoxycarbonyl, t-pentyloxycarbonyl and heptyloxycarbonyl; cycloalkylcarbonyl such as cyclopropylcarbonyl cyclobutylcarbonyl, cyclopentylcarbonyl and cyclohexylcarbonyl; alkylsulfonyl such as methylsulfonyl and ethylsulfonyl; alkoxysulfonyl such as methoxysulfonyl and ethoxysulfonyl; aroyl such as benzoyl, toluoyl and naphthoyl; aralkanoyl such as phenylalkanoyl (e. g. phenylacetyl, phenylpropanoyl, phenylbutanoyl, phenylisobutyl, phenylpentanoyl and phenylhexanoyl) and naphthylalkanoyl (e. g. naphthylacetyl, naphthlpropanoyl and naphthylbutanoyl); aralkenoyl such as phenylalkenoyl (e. g. phenylpropenoyl, phenylbutenoyl, phenylmethacrylyl, phenylpentenoyl and phenylhexenoyl and naphthylalkenoyl (e. g. naphthylpropenoyl, naphthylbutenoyl and naphthylpentenoyl); aralkoxycarbonyl such as phenylalkoxycarbonyl (e. g. benzyloxycarbonyl); aryloxycarbonyl such as phenoxycarbonyl and naphthyloxycarbonyl; aryloxyalkanoyl such as phenoxyacetyl and phenoxypropionyl; arylcarbamoyl such as phenylcarbamoyl; arylthiocarbamoyl such as phenylthiocarbamoyl; arylglyoxyloyl such as pnenylglyoxyloyl and naphthylglyoxyloyl; arylsulfonyl such as phenylsulfonyl and naphthylsulfonyl; heterocycliccarbonyl; heterocyclicalkanoyl such as thienylacetyl, thienylpropanoyl, thienylbutanoyl, thienylpentanoyl, thienylhexanoyl, thiazolylacetyl, thiadiazolylacetyl and tetrazolylacetyl; heterocyclicalkenoyl such as heterocyclicpropenoyl, heterocyclicbutenoyl, heterocyclicpentenoyl and heterocyclichexenoyl; and heterocyclicglyoxyloyl such as thiazolylglyoxyloyl and thienyglyoxyloyl.
The term "aryl" used either alone or in compound words such as "optionally substituted aryl", "arylalkyl" or "heteroaryl" denotes single, polynuclear, conjugated and fused residues of aromatic hydrocarbons or aromatic heterocyclic ring
systems. Examples of aryl include phenyl, biphenyl, terphenyl, quaterphenyl, phenoxyphenyl, naphthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl, benzanthracenyl, dibenzanthracenyl, phenanthrenyl, fluorenyl, pyrenyl, indenyl, azulenyl, chrysenyl, pyridyl, 4-phenylpyridyl, 3-phenylpyridyl, thienyl, furyl, pyrryl, pyrrolyl, furanyl, imadazo'lyl, pyrrolydinyl, pyridinyl, piperidinyl, indolyl, pyridazinyl, pyrazolyl, pyrazinyl, thiazolyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothienyl, purinyl, quinazolinyl, phenazinyl, acridinyl, benzoxazolyl, benzothiazolyl and the like. Preferably, the aromatic heterocyclic ring system contains 1 to 4 heteroatoms independently selected from N, O and S and containing up to 9 carbon atoms in the ring.
The term "heterocycle" used ejther alone or in compound words as "optionally substituted heterocycle" denotes monocyclic or polycyclic heterocyclyl groups containing at least one heteroatom atom selected from nitrogen, sulphur and oxygen. Suitable heterocyclyl groups include N-containing heterocyclic groups, such as, unsaturated 3 to 6 membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl or tetrazolyl; saturated to 3 to 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, such as, pyrrolidinyl, imidazolidinyl, piperidin or piperazinyl ; unsaturated condensed heterocyclic groups containing 1 to 5 nitrogen atoms, such as, indolyl, isoindolyl, indolizinyl, benzimidazoyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl or tetrazolopyridazinyl; unsaturated 3 to 6-membered heteromonocyclic group containing an oxygen atom, such as, pyranyl or furyl; unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulphur atoms, such as, thienyl; unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as, oxazolyl, isoxazolyl or oxadiazolyl; saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as, morpholinyl; unsaturated condensed heterocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as, benzoxazolyl or benzoxadiazolyl; unsaturated 3 to 6-membered
heteromonocyclic group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as, thiazolyl or thiadiazolyl; saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as thiazolidinyl; and unsaturated condensed heterocyclic group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as, benzothiazolyl or benzothiadiazolyl.
In a preferred embodiment, the invention relates tq said first method wherein the compounds are synthesized.
In a preferred embodiment, the invention relates to said first method wherein the biological assays are selected from peptide ligand class of GPCRs.
In another aspect the invention provides a compound according to formula 1 in which at least one X is nitrogen, and said X is combined with the corresponding R2-R5 to form a heterocycle. The synthesis of the heterocyclic components of the present invention is disclosed in WO 2004/022572.
In a preferred embodiment, the invention provides a compound according to formula 1 wherein X and R2 combine to form a heterocycle.
In a preferred embodiment, the invention provides a compound according to formula 1 wherein the heterocycle is heteroaryl, includ ing triazoles, benzimidazoles, benzimidazolone, benzimidazolothione, imidazole, hydantoine, thiohydantoine and purine.
Detailed Description of the invention
The embodiments of the invention will be described with reference to the foilowing examples. Where appropriate, the foilowing abbreviations are used.
Ac Acetyl
DTPM 5-Acyl-1,3-dimethylbarbiturate
Ph Phenyl
TBDMS t-Butyldimethylsilyl
TBDPS t-Butyldiphenylsilyl
Bn benzyl
Bz benzoyl
Me methyl
DCE 1,2-dichloroethane
DCM dichloromethane, methylene chloride
Tf trifluoromethanesulfonyl
Ts 4-methylphenylsulfonyl, p-toluenesulfonyl
DMF N,N-dimethylformamide
DMAP N,N-dimethylaminopyridine
α,α-DMT α,α-dimethoxytoluene, benzaldehyde dimethyl acetal
DMSO dimethylsulfoxide
DTT dithiothreitol
DMTST Dimethyl(methylthio)sulphoniumtrifluoro-methanesulphonate
TBAF tetra-n-butylammonium fluoride
Part A: Preparation of building blocks:
In order to fully enable the invention, there is described below methods for the preparation of certain building blocks used in the preparation of the compounds of the invention. The building blocks described are suitable for both solution and solid phase synthesis of the compounds of the invention.
Example A: Synthesis of a 2.4 dinitroaen containina Galactopyranoside Buildina Block (Formula Removed)
Conditions: (i) a,a-dimethoxytoluene (α,α-DMT), p-toluenesulphonic acid (TsOH), acetonitrile (MeCN), 76°C, 85%; (ii) Benzoylchloride (BzCI), triethylamine; DCM, 99%; (iii) methanol (MeOH)/MeCN/water, TsOH, 75°C, 98%; (iv) t-butyldiphenylsilylchloride (TBDPS-CI), imidazole, pyridine, 120°C, 99% ; (v) Tf2O, pyridine, DCM, 0°C, 100%;(b) NaN3, DMF, 16hr, RT, 99%.
Example B: Synthesis of a 3-nitroaen containina Gulopyranoside Buildina Block(Formula Removed)
Conditions: (i) (a) trifluoromethanesulfonic anhydride (Tf2O), pyridine, -20°C, dichloromethane (DCM), 1 hour, 100%, (b) sodium azide (NaN3), N,N-dimethylformamide (DMF), 50°C, 5 hours, quantitative; (ii) TsOH, MeCN/ MeOH/water (12:3:1), 90°C, 6 hours, 88%(iii) TBDPSCI, DMAP, pyridine, 120°C, 12 hours, 93%
Examole C: Synthesis of a 2,6-dinitroaen substituted Glucopvranoside Building Block (Formula Removed)
Conditions: (i) (a) Tosylchlodride, pyridine, RT, 24 hours, 33%(b) NaN3, DMF, RT, 168 hours. (Formula Removed)
Example D: Synthesis of a 2-nitrogen containing Tallopyranoside Building Block
Conditions: (i) TBDPSCI, imidazole, 1,2-DCE, reflux; (ii) NaOMe/MeOH; (iii) (a) Tf2O, pyridine, -20°C, DCM, 1 hour, (b) NaN3, DMF, 50°C, 5 hours; (iv) TsOH, MeCN/MeOH/water; (v) benzoylchloride, DMAP, 1,2-DCE, -20°C.
Examole E: Synthesis of two 3-nitroaen containina Altropyranoside Buildina Block (Formula Removed)
Conditions: (i) cyclohexanone dimethylacetal, TsOH, MeCN; (ii) p-methoxybenzaldehyde dimethylacetal, TsOH, MeCN; (iii) DIBAL, -78°C, diethyl ether; (iv) (a) Tf2O, pyridine, -20°C, DCM, 1 hour, (b) NaN3, DMF, 50°C, 5 hours; (v) TsOH, MeCN/MeOH/water; (vi) TBDPSCI, DMAP, 1,2-DCE; (vii) (a) CAN, (b) BzCI, DMAP, 1,2-DCE, (c) TsOH, MeCN/MeOH/water; (viii) TBDPSCI, DMAP, 1,2-DCE.
Examole F: Svnthesis of a 2-nitroaen containina Glucopvranoside Buildina Block
(Formula Removed)
Conditions: (i) α,α-DMT, TsOH, MeCN; (ii) 1,2-DCE, BzCI, DMAP; (iii) TsOH, MeOH/MeCN; (iv) TBDPS-CI, DMAP, 1,2-DCE. (Formula Removed)
Conditions: (i) TBDPSCI, DMAP, pyridine, 120°C, 0.5 hours, 81%; (ii) a. (Bu)2SnO, MeOH; b. Benzoylchloride, RT, 24 hour;
Example G: Svnthesis of a 2-nitrogen containinq Allopvranoside Building Block
(Formula Removed)
Conditions: (i) DCM/pyridine, MsCI, DMAP, 0°C; (ii) sodium benzoate, dimethylsulphoxide (DMSO), 140°C; (iii) TsOH, MeOH/MeCN/water; (iv) TBDPS-Cl, imidazole, DCM, 1 hour, reflux.
The Solid Phase Library Synthesis of Sugars is illustrated in Scheme 1. The reaction conditions are as follows:
(A) 2P compound synthesis: R1=R2=OMe;
i) 2-naphthalene methanol, DMTST, DCM; ii) TCA-Wang resin, BF3Et2O, DCM; iii) NaOMe, methanol; iv) a. KOtBu, DMF; b. Mel, DMF; v) HF 'proton sponge', AcOH, DMF, 65°C; vi) a. KOtBu, DMF; b. Mel, DMF; vii) 1,4-dithio-DL-threitol, KOtBu, DMF; viii) HBTU, Fmoc-ß-Ala-OH, DIPEA, DMF; ix) piperidine/DMF (1/4); x) TFA, Et3SiH, DCM
(B) 3P compound synthesis: Ri=methyl-2-naphthyl, R2=OMe;
i) 2-naphthalene methanol, DMTST, DCM; ii) TCA-Wang resin, BF3 Et2O, DCM; iii)
NaOMe, methanol; iv) a. KOtBu, DMF; b. 2-bromomethyl-naphthalene, DMF; v)
HF' proton sponge1, AcOH, DMF, 65°C; vi) a. KOtBu, DMF; b. Mei, DMF; vii) 1,4-
dithio-DL-threitol, KOtBu, DMF; viii) HBTU, Fmoc-ß-Ala-OH, DlPEA, DMF; ix)
piperidine/DMF (1/4); x) TFA, Et3SiH, DCM
(C) 4P compound synthesis: Ri=methyl-2-naphthyl, R2=4-chlorobenzyl
i) 2-naphthalene methanol, DMTST, DCM; ii) TCA-Wang resin, BF3Et2O, DCM; iii)
NaOMe, methanol; iv) a. KOtBu, DMF; b. 2-bromomethyl-naphthalene, DMF; v)
HF'proton sponge', AcOH, DMF, 65°C; vi) a. KOtBu, DMF; b. 4-
chlorobenzylbromide, DMF; vii) 1,4-dithio-DL-threitol, KOtBu, DMF; viii) HBTU,
Fmoc-p-Ala-OH, DIPEA, DMF; ix) piperidine/DMF (1/4); x) TFA, Et3SiH, DCM
(Formula Removed)
Scheme 1 Solid Phase library Synthesis ofsugars
Scheme 2 Synthesis of allose 2,6 building block; an example of synthesis of 2P, 3P and 4P type compound
The synthesis of the Allose 2,6N building block is illustrated in Scheme 2. The reaction conditions are as follows:
i) p-methoxybenzaldehyde dimethylacetal, camphorsulfonic acid, N,N-dimethylformamide (DMF); ii) Tf20, pyridine, dichloromethane (DCM); iii) tetrabutylammonium benzoate, DMF, 55°C; iv) BH3THF, Bu2BOTf, DCM; v) methanesulfonylchloride, pyridine, DCM; vi) sodium azide, DMF, 85°C; vii) sodium methanolate (NaOMe), methanol; viii) n-butanol, ethylene diamine, reflux; ix) DTPM reagent, methanol; x) benzoic anhydride, pyridine; xi) trifluoroacetic acid, triethylsilane, DCM
Designing libraries:
The design of the libraries is based on the presentation of a positive charge and a varying number of aromatic substituents in different spaţial arrangements on a monosaccharide scaffold. Starting with a positive charge and one aromatic displayed on the core scaffold, actives from this first library were elaborated on by further variation and addition of more aromatic substituents to quickly identify highly active molecules.
The first library of compounds comprises two pharmacophoric groups, known as a 2P library, in particular, one containing an aromatic and a positive charge. The library was designed such that each molecule presents two pharmacophoric groups in different relative orientation or presentation (e.g., distance, relative angle, i.e. relative position in space is different).
Actives from this library were identified and SAR information from this library was used to design subsequent library of compounds wherein each compound may include three pharmacophoric groups, known as a 3P library. Subsequent libraries with four pharmacophoric groups are called 4P library, etc.
Members of significantly improved activity were identified out of the second library and were selected for further drug development.
The method of the invention includes real and virtual libraries.
Thus, the molecules according to formula 1 are well suited for generating iterative scanning libraries, starting from a selected number of pharmacophores (eg, two) in the first library and designing subsequent libraries with additional pharmacophores based on SAR Information from the first library, thereby assisting in delineating pharmacophores.
The 2P and 3P library of compounds were synthesized according to the building blocks as described in Examples A-G.
The 2P library (Table 1) was designed to scan molecular diversity for 2P
molecules, comprising an aromatic and a positive charge.
The 2P library was screened for biological activity and the results are given in
Table 1.
Similarly, the 3P library was designed to scan molecular diversity for 3P molecules. Design of 3P library resulted from SAR obtained from 2P library in Table 1.
The 3P library was screened for biological activity and the results are given in Table 2.
A visual analysis of the results according to Table 1 (2P library) and Table 2 (3P Library) indicates that:
1.1,2 allose substitution according to formula 3 (and Scaffold C/D) presents the most active arrangement of molecules in the library wherein Z is oxygen, R-\ is naphthyl and R2 is propylamine or ethylamine.
These compounds represent most actives at low nM range, and are suitable candidates for further drug development.
2. R-i as naphthyl is more active than the corresponding p-chlorobenzyl
substituent.
3. 1, 2 allose according to formula 3 (Scaffold C/D) is more active than the
corresponding 1, 2 glucose conformation (Scaffold A/B).
4. 1, 2 substitution according to formula 3 (Scaffold C/D) is more active than the
corresponding 2, 6 substitution according to formula 4 (Scaffold G)
5. R2 as propylamine and ethylamine are more active than
methylamine wherein Z, R1 and R2 are as described above.
6. 2, 3 allose substitution according to formula 3 (Scaffold C/D) presents the more
actives wherein R2 is ethylamine, and R3 is p-chlorobenzyl compared to
corresponding R2 as propylamine and ethylamine wherein R3 is p-chlorobenzyl
substituent, and also wherein R2 is methylamine, ethylamine or
propylamine and R3 is naphthyl.
7. 2, 3 glucose substitution according to formula 3 (scaffold A/B) presents the
more actives wherein R2 is propylamine and R3 is naphthyl compared to
corresponding R2 as methylamine or ethylamine, and also wherein R2 is
methylamine, ethylamine or propylamineand R3 is p-chlorobenzyl.
8. 2, 4 and 3, 4 substitutions according to formula 3 (Scaffold G) present the least
actives.
Part B: Biological assays
Example H. In vitro screening of compounds against somatostatin subtypes SSTR-1 to SSTR-5
General method
Receptor membrane preparations containing the desired cloned receptor (for example cloned human somatostatin receptor subtype 5, SSTR5) and radio-labeled ligand were diluted at the concentration required for testing and according to the specific parameters associated with the selected receptor-ligand combination, including receptor Bmax, ligand Kd and any other parameters necessary to optimize the experimental conditions. When tested for competition activity to the reference ligand, "compound " was mixed with membrane suspension and the radiolabeled reference ligand (with or without an excess of unlabeled ligand to the receptor for determination of non-specific binding) and incubated at the temperature required by internai standard operating procedures. Following incubation, the binding reaction was stopped by the addition of ice-cold washing buffer and filtered on appropriate filters, which are then counted. Data analysis and curve-fitting was performed with XLfit (lDBS).
Preparation ofcompounds
10mM solutions of test compounds in 100% DMSO were prepared. ~160 ni was used for each dilution (20 nl/well in triplicate).
A 1.25 mM assay stock was prepared by making a 1:8 dilution of the 10 mM solution. To 30 ^L of the 10 mM solution was added 210 µL milli-Q H2O. A 1:5 dilution series in milli-Q H20 was then prepared.
Final concentration Final concentration
in SST4 assay in S ST 5 assay
A. 240 µL of 1.25 mM 0.25 mM 0.125 mM
B. 48 µL A+ 192 µLmQ 0.05 mM 0.025 mM
C. 24 µL B+ 192 µLmQ 0.01 mM 0.005 mM
etc
Assays were performed in triplicate at each concentration within the 1:5 dilution series: 250µM, 50µM, 10µM, 2mM, 0.4µM, 0.08µM, 0.016µM, 0.0032 µM, etc. (for SST4 assay) and 125µM, 10µM, 2µM, 1uM, 0.5. µM, etc (for SST5 assay).
Filter plate assay for SST5 receptor
Human SST5 somatostatin receptor was transfected into HEK-293 EBNA cells. Membranes were suspended in assay buffer (50 mM Tris-HCI, 1 mM EGTA, 5 mM MgCI2, 10% sucrose, pH 7.5). The receptor concentration (Bmax) was 0.57 pmol/mg proteinKd for [125I]SST-14 Binding 0.31 nM, volume 0.4 ml per vial (400 microassays/vial), and protein concentration 1.03 mg/ml.
After thawing the frozen receptor preparation rapidly, receptors were diluted with binding buffer, homogenized, and kept on ice.
1. Use Multiscreen glass fiber filter plates (Millipore, Cat No MAFCNOB10)
precoated with o.5 % PEI for ~ 2hr at 4°C. Before use add 200 ul/well
assay buffer and filter using Multiscreen Separation System.
2. Incubate 5.5 µg of membranes (40 ui of a 1:40 dilution), buffer and
[125I]SST-14 (4 nM, -80 000 cpm, 2000 Ci/mmol) in a total volume of 200 ui
for 60 min at 25°C. Calculate IC50 for SST-14 (a truncated version of the
natural ligand SST-28) (Auspep, Cat No 2076) and SST-28 (Auspep, Cat
No 1638). Prepare serial dilutions (1:5) of compounds, as descnbed above
and instead of adding SST-14 in well, add 20 µl of compounds (Table 3).
3. Filter using Multiscreen Separation System with 5 x 0.2 ml ice-cold Assay
buffer.
4. Remove the plastic underdrain and dry plate in oven for 1 hr at 40°C.
5. Seal tape to the bottom of the plate.
6. Add 50 ul/well scintillant (Supermix, Wallac, Cat No 1200-439).
7. Seal and count in the BJET, program 2.
Table 3 (Table Removed)
TB: total binding
NSB: non-speciflc binding
Part C: General Experimental Methods
Example I: HPLC Method for compounds in Tables 1 and 2
The HPLC separation of compounds in Tables 1 and 2 was conducted under Method A or Method B as shown below.
Method A
Column: Agilent SB Zorbax C18 4.6x50mm (5um, 80 Â)
LC mobile phase:
5% aqueous MeCN /1 min
100%MeCN/7-12min
Method B
Column: Agilent SB Zorbax C18 4.6x50mm (5um, 80 Â)
LC mobile phase: 5% aq MeCN/1 min 30% aq MeCN/3min 40%aqMeCN/12 min 100%MeCN/13-15 min
Key to Building Blocks for Tables 1 and 2
Table 1: * % SST5 radio- ligand binding displaced at conc (µM) for 2P library of compounds
Table 2: * % SST5 radio- ligand binding displaced at conc (µM) for 3P library of compounds; ; R4 = X30; compounds 60-63, 119 and 156-159 are comparative compounds from 2P library
"+ +": % SST5 radio- ligand binding displaced at conc (µM) >60% "+": % SST5 radio- ligand binding displaced at conc (µM) 60>+>40% "-": % SST5 radio- ligand binding displaced at conc (µM) - Table 1: Biological activity of example 2P library (Table Removed)
Table 2: Biological activity of example 3P library (Table Removed)
Throughout the specification and the claims (if present), unless the context requires otherwise, the term "comprise", or variations such as "comprises" or "comprising", will be understood to apply the inclusion of the stated integer or group of integers but not the exclusion of any other integer or group of integers.
Throughout the specification and claims (if present), unless the context requires otherwise, the term "substantially" or "about" will be understood to not be limited to the value for the range qualified by the terms.
It should be appreciated that various other changes and modifications can be made to any embodiment described without departing from the spirit and scope of the invention.
References
[1] Patel, Y.C. (1999) Somatostatin and its receptor family. Front. Neuroendocr.
20, 157-198
[2] Csaba, Z. and Dournaud, P. (2001) Cellular biology of somatostatin receptors.
Neuropeptides 35, 1-23
[3] T Reisine, T. (1995) Somatostatin receptors; Am. J. Pysiol. (Gastrointest. Liver
Physiol. 32) 269, G813-G820
[4] Bauer, W. et al. (1982) SMS201-995: A very potent and selective octapeptide
analogue of somatostatin with prolonged action. Life Sci. 31, 1133-1140
[5] Lamberts, S.W.J. et al. (1996) Drug therapy: Octreotide. N. Eng. J. Med. 334,
246-254
[6] Robinson, C. and Castaner, J. (1994) Lanreotide acetate. Drugs Futune 19,
992-999
[7] Reisine, T. and Bell. G.l. (1995) Molecular biology of somatostatin receptors.
Endocr. Rev. 16, 427-442

CLAIMS:
1. A method of identifying biologically active compounds comprising:
(a)designing a first library of compounds of formula 1 to scan molecular diversity wherein each compound of the library has at least two pharmacophoric groups R1 to R5 as defined below and wherein compound of the library has same number of pharmacophoric groups; (b)assaying the first library of compounds in one or more biological assay(s); and
(c)designing a second library wherein each compound of the second library contains one or more additional pharmacophoric groups with respect to the first library
such that the/each component of the first and second library is a compound of formula 1: (Formula Removed)
wherein the ring may be of any configuration;
Z is sulphur, oxygen, CH2, C(0), C(0)NRA, NH, NRA or hydrogen, in the case where Z is hydrogen then R1 is not present, RA is selected from the set defined for R1 to R5, or wherein Z and R1 together form a heterocycle,
X is oxygen or nitrogen providing that at least one X of Formula l is nitrogen, X may also combine independently with one of R1 to R5 to form an azide, R1 to R5 are independently selected from the foilowing non-pharmacophoric groups H, methyl and acetyl, and pharmacophoric groups R1 to R5 are independently selected from the group which includes but is not limited to C2 to C20 alkyl or acyl excluding acetyl; C2 to C20 alkenyl, alkynyl, heteroalkyl; C5 to C20 aryl, heteroaryl, arylalkyl or heteroarylalkyl, which is optionally substituted, and can be branched or linear, or wherein X and the corresponding R moiety, R2 to R5 respectively, combine to form a heterocycle.
2. A method according to claim 1 wherein in the first library, three of the
substituents R1-R5 are non-pharmacophoric groups and are selected from hydrogen
or methyl or acetyl.
3. A method according to claim 1 wherein in the first library, two of the
substituents R1-R5 are non-pharmacophoric groups and are selected from hydrogen
or methyl or acetyl.
4. A method according to claim 1 wherein Z is sulphur or oxygen.
5. A method according to any one of claims 1-4 wherein at least one of the
pharmacophoric groups is selected from aryl, arylalkyl, heteroaryl, heteroarylalkyl or
acyl.
6. A library of compounds selected from compounds of formula 1 when used
according to claim 2.
7. A library of compounds selected from compounds of formula 1 when used
according to claim 3.
8. A method according to any of claims 1 to 5 wherein the/each component of the
library is a compound selected from formula 2 or formula 3 or formula 4.
(Formula Removed)
9. A method according to claim 8 wherein the/each compound is of the gluco- or
galacto- or allo- configuration.
10. A method according to claim 9 wherein the/each compound is of the gluco-
configuration.
11. A method according to claim 9 wherein the/each compound is of the allo-
configuration.
12. A method according to claim 9 wherein the/each compound is of the galacto-
configuration.
13. A method according to claim 1 wherein designing the library comprises
molecular modeling to assess molecular diversity.
14. A method according to claim 1 wherein R1 to R5 opţional substituents are
selected from OH, NO, NO2, NH2, N3, halogen, CF3, CHF2, CH2F, nitrile, alkoxy,
aryloxy, amidine, guanidiniums, carboxylic acid, carboxylic acid ester, carboxylic acid
amide, aryl, cycloalkyl, heteroalkyl, heteroaryl, aminoalkyl, aminodialkyl, aminotrialkyl,
aminoacyl, carbonyl, substituted or unsubstituted imine, sulfate, sulfonamide,
phosphate, phosphoramide, hydrazide, hydroxamate, hydroxamic acid, heteroaryloxy,
aminoaryl, aminoheteroaryl, thioalkyl, thioaryl or thioheteroaryl, which may optionally
be further substituted.
15. A method according to any of claims 1 to 5 wherein the compounds are
synthesized.
16. A method according to claim 1 wherein the biological assays involve peptide
ligand class of GPCRs.
17. A compound according to formula 1 in which at least one X is nitrogen, and
said X is combined with the corresponding R2-R5 to form a heterocycle.
18. A compound according to claim 17 wherein X and R2 combine to form a
heterocycle.
19. A compound according to any one of claims 17 and 18 wherein the heterocycle
is heteroaryl.
20. A compound according to any one of claims 17-19 wherein the heteroaryl is
selected from triazoles, benzimidazoles, benzimidazolone, benzimidazolothione,
imidazole, hydantoine, thiohydantoine and purine.

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http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=uY5t4ota6cn1DhqEY5YtZQ==&loc=+mN2fYxnTC4l0fUd8W4CAA==

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Patent Number

277226

Indian Patent Application Number

1862/DELNP/2008

PG Journal Number

48/2016

Publication Date

18-Nov-2016

Grant Date

15-Nov-2016

Date of Filing

03-Mar-2008

Name of Patentee

VAST BIOSCIENCE PTY LTD

Applicant Address

311 SWANN RD,ST LUCIA, QUEENSLAND 4067, AUSTRALIA

Inventors:

#	Inventor's Name	Inventor's Address
1	WIM MEUTERMANS	293 BIRDWOOD TERRACE, TOOWONG, QLD 4066, AUSTRALIA
2	GERALD B. TOMETZKI (DESEASED)	38 SAMUEL WEST, MANLY WEST, QLD 4179, AUSTRALIA
3	JOHANNES ZUEGG	43 WASSELL ST, WYNNUM, QLD 4178, AUSTRALIA.

PCT International Classification Number

C40B 30/04

PCT International Application Number

PCT/AU2006/001431

PCT International Filing date

2006-10-03

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	2005905465	2005-10-04	Australia