WO2009023495A2

WO2009023495A2 - Bisubstrate inhibitors of adenylosuccinate synthetase

Info

Publication number: WO2009023495A2
Application number: PCT/US2008/072375
Authority: WO
Inventors: Wolfgang Wrasidlo; Gregory I. Elliott; Dennis A. Carson; Michael Rosenbach
Original assignee: The Regents Of The University Of California
Priority date: 2007-08-10
Filing date: 2008-08-06
Publication date: 2009-02-19
Also published as: WO2009023495A3

Abstract

The present invention provides compound having the general structure (A) or pharmaceutically acceptable salts thereof: (A) wherein each of R1 and R2 can be, independently, any one of-H, a halogen, -NH2, -OH, - NH-R3, and -O-R3; each of G1, G2, and G4, can be, independently any of CH, N, O, and S; G3 can be any Of CH2, NH, O; and S; or G3 and/or G4 can be C=O; G5 can be any of C and N; L can be absent or be any of O, NH, and S; R3 can be any of-H, an C1-C18 alkyl, an aryl,- C(O)-H, and -C(O)-alkyl; R4 can be any of -H, -C(O)O-; and -C(O)-R3; R5 can be any of - H, an C1 -C18 alkyl, or an aryl; M can be absent or be any Of -CH2-; -NH-; -NH-C(O)-; -O-, and -S-; and n is an integer having the value between 1 and 6. The compounds having the general structure A can serve as inhibitors of adenylosuccinate synthetase.

Description

BISUBSTRATE INHIBITORS OF ADENYLOSUCCINATE SYNTHETASE

BACKGROUND

FIELD OF THE INVENTION

[0001] The present invention relates generally to compounds useful for the inhibition of AMP biosynthesis. More specifically, the invention relates to compounds that inhibit the activity of adenylosuccinate synthetase.

BACKGROUND INFORMATION

[0002] Cells in approximately 25 to 40% of many common human cancers have homozygous deletions of the gene that encodes the adenine nucleotide salvage enzyme, methylthioadenosine phosphorylase (MTAP). Such tumors are often aggressive and chemotherapy-resistant due to the co-deletion of the chromosomal CDKN2A locus, which encodes proteins (pl6INK4A and pl4ARF) that regulate the pRB and p53 pathways.

[0003] During cell proliferation, adenine nucleotides for nucleic acid synthesis are generated through a de novo biosynthetic route that requires the key enzyme adenylosuccinate synthetase (AdSS) for completion. AdSS acts by converting inosine monophosphate (IMP) to adenosine monophosphate (AMP). Adenine nucleotides are salvaged by a pathway that requires the presence of active MTAP. In particular, MTAP cleaves a by-product of polyamine synthesis, 5 '-deoxy-5 '-methylthioadenosine (MTA) into adenine and 5-methylthioribose-l -phosphate. Adenine is then salvaged to AMP by adenine phosphoribosyltransferase.

[0004] When treated with an inhibitor of de novo adenine synthesis, division of cells is arrested, followed by cell death. Cells containing active MTAP can be rescued by treatment with MTA as a purine source, but MTAP-deficient cells cannot. Hence, MTAP-deficient cancers are hypersensitive to the cytotoxic effects of AdSS inhibitors, such as L-alanosine, lometrexol and AG2037. However, these existing AdSS inhibitors are not sufficiently potent to kill all tumor cells. There is a need for more potent AdSS inhibitors that bind directly to the AdSS molecule and therefore provide means for practicing targeted, AdSS-specific therapy. SUMMARY OF THE INVENTION

[0005] The invention provides compounds which bind Adss and inhibit its enzymatic activity for more efficient depletion adenine nucleotides in cells than is presently provided by the art. Such depletion can be expected to induce cell death in MTAP-defϊcient tumors. The inhibitors are designed to span the catalytic subdomain from the IMP loop into the aspartic acid site and to the magnesium center of human monomeric AdSS. The inhibitors are therefore preferably bisubstrate or multisubstrate binding compounds specific for Adss. Methods for predicting the binding energies of similarly designed inhibitors, toward identifying candidate molecules of greater potency, are also provided.

[0006] According to embodiments of he present invention, there are provided compounds having the general structure A or pharmaceutically acceptable salts or tautomers thereof are provided:

wherein each of Ri and R₂ can be, independently, any one of-H, a halogen, -NH₂, -OH, - NH-R₃, and -0-R₃; each OfG₁, G₂, and G₄, can be, independently any of CH, N, O, and S; G₃ can be any of CH₂, NH, O; and S; or G₃ and/or G₄ can be C=O; G₅ can be any of C and N; L can be absent or be any of O, NH, and S; R₃ can be any of-H, an Ci-Ci₈ alkyl, an aryl,- C(O)-H, and -C(O)-alkyl; R₄ can be any of-H, -C(O)O-; and -C(O)-R₃; R₅ can be any of - H, an Ci-Ci₈ alkyl, or an aryl; M can be absent or be any Of-CH₂-; -NH-; -NH-C(O)-; -O, and -S-; and n is an integer having the value between 1 and 6. Alkyl and aryl substitutents can be themselves substituted.

[0007] Pharmaceutical compositions of inhibitory compounds of the invention and methods for their use are also provided. The compounds can be expected to be therapeutically useful in the treatment of disease, disorders and pathologies clinically related to MTAP-deficiency in cells. More particularly, the compounds are to be used in treatment of cancers with MTAP-deficient tumors, such as certain acute leukemias, lung cancers, pancreatic cancers, hand and neck cancers, bladder cancers and glioblastomas (hereafter, MTAP-deficient cancers").

BRIEF DESCRIPTION QF THE DRAWINGS

[0008] FIG. 1 illustrates the crossed stereoview of an exemplary AdSS inhibitor of the invention in the catalytic domain of human monomeric AdSS. The inhibitory compound is depicted in the subdomain of the enzyme extending from the IMP into the aspartate loop with nearest neighbor interactions including the magnesium center. The flexible ether-alkyl bridge contributes to a sterically favorable orientation of the heterocycle in the IMP loop. The conformation shown allows for hydrogen bonding interaction of the compound with residues Lys 334, Arg 335, Asn 64 and magnesium complexing with the terminal carboxylic acid grou of the hadacidin motif.

[0009] FIG. 2 is a graph depicting the in vitro inhibitory activity of a compound according to the invention (compound I, labeled "GE- 109" in the Figure), as compared to a hadacidin control and a non-heterocyclic AdSS inhibitor.

[0010] FIG. 3 illustrates the structural features of compounds identified as having inhibitory activity against AdSS in an in vitro assay and compounds which did not demonstrate such activity.

DETAILED DESCRIPTION OF THE INVENTION

A. Non-limiting Definitions.

[0011] The following definitions are provided for ease of reference, and are intended to illustrate, rather than limit, the scope of this disclosure.

[0012] The term "alkyl" refers to either substituted or unsubstituted Ci-C]₀ straight chain saturated aliphatic hydrocarbon groups, substituted and unsubstituted C₂-Ci₀ straight chain unsaturated aliphatic hydrocarbon groups, substituted and unsubstituted C₄-Ci_O branched saturated aliphatic hydrocarbon groups, substituted and unsubstituted C₄-Ci₀ branched unsaturated aliphatic hydrocarbon groups, substituted and unsubstituted C₃-C₈ cyclic saturated aliphatic hydrocarbon groups, substituted and unsubstituted C₅-C₈ cyclic unsaturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, the definition of "alkyl" shall include but is not limited to: methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, ethenyl, propenyl, butenyl, penentyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, isopropyl, isobutyl, tert-butyl, sec- butyl, isopentyl, neopentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, methylcyclopropyl, ethylcyclohexenyl, butenylcyclopentyl, adamantyl, norbornyl and the like.

[0013] Alkyl substituents are independently selected from a group consisting of halogen, -OH, -SH, -NH₂, -CN, -NO₂, =O, =CH₂, trihalomethyl, carbamoyl, arylC_o-i_Oalkyl, heteroarylCo_-10alkyl, Ci-ioalkyloxy, arylCo-ioalkyloxy, Ci-ioalkylthio, arylCo-ioalkylthio, Ci-ioalkylamino, arylC_0-ioalkylamino, N-aryl-N-C_0-ioalkylamino, Ci-ioalkylcarbonyl, arylCo-ioalkylcarbonyl, Ci_-10alkylcarboxy, arylCo_-10alkylcarboxy, Ci-iQalkylcarbonylamino, arylCo-ioalkylcarbonylamino, tetrahydrofuryl, morpholinyl, piperazinyl, hydroxypyronyl, -C_0- ioalky ICOORa and -Co-ioalkylCONRbRc, wherein R₃, Rb and R₀ are independently selected from hydrogen, alkyl, aryl, or R_b and R₀ are taken together with the nitrogen to which they are attached forming a saturated cyclic or unsaturated cyclic system containing 3 to 8 carbon atoms, with at least one substituent.

[0014] The term "aryl" refers to an unsubstituted, mono-, di- or trisubstituted monocyclic, polycyclic, biaryl aromatic groups covalently attached at any ring position capable of forming a stable covalent bond, certain preferred points of attachment being apparent to those skilled in the art (e.g., 3-phenyl, 4-naphtyl and the like). The aryl substituents are independently selected from a group consisting of halogen, -OH, -SH, -CN, -NO₂, trihalomethyl, hydroxypyronyl, Ci-ioalkyl, arylC_o-ioalkyl, Co-ioalkyloxyCo-ioalkyl, arylCo-ioalkyloxyC_0- i_Oalkyl, Co-ioalkylthioCo-ioalkyl, arylCo-ioalkylthioCo-ioalkyl, Co-ioalkylaminoCQ-ioalkyl, ary IC_0- 1 _oalky laminoCo- 1 _oalky 1, N-aryl-N-Co- 1 oalkylaminoC_0- 1 oalky 1, Ci_-I oalkylcarbony IC_0- loalkyl, arylCo-ioalkylcarbonylCo-ioalkyl, Ci-ioalkylcarboxyCo-ioalkyl, arylCo- i oalkylcarboxyCo- 1 _oalkyl, C I- loalkylcarbonylaminoCo. i _oalkyl, ary IC_{0- 10}alkylcarbonylaminoCo_ loalkyl, -Co-ioalkylCOORa, and -Co-ioalkylCONRbRc, wherein R_a, R_b and R₀ are independently selected from hydrogen, alkyl, aryl or R_b and R₀ are taken together with the nitrogen to which they are attached forming a saturated cyclic or unsaturated cyclic system containing 3 to 8 carbon atoms with at least one substituent.

[0015] The definition of "aryl" includes, but is not limited to, such specific groups as phenyl, biphenyl, naphthyl, dihydronaphthyl, tetrahydronaphthyl, indenyl, indanyl, azulenyl, anthryl, phenanthryl, fluorenyl, pyrenyl and the like.

[0016] The terms "halogen", "halide" or "halo" refer to fluorine, chlorine, bromine, and iodine.

[0017] The term "AdSS" refers to the human adenylosuccinate synthetase enzyme, and analogs thereof in other mammalian species.

[0018] The term "effective amount" of a compound refers a non-toxic but sufficient amount of the compound that provides a desired effect. This amount may vary from subject to subject, depending on the species, age, and physical condition of the subject, the severity of the disease that is being treated, the particular compound used, its mode of administration, and the like. Therefore, it is difficult to generalize an exact "effective amount," yet, a suitable effective amount may be determined by one of ordinary skill in the art.

[0019] The term "pharmaceutically acceptable" refers to a compound, additive or composition that is not biologically or otherwise undesirable. For example, the additive or composition may be administered to a subject along with a compound of the invention without causing any undesirable biological effects or interacting in an undesirable manner with any of the other components of the pharmaceutical composition in which it is contained.

[0020] The term "pharmaceutically acceptable salts" includes hydrochloric salt, hydrobromic salt, hydroiodic salt, hydrofluoric salt, sulfuric salt, citric salt, maleic salt, acetic salt, lactic salt, nicotinic salt, succinic salt, oxalic salt, phosphoric salt, malonic salt, salicylic salt, phenylacetic salt, stearic salt, pyridine salt, ammonium salt, piperazine salt, diethylamine salt, nicotinamide salt, formic salt, urea salt, sodium salt, potassium salt, calcium salt, magnesium salt, zinc salt, lithium salt, cinnamic salt, methylamino salt, methanesulfonic salt, picric salt, tartaric salt, triethylamino salt, dimethylamino salt, tris(hydroxymethyl)aminomethane salt and the like. Additional pharmaceutically acceptable salts are known to those of skill in the art. [0021] As used herein, the term "patient" refers to organisms to be treated by the methods of the present invention. Such organisms include, but are not limited to, humans. In the context of the invention, the term "subject" generally refers to an individual who will receive or who has received treatment for the treatment of a disease, disorder or pathology.

B. Design Criteria for AdSS Inhibitors of the Invention.

[0022] Inhibitory compounds according to the invention will generally be those with structures that span both the IMP and aspartate loops in the AdSS enzyme. Such compounds can be expected to function as bisubstrate or multisubstrate inhibitors. The bisubstrate binding interaction between a particular compound of the invention having structure A as described hereinbelow with the AdSS monomer is depicted in Figure 1.

[0023] To that end, pharmacophores in the catalytic domain of the enzyme capable of competitive inhibition of substrate binding are identified by computational receptor mapping of the domain using natural inhibitors of the enzyme with known affinities therefore, such as aspartic acid (IC50 30 mM), hadacidin (IC50 6 μM), L-alanosine (IC50 3 μM) and hydantocidin (0.6 μM). This yields three-dimensional receptor-ligand interaction complexes as recognition events from which a set of structural elements can be evolved to suggest the bi- to multi- substrate structures of potential use as AdSS inhibitors of the invention.

[0024] More particularly, a trifunctional purine (IMP mimic) is used as starting material for reactions with several peptide-like structures, such as hadacidin. Coupling reactiosn incorporating flexible linkers are used to bridge the IMP with the Asp-loop in the AdSS monomer by incorporating ether or thioether groups, which allow for free rotation between the heterocycle and peptide segments, thus reducing steric hindrance. Fine tuning of the structures is performed by varying the hydrocarbon chain length, and incorporating various hydrogen bond donor and receptor moieties.

[0025] The resulting compounds are synthesized and tested for nhibition of de novo adenine nucleotide synthesis, and for selective toxicity toward MTAP-defϊcient cancer cells in vitro and/or in vivo, as described hereinbelow. Particularly preferred inhibitors will be those that are cell-permeable and have cytotoxic activities at concentrations in the sub- micromolar, and especially the nanomolar range. C. Exemplary AdSS Inhibitors of the Invention.

[0026] According to embodiments of the present invention, compounds having the general structure A are provided, and pharmaceutically acceptable salts or tautomers thereof:

[0027] In the compounds of the general structure A, each of Ri and R₂ can be, independently, any one of-H, a halogen (e.g., Cl, F, or Br), -NH₂, -OH, -NH-R₃, or -0-R₃; each of Gi, G₂, and G₄, can be, independently any of CH, N, O, or S; G₃ can be any Of CH₂, NH, O, or S; or G₃ and/or G₄ can be C=O; G₅ can be any of C or N; L can be absent or be any of O, NH, or S; R₃ can be any of-H, an Ci-Ci ₈ alkyl, an aryl,-C(O)-H, or -C(O)-alkyl; R₄ can be any of-H, -C(O)O-, or -C(O)-R₃; R₅ can be any of-H, an Ci-Ci₈ alkyl, or an aryl; M can be absent or be any of -CH₂-; -NH-; -NH-C(O)-; -0-, or -S-; and n is an integer having the value between 1 and 6. Alkyl and aryl substitutents can be themselves further substituted.

[0028] In some embodiments, compounds of the present invention can comprise a substituted purin moiety. In such purin moiety, the available carbon in the five-member ring of purin can carry a variety of substitutents, such as a substituted methoxy group, a substituted methyl thio group, and a substituted secondary amino group. Furthermore, in such a purin moiety, one or both available carbons in the six-member ring of purin can optionally carry such a substitutent as a primary or secondary amino group, hydroxyl group, alkoxy group, or aryloxy group.

[0029] Another example of a substitutent that can be present in any of the above- mentioned substituted methoxy group, the substituted methylthio group, and the substituted secondary amino group is a moiety having the structure B:

B

[0030] In moiety B, R₃ can be any of -H, an Ci-Ci₈ alkyl, an aryl,-C(O)-H, or -C(O)- alkyl; R₄ can be any of -H, -C(O)O-; or -C(O)- R₃; M can be absent or be any of -CH₂-, NH-, -NH-C(O)-, -O-, or -S-; and n is an integer having the value between 1 and 5.

[0031] Non-limiting examples of specific compounds encompassed by the general structure A include compounds I- VIII, and pharmaceutically acceptable salts or, where applicable, tautomers, such as keto-enol tautomers, thereof:

II

III

IV

VI

VII

[0032] The compounds of the present invention are capable of inhibiting AdSS and may, therefore, be used for the treatment of various disorders, diseases, and pathologies clinically related to AdSS activity in vivo. The inhibitors can be expected to have particular application in treatment of MTAP-deficient cancers. Accordingly, the compounds having the structure A, including the specific compounds I- VII, or pharmaceutically acceptable salts thereof can be used for preparing pharmaceutical compositions, e.g., by combining these compounds and pharmaceutically acceptable carriers. The pharmaceutical compositions can then be used in pharmacologically effective doses for the treatment of various disorders, diseases, and pathologies in which AdSS plays a causative role, especially MTAP-deficient cancers.

[0033] Various synthetic schemes can be designed for manufacturing the products having the structure A , including the specific compounds I- VII. To exemplify, but not limit, the present invention, in one embodiment, the reaction scheme (A) shown below can be employed for making such compounds. If desired, other synthetic processes can be designed by those having ordinary skill in the art.

(A)

D. Pharmaceutically Acceptable Compositions of the Invention and Dosages Thereof.

[0034] Pharmaceutically acceptable salts of the compounds of the present invention may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

[0035] The above-described compounds A, including the sub-genera I- VII, can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, e.g., orally or parenterally, by intravenous, intraperitoneal, intramuscular, topical or subcutaneous routes. Dosages may be similar to those utilized in the art for existing inhibitors of de novo adenine synthesis, but at lower doses to accommodate the higher potencies of compounds of the invention. Such dosing adjustments should be within the ordinary skill of those in the clinical arts. [0036] Useful dosages of the compounds A, including the species I- VII can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to those having ordinary skill in the art who can, for example, be guided by the procedures described in U.S. Patent No. 4,938,949.

[0037] Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0038] The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol.

[0039] Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

[0040] The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0041] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.

[0042] The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0043] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0044] For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

[0045] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.

[0046] Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

[0047] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0048] Generally, the concentration of the compounds A, including the species I-IV in a liquid composition, such as a lotion, can be between about 0.1 and 25 mass %, such as between about 0.5 and 10 mass %. The concentration in a semi-solid or solid composition such as a gel or a powder can be between about 0.1 and 25 mass %, such as between about 0.5 and 2.5 mass %.

[0049] The amount of the compounds A, including the species I- VII, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. [0050] In general, however, a suitable dose can be in the range of between about 0.5 and 100 mg/kg, e.g., between about 10 and 75 mg/kg of body weight per day, such as between about 15 and 60 mg/kg/day. The compounds A, including the species I- VII can be conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, such as 10 to 750 mg, for example, 50 to 500 mg of active ingredient per unit dosage form.

[0051] The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub- doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations. Optionally, the compositions of the present invention can be administered to a patient in need thereof in combination with other therapeutically beneficial agent(s).

E. Methods for Evaluating the AdSS Inhibitory Activity of a Candidate Inhibitor

Compound.

[0052] AdSS inhibitory activity may be measured against an catalytically active reaction mixture comprised of GTP and/or ATP, IMP, MgCl₂, AdSS (which may be partially purified from pellets of human Molt3 leukemia cells or commercially obtained), with addition of Asp as a reaction initiator. The test compounds are diluted into the reaction mixtures from stock solutions in DMSO (e.g., 10 mM solutions). The formation of product is monitored spectrophotometrically as a difference in absorbance between 282 and 320 nm.

[0053] Cell culture assays for in vitro evaluation of the AdSS inhibitory activity of test compounds may be performed as previously described for testing of L-alanosine. Briefly, MTAP-positive cancer cells (e.g., Molt3 leukemia cells, lung cancer cells [A427 and MTAP transfected A549]) as well as MTAP-deficient cancer cells (e.g., CEM leukemia cells and A549 lung cancer cells) are cultured with test compounds.

[0054] Inhibitors having potential for in vivo use are preferably those which induce > 90% cell killing in MTAP-deficient cells at concentrations 10 fold lower than those that similarly kill MTAP positive cells, and will have activity at sub-micromolar (preferably nanomolar) concentrations. Further, toxicity of preferred inhibitors of the invention will be preventable by MTA and adenine addition to eels expressing MTAP, but only preventable by adenine in cells with MTAP deficiency. [0055] In vivo activity of inhibitory compounds of the invention may be evaluated in a xenograft murine model. To that end, the compounds are suspended at varying concentrations in saline or DMSO and given to normal mice 2 times daily by intraperitoneal injections for up to 2 weeks, to determine maximally tolerated dosages. The xenograft model mice will then be crafted by implanting either A549, A549/MTAP-transfected, or A427 cells subcutaneously into groups of SCID mice.

[0056] When tumor areas have reached a sufficient phase of growth (e.g., 0.3cm²), treatment with doses of the candidate inhibitor compounds or a control is performed to the maximally tolerable dosage level. Animals are then sacrificed when moribund or after a predetermined period of time; e.g., 14 days. Histological examinations of tumor tissue from the animals may then be made for evidence of residual tumor and tissue toxicity.

[0057] The following examples are intended to further illustrate but not limit the scope of the invention. Standard abbreviations (e.g., "ml" for milliters, and "min." for minutes) are used.

EXAMPLES

EXAMPLE 1. Synthesis of 8-(2-aminoethylthioMH-purin-6(9H)-one

[0058] The title compound shown above was synthesized according to the following reaction scheme:

[0059] 8-mercapto- 1 H-purin-6(9H)-one (50 mg, 0.3 mmol) was stirred in anhydrous DMF (3 ml) and solid 2-bromoethylamine hydrogen bromide was added to the solution (61 mg, 0.3 mmol), followed by adding triethylamine (90 μL, 0.65 mmol). The reaction mixture was stirred for 18 hours at room temperature and was concentrated under reduced pressure, followed by purification on C18 reverse phase to give the title compound. Yield 55 mg (88

%).

EXAMPLE 2. Synthesis of 2-(N-hvdroxyformamido)-N-(2-(6-oxo-6,9-dihvdroMH- purin-8-ethvIthio)ethvl)acetamide

[0060] The title compound shown above was synthesized from 8-(2-aminoethylthio)-lH- purin-6(9H)-one shown in Example 1, according to the following procedure. 8-(2- aminoethylthio)-lH-purin-6(9H)-one (55 mg, 0.26 mmol) was stirred in 2 ml of anhydrous DMF at room temperature.

[0061] To this solution was added 2-(hydroxyamino)acetic acid (59 mg, 0.65 mmol), EDCI (124 mg, 0.65 mmol) and DMAP (79 mg, 0.65 mmol). The reaction mixture was stirred for 16 hours and was concentrated and re-dissolved in formic acid (85 %, 1 mL) and acetic anhydride (250 μL). Purification on Cl 8 reverse phase to yielded the title compound. EXAMPLE 3. Synthesis of 5-[-(6-hvdroxy-9H-purin-8-ylthio)-2-(N- hydroxyformaidyl)! -pentanoic acid

[0062] The title compound shown above was synthesized according to the following 12- step reaction scheme shown below. This reaction scheme also indicates the quantities of intermediate compounds used at every step of the process as well as conditions that were used.

33 g 33 g 17 g I 17 g 12 g 12 g 8 g 8 g

12 500 mg

EXAMPLE 4. /« viϊro Inhibitory Activity of an AdSS Inhibitor of the Invention

[0063] An AdSS assay was performed as described in the Specification, using hadacidin as a positive control. The reaction mixture contained (in μM) 250 Hepes, 50 IMP, 25 GTP, 500 MgCl₂, and 730 ng AdSS, and the reaction was initiated by the addition of 500 μM Asp. The formation of the product was monitored spectrophotometrically as a difference in absorbance between 282 and 320 nm. As shown in Figure 2, compound I, labeled GE- 109, was significantly more potent than the hadacidin control or a non-heterocyclic inhibitor, labeled GIE 1-25.

[0064] Other heterocyclic bisubstrate inhibitors made according to the invention with activity in the assay were compounds II through VII. In contrast, inactive compounds (those that did not significantly inhibit AdSS activity in the assay, as compared to the control), including GIE 1-25, are illustrated in Figure 3. With the exception of GE-75, substituent L in structure A was not S in the inactive compounds tested.

[0065] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

CLAIMSWhat is claimed is:

1. A compound having the general structure A, and pharmaceutically acceptable salts or tautomers thereof:

wherein:

each of Ri and R₂ is independently selected from the group consisting of -H, a halogen, -NH₂, -OH, -NH-R₃, and -O-R₃;

each of Gi, G₂, and G₄, is independently selected from the group consisting of CH, N, O, and S, or G₄ is independently C=O group;

G₃ is independently selected from the group consisting of CH₂, NH, O, C=O group and S;

G₅ is independently selected from the group consisting of C and N;

L is absent or is selected from the group consisting of O, NH, and S;

R₃ is selected from a group consisting of -H, an C]-Ci₈ alkyl, an aryl,-C(O)-H, and -C(O)-alkyl;

R₄ is selected from a group consisting of -H, -C(O)O-; and -C(O)-R₃;

R₅ is selected from a group consisting of -H, an Ci-Ci₈ alkyl, and an aryl; M is absent or is selected from the group consisting Of-CH₂-; -NH-; -NH-C(O)-; - 0-, and -S-; and

n is an integer having the value between 1 and 6.

2. The compound of claim 1, wherein each of G₁, G₂, and G₄ is N, G₃ is NH, and G₅ is C.

3. The compound of claim 1, wherein the compound has the formula I or a pharmaceutically acceptable salt or the keto tautomer thereof:

4. The compound of claim 1, wherein the compound is selected from a group consisting of compounds

II, a pharmaceutically acceptable salt or the keto tautomer thereof;

in, a pharmaceutically acceptable salt or the keto tautomer thereof;

IV or a pharmaceutically acceptable salt thereof;

V, a pharmaceutically acceptable salt or the keto tautomer thereof;

VI, or a pharmaceutically acceptable salt thereof; and

VII, or a pharmaceutically acceptable salt thereof.

II

in

IV

VI

VII

5. A compound, and pharmaceutically acceptable salts and tautomers thereof, the compound comprising a substituted purin moiety, wherein the available carbon in the five- member ring of purin carries a substitutent selected from a group consisting of a substituted methoxy group, a substituted methylthio group, and a substituted secondary amino group,

with the further proviso that one or both available carbons in the six-member ring of purin optionally carries a substitutent selected from a group consisting of a halogen, a primary or secondary amino group, hydroxyl group, alkoxy group, or aryloxy group.

6. The compound of claim 5, wherein the substitutent in any of the substituted methoxy group, the substituted methylthio group, and the substituted secondary amino group is independently a moiety having the structure B:

B

wherein:

R₃ is selected from a group consisting of -H, an Ci-C₁₈ alkyl, an aryl,-C(O)-H, and -C(O)-alkyl;

R₄ is selected from a group consisting of -H, -C(O)O-; and -C(O)- R₃;

M is absent or is selected from the group consisting Of-CH₂-; -NH-; -NH-C(O)-; - O-, and -S-; and

n is an integer having the value between 1 and 5.

7. A pharmaceutical composition comprising a compound of any one of claims 1-6 and a pharmaceutically acceptable carrier therefor.

8. A method of treatment of a disorder, disease, or pathology that is clinically related to the activity of AdSS, comprising administering a pharmacologically effective dose of a pharmaceutical composition of claim 7 to a subject in need thereof, thereby treating the disorder, disease, or pathology.

9. The method of claim 8, wherein the disorder, disease, or pathology is a MTAP-defϊcient cancer.

10. A kit comprising a packaging material and a pharmaceutical composition according to claim 7 contained within the packaging material, wherein the packaging material comprises a label which indicates that the composition can be used for treating a disorder, disease, or pathology in a subject in need thereof.