Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
  • C07D487/04—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C255/00—Carboxylic acid nitriles
  • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
  • C07C255/31—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing rings other than six-membered aromatic rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C255/00—Carboxylic acid nitriles
  • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
  • C07C255/32—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring
  • C07C255/35—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by halogen atoms, or by nitro or nitroso groups
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C255/00—Carboxylic acid nitriles
  • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
  • C07C255/32—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring
  • C07C255/40—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by doubly-bound oxygen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C255/00—Carboxylic acid nitriles
  • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
  • C07C255/32—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring
  • C07C255/42—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by singly-bound nitrogen atoms, not being further bound to other hetero atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
  • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
  • C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
  • C07D207/34—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms

Definitions

• the present invention relates to derivatives of 2-amino-
• 4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidine it also relates to 4-oxo-3H, 5H.-pyrrolo [ 3 , 2-d pyrimidine derivatives substituted at the 7-position.
• Purine nucleoside phosphorylase catalyzes the phosphorolysis of purine nucleosides in a reversible reaction. Individuals who are deficient in PNP exhibit impaired T-cell development, resulting in lowered cell- mediated immunity, but normal B-cell development, resulting in normal humoral immunity. Accordingly, specific inhibitors of PNP that selectively inhibit T-cell development without damaging humoral immunity could be potentially effective against disorders in which activated T-cells are pathogenic.
• the present invention provides a 2- amino-7-(R)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-one wherein R is optionally substituted cyclohexenyl, cyclohexyl, or -CH 2 -R ⁇ , wherein R- is an optionally substituted heteroalicyclic, pyridinyl or alicyclic group.
• the present invention is also directed to a compound of the formula
• R 1 is H, NH 2 , or OCH 3
• R 2 is an optionally substituted cyclic group of 5-7 carbon atoms optionally containing one or more heteroatoms
• R 3 and R 4 are independently H or C 1-4 alkyl
• m is 0-4, n is 0-6, p is 0- 1
• X is CN, CSNH 2 , PO(OH) 2/ COOH, S0 2 NH 2 , NH 2 , OH, CNHNH 2 , tetrazole, triazole or COR 5 where R 5 is C!_ 4 alkyl, CF3, NH 2 , or 0C!_4 alkyl, and Y is O or NH.
• the compound of the present invention is useful as a PNP inhibitor.
• a pharmaceutical composition for the selective suppression of mammalian T-cell immunity comprising an pharmaceutically effective amount of the compound of the present invention and a pharmaceutically acceptable carrier or diluent and a method for the selective suppression of mammalian T-cell immunity without diminished effect on humoral immunity comprising administering to a subject a pharmaceutically effective amount of the compound of the present invention.
• the heteroalicyclic group is a 5 or 6 embered saturated ring having oxygen, nitrogen, or sulfur as the heterocyclic atom. More preferably the heteroalicyclic group is 2- or 3-tetrahydrothienyl, 2-, 3-, or 4- piperidinyl, 2- or 3-tetrahydrofuranyl, 2-, or 3- pyrrolidinyl, or 2-, 3-, or 4-tetrahydropyranyl.
• R- ⁇ is unsubstituted, e.g., the compound (I) is 2-amino-7-(2-piperidinylmethyl)-3H,5H-pyrrolo[- ,2-d]pyrimidin-4-one (IB), 2-amino-7-(3-piperidinyl- methyl)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-one (IC) , 2-amino- 7-(4-piperidinylmethyl)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-one (ID) , 2-amino-7-(2-tetrahydrofuranylmethyl) -3H,5H-pyr- rolo[3,2-d]pyrimidin-4-one (IE), 2-amino-7-(3-tetrahy- drofuranylmethyl)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-one (IF) , 2-amino-7-(2-piperidin
• the R-_ has one or two substituents selected from the group consisting of halogen, hydroxy, alkoxy, alkyl, or trifluoromethyl.
• halogen is preferably mentioned chloro or fluoro.
• alkoxy is preferably mentioned lower alkoxy, including ethoxy, ethoxy, propoxy and butoxy.
• alkyl is preferably mentioned lower alkyl, including methyl, ethyl, propyl and butyl.
• R-_ is unsubstituted, i.e., the compound (II) is 2- amino-7-(3-pyridinylmethyl)-3H,5H-pyrrolo[3,2-d]pyrimidin- 4-one (IIA) , 2-amino-7-(2-pyridinylmethyl) -3H,5H- pyrrolo[3,2-d]-pyrimidin-4-one (IIB) , or .2-amino-7-(4- pyridinylmethyl) -3H,5H-pyrrolo[3 ,2-d]-pyrimidin-4-one (IIC) .
• the R ⁇ group has one or two substituents selected from the group consisting of halogen, hydroxy, alkoxy, alkyl, or trifluoromethyl.
• halogen is preferably mentioned chloro or fluoro.
• alkoxy is preferably mentioned lower alkoxy, including methoxy, ethoxy, propoxy and butoxy.
• alkyl is preferably mentioned lower alkyl , including methyl , ethyl , propyl and butyl .
• R is unsubstituted, i.e., the compound
• (III) is 2-amino-7-(l-cyclohexenyl)-3H,5H-pyrrolo[3,2-d]- pyrimidin-4-one (IIIA) , 2-amino-7-(2-cyclohexenyl)-3H,5H- pyrrolo[3 ,2-d]pyrimidin-4-one (IIIB) , 2-amino-7-(3- cyclohexenyl)-3H,5H-pyrrolo[3,2-d]-pyrimidin-4-one (IIIC) , or 2-amino-7-(cyclohexyl)-3H,5H-pyrrolo[3,2-d]-pyrimidin-4- one (HID) .
• the R has at least one substituent selected from the group nsisting of halogen, hydroxy, alkoxy, alkyl, or
• halogen is preferably mentioned chloro or fluoro.
• alkoxy is preferably lower alkoxy, including methoxy, ethoxy, propoxy and butoxy.
• alkyl is preferably mentioned lower alkyl, including methyl, ethyl, propyl and butyl.
• alicyclic groups include, e.g., single-ring cycloparafins such as cyclopentyl, cyclohexyl, and cycloheptyl, multi-ring cycloparafins such as 1- and 2- adamantyl, 1-norbornanyl, 2-exo-norbornanyl, 2-endo- norbornanyl, 1- and 2-bicyclo[2.2.2]-octanyl, 1-, 2-, 3-, 6-, and 8-bicyclo[3.2.1]octanyl, and 1-, 2-, and 3- bicyclo[3.3.1]nonanyl and cycloolefins such as 1- and 2- norbornenyl.
• single-ring cycloparafins such as cyclopentyl, cyclohexyl, and cycloheptyl
• multi-ring cycloparafins such as 1- and 2- adamantyl, 1-norbornanyl, 2-exo-norbornanyl,
• Examples of the preferred compound (IV) are 2-amino-7-(2-adamantylmethyl) -3H, 5H-pyrrolo[3 , 2-d]- pyrimidin-4-one (IVA) , 2-amino-7- (1-adamantylmethyl)- 3H,5H-pyrrolo[3,2-d]pyrimidin-4-one (IVB) , 2-amino-7- (cyclopentyl ethyl ) -3H , 5H-pyrrolo [ 3 , 2-d] -pyrimidin-4-one (IVC) , 2-amino-7-(cyclohexylmethyl)-3H,5H-pyrrolo[3,2-d]- pyrimidin-4-one (IVD) , 2-amino-7-(cycloheptylmethyl) -3H,5H- pyrrolo[3 , 2-d]pyrimidin-4-one (IVE), 2-amino-7-(l- norbornanylmethyl ) -3H, 5
• the R group has one or two substituents selected from the group consisting of halogen, hydroxy, alkoxy, alkyl, or trifluoromethyl.
• halogen is preferably mentioned chloro or fluoro.
• alkoxy is preferably mentioned lower alkoxy, including methoxy, ethoxy, propoxy and butoxy.
• alkyl is preferably mentioned lower alkyl, including methyl, ethyl, propyl and butyl.
• the present invention is also directed to a compound of the formula
• R 1 is H, NH 2/ or OCH 3
• R 2 is an optionally substituted cyclic group optionally containing one or more heteroatoms
• R 3 and R 4 are independently H or C - ⁇ alkyl
• m is 0-4, n is 0-6, p is 0-1,
• X is CN, CSNH 2 , PO(OH) 2 , COOH, S0 2 NH 2 , NH 2 , OH, CNHNH 2 , tetrazole, triazole or COR 5 where R 5 is Ci-,4 alkyl, CF 3 , NH 2 , or alkyl, and Y is 0 or NH.
• the optionally substituted cyclic group (hereinafter referred to as cyclo) recited for the above formula includes aromatic, heteroaromatic, alicyclic, and heteroalicyclic groups preferably containing five to nine atoms.
• Preferred optional substituents include halogen, hydroxy, alkoxy, alkyl, and trifluoromethyl.
• Exemplary substituents include chloro, fluoro, methoxy, ethoxy, propoxy, butoxy, methyl, ethyl, propyl, and butyl.
• Preferred heteroatoms include oxygen, nitrogen, and sulfur, which can be present in combination in the same group.
• the preferred aromatic and heteroaromatic groups are phenyl, 2- or 3-thienyl, 2- or 3-furanyl, 2-, 3-, or 4-pyridinyl, 2- or 3-pyrrolyl, 2-, 4-, or 5-thiazolyl, 2-pyrazinyl, 3- or 4-pyridazinyl, and 3-, 4-, or 5-pyrazolyl.
• the preferred alicyclic and heteroalicyclic groups are 1- or 2-adamantyl, cyclohexyl, cycloheptyl, 2- or 3-tetrahydrofuranyl, 2- or 3-tetrahydrothienyl, 2- or 3-tetrahydropyranyl, 2-, 3-, or 4-piperidinyl, 3- or 4-pyrazolidinyl, 2-, 4-, or 5-thiazolidinyl, 2- or 3-piperazinyl, 2- or 3-morpholinyl, or 3- or 4-hexahydropyridazinyl.
• a method for the selective suppression of mammalian T-cell function without diminished effect on humoral immunity comprises administering to a mammal the compound (I) , whereby said compound inhibits purine nucleoside phosphorylase and thereby T-cell formation.
• a pharmaceutical composition for the selective suppression of mammalian T-cell function without diminished effect on humoral immunity which comprises an effective amount of the compound and a pharmaceutically acceptable diluent therefor.
• the invention further relates to pharmaceutical compositions suitable for enteral, such as oral or rectal, transdermal and parenteral administration to mammals including man, which are useful to inhibit purine nucleoside phosphorylase activity and for the treatment of disorders responsive thereto, comprising an effective amount of a pharmacologically active compound of the invention, alone or in combination, with one or more pharmaceutically acceptable carriers.
• Preferred pharmaceutical compositions are tablets and gelatin capsules comprising the active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, ethylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners.
• diluents e.g., lactose, dext
• compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions.
• Said compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In 8 addition,they may also contain other therapeutically valuable substances.
• Said compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, 5 preferably about 1 to 50%, of the active ingredient.
• transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at 5 a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.
• the present invention provides a method of inhibiting purine nucleoside phosphorylase activity in mammals and treating diseases and conditions responsive thereto, e.g., 0 autoimmune disorders, rejection of transplantation, or psoriasis, which comprises administering to a mammal in need thereof an effective amount of a compound of the invention or of a pharmaceutical composition comprising a said compound in combination with one or more 5 pharmaceutically acceptable carriers.
• diseases and conditions responsive thereto e.g., 0 autoimmune disorders, rejection of transplantation, or psoriasis
• a further aspect of the invention relates to a method of inhibiting the phosphorolysis and metabolic breakdown of antiviral or antitumor purine nucleosides in mammals which comprises administering in conjunction therewith to a 0 mammal in need thereof, either separately or in combination therewith, an effective purine nucleoside phosphorylase inhibiting amount of a compound of the invention or of a said compound in combination with one or more pharmaceutically acceptable carriers. More particularly.
• Such relates to a method of inhibiting the phosphorolysis and metabolic breakdown of purine nucleosides known in the art, e.g., of 2'-deoxyguanosine, 2' ,3'-dideoxyinosine, 2' ,3'-dideoxyguanosine or 2' ,3'-dideoxyadenosine.
• the invention thus relates to a method of potentiating the antiviral or antitumor effect of 2* or 3*- monodeoxypurine nucleosides or of 2' ,3•-dideoxypurine nucleosides in mammals which comprises administering in conjunction therewith to a mammal in need thereof, either separately or in combination with a said nucleoside, an effective purine nucleoside phosphorylase inhibiting amount of a compound of the invention preferably in combination with one or more pharmaceutically acceptable carriers.
• such relates to a method of enhancing or potentiating the effect of 2 ⁇ ,3'-dideoxypurine nucleosides known in the art, e.g., of 2' ,3'-dideoxyinosine, 2',3 I - dideoxyguanosine or 2 '-3 •-dideoxyadenosine for the treatment of retrovirus infections, e.g., HIV-retrovirus infections (acquired immunodeficiency syndrome, AIDS) .
• retrovirus infections e.g., HIV-retrovirus infections (acquired immunodeficiency syndrome, AIDS) .
• 2',3 • -Dideoxypurine nucleosides are known in the art as inhibitors of HIV retrovirus infectivity and to be metabolically degraded by PNP, e.g., as described in Biochemical Pharmacology 22, 3797 (1987) . Such are administered at a pharmaceutically acceptable dose which is effective in inhibiting HIV-retrovirus infections. Preferably the lowest possible effective dose is used.
• the pharmaceutically acceptable effective dosage of active compound of the invention to be administered is dependent on the species of warm-blooded animal (mammal) , the body weight, age and individual condition, and on the form of administration.
• the pharmaceutical composition may be oral, parenteral, suppository or other form which delivers the compound into the bloodstream of a mammal to be treated.
• An oral form 10 has from about 1 to about 150 mg of the compound for an adult (50 to 70 kg) which is mixed together with pharmaceutically acceptable diluents such as lactose. In a typical capsule, 25 mg of the compound are mixed together with 192 mg lactose, 80 mg modified starch and 3 mg magnesium stearate. Injectable forms of the compound are also contemplated for administration.
• the present invention is also useful with other therapeutic agents.
• a daily dosage for a human weighing 50 to 70 kg of 1-50 mg/kg inhibits metabolic destruction of certain anticancer agents such as beta-2 '-deoxy-6- thioguanosine and antiviral agents such as 2*,3*- dideoxyinosine, an anti-AIDS drug.
• anticancer agents such as beta-2 '-deoxy-6- thioguanosine and antiviral agents such as 2*,3*- dideoxyinosine, an anti-AIDS drug.
• anticancer agents such as beta-2 '-deoxy-6- thioguanosine and antiviral agents such as 2*,3*- dideoxyinosine, an anti-AIDS drug.
• anticancer agents such as beta-2 '-deoxy-6- thioguanosine
• antiviral agents such as 2*,3*- dideoxyinosine, an anti-AIDS drug.
• One method of making the compound (I) of the present invention uses 3-substituted propionitriles as starting materials. Such starting materials can be obtained by a variety of methods that are well documented in the literature.
• the compound (I) is then prepared from the starting material by an adaptation of the synthetic methodology disclosed in M. I. Lim, R. S. Klein, and J. J. Fox, J. Pro. Chem.. 44. 3826 (1979); M.I. Lim, R.S. Klein, and J.J. Fox, Tetrahedron Lett.. 21. 1013 (1980); M.I. Lim and R.S. Klein, Tetrahedron Lett.. 22. 25 (1981) ; M. I. Lim, W. Y. Ren, B.A. Otter, and R.S. Klein, J. Or ⁇ . Chem.. 48., 780 (1983).
• a method of making the compound (II) of the present invention uses a 3-(pyridinyl)propionitrile as the starting material to make the compound (II) .
• the appropriate 3- (pyridinyl)propionitrile can be produced by converting the corresponding 3-(pyridinyl)propionyl chloride to the corresponding amide by ammonolysis with, e.g., ammonium hydroxide, which is then dehydrated to the desired nitrile by distillation with a dehydrating agent, such as P0C1 3 or S0C1 2 .
• the starting material is produced by condensation of the 3-aldehyde with cyanoacetic acid followed by decarboxylation to give the corresponding substituted acrylonitrile, which is hydrogenated to give the corresponding 3-(pyridinyl)propionitrile by either catalytic hydrogenation or magnesium metal dissolving in methanol at 0 ' C, such as disclosed in Profitt, J. , et al., J. Qr ⁇ . Chem. , 4O, 127 (1975) .
• the compound (II) is then prepared from the starting material by an adaptation of the synthetic methodology disclosed in M. I. Lim, R. S. Klein, and J. J. Fox, J. Orq. Chem.. 44. 3826 (1979) ; M.I.
• Another method of making the compound of the present invention uses a known compound, i.e., cyclohexenyl- acetonitrile, as the starting material.
• the compound (III) of the present invention is made by reacting the starting material in an adaptation of the synthetic methodology disclosed in M. I. Lim, R. S. Klein, and J. J. Fox, J.
• a method of making the compound (IV) of the present invention uses 3-substituted propionitriles as starting materials. Such starting materials can be obtained by a 12 variety of methods that are well documented in the literature.
• the compound (IV) is then prepared from the starting material by an adaptation of the synthetic methodology disclosed in M. I. Lim, R. S. Klein, and J. J. Fox, J. Orq. Chem.. 44. 3826 (1979); M.I. Lim, R.S. Klein, and J.J. Fox, Tetrahedron Lett.. 21. 1013 (1980) ; M.I. Lim and R.S. Klein, Tetrahedron Lett.. 22. 25 (1981) ; M. I. Lim, W. Y. Ren, B.A. Otter, and R.S. Klein, J. Orq. Chem..
• the first step of the method involves reacting an optionally substituted cyclic aldehyde with cyanoacetic acid at a molar ratio of about 1/1 to 1/5 in the presence of ammonium acetate at about reflux temperature for about 10 hours to 8 days to make a 3-cyclo- substituted pentanedinitrile as an intermediate.
• the 3-cyclo-pentanedinitrile is reacted with an alkyl formate such as ethyl formate and a strong base such as the metal-containing bases sodium hydride or sodium alkoxide, e.g., sodium methoxide, at a molar ratio of about 1-2/3-6/1-3 and at a temperature of about 20-65°C for about 10 hours to 8 days to make a 3-cyclo-2-formylpentanedini- trile as a further intermediate.
• an alkyl formate such as ethyl formate
• a strong base such as the metal-containing bases sodium hydride or sodium alkoxide, e.g., sodium methoxide
• the next step involves reacting the 3-cyclo-2-formylpentanedinitrile with a glycine alkyl ester hydrochloride and sodium or ammonium acetate at a molar ratio of about 1-2/1.5-4/1.5-4 and at a temperature of about 20-60"C for about 10-48 hours to make methyl N-[ (3-cyclo-2,4-dicyano)-2-butenyl]glycine as an intermediate.
• the methyl N-[(3- cyclo-2,4-dicyano)-2-butenyl]glycine is reacted with an alkyl chloroformate such as ethyl chloroformate and 1,5- diazabicyclo[4.3.0]non-5-ene (DBN) or 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU) at a molar ratio of about 1-2/1.5- 5/1.5-4 and at a temperature of about 0-50°C for about 10 hours to 10 days to make methyl 3-amino-4-(2-cyano-l-cyclo- ethyl) -1-ethyl-lH-pyrrole-l , 2-dicarboxylate as an intermediate.
• an alkyl chloroformate such as ethyl chloroformate and 1,5- diazabicyclo[4.3.0]non-5-ene (DBN) or 1,8-diazabicyclo [5.4.0]unde
• the next step involves reacting the methyl 3-amino-4-(2-cyano-1-cyclo-ethyl)-1-ethyl-lH-pyrrole-l,2- dicarboxylate with a base such as sodium carbonate at a molar ratio of about 2/1 to 1/5 and at about room temperature for about 10-48 hours to make methyl 3-amino-4- (2-cyano-l-cyclo-ethyl)-lH-pyrrole-2-carboxylate as an intermediate.
• a base such as sodium carbonate
• the methyl 3-amino-4-(2- cyano-l-cyclo-ethyl)-lH-pyrrole-2-carboxylate is reacted with benzoylisothiocyanate at a molar ratio of about 2/1 to 1/2 and at about room temperature for about 30 minutes to 3 hours to make N-benzoyl-N—[4-(2-cyano-l-cyclo-ethyl)-2- methoxycarbonyl-lH-pyrrol-3-yl]thiourea as an intermediate.
• the next step reacts the N-benzoyl-N—[4-(2-cyano-l-cyclo- ethyl)-2-methoxycarbonyl-lH-pyrrol-4-3-yl]thiourea with an alkyl halide such as methyl iodide at a molar ratio of about 1/1 to 1/6 and at a temperature of about 0-30"C for about 10 minutes to 10 hours to make N-benzoyl-N—[4-(2- cyano-1-cyclo-ethyl) -2-methoxycarbonyl-lH-pyrrol-3-yl]S- ethylthiourea as an intermediate.
• an alkyl halide such as methyl iodide
• the N-benzoyl-N' -[4- (2-cyano-l-cyclo-ethyl) -2- methoxycarbonyl-lH-pyrrol-3-yl]-S-methylthiourea (about 1- 2 mol) is reacted with methanolic or ethanolic ammonia at a ratio of about 1/1 to 1/20 and at a temperature of about 20-130 ⁇ C for about 16-60 hours to make a mixture of a 2- amino compound of the present invention 3-cyclo-3-[2-amino- 4-oxo-3H-5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile and a 3-cyclo-3-[2-methylmercapto-4-oxo-3H,5H-pyrrolo[3,2- d]pyrimidin-7-yl]propanenitrile as an intermediate in making another compound of the present invention.
• the 3-cyclo-3-[2-methylsulfonyl-4-oxo-3H,5H-pyrrolo[3,2- d]pyrimidin-7-yl]propanenitrile is reacted with a sodium alkoxide such as sodium methoxide at a molar ratio of about 1/1 to 1/10 and at a temperature of about 25-100°C for about 1-48 hours to make a 2-methoxy compound of the present invention 3-cyclo-3-[2-methoxy-4-oxo-3H,5H- pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile.
• a method of making a compound wherein R 1 is hydrogen.
• the methyl 3-amino-4- (2-cyano-l-cyclo-ethyl)-lH-pyrrole-2-carboxylate intermediate described supra is reacted with dimethylformamide dimethyl acetal at a molar ratio of about 1/1 to 1/4 and at a temperature of about 25-100 " C for about 1-10 days to make methyl 4-(2-cyano-l-cyclo-ethyl)-3-[N- (dimethylaminomethylene)amino]-lH-pyrrole-2-carboxylate as an intermediate.
• the next step involves reacting the methyl 4-(2-cyano-l-cyclo-ethyl) -3-[N-(dimethylamino- methylene)amino]-lH-pyrrole-2-carboxylate with methanolic or ethanolic ammonia at a molar ratio of about 1/1 to 1/20 and at a temperature of about 20-130°C for about 10-68 hours to make the compound of the present invention 3- cyclo-3-[4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7- yl]propanenitrile.
• variations of the aforesaid procedures are useful in making the variety of compounds of the present invention without departing from the spirit thereof.
• EXAMPLE 1 3-(3-Pyridinyl)propionitrile is prepared in this example.
• a three-neck flask carrying a magnetic stir bar is fitted with a thermometer, pressure equalizing addition funnel, and a reflux condenser carrying an argon inlet.
• Freshly powdered potassium hydroxide (6.6 g, 0.1 mol) and anhydrous acetonitrile (150 ml) are charged into the flask and heated at reflux while 3-pyridinecarboxaldehyde (10.7 g, 0.1 mol) in anhydrous acetonitrile (50 ml) is added dropwise over a period of about five minutes and refluxing continued for about another three minutes.
• EXAMPLE 2 3-(3-Pyridinyl)propionitrile of Example 1 is further treated in the synthesis of the present invention. Under 17 an atmosphere of dry N 2 , a mixture of 3- (3- pyridinyl)propionitrile (0.661 g, 5.0 mmole), sodium hydride (0.240 g, 10.0 mmole), and ethyl formate (l.ll g, 15.0 mmole) in anhydrous tetrahydrofuran (20 ml) is stirred for 48 hours with protection from air and moisture. Volatile matter is evaporated, and a solution of the solid residue in 15 ml of cold water is adjusted at 0 ⁇ C to a pH of 6 with cold 6N HC1.
• EXAMPLE 4 Under a nitrogen atmosphere, ethyl chloroformate (0.521 g, 4.8 mmole) is added dropwise to a solution of the enamine of Example 3 (0.513 g, 3.2 mmole) and 1,5- diazabicyclo[4.3.0]non-5-ene (DBN, 1.37 g, 11.1 mmole) in dry CH 2 C1 2 (15 ml) with external cooling in an ice bath. After stirring at 0 °C for one hour, the solution is allowed to a stand at room temperature overnight. After checking progress by TLC, additional ClC0 2 Et (0.1 ml) and DBN (1.0 ml) are added to complete the conversion, and the solution is allowed to stand for 24 hours.
• DBN 1,5- diazabicyclo[4.3.0]non-5-ene
• Volatile matter is evaporated in vacuo and the viscous residue is purified on a short silica gel column (whose main purpose is to remove the less-mobile DBN) to give an N-blocked pyrrole; which is used for the next step without further purification.
• Methyl iodide (0.228 g, 1.61 mmole) is added to a solution of the thioureido product of Example 6 (0.383 g. 0.94 mmole) and DBN (0.140 g, 1.12 mmole) in dry CH C1 2 (10 ml) at 0 "C. The solution is stirred at 0 "C for 15 min., at ambient temperature for 1 h, and then evaporated in vacuo. A solution of the residue in CHC1 3 is chromatographed on a silica gel column with CHCl 3 /methanol (97:3) as eluent to give homogeneous fractions of the methylthio intermediate compound.
• EXAMPLE 8 A solution of the methylthio compound of Example 7 (0.358 g, 0.85 mmole) in 100 ml of MeOH that has been saturated with NH 3 at 0 "C is heated at 90-95 "C for 24 hours in a glass-lined stainless steel bomb. The contents of the chilled bomb are evaporated in vacuo to give a mixture of the desired 2-amino intermediate compound, benzamide, and a by-product that is a 2-methylthio derivative, as opposed to the 2-amino compound. The mixture is dissolved in methanol and the solution is evaporated with silica gel (about 5 g) .
• a solution of the pyridinylmethyl intermediate in 0.1N HC1 is hydrogenated with a platinum catalyst at 60 lb/in 2 H pressure.
• the catalyst is generated by brief hydrogenation of Pt0 2 in 0.1N HC1.
• the catalyst is removed by filtration under N pressure, and the filtrate is evaporated.
• a solution of the residue in the minimum amount of ethanol is diluted slowly with a large amount of Et0 2 , and the hydrochloride salt is obtained as a white hygroscopic solid of the compound (IC) .
• Example 8 The compound (IC) of Example 8 is tested for enzyme inhibition activity.
• a purine nucleoside phosphorylase A purine nucleoside phosphorylase
• PNP enzyme assay is performed in which PNP activity
• IC50 for the compound is observed, which is determined radiochemically by measuring the formation of [ 14 C]- hypoxanthine from [ 1 C]-inosine (see Biomedicine. 33, 39 (1980)) using calf spleen as the enzyme source.
• EXAMPLE 15 3-(2-Pyridinyl)propionitrile is prepared in this example using the procedure of V. Boekelheide, et al., J. Am. Chem. Soc.. 75_, 3243 (1953) .
• a solution of potassium cyanide (83.74 g) in water (160 ml) is added to a solution of freshly distilled 2-vinylpyridine (67.59 g) in acetic anhydride (131.30 g) with the rate of addition adjusted to maintain gentle refluxing.
• the resulting dark red mixture is heated for about 17 hours at 105 ⁇ C in an oil bath with vigorous stirring.
• the cooled reaction mixture is adjusted to pH 8 with a saturated Na 2 C ⁇ 3 solution.
• a pharmaceutical composition for intraperitoneal injection is prepared for testing the compound (IC) .
• An intraperitoneal injection solution containing the compound of Example 8 is dissolved in an aqueous carrier that contains ten percent DMSO.
• EXAMPLE 22 The compound (IC) is intraperitoneally injected into Lewis Rats via the test composition of Example 21 to provide 30 mg of the compound (IC) , with an injection given twice per day. Controls are used, which receive only the vehicle. At specific times after administration, the animals are sacrificed and plasma samples are prepared. 22
• the plasma is extracted with cold 0.5 N HCIO4 and neutralized with solid NH4HCO3. After removal of perchlorate salts, the extract is subjected to HPLC on a reversed phase column (Spherisorb ODSI) . A significant increase in plasma inosine is observed in the plasma taken from animals receiving the compound (IC) .
• EXAMPLES 23-33 Compounds prepared as in Examples 10-20 are each made into a pharmaceutical formulation in accordance with the preparation of Example 21 and the resultant injectable solutions are tested in accordance with the procedure of Example 22. A significant increase in plasma inosine is observed in the plasma taken from animals receiving the compounds of the present invention.
• EXAMPLE 34
• 3-(2-Furanyl)propionitrile is prepared in this example.
• magnesium turnings (20 g) are added to a solution of 3-(2-furanyl)acrylonitrile (67.2 g) in dry methanol (2 1). Additional magnesium is added in parts as the reaction rate would allow until a total of 145 g has been added.
• the solvent is evaporated until the contents set to a solid paste, which is adjusted to a pH of about 6.5 with 6N HC1 with cooling.
• the mixture is extracted with several portions of CHC1 3 , dried with Na 2 S0 4 , and concentrated to a dark oil. Distillation in vacuo through a short Vigreaux column gives the propionitrile as a colorless oil; yield 49.5 g (72%); bp 46.0-46.5°C at 0.5 mm.
• 3-(2-Furanyl)propionitrile of Example 34 is further treated in the synthesis of the present invention.
• the propionitrile (48.7 g) , sodium hydride (10.28 g) , ethyl formate (32.74 g) , and anhydrous tetrahydrofuran (200 ml) are stirred at room temperature with protection from moisture for about 18 hours.
• the volatile matter is then evaporated, the resulting yellow solid is dissolved in about 20 ml of cold water with ice- bath cooling, and the solution is adjusted to a pH of 6.0 with cold 6N HC1.
• the resulting heavy oil precipitate is extracted into CHC1 3 , and the extract is washed with water, dried with Na 2 S04, and evaporated to give a thin oil which contains the crude formyl compound; yield 53.06 g.
• EXAMPLE 37 Under a nitrogen atmosphere, ethyl chloroformate (43.68 g) is added dropwise to a solution of the amber oil of Example 36 (59.1 g) and DBN (133 g) in dry CH 2 Cl 2 (400 ml) with external cooling in an ice bath. After stirring at 0 °C for one hour, the solution is allowed to stand at room temperature overnight. After checking progress by TLC, additional ClC0 2 Et and DBN are added to complete the conversion, and the solution is allowed to stand for 24 hours.
• Benzoyl isothiocyanate (4.58 g) is added dropwise to a solution of the unblocked pyrrole of Example 38 (5.15 g) in dry CH 2 C1 2 (75 ml) . After 1 h at room temperature, solution is evaporated, and the gummy residue is dissolved in Et 2 0 (100 ml) with almost immediate separation of the crystalline solid, which is collected by filtration. The Et 2 0 filtrate is heated to boiling and diluted with an equal volume of warm cyclohexane. On cooling slowly the solution gives additional thioureido product.
• EXAMPLE 40 EXAMPLE 40
• Methyl iodide (8.70 g) is added to a solution of the thioureido product of Example 39 (10.68 g) and DBN (4.15 g) in dry CH 2 C1 2 (250 ml) at 0 °C. The solution is stirred at 0 °C for 15 min., at ambient temperature for 1 h, and then evaporated in vacuo. A solution of the residue in CHC1 3 is ⁇ hromatographed on a silica gel column with CHC1 3 as eluent to give homogeneous fractions of the methylthio intermediate compound. 25
• EXAMPLE 41 A solution of the methylthio compound of Example 40 (0.80 g, 2.0 mmole) in 25 ml of MeOH that has been saturated with NH 3 at 0 ⁇ C is heated at 90-95 °C for 24 hours in a glass-lined stainless steel bomb. The contents of the chilled bomb are evaporated in vacuo to give a mixture of the 2-amino compound, benzamide and a by-product that is a 2-methylthio derivative, as opposed to the 2- amino compound. The mixture is stirred vigorously for several minutes with 30 ml of 2:1 Et 2 0/cyclohexane, and the insoluble white solid is filtered off and washed with Et 2 0. The filtrate contained most of the benzamide and 2- methylthio components.
• a pharmaceutical composition for intraperitoneal injection is prepared for testing the compound (IE) .
• An intraperitoneal injection solution containing the compound of Example 41 is dissolved in an aqueous carrier that contains ten percent DMSO.
• EXAMPLE 44 Using the procedure of Example 16, the compound (IE) is intraperitoneally injected into Lewis Rats via the test composition of Example 43 to provide 30 mg of the compound (IE) and the results are analyzed compared to controls. A significant increase in plasma inosine is observed in the plasma taken from animals receiving the compound (IE) .
• a solution of the bromoalcohol in anhydrous di ethyl- acetamide is chilled in an ice bath and treated with an equimolar portion of PBr3 « The solution is allowed to stir at room temperature for one hour, then the dimethylacetamide is evaporated n vacuo at low temperature using an air pump and dry-ice trap.
• a suspension of the residue in ice/water is adjusted to pH 7 using IN NaOH to give the crude reaction product containing the 1,4-dibromo compound.
• the resulting product is refluxed with sodium sulfide in 50% ethanol/water, or alternatively warmed with Na 2 s in N,N-dimethylacetamide solution; the solvent removed under vacuum, the residue washed with water to remove NaBr, and the pH adjusted to about 7 to precipitate the 2-tetrahydrothienyl compound (IG).
• EXAMPLE 51 The compound of Example 50 is tested for enzyme inhibition activity as in Example 9, and PNP activity (IC50) for the compound is observed.
• EXAMPLE 52 A pharmaceutical composition for intraperitoneal injection is prepared for testing the compound (IG) .
• An intraperitoneal injection solution is made containing the compound of Example 50 is dissolved in an aqueous carrier that contains ten percent DMSO.
• EXAMPLE 53 Using the procedure of Example 16, the compound (IG) is intraperitoneally injected into Lewis Rats via the test composition of Example 52 to provide 30 mg of the compound (IG) and the results are analyzed compared to controls. A significant increase in plasma inosine is observed in the plasma taken from animals receiving the compound (IG) .
• EXAMPLE 60 Using the procedure of Example 1, 3-(3-pyrrolyl)- propionitrile is prepared using pyrrole-3-carboxaldehyde as the starting material. Following Examples 2-8, the intermediate 2-amino-7-(3-pyrrolylmethyl)-3H,5H-pyrrolo- [3,2-d]pyrimidin-4-one is prepared from the propionitrile. 29 from which the 3-pyrrolidinyl compound (IJ) is obtained by reduction of the intermediate.
• [3,2-d]pyrimidin-4-one is prepared from the propionitrile, from which the 2-tetrahydro-pyranyl compound (IL) is obtained by reduction of the intermediate.
• EXAMPLE 62 Using the procedure of Example 1, 3- (3-pyranyl) propionitrile is prepared using 3-pyrancarboxaldehyde as the starting material. Following Examples 2-8, the intermediate 2-amino-7- (3-pyranylmethyl ) -3H. / 5I£- pyrrolo[3 ,2-d]pyrimidin-4-one is prepared from the propionitrile, from which the 3-tetrahydro-pyranyl compound (IM) is obtained by reduction of the intermediate.
• IM 3-tetrahydro-pyranyl compound
• EXAMPLE 64 A solution of the methylthio compound of Example 7 (0.358 g, 0.85 mmole) in 100 ml of MeOH that has been saturated with NH 3 at 0 ⁇ C is heated at 90-95 °C for 24 hours in a glass-lined stainless steel bomb. The contents of the chilled bomb are evaporated in vacuo to give a mixture of the desired 2-amino intermediate compound, benzamide, and a by-product that is a 2-methylthio derivative, as opposed to the 2-amino compound. The mixture is dissolved in ethanol and the solution is evaporated with silica gel (about 5 g) .
• EXAMPLE 65 The compound (HA) of Example 64 is tested for enzyme inhibition activity.
• a purine nucleoside phosphorylase (PNP) enzyme assay is performed in which PNP activity (IC50) for the compound is observed, which is determined radiochemically by measuring the formation of [ 14 C]- hypoxanthine from [ 1 C]-inosine (see Biomedicine. 33, 39 (1980) using calf spleen as the enzyme source.
• PNP activity IC50
• a pharmaceutical composition for intraperitoneal injection is prepared for testing the compound (HA) .
• An intraperitoneal injection solution containing the compound of Example 64 is dissolved in an aqueous carrier that contains ten percent DMSO.
• EXAMPLE 70 The compound (HA) is intraperitoneally injected into Lewis Rats via the test composition of Example 69 to provide 30 mg of the compound (IIA) , with an injection given twice per day. Controls are used, which receive only the vehicle. At specific times after administration, the animals are sacrificed and plasma samples are prepared. The plasma is extracted with cold 0.5 N HC10 4 and neutralized with solid NH 4 HC0 3 . After removal of perchlorate salts, the extract is subjected to HPLC on a reversed phase column (Spherisorb ODSI) . A significant increase in plasma inosine is observed in the plasma taken from animals receiving the compound (IIA) .
• Spherisorb ODSI reversed phase column
• EXAMPLES 71-73 Compounds prepared as in Examples 66-68 are each made into a pharmaceutical formulation in accordance with the preparation of Example 69 and the resultant injectable solutions are tested in accordance with the procedure of Example 70. A significant increase in plasma inosine is observed in the plasma taken from animals receiving the compounds of the present invention.
• EXAMPLE 74 1-Cyclohexenylacetonitrile is treated in the synthesis of the present invention. Under an atmosphere of dry N 2 , a solution of 1-cyclohexenylacetonitrile (9.2 g; 75.92 mmole) in anhydrous tetrahydrofuran (THF, 10 ml) is added to a stirred mixture of sodium hydride (3.18 g; 132.86 mmole) and ethylformate (30.14 g; 406.93 mmole) in 50 ml THF, and the resulting mixture is stirred at room temperature for about 18 hours. Volatile matter is evaporated in vacuo at room temperature.
• THF tetrahydrofuran
• the extract is dried (Na 2 S0 ) and evaporated to give a viscous gum, which is purified on a silica gel column using CHC1 3 as the eluent; yield 2.04 g; m.p. 125 ⁇ C.
• EXAMPLE 78 Benzoyl isothiocyanate (0.76g, 4.65 mmole) is added dropwise to a solution of the unblocked pyrrole of Example 77 (0.91 g, 4.13 mmole) in dry CH 2 C1 2 (30 ml). After 1 h at room temperature, the solution is evaporated, and the gummy residue is triturated with methanol to give a thioureido product; yield 0.70 g; m.p. 170°C EXAMPLE 79
• Methyl iodide (0.678 g, 4.78 mmole) is added to a solution of the thioureido product of Example 78 (0.630 g, 1.64 mmole) and DBN (0.230 g, 1.85 mmole) in dry CH 2 C1 2 (50 ml) at 0 °C. The solution is stirred at 0 °C for 15 min., at ambient temperature for 1 h, and then evaporated in vacuo. A solution of the residue in CHCI3 is chromatographed on a silica gel column with CHC1 3 as eluent to give homogeneous fractions of the methylthio intermediate; yield 0.7 g. EXAMPLE 80
• the compound of Example 80 is tested for enzyme- inhibition activity.
• a purine nucleoside phosphorylase (PNP) enzyme assay is performed in which PNP activity for the compound is determined radiochemically by measuring the formation of [ 14 C]-hypoxanthine from [ 14 C]-inosine (see Biomedicine. 33, 39 (1980) using calf spleen as the enzyme source.
• PNP activity for the compound is determined radiochemically by measuring the formation of [ 14 C]-hypoxanthine from [ 14 C]-inosine (see Biomedicine. 33, 39 (1980) using calf spleen as the enzyme source.
• PNP activity for the compound is determined radiochemically by measuring the formation of [ 14 C]-hypoxanthine from [ 14 C]-inosine (see Biomedicine. 33, 39 (1980) using calf spleen as the enzyme source.
• the IC 50 is 1.9 ⁇ M
• 50 mM phosphate the IC 50 is 19 ⁇ M.
• EXAMPLE 82 Following the procedures set forth in Examples 74-80, compounds (IIIB) and (IIIC) are made using 2- and 3- cyclohexenyl-acetonitrile, respectively, as starting materials. The compounds are tested as in Example 81 and significant enzyme-inhibition activity is observed. EXAMPLES 83-87
• a pharmaceutical composition for intraperitoneal injection is prepared for testing the compound (IIIA) .
• An intraperitoneal injection solution containing the compound (IIIA) is prepared for testing the compound (IIIA) .
• (IIIA) is dissolved in an aqueous carrier that contains ten percent DMSO.
• EXAMPLE 89 The compound (IIIA) is intraperitoneally injected into Lewis Rats via the test composition of Example 88 to provide 30 mg of the compound (IIIA) , with an injection given twice per day. Controls are used, which receive only the vehicle. At specific times after administration, the animals are sacrificed and plasma samples are prepared.
• the plasma is extracted with cold 0.5 N HC10 and neutralized with solid NH4HCO 3 . After removal of perchlorate salts, the extract is subjected to HPLC on a reversed phase column (Spherisorb ODSI) . A significant increase in plasma inosine is observed in the plasma taken from animals receiving the compound (IIIA) .
• EXAMPLES 90-94 Compounds prepared as in Examples 83-87 are each made into a pharmaceutical formulation in accordance with the preparation of Example 88 and the resultant injectable solutions are tested in accordance with the procedure of Example 89. A significant increase in plasma inosine is observed in the plasma taken from animals receiving the compounds of the present invention.
• EXAMPLE 95
• the compound (HID) is prepared using 2-amino-7-(l- cyclohexenyl) -3H_,5H-pyrrolo[3,2-d]pyrimidin-4-one as an intermediate.
• a solution of the intermediate (0.2 g; 0.86 mmole) in ethanol (50 ml) is hydrogenated with 10% Pd-C (50 mg) at 45 lb/in 2 for 16 h and filtered hot through Celite.
• EXAMPLE 96 The compound (HID) prepared in Example 95 is tested for enzyme-inhibition activity as in Example 81. At 1 mM phosphate the IC 50 is 1.3 ⁇ M, and at 50 mM phosphate the IC 50 is 145 ⁇ M.
• 3-(2-Adamantyl)propionitrile is prepared in this example using a modification of the procedure of M. Ohno, et al., J. Org. Chem. 53, 1285 (1988).
• a Solution of 2- bromoadmantane (20 g; 92.96 mmole); Bu 3 SnH (32.46 g; 111.5 mmole), acrylonitrile (9,86 g; 185.92 mmole), and AIBN (740 mg) in toluene (280 ml) is stirred at reflux temperature for 3 h.
• reaction mixture is washed with ammonia water (0.4 M, 500 ml), the organic layer is washed with H 2 0 and dried over MgS0 4 and evaporated.
• the residue is distilled between 110-118 ⁇ C (and about 0.2 mmHg) ; fractions are combined to give a crude sample of contaminated 3-(2- ada antyl)propionitrile with tin complexes, which is purified on silica gel column with hexanes; followed by hexanes/ethylacetate 97:3 and hexanes/ethylacetate 95:5, yield 9.4 g (53.4%); mp semi-solid.
• EXAMPLE 98 3-(2-Adamantyl)propionitrile of Example 97 is further treated in the synthesis of the present invention. Under an atmosphere of dry N 2 , a mixture of 3-(2-adamantyl) propionitrile (7.0 g, 36.99 mmole), sodium hydride (1.7 g, 73.95 mmole), and anhydrous tetrahydrofuran (75 ml) is heated at 52"C in a water bath for 15 min., and a solution of ethyl formate (13.69 g, 184.89 mmole) in THF (100 ml) is added over a period of 45 min.
• EXAMPLE 104 A solution of the methylthio compound of Example 103 (2.78 g, 5.18 mmole) in 150 ml of MeOH that has been saturated with NH 3 at 0 ⁇ C is heated at 90-95 °C for 24 hours in a glass-lined stainless steel bomb. The contents of the chilled bomb are evaporated in vacuo to give a mixture of the compound (IVA) , benzamide and a by-product that is a 2-methylthio derivative, as opposed to the 2- amino compound (IVA) . The mixture is stirred vigorously for several minutes with appr. 75 ml of Et 2 0, and the insoluble white solid is filtered off and washed with Et 2 0.
• EXAMPLE 105 The compound of Example 104 is tested for enzyme inhibition activity.
• a purine nucleoside phosphorylase A purine nucleoside phosphorylase
• IC 50 for the compound is found, which is determined radiochemically by measuring the formation of [ 14 C]- hypoxanthine from [ 1 C]-inosine (see Biomedicine. 33, 39
• IC 50 is 2.5 ⁇ M.
• Example 106 i 2-(l-methyl)-adamantyl
• Example 107 Ri 2-(l-chloro)-adamantyl
• a pharmaceutical composition for intraperitoneal injection is prepared for testing the compound (IVA) .
• An intraperitoneal injection solution containing the compound (IVA) is prepared for testing the compound (IVA) .
• the compound (IVA) is intraperitoneally injected into Lewis Rats via the test composition of Example 117 to provide 30 mg of the compound (IVA) , with an injection given twice per day. Controls are used, which receive only the vehicle. At specific times after administration, the animals are sacrificed and plasma samples are prepared. The plasma is extracted with cold 0.5 N HC10 4 and neutralized with solid NH4HCO3. After removal of perchlorate salts, the extract is subjected to HPLC on a reversed phase column (Spherisorb ODSI) . A significant increase in plasma inosine is observed in the plasma taken from animals receiving the compound (IVA) .
• 3-Cyclopentylpropionitrile is prepared in this example.
• 3-Cyclopentylpropionyl chloride (57.7 g, 0.36 mole) is added dropwise to a large excess of concentrated ammonium hydroxide (400 ml) cooled in an ice/salt bath.
• the heavy suspension of white solid is stirred overnight, collected by filtration, washed with cold water, and recrystallized from about 2 liters of boiling water.
• the lustrous white plates of the amide are dried in vacuo over P 2 Os; yield 31.6 g (62.3%); mp 122 *C.
• 3-Cyclopentylpropionitrile of the previous example is further treated in the synthesis of the present invention.
• a mixture of 3-cyclopentyl- propionitrile (14.8 g, 0.12 mole), sodium hydride (5.8, 0.24 mole), and anhydrous tetrahydrofuran (300 ml) is heated at 52 °C in a water bath for 15 min. , and a solution of ethyl formate (13.3 g, 0.18 mole) in THF (100 ml) is added over a period of 45 min.
• EXAMPLE 132 Glycine methyl ester hydrochloride (19.31 g, 0.154 mole) and anhydrous sodium acetate (12.61 g, 0.154 mole) are added to a solution of the crude formyl compound of the previous example (15.6 g) in MeOH/H 0 (4:1, 500 ml). After 24 hours, the MeOH is evaporated in vacuo, and the mixture of water and oil is extracted with CHC1 3 . The CHC1 3 layer is dried (Na 2 S0 4 ) and evaporated to give an amber oil which is applied to a silica.gel column.
• Benzoyl isothiocyanate (2.62 g, 16.03 mmole) is added dropwise to a solution of the unblocked pyrrole of Example 134 (2.97 g, 13.36 mmole) in dry CH 2 C1 2 (100 ml). After 1 h at room temperature, solution is evaporated, and the 0 gummy residue is dissolved in Et 2 0 (100 ml) with almost immediate separation of crystalline solid; yield 1.75 g. The Et 2 0 filtrate is heated to boiling and diluted with an equal volume of warm cyclohexane. On cooling slowly the solution gave an additional 1.58 g of thioureido product; total yield 3.33 g (64.6%).
• EXAMPLE 136 Methyl iodide (2.60 g, 18.32 mmole) is added to a solution of the thioureido product of Example 135 (3.21 g, 8.33 mmole) and DBN (1.24 g, 9.99 mmole) in dry CH 2 C1 2 (80 ml) at 0 °C. The solution is stirred at 0 °C for 15 min., at ambient temperature for 1 h, and then evaporated in vacuo. A solution of the residue in CHC1 3 is chromatographed on a silica gel column with CHC1 3 as eluent to give homogeneous fractions of the methylthio intermediate compound; yield, 2.46 g (74%).
• EXAMPLE 137 A solution of the methylthio compound of Example 136 (2.07 g, 5.18 mmole) in 150 ml of MeOH that has been saturated with NH 3 at 0 ⁇ C is heated at 90-95 °C for 24 hours in a glass-lined stainless steel bomb. The contents of the chilled bomb are evaporated in vacuo to give a mixture of the compound (IVC) , benzamide and a by-product that is a 2-methylthio derivative, as opposed to the 2- amino compound (IVC) . The mixture is stirred vigorously for several minutes with appr. 75 ml of Et 2 0, and the insoluble white solid is filtered off and washed with Et 2 0.
• the filtrate contained most of the benzamide and 2- methylthio components.
• a solution of the Et 2 0-insoluble solid (1.13 g) in MeOH is evaporated with appr. 10 g of silica gel.
• the powdered residue is layered evenly onto the top of a silica gel column, which is then eluted with CHCl 3 /MeOH/HOAc (95:5:1) to give the 2-methylthio by ⁇ product (252 mg; MS (FAB): 264 (M + H) + ) and the desired 2-amino product (IVC) (679 mg, 56.4%).
• (IVC) is recrystallized by extraction into boiling isopropyl acetate in a Soxhlet apparatus.
• EXAMPLE 138 The compound of Example 137 is tested for enzyme- inhibition activity in accordance with the procedure of Example 9. At 1 mM phosphate the IC 50 is 0.029 ⁇ M, and at 50 mM phosphate the IC 50 is 1.8 ⁇ M.
• the compounds are prepared following the procedures set forth in Examples 130-137 using the appropriate 3- (substituted cyclopentyl) -propionitriles as starting materials.
• a pharmaceutical composition for intraperitoneal injection is prepared for testing the compound (IVC) .
• An intraperitoneal injection solution is prepared containing the compound (IVC) is dissolved in an aqueous carrier that contains ten percent DMSO.
• EXAMPLE 144 Using the procedure of Example 112, the compound (IVC) is intraperitoneally injected into Lewis Rats via the test composition of Example 143 and the results compared with controls. A significant increase in plasma inosine is observed in the plasma taken from animals receiving the compound (IVC) .
• 3-Cyclohexylpropionitrile is prepared in this example.
• a solution of cyclohexanepropionic acid (50 g; 0.32 mole) and thionyl chloride (152 g; 1.28 mole) in 100 ml benzene is allowed to stand overnight and is then evaporated to an oily residue.
• the residue is added in portions to 28% aqueous ammonia (270 ml) at 25°C and the mixture stirred for about two hours.
• the resulting product is collected by filtration, washed with cold water, and recrystallized from about 2 liters of boiling water.
• the lustrous white plates of the amide are dried in vacuo over P 2 ⁇ 5,* yield 45.5 g.
• EXAMPLE 146 3-Cyclohexylpropionitrile of Example 145 is further treated in the synthesis of the present invention. Under an atmosphere of dry N 2 , a mixture of 3-cyclohexyl- propionitrile (22.3 g, 0.16 mole), sodium hydride (5.38, 0.224 mole), and anhydrous tetrahydrofuran (120 ml) is heated at 52 'C in a water bath for 15 min., and a solution of ethyl formate (55.4 g, 0.75 mole) in THF (50 ml) is added over a period of 45 min.
• EXAMPLE 148 Under a nitrogen atmosphere, ethyl chloroformate (1.38 g, 12.7 mmole) is added dropwise to a solution of the enamine of Example 147 (2.0 g, 8.46 mmole) and DBN (2.1 g, 16.9 mmole) in dry CH C1 (50 ml) with external cooling in an ice bath. After stirring at 0 °C for one hour, the solution is allowed to a stand at room temperature overnight. After checking progress by TLC, additional ClC0 2 Et (0.5 ml) and DBN (1.5 ml) are added to complete the conversion, and the solution is allowed to stand for 24 hours.
• Example 148 To a solution of the N-blocked pyrrole of Example 148 (2.6 g, 8.43 mmole) in MeOH (100 ml) is added solid Na 2 C0 3 (2.23 g, 21.07 mmole), and the reaction mixture is stirred at room temperature for 48 hr with separation of the resultant deblocked pyrrole. The mixture is evaporated to dryness, and the residue is triturated thoroughly with H 2 0 (50 ml) to dissolve inorganics and extracted with CHC1 3 (3 x 100 ml) .
• the extract is dried (Na 2 S0 4 ) and evaporated to give a viscous gum, which is purified on a silica gel column using CHC1 3 as the eluent; yield 1.67 g (84%); m.p. 73-74°C.
• EXAMPLE 150 Benzoyl isothiocyanate (0.74 g, 4.02 mmole) is added dropwise to a solution of the unblocked pyrrole of Example 149 (0.95 g) in dry CH 2 C1 2 (20 ml). After one hour at room temperature, the solution is evaporated, and the gummy residue is dissolved in Et 2 0 (100 ml) with almost immediate separation of crystalline solid. The Et 2 0 filtrate is heated to boiling and diluted with an equal volume of warm cyclohexane. On cooling slowly the solution gives additional thioureido product; total yield 1.41 g (88%); m.p. 156-157 ⁇ C.
• Methyl iodide (1.1 g, 7.6 mmole) is added to a solution of the thioureido product of Example 150 (0.96 g, 2.61 mmole) and l,5-diazabicyclo[4.3.0]non-5-ene (0.38 g, 3.0 mmole) in dry CH 2 C1 2 (20 ml) at 0°C. The solution is stirred at 0*C for 15 min., at ambient temperature for 1 h, and then evaporated in vacuo. A solution of the residue in CHC1 3 is chromatographed on a silica gel column with CHC1 3 as eluent to give homogeneous fractions of the methylthio intermediate compound; yield 0.92 g.
• EXAMPLE 152 A solution of the methylthio compound of Example 151 (0.8 g, 1.93 mmole) in 50 ml of MeOH that has been saturated with NH 3 at 0 °C is heated at 90-95 °C for 24 hours in a glass-lined stainless steel bomb. The contents of the chilled bomb are evaporated in vacuo to give a mixture of the compound (IVD) , benzamide and a by-product that is a 2-methylthio derivative, as opposed to the 2- amino compound (IVD) . The mixture is stirred vigorously for several minutes with appr. 75 ml of Et 2 0, and the insoluble white solid is filtered off and washed with Et 2 0.
• the filtrate contained most of the benzamide and 2- methylthio components.
• a solution of the Et 2 0-insoluble solid (0.390 g) in MeOH is evaporated with appr. 10 g of silica gel.
• the powdered residue is layered evenly onto the top of a silica gel column, which is then eluted with CHCl 3 /MeOH/HOAc (95:5:1) to give the 2-methylthio by ⁇ product and the desired 2-amino product (IVD) .
• IVD is recrystallized by extraction into boiling isopropyl acetate in a Soxhlet apparatus.
• Example 153 The compound of Example 152 is tested for enzyme inhibition activity as in Example 105. At 1 mM phosphate the IC 50 is 0.037 ⁇ M, and at 50 mM phosphate the IC50 is 2.2 ⁇ M.
• Example 152 The compound of Example 152 is tested to determine its effectiveness in potentiation of the toxicity of 2'- deoxyguanosine (d-Guo) (see D. A. Schewach et al., Cancer
• CCRF-CEM cells are grown in
• IC 50 is calculated to be
• the compound 2-amino-7-(3-methylcyclohexylmethyl)-3H,5H- pyrrolo[3,2-d]pyrimidin-4-one is prepared.
• the aryl derivative 2-amino-7-(3-methylbenzyl)- 3H,5H-pyrrolo[3,2-d]-pyrimidin-4-one is made.
• a solution of the aryl derivative (0.2 g, 0.78 mmole) in trifluoroacetic acid (TFA) (20 ml) is hydrogenated with Pto 2 at 60 lb/in 2 for 24 h.
• EXAMPLE 156 The procedure described in Example 155 is repeated to prepare 2-amino-7-(3-trifluoromethylcyclohexyl-methyl)- 3H, 5H.-pyrrolo- [3 , 2-d]pyrimidin-4-one using 3-(3- trifluoromethylbenzyl) -propionitrile as the starting compound: yield 69%; mp 165 °C. Anal, calcd. for c 14 H 17 N 4 OF 3 * 0 - 6H 2 O ⁇ c ' 51.72; H, 5.64; N, 17.23. Found: C, 51.82; H, 5.71; N, 16.81%.
• EXAMPLE 157 The compound prepared in Example 155 is tested for enzyme-inhibition activity as in Example 105. At 1 mM phosphate the IC 50 is 0.025 ⁇ M, and at 50 mM phosphate the IC 50 is 0.0.820 ⁇ M.
• EXAMPLE 158 The compound prepared in Example 156 is tested for enzyme-inhibition activity as in Example 105. At 1 mM phosphate the IC50 is 0.020 ⁇ M, and at 50 mM phosphate the IC 50 is 0.740 ⁇ M.
• EXAMPLE 159 3-Cycloheptylpropionitrile is prepared in this example according to the procedure of Example 97 using a solution of 2-bromocycloheptane (25.57 g; 144.38 mmole) ; Bu 3 SnH (50.42 g; 173.26 mmole), acrylonitrile (15.32 g; 288.77 mmole), and AIBN (1.13 g) in toluene (300 ml). Yield is 16 g; mp oil.
• EXAMPLE 160 3-Cycloheptylpropionitrile of Example 159 is further treated in the synthesis of the present invention. Under an atmosphere of dry N 2 , a mixture of 3-cycloheptyl- propionitrile (8.5 g, 56.19 mmole), sodium hydride (2.6 g, 112.39 mmole), and anhydrous tetrahydrofuran (100 ml) is heated at 52 ⁇ C in a water bath for 15 min., and a solution of ethyl formate (20.81 g, 280.99 mmole) in THF (100 ml) is added over a period of 45 min.
• ethyl formate 20.81 g, 280.99 mmole
• EXAMPLE 161 Glycine methyl ester hydrochloride (9.35 g, 74.47 mmole) and anhydrous sodium acetate (6.10 g, 74.47 mmole) are added to a solution of the crude formyl compound of Example 160 (8.9 g; 49.65 mmole) in MeOH/H 2 0 (4:1, 250 ml). After 24 hours, the MeOH is evaporated in vacuo, and the mixture of water and oil is extracted with CHC1 3 . The CHC1 3 layer is dried (Na 2 S0 4 ) and evaporated to give an amber oil which is applied to a silica gel column.
• EXAMPLE 162 Under a nitrogen atmosphere, ethyl chloroformate (4.01 g, 37.03 mmole) is added dropwise to a solution of the enamine of Example 161 (6.18 g, 24.69 mmole) and DBN (9.19 g, 74.04 mmole) in dry CH 2 C1 2 (100 ml) with external cooling in an ice bath. After stirring at 0 °C for one hour, the solution is allowed to a stand at room temperature overnight. After checking progress by TLC, additional ClC0 2 Et (0.5 ml) and DBN (3.0 ml) are added to complete the conversion, and the solution is allowed to stand for 24 hours.
• Volatile matter is evaporated in vacuo, the viscous residue purified on a short silica gel column (whose main purpose is to remove the less-mobile DBN) to give an N- blocked pyrrole, which is used for the next step without further purification.
• Benzoyl isothiocyanate (1.5 g, 8.96 mmole) is added dropwise to a solution of the unblocked pyrrole of Example 163 (1.99 g, 7.95 mmole) in dry CH 2 C1 2 (50 ml). After 1 h at room temperature, solution is evaporated, and the gummy residue is dissolved in Et 2 0 (100 ml) with almost immediate separation of the crystalline solid. The Et 2 0 filtrate is heated to boiling and diluted with an equal volume of warm cyclohexane. On cooling slowly the solution gives 55 additional thioureido product; yield 2.89 g (88%); m.p. 158-159°C.
• Methyl iodide (1.7 g, 11.96 mmole) is added to a solution of the thioureido product of Example 164 (1.7 g, 4.1 mmole) and DBN (0.56 g, 4.52 mmole) in dry CH 2 C1 2 (80 ml) at 0 ⁇ C. The solution is stirred at 0*C for 15 min., at ambient temperature for 1 h, and then evaporated in vacuo. A solution of the residue in CHCI3 is chromatographed on a silica gel column with CHCI3 as eluent to give homogeneous fractions of the methylthio intermediate compound.
• EXAMPLE 166 A solution of the methylthio compound of Example 165 (1.72 g, 4.02 mmole) in 50 ml of MeOH that has been saturated with NH 3 at 0 ⁇ C is heated at 90-95 °C for 24 hours in a glass-lined stainless steel bomb. The contents of the chilled bomb are evaporated in vacuo to give a mixture of the compound (IVE) , benzamide and a by-product that is a 2-methylthio derivative, as opposed to the 2- amino compound (IVE) . The mixture is stirred vigorously for several minutes with appr. 75 ml of Et 2 ⁇ , and the insoluble white solid is filtered off and washed with Et 2 0.
• the filtrate contained most of the benzamide and 2- ethylthio components.
• a solution of the Et 2 0-insoluble solid (0.850 g) in MeOH is evaporated with appr. 10 g of silica gel.
• the powdered residue is layered evenly onto the top of a silica gel column, which is then eluted with CHCl 3 /MeOH/HOAc (95:5:1) to give the 2-methylthio by ⁇ product and the desired 2-amino product (IVE) .
• (IVE) is recrystallized by extraction into boiling isopropyl acetate in a Soxhlet apparatus.
• EXAMPLE 167 The compound of Example 166 is tested for enzyme inhibition activity as in Example 105. At 1 mM phosphate the IC50 ⁇ s 0 -°3° M, and at 50 mM phosphate the IC 50 is 0.840 ⁇ M.
• EXAMPLE 168 Using the procedure of Example 97, 3-(l-norbornanyl)- propionitrile is made from 1-bromonorbornane, and 3- (2- norbornanyl) -propionitrile (mixture of 2-exo and 2-endo) is made from 2-bromonorbornane. Following Examples 98-104, the propionitriles are converted to the compounds (IVF) , IV (G) , and (IVH) .
• EXAMPLE 169 Using the procedure of Example 97, 3-(l-norbornanyl)- propionitrile is made from 1-bromonorbornane, and 3- (2- norbornanyl) -propionitrile (mixture of 2-exo and 2-endo) is made from 2-bromonorbornane. Following Examples 98-104, the propionitriles are converted to the compounds (IVF) , IV (G) , and (IVH) .
• EXAMPLE 169 Using the procedure of Example 97, 3-(l
• EXAMPLE 172 Following the procedure of Example 171, bicyclo[3.3.1]- nonane-9-one is reacted to form the corresponding aldehyde, from which is made the corresponding 3-substituted propionitrile, which is then converted into the 9- bicyclof3.3.1]nonanyl derivative related to the compound (IVM) .
• EXAMPLE 176 Following the procedure of Example 170, noradamantane-7- one is reacted to form the corresponding aldehyde, from which is made the corresponding 3-substituted propionitrile, which is then converted into the 7- noradamantyl derivative related to the compound (IVN) .
• EXAMPLE 177 Following the procedure of Example 170, noradamantane-7- one is reacted to form the corresponding aldehyde, from which is made the corresponding 3-substituted propionitrile, which is then converted into the 7- noradamantyl derivative related to the compound (IVN) .
• Volatiles are evaporated .in vacuo to give a viscous dark gum which is purified by flash column chromatography over silica gel using CHC1 3 as the eluent. All the fractions containing the desired N-protected pyrrole are pooled and evaporated to give a foamy light pale yellow material which is stirred in MeOH (100 ml) to give the crystalline material which is recrystallized from CHC1 3 - MeOH, yield 8.92 g (74.7%), mp 160-161 °C.
• the above compounds A and B are prepared in this Example.
• the compound A is a compound of the present invention and the compound B is an intermediate.
• a solution of the methylthio intermediate of Example 186 (6.90 g, 12.54 mmol) in MeOH (200 ml) is saturated at 0 °C with ammonia and heated at 100 °C for 20 h in a glass-lined stainless steel bomb. The reaction mixture is brought to room temperature and the solvent is evaporated at room temperature.
• the compound of the present invention of Example 187 is tested for enzyme inhibition activity.
• a purine nucleoside phosphorylase (PNP) enzyme assay is performed in which the PNP activity (IC 50 ) for the compound (8A) is found, which is determined radiochemically by measuring the formation of
• the IC50 is 0.64 ⁇ M and at 50 mM phosphate the IC 50 is 10 ⁇ M.
• (3-chlorophenyl) -3- (2-amino-4-oxo-3H,5H-pyrrolo[3,2- d]pyrimidin-7-yl)propanenitrile is prepared using 3-(3- chlorophenyl)-pentanedinitrile as the starting material, yield 54.5%, mp 157-158 °C.
• Ar is each of the following: (1) phenyl, 2,3- dichlorophenyl, 3-methylphenyl, and 3-methoxyphenyl, (2) thienyl (2- and 3-), (3) furanyl (2- and 3-), (4) pyridinyl (2-, 3-, and 4-) , (5) pyrrolyl (2- and 3-) , (6) thiazolyl (2-, 4-, and 5-), (7) 2-pyrazinyl, (8) pyridazinyl (3- and 4-) , and (9) pyrazolyl.
• EXAMPLE 191 Following the procedure set forth in Examples 180-187, the following compounds 10-14 and 21 are prepared starting from the appropriately substituted pentanedinitrile. Compounds 15-20, and 22 are prepared from the corresponding unsaturated Ar analogues in Example 190. In this procedure, the nitrile group of the unsaturated analogue is first converted to an amide group by acid- or base- catalyzed hydrolysis, then the unsaturated Ar group is converted to the saturated R 2 group by known catalytic hydrogenation, followed by reconverting the amide back to the nitrile by known dehydration procedures.
• R 2 is each of : 10) 1-adamantyl , 11) 2 -adamantyl , 12 ) cyclohexyl , 13 ) cycloheptyl , 14 ) cyclopentyl , 15 ) tetrahydro furany l , 16 ) t etrahydr oth i enyl , 17 ) tetrahydropyranyl , 18) pyrazolidinyl , 19) thiazolidinyl .
• EXAMPLE 193 The compound prepared in Example 192 is tested for enzyme inhibition activity as in Example 188. At 1 mM phosphate the IC 50 is 0.023 ⁇ M and at 50 mM phosphate the IC 50 is 4.7 ⁇ M.
• EXAMPLE 195 The compound prepared in Example 194 is tested for enzyme inhibition activity as in Example 188. At 1 mM phosphate the IC 50 is 0.012 ⁇ M and at 50 M phosphate the IC 50 is 0.19 ⁇ M.
• Example 196 The above compound, 3-(2-amino-4-oxo-3H,5H-pyrrolo[3,2- d]pyrimidin-7-yl)-3-phenylpropanamide, is prepared in this example.
• a solution of the compound obtained in Example 192 (0.200 g, 0.72 mmol) in cone H 2 S0 4 (0.5 ml) is stirred at room temperature for 20 h and then poured onto crushed ice (5.0 g) and adjusted to pH 6.8 by cone NH 4 OH. The precipitated solid is collected, washed with H 2 0 and dried, yield 0.180 g, mp 199-201 °C dec EXAMPLE 197
• the compound prepared in Example 196 is tested for enzyme inhibition activity as in Example 188. At 1 mM phosphate the IC 50 is 0.20 ⁇ M and at 50 mM phosphate the IC 50 is 6.6 ⁇ M.
• the solvent is removed on a water aspirator (30°C) and vacuum pump (lyophilize) to give a semisolid mass which is purified on a silica gel column using CHCl 3 -MeOH as the eluent, yield 0.1 g.
• EXAMPLE 199 The compound prepared in Example 198 is tested for enzyme inhibition activity. Significant activity (IC 50 ) is found.
• EXAMPLE 201 The compound prepared in Example 200 is tested for enzyme inhibition activity as in Example 188. At 1 mM phosphate the IC 50 is 0.097 ⁇ M and at 50 mM phosphate the IC 50 is 1.0 ⁇ M.
• EXAMPLE 202 A compound of the present invention is prepared wherein X is PO(OH) 2 .
• X is PO(OH) 2 .
• 192 is converted to the corresponding amide by treatment with sulfuric acid.
• the amide is converted to the corresponding amine, which is then converted to the corresponding pyridinium salt using a pyrillium salt.
• Conversion of the salt to the corresponding halide is accomplished using sodium bromide, which is then converted to the phosphonic ester using triethyl phosphite. Hydrolysis of the ester using trimethylsilylbromide yields the corresponding phosphonic acid wherein "n" is 1 and "m" is 0.
• EXAMPLE 203 This Example makes a compound of the present invention by stepping up the number of carbon atoms from “m” is 0 to "m” is 1.
• the nitrile group of the compound of Example 192 is reduced to the corresponding aldehyde, which is then converted to the corresponding alcohol.
• phosphorous tribromide the alcohol is converted to the corresponding alkyl bromide, which is then converted to the nitrile compound of the present invention wherein m is 1 using potassium cyanide.
• EXAMPLE 204 In this example a compound of the present invention is prepared wherein "p" is 1 and "Y" is oxygen.
• the alcohol prepared as an intermediate in the previous example is converted to the corresponding diethyl phosphonomethyl ether using diethylchloromethyl phosphonate. Removal of the ethyl groups of the ester is accomplished using trimethylsilylbromide to give the phosphonic acid.
• a compound of the present invention is made wherein "Y" is NH and "X" is s 0 NH .
• the nitrile group of the compound of Example 192 is reduced to the amine using standard catalytic hydrogenation with palladium in acidic media (usually 0.01 N to 1 N HCl), which is then converted to the sulfamide using sulphamoyl chloride.
• EXAMPLE 206 In this example a compound of the present invention is prepared wherein "X" is COOH and "Y" is NH by reacting the methyl amine intermediate prepared in the previous example with chloroacetic acid.
• a compound of the present invention is prepared wherein "X” is PO(OH) 2 and "Y” is NH by reacting the methyl amine intermediate prepared in Example 206 with diethylchloromethyl phosphonate, and reacting the resulting product with trimethylsilylbromide.
• EXAMPLE 208 In this example a compound of the present invention is prepared wherein "X” is S0 2 NH 2 and "Y” is oxygen by reacting the alcohol intermediate prepared in Example 203 with sulphamoyl chloride.
• a compound of the present invention is prepared wherein R 1 is H, R 2 is phenyl, R 3 and R 4 are hydrogen, m is 0, n is 1, p is 0, and X is CN.
• R 1 is H
• R 2 is phenyl
• R 3 and R 4 are hydrogen
• m is 0,
• n is 1
• p is 0,
• X is CN.
• EXAMPLE 210 In this example a compound of the present invention is prepared wherein R 1 is OCH 3 , R 2 is phenyl, R 3 and R 4 are hydrogen, m is 0, n is 1, p is 0, and X is CN.
• R 1 is OCH 3
• R 2 is phenyl
• R 3 and R 4 are hydrogen
• m is 0,
• n is 1
• p is 0,
• X is CN.
• a compound of the present invention is prepared wherein X is tetrazole.
• the compound of Example 192 is treated with lithium azide in the presence of ammonium chloride as a catalyst in dimethylformamide (DMF) at 100 degrees C to give the desired tetrazole.
• DMF dimethylformamide
• EXAMPLE 213 The compound prepared in Example 189 is tested for enzyme inhibition activity as in Example 188. At 1 mM phosphate the IC 50 is 0.012 ⁇ M and at 50 mM phosphate the IC 50 is 2,0 ⁇ M.
• EXAMPLE 214 In this example an amidine compound of the present invention is prepared, i.e., wherein X in the recited generic formula is CNHNH 2 .
• the compound A from Example 187 is reacted with sodium methoxide in methanol at room temperature for about 2 days to give a methyl-imidate intermediate.
• the intermediate is then reacted with ammonia in methanol to give the amidine product.

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