CA2133346A1 - 7-disubstituted-methyl-4-oxo-3h,5h-pyrrolo[3,2-d]pyrimidine and pharmaceutical uses and compositions containing the same - Google Patents
7-disubstituted-methyl-4-oxo-3h,5h-pyrrolo[3,2-d]pyrimidine and pharmaceutical uses and compositions containing the sameInfo
- Publication number
- CA2133346A1 CA2133346A1 CA002133346A CA2133346A CA2133346A1 CA 2133346 A1 CA2133346 A1 CA 2133346A1 CA 002133346 A CA002133346 A CA 002133346A CA 2133346 A CA2133346 A CA 2133346A CA 2133346 A1 CA2133346 A1 CA 2133346A1
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- Prior art keywords
- cyclo
- inhibitor
- ethyl
- mammalian
- effective amount
- Prior art date
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- 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
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- 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
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- 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
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- 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
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- 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
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
Abstract
Disclosed is a compound of formula (I) where R1 is H, NH2, or OCH3, R2 is an optionally substituted cyclic group optionally containing one or more heteroatoms, R3 and R4 are independently H or C1-4 alkyl, m is 0-4, n is 0-6, p is 0.1, X is CN, CSNH2, PO(OH)2, COOH, SO2NH2, NH2, OH, CNHNH2, tetrazole, triazole, or COR5 where R5 is C1-4 alkyl, CF3, NH2, or OC1-4 alkyl, and Y is O or NH that is useful as a pharmaceutical.
Description
r`W0 93/21187 2 1 3 ~ 3 ~ ~ PCr/U~93/02~41 7 DI~ 3TI~TEI)-Nl ~Yh- 4--0~0--3H~
PYRROLOL3,2-d~PY~I~IDINE AND P~R~ E~TICAL ~8E~
C~Ol~POSITION8 CONTAINING q~
The present inventlon relates to derivatives of 4-oxo-3H,5H-pyrrolo[3,2~d]pyrimidine. In particular, it rela~es to 4-oxo-3H,5H-pyrrolo[3,2-dJpyrimidinederi~ativessubstitutedatthe 7-position.
Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of purine nuoleosides in a reversible reaction.
Individuals who are deficient in PNP exhibit impaired T-c~ll development, resulting in lowered cell-mediated immunity, but normal ~-cell de~elopment, 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-cel-s are pathogenic.
Accordingly, the present invention is a compound of the formula HN
R~
R2~ / C ~ ')p-(CH~)m-X
\ R3 F~ ,/n i' wherein R1 is H, NH~, or OCH~, R2 is an optionally substituted cyclic group optionally containing one or more heteroatoms, R3 and R4 are independently H or C14 alkyl, m is 0-4, n is 0-6, p is 0-~, X is CN, CSNH2, PO~OH)2, COOH, SOzMH2~ NH2, OH, CN~N~2, tetrazole, triazole or CoR5 wher~ Rs is C~ 4 alkyl, CF3, NH2~ or ~, . ' ~ !
; OC~,4 allcyl, and Y is O or NH. The compound of the present 3 0 in~rention is u5eful as a PNP inhi~itor . Also coIltempla~ed . according to the present invention a~e a pharmac:eutical composition for the selective suppression of mammalian T-cell immunity comprising an pharmaceutic:ally effective amount of the t :: compound of the pre5ent invent~ on and a pharmaceutirally acceptable carrier or diluent and a method for ~he s lective 93/21187 ~ 3 3 3 il~ PCT/US93/02~
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 optionally substituted cyclic group (he~einafter r~ferred to as cyclo) recited for the above formula includes aromatic, heteroaromatic, alicyclic, and heteroalicyclic groups prefera~ly containing five to nine atoms. Preferred optional substituents include halogen, hydroxy, alkoxy, alkyl, and trifluoromethyl. Exemplary su~stituents 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 axomatic 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 S-pyrazolyl. The preferred alicyclic and heteroalicyclic groups are 1- or 2-adamantyll 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,
PYRROLOL3,2-d~PY~I~IDINE AND P~R~ E~TICAL ~8E~
C~Ol~POSITION8 CONTAINING q~
The present inventlon relates to derivatives of 4-oxo-3H,5H-pyrrolo[3,2~d]pyrimidine. In particular, it rela~es to 4-oxo-3H,5H-pyrrolo[3,2-dJpyrimidinederi~ativessubstitutedatthe 7-position.
Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of purine nuoleosides in a reversible reaction.
Individuals who are deficient in PNP exhibit impaired T-c~ll development, resulting in lowered cell-mediated immunity, but normal ~-cell de~elopment, 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-cel-s are pathogenic.
Accordingly, the present invention is a compound of the formula HN
R~
R2~ / C ~ ')p-(CH~)m-X
\ R3 F~ ,/n i' wherein R1 is H, NH~, or OCH~, R2 is an optionally substituted cyclic group optionally containing one or more heteroatoms, R3 and R4 are independently H or C14 alkyl, m is 0-4, n is 0-6, p is 0-~, X is CN, CSNH2, PO~OH)2, COOH, SOzMH2~ NH2, OH, CN~N~2, tetrazole, triazole or CoR5 wher~ Rs is C~ 4 alkyl, CF3, NH2~ or ~, . ' ~ !
; OC~,4 allcyl, and Y is O or NH. The compound of the present 3 0 in~rention is u5eful as a PNP inhi~itor . Also coIltempla~ed . according to the present invention a~e a pharmac:eutical composition for the selective suppression of mammalian T-cell immunity comprising an pharmaceutic:ally effective amount of the t :: compound of the pre5ent invent~ on and a pharmaceutirally acceptable carrier or diluent and a method for ~he s lective 93/21187 ~ 3 3 3 il~ PCT/US93/02~
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 optionally substituted cyclic group (he~einafter r~ferred to as cyclo) recited for the above formula includes aromatic, heteroaromatic, alicyclic, and heteroalicyclic groups prefera~ly containing five to nine atoms. Preferred optional substituents include halogen, hydroxy, alkoxy, alkyl, and trifluoromethyl. Exemplary su~stituents 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 axomatic 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 S-pyrazolyl. The preferred alicyclic and heteroalicyclic groups are 1- or 2-adamantyll 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. Examples include compounds wherein R1 is NHz or H, R2 is phenyl, 3-chlorophenyl, or 3,4-dicholorophenyl, and (C~3R4)n-(Y)p-(CH2)~-X is CH2C~2CN; CH2CH2CQOH; CH2CH2CH2OH;
C}I2CH2CH2CN; CH2CH2CHzCOOH C~I2C~I2CH2CX20H, or sub~tituents where an oxygen atom replaces one or more of the methylene groups.
The present invention contemplates pharmaceutical : compositions suitable for enteral, such as oral or rectal, transdermal and parentera} administration to m~mmals including man~ which are useful to inhibit purine nucleoslde phosphorylase acti~ity and for the treatment of disorders responsive thereto, comprising an effectiYe amount of a pharmacologically ac~i~e compound of the invention, alone or in com~ination, with one or more pharmaceutically acc~ptable carriers~
Preferred phaxmaceutical compositions are tablets and gelatin capsules compr_sing the active ingredient together with ~ wo g~/2ll87 2 ~ 3 3 3 'i ~ PCT/US93/0~
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 pas~e, gelatin, tr~gacanth, methylcellulose, sodium carbox~me~hylcellulose and/or polyvinylpyrrolidone; if desired d) disintegrants, e.g., .
starches, agar, alginic a~id or its sodium salt, or efferYescent mixtures; and/or e) abs~rbents, colorants, flavors and sweeteners. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are ad~antageously prepared from fatty emulsions or suspensions.
Said compositions may be sterilized and/or contain adjuvants, such as preser~ing, stabilizing, wetting or emulsifyin~ agents/
solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition,they may also contain ot~er t~erapeutically valuable substances. Said compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 7~%, pref2rahly about 1 to 50%, of the active ingredient.
Suitable formulations for transdermal application in~lude an effective amount of a compound of the invention with a carrier. Advantageous carriers include absorbable pharmacologically acceptable solvents to as~ist passage through ~he skin of the host. Characteristically, 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 a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.
nother aspect o~ the present invention provides a method of making a 2amino compoun~ (R1 = NX2) of the presen~ invention and int~irmediates thereof. The first step of the met~od involves reacting an~ optionally substituted cyclic aldehyde with cyanaacetic acid at a molar ratio of about 1/1 to 1/5 in the presence of ammonium ~acetate at about reflux temperature for
C}I2CH2CH2CN; CH2CH2CHzCOOH C~I2C~I2CH2CX20H, or sub~tituents where an oxygen atom replaces one or more of the methylene groups.
The present invention contemplates pharmaceutical : compositions suitable for enteral, such as oral or rectal, transdermal and parentera} administration to m~mmals including man~ which are useful to inhibit purine nucleoslde phosphorylase acti~ity and for the treatment of disorders responsive thereto, comprising an effectiYe amount of a pharmacologically ac~i~e compound of the invention, alone or in com~ination, with one or more pharmaceutically acc~ptable carriers~
Preferred phaxmaceutical compositions are tablets and gelatin capsules compr_sing the active ingredient together with ~ wo g~/2ll87 2 ~ 3 3 3 'i ~ PCT/US93/0~
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 pas~e, gelatin, tr~gacanth, methylcellulose, sodium carbox~me~hylcellulose and/or polyvinylpyrrolidone; if desired d) disintegrants, e.g., .
starches, agar, alginic a~id or its sodium salt, or efferYescent mixtures; and/or e) abs~rbents, colorants, flavors and sweeteners. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are ad~antageously prepared from fatty emulsions or suspensions.
Said compositions may be sterilized and/or contain adjuvants, such as preser~ing, stabilizing, wetting or emulsifyin~ agents/
solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition,they may also contain ot~er t~erapeutically valuable substances. Said compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 7~%, pref2rahly about 1 to 50%, of the active ingredient.
Suitable formulations for transdermal application in~lude an effective amount of a compound of the invention with a carrier. Advantageous carriers include absorbable pharmacologically acceptable solvents to as~ist passage through ~he skin of the host. Characteristically, 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 a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.
nother aspect o~ the present invention provides a method of making a 2amino compoun~ (R1 = NX2) of the presen~ invention and int~irmediates thereof. The first step of the met~od involves reacting an~ optionally substituted cyclic aldehyde with cyanaacetic acid at a molar ratio of about 1/1 to 1/5 in the presence of ammonium ~acetate at about reflux temperature for
-3-~ hO93~21187 ~13 3 3 ~ ~ PCT/US93/~2~1 about 10 hours to ~ days to make a 3-cyclo~substituted pentanedinitrile as an intermediate~3 In the second step, 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 me~hoxide, 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 B days to make a 3-cyclo-2-formylpentanedinitrile as a further intermediate. The next step involves reacting the 3-cyclo-2~formylpentanedinitrile with a glycine alkyl ester hydrochloride and sodium or ammonium acetate at a molar ratlo of about 1-2/1.5-4~1~5-~ and at a temperature of about 20-60-C for about 10-48 hours to make methyl N-~(3-cyclo-2,4-~icyano)-2-butenyl]glycine as an intermediate.
In the subse~uent step, the~methyl N-[(3-cyclo-2,4~dicyano)-2-15 butenyl ]glycine is reacted with an alkyl chloroformate such asethyl chloroformate and l,S-diazabicyclo[ 4.~.O]non-5-ene (DBN) or 1,8-diazabicyclo~S.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 abou~ 10 hours to 10 days to make methyl 3-amino-4-(2-cyano-l-cyclo-ethyl)-1-ethyl-lH-pyrrole-1,2-dicarboxylate as an intexmediate. The next step involves reacting the methyl 3-~ amino-4-(2-cyano-l~-cyclo-ethyl)-1-ethyl-lH-pyrrole-1,2-r ~ ~ dicarboxylate with a ba5e such as sodium car~onate at a molar ratio o~ about 2~1 to 1~5 and at about room temperature for abou~
}0-48 hours to make methyl 3-amino-4-(2-cyano-1-cyclo-ethyl~-~; ; lH-pyrrole-2-car~oxylate as an interm3ediate. In the next step, the methyl 3-amino-4-(2-cyano~1-cyclo-ethyl)-lH-pyrrole-2-`; carboxylate is reacted with ~enzoylisothiocyanate at a molar ratic of about 2/1 to~ 1/2 and at about room temperature for about 3 0 3 0 minutes to 3 hours to make N-benzoyl-N'-~4-(2-cyano 1-cyclo-ethyl~2-methoxy~arbonyl-lH-pYrrol-3-yl~thio~urea as an intermediate. The next step~ reacts the N-benzoyl-N'-~4-(2-cyano-l-cyclo33333ethyl~ 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 an~ at a temperature of about 0-30OC for a~out 10 min~tes to 10~hours to make N-~enzoyl-N -~ 2-cyano-1-cyclo-ethyl)-2-methoxycarbonyl-}H-pyrrol-3 yl]S-m2thylthiourea as an
In the subse~uent step, the~methyl N-[(3-cyclo-2,4~dicyano)-2-15 butenyl ]glycine is reacted with an alkyl chloroformate such asethyl chloroformate and l,S-diazabicyclo[ 4.~.O]non-5-ene (DBN) or 1,8-diazabicyclo~S.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 abou~ 10 hours to 10 days to make methyl 3-amino-4-(2-cyano-l-cyclo-ethyl)-1-ethyl-lH-pyrrole-1,2-dicarboxylate as an intexmediate. The next step involves reacting the methyl 3-~ amino-4-(2-cyano-l~-cyclo-ethyl)-1-ethyl-lH-pyrrole-1,2-r ~ ~ dicarboxylate with a ba5e such as sodium car~onate at a molar ratio o~ about 2~1 to 1~5 and at about room temperature for abou~
}0-48 hours to make methyl 3-amino-4-(2-cyano-1-cyclo-ethyl~-~; ; lH-pyrrole-2-car~oxylate as an interm3ediate. In the next step, the methyl 3-amino-4-(2-cyano~1-cyclo-ethyl)-lH-pyrrole-2-`; carboxylate is reacted with ~enzoylisothiocyanate at a molar ratic of about 2/1 to~ 1/2 and at about room temperature for about 3 0 3 0 minutes to 3 hours to make N-benzoyl-N'-~4-(2-cyano 1-cyclo-ethyl~2-methoxy~arbonyl-lH-pYrrol-3-yl~thio~urea as an intermediate. The next step~ reacts the N-benzoyl-N'-~4-(2-cyano-l-cyclo33333ethyl~ 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 an~ at a temperature of about 0-30OC for a~out 10 min~tes to 10~hours to make N-~enzoyl-N -~ 2-cyano-1-cyclo-ethyl)-2-methoxycarbonyl-}H-pyrrol-3 yl]S-m2thylthiourea as an
4-`:; :
~ :
WO 93/211~7 213 3 3 ~ ~ PCI/US93/02841 intermediate . In the following step, the N-benzoyl-N ' - [ 4 - ( 2 -cyano- l-cyclo-ethyl ) -2 -methoxycarbonyl-l~-pyrrol-3 -yl ] -S-meth~ylthiourea ( about 1-2 mol ) is reacted with methanolic or ethanolic ammonia at a ratio of a}:~out l/l to l/20 and at a temperature of about 20-130'C for about 16~60 hour~ to make a mixture of a 2-amino compound of t~e present invention 3-cyclo-3 ~ 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 ~ ~ pyrimidin-7 -yl ] propanenitril e as an intermediate in making another compound of the present invention.
In a further aspect of the present inYention there is provided a method of making a 2-methoxy compound (R1 = OCH3) and intermediates thereof. The intermediate 3-cyclo~3-[2-methylmercapto-4-oxo-3H,5H-pyrrolo~3,2-d~pyrimidin-7-yl]propanenitrile is reacted with an oxidi~ing agent such as permanganate or hydrogen peroxide at a molar ratio of about 1/1 to 1/10 a~d at a temperature of about 25-120-C for about 3-48 hours to make 3~cyclo-3-~2-methylsulfonyl-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile as an intermediate.
In the next step, the 3-cyolo-3-[2-methylsul~onyl-4-oxo-3H~5H-pyrrolo[3,2-d]pyrimidin-7-~lJpropanenitrile is reacted with a sodium alkoxide such as sodium methoxide at 2 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.
In a further aspect of the present invention there is provided a method of making a compound of the present invention wherein Rl is hy~rogen. The methyl 3 a~ino-4-(2-cyano~l-cyclo-3 0 ethyl ) -lH~-pyrrole~2 -carboxylate intermediate described su~ra is reacted with dlmethylformamide dime~hyl acetal at a molar ratio of about 1/1 to 1/4 and at a temperature of about 25-lOODC for about 1-10 days to make methyl 4-(2 cyano-l-cyclo-ethyl)-3-[N
(dimethylaminomethylene)amino~ pyrrole-2-car~oxylate as an intermedi~te. The next step involves reacting the methyl 4--~2-cyano-l-cyclo-ethyl)-3-~N-(dimethylaminomethylene~amino)-lH-pyrrole-2-carboxylate with methanolic or ethanolic ammonia at a ~`i` 213~3~
0g3/2~187 PCT/US93/~2~1 molar ratio of about 1/1 to 1/20 and at a temperature of about 20-130C 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~
As will be apparent to the skilled artisan, variations of the aforesaid procedures are useful in making the variety of compounds of th~ present in~ention without departing frcm 'he spirit thereof. For example, compounds having different values for "n" and "~" in accordance with the present invention are obtained by either stepping up or stepping down the series by.the necessary number of carbon atoms in accoxdance with known procedures. Also, reactions involving some intermediates require protection of nitrogen or oxygen atoms on the intermediates using known procedures.
The present invention provides a method of inhibiting purine nucleoside phosphorylase activity in mammals and treating diseases and conditions responsive thereto, e.g., autoimmune disorders, rejection of transplantation, or psoriasis, which comprises administaring to a mammal in need thereof an effective amount of a compound of the in~ention or of a pharmaceutical ~ composition comprising a said compound in combination with one .~ or more pharmaceutically acceptable carriers.
A ~urther aspect of the invention relates to a method of ~: inhibiting the phosphorolysis and metabolic breakdown of 2S anti~iral or antitumor purine nucleosides ~n mammals which compris~s administering in conjunction therewith to a mammal in need thereof, either separately or in combination therewith, an :~ effecti~e purine nucleoside phosphorylase inhi~iting amount of ~: a compound of the invention or of a said compound in combination with one or more pharmaceutically acc~ptable carriers. M~re particularly,j such relates to a ~ethod o~ inhibiting Ithe phosphorolysis and metabolic breakdown of purine nucleosides known in the art, e.~, of 2' deoxyguanosine, 2',3'-~id~oxyinosine, 2',3'-dideoxyguano~ine or 2',3'-dideoxyadenosine.
Furthermore, the in~ent1on thus relates to a method of ~ potentiating the antiviral or antitumor eff~ct of 2' or 3' ;~ monodeoxypurine nucleosides or of2',3' dideoxypurine nucleosides ': :
~ 6-WO93/211~7 Z1 3 3 ~ ~ ~ PCT/U593/02~1 in mammals which comprises administering in conjunction ~herewith to a mammal in need thereof, either separately or in combina~ion with a said nucleoside, an effective purine nucleosidP
. phosphorylase inhibiting amount of a compound of the invention preferably in combination wit~ one or more pharmaceutically acceptable carriers. More particularly, 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'~dideoxyguanosine or 2'-3'-dideoxyadenosine for the treatment of retrovirus infections, eOg., HIV-retrovirus infections (ac~uired immunode~iciency syn~rome, AI~S). 2',3'-Dideox~purine nucleosides are kn~wn in the art as inhibitors of HIV retrovirus infectivity and to be metaboli~ally degraded by PNP, e.g., as described in Biochem~cal Pharmacoloqy 22, 3797 (1987). Such are administered at a pharmaceutically acceptable dose which is effective in i~hibiting HIV-retro~irus infections.
Preferably the lowest possible effective dose is used.
The pharmaceutically acceptable ef~ective dosage of active compound ~f the invention to be administered is dependent on the species of warm-blooded animal (mammal), the b~dy weight, age and individual condition, and on ~he fo~m of administration.
The pharmaceutical composition may be aral, parenteral~
suppository or ~ther form which delivers the compound of the present invention into the bloodstream of a mammal to be treated.
An oral ~orm has from about 1 to about 150 mg of the compound of the pr~sent invention for an adult (50 to 70 kg) which is mixed together with pharmaceutically acceptable diluents such as lactose. In a typ~cal capsule, 25 mg of the compound of the present invention is mixed together with 192 m~ lactose, 80 mg modified 5tarch and 3 mg magnesium stearate. Injectable forms of the compound are also contemplated for administratio~. ~
'The present invention is also useful with other therapeutic : agents. A daily dosage of the compound of the present invention for a human weighing 50 to 70 kg of l-~0 mg/kg inhibits meta~olic 3S d~struction of certain anticancer agents such as ~-2'-deoxy-6-thio~uanosine and anti~iral agents such as 2'j3'-dideoxyinosine, an anti-AIDS drug. These t~pes of agents are known ~o be `~ ~093~21187 ~ 1 3 3 3 ~ ~ PCT/US93/02~]
susceptible to cleava~e. Upon cleavage, the agents lose ef~ectiveness~ The compounds of the present invention are capable of reducing such cle~vage. This protection, therefore, enhances the efficacy of other chemotherapeutic agents.
In order to more full~ describe the present invention`~the following non-limiting examples are provided. In the examples all parts and percentages are by weight unless indicated otherwise. Proportions of so.Lvent mixtures used as chromatographic eluents are by vol~me.
XA~PLE 1 ~3 cl ~
cl , t:~ The a~ove intermediate compound is prepared in this Example ~!''' by the modi~ication o~ the procedure of Schiemenz, G. P.;
;~ Engelhard, H~ (Chem. Ber., l962, 95, 195).
t~ O A mixture of cyanoacetic acid t25.38 g, 298.38 mmol), 2,3,5-.. t~ichlorobenzaldehyde (25.0 g, 119.35 mmol), ammonium acetate ,~,; (500 mg), toluene (120 mL), and pyridine (65 ml) is heated at ,.~ reflux for 16 h in a flask fitted with Dean Stark trap and . condens~r. The solvents are evaporated in vacuo, residue is ~.,, 5 extracted with ~HCl~, which is washed with H20~ ~ried ~Na2SO~), .~ and e~aporated to give the crude product, which is purified by silica gel column chromatography using hexaneEtOAc mixture as :;~' ~he eluent. Yield 23.69 g (73%); mp 90-91 C.
~A~PL~ 2 ~CN~
C~ ~ ~HO
; ii Cl The a~ove intermediate compound is prepared in this Example.
To a stirred mixture of NaH (l.S6 g, 65005 mmol) and ethyl ?
,r'~ :`
':
,.~,j r~ , . WO 93~2l~X7 2 ~ 3 3 ~ ~ J PCT/U593/02~1 ." formate (14.~8 g, 199.51 mmol) in THF (100 ml) is added substituted pentanedinitrile of Example 1 (10.17 g, 37.17 mmol) at r~om temperature under a nitrogen atmosphere, and the ~ resulting reaction mixture is stirred for 24 h. Volatile matter ,.~ 5 is evaporated ln vacuo at room temperature. Water (50 ml) is ~ added to t~e residue at 0-5 C, and the solution is adjusted to : pH 5-6 by 20% conc. HCl (_~v). The heavy oil is extracted into ethyl acetate, washed with H20 (1 x 100 ml) and dried (~gSO4).
. The ethyl acetate layer is evaporated to give a red-brown oil ~ 0 g) that is used in the next step without further . purification.
""
;.', ~AMP~ 3 ~ j O~e The above intermediate compound is prepared in this Example.
2Q Glycine met~yl ester hydrochloride (8.17 gl 65.06 mmol~ and sodium acetate (5.33 g, 65.06 mmol) are added to a solution of the crude formyl compound of Example 2 (11.0 g) in a mixture o~
; MeOH (80 m~) and H20 (20 ml), and the resulting solution is .~ stirred at room temperatur~ for 22 h. A~ter evaporation of solvent at room temperature, the residue is extracted with ethyl acetate. The washed (H20) and dried (MgS04) organic layer is evaporated to gi~e an oil. Flash column chromatography (silica .. gel) using CHCl3 as eluent gave the pure desired enamine as a mixture of ~rtra~s isomers which is recrystallized from MeOH, ;, 30 yield 10.41 g (75%), mp 142-143 C~
~AMPLE 4 !.'¦
Cl E~70C 0 .,¦ Cl ~ ~NH~
Cl ~ N
~, _g_ , ,~
2~333~G
';~ VO93/XllX7 PCT/US93/02~1 The above intermediate compound is prepared in this Example.
solution of enamine of Example 3 (10.0 g, 26.84 mmol) in dry C~2Cl2 (100 ml) is cooled to 0 ~C and treated with 1,5 diazabicyclo[4.3.0~non-5-ene (10.53 g, 84.79 mmol) under a nitrogen atmosphere followed by ethyl chloroformate (6.90 g, 63~57 mmol). The solution is stirred at 0 ~C for 1 h and then at room temperature for 48 h. Volatiles are evaporated in vacuo to give a viscaus dark gum which is purified by flash column chromatography over silica gel using CHC13 as the eluent. All the fractions containing the desired ~-protected pyrrole are pooled and evaporated to give a foamy light pale yellow material which is stirred in MeOH (100 ml) to give ~he crystalline material which is recrystallized from CHCl3-MeOH, yield 8.92 g (74.7%), mp 160-16~ C.
~MP~ 5 ~ o M e ;20 Cl'``~ `NH2 Cl C~
The above intermediate compound is prepared in this Example.
A suspension of N-protected pyrrole of Example 4 (8.6 g, 19.34 2S .mmol) in MeOH (300 ml) is treated with NazCO3 (5.12 g, 48.34 mmoI) and the reaction mixture is stirred at room temperature for 17 h with separation of the deblocked pyrrole during the f irst hour~ Solid sodium carbonate is removed by filtration and washed well with MeOH. The filtrate is reduced to a ~olume of -25 ml and kept in a refrigerator for 1 h to give 5.23 g of crystalline prqduct. Fur~her concentration of the mother liquor Igave an additional 0.14 ~ o~ pure product; total yield 6.45 g (89.5 ~), mp 178-1~1 C.
"
: :, ~ `. ~og3/~l~$7 2~ ~ 3~ PCT/~S~3~2~1 PL~_6 C~ ~ o Cl'" ,' ~ NH-ICl-NHC~Ph .
Cl CN S
The above inte~nediate compound is prepared in this Example.
To a suspension of pyrrole of Example ~ (5~83 g, 15.64 mmol) in dichloromethane (lO0 ml) is added benzoylisothiocyanate (2.88 g, 17.64 mmol) at room temperature un~er nitrogen. The reaction mixture is stirred for 30 min with the separation of the desired t~ioureido compound. Additional benzoyl isothlocyanate (0.5 ml) is added to it and again stirred for 30 min~ T~e solvent is ev~porated to d~yness, and the light yellow residue is triturated with methanol. The white crystalline material is isolated by filtration and recrystallized from a chlorofo~m-ether mixture to give the required thioureido compound as an analytically pure sample, yield 7.71 g (92%), mp 210-211 DC.
_-S~MPL~ 7 ClH 0 ' i ' \~ /i Cl~ .~ `N=C-NHCOPh Cl ~CN SMe The above intermediate compound is prepared in this Example, A solution of thioureido campour~d of Ex~mple 6 ( 6 . 75 g, 12 . 6 mmol ) arld 1, 5-diaæabicyclo [ 4 . 3 . O ] non-5-ene ~1. 7 ~ g, 14 . 2 0 ~ol ) in dry CHzC12 (200 ml) is cooled to o oc and treated with methyl iodi`de (So20 g; 36.65 ~ol~ . The reaction mix~ure is istirred at O C f or 10 min and then f or 1 h at room temperature . The solvent is evaporat~d at room temperature, and the residue is ~ extracted with CHC13, washed with H20 (2 x 50 ml), dried (Na2S~4~
: 35 and evaporated to gi~e a glas5y foam (6095 g) which is used in the next step without purification.
:
:
O 93/21~87 ~ ~ 3 ~ 3 ~ ~ PCr/US93/~)2~41 PLE ~
.~
O O
I, H 1I H
HN~--~> H I ~\1\
H 2 N~ M e S~\N--~/
~ /CN Cl \~CN
Cl Cl A B
The above compo~nds A and B are prepared in this Example.
The compound A is a compound of the present in~ention and the compound B is an intermediate. A solu~ion of the methylthio intermediate of Example 7 (6.90 gl 12.~4 mol) ln MeOH (200 ml) is saturated at o D C with ammonia and hPated at 100 ~C for 20 h in a glass-lined stainless steel bo~h. The reaction mixture is brought to room kemperature and the solvent is evaporated at room temperature~ Purification of the crude mixtur~ by flash column chromatoyraphy oYer silica gel using CHC13 as eluent gave 8B (l.l g, 21%), mp 290-291 C then CHCl3-MeOH (95:5) gave pure ~ (2.76 gj 57.~), mp 284-285 C.
: :
P~E 9 The compound of the present invention of Example 8 is tested for~ enzyme inhibition activity. A purine nucleoside:
~` phosphoxylas~ (~NP) enzyme a~say is performed in whlch the PNP
.30 activity (IC50) for the compound (8A) is Eound, which i5 det~mine~ radiochemicall.y by:measuring the formation ~f [1~.C]-~:~ hyp~xanthine from ~14C~-in~sine (see Biomedic ne, 19~0, 3~, 39) usi~g cal~ spleen as the enzyme source. At 1 mM phosphate the IC5~ is 0. 64 ~M and at 50 mM pho5phate the IC50 is 10 ~M. ~ ;
.~ 3~:
,, ~
: ~ ~ : : :
` wog3/2ll~7 ~3.~ PCr/US93/0284~
E~PLE: l o , ~
S H~
H2Nl\~
J~ CN
lC
Following the procedure set forth in Examples 1-8, 3 - ( 3-15chlorophenyl ) -3 - ( 2 -amino-4-oxo-3H, 5H-pyrrclo i' 3, 2 -d ] pyrimidin-7-yl ) propanenitrile ( IC) is prepared using 3- ( 3-chlorophenyl ) -pentanedinltrile as the starting material, yield 54 . 5%, mp 157-158 ~ C.
2 OE:~PLB 11 Following the procedure set forth in Examples 1-8, the ~;
~o}lowing compounds are also prepared ( 1-9 ) .
O ,,.
;: 2 5H N~--H~N'~,T~, 3-Aryl 3-i~2-amino -4-oxo-3H,5E~-pyrrolc: [3,2-d]-pyrimidin 7-yl) propanenitrile Where Ar is each of the following. (1,~ phenyl, 2,3 dich~orophenyl, 3-methylphenyl, and 3-methoxyphenyl, (2) thienyl an~ 3 ~ ~, ( 3 ) furarlyl ( 2 - and 3 - ), ( 4 ) pyridinyl ( 2 - , 3 - , and .
~: 35 4~ 5) pyrrolyl (2~ and 3-), (6) thiazolyl (2-, 4-, and 5-), ~ . . ., (7~ 2-pyrazinyl, (8) pyridazinyl (3- and 4-), and (9) pyrazolyl. ¦-- 1 3 - .
:
;`: WO93/2~187 ~ 3 3 1 ~ PCT/US93/02~1 Eæ~MPLE 1~
Following th procedure set forth in Examples 1-8, 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 }1. 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 unsat1~rated Ar group is converted to the saturated R2 group by Xnown catalytic hydrogenation, followed by reconverting the amidei bac~ to the nitrile by known dehydration procedures.
O
H
H 2 N ~/~
~;: 15R ~ ~`--~
3-(Substituted)-3-(2-amino-4-oxo-3H,5H-pyrrolo[3,2-d]- :
~`` pyrimidin-7-yl)propanenitrile Where R2 is each of; 10) 1-adamantyl, 11) 2~-adamantyl, 12) 20cycloh~xyl, 13) cycloheptyl, 14) cyclopenkyl, 15 .
tetrahydro~uranyl, 16) tetrahydrothienyl, 17) tetrahydropyranyl, 18) pyrazolidinyl, ~19) thiazolidinyl, 20) piperazinyl, 21) mo~pholinyl, and 22) hexahydropyridazinyl. ~:
:: :
~ 25 ,. :
. ~
. ~ :
~ 14- ~
WO93121187 ~1 ~ 3 3 ~ 5 PCT/US93/02~1 E~n~E 13 .
~. O
S
The abo~e compound, 3-~2-amino-4~oxo-3H,SH-pyrrolo~3,2-d]pyrimidin-7-yl)-3-phenylpropanenitrile, is prepared i~ this Example. A solution of the compound A obtained in ExamplP 8 ~2.0 g, 5.22 mmol) in warm ethanol ~250 ml) and dimethylformamide (DMF) (lS0 ml) is hydrogenated over 30% Pd/C catalyct (1.0 g~ in the presence of triethylamine ~2.64 g, 5.O equivalent~ at atmospheric pressure. After 5 h, the reaction is complete, and the catalyst is filtered off u~der N2 pressure. The solid obtained by evaporation o~ the filtrate is triturated and sonicated w'th H20 and dried, yield 1.28 g (88%), mp 168-170 C.
20~MP~ 14 The compound prepared in Example 13 is tested for enzyme inhibition activity as in Example 9. At 1 ~M phospha~e the IC5 , is 0.023 ~M and at 50 mM phosphate the IC50 is 4.7 ~M.
I 1` ~ , ~
~
,;
`:, :
; :: :
: ~ : :
'.`~093/~1187 2 1 3 3 3 ~ ~ P~T/US93/02~
E~MFL~ ~s o . ~ H
~ ~ H
The above compoundl 3-(2-amino-4-o~o-3H r 5H-pyrrolo[3,2-dJpyrimidin-7-yl)-3-phenylpropanoic acid, is prepared in thiis example~ A solution of the compound obtainPd in Example 13 (0.200 g, 0.72 mmol) in 6N HCl (3,.~ ml) lS heated at reflux for 18 h. The solvent is evaporated ln vacuo and the residue is triturated with H20 (6 ml), adjusted to pH ~lO by conc. ammonium hydroxidei. Insoluble material is collected by filtration and the filtrate is readjusted to pH ~6.8. Whiite material which is pxecipitated out i5 colle ted, washed with wate~ and dried, yield O.l9 g (89~), mp 290 C dec.
:32U~P~
: 20 The compound prepared in Example lS is tested for enzyme :~ inhiibition activity as in Exampl~ 9. At 1 ~M phosphate the IC5 iig 0.012 ~ and`at 5Q mM phosphate the ICso is O.l9 ~M.
' ~
~ , ., :
~ `W~93/~lY~7 2 1 3 3 3 '1 S PCT/U~93/0~l ,, E~PL~ 17 H ~ ..
"~ ~,J~ NH2 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 13 (0.200 g, 0.72 mmol) in conc. HzS04 (0.5 ml) is stirred at room temperature for 20 h and then poured onto crushed ice (5.0 g) and adjust~d to pH ~6.8 by conc. NH40H. Th preoipitat~d solid is collected, washed with H20 and dried, yield 0,180 g, mp 199-20 D C dec.
~P~ l ~
The compound prepared in Example 17 is tested for enzyme inhibition activity as in Example 9. At 1 mM phosphate the IC50 is O.20 ~M and at 50 mM phosphate the IC50 is 6.6 ~M.
.
~ 17-:
-;
. NO 93/21187 213 3 3 ~ ~ P~/US~3/0~2841 E~MPL}: l9 ~2N ~ o OMe .
' The above compound, 3-(2-amino-4-oxo-3~,5H-pyrrolo~3,2-:: d}pyrimidin-7-ylj-3-phenylpropanoic acid, methyl ester, ~is prepared in this example.~ Thionyl chloride (O.2 g, 0.l17 mmol) is added to stirred mathanol (4.0 ml) a~ 0 C. The ~ompound obtained in Example lS (0.~ g, 0.67 mmol) is added and the mixture is stirred at~ro~m temperature ~or 18 h. The solvent is
~ :
WO 93/211~7 213 3 3 ~ ~ PCI/US93/02841 intermediate . In the following step, the N-benzoyl-N ' - [ 4 - ( 2 -cyano- l-cyclo-ethyl ) -2 -methoxycarbonyl-l~-pyrrol-3 -yl ] -S-meth~ylthiourea ( about 1-2 mol ) is reacted with methanolic or ethanolic ammonia at a ratio of a}:~out l/l to l/20 and at a temperature of about 20-130'C for about 16~60 hour~ to make a mixture of a 2-amino compound of t~e present invention 3-cyclo-3 ~ 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 ~ ~ pyrimidin-7 -yl ] propanenitril e as an intermediate in making another compound of the present invention.
In a further aspect of the present inYention there is provided a method of making a 2-methoxy compound (R1 = OCH3) and intermediates thereof. The intermediate 3-cyclo~3-[2-methylmercapto-4-oxo-3H,5H-pyrrolo~3,2-d~pyrimidin-7-yl]propanenitrile is reacted with an oxidi~ing agent such as permanganate or hydrogen peroxide at a molar ratio of about 1/1 to 1/10 a~d at a temperature of about 25-120-C for about 3-48 hours to make 3~cyclo-3-~2-methylsulfonyl-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile as an intermediate.
In the next step, the 3-cyolo-3-[2-methylsul~onyl-4-oxo-3H~5H-pyrrolo[3,2-d]pyrimidin-7-~lJpropanenitrile is reacted with a sodium alkoxide such as sodium methoxide at 2 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.
In a further aspect of the present invention there is provided a method of making a compound of the present invention wherein Rl is hy~rogen. The methyl 3 a~ino-4-(2-cyano~l-cyclo-3 0 ethyl ) -lH~-pyrrole~2 -carboxylate intermediate described su~ra is reacted with dlmethylformamide dime~hyl acetal at a molar ratio of about 1/1 to 1/4 and at a temperature of about 25-lOODC for about 1-10 days to make methyl 4-(2 cyano-l-cyclo-ethyl)-3-[N
(dimethylaminomethylene)amino~ pyrrole-2-car~oxylate as an intermedi~te. The next step involves reacting the methyl 4--~2-cyano-l-cyclo-ethyl)-3-~N-(dimethylaminomethylene~amino)-lH-pyrrole-2-carboxylate with methanolic or ethanolic ammonia at a ~`i` 213~3~
0g3/2~187 PCT/US93/~2~1 molar ratio of about 1/1 to 1/20 and at a temperature of about 20-130C 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~
As will be apparent to the skilled artisan, variations of the aforesaid procedures are useful in making the variety of compounds of th~ present in~ention without departing frcm 'he spirit thereof. For example, compounds having different values for "n" and "~" in accordance with the present invention are obtained by either stepping up or stepping down the series by.the necessary number of carbon atoms in accoxdance with known procedures. Also, reactions involving some intermediates require protection of nitrogen or oxygen atoms on the intermediates using known procedures.
The present invention provides a method of inhibiting purine nucleoside phosphorylase activity in mammals and treating diseases and conditions responsive thereto, e.g., autoimmune disorders, rejection of transplantation, or psoriasis, which comprises administaring to a mammal in need thereof an effective amount of a compound of the in~ention or of a pharmaceutical ~ composition comprising a said compound in combination with one .~ or more pharmaceutically acceptable carriers.
A ~urther aspect of the invention relates to a method of ~: inhibiting the phosphorolysis and metabolic breakdown of 2S anti~iral or antitumor purine nucleosides ~n mammals which compris~s administering in conjunction therewith to a mammal in need thereof, either separately or in combination therewith, an :~ effecti~e purine nucleoside phosphorylase inhi~iting amount of ~: a compound of the invention or of a said compound in combination with one or more pharmaceutically acc~ptable carriers. M~re particularly,j such relates to a ~ethod o~ inhibiting Ithe phosphorolysis and metabolic breakdown of purine nucleosides known in the art, e.~, of 2' deoxyguanosine, 2',3'-~id~oxyinosine, 2',3'-dideoxyguano~ine or 2',3'-dideoxyadenosine.
Furthermore, the in~ent1on thus relates to a method of ~ potentiating the antiviral or antitumor eff~ct of 2' or 3' ;~ monodeoxypurine nucleosides or of2',3' dideoxypurine nucleosides ': :
~ 6-WO93/211~7 Z1 3 3 ~ ~ ~ PCT/U593/02~1 in mammals which comprises administering in conjunction ~herewith to a mammal in need thereof, either separately or in combina~ion with a said nucleoside, an effective purine nucleosidP
. phosphorylase inhibiting amount of a compound of the invention preferably in combination wit~ one or more pharmaceutically acceptable carriers. More particularly, 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'~dideoxyguanosine or 2'-3'-dideoxyadenosine for the treatment of retrovirus infections, eOg., HIV-retrovirus infections (ac~uired immunode~iciency syn~rome, AI~S). 2',3'-Dideox~purine nucleosides are kn~wn in the art as inhibitors of HIV retrovirus infectivity and to be metaboli~ally degraded by PNP, e.g., as described in Biochem~cal Pharmacoloqy 22, 3797 (1987). Such are administered at a pharmaceutically acceptable dose which is effective in i~hibiting HIV-retro~irus infections.
Preferably the lowest possible effective dose is used.
The pharmaceutically acceptable ef~ective dosage of active compound ~f the invention to be administered is dependent on the species of warm-blooded animal (mammal), the b~dy weight, age and individual condition, and on ~he fo~m of administration.
The pharmaceutical composition may be aral, parenteral~
suppository or ~ther form which delivers the compound of the present invention into the bloodstream of a mammal to be treated.
An oral ~orm has from about 1 to about 150 mg of the compound of the pr~sent invention for an adult (50 to 70 kg) which is mixed together with pharmaceutically acceptable diluents such as lactose. In a typ~cal capsule, 25 mg of the compound of the present invention is mixed together with 192 m~ lactose, 80 mg modified 5tarch and 3 mg magnesium stearate. Injectable forms of the compound are also contemplated for administratio~. ~
'The present invention is also useful with other therapeutic : agents. A daily dosage of the compound of the present invention for a human weighing 50 to 70 kg of l-~0 mg/kg inhibits meta~olic 3S d~struction of certain anticancer agents such as ~-2'-deoxy-6-thio~uanosine and anti~iral agents such as 2'j3'-dideoxyinosine, an anti-AIDS drug. These t~pes of agents are known ~o be `~ ~093~21187 ~ 1 3 3 3 ~ ~ PCT/US93/02~]
susceptible to cleava~e. Upon cleavage, the agents lose ef~ectiveness~ The compounds of the present invention are capable of reducing such cle~vage. This protection, therefore, enhances the efficacy of other chemotherapeutic agents.
In order to more full~ describe the present invention`~the following non-limiting examples are provided. In the examples all parts and percentages are by weight unless indicated otherwise. Proportions of so.Lvent mixtures used as chromatographic eluents are by vol~me.
XA~PLE 1 ~3 cl ~
cl , t:~ The a~ove intermediate compound is prepared in this Example ~!''' by the modi~ication o~ the procedure of Schiemenz, G. P.;
;~ Engelhard, H~ (Chem. Ber., l962, 95, 195).
t~ O A mixture of cyanoacetic acid t25.38 g, 298.38 mmol), 2,3,5-.. t~ichlorobenzaldehyde (25.0 g, 119.35 mmol), ammonium acetate ,~,; (500 mg), toluene (120 mL), and pyridine (65 ml) is heated at ,.~ reflux for 16 h in a flask fitted with Dean Stark trap and . condens~r. The solvents are evaporated in vacuo, residue is ~.,, 5 extracted with ~HCl~, which is washed with H20~ ~ried ~Na2SO~), .~ and e~aporated to give the crude product, which is purified by silica gel column chromatography using hexaneEtOAc mixture as :;~' ~he eluent. Yield 23.69 g (73%); mp 90-91 C.
~A~PL~ 2 ~CN~
C~ ~ ~HO
; ii Cl The a~ove intermediate compound is prepared in this Example.
To a stirred mixture of NaH (l.S6 g, 65005 mmol) and ethyl ?
,r'~ :`
':
,.~,j r~ , . WO 93~2l~X7 2 ~ 3 3 ~ ~ J PCT/U593/02~1 ." formate (14.~8 g, 199.51 mmol) in THF (100 ml) is added substituted pentanedinitrile of Example 1 (10.17 g, 37.17 mmol) at r~om temperature under a nitrogen atmosphere, and the ~ resulting reaction mixture is stirred for 24 h. Volatile matter ,.~ 5 is evaporated ln vacuo at room temperature. Water (50 ml) is ~ added to t~e residue at 0-5 C, and the solution is adjusted to : pH 5-6 by 20% conc. HCl (_~v). The heavy oil is extracted into ethyl acetate, washed with H20 (1 x 100 ml) and dried (~gSO4).
. The ethyl acetate layer is evaporated to give a red-brown oil ~ 0 g) that is used in the next step without further . purification.
""
;.', ~AMP~ 3 ~ j O~e The above intermediate compound is prepared in this Example.
2Q Glycine met~yl ester hydrochloride (8.17 gl 65.06 mmol~ and sodium acetate (5.33 g, 65.06 mmol) are added to a solution of the crude formyl compound of Example 2 (11.0 g) in a mixture o~
; MeOH (80 m~) and H20 (20 ml), and the resulting solution is .~ stirred at room temperatur~ for 22 h. A~ter evaporation of solvent at room temperature, the residue is extracted with ethyl acetate. The washed (H20) and dried (MgS04) organic layer is evaporated to gi~e an oil. Flash column chromatography (silica .. gel) using CHCl3 as eluent gave the pure desired enamine as a mixture of ~rtra~s isomers which is recrystallized from MeOH, ;, 30 yield 10.41 g (75%), mp 142-143 C~
~AMPLE 4 !.'¦
Cl E~70C 0 .,¦ Cl ~ ~NH~
Cl ~ N
~, _g_ , ,~
2~333~G
';~ VO93/XllX7 PCT/US93/02~1 The above intermediate compound is prepared in this Example.
solution of enamine of Example 3 (10.0 g, 26.84 mmol) in dry C~2Cl2 (100 ml) is cooled to 0 ~C and treated with 1,5 diazabicyclo[4.3.0~non-5-ene (10.53 g, 84.79 mmol) under a nitrogen atmosphere followed by ethyl chloroformate (6.90 g, 63~57 mmol). The solution is stirred at 0 ~C for 1 h and then at room temperature for 48 h. Volatiles are evaporated in vacuo to give a viscaus dark gum which is purified by flash column chromatography over silica gel using CHC13 as the eluent. All the fractions containing the desired ~-protected pyrrole are pooled and evaporated to give a foamy light pale yellow material which is stirred in MeOH (100 ml) to give ~he crystalline material which is recrystallized from CHCl3-MeOH, yield 8.92 g (74.7%), mp 160-16~ C.
~MP~ 5 ~ o M e ;20 Cl'``~ `NH2 Cl C~
The above intermediate compound is prepared in this Example.
A suspension of N-protected pyrrole of Example 4 (8.6 g, 19.34 2S .mmol) in MeOH (300 ml) is treated with NazCO3 (5.12 g, 48.34 mmoI) and the reaction mixture is stirred at room temperature for 17 h with separation of the deblocked pyrrole during the f irst hour~ Solid sodium carbonate is removed by filtration and washed well with MeOH. The filtrate is reduced to a ~olume of -25 ml and kept in a refrigerator for 1 h to give 5.23 g of crystalline prqduct. Fur~her concentration of the mother liquor Igave an additional 0.14 ~ o~ pure product; total yield 6.45 g (89.5 ~), mp 178-1~1 C.
"
: :, ~ `. ~og3/~l~$7 2~ ~ 3~ PCT/~S~3~2~1 PL~_6 C~ ~ o Cl'" ,' ~ NH-ICl-NHC~Ph .
Cl CN S
The above inte~nediate compound is prepared in this Example.
To a suspension of pyrrole of Example ~ (5~83 g, 15.64 mmol) in dichloromethane (lO0 ml) is added benzoylisothiocyanate (2.88 g, 17.64 mmol) at room temperature un~er nitrogen. The reaction mixture is stirred for 30 min with the separation of the desired t~ioureido compound. Additional benzoyl isothlocyanate (0.5 ml) is added to it and again stirred for 30 min~ T~e solvent is ev~porated to d~yness, and the light yellow residue is triturated with methanol. The white crystalline material is isolated by filtration and recrystallized from a chlorofo~m-ether mixture to give the required thioureido compound as an analytically pure sample, yield 7.71 g (92%), mp 210-211 DC.
_-S~MPL~ 7 ClH 0 ' i ' \~ /i Cl~ .~ `N=C-NHCOPh Cl ~CN SMe The above intermediate compound is prepared in this Example, A solution of thioureido campour~d of Ex~mple 6 ( 6 . 75 g, 12 . 6 mmol ) arld 1, 5-diaæabicyclo [ 4 . 3 . O ] non-5-ene ~1. 7 ~ g, 14 . 2 0 ~ol ) in dry CHzC12 (200 ml) is cooled to o oc and treated with methyl iodi`de (So20 g; 36.65 ~ol~ . The reaction mix~ure is istirred at O C f or 10 min and then f or 1 h at room temperature . The solvent is evaporat~d at room temperature, and the residue is ~ extracted with CHC13, washed with H20 (2 x 50 ml), dried (Na2S~4~
: 35 and evaporated to gi~e a glas5y foam (6095 g) which is used in the next step without purification.
:
:
O 93/21~87 ~ ~ 3 ~ 3 ~ ~ PCr/US93/~)2~41 PLE ~
.~
O O
I, H 1I H
HN~--~> H I ~\1\
H 2 N~ M e S~\N--~/
~ /CN Cl \~CN
Cl Cl A B
The above compo~nds A and B are prepared in this Example.
The compound A is a compound of the present in~ention and the compound B is an intermediate. A solu~ion of the methylthio intermediate of Example 7 (6.90 gl 12.~4 mol) ln MeOH (200 ml) is saturated at o D C with ammonia and hPated at 100 ~C for 20 h in a glass-lined stainless steel bo~h. The reaction mixture is brought to room kemperature and the solvent is evaporated at room temperature~ Purification of the crude mixtur~ by flash column chromatoyraphy oYer silica gel using CHC13 as eluent gave 8B (l.l g, 21%), mp 290-291 C then CHCl3-MeOH (95:5) gave pure ~ (2.76 gj 57.~), mp 284-285 C.
: :
P~E 9 The compound of the present invention of Example 8 is tested for~ enzyme inhibition activity. A purine nucleoside:
~` phosphoxylas~ (~NP) enzyme a~say is performed in whlch the PNP
.30 activity (IC50) for the compound (8A) is Eound, which i5 det~mine~ radiochemicall.y by:measuring the formation ~f [1~.C]-~:~ hyp~xanthine from ~14C~-in~sine (see Biomedic ne, 19~0, 3~, 39) usi~g cal~ spleen as the enzyme source. At 1 mM phosphate the IC5~ is 0. 64 ~M and at 50 mM pho5phate the IC50 is 10 ~M. ~ ;
.~ 3~:
,, ~
: ~ ~ : : :
` wog3/2ll~7 ~3.~ PCr/US93/0284~
E~PLE: l o , ~
S H~
H2Nl\~
J~ CN
lC
Following the procedure set forth in Examples 1-8, 3 - ( 3-15chlorophenyl ) -3 - ( 2 -amino-4-oxo-3H, 5H-pyrrclo i' 3, 2 -d ] pyrimidin-7-yl ) propanenitrile ( IC) is prepared using 3- ( 3-chlorophenyl ) -pentanedinltrile as the starting material, yield 54 . 5%, mp 157-158 ~ C.
2 OE:~PLB 11 Following the procedure set forth in Examples 1-8, the ~;
~o}lowing compounds are also prepared ( 1-9 ) .
O ,,.
;: 2 5H N~--H~N'~,T~, 3-Aryl 3-i~2-amino -4-oxo-3H,5E~-pyrrolc: [3,2-d]-pyrimidin 7-yl) propanenitrile Where Ar is each of the following. (1,~ phenyl, 2,3 dich~orophenyl, 3-methylphenyl, and 3-methoxyphenyl, (2) thienyl an~ 3 ~ ~, ( 3 ) furarlyl ( 2 - and 3 - ), ( 4 ) pyridinyl ( 2 - , 3 - , and .
~: 35 4~ 5) pyrrolyl (2~ and 3-), (6) thiazolyl (2-, 4-, and 5-), ~ . . ., (7~ 2-pyrazinyl, (8) pyridazinyl (3- and 4-), and (9) pyrazolyl. ¦-- 1 3 - .
:
;`: WO93/2~187 ~ 3 3 1 ~ PCT/US93/02~1 Eæ~MPLE 1~
Following th procedure set forth in Examples 1-8, 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 }1. 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 unsat1~rated Ar group is converted to the saturated R2 group by Xnown catalytic hydrogenation, followed by reconverting the amidei bac~ to the nitrile by known dehydration procedures.
O
H
H 2 N ~/~
~;: 15R ~ ~`--~
3-(Substituted)-3-(2-amino-4-oxo-3H,5H-pyrrolo[3,2-d]- :
~`` pyrimidin-7-yl)propanenitrile Where R2 is each of; 10) 1-adamantyl, 11) 2~-adamantyl, 12) 20cycloh~xyl, 13) cycloheptyl, 14) cyclopenkyl, 15 .
tetrahydro~uranyl, 16) tetrahydrothienyl, 17) tetrahydropyranyl, 18) pyrazolidinyl, ~19) thiazolidinyl, 20) piperazinyl, 21) mo~pholinyl, and 22) hexahydropyridazinyl. ~:
:: :
~ 25 ,. :
. ~
. ~ :
~ 14- ~
WO93121187 ~1 ~ 3 3 ~ 5 PCT/US93/02~1 E~n~E 13 .
~. O
S
The abo~e compound, 3-~2-amino-4~oxo-3H,SH-pyrrolo~3,2-d]pyrimidin-7-yl)-3-phenylpropanenitrile, is prepared i~ this Example. A solution of the compound A obtained in ExamplP 8 ~2.0 g, 5.22 mmol) in warm ethanol ~250 ml) and dimethylformamide (DMF) (lS0 ml) is hydrogenated over 30% Pd/C catalyct (1.0 g~ in the presence of triethylamine ~2.64 g, 5.O equivalent~ at atmospheric pressure. After 5 h, the reaction is complete, and the catalyst is filtered off u~der N2 pressure. The solid obtained by evaporation o~ the filtrate is triturated and sonicated w'th H20 and dried, yield 1.28 g (88%), mp 168-170 C.
20~MP~ 14 The compound prepared in Example 13 is tested for enzyme inhibition activity as in Example 9. At 1 ~M phospha~e the IC5 , is 0.023 ~M and at 50 mM phosphate the IC50 is 4.7 ~M.
I 1` ~ , ~
~
,;
`:, :
; :: :
: ~ : :
'.`~093/~1187 2 1 3 3 3 ~ ~ P~T/US93/02~
E~MFL~ ~s o . ~ H
~ ~ H
The above compoundl 3-(2-amino-4-o~o-3H r 5H-pyrrolo[3,2-dJpyrimidin-7-yl)-3-phenylpropanoic acid, is prepared in thiis example~ A solution of the compound obtainPd in Example 13 (0.200 g, 0.72 mmol) in 6N HCl (3,.~ ml) lS heated at reflux for 18 h. The solvent is evaporated ln vacuo and the residue is triturated with H20 (6 ml), adjusted to pH ~lO by conc. ammonium hydroxidei. Insoluble material is collected by filtration and the filtrate is readjusted to pH ~6.8. Whiite material which is pxecipitated out i5 colle ted, washed with wate~ and dried, yield O.l9 g (89~), mp 290 C dec.
:32U~P~
: 20 The compound prepared in Example lS is tested for enzyme :~ inhiibition activity as in Exampl~ 9. At 1 ~M phosphate the IC5 iig 0.012 ~ and`at 5Q mM phosphate the ICso is O.l9 ~M.
' ~
~ , ., :
~ `W~93/~lY~7 2 1 3 3 3 '1 S PCT/U~93/0~l ,, E~PL~ 17 H ~ ..
"~ ~,J~ NH2 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 13 (0.200 g, 0.72 mmol) in conc. HzS04 (0.5 ml) is stirred at room temperature for 20 h and then poured onto crushed ice (5.0 g) and adjust~d to pH ~6.8 by conc. NH40H. Th preoipitat~d solid is collected, washed with H20 and dried, yield 0,180 g, mp 199-20 D C dec.
~P~ l ~
The compound prepared in Example 17 is tested for enzyme inhibition activity as in Example 9. At 1 mM phosphate the IC50 is O.20 ~M and at 50 mM phosphate the IC50 is 6.6 ~M.
.
~ 17-:
-;
. NO 93/21187 213 3 3 ~ ~ P~/US~3/0~2841 E~MPL}: l9 ~2N ~ o OMe .
' The above compound, 3-(2-amino-4-oxo-3~,5H-pyrrolo~3,2-:: d}pyrimidin-7-ylj-3-phenylpropanoic acid, methyl ester, ~is prepared in this example.~ Thionyl chloride (O.2 g, 0.l17 mmol) is added to stirred mathanol (4.0 ml) a~ 0 C. The ~ompound obtained in Example lS (0.~ g, 0.67 mmol) is added and the mixture is stirred at~ro~m temperature ~or 18 h. The solvent is
5:~ removed on a water aspirator (30 C) and vacuum pump (lyophiIize) to give a semisolid mass which iS purified on a silica gel column ~ : j using~CHCl3-MeOH as the eluent, yield 0.1 g. :;~
20: ~ 7rJ~ 20 ;: , The compound: prepared i~:Example 19 is tested ~or enzyme inhibition acti~ity . S ignlf icant activity (ICso) is ~ound.
" W093~211~7 2 1 3 3 3 '~ J~ PCT/US93fO2~]
E~PL~
ll H
~ ~ OH
~ Amino-4-oxo~3H,SH-pyrrolo[3,2-d~p~rimidin-7-yl)-3-cyclohexylpropanoic acld is prepared in this example. A solution of the compound obtained in Example 15 ~83 mg, O.28 mmol) in trifluoroacPtic acid ~TFA) (15 ml) is hydrogenated with PtO2 (83 mg) at 60 lb/in2 for 24 h. The catalyst is filtered off throu~h 15 a Celite bedt and t~e filtrate is evaporated at 25 C. The residue is susp~nded in H20 (8 ml), and adjusted to pH 8.5 by conc. N~OH and filtered through a Whatman filter paper to remove brown colored impurities. The colorless filtrate is adjusted tG
pH -6.8, and t~e precipitated compound is filtered, washed with ~0 HzO, and dxied, yield 65 mg (77%), mp >300 C.
~ PLE 2 2 The compound prepared in Example 2l is tested for enzyme inhibition activity as in Example 9. At l mM phosphat'e the'I~5 25 is 0.097 ~M and at 50 mM phosphate the IC~o is l.O ~M.
:
' . ~093~21187 2 ~ 3 3 d ~ ~ PCT/US~3/02~1 ~MP~ 23 A compound of the present invention is prepared wherein X
is PO(OH)2~ The nitrile group of the compound of Example 13 is converted to the corresponding amide by treatment with sulfuric S acid. Using a Hoffman degradation reaction, the amide is converted to the corresponding amine, which is then converted to the corresponding pyridinium salt using a pyrillium salt.
Canversion of the salt to the corresponding halide is accomplished using sodium ~romide, which is then converted to the phosphonic ester using triethyl phosphite. Hydrolysis of the ester using trimethylsilylbromide yields the correspondinq phosphonic acid wherein "n" is l and "m" is O.
`:
M~L~ 2 This Example makes a compound of the present invention ~y~
: stepping up the number of car~on atoms ~rom "m" is 0 to "m" is:
;~ l. The nitrile yroup oP the compound o~ Example 13 is reduced to;~the corresponding aldehyde, which is then converted to the corresponding alcohol. Using phosphorous tribromide the alcohol ;:
is converted to ~he corresponding alkyl bromide, which is the~n : conYerted to the nitrile compound of the present invention wher~in m is 1 using~p:otassium cyanide.
In this example a compound of the present invention LS~
prepared whereln "p"~ is 1 and "Y" is oxygen. The alcohol prepared as~an intermedlate in the previous example is converted to ~he corresponding :~diethy1 phosphonomethyl ether using~:
~ `;WO93~211~7 ~ 1 3 3 3 ~ ~ PCT/VSg3/02~1 ~
:C
diethy1chloromethyl phosphonate. Removal of ~he ethyl groups of the ester is accomplished using trimethylsilylbromide to gi~e the phosp~onic acid.
S~S~P~ 26 In this example a comp~und of the pre ent .invention is made wherein "Y" is NH and "X" is SO~NH2. The nitrile group of the compound of Example 13 is reduced to the amine using standard catalytic hydrogenation wit~ palladium in acidic media (usually l00.0l N to l N HCl), which is then converted to the sulfamide uslng sulphamoyl chloride.
~MPL~ 27 : In this example a compound of the present invention is 15preparad wherein "X" is COOH and "Y" is NH by reacting the methyl amine intermediate prepared in the previous example with chloroacetic acid.
.
~SPIIE 2 8 ;~ 20In this example a compound of the present invention is prepared wherein "Xl' is PO(OH)z and "Y" is NH by reacting the methyl amine intermediate prepared in Example 27 with ~.
diethylchloromethyl phosphonate, and reacti~g the resulting p~od~uct with trimethylsiiylbromide.
~;~ 25~ :
~In this example a compound of the present invention is :~:prepared wherein l'X7' is 5O2NH2 and "Y" is oxygen by reacting the ~ .
NO 93t21~i7 2 1 3 3 3 ~ ~ ~CrlUS93/0~84}
alcohol intermediate prepared in Example 24 with sulphamoyl chloride.
i ' , P I~ 3 0 r ~
In this example a compnund of t~e present invention is prepared wherein R1 is H, R2 is phenyl, R3 and R4 are hydrogen, m is 0, n is 1, p is 0, a~d X is CN~ A modifiGation of the proceduxe disclosed in Mu-Ill Lim, et al., J. Or~. Chem., VOlr 44, No. 22, 3826 (1979) is used. A mixture of the compound of Example S and dimethylformamide dimethyl acetal is reacted at room temperature ~or two days and then evaporated to dryness in vacuo. The residue is crystallized to~ give the Ipure N-(dimethylaminojmethylene deri~ative, which is cyclized with saturated methanolic a~monia tQ give the desired end product.
~ 15 ~ B~P1 31 ~ -, ."~ In this example a compound of thç present in~ention is ~` prepared wherein R1 is 0C~3, R2 is phenyl, R3 and R4 are hydrogen;
m is 0, n is 1, p is 0, and X is CN. Usin~ the compound B of ; 20 Example 8, the S-methyl group is oxidized to methylsulfone, whlch hen is converted to the final methoxy compound ~y treatment with :
sodium methoxid`e in methanol. ~ :
:
F~l~ 3 ~
; ~25 In this example a ~-compound of the pre~ent invention lS
`~ preparéd wherein X is tetrazole. The compound~of Exa~ple 13~is ~ : treated with= lithium:azide in the:presence of ammonium chloride ; : : :
. .~
; ~
t; wo g3,21 ,87 2 ~ 3 3 ~ 4 ~ PCT/US93~2~1 as a catalyst in dimethylformamide ( DMF) at lO0 degrees C to give t~e desired tetrazole.
.,.
In this example a compound of the present invention is prepared wherein X is triazole. The campound of Example l9 is treated with hydrazine hydrate to give the corresponding hydrazide, which is then treated with imino ether to give the desired triazole.
X~PL~_3~
The compound prepared in Example lO is tested $or en2yme inhibition acti~ity as in Example 9. A~ 1 mM phosphate the ICso is 0. 012 ~M and at 50 mM phosphate the ICso is 2 . 0 ~.
E~AMPL~ 35 In this example an amidine compound of the present invention is prepared, i.e., wherein X in the recited generic formula is CNHNH2. The compound A from Example 8 is reacted with sodium methoxide in methanol at room temperature ~or abou~ 2 days to ~:~ give a methyl-imidate intermediate. The intermediate is then reacted with ammonia in methanol to ~iYe the amidine product.
:, ~
` . WO g3/211~7 ~ ~ 3 3 ~ PCT/~S93/02~1 E~NPL~ 36-42 The followlng table gives the formula~ for the compounds made in Examples 36-42 and the IC50 (nM) values obtained for these compounds.
S ~ ~ H
--~R
CHR~
Table No. _ Rl_ R2 - . R~ __ IC~ a Ex~ 36 H2N 3-Chlorophenyl C~2Co2H 7 Ex. 37 H2N 3-Chlorophenyl CH2co2H (S)5-9 Exc 38 H2N 3-Chlorophenyl CH2co2H (R~160 Ex. 39 SMe 3-Chlorophenyl C~2C~ --Ex. 40 H 3 Chlorophenyl CH2CN . 10 Ex~ 41 H2N 3-Chlorophenyl CH2CH20H 25 Ex. 42 H2N 3-Chlorophenyl CH2CO~Me 85 2,6-diamino~3,5-dihydro-7-(2-thienylmethyl)-4H-pyrollo-[3~2-d]pyrimidine-4-one (available from ~arner-Lambert) 160 ~a~PL~ 36 HN
~ H 2N
0!1 ~ Cl '~`' W093/~i87 2 1 3 ~ 3 ~ ù PCT/US93/02~1 The compound prepared in Example lO is hydrolyzed to the cQrxesponding acid of the abo~e formula in this example. A
f ' solution of 3-~3-chlorophenyl)-3-(2-amino-4-oxo-3H,5H-¦ pyrrolo(3,2-d~pyrimidin-~-yl)prop~nenitrile (2.0 g; 63.75 mmol) in 6N HCl (60 ml) is heated at reflux for 8 h. T~e solvent is evaporated in vacuo and the residue is dissol~ed in H2O (18 ml).
The resulting solution is adjusted to pH ~lO by conc. ammonium hydroxide and any i.n~oluble material is removed by filtration.
The filtrate is then readj-~sted to pH ~6.8. The white 10 precipitated material was collected, washed with H2O, and dried to yield 1.8 g of desired compound, m.p. 295-96-C dec, as a dl racemic mixture.
~AMPLE 37 H~N
i ~N~<~
~ 20 C1 .
The above ~ompound, 3-(2-amino-3H,5H 4-oxo-py~rolo[3,2-d]pyrimidin-7-yl1-3-(3-chlorophenyl-N-:. (phenylethyl;)propanamide, is prepared in this example. A
solution of diphenylphosphoryl a~ide (O.72 g, 2.6 mmol) in D~F
(lO ml) is added dropwise during lO min to a mechanically-stirred, cold (-5 to O'C) solution of the compound obtained in ~xample 36 t0.7~0 g; 2.4 mmol) and (R)d-~+)-~-methylbenzylamine ~::
. 093/21187 w~ 3 3 ~ ~ 5 PCT/US93/02~1 (0.32 g, 2.6 mmol) in D~F (lO0 ml). A solution of N-methylmorpholine (0.48 g, 4.7~ mmol) in DMF (5 ml) is then added dropwise during 5-lO min, and the solution is kept near O C for 5 h. It is then allowed to warm to room temperatur-~ and is stirred overnight (18 h). A second portion of diphenylphosphonyl axide (0~36 g), (R)d~ a~mQthylbenzylamine (0.16 g) and N-methylmorpholine (O.24 g) is added at 0C and the reaction mixture is stirred for 2 days. The solvent is removed in vacuo and the residue is dissolved in an 8: 2 mixture of acetonitrile and ammonium hydroxide (lM). The crude produc-t is adsorbed on silica gel and dried in vacuo to remove the last traces of solvent. Flash column chromatographic purification using acetonitrile and lM ammonium hydroxide (95:5) gives the pure desired material as a mixture of diastereomers (yield 0.630 g).
~: 15 These isomers are separated ~y repeated flash calumn chxomatography on silica gel using acetonitrile and lM ammonium ~: hydroxide (98:2) as the eluent to yield 0~18 g of S,R-isomer (Compound A), m.p. 170~75C dec. and 0.120 g of R,R-isome~
(Compound B), m.p. 155-60-C dec.
~NN~ ~NNz ~N--C--HzCJ~ N2C~
` ' Cl .
-2~-~ ` WO93/2~1~7 2 1 3 J 3 ~ s PCT/~S93/0~1 o ~I 2N
S
S-i~omer I The above compound labeled "S-isomer," (S)-3-(2-amino-4-ox o-3H,5H-pyrroloC3,2-d]py rimidin-7-yl) -3-(3 -chlorophenyl)propanoic acid, is prepared in this example. A
10solution of the compound A (S,R~isomer) (0.170 g), obtained above, in 6N HCl (30 ml) is heated at reflux for 6 h and then le~t at room temperature for 6 h. The sol~ent is evaporated in vacuo and the residue is dissolved in H20 (5 ml). T~e resulting solution is adjusted to pH ~10 by conc. ammonium hydroxide~and 15any ins~luble material is removed hy flltration. The filtrate ~:; is then readjusted to pH -6.8 by ammonium hydroxide~ The white precipitated material is collected, washed with H20, and dried to yield 0.0~0 g o~ the crude material which was purified by flash column chromatoyraphy usin~ a 98:2 mixture of acetonitrile~and 20ammonium hydroxide (1 M). Yield 48 mg, m~p. ~ 285~C dec.
: A purine nucleoside phosphorylase (PNP) enzyme assay is performed in which the inhibitory activity (ICso) of the S-; isomer compound is dete ~ ined by measuring the formation of 4C~-hypoxanthi~e from [14C]-inosine ~see Bigmedic~ne, 1980, 33~
253~) using calf spleen PNP in the presence and absence of inhi~itor. At 50 mM phosphate the IC50 is 0.031 ~M and at 1 mM
~`~phosphate, it is 0.0059 ~.
: :
` :~
:
` :~
~::
3 '~ ~
~093/~1187 PCT/US93/~2~1 E~l~ 38 o HN
_ H2N ~
H
ao~c~2c i R-i~om~r Cl The procedure described in Example 37 is repeated to prepare 10 theabove compound, (R)-3-~2-amino~4~oxo-3H,5H-pyrrolo~3,2-d]pyrimidin 7-yl)-3-(3-chlorophenyl)propanoic acid from Compound B (R,R-isomer), obtained in Example 37. Yield 40%~
m.p. > 280 C dec. The compound prepared in Example 38 is tested for enzyme inhibiti~n activity as in Example 37. At 50 mM
15 ph~sphate the IC50 is 0.900 ~M and at 1 mM phosphate the IC~o is 0.160 ~M. Thus the S-isomer (Example 383 .is ca. 30X as potent as the R-isomer in the inhibition of PNP~ X ray crystalloyraphic analysis of the enzyme-inhibitor complex ~ormed from a soak of a crystal of the enzyme in a solution containing the unresolved racemic mlxture (Example 36) showed that the S-isomer excluslvely bound to the active site of the enzyme.
~3~Ph~ 3 9 The procedures of Examples 1 8 are followed, except that the starting material used is the 3-chlorophenyl deriYative rather than the 2,3,5-trichlorophenyl derivative used in the pre~iou5 Examples. The SMe derivative as shown in the Table is obtaine~.
.
~ 093/21~87 2 1 ~ 3 3 ~1 5 PCT/US93/0~l ~PLE 4~
The compound from Example 39 (1 g) in ethanol~(loO ml) is suspended in in 30% palladium on carbon (1 g) and subjected to reflux for a few minutes. Hydrazine hydrate (0.3 ml)-~s added with stirrlng an the mixture refluxed for two days~ Additional hydrazine hydrate (O.3 ml) and palladium on carbon (O.5 g) are added and the mixture refluxed for an additional four days. The catalyst is remo~ed by filtration, and the filtrate reduced to 25 ml and filtered vn Whatman filter paper and evaporated to give the final product.
An altern~tive way of making the final product begins by using the present Examples 1-5 except t~at the 3-chlorophenyl derivative i5 used as the starting material. The resulting material, 3-amino-4-~(3-chlorophenyl)methyl]methylester-1-H-pyrolle-2-carboxylic acid, ~5 g) is disolved in dimethylformamide dimethylacetal (50 ml) under ar~on and heated for 24 hours at 60 70 C. After e~aporation to dryness, the material is disol~ed in dichloromethane (50 ml), filtered and diluted with patroleum et~er until cloudy, triturated to induce crystallization, and slowly diluted with and additional 40 ml of patroleum ether.
This mono-chloro intermediate is collected, washed with patroleum eth~r and dried~ Yield 5 g (88%)~ mp 122-124 C. The resulting : intermedi~te is heat~d in methanolic ammonia at 95-100 ~C for 24 hour~ in a s~ainles5 steel bomb, evaporated to a yellowish solid crude product. The yellowish solid crude product (3 ~) in 175 ml ho~ methano~ yiel~s a final pro~uct of 2.2 g (8B% yield~.
21 333~
, .
W093/2~1~7 PCT/US93/~2~1 E~Ph~ 41 T~e compound of Example 10 (6.80 g) in 6N HCl (400 ml) is re~luxed for 10 h, cooled overnight, and evaporated under reduced ¦ pressure. The residue is added to methanol and evaporated and then added to toluene, which results in a white foam in nearly quantitative yield. A solution of the dried white foam in an~ydrous methanol (400 ml) is cooled below O C in an ice salt bath under dry conditionc. Thienyl chloride tlO.31 g) is added 510wly dropwisa, and the solution allowed to come to ambient lQ temperature and stand overnight. The solvent is evaporated in vacuo, ~resh methanol and toluene are added and then evaporated to aid in the removal of acid vapors. A suspension of the solid in cold water (200 ml) is neutralized in lN NaOH and the solid is collected by filtration, washed ~ith cold water, and dried in vacuo o~er P205 at llO-C. Yield 6.08 g (81% from the material of Example 10)~ Thi5 product is of sufficient purity for use in.the n~ixt step, but may recrystallize in methanol using Soxhlet apparatus to fine white crystals having a m.p. o~ 302-303C
(dècompose~. An amount of 6g of the product from the pre~ious paraqraph with 100 mg dry a~monium sulfate in h~xamethyldisilazane (400 ml) is refluxed for 8 h under dry condi~ions. The resulting clear solu~ion is evaporated in ~acuo to a viscous gum that is further dried over P20~, which is used in the next Example without further treatment.
~5 ,~M~ 42 Under nitroqen, a s~lution of the product from the preivious paragraph in anhydrous THF or ether (200 ml) is treated dropwise : -3~-' ; wo g3/211g7 213 3 3 ~1 a PCT/US93~02~1 with a 1 molar solution of lithium aluminum hydride (26 ml) in T~F. After 1 h at room temperature, excess hydride is des~royed by dropwise addition of ethyl acetate (50 ml), and the solvent evaporated in vacuo. The residue is suspended in cold water (200 ml), adjusted to a pH of 1 with HCl, stirred for 15 min, adjusted to a pH 7 with dilute sodium hydroxide, and filtered r The resulting filter cake is wa~hed with cold water, dried, and washed with ethyl to remove TMS by-products. Silica gel (50 g) is added to a hot solution of the resulting crude solid (~ 8 g) in a large volume of methanol, and tAe resulting slur~y is evaporated to dryness. The resulting material is layered carefully onto a silica gel column that is eluted with a chloroform~methanol mixture (85/15) to give the desired alcohol final product. Yield 4.65 g (84%). Two recrystallizations from ethanol/water gives a white crystalline materiai. Yield 3036 g (61~), m.p. 2S5-277~C (decompose).
20: ~ 7rJ~ 20 ;: , The compound: prepared i~:Example 19 is tested ~or enzyme inhibition acti~ity . S ignlf icant activity (ICso) is ~ound.
" W093~211~7 2 1 3 3 3 '~ J~ PCT/US93fO2~]
E~PL~
ll H
~ ~ OH
~ Amino-4-oxo~3H,SH-pyrrolo[3,2-d~p~rimidin-7-yl)-3-cyclohexylpropanoic acld is prepared in this example. A solution of the compound obtained in Example 15 ~83 mg, O.28 mmol) in trifluoroacPtic acid ~TFA) (15 ml) is hydrogenated with PtO2 (83 mg) at 60 lb/in2 for 24 h. The catalyst is filtered off throu~h 15 a Celite bedt and t~e filtrate is evaporated at 25 C. The residue is susp~nded in H20 (8 ml), and adjusted to pH 8.5 by conc. N~OH and filtered through a Whatman filter paper to remove brown colored impurities. The colorless filtrate is adjusted tG
pH -6.8, and t~e precipitated compound is filtered, washed with ~0 HzO, and dxied, yield 65 mg (77%), mp >300 C.
~ PLE 2 2 The compound prepared in Example 2l is tested for enzyme inhibition activity as in Example 9. At l mM phosphat'e the'I~5 25 is 0.097 ~M and at 50 mM phosphate the IC~o is l.O ~M.
:
' . ~093~21187 2 ~ 3 3 d ~ ~ PCT/US~3/02~1 ~MP~ 23 A compound of the present invention is prepared wherein X
is PO(OH)2~ The nitrile group of the compound of Example 13 is converted to the corresponding amide by treatment with sulfuric S acid. Using a Hoffman degradation reaction, the amide is converted to the corresponding amine, which is then converted to the corresponding pyridinium salt using a pyrillium salt.
Canversion of the salt to the corresponding halide is accomplished using sodium ~romide, which is then converted to the phosphonic ester using triethyl phosphite. Hydrolysis of the ester using trimethylsilylbromide yields the correspondinq phosphonic acid wherein "n" is l and "m" is O.
`:
M~L~ 2 This Example makes a compound of the present invention ~y~
: stepping up the number of car~on atoms ~rom "m" is 0 to "m" is:
;~ l. The nitrile yroup oP the compound o~ Example 13 is reduced to;~the corresponding aldehyde, which is then converted to the corresponding alcohol. Using phosphorous tribromide the alcohol ;:
is converted to ~he corresponding alkyl bromide, which is the~n : conYerted to the nitrile compound of the present invention wher~in m is 1 using~p:otassium cyanide.
In this example a compound of the present invention LS~
prepared whereln "p"~ is 1 and "Y" is oxygen. The alcohol prepared as~an intermedlate in the previous example is converted to ~he corresponding :~diethy1 phosphonomethyl ether using~:
~ `;WO93~211~7 ~ 1 3 3 3 ~ ~ PCT/VSg3/02~1 ~
:C
diethy1chloromethyl phosphonate. Removal of ~he ethyl groups of the ester is accomplished using trimethylsilylbromide to gi~e the phosp~onic acid.
S~S~P~ 26 In this example a comp~und of the pre ent .invention is made wherein "Y" is NH and "X" is SO~NH2. The nitrile group of the compound of Example 13 is reduced to the amine using standard catalytic hydrogenation wit~ palladium in acidic media (usually l00.0l N to l N HCl), which is then converted to the sulfamide uslng sulphamoyl chloride.
~MPL~ 27 : In this example a compound of the present invention is 15preparad wherein "X" is COOH and "Y" is NH by reacting the methyl amine intermediate prepared in the previous example with chloroacetic acid.
.
~SPIIE 2 8 ;~ 20In this example a compound of the present invention is prepared wherein "Xl' is PO(OH)z and "Y" is NH by reacting the methyl amine intermediate prepared in Example 27 with ~.
diethylchloromethyl phosphonate, and reacti~g the resulting p~od~uct with trimethylsiiylbromide.
~;~ 25~ :
~In this example a compound of the present invention is :~:prepared wherein l'X7' is 5O2NH2 and "Y" is oxygen by reacting the ~ .
NO 93t21~i7 2 1 3 3 3 ~ ~ ~CrlUS93/0~84}
alcohol intermediate prepared in Example 24 with sulphamoyl chloride.
i ' , P I~ 3 0 r ~
In this example a compnund of t~e present invention is prepared wherein R1 is H, R2 is phenyl, R3 and R4 are hydrogen, m is 0, n is 1, p is 0, a~d X is CN~ A modifiGation of the proceduxe disclosed in Mu-Ill Lim, et al., J. Or~. Chem., VOlr 44, No. 22, 3826 (1979) is used. A mixture of the compound of Example S and dimethylformamide dimethyl acetal is reacted at room temperature ~or two days and then evaporated to dryness in vacuo. The residue is crystallized to~ give the Ipure N-(dimethylaminojmethylene deri~ative, which is cyclized with saturated methanolic a~monia tQ give the desired end product.
~ 15 ~ B~P1 31 ~ -, ."~ In this example a compound of thç present in~ention is ~` prepared wherein R1 is 0C~3, R2 is phenyl, R3 and R4 are hydrogen;
m is 0, n is 1, p is 0, and X is CN. Usin~ the compound B of ; 20 Example 8, the S-methyl group is oxidized to methylsulfone, whlch hen is converted to the final methoxy compound ~y treatment with :
sodium methoxid`e in methanol. ~ :
:
F~l~ 3 ~
; ~25 In this example a ~-compound of the pre~ent invention lS
`~ preparéd wherein X is tetrazole. The compound~of Exa~ple 13~is ~ : treated with= lithium:azide in the:presence of ammonium chloride ; : : :
. .~
; ~
t; wo g3,21 ,87 2 ~ 3 3 ~ 4 ~ PCT/US93~2~1 as a catalyst in dimethylformamide ( DMF) at lO0 degrees C to give t~e desired tetrazole.
.,.
In this example a compound of the present invention is prepared wherein X is triazole. The campound of Example l9 is treated with hydrazine hydrate to give the corresponding hydrazide, which is then treated with imino ether to give the desired triazole.
X~PL~_3~
The compound prepared in Example lO is tested $or en2yme inhibition acti~ity as in Example 9. A~ 1 mM phosphate the ICso is 0. 012 ~M and at 50 mM phosphate the ICso is 2 . 0 ~.
E~AMPL~ 35 In this example an amidine compound of the present invention is prepared, i.e., wherein X in the recited generic formula is CNHNH2. The compound A from Example 8 is reacted with sodium methoxide in methanol at room temperature ~or abou~ 2 days to ~:~ give a methyl-imidate intermediate. The intermediate is then reacted with ammonia in methanol to ~iYe the amidine product.
:, ~
` . WO g3/211~7 ~ ~ 3 3 ~ PCT/~S93/02~1 E~NPL~ 36-42 The followlng table gives the formula~ for the compounds made in Examples 36-42 and the IC50 (nM) values obtained for these compounds.
S ~ ~ H
--~R
CHR~
Table No. _ Rl_ R2 - . R~ __ IC~ a Ex~ 36 H2N 3-Chlorophenyl C~2Co2H 7 Ex. 37 H2N 3-Chlorophenyl CH2co2H (S)5-9 Exc 38 H2N 3-Chlorophenyl CH2co2H (R~160 Ex. 39 SMe 3-Chlorophenyl C~2C~ --Ex. 40 H 3 Chlorophenyl CH2CN . 10 Ex~ 41 H2N 3-Chlorophenyl CH2CH20H 25 Ex. 42 H2N 3-Chlorophenyl CH2CO~Me 85 2,6-diamino~3,5-dihydro-7-(2-thienylmethyl)-4H-pyrollo-[3~2-d]pyrimidine-4-one (available from ~arner-Lambert) 160 ~a~PL~ 36 HN
~ H 2N
0!1 ~ Cl '~`' W093/~i87 2 1 3 ~ 3 ~ ù PCT/US93/02~1 The compound prepared in Example lO is hydrolyzed to the cQrxesponding acid of the abo~e formula in this example. A
f ' solution of 3-~3-chlorophenyl)-3-(2-amino-4-oxo-3H,5H-¦ pyrrolo(3,2-d~pyrimidin-~-yl)prop~nenitrile (2.0 g; 63.75 mmol) in 6N HCl (60 ml) is heated at reflux for 8 h. T~e solvent is evaporated in vacuo and the residue is dissol~ed in H2O (18 ml).
The resulting solution is adjusted to pH ~lO by conc. ammonium hydroxide and any i.n~oluble material is removed by filtration.
The filtrate is then readj-~sted to pH ~6.8. The white 10 precipitated material was collected, washed with H2O, and dried to yield 1.8 g of desired compound, m.p. 295-96-C dec, as a dl racemic mixture.
~AMPLE 37 H~N
i ~N~<~
~ 20 C1 .
The above ~ompound, 3-(2-amino-3H,5H 4-oxo-py~rolo[3,2-d]pyrimidin-7-yl1-3-(3-chlorophenyl-N-:. (phenylethyl;)propanamide, is prepared in this example. A
solution of diphenylphosphoryl a~ide (O.72 g, 2.6 mmol) in D~F
(lO ml) is added dropwise during lO min to a mechanically-stirred, cold (-5 to O'C) solution of the compound obtained in ~xample 36 t0.7~0 g; 2.4 mmol) and (R)d-~+)-~-methylbenzylamine ~::
. 093/21187 w~ 3 3 ~ ~ 5 PCT/US93/02~1 (0.32 g, 2.6 mmol) in D~F (lO0 ml). A solution of N-methylmorpholine (0.48 g, 4.7~ mmol) in DMF (5 ml) is then added dropwise during 5-lO min, and the solution is kept near O C for 5 h. It is then allowed to warm to room temperatur-~ and is stirred overnight (18 h). A second portion of diphenylphosphonyl axide (0~36 g), (R)d~ a~mQthylbenzylamine (0.16 g) and N-methylmorpholine (O.24 g) is added at 0C and the reaction mixture is stirred for 2 days. The solvent is removed in vacuo and the residue is dissolved in an 8: 2 mixture of acetonitrile and ammonium hydroxide (lM). The crude produc-t is adsorbed on silica gel and dried in vacuo to remove the last traces of solvent. Flash column chromatographic purification using acetonitrile and lM ammonium hydroxide (95:5) gives the pure desired material as a mixture of diastereomers (yield 0.630 g).
~: 15 These isomers are separated ~y repeated flash calumn chxomatography on silica gel using acetonitrile and lM ammonium ~: hydroxide (98:2) as the eluent to yield 0~18 g of S,R-isomer (Compound A), m.p. 170~75C dec. and 0.120 g of R,R-isome~
(Compound B), m.p. 155-60-C dec.
~NN~ ~NNz ~N--C--HzCJ~ N2C~
` ' Cl .
-2~-~ ` WO93/2~1~7 2 1 3 J 3 ~ s PCT/~S93/0~1 o ~I 2N
S
S-i~omer I The above compound labeled "S-isomer," (S)-3-(2-amino-4-ox o-3H,5H-pyrroloC3,2-d]py rimidin-7-yl) -3-(3 -chlorophenyl)propanoic acid, is prepared in this example. A
10solution of the compound A (S,R~isomer) (0.170 g), obtained above, in 6N HCl (30 ml) is heated at reflux for 6 h and then le~t at room temperature for 6 h. The sol~ent is evaporated in vacuo and the residue is dissolved in H20 (5 ml). T~e resulting solution is adjusted to pH ~10 by conc. ammonium hydroxide~and 15any ins~luble material is removed hy flltration. The filtrate ~:; is then readjusted to pH -6.8 by ammonium hydroxide~ The white precipitated material is collected, washed with H20, and dried to yield 0.0~0 g o~ the crude material which was purified by flash column chromatoyraphy usin~ a 98:2 mixture of acetonitrile~and 20ammonium hydroxide (1 M). Yield 48 mg, m~p. ~ 285~C dec.
: A purine nucleoside phosphorylase (PNP) enzyme assay is performed in which the inhibitory activity (ICso) of the S-; isomer compound is dete ~ ined by measuring the formation of 4C~-hypoxanthi~e from [14C]-inosine ~see Bigmedic~ne, 1980, 33~
253~) using calf spleen PNP in the presence and absence of inhi~itor. At 50 mM phosphate the IC50 is 0.031 ~M and at 1 mM
~`~phosphate, it is 0.0059 ~.
: :
` :~
:
` :~
~::
3 '~ ~
~093/~1187 PCT/US93/~2~1 E~l~ 38 o HN
_ H2N ~
H
ao~c~2c i R-i~om~r Cl The procedure described in Example 37 is repeated to prepare 10 theabove compound, (R)-3-~2-amino~4~oxo-3H,5H-pyrrolo~3,2-d]pyrimidin 7-yl)-3-(3-chlorophenyl)propanoic acid from Compound B (R,R-isomer), obtained in Example 37. Yield 40%~
m.p. > 280 C dec. The compound prepared in Example 38 is tested for enzyme inhibiti~n activity as in Example 37. At 50 mM
15 ph~sphate the IC50 is 0.900 ~M and at 1 mM phosphate the IC~o is 0.160 ~M. Thus the S-isomer (Example 383 .is ca. 30X as potent as the R-isomer in the inhibition of PNP~ X ray crystalloyraphic analysis of the enzyme-inhibitor complex ~ormed from a soak of a crystal of the enzyme in a solution containing the unresolved racemic mlxture (Example 36) showed that the S-isomer excluslvely bound to the active site of the enzyme.
~3~Ph~ 3 9 The procedures of Examples 1 8 are followed, except that the starting material used is the 3-chlorophenyl deriYative rather than the 2,3,5-trichlorophenyl derivative used in the pre~iou5 Examples. The SMe derivative as shown in the Table is obtaine~.
.
~ 093/21~87 2 1 ~ 3 3 ~1 5 PCT/US93/0~l ~PLE 4~
The compound from Example 39 (1 g) in ethanol~(loO ml) is suspended in in 30% palladium on carbon (1 g) and subjected to reflux for a few minutes. Hydrazine hydrate (0.3 ml)-~s added with stirrlng an the mixture refluxed for two days~ Additional hydrazine hydrate (O.3 ml) and palladium on carbon (O.5 g) are added and the mixture refluxed for an additional four days. The catalyst is remo~ed by filtration, and the filtrate reduced to 25 ml and filtered vn Whatman filter paper and evaporated to give the final product.
An altern~tive way of making the final product begins by using the present Examples 1-5 except t~at the 3-chlorophenyl derivative i5 used as the starting material. The resulting material, 3-amino-4-~(3-chlorophenyl)methyl]methylester-1-H-pyrolle-2-carboxylic acid, ~5 g) is disolved in dimethylformamide dimethylacetal (50 ml) under ar~on and heated for 24 hours at 60 70 C. After e~aporation to dryness, the material is disol~ed in dichloromethane (50 ml), filtered and diluted with patroleum et~er until cloudy, triturated to induce crystallization, and slowly diluted with and additional 40 ml of patroleum ether.
This mono-chloro intermediate is collected, washed with patroleum eth~r and dried~ Yield 5 g (88%)~ mp 122-124 C. The resulting : intermedi~te is heat~d in methanolic ammonia at 95-100 ~C for 24 hour~ in a s~ainles5 steel bomb, evaporated to a yellowish solid crude product. The yellowish solid crude product (3 ~) in 175 ml ho~ methano~ yiel~s a final pro~uct of 2.2 g (8B% yield~.
21 333~
, .
W093/2~1~7 PCT/US93/~2~1 E~Ph~ 41 T~e compound of Example 10 (6.80 g) in 6N HCl (400 ml) is re~luxed for 10 h, cooled overnight, and evaporated under reduced ¦ pressure. The residue is added to methanol and evaporated and then added to toluene, which results in a white foam in nearly quantitative yield. A solution of the dried white foam in an~ydrous methanol (400 ml) is cooled below O C in an ice salt bath under dry conditionc. Thienyl chloride tlO.31 g) is added 510wly dropwisa, and the solution allowed to come to ambient lQ temperature and stand overnight. The solvent is evaporated in vacuo, ~resh methanol and toluene are added and then evaporated to aid in the removal of acid vapors. A suspension of the solid in cold water (200 ml) is neutralized in lN NaOH and the solid is collected by filtration, washed ~ith cold water, and dried in vacuo o~er P205 at llO-C. Yield 6.08 g (81% from the material of Example 10)~ Thi5 product is of sufficient purity for use in.the n~ixt step, but may recrystallize in methanol using Soxhlet apparatus to fine white crystals having a m.p. o~ 302-303C
(dècompose~. An amount of 6g of the product from the pre~ious paraqraph with 100 mg dry a~monium sulfate in h~xamethyldisilazane (400 ml) is refluxed for 8 h under dry condi~ions. The resulting clear solu~ion is evaporated in ~acuo to a viscous gum that is further dried over P20~, which is used in the next Example without further treatment.
~5 ,~M~ 42 Under nitroqen, a s~lution of the product from the preivious paragraph in anhydrous THF or ether (200 ml) is treated dropwise : -3~-' ; wo g3/211g7 213 3 3 ~1 a PCT/US93~02~1 with a 1 molar solution of lithium aluminum hydride (26 ml) in T~F. After 1 h at room temperature, excess hydride is des~royed by dropwise addition of ethyl acetate (50 ml), and the solvent evaporated in vacuo. The residue is suspended in cold water (200 ml), adjusted to a pH of 1 with HCl, stirred for 15 min, adjusted to a pH 7 with dilute sodium hydroxide, and filtered r The resulting filter cake is wa~hed with cold water, dried, and washed with ethyl to remove TMS by-products. Silica gel (50 g) is added to a hot solution of the resulting crude solid (~ 8 g) in a large volume of methanol, and tAe resulting slur~y is evaporated to dryness. The resulting material is layered carefully onto a silica gel column that is eluted with a chloroform~methanol mixture (85/15) to give the desired alcohol final product. Yield 4.65 g (84%). Two recrystallizations from ethanol/water gives a white crystalline materiai. Yield 3036 g (61~), m.p. 2S5-277~C (decompose).
Claims (37)
1. A PNP inhibitor comprising a compound of the formula wherein R1 is H, NH2, or OCH3, R2 is an optionally substituted cyclic group optionally containing one or more heteroatoms, R3 and R are independently H or C1-4 alkyl, m is 0 4, n is 0-6, p is 0-1, X is CN, CSNH2, PO(OH)2, COOH, SO2NH2, NH2, OH, CNHNH2, tetrazole, or triazole, COR5 where R5 is C1-4 alkyl, CF3, NH2, or OC1-4 alkyl, and Y is O or NH.
2. The inhibitor of claim 1 wherein R2 is unsubstituted.
3. The inhibitor of claim 2 wherein R1 is NH2, R3 and R4 are H, m is 0 and n is 1.
4. The inhibitor of claim 3 wherein R2 is phenyl.
5. The inhibitor of claim 4 wherein X is CN.
6. The inhibitor of claim 4 wherein X is COOH.
7. The inhibitor of claim 4 wherein X is CONH2.
8. The inhibitor of claim 3 wherein R2 is 2- or 3-thienyl, 2-or 3-furanyl, 2-, 3-, or 4-pyridinyl, 2- or 3-pyrrolyl, 2-, 4-, or 5-thiazolyl, 2- or 3-pyrazinyl, 3- or 4-pyridazinyl, or pyrazolyl.
9. The inhibitor of claim 8 wherein X is CN, COOH, or CONH2.
10. The inhibitor of claim 3 wherein R2 is 1- or 2-adamantyl, cyclopentyl, 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-thizolidinyl, 2- or 3-piperazinyl, 2- or 3-morpholinyl, or hexahydropyridazinyl.
11. The inhibitor of claim 10 wherein X is CN, COOH, or CONH2.
12. The inhibitor of claim 3 wherein RZ is cyclohexyl and X
is COOH.
is COOH.
13. The inhibitor of claim 1 wherein R2 is an optionally substituted 5- or 6-membered aromatic or heteroaromatic group.
14. The inhibitor of claim 1 wherein R2 is an optionally substituted alicyclic group or heteroalicyclic group of 5-9 members.
15. The inhibitor of claim 1 wherein R2 is substituted with at least one of halogen, hydroxy, C1-4 alkoxy, C1-4 alkyl, or trifluoromethyl.
16. A method for the selective suppression of mammalian T-cell function without diminished effect on humoral immunity comprising administering to a subject an effective amount of the PNP inhibitor of claim 1.
17. A method for the selective suppression of mammalian T-cell function without diminished effect on humoral immunity comprising administering to a subject an effective amount of the PNP inhibitor of claim 5.
18. A method for the selective suppression of mammalian T-cell function without diminished effect on humoral immunity comprising administering to a subject an effective amount of the PNP inhibitor of claim 6.
19. A method for the selective suppression of mammalian T-cell function without diminished effect on humoral immunity comprising administering to a subject an effective amount of the PNP inhibitor of claim 7.
20. A method for the selective suppression of mammalian T-cell function without diminished effect on humoral immunity comprising administering to a subject an effective amount of the PNP inhibitor of claim 8.
21. A method for the selective suppression of mammalian T-cell function without diminished effect on humoral immunity comprising administering to a subject an effective amount of the PNP inhibitor of claim 10.
22. A method for the selective suppression of mammalian T-cell function without diminished effect on humoral immunity comprising administering to a subject an effective amount of the PNP inhibitor of claim 12.
23. A pharmaceutical composition for the selective suppression of mammalian T-cell function without diminished effect on humoral immunity comprising an effective amount of the PNP inhibitor of claim 1 and a pharmaceutically acceptable carrier or diluent.
24. A pharmaceutical composition for the selective suppression of mammalian T-cell function without diminished effect on humoral immunity comprising an effective amount of the PNP inhibitor of claim 5 and a pharmaceutically acceptable carrier or diluent.
25. A pharmaceutical composition for the selective suppression of mammalian T-cell function without diminished effect on humoral immunity comprising an effective amount of the PNP inhibitor of claim 6 and a pharmaceutically acceptable carrier or diluent.
26. A pharmaceutical composition for the selective suppression of mammalian T-cell function without diminished effect on humoral immunity comprising an effective amount of the PNP inhibitor of claim 7 and a pharmaceutically acceptable carrier or diluent.
27. A pharmaceutical composition for the selective suppression of mammalian T-cell function without diminished effect on humoral immunity comprising an effective amount of the PNP inhibitor of claim 8 and a pharmaceutically acceptable carrier or diluent.
28. A pharmaceutical composition for the selective suppression of mammalian T-cell function without diminished effect on humoral immunity comprising an effective amount of the PNP inhibitor of claim 10 and a pharmaceutically acceptable carrier or diluent.
29. A pharmaceutical composition for the selective suppression of mammalian T-cell function without diminished effect on humoral immunity comprising an effective amount of the PNP inhibitor of claim 12 and a pharmaceutically acceptable carrier or diluent.
30. A method for making a chemical compound comprising the steps of:
a) reacting an optionally substituted cyclic aldehyde with cyanoacetic acid in the presence of ammonium acetate to make a 3-cyclo-substituted pentanedinitrile;
b) reacting the 3-cyclo-pentanedinitrile with an alkyl formate and a base to make a 3-cyclo-2-formylpentanedinitrile;
c) reacting the 3-cyclo-2-formylpentanedinitrile with glycine methyl ester hydrochloride and sodium or ammonium acetate to make methyl N-(3-cyclo-2,4-dicyano)-2-butenyl]glycine;
d) reacting the methyl N-(3-cyclo-2,4-dicyano)-2-butenyl]glycine with an alkyl chloroformate and DBN or DBU to make methyl 3-amino-4-(2-cyano-1-cyclo-ethyl)-1-ethyl-1H-pyrrole-1,2-dicarboxylate; and e) reacting the methyl 3-amino-4-(2-cyano-1-cyclo-ethyl)-1-ethyl-1H-pyrrole-1,2-dicarboxylate with a base to make methyl 3-amino-4-(2-cyano-1-cyclo-ethyl)-1H-pyrrole-2-carboxylate.
a) reacting an optionally substituted cyclic aldehyde with cyanoacetic acid in the presence of ammonium acetate to make a 3-cyclo-substituted pentanedinitrile;
b) reacting the 3-cyclo-pentanedinitrile with an alkyl formate and a base to make a 3-cyclo-2-formylpentanedinitrile;
c) reacting the 3-cyclo-2-formylpentanedinitrile with glycine methyl ester hydrochloride and sodium or ammonium acetate to make methyl N-(3-cyclo-2,4-dicyano)-2-butenyl]glycine;
d) reacting the methyl N-(3-cyclo-2,4-dicyano)-2-butenyl]glycine with an alkyl chloroformate and DBN or DBU to make methyl 3-amino-4-(2-cyano-1-cyclo-ethyl)-1-ethyl-1H-pyrrole-1,2-dicarboxylate; and e) reacting the methyl 3-amino-4-(2-cyano-1-cyclo-ethyl)-1-ethyl-1H-pyrrole-1,2-dicarboxylate with a base to make methyl 3-amino-4-(2-cyano-1-cyclo-ethyl)-1H-pyrrole-2-carboxylate.
31. The method of claim 30 further comprising the steps of:
f) reacting the methyl 3-amino-4-(2-cyano-1-cyclo-ethyl)-1H-pyrrole-2-carboxylate with benzoylisothioyanate to make N-benzoyl-N'-[4-(2-cyano-1-cyclo-ethyl)-2-methoxycarbonyl-1H-pyrrol-3-yl]thiourea;
g) reacting N-benzoyl-N'-[4-(2-cyano-1-cyclo-ethyl)-2-methoxycarbonyl-1H-pyrrol-3-yl]thiorea with an alkyl halide to make N-benzoyl-N'-[4-(2-cyano-1-cyclo-ethyl)-2 methoxycarbonyl-1H-pyrrol-3-yl]S-methylthiourea; and h) reacting N-benzoyl-N'-[4-(2-cyano-1-cyclo-ethyl)-2 methoxycarbonyl-1H-pyrrol-3-yl]-S-methylthiourea with methanolic or ethanolic ammonia to make a mixture of 3-cyclo-3-[2-amino-4-oxo-3H-5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile and 3-cyclo-3-[2-methylmercapto-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile.
f) reacting the methyl 3-amino-4-(2-cyano-1-cyclo-ethyl)-1H-pyrrole-2-carboxylate with benzoylisothioyanate to make N-benzoyl-N'-[4-(2-cyano-1-cyclo-ethyl)-2-methoxycarbonyl-1H-pyrrol-3-yl]thiourea;
g) reacting N-benzoyl-N'-[4-(2-cyano-1-cyclo-ethyl)-2-methoxycarbonyl-1H-pyrrol-3-yl]thiorea with an alkyl halide to make N-benzoyl-N'-[4-(2-cyano-1-cyclo-ethyl)-2 methoxycarbonyl-1H-pyrrol-3-yl]S-methylthiourea; and h) reacting N-benzoyl-N'-[4-(2-cyano-1-cyclo-ethyl)-2 methoxycarbonyl-1H-pyrrol-3-yl]-S-methylthiourea with methanolic or ethanolic ammonia to make a mixture of 3-cyclo-3-[2-amino-4-oxo-3H-5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile and 3-cyclo-3-[2-methylmercapto-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile.
32. The method of claim 31 further comprising the steps of:
i) reacting the 3-cyclo-3-[2-methylmercapto-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile with an oxidizing agent to make 3-cyclo-3-[2-methylsulfonyl-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile; and j) reacting the 3-cyclo-3-[2-methylsulfonyl-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile with sodium alkoxide to make 3-cyclo-3[2-methoxy-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile.
i) reacting the 3-cyclo-3-[2-methylmercapto-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile with an oxidizing agent to make 3-cyclo-3-[2-methylsulfonyl-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile; and j) reacting the 3-cyclo-3-[2-methylsulfonyl-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile with sodium alkoxide to make 3-cyclo-3[2-methoxy-4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile.
33. The method of claim 30 further comprising the steps of:
f) reacting the methyl 3-amino-4-(2-cyano-1-cyclo-ethyl)-1H-pyrrole-2-carboxylate with dimethylformamide dimethyl acetal to make methyl 4-(2-cyano-1-cyclo-ethyl)-3-[N
(dimethylaminomethylene)amino]-1H-pyrrole-2-carboxylate; and g) reacting the methyl 4-(2-cyano-1-cyclo-ethyl)-3-[N-(dimethylaminomethylene)amino]-1H-pyrrole-2-carboxylate with methanolic ammonia to make 3-cyclo-3-[4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile.
f) reacting the methyl 3-amino-4-(2-cyano-1-cyclo-ethyl)-1H-pyrrole-2-carboxylate with dimethylformamide dimethyl acetal to make methyl 4-(2-cyano-1-cyclo-ethyl)-3-[N
(dimethylaminomethylene)amino]-1H-pyrrole-2-carboxylate; and g) reacting the methyl 4-(2-cyano-1-cyclo-ethyl)-3-[N-(dimethylaminomethylene)amino]-1H-pyrrole-2-carboxylate with methanolic ammonia to make 3-cyclo-3-[4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile.
34. The method of claim 30 wherein the cyclic substituent is phenyl.
35. The method of claim 30 wherein the cyclic substituent is 2- or 3-thienyl, 2- or 3-furanyl, 2-, 3-, or 4-pyridinyl, 2- or 3-pyrrolyl, 2-, 4-, or 5-thiazolyl, 2-or 3-pyrazinyl, 3- or 4 pyridazinyl, or 3-, 4-, or 5-pyrazolyl.
36. The method of claim 30 wherein the cyclic substituent is 1- or 2- adamantyl, cyclopentyl, cyclohexyl, cycloheptyl, and morpholinyl.
37. A compound of the formula or wherein R1 and R2 are as defined claim 1.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US87144192A | 1992-04-21 | 1992-04-21 | |
| US07/871,441 | 1992-04-21 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2133346A1 true CA2133346A1 (en) | 1993-10-28 |
Family
ID=25357439
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002133346A Abandoned CA2133346A1 (en) | 1992-04-21 | 1993-03-25 | 7-disubstituted-methyl-4-oxo-3h,5h-pyrrolo[3,2-d]pyrimidine and pharmaceutical uses and compositions containing the same |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP0638080A4 (en) |
| JP (1) | JPH07507062A (en) |
| KR (1) | KR950701335A (en) |
| AU (1) | AU4276693A (en) |
| CA (1) | CA2133346A1 (en) |
| FI (1) | FI944933A (en) |
| HU (1) | HUT71559A (en) |
| NO (1) | NO303637B1 (en) |
| RU (1) | RU94046387A (en) |
| WO (1) | WO1993021187A1 (en) |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU8210798A (en) * | 1997-05-29 | 1998-12-30 | Novartis Ag | 2-amino-7-(1-substituted-2-hydroxyethyl)-3,5-dihydro-pyrrolo (3,2-d)pyrimidin-4-ones |
| US6355244B1 (en) | 1997-11-17 | 2002-03-12 | University Of Kentucky Research Foundation | Methods and compositions for the treatment of psoriasis |
| US6174888B1 (en) | 1998-05-28 | 2001-01-16 | Novartis Ag | 2-amino-7-(1-substituted-2-hydroxyethyl)-3,5-dihydropyrrolo[3,2-D]pyrimidin-4-ones |
| GB0215392D0 (en) * | 2002-07-03 | 2002-08-14 | Glaxo Group Ltd | Chemical compounds |
| US7557113B2 (en) | 2003-08-26 | 2009-07-07 | Teijin Pharma Limited | Substituted pyrrolo[3,2-d]pyrimidine derivatives |
| US20050153992A1 (en) * | 2003-08-26 | 2005-07-14 | Teijin Pharma Limited | Pyrrolopyrimidine thion derivatives |
| MY140748A (en) | 2004-12-06 | 2010-01-15 | Astrazeneca Ab | Novel pyrrolo [3,2-d] pyrimidin-4-one derivatives and their use in therapy |
| US7763257B2 (en) | 2004-12-09 | 2010-07-27 | Christina Juneau | Compositions comprising transforming growth factor (TGF)-β1 and TGF-β2 in admixture of proteins obtained from dairy products |
| US8138191B2 (en) | 2007-01-11 | 2012-03-20 | Critical Outcome Technologies Inc. | Inhibitor compounds and cancer treatment methods |
| EP2225226B1 (en) | 2007-12-26 | 2016-08-17 | Critical Outcome Technologies, Inc. | Compounds and their use in a method for treatment of cancer |
| CA2999435A1 (en) | 2010-04-01 | 2011-10-06 | Critical Outcome Technologies Inc. | Compounds and method for treatment of hiv |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| HUT61765A (en) * | 1989-10-31 | 1993-03-01 | Biocryst Inc | Process for producing purine nucleoside phosphorylase inhibitors and pharmaceutical compositions comprising same as active ingredient |
| US5189039A (en) * | 1989-11-29 | 1993-02-23 | Biocryst, Inc. | 7-disubstituted-methyl-4-oxo-3H,5H-pyrrolo[3,2d]pyrimidine and pharmaceutical uses and compositions containing the same |
-
1993
- 1993-03-25 CA CA002133346A patent/CA2133346A1/en not_active Abandoned
- 1993-03-25 WO PCT/US1993/002841 patent/WO1993021187A1/en not_active Application Discontinuation
- 1993-03-25 EP EP93912081A patent/EP0638080A4/en not_active Withdrawn
- 1993-03-25 RU RU94046387/04A patent/RU94046387A/en unknown
- 1993-03-25 HU HU9403021A patent/HUT71559A/en unknown
- 1993-03-25 JP JP5518357A patent/JPH07507062A/en active Pending
- 1993-03-25 KR KR1019940703788A patent/KR950701335A/en not_active Application Discontinuation
- 1993-03-25 AU AU42766/93A patent/AU4276693A/en not_active Abandoned
-
1994
- 1994-10-20 NO NO943988A patent/NO303637B1/en unknown
- 1994-10-20 FI FI944933A patent/FI944933A/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| AU4276693A (en) | 1993-11-18 |
| FI944933A0 (en) | 1994-10-20 |
| KR950701335A (en) | 1995-03-23 |
| RU94046387A (en) | 1996-09-27 |
| HU9403021D0 (en) | 1994-12-28 |
| NO303637B1 (en) | 1998-08-10 |
| JPH07507062A (en) | 1995-08-03 |
| NO943988L (en) | 1994-10-20 |
| FI944933A (en) | 1994-10-20 |
| EP0638080A4 (en) | 1995-08-23 |
| EP0638080A1 (en) | 1995-02-15 |
| NO943988D0 (en) | 1994-10-20 |
| HUT71559A (en) | 1995-12-28 |
| WO1993021187A1 (en) | 1993-10-28 |
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