LV10100B - Inhibitors of purine nucleoside phosphorylase - Google Patents

Inhibitors of purine nucleoside phosphorylase Download PDF

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LV10100B
LV10100B LVP-93-342A LV930342A LV10100B LV 10100 B LV10100 B LV 10100B LV 930342 A LV930342 A LV 930342A LV 10100 B LV10100 B LV 10100B
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compound
cyclo
ethyl
substituted
amino
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LVP-93-342A
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LV10100A (en
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Secrist John A Iii
Erion Mark David
John A Montgomery
Steven E Ealick
Wayne C Guida
Niwas Shri
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Biocryst Pharm Inc
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Priority claimed from US07/429,097 external-priority patent/US5008270A/en
Priority claimed from US07/429,099 external-priority patent/US5008265A/en
Priority claimed from US07/429,098 external-priority patent/US4985434A/en
Priority claimed from US07/429,100 external-priority patent/US4985433A/en
Application filed by Biocryst Pharm Inc filed Critical Biocryst Pharm Inc
Publication of LV10100A publication Critical patent/LV10100A/en
Publication of LV10100B publication Critical patent/LV10100B/en

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

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  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Virology (AREA)
  • Communicable Diseases (AREA)
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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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Description

1 LV 10100
INHIBITORS OF PURINE NUCLEOSIDE PHOSPHORYLASE
The present invention relates to derivatives of 2-amino-3H,5H-pyrrolo[3,2-d]pyrimidin-4-one and to derivatives of 4-oxo-3H,5H-pyrrolo[3i2-d]pyrimidine. It also relates to 5 4-ΟΧΟ-3Η, 5H-pyrrolo [ 3,2-d.]pyrimidine derivatives substituted at the 7-position.
Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of purine nucleosides in a reversible reaction. Individuāls who are deficient in PNP exhibit 10 impaired T-cell development, resulting in lovered cell-mediated immunity, but normai B-cell development, resulting in normai humoral immunity. Accordingly, specific inhibitors of PNP that selectively inhibit T-cell development vithout damaging humoral immunity could be 15 potentially effective against disorders in vhich activated T-cells are pathogenic.
As a PNP inhibitor, the present invention provides a 2-amino-7-(R)-3H, 5H-pyrrolo[3,2-^]pyrimidin-4-one vherein R is optionally substituted cyclohexenyl, cyclohexyl, or 20 -CH2”Rit vherein is an optionally substituted heteroalicyclic, pyridinyl or alicyclic group.
The present invention is also directed to a compound of the formula
25 30 vherein R1 is H, NH2, or OCH3, R2 is an optionally 35 substituted cyclic group of 5-7 carbon atoms optionally 2 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 ΟΙ, X is CN, CSNH2/ PO(OH)2, cooh, so2nh2, nh2, oh, cnhnh2, tetrazole, triazole or COR3 where R5 is 0^.4 alkyl, CF3, 5 NH2, or OCļ__4 alkyl, and Y is O or NH. The compound of the present invention is useful as a PNP inhibitor. Also contemplated according to the present invention is a pharmaceutical composition for the selective suppression of mammalian T-cell immunity comprising an pharmaceutically 10 effective amount of the compound of the present invention and a pharmaceutically acceptable carrier or diluent and a method for the selective suppression of mammalian T-cell immunity vithout diminished effect on humoral immunity comprising administering to a subject a pharmaceutically 15 effective amount of the compound of the present invention.
In one aspect of the invention there is provided a . compound 2-amino-7-(R)-3H#5H-pyrrolo[3,2-d3-pyrimidin-4-one (I) vherein R is -ch2-Ri 20 and R^ is an optionally substituted heteroalicyclic group. Preferably, the heteroalicyclic group is a 5 or 6 membered saturated ring having oxygen, nitrogen, or sulfur as the heterocyclic atom. More preferably the heteroalicyclic . group is 2- or 3-tetrahydrothienyl, 2-, 3-, or 4-25 piperidinyl, 2- or 3-tetrahydrofuranyl, 2-, or 3-pyrrolidinyl, or 2-, 3-, or 4-tetrahydropyranyl. In a preferred aspect, Rļ is unsubstituted, e.g., the compound (I) is 2-amino-7-(2-piperidinylmethyl)-3H,5H-pyrrolo[-3,2-d]pyrimidin-4—one (IB), 2-amino-7-(3-piperidinyl- 30 methyl)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-one (IC) , 2-amino-7 - (4 -piperidinylmethyl) -3 H, 5H-pyrrolo [3,2 -d ] pyrimidin-4 -one (ID), 2-amino-7-(2-tetrahydrofuranylmethyl)-3H,5H-pyr-rolo[3,2-d]pyrimidin-4-one (IE), 2-amino-7-(3-tetrahy-drofuranylmethyl)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-one (IF) , 3 LV 10100 2 - amino-7 - (2-tetrahydrothienylmethyl) - 32, 5H-pyrrolo(-3,2-d]pyrimidin-4-one (IG), 2-amino-7-(3-tetrahydrothieny-lmethyl)-32,5g-pyrrolo[3,2-2]pyrimidin-4-one (IH), 2-amino-7 - (2-pyrrolidinylmethyl)-32, 5g-pyrrolo[3,2-d)pyrimidin-4-one (II), 2-amino-7-(3-pyrrolidinylmethyl)-3H,5H-pyr-rolo[3,2-d]pyrimidin-4-one (IJ), 2-amino-7-(2-tetrahydro-pyranylmethyl)-32,5H-pyrrolo[3,2-d]pyrimidin-4-one (IL), 2- amino-7-(3-tetrahydropyranylmethyl) -32,5H“,pyrrolo( 3,2-d]py-rimidin-4-one (IM) , or 2-amino-7-(4-tetrahydropyranyl-Taethyl)-32,52“Pyrrolo[3,2-d]pyrimidin-4-one (IN). In an altemative preferred embodiment the R^ has one or two substituents selected from the group consisting of halogen, hydroxy, alkoxy, alkyl, or trifluoromethyl. As halogen is preferably mentioned chloro or fluoro. As alkoxy is preferably mentioned lover alkoxy, including methoxy, ethoxy, propoxy and butoxy. As alkyl is preferably mentioned lover alkyl, including methyl, ethyl, propyl and butyl.
In another aspect of the invention there is provided a compound 2-amino-7-(R)-3g,5H-pyrrolo[3,2-d]-pyrimidin-4-one (II) vherein R is -CH2-Ri and R]_ is optionally substituted pyridinyl. In a preferred aspect, Rļ is unsubstituted, i.e., the compound (II) is 2-amino-7- (3 -pyridinylmethyl) -32,5g-pyrrolo [ 3,2-d]pyrimidin-4-one (IIA), 2-amino-7-(2-pyridinylmethyl)-32,5H-pyrrolo[3,2-d]-pyrimidin-4-one (IIB), or .2-amino-7-(4-pyridinylmethyl) -32, 52~pyrrolo[3,2-d]-pyrimidin-4-one (IIC). In an altemative preferred embodiment the Rji group has one or tvo substituents selected from the group consisting of halogen, hydroxy, alkoxy, alkyl, or trifluoromethyl. As halogen is preferably mentioned chloro or fluoro. As alkoxy is preferably mentioned lover alkoxy, including methoxy, ethoxy, propoxy and butoxy. As alkyl is 4 preferably mentioned lover alkyl, including methyl, ethyl, propyl and butyl.
In another aspect of the invention there is provided a compound (III) 2-amino-7-(R)-3H,5H-pyrrolo[3,2-d]-pyrimidin-4-one vherein the R group is unsubstituted or substituted 12-, or 3-cyclohexenyl or cyclohexyl. In a preferred aspect, R is unsubstituted, i.e., the compound (III) is 2-amino-7-(l-cyclohexenyl)-3H,5H-pyrrolo[3,2-d]- pyrimidin-4-one (IIIA), 2-amino-7-(2-cyclohexenyl)-3g,5H*" pyrrolo(3,2-d ] pyrimidin-4-one (IIIB) , 2-amino-7-(3- cyclohexenyl)-3H/5H-pyrrolo(3,2-d]-pyrimidin-4-one (IIIC), or 2-amino-7-(cyclohexyl)-3H,52-pyrrolo(3,2-d]-pyrimidin-4-one (IIID). In an altemative preferred embodiment the R has at least one substituent selected from the group onsisting of halogen, hydroxy, alkoxy, alkyl, or .-rifluoromethyl. As halogen is preferably mentioned chloro or fluoro. As alkoxy is preferably lover alkoxy, including methoxy/ ethoxy, propoxy and butoxy. As alkyl is preferably mentioned lover alkyl, including methyl, ethyl, propyl and butyl.
In another aspect of the invention there is provided a compound 2-amino-7-(R)-3H,5H-pyrrolo[3,2-d]-pyrimidin-4-ono (IV) vherein R is -CH2-Ri and Rj_ is an optionally substituted alicyclic group. Preferable alicyclic groups include, e.g., single-ring cycloparafins such as cyclopentyl, cyclohexyl, and cycloheptyl, multi-ring cycloparafins such as l- and 2-adamantyl, l-norbornanyl, 2-exo-norbornanyl, 2-endo-norbornanyl, 1- and 2-bicyclo[2.2.2]-octanyl, 1-, 2-, 3-, 6-, and 8-bicyclo(3.2.1] octanyl, and 1-, 2-, and 3- bicyclo[3.3.l]nonanyl and cycloolefins such as 1- and 2-norbomenyl. Examples of the preferred compound (IV) are 2-amino-7-(2-adamantylmethyl)-3H,5H-pyrrolo(3,2-d]- 5 LV 10100 pyrimidin-4-one (IVA), 2-anino-7-(l-adamantylmethyl)-3lļ#5H“pyrrolo[3f2-d3pyrinidin-4-one · (IVB), 2-amino-7-(cyclopentylmethyl) -3&, 5fi”pyrrolo[ 3,2-d]-pyrimidin-4-one (IVC), 2-amino-7-(cyclohexylmethyl)-3H,5H-pyrrolo[3,2-d]-5 pyrimidin-4-one (IVD), 2-amino-7-(cycloheptylmethyl)-32,52“* pyrrolo[3,2-d]pyrimidin-4-one (IVE), 2-amino-7-(1-norbornanylmethyl)-32*5H-pyrrolo[3,2-d]pyrimidin-4-one (IVF) , 2-amino-7-(2-exo-norbornanylmethyl)-32/52-pyrrolo[3,2-d]pyrimidin-4-one (IVG), 2-amino-7-(2-endo-10 norbornanylmethyl)-3Ū,52-pyrrolo(3,2-d]pyrimidin-4-ane (IVH) , 2-amino-7-(l-norbornenylmethyl)-32/5H-pyrrolo(3,2-d]-pyrimidin-4-one (ĪVI), 2-amino-7-(2-norbornenylmethyl)-32/ 52-pyrrolo[3,2-d]pyrimidin-4-one (IVJ) , 2-amino-7-(l-bicyclo[2.2.2]-octanylmethyl)-3H/5H·" 15 pyrrolo[3,2-d]-pyrimidin-4-one (IVK), 2-amino-7-(l-bicyclo-[3.2.1]octanyl- methyl)-32* 52-pyrrolo[3,2-d]pyrimidin-4-one (IVL), and 2-amino-7-(l-bicyclo[3.3.1]nonanylmethyl)-32/52** pyrrolo[3/2-d]-pyrimidin-4-one (IVH), and 2-amino-7-(l-noradamantyl-methyl)-32/ 52“*pyrrolo[3,2-d]-pyrimidin-4-one 20 (IVN). In an altemative preferred embodiment the R^ group has one or two substituents selected from the group consisting of halogen, hydroxy, "alkoxy, alkyl, or trifluoromethyl. As halogen is preferably mentioned chloro or fluoro. As alkoxy is preferably nentioned lower alkoxy, 25 including methoxy, ethoxy, propoxy and butoxy. As alkyl is preferably nentioned lover alkyl, including methyl, ethyl, propyl and butyl.
The present invention is also directed to a compound of the formula 30 6 vherein R1 is Η, NH2, or OCH3, R2 is an optionally substituted cyclic group optionally containing one or more heteroatoms, R3 and R4 are independently H or C^_4 alkyl, m is 0-4, n is 0-6, p is 0-1, X is CN, CSNH2, PO(OH)2, COOH, 5 S02NH2, NH2, OH, CNHNH2, tetrazole, triazole or COR5 where R5 is C31.4 alkyl, CF3, NH2, or OC^ alkyl, and Y is 0 or NH.
The optionally substituted cyclic group (hereinafter referred to as cyclo) recited for the above formula 10 includes aromatic, heteroaromatic, alicyclic,_ and heteroalicyclic groups preferably containing five to nine atoms. Preferred optional substituents include halogen, hydroxy, alkoxy, alkyl, and trifluoromethyl. Exemplary substituents include chloro, fluoro, methoxy, ethoxy, 15 propoxy, butoxy, methyl, ethyl, propyl, and butyl. Preferred heteroatoms include oxygen, nitrogen, and sulfur, which can be present in combination in the same group. The preferred aromatic and heteroaromatic groups are phenyl, 2-or 3-thienyl, 2- or 3-furanyl, 2-, 3-, or 4-pyridinyl, 2-20 or 3-pyrrolyl, 2-, 4-, or 5-thiazolyl, 2-pyrazinyl, 3- or 4- pyridazinyl, and 3-, 4-, or 5-pyrazolyl. The preferred alicyclic and heteroalicyclic groups are 1- or 2-adamantyl, cyclohexyl, cycloheptyl, 2- or 3-tetrahydrofuranyl, 2- or 3-tetrahydrothienyl, 2- or 3-tetrahydropyranyl, 2-, 3-, or 25 4-piperidinyl, 3- or 4-pyrazolidinyl, 2-, 4-, or 5- thiazolidinyl, 2- or 3-piperazinyl, 2- or 3-morpholinyl, or 3- or 4-hexahydropyridazinyl. * In another aspect of the invention there is provided a method for the selective suppression of mammalian T-cell 30 function vithout diminished effect on humoral immunitv which comprises administering to a mammai the compound (I), whereby said compound inhibits purine nucleoside phosphorylase and thereby T-cell formation. 7 LV 10100
In a further aspect of the present invention there is provided a pharmaceutical composition for the selective suppression of mammalian T-cell function without diminished effect on humoral immunity vhich comprises an effective 5 amount of the compound and a pharmaceutically acceptable diluent therefor.
The invention further relates to pharmaceutical compositions suitable for enteral, such as oral or rectal, transdermal and parenteral administration to mammals 10 including man, vhich are useful to · inhibit _purine nucleoside phosphorylase activity and for the treatment of disorders responsive thereto, comprising an effective amount of a pharmacologically active compound of the invention, alone or in combination, with one or more 15 pharmaceutically acceptable carriers.
Preferred pharmaceutical compositions· are tablets and gelatin capsules comprising the active ingredient together vith a) diluents, e.g., lactose, dextrose, sucrose, • mannitol, sorbitol, cellulose and/or glycine; b) 20 lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone; if 25 desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures? and/or e) absorbents, colorants, flavors and s.veeteners. Injectable compositions are preferably agueous isotonic • Solutions or suspensions, and suppos itories are 30 advantageously prepared from fatty emulsions or suspensions. Said compositions raay be sterilized and/or contain adjuvants, such as preserving, stabilizing, vetting or emulsifying aģents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In 8 addition, they may also contain other therapeutically valuable substances. Said compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient.
Suitable formulations for transdermal application include an effective amount of a compound of the invention vith a carrier. Advantageous carriers include absorbable pharmacologically acceptable solvents to assist passage f \ through the skin of the host. Characteristically, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally vith carriers, optionally a rāte controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rāte over a prolonged period of time, and means to secure the device to the skin.
The present invention provides a method of inhibiting purine nucleoside phosphorylase activity in mammals and treating diseases and conditions responsive thereto, e.g., autoimmune disorders, rejection of transplantation, or psoriasis, which comprises admihistering to a mammai in need thereof an effective amount of a compound of the '· invention or of a pharmaceutical composition comprising a said compound in combination vith one or more pharmaceutically acceptable carriers. A further aspect of the invention relates to a method of inhibiting the phosphorolysis and metabolic breakdovn of antiviral or antitumor purine nucleosides in mammals vhich comprises administering in conjunction therevith to a mammai in need thereof, either separately or in combination therevith, an effective purine nucleoside phosphorylase inhibiting amount of a compound of the invention or of a said compound in combination vith one or more pharmaceutically acceptable carriers. More particularly, 9 LV 10100 such relates to a method of inhibiting the phosphorolysis and metabolic breakdovn of purine nucleosides knovn in the art, e.g., of 2*-deoxyguanosine, 2',3'-dideoxyinosine, 2', 3 '-dideoxyguanosine or 2 *, 3 ' -dideoxyadenosine.
Furthermore, the invention thus relates to a method of potentiating the antiviral or antitumor effect of 2' or 31-monodeoxypurine nucleosides or of 2 ', 3 1-dideoxypurine nucleosides in mammals vhich comprises administering in conjunction therevith to a mammai in need thereof, either separately or in combination with a said nucleoside, an effective purine nucleoside phosphorylase inhibiting amount of a compound of the invention preferably in combination with one or more pharmaceutically acceptable carriers. More particularly, such relates to a method of enhancing or potentiating the effect of 21,3'-dideoxypurine nucleosides knovn in the art, e.g., of 2*,3*-dideoxyinosine, 2',3'-dideoxyguanosine or 2'-3'-dideoxyadenosine for the treatment of retrovirus infections, e.g., HlV-retrovirus infections (acquired immunodeficiency syndrome, AIDS). 2^31 -Dideoxypurine nucleosides are knovn in the art as inhibitors of ΗΓ7 retrovirus infectivity and to be metabolically degraded by PNP, e.ģ., “as described in Biochemical Pharmacolocry 22. ‘3797 (1987) . Such are administered at a pharmaceutically acceptable dose vhich is effective in inhibiting HlV-retrovirus infections. Preferably the lovest possible effective dose is used.
The pharmaceutically acceptable effective dosage of active compound of the invention. to be administered is dependent on the species of varm-blooded animal (mammai), the body veight, age and individual condition, and on the form of administration.
The pharmaceutical composition may be oral, parenteral, suppository or other form vhich delivers the compound into the bloodstream of a mammai to be treated. An„ oral form 10 has from about 1 to about 150 mg of the compound for an adult (50 to 70 kg) vhich is mixed together vith pharmaceutically acceptable diluents such as lactose. In a typical capsule, 25 mg of the compound are mixed together 5 vith 192 mg lactose, 80 mg modified . starch and 3 mg magnesium stearate. Injectable forms of the compound are also contemplated for administration.
The present invention is also useful vith other therapeutic aģents. A daily dosage for a human veighing 50 10 to 70 kg of 1-50 mg/kg inhibits metabolic destruction of certain anticancer aģents such as beta-2' -deoxy-6-thioguanosine and antiviral aģents such as 2·, 3'-dideoxyinosine, an anti-AIDS drug. These types of aģents are known to be susceptible to cleavage. Upon cleavage, 15 the aģents lose effectiveness. The compounds of the present invention are capable of reducing such cleavage. This protection, therefore, enhances the efficacy of other chemotherapeutic aģents.
One method of making the compound (I) of the present 20 invention uses 3-substituted propionitriles as starting materiāls. Such starting materiāls can be obtained by a variety of methods that are veli documented in the literature., The compound (I) is then prepared from the starting material by an adaptation of the synthetic 25 methodology disclosed in Μ. I. Lim, R. S. Klein, and J. J. Fox, J. Ore. Chem. . 44 . 3826 (1979) ; M.I. Lim, R.S. Klein, and J.J. Fox, Tetrahedron Lett.f 21. 1013 (1980); M.I. Lim and R.S. Klein, Tetrahedron. Lett., 22, 25 (1981); Μ. I.
Lim, W. Y. Ren, B.A. Otter, and R.S. Klein, J. Orcr. Chem.. 30 48, 780 (1983). A method of making the compound (II.) of the present invention uses a 3-(pyridinyl)propionitrile as the starting material to make the compound (II). The appropriate 3-(pyridinyl)propionitrile can be produced by converting the 11 LV 10100 corresponding 3-(pyridinyl) propionyl chloride to the corresponding amide by ammonolysis with, e.g., ammonium hydroxide, which is then dehydrated to the desired nitrile by distillation with a dehydrating aģent, such as POCl3 or S0C12. Altematively, the starting material is produced by condensation of the 3-aldehyde with cyanoacetic acid folloved by decarboxylation to give the corresponding substituted acrylonitrile, Which is hydrogenated to give the corresponding 3-(pyridinyl)propionitrile by either catalytic hydrogenation or magnesium mētai dissolving in methanol at 0 *C, such as disclosed in Profitt, J., et al., J. Oro. Chem. . 40. 127 (1975). The compound (II) is then prepared from the starting material by an adaptation of the synthetic methodology disclosed in Μ. I. Lim, R. S. Klein, and J. J. Fox, J. Oro. Chem.. 44, 3826 (1979); M.I. Lim, R.S. Klein, and J.J. Fox, Tetrahedron Lett.. 21, 1013 (1980) ; M.I. Lim and R.S. Klein, Tetrahedron Lett.. 22, 25 (1981) ; Μ. I. Lim, W. Y. Ren, B.A. Otter, and R.S. Klein, J. Ora. Chem.. 48, 780 (1983).
Another method of making the compound of the present invention uses a knovn compound, i.e., cyclohexenyl-acetonitrile, as the starting material.' The compound (III) of the present invention is made by reacting the starting material in an adaptation of the synthetic methodology disclosed in Μ. I. Lim, R. S. Klein, and J. J. Fox, J. Oro. Chem.. 44, 3826 (1979); M.I. Lim, R.S. Klein, and J.J. Fox, Tetrahedron Lett.. 21, 1013 (1980); M.I. Lim' and R.S.
Klein, Tetrahedron Lett.. 22, 25 (1981); Μ. I. Lim, W. Y. Ren, B.A. Otter, and R.S. Klein, J. Oro. Chem. . 48, 780 (1983). Catalytic hydrogenation of either (IIIA), (IIIB), or (IIIC) yields the compound (IIID). A method of making the compound (IV) of the present invention uses 3-substituted propionitriles as starting materiāls. Such starting materiāls can be obtained by a 12 variety of methods that are veli documented in the literature. The compound (IV) is then prepared from the starting material by an adaptation of the·. synthetic methodology disclosed in Μ. I. Lim, R. S. Klein, and J. J. Fox, J. Ore. Chem.. ££/ 3826 (1979); M.I. Lim, R.S. Klein, and J.J. Fox, Tetrahedron Lett.. 21, 1013 (1980); M.I. Lim and R.s. Klein. .Tetrahedron Lett.. 22, 25 (1981)? Μ. I.
Lim, W. Y. Ren, B.A. Otter, and R.S. Klein, J. Ora. Chem.. 41/ 780 (1983). ·,
Another aspect· of the present invention conceming a compound of the’ formula
provides a method of making a 2-amino compound (Rl = NH2) and intermediates thereof. The first step of the method involves reacting·; an optionally substituted cyclic aldehyde with cyanoaceticyacid at a molar ratio of about 1/1 to 1/5 in the presence of ammonium acetate at about reflux temperature for about 10 hours to 8 days to make a 3-cyclo-substituted -pentanedinitrile as an intermediate. 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 raetal-containing bases sodium hydride or sodium alkoxide, e.g., sodium methoxide, at a molar ratio of about 1-2/3-6/1-3 aļnd at a temperature of about 20-65*c for about 10 hours to 8 days to make a 3-cyclo-2-formylpentanedini- 13 LV 10100 trile 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 ratio of about 1-2/1.5-4/1.5-4 and at a 5 temperature of about 20-60*C for about 10-48 hours to make methyl N-[.(3-cyclo-2,4-dicyano)-2-butenyl]glycine as an intermediate. In the subseguent step, the methyl N-[{3-cyclo-2,4-dicyano)-2-butenyl]glycine is reacted with an alkyl chloroformate such as ethyl chloroformate and 1,5-10 diazabicyclo[4.3.0]non-5-ene (DBN) or l,8-diazabicyclo [5.4.0]undec-7-ene (DBU) at a molar ratio of about 1-2/1.5-5/1.5-4 and at a temperature of about 0-50*C for about 10 hours to 10 days to raake methyl 3-amino-4-(2-cyano-l-cyclo-ethyl)-l-ethyl-lH-pyrrole-l,2-dicarboxylate as an 15' intermediate. The next step involves reacting the methyl 3-amino-4- (2-cyano-l-cyclo-ethyl) -l-ethyl-lH-pyrrole-l,2-dicarboxylate vith a base such as sodium carbonate at a molar ratio of about 2/1 to 1/5 and at about room temperature for about 10-48 hours to make methyl 3-amino-4-30 (2-cyano-l-cyclo-ethyl)-lH-pyrrole-2-carboxylate as an intermediate. In the next step, the methyl 3-amino-4-(2-cyano-l-cyclo-ethyl)-lH-pyrrole-2-carboxylate is reacted with benzoylisothiocyanate at a molar ratio of about 2/1 to 1/2 and at about room temperature for about 3 0 minūtes to 3 25 hours to make N-benzoyl-N—[4-(2-cyano-l-cyclo-ethyl)-2-methoxycarbonyl-lH-pyrrol-3-yl]thiourea as an intermediate. The next step reacts the N-benzoyl-N—[4-(2-cyano-l-cyclo-ethyl)-2-methoxycarbonyl-lH-pyrrol-.4-3-yl] thiourea with an alkyl halide such as iaethyl iodide at a molar ratio of 3 0 about 1/1 to 1/6 and at a temperature of about 0-30*C for about 10 minūtes to 10 hours to make N-benzoyl-N—[4-(2-cyano-l-cyclo-ethyl) -2-methoxycarbonyl-lH-pyrrol-3-yl]S-methylthiourea as an intermediate. In the folloving step, the N-benzoyl-N,-(4-(2-cyano-l-cyclo-ethyl) - 2- 14 methoxycarbonyl-lHrpyrrol-3-yl] -S-methylthiourea (about 1-2 mol) . is reacted v?ith methanolic or ethanolic ammonia at a ratio of about 1/1 to 1/20 and at a temperature of about 20-130*C '· for about 16-60 hours to make a mixture of a 2-5 amino compound of the present invention 3-cyclo-3-[2-amino-4-oxo-3H-5H-pyrrolo[3,2-d]pyrimidin-7-y1]propanenitrile and a 3-cyclo-3-[2rmethylmercapto-4-oxo-3H, 5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile as an intermediate in making another compound of the present invention. 10 in a' further a!spect conceming the compound of the formula”
15 20 there is provided a method.pf making a 2-methoxy compound (R1 = OCH3) and intermediates thereof. The intermediate 3-cyclo-3-[2-methylmercapto-4-oxo-3H,5H-pyrrolo[3,2-25 d.] pyrimidin-7-yl Jpropanenitrile is reacted vith an oxidizing aģent such as permanganate or hydrogen peroxide at a molar ratio of about 1/1 to 1/10 and at a temperature of about 25-120‘C for about 3-48 hours to make 3-cyclo-3-[2-methylsulfonyl-4-oxo-3ii,5H"Pyrrolo[3,2-d]pyrimidin-7-3 0 yl]propanenitrile as an intermediate. In the next step, the 3 -cyclo-3- [ Z-meth/lsulf onyl-4-oxo-3H, 5H-pyrrolo [3,2-d]pyrimidin-7-yl]propanenitrile is reacted vith a sodium alkoxide such sodium methoxide at a molar ratio of about 1/1 to 1/10 and at a temperature of about 25-100’ C for 15 LV 10100 about 1-48 hours to make a 2-methoxy compound of the present invention 3-cyclo-3-(2-methoxy-4-oxo-3īi, 5&-pyrr ol o [ 3 , 2 -d ] pyrimidin-7 -y 1 ] propaneni tril e.
In a further aspect, there is provided a method of making 5 a compound vherein R*· is hydrogen. The methyl 3-amino-4-(2-cyano-l-cyclo-ethyl)-lH-pyrrole-2-carboxylate intermediate · described supra is reacted vith dimethylformaaide dimethyl acetal at a molar ratio of about 1/1 to 1/4 and at a temperature of about 25-100*C for about 10 1-10 days to make methyl 4-(2-cyano-l-cyclo-ethyl)-3-[N- (dimethylaminomethylene) amino] -iH-pyrrole-2-carboxylate as an intermediate. The next step involves reacting the methyl 4 - {2-cyano-l-cyclo-ethyl)-3 -[N-(dimethylamino-methylene) amino] -lH-pyrrole-2-carboxylate vith methanolic 15 or ethanolic ammonia at a molar ratio of about 1/1 to 1/20 and at a temperature of about 20-130*C for about 10-68 hours to make the compound of the present invention 3-cyclo-3-[4-oxo-3H,5H-pyrrolo[3,2-d]pyrimidin-7-yl]propanenitrile. 20 As will be apparent to the skilled artisan, variations of the aforesaid procedures are useful in making the variety of compounds of the present -'invention vithout departing from the spirit thereof. -For examplei compounds having different values for "n" and "m" in accordance vith the 25 formula of the present invention are obtained 'by either stepping up or stepping dovm the series by the necessary number of carbon atoms in accordance vith knovn procedures. Also, reactions involving some intermediates reguire protection of nitrogen or oxygen atoms on the intermediates 30 using knovn procedures.
In order to more fully describe the present invention the folloving non-limiting examples are provided. In the examples ali parts and percentages are by veight unless 16 indicated othervise. Proportions of solvent mixtures used as chromatographic eluents are by volume. EXAMPLE 1 3-(3-Pyridinyl)propionitrile is prepared in this example. A three-neck flask carrying a magnetic stir bar is fitted vith a thermometer, pressure equalizing addition funnel, and a reflux condenser carrying an argon inlet. Freshly powdered potassium hydroxide (6.6 g, 0.1 mol) and anhydrous acetonitrile (150 ml) are charged into the flask and heated at reflux vhile 3-pyridinecarboxaldehyde (10.7 g, 0.1 mol) in anhydrous acetonitrile (50 ml) is added dropvise over a period of about five minūtes and refluxing continued for about another three minūtes. The resulting hot reaction mixture is poured into an ice/water mixture (100 g), and the resulting solution is extracted vith CH2CI2 (3 x 100 ml), dried vith Na2S04, and evaporated to give crude 3-(3-pyridinyl)acrylonitrile, vhich is purified by column chromatography over silica gel using CHCI3 as the eluent; yield 3.3 g (25.6%).
Under an argon atmosphere, a stirred solution of the acrylonitrile (2.662 g, 0.02 mol) in 99% ethanol (100 ml) is treated vith a drop of 4% aqueous sodium hydroxide folloved by sodium borohydride (0.378 g, 0.01 mol). Additional sodium borohydride (0.378 g) is added tvice more at four-hour intervāls. The mixture is stirred at room temperatūre overnight, diluted vith vater, extracted vith EtOAc and dried vith Na2S04. The solvent is removed under reduced pressure and the crude_ product obtained is chromatographed over a column of silica using chloroform/methanol (40/1) as the eluent to give 2.2 g (84.6%) of the product as a colorless oil. EKAHPLE 2 3-(3-Pyridinyl)propionitrile of Example 1 is further treated in the synthesis of the present invention. Under 17 LV 10100 an atmosphere of dry N2, a mixture of 3-(3-pyridinyl)propionitrile (0.661 g, 5.0 mmole), sodium hydride (0.240 g, 10.0 mmole), and ethyl formate (1.11 g, 15.0 mmole) in anhydrous tetrahydrofuran (20 ml) is stirred for 48 hours vith protection from air and moisture. Volatile matter is evaporated, and a solution of the solid residue in 15 ml of cold water is adjusted at 0*C to a pH of 6 vith cold 6N HC1. The resulting oily mixture is extracted vith CHCI3, and the extract is vashed vith vater, dried using Na2S04, and evaporated to give a dark oil, vhich is a mixture of 2-formyl-3-(3-pyridinyl)propionitrile and the nitrile starting material. This crude product is .ised in the next reaction vithout further purification. EXAMPLE 3
Glycine methyl ester hydrochloride (0.942 g, 7.5 mmole) and anhydrous sodium acetate (0.615 g, 7.5 mmole) airē added to a solution of the crude formyl compound (0,.89 g) in MeOH/H20 (4:1, 50 ml). After 24 hours, the MeOH is evaporated in vacuo. and the mixture of water and oil is extracted vith CHCI3. The CHCI3 layer is dried (Na2S04) and evaporated to give an amber oil vhich is applied to a silica gel column. Elution vith CHCI3 'gavē' tvo major bands: (1) 3-(3-pyridinyl)propionitrile (used as’ starting material in the previous step), and (2) the desired enamine. EXAMPLE 4
Under a nitrogen atmosphere, ethyl chloroformate (0.521 g, 4.8 mmole) is added dropvise to a solution of the enamine of £xample 3 (0.513 g, 3.2 mmole) and 1,5- diazabicyclo[4.3.0]non-5-ene (DBN‘, 1.37 g, 11.1 mmole) in dry CH2Cl2 (15 ml) vith external cooling in an ice bath. After stirring at 0 *C for one hour, the solution is alloved to a stand at room temperature ovemight. After checking progress by TLC, additional ClC02Et (0.1 ml) and 18 DBN (1.0 ml) are added to complete the conversion, and the solution is alloved to stand for 24 hours. Volatile matter is evaporated in vacuo and the viscous residue is purified on a short silica gel column (vhose main purpose is to 5 remove the less-mobile DBN) to give an N-blocked pyrrole; vhich is used for the next step vithout further purification. EXAMPLE 5
To a solution of the N-blocked pyrrole of Example 4 10 (0.635 g, 2.0 mmole) in MeOH (50 ml) is added solid Na2C03 (0.212 g, 2.0 mmole), and the reaction mixture is .stirred at room temperature for 48 hr with separation of the resultant deblocked pyrrole. The mixture is evaporated to dryness, and the residue is triturated thoroughly with H2O 15 (25 ml) to dissolve inorganics and extracted vith CHC13 (3 x 100 ml). The extract is dried (Na2S04) and evaporated to give a viscous gum that crystallized upon drying in vacuo for use as an intermediate vithout further purification. More extensive purification can, hovever, be effected by 20 using either column chromatography employing silica gel/CHCl3 or recrystallization from toluene/cyclohexane (1:3) . ... . ... ... EXAMPLE 6
Benzoyl isothiocyanate (0.232 g, 1.42 mmole) is added 25 dropvise to a solution of the unblocked pyrrole of Example 5 (0.290 g, 1.18 mmole) in dry ČH2Cl2 (100 ml). After 1 h at room temperature, solution is evaporated, and the gummy residue is stirred in Et20/cyclohexane (1:1, 20 ml). The resulting suspension of yellow solid is filtered under N3 30 pressure, and the thioureido product is dried in vacuo over p2°5* EXAMPLE 7
Methyl iodide (0.228 g, 1.61 mmole) is added to a solution of the thioureido product of Example 6 (0.383 g,‘ 19 LV 10100 0.94 mmole) and DBN (0.140 g, 1.12 mmole) in dry CH2CI2 (10 ml) at 0 *C. The solution is stirred at 0 *C for 15 min., at ambient temperature for 1 h, and then evaporated in vacuo. A solution of the residue in CHC13 is chromatographed on a silica gel column with CHC^/methanol (97:3) as eluent to give homogeneous fractions of the methylthio intermediate compound. EXAMPLE 8 A solution of the methylthio compound of Example 7 (0.358 g, 0.85 mmole) in 100 ml of MeOH that has been saturated with NH3 at 0 *C is heated at 90-95 ’C for 24 hours in a glass-lined stainless Steel bomb. The contents of the chilled bomb are evaporated in vacuo to give a mixture of the desired 2-amino intermediate compound, benzamide, and a by-product that is a 2-methylthio derivative, as opposed to the 2-amino compound. The mixture is dissolved in methanol and the solution is evaporated with silica gel (about 5 g) . The mixture is then carefully layered onto the top of a silica-gel chromatography column, vhich is then eluted with CHC^/MeOH (9:1) to give the methylthio byrproduct and the desired 2-amino-7- (3-pyridinylmethyl)-3H,5H-pyrrolo(3,2-d]pyrimidin-4-one intermediate. Further - purification is obtained by recrystallization from boiling isopropyl acetate in a Soxhlet apparatus. A solution of the pyridinylmethyl intermediate in 0.1N HC1 is hydrogenated with a platinum catalyst at 60 lb/in2 H2 pressure. The catalyst is generated by brief hydrogenation of ,Pto2 in 0.1N HC1. When the reaction is complete, the catalyst is removed by filtratiori under N2 pressure, and the filtrate is evaporated. A solution of the residue in the minimum amount of ethanol is diluted slowly with a large amount of Eto2, and the hydrochloride 20 salt is obtained as a white hygroscopic solid of the compound (IC). £XAMPLE 9
The compound (IC) of Example 8 is tested for enzyme inhibition activity. A purine nucleoside phosphorylase (PNP) enzyme assay is performed in vhich PNP activity (IC50) for the compound is observed, vhich is determined radiochemically by measuring the formation of (14CJ-hypoxanthine from (14C]-inosine (see Blomedicine. 33, 39 (1980)) using calf spleen as the enzyme source. EXAMPLES 10-14
The folloving compounds of the present invention are prepared that are 2-amino-7-(R) -3H,5g-pyrrolo(3,2-d]-pyrimidin-4-ones vherein R is -CH2R1 it whicil the R^ group is as follovs: £xample 11 Example 12 Example 13 Example 14
Example 10 Rļ « 2-methyl-3-piperidinyl Rļ = 2-chloro-3-piperidinyl Rļ = 2-trifluorooethyl-3-piperidinyl· R«L = 2-methoxy-3-piperidinyl Rļ_ = 2-fluoro-3-piperidinyl The compounds are prepared following the procedures set forth in Examples 1-8 -using the appropriate 3-(substituted 3-pyridinyl)-propionitriles as-starting materiāls. EXAMPLE 15 3-(2-Pyridinyl)propionitrile is prepared in this example using the procedure of V. Boekelheide, et al., J. Am. Chem. Soc. . 75. 3243 (1953). A solution of potassium cyanide (83.74 g) in vater (160 ml) is added to a solution of freshly distilled 2-vinylpyridine (67.59 g) in acetic anhydride (131.30 g) vith the rāte of addition adjusted to maintain gentle refluxing. When the addition is complete, the resulting dark red mixture is heated for about 17 hours at 105*c in an oil bath vith vigorous stirring. The cooled reaction mixture is adjusted ' to pH 8 vith a saturated 21 LV 10100
Na2C03 solution. The mikture is extracted vith CHCI3 (4 x 150 ml) , vashed with waterr dried with Na2SC>4, and evaporated to a dark viscous oil. The oil is fractionally distilled in vacuo through a Vigreaux column. After a 5 small forerun containing 2-vinylpyridine (0.35 g), the desired 3-(2-pyridinyl)propionitrile is collected as a clear, fluorescent yellow-green, viscous oil: yield 59.8 g; 70.4%; bp 86'C at 1.0 mm Hg. EXAKPLES 16-20 10 15 20
The folloving compounds of the present invention are prepared that are 2-amino-7-{R)-3ū,5H-pyrrolo[3,2-£] pyrimidin-4-ones vherein R is -CH2Rļ the Rļ group is as follovs: 2- piperidinyl 4-piperidinyl 3- trifluoromethyl-4-piperidinyl 3-methoxy-2-piperidiny1 3-fluoro-4-piperidinyl The compounds are prepared folloving the procedures set forth in Examples 1-8 and 15 using the appropriate 3-(2- or 4-pyridinyl)-propionitriles as starting materiāls. •EXAMPLE'21
Example 16 Example 17 Example 18 Example 19 Example 20
Rl = Rl = Rl = Rl = Rl = 25 A pharmaceutical composition for intraperitoneal injection is prepared for testing the compound (IC). An intraperitoneal injection solution containing the compound of Example 8 is dissolved in an agueous carrier that contains ten percent DMSO. EXAMPLE 22..
The compound (IC) is intraperitoneally injected into Levis Rats via the tēst composition of Example 21 to provide 30 mg of the compound (IC), vith an injection given tvice per day. Controls are used, vhich receive only the vehicle. At specific times after administration, the animals are sacrificed and plasma samples are prepared. 30 22
The plasma is extracted with cold 0.5 N HC104 and neutralized vith solid NH4HCO3. After removal of perchlorate salts, the extract is subjected to HPLC on a reversed phase column (Spherisorb ODSI). A significant increase in plasma inosine is observed in the plasma taken from animals receiving the compound (IC). EXAMPLES 23-33
Compounds prepared as in Examples 10-20 are each made into a pharmaceutical formulation in accordance with the preparation of Example 21 and the resultant injectable Solutions are tested in accordance with the procedure of Example 22. A significant increase in plasma inosine is observed in the plasma taken from animals receiving the compounds of the present invention. EXAMPLE 34 3-(2-Furanyl)propionitrile is prepared in this example. In a three-neck flask fitted with a condenser and drying tube, magnesium tumings (20 g) are added to a solution of 3-(2-furanyl)acrylonitrile (67.2 g) in dry methanol (2 1). Additional magnesium is added in parts as the reaction rāte vould allov until a total of 145 g has been added. After .about five · hours.,-* the solvent is evaporated until the .contents set_ to. a solid .paste, which is adjusted to a pH of about 6.5 with 6N HC1 vith cooling. The mixture is extracted vith several portions of CHCI3, dried vith Na2S04, and concentrated to a dark pil. Distillation in vacuo through a short Vigreaux column gives the propionitrile as a colorless oil;.yield 49.5 g (72%); bp 46.0-46.5*C at 0.5 mm. EXAMPLE 35 3-(2-Furanyl)propionitrile of Example 34 is further treated in the synthesis of the present invention. Under an atmosphere of dry N2, the propionitrile (48.7 g), sodium hydride (10.28 g), ethyl formate (32.74 g), and anhydrous 23 LV 10100 tetrahydrofuran (200 ml) are stirred at room temperature vith protection from moisture for about 18 hours. The volatile matter is then evaporated, the resulting yellow solid is dissolved in about 20 ml of cold water with ice-5 bath cooling, and the solution is adjusted to a pH of 6.0 with cold 6N HCl. The resulting heavy oil precipitate is extracted into -CHC^, and the extract is vashed with vater, dried with Na2S04, and evaporated to give a thin oil vhich contains the crude formyl compound; yield 53.06 g. 10 EXAMPLE 36
Glycine methyl ester hydrochloride (67.05 g) and anhydrous sodium acetate (43.79 g) are added to a solution of the crude formyl compound of the previous example (53.05 g) in MeOH/H20 (4:1, 1500 ml). After 24 hours, the 15 MeOH is evaporated in vacuo, and the mixture of vater and oil is extracted vith CHCI3. The CHCI3 layer is dried (Na2S04) and evaporated to give an amber oil (59.1 g). EXAMPLE 37
Under a nitrogen atmosphere, ethyl chloroformate (43.68 g) is added dropvise to a solution of the amber oil of Example 36 (59.1 g) and DBN (133 g) in dry CH2CI2 (400 ml) vith extemal cooling 'in an ice'bath.' ‘After stirring at 0 *C for one hour, the solution is alloved to stand at room temperature ovemight. After checking progress by 25 TLC, additional ClC02Et and DBN are added to complete the conversion, and the solution is alloved to stand for 24 hours. Volatile matter is evaporated in vacuo, the viscous residue purified on a silica gel column (vhose main purpose is to remove the less-mobile DBN) to give an N-blocked 30 pyrrole, a corresponding pyrrole vithout the ethoxycarbonyl blocking group on the pyrrole nitrogen, and the starting propionitrile. 24 EXAMPLE 38
To a solution of the N-blocked pyrrole of Example 37 (11.66 g) in MeOH (200 ml) is added solid Na2C03 (4-23 g), and the reaction mixture is stirred at room temperature for 5 24 hr vith separation of the resultant deblocked pyrrole.
The mixture is evaporated to drynessf and the residue is triturated thoroughly vith H20 (200 ml) to dissolve inorganics and extracted vith CHCI3 (3 x 200 ml), The extract is dried (Na2S04) and evaporated to give a viscous 10 gum that crystallized upon drying in vacuo for use as an intermediate vithout further purification. More extensive purification can, hovever, be effected by using either column chromatography employing silica gel/CHCl3 or recrystallization from toluene/cyclohexane (1:3). 15 EXAMPLE 39
Benzoyl isothiocyanate (4.58 g) is added dropvise to a solution of the unblocked pyrrole of £xample 38 (5.15 g) in dry CH2C12 (75 ml) . After 1 h at room temperature, solution is evaporated, and the gummy residue is dissolved 2 0 in Et20 (100 ml) vith almost immediate separation of the crystalline solid, vhich is collected by filtration. The Et20 filtrate is heatcd to boiling and diluted vith an egual volume. of .varm..cyclchexane. On cooling slovly the solution gives additional thioureido product. 25 EXAMPLE 40
Methyl iodide (8.70 g) is added to a solution of the thioureido product of Example 39 (10.68 g) and DBN (4.15 g) in dry CH2C12 (250 ml) at 0 *C. The solution is stirred at 0 ’C for 15 min., at ambient temperature for 1 h, and then 30 evaporated in vacuo. A solution of the residue in CHCI3 is chromatographed on a silica gel column vith CHCI3 as eluent to give homogeneous fractions of the methylthio intermediate compound. 25 LV 10100 EKAMPLE 41 A solution of the methylthio compound of' Example 40 (0.80 g, 2.0 mmole) in 25 ml of MeOH that has been satur atēd with NH3 at 0 *C is heated at 90-95 ’C for 24 hours in a glass-lined stainless Steel bomb. The contents of the chilled bomb are evaporated in vacuo to give a mixture of the 2-amino compound, benzamide and a by-product that is a 2-methylthio derivative, as opposed to the 2-amino compound. The mixture is stirred vigorously for several minūtes vith 30 ml of 2:1 Et20/cyclohexane/ and the insoluble vhite solid is filtered off and vashed vith Et20. The filtrate contained most of the benzamide and 2-methylthio components. A solution of the Et20/cyclohexane-insoluble solid (0.425 g) in MeOH is evaporated vith appr. 10 g of silica gel. The powdered residue is layered evenly onto the top of a silica gel column, vhich is then eluted vith CHCl3/MeOH/HOAc (95:5:1) to give the 2-methylthio by-product and the desired furanyl intermediate. The intermediate is recrystallized by extraction into boiling isopropyl acetate in a Soxhlet apparatus. The vhite crystals are collected in three crops and dried in vacuo over P2O5 at 110*C for-7h.···' -· A solution of the intermediate (116 g, 0.5 mmole.) in methanol (50 ml) is hydrogenated vith 30% palladium-on-charcoal (40 mg) at 62 lb/in2 H2 pressure for about 36 hours. The catalyst is removed by filtration under N2 pressure, and the filtrate is evaporated and reevaporated vith toluene. The solid residue is recrystallized by extraction into boiling isopropyl acetate in a Soxhlet apparatus. The 2-tetrahydrofuranyl compound (IE) is obtained as a vhite crystalline solid, vhich is dried iri vacuo over P205 at 110*C for about s'ix hours; yield 73 mg (61.8%); m.p. 284-286*C dec. 26 EXAMPLE 42
The product of Example 41 is tested for enzyme inhibition activity according to the procedure of Example 9, and PNP activity (IC50) for the compound is observed. 5 EXAMPLE 43 A pharmaceutical composition for intraperitoneal injection is prepared for testing the compound (IE). An intraperitoneal injection solution containing the compound of Example 41 is dissolved in an aqueous carrier that 10 contains ten percent DMSO. EXAMPLE 44
Using the procedure of Example 16, the compound (IE) is intraperitoneally injected into Lewis Rats via the tēst composition of Example 43 to provide 30 mg of the compound 15 (IE) and the results are analyzed compared to Controls. A significant increase in plasma inosine is observed in the plasma taken from animals receiving the compound (IE). EKAMPLES 45-49
The following compounds of the present invention are 20 prepared that are 2-amino-7-(R) -3H,5H-pyrrolo[3,2-d]-pyrimidin-4-ones vherein R is -CH2-R1 in vhich the R^ group is as follovs: i r> li t · Λ Ķ
Example 45 = 3-tetrahydrofuranyl
Example 46 R^ = 3-chloro-2-tetrahydrofuranyl 25 Example 47 Rj_ = 3-trifluoromethyl-2-tetrahydrofuranyl Example 48 R]_ = 3-methoxy-3-tetrahydrofuranyl
Example 49 R^ = 3-fluoro-2-tetrahydrofuranyl
The compounds are prepared folloving the procedures set forth in Examples 34-41 using the appropriate 3-(furanyl)-30 acrylonitriles as starting materiāls. EXAMPLE 50
Under a nitrogen atmosphere, a solution of the tetrahydrofuranyl compound (IE) (500 mg) obtained from Example 41 and a crystal of phenol in 2N HBr (20 ml) is 27 LV 10100 stirred for 18 hours at 40*C. The solvent is evaporated in vacuo at low tempera tūre, and the residue is vashed with a few ml of Et20 by decantation to remove any tribromophenol. A suspension of the residue in H20 is adjusted to pH 7 vith 1N NaOH to give the bromoalcohol as its free base. The solid is collected, vashed vith cold vater, and dried at room temperatūre.. A solution of the bromoalcohol in anhydrous dimethyl-acetamide is chilled in an ice bath and treated vith an equimolar portion of ΡΒγ3· The solution is allowed to stir at room temperature for one hour, then the dimethylacetamide is evaporated in vacuo at lov temperature using an air pump and dry-ice trap. A suspension of the residue in ice/vater is adjusted to pH 7 using IN NaOH to give the crude reaction product containing the 1,4-dibromo compound. After filtration, vashing vith cold vater, and drying at room temperature, the resulting product is refluxed vith sodium sulfide in 50t ethanol/vater, or altematively varmed vith Na2S in N,N-dimethylacetamide solution; the solvent removed under vacuum, the residue· vashed vith vater to remove NaBr, and the pH adjusted to about 7 to precipitate the 2-tetrahydrothienyl compound (IG). EXAMPLE 51
The compound of Example 50 is tested for enzyme inhibition activity as in Example 9, and PNP activity (IC5o) for the compound is observed. EXAMPLE 52 A pharmaceutical composition for intraperitoneal injection is prepared for testing the compound (IG). An intraperitoneal injection solution is made containing the compound of Example 50 is dissolved in an aqueous carrier that contains ten percent DMSO. 28 EXAMPLE 53
Using the procedure of Example 16, the compound (IG) is intraperitoneally injected into Levis Rats via the tēst composition of Example 52 to provide 30 mg of the compound 5 (IG) and the results are analyzed compared to Controls. A significant increase in plasma inosine is observed in the plasma taken from -animals receiving the compound (IG). EXAMPLES 54-58
The folloving compounds of the present invention are 10 prepared that are 2-amino-7-(R)-3ii,5fi-pyrrolo(3,2-d]-pyrimidin-4-ones vherein R is -0¾-Ri in the Rļ group is as follovsi
Example 54 Rļ = 3-tetrahydrothienyl Example 55 R^ = 3-chloro-2-tetrahydrothienyl 15 Example 56 R^ = 3-trifluoromethyl-2-tetrahydrothienyl Example 57 R^ = 3-methoxy-2-tetrahydrothienyl Example 58 Rļ « 3-fluoro-2-tetrahydrothienyl
The compounds are prepared folloving the procedures set forth in Examples 34-41 and 50 using the appropriate 3-20 (furanyl)-acrylonitriles as starting materiāls. EXAMPLE 59
Using ..the.. . procedure of Example 1, 3-(2-pyrrolyl)-propionitrile is prepared using pyrrole-2-carboxaldehyde as the starting material. Folloving Examples 2-8, the 25 intermediate 2-amino-7 - ( 2-pyrrolylmethyl)-3H., 5&-pyrrolo[3,2-d]pyrimidin-4-one is prepared from the propionitrile, from vhich the 2-pyrrolidinyl compound (II) is obtained by reduction of the intermediate. EXAMPLE 60 30 Using the procedure of Example 1, 3-(3-pyrrolyl)-propionitrile is prepared using pyrrole-3-carboxaldehyde as the starting material. Folloving Examples 2-8, the intermediate 2-amino-7- (3-pyrrolylmethyl) -3H, 5H-pyrrolo-(3,2—d]pyrimidin-4—one is prepared from the propionitrile, 29 LV 10100 from vhich the 3-pyrrolidinyl compound (IJ) is obtained by reduction of the intermediate. EXAMPLE 61
Using the procedure of Example 1, 3-(2-pyranyl) propionitrile is prepared using 2-pyrancarboxaldehyde as the starting material. Folloving Examples 2-8, the intermediate 2-amino-7-(2-pyranylmethyl) -3H,5H-pyrrolo-[3,2-d]pyrimidin-4-one is prepared from the propionitrile, from vhich the 2-tetrahydro-pyranyl compound (IL) is obtained by reduction of the intermediate. EXAMPLE 62
Using the procedure of Example 1, 3-(3-pyranyl) propionitrile is prepared using 3-pyrancarboxaldehyde as the starting material. Folloving Examples 2-8, the intermediate 2-amino-7-( 3 - pyranylmethyl )-32./ 52-pyrrolo[3,2-2]pyrimidin-4-one is prepared from the propionitrile, from vhich the 3-tetrahydro-pyranyl compound (IM) is obtained by reduction of the intermediate. EXAHPLE 63
Using the procedure of Example 1, 3-(4-pyranyl) propionitrile is prepared using 4-pyrancarboxaldehyde as the starting material. Folloving - Examples 2-8, the intermediate 2-amino-7-(4-pyranylmethyl) -3H, 5H-pyrrolo-[3,2-d]pyrimidin-4-one is prepared from the propionitrile, from vhich the 4-tetrahydro-pyranyl compound (IN) is obtained by reduction of the intermediate. EXAHPLE 64 A solution of the methylthio compound of Example 7 (0.358 g, 0.85 mmole) in 100 ml of MeOH that has been saturated vith NH3 at 0 *C is heated at 90-95 *C for 24 hours in a glass-lined stainless Steel bomb. The contents of the chilled bomb are evaporated in vacuo to give a mixture of the desired 2-amino intermediate compound, benzamide, and a by-product that is a 2-methylthio 30 derivative, as opposed to the 2-amino compound. The mixture is dissolved in methanol and the solution is evaporated with silica gel (about 5 g) . The mixture is then carefully layered onto the top of a silica-gel 5 chromatography column, vhich is then eluted with CHCl3/HeOH (9:1) to give the methylthio by-product and the desired 2-amino-7- (3-pyridinylmethyl) -3H,5H-pyrrolo[3,2-d]pyrimidin-• 4-one, compound (IIA). Further purification is obtained by recrystallization from boiling isopropyl acetate in a 10 Soxhlet apparatus, EXAMPLE 65
The compound (IIA) of Example 64 is tested for enzyme inhibition activity. A purine’' nucleoside phosphorylase (PNP) enzyme assay is performed in vhich PNP activity 15 (IC50) for the compound is observed, vhich is determined radiochemically by measuring the formation of (14C]-hypoxanthine from (14C]-inosine (see Biomedicine. 33, 39 (1980) using calf spleen as the enzyme source. EXAMPLES 66-68 2r The folloving compounds of the present invention are prepared that are 2-amino-7-(R)—3 ϋ,5H-pyrrolo(3,2-d] pyrimidiin-47ones vherain R is -Ο^-Κχ in vhich the Rļ group is as follovs:
Example 66 Rļ = 2-pyridinyl 25 Example 67 Rx = 4-pyridinyl
Example 63 Ri = 3-chloro-2-pyridinyl
The compounds are prepared folloving the procedures set forth in Examples 1-7 and 64 using the appropriate 3-(pyridinyl)-propionitriles as starting materiāls. 30 . EXAMPLE 69 A pharmaceutical composition for intraperitoneal injection is prepared for testing the compound (IIA). An intraperitoneal injection solution containing the compound 31 LV 10100 of Example 64 is dissolved in an agueous carrier that contains ten percent DMSO. EXAMPLE 70
The compound (IIA) is intraperitoneally injected into 5 Levis Rats via the tēst composition of Example 69 to provide 3 0 mg of the compound (IIA) , with an injection given tvice per day. Controls are used, vhich receive only the vehicle. At specific times after administration, the animals are sacrificed and plasma samples are prepared. 10 The plasma is extracted vith cold 0.5 N HCIO^ and neutralized with solid NH4HCO3. After removal of perchlorate salts, the extract is subjected to HPLC on a reversed phase column (Spherisorb ODSI). A significant increase in plasma inosine is observed in the plasma taken 15 from animals receiving the compound (IIA). EXAMPLES 71-73
Compounds prepared as in Examples 66-68 are each made into a pharmaceutical formulation in accordance with the preparation of Example 69 and the resultant injectable 2 0 Solutions are tested in accordance with the procedure of Example 70. A significant increase in plasma inosine is observed in the plašina taken from animals receiving the compounds of the present invention. EXAMPLE 74 25 l-Cyclohexenylacetonitrile is treated in the synthesis of the present invention. Under an atmosphere of dry H2» a solution of l-cyclohexenylacetonitrile (9.2 g,· 75.92 mmole) in anhydrous tetrahydrofuran (THF,. 10 ml) is added to a stirred mixture of sodium hydride (3.18 g; 132.86 mmole) 30 and ethylformate (30.14 g; 406.93 mmole) in 50 ml THF, and the resulting mixture is stirred at room temperature for about 18 hours. Volatile matter is evaporated in vacuo at room temperature. Water (30 ml) is added to the residue at 0*C, and the solution adjusted to a pH of 6 by dropvise addition of 6N HC1. The resulting precipitate of heavy oil is extracted into CHC13. The extract is washed with water and dried with Na2S04, and the resulting organic layer evaporated to give a crude fonnyl compound as a red-brovn 5 oil (9.6 g). EXAMPLE 75
Glycine methyl ester hydrochloride (16.68 g, 132.85 mmole) and anhydrous sodium acetate (10.89 g, 132.85 mmole) are added to a solution of the crude formyl compound (9.6 10 g) of Example 74 vithout further purification in MeOH/^O (4:1, 150 ml). After 24 hours, the MeOH is evaporated in vacuo, and the mixture of water and oil is extracted with CHCI3. The CHCI3 layer is dried (Na2S04) and evaporated to give an amber oil vhich is applied to a silica gel column. 15 Elution with CHCI3 gavē the desired enamine: yield 4.5 g. EXAMPLE 76
Under a nitrogen atmosphere, ethyl chloroformate (3.04 g,· 28.06 mmole) is added dropvise to a solution of the enamine of Example 75 (4.12 g, 18.70 mmole) and 1,5-di- 20 azabicyclo(4.3.0]-non-5-ene (DBN, 6.96 g, 56.11 mmole) in dry CH2C^2 (1ūū extemal cooling in an ice bath.
After stirring at 0 .. *C for · one hour, the solution is alloved to stand at . room temperatūra overnight. After checking progress by TLC, additional ClC02Et (0.5 ml) and 25 DBN (3.0 ml) are added to complete the conversion, and the, solution is allowed to stand for 24 hours. Volatile matter is evaporated in vacuo, the viscous residue purified on a short silica gel column (vhose main purpose is to remove the less-mobile DBN) to give an N-blocked pyrrole, vhich is 3 0 used for the next step vithout further purification. EKAMPLE 77 ~
To a solution of the N-blocked pyrrole of Example 76 (3.0 g, 10.26 mmole) in MeOH (100 ml) is added solid Na2C03 (2.71 g, 25.65 mmole), and the reaction mixture is stirred 33 LV 10100 at room temperature for 48 hr with separation of the resultant deblocked pyrrole. The mixture is evaporated to dryness, and the residue is triturated thoroughly with H20 (50 ml) to dissolve inorganics and extracted vith CHC13 (3 x 50 ml). The extract is dried (Na2S04) and evaporated to give a viscous gum, vhich is purified oir a silica gel column using CHCI3 as the eluent; yield 2.04 g; m.p. 125*C. EXAMPLE 78
Benzoyl isothiocyanate (0.76g, 4.65 mmole) is added dropvise to a solution of the unblocked pyrrole of Example 77 (0.91 g, 4.13 mmole) in dry CH2C^2 (30 * After 1 h at room temperature, the solution is evaporated, and the gummy residue is triturated with methanol to give a thioureido product; yield 0.70 g? m.p. 170*C. EXAMPLE 79
Methyl iodide (0.678 g, 4.78 mmole) is added to a solution of the thioureido product of Example 78 (0.630 g, 1.64 mmole) and DBN (0.230 g, 1.85 mmole) in dry CH2C12 (50 ml) at 0 *C. The solution is stirred at 0 *c for 15 min., at ambient temperature for 1 h, and then evaporated in vacuo. A solution of the residue in 0Η013 is chromatographed on a silica 'gel čolumn' vith CHCI3 as eluent to give homogeneous · fractio’ns" of the methylthio intermediate; yield 0.7 g. EXAMPLE 80 A solution of the methylthio compound of Example 79 (0.70 g, 1.76 mmole) in 50 ml of MeOH that has been saturated vith NH3 at 0 ’C is heated at 90-95 ’C for 24 hours in a glass-lined stainless Steel bomb. The contents of the chilled bomb are evaporated in vacuo to give a mixture of the compound (IIIA), benzamide and a by-product that is a 2-methylthio derivative, ' as opposed to the 2-amino compound (IIIA) . The mixture is stirred vigorously for several minūtes vith appr. 50 ml of Et20, and the 34 insoluble vhite solid is filtered off and vashed with EtjO. The filtrate contained most of the benzamide and 2-methylthio components. A solution of the Et20-insoluble solid (0.342 g) in MeOH is evaporated with appr. 10 g of 5 silica gel. The povdered residue is layered evenly onto the top of a silica gel column, vhich is then eluted vith CHClj/MeOH/HOAc (95ī5:1) to give the 2-methylthio by-product and the desired 2-amino product (IIIA). (IIIA) is recrystallized by extraction into boiling isopropyl acetate 10 in a Soxhlet apparatus. The white crystals are collected in three crops and dried in vacuo over P2O5 at 110 *C for 7h; yield 444; mp 280 *C dec., anal. calcd. for C12H14N40’°*6H20: c' 59.78; H. 6.35; N, 23.23. Found: C, 59.98; H, 6.46; N, 23.15. 15 EXAKPLE 81
The compound of Example 80 is tested for enzyme-inhibition activity. A purine nucleoside phosphorylase (PNP) enzyme assay is performed in vhich PNP activity for the compound is determined radiochemically by measuring the 20 formation of (14C]-hypoxanthine from [14C]-inosine (see Biomedicine. 33, 39 (1980) using calf spleen as the enzyme source. At. ļ mM phosphat». th.e IC2q is 1.9 μϋ, and at 50 mM phosphate the IC50 is 19 μΜ. EXAMPLE 82 25 Folloving the procedures set forth in Examples 74-80, compounds (IIIB) . and (IIIC) are raade using 2- and 3-cyclohexenyl-acetonitrile, respectively, as starting materiāls. The compounds are tested as in Example 81 and significant enzyme-inhibition activity is observed. 30 EXAMPLES 83-87
The folloving compounds of the present invention are prepared that are 2-amino-7-(R) -3Jļ, 5H-pyrrolo(3,2-d] pyrimidin-4-ones in vhich the R group is as follovs: .Example 83 R « 3-raethyl-2-cyclohexenyl 35 LV 10100
Example 84 R = 2-chloro-3-cyclohexenyl
Example 85 R = 3-trifluoromethyl-l-cyclohexenyl
Example 86 R = 3-methoxy-l-cyclohexenyl
Example 87 R *= 2-fluoro-3-cyclohexenyl
The compounds are prepared folloving the procedures set forth in Examples 74-81 using the appropriate substituted cyclohexenyl acetonitriles as starting materiāls. EXAMPLE 88 A pharmaceutical composition for intraperitoneal injection is prepared for testing the compound (IIIA). An intraperitoneal injection solution containing the compound (IIIA) is dissolved in an agueous carrier that contains ten percent DMSO. EXAMPLE 89
The compound (IIIA) is intraperitoneally injected into Lewis Rats via the tēst composition of Example 88 to provide 30 mg of the compound (IIIA), vith an injection given tvice per day. Controls are used, vhich receive only the vehicle.' At specific times after adroinistration, the animals are sacrificed and plasma samples are prepared. The plasma is extracted vith cold 0.5 N HC104 and neutralized vith'·' solid NH4HCO3'. After removal of perchlorate· salts, the extract is ''subjected to HPLC on a reversed phase column (Spherisorb ODSI). A significant increase in plasma inosine is observed in the plasma taken from animals receiving the compound (IIIA). EXAMPL£S 90-94
Compounds prepared as in Examples 83-87 are each made into a pharmaceutical formulation in accordance vith the preparation of Example 88 and the resultant injectable Solutions are tested in accordance vith the procedure of Example 89. A significant increase in plasma inosine is observed in the plasma taken from animals receiving the compounds of the present invention. 36 EXAMPLE 95
The compound (IIID) is prepared using 2-amino-7-(l-cyclohexenyl)-3ū, 5g-pyrrolo[3,2-d]pyrimidin-4-one as an intermediate. A solution of the intermediate (0.2 g? 0.86 mmole) in ethanol (50 ml) is hydrogenated with 10% Pd-C (50 mg) at 45 lb/in2 for 16 h and filtered.hot through Celite. The filtrate is evaporated to dryness, and the residue is crystallized from hot ethanol to give the compound (IIID) ? yield 157 mg (78%), mp >300 *C dec. anal. calcd. for C12H16N40*0· EtOH: C, 61.80? H, 7.10? N, 23.51. Found: C, 61.95? Hf 7.43? N, 23.56. EXAMPLE 96
The compound (IIID) prepared in Example 95 is tested for enzyme-inhibition activity as in Example 81. At 1 mM phosphate the IC50 is 1.3 μΜ, and at 50 mM phosphate the IC50 is 145 μΜ. EXAMPLE 97 3-(2-Adamantyr)propionitrile is prepared in this example using a modification of the procedure of M. Ohno, et al., J. Org. Chem. 53, 1285 (1988). A Solution of 2-bromoadmantane (20 g; 92.96 mmole); Bu3SnH (32.46 g; 111.5 mmole),.aprylonitrile (9,86 g? 185,92 mmole), and AIBN (740 mg) _iņ toluene.. (280 ml) is stirred at reflux temperature for 3 h. The reaction mixture is vashed with ammonia water (0.4 M, 500 ml), the organic layer is vashed vith HjO and dried over MgS04 and evaporated. The residue is distilled betveen 110-118‘C (and about 0.2 mmHg); fractions are combined to give a crude sample of contaminated 3-(2-adamantyl) propionitrile vith tin complexes, vhich is purified on silica gel column vith hexanes; f.olloved by hexanes/ethylacetate 97:3 and hexanes/ethylacetate 95:5, yield 9.4 g (53.4%); mp semi-solid. - 37 LV 10100 EXAMPLE 98 3-(2-Adamantyl)propionitrile of Example 97 is further treated in the synthesis of the present invention. Under an atmosphere of dry N2, a mixture of 3-(2-adamantyl) propionitrile (7.0 g, 36.99 mmole), sodium hydride (1.7 g, 73.95 mmole), and anhydrous tetrahydrofuran (75 ml) is heated at 52*C in a vater bath for 15 min., and a solution of ethyl formate (13.69 g, 184.89 mmole) in THF (100 ml) is added over a period of 45 min. After two hours at 50 - 55 *C, a second portion of NaH (0.8 g) and HC02Et (7.5 ml) are added, and the reaction miacture is stirred for about tvo days. A third portion of HC02Et (7.5 ial) and NaH (0.8 g) are added and left at room temperature for about 24 hours (unreacted nitrile is inert in the next step and can be recovered at the first purification step). The thick paste is stirred overnight and alloved to cool ‘ to room temperature. Volatile matter is evaporated under reduced pressure, and the residual pale yellov crust is dissolved in the minimum volume of cold vater (appr. 150 ml) at 0 *C. The solution is adjusted to pH 6.0 by addition of 6N HC1 and extracted vith CHCI3 x 100 ml) · The extract is vashed vith H20,.dried, over Na2S04, and evaporated in vacuo to a .thick amber. oil. This crude product is used in the next reaction vithout further purification. EXAMPLE 99
Glycine methyl ester hydrochloride (6.96 g, 55.46 mmole) and anhydrous sodium acetate (4.55 g, 55.46 mmole) are added to a solution of the crude formyl compound (8.0 g) in MeOH/H20 (4:1, 500 ml). After 24 hours, the MeOH is evaporated in vacuo, and the mixture of vater and oil is extracted vith CHCI3. The CHC13 layer is dried (Na2S04) and evaporated to give an amber oil vhich is applied to a silica gel column. Elution vith CHCI3 gavē ,.tvo major bands: (1) 3-(2-adamantyl)-propionitrile (used as starting 38 material in the previous step), and (2) the desired enamine; yield 6 g. EXAMPLE 100
Under a nitrogen atmosphere, ethyl chloroformate (2.82 g, 5' 26.0 mmole) is added dropvise to a solution of the enamine of Example 99 (5.0 g, 17.34 mmole) and l,5-diazabicyclo-[4.3.0]non-5-ene ("DBN," 6.46 g, 52.0 mmole) in dry CH2Cl2 (50 ml) with ejctemal cooling in an ice bath. After stirring at 0 *C for one hour, the solution is alloved to a 10 stand at room tempera tūre ovemight. After checking progress by TLC, additional ClC02Et (1.81 ml) and DBN (3.23 g) are added to complete the conversion, and the solution is alloved to stand for 24 hours. Volatile matter is evaporated in vacuo, the viscous residue purified on a 15 short silica gel column (vhose main purpose is to remove the less-mobile DBN) to give an N-blocked pyrrole, vhich is used for the next step vithout further purification. EXAMPLE 101
To a solution of the crude N-blocked pyrrole of Example 100 (6.0 g, 16.59 mmole) in MeOH (100 ml) is added solid Na2C03 (4.39 g, 41.49 mmole), and the reaction mixture is stirred at room temperatūra for 4 8 hr vith separation of the resultant deblocked pyrrole. The mixture is evaporated to dryness, and the residue is triturated thoroughly vith 25 H20 (50 ml) to dissolve inorganics and extracted vith CHCI3 (3 x 100 ml). The extract is dried (Na2SŪ4) and evaporated to give a viscous gum, vhich crystallized by triturating vith ether; yield 4 g; m.p. 162-163*C. EXAMPLE 102 30 Benzoyl isothiocyanate (1.22 g, 7.47 mmole) is added dropvise to a solution of the unblocked pyrrole of Example 101 (1.91 g, 6.62 mmole) in dry CH2C12 (50 ml). After 1 h at room temperature, solution is evaporated, and the gummy residue is dissolved in Et20 (100 ml) vith almost immediate 39 LV 10100 separation of the crystalline solid. The Et20 filtrate is heated to boiling and diluted with an equal volume of vara cyclohexane. On cooling slovly the solution gives additional thioureido product; yiled 2.81 g (95%); m.p. 5 193-194 *C. EXAKPLE 103
Methyl iodide· (2.46 g, 17.39 mmole) is added to a solution of the thioureido product of Example 102 (2.7 g, 5.98 mmole) and DBN (0.82 g, 6.57 nunole) in dry CH2Cl2 (50 10 ml) at 0 *C. The solution is stirred at 0 *C for 15 min., at ambient tempera tūre for 1 h, and then evaporated in vacuo to give a crude sample of the methythio intermediate compound; yield 2.78 g (crude). EKAMPLE 104 15 A solution of the methylthio compound of Example 103 (2.78 g, 5.18 mmole) in 150 ml of MeOH that has been saturated vith NH3 at 0 *C is heated at 90-95 ’C for 24 hours in a glass-lined stainless Steel bomb. The contents of the chilled bomb are evaporated in vacuo to give a 20 mixture of the compound (IVA), benzamide and a by-product that is a 2-methylthio derivative, as opposed to the 2-amino compound (IVA); · The’j mixture is stirred vigorously for several minūtes vith appr. 75 ml of Et2o, and the insoluble vhite solid is filtered off and vashed vith Et20. 25 The filtrate contained most of the benzamide and 2-methylthio components. A solution of the Et20-insoluble solid (1.38 g) in MeOH is evaporated vith appr. 25 g of silica gel. The povdered residue .is layered evenly onto the top of a silica gel column, vhich is then eluted vith 30 CHClj/MeOH/HOAc (95:5:1) to give the 2-methylthio by-product and the desired 2-amino product (IVA). (IVA) is recrystallized by extraction into boiling isopropyl acetate in a Soxhlet apparatus. The vhite crystals are collected in three crops and dried in vacuo over Ρ2θ5 at 110 ’C for 40 7h? yield 51.6%; mp >350 *C dec.; anal. calcd. for c17H22N4°*°*21MeOH,0*22H2O: cr 66.88; H, 7.59 ;·· N, 18.12.
Found: C, 66.86? Hf 7.59; N, 18.12. EXAKPLE 105 5 The compound o£ Example 104 is tested for enzyme inhibition activity. A purine nucleoside phosphorylase (PNP) enzyme assay is performed in vhich the PNP activity (IC50) for the compound is found, vhich is determined radiochemically by measuring the formation of [14C]-10 hypoxanthine from [14C]-inosine (see Blomedicine. 33, 39 (1980)) using‘calf spleen as the enzyme source. At 1 mM phosphate the IC50 is 0.090 μΜ, and at 50 mM phosphate the IC50 is 2.5 μΜ. . EXAMPLES 106-110 15 The folloving compounds of the present invention are prepared that are 2-&aino-7-(R)-3H,5H-pyrrolo(3,2-d]-pyrimidin-4-ones vherein R is -0¾-Ri in vhich the Rļ group is a 2-adamantyl group as follovs:
Example 106 Rļ = 2-(l-methyl)-adamantyl 20 Example 107 R^ = 2-(l-chloro)-adamantyl
Example 108 R^ = 2-(l-trifluoromethyl)-adamantyl Example 109 Rļ = 2-(l-methoxy) -adamantyl Example 110 Rļ =. 2-(l-fluoro)-adamantyl ..... The compounds are prepared folloving the procedures set 25 forth in Examples 98-104 using the appropriate 3-(2-adamantyl)-propio-nitriles as starting materiāls. EXAMPLES 111-116
The folloving compounds of the _ present invention are prepared that are 2-amino-7-(R)-3H,5H-pyrrolo[3,2-d]-30 pyrimidin-4-ones vherein R is -CH2-R1 which the Ri group is a l-adamantyl group as follovs:
Example 111 R]_ * l-(2-methyl) -adamantyl Example 112 Rj_ * l-(2-chloro)-adamantyl Example 113 R^ = 1-(2-trifluoromethyl)-adamantyl 41 LV 10100
Example 114 R]_ = l-(2-methoxy)-adamantyl
Example 115 R^ » l-(2-fluoro) -adamantyl
Example 116 Rļ = l-adamantyl
The compounds are prepared folloving the procedures set 5 forth in Examples 98-104 using the appropriate 3-(l-adamantyl)-propio-nitrilee as starting materiāls. ΕΧΑΜΡΙΖ 117 A pharmaceutical composition for intraperitoneal injection is prepared for testing the compound (IVA). An 10 intraperitoneal injection solution containing the compound (IVA) is dissolved in an agueous carrier that contains ten percent DMSO. EXAMPLE 118
The compound (IVA) is intraperitoneally injected into 15 Lewis Rats via the tēst composition of Example 117 to provide 30 mg of the compound (IVA), with an injection given tvice per day. Controls are used, vhich receive only the vehicle. At specific times after administration, the animals are sacrificed and plasma samples are prepared. 20 The plasma is extracted with cold 0.5 N HCIO4 and neutralized with solid NH4HCO3. After removal of perchlorate salts,· the extract‘ is subjected to HPLC on a reversed phase column (Spherisorb ODSI). A significant increase in plasma inosine is observed in the plasma taken 25 from animals receiving the compound (3TVA). E3CAMPLES 119-129
Compounds prepared as in Examples 106-116 are each made into a pharmaceutical formulation in accordance with the preparation of Example 117 and the resultant injectable 30. Solutions are tested in accordance with the procedure of Example 118. A significant increase in plasma inosine is observed in the plasma taken from animals receiving the compounds of the present invention. 42 ΕΧΑΜΡ1Ε 130 3-Cyclopentylpropionitrile is prepared in this example. 3-Cyclopentylpropionyl chloride (57.7 g, 0.36 mole) is added dropvise to a large excess of concentrated ammonium 5 hydroxide (400 ml) cooled in an ice/salt bath. The heavy suspension of vhite solid is stirred ovemight, collected by filtration, vashed with cold vater, and recrystallized from about 2 liters of boiling vater. The lustrous vhite plates of the amide are dried in vacuo over P205; yield 10 31.6 g (62.3t); mp 122 *C.
With protection from atmospheric moisture, a solution of the amide (23.5 g, 0.166 mole) in POCl3 (150 ml) is heated at 120 *C for 1 h. The oil bath is cooled to about 70 *C, and excess POCI3 distilled off under vacuum, and the 15 cooled residue is poured onto ice (about 300 g), The mixture is neutralized by cautious addition of solid Na2C03 and extracted vith several portions of Et20. The dried (Na2S04) extract is evaporated to give a clear, pale yellov oil vhich is distilled in vacuo to give the desired 20 nitrile; yieldf 16.86 g (82%) bp 88.0 - 88.5 *C/8.7 mm). MS (EI): m/z 122 (M-H)+; IR (cap. film), 2245cm"1(CN)? ^ NMR, S 1.67 (qf 2, -CH2CH2CN), 2.36 (t, 2, -CH2CN), complex "multiplets centered about 1.11, 1.63, 1.86 (cyclopentyl protons). 25 EXAMPLE 131 3-Cyclopentylpropionitrile of the previous example is further treated in the synthesis of the present invention. Under an atmosphere of dry N2/ a mixture of 3-cyclopentyl-propionitrile (14.8 g, 0.12 mole), sodium hydride (5,8, 30 0.24 mole), and anhydrous tetrahydrofuran (300 ml) is heated at 52 *C in a vater bath for 15 min., and a solution of ethyl formate (13.3 g, 0.18 mole) in THF (100 ml) is added over a period of 45 min. After tvo hours at 50 - 55 *C, a second portion of NaH (1.9 g) and HC02Et (5.0 ml) are 43 LV 10100 added, folloved in 30 min. by a third portion of HC02Et (unreacted nitrile is inert in the next step and can be recovered at the first purification step). The thick paste is stirred overnight and alloved to cool to room temperature. Volatile matter is evaporated under reduced pressure, and the residual pale yellov crust is dissolved in the minimum volume of cold vater (about 150 ml) at 0*C. The solution is adjusted to pH 6.0 by addition_ of 6N HC1 and extracted with CHCI3 (3 x 100 ml) * The extract is vashed vith H20, dried over Na2S04, and evaporated in vacuo to a thick amber oil, This crude product (15.6 g) is used in the next reaction vithout further purification. EXAMPLE 132
Glycine methyl ester hydrochloride (19.31 g, 0.154 mole) and anhydrous sodium acetate (12.61 g, 0.154 mole) are added to a solution of the crude formyl compound of the previous example (15.6 g) in MeOH/H20 (4:1, 500 ml). After 24 hours, the MeOH is evaporated in vacuo, and the mikture of vater and oil is extracted vith CHC13. The CHCI3 layer is dried (Na2S04) and evaporated to give an amber oil vhich is applied to a silica gel column. Elution vith CHC13 gavē tvo major bands:‘ '(1) 3-čyclopentylpropionitrile (8.22 g, 66.7 mmole or 55.6% of ' the nitrile used as starting material in the previous step), and (2) the desired enamine (3.45 g; 29.1% based on theoretical yield corrected for amount of nitrile present in starting material? MS (FAB): 223 (M + H)+). EXAMPLE 133 .
Under a nitrogen atmosphere, ethyl chloroformate (2.53 g, 23.3 mmole) is added dropvise to a solution of the enamine of the previous example (3.45 g, 15,5 mmole) and DBN (5.78 g, 46.6 mmole) in dry CH2C12 (50 ml) vith extemal cooling in an ice bath. After stirring at 0 *c for one hour, the solution is alloved to a stand at room temperature 44 ovemight. After checking progress by TLC, additional ClC02Et (0.5 ml) and DBN (3.0 al) are added to complete the conversion, and the solution is alloved to stand for 24 hours. Volatile matter is evaporated in vacuo, the viscous 5 residue purified on a short silica gel column (vhose main purpose is to remove the less-mobile DBN) to give an N- blocked pyrrole (4.50 g; 98%), vhich is used for the next step vithout further purification. ΕΧΑΜΡ1Ε 134 10 To a solution of the N-blocked pyrrole of the previous example (4.50 g, 15.3 mmole) in MeOH (100 ml) is added solid Na2C03 (1.62 g, 15.3 mmole), and the reactļon mixture is stirred at room tempera tūre for 48 hr vith separation of the resultant deblocked pyrrole. The mixture is evaporated 15 to dryness, and the residue is triturated thoroughly vith H20 (50 ml) to dissolve the inorganics and extracted vith CHC13 (3 x 100 ml). The extract is dried (Na2S04) and evaporated to give a viscous gum that *crystallized upon drying in vacuo; yield 2.97 g (87.4%) of material suitable 20 for use as an intermediate vithout further purification. More extensive purification can, hovever, be effected by using either column chromatogr?phy employing silica gel/CHCl3 or recrystallization from toluene/cyclohexane ^’(Vi3). 25 EXAMPLE 135
Benzoyl isothiocyanate (2.62 g, 16.03 mmole) is added dropvise to a solution of the unblocked pyrroleof Example 134 (2.97 g, 13.36 mmole) in dry CH2C12 (100 ml). After 1 h at room temperatūra, solution is evaporated, and the 3 0 gummy residue is dissolved in Et20 (100 ml) vith almost immediate separation of crystalline solid; yield 1.75 g. The Et20 filtrate is heated to boiling and diluted vith an equal volume of varm cyclohexane. On cooling slowly the solution gavē an additional 1.58 g of thioureido product; 45 uv 10100 total yield 3.33 g (64.6%). A small amount of the thioureido product is recrystallized from vara Et20/cyclohexane (15 ml each) ? mp 123 - 125 *c. MS (FAB): 386 (M‘ + H) + . Anal. Calcd, for C20H23N303S*0.45C6H12: C, 64.40; Hf 6.76; N, 9.93. Found: C, 64.51; H, '7.10; N, 9.93. EXAMPLE 136
Methyl iodide (2.60 g, 18.32 mmole) is added to a solution of the thioureido product of Example 135 (3.21 g, 8.33 mmole) and DBN (1.24 g, 9.99 mmole) in dry CH2Cl2 (80 ml) at 0 *C. The solution is stirred at 0 *C for 15 min., at ambient temperature for 1 h, and then evaporated in vacuo. A solution of the residue in CHC13 is chromatographed on a silica gel column with CHC13 as eluent to give homogeneous fractions of the methylthio intermediate compound; yield, 2.46 g (74%). EXAKPLE 137 A solution of the methylthio compound of Example 136 (2.07 g, 5.18 mmole) in 150 ml of MeOH that has been saturated with KH3 at 0 *C is heated at 90-95 *C for 24 hours in a glass-lined stainless Steel bomb. The contents of the .chilled bomb· are evaporated in vacuo to give a mixture of the ··compound (IVC), benzamide and a by-product that is a 2-methylthio derivative, as opposed to the 2-amino compound (IVC). The mixture is stirred vigorously for several minūtes with' appr. 75 ml of Et2o, and the insoluble vhite solid is filtered off and vashed with Et20. The filtrate contained most of the benzamide and 2-methylthio components. A solution of the Et20-insoluble solid (1.13 g) in MeOH is evaporated with appr. 10 g o’f silica gel. The povdered residue is layered evenly onto the top of a silica gel column, which is then eluted with CHCl3/MeOH/HOAc (95:5:1) to give the 2-methylthio by-product (252 mg; MS (FAB): 264 (M + H)+) and the desired 46 2-amino product (IVC) (679 mg, 56.4%). (IVC) is recrystallized by extraction into boiling isopropyl acetate in a Soxhlet apparatus. The vhite crystals are collected in three crops and dried in vacuo over Ρ205 at 110 *C for 5 7h; yield 540 mg (44.9%); mp 324 - 326 *C dec.; MS (FAB) i 233 ( M + H) + ; anal. calcd. for C12H16N40: C, 62.05; H, 6.94; N, 24.12. Found: C, 62.04; H, 7.11; N, 24.48. EXAMPLE 138
The compound of Example 137 is tested for enzyme-10 inhibition activity in accordance with the procedure of Example 9. At 1 mM.phosphate the IC50 is 0.029 μΜ, and at 50 mM phosphate the IC50 is 1.8 μΜ. EXAMPLES 139-142
The folloving compounds of the present invention are 15 prepared that are 2-amino-7-(R) -3H,5H-pyrrolo(3, 2-d]-pyrimidin-4-ones vherein R is -CH2-Rļ in the Rl gvoup is as follovs:
Example 139 R^ * 3-methylcyclopentyl Example 140 R^ = 2-chlorocyclopentyl 20 Example 141 Rļ = ‘3-triflouromethylcyclopentyl Example 142 R^ * 3-methoxycyclopentyl
The compounds are prepared folloving the procedures·set forth in Examples 130-137 using the appropriate 3-(substituted cyclopentyl) -propionitriles as starting 25 materiāls. EXAMPLE 143 A pharmaceutical composition for intraperitoneal injection is prepared for testing the compound (IVC). An intraperitoneal injection solution is prepared.containing 3 0 the compound (IVC) is dissolved in an aqueous carrier that contains ten percent DMSO. 47 LV 10100 EXAMPLE 144
Using the procedure of Example 112, the'compound (IVC) is intraperitoneally injected into Lewis Rats via the tēst composition of Example 143 and the results compared vith 5 Controls. A significant increase in plasma inosine is observed in the plasma taken from animals receiving the compound (IVC). ΕΧΑΜΡΙΕ 145 3-cyclohexylpropionitrile is prepared in this example. A 10 solution of cyclohexanepropionic acid (50 g; 0.32 mole) and thionyl chloride (152 g; 1.28 mole) in 100 ml benzene is alloved to stand overnight and is then evaporated to an oily residue. The residue is added in portions to 28% aqueous ammonia (270 ml) at 25*C and the mixture stirred 15 for about two hours. The resulting product is collected by filtration, vashed vith cold vater, and recrystallized from about 2 liters of boiling vater. The lustrous vhite plates of the amide are dried in vacuo over P205,· yield ·45.5 g.
With protection from atmospheric moisture, a solution of 20 the amide (45.5 g, 0.293 mole) in SOCI2 (200.3 g? 1.68 mole) refluxed for about six hours. The oil bath is cooled to about. 70 ..r,C, · and excess SOCl2 is distilled off under vacuum, and the cooled residue is poured onto ice (about 300 g). The mixture is neutralized by cautious addition of 25 solid Na2C03 and extracted vith several poirtions of Et20. The dried (Na2S04) extract is evaporated to give a clear, pale yellov oil vhich is distilled in vacuo to give the desired nitrile; yield 42.0 g. EXAMPLE 146 3 0 3-Cyclohexylpropionitrile of Example 14 5 is further treated. in the synthesis of the present invention. Under an atmosphere of dry N2, a mixture of 3-cyclohexyl-propionitrile (22.3 g, 0.16 mole), sodium hydride (5.38, 0.224 mole), and anhydrous tetrahydrofuran (120 ml) is 48 heated at 52 *C in a vater bath for 15 min., and a solution of ethyl formate (55.4 g, 0.75 mole) in THF (50 ml) is added over a period of 45 min. After two hours at 50 - 55 *C, a second portion of NaH (2.0 g) and HC02Et (5.0 ml) are 5 added (unreacted nitrile is inert in the next step and ca be recovered at the first purification step), and the reaction mixture·is stirred for about three days at 55’C and then alloved to cool to room tempera tūre. Volatile matter is evaporated under reduced pressure, and the 10 residual pale yellow crust is dissolved in the minimum volume of cold vater (about 75 ml) at 0 *C. The solution is adjusted to pH 6.0 by addition of 6N HC1 and extracted with CHCI3 (3 x 100 ml) . The extract is vashed with H20, dried over Na2S04, and evaporated in vacuo to a thick amber 15 oil. This crude product is used in the next reaction vithout further purification. EXAMPLE 147
Glycine methyl ester hydrochloride (30.60 g, 0.24 mole) and anhydrous sodium acetate (19.99 g, 0.24 mole) are added 20 to a solution of the crude formyl compound of the previous example (25.22 g) in Me0H/H20 (4:1, 500 ml). .After 24 hours, the MeOH is evaporated in vacuo, and the mixture of water and oil is extracted with CHCI3 · The CHC^-3 iayer is dried (Na2S04) and evaporated to give an amber oil vhich is 25 applied to a silica gel column. Elution vith CHCI3 gavē tvo major bands: (1) 3-cyclohexylpropionitrile (used as starting material in the previous step), and (2) the desired enamine; yield 16 g. EXAMPLE 148 30 Under a nitrogen atmosphere, ethyl chloroformate (1.38 g, 12.7 mmole) is added dropwise to a solution of the enamine of Example 147 (2.0 g, 8.46 mmole) and DBN (2.1 g, 16.9 mmole) in dry CH2C12 (50 ml) vith external cooling in an ice bath. After stirring at 0 *c for one hour, the 49 LV 10100 solution is alloved to a stand at room temperature overnight. After checking progress by TLC, additional ClC02Et (0.5 ml) and DBN (1.5 ml) are added to complete the conversion, and the solution is alloved to stand for 24 hours. Volatile matter is evaporated in vacuo, the viscous residue purified on a short silica gel column (vhose main purpose is to rernove the less-mobile DBN) to give an N-blocked pyrrole, vhich is used for the neiet step vithout further purification. EXAMPLE 149
To a solution of the N-blocked pyrrole of Example 148 (2.6 g, 8.43 mmole) in MeOH (100 ml) is added solid Na2C03 (2.23 g, 21.07 mmole), and the reaction mixture is stirred at room temperature for 48 hr with separation of the resultant deblocked pyrrole. The mixture is evaporated to dryness, and the residue is triturated thoroughly with H20 (50 ml) to dissolve inorganics and extracted vith CHCI3 (3 x 100 ml). The extract is dried (Na2S04) and evaporated to give a viscous gum, vhich is purified on a silica gel column using CHCI3 as the eluent; yield 1.67 g (84%); m.p. 73-74 *C. £XAMPLE 150
Berizoyl isothiocyanate (0.74 g, 4.02 mmole) " is added dropvise to a solution of the unblocked pyrrole of Example 149 (0.95 g) in dry CH2Cl2 (20 ml). After one hour at room temperature, the solution is evaporated, and the gummy residue is dissolved in Et20 (100 ml) with almost immediate separation of crystalline solid. The Et2o filtrate is heated to boiling and diluted vith an egual volume of varm cyclohexane. On cooling slowly the solution gives additional thioureido product; total yield 1.41 g (88%); m.p. 156-157 * C. 50 EXAMPLE 151
Methyl iodide (1.1 g, 7.6 mmole) is added to a solution of the thioureido product of Example 150 (0.96 g, 2.61 mmole) and l,5-diazabicyclo[4.3.0]non-5-ene (0.38 g, 3.0 5 mmole) in dry CH2CI2 (20 ml) at 0*C. The solution is stirred at 0*C for 15 min., at ambient temperature for 1 h, and then evaporated in vacuo. A solution of the residue in CHCI3 is chromatographed on a silica gel column with CHCI3 as eluent to give homogeneous fractions of the methylthio 10 intennediate compound; yield 0.92 g. EXAMPLE 152 A solution of the methylthio compound of Example 151 (0.8 g, 1.93 mmole) in 50 ml of HeOH that has been saturated with NH3 at 0 *C is heated at 90-95 *C for 24 15 hours in a glass-lined stainless Steel bomb. The contents of the chilled bomb are evaporated in vacuo to give a mixture of the compound (IVD), benzamide and a by-product that is a 2-aethylthio derivative, as opposed to the 2-amino compound (IVD). The mixture is stirred vigorously 2 0 for several minūtes vith appr. 75 ml of Et20, and the insoluble white solid is filtered off and vashed vith Et20. The filtrate contained most of the benzamide and 2-methylthio components. A solution of the Et20-insoluble solid (0.390 g) in MeOH is evaporated vith appr. 10 g of 25 silica gel. The povdered residue is layered evenly onto the top of a silica gel column, vhich is then eluted vith CHC13/MeOH/HOAc (95:5:1) to give the 2-methylthio by-product and the desired 2-amino product (IVD). (IVD) is recrystallized by extraction into boiling isopropyl acetate 3 0 in a Soxhlet apparatus. The vhite crystals are collected in three crops and dried in vacuo over P2O5 at 110 *C for 7h; yield 49%, mp >300 *C; anal. calcd. for C13H18N4O: C, 63.39, H, 7.36; N, 22.74. Found: C, 63.50; H, 7.74? N, 22.67. 51 LV 10100 EXAMPLE 153
The compound of Example 152 is tested for enzyme inhibition activity as in Example 105. At 1 mM phosphate the IC50 is 0.037 μΜ, and at 50 mM phosphate the IC5q is 2.2 μΜ. EXAMPLE 154
The compound of Example 152 is tested to determine its effectiveness in potentiation of the toxicity of 2'-deoxyguanosine (d-Guo) (see D. A. Schevach et al.( Cancer “es.. 46, 519 (1986), and J. C. Sircar et al., Aģents and i^ions. 21, 253 (1987)). CCRF-CEM celis are grovn in •RPMI-1640 raedium. To a suspension cultures of these celis, d-Guo at a fixed concentration (5.62 μΜ) and the compound at varied concentrations are added and the number of celis are determined in a Coulter counter 24, 48, and'72 hours thereafter. From these data, the IC50 is calculated to be 2.0 μΜ as the concentration of the compound required to reduce the increase in celi number betveen 0 and 72 hours to 50% of that of control cultures. . EXAMPLE 155
The compound 2-amino-7-(3-methylcyclohexylmethyl)-3g, 5Jī-pyrrolo(3,2-d]pyrimidin-4-one is prepared. First, using the procedures set forth in Examples 146-152 above, but with 3-(3-roethylbenzyl)-propionitrile as the starting material, the aryl derivative 2-amino-7-(3~methylbenzyl)-3H,5H-pyrrolo[3,2-d]-pyrimidin-4-one is made. A solution of the aryl derivative (0.2 g, 0.78 mmole) in trifluoroacetic acid (TFA) (20 ml) is hydrogenated with Pt02 at 60 lb/in2 for 24 *h. Catalyst is filtered off through a Celite bed, and the filtrate is evaporated. The residue is triturated with methanol and left in the refrigerator overnight. The resulting crystallized trifluoroacetate salt precipitates from the solution and is collected by filtration. The TFA salt is suspended in 8 ml 52 of H2O, adjusted to pH8 by conc. NH4OH and sonicated. The pure product is collected, vashed with H2O and dried: yield 165 mg (81%) ; jnp 282 *C. Anal: Calcd. for C14H20N4O: c, 64.60; H, 7.74; N, 21.52. Found: C, 64.24? H, 7.96? N, 5 21.51%. EXAMPLE 156
The procedure · described in Example 155 is repeated to prepare 2-amino**7- (3-trifluoromethylcyclohexyl-methyl) -3Η.* 5H.-pyrrolo-(3,2-d]pyrimidin-4-one using 3-(3- 10 trifluoromethylbenzyl)-propionitrile as the starting compound: yield 69%; mp 165 *C. Anal. calcd. for C14H17N40F3'°-6H20: c* 51.72? H, 5.64? N, 17.23. Found: C, 51.82? H, 5.71? N, 16.81%. EKAMPLE 157 15 The compound prepared in Example 155 is tested for enzyme-inhibition activity as in Example 105. · At 1 mM phosphate the IC50 is 0.025 μΜ, and at 50 mM phosphate the IC513 is 0.0.820 μΜ. EXAMPLE 158 2 0 The compound prepared in Example 156 is tested for enzyme-inhibition activity as in Example 105. At 1 mM phosphate the ICcn is 0.020 μΜ, and at 50 mM phosphate the ( ^ f Λ .,· V w IC50 is 0.740 μΜ. ΕΧΑΜΡΙΕ 159 25 ^ 3-Cycloheptylpropionitrile is prepared in this example according to the procedure of Example 97 using a solution of 2-bromocycloheptane (25.57 g; 144.38 mmole)? Bu3SnH (50.42 g? 173.26 mmole), acrylonitrile (15.32 g? 288.77 mmole), and AIBN (1.13 g) in toluene (300 ml). Yield is 30 16 g; mp oil. EXAMPLE 160 3-Cycloheptylpropionitrile of Example 159 j.s further treated in the synthesis of the present invention. Under an atmosphere of dry N2, a mixture of 3-cycloheptyl- 53 LV 10100 propionitrile (8.5 g, 56.19 mmole}, sodium hydride (2.6 g, 112.39 mmole), and anhydrous tetrahydrofuran (100 ml) is heated at 52 ’C in a vater bath for 15 min., and a solution of ethyl formate (20.81 g, 280.99 mmole) in THF (100 ml) is 5 added over a period of 45 min. After two hours at 50-55’C, a second portion of NaH (1.35 g) and HC02Et (10.4 g) are added, folloved in 30 min. by a third portion of HC02Et (unreacted nitrile is inert in the next step and can be recovered at the first purification step). The thick paste 1 is stirred overnight and allowed to cool to room temperature. Volatile matter is evaporated under reduced pressure, and the residual pale yellow crust is dissolved in the minimum volume of cold vater (about 150 ml) at 0*C. The solution is adjusted to pH 6.0 by addition of 6N HC1 15 and extracted with CHCI3 (3 x 100 ml). The extract is vashed vith H20, dried over Na2S04/ and evaporated in vacuo to a thick amber oil. This crude product is used in the next reaction vithout further purification. ΕΧΑΜΡΙΕ 161 20 Glycine methyl ester hydrochloride (9.35 g, 74.47 mmole) and anhydrous sodium acetate (6.10 g, 74.47 mmole) are added -to a solution of the crude formyl compound of Example 160 (8.9 g? 49.65 mmole) in MeOH/H20 (4:1, 250 ml). After 24 hours, the MeOH is evaporated in vacuo, and the mixture 25 of vater and oil is extracted vith CHCl3. The CHCI3 layer is dried (Na2S04) and evaporated to give an amber oil vhich is applied to a silica gel column. Elution vith CHCI3 gavē tvo major bands: (1) 3-cycloheptylpropionitrile (used as starting material in the previous step), and (2) the 30 desired enamine, vhich is recrystallized from a CHCl3/Et20 mixture; yield 6.18 g; m.p. 57-58*C. EXAMPLE 162
Under a nitrogen atmosphere, ethyl chloroformate (4.01 g, 37.03 mmole) is added dropvise to a solution of the enamine 54 of Example 161 (6.18 g, 24.69 mmole) and DBN (9.19 g, 74.04 mmole) in dry CH2C12 (100 ml) with external cooling in an ice bath. After stirring at 0 *C for one hour, the solution is alloved to a stand at room temperature 5 ovemight. After checking progress by TLC, .. additional ClC02Et (0.5 ml) and DBN (3.0 ml) are added to complete the conversion, and the solution is alloved to stand for 24 hours. Volatile matter is evaporated in vacuo, the viscous residue purified on a short silica gel column (whose main 10 purpose is to remove the less-mobile DBN) to give an N-blocked pyrrole, which is used for the next step vithout further purification.
EkAMPLE 163
To a solution of the N-blocked pyrrole of Example 162 15 (7.8 g, 24.19 mmole) in MeOH (100 ml) is added solid Na2C03 (6.41 g, 60.48 mmole), and the reaction mixture is stirred at room temperature for 48 hr with separation of the resultant deblocked pyrrole. The mixture is evaporated to dryness, and the residue is triturated thoroughly with H20 20 (50 ml) to dissolve inorganics and extracted with CHC13 (3 x 100 ml). The extract is dried (Na2S04) and evaporated to give · a viscous gum, which was purified" ’by column 'Chromatography employing silica gel/CHCl3; yie'ld 4 g; m.p. 88-89*0. 25 EXAMPLE 164
Benzoyl isothiocyanate (1.5 g; 8.96 mmole) is added dropvise to a solution of the unblocked pyrrole of Example 163 (1.99 g, 7.95 mmole) in dry CH2C12 (50 ml). After 1 h at room temperature, solution is evaporated, and the gummy 3 0 residue is dissolved in Et20 (100 ml) with almost immediate separation of the crystalline solid. The Et20 filtrate is heated to boiling and diluted with an equal volume of varm cyclohexane. On cooling slowly the solution gives 55 LV 10100 additional thioureido product; yield 2.89 g (88%); m.p. 158-159 *C. EXAHPLE 165
Hethyl iodide (1.7 g, 11.96 mmole) is added to a solution 5 of the thioureido product of Example 164 (1.7 g, 4.1 mmole) and DBN (0.56 g, 4.52 mmole) in dry CH2CI2 (30 ml) at 0 *C. The solution is stirred at 0*C for 15 min., at ambient temperature for 1 h, and then evaporated in vacuo. A solution of the residue in CHCI3 is chromatographed on a 10 silica gel column with CHCI3 as eluent to give homogeneous fractions of the methylthio intennediate compound. EXAMPLE 166 A solution of the methylthio .compound of Example 165 (1.72 g, 4.02 mmole) in 50 ml of MeOH that has been 15 saturated with HH3 at 0 *C is heated at 90-95 ’C for 24 hours in a glass-lined stainless Steel bomb. The contents of the chilled bomb are evaporated in vacuo to give a mixture of the compound (IVE), benzamide and a by-product that is a 2-methylthio derivative, as opposed to the 2-20 amino compound (IVE). The mixture is stirred vigorously· for several minūtes with appr. 75 ml of Et20, and the insoluble vhite solid is filtered off and vashed vith Et20. The filtrate .contained most of the benzamide and 2-methylthio components. A solution of the Et20-insoluble 25 solid (0.850 g) in MeOH is evaporated with appr. 10 g of silica gel. The povdered residue is layered evenly onto the top of a silica gel column, vhich is then eluted with CHClj/MeOH/HOAc (95:5:1) to give the 2-methylthio by-product and the desired 2-amino product (IVE). (IVE) is 30 recrystallized by extraction into boiling isopropyl acetate in a Soxhlet apparatus. The vhite crystals are collected in three crops and dried in vacuo over P205 at 110 *C for 7h; yield 54%, mp >300 *c dec.; anal. calcd. for C14H2oN40: 56 C, 64.60? Η, 7.74? N, 21.52. Found: C, 64.78? H, 8.01? N, 21.61. EXAMPLE 167
The compound of Example 166 , is tested for enzyme 5 inhibition activity as in Example 105. At 1 oM phosphate the IC50 is 0.030 μΜ, and at 50 mM phosphate the IC50 is 0.840 μΜ. EXAMPLE 168
Using the procedure of Example 97, 3-(l-norbomanyl)-10 propionitrile is made from 1-bromonorbomane, and 3-(2-norbornanyl)-propionitrile (mixture of 2-exo and 2-endo) is made from 2-bromonorbomane. Folloving Examples 98-104, the propionitriles are converted to the compounds (IVF), IV(G), and (IVH). 15 EXAMPLE 169
Using the procedure of Example 97, 3-(l-bicyclo-« ( 3 . 2.1]octanyl)-propionitrile, 3-(2-bicyclo-[3.2.1]octanyl)-propionitrile, 3-(3-bicyclo-[ 3.2.1]octanyl)-propionitrile, and 3-(8-bicyclo-20 [3.2.l]octanyl)-propionitrile are made respectively from 1- bromo-bicyclo [3.2.1] octane, 2-bromo-bicyclo [3.2.1] octane, 3-bromo-bicyclo(3.2.1]octane, and· 8-bromo-^. bicyclo[3.2.1]octane. Folloving Examples 98-104, the propionitriles are converted to the compound (IVL) and the 25 related 2-bicyclo[3.2.1]octanyl, 3-bicyclo[3.2.1]octanyl, and 8-bicyclo[3.2.1]octanyl derivatives. EXAMPLE 170
Using a modification of the procedure disclosed in D. Farcasiu, Svnthesis. 615 (1972), 6-bicyclo[3.2.l]octane-30 carboxaldehyde is prepared by reacting bicyclo[3.2.1]octan- 6-one vith trimethylsulfoxonium iodide, giving an intermediate epoxide, vhich is then converted to the aldehyde by treatment vith boron trifluoride etherate. Folloving the procedure of Netherlands Pat. 6,610,204, the 57 L.V 10100 aldehyde is condensed vith cyanoacetic acid by refluxing in a pyridene/toluene solution vith a catalytic quantity of ammonium acetate for 48-72 hours to give the corresponding acrylonitrile. The acrylonitrile is then hydrogenated 5 using a palladium-on-carbon catalyst in roethanol as taught in Profitt, et al, J. Org, Chem.. 40. 127 (1975) to give 3-(6-bicyclo-(3.2.l]octanyl)-propionitrile. Folloving Examples 98-104, the propionitrile is converted to the 6-bicyclo[3.2.1]octanyl derivative related to the compound .10 (IVL). EXAMPLE 171
Using the procedure of Example 97, 3-(l-bicyclo[3.3.1) nonanyl) -propionitrile and 3-(3-bicyclo-[3.3.1]nonanyl)-propionitrile are respectively made from l-bromo-15 bicyclo( 3.3.1] nonane and 3-bromo-bicyclo[3.3.1]nonane. Folloving Examples 98-104, the propionitriles are converted to the compound (IVM) and the related 3-bicyclo(3.3.1)-nonanyl derivative. EKAMPLE 172 20 Folloving the procedure of Example 171, bicyclo[3.3.1]-nonane-9-one is reacted to fonn the corresponding aldehyde, froro vhich is .. made the corresponding 3-substitutcd propionitrile, ,vhich is- then converted into the 9-• bicyclo(3.3.1]nonanyl derivative related to the compound 25 (IVM). EXAMPLE 173
Folloving the procedure of Example 170, 2-bicyclo[3.3.1]-nonanecarboxaldehyde is reacted to form the corresponding 3-substituted propionitrile, vhich is then converted into 30 the 2-bicyclo[3.3.1]nonanyl derivative related to the compound (IVM), EXAMPLE 174
Using the procedure of Example 97, 3-(l-noradamantyl)-propionitrile is made frora 1-bromonoradamantane, and 3-(2- « 58 noradamantyl)-propionitrile is made from 2-bromonoradamantane. Folloving Examples 98-104, the propionitrile is converted to the final compound (IVN). EXAMPLE 175
Folloving the procedure of Example 170, 3-noradamantane-carboxaldehyde is reacted to form the corresponding 3-substituted propionitrile, vhich is then converted into the 3-noradamantyl derivative related to the compound (IVN). EXAMPLE 176
Folloving the procedure of Example 170, noradamantane-7-one is reacted to form the corresponding aldehyde, from vhich is made the corresponding 3-substituted propionitrile, vhich is then converted into the 7-noradamantyl derivative related to the compound (IVN). EXAMPLE 177
Using the procedure of Example 97, 3-(l-bicyclo(2.2.2]-octanyl)propionitrile is made from l-bromobicyclo[2,2.2]-octane and 3-(2-bicyclo[2.2.2]octanyl)propionitrile is made from 2-bromobicyclo(2.2.2]octane. Folloving Examples 98-104, the propionitriles are converted to the compound (IVK) and the related 2-bicyclo[2.2.2]octanyl derivative. EXAMPLE 178
Using the procedure of Example 97, 3-(l-norbornenyl)-propionitrile is made from 1-bromonorbornene. Folloving Examples 98-104, the propionitrile is converted to the compound (IVI). EXAMPLE 179
Folloving the procedure of Example 170, 5-norborene-2-carboxaldehyde (a mixture of 2-endo and 2-exo) is reacted to form the corresponding 3-substituted propionitrile, vhich is then converted into the compound (IVJ). 59 LV10100 EXAMPLE 180 Cl
CN N Cl
The above intermediate compound is prepared" in this Example by the modification of the procedure of Schiemenz, G. P.; Engelhard, H. (Chem. Ber.. 1962, 2J5, 195). A mixture of cyanoacetic acid (25.38 g, 298.38 mmol) 2,3,5-trichlorobenzaldehyde (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 condenser. The solvents are evaporated in vacuo, residue is extracted with CHCI3, which is vashed with H20, ^ried (Na2S04), and evaporated to give the crude product, which is purified by silica gel column chromatography using hexane-EtOAc mixture as the eluent. Yield 23.69 g (73%); rap 90-91 ’C. ;EXAMPLE181
Cl
The above intermediate compound is prepared in this Example. To a stirred mixture of NaH (1.56 g, 65.05 mmol) and ethyl formate (14.78 g, 199.51 mmol) in THF (100 ml) is added substituted pentanedinitrile of Example 180 (10.17 g, 37.17 mmol) at room temperature under a nitrogen 60 atmosphere, and the resulting reaction mixture is stirred for 24 h. Volatile matter 1s evaporated in vacuo at room tempera tūre. Water (50 ml) is added to the residue at 0-5 *C, and the solution is adjusted to pH 5-6 by 20% conc. HC1 (v/v). The heavy oil is extracted into ethyl acetate, vashed with H2o (1 x 100 ml) and dried (MgS04). The ethyl acetate layer is .evaporated to give a red-brown oil (11.0 g) that is used in the next step vithout further purification. 60 EXAMPLE 182 Cl·
Cl Vn
The above intermediate compound is prepared in this Example. Glycine methyl ester hydrochloride (8.17 g, 65.06 mmol) and sodium acetate (5.33 g, 65.06 mmol) are added to a solution of the crude formyl compound of Example 181 (11.0 g) in a mixture of HeOH (80 ml) and H20 (20 ml), and the resulting solution is stirred at room tempsrature for 22 h. After evaporation of solvent at room tempera tūre, the residue is extracted with ethyl acetate. The vashed (H20) and dried (MgS04) organic layer is evaporated to give an oil. Flash column chromatography (silica gel) using CHCI3 as eluent gavē the pure desired enamine as a mixture of cis-trans isomers which is recrystallized from MeOH, yield 10.41 g (75%), mp 142-143 ’C. 61 LV10100 EXAMPLE 183
The above intermediate compound is prepared in this Exaimple. A solution of enamine of Example 182 (10.0 g, 26.84 mmol) in dry CH2CI2 (100 ml) is cooled to 0 ’C and treated with l,5-diazabicyclo[4.3.0]non-5-ene (10.53 g, 84.79 mmol) under a nitrogen atmosphere folloved by ethyl chloroformate (6.90 g, 63.57 mmol). The solution is stirred at 0 *C for 1 h and then at room tempera,tūre for 48 h. Volatiles are evaporated in vacuo to give a viscous dark gum- which is purified by flash column chromatography over silica gel using CHCI3 as the eluent. Ali the fractions containing the desired N-protected pyrrole are pooled and evaporated to give a foamy light pale yellov material which is stirred in MeOH (100 ml) to give the crystalline material which is recrystallized from CHCI3-MeOH, yield 8.92 g (74.7%), mp 160-161 *C. EXAMPLE 184- ··
The above intermediate compound is prepared in this Example. A suspension of N-protected pyrrole of Example 183 (8.6 g, 19.34 mmol) in MeOH (300 ml) is treated with
Na2C03 (5.12 g, 48.34 mmol) and the reaction mixture is stirred at room temperature for 17 h with separation of the deblocked pyrrole during the first hour. Solid sodium 62 carbonate is removed by filtration and vashed veli with MeOH. The filtrate is reduced to a volume of 25 ml and ķept in a refrigerator for 1 h to give 5.23 g of crystalline product. Further concentration of the mother liguor gavē an additional 0.14 g of pure product; total yield 6.45 g (89.5 %) , mp 178-181 *C. EXAMPLE 185 Cl H 0 ģ 1 t '’N. \ “NH- 1 Cl ‘OMe 10
The above intermediate compound is prepared in this 15 Example. To a suspension of pyrrole of Example 184 (5.83 g, 15.64 mmol) in dichloromethane (100 ml) ' is added benzoylisothiocyanate (2.88 g, 17.64 mmol) at room temperature under nitrogen. The reaction mixture is stirred for 30 min with the separation of the desired 20 thioureido compound. Additional benzoyl isothiocyanate (0.5 ml) is added to it and again stirred for 3 0 min. The solvent is evaporated to dryness, and the light yellow residue is triturated with methanol. The vhite crystalline material is isolated by filtration and recrystallized from 25 a chloroform-ether mixture to give the reguired thioureido compound as an analytically pure sample, yield 7.71 g (92%) , mp 210-211 *C. EXAMPLE 186 Cl un
Cl 30 63 LV 10100
The above intermediate compound is prepared in this Example. A solution of thioureido compound of Example 185 (6.75 g, 12.6 mmol) and l,5-diazabicyclo[4.3.0]non-5-ene (1.76 g, 14.20 mmol) in dry CH2CI2 (200 ml) is cooled to 0 •c and treated with methyl iodide (5.20 g, 36.65 mmol). The reaction mixture is stirred at 0 *C for 10 min and then for 1 h at room temperature. The solvent is evaporated at room temperature, and the residue is extracted with CHCI3, vashed with H2O (2 x 50 ml), dried (Na2S04) and evaporated to give a glassy foam (6.95 g) vhich is used in the next step vithout purification. EXAMPLE 187
CN
The above compounds A and B are prepared in this Example. The compound A is a compound of the present invention and the compound B is an intermediate. A solution of the methylthio intermediate of Example 186 (6.90 g, 12.54 mmol) in MeOH (200 ml) is saturated at 0 *C with ammonia and heated at 100 *C for 20 h in a glass-lined stainless Steel bomb. The reaction mixture is brought to room temperature and the solvent is evaporated at room temperature. Purification of the crude mixture by flash column chromatography over silica gel using CHCI3 as eluent gavē 8B (1.1 g, 21%), mp 290-291 *C then CHCl3-MeOH (95:5) gavē pure 8A (2.76 g, 57.5%), mp 284-285 *C. 64 EJCAMPLE 188
The compound of the present invention of Example 187 is tested for enzyme inhibition activity. A purine nucleoside phosphorylase (PNP) enzyme assay is performed in which the 5 PNP activity (ĪC5q) for the compound (8A) is found, which is determined radiocheaically by measuring the formation of [14C]-hypoxanthine fron [14C]-inosine (see Bloinedicine. 1980, 21, 39) using calf spleen as the enzyme source. At 1 mM phosphate the IC50 is 0.64 μΜ and at 50 mM phosphate the 10 IC5q is 10 μΜ. EXAMPLE 189
IC 20 —
Folloving the procedure set forth in Examples 180-187, 3-(3-chloropheny 1)-3-(2-amino-4-οκό-3 H, 5H-pyrrolo [3,2-d]pyrinidin-7-yl)propanenitrile is prepared using 3-(3-chlorophenyl)-pentanedinitrile as the starting material, 25 yield 54.5%, Op 157-158 *C. EXAMPLE 190
Folloving the procedure set forth in Examples 180-187, the folloving compounds are also prepared (1-9).
Ar N 30 65 LV 10100 3-Aryl-3-(2-amino-4-oxo-3H,5H-pyrrolo[3,2-d ]-pyrimidin-7-yl)propanenitrile Where Ar is each of the folloving: (1) phenyl, 2,3-dichlorophenyl, 3-methylphenyl, and 3-methoxyphenyl, (2) 5 thienyl (2- and 3-), (3) furanyl (2- and 3-), (4) pyridinyl (2-, 3-, and 4-), (5) pyrrolyl (2- and 3-), (6) thiazolyl (2-, 4-, and 5-), (7) 2-pyrazinyl, (8) pyridazinyl (3- and 4-), and (9) pyrazolyl. EXAMPLZ 191 10 Folloving the procedure set forth in Examples 180-187, the folloving compounds 10-14 and 21 are prepared starting from the appropriately substituted pentanedinitrile. Compounds 15-20, and 22 are prepared from the corresponding unsaturated Ar analogues in Example 190. In this 15 procedure, the nitrile group of the unsaturated analogue is first converted to an amide group by acid- 6r base-catalyzed hydrolysis, then the unsaturated Ar group is converted to the saturated R2 group by knovn catalytic hydrogenation, folloved by reconverting the amide back to 20 the nitrile by knovn dehydration procedures.
N 25 3-(Substituted)-3-(2-amino-4-oxo-3H,5H-pyrrolo[3,2-d]-30 pyrimidin-7-yl)propanenitrile
Where R2 is each of: 10) l-adamantyl, 11) 2-adamantyl, 12) cyclohexyl, 13) cycloheptyl, 14) cyclopentyl, 15) tetrahydrofurany1, 16) tetrahydrothienyl, 17) tetrahydropyranyl, 18) pyrazolidinyl, 19) thiazolidinyl, 5 66 20) piperazinyl, hexahydropyridazinyl. 21) morpholinyl, and 22) EXAMPLE 192
H
10 The above compound, d]pyrimidin-7-yl)-3“phenylpropanenitrile, is prepared in this Example. A solution of the compound A obtained in Example 187 (2.0 g, 5.22 mmol) in warm ethanol (250 ml) and 15 dimethylformamide (DMF) (150 ml) is hydrogenated over 30% Pd/C catalyst (1.0 g) in the presence of triethylamine (2.64 g, 5.0 equivalent) at atmospheric pressure. After 5 h, the reaction is complete, and the catalyst is filtered off under N2 pressure. The solid obtained by evaporation 20 of the filtrate is triturated and sonicated vith H2O and dried, yield 1.28 g (88%), mp 168-170 *C. EXAMPLE 193 i\ 3—(2—amino-4-oxo-3fi,5H-pyrrplo(3,2 25
The compound prepared in Example 192 is tested for enzyme inhibition activity as in Example 188. At 1 mM phosphate the IC50 is 0.023 μΜ and at 50 mM phosphate the IC50 is 4.7
30 67 LV1010C?
The above compound, 3-(2-amino-4-oxo-3H,5H-pyrrolo(3,2-d]pyrimidin-7-yl)-3-phenylpropanoic acid, is prepared in this example. A solution of the compound obtained in Example 192 (0.200 g, 0.72 mmol) in 6N HCl (3.0 ml) is 5 heated at reflux for 18 h. The solvent is evaporated ίη vacuo and the residue is triturated with H2O (6 ml), adjusted to pH 10 by conc. ammonium hydroxide. Insoluble material is collected by filtration and the filtrate is readjusted to pH 6.8. White material vhich is 10 precipitated out is collected, vashed vith vater and dried, yield 0.19 g (89%), mp 290 ’C dec. EXAMPLE 195
The compound prepared in Example 194 is tested for enzyme inhibition activity as in Example 188. At 1 mM phosphate 15 the IC5q is 0.012 μΜ and at 50 mM, phosphate the IC5q is 0.19 μΜ. EXAMPLE 196
20 H, 25 The above compound; 3-(2-amino-4-oxo-3H,5H-pyrrolo(3,2-d]pyrimidin-7-yl)-3-phenylpropanamide, is prepared in this example. A solution of the compound obtained in 'Example 192 (0.200 g, 0.72 mmol) in conc. H2SO4 (0.5 ml) is stirred at room temperature for 20 h and then poured onto crushed 30 ice (5.0 g) and adjusted to pH 6.8 by conc. NH4OH. The precipitated solid is collected, vashed vith H2O and dried, yield 0.180 g, mp 199-201 *C dec. 68 EXAMPLE 197
The compound prepared in Example 196 is tested for enzyme inhibition activity as in Example 188. At 1 mM phosphate the IC50 is 0.20 μΜ and at 50 mM phosphate the IC50 is 6.6 μΜ. EXAMPLE 198
OMe
The above compound, 3-(2-amino-4-oxo-3H,5H-pyrrolo(3,2-d]pyrimidin-7-yl)-3-phenylpropanoic acid, methyl ester, is prepared in this example. Thionyl chloride (0.2 g, 0.17 mmol) is added to stirred methanol (4.0 ml) at 0 *C. The compound obtained in Example 194 (0.2 g, 0.67 mmol) is added and the mixture is stirred at room temperature for 18 h. The solvent is removed on a vater aspirator (30*C) and vacuum pump (lyophilize) to give a semisolid mass vhich is purified on a silica gel column using CHClj-MeOH as the eluent, yield 0.1 g. EXAMPLE 199
The compound prepared in Example 198 is tested for enzyme inhibition activity. Significant activity (IC50) is found. EXAMPLE 200
69 LV10100 *» 3- (2-Amino-4-oxo-3ii, 5H-pyrrolo[3,2-d]pyrimidin-7-yl) 3-cyclohexylpropanoic acid is prepared in this example. A solution of the compound obtained in Example 194 (83 mg, 0.28 mmol) in trifluoroacetic acid (TFA) (15 ml) is 5 hydrogenated vith Pt02 (83 mg) at 60 lb/in2 for 24 h. The catalyst is filtered off through a Celite bed, and the filtrate is evaporated at 25 *C. The residue is suspended in H2O (8 ml), and adjusted to pH 8.5 by conc. NH40H and. filtered through a Whatman filter paper to remove brovn 10 colored impurities. The colorless filtrate is adjusted to pH 6.8, and the precipitated compound is filtered, vashed vith H2O, and dried, yield 65 mg (77%), mp >300 *C. EXAHPLE 201
The compound prepared in Example '200 is tested for enzyme 15 inhibition activity as in Example 188. At 1 mM phosphate the IC50 is 0.097 μΜ and at 50 mM phosphate the IC50 is 1.0 MM. EXAMPLE 202 A compound of the present invention is prepared vherein X 20 is PO(OH)2· The nitrile group of the compound of Example 192 is converted to the corresponding amide by treatment vith sulfuric acid. Using a Hoffman degradation reaction, the amide is converted to the corresponding amine, vhich is then converted to the corresponding pyridinium salt using a 25 pyrillium salt. Conversion of the salt to the corresponding halide is accomplished using sodium bromide, vhich is then converted to the phosphonic ester using triethyl phosphite. Hydrolysis of the ester using trimethylsilylbromide yields the corresponding phosphonic 30 acid vherein "n" is 1 and "mH is 0. EXAMPLE 203
This Example makes a compound of the present invention by stepping up the number of carbon atoms from "m" is 0 to "m" is 1. The nitrile group of the compound of Example 192 is 70 reduced to the corresponding aldehydef which is then converted to the corresponding alcohol. Using phosphorous tribromide the alcohol is converted to the corresponding alkyl bromide, which is then converted to the nitrile compound of the present invention vherein m is 1 using potassium cyanide. EXAMPLE 204
In this example a compound of the present invention is prepared vherein "p" is 1 and "Y" is oxygen. The alcohol prepared as an intermediate in the previous example is converted to the corresponding diethyl phosphonomethyl ether using diethylchloromethyl phosphonate. Removal of the ethyl groups of the ester is accomplished using trimethylsilylbromide to give the phosphonic acid. ΕΧΑΜΡΚΕ 205
In this example a compound of the present invention is made vherein "Y" is NH and "X” is s°2NH2 · The nitrile group of the compound of Example 192 is reduced to the amine using Standard catalytic hydrogenation vith palladium in acidic media (usually 0.01 N to 1 N HCl), vhich is then converted to the sulfamide using sulphamoyl chloride. EXAMPLE 206 '
In this example a compound of the present invention is prepared vherein "X" is COOH and MY" is NH by reacting the methyl amine intermediate prepared in the previous example vith chloroacetic acid. EXAMPLE 207
In this example a compound of the present invention is prepared vherein "X" is PO(OH)2 and "Υ" is NH by reacting the methyl amine intermediate prepared in Example 206 vith diethylchloromethyl phosphonate, and reacting the resulting product vith trimethylsilylbromide. 71 LV 10100 EXAMPLE 208
In this example a compound of the present invention is prepared vherein MXM is S02NH2 and UY" is oxygen by reacting the alcohol intermediate prepared in Example 203 5 with sulphamoyl chloride. EXAMPLE 209
In this example a compound of the present invention is prepared vherein R1 is H, R2 is phenyl/ R3 and R4 are hydrogen, n is 0, n is 1, p is 0, and X is CN. A 10 modification of the procedure disclosed in Mu-Ill Lim, et al., J. Ore. Chem.. Vol. 44. No. 22, 3826 (1979) is used. A mixture of the compound of Example 184 and dimethylforaamide dimethyl acetal is reacted at room temperature for tvo days and then evaporated to dryness ln 15 vacuo. The residue is crystallized to give the'pure N-(dimethylamino)methylene derivative, vhich is cyclized vith saturated methanolic ammonia to give the desired end product. EXAMPLE 210 20 In this example a compound of the present invention is prepared vherein R1 is 0CH3, R2 is phenyl, R3 and R4 are hydrogen, m is 0, n is 1, p is 0, and X is CN. Using the compound.B of Example 187, the S-methyl group is oxidized to methylsulfone, vhich then is converted to the final 25 methoxy compound by treatment vith sodium methoxide in methanol. EXAMPLE 211
In this example a compound of the present invention is prepared vherein X is tetrazole. The compound of Example 30 192 is treated vith lithium azide in the presence of ammonium chloride as a catalyst in dimethylformamide (DMF) at 100 degreeš C to give the desired tetrazole. 72 EXAMPLE 212
In this example a compound of the present invention is prepared vherein X is triazole. The compound of Example 198 is treated with hydrazine hydrate' to give the 5 corresponding hydrazide, vhich is then treated vith imino ether to give the desired triazole. EXAMPLE 213
The compound prepared in Example 189 is tested for enzyme inhibition activity as in Exaaple 188. At 1 bM phosphate 10 the IC50 is 0.012 μΜ and at 50 mM phosphate the IC50 is 2.0 μΜ. EXAMPLE 214
In this example an amidine compound of the present invention is prepared, i.e., vherein X in the recited 15 generic formula is CNHNH2. compound A from Example 187 is reacted vith sodium methoxide in methanol at room temperature for about 2 days to give a methyl-imidate intermediate. The intermediate is then reacted vith ammonia in methanol to give the amidine product. 73 LV 10100 WHAT IS CLAIKED IS: 1. A compound 2-amino-7- (R) — 3H; 5E“Pyrrolo [ 3,2-d] -pyrimidin-4-one vherein R is cyclohexenyl, cyclohexyl, or-Ci*2"Rl» and vherein Rj is an optionally substituted 5 heteroalicyclic, pyridinyl or alicyclic group. 2. The compound of claim 1 vherein R is -CH2“Ri and R^ is optionally substituted 2-, 3-, or 4-piperidinyl, 2- or 3-tetrahydrofuranyl, 2- or 3-tetrahydrothienyl, 2- or 3-pyrrolidinyl, 2-, 3-, or 4-tetrahydropyranyl, or 2-, 3-, 4- 10 pyridinyl. 3. The compound of claim 1 vherein R is optionally substituted cyclohexyl or 1-, 2-, or 3-cyclohexenyl. 4. The compound of claim 1 vherein R is -CH2-Ri and R^ is optionally substituted cyclopentyl, cycl'ohexyl, 15 cycloheptyl, l-or 2-adamantyl, 1- or 2-noradamantyl, 1-norbomanyl, 2-exo-norbomanyl, 2-endo-norbornanyl; 1- or 2-bicyclo[2.2.2]octanyl, 1-, 2-, 3-, 6-, or 8- bicyclo[3,2.1]octanyl, 1-, 2-, 3-, or 9- bicyclo[3.3. l]nonanyl, or 1- or 2- norbomenyl. 20 5. A compound of the formula
vherein R1 is H, NH2, or OCH3, R2 is an optionally 30 substituted cyclic group of 5-7 carbon atoms optionally containing one or more heteroatoms, R3 and R4 are independently H or Cļ^ alkyl, m is 0-4, n is 0-6, p is ΟΙ, X is CN, CSNH2, PO(OH)2, cooh, so2nh2, nh2, oh, cnhnh2, 74 tetrazole, or triazole, COR5 where R^ is Cļ.4 alkyl, CF3, NH2# or OCļ.4 alkyl, and Ϊ is O or NH. 6. The compound of claim 5 vherein R1 is NH2/ R2 and R4 are H, m is 0 and n is 1. 5 7. The compound of claim 5 vherein R2 is phenyl. 8. The compound of claim 5 vherein X is CN, COOH, or CONH2. 9. The compound of claim 5 vherein R2 is an optionally substituted 5- or 6-membered aromatic or heteroaromatic 10 group or an optionally substituted alicyclic group or heteroalicyclic group of 5-9 members. 10. λ method for the selective suppression of aammalian T-cell function vithout diminished effect on„ humoral immunity vhich comprises administering to a mammai the 15 compound of any of claims 1-8 or 9 vhereby said compound inhibits purine nucleoside phosphorylase and thereby T-cell formation. 11. A method for making a Chemical compound comprising the steps of: 20 a) reacting an optionally substituted cyclic aldehyde vith cyanoacetic acid in the presence of ammonium acetate to make a 3-cyclo-substituted pe'ntanedinitrile; b) reacting the 3-cyclo-pentanedinitrile vith an alkyl formate and a base to make a 3-cyclo-2- 25 formylpentanedinitrile; c) reacting the 3-cyclo-2-formylpentanedinitrile vith glycine methyl ester hydrochloride and sodium or ammonium acetate to make methyl K-[(3-cyclo-2,4-dicyano) -2-butenyl]glycine; 30 d) reacting the methyl N-[ (3-cyclo-2,4-dicyano) -2-butenyl]glycine vith an alkyl chloroformate and DBN or DBU to make methyl 3-amino-4-(2-cyano-l-cyclo-ethyl)-l-ethyl-lH-pyrrole-l,2-dicarboxylate; and 75 LV 10t e) reacting the methyl 3-amino-4-(2-cyano-l-cyclo-ethyl) = l-ethyl-lH-pyrrole-l,2-dicarboxylate with a base to Kaķe raethyl 3-amino-4- (2-cyano-l-cyclo-ethyl) -lH-pyrro],e“2 ·' carboxylate. 12. The method of claim 11 further comprising the of: f) reacting the.methyl 3-amino-4-(2-cyano-l-cyclo-ethyl)~ lH-pyrrole-2-carboxylate vith benzoylisothiocyanate to mako N-benzoyl-N ’ -[4-(2-cyano-l-cyclo-ethyl) -2-methoxycarbonyl° lH-pyrrol-3-yl3thiourea; g) reacting N-benzoyl-N'-[4-(2-cyano-l-cyclo-ethyi) -2° methoxycarbonyl-lH-pyrrol-3-yl}thiourea vith an alkyl halide to make N-benzoyl-N,-[4-(2-cyano-l-cyclo-ethyl)-2‘= methoxycarbonyl-lH-pyrrol-3-yl3S-methylthiourea? and h) reacting N-benzoyl-N,-[4-(2-cyano-l-cyclo-ethyl)-2=· methoxycarbonyl-lH-pyrrol-3-yl3-S-methylthiourea vith 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 Jpropanenitrile and 3-cyclo-3-[2-methylmercapto-4-oxo-3H,5H-pyrrolo[3,2-d 3pyrimidin-7-ylJ propanenitrile. 13. The method of claim 12 further comprising the steps of: i) reacting the 3-cyclo-3-[2-methylmercapto-4-oxo-3H,5H“· pyrrolo(3 , 2-d } pyriraidin-7-ylļpropanenitrile vith an oxidizing aģent 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 Jpropanenitrile vith sodium alkoxide to make 3-cyclo-3-[2-methoxy-4-oxo-3H,5H-pyrrolo[3,2-dJpyrimidin-7-yl]propanenitrile. 14. The method of claim 11 further comprising the steps of; f) reacting the methyl 3-amino-4-(2-cyano-l-cyclo-ethyl) -lH-pyrrole-2-carboxylate vith dimethylformamide dimethyl 76 acetal to make methyl 4-(2-cyano-l--cyclo-ethyl) (dimethylaminomethylene) anino] -lH-pyrrole-2-carboxylate; and g) reacting the methyl 4-(2-cyano-l-cyclo-ethyl)-3-[N-5 (dimethylaminoaethylene)aaino]-lH-pyrrole-2-carboxylate with aethanolic aaaonia to make 3-cyclo-3-[4-oxo-3H,5H-pyrrolo[3,2-d]pyriaidin-7-yl]propanenitrile. 15. The method of claia 11 vherein the cyclic substituent is phenyl, 2- or 3-thienyl, 2- or 3-furanyl, 10 2-, 3-, or 4-pyridinyl, 2- or 3-pyrrolyl, 2-, 4-, or 5-thiazolyl, 2-or 3-pyrazinyl, 3- or 4-pyridazinyl, 3-, 4-, or 5-pyrazolyl 1- or 2-adaaantyl, cyclopentyl, cyclohexyl, cycloheptyl, or aorpholinyl. 16. A coapound of the formula 15 20
HO CN
Et20C 0 25 77LV 10100
Me
78
vherein R1 and R2 are as defined claim 5

Claims (16)

LV 10100 Patentformulas punkti 1. Savienojums 2-amino-7-(R)-3H,5H-pirolo[3,2-d]pirimidīn-4-ons, kurā R ir cikloheksenilgrupa, cikloheksilgrupa vai grupa -CH^-R* (kur R* ir neobligāti nenoteikti aizvietota alicikliska, he-teroalicikliska grupa vai piridinilgrupai). 1 1Claims 1. Compounds of 2-amino-7- (R) -3H, 5H-pyrrolo [3,2-d] pyrimidin-4-one, wherein R is cyclohexenyl, cyclohexyl or -CH2 -R * ( wherein R * is an optionally unsubstituted alicyclic, heteroalicyclic or pyridinyl group). 1 1 2. Savienojums pēc punkta 1, kurā R ir grupa -CH^-R un R ir nenoteikti aizvietota 2-, 3- vai 4-piperidinilgrupa, 2- vai 3-tetra-hidrofuranilgrupa, 2- vai 3-tetrahidrotienilgrupa, 2- vai 3-pirolidi-nilgrupa, 2-, 3- vai 4-tetrahidropiranilgrupa, 2-, 3- vai 4-piridi-nilgrupa.A compound according to claim 1, wherein R is -CH 2 -R and R is an unsubstituted 2-, 3- or 4-piperidinyl, 2- or 3-tetrahydrofuranyl, 2- or 3-tetrahydrothienyl, 2- or 3-pyrrolidinyl, 2-, 3- or 4-tetrahydropyranyl, 2-, 3- or 4-pyridyl. 3. Savienojums pec punkta 1, kura R ir nenoteikti aizvietota cikloheksilgrupa vai 1^, 2- vai 3-cikloheksenilgrupa. 11The compound of claim 1, wherein R is an unsubstituted cyclohexyl group or a 1, 2, or 3-cyclohexenyl group. 11 4. Savienojums pēc punkta 1, kura R ir grupa -CH^-R un R . ir neobligāti aizvietota ciklopentilgrupa, cikloheksilgrupa, ciklohep-tilgrupa, 1- vai 2-adamantilgrupa, 1- vai 2-noradamantilgrupa, 1-nor-bornanilgrupa, 2-ekso-norbornanilgrupa, 1- vai 2-biciklo^.2.2Jokta- nilgrupa, 1-, 2-, 3-, 6- vai 8-biciklo[3.2. ljoktanilgrupa, 1-, 2-, 3-vai 9-biciklo[3.3.1]nonanilgrupa, 1- vai 2-norbornenilgrupa.The compound of claim 1, wherein R is -CH 2 -R and R 1. is optionally substituted cyclopentyl, cyclohexyl, cyclohep-til, 1- or 2-adamantyl, 1- or 2-noradamantyl, 1-nor-bornanyl, 2-exo-norbornanyl, 1- or 2-bicyclo [2.2.2] octane, 1-, 2-, 3-, 6- or 8-bicyclo [3.2. 1-octanyl, 1-, 2-, 3-or 9-bicyclo [3.3.1] nonanilyl, 1- or 2-norbornenyl. 5. Savienojums ar formulu (I)5. A compound of formula (I) kura: R1 ir H, NH2 vai 0CH3; 2 R ir neobligāti aizvietota cikliska grupa ar 5 - 7 oglekļa atomiem, kas neobligāti satur vienu vai vairākus heteroatomus; 3 4 R un R neatkarīgi viens no otra ir ūdeņraža atoms vai C^_^-al-kilgrupa; m ir 0 - 4; n ir 0 - 6; p ir 0 - 1; X ir CN, CSNH2,P0(0H)2, COOH, SC>2NH2, NH2> OH, C(=NH)NH2, tetr- azolilgrupa, triazolilgrupa, grupa COR^, kurā s R ir C^^-alkilgrupa, CF^, NH2» OC^-alkilgrupa; Y ir 0 vai NH. LV 10100 1 3 4wherein: R 1 is H, NH 2 or OCH 3; 2 R is an optionally substituted cyclic group of 5 to 7 carbon atoms optionally containing one or more heteroatoms; R 4 and R 4 are independently hydrogen or C 1-4 alkyl; m is 0 to 4; n is 0 to 6; p is 0 to 1; X is CN, CSNH2, P0 (O) 2, COOH, SC > 2NH2, NH2 > OH, C (= NH) NH 2, tert-azolyl, triazolyl, COR 1, wherein s R is C 1-4 alkyl, CF 4, NH 2, OC 4 -alkyl; Y is 0 or NH. EN 10100 1 3 4 6. Savienojums pēc punkta 5, kura R ir NH^, R un R ir H, Jv m ir 0 unMr 1. 26. A compound according to claim 5, wherein R is NH 4, R and R are H, Jv m is 0 and Mo 1 2 7. Savienojums pēc punkta 5, kurā R ir fenilgrupa.The compound of claim 5, wherein R is phenyl. 8. Savienojums pēc punkta 5, kurā X ir CN, C00H vai C0NH2. 2A compound according to claim 5 wherein X is CN, C00H or C0NH2. 2 9. Savienojums pec punkta 5, kurā R ir nenoteikti aizvietota 5- vai 6-locekļu aromātiska vai heteroaromātiska grupa, vai nenoteikti aizvietota alicikliska grupa vai heteroalicikliska grupa ar 5 - 9 atomiem ciklā.The compound of claim 5, wherein R is an unsubstituted 5- or 6-membered aromatic or heteroaromatic group, or an unsubstituted alicyclic group or heteroalicyclic group having 5 to 9 atoms per cycle. 10. selektīvai T-limfocītu funkciju pavājināšanai zīdītāju organismā bez humorālās imunitātes pavājināšanas, kas atšķiras ar to, ka zīdītājam ievada savienojumu pēc jebkura no punktiem 1-9, kas inhibē purinu nukleozīdfosforilāzi un līdz ar to T-limfocītu veidošanos.10. selectively attenuating T-lymphocyte functions in a mammal without impairing humoral immunity, characterized in that the compound is administered to a mammal according to any one of claims 1-9, which inhibits purine nucleoside phosphorylase and hence T-lymphocyte formation. 11. ķīmiskā savienojuma iegūšanai, kas atšķiras ar to, ka veic: a) nenoteikti aizvietota cikliskā aldehīda reakciju ar ciān-etiķskābi amonija acetāta klātienē, iegūstot 3-cikloaizvietotu pentāndinitrilu; b) 3-cikloaizvietota pentāndinitrila reakciju ar alkilformiātu bāzes klātienē, iegūstot 3-cikloaizvietotu-2-formilpentāndinitrilu; c) 3-cikloaizvietota-2-formilpentāndinitrila reakciju ar gli-cīna metilestera hidrogenhlorīdu un nātrija vai amonija acetātu, ie- gūstot N-[(3-cikloaizvietotu-2,4-diciān)-2-butenil]glicīna metil-esteri; d) N-[(3-cikloaizvietota-2,4-diciān)-2-butenil]glicīna m$til-estera reakciju ar hlorskudrskābes alkilesteri DBN vai DBU klātienē, iegūstot 3-amino-4-(2-ciān-l-cikloaizvietotu-etil)-l-etil-lH-plrol- 1.2- dikarbonskābes metilesteri; e) 3-amino-4-(2-ciān-l-cikloaizvietota-etil)-l-etil-lH-pirol- 1.2- dikarbonskābes metilestera reakciju ar bāzi, iegūstot 3-amino-4-(2-ciān-l-cikloaizvietotu-etil)-l-etil-lH-pirol-2-karbonskābes metilesteri.11. For the preparation of a chemical compound, which comprises: a) reacting an indeterminate substituted cyclic aldehyde with cyanoacetic acid in the presence of ammonium acetate to give 3-cyclo-substituted pentanenitrile; b) reacting the 3-cyclo-substituted pentanenitrile with an alkylformate-based in situ to give 3-cyclo-substituted-2-formylpentanenitrile; c) reaction of 3-cyclo-substituted-2-formylpentanenitrile with glycine methyl ester hydrogen chloride and sodium or ammonium acetate to give N - [(3-cyclo-substituted-2,4-dicyano) -2-butenyl] glycine methyl ester; d) Reaction of N - [(3-cyclo-substituted-2,4-dicyano) -2-butenyl] glycine m $ tert ester with chloro-acid acid alkyl ester in DBN or DBU to give 3-amino-4- (2-cyano-l- cyclo-substituted-ethyl) -1-ethyl-1H-plrol-1,2-dicarboxylic acid methyl ester; e) Reaction of 3-amino-4- (2-cyano-1-cyclo-substituted-ethyl) -1-ethyl-1H-pyrrole-1,2-dicarboxylic acid with a base to give 3-amino-4- (2-cyano-l- cyclo-substituted-ethyl) -1-ethyl-1H-pyrrole-2-carboxylic acid methyl ester. 12. Pēc punkta 11, kas atšķiras ar to, ka veic šādas papildreakcijas: f) 3-amino-4- (2-ciān-l-cikloaizvietota-etil)-l-etil-lH-pirol- 2-karbonskābes metilestera reakciju ar benzoilizotiocianātu, iegūstot N-benzoil-N’ -[4-(2-ciān-l-cikloaizvietotu-etil)-2-metoksi-karbonil-lH-pirol-3-il] tiourīnvielu; g) N-benzoil-N’-[4-(2-ciān-l-cikloaizvietotas-etil)-2-metoksi-karbonil-lH-pirol-3-il]tiourīnvielas reakciju ar alkilhalogenīdu, iegūstot N-benzoil-N’ -[4-(2-ciān-l-cikloaizvietotu-etil)-2-metoksikar-bonil-lH-pirol-3-il]-S-alkil tiourīnvielu; h) N-benzoil-N’-[4-(2-cian-l-cikloaizvietotas-etil)-2-metoksi-karbonil-lH-pirol-3-il]-S-alkiltiourīnvielas reakciju ar amonjaka šķīdumu metanolā vai etanolā, iegūstot 3-cikloaizvietota-3-(2-amino-4-okso-3H,5H-pirolo[3,2-d]pirimidin-7-il)propānnitrila un 3-cikloaiz-vietota-3-(2-metilmerkapto-4-okso-3H, 5H-pirolo[3,2-d]pirimidin-7-il)-propānnitrila maisījumu.12. The method of claim 11, further comprising the following reaction: f) reaction of 3-amino-4- (2-cyano-1-cyclohexyl-ethyl) -1-ethyl-1H-pyrrole-2-carboxylic acid with methyl ester; benzoyl isothiocyanate to give N-benzoyl-N '- [4- (2-cyano-1-cycloalkyl-ethyl) -2-methoxy-carbonyl-1H-pyrrol-3-yl] -thiourea; g) Reaction of N-benzoyl-N '- [4- (2-cyano-1-cyclo-substituted-ethyl) -2-methoxy-carbonyl-1H-pyrrol-3-yl] -thiourea with alkyl halide to give N-benzoyl-N' - [4- (2-cyano-1-cycloalkyl-ethyl) -2-methoxycarbonyl-1H-pyrrol-3-yl] -S-alkyl-thiourea; h) Reaction of N-benzoyl-N '- [4- (2-cyano-1-cyclo-substituted-ethyl) -2-methoxy-carbonyl-1H-pyrrol-3-yl] -S-alkylthiourea with ammonia solution in methanol or ethanol; to obtain 3-cyclo-substituted-3- (2-amino-4-oxo-3H, 5H-pyrrolo [3,2-d] pyrimidin-7-yl) propanenitrile and 3-cyclohexyl-3- (2-methyl-mercapto-4) -oxo-3H, 5H-pyrrolo [3,2-d] pyrimidin-7-yl) -propanenitrile. 13. Panemiens pēc punkta 12, kas atšķiras ar to, ka veic šādas papildreakcijas: i) 3-cikloaiZ/'Vietota-3-(2-metilmerkapto-4-okso-3H, 5H-pirolo-[3,2-d]pirimidin-7-il)propānnitrila reakciju ar oksidētāju, iegūstot 3-cikloaizvietotu-3-(2-metilsulfonil-4-okso-3H,5H-pirolo [3,2-dlpiri-midin-7-il)propānnitrilu; j) 3-cikloaizvietota-3-(2-metilsulfonil-4-okso-3H,5H-pirolo-[3,2-d]pirimidin-7-il)propānnitrila reakciju ar nātrija alkoksīdu, iegūstot 3-cikloaizvietotu-3-(2-metoksi-4-okso-3H,5H-pirolo[3,2-d]-pirimidin-7-il)propānnitrilu.13. The method of claim 12, further comprising the following steps: i) 3-CycloheZ / 'Vietota-3- (2-methylcapto-4-oxo-3H, 5H-pyrrolo [3,2-d]] pyrimidin-7-yl) -propanenitrile reaction with an oxidant to give 3-cyclo-substituted-3- (2-methylsulfonyl-4-oxo-3H, 5H-pyrrolo [3,2-d] pyrimidin-7-yl) -propanenitrile; j) reaction of 3-cyclo-substituted-3- (2-methylsulfonyl-4-oxo-3H, 5H-pyrrolo [3,2-d] pyrimidin-7-yl) -propanenitrile with sodium alkoxide to give 3-cyclo-substituted-3- ( 2-Methoxy-4-oxo-3H, 5H-pyrrolo [3,2-d] -pyrimidin-7-yl) -propanenitrile. 14. Paņēmiens pēc punkta 11, kas atšķiras ar to, ka veic šādas papildreakcijas: f) 3-amino-4- (2-ciān-l-cikloaizvietota-etil)-l-etil-lH-pirol- 2-karbonskābes metilestera reakciju ar dimētilformamīda dimēti lace tālu, iegūstot 4-(2-ciān-l-cikloaizvietotu-etil)-3-[N-(dimetil-aminometi 1 ēn)amino]-1 H-pirο 1 -2-karbonskābes metilesteri; g) 4-(2-ciān-l-cikloaizvietota-etil)-3-[N-(dimetil-aminometi-lēn)amino]-lH-pirol-2-karbonskābes metilestera reakciju ar amonjaka šķīdumu metanolā, iegūstot 3-cikloaizvietotu-3-(4-okso-3H,5H-pirolo-[3,2-d]pirimidin-7-il )propānnitrilu.14. A process according to claim 11, wherein the following additional reactions are performed: f) reaction of 3-amino-4- (2-cyano-1-cyclohexyl-ethyl) -1-ethyl-1H-pyrrole-2-carboxylic acid methyl ester with dimethylformamide dimmed lace to give 4- (2-cyano-1-cyclohexyl-ethyl) -3- [N- (dimethylaminomethyl-ene) amino] -1H-pyrrole-2-carboxylic acid methyl ester; g) Reaction of 4- (2-cyano-1-cyclohexyl-ethyl) -3- [N- (dimethylaminomethylene) amino] -1H-pyrrole-2-carboxylic acid with methyl ammonia solution to give 3-cyclo-substituted. 3- (4-Oxo-3H, 5H-pyrrolo [3,2-d] pyrimidin-7-yl) propanenitrile. 15. Paņēmiens pēc punkta 11, kas atšķiras ar to, ka cikliskais aizvietotājs ir fenilgrupa, 2- vai 3- tienilgrupa, 2- vai 3-furanilgrupa, 2-, 3- vai 4-piridinilgrupa, 2- vai 3-pirolilgrupa, 2-, 4- vai 5-tiazolilgrupa, 2- vai 3-pirazinilgru'pa, 3- vai 4-pirid-azinilgrupa, 3-, 4- vai pirazolilgrupa, 1- vai 2-adamantilgrupa, cik- lopentilg'rupa, cikloheksilgrupa, cikloheptilgrupa vai morfolinilgrupa.15. A process according to claim 11, wherein the cyclic substituent is phenyl, 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-pyrazinylgrip, 3- or 4-pyridazinyl, 3-, 4- or pyrazolyl, 1- or 2-adamantyl, cyclopentyl, cyclohexyl, cycloheptyl or morpholinyl. 16. Savienojumi, kuros R 1 un R nozīmes atbilst punkta 5 dotām: R2 :nr* CN HO16. Compounds in which R 1 and R have the meanings given in 5: R 2: NO * CN HO CN LV 10100CN LV 10100 MeWe
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US07/429,097 US5008270A (en) 1989-10-31 1989-10-31 2-amino-7-(heterocyclomethyl)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-ones and pharmaceutical uses and compositions containing the same
US07/429,099 US5008265A (en) 1989-10-31 1989-10-31 2-amino-7-(alicyclomethyl)-3H,5H,-pyrrolo[3,2-d]pyrimidin-4-ones and pharmaceutical uses and compositions containing the same
US07/429,098 US4985434A (en) 1989-10-31 1989-10-31 7-substituted derivatives of 2-amino-3H,5H-pyrrolo(3,2-d)pyrimidin-4-ones and pharamceutical uses and compositions containing the same
US07/429,100 US4985433A (en) 1989-10-31 1989-10-31 2-amino-7-(pyridinylmethyl)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-ones and pharmaceutical uses and compositions containing the same
US44279889A 1989-11-29 1989-11-29
PCT/US1990/005756 WO1991006548A1 (en) 1989-10-31 1990-10-12 Inhibitors of purine nucleoside phosphorylase

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