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WO1998030563A1 - 2-hydroxymethylecyclopropyli- denemethylpurines et -pyrimidines utilises comme agents antiviraux - Google Patents

2-hydroxymethylecyclopropyli- denemethylpurines et -pyrimidines utilises comme agents antiviraux Download PDF

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Publication number
WO1998030563A1
WO1998030563A1 PCT/US1998/000440 US9800440W WO9830563A1 WO 1998030563 A1 WO1998030563 A1 WO 1998030563A1 US 9800440 W US9800440 W US 9800440W WO 9830563 A1 WO9830563 A1 WO 9830563A1
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WIPO (PCT)
Prior art keywords
amino
purine
syn
adenine
hydroxymethylcyclopropylidenemethyl
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PCT/US1998/000440
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English (en)
Inventor
Jiri Zemlicka
Yao-Ling Qiu
John C. Drach
Roger G. Ptak
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The Regents Of The University Of Michigan
Wayne State University
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Application filed by The Regents Of The University Of Michigan, Wayne State University filed Critical The Regents Of The University Of Michigan
Publication of WO1998030563A1 publication Critical patent/WO1998030563A1/fr
Priority to US09/267,839 priority Critical patent/US6352991B1/en
Priority to US10/047,202 priority patent/US6790841B2/en
Priority to US10/913,219 priority patent/US6958345B2/en
Priority to US11/257,776 priority patent/US20060122203A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more 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, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/47—One nitrogen atom and one oxygen or sulfur atom, e.g. cytosine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more 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, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
    • C07F9/6503—Five-membered rings
    • C07F9/6506—Five-membered rings having the nitrogen atoms in positions 1 and 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs

Definitions

  • the present invention relates generally to novel purine and pyrimidine compounds which have antiviral activity and methods of making and using same.
  • herpes viruses such as herpes simplex 1 (HSV-1 ), herpes simplex 2 (HSV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella zoster virus (VZV) and human herpes virus 6 (HHV 6) which are associated with many common viral illnesses.
  • HSV-1 and HSV-2 infections are characterized by cold sores of skin, mouth or genital region. After primary infection the virus is harbored in neural cells and can reappear later in the life of a patient.
  • HCMV infection Human CMV (HCMV) infection is a life-long affliction which can result in morbidity and mortality. These pathologies include microcephaly, hepatosplenomegaly, jaundice, encephalitis, infections of the newborn infants or fetuses in utero, and infections of immunocompromised hosts.
  • the HCMV infection is responsible for retinitis, gastritis and pneumonitis in AIDS patients and HCMV-induced pneumonias or hepatitis are frequent and serious complications of bone marrow transplants.
  • EBV causes infectious mononucieosis and it is considered as the etiologic agent of nasopharyngeal cancer, immunoblastic lymphoma, Burkitt's lymphoma and hairy leukoplakia.
  • VZV causes chicken pox and shingles. Although in children the chicken pox is usually a non-fatal disease, the recurrent form of this infection, shingles, may in advanced stage lead to paralysis, convulsions and ultimately death. Again, in immunocompromised patients the infection with VZV is a serious complication.
  • Human herpes virus 6 which is commonly associated with children's rash was also identified in acquired immunodeficiency syndrome (AIDS) patients and it may be a cofactor in the pathogenesis of AIDS in hosts infected with human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • Levine, A.J. Viruses, Ch. 4, W. H. Freeman & Co., New York, pp. 67-85 (1992); Human Herpesvirus Infections, Raven Press, New York (1986).
  • HIV is the underlying cause of AIDS, a world-wide epidemic with fatal consequences. According to the World Health Organization, over 4.5 million AIDS cases were recorded by late 1994 and 19.5 million people had been infected with HIV.
  • acyclovir Zovirax
  • ganciclovir Cytovene
  • Acyclovir Therapy for Herpesvirus Infections (Baker, Ed.), M. Dekker, New York, (1990); Ganciclovir Therapy for Cvtomegalovirus Infection (Spector, S. S., Ed.), M. Dekker, New York (1991).
  • a considerable effort went into design, synthesis and biological investigation of analogues of these drugs as well as in development of new antiviral agents.
  • viruses including HCMV, HSV-1 , HSV-2, HHV-6, HIV, hepatitis B virus (HBV), and other mammalian viruses.
  • the present invention provides novel 2-hydroxymethylcyclopropylidenemethyl- derivatives of heterocyclic compounds, prodrugs and pharmacologically acceptable acid salts thereof. These compounds have been found to be useful antiviral agents and are effective against HCMV, HSV-1 , HSV-2, HHV-6, HIV and HBV, as well as against other mammalian viruses.
  • the compounds of the present invention have the following Formulas:
  • B is a heterocyclic ring derived from a purine or pyrimidine moiety.
  • the purine moieties include 6-aminopurine (adenine), 2,6- diaminopurine, 2-amino-6-cyclopropylaminopurine, 6-hydroxypurine (hypoxanthine), 2-amino-6-halo substituted purines, such as 2-amino-6-chloropurine, 2-amino-6- alkoxy substituted purines, such as 2-amino-6-methoxypurine, 2-amino-6- hydroxypurine (guanine), 3-deazapurines, 7-deazapurines, 8-azapurines, cytosine, 5-halo substituted cytosines, 5-alkyl substituted cytosines, thymine, uracil and 6- azapyrimidines.
  • Prodrugs of the antiviral nucleoside analogues of the present invention include lipophilic phosphate esters or amidates capable of penetrating the cell membrane. Those skilled in the art will appreciated that the aim of prodrugs is to provide effective therapeutic agents for resistant strains of viruses (McGuigan, C, et al., J. Med. Chem. 36:1048-1052 (1993)) or activate inactive analogues. Franchetti, P., et al., J. Med. Chem. 37:3534-3541 (1994).
  • the compounds of the present invention therefore also include prodrugs of the novel compounds, wherein the prodrugs have the following Formulas:
  • B is a heterocyclic ring as defined above for Formulas 1 and 2; X is O; and
  • R 1 and R 2 are alkyl or aryl.
  • the R.,X or R 2 X may also be amino acid residues with X as NH.
  • compositions useful for treating viral infections such as HCMV, HSV-1 , HSV- 2, HHV-6, HIV and HBV contain an effective amount of at least one compound of Formulas 1 to 4 or a pharmaceutically acceptable salt thereof.
  • the present invention also includes methods for synthesizing compounds of Formulas 1 and 2 wherein B is a heterocyclic ring derived from purine or pyrimidine moiety such as 6-aminopurine (adenine), 2,6-diaminopurine, 2-amino-6- cyclopropylaminopurine, 6-hydroxypurine (hypoxanthine), 2-amino-6-halo substituted purines, such as 2-amino-6-chloropurine, 2-amino-6-alkoxy substituted purines, such as 2-amino-6-methoxypurine, 2-amino-6-hydroxypurine (guanine), 3- and 7- deazapurines, such as 3- and 7-deazaadenine, 8-azapurines such as 8-azaadenine, cytosine, 5-halocytosine (wherein halo is bromo, chloro, iodo or fluoro) and related alkyl derivatives containing a saturated or unsaturated alkyl group at
  • the present invention also provides methods for synthesizing prodrugs of the compounds as set forth in Formulas 3 and 4.
  • Figure 1 shows the synthesis of the mixture of c/s-ethyl 2-bromo-2-bromo- methylcyclopropane 1-carboxylate (6) and trans-ethyl 2-bromo-2-bromomethylcyclo- propane 1 -carboxylate (7);
  • Figure 2 shows the synthesis of purine and pyrimidine 2-hydroxymethyl- cyclopropylidenemethylpurines and -pyrimidines of Formulas 1 and 2;
  • the compounds of the present invention which have been found to be effective against herpes viruses, human immunodeficiency virus, and hepatitis B virus, are compounds of Formulas 1 to 4, wherein B represents a heterocyclic ring derived from purine or pyrimidine moiety such as 6-aminopurine (adenine), 2,6- diaminopurine, 2-amino-6-cyclopropylaminopurine, 6-hydroxypurine (hypoxanthine), 2-amino-6-halo substituted purines, such as 2-amino-6-chloropurine, 2-amino-6- alkoxy substituted purines, such as 2-amino-6-methoxypurine, 2-amino-6- hydroxypurine (guanine), 3- and 7-deazapurines, such as 3- and 7-deazaadenine, 8-azapurines such as 8-azaadenine, cytosine, 5-halocytosine (wherein halo is bromo, chloro, iodo,
  • the preferred compounds of the present invention are syt?-N 9 -(2-hydroxy- methylcyclopropylidenemethyl)adenine (synadenol), wherein in Formula 1, B is adenin-N 9 -yl, syn-N -(2-hydroxymethylcyclopropylidenemethyl)guanine (synguanol), wherein B is guanin-N 9 -yl, sy/?-N 1 -(2-hydroxymethylcyclopropylidenemethyl)cytosine
  • B is cytosin-N 1 -yl, sy/7-2,6-diamino-N 9 -(2- hydroxymethylcyclopropylidenemethyl)purine wherein Formula 1, B is 2,6- diaminopurine, syt7-2-amino-6-cyclopropylamino-N 9 -(2- hydroxymethylcyclopropylidenemethyl)purine wherein in Formula 1, B is 2-amino-6- cyclopropylaminopurin-N 9 -yl and syt7-2-amino-6-chloro-N 9 -(2- hydroxymethylcyclopropylidenemethyl)purine wherein in Formula 1 , B is 2-amino-6- chloropurin-N 9 -yl.
  • the preferred compounds of the present invention are also methyl phenyl- phosphoro-L-alaninate of syn-N 9 -(2-hydroxymethylcyclopropylidenemethyl)adenine
  • heterocyclic rings containing hydroxy and amino groups are tautomeric with the corresponding oxo and imino compounds.
  • the compounds described by Formulas 1 through 4 contain an asymmetric carbon atom marked in the Formulas 1 and 2 by an asterisk.
  • Compounds of Formula 1 and 2 of the present invention are therefore racemic mixtures of two optical antipodes which may be resolved by conventional methods such as chromatography or fractional crystallization of suitable diastereoisomeric derivatives such as salts or esters with optically active acids (camphor 10-sulfonic acid, methoxyacetic acid, dibenzoyltartaric acid, 6-methoxy-2-naphthyl-2-propanoic acid, etc.), by an enantioselective enzymic synthesis of esters of one antipode such as acetates or butyrates or by an enantioselective enzymic hydrolysis of esters of compounds of Formulas 1 and 2 such as acetates or butyrates.
  • the suitable enzymes include, but are not limited to, lipases such as lipase AK, lipase P30 or esterases such as pig liver esterase. Racemic compounds containing adenine moiety may also be resolved by the action of adenosine deaminase.
  • alkyl cis- and trat7s-2-halo-2-halomethylcyclopropane 1- carboxylates can be used as suitable alkylating agents.
  • Ethyl cis- and trans-2-chloro- 2-chloromethylcyclopropane 1-carboxylates were previously described. Dyakonov, I.A., et al., J. Gen. Chem. USSR (English translation) 25:1435-1440 (1955).
  • an appropriate base e.g., potassium carbonate or sodium hydride
  • organic solvent e.g., N,N-dimethylformamide
  • a strong base e.g., potassium tert.-butoxide in an appropriate solvent such as N,N-dimethylformamide as shown in Figure 2.
  • the alkylation and elimination can be combined into a single step.
  • a suitable benzoylating agent such as benzoic anhydride
  • an appropriate solvent such as ethanol
  • a suitable catalyst e.g., N- methylimidazole
  • compositions within the scope of invention include those comprising a novel compound of the present invention in an effective amount to achieve an intended purpose. Determination of an effective amount and intended purpose is within the skill of the art.
  • Preferred dosages which are dependent for example, on the severity of the infection and the individual patient's response to the treatment, can range from about 0.01 to about 100 mg/kg of body weight to give a blood concentration ranging from about 0.05 ⁇ g to about 5 mg per mL of whole blood.
  • pharmaceutically acceptable salts is intended to mean salts of the compounds of the present invention with pharmaceutically acceptable acids, e.g., inorganic acids such assulfuric, hydrochloric, phosphoric, etc. or organic acids such as acetic.
  • compositions of the present invention may also include suitable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which may be used pharmaceutically.
  • suitable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which may be used pharmaceutically.
  • Such preparations preferably those which can be administered orally, include tablets, dragees and capsules.
  • Further preferred preparations are those which can be administered rectally, such as suppositories, as well as suitable solutions for administration by injection pr orally, contain from about 0.1 to about 99%, preferably about 25 to about 85%, of the active compound of the present invention, together with the excipient.
  • compositions of the present invention are manufactured in a manner which is itself known, e.g., using the conventional mixing, granulating, dragee-making, dissolving or lyophilizing processes.
  • pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding a resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, e.g., lactose or sucrose, mannitol orsorbitol, cellulose preparations and/or calcium phosphates, e.g., tricalcium diphosphate or calcium hydrogen phosphate, as well as binders such as starch paste, using, e.g., maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose and/or polyvinylpyrrolidone.
  • fillers such as sugars, e.g., lactose or sucrose, mannitol orsorbitol, cellulose preparations and/or calcium phosphates, e.g., tricalcium diphosphate or calcium hydrogen phosphate, as well as binders such as starch paste, using, e.g., maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl
  • disintegrating agents may be added such as the above-mentioned starches and also carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
  • Auxiliaries are, above all, flow-regulating agents and lubricants, e.g., silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol.
  • Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices.
  • concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvent or solvent mixtures.
  • suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate, are used.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings, e.g., for identification or in order to characterize different combinations of active compound doses.
  • Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol.
  • the push-fit capsules may contain the active compounds in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds are preferably dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be used.
  • Possible pharmaceutical preparations which can be used rectally include, e.g., suppositories, which consist of a combination of the active compounds with a suppository base.
  • Suitable suppository bases are, e.g., natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alkanols.
  • gelatin rectal capsules which consist of a combination of the active compounds with a base.
  • Possible base materials include, e.g., liquid triglycerides, polyethylene glycols or paraffin hydrocarbons.
  • Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, e.g., water-soluble salts.
  • suspension of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, e.g., ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension such as sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the active compounds of the present invention may be administered in the form of liposomes, pharmaceutical compositions wherein the active compound is contained either dispersed or variously present in corpuscles consisting of aqueous concentrate layers adherent to hydrophobic lipidic layer.
  • the active compound may be present both in the aqueous layer and in the lipidic layer or in the non-homogeneous system generally known as a lipophilic suspension.
  • the active compounds of the present invention may be administered in combination with known antiviral agents, e.g., acyclovir, ganciclovir, zidovudine, AZT, ddl, ddC and d4T.
  • EI-MS 288 (M, 0.3), 286 (M, 1.9) and 284 (M, 0.5), 256 (1.6), 258 (2.6) and 260 (1.2), 243 (5.0), 241 (10.2) and 239 (5.3), 211 (7.0), 213 (13.8) and 215 (7.4), 205 (52.2) and 207 (51.0), 177 (96.7) and 179 (94.4), 159 (7.1) and 161 (7.0), 131 (15.2) and 133 (16.8), 97 (30.4), 81 (15.3), 69 (16.3), 53 (70.8), 39 (15.9), 28 (CO, 100.0);
  • FAB-MS thioglycerol matrix 341 and 343 (M+2H , 20.3, 18.6), 340 and 342
  • FAB-MS thioglycerol matrix 341 and 343 (M + 2H, 19.0, 18.2), 340 and 342 (M + H, 96.3, 100.0), 262 (32.1), 214 (12.1), 188 (12.5), 136 (78.3).
  • FAB-MS thioglycerol matrix 381 (M + thioglycerol + H, 51.7), 273 (M + H, 100.0), 191 (69.8).
  • EI-MS 293 and 295 (M 89.4, 29.9), 264 and 266 (30.8, 11.5), 248 and 250 (22.7, 8.7), 236 and 238 ( 10.9, 4.0), 221 and 223 (100.0, 32.9), 208 and 210 (5.9, 3.2), 184 (25.3), 169 and 171 (39.1 , 14.1).
  • EI-MS 251 and 253 (M, 41.4, 15.1), 234 and 236 (50.9, 19.8), 221 and 223 (18.6, 8.9), 198 ( 26.2), 170 and 172 (100.0, 38.5).
  • EI-MS 251 and 253 (M, 36.1 , 12.3), 234 and 236 (84.0, 29.7), 222 and 224 (8.6, 3.4), 198 (24.6), 170 and 172 (100.0, 37.8).
  • EI-MS 232 (M, 45.2), 215 (39.7), 202 (18.5), 198 (10.8), 173 (7.9), 163 (10.9), 159 (9.5), 150 (100.0).
  • the mixture was cooled to 50°C methanol ⁇ W mL) was added with stirring which was continued at 50°C for another hour.
  • the insoluble portion was filtered off using a Celite bed and it was washed with dichloromethane - methanol (4 : 1 , 3 x 30 mL). The filtrate was evaporated and the residue was chromatographed on a silica gel column (85 g) using dichloromethane - methanol
  • UV (ethanol) max 331 (e 14,800), 270 nm (e 19,200), 220 nm (e 15,400), 206 nm (e 17,200).
  • FAB-MS thioglycerol matrix 567 (M + thioglycerol + H, 41.0), 459 (M + H, 11.9), 308 (17.5), 281 (14.2), 234 (21.1), 200 (100.0), 136 (adenine + H, 60.5).
  • the reaction mixture was stirred at room temperature for 4 h. After removal of solvents, the residue was dissolved in methanol (1 mL) and it was partitioned between ethyl acetate (250 mL) and water (100 mL). The aqueous phase was extracted once with ethyl acetate (60 L). The combined organic phases were washed with water (4 x 80 mL) and brine (80 mL). They were dried over Na ⁇ SO 4 and evaporated to leave a syrup.
  • FAB-MS thioglycerol matrix 582 (M + thioglycerol +H, 48.6), 474 (M + H, 11.6), 323 (19.4), 281 (15.7), 249 (27.5), 215 (43.7), 200 (36.2), 151 (2,6- diaminopurine + H, 100.0).
  • HFF human foreskin fibroblasts
  • MRC-5 medium consisting of MEM(E) with 10% fetal bovine serum.
  • HBS HEPES buffered salt solution
  • HCMV Virological procedures.
  • Stock HCMV was prepared by infecting HFF cells at a multiplicity of infection (m.o.i.) of ⁇ 0.01 plaque-forming units (p.f.u.) per cell. Cell growth medium was changed every four days until cytopathology was evident in all cells (approximately 21 days). Supernatant fluids were retained as the virus stock.
  • High titer HSV-1 stocks were prepared by infecting KB cells at an m.o.i. of ⁇ 0.1 as detailed previously. Turk, S.R., et al., Antimicrob. Agents Chemother. 31 :544-550 (1987).
  • Virus titers were determined using monolayer cultures of HFF cells for HCMV and monolayer cultures of BSC-1 cells for HSV-1 as described earlier. Prichard, M.N., et al., J. Virol. Methods 28:101-106 (1990). Briefly, HFF or BSC-1 cells were planted as described above in 96-well cluster dishes and incubated overnight at 37°C in a humidified 3% CO 2 - 97% air atmosphere. The next day cultures were inoculated with HCMV or HSV-1 and serially diluted 1 : 3 across the remaining eleven columns of the 96-well plate.
  • Cultures were incubated at 37°C for 2 hr to permit virus adsorption and then virus inoculum was replaced with 0.2 mL of fresh medium. Cultures were incubated for seven days for HCMV, two or three days for HSV-1, medium was removed, and the cell sheets were stained with 0.1% crystal violet in 20% methanol. Plaques were enumerated under 20-fold magnification in wells having the dilution which gave 5 to 20 plaques per well.
  • HFF cells were planted as described above in 96-well cluster dishes, incubated overnight, medium removed and the cultures were inoculated with HCMV at a m.o.i. of 0.5 to 1 p.f.u. per cell as reported elsewhere.
  • inoculum was replaced with 0.2 mL of fresh medium containing test compounds.
  • the first row of 12 wells was left undisturbed and served as virus controls.
  • Each well in the second row received an additional 0.1 mL of medium with test compound at three times the desired final concentration.
  • the contents of the 12 wells were mixed by repeated pipetting and then serially diluted 1 : 3 along the remaining wells. In this manner, six compounds could be tested in duplicate on a single plate with concentrations from 100 ⁇ M to 0.14 ⁇ M.
  • Plates were incubated at 37°C for seven days, subjected to one cycle of freezing and thawing; aliquots from each of the eight wells of a given column were transferred to the first column of a fresh 96-well monolayer culture of HFF cells. Contents were mixed and serially diluted 1 : 3 across the remaining eleven columns of the secondary plate. Each column of the original primary plate was diluted across a separate plate in this manner. Cultures were incubated, plaques were enumerated, and titers calculated as described above.
  • HSV-1 An enzyme-linked immunosorbent assay (ELISA) was employed to detect HSV-1.
  • ELISA enzyme-linked immunosorbent assay
  • 96-well cluster dishes were planted with BSC-1 cells at 10,000 cells per well, in a total volume of 200 ⁇ L per well of MEM(E) plus 10% calf serum. After overnight incubation at 37°C, drug and HSV-1 was added at the rate of 100 PFU/well.
  • ELISA plates were blocked with 200 ⁇ L per well of 10% calf serum and 0.05% tween in HBS. After incubation for 30 minutes, the blocking agent was rinsed two times with HBS-T. A 1 : 400 dilution of AP conjugated rabbit anti-HSV-1 antibody in HBS-F was added.
  • the plates were activated by the addition of a homobifunctional crosslinking agent, bis(sulfosuccinimidyl) suberate, which was dissolved at 1 mg/mL in 30 mL of phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCI, 4.3 mM Na 2 HP0 4 , 1.4 mM KH 2 PO 4 , pH 7.4) and 300 ⁇ L of the crosslinker was added to each well in the covalent plate. The crosslinker reacted with the amine function on the plate for 30 min at room temperature. The byproduct, sodium N-hydroxysuccinimide sulfite, was removed by decanting and washing the plate twice with PBS.
  • PBS phosphate buffered saline
  • Samples consisting of 150 ⁇ of mixed suspended HSB 2 cells from the original drug-treated plate were solubilized in an equal volume of 10% Triton X-100 in coating buffer (15 mM Na 2 CO 3 , 3.5 mM NaHCO 3 , pH 9.6). The plate was covered and then incubated for 1 h at 37°C in a 5% CO 2 atmosphere. These binding conditions facilitated covalent attachment of the antigen to the free end of the crosslinker.
  • the antigen solution was decanted and the plate was washed six times in HEPES buffered saline (Shipman, C, Jr., Proc. Soc. Exp. Biol. 130:305-310 (1969)) with 0.05% Tween 20 (HBS-T ), soaking for three min for each wash. Unbound sites on the plate were blocked with 300 ⁇ L per well of 2% lowfat dry milk in PBS (blocker) for 30 min at room temperature on a shaker. The blocker was decanted and 50 ⁇ L of the diluted primary monoclonal antibody, specific for HHV-6 (GS) glycoprotein gp116, was added.
  • HEPES buffered saline Chipman, C, Jr., Proc. Soc. Exp. Biol. 130:305-310 (1969)
  • HBS-T Tween 20
  • the antibody solution consisted of antibody diluted 1 : 400 in equal volumes of blocker and 10% Triton X-100 in coating buffer. The presence of both blocker and detergent in the antibody solutions was necessary to reduce background signal.
  • the plate was then covered and incubated for 1 h at 37°C. The plate was washed again, as described above, then blocker was added again, as before. Next, each well received 100 ⁇ L of a solution of the secondary antibody, horse radish peroxidase-labeled rabbit anti-mouse antibody, diluted to 1 : 400 (as above). The plate was incubated for 1 h at 37°C.
  • the plate was washed again as described above, and developed using 100 ⁇ L/well of TMB- Turbo (Pierce, Rockford, IL) for 30 min at room temperature. The reaction was stopped with 50 ⁇ L/well 2 M H 2 SO 4 . Absorbance in each well was determined at 450/570 nm.
  • RT Reverse transcriptase
  • This assay measured the presence of HIV in supernatants of CEM cells infected with strain lll B of HIV-1 by the amount of RT activity. Cells were grown, infected, and incubated in the presence of seven concentrations (one-half Iog 10 dilutions) beginning at 1 or 100 ⁇ M of compounds to be assayed. Procedures and the RT assay were performed as detailed previously. Kucera, L.S., et al., AIDS Res. Human Retroviruses 9:307-314 (1993); White, E.L., et al., Antiviral Res. 16:257-266 (1991).
  • Cytotoxicity assays Two different assays were used to explore cytotoxicity of selected compounds as we have detailed previously.
  • Dose-response relationships were constructed by linearly regressing the percent inhibition of parameters derived in the preceding sections against log drug concentrations. Fifty-percent inhibitory (IC 50 ) concentrations were calculated from the regression lines. Samples containing positive controls (acyclovir for HSV-1 , ganciclovir for HCMV, and 2-acetylpyridine thiosemicarbazone for cytotoxicity) were used in all assays. Testing Results. Compounds of Formula 1 exhibit a significant activity against herpes viruses.
  • Compounds of the present invention also strongly inhibit HHV 6 to a greater extent than current drug foscarnet (Foscavir) as determined by enzyme-linked immunosorbent assay (ELISA) in HSB-2 cells by the method described above.
  • current drug foscarnet Foscavir
  • ELISA enzyme-linked immunosorbent assay
  • Compounds of the present invention also inhibit HIV-1 when tested in a reverse transcriptase assay as described above.
  • MCMV murine cytomegalovirus
  • Compound 1 (B adenin-N 9 -yl) reduced mortality from 100% in placebo treated mice to 33% in mice treated with 50 mg/kg of the drug. Decreasing effects were noted at lower doses and when drug administration was delayed until 24 or 48 hours after infection. No deaths were observed in uninfected mice that received only drug.
  • a pharmaceutical composition comprising the compounds listed above and pharmaceutically acceptable salts thereof. Prodrugs of the compounds listed above and pharmaceutically acceptable salts thereof.
  • a method of treatment of mammals infected with a virus comprising administering an effective amount of a compound listed above. The method above wherein said mammal is a human and said virus is a human herpes virus. The method above wherein said mammal is a human and said virus is human immunodeficiency virus. The method above wherein said mammal is a human and said virus is hepatitis B virus. The method above wherein the compounds listed above are used in combination with other antiviral drugs such as acyclovir, ganciclovir and zidovudine.

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Abstract

L'invention concerne des composés ayant une activité antivirale et répondant aux formules (I), (II), (III) et (IV) dans lesquelles B est un noyau hétérocyclique de purine ou de pyrimidine, sélectionné de préférence dans le groupe qui comprend 6-aminopurine (adénine), 2,6-diaminopurine, 2-amino-6-cyclopropylaminopurine, 6-hydroxypurine (hypoxanthine), purines à substitution 2-amino-6-halo, purines à substitution 2-amino-6-alcoxy, 2-amino-6-hydroxypurine (guanine), 3-déazapurines, 7-déazapurines, 8-azapurines, cytosine, cytosines à substitution 5-halo, cytosines à substitution 5-alkyle, thymine, uracile; dans lesquelles X est O, R1 et R2 étant des groupes alkyle ou aryle. R1X et/ou R2X peuvent également être des restes d'acides aminés avec X représentant NH.
PCT/US1998/000440 1997-01-08 1998-01-07 2-hydroxymethylecyclopropyli- denemethylpurines et -pyrimidines utilises comme agents antiviraux WO1998030563A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US09/267,839 US6352991B1 (en) 1997-01-08 1999-03-12 2-hydroxymethylcyclopropylidenemethylpurines and -pyrimidines as antiviral agents
US10/047,202 US6790841B2 (en) 1997-01-08 2002-01-14 2-hydroxymethylcyclopropylidenemethylpurines and -pyrimidines as antiviral agents
US10/913,219 US6958345B2 (en) 1997-01-08 2004-08-06 2-hydroxymethylcyclopropylidene methylpurines and -pyrimidines as antiviral agents
US11/257,776 US20060122203A1 (en) 1997-01-08 2005-10-25 2-Hydroxymethylcyclopropylidenemethylpurines and - pyrimidines as antiviral agents

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US3582697P 1997-01-08 1997-01-08
US60/035,826 1997-01-08
US4567697P 1997-05-06 1997-05-06
US60/045,676 1997-05-06

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1165560A4 (fr) * 1999-03-12 2002-04-17 Univ Michigan 2-hydroxy methylcyclopro pylidenemethyl purines et pyrimidines comme agents antiviraux
EP1389461A1 (fr) * 2002-08-14 2004-02-18 Axxima Pharmaceuticals Aktiengesellschaft Pyrimidones avec des proprietés antivirales
WO2004016271A1 (fr) * 2002-08-14 2004-02-26 Axxima Pharmaceuticals Ag Pyrimidones en tant qu'agents antiviraux
JP2005524720A (ja) * 2002-03-15 2005-08-18 ウエイン・ステイト・ユニバーシテイ 抗ウイルス薬としての新規２−アミノ−９−［（２−ヒドロキシメチル）シクロプロピリデンメチル］プリン
JP2021516700A (ja) * 2018-03-09 2021-07-08 メディヴィル・アクチエボラーグ （２，２−ビスヒドロキシメチル）メチレンシクロプロパンヌクレオチドを用いたがんの治療
CN113185514A (zh) * 2021-03-11 2021-07-30 杨锦飞 一种抗乙型肝炎病毒药物的合成方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
QIU Y.-L., ET AL.: "(Z)- AND (E)-2-((HYDROXYMETHYL)CYCLOPROPYLIDENE)METHYLADENINE AND -GUANINE. NEW NUCLEOSIDE ANALOGUES WITH A BROAD-SPECTRUM ANTIVIRAL ACTIVITY.", JOURNAL OF MEDICINAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, US, vol. 41., no. 01., 1 January 1998 (1998-01-01), US, pages 10 - 23., XP002913470, ISSN: 0022-2623, DOI: 10.1021/jm9705723 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6790841B2 (en) 1997-01-08 2004-09-14 Wayne State University 2-hydroxymethylcyclopropylidenemethylpurines and -pyrimidines as antiviral agents
EP1165560A4 (fr) * 1999-03-12 2002-04-17 Univ Michigan 2-hydroxy methylcyclopro pylidenemethyl purines et pyrimidines comme agents antiviraux
JP2002539125A (ja) * 1999-03-12 2002-11-19 ザ・リージエンツ・オブ・ザ・ユニバーシテイ・オブ・ミシガン 抗ウイルス剤としての２−ヒドロキシメチルシクロプロピリデンメチルプリンおよび−ピリミジン類
JP2005524720A (ja) * 2002-03-15 2005-08-18 ウエイン・ステイト・ユニバーシテイ 抗ウイルス薬としての新規２−アミノ−９−［（２−ヒドロキシメチル）シクロプロピリデンメチル］プリン
EP1389461A1 (fr) * 2002-08-14 2004-02-18 Axxima Pharmaceuticals Aktiengesellschaft Pyrimidones avec des proprietés antivirales
WO2004016271A1 (fr) * 2002-08-14 2004-02-26 Axxima Pharmaceuticals Ag Pyrimidones en tant qu'agents antiviraux
JP2021516700A (ja) * 2018-03-09 2021-07-08 メディヴィル・アクチエボラーグ （２，２−ビスヒドロキシメチル）メチレンシクロプロパンヌクレオチドを用いたがんの治療
CN113185514A (zh) * 2021-03-11 2021-07-30 杨锦飞 一种抗乙型肝炎病毒药物的合成方法

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