Absence of adenosine deaminase activity in a mammalian cell line transformed by Rous sarcoma virus.

No detectable adenosine deaminase activity was found in whole cells or 105,000g cytosol preparations of B-mix K-44/6 cells when either [3H]adenosine or [3H]arabinosyladenine was used as substrate. When grown in tissue culture medium supplemented with horse serum these cells provide a deaminase-free system not requiring the use of an adenosine deaminase inhibitor.

Tritiated thymidine incorporation does not measure DNA synthesis in ribavirin-treated human cells.

When the incorporation of tritiated thymidine into acid insoluble material was measured, ribavirin appeared to be a potent inhibitor of DNA synthesis in KB cells and human lymphocytes. Inhibition was nearly 100-fold less, however, when DNA synthesis was measured by incorporation of phosphorus-32-labeled phosphate or by DNA fluorescence. The potent inhibition detected by incorporation of tritiated thymidine into DNA actually was the result of a potent effect on the labeling of deoxythymidine triphosphate, not on the synthesis of DNA.

Deoxyadenosine antagonism of the antiviral activity of 9-beta-D-arabinofuranosyladenine and 9-beta-D-arabinofuranosylhypoxanthine.

Deoxyadenosine but not adenosine reversed the antiviral activity of 9-beta-D-arabinofuranosyladenine (ara-A) and 9-beta-D-arabinofuranosylhypoxanthine (ara-H) when used in the presence of coformycin, an inhibitor of adenosine deaminase. In suspension cultures of KB cells, 10 muM ara-A inhibited the replication of herpes simplex virus type 1 by 80%. Concomitant addition of 50 muM deoxyadenosine reduced the antiviral activity of 10 muM ara-A to only 40% inhibition. Adenosine failed to antagonize the antiviral activity. In monolayer cultures of KB cells, the 50% inhibitory concentration of ara-A was increased from 1.5 to 2.9 muM by 2 muM deoxyadenosine and to 8.5 muM by 10 muM deoxyadenosine. Analysis of the dose-response data by a double reciprocal plot method indicated that the antagonism was competitive. The antiviral activity of ara-H also was antagonized by deoxyadenosine. The 50% inhibitory concentration of ara-H was increased from 42 muM to 70, 91, or 121 muM by the concurrent addition of 5, 10, or 20 muM deoxyadenosine. Competitive antagonism could not be demonstrated. In the absence of the adenosine deaminase inhibitor, neither ara-A nor ara-H was antagonized by deoxyadenosine. Since such inhibitors were not available unitl recently, previous investigators were unable to observe the antagonistic capacity of deoxyadenosine.

DNA polymerase in nuclei isolated from herpes simplex virus type-2-infected cells. Characterization of the reaction product and inhibition by substrate analogs.

Nuclei isolated from herpes simplex virus (HSV) type 2-infected KB cells were examined for their capacity to serve as an in situ source of herpes DNA polymerase. In contrast to purified enzymes with added template, approx. 80% of the DNA synthesized in isolated nuclei was viral. The average size of DNA fragments labeled in vitro was 3.2 X 10(6) Da. Based on an increase in DNA density when nuclei were incubated in the presence of BrdUTP rather than dTTP, 16% of the nucleotides were added during the in vitro reaction. Sucrose gradient analysis of DNA polymerase activity in extracts of isolated nuclei demonstrated the nearly exclusive presence of herpes DNA polymerase. Km concentrations for the four dNTPs were from 0.14 to 0.55 microM. DNA synthesis was inhibited competitively by the 5'-triphosphates of ara-A and ara-C (Ki = 0.03 and 0.22 microM, respectively) but not by the 5'-triphosphate of dideoxythymidine. aATP also served as a substrate (Km = 0.014 microM) for the reaction. We conclude that nuclei from HSV-infected cells have significant advantages for the detailed study of inhibitors of herpesvirus replication.

Design, synthesis, and antiviral evaluation of 2-substituted 4,5-dichloro- and 4,6-dichloro-1-beta-D-ribofuranosylbenzimidazoles as potential agents for human cytomegalovirus infections.

The syntheses of 2,4,6-trichlorobenzimidazole (4a) and 2-bromo-4,6-dichlorobenzimidazole (4b) were accomplished via the 2-amino intermediate (3) using a mild diazotization procedure. Ribosylation of 4a and 4b and subsequent deprotection afforded the corresponding 2,4,6-trichloro-1-beta-D-ribofuranosylbenzimidazole (7a) and 2-bromo-4,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (7b). The 2-azido (10), 2-amino (11), 2-thione (13), 2-methylthio (14a), and 2-benzylthio (14b) derivatives were prepared via displacement reactions at the 2-position of the 2,3,5-tri-O-acetyl derivative of 7a. 2,4,5-Trichlorobenzimidazole (17a) and 2-bromo-4,5-dichlorobenzimidazole (17b) were synthesized from the corresponding 1,2-phenylenediamines via successive cyclization with cyanogen bromide and diazotization in the presence of an appropriate cupric halide. Ribosylation of compounds 17a and 17b was followed by deprotection to afford 2,4,5-trichloro-1-beta-D-ribofuranosylbenzimidazole (20a), and 2-bromo-4,5-dichloro-1-beta-D-ribofuranosylbenzimidazole (20b). Heterocycles (3, 4a, 17a) and nucleosides (7a, b, 8, 10, 11, 13, 14a,b, 20a,b) were evaluated for activity against human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) and for cytotoxicity. The 2-chloro but not the 2-amino heterocycles were active against HCMV (IC50's = 5-8 microM) but not HSV-1; both also were somewhat cytotoxic to uninfected cells (IC50's = 32-100 microM). Among the nucleosides, the 2-chloro and 2-bromo analogs in both the 4,5- and 4,6-dichloro series (20a,b, 7a,b, respectively) were active against HCMV (IC50's = 1-10 microM) and noncytotoxic in their antiviral dose ranges. The 2-bromo compounds were more active than the 2-chloro analogs; the 2-azido and 2-thiobenzyl analogs (10, 14b) were weakly active against HCMV, but this activity was not well separated from cytotoxicity. None of the nucleosides were active against HSV-1. This pattern of activity and cytotoxicity is similar to that of the 2-chloro- and 2-bromo-5,6-dichloro analogs (TCRB, BDCRB) which we reported previously. Although these new 4,5- and 4,6-dichloro analogs are potent and selective inhibitors of HCMV, they are not as potent at TCRB and BDCRB.

Design, synthesis, and antiviral evaluation of 2-chloro-5,6-dihalo-1-beta-D-ribofuranosylbenzimidazoles as potential agents for human cytomegalovirus infections.

2-Chloro-5,6-difluorobenzimidazole (8) was prepared from 4,5-difluoro-2-nitroaniline (5) via successive reduction, cyclization, and diazotization reactions. 2-Chloro-5,6-dibromobenzimidazole (10) was obtained by a direct bromination of 2-chlorobenzimidazole (9) with bromine-water. 2-Chloro-5,6-diiodobenzimidazole (15) was synthesized by a stepwise transformation of the nitro functions of 2-chloro-5,6-dinitrobenzimidazole (11) into iodo groups via diazotization reactions. Ribosylation of 8, 10, and 15 gave the respective beta nucleosides 16a-c as the major products along with a small amount of the alpha anomers 17a-c. Deprotection of 16a-c afforded the corresponding free beta nucleosides 2-chloro-5,6-difluoro-1-beta-D-ribofuranosylbenzimidazole (2), 2-chloro-5,6-dibromo-1-beta-D-ribofuranosylbenzimidazole (3), and 2-chloro-5,6-diiodo-1-beta-D-ribofuranosylbenzimidazole (4). Similar deprotection of the alpha anomers (17a-c) resulted in a removal of the acetyl protecting groups and a concomitant cyclization to give the 2,2'-O-cyclonucleosides (18a-c). Most of the benzimidazole heterocycles, but not the difluoro analog, were active against human cytomegalovirus (HCMV) (IC50's = 3-40 microM) and herpes simplex virus type 1 (HSV-1) (IC50's = 50-90 microM). This activity, however, was not well separated from cytotoxicity, IC50's = 10-100 microM. The corresponding unsubstituted, the 5,6-dimethyl, and the 5,6-difluoro ribonucleosides (19, 20, and 2, respectively), were inactive against both viruses. Similar to the previously reported 2,5,6-trichloro analog (TCRB), the 5,6-dibromo ribonucleoside 3 was active against HCMV (IC50 approximately 4 microM) but more cytotoxic than TCRB. The 5,6-diiodo analog 4 also was active (IC50 approximately 2 microM) but more cytotoxic (IC50 = 10-20 microM) than either 3 or TCRB. The cyclonucleosides were inactive against both viruses and not cytotoxic, or slightly active with corresponding cytotoxicity. The order of activity against HCMV of the dihalobenzimidazole ribonucleosides was I approximately equal to Br approximately equal to CI > > F > H = CH3. The order of cytotoxicity among the most active compounds, however, was I > Br > Cl, thereby establishing that TCRB had the best antiviral properties.

Synthesis and antiviral evaluation of certain disubstituted benzimidazole ribonucleosides.

Ribosylation of 2-chloro-5(6)-nitrobenzimidazole (3) gave 2-chloro-5-nitro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)benzimidazol e (4a) and 2-chloro-6-nitro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)benzimidazol e (4b) as a mixture of positional isomers. Subsequent hydrogenation of this mixture over Raney Nickel afforded the corresponding 5-amino and 6-amino derivatives 5 and 6. At this stage the products were readily resolved via silica column chromatography into pure isomeric forms, and the pure isomers 5 and 6 were diazotized with tert-butyl nitrite and cupric chloride to furnish the isomerically pure 5-chloro derivative 2a and 6-chloro derivative 2b. Deprotection of 5, 6, 2a, and 2b with methanolic ammonia yielded the free nucleosides 5-amino-2-chloro-1-(beta-D-ribofuranosyl)benzimidazole (7), 6-amino-2-chloro-1-(beta-D-ribofuranosyl)-benzimidazole (8), 2,5-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (9), and 2,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (10), respectively. Treatment of 10 with thiourea afforded 6-chloro-1-(beta-D-ribofuranosyl)benzimidazole-2-thione (14). Alkylation of 14 with methyl iodide and benzyl bromide gave good yields of the corresponding 2-methylthio (12) and the 2-benzylthio (13) analogs. The synthesis of 6-chloro-2-methoxy-1-(beta-D-ribofuranosyl)benzimidazole (11) was accomplished by the treatment of 2b with sodium methoxide in methanol. A difference NOE spectroscopic experiment was conducted to allow unequivocal assignment of regiochemistry to the positional isomers 5 and 6. Evaluation of compounds for activity against human cytomegalovirus (HCMV) and herpes simplex virus type 1 revealed that the heterocycle 3 was active against both viruses but also was cytotoxic. Only the dichloro compounds 9 and 10 were weakly active against HCMV and noncytotoxic in their antiviral dose range. These data further substantiate the conclusion that activity against HCMV at noncytotoxic concentrations by benzimidazole ribonucleosides requires a halogen not only at the 2-position, but also more than one halogen on the benzene moiety.

Design, synthesis, and biological evaluation of tricyclic nucleosides (dimensional probes) as analogues of certain antiviral polyhalogenated benzimidazole ribonucleosides.

The polyhalogenated benzimidazole nucleosides 2,5, 6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) and the 2-bromo analogue (BDCRB) were synthesized in our laboratory and established as potent and selective inhibitors of human cytomegalovirus (HCMV) with a novel mode of action. In an effort to study the behavior of the key substructure in a dimensionally extended manner and probe the spatial limitation of the target enzyme(s), a series of 2-substituted 6, 7-dichloro-1-(beta-D-ribofuranosyl)naphtho¿2,3-dimidazoles and the N1- and N3-ribonucleosides of 2-substituted 6,7-dichloroimidazo¿4, 5-bquinolines were prepared. The nucleosides 6, 7-dichloro-1-(beta-D-ribofuranosyl)imidazo¿4,5-bquinolin-2-one and 6,7-dichloro-3-(beta-D-ribofuranosyl)imidazo¿4,5-bquinolin-2-one were selected and used as the key synthetic intermediates in the imidazo¿4,5-bquinoline series. Evaluation of the compounds for activity against HCMV and herpes simplex virus type 1 revealed that the trichloro analogues of TCRB (2a, 3a) were nearly as active against HCMV as TCRB but were more cytotoxic. The results suggest that extending the heterocycle of TCRB affected the affinity for the HCMV target only slightly but increased the affinity for cellular enzymes.

Synthesis and antiviral evaluation of trisubstituted indole N-nucleosides as analogues of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB).

2,5,6-Trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) and 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (BDCRB) are nucleosides that exhibit strong and selective activity against human cytomegalovirus (HCMV). Selected polyhalogenated indole nucleosides have now been synthesized as 3-deaza analogues of the benzimidazole nucleosides using the sodium salt glycosylation method. 2-Benzylthio-1-¿2-deoxy-3, 5-bis-O-(4-methylbenzoyl)-beta-D-erythro-pentofuranosyl-5, 6-dichloroindole (8) was prepared stereoselectively via the coupling of a 2-deoxyribofuranosyl alpha-chloride derivative with the sodium salt of 2-benzylthio-5,6-dichloroindole (5). Compound 8 was then elaborated into the targeted 2-benzylthio-1-(beta-D-ribofuranosyl)-5, 6-dichloroindole (18) in five steps. 2,5, 6-Trichloro-(1-beta-D-ribofuranosyl)indole (19) was prepared using the same synthetic route with 2,5,6-trichloroindole (6) as the starting material. We were subsequently able to prepare 19 in three steps using a modification of the sodium salt glycosylation method. 2-Bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)indole (25) was also prepared using the same procedures. Target compounds were tested for activity against HCMV, herpes simplex virus type 1 (HSV-1), and human herpes virus six (HHV-6) and for cytotoxicity. All of the compounds were less active against HCMV than TCRB and weakly active or inactive against HSV-1 and HHV-6.

Structure-activity relationships among 2-substituted 5,6-dichloro-, 4,6-dichloro-, and 4,5-dichloro-1-[(2-hydroxyethoxy) methyl]- and -1-[(1,3-dihydroxy-2-propoxy) methyl]benzimidazoles.

The sodium salt of 2,5,6-trichlorobenzimidazole (8a) was condensed with [2-(benzyloxy)ethoxy]-methyl chloride (9) and [1,3-bis(benzyloxy)-2-propoxy]methyl chloride (18) to provide the corresponding protected acyclic nucleosides 10a and 19a, which on debenzylation afforded 2,5,6-trichloro-1-[(2-hydroxyethoxy)methyl]benzimidazole (11a) and 2,5,6-trichloro-1-[(1,3-dihydroxy-2-propoxy)methyl] benzimidazole (20a), respectively. A similar condensation of 2,4,6-trichlorobenzimidazole (2a) and 2,4,5-trichlorobenzimidazole (7a) followed by debenzylation yielded 11b, 20b, 11c, and 20c, respectively. A nucleophilic displacement of the 2-chloro group of 11a-c and 20a-c with liquid ammonia, methylamine, dimethylamine, and thiourea furnished several interesting 2-substituted compounds in good yields, e.g., 12-14(a-e), 21-23(a-e), 15-17, and 24-26. Alkylation of the 2-thio analogs 15-17 and 24-26 with benzyl chloride furnished the 2-alkylthio acyclic nucleosides 12d-14d and 21d-23d. Desulfurization of 15 and 24 with Raney Ni furnished 5,6-dichloro-1[(2-hydroxyethoxy)methyl]benzimidazole (12e) and 5,6-dichloro-1-[1,3-dihydroxy-2-propoxy)methyl]benzimidazole (21e), respectively (acyclic analog of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole). Similarly the dihalo compounds 13e, 14e, and 23e were prepared in moderate yields from the 2-thio analogs 16,17, and 26. Treatment of 2-bromo-5,6-dichlorobenzimidazole (8b) with 27 and 30 gave the protected acyclic compounds 28a and 31a, which on deacetylation with sodium carbonate and potassium cyanide yielded 2-bromo-5,6-dichloro-1-[(2-hydroxyethoxy)methyl]benzimidazole (29a) and 2-bromo-5,6-dichloro-1-[(1,3-dihydroxy-2-propoxy)methyl]benzimidazole (32a), respectively, in moderate yields. The 2-bromo-4,6-dichlorobenzimidazole and 2-bromo-,5-dichlorbenzimidazole analogs 29b,c and 32b,c were prepared in a similar manner. Compounds were tested for activity against human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) and for cytotoxicity. In marked contrast to the ribosylbenzimidazoles, none of the acyclic analogs were specific and potent inhibitors of HCMV. Only the 2-thiobenzyl analogs 12d, 13d, 14d, and 23d and the 2-Br analogs 32a,b were active, but activity was not well separated from cytotoxicity. The lack of specific and potent antiviral activity strongly suggests that these acyclic nucleoside analogs are not phosphorylated by HCMV or HSV-1 gene products and that the ribosylbenzimidazoles do not require phosphorylation for antiviral activity.

Synthesis and antiviral activity of some 7-[(2-hydroxyethoxy)methyl]pyrazolo[3,4-d]pyrimidine analogues of sangivamycin and toyocamycin.

The sodium salt of 4-amino-3-cyanopyrazolo[3,4-d]pyrimidine (1) was condensed with (2-acetoxyethoxy)methyl bromide (2) to provide the corresponding protected acyclic nucleoside, 4-amino-3-cyano-1-[(2-acetoxyethoxy)methyl]-pyrazolo[3,4-d]pyrimid ine (3). Treatment of 3 with sodium methoxide in methanol provided a good yield of methyl 4-amino-1-[(2-hydroxyethoxy)methyl]pyrazolo[3,4-d]pyrimidine-3- formimidate (4). Treatment of the imidate (4) with sodium hydrogen sulfide gave the thiocarboxamide derivative 5. Aqueous base transformed 4 into 4-amino-1-[(2-hydroxyethoxy)methyl]pyrazolo[3,4-d]pyrimidine-3- carboxamide (6) in good yield. Treatment of 5 with mercuric chloride furnished the toyocamycin analogue 7. Evaluation of compounds 1, 3-7 revealed that only the heterocycle (1) and the thiocarboxamide acyclic nucleoside (5) were active. Compound 5 was the more potent with activity against human cytomegalovirus and herpes simplex virus type 1.

7-Aminoquinolines. A novel class of agents active against herpesviruses.

A series of 7-aminoquinoline derivatives was synthesized and evaluated for their capacity to produce cytotoxicity in KB cells and to inhibit the replication of herpes simplex virus (HSV) type 1. All compounds tested inhibited the replication of HSV-1 with 50% inhibitory concentrations in the range of 2-50 micrograms/mL. The antiviral activity of many compounds, however, was separated from cytotoxicity to replicating uninfected cells by only two- to fivefold higher than those required for antiviral activity. Nonetheless, six compounds (10, 28, 29, 32, 34, and 36) were identified in which the separation was greater than fivefold. All compounds examined were more potent inhibitors of viral DNA synthesis than the cellular DNA synthesis.

Synthesis, antiproliferative and antiviral activity of imidazo[4,5-d]isothiazole nucleosides as 5:5 fused analogs of nebularine and 6-methylpurine ribonucleoside.

A series of imidazo[4,5-d]isothiazole nucleosides related to the antibiotic nebularine and the highly cytotoxic 6-methyl-9-beta-D-ribofuranosylpurine have been synthesized from the corresponding heterocycles. The sodium salt glycosylation of the imidazo[4,5-d]isothiazoles proceeded smoothly, giving mixtures of N-4 and N-6 regioisomers in generally good yields. The protected derivatives were deblocked using standard conditions to afford the desired imidazo[4,5-d]-isothiazole nucleosides, usually as crystalline solids. None of the new nucleosides or heterocycles displayed selective activity against human cytomegalovirus (HCMV) or herpes simplex virus type 1 (HSV-1). The N-6 glycosylated imidazo[4,5-d]isothiazoles were completely inactive up to the highest concentration tested. The N-6 glycosylated imidazo[4,5-d]isothiazoles also were inactive in antiproliferative and cytotoxicity assays, except for 3-methyl-6-beta-D-ribofuranosylimidazo[4,5-d]isothiazole (15a) and 5-(benzylthio)-6-(2-deoxy-beta-D-ribofuranosyl)imidazo[4,5-d]isothiaz ole (5e), which showed moderate inhibition of L1210 cell growth. However, the heterocycles and several of the N-4 glycosylated derivatives were toxic to HFF, KB and L1210 cells; compounds with 5-benzylthio substituents were the most cytotoxic agents in this series.

Synthesis and antiviral activity of certain 5'-modified analogs of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole.

A series of 5'-modified 2,5,6-trichlorobenzimidazole ribonucleosides has been synthesized and tested for activity against two human herpesviruses and for cytotoxicity. The 5'-methoxy, 5'-ethoxy, and 5'-butoxy analogs of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) were prepared by coupling the appropriate 5-O-alkyl-1,2,3-tri-O-acetyl-beta-D-ribose derivatives with 2,5,6-trichlorobenzimidazole followed by removal of the protecting groups. The 5'-deoxy-5'-fluoro, -5'-chloro, -5'-bromo, -5'-iodo, -5'-azido, and -5'-thiomethyl derivatives were synthesized in a similar fashion. All of these 5'-modified derivatives had significant activity against HCMV in plaque and yield reduction assays (IC50's = 0.5-14.2 microM) but had little activity (IC50's > 100 microM) against HSV-1. This pattern in similar to the antiviral activity profile observed for TCRB. The 5'-halogenated derivatives were more active than the other 5'-modified derivatives with antiviral activity well separated from cytotoxicity. In general, cytotoxicity of all the 5'-modified derivatives was greater in human foreskin fibroblasts (HFF cells) than in L1210 or KB tumor cells. These results indicate that the viral target tolerates significant modifications of TCRB at the 5'-position without adversely affecting activity against HCMV, whereas the 5'-modifications increased cytotoxicity in human diploid cells.

Synthesis and antiproliferative and antiviral activity of carbohydrate-modified pyrrolo[2,3-d]pyridazin-7-one nucleosides.

Sugar-modified analogs of 4-amino-1-(beta-D-ribofuranosyl)pyrrolo[2,3-d]pyridazin-7-one (1) and 4-amino-3-bromo-1-(beta-D-ribofuranosyl)pyrrolo[2,3-d]pyridazin-7- one (3) were prepared in an effort to obtain selective antiviral agents. Treatment of ethyl 3-cyano-1-(2,3,5-tri-O-benzyl-1-beta-D-arabinofuranosyl)pyrrole-2- carboxylate (6) with hydrazine afforded 4-amino-1-(2,3,5-tri-O-benzyl-1-beta-D- arabinofuranosyl)pyrrolo[2,3-d]pyridazin-7-one (7). Treatment of 7 with bromine afforded 4-amino-3-bromo-1-(2,3,5-tri-O-benzyl-beta-D-arabinofuranosyl) pyrrolo[2,3-d]pyridazin-7-one hydrobromide (9). The benzyl ether functions of 7 and 9 were removed with boron trichloride to afford 4-amino-1-(beta-D-arabinofuranosyl)pyrrolo[2,3-d]pyridazin-7-one (8) and its 3-bromo analog 10. 4-Amino-1-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrida zin-7- one (13) was prepared by the sodium salt condensation of ethyl 3-cyanopyrrole-2-carboxylate (5) with 2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranosyl chloride (11) followed by ring annulation with hydrazine. Deprotection of ethyl 3-cyano-1-(2-deoxy-3,5-di-O-p-toluoyl-beta-D-erythro-pentofuranosyl)pyrr ole- 2-carboxylate (12) using sodium ethoxide furnished ethyl 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-3-cyanopyrrole-2-carboxy late (14) which served as the starting material for the preparation of 4-amino-1-(2,3-dideoxy-beta-D-glycero-pentofuranosyl)pyrrolo[2,3-d] pyridazin-7-one (20). Selective protection of the 5'-hydroxyl group of 14 with tert-butyldimethylsilyl chloride followed by a Barton type deoxygenation sequence of the 3'-hydroxyl groups afforded ethyl 3-cyano-1-[2,3-dideoxy-5-O-tert-butyldimethylsilyl)-beta-D-glycero- pentofuranosyl]pyrrole-2-carboxylate (18). Deprotection of 18 with tetra-n-butylammonium fluoride and ring annulation with hydrazine afforded 20. The acyclic analog 4-amino-1-[(1,3-dihydroxy-2-propoxy)methyl]pyrrolo[2,3-d]pyridazin -7-one (24) was prepared via the sodium salt glycosylation of 5 with (1,3-dihydroxy-2-propoxy)methyl bromide (22) followed by a ring annulation with hydrazine. N-Bromosuccinimide treatment of 13, 20, and 25 afforded the 3-bromo derivatives 15, 21, and 25. Evaluation of these compounds in L1210, HFF, and KB cells showed that the sugar-modified analogs all were less cytotoxic than their corresponding ribonucleoside analogs. The compounds also were less active against human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1). The 3-bromo derivatives were much more active than the 3-unsubstituted analogs in both the cytotoxicity, and antiviral assays. However, there was only modest separation between activity against HCMV and cytotoxicity and there was virtually no selectivity for activity against HSV-1.

Dehydrative metabolites of 1-(3-chlorophenyl)-1methyl-2-phenyl-2-(2-pyridine)ethanol as potential hypocholesteremic agents.

The E and Z isomers of 2-[2-(3-chlorophenyl)-1-phenyl-1-propenyl]pyridine (2a,b) and 2-[2-(3-chlorophenyl)-1-(4-hydroxyphenyl)-1-propenyl]pyridine (4a,b) were synthesized and separated as possible metabolites of 1-(3-chlorophenyl)-1-methyl-2-phenyl-2-(2-pyridine)ethanol (1a). Following administration of 1a to rats, a HPLC system was used to examine urine and serum specimens for the less polar metabolites of 1a. Isomers 2a and 2b were not detected but their hydroxylated derivatives 4a and 4b were observed as minor metabolites. Compounds 2a,b and 4a,b exhibited hypocholesteremic activity in rats; compounds 4a and 4b are of special interest because they possessed relatively low estrogenicity.

Methylene-gem-difluorocyclopropane analogues of nucleosides: synthesis, cyclopropene-methylenecyclopropane rearrangement, and biological activity.

Alkylation-elimination of adenine and 2-amino-6-chloropurine with gem-difluorocyclopropane dibromide 10 gave E- and Z-methylene-gem-difluorocyclopropanes 11a, 11b, 12a, and 12b and gem-difluorocyclopropenes 13a and 13b. Debenzylation of intermediates 11a, 11b, 12a, and 12b afforded E- and Z-methylenecyclopropanes 4a, 4b, 5a, and 5b. Hydrolysis of 2-amino-6-chloropurine derivatives 4b and 5b afforded guanine analogues 4c and 5c. Composition of products (except 14b) obtained from alkylation-elimination reflects thermodynamically controlled cyclopropene-methylenecyclopropene rearrangement. The E-isomer 4a was moderately active against human cytomegalovirus and along with the Z-isomer 5a was active against leukemia L1210 and solid tumors in vitro.

Design, synthesis, and antiviral activity of certain 2,5,6-trihalo-1-(beta-D-ribofuranosyl)benzimidazoles.

A new series of 2-substituted 5,6-dichlorobenzimidazole ribonucleosides has been synthesized and tested for activity against two human herpes viruses and for cytotoxicity. 2,5,6-Trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) was prepared by ribosylation of the heterocycle 2,5,6-trichlorobenzimidazole followed by a removal of the protecting groups. The 2-bromo derivative (BDCRB) was made in a similar fashion from 2-bromo-5,6-dichlorobenzimidazole. In contrast, the 2-iodo derivative presented a more difficult problem since the appropriate heterocycle was unavailable. This prompted us to prepare the 2-amino derivative followed by nonaqueous diazotization and removal of the blocking groups. Biological evaluation revealed marked differences in the activities of these compounds and the closely related known compound 5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB). DRB was weakly active against both human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1), (IC50's = 42 and 30 microM, respectively) but was cytotoxic to uninfected human foreskin fibroblasts and KB cells in the same dose range. Similar results were obtained with the heterocycle 2,5,6-trichlorobenzimidazole. In marked contrast, the ribonucleoside of 2,5,6-trichlorobenzimidazole (TCRB) was active against HCMV (IC50 = 2.9 microM, plaque assay; IC90 = 1.4 microM, yield assay) but only weakly active against HSV-1 (IC50 = 102 microM, plaque assay). Little to no cytotoxicity was observed in HFF and KB cells at concentrations up to 100 microM. By changing the substituent at the 2-position from chlorine to bromine (BDCRB), a 4-fold increase in activity against HCMV was observed without any significant increase in cytotoxicity. In contrast, the 2-I and 2-NH2 derivatives were only weakly active against HCMV and HSV-1 with activity not well-separated from cytotoxicity. These data establish that for maximum activity against HCMV with separation from cytotoxicity, ribose is preferred at the 1-position and that Cl or Br is apparently preferred at the 2-position. The activity and selectivity of both TCRB and BDCRB were better than that observed with either ganciclovir or foscarnet.

Synthesis of fluorosugar analogues of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole as antivirals with potentially increased glycosidic bond stability.

The metabolic instability in vivo of the glycosidic bond of 2,5, 6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) prompted us to design and synthesize the hitherto unreported fluorinated benzimidazole nucleosides 2,5, 6-trichloro-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)benzimidazole , 2,5, 6-trichloro-1-(3-deoxy-3-fluoro-beta-D-xylofuranosyl)benzimidazole, and 2-bromo-5, 6-dichloro-1-(2-deoxy-2-fluoro-beta-D-ribofuranosyl)benzimidazole. TCRB was converted into the 2',5'-ditrityl and 3',5'-ditrityl derivatives, which were fluorinated with DAST and deprotected to yield 2,5, 6-trichloro-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)benzimidazole and 2,5, 6-trichloro-1-(3-deoxy-3-fluoro-beta-D-xylofuranosyl)benzimidazole. The resulting low overall yield (5%) of 2,5, 6-trichloro-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)benzimidazole encouraged us to develop an alternative route. The heterocycle 2,5, 6-trichlorobenzimidazole was condensed with 1-bromo-3, 5-di-O-benzoyl-2-deoxy-2-fluoro-alpha-D-arabinofuranose to give, after deprotection, 2,5, 6-trichloro-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)benzimidazole in a 50% overall yield. The 2'-deoxy-2'-fluoro-beta-D-ribofuranosyl compounds were prepared using 2'-deoxy-2'-fluorouridine, N-deoxyribofuranosyl transferase, and 5,6-dichlorobenzimidazole. Functionalization of the C2 position then gave the desired derivatives. Antiviral and cytotoxicity testing revealed that the deoxy fluoro arabinofuranosyl, xylofuranosyl, and ribofuranosyl derivatives were less active against human cytomegalovirus and more cytotoxic than TCRB.

Synthesis, antiproliferative, and antiviral activity of 4-amino-1-(beta-D-ribofuranosyl)pyrrolo[2,3-d]pyridazin-7(6H)-one and related derivatives.

The synthesis of 4-amino-1-beta-D-ribofuranosylpyrrolo[2,3-d]pyridazin-7 (6H)-one (3) from the reaction of ethyl 3-cyano-1-beta-D-ribofuranosylpyrrole-2-carboxylate (10) and hydrazine is described. The 5:6 pyrrolo[2,3-d]pyridazin-7(6H)-one structure of 3 was established via a three-step conversion of 3 into 1-beta-D-ribofuranosylpyrrolo[2,3-d]pyridazin-4,7(5H,6H)- dio ne (14). 4-Amino-3-chloro-1-beta-D-ribofuranosylpyrrolo[2,3-d]pyridazin+ ++-7(6H)-one (16) 4-amino-3-bromo-1-beta-D-ribofuranosylpyrrolo[2,3-d]pyridazin++ +-7(6H)-one (18) were prepared via N-chlorosuccinimide or N-bromosuccinimide treatment of 4-amino-1-(2,3,5-tri-O-benzyl-beta- D-ribofuranosyl)pyrrolo[2,3-d]pyridazin-7(6H)-one (7) followed by a removal of the benzyl groups with boron trichloride. Direct treatment of 3 with N-iodosuccinimide furnished 4-amino-3-iodo-1-beta-D-ribofuranosylpyrrolo[2,3-d]pyridazin -7(6H)-one (19). The antiproliferative activity of the compounds was determined in L1210, H. Ep. 2 and several additional human tumor cell lines. In L1210 cells, the 3-halo-substituted compounds 16, 18, and 19 exhibited significant cytotoxicity (IC50 = 0.2, 0.1, 0.08 microM, respectively), in contrast to the 3-unsubstituted compound 3, which had only slight activity. The greater antiproliferative activity of 18 and 19 in contrast to 3 was confirmed in H. Ep. 2 cells and KB cells. The antiviral evaluation of these compounds revealed that compounds 16, 18, and 19 were active against human cytomegalovirus in both plaque- and yield-reduction assays. However, this activity was only partially separated from cytotoxicity in human cell lines.