Activity of acyclic halogenated tubercidin analogs against human cytomegalovirus and in uninfected cells.

Novel acyclic halogenated tubercidins (4-amino-5-halo-7-[(2-hydroxyethoxy)-methyl]pyrrolo[2,3-d]pyrimidines) were examined for their ability to inhibit human cytomegalovirus (HCMV) in yield reduction assays. 5-Bromo acyclic tubercidin (compound 102) was a more potent inhibitor of virus replication than the chloro- and iodo-substituted analogs (compounds 100 and 104). At a 100 microM concentration, the bromo and chloro compounds were more potent than acyclovir but not ganciclovir. Virus titers were reduced more than 99% by compounds 102 and 104 whereas compound 100 and the equally potent acyclovir reduced titers by only 90%. Quantitation of viral DNA by DNA hybridization demonstrated strong inhibition of HCMV DNA synthesis by these compounds. The most potent inhibitor, compound 102, had a 50% inhibitory (I50) concentration (1.6 microM) comparable to that of ganciclovir (1.8 microM). Cytotoxicity in uninfected human cells was evaluated and revealed the following: cell growth rates slowed markedly in the presence of 10 microM compound 102 whereas the same concentration of compounds 100 and 104 led to only a slight prolongation of population doubling time; these compounds inhibited cellular DNA synthesis but not RNA or protein synthesis, as measured by incorporation of radiolabeled precursors into acid-precipitable macromolecules; flow cytometry indicated that compound 102 was a mid-S phase blocker, and adenosine antagonized the inhibition of [3H]dThd incorporation by compound 102. Together, these results demonstrate that compound 102 is a potent and selective inhibitor of viral and cellular DNA synthesis and that acyclic halogenated pyrrolo-pyrimidine nucleosides may have therapeutic potential.

Synthesis, antiproliferative, and antiviral activity of certain 4-substituted and 4,5-disubstituted 7-[(1,3-dihydroxy-2-propoxy)methyl]pyrrolo[2,3-d]pyrimidines.

The sodium salts of 4-chloro- and several 4-chloro-5-substituted-7H-pyrrolo[2,3-d]pyrimidines were treated with [1,3-bis(benzyloxy)-2-propoxy]methyl chloride (6) to provide the corresponding 4-chloro- and 4-chloro-5-substituted-7-[[1,3-bis(benzyloxy)-2-propoxy]methyl]pyrrolo [2,3-d]pyrimidines (7-11). Debenzylation with boron trichloride at -78 degrees C furnished 4-chloro- and several 4-chloro-5-substituted-7-[(1,3-dihydroxy-2-propoxy)methyl]pyrrolo[2,3- d]pyrimidines (12.16). Subsequent amination of 12-16 yielded the 4-amino-5-substituted-7-[(1,3-dihydroxy-2-propoxy)methyl]pyrrolo[2,3- d]pyrimidines (17-21). Treatment of 14 with methylamine and 13 and 14 with ethylamine yielded the 4-(alkylamino)-5-halo-7-[(1,3-dihydroxy-2- propoxy)methyl]pyrrolo[2,3-d]pyrimidines (22-24). Treatment of 12-15 with hydroxylamine in refluxing 2-propanol yielded the 5-substituted-4-(hydroxyamino)-7-[(1,3-dihydroxy-2-propoxy)methyl]pyrrol o [2,3-d]pyrimidines (25-28). Treatment of compound 12 with Pd/C under a hydrogen atmosphere has furnished the nebularine analogue 31. The antiproliferative activity of compounds 17-28 and 31 was studied using L1210 cells in vitro. The 4-amino- and 4-(hydroxyamino)-5-halogenated derivatives (compounds 18-20, 26-28) inhibited cell growth. Although the effect of compounds 18-20 and 27 on final growth rate was pronounced (IC50 = 2.3, 0.7, 2.8, and 3.7 microM, respectively), cells underwent at least one doubling before cell division stopped. The remaining compounds were less cytotoxic, with IC50's greater than 30 microM for 21, 23, 26, and 28, whereas no inhibition of L1210 cell growth was observed with compounds 17, 22, 24, 25, and 31 at 100 microM. The antiviral activity of these compounds also was tested. Compounds 18-20 and 26-28 were active against human cytomegalovirus and herpes simplex type 1. The 4-amino derivatives (18-20) were more active than the 4-hydroxyamino derivatives (26-28), the 4-amino-5-bromo and 4-amino-5-iodo derivatives produced more than five log reductions in virus titer at concentrations of 10-100 microM. Although some cytotoxicity was observed at these concentrations, compound 19 was active against murine cytomegalovirus in vivo. At 5.6 mg/kg, 14/15 animals survived compared to 10/15 treated with 5.6 mg/kg of ganciclovir or 1/15 treated with placebo.

Flow cytometric evaluation of the cytotoxicity of novel antiviral compounds.

Two acyclic analogs of bromotubercidin were tested for cytotoxic effects on uninfected cells by monitoring cell growth and measuring cell cycle perturbations using flow cytometry. As reported elsewhere, 5-bromotubercidin analogs in which ribose was replaced by 2-hydroxyethoxymethyl (compound 102) or by 1,3-dihydroxypropoxymethyl (compound 183) were potent inhibitors of human cytomegalovirus (HCMV) replication in vitro (Pudlo et al.: Journal of Medicinal Chemistry 31:2086-2092, 1988). Because these compounds also inhibited the growth of uninfected cells, we performed kinetic studies with an established neoplastic line of human cells (KB) using flow cytometry. Growth of KB cells treated with either compound 102 or 183 were inhibited in a dose-dependent manner. Growth inhibition by compound 183, however, was not fully expressed for at least 24 h. DNA analysis by flow cytometry showed that a 4-h incubation with 10 microM compound 102 caused a decrease of cells in G2/M phase. Cells began to accumulate in early S phase by 12 h of incubation, leading to mid S phase accumulation at 21 h. Compound 183 at 10 microM slightly decreased the number of cells in G2/M phase after a 4-h incubation, and led to accumulation of DNA in S phase after a 12-h incubation. By 24 and 30 h, DNA histograms appeared similar to those of control cells but with a slight accumulation of the population in early S phase. In separate experiments, drugs were removed following a 24-h incubation. After removal of compound 102, KB cell growth resumed with a normal population doubling time. In contrast, the effects of compound 183 were not reversible, suggesting the two compounds acted by different biochemical mechanisms.

Synthesis and antiviral activity of certain 4- and 4,5-disubstituted 7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidines.

In vitro evaluation of a series of previously prepared tubercidin analogues revealed that certain 5-halogen-substituted analogues were active against human cytomegalovirus (HCMV) at concentrations lower than those that produced comparable cytotoxicity in uninfected cells. In contrast, tubercidin was cytotoxic at all antiviral concentrations. Even though the antiviral selectivity of the 5-substituted compounds was slight, this observation led us to prepare a series of acyclic analogues. Treatment of the sodium salt of 4-chloropyrrolo[2,3-d]pyrimidine (2) with (2-acetoxyethoxy)methyl bromide (2a) provided the acyclic nucleoside 4-chloro-7-[(2-acetoxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine (3). A nucleophilic displacement of the 4-chloro group with methoxide, methylamine, and dimethylamine yielded the corresponding 4-substituted compounds 4, 5, and 6, respectively, in good yield. Electrophilic substitution (chlorination, bromination, and iodination) was effected at the C-5 position of compound 3 with N-chlorosuccinimide, N-bromosuccinimide, and iodine monochloride, respectively, in methylene chloride. Removal of the acetyl group from these intermediates (7a-9a) with methanolic ammonia at room temperature afforded the 5-chloro (7b), 5-bromo (8b), and 5-iodo (9b) derivatives of 4-chloro-7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine. Treatment of compounds 7b-9b with methanolic ammonia at an elevated temperature produced the corresponding 5-halotubercidin analogues 10, 11, and 12, respectively. An alternate procedure for the preparation of these 4,5-disubstituted 7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidines involved an electrophilic substitution prior to the condensation of the heterocycle with 2a. Treatment of 2 with N-chlorosuccinimide and N-bromosuccinimide gave compounds 13a and 13b, respectively. The condensation of 13a and 13b with 2a and subsequent treatment with methylamine and ethylamine furnished the corresponding 5-halo-4-substituted-pyrrolo[2,3-d]pyrimidines 14a, 14b, 14c, and 14d, respectively. Evaluation of the target compounds (4-6, 7b-9b, 10-12, and 14a-14d) for cytotoxicity and activity against HCMV and herpes simplex virus type 1 (HSV-1) revealed that all compounds except the 5-halogen-substituted compounds 10, 11, and 12 were inactive. Compounds 10, 11, and 12 were active against both viruses at noncytotoxic concentrations. The activity of compound 11 was particularly noteworthy, being at least 10-fold more potent than acyclovir.