Inhibition of purine nucleoside phosphorylase by 8-aminoguanosine: selective toxicity for T lymphoblasts.

The guanosine analog 8-aminoguanosine is an effective inhibitor of the purine degradative enzyme purine nucleoside phosphorylase, both in vitro and in intact lymphoid cells. In a human lymphoblast tissue culture system, 8-aminoguanosine, in combination with low concentrations of 2'-deoxyguanosine, causes toxicity toward T cells but not B cells. The selective T cell toxicity correlates with increased accumulation of deoxyguanosine triphosphate in the treated T lymphoblasts.

Identification of 6-azauridine triphosphate in L1210 cells and its possible relevance to cytotoxicity.

L1210 cells treated with 1 mM 6-azauridine (AzUrd) (concentration causing 50% inhibition of cell growth, 3 microM) continued to divide at a reduced rate for 72 h before stopping. However, a 24-h treatment was lethal to 99% of the cells, as determined by colony formation. To investigate the mechanism for this delayed cytotoxicity, the metabolism of AzUrd was studied. Cells incubated with AzUrd contained a new 254 nm-absorbing component, not found in control cells. It appeared to be 6-azauridine-5'-triphosphate, since it was the only peak in the triphosphate region of the chromatogram which contained 3H after incubation of cells with [3H]AzUrd. Incorporation of [3H]AzUrd into the acid-insoluble fraction (nucleic acids) was also detected. A role for this incorporation in the mechanism of AzUrd cytotoxicity was strongly suggested by the observation that cordycepin (0.01 mM) partially protected cells from the lethality of AzUrd, presumably by preventing its incorporation into RNA. The previously known inhibition of pyrimidine de novo synthesis by AzUrd was confirmed by a decrease in the intracellular contents of UTP and CTP in AzUrd-treated cells. Therefore, we propose that the inhibition of pyrimidine de novo synthesis and the incorporation into nucleic acid(s) may act in concert to produce the cytotoxic effects of AzUrd.

Study of the cytotoxicity and metabolism of 4-amino-3-carboxamido-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine using inhibitors of adenosine kinase and adenosine deaminase.

The greater in vivo antitumor activity of 4-amino-3-carboxamido-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (APPCR) in comparison to the parent compound 4-amino-1-(beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (APPR) may involve intrinsic differences in the effects of these two compounds on cells, as well as in their metabolisms. In studies with L1210 cells in vitro, growth inhibition by APPCR for 36 hr or more was found to be cytocidal, while growth inhibition by APPR was cytostatic in that a substantial fraction of the cells survived 36 or 72 hr of exposure to growth-inhibiting concentrations of APPR. It appears that this difference in the cellular effects of APPCR and APPR is not related to differences in the requirement for activation by phosphorylation or in susceptibility to inactivation by deamination. However, deamination may limit the effectiveness of APPR in vivo since it is a substrate for adenosine deaminase, while deamination of APPCR is not detectable.

Thioguanine-induced S and G2 blocks and their significance to the mechanism of cytotoxicity.

The delayed cytotoxic effect of 6-thioguanine (TG) was studied using L1210 mouse leukemic cells in culture. The cell cycle distribution of a population treated continuously with 10(-5) M TG was compared to that of control cells using flow cytometric analysis. The TG-treated cells had an increase in the fraction of the population in G2-M, a decrease in G1, and a constant level in S phase. However, the [methyl-3H]thymidine-labeling index decreased dramatically during TG treatment. Thus, it appeared that some cells were arrested in S phase and that G1 cells did not enter S phase, due to failure to synthesize DNA. To examine the importance of the G2 and S cell progression blocks, cells were exposed to a lethal treatment of 10(-5) M TG for 12 hr and returned to normal medium. Under these conditions, the fraction of the population in both S and G1 decreased, and nearly one-half of the cells accumulated in G2 by 60 hr after TG addition, compared to a G2 fraction of less than one-tenth for the control cells. These results showed that the delayed cytotoxic effect of TG was associated with a cell progression block in the second G2 phase after TG addition, whereas the retention of cells in S phase appeared to be due to readily reversible secondary effects of TG.

Cytotoxicity of a new uridine analog, 4-hydroxy-1-(beta-D-ribofuranosyl)-pyridazine-6-one, and its interaction with uridine kinase.

A new uridine analog, 4-hydroxy-1-(beta-D-ribonfuranosyl)-pyridazin-6-one (3-deaza-6-azaUrd), inhibited the growth of L1210 cells in culture, with a concentration to reduce growth rate to 50% of control of 7 X 10(-5) M. After treatment for 24 or 48 h with 5 X 10(-4) M 3-deaza-6-azaUrd, 80% of the cells were unable to resume growth when the analog was removed from the cultures; also, 99% of the cells were killed, as determined by colony formation in soft agar. Studies on the prevention of the cytotoxic effects of 5 X 10(-4) M 3-deaza-6-azaUrd showed that uridine or cytidine gave complete protection. 2'-Deoxycytidine also gave partial protection, but orotic acid or thymidine had no effect on the growth inhibition by 3-deaza-6-azaUrd. These results suggested that growth inhibition by 3-deaza-6-azaUrd might be due to interference in pyrimidine biosynthesis. Activation of 3-deaza-6-azaUrd to its 5'-phosphate derivative appeared to be catalyzed by uridine kinase. 3-Deaza-6-azaUrd was shown to complete with uridine for phosphorylation (Ki = 4.7 mM) and, therefore, to be a possible alternative substrate for uridine kinase from mouse kidney (Km for uridine = 82 microM). The enzyme was partially purified by streptomycin sulfate precipitation, ammonium sulfate fractionation, and gel filtration. This preparation was found to be free of pyrimidine nucleoside phosphorylase and uridine monophosphate kinase.

Effects of the tricyclic nucleoside 6-amino-4-methyl-8-(beta-D-ribofuranosyl)- pyrrolo[4,3,2-de]pyrimido[4,5-c]pyridazine on the viability and cell cycle distribution of L1210 cells in vitro.

TCN (1 microM) totally inhibited the growth of L1210 cells in culture and caused progressive loss of cellular viability, as indicated by a decreased clonogenicity and nigrosin dye exclusion. After 24 h or more of TCN treatment, a significant fraction of the cells had shrunk in size but did not fragment into dye-impermeable vesicles as reported previously for N1S1-67 hepatoma cells (P. G. W. Plagemann, J. Natl. Cancer Inst., 57: 1283-1295, 1976). TCN-induced growth inhibition was accompanied by a block of cell cycle progression in G1 or at the G1-S boundary. At all TCN concentrations studied, progression of cells out from behind this block was evident as a depletion of the early S-phase population in comparison to controls, while increasing the concentration of TCN (0.1 to 1 microM) led to a progressive retention of cells in S phase, suggesting a slowing of progression through S phase. The fraction of S-phase cells incorporating [methyl-3H]thymidine and the amount of [methyl-3H]thymidine incorporated per labeled cell were both decreased by TCN treatment. Increasing the concentration of TCN (0.1 to 1 microM) progressively decreased DNA synthesis and increased cell lethality. Thus it appeared that inhibition of DNA synthesis might cause the retention of cells in S phase which is associated with TCN lethality.

Dual mechanisms of inhibition of DNA synthesis by triciribine.

The inhibition of DNA synthesis in triciribine (TCN)-treated L1210 cells was shown to involve two mechanisms, with different concentration dependence. (a) Initiation of new replicons and possibly of Okazaki fragments was inhibited when the cells were treated with 0.1 microM TCN. The inhibition of replicon initiation was shown by the rate of alkaline elution of [3H]DNA from 15-min-[3H]thymidine-labeled cells being slower if the cells had been pretreated with TCN, indicating that the average size of actively replicating DNA strands was increased. (b) At 1 microM TCN elongation of previously initiated DNA chains was also inhibited. This conclusion was suggested by the decrease in the rate of alkaline elution of [3H]DNA, during postlabeling incubation, being less if TCN was included in the medium. The mechanism of inhibition of DNA synthesis by TCN was shown not to involve DNA strand breakage or cross-linking, inhibition of polyamine biosynthesis, inhibition of purine de novo biosynthesis, inhibition of DNA polymerase alpha or DNA primase, or inhibition of ligation of Okazaki fragments. The effects of TCN on the incorporation of [3H]thymidine into Okazaki fragments and higher molecular weight DNA suggested the possibilities of inhibition of Okazaki fragment initiation and/or DNA polymerase delta.

Pyrrolopyrimidine nucleosides. 18. Synthesis and chemotherapeutic activity of 4-amino-7-(3-deoxy-beta-D-ribofuranosyl)pyrrolo[2,3-d] pyrimidine-5-carboxamide (3'-deoxysangivamycin) and 4-amino-7-(2-deoxy-beta-D-ribofuranosyl)pyrrolo[2,3-d] pyrimidine-5-carboxamide (2'-deoxysangivamycin).

A multistep synthesis, using the nucleoside antibiotic toyocamycin as the starting material, has furnished 6,2'-S-cyclosangivamycin (6). Desulfurization of 6,2'-S-cyclosangivamycin (6) with Raney nickel has provided 2'-deoxysangivamycin (7). Treatment of sangivamycin (1c) with sodium iodide and alpha-acetoxyisobutyryl chloride has furnished 4-amino-7-[2-O-acetyl-3-deoxy-3-iodo-5-O-(2,5,5-trimethyl-4-oxo-1, 3-dioxolan-2-yl)-beta-D-xylofuranosyl]-pyrrolo[2,3-d] pyrimidine-5-carboxamide (8a). Dehalogenation of 8a with 10% palladium on charcoal was followed by a removal of the blocking groups with ammonium hydroxide to give 3'-deoxysangivamycin (9) in 49% overall yield. The reaction of sangivamycin (1c) with diphenyl disulfide and tributylphosphine gave 5'-(phenylthio)-5'-deoxysangivamycin (10). Treatment of 10 with Raney Nickel afforded 5'-deoxysangivamycin (11). Antitumor evaluation showed that 3'-deoxysangivamycin had significant activity against the murine leukemia L1210 both in vivo and in vitro, although it was less potent on a molar basis than the parent compound sangivamycin. The 2'- and 5'-deoxysangivamycins did not show significant antitumor activity.

Synthesis and cytotoxicity studies of 8-amino-6-methyl-2-beta-D-ribofuranosyl-1,2,3,5,6,7-hexaazaacenaphthyle ne (7-aza-TCN) and the corresponding 2'-deoxy- and arabinonucleoside analogues.

The tricyclic nucleoside 8-amino-6-N-methyl-2-beta-D-ribofuranosyl-1,2,3,5,6, 7-hexaazaacenaphthylene was synthesized from 8-amino-6-N-methyl-4-(methylthio)-2-beta-D-ribofuranosyl-1,2,3,5,6, 7-hexaazaacenaphthylene. The 2'-deoxy analogue of 5, 8-amino-6-N-methyl-2-(2-deoxy-beta-D-ribofuranosyl)-1,2,3,5,6, 7-hexaazaacenaphthylene (11), and the arabino analogue of (5), 8-amino-6-N-methyl-2-beta-D-arabinofuranosyl-1,2,3,5,6, 7-hexaazaacenaphthylene (14) were synthesized from 5. Nucleosides 2,3,4,5,11, and 14 were evaluated for potential anticancer activity by measuring their ability to inhibit the growth of L1210 and H. Ep. 2 tumor cells in vitro.

Synthesis and evaluation of 6-(dibromomethyl)-5-nitropyrimidines as potential antitumor agents.

A series of 2,4,6-trisubstituted-5-nitropyrimidines have been prepared and evaluated for inhibition of proliferation of L1210 and H.Ep.2 cells in vitro. The most potent compound was 6-(dibromomethyl)-2-methoxy-4-morpholino-5-nitropyrimidine (11) (L1210, IC50 = 0.32 microM; H.Ep.2, IC50 = 1.6 microM). Of the 6-substituents incorporated, only CHBr2, CH2Br, and CHO were compatible with antiproliferative activity, while a wider variety of 4-substituents were tolerated. At concentrations near the IC50 for antiproliferative activity, a delayed resumption of cell proliferation in L1210 cultures indicated that the activity of the compounds was short-lived and suggested they might act by an alkylation mechanism.

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.

Pyrazolopyrimidine nucleosides. 12. Synthesis and biological activity of certain pyrazolo[3,4-d]pyrimidine nucleosides related to adenosine.

The chemical synthesis of certain 4-substituted pyrazolo[3,4-d]pyrimidine nucleosides is described. Using 1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidin-4-one (1) as the starting material, the reactive intermediate 4-chloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (2) was prepared in excellent yield. Compound 2 served as a versatile precursor for the synthesis of a number of 4-substituted pyrazolo[3,4-d]pyrimidine nucleosides. In antitumor studies of these nucleosides, in vitro and in vivo, it was found that any alteration of the 4-amino substituent of 4-amino-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidine (3) was accompanied by a significant decrease or loss of antitumor activity. On the other hand, introduction of certain substituents at the 3 position of 3 (synthesis reported previously) led to a dramatic increase in antitumor activity in comparison to the parent compound.

Synthesis of non-nucleoside analogs of toyocamycin, sangivamycin, and ++thiosangivamycin: influence of various 7-substituents on antiviral activity.

A number of 7-substituted 4-aminopyrrolo[2,3-d]pyrimidine-5-carbonitrile, -5-carboxamide, and -5-thiocarboxamide derivatives related to the nucleoside antibiotics toyocamycin and sangivamycin were prepared and tested for their activity against human cytomegalovirus (HCMV) and herpes simplex virus type-1 (HSV-1). Treatment of 2-amino-5-bromo-3,4-dicyanopyrrole (1) with triethyl orthoformate followed by alkylation via the sodium salt method with a variety of alkylating agents furnished the corresponding 1-substituted pyrroles 2a-k. Ring annulation was achieved with methanolic ammonia affording the 7-substituted 4-amino-6-bromopyrrolo++-[2,3-d]pyrimidine-5-carbonitrile derivatives 3a-k. Debromination of 3a-k, via catalytic hydrogenation, gave the corresponding 7-substituted 4-aminopyrrolo[2,3-d]pyrimidine-5-carbonitrile analogs 4a-j,l. A selective reduction of 4-amino-6-bromo-7-allylpyrrolo[2,3-d]-pyrimidine-5-carbon ril e (3k) in zinc and acetic acid furnished 4-amino-7-allylpyrrolo-[2,3-d]pyrimidine-5-carbonitrile (4k). Conventional functional group transformations involving the 5-cyano group of 4 furnished the 5-carboxamide derivatives 5a-1 and the 5-thio-amide analogs 6a-l. A similar transformation of the aglycone of toyocamycin (4m) furnished the corresponding aglycone of thiosangivamycin (6m). Several of the new compounds (4-6a-ej-l) were evaluated for their ability to inhibit the growth of L1210 murine leukemic cells. Whereas a number of the carboxamide (5) and thioamide (6) derivatives had modest activity, the corresponding nitrile analogs (4) were all inactive. All compounds were tested for activity against HCMV and HSV-1. The non-nucleoside nitrile analogs 4a-m and carboxamide derivatives 5a-l were, with a few exceptions, essentially inactive against HCMV and HSV-1 and relatively nontoxic. In direct contrast, nearly all of the thioamide derivates 6a-1, including the aglycone of thiosangivamycin (6m), were good inhibitors of HCMV and HSV-1. Most were noncytotoxic in their antiviral concentration range. Cytotoxicity which was observed appeared to be a consequence of DNA synthesis inhibition. Several of these compounds, such as 6b,e, were particularly interesting inhibitors of HCMV with IC(50)'s ranging from 0.1 to 1.3 muM. The antiviral activity of both compounds was well separated from cytotoxicity in KB, HFF, and L1210 cells.

Synthesis and biological activity of a novel adenosine analogue, 3-beta-D-ribofuranosylthieno[2,3-d]pyrimidin-4-one.

The title nucleoside 5 was prepared by a condensation of the silylated heterocycle thieno[2,3-d]pyrimidin-4-one (1) with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose (2a) in the presence of a Lewis acid or with 2,3,5-tri-O-acetyl-D-ribofuranosyl bromide (2b) in the presence of mercuric oxide and mercuric bromide. The site of ribosylation and anomeric configuration of this nucleoside were established by 1H NMR. The synthesis of 3-beta-D-ribofuranosylpyrrolo[2,3-d]pyrimidin-4-one (8), 1-phenyl-5-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4-one (9), 5-methyl-3-beta-D-ribofuranosylthieno[2,3-d]pyrimidin-4-one (10), and 2-methyl-6-beta-D-ribofuranosyltriazolo[5,4-d]pyrimidin-7-one (11) is also described. The title compound inhibited the growth of murine L-1210 leukemic cells in vitro with an ID50 of 3 X 10(-5)M. The growth inhibition could not be prevented by uridine, cytidine, thymidine, deoxycytidine, cytosine, hypoxanthine, or uridine and hypoxanthine together. On the other hand, inhibition of adenosine kinase by 10(-7) M 5-iodotubercidin prevented the cytotoxic effect. Also a subline of L-1210 cells resistant to several cytotoxic adenosine analogues was also resistant to this nucleoside. Thus it appears that this compound 5 may act as an adenosine analogue.

Synthesis of 2,4-disubstituted thiazoles and selenazoles as potential antitumor and antifilarial agents: 1. Methyl 4-(isothiocyanatomethyl)thiazole-2-carbamates, -selenazole-2- carbamates, and related derivatives.

Methyl 4-(isothiocyanatomethyl)thiazole-2-carbamate and methyl 4-(isothiocyanatomethyl)selenazole-2-carbamate have been prepared via chemical transformations involving 2-amino-4-(chloromethyl)thiazole (1) and 2-amino-4-(chloromethyl)selenazole (2), respectively, as starting materials. The homoanalog, methyl 4-(2-isothiocyanatoethyl)thiazole-2-carbamate, was prepared from (2-aminothiazol-4-yl)acetic acid. All compounds prepared were evaluated for their ability to inhibit leukemia L1210 cell proliferation. Methyl 4-(isothiocyanatomethyl)thiazole-2-carbamate (7) was the most active compound in this screen, inhibiting the growth of L1210 leukemic cells with an IC50 = 3.2 microM. Mitotic blocking appears to be its primary mechanism of cytotoxic activity. Compound 7 also was the only compound which demonstrated significant in vivo antifilarial activity against the adult worms of Acanthocheilonema viteae in experimentally infected jirds. This compound was inactive against Brugia pahangi at a dosage of 100 mg/kg x 5 days.

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.

Synthesis of 2,4-disubstituted thiazoles and selenazoles as potential antifilarial and antitumor agents. 2. 2-Arylamido and 2-alkylamido derivatives of 2-amino-4-(isothiocyanatomethyl)thiazole and 2-amino-4-(isothiocyanatomethyl)selenazole.

The synthesis of a series of 2-arylamido and 2-alkylamido derivatives of 2-amino-4-(isothiocyanatomethyl)thiazole and 2-amino-4-(isothiocyanatomethyl)selenazole is described. In vitro antiproliferative evaluations were carried out using L1210 cells. The 2-(alkylamido)thiazole derivatives were moderately antiproliferative, with IC50's of 4-8 microM. A significant increase in activity was obtained for the arylamido derivatives, with IC50's of 0.2-1 microM. The results obtained for the selenazoles were similar to those for the thiazoles. 2-Benzamido-4-(isothiocyanatomethyl)-thiazole (19) was found to be a potent inhibitor of GMP synthetase. None of the compounds prepared in this study demonstrated antifilarial activity.

Synthetic approaches to the guanosine and xanthosine analogues 5-amino-3-beta-D-ribofuranosylpyrazolo[3,4-e][1,3]oxazin-7-one and 3-beta-D-ribofuranosylpyrazolo[3,4-e][1,3]oxazine-5,7-dione and studies of their antitumor potential.

5-Amino-3-beta-D-ribofuranosylpyrazolo[3,4-e][1,3]oxazin-7-o ne has been synthesized via cyclization of the appropriately protected pyrazofurin derivatives and subsequent transformations of the heterocyclic moiety. This guanosine analogue was marginally cytotoxic to L1210 cells in vitro. The xanthosine analogue 3-beta-D-ribofuranosylpyrazolo[3,4-e][1,3]oxazine-5,7-dione was also synthesized, and was found to be highly cytotoxic. It appeared to act as a prodrug of pyrazofurin.