Deoxy sugar analogues of triciribine: correlation of antiviral and antiproliferative activity with intracellular phosphorylation.

Triciribine (TCN) and triciribine monophosphate (TCN-P) have antiviral and antineoplastic activity at low micromolar or submicromolar concentrations. In an effort to improve and better understand this activity, we have conducted a structure-activity relationship study to explore requirements for the number of hydroxyl groups on the ribosyl moiety for biological activity. 2'-Deoxytriciribine (2'-dTCN), 3'-deoxytriciribine (3'-dTCN), 2', 3'-epoxytriciribine (2',3'-epoxyTCN), 2',3'-dideoxy-2', 3'-didehydrotriciribine (2',3'-d4TCN), and 2',3'-dideoxytriciribine (2',3'-ddTCN) were synthesized and evaluated for activity against human immunodeficiency virus (HIV-1), herpes simplex virus type 1 (HSV-1), and human cytomegalovirus (HCMV). Antiproliferative activity of the compounds also was tested in murine L1210 cells and three human tumor cell lines. All compounds were either less active than TCN and TCN-P or inactive at the highest concentration tested (100 microM) in both antiviral and antiproliferative assays. Reverse-phase HPLC of extracts from uninfected cells treated with the deoxytriciribine analogues only detected the conversion of 3'-dTCN and 2',3'-ddTCN to their respective monophosphates. Therefore, either the deoxytriciribine analogues were not transported across the cell membrane or, more likely, they were not substrates for a nucleoside kinase or phosphotransferase. We have concluded that the hydroxyl groups on the ribosyl ring system of TCN and TCN-P must be intact in order to obtain significant antiviral and antineoplastic activity.

Acyclic sugar analogs of triciribine: lack of antiviral and antiproliferative activity correlate with low intracellular phosphorylation.

Triciribine and triciribine monophosphate have antiviral and antiproliferative activity at low or submicromolar concentrations. In an effort to improve and better understand this activity, we have synthesized a series of acyclic analogs and evaluated them for activity against select viruses and cancer cell lines. We conclude that the rigid ribosyl ring system of triciribine must be intact in order to be phosphorylated and to obtain significant antiviral and antiproliferative activity.

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.

Adrenal proteins bound by a reactive intermediate of mitotane.

PURPOSE: Mitotane (o,p'-DDD), is the only adrenolytic agent available for the treatment of adrenocortical carcinoma. Previous studies have shown that mitotane covalently binds to adrenal proteins following its metabolism in adrenocortical tissue to a reactive acyl chloride intermediate. It was the objective of this study to compare the electrophoresis separation patterns of such adducts following activation of mitotane by various adrenocortical sources. METHODS: With the use of a 125I-labeled analog of mitotane, 1-(2-chlorophenyl)-1-(4-iodophenyl)-2,2-dichloroethane, gel electrophoresis patterns were obtained for homogenates from bovine, canine and human adrenocortical preparations as well as from a human adrenal preparation. Western immunoblotting analysis was used to test the resulting patterns for adducts of cytochrome P-450scc and adrenodoxin. RESULTS: The electrophoresis separations were similar for all preparations, with bands at apparent molecular weights of 49.5 and 11.5 kDa being the most pronounced. Radiolabeling of the proteins of a human adrenal cancer cell line NCI H-295 was weak, but a band at 11.5 kDa was detected. Western immunoblotting analyses indicated that the band at 49.5 kDa corresponded in molecular weight to that of adrenal cytochrome P-450scc, but the band at 11.5 kDa did not correspond to adrenodoxin. CONCLUSIONS: The similarity of the results with canine and bovine adrenal preparations to that of human material offers useful systems for studying mitotane and its analogs. This should aid in understanding the mechanism of action of mitotane and in the design of compounds for the treatment of adrenocortical carcinoma.

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 antiproliferative and antiviral activity of 2'-deoxy-2'-fluoroarabinofuranosyl analogs of the nucleoside antibiotics toyocamycin and sangivamycin.

The glycosylation of 3,4-dicyano-2-[(ethoxymethylene)amino]pyrrole (7) with 2-deoxy-2-fluoro-alpha-D-erythro-pentofuranosyl bromide (2) furnished an anomeric mixture of nucleosides (8a,b). This mixture was separated, and the individual anomers were treated with methanolic ammonia to effect a concomitant deblocking and ring closure. This furnished both anomers of 2'-deoxy-2'-fluoro-ara-toyocamycin (9a,b). The cyano moiety of 9b was converted to the carboxamide moiety to furnish 2'-deoxy-2'-fluoro-ara-sangivamycin (10) and to the thiocarboxamide moiety to furnish 2'-deoxy-2'-fluoro-ara-thiosangivamycin (11). The target compounds 10 and 11 showed similar antiproliferative activity against L1210 cells in vitro, with IC50's of 3 and 5 microM. Antiviral evaluation revealed a somewhat different pattern of activity. All analogs, both alpha and beta anomers, were active against human cytomegalovirus (HCMV), albeit the beta anomers were most active. The beta anomers also were active against herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus (HIV). Compound 10 was most active in the series, ca. 10-fold more potent than 11; IC50's for 10 ranged from 4 to 25 nM for HCMV, HIV, and varicella zoster virus (VZV) and from 30 to 500 nM for HSV-1. Even though compound 10 was cytotoxic, which will probably preclude its use as an antiviral drug (IC50's = 0.2-5.5 microM), the difference between cytotoxicity and activity against HCMV, HIV, and VZV was sufficient to indicate specific activity against a viral target.

Synthesis and evaluation of certain thiosangivamycin analogs as potential inhibitors of cell proliferation and human cytomegalovirus.

A series of 7-substituted 4-aminopyrrolo[2,3-d]pyrimidines related to the nucleosides toyocamycin and thiosangivamycin were prepared and tested for their activity against human cytomegalovirus (HCMV). The nucleosides 2'-deoxytoyocamycin (1), xylo-toyocamycin (2), 3'-deoxytoyocamycin (3), 2',3'-dideoxy-2',3'-didehydrotoyocamycin (4), 2',3'-dideoxytoyocamycin (5), ara-toyocamycin (6), 2'-deoxy-2'-amino-ara-toyocamycin (7), and 5'-deoxytoyocamycin (8) were treated with sodium hydrogen sulfide generated in situ to afford the corresponding thiosangivamycin analogs (9-16). The cyano derivatives 1-8 were synthesized by modifications of literature procedures. All of the thioamide derivatives (9-16) were active against HCMV with IC50's ranging from 0.5 to 6 microM. Most also were active against herpes simplex virus type 1 (HSV-1) but at higher concentrations. The antiviral activity was not completely separated from cytotoxicity in two human cell lines. The antiproliferative activity was strongly influenced by the position of the modification on the carbohydrate moiety. The xylosyl and 3'-deoxy derivatives were significantly more potent than those with modifications at the 2', 5', or 2',3' position(s). Interestingly, 5'-deoxythiosangivamycin (16) possessed both antiviral and antiproliferative activity suggesting that phosphorylation of the 5'-hydroxyl may not be required for these compounds to have biological activity.

Bovine adrenal cortex transformations of mitotane [1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane; o,p'-DDD] and its p,p'- and m,p'-isomers.

The adrenalytic activity of mitotane (o,p'-DDD) has made it useful in the treatment of adrenocortical carcinoma and Cushing's syndrome. In support of a study to develop mitotane analogs as more effective therapeutic agents and as a basis for understanding the toxicity of related compounds in the adrenals, the biotransformations of o,p'-DDD in adrenocortical homogenate preparations have been studied and compared with those of its m,p'- and p,p'-isomers. Aliphatic oxidation to the corresponding acetic acid derivative, o,p'-, m,p'- or p,p'-DDA, was the major transformation for all the preparations. In the comparisons of the DDD isomers, the order of both DDA formation and apparent covalent binding was o,p'- > m,p'- > p,p'-DDD. There was also evidence for alpha-hydroxylation at the benzylic carbon with subsequent loss of water to form ethylene derivatives. This was a minor pathway for o,p'-DDD, but was the major pathway for the other two isomers. Thus, while the total yields of metabolites of o,p'- and m,p'-DDD were similar and at least twice that of the p,p'-isomer, their distribution of metabolites differed significantly. The effects of the three isomers on cell growth and cortisol production with the human adrenocortical carcinoma cell line, NCI H-295, followed the same order as their DDA formation and tissue binding. It is proposed that hydroxylation by the adrenal cortex at the beta-carbon leads to an adrenalytic effect, whereas hydroxylation at the alpha-carbon would represent an alternate deactivation pathway.

Structural and stereochemical studies of potent inhibitors of glucosylceramide synthase and tumor cell growth.

Analogs and homologs of PDMP were synthesized, based on its structure (D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol). This compound had previously been found to block the synthesis of GlcCer (glucosylceramide). Increasing the acyl chain length from 10 to 16 carbon atoms greatly enhanced the efficacy of the enzyme inhibitor, as did the use of a less polar cyclic amine, especially a pyrrolidine instead of a morpholine ring. Replacement of the phenyl ring by a chain corresponding to sphingosine also yielded a strongly inhibitory material. By using a chiral synthetic route, we showed that the isomers active against GlcCer synthase had the R,R-(D-threo)-configuration. However, strong inhibition of the growth of human cancer cells in plastico was produced by both the threo and erythro racemic compounds, showing involvement of an additional factor (beyond simple depletion of cell glycosphingolipids by blockage of GlcCer synthesis). The growth arresting effects could be correlated with increases in cellular ceramide and diglyceride levels. The aliphatic pyrrolidino compound was strongly inhibitory toward the glucosyltransferase and produced almost complete depletion of glycolipids, but did not inhibit growth or cause an accumulation of ceramide. Attempts were made to see whether the differences in growth effects could be attributed to the influence of the inhibitors on related enzymes (ceramide and sphingomyelin synthase and ceramidase and sphingomyelinase). While some stimulation of enzyme activity was noted, particularly at high inhibitor concentrations (50 microM), these findings did not explain the differing effects of the different inhibitors. The best inhibitors of GlcCer synthase compared favorably in efficacy with some cancer chemotherapeutic drugs in current use when tested with a battery of human cancer cells.

Metabolic activation and binding of mitotane in adrenal cortex homogenates.

Mitotane [1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane, o,p'-DDD] is an adrenocorticolytic agent of value in the treatment of adrenocortical carcinoma and Cushing's syndrome. In support of a program to develop agents superior to mitotane, it is the purpose of this study to explore the relationship of the metabolism of mitotane to its binding to adrenal cortex tissue from several sources. The objective was to detect the mitotane moiety responsible for its covalent binding in various test systems. Studies were conducted with an 125l-labeled analog of mitotane, 1-(2-chlorophenyl)-1-(4-iodophenyl)-2,2-dichloroethane, prior to a comparison to results with lower specific activity [14C]mitotane. With dog adrenal cortical whole homogenates, the majority of covalent binding was to proteins with an additional one-sixth of the total bound radioactivity associated with a phospholipid fraction. No radioactivity was associated with DNA. The rank order of species in regard to metabolism and protein binding was bovine > dog > rat adrenal homogenates > human normal adrenal or tumor homogenates. The percentage of radioactivity recovered from the hydrolysates of those fractions was uniformly high. In addition, the only metabolite present in the hydrolysates corresponded to 1-(2-chlorophenyl)-1-(4-iodophenyl)acetic acid from the iodo analog of o,p'-DDD and the corresponding o,p'-dichlorodiphenylacetic acid (o,p'-DDA) from o,p'-DDD. Our results are consistent with an acyl chloride being the reactive intermediate formed from the dichloromethyl moiety of mitotane, which leads to both DDA metabolite formation and binding to adrenal cortical bionucleophiles.

Relationship between cytotoxicity and conversion of thiosangivamycin analogs to toyocamycin analogs in cell culture medium.

Non-nucleoside analogs of the pyrrolopyrimidine nucleosides toyocamycin, sangivamycin and thiosangivamycin have been synthesized and their cytotoxicity in mammalian cells determined. While studying the effects of 5-thioamide-substituted analogs on cell growth, we observed an interesting phenomenon in which cells recovered spontaneously from growth inhibition during extended incubations. HPLC studies demonstrated that the 5-thioamide moiety of several structurally dissimilar 7-substituted 4-aminopyrrolo[2,3-d]pyrimidines, including thiosangivamycin, is unstable in cell culture medium and is converted to the corresponding 5-nitrile with a half-life of approximately 48 h. In contrast, different substituents at the 4-position of the heterocycle significantly affected the stability of the 5-thioamide moiety. Conversion of the thioamide to the nitrile was caused by components in the cell culture medium, not components of serum. The above observations demonstrate that caution should be exercised in interpreting biological data obtained in vitro for 5-thioamide pyrrolo[2,3-d]pyrimidines.

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.

Comparison of the adrenalytic activity of mitotane and a methylated homolog on normal adrenal cortex and adrenal cortical carcinoma.

Mitotane is an important adrenalytic drug for the treatment of adrenal cancer whose use is limited by toxicity. Reports from another laboratory indicated that a methylated homolog of Mitotane (Mitometh) tested in guinea pigs possessed comparable adrenalytic activity but was less toxic than Mitotane. This observation prompted us to undertake a comparative study of these two drugs on the basis that Mitometh may be a superior agent for the treatment of adrenal cancer. Preliminary studies in guinea pigs failed to show a significant adrenalytic effect for either Mitotane or Mitometh. Thus, we extended the study to 13 mongrel dogs weighing 12-15 kg that were treated daily with Mitometh or Mitotane (50-100 mg/kg) for 6 or 12 days. Cortisol decreased to undetectable levels and adrenocorticotropic hormone (ACTH) rose to 10 times the baseline levels within 72 h in Mitotane-treated animals. Despite the achievement of similar drug levels, Mitometh treatment in dogs failed to suppress cortisol or increase ACTH. To determine whether these differences were due to differences in bioavailability, we measured the relative concentration of Mitotane and Mitometh in homogenates of adrenal cortex obtained from Mitotane- and Mitometh-treated dogs. The adrenal concentration of Mitometh determined in Mitometh-treated dogs was 5 times higher than the concentration of Mitotane measured in Mitotane-treated animals. Whereas the adrenal glands of Mitotane-treated dogs showed hemorrhage and necrosis, the Mitometh-treated animals showed no adrenal damage. Despite the lack of adrenalytic activity, Mitometh maintained its toxicity as demonstrated by microscopic evidence of hepatic necrosis and an increase in hepatic enzymes. The adrenalytic effects of both agents was also studied in vitro using a human functioning adrenal cortical carcinoma cell line, NCI-H295. Whereas Mitotane strongly suppressed cell growth, Mitometh had a weaker effect. We conclude that Mitometh is not likely to be effective in the therapy of adrenal cancer. Moreover, the results of this study are supportive of the view that metabolic transformation of Mitotane is in some way linked to its adrenalytic action.

Synthesis and biological activity of certain alkyl 5-(alkoxycarbonyl)-1H-benzimidazole-2-carbamates and related derivatives: a new class of potential antineoplastic and antifilarial agents.

A series of methyl and ethyl 5-(alkoxycarbonyl)-1H-benzimidazole-2-carbamates (7-19) and methyl 5-carbamoyl-1H-benzimidazole-2-carbamates (24-34) have been synthesized via the reaction of an appropriate alcohol or amine with the acid chloride derivatives 6a or 6b at room temperature. Reaction of an alcohol with acid chloride 6a at reflux temperature afforded transesterified products 20-23 in good yield. Treatment of methyl 5-amino-1H-benzimidazole-2-carbamate with substituted benzoyl chlorides furnished the methyl 5-benzamido-1H-benzimidazole-2-carbamates (36-38). Compounds 9, 16, 20, and 22 demonstrated significant growth inhibition in L1210 cells with IC50's less than 1 microM. Growth inhibition by this series of compounds appears to be associated with mitotic spindle poisoning. All the compounds tested, 9, 10, 19, 20, 22, and 23, caused significant accumulation of L1210 cells in mitosis. Compounds 7, 9, 19, 25, 26, 27, and 36 showed significant in vivo antifilarial activity against adult worms of Brugia pahangi, Litomosoides carinii, and Acanthocheilonema viteae in experimentally infected jirds.

Synthesis, antiproliferative, and antiviral activity of certain 4-aminopyrrolo[2,3-d]pyridazine nucleosides: an entry into a novel series of adenosine analogues.

The preparation of novel heterocyclic base modified adenosine analogues, the 4-aminopyrrolo[2,3-d]pyridazine nucleosides, is described. Crucial to their successful preparation was the use of the pyrrole glycoside intermediates 3-cyano-2-formyl-1-(2,3,5-tri-O-benzyl-beta-D-ribofuranosyl)pyrrole (11) and 3-cyano-2-formyl-1-(2,3,5-tri-O-benzyl-beta-D-arabinofuranosyl)pyrrole (17). Treatment of 11 and 17 with hydrazine dihydrochloride followed by treatment with boron trichloride provided 4-amino-1-beta-D-ribofuranosylpyrrolo[2,3-d]pyridazine (2) and 4-amino-1-beta-D-arabinofuranosylpyrrolo[2,3-d]pyridazine (3), respectively. 4-Amino-3-bromo-1-beta-D-ribofuranosylpyrrolo[2,3-d]-pyridazine (4) was prepared by a bromination of 4-amino-1-(2,3,5-tri-O-benzyl-beta-D-ribofuranosyl)pyrrolo[2,3-d]pyri daz ine (12) and subsequent removal of the benzyl groups with boron trichloride. Compounds 2-4 were evaluated for antiproliferative and antiviral activity. The tubercidin analogue (2) and its arabinosyl derivative (3) were virtually inactive in all assays. In contrast, the 3-bromo analogue 4 inhibited growth of L1210 and H. Ep. 2 cells. Compound 4 was also active against human cytomegalovirus and herpes simplex virus type 1, but the antiviral activity was not completely separated from cytotoxicity.