Synergistic inhibition of growth of breast and colon human cancer cell lines by site-selective cyclic AMP analogues.

Our past studies on the mechanism of cyclic AMP (cAMP)-mediated control of tumor growth, using the experimental rat mammary tumor models as well as human breast cancer cell lines, indicated that the action of cAMP is mediated by the RII cAMP receptor protein, the regulatory subunit of cAMP-dependent protein kinase type II (Y. S. Cho-Chung, J. Cyclic Nucleotide Res., 6: 163, 1980). We now shown that the site-selective cAMP analogues, which are manyfold more active in binding to the cAMP receptor protein than previously studied analogues, demonstrate a potent growth inhibition of seven breast and three colon human cancer cell lines. The cAMP receptor protein has two different cAMP binding sites, and cAMP analogues that selectively bind to either one of the two binding sites are known as either site 1 selective (C-8 analogues) or site 2 selective (C-6 analogues). Nineteen site-selective analogues, C-6 and C-8 monosubstituted and C-6,-8 disubstituted, were tested for their growth regulatory effect. The majority of these analogues demonstrated an appreciable growth inhibition, with no sign of toxicity in all 10 cancer lines at micromolar concentrations. The three most potent inhibitors were 8-Cl-, N6-benzyl-, and N6-phenyl-8-thio-p-chlorophenyl-cAMP, demonstrating 50% growth inhibition at 5-25 microM concentrations (IC50). Furthermore, N6-analogues, in combination with halogen or thio derivatives of C-8 analogues, demonstrated synergistic enhancement of growth inhibition. The growth inhibition paralleled a change in cell morphology, an augmentation of the RII cAMP receptor protein, and a reduction in p21 ras protein. The growth inhibition by 8-Cl-cAMP was not due to its metabolite, 8-Cl-adenosine, since: (a) the growth inhibition by 8-Cl-cAMP was released upon cessation of treatment, whereas that by 8-Cl-adenosine was not released; (b) 8-Cl-cAMP treatment did not affect cell cycle progression, whereas 8-Cl-adenosine brought about G1 synchronization; (c) 8-Cl-cAMP treatment caused reduction of p21 ras protein, whereas 8-Cl-adenosine did not affect p21 levels; and (d) 8-Cl-adenosine was not detected in either cell extracts or medium from the cells treated with 8-Cl-cAMP for 48-72 h. Site-selective cAMP analogues thus provide a new physiological means to control the growth of breast and colon human cancer cells.

Chemotherapeutic characterization in mice of 2-amino-9-beta-D-ribofuranosylpurine-6-sulfinamide (sulfinosine), a novel purine nucleoside with unique antitumor properties.

In preclinical investigations performed in mice, 2-amino-9-beta-D-ribofuranosyl purine-6-sulfinamide (sulfinosine), a novel derivative of 6-thioguanosine (6TGR), was active against six solid tumors and four strains of experimental leukemia. Sulfinosine penetrated the central nervous system more readily than did 6TGR and, when given repeatedly, was much more effective in the treatment of L1210 leukemia, being curative for some mice. Other findings of major interest to us were the different dosing characteristics of sulfinosine and 6TGR, the divergent efficiencies of the two drugs in generating cellular resistance, and the activity of sulfinosine against experimental leukemias refractory to 6TGR and other experimental or clinically used chemotherapeutic agents. The chemotherapeutic characterization of sulfinosine that evolved from these studies suggests that this agent may have unique properties that deserve clinical consideration. Both the dosing characteristics of the drug and its pronounced activity against thiopurine-resistant experimental leukemia favor the possibility that sulfinosine could be used to advantage in the treatment of human leukemia unresponsive to 6-mercaptopurine or 6-thioguanine.

Synthesis and biological evaluation of certain 3-beta-D-ribofuranosyl-1,2,4-triazolo[4,3-b)pyridazines related to formycin prepared via ring closure of pyridazine precursors.

All three amino-substituted 3-beta-D-ribofuranosyl-1,2,4-triazolo[4,3-b]pyridazines (5, 19, and 20) structurally related to formycin A were prepared and tested for their antitumor and antiviral activity in cell culture. Dehydrative coupling of 4-amino-5-chloro-3-hydrazinopyridazine (7) with 3,4,6-tri-O-benzoyl-2,5-anhydro-D-allonic acid (6) in the presence of DCC and subsequent thermal ring closure of the reaction product (8) provided 8-amino-7-chloro-3-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)- triazolo[4,3-b]pyridazine (9). Dehalogenation of 9, followed by debenzoylation, gave the formycin congener 8-amino-3-beta-D-ribofuranosyl-1,2,4- triazolo[4,3-b]pyridazine (5). Similar condensation of 5-amino-4-chloro-3-hydrazinopyridazine (13) with 6 and dehalogenation of the cyclized product (16), followed by debenzoylation, gave the isomeric 7-amino-3-beta-D-ribofuranosyl-1,2,4- triazolo[4,3-b]pyridazine (19). DCC-mediated coupling of 6 with 6-chloro-3-hydrazinopyridazine (12), followed by ammonolysis of the cyclized product (21) with liquid NH3, provided a convenient route to 6-amino-3-beta-D-ribofuranosyl-1,2,4-triazolo[4,3-b]pyridazine (20). The structural assignment of 5 was made by single-crystal X-ray diffraction analysis. Compounds 5, 19, 20, and certain deprotected nucleoside intermediates were evaluated against L1210, WI-L2, and CCRF-CEM tumor cell lines, as well as against DNA and RNA viruses in culture. These compounds did not exhibit any significant antitumor or antiviral activity in vitro.

Synthesis and biological activity of nucleosides and nucleotides related to the antitumor agent 2-beta-D-ribofuranosylthiazole-4-carboxamide.

Phosphorylation of 2-beta-D-ribofuranosylthiazole-4-carboxamide (1) provided the 5'-phosphate 2, which was converted to the corresponding 5'-triphosphate 4 and the cyclic 3',5'-phosphate 5. Treatment of 2-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)thiazole-4-carbonitrile (6) with NH3-NH4Cl provided 2-beta-D-ribofuranosylthiazole-4-carboxamidine hydrochloride (7), and treatment with H2S-pyridine provided the corresponding 4-thiocarboxamide 9. Compound 9 was treated with ethyl bromopyruvate, followed by treatment with methanolic ammonia, to yield 2'-(2-beta-D-ribofuranosylthiazol-4-yl)thiazole-4'-carboxamide (11). 5'-Phosphate 2 was cytotoxic to L1210 cells in culture and significantly effective against the intraperitoneally implanted murine leukemias in mice. Amidine 7 was slightly toxic to L1210 in culture and inhibitory to purine nucleoside phosphorolysis. The cyclic 3',5'-phosphate 5 was less effective than the corresponding 5'-phosphate 2 or the parent nucleoside 1 as an antitumor agent.

Synthesis and antitumor activity of certain 3-beta-D-ribofuranosyl-1,2,4-triazolo[3,4-f]-1,2,4-triazines related to formycin prepared via ring closure of a 1,2,4-triazine precursor.

Several 3-beta-D-ribofuranosyl-1,2,4-triazolo[3,4-f]-1,2,4-triazines related to formycin were prepared and tested for their antitumor activity in cell culture. Dehydrative coupling of 3-amino-6-hydrazino-1,2,4-triazin-5(4H)-one (5) with 3,4,6-tri-O-benzoyl-2,5-anhydro-D-allonic acid (6a) and further ring closure of the reaction product (7) provided 6-amino-3-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-1,2,4-triazolo[3,4- f]-1,2,4-triazin-8(7H)-one (8). Condensation of 5 with 3,4,6-tri-O-benzoyl-2,5-anhydro-D-allonic acid chloride (6b), followed by ring annulation, also gave 8 in good yield. Debenzoylation of 8 furnished the guanosine analogue 6-amino-3-beta-D-ribofuranosyl-1,2,4-triazolo[3,4-f]-1,2,4-triazin -8(7H)-one (4b). Thiation of 8 with P2S5, followed by debenzoylation of the thiated product (11a), afforded 6-amino-3-beta-D-ribofuranosyl-1,2,4-triazolo[3,4-f]-1,2,4-triazin -8(7H)- thione (11b). Methylation of the sodium salt of 11a gave the 8-methylthio derivative (10), which on ammonolysis furnished 6,8-diamino-3-beta-D-ribofuranosyl-1,2,4-triazolo[3,4-f]-1,2,4-triazine (9). Diazotization of 10 with tert-butyl nitrite (TBN) and SbCl3 in 1,2-dichloroethane gave the corresponding 6-chloro derivative (12a). Reaction of 10 with TBN in THF in the absence of a halogen source gave 8-(methylthio)-3-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-1,2,4- triazolo[3,4-f]-1,2,4-triazine (12b). Ammonolysis of 12b gave the azaformycin A analogue 8-amino-3-beta-D-ribofuranosyl-1,2,4- triazolo[3,4-f]-1,2,4-triazine (3), which on deamination afforded 3-beta-D-ribofuranosyl-1,2,4-triazolo[3,4- f]-1,2,4-triazin-8(7H)-one (4a). The azaformycin A analogue (3) showed pronounced inhibitory effects against L1210, WIL2, and CCRF-CEM cell lines with ID50 values ranging from 5.0 to 7.3 microM.

Synthesis and biological activity of 6-azacadeguomycin and certain 3,4,6-trisubstituted pyrazolo[3,4-d]pyrimidine ribonucleosides.

Several 3,4,6-trisubstituted pyrazolo[3,4-d]pyrimidine ribonucleosides were prepared and tested for their biological activity. High-temperature glycosylation of 3,6-dibromoallopurinol with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose in the presence of BF3 X OEt2, followed by ammonolysis, provided 6-amino-3-bromo-1-beta-D-ribofuranosylpyrazolo-[3,4-d]pyrimidin-4(5H)-on e. Similar glycosylation of either 3-bromo-4(5H)-oxopyrazolo [3,4-d]pyrimidin-6-yl methyl sulfoxide or 6-amino-3-bromopyrazolo [3,4-d]pyrimidin-4(5H)-one, and subsequent ammonolysis, also gave 7a. The structural assignment of 7a was on the basis of spectral studies, as well as its conversion to the reported guanosine analogue 1d. Application of this glycosylation procedure to 6-(methylthio)-4(5H)-oxopyrazolo[3,4-d]pyrimidine-3-carboxamide gave the corresponding N-1 glycosyl derivative. Dethiation and debenzoylation of 16a provided an alternate route to the recently reported 3-carbamoylallopurinol ribonucleoside thus confirming the structural assignment of 16a and the nucleosides derived therefrom. Oxidation of 16a and subsequent ammonolysis afforded 6-amino-1-beta-D-ribofuranosyl-4(5H)-oxopyrazolo[3, 4-d]pyrimidine-3-carboxamide. Alkaline treatment of 15a gave 6-azacadeguomycin. Acetylation of 15a, followed by dehydration with phosgene, provided the versatile intermediate 6-amino-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)-4(5H)-oxopyrazolo [3, 4-d]pyrimidine-3-carbonitrile. Deacetylation of 19 gave 6-amino-1-beta-D-ribofuranosyl-4(5H)-oxopyrazolo[3, 4-d]pyrimidine-3-carbonitrile. Reaction of 19 with H2S gave 6-amino-1-beta-D-ribofuranosyl-4(5H)-oxopyrazolo[3, 4-d]pyrimidine-3-thiocarboxamide. All of these compounds were tested in vitro against certain viruses and tumor cells. Among these compounds, the guanosine analogues 7a and 20a showed significant activity against measles in vitro and were found to exhibit moderate antitumor activity in vitro against L1210 and P388 leukemia. 6-Azacadeguomycin and all other compounds were inactive against the viruses and tumor cells tested in vitro.

Synthesis and anti-DNA viral activities in vitro of certain 2,4-disubstituted-7-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)pyrrolo[2,3-d d pyrimidine nucleosides.

Several novel 2,4-disubstituted-7-(2-deoxy-2-fluoro-beta-D- arabinofuranosyl)pyrrolo[2,3-d]pyrimidines have been synthesized and evaluated for their anti-human cytomegalovirus (HCMV), anti-hepatitis B virus (HBV), and anti-herpes simplex virus (HSV) activities in vitro. These nucleosides were prepared starting from 2-amino-4-chloro-7-(2-deoxy-2-fluoro- 3,5-di-O-benzoyl-beta-D-arabinofuranosyl)pyrrolo[2,3-d]pyrimidine (3), which in turn was synthesized by direct glycosylation of the sodium salt of 2-amino-4-chloropyrrolo[2,3-d]pyrimidine (1) with 2-deoxy-2-fluoro-3,5-di-O-benzoyl-alpha-D-arabinofuranosyl bromide (2). Displacement of the 4-chloro group of 3 with OH, NH2, NHOH, SH, and SeH nucleophiles furnished the corresponding nucleosides 6-8, 12, and 14, respectively. The 3'-deoxygenation of 2-amino-4-chloro-7- (2-deoxy-2-fluoro-beta-D-arabinofuranosyl)pyrrolo[2,3-d]pyrimidine (4) and subsequent amination gave 2,4-diamino-2',3'-dideoxy derivative 19. Catalytic hydrogenation of 3 followed by debenzoylation afforded 2-aminopyrrolo[2,3-d]pyrimidine nucleoside 23. Among the compounds evaluated for their ability to inhibit the growth of HCMV (strain AD169) in MRC-5 cells using a plaque reduction assay, only 7 was significantly active in vitro with a 50% inhibitory concentration (IC50) of 3.7 micrograms/mL (TI > 125), whereas the IC50 value of ganciclovir (DHPG) was 3.2 micrograms/mL. Strain D16 of HCMV was more resistant to 7 (IC50 11 micrograms/mL) than the AD169 strain. When 7 was tested in combination with DHPG, the resultant anti-HCMV activity was found to be moderately synergistic with no evidence of antagonism. Nucleoside 7 also reduced episomal HBV replication in human hepatoblastoma 2.2.15 cells with an IC50 of 0.7 micrograms/mL (TI > 143). Development of cells harboring HBV which had become resistant to the drug was not observed with 7. Compound 7 also exhibited significant activity against herpes simplex virus types 1 and 2 (IC50 of 4.1 and 6.3 micrograms/mL, respectively) in Vero cells.

Sulfinosine congeners: synthesis and antitumor activity in mice of certain N9-alkylpurines and purine ribonucleosides.

A number of N9-alkyl-substituted purines and purine ribonucleosides have been synthesized as congeners of sulfinosine and evaluated for their antileukemic activity in mice. NaH-mediated alkylation of 6-chloropurine (4) and 2-amino-6-chloropurine (5) with certain alkyl bromides gave N7- and N9-alkylated derivatives (7a-d and 6a-d), the N9-isomer being the major product. Treatment of 6a-d and 7a-d with thiourea furnished the corresponding 6-thio derivatives (9a-d and 8a-d). Amination of 9a-e with aqueous chloramine solution afforded the corresponding purine-6-sulfenamides (10-a-e), which on controlled oxidation with 3-chloroperoxbenzoic acid (MCPBA) gave the respective (R,S)-9-alkylpurine-6-sulfinamides (11a-e). A similar oxidation of 2-amino-6-(methyl/benzylthio)-9-beta-D-ribofuranosylpurine (12a and 12b) and 2-amino-9-(2-deoxy-beta-D-erythro-pentofuranosyl)-6- (methylthio)-purine (12c) with MCPBA gave the corresponding sulfoxides (13a-c), which on further oxidation furnished the respective sulfones (14a-c). Of the 20 compounds evaluated, six exhibited biologically significant anti-L1210 activity in BD2F1 mice and reduced body burdens of viable L1210 cells more than 90-97% by single treatment. Although compounds 9b and 9c at 44 mg and 40 mg/kg per day x 1 showed a T/C of 147 and 149, respectively, this group of compounds was found to be less effective than some of the sulfur-containing drugs that we previously described (e.g. sulfenosine and sulfinosine).

Synthesis and biological activity of certain 6-substituted and 2,6-disubstituted 2'-deoxytubercidins prepared via the stereospecific sodium salt glycosylation procedure.

A number of 6-substituted and 2,6-disubstituted pyrrolo[2,3-d]pyrimidine 2'-deoxyribonucleosides were prepared by the direct stereospecific sodium salt glycosylation procedure. Reaction of the sodium salt of 4-chloro-6-methyl-2-(methylthio)pyrrolo[2,3-d]pyrimidine (6a) or 4,6-dichloro-2-(methylthio)pyrrolo[2,3-d]pyrimidine (6b) with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranose (9) provided the corresponding N7 2'-deoxy-beta-D-ribofuranosyl blocked derivatives (8a and 8c) which, on ammonolysis, gave 4-amino-6-methyl-2-(methylthio)-7-(2-deoxy-beta-D-erythro-pentofuranosyl )pyrrolo[2,3-d]pyrimidine (11a) and 4-amino-6-chloro-2-(methylthio)-7-(2-deoxy-beta-D-erythro-pentofuranosyl )pyrrolo[2,3-d]pyrimidine (11b), respectively. Dethiation of 11a and 11b afforded 6-methyl-2'-deoxytubercidin (10a) and 6-chloro-2'-deoxytubercidin (10b), respectively. Dehalogenation of 10b provided an alternate route to the reported 2'-deoxytubercidin (3a). Application of this glycosylation procedure to 4,6-dichloro and 4,6-dichloro-2-methyl derivatives of pyrrolo[2,3-d]pyrimidine (15a and 15b) gave the corresponding blocked 2'-deoxyribonucleosides (18a and 18b), which on ammonolysis furnished 10b and 4-amino-6-chloro-2-methyl-7-(2-deoxy-beta-D-erythro- pentofuranosyl)pyrrolo[2,3-d]pyrimidine (17), respectively. This stereospecific attachment of the 2-deoxy-beta-D-ribofuranosyl moiety appears to be due to a Walden inversion at the C1 carbon by the anionic heterocyclic nitrogen. Controlled deacylation of 4-chloro-7-(2-deoxy-3,5-di-O-p-toluoyl-beta-D-erythro-pentofuranosyl) pyrrolo[2,3-d]pyrimidine (20a) gave 4-chloro-7-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrrolo[2,3-d] pyrimidine (20b). Dehalogenation of 20b gave the 2'-deoxynebularin analogue 7-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidine (19), and reaction of 20b with thiourea gave 7-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidine-4(3H)- thione (21). All of these compounds were tested in vitro against certain viruses and tumor cells. Only compounds 12a, 20b, and 21 showed significant activity against measles in vitro, and the activity is comparable to that of ribavirin. Although compounds 3a and 12b are slightly more active than ribavirin against HSV-2 in vitro, they are relatively more toxic to Vero cells. Compounds 3a and 20b exhibited moderate cytostatic activity against L1210 and P388 leukemia in vitro but are considerably less active than 2-chloro-2'-deoxyadenosine (1).

Imidazo[1,2-a]-s-triazine nucleosides. Synthesis and antiviral activity of the N-bridgehead guanine, guanosine, and guanosine monophosphate analogues of imidazo[1,2-a]-s-triazine.

The first chemical synthesis of 2-aminoimidazo[1,2-a]-s-triazin-4-one (8), the corresponding nucleoside and nucleotide, and certain related derivatives of a new class of purine analogues containing a bridgehead nitrogen atom is described. Condensation of 2-amino-4-chloro-6-hydroxy-s-triazine (2) with aminoacetaldehyde dimethyl acetal followed by the ring annulation gave the guanine analogue 8. A similar ring annulation of 4-(2,2-dimethoxyethylamino)-s-triazine-2,6-dione (5) gave imidazo[1,2-a]-s-triazine-4,6-dione (9). Direct glycosylation of the trimethylsilyl derivative of 8 with 1-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose in the presence of stannic chloride, followed by debenzoylation, gave the guanosine analogue 2-amino-8-(beta-D-ribofuranosyl)imidazo[1,2-a]-s-triazin-4-one (12b), which on deamination gave the xanthosine analogue 13. Phosphorylation of 12b gave 2-amino-8-(beta-D-ribofuranosyl)imidazo[1,2-a]-s-triazin-4-one 5'-monophosphate (II). The anomeric configuration has been determined unequivocally by using NMR of the 2',3'-O-isopropylidene derivate 10 and the site of ribosylation has been established by using 13C NMR spectroscopy. These compounds were tested against type 1 herpes, type 13 rhino, and type 3 parainfluenza viruses in tissue culture. Moderate rhinovirus activity was observed for several compounds at nontoxic dosage levels.

Synthesis and biological evaluation of certain 2'-deoxy-beta-D-ribo- and -beta-D-arabinofuranosyl nucleosides of purine-6-carboxamide and 4,8-diaminopyrimido[5,4-d]pyrimidine.

The key intermediate 9-(2,3,5,-tri-O-acetyl-beta-D-arabinofuranosyl)purine-6-carbonitrile (7) was synthesized in four steps from 9-beta-D-arabinofuranosylpurine-6-thione (3) via 6-(methylsulfonyl)-9-(2,3,5-tri-O-acetyl-beta-D-arabinofuranosyl)purine (6). Reaction of compound 7 with methanolic ammonia provided the rearranged compound 4-amino-8-(beta-D-arabinofuranosylamino)pyrimido[5,4-d]pyrimidine (8). Treatment of 7 with ammonium hydroxide and hydrogen peroxide provided 9-beta-D-arabinofuranosylpurine-6-carboxamide (9). Compound 7 was also treated with sodium hydrosulfide to yield 9-beta-D-arabinofuranosylpurine-6-thiocarboxamide (10). Similarly, 9-(2-deoxy-3,5-di-O-acetyl-beta-D-erythro-pentofuranosyl)purine 6-carbonitrile (17) was prepared from 6-chloro-9-(2-deoxy-beta-D-erythro-pentofluranosyl)purine (11) via 9-(2-deoxy-beta-D-erythro-pentofuranosyl)purine-6-thione. Compound 17 was converted into 4-amino-8-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]pyrimido[5,4-d]pyrimidi ne (18) and 9-(2-deoxy-beta-D-erythro-pentofuranosyl)purine-6-carboxamide (20), respectively. Compound 2 showed immunosuppressive activity and also inhibited the growth of L-1210 leukemia in mice. Arabinonucleoside analogues 8-10 were inactive when tested against RNA and DNA viruses in cell culture.

Imidazo(1,2-c)pyrimidine nucleosides. Synthesis and biological evaluation of certain 1-(beta-D-arabinofuranosyl)imidazo(1,2-c)pyrimidines.

The first chemical syntheses of the arabinosylhypoxanthine and arabinosylguanine analogues of the imidazo-[1,2-c]pyrimsdine series are described. Condensation of trimethylsilyl-7-chloroimidazo[1,2-c]pyrimidin-5-one (1) with 2,3,5-tri-O-benzyl-alpha-D-arabinofuranosyl chloride (2) gave 7-chloro-1-(2,3,5-tri-O-benzyl-beta-arabinofuranosyl)imidazo[1,2-c]pyrimidin-5-one (3) which on catalytic dehalogenation furnished 1-(2,3,5-tri-O-benzyl-beta-D-arabinofuranosyl)imidazo[1,2-c]pyrimidin-5-one (4). Amination of 3 gave 7-amino-1-(2,3,5-tri-O-benzyl-beta-D-arabinofuranosyl)imidazo[1,2-c]pyrimidin-5-one (5). Reductive hydrogenolysis of 4 and 5 gave 1-(betaD-arabinofuranosyl)imidazo[1,2-c]pyrimidin-5-one (6), the arabinosylhypoxantine analogue, and the corresponding 7-amino isomer 7, the arabinoosylguanine analogue, respectively. The unequivocal assignment of the site of glycosylation and the anomeric configuration have been established. None of the compounds exhibited significant antiviral or antimicrobial activity in vitro.

Synthesis and biological evaluation of certain C-4 substituted pyrazolo[3,4-b]pyridine nucleosides.

A series of C-4 substituted pyrazolo[3,4-b]pyridine nucleosides have been synthesized and evaluated for their biological activity. Successful synthesis of various C-4 substituted pyrazolo[3,4-b]pyridine nucleosides involves nucleophilic displacement by a suitable nucleophile at the C-4 position of 4-chloro-1H-pyrazolo[3,4-b]pyridine (5), followed by glycosylation of the sodium salt of the C-4 substituted pyrazolo[3,4-b]pyridines with a protected alpha-halopentofuranose. Use of this methodology furnished a simple and direct route to the beta-D-ribofuranosyl, beta-D-arabinofuranosyl, and 2-deoxy-beta-D-erythro-pentofuranosyl nucleosides of C-4 substituted pyrazolo[3,4-b]pyridines, wherein the C-4 substituent was azido, amino, methoxy, chloro, or oxo. The regiospecificity of these glycosylations was determined on the basis of UV data and the anomeric configuration was established by 1H NMR analysis. Conclusive structural assignment was made by a single-crystal X-ray diffraction study of three compounds, 15, 31, and 42, as representatives of ribo-2'-deoxy-, and aranucleosides, respectively. The stereospecific attachment of all three alpha-halogenoses appears to occur by a Walden inversion (SN2 mechanism) at the C-1 carbon of the halogenose by the anionic N-1 of pyrazolo[3,4-b]pyridine. All deprotected nucleosides were tested against various viruses and tumor cells in culture. The effects of these compounds on de novo purine and pyrimidine nucleotide biosynthesis was also evaluated. Among the compounds tested, 4-chloro-1-beta-D-ribofuranosylpyrazolo[3,4-b]pyridine (16) and 1-beta-D-ribofuranosyl-4,7-dihydro-4-oxopyrazolo[3,4-b]pyridine (19) were found to be moderately cytotoxic to L1210 and WI-L2 in culture.

Nucleoside peptides. 10. Synthesis and T-cell immunostimulatory properties of certain peptide derivatives of 6-azacadeguomycin.

Several amino acid and peptide conjugates of 6-azacadeguomycin (6-amino-1-beta-D-ribofuranosyl-4,5-dihydro-4-oxopyrazolo[3,4-d]py rimidine- 3-carboxylic acid, 2) have been prepared in good yields, via a two-step procedure involving 1-hydroxybenzotriazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride mediated coupling of 2 with an appropriately protected amino acid or peptide, followed by ammonolysis. Thus, condensation of 2 with L-phenylalanine methyl ester, glycine ethyl ester, and L-glutamic acid diethyl ester gave the corresponding protected linear nucleoside peptides (3, 5 and 7, respectively). Subsequent ammonolysis of 3, 5 and 7 furnished L-phenylalanine amide (4), glycine amide (6) and L-glutamic acid diamide (8) conjugates of 6-azacadeguomycin, respectively. Saponification of 7 gave the corresponding L-glutamic acid derivative 9. A similar coupling of 2 with L-phenylalaninyl-N epsilon-nitro-L-arginine methyl ester trifluoroacetate and subsequent ammonolysis (after catalytic hydrogenation) gave L-phenylalaninyl-L-arginine amide conjugate (12) of 6-azacadeguomycin. Compounds 2, 4, 6, 8, 9, and 12 were evaluated for their ability to potentiate T-cell responses to plant mitogens, in comparison with cadeguomycin (1). Compounds 4, 6, and 9 exhibited an increase in the T-cell proliferation in a dose-dependent manner.

Synthesis and antiviral/antitumor activities of certain pyrazolo[3,4-d]pyrimidine-4(5H)-selone nucleosides and related compounds.

Several pyrazolo[3,4-d]pyrimidine-4(5H)-selone ribonucleosides were prepared as potential antiparasitic agents. Treatment of 4-chloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo [3,4-d]pyrimidine (5a) with selenourea and subsequent deacetylation gave 1-beta-D-ribofuranosylpyrazolo[3,4-d] pyrimidine-4(5H)-selone (6a). A similar treatment of 3-bromo-4-chloro-1-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)pyrazolo [3,4-d]pyrimidine (5b) with selenurea, followed by debenzoylation, gave the 3-bromo derivative of 6a (6b). Glycosylation of persilylated 4-chloro-6-methyl-pyrazolo [3,4-d]pyrimidine (7) with tetra-O-acetylribofuranose (8) provided the key intermediate 4-chloro-6-methyl-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl) pyrazolo[3,4-d]pyrimidine (9). Ammonolysis of 9 gave 4-amino-6-methyl-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidine (10), whereas treatment with sodium hydroxide gave 6-methylallopurinol ribonucleoside (11a). Reaction of 9 with either thiourea or selenourea, followed by deacetylation, provided 6-methylpyrazolo[3,4-d]pyrimidine-4(5H)-thione ribonucleoside (11c) and the corresponding seleno derivative (11d), respectively. The structural assignment of these nucleosides was made on the basis of spectral studies. These compounds were tested in vitro against certain viruses and tumor cells. All the compounds except 11c exhibited significant activity against HSV-2 in vitro, whereas 11c exhibited the most potent activity against measles and has a very low toxicity. Compounds 6a, 6b, and 11d were found to be potent inhibitors of growth of L1210 and P388 leukemia in vitro.

Synthesis and antiviral activity of certain guanosine analogues in the thiazolo[4,5-d]pyrimidine ring system.

Several sugar-modified nucleoside derivatives of the purine analogue 5-amino-3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,7-dione (1) were synthesized. Phosphorylation of 1 using POCl3 resulted in 5'-monophosphate 2, which was subsequently converted to 3',5'-cyclic phosphate 3, by reported methods. 5'-Sulfamoyl derivative 4 was synthesized by treatment of the 2,3-O-isopropylidene derivative of 1 with chlorosulfonamide followed by acid deprotection. Compounds 5-7, the 5'-deoxy, the tri-O-acetyl, and the 2'-deoxy derivatives of 1, respectively, were synthesized by glycosylation of 5-aminothiazolo[4,5-d]pyrimidine-2,7-dione, the aglycon of 1, with the appropriate sugar moieties, utilizing the Vorbruggen procedure. Oxidative cleavage of the C2'-C3' bond in 1 followed by reduction with sodium borohydride led to "seco" analogue 8. Nucleosides 2-8 were evaluated for antiviral activity in vivo against the Semliki Forest virus. The activity of compounds 2, 5, and 7 were similar to that of 1. Cyclic phosphate 3 was toxic at the high dose and weakly active at the lower dose. Compounds 4, 6, and 8 were inactive in this system.

Analysis of the in vitro antitumor activity of novel purine-6-sulfenamide, -sulfinamide, and -sulfonamide nucleosides and certain related compounds using a computer-aided receptor modeling procedure.

The comparative antileukemic activities of 21 novel nucleosides were determined in vitro by using cultured L1210 cells and analyzed for structure-related efficacy by a computer-aided receptor modeling method (REMOTEDISC) as recently described (Ghose, A. K.; et al. J. Med. Chem. 1989, 32, 746). The algorithm can be classified as a 3D-QSAR method and consists of the following steps: selection of a reference structure from the low-energy conformations of the active compounds; an automated superposition of the low-energy conformations of the other compounds so that there is maximum matching (or overlapping) of the atom-based physicochemical properties; construction of the binding-site cavity from the location of the atoms of the superimposed molecules; and determinations of the relative importance of the various physicochemical properties at different regions of the site cavity using reverse stepwise regression analysis. The model was based on the minimum energy conformation of (R,S)-2-amino-9-beta-D-ribofuranosylpurine-6-sulfinamide (sulfinosine, 5), an effective antileukemic agent in vivo, in the data set. The model fit the biological data with a standard deviation of 0.363, a correlation coefficient of 0.933 and a explained variance of 0.815. The method targeted a syn conformation as the probable active form and the 2'-OH, 5'-OH as well as C2-NH2 group of the purine ring as favoring the stability of the syn conformation, thereby establishing the major contributions of these three molecular entities to overall antitumor activity.

Synthesis and anti-DNA -virus activity of the 5'-monophosphate and the cyclic 3',5'-monophosphate of 9-(beta-D-xylofuranosyl) guanine.

9-(beta-TD-xylofuranosyl)guanine (xylo-G) was converted chemically to the 9-(beta-D-xylofuranosyl)guanine 5'-monophosphate (xylo-GMP) and 9-(beta-D-xylofuranosyl)guanine cyclic 3',5'-monophosphate (c-xylo-GMP). These compounds were tested against a variety of DNA viruses in tissue culture in parallel with 9-(beta-D-arabinofuranosyl)adenine (ara-A). This evaluation revealed that xylo-G, xylo-GMP, and c-xylo-GMP were all moderately active but less effective than ara-A. When the four compounds were administered intracerebrally as a treatment for herpes virus, type 1 induced encephalitis in mice, c-xylo-GMP exhibited superior activity to that shown by the other three. When administered intraperitoneally, c-xylo-GMP was found to have a therapeutic index of about 4, which is less than that for ara-A (approximately 30) in the same system.

Synthesis and antiviral activity of certain carbamoylpyrrolopyrimidine and pyrazolopyrimidine nucleosides.

Following our recent discovery that 9-beta-D-ribofuranosylpurine-6-carboxamide (1) exhibits potent antiviral activity, we were prompted to synthesize certain pyrrolopyrimidine and pyrazolopyrimidine nucleosides containing a carbamoyl function (7a,b and 13). The key precursor, 7-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine-4- carbonitrile (8a), required for the synthesis of 7a was prepared from the corresponding 4-chloro analogue (4a). Reaction of 4a with methanethiol, followed by oxidation, gave the 4-methylsulfonyl derivative (6a), which with NaCN in DMF gave 8a. Alkaline hydrolysis of 8a provided 7a. Similarly, 7b was prepared from 4-chloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo[3,4-d] pyrimidine (4b) via the carbonitrile intermediate 8b. Starting with thioformycin B or 7-chloro-3-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo[4,3-d]pyrimidine (10) and following the similar sequence of reactions, we obtained compound 13. The in vitro antiviral studies of these carbamoyl and certain related nucleosides indicated 7a to be a potent antiviral agent against vaccinia virus, whereas 13 was moderately active. 4-Chloro-7-beta-D-ribofuranosylpyrrolo[2,3-d]pyrimidine was found to be one of the most active compounds against RVF, PICH, YF, and SF viruses in culture.

Benzimidazole ribonucleosides: design, synthesis, and antiviral activity of certain 2-(alkylthio)- and 2-(benzylthio)-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazoles.

Several 2-alkylthio- and 2-benzylthio derivatives of 5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (DRB) have been designed and synthesized from 5,6-dichloro-1-(beta-D-ribofuranosyl)-benzimidazole-2-thione. All compounds were evaluated for activity against human cytomegalovirus (HCMV) and/or herpes simplex virus type-1 (HSV-1). Three different cytotoxicity assays were used to determine if the compounds were toxic to uninfected cells. Most of the 2-alkylthio compounds were either inactive against HCMV and HSV-1 or were active only at concentrations at or near those which produced toxicity in uninfected cells. The best separation between activity against HCMV and cytotoxicity was observed with the 2-benzylthio analog 7. This prompted us to synthesize the substituted 2-benzylthio analogs 11-23 using a Topliss Tree approach. None of these compounds were more active than compound 7; most of the analogs were weakly active against both HCMV and HSV-1, but the activity was not separated from cytotoxicity. On the basis of both antiviral and cytotoxicity data, compound 7 was the best compound in the series. It was more active against HCMV than DRB (the 2-unsubstituted analog), acyclovir, and foscarnet, but it was less active than ganciclovir.