Inhibitors of purine nucleoside phosphorylase: effects of 9-deazapurine ribonucleosides and synthesis of 5'-deoxy-5'-iodo-9-deazainosine.

9-Deazapurine ribonucleosides constitute a new class of noncleavable purine nucleoside phosphorylase inhibitors that have at least 30-fold greater affinity for the enzyme than the corresponding C-nucleosides of the formycin B series. 9-Deazaguanosine, 9-deazainosine, and 5'-deoxy-5'-iodo-9-deazainosine competitively inhibited human erythrocytic purine nucleoside phosphorylase with Ki values of 29, 20, and 1.8 X 10(-7) M. The last compound is the most potent nucleoside inhibitor of the enzyme presently available and its synthesis is described. In contrast, 7,9-dideaza-7-thiainosine is a very weak inhibitor of the enzyme. When tested as an inhibitor of 2'-deoxyguanosine phosphorolysis in intact human erythrocytes and MOLT-3 human T-cell lymphoblastic leukemia cells, 5'-deoxy-5'-iodo-9-deazainosine was equipotent with 8-aminoguanosine (which is a precursor for 8-aminoguanine, Ki = 2 X 10(-7) M). Similarly, 5'-deoxy-5'-iodo-9-deazainosine and 8-aminoguanosine both potentiated the growth inhibition of human T-lymphocytic MOLT-3 cells by 2'-deoxyguanosine, reducing the 50% inhibitory concentration from approximately 2 X 10(-5) to approximately 2 X 10(-6) M.

Cyclopentenylcytosine. A carbocyclic nucleoside with antitumor and antiviral properties.

Cyclopentenylcytosine (CPE-C, 2), a pyrimidine analogue of the fermentation derived carbocyclic nucleoside neplanocin A, has been synthesized from the optically active cyclopentenylamine 3b by two synthetic routes. CPE-C demonstrates significant antitumor activity against both the sensitive and ara-C resistant lines of L1210 leukemia in vivo. Multiple long term survivors are produced in both tumor models. The compound also gives 100% growth inhibition of the solid human A549 lung and MX-1 mammary tumor xenografts grown in athymic mice. Good activity is also observed against a third human tumor xenograft model, metastatic LOX melanoma. CPE-C has significant activity against both DNA and RNA viruses in vitro. Potent activity is observed against HSV-1 (TK+ and TK-), HSV-2, vaccinia, cytomegalovirus, and varicella-zoster virus. Good activity is also found against a strain of influenza virus (Hong Kong flu), vesicular stomatitis virus, Japanese encephalitis virus, and Punta Toro virus.

Cyclopentenyl cytidine analogue. An inhibitor of cytidine triphosphate synthesis in human colon carcinoma cells.

The mechanism of action of the cyclopentenyl analogue of cytidine, cCyd, was investigated in human colon carcinoma cell line HT-29. Upon exposure of cells to 10(-6)M cCyd, cell viability was reduced to 20% of control, whereas cytocidal activity was not present after 2 hr of drug exposure. Cell lethality was partially reversible by Urd, Cyd or dCyd at 10(-6)M cCyd, and fully reversible by these nucleosides at 2.5 X 10(-7)M cCyd. The incorporation of [14C]dThd and [3H]Urd into DNA and RNA was inhibited by 50% by exposure for 2 hr to 2.5 X 10(-7) and 1.5 X 10(-6)M cCyd respectively. Upon 24 hr of drug exposure, the IC50 for RNA synthesis was reduced 2.5-fold, whereas DNA synthesis was almost totally inhibited. cCyd produced a rapid and preferential reduction of CTP synthesis with a half-life of 1 hr at 10(-6)M drug. The IC50 of cCyd for reducing CTP concentrations after 2 hr of drug exposure was 4 X 10(-7)M. Concomitant with the reduction of CTP levels was the inhibition of transcription of rRNA and, to a lesser extent, tRNA, without changes in the processing nucleolar RNA. No changes in the size of DNA were produced following treatment with cCyd. These results indicate that cCyd is a potent and rapid inhibitor of CTP synthesis and that this effect correlates with its cytocidal activity.

9-Deazaadenosine--a new potent antitumor agent.

-Deazaadenosine (9-DAA), a novel purine analog, was found to be a potent inhibitor of the growth of nine different human solid tumor cell lines in vitro and of pancreatic carcinoma (DAN) in antithymocyte serum (ATS)-immunosuppressed mice. In culture, IC50 values ranged from 1.1 to 8.5 X 10(-8)M. Ovarian carcinoma (MR) was the only cell line in which the activity of 9-DAA was potentiated (about 10-fold) by pretreatment with the adenosine deaminase inhibitor 2'-deoxycoformycin (dCF). After incubation of cultured pancreatic DAN cells with 9-DAA (10(-5)M) for 2 hr, a peak appeared in the triphosphate region of HPLC nucleotide profiles that was identified tentatively as 9-deazaATP. Under the same incubation conditions, the incorporation of [3H]uridine into RNA and of [3H]thymidine into DNA was inhibited by 34 and 80% respectively. In vivo studies using ATS-immunosuppressed mice showed that 9-DAA at 0.4 mg/kg/day for 3 consecutive days reduced pancreatic carcinoma (DAN) tumor weights to approximately 50% of untreated controls. The nucleoside transport inhibitor p-nitrobenzyl-6-thioinosine (NBMPR) was shown to selectively protect host tissues from 9-DAA toxicity and, thereby, potentiated the antitumor activity of 9-DAA in vivo at optimal dosages.

Induction of differentiation in the human promyelocytic leukemia cell line HL-60 by the cyclopentenyl analogue of cytidine.

The effects of the cyclopentenyl (cCyd) and cyclopentyl (carbodine) analogues of cytidine on differentiation, and nucleic acid and nucleotide biosynthesis, were examined in the human promyelocytic leukemia cell line HL-60. Continuous exposure for 5 days to 10(-8) to 10(-6) M cCyd or 10(-6) to 10(-5) M carbodine produced progressive inhibition of cell growth. During this exposure interval, pronounced differentiation to mature myeloid cells occurred wherein 95% of the cell population reduced nitroblue tetrazolium 4 days after exposure to 10(-7) M cCyd or 10(-5) M carbodine. Preceding differentiation was the inhibition of DNA synthesis which reached 10% of control levels 24 hr after exposure to 10(-7) M cCyd or 10(-5) M carbodine, while RNA synthesis was inhibited to a lesser extent. The induction of mature myeloid cells by cCyd was preceded by the inhibition of c-myc mRNA levels which was more pronounced than the reduction in total cellular RNA synthesis. During the interval of cCyd treatment, there was a rapid and pronounced inhibition in the level of CTP, but not of UTP, ATP or GTP, where the half-life for the disappearance of CTP was 1.5 to 2 hr. Following drug removal, cells treated with cCyd showed a sustained reduction in CTP levels, whereas cells treated with carbodine showed almost complete recovery of CTP levels within 48 hr. These results indicate that the reduction in CTP levels leads to rapid inhibition of DNA synthesis and reduction in c-myc mRNA levels which precede the appearance of differentiated cells.

Inhibition of lymphocyte function by 9-deazaadenosine.

9-Deazaadenosine (c9Ado), a novel C-nucleoside, has been found to inhibit lymphocyte-mediated cytolysis (LMC) in a time-dependent manner. c9Ado inhibited LMC by 50% at concentrations of 10 and 0.07 microM after drug-pretreatment periods of 3 and 22 hr, respectively, although a 1-hr pretreatment of cytolytic lymphocytes with 100 microM c9Ado had no effect upon this lymphocyte function. c9Ado was metabolized rapidly and extensively to 9-deazaadenosine 5'-triphosphate (c9ATP) both by mouse cytolytic lymphocytes and by human erythrocytes. Adenosine kinase purified from rabbit liver phosphorylated c9Ado with a Km of 200 microM and a Vmax of 8% that for adenosine. The metabolic buildup of c9ATP in lymphocytes was accompanied by a large, time-dependent decrease in cellular ATP and by smaller percentage decreases in CTP, UTP and GTP. Among other biochemical effects examined, c9Ado was found to cause a decrease in lymphocyte cAMP content and appeared to be neither an inhibitor nor a substrate for S-adenosylhomocysteine hydrolase. Consistent with this latter result, L-homocysteine thiolactone had no effect on the inhibition of LMC by c9Ado. Neither the inhibition of LMC by c9Ado nor the metabolic formation of c9ATP in lymphocytes was affected by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), indicating that c9Ado is not a substrate for adenosine deaminase. 5-Iodotubercidin, a non-competitive inhibitor (Kis = 9 nM, Ku = 20 nM) of adenosine kinase, prevented the above effects of c9Ado on lymphocyte function, c9ATP formation, and ATP levels. Either complete preservation (with coformycin) or partial replenishment (with adenosine plus EHNA) of ATP levels in c9Ado-treated lymphocytes resulted in partial restoration of cytolytic function to cells containing large amounts of c9ATP. These results suggest that c9Ado is inhibitory to LMC both because it causes a decrease in the absolute concentration of ATP within the cytolytic lymphocytes and because it permits the establishment within these cells of an unfavorable c9ATP:ATP ratio which impedes the utilization of ATP in a reaction essential to the execution of this lymphocyte function.