Role of deoxycytidine kinase in the inhibitory activity of 5-substituted 2'-deoxycytidines and cytosine arabinosides on tumor cell growth

A number of 5-substituted derivatives of dCyd and 1-beta-D-arabinofuranosylcytosine (araC) have been evaluated for their inhibitory effects on the growth of three murine leukemia cell lines (L1210/0, L1210/BdUrd, and L1210/araC). The L1210/BdUrd and L1210/araC cell lines were selected from the parental L1210/0 cell line by their ability to grow at high concentrations of 5-bromo-2'-deoxyuridine and araC, respectively; the L1210/BdUrd cell line was deficient in dThd kinase activity, whereas the L1210/araC cell was deficient in dCyd kinase activity. The most effective inhibitors of L1210/0 cell proliferation were 5-fluoro-dCyd, araC, and 5-fluoro-araC. Their 50% inhibitory dose fell within the 0.001-0.015 micrograms/ml range. The 5-substituted araC analogues were much less inhibitory for L1210/araC cells but equally inhibitory for L1210/BdUrd as for the parental L1210/0 cell line. The role of dCyd kinase in the antitumor activity of the dCyd and araC analogues was further assessed by kinetic studies with dCyd kinase extracted from L1210/0 cells. All dCyd and araC analogues caused a competitive inhibition of dCyd kinase, the most potent inhibitor being 5-fluoro-dCyd (Ki/Km value 0.24). The Km of dCyd kinase from L1210/0 cells for dCyd was 23.1 microM as compared with 50 microM for araC. These values were increased to 53 and 182 microM, respectively, for the dCyd kinase isolated from L1210/araC cells.     

5-substituted 2′-deoxyuridines: Correlation between inhibition of tumor cell growth and inhibition of thymidine kinase and thymidylate synthetase

A large variety of 5-substituted 2′-deoxyuridines (dUrds) and 2′-deoxyuridylates (dUMPs) have been evaluated for their inhibitory effects on the thymidine (dThd) kinase or thymidylate (dTMP) synthetase isolated from mouse leukemia L1210 cells. The most potent inhibitors of dThd kinase were 5-chloro-, 5-bromo- and 5-iodo-dUrd. Their K_i/K_m values ranged from 0.57 to 0.82. All dUrd analogs tested showed competitive kinetics with respect to dThd. However, there was little, if any, correlation between the inhibitory effects of the compounds on L1210 cell growth and their inhibitory activities against dThd kinase (r = 0.16). The most potent inhibitors of dTMP synthetase were (in order of decreasing activity): 5-nitro-dUMP > 5-formyl-dUMP > 5-fluro-dUMP > 5-oxime of 5-formyl-dUMP > 5-azidomethyl-dUMP > (E)-5-(2-bromovinyl)-dUMP. The k_i/K_m values for these compounds ranged from 0.001 to 0.665. All dUMP analogs tested showed competitive kinetics with respect to dUMP (if not preincubated with the enzyme at 37°). There was a strong correlation (r = 0.833) between the inhibitory effects of these compounds on L1210 cell growth and their inhibitory activities against dTMP synthetase. Thus, the suppressive action of 5-substituted dUrd derivatives on tumor cell growth would involve prior conversion of the nucleoside analogs to the corresponding 5′-monophosphates followed by an inhibition of dTMP synthetase.     

Potentiation by 2'-deoxycoformycin of the inhibitory effect of xylosyladenine on nuclear RNA synthesis in L1210 cells in vitro

The effect of the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), on the inhibitory effect of 9-β-d-xylofuranosyladenine (XA) on nuclear RNA synthesis was examined in L1210 cells in vitro. Pretreatment of cells for 15 min with a 100 per cent inhibitory dose (1 × 10^?6 M) of dCF resulted in approximately a 3- to 8-fold reduction in the 50 per cent inhibitory dose (id₅₀) of XA for [³H]uridine and [³H]thymidine incorporation into RNA and DNA respectively. The id₅₀ for XA for RNA synthesis vs DNA synthesis was 5-fold lower in the absence of dCF and 20-fold lower in the presence of dCF, indicating the greater sensitivity of RNA synthesis to this inhibitor. Fractionation of nuclear RNA into rRNA, non-poly(A) heterogeneous RNA and poly(A)heterogeneous RNA revealed the latter species of RNA to be less sensitive to XA in the absence of dCF; however, in the presence of dCF, all three species of nuclear RNA showed similar sensitivities. Nuclear polyadenylic acid synthesis was among the most sensitive RNA fractions to XA, and was also inhibited to a greater degree by pretreatment of cells with dCF. These results indicate that XA is potentiated markedly by inhibition of adenosine deaminase, and that deamination serves as a major catabolic route for this drug.     

In vivo inhibition of adenosine deaminase by 2'-deoxycoformycin in mouse blood and leukemia L1210 cells

Adenosine deaminase (ADA) activities in mouse whole blood, washed erythrocytes and L1210 cells were 0.48, 0.93 and 4.76 units/ml respectively. Methods were developed to determine the second-order association rate constant (k₁) of a tight-binding ADA inhibitor, deoxycoformycin (DCF), and ADA in mouse blood and L1210 cells in vivo. After i.v. injection of DCF, the inhibition of the enzyme was of a monophasic pseudo-first-order nature in blood and biphasic (with an initial lag of 3–5 min) in L1210 cells. In contrast, i.p. injection of DCF produced the opposite pattern, monophasic in L1210 cells and biphasic in blood. The apparent k₁ values determined from the linear portions of these curves were compared with the k₁ values obtained in vitro. The mean k₁ values in vivo were: 4.2 × 10⁴ and 1.4 × 10⁴M^?1 sec^?1 in blood after i.v. and i.p. injections, respectively, and 2.6 × 10³ and 2.2 × 10⁴ M^?1 sec^?1 in L1210 cells after i.v. and i.p. injections respectively. The k₁ values with either whole blood or L1210 in vitro (3.1 × 10⁴ and 5.5 × 10³ M^?1 sec^?1, respectively) were of the same order of magnitude as those obtained with these tissues in vivo. In contrast, the k₁ values were about 150 to 1400-fold higher when either blood hemolysates (4.8 x 10^?6M^?1 sec^?1) or homogenized L1210 cells (7.5 x 10^6?1 sec^?1) were used. The 150 to 1400-fold higher k₁ values for blood hemolysates and homogenized L1210 cells than for intact cell samples (whole blood or whole L1210 ascitic fluid) suggest that the cell membrane plays a role in the interaction of DCF and ADA in these cell lines. The similarity of the rates of association of DCF and ADA in vivo and in vitro for mouse blood and ascites L1210 cells suggests that data obtained in vitro may be used to estimate the k₁ values in in vivo conditions.     

Antitumor effects of bis-thiosemicarbazones— inhibition of deoxyribonucleic acid synthesis in vitro

A group of bis-thiosemicarbazones was evaluated for potential antitumor activity, using the L1210 murine leukemia in cell culture. Drug levels required to inhibit DNA synthesis by 50 per cent, under standard conditions, were determined. The most potent of the agents examined had the structure X[CH₂CR₁=NNHCSNHR₂]₂ where X = C or S and R₁ = R₂ = CH₃. Optimal activity was also obtained with R₁ = H and R₂ = CH₃ only when X = S. The most potent derivatives inhibited DNA synthesis by 50 per cent within 10 min at 10^?6 M levels (id₅₀). Metal chelates of several compounds tested were extremely potent inhibitors of DNA synthesis ($\text{id}{}_{50}\text{= 10}{}^{\mathrm{?7}}\text{M or less}$). Insolubility in water and short duration of action in vivo may limit effectiveness of the bis-thiosemicarbazones.     

Inhibitory effects of cordycepin on cyclic nucleotide-dependent and cyclic nucleotide-independent protein kinases

The in vitro effect of cordydepin was tested using various protein kinase preparations. These included cyclic AMP-dependent protein kinase (A-PK) from bovine heart, cyclic GMP-dependent protein kinase (G-PK) from fetal guinea pig lung, and two cyclic nucleotide-independent nuclear protein kinases (PK-I and PK-II) prepared from rat hepatoma 3924A and rat liver. The 50 per cent inhibitory concentrations (id₅₀) of cordycepin for A-PK and G-PK ranged from 1.5–5.0 × 10^?4M and 2.5–8.0 × 10^?4 M, respectively, depending on the presence or absence of cyclic AMP and cyclic GMP in the assay. The id₅₀ of cordycepin with either hepatoma 3924A or rat liver PK-I and PK-II was 4.5 × 10^?5 M and 1.0 × 10^?3 M. respectively. The inhibitory effect of cordycepin was competitive with respect to ATP in all cases. The K_{m} for ATP was increased 3-fold and 5-fold by 5 × 10^?4 M cordycepin for G-PK and A-PK, respectively, while the K_m for ATP was increased 10-fold and 4-fold by 1 × 10^?3 M cordycepin for PK-I and PK-II, respectively.     

Inhibitory activity of diarylamidine derivatives on murine leukemia L1210 cell growth

Summary A series of 96 diarylamidine (and diarylimidazoline) derivatives were evaluated for their inhibitory effects on the growth and DNA synthesis of murine leukemia L1210 cells. The amidino- and imidazolino-substituted aryl moieties of the compounds consisted of phenyl, indole, indene, benzofuran, benzo[b]thiophene or benzimidazole. Several of these compounds were found to inhibit L1210 cell proliferation with an ID₅₀ (50% inhibitory dose) of 1 g/ml or lower. Structure-function analysis revealed that the antitumor cell activity of the diarylamidines depended on the planarity of the molecule, the presence of amidino- (or, preferably, imidazolino-) groups on both aryl moieties, the nature of the bridge connecting the two aryl moieties (preferably no bridge at all, phenoxy or ethene) and, finally, the nature of the aryl moieties (preferably, benzofuran or benzo[b]thiophene). Hence, compound 20 (6-(2-imidazolin-2-yl)-2-[4-(2-imidazolin-2-yl)phenyl] benzo[b]thiophene) emerged as the most potent inhibitor of L1210 cell growth (ID₅₀: 0.21 g/ml). Its inhibitory potency was similar to that of the well-known trypanocidal drug ethidium bromide (compound 98). For all diarylamidine derivatives taken together, some correlation (r = 0.612) was noted between the log ID₅₀ for L1210 cell proliferation and the log ID₅₀ for L1210 cell DNA synthesis (as monitored by [methyl ³H]dThd incorporation). These findings suggest that the inhibitory effects of the diarylamidines on L1210 cell proliferation may at least partially reside in an inhibition of DNA synthesis. Compound 41 (2,2-vinylenedi-1-benzofuran-5-carboxamidine), that exhibited a potent antitumor activity in vitro (ID₅₀: 1.5 g/ml), was further evaluated for its antitumor efficacy in vivo and found to increase the median survival time of L1210 cell-inoculated BDF₁ mice up to 204%, if administered at a dose of 200 mg/kg.     

Effects of bis(6-mercaptopurine-9-beta-D-ribofuranoside)-5',5"-phosphate and its butyryl derivative on mouse leukaemia L1210 and a 6-mercaptopurine-resistant subline in culture

Bis(6-mercaptppurine-9-β-d-ribofuranoside)-5′-5″′-monophosphate (bis(MPR)P) and its butyryl derivative, bis(O²,$\text{O}{}^3\text{-}\text{dibutyryl}\text{-6-}\text{mercaptopurine}\text{-9-β-}\text{d}\text{-}\text{ribofuranoside}$)-5′,5″′-monophosphate (bis(dibutyrylMPR)P) were synthesized from 6-mercaptopurine-9-β-d-ribofuranoside (MPR). Bis(MPR)P (ec₅₀ = 0.014 μM) and MPR (ec₅₀ = 0.022 μM) were essentially equivalent in their growth inhibitory activities against L1210/0 cell cultures, whilst bis(dibutyrylMPR)P (ec₅₀ = 1.1 μM) was considerably less effective. L1210/MPR cells grew normally in the presence of 1 mM MPR but were inhibited by bis(MPR)P (ec₅₀ = 580 μM) and (bis(dibutyrylMPR)P (ec₅₀ = 42 μM). Bis(dibutyrylMPR)P was less readily broken down to MPR by enzymes in the serum component of the culture medium than was bis(MPR)P, and leukaemia cells did not contribute to the extracellular degradation of the acylated derivative. The delayed cytotoxic effects of bis(MPR)P and bis(dibutyrylMPR)P on L1210/0 cells were those of the MPR breakdown product. Exposure to bis(MPR)P resulted in delayed cytotoxicity in L1210/MPR cultures whilst bis(dibutyrylMPR)P produced only acute growth inhibition and no delayed effect on the MPR-resistant subline. MPR was incorporated into DNA of L1210/0 cells as 6-thioguanine deoxyribonucleotide whilst bis(MPR)P was not incorporated into L1210/MPR cell DNA. These results suggested that the ultimate mechanisms of action of bis(MPR)P and bis(dibutyrylMPR)P in L1210/ MPR cells may have been different from that of MPR in sensitive L1210/0 cells and therefore might not represent true circumvention of resistance to MPR.     

Analog specific aberrancies in antifolate inhibition of L1210 cell dihydrofolate reductase

During studies with L1210 cells and a variety of folate analogs, large discrepancies were revealed between data on membrane transport, on inhibition of dihydrofolate reductase in cell-free extracts, and on inhibition of growth in culture for 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin, and for 3-deaza, 10-methyl-aminopterin. While aminopterin, 10-methyl (methotrexate)-, 10-ethyl- and 10-propyl-aminopterin were tight binding inhibitors (K_i: 2–3 × 10^?12M) of dihydrofolate reductase in cell-free extracts from L1210 cells, the other four analogs were only weak competitive inhibitors (K_i = 3–300 × 10^?8M). Similar differences among analogs were observed for inhibition of dihydrofolate reductase in cell-free extracts from Sarcoma 180 and Ehrlich cells, but not for this enzyme in microbial cell-free extracts. There were only small differences in the transport of all of the analogs by L1210 cells. Inhibition of L1210 cell growth in culture by 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin and by 3-deaza, 10-methyl-aminopterin, in contrast to the other analogs, was several orders of magnitude greater than that predicted from the data on dihydrofolate reductase inhibition. The extent of binding of 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin, and of 3-deaza and 10-methyl-aminopterin to dihydrofolate reductase in intact L1210 cells, in contradistinction to that seen for the cell-free enzyme preparations, approached that observed for methotrexate; these estimates of drug-enzyme interaction in situ were more predictive of the extent of inhibition by these analogs of L1210 cell growth in culture.     

N-Methylformycins. Reactivity with adenosine deaminase, incorporation into intracellular nucleotides of human erythrocytes and L 1210 cells and cytotoxicity to L 1210 cells.

The N-methyl derivatives of the C-nucleoside, formycin (7-amino-3(β-d-ribofurano-syl)pyrazolo[4, 3-d]pyrirnidine) were compared to formycin and adenosine with regard to their substrate activity with human erythrocytie adenosine deaminase (ADA), their ability to form intracellular nucleotides and their cytotoxicity to L1210 cells. Only 2-methylformycin (K_m = 6.1 mM, relative V_max = 396) and N^? -methylformycin (K_m = 0.1 mM, relative V_max = 3) showed substrate activity with ADA (corresponding kinetic parameters for adenosine were: K_m = 0.025 mM, relative V_max = 100). In contrast to previous hypotheses, these results suggest that the conformation (either syn or anti) of an adenosine analog is not a major factor in determining substrate activity with ADA. Neither 4-methylformycin nor 6-methylformycin formed their corresponding nucleotides when incubated with human erythrocytes, whereas both 1-methylfor-mycin and 2-methylformycin formed large amounts of their corresponding mono-, di- and triphosphate nucleotides. Inhibition of ADA by pretreatment of the erythrocytes with the potent ADA inhibitor, 2'-deoxycoformycin, had no effect on the incorporation of 1-methylformycin into erythrocytic nucleotides but greatly increased the incorporation of 2-methylformycin and N⁷-methylformycin. The conversion of both 1-methylformycin and 2-methylformycin into nucleotides was almost complete after 18 hr of incubation (in the presence of 2'-deoxycoformycin in the case of 2 methylformycin), whereas that of N⁷-methylformycin was only partially complete in the presence of 2'-deoxycoformycin. With both 1-methylformycin and N⁷-methylformycin, transient accumulation of the corresponding nucleoside 5'-monophosphate derivative was observed prior to the accumulation of the triphosphate nucleotide. Results, qualitatively similar to those found with erythrocytes, were obtained when the effects of 2'-deoxycoformycin on the incorporation of 1-methyl- and 2-methylformycins into the nucleotide pools of L 1210 cells in vitro were examined. Compounds capable of forming analog nucleotides in human erythrocytes or L1210 cells if deamination is prevented either by the molecular structure of the analog or by pretreatment of the cells with 2'-deoxycoformycin, also showed marked cytotoxicity to L1210 cells in culture, i.e. 1-methyl-, 2-methyl- and N⁷-methylformycin exhibited id₅₀ values of 0.5 to 2 μM, whereas 4-methyl- and 6-methylformyein were not significantly growth inhibitory. The potential usefulness of the various N-methyl derivatives of formycin (alone or in combination with an ADA inhibitor) as cytotoxic or antiviral agents is discussed.     

Studies with a 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP)-resistant L1210 leukemia cell line without cross-resistance to methotrexate

An L1210 leukemia cell line resistant to 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP) (L1210/DDMP) was developed in vivo by treatment of tumor-bearing mice. Resistance to DDMP was confirmed by subsequent in vivo survival experiments and by in vitro dose-response curves. The L1210/DDMP line demonstrated little cross-resistance to another folate analog, methotrexate (MTX). This was confirmed both in vivo, with survival experiments, and in vitro, using dose-response curves. A statistical analysis of the in vivo data confirmed DDMP resistance with lack of MTX cross-resistance. Dihydrofolate reductase (DHFR) activity in the L1210/DDMP/R₅ line was no greater than in the parent cell line (L1210/S). and the K_m of DHFR for dihydrofolate was the same in the L1210/DDMP/R₅ and L1210/S lines. The K_i for DHFR of the L1210/DDMP/R₅ cell line versus the L1210/S cell line was increased 3.0-fold for MTX and 3.5-fold for DDMP. Total accumulation of [¹⁴C]DDMP was identical in the two cell lines. The explanation for the lack of MTX cross-resistance in the L1210/DDMP/R₅ line is unknown.     

Antiviral, antitumor, and thymidylate synthetase inhibition studies of 5-substituted styryl derivatives of 2'-deoxyuridine and their 5'-phosphates

2′-Deoxyuridine derivatives containing styryl, 3-nitrostyryl, 4-nitrostyryl, and phenylethyl groups substituted at the 5-position of the pyrimidine ring have been evaluated for their effects on vaccinia and herpes simplex virus replication (in primary rabbit kidney cell cultures) and mouse leukemia L-1210 cell culture growth. 5-Phenylethyl-2′-deoxyuridine inhibited herpes simplex (type 1 and 2) virus-induced cytopathogenicity by 50 per cent at a dose (id₅₀) of 10–30 μg/ml. It was inactive against tumor cell growth. The corresponding styryl derivative showed an id₅₀ of 30–70 μg/ml for herpes simplex virus, 20 μg/ml for vaccinia virus, and 280 μg/ml for L-1210 cell growth. 5(E)-(3-Azidostyryl)-2′-deoxyuridine 5′-phosphate inhibited vaccinia replication with an IC₅₀ of 20 μg/ml and L-1210 cell culture growth with an id₅₀ of 80 μg/ml. The nucleotides of these compounds were all potent reversible inhibitors of thymidylate synthetase (Lactobacillus casei) with the following $\text{K}{}_{\mathrm{i}}\text{K}{}_{\mathrm{m}}$ ratios: 3-nitrostyryl, 0.035; 4-nitrostyryl, 0.05; 3-azidostyryl, 0.06; styryl, 0.08; and phenylethyl, 0.31. The photodecomposition of the azidostyryl derivative, a photoaffinity labeling reagent for thymidylate synthetase, was examined at two wavelengths.     

Modification of in vivo methotrexate antitumor effect in L1210 leukemia by induced impairment of purine salvage

Studies with murine cells have shown that the antitumor action of methotrexate (MTX) may be through a purineless mechanism. If the MTX effect depends, in part, on inhibition of de novo purine synthesis, then the ability of tumor cells to salvage available purine precursors could reduce the cell kill. In the present study, we produced L1210 murine leukemia mutants with impaired purine salvage to determine whether this would affect responsiveness to MTX. Mutant lines L1210/MP, L1210/FAMP, and L1210/555 were produced by developing resistance to the purine analogs 6-mercaptopurine (6-MP), 6-MP + 2-fluoroadenine (2-FA), and 6-MP + 2-FA + 6-methylmercaptopurine riboside respectively. The purine salvage capability of the cell lines was confirmed in vitro by testing the ability of various purines to reverse the growth inhibitory and biochemical effects of MTX in the presence of thymidine. Dose-response curves demonstrated identical in vitro MTX sensitivity for L1210/MP, L1210/FAMP, and the parent line, L1210/S. Despite identical in vitro MTX sensitivity, the cell lines L1210/MP and L1210/FAMP displayed increased sensitivity to the biochemical effects of MTX in an in vivo model, and this was translated into enhanced sensitivity as measured by survival experiments in tumor-bearing mice. The results indicate that impairment of purine salvage sensitizes cells to the antitumor effect of MTX in vivo. This has implications for the clinical use of MTX in view of the variety of rescue techniques that is available.     

Synthesis of ester and amide derivatives of 1-phenyl-3-(thiophen-3-yl)-1H-pyrazole-4-carboxylic acid and study of their anticancer activity

A series of novel thiophene containing 1,3-diarylpyrazole derivatives were synthesized and the structures were determined by IR, ¹H-NMR, and HRMS analysis. The anticancer activity of the title compounds against MCF7, MDA-MB-231, HeLa, Raji, and HL60 human cancer cells growth were investigated by MTT assay. Interestingly, Raji and HL60 cells exhibited more sensitivity to synthesized compounds. (4-Benzyl-piperidin-1-yl)-(1-phenyl-3-thiophen-3-yl-1H-pyrazol-4-yl)-methanone (4c) possessed the highest growth inhibitory effect on Raji and HL60 cancer cells (GI₅₀ 25.2 ± 3.2 and 28.3 ± 1.53 μM, respectively). Compound 4f, 4-trifluoromethylphenyl piperazine at the amide part, also showed potent activity on Raji and HL60 cancer cell lines with a GI₅₀ value of 32.0 ± 1.27 and 36.7 ± 2.15 μM, respectively. Physicochemical properties of the synthesized compounds were evaluated in silico, and it was found that all compounds should present good passive oral absorption. All synthesized compounds demonstrated good drug-likeness values. The data suggested that these compounds might be promising for further development.     

2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline (tmq), a potent non-classical folate antagonist inhibitor--I effect on dihydrofolate reductase and growth of rodent tumors in vitro and in vivo.

Seventeen non-classical 2,4-diamino-6-[(anilino)methyl]quinazoline antifolates were tested as inhibitors of dihydrofolate reductase from L1210 leukemia cells and from human leukemia cells (acute lymphocytic leukemia). Several potent inhibitors of this enzyme were found, some with I₅₀ values of 10^?9 M, thus displaying activity comparable to that of methotrexate. In general, the potency of dihydrofolate reductase inhibition correlated with the inhibition of cell growth in vitro against L1210 cells. Two of these compounds, compound 14 (2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline; TMQ, JB-11, NSC 249008) and compound 3 (2,4-diamino-5-chloro-6-[(3,4-dichloroanilino)methyl]quinazoline; NSC 208652), were further evaluated against murine tumors in vivo and both showed a broad spectrum of antitumor effects. The results of these studies encourage further evaluation of these compounds, in particular compound 14, as possible anti-neoplastic agents in the treatment of human disease.     

Studies on the mechanism of cytotoxicities of polyacetylenes against L1210 cell

This study was performed to investigate the mechanism ofin vitro cytotoxic actions of polyacetylenes which are panaxydol, panaxynol and panaxytriol isolated fromPanax ginseng C.A. Meyer. DNA synthesis of L1210 cells was significantly inhibited with dose-dependent pattern when L1210 cells were treated for 1 hour with over 5 μg/ml of polyacetylenes. Panaxydol which had the most potent cytotoxicity among three polyacetylenes showed also the strongest inhibitory effect on DNA synthesis. Intracellular cyclic AMP levels of L1210 cells treated with 2.5 μg/ml of panaxydol or panaxytriol were significantly elevated on the incubation duration. The elevation of cyclic AMP levels by panaxytriol was higher than that by panaxydol, but no significant increase in cyclic AMP by panaxynol was observed. All three polyacetylenes had no effect on glycolysis of L1210 cells. Electron microscopic observations revealed that polyacetylenes caused damage to plasma membranes of L1210 cells in proportion to their cytotoxicities at each ED₅₀ value (panaxydol>panaxynol>panaxytriol). These results suggest that cytotoxicities of polyacetylenes against L1210 cells might be mediated by elevated cyclic AMP level, even though the relationship among their cytotoxicities, inhibitory effect on DNA synthesis and ability to elevation of cyclic AMP level are not fully agreed, and might be also related to membrane damage.     

Synthesis, structure, and antitumor and antiviral activities of a series of 5-halouridine cyclic 3',5'-monophosphates

A series of potential prodrug 5-halouridine 3',5'-cyclic monophosphates (5-X-cUMPs, X = F, Cl, Br, I, 1-4) has been prepared and tested for antitumor activity against murine leukemia L1210/0 and human lymphoblast Raji/0 cells and their deoxythymidine kinase deficient (TK-) counterparts, as well as for antiviral activity in primary rabbit kidney cells infected with herpes simplex virus type 1 or 2, vaccinia virus, or vesicular stomatitis virus. The 5-halopyrimidine bases, nucleosides (5-X-U), and 5'-monophosphates (5-X-UMP) were tested for comparison. 5-F-cUMP (1) showed reasonably potent inhibition of tumor cell proliferation (ID50 = 0.33-1.6 micrograms/mL), while the remaining diesters displayed ID50's ranging from 210 to greater than 1000 micrograms/mL. 5-F-cUMP was 70- to 300-fold less active than 5-F-dU in the same systems. With TK- L1210 cells, 5-F-cUMP was as potent as with the normal (L1210/0) line but was about fourfold less active with TK- Raji cells compared to Raji/0 cells. The 5-X-cUMPs showed little potency as antivirals. A single-crystal X-ray analysis of the ammonium salt of 5-I-cUMP confirmed its structure and showed the conformation of the phosphate ring to be the expected chair. The ribose pucker is near 3(4)T, and the torsion angle about the beta-glycosidic N(1)-C(1') bond is in the syn range (-84.8 degrees).     

Synthesis and biologic studies of arabinosylcytosine 5'-methylphosphonate

Arabinosylcytosine (ara-C), a clinically useful antitumor agent, is ineffective against cells that have deleted deoxycytidine kinase, the enzyme necessary for conversion of ara-C to its active nucleotide form. To circumvent this resistance, arabinosylcytosine-5'-methylphosphonate (ara-CMeP) was synthesized as an analogue of ara-CMP that would be membrane-permeable, resistant to serum phosphatase attack, and resistant to nucleoside deaminase inactivation. Ara-CMP was inhibitory to leukemia P388 in vitro but required concentrations 90-fold greater than that of ara-C for comparable cell inhibition. Both ara-CMeP and ara-CMP were competitive inhibitors of dCMP kinase from leukemia L1210 with K_i values of 4.0 × 10^?3 and 4.4 × 10^?3 M respectively. However, ara-CMP is a substrate for dCMP kinase, whereas ara-CMeP was not. Thus, the inability of ara-CMeP to be phosphorylated precludes its usefulness as a functional analogue of ara-CMP.     

Antiviral activity of 5-methylthiomethyl-2'-deoxyuridine and other 5-substituted 2'-deoxyuridines.

Of a series of eight 5-substituted 2'-deoxyuridine (dUrd) derivatives, which were evaluated for their antiviral and antimetabolic activities in primary rabbit kidney or human skin fibroblast cell cultures, five dUrd derivatives, 5-dimethylaminomethyl-dUrd, 5-chloroacetamidomethyl-dUrd, 5-iodoacetamidomethyl-dUrd, 5-pyrrolidinylmethyl-dUrd and 5-N-methylpiperazinylmethyl-dUrd, showed little, if any, activity. The three others, 5-formyl-dUrd, 5-azidomethyl-dUrd and 5-methylthiom-ethyl-dUrd, were found to inhibit the replication of various HSV (herpes simplex virus) strains (whether type 1 or 2) at a concentration of approximately 1–10 μg/ml. The antiviral activity of 5-formyl-dUrd may be accounted for by an inhibition of thymidylate synthetase. The inhibitory effect of 5-formyl-dUrd on HSV multiplication was readily reversed by adddition of 2'-deoxythymidine (dThd), and, in analogy with other established thymidylate synthetase inhibitors, 5-formyl-dUrd blocked the incorporation of [2-¹⁴C]dUrd into cellular DNA to a markedly greater extent than the incorporation of [methyl-³H]dThd. Unlike 5-formyl-dUrd, 5-azidomethyl-dUrd and 5-methylthiomethyl-dUrd did not preferentially inhibit the incorporation of [2-¹⁴C]dUrd. Antiviral indexes, defined as the id₅₀ for [2-¹⁴C]dUrd incorporation divided by the id₅₀ for HSV (type 1, strain KOS) replication, were 0.25, 43 and > 100 for 5-formyl-dUrd, 5-azidomethyl-dUrd and 5-methylthiomethyl-dUrd, respectively. The latter two compounds may therefore be considered as rather selective anti-herpes agents.     

海南粗榧新碱衍生物HH07A的抗肿瘤作用

用细胞生长曲线测定法及软琼脂集落形成分析法研究了HH07A对几种肿瘤及正常细胞生长的影响。结果表明,1.5ug·ml^-1及3μg·ml^-1HH07A能分别明显抑制L1210和HL-60细胞的生长。3种肿瘤细胞对HH07A的敏感性依次为L1210>KB>HL-60,而正常小鼠粒系祖细胞GM-CPC对药物的敏感性则低于前三者,且HH07A3.5μg·ml^-1对HL-60细胞无分化诱导作用。HH07A对腹水型L1210白血病小鼠、S180小鼠均有较明显的治疗作用,使L1210荷瘤小鼠、S180荷瘤小鼠存活时间延长。也能抑制S180实体瘤的生长。