Polycytidylate and poly(7-deazaguanylate): a pair of complementary templates.

We have studied the template-directed oligomerization on polycytidylic acid of the 5'-phosphoro(2-methyl)imidazolides of a number of analogues of guanosine. None of the analogues reacted as efficiently as the original guanosine compound, and only the 7-deazaguanosine analogue gives a detectable yield of oligomers. Similar results are described for a reaction involving the intramolecular template-directed elongation of a short oligocytidylate primer. Oligocytidylates containing five or more cytidylate residues are extended on the single-stranded regions of poly(G). In the present study we show that these oligocytidylates are extended efficiently by reaction with cytidine-5'-phosphoro(2-methyl)imidazolide on a poly(7-deazaguanylic acid) template. The products are considerably longer than those obtained using a polyguanylic acid template. We believe that the formation of a tetrahelix inhibits the latter reaction, while poly(7-deazaguanylate) does not aggregate and, therefore, acts as a more efficient template. This work identifies for the first time a pair of homopolymers each of which facilitates the template-directed elongation of the other.

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).

Alkylpurines as immunopotentiating agents. Synthesis and antiviral activity of certain alkylguanines.

Several simple 8-substituted 9-alkyl- and 7,8-disubstituted 9-alkylguanine derivatives were synthesized as potential antiviral agents. These were tested for antiviral protection against a lethal Semliki Forest virus (SFV) infection in mice, and their antiviral properties were evaluated from a structure-activity standpoint. In this model system, 9-alkylguanines with the alkyl chain consisting of four to six carbons were found to be the most active. Substitution of the 8-position of the purine ring did not enhance activity, with the exception of the 7-alkyl-8-oxo substituent. These data were found to support the hypothesis that guanines need not contain an intact carbohydrate moiety in order to exhibit antiviral activity by virtue of immune potentiation. Hence, phosphorylation of guanosine analogs that exhibit antiviral activity by a similar mechanism does not play a significant role.

Inhibition of human pancreatic cancer cell (MIA PaCa-2) growth by cholera toxin and 8-chloro-cAMP in vitro.

The effects of cholera toxin (CT) and 8-chloro-cAMP (8-Cl-cAMP) on cell growth were investigated using two human pancreatic carcinoma cell lines (MIA PaCa-2, Panc-1). CT, which catalyses the ADP ribosylation of Gs, suppresses the proliferation of MIA PaCa-2(PC) cells. CT at the low dose of 0.1 pg ml-1 was inhibitory of PC cell growth, and the maximum suppression (70%) was achieved at a CT concentration of 100 pg ml-1. This phenomenon was reversible. The production of cAMP by CT (100 pg ml-1) in PC cells was enhanced 320-fold compared with the control. In addition, cAMP analogues (8-Cl-cAMP, 8-Br-cAMP) and forskolin decreased the growth rate of PC cells in a dose-dependent manner. These results support the view that CT suppresses PC cell growth by stimulating cAMP production. Conversely, Panc-1 cells were far less sensitive to CT in cell growth and cAMP production. 8-Cl-cAMP was also less effective on Panc-1 cell growth. The binding of an insulin-like growth factor (IGF)-I and transforming growth factor (TGF)-alpha, which has been shown to stimulate PC cell growth in an autocrine manner, to PC cells was not modified in cells treated with CT or 8-Cl-cAMP. The results suggest that the inhibitory actions of these substances do not occur at the level of the receptor for IGF-I or EGF/TGF-alpha. We have previously shown that phorbol esters, which decrease the binding of TGF-alpha to PC cells, has an anti-proliferative activity on these tumour cells. Inhibited cell growth by maximum suppressive dose of CT or 8-Cl-cAMP was further inhibited by TPA. In addition, an oncogene product of K-ras which is commonly activated in pancreatic cancer, was increased by CT and 8-Cl-cAMP. It is concluded that CT and 8-Cl-cAMP inhibit PC cell growth, presumably in a similar manner, and their mechanism(s) of action may be different from that of TPA. The anti-proliferative effect of CT or 8-Cl-cAMP was enhanced by TPA, implying that the combination of these substances results in increased inhibition of the PC cell growth.

A novel non-radioactive method for detection of nucleoside analog phosphorylation by 5'-nucleotidase.

Cytosolic 5'-nucleotidase has been implicated in the phosphorylation of certain nucleosides of therapeutic interest. In vitro, IMP and GMP serve as the optimal phosphate donors for this nucleoside phosphotransferase reaction. Existing assays for nucleoside phosphorylation effected by 5'-nucleotidase require a radiolabeled nucleoside as the phosphate acceptor and separation of the substrate-nucleoside from product-nucleotide has been accomplished either by a filter binding method or HPLC. However, detection of the phosphorylation of unlabeled nucleoside by HPLC is difficult since the ultraviolet absorbance of the phosphate donor, IMP, frequently obscures the absorbance of newly formed nucleotide. The use of ribavirin 5'-phosphate (RMP, 1,2,4-triazole-3-carboxamide riboside 5-monophosphate) as the phosphate donor obviates this difficulty since this triazole heterocycle does not significantly absorb at the wavelengths used to detect most nucleoside analogs. Using this procedure, a 5'-nucleotidase activity from the 100,000 x g supernatant fraction of human T-lymphoblasts deficient in adenosine kinase, hypoxanthine-guanine phosphoribosyltransferase, and deoxycytidine kinase, was characterized with regard to structure-activity relationships for certain inosine and guanosine analogs.

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.

Successful immunotherapy of murine melanoma metastases with 7-thia-8-oxoguanosine.

We have recently reported that a synthetic nucleoside, 7-thia-8-oxoguanosine (7T8OG) is a potent activator of a number of effectors which are involved in anti-tumor immune responses. 7T8OG was found to induce interferon (IFN) production, to activate asialo-GM1 positive (AGM+1) killer cells, and to enhance specific antibody responses. In the present study, we investigated the effect of 7T8OG on growth of the murine pulmonary B16 melanoma and on formation of metastases. C57BL/6 mice were injected i.p. with 50-150 mg/kg 7T8OG before or after i.v. inoculation of B16 melanoma tumor cells, and 17-19 days after tumor inoculation, the number of metastases in the lungs were counted. 7T8OG given systemically in a single or a divided dose 24 h prior to the challenge of tumor cells reduced the number of lung tumor metastases by 89-99% which is highly significant as compared to untreated control (P less than 0.001). Occasional extra pulmonary tumor growth in the thoracic cavity and neck lymph node was also completely inhibited. The reduction in the number of tumor nodules was dose dependent. A single dose of 150 mg/kg of 7T8OG was also effective in inhibiting the growth of 3-5 day old metastatic tumors. The cytotoxic activity of killer cells induced in vivo by 7T8OG was completely abolished by in vitro treatment of cells with anti-AGM1 antibody plus complement. Administration of anti-AGM1 antibody following the 7T8OG treatment completely abrogated the anti-tumor effect of 7T8OG, resulting in a massive increase in the number of tumor foci in the lungs. Administration of carageenan or silica followed by injection of 7T8OG caused a significant increase (P less than 0.01) in the number of pulmonary tumor nodules compared to treatment with 7T8OG only. These findings indicate that activated macrophages or perhaps their cytokine (tumor necrosis factor) also contribute to the host tumor defense by 7T8OG.

Aerosol and intraperitoneal administration of ribavirin and ribavirin triacetate: pharmacokinetics and protection of mice against intracerebral infection with influenza A/WSN virus.

Ribavirin is active in vitro but not in vivo against a number of viruses capable of causing encephalitis. Ribavirin triacetate (RTA), a lipophilic derivative, has been reported to be more effective than ribavirin in protecting animals from encephalitis. By using an influenza A/WSN virus encephalitis model, we demonstrated that RTA administered by small-particle aerosol was able to decrease the death rate and increase the time of survival. To determine if this beneficial effect was due to increased delivery of drug, the pharmacokinetic properties of ribavirin and RTA when administered as an aerosol or by intraperitoneal injection were examined. Aerosol administration of ribavirin or RTA gave significantly higher concentrations of ribavirin in the lungs and serum of mice than did intraperitoneal injection. There was no difference, however, in ribavirin levels when either ribavirin or RTA was administered by small-particle aerosol. In brain tissue, ribavirin concentrations increased with time and did not appear to decrease as rapidly as in lungs and serum. Mean peak ribavirin concentrations in the brain were higher following aerosol administration of ribavirin than RTA, and both were higher than that following intraperitoneal injection of either drug. Administration of ribavirin or RTA by intraperitoneal injection failed to protect mice from a lethal intracerebral inoculation of influenza A/WSN virus, while aerosolized RTA did protect mice. The pharmacokinetics of ribavirin in brain tissue following aerosol administration of either drug did not explain the advantage of RTA over ribavirin in protecting mice from intracerebral infection with influenza A/WSN virus.

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.

Potentiation of the efficacy of murine L1210 leukemia vaccine by a novel immunostimulator 7-thia-8-oxoguanosine: increased survival after immunization with vaccine plus 7-thia-8-oxoguanosine.

We have investigated the ability of a novel immunopotentiator, 7-thia-8-oxoguanosine (7T8OG) to increase the efficacy of a weakly immunogenic murine L1210 leukemia vaccine. The vaccine was prepared by irradiating L1210 leukemia cells in a cesium source with a total of 6000-R dose. DBA/2 mice were treated with 150 mg/kg 7T8OG and/or with vaccine consisting of 10(7) irradiated cells. In combination therapy, mice first received the vaccine and then were injected with 75 mg/kg 7T8OG 2 h and 4 h after vaccination. One week after the last treatment all mice were inoculated with 10(4) live leukemia cells intraperitoneally. Control, untreated mice (n = 66) injected with 10(4) live leukemia cells had a mean survival time +/- standard error of 10.5 +/- 0.2 days. Treating mice (n = 66) with one, two or three doses of 7T8OG administered i.p. 1 week apart did not increase survival (mean survival time = 10.7 days). Mice immunized with one, two or three doses of vaccine had 14.5 +/- 1.1, 45.4 +/- 6.2 and 68.3 +/- 10.6 days mean survival, respectively. 7T8OG-stimulated vaccination increased the survival dramatically. The best survival was noted when the mice were treated with 2x (vaccine + 7T8OG). Immunization of mice (n = 30) with this treatment regimen increased the mean survival to 156 +/- 10.0 days. Over 90% of mice that were treated this way had a cumulative survival time greater than 160 days. In contrast, only 12% of the mice immunized twice with the leukemia vaccine alone survived over 160 days. These results suggest a rationale for the use of this immuno-potentiator with various vaccines for a more effective immunization.

Direct C-glycosylation of guanine analogues: the synthesis and antiviral activity of certain 7- and 9-deazaguanine C-nucleosides.

C-Glycosylation of two guanine analogues, 9-deaza- and 7-deazaguanine, has been achieved under Friedel-Crafts conditions, providing a direct synthetic route to 9-deazaguanosine (4; 2-amino-7-beta-D-ribofuranosyl-5H-pyrrolo[3,2-d]pyrimidin-4(3H)-one) and 8-beta-D-ribofuranosyl-7-deazaguanine (16), respectively. This electrophilic C-glycosylation was applied successfully to six guanine and substituted-guanine analogues resulting in yields of approximately 50%. This represents the first reported C-ribosylation of preformed nitrogen heterocycles isosteric with guanine. These C-nucleosides were evaluated for their ability to provide protection against a lethal Semliki Forest virus infection in mice, relative to 7-thia-8-oxoguanosine which was used as a positive control. Two of the C-nucleosides, 2-amino-6-chloro-5-methyl-7-beta-D-ribofuranosyl-5H-pyrrolo [3,2-d]pyrimidin-4(3H)-one (12) and the corresponding 6-bromo derivative (13), showed good prophylactic activity in this virus model system.

Tiazofurin is phosphorylated by three enzymes from Chinese hamster ovary cells.

The growth inhibitory activity of tiazofurin toward adenosine kinase deficient Chinese hamster ovary (CHO) cells was partially reversed by the presence of nicotinamide riboside. Similarly, the formation of tiazofurin 5'-monophosphate and the active metabolite, tiazofurin 5'-adenine dinucleotide could be partially inhibited by 100 microM nicotinamide riboside in CHO cells and substantially inhibited (80-90%) in adenosine kinase deficient cells. Tiazofurin phosphorylating activity from CHO cell extracts was resolved into two peaks by DEAE-cellulose chromatography. The first peak of activity was identified as adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20). The second peak of activity correlated with a previously described 3-deazaguanosine phosphorylating activity that was identified as a nicotinamide ribonucleoside kinase. Contaminating purine nucleoside phosphorylase was removed by sedimentation through a sucrose density gradient which also resolved the tiazofurin phosphorylating activity into two peaks, one requiring just ATP and the other requiring both ATP and IMP. Of the substrates tested with the lower density peak, nicotinamide riboside was most efficient and was the only natural substance that competed well with tiazofurin for phosphorylation, substantiating its suggested identity as a nicotinamide ribonucleoside kinase. The apparent Km value for nicotinamide riboside (2 microM) was significantly less than that for tiazofurin (13.6 microM). ATP was the best phosphate donor; CTP and UTP were utilized less efficiently and IMP did not support the reaction. The best substrate for the higher density peak of tiazofurin phosphorylation was inosine and both ATP and IMP were required for the reaction, suggesting its identity as a 5'-nucleotidase. In summary, it appears that adenosine kinase, nicotinamide ribonucleoside kinase, and 5'-nucleotidase may all contribute to the phosphorylation of tiazofurin in CHO cells.

Synthesis and broad-spectrum antiviral activity of 7,8-dihydro-7-methyl-8-thioxoguanosine.

2,6,8-Trichloro-7-methylpurine (3) was converted to 2-chloro-8,9-dihydro-7-methyl-8-thioxopurin-6(1H)-one (5) by utilizing the difference in reactivity of the 2-, 6-, and 8-positions in the trichloropurine ring system to nucleophilic displacement. Compound 5 was subsequently glycosylated with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose according to the Vorbrüggen procedure to yield 2-chloro-8,9-dihydro-7-methyl-9-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosy l)-8- thioxopurin-6(1H)-one (6). Removal of the benzoyl protecting groups, followed by amination of 7 with liquid ammonia at 150 degrees C, gave 7,8-dihydro-7-methyl-8-thioxoguanosine (2). The structure of compound 2 was confirmed by X-ray crystallographic analysis. Compounds 1 (7,8-dihydro-7-methyl-8-oxoguanosine) and 2 were evaluated for activity in various animal virus infection models. Against banzi, Semliki Forest, and San Angelo viruses in mice, 2 was highly active when administered before virus inoculation.

Guanosine analogues. Synthesis of nucleosides of certain 3-substituted 6-aminopyrazolo[3,4-d]pyrimidin-4(5H)-ones as potential immunotherapeutic agents.

Several guanosine analogues were synthesized in the pyrazolo[3,4-d]pyrimidine ring system with various substituents at the 3-position. The new analogues prepared here include the CH3 (2-amino-3-methyl-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4 (5H)-one, 13a), the phenyl (2-amino-3-phenyl-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4 (5H)-one, 13b), and the NH2 (3,6-diamino-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidin-4(5H)- one, 17) substituted derivatives. These new agents, as well as several other 3-substituted derivatives including H, Br, OCH3, COOH, and oxo, were evaluated for their ability to potentiate certain murine immune functions relative to the known active agent 5-amino-3-beta-D-ribofuranosylthiazolo[4,5-d]pyrimidine-2,7(3H,6H) -dione (4, 7-thia-8-oxoguanosine). The biological evaluation included the (1) ex vivo determination of increased natural killer cell function and (2) in vivo antiviral protection against a lethal challenge of Semliki Forest virus. The 3-unsubstituted (5a) and the 3-bromo (5c) derivatives were found to be the most active immunopotentiators in this series.

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 antitumor evaluation in mice of certain 7-deazapurine (pyrrolo[2,3-d]pyrimidine) and 3-deazapurine (imidazo[4,5-c]pyridine) nucleosides structurally related to sulfenosine, sulfinosine, and sulfonosine.

7-Deaza (pyrrolo[2,3-d]pyrimidine) and 3-deaza (imidazo[4,5-c]pyridine) congeners of sulfenosine (5a and 9), sulfinosine (6a and 10), and sulfonosine (7a) have been prepared and evaluated for their antileukemic activity in mice. Amination of 2-amino-7-beta-D-ribofuranosylpyrrolo[2,3-d]pyrimidine-4(3H)-th ion e (4a) and its 2'-deoxy analogue (4c) with a chloramine solution gave the corresponding 4-sulfenamides (5a and 5c, respectively), which on selective oxidation with m-chloroperoxybenzoic acid (MCPBA) gave the respective diastereomeric 2-amino-7-beta-D-ribofuranosyl-pyrrolo[2,3-d]pyrimidine-4-sulfinamide (7-deazasulfinosine, 6a) and its 2'-deoxy derivative (6c). A similar amination of 7-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidine-4(3H)- thione (4b) gave the corresponding 4-sulfenamide derivative (5b). Oxidation of 5b with 1 molar equiv of MCPBA furnished (R,S)-7-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidine- 4- sulfinamide (6b), whereas use of excess of MCPBA afforded the corresponding sulfonamide derivative (7b). Treatment of 3-deaza-6-thioguanosine (8) with a chloramine solution gave 3-deazasulfenosine (6-amino-1-beta-D- ribofuranosylimidazo[4,5-c]pyridine-4-sulfenamide, 9). Controlled oxidation of 9 with MCPBA afforded 3-deazasulfinosine (10). As gauged by increases in the mean postinoculation life spans of L1210 inoculated mice, none of these nucleosides exhibited biologically significant activity (T/C greater than or equal to 125). Even so, antileukemic activity appeared to be influenced, albeit not uniformly, by structural modifications in the base and carbohydrate moieties of sulfenosine and sulfinosine. Thus, while several of the compounds were lacking in cytotoxic activity, eight others (4c, 5a, 5c, 6a, 6b, 7b, 9, and 10) were estimated to have reduced body burdens of viable L1210 cells by 16-77%.

Oxidation of 2-amino-9-beta-D-ribofuranosylpurine-6-sulfenamide to the corresponding 6-sulfonamide facilitates changes in biologic characterization that include activity against thiopurine-refractory experimental leukemia.

Preclinical investigations in vivo revealed unexpected differences in the biological characteristics of 2-amino-9-beta-D-ribofuranosylpurine-6-sulfenamide (sulfenosine, 1) and 2-amino-9-beta-D-ribofuranosylpurine-6-sulfonamide (sulfonosine, 2), two novel but structurally related derivatives of 6-thioguanosine (6TGR). Strikingly, the addition of a fully oxidized sulfur atom at the 6 position of sulfenosine produced a purine derivative (sulfonosine) that was remarkably active against experimental leukemia resistant to treatment with either sulfenosine or 6TGR. This slight structural modification also appeared to influence solubility, scheduling capability, and oral activity as well as penetration of the central nervous system (CNS) and the onset of cellular resistance. These findings underscore the dramatic changes in biologic activity that can be produced by subtle modifications in molecular structure. We trust they may also contribute to the development of improved clinical therapy.

A novel guanosine analog, 7-thia-8-oxoguanosine, enhances macrophage and lymphocyte antibody-dependent cell-mediated cytotoxicity.

Intraperitoneal treatment of mice with a novel guanosine analog, 7-thia-8-oxoguanosine (7-thia-8-oxoGuo), gives rise to activated splenic lymphocytes and peritoneal macrophages with enhanced capacity to mediate antibody-dependent cellular cytotoxicity (ADCC). ADCC activities against both chicken red blood cells and P815 murine plasmacytoma cells were enhanced, indicating that macrophages as well as lymphocytes functioning as K-cells in the two distinct cytolytic systems, were activated by 7-thia-8-oxoGuo. Furthermore, 7-thia-8-oxoGuo enhanced lymphocyte-mediated ADCC activity in beige (bgJ/bgJ) mice against P815, thus indicating the ability of 7-thia-8-oxoGuo to function as a potent immunomodulator even in an animal that is known to possess selective impairment of naturally occurring killer lymphocytes. These results suggest that 7-thia-8-oxoGuo could serve as an agent for immunomodulation and immunorestoration.

Changes in diacylglycerol and membrane associated protein kinase C activity reflect the growth status of xenografted human mammary carcinoma treated with 8-Cl-cAMP.

The intracellular accumulation of cAMP inhibits the growth of transformed cells in vitro and in vivo, and exposure to various cAMP analogs produces similar results. The influence of such analogs on the growth of neoplastic cells in vivo is less well defined, and the relevance of these analogs for the phosphoinositide pathway has not been established. The present report details the inhibition of tumor growth that occurred when human mammary xenografts were treated with 8-Cl-cAMP, the subsequent rebound in tumor growth that occurred when treatment ceased, and the levels of diacylglycerol and membrane-associated protein kinase C activity that characterized tumors in different growth states. Tumor levels of diacylglycerol and particulate PKC activity appeared to be influenced not only by treatment but also by treatment withdrawal. Changes in these entities tended to coincide with tumor growth rate, being relatively suppressed during growth stasis and markedly elevated during periods of rapid growth. The data presented do not establish a causal relationship. Thus, the concomitant changes noted in tumor growth and tumor levels of either diacylglycerol and membrane-associated protein kinase C may only be coincidental. Alternatively, they may indicate that cAMP analogs inhibit tumor growth in vivo by modulating the phosphoinositide pathway.