Pheromone-induced changes in the acidophil concentration of mouse pituitary glands.

Pituitaries of female mice in anestrus resulting from colony housing were characterized by a 58.0-percent (acidophil content. Subsequent exposure to restrained male mice for one and two nights failed to evoke significant acidophilic degranulation and resulted in pituitary acidophil values of 57.4 and 55.1 percent respectively. Exposure to released males on the third night produced marked acidophilic degranulation resulting in a significant decline in pituitary acidophils to 38.0 percent. These findings support the view that female pheromone suppresses and male pheromone favors the secretion of follicle-stimulating hormone and indicate that luteotrophic hormone is secreted at its assigned time in the sequence of cyclic ovarian events initiated by the secretion of follicle-stimulating hormone.

Adriamycin and cyclophosphamide in combination chemotherapy of L1210 leukemia.

Combination of cyclophosphamide and adriamycin, encompassing a wide range of dosages, were administered on five different schedules to C57BL/6J X DBA/2J F1 female mice inoculated i.p. with L1210 ascites tumor cells. Among the resulting 85 treatment groups, the mean postinoculation survival of mice that died with tumor was 11.4 to 51.3 days; this represented increases of 62 to 628% over the survival of untreated controls. In some groups, tumor cells were eradicated in 9 of 10 treated mice. By contrast, neither drug given alone cured leukemic mice or extended their survival beyond controls by more than 96%. Adriamycin and cyclophosphamide were most effective when administered simultaneously on Day 1; additional treatment with adriamycin on Days 4 and 7 produced a significant increase in the survival of mice that died with tumor, but this regimen did not increase the incidence of cures. Combination therapy with these agents reduced the cytotoxic response of the host to subsequently inoculated L1210 cells. The pronounced therapeutic effectiveness of this drug combination is attributed to a true potentiation of the independent oncolytic action of each agent.

Cytotoxicity, uptake, polyglutamate formation, and antileukemic effects of 8-deaza analogues of methotrexate and aminopterin in mice.

In contrast to methotrexate (MTX) and aminopterin (AMT), the 8-deaza analogues of these antifolates are not substrates for rabbit liver aldehyde oxidase. Since they are not converted to 7-hydroxy derivatives, they have been investigated with regard to their cytotoxicity for CCRF-CEM cells, transport into these cells, and conversion to polyglutamate forms. For this purpose 3H-labeled analogues were synthesized. The drug concentrations of the analogues required to inhibit cell growth by 50% are significantly lower than for the parent compounds particularly for a short exposure of cells to the drug. Vmax and Km for unidirectional influx do not differ greatly among the four drugs, but amounts of uptake over 1 h are markedly different and increase in the order MTX less than 8-deazaMTX less than AMT less than 8-deazaAMT. During 1 h of uptake a much greater proportion of the 8-deaza analogues is converted to polyglutamate forms than in the case of parent drugs. Only 52% of MTX is converted to polyglutamates, whereas in the case of the other three compounds the conversion is greater than or equal to 90%. However, MTX is relatively efficient in adding two glutamate residues, whereas the other drugs predominantly accumulate as forms with only one additional glutamate (+Glu1). During 1 h of efflux the drugs without additional glutamates decrease to low concentrations and there is also a major loss of +Glu1 form, but there is also an increase in longer chain forms, especially in the case of MTX. The net result is a still greater disparity in total intracellular levels of the four drugs after the period of efflux. MTX has much lower toxicity in mice in vivo than the other three compounds, 8-deazaAMT being the most toxic. At the maximum tolerated dose MTX produced a considerably greater increase in life span for mice bearing P388 than any of the other drugs, and a somewhat greater increase for mice bearing L1210. Thus the 8-deaza analogues do not offer a therapeutic advantage over MTX against leukemias in the mouse, primarily due to their much greater toxicity.

Effects of cytotoxicity of 2-chloro-2'-deoxyadenosine and 2-bromo-2'-deoxyadenosine on cell growth, clonogenicity, DNA synthesis, and cell cycle kinetics.

The cytotoxic effects of the adenosine deaminase resistant analogues 2-bromo-2'-deoxyadenosine (2-BrdAdo) and 2-chloro-2'-deoxyadenosine (2-CldAdo) have been compared with those of deoxyadenosine (dAdo). Like 2-CldAdo, 2-BrdAdo is highly effective in inhibiting the growth of many T-lymphoblastoid, B-lymphoblastoid, and myeloid cell lines in culture. Concentrations required to inhibit growth of CCRF-CEM human T-lymphoblastoid cells by 50% (IC50) are: 2-CldAdo, 0.045 microM; 2-BrdAdo, 0.068 microM; dAdo, 0.9 microM in the presence of 5 microM erythro-9-(2-hydroxy-3-nonyl)adenine. Like dAdo, 2-BrdAdo causes a much greater decrease in DNA synthesis than in RNA and protein synthesis. For each of the nucleosides the concentration required to cause 50% inhibition of DNA synthesis (as measured by thymidine incorporation) in an 18-h exposure is very similar to the IC50 for growth and to the concentration required to decrease viability (clonogenicity) over 18 h by 50% (EC50). A fraction of CCRF-CEM cells (approximately equal to 30%) is resistant to killing by exposure to 2-BrdAdo or 2-CldAdo for 4 h at concentrations 100 times the EC50, but 3% of cells are resistant to exposure for 4 h to a concentration of dAdo 3 times the EC50. Each of the three nucleosides causes accumulation of cells in S phase, the accumulation becoming more marked with longer periods of exposure and with higher concentrations of nucleoside. During exposures for 18-24 h at a concentration of nucleoside near the EC50 most cells accumulate in S, with most in early S, whereas exposure to concentrations greater than EC95 accumulates cells at the G1/S border. This suggests that loss of viability is associated with a blockade of some process specifically occurring at the initiation of S phase. At an optimum dosage schedule, 2-BrdAdo and 2-CldAdo have similar therapeutic effects against L1210 in vivo, both producing over 99% cell kill, but the optimum dosage of 2-CldAdo is lower.

Biochemical pharmacology of 2-chlorodeoxyadenosine in malignant human hematopoietic cell lines and therapeutic effects of 2-bromodeoxyadenosine in drug combinations in mice.

Growth of human hematopoietic cell lines showed a 100-fold range of sensitivity to inhibition by 2-chloro-2'-deoxyadenosine (CldAdo), with highly sensitive lines in all three groups: T-lymphoblastic, B-lymphoblastic, and non-T, non-B. Formation of nucleotides from [8-3H]CldAdo was investigated in ten lines. In cells exposed to 0.15 microM CldAdo, CldAdo 5'-phosphate (CldAMP) reached 0.7-14 microM and CldAdo 5'-triphosphate (CldATP) reached 0.05-6 microM in 1 h. In most cases these nucleotide concentrations at 1 h were close to the steady-state concentrations, and the latter concentrations were approximately proportional to extracellular CldAdo concentration. On removal of extracellular CldAdo, intracellular CldAMP and CldATP declined rapidly with half times of 0.56-0.9 and 0.64-1.46 h, respectively. There was no correlation between these rates of catabolism and steady-state levels. The different sensitivities of the lines to CldAdo is explained only in part by the different steady-state concentrations of CldATP, and must be more directly related to differential effects on target enzymes. Mice inoculated with L1210 leukemia were treated with 2-bromo-2'-deoxyadenosine (BrdAdo) paired with one of 18 other therapeutic agents. Eight of the drugs paired with BrdAdo gave therapeutic responses from the combination greater than the sum of the responses of members of the pair. They included alkylating agents, antimetabolites blocking deoxyribonucleotide synthesis, and DNA polymerase inhibitors. Toxic dosages of CldAdo caused damage chiefly to the hemic-lymphatic systems and the kidneys.

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.

Inhibition of growth and modulation of gene expression in human lung carcinoma in athymic mice by site-selective 8-Cl-cyclic adenosine monophosphate.

Site-selective cyclic AMP (cAMP) analogues inhibit growth and induce changes in morphology in a spectrum of human cancer cell lines (D. Katsaros et al., FEBS Lett., 223:97, 1987). The cellular events underlying such effects of cAMP analogues include differential regulation of type I versus type II cAMP-dependent protein kinase isozymes (S. Ally et al., Proc. Natl. Acad. Sci. USA, 85: 6319, 1988). Infusion (i.p.) of 8-Cl-cAMP, the most potent site-selective cAMP analogue, for 7 days produced regression of LX-1 lung carcinoma in athymic mice in a dose-dependent manner. The tumor regression correlated with the changing levels of cAMP receptor proteins, RI alpha and RII beta, the regulatory subunits of cAMP-dependent protein kinase type I and type II, respectively. By photoaffinity labeling with 8-N3-[32P]cAMP and immunoblotting with a monospecific anti-RII antibody, RI alpha (Mr 49,000) and RII beta (Mr 51,000) were identified in the untreated control tumors. 8-Cl-cAMP treatment induced a rapid increase of both RI alpha and RII beta in tumor cytosols and translocation (within 1 h) of only RII beta from the cytosol to the nucleus. RII beta in both cytosols and nuclei remained elevated during 8-Cl-cAMP treatment, whereas RI alpha in the cytosols gradually decreased with time of treatment after its initial transient increase. Northern blot analyses demonstrated that the RII beta mRNA level increased within 6 h of 8-Cl-cAMP treatment and remained elevated during treatment, whereas the RI alpha mRNA level decreased to below that of the untreated control tumor level after its transient increase during 1-6 h of treatment. 8-Cl-cAMP treatment also caused a sharp decrease in both N-ras and c-myc mRNA levels. These results suggest that the fundamental basis for the antineoplastic activity of 8-Cl-cAMP may reside in the restoration of normal gene regulation in neoplasms in which cAMP receptor proteins play a role.

2'-O-nitro-1-beta-D-arabinofuranosylcytosine. A new derivative of 1-beta-D-arabinofuranosylcytosine that resists enzymatic deamination and has antileukemic activity.

To overcome the susceptibility of the anticancer drug 1-beta-D-arabinofuranosylcytosine (ara-C) to enzymatic deamination, and hence deactivation, we prepared the 2'-O-nitro-1-beta-D-arabinofuranosylcytosine (termed nitrara-C) and evaluated it for biological activity. Nitrara-C was resistant to enzymatic deamination and inhibited the proliferation of several strains of human leukemic T and B lymphoblasts grown in culture. Moreover, it substantially extended the life spans of mice with L1210 leukemia. Studies with ara-C-resistant human leukemic lymphoblasts deficient in deoxycytidine kinase activity disclosed that the inhibitory activity of the new compound depends on its phosphorylation.

Synthesis and antitumor activity of ribavirin imidates. A new facile synthesis of ribavirin amidine (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine hydrochloride).

Methyl 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboximidate (4) and ethyl 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamidate (6) were synthesized and tested for antitumor and antiviral activity. A new facile synthesis of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine hydrochloride (5), starting with imidate 4, was also developed. The imidates 4 and 6 differed greatly in solubility and dosing requirements. Even so, both compounds exhibited significant activity in vivo against murine leukemia L1210. Nontoxic dosing with 4 also significantly diminished Friend leukemia induced splenomegaly. In contrast, neither imidate was active in vitro.

Improved synthesis and antitumor activity of 2-bromo-2'-deoxyadenosine.

A more convenient synthetic route to 2-bromo-2'-deoxyadenosine (5) is reported, and results indicating significant antitumor activity of 5 against three murine tumors (L1210 leukemia, B16 melanoma, and M5076 ovarian carcinoma) are presented. The antitumor activity is very schedule dependent, being much greater when the drug is given q 3 h (X8) every 3rd or 4th day than when given by single daily administration. Toxicity of 5 for the tumor-bearing host is also very schedule dependent. Thus, on the q 3 h schedule of administration, a greater cumulative dose is tolerated by the host, and the therapeutic effectiveness of 5 is enhanced accordingly.

1,2,4-Diazaphosphole nucleosides. Synthesis, structure, and antitumor activity of nucleosides with a lambda 3 phosphorus atom.

Glycosylation of 1,2,4 lambda 3-diazaphosphole (4) under Lewis acid catalyzed conditions gave 1-alpha-D-ribofuranosyl-1,2,4 lambda 3-diazaphosphole (5) as the only product. Ethyl 1,2,4 lambda 3-diazaphosphole-3-carboxylate (10) was synthesized by the cyclocondensation of ethyl (chlorophosphinidene)(trimethylsilyl)acetate (8) with (trimethylsilyl)diazomethane and subsequent desilylation with tetra-n-butylammonium fluoride. Reaction of 10 with methanolic ammonia at 80 degrees C gave 1,2,4 lambda 3-diazaphosphole-3-carboxamide. Glycosylation of 10 using trimethylsilyl triflate catalyst followed by ammonlysis gave the ribavirin (1) analogue 1-beta-D-ribofuranosyl-1,2,4 lambda 3-diazaphosphole-3-carboxamide (11). Acetylation of 11 and subsequent treatment with phosphorus pentasulfide gave 2',3',5'-tri-O-acetyl-1-beta-D-ribofuranosyl-1,2,4 lambda 3-diazaphosphole-3- thiocarboxamide (13). Deprotection with methanolic ammonia gave 1-beta-D-ribofuranosyl-1,2,4 lambda 3-diazaphosphole-3-thiocarboxamide (14). Compound 14 gave a 25% increase in life span (ILS) against L1210 in female BDF1 mice. The anomeric configuration and site of glycosylation of 5 and 13 were established by single-crystal X-ray crystallography.

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 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%.

Synthesis and in vivo antitumor activity of 2-amino-9H-purine-6-sulfenamide, -sulfinamide, and -sulfonamide and related purine ribonucleosides.

A number of 6-sulfenamide, 6-sulfinamide, and 6-sulfonamide derivatives of 2-aminopurine and certain related purine ribonucleosides have been synthesized and evaluated for antileukemic activity in mice. Amination of 6-mercaptopurine ribonucleoside (7a) and 6-thioguanosine (7b) with chloramine solution gave 9-beta-D-ribofuranosylpurine-6-sulfenamide (8a) and 2-amino-9-beta-D-ribofuranosylpurine-6-sulfenamide (sulfenosine, 8b), respectively. Selective oxidation of 8a and 8b with 3-chloroperoxybenzoic acid (MCPBA) gave (R,S)-9-beta-D-ribofuranosylpurine-6-sulfinamide (9a) and (R,S)-2-amino-9-beta-D-ribofuranosylpurine-6-sulfinamide (sulfinosine, 9b), respectively. However, oxidation of 8a and 8b with excess of MCPBA gave 9-beta-D-ribofuranosylpurine-6-sulfonamide (10a) and 2-amino-9-beta-D-ribofuranosylpurine-6-sulfonamide (sulfonosine, 10b), respectively. Similarly, amination of 5'-deoxy-6-thioguanosine (7c) afforded the 6-sulfenamide derivative (8c), which on controlled oxidation gave (R,S)-2-amino-9-(5-deoxy-beta-D-ribofuranosyl)purine-6-sulfinamide (9c) and the corresponding 6-sulfonamide derivative (10c). Treatment of 6-thioguanine (12) with aqueous chloramine solution gave 2-amino-9H-purine-6-sulfenamide (13). Oxidation of 13 with 1 molar equiv of MCPBA afforded (R,S)-2-amino-9H-purine-6-sulfinamide (14), whereas the use of 4 molar equiv of MCPBA furnished 2-amino-9H-purine-6-sulfonamide (15). The resolution of R and S diastereomers of sulfinosine (9b) was accomplished by HPLC techniques. The structures of (R)-9b and 10b were assigned by single-crystal X-ray diffraction studies. (R)-9b exists in the crystal structure in four crystallographically independent conformations. Of the 18 compounds evaluated, 13 exhibited very significant anti-L1210 activity in mice. Sulfenosine (8b) at 22 mg/kg per day X 1 showed a T/C of 170, whereas sulfinosine (9b) at 173 mg/kg per day X 1 showed a T/C of 167 against L1210 leukemia. The 5'-deoxy analogue of sulfinosine (9c) at 104 mg/kg per day also showed a T/C of 172. A single treatment with 8b, 9b, and 9c reduced body burdens of viable L1210 cells by more than 99.8%.

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.

Antitumor and antiviral activity of synthetic alpha- and beta-ribonucleosides of certain substituted pyrimido[5,4-d]pyrimidines: a new synthetic strategy for exocyclic aminonucleosides.

A novel and direct synthesis of the antiviral and antitumor agent 4-amino-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (ARPP, 8) and its alpha-anomer (11) has been developed. Treatment of 2,4,6,8-tetrachloropyrimido[5,4-d]pyrimidine (1) with 2,3-O-isopropylidene-D-ribofuranosylamine gave an anomeric mixture of 2,4,6-trichloro-8-(2,3-O-isopropylidene-beta- and -alpha-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidines (3 and 4) in a ratio of 1.0:0.7. A nucleophilic displacement of the 4-chloro group of 3 and 4 with NH3 furnished 4-amino-2,6-dichloro-8-[(2,3-O-isopropylidene-beta-D-ribofuranosyl)amino ] pyrimido[5,4-d]pyrimidine (6) and its alpha-anomer (9), respectively. Catalytic hydrogenation of 6 and 9, followed by deisopropylidenation gave ARPP (8) and the alpha-anomer 11, respectively. Similarly, 3 and 4 have been transformed to 4-methoxy-8-(beta-D-ribofuranosylamino)pyrimido-[5,4-d]pyrimidine (MRPP, 14) and its alpha-anomer (17). Application of this procedure to 3 with NH2Me or NHMe2 resulted in the synthesis of 4-(methylamino)- and 4-(dimethylamino)-8-(beta-D-ribofuranosylamino)pyrimido [5,4-d]pyrimidine (24 and 27, respectively). A synthesis of 8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidin-4(3H)-one (21) has also been accomplished from 3 in three steps. Selective hydrogenation of 6 furnished 4-amino-6-chloro-8-[(2,3-O-isopropylidene-beta-D-ribofuranosyl)amino] pyrimido[5,4-d]pyrimidine (36), the structure of which was established by single-crystal X-ray diffraction analysis. Deisopropylidenation of 36 gave 6-chloro-ARPP (37). Extended treatment of 36 with NH3 furnished 4,6-diamino-8-[(2,3-O-isopropylidene-beta-D-ribofuranosyl)amino]pyrimido [5,4-d]pyrimidine (34), which on deisopropylidenation gave 6-amino-ARPP (35). An unambiguous synthesis of 34 and 36 has also been accomplished by the reaction of 4,6,8-trichloropyrimido[5,4-d]pyrimidine (28) with 2, followed by the treatment with NH3. Nucleophilic displacement studies with 1, 6, and 28 indicated the reactivity of the halogens in these compounds is in the order of 8 greater than 4 greater than 6 greater than 2. The structures of 3 and 9 have been assigned on the basis of 1H NMR data and further confirmed by single-crystal X-ray diffraction analysis. The exocyclic aminonucleosides synthesized during this study were tested for their activity against several RNA and DNA viruses in vitro and against L1210, WI-L2, and LoVo/L in cell culture. The effect of these compounds on the de novo nucleic acid biosynthesis has been studied. Compound 14 (MRPP) exhibited enhanced activity against L1210 in vivo, when compared to ARPP (8).

Activation of the respiratory burst in murine phagocytes by certain guanine ribonucleosides modified at the 7 and 8 positions: possible involvement of a pertussis toxin-sensitive G-protein.

The capacity of certain guanine ribonucleosides (modified at the 7 and/or 8 positions) to enhance the respiratory burst of murine peritoneal phagocytes was evaluated. The results show that 8-mercaptoguanosine, 8-bromoguanosine, 7-methyl-8-oxoguanosine and 7-thia-8-oxoguanosine, when injected intraperitoneally into mice, induced peritoneal phagocytes to generate reactive oxygen species as early as 1 h after injection. In vivo administration of the nucleosides induced higher levels of phagocyte activation than in vitro treatment with the same nucleosides. However, the addition of interferon alpha/beta in vitro significantly increased the magnitude of phagocyte activation by the nucleosides, suggesting an important role for cytokines/lymphokines in the nucleoside-induced phagocyte activation in vivo. Furthermore, pre-treatment of phagocytes in vitro with Bordetella pertussis toxin, before treatment with the guanosines, inhibited their capacity to induce the respiratory burst. These observations establish these low-molecular-weight compounds as interesting probes for the study of stimulus-response coupling in phagocytes.

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.

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.

Inhibition of phosphoribosylpyrophosphate synthetase by 4-methoxy-(MRPP) and 4-amino-8-(D-ribofuranosylamino) pyrimido[5,4-d]pyrimidine (ARPP).

The basis for the antitumor activities of the exocyclic amino nucleosides 4-amino-(ARPP) and 4-methoxy-8-(D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (MRPP) was investigated. The primary target of these nucleosides appeared to be 5-phospho-alpha-D-ribofuranose-1-pyrophosphate (PRPP) synthetase. MRPP-5'-monophosphate was a competitive inhibitor (Ki = 40 microM) of the activation of this enzyme by the cofactor inorganic phosphate (K alpha = 2.2 mM). Consequently, ARPP and MRPP treatment of WI-L2 cultures rapidly inhibited both de novo pyrimidine and purine synthesis as well as the nucleotide salvage reactions dependent on PRPP, ARPP or MRPP treatment completely prevented [14C]bicarbonate incorporation into acid-soluble pyrimidine and purine nucleotides. The rate of salvage of [8-14C]hypoxanthine to form IMP was decreased by 85%. Treatment of cells with these agents caused a 50% reduction in the steady-state level of PRPP. When the capacity of the treated cells for sustained synthesis of PRPP was examined by adenine incorporation, the rate of adenine uptake was inhibited by greater than 50%. In vivo treatment of BDF1 mice with a single dose of ARPP (173 mg/kg) or MRPP (62 mg/kg) extended the mean life span of the mice, which had been inoculated intraperitoneally 1 day earlier with 1 x 10(6) L1210 murine leukemia cells, by 62 and 82% respectively. These studies indicate that MRPP and ARPP inhibit PRPP synthetase, and that PRPP synthetase may be a viable target in the development of certain antitumor agents.