Conservation of antiviral activity and improved selectivity in PMEO-DAPym upon pyrimidine to triazine scaffold hopping.

Acyclic nucleoside phosphonates incorporating 2,4-diaminotriazine (DAT) as a 5-aza-analog of the 2,4-diamino-pyrimidine (DAPym) nucleobase present in PMEO-DAPyms have been synthesized. The lead PMEO-DAT is as inhibitory against HIV, HBV, MSV and VZV replication as the parent PMEO-DAPym and equally inefficient at markedly affecting replication of HSV-1, HSV-2 and HCMV. A rationale for this similar biological profile is proposed on the basis of structural differences in the active site of the viral DNA polymerases. PMEO-DAT is, however, more selective because, unlike PMEO-DAPym, it does not stimulate secretion of ß-chemokines in cultured PBMC.

Synthesis and antiviral activity of some 5'-N-phthaloyl-3'-azido-2',3'-dideoxythymidine analogues.

A variety of substituted 5'-N-phthaloyl-3'-azido-2',3'-dideoxythymidine derivatives has been evaluated for their activity against HIV-1, HIV-2 and Moloney murine sarcoma virus (MSV) in cell culture. Most of the 3'-azido-2',3'-dideoxythymidine (AZT, zidovudine) derivatives showed antiviral activity in the lower micromolar concentration range and there was a close correlation between their anti-HIV and anti-MSV activity (r = 0.99). The adamantyl phthaloyl derivative was active at submicromolar concentrations. None of the compounds showed marked cytostatic activity. They did not inhibit recombinant HIV-1 reverse transcriptase. All compounds were inactive against HIV in thymidine kinase-deficient cells, pointing to the compounds' requirement to release free AZT to afford antiviral efficacy.

Acyclic nucleotide analogs derived from 8-azapurines: synthesis and antiviral activity.

Reaction of phosphoroorganic synthons with 8-azaadenine, 8-aza-2, 6-diaminopurine, and 8-azaguanine using cesium carbonate yielded regioisomeric 8-azapurine N7-, N8-, and N9-(2-(phosphonomethoxy)alkyl) derivatives. This reaction followed by deprotection afforded isomeric 2-(phosphonomethoxy)ethyl (PME), (S)-(3-hydroxy-2-(phosphonomethoxy)propyl) [(S)-HPMP], (S)-(3-flouro-2-(phosphonomethoxy)propyl) [(S)-FPMP], (S)-(2-(phosphonomethoxy)propyl) [(S)-PMP], and (R)-(2-(phosphonomethoxy)propyl) [(R)-PMP] derivatives. 13C NMR spectra were used for structural assignment of the regioisomers. None of the 8-isomers exhibited any antiviral activity against herpesviruses, Moloney murine sarcoma virus (MSV), and/or HIV. 9-(S)-HPMP-8-azaadenine (23) and PME-8-azaguanine (65) were active against HSV-1, HSV-2, and CMV at 0.2-7 micrograms/mL, VZV at 0.04-0.4 microgram/mL, and MSV (at 0.3-0.6 microgram/mL). PME-8-azaguanine (65) and (R)-PMP-8-azaguanine (71a) protected MT-4 and CEM cells against HIV-1- and HIV-2-induced cytopathicity at a concentration of approximately 2 micrograms/mL.

Potentiating effect of ribavirin on the anti-retrovirus activity of 3'-azido-2,6-diaminopurine-2',3'-dideoxyriboside in vitro and in vivo.

3'-Azido-2,6-diaminopurine-2',3'-dideoxyriboside (AzddDAPR) is a potent and selective inhibitor of human immunodeficiency virus (HIV) replication in vitro. It also inhibits Moloney murine sarcoma virus (MSV)-induced transformation of murine C3H/3T3 embryo fibroblasts. AzddDAPR causes a marked dose-dependent suppression of MSV-induced tumor formation and mortality therewith associated in newborn mice infected with MSV. Combination of AzddDAPR with ribavirin resulted in a marked potentiation of its anti-retrovirus activity in vitro and a significant enhancement of its inhibitory effect on MSV-induced tumor formation in vivo. A slight increase in the in vivo toxicity of AzddDAPR was noted when combined with ribavirin.

Differential patterns of intracellular metabolism of 2',3'-didehydro-2',3'-dideoxythymidine and 3'-azido-2',3'-dideoxythymidine, two potent anti-human immunodeficiency virus compounds.

3'-Azido-2',3'-dideoxythymidine (AZT) and 2',3'-didehydro-2',3'-dideoxythymidine (D4T) are potent and selective inhibitors of human immunodeficiency virus replication in MT-4 and ATH8 cells. They are also inhibitory to the replication of murine retroviruses, i.e. Moloney murine sarcoma virus-induced transformation of C3H cells. In MT-4 cells AZT is readily phosphorylated to its 5'-monophosphate, while the 5'-di- and 5'-triphosphates are generated to a 200-600-fold lower extent than the 5'-monophosphate. D4T is phosphorylated in MT-4 cells to its 5'-monophosphate at a 300-600-fold lower extent than AZT. The phosphorylation of AZT in the thymidine kinase-deficient cell line (Raji/TK-) is severely depressed, while D4T phosphorylation is only slightly diminished in Raji/TK- as compared to Raji/0 cells. D4T has a 10-fold lower affinity for phosphorylation by crude MT-4 cell extracts than AZT (Km, 142 and 14 microM, respectively), and the Vmax for phosphorylation of D4T is only 5% that of AZT. D4T is phosphorylated by MT-4 cell extracts about 180-fold less efficiently than AZT (Vmax/Km, 0.06 for D4T, as compared to 11 for AZT), and this is consistent with the differences found in the amounts of phosphorylated products of D4T and AZT formed in intact MT-4 cells. The 5'-triphosphates of AZT and D4T are equipotent in their inhibitory effects on the reverse transcriptases from human immunodeficiency virus and Moloney murine leukemia virus.

Phosphonylmethoxyethyl purine derivatives, a new class of anti-human immunodeficiency virus agents.

A study of the structure-activity relationship of a series of newly synthesized phosphonylmethoxyalkyl purine and pyrimidine derivatives revealed that several adenine derivatives substituted at the N9 position by a 2-phosphonylmethoxyethyl (PME) group inhibited human immunodeficiency virus (HIV)-induced cytopathogenicity and HIV antigen expression in vitro at concentrations significantly below the toxicity threshold for the host cells. In terms of anti-HIV potency in MT-4 cells, the PME 2,6-diaminopurine derivative (50% effective dose [ED50], 1 microM) ranked first, followed by the PME adenine derivative (ED50, 2 microM [MT-4]) and the PME 2-monoaminopurine derivative (ED50, 45 microM). Antiretroviral activity was also demonstrated in ATH8 and H9 cells, which were de novo infected with HIV, and extended to C3H mouse fibroblasts infected with Moloney murine sarcoma virus. Unlike 2',3'-dideoxyadenosine, these compounds were not found to be degraded by deaminases derived from bovine intestine.

Butyrate prevents Harvey Sarcoma virus focus formation but permits oncogene expression.

Since butyrate inhibits DNA synthesis and cell growth, we adapted NIH 3T3 cells to grow in butyrate to study its effects on retroviral cell transformation. In cultures of butyrate-adapted NIH 3T3 cells infected with Harvey Sarcoma Virus, no foci were formed; however, there was evidence of retroviral replication, and the P21 oncogene product was demonstrated by immunofluorescence in its characteristic localization beneath the cytoplasmic membrane of single cells and pairs of cells. When the butyrate was removed, these cells proliferated to form foci. Butyrate therefore does not prevent oncogene expression but does reversibly inhibit focus formation.

Synthesis and biological investigations of new tuftsin analogs with elongated peptide chain.

In this paper the synthesis of the following elongated tuftsin analogs: Thr-Lys-Pro-Lys-Thr-Lys-Pro-Lys (I), Thr-Lys-Pro-Lys-Thr-Lys-Pro-Arg (II) and Ala-Lys-Thr-Lys-Pro-Arg-Glu-Gln (III) by the classical method is described. The compound II markedly inhibited the growth of murine sarcoma viruses (MSV).

The mechanism of interferon effect on cell transformation by murine sarcoma virus.

Mouse interferon (IFN) was found to inhibit murine sarcoma virus (MSV)-induced neoplastic transformation of normal rat kidney (NRK) cells. This effect was observed upon examining the formation of foci of morphologically altered cells and colonies of anchorage-independent cells. IFN had no cytotoxic effect on MSV-transformed NRK cells, nor on their focus or colony-forming ability. It was therefore apparent that its inhibitory effect was directed against the viral role in cell transformation. In attempts to define the mechanism of this effect, we found that IFN delayed the initiation of the cytoplasmic viral DNA synthesis. However, the amount of this DNA eventually formed in IFN-treated cells was the same as in the control cells. Furthermore, the transport of this DNA to the nucleus was slower in IFN-treated cells, although all of it was finally transferred. However, while most of the viral DNA integrated into the genome of the control cells, very little integration occurred in IFN-treated cells. The unintegrated viral DNA of these cells was slowly degraded. Therefore, if the cells recovered from the antiviral effect of IFN when intact viral DNA molecules still existed in their nucleus, they could resume viral DNA integration and cell transformation. IFN was found to block viral DNA supercoiling. Since supercoiled viral DNA is considered to be a precursor to integrated provirus, it seems that the inhibition of both integration and cell transformation is due to this impaired coiling.

2-l-Rhamnopyranosyl[1,2,4]triazolo[1,5-a]pyridine. 4' and 3' Oxidation products. Synthesis and structure-activity relationships.

A series of 2-alpha-L-rhamnopyranosylnitro[1,2,4]triazolo[1,5-a] pyridine C-nucleosides was synthesized from the condensation oa thioiminoether with nitro-2-pyridylhydrazines. Catalytic reduction afforded the corresponding amino derivative. A 1',2' unsaturated C-nucleoside was also obtained by two different routes. Selective oxidation gave the 3'- and 4'-ketonucleosides. The cytotoxic properties of the nucleosides, as well as their effect on viral transformation and replication, were described. The nitro derivatives inhibit viral replication, but at toxic doses; the introduction of a keto function leads to a product which inhibits the replication of murine leukemia virus (MuLV) at noncytotoxic concentrations. The amino derivatives have no significant antiviral effect.

Inhibition of endogenous murine retrovirus expression by L-beta-3,4-dihydroxyphenylalanine (L-dopa) methyl ester.

Induction of endogenous xenotropic type-C virus from Kirsten sarcoma-virus-transformed BALB/c (K-BALB) mouse cells was inhibited by short-term exposure to L-beta-3,4-dihydroxyphenylalanine (L-dopa) methyl ester. Partially synchronized cells cultured for 1-4 h with 0.8-1.6 mM L-dopa methyl ester and subsequently induced with 5-iododeoxyuridine (IdUrd), cycloheximide and histidinol showed inhibition of virus activation. Incorporation of thymidine, uridine and leucine was measured at the end of the drug treatment and during the subsequent induction period. L-dopa methyl ester had a pronounced effect on DNA synthesis, reducing it by more than 85% during a 4-h incubation period, whereas RNA and protein synthesis remained largely unaffected. Removal of the drug and replacement with fresh medium did not reverse DNA synthesis or virus activation during the subsequent induction interval. L-dopa methyl ester was also shown to potentially function as an analogue of tyrosine in this cell system. These results suggest that inhibition of virus induction may be caused by inhibiting the normal progressing of cells through the S phase of the cell cycle.

Effect of sodium butyrate on the antiviral and anticellular action of interferon in normal and MSV-transformed cells.

Sodium butyrate enhances the antiviral and anticellular action of interferon in MSV-transformed cells but has no such effect in normal cells. The increased sensitivity could be mediated at least partially by the butyrate-induced elaboration of the microtubule and microfilament network.

Antiviral and anticellular effects of interferon on the mouse embryonic cells transformed by viruses and/or chemical carcinogen.

The antiviral and anticellular activity of partially purified mouse interferon has been tested in cell lines transformed with Simian Virus 40 (MEB-SV 40), mouse sarcoma virus (Harvey strain) (MEB-MSV), and 20-methylcholanthrene (MEB-MCH), respectively. The transformed lines were derived from C3H mouse embryonic primary cells. It has been shown that the MEB-MSV cells were 10 to 50 times less sensitive to the antiviral effect of interferon than the MEB-SV 40 or MEB-MCH cells. A 30% reduction of the number of treated cells as compared with untreated control cells was taken as basis for comparison of anticellular activity of interferon in transformed lines. While the MEB-MCH cells required 1000 units of interferon for a 30% growth inhibition, about 3000 units were necessary for a comparable suppression of MEB-SV 40 cells and/or MEB-MSV cells.

Inhibition of murine sarcoma virus-induced foci formation by cytidine analogues and other drugs chemotherapeutically effective in human malignancies.

The present report describes an in vitro culture system using the Kirsten murine sarcoma virus transformation focus assay to evaluate the antiviral activities of 12 commonly used chemotherapeutically effective drugs. Since these drugs are cytotoxic, the plating efficiencies of the cells treated were monitored simultaneously. Using this procedure, Adriamycin, Daunorubicin, Bleomycin Camptothecin, Mithramycin, hydroxyurea, 5-fluorouracil, thioguanine, 9-beta-D-arabinofuranosyladenine, 1-beta-D-arabinofuranosylcytosine (ara-C), azacytidine and cyclocytidine were tested. Of all these compounds tested only the cytidine analogues - cyclocytidine, ara-C and, to a lesser extent, azacytidine - showed selective effect on inhibition of viral foci over cytotoxicity. Studies on the duration of exposure to ara-C indicated that an exposure time of 10-30 h produced the most pronounced effect on the inhibition of foci formation over that of cytotoxicity.

Glucocorticoids induce focus formation and increase sarcoma viral expression in a mink cell line that contains a murine sarcoma viral genome.

Dexamethasone (3 X 10(-10) to 3 X 10(-6) M) induced foci of morphologically transformed cells in a small proportion of a mink cell line that contains the Moloney murine sarcoma viral genome (S+L-). The induction was glucocorticoid specific, since other steroids with glucocorticoid activity (prednisolone, cortisol, and aldosterone) induced foci with an efficiency that paralleled their glucocorticoid activity, and steroids lacking glucocorticoid activity (17B-estradiol, testosterone, and progesterone) failed to induce foci. Viral antigen, as measured by specific immunofluorescence, was localized to the foci. The induction of foci by dexamethasone (3 X 10(-7)) was accompanied by an approximately 10-fold increase in intracellular Moloney murine sarcoma virus-specific RNA and viral p30 antigen. Removal of dexamethasone was followed by the disappearance of foci and a decrease in viral RNA and p30. In this cell system, therefore, glucocorticoids can affect the intracellular levels of type C viral RNA and protein.

3-N-Substituted aminomethyl derivatives of rifamycin SV. A convenient method of synthesis, cyclization of certain derivatives, and anticellular and antiviral activities of several derivatives.

A new synthesis of Mannich bases of rifamycin SV using the Borch2 procedure with rifaldehyde is described. This new synthesis offers two advantages over the previously published method. It provides a route to monoalkyl-aminomethylrifamycins (le-h) and to unsubstituted aminomethylrifamycins that were not accessible by the old procedure. The new method also offers a preparative route to Mannich bases 1a and 1b were needed in multigram quantities for biological testing. In addition, the cyclization of certain of the monoalkylaminomethylrifamycins to the novel N,15-didehydro-15-epi[methano(alkylimino)]rifamycin SV derivatives (2) is described. The anticellular and antiviral effects of representatives of both series of compounds against cultured mouse cells and murine oncornavirus are are discussed.

Interruption of oncornavirus replication by modified rifamycin antibiotics.

Thirteen rifamycin SV derivatives containing 3'-alkylaminomethyl substituents fail to inhibit the activities of the simian sarcoma virus Type 1 DNA polymerase, and of cellular DNA, RNA, and poly(A) polymerases prepared from NIH Swiss mouse embryos. These compounds show a range in their toxicities for NIH Swiss mouse 3T3 cells and in their capacities to inhibit production of foci of morphologically altered cells by murine sarcoma virus (MSV). Three compounds--the N-methyl-N-hydroxyethylaminomethyl, the N,N-dimethyl-aminomethyl, and the N4-methylpiperazinomethyl rifamycin derivatives--are comparable to adenine arabinoside and ribavirin in their toxicity for 3T3 cells, but these compounds show superior focus inhibition. These compounds inhibit oncornavirus production apparently by exacerbation of a delay in growth that results from infection of 3T3 cells with MSV.