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Suligovir is a 35-mer homo-oligonucleotide, containing exclusively 4-thio deoxyuridylate, proved to be a potent inhibitor of HIV entry. In this paper, we described the effect of extent of thiolation and the introduction of nuclease-resistant phosphorothioate linkages on the anti-HIV activity of Suligovir. We found that the decreased thiolated nucleotide content decreases the anti-HIV potency of the compound and the introduction of phosphorothioate linkages does not improve its antiviral activity.

We have previously reported the potent in vitro HIV-1 anti-reverse transcriptase activity of a 35-mer of 4-thio-deoxyuridylate [(s(4)dU)(35)]. In efforts to define its activity in a more physiological system, studies were carried out to determine the stage of viral infection that this compound mediates its anti-viral effect. Results of the studies reported herein show that (s(4)dU)(35) is nontoxic and is capable of inhibiting both single and multi-drug resistant HIV strains (IC(50): 0.8-25.4 microg/ml) in vitro. Besides its previously reported anti-RT activity, (s(4)dU)(35) mediated its antiviral action by preventing virus attachment (IC(50): 0.002-0.003 microg/ml), and was stable in vitro and slowly degraded by DNAses. Competition studies and fluorescence resonance energy transfer (FRET) experiments indicated that (s(4)dU)(35) preferentially binds to CD4 receptors, but not to CD48. Confocal laser scanning microscopy (CLSM) studies showed that (s(4)dU)(35) did not penetrate into the cells and colocalized with cell surface thioredoxin. Our studies identify (s(4)dU)(35) as a potential novel HIV entry inhibitor that may have utility as either a systemic antiretroviral or as a preventing agent for HIV transmission.

Double-stranded RNAs induce interferons and cause the development of antiviral and antiproliferative activities. Antiviral activity is related to the production of interferons and other proteins that stimulate various immunologic activities, which appear to contribute to their overall antiproliferative activity. The most active double-stranded RNA, polyI:polyC, was shown to be too toxic for therapeutic use. We conducted selective thiolation of the polyC strand at the five position of the cytosine bases, generating a partially thiolated polyC (MPC) which after annealing with a complimentary unmodified polyI, gave the thiolated double-stranded RNA, pI:MPC. We have explored antiviral and antiproliferative activities at various levels of thiolation and found that optimal responses can be obtained at 7.4% level of thiolation. This compound deserves further study of antiviral and antiproliferative responses in vivo, and eventually clinical exploration. Earlier studies have shown that this and related compounds are active against HIV-1, in human cells, and against DNA polymerases of DNA and RNA tumor viruses.

This is a brief review on studies of attacking HIV through a new angle. In previous studies, we have found that many patients with AIDS are resistant to interferon (IFN) therapy, and some develop resistance during therapy. Four factors were found to be responsible for the resistance of untreated patients: (a). release of free-circulating IFN-alpha/beta type 1 receptors, (b). a newly detected IFN inhibitory protein, (c). high prostaglandin E2, and (d). high levels of cAMP phosphodiesterases, particularly in AIDS-related neoplasms. This may interfere with intrinsic disease resistance and with the efficacy of IFN therapy. In an attempt to overcome this resistance, new compounds were synthesized which increase endogenous production of alpha, beta and gamma IFNs, have anti-template activity against DNA and RNA polymerases, inhibit reverse transcriptases and activates IFN-induced double-stranded RNA (dsRNA)-dependent protein kinase. It is expected that planned nonhuman primate and clinical studies will support preliminary findings. Preliminary in vitro and animal studies suggest that these new compounds may be effective against HIV, including multi-drug resistant strains.

A polymerase chain reaction (PCR)-based radioactive telomerase assay was developed in our laboratory which is quantitative and does not require electrophoretic evaluation (designated as TP-TRAP; it utilizes two reverse primers). The main steps of the assay include (1) extension of a 20-mer oligonucleotide substrate (MTS) by telomerase, (2) amplification of the telomerase products in the presence of [(3)H]dTTP using the substrate oligonucleotide and two reverse primers (RPC3, 38 mer; RP, 20 mer), (3) isolation of the amplified radioactive dsDNA by precipitation and filtration, (4) determination of the radioactivity of the acid-insoluble DNA. The length of the telomerase products does not increase on amplification. This valuable feature of the assay is achieved by utilization of the two reverse primers and a highly specific PCR protocol. The assay is linear, accurate, and suitable for cell-biological studies where slight quantitative differences in telomerase activity must be detected. The assay is also suitable for screening and characterization of telomerase inhibitors, as shown with a chemically modified oligonucleotide reverse transcriptase inhibitor [(s(4)dU)(35)]., (Copyright 2000 Academic Press.)

Partially thiolated analogs of the biological response modifier poly I.poly C (pI.pC) were synthesized. Each of these analogs (pI.MPC) contained a partially thiolated polycytidylate (MPC) strand containing either 1.2%, 4.6%, or 17% 5-mercaptocytosine (%SH) randomly introduced throughout the polynucleotide. The ability of these double stranded RNAs (dsRNAs) to activate murine peritoneal macrophages in vitro and augment natural killer (NK) cell activity in mice following intraperitoneal (i.p.) administration was determined. Macrophages were treated in vitro for 24 hours with pI.pC, or pI.MPC, washed, and then overlayed with exponentially growing L1210 leukemia cells at an effector to target (E:T) ratio of 25:1. The cytostatic effect of the macrophages on the L1210 cells was determined by 3H.thymidine pulse labelling. The rank order of potency for macrophage activation was determined to be: pI.pC>pI.MPC(1.2% SH)>pI.MPC(4.6% SH)pI.MPC(17% SH). Twenty hours following i.p. administration of 5 mg/kg of each pI.MPC analog, splenic NK cell activity was assessed in a standard 51Cr release assay using the murine tumor target cell line YAC- 1. The rank order of potency observed for NK cell activation was determined to be; pI.pCpI MPC(1.2% SH)>pI.MPC(4.6% SH)>pI.MPC(17% SH). These dsRNAs activated NK cells in a dose dependent manner. The efficacy and time course for NK cell activation following i.p. administration of pI.MPC (1.2% SH) at a dose of 10 mg/kg was directly compared to an equivalent 10 mg/kg i.p. dose of pI.pC. NK cell activation took place within three hours following treatment with pI-pC whereas the onset of NK cell activation by pI.MPC (1.2%) occurred between 8 and 20 hours post treatment. NK cell activity steadily declined from 24 to 50 hours post treatment at which time the NK activity in both treatment groups was similar. There was a significant correlation between the immunostimulatory potency of these dsRNAs and their experimentally determined melting temperatures (r2 = 0.88) and percent hyperchromicity upon thermal denaturation (r2 = 0.99). At the lower %SH, pI.MPC retains most of the immunostimulatory activities of p1.pC and may serve as a useful and potent biological response modifier.

Introduction of a reactive 5-mercapto group into some of the cytosine and/or uracil bases of various oligo- and polynucleotides by partial thiolation resulted in several potent inhibitors of the replication of human immunodeficiency virus type 1 (HIV-1) in primary human lymphocytes. These compounds exhibited little if any toxicity against uninfected peripheral blood mononuclear cells and showed 15 to 75 times higher antitemplate activity against a p66/p51 HIV-1 recombinant reverse transcriptase (RT) than against the DNA polymerase alpha from human lymphocytes. In contrast, the unthiolated oligo- and polynucleotides are void of antitemplate activity, and their apparent inhibitory effect on HIV-1 closely paralleled their toxicity for the cells. Partially thiolated poly(dC) (MPdC) was the most potent of all the compounds tested against HIV-1 in peripheral blood mononuclear cells (50% effective concentration, 1.8 micrograms/ml or 0.019 microM), while showing low cytotoxicity (greater than 100 micrograms/ml). The corresponding unmodified poly(dC) showed no anti-HIV-1 activity at 50 micrograms/ml but had pronounced cytotoxicity. MPdC was also a potent inhibitor of HIV-1 RT (50% inhibitory concentration, 0.30 micrograms/ml). The inhibitory activities of thiolated homooligo(dCs) against both HIV-1 replication and HIV-1 RT increased with increasing chain length. The heterooligonucleotides included in this study were designed as structural analogs of portions of the natural primer of HIV-1 RT, i.e., tRNA(3Lys). An 18-mer analog of the 3' terminus, complementary (antisense) to the primer-binding site of the HIV-1 genome, was attached to an oligo(dC) tail and 5-thiolated; this increased its activity and decreased its toxicity. This compound will serve as a new lead in the development of more effective antitemplates against HIV-1.

In view of the in vivo demonstrated radiation-potentiating activities of several previously studied 2,2-dimethylphosphoraziridines, six new compounds incorporating the bis(2,2-dimethyl-1-aziridinyl)phosphinyl moiety, together with an electron-affinic group such as 2-nitroimidazole or nitrobenzyl, have been synthesized and tested (1) in vitro for ability to increase the effect of X-irradiation under hypoxic conditions on V-79 Chinese hamster lung fibroblast cells, (2) in vivo for antitumor activity in the absence of radiation against P388 leukemia in mice, and (3) in a preliminary experiment with compound 10 only, in combination with whole-body gamma-radiation, using the P388 leukemia mouse model for in vivo radiation-potentiating activity. The chemical-alkylating activities and hydrolytic behavior of these compounds, as well as their antitumor activities without radiation, were found to be comparable to those of other 2,2-dimethylphosphoraziridines, while their in vitro radiosensitizing activities were at low concentrations generally comparable to that of misonidazole, with compound 8 showing superior activity. At higher concentrations, only compound 10 was sufficiently soluble and nontoxic to the cells for evaluation in this assay. Thus, the bis(2,2-dimethyl-1-aziridinyl) phosphinyl moiety does not seem to have contributed to the hypoxic radiosensitizing activities (only to the cytotoxicities) of the electron-affinic moieties in this in vitro assay. In comparison, the prototype 2,2-dimethylphosphoraziridine, ethyl [bis(2,2-dimethyl-1-aziridinyl) phosphinyl]carbamate (AB-132), showed at nontoxic doses no radiosensitizing activity in this assay, and at cytotoxic doses increased the cell-killing effect of each given dose of X-radiation additively under both hypoxic and oxic conditions. Conversely, only the 2,2-dimethylphosphoraziridine moiety appeared to participate in the moderate "therapeutic radiation-potentiating" activity indicated by compound 10 in the in vivo experiment using the P388 leukemia model (on day 1), as the misonidazole standard was inactive in this nonhypoxic system. Clearly, the mechanism of the in vivo observed radiation-potentiating effect of AB-132 and other 2,2-dimethylphosphoraziridines is different from that of the hypoxic radiosensitizers, but the possible synergism between the two biologically active moieties of the new compounds could not be demonstrated with the experimental models so far employed.

The chemical alkylating activities of four prototype phosphoraziridine antineoplastic agents were compared with their biological effects on V-79 Chinese hamster lung fibroblasts. It was found that the chemical reactivity patterns correlate well with all of the biological parameters examined in this study, i.e. cytotoxicity, DNA synthesis, and production of alkali labile strand breaks. Specifically, the 2,2-dimethylaziridine derivatives (AB-132 and AB-163) showed higher initial activities reaching a plateau after a short reaction time in all of the systems used in this study while the unsubstituted aziridine derivatives (AB-100 and D-63) reacted more slowly but continued to exert their action in a linear fashion to produce greater overall effects. These findings are consistent with the conclusion that the difference between the time-dependent biological activities of these drugs closely follows the different chemical mechanisms of their alkylating reactions (SN1 vs SN2). The more rapid action and subsequent hydrolytic inactivation of the 2,2-dimethylphosphoraziridines as effective alkylators could be the basis of their lower hemopoietic toxicity compared to conventional alkylating agents including their own C-unsubstituted aziridine analogs. The much more rapid action of the 2,2-dimethylphosphoraziridines on DNA inside the cell may have some bearing on their radiation potentiating activity, but this aspect and the cholinesterase inhibitory activity of these agents (which may depend on phosphorylation) were not investigated in the present study.

Partially thiolated polycytidylic acid (5-mercaptopolycytidylic, MPC) and its double-stranded complex with polyinosinic acid [poly (I)].poly(I).MPC, were assayed in both antiproliferative and cytotoxicity tests against human cell lines: lung carcinoma A549, colon carcinoma HT-29, osteosarcoma HOS, and amnion cells (WISH). Inhibitory effects of MPC were noted in the antiproliferative assay with ID50 of 7, 24, 33, and 35 micrograms.ml-1, and in the cytotoxicity test with ID50 of 164, 174, 210, and 290 micrograms.ml-1 against the HOS, A549, HT-29, and WISH cells respectively. Comparison with the corresponding partially thiolated mononucleotide (5-mercapto-CMP + CMP) and the nucleoside (5-mercapto-cytidine) demonstrated that MPC was a more potent antiproliferative agent than either of its monomeric constituents. The inhibitory effect of MPC upon the incorporation of [3H]thymidine into the DNA of growing A549 cells paralleled its antiproliferative activity.

With the aim of increasing the selectivity of the 2,2-dimethylphosphoraziridine type antitumor agents toward the intracellular site of DNA synthesis, a series of new compounds was synthesized in which the reactive bis(2,2-dimethyl-1-aziridinyl)phosphinyl (2,2-DMAP) group was linked through a carbamate or amide linkage to thymidine or cytosine nucleoside moieties. The 3'- and 5'-(2,2-DMAP)carbamates of thymidine (1 and 2) were found to be highly unstable, therefore the corresponding O-acetyl derivatives 5 and 6 were prepared by reacting 5'- and 3'-acetylthymidine, respectively, with dichloroisocyanatophosphine oxide followed by the addition of 2,2-dimethylaziridine and triethylamine. The 3'- and 5'-(2,2-DMAP)amides of thymidine 14 and 15 were prepared by reacting the appropriate thymidinylamines with bis(2,2-dimethyl-1-aziridinyl)phosphinyl chloride (17). The N4-(2,2-DMAP)amides of cytidine, 2'-deoxycytidine, and cytosine arabinoside (18, 19, and 20, respectively) were prepared by reacting the hydrochlorides of the O-peracetylated cytosine nucleosides with triethylamine and POCl3 and, subsequently, with 2,2-dimethylaziridine and triethylamine, to give the corresponding N4-(2,2-DMAP)cytosine nucleoside peracetates 21, 22, and 23, respectively, which were then deacetylated by aminolysis. However, the peacetate intermediates were found to be more stable and, probably for the same reason, also more active against P388 leukemia in mice than the deacetylated products. Particularly, 22 and 23 showed sufficient activity in this in vivo assay system to warrant further evaluation. The relationships between the antitumor activities, the chemical alkylating activities, and the cholinesterase inhibitory activities of these agents are discussed.

1,3,4,6-Tetra-O-acetyl-2-(di-2-chloroethyl)amino-2-deoxy-D-glucopyranose is active against L1210 leukemia, giving over 100% increased life-span at optimal dose. Against P388 leukemia, it gives 200% increased life-span with long-term survivors. The compound is most active when given i.p., but shows some activity when given s.c. than p.o., and is more potent (therapeutic and toxic effect) than mechlorethamine on both a molar and a mg basis. Of importance, the schedule dependency for the administration of 1,3,4,6-tetra-O-acetyl-2-(di-2-chloroethyl)amino-2-deoxy-D-glucopyranose in L1210 leukemia differs from most alkylating agents in that it is best given by multiple daily injections rather than as a single large injection on Day 1. This characteristic can be attributed to the amino-glucose moiety.

Thiolation of position 5 of some of the cytosine bases in polycytidylic acid results in the formation of mercaptopolycytidylic acid (MPC). Annealing of MPC to polyinosinic acid (Poly I) results in the formation of double-stranded Poly I-MPC. In this study we investigated the interferon inducing ability, in vivo toxic effects, effect on DNA synthesis, and the effects in human tumor cell lines of Poly I-MPC. Poly I-MPC was capable of inducing human alpha, beta and gamma interferons in the appropriate cell systems. In vivo toxicity was measured in mice, guinea pigs, and rabbits according to FDA guidelines. Weight loss and lethal and pyrogenic effects were markedly lower in Poly I-MPC treated animals than in those that received unmodified Poly I-Poly C. In contrast to the lack of an effect of Poly I-Poly C in human lymphocytes, Poly I-MPC inhibited DNA synthesis. It also inhibited colony formation and was cytotoxic in several human tumor cell lines. Poly I-MPC's ability to induce human alpha, beta and gamma interferons, to inhibit DNA synthesis and its effects in human tumor cell lines demonstrate the potential of this drug for future clinical studies, both as an antiviral and antitumor agent.

1-(2-Deoxy-beta-D-ribofuranosyl)-5-bromo-2-pyrimidinone (BrPdR) and 1-(2-deoxy-beta-D-ribofuranosyl)-5-iodo-2-pyrimidinone (IPdR) have been synthesized by condensation of the appropriate silylated bases 2a and 2b, respectively, with 3,5-bis-O-(p-chlorobenzoyl)-2-deoxy-alpha-D-ribofuranosyl chloride (8) in 1,2-dichloroethane, in the presence of SnCl4, followed by separation of the anomeric blocked nucleosides via column chromatography and subsequent deprotection with methanolic ammonia. Both BrPdR and IPdR exhibited significant antiherpes activities against various strains of HSV-1 and HSV-2, the latter compound (IPdR) showing the higher activity as well as the stronger binding to the virus-specific thymidine kinase.

1-(2-Deoxy-beta-D-ribofuranosyl)-5-(methylmercapto)-2-pyrimidinone (1b) was synthesized via modification of the silyl method. 1b inhibits the Herpes simplex virus type 1 (98%) and type 2 (97%) at a concentration which is nontoxic to human HeLa cells. The compound shows 50 times greater binding affinity (lower Ki) to the virus-specific thymidine kinase than to the thymidine kinase of uninfected HeLa cells.

Radioactive (35S-labeled) partially thiolated polycytidylic acid (MPC) was administered i.v. to male Sprague-Dawley rats. Blood samples were taken at various intervals, and the radioactivity in plasma was determined. The concentration of total radioactivity in plasma decreased rapidly postinjection, independently of the dose, and could not be readily resolved into a series of exponential terms with a high degree of confidence. Coadministration with polyinosinic acid in a 1:1 ratio significantly decreased the clearance of radioactive compounds from the plasma; moreover, the clearance of radioactivity decreased with increasing dose. Complexing with polyinosinic acid also decreased the rate of degradation of [35S]MPC as evidenced by an increase of the trichloroacetic acid-precipitable fraction (i.e., oligonucleotides larger than five to ten nucleotide units), from 0.45 to 0.92 of the total radioactivity in plasma 60 min postinjection. The plasma clearance and organ distribution of radioactivity following injection of [35S]MPC were determined in normal and leukemic RFM/Un mice. About 90% of the 35S radioactivity was removed from the plasma in 5 and 10 min, respectively, in these two groups of mice, and the residual plasma levels of radioactivity at any given time were twice as high in the leukemic group throughout an observation period of 1 hr. Organ distribution studies demonstrated significantly greater (per mg tissue) accumulation of radioactivity in the livers and spleens of the leukemic versus normal mice at all time points, while the corresponding data for the kidneys were similar for the two groups. Another study, comparing the radioactivity in suspended and washed spleen cells harvested 60 min postinjection, indicated that 4 to 10 times more MPC and/or 35S-labeled oligonucleotides were localized and bound intracellularly in the spleens of the leukemic mice. These studies of the pharmacokinetic properties and metabolic degradation of [35S]MPC suggest that this polynucleotide may be protected from degradation by complexing with polyinosinic acid and that preferential accumulation of [35S]MPC occurs in organs infiltrated by leukemic cells.

Patients with bronchogenic carcinoma or with advanced pediatric solid tumours were treated with various radiation therapy schedules alone or with a new alkylating carbamate: AB 132. Drug therapy appeared to potentiate radiation therapy results without significant toxicity except for certain neurologic symptoms (myasthenia, nightmares, disturbances of taste and smell). The latter appeared to be related to acetylcholinesterase and pseudocholinesterase inhibition.

Several 5-substituted 2-pyrimidinone 2'-deoxyribonucleoside (PdR) analogs were examined for their anti-herpes simplex virus (HSV) activity in cell culture. The order of potency of their antiviral activities against HSV type 1 (HSV-1) and HSV-2 was iodo PdR approximately ethynyl PdR approximately propynyl PdR. The antiviral action of iodo PdR is dependent on the ability of HSV to induce virus-specified thymidine kinase in infected cells. Several HSV-1 variants with altered thymidine kinase changed their sensitivity to iodo PdR, whereas HSV-1 variants with altered DNA polymerase were as sensitive as the parental virus to iodo PdR. Continuous presence of iodo PdR for more than one virus replication cycle was required for optimal antiviral activity. Iodo PdR (100 microM) had no activity against Epstein-Barr virus DNA replication in P3HR-1 cells. With an oral, an intraperitoneal, or a subcutaneous route of injection, iodo PdR administered twice a day for 2.5 days could prevent the death of mice infected with HSV-2. This in vivo activity is unlikely to be related to the potential conversion of iodo PdR to iododeoxyuridine, since iodo PdR is not a substrate of xanthine oxidase.

A series of oligomers of cytidylic acid were prepared and partially (6-9% of the bases) thiolated in the 5 positions. The modified oligomers showed increasing inhibition with increasing chain length of both the DNA polymerase-alpha from regenerating rat liver and the DNA-dependent RNA polymerase of E. coli, but the minimum chain length for observable inhibitory activity was 5 nucleotide units for the DNA polymerase-alpha and 16 units for the RNA polymerase.

Reaction of 3'-acetylthymidine with phosphorus oxychloride in trimethyl phosphate yielded the phosphorodichloridate 5, which was subsequently reacted with aziridine, or 2,2-dimethylaziridine to give compounds 6 and 7, respectively. The 2,2-dimethylaziridine derivative 7 was considerably more active than 6 against leukemia L1210 and P-388 in mice but less active than the previously synthesized, simpler phosphinate derivatives 2 and 3. It appears that the thymidine moiety did not enable these compounds to use the nucleoside transport mechanism of the cells and also failed to increase the selectivity of the 2,2-dimethylaziridine analogues by interference with their binding to cholinesterase. Compound 7 strongly inhibited horse serum cholinesterase, while 6 was inactive.

Ethyl-N-bis (2,2-dimethylethylamidinophosphoro) carbamate (AB-132) has been shown to potentiate the effect of whole-body radiation on inhibition of splenomegaly induced by Friend leukemia virus in ICR/H Swiss mice. The combined effect of AB-132 and radiation does not appear to be related to Friend virus inhibition but seems to act on the proliferating tumor in the spleen. Nine children with advanced cancer were treated with combined administration of local radiation and systemic AB-132. Although regression of tumor was seem, no dramatic effect on survival was apparent. One case of metastatic Ewing's sarcoma showed systemic tumor response to AB-132 in addition to localized response to radiation. Bone marrow depression appeared to be the main side effect of combination therapy.

Partially thiolated polycytidylic acid (MPC), an antileukemic agent, when administered to leukemic RF/UN mice inhibited the clonogenicity of bone marrow progenitor cells in a time- and dose-dependent manner. The effect of a single dose of MPC disappeared within 40 hr due to the rapid degradation of this compound in mice. When MPC was encapsulated in liposomes before injection, its activity at 19 hr after inoculation was similar to that of free MPC. The inhibitory effect of this liposome-MPC complex, however, persisted for at least 40 hr, indicating that the MPC was protected from hydrolysis by the nucleases present in blood. Drug-free liposomes increased the number of clonogenic progenitor cells, whereas a mixture of plain liposomes and MPC decreased the number of clonogenic cells to a greater extent than did MPC alone or MPC within liposomes. A possible explantation for these observations is that the liposomes per se altered the clearance function of the reticuloendothelial system and completed with MPC for uptake by the reticuloendothelial system cells, thereby resulting in increased plasma levels of MPC which in turn resulted in greater killing of the target cells.

The effects of 5-mercapto-2'-deoxyuridine (MUdr) on DNA synthesis in a primary murine spleen lymphocyte culture system stimulated by phytohemagglutinin (PHA) were studied. Inhibition of thymidine incorporation into acid insoluble nucleic acid material was 50% at 0.5 mM MUdR concentration, while inhibition of deoxyuridine incorporation into acid-insoluble nucleic acids was 50% at 0.01 mM MUdR. Time course studies, at 0.5 and 0.05 mM MUdR, showed that the magnitude of inhibition of incorporation for thymidine and deoxyuridine, respectively, increased from a time point after PHA stimulation when increased synthesis of thymidine kinase and thymidylate synthetase had leveled off. At 1 mM MUdR, total cellular DNA in cultures was decreased 43% at 42 hr after PHA stimulation. Neither the total number of cells nor the percentage of PHA-transformed cells was decreased in comparison to that of controls. MUdR therefore blocks the increase in DNA content of lymphocytes that is initiated during the S phase of the cell cycle. Millimolar levels of MUdR inhibited incorporation or uridine, adenosine, and cytidine into acid-insoluble material in pha-stimulated primary murine lymphocyte cultures. Total cellular RNA synthesis was inhibited at these levels of MUdR, with no differential effects on 4, 18, or 28 S RNA species observed. Uptake of these nucleosides into the total cellular acid-soluble material was not blocked. Uptake of different labeled nucleosides into cellular, acid-soluble pools occurs at different rates. Thus, choice of a suitable minimum pulse time to achieve saturation for different labeled nucleosides must relate to this consideration. Thymidine kinase from whole-cell sonic extracts of PHA-stimulated lymphocytes was inhibited 65% by 1 mM MUdR at 24 and 48 hr after stimulation. Uridine kinase extracted from the PHA-stimulated cells was also significantly inhibited by 1mM MUdR at 24 hr (56%). Exogenous guanosine incorporation into lympohcyte acid-insoluble material is increased by MUdR. This increased utilization of exogenous nuceloside is apparently the result of MUdR inhibition of conversion of adenosine to guanine nucleotides within the lymphocytes and a consequent diminution of the total intracellular guanine nucleotide pool size. The active inhibitory compound is the deoxyribonucleoside or deoxyribonucleotide. Comparison with the riboside analog 5-mercaptouridine showed that MUdR was a more efficient inhibitor of nucleoside incorporation.

The synthesis of the 2,4-diamino analogue of riboflavin is described. Inhibition analysis in a microbial assay system indicated that this compound has a weak antifolate activity that could be overcome with a minimal amount of folinic acid, but at higher concentrations both folinic acid and riboflavin were required for the reversal of its inhibitory effect.

Partially thiolated polycytidylic acids MPC I-III, containing 1.7%, 3.5% and 8.6% 5-mercaptocytidylate units, respectively) inhibited the DNA polymerase of Friend leukemia virus (FLV) in the endogenic reaction as well as in the presence of poly(A)-(dT)14 or poly[d(a-T)] templates; the inhibitory activities were directly related to the percent of thiolation. Various partially thiolated RNA and DNA isolates from Ehrlich ascites cells (containing one 5-mercaptopyrimidine nucleotide/50-100 nucleotide units) also inhibited the DNA polymerases of FLV in the endogenic reaction, and also in the presence of the synthetic templates. The thiolated DNA was the most active, but the thiolated tRNA also showed substantial inhibitory effects, while the thiolated ribosomal RNA was less effective. In a bacterial DNA polymerase (E. coli-K12, using denatured DNA as template), MPC I-III showed no activity. By contrast, MPC III and several partially thiolated nucleic acid isolates significantly inhibited a regenerating rat liver DNA polymerase (I) system; among those tested, the thiolated DNA from Ehrlich ascites cells showed the highest activity. Kinetic analysis of the inhibitory action of this thiolated DNA in the rat liver enzyme system, using as template the corresponding unmodified DNA, demonstrated that the thiolated DNA acts as a competitive inhibitor of the template, with a Ki/Km ratio of 0.5.

The synthesis of 4-amino-4-deoxy-N10-methylpteroyl-(6-diazo-5-oxo)-L-norleucine and 4-amino-4-deoxy-N10-methylpteroyl-(6-chloro-5-oxo)-L-norleucine, analogs of methotrexate in which the gamma-carboxyl group is replaced by a diazoketone and a chloromethylketone, respectively, was carried out. The analogs inhibited the growth of leukemia L-1210 cells in culture by 50% at 4 X 10(-7) M and 2 X 10(-7) M, respectively, and were effective inhibitors of the synthesis of thymidylate in L-1210 cells in vitro (I50 = 3 X 10(-6) M), exhibiting significant antifolate activity. The results demonstrated the feasibility of introducing chemically reactive groups at the gamma-position of pteroyl glutamates with retention of biological activity. However, in the systems investigated thus far, there was no evidence of covalent bond formation due to these reactive groups at the active sites of the enzymes.

In continuation of efforts to correlate the antitemplate activities of chemically modified polynucleotides with their base composition and structure, four synthetic copolymers, poly(A,C), poly(C,U), poly(A,C,U), and poly(A,C,G) were modified by thiolation of 2.6-4.8% of their pyrimidine bases. The resulting 5-mercaptoheteropolynucleotides and the previously described 5-mercapto-polycytidylate (MPC) and -polyuridylate (MPU) were tested in a comparative manner as inhibitors of the DNA polymerase-alpha from rat hepatoma. A wide scale of inhibitory potencies was obtained in the following (decreasing) order: MPU greater than M-poly(A,C,U) greater than M-poly(C,U) greater than MPC greater than M-poly(A,C) greater than or equal to M-poly(A,C,G). The sensitivity of the hepatoma DNA polymerase toward these antitemplates increased upon further purification of the enzyme through the DNA-agarose step. Partially thiolated DNA-isolates from rat hepatoma and calf thymus, respectively, showed significant inhibition of the hepatoma DNA polymerase, the thiolated hepatoma DNA being the more active inhibitor.

The effects of partially thiolated polynucleotides on the DNA polymerase alpha from regenerating rat liver were investigated. The enzyme was isolated from the nuclear fraction essentially according to the method of Baril et al.; it was characterized as the alpha polymerase on the basis of its response to synthetic templates and its inhibition with N-ethylmaleimide. Although polycytidylic acid had no effect on the DNA polymerase alpha either as a template or as an inhibitor, partially thiolated polycytidylic acid (MPC) was found to be a potent inhibitor, its activity being directly related to its extent of thiolation (percentage of 5-mercaptocytidylate units in the polymer). In comparison, the DNA polymerase beta which was purified from normal rat liver nuclear fraction, was much less sensitive to inhibition by MPC. Analysis of the inhibition of the alpha polymerase by the method of Lineweaver and Burk showed that the inhibitory action of MPC was competitively reversible with the DNA template, but the binding of the 7.2%-thiolated MPC to the enzyme was much stronger than that of the template (Ki/Km less than 0.03). Polyuridylic acid as such showed some inhibitory activity which increased on partial thiolation, but the 8.4%-thiolated polyuridylic acid was less active than the 7.2% MPC. When MPC was annealed with polyinosinic acid, it lost 80% of its inhibitory activity in the double-stranded configuration. However, 1 to 2%-thiolated DNA isolates were significantly more potent inhibitors than were comparable (1.2%-thiolated) MPC and showed competitive reversibility with the unmodified (but "activated") DNA template. These results indicate that the inhibitory activities of partially thiolated polynucleotides depend not only on the percentage of 5-mercapto groups but also on the configuration, base composition, and other specific structural properties.

5-Selenium-substituted derivatives (diselenides) or uracil, 2'-deoxyuridine, and 2'-deoxyuridylic acid were synthesized via the addition of methyl hypobromite to the 5,6 double bond, followed by reaction of the adducts with sodium diselenide. The physical and chemical properties of these compounds (including their facile reduction by dithiothreitol and rapid reoxidation) were similar to those of the corresponding 5-sulfur analogues. 5-Hydroseleno-2'-deoxyuridylic acid was as potent as 5-mercapto-2'-deoxyuridylate in inhibiting thymidylate synthetase from L. casei (ki approximately 6 X 10(-8) M) but the nucleoside III was considerably less active than 5-mercapto-2'-deoxyuridine in the inhibition of growth of the leukemia L1210 cell in culture.

In continuation of efforts to improve the antitumor selectivity of the 2,2-dimethylaziridine class of alkylating agents, a series of N-substituted bis(2,2-dimethyl-1-aziridinyl)phosphinic amides has been synthesized and evaluated. All of these compounds (3-15) were tested in vivo against leukemia P-388 in mice, where most of them caused significant increase of survival time at nontoxic dose levels. Some of the most active compounds were also tested against leukemia L1210, B16 melanoma, and colon 26 carcinoma; in the latter tests, the parent unsubstituted amide 3 appeared to show the highest antitumor activity. Since the dose-limiting toxicity of the clinically tested prototypes of this class of anticancer agents AB-132 (1) and AB-163 (2) had been found to be CNS toxicity attributable mainly to the inhibition of cholinesterase, the compounds were tested in vitro against the cholinesterases from horse serum, electric eel, and bovine erythrocytes, as well as in vivo for the inhibition of the cholinesterase present in the whole blood of mice. In all of these assays, the various members of the present series showed a wide range of anticholinesterase activities, ranging from almost zero (for 3) to even higher potency than that of the prototype 2. A similarly wide range of stability was observed toward hydrolytic ring opening of the 2,2-dimethylaziridine moieties. Several of the compounds, particularly 3, deserve further study.

5-Methylmercapto-2'-deoxyuridine (MeMUdR), a structural analogue of thymidine (TdR), inhibits herpes simplex virus type 1 production in mouse L (Lb) cells at concentrations that are not inhibitory to viral growth in monkey kidney (CV-1) cells. It is moderately toxic to Lb cells but not to CV-1 cells at a concentration that causes 95% inhibition of viral replication in Lb cells. MeMUdR is incorporated into cellular and viral deoxyribonucleic acid (DNA) in both systems, but to a significantly higher level (compared with thymidine) in Lb cells. These results indicate that MeMUdR is a substrate for enzymes leading to DNA synthesis and suggest that the biological function of herpes simplex virus type 1 DNA is impaired only when the incorporation of MeMUdR into the DNA reaches a relatively high level.

The uptake and intracellular localization by Ehrlich ascites cells of partially [35S]thiolated homologous DNA ("antitemplate") were studied in comparison with that of the corresponding unmodified [3H]DNA, at 37 degrees and 0 degrees, under standardized conditions. For the unmodified DNA, washing the cells after incubation with 0.08 M iodoacetate (in 0.15 M NaCl) alone gave high but reproducible uptake values (23%); washing with 1 M NaCl reduced the cell-associated DNA to 12% (less than 1% at 0 degrees). It appears that 1 M NaCl is able to remove DNA reversibly bound to the cells, similarly to DNase treatment. Approximately 5% of the input [3H]DNA was taken up into the cell nuclei. Diethylaminoethyl dextran (1:1, by weight) greatly enhanced the cellular uptake of [3H]DNA. In the case of [35S]thiolated DNA, the rate as well as the extent of uptake was significantly higher (33%). Washing the cells with 1 M NaCl or treatment with DNase caused relatively small decrease in the total cell-associated [35S]thiolated DNA, the bulk of which (22% of input) was recovered in the isolated nuclei. Stimulation by diethylaminoethyl dextran of the uptake of [35S]thiolated DNA could not be established because of the insolubility of the 1:1 complex in 1 M NaCl. Excess calcium ions during incubation dramatically increased the uptake of the thiolated DNA at 37 degrees (but not at 0 degrees) by the cells (to 90 to 100%) and into the nuclear fraction (to 70% of the total [35S]DNA input). The calcium salt procedure appears to be applicable to the in vivo testing of thiolated DNA's as potential chemotherapeutic agents.

Several new "dual antagonists" were synthesized in which the 2,2-dimethyl (or ring C unsubstituted) aziridine phosphinyl function is linked to N-hydroxyurethane rather than the urethane moiety. Three of the new compounds showed very high activities against leukemia L1210 in mice.

The effects of partially thiolated polyuridylic acid (MPU), partially thiolated polycytidylic acid (MPC), and their unmodified counterparts, poly(U) and poly(C), respectively, on the reverse transcriptase of avian myeloblastosis is virus (AMV) have been determined, using different template-primers. Poly(C) stimulated and MPC inhibited the polymerization reaction catalyzed by the AMV reverse transcriptase in the presence of the natural viral template (endogenous RNA or purified 70S RNA), or with poly(C) . oligo(dG)12-18 but not with poly(A) . oligo(dT)12-18 as the only template-primer present in the purified enzyme system. Both poly(U) and MPU inhibited the reaction in the presence of either of the synthetic or natural templates. Using the purified enzyme with the 70S RNA template, MPU was by far the most potent inhibitor, with I50 = 0.8 microM based on the epsilon (P) value, or I50 less than 3 x 10(-9) M based on the actual, macromolecular weight of the polynucleotide.

A series of S-alkylated derivatives of 5-mercapto-2'-deoxyuridine have been prepared by alkylation of the preformed nucleoside. Two of these compounds, the S-propargyl and S-allyl derivatives, have shown significant antiviral activity against Herpes simplex type 1 in HeLa TK- cells but appear to be less effective in this assay system than some previously reported 5-substituted 2'-deoxyuridines.

DNA polymerase alpha from calf thymus was relatively insensitive to the action of partially thiolated polycytidylic acid (MPC) which had been shown previously to be a potent inhibitor of the corresponding enzyme from regenerating rat liver, competitive with the activated DNA template. In contrast, partially thiolated polyuridylic acid (MPU) strongly inhibited the calf thymus enzyme as well, but showed non-competitive kinetics with respect to the activated DNA template. The much more potent inhibitory activity of MPU compared to MPC was attributed to the less rigid conformation of the former. Methyl substitution on the 5-mercapto groups of MPU substantially decreased but did not abolish its inhibitory activity. MPU was also a potent inhibitor of the herpes virus (HSV-1) induced DNA polymerase which, too, showed little sensitivity toward MPC; in this case, the inhibition by MPU was uncompetitive with respect to the DNA template. In preliminary experiments, MPU showed significant (61%) inhibition of the replication of HSV-1, while MPC was inactive. The results demonstrate that the inhibitory activity of partially thiolated synthetic polynucleotides toward certain DNA polymerases is dependent on the base composition.

Two new drug candidates, in which a quinonoid moiety is linked to the reactive bis(2,2-dimethyl-1-aziridinyl)phosphinyl function, have been prepared and tested in vivo for antitumor activity and in vitro as potentiators of the cytotoxic effect of X-irradiation. Without irradiation only moderate effectiveness against leukemia P-388 in mice was exhibited by one of the quinonoid compounds that had sufficient water solubility to be used in the in vivo screening. However, both compounds were shown to potentiate the effect of X-irradiation in vitro by a colony-forming cell culture assay under hypoxic conditions.

5'Mercaptouridine-5'-diphosphate (hs5UDP) has been synthesized and investigated as a substrate of the polynucleotide phosphorylase of Micrococcus luteus. While hs5UDP is not utilized alone, it can be copolymerized with UDP; however, unusually for this enzyme, the ratio of 5'mercaptouridylate vs. uridylate residues in the polynucleotide product (MPU) is always lower than the ratio of hs5UDP v. UDP in the substrate mixture. Furthermore, hs5UDP decreases the rate of the enzymic polymerization reaction. The MPU product forms two-stranded and three-stranded complexes with poly(A). The circular dichroic spectra of these complexes are similar to those formed between poly(U) and poly(A), but their melting profiles indicate somewhat lower stability. The physicochemical and biochemical properties of the enzymic product are qualitatively similar to those of MPU prepared by chemical modification; both are potent inhibitors of a DNA-dependent RNA polymerase.

In continuing search for exploitable biochemical differences between cancer and normal cells at the level of DNA replication, leukemic and "normal" hematopoietic cells from four different, established human cell lines were grown in culture flasks, and both the DNA and the DNA polymerase alpha were isolated in each case from the harvested (5-10 g wet weight) cell pellets. The four selected cell lines included a "normal" lymphoblastoid B-cell line (RPMI-1788), a pre-B cell (NALM-6) and a T-cell (MOLT-4) acute lymphoblastic leukemias, and a promyelocytic leukemia (HL-60). The DNA polymerase alpha enzyme of the two B-cell lines (both the leukemic and the "normal") showed the usual sensitivity toward inhibition by aphidicolin, while those from the two other leukemic cell lines were remarkably resistant to the antibiotic. Partially thiolated polycytidylic acid (MPC) strongly inhibited only the DNA polymerase alpha of the "normal" cell line, whereas the corresponding enzymes of all three leukemic cell lines were relatively insensitive to MPC. In contrast, the partially thiolated DNAs derived from the leukemic cell lines more strongly inhibited the DNA polymerase alphas of the leukemic cell lines than that of the "normal" cell line. These results indicate the existence of some structural differences between the DNA polymerase alpha enzymes (as well as between the DNAs) of human cells of different lineage and, particularly, of leukemic vs. "normal" character; such differences could be exploited in the design of selective antitemplates for chemotherapy.

The effect of partially thiolated polycytidylic acid (MPC) on the colony-forming ability of the progenitor cells (CFUC) of RF/Un leukemic mice was investigated using the plasma clot method in order to study the mode of action of the modified polynucleotide. The results showed that MPC inhibited the CFUC in a dose-dependent and time-dependent manner. Once a maximum level of inhibition of CFUC (approximately 40%) was observed, no further inhibition occurred whether the concentration of MPC was increased or whether the duration of incubation was lengthened. High-specific-activity [3H]thymidine, an S-phase-specific agent, showed a similar inhibition profile on the CFUC as did MPC. When MPC and high-specific-activity [3H]thymidine were incubated together with the bone marrow cells, there was no additive or synergistic inhibitory effect on the CFUC. Thus, it appears that MPC is an S-phase-specific agent. When injected i.v. into the mice, MPC decreased the number of CFUC of both the bone marrow and the spleen significantly.