Effects of partially thiolated polycytidylic acid on the clonogenicity of murine leukemic stem cells.

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.

Uptake of partially thiolated DNA by ascites tumor cells.

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.

Action of partially thiolated polynucleotides on the DNA polymerase alpha from regenerating rat liver.

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.

Effects of 5-mercapto-2'-deoxyuridine on the incorporation of nucleosides into RNA and DNA in a primary lymphocyte culture system.

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.

Plasma clearance and tissue distribution of partially thiolated polycytidylic acid and its degradation products in rodents.

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.

Effects of partially thiolated polycytidylic acid and liposomes on in vitro colony-forming cells of leukemic mice.

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.

Antitumor activity of tetraacetylglucosamine mustard.

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.

Synthesis of 5'-thymidinyl bis(1-aziridinyl)phosphinates as antineoplastic agents.

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.

Synthesis of some new S-alkylated derivatives of 5-mercapto-2'-deoxyuridine as potential antiviral agents.

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.

Synthesis and inhibition analysis of 2(4)-imino-4(2)-amino-2,4-dideoxyriboflavin, a dual antagonist of riboflavin and folinic acid.

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.

Synthesis of 5-selenium-substituted uracil derivatives. Inhibition of thymidylate synthetase by 5-hydroseleno-2'-deoxyuridylate.

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.

Synthesis and testing of quinone-based bis(2,2-dimethyl-1-aziridinyl)phosphinyl carbamates as radiation-potentiating antitumor agents.

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.

Synthesis and antiviral activity of 1-(2-deoxy-beta-D-ribofuranosyl)-5-(methylmercapto)-2-pyrimidinone.

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.

Synthfsis of bis(aziridinyl)phosphinyl-N-hydroxyurethane derivatives as antineoplastic 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.

Synthesis of new nucleoside phosphoraziridines as potential site-directed antineoplastic agents.

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.

Synthesis and biological activities of 2-pyrimidinone nucleosides. 2. 5-Halo-2-pyrimidinone 2'-deoxyribonucleosides.

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.

Synthesis of potential dual-acting radiation sensitizer antineoplastic agents: 2,2-dimethylphosphoraziridines containing 2-nitroimidazoles or other electron-affinic moieties.

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.

Synthesis and properties of bis(2,2-dimethylaziridinyl)phosphinic amides: a series of new antineoplastic agents.

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.

Comparative chemical and biological studies of four prototype phosphoraziridine antineoplastic agents.

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.

Selectivity of antitemplates as inhibitors of deoxyribonucleic acid polymerases.

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.