Chloroethylating and methylating dual function antineoplastic agents display superior cytotoxicity against repair proficient tumor cells.

Two new agents based upon the structure of the clinically active prodrug laromustine were synthesized. These agents, 2-(2-chloroethyl)-N-methyl-1,2-bis(methylsulfonyl)-N-nitrosohydrazinecarboxamide (1) and N-(2-chloroethyl)-2-methyl-1,2-bis(methylsulfonyl)-N-nitrosohydrazinecarboxamide (2), were designed to retain the potent chloroethylating and DNA cross-linking functions of laromustine, and gain the ability to methylate DNA at the O-6 position of guanine, while lacking the carbamoylating activity of laromustine. The methylating arm was introduced with the intent of depleting the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT). Compound 1 is markedly more cytotoxic than laromustine in both AGT minus EMT6 mouse mammary carcinoma cells and high AGT expressing DU145 human prostate carcinoma cells. DNA cross-linking studies indicated that its cross-linking efficiency is nearly identical to its predicted active decomposition product, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (90CE), which is also produced by laromustine. AGT ablation studies in DU145 cells demonstrated that 1 can efficiently deplete AGT. Studies assaying methanol and 2-chloroethanol production as a consequence of the methylation and chloroethylation of water by 1 and 2 confirmed their ability to function as methylating and chloroethylating agents and provided insights into the superior activity of 1.

Confirmation of N-(nitrosomethyl) urea as a nitrosourea derived by nitrosation of fish sauce.

N-(nitrosomethyl) urea (NMU) was characterized in the carcinogenic nitrosated fish sauce (NFS) with high performance liquid chromatography (HPLC)-photohydrolysis-pyrolysis-thermal energy analysis recently. We used HPLC-electronic spray ionization-mass spectrometry and HPLC-diode array detection to confirm NMU in NFS further. It was observed that the corresponding chromatographic fraction of NMU of NFS showed the same mass spectrum (m/z 64, 102, and 145) and ultraviolet-absorbance (lambda max = 230 nm) as those of authentic NMU. These results confirmed that the component of NFS was NMU.

Stereochemistry of the in vitro and in vivo methylation of DNA by (R)- and (S)-N-[2H1,3H]methyl-N-nitrosourea and (R)- and (S)-N-nitroso-N-[2H1,3H]methyl-N-methylamine.

Reaction of DNA with the carcinogens N-methyl-N-nitrosourea and N-nitroso-N,N-dimethylamine produces several methylated species including the premutagenic O6-methylguanine. The mechanism of methylation is believed to be through a methanediazonium ion. We have studied the mechanism of methylation of DNA by these carcinogens by analyzing the stereochemistry of the methyl transfer. DNA was methylated in vitro by (R)- and (S)-N-[2H1,3H]methyl-N-nitrosourea and in vivo by (R)- and (S)-N-[2H1,3H]methyl-N-methyl-N-nitrosamine and (R)- and (S)-N-[2H1,3H]methyl-N-nitrosourea. 7-Methylguanine, 3-methyladenine, O6-methylguanine, and the methylated phosphate backbone were isolated. The methyl groups were converted into acetic acid, and the stereochemistry was analyzed. The identity of the nucleophile did not influence the stereochemistry of the methylation reaction. It was found that the methyl group was transferred with an average of 73% inversion and 27% retention of configuration. The most likely mechanism for the retention of configuration is through multiple methylation events in which nucleophiles which initially react with the methanediazonium ion react as electrophiles with DNA.

[Derivatives of antineoplastic antibiotics of the daunorubicin series containing methylurea or nitrosomethylurea residues]. / Proizvodnye protivoopukholevykh antibiotikov riada daunorubitsina, soderzhashchie ostatok metilmocheviny ili nitrozometilmocheviny.

Derivatives of antitumour anthracycline antibiotics containing N-methylurea moiety in the carbohydrate ring were obtained by the interaction of methyl isocyanate with daunorubicin, doxorubicin, carminomycin and daunorubicin derivatives, substituted at C-13 or C-14 positions. N-Nitrosation of these compounds yielded modified anthracycline antibiotics containing the N-methyl-N-nitrosourea substituent at C-3' position. Alkaline degradation of these derivatives produced, through corresponding isocyanates cyclic 3'-N,4'-carbonylderivatives. In these anthracycline derivatives with sugar cycles conjugated with oxazoline-2-ones the predominant conformations of sugar ring has changed from 1C4 to 4C1, 2,5B, or B0,3 (shown by 1H NMR spectroscopy). It was demonstrated, both in vitro and in vivo, that introduction of methylurea or cytotoxic methylnitrosourea moieties does not potentiate antimicrobial, cytotoxic or antitumour properties of these compounds.

Synthesis of an N-methyl-N-nitrosourea linked to a methidium chloride analogue and its reactions with 32P-end-labeled DNA.

The synthesis and characterization of an N-methyl-N-nitrosourea (MNU) analogue that is covalently linked to a methidium nucleus is described. At 37 degrees C in pH 8.0 buffer 9 hydrolyzes via pseudo-first-order kinetics, with a calculated t1/2 = 77 min. By use of polyacrylamide sequencing gels the formation of piperidine-labile N7-methylguanine adducts from the reaction of 9 and MNU with 5'-32P-end-labeled DNA restriction fragments is reported. DNA methylation by 9 in 10 mM Tris buffer is enhanced with increasing ionic strength (50-200 mM NaCl), which contrasts to the inhibition of MNU-induced cleavage with increasing salt. In addition, 9 methylates all G sites equally, while MNU shows a clear preference for d(G)n (n greater than or equal to 3) runs and an asymmetrical methylation pattern within these G-rich regions. The results are discussed in terms of the delivery of the MNU moiety to the DNA target by a non-sequence-specific intercalation process and the subsequent hydrolytic generation of a nondiffusible alkylating intermediate.