Nitric oxide and free stable nitroxyl radicals in the mechanism of biological action of the spin-labeled compounds.

A comparison of more important physical, chemical and biological properties of the nitric oxide (NO) and free stable nitroxyl radicals (nitroxides) on the base of their structural similarity is made in the article. The active moiety in the nitroxide molecule represents a sterically hindered nitric oxide. The mechanisms of biological action of the nitroxides and especially of their derivatives with antitumor agents from the groups of nitrogen mustards, nitrosoureas, aziridines and triazenes (spin-labeled compounds) is explained through the biological activities of sterically hindered NO. Similarly to NO, nitroxides also can react with superoxide anion radical (O(2)(-)), they possess superoxide dismutase (SOD) mimetic action. While the interaction of NO with O(2)(-)yields very toxic peroxynitrite (ONOO(-)), its formation is strongly limited in the presence of a nitroxide. It is known that the nitrosourea antitumor drugs, like lomustine (CCNU) and carmustine (BCNU), showed high general toxicity, one of the reasons for that probability is the formation of NO, and subsequently of ONOO(-), during their metabolism. The biological investigations of the nitroxides showed their considerably lower general toxicity that could be explained with the SOD-mimetic action of the nitroxide present in their molecule.

New method to measure the carbamoylating activity of nitrosoureas by electron paramagnetic resonance spectroscopy.

A new method for measuring the carbamoylating activity of nitrosoureas and isocyanates using electron paramagnetic resonance (EPR) spectroscopy is described. The extent and time course of carbamoylation reaction of chloroethyl isocyanate and a series of 9 nitrosoureas toward amino group of 4-amino-2,2,6,6-tetramethyl-piperidine-1-oxyl were examined with both the EPR method and the HPLC method which has been proposed by Brubaker et al. [Biochem. Pharmacol. 35:2359 (1986)]. Spin-labeled nitrosoureas we synthesized are included in this study since they have less toxicity or more efficiency than commercially available drug in some cases. The concentration of carbamoylated product was easily determined with the EPR spectra. There is a very high correlation (r = 0.982, t = 2.58, N = 10, p < 0.001) between the EPR and HPLC methods. Spin-labeled nitrosoureas showed lower carbamoylating activity than non-labeled analogues. The carbamoylating activity for these nitrosourea depended on the reactivity of isocyanate intermediate and almost independent of their half life. This rapid and simple EPR method is suitable for the detailed investigation of the rate and extent of carbamoylation reaction.

Spin labeled amino acid nitrosourea derivatives--synthesis and antitumour activity.

The synthesis of three spin labeled derivatives of N-[N'-(chloroethyl)-N'-nitrosocarbamoyl] amino acids is reported. The new nitrosoureas are obtained by condensation of the corresponding N-[N'-(2-chloroethyl)-N'-nitrosocarbamoyl] amino acid with 2,2,6,6-tetramethyl-1-oxyl-4-aminopiperidine using dicyclohexylcarbodiimide. Their chemical structures are confirmed by elemental analysis, IR, MS, and EPR spectroscopy. All newly synthesized compounds showed high antitumour activity against the lymphoid leukemia L1210 in BDF1 mice.

Superoxide scavenging activity of spin-labeled nitrosourea and triazene derivatives.

Superoxide scavenging activities (SSA) of newly synthesized spin-labeled nitrosourea and triazene derivatives, and their precursor nitroxides were investigated by the ESR/spin-trapping method using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and hypoxanthine/xanthine oxidase as the superoxide-generating system. The spin-labeled nitrosoureas, triazenes and their precursor nitroxides exhibited excellent SSA, whereas clinically used nitrosourea and triazene, which do not contain the nitroxide moiety, did not show any SSA. Furthermore, it was deduced that these nitroxides scavenge superoxide by redox cycling between nitroxide and corresponding hydroxylamine.

Tissue distribution of polybutylcyanoacrylate nanoparticles loaded with spin-labelled nitrosourea in Lewis lung carcinoma-bearing mice.

The tissue distribution of polybutylcyanoacrylate nanoparticles (PBCN) with a diameter of 127 nm, loaded with 1-(2-chloroethyl)-3-(1-oxyl-2,2,6,6-tetramethylpiperidinyl)-1- nitrosourea (spin-labelled nitrosourea, SLCNU) is described. PBCN-suspensions were intraperitoneally (i.p.) injected into Lewis lung carcinoma bearing mice. The biodistribution of PBCN in the visceral organs, blood and tumor was studied by electron spin resonance (ESR) spectroscopy. A relatively low accumulation of nanoparticles in the liver and spleen was found. The accumulation was negligible in the i.m. implanted primary tumor. SLCNU-loaded nanoparticles were mainly found in the lungs, kidneys, and heart. The highest content of the particles studied was observed in the lungs of tumor bearing experimental animals damaged by metastases. These findings suggest that PBCN offer some opportunities in the targeting of SLCNU to lung metastases.

New nitrosoureas and their spin-labeled derivatives influence dopa-oxidase activity of tyrosinase.

Tyrosinase is a key enzyme in melanine biosynthesis. The modulating effect of cytostatic agents on DOPA-oxidase activity of tyrosinase could be linked with the drug treatment of melanoma tumors. Two groups of nitrosoureas which influence DOPA-oxidase activity of tyrosinase were studied: new nitrosoureas and their spin-labeled derivatives synthesized in our laboratory. Using Burnett's spectrophotometric method (Burnett et al., 1967) the following effects were established: inhibition by CCNU, inhibition and the activating effects of the other investigated nitrosoureas depend on their physicochemical half-life. The predominant activating effect of the spin-labeled derivatives is due to the nitroxyl radical present in these compounds.

Activation of the anticancer drug CCNU into a free radical intermediate via UV irradiation--an ESR study.

We studied the formation of a free radical induced by UV irradiation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in benzene. It was determined a stable nitroxide radical by ESR spectroscopy. We confirmed that sterically hindered cyclic amine 2,2,6,6-tetramethyl-4-piperidone (TMPone) was transformed into a corresponding stable free nitroxyl radical during UV irradiation. When CCNU was present, the rate of free radical formation from TMPone increased considerably.

Synthesis and antitumour activity of 4-nitroso-4-(2-chloroethyl)semicarbazides.

1-Methyl-1-(2-chloroethyl)-4-nitroso-4-(2-chloroethyl)semicarbazide, 1,1-di-(2-chloroethyl)-4-nitroso-4-(2-chloroethyl)semicarbazide and 1,1-di-(2-bromoethyl)-4-nitroso-4-(2-chloroethyl)semicarbazide, which are a novel class of nitrosoureas containing hydrazinomustard residue, have been synthesized. A dose-dependent antitumour activity was found with the three tested compounds.

EPR spectroscopic analysis of binding sites of a cancerostatic agent on erythrocyte membranes.

A biologically highly active antitumor agent, chloroethyl-nitrosourea-piperidine-N-oxyl, was found to bind covalently to sulfhydryl groups of membrane proteins of erythrocytes. The agent penetrates the erythrocyte membrane very easily within three minutes. Mainly two different binding sites are distinguishable with rather high rotational mobility of the NO-moiety and high polarity of their environment. The lowered broadening of bound label suggests pocket-like binding sites with a limited accessibility for paramagnetic ions. Besides common concepts that antitumor agents often bind to DNA and inhibit cell proliferation, further targets on plasma membranes are taken into consideration for antitumor agents.

Nucleophilic targets in carcinogenesis, mutagenesis and chemotherapy of cancer.

A hypothesis is suggested, which emphasizes the role in carcinogenesis of the attack on low molecular nucleophilic substances (LMN) by electrophilic agents - chemical carcinogens, phisical factors, and antitumor alkylating agents. The significance of the degree of nucleophilicity (electronic charge, order of bonds, index of valence) as a locus minoris resistentiae of the LMN in the electrophilic attack on the latter is emphasized as well as the probable role of the hydrogenated pteridines in influencing carcinogenesis by means of ascorbate, tocopherol, SH-containing compounds etc. In support of this hypothesis the preference of electrophilic agents (derivatives of nitrogen mustard and nitrosoureas) for the places with highest degree of nucleophilicity as targets, in experiments in vitro with nucleic bases and pteridines is emphasized.

Synthesis and study of spin-labeled nitrosoureas.

For the first time we have synthesized spin-labeled nitrosoureas and have studied their properties--reduction of the iminoxyl group by vitamin C leading to the formation of the corresponding hydroxylamine derivatives and degradation in the presence of an aminoradical, leading to biradicals. The ESR spectra of biradicals in methanol have nine hyperfine resonance lines. The spin-labeled nitrosoureas have shown a high antitumor activity against the L 1210 lymphoid leukemia and P 388 lymphocytic leukemia in BDF1 mice. A study of a broad range of transplantable tumors is in progress.

Spin-labelling of DNA with hydrazine mustard spin label (HMSL).

1. The hydrazine mustard spin label (HMSL), recently synthesized in our laboratory (Raikova, 1977) was used for spin-labelling of DNA. 2. It alkylates both double- and single-stranded DNAs. 3. The reaction of HMSL with DNA was studied with respect to the kinetics of alkylation, dependence on salt concentration and base specificity. 4. It was found that HMSL is a base-specific reagent, alkylating preferentially guanine. According to their ability to bind HMSL, the four deoxyribonucleotides are ordered in the following way: G greater than A greater than C greater than T. 5. The EPR spectra obtained strongly depended on the secondary structure of the spin-labelled DNA: unlike the immobilized spectra of the double-stranded DNAs (2AZZ = 44.8G), the EPR spectra of single-stranded DNAs were non-immobilized (2AZZ = 32.8 G). 6. When sheared double-stranded DNA was spin-labelled, the parameters of the EPR spectrum depended also on the GC content of DNA.

Distribution of 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl free radical in normal and neoplastic animal tissues.

The free radical 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (TMPO) was injected i.p. in doses of 100 mg/kg bw into Syrian hamsters: untreated, partially hepatectomized and grafted with transplantable tumours as well as into mice with B16 melanotic melanoma or with an adenocarcinoma. One hour after application of the compound its content was determined spectrometrically in livers, kidneys, lungs and all tumours. The characteristic triplet signal of TMPO was registered in the hamster and mouse melanotic melanomas but not in all other animal tissues and tumours. This may be the result of an enzyme defect in the melanotic melanoma cells or of a retention of TMPO by melanin. It was also found that TMPO is metabolized predominantly in the livers of the animals.