An analysis of 1-(2-chloroethyl)-1-nitrosourea activity at the cellular level

The effect of five different 1-(2-chloroethyl)-1-nitrosoureas on the growth of cultured P388 cells has been analyzed in terms of physical, chemical, and kinetic parameters that are related to the mechanism of action of this class of cancer chemotherapeutic agent. This study correlates structure with activity at the cellular level by using a dose function that is related to the amount of active species, the (2-chloroethyl)diazonium ion, that is formed during the period of exposure of cells to drug rather than to the initial drug dose. 1-(2-Chloroethyl)-1-nitrosourea analogues that rapidly enter the P388 cells are shown to have the same activity relative to the amount of active species formed. When analyzed in this way, activity is not influenced by the structure of the N-3 substituent, lipophilicity, or carbamoylating activity. The agents 1-(2-chloroethyl)-1-nitrosourea (CNU), 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea (PCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) all produce a 50% cell growth inhibition at 6 to 7 microM active species formed per cell volume. Chlorozotocin required a twofold higher effective dose to produce the same toxic effect. This decreased activity is attributed to the slow uptake of the water-soluble chlorozotocin into P388 and L1210 cells relative to the rate of chlorozotocin conversion to active species in medium. The yields to 2-chloroethanol from CNU, BCNU, and chlorozotocin were shown to be the same, indicating that these agents generate the same yield of alkylating intermediate at 37 degrees C and pH 7.4.     

Carbamoylating chemoresistance induced by cobalt pretreatment in C6 glioma cells: putative roles of hypoxia-inducible factor-1

1. We tested whether pretreatment of reagents known to induce hypoxia-inducible factor-1 (HIF-1) may confer chemoresistance against cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to rat C6 glioma cells. We also studied which cytotoxic mechanism(s) of chloroethylnitrosoureas could be neutralized by cobalt preconditioning. 2. Preconditioning of rat C6 glioma cells with cobalt chloride (300 microm, 2 h) induced HIF-1 binding activity based on electrophoretic mobility shift assay (EMSA). Results from Western blotting confirmed a heightened HIF-1alpha level upon cobalt chloride exposure (300-400 microm, 2 h). Cobalt chloride (300 microm) pretreatment for 2 h substantially neutralized BCNU toxicity, leading to increases in glioma cell survival based on MTT assay. In addition, pre-exposure of C6 cells with desferrioxamine (DFO; 400 microm, 3 h), an iron chelator known to activate HIF-1, also induced HIF-1 binding and rendered the glioma cells resistant to cytotoxicity of BCNU. 3. Pre-incubation with cobalt chloride abolished the cytotoxicity of several carbamoylating agents including 2-chloroethyl isocyanate and cyclohexyl isocyanate, the respective carbamoylating metabolites of BCNU and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. The protective effect of cobalt exposure, however, was not observed when cells were challenged with alkylating agents including temozolomide. 4. Cadmium chloride (50 microm) effectively reversed cobalt-induced HIF-1 activation. Correspondingly, cadmium chloride suppressed carbamoylating chemoresistance mediated by cobalt chloride pretreatment. Furthermore, both double-stranded oligodeoxynucleotide (ODN) decoy with HIF-1 cognate sequence and antisense phosphorothioate ODNs against HIF-1alpha partially abolished the carbamoylating chemoresistance associated with cobalt preconditioning. 5. Our results suggest that cobalt- or DFO-preconditioning may enhance glioma carbamoylating chemoresistance that is dependent, at least in part, on induction of HIF-1.     

Differential inhibition of cellular glutathione reductase activity by isocyanates generated from the antitumor prodrugs Cloretazine and BCNU

The antitumor, DNA-alkylating agent 1,3-bis[2-chloroethyl]-2-nitrosourea (BCNU; Carmustine), which generates 2-chloroethyl isocyanate upon decomposition in situ, inhibits cellular glutathione reductase (GR; EC 1.8.1.7) activity by up to 90% at pharmacological doses. GR is susceptible to attack from exogenous electrophiles, particularly carbamoylation from alkyl isocyanates, rendering the enzyme unable to catalyze the reduction of oxidized glutathione. Evidence implicates inhibition of GR as a cause of the pulmonary toxicity often seen in high-dose BCNU-treated animals and human cancer patients. Herein we demonstrate that the prodrug Cloretazine (1,2-bis[methylsulfonyl]-1-[2-chloroethyl]-2-[(methylamino)carbonyl]hydrazine; VNP40101M), which yields methyl isocyanate and chloroethylating species upon activation, did not produce similar inhibition of cellular GR activity, despite BCNU and Cloretazine being equally potent inhibitors of purified human GR (IC(50) values of 55.5 microM and 54.6 microM, respectively). Human erythrocytes, following exposure to 50 microM BCNU for 1h at 37 degrees C, had an 84% decrease in GR activity, whereas 50 microM Cloretazine caused less than 1% inhibition under the same conditions. Similar results were found using L1210 murine leukemia cells. The disparity between these compounds remained when cells were lysed prior to drug exposure and were partially recapitulated using purified enzyme when 1mM reduced glutathione was included during the drug exposure. The superior antineoplastic potential of Cloretazine compared to BCNU in animal models could be attributed in part to the contribution of the methyl isocyanate, which is synergistic with the co-generated cytotoxic alkylating species, while at the same time unable to significantly inhibit cellular GR.     

Main metabolites of 1-(2-chloroethyl)-3-[1'-(5'-p-nitrobenzoyl-2',3'-isopropylidene)-alpha, beta-D-ribofuranosyl]-1-nitrosourea and 1-(2-chloroethyl)-3-(2',3', 4'-tri-O-acetyl-alpha, beta-D-ribopyranosyl)-1-nitrosourea in rats

The metabolism of two glycosylnitrosoureas, 1-(2-chloroethyl)-3-[1'-(5'-p-nitrobenzoyl-2',3'-isopropylidene)-alpha, beta-D-ribofuranosyl]-1-nitrosourea (RFCNU) and 1-(2-chloroethyl)-3-(2',3',4'-tri-O-acetyl-alpha, beta-D-ribopyranosyl)-1-nitrosourea (RPCNU), has been investigated in the rat. With the label on the carboxyl moiety of RFCNU, we have shown that hydrolysis of the 4-nitrobenzoyl ester occurred to a large extent in vivo; 4-nitrobenzoic acid and its glucuronide were the major urinary metabolites. Two other minor metabolites and their glucuronides were identified as 4-aminobenzoic acid and 4-acetamidobenzoic acid. With the label on the chloroethyl moieties of RFCNU and RPCNU, we have shown that chloroethanol was a major degradation product of this alkylating part of the molecule. The concentration of chloroethanol in plasma vs. time has been determined. In urine, four metabolites derived from alkylated glutathione, namely thiodiacetic acid and its sulfoxide, N-acetylcarboxymethylcysteine, and N-acetylhydroxyethylcysteine, have been identified.     

Early effect of BCNU on rat astrocytes. Inhibition of S6 kinase activation by growth factors.

In primary cultures of astrocytes, methylmethane, 2-N-methyl 9-hydroxy-ellepticinium acetate, ditercalinium, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and 1,3 bis (2-chloroethyl)-1-nitrosourea (BCNU) blocked to various extents the activation of S6 kinase by acidic fibroblast growth factor and insulin [or insulin-like growth factor 1 (IGF1)]. The effects of the most active agent, BCNU, were time and concentration dependent. Pretreatment of cells with 50 microM BCNU for 1 hr completely prevented S6 kinase activation by growth factors for at least 2 days. The S6 kinase activity of unstimulated cells was slightly affected. S6 kinase activation by 12-O-tetradecanoylphorbol 13 acetate was also strongly impaired by treating cells with BCNU whereas activation by 8-bromo-cyclic AMP was slightly reduced. Cyclic AMP-dependent protein kinase and phospholipid and Ca(2+)-dependent protein kinase were unaffected. BCNU had no direct effect on IGF1 binding to cell surface receptors or on the S6 kinase activity of cell cytosols.     

The lethal activity and repair inhibition capacity of 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea

Summary The survival response of human colorectal carcinoma cells treated in vitro for 1 h with PCNU was characterized by a threshold exponential curve, Dq = 8 g/ml (1 h) and D ₀ = 22 g/ml (1 h). Continuous treatment induced decreasing degrees of cell kill although PCNU was biologically stable in solution for at least 24 h. Cells treated with PCNU were unable to recover from potentially lethal damage but were quite capable of repairing PCNU-induced sublethal damage. Thus, PCNU with different alkylating and carbamoylating than other nitrosourea congeners had similar cytotoxic and repair inhibition capacities. Any therapeutic gain in the clinical use of PCNU must derive only from its lipophilic properties and not from its superior activity at the cellular level.Abbreviations PCNU 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl-1-nitrosourea (NSC 95466) - CCNU 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (NSC 79037) - BCNU 1,3-bis(2-chloroethyl)-1-nitrosourea (NSC 409962) cis-acid, 4-(3-(2-chloroethyl)-3-nitrosoureido)-cis-cyclohexane-carboxylic acid (NSC 153174) - MeCCNU 1-(2-chloroethyl)-3-trans-(4-methyl-cyclohexyl)-1-nitrosourea (NSC 95441) - PE plating efficiency - Dq quasithreshold dose equal to the intercept with the abscissa (at 100% survival) of the exponential part of survival curve - D ₀ mean lethal dose equal to the concentration required to reduce survival by 63% on exponential part of survival curve     

Pronounced damage of intestinal tract mucosa by the combination of 1-methyl-1-nitrosourea plus 1,3-bis-(2-chloroethyl)-1-nitrosourea. Effect of drug sequence and time interval of administration

The combination of 1-methyl-1-nitrosourea (MNU) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) has an overadditive toxicity in rats. This overadditive effect is dependent on drug sequence and time interval between the administration of both compounds. Application of MNU within 2 h prior to BCNU or simultaneous application of both compounds displayed the highest toxicity. The dose-limiting toxicity appears to be a severe damage of the intestinal mucosa.     

Comparison of DNA lesions produced by tumor-inhibitory 1,2-bis(sulfonyl)hydrazines and chloroethylnitrosoureas

1,2-Bis(sulfonyl)hydrazine derivatives, designed to generate several of the electrophilic species classically believed to be responsible for the alkylating (chloroethylating) and/or carbamoylating activities of the chloroethylnitrosoureas (CNUs), were compared with respect to the cross-linking and nicking of T7 DNA to that caused by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), and 1-(2-chloroethyl)-3-(4-trans-methylcyclohexyl)-1-nitrosourea (MeCCNU). In the case of BCNU, a large proportion of T7 DNA strand nicking was found to be due to the generation of 2-chloroethylamine, produced from the hydrolysis of 2-chloroethylisocyanate, in turn formed during the decomposition of the parental nitrosourea. 1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine (compound 1) gave a greater yield of DNA cross-links than the CNUs. Compound 1, as well as its derivatives that were incapable of generating 2-chloroethylisocyanate, did not produce detectable levels of strand nicking, indicating that N7-alkylation of guanine did not occur to a significant extent with these agents. Since compound 1 and its derivatives are believed to generate chloronium and chloroethyldiazonium ions, it would appear that these species could not be significantly involved in the N7-alkylation of guanine caused by the CNUs. The relatively low level of N7-alkylation of guanine residues and the relatively high yield of cross-links generated by some of the 1,2-bis(sulfonyl)-1-(2-chloroethyl)hydrazine derivatives implies that they are more exclusive O6-guanine chloroethylating agents than the CNUs. O6-Guanine chloroethylation is believed to be the therapeutically relevant event produced by the CNUs; therefore, compound 1 derivatives represent promising new cancer chemotherapeutic agents, since they appear to generate lower quantities of therapeutically unimportant, yet carcinogenic lesions, and more of the therapeutically relevant O6-guanine chloroethylation than the CNUs.     

alpha-Difluoromethylornithine effects on nitrosourea-induced cytotoxicity and crosslinking in a methylation excision repair positive (MER+) human cell line

We investigated the cytotoxic effects of nitrosoureas with and without a 42-hr preincubation with the ornithine decarboxylase (EC 4.1.1.17) inhibitor alpha-difluoromethylornithine (DFMO, 1 mM) in a MER+ (methylation excision repair positive) human cell line. DFMO combined with a chloroethyl nitrosourea [1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) or 1-(2-chloroethyl)-1-nitrosourea (CNU)] yielded increased toxicity with D37 ratios of 1.9 and 3.3 respectively. There was no enhanced toxicity with the monofunctional nitrosourea 1-ethyl-1-nitrosourea (ENU). BCNU or CNU did not induce DNA-DNA interstrand crosslinks in cells with or without a DFMO pretreatment. DNA single-strand breakage was not increased by addition of DFMO. BCNU-induced DNA-protein crosslinking was decreased in cells pretreated with DFMO. These findings are similar to those in MER- cells in that the chloroethyl carbonium alkylating species is required for the enhanced cytotoxicity seen with DFMO. The ability to form DNA interstrand crosslinks, however, does not appear to be necessary for this toxicity enhancement.     

In vitro cellular characteristics and survival responses of human astrocytoma clones to chloroethyl-nitrosoureas and dianhydrogalactitol

Three permanent clones were derived from a single astrocytoma cell line and were characterized for in vitro cell kinetics, chromosomal properties and for their responses to the anticancer drugs: 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU); 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (MeCCNU); 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl-1-nitrosourea) (PCNU); and 1,2:5,6-dianhydrogalactitol (GAL); all of which have been shown to cross the blood brain barrier. The clones showed different population doubling times, saturation densities, plating efficiencies, chromosome counts, ploidy, cell cycle phase distributions and DNA indices. The only positive correlation among these parameters was between the population doubling times and the modal chromosome numbers; the lower the chromosome number, the shorter the doubling time. No correlation was observable between any of the cellular properties and responses to the four drugs. The clones showed a differential sensitivity to the nitrosoureas, seen maximally as a 600-fold difference in survival between two of the clones treated with the same dose of BCNU. In contrast, the clones exhibited almost identical and uniform sensitivities to galactitol, suggesting that this agent exerted its cytotoxic effects by similar mechanisms in each of the clones. By comparison BCNU (at the tested doses and duration of drug exposure used in this study) was found to be the most effective of the agents tested.     

Glutathione reductase inhibitors as potential antimalarial drugs. Effects of nitrosoureas on Plasmodium falciparum in vitro

Malarial parasites are believed to be more susceptible to oxidative stress than their hosts. BCNU(1,3-bis(2-chloroethyl)-1-nitrosourea) and HeCNU(1-(2-chloroethyl)-3-(2-hydroxythyl)-1-nitrosourea), inhibitors of the antioxidant enzyme glutathione reductase, were found to prevent the growth of Plasmodium falciparum in all intraerythrocytic stages. When exposing infected red blood cells to 38 microM BCNU or 62 microM HeCNU for one life cycle of synchronously growing parasites, the parasitemia decreased by 90%. During the formation of new ring forms, the parasites are even more susceptible to these drugs. The treatment with BCNU or HeCNU produced a rapid depletion of GSH in the parasites and their host cells; in addition, protection against lipid peroxidation was impaired in these cells. Possible mechanisms for the antimalarial action of the inhibitors are discussed. Our results suggest that erythrocyte glutathione reductase, an enzyme of known structure, might be considered as a target for the design of antimalarial drugs.     

Correlation of nitrosourea murine bone marrow toxicity with deoxyribonucleic acid alkylation and chromatin binding sites

All of the clinically available nitrosourea antitumor agents produce serious treatment-limiting bone marrow toxicity. A reduction in this toxicity can be achieved by attaching the chloroethylnitrosourea cytotoxic group to C2 (chlorozotocin) or C1 (1-(2-chloroethyl)-3-(β-d-glucopyranosyl)-1-nitrosourea, GANU) of glucose. Both glucose analogs are less myelotoxic in mice than 1-(2-chloroethyl)-3-cyclohepyl-1-nitrosourea (CCNU) or 1-(4-amino-2-methylpyrimidin-5-yl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), while retaining comparable antitumor activity against the murine L121o leukemia. To define the nuclear mechanisms for this reduced myelotoxicity, alkylation of L1210 and murine bone marrow DNA was quantitated. With the use of the endonucleases micrococcal nuclease and DNase I, the sites of alkylation within the chromatin substructure were determined. Experiments were performed on L1210 leukemia or bone marrow cells that had been incubated in vitro for 2 hr with 0.1 mM [¹⁴C]chloroethyl drug. The quantitative alkylation of DNA by GANU was 1.3-fold greater in L1210, as compared to bone marrow, cells. This ratio of DNA alkylation is comparable to the 1.3 ratio we previously reported for chlorozotocin [L. C. Panasci, D. Green and P. S. Schein, J. clin. Invest.64, 1103 (1979)]. In contrast, the ratio of alkylation (L1210: bone marrow DNA) for the myelotoxic ACNU was 0.66, similar to 0.59 for CCNU. Nuclease digestion experiments demonstrated that chlorozotocin and GANU preferentially alkylated internucleosomal linker regions of bone marrow chromatin, while nucleosome core particles were the preferred targets of CCNU and ACNU. The reduced myelotoxicity of chlorozotocin and GANU may be correlated with the advantageous ratio of L1210: bone marrow DNA alkylation and preferential alkylation of internucleosomal regions of bone marrow chromatin.     

Synthesis and cytotoxic activity of N-(2-chloroethyl)-N-nitroureas and N-(2-chloroethyl)-N-nitrocarbamates

As analogs of the widely used anti-tumor agents, N-(2-chloroethyl)-N-nitrosoureas, N-(2-chloroethyl)-N-nitroureas and N-(2-chloroethyl)-N-nitrocarbamates were synthesized by nitration following the reaction of the appropriate amines or alcohols with 2-chloroethyl isocyanate. All tested compounds exert cytotoxic effect with IC50 values of 10(-4) to 10(-6) M and most of them show somewhat higher cytotoxicity in nitrogen than in air.     

Synthesis and evaluation of 1,2,2-tris(sulfonyl)hydrazines as antineoplastic and trypanocidal agents

Several 1,2,2-tris(sulfonyl)hydrazines, conceived as prodrugs of 1,2-bis(sulfonyl)hydrazines, were synthesized and evaluated for antineoplastic and trypanocidal activities in mice. 1-Methyl-1,2,2-tris(methylsulfonyl)hydrazine emerged as an extremely efficacious antitrypanosomal agent, whereas 1-(2-chloroethyl)-1,2,2-tris(methylsulfonyl)hydrazine was inactive. In contrast, 1-(2-chloroethyl)-1,2,2-tris(methylsulfonyl)hydrazine displayed potent antineoplastic activity, producing several 60-day "cures" of mice bearing leukemia L1210, leukemia P388, or Sarcoma 180. Furthermore, the fact that the tris(sulfonyl) derivatives will not generate isocyanates, which contribute to the host toxicity of nitrosoureas like 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), makes them agents of significant promise in trypanosomal and cancer chemotherapy.     

Changes in drug sensitivity of a human astrocytoma clone previously treated with 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea in vitro

Summary We have shown in earlier studies that repeated weekly exposures of a human astrocytoma clone (AST 3–4) to MeCCNU (10 g/ml for 1 h per week) produced a linear decrease in survival over the first 3 weekly treatments. But, after that time these cells became progressively more resistant to the 10 g/ml concentration of the agent. In the studies reported here we show that these previously treated cells were also less responsive to other doses ranging from 1 to 30 g MeCCNU/ml. This change in sensitivity to MeCCNU was accompanied by collateral changes in response to other agents: resistance to BCNU and Galactitol, increased sensitivity to Adriamycin, and no change to ionizing radiation. These experiments suggest that once repeated treatments with a single agent cause a tumor cell population to become more resistant, sensitivity to other agents may also change unpredictably.Abbreviations DAG Galactitol - Adria Adriamycin - PCNU 1-(2-Chloroethyl)-3-(2,6 dioxo-3-piperidyl-1-nitrosourea) - MeCCNU 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea - BCNU 1,3-bis(2-chloroethyl)-1-nitrosourea - FMF Flow microfluorometry Dedicated to the memory of Benjamin Drewinko, Physician, Scientist and friend.     

Cytochrome P-450-dependent formation of alkylating metabolites of the 2-chloroethylnitrosoureas MeCCNU and CCNU

Rat liver microsomes catalyzed the biotransformation of the clinically important nitrosourea anticancer agents 1-(2-chloroethyl)-3-(trans-4-methyl-cyclohexyl)-1-nitrosourea (MeCCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) to alkylating metabolites that bound covalently to microsomal protein and to DNA. The enzyme-mediated microsomal alkylation required NADPH and oxygen and was inhibited by carbon monoxide, indicating the participation of a cytochrome P-450-dependent monooxygenase. Additional studies with inhibitors such as piperonyl butoxide and with the inducers 3-methylcholanthrene and phenobarbital were consistent with this view. In contrast to these observations on the formation of alkylating metabolites, carbamylation reactions were not affected significantly by microsomal metabolism. Reduced glutathione, cysteine or N-acetylcysteine decreased the microsomal alkylation by MeCCNU and produced a corresponding increase in the formation of polar metabolites that was resolved by HPLC as three distinct N-acetylcysteine-MeCCNU adducts. The addition of semicarbazide to the reaction decreased microsomal alkylation by 30%, indicating that the formation of the alkylating species may proceed via an aldehyde intermediate. Renal microsomes were not found to catalyze the alkylation reaction. Moreover, MeCCNU inhibited the renal slice accumulation of p-aminohippuric acid only in the presence of liver microsomes and NADPH, suggesting that a liver metabolite may be responsible for the renal toxicity of the parent nitrosourea.     

Examination of newly synthesized 2-chloroethylnitrosoureas in preterminal leukemia L 5222 and in two transplanted neurogenic tumors

The chemotherapeutic activities of 11 chloroethylnitrosoureas, among them 10 newly synthesized compounds, were investigated in rat leukemia L 5222 and in two neurogenic rat tumors. 1-(4-Amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU, compound 1) and cyclophosphamide (12) served as reference substances. The newly synthesized compounds were 1-(2-chloroethyl)-1-nitroso-3-(2-carboxyethyl)-urea (2-carboxyethyl-CNU, 2), 1-(2-chloroethyl)-1-nitroso-4-methyl-4-formyl-semicarbazide (methyl-formyl-amino-CNU, 3), 1-(2-chloroethyl)-1-nitroso-3-(methylene-2-pyridyl)-urea(methylene-2-pyridyl- CNU, 4), 1-(2-chloroethyl)-1-nitroso-3-(methylene-2-pyridyl)-urea hydrochloride (methylene-2-pyridyl-CNU . HCl, 5) 1-(2-chloroethyl)-1-nitroso-3-(methylene-4-pyridyl)-urea hydrochloride (methylene-4-pyridyl-CNU . HCl, 6), 1-(2-chloroethyl)-1-nitroso-3-(3-pyridino)-urea (pyridyl-3-CNU, 7), 1-(2-chloroethyl)-1-nitroso-3-(3-pyridyl)-urea hydrochloride (pyridyl-3-CNU . HCl, 8), 1-(2-chloroethyl)-1-nitroso-3-[4(2,6-dimethyl-morpholino)]-urea (dimethyl-morpholino-CNU, 9), 1-(2-chloroethyl)-1-nitrosocarbamoyl-morpholine (chloroethyl-nitroso-carbamoyl-morpholine, 10), 1-(2-chloroethyl)-1-nitroso-carbamoyl-2,6-dimethyl-morpholine (chloroethyl-nitroso-carbamoyl-dimethylmorpholine, 11). Against both neurogenic tumors cyclophosphamide was distinctly superior to all nitrosoureas. In leukemia L 5222 all nitrosoureas except compounds 7, 8, 11 effected cures. Remarkable differences in toxicity could be observed between the nitroso compounds investigated.     

Spin-labeled 1-alkyl-1-nitrosourea synergists of antitumor antibiotics

A new method for synthesis of four spin-labeled structural analogues of the antitumor drug 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), using ethyl nitrite for nitrosation of the intermediate spin-labeled ureas has been described. In vitro synergistic effects of 1-ethyl-3-[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)]-1-nitrosourea (3b) on the cytotoxicity of bleomycin and farmorubicin were found in human lymphoid leukemia tumor cells. We measured the tissue distribution of 3b in organ homogenates of C57BL mice by an electron paramagnetic resonance method. The spin-labeled nitrosourea was mainly localized in the lungs. Our results strongly support the development and validation of a new approach for synthesis of less toxic nitrosourea derivatives as potential synergists of antitumor drugs.     

Mechanism of action of 2-haloethylnitrosoureas on deoxyribonucleic acid. Nature of the chemical reactions with deoxyribonucleic acid.

1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) covalently cross-links DNA under physiological conditions. Methyl substitution at either carbon atom of the 2-chloroethyl portion of the molecule prevents cross-linking. Haloalkyi nitrosoureas including 3-chloropropyl, 4-chlorobutyl and 5-chloropentyl, although they readily alkylate DNA, exhibit no ability to cross-link DNA. 3-(2-Chloroethyl)-1-methylcytosine hydrochloride and N⁴-(2-chloroethyl)- 1-methylcytosine hydrochloride, similar to intermediates suggested in the cross-linking process, alkylate PM2-CCC-DNA readily. These two cytosine derivatives also cyclize readily to give 3,N⁴-ethano-1-methylcytosine closely similar to a species isolated from the treatment of poly-C with BCNU. A number of processes including the extent of DNA alkylation, measured with [¹⁴C]CCNU labeled in the ethylene portion of the molecule, as well as concomitant DNA single strand scission, and intramolecular alkylation and/or hydrolysis of the chloroethyl cytidine intermediate were investigated as to their effects upon the interstrand cross-linking process.     

Biochemical and cytotoxic properties of the isomeric forms of N,N'-bis[N-2-chloroethyl)-N-nitrosocarbamoyl] cystamine

Three isomeric forms of a cystamine-containing chloroethylnitrosourea, N,N'-bis[N-(2-chloroethyl)-N-nitrosocarbamoyl]cystamine (CNCC), have been identified and separated by high pressure liquid chromatography. Isomer S, 3,3'-bis[N-(2-chloroethyl)-N-nitrosocarbamoyl] ethyl disulfide, was significantly less cytotoxic than isomer C, 1,1'-bis [N-(2-chloroethyl)-N-nitrosocarbamoyl] ethyl disulfide, or isomer M, 1,3'-bis[N-(2-chloroethyl)-N-nitrosocarbamoyl] ethyl disulfide, in either a human Namalva lymphoblastoid or a rat Walker 256 carcinoma cell line. Both isomers S and C inhibited DNA synthesis at a 50 microM concentration. A structural analysis of the isomeric forms suggested that bioreduction of the disulfide bond would permit both isomers to produce isocyanate moieties which would carbamoylate intracellular proteins and depress nucleic acid synthesis. The reduced cytotoxic potential of isomer S is consistent with a prolongation in the half-life of production of alkylating carbonium species that lack the capacity to cross-link macromolecules. Overall, the relative position of the NH group within each of the nitrosourea isomers appears critical to the biological properties of the drug.