Reactions of 1,3-bis(2-chloroethyl)-1-nitrosourea and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in aqueous solution

Products formed from the reaction of two chloroethylnitrosoureas in neutral aqueous solution have been identified and quantified. Mixture components recovered after a 1-h incubation period accounted for 75--85% of the starting nitrosourea. Approximately 65--85% of the reaction products were formed by an initial cleavage of the nitrosourea to the proposed intermediates 2-chloroethyl azohydroxide and an isocyanate and by subsequent hydrolytic reactions. A minor pathway, 5--10% of products, involves denitrosation of the nitrosourea with oxazoline formation. Stable isotope labeling and mass spectrometry have been used to determine the reaction sequence and product origins. Reaction product identification has been made using high-performance LC isolation and comparison with synthetic material.     

Enhanced Oxygen Toxicity following Treatment with 1,3-Bis(2-chloroethyl)-1 -nitrosourea

Enhanced Oxygen Toxicity following Treatment with l,3-Bis(2-chloroethyi)-l-nitrosourea.Kehrer, J. P., AND PARAIDATHATHU, T. (1984). Fundam. Appl. Toxicol.4, 760–l767. The anticancer drug l,3-bis(2-chloroethyl)-l-nitrosourea(BCNU) inhibits glutathione reductase, an enzyme involved inoxidant defense systems. The 30-day LD50 for BCNU in male andfemale BALB/c mice was 52 and 46 mg/kg, respectively. A 35-mg/kgBCNU dose was not lethal to any animal. Glutathione reductasewas inhibited in lung tissue by about 50% for 4 days followinga single 35 mg/kg dose of BCNU. The prolonged inhibition ofglutathione reductase by BCNU suggested this drug might enhancepulmonary oxygen toxicity by diminishing the lung's antioxidantcapacity. Exposing mice treated with 35 or 50 mg/kg BCNU tocontinuous 85% oxygen decreased the LT50 from 13.1 to 6.3 and5.3 days, respectively, compared to vehicle-treated controls.All mice treated with 35 mg/kg BCNU or vehicle and exposed to85% oxygen only on Days 0–4 survived to Day 30. Extendingthe hyperoxic exposure 1 additional day resulted in the deathof all BCNU-treited mice, while 70% of the vehicle-treated micesurvived to Day 30. Pulmonary glutathione peroxidase, catalase,and superoxide dismutase activities were unaffected up to 6days following 35 mg/kg BCNU, 85% oxygen, or both. Pulmonaryglutathione reductase activity was unaffected by 85% oxygenalone, although hyperoxia extended the BCNU-induced inhibitionof this enzyme to Day 6. BCNU, 35 mg/kg, had little effect onlung reduced glutathione (GSH) levels. A significant decreasewas only measured on Day 4. Hyperoxia, either alone or withBCNU, had no effect on lung GSH content The total lung contentof hydroxyproline, an index of fibrosis, was unchanged by BCNU-treatmentseither alone or in combination with oxygen. These data showthat single BCNU doses of 35 mg/kg or more can increase thelethal effect of hyperoxia. The mechanism of this effect remainsunclear since, while glutathione reductase was the only antioxidantenzymatic activity inhibited, GSH levels remained unchanged.     

Urinary metabolites of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea

Urinary metabolites of ring 14C-labeled 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (Methyl CCNU) from rats have been isolated and characterized by high-performance liquid chromatography and mass spectrometry. About 44% of the cyclohexyl moiety of CCNU was excreted in 24 hr and included approximately 10% of the excreted dose as free amines and 40% as conjugates that could be converted to amines by hydrolysis. Amine composition of free base plus hydrolyzable conjugates was 55% hydroxycyclohexylamines (3-trans, 3-cis, 4-cis, and 4-trans) and 30% cyclohexylamine. This strongly supports previous studies which indicated that CCNU is largely hydroxylated in vivo as well as in vitro. Rats pretreated with phenobarbital excreted high relative amounts of cis-4-hydroxy derivatives (41%), again showing a high degree of correlation between in vitro and in vivo results. Treatment of urine with beta-glucuronidase gave no apparent increase in free amines. However, sulfatase was about 25% as effective as alkaline hydrolysis for releasing free amines from whole urine. Major urinary metabolites were found to have m.w. of about 629, 413, 329, and 243 and represented 55%, 20%, 20%, and 5% of total excreted 14C, respectively. It was concluded that the higher m.w. metabolites may be conjugates of peptides possibly derived from active site-directed inactivation of specific enzymes. Previous work has shown that enzymes such as chymotrypsin and glutathione reductase are inhibited by isocyanates in this manner. Hydroxylated metabolites of Methyl CCNU had a pattern similar to that of CCNU. The major free (12%) and conjugated amine (54%) metabolites of Methyl CCNU in the urine in decreasing order of quantity present were cis-3-hydroxy-trans-4-methylcyclohexylamine, trans-4-methylcyclohexylamine, trans-4-hydroxymethylcyclohexylamine, and trans-3-hydroxy-trans-4-methyl-cyclohexylamine.     

In vitro pharmacokinetics and pharmacodynamics of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)

The relationship between treatment efficacy and the pharmacokinetics (PK) and pharmacodynamics (PD) of anticancer drugs is poorly defined. 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU) is an alkylating agent used in the treatment of brain and other forms of cancer. It is postulated that BCNU kills cells by forming DNA interstrand cross-links. The present study was undertaken to characterize the PK and PD of BCNU in mouse L1210 cells. L1210 cells were exposed to BCNU (0-160 microM) and analyzed for intracellular BCNU concentrations, DNA interstrand cross-links, cell cycle phase, and cytotoxicity. The half-life of BCNU in cells was approximately 40 min. The maximum reduction of mitochondrial enzyme activity (maximum cell death) achieved within 24 hr after exposure to BCNU was concentration-dependent and could be described by a Hill equation. At lower concentrations, the area under the DNA interstrand cross-link-time curve linearly correlated with the maximum cell death and the area under the BCNU concentration-time curve. BCNU induced cell accumulation in the G(2)/M phase of the cell cycle, which continued even after apparent completion of cross-link repair. Loss of membrane permeability was minimal (approximately 2%) during the first 24 hr. Thereafter, cells died exponentially over the next 9 days, primarily by necrosis. In conclusion, while cytotoxicity was concentration-dependent, an indirect relationship was found among the time-course of BCNU concentrations, DNA interstrand cross-links, and cell death. Because of the disparity between the time-scale of PK and PD, focusing only on the early events may provide limited information about the process of anticancer drug-induced cell death.     

Stereoselective monooxygenation of carcinostatic 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea by purified cytochrome P-450 isozymes

Three highly purified forms of liver microsomal cytochrome P-450 (P-450a, P-450b and P-450c) from Aroclor 1254-treated rats catalyzed 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea (CCNU) and 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU) monooxygenation in the presence of purified NADPH-cytochrome P-450 reductase, NADPH, and lipid. Differences in the regioselectivity of CCNU and MeCCNU monohydroxylation reactions by the cytochrome P-450 isozymes were observed. Cytochrome P-450-dependent monooxygenation of CCNU gave only alicyclic hydroxylation products, but monooxygenation of MeCCNU gave alicyclic hydroxylation products, an αhydroxylation product on the 2-chloroethyl moiety, and a trans-4-hydroxymethyl product. A high degree of stereoselectivity for hydroxylation of CCNU and MeCCNU at the cis-4 position of the cyclohexyl ring was demonstrated. All three cytochrome P-450 isozymes were stereoselective in primarily forming the metabolite cis-4-hydroxy-trans-4-Methyl-CCNU from MeCCNU. The principal metabolite of CCNU which resulted from cytochromes P-450a and P-450b catalysis was cis-4-hydroxy CCNU, whereas the principal metabolites from cytochrome P-450c catalysis were the trans-3-hydroxy and the cis-4-hydroxy isomers. Total amounts of CCNU and MeCCNU hydroxylation with cytochrome P-450b were twice that with hepatic microsomes from Aroclor 1254-treated rats. Catalysis with cytochromes P-450a and P-450c was substantially less effective than that observed with either cytochrome P-450b or hepatic microsomes from Aroclor 1254-treated rats.     

Chemico-Pharmacological Investigation of a New Medicinal Form Of 1,3-Bis(2-chloroethyl)-1-nitrosourea

Pharmaceutical Chemistry Journal -     

Hydroxylation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea.

Rat liver microsomal mixed function oxidase catalyzes the hydroxylation of the cyclohexyl moiety of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) to give at least five metabolites. When exposed to alkaline pH at 100° CCNU and its metabolites quantitatively release their cyclohexyl moiety as cyclohexylamine and aminocyclohexanol respectively. The N-(2,4-dinitrophenyl) derivatives of cyclohexylamine and aminocyclohexanols were separated by high pressure liquid chromatography. The metabolites in vitro and in vivo have been identified as trans-2-hydroxy CCNU, cis-3-hydroxy CCNU, trans-3-hydroxy CCNU, cis-4-hydroxy CCNU and trans-4-hydroxy CCNU. Ring hydroxylation axial to the 1-(2-chloroethyl)-1-nitrosourea group (cis-2-, trans-3-, cis-4-) is favored over equatorial attack (trans-2-, cis-3-, trans-4-). Pretreatment of rats with phenobarbital leads to an increased rate of hydroxylation and a change in the relative amounts of the hydroxylated products. The significance of hydroxylation in relation to the antitumor activity of CCNU is discussed.     

Effect of 1,3-bis(2-chloroethyl)-1-nitrosourea on newly synthesized DNA in L-1210 cells.

The synthesis of DNA in L-1210 cells was selectively inhibited by BCNU [1,3-bis-(2-chloroethyl)-1-nitrosourea, NSC 409962]. When the DNA chain growth in exponentially grown L-1210 cells was analyzed by alkaline sucrose gradient centrifugation, the initially synthesized DNA had short segments (approximately 5S) which increased in size to 30, 70 and to over 100S. When treated with BCNU, the short, newly synthesized DNA segments accumulated; the molecular weights were 5-30S. While it appears that BCNU inhibits the early elongation steps of newly synthesized DNA, the chasing experiments suggest that the drug does not affect the joining process from replicon-sized DNA (70S) to bottom peaks with a molecular weight of over 100S.     

Protection of CHO cells by mutant forms of O6-alkylguanine-DNA alkyltransferase from killing by 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) plus O6-benzylguanine or O6-benzyl-8-oxoguanine.

O6-Benzylguanine (BG) is an inactivator of human O6-alkylguanine-DNA alkyltransferase (AGT) currently undergoing clinical trials to enhance cancer chemotherapy by alkylating agents. Mutant forms of AGT resistant to BG in vitro were expressed in CHO cells to determine if they could impart resistance to killing by the combination of BG and 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU). All the BG-resistant mutant proteins tested (P140A, P140K, P138M/V139L/P140K, G156A, P140A/G160R, and G160R) showed a reduced rate of reaction with methylated DNA substrates in vitro. However, when expressed in equal amounts in CHO cells, mutants P140A, P140K, P138M/V139L/P140K, and G160R gave levels of protection from the chloroethylating agent BCNU equivalent to that of wild-type AGT. This indicates that a 10-fold reduction in rate constant did not prevent their ability to repair chloroethylated DNA in the cell. AGT activity was readily lost when CHO cells expressing wild-type AGT were exposed to BG or its 8-oxo metabolite (O6-benzyl-8-oxoguanine), but cells expressing mutants P140A or G160R required 30-fold higher concentrations and cells expressing mutants P140K or P138M/V139L/P140K were totally resistant. When cells were treated with 80 microM BCNU plus BG or 8-oxo-BG, those expressing wild-type AGT were killed when inhibitor concentrations of up to 500 microM were used, whereas cells expressing P140K or P138M/V139L/P140K showed no effect, and cells expressing P140A or G160R showed an intermediate resistance. These results suggest that: (i) appearance of BG-resistant mutant AGTs may be a problem during therapy, and (ii) the P140K mutant AGT is an excellent candidate for gene therapy approaches where expression of a BG-resistant AGT in hematopoietic cells is used to reduce toxicity.     

Synthesis and characterization of DNA containing an N1-2'-deoxyinosine-ethyl-N3-thymidine interstrand cross-link: a structural mimic of the cross-link formed by 1,3-bis-(2-chloroethyl)-1-nitrosourea

Interstrand cross-links, which are generated by chemotherapeutic treatment with bis-alkylating agents, exert their therapeutic effect by connecting the nucleobases of adjacent DNA strands together and represent some of the most threatening forms of damage suffered by genomic DNA. However, one of the reasons for treatment failure using these agents is due to enhanced repair of this DNA damage. The pursuit of understanding the repair of interstrand cross-links by repair systems has necessitated the synthesis of sufficient quantities of such damaged DNA. We report the synthesis of a site-specific interstrand cross-linked duplex containing an ethylene-bridged N (1)-2'-deoxyinosine- N (3)-thymidine base pair prepared by solution and solid-phase synthesis as a mimic for the lesion formed by the therapeutic agent 1,3-bis-(2-chloroethyl)-1-nitrosourea using both a phosphoramidite and a bis-phosphoramidite approach. UV thermal denaturation experiments revealed that this cross-linked duplex was stabilized by 52 degrees C relative to the noncross-linked control, and circular dichroism studies indicated little deviation from a B-form structure compared to a duplex that contained a G-C base pair at the same position. Molecular models of the cross-linked duplex that were geometry optimized using the AMBER forcefield also suggest that this lesion induces minimal distortion in B-form DNA. This modified oligonucleotide will be useful for studies related to the investigation of interstrand cross-linked DNA repair.     

Molecular structure of 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea

The three-dimensional structure of 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea (MeCCNU, NSC-95441), an effective antitumor agent, has been determined by single-crystal x-ray diffraction. MeCCNU crystallizes in monoclinic space group P21/c, with cell dimensions a = 12.387, b = 10.810, and c = 10 .198 A , beta = 102.62 degrees , and Z = four molecules per unit cell. The structure was solved by direct phasing procedures and refinement by anisotropic least squares converged at a discrepancy index R = 0.065. The cyclohexyl ring is in the chair conformation with the plane of the nitrosourea moiety twisted approximately 90 degrees from the cyclohexyl ring. The carbon-nitrogen bonds of the urea group are significantly asymmetric.     

Preferential alkylation by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) of guanines with guanines as neighboring bases in DNA

The base sequence of DNA has been shown to influence the kinds and amounts of alkylation of purine bases by N-methyl-N-nitrosourea [W. T. Briscoe and L-E. Cotter, Chem. Biol. Interact. 56, 321 (1985)]. In the present study, the alkylation of DNA polymers of defined sequence by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) has been investigated. The assay involved treating poly (dG).poly(dC), poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT), poly(dA-dG).poly(dC-dT), and calf thymus DNA with BCNU, followed by hydrolysis to release the modified purine bases and separation and quantitation of these by HPLC. Analysis of the results revealed that there was a 24-fold increase of 7-(beta-hydroxyethyl)guanine (HOEtG) in poly(dG).poly(dC) relative to poly(dA-dG).poly(dC-dT). There was also a 3-fold increase in HOEtG in poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT) and calf thymus DNA relative to poly(dA-dG).poly(dC-dT). A 2- to 4-fold increase of 7(beta-aminoethyl)guanine (AmEtG) was observed in poly(dG-dC).poly(dG-dC) relative to the other polymers tested. This study has determined that guanines in certain base sequences in polydeoxyribonucleotides are more susceptible to BCNU alkylation at the N-7 position than guanines in other sequences.     

Effect of 7-hydroxymethyl-12-methylbenz[a]anthracene and 1,3-bis-(2-chloroethyl)-1-nitrosourea on enzyme activities and oxidation of glutathione in cultured rat adrenal cells

1. The activities of enzymes participating in the regeneration of reduced glutathione (GSH), and their subcellular distribution were studied in cultured rat adrenal cells. 2. It has previously been shown that the adrenocorticolytic agent 7-hydroxymethyl-12-methylbenz[a]anthracene (7-hydroxymethyl-12-MBA) causes a drastic and selective oxidation of mitochondrial GSH in rat adrenal cells. Treatment of the adrenal cells with 7-hydroxymethyl-12-MBA, resulted in a minor decrease in the content of cytochrome c oxidase, nicotinamide nucleotide transhydrogenase, isocitrate dehydrogenase and cytosolic GSH reductase, whereas the activity of lactate dehydrogenase and citrate synthase was unaffected. None of these effects were considered to be responsible for the massive oxidation of mitochondrial GSH induced by 7-hydroxymethyl-12-MBA. 3. 1,3-Bis-(2-chloroethyl)-1-nitrosourea (BCNU) was used to obtain rat adrenal cells cultures with inactivated cytosolic and mitochondrial GSH reductase. The oxidation of mitochondrial GSH, induced by 7-hydroxymethyl-12-MBA, was not dramatically enhanced by the inactivation of GSH reductase, indicating that this enzyme was not rate-limiting in the regeneration of GSH. 4. Fractionation of rat adrenal cells with increasing concentrations of digitonin resulted in an earlier release of citrate synthase in cells treated with 7-hydroxymethyl-12-MBA compared with controls. These results may indicate damage to mitochondrial membranes as a result of 7-hydroxymethyl-12-MBA treatment.     

Potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-induced cytotoxicity in 9L cells by pretreatment with 6-thioguanine

9L Rat brain tumor cells were treated with 0.2 microM 6-thioguanine for 48 hr, which produced a 40% cell kill, a small (15%) inhibition of cell growth, and an accumulation of cells in S-phase. Maximum incorporation of [14C]6-thioguanine into cellular DNA occurred after 24 hr of incubation; 70% of the label was incorporated into DNA as 6-thio-2'-deoxyguanosine. Pretreatment of 9L cells for 48 hr with 0.2 microM 6-thioguanine potentiated the cytotoxicity of 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) by 50% with a dose enhancement ratio of 1.5, and caused a 30% increase in the number of BCNU-induced sister chromatid exchanges (SCEs) and a 50% increase in DNA crosslinks formed, compared to treatment with BCNU alone. Used as a single agent, 6-thioguanine induced a significant number of SCEs. Results suggest that these effects may be related to the increased formation of DNA crosslinks, possibly as the result of the formation of S6-(2-chloroethyl)-6-thioguanine in cellular DNA.