2-[N1-2-pyrimidyl-aminobenzenesulfonamido] ethyl 4-bis(2-chloroethyl) aminophenyl butyrate: a potent antitumor agent

2-[N'-2-Pyrimidyl-aminobenzenesulfonamido] ethyl 4-bis(2-chloroethyl) aminophenyl butyrate has been prepared by reaction of chlorambucil with sulfadiazine derivative. Schiffs base has been used as the protective group of the aromatic amine in the synthesis. It can be completely removed by the irradiation of 365 nm UV light at room temperature. The title compound exhibits a high antitumor activity with a therapeutic index (TI) of 47.55 which is twice that of chlorambucil's (TI: 22.84).     

Synthesis and biological activity of stable and potent antitumor agents, aniline nitrogen mustards linked to 9-anilinoacridines via a urea linkage

To improve the chemical stability and therapeutic efficacy of N-mustard, a series of phenyl N-mustard linked to DNA-affinic 9-anilinoacridines and acridine via a urea linker were synthesized and evaluated for antitumor studies. The new N-mustard derivatives were prepared by the reaction of 4-bis(2-chloroethyl)aminophenyl isocyanate with a variety of 9-anilinoacridines or 9-aminoacridine. The antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro without cross-resistance to taxol or vinblastine and showed potent antitumor therapeutic efficacy in nude mice against human tumor xenografts. It also showed that 24d was capable of inducing marked dose-dependent levels of DNA cross-linking by comet assay and has long half-life in rat plasma.     

Rapid determination of the anti-cancer drug chlorambucil (Leukeran™) and its phenyl acetic acid mustard metabolite in human serum and plasma by automated solid-phase extraction and liquid chromatography–tandem mass spectrometry

A bioanalytical method for the determination of the anticancer drug chlorambucil (Leukeran™) and its phenyl acetic acid mustard metabolite in human serum and plasma is described. Automated solid-phase extraction of the analytes is carried out with C₁₈ sorbent packed in a 96 well format microtitre plate using a robotic sample processor. The extracts are analysed by isocratic reversed-phase liquid chromatography using pneumatically and thermally assisted electrospray ionisation (TurboIonspray) with selected reaction monitoring. The method is specific and sensitive, with a range of 4–800 ng/ml in human serum and plasma for both parent drug and metabolite (sample volume 200 μl). The method is accurate and precise with intra-assay and inter-assay precision (C.V.) of <15% and bias <15% for both analytes. The automated extraction procedure is significantly faster than manual sample pre-treatment methods, a batch of 96 samples is extracted in 50 min allowing for faster sample turnaround. The method has been used to provide pharmacokinetic support to biocomparability studies of Leukeran™ following single doses of oral tablet formulations.     

A new approach for evaluating in vivo anti-leukemic activity using the SCE assay. An application on three newly synthesised anti-tumour steroidal esters

Three newly synthesised steroidal esteric derivatives of nitrogen mustard (compounds 1-3) were comparatively studied on a molar basis regarding their ability to induce sister chromatid exchanges (SCEs) in normal human lymphocytes in vitro and therapeutic effects on leukemia P388 bearing mice. Compounds 1 and 3 are modified steroidal esters of p-methyl-m-N,N-bis(2-chloroethyl)amino benzoic acid, and compound 2 is a modified steroidal ester of chlorambucil. All compounds induced statistically significant increases in SCEs and decreases in proliferation rate indices (PRIs) of cultured human lymphocytes and significantly increased the life span of P388 bearing mice. In this study, the doses applied for therapeutic purposes upon leukemia P388 bearing mice in vivo were derived from cytogenetic observations in normal human lymphocytes in vitro. A substantially better therapeutic effect was obtained compared to the effect achieved after the use of quite higher doses related with LD(10) values. We have demonstrated that the order of anti-tumour effectiveness of the treatment schedules of the three newly synthesised compounds tested (at doses derived from cytogenetic observations) coincides with the order of the cytogenetic effects they induce. The SCE assay appears to have an application in the clinical prediction of tumour sensitivity to potential chemotherapeutics.     

Synthesis, characterization and antitumor activity of copper(II) complex with nicotinamido-4-bis(2-chloroethyl)aminobenzaldimine.

Nicotinamido-4-bis(2-chloroethyl)aminobenzaldimine (NBAB) was synthesized and characterized by elemental analysis, IR and 1H NMR spectra. The complex of Cu(NBAB)2(NO3)2 was prepared in ethanol and characterized by elemental analysis, conductivity, cyclic voltammetry, IR, UV-Vis, fluorescence, CD and EPR spectra. The characteristic data suggest that the complex has an elongated octahedral structure, and NBAB behaves as bidentate in the keto form. The antitumor activities of NBAB and the complex against L1210 murine leukemia and K562 were investigated with both the MTT method and the colony formation test. The results in vitro indicate that antitumor activities of NBAB are superior to 2,2'-chlorodiethylamine hydrochloride (nitrogen mustard) for L1210, and inferior to nitrogen mustard for K562, but the antitumor activities of the complex for both cell lines are superior to nitrogen mustard.     

Aneugenic potential of the nitrogen mustard analogues melphalan, chlorambucil and p-N,N-bis(2-chloroethyl)aminophenylacetic acid in cell cultures in vitro

Melphalan (MEL), chlorambucil (CAB) and p-N,N-bis(2-chloroethyl)aminophenylacetic acid (PHE) are nitrogen mustard analogues, which are clinically used as chemotherapeutic agents. They also exert carcinogenic activity. The aim of this study was to investigate the aneugenic potential of the above drugs and the possible mechanism responsible for this activity. The Cytokinesis Block Micronucleus (CBMN) assay in combination with fluorescence in situ hybridization (FISH) was used in human lymphocyte cultures to evaluate micronucleus (MN) frequency. Pancentromeric probe (alpha-satellite) was applied to identify chromosomes in micronuclei and an X-chromosome specific centromeric probe was used to asses micronucleation and non-disjunction of this chromosome in binucleated cells. The effect of the above compounds on the organization of mitotic apparatus, as a possible target of chemicals with aneugenic potential, was investigated in C(2)C(12) mouse cell line by double immunofluorescence of alpha- and gamma-tubulin. We found that the studied drugs increased MN frequency in a linear dose-dependent manner primarily by chromosome breakage and in a lesser extent by an aneugenic mechanism. Non-disjunction and micronucleation of X-chromosome were also induced. Abnormal metaphase cells were linearly increased with concentration and characterized by abnormal centrosome number. Interphase cells with micronuclei and abnormal centrosome number were also observed. Since nitrogen mustards are highly reactive agents, with low selectivity and form covalent bonds with different nucleophilic sites in proteins and nucleic acids, it is reasonable to consider that one possible pathway for nitrogen mustard analogues to exert their aneugenic activity is through reaction with nucleophilic moieties of proteins or genes that are involved in the duplication and/or separation of centrosomes, resulting in abnormal centrosome number. Based on our results the carcinogenicity of nitrogen mustard analogues studied may be attributed not only to their activity to trigger gene mutation and chromosome breakage, but also to their aneugenic potential. Further studies are warranted to clarify the above two hypotheses.     

Relationships between structure, toxicity and genetic effects in Salmonella typhimurium and Saccharomyces cerevisiae for substituted aniline mustards

A series of 4-substituted aniline mustards of widely varying reactivities have been evaluated for their mutagenic effects in Salmonella typhimurium strains of varying uvrB gene and plasmid status, and for their ability to cause mitotic crossing-over in Saccharomyces cerevisiae. The 4-methyl aniline mustard N,N-bis(2-chloroethyl)-4-methylaniline and its corresponding half-mustard N-(2-chloroethyl)-4-methylaniline showed widely different effects in the various bacterial strains, with the half-mustard being much less toxic than the full mustard in the uvrB- strain TA100. However, in the uvrB+ strain TA1978+, possessing an intact excision repair system, both compounds were equally toxic and the full mustard was the more mutagenic. Both compounds were equally effective in promoting mitotic crossing-over in yeast. For a series of 4-substituted full mustards, the toxicity in S. typhimurium strain TA100 correlated with substituent electronic parameters in the same way as does mammalian cell toxicity, supporting the view that the primary mode of toxicity is via DNA cross-linking, even for unreactive analogues. However, there were no obvious correlations between substituent physiochemical properties and mutagenic potential in bacteria, suggesting that mutagenic events are subject to a variety of influences other than the reactivity of the mustard group. In contrast, the most chemically reactive compounds were the most toxic and most recombinogenic in yeast.     

Carcinogenic potency in rodents versus genotoxic potency in E. coli: a correlation analysis for bifunctional alkylating agents

The mutagenic (M), recombinagenic (R) and SOS inducing (I) potencies of 6 bifunctional directly acting alkylating agents (mitomycin C, thiotepa, chlorambucil, nitrogen mustard, bis(2-chloroethyl)ether and bis(2-chloroethyl)nitrosourea) were measured in an E. coli test system (E. coli multitest) as the integral under the yield-dose curve obtained for each event. This potency crresponds to the cumulative yield of the affected cell population over the entire effective dose range of the chemical treatment.

A weak mutagenic activity was detected only for mitomycin C and thiotepa. Except for bis(2-chloroethyl)ether, all agents were recombinagenic and SOS inducing.

When the 3 genotoxic potencies (M, R and I) of these bifunctional alkylating agents were correlated, separately or in combination, with the respective carcinogenic potencies in rodents, a highly significant correlation was obtained with both the recombinagenic and SOS inducing potencies.     

Participation of peroxisomal beta-oxidation system in the chain-shortening of a xenobiotic acyl compound

A drug, (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoic acid, was metabolized to 4-(1-imidazolylmethyl)benzoic acid in isolated hepatocytes of rats, which was enhanced markedly by the pretreatment of rats with clofibrate. With liver homogenates, the formation of the CoA-ester of this drug and its subsequent chain-shortening were demonstrated. In the series of these reactions, acyl-CoA synthetase, CoA, ATP and NAD were required, whereas cyanide did not inhibit the reaction. These results indicate that peroxisomes are capable of shortening the acyl side-chains of drugs by the beta-oxidation, giving an additional suggestion on the functions of peroxisomes.     

Role of O6-alkylguanine-DNA alkyltransferase in the resistance of mouse spermatogenic cells to O6-alkylating agents

The O(6)-alkylguanine-DNA alkyltransferase inactivator O(6)-benzylguanine was administered to BALB/c mice either alone or before exposure to 1,3-bis(2-chloroethyl)-1-nitrosourea to study the role of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase in the protection of the testis against anti-cancer O(6)-alkylating agents. Exposure of the mice to 1, 3-bis(2-chloroethyl)-1-nitrosourea or O(6)-benzylguanine alone did not produce any marked testicular toxicity at the times studied. Testicular O(6)-alkylguanine-DNA alkyltransferase concentrations were assayed between 0 and 240 min after O(6)-benzylguanine treatment and were shown to be > 95% depleted 15 min after treatment with O(6)-benzylguanine and remained at > 95% at all the times assayed. Histological examination, the reduction in testicular mass and the induction of spermatogenic cell apoptosis showed that this depletion significantly potentiated 1, 3-bis(2-chloroethyl)-1-nitrosourea-induced testicular damage after treatment. Major histological damage was apparent 42 days after treatment, demonstrating that the stem spermatogonia were significantly affected by the combination. These results demonstrate that O(6)-alkylguanine-DNA alkyltransferase plays a significant role in protecting the spermatogenic cells from damage caused by DNA alkylation and indicate that the observed toxicity may result from damage to stem spermatogonia.     

Effects of nitrosoureas on human DNA polymerase activities from acute and chronic granulocytic leukemia cells.

In vitro DNA synthesis by isolated cytoplasmic DNA polymerases of human leukemic cells was found to be inhibited by 1,3-bis(2-chloroethyl)-1-nitrosourea, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and 1-(2-chloroethyl)-3-(trans-4-methyl-cyclohexyl)-1-nitrosourea. 2-Chloroethyl isocyanate and cyclohexyl isocyanate, the decomposition products of 1,3-bis(2-chloroethyl)-1-nitrosourea and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, respectively, are as effective as their parent nitrosoureas in inhibiting the enzyme activity. Preincubation studies indicated that these compounds inhibit DNA synthesis primarily by altering the enzyme DNA polymerases without significantly affecting the DNA template activities.     

Utilization of Plasma Fatty Acid in Rat Brain: Distribution of [14C]Palmitate Between Oxidative and Synthetic Pathways

Radioactivity within individual brain compartments was determined from 5 min to 44 h after intravenous injection of [14C]palmitate into awake Fischer-344 rats, aged 21 days or 3 months. Total radioactivity peaked broadly between 15 min and 1 h after injection, declined rapidly between 1 and 2 h, and then more slowly. In 3-month-old rats, the lipid and protein brain fractions were maximally labeled within 15 min after [14C]palmitate injection, then retained approximately constant label for up to 2 days. Radioactivity in the aqueous brain fraction comprised mainly radioactive glutamate and glutamine, and peaked at 45 min, when it comprised 48% of total brain radioactivity, then decreased to 27% of the total at 4 h, 15% at 20 h, and 10% at 44 h. Percent distribution of radioactivity within the different brain compartments, 4 h after intravenous injection of [14C]palmitate, was similar in 21-day-old and 3-month-old rats, despite higher net brain uptake in the younger animals. The results indicate that about 50% of plasma [14C]palmitate that enters the brain of adult rats is incorporated rapidly into stable protein and lipid compartments. The remaining [14C]palmitate enters the aqueous fraction after beta-oxidation, and is slowly lost. At 4 h after injection, 73% of brain radioactivity is within the stable brain compartments; this fraction increases to 86% by 20 h.     

Halothane metabolism. Impairment of hepatic omega-oxidation of leukotrienes in vivo and in vitro.

Omega-oxidation of leukotrienes is the initial step of hepatic degradation and thus inactivation of these proinflammatory mediators. Omega-oxidation is followed by beta-oxidation of leukotrienes from the omega-end. After exposure of rats to a single dose of the anesthetic agent halothane, a transient decrease in leukotriene omega-oxidation was induced both in vivo and in vitro. In untreated rats, 44.1 +/- 6.0% of N-[3H]acetylleukotriene E4 injected intravenously was recovered unchanged in bile collected for 60 min in vivo; 46.5 +/- 3.0% was recovered as omega-/beta-oxidation products, of which 24.7 +/- 4.5% were associated with beta-oxidation products only (mean +/- SEM; n = 5). In rats receiving a single dose of halothane 18 h before the experiment, recovery of unchanged N-[3H]acetylleukotriene E4 was significantly increased to 79.8 +/- 4.8%, while the fraction of omega-/beta-oxidation products decreased to 9.0 +/- 1.7% (n = 5); 90 h after exposure to halothane, N-[3H]acetylleukotriene E4 recovery decreased to 30.0 +/- 3.0% and omega-/beta-oxidation products amounted to 49.1 +/- 3.8%; the fraction of beta-oxidation products was significantly increased to 43.1 +/- 3.4% (n = 5). Ten days after exposure of rats to halothane, the recoveries of N-[3H]acetylleukotriene E4, of omega-/beta-oxidation products, and of beta-oxidation products alone, returned to almost normal values. Microsomal fractions obtained from rat hepatocytes catalyzed the NADPH- and O2-dependent leukotriene omega-oxidation in vitro. The formation of omega-hydroxy-metabolites of leukotriene B4, leukotriene E4, and N-acetylleukotriene E4 was decreased by 50% in microsomal fractions obtained from rats 18 h and 90 h after halothane treatment, and returned back to control levels in microsomal fractions obtained 10 days after halothane treatment. The Km value of leukotriene B4 omega-oxidation revealed no significant change in enzyme affinity towards leukotriene B4; in contrast, as reflected by the reduction of the Vmax value by 65%, a decrease in the amount of the active enzyme in microsomes obtained from rats 18 h after halothane treatment was observed. Halothane-metabolism-dependent trifluoroacetylation of hepatic proteins may mediate this process. Thus, the time course of the density on immunoblots of trifluoroacetylated protein adducts paralleled that of the transient decrease in leukotriene omega-oxidation. In contrast to its omega-oxidation, leukotriene B4 synthesis from 5-hydroperoxyeicosatetraenoate was not inhibited in hepatocyte homogenates obtained from rats pretreated with halothane. The data suggest that metabolism of halothane causes a transient derangement of hepatic leukotriene homeostasis in vivo.     

Hydroxylation of the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) by rat liver microsomes

Ring hydroxylation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea was shown to occur in the presence of liver microsomes prepared from both normal and phenobarbital induced rats. The metabolite was identified by mass spectrometry after selective extraction and purification by liquid chromatography. The microsomal catalyzed reaction was oxygen and NADPH dependent, inhibited by carbon monoxide and induced 4–5 fold by $\text{in vivo}$ phenobarbital pre-treatment. Phenobarbital induced microsomes hydroxylated the substrate at a rate of 17.6 nmoles/min/mg protein at 37°. A Type I difference spectrum was observed with phenobarbital induced microsomes that also displayed a substrate binding constant (K_s of 4 × 10^?5 M.     

Induction of cytogenetic damage by modified steroidal derivatives of p-bis(2-chloroethyl)aminophenylacetic acid in human lymphocytes

The effect of modified steroids, containing alkylating agents, on SCE rates and on cell kinetics in cultured human lymphocytes was studied. The homo-aza-steroidal ester of p-bis(2-chloroethyl)aminophenylacetic acid (ASE) was found to be the most effective in causing markedly increased SCE rates and cell division delays. The androsterone ester of p-bis(2-chloroethyl)aminophenylacetic acid (AE-CAPA) was found to be next in order of effectiveness with the lactone ester (LE-CAPA), chlorambucil ester 3 beta-hydroxy-13a-amino-13,17-seco-5a-androstan-17-oic-13,17-lactam (CBC-HAAL) and chlorambucil (CBC) following. p-Bis(2-chloroethyl)aminophenylacetic acid (CAPA) had only a small effect and 3 beta-hydroxy-13a-amino-13,17-seco-5a-androstan-17-oic-13,17-lactam (HAAL) had no effect at all. A correlation between potency for SCE induction, effectiveness in cell division delay and previously established antitumor activity of these drugs was observed.     

Rapid assay for plasma chlorambucil and phenyl acetic mustard using reversed-phase liquid chromatography

A rapid assay for chlorambucil, a drug used for the treatment of chronic lymphocytic leukemia, and its major metabolite is described. Chromatographic behaviour of the two compounds on two different reversed-phase columns is discussed, as well as the kinetics of their hydrolysis in aqueous medium. The developed analysis can be applied to the determination of the plasma levels of the drug and its metabolite. No sample preparation is required and the spectrophotometric detection affords the sensitivity in the picomole range. Total analysis time is between 10 and 15 min.     

Evidence that O6-alkylguanine-DNA alkyltransferase becomes covalently bound to DNA containing 1,3-bis(2-chloroethyl)-1-nitrosourea-induced precursors of interstrand cross-links

The reaction of partially purified human O6-alkylguanine-DNA alkyltransferase with 1,3-bis(2-chloroethyl)-1-nitrosourea-treated DNA resulted in formation of a DNA-protein covalent complex. Complex formation required active alkyltransferase and brief treatment of DNA with the drug. DNA lost its capacity to form the complex once drug-induced DNA interstrand cross-links were completely formed. These results are consistent with a model in which the transferase catalyzes cleavage at O6-guanine and transfer of the alkyl moiety in a putative O6, N1-ethanoguanine intermediate of cross-link formation. DNA-protein complex formation presumably results when the transferase accepts the N1-ethanoguanine-DNA structure, analogous to its acceptance of simple alkyl groups.     

Melanocyte-Directed enzyme prodrug therapy (MDEPT): development of second generation prodrugs for targeted treatment of malignant melanoma.

Evaluation of second generation prodrugs for MDEPT, by oximetry, has highlighted structural properties that are advantageous and disadvantageous for efficient oxidation using mushroom tyrosinase. In particular, a sterically undemanding prodrug bis-(2-chloroethyl)amino-4-hydroxyphenylaminomethanone 28 was synthesised and found to be oxidised by mushroom tyrosinase at a superior rate to tyrosine methyl ester, the carboxylic acid of which is the natural substrate for tyrosinase. The more sterically demanding phenyl mustard prodrugs 9 and 10 were oxidised by mushroom tyrosinase at a similar rate to tyrosine methyl ester. In contrast, tyramine chain elongation via heteroatom insertion was detrimental and the rate of mushroom tyrosinase oxidation of phenyl mustard prodrugs 21 and 22 decreased by 10 nanomol/min.     

Alkylthioacetic acids (3-thia fatty acids) as non-beta-oxidizable fatty acid analogues: a new group of hypolipidemic drugs. III. Dissociation of cholesterol- and triglyceride-lowering effects and the induction of peroxisomal beta-oxidation

Previous work in this laboratory indicated that sulfur-substituted fatty acid analogues, 1.10-bis(carboxymethylthio)decane and alkylthioacetic acid, both non-beta-oxidizable compounds, and the beta-oxidizable alkylthiopropionic acid (1) caused, to different extents, dose-related hepatomegaly and proliferation of peroxisomes and enhanced peroxisomal fatty acid beta-oxidation. In the present study, treatment of normolipidemic rats with alkylthioacetic acid resulted in a dose- and time-dependent decrease in serum cholesterol and serum and liver triglycerides to an extent comparable to that of the 3-thiadicarboxylic acid. At hypolipidemic doses, alkylthioacetic acid caused no hepatomegaly, did not significantly alter peroxisome morphology, and only marginally affected peroxisomal beta-oxidation activity. Only at the highest, nonpharmacological doses of alkylthioacetic acid were these hepatic parameters increased, although to a lesser extent than by the 3-thiadicarboxylic acid. Hence, on the basis of dose- and time-related studies of the two compounds, data indicate that the hypotriglyceridemia and hypocholesterolemia were dissociated from induction of peroxisomal beta-oxidation and peroxisome proliferation. Palmitic acid and hexadecanedioic acid, both beta-oxidizable fatty acids, only marginally affected the serum and liver parameters. The beta-oxidizable fatty acid analogue, alkylthiopropionic acid lowered the serum triglycerides in normolipidemic rats. In contrast to the 3-thiadicarboxylic acid and alkylthioacetic acid, alkylthiopropionic acid treatment at hypolipidemic doses caused accumulation of triglycerides in the liver.