Induction of chromosomal aberrations in human lymphocytes by the antitumour alkylating agent homo-aza-steroidal ester of p-bis(2-chloroethyl)aminophenoxy acetic acid, in vitro

The clastogenic activity of the antineoplastic alkylating agent 3β-hydroxy-13-amino-13,17-seco-5-androstan-17-oic- 13,17-lactam-p-bis (2-chloroethyl) aminophenoxy acetic acid (NSC 294859) and its congeners was studied in human lymphocyte cultures in vitro. Cells were exposed to several concentrations of the drugs for 24 h. It was found that NSC 294859 reduces the mitotic index and causes chromosome- as well as chromatid-type aberrations in a dose-dependent way. From its congeners, the alkylating agent (p-bis (2-chloroethyl) aminophenoxy acetic acid) induces the same phenomena but to a lesser extent, while the modified steroid (3β-hydroxy-13-amino-13,17-seco-5-androstan-17-oic-13,17-lactam) causes cytogenetic damages at the control level. These results favour the assumption that the antitumor activity of NSC 294859 is mainly based on its cytogenetic effects.     

Altered protein synthesis rate in ovaries of D. melanogaster caused by new antitumour alkylating agents.

1. The effect of two homo-aza-steroidal esters with antineoplastic activity, namely 3 beta-hydroxy-13 alpha-amino-13,17-seco-5 alpha-androstan-17-oic-13,17-lactam-p-bis(2-chloroethyl)aminoph enoxyacetate (NSC 294859) and 3 beta-hydroxy-13 alpha-amino-13,17-seco-5 alpha-androstan-17-oic-13,17-lactam-p-bis(2-chloroethyl)aminoph enylacetate (ASE) on protein synthesis rate was studied in ovaries of Drosophila melanogaster females. 2. Two different concentrations for each compound were examined. 3. Both esters containing the same alkylating agent have been shown to decrease protein synthesis in relation to control.     

Comparative study on cytogenetic damage induced by homo-aza-steroidal esters in human lymphocytes

The effect of P[N,N-bis(2-chloroethyl)amino]phenylacetate esters of $\text{3β-}\text{hydroxy}\text{-}\text{methyl}\text{-17α-}\text{aza-}\text{d}\text{-}\text{homo}\text{-5α-}\text{androstan}\text{-17-}\text{one}$ (compound 3) and 3β-hydroxy-17α-aza-d-homo-5α-androstane (compound 2) on sister-chromatid exchange (SCE) frequencies and on human lymphocytes proliferation kinetics was studied. The results are compared with those of the P[N,N-bis(2-chloroethyl)phenylacetate esters of 3β-hydroxy-17α-aza-d-homo-5α-androstan-17-one (compound 1). All compounds were found to be active in inducing markedly increased SCE rates and cell division delays. A correlation between potency for SCE induction, effectiveness in cell division delay and previously established antitumour activity of these compounds was observed.     

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.     

Studies on anabolic steroids--8. GC/MS characterization of unusual seco acidic metabolites of oxymetholone in human urine.

One of the biotransformation routes of oxymetholone (17 beta-hydroxy-2-hydroxymethylene-17 alpha-methyl-5 alpha-androstan-3-one) in man leads to the formation of 17 beta-hydroxy-17 alpha-methyl-5 alpha-androstan-3-one (mestanolone). To demonstrate that this latter steroid may be formed by decarboxylation of an intermediate metabolite of oxymetholone bearing a 2-carboxylic group, we studied the urinary excretion of oxymetholone acidic metabolites. Five new acidic metabolites are reported here for the first time, among which four are unusual seco steroids resulting from the oxidative cleavage of the A-ring. The most abundant compound is 17 beta-hydroxy-17 alpha-methyl-2,3-seco-5 alpha-androstane-2,3-dioic acid 1, the cumulative excretion of which accounted for 1.52% of the dose. Three other seco diacids were produced in smaller amounts, namely 17 beta-hydroxy-17 alpha-methyl-2,3-seco-5 alpha-androstane-2,4- dicarboxylic acid 3, 17 beta-hydroxy-17 alpha-methyl-1,3-seco-5 alpha-androstane-1,3-dioic acid 4 and 17 beta-hydroxy-17 alpha-methyl-2,4-seco-5 alpha-androstane-2,4-dioic acid 5. The fifth acidic metabolite was identified as 3 alpha, 17 beta-dihydroxy-17 alpha-methyl-5 alpha-androstane-2 beta-carboxylic acid 2. The excretion in urine of these acidic metabolites suggests that the 2-hydroxymethylene group in oxymetholone is readily oxidized to yield the corresponding beta-keto acid which can be (1) decarboxylated to form mestanolone; (2) reduced at C-3 to give compound 2; and (3) further oxidized to afford the unexpected seco diacids 1, 3, 4 and 5. The identity of compounds 1 and 2 was ascertained by GC/MS and 1H and 13C-NMR analysis of reference compounds. The other metabolites were characterized by GC/MS analysis.     

Comparative study of SCE induction and cytostatic effects by homo-azasteroidal esters of N,N-bis(2-chloroethyl)aminobenzoic acid in human lymphocytes

The effect of homo-azasteroidal esters of benzoic acid mustard isomers and the 4-methyl derivatives, which have steroidal lactams as a biological basis, on cytogenetic damage was studied. Twenty compounds were comparatively studied, on a molar basis, as regards their ability to induce sister-chromatid exchanges (SCEs) and cell division delays.A correlation between potency for SCE induction, effectiveness in cell division delay and previously established antitumor activity of these compounds was observed.     

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.     

Enhancement by methylxanthines of sister-chromatid exchange frequency induced by cytostatics in normal and leukemic human lymphocytes

The effect of theobromine (TB) and diphylline (DP) or (1,2-dihydroxy-3-propyl)theophylline on SCE rates induced in vitro by mitomycin C (MMC), and the effect of caffeine on SCE rates induced in vitro by cytosine arabinoside (Ara-C) was studied. The combined treatments with MMC plus TB or DP showed the potentiating ability of the latter drugs. Caffeine also enhanced SCEs induced by Ara-C in cultured human lymphocytes. Caffeine and adriamycin (ADR) did not act synergistically on induction of SCEs. In a combined study, in vivo and in vitro, lymphocytes taken from 2 leukemic patients who had been given chlorambucil (CBC) or Ara-C by injection 3 h before, and then treated with caffeine in vitro, were found to have synergistically increased exchange frequencies.     

Esters of 2-(1-hydroxyalkyl)-1,4-dihydroxy-9,10-anthraquinones with melphalan as multifunctional anticancer agents

Eight esters of 2-(1-hydroxyalkyl)-1,4-dihydroxy-9,10-anthraquinone with melphalan were prepared and tested for their antitumor activity (S-180) and cytotoxicity. 2-[1-[4-(p-Bis(2-chloroethyl)-aminophenyl)-butanoyloxy]methyl]-1,4-dihydroxy-9,10-anthraquinone and 2-[1-[4-(p-bis(2-chloroethyl)-aminophenyl)-butanoyloxy]ethyl]-1,4-dihydroxy-9,10-anthraquinone showed remarkable antitumor activity (T/C, 265 and 272%).     

The metabolism of deuterated analogues of chlorambucil by the rat

The antitumour agent chlorambucil (4[4-bis(2-chloroethyl)aminophenyl]-butyric acid) is converted by beta-oxidation in vivo into phenylacetic mustard (2[4-bis(2-chloroethyl)aminophenyl]acetic acid). This process may be disadvantageous from a therapeutic viewpoint since the metabolite has half the therapeutic index of the parent drug against the Walker 256 carcinoma in rats. In seeking to retard beta-oxidation, selectively deuterated analogues have been synthesised and administered to rats. Plasma levels of phenylacetic mustard after giving chlorambucil-beta-d2 were lower than those given by unlabelled drug, but the therapeutic activity was not significantly altered by deuteration. A dehydro derivative of chlorambucil was detected as an intermediate in the beta-oxidation pathway. The isotopic compositions of this metabolite, and of recovered chlorambucil, were measured in plasma samples taken after giving labelled chlorambucil (alpha-d2 and beta-d2 variants) to rats. Deuterium was almost totally lost from the alpha-d2 form and from its metabolite after 30 min and partially lost in 10 min. The beta-d2 variant and its dehydro-derivative retained the label. Possible mechanisms for deuteration loss are discussed. The design of novel analogues, based on these metabolic studies, is proposed.     

Metabolism of chlorambucil by rat liver microsomal glutathione S-transferase

Clinical efficacy of alkylating anticancer drugs, such as chlorambucil (4-[p-[bis [2-chloroethyl] amino] phenyl]-butanoic acid; CHB), is often limited by the emergence of drug resistant tumor cells. Increased glutathione (gamma-glutamylcysteinylglycine; GSH) conjugation (inactivation) of alkylating anticancer drugs due to overexpression of cytosolic glutathione S-transferase (GST) is believed to be an important mechanism in tumor cell resistance to alkylating agents. However, the potential involvement of microsomal GST in the establishment of acquired drug resistance (ADR) to CHB remains uncertain. In our experiments, a combination of lipid chromatography/electrospray ionization mass spectrometry (LC/ESI/MS) was employed for structural characterization of the resulting conjugates between CHB and GSH. The spontaneous reaction of 1mM CHB with 5 mM GSH at 37 degrees C in aqueous phosphate buffer for 1 h gave primarily the monoglutathionyl derivative, 4-[p-[N-2-chloroethyl, N-2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG) and the diglutathionyl derivative, 4-[p-[2-S-glutathionylethyl] amino]phenyl]-butanoic acid (CHBSG2) with small amounts of the hydroxy-derivative, 4-[p-[N-2-S-glutathionylethyl, N-2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBSGOH), 4-[p-[bis[2-hydroxyethyl] amino]phenyl]-butanoic acid (CHBOH2), 4-[p-[N-2-chloroethyl, N-2-S-hydroxyethyl]amino]phenyl]-butanoic acid (CHBOH). We demonstrated that rat liver microsomal GST presented a strong catalytic effect on these reactions as determined by the increase of CHBSG2, CHBSGOH and CHBSG and the decrease of CHB. We showed that microsomal GST was activated by CHB in a concentration and time dependent manner. Microsomal GST which was stimulated approximately two-fold with CHB had a stronger catalytic effect. Thus, microsomal GST may play a potential role in the metabolism of CHB in biological membranes, and in the development of ADR.     

Mutagenicity and clastogenicity of the antineoplastic agents homo-azasteroidal ester of p-bis(2-chloroethyl)aminophenyl acetic acid and chlorambucil

The mutagenic and clastogenic effects of the antineoplastic agents homo-aza-steroidal ester (ASE) and chlorambucil (CBC) were tested for their ability to induce mutations in the Salmonella/microsome system and SCE in CHO cells in culture. ASE was found to be positive in strains TA1535 and TA100 and in the newer strain TA102 with and without metabolic activation, while CBC caused histidine reversion in strain TA102 after the addition of mammalian liver microsomal extract (S9). In addition, both agents were found to be strongly positive for SCE induction. The mutagenic and clastogenic actions of both agents were of a dose-response type.     

Mass spectra of methyl-branched hydrocarbons from eggs of the tobacco hornworm

Hydrocarbons from three homologous series of branched alkanes from the eggs of the tobacco hornworm, Manduca sexta (L.), were identified by mass spectrometry. Gas-liquid chromatography (GLC) peaks 37-A (equivalent chain length of 37.2) and 39-A (equivalent chain length of 39.2) were mixtures of 13-, 15-, 17-, and 19-methylheptatriacontane and 13-, 15-, 17-, and 19-methylnonatriacontane, respectively. GLC peaks 33-B, 37-B, and 39-B with equivalent chain lengths of 33.4, 37.4, and 39.4, respectively, were mixtures of 13,17- and 15,19-dimethyltritriacontane, 13,17-, 15,19-, and 17,21-dimethylheptatriacontane, and 13,17-, 15,19-, and 17,21-dimethylnonatriacontane, respectively. GLC peak 37-C (equivalent chain length of 37.6) was a mixture of 11,15,19-, 13,17,21-, and 15,19,23-trimethylheptatriacontane.     

The anticancer drug chlorambucil interacts with the human erythrocyte membrane and model phospholipid bilayers

The plasma membrane has gained increasing attention as a possible target of antitumor drugs. It has been reported that they act as growth factor antagonists, growth factor receptor blockers, interfere with mitogenic signal transduction or exert direct cytotoxic effects. Chlorambucil (4-[p-(bis[2-chloroethyl]amino)phenyl]butyric acid) is an alkylating agent widely used in the treatment of chronic lymphocytic leukaemia. Contradictory reports have been published concerning its interaction with cell membranes. Whereas a decrease in the fluidity of Ehrlich ascite tumor cells has been adduced, no evidences were found that chlorambucil changes membrane lipid fluidity and alkylating agents had effects in these systems even at highly toxic concentrations. Our results showed that chlorambucil at a dose equivalent to its therapeutical concentration in the plasma (3.6 microM) caused the human erythrocyte membrane to develop cup-shaped forms (stomatocytes). Accordingly to the bilayer couple hypothesis, this means that the drug is inserted into the inner monolayer of the erythrocyte membrane, a conclusion supported by X-ray diffraction performed on multilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. It is concluded that the cytotoxic effect of chlorambucil might be due to alteration of the structure and therefore of the physiological properties of cell membranes such as fluidity, permeability, receptor and channel functions.     

Production of 19-oic-11-deoxycorticosterone from 19-oxo-11-deoxycorticosterone by cytochrome P-450(11)beta and nonenzymatic production of 19-nor-11-deoxycorticosterone from 19-oic-11-deoxycorticosterone

19-Oxo-11-deoxycorticosterone was incubated with a cytochrome P-450(11)beta-reconstituted system, and the metabolites were analyzed by high performance liquid chromatography(HPLC). The main product found after chromatography was collected and treated with diazomethane. HPLC and 1H-NMR analysis of the methylated derivative indicated that it was 19-oic-11-deoxycorticosterone methyl ester. When 19-oic-11-deoxycorticosterone was stored at -20 degrees C for 1 month, it was spontaneously converted to other steroids. Structural analysis of the main degradation product indicated that it was 19-nor-11-deoxycorticosterone. These results suggest that the conversion of 19-oxo-11-deoxycorticosterone to 19-oic-11-deoxycorticosterone occurs through the P-450(11)beta-catalyzed reaction, and that the 19-oic-11-deoxycorticosterone thus formed is nonenzymatically converted into 19-nor-11-deoxycorticosterone.     

Studies of the synthesis of biomarkers. X. Synthesis of 13,17-secodiacholestanes.

13,17-Secodiacholestanes (6) were synthesized from cholesterol (1) in six steps. The key intermediates, (20R)- and (20S)-diacholest-13(17)-enes (3a and 3b), underwent ozonization and reduction to provide (20R)- and (20S)-13,17-secodiacholesta-13,17-dione (5a and 5b), respectively. On Clemmensen reduction, the diones (5a and 5b) yielded the target molecule 6. The structure of an unknown biomarker was shown to be different from the proposed 6 by gas chromatographic/mass spectrometric comparison.     

Synthesis and cytogenetic studies of structure--biological activity relationship of esters of Hecogenin and aza-homo-Hecogenin with N,N-bis(2-chloroethyl)aminocinnamic acid isomers

The esters of Hecogenin and aza-homo-Hecogenin with N,N-bis(2-chloroethyl)aminocinnamic acid isomers have been prepared and their cytogenetic studies of structure-biological activity relationship were evaluated. The cytogenetic effects (sister chromatid exchanges (SCEs) induction and proliferation rate indices (PRIs) depression) by o-, m- and p-[N,N-bis(2-chloroethyl)amino] cinnamic acid were also investigated. Among the above compounds tested, those of the m-[N,N-bis(2-chloroethyl)amino] cinnamic acid and of the o-[N,N-bis(2-chloroethyl)amino] cinnamic acid ester of aza-homo-Hecogenin were more active in comparison to the others.     

Enhancement of cytogenetic damage by chlorpromazine in human lymphocytes treated with alkylating antineoplastics and caffeine

In cultured human lymphocytes chlorpromazine (CPZ) was found to induce cell division delays and to have no effect on sister-chromatid exchanges (SCEs) or on mitotic indices (MIs). CPZ induces cytotoxic effects in combination with caffeine (CAF) and alkylating agents. In combination with CAF it induced cell division delays and suppression of MIs. In combination with melphalan (MEL) and CAF, CPZ synergistically induced SCEs, caused cell division delay and suppressed MIs. In combination with chlorambucil (CBC) and CAF, CPZ produced synergism on induction of SCEs, enhanced cell division delays and reduced MIs.     

Changing the specificity of α-amino acid ester hydrolase toward para-hydroxyl cephalosporins synthesis by site-directed saturation mutagenesis

α-Amino acid ester hydrolases (AEHs) catalyze the synthesis of β-lactam antibiotics containing an α-amino group with decreased activity toward antibiotics with a p-hydroxyl group. The AEH gene from Xanthomonas rubrillineans was cloned and expressed in Escherichia coli. Based on the crystal structure of the AEH and cefprozil complex, 13 residues not directly involved in substrate recognition were mutated individually. The resulting ~1,300 mutants were screened for activity using cefprozil as a model product based on spectrophotometric assay in a 96-well format. Mutants with improved cefprozil synthetic activity revealed the particular importance of positions 87, 131 and 175 for specificity. The mutant V131S with the highest initial rates of synthesis toward three p-hydroxyl cephalosporins showed 23?%, 17?% and 64?% increase in maximum product accumulation of cefadroxil, cefprozil and cefatrizine, respectively.     

Activity of the diastereoisomers of 13,23-dimethylpenta-triacontane, the sex pheromone of Glossina pallidipes, and comparison with the natural pheromone

ABSTRACT. All three synthetic diastereoisomers of 13,23-dimethyl-pentatriacontane, the pheromone of Glossina pallidipes (Austen), have been tested in the G. pallidipes sex pheromone bioassay, together with one of the four diastereoisomers of 13,17-dimethylpentatriacontane, originally proposed as a candidate pheromone of G. pallidipes. Of the three isomers of 13,23-dimethylpentatriacontane only the (13R,23S) isomer was active with an ED-50 of 4.47±1.09/μg (mean±SD). The (13R,17R)-13,17-dimethylpentatriacontane was inactive. The diastereo-isomeric mixtures of 13,17- and 13,23-dimethylpentatriacontane were active with ED-50 values of 17.88±1.25/μg and 8.88±1.15μg respectively. The natural pheromone was active with an ED-50 of 8.07±1.17 μg indicating it to be a diastereoisomeric mixture.