Thomas A. Connors

Some studies of the active intermediates formed in the microsomal metabolism of cyclophosphamide and isophosphamide

Abstract Evidence is presented in support of the following metabolic pathways, in the liver, of the antitumour agent cyclophosphamide 2-[bis(2-chloroethyl)amino]-tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide. The drug is first converted, presumably by the mixed-function oxidases, into 4-hydroxycyclophosphamide which may then break down by elimination of acrolein from its tautomeric form, aldophosphamide, to yield phosphoramide mustard [N,N-bis(2-chloroethyl)phosphorodiamidic acid], a known cytotoxic agent. In competition with this process is the enzymic conversion of 4-hydroxycyclophosphamide (by dehydrogenation) and aldophosphamide (by oxidation) into the known in vivo metabolites of cyclophosphamide, 4-ketocyclophosphamide and carboxyphosphamide, respectively, each of which has low cytotoxicity. 4-Hydroxycyclophosphamide, which was too unstable to allow identification directly by conventional procedures, was trapped by reaction with ethanol. The resulting two, apparently isomeric, ethyl derivatives, (1) were amenable to mass spectrometry, (2) yielded acrolein 2,4-dinitrophenylhydrazone on treatment with acidic 2,4-dinitrophenylhydrazine, (3) were hydrolysed in water (pH 4.3), each isomer apparently regenerating 4-hydroxycyclophosphamide, (4) were highly toxic to Walker tumour cells in culture. Phosphoramide mustard was also isolated after in vitro metabolism of cyclophosphamide. On the basis of a bioassay involving Walker tumour cells in whole animals it appeared that, of the known metabolites of cyclophosphamide, only phosphoramide mustard possesses the cytoxicity and biological half-life appropriate to the active antitumour metabolite. Four other metabolites of low cytotoxicity were isolated and identified, namely, 4-ketocyclophosphamide, carboxyphosphamide, 2-(2-chloroethylamino)tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide, and 3-hydroxypropyl-N,N-bis(2-chloroethyl)phosphorodiamidate. The significance of metabolic detoxification processes in relation to the selective cytotoxicity of cyclophosphamide towards tumour cells in vivo is discussed. The metabolic activation of isophosphamide appears to follow a pathway similar to that of cyclophosphamide.

Tumour inhibitory triazenes: Structural requirements for an active metabolite

Abstract A series of aryldialkyltriazenes and some related compounds have been investigated for their anti-tumour properties. Unlike the imidazole-triazenes in clinical use, aryltriazenes are stable in light and do not undergo photodecomposition to toxic diazonium salts. Some of the triazenes investigated had good anti-tumour activity yet did not form diazonium salts under physiological conditions, implying that this pathway is not important for anti-tumour action. Evidence has been obtained that only aryltriazenes that can be metabolized in vivo to an aryl- N 3 -monomethyltriazene have anti-tumour properties. It was also found that the aryltriazenes were dose-schedule dependent in their anti-tumour action, probably a consequence of their short biological half-lives.

Studies on the mechanism of action of the tumour inhibitory triazenes.

Abstract A series of imidazole and phenyl dialkyi triazenes were synthesized and investigated for their anti-cancer activity in experimental animals. Active triazenes had a broad spectrum of anti-tumour action and like bischloroethylnitrosourea (BCNU) were active against tumours not sensitive to conventional alkylating agents. It was confirmed that at least one N -methyl group was necessary for anti-cancer activity but there was no correlation between dealkylation by liver microsomes and activity since a diethyl triazene was readily dealkylated but not active. A factor appears to be present in normal cell cytoplasm which can detoxify triazenes but which is absent from tumours sensitive to these agents.

Metabolism of aniline mustard [N,N-di-(2-Chloroethyl)-aniline]

The possible metabolic activation of the antineoplastic agent N,N-di-(2-chloroethyl) aniline (aniline mustard) is discussed. Conversion of aniline mustard into the glucuronide (p-di-2-chloroethylaminophenyl-β-d-glucopyranosid)uronic acid was mediated by a rat liver homogenate containing the appropriate cofactors. The glucuronide was a major metabolite in the serum and bile after administration of aniline mustard to rats and after isolation and purification it was identified as its methyl ester by mass spectrometry. The use of Amberlite XAD-2 resin facilitated the isolation from serum of the glucuronide and another metabolite, N-(2-chloroethyl)-4-hydroxyaniline. The implication of these findings for the clinical application of aniline mustard is discussed.

Chemical trapping of a reactive metabolite: The metabolism of the azo-mustard 2′-carboxy-4-di-(2-chloroethyl)amino-2-methylazobenzene

Abstract A general approach of potential value for the identification of reactive metabolites derived from anti-tumour (or other) agents involves chemical trapping with suitable radioactive reagents. The method, which may preclude the need for radioactively labelled drugs, is exemplified by the identification of the reactive metabolite formed on reduction of the azo-mustard, 2′-carboxy-4-di-(2-chloroethyl)amino-2-methylazobenzene (CB 1414) using a rat liver homogenate. The metabolite was shown to be 4-di-(2-chloroethyl)amino-2-methylaniline since trapping with sodium sulphide- 35 S gave the corresponding radioactively labelled and chemically stable thiazan, 2-methyl-4-(thiazan-4-yl)aniline which was characterized by mass spectrometry. The application of radioactive thiazan formation as a general method for the identification of nitrogen mustards in biological systems is discussed.

Studies of the mechanism of action of the tumour- -inhibitory nitrosoureas.

Abstract BCNU [1,3 bis-(2-chloroethyl)-1-nitrosourea] and some related nitrosoureas have been shown to have a wide spectrum of action against a number of transplanted rodent tumours. No correlation was found between the chemical instability of a nitrosourea and its antitumour activity. Unlike difunctional alkylating agents, the nitrosoureas inhibit the incorporation of tritiated precursors in DNA, RNA and protein to equal extents, the inhibition of tritiated thymidine incorporation into DNA occurring within 5 min of incubating cells with BCNU. Although the biological half life of BCNU was found to be very short (15 min by bioassay) a single injection was as effective against the established and widely-disseminated TLX5 lymphoma as against the early transplant. BCNU interfered specifically with the incorporation of labelled thymidine triphosphate into DNA, but no inhibition of DNA polymerase could be demonstrated at physiological dose levels. In their mechanism of action and in their biological properties the tumour inhibitory nitrosoureas are quite distinct from the bifunctional alkylating agents.

Molecular Structure and Antitumour Activity of Alkylating Agents

An alkylating agent is a compound capable of replacing a proton in another molecule by an alkyl radical. When the alkyl radical has the form RCH2—, the overall reaction may be written as follows: Open image in new window Although R may be a complex, multifunctional species, and may even contain one or more aromatic rings, the final attachment of R to the substrate R’H must be through a saturated carbon atom. The alkylation reaction can occur by two extreme mechanisms.