1-Aryl-3-(2-chloroethyl) ureas: synthesis and in vitro assay as potential anticancer agents

1-Aryl-3-(2-chloroethyl) ureas and 1-aryl-3-nitroso-3-(2-chloroethyl) ureas, derived from 4-phenylbutyric acid and alkylanilines, were synthesized and their cytotoxicity was evaluated on human adenocarcinoma cells in vitro. Methyl 4-[p-[3-(2-chloroethyl)ureido]-phenyl]butyrate, 4-methyl [3-(2-chloroethyl)ureido]benzene, and 4-butyl[3-(2-chloroethyl)ureido]benzene were found to be at least as cytotoxic as 4-[p-[bis-(2-chloroethyl)amino]phenyl]butyric acid (chlorambucil), while their N-nitroso derivatives were inactive.     

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

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.     

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 $\text{(p-}\text{di}\text{-2-}\text{chloroethylaminophenyl}\text{-β-}\text{d}\text{-glucopyranosid}\text{)}$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.     

肿瘤化学治疗的研究 Ⅴ.某些具有双(2-氯乙基)氨基的苯丙氨酸的合成

本文报导了溶肉瘤素的三种异构体,卽β-[间双(2-氯乙基)氢基苯基]-α-氨基丙酸(Ⅰ_b)、β-[对双(2-氯乙基)氨基苯基]-β-氨基丙酸(Ⅱ_α)及β-[间双(2-氯乙基)氨基苯基]-β-氨基丙酸(Ⅱ_b)的合成和它们的抗肿瘤作用,并对它们的化学结构与生理作用的关系进行了讨论。上述化合物对多种动物实验肿瘤的生长皆具显著的、不同程度的抑制作用。化合物Ⅱ_a(编号合14)已在临床研究中。     

Synthesis of deuterium-labeled analogs of cyclophosphamide and its metabolites.

Convenient syntheses are described of d4 analogs of cyclophosphamide and some of its metabolites, potential standards for the quantitative analysis of the drug and its metabolites in human body fluids by stable isotope dilution-mass spectrometry. Base-catalyzed H-D exchange on N-nitrosobis(2-hydroxyethyl)amine gave N-nitrosobis(1,1-dideuterio-2-hydroxyethyl)amine from which bis(2-chloro-1,1-dideuterioethyl)amine (nor-HN2-d4) was readily obtained. Established synthetic routes were then used to convert nor-HN2-d4 into d4 analogs of cyclophosphamide [2-[bis(2-chlorethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide], 4-ketocyclophosphamide [2[BIS(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorin-4-one 2-oxide], and carboxyphosphamide [2-carboxyethyl N-N-bis(2-chloroethyl)phosphorodiamidate], and these analogs were used in a preliminary investigation into the quantitation of the appropriate components in human plasma and urine. Also prepared were d4 analogs of phosphoramide mustard [N,N-bis(2-chloroethyl)phosphorodiamidic acid (cyclohexylammonium salt)] and 3-(2-chloroethyl)oxazolidone and the methyl and trideuteriomethyl esters of phosphoramide mustard.     

Studies on the pharmacokinetics of chlorambucil and prednimustine in man.

1 Chlorambucil (10 mg) and prednimustine (20 mg), the prednisolone ester of chlorambucil, were administered orally on separate occasions to six patients. 2 Chlorambucil was rapidly absorbed such that the parent compound was observed in the plasma 30 min after administration. 3 A preliminary comparison of chlorambucil levels following oral and intravenous administration, and after repeat oral dosage indicated that chlorambucil was well (greater than 70%) and consistently absorbed. 4 Following prednimustine no parent drug or alkylating metabolites (chlorambucil or phenyl acetic mustard) could be detected in the plasma. 5 In studies with intravenously administered chlorambucil plasma levels of the parent drug were described by a two-compartment open model with first-order kinetics. Significant levels of the cytotoxic metabolite phenyl acetic mustard were detected. 6 It is concluded that: a. the bioavailability of orally administered prednimustine is much lower than that of chlorambucil. Thus the use of prednimustine in routine combination therapy is not recommended. b. due to the lower therapeutic index of phenyl acetic mustard in experimental systems, the production of this metabolite in man may be disadvantageous. Thus research aimed at producing chlorambucil analogues, which cannot be metabolised, seems justified.     

The metabolism of the anti-tumour agent 1(1-aziridinyl)- 2,4-dinitrobenzene (CB 1837).

1-(1-Aziridinyl)-2,4-dinitrobenzene (CB 1837) is the parent member of a series of nitrophenyla-ziridines having a highly specific action against the Walker tumour in the rat. CB 1837 was much less cytotoxic in vitro than was predicted on the basis of its antitumour activity in vivo, but its activity was enhanced in the presence of liver 9000-g supernatant and cofactors, which also converted it into a metabolite, 2-amino-1-(1-aziridinyl)-4-nitrobenzene. This metabolite was more active in the in vitro test than was CB 1837.CB 1837 is extensively metabolised in vivo by the rat, and the following urinary metabolites have been identified by mass spectrometry and quantitatively determined after administration of the tritium-labelled drug: 1-(2-chloroethyl)amino-2,4-dinitrobenzene and its 2-hydroxyethylamino analogue; 2,4-dinitroaniline: 2-amino-1-(1 -aziridinyl)-4-nitrobenzene,2-amino-1-(2-chloroethyl)amino-4-nitrobenzene and their respective N(2)-acetyl derivatives; and S-[N-(2,4-dinitrophenyl)-2-aminoethyl] mercapturic acid.     

Synthesis and primary screening for growth inhibitors of L1210 cells of cholesterylp-[3-(2-chloroethyl)-3-nitrosoureido] phenylthioacetate

Cholesterylp-[3-(2-chloroethyl)-3-nitrosoureido] phenylthioacetate (2) was synthesizedian intermediate,p-[3-(2-chloroethyl)-3-nitrosoureido] phenylthioacetic acid (1) is a congener of an antitumor chlorambucil which both the-CH₂CH₂-linkage and-N(CH₂CH₂Cl)₂ group of chlorambucil molecule is doubly modified into the respective-S-linkage and-NH?CO?NNO?CH₂CH₂Cl group. The attackment of cholesterol moiety as a carrier group top-[3-(2-chloroethyl)-3-nitrosoureido] phenylthioacetic acid was accomplished through the esterification of cholesterol withp-[3-(2-chloroethyl)-3-nitrosoureido] phenylthioacetyl chloride which was obtained from the treatment ofp-[3-(2-chloroethyl)-3-nitrosoureido] phenylthioaceticacid with SOCl₂.p-[3-(2-chloroethyl)ureido]-phenylthioacetic acid was nitrosated with NaNO₂ in 98≈100% HCOOH to give exclusivelyp-[3-(2-chloroethyl)-3-nitrosoureido]phenylthioacetic acid. Antitumor evaluation of compounds, 1 and 2 on L 1210 leukemia did show significant activity (ED₄₀:I. 14μg/ml and 8.4μg/ml. respectively). Further studies were subjected.     

Nitro analogues of chlorambucil as potential hypoxia-selective anti-tumour drugs

The chlorambucil isomer 4-[3-[N,N-bis(2-chloroethyl)amino]phenyl] butanoic acid (m-chlorambucil) has been synthesized for the first time, and the two isometric nitro derivatives of both m-chlorambucil and chlorambucil itself have been prepared as potential hypoxia-selective cytotoxins. Reduction potentials (E1/2) of the two nitro compounds were determined by cyclic voltammetry, and one-electron reduction potentials (E(1] were estimated. Both the chlorambucil isomers and the derived nitro compounds crosslink DNA, as determined by their cytotoxicity ratios in DNA repair-proficient and -deficient cell lines, but neither of the nitro derivatives showed selective toxicity under hypoxic conditions, probably due to their rather low reduction potentials.     

Identification of the in vitro N-oxidized metabolites of (+)- and (−)-N-benzylamphetamine

This study has identified (+)- and (-)-N-benzyl-N-hydroxyamphetamine as metabolites after incubation of both (+)- and (-)-N-benzylamphetamine with fortified rabbit liver homogenates. The isomeric hydroxylamine metabolites were identified using the techniques of g.l.c., t.l.c. and combined g.l.c.-mass spectrometry (ms) and by comparison with results from reference samples. An additional novel metabolic product was identified after incubation of N-benzylamphetamine which had properties consistent with that of N-benzyl-amphetamine nitrone.     

Biotransformation of a new pyrrolidinone cognition-enhancing agent: isolation and identification of metabolites in human urine

1. The metabolites of N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl)acetamide (DMPPA; MH-1), in the urine of human volunteers have been investigated.

2. Ten metabolites together with the unchanged drug (MH-1) were isolated by?h.p.l.c. and identified by n.m.r. and mass spectrometry as: three metabolites hydroxylated in the pyrrolidine ring of MH-1 (MH-2, MH-3 and MH-4), three metabolites hydroxylated in the dimethylphenyl ring of MH-1 (MH-6, MH-7 and MH-8), N-[(2,6-dimethyl-phenylcarbamoyl)methyl]-4-hydroxybutyrylamide (MH-5), N-[(2,6-dimethyl-phenyl-carbamoyl)methyl]succinamic acid (MH-9), the 3-O-sulphate of MH-6 (MH-10) and the 3-O-sulphate of N-(2,6-dimethyl-3-hydroxyphenyl)-2-(5-hydroxy-2-oxo-1-pyrrolidinyl)acetamide (MH-11).

3. DMPPA was extensively metabolized. The principal metabolic transformations were hydroxylation of the pyrrolidine ring at the C5 carbon followed by oxidative C-N cleavage, and hydroxylation of the phenyl ring followed by sulphate conjugation.     

Studies related to the metabolism of anabolic steroids in the horse: the identification of some 16-oxygenated metabolites of 19-nortestosterone

1. The metabolism of 19-nor[4-¹⁴C]testosterone in a thoroughbred horse has been studied and neutral urinary metabolites obtained after enzyme hydrolysis have been investigated by g.l.c.-mass spectrometry.

2. 3-Hydroxyestran-17-one, 17α- and 17β-19-nortestosterone, estrane-3,17-diol (two isomers), 3,16-dihydroxyestran-17-one (two isomers), 3,17-dihydroxyestran-16-one (two isomers) and estrane-3,16,17-triol were identified in the neutral urinary extracts.     

Syntheses of Potentially Antineoplastic Amides and Esters of N-[N'-(2-Chloroethyl)-N'-nitrosocarbamoyl]amino Acids,II

Syntheses of N-[N'-(2-chloroethyl)-N'-nitrosocarbamoyl]amino acid amides and esters as potential antineoplastic substances are reported. N-[N'-(2-chloroethyl)-N'-nitrosocarbamoyl]amino acids (with the exception of the glycine derivative) were prepared by reaction of 2-chloroethyl isocyanate with the sodium salt of an amino acid in a heterogenous medium followed by nitrosation with sodium nitrite under acidic conditions. Condensation with amines or alcohols using 1,1-carbonyldiimidazole led to the amides or esters.     

Biological fate of sulphur mustard, 1,1'-thiobis(2-chloroethane): isolation and identification of urinary metabolites following intraperitoneal administration to rat

1. The metabolism of sulphur mustard, 1,1'-thiobis(2-chloroethane), in vivo was investigated following i.p. administration to rat.

2. Approx. 60% of dose was excreted in the 24 h urine. Many metabolites were present; nine have been isolated by h.p.l.c. and characterized by mass spectrometry. Structural assignments were confirmed by comparison with authentic synthetic standards.

3. Some metabolites result from initial hydrolysis of the sulphur mustard, but the majority are formed by conjugation with glutathione. These are further metabolized to N-acetylcysteine conjugates, or to methylthio/methylysulphinyl derivatives by a pathway probably involving β-lyase, accompanied by oxidation of the mustard sulphur atom to sulphoxide or sulphone.

4. Thiodiglycol sulphoxide, 1,1'-sulphonylbis[2-S (N-acetylcysteinyl)ethane] and 1,1'-sulphonylbis[2-methylsulphinyl)ethane] or 1-methylsulphinyl-2-[2-(methylthio ethylsulphonyl]ethane were the most prevalent metabolites resulting from the three major pathways. Metabolic pathways for the formation of the excretion products are proposed.     

Biotransformation of diethenylbenzenes. III: Identification of metabolites of 1,3-diethenylbenzene in rat

1. Biotransformation of 1,3-diethenylbenzene (1) in rat gave four major metabolites, namely, 3-ethenylphenylglyoxylic acid (2), 3-ethenylmandelic acid (3), N-acetyl-S-[2-(3-ethenylphenyl)-2-hydroxyethyl]-L-cysteine (4) and N-acetyl-S-[1-(3-ethenylphenyl)-2-hydroxyethyl]-L-cysteine (5) were isolated from urine and identified by n.m.r. and mass spectrometry.

2. Four minor metabolites, 3-ethenylbenzoic acid (6), 3-ethenylphenylacetic acid (7), 3-ethenylbenzoylglycine (8) and 2-(3-ethenylphenyl)ethanol (9) were identified by g.l.c.-mass spectrometric analysis of urine extract derivatized in two different ways.

3. All identified metabolites are derived from 3-ethenylphenyloxirane (10), a reactive metabolic intermediate. No product of any metabolic transformation of second ethenyl group has been identified. However, several minor unidentified metabolites were detected by g.l.c.-mass spectrometry.

4. Total thioether excretion in 24h urine after a single i.p. dose of 1 amounted to 28·3±3·5 dose (mean±SD). No significant differences in the thioether fraction were observed in the dose range 100-300mg/kg.

5. Thioether metabolites consisted mainly of mercapturic acids 4 and 5. The ratio of metabolites 5 to 4 was 62:38. Each mercapturic acid consisted of two diastereomers. Their ratio, as determined by quantitative ¹³C-n.m.r. measurement was 95:5 and 79:21 for mercapturic acids 4 and 5, respectively.     

Dithiazepane formation from reactive bifunctional alkylating agents

The technique of trapping a reactive alkylating metabolite with sodium sulphide-³⁵S, previously applied to the azo-mustard, 2′-carboxy-4-di-(2-chloroethyl)amino-2-methylazobenzene (CB 1414) has been extended to the related di-(2-methanesulphonyloxyethyl)amino analogue (CB 10221). In addition to the previously reported 4-(4-amino-3-methylphenyl)-1,2-thiazane, mass spectrometry afforded evidence for the additional formation of 5-(4-amino-3-methylphenyl)-1,2-dithia-5-azepane when CB 10221 was incubated with a rat liver homogenate in the presence of sodium sulphide. Dithiazepane formation was further exemplified by the production of 4-(4-hydroxyphenyl)-1,4-thiazane and 5-(4-hydroxyphenyl)-1,2-dithia-5-azepane from N,N-di-(2-chloroethyl)-4-hydroxyaniline (p-hydroxyaniline mustard) and sodium sulphide. The preparation of thiazanes of some additional compounds of interest in relation to metabolic studies is also reported. The implications of dithiazepane formation for the identification and quantitative determination of alkylating metabolites by this trapping procedure are discussed.     

Degradation and disposal of some antineoplastic drugs

Bulk quantities and pharmaceutical preparations of the antineoplastic drugs carmustine (BCNU), lomustine (CCNU), chlorozotocin, N-[2-chloroethyl]-N'-[2,6-dioxo-3-piperidinyl]-N-nitrosourea (PCNU), methyl CCNU, mechlorethamine, melphalan, chlorambucil, cyclophosphamide, ifosfamide, uracil mustard, and spiromustine may be degraded using nickel-aluminum alloy in KOH solution. The drugs are completely destroyed and only nonmutagenic reaction mixtures are produced. Destruction of cyclophosphamide in tablets requires refluxing in HCl before the nickel-aluminum alloy reduction. Streptozotocin, chlorambucil, and mechlorethamine may be degraded using an excess of saturated sodium bicarbonate solution. The nitrosourea drugs BCNU, CCNU, chlorozotocin, PCNU, methyl CCNU, and streptozotocin were also degraded using hydrogen bromide in glacial acetic acid. The drugs were completely destroyed but some of the reaction mixtures were mutagenic and the products were found to be, in some instances, the corresponding mutagenic, denitrosated compounds.     

Synthesis and antiestrogenic activity of [3,4-dihydro-2-(4-methoxyphenyl)-1-naphthalenyl][4-[2-(1-pyrrolidinyl)ethoxy]-phenyl]methanone, methanesulfonic acid salt.

Acylation of the sodio anion of beta-tetralone with phenyl anisoate, followed by a Grignard reaction of the resultant 4 with 4-methoxyphenylmagnesium bromide, gave rise to two novel dihydronaphthalene isomers 5 and 6. Regioselective demethylation of either 5 or 6 by NaSEt produced [3,4-dihydro-2-(4-methoxyphenyl)-1-naphthalenyl](4-hydroxyphenyl)methanone (7). Etherification of the phenolic group of 7 by N-(2-chloroethyl)pyrrolidine and subsequent methanesulfonate salt formation provided [3,4-dihydro-2-(4-methoxyphenyl)-1-maphthalenyl]]4-]2-(1-pyrrolidinyl)ethoxy]phenyl]methanone, methane sulfonic acid salt (3). Potent antiestrogenic activity of 3 was demonstrated by both oral and subcutaneous administration to rats and mice. In vitro binding studies with rat uterine cytosol estrogen receptors indicate compound 3 has a very high binding affinity which exceeds that of estradiol.     

Synthesis of Potentially Antineoplastic Derivatives of N-[N-(2-Chloroethyl)-N-nitrosocarbamoyl]amino Acids

The synthesis of N-[N-(2-chloroethyl)-N-nitrosocarbamoyl]amino acids and their anilides, congeners of N-(2-chloroethyl)-N-nitrosoureas, as potential antineoplastic substances is reported. N-[N-(2-chloroethyl)-N-nitrosocarbamoyl]amino acids are prepared by reaction of amino acids with N-(2-chloroethyl)-N-nitrosocarbamoyl azide. Corresponding anilides and toluidides are obtained by condensation of the primary reaction products with aniline and toluidine using dicyclohexylcarbodiimide (DCC).     

The microsomal metabolism of some analogues of cyclophosphamide: 4-methylcyclophosphamide and 6-methylcyclophosphamide

4-Methylcyclophosphamide and 6-methylcyclophosphamide are, like cyclophosphamide, converted by rat liver microsomes into 4-hydroxy derivatives. 4-Hydroxy-4-methylcyclophosphamide was isolated directly, in admixture with the product [2-(2-chloroethylamino)tetrahydro-4-methyl-2H-1,3,2-oxazaphosphorine 2-oxide] of dechloroethylation. P.m.r. data for the hydroxy derivative, which was also formed when 4-methylcyclophosphamide was treated with aqueous KMnO₄, indicated that it exists in aqueous solution as the acyclic tautomer, 2-oxopropyl-N, N-bis-(2-chloroethyl)phosphorodiamidate. 4-Hydroxy-6-methylcyclophosphamide was trapped by reaction with ethanol, and afforded two isomeric ethoxy derivatives analogous to those previously reported from cyclophosphamide.Treatment of the products of metabolism of 4-methyl- and 6-methylcyclophosphamide with 2,4-dinitro-phenylhydrazine afforded, respectively, the 2,4-dinitrophenylhydrazones of methyl vinyl ketone and of crotonaldehyde.4-Methylcyclophosphamide cannot form metabolites analogous to 4-ketocyclophosphamide and carboxyphosphamide, the relatively non-toxic metabolites of cyclophosphamide. The significance of this fact is discussed in relation to a mechanism which could account for the relatively selective cytotoxicity of cyclophosphamide in vivo towards neoplastic tissue.Conventional electron impact mass spectrometry has played an important role in the characterization of the products described in this study. 4-Hydroxy-4-methylcyclophosphamide was additionally characterized by the relatively novel technique of field desorption mass spectrometry.