The allylic 7-ketone at the steroidal skeleton is crucial for the antileukemic potency of chlorambucil's active metabolite steroidal esters

We have investigated the role of the allylic 7-ketone in oxidized Delta5-steroids on antileukemic activity. We synthesized and studied a series of oxidized and non-oxidized steroidal esters of p-N,N-bis(2-chloroethyl)aminophenylacetic acid (PHE), chlorambucil's active metabolite. In a comparative study of these 7-keto derivatives, on a molecular basis, regarding their ability to induce sister chromatid exchanges (SCEs) and to inhibit cell proliferation in normal human lymphocytes in vitro, the results with these 7-keto derivatives, on a molecular basis, correlated well with their antileukemic potency against leukemia P388- and L1210-bearing mice, which proved to be significantly increased compared to that of the non-oxidized derivatives. Our results indicate that the role of the steroidal skeleton it is not only for the transportation of the alkylating agent into the cell, but also contributes directly to the mechanism of antileukemic action, by an as-yet unknown way. The main conclusion from this study is that the existence of the allylic 7-keto group in the skeleton of the Delta5-steroidal esters impressively enhances their antileukemic activity, while the toxicity remains at clinically acceptable levels, suggesting that this structural modification should be further investigated.     

苯甲酸氮芥衍生物的合成及抗肿瘤活性

氮芥与苯环相连时称为芳香氮芥，芳香氮芥类药物广泛应用于临床，如苯丁酸氮芥、美法仑等。目前使用的芳香氮芥类抗肿瘤药物中，虽然有较高的烷化效率，但毒性较大。苯甲酸氮芥毒性相对较小，是最早作为氮芥类的抗体导向酶前药疗法药物之一，而且近几年有报道苯甲酸氮芥衍生物如CMDA和CJS1050活性较好，正处在临床实验阶段。     

On the formation of 4-[N,N-bis(2-chloroethyl)amino]phenyl acetic acid esters of hecogenin and aza-homo-hecogenin and their antileukemic activity

The p-[N,N-bis(2-chloroethyl)amino]phenylacetic acid esters of hecogenin and aza-homo-hecogenin have been prepared and their antineoplastic activity was evaluated against two basic drug screening systems in rodents, P388 lymphocytic and L1210 lymphoid murine leukemias. Among the compounds tested, the p-[N,N-bis(2-chloroethyl)amino]phenylacetic acid ester of aza-homo-hecogenin was appeared to possess a significant higher antileukemic effect. These results support that the alkylating esters of hecogenin produce important antitumor activity as well as, indicate that the aza-homo-hecogenin ester exhibits significantly higher activity due to lactam group (-NHCO-) modification.     

肿瘤的化学治疗——ⅩⅩⅩⅧ.氯霉素同型物的合成

In view of the tumour inhibitory effect of AT-16, a Schiff's base prepared by condensing 1-p-nitrophenyl-2-aminopropandiol (1, 3) with p-bis (2-chloroethyl) aminobenzaldehyde, several other Schiff's bases were prepared by condensing 1-p-methoxyphenyl2-aminopropane diol (1, 3) with aromatic aldehyde containing N, N, -bis (2-chloroethyl) amino group. Analogys of chloramphenicol in which the nitro group was replaced by methoxy group and the dichloroacetyl group by the moiety of plant growth inhibitors, 2, 4-dichlorophenoxyacetic acid and 2, 4, 5-trichlorophenoxyacetic acid, were also reported.Among these compounds, it was found that the Schiff's base of 4-N, N-bis (2-chloroethyl) amino-3-methyl-benzaldehyde(Ⅳb) possessed a moderate inhibiting action against S-180 in mice.     

Effect of an homo-aza-steroidal ester on estrogen receptor

A new modified steroid esterified with the cytotoxic moiety, p-[N,N-bis(2-chloroethyl)amino]phenyl-acetic acid, has been synthesized and tested for interaction with estrogen receptor and cytotoxic activity on the MCF-7 cell line.     

7-Keto hybrid steroidal esters of nitrogen mustard: cytogenetic and antineoplastic effects

Four newly synthesized antitumor steroidal compounds were compared, on a molar basis, regarding their ability to induce sister chromatid exchanges (SCEs) and cell division delays. The concept of designing and developing these compounds (1-4) is to enhance the anticancer activity of esteric steroidal derivatives of nitrogen mustard by introduction of a keto group at the 7-position of the D5 steroidal skeleton, and a double bond between positions 6 and 7 of the B ring of the steroidal nucleus. In our study, the cytogenetic and antileukemic effects of these newly synthesized compounds are reported. The four substances induced statistically significant enhancement of SCEs and of cell division delays, and in both schedules used, therapeutic effects. However, compounds 1 and 3 showed increased genotoxicity towards human lymphocytes (p<0.001) and antileukemic activity towards P388 leukemias (p<0.001), compared to compounds 2 and 4. It seems that the introduction of a keto group at the 7-position of the steroidal skeleton enhances the antitumor effect of these substances in comparison with our previous studies with the corresponding compounds characterized by the absence of the 7-keto group. Therefore, the in vivo antitumor effect of the four compounds appears to correlate well with the in vitro cytogenetic effect caused by these chemicals.     

Comparison of half-lives and cytotoxicity of N-chloroethyl-4-amino and N-mesyloxyethyl-benzoyl compounds, products of prodrugs in antibody-directed enzyme prodrug therapy (ADEPT).

The synthesis of two novel drugs, 4-[bis[2-(mesyloxy)ethyl]amino]benzoic acid (7) and 4-[(2-chloroethyl)[2-(mesyloxy)ethyl]amino]benzoic acid (8) is described here. They are the active drugs of two prodrugs (9 and 10) designed for use as anti-cancer agents. The prodrugs (9, 10 and 11) were made as a series of compounds which are bifunctional alkylating agents in which the activating effect of the ionized carboxyl function is masked through an amide bond to a glutamic acid residue. These relatively inactive prodrugs were designed to be activated to their corresponding alkylating agent active drugs (7, 8 and 12 respectively) at a tumour site by prior administration of a monoclonal antibody conjugated to a bacterial enzyme. This system is called antibody-directed enzyme prodrug therapy (ADEPT). The chemical half-lives of the prodrugs and their active drugs were measured in order to determine their relative reactivities. The half-lives ranged from 21 to 324 min for the active drugs and from 42 to 1158 min for the prodrugs. The viability of two different tumour cell lines was monitored with each active drug and prodrug. The IC50 values varied from 65 to 625 microM for the active drugs: no IC50 values could be obtained for the prodrugs, using a rapid incubation procedure. Each in vitro technique demonstrated the ability of the glutamic acid moiety to deactivate the drugs, forming effective prodrugs.     

Structure-anti-leukemic activity relationship study of B- and D-ring modified and non-modified steroidal esters of chlorambucil's active metabolite

We have studied the effect of modification of the B-steroidal ring to lactamic on the anti-leukemic potency of D-modified and D-non-modified steroidal esters of chlorambucil's active metabolite. The compounds synthesized were studied against leukemias P388 and L1210 after the subsequent estimation of their toxicity in vivo, and for their ability to induce sister chromatid exchanges (SCEs) and to inhibit cell proliferation in normal human lymphocytes in vitro. The in vitro results correlated well, on a molar basis, with the results obtained from the study of the anti-leukemic potency. In a comparative study, the B-lactamic steroidal derivatives proved less active than the 7-oxidized ones against both leukemias. The presence of the -NHCO- group in the B-steroidal ring did not have the same positive effect on the biological action of chlorambucil's active metabolite esters as in the D-lactamic ring. However, this new modification of the B-ring rendered the final esteric derivatives much more toxic, compared with to the corresponding esters with a simple B-ring. This loss of the anti-leukemic specificity, which occurs from the modification of the B-ring, is additional evidence for the role of the steroidal part on the mechanism of action of these promising compounds. This provides support for the notion that the steroidal part of these molecules is not just a simple biological carrier, as has been speculated for many years.     

抗肿瘤药物的研究 Ⅲ.含氨基酸的氮芥磷酰胺衍生物的合成

以N-磷酰氨基酸为载体而形成的氮芥磷酰胺衍生物,可能具有较好的抗肿瘤专属性。双-(β-氯乙基)氨基磷酰二氯(Ⅰ)与二当量的甘、丙、缬、亮、笨丙、天冬及谷氨酸乙酯(Ⅱ)缩合,生成相应的N,N-双-(β-氯乙基)-N′,N″-二-(乙氧羰甲基)磷三酰胺衍生物(Ⅲ);与当量的丝氨酸乙酯(Ⅹ)缩合时生成环状衍生物,2-双-(β-氯乙基)氨基-4-乙氧羟基-四氢-1,2,3-氧膦氮茂,2-氧化物(Ⅺ)。这类化合物经动物实验有显著的抗肿瘤活性。     

Novel inhibitors of carboxypeptidase G2 (CPG2): potential use in antibody-directed enzyme prodrug therapy

The design and synthesis of potent thiocarbamate inhibitors for carboxypeptidase G2 are described. The best thiocarbamate inhibitor N-(p-methoxybenzenethiocarbonyl)amino-L-glutamic acid 6d, chosen for preliminary investigations of in vitro antibody-directed enzyme prodrug therapy (ADEPT), abrogated the cytotoxicity of a combination of A5B7-carboxypeptidase G2 conjugate and prodrug PGP (N-p-{N,N-bis (2-chloroethyl)amino}phenoxycarbonyl-L-glutamate) toward LS174T cells. This is the first report of a small-molecule enzyme inhibitor proposed for use in conjunction with the ADEPT approach.     

Synthesis, in vivo antileukemic evaluation and comparative study of novel 5alpha-7-keto steroidal esters of chlorambucil and its active metabolite

Recent structure-antileukemic activity studies showed that the steroidal part of complex molecules containing DNA alkylators does not play only the role of the "biological carrier". New such compounds designed to possess an allylic 7-ketone showed enhanced antileukemic potency compared with derivatives with a simple steroidal skeleton. In order to investigate whether the enhancement of the antileukemic potency is attributed to the introduction of the 7-ketone or to the Delta5-7-keto conjugated steroidal system we decided to reduce the Delta5 double bond. The 5alpha-7-keto-steroidal skeletons synthesized were tethered to chlorambucil and phenyl acetic acid's nitrogen mustard and studied against leukemia P338 in vivo. The reduction of the double bond had a negative impact on the antileukemic potency since the comparative study of the novel derivatives showed that a series of very potent Delta 5-7-keto-steroidal esters were converted by this modification to compounds with marginally accepted activity.     

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.     

Selective cytotoxicity of a system L specific amino acid nitrogen mustard

The synthesis and characterization of DL-2-amino-7-bis[(2-chloroethyl)amino]-1,2,3,4-tetrahydro-2-naphthoic acid and DL-2-amino-5-bis[(2-chloroethyl)amino]-1,2,3,4-tetrahydro-2-napthoic+ ++ acid were accomplished. The correct assignment of the site of attachment of the bis(2-chloroethyl)amino side chain was ascertained by selective proton decoupling of the 13C NMR spectra performed on the corresponding nitrospirohydantoin precursors 2 and 3, which were obtained from the nitration of beta-tetralone hydantoin. The two target compounds 6 and 7 were designed as tumor-specific agents capable of being selectively transported into tumor cells by the leucine-preferring transport system (system L). Inhibition analysis of the initial rate of transport of the system L specific substrate 2-amino-bicyclo[2.2.1]heptane-2-carboxylic acid (BCH) by 6 and 7 indicated that the 7-substituted isomer 6 was an extremely potent competitive inhibitor of that transport system in murine L1210 leukemic cells (Ki = 0.2 microM). Evaluation of the selectivity of this compound indicated that it possessed enhanced in vitro antitumor activity and reduced myelosuppressive activity when compared to its prototype amino acid nitrogen mustard, L-phenylalanine mustard (L-PAM). In addition to being more selectively toxic to tumor cells, this compound differs from L-PAM in having a 2-3-fold shorter half-life (t1/2).     

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.     

Folate analogues. 21. Synthesis and antifolate and antitumor activities of N10-(cyanomethyl)-5,8-dideazafolic acid

A close analogue of the antileukemic agent 5,8-dideaza-N10 propargylfolic acid (2) was synthesized by replacing the propargyl moiety of 2 with a cyanomethyl group. This compound, N10-(cyanomethyl)-5,8-dideazafolic acid (3), was evaluated for its antifolate and antitumor activities in several biological test systems. Alkylation of diethyl N-(4-aminobenzoyl)-L-glutamate with bromoacetonitrile gave diethyl N-[4-[(cyanomethyl)amino]benzoyl]-L-glutamate (7). Reaction of 7 with 2 amino-6-(bromomethyl)-4-hydroxyquinazoline (9) in dimethylacetamide gave the corresponding diethyl ester 11, which was hydrolyzed to the target compound 3. The known antileukemic agent 2 was also synthesized for comparative studies by employing a modified procedure, which resulted in a better yield of this product. Both compounds 2 and 3 were evaluated for their antifolate activities by using two folate-requiring microorganisms, Streptococcus faecium and Lactobacillus casei. They were further evaluated as inhibitors of thymidylate synthase and dihydrofolate reductase derived from the above organisms, as well as for their antitumor activity by using selected tumor cells in culture. Compound 2 was found to be as equally potent as methotrexate (MTX) against S. faecium, and it was an excellent inhibitor of L. casei thymidylate synthase. The cyanomethyl analogue 3 was less active than 2 in all the test systems, except the inhibition of dihydrofolate reductase.     

肿瘤化学治疗的研究 ⅩⅨ.N,N-双(2-氯乙基)腙化合物的合成

合成了睾丸素、甲基睾丸素和其他一系列羰基化合物的N,N-双(2-氯乙基)腙化合物(Ⅱ)、(Ⅲ)、(Ⅳ)和(Ⅴ_1-10),以进行抗肿瘤试验。它们的制备,系由N,N-双(2-氯乙基)肼盐酸盐(Ⅰ)与相应的羰基化合物,在碱存在或不存在的醇溶液中反应而得。此外又自17-氢基Δ⁵孕-3醇,与对双(2-氯乙基)氨基苯甲醛缩合,制得了另一甾体氮芥化合物——17-[对双(2-氯乙基)氨基苯亚甲基]氨基-Δ⁵-孕-3醇(Ⅵ)。     

Synthesis and antileukemic activity of new 3-(1-phenyl-3-methylpyrazol-5-yl)-2-styrylquinazolin-4(3H)-ones

3-(1-Phenyl-3-methylpyrazol-5-yl)-2-styrylquinazolin-4(3H)-ones 14a-q and 15a-q were synthesized by refluxing in acetic acid the corresponding 2-methylquinazolinones 12 and 13 with the opportune benzoic aldehyde for 12 h. The synthesized styrylquinazolinones 14a-q and 15a-q were tested in vitro for their antileukemic activity against L1210 (murine leukemia), K562 (human chronic myelogenous leukemia) and HL60 (human leukemia) cell lines showing in some cases good activity.     

Alkylation of beta-tubulin on Glu 198 by a microtubule disrupter

We have shown that beta-tubulin was alkylated by a microtubule disrupter, N-4-iodophenyl-N'-(2-chloroethyl)urea (ICEU), on a glutamic acid residue at position 198 and not on the previously proposed reactive cysteine 239. ICEU belongs to the 4-substituted-phenyl-N'-(2-chloroethyl) urea class that alkylates mainly cellular proteins. Previous studies have shown that the tert-butyl (tBCEU) and iodo (ICEU) derivatives induce microtubule disruption because of beta-tubulin alkylation. tBCEU was supposed to bind covalently to cysteine 239 of beta-tubulin, but this binding site was not clearly confirmed (Cancer Res 60:985-992, 2000). We have isolated and analyzed beta-tubulin after two-dimensional gel electrophoresis of proteins from B16 cells incubated with ICEU. Alkylated beta-tubulin had a lower apparent molecular weight and a more basic isoelectric point than the unmodified protein. Labeled N-4-[125I]CEU was effectively bound to the modified beta-tubulin but using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry, we demonstrated that none of the cysteine residues of beta-tubulin was linked to the alkylating agent. In contrast, peptide masses at m/z 4883 and 1792 in trypsin or Asp-N digestions of beta-tubulin confirmed binding of iodophenylethylureido moiety to peptides [175-213] or [197-208] respectively. Fragmentation analyses by electrospray mass spectrometry using triply charged ions of peptide [175-213] identified a glutamic acid at position 198 as target for alkylation via an ester bond with ICEU. This amino acid located in the intermediate domain of the beta-tubulin should play an essential role in the conformational structure necessary for the interaction between dimers in the protofilament.     

Identification of cyclophosphamide-DNA adducts in rat embryos exposed in vitro to 4-hydroperoxycyclophosphamide.

Cyclophosphamide and other bifunctional alkylating agents are potent animal teratogens inducing a variety of malformations. Although cyclophosphamide-induced DNA damage is implicated as a primary mechanism underlying the teratogenesis initiated by cyclophosphamide, additional insights into the complex nature of the teratogenic process have been hampered by the inability to analyze the primary teratogenic lesions, i.e., cyclophosphamide-DNA adducts. Using tandem mass spectrometry, we show that the monofunctional adduct N-(2-chloroethyl)-N-[2-(7-guaninyl)ethyl]amine (NOR-G) and bifunctional adduct N,N-bis[2-(7-guaninyl)ethyl]amine (G-NOR-G) can be detected in the DNA of organogenesis-stage rat embryos after an in vitro exposure to an embryotoxic concentration of activated cyclophosphamide, i.e., 4-hydroperoxycyclophosphamide.