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On the basis of the results of in-silico predictions and in an effort to extend our structure-activity relationship studies, the aromatic nitrogen mustard 2-[4-N,N-bis(2-chloroethyl) amino-phenyl]butanoic acid (2-PHE-BU) was synthesized and conjugated with various steroidal alcohols. The resulting steroidal esters were evaluated for their in-vivo toxicity and antileukemic activity in P388-leukemia-bearing mice. The new derivatives showed significantly reduced toxicity and marginally improved antileukemic activity compared with free 2-PHE-BU. Nevertheless, they did not prove to be superior either to the template steroidal ester used for in-silico predictions or to previously synthesized steroidal esters of aromatic nitrogen mustards. The results obtained indicate that in-silico design predictions may guide the design and synthesis of new bioactive steroidal esters, but further parameters should be considered aiming at the discovery of compounds with optimum activity.

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.

The synthesis and the in vivo evaluation against leukemias P388 and L1210 of six new alkylating steroidal esters are described. The esteric derivatives incorporating the 17beta-acetamido-B-lactamic steroidal skeleton exhibited increased antileukemic activity and lower toxicity, compared to the 17beta-acetamido-7-keto analogs. Among the 17beta-acetamido-B-lactamic steroidal esters, the most potent compound afforded four out of six cures in leukemia P388 and was measured to be almost non-toxic, producing significant low levels of toxicity.

This study was designed as a rational continuation of our research regarding the functional requirements essential for the antileukemic activity of compounds comprising an alkylating moiety and a modified steroid. The steroidal esteric derivatives of 4-methyl-3-N,N-bis(2-chloroethyl)amino benzoic acid were tested on leukemias P388 and L1210 in vivo and in normal human lymphocytes in vitro. Among them the B-lactamic steroidal esters proved more potent antileukemic agents than the 7-oxidized and those with a simple B-ring, but not more effective inducers of DNA damage and cell cycle arrest in vitro. We speculate that these results indicate a different mechanism of action induced by the lactamized B steroidal ring, in comparison to the 7-keto or the D-lactamic groups, which involves the interaction of the -NHCO- moiety with cellularcomponents essential for tumor growth. 4-Methyl-3-N,N-bis(2-chloroethyl)amino benzoic acid proved a more proper module for the B-lactams than chlorambucil and phenyl acetic acid's nitrogen mustard probably because the esteric bond is less cleaved by the esterases, resulting in an increased concentration of the drug in the vinicity of the target site essential for an antineoplasmatic response.

Combination chemotherapy is widely and routinely used for most cancer patients. The main objective of this study is an effort to develop new anticancer drugs and procedures with enhanced antitumor activity and reduced toxicity. This study was designed to determine the antileukemic and cytogenetic activity of five mixtures of three specific steroidal esters of aromatic nitrogen mustards in different proportions. This is the next step of two previous studies where the combination of two such esteric analogues was investigated with promising results. All of the five mixtures used proved active against leukemia P388 and in the induction of sister chromatid exchanges, indicating that the combination of the same class of compounds can be successful, especially when a highly potent agent is combined with another less active but probably mechanistically supplementary one. These results can be used in future experiments in order to further scout the specific role of the steroidal part of these molecules in the antileukemic potency of them., (Copyright 2007 S. Karger AG, Basel.)

Recent studies have indicated that minor functional changes on the steroidal part of complex molecules, comprising of an alkylating moiety and a steroidal congener, lead to compounds with enhanced biological activity. The observed induction of the genotoxic, cytotoxic and antileukemic effects suggest a determinative role of the steroidal congener on the mechanism of action. In order to further elucidate the structural requirements responsible for this, we designed and synthesized a new modified steroid, carrying a 17beta-acetamide substituent and a B lactamic ring, and studied the ability of its esters with three potent nitrogen mustards to induce sister chromatid exchange (SCEs) and to inhibit cell proliferation in normal human lymphocytes in vitro. The role of the steroidal skeleton was clearly stated by the results of the in vitro evaluation of the final compounds, as all three derivatives proved better inducers of SCE (58-102 SCE/cell) and cell division delays (1.18-1.25 PRI) than the simple nitrogen mustards (24-38 SCE/cell and 1.51-1.62 PRI). Obviously, the steroidal module enhances the formation of DNA adducts that cannot be repaired by excision repair enzymes probably through the induction of the interaction of these complex compounds with different base sequences or by disabling the repair mechanisms through the blockage of the enzymes responsible for excision repair. On the other hand, it seems that these compounds also act through a parallel site of action responsible for cell death when their primary binding site becomes saturated, as in higher concentrations two of the derivatives tested showed enhanced cytotoxicity while their ability to induce SCE stabilized.

In order to study the role of the steroidal moiety on the expression of anti-leukemic activity, we synthesized six derivatives of chlorambucil (CHL), and tested them on leukemias P388 and L1210 in vivo and in normal human lymphocytes in vitro. Five of the six tested compounds produced submultiple toxicity, while the measured anti-leukemic potency was significantly increased. The lactamization of the B-steroidal ring rendered the molecules more potent, but the corresponding 7-oxidized derivatives proved better in both leukemias tested. The lactamization of the D-steroidal ring afforded potent compounds, regardless of the configuration of the B-ring. The best among all derivatives contains both chemical modifications and is intended as a promising key molecule that must be further studied. We speculate that in leukemic cells a tumor-specific protein is overexpressed, the steroid has the ability to bind and block this protein from carrying out its normal function, and the drug-protein complex prevents the repair of the adducts. The synthesis, physicochemical and spectroscopic data of these compounds and a modified route for the synthesis of CHL are also reported.

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.

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.

A new synthetic procedure and a modification of the original method described in the literature for the synthesis of the steroidal B-D bilactam, 3 beta-hydroxy-7 alpha,17 alpha-diaza-B,D-dihomo-5-androsten-7,17-dione are reported. The key step in the modified method involved protection of the D-lactamic nitrogen atom of 3 beta-acetoxy-17 alpha-aza-D-homo-5-androsten-17-one using a reagent of specific electrophilicity (due to the stereoelectronic properties of the cyclic amide), as Beckmann rearrangement of the B-steroidal ring was hindered, possibly via long range effects, by the presence of the unprotected D-lactamic moiety. Using the 3 beta-acetoxy-5-androsten-17-one as starting material, a new synthetic procedure was developed through ketalization of the 17-ketone and allylic oxidation to the 7-ketone, which was subsequently followed by Beckmann rearrangement of the B- and D-steroid rings. Both approaches resulted in 45 and 67% yields of the desired B,D-bilactam, respectively, in contrast to the 15% yield, which has been reported in the literature.

7-keto-Delta(5)-steroids have been suggested for the treatment of several diseases. Their significant biological profile resulted in the development of a great number of methods and reagents for the allylic oxidation of Delta(5)-steroids. These methods and the biological evaluation of the main oxidized Delta(5)-steroids are summarized.

A variety of delta5-steroids were converted into alpha, beta-unsaturated 7-ketones using a modification of the already known method of t-butyl hydroperoxide in the presence of copper iodide in acetonitrile. The same alteration was applied to another oxidative procedure, which had never been used before on steroidal substrates. The same oxidative agent was used in the presence of copper iodide, and tetra-n-butylammonium bromide was used as a phase-transfer catalyst in a two-phase system of water/methylene chloride. It was found that the allylic oxidation proceeded more efficiently when t-butyl hydroperoxide was added to the reaction mixture in portions. The initial addition of the total amount of oxidant or its dropwise addition afforded low yields. This observation contributes to the investigation of the reaction mechanism, and high-yield conversions of steroidal 5,6-enes into the corresponding conjugated 7-ones in short reaction times are reported.

The increase of the damaging effects on specific DNA sequences and the reduction of the subsequent toxicity of nitrogen mustards has been achieved by their chemical conjugation with modified steroids through an esteric bond. In an attempt to study the structure-activity relationships of these compounds, we synthesized eight steroidal esters of 4-methyl-3-bis(2-chloroethyl)aminobenzoic acid (4-Me-CABA). The anti-leukemic and cytogenetic effects of the parent alkylating agent were compared with those produced by the steroidal compounds, in vivo against leukemias P388 and L1210 and in vitro for induction of Sister Chromatid Exchanges (SCE) and on proliferation rate indices (PRI). The results demonstrate that the existence of the NH-CO group, either as an endocyclic lactamic or as an out of the ring amidic one but at axial conformation, at the steroid-carrier moiety is necessary for the expression of the antileukemic activity. The synthetic route for the preparation of the steroidal esters and their physicochemical data are also reported.