Evaggelia S. Arsenou

Optimization of the allylic oxidation in the synthesis of 7-keto-Δ5-steroidal substrates

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.

Synthetic approaches for the synthesis of a cytostatic steroidal B–D bilactam

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.

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 &Dgr;5-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), chlorambucils 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 &Dgr;5-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 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.

Structure–anti-leukemic activity relationship study of B- and D-ring modified and non-modified steroidal esters of chlorambucil

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.

Antileukemic and Cytogenetic Activity by Triple Administration of Three Modified Steroidal Derivatives of Nitrogen Mustards

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.

Effects of retinoic acid steroidal analogs on human leukemic HL60 cell proliferation in vitro and on angiogenesis in vivo.

Retinoic acid (RA) can be regarded as a pharmacological agent commonly used for its ability to affect growth and differentiation of a variety of cell types, such as acute promyelocytic leukemic and endothelial cells. In the present work we studied the effect of all-trans-RA (ATRA) and its steroidal analogs EA-4, EA-136 and EA-137 on the growth of human promyelocytic HL-60 cells in vitro. The specific steroidal substrates were chosen in order to further investigate their ability to improve the pharmacological properties of conjugated antileukemic agents. ATRA decreased the number of HL60 cells from the first 24 h after its addition to the cell culture medium. The decrease was significant at concentrations higher than 10−5 M. All the analogs tested also decreased the number of HL60 cells with an IC50 similar to that of ATRA, except for EA-4 whose IC50 was almost two orders of magnitude lower than that of ATRA, 72 h after its addition to the cell culture medium. Since angiogenesis is important for the growth of hematological malignancies, we furthermore studied the effect of ATRA and its analogs on the formation of new capillaries in the in vivo chicken embryo chorioallantoic membrane (CAM). ATRA, EA-136 and EA-137 induced angiogenesis in the CAM, increased the layer of CAM keratinocytes, and resulted in a significant degree of extravasation. EA-4 had no effect on either angiogenesis or tissue structure in general. It seems that the retinoid EA-4 is a promising agent for the inhibition of human leukemia cell growth.