Steroid Esters Research Articles

Genotoxic, cytostatic, antineoplastic and apoptotic effects of newly synthesized antitumour steroidal esters

In this study, we have investigated the genotoxic, cytostatic, antineoplastic and apoptotic effects of three newly synthesized modified steroidal esters, having as alkylating agent p- N,N-bis(2-chloroethyl) aminophenyl butyrate (CHL) or p- N,N-bis(2-chloroethyl) aminophenyl acetate (PHE) esterified with the steroidal nucleus modified in the B- and D-ring. The genotoxic and cytotoxic effects of the compounds were investigated both in vitro, in lymphocyte cultures obtained from blood samples of healthy donors and in vivo, in ascites cells of P388 leukemia obtained from the peritoneal cavity of DBA/2 mice. Preparations were scored for sister-chromatid exchange (SCE) and proliferation-rate indices (PRI). The newly synthesized compounds were also studied for antineoplastic activity against lymphocytic P388 and lymphoid L1210 leukemias in mice, by calculating the mean of the median survival of the drug-treated animals (T) versus the untreated control (C) (T/C%). The activity of caspase-2 and caspase-3, indicators of apoptosis, was assessed biochemically in primary cultures of human lymphocytes. Our results show that the newly synthesized compounds caused severe genotoxic effects by significantly increasing the frequency of SCE and decreasing the PRI values in cultures of peripheral lymphocytes in vitro and in ascites cells of lymphocytic P388 leukemia in vivo. A significant correlation was also observed in both the in vitro and in vivo experiments: the higher the SCE frequency the lower the PRI value ( r = −0.65, P < 0.001 and r = −0.99, P < 0.01, respectively). The measured antileukemic potency was statistically increased by all test compounds in both types of tumours, while the activity of caspase-2 and caspase-3 showed a statistically significant increase after two periods of exposure. The genotoxic (increase of SCE), cytostatic/cytotoxic (decrease of PRI) and antileukemic effects (increase of T/C%) in combination with the induction of apoptosis (activation of caspase-2 and caspase-3) caused by the newly synthesized compounds, lead us to propose them as agents with potentially antineoplastic properties.

Mass spectrometric profiling of saturated fatty acid esters of steroids separated by high-temperature gas chromatography

An efficient analytical method for simultaneous determination of 12 SFEs in serum is described. The method involves solid-phase extraction to isolate of SFEs from interfering species, especially cholesteryl esters, conversion to trimethylsilyl (TMS) ether derivatives for the direct analysis by gas chromatography–mass spectrometry (GC–MS) using a high temperature MXT-1 (Silcosteel-treated stainless steel) capillary column. All SFEs as their TMS derivatives were well separated with excellent peak shapes within 12min. Overall recoveries ranged from 88% to 119%, with a detection limits for SFEs ranged from 2 to 30μgL−1. The linearity as correlation coefficient was higher than 0.99 except for pregnenolone-3-arachidate (r2=0.98) in the concentration range of 5–3000μgL−1. Ten serum samples obtained from volunteers were also analyzed and quantitatively determined of DHEA-3-palmitate and pregnenolone-3-stearate in 1.8–1195.8μgL−1 concentration. The devised high temperature GC–MS method could be useful for identification of SFEs in biological specimens including serum.

Full-Scan Accurate Mass Selectivity of Ultra-Performance Liquid Chromatography Combined with Time-of-Flight and Orbitrap Mass Spectrometry in Hormone and Veterinary Drug Residue Analysis

The applicability of ultra-performance liquid chromatography (UPLC) combined with full-scan accurate mass time-of-flight (TOF) and Orbitrap mass spectrometry (MS) to the analysis of hormone and veterinary drug residues was evaluated. Extracts from blank bovine hair were fortified with 14 steroid esters. UPLC-Orbitrap MS performed at a resolving power of 60,000 (FWHM) enabled the detection and accurate mass measurement (<3 ppm error) of all 14 steroid esters at low ng/g concentration level, despite the complex matrix background. A 5 ppm mass tolerance window proved to be essential to generate highly selective reconstructed ion chromatograms (RICs) having reduced background from the hair matrix. UPLC-Orbitrap MS at a lower resolving power of 7500 and UPLC-TOFMS at mass resolving power 10,000 failed both to detect all of the steroid esters in hair extracts owing to the inability to mass resolve analyte ions from co-eluting isobaric matrix compounds. In a second application, animal feed extracts were fortified with coccidiostats drugs at levels ranging from 240 to 1900 ng/g. UPLC-Orbitrap MS conducted at a resolving power of 7500 and 60,000 and UPLC-TOFMS detected all of the analytes at the lowest investigated level. Thanks to the higher analyte-to-matrix background ratio, the utilization of very narrow mass tolerance windows in the RIC was not required. This study demonstrates that even when the targeted sample preparation from conventional LC-MS/MS is applied to UPLC with full-scan accurate mass MS, false compliant (false negative) results can be obtained when the mass resolving power of the MS is insufficient to separate analyte ions from isobaric co-eluting sample matrix ions. The current trend towards more generic and less selective sample preparation is expected to aggravate this issue further.

3D QSAR/CoMFA and CoMSIA Studies on Antileukemic Steroidal Esters Coupled with Conformationally Flexible Nitrogen Mustards

Thirty-eight antileukemic steroidal esters possessing conformationally flexible nitrogen mustards were studied, and the 3D QSAR/CoMFA and CoMSIA methodologies were applied in order to derive the correlation between their structure and the in vivo antileukemic activity. These compounds show significantly reduced toxicity and possibly increased bioavailability compared to free nitrogen mustards and therefore constitute potent antileukemic drugs. Both the CoMFA and CoMSIA studies gave similar results indicating that the steric effect and the hydrophobic/hydrophilic balance especially in the steroidal part of the molecules probably determined their bioactivity. Of paramount interest is the observation that the orientation of the alkylating part of the SMEs toward the surface of ring B of the steroidal skeleton was related with increased activity. Concerning the steroidal part, the presence of hydrophobic groups in rings B and D was found to be important for enhanced activity. Enhancement of antileukemic potency is further observed if hydrophilic/H-bond acceptor groups are present at the positions 7 and 17 of the steroidal skeleton. Leapfrog simulations provided novel compounds which lead our future synthetic endeavor for obtaining SMEs with optimum bioactivity.

Kinetics of drug release from a hyaluronan–steroid conjugate investigated by NMR spectroscopy

The methylprednisolone steroid ester of hyaluronan was hydrolyzed under physiological conditions in vitro, and the kinetics of drug release was investigated by NMR spectroscopy. Transverse relaxation times are correlated with the molecular rotational freedom, which undergoes large changes for methylprednisolone when released. Multi-exponential decays were observed, which together with the corresponding population gave valuable insights into the conformational changes that occur in the biopolymer during hydrolysis. The biomaterial exists in aqueous solution in two conformations, ‘collapsed’ and ‘water-exposed’, in equilibrium. Under physiological conditions, the methylprednisolone is completely released within 48 h. Transverse relaxation times proved to be an appropriate tool for monitoring the drug release in vitro.

Determination of hormonal growth promoters in bovine hair: Comparison of liquid chromatography–mass spectrometry and gas chromatography–mass spectrometry methods for estradiol benzoate and nortestosterone decanoate

The detection of steroid residues in hair is a powerful strategy to demonstrate long-term administration of these growth promoters in meat production animals. Analysis of the ester form of administered steroids is an unambiguous approach to prove the illegal use of natural hormones. For detection, gas chromatography–mass spectrometry (GC–MS/MS) was generally used. However, recent advances in liquid chromatography–tandem mass spectrometry (LC–MS/MS) technology have improved the robustness and potential sensitivity of this method. This paper describes development and validation, according to Commission Decision 2002/657/EC, of LC–MS/MS and GC–MS/MS methods, in two separate laboratories, for determination of steroid esters in bovine hair. Bovine hair samples taken from animals treated with estradiol-3-benzoate and nortestosterone decanoate, as well as from an untreated animal, were used to evaluate the comparability of the results of the two validated methods. The results of the inter-comparison demonstrate that both the LC–MS/MS and the GC–MS/MS methods are fit for purpose and capable of determining steroid esters in hair samples from treated bovine animals.

Rational design, synthesis, and in vivo evaluation of the antileukemic activity of six new alkylating steroidal esters

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 17β-acetamido-B-lactamic steroidal skeleton exhibited increased antileukemic activity and lower toxicity, compared to the 17β-acetamido-7-keto analogs. Among the 17β-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.

Platinum(II) complexes with steroidal esters of l-methionine and l-histidine: Synthesis, characterization and cytotoxic activity

Twelve steroidal platinum(II) complexes were synthesized by reaction of potassium tetrachloroplatinate with steroidal esters of l-methionine and l-histidine. The steroidal esters coordinated as bidentate ligands via S and N donor atoms of l-methionine and via two N donor atoms of l-histidine. Cholesterol, cholestanol, diosgenine, pregnenolone, dehydroepiandrosterone, testosterone, estrone, and estradiol were used as the steroidal compounds. The esters and complexes prepared were characterized by infrared, mass, and 1H NMR spectroscopy and elemental analysis. Platinum complexes were tested for in vitro cytotoxicity against several cancer cell lines: T-lymphoblastic leukemia CEM, breast carcinoma MCF-7, lung carcinoma A-549, multiple myeloma RPMI 8226, and one normal cell line human fibroblast BJ.

New Lipoxygenase‐Inhibiting Constituents from Calligonum polygonoides.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Comparative study on the chemical composition of lipophilic fractions from three wood tissues of Eucalyptus species by gas chromatography-mass spectrometry analysis

Abstract The chemical compositions of lipophilic fractions from Eucalyptus urograndis and Eucalyptus urophylla cultivated in Brazil and Eucalyptus camaldulensis from Mexico were determined by gas chromatography-mass spectrometry (GC-MS) before and after alkaline hydrolysis followed by derivatization. In all fractions, fatty acids (including small amounts of α-and ω-hydroxy fatty acids) and sterols were the most abundant components followed by smaller amounts of long-chain aliphatic alcohols, phenolic acids, and hydrocarbons. The presence of steroid esters and triacylglycerols in all three species was indirectly confirmed by the increased amount of fatty acids and sterols (manly β-sitosterol) in the hydrolyzed fractions compared with the corresponding nonhydrolyzed fractions. The amount of liphophilic compounds (mainly fatty acids and sterols) identified in hydrolyzed fractions of E. urograndis, E. camaldulensis, and E. urophylla corresponded to 1921, 1915, and 634 mg kg−1 of dry matter, respectively. The lower abundance of fatty acids and sterols in the fractions from E. urophylla indicates that problems related to pitch formation will be less severe for this species than for the other two.

Structure–antileukemic activity relationship study of B- and D-ring modified and nonmodified steroidal esters of 4-methyl-3-N,N-bis(2-chloroethyl)amino benzoic acid: a comparative study

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.

Inactivation of recombinant human steroid sulfatase by KW-2581

Steroid sulfatase (STS) catalyses the hydrolysis of the sulfate esters of 3-hydroxy steroids, which are inactive transport or precursor forms of the active 3-hydroxy steroids. STS inhibitors are expected to block the local production and, consequently to reduce the active steroid levels; therefore, they are considered as potential new therapeutic agents for the treatment of estrogen- and androgen-dependent disorders such as breast and prostate cancers. KW-2581 is a novel steroidal STS inhibitor. In the present study, we found KW-2581 inhibited recombinant human STS (rhSTS) activity with an IC 50 of 2.9 nM when estrone sulfate was used as a substrate. The potency of KW-2581 was approximately 5-fold higher than that of a non-steroidal STS inhibitor, 667 COUMATE. KW-2581 was able to equally inhibit rhSTS activity when dehydroepiandrosterone sulfate was used as another substrate. KW-2581 inhibited rhSTS activity in a time- and concentration-dependent manner ( k inact, 0.439 min −1; K i, app , 15 nM), suggesting that it is an active site-directed irreversible inhibitor. Both decrease of KW-2581 concentration and increase of the des-sulfamoylated form's concentration were simultaneously observed during the reaction in a time-dependent manner with corresponding to the decrease of STS activity. Our findings for the first time demonstrated the production of des-sulfamoylated form of the compound as a consequence of STS inactivation.

6538 POSTER Preclinical studies on the antitumor activity induced by novel modified steroidal alkylating esters of propenoic acid against murine Lewis lung carcinoma (LLC)

6538 POSTER Preclinical studies on the antitumor activity induced by novel modified steroidal alkylating esters of propenoic acid against murine Lewis lung carcinoma (LLC)

Mesomorphic chiral non‐symmetrical dimers: synthesis and characterization

The preparation and phase transitional properties of several optically active non‐symmetrical dimers are reported. Two types of dimeric mesogens formed by covalently combining pro‐mesogenic cholesterol with either phenyl 4‐cyanobenzoate (polar) or phenyl trans‐4‐pentylcyclohexanecarboxylate (apolar) cores in an end‐to‐end fashion through an ω‐oxyalkanoyl spacer were investigated. The phase behaviour of these two types of dimers differs, indicating that the nature and extent of mesomorphism are sensitive to the structure of the mesogen attached to the cholesterol. However, within the series, the spacer parity greatly influences the clearing temperatures of the materials, as expected. In general, all the compounds display an enantiotropic chiral nematic (N*) phase; several of them also stabilize smectic and/or twist grain boundary phases. The optical properties of the N* phase of all the compounds were investigated with the aid of both UV–visible and chiroptical spectroscopic techniques. These complementary studies revealed that the dimers exhibit an odd–even effect in the wavelength of selective reflection, with even‐parity members having higher values, similar to previous reports. The chiroptical spectroscopy indicated that the N* pitch is right‐handed for all the investigated dimers, which is in accordance with the fact that most steroidal esters are right‐handed.

New Lipoxygenase‐Inhibiting Constituents from Calligonum polygonoides

Calligonolides A (1) and B (2), two new butanolides, and a new steroidal ester, 3, have been isolated from the whole plant of Calligonum polygonoides, together with four known compounds, tetracosan-4-olide, beta-sitosterol and its glucoside, and ursolic acid. Their structures were elucidated by spectroscopic and mass-spectrometric studies. Compounds 1-3 showed moderate inhibitory potential against lipoxygenase from soybean.

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.

Transformations of Steroid Esters by Fusarium culmorum

The course of transformations of the pharmacological steroids: testosterone propionate, 4-chlorotestosterone acetate, 17beta-estradiol diacetate and their parent alcohols in Fusarium culmorum AM282 culture was compared. The results show that this microorganism is capable of regioselective hydrolysis of ester bonds. Only 4-ene-3-oxo steroid esters were hydrolyzed at C-17. 17beta-Estradiol diacetate underwent regioselective hydrolysis at C-3 and as a result, estrone--the main metabolite of estradiol--was absent in the reaction mixture. The alcohols resulting from the hydrolysis underwent oxidation at C-17 and hydroxylation. The same products (6beta- and 15alpha-hydroxy derivatives) as from testosterone were formed by transformation of testosterone propionate, but the quantitative composition of the mixtures obtained after transformations of both substrates showed differences. The 15alpha-hydroxy derivatives were obtained from the ester in considerably higher yield than from the parent alcohol. The presence of the chlorine atom at C-4 markedly reduced 17beta-saponification in 4-chlorotestosterone acetate. Only 3beta,15alpha-dihydroxy-4alpha-chloro-5alpha-androstan-17-one (the main product of transformation of 4-chlorotestosterone) was identified in the reaction mixture. 6beta-Hydroxy-4-chloroandrostenedione, which was formed from 4-chlorotestosterone, was not detected in the extract obtained after conversion of its ester.

Identification of the steroid fatty acid ester conjugates formed in vivo in Mytilus edulis as a result of exposure to estrogens

Vertebrate-type sex steroids have been detected in a number of mollusk species and may play a role in the reproductive physiology of the animal. Mollusks are also exposed to exogenous estrogenic steroids that are present in sewage effluents, and these may add to the estrogenic burden of exposed animals. We investigated the uptake of estrogens in the blue mussel, Mytlius edulis and report for the first time the identity of estrogen fatty acid ester metabolites formed in vivo in an invertebrate. We exposed mussels to waterborne radiolabeled [ 14C]-17β-estradiol (E2) or estrone (E1) and determined the nature of their metabolites using radio-HPLC and mass spectrometry (MS). After 13 days of exposure to 10 ng/L E2, concentrations of radiolabeled residues were 2428-fold higher in M. edulis soft tissues compared with the ambient water concentration of E2. All the E2 residues in the mussel were present as a lipophilic ester which, in depuration studies, had a half-life of 8.3 days. Exposure of mussels to [ 14C]-E1 (70 ng/L) resulted in formation of a similar lipophilic metabolite that after hydrolysis released [ 14C]-E2. Tandem MSMS analyses of the purified steroid ester fraction isolated from mussels exposed to either E2 or E1 revealed that they had the same composition and comprised C16:0, C16:1 and C16:2 esters of E2. This work reveals that in vivo E1 is rapidly metabolized to E2 in mussels prior to conjugation to C16 fatty acid esters, proving that C17-ketoreductase and C16 fatty acid acyl-CoA:E2 acyltransferase are important enzymes for the metabolism of estrogens in M. edulis.

Rational Design, Synthesis and In Vitro Evaluation of Three New Alkylating Steroidal Esters

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.

Steroid and Aliphatic Esters from the Seeds of Nigella sativa.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.