M. Luisa Sá e Melo

Sterols as Anticancer Agents: Synthesis of Ring-B Oxygenated Steroids, Cytotoxic Profile, and Comprehensive SAR Analysis

The cytotoxicity of oxysterols was systematically studied in tumor and normal cells. Synthetic strategies to prepare this library included oxidations at ring B and a new method to yield 6β-hemiphthalates directly from Δ(5)-steroids. Most oxysterols were cytotoxic and showed selectivity toward cancer cells, LAMA-84 cells (leukemia) being particularly sensitive to 4, 8, 22, and 27 (IC(50) < 5.6 μM). The structural requirements to induce selective toxicity are discussed to shed light on the development of new anticancer drugs.

An efficient steroid pharmacophore-based strategy to identify new aromatase inhibitors.

Aromatase, an enzyme involved in the conversion of androgens into estrogens, is an important target for the endocrine treatment of breast cancer. Aromatase inhibition is usually achieved with steroids structurally related to the substrate of catalysis or, alternatively, with azole non-steroid compounds. Substituted androstenedione derivatives with Delta(1), Delta(6) and Delta(1,6) unsaturations and 6-alkyl/6-phenyl aliphatic substitutions, are among the most potent steroid aromatase inhibitors known to date. In this paper we have combined the common pharmacophoric and shape features of these molecules into a new pharmacophore model, useful for virtual screening of large compound databases. Small subsets of the best fitting anti-aromatase candidates were extracted from the NCI database and experimentally tested on an in vitro assay with human placental aromatase. New potent aromatase inhibitors were identified such as compounds 8 and 14. Considering the lack of a crystal structure for the aromatase enzyme, this ligand-based method is a valuable tool for the virtual screening of new aromatase inhibitors.

Fast three dimensional pharmacophore virtual screening of new potent non-steroid aromatase inhibitors.

Suppression of estrogen biosynthesis by aromatase inhibition is an effective approach for the treatment of hormone sensitive breast cancer. Third generation non-steroid aromatase inhibitors have shown important benefits in recent clinical trials with postmenopausal women. In this study we have developed a new ligand-based strategy combining important pharmacophoric and structural features according to the postulated aromatase binding mode, useful for the virtual screening of new potent non-steroid inhibitors. A small subset of promising drug candidates was identified from the large NCI database, and their antiaromatase activity was assessed on an in vitro biochemical assay with aromatase extracted from human term placenta. New potent aromatase inhibitors were discovered to be active in the low nanomolar range, and a common binding mode was proposed. These results confirm the potential of our methodology for a fast in silico high-throughput screening of potent non-steroid aromatase inhibitors.

Selective Cytotoxicity of Oxysterols through Structural Modulation on Rings A and B. Synthesis, in Vitro Evaluation, and SAR

Chemically diverse oxysterols were prepared and evaluated for cytotoxicity, aiming to push forward potency and selectivity. They were tested against seven cancer (HT-29, HepG2, A549, PC3, LAMA-84, MCF-7, and SH-SY5Y) and two noncancerous cell lines (ARPE-19 and BJ). The influence of the oxidation pattern on rings A and B was studied. Oxygen functionalities on ring B, such as oxo, oxime, acetamide, acetate, and alkoxy, were evaluated. Most oxysterols were cytotoxic in the low micromolar range, with emphasis to the tetrols 14 and 34, the 6β methoxy and acetoxy derivatives 21 and 45, and the oxime 28. In general, the oxysterols were more toxic to cancer cells and a set of compounds (9, 14, 21, 28, 45) with very high selectivity was identified. The cytotoxicity of 3β-acetates was lower than that of the parent alcohols, although incubation for a longer period rendered them equally cytotoxic, pointing them as potential prodrugs of oxysterols.

Synthesis and biological evaluation of two new radiolabelled estrogens: [125I](E)-3-methoxy-17α-iodovinylestra-1,3,5(10),6-tetraen-17β-ol and [125I](Z)-3-methoxy-17α-iodovinylestra-1,3,5(10),6-tetraen-17β-ol

The synthesis of two novel radiolabelled estrogen derivatives, [125I](E)-3-methoxy-17alpha-iodovinylestra-1,3,5(10),6-tetraen-17beta-ol (E[125I]IVDE) and [125I](Z)-3-methoxy-17alpha-iodovinylestra-1,3,5(10),6-tetraen-17beta-ol (Z[125I]IVDE), was carried out aiming to study the influence of the introduction of a C6-C7 double bond on the biological properties of the estradiol molecule. 3-Methoxyestra-1,3,5(10),6-tetraen-17-one was synthesised starting from a suitably protected estrone and subsequently converted into the 17alpha-ethynyl derivative. The radioiodinated derivatives were stereoselectively formed by radioiododestannylation of the corresponding tributylstannyl precursors. The biodistribution of the novel [125I]iodovinylestradiol derivatives was evaluated in immature female mice. Biological data indicated that the Z-isomer, owing to its higher in vivo uptake by the target tissue, has the preferable configuration for further development of similar compounds for estrogen receptor detection.

Combining Computational and Biochemical Studies for a Rationale on the Anti-Aromatase Activity of Natural Polyphenols

Aromatase, an enzyme of the cytochrome P450 family, is a very important pharmacological target, particularly for the treatment of breast cancer. The anti‐aromatase activity of a set of natural polyphenolic compounds was evaluated in vitro. Strong aromatase inhibitors including flavones, flavanones, resveratrol, and oleuropein, with activities comparable to that of the reference anti‐aromatase drug aminoglutethimide, were identified. Through the application of molecular modeling techniques based on grid‐independent descriptors and molecular interaction fields, the major physicochemical features associated with inhibitory activity were disclosed, and a putative virtual active site of aromatase was proposed. Docking of the inhibitors into a 3D homology model structure of the enzyme defined a common binding mode for the small molecules under investigation. The good correlation between computational and biological results provides the first rationalization of the anti‐aromatase activity of polyphenolic compounds. Moreover, the information generated in this approach should be further exploited for the design of new aromatase inhibitors.

Ligand-guided optimization of CXCR4 homology models for virtual screening using a multiple chemotype approach.

CXCR4 is a G-protein coupled receptor for CXCL12 that plays an important role in human immunodeficiency virus infection, cancer growth and metastasization, immune cell trafficking and WHIM syndrome. In the absence of an X-ray crystal structure, theoretical modeling of the CXCR4 receptor remains an important tool for structure–function analysis and to guide the discovery of new antagonists with potential clinical use. In this study, the combination of experimental data and molecular modeling approaches allowed the development of optimized ligand-receptor models useful for elucidation of the molecular determinants of small molecule binding and functional antagonism. The ligand-guided homology modeling approach used in this study explicitly re-shaped the CXCR4 binding pocket in order to improve discrimination between known CXCR4 antagonists and random decoys. Refinement based on multiple test-sets with small compounds from single chemotypes provided the best early enrichment performance. These results provide an important tool for structure-based drug design and virtual ligand screening of new CXCR4 antagonists.

Ultrasound assisted remote functionalization of non-activated carbon atoms: efficient in situ formation of tetrahydrofurans by sonolysis of bromohydrins with (diacetoxyiodo)benzeneI2

Abstract Ultrasonic irradiation of bromohydrins in the presence of (diacetoxyiodo)benzene and I 2 generates alkoxyl radicals which lead in situ to intramolecular hydrogen abstraction on the C-19 non-activated carbon atoms in steroids producing tetrahydrofurans selectively in very good yields. Photolysis was less effective in promoting such conversions.

Efficient Chemoenzymatic Synthesis, Cytotoxic Evaluation, and SAR of Epoxysterols

A library of diastereomerically pure epoxysterols, prepared by combining chemical and enzymatic methodologies, was evaluated for cytotoxicity toward human cancer and noncancer cell lines. Unsaturated steroids were oxidized by magnesium bis(monoperoxyphthalate) hexahydrate in acetonitrile, and the resulting epimeric epoxides were enzymatically separated using Novozym 435 or lipase AY. Some of the synthesized epoxysterols have potent cytotoxicity and higher activity on cancer cell lines HT29 and LAMA-84.

X-ray and deuterium labeling studies on the abnormal ring cleavages of a 5β-epoxide precursor of formestane

A new convergent synthesis of the antitumor steroid formestane (4-OHA) 5 has been performed from the easily available epimeric mixture of 5 alpha- and 5 beta-androst-3-en-17-one 1a and 1b in order to attempt a yield improvement. A two-step oxidative route followed by base-catalyzed isomerization was applied to the 5 alpha- and 5 beta-epimers 1a and 1b, either as a mixture or separately, leading to the title compound 5. From epimer 1a an efficient process was attained to prepare the desired aromatase inhibitor formestane. Epimer 1b led to the formation of the same compound 5. Additionally, 1b have also been converted in 5 beta-hydroxyandrostane-3,17-dione 12 and androst-4-ene-3,17-dione 13, revealing an unexpected reactivity of the 3 beta,4 beta-epoxy-5 beta-androstan-17-one intermediate 6 formed from 1b during the first oxidative step with performic acid. Cleavage of the epoxide 6 led to the trans-diaxial and the trans-diequatorial vic-diols 7 and 8 and to the 1,3-diol 9. The formation of the abnormal products 8 and 9 were investigated through X-ray and deuterium labeling studies. Diol 8 was formed through a trans-diequatorial epoxide ring opening and the 1,3-diol 9 was formed through an intramolecular rearrangement involving a 1,2-hydride shift. All the vic-diols 3, 7 and 8 formed, proved to be good precursors for the synthesis of the target compound 5.