Parallel Solid-Phase Synthesis using a New Diethylsilylacetylenic Linker and Leading to Mestranol Derivatives with Potent Antiproliferative Activities on Multiple Cancer Cell Lines.

BACKGROUND: RM-133 belongs to a new family of aminosteroid derivatives demonstrating interesting anticancer properties, as confirmed in vivo in four mouse cancer xenograft models. However, the metabolic stability of RM-133 needs to be improved. After investigation, the replacement of its androstane scaffold by a more stable estrane scaffold led to the development of the mestranol derivative RM-581. METHODS: Using solid-phase strategy involving five steps, we quickly synthesized a series of RM-581 analogs using the recently-developed diethylsilylacetylenic linker. To establish structure-activity relationships, we then investigated their antiproliferative potency on a panel of cancer cell lines from various cancers (breast, prostate, ovarian and pancreatic). RESULTS: Some of the mestranol derivatives have shown in vitro anticancer activities that are close to, or better than, those observed for RM-581. Compound 23, a mestranol derivative having a ((3,5-dimethylbenzoyl)- L-prolyl)piperazine side chain at position C2, was found to be active as an antiproliferative agent (IC50 = 0.38 ± 0.34 to 3.17 ± 0.10 µM) and to be twice as active as RM-581 on LNCaP, PC-3, MCF-7, PANC-1 and OVCAR-3 cancer cells (IC50 = 0.56 ± 0.30, 0.89 ± 0.63, 1.36 ± 0.31, 2.47 ± 0.91 and 3.17 ± 0.10 µM, respectively). CONCLUSION: Easily synthesized in good yields by both solid-phase organic synthesis and classic solution-phase chemistry, promising compound 23 could be used as an antiproliferative agent on a variety of cancers, notably pancreatic and ovarian cancers, both having very bad prognoses.

Design of a Mestranol 2-N-Piperazino-Substituted Derivative Showing Potent and Selective in†vitro and in†vivo Activities in MCF-7 Breast Cancer Models.

Anticancer structure-activity relationship studies on aminosteroid (5α-androstane) derivatives have emerged with a promising lead candidate: RM-133 (2ß-[1-(quinoline-2-carbonyl)pyrrolidine-2-carbonyl]-N-piperazine-5α-androstane-3α,17ß-diol), which possesses high in vitro and in vivo activities against several cancer cells, and selectivity over normal cells. However, the relatively weak metabolic stability of RM-133 has been a drawback to its progression toward clinical trials. We investigated the replacement of the androstane backbone by a more stable mestranol moiety. The resulting compound, called RM-581 ({4-[17α-ethynyl-17ß-hydroxy-3-methoxyestra-1,3,5(10)-trien-2-yl]piperazin-1-yl}[(2S)-1-(quinolin-2-ylcarbonyl)pyrrolidin-2-yl]methanone), was synthesized efficiently in only five steps from commercially available estrone. In comparison with RM-133, RM-581 was found to be twice as metabolically stable, retains potent cytotoxic activity in breast cancer MCF-7 cell culture, and fully blocks tumor growth in a mouse xenograft model of breast cancer. Advantageously, the selectivity over normal cells has been increased with this estrane version of RM-133. In fact, RM-581 showed a better selectivity index (15.3 vs. 3.0) for breast cancer MCF-7 cells over normal breast MCF-10A cells, and was found to be nontoxic toward primary human kidney proximal tubule cells at doses reaching 50 µm.

Amphidynamic crystals of a steroidal bicyclo[2.2.2]octane rotor: a high symmetry group that rotates faster than smaller methyl and methoxy groups.

The synthesis, crystallization, single crystal X-ray structure, and solid state dynamics of molecular rotor 3 provided with a high symmetry order and relatively cylindrical bicyclo[2.2.2]octane (BCO) rotator linked to mestranol fragments were investigated in this work. By use of solid state (13)C NMR, three rotating fragments were identified within the molecule: the BCO, the C19 methoxy and the C18 methyl groups. To determine the dynamics of the BCO group in crystals of 3 by variable temperature (1)H spin-lattice relaxation (VT (1)H T1), we determined the (1)H T1 contributions from the methoxy group C19 by carrying out measurements with the methoxy-deuterated isotopologue rotor 3-d6. The contributions from the quaternary methyl group C18 were estimated by considering the differences between the VT (1)H T1 of mestranol 8 and methoxy-deuterated mestranol 8-d3. From these studies it was determined that the BCO rotator in 3 has an activation energy of only 1.15 kcal mol(-1), with a barrier for site exchange that is smaller than those of methyl (E(a) = 1.35 kcal mol(-1)) and methoxy groups (E(a) = 1.92 kcal mol(-1)), despite their smaller moments of inertia and surface areas.

Synthesis, characterization and biological evaluation of some novel 17-isoxazoles in the estrone series.

Regioselective 1,3-dipolar cycloadditions of different aryl nitrile oxides to mestranol were carried out to furnish novel steroidal 17α-isoxazoles in good to excellent yields. Copper(I) was found to be an efficient catalyst, accelerating the intermolecular ring-closures and leading exclusively to 3,5-disubstituted isoxazoles. The yields of the cycloadducts, however, were influenced by the substituents on the aromatic moiety of the 1,3-dipoles. Moreover, dehydration of the primary products resulted in the corresponding Δ(16,17)exo-heterocyclic derivatives. All the synthesized compounds were subjected to in vitro pharmacological studies of their antiproliferative effects relative to three human malignant cell lines (HeLa, MCF7 and A2780).

Improved synthesis of mestranol and ethinyl estradiol (EE) related degradation products as authentic references.

Preparative chemical methods for the synthesis of 10 degradation or photodecomposition products of mestranol and ethinyl estradiol (EE) are described. The synthesized compounds are useful as reference materials and standards for pharmaceutical analysis of mestranol and EE as bulk chemical or in formulated product. New synthetic methods were presented and the known synthetic procedures were improved. Detailed structural characterization of the degradation or photodecomposition products of mestranol and EE and related compounds was reported.

Microwave-accelerated derivatization for the simultaneous gas chromatographic-mass spectrometric analysis of natural and synthetic estrogenic steroids.

A rapid microwave-accelerated derivatization process for the GC-MS analysis of steroid estrogens, estrone (E1), 17beta-estradiol (E2), estriol (E3), 17alpha-ethynylestradiol (EE2) and mestranol (MeEE2), was developed. Under microwave irradiation, the five estrogenic hormones studied were simultaneously derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA)+trimethylchlorosilane (TMCS) in pyridine solution. Effects of irradiation time (15-120 s) and power level (240-800 W) on the yield of the derivatization were investigated. The derivatization under the irradiation of 800 W microwave for 60s produced comparable results when compared with the conventional heating process in a sand bath for 30 min at 80 degrees C in terms of derivatization yield, linearity and precision for all steroid hormones tested. The calibration curves are linear between 3.00 and 3.00 x 10(2) microg mL(-1). The square of the regression coefficients (R(2)) range from 0.979 to 1.000. The applicability of the method was evaluated on spiked river and distilled water samples at two concentrations, 25.0 and 2.00 x 10(2) ng mL(-1). The recoveries obtained by using microwave heating (60s, 800 W) were similar to those by conventional heating. When combined solid-phase extraction (SPE) with the application of the microwave-accelerated derivatization proposed here, the detection limits of 0.02-0.1 ng L(-1) for the steroid hormones have been achieved. The results demonstrated that microwave-accelerated derivatization is an efficient and suitable sample preparation method for the GC-MS analysis of estrogenic steroids.

Steroids 49. Investigations on the dehydration of 17 alpha-ethynyl-17 beta-hydroxysteroids.

The acidic dehydration of 17 alpha-ethynyl-17 beta-hydroxysteroids (1-3) was investigated. On reaction with thionyl chloride, phosphorus oxychloride, and formic acid, the desired dehydration was accompanied by chlorination, Wagner-Meerwein rearrangement, and D-homoaromatic rearrangement. The structure of the product from the transformation of 17 beta-hydroxypregn-20-yne derivative (3) on reaction with formic acid was misjudged. It was regarded as a pregn-16-en-20-yne (10) instead of the actually rearranged D-homoaromatic compound (11). As a consequence, physical data corresponding to this latter structure have been cited in the literature as those of pregn-16-en-20-yne derivative (10). This confusion prompted us to prepare compounds of both types (4, 9, 10, and 11), the characterization of which is here described.