Synthesis and Physicochemical Characterization of the Process-Related Impurities of Eplerenone, an Antihypertensive Drug.

Two unknown impurities were observed during the process development for multigram-scale synthesis of eplerenone (Inspra®). The new process-related impurities were identified and fully characterized as the corresponding (7ß,11α,17α)-11-hydroxy- and (7α,11ß,17α)-9,11-dichloroeplerenone derivatives 12a and 13. Seven other known but poorly described in the literature eplerenone impurities, including four impurities A, B, C and E listed in the European Pharmacopoeia 8.4 were also detected, identified and fully characterized. All these contaminants result from side reactions taking place on the steroid ring C of the starting 11α-hydroxy-7α-(methoxycarbonyl)-3-oxo-17α-pregn-4-ene-21,17-carbolactone (12) and the key intermediate (7α,17α)-9(11)-enester 7, including epimerization of the C-7 asymmetric center, oxidation, dehydration, chlorination and lactonization. The impurities were isolated and/or synthesized and fully characterized by infrared spectroscopy (IR), nuclear magnetic resonance spectroscopy (NMR) and high-resolution mass spectrometry/electrospray ionization (HRMS/ESI). Their ¹H- and 13C-NMR signals were fully assigned. The molecular structures of the eight impurities, including the new (7ß,11α,17α)-11-hydroxy- and (7α,11ß,17α)-9,11-dichloroeplerenone related substances 12a and 13, were solved and refined using single-crystal X-ray diffraction (SCXRD). The full identification and characterization of these impurities should be useful for the quality control and the validation of the analytical methods in the manufacture of eplerenone.

Synthesis and comprehensive structural and physicochemical characterization of dutasteride hydrochloride hydrate solvates.

Four crystalline dutasteride hydrochloride hydrate solvates containing respectively methanol, ethanol, acetone and acetonitrile molecules were obtained. All samples were characterized by extensive spectroscopic analysis with infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and 1H as well as 13C NMR techniques. For three solvates, i.e. methanol, ethanol and acetone solvates, the single crystal X-ray diffraction (SCXRD) experiments were possible, and their respective crystal and molecular structures were determined. The present study allowed to unambiguously establish the molecular composition of solvates as consisting of a dutasteride : hydrogen chloride : water : solvent in a molar ratio of 1:1:1:1 and confirm that they are isostructural. Beyond providing the full spectroscopic characteristic of the compounds, the results obtained have also allowed clarifying of some appearing inconsistencies in published literature regarding the appropriate attribution of IR absorption bands to the relevant molecular vibrations.

A Novel Convergent Synthesis of the Potent Antiglaucoma Agent Tafluprost.

Tafluprost (AFP-168, 5) is a unique 15-deoxy-15,15-difluoro-16-phenoxy prostaglandin F2α (PGF2α) analog used as an efficacious ocular hypotensive agent in the treatment of glaucoma and ocular hypertension, as monotherapy, or as adjunctive therapy to ß-blockers. A novel convergent synthesis of 5 was developed employing Julia-Lythgoe olefination of the structurally advanced prostaglandin phenylsulfone 16, also successfully applied for manufacturing of pharmaceutical grade latanoprost (2), travoprost (3) and bimatoprost (4), with an aldehyde ω-chain synthon 17. The use of the same prostaglandin phenylsulfone 16, as a starting material in parallel syntheses of all commercially available antiglaucoma PGF2α analogs 2-5, significantly reduces manufacturing costs resulting from its synthesis on an industrial scale and development of technological documentation. Another key aspect of the route developed is deoxydifluorination of a trans-13,14-en-15-one 30 with Deoxo-Fluor. Subsequent hydrolysis of protecting groups and final esterification of acid 6 yielded tafluprost (5). The main advantages are the preparation of high purity tafluprost (5) and the application of comparatively cheap reagents. The preparation and identification of two other tafluprost acid derivatives, tafluprost methyl ester (32) and tafluprost ethyl amide (33), are also described.

Synthesis and Physicochemical Characterization of the Process-Related Impurities of Olmesartan Medoxomil. Do 5-(Biphenyl-2-yl)-1-triphenylmethyltetrazole Intermediates in Sartan Syntheses Exist?

During the process development for multigram-scale synthesis of olmesartan medoxomil (OM), two principal regioisomeric process-related impurities were observed along with the final active pharmaceutical ingredient (API). The impurities were identified as N-1- and N-2-(5-methyl-2-oxo-1,3-dioxolen-4-yl)methyl derivatives of OM. Both compounds, of which N-2 isomer of olmesartan dimedoxomil is a novel impurity of OM, were synthesized and fully characterized by differential scanning calorimetry (DSC), infrared spectroscopy (IR), nuclear magnetic resonance spectroscopy (NMR) and high-resolution mass spectrometry/electrospray ionization (HRMS/ESI). Their ¹H, (13)C and (15)N nuclear magnetic resonance signals were fully assigned. The molecular structures of N-triphenylmethylolmesartan ethyl (N-tritylolmesartan ethyl) and N-tritylolmesartan medoxomil, the key intermediates in OM synthesis, were solved and refined using single-crystal X-ray diffraction (SCXRD). The SCXRD study revealed that N-tritylated intermediates of OM exist exclusively as one of the two possible regioisomers. In molecular structures of these regioisomers, the trityl substituent is attached to the N-2 nitrogen atom of the tetrazole ring, and not to the N-1 nitrogen, as has been widely reported up to the present. This finding indicates that the reported structural formula of N-tritylolmesartan ethyl and N-tritylolmesartan medoxomil, as well as their systematic chemical names, must be revised. The careful analysis of literature spectroscopic data for other sartan intermediates and their analogs with 5-(biphenyl-2-yl)tetrazole moiety showed that they also exist exclusively as N-2-trityl regioisomers.

Asthmatic bronchial fibroblasts demonstrate enhanced potential to differentiate into myofibroblasts in culture.

BACKGROUND: Chronic inflammation and remodeling of the bronchial wall are basic hallmarks of asthma. It is known that mesenchymal cells in the lamina reticularis underlying the basement membrane of the thickened airway wall of asthmatics predominantly display the phenotype of myofibroblasts and express alpha-smooth muscle actin (alpha-SMA). Human bronchial fibroblasts (HBFs) transform in vitro into myofibroblasts under the influence of transforming growth factor (TGF-beta). Differences in the reactivity of fibroblasts to TGF-beta in cultures derived from healthy and asthmatic donors are elucidated here. MATERIAL/METHODS: Primary human bronchial fibroblasts (HBFs) were cultured from bronchial biopsies from non-asthmatic (n=7) and asthmatic (n=7) donors and treated with TGF-beta1 or TGF-beta2 to induce myofibroblast differentiation. Expression of alpha-smooth muscle actin (alpha-SMA) was assessed by immunocytochemistry and Western blotting. The cell size and shape parameters were measured by computer-aided methods. RESULTS: Regardless of whether TGF-beta1 or TGF-beta2 was used, asthmatic cells showed enhanced expression of the myofibroblast marker as confirmed by immunocytochemistry and immunoblotting. Analysis of the shape parameters of cells incubated in the presence of TGF-beta1 revealed that HBFs of asthmatics differ from those of non-asthmatics. CONCLUSIONS: It is concluded that asthmatic HBFs cultured in vitro display some inherent features which facilitate their differentiation into myofibroblasts. These data indicate that increased reactivity of asthmatic fibroblasts to TGF-beta may play a crucial role in asthma.