Mengmeng Zhao,Xiaoxia Wu,Zengda Yu,Yunkai Sun,Zhao Liu,Jinqiao Yuan,Hu Liu,Yiping Jin
Avanafil is a phosphodiesterase type 5 inhibitor which is used to treat erectile dysfunction in men. The process-related impurities of avanafil were investigated, and four kinds of impurities in several laboratory batches with a content of 0.29–1.63% were detected by the newly developed gradient ultra-high performance liquid chromatography (UPLC). Based on the synthesis route and UPLC-MS research, the impurities are inferred as Imp-A, Imp-B, Imp-C and Imp-D. The structures of the impurities were inferred from LC-MS studies and confirmed by synthesis, followed by spectroscopic characterization such as NMR and mass spectrometry. Especially, the synthesis of Imp-D is firstly reported. The drug-related substances can be separated well by efficient and selective ultra-high performance liquid chromatography on a Waters ACQUITY HSS C18 (50 × 2.1 mm, particle size 1.8 μm) column at 35 °C, with the mobile phase consisting of ammonium formate (20 mM) and acetonitrile, and the detection at 239 nm with a DAD detector. The method was validated in terms of specificity, linearity, precision, accuracy and sensitivity, and satisfactory results were obtained. The results indicated this developed UPLC method for avanafil and the proposed synthesis mechanism can be used for quality control purposes as required by regulatory agencies to ensure the safety and efficacy of the product.
Yunkai Sun,Xiaoxia Wu,Meng Cao,Xiaoqiang Xue,Dongfang Jiang,Zhao Liu
In the classical route of synthesizing of avanafil, the last step of the reaction uses EDCI and HOBT as an acid and amine binding agent. HOBT may contain residues of hydrazine, and potential mutagenic impurities containing acylhydrazide structures may be formed. The intermediate product (M6) of avanafil was synthesized by 6 steps using diethyl ethoxylmethylmalonate and S-methyl isothiourea semisulfide as starting materials. The potentially genotoxic impurity E (Imp-E) of avanafil was given by the reaction of M6 and hydrazine sulfate acid amine, yield of 20%. The single crystal of Imp-E was cultivated by solvent volatilization method, and its structure was analyzed by high resolution mass spectrometry (HRMS) and nuclear magnetic resonance spectroscopy (NMR). The Flack parameter of Imp-E was -0.03(3) by X-ray single crystal diffraction analysis, and the absolute configuration of Imp-E was determined as S configuration. The intramolecular / intermolecular hydrogen bonds of Imp-E make the molecules maintain a stable arrangement in space.
In the technical route for the synthesis of avanafil, 1-ethyl-(3-dimethylaminopropyl)carbamyldiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBT) are used as reactive acid–amine binding agents. HOBT contains trace amounts of hydrazine residue, and there is a risk of introducing potentially mutagenic impurities with hydrazide-containing structures. The potentially genotoxic impurities E (Imp-E) and F (Imp-F) of avanafil with altering hydrazide-structure were synthesized by chemical method; subsequently, the impurities were evaluated and classified according to ICH M7 guidelines. Two complementary quantitative structure–activity relationship (QSAR) evaluation systems (Derek and Sarah) based on expert rules and statistics were used to preliminarily predict the genotoxicity of Imp-E and Imp-F, and the prediction result of E was suspected to be positive. In the Ames test of Imp-E and Imp-F, in the dose range of 62.5–1000 μg per plate, with or without the presence of metabolic activation system S9, the number of revertant colonies did not exceed 2 times the number of colonies in the solvent control group and did not show a dose–response relationship, and the test results were negative. Imp-E and Imp-F were determined to be negative for genotoxicity, which could be controlled as class 5 in ICH M7, that is, non mutagenic impurity.
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