Synthesis of High Purified Tulobuterol and Its Study of the Organic Impurities

Journal of Chromatographic Science, 2019, Vol. 57, No. 4, 299–304 doi: 10.1093/chromsci/bmy083 Advance Access Publication Date: 4 February 2019 Article Article Synthesis of High Purified Tulobuterol and Its Study of the Organic Impurities 1 Beijing Key Laboratory of Drug Delivery and Novel Formulation, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China, 2Pesent address: Hui Song, master degree candidate, postal address: No.1 Xiannongtan, Xicheng District, Beijing, 10050, China. Email: , and 3 Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China * Author to whom correspondence should be addressed. Wensheng Zheng, Professor, No.1 Xiannongtan Street, Xicheng District, Beijing 10050, China. Email: Received 11 October 2017; Revised 18 June 2018; Editorial Decision 30 July 2018 Abstract The synthetic condition of tulobuterol was optimized to gain lower impurity content. Two intermediates were analyzed, and three degradation impurities were isolated using preparative liquid chromatography for the first time and subsequently characterized by various techniques. Possible degradation impurities were deduced by an oxidative mechanism. Two intermediate impurities were detected: α-bromo-2-chloroacetophenone and 1-(2-chlorophenyl)-2-bromoethanol. Three unreported degradation impurities were found and characterized as N-tert-butyl glycine, o-chlorobenzoic acid and chlorobenzene. The single crystal structure of tulobuterol was firstly reported. Introduction Impurity is any component of the new drug substance that is not the chemical entity defined as the new drug substance (1). The drug regulatory agencies, such as the United States Food and Drug Administration (FDA), the International Council for Harmonisation (ICH) have specific guidelines for registration on the quantification and limitation of impurities. Generally, impurities can be classified into three categories: organic impurities, inorganic impurities and residual solvents and the quantification of organic impurities is the most complicated (2–4). The drug researchers need to take the actual and potential impurities into consideration, which is most likely to arise during the synthesis, purification and storage. Then impurities beyond the certain threshold or in forced degradation study remain challenging. Hence, it is of often difficulty to obtain enough pure impurity for characterization. Tulobuterol, 2-tert-butylamino-1-(2-chloro-phenyl)-ethanol, is a β2-adrenergic agonist which has been used extensively in the longterm management of asthma and chronic obstructive pulmonary disease (COPD) (5–7). Since tulobuterol was approved by Japanese drug regulatory agency in 1990s, its clinical efficiency and safety have been widely confirmed. However, there is no data about the chemical formula of actual and potential impurities in the Japanese Pharmacopoeia (JP), United States Pharmacopoeia (USP), European Pharmacopeia (EP) and Chinese Pharmacopoeia (ChP). The data is not reported in the literature so far, which brings great difficulties to quality control of impurity profile. There are three reported synthetic routes for tulobuterol: selenium dioxide used in route 1 is too expensive and toxic to purchase (8); the route 2 of o-chloroacetophenone being converted into o-chlorostyrene as raw materials has four-step reaction and total yield is low (9); route 3 has high yield by bromination, reduction and amination in three steps (10). Route 3 is relatively easy to get raw materials. Through appropriate improvement of the method, the related substances are reduced with easy purification process and the method is suitable for large scale production. © The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: 299 Hui Song1,2, Jingjing Lin3, Huajin Tan1, Longying Shen3, Nan Zhang1, Yujia Zhang1, Xiaochuan Tan1, Yajun Yang3, Xiandao Pan3, and Wensheng Zheng1,* 300 In present study, synthetic tulobuterol crude and purified product were analyzed and the forced degradation impurities were conducted under the different conditions (hydrolysis under acid and alkali condition, oxidation, heat and photolysis). Tulobuterol has been found to remain stable to hydrolysis, heat and photolysis and be oxidated under slight condition. The analysis of crude product shows two intermediates with higher concentration. Ultimately, two intermediate impurities (Imp A and Imp B) and three degradation impurities (Imp C, Imp D and Imp E) are characterized through appropriate analysis method, which offers the reference for further drug development for clinical efficiency and safety. Reagents and methods The chemicals and reagents used for the synthesis, analysis, isolation, purification and characterization purpose of impurities. o-Chloroacetophenone and tulobuterol (CP grade): Hubei Prosperity Galaxy Chemical Co., Ltd., China; tert-butylamine (CP grade) and chloroform-d: Sigma-Aldrich Co. LLC.; Br2, hydrogen peroxide (H2O2), hydrochloric acid, sodium hydroxide, sodium dihydrogen phosphateanhydrous (AR grade): Beijing Chemical Works, China; N-tert-butyl glycine (AR grade, 97%): Ark Pharm, Inc., USA; 1-pentanesulfonic acid sodium salt (HPLC grade), methanol and acetonitrile (HPLC grade): Fisher Scientific. Conditions of analysis and isolation The chromatographic separation was performed on Waters 2695 separation module with automatic sampler and Waters 2487 dual λ UV detector. The data was processed and analyzed by using Empower 3 Chemstation purchased from Waters Corporation. The separation was performed on an Inertsil ODS-3 column (4.5 × 250 mm, 5 μm particle size). The mobile phase was 3.3 g/L sodium pentanesulfonate and 0.02 mol/L sodium dihydrogen phosphate (pH = 3.0)-acetonitrile (750:250) at the flow of 1.0 mL/min. The column temperature was 35°C and the detection wavelength was set at 215 nm. The test was performed with exactly 20 μL each of the sample solution at 1 mg/mL and standard solution at 1 μg/mL according to the above conditions. Each impurity percentage in the portion of tulobuterol was calculated by the equation: content of impurity = (RU/RS) × (CS/CU) × 100 (RU = peak response of any impurity from the sample solution; RS = peak Figure 1. Synthetic pathway of tulobuterol. response of tulobuterol from the standard solution; CS = concentration of tulobuterol from the standard solution (mg/mL); CU = concentration of tulobuterol from the sample solution (mg/mL)). Signal of tulobuterol and impurities could be effectively separated. Tulobuterol had good linearity in the range of 20–500 μg/mL and the linear regression equation was A = 1546.4C-32.567 (r = 0.9998); the mean absolute recovery and relative recovery were 97.5% and 98.8%, respectively; the lower limit of detection was 16 ng; the RSD of intra- (...truncated)