Characterization of two new degradation products of atorvastatin calcium formed upon treatment with strong acids

Abstract

Atorvastatin calcium (Lipitor®, Sortis®) is a well-established cholesterol synthesis enzyme (CSE) inhibitor commonly used in the therapy of hypercholesterolemia. This drug is known to be sensitive to acid treatment, but only little data has been published on the structures of the degradation products. Here we report the identification of two novel degradation products of atorvastatin, which are formed only under drastic acidic conditions. While treatment with conc. sulfuric acid led to a loss of the carboxanilide residue (accompanied by an expectable lactonization/dehydration process in the side chain), treatment with conc. aqueous hydrochloric acid gave a complex, bridged molecule under C–C-bond formation of the lactone moiety with the pyrrole, migration of the isopropyl group and loss of the carboxanilide residue. The novel degradation products were characterized by NMR spectroscopy, HRMS data and X-ray crystal structure analysis. Introduction Over the past decades, the general trend toward globalization of the supply chains for active pharmaceutical ingredients has created new challenges for the authorities in ensuring the safety and quality of the drug supply [1]. Unprecedented impurities can appear, most likely if limited information is available about details (or alterations) of production processes of drugs. On the one hand, it is impossible to check drug substances routinely for all imaginable impurities, on the other hand it is desirable to identify as much as possible degradation products of drugs resulting from inappropriate exposition to potentially harmful conditions during production, manufacturing and storage. For being able to provide relevant data in a manageable time frame, two kinds of stress tests have found broad application: accelerated storage conditions (higher temperatures, higher Beilstein J. Org. Chem. 2019, 15, 2085–2091. 2086 humidity, ...) typically provide reliable data on the stability of a drug, but are still time-consuming; on the other hand in “forced degradation experiments” the drug is submitted to more drastic conditions (e.g., strong acid or base, strong oxidant, very high temperature), and potential degradation products can be identified in very short time [2,3]. However, forced degradation experiments are highly artificial in nature, and thus one has to keep in mind that these extremely drastic conditions are prone to lead to results that might be out of proportion for daily quality control [3]. Nevertheless, knowledge about the outcome of stress tests under extreme conditions helps to get insight into the overall reactivity of drug substances. Atorvastatin calcium (1, marketed as the trihydrate in Lipitor®, Sortis®), is a well-established drug for treatment of hypercholesterolemia [4]. This drug is monographed in the leading pharmacopoeias (Ph. Eur., USP), and a couple of impurities are listed there. Most of these impurities result from the synthesis process (stereoisomers, products resulting from impure starting materials or side reactions), and only one of these impurities, lactone 2, is most likely a degradation product, resulting from acid-mediated lactonization of the 3,5-dihydroxyheptanoate side chain. A couple of previous publications deal with stress tests on atorvastatin (and its salts), and an overview has been published by Sirén [5]. Hereby, atorvastatin was found to be sensitive to acidic, oxidative, photochemical and thermal stress. Acidic degradation of atorvastatin was reported to follow first order kinetics, but decomposition products were not characterized in this [6] and several other reports, which only determined the downsizing of the atorvastatin peak in HPLC after treatment with acid [7-10]. The most prominent decomposition product upon acidic treatment, compound 2, results from lactonization of the 3,5-dihydroxyheptanoate side chain under moderately acidic conditions (0.1 M HCl) [11-14]. Shah et al. [15] identified six additional decomposition products upon treatment with 0.1 M HCl at 80 °C for 24 h, among which the dehydrated lactone 3 was dominating, accompanied by minor amounts of products arising from dehydration of the δ-hydroxy group and some epimers resulting from acid-catalyzed isomerization reactions. In contrast, Vukkum et al. [13] describe, besides lactones 2 and 3, an α,β-unsaturated carboxylic acid 4. Treatment under more drastic conditions (6 M HCl, reflux, 3 h) was reported to result mainly in hydrolysis of the anilide moiety to give carboxylic acid 5 [16,17] (Figure 1). Here we report on the results of our investigations on the decomposition of atorvastatin calcium (1) under strongly acid conditions. Results and Discussion Stress tests Since the lability of atorvastatin towards moderately acidic conditions is well-documented, we aimed at investigating the outcome of incubation with acids under more drastic conditions. Treatment of atorvastatin calcium trihydrate (1) with 2 M aqueous hydrochloric acid at room temperature (Table 1, entry 1) gave, in accordance with previous reports, only hydroxylactone 2 (55% yield). This outcome was confirmed by comparison with published NMR data [18,19]. At elevated temperature (reflux, 4 h; Table 1, entry 2) a mixture of lactone 2 and known unsaturated lactone 3 [15] (arising from acid-catalyzed dehydration of 2) was obtained. Under even more drastic acidic conditions (refluxing with 6 M hydrochloric acid for 3 h, with 20% aqueous H2SO4 for 2 h, or with p-toluenesulfonic acid in toluene for 5 h; Table 1, entries 3–5) unsaturated lactone 3 was formed exclusively and in high to almost quantitative yields (Table 1). When atorvastatin calcium trihydrate (1) was submitted to extremely strong acidic conditions by refluxing with concentrated (37%) aqueous hydrochloric acid (entry 6), a new product 6 was formed in almost quantitative yield. The 1H NMR analysis clearly indicated that the entire carboxanilide partial structure got lost under these conditions. However, no signal was observed which could be attributed to a C–H group at the pyrrole ring. The 13C NMR data showed one carbonyl resonance at 170.3 ppm, assignable to a lactone moiety. The HMBC experiment showed a cross peak between the proposed lactone carbonyl carbon and a neighboring CH-O group, confirming the lactone moiety, and the DEPT spectrum showed a new aliphatic methine resonance at 25.2 ppm. By HRESIMS mass data (found: 404.2020 for [M + H]+) a molecular formula of C26H26FNO2 was confirmed, excluding incorporation of HCl into this artefact. Finally, X-ray crystallography structure analysis (see Figure 2 and Supporting Information File 1) disclosed the structure of 6, bearing a novel, bridged tricyclic 1,5methanopyrrolo[1,2-e][1,5]oxazonin-3-one ring system (Scheme 1). In contrast, submission of atorvastatin calcium trihydrate (1) to concentrated sulfuric acid for two hours at 60 °C (Table 1, entry 7) led to the degradation product 7 in low yield (18%) (Scheme 1). No further decomposition products could be isolated. Here, lactonization and dehydration steps in the side chain took place as observed before in the other acid treatments, however, under these extremely strong, virtually anhydrous acid conditions, the entire carboxanilide residue was removed to give the (S)-configured 4-unsubstituted pyrrole 7, as exemplified by a typical CH resonance at 6.20 ppm in the 1H NMR spectrum. This structure was further Beilstein J. Org. Chem. 2019, 15, 2085–2091. 2087 Figure 1: Atorvastatin calcium trihydrate (1) and previously published decomposition products arising from treatment with acids: lactone 2, dehydrated lactone 3, α,β-unsaturated carboxylic acid 4, and carboxylic acid 5 (resulting from postulated anilide hydrolysis). Table 1: Acidic stress conditions and decomposition products formed. Entry no. Acidic conditions Decomposition products (yield) 2 3 6 7 1 2 M HCl, 20 °C, 2 h 55% – – – 2 2 M HCl, reflux, 4 h 65% 14% – – 3 6 M HCl, reflux, 3 h – 70% – – 4 20% H2SO4, reflux, 2 h – >98% – – 5 p-toluenesulfonic acid, toluene, reflux, 5 h – 95% – – 6 37% HCl, reflux, 5 h – – 96% – 7 conc. H2SO4, 60 °C, 2 h – – – 18% confirmed by X-ray data (see Figure 2 and Supporting Information File 1). HPLC method for the detection of the novel impurities In order to provide a convenient method for including our new findings into quality control of atorvastatin batches, we worked out an isocratic HPLC protocol, which prettily separates the four artefacts 2, 3, 6 and 7 from atorvastatin (1). This method uses an RP18 stationary phase (Eurospher 100–C18), isocratic elution with 0.01 M ammonium acetate buffer (pH 4)-acetonitrile 54:46 (v/v) at a flow rate of 1 mL/min at 40 °C, with UV detection at 246 nm (Figure 3). Beilstein J. Org. Chem. 2019, 15, 2085–2091. 2088 Figure 3: Separation of atorvastatin (1; retention time: 5.8 min) from the four decomposition products 2 (retention time: 9.2 min), 3 (retention time: 15.6 min), 6 (retention time: 21.4 min) and 7 (retention time: 24.9 min). Chromatogram obtained with a solution containing 10 mg each in 5.0 mL DMF (retention time 2.3 min), diluted 1:5 with the eluent buffer before injection. Scheme 1: Formation of novel artefacts 6 and 7 under extremely strong acidic conditions. Discussion In this investigation we first confirmed some pathways of decomposition of atorvastatin under acidic conditions. With dilute mineral acids at room temperature, atorvastatin is conveniently converted into the lactone 2 under retention at the C5–O bond of the aliphatic chain [13,20], whereas treatment under more drastic conditions (e.g., 6 M HCl or heating) causes expectable subsequent dehydration to give the unsaturated lactone 3 [13,15]. In contrast to previous reports [16,17] we could not find any indication for a cleavage of the carboxanilide partial structure to give free pyrrolecarboxylic acid 5 under treatment with 6 M HCl under reflux. Figure 2: Top: Molecular structure of artefact 6. Shown here is the molecular structure of on