Refined Preparation of 1α,3-Dipyrrolidino-Androsta-3,5-Diene-17-One, a Key Intermediate in the Exemestane Synthesis
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ORIGINAL ARTICLES Pharmaceutical Research Institute1; Faculty of Chemistry, University of Warsaw, Warsaw, Poland Refined preparation of 1␣,3-dipyrrolidino-androsta-3,5-diene-17-one, a key intermediate in the exemestane synthesis Ł. Kaczmarek 1, M. Cybulski 1, M. Kubiszewski 1,A.Les´ 1,2 Received February 3, 2012, accepted March 31, 2012 Prof. Andrzej Le´s, Pharmaceutical Research Institute, Rydygiera 8, 01-793 Warsaw, Poland [email protected] Pharmazie 67: 899–905 (2012) doi: 10.1691/ph.2012.2025 A significant improvement of the patent route for exemestane synthesis has been achieved. The key inter- mediate 1␣,3-dipyrrolidino-androsta-3,5-diene-17-one (7) was obtained in a good yield and effectively used without further isolation in the next reaction step. The original analytical method for the identification and quantification of the substrate androsta-1,4-diene-3,17-dione (ADD, 6), intermediate 7 and 1-pyrrolidino- androsta-1,3,5-triene-17-one (9) impurity in the reaction mixture was applied. Due to the newly developed process, the economical synthesis of the final pharmaceutical product in a large scale was possible. In addition, the complete NMR characteristics of 7 was described for the first time. The experiments were also analyzed with the theoretical quantum mechanical density functional B3LYPcalculations for the energy outputs in model reactions. Based on these studies hypothetical routes of key intermediate (7) formation have been suggested. These predictions were consistent with the solutions of kinetic equations fitted to the experimental curves for time-dependence of three components of the reaction mixture. 1. Introduction Breast cancer is the leading cause of deaths among women (World Cancer Report 2008). One-third of this type of cancer is hormone-dependent and uses estrogen to proliferate (Segaloff 1978). The major source of estrogens in post-menopausal women is enzymatic conversion of circulating androgens. The final step in this process is catalyzed by the aromatase enzyme (Miller 1990). Therefore, the inhibition of this enzyme activ- ity causes tumor regression in post-menopausal women with estrogen-dependent breast cancer (Bajetta et al. 1999). There are two types of aromatase inhibitors. Type II (reversible inhibitors), such as aminoglutethimide, rogletimide, fadrosole, anastrozole, Scheme 1: Two common routs of synthesis of exemestane (1) letrozole and vorozole, act by reversibly binding to the enzyme. Hence ongoing estrogen deprivation needs the continued pres- ence of the drug. This results in enhanced toxicity. Type I with DDQ (Buzzetti et al. 1987) or Jones reagent (Longo and aromatase inhibitors, having androstenedione structure, inac- Lombardi 1987), respectively (Scheme 1). tivate the enzyme irreversibly. In effect, the renewed estrogen The second route, proposed by a German group in 1986, synthesis requires biosynthesis of new enzyme, which results in starts from androsta-1,4-diene-3,17-dione (ADD)(6). Com- the reduced toxicity of the drug, since the enzyme inhibition per- pound 6 reacts with pyrrolidine to give the corresponding sists after the drug has been removed from the system (Lombardi 1,3-dipyrrolidino derivative 7 which readily transforms into 2002). One of the Type I aromatase inhibitors is exemestane (6- 6-(hydroxymethyl)androsta-1,4-dien-3,17- dione (8) through methyleneandrosta-1,4-dione) (1) which was launched by the stirring with the aqueous formaldehyde (Wagner 1986a, b). The Farmitalia Carlo Erba group (Giudici et al. 1988). Exemestane acid-catalyzed dehydration of 8 yields the final exemestane (1) is significant as the only orally active irreversible steroidal aro- (Wagner 1986a, b) (Scheme 2). matase inhibitor, effectively used in postmenopausal women The analysis of literature and patent data reveals, that the meth- with advanced breast cancer (Clemett and Lamb 2000). More- ods disclosed in patents of Farmitalia group (Buzzetti et al. 1987; over, it appears that exemestane is also effective for the Longo and Lombardi 1987) are unfavorable because of toxic and chemoprevention of breast cancer (Barton 2011). environmentally unsafe reagents (e.g. pyridine chromate, DDQ, In general, there are two different routes of obtaining exemes- POCl3 or bromine) as well as harsh reaction conditions. From tane. The first, applied by Farmitalia Carlo Erba originator, this point of view the exemestane synthesis described in German exploits 6-methylation of androstenedione (2) according to the patents (Wagner 1986a, b) appears to be an advantageous alter- general method of Annen et al. (1982) or boldenone (3)in native. This method of exemestane synthesis was used formerly Mannich reaction. Then the intermediates 4 or 5 are oxidized (Wojciechowska 2001; Wojciechowska et al. 2004) and follow- Pharmazie 67 (2012) 899 ORIGINAL ARTICLES Scheme 2: The exemestane (1) synthesis from ADD (6) ing some modifications, by Kunnen et al. (2005) from Cedarburg Pharmaceuticals Inc. In both cases the crucial point of the syn- thesis is the formation of dipyrrolidino derivative 7 followed by its transformation into 6-hydroxymethyl-androstanodienone 8. In order to develop the technology of exemestane in a pilot scale we examined these reactions, starting from the verification Scheme 3: The transformation of an impurity 9 into the desired intermediate 8 of literature data. Cedarburg Pharmaceuticals Inc. (Kunnen 2005) was revisited. Actually, it was confirmed that the addition of a catalytic amount 2. Investigations, results and discussion of acetic acid accelerates the reaction in mild temperature con- ◦ 2.1. Exploration of reaction parameters ditions (40 C), but the product cannot be easily isolated - as it was described in the patent application example - from the According to the procedure described by Wojciechowska et al. ethanol medium. Therefore, our next experiments with the acid (2004), which was based on the previously published procedure catalyst were continued without a solvent, in the excess of pyrro- from the German patent (Wagner 1986a, b), the compound 7 was lidine. Initially it was found that the acid catalyzed reaction at slowly formed by refluxing ADD (6) for 68–80 h in an excess of 50 ◦C needs only 7 h to yield compound 7. Then it was demon- pyrrolidine (3 mL/g) under inert atmosphere. When the quantity strated that the acid catalyzed reaction may be carried out even of the starting 6 fell below 10%, the reaction was completed. The at ambient temperature under normal atmospheric conditions reaction could not be controlled by HPLC or TLC techniques but its progress depends strongly on the amount of acetic acid because of the instability of 7 during chromatography, so the IR added, see Fig. 1. spectroscopy control was applied as a method of choice. The The acid addition range was established by determining (with dipyrrolidine adduct 7 was isolated in the yield of 60–70% by NMR spectroscopy) seven single measurement points obtained removing pyrrolidine excess and crystallization of the residue through adding from 0 to 7 mL of acid per 10 g of ADD. with methanol. Although the precision of ADD content determination is gen- An attempt to repeat the above procedure revealed that the reac- erally moderate, about 1–2 %, we are convinced that the acid tion time increased rapidly when the reaction was scaled up. range of 0.15–0.30 mL per 1 gram of starting ADD is optimal The consumption of the starting ADD exceeding 90% could for practical purposes. be reached in 48 h at the 10 g scale, while scaling the reac- At that point the procedure suitable for the reaction carried out in tion up to 60 g of ADD prolonged this time to 70 h. In the a large scale was achieved. It included the ADD (6) reaction with 1 course of experiment it appeared that the H NMR spectroscopy an excess of pyrrolidine and without a solvent in the presence of is an excellent alternative technique for the reaction control the catalytic amount of the acetic acid. The reaction was carried (Kubiszewski et al. 2012). Satisfactory separation of the pro- out in ambient temperature without the inert gas atmosphere for ␦ ton signals in the region of chemical shifts 4.5–7.5 allowed 24 h. This time was sufficient for more than 98% transforma- to calculate the quantity of the compound 7 and the substrate tion of 100 g ADD (6). However, there was still a problem of 1 6 in the reaction mixture. Additionally, the usage of H NMR isolating the intermediate 7 from the reaction mixture. Some let us detect the unexpected impurity 1-pyrrolidino-androsta- authors even reported that despite varying reaction conditions, 1,3,5-triene-17-one (9) (12–17%) which was not monitored by compound 7 could not be isolated (Goerlitzer et al. 2002; Bon- other authors. We observed that the impurity 9 content, both nekessel and Görlitzer 2002). In our opinion the essential reason in the crude reaction mixture and the isolated yellow adduct of their failure was the application of column chromatography 7, strongly depended on the process temperature and increased in the reaction mixture work-up, which led to the arrangement with the temperature of the reaction. Fortunately, the formation of an estrone derivative instead of 7 purification. It was found of 9 during the condensation of ADD with pyrrolidine was not a problem. It was experimentally confirmed that the compound 9, like 7, transformed into the desired intermediate 8 in the next reaction with formaldehyde (Scheme 3). Afterwards, to elimi- nate the impurity 9 from the process, the reaction temperature was lowered in the succesive experiments from about 90 ◦C (reflux) to 50 ◦C. It allowed us to obtain the anticipated com- position of the crude reaction mixture with 98% conversion to 7. Moreover, no 9 was detected. It occured, however, that lower temperature extended the reaction time (up to 120 h). This was technologically not acceptable and obliged us to perform further optimization of the reaction.