Regioselective Enzymatic Acylation of Vicinal Diols of Steroids
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Tetrahedron 61 (2005) 3065–3073 Regioselective enzymatic acylation of vicinal diols of steroids M. Manuel Cruz Silva,a Sergio Rivab and M. Luisa Sa´ e Meloa,* aCentro de Estudos Farmaceˆuticos, Lab. Quı´mica Farmaceˆutica, Faculdade de Farma´cia, Universidade de Coimbra, Rua do Norte 3000-295 Coimbra, Portugal bIstituto di Chimica del Riconoscimento Molecolare, C.N.R., Via Mario Bianco 9, 20131 Milano, Italy Received 28 October 2004; revised 29 November 2004; accepted 25 January 2005 Abstract—Monoacylated derivatives of a complete set of 2,3- and 3,4-vicinal diols of steroids were prepared by regioselective lipase- catalysed transesterification reactions. The enzymes displayed different selectivities towards the vicinal diols depending on the configuration of the hydroxyl groups. q 2005 Elsevier Ltd. All rights reserved. 1. Introduction Studies on the transesterification of polyfunctionalyzed steroids have shown that hydrolases can have access to Polyhydroxylated steroids bearing vicinal diols on the substituents either on the A-ring or on the D-ring and/or on A-ring are frequently found in Nature and some of them the side-chain of steroids. Several lipases showed a have relevant biological activities. For instance, the trans- preference for C-3 hydroxyl groups,13,14 whereas the diaxial 2b,3a-di-hydroxy pattern is present in natural protease subtilisin Carlsberg catalysed the acylation of 14 sulphated sterols with antiviral1 or anti-angiogenic2 action, C-17 OH. Moreover, stereoselective resolutions of while 2a,3a-diols isolated from marine sources hold epimeric alcohols located on the steroid side-chain have 15 cytotoxic activity.3 Steroidal saponins displaying trans- been carried out by lipase PS and, more recently, by 16 diequatorial 2a,3b-vicinal diols are quite frequent as subtilisin. gitogenin derivatives with antitumor properties.4 In turn, the 3b,4b-diol functionality is present in a variety of steroids Concerning the modifications of A-ring substituents, the like in the agosterols, which induce reversal of multidrug selectivity of lipases for 3-hydroxysteroids has been applied 5 6 to the chemoenzymatic synthesis of pharmacologically resistance and proteasome inhibition, as well as in 17 formestane metabolites7 and in volkendousins, which are relevant tibolone metabolites. Quite recently, we have potent antitumor agents.8 Finally, the 3a,4b-vicinal diol reported a highly selective lipase-catalysed preparation of epimerically pure 5a,6a- and 5b,6b-epoxysteroids through pattern has been identified in contignasterol, a natural anti- acylation or deacylation reactions at the C-3 OH.18 Finally, inflammatory compound9 and, recently, in a steroid the ability to discriminate among different hydroxyl groups possessing chemotaxis activity.10 on the A-ring has been demonstrated in the esterification of ecdysteroids catalysed by Candida antarctica lipase, which The discovery that lipases and proteases are able to act in afforded the 2b-monoacyl derivatives in good yields.19 organic solvents opened the way to an intensive synthetic exploitation of these biocatalysts, which, as shown in In this context, to further explore the enzymatic transform- hundreds of papers and several industrial applications, 11 ations of steroids, we endeavoured a systematic study on the display remarkable chemo-, regio- and stereoselectivity. selectivity of commercially available lipases towards a complete set of stereoisomeric 2,3- and 3,4-vicinal diols, to Specifically in the steroids field, enzyme catalysis can play provide a new tool for the selective transformation of these an important role for the mild and selective interconversion molecules and of related natural compounds. of functional groups via regioselective transformations.12–19 Keywords: Lipases; Steroids; Diols; Regioselectivity; Enzymatic esterification. 2. Results and discussion * Corresponding author. Tel.: C351 239 859990; fax: C351 239 827030; e-mail: [email protected] Different synthetic strategies were used to afford the 0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tet.2005.01.104 3066 M. M. Cruz Silva et al. / Tetrahedron 61 (2005) 3065–3073 Table 1. Lipase-catalysed monoacylation of vicinal diolsa Substrate Novozym C. rugosa Lipase Lipase Lipase C. viscosum Porcine pancreatic Lipozyme Lipase 435 lipase AY PS AK lipase lipase IM 20 CE 1 K CCCCCC K K K 2 KCCCCCCK KK 3 CCK KKKK K K K 4 CC Cb Cb CC K K K K K 5 C CCCCCCC K K K 6 C C CCCCCK K K 7 KCKCCCCK KK 8 K K KKKK K K a Conversion and product(s) formation was evaluated by TLC. b The formation of two products was observed. requested stereoisomeric vicinal diols 1–8. Woodward’s cis- notable exception of compound 3 (2a,3a-diol) acylated only dihydroxylation method, which is known to be selective for by Novozym 435 (immobilized lipase B from Candida the more hindered b-face of 5a-steroids,20 was applied to antarctica). Enzymatic acylations were highly regioselec- D2-orD3-unsaturated precursors affording the cis-b diols tive, showing the formation of only one product by TLC, 1 and 5 (Scheme 2). Complementarily, the cis-a diols 3 and with the exception of Candida rugosa lipase (from Sigma or 7 were accessed through osmium tetroxide-mediated Amano) acting on compound 4. Noteworthy, lipase PS and dihydroxylation on the same olefins.21 Trans-diequatorial Novozym 435 showed complementary regioselectivity diols 2 and 6 were obtained by two different approaches. towards compound 4. Starting from cholestan-3-one, a-acetoxylation by lead tetracetate,22 followed by stereoselective reduction by For each substrate the best performing lipase (evaluated by NaBH4/CeCl3 and deacetylation rendered the diol 2.On TLC) was chosen for scale-up reactions, allowing the the other hand, the 3b,4a-diol 6 was directly accessed by isolation of the corresponding monoester in good yields hydroboration of the D4-3-one precursor.23 Finally, trans- (Scheme 1). Products identification was easily done by diaxial diols 4 and 8 were obtained by epoxide opening NMR analysis (downfield shift of the signals due to the reactions. proton geminal to the acylated OH) and, when possible, by comparison with literature data. As shown in the formula, the 2,3-diols were prepared by modifying the cholestane skeleton, whereas the diffi- The general preference of lipases for the C-3 OH13,14 was culties found on separating the 5a- and 5b-epimers of observed with most of the substrates (Scheme 2). Specifi- D3-cholestane by sequential crystallizations led us to cally, the diequatorial vicinal diols 2 and 6 were converted prepare the set of 3,4-diols in the androstane series. The into the corresponding 3b-acetate, showing a common cholestane derivative 9 was also synthesised to confirm that preference of different lipases toward a 3b-equatorial OH lipases selectivity towards the A-ring OH was not affected in the presence of 2a-equatorial OH (substrate 2)orof by different substituents on the D-ring. 4a-equatorial OH (substrate 6). The performances of a panel of 9 commercial lipases were Concerning the diaxial 2b,3a- and 3a,4b-diols (substrates 4 evaluated for the esterification of the vicinal diols 1–9, using and 8), different outcomes were noticed. The diaxial 2b,3a- vinyl acetate as the acyl donor and toluene or acetone/THF diol was differently accepted by the lipases tested. Whereas as solvents for the cholestane or the androstane derivatives, Novozym 435 converted this diol exclusively into the respectively. TLC monitoring allowed the identification, 3a-acetate 4a, lipase PS showed opposite selectivity for each substrate, of the lipase(s) able to promote the rendering the 2b-acyl derivative 4b as the only product. monoacylation of the substrates. As shown in Table 1, all Moreover, acylations catalysed by Candida rugosa lipases the stereoisomeric vicinal diols were accepted as substrates were not regioselective with this substrate. Conversely, the by some of the enzymes tested with the exception of diaxial 3a,4b diol (8) was not accepted by any of the compound 8. enzymes tested. Lipases from different sources (Candida antarctica, column Finally, the equatorial/axial 2a,3a-diol (3), only accepted 2; Candida rugosa, columns 3 and 4; Pseudomonas strains, by Novozym 435, was acylated at the axial 3a-position, columns 5 and 6; Chromobacterium viscosum, column 7) while, at variance, the 3a,4a-dihydroxy steroid (7) display- were able to acylate the target compounds. Usually more ing axial/equatorial configuration, was acylated by different than one enzyme was acting on the same substrate, with the lipases at its equatorial 4a-OH. Scheme 1. M. M. Cruz Silva et al. / Tetrahedron 61 (2005) 3065–3073 3067 Scheme 2. Moreover, in agreement with previous observations, the Specifically, this enzyme catalysed the acetylation of lipases tested did not catalyse any acylation of the 17b-OH substrates 1, 2, 5 and 6 at their 3b-OH, as was expectable (substrates 5–8), that was found unaffected in each of the from previous studies.13b,c However, the 3a,4a-diol 7 was isolated products. also acylated by Candida rugosa lipase at the C-4 OH, and the 2b,3a-diol 4 was acylated at both its hydroxy groups, The ability of Novozym 435 to accept axial 3a-OH as a showing that this enzyme has a quite variable affinity and nucleophile has been reported previously.13d Herein, we selectivity pattern. noticed that this lipase catalyses the acylation of the 3a-OH even when a second hydroxyl is located at C-2 (substrates 3 Chromobacterium viscosum lipase, which is known to have and 4), whereas the enzyme is inactive when the other OH is a strict selectivity for the 3b-hydroxyl of 5a-steroids,14 located at C-4 (substrates 7 and 8). showed the expected preference for diols carrying this OH (substrates 1, 2, 5 and 6). In addition, acylation of the Concerning lipase PS, the ability of this enzyme to esterify substrate 7 was also noticed, but at the 4a-OH.