Integrated Biocatalysis in Multistep Drug Synthesis without

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Integrated Biocatalysis in Multistep Drug Synthesis without Intermediate Isolation: A de Novo Approach toward a Rosuvastatin Key Building Block Richard Metzner,†,⊥ Werner Hummel,‡,# Frank Wetterich,*,§ Burghard König,∥ and Harald Gröger*,† †

Faculty of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany Institute of Molecular Enzyme Technology, Jülich Research Center, Heinrich-Heine-University Düsseldorf, Stetternicher Forst, 52426 Jülich, Germany § Sandoz International GmbH, Industriestr. 18, 83607 Holzkirchen, Germany ∥ Sandoz Industrial Products GmbH, Brüningstraße 50, 65929 Frankfurt am Main, Germany ‡

S Supporting Information *

ABSTRACT: In this contribution, we report the chemoenzymatic preparation of a key building block for the active pharmaceutical ingredient rosuvastatin, one of the “top 5 blockbuster drugs” with a worldwide market value of 6.25 billion USD in 2012, via a seven-step synthesis without isolation of intermediates and with incorporation of two highly efficient biotransformations. This chemoenzymatic process reaches excellent space-time yields by using high substrate concentrations (several hundred grams per liter), emphasizing the potential of biocatalysis for industrial processes related to pharmaceutical drug synthesis and the compatibility of enzyme chemistry with classical organic synthesis.

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herein we report a seven-step synthesis of an alkene key intermediate of 1 that is prepared without purifying the intermediates and with incorporation of two biotransformations. This chemoenzymatic process was developed to reach an excellent space-time yield by using high substrate concentrations of several hundred grams per liter. Since this process met and surpassed the required high productivity, it was recently transferred to the industrial partner Sandoz for internal work on process scale-up.6 Our initial retrosynthetic approach was based on the development of an economically attractive synthesis of alkene 2 (Scheme 1). This compound is an industrial key intermediate, since it is subsequently converted to the final rosuvastatin

ith a worldwide market value of 6.25 billion USD in 2012 and a U.S. market value of 5.31 billion USD, rosuvastatin (1) belongs to the top 5 of the so-called “blockbuster drugs”, hence encouraging the development of more efficient and sustainable processes for its production.1 The expiration of relevant patents in 2016 further increases the ongoing interest in novel synthetic approaches, especially for generic drug manufacturers. In the past, this issue of establishing the most efficient process and the most favorable synthetic route toward active pharmaceutical ingredients was often focused on a multistep retrosynthesis depending entirely on chemical reaction sequences.2 In addition, industrial syntheses are also challenging in regard to minimizing waste and maximizing the space-time yield of the overall process. Although currently biotransformations have been gaining increasing attention as key steps for novel industrial drug syntheses,3 as illustrated by the term biocatalytic retrosynthesis coined for this field by Turner et al.,3c enzymatic reactions are often still regarded as being incompatible with chemical reactions, and usually the two are strictly separated from each other with purification steps for most intermediates. Accordingly, retrosynthetic approaches based on chemoenzymatic one-pot processes for key intermediates remain rare. However, even processes relying solely on chemical reactions often tend to include purification steps of every intermediate. This contradicts the aims of a sustainable process chemistry for the production of pharmaceutical drugs as advised by the ACS GCI Pharmaceutical Roundtable, established by the ACS Green Chemistry Institute (GCI) and several of the world’s leading pharmaceutical companies, as well as Pfizer’s “Green Chemistry Metrics Programme”.4,5 Underlining the tremendous potential of biocatalysis for the design of novel retrosynthetic approaches in drug synthesis, © XXXX American Chemical Society

Scheme 1. Retrosynthetic Wittig approach to rosuvastatin calcium

Received: February 25, 2015

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DOI: 10.1021/acs.oprd.5b00057 Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development

Article

As a result, none of the three initial routes (A, B, or C) could be used as an overall process. It is, however, noteworthy that the successful parts of the processes, i.e., the synthesis of chiral monoester 9 starting from the prochiral bulk chemical 7 (route A) and the synthesis of the chiral phosphorus ylide (R)-6b from free 3-hydroxy monoester 14 (route B/C), are complementary to each other. Thus, the missing “link” was the hydrolytic cleavage of the O-acetyl group of monoester 9 to obtain the required unprotected compound 14. At first glance this reaction seemed trivial, but it became in fact quite challenging because of the similar reactivities of the two carboxylic ester moieties in this molecule. During a classical base-catalyzed saponification, most of the product was lost through the formation of the prochiral diacid (data not shown). Even enzymatic hydrolysis turned out to be difficult. A range of examined hydrolases showed no measurable activity for this compound. Fortunately, a biocatalyst used for the synthesis of antibiotics, namely, a cephalosporin C acetyl esterase,14 showed the desired selectivity for the O-acetyl cleavage paired with excellent efficiency. Hence, the missing “link” (Scheme 2, conversion of 9 to 14) was realized and successfully integrated into the process, thus completing the overall reaction sequence starting from the readily accessible and industrially attractive diester 7 to the desired chiral ylide intermediate (R)-6b. Next we focused on the development of a highly efficient overall synthesis featuring excellent productivities, high substrate concentrations, and facile catalyst separation while avoiding isolation and purification steps. At first, we combined the initial three reaction steps, especially the two consecutive biotransformations for the preparation of chiral monoester 14 from acetylated diester 8. We were pleased to find that the initial process development successfully reached an initial substrate 8 concentration of 2.0 M, equivalent to 492 g/L, with quantitative conversion and a very high enantioselectivity of 97% ee (Scheme 3). Since the resulting acid 9 has to be

calcium by well-established procedures (silyl ether cleavage, diastereoselective Narasaka−Prasad reduction,7 ester hydrolysis, salt formation).8 The formation of the CC double bond in 2 can readily be achieved via Wittig reaction. Among the two possibilities depicted in Scheme 1, the synthesis starting from heteroaromatic aldehyde 5 and phosphorus ylide 6 appeared to be more favorable for us, e.g., due to the presence of the oxostabilized phosphorus ylide 6.8,9 However, while aldehyde 5 is an easily accessible10 and commercial starting material, finding an efficient synthetic route to the chiral phosphorus ylide 6 remained a challenge. From a retrosynthetic point of view, such a type of stabilized phosphorus ylide can be prepared by direct alkylation of triphenylphosphine with a corresponding α-halo ketone11 or an acylation of methyltriphenylphosphonium ylide with activated carboxylic acid derivative 10 (as depicted in Scheme 2, route Scheme 2. Chemoenzymatic routes A, B, and C and their “bottlenecks”

A).12 An advantage of the latter route is the possibility of obtaining this compound 10 by a straightforward enantioselective ester hydrolysis of the readily available prochiral dialkyl 3-hydroxyglutarate of type 7. In this asymmetric step, the use of a biocatalyst seems favorable. While the enzymatic desymmetrization using α-chymotrypsin was already proven to be Rselective for different O-acyl derivatives of 7 and highly enantioselective for the O-acetyl derivative 8 (in step 2 of route A),13 the subsequent steps of route A in Scheme 2 became troublesome. This was attributed to side reactions, e.g., elimination of acetic acid with formation of enoates due to the lability of the O-acetyl group under strongly basic reaction conditions. To suppress this undesired side reaction, different derivatives of the chiral monoester 14 with an exchanged Oprotecting group were examined, revealing the tert-butyldimethylsilyl (TBS) ether as the most promising candidate (Scheme 2, route B/C). Starting from this TBS-protected monoester 12, which is usually prepared over three steps via its L-mandelate diastereomer,2b ylide 6b was prepared in two steps in high yield. The drawback of this route B was the initial desymmetrization reaction of silylated diester 11, since the bulky O-substituent inhibited the enzymatic conversion of this substrate. Furthermore, another alternative route C via desymmetrization of the free hydroxy diester 7 itself could not be established in an efficient fashion, since it was inferior in terms of enantioselectivity. The enzymatic hydrolysis of 7 yielded the corresponding monoester 14 with only 60% ee, even with the purified biocatalyst.

Scheme 3. Combination of two biotransformations for the production of the chiral unprotected monoester

neutralized during the biotransformation step, the added volume of the aqueous base lowers the “overall” substrate concentration accordingly. The O-acetylated monoester 9 was obtained in 95% isolated yield. In addition, the subsequent regioselective ester hydrolysis with the cephalosporin C acetyl esterase proceeded in a highly efficient manner, and the monoester 14 was obtained with >95% conversion in 77% isolated yield. This second enzymatic reaction was also done with a high substrate concentration of 0.8 M, corresponding to 174 g/L (Scheme 3). Even with these yields of 95% and 77% (obtained without the need of column chromatography), isolation steps on a technical scale are disadvantageous from both economic and ecological perspectives due to, e.g., the large amount of extraction solvent, the resulting amount of waste, and the B



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was added under the same pH-controlled reaction conditions. This heterogeneous biocatalyst was removed via filtration over a glass frit after quantitative formation of free hydroxy monoester (R)-14. With this enzyme and its outstanding chemoselectivity for the acetyl group, it was therefore possible to produce chiral monoester (R)-14 with a substrate loading of more than 430 g/L, an overall yield of 95% over three steps starting from diester 7, and a very high enantioselectivity of >97% ee. These three consecutive steps of chemical acetylation followed by two complementary biotransformations proceeded in a highly efficient way, with the (initial) 980 g/L concentration of 8 being one of the highest substrate concentrations reported to date in biocatalysis. In addition to the avoidance of any isolation steps between these three reaction steps, both enzymes could be reused after their separation from the corresponding product mixture. The catalyst recycling was examined over five reaction cycles, with each cycle being stopped after reaching >95% conversion as determined by the consumption of aqueous base to maintain the reaction pH (Table 1). While the reaction time for αchymotrypsin increased slowly within each cycle (due to partial inactivation), the cephalosporin C acetyl esterase demonstrated impressive stability and high efficiency with a negligible decrease in activity over five cycles. Furthermore, on the basis of this efficient three-step synthesis of (R)-14 from 7 without intermediate isolation, a seven-step overall process for alkene key intermediate (R)-2b was developed that does not require isolation procedures for purified intermediates (Scheme 4). Following the described three-step chemoenzymatic synthesis of (R)-14, the crude product was silylated by chemical means with tert-butyldimethylchlorosilane and 1H-imidazole. Although purification of silylated monoester (R)-12 can be avoided as well, the isolation of (R)-12 is beneficial and represents the only purification step that improved the overall yield of the route by removing the residual tert-butyldimethylsilanol byproduct, which forms a hemihydrate16 that subsequently hydrolyzes parts of the following reagents. The conversion of (R)-12 into mixed anhydride (S)-13 and the acylation of methyltriphenylphosphonium ylide to obtain chiral ylide (R)-6b were again conducted without isolation steps of the respective products (Scheme 4). The crude product (R)-6b was reacted in situ with heteroaromatic aldehyde 5 in a Wittig reaction, which gave the key intermediate (R)-2b in 59% overall conversion relative to diester 8. It should be noted that all of these reactions ran with high and in many cases even

increased time and reactor capacity. Thus, the next aim was the combination of the three initial process steps (O-acetylation of diester 7, α-chymotrypsin-catalyzed desymmetrization of 8, and cephalosporin C acetyl esterase-catalyzed deacetylation of 9) with practical unit operations steps for separation of the enzymes from the reaction mixture without the necessity of isolation or purification procedures of the formed products (Table 1). After the initial solvent-free acetylation of diester 7 Table 1. Three-step synthesis with enzyme recycling

productivity [mg g−1 day−1]a

conv. [%] cycle

B

C

CHY (B)

CAE (C)

1 2 3 4 5

>95 >95 >95 >95 >95

>95 >95 >95 >95 >95

31.51 32.12 38.27 68.34 124.43

44.35 44.64 49.36b 49.65b 49.91b

a

Milligrams of biocatalyst per gram product and a reaction time of 24 h. bThe immobilized enzyme was stored for 14 days at 4−8 °C between cycles 2 and 3.

in acetic anhydride, the subsequent desymmetrization reaction was realized without isolation of 8 and with an excellent (initial) substrate concentration of 4.0 M, corresponding to more than 980 g/L. Because of the addition of aqueous base in the biotransformation step for neutralization purposes, the reaction volume is increased and the “overall” substrate concentration is lowered accordingly. The homogeneous biocatalyst α-chymotrypsin was subsequently separated via ultrafiltration15 (and recycled), while the filtrate containing chiral monoester (R)-9 was directly used for the next reaction. For this step, the immobilized cephalosporin C acetyl esterase

Scheme 4. Final seven-step reaction sequence leading to the rosuvastatin key intermediate (R)-2b

C



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(5) Assaf, G.; Checksfield, G.; Critcher, D.; Dunn, P. J.; Field, S.; Harris, L. J.; Howard, R. M.; Scotney, G.; Scott, A.; Mathew, S.; Walker, G. M. H.; Wilder, A. Green Chem. 2012, 14, 123−129. (6) König, B.; Wetterich, F.; Gröger, H.; Metzner, R. Process for enantioselective synthesis of 3-hydroxy-glutaric acid monoesters and use thereof. PCT Int. Appl. WO 2014/140006, Sept 18, 2014. (7) (a) Narasaka, K.; Pai, F.-C. Tetrahedron 1984, 40, 2233−2238. (b) Chen, K.-M.; Hardtmann, G. E.; Prasad, K.; Repič, O.; Shapiro, M. J. Tetrahedron Lett. 1987, 28, 155−158. (8) For overviews of the industrial synthetic routes to rosuvastatin, see: (a) Kleemann, A.; Engel, J.; Kutscher, B.; Reichert, D. Pharmaceutical Substances: Syntheses, Patents, Applications; Thieme: Stuttgart, Germany, 2001; pp 1234 ff. (b) Casar, Z. Curr. Org. Chem. 2010, 14, 816−845. (9) For recent developments regarding an alternative route using a formylated statin side chain, see: (a) Ošlaj, M.; Cluzeau, J.; Orkić, D.; Kopitar, G.; Mrak, P.; Č asar, Z.; Marr, A. C. PLoS One 2013, 8, No. e62250. (b) Č asar, Z.; Steinbücher, M.; Košmrlj, J. J. Org. Chem. 2010, 75, 6681−6684. (10) Šterk, D.; Č asar, Z.; Jukič, M.; Košmrlj, J. Tetrahedron 2012, 68, 2155−2160. (11) Lin, W.; Zheng, H.; Liu, X. PCT Int. Appl. WO 2011/124050 A1, Oct 13, 2011. (12) Watanabe, M.; Koike, H.; Ishiba, T.; Okada, T.; Seo, S.; Hirai, K. Bioorg. Med. Chem. 1997, 5, 437−444. (13) (a) Santaniello, E.; Chiari, M.; Ferraboschi, P.; Trave, S. J. Org. Chem. 1988, 53, 1567−1569. (b) Ö hrlein, R.; Baisch, G. Adv. Synth. Catal. 2003, 345, 713−715. (14) Riethorst, W.; Reichert, A. Chimia 1999, 53, 600−607. (15) For more examples of enzyme separation via ultrafiltration, see: (a) Liese, A.; Kragl, U.; Kierkels, H.; Schulze, B. Enzyme Microb. Technol. 2002, 30, 673−681. (b) Haberland, J.; Hummel, W.; Daussmann, T.; Liese, A. Org. Process Res. Dev. 2002, 6, 458−462. (16) Barry, S. M.; Mueller-Bunz, H.; Rutledge, P. J. Acta Crystallogr. 2008, E64, o1174.

quantitative conversions, which emphasizes the high compatibility of the chemical and enzymatic reaction steps (Scheme 4). In summary, a chemoenzymatic process for the production of key intermediate (R)-2b leading to the “blockbuster” drug rosuvastatin (1) was developed, comprising a seven-step reaction sequence without mandatory isolation steps. The two consecutive biotransformations run on excellent substrate concentrations of up to 4.0 and 2.0 M respectively, to afford the desired chiral monoester (R)-14 with a final product concentration of 220 g/L. Recently this process was transferred to the industrial partner Sandoz for internal work on process scale-up, and a patent application on the biocatalytic enantioselective synthesis of 3-hydroxy-glutaric acid monoesters and their use was filed.6 The successful implementation of two recyclable biocatalysts for the enantioselective synthesis of the chiral monoester (R)-14 in combination with organicchemical derivatization under retention of the stereogenic information underlines the great potential of biocatalysis in the development of novel industrially applicable retrosynthetic chemoenzymatic routes to active pharmaceutical ingredients.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental procedures and analytical methods. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.oprd.5b00057.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Present Addresses ⊥

R.M.: Asano Active Enzyme Molecule Project, ERATO, JST Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan. # W.H.: Faculty of Chemistry, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Sandoz AG for financial and technical support and student Susann Rath for experimental support.

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REFERENCES

(1) Brooks, M. List of Top 100 Selling Drugs in 2013. Medscape Medical News, Jan 14, 2014; http://www.medscape.com/viewarticle/ 820011 (accessed Feb 25, 2015). (2) (a) Kleemann, A.; Engel, J.; Kutscher, B.; Reichert, D. Pharmaceutical Substances: Syntheses, Patents, Applications; Thieme: Stuttgart, Germany, 2001. (b) Konoike, T.; Araki, Y. J. Org. Chem. 1994, 59, 7849−7854. (3) (a) Bornscheuer, U. T.; Huisman, G. W.; Kazlauskas, R. J.; Lutz, S.; Moore, J. C.; Robins, K. Nature 2012, 485, 185−194. (b) Renata, H.; Wang, Z. J.; Arnold, F. H. Angew. Chem., Int. Ed. 2015, 54, 3351− 3367. (c) Turner, N. J.; O’Reilly, E. Nat. Chem. Biol. 2013, 9, 285−288. (d) Wohlgemuth, R. Curr. Opin. Biotechnol. 2010, 21, 713−724. (e) Liese, A.; Seelbach, K.; Wandrey, C. Industrial Biotransformations; Wiley-VCH: Weinheim, Germany, 2006. (4) Constable, D. J. C.; Dunn, P. J.; Hayler, J. D.; Humphrey, G. R.; Leazer, J. L.; Linderman, R. J.; Lorenz, K.; Manley, J.; Pearlman, B. A.; Wells, A.; Zaksh, A.; Zhang, T. Y. Green Chem. 2007, 9, 411−420. D


Integrated Biocatalysis in Multistep Drug Synthesis without

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