Organic Process Research & Development 2008, 12, 831–836
Scale-Up Synthesis of Swainsonine: A Potent r-Mannosidase II Inhibitor† Pradeep K. Sharma,* Rajan N. Shah, and Jeremy P. Carver GLYCO Design Inc., 480 UniVersity AVenue, Toronto, Ontario, Canada
Abstract: The large-scale synthesis of Swainsonine 1, a potent r-mannosidase II inhibitor, has been achieved with several improvements. The key modifications were (a) performing the Wittig olefination under mild conditions and isolation of the product 4 with modified workup conditions, (b) introduction of the azido group on a large scale under Mitsunobu conditions to produce 12, (c) performing the 1, 3-dipolar cycloaddition of an unactivated azide 12 to afford the imino carboxylic ester 7, (d) formation of amide 10 from 7 under mild acidic conditions, and (e) isolation of the final compound 1 as a stable hydrochloride salt. In addition, synthesis of 11 was accomplished from 12 by telescoping the four steps. Introduction Polyhydroxy indolizidine alkaloids possess a wide range of biological activities such as immunoregulatory activity, antiHIV activity and anticancer activity.1,2 Swainsonine 1 is an indolizidine alkaloid isolated from the Australian plant Swainsona canescens,3a North American plants of genera Astragalus and Oxytropis,3b and from the fungus Rhizoctonia leguminocola.3c Compound 1 (Figure 1.) is a potent inhibitor of the Golgi enzyme R-mannosidase II, an enzyme required for maturation of N-linked oligosaccharides of newly synthesized glycoproteins. Compound 1 also blocks lysosomal Rmannosidase resulting in the accumulation of oligomannoside chains in cells exposed to the drug.4 † Part of this work has been incorporated in the patent application (U.S. Patent 6,051,711, WO992185, CAN 130:338281 AN 1999: 297421). * Author for correspondence. E-mail:
[email protected].
(1) (a) For selected reviews see: deMelo, E. B.; de Silveria Gomes, A.; Carvalho, I. Tetrahedron 2006, 62, 10277. (b) Lillelund, V. H.; Jensen, H. H.; Liang, X. F.; Bols, M. Chem. ReV. 2002, 102, 515. (c) Heihtman, T. D.; Vasella, A. T. Angew. Chem., Int. Ed. 1999, 38, 750. (d) deMelo, E. B.; de Silveria Gomes, A.; Carvalho, I. Tetrahedron 2006, 62, 10277. (2) (a) Asano, N.; Nash, R. J.; Molyneux, R. J.; Fleet, G. W. J. Tetrahedron: Asymmetry 2000, 11, 1645. (b) Asano, N. Glycobiology 2003, 13, 93R. (c) Watson, A.; Fleet, G. W. J.; Asano, N.; Molyneux, R. J.; Nash, R. J. Phytochemistry 2001, 56, 265. (d) Gerber-Lemaire, S.; Juillerat-Jeanneret, L. Mini-ReV. Med.Chem. 2006, 6, 1043. (3) (a) Colegate, S. M.; Dorling, P. R.; Huxtable, C. R. Aust. J. Chem. 1979, 32, 2257. (b) Molyneux, R. J.; James, L. F. Science (Washington, D.C.) 1982, 216, 190. (c) Davis, D.; Schwarz, P.; Hernandez, T.; Mitchell, M.; Warnock, B.; Elbein, A. D. Plant Physiol. 1984, 76, 972. (d) Skelton, B. W.; White, A. H. Aust. J. Chem. 1980, 33, 435. (4) (a) Taylor, P. C.; Winchester, B. G. Iminosugars as Glycosidase Inhibitors 1999, 125. (b) Burgess, K.; Henderson, I. Tetrahedron 1992, 48, 4045. (c) Fellow, L. E.; Kite, G. C.; Nash, R. J.; Simmonds, M. S. J.; Scofield, A. M. Rec. AdV. Phytochem. 1989, 23, 395. (d) Goss, P. E.; Reid, C. L.; Bailey, D.; Dennis, J. W. Clin. Cancer Res. 1997, 3, 11077. (e) Elbein, A. D. FASEB J. 1991, 5, 3055. (f) Galustian, C.; Foulds, S.; Dye, J. F.; Guillou, P. J. Immunopharmacology 1994, 27, 165. (g) Grzegorzewski, K.; Newton, S. A.; Akiyama, S. K.; Sharrow, S.; Olden, K.; White, S. L. Cancer Commun. 1989, 1, 373. (h) Elbein, A. D. Annu. ReV. Biochem. 1987, 56, 497. (i) Di Bello Cenci, I.; Fleet, G.; Tadano, S. K.; Winchester, B. Biochem. J. 1989, 259, 855. 10.1021/op800059y CCC: $40.75 2008 American Chemical Society Published on Web 07/26/2008
Figure 1.
Various syntheses5 reported in the literature have described the preparation of 1 in milligram quantities, which were sufficient for the initial structure-activity relationships. However, to fully develop the potential clinical applications of 1 and address initial pharmacological and toxicological requirements, 100 g to kilogram levels of 1 as the hydrochloride salt were required. To this end we report process conditions required for the large-scale synthesis of 1 · HCl salt. Results and Discussions The medicinal chemistry route used to prepare 1 is based on the synthesis described by Cha et al. as depicted in Scheme 1.6 Treatment of lactol 3 with the ylide of 3-(carbethoxypropyl)triphenylphosphonium bromide, generated in situ by treatment with KN(TMS)2 at -78 °C, produced the olefinic alcohol 4 in 58% yield after purification by silica gel chromatography. Conversion of alcohol 4 with TsCl/Et3N/CH2Cl2 gave 5. Subsequent treatment of 5 with excess NaN3 in DMF at elevated temperature gave 7 in 72% yield. It is believed that triazole 6 was the intermediate, which upon loss of a mole of N2 gave 7. The alkaline hydrolysis of the ester group of 7 resulted in 8, which upon heating in a solution of toluene using a Dean-Stark apparatus produced the enamide 10 Via the intermediate 9. Hydroboration of 10 followed by oxidation resulted in a single diastereomer 11. The removal of the acetonide linkage under acidic conditions using HCl followed by treatment with Dowex -OH afforded the desired compound 1 as an off-white solid. The medicinal chemistry route is elegant, but a number of factors (such as the use of multiple column chromatography steps for purifications, excess NaN3 at elevated temperature, basic resins, etc.) rendered the medicinal chemistry approach unsuitable for scale-up conditions to deliver the quantities required for development activities. In addition, 1 was isolated as a free base from its corresponding hydrochloride salt. As a consequence, we report a newly developed process for the largescale synthesis of 1 · HCl with several modifications. Synthesis of 4. Wittig reaction between lactol 3 with 3-(carbethoxypropyl)triphenylphosphonium bromide in the pres(5) (a) Nemr, A. E. Tetrahedron 2000, 56, 8579. (b) Martin, R.; Murruzzu, C. A; Riera, M. A. J. Org. Chem. 2005, 70, 2325, and references cited there-in. (c) Heimgaertner, G.; Raatz, D.; Reiser, O. Tetrahedron 2005, 61, 643. (d) Pearson, W. H.; Ren, Y.; Powers, J. D. Heterocycles 2002, 58, 421. (e) Lindsay, K. B.; Pyne, S. G. Aust. J. Chem. 2004, 57, 669. (f) Guo, H.; O’Doherty, G. A. Org. Lett. 2006, 8, 1609. (6) Bennett, R. B.; Choi, J. R.; Montgomery, W. D.; Cha, J. K. J. Am. Chem. Soc. 1989, 111, 2580. Vol. 12, No. 5, 2008 / Organic Process Research & Development
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Scheme 1 a
a Reagent and conditions: (a) BrPPh CH CH CH CO Et, KN(TMS) , THF, -78 °C; (b) TsCl, Et N, CH Cl ; (c) NaN , DMF; (d) NaOH, H O, CH Cl ; (e) toluene 3 2 2 2 2 2 3 2 2 3 2 2 2 (f) BH3.THF, H2O2, H2O, NaOH (g) HCl, Dowex -OH resin.
Table 1 entry
3 (equiv)
phosphonium salt (equiv)
solvent
base (equiv)a
reaction temp ( °C)
1 2 3 4 5 6 7
1 1 1 1 1 1 1
2.5 2.5 3.5 3.5 2.0 1.0 2.0
THF THF THF toluene THF THF THF
KN(TMS)2(2.4) KN(TMS)2(2.5) tBuOK(3.5) tBuOK(2.4) tBuOK(2.2) tBuOK(1.0) tBuOK(2.2)
-78 -78 -78 -5 to 10 -5 to 10 -5 to 10 -5 to 10
a
58c 45 40 gel 43 10 80
The amount of the base is based upon 3. b All values are isolated yields. c Reference 6.
ence of KN(TMS)2, produced alcohol 4 in 45% yield as compared to 58% after purification by chromatography.6 In the reaction, a large excess of the phosphonium salt (2.5 equiv) [compared to lactol 3 (1 equiv)] and an expensive base were used. Under these conditions, it was observed that during the aqueous workup, the ester group of 4 was hydrolyzed and remained in the aqueous layer, to result in the loss of product. In order to optimize the amount of the phosphonium salt and base and the workup conditions, various modifications were tried and are summarized in Table 1. When the base KN(TMS)2, was replaced with tBuOK under similar reaction conditions (entry 2, Scheme 1), the desired alcohol 4 was obtained in comparable yield (entry 3). This indicated that tBuOK could be used as a replacement for KN(TMS)2. In addition, various temperature controlled experiments revealed that the ylide was stable at a much higher temperature. This allowed the reaction to be performed at (-5 to 10 °C) instead of -78 °C. Next, the reaction conditions were modified. Replacing THF with toluene (entry 4) did not have any effect on the reaction. However, a gel was obtained during the aqueous workup, and the product could not be isolated by repeated extraction and washings. This indicated that toluene was a poor choice of solvent. Reducing the amount of phosphonium salt with an equivalent amount of the base (entry 5) followed by aqueous workup and column chromatography, allowed for the recovery of compound 4 in low yield. In a different experiment where 3 and equimolar amounts of 832
yieldb (%)
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phosphonium salt (1 equiv) and base (1.0 equiv) were used under similar reaction conditions (entry 6), 3 remained in the reaction mixture even after an extended reaction time. When 3-(carbethoxypropyl)triphenylphosphonium bromide (2 equiv) and tBuOK (2.2 equiv) were reacted in situ, followed by the addition of 3 (1 equiv) at -5 °C and the reaction mixture maintained at 98% (HPLC area %) purity. Synthesis of 12. In the medicinal chemistry route6 alcohol 4 was first converted to the corresponding tosylate 5 followed by its further transformation to imino ester 7 in a single pot. The use of a large excess of NaN3 at elevated temperature represented a significant safety hazard. In addition, the displacement of the tosylate group followed by the formation of 7 resulted in no control of the reaction i.e. the intermediate 6 was not stable and converted to 7 immediately under the reaction conditions. Also, any presence of excess NaN3 in the reaction mixture could result in a significant safety issue at the large
scale. Thus, the replacement of NaN3 with an equivalent reagent was required, which could be scaled up under normal handling conditions.7 An alternative method was proposed where the alcohol 4 was directly converted to the olefinic azide 12 (Scheme 2) under Mitsunobu reaction conditions. In general, the conversion of an alcohol to the corresponding azido group required the use of PPh3 and diisopropyl azodicarboxylate (DIAD) along with HN3 as the source of the azide group.8 Because of the hazardous nature, stability issues, and the difficulty in obtaining large quantities of HN3, an alternative to this reagent was required. It has been reported that TMSN3 could be used as a source of azide ion in the Mitsunobu reaction.9 When alcohol 4 was treated with an equimolar amount of PPh3 and DIAD in THF at 0 °C, followed by the addition of TMSN3, complete consumption of alcohol 4 was observed along with the formation of two products. After extractive workup and chromatographic purification, the desired azide 12 (Scheme 2) was isolated in 45% yield. The alcohol 4 was recovered in 51% yield. Scheme 2
Upon careful analysis of the reaction mixture, it was observed that the corresponding OTMS product 13 (Figure 2.) was also formed and contributed to the low yield of the desired 12.
Figure 2.
The course of the reaction could not be altered after many attempts at modifying the reaction conditions, rate and sequence of additions. To overcome this difficulty, we focused at regenerating the alcohol 4 in situ, and then performed the Mitsunobu reaction to obtain the desired compound 12. After
the addition of the first set of reagents and monitoring for the completion of the reaction by HPLC, the OTMS group of 13 was cleaved in situ by the addition of a calculated amount of n Bu4NF solution.10 The mixture was then reacted under Mitsunobu reaction conditions using 0.5 equiv of the reagents (Scheme 3). Approximately 75% (HPLC area %) of the desired product 12 was formed. Further, addition of nBu4NF solution (0.25 equiv) and treatment of the mixture under Mitsunobu reaction conditions resulted in the transformation to 12 in >90% by HPLC area % (Note: If the transformation did not reach an acceptable conVersion, the aboVe steps could be repeated.). After removing half the reaction solvent in Vacuo, the reaction mixture was diluted with MTBE/heptane (8/2, v/v). This resulted in the precipitation of the byproducts, [PPh3dO and Me2CHOC(O)NHNHC(O)OCHMe2], which were removed by filtration. The desired compound was then isolated by silica gel chromatography in >85% yield as a colorless viscous oil. During the addition of DIAD and nBu4NF solutions, mild exotherms (98%.
Scheme 3
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Scheme 4 a
Table 2 entry solvent 1 2 3 4 5 6
PhH PhH PhH PhMe PhMe PhMe a
concentration reaction time yield puritya (M) temp (°C) (h) (%) (%) 0.5 1 0.05 1 0.5 0.25
80 80 80 110 110 110
30 30 30 20 20 12
81 86 86 90 96 98
97 93 96 92 94 98
Purity is determined by HPLC area %.
In the original protocol, the hydrolysis of 7 was accomplished under mild basic conditions (K2CO3/CH3OH), and then the free acid 8 was isolated after repeated extractive workup. Because the acid 8 has good solubility in water, it was difficult to completely recover 8 in the organic solvent(s) even after repeated extractions. In the modified procedure, K2CO3 was replaced with a 2 N NaOH solution, and methanol, with ethanol. When a solution of 7 in ethanol was treated with a 2 N NaOH solution (
