Organic Process Research & Development 2010, 14, 815–819
First Scale-Up Synthesis of FU-23, a Novel Water-Soluble Pleuromutilin-Derived Antibiotic Liqiang Fu, Xin Liu, Jianjun Cheng, Huili He, Zhan Cai, Xingsheng Guo, Shi Ding, and Yushe Yang* State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, P.R. China
Abstract: A first scale-up synthesis of FU-23 (1), a potent and effective watersoluble pleuromutilin-derived antibiotic, is described. The original synthesis from the medicinal chemistry group provided 1 in seven steps and 10.9% overall yield, required four chromatographies and employed expensive reagents such as AgOCN and 4-acetylsalicoyl chloride. The optimized synthetic route for the preparation of phosphate salt 1 consists of seven linear steps with a 42.8% overall yield. Significant cost reduction and more robust reaction conditions have been developed with no chromatography required at any stage.
Introduction Owing to the rapid emergence of multidrug-resistant Grampositive bacteria, there is an urgent need to identify and develop new antibacterial agents with novel mechanisms of action against drug-resistant bacterial strains. Pleuromutilin was first isolated in 1951 from basidiomycetes Pleurotus and P. passeckerianus and displayed modest activity against Gram-positive organisms in Vitro.1 Further studies have shown that this class of antibiotics interferes with bacterial protein synthesis via a specific interaction with the 23S rRNA of the 50S bacterial ribosome subunit.2 These compounds display a distinct antibacterial profile and show no cross-resistance with any other class of antibiotics.3,4 In 2007, GlaxoSmithKline’s novel pleuromutilin analogue retapamulin, with excellent activity in Vitro, was first approved for human use as a topical antimicrobial agent to treat skin infections.5,6 The emergence of FU-23 (1) as a promising water-soluble pleuromutilin-derived preclinical candidate within our lab discovery program led us to seek a practical and scalable route to synthesize mutigram quantities required for regulatory toxicology studies and preclinical evaluation.7 Herein is described the first scale-up synthesis of FU-23 (1). * Author for correspondence. Telephone: (86-21) 50806786. Fax: (86-21) 50806786. E-mail:
[email protected].
(1) Kavanagh, F.; Hervey, A.; Robbins, W. J. Proc. Natl. Acad. Sci. U.S.A. 1951, 37, 570. (2) (a) Long, K. S.; Hansen, L. H.; Jakobsen, L.; Vester, B. Antimicrob. Agents Chemother. 2006, 50, 1458. (b) Davidovich, C.; Bashan, A.; Auerbach, N. T.; Yaggie, R. D.; Gontarek, R. R.; Yonath, A. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 429. (3) Fukuda, Y. Drug Future 2009, 34, 127. (4) Phillips, O. A.; Sharaf, L. H. Expert Opin. Ther. Pat. 2007, 17, 429. (5) Parish, L. C.; Parish, J. L. Drugs Today. 2008, 44, 91. (6) Daum, R. S.; Kar, S.; Kirkpatrick, P. Nat. ReV. Drug DiscoVery 2007, 6, 865. (7) Fu, L. Q.; Jiang, Z.; Cai, Z.; Liu, X.; He, H.; Yang, Y. S. Bioorg. Med. Chem. Lett. 2009, 19, 5407. 10.1021/op100063h 2010 American Chemical Society Published on Web 05/12/2010
Figure 1. Chemical structure of FU-23 (1).
Results and Discussion The initial medicinal chemistry approach to the synthesis of the title compound 1 is depicted in Scheme 1, which provided 1 in 10.9% overall yield. The key intermediate 7 was prepared successfully, following the method reported by Jeremy D. Hinks.8 The synthesis of 7 started with commercially available pleuromutilin 2, which was treated with trimethyl orthoformate and concd H2SO4 in methanol at 35 °C to give 3, followed by the subsequent hydrolysis to afford 4. AgOCN was suspended in a solution of 4-acetoxybenzoyl chloride 5 in dry dichloromethane under an atmosphere of argon and stirred at reflux for 4 h; then, 4 was added at room temperature for 1 h to give compound 6, which was obtained in high yield after chromatographic isolation. Then, 6 in dioxane was treated with a saturated solution of zinc chloride in concd HCl to afford the intermediate 7 in 27% yield after chromatographic purification.8 The regioselective phosphorylation of the phenolic hydroxyl group of 79,10 and hydrogenolysis of the benzyl and vinyl moieties of 8 provided the free acid of 1. Methanol and water were then added to the free acid of 1, and the pH was adjusted to 9 by addition of 10% Na2CO3 solution. The salt was purified by chromatography (C18), eluting with a gradient of 0-100% methanol in water to give 1 in about 92% yield for two steps.7 As noted above, the original synthesis was judged not suitable for scale-up, as it involved several chromatographic purifications and provided very low overall yield (10.9%). Moreover, it also required the use of 4-acetoxybenzoyl chloride and AgOCN which are expensive and have processing issues. Alternative Approaches to 1. We first looked at the possibility of a more efficient process for the synthesis of 1. As a result, the alternative approach shown in Scheme 2 was implemented. (8) (a) Hinks, J. D.; Takle, A. K.; Hunt, E. Chem. Abstr. 1997, 127, 161997. Hinks, J. D.; Takle, A. K.; Hunt, E. WO/97/25309, 1997. (b) Brooks, G.; Burgess, W.; Colthurst, D.; Hinks, J. D. Bioorg. Med. Chem. 2001, 9, 1221. (9) Silverberg, L. J.; Dillon, J. L.; Vemishetti, P. Tetrahedron Lett. 1996, 37, 771. (10) Silverberg, L. J.; Dillon, J. L.; Vemishetti, P.; Sleezer, P. D.; Discordia, R. P.; Hartung, K. B. Org. Process Res. DeV. 2000, 4, 34. Vol. 14, No. 4, 2010 / Organic Process Research & Development
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Scheme 1. Medicinal chemistry synthesis of compound 1a
a Reagents and conditions: (a) (CH O) CH, MeOH, H SO , 75%; (b) NaOH, MeOH, H O, chromatography, 90%; (c) CH Cl , AgOCN (4.6 equiv), chromatography, 3 3 2 4 2 2 2 80%; (d) dioxane, concd HCl, ZnCl2, 27%; (e) (BnO)2POH, DIPEA, DMAP, CCl4, CH3CN, chromatography, 82%; (f) 10%Pd/C, MeOH; (g) Na2CO3, 92% for 2 steps.
As reported by Joseph Sisko,11 there were clear benefits in the conversion of 2 to 4 without isolation of 3 with good overall yields. However, intermediate 4 was difficult to crystallize, probably owing to impurities derived from fermentation product 2. Despite this, we refocused our efforts on the purification of intermediate 4 by recrystallization and found that 4 could be crystallized in petroleum ether at 0 °C. After a wash with cold petroleum ether, compound 4 was isolated as a fine crystalline solid in high purity (98%, HPLC). This method avoids the need for isolating the intermediate 3 and offers advantages in terms of practicality. To avoid 4-acetoxybenzoyl chloride and AgOCN owing to cost and processing issues, we turned our attention to 4-benzyloxybenzamide 9 as an alternative starting material. This compound is readily available in bulk quantities.12 4-Benzyloxybenzoyl isocyanate 10 was prepared by treating amide 9 with oxalyl chloride in dichloromethane at 50 °C in almost quantitative yield.13 Without any isolation, slow addition of 4 in dichloromethane to the solution of 10 generated intermediate 11 in high yield (92%) and high purity (>98%). The skeletal rearrangement of intermediate 11 to generate 12 proved to be more difficult than expected. When a solution of 11 in dioxane was treated with a saturated solution of zinc chloride in concd HCl, up to 10% of impurities formation was observed, and the major one is amide 9 as identified by HPLC and NMR. We speculated that the presence of zinc chloride accelerated the decomposition, perhaps owing to the high concentration of nucleophilic chloride ion. However, when (11) Andemichael, Y.; Chen, J.; Clawson, J. S.; Dai, W.; Diederich, A.; Downing, S. V.; Freyer, A. J.; Liu, P.; Oh, L. M.; Patience, D. B.; Sharpe, S.; Sisko, J.; Tsui, J.; Vogt, F. G.; Wang, J.; Wernersbach, L.; Webb, E. C.; Wertman, J.; Zhou, L. Org. Process Res. DeV. 2009, 13, 729. (12) Compound 9 was readily prepared in high yield from commercially available 4-benzyloxybenzoic acid by sequential treatment with oxalyl chloride and concentrated aqueous ammonia. (13) (a) McGrew, L. A.; Sweeny, W.; Campbell, T. W.; Foldi, V. S. J. Org. Chem. 1964, 29, 3002. (b) Anchoori, R. K.; Kortenhorst, M. S.; Hidalgo, M.; Sarkar, T.; Hallur, G.; Bai, R.; Van Diest, P. J.; Hamel, E.; Khan, S. R. J. Med. Chem. 2008, 51, 5953. 816
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concd HCl without zinc chloride was used, a longer reaction time was needed, and the reaction stalled at 90-95% conversion. When a temperature greater than 30 °C was employed, more than 10% of impurity formation was observed, and further recrystallization from a variety of solvents did not remove the impurity. Finally, we found that a half-saturated solution of zinc chloride in concd HCl (250 g ZnCl2 in 500 mL concd HCl) at room temperature effected the conversion of 11 to 12 in good yield with low levels of impurity formation (Table 1). Furthermore, under these reaction conditions the poor solubility of 12 could be used advantageously in the isolation. In fact 12 was obtained directly from the reaction mixture by simple addition of a 1/3 equivalent volume of water followed by filtration and washing with water and ethanol, giving 12 as a colourless solid in high yield (93.4%) and high purity (>99.0%). Hydrogenation of the double bond and debenzylation to deprotect the hydroxy group could be done simultaneously to afford alcohol 13 in near quantitative yield. The regioselective phosphorylation of the phenolic hydroxyl group of 13 at the C-14 side chain without protecting the C-11 skeleton hydroxyl group relied on the higher acidity of the phenolic proton and the steric hindrance of the C-11 hydroxyl group.9 Treatment of 13 with N,N-dimethylaminopyridine (DMAP), carbon tetrachloride, N,N-diisopropylethylamine (DIPEA) and dibenzyl phosphite (BnO)2P(O)H successfully produced the phosphoric acid dibenzyl ester compound 14 in high yield and high purity after recrystallization from hexane and ethyl acetate. Various conditions were tested before the suitable reaction conditions were found. The results from the screening studies are shown in Table 2. A critical parameter in the reaction is to control the reaction temperature between -20 and -25 °C (entries 5-9, Table 2). At this low temperature, impurity formation was greatly reduced. The number of equivalents of (BnO)2P(O)H is also very important. The reaction was best run using 1.15 equiv of (BnO)2P(O)H at -25 °C, under which conditions the byproducts were minimised (entry 7, Table 2).
Scheme 2. First scale-up synthesis of compound 1a
a Reagents and conditions: (a) (CH O) CH, MeOH, H SO , 35 °C; (b) NaOH, MeOH, H O, 65 °C, 81% for 2 steps; (c) ClCOCOCl, CH Cl , rt to 50 °C; (d) CH Cl , 3 3 2 4 2 2 2 2 2 rt, 92% for 2 steps; (e) dioxane, concd HCl, ZnCl2, 25 °C, 93%; (f) H2, 10% Pd/C, THF, rt, 95%; (g) (BnO)2P(O)H, DIPEA, DMAP, CCl4, CH3CN, -25 °C, 82%; (h) H2, 5% Pd/C, THF, rt, 96%; (i) H2O, MeOH, Na2CO3, i-PrOH, 82%.
Table 1. Optimization of skeletal rearrangement conditions reaction
mixture
composition (%)
entry
reaction conditions
12
impurity 9
other impurities
yield (%)
1 2 3 4
concd HCl, 10 h, 35 °C saturated ZnCl2-concd HCl, 2.5 h, 30 °C half-saturated ZnCl2-concd HCl, 3 h, 30 °C half-saturated ZnCl2-concd HCl, 5 h, 25 °C
85.0 86.2 93.0 96.5
10.0 8.0 4.3 2.0
5.0 5.8 2.7 1.5
73.9 81.5 86.8 93.4
Table 2. Effects of (BnO)2P(O)H and temperature on formation of phosphate 14 reaction condition entry
T (°C)
(BnO)2P(O)H (equiv)
1 2 3 4 5 6 7 8 9
-5 -10 -15 -20 -20 -25 -25 -25 -40
1.5 1.5 1.5 1.5 1.2 1.2 1.15 1.10 1.15
impurity (%)
yield (%)
puritya (%)
15.0 13.2 10.3 9.1 8.0 6.0 4.2 7.8 10.6b
55.2 66.1 72.8 75.0 75.1 77.5 82.0 76.5 73.6
90.0 93.0 93.8 95.0 97.1 98.0 98.8 98.0 96.5
a The single recrystallization purity by HPLC. material 13 (4.0%).
b
screened various solvent systems such as methanol, methanol/ THF, methanol/ethyl acetate, and THF for the hydrogenolysis of 14. In methanol the rate of reaction was fast; however, the purity of product 15 was less than 98.0%. Ultimately, it was determined that the preferred reaction solvent was THF, and further recrystallization of 15 from water and CH3CN gave a purity over 99.2%. For salt formation, 15 was dissolved in methanol and water, and a solution of Na2CO3 (1.05 equiv) was added. After a small quantity of insoluble material was removed by filtration, the filtrate was concentrated under reduced pressure, and the residue was recrystallized from methanol/isopropanol to give 1 in 82% yield with >99.5% purity and no single impurity >0.1%.
Including unreacted starting
Hydrogenolysis of the benzyl moieties of 14 smoothly gave 15 in excellent yield. As a part of our optimization study, we
Conclusion In conclusion, we have developed an improved and more efficient process for the synthesis of 1, which offers distinctive advantages over the published procedures.7,8 The present Vol. 14, No. 4, 2010 / Organic Process Research & Development
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synthesis is free of column chromatography in all stages and greatly improves the overall yield from 10.9% to 42.8%. This process is amenable for the large-scale laboratory production of FU-23 (1). Experimental Section General Methods. All reagents and solvents were commercially available and were used without further purification unless otherwise stated. NMR spectra of the intermediates were recorded on a Bruker 300 NMR spectrometer using TMS as an internal standard. EI-MS spectra were obtained on a Finnigan MAT 95 mass spectrometer and ESI-MS spectra were obtained on a Kratos MS 80 mass spectrometer. Elemental analysis was obtained using a vario EL spectrometer. HPLC analysis of the intermediates and reaction monitoring was performed on an Agilent 1100 liquid chromatograph equipped with a Dikma Technologies ODS 250 mm ×4.6 mm 5 µM column; flow rate 1 mL/min, wavelength: 210 nm, temperature: 25 °C. Standard mobile phase composition: 30:70 to 10:90 gradient of water/ acetonitrile. Product 1 (FU-23) mobile phase composition: 30% water with 0.1% TFA, 70% methanol, flow rate 1 mL/min. (3R)-3-Deoxo-11-deoxy-3-methoxy-11-oxo-4-epi-mutilin (4). Pleuromutilin (400 g, 1.06 mol) was dissolved in methanol (1.5 L) and trimethyl orthoformate (504 g, 520 mL, 4.75 mol) at 20 °C. Concentrated sulphuric acid (198 g, 100 mL, 2.06 mol) was added over 10 min at
