The Story of LIPITOR® - A Peek into the World of Pharmaceutical Process Chemistry Ca2+ HO HO F N NH O Chemical Synthesis CO 2 LIPITOR® – $12 billion/year sales (2005) Chiral side chain (circled) – 220 ton/year Atorvastatin calcium LIPITOR ® 2 Biocatalysis Aman Desai 7th Feb. 2007 The Problem – The “Bad” Cholesterol • • Cholesterol – a very important biological molecule. Most cholesterol is not dietary, it is synthesized internally. H HO H H • • Cholesterol is bound to lipoproteins and transported Cholesterol through blood. High Density Lipoprotein (HDL) – “good” 2 kinds of lipoproteins Low Density Lipoprotein (LDL) – “bad” atherosclerosis coronary heart disease & other cardiovascular diseases One of the leading causes of death in the world today! www.wikipedia.org www.americanheart.org The Solution – Suppressing Cholesterol Biosynthesis O O SCoA + O SCoA HSCoA OH HMG-CoA-Synthetase O O HMG-CoA 2 NADPH + 2H + HMG-CoA-Reductase 2 NADP + + HSCoA Rate Limiting Step O SCoA OH H H HO Cholesterol >25 steps H H O O OH Mevalonic Acid http://www.med.unibs.it/~marchesi/cholest.html The Solution – Suppressing Cholesterol Biosynthesis HO O O 9' H 9' O O O 2'' HO O O H O OH O SCoA O O HMG-CoA 2 NADPH + 2H+ HMG-CoA-Reductase 2 NADP + + HSCoA Inhibition Rate Limiting Step Mevastatin Lovastatin (MEVACOR®) MERCK HO O O 9' HO HO CO 2Na O HO O O O H 2'' OH H OH O O Pravastatin (PRAVACOL®) BRISTOL - MYERS SQUIBB Simvastatin (ZOCOR®) MERCK Mevalonic Acid Endo, A. J. Lipid Res. 1992, 33, 1569-1582. Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22. Mechanism of Action of Statin Drugs HO O O H O O O O H HO O O HO O O SCoA HMG-CoA 2 NADPH + 2H+ Rate Limiting Step O Mevastatin O Lovastatin (MEVACOR ) MERCK ONa ® HMG-CoA-Reductase 2 NADP + + HSCoA Inhibition HO O O H HO O O H O O OH OH OH O O HO Pravastatin (PRAVACOL®) BRISTOL - MYERS SQUIBB Simvastatin (ZOCOR ) MERCK ® Mevalonic Acid http://www.med.unibs.it/~marchesi/cholest.html Mechanism of Action of Statin Drugs HO O O H O O O O H HO O O HO O O Ca 2+ 2 OH Mevastatin O Lovastatin (MEVACOR®) MERCK ONa F N NH O HO O O H HO O O H O O OH Atorvastatin calcium (LIPITOR®) PFIZER HO Pravastatin (PRAVACOL®) BRISTOL - MYERS SQUIBB Simvastatin (ZOCOR®) MERCK http://www.med.unibs.it/~marchesi/cholest.html Human HMGR with Natural Substrates HO O O SCoA HMG-CoA 2 NADPH + 2H+ HMG-CoA-Reductase 2 NADP + + HSCoA Inhibition Rate Limiting Step O OH OH O O Mevalonic Acid Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164. Human HMGR with Natural Substrates HO O O SCoA HMG-CoA O HO O O Ca 2+ 2 OH F N NH O LIPITOR ® Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164. Human HMGR with Natural Substrates HO O O O HMG-CoA S CH2 CH2 H N C O CH2 pantothenic CH2 acid H N C O H C OH H3C C CH3 CH2 O R R = 3'-phosphoadenosine diphosphate HO O Ca 2+ O 2 OH F N NH O LIPITOR® Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164. Human HMGR with LIPITOR® HO O O Ca 2+ 2 OH F N NH O LIPITOR® Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164. Human HMGR with LIPITOR® HO O O Ca 2+ 2 OH F N NH O LIPITOR® Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164. Circa 1995 – The Statin Drugs Market HO O O H O O O O H HO O O • Merck = cholesterol control • At 20 mg, ZOCOR® lowered LDL by -29%. Lovastatin (MEVACOR®) MERCK Simvastatin (ZOCOR®) MERCK Thayer, A. M. Chem. Eng. News, 2006, 84, 33, 26-27. Jones P.; Kafonek, S.; Laurora, I.; Hunninghake, D. Am. J. Cardiol. 1998, 81, 582-587. Circa 1995 – The Statin Drugs Market HO O O H O O O O H HO O O • Merck = cholesterol control. • At 20 mg, ZOCOR® lowered LDL by -29%. Lovastatin (MEVACOR®) MERCK Simvastatin (ZOCOR®) MERCK Spring 1997 – Pfizer launches LIPITOR®! Ca 2+ HO HO F CO 2 • At 20 mg, LIPITOR® lowered LDL by -46%. • 2005 – $12 billion sales, used by over 45 million people. N NH O Atorvastatin calcium LIPITOR® 2 The Story of LIPITOR® - a Peek into the World of Pharmaceutical Process Chemistry Ca2+ HO HO F N NH O CO 2 Chemical Synthesis Drug Discovery Process Development Biocatalysis 2 Atorvastatin calcium LIPITOR ® The Story of LIPITOR® - a Peek into the World of Pharmaceutical Process Chemistry Ca+2 HO HO F N NH O CO 2 Chemical Synthesis Drug Discovery Process Development Biocatalysis 2 Atorvastatin calcium LIPITOR ® The Drug Discovery HO O O H O O The Decision of the Core Template HO O O O O Mevastatin Lovastatin (MEVACOR®) MERCK CO 2Na HO O F O ? H HO HO ? O O H HO Pravastatin (PRAVACOL®) BRISTOL - MYERS SQUIBB MERCK Willard, A. K.; Novello, F. C.; Hoffmann, W. F.; Cragoe, E.; E. J. Jr. USP 4459422, 1984. Fortune, 2003, January 20. Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22. Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31. The Drug Discovery HO O O H O O The Decision of the Core Template correct spatial relationship HO O O Mevastatin hydrophobic group X template HO O F O Hydrolysis – 100-fold loss in potency A Potent HMGR Inhibitor MERCK Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31. The Drug Discovery The Pyrrole Template HO O X N O R1 R2 Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31. The Drug Discovery F NEt3 + O HO 58% O F Bn N Cl S The Pyrrole Template H2 N O O CN F CN N HOAc, reflux 73% 20 mol% DIBAL-H 92% O HO CO 2CH 3 CH 3COCH 2CO 2CH3 NaH, n-BuLi F THF, -78 °C 64% CHO N 1) Bu3B, NaBH4 THF, -78 °C 2) NaOH, H2O 2 F N HO HO F CO 2H HO O toluene, reflux 52% tr ans:cis 97:3 F O N N Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31. The Drug Discovery F NEt3 + O HO 58% O F Bn N Cl S The Pyrrole Template H2 N O O CN F CN N HOAc, reflux 73% 20 mol% DIBAL-H 92% O HO CO 2CH 3 CH 3COCH 2CO 2CH3 NaH, n-BuLi F THF, -78 °C 64% CHO N 1) Bu 3B, NaBH4 THF, -78 °C 2) NaOH, H2O 2 F N HO HO F CO 2H HO O toluene, reflux 52% tr ans:cis 97:3 R1 X N O • Over 40 analogs prepared. • Optimization studies for X, R1 and R2. N R2 Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31. The Drug Discovery The Need for Additional Functionality HO O O O HO O F O O H N • IC50 (analog): 0.40 µM. • Limit of current synthetic route. Mevastatin MERCK IC50 – 0.030 µM. Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31. The Drug Discovery The Need for Additional Functionality HO O O F HO O F O N An overlay HO O F F O O MERCK N Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22. The Drug Discovery Incorporation of Additional Functionality HO O O HO O F O TBDMSO TBDMSCl imidazole, DMF 25 ºC 100% F N O O 1) 2 eq NCS or NBS DMF, 0 ºC 100% 2) TBAF, HOAc THF, 25 ºC 35% F N X X = Cl X = Br X N X H Cl Br IC50 (µM) 0.23 0.028 0.028 HO O O H O O Mevastatin MERCK IC50 – 0.030 µM. Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366. The Drug Discovery Incorporation of Additional Functionality HO O O HO O F O TBDMSO TBDMSCl imidazole, DMF 25 ºC 100% F N O O 1) 2 eq NCS or NBS DMF, 0 ºC 100% 2) TBAF, HOAc THF, 25 ºC 35% F N X X = Cl X = Br X N X H Toxicity in early preclinical development! Cl Br IC50 (µM) 0.23 0.028 0.028 HO O O H O O Mevastatin MERCK IC50 – 0.030 µM. Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22. The Drug Discovery O F Br CO2 Et H2N Penta-substituted Pyrroles via [3+2] O O F NH CO2 Et O 1) O Cl F NEt3 , CH2Cl2, 0 ºC 2) NaOH 97% HO2C NHPh Ph O Ac2O, 90 ºC 43% O O NEt3, CH 3CN, 25 ºC 82% N O HO O O CHO F N Ph NHPh O F N Ph O NHPh 1) HCl, EtOH reflux 2) p-TSA, acetone-water reflux 87% O F O N Ph O NHPh Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366. The Drug Discovery HO O F O Penta-substituted Pyrroles via [3+2] HO O O 20 analogs tested N X Y F N NH O (racemic) HO O O H O O IC50 – 0.025 µM Mevastatin MERCK IC50 – 0.030 µM. Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366. The Drug Discovery HO O F O Penta-substituted Pyrroles via [3+2] HO O O HO O F O 20 analogs tested N X Y F N NH O (racemic) resolution N NH O (R, R) HO O O H O O IC50 – 0.025 µM IC50(µM) (R, R) – 0.007 (S, S) – 0.44 Mevastatin MERCK IC50 – 0.030 µM. Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366. The Drug Discovery HO O F 20 analogs tested N X Y F The Birth of LIPITOR®! O HO O F O HO O O N NH O (racemic) resolution N NH O (R, R) Ca2+ HO HO F N NH O CO 2 Atorvastatin calcium LIPITOR ® 2 The Story of LIPITOR® - a Peek into the World of Pharmaceutical Process Chemistry Ca2+ HO HO F N NH O CO 2 Chemical Synthesis Drug Discovery Process Development Biocatalysis 2 Atorvastatin calcium LIPITOR ® The Story of LIPITOR® - a Peek into the World of Pharmaceutical Process Chemistry Ca2+ HO HO F N NH O CO 2 Chemical Synthesis Drug Discovery Process Development Biocatalysis 2 Atorvastatin calcium LIPITOR ® The Process Development Scale-up Issues and Potential Solutions O F N HO2C O O [3+2] Cycloaddition Route 1.6 eq Ph NHPh O F O O Ac 2O, 90 ºC N Ph O NHPh 43% Paal-Knorr Route: Penta-substituted Pyrroles HO HO CO 2R H2N O O CO2 R + F N NH O F O O Failed Model Study H 2N F NH O Paal-Knorr Route: Tetra-substituted Pyrroles Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22. EtO F OEt OEt OEt + O O N The Process Development From Tetra- to Penta-substituted Pyrroles F F O O O NEt3 Bn N Cl OEt 71% H2 N OEt O O O PhCHO, p-TSA toluene, reflux 80% O O O 20 mol% S HO 2) NaOH, CH3 OH, rt 30% p-TSA, toluene reflux CHO F N CONHPh 1) NBS, DMF 0 ºC, 100% 2) n-BuLi, THF, PhNCO, 69% 3) H3O +, 86% EtO F OEt N Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366. Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22. The Process Development CHO F N CONHPh From Tetra- to Penta-substituted Pyrroles 1) O O Ph OH Ph Ph 2 eq. LDA, MgBr2 2) Recrystallization dr 98:2 38% Ph HO F -78 ºC -10 ºC O O Ph Ph OH N CONHPh 1) NaOCH 3 0 ºC 68% 2) OLi -70 ºC 78% HO O F 1) Et3 B. NaBH 4 -78 ºC 2) H 2O2, NaOH 3) toluene, reflux F O Ot Bu HO O N CONHPh CO 2t Bu N CONHPh >99% ee Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366. The Process Development CHO F N CONHPh From Tetra- to Penta-substituted Pyrroles 1) O O Ph OH Ph Ph 2 eq. LDA, MgBr2 2) Recrystallization dr 98:2 38% Ph HO F -78 ºC -10 ºC O O Ph Ph OH N CONHPh 1) NaOCH 3 0 ºC 68% 2) OLi -70 ºC 78% HO O F 1) Et3 B. NaBH 4 -78 ºC 2) H 2O2, NaOH 3) toluene, reflux F O Ot Bu HO O N CONHPh CO 2t Bu N CONHPh >99% ee Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366. The Process Development CHO F N CONHPh From Tetra- to Penta-substituted Pyrroles 1) O O Ph OH Ph Ph 2 eq. LDA, MgBr2 2) Recrystallization dr 98:2 38% Ph HO F -78 ºC -10 ºC O O Ph Ph OH N CONHPh 1) NaOCH 3 0 ºC 68% 2) OLi -70 ºC 78% HO O F 1) Et3 B. NaBH 4 -78 ºC 2) H 2O2, NaOH 3) toluene, reflux 3 columns & 1 crystallization 7% yield Ot Bu O HO F O N CONHPh CO 2t Bu N CONHPh >99% ee Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366. The Process Development A Recap – The Failures O F N HO2C O O [3+2] Cycloaddition Route 1.6 eq Ph NHPh O F O O N Ph O NHPh Ac2O, 90 ºC 43% Paal Knorr Route: Tetra-substituted Pyrroles F OEt O O H2N OEt F EtO OEt HOAc, reflux 71% N Paal Knorr Route: Penta-substituted Pyrroles F HO HO CO 2R H2N O O CO2 R + N NH O F O O Failed Model Study NH O The Process Development Paal Knorr Route: Penta-substituted Pyrroles OEt H2 N + OEt O O 1 eq O OH THF, reflux 43% NH O O F EtO OEt F N NH R2O R2O R1O F NH 2 + O O CO 2R R1O B CO 2R N NH O F NH O A Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22. The Process Development Pfizer’s Commercial Route: Fragment A O NHPh PhCHO β -Alanine, AcOH hexane, heat 85% O O NHPh Ph O F CHO NEt3 , EtOH, heat 80% 20 mol% N HO S Br F O O NH O A Dr. Bruce D. Roth (VP, Global Research & Development, Pfizer), personal communication. Baumann, K. L. et al. Tetrahedron Lett. 1992, 33, 2283-2284. The Process Development Pfizer’s Commercial Route: Fragment B H2 O2, CaCO 3 K2CO3 O HBr, HOAc CH3 OH HO OH H O OH HO CO2 K OH OH Br Br CO2 CH3 HO OH R2O R 1O B NH2 CO2R H 2, Pd/C CH 3COONa/CH3COOH 60% (over 3 steps) NC OTBDMS CO2 CH 3 1) TBDMS-Cl imid., 4-DMAP Br 2) NaCN, DMSO OH CO2 CH 3 Browser, P. L. et al. Tetrahedron Lett. 1992, 33, 2279-2282. Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22. The Process Development Pfizer’s Commercial Route: Fragment B 1) NaOH 2) CDI Mg(O 2CCH2 CO2 t Bu) NC OH O CO 2t Bu NC OTBDMS CO 2CH 3 3 eq LiCH2 CO 2t Bu THF 75% OH NC CO2CH3 3) TBAF, HOAc, THF R2O R 1O B NH2 CO2R Dr. Donald E. Butler (Former Process Development Leader, Pfizer), personal communication. Browser, P. L. et al. Tetrahedron Lett. 1992, 33, 2279-2282. The Process Development Pfizer’s Commercial Route: Fragment B 1) NaOH 2) CDI Mg(O 2CCH2 CO2 t Bu) 3) TBAF, HOAc, THF 1) NaBH4, Et2 BOMe CH3OH, -90 °C 2) (CH 3)2C(OCH 3)2 CH3SO3 H NC OTBDMS CO 2CH 3 OH NC O CO 2t Bu 3 eq LiCH 2CO 2t Bu THF 75% OH NC CO2CH3 Cryogenic reactor employed O H 2N B O CO 2t Bu Raney-Ni, MeOH 50 psi H2 95% NC O O CO 2t Bu >99.5% ee dr 350:1 ee >99.5% liquid N2 liquid N2 Dr. Donald E. Butler (Former Process Development Leader, Pfizer), personal communication. Browser, P. L. et al. Tetrahedron Lett. 1992, 33, 2279-2282. The Process Development F O O + O O Pfizer’s Commercial Route O CO2t Bu 1 eq O OH F N NH O O CO2t Bu NH O NH 2 A B 1:4:1 toluene:heptane:THF reflux 75% • Highly convergent. • Yields >75% at each step. • One low temperature reaction. • One special equipment requirement. • No chromatography. • Scalable to ton quantities. F a) HCl, MeOH b) NaOH c) Ca(OAc)2 HO HO 84% Ca2+ CO2 2 N NH O Atorvastatin calcium LIPITOR ® >99.5% ee Baumann, K. L. et al. Tetrahedron Lett. 1992, 33, 2283-2284. Dr. Donald E. Butler (Former Process Development Leader, Pfizer), personal communication. LIPITOR®: Drug tackled, the struggles remained… “The number of factors, internal and external, that had to come together for the drug to be a success really boggles the mind" – Bruce D. Roth The problems: • By 1987, three statins in market. • A decade since the first statin introduced. • Warner-Lambert was floundering. • Come on, how good could it be? • On the verge of terminating LIPITOR®. And the facts now: • #1 statin in the market. • Top selling drug in history. • 2005: $12 billion sales & used by >45 million people. • “LIPITOR® is on track to have greater benefit for more people than any other drug in the history of the industry in terms of lives improved and saved.” - Nobel Laureate Michael Brown Fortune, 2003, January 20. The Story of LIPITOR® “The story of how Pfizer acquired the rights to an improved statin and turned it into the all-time biggest blockbuster is a tale of hyperaggresive marketing, deft timing, financial power and plain dumb luck!” Fortune, 2003, January 20. The Story of LIPITOR® - a Peek into the World of Pharmaceutical Process Chemistry Ca2+ HO HO F N NH O CO 2 Chemical Synthesis Drug Discovery Process Development Biocatalysis 2 Atorvastatin calcium LIPITOR ® The Story of LIPITOR® - a Peek into the World of Pharmaceutical Process Chemistry Ca2+ HO HO F N NH O CO 2 Chemical Synthesis 220 ton/year market ($500 million) Biocatalysis 2 Drug Discovery Process Development Atorvastatin calcium LIPITOR ® The Story of LIPITOR® - a Peek into the World of Pharmaceutical Process Chemistry Ca2+ HO HO F N NH O CO 2 Chemical Synthesis 220 ton/year market ($500 million) Biocatalysis 2 Drug Discovery Process Development Atorvastatin calcium LIPITOR ® OH NC OH O OR Existing Route & the Need for Improvement H2O 2, CaCO3 K 2CO 3 OH HO CO2K OH HBr, HOAc CH3 OH HO OH H OH Br Br CO 2CH3 O O HO OH H 2, Pd/C CH3COONa/CH 3COOH 60% (over 3 steps) OH NC O CO2 t Bu 3 eq LiCH 2CO 2t Bu THF 75% OH NC CO2 CH 3 NaCN, DMSO OH Br CO2 CH 3 1) NaBH4, Et2BOMe CH3OH, -90 °C 2) (CH 3)2C(OCH 3)2 CH3SO3 H O NC O CO 2t Bu >99.5% ee OH NC OH O OR Existing Route & the Need for Improvement H2O 2, CaCO3 K 2CO 3 OH HO CO2K OH HBr, HOAc CH3 OH HO OH H OH Br Br CO 2CH3 O O HO OH H 2, Pd/C CH3COONa/CH 3COOH 60% (over 3 steps) OH NC O CO2 t Bu 3 eq LiCH 2CO 2t Bu THF 75% OH NC CO2 CH 3 NaCN, DMSO OH Br CO2 CH 3 1) NaBH4, Et2BOMe CH3OH, -90 °C 2) (CH 3)2C(OCH 3)2 CH3SO3 H • SHE issues. • Cryogenic step – special requirements/waste. • With purification – 11 steps. • Month’s supply: $66 LIPITOR® vs $120 ZOCOR®. • But patent expires ~ 2010. O NC O CO 2t Bu >99.5% ee OH NC OH O OR Biocatalytic Routes for the Chiral Side Chain KANEKA OH Cl O OEt ADH GDH O Cl O OEt CIBA O HO OH O OEt Lipase EtO O OR1 O OEt OH OH NC OH O OR DOWPHARMA (DIVERSA) OH NC O OH Nitrilase NC CN CODEXIS OH NC O OEt 1) ADH 2) Dehalogenase Aldolase O Cl O OEt DIVERSA X O OH O X H + 2 O H OH Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27. Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365. OH NC OH O OR Biocatalytic Routes for the Chiral Side Chain KANEKA OH Cl O OEt ADH GDH O Cl O OEt CIBA O HO OH O OEt Lipase EtO O OR1 O OEt OH OH NC OH O OR DOWPHARMA (DIVERSA) OH NC O OH Nitrilase NC CN CODEXIS OH NC O OEt 1) ADH 2) Dehalogenase Aldolase O Cl O OEt DIVERSA X O OH O X H + 2 O H OH Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27. Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365. OH NC OH O OR Biocatalytic Routes for the Chiral Side Chain KANEKA OH Cl O OEt ADH GDH O Cl O OEt CIBA O HO OH O OEt Lipase EtO O OR1 O OEt OH OH NC OH O OR DOWPHARMA (DIVERSA) OH NC O OH Nitrilase NC CN CODEXIS OH NC O OEt 1) ADH 2) Dehalogenase Aldolase O Cl O OEt DIVERSA X O OH O X H + 2 O H OH Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27. Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365. OH NC OH O OR Biocatalytic Routes for the Chiral Side Chain KANEKA OH Cl O OEt ADH GDH O Cl O OEt CIBA O HO OH O OEt Lipase EtO O OR1 O OEt OH OH NC OH O OR DOWPHARMA (DIVERSA) OH NC O OH Nitrilase NC CN CODEXIS OH NC O OEt 1) ADH 2) Dehalogenase Aldolase O Cl O OEt DIVERSA X O OH O X H + 2 O H OH Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27. Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365. OH NC OH O OR Biocatalytic Routes for the Chiral Side Chain KANEKA OH Cl O OEt ADH GDH O Cl O OEt CIBA O HO OH O OEt Lipase EtO O OR1 O OEt OH OH NC OH O OR DOWPHARMA (DIVERSA) OH NC O OH Nitrilase NC CN CODEXIS OH NC O OEt 1) ADH 2) Dehalogenase Aldolase O Cl O OEt DIVERSA X O OH O X H + 2 O H OH Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27. Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365. OH NC OH O OR Biocatalytic Routes for the Chiral Side Chain KANEKA OH Cl O OEt ADH GDH O Cl O OEt CIBA O HO OH O OEt Lipase EtO O OR1 O OEt OH OH NC OH O OR DOWPHARMA (DIVERSA) OH NC O OH Nitrilase NC CN CODEXIS OH NC O OEt 1) ADH 2) Dehalogenase Aldolase O Cl O OEt DIVERSA X O OH O X H + 2 O H OH Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27. Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365. OH NC OH O OR Kaneka’s Route NADP+ OH Cl O O Et D-glucose glucose dehydrogenase recombinant E. coli NADPH carbonyl reductase O D-gluconolactone spontaneous Aqueous phase D-glutonic acid Organic phase (n-butyl acetate) Cl O O Et • >99.9% ee, 89% yield. • Product concentration: 450 g/L. • NADP+ TON: 16,200 mol/mol. • Problematic product separation. Kizaki, N. et al. Appl. Microbiol. Biotechnol. 2001, 55, 590-595. Yasohara, Y. et al. Tetrahedron: Asymmetry 2001, 12, 1713-1718. OH NC OH O OR Daicel’s Route NADP+ OH Cl O O Et formic acid formate dehydrogenase recombinant E. coli NADPH carbonyl reductase O CO2 Cl O O Et • >99% ee. • Product concentration: 50 g/L. • Easy product separation. • Commercially used: >100 ton/year. Rouhi, A. M. Chem. Eng. News 2004, 82, (24), 47-62. OH NC OH O OR Ciba’s Route O O O EtO O O Cl O O OEt O α-chymotrypsin 0.008 mol% 24-30 h 94% 98.2% ee HO O O O O OEt 6 steps 44% overall yield O EtO OH O OEt pyridine 0 °C to rt ~ 12 h 98% O N3 O O OEt The good things • 94% yield, 98.2% ee. • Substrate concentration: 285 g/L. • Kilogram scale. • Cheap & robust biocatalyst. The bad things • Follow up chemistry – long. • Low temperature reactions. • Column chromatography. Öhrlein, R.; Baischf, G. Adv. Synth. Catal. 2003, 345, 713-715. OH NC OH O OR Diversa/Dowpharma’s Route OH NC CN nitrilase pH 7.5 NaH2PO4 27 °C, 16 h 81% 98.8% ee NC OH COOH DeSantis, G. et al. J. Am. Chem. Soc. 2002, 124, 9024-9025. DeSantis, G. et al. J. Am. Chem. Soc. 2003, 125, 11476-11477. OH NC OH O OR Diversa/Dowpharma’s Route OH NC Cl NaCN (aq.) pH=10, 4 h, 50 °C NC 45% nitrilase EtOH, H+ 3 h, 80 °C CO 2Et 62% pH 7.5 NaH 2PO4 27 °C, 16 h 81% 98.8% ee OH NC COOH OH CN Cl O HCN, base (n-Bu)4N+Br12 h, 45 °C 92% OH NC The good things • Cheap starting material. • Efficient enzymatic step: 3 M [substrate] & 619 g L-1 d-1. • Low cost of catalyst by expression in Pseudomonas fluorescens developed by Dow. Scale-up economics good! The bad things • HCN under heated alkaline conditions. • Special equipment for purification. • Some low yield steps. Bergeron, S. et al. Org. Process Res. Dev. 2006, 10, 661-665. OH NC OH O OR Diversa’s Route O DERA 25 °C Cl OH O DERA OH Cl OH O Aldol reaction O Cl + O Wild Type DERA Catalyst Load Product Isolation Reaction Time [chloroacetaldehyde] Volumetric Productivity ee (de) Practical? 20% w/w Difficult 6 days 100 mM 2 g L-1 d-1 DERA = deoxyribose5-phosphate aldolase Cl O OH OH OH Cl OH O OR Unknown NO Gijsen, H. J. M.; Wong, C.-H. J. Am. Chem. Soc. 1994, 116, 8422-8423. Wong, C.-H. et al. J. Am. Chem. Soc. 1995, 117, 3333-3339. OH NC OH O OR Diversa’s Route O DERA 25 °C Cl OH O DERA OH Cl OH O Aldol reaction O Cl + O Wild Type DERA Catalyst Load Product Isolation Reaction Time [chloroacetaldehyde] Volumetric Productivity ee & de Practical? 20% w/w Difficult 6 days 100 mM 2 g L-1 d-1 Unknown NO Improved DERA 2% w/w Simple 3h Fed-batch process 735 g L-1 d-1 ≥ 99.9% YES DERA = deoxyribose5-phosphate aldolase Cl O OH OH O NC O O OMe 23% yield 3 overall steps Greenberg, W. A. et al. Proc. Natl. Acad. Sci. USA 2004, 101, 5788-5793. OH NC OH O OR Codexis Route OH Cl not purified O OEt OH NC O OEt O Cl O OEt KRED/GDH HHDH oil, not purified - HCl + HCl KRED = Ketoreductase GDH = Glucose dehydrogenase HHDH = Halohydrin dehalogenase O O OEt + HCN pK a ~9 HCNaq NaClaq HClaq pKa 99.9% ee 99.8% de OH oil, not purified O ONa OH NC OH O ONa OH Cl OH O ONa O OH O H2N O O OtBu O NC O O OMe Coupling partner for the Paal-Knorr oil, column chromatography 48% (over 4 steps) Greenberg, W. A. et al. Proc. Natl. Acad. Sci. USA 2004, 101, 5788-5793. Diversa’s Fluorogenic Activity Based Screen 1) O TosO HO O O O 4-methylumbelliferone OH O HO K 2CO 3, DMF, 75 °C, 16 h 2) H 2O / 20% CH3CN DOWEX 50WX8-100 2 days 62% O O O OH 5-toluenesulfonyl2-deoxyribose O O O O OH spontaneous + O H DERA O O O OH O H OH O + O O fluorescent OH O Greenberg, W. A. et al. Proc. Natl. Acad. Sci. USA 2004, 101, 5788-5793. OH NC OH O OR Codexis Biocatalyst Improvement O Cl O OEt NADPH Na-gluconate NADP + glucose KRED OH O Cl OEt GDH Parameter Substrate loading Reaction time Enzyme loading Isolated yield Phase separation time Volumetric productivity Initial 80 g/L 24 h 10 g/L ~80% >1 h 80 g/L.day Final 180 g/L 8h 0.7 g/L 97% ~ 1 min 540 g/L.day Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication. OH NC OH O OR Codexis Biocatalyst Improvement OH Cl O OEt - HCl + HCl O O OEt pKa ~9 HCNaq NaClaq HClaq pKa
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Lipitor Development

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The Story of LIPITOR® - A Peek into the World of Pharmaceutical Process Chemistry Ca2+ HO HO F N NH O Chemical Synthesis CO 2 LIPITOR® – $12 billion/year sales (2005) Chiral side chain (circled) – 220 ton/year Atorvastatin calcium LIPITOR ® 2 Biocatalysis Aman Desai 7th Feb. 2007 The Problem – The “Bad” Cholesterol • • Cholesterol – a very important biological molecule. Most cholesterol is not dietary, it is synthesized internally. H HO H H • • Cholesterol is bound to lipoproteins and transported Cholesterol through blood. High Density Lipoprotein (HDL) – “good” 2 kinds of lipoproteins Low Density Lipoprotein (LDL) – “bad” atherosclerosis coronary heart disease & other cardiovascular diseases One of the leading causes of death in the world today! www.wikipedia.org www.americanheart.org The Solution – Suppressing Cholesterol Biosynthesis O O SCoA + O SCoA HSCoA OH HMG-CoA-Synthetase O O HMG-CoA 2 NADPH + 2H + HMG-CoA-Reductase 2 NADP + + HSCoA Rate Limiting Step O SCoA OH H H HO Cholesterol >25 steps H H O O OH Mevalonic Acid http://www.med.unibs.it/~marchesi/cholest.html The Solution – Suppressing Cholesterol Biosynthesis HO O O 9' H 9' O O O 2'' HO O O H O OH O SCoA O O HMG-CoA 2 NADPH + 2H+ HMG-CoA-Reductase 2 NADP + + HSCoA Inhibition Rate Limiting Step Mevastatin Lovastatin (MEVACOR®) MERCK HO O O 9' HO HO CO 2Na O HO O O O H 2'' OH H OH O O Pravastatin (PRAVACOL®) BRISTOL - MYERS SQUIBB Simvastatin (ZOCOR®) MERCK Mevalonic Acid Endo, A. J. Lipid Res. 1992, 33, 1569-1582. Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22. Mechanism of Action of Statin Drugs HO O O H O O O O H HO O O HO O O SCoA HMG-CoA 2 NADPH + 2H+ Rate Limiting Step O Mevastatin O Lovastatin (MEVACOR ) MERCK ONa ® HMG-CoA-Reductase 2 NADP + + HSCoA Inhibition HO O O H HO O O H O O OH OH OH O O HO Pravastatin (PRAVACOL®) BRISTOL - MYERS SQUIBB Simvastatin (ZOCOR ) MERCK ® Mevalonic Acid http://www.med.unibs.it/~marchesi/cholest.html Mechanism of Action of Statin Drugs HO O O H O O O O H HO O O HO O O Ca 2+ 2 OH Mevastatin O Lovastatin (MEVACOR®) MERCK ONa F N NH O HO O O H HO O O H O O OH Atorvastatin calcium (LIPITOR®) PFIZER HO Pravastatin (PRAVACOL®) BRISTOL - MYERS SQUIBB Simvastatin (ZOCOR®) MERCK http://www.med.unibs.it/~marchesi/cholest.html Human HMGR with Natural Substrates HO O O SCoA HMG-CoA 2 NADPH + 2H+ HMG-CoA-Reductase 2 NADP + + HSCoA Inhibition Rate Limiting Step O OH OH O O Mevalonic Acid Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164. Human HMGR with Natural Substrates HO O O SCoA HMG-CoA O HO O O Ca 2+ 2 OH F N NH O LIPITOR ® Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164. Human HMGR with Natural Substrates HO O O O HMG-CoA S CH2 CH2 H N C O CH2 pantothenic CH2 acid H N C O H C OH H3C C CH3 CH2 O R R = 3'-phosphoadenosine diphosphate HO O Ca 2+ O 2 OH F N NH O LIPITOR® Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164. Human HMGR with LIPITOR® HO O O Ca 2+ 2 OH F N NH O LIPITOR® Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164. Human HMGR with LIPITOR® HO O O Ca 2+ 2 OH F N NH O LIPITOR® Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164. Circa 1995 – The Statin Drugs Market HO O O H O O O O H HO O O • Merck = cholesterol control • At 20 mg, ZOCOR® lowered LDL by -29%. Lovastatin (MEVACOR®) MERCK Simvastatin (ZOCOR®) MERCK Thayer, A. M. Chem. Eng. News, 2006, 84, 33, 26-27. Jones P.; Kafonek, S.; Laurora, I.; Hunninghake, D. Am. J. Cardiol. 1998, 81, 582-587. Circa 1995 – The Statin Drugs Market HO O O H O O O O H HO O O • Merck = cholesterol control. • At 20 mg, ZOCOR® lowered LDL by -29%. Lovastatin (MEVACOR®) MERCK Simvastatin (ZOCOR®) MERCK Spring 1997 – Pfizer launches LIPITOR®! Ca 2+ HO HO F CO 2 • At 20 mg, LIPITOR® lowered LDL by -46%. • 2005 – $12 billion sales, used by over 45 million people. N NH O Atorvastatin calcium LIPITOR® 2 The Story of LIPITOR® - a Peek into the World of Pharmaceutical Process Chemistry Ca2+ HO HO F N NH O CO 2 Chemical Synthesis Drug Discovery Process Development Biocatalysis 2 Atorvastatin calcium LIPITOR ® The Story of LIPITOR® - a Peek into the World of Pharmaceutical Process Chemistry Ca+2 HO HO F N NH O CO 2 Chemical Synthesis Drug Discovery Process Development Biocatalysis 2 Atorvastatin calcium LIPITOR ® The Drug Discovery HO O O H O O The Decision of the Core Template HO O O O O Mevastatin Lovastatin (MEVACOR®) MERCK CO 2Na HO O F O ? H HO HO ? O O H HO Pravastatin (PRAVACOL®) BRISTOL - MYERS SQUIBB MERCK Willard, A. K.; Novello, F. C.; Hoffmann, W. F.; Cragoe, E.; E. J. Jr. USP 4459422, 1984. Fortune, 2003, January 20. Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22. Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31. The Drug Discovery HO O O H O O The Decision of the Core Template correct spatial relationship HO O O Mevastatin hydrophobic group X template HO O F O Hydrolysis – 100-fold loss in potency A Potent HMGR Inhibitor MERCK Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31. The Drug Discovery The Pyrrole Template HO O X N O R1 R2 Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31. The Drug Discovery F NEt3 + O HO 58% O F Bn N Cl S The Pyrrole Template H2 N O O CN F CN N HOAc, reflux 73% 20 mol% DIBAL-H 92% O HO CO 2CH 3 CH 3COCH 2CO 2CH3 NaH, n-BuLi F THF, -78 °C 64% CHO N 1) Bu3B, NaBH4 THF, -78 °C 2) NaOH, H2O 2 F N HO HO F CO 2H HO O toluene, reflux 52% tr ans:cis 97:3 F O N N Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31. The Drug Discovery F NEt3 + O HO 58% O F Bn N Cl S The Pyrrole Template H2 N O O CN F CN N HOAc, reflux 73% 20 mol% DIBAL-H 92% O HO CO 2CH 3 CH 3COCH 2CO 2CH3 NaH, n-BuLi F THF, -78 °C 64% CHO N 1) Bu 3B, NaBH4 THF, -78 °C 2) NaOH, H2O 2 F N HO HO F CO 2H HO O toluene, reflux 52% tr ans:cis 97:3 R1 X N O • Over 40 analogs prepared. • Optimization studies for X, R1 and R2. N R2 Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31. The Drug Discovery The Need for Additional Functionality HO O O O HO O F O O H N • IC50 (analog): 0.40 µM. • Limit of current synthetic route. Mevastatin MERCK IC50 – 0.030 µM. Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31. The Drug Discovery The Need for Additional Functionality HO O O F HO O F O N An overlay HO O F F O O MERCK N Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22. The Drug Discovery Incorporation of Additional Functionality HO O O HO O F O TBDMSO TBDMSCl imidazole, DMF 25 ºC 100% F N O O 1) 2 eq NCS or NBS DMF, 0 ºC 100% 2) TBAF, HOAc THF, 25 ºC 35% F N X X = Cl X = Br X N X H Cl Br IC50 (µM) 0.23 0.028 0.028 HO O O H O O Mevastatin MERCK IC50 – 0.030 µM. Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366. The Drug Discovery Incorporation of Additional Functionality HO O O HO O F O TBDMSO TBDMSCl imidazole, DMF 25 ºC 100% F N O O 1) 2 eq NCS or NBS DMF, 0 ºC 100% 2) TBAF, HOAc THF, 25 ºC 35% F N X X = Cl X = Br X N X H Toxicity in early preclinical development! Cl Br IC50 (µM) 0.23 0.028 0.028 HO O O H O O Mevastatin MERCK IC50 – 0.030 µM. Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22. The Drug Discovery O F Br CO2 Et H2N Penta-substituted Pyrroles via [3+2] O O F NH CO2 Et O 1) O Cl F NEt3 , CH2Cl2, 0 ºC 2) NaOH 97% HO2C NHPh Ph O Ac2O, 90 ºC 43% O O NEt3, CH 3CN, 25 ºC 82% N O HO O O CHO F N Ph NHPh O F N Ph O NHPh 1) HCl, EtOH reflux 2) p-TSA, acetone-water reflux 87% O F O N Ph O NHPh Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366. The Drug Discovery HO O F O Penta-substituted Pyrroles via [3+2] HO O O 20 analogs tested N X Y F N NH O (racemic) HO O O H O O IC50 – 0.025 µM Mevastatin MERCK IC50 – 0.030 µM. Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366. The Drug Discovery HO O F O Penta-substituted Pyrroles via [3+2] HO O O HO O F O 20 analogs tested N X Y F N NH O (racemic) resolution N NH O (R, R) HO O O H O O IC50 – 0.025 µM IC50(µM) (R, R) – 0.007 (S, S) – 0.44 Mevastatin MERCK IC50 – 0.030 µM. Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366. The Drug Discovery HO O F 20 analogs tested N X Y F The Birth of LIPITOR®! O HO O F O HO O O N NH O (racemic) resolution N NH O (R, R) Ca2+ HO HO F N NH O CO 2 Atorvastatin calcium LIPITOR ® 2 The Story of LIPITOR® - a Peek into the World of Pharmaceutical Process Chemistry Ca2+ HO HO F N NH O CO 2 Chemical Synthesis Drug Discovery Process Development Biocatalysis 2 Atorvastatin calcium LIPITOR ® The Story of LIPITOR® - a Peek into the World of Pharmaceutical Process Chemistry Ca2+ HO HO F N NH O CO 2 Chemical Synthesis Drug Discovery Process Development Biocatalysis 2 Atorvastatin calcium LIPITOR ® The Process Development Scale-up Issues and Potential Solutions O F N HO2C O O [3+2] Cycloaddition Route 1.6 eq Ph NHPh O F O O Ac 2O, 90 ºC N Ph O NHPh 43% Paal-Knorr Route: Penta-substituted Pyrroles HO HO CO 2R H2N O O CO2 R + F N NH O F O O Failed Model Study H 2N F NH O Paal-Knorr Route: Tetra-substituted Pyrroles Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22. EtO F OEt OEt OEt + O O N The Process Development From Tetra- to Penta-substituted Pyrroles F F O O O NEt3 Bn N Cl OEt 71% H2 N OEt O O O PhCHO, p-TSA toluene, reflux 80% O O O 20 mol% S HO 2) NaOH, CH3 OH, rt 30% p-TSA, toluene reflux CHO F N CONHPh 1) NBS, DMF 0 ºC, 100% 2) n-BuLi, THF, PhNCO, 69% 3) H3O +, 86% EtO F OEt N Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366. Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22. The Process Development CHO F N CONHPh From Tetra- to Penta-substituted Pyrroles 1) O O Ph OH Ph Ph 2 eq. LDA, MgBr2 2) Recrystallization dr 98:2 38% Ph HO F -78 ºC -10 ºC O O Ph Ph OH N CONHPh 1) NaOCH 3 0 ºC 68% 2) OLi -70 ºC 78% HO O F 1) Et3 B. NaBH 4 -78 ºC 2) H 2O2, NaOH 3) toluene, reflux F O Ot Bu HO O N CONHPh CO 2t Bu N CONHPh >99% ee Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366. The Process Development CHO F N CONHPh From Tetra- to Penta-substituted Pyrroles 1) O O Ph OH Ph Ph 2 eq. LDA, MgBr2 2) Recrystallization dr 98:2 38% Ph HO F -78 ºC -10 ºC O O Ph Ph OH N CONHPh 1) NaOCH 3 0 ºC 68% 2) OLi -70 ºC 78% HO O F 1) Et3 B. NaBH 4 -78 ºC 2) H 2O2, NaOH 3) toluene, reflux F O Ot Bu HO O N CONHPh CO 2t Bu N CONHPh >99% ee Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366. The Process Development CHO F N CONHPh From Tetra- to Penta-substituted Pyrroles 1) O O Ph OH Ph Ph 2 eq. LDA, MgBr2 2) Recrystallization dr 98:2 38% Ph HO F -78 ºC -10 ºC O O Ph Ph OH N CONHPh 1) NaOCH 3 0 ºC 68% 2) OLi -70 ºC 78% HO O F 1) Et3 B. NaBH 4 -78 ºC 2) H 2O2, NaOH 3) toluene, reflux 3 columns & 1 crystallization 7% yield Ot Bu O HO F O N CONHPh CO 2t Bu N CONHPh >99% ee Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366. The Process Development A Recap – The Failures O F N HO2C O O [3+2] Cycloaddition Route 1.6 eq Ph NHPh O F O O N Ph O NHPh Ac2O, 90 ºC 43% Paal Knorr Route: Tetra-substituted Pyrroles F OEt O O H2N OEt F EtO OEt HOAc, reflux 71% N Paal Knorr Route: Penta-substituted Pyrroles F HO HO CO 2R H2N O O CO2 R + N NH O F O O Failed Model Study NH O The Process Development Paal Knorr Route: Penta-substituted Pyrroles OEt H2 N + OEt O O 1 eq O OH THF, reflux 43% NH O O F EtO OEt F N NH R2O R2O R1O F NH 2 + O O CO 2R R1O B CO 2R N NH O F NH O A Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22. The Process Development Pfizer’s Commercial Route: Fragment A O NHPh PhCHO β -Alanine, AcOH hexane, heat 85% O O NHPh Ph O F CHO NEt3 , EtOH, heat 80% 20 mol% N HO S Br F O O NH O A Dr. Bruce D. Roth (VP, Global Research & Development, Pfizer), personal communication. Baumann, K. L. et al. Tetrahedron Lett. 1992, 33, 2283-2284. The Process Development Pfizer’s Commercial Route: Fragment B H2 O2, CaCO 3 K2CO3 O HBr, HOAc CH3 OH HO OH H O OH HO CO2 K OH OH Br Br CO2 CH3 HO OH R2O R 1O B NH2 CO2R H 2, Pd/C CH 3COONa/CH3COOH 60% (over 3 steps) NC OTBDMS CO2 CH 3 1) TBDMS-Cl imid., 4-DMAP Br 2) NaCN, DMSO OH CO2 CH 3 Browser, P. L. et al. Tetrahedron Lett. 1992, 33, 2279-2282. Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22. The Process Development Pfizer’s Commercial Route: Fragment B 1) NaOH 2) CDI Mg(O 2CCH2 CO2 t Bu) NC OH O CO 2t Bu NC OTBDMS CO 2CH 3 3 eq LiCH2 CO 2t Bu THF 75% OH NC CO2CH3 3) TBAF, HOAc, THF R2O R 1O B NH2 CO2R Dr. Donald E. Butler (Former Process Development Leader, Pfizer), personal communication. Browser, P. L. et al. Tetrahedron Lett. 1992, 33, 2279-2282. The Process Development Pfizer’s Commercial Route: Fragment B 1) NaOH 2) CDI Mg(O 2CCH2 CO2 t Bu) 3) TBAF, HOAc, THF 1) NaBH4, Et2 BOMe CH3OH, -90 °C 2) (CH 3)2C(OCH 3)2 CH3SO3 H NC OTBDMS CO 2CH 3 OH NC O CO 2t Bu 3 eq LiCH 2CO 2t Bu THF 75% OH NC CO2CH3 Cryogenic reactor employed O H 2N B O CO 2t Bu Raney-Ni, MeOH 50 psi H2 95% NC O O CO 2t Bu >99.5% ee dr 350:1 ee >99.5% liquid N2 liquid N2 Dr. Donald E. Butler (Former Process Development Leader, Pfizer), personal communication. Browser, P. L. et al. Tetrahedron Lett. 1992, 33, 2279-2282. The Process Development F O O + O O Pfizer’s Commercial Route O CO2t Bu 1 eq O OH F N NH O O CO2t Bu NH O NH 2 A B 1:4:1 toluene:heptane:THF reflux 75% • Highly convergent. • Yields >75% at each step. • One low temperature reaction. • One special equipment requirement. • No chromatography. • Scalable to ton quantities. F a) HCl, MeOH b) NaOH c) Ca(OAc)2 HO HO 84% Ca2+ CO2 2 N NH O Atorvastatin calcium LIPITOR ® >99.5% ee Baumann, K. L. et al. Tetrahedron Lett. 1992, 33, 2283-2284. Dr. Donald E. Butler (Former Process Development Leader, Pfizer), personal communication. LIPITOR®: Drug tackled, the struggles remained… “The number of factors, internal and external, that had to come together for the drug to be a success really boggles the mind" – Bruce D. Roth The problems: • By 1987, three statins in market. • A decade since the first statin introduced. • Warner-Lambert was floundering. • Come on, how good could it be? • On the verge of terminating LIPITOR®. And the facts now: • #1 statin in the market. • Top selling drug in history. • 2005: $12 billion sales & used by >45 million people. • “LIPITOR® is on track to have greater benefit for more people than any other drug in the history of the industry in terms of lives improved and saved.” - Nobel Laureate Michael Brown Fortune, 2003, January 20. The Story of LIPITOR® “The story of how Pfizer acquired the rights to an improved statin and turned it into the all-time biggest blockbuster is a tale of hyperaggresive marketing, deft timing, financial power and plain dumb luck!” Fortune, 2003, January 20. The Story of LIPITOR® - a Peek into the World of Pharmaceutical Process Chemistry Ca2+ HO HO F N NH O CO 2 Chemical Synthesis Drug Discovery Process Development Biocatalysis 2 Atorvastatin calcium LIPITOR ® The Story of LIPITOR® - a Peek into the World of Pharmaceutical Process Chemistry Ca2+ HO HO F N NH O CO 2 Chemical Synthesis 220 ton/year market ($500 million) Biocatalysis 2 Drug Discovery Process Development Atorvastatin calcium LIPITOR ® The Story of LIPITOR® - a Peek into the World of Pharmaceutical Process Chemistry Ca2+ HO HO F N NH O CO 2 Chemical Synthesis 220 ton/year market ($500 million) Biocatalysis 2 Drug Discovery Process Development Atorvastatin calcium LIPITOR ® OH NC OH O OR Existing Route & the Need for Improvement H2O 2, CaCO3 K 2CO 3 OH HO CO2K OH HBr, HOAc CH3 OH HO OH H OH Br Br CO 2CH3 O O HO OH H 2, Pd/C CH3COONa/CH 3COOH 60% (over 3 steps) OH NC O CO2 t Bu 3 eq LiCH 2CO 2t Bu THF 75% OH NC CO2 CH 3 NaCN, DMSO OH Br CO2 CH 3 1) NaBH4, Et2BOMe CH3OH, -90 °C 2) (CH 3)2C(OCH 3)2 CH3SO3 H O NC O CO 2t Bu >99.5% ee OH NC OH O OR Existing Route & the Need for Improvement H2O 2, CaCO3 K 2CO 3 OH HO CO2K OH HBr, HOAc CH3 OH HO OH H OH Br Br CO 2CH3 O O HO OH H 2, Pd/C CH3COONa/CH 3COOH 60% (over 3 steps) OH NC O CO2 t Bu 3 eq LiCH 2CO 2t Bu THF 75% OH NC CO2 CH 3 NaCN, DMSO OH Br CO2 CH 3 1) NaBH4, Et2BOMe CH3OH, -90 °C 2) (CH 3)2C(OCH 3)2 CH3SO3 H • SHE issues. • Cryogenic step – special requirements/waste. • With purification – 11 steps. • Month’s supply: $66 LIPITOR® vs $120 ZOCOR®. • But patent expires ~ 2010. O NC O CO 2t Bu >99.5% ee OH NC OH O OR Biocatalytic Routes for the Chiral Side Chain KANEKA OH Cl O OEt ADH GDH O Cl O OEt CIBA O HO OH O OEt Lipase EtO O OR1 O OEt OH OH NC OH O OR DOWPHARMA (DIVERSA) OH NC O OH Nitrilase NC CN CODEXIS OH NC O OEt 1) ADH 2) Dehalogenase Aldolase O Cl O OEt DIVERSA X O OH O X H + 2 O H OH Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27. Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365. OH NC OH O OR Biocatalytic Routes for the Chiral Side Chain KANEKA OH Cl O OEt ADH GDH O Cl O OEt CIBA O HO OH O OEt Lipase EtO O OR1 O OEt OH OH NC OH O OR DOWPHARMA (DIVERSA) OH NC O OH Nitrilase NC CN CODEXIS OH NC O OEt 1) ADH 2) Dehalogenase Aldolase O Cl O OEt DIVERSA X O OH O X H + 2 O H OH Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27. Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365. OH NC OH O OR Biocatalytic Routes for the Chiral Side Chain KANEKA OH Cl O OEt ADH GDH O Cl O OEt CIBA O HO OH O OEt Lipase EtO O OR1 O OEt OH OH NC OH O OR DOWPHARMA (DIVERSA) OH NC O OH Nitrilase NC CN CODEXIS OH NC O OEt 1) ADH 2) Dehalogenase Aldolase O Cl O OEt DIVERSA X O OH O X H + 2 O H OH Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27. Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365. OH NC OH O OR Biocatalytic Routes for the Chiral Side Chain KANEKA OH Cl O OEt ADH GDH O Cl O OEt CIBA O HO OH O OEt Lipase EtO O OR1 O OEt OH OH NC OH O OR DOWPHARMA (DIVERSA) OH NC O OH Nitrilase NC CN CODEXIS OH NC O OEt 1) ADH 2) Dehalogenase Aldolase O Cl O OEt DIVERSA X O OH O X H + 2 O H OH Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27. Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365. OH NC OH O OR Biocatalytic Routes for the Chiral Side Chain KANEKA OH Cl O OEt ADH GDH O Cl O OEt CIBA O HO OH O OEt Lipase EtO O OR1 O OEt OH OH NC OH O OR DOWPHARMA (DIVERSA) OH NC O OH Nitrilase NC CN CODEXIS OH NC O OEt 1) ADH 2) Dehalogenase Aldolase O Cl O OEt DIVERSA X O OH O X H + 2 O H OH Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27. Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365. OH NC OH O OR Biocatalytic Routes for the Chiral Side Chain KANEKA OH Cl O OEt ADH GDH O Cl O OEt CIBA O HO OH O OEt Lipase EtO O OR1 O OEt OH OH NC OH O OR DOWPHARMA (DIVERSA) OH NC O OH Nitrilase NC CN CODEXIS OH NC O OEt 1) ADH 2) Dehalogenase Aldolase O Cl O OEt DIVERSA X O OH O X H + 2 O H OH Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27. Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365. OH NC OH O OR Kaneka’s Route NADP+ OH Cl O O Et D-glucose glucose dehydrogenase recombinant E. coli NADPH carbonyl reductase O D-gluconolactone spontaneous Aqueous phase D-glutonic acid Organic phase (n-butyl acetate) Cl O O Et • >99.9% ee, 89% yield. • Product concentration: 450 g/L. • NADP+ TON: 16,200 mol/mol. • Problematic product separation. Kizaki, N. et al. Appl. Microbiol. Biotechnol. 2001, 55, 590-595. Yasohara, Y. et al. Tetrahedron: Asymmetry 2001, 12, 1713-1718. OH NC OH O OR Daicel’s Route NADP+ OH Cl O O Et formic acid formate dehydrogenase recombinant E. coli NADPH carbonyl reductase O CO2 Cl O O Et • >99% ee. • Product concentration: 50 g/L. • Easy product separation. • Commercially used: >100 ton/year. Rouhi, A. M. Chem. Eng. News 2004, 82, (24), 47-62. OH NC OH O OR Ciba’s Route O O O EtO O O Cl O O OEt O α-chymotrypsin 0.008 mol% 24-30 h 94% 98.2% ee HO O O O O OEt 6 steps 44% overall yield O EtO OH O OEt pyridine 0 °C to rt ~ 12 h 98% O N3 O O OEt The good things • 94% yield, 98.2% ee. • Substrate concentration: 285 g/L. • Kilogram scale. • Cheap & robust biocatalyst. The bad things • Follow up chemistry – long. • Low temperature reactions. • Column chromatography. Öhrlein, R.; Baischf, G. Adv. Synth. Catal. 2003, 345, 713-715. OH NC OH O OR Diversa/Dowpharma’s Route OH NC CN nitrilase pH 7.5 NaH2PO4 27 °C, 16 h 81% 98.8% ee NC OH COOH DeSantis, G. et al. J. Am. Chem. Soc. 2002, 124, 9024-9025. DeSantis, G. et al. J. Am. Chem. Soc. 2003, 125, 11476-11477. OH NC OH O OR Diversa/Dowpharma’s Route OH NC Cl NaCN (aq.) pH=10, 4 h, 50 °C NC 45% nitrilase EtOH, H+ 3 h, 80 °C CO 2Et 62% pH 7.5 NaH 2PO4 27 °C, 16 h 81% 98.8% ee OH NC COOH OH CN Cl O HCN, base (n-Bu)4N+Br12 h, 45 °C 92% OH NC The good things • Cheap starting material. • Efficient enzymatic step: 3 M [substrate] & 619 g L-1 d-1. • Low cost of catalyst by expression in Pseudomonas fluorescens developed by Dow. Scale-up economics good! The bad things • HCN under heated alkaline conditions. • Special equipment for purification. • Some low yield steps. Bergeron, S. et al. Org. Process Res. Dev. 2006, 10, 661-665. OH NC OH O OR Diversa’s Route O DERA 25 °C Cl OH O DERA OH Cl OH O Aldol reaction O Cl + O Wild Type DERA Catalyst Load Product Isolation Reaction Time [chloroacetaldehyde] Volumetric Productivity ee (de) Practical? 20% w/w Difficult 6 days 100 mM 2 g L-1 d-1 DERA = deoxyribose5-phosphate aldolase Cl O OH OH OH Cl OH O OR Unknown NO Gijsen, H. J. M.; Wong, C.-H. J. Am. Chem. Soc. 1994, 116, 8422-8423. Wong, C.-H. et al. J. Am. Chem. Soc. 1995, 117, 3333-3339. OH NC OH O OR Diversa’s Route O DERA 25 °C Cl OH O DERA OH Cl OH O Aldol reaction O Cl + O Wild Type DERA Catalyst Load Product Isolation Reaction Time [chloroacetaldehyde] Volumetric Productivity ee & de Practical? 20% w/w Difficult 6 days 100 mM 2 g L-1 d-1 Unknown NO Improved DERA 2% w/w Simple 3h Fed-batch process 735 g L-1 d-1 ≥ 99.9% YES DERA = deoxyribose5-phosphate aldolase Cl O OH OH O NC O O OMe 23% yield 3 overall steps Greenberg, W. A. et al. Proc. Natl. Acad. Sci. USA 2004, 101, 5788-5793. OH NC OH O OR Codexis Route OH Cl not purified O OEt OH NC O OEt O Cl O OEt KRED/GDH HHDH oil, not purified - HCl + HCl KRED = Ketoreductase GDH = Glucose dehydrogenase HHDH = Halohydrin dehalogenase O O OEt + HCN pK a ~9 HCNaq NaClaq HClaq pKa 99.9% ee 99.8% de OH oil, not purified O ONa OH NC OH O ONa OH Cl OH O ONa O OH O H2N O O OtBu O NC O O OMe Coupling partner for the Paal-Knorr oil, column chromatography 48% (over 4 steps) Greenberg, W. A. et al. Proc. Natl. Acad. Sci. USA 2004, 101, 5788-5793. Diversa’s Fluorogenic Activity Based Screen 1) O TosO HO O O O 4-methylumbelliferone OH O HO K 2CO 3, DMF, 75 °C, 16 h 2) H 2O / 20% CH3CN DOWEX 50WX8-100 2 days 62% O O O OH 5-toluenesulfonyl2-deoxyribose O O O O OH spontaneous + O H DERA O O O OH O H OH O + O O fluorescent OH O Greenberg, W. A. et al. Proc. Natl. Acad. Sci. USA 2004, 101, 5788-5793. OH NC OH O OR Codexis Biocatalyst Improvement O Cl O OEt NADPH Na-gluconate NADP + glucose KRED OH O Cl OEt GDH Parameter Substrate loading Reaction time Enzyme loading Isolated yield Phase separation time Volumetric productivity Initial 80 g/L 24 h 10 g/L ~80% >1 h 80 g/L.day Final 180 g/L 8h 0.7 g/L 97% ~ 1 min 540 g/L.day Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication. OH NC OH O OR Codexis Biocatalyst Improvement OH Cl O OEt - HCl + HCl O O OEt pKa ~9 HCNaq NaClaq HClaq pKa
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