Lipitor Development

  • Published on
    10-Oct-2014

  • View
    126

  • Download
    3

Transcript

The Story of LIPITOR - A Peek into the World of Pharmaceutical Process ChemistryCa2+ HO HO F N NH OChemical SynthesisCO 2LIPITOR $12 billion/year sales (2005) Chiral side chain (circled) 220 ton/yearAtorvastatin calcium LIPITOR 2BiocatalysisAman Desai 7th Feb. 2007The Problem The Bad Cholesterol Cholesterol a very important biological molecule. Most cholesterol is not dietary, it is synthesized internally.H HOH HCholesterol 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.orgThe Solution Suppressing Cholesterol BiosynthesisO 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 SCoAOH H H HO Cholesterol >25 steps H H O OOHMevalonic Acidhttp://www.med.unibs.it/~marchesi/cholest.htmlThe Solution Suppressing Cholesterol BiosynthesisHO O O 9' H9'O OO 2''HO O O HOOHO SCoAOO HMG-CoA2 NADPH + 2H+ HMG-CoA-Reductase 2 NADP + + HSCoA Inhibition Rate Limiting StepMevastatinLovastatin (MEVACOR) MERCKHO O O 9' HO HOCO 2NaOHO O OOH2''OHHOHOOPravastatin (PRAVACOL) BRISTOL - MYERS SQUIBBSimvastatin (ZOCOR) MERCKMevalonic AcidEndo, A. J. Lipid Res. 1992, 33, 1569-1582. Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.Mechanism of Action of Statin DrugsHO O O H O OO O H HO O OHO O O SCoA HMG-CoA 2 NADPH + 2H+ Rate Limiting Step OMevastatinOLovastatin (MEVACOR ) MERCKONaHMG-CoA-Reductase 2 NADP + + HSCoA InhibitionHO O O HHO O O H OOOH OHOHOOHOPravastatin (PRAVACOL) BRISTOL - MYERS SQUIBBSimvastatin (ZOCOR ) MERCKMevalonic Acidhttp://www.med.unibs.it/~marchesi/cholest.htmlMechanism of Action of Statin DrugsHO O O H O OO O H HO O OHOOOCa 2+ 2OHMevastatinOLovastatin (MEVACOR) MERCKONaF N NH OHO O O HHO O O H OOOHAtorvastatin calcium (LIPITOR) PFIZERHOPravastatin (PRAVACOL) BRISTOL - MYERS SQUIBBSimvastatin (ZOCOR) MERCKhttp://www.med.unibs.it/~marchesi/cholest.htmlHuman HMGR with Natural SubstratesHO O O SCoA HMG-CoA 2 NADPH + 2H+ HMG-CoA-Reductase 2 NADP + + HSCoA Inhibition Rate Limiting Step OOHOHOOMevalonic AcidIstvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.Human HMGR with Natural SubstratesHO O O SCoA HMG-CoA OHOOOCa 2+ 2OH F N NH O LIPITOR Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.Human HMGR with Natural SubstratesHOO OOHMG-CoAS 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 OCa 2+O2OH FN NH O LIPITORIstvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.Human HMGR with LIPITORHOOOCa 2+ 2OH FN NH OLIPITORIstvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.Human HMGR with LIPITORHOOOCa 2+ 2OH FN NH OLIPITORIstvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.Circa 1995 The Statin Drugs MarketHO O O H O OO O H HO O O Merck = cholesterol control At 20 mg, ZOCOR lowered LDL by -29%.Lovastatin (MEVACOR) MERCKSimvastatin (ZOCOR) MERCKThayer, 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 MarketHO O O H O OO O H HO O O Merck = cholesterol control. At 20 mg, ZOCOR lowered LDL by -29%.Lovastatin (MEVACOR) MERCKSimvastatin (ZOCOR) MERCKSpring 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 OAtorvastatin calcium LIPITOR2The Story of LIPITOR - a Peek into the World of Pharmaceutical Process ChemistryCa2+ HO HO F N NH OCO 2Chemical SynthesisDrug Discovery Process DevelopmentBiocatalysis2Atorvastatin calcium LIPITOR The Story of LIPITOR - a Peek into the World of Pharmaceutical Process ChemistryCa+2 HO HO F N NH OCO 2Chemical SynthesisDrug Discovery Process DevelopmentBiocatalysis2Atorvastatin calcium LIPITOR The Drug DiscoveryHO O O H O OThe Decision of the Core TemplateHO O O OOMevastatinLovastatin (MEVACOR) MERCKCO 2NaHO O F O?HHOHO?O O HHOPravastatin (PRAVACOL) BRISTOL - MYERS SQUIBBMERCKWillard, 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 DiscoveryHO O O H O OThe Decision of the Core Templatecorrect spatial relationshipHO O OMevastatinhydrophobic groupXtemplateHO O F OHydrolysis 100-fold loss in potencyA Potent HMGR InhibitorMERCKRoth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.The Drug DiscoveryThe Pyrrole TemplateHO O X NOR1R2Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.The Drug DiscoveryF NEt3 + O HO 58% O F Bn N Cl SThe Pyrrole TemplateH2 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 N1) Bu3B, NaBH4 THF, -78 C 2) NaOH, H2O 2FNHO HO FCO 2HHO O toluene, reflux 52% tr ans:cis 97:3 FONNRoth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.The Drug DiscoveryF NEt3 + O HO 58% O F Bn N Cl SThe Pyrrole TemplateH2 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 N1) Bu 3B, NaBH4 THF, -78 C 2) NaOH, H2O 2FNHO HO FCO 2HHO O toluene, reflux 52% tr ans:cis 97:3 R1 X NO Over 40 analogs prepared. Optimization studies for X, R1 and R2.NR2Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.The Drug DiscoveryThe Need for Additional FunctionalityHO O O OHO O FOO HN IC50 (analog): 0.40 M. Limit of current synthetic route.Mevastatin MERCKIC50 0.030 M.Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.The Drug DiscoveryThe Need for Additional FunctionalityHO O O FHO O FONAn overlayHO O F F O OMERCKNRoth, B. D. Prog. Med. Chem. 2002, 40, 1-22.The Drug DiscoveryIncorporation of Additional FunctionalityHO O OHO O FOTBDMSO TBDMSCl imidazole, DMF 25 C 100% F N OO 1) 2 eq NCS or NBS DMF, 0 C 100% 2) TBAF, HOAc THF, 25 C 35%FN X X = Cl X = Br XNX H Cl BrIC50 (M) 0.23 0.028 0.028HO O O H OOMevastatin MERCKIC50 0.030 M.Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.The Drug DiscoveryIncorporation of Additional FunctionalityHO O OHO O FOTBDMSO TBDMSCl imidazole, DMF 25 C 100% F N OO 1) 2 eq NCS or NBS DMF, 0 C 100% 2) TBAF, HOAc THF, 25 C 35%FN X X = Cl X = Br XNX H Toxicity in early preclinical development! Cl BrIC50 (M) 0.23 0.028 0.028HO O O H OOMevastatin MERCKIC50 0.030 M.Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.The Drug DiscoveryO F Br CO2 Et H2NPenta-substituted Pyrroles via [3+2]O O F NH CO2 Et O 1) O Cl F NEt3 , CH2Cl2, 0 C 2) NaOH 97% HO2CNHPh Ph O Ac2O, 90 C 43%OONEt3, CH 3CN, 25 C 82%N OHO OOCHO F N Ph NHPh OFN Ph O NHPh1) HCl, EtOH reflux 2) p-TSA, acetone-water reflux 87%O FON Ph O NHPhRoth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.The Drug DiscoveryHO O F OPenta-substituted Pyrroles via [3+2]HO O O20 analogs tested N X YFN NH O (racemic)HO O O H OOIC50 0.025 MMevastatin MERCKIC50 0.030 M.Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.The Drug DiscoveryHO O F OPenta-substituted Pyrroles via [3+2]HO O OHO O F O20 analogs tested N X YFN NH O (racemic)resolutionN NH O (R, R)HO O O H OOIC50 0.025 MIC50(M) (R, R) 0.007 (S, S) 0.44Mevastatin MERCKIC50 0.030 M.Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.The Drug DiscoveryHO O F 20 analogs tested N X YFThe Birth of LIPITOR!OHO OFOHO OON NH O (racemic)resolutionN NH O (R, R)Ca2+HO HO F N NH OCO 2Atorvastatin calcium LIPITOR 2The Story of LIPITOR - a Peek into the World of Pharmaceutical Process ChemistryCa2+ HO HO F N NH OCO 2Chemical SynthesisDrug Discovery Process DevelopmentBiocatalysis2Atorvastatin calcium LIPITOR The Story of LIPITOR - a Peek into the World of Pharmaceutical Process ChemistryCa2+ HO HO F N NH OCO 2Chemical SynthesisDrug Discovery Process DevelopmentBiocatalysis2Atorvastatin calcium LIPITOR The Process DevelopmentScale-up Issues and Potential SolutionsO F N HO2C O O[3+2] Cycloaddition Route1.6 eq PhNHPh OFOOAc 2O, 90 CN Ph O NHPh43%Paal-Knorr Route: Penta-substituted PyrrolesHO HOCO 2R H2NOO CO2 R +F N NH OFO OFailed Model StudyH 2N FNH OPaal-Knorr Route: Tetra-substituted PyrrolesRoth, B. D. Prog. Med. Chem. 2002, 40, 1-22.EtO FOEtOEt OEt + O ONThe Process DevelopmentFrom Tetra- to Penta-substituted PyrrolesF F O O O NEt3 Bn N Cl OEt 71% H2 N OEt O OOPhCHO, p-TSA toluene, reflux 80%OOO20 mol%S HO 2) NaOH, CH3 OH, rt 30%p-TSA, toluene refluxCHO F N CONHPh 1) NBS, DMF 0 C, 100% 2) n-BuLi, THF, PhNCO, 69% 3) H3O +, 86%EtO FOEtNRoth, B. D. et al. J. Med. Chem. 1991, 34, 357-366. Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.The Process DevelopmentCHO F N CONHPhFrom Tetra- to Penta-substituted Pyrroles1) O O Ph OH Ph Ph 2 eq. LDA, MgBr2 2) Recrystallization dr 98:238%Ph HO F-78 C -10 CO O PhPh OHN CONHPh1) NaOCH 3 0 C 68% 2) OLi -70 C 78% HO O F 1) Et3 B. NaBH 4 -78 C 2) H 2O2, NaOH 3) toluene, reflux F O Ot BuHO O N CONHPhCO 2t BuN CONHPh >99% eeRoth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.The Process DevelopmentCHO F N CONHPhFrom Tetra- to Penta-substituted Pyrroles1) O O Ph OH Ph Ph 2 eq. LDA, MgBr2 2) Recrystallization dr 98:238%Ph HO F-78 C -10 CO O PhPh OHN CONHPh1) NaOCH 3 0 C 68% 2) OLi -70 C 78% HO O F 1) Et3 B. NaBH 4 -78 C 2) H 2O2, NaOH 3) toluene, reflux F O Ot BuHO O N CONHPhCO 2t BuN CONHPh >99% eeRoth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.The Process DevelopmentCHO F N CONHPhFrom Tetra- to Penta-substituted Pyrroles1) O O Ph OH Ph Ph 2 eq. LDA, MgBr2 2) Recrystallization dr 98:238%Ph HO F-78 C -10 CO O PhPh OHN CONHPh1) NaOCH 3 0 C 68% 2) OLi -70 C 78% HO O F 1) Et3 B. NaBH 4 -78 C 2) H 2O2, NaOH 3) toluene, reflux3 columns & 1 crystallization 7% yieldOt BuOHO F O N CONHPhCO 2t BuN CONHPh >99% eeRoth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.The Process DevelopmentA Recap The FailuresO F N HO2C O O[3+2] Cycloaddition Route1.6 eq PhNHPh OFOON Ph O NHPhAc2O, 90 C43%Paal Knorr Route: Tetra-substituted PyrrolesFOEt O O H2N OEt FEtOOEtHOAc, reflux 71%NPaal Knorr Route: Penta-substituted PyrrolesFHO HOCO 2R H2NOO CO2 R +N NH OFO OFailed Model StudyNH OThe Process DevelopmentPaal Knorr Route: Penta-substituted PyrrolesOEt H2 N + OEt O O 1 eq O OH THF, reflux 43% NH O O F EtO OEtFN NHR2O R2O R1O F NH 2 + O O CO 2R R1O BCO 2RN NH OFNH O ARoth, B. D. Prog. Med. Chem. 2002, 40, 1-22.The Process DevelopmentPfizers Commercial Route: Fragment AO NHPh PhCHO -Alanine, AcOH hexane, heat 85% O O NHPh PhOF CHO NEt3 , EtOH, heat 80%20 mol%N HO SBrFO ONH O ADr. Bruce D. Roth (VP, Global Research & Development, Pfizer), personal communication. Baumann, K. L. et al. Tetrahedron Lett. 1992, 33, 2283-2284.The Process DevelopmentPfizers Commercial Route: Fragment BH2 O2, CaCO 3 K2CO3 O HBr, HOAc CH3 OHHOOH HOOH HO CO2 K OHOH Br Br CO2 CH3HOOHR2O R 1O B NH2CO2RH 2, Pd/C CH 3COONa/CH3COOH60% (over 3 steps)NCOTBDMS CO2 CH 31) TBDMS-Cl imid., 4-DMAP Br 2) NaCN, DMSOOH CO2 CH 3Browser, P. L. et al. Tetrahedron Lett. 1992, 33, 2279-2282. Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.The Process DevelopmentPfizers Commercial Route: Fragment B1) NaOH 2) CDI Mg(O 2CCH2 CO2 t Bu)NC OH O CO 2t BuNCOTBDMS CO 2CH 33 eq LiCH2 CO 2t Bu THF 75%OH NC CO2CH33) TBAF, HOAc, THFR2O R 1O B NH2CO2RDr. Donald E. Butler (Former Process Development Leader, Pfizer), personal communication. Browser, P. L. et al. Tetrahedron Lett. 1992, 33, 2279-2282.The Process DevelopmentPfizers Commercial Route: Fragment B1) NaOH 2) CDI Mg(O 2CCH2 CO2 t Bu) 3) TBAF, HOAc, THF1) NaBH4, Et2 BOMe CH3OH, -90 C 2) (CH 3)2C(OCH 3)2 CH3SO3 HNCOTBDMS CO 2CH 3OH NCO CO 2t Bu3 eq LiCH 2CO 2t Bu THF 75%OH NC CO2CH3Cryogenic reactor employedO H 2N BO CO 2t BuRaney-Ni, MeOH 50 psi H2 95% NCOO CO 2t Bu>99.5% eedr 350:1 ee >99.5%liquid N2 liquid N2Dr. Donald E. Butler (Former Process Development Leader, Pfizer), personal communication. Browser, P. L. et al. Tetrahedron Lett. 1992, 33, 2279-2282.The Process DevelopmentF O O + O OPfizers Commercial RouteO CO2t Bu 1 eq O OH F N NH O O CO2t BuNH ONH 2 A B1:4:1 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.Fa) HCl, MeOH b) NaOH c) Ca(OAc)2HO HO84%Ca2+ CO2 2N NH OAtorvastatin calcium LIPITOR >99.5% eeBaumann, 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 remainedThe 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 BrownFortune, 2003, January 20.The Story of LIPITORThe 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 ChemistryCa2+ HO HO F N NH OCO 2Chemical SynthesisDrug Discovery Process DevelopmentBiocatalysis2Atorvastatin calcium LIPITOR The Story of LIPITOR - a Peek into the World of Pharmaceutical Process ChemistryCa2+ HO HO F N NH OCO 2Chemical Synthesis 220 ton/year market ($500 million) Biocatalysis2Drug Discovery Process DevelopmentAtorvastatin calcium LIPITOR The Story of LIPITOR - a Peek into the World of Pharmaceutical Process ChemistryCa2+ HO HO F N NH OCO 2Chemical Synthesis 220 ton/year market ($500 million) Biocatalysis2Drug Discovery Process DevelopmentAtorvastatin calcium LIPITOR OH NCOH O ORExisting Route & the Need for ImprovementH2O 2, CaCO3 K 2CO 3 OH HO CO2K OHHBr, HOAc CH3 OHHOOH HOH Br Br CO 2CH3OOHOOH H 2, Pd/C CH3COONa/CH 3COOH 60% (over 3 steps)OH NCO CO2 t Bu3 eq LiCH 2CO 2t Bu THF 75%OH NC CO2 CH 3NaCN, DMSOOH Br CO2 CH 31) NaBH4, Et2BOMe CH3OH, -90 C2) (CH 3)2C(OCH 3)2 CH3SO3 HO NCO CO 2t Bu>99.5% eeOH NCOH O ORExisting Route & the Need for ImprovementH2O 2, CaCO3 K 2CO 3 OH HO CO2K OHHBr, HOAc CH3 OHHOOH HOH Br Br CO 2CH3O OHOOH H 2, Pd/C CH3COONa/CH 3COOH 60% (over 3 steps)OH NCO CO2 t Bu3 eq LiCH 2CO 2t Bu THF 75%OH NC CO2 CH 3NaCN, DMSOOH Br CO2 CH 31) NaBH4, Et2BOMe CH3OH, -90 C2) (CH 3)2C(OCH 3)2 CH3SO3 H SHE issues. Cryogenic step special requirements/waste. With purification 11 steps. Months supply: $66 LIPITOR vs $120 ZOCOR. But patent expires ~ 2010.O NCO CO 2t Bu>99.5% eeOH NCOH O ORBiocatalytic Routes for the Chiral Side ChainKANEKAOH ClO OEtADH GDHO ClO OEtCIBAO HOOHO OEtLipase EtOOOR1 O OEt OHOH NCOH O ORDOWPHARMA (DIVERSA)OH NCO OHNitrilase NCCNCODEXISOH NCO OEt1) ADH 2) Dehalogenase AldolaseO ClO OEtDIVERSAXOOHO X H+ 2O HOHThayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27. Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.OH NCOH O ORBiocatalytic Routes for the Chiral Side ChainKANEKAOH ClO OEtADH GDHO ClO OEtCIBAO HOOHO OEtLipase EtOOOR1 O OEt OHOH NCOH O ORDOWPHARMA (DIVERSA)OH NCO OHNitrilase NCCNCODEXISOH NCO OEt1) ADH 2) Dehalogenase AldolaseO ClO OEtDIVERSAXOOHO X H+ 2O HOHThayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27. Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.OH NCOH O ORBiocatalytic Routes for the Chiral Side ChainKANEKAOH ClO OEtADH GDHO ClO OEtCIBAO HOOHO OEtLipase EtOOOR1 O OEt OHOH NCOH O ORDOWPHARMA (DIVERSA)OH NCO OHNitrilase NCCNCODEXISOH NCO OEt1) ADH 2) Dehalogenase AldolaseO ClO OEtDIVERSAXOOHO X H+ 2O HOHThayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27. Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.OH NCOH O ORBiocatalytic Routes for the Chiral Side ChainKANEKAOH ClO OEtADH GDHO ClO OEtCIBAO HOOHO OEtLipase EtOOOR1 O OEt OHOH NCOH O ORDOWPHARMA (DIVERSA)OH NCO OHNitrilase NCCNCODEXISOH NCO OEt1) ADH 2) Dehalogenase AldolaseO ClO OEtDIVERSAXOOHO X H+ 2O HOHThayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27. Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.OH NCOH O ORBiocatalytic Routes for the Chiral Side ChainKANEKAOH ClO OEtADH GDHO ClO OEtCIBAO HOOHO OEtLipase EtOOOR1 O OEt OHOH NCOH O ORDOWPHARMA (DIVERSA)OH NCO OHNitrilase NCCNCODEXISOH NCO OEt1) ADH 2) Dehalogenase AldolaseO ClO OEtDIVERSAXOOHO X H+ 2O HOHThayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27. Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.OH NCOH O ORBiocatalytic Routes for the Chiral Side ChainKANEKAOH ClO OEtADH GDHO ClO OEtCIBAO HOOHO OEtLipase EtOOOR1 O OEt OHOH NCOH O ORDOWPHARMA (DIVERSA)OH NCO OHNitrilase NCCNCODEXISOH NCO OEt1) ADH 2) Dehalogenase AldolaseO ClO OEtDIVERSAXOOHO X H+ 2O HOHThayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27. Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.OH NCOH O ORKanekas RouteNADP+ OH Cl O O EtD-glucoseglucose dehydrogenase recombinant E. coliNADPHcarbonyl reductaseO D-gluconolactone spontaneous Aqueous phase D-glutonic acid Organic phase (n-butyl acetate) ClO 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 NCOH O ORDaicels RouteNADP+ OH Cl O O Etformic acidformate dehydrogenaserecombinant E. coliNADPHcarbonyl reductaseO CO2 ClO 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 NCOH O ORCibas RouteO O O EtO O O Cl O O OEt O -chymotrypsin 0.008 mol% 24-30 h 94% 98.2% ee HO O O O O OEt 6 steps 44% overall yieldO EtOOHO OEtpyridine 0 C to rt ~ 12 h 98%O N3OO OEtThe 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.hrlein, R.; Baischf, G. Adv. Synth. Catal. 2003, 345, 713-715.OH NCOH O ORDiversa/Dowpharmas RouteOH NC CNnitrilase pH 7.5 NaH2PO4 27 C, 16 h 81% 98.8% ee NCOH COOHDeSantis, G. et al. J. Am. Chem. Soc. 2002, 124, 9024-9025. DeSantis, G. et al. J. Am. Chem. Soc. 2003, 125, 11476-11477.OH NCOH O ORDiversa/Dowpharmas RouteOH 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 CNClOHCN, base (n-Bu)4N+Br12 h, 45 C 92%OH NCThe 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 NCOH O ORDiversas RouteO DERA 25 C Cl OH O DERA OH Cl OH O Aldol reactionO Cl+OWild 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-1DERA = deoxyribose5-phosphate aldolaseCl O OHOHOH ClOHO ORUnknown NOGijsen, 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 NCOH O ORDiversas RouteO DERA 25 C Cl OH O DERA OH Cl OH O Aldol reactionO Cl+OWild 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 NOImproved DERA 2% w/w Simple 3h Fed-batch process 735 g L-1 d-1 99.9% YESDERA = deoxyribose5-phosphate aldolaseCl O OHOHO NCOO OMe23% yield 3 overall stepsGreenberg, W. A. et al. Proc. Natl. Acad. Sci. USA 2004, 101, 5788-5793.OH NCOH O ORCodexis RouteOH Cl not purified O OEt OH NC O OEtO ClO OEtKRED/GDHHHDHoil, not purified- HCl + HClKRED = Ketoreductase GDH = Glucose dehydrogenase HHDH = Halohydrin dehalogenaseOO OEt+ HCNpK a ~9 HCNaq NaClaqHClaq pKa 99.9% ee 99.8% deOHoil, not purifiedO ONa OH NC OH O ONaOH ClOHO ONaOOHO H2NOO OtBuO NCOO OMeCoupling partner for the Paal-Knorroil, column chromatography 48% (over 4 steps)Greenberg, W. A. et al. Proc. Natl. Acad. Sci. USA 2004, 101, 5788-5793.Diversas Fluorogenic Activity Based Screen1) O TosO HO O O O4-methylumbelliferoneOH O HOK 2CO 3, DMF, 75 C, 16 h 2) H 2O / 20% CH3CN DOWEX 50WX8-100 2 days 62% O O O OH5-toluenesulfonyl2-deoxyriboseO O O O OH spontaneous+O HDERA O O OOHO HOHO + O O fluorescent OH OGreenberg, W. A. et al. Proc. Natl. Acad. Sci. USA 2004, 101, 5788-5793.OH NCOH O ORCodexis Biocatalyst ImprovementO Cl O OEt NADPH Na-gluconate NADP + glucose KRED OH O Cl OEtGDHParameter Substrate loading Reaction time Enzyme loading Isolated yield Phase separation time Volumetric productivityInitial 80 g/L 24 h 10 g/L ~80% >1 h 80 g/L.dayFinal 180 g/L 8h 0.7 g/L 97% ~ 1 min 540 g/L.dayDr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.OH NCOH O ORCodexis Biocatalyst ImprovementOH Cl O OEt - HCl + HCl O O OEt pKa ~9 HCNaq NaClaq HClaq pKa

Recommended

View more >