the story of lipitor - seminar · the story of lipitor ... atorvastatin calcium lipitor

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The Story of LIPITOR ® - A Peek into the World of Pharmaceutical Process Chemistry Aman Desai 7 th Feb. 2007 Chemical Synthesis Biocatalysis LIPITOR ® – $12 billion/year sales (2005) Chiral side chain (circled) – 220 ton/year Atorvastatin calcium LIPITOR ® N CO 2 HO HO F O NH 2 Ca 2+

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Page 1: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The Story of LIPITOR® - A Peek into the World

of Pharmaceutical Process Chemistry

Aman Desai

7th Feb. 2007

Chemical Synthesis

Biocatalysis

LIPITOR® – $12 billion/year sales (2005)

Chiral side chain (circled) – 220 ton/year

Atorvastatin calciumLIPITOR®

N

CO2

HO

HO

F

O

NH

2

Ca2+

Page 2: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The Problem – The “Bad” Cholesterol

• Cholesterol – a very important biological molecule.

• Most cholesterol is not dietary, it is synthesized internally.

• Cholesterol is bound to lipoproteins and transported through blood.

• 2 kinds of lipoproteins

www.wikipedia.orgwww.americanheart.org

High Density Lipoprotein (HDL) – “good”

Low Density Lipoprotein (LDL) – “bad”

atherosclerosis

coronary heart disease & other cardiovascular diseases

One of the leading causes of death in the world today!

HOH

H

H

Cholesterol

Page 3: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The Solution – Suppressing Cholesterol Biosynthesis

http://www.med.unibs.it/~marchesi/cholest.html

O

SCoA

OHMG-CoA-Synthetase

SCoA

O

HSCoA

OH

SCoA

O

OO

HMG-CoA

2 NADPH + 2H+

2 NADP+ + HSCoA

HMG-CoA-Reductase

OH OH

OO

Mevalonic Acid

+

RateLimiting

Step

HHO

H

H

Cholesterol

H

>25 steps

Page 4: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The Solution – Suppressing Cholesterol Biosynthesis

OH

SCoA

O

OO

HMG-CoA

2 NADPH + 2H+

2 NADP+ + HSCoA

HMG-CoA-Reductase

OH OH

OO

Mevalonic Acid

RateLimiting

Step

Inhibition

Endo, A. J. Lipid Res. 1992, 33, 1569-1582.Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.

H

O

OHO

O

O

Lovastatin (MEVACOR®)MERCK

9'

2''

HO

O

HO

CO2Na

HO

HO

Pravastatin (PRAVACOL®)BRISTOL - MYERS SQUIBB

9'

H

O

OHO

O

O

Mevastatin

9'

H

O

OHO

O

O

Simvastatin (ZOCOR®)MERCK

2''

Page 5: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Mechanism of Action of Statin Drugs

O

HMG-CoA

2 NADPH + 2H+

2 NADP+ + HSCoA

HMG-CoA-Reductase

OH OH

OO

Mevalonic Acid

RateLimiting

Step

Inhibition

SCoA

HO O

O

H

O

OHO

O

O

Lovastatin (MEVACOR®)MERCK

HO

O

HO

Pravastatin (PRAVACOL®)BRISTOL - MYERS SQUIBB

ONaO

HO

OH

H

O

OHO

O

O

Mevastatin

H

O

OHO

O

O

Simvastatin (ZOCOR®)MERCK

http://www.med.unibs.it/~marchesi/cholest.html

Page 6: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Mechanism of Action of Statin Drugs

Atorvastatin calcium (LIPITOR®)PFIZER

NF

O

NH

2

Ca2+

OH

OO

HO

http://www.med.unibs.it/~marchesi/cholest.html

H

O

OHO

O

O

Lovastatin (MEVACOR®)MERCK

HO

O

HO

Pravastatin (PRAVACOL®)BRISTOL - MYERS SQUIBB

ONaO

HO

OH

H

O

OHO

O

O

Mevastatin

H

O

OHO

O

O

Simvastatin (ZOCOR®)MERCK

Page 7: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Human HMGR with Natural Substrates

Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.

O

HMG-CoA

2 NADPH + 2H+

2 NADP+ + HSCoA

HMG-CoA-Reductase

OH OH

OO

Mevalonic Acid

RateLimiting

Step

Inhibition

SCoA

HO O

O

Page 8: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Human HMGR with Natural Substrates

Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.

O

HMG-CoA

SCoA

HO O

LIPITOR®

NF

O

NH

2

Ca2+

OH

OO

HO

O

Page 9: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Human HMGR with Natural Substrates

Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.

O

HMG-CoAS

OHO

O

CH2

CH2

NC OCH2

CH2

NC OC

H

H

OHHCH3C CH3

CH2

OR

R = 3'-phosphoadenosinediphosphate

LIPITOR®

NF

O

NH

2

Ca2+

OH

OO

HO

pantothenicacid

Page 10: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Human HMGR with LIPITOR®

Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.

LIPITOR®

NF

O

NH

2

Ca2+

OH

OO

HO

Page 11: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Human HMGR with LIPITOR®

Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.

LIPITOR®

NF

O

NH

2

Ca2+

OH

OO

HO

Page 12: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Circa 1995 – The Statin Drugs Market

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.

• Merck = cholesterol control

• At 20 mg, ZOCOR® lowered LDL by -29%.H

O

OHO

O

O

Lovastatin (MEVACOR®)MERCK

H

O

OHO

O

O

Simvastatin (ZOCOR®)MERCK

Page 13: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Circa 1995 – The Statin Drugs Market

• Merck = cholesterol control.

• At 20 mg, ZOCOR® lowered LDL by -29%.H

O

OHO

O

O

Lovastatin (MEVACOR®)MERCK

H

O

OHO

O

O

Simvastatin (ZOCOR®)MERCK

Spring 1997 – Pfizer launches LIPITOR®!

• At 20 mg, LIPITOR® lowered LDL by -46%.

• 2005 – $12 billion sales, used by over 45 million people.

Atorvastatin calciumLIPITOR®

N

CO2

HO

HO

F

O

NH

2

Ca2+

Page 14: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The Story of LIPITOR® - a Peek into the World

of Pharmaceutical Process Chemistry

Chemical Synthesis

Biocatalysis

Drug Discovery

Process Development

Atorvastatin calciumLIPITOR®

N

CO2

HO

HO

F

O

NH

2

Ca2+

Page 15: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The Story of LIPITOR® - a Peek into the World

of Pharmaceutical Process Chemistry

Chemical Synthesis

Biocatalysis

Drug Discovery

Process Development

Atorvastatin calciumLIPITOR®

N

CO2

HO

HO

F

O

NH

2

Ca+2

Page 16: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

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.

H

O

OHO

O

O

Mevastatin

H

O

OHO

O

O

Lovastatin (MEVACOR®)MERCK

HO

O

HO

CO2Na

HO

HO

Pravastatin (PRAVACOL®)BRISTOL - MYERS SQUIBB

?

?

The Decision of the Core TemplateThe Drug Discovery

F

O

OHO

MERCK

Page 17: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The Decision of the Core Template

H

O

OHO

O

O

Mevastatin

The Drug Discovery

Hydrolysis – 100-fold

loss in potency

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

hydrophobic group

template

correct spatial relationship

A Potent HMGR Inhibitor

X

O

OHO

F

O

OHO

MERCK

Page 18: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

The Pyrrole TemplateThe Drug Discovery

N

X

R2R1

O

OHO

Page 19: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The Pyrrole TemplateThe Drug Discovery

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

O+

O

FNEt3

S

N

HO

BnCl

58%

H2NCN

HOAc, reflux

73%N

CN

F

DIBAL-H92%

N

CHO

F

CH3COCH2CO2CH3

NaH, n-BuLiTHF, -78 °C

64%N

F

CO2CH3

O

HO

NF

CO2HHO

HO

1) Bu3B, NaBH4

THF, -78 °C2) NaOH, H2O2

toluene, reflux

52%tr ans:cis 97:3

NF

O

OHO

F O O

20 mol%

Page 20: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

The Pyrrole TemplateThe Drug Discovery

• Over 40 analogs prepared.

• Optimization studies for X, R1 and R2.

O+

O

FNEt3

S

N

HO

BnCl

58%

H2NCN

HOAc, reflux

73%N

CN

F

DIBAL-H92%

N

CHO

F

CH3COCH2CO2CH3

NaH, n-BuLiTHF, -78 °C

64%N

F

CO2CH3

O

HO

NF

CO2HHO

HO

1) Bu3B, NaBH4

THF, -78 °C2) NaOH, H2O2

toluene, reflux

52%tr ans:cis 97:3

N

X

R2R1

O

OHO

F O O

20 mol%

Page 21: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

The Need for Additional FunctionalityThe Drug Discovery

N

O

OHO

F

• IC50

(analog): 0.40 µM.

• Limit of current synthetic route.

H

O

OHO

O

O

MevastatinMERCK

IC50

– 0.030 µM.

Page 22: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The Need for Additional FunctionalityThe Drug Discovery

N

O

OHO

F

Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.

F

O

OHO

MERCK

F

O

OHO

An overlay

N

O

OHO

F

Page 23: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.

Incorporation of Additional FunctionalityThe Drug Discovery

N

O

OHO

F

X X

X = ClX = Br

TBDMSClimidazole, DMF

25 ºC100%

N

O

OTBDMSO

F

1) 2 eq NCS or NBSDMF, 0 ºC100%

2) TBAF, HOAcTHF, 25 ºC35%

0.028Br

0.028Cl

0.23H

IC50

(µM)X

N

O

OHO

F

H

O

OHO

O

O

MevastatinMERCK

IC50

– 0.030 µM.

Page 24: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Incorporation of Additional FunctionalityThe Drug Discovery

N

O

OHO

F

X X

X = ClX = Br

TBDMSClimidazole, DMF

25 ºC100%

N

O

OTBDMSO

F

1) 2 eq NCS or NBSDMF, 0 ºC100%

2) TBAF, HOAcTHF, 25 ºC35%

N

O

OHO

F

Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.

Toxicity in early preclinical development! 0.028Br

0.028Cl

0.23H

IC50

(µM)X

H

O

OHO

O

O

MevastatinMERCK

IC50

– 0.030 µM.

Page 25: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Penta-substituted Pyrroles via [3+2]The Drug Discovery

Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.

Br

CO2Et

F H2N O

O

NEt3, CH3CN, 25 ºC82%

NH

CO2Et

F

OO1) O

Cl

NEt3, CH2Cl2, 0 ºC

2) NaOH

N

HO2C

F

OO

O97%

O

NHPhPh

Ac2O, 90 ºC

NF

PhO

NHPh

OO1) HCl, EtOHreflux

2) p-TSA,acetone-waterreflux

N

CHO

F

PhO

NHPh

43%

87%

NF

PhO

NHPh

O

OHO

Page 26: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Penta-substituted Pyrroles via [3+2]The Drug Discovery

Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.

NF

X Y

O

OHO

20 analogstested

NF

O

OHO

O

NH

(racemic)

IC50

– 0.025 µM

H

O

OHO

O

O

MevastatinMERCK

IC50

– 0.030 µM.

Page 27: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Penta-substituted Pyrroles via [3+2]The Drug Discovery

Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.

NF

O

OHO

O

NH

(racemic)

resolution NF

O

OHO

O

NH

(R, R)

NF

X Y

O

OHO

20 analogstested

H

O

OHO

O

O

MevastatinMERCK

IC50

– 0.030 µM.

IC50

– 0.025 µM

(S, S) – 0.44

(R, R) – 0.007

IC50

(µM)

Page 28: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The Birth of LIPITOR®!The Drug Discovery

NF

O

OHO

O

NH

(racemic)

resolution NF

O

OHO

O

NH

(R, R)

Atorvastatin calciumLIPITOR®

N

CO2

HO

HO

F

O

NH

2

Ca2+

NF

X Y

O

OHO

20 analogstested

Page 29: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The Story of LIPITOR® - a Peek into the World

of Pharmaceutical Process Chemistry

Chemical Synthesis

Biocatalysis

Drug Discovery

Process Development

Atorvastatin calciumLIPITOR®

N

CO2

HO

HO

F

O

NH

2

Ca2+

Page 30: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The Story of LIPITOR® - a Peek into the World

of Pharmaceutical Process Chemistry

Chemical Synthesis

Biocatalysis

Drug Discovery

Process Development

Atorvastatin calciumLIPITOR®

N

CO2

HO

HO

F

O

NH

2

Ca2+

Page 31: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

[3+2] Cycloaddition Route

N

HO2C

F

OO

O

O

NHPhPh1.6 eq

Ac2O, 90 ºC

43%

NF

PhO

NHPh

OO

Paal-Knorr Route: Penta-substituted

Pyrroles

Scale-up Issues and Potential SolutionsThe Process Development

Failed Model Study

Paal-Knorr Route: Tetra-substituted

Pyrroles

Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.

H2N

NF

OEt

OEtOEtEtO

F O O+

F

O

NH

O ONF

CO2RHO

HO

+

H2NCO2R

O O

NH

O

Page 32: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

From Tetra- to Penta-substituted PyrrolesThe Process Development

Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.

NF

OEtEtO1) NBS, DMF 0 ºC, 100%2) n-BuLi, THF, PhNCO, 69%3) H3O+, 86%N

CHO

F

CONHPh

H2N

p-TSA, toluenereflux

OEt

OEt

71%

O PhCHO, p-TSAtoluene, reflux

80%

OO

F

NEt3

S

N

HO

BnCl

30%

2) NaOH, CH3OH, rt

O O O O

F O O

20 mol%

Page 33: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

From Tetra- to Penta-substituted PyrrolesThe Process Development

Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.

38%

-78 ºC

-10 ºC

0 ºC

-78 ºC

-70 ºC

O

O

2 eq. LDA, MgBr2N

F

CONHPh

O

OHO1)

2) Recrystallization

N

CHO

F

CONHPh

1) NaOCH368%

2)

OtBu

OLi

78%

NF

CONHPh

OCO2

tBuHO

1) Et3B. NaBH42) H2O2, NaOH3) toluene, refluxN

F

CONHPh

O

OHO

>99% ee

dr 98:2

Ph

Ph

OHPh Ph

OH

Ph

Ph

Page 34: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

From Tetra- to Penta-substituted PyrrolesThe Process Development

Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.

38%

-78 ºC

-10 ºC

0 ºC

-78 ºC

-70 ºC

O

O

2 eq. LDA, MgBr2N

F

CONHPh

O

OHO1)

2) Recrystallization

N

CHO

F

CONHPh

1) NaOCH368%

2)

OtBu

OLi

78%

NF

CONHPh

OCO2

tBuHO

1) Et3B. NaBH42) H2O2, NaOH3) toluene, refluxN

F

CONHPh

O

OHO

>99% ee

dr 98:2

Ph

Ph

OHPh Ph

OH

Ph

Ph

Page 35: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

From Tetra- to Penta-substituted PyrrolesThe Process Development

Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.

38%

-78 ºC

-10 ºC

0 ºC

-78 ºC

-70 ºC

3 columns & 1 crystallization7% yield

O

O

2 eq. LDA, MgBr2N

F

CONHPh

O

OHO1)

2) Recrystallization

N

CHO

F

CONHPh

1) NaOCH368%

2)

OtBu

OLi

78%

NF

CONHPh

OCO2

tBuHO

1) Et3B. NaBH42) H2O2, NaOH3) toluene, refluxN

F

CONHPh

O

OHO

>99% ee

dr 98:2

Ph

Ph

OHPh Ph

OH

Ph

Ph

Page 36: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

A Recap – The FailuresThe Process Development

N

HO2C

F

OO

O

O

NHPhPh1.6 eq

Ac2O, 90 ºC

43%

NF

PhO

NHPh

OO[3+2] Cycloaddition Route

Paal Knorr Route: Tetra-substituted

Pyrroles

Paal Knorr Route: Penta-substituted

Pyrroles

H2N

HOAc, reflux71%

NF

OEt

OEt OEtEtOF O O

F

O

NH

O ONF

CO2RHO

HO

+

H2NCO2R

O O

NH

O

Failed Model Study

Page 37: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Paal Knorr Route: Penta-substituted PyrrolesThe Process Development

Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.

N

CO2RR2O

R1O

F

O

NH

NH2

CO2RR2O

R1O

F

O

NH

O O

+

B

A

F

O

NH

O O

+

H2N OEt

OEt

NF

THF, reflux

OEtEtO

43%

OH

O1 eq

O

NH

Page 38: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Pfizer’s Commercial Route: Fragment AThe Process Development

Dr. Bruce D. Roth (VP, Global Research & Development, Pfizer), personal communication.Baumann, K. L. et al. Tetrahedron Lett. 1992, 33, 2283-2284.

F

ONH

O O

A

O

NHPh

O ββββ -Alanine, AcOHhexane, heat

85%

O

NHPh

O

F

CHONEt3, EtOH, heat

80%

S

NBr

HO

Ph

PhCHO

20 mol%

Page 39: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Pfizer’s Commercial Route: Fragment BThe Process Development

Browser, P. L. et al. Tetrahedron Lett. 1992, 33, 2279-2282.Roth, B. D. Prog. Med. Chem. 2002, 40, 1-22.

NH2

CO2RR2O

R1O

B

OO

OHHO

HOOH

HH2O2, CaCO3

K2CO3HO CO2K

OH

OH

HBr, HOAcCH3OH

Br CO2CH3

OH

Br

H2, Pd/CCH3COONa/CH3COOH

60%(over 3 steps)

Br CO2CH3

OH1) TBDMS-Cl

imid., 4-DMAP

2) NaCN, DMSONC CO2CH3

OTBDMS

Page 40: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Pfizer’s Commercial Route: Fragment BThe Process Development

Dr. Donald E. Butler (Former Process Development Leader, Pfizer), personal communication.Browser, P. L. et al. Tetrahedron Lett. 1992, 33, 2279-2282.

1) NaOH2) CDI

Mg(O2CCH2CO2tBu)

3) TBAF, HOAc, THFNC CO2CH3

OTBDMSNC

OH O

CO2tBu

NH2

CO2RR2O

R1O

B

NC CO2CH3

OH3 eq LiCH2CO2tBu

THF

75%

Page 41: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Pfizer’s Commercial Route: Fragment BThe Process Development

Dr. Donald E. Butler (Former Process Development Leader, Pfizer), personal communication.Browser, P. L. et al. Tetrahedron Lett. 1992, 33, 2279-2282.

1) NaOH2) CDI

Mg(O2CCH2CO2tBu)

3) TBAF, HOAc, THFNC CO2CH3

OTBDMS

liquid N2

liquid N2

Cryogenic reactor employed

NC

OH O

CO2tBu NC CO2CH3

OH3 eq LiCH2CO2tBu

THF

75%

1) NaBH4, Et2BOMeCH3OH, -90 °C

2) (CH3)2C(OCH3)2CH3SO3H

NC

O O

CO2tBu

Raney-Ni, MeOH50 psi H2

95%

O O

CO2tBu

H2N

>99.5% ee

B

dr 350:1ee >99.5%

Page 42: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Pfizer’s Commercial RouteThe Process Development

Baumann, K. L. et al. Tetrahedron Lett. 1992, 33, 2283-2284.Dr. Donald E. Butler (Former Process Development Leader, Pfizer), personal communication.

NH2

CO2tBu

O

O

F

ONH

O O

+ 1 eq OH

O

1:4:1 toluene:heptane:THFreflux75%

N

CO2tBu

O

O

F

O

NH

a) HCl, MeOHb) NaOHc) Ca(OAc)2

84%

A B

Atorvastatin calciumLIPITOR®

N

CO2

HO

HO

F

O

NH

2

Ca2+

>99.5% ee

• Highly convergent.

• Yields >75% at each step.

• One low temperature reaction.

• One special equipment requirement.

• No chromatography.

• Scalable to ton quantities.

Page 43: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

LIPITOR®: Drug tackled, the struggles remained…

Fortune, 2003, January 20.

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®.

“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

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

Page 44: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

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 plaindumb luck!”

Fortune, 2003, January 20.

Page 45: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The Story of LIPITOR® - a Peek into the World

of Pharmaceutical Process Chemistry

Chemical Synthesis

Biocatalysis

Drug Discovery

Process Development

Atorvastatin calciumLIPITOR®

N

CO2

HO

HO

F

O

NH

2

Ca2+

Page 46: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The Story of LIPITOR® - a Peek into the World

of Pharmaceutical Process Chemistry

Chemical Synthesis

Biocatalysis

Drug Discovery

Process Development

220 ton/year market ($500 million)

Atorvastatin calciumLIPITOR®

N

CO2

HO

HO

F

O

NH

2

Ca2+

Page 47: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The Story of LIPITOR® - a Peek into the World

of Pharmaceutical Process Chemistry

Chemical Synthesis

Biocatalysis

Drug Discovery

Process Development

220 ton/year market ($500 million)

Atorvastatin calciumLIPITOR®

N

CO2

HO

HO

F

O

NH

2

Ca2+

Page 48: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Existing Route & the Need for ImprovementNC

OHOH O

OR

NaCN, DMSO

HBr, HOAcCH3OH

1) NaBH4, Et2BOMeCH3OH, -90 °C

OO

OHHO

HOOH

H H2O2, CaCO3K2CO3 HO CO2K

OH

OH

NC CO2CH3

OH

NC

OH O

CO2tBu

3 eq LiCH2CO2tBu

THF

75%

NC

O O

CO2tBu

>99.5% ee

Br CO2CH3

OH

Br

60%(over 3 steps)

Br CO2CH3

OH

H2, Pd/CCH3COONa/CH3COOH

2) (CH3)2C(OCH3)2CH3SO3H

Page 49: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Existing Route & the Need for ImprovementNC

OHOH O

OR

• SHE issues.

• Cryogenic step – special requirements/waste.

• With purification – 11 steps.

• Month’s supply: $66 LIPITOR® vs $120 ZOCOR®.

• But patent expires ~ 2010.

OO

OHHO

HOOH

H H2O2, CaCO3K2CO3 HO CO2K

OH

OH

NC CO2CH3

OH

NC

OH O

CO2tBu

3 eq LiCH2CO2tBu

THF

75%

NC

O O

CO2tBu

>99.5% ee

Br CO2CH3

OH

Br

60%(over 3 steps)

Br CO2CH3

OH

H2, Pd/CCH3COONa/CH3COOH

2) (CH3)2C(OCH3)2CH3SO3H

1) NaBH4, Et2BOMeCH3OH, -90 °C

NaCN, DMSO

HBr, HOAcCH3OH

Page 50: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Biocatalytic Routes for the Chiral Side Chain

Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.

NC

OHOH O

OR

OEt

OOH

Cl

HO OEt

OO OH

NCOH

OOH

NCOEt

OOH

O OHX

OH

OEt

OO

Cl

ADHGDH

Lipase

EtO OEt

OO OR1

NitrilaseCNNC

OH

1) ADH

2) Dehalo-genase

ClOEt

OO

AldolaseX

H

O+

H

O2

KANEKA

CIBA

DOWPHARMA(DIVERSA)

CODEXIS

DIVERSA

NC

OHOH O

OR

Page 51: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Biocatalytic Routes for the Chiral Side Chain

OEt

OOH

Cl

HO OEt

OO OH

NCOH

OOH

NCOEt

OOH

O OHX

OH

OEt

OO

Cl

ADHGDH

Lipase

EtO OEt

OO OR1

NitrilaseCNNC

OH

1) ADH

2) Dehalo-genase

ClOEt

OO

AldolaseX

H

O+

H

O2

NC

OHOH O

OR

NC

OHOH O

OR

KANEKA

CIBA

DOWPHARMA(DIVERSA)

CODEXIS

DIVERSA

Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.

Page 52: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Biocatalytic Routes for the Chiral Side Chain

OEt

OOHCl

HO OEt

OO OH

NCOH

OOH

NCOEt

OOH

O OHX

OH

OEt

OO

Cl

ADHGDH

Lipase

EtO OEt

OO OR1

NitrilaseCNNC

OH

1) ADH

2) Dehalo-genase

ClOEt

OO

AldolaseX

H

O+

H

O2

NC

OHOH O

OR

NC

OHOH O

OR

KANEKA

CIBA

DOWPHARMA(DIVERSA)

CODEXIS

DIVERSA

Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.

Page 53: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Biocatalytic Routes for the Chiral Side Chain

OEt

OOHCl

HO OEt

OO OH

NCOH

OOH

NCOEt

OOH

O OHX

OH

OEt

OO

Cl

ADHGDH

Lipase

EtO OEt

OO OR1

NitrilaseCNNC

OH

1) ADH

2) Dehalo-genase

ClOEt

OO

AldolaseX

H

O+

H

O2

NC

OHOH O

OR

NC

OHOH O

OR

KANEKA

CIBA

DOWPHARMA(DIVERSA)

CODEXIS

DIVERSA

Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.

Page 54: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Biocatalytic Routes for the Chiral Side Chain

OEt

OOHCl

HO OEt

OO OH

NCOH

OOH

NCOEt

OOH

O OHX

OH

OEt

OO

Cl

ADHGDH

Lipase

EtO OEt

OO OR1

NitrilaseCNNC

OH

1) ADH

2) Dehalo-genase

ClOEt

OO

AldolaseX

H

O+

H

O2

NC

OHOH O

OR

NC

OHOH O

OR

KANEKA

CIBA

DOWPHARMA(DIVERSA)

CODEXIS

DIVERSA

Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.

Page 55: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Biocatalytic Routes for the Chiral Side Chain

OEt

OOHCl

HO OEt

OO OH

NCOH

OOH

NCOEt

OOH

O OHX

OH

OEt

OO

Cl

ADHGDH

Lipase

EtO OEt

OO OR1

NitrilaseCNNC

OH

1) ADH

2) Dehalo-genase

ClOEt

OO

AldolaseX

H

O+

H

O2

NC

OHOH O

OR

NC

OHOH O

OR

KANEKA

CIBA

DOWPHARMA(DIVERSA)

CODEXIS

DIVERSA

Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.

Page 56: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Kaneka’s Route

Kizaki, N. et al. Appl. Microbiol. Biotechnol. 2001, 55, 590-595.Yasohara, Y. et al. Tetrahedron: Asymmetry 2001, 12, 1713-1718.

• >99.9% ee, 89% yield.

• Product concentration: 450 g/L.

• NADP+ TON: 16,200 mol/mol.

• Problematic product separation.

ClO

O

OEt

Cl

OH

O

O

EtNADP+

NADPH

D-glucose

D-gluconolactone

spontaneous

D-glutonic acidAqueous phase Organic phase

(n-butyl acetate)

NC

OHOH O

OR

glucose dehydrogenase

carbonyl reductase

recombinant E. coli

Page 57: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Daicel’s Route

• >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.

Cl

O

O

O

Et

Cl

OH

O

O

EtNADP+

NADPH

formic acid

CO2

NC

OHOH O

OR

formate dehydrogenase

carbonyl reductase

recombinant E. coli

Page 58: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Ciba’s Route

Öhrlein, R.; Baischf, G. Adv. Synth. Catal. 2003, 345, 713-715.

The good things

• 94% yield, 98.2% ee.

• Substrate concentration: 285 g/L.

• Kilogram scale.

• Cheap & robust biocatalyst.

EtO OEt

OO OH Cl

O

O

pyridine0 °C to rt

~ 12 h98%

EtO OEt

OO O

O

αααα-chymotrypsin0.008 mol%

HO OEt

OO O

O

24-30 h

O O

6 steps44% overall

yield

OEt

OO O

N3

94%98.2% ee

NC

OHOH O

OR

The bad things

• Follow up chemistry – long.

• Low temperature reactions.

• Column chromatography.

Page 59: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Diversa/Dowpharma’s Route

DeSantis, G. et al. J. Am. Chem. Soc. 2002, 124, 9024-9025.DeSantis, G. et al. J. Am. Chem. Soc. 2003, 125, 11476-11477.

NC

OHOH O

OR

NC CNOH nitrilase

pH 7.5 NaH2PO427 °C, 16 h

81%98.8% ee

NC COOHOH

Page 60: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Diversa/Dowpharma’s RouteNC

OHOH O

OR

Bergeron, S. et al. Org. Process Res. Dev. 2006, 10, 661-665.

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.

NC CN

OH

nitrilase

ClO

HCN, base(n-Bu)4N+Br-

NaCN (aq.)pH=10, 4 h, 50 °C

pH 7.5 NaH2PO427 °C, 16 h

81%98.8% ee

EtOH, H+

3 h, 80 °CNC CO2Et

OH

62%

92%

NC Cl

OH

45%

NC COOH

OH

12 h, 45 °C

Page 61: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Diversa’s Route

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.

OCl

OH

OH

Cl

OH OH O

OR

DERA = deoxyribose-5-phosphate aldolase

ClO DERA

+O

ClOH O

O

DERACl

OH OH OAldol

reaction25 °C

NC

OHOH O

OR

Unknownee (de)

NOPractical?

2 g L-1 d-1Volumetric Productivity

100 mM[chloroacetaldehyde]

6 daysReaction Time

DifficultProduct Isolation

20% w/wCatalyst Load

Wild Type DERA

Page 62: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Diversa’s Route

OCl

OH

OH

DERA = deoxyribose-5-phosphate aldolase

NC

OHOH O

OR

≥ 99.9%Unknownee & de

YESNOPractical?

735 g L-1 d-12 g L-1 d-1Volumetric Productivity

Fed-batch process

100 mM[chloroacetaldehyde]

3 h6 daysReaction Time

SimpleDifficultProduct Isolation

2% w/w20% w/wCatalyst Load

Improved DERA

Wild Type DERA

Greenberg, W. A. et al. Proc. Natl. Acad. Sci. USA 2004, 101, 5788-5793.

NC

O O O

OMe

23% yield3 overall steps

ClO DERA

+O

ClOH O

O

DERACl

OH OH OAldol

reaction25 °C

Page 63: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Codexis Route

Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.

NC

OHOH O

OR

NCOH O

CO2tBu

oil, not purified

KRED/GDHNC

O O

CO2tBu

first crystalline intermediate

NC

OH OH

CO2tBu

oil, not purified

chemicalchain

elongation

chemicalprotection

Cl

O

OEt

OKRED/GDH

ClOH

OEt

O HHDHNC

OH

OEt

O

not purif ied oil, not purified

KRED = KetoreductaseGDH = Glucose dehydrogenaseHHDH = Halohydrin dehalogenase

- HCl

+ HClOEt

OO

pKa ~9 HCNaq HClaq pKa <0

NaCNaqNaClaqneutral pH

two phase, no solvent

+ HCN

Page 64: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Codexis Route

Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.

NC

OHOH O

OR

NC

OH O

CO2tBu

oil, not purified

KRED/GDHNC

O O

CO2tBu

first crystalline intermediate

NC

OH OH

CO2tBu

oil, not purif ied

chemicalchain

elongation

chemicalprotection

Cl

O

OEt

OKRED/GDH

Cl

OH

OEt

O HHDHNC

OH

OEt

O

not purified oil, not purified

KRED = KetoreductaseGDH = Glucose dehydrogenaseHHDH = Halohydrin dehalogenase

- HCl

+ HClOEt

OO

pKa ~9 HCNaq HClaq pKa <0

NaCNaqNaClaqneutral pH

two phase, no solvent

+ HCN

Page 65: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Codexis Route

Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.

• Excellent selectivities and yields.

• Purification only at the last step – reduced requirements.

• Green and safe.

• Reduced by-products.

• Mild reaction conditions.

• Efficient, scalable and cost effective.

• Commercialized: (1) Arch Pharmalabs, India – Codexis production partner.

(2) Lonza – a Pfizer supplier.

Cl

O

OEt

O

NC

O O

CO2tBu

1) KRED/GDH2) HHDH

3) Chemical chainelongation

4) KRED/GDH5) Protection

"TBIN"First crystalline intermediate

NC

OHOH O

OR

Page 66: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Biocatalytic Routes for the Chiral Side Chain

Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.

NC

OHOH O

OR

OEt

OOH

Cl

HO OEt

OO OH

NCOH

OOH

NCOEt

OOH

O OHX

OH

OEt

OOCl

ADHGDH

Lipase

EtO OEt

OO OR1

NitrilaseCNNC

OH

1) ADH

2) Dehalo-genase

ClOEt

OO

AldolaseX

H

O+

H

O2

KANEKA

CIBA

DOWPHARMA(DIVERSA)

CODEXIS

DIVERSA

NC

OHOH O

OR

Page 67: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Biocatalytic Routes for the Chiral Side Chain

OEt

OOHCl

HO OEt

OO OH

NCOH

OOH

NCOEt

OOH

O OHX

OH

OEt

OO

Cl

ADHGDH

Lipase

EtO OEt

OO OR1

NitrilaseCNNC

OH

1) ADH

2) Dehalo-genase

ClOEt

OO

AldolaseX

H

O+

H

O2

NC

OHOH O

OR

NC

OHOH O

OR

KANEKA

CIBA

DOWPHARMA(DIVERSA)

CODEXIS

DIVERSA

Thayer, A. M. Chem. Eng. News 2006, 84, (33), 26-27.Muller, M. Angew. Chem. Int. Ed. 2005, 44, 362-365.

Page 68: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

An Extremely Attractive Chemo-enzymatic Approach

NH2

CO2tBu

O

O

F

ONH

O O

LIPITOR®

N

CO2

HO

HO

F

O

NH

2

Ca2+

+

CHEMICALSYNTHESISat PFIZER

BIOCATALYSISat CODEXIS

Cl

O

O

O

CHEMICALSYNTHESISat PFIZER

O

NHPh

O

Page 69: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

An Extremely Attractive Chemo-enzymatic Approach

NH2

CO2tBu

O

O

F

O

NH

O O

LIPITOR®

N

CO2

HO

HO

F

O

NH

2

Ca2+

+

CHEMICALSYNTHESISat PFIZER

BIOCATALYSISat CODEXIS

Cl

O

O

O

CHEMICALSYNTHESISat PFIZER

O

NHPh

O

Why not other pharmaceuticals and natural products also?

Page 70: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The Take-Home Message

The Story of LIPITOR®: Drug Discovery & Chemical Development

• The world of process chemistry works on the same basic principles as us here in this building.

• But in many respects, it is a vastly different world, driven by an entirely different set of considerations.

The Story of LIPITOR®: Biocatalytic Routes for the Sidechain

• State-of-art enzymatic transformations have reached an extraordinary level, making them valuable and competitive methods for use in the pharmaceutical industry.

• Biocatalysis has been emerging

Page 71: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The Take-Home Message

The Story of LIPITOR®: Drug Discovery & Chemical Development

• The world of process chemistry works on the same basic principles as us here in this building.

• But in many respects, it is a vastly different world, driven by an entirely different set of considerations.

The Story of LIPITOR®: Biocatalytic Routes for the Sidechain

• State-of-art enzymatic transformations have reached an extraordinary level, making them valuable and competitive methods for use in the pharmaceutical industry.

• Biocatalysis has been emerging clearly emerged as a very attractive tool in a synthetic chemist’s toolkit.

Page 72: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Acknowledgements

Dr. WulffDr. Walker Dr. Borhan Dr. Maleczka

Dr. Bruce Roth (VP, Global R&D, Pfizer)Dr. Donald Butler (Former Process Development Leader, Pfizer

Chief Science Advisor, Austin Chemical Company)Dr. Peter Seufer-Wasserthal (VP, Head of Pharma Services, Codexis)

Dr. Stephen Ritter (Senior Editor, C&EN)

Dr. Christopher Schmid (Lilly) Professor Samir ZardDr. Jos Brands (Merck) Dr. Michael Lipton (SPCorp)

Keith, Ding, Cory, Zhenjie, Gang, Chunrui, Alex, Kostas, Dima, Nilanjana, Anil, Munmun, Li, Yong

Janelle

Aman K., Toyin, Luis, Brian and Alli & Dan

Page 73: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Other Beneficial Effects of Statin Drugs

Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.Rosanoff, A.; Seelig, M. S. Journal of the American College of Nutrition 2004, 23, 501S-505.

• Promotion of new blood vessel growth.

• Stimulation of bone formation.

• Protection against modification of low density lipoproteins.

• Reduction of clot-forming process so important in plaque formation.

• Improvement in endothelial function.

• Reduction of inflammation in blood vessel tissue.

Page 74: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Some Rare Adverse Effects of Statin Drugs

www.lipitor.comRosanoff, A.; Seelig, M. S. Journal of the American College of Nutrition 2004, 23, 501S-505.

• The most common side effects are gas, constipation, stomach pain and heartburn.

• Rare Myalgia (muscle pain).

• Rare Rhabdomyolosis (skeletal muscle failure – loss of kidney function).

possibly life threatening!

Uncommon side reactions occur mainly when statins are co-prescribed with other

interacting drugs.

Page 75: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Statin Drugs

N

OO

HO

HO

H

O

OHO

O

O

Lovastatin (MEVACOR®)MERCK

H

O

OHO

O

O

Simvastatin (ZOCOR®)MERCK

HO

O

HO

C

HO

HO

Pravastatin (PRAVACOL®)BRISTOL - MYERS SQUIBB

Atorvastatin (LIPITOR®)PFIZER

N

CO2

HO

HO

F

O

NH

N

OO

HO

OH

F

Fluvastatin (LESCOL®)NOVARTIS

N

F

OO

HO

OH

O

Cerivastatin (LIPOBAY®)BAYCOL

withdrawn in 2001

N N

OO

HO

OH

N

F

SO

O

Rosuvastatin (CRESTOR®)ASTRAZENECA

F

Pitavastatin (LIVALO®)KOWA

O

O

Page 76: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Chemo-catalytic vs. Bio-catalytic: Wacker Specialty Chemicals

OR

OOH

Cl

Asymmetric Hydrogenation(Ruthenium Catalyst +Diphosphine Ligand)

OR

OO

Cl

Biocatalytic Reduction(Alcohol Dehydrogenase

Enzyme)

OR

OO

Cl

Standard StandardEquipment

2 L< 100 gWaste/kg (product)

1 (dilute conditions)3Relative Throughput

None – ambient temp. & pressure

100 °C, MeOH solvent, handling H2

SHE issues

< $100< $85Cost/kg (product)

Multi-tonMulti-tonScale

>99.9 (97)98 (95)%ee (yield)

Bio-catalysisChemo-catalysis

Requirement of price & purity will dictate the route.

Rouhi, A. M. Chem. Eng. News 2004, 82, (24), 47-62.

Page 77: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Sliskovic, D. R. et al. J. Med. Chem. 1991, 34, 367-373.Sliskovic, D. R. et al. J. Med. Chem. 1990, 33, 31-38.

Other Templates AttemptedThe Drug Discovery

0.018F3 (N-oxide)

0.013OCH32

0.032Cl1

IC50 (µM)R1Compound

NNR

F

O

OHO

H

R = Ph (IC50 = 0.035 µM)

N

R1

F

O O

OH

H

mevastatin: IC50 = 0.030 µM

H

O

OHO

O

O

Page 78: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Human HMGR with LIPITOR

Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.

The specificity & tight binding achieved by:

• hydrogen bonds

• ionic interactions

• polar interactions

LIPITOR®

NF

O

NH

2

Ca2+

OH

OO

HO

Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.

Page 79: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Human HMGR with Statin Drugs

Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160-1164.

Page 80: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The solution – suppressing cholesterol biosynthesis

www.cellml.org

Cholesterol Biosynthesis

Acetyl-CoA

Acetoacetyl-CoA

Hydroxymethylglutaryl-CoA(HMG-CoA)

Mevalonate

5-phosphomevalonate

5-pyrophosphomevalonate

Dimethylallylpyrophosphate

Geranylpyrophosphate

Farnesylpyrophosphate

Squalene

Squalene-2,3-epoxideLanosterol18 reactions

DesmosterolCholesterol

Page 81: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

The solution – suppressing cholesterol biosynthesis

Steinberg, D.; Avigan, J. J. Biol. Chem. 1960, 235, 3127-3129.

Cholesterol Biosynthesis

OH

Cl

CH3

OH2N

Triparanol (MER-29)

DesmosterolCholesterol

Severe side effects – failed!

Acetyl-CoA

Acetoacetyl-CoA

Hydroxymethylglutaryl-CoA(HMG-CoA)

Mevalonate

5-phosphomevalonate

5-pyrophosphomevalonate

Dimethylallylpyrophosphate

Geranylpyrophosphate

Farnesylpyrophosphate

Squalene

Squalene-2,3-epoxideLanosterol18 reactions

Page 82: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

Structure-Activity Relationship StudiesThe Drug Discovery

F

O

OHO

1MERCK

N

X

R2R1

O

OHO

Page 83: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Structure-Activity Relationship StudiesThe Drug Discovery

N

X

O

OHO

F

X =

-CH2CH2CH2

-CH2CH2

N

O

OHO

F

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

Page 84: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Structure-Activity Relationship StudiesThe Drug Discovery

N

X

O

OHO

FNR1

O

OHO

R1 =

Ph

4-PhC6H4

4-MeOC6H4

4-ClC6H4

4-HOC6H4

4-FC6H4

3-F3CC6H4

3-MeOC6H4

3-HOC6H4

2-MeOC6H4

2-HOC6H4

2-naphthyl

1-naphthyl

cyclohexyl

N

O

OHO

F

N R2

O

OHO

R1

< 5.9 Å

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

Page 85: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Structure-Activity Relationship StudiesThe Drug Discovery

N

X

O

OHO

FNR1

O

OHO

N R2

O

OHO

F

R2 =

-CH3

N

O

OHO

F

N R2

O

OHO

R1

< 5.9 Å

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

Page 86: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Structure-Activity Relationship StudiesThe Drug Discovery

N

X

O

OHO

FNR1

O

OHO

N R2

O

OHO

F

R2 =

-CH3

N

O

OHO

F

N R2

O

OHO

R1

< 5.9 Å < 3.3 Å

< 10.6 Å

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

Page 87: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Structure-Activity Relationship StudiesThe Drug Discovery

N

X

O

OHO

FNR1

O

OHO

N R2

O

OHO

F

N R2

O

OHO

R1

< 5.9 Å < 3.3 Å

< 10.6 Å

3-FC6H4

2-FC6H4

2,4-F2C6H3

2-MeOC6H4

2-ClC6H4

2,6-(MeO)2C6H3

2,5-Me2C6H3

2-iPrOC6H4

O

N

O

OHO

FNR1

O

OHO

Roth, B. D. et al. J. Med. Chem. 1990, 33, 21-31.

Page 88: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Pentasubstituted Pyrroles via [3+2]The Drug Discovery

Roth, B. D. et al. J. Med. Chem. 1991, 34, 357-366.

“Munchnone”

N

OHOOC

F

OO

CO2Et

Ac2O, reflux

N

CO2Et

OO

F

N

OHOOC

OO

CO2Et

Ac2O, reflux

N

EtO2C

OO

F

N

R2

O

R3R1

HOOC

N

R2

R1

R4 R5

R3

Ac2Oreflux N

OO

R2

R3

R1 R4 R5

Regiocontrol:

Page 89: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Ciba’s Route

EtO OEt

OO OH Cl

OO

pyridine0 °C to rt

~ 12 h98%

EtO OEt

OO O

O

α-chymotrypsin0.008 mol%

HO OEt

OO O

O

24-30 h

(COCl)2, 0 °C100%

Cl OEt

OO O

Oethylene, AlCl3

DCE, 0 °C

89%OEt

OO O

O

Cl

pig-leveresterase

pH=776%OEt

OO OH

Cl

1) BEt3, NaBH4-78 °C, 91%

2) dimethoxypropaneH+, 98%

3) NaN3, DMF94% y, 98% ee, 97% de

OEt

OO O

N3

+

F O O

Ph CONHPh

O OH

PBu370%

N

CO2EtO

O

F

Ph CONHPh

O O

OO

Öhrlein, R.; Baischf, G. Adv. Synth. Catal. 2003, 345, 713-715.

NC

OHOH O

OR

Page 90: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Diversa/Dowpharma’s Route

DeSantis, G. et al. J. Am. Chem. Soc. 2002, 124, 9024-9025.DeSantis, G. et al. J. Am. Chem. Soc. 2003, 125, 11476-11477.

Screen genomic libraries

Most effective WT nitrilase

0.24 M [substrate] – 98% y, 95% ee

3 M [substrate] – 88% ee

NC

OHOH O

OR

NC CN

OH nitrilaseNC COOH

OH

pH 7.5 NaH2PO427 °C, 16h

81% y, 98.8% ee

Page 91: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Diversa/Dowpharma’s Route

DeSantis, G. et al. J. Am. Chem. Soc. 2002, 124, 9024-9025.DeSantis, G. et al. J. Am. Chem. Soc. 2003, 125, 11476-11477.

Screen genomic libraries

Most effective WT nitrilase

0.24 M [substrate] – 98% y, 95% ee

3 M [substrate] – 88% ee

NC

OHOH O

OR

NC CN

OH nitrilaseNC COOH

OH

pH 7.5 NaH2PO427 °C, 16h

81% y, 98.8% ee

Genetical Engineering gave another library

Best mutant

2.25 M [substrate] – 98% ee in 15 h

Page 92: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Diversa’s GSSM™: Gene Site Saturation Mutagenesis

1. Target Protein forImprovement

2. Change Amino Acid(s)

A new gene variant library isborn containing genes with everysingle site variation in sequence.

Identify protein for optimization through extensive screening of gene libraries.

Evolve the gene encoding the protein by systematically changing each amino acid in the sequence to every other possible amino acid.

3. Complete Variant Library

4. Select Variants

Through screening, identify the variants that demonstrate improved characteristics.

5. Combine Mutations

Tests all potential combinations of the single amino acid changes that demonstrated improved characteristics and select the optimal combination.

6. Generate Optimized Protein

www.diversa.com

Page 93: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Diversa’s High Throughput Screen for Improved Selectivity

DeSantis, G. et al. J. Am. Chem. Soc. 2003, 125, 11476-11477.

NC C15N

OH

15N-(R)-12

R-selectivenitrilase

S-selectivenitrilase

NC COOHOH

(R)-13m/z = 129

HOOC C15NOH

15N-(S)-13

m/z = 130

Page 94: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Diversa’s Route

Greenberg, W. A. et al. Proc. Natl. Acad. Sci. USA 2004, 101, 5788-5793.

OCl

OH

OH

DERA = deoxyribose-5-phosphate aldolase

Cl

O DERA+

OCl

OH O

O

DERACl

OH OH OAldol

reaction25 °C

NaOCl, HOAcH2O, 25 °C, 3 h

45% (over2 steps)

OCl

OH

O

white crystals>99.9% ee99.8% de

a) NaCN, DMF, 5% H2O40 °C, 16 h

b) H2SO4c) (MeO)2CMe2d) TMSCHN2

oil, not purified

Cl

OH OH O

ONa

OH O

ONa

O NC

OH OH O

ONa

NC

O O O

OMe

oil, column chromatography48% (over 4 steps)

O O O

OtBuH2NCoupling partner

for the Paal-Knorr

NC

OHOH O

OR

Page 95: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Diversa’s Fluorogenic Activity Based Screen

Greenberg, W. A. et al. Proc. Natl. Acad. Sci. USA 2004, 101, 5788-5793.

OTosO

HO

O

OO OH

K2CO3, DMF, 75 °C, 16 h

OO O O

1)

2) H2O / 20% CH3CNDOWEX 50WX8-1002 days

62%

OH

HO

OO O H

O

OH

OHDERA

spontaneous

OO OH

+

O

O

fluorescent

OO O

OH

O+

H

O

5-toluenesulfonyl-2-deoxyribose

4-methylumbelliferone

Page 96: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Codexis Biocatalyst Improvement

Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.

540 g/L.day80 g/L.dayVolumetric productivity

~ 1 min>1 hPhase separation time

97%~80%Isolated yield

0.7 g/L10 g/LEnzyme loading

8 h24 hReaction time

180 g/L80 g/LSubstrate loading

FinalInitialParameter

Cl

O

OEt

OKRED

Cl

OH

OEt

O

NADPH NADP+

GDHglucoseNa-gluconate

NC

OHOH O

OR

Page 97: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Codexis Biocatalyst Improvement

Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.

92%~60%Isolated yield

1.2 g/L130 g/LEnzyme loading

5 h72 hReaction time

140 g/L20 g/LSubstrate loading

FinalInitialParameter

Cl

OH

OEt

O

NC

OH

OEt

O

- HCl

+ HClOEt

OO

pKa ~9 HCNaq HClaq pKa <0

NaCNaqNaClaqneutral pH

two phase, no solvent

+ HCN

halohydrindehalogenase

NC

OHOH O

OR

Page 98: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Codexis Biocatalyst Improvement

Dr. Peter Seufer-Wasserthal (VP, Head of Codexis Pharma Services), personal communication.

faciledifficultWork-up

100%100%Diastereomeric excess

99.3%87%Conversion

22 h65 hReaction time

300 g/L120 g/LSubstrate loading

FinalInitialParameter

KRED

NADPH NADP+

GDHglucoseNa-gluconate

NC

OH O

CO2tBu NC

OH OH

CO2tBu

NC

OHOH O

OR

Page 99: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Stetter Reaction Mechanism

Hermann, S. Angew. Chem., Int. Ed. Engl. 1976, 15, 639-647.

S

N

HO

BnCl

+ NEt3S

N

HO

BnCl HNEt3

O

H

Hp.t. S

N

HO

Bn

R

OH

R

R1

Op.t.

S

N

HO

BnO

RR1

OHR

OR1

OH

R

O

R1

O

Irreversible addition to the Michael Acceptor

R

O

Hp.t.

S

N

HO

BnO

R

OH

R

R

O

R

OH

Reversible benzoin formation

Page 100: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Syn-selective reduction of β-hydroxy ketones

Narasaka, K.; Pai, H. C. Chem. Lett. 1980, 1415-1418.

R1 R2

OH O

Bu3B, THF

OR2

BO

R1

HBu

Bu

favored

OR2

OB

Bu H

R1

Bu

attack fromless hindered

side

cis - diol+

O

BO

Bu

Bu

R2

R1

favored

R1 R2

O OB

BuBu

OB

O

Bu

Bu

H

H

R1

R2

unfavored

Htop bottom

Page 101: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Mechanism for H2O2 Oxidation of Ascorbic Acid

Isbell, H. S.; Frush, H. L. Carbohyd. Res. 1979, 72, 301-304.

OO

OHHO

HOOH

H-O-O-H OO

OHO

HOOH

OH+

H2O

H2O

OH

OO

OO

HOOH

H-O-O-H

OH

OO

O

HOOH

OOHOH

OO

HOOH

OHHO

OO

OH

OO

HOOH

OHHO

OO

OH

H2O(CO2H)2

HOOH

CO2H

HO

Page 102: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Mechanism for Hydrolysis by Chymotrypsin/PLE

Asp102

O

O

HN

N

His57

HO Ser195

R

O

O

H

N

Ser195

HN

Gly193

Asp102

O

O

HN

N

His57

O Ser195

R

O

O

H

N

Ser195

HN

Gly193

HH2O

OH

Asp102

O

O

HN

N

His57

O Ser195

R

O

H

N

Ser195

HN

Gly193

H

O

H

O

O

HN

N

His57

O Ser195

R

O

H

N

Ser195

HN

Gly193

Asp102

HO

H

O

O

HN

N

His57

HO Ser195

R

O

HO

H

N

Ser195

HN

Gly193

Asp102

Page 103: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

Mechanism for Hydrolysis by Nitrilase

Faber, K. Biotransformations in Organic Chemistry, Springer-Verlag, 2004.

R C N + Enz SH RNH

S EnzH2O

R

OH

NH2

SEnz

NH3

RO

S EnzH2O

ROH

O

+ Enz SH

Page 104: The Story of Lipitor - Seminar · The Story of LIPITOR ... Atorvastatin calcium LIPITOR

DERA Mechanism

Bioorg. Med. Chem. 11, 2003, 43-52.

H

O

H

O

OPO3

OH

+H

O

OPO3

OH

OHDERA

NH3

Lys 201

O

CH3

HO H

Lys 167

HNH

O

O

Asp102 NH3

Lys 201

H

CH3

HO H

Lys 167

O

OH

Asp102

HN

HO

NH2

Lys 201

H

CH3

HO H

Lys 167

O

OH

Asp102

N

NH3

Lys 201

H

CH2

HO H

Lys 167

O

O

Asp102

HN

O

OPO3

OH

NH3

Lys 201

H

Lys 167

O

O

Asp102

N OPO3OHH

OH

H2O hydrophilicsite

Ser238, 239Gly205

Thr170, Lys172

hydrophobicsite