ABCD

The Process Development of New Hepatitis C Protease Inhibitors

Vittorio Farina,

Dept. of Chemical Development,

Boehringer Ingelheim, Ridgefield CT

New Methods in Process Chemistry, Univ. of Zurich, 14 Oct. 2004

ABCDTherapeutic Need

• Over 170 million people are infected with hepatitis C virus (HCV) worldwide (WHO 1997).

• > 70% of infected individuals will develop chronic hepatitis C. Chronic hepatitis C is a progressive condition leading to end-stage liver disease (cirrhosis and hepatocellular carcinoma).

• Available interferon-containing therapies have limited efficacy and have significant side effects.

ABCDHepatitis C - Standard Therapy

• Interferon or (subcutaneous administration).

• Rebetron: Intron A + oral ribavirin, launched in 1998 (USA).

• New standard therapy: 50% sustained response rate(genotype I virus).

• Treatment cost: ~ $17,000/year ($1,440/month) per patient.

ABCDHepatitis C Virus NS3 Protease

•Cleaves at NS3/4A; NS4A/4B; NS4B/5A and NS5A/5B. Good medicinal target.

•In in vitro assays, it was found that protease is inhibited by a hexapeptide (N-terminal) produced by cleavage of substrate from NS5A/5B [DDIVPC].

•This can be the basis for rational drug design.

•Rapid peptide screening shows: DDIVPC IC50 = 71MDdIVPC IC50 = 4 M (replaced L-Asp with D-Asp)

M. Llinàs-Brunet et al. Bioorg. Med. Chem. Lett. 1998, 8, 1713

ABCD

H2NNH

HN

NH

N

HO2C

O

O

O

O

CO2H

O NH

CO2H

SH

PEPTIDE LEAD / DDIVPC / IC50= 71 M

AcHNNH

HN

NH

N

HO2C

O

O

O

OO N

HCO2H

CO2H

O

MODIFIED PEPTIDE IC50 = 0.013 M

Amino Cap

Modified P4

Huge LipophilicPocket at P2

Modified P1

L-Aspto D-Glu

Initial SAR Work

M. Llinàs-Brunet et al. Bioorg. Med. Chem. Lett. 2000, 10, 2267

ABCDFrom Peptides to Peptidomimetics

NHN

OH

O

OO

NH

O

O

O

N

MeO

S

N HN

S

S

S

R

R

BILN 2061Clinical Candidate

Reversible inhibitor of HCV genotypeIa and Ib, Ki = 0.3-0.66 nM.Good PK, metabolic stability.

Structure-Activity Relationships:Angew. Chem. Int. Ed. Engl. 2003, 42, 1356J. Med. Chem. 2004, 47, 123J. Med. Chem. 2004, 47, 1605J. Med. Chem. 2004, 47, 2511Nature 2003, 426, 186

Discovery Synthesis:Org. Lett. 2004, 4, 2901.

ABCDBILN 2061: Clinical Proof of Principle

•Administered in solution (Water, PEG 400, EtOH) at 200 mg bid, for 2 days.

•Viral titer dropped from an average of 106 copies/mL to <1,500 (LOD) in most patients.

•Viral titer rebounded in 6-13 days after treatment was discontinued (as expected).

H. Hinrichsen et al. Gastroenterology, in press.

ABCDBILN 2061: Retrosynthetic Analysis

NHN

OH

O

OO

NH

O

O

O

N

MeO

S

N HN

H2NOH

OO

NHO

O

HO

N

MeO

S

N HN

OH

N

O

OH

OH

S

S

S

R

R

BILN 2061

H

INRF3 (P1) INRF15 (P3) INRF13INRF1 (P2)

C40H50N6O8SMW 774.955 Asymmetric Centers(Z) -Alkene15-Membered Ring

Amenable to convergentassembly

ABCDProgram Goals

• Develop “Expedient Route” in order to supply early batches for toxicologyand phase Ia studies (0.5 – 1.0 Kg).

• Develop cost-effective “Practical Route” and develop it into a process that can form the basis for commercial manufacturing:

- Highly Reproducible.- No chromatographies.- Control of all organic impurities > 0.1 %.- Control of all residual organic volatiles (ICH guidelines).- Control of inorganic impurities (Ru?).- Control of solid-state properties of Active Pharmaceutical Ingredients (API) (polymorph, crystal habit, particle size).

N.G. Anderson “Practical Process Research and Development”, Academic Press,San Diego, 2000.

ABCDThe BILN 2061 Chemistry Team

BIPI Process ChemistryDr. Nathan YeeDr. Yannis HoupisDr. Vittorio FarinaDr. Nizar HaddadDr. Rogelio FrutosDr. Fabrice GallouDr. Xiao-jun WangDr. Xudong WeiDr. Robert SimpsonDr. XuWu FengVictor FuchsYibo XuJonathan TanLi ZhangJinghua XuLana SmithJana VitousEarl Spinelli

BIKG Biberach, ProcessDr. Volker EhrigDr. Rolf HerterDr. Juergen SchnaubeltDr. Rainer Soyka

BI Laval Discovery ChemistryDr. Pierre BeaulieuDr. Anne-Marie FaucherDr. Youla TsantritzosDr. Montse Llinàs-BrunetDr. Murray BaileyDr. Stephen KawaiDr. Bruno SimoneauDr. Jean-Marie FerlandChristian BrochuJean-Simon DuceppeElise GhiroVida GorysTed HalmosMartin PoirierJames GillardBruno HachéColette Boucher

BIKG Ingelheim, ProcessDr. Wolfgang DerschDr. Wendelin SamstagDr. Thomas NicolaDr. Kai Donsbach

ABCDSynthesis of Racemic P1 Unit

O

OEtNPh

CO2EtNPh

PhPh

+CO2EtN

Ph Ph

BrBr

tBuOK, THF

-78 oC

NPh

Ph

[3,3]

CO2Et

HN

PhPh

CO2Et

organic soluble

2:1HCl

CO2EtH2N

E, isolated from aqueous(ca. 30%)

E

Z

Very rapid entry into racemicINRF3 Et ester.• Diastereoselectivity is modest.• Yield must be increased.• Cryogenic conditions must be removed.• Needs resolution.

Discovery Chemistry, Laval

ABCDRoute to P1: Optimization

Imine: Benzaldehyde best (selected).

Counterion effect (tBuO base) on diastereoselectivity: Li > Na > K (d.e.=20:1 with Li, 10:1 with Na, 2:1 with K).

Solvents: Non-polar solvents (PhMe) give best yields.

Esters: Et best, Me 5-8% lower yield.

Concentration: No difference in yield over practicable range (0.28-0.7 M).

Temperature: No difference in range 20-45 0C . No improvements at lower temperature, just prolonged reaction times.

ABCDP1:Scalable Process

Process Chemistry, Ridgefield and BIKG Biberach

CO2MeN

ClH3N CO2Me

PhCHO

+

1 eq HC(OMe)3

PhMe, NMP (8:1)NEt3, 15-25oC 5h

N CO2MePh

aqueous wash, concentrate vol. 60%

BrBr

tBuOLi

MTBE, 15-20 oC

3h

Ph

washed with aqueous

aq. HCl / pH=2

2.5 h , rtCO2MeH2N

NaOH, Boc2O

acetone

CO2MeBocHN

Solvent change to acetone

1.7 M conc.

distil acetone

work-up MTBE

waterRacemate

ABCDP1: Scalable Process

Process Chemistry, Ridgefield and Biberach

CO2MeBocHN

Alcalase, pH 8.15

Na2HPO4

10:1 water / acetone, 40 oC

70 h

0.12 - 0.13M

Racemate

CO2MeBocHN

31-32%in situ yield overall

>98% ee

work-up

evap. acetone,extractionwater/MTBE

1 equiv pTsOH

MIBK

40 oC, 2h.the xtal at 2-3 oC

CO2MeTsOH . H2N

>99.9 % ee

28.7% overall

Features:• No isolations till the end.• Use of NMP in first step to increase solubility and throughput (>10 fold).• Scalable to >100 Kg•Tosylate aids isolation.

ABCDP3 Unit: Scalable Synthesis

ClBr

MgBr ClNHAc

CO2Et

CO2Et

NHAc

CO2H

NH2

CO2H

O

ClOHN

CO2H / HNCy2

O

O

Li2CuCl4ether/THF

1. KOHH2O/EtOH

Acylase 1 (0.5% w/w)pH = 7.6 - 7.75

2. citric acid

+

90% assay

NHAc

EtO2C CO2Et

DMF, K2CO3, Cs2CO3

80 oC

85% assay12

3 (isolated ,cryst. IPA/H2O)

4 (>99% ee)

46%

1.

NaOH, H2O

2. HNCy2, MTBE

85%

• Preparation of 2 using 1,4-dibromobutane gives lower yield in Grignard coupling.• Recycle of (R)-3 possible by racemization with Ac2O at reflux followed by resolutionas above (71% total yield after one recycle).• DCHA salt used for crystallinity. Process smoothly scalable to >100 Kg.

Process Chemistry, Ridgefield

ABCDSynthesis of Quinolone Unit

MeO NH2

AcCl, BCl3, AlCl3, CH2Cl2

-50 to 40 oCMeO NH2 HO NH2

O O

1, 35% 2, 10%

+

• Literature procedure: J. Org. Chem. 1979, 44, 578; J. Am. Chem. Soc. 1978, 100, 4842.• Reported yield: 40%. Obtained on 300g scale: 35%.• Difficulty in scale-up due to further reaction (decomposition) of 1 and emulsions during work-up.• m-Anisidine is a very attractive starting material.

Discovery Chemistry, Laval, and Process Chemistry, Ridgefield

Step 1: Friedel-Crafts Reaction

ABCDAn Alternative: Sugasawa Reaction

OMe

NH2O

MeO NH2

CH3CN, BCl3, AlCl3

MeO NH2

O

+

1 2

PhMe: CH2Cl2 = 1: 1.6

Solvent Ratio 1/2

CH2Cl2 1.4 : 1

PhMe 6 : 1

Highlight of solvent effects:

• Order of addition important: preform anisidine/BCl3 complex, then CH3CN, then AlCl3

• Optimum temperature: 40 oC. Optimum pH for work-up: 3.0• Purified by MTBE slurry at reflux.• Typical isolated yields: 42-47% on multikilo scale.

Discovery Chemistry, Laval, and Process Chemistry, Ridgefield

ABCDSynthesis of Quinolone Unit (cont.)

Discovery Chemistry, Laval, and Process Chemistry, Ridgefield

HN

S

NH2 +HO

O

O

Brdioxane , 100 oC

94%

NS

NHiPr

CO2H

. HBr

Oxalyl Chloride

CH2Cl2, NEt3, 20 oC

NS

NHiPr

COCl

MeO NH2

O

0 - 14 oC

89% isolated

3, crystalline solid

MeO NH

O

O

S

N NHiPr

tBuOK, tBuOH, THF

78-84 oC NH

O

MeO

S

NNHiPr

75% isol.

1 24

5

6 7

ABCDScale-up Problems

Process Chemistry, Ridgefield

MeO NH

O

O

S

N NHiPr

tBuOK, tBuOH, THF

78-84 oC NH

O

MeO

S

NNHiPr

6

7

NH

O

MeO

S

NNHiPr

HN

O

MeO

S

NNHiPr

NH

N

S NHiPr

OMeO

Early lots had only 64-78%purity (HPLC)Major impurities Isolated:

A: formed from CH2Cl2(incomplete removal)

B: Formed at lower temperatures.Ratio (Product/B) is solvent dependent

ABCDSolution to Scale-up Problems

MeO NH

O

O

S

N NHiPr

tBuOK, DME

89-90 oC NH

O

MeO

S

NNHiPr

6

7

• DME best solvent to minimize formation of B (probably minimizes deprotonation of NH-i Pr group).• Dichloromethane “chased” with DME prior to cyclization.• Crude product is purified by slurry in DME / water.• HPLC Assay yield 82%, isolated 77% .• Reproduced on multikilo scale.

Process Chemistry, Ridgefield

ABCDASSEMBLING THE PUZZLE

1. Assemble quickly.2. Find the assembly that minimizes number of “moves” and total cost.