Sarah Burke, Andrew Krasley, Maria Winters

Hepatitis C Virus (HCV):

Serine Protease Inhibitors

Some Statistics

3% of the world population and 1.3% of the United States are infected with HCV.

HCV is the leading cause of liver transplantation.

HCV is 5x more widespread than HIV.

http://www.hivandhepatitis.com/2008icr/aasld/docs/111808_c.html

HCV Basics

HCV has surface proteins which recognize the liver cells it ultimately infects.

HCV has an icosohedral core like HIV.

HCV contains single-stranded positive sense RNA. This means that it is

similar to mRNA and can therefore be immediately translated by the host cell.

http://people.rit.edu/japfaa/infectious.html

How To Contract HCV

http://www.youtube.com/watch?v=tQIUV_cSll0

HCV in the Body

HCV targets hepatocytes (liver cells) because the liver is its main replication site.

HCV is very similar to HIV in that it attaches itself to the hepatocyte and releases its genetic material into the cell. It then “hijacks” the cell and replicates.

Ultimately the liver becomes inflamed and cirrhosis can occur and lead to death.

Effect of HCV on Liver

Uniform and smooth healthy liver.

Liver from individual who died from cirrhosis.

Current Treatments: Interferons

Interferons (IFN) are glycoproteins which stimulate the immune system. IFN-α can be injected into the bloodstream as a treatment of HCV.

More recently PEGylated-IFN-α’s have been used.

PEG gives the IFN- α better PK properties such as solubility and half-life.

Current Treatments: Ribavirin

Ribavirin is a prodrug which resembles RNA nucleotides.

The mechanism of action is unknown, but it somehow interferes with production of viral RNA and prevents HCV from replicating.

Downfalls of Current Therapies

Only effective on 40-50% of patients with the most common form of HCV.

IFNs not directly act on the virus and the virus can quickly replicate to overcome ribavirin.

They cause side effects such as: Fatigue, fever, and headaches Depression and insomnia Cough, skin rash, and chest pain

New Target: NS3 Protease

Hepatitis C RNA encodes for a polyprotein that is 3000 amino acids long.

This is cleaved by viral and host proteases to form structural and nonstructural proteins.

The nonstructural (NS) proteins are further processed by two viral proteases, NS2/3 and NS3.

More About the NS3 Protease

180 amino acid serine protease with a His-57, Asp-81, and Ser-139 catalytic triad.

In order to be actived it must be bound to an NS4a cofactor.

Located at the N-terminal end of the NS3 protein.

Contains a shallow active site on the surface of the enzyme.

http://www.biomedcentral.com/1472-6807/5/1

Two types of NS3 Protease Inhibitors

Form a reversible covalent bond with serine in the active site.

Aim to bind more strongly to active site than substrate through noncovalent interactions.

α-ketoamides Competive, noncovalent inhibitors

NH

O

O

Mechanism of Action of α-ketoamides

These are typically peptides containing an electrophile that form reversible covalent bonds with serine139 in the active site.

Serine Protease Mechanism

Alpha-Ketoamide Inhibitors

http://www.rcsb.org/pdb/explore/jmol.do?structureId=2OC0&bionumber=1

General PK Information

Challenges in Modern Drug Discovery: A Case Study of Boceprevir, an HCV Protease Inhibitor for the Treatment of Hepatitis C Virus Infection

F. George Njoroge et al.

Selecting a Target

NS3-NS4A protease Screened 4 million compounds

No leads generated Structure based design

Look at substrate-enzyme active site interactions

Account for oral bioavailability/PK properties

Exploit H-bonding and hydrophobic interaction

Testing Inhibitors

Cell-based replicon system of HuH-7 cells http://huh7.com/

HCV NS3 protease continuous assay Lack of small animal model

Severe immunodeficiency disease (SICD) mice

Chronically infected chimpanzees Many are compared against human

neutrophil elastase (HNE) Many are compared against CYPs

Creating a Drug

cyclopropylalanine

cyclopropylalanine

cyclohexylglycine

isobutyl carbamate cap

phenylglycine

carboxylic acid

gem-dimethylcyclopropylproline

tert-butylglycine

tert-butyl carbamate

dimethylamide

P2’

substituted hydrazine urea

macrocyclizationtert-alkyl ether linkage

dimethylcyclopropyl-proline

macrocyclization

Boceprevir

cyclobutylalanineurea

α-center

Performance

X-ray of Bioceprevir Bound in Pocket

cyclobutylalanine

dimethylcyclopropylproline

tert-butylglycine

urea capping group

H-bond

Discovery of Narlaprevir (SCH 900518): A Potent, Second Generation HCV NS3 Serine Protease Inhibitor

Ashok Arasappan et al.

Major Goals

Design a second generation HCV NS3 Serine Protease Inhibitor (α-ketoamide class of slow-binding reversible inhibitors)

Eliminate purification issues by developing a molecule that exists as a single isomer

Focus on in vitro potency and PK profileBoceprivir Narlaprivir

Crystal Structure of Bound Boceprivir

SAR Studies Lead to t-butyl Sulfone Initial attempts showed improvement in

replicon potency and rat PK properties Monkey exposure was more of a

challenge t-butyl sulfone moiety showed an EC90 =

100 nM with good bioavailability in monkey plasmaHNHN

Potency ImprovementHN

SO

O

Potency and MonkeyExposure Improvement

SAR Toward new P4 Moiety

EC90 = 350 nM EC90= 200 nM EC90 = 100 nM

Synthesis: Route 1

CO2MeKHMDS/THF

-78oC

O Cl

CO2MeBnOH2C H2, 10% Pd/C THF

MsCl, Et3N, CH2Cl2

CO2MeMsOH2C

NaStBu, EtOH, H2O

100oC

CO2MeLiOH, THF, MeOH, H2O, 80oC

Oxone, MeOH, H2O

CO2H

SO

OS

87

H

O P O

O

N3

Diphenylphosphoryl azide (DPPA)

CO2H

SO

O

8

Et3N, toluene, 100oC

SO

O

9

N

O

N+ N- SO

ON-

O

N+

N

Curtis Rearrangement

S

O

O

N-

O

N+

NN

S

O

O

-N2(g) CO

isocyanate

9

N

SO

OC

O

SO

OHN

HN

O

N

O

HN

O

NHO

O

H2NN

O

HN

O

NH

O

1) Et3N, CH2Cl22) Dess Martin Periodinane

11

10

9

P1

P1

HO

Mixture of P1 Diastereomers

Synthesis: Route 2

N

SO

OC

O

9

H2N

N

OO

OMe

1) DIPEA, CH2Cl2,0oC

2) LiOH, MeOH, THF

SO

OHN

HN

O

N

OO

OH

13

NH2

NH

O

nBu

OH

P'

1) EDCl, HOOBt, NMM

CH2Cl2/DMF -20oC to 0oC

2) Dess Martin Periodinane

CH2Cl2 0oC to RT

SO

OHN

HN

O

N

OO

HN

HN

O

O

P'

15HPLC separation affordedS-P1 diastereomer

Optimized Stereoselective Synthesis of 37

CO2Me CO2MeR1

LiOH, THF, MeOH, H20

80oC

CO2HR1

Oxone, MeOH, H2ONCO

9

1) LDA, TMS-Cl

2) ZnBr2, CH2CL2

SCl

R1 = tBuSCH2 R1 = tBuSCH2

R1 CO2H

4 7 31

R1 = tBuSO2CH2

DPPA, Et3N, Toluene

100oC

8

R1

R1 = tBuSO2CH2

OH

O

NH2

L-ter t-leu

Et3N, toluene, H2O

SO

OHN

HN

O

O

OH

NH O

OMe

EDCl, HOBt, NMM, MeCN

0oC to RT

SO

OHN

HN

O

N

OO

OMe

LiOH, THF, MeOH, H2O

0oC to RT

SO

OHN

HN

O

N

OO

OH

33

35 13

21

EDCl, HOBt, DIPEA, MeCN

0oC to RT

N

HN

O

HN

OH

O

36

NaOCl, TEMPO, KBr, NaHCO3

EtOAc, H2O

SO

O HN H

N

O

N

O

HN

O

HN

O

O

37

SAR Studies Investigating P1 P’ 2o allyl amides showed desirable rat

PK profile Allyl (or small alkyl groups) were not

tolerated well with certain P1 groups Identification of the best P’-P1

combination was desirable.

SO

OHN

HN

O

N

OO

HN

HN

O

O

P'

P1

P’ SAR of Inhibitors of Type 15P’ SAR of Inhibitors of Type 15

SO

OHN

HN

O

N

OO

HN

HN

O

O

P'

15

Table 3 : Discovery of P1 Moiety

Table 3

Synthesis of P1-P’ intermediate

Synthesis of Intermediate Passerini Reaction Mechanism

H2NOH

O

1) LAH, THF, relflux

2) BOC2O, THF

BOCHNOH

NaOCl, TEMPO, LiBr, NaHCO3

EtOAc/H2O

BOCHNH

OAcOH, EtOAc

0oC to RT

NC

BOCHN

OAcHN

O

1) LiOH, THF, H2O

2) 4.0 M HCl/Dioxane

H2N

OHHN

O

21

NC

R H

O

H3C OH

O

H

O

R

HO

CH3O

H

O

R

HO

CH3O

+

H

O

R

HO

CH3O

N

C

O

CH3

R

H

O

N

OH

H3C ON

O

O

H R H

R= BOCHN

Comparison of 37 & 70

Compound AUC (monkey/rat/dog)µM

Bioavailability (monkey/rat/dog)

Boceprivir 0.1/1.5/3.1 4-11%/26%/30%

37 1.1/6.5/0.9 46%/46%/29%

70 -/2.1/0.5 -/21%/16%

SO

OHN

HN

O

N

OO

HN

HN

O

O

37

SO

OHN

HN

O

N

OO

HN

HN

O

O

70

NS3 Protease Bound X-ray Structure Containing 37

Montse Llinas-Brunet* et al

Discovery of a Potent and Selective Noncovalent Linear Inhibitor of the Hepatitis C Virus NS3 Protease

Major Goals of this Research

After the failure of their initial macrocyclic lead compound, these authors set out to find a new series of potent noncovalent inhibitors.

Goal was to develop a linear tripeptide with single digit nM EC50 and IC50 values.

They also wanted to study the PK properties of their compounds, specifically plasma concentrations and oral bioavailability.

Previous Studies: Macrocyclic Inhibitors

Previously reported C-terminus carboxylic acid containing noncovalent inhibitor of NS3 protease.

P1 residue: 1-amino-2-vinylcyclopropylcarboxylic acid (vinyl-ACCA).

Advancement of this compound was discontinued because of cardiotoxicity in monkeys.

Vinyl-ACCA

Previous Studies: Linear Inhibitors

The authors have previously published on linear inhibitors like compound 2 which are easier and less costly to synthesize.

This peptide backbone forms many favorable interactions with the active site.

Synthesis

O NH

OOH

O

O

OO2N

HNNH

O

OMe

O

+TBTU, DIPEA, DCM

RT, 14 hrs

OPNB

NNH

O

OMe

OO N

H

O

O

LiOH, H2O

THF, 0 C, 3hrs

OH

NNH

O

OMe

OO N

H

O

O

DMAP,

DCM, 0 C to RT

SCl

OO

Br

OBrs

NNH

O

OMe

OO N

H

O

O

NMP, 70 C, 7 hrs

Cs2CO3

NMeO

R1

OH

OMe

O

NNH

O

OMe

OO N

H

O

O

NMeO

R1

O

OMe

O

Synthesis

NNH

O

OMe

OO N

H

O

O

NMeO

R1

O

OMe

O

1. NaOH, H2O-THF, 0 C

2. IBCF, TEA, THF, 0 C to RT

NNH

O

OMe

OO N

H

O

O

NMeO

R1

O

O

O

CH2N2, Et2O

0 C to RT

NNH

O

OMe

OO N

H

O

O

NMeO

R1

O

O

N

N

HBr, THF

NNH

O

OMe

OO N

H

O

O

NMeO

R1

O

O

Br

O

Synthesis

NNH

O

OMe

OO N

H

O

O

NMeO

R1

O

O

Br iPrOH, 50 C, 1 hr

H2N R2

S

NNH

O

OMe

OO N

H

O

O

NMeO

R1

O

NNH

O

OH

OO N

H

O

O

NMeO

R1

O

N

S

N

S

R2

R2LiOH, H2O-MeOH-THF

SAR Studies on Capping Group

SAR Studies on Aminothiazol and Quinoline Moieties

PK Studies

NNH

O

OH

OO N

H

O

O

NMeO

H

O

N

SNH

Compound 19

O

NNH

O

OH

OO N

H

O

O

NMeO

H

O

N

SNH

Compound 3

NNH

O

OH

OO N

H

O

O

NMeO

O

N

SNH

Compound 23

O

NNH

O

OH

OO N

H

O

O

NMeO

H

O

N

SNH

Compound 20

O

More SAR Studies

PK Studies

NNH

O

OH

OO N

H

O

O

NMeO

O

N

SNH

Compound 23

O

NNH

O

OH

OO N

H

O

O

NMeO

Br

O

N

SNH

Compound 26

O

NNH

O

OH

OO N

H

O

O

NMeO

Br

O

N

SNH

Compound 28

O

O

NNH

O

OH

OO N

H

O

O

NMeO

Br

O

N

SNH

Compound 29

O