A Tale of Two HCV Inhibitors: Discovery of Victrelis™ (Boceprevir)

and the Thiazolide RM5038

J. Edward Semple, Ph.D.

Romark Laboratories, L.C., Tampa, FL 33606

&

Corvas International, Inc., La Jolla, CA 92121

Originally presented June, 2011,

with minor updates June, 2016

TIZ X-ray structure:

J. N. Lisgarten

Dept of Crystallography

Birkbeck College,

London, UK

2.03 Å

2

What is Hepatitis C Virus?

Infectious disease affecting the liver, often asymptomatic chronic infection can progress to fibrosis and cirrhosis, apparent after many

years

liver failure, liver cancer, esophageal and gastric varices (extremely dilated

sub-mucosal veins)

leading cause of liver transplantation.

5x more widespread than HIV

Small (60 nm), enveloped, single-stranded, positive sense RNA

virus: only known member of the hepacivirus genus in the family Flaviviridae

has an icosohedral core like HIV

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

11 major genotypes: G1-G11, each with 1-4 subtypes (e.g.G1a,

G1b, G1c, etc.)

EM image, Scale = 60 nmhttp://www.cdc.gov/hepatitis/HCV/index.htm

http://www.hepatitis-central.com/hcv/genotype/explained.html

3

Global HCV Infection: Prevalence

~ 270-300 million people worldwide are infected

~ 4-5 million in US alone

http://www.cdc.gov/hepatitis/HCV/index.htm

4

Transmission: Blood-to-Blood Contact

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

http://thehepatitisblog.com/?p=753

5

HCV: Structure & Replication

HCV Replication

Video link: http://www.youtube.com/watch?v=eI5t0PbgnBk

http://www.rmgh.net/wiki/index.php?title=HCV

http://wiki.verkata.com/en/wiki/Hepatitis_C_virus

IRES (ribosome)-> RNA translation -> HCV polyprotein

-> 10 proteins -> replication complex -> (+)RNA

6

Treatment: Current Standard of Care (SOC)

Dual therapy with peginterferon (IFN) and ribavirin

(RBV): Weekly s.c. injections of PEG-IFN-a-2a (Pegasyss®) or -a2

(Pegintron®) in combination with RBV b.i.d:

treatment of 24 weeks for G2 &G3; 48 weeks for G1 patients

regimen poorly tolerated due to significant side effects

side effects make it difficult to complete treatment (low compliance)

~40% Cure rate in US (naïve, G1a/1b), ~40-50% (other genotypes)

Cure = sustained viral response (SVR) = HCV RNA <10 IU/ml @ 6

months post treatment

rIFNa (h) a/b domain

O

NN

N NH2

O

OHOH

HO

RBV

A J. Sadler, B.R.G. Williams Nature Rev. Immunology 2008 8, 559-568.

7

HCV Drugs: Clinical Factors & Endpoints

Patient populations: naïve, non-naïve, non-

responders, relapsers; low to very high viral loads

Goal to produce >2–6 log10 reduction in plasma HCV

RNA in patients (SVR) RVR: undetectable at week 4

ETR: undetectable at end of Tx

SVR: undetectable (<10 IU/ml) @ 6 months post treatment

= long-term efficacy ~ cure

Issues: emergence of resistant HCV strains combination therapy (3 drugs) reduces risk

8

Current HCV Therapies: Challenges & Opportunities

Both IFN and RBV are indirect antivirals- do not target a

specific HCV protein or RNA element: low efficacy

severe side effects

many patients forego treatment- compliance issues

emergence of drug resistant HCV strains

Large unmet medical need and high market demand for

new therapies: market potential of new protease inhibitors alone is projected at 3-5 billion dollars

annually

triple combination therapy > SVR and < development of resistance

impetus for development of specifically targeted antiviral therapies for HCV (STAT-C)

ultimate goal to develop monotherapy…feasible?

M.G. Ghany, D.B. Strader, D.L..Thomas, L.B. Seeff Hepatology 2009, 49, 1335–1374.

Y.J. Li Annu Rev Immunol. 2005, 23, 275–306.

RBV Review: Hartwell D, Jones J, Baxter L, Shepherd J. Health Technology Assessment 2011, 17, 1-210.

9

HCV NS3/4A Protease

Trypsin/chymotrypsin-like serine protease- heterodimer consisting of a catalytic subunit (the N-terminal one-third of NS3

protein) and an activating cofactor (NS4A protein)

cleavage of HCV polyprotein at the NS3/NS4A, NS4A/NS4B, NS4B/NS5A,

and NS5A/NS5B sites by the viral NS3 protease releases functional viral

proteins essential for viral replication -> “replication complex”

considered one of the most attractive targets for developing novel anti-HCV

therapies

proof-of-concept demonstrated for several classes of small mol inhibitors in

human clinical trials

Lin, C. HCV NS3-4A Serine Protease.In Hepatitis C Viruses

Genomes and Molecular Biology; Tan S.L., Ed.; Horizon

Bioscience: Norfolk (UK), 2006, Chapter 6.

Link: http://www.ncbi.nlm.nih.gov/books/NBK1623/

10

X-Ray Structure of HCV NS3/4A Serine Protease

C-terminal sub-domain

of the NS3 protease

N-terminal sub-domain

of the NS3 protease

NS4A β-strand

Asp81

Cys145

His57

Asp81Ser139

H20

Cys97

Cys99

Zn

Bartenschlager, R. J. Vir. Hepatitis 1999, 6, 165-181.

Lin, C. HCV NS3-4A Serine Protease.In Hepatitis C Viruses Genomes and Molecular

Biology; Tan S.L., Ed.; Horizon Bioscience: Norfolk (UK), 2006, Chapter 6.

Link: http://www.ncbi.nlm.nih.gov/books/NBK1623/

Heterodimer -> catalytic subunit and activating cofactor

AS region

11

Current Classes of HCV Inhibitors

Entry Inhibitors

Helicase Inhibitors

Internal Ribosome Entry Site (IRES) Inhibitors

RdRp (NS5B) Polymerase Inhibitors-NI, NNI

NS5A Inhibitors

NS3/4A Protease Inhibitors

Cyclophilin Inhibitors

Novel Immune-based Inhibitors

Thiazolide Small Molecule Modulators

Glucosidase Inhibitors

12

Current Clinical HCV NS3/4A Protease Inhibitors

DanoprevirBI 201335

MK-5172

Narlaprevir® (SCH900518) VictrelisTM (boceprevir, SCH503034) IncivekTM (telaprevir, VX-950)

TMC-435350

Vaniprevir™ (MK-7009)

13

Current Clinical RdRp (NS5B) Inhibitors

R-7128 Valopicitabine

GL-60667

Filibuvir

Nesbuvir (HCV-796) ANA-598

Sovaldi

(Sofosbuvir)

14

Current Clinical HCV Inhibitors: Other Classes

Alisporivir® (Debio 025)

Elvitegravir® (GS-9190)

BMS-790052

Alinia® (Nitazoxanide)

15

Romark Laboratories, L.C.: Thiazolides

TIZ X-ray structure:

J. N. Lisgarten

Dept of Crystallography

Birkbeck College,

London, UK

2.03 Å

Note: Thiazolides may appear to be Mickey Mouse

molecules but they are nearly as potent in cell culture

as other classes of more structurally complex HCV inhibitors!

1616

Thiazolide Class: Antiviral Activity of Nitazoxanide Confirmed in Human Studies

Virus Test System Stage

Rotavirus Humans Phase 2 (200 patients)

Hepatitis B Humans Phase 1b (12 patients)

Hepatitis C G4 Humans Phase 2 (251 patients)

Hepatitis C G1 Humans Phase 2 (179 patients)

Influenza A Humans Phase 2 (440 patients)

Alinia® (Nitazoxanide)

Broad spectrum antibacterial, antiparasitic, antiviral

FDA-approved for treatment of C. parvum and G. lamblia

OBA (h), F ~35%, improved with food.

17

Structural Evolution of the Thiazolides

Broad spectrum: antiviral,

antiparasitic & antibacterial

NTZ/TIZ

(R = Ac, H)

TIZ Prodrugs

(dual therapy)Salicylanilides

Current Targets

• Antiviral-selective

• R6, R7 ≠ NO2

2nd Generation

Thiazolide

Prodrugs

Thiazolides as Novel Antiviral Agents: I. Inhibition of Hepatitis B Virus Replication. A. V. Stachulski,

B. E. Korba, J. E. Semple, J. F. Rossignol, et al. J. Med. Chem. 2011, 54, 4119-4132.

Thiazolides as Novel Antiviral Agents. 2. Inhibition of Hepatitis C Virus Replication. A. V. Stachulski,

B. E. Korba, J. E. Semple, J. F. Rossignol, et al. J. Med. Chem. 2011, 54, 8670-8680.

18

RM5038HCV (Huh7.5,G1b): EC50= 0.23 M, SI = 19

RSV (A2): EC50= 1.04 M, SI = 22

CcoV (A72): EC50= 2.02 M, SI >83.3

RM4829HBV (VIR): EC50= 0.22 M, SI >137

HBV (RI): EC50= 1.20 M, SI >25

RM5021Influenza A (PR8, MDCK):

EC50= 0.028 M, SI >5000

Parainfluenza (SV, 37RC):

EC50= 0.085 M, SI >1670

RM4804Parainfluenza (SV, 37RC):

EC50= 0.97 M, SI >167

RM4860Rotavirus (SA11):

EC50= 0.026 M, SI >5000

RM5034HSV-1(Hep-2):

EC50= 0.091 M, SI >1667

Broad Spectrum Antivirals: Recent Thiazolide Leads

19

SAR of Core Thiazolides in HCV Replicon Assay (G1b)

SAR of 28 prototypes indicates tight, specific structural requirements for high potency

and selectivity

pKa, steric environment & polarizability impact potency: EWG favored

TIZ is most acidic analog- amide moiety pKa 5.7 will be fully deprotonated

at physiological pH (cf. TIZ vs. isomer RM5048)

Several non-nitro thiazolide and salicylanilide acetates are reproducibly active and

demonstrate good SI

Thiazolides as Novel Antiviral Agents. 2. Inhibition of Hepatitis C Virus Replication. A. V. Stachulski, B. E. Korba, J. E. Semple, J. F.

Rossignol, et al. J. Med. Chem. 2011, 54, 8670-8680.

Substitution leads to

< potency, selectivity

OH ~OAc > NH2, NHAc >> OMe

When R6 = H, EC50 (M):

NO2 (0.15) > Cl (0.23) > SO2Me (1.5) >

CN (3.7) > Br (4.9) > 14 other FGs

(>10)

When R7 = H, EC50 (M):

CH2SO2Me (0.37) > SOMe (2.2) > Ph

(3.5) > NO2 and 5 other FGs (>10)

20

Thiazolide Core Pharmacophore & MOA

NH

L2

OH OEWG/HBA

Minimal pharmacophore Metal complex at active or allosteric binding site

vs. classical protein backbone H-bonds and/or

water-mediated H-bonds?

NH

L2

O OEWG/HBA

M

N

M

Human VAP-B Is Involved in Hepatitis C Virus Replication

Through Interaction with NS5A and NS5B.

Y. Matsuura et al. J. Virol. 2005, 79, 13473

HCV Replication Complex

& Host Protein VAP-B

21

HCV Lead RM5038: Preclinical Data

N

SNH

OO

Cl

O

RM50382-(5-Chlorothiazol-2-ylcarbamoyl)phenyl acetate

C12H9ClN2O3S

Mol. Wt. 296.73

LogD (octanol/PBS, pH7.4) = 2.66

cLogP = 2.08

MR = 71.3 cm3/ mol

TPSA = 67.8 Å2

Nrot = 3

Solubility (Cerep):

PBS (pH7.4) = 8.5 mg/L

SGF = 36.3 mg/L

SIF = 17.4 mg/L

Cerep ADME-Tox and in vitro pharmacology panels

Patent Family:PCT WO 2006/031566 A2, March 23, 2006

JP2008512474 T, April 24, 2008

US 2006/0089396 A1, April 27, 2006

US 2008/0096941 A1, April 24, 2008

US 7645783 B2, January 12, 2010.

Bacteriology:Inactive (MIC > 64 g/ml) against 110 anaerobes.

Against 53 aerobes, modest inhibitory activity against only a few

MRSA strains, where MIC’s ranged from 4 to >64 g/ml.

Parasitology:Against Cryptosporidium parvum, IC50 = 1 g/ml, with very low

cytotoxicity (10% of control)

Not effective against Giardia lamblia, three strains of Candida

spp. and two strains of Trichophyton spp. except

at the highest test concentration of 10 g/ml.

PK, Toxicology, [14C]-RM5038 Distribution:

Studies in mice, rats, and dogs in progress.

Virus IC50

IC90 LD50 SI Cell Line HCV (genotype 1b) 0.23 µM 1.10 µM 4.3 µM 18.9 AVA5 HCV (genotype 1a) 0.40 µM 1.90 µM 5.7 µM 14.0 AVA5

Hepatitis B (virion) >10.0 µM >10.0 µM > 100 µM - 2.2.15

Influenza A (PR8) 1 µg/ml 7 µg/ml 20 µg/ml 20 MDCK

Avian Influenza (A/Ck) 0.5 µg/ml 6.0 µg/ml >50 µg/ml >100 MDCK

Parainfluenza (Sendai) 0.5 µg/ml 5 µg/ml >50 µg/ml >100 37RC

Coronavirus (CcoV) 0.6 µg/ml 4 µg/ml >50 µg/ml >83.3 A72

Rotavirus (SA-11) 1 µg/ml 15 µg/ml >50 µg/ml >50 MA104

HSV-1 0.15 µg/ml 0.8 µg/ml >50 µg/ml >333 Hep-2

Rhabdovirus (VSV) 1 µg/ml 10 µg/ml 50 µg/ml >50 MA104

Adenovirus (type 5) pending HeLa

Rhinovirus (type 2) pending HeLa R19

RSV (A2) 0.31 µg/ml 5.0 µg/ml 6.8 µg/ml 22 HeLa-ATCC

22

Romark: New Patents (Updated 6/16)

“Compounds and Methods for Treating Influenza.” J. F. Rossignol and J. E. Semple. U.S.

Pat. Appl. Publ. US 20150250768 A1, September 2015. (MOU)

“Haloalkyl Heteroaryl Benzamide Compounds.” J. F. Rossignol and J. E. Semple. US

9126992 B2, September 2015. (PC/MOU)

“Compounds and Methods for Treating Influenza.” J. F. Rossignol and J. E. Semple. US

9023877 B2, May 2015. (PC/MOU)

“Compounds and Methods for Treating Influenza.” J. F. Rossignol and J. E. Semple. US

9345690 B2, May 2015. (MOU)

“Alkylsulfonyl-Substituted Thiazolide Compounds.” J. F. Rossignol and J. E. Semple. US

8895752 B2, November 2014. (COM/PC)

“Haloalkyl Heteroaryl Benzamide Compounds.” J. F. Rossignol and J. E. Semple. US

8846727 B2, September 2014 (COM/PC)

“Alkylsulfinyl-Substituted Thiazolide Compounds.” J. E. Semple and J. F. Rossignol. US

8772502 B2, July 2014. (COM)

“Alkylsulfonyl-Substituted Thiazolide Compounds.” J. F. Rossignol and J. E. Semple. US

8124632 B2, February 2012. (MOU)

“Pharmaceutical Compositions and Methods of Use of Salicylanilides for Treatment of

Hepatitis Viruses.” J. E. Semple and J. F. Rossignol. PCT Int. Appl. WO 2012058378 A1

May 2012. (PC, MOU).

Key: COM = composition of matter claims, PC = pharmaceutical composition claims,

MOU = method of use claims.

23

Romark Acknowledgements

Romark Laboratories, L.C.:Jean-Francois Rossignol, M.D., Ph.D.

Mark Ayers

Emmet B. Keeffe, M.D.

Maria Carrion, M.D.

Matthew Bardin, Ph.D.

Raymond Pasinski

Chemistry:

University of Liverpool:

Andrew V. Stachulski, Ph.D

Chandrakala Pidathala, Ph.D

Mazhar Iqbal, Ph.D.

Kalexsyn, Inc:

Brian Eklov, Ph.D

Mel Schroeder, M.S.

Virology and MOA:Brent E. Korba, Ph.D. (Georgetown University Medical

Center, Rockville, MD)

Gabriella Santoro, Ph.D. (Department of Biology,

University of Rome, Italy)

Jeffrey S. Glenn, M.D., Ph.D.(Division of

Gastroenterology & Hepatology, Stanford University

School of Medicine, Palo Alto. CA)

Parasitology:Gilles Gargala, Ph.D. & Loic Favennec, Ph.D. (Faculty of

Medicine & Pharmacy, University of Rouen, FR)

Computational Chemistry:John H. Van Drie (Van Drie Research LLC, Andover, MA)

24

Corvas Collaboration with Schering Plough Research Institute

CVS 4083

Victrelis™ (Boceprevir)

25

Corvas-Schering Plough Research Institute Collaborations

Oral antithrombotics- FIIa, FXa protease inhibitors

HCV NS3/4A protease inhibitors

Corvas received >$50M in research funding and milestones from SPRI

26

Key Stages of HCV Drug Development Program

Target validation

Assay development- In vitro potency Ki* assay

Cell-based replicon assay

Med. chem. identification of lead compounds

Lead optimization- SAR <-> SBDD <-> structural biology

PK, ADME-Tox & HT-DMPK screens

Identification of a drug candidate- Preclinical animal tox and [14C]-drug disposition studies,

GMP manufacture, COG

Clinical trials and drug launch- Safety, efficacy, resistance issues

27

Iterative Process of Lead Optimization Leading to a Clinical Candidate

SBDDX-Ray &

Structural biology

28

Corvas Chemistry Tools

• Structure-based drug design (X-ray, structural biology)

• SAR optimization (QSAR, computational chem/models)

• Analytical (PK, ADME/Tox, cassette dosing, phys. props., stability, etc.)

• Peptides & peptidomimetics (scaffold morphing)

• Cancer drug conjugates- Targeted drug delivery

• Heterocyclic, Aromatic, and Organometallic chemistry

• Asymmetric synthesis

• Combinatorial chemistry platforms:

- proprietary and known SPS and solution phase technologies

• Novel synthetic technology:

- Multiple-component reactions

- Natural products: semi-synthesis, total synthesis

29

Peptides:Substrate

Motifs e.g.

- dFPR

- dRGR

- dSAR

Hirudin

Hirulogs

TAP

NAPc2

NAP5

HCV NSPs

Antithrombotic Peptidomimetics:Mono-, Bicyclic- and Tricyclic Lactams

Aromatics

Heterocycles

Achiral

(Hetero)Aromatic

Inhibitor Scaffolds

HepC

PAI-1

Cancer Proteases

PACT Prodrugs

Evolution of Corvas Protease Inhibitors

Leverage

Technology

Leverage

Technology Scaffold Morphing II

Peptide & Scaffold

Morphing I

30

Corvas P3-Lactam Scaffold Morphing

31

Chronology of Corvas Thrombin Inhibitors

32

Sequence Alignment of HCV NS3/4A Serine Protease and its Substrates

Scissile bond

C. Lin. HCV NS3-4A Serine Protease. In Hepatitis C Viruses

Genomes and Molecular Biology; Tan S.L., Ed.; Horizon

Bioscience: Norfolk (UK), 2006, Chapter 6.

Link: http://www.ncbi.nlm.nih.gov/books/NBK1623/

Starting point for design of

peptidic P1-aldehyde and

a-ketoamide inhibitors

(cf. next slide)

33

Peptides and a-Ketoamides

Peptide Substrate Peptidic a-ketoamide

Schechter-Berger Notation:

G Barbato et al. EMBO, 2000, 19, 1195.

I. Schechter and A. Berger Biochem. Biophys.

Res. Commun. 1967, 27, 157.

34

Undecapeptide Ketoamide Lead

Using substrate and early P1- aldehyde inhibitor SAR data, a 64-

member ketoamide library was prepared by SPS methods (HCAM,

PAM, AM, MBHA, et al.)*

CVS 4083, a potent inhibitor lead was discovered:

AcEEVVPnV(CO)GMSYS-NH2, Ki* = 2.8 nM, HNE/HCV = 7

CVS 4083Mol Wt = 1265

17 H-bond donors

18 H-bond acceptors

2 negative charges

…..not quite drug-like as per

Lipinski, Weber, et al.

*Combichem Library Technology:

D. V. Siev, J. E. Semple, M.I. Weinhouse. US 6787612B1 (2004).

D. V. Siev, J. A. Gaudette, J. E. Semple Tetrahedron Lett. 1999, 40, 5123.

HCAM resin: D. V. Siev, J. E. Semple Org. Lett. 2000, 2, 19.

J. Z. Ho, O.E. Levy, T. S. Gibson, K. Nguyen, J. E. Semple. Bioorg. Med.

Chem. Lett. 1999, 9, 3459.

35

CVS 4083: Lead Optimization Goals

• Drug Candidate Criteria:

• <10 nM inhibitor

• >1000-fold selective vs. elastase

• Active in cell-based assay

• Oral bioavailable

• Good pharmacokinetics

• Low toxicity

• Absence of reactive metabolites

• IC50 > 5 uM for CYPs 3A4, 2D6, 2C8,

and 2C9

• Moderate human hepatocyte

clearance

• No CYP induction liability.

• Chemistry Objectives:

• Reduce MW

• Maintain Potency

• Increase selectivity

• Reduce hydrogen bonding groups

• Eliminate charges

• LogP of approximately 3

36

Taming the Beast: CVS 4083 Truncation Effects

CVS# Structure Ki* (nM) CVS# Structure Ki* (nM)

4083 Ac-EEVVPnV(CO)-GMSYS-NH2 2.8 4083 Ac-EEVVPnV(CO)-GMSYS-NH2 2.8

4437 Ac-EVVPnV(CO)-GMSYS-NH2 96 4488 Ac-EEVVPnV(CO)-GMSY-NH2 0.6

4438 Ac-VVPnV(CO)-GMSYS-NH2 544 4489 Ac-EEVVPnV(CO)-GMS-NH2 5.4

4439 Ac-VPnV(CO)-GMSYS-NH2 3100 4490 Ac-EEVVPnV(CO)-GM-NH2 11

4441 Ac-PnV(CO)-GMSYS-NH2 >100000 4476 Ac-EEVVPnV(CO)-G-NH2 50

4445 Ac-EEVVPnV(CO)-NH2 760

• CVS 4476 important truncated P6-P1’- heptapeptide

analog with moderate potentcy

• Potency (via binding efficiency) dependent upon P6 to P1’

residues-electrostatic and hydrophobic

Truncate P region: Truncate P’ region:

37

CVS 4083 P1 SAR Studies

AcEEVVP-P1-(CO)GMSYS-NH2 AcEEVVP-P1-(CO)G-OAllyl

CVS# P1 Ki* (nM) CVS# P1 Ki* (nM)

4083 nV 2.8 2436 nV 60

4470 G(propynyl) 9 2435 nL 110

4436 aT 60 2443 V 160

4432 L 66 2429 L 220

4433 nL 100 4487 G(propynyl) 230

4434 Abu 130 4469 G(allyl) 360

4431 V 130

• S1 pocket of HCV NS3 protease is shallow and only tolerates small

(~3-4C) P1-side chains

• Larger P1 moieties destabilize E-I* due to steric clash at S1 pocket

and result in diminished activity

• In both series, P1 -norVal is optimal; in other series Leu, c-Bua and

c-Pra are optimal

38

P1’-C-Terminal Cap SAR Studies

AcEEVVPnV(CO)G-CAP

CVS# CAP Ki* (nM)

4476 NH2 43

4453 OH 8.3

4485 NHPropyl 47

4475 NHPropynyl 60

4474 NHAllyl 140

4454 OtBu 570

4443 OEt 1400

4444 NHCH2CH2Ph 1500

• Potency of CVS 4453 > CVS 4476, however amide deriv. more attractive

• CVS4453 and CVS4476 demonstrated moderate elastase (HNE) selectivity

39

CVS 4453: Lead Compound

Ki* = 8.3 nM

Moderate elastase (HNE) selectivity

Molecular Weight: 798

9 H-bond donors

11 H-bond acceptors

3 negative charges

CVS 4453

40

Solid-Phase Synthesis of CVS 4453 Analogs

PAM-OH =

via Passerini chemistry

41

CVS 4453: P2 Library

Ac-EEVV-P2-nV-(CO)-G-OH

CVS# P2 Ki* (nM)

4524 Tic 3.2

4581 P(3-trans-Me) 4.5

4528 C(SO2Me) 6.8

4453 P 8.3

4529 C(S-CH2CO2H) 8.6

4507 P(4-trans-OCH2CO2H) 10

4523 Pip 12

4561 F 12

4560 E 21

4527 C(Me) 38

4525 thioP 68

4504 Aze 87

4503 Sar 190

4559 D 270

4531 M(O2) 360

• P2 position tolerant of several proline and S-subst’d. cysteine variants

• P2 analogs gleaning hydrophobic and/or anionic contacts at S2 pocket in NS3

42

CVS 4453: 4-(trans-Subst’d)-Proline P2-Analogs

CVS # R Ki* (nM) CVS # R Ki* (nM)

4453 H 8 4555 NHSO2Ph(4-OMe) 4.2

4580 Ph 5 4556 NHCONHPh 7.1

4563 CH2COOH 10 4553 NHiBoc 9.3

4550 Allyl 10 4541 NH-Fmoc 14

4549 4-MeOBn 13 4557 NHCONHPh(4-OMe) 10

4548 Bn 15 4547 NHCOPh(3-OPh) 13

4545 NHCOPh(4-OMe) 20

4542 CH2NHCONHPh 4.4 4544 NHCOPh 22

4537 CH2NHCOPh 4.7 4551 NHCOPh(3,4-OMe) 52

4539 CH2NHCOPh(3-OPh) 5.2 4552 NHCOPh(4-F) 54

4540 CH2NHSO2Ph 7.5 4554 NHSO2Ph 74

4538 CH2NH-Fmoc 8 4546 NHBzl(4-OPh) 76

4536 CH2NHCO2Ph 8.5 4562 NH2 160

• Several potent P2-Pro analogs identified featuring lipophilic, aromatic, hydrophilic & anionic groups

43

CVS 4453: P3 Library

• Incorporation of more hydrophobic cyclohexylglycine and isoleucine residues afforded improved potency

• tBu-glycine, while slightly less potent, showed increased elastase selectivity

• P3 analogs glean additional productive hydrophobic contacts at S3 pocket in NS3.

Ac-EEV-P3-P-nV-(CO)-G-OH

CVS# P3 Ki* (nM) CVS# P3 Ki* (nM)

4516 G(Chx) 1.9 4671 S(O-Me) 1100

4518 I 4.5 4686 Q 2100

4453 V 8.3 4667 S 5900

4666 G(tBu) 26 4668 T 6700

4672 N 10500

4500 Phg 120 4520 dC(2-AcOH) 54000

4698 M 180 4521 dN(MeTzl) 54000

4699 C 210 4522 dQ(MeTzl) 80000

4665 L 300 4502 dD >100000

4517 F 310 4519 dE >100000

4670 Dif 310

4669 nL 460

44

P2-3,4-(Isopropylidene)Proline series

CAP-P3-P[3,4-(diMe-cyclopropyl)]-P1-(CO)-G-G(Ph)-NMe2

• CVS 4845 demonstrated good potency in replicon cell assay (G1b)

• P3 -G(1-MeChx) with P1 nV or L confers good potency and selectivity

• P3 -G(tBu) with P1 nV or L confers excellent potency and selectivity

Replicon

assay

CVS# CAP P3 P1 K i * (nM)

HNE K i

(nM)

HNE/

HCV EC90 (nM)

4845 iBoc Chg nV 10 46 4.6 200

4858 iBoc Chg c-Pra 66 18 0.3

4893 iBoc G(1-MeChx) nV 12 170 14

4894 iBoc G(1-MeChx) L 15 1100 73

4899 ((R)-1-Me)iBoc G(1-MeChx) nV 6 270 45

4895 iBoc G(tBu) L 13 1100 85

4901 iPoc G(tBu) nV 2 320 160

4902 iPoc G(tBu) L 8 3000 375

CVS 4453

45

Synthesis of 4,4-Dialkylproline Derivatives

S. Kemp, M. Lawrence, K. Matthews

46

Synthesis of 4,4-Spiropentylproline Derivatives

S. Kemp, M. Lawrence, K. Matthews

47

Synthesis of 3,4-Isopropylideneproline Peptides

S. Kemp, M. Lawrence, K. Matthews

48

Novel Modifications of the

Passerini Reaction & Applications

to HCV Protease Inhibitors

49

The Passerini Reaction

N

OH

O

R1

R4

O O

NH

O

R1R2

R1NC + R 2R3CO + R 4CO 2H

acyl

H+

O O

HO

R3

R4

R2R1 N

R3R2

R4

O

R3

a-Acyloxyamide ProductM. Passerini, Gazz. Chim. Ital. 1921, 51, 126.

M. Passerini and G. Ragni, Gazz. Chim. Ital. 1931, 61, 964.

I. Ugi et al. in Isonitrile Chemistry, I. Ugi, Ed.; Academic:

New York, 1971; Chapter 7.

A. Dömling and I. Ugi, Angew. Chem. Intl. Ed. 2000, 39, 3168.

shift

50

Passerini Reactions of a-Amino Aldehydes with TFA and Pyridine-Type Bases

PG1NHN

OH

O

R1

CF3

O

PGNH

PGNH

O2CCF3

NH

O

R1

H

PGNH

OH

NH

O

R1NH

OH

NH

O

R1R3

O

NH

NH

O

R1R3

O

O

H+

PGNH

OH

OH

O

R2

O

R2

R2

R2R2

R2R2

CF3CO2H,R1NC,Pyridine,CH2Cl2

acyl

shift

Hydrolyticwork-up

a-Hydroxy-b-amino amide derivatives:• ca. 1:1 mixture @ new hydroxy center • retention of chirality at original centers

*

Elaboration

1. Optional sidechain deprotection2. Oxidation

Hydrolysis

a-Ketoamide Derivativesa-Hydroxy-b-amino acid "norstatine" derivatives

J. E. Semple, T. D. Owens, K. Nguyen and

O. E. Levy Organic Lett. 2000, 2, 2769.

J. E. Semple and O. E. Levy. WO 0035868 A2,

June, 2000; Priority: December 1998;

U.S. Patent 6376649 B1, April 2002.

J. E. Semple et al. Abstracts of Papers, 218th

American Chemical Society National Meeting,

New Orleans, LA, August 22-26, 1999;

ORGN-419, MEDI-240.

Passerini reaction with TFA and pyridine:

W. Lumma J. Org. Chem. 1981, 46, 3668.

TiCl4-catalyzed Passerini-type reactions:

D. Seebach et al. Chem. Ber. 1988, 121, 507;

Helv. Chim. Acta 1983, 66, 1618.

51

Passerini Reactions of a-Amino Aldehydes with TFA: Effect of Bases

Organic Base Additive pKa % Yield 3

2,6-di-t-Butyl Pyridine ~ 9 72

2,4,6-Collidine 7.4 71

2,6-Lutidine 6.6 68

Pyridine 5.2 60

N-Methylmorpholine 7.5 41

DABCO 8.2 33

4-N,N-Dimethylaminopyridine 9.7 18

N,N-Diisopropylethylamine 11 15

FmocNH

CHO CN

O

O FmocNH

O

O

OH

NH

OOrganic base, TFA, DCM, 0 °C to RT

1 2 3

J. E. Semple, T. D. Owens,

K. Nguyen and O. E. Levy

Organic Lett. 2000, 2, 2769.

52

Passerini Reactions of a-Amino Aldehydes with TFA

Variation 1: PGNHCH(R 2)CHO + R 1 NC + CF 3CO 2H = PGNHCH(R 2)CH(OH)CONHR 1

Cmpd PG Amino Acid SC R2 R1 % Yield

a Boc Cys(Me) CH2SMe CH2CO 2Me 62

b Fmoc Val CH(CH 3)2 CH2CO 2t -Bu 68

c Fmoc Tyr(t -Bu) CH2Ph-4-(t -BuO) CH2CO 2Et 69

d Boc Arg(NO2) (CH2)3NHC(=NH)NHNO 2 CH2CO 2Et 38

e Fmoc Arg(Pmc) (CH2)3NHC(=NH)NHPmc CH2CH2Ph 75

f Boc Arg(NO2) (CH2)3NHC(=NH)NHNO 2 t -Bu 92

g Boc Phe CH2Ph CH2CO 2Allyl 67

h Boc Phe CH2Ph t -Bu 24-71

i Cbz d -Phe CH2Ph (S )-CH(i -Bu)CO 2Bn 65

j Boc ChxAla CH2Chx t -Bu 46

k Fmoc Gly H CH2CO 2Allyl 77

l Fmoc Ala CH3 CH2CO 2Allyl 83

m Fmoc Abu CH2CH3 CH2CO 2Allyl 73

n Fmoc Val CH(CH 3)2 CH2CO 2Allyl 68

o Fmoc nor-Val (CH2)2CH3 CH2CO 2Allyl 87

p Fmoc Leu CH2CH(CH 3)2 CH2CO 2Allyl 85

q Fmoc nor-Leu (CH2)3CH3 CH2CO 2Allyl 69

r Fmoc Phe CH2Ph CH2CO 2Allyl 67

s Fmoc Tyr(t -Bu) CH2Ph-4-(t -BuO) CH2CO 2Allyl 66

t Fmoc Ser(t -Bu) CH2Ot -Bu CH2CO 2Allyl 68

u Fmoc Asp(t -Bu) CH2CO 2t -Bu CH2CO 2Allyl 60

v Fmoc Arg(Pmc) (CH2)3NHC(=NH)NHPmc CH2CO 2Allyl 76

w Fmoc Lys(Boc) (CH2)4NHBoc CH2CO 2Allyl 79

x Fmoc Thr CH3(CH)O t -Bu CH2CO 2Allyl 62

y Fmoc allo -Thr CH3(CH)O t -Bu CH2CO 2Allyl 74

P1-P1’of

HCV

Inhibitor

libraries

Thrombin

and FXa

Inhibitors,

Libraries,

Bestatin

NH

NH

R2

OH

O

PG R1

J. E. Semple, T. D. Owens,

K. Nguyen and O. E. Levy

Organic Lett. 2000, 2, 2769.

J. E. Semple and O. E. Levy,

WO 0035868 A2, 2000; US

Patent 6376649 B1, 2002.

53

Passerini Reactions of a-Amino Aldehydes with Carboxylic Acids

L. Banfi, G. Guanti, R. Riva, A. Basso, E. Calcagno

Tetrahedron Lett. 2002, 43, 4067.

L. Banfi, G. Guanti, and R. Riva Chem. Commun. 2000, 985.

J. E. Semple and O. E. Levy. WO 0035868A2, June, 2000

(Priority: 12/18/98); US Patent 6376649 B1, April 2002.

J. E. Semple et al. Abstracts of Papers, 218th American

Chemical Society National Meeting, New Orleans, LA,

August 22-26, 1999; ORGN-419, MEDI-240. J. E. Semple, T. D. Owens, K. Nguyen and O. E. Levy 16th International Symposium for Synthesis in Organic Chemistry, Cambridge, UK, July 19–22, 1999; P.4.O. E. Levy, K. Nguyen, T. D. Owens and J. E. SempleAbstracts of Papers, 16th American Peptide Symposium, Minneapolis, MN, June 26–July 1, 1999; P-6653.

PGNH CHO PGNH

R2

NHR

O

O

R1

O

H2N

R2

NHR

O

O

R1

O

NH

R2

NHR

O

OH

R1

O

NH

R2

NHR

O

R1

O

O

R2

acyl migration

a-Acyloxy-b-aminoamide:• Ca. 1,1 mixture @ new acyloxy center.• Retention of chirality @ *.

N

OH

O

R

R1

O

PGNH

R2

*

RNC, R1CO2H, solvent

-PG

Cleaveacyl moiety

NH

R2

NHR

O

OH

Ketoamide target or advanced intermediate

PG

H+

Further chemistry

[O]

Further chemistry

54

Concise Synthesis of Potent HCV Lead CVS4845 via Passerini-Deprotection-Acyl Migration (PADAM) Strategy

J. E. Semple, S.J. Kemp and T.D. Owens, unpublished

J. E. Semple, T. D. Owens Organic Lett. 2001, 3, 3301.

J. E. Semple, 219th American Chemical Society National Meeting,

San Francisco, CA March 26-30, 2000; ORGN.667.

J. E. Semple, T. D. Owens, K. Nguyen and O. E. Levy

Organic Lett. 2000, 2, 2769.

55

HCV Drug DMPK Screening Paradigm

K.-C. Cheng, W.A. Korfmacher,

R.E. White, F.G. Njoroge,

Perspectives in Medicinal

Chemistry 2007, 1, 1-9.

1000 compounds

EC90 < 1 M (rep)

3 compounds

Boceprevir (SCH503034)

56

Pathway to Discovery of Victrelis™(Boceprevir):Part I

64-member Library

P1-Library

Truncate

P2’-P5’

Truncate P4-P6

Focused CAP- and P3-Library

P3CAP

57

Pathway to Discovery of Victrelis™(Boceprevir):Part II

P2-SAR

SAR

optimize

P2-P3

CVS4704

Step 1: 13-membered P2’-Library w/ P2-Leu

Step 2: 14-membered P2’-Library w/ P2-Pro (Ki* = 360 nM)*

Iterate/

optimize

P4-CAP

(X-ray)

(X-ray)

*A. Arasappan, S. Kemp, O. Levy, M. Lim-Wilby, S. Tamura, et al.

Bioorg. Med. Chem. Lett. 2005, 15, 4180–4184.

58

X-Ray of SCH225724, iBoc-Chg-L-nV(CO)-G-Phg-NH2

P2’-residue wraps over Lys136 side chain

P1–P2’ moiety forms C-clamp, locking Lys136 in place

Extensive hydrophobic interactions translated into enhanced

binding potency (Ki* = 66 nM).

A. Arasappan, S. Kemp, O. Levy, M. Lim-Wilby, S. Tamura,

et al. Bioorg. Med. Chem. Lett. 2005, 15, 4180–4184.

59

Key Interactions of CVS4901-NS3/4A Complex Based on X-Ray Crystal Structure

Val 158

P1–P2’ moiety forms C-clamp with Lys136

60

Pathway to Discovery of Victrelis™(Boceprevir):

Part III

Final optimization

selectivity,

cell activity,

ADME/Tox & PK

EC90 = 290 nM

4 HBD

7 HBA

X-ray

EC90 = 350 nM

5 HBD

5 HBA

X-ray

A. K. Saksena, T. K. Brunck, S. J. Kemp, O. E. Levy, M. Lim-Wilby, et al. US 6800434B2 (2004).

A. K. Sakena, T. K. Brunck, S. J. Kemp, O. E. Levy, M. Lim-Wilby, et al. US 7012066B2 (2006).

S. Venkatraman et al. J. Med. Chem. 2006, 49, 6074.

F. G. Njoroge, K. X. Chen, N.-Y. Shih, J. J. Piwinski, Acc. Chem. Res. 2008, 41, 50.

A. J. Prongay et al. J. Med. Chem. 2007, 50, 2310.

N.A. Meanwell, J. F. Kadow, P. M. Scola. Annual Reports in Medicinal

Chemistry; J. E. Macor, Ed.; Academic Press: New York, 2009; Vol. 44, Ch. 20.

61

X-Ray Structure of Boceprevir (R = 2.3 A)

V. Madison et al. J. Synchrotron Rad. 2008, 15, 204–207

Crystal structure of the covalent Boceprevir (SCH503034)-

NS3/4A complex, generated using Pymol

62

Victrelis™ (boceprevir, SCH503034), a First-in-Class FDA-Approved HCV NS3/4A Inhibitor

VictrelisTM (boceprevir, SCH503034)

(1R,2S,5S)-N-((S)-4-amino-1-cyclobutyl-3,4-dioxobutan-2-yl)-

3-((S)-2-(3-tert-butylureido)-3,3-dimethylbutanoyl)-6,6-

dimethyl-3-azabicyclo[3.1.0]hexane-2-carboxamide

Chemical Formula: C27H45N5O5

Molecular Weight: 519.7

Log P: 0.96

CLogP: 3.34

MR: 141 [cm3/mol]

tPSA: 150.7

White to off-white amorphous powder, freely soluble in

MeOH, EtOH, iPrOH, slightly soluble in water.

A. K. Saksena, T. K. Brunck, S. J. Kemp, O. E. Levy, M. Lim-Wilby, et al. US 6800434B2 (2004).

A. K. Saksena, T. K. Brunck, S. J. Kemp, O. E. Levy, M. Lim-Wilby, et al. US 7012066B2 (2006).

S. Venkatraman et al. J. Med. Chem. 2006, 49, 6074.

F. G. Njoroge, K. X. Chen, N.-Y. Shih, J. J. Piwinski, Acc. Chem. Res. 2008, 41, 50.

A. J. Prongay et al. J. Med. Chem. 2007, 50, 2310.

N. A. Meanwell, J. F. Kadow, P. M. Scola. Annual Reports in Medicinal

Chemistry; J. E. Macor, Ed.; Academic Press: New York, 2009; Vol. 44, Ch. 20.

63

Victrelis™ (boceprevir, SCH503034): ADME/Tox and PK

VictrelisTM (boceprevir, SCH503034)

Ki* = 14 nM; EC90 = 350 nM (replicon)

Potent, selective, mechanism-based inhibitor of

NS3/4A enzyme

Binding studies conducted with a G1a HCV protease

indicate that dissociation of the E-I complex occurs

slowly, with a t1/2~ 1 hr

Low to moderate OBA in mouse (34%), rat (26%), dog

(30%) and cyno (4-11%),with liver exposure in the rat

liver/plasma ratio >30 (AUC ratios)

Human plasma protein binding is ~ 75%.

In humans dosed @ 800 mg t.i.d., AUC(т) = 5.41

g.hr/mL (n=71), Cmax of 1.72 g/mL (n=71), Cmin of

0.088 g/mL (n=71), median Tmax = 2 hours, Vd/Fss ~

772 L

Absolute bioavailability (F) in humans was not

determined (as of ca. 2011).

IC50’s CYP2D6, 2C9, 2C19 >30/>30 M (co/pre)

CYP3A4 > 30/8.5 M (co/pre).N. A. Meanwell, J. F. Kadow, P. M. Scola. Annual Reports in Medicinal

Chemistry; J. E. Macor, Ed.; Academic Press:New York, 2009; Vol. 44, Ch. 20.

F. G. Njoroge, K. X. Chen, N.-Y. Shih,J. J. Piwinski, Acc. Chem. Res. 2008, 41, 50.

A. J. Prongay et al. J. Med. Chem. 2007, 50, 2310.

MAT = mean absorption time

64

Victrelis™ Human PK Profiles (cont’d).

VICTRELIS capsules contain a 1:1 mixture of two diastereomers-

In plasma the ratio changes to 2:1, favoring the active (a-S)-diastereomer.

Accumulation is minimal (0.8- to 1.5-fold) and pharmacokinetic steady state is achieved

after approximately 1 day of t.i.d. dosing.

Food enhanced the exposure of boceprevir by up to 65% at the 800 mg t.i.d. dose,

relative to the fasting state.

Primarily undergoes metabolism via the aldoketoreductase (AKR)-mediated pathway to

ketone-reduced metabolites that are inactive against HCV.

After a single 800-mg oral dose of 14C-boceprevir, the most abundant circulating

metabolites were a diasteriomeric mixture of ketone-reduced metabolites with a mean

exposure approximately 4-fold greater than that of boceprevir.

65

Clinical Efficacy in Phase IIb Trials

Sustained virologic response rates in phase IIb trials of telaprevir and boceprevir. B indicates

boceprevir; P, peginterferon alfa; r, low-dose (400-1000 mg) ribavirin; R, expanded dose (800-

1400 mg) ribavirin; T, telaprevir. Numerals in regimens indicate weeks of treatment. Numerals atop

bars indicate relapse rate. Based on data from Hézode et al, N Engl J Med, 2009; Kwo et al, EASL,

2009; McHutchison et al, N Engl J Med, 2009.

66

Summary & Conclusions Starting with HCV substrates, a series of focused combinatorial a-ketoamide libraries were

prepared that elucidated SAR at each of the P6-P5’ positions: Developed novel SPPS methods (HCAM) for early P1-aldehyde libraries and Passerini MCR

methodology for rapid assembly of key intermediates and a-ketoamide inhibitors.

Probed each of the P6-P5’ moieties with novel types of bioisosteres, unnatural amino acids,

and peptidomimetics, i.e. identified more “drug-like” scaffolds: Corvas discovered P3-t-BuGly and P2-3,4-(Isopropylidene)Pro moieties found in boceprevir.

Truncation efforts coupled with iterative SAR and SBDD optimization led to CVS4083 (11-

mer, Ki* = 2.8 nM, HNE/HCV = 7), CVS4453 (7-mer, Ki* = 8.3 nM), CVS4704 (4-mer, Ki* =

2900 nM), SCH225724 (5-mer, Ki* = 66 nM), CVS4845 (5-mer, Ki* = 10 nM, HNE/HCV = 5),

CVS4882 (5-mer, Ki* = 6 nM, HNE/HCV = 200) and CVS4901 (5-mer, Ki* = 2 nM, HNE/HCV

= 160).

Optimized drug potency (Ki* ~1-10 nM), selectivity, and oral efficacy profiles in later

generations of inhibitors.

Multiple HT ADME/Tox and PK studies expedited selection, elimination, and optimization of

several lead classes.

Final med chem optimization of CVS4901 at SRPI led to the identification of boceprevir (Ki*

= 14 nM, HNE/HCV = 2200, EC90 = 350 nM): Found that potency, selectivity, OBA, PK, and efficacy are sensitive to nature of inhibitor structure.

Total efforts at SPRI led to screening of ~10K compounds, ~1K of which had EC90 < 1 M in

cell assays. Three main classes were identified, which through attrition in ADME/Tox, PK

and other screens afforded three preclinical candidates, one of which was boceprevir.

67

Acknowledgements

Analytical Chemistry

Kirk Kozminsky

Michael Ma

Thomas G. Nolan, Ph.D.

Molecular Modeling,

and NMR Support:

Marguerita S. Lim-Wilby, Ph.D.

Terence K. Brunck, Ph.D.

X-Ray Crystallography (SPRI):

Vincent Madison, Ph.D.

Patricia Weber, Ph.D.

Academic Consultants:

Prof. Henry Rapoport (UC Berkeley)

Prof. Andrew B. Holmes (Cambridge)

Prof. Victor A. Snieckus (Queen’s)

Prof. William Lubell (Montreal)

Tea and Sympathy:

Grace M. Semple

Eric J. Semple

Medicinal Chemistry:

Susan Y. Tamura, Ph.D.

Odile E. Levy, Ph.D.

Scott Kemp, Ph.D.

Max Lawrence, M.S.

Timothy D. Owens

Nathaniel K. Minami

Daniel V. Siev

Erick A. Goldman

John Gaudette

Christopher Roberts

Kenneth Matthews

George P. Vlasuk, Ph.D.

Ruth F. Nutt, Ph.D.

William C. Ripka, Ph.D.

J. Edward Semple, Ph.D.

SPRI (now Merck):F. George Njoroge, Ph.D.

Bruce Malcolm, Ph.D.

Brian McKittrick, Ph.D.

Anil Saksena, Ph.D.

Kevin X. Chen, Ph.D.

Neng-Yang Shih, Ph.D.

K.-C. Cheng, Ph.D.

Walter A. Korfmacher, Ph.D.

Ronald E. White, Ph.D.

Srikanth Venkatraman, Ph.D.

Frank Bennett, Ph.D.

John Pichardo, Ph.D.

Viyyoor Girijavallabhan, Ph.D.

John J. Piwinski, Ph.D.

Ashit Ganguly, Ph.D

…and many others