1. 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. 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. 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
6. 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. 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. 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. 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. 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
Asp81
Ser139
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. 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
14. 14
Current Clinical HCV Inhibitors: Other Classes
Alisporivir® (Debio 025)
Elvitegravir® (GS-9190)
BMS-790052
Alinia® (Nitazoxanide)
15. 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!
16. 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. 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. 18
RM5038
HCV (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
RM4829
HBV (VIR): EC50= 0.22 M, SI >137
HBV (RI): EC50= 1.20 M, SI >25
RM5021
Influenza A (PR8, MDCK):
EC50= 0.028 M, SI >5000
Parainfluenza (SV, 37RC):
EC50= 0.085 M, SI >1670
RM4804
Parainfluenza (SV, 37RC):
EC50= 0.97 M, SI >167
RM4860
Rotavirus (SA11):
EC50= 0.026 M, SI >5000
RM5034
HSV-1(Hep-2):
EC50= 0.091 M, SI >1667
Broad Spectrum Antivirals:
Recent Thiazolide Leads
19. 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. 20
Thiazolide Core Pharmacophore & MOA
N
H
L2
OH O
EWG/HBA
Minimal pharmacophore Metal complex at active or allosteric binding site
vs. classical protein backbone H-bonds and/or
water-mediated H-bonds?
N
H
L2
O O
EWG/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. 21
HCV Lead RM5038: Preclinical Data
N
SN
H
OO
Cl
O
RM5038
2-(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. 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. 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)
25. 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. 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. 27
Iterative Process of Lead Optimization
Leading to a Clinical Candidate
SBDD X-Ray &
Structural biology
28. 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. 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
32. 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. 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. 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 4083
Mol 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. 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. 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. 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. 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
49. 49
The Passerini Reaction
N
OH
O
R1
R4
O O
N
H
O
R1R2
R1NC + R2R3CO + R 4CO 2H
acyl
H+
O O
H
O
R3
R4
R2
R1 N
R3
R2
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. 50
Passerini Reactions of a-Amino Aldehydes with TFA
and Pyridine-Type Bases
PG1NH
N
OH
O
R1
CF3
O
PGNH
PGNH
O2CCF3
N
H
O
R1
H
PGNH
OH
N
H
O
R1
N
H
OH
N
H
O
R1
R3
O
N
H
N
H
O
R1
R3
O
O
H+
PGNH
OH
OH
O
R2
O
R2
R2
R2R2
R2
R2
CF3CO2H,
R1NC,
Pyridine,
CH2Cl2
acyl
shift
Hydrolytic
work-up
a-Hydroxy-b-amino amide derivatives:
• ca. 1:1 mixture @ new hydroxy center
• retention of chirality at original centers
*
Elaboration
1. Optional sidechain
deprotection
2. Oxidation
Hydrolysis
a-Ketoamide Derivatives
a-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. 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
Fmoc
N
H
CHO CN
O
O Fmoc
N
H O
O
OH
N
H
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. 52
Passerini Reactions of a-Amino Aldehydes with TFA
Variation 1: PGNHCH(R 2)CHO + R 1 NC + CF 3CO2H = PGNHCH(R 2)CH(OH)CONHR 1
Cmpd PG Amino Acid SC R2 R1 %Yield
a Boc Cys(Me) CH2SMe CH2CO2Me 62
b Fmoc Val CH(CH3)2 CH2CO2t -Bu 68
c Fmoc Tyr(t -Bu) CH2Ph-4-(t -BuO) CH2CO2Et 69
d Boc Arg(NO2) (CH2)3NHC(=NH)NHNO2 CH2CO2Et 38
e Fmoc Arg(Pmc) (CH2)3NHC(=NH)NHPmc CH2CH2Ph 75
f Boc Arg(NO2) (CH2)3NHC(=NH)NHNO2 t -Bu 92
g Boc Phe CH2Ph CH2CO2Allyl 67
h Boc Phe CH2Ph t -Bu 24-71
i Cbz d -Phe CH2Ph (S )-CH(i -Bu)CO2Bn 65
j Boc ChxAla CH2Chx t -Bu 46
k Fmoc Gly H CH2CO2Allyl 77
l Fmoc Ala CH3 CH2CO2Allyl 83
m Fmoc Abu CH2CH3 CH2CO2Allyl 73
n Fmoc Val CH(CH3)2 CH2CO2Allyl 68
o Fmoc nor-Val (CH2)2CH3 CH2CO2Allyl 87
p Fmoc Leu CH2CH(CH3)2 CH2CO2Allyl 85
q Fmoc nor-Leu (CH2)3CH3 CH2CO2Allyl 69
r Fmoc Phe CH2Ph CH2CO2Allyl 67
s Fmoc Tyr(t -Bu) CH2Ph-4-(t -BuO) CH2CO2Allyl 66
t Fmoc Ser(t -Bu) CH2Ot -Bu CH2CO2Allyl 68
u Fmoc Asp(t -Bu) CH2CO2t -Bu CH2CO2Allyl 60
v Fmoc Arg(Pmc) (CH2)3NHC(=NH)NHPmc CH2CO2Allyl 76
w Fmoc Lys(Boc) (CH2)4NHBoc CH2CO2Allyl 79
x Fmoc Thr CH3(CH)Ot -Bu CH2CO2Allyl 62
y Fmoc allo -Thr CH3(CH)Ot -Bu CH2CO2Allyl 74
P1-P1’of
HCV
Inhibitor
libraries
Thrombin
and FXa
Inhibitors,
Libraries,
Bestatin
N
H
N
H
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. 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. Semple
Abstracts 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
N
H
R2
NHR
O
OH
R1
O
N
H
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
Cleave
acyl moiety
N
H
R2
NHR
O
OH
Ketoamide target or
advanced intermediate
PG
H+
Further
chemistry
[O]
Further
chemistry
54. 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.
56. 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
P3
CAP
57. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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
