Sarah Burke, Andrew Krasley, Maria Winters Hepatitis C Virus (HCV): Serine Protease Inhibitors
Apr 01, 2015
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