Achiral pyrazinone-based inhibitors of the hepatitis C virus NS3 protease and drug-resistant variants with elongated substituents directed toward the S2 pocket

Article abstract of DOI:10.1021/jm301887f

Herein we describe the design, synthesis, inhibitory potency, and pharmacokinetic properties of a novel class of achiral peptidomimetic HCV NS3 protease inhibitors. The compounds are based on a dipeptidomimetic pyrazinone glycine P3P2 building block in combination with an aromatic acyl sulfonamide in the P1P1′ position. Structure-activity relationship data and molecular modeling support occupancy of the S2 pocket from elongated R⁶ substituents on the 2(1H)-pyrazinone core and several inhibitors with improved inhibitory potency down to K_i = 0.11 μM were identified. A major goal with the design was to produce inhibitors structurally dissimilar to the di- and tripeptide-based HCV protease inhibitors in advanced stages of development for which cross-resistance might be an issue. Therefore, the retained and improved inhibitory potency against the drug-resistant variants A156T, D168V, and R155K further strengthen the potential of this class of inhibitors. A number of the inhibitors were tested in in vitro preclinical profiling assays to evaluate their apparent pharmacokinetic properties. The various R⁶ substituents were found to have a major influence on solubility, metabolic stability, and cell permeability.

Full text of DOI:10.1021/jm301887f

Article
pubs.acs.org/jmc
Achiral Pyrazinone-Based Inhibitors of the Hepatitis C Virus NS3
Protease and Drug-Resistant Variants with Elongated Substituents
Directed Toward the S2 Pocket
Johan Gising,†,# Anna Karin Belfrage,†,# Hiba Alogheli,† Angelica Ehrenberg,‡ Eva Åkerblom,†
Richard Svensson,§,∥ Per Artursson,§,∥ Anders Karlen,† U. Helena Danielson,‡ Mats Larhed,†
́
and Anja Sandstrom*,†
̈
†Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, BMC, Uppsala University, Box 574, SE-751 23 Uppsala,
Sweden
‡Department of Chemistry, BMC, Uppsala University, BMC, Box 576, SE-751 23 Uppsala, Sweden
§Department of Pharmacy, Uppsala University, Box 580, SE-751 23 Uppsala, Sweden
∥The Uppsala University Drug Optimization and Pharmaceutical Profiling Platform, Chemical Biology Consortium Sweden (CBCS),
Uppsala University, Box 580, SE-751 23 Uppsala, Sweden
S
* Supporting Information
ABSTRACT: Herein we describe the design, synthesis,
inhibitory potency, and pharmacokinetic properties of a
novel class of achiral peptidomimetic HCV NS3 protease
inhibitors. The compounds are based on a dipeptidomimetic
pyrazinone glycine P3P2 building block in combination with
an aromatic acyl sulfonamide in the P1P1′ position.
Structure−activity relationship data and molecular modeling
support occupancy of the S2 pocket from elongated R6
substituents on the 2(1H)-pyrazinone core and several
inhibitors with improved inhibitory potency down to Ki =
0.11 μM were identified. A major goal with the design was to
produce inhibitors structurally dissimilar to the di- and
tripeptide-based HCV protease inhibitors in advanced stages
of development for which cross-resistance might be an issue. Therefore, the retained and improved inhibitory potency against the
drug-resistant variants A156T, D168V, and R155K further strengthen the potential of this class of inhibitors. A number of the
inhibitors were tested in in vitro preclinical profiling assays to evaluate their apparent pharmacokinetic properties. The various R6
substituents were found to have a major influence on solubility, metabolic stability, and cell permeability.
treatment is associated with severe adverse effects and
inconvenient dosing regimens.4,5
INTRODUCTION
■
Hepatitis C virus (HCV) is a widespread disease affecting
approximately 130−200 million people worldwide.1 Each year,
3−4 million people are newly infected and more than 350000
people die from HCV related liver diseases yearly.1,2 Despite
significant effort in the area, there is still no vaccine available.3
About 70% of all infections will develop into a chronic HCV
infection, which in one-fourth of the cases leads to cirrhosis and
ultimately hepatocellular carcinoma or end-stage liver disease.4
The standard treatment has long consisted of a combination
therapy of pegylated interferon-α, which boosts the patient’s
immune system, and ribavirin, which inhibits the virus RNA
replication (pegIFNα/RBV). The efficacy has been highly
dependent on the genotype of the virus. Among individuals
infected with HCV genotypes 1 or 4 less than 50% obtain a
sustained virological response (SVR), whereas 85% of patients
infected with genotypes 2 or 3 obtain SVR.4,5 Furthermore, this
As an alternative or a complement to pegIFNα/RBV, direct
acting antivirals (DAA), especially those targeting the NS3
protease, the NS5B polymerase, and the NS5A protein, which
are all part of the replication complex, have been intensively
explored as promising future drugs. Several DAAs are currently
in the final stage of clinical development,4 and the reversible
covalent peptidomimetic NS3 protease inhibitors 1 (Telapre-
vir)6 and 2 (Boceprevir)7 were recently approved for the
treatment of chronic HCV infection. These drugs (1 or 2) are
now included in the standard therapy, in combination with
pegIFNα/RBV (Figure 1), for people infected with genotype
1.4−6 Besides positive qualities in terms of more efficient
Special Issue: HCV Therapies
Received: December 21, 2012
© XXXX American Chemical Society
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Figure 1. Direct acting antivirals of the hepatitis C virus NS3 protease. Telaprevir (1) and Boceprevir (2), approved covalent reversible inhibitors.
Simeprevir/TMC435350 (3) and Faldaprevir/BI201335 (4), examples of clinically evaluated, noncovalent product-based inhibitors.
outcome and shorter treatment periods, both of these drugs are
accompanied with a high pill burden and side effects, like rash
and anemia, in addition to those caused by pegIFNα/RBV.
Among the HCV NS3 protease inhibitors under clinical
investigation, the most advanced are 3 (Simeprevir/
TMC435350),8 a noncovalent, macrocyclic inhibitor, and 4
(Faldaprevir/BI201335),9 a noncovalent linear inhibitor
(Figure 1). Both of the compounds are currently in phase 3
clinical studies. These promising drug candidates are dosed
once daily instead of three times daily as required for 1 or 2 and
are associated with fewer side effects.10
One major concern in HCV therapy, and especially in cases
of low compliance, is potential development of drug resistance.
This is a consequence of the high replication rate and the error-
prone nature of the HCV RNA-dependent RNA polymerase.11
Indeed, drug resistance has been observed frequently in in vivo
and in vitro studies during treatment with 1 and 2 and several
of the HCV NS3 protease inhibitors in advanced clinical
development.12,13 Critically, several of the amino acid
substitutions observed in NS3 (i.e., A156T, D168V, and
R155K etc.) convey some cross-resistance because the
Figure 2. (I) Possible hydrogen bond interactions of the peptide
backbone. (II) A schematic representation of the substituents on the
pyrazinone scaffold. 5, Pyrazinone comprising inhibitor.21
structures of 1, 2, and advanced drug candidates are somewhat
similar; often based on a P2 proline/proline mimic.10,12,14−17
To combat potential future problems with drug resistance,
beside the obvious use of a combination therapy, there is a need
for novel protease inhibitors based on unique structural motifs.
Aiming at new types of HCV NS3 protease inhibitors, we
have previously explored macrocyclic and linear inhibitors
based on P2 phenylglycine18−20 and preliminary efforts to
include a P3 2(1H)-pyrazinone scaffold (Figure 2).21 The
2(1H)-pyrazinone scaffold possesses structural features useful
in peptidomimetic compounds due to its ability to act as a β-
strand inducer and retain the H-bonding pattern of the peptide
backbone (Figure 2, I).22,23 Compound 5 displayed single digit
μM inhibitory potency against the full-length wild-type NS3
protease as well as promising activity against the resistant NS3
variants A156T and D168A (Figure 2), prompting further
exploration of this class of inhibitors.21 Preliminary results
indicated that the large, not fully occupied, S2 pocket could be
reached from the R6 position of the pyrazinone (Figure 2, II).21
We herein demonstrate that lead optimization of achiral
pyrazinone-based HCV NS3 protease inhibitors is possible
using elongated R6 substituents directed toward the S2 pocket.
This regards both inhibitory potency against wild-type and
drug-resistant variants, as well as the pharmacokinetic (PK)
properties.
CHEMISTRY
■
The 2(1H)-pyrazinone comprising HCV NS3 inhibitors were
assembled in an altogether 5−7-step procedure (Scheme 1−3)
via coupling of the P1P1′ amine (8 and 10, Scheme 1) with the
P3P2 pyrazinone building blocks (Scheme 2). Final function-
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a
Scheme 1. Synthesis of P1P1′ Building Blocks 8 and 10
a
Reagents and conditions: (a) KCN, acetic acid, MeOH, DCM, 0 °C, 3h (88%); (b) H2O2, LiOH, MeOH, 0 °C, 2 h (34%); (c) HCl in dioxane, rt,
30 min (quant).
a
Scheme 2. Synthesis of HCV Inhibitors 68−86
a
Reagents and conditions: (a) LiAlH4, THF, −78 °C to rt; (b) SO3−Py, Et3N, DMSO, DCM, rt, 1 h; (c) Dess−Martin periodinane, DCM, rt, 30
min; (d) 21, TMSCN, DIPEA, reflux, 1 h; (e) TMSCN, DIPEA, DME, MW, 110−170 °C, 10 min or reflux, 30 min; (f) HCl gas, Et2O, then oxalyl
chloride, DME, MW, 145 °C for 25 min or reflux on (23−66% from 20 or 21); (g) tert-butylurea, Pd(OAc)2, Xantphos, Cs2CO3, DME, MW, 100−
110 °C, 15−20 min or reflux, 0.5−1 h (46−89%); (h) K2CO3, MeCN, H2O, MW, 110 °C, 15 min; (i) 8, HATU, DIPEA, DMF, 0 °C, 2.5 h (67,
62%); (j) 8, HATU, DIPEA, DCM, ultra sonic bath, rt, 5 min (66, 85%); (k) EDCI, DMSO, dichloroacetic acid, DCM, rt, 4−6 h (13−64%); (l) 10,
HATU, DIPEA, DCM, rt to 40 °C, 2.5 h (71, 22%); (m) 10, POCl3, pyridine, −15 °C to rt, 0.5−3 h (20−66%).
alizations of the R6-group were done using Suzuki−Miyaura
couplings (Scheme 3).
The achiral acylsulfonamide building block 10 was prepared
from the known carbamate 921 via Boc removal in acidic
dioxane.
The commercially available aldehydes 22−31 were used in a
Strecker type reaction involving imine formation with phenyl-
glycine methyl ester (20) or glycine benzyl ester (21) followed
by cyanide addition to afford the α-aminonitriles (Scheme 2).
Full consumption of 20 and 21 was reached after microwave
The P1 subunit 8 was synthesized starting from aldehyde 6
that was treated with potassium cyanide and acetic acid in
MeOH/DCM24 to give cyanohydrin 725 (88% yield, 1:1
diastereomeric ratio, Scheme 1). Partial hydrolysis of the nitrile
in a basic hydrogen peroxide solution followed by Boc
deprotection gave the α-hydroxyamide unit 8 in 34% yield.25
C
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irradiation26,27 at 110−120 °C for 10 min or reflux for 30 min,
with the exception of reactions utilizing paraformaldehyde (24).
In this case, microwave heating at 170 °C for 10 min was
required to give full consumption of the amine. Alcohols 13−
19 were used to incorporate other R6 functionalities and 2- and
1-naphthyl ethanols 13 and 14 were obtained through
reduction of the corresponding carboxylic acids 11 and 12.
Pyridine sulfur trioxide was identified as a convenient oxidizing
agent that allowed a straightforward one-pot transformation of
the alcohols into α-aminonitriles. Thus, the alcohols 13−18
were oxidized to desired aldehydes and then trapped in situ by
the addition of amine 21 and trimethylsilyl cyanide to afford the
desired α-aminonitriles. This protocol was used for all alcohols
with the exception of the 3-cyclohexylpropanol (19), which was
instead oxidized using the Dess−Martin periodinane reagent.
The crude α-aminonitriles were then enriched with HCl gas in
diethylether, followed by a solvent exchange to 1,2-dimethoxy-
ethane (DME) and the addition of oxalyl chloride. Subsequent
cyclization to the N-1, C-6-disubstituted 3,5-dichloro-2(1H)-
pyrazinones 32−48 was achieved either by microwave
irradiation at 145 °C for 25 min in sealed vials28 or reflux
overnight in comparable yields (25−66% over 2 or 3 steps).
Unfortunately, when phenylglycine methyl ester 20 was used in
the synthesis, racemization occurred during the cyclization to
the 2(1H)-pyrazinones (32 and 33). Next, a chemoselective
palladium-catalyzed urea N-arylation coupling was used to
install the C-3 urea moiety.28,29 By using Pd(OAc)2 as a
palladium source, Xantphos as ligand, Cs2CO3 as base, and tert-
butylurea as nucleophile, the products 49−65 were isolated in
moderate to good yields (ranging from 46 to 89%) after
microwave heating at 100−110 °C for 15−20 min or reflux for
30−60 min. It is well-known that the chlorine in position C-3
can be readily substituted in numerous reactions due to the low
electron density on the carbon, leaving the C-5 chlorine moiety
intact.30,31 However, it was surprising to note that complete C-
3 selectivity also was achieved in the synthesis of compounds
60−65 (precursors to target compounds 81−86, Scheme 3),
comprising potentially Pd(0) reactive aryl bromide moieties,
even though an excess of tert-butylurea (3 equiv) was used.
After urea N-arylation, the methyl and benzyl esters were
hydrolyzed, without any decomposition of the urea function-
ality, by microwave irradiation at 110 °C for 15 min in an
aqueous solution of acetonitrile and potassium carbonate.21
The HATU promoted peptide coupling between the carboxylic
acid derived from 49, and the building block 8 in DCM was
slow and gave low yields. To better solubilize the starting
materials, the use of an ultrasonic bath32 was evaluated. This
resulted in full conversion after only 5 min, and the product 66
was isolated in 85% yield. The corresponding peptide coupling
to 67 was performed in DMF, which gave the α-hydroxyamide
product in 62% yield. Finally, oxidation with EDCI, DMSO,
and dichloroacetic acid in DCM gave the final α-ketoamides 68
and 69 in low to modest yields (13% and 64%, respectively).
The low yield of 68 was mainly due to dimerization of the
product during workup. To couple the acyl sulfonamide 10
with the free carboxylic acids derived from esters 49−65, two
peptide coupling protocols were evalutated. Because of the
poor nucleophilicity of 10, much of the starting material
remained intact in the HATU promoted couplings (71, 22%
yield). Instead, phosphoryl chloride in pyridine at low
temperatures provided a more robust protocol, enabling
isolation of the final inhibitors 70 and 72−86 in 23−66% yield.
Scheme 3 illustrates the final decoration of the aryl-bromide
containing pyrazinones 81−86. The final products were
prepared via a microwave-assisted Suzuki−Miyaura coupling
with 3-pyridylboronic acid, which gave 87−92 in poor to
moderate yields (20−69%).33 Even though a large excess of the
boronic acid was used (5 equiv), we never observed the
corresponding C-5 substitution and, furthermore, only traces of
the hydrolyzed tert-butyl urea could be detected.
RESULTS
■
Biochemical Evaluation. The expression and purification
of all enzyme variants were performed according to previously
published methods.14,34 An R155K variant (NS3fl R155K1a), in
which the arginine (R) to lysine (K) mutation was introduced
by PCR at position 155, was cloned in a similar way as has been
described previously.14 The Ki values of the inhibitors 5, 68−92
were obtained in an in vitro assay for the full-length NS3
protein and the central part of the NS4A as cofactor (Tables
1−2).34 The inhibitors in this series have Ki values between
0.11−7.1 μM. Compounds 70, 80, and 87 were also evaluated
in an in vitro assay using the full length protein with amino acid
substitution A156T, D168V, or R155K, presented in Table 3.
Vitality values were calculated to normalize the inhibitory
effects of the inhibitors with respect to the effects from amino
acid substitutions on catalytic efficiency (kcat/Km) of the
enzyme variants. A vitality value less than 1 demonstrates a
more efficient inhibitor against the mutated variant compared
to the wild-type enzyme, while if V > 1, the inhibitor is less
efficient against the mutated virus.35,36 The three inhibitors
evaluated possess vitality values between 0.6 and 2.0.
Scheme 3. Suzuki−Miyaura Cross-Couplings to Give HCV
a
NS3 Inhibitors 87−92
Calculated pKa and log D7.4 Values. Predictions of pKa
and log D7.4 were performed using ADMET Predictor v.5.5.
Calculated pKa values for the acyl sulfonamide of six selected
compounds in this series are found in the range of 4.7−5.1, and
log D7.4 values stretch between 3.8 and 5.0 (Table 4). From
these log D7.4 values, one could expect low solubility and
moderate to high permeability for rule-of-5 compliant
compounds.37
a
Reagents and conditions: (a) 3-pyridylboronic acid, Pd(PPh3)2Cl2,
Na2CO3, H2O, EtOH, DME, MW, 120 °C, 15 min (20−69%).
D
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Table 1. Inhibition of the Full-Length Wild-Type NS3
Protease: Evaluation of the Carbamate to Urea Exchange
and an Electrophilic versus an Acidic Product-Based P1P1′
Residue
Table 2. Inhibition of the Full-Length Wild-Type NS3
Protease: Pyrazinone Containing HCV Inhibitors with
Elongated R6 Substituents
a
Ki values are the average of three separate experiments, with the
a
Ki values are the average of three separate experiments, with the
standard deviation.
standard deviation.
In Vitro Physicochemical and Pharmacokinetic Profil-
ing. Six representative compounds were selected for in vitro
pharmacokinetic profiling: one from the phenylglycine series
(70, Table 1) and five with different R6 substituents from the
glycine series (74, 76, 79, 80, and 87, Table 2). Solubility was
determined in phosphate buffered saline (PBS) at pH 7.4 with a
final concentration of DMSO of 1%. The compounds were
soluble in the lower region, with solubility ranging between
21−53 μM (Table 4). The pyridine containing compound 87
was the most soluble (53 μM) in the series.
The metabolic stability was determined by incubating the
compounds with pooled human liver microsomes (0.5 mg/mL,
0−40 min). In vitro half-life (t1/2) and in vitro intrinsic
clearance (Clint) were calculated using previously published
models,38,39 and the results are presented in Table 4. The Clint
in microsomal incubations gives an estimation of the risk of
oxidative first pass metabolism in vivo. A general classification
is: Clint < 30 (μL/min/mg), no risk for high first metabolism in
vivo; 30 < Clint < 92, moderate risk; Clint > 92, high risk. The
inhibitors evaluated in this series showed in vitro half-life values
(t1/2) between 27 and >100 min and in vitro intrinsic clearance
(Clint) between 13−52 μL/min/mg (Table 4). Thus, the
pyrazinone-based HCV inhibitors tested herein show low to
moderate risk of oxidative first pass metabolism.
The intestinal epithelial permeability, expressed as apparent
permeability coefficients (Papp), was determined from transport
rates across Caco-2 cell monolayers, essentially as described
previously.40 However, a modified assay based on more
physiological media had to be devised because all tested
compounds displayed very strong adsorption to the plastic
ware. Briefly, to minimize the nonspecific binding, fasted state
simulated intestinal fluid (FASSIF)41 was used in the apical
(donor) compartment and a 1% (w/v) bovine serum albumin
(BSA) solution of Hank’s Buffered Salt Solution (HBSS)42 was
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Table 3. Inhibition of the Protease Activity of Wild-Type (wt) and the A156T, D168V, and R155K Variants of the Full-Length
NS3 Protease and Corresponding Vitality Values (V)
a
b
Vitality values (V) were calculated using the equation: V = [Ki × (kcat/Km)]variant/[Ki × (kcat/Km)]wild‑type as described by Dahl et al.14 Previously
reported in Dahl et al.14 Ciluprevir/BILN2061 (93).
c
Table 4. In Vitro Pharmacokinetic Properties and in Silico pKa Values (Acidic Acyl Sulfonamides) and log D7.4 Values of
Compounds 70, 74, 76, 79, 80, and 87
in vitro
in silico
metabolic stability
a
b
c
d
compd
solubility (μM) pH 7.4
Caco-2 Permeability Papp (10‑6 cm/s) a−b
Clint (μL/min/mg)
t1/2 (min)
54
>100
pKa
log D7.4
70
74
76
79
80
87
38
23
22
21
31
53
1.1 0.4
0.3 0.2
26
<13
17
2
3
4.7
4.7
4.7
4.7
4.8
5.1
4.2
4.4
3.8
4.3
5.0
4.1
4.0 1.1
4
81 19
42 16
0.04 0.02
0.12 0.09
7.0 1.5
33 13
52
17
3
27
2
3
82 13
a
b
c
d
Papp = apparent permeability coefficient. a−b = apical to basolateral. Clint = in vitro clearance. t1/2 = in vitro half-life.
used in the basolateral (receiver) compartment. Using this
format, the experiments could be run with good mass balances
above 64%. In our calibrated assay setup, a Papp value below 0.2
× 10−6 cm/s indicates low predicted intestinal permeability, a
Papp value ranging from 0.2 × 10−6 cm/s to 1.6 × 10−6 cm/s
moderate predicted intestinal permeability, and a Papp value
above 1.6 × 10−6 cm/s indicates high predicted intestinal
permeability.43 The permeability data in the a−b direction
showed that compounds (70, 74, 76, and 87) displayed
moderate to high permeability (ranging from 0.3 × 10−6 cm/s
to 7.0 × 10−6 cm/s), while the two R6-alkyl compounds (79
and 80) were poorly permeable compounds. The experimental
setup prevents proper estimation of the potential efflux and P-
glycoprotein interaction due to unknown interaction of the
compounds with excipients and BSA, which will reduce the free
concentrations of the compounds. On the other hand, the
reduction in free concentrations will also result in lower
estimates of the Papp values. Thus, the data reported in Table 4
are under rather than over predictions of the true intestinal
permeability.
Molecular Modeling. The recently released HCV NS3/4A
protease-helicase crystal structure in complex with a macro-
cyclic protease inhibitor (PDB code 4A92) was used for the
molecular modeling studies.44 The active site is situated in the
boundary between the protease and the helicase domain. This
is the first published cocrystal structure of the full-length NS3
protein structure and an inhibitor. The inhibitor displays not
only intermolecular interactions with the protease domain but
also to the helicase domain. This data is in agreement with our
previous docking protocols and studies, which have often
indicated some influence from the helicase domain on protease
inhibitor binding.45 Thus, this X-ray structure was particularly
useful for studying possible interactions between the R6
substituents and the S2 pocket of the protein, which is partly
F
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Figure 3. (A) Compound 88 modeled into the NS3 protease active site (gray surface) of the 4A92 crystal structure. The NS3 helicase domain is
shown in blue surface. Key hydrogen bond interactions are highlighted as red dashed lines. The inhibitor shows π-interactions with Phe154 and
Gln41. (B) Compound 88 (orange carbons) overlaid with the cocrystallized macrocyclic inhibitor (green carbons).
comprised of the helicase domain.44 The inhibitors were
modeled into the protease active site according to the protocol
described in the Supporting Information. In the validation
process of the modeled inhibitors, β-strand mimetics with the
protein backbone were considered. This mimetic is maintained
by a hydrogen-bond pattern between the CO and NH of the
pyrazinone ring and the backbone of Ala157. Molecular
modeling showed that the substituents in the R6 position fit
nicely into the S2 pocket. Compound 88 in Figure 3A shows a
representative binding mode of the modeled inhibitors into the
protein active site. The binding mode shows maintenance of
the hydrogen-bond pattern between the CO and NH of the
pyrazinone ring of compound 88 and the backbone of Ala157,
edge-to-face interaction between the aromatic P1 moiety and
Phe154, a possible π-stacking interaction between the P1′
moiety and Gln41, and an internal hydrogen bond from the
urea NH to the pyrazinone. Furthermore, compound 88 was
overlaid with the macrocyclic inhibitor in the X-ray structure
(Figure 3B), where it can be seen that the R6 substituent of
inhibitor 88 reaches up to the S2 pocket in consistency with the
P2 moiety of the cocrystallized inhibitor.
most promising capping group (R3 group) of the pyrazinone in
terms of inhibition potency but it was accompanied with
instability problems.21
Consequently, the first modification made on this series of
compounds was to replace the carbamate group with a more
stable urea functionality (Table 1). The exchange resulted in
compound 70, containing a tert-butyl urea, which not only
proved to be more robust but also showed an impressive 10-
fold improvement in potency (Ki = 0.39 μM) compared to
compound 5 (Ki = 3.8 μM). Part of the potency enhancement
could be related to entropy gain from potential hydrogen
bonding between the urea and the pyrazinone nitrogen, as
suggested from molecular modeling (see Figure 3A). Hence,
the tert-butyl urea was successfully used throughout the series
synthesized in this study. Regarding the P1P1′ unit, we decided
to further explore the aromatic acyl sulfonamide (5, 70−72,
Table 1), and the same electrophilic α-ketoamide as in
compound 2 (68 and 69, Table 1). In combination with the
pyrazinone structure, the α-ketoamide functionality was less
favorable (10-fold) as compared to the aromatic acyl
sulfonamide residue (68 and 69, Ki = 6.2 and 7.1 μM vs 70
and 71, Ki = 0.39 and 0.66 μM, respectively). These
compounds were also used to confirm the possibility to reach
the S2 pocket from the R6 position of the pyrazinone core (i.e.,
moving the P2 side chain to the R6 position of the P3
pyrazinone), as suggested by modeling (Figure 3). Gratifyingly,
almost equipotent glycine compounds 69 and 71 (Ki = 7.1 μM
and 0.66 μM, respectively) were achieved compared to the
corresponding phenylglycine compounds 68 and 70 (Ki = 6.2
and 0.39 μM, respectively). These results posed an obvious
question; whether it is possible to benefit from both the phenyl
glycine and a benzyl in the R6 position or if they should occupy
the same pocket. Compound 72 (Ki = 0.70 μM) was
synthesized accordingly but showed no advantages over the
glycine-based inhibitors. Therefore, we were encouraged to
further explore achiral inhibitors and improve inhibitory
potency via optimizations of the R6 substituents of the
pyrazinone.
DISCUSSION
■
With the overall aim of developing HCV NS3 protease
inhibitors different from the advanced and widely explored
peptide-based compounds with a central P2 proline or a proline
mimic (see, e.g., compounds 1−4, Figure 1), we wanted to
explore a pyrazinone ring combined with an amino acid as a
central P3P2 scaffold in HCV NS3 protease inhibitors. The
preliminary inhibitors (e.g., compound 5, Figure 2) were
evaluated as product-based inhibitors, meaning encompassing
an acidic P1P1′ group, an acyl sulfonamide, but yet without a
large P2 extension. Potent product-based inhibitors normally
require a big P2 substituent to occupy the large S2 pocket (see
compounds 3 and 4). This is in contrast to the electrophilic
(serine trapping) compounds, such as 1 and 2, having smaller
P2 groups. Therefore, we felt prompted to further explore
pyrazinone-based compounds in two ways: either encompass-
ing an electrophilic P1P1′ group or an acidic P1P1′ group
combined with an expanded P2 substituent. Furthermore, a
carbamate functionality had previously been identified as the
The structure−activity relationship of the R6 group was
studied with preserved functionalities in the rest of the
structure, i.e., the tert-butyl urea on the R3 position in
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combination with the aromatic P1P1′ scaffold (see compounds
73−92, Table 2). The R6 substituents were chosen to address
the preferred length and direction of the substituent in this new
type of inhibitors. Hence, elongated phenyl containing
substituent of different linker lengths and corresponding alkyl
substituents were evaluated, as were naphthyl and various
bromo- and pyridine-substituted phenyl and benzyl substitu-
ents. Overall, it is clear that the R6 substituent occupies a rather
large lipophilic pocket because the inhibitory potency is
enhanced by several of the larger substituents with Ki values
down to 0.11 μM (80). However, interactions are not
completely unspecific because elongations in some directions
are not allowed, exemplified by the ortho-pyridyl comprising 89
and 92 (Ki = 2.1 and 1.1 μM, respectively). In line with this, the
inhibitory potency decreased considerably in the absence of an
R6 group, as demonstrated by compound 73 (Ki = 1.9 μM)
compared to benzyl 71 (Ki = 0.66 μM). Elongation of the
benzyl compound 71 via a methylene spacer gave phenethyl 74
with a 5-fold improved potency (Ki = 0.12 μM), and further
elongation by one carbon resulted in comparable inhibitory
potency (75, Ki = 0.14 μM). Replacing a methylene with an
oxygen gave the less hydrophobic compound 76 (Ki = 0.56
μM), but this alteration resulted in a less potent inhibitor. The
corresponding naphthyl groups gave somewhat less potent
inhibitors (77, Ki = 0.33 μM and 78, Ki = 0.29 μM), however,
compared to benzyl 71, this alteration was well accepted.
Molecular modeling suggested that a R6 phenyl equipped
with bulky substituents (e.g., a pyridyl, Figure 3) could establish
important interactions with the S2 pocket. Consequently, we
decided to synthesize bromoaryls as intermediate inhibitors that
also should allow further functionalization using metal catalyzed
cross coupling reactions. The ortho-, meta-, and para-bromo
phenyl analogues 81−83 (Ki = 0.38, 0.60, and 0.40 μM,
respectively) provided improved or similar inhibitory potencies
as the benzyl compound 71 (Ki = 0.66 μM), although, from
these results, the preferred direction of the substituent on the
aryl could not be identified. The R6 bromo-benzyl analogues
84−86 (Ki = 0.44, 0.29, and 0.25 μM, respectively) showed a
slight overall enhanced or similar inhibitory potencies
compared to the corresponding analogous phenyl. Again,
there was no clear preference for a particular regioisomer. We
anticipated that introduction of a heteroaromatic group in these
positions could be useful in order to reach interactions with the
S2 pocket as well as influence the pharmacokinetic properties
and solubility. The majority of the HCV NS3 protease
inhibitors in clinical studies contain a heteroaromatic P2
substituent which has proven beneficial for activity and
pharmacokinetic properties.8,46 Thus, compounds 87−92
were prepared comprising ortho-, meta-, and para-3-pyridyls,
however, no significant improvement in potency was observed.
Interestingly, an ortho-pyridyl was not well tolerated, giving a
4−5-fold decrease in potency compared with the ortho-bromo
equivalents (e.g., 83, Ki = 0.40 μM vs 89, 2.1 μM, and 86, 0.25
μM vs 92, 1.1 μM). According to molecular modeling (see
Figure S1, Supporting Information), the ortho-bromo com-
pounds seem to direct the bromo-substituent into the S2
pocket in analogy with the majority of the compounds in the
series (i.e., meta and para). However, for the inhibitors with the
pyridyl substituent in the ortho position, modeling suggests
poses where the pyridyl group is directed in the opposite
direction, i.e., out from the S2 pocket, and instead forming
hydrophobic interaction with the inhibitor itself. In these poses,
a distortion of the inhibitor backbone is also seen, which
altogether may account for the loss in inhibitory potency.
The high aromatic ring content raised concerns about
potential pharmacokinetic issues as it is considered to be less
suitable in a bioavailable drug candidate.47−49 Hence, we
speculated if a simple alkyl chain could retain the potency
possessed by their aromatic counterparts. Indeed, the pentyl
containing compound 79 revealed an impressive inhibitory
potency (Ki = 0.16 μM) comparable with the most potent
aromatic analogue (phenethyl 74, Ki = 0.12 μM). In analogy,
the corresponding cyclohexyl variant 80 (Ki = 0.11 μM) turned
out to be the most potent inhibitor in this set of compounds.
The total SAR accomplished from the R6 substituent study in
Table 2 indicates that the potency loss suffered from removal of
the classical P2 side chain indeed could be regained by
elongation of a variety of lipophilic, but not too sterically
hindered, R6 substituents. Consequently, there is a freedom for
the actual lead optimization strategy chosen for the R6 group to
be guided by other drug properties, i.e., resistance profile or
pharmacokinetic data.
Substitutions of the residues at positions 155, 156, and 168 in
the HCV NS3 protease are among the most frequently
observed mutations in vitro and in vivo during clinical studies
evaluating HCV NS3 protease inhibitors.50 These resistance
mutations impair the efficacy of the direct-acting antiviral drugs
on the market as well as those in clinical trials.12,50,51 Because
there is a risk for cross-resistance,12 future NS3 protease
inhibitors should be designed to retain potency against cross-
resistant enzyme variants. Accordingly, three inhibitors 70, 80,
and 87, were selected for evaluation toward the resistant
enzyme variants A156T, D168V, and R155K (Table 3). The
compounds were chosen based on their promising inhibitory
potencies and their structural diversity: one compound from
the phenylglycine series (70) and one alkyl and aromatic
analogue from the achiral glycine series (80 and 87). It is
reported that mutations leading to amino acid substitutions at
Ala156 cause resistance to most of the evaluated HCV NS3
protease inhibitors.12,52,53 These mutations result in increased
bulk and steric repulsion between the enzyme and the P2 and
P4 residues of the inhibitor.53 It is inaccurate to compare
inhibition constants (Ki values) between enzyme variants of
different catalytic efficiencies (kcat/Km). Therefore, vitality
values (V) that normalize for this were calculated (Table 3).
The first proof-of-concept HCV NS3 protease inhibitor 93
(Ciluprevir/BILN2061)54 loses inhibitory potency dramatically
against the A156T variant, showing a vitality value of 1600.
Also, inhibitor 1 loses inhibitory potency substantially, with a
vitality value of 480. According to the vitality values for
inhibition of the A156T variant, the inhibitors 70, 80, and 87
were able to retain most of their inhibition potency in relation
to the wild-type enzyme. Interestingly, the achiral inhibitors 80
(V = 1.3) and 87 (V = 1.4) showed a more promising potency
compared to the racemic inhibitor 70 with a V value of 2.0. The
D168V mutated form of the protease is known to dramatically
affect macrocyclic inhibitors with large P2 substituents.52,55
This is supported by a vitality value of 3200 for macrocyclic
inhibitor 93, comprising a bulky P2 group, and a vitality value
of 0.4 for the linear inhibitor 1 lacking a large P2 group. Asp168
is positioned between the S2 and the S4 pocket and forms salt
bridges to Arg123 in S4 and Arg155 in S2. A mutation at
Asp168 disrupts the stabilizing effect and leads to weaker
interactions of the inhibitor with the S2 and S4 pockets.11,53
Evaluation against the D168V mutated form resulted in vitality
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Article
values close to 1, which implies maintained efficacy compared
to the wild-type enzyme. This may be due to the smaller size of
the P2 substituent, which will suffer less from changes in the S2
pocket. Arg155 is positioned in the S2 pocket and thereby has
the ability to interact with large P2 substituents. Mutations
leading to substitutions at the Arg155 position appear to be
most crucial presently because they lead to cross-resistance to
all HCV NS3 protease inhibitors in advanced clinical
development.12 The potencies for both reference substances,
1 and 93, were indeed negatively affected by amino acid
substitution R155K (V = 24 and 4300, respectively). Amino
acid substitutions at this position could disrupt the previously
mentioned salt bridge with Asp168, resulting in resistance to
inhibitors with interactions in this region.11,53 Evaluation of the
three inhibitors 70, 80, and 87 against R155K produced
encouraging results. The vitality values were determined to be
less than 1, indicating that these early stage inhibitors are more
efficient against the mutated form compared to the wild-type
enzyme. Thus, the pyrazinone-based inhibitors can be regarded
as promising lead compounds in terms of drug resistance
profile. However, further improvements in the overall
inhibitory potencies are still highly warranted.
The in vitro solubility, metabolic stability, and permeability
were determined for six representative compounds: one from
the phenylglycine series (70) and five from the achiral glycine
series (74, 76, 79, 80, and 87), as shown in Table 4. We hoped
that such data could aid future development of the lead
compounds derived herein. All evaluated inhibitors have
solubility higher than 20 μM (corresponds roughly to >15
μg/mL), which imply that solubility is not a major problem for
this series of inhibitors. Nevertheless, the extensive adsorption
seen with plastics in the Caco-2 assay implies that care must be
taken when designing in vitro experiments for this type of
lipophilic peptidomimetics. Considering that the pyridine
comprising analogue 87 was the most soluble (56 μM/44
μg/mL), modifications using pyridines or other heteroaryls
should be considered in future designed inhibitors. The in vitro
intrinsic clearance measurement, performed in human liver
microsomes of the compounds, indicated a low or moderate
risk for high oxidative first pass metabolism in vivo for this class
of compounds. This is pleasing results given their high
lipophilicity (log D7.4 = 3.8−5.0), high molecular weight
(700−785 g/mol), and aromatic nature. In addition, the
pyrazinone-based compounds with alkyls in R6 position (79
and 80) are metabolically less stable than the corresponding
aryl comprising analogues (74, 76, and 87), with intrinsic
clearance values of >30 μL/min/mg (apparent moderate risk)
in the former cases and <20 μL/min/mg (apparent no risk) in
the latter cases. Finally, the permeability data shown in Table 4
indicate widely different permeabilities of the compounds,
although they only differ in the R6 position. The two alkyl
comprising compounds 79 and 80 displayed low permeability,
and the phenylglycine compound 70 and the glycine analogue
with a phenethyl R6 substituent 74 displayed moderate
permeability, whereas the two compounds comprising one
heteroatom in the R6 group, i.e., the ether analogue 76 and the
pyridyl analogue 87 were highly permeable compounds. Thus,
the presence of a pyridine has a positive influence on the in
vitro pharmacokinetic properties in general. Notably, the
modified Caco-2 assay mimics both the intestinal compartment
in the donor chamber and the blood in the receiving
compartment. The tradeoff is that at present accurate
determination of the free compound concentrations, driving
permeability and active efflux of these compounds, cannot be
estimated.
CONCLUSION
■
The evaluated series of peptidomimetic HCV NS3 protease
inhibitors based on a P3 2(1H)-pyrazinone scaffold benefited
from an aryl acyl sulfonamide-based P1P1′ group, in contrast to
corresponding electrophilic α-keto amides. A key improvement
was further achieved by an exchange of an unstable carbamate
P3 capping group to a robust urea, which resulted in a 10-fold
increase in inhibitory potency. Results from the SAR study
indicated that the P2 side chain could be transferred to the R6
position on the P3 pyrazinone, giving achiral structures, and
further optimized using lipophilic but not too sterically
hindered substituents. Furthermore, the same substituents
were found to significantly influence the pharmacokinetic
properties of the compounds. Even though aliphatic R6
substituents were allowed in terms of potency, contrasting
results were achieved from pharmacokinetic evaluations, which
instead revealed a beneficial effect of a 3-pyridyl group.
Accordingly, further elaboration in this position is warranted
and is currently underway in our laboratory. Indeed, the
developed synthetic pathway enables a wide variety of structural
modifications to be performed in the future design of improved
inhibitors. Evaluation of the inhibitors against drug-resistant
variants of the full-length NS3 protease revealed retained
inhibitory potencies compared to the wild-type enzyme. Taken
together, these results combined with the unique structural
features and promising in vitro pharmacokinetic properties
prompt further optimization of this novel class of achiral drug
leads.
EXPERIMENTAL SECTION
■
General Methods. Microwave-assisted reactions were performed
in sealed vials dedicated for microwave processing, using a Smith
synthesizer. NMR spectra were recorded on a Varian Mercury Plus for
1H at 399.9 MHz and for 13C NMR at 100.5 MHz. Analytical HPLC-
UV/MS analysis of pure products were performed on a Gilson HPLC
system with a Chromolith SpeedROD RP-18e column (50 mm × 4.6
mm) equipped with a Finnigan AQA quadrupole mass spectrometer
using a 4 mL/min MeCN/H2O gradient (0.05% HCOOH) and
detection by UV (DAD) and MS (ESI+). All compounds were
determined to be >95% pure by HPLC-UV at 254 nm.
Representative Total Synthesis of an Inhibitor (Compound
80). Benzyl 2-(3,5-Dichloro-6-(2-cyclohexylethyl)-2-oxopyrazin-
1(2H)-yl)acetate (40). A 50 mL round-bottom flask was loaded with
3-cyclohexyl-1-propanol (0.55 g, 3.87 mmol), dry DCM (25 mL), and
Dess−Martin periodinane (1.64 g, 3.87 mmol). The reaction was
stirred in rt for 30 min and then washed with 25 mL of saturated
NaHCO3, dried over MgSO4, and evaporated. A solution of glycine
benzyl ester hydrochloride (0.65 g, 3.22 mmol) and DIPEA (0.70 mL,
4.02 mmol) in DCM (25 mL) was added to the crude residue.
Trimethylsilyl cyanide (0.45 mL, 3.54 mmol) was added after 0.5 min,
and the reaction was refluxed for 1 h. The solvent was removed under
reduced pressure and the residue flushed through a silica plug and
eluted with EtOAc:isohexane (20:80). The crude residue was taken up
in 15 mL of diethyl ether and transferred to a 20 mL Smith vial. HCl
gas was bubbled through the reaction mixture for 5 min, followed by
evaporation of the solvent. Oxalyl chloride (0.85 mL, 9.68 mmol) and
DME (10 mL) was added, and the vial was capped and irradiated with
MW to 145 °C for 25 min.28 The crude product was, after evaporation
of the solvent, purified by silica column flash chromatography using
EtOAc:isohexane (10:90 to 30:70) as eluent and gave 40 in 28% yield,
1
0.38 g as pale-yellow solid. H NMR (CDCl3) δ 7.38−7.30 (m, 5H),
5.21 (s, 2H), 4.80 (s, 2H), 2.58 (m, 2H), 1.73−1.61 (m, 5H), 1.36 (m,
2H), 1.31 (m, 4H), 0.93−0.80 (m, 2H). 13C NMR (CDCl3) δ 166.0,
I
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Journal of Medicinal Chemistry
Article
152.6, 143.2, 139.5, 134.6, 128.8, 128.7, 128.5, 123.5, 68.1, 47.3, 37.5,
34.2, 32.7, 27.8, 26.3, 26.0. ESI-MS (m/z) 423 (M + H+). HRMS calcd
for C21H24Cl2N2O3 (M + H+) 423.1242; found 423.1237.
Notes
The authors declare no competing financial interest.
Benzyl 2-(3-(3-(tert-Butyl)ureido)-5-chloro-6-(2-cyclohexylethyl)-
2-oxopyrazin-1(2H)-yl)acetate (57). A 5 mL microwave process vial
was charged with 40 (210 mg, 0.50 mmol), tert-butylurea (290 mg,
2.50 mmol), Pd(OAc)2 (6 mg, 0.03 mmol), Xantphos (23 mg, 0.04
mmol), Cs2CO3 (325 mg, 1.00 mmol), and DME (5 mL). The vial
was capped under air, and the reaction mixture was irradiated with
MW to 100 °C for 15 min. The solids were filtered off and the residue
concentrated under reduced pressure. The crude product was purified
by silica column flash chromatography and eluted with EtOAc:isohex-
ane (15:85 to 30:70) to afford 57 in 72% yield, 181 mg as white solid.
1H NMR (CDCl3) δ 8.55 (br s, NH), 7.88 (br s, NH), 7.39−7.30 (m,
5H), 5.20 (s, 2H), 4.77 (s, 2H), 2.51 (m, 2H), 1.72−1.61 (m, 5H),
1.41 (s, 9H), 1.32 (m, 2H), 1.27−1.08 (m, 4H), 0.92−0.81 (m, 2H).
13C NMR (CDCl3) δ 166.4, 151.4, 150.7, 143.7, 134.7, 128.9, 128.8,
128.7, 128.5, 122.4, 68.0, 51.0, 46.3, 37.5, 35.0, 32.8, 28.9, 27.1, 26.4,
26.1. ESI-MS (m/z) 503 (M + H+). HRMS calcd for C26H35ClN4O5
(M + H+) 503.2425; found 503.2420.
ACKNOWLEDGMENTS
■
We thank Johan Gustafson for the synthesis of compounds 36,
39 and 40, and Benjamin Schmuck and Alexander Sachse for
their assistance in the construction, expression, and purification
of the R155K HCV NS3 variant and Dr. Luke R. Odell for
constructive proofreading of the manuscript. We also thank
Simulations Plus for providing access to the ADMET Predictor
software and the Swedish Research Council and Alice
Wallenberg Foundation for financial support.
ABBREVIATIONS USED
■
HCV, hepatitis C virus; DME, dimethoxyethane; Xantphos,
4,5-bis(diphenylphosphino)-9,9-dimethylxanthene; HATU, 1-
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxid hexafluorophosphate; EDCI, 3-(ethylimino-
methyleneamino)-N,N-dimethylpropan-1-amine. Clint, intrinsic
clearance; Papp, apparent permeability coefficient
2-(2-(3-(3-(tert-Butyl)ureido)-5-chloro-6-(2-cyclohexylethyl)-2-ox-
opyrazin-1(2H)-yl)acetamido)-N-((4-(trifluoromethyl)phenyl)-
sulfonyl)benzamide (80). A 5 mL microwave process vial was charged
with 57 (125 mg, 0.25 mmol), K2CO3 (70 mg, 0.51 mmol), MeCN
(2.0 mL), and H2O (1.0 mL), and the vial was capped and irradiated
by MW to 110 °C for 15 min. After cooling to rt, 1.0 M HCl (20 mL)
was added and the mixture was extracted with EtOAc (2 × 20 mL).
The organic layers were dried over MgSO4 and evaporated.21 The
crude acid and 6 (143 mg, 0.38 mmol) were dissolved in pyridine (4
mL) and cooled to −15 °C under N2 atmosphere. POCl3 (26 μL, 0.28
mmol) was added, and the reaction mixture was stirred at −15 °C for
15 min before removal of the cold bath. After 30 min stirring in rt,
water (20 mL) was added and the pH adjusted to 1 using 6.0 M HCl
followed by extraction with EtOAc (2 × 20 mL). The organic layer
was dried over MgSO4 and evaporated, and the crude product was
purified by silica column flash chromatography two times, first eluting
with EtOAc:isohexane:HCOOH (20:80:3 to 30:70:3), and in the
second column the product was eluted with MeOH:DCM (1:100 to
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* Supporting Information
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Wickstrom, K.; Hamelink, E.; Edlund, M.; Vrang, L.; Vendeville, S.;
̈
Additional experimental details concerning synthesis, character-
ization, and spectra (1H, 13C NMR, LC-UV/MS) of novel
compounds (33, 34, 36−92), inhibition assay, molecular
modeling, metabolic stability assay, and solubility determi-
nation. This material is available free of charge via the Internet
at http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: 46-18-471-4957. Fax: 46-18-471-4474. E-mail: Anja.
Sandstrom@orgfarm.uu.se.
Author Contributions
#J.G. and A.K.B. contributed equally. The manuscript was
written through contributions of all authors. All authors have
given approval to the final version of the manuscript.
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