Discovery of novel highly potent hepatitis c virus NS5A inhibitor (AV4025)

Article abstract of DOI:10.1021/jm500951r

A series of next in class small-molecule hepatitis C virus (HCV) NS5A inhibitors with picomolar potency containing 2-pyrrolidin-2-yl-5-{4-[4-(2-pyrrolidin-2-yl-1H-imidazol-5-yl)buta-1,3-diynyl]phenyl}-1H-imidazole cores was designed based on the SAR studies available for the reported NS5A inhibitors. Compound 13a (AV4025), with (S,S,S,S)-stereochemistry (EC₅₀ = 3.4 ± 0.2 pM, HCV replicon genotype 1b), was dramatically more active than were the compounds with two (S)- and two (R)-chiral centers. Human serum did not significantly reduce the antiviral activity (<4-fold). Relatively favorable pharmacokinetic features and good oral bioavailability were observed during animal studies. Compound 13a was well tolerated in rodents (in mice, LD₅₀ = 2326 mg/kg or higher), providing a relatively high therapeutic index. During safety, pharmacology and subchronic toxicity studies in rats and dogs, it was not associated with any significant pathological or clinical findings. This compound is currently being evaluated in phase I/II clinical trials for the treatment of HCV infection.

Full text of DOI:10.1021/jm500951r

Article
pubs.acs.org/jmc
Discovery of Novel Highly Potent Hepatitis C Virus NS5A Inhibitor
(AV4025)
Alexandre V. Ivachtchenko,†,‡,§ Oleg D. Mitkin,‡ Pavel M. Yamanushkin,‡ Irina V. Kuznetsova,‡
Elena A. Bulanova,‡ Natalia A. Shevkun,‡ Angela G. Koryakova,‡ Ruben N. Karapetian,‡
Vadim V. Bichko,†,§ Andrey S. Trifelenkov,‡ Dmitry V. Kravchenko,‡ Natalia V. Vostokova,‡
Mark S. Veselov,∥ Nina V. Chufarova,∥ and Yan A. Ivanenkov*,‡,§,∥
†Alla Chem LLC, 1835 East Hallandale Beach Boulevard 442, Hallandale Beach, Florida 33009, United States
‡Chemical Diversity Research Institute, Rabochaya Street 2a, Khimki, Moscow Region 141401, Russia
§ChemDiv, 6605 Nancy Ridge Drive San Diego, California 92121, United States
∥Moscow Institute of Physics and Technology (State University), 9 Institutskiy Lane, Dolgoprudny City, Moscow Region 141700,
Russia
S
* Supporting Information
ABSTRACT: A series of next in class small-molecule hepatitis
C virus (HCV) NS5A inhibitors with picomolar potency
containing 2-pyrrolidin-2-yl-5-{4-[4-(2-pyrrolidin-2-yl-1H-imi-
dazol-5-yl)buta-1,3-diynyl]phenyl}-1H-imidazole cores was de-
signed based on the SAR studies available for the reported
NS5A inhibitors. Compound 13a (AV4025), with (S,S,S,S)-
stereochemistry (EC50 = 3.4 0.2 pM, HCV replicon genotype
1b), was dramatically more active than were the compounds
with two (S)- and two (R)-chiral centers. Human serum did not
significantly reduce the antiviral activity (<4-fold). Relatively
favorable pharmacokinetic features and good oral bioavailability
were observed during animal studies. Compound 13a was well
tolerated in rodents (in mice, LD50 = 2326 mg/kg or higher),
providing a relatively high therapeutic index. During safety, pharmacology and subchronic toxicity studies in rats and dogs, it was
not associated with any significant pathological or clinical findings. This compound is currently being evaluated in phase I/II
clinical trials for the treatment of HCV infection.
HCV protein NS5A,4 have attracted increasing attention. NS5A
is one of the key components in the HCV replication complex
INTRODUCTION
■
Hepatitis C infection caused by HCV is among the most
essential for virus RNA replication in host cells. It has
common liver diseases and is widespread throughout the world.
successfully been validated in clinics as an antihepatitis C
On the basis of annual World Health Organization (WHO)
reports, more than 130−150 million people are infected and
more than 350−500 K individuals die from HCV-related liver
drug target with a high therapeutic potential.5,6 A number of
NS5A inhibitors generally bearing a peptidomimetic core have
pathologies.1 In accordance with the Centers for Disease
recently been developed and show nano- and picomolar activity
in vitro.7−14 Their structures can be roughly generalized by the
Control and Prevention statistical estimation, approximately 3.2
million people are chronically infected in the USA.2 Acute
disease states are frequently observed after long-term and
simplified topological pharmacophore hypothesis (Figure 1).
Their backbones consist of two amino-acid or peptide-based
fragments (Capi, Capj) connected with linkers of different types
asymptomatic periods. Approximately 75−85% of newly
and lengths (L).
infected persons become chronically infected. Among these
Representative examples of reported NS5A inhibitors (1−
patients, 60−70% will suffer chronic liver disease. In 5−20% of
12) fitting this pharmacophore are listed in Table 1.13,16−23 As
cases, cirrhosis or liver cancer is diagnosed, resulting in 1−5%
shown in Table 1, the first-in-class NS5A inhibitor, 1 (BMS-
lethal outcomes. It is not surprising that HCV is the leading
790052),33 contains four (S)-chiral centers. Other known
NS5A ligands share four (2−9), five (10 [GS-5885], 11 [MK-
8742]), and six (12 [ABT-267]) chiral centers. A relatively rigid
indication for liver transplantation.3
Recent years have seen a significant change in HCV
treatment. Much of the challenge in current antiviral chemo-
therapy should address the development of novel direct-acting
agents with a clear mechanism of action. Novel biological
targets with high therapeutic value, including nonstructural
Received: June 23, 2014
Published: August 22, 2014
© 2014 American Chemical Society
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Table 1. Examples of Known NS5A Inhibitors with High
Activity against HCV Replicon GT1b
Figure 1. Common topological pharmacophore model for symmetric
and pseudosymmetric NS5A inhibitors.
spacer of an optimal length is much more appropriate for good
activity than a flexible one. Imidazole (in, e.g., 1 and 2) and its
isosteric and bioisosteric analogues, including simple benzimi-
dazole (e.g., 3 [IDX-719]) or nontrivial carboxamide replace-
ments (e.g., 12), can be reasonably regarded as privileged
substructures. However, the spatial orientations of the
substituents within the cap regions are crucial for tight binding.
A detailed overview of possible binding modes predicted for
compound 13a and other NS5A inhibitors using 3D-molecular
docking studies is discussed below. The main drawback of the
NS5A inhibitors currently being evaluated in clinical trials is in
their relatively poor bioavailability and suboptimal pharmaco-
kinetic properties. For example, in 2009, Arrow Therapeutics
discontinued the phase II development of AZD-2836 and AZD-
7295 (standard oral formulation) against hepatitis C mainly
because of their inappropriate pharmacokinetic profiles.15 On
the other hand, compound 1 is an antiviral drug developed by
Bristol-Myers Squibb that is currently undergoing registration
in Japan for the treatment of chronic hepatitis C infection
(genotype 1b) in combination with asunaprevir.
RESULTS AND DISCUSSION
■
As shown in Table 1, two NS5A inhibitors (7 and 8) possessing
diynyl-imidazole and diynyl-diphenyl-imidazole linkers showed
target activities of 1.332 and 0.384 nM, respectively, with spacer
lengths of 14
1 and 21
1 Å. Interestingly, much more
potent analogues, e.g., 11, 12, and 1, have linker lengths in the
median range of 19 2 Å. On the basis of these observations
and related in silico modeling, we have tentatively suggested
that diynyl-monophenyl-imidazole linkers (19−21 Å) can
provide superior binding. To establish whether novel linkers
with a predefined length could indeed serve as appropriate
bioisosteric replacements for previously reported spacers, a
relatively small pilot library of 17 small molecule potential α-
helix mimetics (13a−k) was synthesized and evaluated against
the HCV genotype 1b replicon in cell-based assays (Table 2). A
detailed description of the performed biological trial is provided
in the Experimental Section (vide infra). On the basis of the
available structure−activity relationship (SAR) information and
our in silico modeling, we have concluded that a rigid
diynylbenzene linker of an optimal length could position the
caps appropriately for good binding and exhibit an appropriate
pharmacokinetic (PK) profile because the obtained compounds
have lower molecular weights (MW = 710.84, 13a) than do
other reported NS5A inhibitors with similar geometries and
structures, e.g., 1 (MW = 738.89) and 11 (MW = 881.05).
With respect to lipophilicity and aqueous solubility, the
following descriptors were calculated: 13a (Log P = 4.99), 1
(Log P = 6.33), and 11 (Log P = 8.61), 13a and 1 (PSA =
174.6 Å), 11 (PSA = 188.8 Å). Therefore, we predicted
comparable or even more beneficial PK features for our
compounds.
a
The distance between two key chiral points (arrow indicated, 1) was
calculated for different minimized (in vacuo) conformations using
MOE Software for all the compounds presented.23 IC50 value
b
The pseudosymmetric (S)-2-(5-{4-[4-((S)-2-pyrrolidin-2-yl-
3H-imidazol-4-yl)-phenyl]-buta-1,3-diynyl}-1H-imidazol-2-yl)-
pyrrolidine dihydrochlorides 13a−f were obtained according to
the synthetic route depicted in Scheme 1. A Sonogashira cross-
coupling reaction between (S)-2-[5-(4-iodo-phenyl)-1H-imida-
c
according to refs 16 and17. Activity determined in this study (see the
Experimental Section and conditions therein). IC50 value according
to ref 18. IC50 value according to ref 19. IC50 value according to ref
d
e
f
g
h
20. IC50 value according to ref 13. IC50 value according to ref 16.
i
j
IC50 value according to ref 21. IC50 value according to ref 22.
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reported previously, the intermediate was subsequently
converted into a series of promising amide derivatives. After
the protecting group was removed in acidic medium, the
acylation of 21a by N-Moc-amino acids 22a−f was conven-
iently performed, providing the final subset of compounds
13a−f in moderate-to-high yields (see Experimental Section).
The desired products were obtained as the optically pure
stereomers 13a−c containing four chiral centers, as mixtures of
the stereoisomers 13d,e containing two pure (S)-chiral centers,
and as the single stereomer 13f bearing two (S)-chiral centers.
The asymmetrically substituted methyl [(S)-1-((S)-2-{5-[4-
(4-{2-[(S)-1-((R)- 13g and methyl [(R)-1-((S)-2-{5-[4-(4-{2-
[(S)-1-((S)-2-methoxycarbonylamino-3-methyl-butyryl)-pyrro-
lidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-phenyl]-1H-imi-
dazol-2-yl}-pyrrolidine-1-carbonyl)-2-methyl-propyl]-carba-
mate dihydrochloride 13h was prepared starting from the
intermediate 18a and methyl {(S)-1-[(S)-19b methyl {(R)-1-
[(S)-2-(5-iodo-1H-imidazol-2-yl)-pyrrolidine-1-carbonyl]-2-
methyl-propyl}-carbamate 19c (Scheme 2) under conditions
similar to those described above (vide supra, Scheme 1).
Methyl [(S)-1-((S)-2-{5-[4-(4-{2-[(R)-1-((S)-2-methoxycar-
bonylamino-3-methyl-butyryl)-pyrrolidin-2-yl]-3H-imidazol-4-
yl}-buta-1,3-diynyl)-phenyl]-1H-imidazol-2-yl}-pyrrolidine-1-
carbonyl)-2-methyl-propyl]-carbamate dihydrochloride 13i was
also prepared starting from the intermediate 18a and tert-butyl
(R)-2-(5-iodo-1H-imidazol-2-yl)-pyrrolidine-1-carbonate 19d
(Scheme 3) under the same reaction conditions.
Table 2. Antiviral Activity of Compounds 13a−k against
HCV Genotype 1b Replicon
Finally, the symmetrically substituted methyl [1-((R)-2-{5-
[4-(4-{2-[(R)-1-(2-methoxycarbonylamino-3-methyl-butyryl)-
pyrrolidin-2-yl]-3H-imidazol-4-yl}-phenyl)-buta-1,3-diynyl]-
1H-imidazol-2-yl}-pyrrolidine-1-carbonyl)-2-methyl-propyl]-
carbamate dihydrochlorides 13j,k were prepared following the
methodology described above (see Scheme 1) starting from
tert-butyl (R)-2-[5-(4-iodo-phenyl)-1H-imidazol-2-yl]-pyrroli-
dine-1-carboxylate 14b and using 5-iodo-1H-imidazol 19d as
an intermediate compound (Scheme 4).
As shown in Table 2, among the tested compounds,
compound 13a was the most active against HCV replicon
GT1b and showed an EC50 value of 0.003 nM, which was
comparable to that observed for other known NS5A inhibitors
with four chiral centers, e.g., 1, 3, 11, and 12 (Table 1, our data
for 1 is 0.003 0.001 nM). A clear relationship between the
target activity and stereospecificity of compounds 13a−k was
revealed. Thus, compound 13h, which contains one (R)-chiral
center at the amino acid residue area, Cap1 and three (S)-chiral
centers, was more than 120-fold less active than compound 13a
under the same biological conditions. Dramatic decreases in
activity (23- and 13814-fold reductions) were observed for
compounds 13g and 13b, bearing one (R)- and two (R)-chiral
points in the Cap1 and Cap2 regions, respectively. An even
greater decrease was observed for compounds with (R)-chiral
centers incorporated into the pyrrolidine ring. For instance,
compound 13i, which contains an (R)-chiral center in the
pyrrolidine moiety of Cap2, was found to be 1580-fold less
active than compound 13a. Molecules with two and four (R)-
chiral centers (13j and 13k) in the pyrrolidine rings (Cap1 and
Cap2) were almost 260000- and 290000-fold less active than
the hit compound. The replacement of the N-Moc-L-valine
moiety in compound 13a by N-Moc-(S)-3-aminotetrahydrofur-
an-3-carboxylic acid (13c) or N-Moc-2-methylalanine (13f) led
to dramatic reductions in the target activity of 7158- and
176176-fold, respectively. Compound 13a showed relatively
low cytotoxicity (CC50 = 17.3 μM, Table 2) and an excellent
a
Three independent tests were performed.
zol-2-yl]-pyrrolidine-1-carboxylic acid tert-butyl ester 14a and
buta-1,3-diynyl-trimethyl-silane 16, obtained from 1,4-bis-
trimethylsilanyl-buta-1,3-diyne 15, yielded the corresponding
tert-butyl (S)-2-{5-[4-(4-trimethylsilanyl-buta-1,3-diynyl)-phe-
nyl]-1H-imidazol-2-yl}-pyrrolidine-1-carbonate 17a. It should
be noted that other synthetic pathways can be applied to
intermediate compound 17a, for example, Suzuki cross-
coupling reaction via dioxaborolane derivatives24 or via the
corresponding boronic acid.25 The trimethylsilyl group in the
intermediate compound 17a was removed, and then Sonoga-
shira cross-coupling was carried out again between the acid tert-
butyl (S)-2-(5-iodo-1H-imidazol-2-yl)-pyrrolidine-1-carboxylate
19a and compound 18a. As a result, the synthetically valuable
chiral intermediate 20a was obtained in good yield and used for
further versatile transformation. Considering the SAR studies
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Scheme 1
Scheme 2
Scheme 3
therapeutic index of >5000 K (SI = CC50/EC50). This
To elucidate the possible mechanisms of action and the
inhibition potencies of the evaluated compounds, we performed
3D molecular docking studies based on available protein
structures and SAR evaluations. The formation of a
homodimeric catalytic subunit between the zinc finger domains
is crucial for the activity of HCV NS5A.27 The relative dimeric
symmetry has recently been revealed for NS5A isoforms28 and
several NS5A inhibitors.14 The range of key amino acid
residues in the N-terminal region was determined within
domain I. Amino acid substitutions in the same positions were
associated with resistance to 1 and thiazolidinone deriva-
tives.29−31 For genotype 1a, resistant mutations included M28T
compound is also highly active against genotypes 1a (EC50
=
59 pM), 2a (EC50 = 51 pM), and 4a (EC50 = 6.5 pM).26
Compound 13a has a log D = 4.46 (pH = 7.4), 14 heteroatoms,
6 potential hydrogen bond acceptors, 4 potential hydrogen
bond donors, and 8 free rotatable bonds. It is a crystalline
powder ranging from nearly white to yellow in shade with a
melting point of 185−186 °C (above 195 °C, degradation
begins). Compound 13a is highly soluble in water (208 μM at
pH = 2, 195 μM at pH = 4, 33 μM at pH = 7.4), alcohol (≥10
μM), dimethyl sulfoxide (≥10 μM), and other solvents.
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Scheme 4
Figure 2. Binding hypothesis for compound 13a; the α-helix domain is not shown. (a) First (orange, Y93 stacks face-to-face with the imidazole) and
alternative (white, t-stacking of Y93 and imidazole) binding modes of 13a, the compound is positioned along the extended cavity formed within the
dimeric interface of NS5A. (b) 2D-supramolecular interface proposed for alternative binding mode of 13a; (′) denotes the other subunit in the dimer
interface.
(ca), Q30H/R (ca), L31M/V (ca, sa), Y93C/H (ca), and
Q54H/R/L (ca), where ca is the cap area, and sa is the spacer
area.22,31,32 A similar resistance profile was reported for 12 and
included M28T, Q30R, Y93C, Y93H, and Y93N for NS5A
GT1a and L28T, L28M + Y93H, L31F + Y93H, and L31V +
Y93H for NS5A GT1b.22,32 The major GT1a resistance
mutations along the dimer interface as well as in the
amphipathic α-helical N-terminus included M28T, L31V/W,
Q30D/E/K/H/R, P32L (sa), H58D, Y93C/H/N, and Q54H/
R/L as well as L31F/V/W, P32L, and Y93C/H/N for
GT1b.22,30,32 The common core point mutations for both
genotypes included Y93C/H/N, L31V/W, and P32L, suggest-
ing the major role of these amino acid residues in drug binding.
The distance between the two cap binding regions within the
dimeric interface is approximately 15−20 Å, as reported by
Makonen Belema and colleagues,14 or 15−18 Å, as reported by
Cordek and co-workers.34 This distance is closely related to the
linker length in the structures of many dimeric inhibitors,
providing a unique binding mode for these compounds. The
common topological pharmacophore includes a spacer (linker)
surrounded by crucial amino acid residues L31 and P32 and
two caps that are in tight interaction with the loop regions on
either side of the dimer interface in proximity to Y93, M28/
L28, L31, and Q54.14,34 In highly potent inhibitors, these caps
generally include Pro, Val (or Phe) residues or their direct
analogues.
We also performed a related in vitro mutagenesis study to
investigate the resistance profile of compound 13a (vide infra).
During the study, we identified the major mutation points
L31F, T56A, and Y93H located in the HCV NS5A gene. These
mutations significantly enhanced the resistance of HCV
replicons toward 13a inhibition activity. The obtained results
confirmed that compound 13a is a direct-acting antiviral agent
that binds to the NS5A protein. The disclosed resistance profile
is similar to that described above for other NS5A inhibitors,
suggesting a similar binding mode.
The molecular docking study for compound 13a was
performed using MOE software23 (Figure 2a) following the
approach reported by Cordek and colleagues.34 The binding
site within the full-length homology dimeric model of GT1b
NS5A domain I and the amphipathic helix anchor domain was
reconstructed based on available crystallographic data (PDB
codes: 3FQM and 1R7C). Compound 13a was then docked
into the defined binding site using the static mode. The
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Figure 3. 2D supramolecular interface predicted for compounds 11, 12, 1, and 3.
compound locates symmetrically across the dimer interface
with the hydrophobic diynyl-phenyl core, lying along T95 and
captured by Q54, T56, and Q62. Y93 stacks face-to-face with
the imidazole ring, while Q62 and/or T56 form hydrogen
bonds with the imidazole N atoms. This binding mode (Figure
2a, colored in orange) is very similar to that reported previously
by Belema and colleagues,14 particularly for compounds 12,22,32
1,14,34 3,35 and 11.21,36,37 We also revealed an alternative
binding mode (Figure 2a, colored in white) in which one
methyl group of the valine moiety is above the imidazole ring,
thereby providing an additional hydrophobic interaction
exclusively in the case of S,S-isomers with Y93, while the
imidazole itself is located appropriately for slanted t-stacking
with Y93 supported by a weak direct (or water-mediated)
hydrogen bond between the OH group of Y93 and the
imidazole NH or the O of the N-acetyl Pro fragment (Figure
2b). The second methyl group of valine provides hydrophobic
interactions with T56 and P58. A similar alternative binding
mode was also observed for the other NS5A inhibitors
mentioned above (Figure 3). These results correlate well with
the obtained biological data and explain the differences in
activity between the diastereomers. Notably, stereospecificity
was found to be much more crucial within the Cap of ethynyl-
imidazole (Cap2) than within the phenyl-imidazole side (Cap1,
Table 2), suggesting that the binding of Cap1 is more flexible
and tolerant toward both drugs and the target. Note also that
the structures and stereochemistries of Cap1 and Cap2 have a
more drastic effect on the activity than does the linker length
(Table 2). This resulted in a relatively high tolerance for linker
length (up to 21 Å) within an extensive group of NS5A
inhibitors, including compounds 13a, 1, 3, 12, and 11. Thus,
the (R,S,S,S)-configuration of 13g (EC50 = 0.079 nM) was
highly active, while the (S,S,S,R)-analogue 13h, with the same
spacer, was less active (EC50 = 0.4089 nM).
in GT1b, L28T/M, L31F/V/M, Y93H, and R30Q were
identified as the most crucial for good binding, while for
GT1a the mutations were Y93H/C/N, M28V/T, Q30R, L31V,
and H58D. Interestingly, inverted resistant mutations (R30Q
and Q30R) were observed for GT1b and GT1a, respectively.
Although arginine and glutamine have comparable lengths,
arginine can provide π−cationic interactions, particularly with
phenyl or heteroaryl fragments, while glutamine provides H-
bonding acceptor functionality. Both residues share H-bonding
donor potential. These amino acids were suggested to be in
close proximity with the cap fragment, providing crucial
contacts as highlighted in Figure 3.
Lambert and colleagues have recently discussed the crystal
structure of GT1a-D1 and suggested a novel mechanism of
action for dimeric HCV inhibitors.38 The obtained crystal
structure is highly exposed to water molecules, having a
Matthews coefficient of 4.73 Å3/Dalton and a solvent content
of 73.97%, similar to the recently disclosed GT1b-D1 dimer
crystal structure.27 Therefore, ligand binding could be depend-
ent largely on the resolvation energy and thermodynamic
terms. Two novel head-to-head dimeric forms of NS5A-D1
were observed, and the cavity surface was described in terms of
the key amino acid content responsible for ligand binding and
symmetry. Molecular dynamic simulations were performed
based on the obtained X-ray and available NMR data to
construct a full-length 3D model. The amino acid residues
Val75, Gly76, and Pro77, located along the spacer area and
capturing the biphenyl core, as well as His54, Arg56, Glu62,
His66, Arg73, and Thr79, surrounding the cap regions, were
suggested to be the most crucial for 1 binding affinity. The
hydrogen bond periodically formed between Glu62 and the
valine carbonyls (or the imidazole NH) was identified in the
docking study. Dynamic modeling (40 ns) was then carried out
and revealed some oscillation along the dimer axis. It was also
revealed through the modeling that the key Tyr93 did not
directly interact with 1 but rather via a tight amino acid frame.
Thus, the salt bridge with Arg56 maintained by Glu62 was
observed and thereby adjusted Tyr93 to complete the network.
This allosteric mode of resistance/binding is partially supported
It was shown that the incorporation of an N-arylated
pyrrolidine core (12 and related compounds) in the center of
the linker area led to dramatic gains in activity against both the
GT1a and GT1b genotypes compared to the unsubstituted
pyrrolidine analogues.31 Among the major resistant mutations
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by the relatively high dynamics observed within the ligand-
capture amino acid interface upon binding, providing tentative
evidence against rigid interactions in a lock and key
mechanism.39 Although in our work the static docking
simulation was actually performed only with a flexible ligand
(not the target), the calculated scores correlated well with the
target activity of the tested compounds. A detailed in silico
study with a relatively wide range of novel NS5A inhibitors
supported by the dynamic docking procedure will be reported
elsewhere (manuscript in preparation).
The flexible 3D alignment of 11 and 12, as revealed using
ICM Pro software,40 out of the target clearly showed that the
cap areas and phenyl moieties in the center of the spacers, as
well as the two-sided aromatic fragments, are well superposed
(Figure 4). In this case, the imidazole ring can be regarded as a
case binding is achieved by the same amino acid sharing by the
second subunit.
The addition of 40% NHS had little effect on the activities of
compounds 13 against the HCV GT1b replicon, suggesting
their weak binding to human plasma proteins.41 The effect of
NHS on compound 13a potency was moderate. Its activity
decreased only 3−4-fold in the presence of 40% NHS. As
mentioned above, 13a has a relatively high TI against HCV
replicons in vitro and was inactive against other flaviviruses,
including West Nile virus, yellow fever virus, and dengue virus,
at the highest concentration tested (30 μM). These data
suggest that compound 13a is a specific HCV inhibitor (Tables
2 and 3).
The 13a-resistant HCV replicons were selected by
continuous passaging of the Huh7 cells harboring the HCV
replicon (GT1b, clone Con1)41 in the presence of the drug at
concentrations exceeding the EC50 values by 5−40-fold for 3−6
weeks. Some drug-resistant cell populations were cloned. The
primary structure of the NS5A gene was determined by direct
sequencing of the HCV RNA pools. As a result, several NS5A
mutations were identified (Table 4). In general, the NS5A
Table 4. HCV NS5A Mutations, Identified after Selection
a
with Compound 13a in Vitro
mutation weight (%)
mutation
20−39
40−59
≥60
total
Figure 4. 3D alignment of 11 (orange) and 12 (yellow); HBA, H-
bond donor; HBD, H-bond acceptor; Aro, aromatic moiety; Hyd,
hydrophobic fragment.
CGA89CCA (Q30P)
TTG93TTT (L31F)
1
1
1
5
3
1
TTG93ATG (L31M)
AAG131AGG (K44R)
ACC166TCC (T56S)
ACC166GCC (T56A)
AGG233AAG (R78K)
TAC277CAC (Y93H)
TAC277AAC (Y93N)
CGG1067CAG (R356Q)
CTG1088CCG (L363P)
ACC1099CCC (T367P)
GTG1103GCG (V368L)
1
1
2
1
5
good bioisosteric replacement for the amide fragment,
providing similar contacts maintained by the H-bond donor
(NH in imidazole and carboxamide) and the H-bond acceptor
points (N in imidazole, O in carboxamide). A detailed binding
picture can be developed on the basis of the 2D- (Figure 3) and
3D-representations (Figure 4) of these compounds. Thus, the
introduction of an aromatic functionality in the linker area of
compound 13a can be reasonably regarded as a beneficial
modification that will, presumably, enhance target activity or/
and selectivity.
The point mutation L31F/V in GT1b NS5A conferred
resistance to compound 12. On the basis of the performed
docking study, it was suggested that the optimal distance for
hydrophobic interaction between res 31 and the iso-butyl
phenyl moiety of compound 12 corresponds to leucine. The
isoform with a bulky Phe can contribute to steric clashes upon
binding, while the small Val residue provides insufficient
hydrophobic contact. The substitution of P32 by L32 is also
associated with resistance types, thereby suggesting that
stacking interactions, presumably with aromatic moieties in
the NS5A inhibitors, are more preferable than a Leu probe.
Similar speculations can be made for the core and side chain
interactions (with phenyl groups) of compound 11, but in this
1
3
1
1
1
6
1
2
1
1
1
1
1
2
1
1
1
a
Numbers of independent cell cultures with a given mutation are
shown.
mutations conferring resistance to compound 13a were similar
to those reported previously for other NS5A inhibitors.30 In the
reference cell culture (not treated with 13a), none of the
mutations described above were found. Then, cultures carrying
100% of one of the mutations (L51F, T56A, or Y93H) were
cloned. All cloned cell cultures harboring mutant HCV
replicons were significantly more resistant to compound 13a
than were the cultures harboring the wild-type HCV replicon
(not shown).
Table 3. Spectrum of Antiviral Activity of Compound 13a in Vero-76 Cells, Infected in Vitro, As Well As Cytotoxicity
West Nile virus (strain NY99)
yellow fever virus
dengue virus
(μM)
(μM)
(μM)
a
a
a
compd
EC50
>26.3
0.011
CC50
SI50
EC50
CC50
SI50
EC50
CC50
28.4
>10
SI50
na
>714
13a
IFN-α (ng/mL)
26.3
>10
na
>23.5
0.11
23.5
>10
na
>28.4
>910
>93
0.014
a
CC50 was determined in Vero-76 cell line.
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a
Table 5. Key Pharmacokinetic Features of 13a and other NS5A Inhibitors after a Single po and iv Administration
b
c
compd
administration route
dose (mg/kg)
Rats
Cmax (mg/L) (μM)
t1/2 (h)
F (%)
13a
po
11.2
30
5
2097 (2950)
318 (360)
3.0
65
9
11
1 (PEG)
1 (solution)
10
50
3
0.538 (0.728)
1.5
4.7
1
58.5
6.2
38
12
3
3 (3.36)
15.9
MK-4882
2
241 (310)
13a
iv
2.8
1
2566 (3610)
3.4
2
MK-4882
11
5
4.2
Dogs
13a
po
10
2
5810 (8174)
256 (290)
148 (190)
4.5
58
35
11 (PEG)
MK-4882
1 (PEG)
10
1
26
2.3
1
108
58.5
57
7.4
7.3
12
2.5
0.64 (0.72)
13a
iv
5
1
1
6900 (9707)
148 (190)
4.9
4.7
7.7
MK-4882
11
Monkeys
MK-4882
po
5
2.5
1
0.14 (0.18)
0.29 (0.32)
12
35
12
10
1
5
10.3
3
58.5
38
3
0.37 (0.50)
MK-4882
iv
1
1
3
11
16
a
b
c
Data for all compounds except 13a was obtained from Integrity Database.42 Concentration was calculated for non saline composition. F = 100%
× [AUC0→∞(po) × dose(iv)]/[AUC0→∞(iv) × dose(po)].
Pharmacokinetics studies performed in mice, rats, and dogs
(Table 5)42 have revealed that compound 13a achieves a
relatively high level in plasma (Cmax) and possesses a long half-
life (t1/2) as well as high oral bioavailability (F %), which in rats
reached 65%, compared to 11% observed for 1.34 Lower
bioavailability was also reported for other NS5A inhibitors,
including 12 (F = 6.2%),22 10 (F = 32.5%),16 and 11 (38%).21
The t1/2 values for compound 13a upon oral administration
were 3 and 4.5 h in rats and dogs, respectively. However, in
dogs (2.5 × 10−3 g/kg, po) and monkeys (2.5 × 10−3 g/kg, po),
the bioavailability of 12 was 57% and 35%, respectively.22
Another compound developed by Abbott containing a
pyrido[2,3-d]pyrimidin-4-amino core showed F = 18.8% in
dogs (2.5 × 10−3 g/kg po sd).43 1 and 11, as PEG formulations,
demonstrated F = 108%44 and F = 35% (in dogs, 2 × 10−3 g/kg
po sd),21 respectively.
Table 6. Affinity of 13a and 1 towards CYP450 Panel
a
compd
13a IC50 (μM)
1
IC50 (μM)
CYP450
1A2
>10
>10
>10
>10
>10
>100
2C9
59.5 13.6
2C19
2D6
>100
3A4
7.20 1.70
a
Data for compound 1 was obtained from Integrity Database.42
Table 7. Metabolic Stability of 13a, Evaluated after
Incubation for 60 min in the Presence of 1 μM Rat, Dog, or
Human Liver Microsomal Fractions
residual substance (%)
substance
rat
dog
human
As shown in Table 6, at a dose of 10 μM, compound 13a did
not inhibit any of the studied human liver CYP450 enzymes.
These results clearly suggest the absence of clinically significant
interactions between 13a and other medicinal products
metabolized by the above-mentioned human cytochromes.
Compound 13a was stable when it was incubated for 60 min in
the presence of 1 μM concentrations of rat, dog, or human liver
microsomal fractions (Table 7).
13a
70.8 + 5.2
84.7 + 4.1
81.5 + 4.6
concentrations using Invitrogen’s Predictor hERG test system.
The binding did not exceed 10% and was not dose-dependent,
suggesting a relatively low risk of cardiotoxicity. We also
investigated the acute toxicity of 13a in mice and rats. It was
found that the LD50 value in mice (intraperitoneal admin-
istration) exceeded the maximum studied dose (1600 mg/kg)
and was equal to 3435.4 502.3 mg/kg in males and 3005.5
The inhibition potency of 13a toward cardiac hERG
potassium channels was investigated at 30 and 3 μM
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678.7 mg/kg in females upon oral administration (Table 8).
The LD50 values in outbred Wistar rats upon the oral
selectivity (EC50 = 3 pM for GT1b, CC50 = 17.3 μM). Human
serum did not significantly reduce the antiviral activity (<4-
fold). No potency was observed against other flaviviruses
(yellow fever, West Nile, dengue) in the in vitro infection
experiments. In vitro studies clearly showed that the 13a
resistance profile overlaps with but is not identical to those of
other known HCV NS5A inhibitors. A 3D molecular docking
study revealed two potential binding modes for the most active
compounds within this series as well as for several other NS5A
inhibitors. The optimal spacer length for ensuring tight binding
was predicted to be 17−20 Å. Animal studies revealed a
favorable pharmacokinetic profile and good oral bioavailability
for compound 13a. It was well-tolerated in rodents (in male
Table 8. LD Values Determined for Compound 13a in SHK
Mice (po)
LD
mice
values (mg/kg)
LD10
males
2320.3
1160.1
females
LD16
LD50
LD84
males
2565.4
1565.7
females
males
3435.4 502.3
3005.5 678.7
mice, LD50 = 3435.4
502.3 mg/kg or higher). In safety
females
pharmacology and subchronic toxicity studies in rats and dogs,
13a was not associated with any significant pathological or
clinical findings. Further clinical development of compound
13a is strongly warranted.
males
4305.3
4445.3
females
administration of compound 13a were as follows: 2902.0
488.1 mg/kg for males, 2829.2 311.4 mg/kg for females, and
2860.8 227.0 mg/kg for both males and females.
The LD10 values for the intraperitoneal administration of 13a
in rats were similar in both males and females, while the LD50
and LD84 were higher in females (Table 9). These differences
EXPERIMENTAL SECTION
■
Chemistry General Procedures. All chemicals and solvents were
used as received from the suppliers without further purification. The
intermediate compounds were obtained following the synthetic
procedures described in the references.10−13,46−48 The crude reaction
mixtures were concentrated under reduced pressure by removing the
organic solvents in a rotary evaporator. Nuclear magnetic resonance
(NMR) spectra were recorded using a Bruker DPX-400 spectrometer
at room temperature (rt) with tetramethylsilane as an internal
standard. The chemical shifts (δ) are reported in parts per million
(ppm), and the signals are reported as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), or br s (broad singlet). The
purities of the final compounds were determined by HPLC and were
greater than 98%. The HPLC conditions for assessing purity were as
follows: Shimadzu HPLC, XBridge C18, 4.6 mm × 250 mm (3.5 μm);
gradient of 0.1% TFA in 5% acetonitrile/water (A) and 0.1% TFA
acetonitrile (B); flow rate, 0.5 mL/min; acquisition time, 20 min;
wavelength, UV 214 and 254 nm. The preparative HPLC system
included two sets of Shimadzu LC-8A pumps, a Shimadzu controller
SCL 10Avp, and a Shimadzu detector SPD 10Avp. A Reprosil-Pur C-
18-AQ 10 μm, 250 mm × 20 mm column was used. The mobile phase
was a gradient of 0.1% TFA in water (A) and 0.1% TFA in acetonitrile
(B). LC/MS was conducted on a PE Sciex API 165 system using
electrospray in positive ion mode, [M + H]+, and a Shimadzu HPLC
system equipped with a Waters XBridge C18 3.5 μm column (4.6 mm
× 150 mm). High resolution mass spectra (HRMS) were acquired on
an Orbitrap Elite mass spectrometer (Thermo, Bremen, Germany)
equipped with an HESI ion source. The N-Moc-amino acids 22a−f
were obtained by reaction between the corresponding amino acid and
methyl chloroformate under basic conditions.49 The 3-amino-
tetrahydrofuran-3-carboxylic acid used for the synthesis of inter-
mediates 22c,d was prepared in accordance with the synthetic
procedure described in ref 50 and was separated to provide
enantiomers by the method published in ref 51. The 2-amino-3-
methoxy-2-methylpropanoic acid used for the synthesis of inter-
mediate compound 22e was prepared from 1-methoxypropan-2-one
by the procedure reported for 3-amino-tetrahydrofuran-3-carboxylic
acid.50
Table 9. LD Values for Intreperitoneal Administration (ip)
of Compound 13a in Rats
lethal dose
LD10
rats
values (mg/kg)
males
228.6 87.7
228.4 63.9
234.2 38.5
females
males and females
LD50
LD84
males
349.4 69.3
412.1 68.4
395.2 40.8
females
males and females
males
485.6 123.7
651.3 87.2
593.2 51.9
females
males and females
were not statistically significant; however, they indicated the
tendency for females to be more tolerant to 13a upon
intraperitoneal administration. These results suggest that
compound 13a could be classified45 as a low toxicity substance
(group I).
In a 6-week toxicity study in dogs and a 12-week study in
rats, no significant signs of toxicity were observed for
compound 13a. In split studies, the compound was found to
be nonmutagenic in vitro and not immunogenic or allergenic in
vivo. No adverse effects on the reproductive systems of male or
female rats were observed. These results will be published
elsewhere. On the basis of the preclinical data obtained during
an extensive preclinical evaluation, compound 13a was selected
for subsequent clinical development.
General Procedure 1. Substituted (S)-2-(5-{4-[4-((S)-2-Pyrrolidin-
2-yl-3H-imidazol-4-yl)-phenyl]-buta-1,3-diynyl}-1H-imidazol-2-yl)-
pyrrolidine Dihydrochlorides (13a−f). Methyllithium lithium bro-
mide complex solution (1.5 M) in ether was added to a solution of
5.83 g (30 mmol) of 1,4-bis(trimethylsilyl)buta-1,3-diyne 15 dissolved
in 40 mL of ether in an argon atmosphere (aa) under vigorous stirring.
The reaction mixture was continuously stirred at rt for 15 h, then
cooled in an ice bath and quenched with 40 mL of a saturated NH4Cl
solution. The organic layer was washed with brine and dried over
Na2SO4, and the solvent was evaporated in a rotovap under vacuum.
tert-Butyl (S)-2-[5-(4-iodophenyl)-1H-imidazol-2-yl]-pyrrolidine-1-
CONCLUSIONS
■
SAR studies with a novel series of hepatitis C virus NS5A
inhibitors containing 5-[4-(4-imidazol-4-yl-phenyl)-buta-1,3-
diynyl]-1H-imidazole linkers led to dramatic improvements in
antiviral activity in the HCV replicon model. Compound 13a,
containing four (S) chiral centers, showed excellent activity and
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carboxylate 14a46 (8.8 g, 20 mmol), triethylamine (20 mL), Pd(PPh)4
(1.16 g, 1 mmol), and CuI (0.19 g, 1 mmol) were added to the
solution of the liquid residual 16 dissolved in THF (70 mL). The
resulting mixture was stirred under aa at 40 °C for 15 h. Then, the
mixture was filtered through Celite, rotovapped, and subjected to
column chromatography on SiO2 (eluent = chloroform: acetone 15:1)
to afford 7.76 g (85%) of 17a. The LC-MS molecular ion peak was at
434 (M + H)+. Potassium carbonate (K2CO3) (7.04 g, 51 mmol) was
added to the solution of compound 17a (7.36 g, 17 mmol) in THF
(120 mL) and methanol (120 mL), and the reaction mixture was
stirred under aa for 2 h then filtered and rotovapped. A mixture of tert-
butyl (S)-2-(5-iodo-1H-imidazol-2-yl)-pyrrolidine-1-carboxylate
(19a)10 (6.17 g, 17 mmol) in triethylamine (20 mL) with Pd(PPh)4
(0.93 g, 0.8 mmol) and CuI (0.15 g, 0.8 mmol) was added to the
solution of the obtained compound 18a dissolved in THF (60 mL).
The resulting mixture was then stirred under aa at 40 °C for 15 h.
After the reaction was completed, the mixture was filtered, the
precipitate was washed with a chloroform:methanol 3:1 solution, the
filtrate was rotovapped, and the residue was boiled in 100 mL of
methanol. The mixture was then cooled to rt and stirred in a
refrigerator for 4 h. The formed precipitate (compound 20a) was then
filtered off, washed with cold methanol and ether, and dried in air. The
desired product, tert-butyl [2-((S)-2-{5-[4-(4-{2-[(S)-1-(tert-buthox-
ycarbonyl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-phe-
nyl]-1H-imidazol-2-yl}-pyrrolidin-1-yl)-carboxylate (20a), was ob-
tained in 57% yield (5.78 g) and purity. The LC-MS molecular ion
peak was at 597 (M + H)+. 1H NMR (DMSO-d6, 400 MHz) δ 12.67,
12.73 (2s, 0.2H), 12.21, 12.28 (2s, 0.9H), 11.94, 12.01 (2s, 0.9H), 7.77
(m, 2H), 7.55 (m, 3.7H), 7.35 (m, 0.3H), 4.77 (m, 2H), 3.51 (m, 2H),
3.34 (m, 2H), 2.18 (m, 2H), 1.95 (m, 3H), 1.84 (m, 3H), 1.39 (s,
7.5H), 1.14, 1.17 (2s, 10.5H).
NMR (DMSO-d6, 400 MHz) δ 14.77 (m, 1.1H), 8.22 (s, 0.75H), 8.20
(s, 0.25H), 7.96 (m, 2.8H), 7.78 (m, 2.2H), 7.63 (d, J = 8.0 Hz,
0.05H), 7.48 (d, J = 8.0 Hz, 0.2H), 7.24 (d, J = 8.0 Hz, 1.5H), 6.73 (m,
0.02H), 5.98 (m, 0.03H), 5.63 (m, 0.2H), 5.22 (m, 1H), 5.07 (m,
0.8H), 4.17 (m, 2H), 3.90 (m, 1H), 3.83 (m, 1H), 3.63 (m, 2H), 3.55,
3.56 (2s, 6H), 2.40 (m, 1H), 2.27 (m, 1H), 2.03 (m, 8H), 0.87 (m,
10.1H), 0.73 (m, 1.1H), 0.43 (m, 0.2H), 0.31 (m, 0.6H). ESIHRMS
m/z calcd for C38H47N8O6 [M + H]+ 711.3613; found 711.3638.
Methyl {(S)-3-{(S)-2-[5-(4-(4-{2-[(S)-1-((S)-3-Methoxycarbonylami-
no-tetrahydro-furan-3-carbonyl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-
buta-1,3-diynyl)-phenyl)-1H-imidazol-2-yl]-pyrrolidine-1-carbonyl}-
tetrahydro-furan-3-yl}-carbamate Dihydrochloride (13c). Yield 39
1
mg (41%). H NMR (DMSO-d6, 400 MHz) δ 14.67 (m, 1.6H), 8.18
(m, 2.8H), 7.97 (m,3H), 7.79 (m, 2.2H), 5.67 (m, 0.05H), 5.44 (m,
0.07H), 5.16 (m, 0.93H), 5.02 (m, 0.91H), 4.14 (m, 2H), 3.87 (m,
1H), 3.72 (m, 9H), 3.60 (s, 3H), 3.56 (s, 3H), 2.34 (m, 3H), 2.14 (m,
6H), 1.95 (m, 3H). ESIHRMS m/z calcd for C38H43N8O8 [M + H]+
739.3198; found 739.3190.
Methyl {(S,R)-3-{(S)-2-[5-(4-(4-{2-[(S)-1-((S,R)-3-Methoxycarbony-
lamino-tetrahydro-furan-3-carbonyl)-pyrrolidin-2-yl]-3H-imidazol-
4-yl}-buta-1,3-diynyl)-phenyl)-1H-imidazol-2-yl]-pyrrolidine-1-car-
bonyl}-tetrahydro-furan-3-yl}-carbamate Dihydrochloride (13d).
1H NMR (DMSO-d6, 400 MHz) δ 14.56 (br s, 1.8H), 8.18 (m,
2.7H), 7.99 (d, J = 8.0 Hz, 2H), 7.88 (br s, 1.3H), 7.77 (d, J = 8.0 Hz,
2H), 5.71 (m, 0.03H), 5.44 (m, 0.06H), 5.18 (m, 0.96H), 5.04 (m,
0.94H), 4.16 (m, 2H), 3.77 (m, 8H), 3.59 (m, 8H), 2.34 (m, 3H), 2.14
(m, 6H), 1.95 (m, 3H). ESIHRMS m/z calcd for C38H43N8O8 [M +
H]+ 739.3198; found 739.3224.
Methyl [2-((S)-2-{5-[4-(4-{2-[(S)-1-(3-Methoxy-2-methoxycarbo-
nylamino-2-methyl-propionyl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-
phenyl)-buta-1,3-diynyl]-1H-imidazol-2-yl}-pyrrolidin-1-yl)-1-me-
thoxymethyl-1-methyl-2-oxo-ethyl]-carbamate Dihydrochloride
1
(13e). H NMR (DMSO-d6, 400 MHz) δ 14.44 (m, 1.8H), 8.19 (d,
Hydrochloric acid (HCl) (4 M, 25 mL) was added dropwise to the
solution of compound 20a (5.78 g, 9.7 mmol) in dioxane (25 mL).
The resulting mixture was then stirred for 15 h. The formed
precipitate was filtered off, washed with ether, and dried under vacuum
to obtain 5.04 g (96%) of 5-[4-((S)-2-pyrrolidin-2-yl-3H-imidazol-4-
yl)-buta-1,3-diynylphenyl]-2-[(S)-pyrrolidin-2-yl]-1H-imidazole tetra-
hydrochloride (21a). The LC-MS molecular ion peak was at 397 (M +
H)+. 1H NMR (DMSO-d6, 400 MHz) δ 10.38 (br s, 1H), 10.27 (br s,
1H), 9.86 (br s, 1H), 9.22 (br s, 1H), 8.18 (s, 1H), 7.98 (d, J = 8.0 Hz,
2H), 7.80 (s, 1H), 7.70 (d, J = 8.0 Hz, 2H), 5.02 (m, 1H), 4.74 (m,
1H), 3.45 (m, 1H), 3.37 (m, 1H), 3.29 (m, 2H), 2.47 (m, 2H), 2.35
(m, 1H), 2.17 (m, 2H), 2.09 (m, 1H), 2.00 (m, 2H). The mixture of
N-Moc-amino acid 22a−f (0.283 mmol), 1-hydroxy-7-azabenzotria-
zole (40 mg, 0.295 mmol), and EDAC (53 mg, 0.277 mmol) in
acetonitrile (1 mL) was stirred in a refrigerator for 1 h, then
compound 21a (64 mg, 0.118 mmol) and DIPEA (61 mg, 0.472
mmol, 82 mL) were added, and the resulting mixture was continuously
stirred in a refrigerator for 15 h. The mixture was rotovapped,
dissolved in dichloromethane, washed twice with a 5% Na2CO3
solution, dried over Na2SO4, rotovapped again, and subjected to
HPLC. Compounds 13a−f were then readily converted into the
corresponding salt compositions by following the procedure described
above and were isolated as the dihydrochloride salts (13a−f·2HCl)
through the addition of acetone.
J = 9.6 Hz, 1H), 7.98 (m, 2H), 7.89 (m, 1.5H), 7.78 (m, 3H), 7.69 (m,
0.5H), 5.22 (m, 1H), 5.06 (m, 1H), 3.85 (m, 1H), 3.77 (m, 2H), 3.68
(m, 4H), 3.58 (m, 5H), 3.43 (m, 2H), 3.24, 3.26 (2s, 6H), 2.31 (m,
1H), 2.14 (m, 3H), 1.94 (m, 4H), 1.35 (m, 6H). ESIHRMS m/z calcd
for C38H47N8O8 [M + H]+ 743.3514; found 743.3546.
Methyl [2-((S)-2-{5-[4-(4-{2-[(S)-1-(2-Methoxycarbonylamino-2-
methyl-propionyl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-phenyl)-buta-
1,3-diynyl]-1H-imidazol-2-yl}-pyrrolidin-1-yl)-1,1-dimethyl-2-oxo-
1
ethyl]-carbamate Dihydrochloride (13f). H NMR (DMSO-d6, 400
MHz) δ 14.44 (br s, 1.7H), 8.19 (s, 1H), 7.98 (d, J = 8.4 Hz, 2H), 7.93
(m, 1H), 7.90 (m, 1H), 7.83 (m, 1H), 7.78 (d, J = 8.4 Hz, 2H), 5.23
(m, 1H), 5.06 (m, 1H), 3.81 (m, 1H), 3.70 (m, 3H), 3.63 (s, 3H), 3.57
(s, 3H), 2.31 (m, 1H), 2.14 (m, 3H), 2.02 (m, 1H), 1.92 (m, 3H), 1.33
(m, 12H). ESIHRMS m/z calcd for C36H43N8O6 [M + H]+ 683.3300;
found 683.3334.
Methyl [(S)-1-((S)-2-{5-[4-(4-{2-[(S)-1-((R)-2-Methoxycarbonyla-
mino-3-methyl-butyryl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-
diynyl)-phenyl]-1H-imidazol-2-yl}-pyrrolidine-1-carbonyl)-2-methyl-
propyl]-carbamate Dihydrochloride (13g). Compound 13g was
prepared according to the procedure described above for compounds
13a−f starting from compound 18a and the corresponding methyl {2-
[(S)-2-(5-iodo-1H-imidazol-2-yl)-pyrrolidin-1-yl]-2-oxo-ethyl}-carbate
19b,g−i, followed by the conversion of the formed product 20b,g−i to
the intermediate compound 21b,g−i and the acylation of amino acid
22a as well as the following transformation of the acylation products to
the corresponding dihydrochlorides 13g−j.
Methyl [(S)-1-((S)-2-{5-[4-(4-{2-[(S)-1-((R)-13g and Methyl [(R)-1-
((S)-2-{5-[4-(4-{2-[(S)-1-((S)-2-Methoxycarbonylamino-3-methyl-bu-
tyryl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-phenyl]-
1H-imidazol-2-yl}-pyrrolidine-1-carbonyl)-2-methyl-propyl]-carba-
mate Dihydrochloride (13h). Compounds 13g,h were prepared
starting from the intermediate compound 18a and methyl {(S)-1-[(S)-
19b methyl {(R)-1-[(S)-2-(5-iodo-1H-imidazol-2-yl)-pyrrolidine-1-
carbonyl]-2-methyl-propyl}-carbamate 19c (see Scheme 2) using the
conditions applied for compounds 13a−f (vide supra).
Methyl [(S)-1-((S)-2-{5-[4-(4-{2-[(S)-1-((R)-2-Methoxycarbonyla-
mino-3-methyl-butyryl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-
diynyl)-phenyl]-1H-imidazol-2-yl}-pyrrolidine-1-carbonyl)-2-methyl-
propyl]-carbamate Dihydrochloride (13g). 1H NMR (DMSO-d6,
Methyl [(S)-1-((S)-2-{5-[4-(4-{2-[(S)-1-((S)-2-(Methoxycarbonyla-
mino)-3-methyl-butyryl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-
1,3-diynyl)-phenyl]-1H-imidazol-2-yl}-pyrrolidine-1-carbonyl)-2-
methylpropyl]-carbamate Dihydrochloride (13a). Yield 62 mg
1
(67%). H NMR (DMSO-d6, 400 MHz) δ 15.49 (br s, 0.3H), 14.95
(br s, 0.8H), 8.23 (s, 0.1H), 8.21 (s, 0.9H), 8.01 (m, 3H), 7.77 (m,
2H), 7.26 (m, 1.8H), 6.84 (m, 0.1H), 5.78 (m, 0.06H), 5.50 (m,
0.05H), 5.19 (t, J = 7.0 Hz, 0.9H), 5.07 (m, 0.9H), 4.06 (m, 2.7H),
3.81 (m, 3H), 3.53 (s, 5.4H), 3.43 (s, 0.4H), 3.30 (s, 0.2H), 2.15 (m,
10H), 0.82 (m, 12H). ESIHRMS m/z calcd for C38H47N8O6 [M + H]+
711.3613; found 711.3590.
Methyl [(R)-1-((S)-2-{5-[4-(4-{2-[(S)-1-((R)-2-Methoxycarbonyla-
mino-3-methyl-butyryl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-phenyl)-
buta-1,3-diynyl]-1H-imidazol-2-yl}-pyrrolidine-1-carbonyl)-2-meth-
yl-propyl]-carbamate Dihydrochloride (13b). Yield 63 mg (68%).1H
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400 MHz) δ 14.79 (m, 0.9H), 8.20 (m, 1.1H), 7.95 (m, 2.3H), 7.85
(br s, 1H), 7.77 (m, 2.1H), 7.60 (m, 0.06H), 7.26 (m, 1.4H), 7.10 (m,
0.04H), 6.86 (m, 0.01H), 6.71 (m, 0.01H), 5.95 (m, 0.06H), 5.37 (m,
0.1H), 5.22 (m, 0.94H), 5.04 (m, 0.9H), 4.19 (m, 1H), 4.06 (m, 1H),
3.90 (m, 1H), 3.80 (m, 2H), 3.67 (m, 1H), 3.54, 3.55 (2s, 6H), 2.39
(m, 1H), 2.23 (m, 1H), 2.11 (m, 3H), 1.99 (m, 5H), 0.85 (m, 11.3H),
0.74 (m, 0.45H), 0.42 (m, 0.25H). ESIHRMS m/z calcd for
C38H47N8O6 [M + H]+ 711.3613; found 711.3601.
[(R)-1-((S)-2-{5-[4-(4-{2-[(S)-1-((S)-2-Methoxycarbonylamino-3-
methyl-butyryl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-diynyl)-
phenyl]-1H-imidazol-2-yl}-pyrrolidine-1-carbonyl)-2-methyl-prop-
yl]-carbamate Dihydrochloride (13h). 1H NMR (DMSO-d6, 400
MHz) δ 15.29 (br s, 0.5H), 14.79 (br s, 0.8H), 8.18, 8.20 (2s, 1H),
7.97 (m, 2.8H), 7.76 (m, 2.3H), 7.47 (m, 0.2H), 7.25 (m, 1.6H), 6.78
(m, 0.1H), 5.72 (m, 0.05H), 5.63 (m, 0.2H), 5.17 (m, 0.95H), 5.07
(m, 0.8H), 4.13 (m, 2H), 3.96 (m, 1H), 3.83 (m, 2H), 3.62 (m, 1H),
3.54, 3.56 (2s, 6H), 2.32 (m, 2H), 2.17 (m, 2H), 2.01 (m, 6H), 0.86
(m, 8H), 0.74 (m, 3.4H), 0.31 (m, 0.6H). ESIHRMS m/z calcd for
C38H47N8O6 [M + H]+ 711.3613; found 711.3602.
Properties Calculation. The selected molecular descriptors were
calculated for 13a and other compounds using SmartMining
Software52 and ChemoSoft Software.53 The Log D was calculated in
Marvin (ChemAxon).54
Solubility of Compound 13a. The aqueous solubility was
determined using a filtration-based kinetic HTS assay with
spectroscopic readout.55 The compound stock was prepared in
DMSO and diluted in pION universal buffer at the desired pH to a
final concentration of 200 μM (DMSO concentration 2%). The
mixture was then incubated at rt for 1 h under constant shaking and
then filtered through a 96w Millipore MultiScreen solubility filter
plate. The optical density spectrum from 250 to 400 nm of the tested
compound in the filtrate was recorded using a SpectraMax UV/V
microplate reader (Molecular Devices, Sunnyvale, CA) after the 1.67-
fold dilution of the filtrate with acetonitrile. To quantify the aqueous
solubility, a standard calibration curve from 0 to 200 μM was
constructed in 40% acetonitrile in buffer. The solubility was calculated
by56
solubility (μM) = 1.67 × (OD (f ) − OD )/k
λ
λ
Methyl [(S)-1-((S)-2-{5-[4-(4-{2-[(R)-1-((S)-2-Methoxycarbonyla-
mino-3-methyl-butyryl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-
diynyl)-phenyl]-1H-imidazol-2-yl}-pyrrolidine-1-carbonyl)-2-methyl-
propyl]-carbamate Dihydrochloride (13i). Compound 13i was
synthesized in accordance with the procedure provided above for
compounds 13a−f starting from compound 18a and 5-iodimidazola
19d. Subsequent removal of the Boc protecting group in 20d resulted
in 21d. The acylation of the formed intermediate compound 21d by
N-Moc-L-valine 22a yielded the desired product 13i, which was easily
transformed into the corresponding dihydrochloride salt by following
where ODλ(f) is the optical density of the filtrate at the selected
wavelength, ODλ is the optical density of the solvent, k is the slope
ratio of the calibration curve (μM−1), and 1.67 is the dilution
coefficient of the filtrate by acetonitrile.
Molecular Modeling. The 3D molecular docking study was
performed in MOE software23 following the approach reported by
Cordek and colleagues.34
Replicon Antiviral Assays. Compounds 13a−k were tested in the
HCV GT1b subgenomic replicon assays with or without the addition
of 40% normal human serum (Table 2).41,57,58 The human hepatoma
cell line Huh7 harboring the HCV replicon (genotype 1b) was used as
the test line to perform the assay. Cells were seeded at a density of 7.5
× 103 cells per well in 96-well plates containing 50 μL of assay media.
The test compound (2× stock solution) was prepared fresh in the
assay medium (DMEM 1×, Cellgro, catalogue no. 10-013-CV). A total
of 11 serial 3-fold dilutions of the test compounds were prepared from
the 2× stock in assay media ranging from 20 nM to 0.2 pM final
concentrations. No earlier than 4 h after the cells were seeded,
compound treatment was initiated by adding the test solution (50 μL)
to the plates. The final concentration of the compound therefore
ranged from 10 nM to 0.1 pM when diluted 1:1 in the existing culture
media. The final DMSO concentration was 0.5%. Test examples were
then incubated for 3 days at 37 °C in a 5% CO2 atmosphere. The
medium was removed from the plates via gentle aspiration. Cells were
fixed with 100 μL of 1:1 acetone:methanol for 1 min, washed three
times with PBS buffer, and then blocked with 150 μL/well 10% FBS in
PBS for 1 h at rt. The cells were washed three times with PBS buffer
and incubated with 100 μL/well antihepatitis C core mAb (Affinity
BioReagents; catalogue no. MA1-080, 1 mg/mL stock diluted 1:4,000
in 10% FBS-PBS) for 2 h at 37 °C. The cells were then washed three
times with PBS and incubated with 100 μL/well HRP-Goat Anti-
Mouse antibody (diluted 1:3,500 in 10% FBS-PBS) for 1 h at 37 °C.
Finally, the cells were washed three times with PBS and developed
with 100 μL/well OPD solution (1 OPD tablet + 12 mL citrate/
phosphate buffer + 5 μL 30% H2O2 per plate) for 30 min in the dark at
rt. The reaction was stopped with 100 μL/well of 2N H2SO4, and the
absorbance was measured at A490 X on a Victor3 V 1420 multilabel
counter (PerkinElmer). The EC50 values for the test compounds were
calculated from the resulting best-fit equations determined by Xlfit
software.
1
the procedure described above. H NMR (DMSO-d6, 400 MHz) δ
15.35 (br s, 0.8H), 14.81 (m, 0.8H), 8.20 (m, 1H), 7.98 (m, 2.8H),
7.76 (m, 2.25H), 7.47 (d, J = 8.0 Hz, 0.2H), 7.26 (m, 1.65H), 6.84 (m,
0.05H), 6.74 (m, 0.05H), 5.76 (m, 0.07H), 5.63 (m, 0.2H), 5.18 (m,
0.94H), 5.08 (m, 0.8H), 4.14 (m, 2H), 4.00 (m, 1H), 3.81 (m, 2H),
3.62 (m, 1H), 3.54, 3.56 (2s, 5.8H), 3.31 (s, 0.2H), 2.37 (m, 1H), 2.26
(m, 1H), 2.17 (m, 2H), 2.02 (m, 5.5H), 1.86 (m, 0.3H), 1.67 (m,
0.2H), 0.86 (m, 8H), 0.74 (m, 3.5H), 0.31 (d, J = 6.8 Hz, 0.5H).
ESIHRMS m/z calcd for C38H47N8O6 [M + H]+ 711.3613; found
711.3638.
General Procedure 2. Methyl [1-((R)-2-{5-[4-(4-{2-[(R)-1-(2-Me-
thoxycarbonylamino-3-methyl-butyryl)-pyrrolidin-2-yl]-3H-imida-
zol-4-yl}-phenyl)-buta-1,3-diynyl]-1H-imidazol-2-yl}-pyrrolidine-1-
carbonyl)-2-methyl-propyl]-carbamate Dihydrochlorides 13j,k.
Compounds 13j,k were prepared according to the procedures
provided above for compounds 13a−f starting from compound 14b
and through the subsequent interaction of intermediate 18b with the
corresponding 5-iodimidazole 19d. The dihydrochlorides 13j,k were
obtained as describe above.
Methyl [(S)-1-((R)-2-{5-[4-(4-{2-[(R)-1-((S)-2-Methoxycarbonyla-
mino-3-methyl-butyryl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-
diynyl)-phenyl]-1H-imidazol-2-yl}-pyrrolidine-1-carbonyl)-2-methyl-
propyl]-carbamate Dihydrochloride (13j). 1H NMR (DMSO-d6, 400
MHz) δ 15.14 (br s, 0.4H), 14.50 (br s, 0.6H), 8.20, 8.22 (2s, 1H),
7.96 (m, 2.9H), 7.79 (m, 2.2H), 7.63 (m, 0.04H), 7.48 (d, J = 8.0 Hz,
0.13H), 7.24 (d, J = 8.0 Hz, 1.1H), 6.73 (m, 0.03H), 5.98 (m, 0.04H),
5.63 (m, 0.19H), 5.22 (m, 0.95H), 5.08 (m, 0.8H), 4.17 (m, 2H), 3.91
(m, 1H), 3.83 (m, 1H), 3.64 (m, 2H), 3.55 (s, 3H), 3.56 (s, 3H), 2.39
(m, 1H), 2.27 (m, 1H), 2.03 (m, 7.9H), 1.66 (m, 0.1H), 0.87 (m,
10.3H), 0.73 (m, 0.9H), 0.43 (m, 0.2H), 0.31 (m, 0.6H). ESIHRMS
m/z calcd for C38H47N8O6 [M + H]+ 711.3613; found 711.3613.
Methyl [(R)-1-((R)-2-{5-[4-(4-{2-[(R)-1-((R)-2-Methoxycarbonyla-
mino-3-methyl-butyryl)-pyrrolidin-2-yl]-3H-imidazol-4-yl}-buta-1,3-
diynyl)-phenyl]-1H-imidazol-2-yl}-pyrrolidine-1-carbonyl)-2-methyl-
propyl]-carbamate Dihydrochloride (13k). 1H NMR (DMSO-d6, 400
MHz) δ 15.28 (br s, 0.4H), 14.78 (br s, 0.6H), 8.20 (s, 1H), 7.95 (m,
3H), 7.77 (d, J = 8.4 Hz, 2H), 7.29 (t, J = 8.0 Hz, 1.3H), 6.87 (m,
0.05H), 5.71 (m, 0.03H), 5.41 (m, 0.05H), 5.16 (t, J = 6.8 Hz, 0.9H),
5.04 (m, 0.9H), 4.09 (m, 2H), 3.95 (m, 1H), 3.82 (m, 3H), 3.54 (s,
5.4H), 3.43 (s, 0.4H), 3.31 (s, 0.2H), 2.37 (m, 1H), 2.25 (m, 1H), 2.16
(m, 3H), 2.01 (m, 5H), 0.81 (m, 12H). ESIHRMS m/z calcd for
C38H47N8O6 [M + H]+ 711.3613; found 711.3635.
Cytotoxicity. The cytotoxicities of the tested compounds were
evaluated in parallel in the same human hepatoma cell line Huh7 as
described previously.45
Antiviral Spectrum Studies. The antiviral activity of compound
13a against West Nile virus, yellow fever virus, and dengue virus
(Table 3) was studied in African green monkey kidney cells (VERO)
obtained from the American Type Culture Collection (Manassas, VA,
USA) and routinely passaged in Minimal Essential Medium (MEM
with 0.15% NaCHO3) supplemented with 5% fetal bovine serum
(FBS, Hyclone). The serum was reduced to a final concentration of
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1%, and then gentamicin, at a final concentration of 50 μg/mL, was
added to the test medium. All viruses were obtained from the
American Type Culture Collection (ATCC, Manassas, VA, USA). The
tested compounds were diluted in MEM using one-half log dilutions,
and the final concentrations were evaluated in an eight-dilution test,
starting with 30 μM. Interferon alphacon-1 (Infergen) was used as a
standard inhibitor. Cells were seeded onto 96-well flat-bottomed tissue
culture plates (Corning Glass Works, Corning, NY), 0.1 mL/well, at
the proper cell concentration, and incubated overnight at 37 °C.
Various dilutions of the test compound were added to each well (3
wells/dilution, 0.1 mL/well). The resulting medium was added to cell
and virus control wells (0.1 mL/well). Virus samples diluted in test
medium were added to the compound test wells (3 wells/dilution of
compound) and to virus control wells at 0.1 mL/well. The test
medium without the virus was added to all the toxicity control wells (2
wells/dilution of each test compound) and cell control wells at 0.1
mL/well. The plates were then incubated at 37 °C in a humidified
incubator under a 5% CO2, 95% air atmosphere until the virus control
wells had developed an adequate cytopathic effect (typically 3 days).
The cell viabilities of the compound-treated and untreated cell cultures
were determined as reported previously,45 and the EC50 and CC50
values were calculated as described above.
Compound 13a-Resistant HCV Replicons. The replicons were
selected by continuous passaging of the Huh7 cells harboring the HCV
replicon (GT1b, clone Con1)41 in the presence of the tested
compound at concentrations exceeding the EC50 values by 5−40-
fold for 3−6 weeks. Some drug-resistant cell populations were cloned.
The primary structure of the NS5A gene was determined by direct
sequencing of the HCV RNA pools. As a result, several NS5A
mutations were identified (Table 4).
Animals. Mice, rats, and dogs were used in the pharmacokinetics
studies, in the acute, subchronic, and chronic toxicity studies, and in
the mutagenicity studies. The main principles of animal housing and
care were in full compliance with the approved guide59 following the
federal and Institutional Animal Care and Use Committee (IACUC)
guidelines.
Pharmacokinetics. Pharmacokinetic studies were performed in
male naive BALB/C mice, male naive Sprague−Dawley rats, and 2
male and 2 female naive English-breed beagle dogs. Intravenous
administration was dosed via infusion over 30 min in a vehicle
containing 20% 2-HP-β-CD in water (mice and rats) and sterile saline
solution (0.9% NaCl in water) (dogs). Oral dosing was administered
by gavage in a vehicle containing 0.5% Tween 80 in water (mice and
dogs) and 0.5% DMSO in PBS, pH = 7.4. Blood samples were
collected over a 24 h period postdose into Eppendorf tubes containing
5% EDTA in water. The plasma was isolated, and the concentrations
of the test compounds in plasma were determined by LC/MS/MS
(QTRAP 5500, Applied Biosystems and chromatograph Agilent 1290,
Agilent Technologies) after protein precipitation with acetonitrile.
Noncompartmental PK was performed on the plasma concentration
data to calculate the PK parameters using WinNonlin Professional 6.1
software (Pharsight Corporation).
Interaction with CYP450 Enzymes. The interaction of
compound 13a with human cytochrome CYP450 was studied on a
cytochrome panel consisting of CYP1A2, CYP2C9, CYP2C19,
CYP2D6, CYP3A4, and CYP3A4 (Invitrogen Vivid CYP450 screening
kits) in 1% DMSO according to the manufacturer’s protocols. The
final compound concentrations were 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and
0.003 μM, and each concentration was tested in duplicate. The EC50
values were determined with Graph Pad Prism 5 sigmoidal dose−
response fitting.
were carried out at 13a concentrations of 3 and 30 μM in accordance
with the manufacturer’s instructions.61 The total reaction volume was
20 μL, and the DMSO content was 0.5%. The Z-factor value was 0.5,
the S/N was 8.3, and the assay window ΔmP was 140. The
antiarrhythmic drug E-4031, which blocks hERG-type potassium
channels, was used as a control sample. The obtained IC50 value
calculated for E-4031 was 17 nM, which correlated well with the data
reported previously.62 For both concentrations, the binding did not
exceed 10%; no dose-dependence was observed.
Acute Toxicity. Acute toxicity studies were performed in mice and
rats according to standard protocols.63 All groups included six males
and six females. The individual body weight variation within each sex
did not exceed 10%. Working solutions were prepared on the day of
administration. For ip administration, physiological saline (0.9% NaCl
in water) was used as a vehicle; hydroxypropyl methylcellulose in
water (0.2%) was used for oral administration. The final solutions of
compound 13a were administered orally using gavage at 10 mL/kg.
Animals from the control group received equivalent volumes of the
vehicle. The clinical signs of toxicity were monitored twice a day, at
approximately 8−10 a.m., and again at the end of the workday, at 3−4
p.m. The doses leading to the death of 50% and 10% of the animals
(LD50 and LD10) and the grade of acute toxicity were derived based on
the mortality reported within 14 days after the administration of 13a.
Statistical processing of the data, obtained by the registration of animal
mortality rate for the purpose of (LD50 standard error), LD10, LD16,
and LD84 determination, was carried out by probit analysis.64
Calculations were performed by Finney’s method using BioStat 2006
software.
Mutagenicity (in Vitro). The mutagenic effect of compound 13a
was estimated using histidine-dependent strains of Salmonella
typhimurium (TA98, TA100, TA1535, and TA1537) Ames MPF
(Xenometrix, Switzerland). The assay was performed in full
compliance with the manufacturer’s manual (Xenometrix, Switzer-
land). Approximately 107 his−bacteria of each strain were treated with
13a at two concentrations (20 and 200 μM) and with positive (specific
mutagen) and negative (vehicle −2% DMSO) controls. The assay was
repeated in triplicate to provide sufficient data for statistical analysis.
The statistically significant increase in the number of colonies in
comparison with the vehicle confirmed the mutagenic action of 13a in
Ames test. The following positive controls recommended by the
manufacturer were used: 2-nitrofluorene, 4-nitroquinolile-N-oxide, N4-
aminocytidine, and 9-aminoacridine for the TA98, TA100, TA1535,
and TA1537 strains, respectively.
Mutagenicity (in Vivo). Male and female C57BL/6 mice weighing
18−20 g at the age of 8−12 weeks were used to estimate the
mutagenicity of compound 13a. In the chromosome aberration test,
13a was administered orally in the form of a suspension in Tween-80.
Single doses of 20 and 200 mg/kg were given to male mice, and the
cell material was preserved 24 h after drug administration; 20 mg/kg
doses were given to male and female mice daily during 5 days with cell
material preservation 6 h after the last drug administration. Mice from
the control group (vehicle control) received equivalent volumes of oral
Tween-80 solution. Cisplatin was used as a positive control. The drug
was administered intraperitoneally in the dose of 5 mg/kg. The
animals were then autopsied 24 h after exposure. Cytogenetic
preparations of femoral marrow were obtained by the standard air-
dry technique.65 Cytogenetic analysis was conducted using a Standart-
20 (Carl Zeiss) microscope in accordance with the principles of
cytogenetic analysis, as previously described.66
ASSOCIATED CONTENT
■
Metabolic Stability of the 13a. The metabolic stability of the hit
compound was evaluated in the presence of 0.5 mg/mL pooled
microsomal protein from rat, dog, and human livers (BD Gentest,
USA) and NADPH over 1 h using 1 μM of the test compound (Table
7). The residual compound was determined by LC-MS/MS analysis.
The t1/2 was calculated as described previously.60
Cardiotoxicity in Vitro. The inhibition potency of compound 13a
toward the K+ channel myocardium of hERG was studied using the
Invitrogen Predictor hERG fluorescence polarization assay kit. Tests
S
* Supporting Information
1H spectra of compounds 13a−k; pharmacokinetic graphs for
13a. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +89057320053. E-mail: yai@chemdiv.com.
■
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Journal of Medicinal Chemistry
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Hepatitis C virus inhibitors. Patent application WO-2008/021936,
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We sincerely thank Nikita Polyakov from the Vernadsky
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ABBREVIATIONS USED
AUC0→∞, area under the concentration−time curve from time
zero to infinity; F, bioavailability (%); Boc, tert-butoxycarbonyl;
■
C
max, maximum plasma concentration; DIPEA, diisopropyle-
thylamine; EDAC, N-(3-(dimethylamino)propyl)-N′-ethylcar-
bodiimide hydrochloride; EDTA, ethylenediaminetetraacetic
acid; FBS, fetal bovine serum; GT1a, hepatitis C virus genotype
1a, a subtype of hepatitis C virus genotype 1; GT1b, hepatitis C
virus genotype 1b, a subtype of hepatitis C virus genotype 1;
̀
hERG, the human ether-a-go-go-related gene; 2-HP-β-CD, 2-
hydroxypropyl-β-cyclodextrin; IFN-α, Infergen; LD, lethal
dose; μM, micromolar; MOE, molecular operating environ-
ment; 40% NHS, normal human serum; N-Moc, N-
metoxycarbonyl; NS5A, hepatitis C virus nonstructural protein
5A; nM, nanomolar; OPD, o-phenylenediamine; pM, picomo-
lar; PSA, molecular polar surface area (Å); RNA, ribonucleic
acid; t1/2, half-lifetime after administration of a drug; THF,
tetrahydrofuran
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