Hepatitis C virus NS5A replication complex inhibitors: The discovery of daclatasvir

Article abstract of DOI:10.1021/jm401836p

The biphenyl derivatives 2 and 3 are prototypes of a novel class of NS5A replication complex inhibitors that demonstrate high inhibitory potency toward a panel of clinically relevant HCV strains encompassing genotypes 1-6. However, these compounds exhibit

Full text of DOI:10.1021/jm401836p

Article
pubs.acs.org/jmc
Hepatitis C Virus NS5A Replication Complex Inhibitors: The
Discovery of Daclatasvir
Makonen Belema,*,† Van N. Nguyen,† Carol Bachand,⊥ Dan H. Deon,⊥ Jason T. Goodrich,†
Clint A. James,⊥ Rico Lavoie,⊥ Omar D. Lopez,† Alain Martel,⊥ Jeffrey L. Romine,† Edward H. Ruediger,⊥
Lawrence B. Snyder,† Denis R. St. Laurent,† Fukang Yang,† Juliang Zhu,‡ Henry S. Wong,‡
David R. Langley,§ Stephen P. Adams,¶ Glenn H. Cantor,∥ Anjaneya Chimalakonda,∥ Aberra Fura,△
Benjamin M. Johnson,¶ Jay O. Knipe,¶ Dawn D. Parker,¶ Kenneth S. Santone,¶ Robert A. Fridell,#
Julie A. Lemm,# Donald R. O’Boyle, II,# Richard J. Colonno,# Min Gao,# Nicholas A. Meanwell,†
and Lawrence G. Hamann†
‡
§
¶
†Departments of Discovery Chemistry, Discovery Chemistry Synthesis, Computer-Assisted Drug Design, and Pharmaceutical
Candidate Optimization, #Virology, Bristol-Myers Squibb Research and Development, 5 Research Parkway, Wallingford, Connecticut
06492, United States
⊥Department of Discovery Chemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, 100 boul. De l’Industrie, Candiac,
Que., Canada J5R 1J1
∥Department of Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Research and Development, 311 Pennington-Rocky
Hill Road, Pennington, New Jersey 08534, United States
△Department of Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Research and Development, Route 206 and Province
Line Road, Princeton, New Jersey 08543, United States
ABSTRACT: The biphenyl derivatives 2 and 3 are prototypes
of a novel class of NS5A replication complex inhibitors that
demonstrate high inhibitory potency toward a panel of
clinically relevant HCV strains encompassing genotypes 1−6.
However, these compounds exhibit poor systemic exposure in
rat pharmacokinetic studies after oral dosing. The structure−
activity relationship investigations that improved the exposure
properties of the parent bis-phenylimidazole chemotype,
culminating in the identification of the highly potent NS5A
replication complex inhibitor daclatasvir (33) are described.
An element critical to success was the realization that the
arylglycine cap of 2 could be replaced with an alkylglycine derivative and still maintain the high inhibitory potency of the series if
accompanied with a stereoinversion, a finding that enabled a rapid optimization of exposure properties. Compound 33 had EC50
values of 50 and 9 pM toward genotype-1a and -1b replicons, respectively, and oral bioavailabilities of 38−108% in preclinical
species. Compound 33 provided clinical proof-of-concept for the NS5A replication complex inhibitor class, and regulatory
approval to market it with the NS3/4A protease inhibitor asunaprevir for the treatment of HCV genotype-1b infection has
recently been sought in Japan.
been focused on inhibitors of the NS3/4A protease and the
NS5B polymerase because these enzymes are readily
recapitulated in biochemical assays that facilitate screening
and evaluation of lead candidates. However, the clinical
validation of NS5A replication complex inhibitors by daclatasvir
(33) generated significant interest in this target class, as
reflected by the increased number of NS5A-targeting
compounds currently undergoing clinical evaluation.2,3
Although HCV NS5A is not associated with any enzymatic
INTRODUCTION
■
The current optimal therapy for the treatment of hepatitis C
virus (HCV) genotype-1 (GT-1) infection, the most prevalent
strain, is a combination of pegylated interferon-α and the
nucleoside analogue ribavirin in conjunction with either the
nonstructural 3/4A (NS3/4A) protease inhibitor simeprevir or
the NS5B nucleotide polymerase inhibitor sofosbuvir.1
Although this drug regimen improves response rates compared
to therapy with pegylated interferon-α and ribavirin alone, it is
difficult to tolerate because of numerous side effects, some of
which present a significant challenge. As part of the effort to
identify direct-acting antiviral agents (DAAs) that exhibit
improved efficacy and tolerability, considerable emphasis has
Special Issue: HCV Therapies
Received: November 27, 2013
Published: February 12, 2014
© 2014 American Chemical Society
2013
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Figure 1. Initial strategies pursued to enhance PK properties of 2 and 3.
a
Table 1. Replicon and Pharmacokinetic Assessment of Aza-Core Analogues 4−6
a
b
c
GT-1b CC50 >10 μM. Amorphous solubility and PAMPA values were determined at pH 6.5 and pH 7.4, respectively. NC = not calculated; ND =
d
e
not detected. Reported previously in ref 6f. Null = compound was not detected by UV plate reader in both the receiver and donor assay wells.
activity, various studies indicate that this protein plays diverse
but not well-defined functions in the replication cycle of the
virus.
imidazole series accomplished with two related analogues,
carbamate 2 and amine 3, toward a broad panel of replicon
strains was attractive, their pharmacokinetic (PK) evaluation in
rats revealed poor systemic exposure following oral dosing.6f
Thus, improving the PK properties of this series while retaining
the antiviral spectrum became the primary focus of the final
phase of the medicinal chemistry campaign. Herein is described
the absorption, distribution, metabolism, and excretion
(ADME) optimization effort that achieved this objective and
culminated in the identification of 33.
Leads that disrupt the function of the NS5A replication
complex were discovered using a phenotypic screen based on a
GT-1b replicon system.4,5 We have recently disclosed the key
milestones that marked the evolution of our HCV NS5A
replication complex inhibitor program, beginning from the
identification of thiazolidinone 1 as a screen hit to the
elucidation of a novel and potent bis-phenylimidazole chemo-
type.4b,c,6 Notable issues that were addressed in this process
included chemical instability, a potential genotoxic liability, and
initial antiviral activity of limited scope toward HCV genotypes.
While the picomolar inhibitory activity of the bis-phenyl-
RESULTS AND DISCUSSION
■
During the earlier phases of the program, the PK properties of
representative analogues from lead chemotypes with distinct
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a
Table 2. Replicon and PK Assessments of Cap-Truncated Analogues 7-14
a
b
GT-1b CC50 >10 μM for all except 9 (>8.1 μM). Amorphous solubility and PAMPA values were determined at pH 6.5 and pH 7.4, respectively.
c
d
e
NC = not calculated; ND = not detected. Reported previously in ref 6f. Null = compound was not detected by UV plate reader in both the
receiver and donor assay wells.
structural compositions were assessed; however, poor oral
exposure was the most frequent observation. Early indications
from in vitro studies including PAMPA permeability assays
were that limited intestinal absorption, likely because of a
combination of poor intrinsic permeability, poor pharmaceut-
ical properties, and P-glycoprotein (P-gp) efflux, might be
playing the dominant role in hampering systemic exposure. The
aqueous solubility of carbamate 2 and amine 3, when assessed
using amorphous material, differed considerably, with marginal
solubility (0.001 mg/mL) noted for 2 and excellent solubility
(>0.61 mg/mL) for the more basic 3. Thus, a decision was
made to implement complementary structure−activity relation-
ship (SAR) optimization strategies concurrently that involved
examining both leads as part of a broader effort aimed at
securing optimal exposure. The first approach focused primarily
on improving the drug-like characteristics of the carbamate cap
series, exemplified by 2, by either increasing its solubility and/
or reducing its molecular footprint, while the second dealt with
improving the permeability properties of the amine cap series,
exemplified by 3, through modulation of electronic and steric
parameters of the benzylic substituent (Figure 1).
(27- to 111-fold) and GT-1b (3- to 5-fold) replicons, there was
an incremental gain in amorphous solubility when assessed in
buffer at pH 6.5. In addition, measurable PAMPA values were
observed, indicative of some level of intrinsic permeability.
However, in rat PK assessmentseither a 4 h screen or a 24 h
study4 and 5 did not exhibit measurable systemic exposure
after oral dosing, whereas 6 showed minimal plasma exposure
(PO-AUC = 21 nM·h; F = 0.5%) but was not detected in either
the liver or plasma at 24 h.7
It was reasoned that some degree of molecular weight
reduction of the leads would be necessary to improve their
exposure properties. With this in mind, an SAR optimization
campaign was embarked upon to further delineate the balance
that may exist between oral exposure and potency (Table 2).
Replacing both of the phenylglycine caps of 2 with the
unnatural enantiomer of valine, which retained the (R)-
stereoconfiguration at the α-carbon, resulted in more than
40000-fold loss in GT-1a inhibitory activity (compare 7 and 2).
A lower but still significant loss in GT-1a inhibitory potency
(410- to 11400-fold) was also observed when one of the caps of
2 was truncated, as in 8−10. It is noteworthy that partial
truncation had minimal impact on GT-1b antiviral potency,
with EC50 values ≤48 pM maintained by these analogues.
Although compound 9 exhibited moderate PAMPA perme-
ability and some gain in amorphous solubility, it had no
meaningful exposure following oral dosing to rats. It soon
As part of the initial SAR exploration, one of the phenyl
groups of the biaryl core of 2 was replaced with six-membered
heterocyclic analogues (Table 1). While the introduction of
polarity to the central scaffold region of 2, as in 4−6, caused
variable levels of loss in inhibitory potency toward both GT-1a
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Table 3. Replicon and PK Assessment of Basic Cap Analogues 15−22
a
Compound 20 and 21 are symmetrical diastereomers but with unknown stereochemistry at their benzylic center [i.e., either (R/R) or (S/S)],
b
whereas the remaining compounds have (R/R)-stereoconfiguration. Compound 22 is a mixed-cap analogue. Amorphous solubility and PAMPA
values were determined at pH 6.5 and pH 7.4, respectively. NC = not calculated; ND = not detected with the exception that one plasma sample
obtained for 3 at 0.17 h indicated 20 nM concentration. Estimated clearance (mL/min·kg) from 4 h IV rat PK screen (2 mg/kg dose; n = 2): 3
(17), 15 (88), 16 (4), 18 (58). Reported previously in ref 6f. Null = compound was not detected by UV plate reader in both the receiver and donor
c
d
e
f
assay wells.
became apparent from structure−activity studies that main-
taining a phenylglycine cap at one end of the molecule and
varying the alternate cap region afforded mixed-cap analogues
exhibiting GT-1a EC50 values near 100 nM but with notable
improvements in PAMPA permeability compared to the
progenitor 2. Assessment of one such analogue, 11, in a rat
PK screen indicated a marked gain in exposure for the molecule
(Table 2). Interestingly, compound 12, a diastereomer of 11
with similar PAMPA permeability value, displayed a lower
exposure in the rat. Compound 11 was advanced into
additional rat, dog, and mouse PK studies in order to gather
information on the cross-species exposure properties of the
chemotype, and data is summarized in Table 4. In a 24 h rat PK
study, 11 exhibited low clearance, a small volume of
distribution, and a half-life of 6.9 h after IV dosing. After oral
dosing, 11 exhibited low bioavailability (6.8%), a favorable
liver/plasma ratio (26), and liver exposure at 24 h (116 nM)
that is below its GT-1a EC50 (138 nM). In light of the
compound’s low IV clearance, its low bioavailability is believed
to reflect incomplete absorption, attributed to P-gp efflux, a
contention supported by results from in vitro Caco-2
bidirectional studies (11: A−B, <15 nm/s; B−A, 105 nm/s).
Compound 11 showed higher oral bioavailability in the mouse
(17%) and dog (45%) when compared to rat (6.8%). Although
a follow-up study to probe into the reasons behind the noted
differences in oral bioavailability was not conducted, it is
noteworthy that there was evidence for enterohepatic
recirculation in the mouse and dog, which may have favorably
impacted the oral bioavailability in these species.
Compounds 13 and 14, the dimethyl amine cap analogues of
11 and 12, respectively, were prepared and profiled in standard
in vitro and rat PK studies to gather comparative data on the
two cap families in order to help chart a path forward. These
compounds exhibited improved solubility, similar inhibitory
activity in the GT-1a replicon, but lower PAMPA permeability,
when compared with their carbamate counterparts 11 and 12,
respectively. In light of the multispecies PK data obtained for
11, the amine cap analogue 13 was advanced directly to 24 h
rat, dog, and mouse PK studies to establish a comparative
benchmark for the two versions of the phenylglycinamide cap
class. Compound 13 exhibited higher clearance and volume of
distribution in all three species and a longer half-life in rat and
mouse when compared to the carbamate counterpart 11. After
oral dosing to rats, 13 was not detected in the plasma or liver at
24 h; however, there was measurable systemic exposure in both
the mouse and dog after PO dosing, although the absolute
levels were lower than that of 11. Enterohepatic recirculation
was evident for 13 across species in both dosing arms, with
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a b
,
Table 4. PK Assessment of 11, 13, and 18 in Rats, Dogs, and/or Mouse
IV (24 h)
PO (24 h)
L-24 h (nM)
compd
animal
CL (mL/min·kg)
Vd (L/kg)
t1/2 (h)
AUC (nM·h)
P-24 h (nM)
% F
11
rat
4.4
22
0.3
7.5
1.9
60
82
14
20
10
6.9
>10
2.9
14
1830
1520
15100
NC
116
4.5
6.0
59
6.8%
45%
17%
dog
mouse
rat
5.3
57
253
ND
13
18
ND
30
c
dog
mouse
rat
102
18
NC
436
60%
7.8
7.7
1422
NC
539
25
6.0%
d
63
ND
ND
8.3
dog
28
>12
272
14%
a
Respective IV/PO dosing levels: rat (2/5 mg/kg), dog (1/3 mg/kg), mouse (5/20 mg/kg). Number of animals per dosing group: rat (n = 3), dog
b
(n = 2), mouse (n = 9; composite design). Vehicle: PEG-400 for mouse-IV/PO, rat-IV/PO, and dog-PO; PEG-400/water (9:1) for dog-IV. NC =
not calculated; ND = not detected. t1/2 could not be calculated because of a rise in plasma level through 24 h. Only 1 of 3 rats had a detectable level
c
d
(318 nM).
rising plasma levels through 24 h in some cases, which
introduced some variability to the data and complicated its
interpretation. Interestingly, in comparing the rat PO exposure
of 11 to 14, there was no correlation between exposure and the
stereoconfiguration of the tetrahydrofuran moiety, because for
the carbamate cap pair 11 and 12, the (R)-isomer exhibited the
better exposure, whereas for the amine cap pair 13 and 14, it
was the (S)-isomer that showed higher exposure. Finally, as
part of the effort to profile lead molecules of interest for off-
target liabilities, 11 and 13 were assessed in a panel of
biochemical screens conducted at a concentration of 10 μM by
MDS Pharma and were found to inhibit the binding of a
radioligand to the Na+ channel site-2 by 82 and 102%,
respectively. In a follow-up study conducted in-house, whole-
cell patch-clamp analysis revealed that 11 and 13 inhibited Na+
channel conductance by 14 and 53%, respectively, at a
concentration of 10 μM in protein-free buffer at pH 7.4.
In a concurrent effort, a complementary SAR survey was
conducted around the second lead, amine 3, in order to
determine the feasibility of improving its PK properties through
modification of the steric and electronic properties of the
benzylic amine substituent (Table 3). Consistent with SAR
findings disclosed previously for the cap region when examined
in the context of an anilide chemotype, there was broad
tolerance to steric bulk around the benzylic site and that
attenuation of the basicity of the amine group had a detrimental
impact on GT-1a inhibitory potency (Table 3).6e For example,
in modifying the dimethylamine moiety of 3 to a piperidine, as
in 18, the GT-1a and GT-1b inhibitory activities remained
essentially unchanged, varying by <4-fold, whereas less basic
analogues, morpholine 19 and fluoro-pyrrolidine 20, exhibited a
44- to 101-fold diminished GT-1a antiviral activity when
compared to pyrrolidine 17. Following oral administration to
rats monitored for 4 h, compounds 15−19 demonstrated low
plasma exposure, with AUC values ranging from 5 to 54 nM·h,
although the liver concentrations at 4 h surpassed 1.0 μM for
three of the five (15, 16, and 18) analogues. Interestingly,
although pyrrolidine 17 exhibited the lowest PAMPA
permeability value and the poorest oral exposure among the
derivatives of 3 advanced to rat PK studies, the reason why this
molecule behaves significantly different from its close
homologues 15, 16, and 18 is not readily apparent. A subset
of the analogues were also assessed in an IV rat PK screen and
the estimated clearance ranged from 4 mL/min·kg for 16 to 88
mL/min·kg for 15.
For 15, 16, and 18, the observed 4 h PO-liver exposure
would provide more than a 25000-fold multiple over their GT-
1a replicon EC50 values, which was much higher than that of
compound 11, 19-fold at 4 h. While the high exposure multiple
achieved after oral dosing by the potent amine cap analogues in
the targeted tissue was encouraging, their plasma 4 h AUC was
>10-fold lower when compared with that of 11. To gain
additional insight into the PK properties of this series, 18 was
advanced into more detailed 24 h rat and dog PK studies
(Table 4). Compound 18 exhibited high clearance, a high
volume of distribution, and a prolonged half-life in rats
following IV dosing. After oral dosing, the compound was
barely detectable in the plasma through 24 h, and only 1 of 3
rats exhibited liver exposure (318 nM) at 24 h. In dog PK
studies, 18 showed moderate to high clearance, a high volume
of distribution, a long half-life after IV dosing, and an oral
bioavailability of 14%. As was the case for mixed-cap analogues
11 and 13 discussed earlier, the homodimeric analogue 18
exhibited better exposure in dogs than rats. It appears that oral
exposure in rats for 18 was subject to considerable variability, a
concern from developability perspective, but the likely cause for
this observation was not investigated.
Compound 18 was characterized further in a select panel of
in vitro assays designed to gather additional information about
the potential off-target liabilities of analogues containing basic
phenylglycine caps. In whole-cell patch-clamp assays, per-
formed at a concentration of 10 μM, 18 inhibited Na+, hERG,
and Ca2+ channels by 94, 82, and 69%, respectively. While no
glutathione (GSH) conjugate was detected following incuba-
tion of 18 with GSH-supplemented human and rat liver
microsomes, a cyanide adduct was detected when KCN was
used in place of GSH, an indication for the formation of a
reactive metabolite. It is noteworthy that the mixed-cap
analogue 22 exhibited cyanide addition only to the piperidine
cap portion when subjected to a similar bioactivation study, an
indication that related basic cap analogues may have differ-
entiated liabilities.
There were a number of important findings from the in vitro
and PK studies discussed above that proved instrumental in
formulating an end-game strategy in search of a candidate
exhibiting optimal virology, ADME, and safety properties. First,
it was demonstrated that reduction of the overall molecular size
of leads 2 and 3, as in 11, 12, and 14, significantly enhanced
oral exposure. Moreover, both carbamate 12 and amine 14
exhibited exposure in a rat PK screen following oral solution
dosing that were within 2-fold of each other, although the
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a
Table 5. Replicon and PK Assessment of Non-Basic Cap-Truncated Analogues 23−31
a
b
GT-1b CC50 >10 μM for all except 9 (>8.1 μM). Amorphous solubility and PAMPA values were determined at pH 6.5 and pH 7.4, respectively.
c
NC = not calculated; ND = not detected.
potential advantage that the relatively more soluble 14 may
have under nonsolution dosing by, for example, minimizing the
likelihood of dissolution-rate limited absorption, was not
investigated. In the case of the homodimeric amine cap series,
although the liver exposure was favorably impacted by increases
in lipophilicity, plasma exposure was low and variable. Second, a
limited correlation was established between PAMPA perme-
ability and oral exposure in rat PK screen for the chemotype. In
addition, it was observed that rat was a more stringent PK
model to guide the SAR evolution because the compounds that
were advanced into dog and/or mouse PK studies hitherto (i.e.,
11, 13, and 18) demonstrated relatively enhanced oral
bioavailabilities in those species. Finally, preliminary in vitro
safety assessments revealed that analogues containing amine-
based caps may possess increased liability for ion channel
inhibition. Taken together, the carbamate series emerged as the
more attractive class for additional optimization where a
combination of PAMPA and rat PK screening were deemed
sufficient to gauge progress toward compounds with targeted
exposure. The key outstanding issue that needed to be
addressed at this juncture was how to regain the significant
(>4900-fold) GT-1a inhibitory potency that was lost in pursuit
of improved PK properties in analogues such as 11.
potency toward GT-1a or GT-1b replicon, the introduction of a
methyl group with the (S)-stereoconfiguration appeared to
have impacted the PAMPA permeability favorably (compare 23
and 25). Because carbamate 9 was among the most potent GT-
1a inhibitors, EC50 of 12 nM, within the cap-truncated
analogues examined hitherto, its (S)-alanine variant was
prepared in anticipation of enhanced PAMPA permeability.
While changing the glycine cap of 9 to the (S)-alanine cap of 26
had the expected favorable impact on PAMPA permeability,
this modification was also associated with a 100-fold enhance-
ment of potency toward the GT-1a replicon, unanticipated
based on the SAR that had been developed to date. Because it
had been demonstrated previously that the GT-1a antiviral
potency of mixed-cap analogues resided between that of the
respective homodimeric cap analogues, the gain in the GT-1a
inhibitory potency with 26 strongly suggested that the
corresponding (S)-alanine cap homodimer would be very
potent toward a GT-1a replicon, which indeed was the case
(27: GT-1a EC50 = 0.282 nM).6f Notably, and in line with the
activity of bis-(R)-valine cap analogue 7 discussed earlier, the
bis-(R)-alanine cap analogue 28 was >2000-fold weaker than its
diastereomer 27 toward the GT-1a replicon. This result
brought into clear focus a difference in stereochemical
requirements at the α-carbon between the phenylglycine and
the alkylglycine cap derivatives. Moreover, this finding
demonstrated that the initial assumption made in retaining
the (R)-stereoconfiguration during the cap truncation exercise
The next phase of the SAR exploration focused around
carbamate 9 and the small set of related analogues 23−25 that
were prepared as part of the initial cap truncation exercise
(Table 5). While acetamides 23−25 had similar inhibitory
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a
Table 6. Replicon and PK Assessment of Advanced Analogues 32−41
a
b
c
GT-1b CC50 >10 μM for all. Amorphous solubility and PAMPA values were determined at pH 6.5 and pH 7.4, respectively. Virology data
d
reported previously in ref 2. NA = not available. In 24 h rat PK study (IV/PO: 2/5 mg/kg; n = 3) 40 showed an oral bioavailability of 4.3%.
Following oral dosing, it had a plasma AUC of 106 nM·h and a 24 h liver level of 132 nM, whereas it was not detected in the plasma at 24 h.
while going from bis-(R)-phenylglycine analogue 2 to bis-(R)-
valine analogue 7, although reasonable based on extant SAR,
was incorrect.8
stereochemical disparity suggests that the respective carbamate
moieties may engage the NS5A proteins in subtly different
fashions.
Securing subnanomolar GT-1a inhibitory potency in the
alkylglycine cap series through stereochemical inversion was a
critical milestone for the program because it allowed a
reduction in the molecular size of the cap moiety and in the
aromatic composition of the chemotype, translating into
improved aqueous solubility and rat oral exposure (compare
27 vs 2).9 The follow-up effort focused on this new cap series in
order to further enhance its exposure properties. Whereas the
modifications illustrated in analogues 29−31 were detrimental
to both GT-1a and GT-1b antiviral activity, incorporation of
other aliphatic moieties at the α-carbon favorably impacted
potency and/or PK, with compound 33 exhibiting the best
combination of replicon potency and oral exposure in the rat
PK screen among related analogues prepared in this final
iteration of SAR study (Tables 5 and 6). Removal of the H-
bond-donating capability of the carbamate moiety of 33, as in
36, caused a significant reduction in GT-1a inhibitory potency
while having minimal impact on the antiviral effect in a GT-1b
replicon. In addition, replacing the carbamate moiety of 33 with
an acetamide, as in 37, which retains H-bonding capability, was
also detrimental to antiviral activity. Despite the apparent
similarity in the carbamate pharmacophoric elements of the
potent (S)-alkyl- and (R)-phenyl-glycine cap classes, their
In a parallel effort directed toward identifying alternate,
structurally differentiated candidates with enhanced pharma-
ceutical properties, a select group of (S)-alkylglycine cap
derivatives was hybridized with (R)-N,N-diethylphenylglycine
to afford the mixed-cap analogues 39−41 (Table 6). This basic
glycinamide cap was chosen over alternate variants (i.e., caps of
15 and 18) because it demonstrated improved properties in
earlier studies, including a lower estimated clearance in the rat
IV PK screen and a decreased liability for bioactivation in an in
vitro assay (see Table 3 and earlier discussions). Though the
replicon potencies of 39−41 were comparable to that of
homodimer 16, the improvement in oral plasma exposure with
39 and 41 in a 4 h rat PK screen was more limited than was the
case for 33. Nevertheless, based on the preliminary virology
profile and rat PK screen assessment results, 39 was selected for
additional characterization along with bis-(S)-valine cap
analogue 33.
Compounds 33 and 39 exhibited potent inhibitory activities
toward a broad panel of HCV genotypes, although 39 was more
potent than 33 toward GT-2a and GT-3a strains (Table 7).2,10
Compound 39 inhibited Na+ and hERG channels in patch-
clamp assays to a greater extent than 33. In rat, dog, and
cynomolgus monkey PK studies, 33 demonstrated higher oral
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100 mg dose, and, most notably, one of these subjects attained
a viral titer below 25 IU/mL, the lower limit of quantification
for the study. This rate and extent of decline of plasma HCV
RNA following a single oral administration of a small molecule
was unprecedented in the field of HCV therapeutics and
provided clinical proof-of-concept for the efficacy of HCV
NS5A replication complex inhibitors. In a 14 day multiple-
ascending dose study, though a mean maximum decline in viral
titer of 2.8 to 4.1 log10 was observed in subjects receiving doses
of 1 to 100 mg QD or 30 mg BID of 33, viral breakthrough
occurred during treatment, indicating that the compound has a
low genetic barrier to resistance.12 The amino acid changes
associated with the resistance correlated with those observed in
the in vitro replicon assay. In this study, the antiviral response
was relatively more pronounced in GT-1b- than GT-1a-infected
subjects, where 4 of 7 GT-1b-infected subjects compared to
none of the 17 GT-1a-infected subjects attained viral titers
below 25 IU/mL at day 14.
Table 7. Replicon and off-Target Liability Assessment of 33
and 39
a
b
compd
33
39
EC50 (nM)
GT-2a JFH-1
GT-2a J6
GT-3a
0.071
7.5
0.020
0.99
0.008
59
0.146
364
GT-3a YH
GT-3a YH*
GT-4a
1451
0.012
0.033
0.054
51%
0.014
0.021
GT-5a
GT-6
% inhibition of Na+ channel at 10 μM
62%
9.1
hERG IC50 (μM)
29.2
a
Except for GT-2a JFH-1, all are hybrid replicons in either GT-2a
JFH-1 or GT-1b Con1 backbone: GT-2a J6 (JFH-1); GT-3a or GT-3a
YH (Con1); GT-3a YH* (JFH-1); GT-4a (Con1); GT-5a (JFH-1);
b
GT-6a (JFH-1). A subset of the replicon data of 33 has been reported
previously in refs 2 and 10.
Compound 33 is currently in phase II and III clinical trials in
combination with other DAA agents targeting the orthogonal
mechanism(s) and/or pegylated-interferon-α/ribavirin regi-
mens.13,14 A noteworthy finding from these studies is the
demonstration that 24 weeks of therapy with a combination of
33 (60 mg QD) and the NS3/4A inhibitor asunaprevir (600
mg BID) was associated with a sustained viral response 24
weeks after cessation of therapy (SVR24) of 36%, providing the
first demonstration that HCV infection can be cured by a drug
bioavailabilities, F = 38−108%, compared to 39, F = 3.6−66%,
and enhanced oral plasma exposure (Table 8). In order to
Table 8. Multispecies Oral 24 h PK Assessment of 33 and
a
39
dose level
P-24h L-24h
compd animals
(mg/kg) AUC (μM·h) (nM) (nM)
% F
regimen that is free of pegylated-interferon-α and ribavirin.13a
A
b
33
39
rat
5.0
2.3
2.8
5.0
3.5
3.0
4.8
11
18
26
103
50%
108%
38%
dog
dual DAA combination therapy involving 33 and asunaprevir
for the treatment of GT-1b interferon-ineligible/-intolerant
subjects or those that are nonresponders to interferon/ribavirin
therapy has completed phase III studies. A request to market 33
and asunaprevir was filed with the Japanese regulatory agency
in October 2013. In addition, a triple DAA combination of 33,
asunaprevir, and the NS5B polymerase inhibitor BMS-791325
is currently undergoing phase II evaluation for the treatment of
HCV GT-1a/1b-infected subjects, with encouraging prelimi-
nary results.15 In addition to the evolving clinical impact that 33
has been demonstrating in the HCV field, it has also served as a
tool compound in a number of mode-of-action studies directed
at deciphering the NS5A protein’s various functions, an effort
that has also shed some light on likely reasons behind the high
inhibitory potency associated with the NS5A-targeting class of
compounds.16
monkey
rat
1.93
0.165
1.2
6.5
ND
9.0
4.2
c
178
3.6%
66%
dog
monkey
0.497
20.9%
a
Number of animals: rat (3), dog (2), monkey (3). See the
b
Experimental Section for details on vehicles. L-24 h data is an
average of 2 values since 33 was not detected in the liver of 1 of 3 rats.
c
ND = not detected.
further differentiate the two compounds, they were examined in
a 4 day mouse toxicology study where male and female animals
were administered doses of 15, 50, and 100 mg/kg orally on a
QD regimen. Both compounds were well-tolerated, and there
were no significant exposure differences between the genders.
However, the 0−24 h plasma AUC and liver and heart
exposures of 39, sampled at 8 and 24 h, approximately doubled
between day 1 and day 4 at each dose level while remaining
unchanged, or slightly decreasing, for 33. Based on these
findings, 33 was deemed the preferred compound, and it was
advanced through additional ADME characterization studies
and an extensive array of standard in vitro and in vivo off-target
liability assessments, including extended multispecies toxicol-
ogy. The results of these studies supported its advancement
into clinical trials.11
Compound 33 was safe and well-tolerated when dosed as a
solution at 1 to 200 mg to healthy volunteers in a placebo-
controlled, double-blind phase I clinical study.2 Plasma
exposure above the protein-binding adjusted GT-1a EC90 of
0.38 nM was observed at all doses through 24 h. In single-
ascending dose studies conducted in HCV-infected subjects,
doses of 1, 10 and 100 mg of 33 were associated with a mean
1.8, 3.2, and 3.3 log10 reduction in viral RNA, respectively,
measured 24 h post administration. The viral decline was more
pronounced in two GT-1b-infected subjects that received the
CHEMISTRY
■
Final compounds were readily prepared according to the
synthetic routes highlighted in Schemes 1−3.17 In the case of
aza-core analogues 4−6, key synthetic fragments 48a, 48c, and
52b were initially assembled as illustrated in Scheme 1.
Bromination of ketone 42, followed by alkylation with Boc-L-
proline and cyclization of the resultant ketoester 44 provided
bromopyridine 48a.18 Regioselective lithiation of dibromopyr-
idine 45a followed by condensation with a Weinreb reagent
furnished carbamate 46a, which was elaborated to 48b via the
intermediacy of ketoamide 47.19 Bromoketone 50, prepared
from 45b through a combination of a Stille coupling and in situ
bromination, was elaborated to chloropyrimidine 52a via
ketoester 51. Whereas 48a was coupled with boronate 53
under standard Suzuki-Miyaura condition to provide 54a,
applying a similar condition to 48b or 52a was not successful
(Scheme 2). However, protection of their imidazole moiety
with SEM ether, as in 48c and 52b, facilitated the Suzuki-
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a
Scheme 1. Preparation of Intermediates 48a, 48c, and 52b
a
Reagents and conditions: (a) Br2, HBr, CH2Cl2; (b) N-Boc-L-proline, Et3N, CH3CN; (c) NH4OAc, xylenes, 140 °C; (d) n-BuLi, t-butyl 2-
(methoxy(methyl)amino)-2-oxoethylcarbamate; −78 °C to −15 °C; (e) 48% HBr, dioxane; (f) N-Boc-L-proline, HATU, DIEA, DMF; (g) NaH,
SEM-Cl, DMF; (h) tributyl(1-ethoxyvinyl)tin, PdCl2(Ph3P)2, DMF, 100 °C; (i) NBS, H2O/THF, 0 °C.
a
Scheme 2. Preparation of Aza-Core Analogues 4−6
a
Reagents and conditions: (a) Pd(Ph3P)4, NaHCO3, DME/H2O, 90 °C; (b) 25% TFA/CH2Cl2; (c) (2R)-2-[(methoxycarbonyl)amino]-2-
phenylacetic acid, HATU, DIEA, DMF; (d) Pd(Ph3P)4, NaHCO3, DME/H2O, 80 °C.
Miyaura coupling. It is noteworthy that the regiochemistry of
the SEM ethers was not determined. Acid-catalyzed depro-
tection of intermediates 54a, 55a, and 56a, followed by
coupling of the resultant pyrrolidines with (2R)-2-
[(methoxycarbonyl)amino]-2-phenylacetic acid under standard
HATU/DIEA condition provided final products 4−6 (Scheme
2). Homodimeric and mixed-cap analogues of the bis-
phenylimidazole template were prepared from 57a and 57b,
respectively, according to general routes described previously
(see Scheme 3).2,6f The majority of the cap moieties used for
the final step were prepared readily from commercially available
precursors through adaptation of literature protocols.17 For a
subset of the cases, such as compounds 11−14, the cap
precursors were obtained from vendors.
CONCLUSION
■
In summary, distinct strategies were implemented toward
enhancing the oral exposure of two lead series of the bis-
phenylimidazole class of HCV NS5A replication complex
inhibitor, represented by the carbamate cap analogue 2 and the
amine cap analogue 3. Although the application of different
approaches favorably impacted the oral exposure properties of
both series to varying degrees, the more promising PK findings
resulted from a cap-truncation exercise that was performed in
the context of 2. The mixed-cap analogue 11 emerged from this
approach as a molecule with improved PK, but the structural
modification unfortunately resulted in >4900-fold loss in GT-1a
inhibitory potency. It was the combination of PAMPA
permeability and 4 h rat PK screen studies that guided
additional SAR exploration toward achieving balanced virology
and oral exposure properties. An unexpected finding with
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a
Scheme 3. Preparation of Homodimeric and Mixed-Cap Analogues
a
Reagents and conditions: (a) R1CO2H, HATU, DIEA, DMF; (b) 25% TFA/CH2Cl2 or 4 N HCl/dioxane; (c) R2CO2H, HATU, DIEA, DMF.
Table 9. LC/MS Conditions for the Characterization of Final Products
method
columns
gradient and run time
flow rate (mL/min)
solvent A
solvent B
1
2
3
4
1
2
1
3
10−100% B; 70 min
10−100% B; 70 min
10−100% B; 70 min
10−50% B; 40 min
50−100% B; 40−70 min
10−100% B; 70 min
10−50% B; 25 min
50−100% B; 25−32 min
0−100% B; 3 min
0−100% B; 3 min
0−100% B; 2 min
0−100% B; 2 min
0−100% B; 4 min
0−100% B; 4 min
0−100% B; 4 min
0.35
0.4
A1
A2
A1
A2
B1
B2
B2
B2
0.3
0.3
5
6
4
5
0.4
0.5
A1
A1
B2
B2
7
8
6
7
4
A3
A3
A3
A4
A3
A3
A3
B3
B3
B3
B4
B3
B3
B3
4
9
7
4
10
11
12
13
8
4
9
0.8
0.8
4
10
6
Columns: column 1: Waters Atlantis C18 (2.1 × 150 mm, 3.5 μm); column 2: Agilent Zorbax Extend C18 (2.0 × 150 mm, 5 μm); column 3: Agilent
Zorbax Extend C18 (2.1 × 150 mm, 3.5 μm); column 4: Waters Sunfire C18 (2.1 × 150 mm, 3.5 μm); column 5: Acquity UPLC BEH C18 (2.1 ×
100 mm, 1.7 μm); column 6: Phenomenex-Luna C18 (4.6 × 50 mm, S10); column 7: Phenomenex Luna C18 (3.0 × 50 mm, S10); column 8:
Primesphere C18-HC (4.6 × 30 mm); column 9: Phenomenex-Luna C18 (2.0 × 50 mm, 3 μm); column 10: Xbridge Phenyl (2.1 × 50 mm, 2.5
μm). Solvent A: solvent A1 = 25 mM NH4OAc in water at pH = 5; solvent A2 = 30 mM NH4HCO3 in water at pH = 10; solvent A3 = 0.1% TFA in
10% MeOH/90% H2O; solvent A4 = 5 mM NH4OAc in 10% CH3CN/90% H2O. Solvent B: solvent B1 = 25 mM NH4OAc in 85% CH3CN/15%
H2O; solvent B2 = CH3CN; solvent B3 = 0.1% TFA in 90% MeOH/10% H2O; solvent B4 = 5 mM NH4OAc in 90% CH3CN/10% H2O.
respect to the stereochemical requirement of the two carbamate
cap series was uncovered that provided the opportunity to
replace the arylglycine cap of 2 with an alkylglycine analog,
enabling the identification of 33. Notable direct-acting antiviral
therapies in clinical development nearing fruition contain NS5A
replication complex inhibitors.20
were performed on a Shimadzu LC instrument coupled to a
Waters Micromass ZQ instrument or Waters 2795 HPLC with
Micromass ZQ MS (electrospray probe) and Waters 996 PDA
detection. All tested compounds exhibited >95% purity under
the LC conditions provided in Table 9. HRMS analyses were
conducted on a Thermo Scientific Finnegan MAT900 or
Fourier Transform Orbitrap spectrometers, calibrated daily.
NMR spectra were recorded on a Bruker Ultrashield 400 MHz
spectrometer or on a Bruker Advance-III 500 MHz
spectrometer, each equipped with a 5 mm TXI cryoprobe.
Residual protio-solvent was used as internal standard for
chemical shift assignments. Coupling constants are provided in
Hz, with the following spectral pattern designations: s, singlet;
d, doublet; t, triplet; q, quartet; quint, quintet; dd, doublet of
doublets; dt, doublet of triplets; m, multiplet; br, broad; app,
EXPERIMENTAL SECTION21
■
Reactions were conducted, purified, and analyzed according to
methods widely practiced in the field, while taking necessary
precautions in the exclusion of moisture and/or oxygen where
appropriate. Final compounds were tested as either TFA salts
or free base. Although, where relevant, the exact TFA content
was not determined, its mole equivalent was assumed to be
equal to the number of basic moieties residing in the molecule
for purposes of yield and EC50 calculations. LC/MS analyses
1
apparent. H NMR analyses of most intermediates and final
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products in DMSO-d6 at ambient temperature indicated the
presence of rotamers and/or tautomers and that the chemical
shift provided is for the dominant rotamer(s) and/or
tautomer(s).
Abbreviations used in the experimental descriptions: DIEA,
N,N-diisopropylethylamine; EtOAc, ethyl acetate; HATU, 1-
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium 3-oxid hexafluorophosphate; MeOH, methanol;
satd. aq, saturated aqueous; RT, room temperature.
dimethoxyethane (18 mL) and H2O (4 mL). The reaction
mixture was flushed with N2, heated at 90 °C for 37 h using an
oil bath, and allowed to cool to RT. The suspension that
formed was filtered and washed with H2O followed by 1,2-
dimethoxyethane, and dried in vacuo. A silica gel mesh was
prepared from the crude solid and submitted to flash
chromatography (silica gel, EtOAc) to afford 54a as a white
solid, which yellowed slightly upon standing at RT (1.124 g,
1
∼68%). H NMR indicated that the sample contained residual
Methyl N-[(1R)-2-[(2S)-2-{5-[6-(4-{2-[(2S)-1-[(2R)-2-
[(methoxycarbonyl)amino]-2-phenylacetyl]pyrrolidin-2-
yl]-1H-imidazol-5-yl}phenyl)pyridin-3-yl]-1H-imidazol-2-
yl}pyrrolidin-1-yl]-2-oxo-1-phenylethyl]carbamate (4). A
solution of Br2 (7.63 g, 47.74 mmol) in CH2Cl2 (10 mL) was
added dropwise over 5 min to an ice water cooled solution of 1-
(6-bromopyridine-3-yl)ethanone (9.496 g, 47.47 mmol) and
48% HBr (0.4 mL) in CH2Cl2 (105 mL). The cooling bath was
removed 40 min later, and stirring was continued at ambient
temperature for 66 h. The cake of solid that formed was
filtered, washed with CH2Cl2, and dried in vacuo to afford
impure bromide 43 as an off-white solid (15.94 g), which was
carried forward directly.
Boc-L-proline (9.70 g, 45.06 mmol) was added in one batch
to a heterogeneous mixture of crude 43 (15.4 g) and CH3CN
(150 mL), followed immediately by Et3N (13.0 mL, 93.2
mmol), which was added dropwise over 6 min. The reaction
mixture was stirred for 50 min, the volatile component was
removed in vacuo, and the residue was partitioned between
CH2Cl2 and H2O. The CH2Cl2 layer was dried (MgSO4),
filtered, and concentrated in vacuo, and the residue was purified
by flash chromatography (silica gel; sample was loaded with
eluting solvent; 25% EtOAc/hexanes) to afford ketoester 44 as
a highly viscous yellow oil (11.44 g, 61%). 1H NMR (400 MHz,
DMSO-d6): δ 8.95 (m, 1H), 8.25−8.21 (m, 1H), 7.88 (d, J =
8.3, 1H), 5.65−5.46 (m, 2H), 4.36−4.31 (m, 1H), 3.41−3.29
(m, 2H), 2.36−2.22 (m, 1H), 2.14−2.07 (m, 1H), 1.93−1.83
(m, 2H), 1.4/1.36 (2 s, 9H). LC/MS (ESI) m/z: [M + Na]+
calcd for C17H21NaBrN2O5, 435.1; found, 435.2. HRMS (ESI)
m/z: [M + H]+ calcd for C17H22BrN2O5, 413.0712; found,
413.0717.
MeOH in a product/MeOH mole ratio of 1.3. LC/MS (ESI)
m/z: [M + H]+ calcd for C35H44N7O4, 626.4; found, 626.6.
HRMS (ESI) m/z: [M + H]+ calcd for C35H44N7O4, 626.3455;
found, 626.3479.
Carbamate 54a (217 mg) was treated with 25% TFA/
CH2Cl2 (3.6 mL) and stirred at RT for 6 h. The volatile
component was removed in vacuo, and the resultant material
was free-based by MCX column (MeOH wash; 2.0 M NH3/
MeOH elution) to afford 54b as a dull, yellow foam that
solidified gradually after it was allowed to stand with no
1
processing (150.5 mg; mass is above theoretical yield). H
NMR (400 MHz, DMSO-d6): δ 11.89 (br s, 2H), 9.01 (d, J =
1.8 Hz, 1H), 8.13 (dd, J = 8.3, 2.2 Hz, 1H), 8.07 (d, J = 8.6 Hz,
2H), 7.92 (d, J = 8.3 Hz, 1H), 7.83 (d, J = 8.5 Hz, 2H), 7.61 (br
s, 1H), 7.50 (br s, 1H), 4.18 (m, 2H), 3.00−2.93 (m, 2H),
2.90−2.82 (m, 2H), 2.11−2.02 (m, 2H), 1.94−1.85 (m, 2H),
1.83−1.67 (m, 4H). Note: the exchangeable pyrrolidine
hydrogens were not observed. LC/MS (ESI) m/z: [M +
H]+ calcd for C25H28N7: 426.2; found, 426.4. HRMS (ESI) m/
z: [M + H]+ calcd for C25H28N7, 426.2406; found, 426.2425.
To a mixture of 54b (23.2 mg, ∼0.0512 mmol), (2R)-2-
[(methoxycarbonyl)amino]-2-phenylacetic acid (24.4 mg,
0.117 mmol), DIEA (40 μL, 0.229 mmol) in DMF (1.5 mL)
was added HATU (41.9 mg, 0.110 mmol). The mixture was
stirred at RT for 1 h and directly submitted to a reverse-phase
preparative HPLC purification (MeOH/H2O/TFA) to afford
1
the TFA salt of 4 as an off-white foam (43.4 mg, 74%). H
NMR (400 MHz, DMSO-d6): δ 14.33 (br s, ∼1H), 9.15−8.95
(series of m, 1H), 8.57−8.51 (m, 0.15H), 8.40−8.04 (m, 5.6H),
8.04−7.92 (m, 1.55H), 7.92−7.76 (m, 0.72H), 7.76−7.66 (m,
1.66H), 7.46−7.25 (m, 9.01H), 7.25−7.10 (m, 0.16H),
7.10−6.94 (m, 1.15H), 5.76−5.63 (2 m, 0.15H), 5.57−5.37
(2 m, 1.97H), 5.26−5.13 (m, 1.88H), 3.99−3.88/3.88−3.74 (2
m, 2H), 3.74−3.66/3.23−3.09 (2 m, 2H), 3.55/3.53 (2 s, 6H),
2.34−2.17 (m, 1.75H), 2.14−1.97 (m, 3.85H), 1.97−1.75 (m,
2.4H). LC (method 1): tR = 31.3 min. LC/MS (ESI) m/z: [M
+ H]+ calcd for C45H46N9O6, 808.4; found, 808.5. HRMS (ESI)
m/z: [M + H]+ calcd for C45H46N9O6, 808.3571; found,
808.3576.
Methyl N-[(1R)-2-[(2S)-2-{5-[5-(4-{2-[(2S)-1-[(2R)-2-
[(methoxycarbonyl)amino]-2-phenylacetyl]pyrrolidin-2-
yl]-1H-imidazol-5-yl}phenyl)pyridin-2-yl]-1H-imidazol-2-
yl}pyrrolidin-1-yl]-2-oxo-1-phenylethyl]carbamate (5). n-
BuLi (12.0 mL of 2.5 M in hexanes, 30 mmol) was added
dropwise over 15 min to a cooled (−78 °C) semisolution of
2,5-dibromopyridine (45a) (6.040 g, 25.5 mmol) in toluene
(300 mL), and the reaction mixture was stirred for 2.5 h. t-
Butyl 2-(methoxy(methyl)amino)-2-oxoethylcarbamate (2.809
g, 12.87 mmol) was added in batches over 7 min, and the
mixture was stirred for 1.5 h at −78 °C. The cooling bath was
replaced with a −60 °C bath, which was allowed to warm up to
−15 °C over 2.5 h. The reaction mixture was quenched with
satd. aq NH4Cl solution (20 mL) and allowed to thaw to
ambient temperature. The organic layer was separated and
A mixture of 44 (1.32 g, 3.19 mmol) and NH4OAc (2.729 g,
35.4 mmol) in xylenes (18 mL) was heated in a microwave at
140 °C for 90 min. The volatile component was removed in
vacuo, and the residue was partitioned between CH2Cl2 and
H2O, where enough satd. aq NaHCO3 solution was added to
neutralize the aqueous medium. The aqueous phase was
extracted with CH2Cl2, and the combined organic phase was
dried (MgSO4), filtered, and concentrated in vacuo. The
residue was purified by flash chromatography (silica gel; 50%
EtOAc/hexanes) to afford imidazole 48a as an off-white foam
1
(1.025 g, 82%). H NMR (400 MHz, DMSO-d6): δ 12.33/
12.09/12.02 (series of br s, 1H), 8.74 (d, J = 2.3 Hz, 0.93H),
8.70 (app br s, 0.07H), 8.03/7.98 (dd for the first peak, J = 8.3,
2.6 Hz, 1H), 7.69/7.67 (2 overlapping app br s, 1H), 7.58/7.43
(d for the first peak, J = 8.3 Hz, 1H), 4.80 (m, 1H), 3.53 (m,
1H), 3.36 (m, 1H), 2.33−2.11 (m, 1H), 2.04−1.79 (m, 3H),
1.39/1.15 (2 br s, 3.9H+5.1H). LC/MS (ESI) m/z: [M +
H]+calcd for C17H22BrN4O2, 393.1; found, 393.2. HRMS (ESI)
m/z: [M + H]+ calcd for C17H22BrN4O2, 393.0926; found,
393.0909.
Pd(Ph3P)4 (115 mg, 0.100 mmol) was added to a mixture of
48a (992 mg, 2.52 mmol), 53 (see ref 2 for preparation; 1.207
g, 2.747 mmol), NaHCO3 (698.8 mg, 8.318 mmol) in 1,2-
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concentrated in vacuo. The crude product was purified by flash
chromatography (silica gel, 15% EtOAc/hexanes) to afford a
reddish-brown semisolid, which was washed with hexanes to
remove some colored residue. Pyridine 46a was isolated as an
ash-colored solid (842 mg, 21%). 1H NMR (400 MHz, DMSO-
d6): δ 8.89 (d, J = 2.3 Hz, 1H), 8.30 (dd, J = 8.4, 2.4 Hz, 1H),
7.90 (d, J = 8.3 Hz, 1H), 7.03(br t, J = 5.7 Hz, 0.88H), 6.63
(app br s, 0.12H), 4.55 (d, J = 5.8 Hz, 2H), 1.40/1.28 (2 app s,
7.83H/1.17H). LC/MS (ESI) m/z: [M + Na]+ calcd for
C12H15BrNaN2O3, 337.0; found, 337.1.
aqueous layer was extracted with CH2Cl2, and the combined
organic phase was dried (MgSO4), filtered, and concentrated in
vacuo. The crude material was purified by flash chromatog-
raphy (silica gel; 20% EtOAc/hexanes) to afford 48c as a
colorless viscous oil (87.5 mg, 84%). The exact regiochemistry
of 48c was not determined, as it was deemed inconsequential
1
for the current purpose. H NMR (400 MHz, CDCl3): δ 8.53
(d, J = 2.2 Hz, 1H), 7.91−7.82 (m, 1H), 7.82−7.72 (m, 1H),
7.52 (s, 1H), 5.87 (m, 0.46H), 5.41 (m, 0.54H), 5.16 (d, J =
10.8 Hz, 1H), 5.03−4.85 (m, 1H), 3.76−3.42 (series of m, 4H),
2.54−1.84 (series of m, 4H), 1.38/1.19 (2 br s, 4.3H/4.7H),
0.97−0.81 (m, 2H), −0.03 (s, 9H). LC/MS (ESI) m/z: [M +
H]+ calcd for C23H36BrN4O3Si, 523.2; found, 523.2.
HBr (48%, 1.0 mL) was added dropwise to a solution of 46a
(840 mg, 2.66 mmol) in dioxane (5.0 mL) over 3 min, and the
reaction mixture was stirred at RT for 17.5 h. The precipitate
was filtered, washed with dioxane, and dried in vacuo to afford
the HBr salt of 46b as an off-white solid (672.4 mg; the precise
Pd(Ph3P)4 (24.4 mg, 0.021 mmol) was added to a mixture of
48c (280 mg, 0.535 mmol), 53 (see ref 2 for preparation; 241.5
mg, 0.55 mmol) and NaHCO3 (148.6 mg, 1.769 mmol) in 1,2-
dimethoxyethane (4.8 mL) and H2O (1.6 mL). The reaction
mixture was flushed with N2, heated with an oil bath at 80 °C
for ∼24 h, and then the volatile component was removed in
vacuo. The residue was partitioned between CH2Cl2 and H2O,
and the organic phase was dried (MgSO4), filtered, and
concentrated in vacuo. The crude material was purified by flash
chromatography (silica gel; 75−100% EtOAc/hexanes) fol-
lowed by a reverse-phase preparative HPLC (H2O/MeOH/
TFA). The HPLC elute was neutralized with 2 M NH3/MeOH
and evaporated in vacuo, and the residue was partitioned
between H2O and CH2Cl2. The organic layer was dried
(MgSO4), filtered, and concentrated in vacuo to afford 55a as a
white foam (162 mg, 40%). LC/MS (ESI) m/z: [M +
H]+ calcd for C41H58N7O5Si, 756.4; found, 756.6.
1
mole equivalent of HBr was not determined). H NMR (400
MHz, DMSO-d6): δ 8.95 (d, J = 2.3 Hz, 1 H), 8.37 (dd, J = 8.4,
2.3 Hz, 1H), 8.20 (br s, 3H), 8.00 (d, J = 8.3 Hz, 1H), 4.61 (s,
2H). LC/MS (ESI) m/z: [M + H]+ calcd for C7H8BrN2O,
215.0; found, 215.0.
DIEA (2.3 mL, 13.2 mmol) was added dropwise over 15 min
to a heterogeneous mixture of 46b (1.365g), (S)-Boc-proline
(0.957 g, 4.44 mmol) and HATU (1.70 g, 4.47 mmol) in DMF
(13.5 mL), and the mixture was stirred at RT for 1 h. The
volatile component was removed in vacuo, and the residue was
partitioned between EtOAc (40 mL) and an aqueous medium
(20 mL H2O + 1 mL satd. aq NaHCO3). The aqueous layer
was washed with EtOAc (20 mL), and the combined organic
phase was dried (MgSO4), filtered, and concentrated in vacuo.
The residue was purified by flash chromatography (silica gel;
40−50% EtOAc/hexanes) to afford 47 as a light-yellow foam
Carbamate 55a (208 mg, 0.275 mmol) was treated with 25%
TFA/CH2Cl2 (4.0 mL), and the mixture was stirred at RT for
10 h. The volatile component was removed in vacuo, and the
residue was first free-based by MCX (MeOH wash; 2.0 M
NH3/MeOH elution) and then purified by a reverse-phase
preparative HPLC (H2O/MeOH/TFA), and the resultant
material was free-based again (MCX) to afford 55b as a film of
1
(1.465g). H NMR (400 MHz, DMSO-d6): δ 8.90 (d, J = 2.3
Hz, 1H), 8.30 (dd, J = 8.5, 2.4 Hz, 1H), 8.21−8.07 (m, 1H),
7.90 (d, J = 8.3 Hz, 1H), 4.80−4.69 (m, 1H), 4.64 (dd, J = 19.1,
5.5 Hz, 1H), 4.24−4.10 (m, 1H), 3.43−3.33 (m, 1H),
3.33−3.26 (m, 1H), 2.20−2.01 (m, 1H), 1.95−1.70 (m, 3H),
1.40/1.35 (2 app s, 9H). LC/MS (ESI) m/z: [M + Na]+ calcd
for C17H22BrN3NaO4, 434.1; found, 434.0.
1
oil (53.7 mg, 46%). H NMR (400 MHz, DMSO-d6): δ 11.86
A mixture of 47 (782.2 mg, 1.897 mmol) and NH4OAc
(800.0 mg, 10.4 mmol) in xylenes (10 mL) was heated in a
microwave (140 °C) for 90 min. The volatile component was
removed in vacuo, and the residue was carefully partitioned
between CH2Cl2 and H2O, where enough satd. aq NaHCO3
was added to neutralize it. The aqueous phase was extracted
with CH2Cl2 (2×), and the combined organic phase was dried
(MgSO4), filtered, and concentrated in vacuo. The crude
material was purified by flash chromatography (silica gel; 50%
CH2Cl2/EtOAc) to afford 48b as an off-white solid (552.8 mg,
74%). 1H NMR (400 MHz, DMSO-d6): δ 12.49/12.39/12.15/
12.06 (series of br s, 1H), 8.62 (app br s, 0.2H), 8.56 (d, J = 2.0
Hz, 0.8H), 8.02 (br d, J = 8.5 Hz, 0.2H), 7.97 (br d, J = 7.8 Hz,
0.8H), 7.77 (d, J = 8.6 Hz, 0.8H), 7.72 (d, J = 8.6 Hz, 0.2H),
7.61−7.49 (m, 1H), 4.93−4.72 (m, 1H), 3.53 (m, 1H), 3.41−
3.32 (m, 1H), 2.33−1.77 (m, 4H), 1.39 (app s, 3.7H), 1.14/
1.12 (2 overlapped app s, 5.3H). LC/MS (ESI) m/z: [M +
Na]+ calcd for C17H21BrN4NaO2, 415.1; found, 415.1.
(app br s, ∼2H), 8.83 (d, J = 2.1 Hz, 1H), 8.07 (dd, J = 8.3, 2.3
Hz, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.84 (d, J = 8.3 Hz, 2H),
7.71 (d, J = 8.3 Hz, 2H), 7.55 (s, 1H), 7.50 (br s, 1H),
4.20−4.15 (m, 2H), 3.00−2.94 (m, 2H), 2.89−2.83 (m, 2H),
2.11−2.02 (m, 2H), 1.95−1.86 (m, 2H), 1.83−1.67 (m, 4H).
Note: the exchangeable pyrrolidine hydrogens were not
observed. LC/MS (ESI) m/z: [M + H]+ calcd for C25H28N7,
426.2; found, 426.3.
Title compound 5 (TFA salt, off-white foam) was prepared
from 55b and (2R)-2-[(methoxycarbonyl)amino]-2-phenyl-
1
acetic acid according to the procedure described for 4. H
NMR (400 MHz, DMSO-d6): δ 14.32 (br s, ∼1H), 9.14−9.00
(m, 1H), 8.45−8.34 (m, 1.82H), 8.30−8.23 (m, 0.15H),
8.23−8.13 (app s, 1.04H), 8.13−7.93 (series of m, 4.81H),
7.93−7.76 (m, 0.72H), 7.76−7.60 (m, 1.5H), 7.44−7.27 (m,
9.08H), 7.07−6.98 (m, 0.88H), 5.75−5.68 (m, 0.12H),
5.57−5.45 (m, 1.6H), 5.45−5.35 (m, 0.34H), 5.26−5.12 (m,
1.94H), 3.99−3.87/3.87−3.77 (2 m, 2H), 3.77−3.66/
3.23−3.06 (2 m, 2H), 3.55/3.53 (2 s, 6H), 2.32−2.16 (m,
1.8H), 2.16−1.96 (m, 4H), 1.96−1.75 (m, 2.2H). LC (method
2): tR = 24.0 min. LC/MS (ESI) m/z: [M + H]+ calcd for
C45H46N9O6, 808.36; found, 808.51.
NaH (60% dispersion in mineral oil; 11.6 mg, 0.29 mmol)
was added in one portion to a heterogeneous mixture of 48b
(80 mg, 0.20 mmol) and DMF (1.5 mL), and stirred at RT for
30 min. SEM-Cl (40 μL, 0.23 mmol) was added dropwise over
2 min to the reaction mixture, and stirring was continued for 14
h. The volatile component was removed in vacuo and the
residue was partitioned between H2O and CH2Cl2. The
Methyl N-[(1R)-2-[(2S)-2-{5-[4-(5-{2-[(2S)-1-[(2R)-2-
[(methoxycarbonyl)amino]-2-phenylacetyl]pyrrolidin-2-
yl]-1H-imidazol-5-yl}pyrimidin-2-yl)phenyl]-1H-imida-
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zol-2-yl}pyrrolidin-1-yl]-2-oxo-1-phenylethyl] carba-
mate (6). Tributyl(1-ethoxyvinyl)tin (10.0 mL, 29.6 mmol)
and PdCl2(Ph3P)2 (1.04 g, 1.48 mmol) were added to a mixture
of 45b (5.72 g, 29.6 mmol) in DMF (80 mL) maintained under
an atmosphere of N2. The mixture was heated at 100 °C for 3 h
before being allowed to stir at RT for 16 h. The reaction
mixture was diluted with Et2O (200 mL) and treated with
aqueous KF solution (25 g of KF in 150 mL of water). The
two-phase mixture was stirred vigorously for 1 h at RT and
filtered through Celite. The organic phase of the filtrate was
separated, washed with saturated NaHCO3 solution, brine and
dried (Na2SO4). The original aqueous phase was extracted
twice with Et2O, and the combined organic extract was treated
as above. The crude product was preabsorbed onto silica gel
and purified by flash chromatography (silica gel, 3−50%
EtOAc/hexanes) to afford 49 (6.1 g) as a white solid which was
slightly impure but was used as is. 1H NMR (500 MHz,
DMSO-d6): δ 8.97 (s, 2H), 5.08 (d, J = 3.7 Hz, 1H), 4.56 (d, J
= 3.4 Hz, 1H), 3.94 (q, J = 7.0 Hz, 2H), 1.35 (t, J = 7.0 Hz,
3H). LC/MS (ESI) m/z: [M + H]+ calcd for C8H10ClN2O,
185.1; found, 185.0. HRMS (ESI) m/z: [M + H]+ calcd for
C8H10ClN2O, 185.0482; found, 185.0490.
NBS (3.37 g, 19.0 mmol) was added in one portion to a
stirred solution of 49 (3.5 g, ∼19.0 mmol) in THF (53 mL)
and H2O (17.5 mL) at 0 °C under N2. The mixture was stirred
for 1 h at 0 °C before it was diluted with H2O and extracted
with EtOAc (2×). The combined extract was washed with satd.
aq NaHCO3, brine and dried (Na2SO4) to afford 50 (4.69 g) as
a pale yellow solid, which was advanced to the next step
without purification. LC/MS (ESI) m/z: [M + H]+ calcd for
C6H5BrClN2O, 236.93; found, 236.78.
phase was extracted with EtOAc (2×), and the combined
extracts were washed with satd. aq NaHCO3, brine and dried
(Na2SO4). The residue was purified by flash chromatography
(silica gel, 5−100% EtOAc/hexanes) to furnish 52b as a pale
yellow foam (2.35 g, 87%). Note that the exact regiochemistry
of 52b was not determined as it was deemed inconsequential
1
for the current purpose. H NMR (500 MHz, DMSO-d6): δ
9.04 (s, 2H), 7.98−7.95 (m, 1H), 5.70−5.31 (3 m, 2H), 5.02−
4.91 (m, 1H), 3.59−3.49 (m, 3H), 3.45−3.35 (m, 1H), 2.30−
2.08 (m, 2H), 1.99−1.83 (m, 2H), 1.36/1.12 (2 s, 9H), 0.93−
0.82 (m, 2H), −0.02 (s, 9H). LC/MS (ESI) m/z: [M+H]+
calcd for C22H35ClN5O3Si, 480.2; found, 480.2. HRMS (ESI)
m/z: [M+H]+ calcd for C22H35ClN5O3Si, 480.2198; found,
480.2194.
Pd(PPh3)4 (0.12 g, 0.103 mmol) was added in one portion to
a stirred suspension of 53 (see ref 2 for preparation; 1.00 g,
2.27 mmol), 52b (0.99 g, 2.06 mmol), and NaHCO3 (0.87 g,
10.3 mmol) in a solution of DME (20 mL) and H2O (6 mL) at
RT under N2. The vessel was sealed and the mixture was heated
at 80 °C with stirring for 16 h before additional Pd(PPh3)4
(0.12 g) was added. After being heated at 80 °C for an
additional 12 h, the mixture was cooled to RT, diluted with
EtOAc, washed with satd. aq NaHCO3, brine and dried
(Na2SO4). The volatile component was removed in vacuo.
Purification of the residue by flash chromatography (silica gel,
40−100% EtOAc/hexanes) furnished 56a as a yellow foam
(1.53 g). A sample of the yellow foam was further purified for
characterization purposes by reverse-phase preparative HPLC
(CH3CN/H2O/NH4OAc) to afford a pure sample as a white
1
solid. H NMR (500 MHz, DMSO-d6): δ 12.30−11.88 (3 m,
1H), 9.17−9.16 (m, 2H), 8.43−8.31 (m, 2H), 7.99−7.35
(series of m, 4H), 5.72−5.30 (3 m, 2H), 5.03−4.76 (2 m, 2H),
3.64−3.50 (m, 4H), 3.48−3.31 (m, 2H), 2.36−2.07 (m, 2H),
2.05−1.80 (m, 4H), 1.46−1.08 (2 m, 18H), 0.95−0.84 (m,
2H), −0.01 (s, 9H). LC/MS (ESI) m/z: [M + H]+ calcd for
C40H57N8O5Si, 757.4; found, 757.4. HRMS (ESI) m/z: [M +
H]+ calcd for C40H57N8O5Si, 757.4221; found, 757.4191.
TFA (8.0 mL) was added in one portion to a stirred solution
of 56a (1.50 g, ∼1.98 mmol) in dry CH2Cl2 (30 mL) at RT.
The flask was sealed, and the mixture was stirred for 16 h
before the volatile component was removed in vacuo. The
residue was dissolved in MeOH and purified by a reverse-phase
preparative HPLC (MeOH/water/TFA). The desired fraction
was concentrated, and the product was dissolved in MeOH and
passed through a resin cartridge (UCT-CHQAX1 Quartnery
amine hydroxide; MeOH elution) to afford the free-base form
of 56b as a pale, mustard yellow-colored solid (307 mg, 36%).
1H NMR (500 MHz, DMSO-d6): δ 12.50−11.80 (br m, 2H),
9.18 (s, 2H), 8.36 (d, J = 8.5 Hz, 2H), 7.89 (d, J = 8.2 Hz, 2H),
7.77 (s, 1H), 7.61 (s, 1H), 4.34−4.24 (m, 2H), 3.09−2.89 (m,
4H), 2.18−2.07 (m, 2H), 2.02−1.89 (m, 2H), 1.88−1.72 (m,
4H). Note: the exchangeable pyrrolidine hydrogens were not
observed. LC/MS (ESI) m/z: [M + H]+ calcd for C24H27N8,
427.2; found, 427.0. HRMS (ESI) m/z: [M + H]+ calcd for
C24H27N8, 427.2359; found, 427.2363.
Crude 50 (4.69 g) was dissolved in anhydrous CH3CN (150
mL) and treated directly with N-Boc-L-proline (4.08 g, 19.0
mmol) and DIEA (3.30 mL, 19.0 mmol). After being stirred for
3 h, the solvent was removed in vacuo, and the residue was
partitioned between EtOAc and H2O. The organic phase was
washed with 0.1 N HCl, satd. aq NaHCO3, brine and dried
(Na2SO4) to afford crude 51, which was advanced to the next
step without purification.
Crude 51 was taken up in xylenes (60 mL) and treated with
NH4OAc (14.6 g, 190 mmol). The mixture was heated at 140
°C for 2 h in a pressure vessel before it was cooled to RT and
suction-filtered. The filtrate was concentrated in vacuo, and the
residue was partitioned between EtOAc and satd. aq NaHCO3.
The organic phase was separated, washed with brine, and dried
over Na2SO4. Purification of the residue by flash chromatog-
raphy (silica gel, 5−40% B/A, where A = CH2Cl2 and B = 25%
MeOH/CH2Cl2) afforded 52a as a pale, yellow foam (2.0 g,
1
30% yield for three steps). H NMR (500 MHz, DMSO-d6): δ
12.24−12.16 (m, 1H), 9.05 (s, 2H), 7.84−7.73 (m, 1H), 4.90−
4.73 (m, 1H), 3.59−3.46 (m, 1H), 3.41−3.31 (m, 1H), 2.32−
2.12 (m, 1H), 2.03−1.77 (m, 3H), 1.39/1.15 (2 s, 9H). LC/MS
(ESI) m/z: [M + H]+ calcd for C16H21ClN5O2, 350.1; found,
350.2. HRMS (ESI) m/z: [M + H]+ calcd for C16H21ClN5O2,
350.1384; found, 350.1398.
NaH (60% dispersion in mineral oil; 0.23 g, 5.72 mmol) was
added in one portion to a stirred solution of 52a (2.00 g, 5.72
mmol) in dry DMF (45 mL) at RT under N2. The mixture was
stirred for 5 min, and SEM-chloride (1.00 mL, 5.65 mmol) was
added gradually in 0.1 mL increments. The mixture was stirred
for 3 h, quenched with satd. aq NH4Cl, and diluted with
EtOAc. The organic phase was separated, washed with satd. aq
NaHCO3, brine and dried (Na2SO4). The original aqueous
To a solution of 56b (60.0 mg, 0.14 mmol), (2R)-2-
[(methoxycarbonyl)amino]-2-phenylacetic acid (64.8 mg, 0.31
mmol), and DIEA (0.20 mL, 1.1 mmol) in DMF (3.5 mL) was
added HATU (123.0 mg, 0.32 mmol). The reaction mixture
was stirred at RT for 2 h. It was then diluted with MeOH (2
mL), filtered through a Whatman 13 mm PVDF 45 μm syringe
filter, and purified by reverse-phase preparative HPLC
(MeOH/H2O/TFA) to afford the TFA salt of 6 as a light
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Journal of Medicinal Chemistry
Article
1
yellow solid (91.4 mg, 63% yield assuming bis-TFA salt). H
NMR (500 MHz, DMSO-d6): δ 9.29/9.22/9.16 (3 s, 2H),
8.60−8.50 (m, 2H), 8.23 (app s, 0.77H), 8.16−8.08 (m,
0.87H), 8.01−7.65 (series of m, 4.27H), 7.44−7.27 (series of
m, 8.8H), 7.05 (app s, 0.97H), 7.00−6.93 (m, 0.32H),
5.75−5.69 (m, 0.09H), 5.62−5.57 (m, 0.19H), 5.55−5.47 (m,
1.52H), 5.43−5.37 (m, 0.42H), 5.23−5.11 (m, 1.78H),
3.98−3.87 (m, 2H), 3.55/3.54/3.53 (3 s, 6H), 3.23−3.05 (m,
2H), 2.33−2.23 (m, 1H), 2.23−2.12 (m, 0.9H), 2.12−1.95 (m,
4H), 1.95−1.72 (m, 2.1H). LC (method 7): tR = 1.99 min. LC/
MS (ESI) m/z: [M + H]+calcd for C44H45N10O6, 809.4; found,
809.2. HRMS (ESI) m/z: [M + H]+ calcd for C44H45N10O6,
809.3524; found, 809.3505.
1.84H), 8.04−7.89 (m, 7.88H), 7.89−7.67 (m, 1.23H),
7.46−7.26 (m, 4.56H), 7.05 (s, 0.49H), 5.76 (m, 0.05H),
5.57−5.48 (m, 0.75H), 5.44−5.36 (m, 0.27H), 5.24−5.12 (m,
1.93H), 4.01−3.85 (m, 1H), 3.82−3.67 (m, 1H), 3.64−3.53
(m, 1.31H), 3.52 (s, 2.94H), 3.22−3.07 (m, 0.75H), 2.44−2.22
(2 m, 2.25H), 2.13−1.85 (series of m with s at 2.04, 8.75H).
LC (method 9): tR = 1.32 min. LC/MS (ESI) m/z: [M + H]+
calcd for C38H40N7O4, 658.3; found, 658.4. HRMS (ESI) m/z:
[M + H]+ calcd for C38H40N7O4, 658.3142; found, 658.3135.
Methyl N-[(1R)-2-oxo-2-[(2S)-2-{5-[4-(4-{2-[(2S)-1-[(2R)-
oxolane-2-carbonyl]pyrrolidin-2-yl]-1H-imidazol-5-yl}-
phenyl)phenyl]-1H-imidazol-2-yl}pyrrolidin-1-yl]-1-
phenylethyl]carbamate (11). Title compound 11 (TFA salt,
1
General Synthesis Description for Analogues of the
Biphenyl Core Series. Homodimeric cap analogues were
prepared from pyrrolidine 57a and appropriate acid precursors
according to the procedure described in the synthesis of 4. The
mixed-cap analogues were prepared from mono-Boc protected
57b through a sequence involving coupling with the first cap,
deprotecting the Boc moiety, and then installing the second
cap, the details of which are provided in ref 6f. Depending on
the HPLC eluting medium, final products were isolated as
either TFA salt or free-base form.
Methyl N-[(2R)-1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-[(2R)-2-
[(methoxycarbonyl)amino]-3-methylbutanoyl]-
pyrrolidin-2-yl]-1H-imidazol-5-yl}phenyl)phenyl]-1H-
imidazol-2-yl}pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-
yl]carbamate (7). Title compound 7 (TFA salt, white solid).
1H NMR (500 MHz, DMSO-d6): δ 14.35 (br s, ∼3H), 8.14
(app br s, 1.83H), 8.02−7.93 (m, 3.77H), 7.93−7.85 (m,
4.59H), 7.60 (app br s, 0.18H), 7.25 (d, J = 8.2 Hz, 1.59H),
6.77 (app br s, 0.04H), 5.87 (app br s, 0.24H), 5.18 (dd, J = 8.1,
3.8 Hz, 1.76H), 4.19 (app t, J = 7.8 Hz, 1.71H), 3.96−3.84 (m,
1.77H), 3.77−3.64 (m, 2.2H), 3.57/3.57 (2 overlapping s, 6H),
3.53−3.47 (m, 0.32H), 2.47−2.28 (m, 2.2H), 2.22−1.87 (series
of m, 7.58H), 1.86−1.66 (m, 0.22H), 0.91 (d, J = 7.0 Hz,
4.9H), 0.87 (d, J = 6.7 Hz, 5.4H), 0.73 (d, J = 6.7 Hz, 0.9H),
0.40 (app br s, 0.8H). LC (method 8): tR = 2.0 min. LC/MS
(ESI) m/z: [M + H]+ calcd for C40H51N8O6, 739.4; found,
739.7. HRMS (ESI) m/z: [M + H]+ calcd for C40H51N8O6,
739.3932; found, 739.3966.
off-white foam). H NMR (500 MHz, DMSO-d6): δ 14.46 (br
s, ∼3H), 8.17/8.15 (2 overlapping app s, 1.63H), 8.11−8.01
(m, 0.34H), 8.01−7.94 (m, 3.57H), 7.94−7.88 (m, 3.94H),
7.88−7.65 (series of m, 1.51H), 7.44−7.27 (series of m,
4.54H), 7.05 (app s, 0.47H), 5.78−5.71 (m, 0.08H), 5.59−5.48
(m, 0.93H), 5.43−5.37 (m, 0.16H), 5.25−5.12 (m, 1.83H),
4.64 (dd, J = 7.9, 5.2 Hz, 0.89H), 4.29 (app t, J = 6.6 Hz,
0.11H), 3.98−3.90 (m, 0.81H), 3.90−3.62 (series of m, 3.25H),
3.62−3.44 (m, 1.17H), 3.54/3.53 (2 s overlapped with m of
same region, 3H), 3.24−3.11 (m, 0.77H), 2.47−2.33 (m,
1.36H), 2.33−2.22 (m, 0.8H), 2.22−2.11 (m, 1.03H),
2.11−1.98 (m, 4.48H), 1.98−1.87 (m, 2.03H), 1.87−1.77 (m,
2H), 1.77−1.64 (m, 0.30H). LC (method 2): tR = 22.5 min.
LC/MS (ESI) m/z: [M + H]+ calcd for C41H44N7O5, 714.3;
found, 714.4. HRMS (ESI) m/z: [M + H]+ calcd for
C41H44N7O5, 714.3404; found, 714.3430.
Methyl N-[(1R)-2-oxo-2-[(2S)-2-{5-[4-(4-{2-[(2S)-1-[(2S)-
oxolane-2-carbonyl]pyrrolidin-2-yl]-1H-imidazol-5-yl}-
phenyl)phenyl]-1H-imidazol-2-yl}pyrrolidin-1-yl]-1-
phenylethyl]carbamate (12). Title compound 12 (TFA salt,
1
off-white foam). H NMR (500 MHz, DMSO-d6): δ 14.51 (br
s, ∼3H), 8.16/8.12 (2 app br s, 2.04H), 8.01−7.93 (m, 3.89H),
7.93−7.87 (m, 3.74H), 7.87−7.65 (series of m, 1.36H), 7.45−
7.25 (m, 4.52H), 7.05 (app s, 0.45H), 5.77−5.67 (m, 0.06H),
5.59−5.49 (m, 0.94H), 5.42−5.39 (m, 0.15H), 5.24−5.14 (m,
1.85H), 4.64 (app dd, J = 7.8, 5.3 Hz, 0.74H), 4.54−4.48 (m,
0.26H), 3.99−3.88 (m, 0.98H), 3.88−3.65 (m, 3.64H), 3.54/
3.53 (2 overlapping s, 3H), 3.50−3.33 (m, 0.75H), 3.23−3.12
(m, 0.63H), 2.45−2.12 (m, 3.52H), 2.12−1.97 (m, 4.82H),
1.97−1.86 (m, 1.21H), 1.86−1.78 (m, 1.56H), 1.78−1.69 (m,
0.89H). LC (method 2): tR = 22.2 min. LC/MS (ESI) m/z: [M
+ H]+ calcd for C41H44N7O5, 714.34; found, 714.24.
Methyl N-[(1R)-2-[(2S)-2-{5-[4-(4-{2-[(2S)-1-{2-
[(methoxycarbonyl)amino]acetyl}pyrrolidin-2-yl]-1H-
imidazol-5-yl}phenyl)phenyl]-1H-imidazol-2-yl}-
pyrrolidin-1-yl]-2-oxo-1-phenylethyl] carbamate (9).
1
Title compound 9 (TFA salt, off-white foam). H NMR (500
(2R)-2-(Dimethylamino)-1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-
[(2R)-oxolane-2-carbonyl]pyrrolidin-2-yl]-1H-imidazol-5-
yl}phenyl)phenyl]-1H-imidazol-2-yl}pyrrolidin-1-yl]-2-
phenylethan-1-one (13). Title compound 13 (TFA salt, light
MHz, DMSO-d6): δ 14.44 (br s, ∼3H), 8.13 (app br s, 1.76H),
8.00−7.87 (2 m, 7.7H), 7.87−7.67 (series of m, 1.51H),
7.44−7.21 (series of m, 5.45H), 7.05 (app s, 0.51H), 6.83 (app
br s, 0.07H), 5.71 (app br s, 0.09H), 5.54−5.49 (m, 0.83H),
5.43−5.37 (m, 0.23H), 5.23−5.26 (m, 1.85H), 3.95−3.89 (m,
1.7H), 3.89−3.80 (m, 1.2H), 3.78−3.68 (m, 1.23H), 3.64−3.56
(m, 0.94H), 3.55/3.53/3.49 (3 s overlapped with m, 6.28H),
3.22−3.14 (m, 0.65H), 2.46−2.32 (m, 1.36H), 2.32−2.17 (m,
0.82H), 2.12−1.96 (m, 4.52H), 1.98−1.83 (m, 1.3H). LC
(method 3): tR = 24.5 min. LC/MS (ESI) m/z: [M + H]+ calcd
for C40H43N8O6, 731.4; found, 731.4. HRMS (ESI) m/z: [M +
H]+ calcd for C40H43N8O6, 731.3306; found, 731.3333.
Methyl N-[(1R)-2-[(2S)-2-{5-[4-(4-{2-[(2S)-1-acetylpyr-
rolidin-2-yl]-1H-imidazol-5-yl}phenyl)phenyl]-1H-imida-
zol-2-yl}pyrrolidin-1-yl]-2-oxo-1-phenylethyl]carbamate
(10). Title compound 10 (TFA salt, off-white foam). 1H NMR
(400 MHz, DMSO-d6): δ 14.48 (br s, ∼3H), 8.23−8.11 (m,
1
yellow solid). H NMR (500 MHz, DMSO-d6): δ 14.55 (br s,
∼3H), 10.24 (br s, ∼1H), 8.14 (app s, 1H), 8.07 (app br s,
1H), 8.00−7.85 (m, 7.73H), 7.72 (app br s, 0.29H), 7.67−7.53
(m, 4.46H), 7.28−7.08 (m, 0.52H), 5.77 (app br d, J = 7.3 Hz,
0.10H), 5.57 (app br d, J = 9.2 Hz, 0.12H), 5.49 (app br s,
0.11H), 5.45 (app s, 0.89H), 5.26−5.14 (m, 1.78H), 4.63 (app
dd, J = 7.9, 5.2 Hz, 0.89H), 4.28 (app t, J = 6.4 Hz, 0.11H),
4.06−3.97 (m, 0.86H), 3.90−3.74 (m, 2.80H), 3.74−3.62 (m,
0.29H), 3.62−3.53 (m, 0.92H), 3.53−3.40 (m, 0.13H), 3.12−
3.00 (m, 1H), 3.00−2.68 (2 overlapping br s, 3H), 2.57−2.11
(m overlapping with DMSO signal, ∼3.7H), 2.30−2.11 (2 m,
2.1H), 2.11−1.98 (m, 4.6H), 1.981−1.67 (series of m, 4.6H).
LC (method 2): tR = 21.6 min. LC/MS (ESI) m/z: [M + H]+
2026
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Article
calcd for C41H46N7O3, 684.4; found, 684.8. HRMS (ESI) m/z:
[M + H]+ calcd for C41H46N7O3, 684.3662; found, 684.3671.
(2R)-2-(Dimethylamino)-1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-
[(2S)-oxolane-2-carbonyl]pyrrolidin-2-yl]-1H-imidazol-5-
yl}phenyl)phenyl]-1H-imidazol-2-yl}pyrrolidin-1-yl]-2-
phenylethan-1-one (14). Title compound 14 (TFA salt, off-
7.71−7.50 (m, 9.35H), 7.50−7.35 (m, 0.37H), 7.24−7.09 (2 m,
1.04H), 5.79−5.73 (m, 0.2H), 5.50 (s, 0.25H), 5.44 (s, 1.75H),
5.24−5.17 (m, 1.8H), 4.04−3.95 (m, 1.77H), 3.88−3.79 (m,
0.23H), 3.75−2.6 (series of overlapping br m, ∼9H), 2.60−2.36
(m overlapped with DMSO signal, ∼1H), 2.30−2.13 (m, 2H),
2.13−1.60 (m, 14H). LC (method 4): tR = 36.9 min. LC/MS
(ESI) m/z: [M + H]+ calcd for C50H55N8O2, 799.44; found,
799.67.
1
white foam). H NMR (400 MHz, DMSO-d6): δ 14.51 (br s,
∼1H), 10.24 (br s, ∼1H), 8.11/8.09 (2 overlapping s, 1.10H),
8.03 (br s, 0.80H), 7.98−7.84 (m, 7.04H), 7.84−7.63 (m,
0.94H), 7.63−7.52 (m, 4.16H), 7.27−7.08 (m, 0.96H), 5.75
(m, 0.07H), 5.57 (m, 0.11H), 5.49−5.42 (m, 1H), 5.23−5.15
(m, 1.82H), 4.64 (app dd, J = 7.7, 5.4 Hz, 0.86H), 4.54−4.48
(m, 0.14H), 4.07−3.95 (m, 0.86H), 3.86−3.70 (overlapping m,
3.74H), 3.48−3.39 (m, 0.21H), 3.34−3.25 (m, 0.19H),
3.07−2.97 (m, 1H), 2.96−2.88 (app br s, 0.34H), 2.82−2.54
(app br s, 4.66H), 2.44−2.32 (m, 1.6H), 2.30−2.11 (m, 1.4H),
2.11−1.96 (m, 6.4H), 1.96−1.65 (series of m, 3.6H). LC
(method 4): tR = 25.7 min. LC/MS (ESI) m/z: [M + H]+ calcd
for C41H46N7O3, 684.4; found, 684.5. HRMS (ESI) m/z: [M +
H]+ calcd for C41H46N7O3, 684.3662; found, 684.3692.
(2R)-2-Phenyl-1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-[(2R)-2-phe-
nyl-2-(piperidin-1-yl)acetyl]pyrrolidin-2-yl]-1H-imidazol-
5-yl}phenyl)phenyl]-1H-imidazol-2-yl}pyrrolidin-1-yl]-2-
(piperidin-1-yl)ethan-1-one (18). Title compound 18 (free
1
base, white solid). H NMR (400 MHz, DMSO-d6): δ 9.98/
9.81 (2 overlapping app br s, ∼2H), 8.07 (app br s, 1.56H),
7.99−7.86 (m, 7.14H), 7.86−7.68 (2 m, 1.17H), 7.68−7.62 (m,
3.16H), 7.62−7.49 (m, 5.67H), 7.45−7.37 (m, 0.40H),
7.26−7.08 (br m, 0.9H), 5.91−5.78 (m, 0.1H), 5.59−5.44 (m
overlapped with app s, 2H), 5.32−5.16 (m, 1.9H), 4.12−3.17
(m, 2H), 3.70−3.24 (m, 1.7H), 3.27−2.59 (series of over-
lapping br m, 6.3H), 2.59−2.30 (m overlapped with DMSO
signal, ∼2H), 2.30−2.13 (m, 1.4H), 2.13−1.98 (m, 3.6H),
1.97−1.77 (m, 3.4H), 1.77−1.50 (m, 8.4H), 1.51−1.28 (m,
3.2H). LC (method 10): tR = 1.85 min. LC/MS (ESI) m/z: [M
+ H]+ calcd for C52H59N8O2, 827.5; found, 827.2. HRMS (ESI)
m/z: [M + H]+ calcd for C52H59N8O2, 827.4761; found,
827.4733.
(2R)-2-[Ethyl(methyl)amino]-1-[(2S)-2-{5-[4-(4-{2-[(2S)-
1-[(2R)-2-[ethyl(methyl)amino]-2-phenylacetyl]-
pyrrolidin-2-yl]-1H-imidazol-5-yl}phenyl)phenyl]-1H-
imidazol-2-yl}pyrrolidin-1-yl]-2-phenylethan-1-one (15).
1
Title compound 15 (TFA salt, yellow foam). H NMR (400
MHz, DMSO-d6): δ 10.13/9.85 (2 overlapping br s, ∼2H),
8.05 (app br s, 1.77H), 7.96−7.83 (m, 7.63H), 7.76−7.69 (m,
0.47H), 7.69−7.61 (m, 3.49H), 7.61−7.45 (m, 5.37H),
7.45−7.35 (m, 0.21H), 7.23−7.08 (m, 1.06H), 5.91 (app br
s, 0.06H), 5.75 (m, 0.09H), 5.52/5.46 (2 overlapping app br s,
2H), 5.24−5.17 (m, 1.85H), 4.12−3.89 (m, 1.65H), 3.89−3.75
(m, 0.2H), 3.68−3.53 (m, 0.15H), 3.47−2.65 (series of
overlapping br m, ∼7.5H), 2.55−2.15 (series of m partially
overlapped with DMSO signal, ∼6.5H), 2.15−1.97 (m, 3.75H),
1.97−1.81 (m, 2H), 1.18−1.74 (m, 0.25H), 1.38−1.10 (m,
6H). LC (method 9): tR = 1.10 min. LC/MS (ESI) m/z: [M +
H]+ calcd for C48H55N8O2, 775.4; found, 775.5. HRMS (ESI)
m/z: [M + H]+ calcd for C48H55N8O2, 775.4448; found,
775.4456.
(2R)-2-(Morpholin-4-yl)-1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-
[(2R)-2-(morpholin-4-yl)-2-phenylacetyl]pyrrolidin-2-yl]-
1H-imidazol-5-yl}phenyl)phenyl]-1H-imidazol-2-yl}-
pyrrolidin-1-yl]-2-phenylethan-1-one (19). Title com-
1
pound 19 (TFA salt, off-white foam). H NMR (400 MHz,
DMSO-d6): δ 8.05 (app br s, 1.84H), 7.99−7.81 (m, 7.63H),
7.77−7.67 (m, 0.61H), 7.67−7.47 (2 m, 8.89H), 7.25−7.10 (m,
1.03H), 5.82−5.77 (m, 0.26H), 5.42 (app br s, 2H), 5.28−5.14
(m, 1.74H), 4.12−3.95 (m, 2.1H), 3.94−3.52 (series of m,
8.1H), 3.52−2.54 (series of overlapping br m, ∼7.8H), 2.50−
2.32 (m overlapped with DMSO signal, ∼1.5H), 2.31−2.14 (m,
2.02H), 2.14−1.97 (m, 4H), 1.97−1.83 (m, 2.05H), 1.83−1.66
(m, 0.43H). LC (method 4): tR = 29.9 min. LC/MS (ESI) m/z:
[M + H]+ calcd for C50H55N8O4, 831.43; found, 831.70.
2-[(3S)-3-fluoropyrrolidin-1-yl]-1-[(2S)-2-{5-[4-(4-{2-
[(2S)-1-{2-[(3S)-3-fluoropyrrolidin-1-yl]-2-phenylacetyl}-
pyrrolidin-2-yl]-1H-imidazol-5-yl}phenyl)phenyl]-1H-
imidazol-2-yl}pyrrolidin-1-yl]-2-phenylethan-1-one (20).
Title compound 20 (TFA salt, light yellow foam; symmetrical
(2R)-2-(Diethylamino)-1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-
[(2R)-2-(diethylamino)-2-phenylacetyl]pyrrolidin-2-yl]-
1H-imidazol-5-yl}phenyl)phenyl]-1H-imidazol-2-yl}-
pyrrolidin-1-yl]-2-phenylethan-1-one (16). Title com-
1
pound 16 (TFA salt, yellow foam). H NMR (400 MHz,
DMSO-d6): δ 9.81 (br s, ∼2H), 8.13−7.98 (m, 1.8H), 7.98−
7.81 (m, 7.4H), 7.81−7.76 (m, 0.3H), 7.76−7.63 (m, 3.85H),
7.63−7.43 (m, 5.59H), 7.28−7.08 (m, 1.06H), 6.02−5.94 (m,
0.2H), 5.60−5.51 (m, 0.26H), 5.44 (s, 1.76H), 5.21−5.11 (m,
1.78H), 4.18−4.01 (m, 1.59H), 3.86−3.79 (m, 0.15H), 3.67−
3.54 (m, 0.13H), 3.35−2.79 (series of overlapping br m,
7.13H), 2.69−2.56 (m, 1H), 2.56−2.40 (m overlapped with
DMSO signal, ∼2H), 2.29−2.14 (m, 1.84H), 2.14−1.98 (m,
3.89H), 1.98−1.80 (m, 1.95H), 1.80−1.65 (m, 0.32H), 1.37−
0.94 (overlapping br m, 12H). LC (method 9): tR = 1.12 min.
LC/MS (ESI) m/z: [M + H]+ calcd for C50H59N8O2, 803.5;
found, 803.6. HRMS (ESI) m/z: [M + H]+ calcd for
C50H59N8O2, 803.4761; found, 803.4728.
1
diastereomer-1). H NMR (400 MHz, DMSO-d6): δ 14.57 (s,
∼1H), 10.94 (br s, ∼1H), 8.13 (br s, 2.02H), 8.05−7.87 (m,
7.79H), 7.80−7.64 (m, 4.11H), 7.64−7.53 (m, 5.35H),
7.34−7.25 (m, 0.28H), 7.18−7.11 (m, 0.45H), 5.83−5.70 (m,
0.22H), 5.60/5.50 (2 overlapped app s, 2.95H), 5.37 (app s,
1H), 5.31−5.23 (m, 1.83H), 4.04−2.78 (series of overlapped br
m, ∼11H), 2.56−2.35 (br m overlapped with DMSO signal,
∼1H), 2.35−2.11 (m, 4.4H), 2.11−1.78 (m, 7.1H), 1.78−1.57
(m, 0.5H). LC (method 4): tR = 36.2 min. LC/MS (ESI) m/z:
[M + H]+ calcd for C50H53F2N8O2, 835.4; found, 835.3. HRMS
(ESI) m/z: [M + H]+ calcd for C50H53F2N8O2, 835.4260;
found, 835.4261.
(2R)-2-Phenyl-1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-[(2R)-2-phe-
nyl-2-(pyrrolidin-1-yl)acetyl]pyrrolidin-2-yl]-1H-imida-
zol-5-yl}phenyl)phenyl]-1H-imidazol-2-yl}pyrrolidin-1-
yl]-2-(pyrrolidin-1-yl)ethan-1-one (17). Title compound 17
(TFA salt, off-white solid). H NMR (400 MHz, DMSO-d6
with D2O exchange) δ 8.06 (s, 1.77H), 7.97−7.82 (m, 7.47H),
2-[(3S)-3-fluoropyrrolidin-1-yl]-1-[(2S)-2-{5-[4-(4-{2-
[(2S)-1-{2-[(3S)-3-fluoropyrrolidin-1-yl]-2-phenylacetyl}-
pyrrolidin-2-yl]-1H-imidazol-5-yl}phenyl)phenyl]-1H-
imidazol-2-yl}pyrrolidin-1-yl]-2-phenylethan-1-one (21).
Title compound 21 (TFA salt, off-white foam; symmetrical
1
1
diastereomer-2). H NMR (400 MHz, DMSO-d6): δ 11.06 (br
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s, ∼1H), 8.15−7.79 (series of overlapped m, 10.5H), 7.65−7.55
(m, 3.3H), 7.55−7.48 (m, 3.2H), 7.48−7.38 (m, 3H), 5.59
(app s, 2.14H), 5.53−5.31 (m, 1.34H), 5.28−5.18 (m, 2.07H),
5.07 (app s, 0.23H), 4.94−4.88 (m, 0.22H), 3.99−2.79 (series
of overlapping br m, ∼11H), 2.5−1.67 (series of overlapping br
m that is partially overlapped with DMSO signal, ∼13H). LC
(method 4): tR = 35.4 min. LC/MS (ESI) m/z: [M + H]+ calcd
for C50H53F2N8O2, 835.4; found, 835.3. HRMS (ESI) m/z: [M
+ H]+ calcd for C50H53F2N8O2, 835.4260; found, 835.4266.
(2R)-2-(Diethylamino)-2-phenyl-1-[(2S)-2-{5-[4-(4-{2-
[(2S)-1-[(2R)-2-phenyl-2-(piperidin-1-yl)acetyl]-
pyrrolidin-2-yl]-1H-imidazol-5-yl}phenyl)phenyl]-1H-
imidazol-2-yl}pyrrolidin-1-yl]ethan-1-one (22). Title com-
salt, off-white foam). 1H NMR (500 MHz, DMSO-d6): δ 14.48
(br s, ∼3H), 8.33 (app d, J = 7.3 Hz, 0.15H), 8.24 (app d, J =
7.3 Hz, 0.11H), 8.22−8.08 (m, 2.79H), 8.04−7.88 (m, 7.62H),
7.88−7.65 (m, 1.36H), 7.46−7.23 (m, 4.54H), 7.10−7.00 (m,
0.43H), 5.81−5.71 (m, 0.1H), 5.57−5.48 (m, 0.82H),
5.48−5.37 (m, 0.27H), 5.26−5.11 (m, 1.81H), 4.58 (app
quint, J = 7.0 Hz, 0.93H), 4.39−4.28 (m, 0.07H), 4.00−3.88
(m, 0.83H), 3.88−3.61 (m, 2.22H), 3.55/3.54/3.53 (3 s, 3H),
3.47−3.34 (m, 0.17H), 3.28−3.10 (m, 0.78H), 2.47−2.34 (m,
1.35H), 2.34−2.19 (m, 0.91H), 2.19−1.84 (2 m, 5.74H), 1.82
(s, 2.64H), 1.69 (s, 0.36H), 1.30−1.19 (m, 3H). LC (method
2): tR = 20.4 min. LC/MS (ESI) m/z: [M + H]+ calcd for
C41H45N8O5, 729.35; found, 729.33.
Methyl N-[(1R)-2-[(2S)-2-{5-[4-(4-{2-[(2S)-1-[(2S)-2-
[(methoxycarbonyl)amino]propanoyl]pyrrolidin-2-yl]-
1H-imidazol-5-yl}phenyl)phenyl]-1H-imidazol-2-yl}-
pyrrolidin-1-yl]-2-oxo-1-phenylethyl]carbamate (26).
Title compound 26 (TFA salt, off-white foam). 1H NMR
(500 MHz, DMSO-d6): δ 14.44 (br s, ∼3H), 8.21−8.00 (m,
2.07H), 8.00−7.85 (m, 7.65H), 7.85−7.59 (m, 1.35H),
7.45−7.27 (m, 5.26H), 7.05 (app br s, 0.67H), 5.75−5.63
(m, 0.11H), 5.51 (app d, J = 7.6 Hz, 0.81H), 5.45−5.31 (m,
0.3H), 5.25−5.09 (m, 1.78H), 4.38 (app quint, J = 7.1 Hz,
0.82H), 4.18−4.09 (m, 0.09H), 4.09−4.01 (m, 0.09H),
3.99−3.88 (m, 0.94H), 3.86−3.78 (m, 0.91H), 3.78−3.59 (m,
1.24H), 3.55/3.53 (2 s, 5.7H), 3.37 (s, 0.3H), 3.24−3.12 (m,
0.91H), 2.47−2.32 (m, 1.37H), 2.32−2.16 (m, 1.04H),
2.16−1.97 (m, 4.24H), 1.97−1.73 (m, 1.35H), 1.21 (d, J =
6.7 Hz, 3H). LC (method 4): tR = 26.9 min. LC/MS (ESI) m/
z: [M + H]+ calcd for C41H45N8O6, 745.3; found, 745.2. HRMS
(ESI) m/z: [M + H]+ calcd for C41H45N8O6, 745.3462; found,
745.3486.
1
pound 22 (free base, yellow solid). H NMR (400 MHz,
DMSO-d6): δ 11.96 (app br s, 1.6H), 9.79 (app br s, 0.4H),
8.03−7.23 (series of overlapping m, 18.1H), 7.23−6.81 (m,
1.9H), 5.82−5.20 (br m, 1.3H), 5.20−4.99 (m, 1.7H),
4.36−2.25 (series of br m overlapping with each other and
that of DMSO signal, ∼11H), 2.23−1.81 (series of m, 8.6H),
1.82−1.27 (series of m, 6.4H), 1.27−0.61 (series of m, 7H). LC
(method 11): tR = 2.53 min. LC/MS (ESI) m/z: [M + H]+
calcd for C51H59N8O2, 815.5; found, 815.2. HRMS (ESI) m/z:
[M + H]+ calcd for C51H59N8O2, 815.4761; found, 815.4763.
Methyl N-[(1R)-2-[(2S)-2-{5-[4-(4-{2-[(2S)-1-(2-
acetamidoacetyl)pyrrolidin-2-yl]-1H-imidazol-5-yl}-
phenyl)phenyl]-1H-imidazol-2-yl}pyrrolidin-1-yl]-2-oxo-
1-phenylethyl]carbamate (23). Title compound 23 (TFA
salt, off-white foam). 1H NMR (500 MHz, DMSO-d6): δ 14.43
(br s, ∼2H), 8.16−8.10 (br m, 2H), 8.08−8.00 (m, 1.46H),
8.00−7.93 (m, 3.67H), 7.93−7.86 (m, 3.92H), 7.86−7.75
(series of m, 0.44H), 7.70 (app br d, J = 7.9 Hz, 0.69H),
7.44−7.28 (m, 4.39H), 7.05 (app s, 0.43H), 5.71 (app br s,
0.09H), 5.55−5.49 (m, 0.82H), 5.43−5.38 (m, 0.27H),
5.22−5.16 (m, 1.82H), 4.03−3.91 (m, 3H), 3.78−3.72 (m,
1.13H), 3.64−3.57 (m, 1.05H), 3.55/3.53 (2 s, 3H), 3.22−3.14
(m, 0.82H), 2.42−2.23 (2 m, 2.41H), 2.11−2.00 (m, 4.59H),
1.96−1.88 (m, 1H), 1.85/1.81 (2 s, 3H). LC (method 5): tR =
21.1 min. LC/MS (ESI) m/z: [M + H]+ calcd for C40H43N8O5,
715.3; found, 715.0. HRMS (ESI) m/z: [M + H]+ calcd for
C40H43N8O5, 715.3356; found, 715.3369.
Methyl N-[(1R)-2-[(2S)-2-{5-[4-(4-{2-[(2S)-1-[(2R)-2-
acetamidopropanoyl]pyrrolidin-2-yl]-1H-imidazol-5-yl}-
phenyl)phenyl]-1H-imidazol-2-yl}pyrrolidin-1-yl]-2-oxo-
1-phenylethyl]carbamate (24). Title compound 24 (TFA
salt, off-white solid). 1H NMR (500 MHz, DMSO-d6): δ 14.32
(br s, ∼3H), 8.31 (app br m, 0.11H), 8.24 (app d, J = 6.7 Hz,
0.77H), 8.21−8.03 (m, 1.78H), 8.03−7.88 (m, 7.48H), 7.87−
7.64 (series of m, 1.7H), 7.46−7.24 (series of m, 4.62H), 7.05
(app s, 0.54H), 5.89−5.66 (m, 0.24H), 5.57−5.46 (m, 0.79H),
5.40 (app d, J = 7.3 Hz, 0.21H), 5.28−5.08 (m, 1.76H), 4.57
(app quint, J = 6.9 Hz, 0.82H), 4.31−4.13 (m, 0.18H), 3.99−
3.91 (m, 1H), 3.91−3.83 (m, 1H), 3.69−3.58 (m, 1.17H),
3.54/3.53 (2 s, 3H), 3.25−3.12 (m, 0.83H), 2.47−2.33 (m,
1.58H), 2.33−2.15 (m, 1.04H), 2.15−1.96 (m, 4.38H), 1.96−
1.88 (m, 1H), 1.87 (s, 2.25H), 1.82/1.81 (2 s, 0.75H), 1.23−
1.21 (m, 2.69H), 0.93 (app d, J = 6.4 Hz, 0.31H). LC (method
2): tR = 20.9 min. LC/MS (ESI) m/z: [M + H]+ calcd for
C41H45N8O5, 729.4; found, 729.0. HRMS (ESI) m/z: [M + H]+
calcd for C41H45N8O5, 729.3513; found, 729.3530.
Methyl N-[(2S)-1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-[(2S)-2-
[(methoxycarbonyl)amino]propanoyl]pyrrolidin-2-yl]-
1H-imidazol-5-yl}phenyl)phenyl]-1H-imidazol-2-yl}-
pyrrolidin-1-yl]-1-oxopropan-2-yl]carbamate (27). Title
1
compound 27 (TFA salt, light yellow foam). H NMR (500
MHz, DMSO-d6): δ 14.50 (br s, ∼3H), 8.14 (app s, 2H), 7.97
(overlap of d & m, J = 8.5 Hz, 4.5H), 7.90 (d, J = 8.5 Hz,
3.5H), 7.48−7.44 (m, 1.85H), 7.03 (app br s, 0.15H), 5.42
(app br d, J = 8.5 Hz, 0.2H), 5.16 (m, 1.8H), 4.38 (app quint, J
= 7.1 Hz, 1.8H), 4.11 (app quint, J = 7.2 Hz, 0.2H), 3.87−3.78
(m, 1.9H), 3.78−3.66 (m, 2.1H), 3.53 (s, 5.5H), 3.35 (s, 0.5H),
2.45−2.32 (m, 2H), 2.17−1.7 (1 major and 2 minor m, 6H),
1.21 (d, J = 7.0 Hz, 6H). LC (method 4): tR = 20.5 min. LC/
MS (ESI) m/z: [M + H]+ calcd for C36H43N8O6, 683.3; found,
682.2. HRMS (ESI) m/z: [M + H]+ calcd for C36H43N8O6,
683.3306; found, 683.3305.
Methyl N-[(2R)-1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-[(2R)-2-
[(methoxycarbonyl)amino]propanoyl]pyrrolidin-2-yl]-
1H-imidazol-5-yl}phenyl)phenyl]-1H-imidazol-2-yl}-
pyrrolidin-1-yl]-1-oxopropan-2-yl]carbamate (28). Title
1
compound 28 (TFA salt, white foam). H NMR (500 MHz,
DMSO-d6): δ 14.32 (br s, ∼3H), 8.16 (s, 1.77H), 8.07 (app br
s, 0.30H), 7.98−7.95 (m, 3.65H), 7.92−7.91 (m, 4.28H), 7.65
(d, J = 4.6 Hz, 0.19H), 7.38 (d, J = 7.3 Hz, 1.66H), 6.89 (app br
s, 0.15H), 5.76 (app br s, 0.2H), 5.17 (m, 1.8H), 4.39 (app
quint, J = 7.0 Hz, 1.59H), 4.30 (br m, 0.18H), 4.13−4.00 (m,
0.23H), 3.91−3.79 (m, 1.76H), 3.71−3.59 (m, 2.07H), 3.54
(overlapped s & m, 6.17H), 2.46−2.32 (m, 2.21H), 2.23−2.00
(m, 5.29H), 1.97−1.77 (2 m, 0.5H), 1.21 (d, J = 7.0 Hz, 5.3H),
0.93 (d, J = 6.4 Hz, 0.7H). LC (method 6): tR = 13.0 min. LC/
MS (ESI) m/z: [M + H]+ calcd for C36H43N8O6, 683.3; found,
Methyl N-[(1R)-2-[(2S)-2-{5-[4-(4-{2-[(2S)-1-[(2S)-2-
acetamidopropanoyl]pyrrolidin-2-yl]-1H-imidazol-5-yl}-
phenyl)phenyl]-1H-imidazol-2-yl}pyrrolidin-1-yl]-2-oxo-
1-phenylethyl]carbamate (25). Title compound 25 (TFA
2028
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683.4. HRMS (ESI) m/z: [M + H]+ calcd for C36H43N8O6,
683.3306; found, 683.3318.
Methyl N-[(2S,3R)-3-methoxy-1-[(2S)-2-{5-[4-(4-{2-
[(2S)-1-[(2S,3R)-3-methoxy-2-[(methoxycarbonyl)
amino]butanoyl]pyrrolidin-2-yl]-1H-imidazol-5-yl}-
phenyl)phenyl]-1H-imidazol-2-yl}pyrrolidin-1-yl]-1-oxo-
butan-2-yl]carbamate (34). Title compound 34 (TFA salt,
Ethyl N-[(2S)-1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-[(2S)-2-
[(ethoxycarbonyl)amino]propanoyl]pyrrolidin-2-yl]-1H-
imidazol-5-yl}phenyl)phenyl]-1H-imidazol-2-yl}-
pyrrolidin-1-yl]-1-oxopropan-2-yl]carbamate (29). Title
1
white solid). H NMR (400 MHz, DMSO-d6): δ 14.66 (br s,
1
∼3H), 8.14 (br s, 1.88H), 8.04−7.80 (m, 8.12H), 7.27 (d, J =
8.3 Hz, 1.88H), 6.80 (app br s, 0.12H), 5.67 (app d, J = 7.0 Hz,
0.15H), 5.16 (app t, J = 7.0 Hz, 1.85H), 4.33 (dd, J = 8.3, 6.3
Hz, 1.65H), 4.28−4.23 (m, 0.35H), 3.97−3.78 (m, 3.7H),
3.64−3.58 (overlapping m & s, 7H), 3.50−3.40 (m, 0.3H), 3.36
(s, 0.5H), 3.29 (s, 0.5H), 3.21/3.19 (2 overlapping s, 6H),
2.47−2.36 (m, 2.22H), 2.25−1.70 (m, 5.78H), 1.12 (d, J = 6.3
Hz, 0.5H), 1.04 (d, J = 6.3 Hz, 5.5H). LC (method 12): tR =
2.82 min. LC/MS (ESI) m/z: [M + H]+ calcd for C40H51N8O8,
771.4; found, 711.2. HRMS (ESI) m/z: [M + H]+ calcd for
C40H51N8O8, 771.3830; found, 771.3802.
compound 29 (TFA salt, white foam). H NMR (400 MHz,
DMSO-d6): δ 14.48 (br s, ∼3H), 8.13 (s, 2H), 7.98−7.94 (m,
4.3H), 7.89 (d, J = 8.5 Hz, 3.7H), 7.42−7.38 (m, 1.85H), 7.02
(app d, J = 6.0 Hz, 0.15H), 5.40 (app d, J = 8.1 Hz, 0.15H),
5.16 (m, 1.85H), 4.37 (app quint, J = 7.1 Hz, 1.8H), 4.16−4.08
(m, 0.2H), 4.05−3.91 (m, 3.8H), 3.87−3.67 (m, 4.2H),
2.48−2.27 (m, 2.1H), 2.20−2.00 (m, 5.5H), 2.00−1.73 (m,
0.4H), 1.20 (app d, J = 6.8 Hz, 5.84H), 1.15 (app t, J = 7.1 Hz,
5.56H), 0.99 (app t, J = 7.1 Hz, 0.6H). LC (method 6): tR =
14.2 min. LC/MS (ESI) m/z: [M + H]+ calcd for C38H47N8O6,
711.4; found, 711.4. HRMS (ESI) m/z: [M + H]+ calcd for
C38H47N8O6, 711.3619; found, 711.3621.
Methyl N-[(1S)-1-cyclopropyl-2-[(2S)-2-{5-[4-(4-{2-
[(2S)-1-[(2S)-2-cyclopropyl-2-[(methoxycarbonyl)
amino]acetyl]pyrrolidin-2-yl]-1H-imidazol-5-yl}phenyl)-
phenyl]-1H-imidazol-2-yl}pyrrolidin-1-yl]-2-oxoethyl]-
carbamate (35). Title compound 35 (TFA salt, off-white
Methyl N-{1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-{2-[(methoxy-
carbonyl)amino]-2-methylpropanoyl}pyrrolidin-2-yl]-
1H-imidazol-5-yl}phenyl)phenyl]-1H-imidazol-2-yl}-
pyrrolidin-1-yl]-2-methyl-1-oxopropan-2-yl}carbamate
(30). Title compound 30 (TFA salt, off-white foam). 1H NMR
(400 MHz, DMSO-d6): δ 14.11 (br s, ∼3H), 8.17 (s, 2H),
8.08−7.81 (m, 10H), 5.22 (m, 2H), 3.87−3.69 (m, 3.45H),
3.67 (s, 6H), 3.55−3.38 (m, 0.55H), 2.46−2.23 (m, 2.1H),
2.21−2.05 (m, 2.1H), 2.05−1.87 (m, 3.8H), 1.34 (s, 12H). LC
(method 9): tR = 1.21 min. LC/MS (ESI) m/z: [M + H]+ calcd
for C38H47N8O6, 711.4; found, 711.5. HRMS (ESI) m/z: [M +
H]+ calcd for C38H47N8O6, 711.3619; found, 711.3652.
Methyl N-{1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-{1-[(methoxy-
carbonyl)amino]cyclopropanecarbonyl}pyrrolidin-2-yl]-
1H-imidazol-5-yl}phenyl)phenyl]-1H-imidazol-2-yl}-
pyrrolidine-1-carbonyl]cyclopropyl}carbamate (31).
Title compound 31 (TFA salt, off-white solid). 1H NMR
(400 MHz, DMSO-d6): δ 14.36 (br s, ∼3H), 8.21−8.06 (m,
3.33H), 8.02−7.88 (m, 8.03H), 7.82 (app br s, 0.36H), 7.73
(app br s, 0.28H), 5.65 (app br s, 0.23H), 5.21−5.02 (m,
1.77H), 3.97−3.83 (m, 1.6H), 3.83−3.67 (m, 1.86H), 3.62 (s,
4.43H), 3.47 (br s, 1.57H), 3.19 (br s, 0.54H), 2.46−2.27 (m,
1.9H), 2.27−1.81 (m, 5.1H), 1.80−1.43 (m, 1H), 1.38−1.21
(m, 2H), 1.21−1.06 (m, 2H), 1.06−0.90 (m, 2.2H), 0.90−0.65
(m, 1.8H). LC (method 9): tR = 1.12 min. LC/MS (ESI) m/z:
[M + H]+ calcd for C38H43N8O6, 707.33; found, 707.45. HRMS
(ESI) m/z: [M + H]+ calcd for C38H43N8O6, 707.3306; found,
707.3309.
Methyl N-[(2S)-1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-[(2S)-2-
[(methoxycarbonyl)amino]butanoyl]pyrrolidin-2-yl]-1H-
imidazol-5-yl}phenyl)phenyl]-1H-imidazol-2-yl}-
pyrrolidin-1-yl]-1-oxobutan-2-yl]carbamate (32). Title
compound 32 (TFA salt, off-white foam). 1H NMR (500
MHz, DMSO-d6): δ 14.62 (br s, ∼3H), 8.11 (br s, 2H), 8.01−
7.92 (m, 4.3H), 7.89 (d, J = 7.9 Hz, 3.7H), 7.42−7.38 (m,
1.87H), 7.04−6.94 (m, 0.13H), 5.48−5.38 (m, 0.15H), 5.15
(dd, J = 7.9, 5.2 Hz, 1.85H), 4.24−4.19 (m, 1.86H), 3.95−3.90
(m, 0.29H), 3.87−3.66 (m, 3.81H), 3.54 (s, 5.34H), 3.45−3.38
(m, 0.26H), 3.36 (s, 0.44H), 2.46−2.30 (m, 2.1H), 2.24−1.98
(m, 5.55H), 1.98−1.90 (m, 0.27H), 1.84−1.63 (m, 2.12H),
1.58−1.35 (m, 1.96H), 0.89−0.84 (m, 6H). LC (method 8): tR
= 1.81 min. LC/MS (ESI) m/z: [M + H]+ calcd for
C38H47N8O6, 711.4; found, 711.4. HRMS (ESI) m/z: [M +
H]+ calcd for C38H47N8O6, 711.3619; found, 711.3643.
1
foam). H NMR (500 MHz, DMSO-d6): δ 14.59 (br s, ∼3H),
8.12 (br s, 2H), 7.96 (app d, J = 8.2 Hz, 3.7H), 7.90 (app d, J =
8.2 Hz, 4.3H), 7.57 (d, J = 7.9 Hz, 1.69H), 7.44 (app br s,
0.2H), 7.13 (app br s, 0.11H), 5.43 (app br d, J = 7.6 Hz,
0.2H), 5.15 (app dd, J = 7.9, 5.2 Hz, 1.8H), 3.87−3.71 (3
overlapping m, 5.64H), 3.54 (s, 5.35H), 3.49−3.40 (m, 0.36H),
3.33 (s, 0.65H), 2.45−2.30 (m, 2.2H), 2.21−1.72 (series of m,
5.8H), 1.26−1.02 (m, 2H), 0.53−0.35 (m, 5.9H), 0.35−0.28
(m, 1.7H), 0.28−0.19 (m, 0.4H). LC (method 8): tR = 1.81
min. LC/MS (ESI) m/z: [M + H]+ calcd for C40H47N8O6,
735.36; found, 735.43. HRMS (ESI) m/z: [M + H]+ calcd for
C40H47N8O6, 735.3619; found, 735.3561.
Methyl N-[(2S)-1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-[(2S)-2-
[(methoxycarbonyl)(methyl)amino]-3-methylbutanoyl]
pyrrolidin-2-yl]-1H-imidazol-5-yl}phenyl)phenyl]-1H-
imidazol-2-yl}pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-
yl]-N-methylcarbamate (36). Title compound 36 (TFA salt,
1
semicrystalline white foam). H NMR (400 MHz, DMSO-d6):
δ 14.57 (br s, ∼3H), 8.11 (app br s, 2H), 7.95 (app d, J = 8.1
Hz, 4H), 7.89 (app d, J = 8.1 Hz, 4H), 5.70−5.55 (2 m, 0.2H),
5.24−5.14 (m, 1.8H), 4.61−4.36 (m, 2H), 3.94−3.73 (2 m,
2.3H), 3.69/3.66 (2 s, 5.4H), 3.64−3.42 (2 m, 1.7H), 3.27/3.19
(2 s, 0.6H), 2.72/2.70/2.63/2.60 (4 s, 6H), 2.46−2.32 (m,
2.06H), 2.24−1.93 (m, 7.68H), 1.81−1.64 (m, 0.26H),
0.91−0.88 (m, 0.63H), 0.81−0.77 (m, 10.49H), 0.74−0.59 (2
m, 0.88H). LC (method 9): tR = 1.46 min. LC/MS (ESI) m/z:
[M + H]+ calcd for C42H55N8O6, 767.4; found, 767.4. HRMS
(ESI) m/z: [M + H]+ calcd for C42H55N8O6, 767.4245; found,
767.4252.
N-[(2S)-1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-[(2S)-2-acetamido-
3-methylbutanoyl]pyrrolidin-2-yl]-1H-imidazol-5-yl}-
phenyl)phenyl]-1H-imidazol-2-yl}pyrrolidin-1-yl]-3-
methyl-1-oxobutan-2-yl]acetamide (37). Title compound
37 (TFA salt, white solid). 1H NMR (500 MHz, DMSO-d6): δ
14.63 (br s, ∼3H), 8.23−8.09 (m, 2.16H), 8.04 (d, J = 8.5 Hz,
1.92H), 8.01−7.92 (m, 4.39H), 7.90 (app d, J = 8.5 Hz, 3.53H),
5.60 (app d, J = 7.3 Hz, 0.23H), 5.14 (app t, J = 7.2 Hz, 1.77H),
4.38 (app t, J = 7.9 Hz, 1.75H), 4.19 (app t, J = 7.9 Hz, 0.25H),
3.97−3.87 (m, 1.73H), 3.87−3.77 (m, 1.78H), 3.76−3.63 (m,
0.28H), 3.54−3.38 (m, 0.21H), 2.49−2.24 (m, 2.42H), 2.23−
1.91 (m, 7.58H), 1.87 (s, 5.41H), 1.74 (s, 0.59H), 0.97−0.88
2029
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(m, 1.15H), 0.83 (d, J = 6.7 Hz, 5.25H), 0.80 (d, J = 6.7 Hz,
5.60H). LC (method 13): tR = 2.05 min. LC/MS (ESI) m/z:
[M + H]+ calcd for C40H51N8O4, 707.40; found, 707.77. HRMS
(ESI) m/z: [M + H]+ calcd for C40H51N8O4, 707.4033; found,
707.4004.
Methyl N-[(2S)-1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-[(2R)-2-(di-
ethylamino)-2-phenylacetyl]pyrrolidin-2-yl]-1H-imida-
zol-5-yl}phenyl)phenyl]-1H-imidazol-2-yl}pyrrolidin-1-
yl]-3-methyl-1-oxobutan-2-yl]carbamate (41). Title com-
1
pound 41 (TFA salt, tan foam). H NMR (500 MHz, DMSO-
d6): δ 14.74 (br s, ∼2H), 9.80 (br s, ∼1H), 8.12 (app br s,
1.24H), 8.09−7.82 (m, 8.71H), 7.82−7.65 (m, 2.05H), 7.65−
7.42 (m, 2.80H), 7.33 (d, J = 8.5 Hz, 0.79H), 7.27−7.19 (m,
0.20H), 7.18−7.12 (m, 0.21H), 5.96 (app br s, 0.09H), 5.64−
5.49 (m, 0.24H), 5.44 (app s, 0.87H), 5.25−5.06 (m, 1.8H),
4.21−4.03 (m, 1.80H), 3.95−3.68 (m, 2.20H), 3.54 (s, 2.75H),
3.34 (s, 0.25H), 3.33−3.13 (m, 2H), 3.13−3.02 (m, 1H), 3.02−
2.79 (m, 1H), 2.68−2.54 (m, ∼1H), 2.46−2.30 (m, 1.3H),
2.30−2.14 (m, 1.79H), 2.14−1.96 (m, 4.76H), 1.96−1.84 (m,
0.98H), 1.84−1.63 (m, 0.17H), 1.38−0.96 (series of m, 6H),
0.96−0.85 (m, 1.18H), 0.84 (d, J = 6.7 Hz, 2.32H), 0.79 (d, J =
6.7 Hz, 2.50H). LC (method 8): tR = 1.76 min. LC/MS (ESI)
m/z: [M + H]+ calcd for C45H55N8O4, 771.4; found, 771.5.
HRMS (ESI) m/z: [M + H]+ calcd for C45H55N8O4, 771.4346;
found, 771.4379.
Virology Assay. The tabulated activity and cytotoxicity data
are mean values of at least two experiments, and in general, the
replicon assay exhibits a maximum of 3-fold variation. GT-1a/-
1b HCV and BVDV EC50 values were obtained from FRET and
Luciferase assays, respectively. Details of the biological assays
used in the characterizations of final compounds are provided
in ref 6d.
N-[(2R)-1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-[(2R)-2-acetamido-
3-methylbutanoyl]pyrrolidin-2-yl]-1H-imidazol-5-yl}-
phenyl)phenyl]-1H-imidazol-2-yl}pyrrolidin-1-yl]-3-
methyl-1-oxobutan-2-yl]acetamide (38). Title compound
1
38 (TFA salt, off-white solid). H NMR (500 MHz, DMSO-
d6): δ 14.33 (br s, ∼3H), 8.30 (br d, J = 6.7 Hz, 0.33H),
8.21−8.03 (m, 3.79H), 8.03−7.79 (m, 7.88H), 6.08−5.88 (m,
0.25H), 5.23 (dd, J = 8.2, 4.0 Hz, 1.75H), 4.43 (app t, J = 7.8
Hz, 1.64H), 3.99−3.87 (m, 2.28H), 3.76−3.62 (m, 1.86H),
3.52−3.40 (m, 0.22H), 2.48−2.26 (m, 2.03H), 2.20−2.00 (m,
5.33H), 2.00−1.91 (m, 2.29H), 1.90 (s, 5.11H), 1.87 (s,
0.89H), 1.84−1.75 (m, 0.35H), 0.97−0.84 (m, 10.44H), 0.73
(d, J = 6.7 Hz, 0.81H), 0.45 (d, J = 6.4 Hz, 0.75H). LC
(method 8): tR = 1.93 min. LC/MS (ESI) m/z: [M + H]+ calcd
for C40H51N8O4, 707.4; found, 707.6. HRMS (ESI) m/z: [M +
H]+ calcd for C40H51N8O4, 707.4033; found, 707.4054.
Methyl N-[(2S)-1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-[(2R)-2-(di-
ethylamino)-2-phenylacetyl]pyrrolidin-2-yl]-1H-imida-
zol-5-yl}phenyl)phenyl]-1H-imidazol-2-yl}pyrrolidin-1-
yl]-1-oxopropan-2-yl]carbamate (39). Title compound 39
1
(TFA salt, white foam). H NMR (500 MHz, DMSO-d6): δ
14.61 (br s, ∼2H), 9.82 (br s, ∼1H), 8.13 (s, 1.26H), 8.06 (br s,
0.9H), 8.00−7.85 (m, 7.89H), 7.81−7.64 (m, 2.04H),
7.63−7.51 (m, 2.68H), 7.49−7.42 (m, 0.77H), 7.23 (m,
0.18H), 7.16 (m, 0.26H), 7.03 (m, 0.02H), 5.99 (app br s,
0.1H), 5.55 (app br s, 0.16H), 5.44 (s, 0.96H), 5.24−5.10 (m,
1.78H), 4.44−4.31 (m, 0.93H), 4.16−4.04 (m, 1H), 3.88−3.70
(2 m, 2.07H), 3.53 (s, 2.73H), 3.35 (s, 0.27H), 3.34−3.12 (m,
2H), 3.12−3.01 (m, 1H), 2.98−2.84 (app br s, 1H), 2.65−2.53
(br m partially overlapped with DMSO signal, 1H), 2.47−2.30
(m, 1.16H), 2.30−2.16 (m, 0.94H), 2.16−1.98 (m, 4.5H),
1.98−1.66 (m, 1.4H), 1.34−1.00 (d overlapped with series of
m, J = 7.0 Hz, 9H). LC (method 6): tR = 12.60 min. LC/MS
(ESI) m/z: [M + H]+ calcd for C43H51N8O4, 743.4; found,
743.5. HRMS (ESI) m/z: [M + H]+ calcd for C43H51N8O4:
743.4033; found, 743.4053.
PK Assessment. Rat PK studies (4 and/or 24 h): Two
animals per dosing group were used in the 4 h rat PK screens,
where plasma was sampled at 0.25, 0.5, 1, 2, and 4 h, and liver
samples were obtained at the 4 h study termination. In the case
of 24 h rat PK studies, a total of three animals per dosing group
were used, where plasma was sampled for a total of eight time
points (0.25, 0.5, 1, 2, 4, 6, 8, and 24 h), and liver samples were
obtained at study termination. Unless noted otherwise, PEG-
400 was the dosing vehicle for the rat PK studies.
Vehicle information for multispecies PO-PK data summar-
ized in Table 8: compound 33, rat (100% PEG-400); dog (60%
PEG-400, 10% ethanol, 30% water); monkey (PEG-400/
povidone K-30/Vitamin E TPGS/Tween-80 in 95:2:2:1 ratio).
Compound 39, rat, dog, and monkey (PEG-400/povidone K-
30/Vitamin E TPGS/Tween-80 in 95:2:2:1 ratio).
Bioactivation Study. The compound (10 μM) was
incubated with human liver microsomes (1 mg protein/mL)
in phosphate buffer (50 mM; pH 7.4) at 37 °C for 30 min. The
incubation was supplemented with NADPH (1 mM) and either
GSH (5 mM) or potassium cyanide (1 mM). The incubation
mixture was treated with a single volume of CH3CN and
centrifuged at 1000g to obtain supernatant, which was stored at
4 °C until LC/MS analysis. Chromatographic separation was
carried out using an HPLC system that consisted of a Waters
(Milford, MA) Alliance 2695 Separations Module and a
Phenomenex Luna C18 column (2 × 150 mm, 3 μm). The
mobile phase consisted of 10 mM NH4OAc (pH 5) in H2O/
CH3CN (95/5 v/v) (solvent-A) and CH3CN (solvent-B) at
200 μL/min with the following gradient conditions: 0% B
isocratic for 5 min, 0−90% B over 25 min, and 90% B isocratic
for 15 min. Assessment for the presence of GSH and cyano
adducts was made on the basis of LC/MS and LC/MS/MS
data.
Methyl N-[(2S)-1-[(2S)-2-{5-[4-(4-{2-[(2S)-1-[(2R)-2-(di-
ethylamino)-2-phenylacetyl]pyrrolidin-2-yl]-1H-imida-
zol-5-yl}phenyl)phenyl]-1H-imidazol-2-yl}pyrrolidin-1-
yl]-1-oxobutan-2-yl]carbamate (40). Title compound 40
1
(TFA salt, light-yellow foam). H NMR (500 MHz, DMSO-
d6): δ 14.71 (br s, ∼3H), 9.78 (br s, ∼1H), 8.13 (app br s,
1.26H), 8.04 (app br s, 0.97H), 7.99−7.82 (m, 7.80H),
7.78−7.64 (m, 2H), 7.63−7.51 (m, 2.67H), 7.44 (app d, J = 8.5
Hz, 0.09H), 7.39 (d, J = 7.9 Hz, 0.75H), 7.29−7.19 (m, 0.19H),
7.19−7.09 (m, 0.24H), 7.03−6.92 (m, 0.03H), 5.98 (app br s,
0.09H), 5.54 (app br s, 0.13H), 5.49−5.40 (m, 0.98H),
5.25−5.08 (m, 1.8H), 4.28−4.16 (m, 0.97H), 4.16−4.04 (m,
0.93H), 3.91−3.64 (m, 2.16H), 3.54 (s, 2.76H), 3.35 (s,
0.24H), 3.33−3.13 (m, 1.8H), 3.13−2.99 (m, 1.11H),
2.99−2.80 (m, 1.03H), 2.68−2.51 (m, ∼1H), 2.45−2.32 (m,
1.44H), 2.28−1.86 (series of m, 6.56H), 1.82−1.68 (m, 1H),
1.53−1.42 (m, 1H), 1.34−0.98 (series of m, 5.97H), 0.90−0.84
(m, 3.03H). LC (method 8): tR = 1.74 min. LC/MS (ESI) m/z:
[M + H]+ calcd for C44H53N8O4, 757.4; found, 757.4. HRMS
(ESI) m/z: [M + H]+ calcd for C44H53N8O4, 757.4190; found,
757.4212.
Solubility Assessment. The compound is incubated in 25
mM aqueous phosphate buffer (pH 6.5) in a sealed 1 fluid
dram glass vial at 22 °C for 24 h, while being agitated with an
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49, 1346−1353. (b) Lemm, J. A.; O’Boyle, D. R., II; Liu, M.; Nower, P.
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C virus NS5A inhibitors with dimeric structures. Antimicrob. Agents
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Orbitron Rotator. The mixture was centrifuged and filtered
through a 0.45 μm PTFE filter membrane. To minimize a
potential filter-adsorption effect, the first 100 μL of the filtrate
was discarded. Sample concentration was determined by HPLC
using an Agilent 1200 equipped with UV detection.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: makonen.belema@bms.com. Tel.: 1-203-677-6928.
(5) For additional chemotypes that emerged from screening efforts
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Notes
The authors declare no competing financial interest.
The discovery chemistry effort at Bristol-Myers Squibb
Pharmaceutical Research Institute, Candiac, Que. lasted until
June 2007.
ACKNOWLEDGMENTS
■
We thank the following members of our analytical and
separation groups for the diverse role they played in enabling
the work described in this manuscript: Jingfang Cutrone, Dieter
Drexler, Xiaohu Huang, Edward S. Kozlowski, and Julia M.
Nielson. We thank Gerry G. Everlof and Lucy Sun for
conducting the solubility and the patch-clamp ion channel
inhibition assessments, respectively, and John G. Houston and
Carl P. Decicco for their guidance and support.
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