Discovery of MK-8742: An HCV NS5A inhibitor with broad genotype activity

Article abstract of DOI:10.1002/cmdc.201300343

The NS5A protein plays a critical role in the replication of HCV and has been the focus of numerous research efforts over the past few years. NS5A inhibitors have shown impressive in vitro potency profiles in HCV replicon assays, making them attractive components for inclusion in all oral combination regimens. Early work in the NS5A arena led to the discovery of our first clinical candidate, MK-4882 [2-((S)-pyrrolidin-2-yl)-5-(2-(4-(5-((S)-pyrrolidin- 2-yl)-1H-imidazol-2-yl)phenyl)benzofuran-5-yl)-1H-imidazole]. While preclinical proof-of-concept studies in HCV-infected chimpanzees harboring chronic genotype 1 infections resulted in significant decreases in viral load after both single- and multiple-dose treatments, viral breakthrough proved to be a concern, thus necessitating the development of compounds with increased potency against a number of genotypes and NS5A resistance mutations. Modification of the MK-4882 core scaffold by introduction of a cyclic constraint afforded a series of tetracyclic inhibitors, which showed improved virologic profiles. Herein we describe the research efforts that led to the discovery of MK-8742, a tetracyclic indole-based NS5A inhibitor, which is currently in phase 2b clinical trials as part of an all-oral, interferon-free regimen for the treatment of HCV infection. Effective treatment of chronic hepatitis C with direct-acting antivirals will require combination therapy with multiple agents that target different steps in the viral replication cycle and impose a high barrier to resistance. MK-8742 is a potent inhibitor of hepatitis C virus non-structural protein 5A (HCV NS5A) that is being developed as a component of an once-daily, all-oral, interferon-free regimen for the treatment of chronic HCV infection. Copyright

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DOI: 10.1002/cmdc.201300343
Discovery of MK-8742: An HCV NS5A Inhibitor with Broad
Genotype Activity
Craig A. Coburn,*[a] Peter T. Meinke,[a] Wei Chang,[e] Christine M. Fandozzi,[c]
Donald J. Graham,[b] Bin Hu,[e] Qian Huang,[b] Stacia Kargman,[d] Joseph Kozlowski,[a]
Rong Liu,[b] John A. McCauley,[a] Amin A. Nomeir,[c] Richard M. Soll,[e] Joseph P. Vacca,[a]
Dahai Wang,[e] Hao Wu,[e] Bin Zhong,[e] David B. Olsen,[b] and Steven W. Ludmerer*[b]
The NS5A protein plays a critical role in the replication of HCV
and has been the focus of numerous research efforts over the
past few years. NS5A inhibitors have shown impressive in vitro
potency profiles in HCV replicon assays, making them attrac-
tive components for inclusion in all oral combination regimens.
Early work in the NS5A arena led to the discovery of our first
clinical candidate, MK-4882 [2-((S)-pyrrolidin-2-yl)-5-(2-(4-(5-((S)-
pyrrolidin-2-yl)-1H-imidazol-2-yl)phenyl)benzofuran-5-yl)-1H-
imidazole]. While preclinical proof-of-concept studies in HCV-
infected chimpanzees harboring chronic genotype 1 infections
resulted in significant decreases in viral load after both single-
and multiple-dose treatments, viral breakthrough proved to be
a concern, thus necessitating the development of compounds
with increased potency against a number of genotypes and
NS5A resistance mutations. Modification of the MK-4882 core
scaffold by introduction of a cyclic constraint afforded a series
of tetracyclic inhibitors, which showed improved virologic pro-
files. Herein we describe the research efforts that led to the
discovery of MK-8742, a tetracyclic indole-based NS5A inhibi-
tor, which is currently in phase 2b clinical trials as part of an
all-oral, interferon-free regimen for the treatment of HCV infec-
tion.
Introduction
The World Health Organization (WHO) estimates that ~3% of
the world’s population are chronically infected with hepatitis C
virus (HCV). Although HCV infection has both acute and chron-
ic forms, most of the morbidity associated with infection is re-
alized through the development of chronic liver disease in
a subset of infected people years after initial acquisition of the
infection.[1] HCV displays a high degree of genetic heterogenei-
ty and can be classified into six major genotypes (GT1–6) and
a series of subtypes (a,b,c,…), with genotypes 1a and 1b ac-
counting for ~60% of all infections.
portant goal of future HCV therapy is to develop direct-acting
antivirals in order to: 1) provide an all-oral interferon-free regi-
men, 2) decrease overall treatment durations, 3) provide broad-
er coverage of HCV patient populations (e.g., GT2/GT3/GT4 in-
fections, interferon-intolerant patients, and other difficult-to-
treat populations), and 4) improve the overall safety and toler-
ability of HCV treatment regimens.[2] Given the propensity of
HCV for generating genetic diversity, an interferon-free regi-
men will likely require two or more potent direct-acting com-
pounds with non-overlapping resistance profiles in order to
suppress the broadest-spectrum infections.
Current interferon-based therapy for chronic hepatitis C is
limited by both efficacy and tolerability. Furthermore, the abili-
ty to achieve a sustained virologic response is largely depen-
dent on the HCV genotype and duration of treatment. An im-
The HCV NS5A protein is a multifunctional RNA binding pro-
tein essential for viral replication. NS5A has no known enzy-
matic function, but has been shown to serve multiple func-
tions at various stages of the life cycle, including viral replica-
tion and virion assembly.[3]
[a] Dr. C. A. Coburn, Dr. P. T. Meinke, Dr. J. Kozlowski, Dr. J. A. McCauley,
Dr. J. P. Vacca
A landmark proof-of-concept study validated NS5A as a ther-
apeutic target when it was demonstrated that a single dose of
the NS5A inhibitor daclatasvir could effect a rapid virologic re-
sponse in GT1-infected patients.[4] This result precipitated activ-
ity in the field and made NS5A a target for many research
groups.[5]
Department of Medicinal Chemistry, Merck and Company, Inc.
West Point, PA 19486 (USA)
E-mail: craig_coburn@merck.com
[b] D. J. Graham, Q. Huang, Dr. R. Liu, Dr. D. B. Olsen, Dr. S. W. Ludmerer
Department of Antiviral Research, Merck and Company, Inc.
West Point, PA 19486 (USA)
E-mail: steven_ludmerer@merck.com
Improvements in potency across a broad spectrum of HCV
genotypes and mutants while maintaining favorable physical
and pharmacokinetic parameters may lead to improved NS5A
inhibitors with a higher barrier to resistance. Toward this end,
a concerted research effort aimed at combining the structural
components from both internal and literature compounds led
to a series of potent NS5A inhibitors containing a central
[c] Dr. C. M. Fandozzi, Dr. A. A. Nomeir
Department of Preclinical Pharmacokinetics and Drug Metabolism (Merck)
[d] Dr. S. Kargman
Department of In Vitro Pharmacology (Merck)
[e] W. Chang, Dr. B. Hu, Dr. R. M. Soll, Dr. D. Wang, Dr. H. Wu, Dr. B. Zhong
Department of Medicinal Chemistry, WuXi AppTec
Shanghai, 200131 China
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indole or benzofuran scaffold from which advanced structures
were derived. Herein we describe research efforts and high-
light the preclinical properties of our first clinical NS5A inhibi-
tor MK-4882, including efficacy studies in infected chimpanzees
which ultimately led to the discovery of the current clinical
compound MK-8742.
indole nitrogen atom resulted in a ~10-fold loss in replicon ac-
tivities (4 f), whereas the isomeric 2,6-disubstituted indole 4g
resulted in a substantial loss in potency relative to 4e.
Within the context of the indole-based scaffold, replacement
of the proline N-Cbz group by either the isosteric hydrocinna-
mate ligand (5) or an (S)-N-Boc-phenylalanine group (6) did
not have a significant influence on potency. Synthesis and eval-
uation of the (S)-N-Boc-phenylglycine homologue 7 showed
a 10-fold increase in genotype 1b and 2a potencies, while the
epimeric (R)-N-Boc-Phg-containing diastereomer 8 afforded
a 100–400-fold increase in GT1b and GT2a potencies and a 20-
fold improvement in GT1a potency (Table 1).
Results and Discussion
Our initial efforts focused on the synthesis and SAR develop-
ment of a series of N-arylpiperazines 1.[6] The incorporation of
a cyclic constraint within these structures afforded indole 2,
which showed similar potency in the replicon assay (GT1b EC50
~150 nm) and was an attractive entry toward modulating this
target. Concurrent to these efforts, other NS5A inhibitors
began to appear in the patent literature, but their SAR had not
been fully delineated. Because of the structural similarity be-
tween compound 1 and the stilbene inhibitors 3,[7] a lead-hop-
ping effort was initiated with the goal of applying a similar
cyclization strategy in order to explore the SAR of the pseudo-
symmetric isosteres 4. Subsequent reports revealed that com-
pound 3 was also pivotal in the design of daclatasvir
(Figure 1).[8]
Additional profiling of inhibitor 8 showed bioavailability to
be low (<2%) in preclinical animals. The poor oral absorption
of this compound was attributed to the high peptidic nature
of the compound. To address this limitation, the C2’-phenyl
amide bond was replaced by a variety of known amide iso-
steres (Table 2; X=oxazole, oxadiazole, pyrazole, thiazole).
These modifications, however, resulted in compounds that dis-
played inferior replicon profiles relative to the parent amide 8.
Pyrazole analogue 11 appeared to have the best profile and it
was speculated that the NH group was important for maintain-
ing genotype 1a and 1b potencies. As such, imidazole 13 was
synthesized, and whereas EC50 values versus GT1b and 2a were
similar to those of amide 8, this modification resulted in
a ~20-fold improvement in genotype 1a potency (EC50 =3 nm).
Further exploration into the SAR of NH-containing heterocy-
clic amide isosteres resulted in the synthesis of the isomeric 2-
prolyl-5-phenylimidazole analogue 14. This modification gave
an additional ~20-fold increase in potency against GT1a while
maintaining low-picomolar EC50 values in the GT1b and GT2a
Several heterocyclic scaffolds (4a–4 f) were synthesized and
incorporated into the final inhibitor structures. The cellular ac-
tivity of each new compound was determined using the repli-
con-system-expressing genotypes 1b, 2a, and 1a. Initial results
showed that benzimidazole 4a, benzothiazole 4b, and benzox-
azole 4c each had in vitro profiles worse than the parent stil-
bene inhibitor 3, whereas benzofuran 4d and indole 4e had
profiles similar to the reference compound. Alkylation of the
Figure 1. Strategy for the development of NS5A inhibitors.
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of the 2-prolyl-5-arylimidazole substitution on both
potency and pharmacokinetics. Table 3 shows the re-
sults from imidazole incorporation first on the C5
benzofuran side (17; A=imidazole), then the C2’
phenyl side (18; B=imidazole). In each case, geno-
type 1b and 2a potencies remained the same while
genotype 1a potency improved by a factor of 10. In-
corporation of both imidazole amide isosteres (19;
A=B=imidazole) resulted in an additional 20-fold
improvement in GT1a EC50 values. On the basis of the
low-picomolar EC50 values in GT1a, 1b, and 2a repli-
con assays, compound 19 became an important lead.
Subsequent research efforts focused on the SAR of
the terminal amino acid groups in order to address
the problematic oral absorption profile without per-
turbing the virologic profile.
Table 1. Central scaffold SAR.
Compd
Substituent
Genotype, EC50 [nm][a]
R
X, Y
1b
2a
1a
3
4a
4b
4c
4d
4e
4 f
4g
5
5
60
>20000
>20000
1500
>20000
>20000
>20000
>20000
>20000
1600
>20000
>20000
2100
Cbz
Cbz
Cbz
Cbz
Cbz
NH, N
S, N
O, N
>20000
>20000
160
5
16
170
O, CH
230
290
990
NH, CH
NCH3, CH
CH, NH
NH, CH
NH, CH
NH, CH
NH, CH
Cbz
Cbz
>20000
4
>20000
290
PhCH2CH2
(S)-Boc-Phe
(S)-Boc-Phg
(R)-Boc-Phg
6
7
8
14
ND
ND
1500
Table 4 shows the area under the curve (AUC)
values after 10 mgkgꢀ1 oral dosing to fasted male
Sprague–Dawley rats for some of the amino acids
surveyed. With the exception of compound 21, little
difference in GT1a potency was observed upon incor-
poration of various alkyl and cycloalkyl substituents.
Conversely, plasma drug exposure depended heavily
on the nature of the amino acid substituent. For ex-
ample, replacement of the C2’ phenyl side d-Phg res-
idue with an l-Val subunit (20; Y=(S)-iPr) resulted in
20-fold higher compound exposure than analogue
19 (Y=(R)-Ph).
0.4
0.004
20
0.05
70
[a] nꢁ3; ND: not determined
Table 2. Amide isosteres.
Genotype, EC50 [nm][a]
The addition of a second l-Val group (X=(S)-iPr)
resulted in even higher plasma drug levels after
10 mgkgꢀ1 oral dosing. Additionally, compound 22,
which has an S,S,S,S configuration, showed 420-fold
higher plasma AUC values than its R,S,S,S diastereo-
mer 21. Higher plasma exposures of 22 are presuma-
bly due to a transporter that recognizes the l-Pro-l-
Val dipeptide subunit. Further SAR on a variety of ho-
mologated valine analogues (that is, cyclobutylgly-
cine 24, tert-butylglycine 25, homoalanine 26)
showed better exposure than 19, although each
were inferior to 22.
Cyclopropylglycine analogue 23 displayed similar
plasma drug exposure and oral bioavailability in the
rat while maintaining good potency in the GT1a and
GT1b replicon assays. However, examination of the
overall potency profiles of 22 versus 23 showed a sig-
nificant loss in potency against both GT2a and the
key genotype 1a Y93H mutant. As such, compound
22 was selected for early clinical development under
the moniker MK-4882 (Table 5).
Compd
X
1b
2a
1a
70
8
9
–NHC(O)–
0.004
0.05
500
0.44
0.09
0.016
0.2
>266
>266
47
10
11
12
86
1.3
26
>266
13
14
0.01
0.01
0.07
3
0.015
0.17
15
0.06
>266
150
[a] n=3
replicon assays. The importance of the heterocyclic NH sub-
stituent was further proven by the loss in replicon potency of
the N-methylated analogue 15. Altogether, the incorporation
of the imidazole amide isostere maintained both GT1b and 2a
potency while improving GT1a potency by ~400-fold relative
to the amide 8.
The synthesis of MK-4882 is straightforward and is illustrated
in Schemes 1 and 2. N-Boc-l-proline aldehyde was converted
into the 2-substituted imidazole 22a using a Radziszewski imi-
dazole synthesis in good yield. Imidazole 22a was subsequent-
ly treated with excess NBS in THF to give the C4,C5-dibromi-
nated intermediate, which was reduced with sodium sulfite to
provide the key monobrominated imidazole intermediate 22b.
Both single and double imidazole amide isosteres in the
benzofuran series were prepared to further explore the effects
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and showed only a three- to
fourfold shift in the presence of
40% normal human serum. A
number of clinical, in vivo, and
in vitro studies have identified
key mutations that confer resist-
ance to NS5A inhibitors.[9,10]
These mutations arise principally
at residues 28 (1a and 1b), 30
(GT1a), 31 (GT1a and GT1b), and
93 (GT1a and GT1b). MK-4882
potency is shifted to low nano-
molar against many mutations at
the key NS5A residues 30, 31,
and 93. Typically, the magnitude
of the potency shift was greater
in the GT1a background. For ex-
Table 3. Imidazole SAR.
Compd
Substituent[a]
Genotype, EC50 [nm][b]
Cmax [mm][c]
A
B
1b
2a
1a
27
16
17
18
19
–(O)CHN–
imidazole
–(O)CHN–
imidazole
–NHC(O)–
–NHC(O)–
imidazole
imidazole
0.01
0.01
0.03
0.01
0.03
0.02
0.002
0.006
0.004
0.004
0.002
0.004
2
0.3
0.015
[a] Imidazole=2-prolyl-substituted imidazole. [b] nꢁ3. [c] Peak plasma concentration following 10 mgkgꢀ1 p.o.
dose in 10% Tween to fasted male Sprague–Dawley rats (average of n=2).
Table 4. Amino acid SAR.
ample,
MK-4882
potencies
against L31V and Y93H mutants
in the GT1b background were
0.5 and 3.0 nm, respectively,
whereas in the genotype 1a
background they were 2 and
27 nm. Data are summarized in
Table 6.
[b]
Compd
X
Y
GT1a EC50 [nm][a]
AUC [mmhꢀ1
]
19
20
21
22
23
24
25
26
(R)-phenyl
(R)-phenyl
(R)-phenyl
(S)-isopropyl
(S)-isopropyl
(S)-isopropyl
(S)-cyclopropyl
(S)-cyclobutyl
(S)-tert-butyl
(S)-ethyl
0.015
0.018
25
0.01
0.07
0.14
0.15
0.02
0.5
10
0.1
42
38
10
The pharmacokinetic proper-
ties of MK-4882 were studied in
Sprague–Dawley rats, beagle
dogs, and rhesus monkeys. MK-
4882 demonstrated low clear-
ance and moderate half-life (2–
5 h) in the three species. The
oral bioavailability was 26% in
dog and 38% in rat, demonstrat-
(R)-isopropyl
(S)-isopropyl
(S)-cyclopropyl
(S)-cyclobutyl
(S)-tert-butyl
(S)-ethyl
10
7.5
[a] Values are the average of nꢁ3 experiments. [b] 10 mgkgꢀ1 p.o. dosed as a homogeneous solution in 10%
Tween to fasted male Sprague–Dawley rats (average of n=2 animals).
Table 5. Profiles of compound 22 (MK-4882) and 23.
Compd
Genotype, EC50 [nm][a]
Rat p.o.[b]
AUC [mmhꢀ1
1b
2a
1a
1aY93H
]
F [%]
22
23
0.001
0.003
0.04
6.2
0.009
0.07
27
230
42
38
38
45
Scheme 1. Synthesis of bromoimidazole fragment 22c. Reagents and condi-
tions: a) glyoxal, 7n NH3 in MeOH; b) 1. NBS, THF, 2. Na2SO3, EtOH, H2O,
reflux.
[a] Nꢁ3; [b] 10 mgkgꢀ1 p.o. dosed in 10% Tween to fasted male SD rats
(average of N=2).
ing that the compound was moderately absorbed in preclinical
species. The tmax in both rat and dog was somewhat long, oc-
curring at 4–6 h. MK-4882 demonstrated slightly greater than
dose-proportional exposures in rat when dosed at 2 and
100 mgkgꢀ1 p.o., and also in dog between doses of 1 and
50 mgkgꢀ1 p.o. Unlike many of the HCV protease inhibitors,
MK-4882 did not appear to undergo transport-mediated
uptake into liver, as the liver-to-plasma ratio averaged <10
after oral dosing to rats.
The 2-phenylbenzofuran scaffold was easily prepared via
one-pot Cs2CO3-mediated alkylation/intramolecular cyclocon-
densation between 5-bromosalicylaldehyde and ethyl-2-
bromo-(4-bromophenyl)acetate (22c). Metal–halogen ex-
change of 5-bromo-2-(4-bromophenyl)benzofuran (22d) af-
forded the bis-pinacolboronate ester, which was subsequently
coupled to bromoimidazole 22b. Deprotection of the proline
Boc groups afforded penultimate compound 22 f, which was
subjected to an amide coupling protocol using the BOP re-
agent and two equivalents of N-Moc-l-Val.
The oral absorption profile in chimpanzees was also evaluat-
ed in preparation for in vivo efficacy studies. As such, two
male chimpanzees were dosed orally with MK-4882 at
1 mgkgꢀ1 as a suspension in Tang, and both plasma and liver
MK-4882 was found to be highly potent against both geno-
type 1a and 1b, with EC50 values in the low-picomolar range
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A single dose of MK-4882 was
orally administered at 1 mgkgꢀ1
as a suspension in Tang to three
chronically infected chimpan-
zees. Two carried high viral load
infections (~106 IUmLꢀ1) of GT1a
or GT1b. The third had a GT1a
viral load of 104 IUmLꢀ1 that was
homogeneous for the NS3 pro-
tease R155K mutation.
Scheme 2. Synthesis of MK-4882 (22). Reagents and conditions: a) NBS, HBr, CCl4; b) 5-bromosalicylaldehyde,
Cs2CO3, DMF, reflux; c) bis(pinacolato)diboron, KOAc, Pd(dppf)Cl2, dioxane, reflux; d) 1. 22b, Na2CO3, Pd(dppf)Cl2,
THF, H2O, reflux, 2. HCl, MeOH; e) N-Moc-l-valine, BOP, DIPEA, DMF.
All three animals responded
rapidly after the single dose;
viral load was suppressed an
average 2.15 log units within
12 h, with continued suppres-
Table 6. MK-4882 in vitro potency profile.
sion to an average nadir of 2.91 log units at 48 h before re-
bounding. Initial 12 h viral load decreases were similar for both
the GT1a and GT1b infections, but an additional one-log sup-
pression was observed with the GT1b infection by 24 h. Sup-
pression of the GT1a infection was maintained through this
time but did not increase further (Figure 2).
Genotype
EC50 [nm]
Genotype
EC50 [nm]
1a WT
0.009ꢂ0.006
7.6ꢂ1.1
1b WT
0.001ꢂ0.001
0.003ꢂ0.001
0.39ꢂ0.24
0.004ꢂ0.001
3.8ꢂ1.8
1a Q30R
1a L31V
1a Y93C
1a Y93H
2a WT
1b R30Q
1b L31V
1b Y93C
1b Y93H
2b(JFH)[a]
4a(con1)[a]
4.4ꢂ3.5
6.6ꢂ2.6
21ꢂ3.6
0.04ꢂ0.01
4.4ꢂ0.76
286
3a(con1)[a]
0.31ꢂ0.07
[a] Chimeric replicons with indicated NS5A genotype cloned into GT1b
(con) or GT2a (JFH) background; see ref. [12] for assay details; values are
the means ꢂSD of n>3 experiments.
levels were determined. Twelve-hour average plasma and liver
concentrations were 0.19 and 1.3 mm, respectively. Total free
drug concentration in plasma at C24h (~3 nm) was greater than
the genotype 1b wild-type and genotype 1b mutant EC50
values (Table 7).
Figure 2. MK-4882 single-dose efficacy study: MK-4882 was dosed orally at
1 mgkgꢀ1 as a suspension in Tang to chronic-HCV-infected chimpanzees har-
boring GT1a, GT1b, or GT1a NS3 R155K infections. Blood samples were col-
lected periodically, processed to plasma, and evaluated for HCV viral load.
Pharmacological activity
Both single-dose and multiple-dose studies in chimpanzees
chronically infected with HCV were conducted to determine
the antiviral efficacy of MK-4882. Study protocols were re-
viewed and approved by the Institutional Animal Care and Use
Committee at both Merck Research Laboratories and at the
New Iberia Research Center (University of Louisiana at Lafay-
ette), where the experiments were conducted, to ensure com-
pliance with all federal regulations.
Plasma concentrations of MK-4882 in this study were similar
to those found in the satellite study and ranged from 0.06 to
0.17 mm at 12 h, diminishing approximately by half at 24 h.
Drug was cleared from plasma and was below the level of
quantification (LOQ ~25 nm) by 48 h. The potency of the drug
was sufficient to maintain viral suppression through at least
48 h. Drug concentration in the
liver, as determined from liver
biopsy samples, ranged be-
Table 7. MK-4882 pharmacokinetics.[a]
tween 0.77 and 1.56 mm at 12 h.
The results are consistent with
the satellite PK study in unin-
fected chimps.
Species
Cl [mLminꢀ1 kgꢀ1
]
t1/2 [h]
p.o. Cmax [mm]
p.o. AUC [mmhꢀ1
]
F [%]
rat
dog
rhesus
chimp
9.3ꢂ0.4
6.4ꢂ4.0
9.1
2.0ꢂ0.1
4.7ꢂ0.5
3.0
0.31ꢂ0.11
0.19ꢂ0.03
0.18
1.74ꢂ0.27
1.06ꢂ0.06
1.44
38
26
12
Resistance analysis was con-
ducted by population sequenc-
ing of the NS5A gene of viral
RNA isolated from serially col-
lected plasma samples. The
ND
ND
0.45
5.1
ND
[a] Rat, dog, monkey i.v. (1 mgkgꢀ1, n=3, 30% captisol + 2 equiv HCl); rat p.o. (2 mgkgꢀ1, n=3, 0.5% methyl-
cellulose); dog p.o. (1 mgkgꢀ1, n=3, 0.5% methylcellulose); rhesus p.o. (5 mgkgꢀ1, n=2, 0.5% methylcellu-
lose); chimpanzee p.o. (1 mgkgꢀ1, n=2, Tang); ND: not determined.
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GT1b-infected chimp became homogenous for the Y93H muta-
tion after 24 h (sequence could not be generated for the 12 h
time point, as no additional sample was available at this time).
Viral load was further suppressed another 0.6 log units by
48 h, which suggests suppression of mutant virus. Early re-
bound virus at day 4 was also homogenous for Y93H, but wild-
type virus became the predominant population by day 7. An
additional K44R polymorphism, co-encoded with the Y93H
virus, was no longer observed at day 10 upon re-emergence of
wild-type virus, suggesting that mutant and wild-type virus are
two distinct populations. Sequencing of a sample collected on
day 28 (four weeks post-dose) showed the emergence of
a new mixture of L31V/L virus. A similar late emergence of ap-
parently distinct resistant virus was also observed in the GT1a
(wild-type) infected chimpanzee (see below). The reason for
these phenomena is currently not understood, and the timing
of the emergence of L31L/V virus cannot be further pinpoint-
ed, as plasma samples were not collected between days 10
and 28.
Figure 3. Resistance analysis of the MK-4882 seven-day efficacy study in
GT1a- and GT1b-infected chimpanzees.
during dosing, although viral load was still suppressed greater
than 1 log from pre-dose levels. Sequence analysis showed
that at day 6 virus collected from this animal was heteroge-
nous for both Q30R and L31M/L. A similar viral mixture was
observed at day 10 (three days post-dosing). Eventually wild-
type virus emerged as the principle population (data not
shown).
Rebound virus from the GT1a (wild-type)-infected chimpan-
zee was heterogenous for both Q30E and Y93H. By day 7,
Y93H was the only mutation detectable, and as a mixed popu-
lation with wild-type virus. By day 10 this evolved to a mixed
population of Y93C and wild-type virus. The shift from Y93H to
Y93C coincides with diminishing drug plasma levels and is con-
sistent with the greater loss of potency observed with Y93H
than Y93C in vitro. However, by day 28 virus evolved further to
an L31M/V mixture. For both the GT1a and 1b infections, wild-
type eventually re-emerged as the principle viral population
(data not shown).
New analogues with improved potency profiles against
resistant mutants
The viral breakthrough evidenced in the preclinical proof-of-
concept efficacy studies for MK-4882 necessitated the design
of newer NS5A inhibitors which had improved virologic pro-
files against the various genotypes and resistance mutations.
SAR of the benzofuran core structure suggested that the intro-
duction of a cyclic constraint could result in more potent in-
hibitors. The initial strategy involved linking the C3 benzofuran
carbon to the C2’ phenyl carbon to give tetracyclic core struc-
tures 27 and 28 (mode a; Figure 4). Evaluation of these com-
pounds in the replicon assay showed that these modifications
resulted in a loss in potency versus the wild-type forms of
GT1a and 1b as well as the key mutants L31V and Y93H.
For the chimpanzee encoding the GT1a NS3 R155K infection,
L31M/L was detected as a mixed population at day 10, and
only wild-type virus was detected on day 28.
Although MK-4882 exhibited a robust virologic response
after a single 1 mgkgꢀ1 dose, the emergence of mutant virus
in the rebound phase warranted further evaluation of efficacy
following multi-dose administration. Two different HCV-infect-
ed chimpanzees (GT1a and GT1b), both treatment-na¨ıve to
NS5A inhibitors, were dosed orally at 1 mgkgꢀ1 once daily for
seven days. The pharmacodynamic responses are shown in
Figure 3. Liver biopsies were collected 12 h following the final
dose; drug concentrations in liver were 7–15-fold higher than
plasma levels, consistent with the findings in the Sprague–
Dawley study and suggests that MK-4882 is not selectively re-
tained in liver tissue.
The results from the study showed a rapid and robust re-
sponse immediately following the initial dose, with an average
maximal decrease in viral load by ~3.5 log units. The GT1b-in-
fected chimpanzee showed a further decline in viral titer
through the duration of the study, reaching a maximal 3.8-log
suppression of virus by day 7 before rebounding. Virus was
mixed Y93H/Y population at day 10 (three days post-dosing),
and eventually became homogenous for wild-type (data not
shown).
Although the initial response in the GT1a-infected chimpan-
zee was robust, viral breakthrough was noted beginning at
day 2. This eventually led to a 2-log increase in viral titer
Figure 4. Design strategy for second-generation NS5A inhibitors.
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An alternative mode of cyclization (mode b) was examined
and was made possible by converting the benzofuran core to
an indole scaffold which allowed cyclization from the indole ni-
trogen to the phenyl C2’ carbon through either an ethylene
bridge (29) or an oxygen-containing bridge (30). These modifi-
cations afforded inhibitors that possessed a tetracyclic indole
scaffold which proved to be equipotent to MK-4882 versus
GT1a and GT1b wild-type replicon systems. Further evaluation
showed that the GT1a Y93H potency was weakened with the
carbon analogue 29 (EC50 =170 nm). This activity was im-
proved by incorporating an oxygen atom in the two-atom
bridge of compound 30 (Y=O; GT1a Y93H EC50 =5 nm). Note-
worthy is the fact that the aminal linkage was extremely stable
to hydrolytic cleavage even under forcing conditions.
Figure 5. Dimethyl ((2S,2’S)-((2S,2’S)-2,2’-(5,5’-((S)-6-phenyl-6H-benzo[5,6]-
[1,3]oxazino[3,4-a]indole-3,10-diyl)bis(1H-imidazole-5,2-diyl))bis-(pyrrolidine-
2,1-diyl))bis(3-methyl-1-oxobutane-2,1-diyl))dicarbamate (MK-8742).
Despite having an improved virologic profile, the unique tet-
racyclic indole-containing compound 30 proved to be cytotox-
ic in the low-micromolar range (CC50 ~1 mm). SAR analysis
showed that the addition of a phenyl substituent at C6 abro-
gated the cytotoxicity. Chiral SFC separation of the two diaste-
reomers afforded the (S)-phenyl compound 31 and its R-con-
figured epimer 32. While the virologic profiles of the two dia-
stereomers showed equivalent potency values in the wild-type
replicons, compound 31 proved to be 25-fold more potent
versus the GT1a Y93H mutant. After extensive profiling, com-
pound 31, renamed MK-8742, supplanted MK-4882 as our lead
clinical compound (Table 8).
Table 9. MK-8742 in vitro potency profile GT1-4 and key GT1 mutants.
Genotype
EC50 ꢂSD [nm]
Genotype
EC50 ꢂSD [nm]
1a WT
0.004ꢂ0.002
0.03ꢂ0.002
0.5ꢂ0.5
1b WT
0.003ꢂ0.001
0.004ꢂ0.001
0.009ꢂ0.003
0.05ꢂ0.02
1a Q30H
1a Q30R
1a L31F
1a L31V
1a Y93C
1a Y93H
2a WT
1b L28V
1b R30Q
1b L31F
1b L31V
1b Y93C
1b Y93H
2b(JFH)[b]
4a(con1)[b]
0.08ꢂ0.04
0.5ꢂ0.3
0.01ꢂ0.01
0.2ꢂ0.07
2.4ꢂ1.3
0.005ꢂ0.001
0.05ꢂ0.03
0.003ꢂ0.001
0.03ꢂ0.01
3.4ꢂ2.6
3a(con1)[b]
0.003ꢂ0.001
[a] SD is calculated from n>3 independent experiments. [b] Chimeric re-
plicons with indicated NS5A genotype cloned into GT1b (con1) or GT2a
(JFH) background; see ref. [12] for assay details.
MK-8742 was found to be highly potent against most HCV
genotypes tested with EC50 values in the low-picomolar range
and modest potency shifts in the presence of 40% NHS
(Figure 5).
MK-8742 maintained significant potency against most of the
NS5A mutations in the screening panel and showed (on aver-
age) an order of magnitude improvement relative to MK-4882.
MK-8742 potencies against the key L31V and Y93H mutants in
the GT1b background were 0.01 and 0.05 nm, respectively,
while in the genotype 1a background they were 0.5 and 2 nm.
MK-8742 also demonstrated a favorable genotypic virologic
profile (Table 9). The decreased potency in the GT2b cell line is
attributed to the presence of methionine residue at position
31 of NS5A versus a leucine present in GT2a.[11]
longer (4–16 h) than the effective t1/2, suggesting rate-limited
return from tissue compartments. The oral bioavailability was
low to moderate (9–35%) for all three species. The low to
moderate bioavailability is likely due to limited absorption
which is consistent with low passive permeability in MDCKII
cells of 47 nmsꢀ1. Preclinical modeling of MK-8742 suggested
a high potential for low-dose once-daily dosing in the clinic
(Table 10).
The first-generation synthesis of MK-8742 proceeded in
eight linear steps (12 total steps) from commercially available
materials with a 3% non-optimized yield from the longest
linear sequence (Scheme 3). An improved synthesis which sup-
ported early-stage clinical trials has been developed and will
be reported elsewhere. Thus, the C2’ phenylindole intermedi-
ate 31a was prepared by starting from 5-bromoacetophenone
and p-bromophenylhydrazine using well-established
two-step Fisher indole conditions. The indole NH
group was cyclized onto the C2’ phenolic group
using standard alkylating conditions to give the race-
The pharmacokinetics of MK-8742 was studied in Wistar Han
rats, beagle dogs and cynomolgus monkey. The i.v. clearance
was moderate and constituted ~14–29% of hepatic blood flow
in all three species. The Vdss was moderate, and the effective
half-life was also moderate (2.5–5.9 h). The terminal t1/2 was
Table 8. SAR for tetracyclic inhibitors.[a]
mic tetracyclic scaffold 31b in good overall yield. The
dibromide intermediate was subsequently converted
into the corresponding pinacolboronate ester 31c
using standard procedures.
Compd
Genotype, EC50 [nm]
CC50 [mm]
1b
2a
1a
1aL31V
1aY93H
22
29
30
31
32
0.003
0.02
0.001
0.003
0.005
0.04
0.90
0.16
0.003
0.010
0.01
6
24
170
5
2.4
60
9
1
1
0.002
0.002
0.004
0.002
ND
2
0.5
ND
Boronate ester 31c was then coupled with two
equivalents of the heterocyclic bromide 22b in the
presence of a catalytic amount of Pd(dppf)Cl2 fol-
lowed by work-up and deprotection of the Boc
>25
>25
[a] Nꢁ3; ND: not determined.
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ly relevant resistance variants.
MK-8742 exhibits antiviral activi-
ty against several HCV geno-
types (including 1a, 1b, 2a, 3a,
4a) with decreased potency
against genotype 2b. Based on
its preclinical characterization,
this clinical candidate has the
potential to retain significant ac-
tivity against many resistant HCV
mutants in both the GT1a and
GT1b backgrounds. MK-8742 is
currently being evaluated as
Table 10. Preclinical pharmacokinetics of MK-8742.
Species
Cl [mLminꢀ1 kgꢀ1
]
t1/2 [h]
p.o. Cmax [mm]
p.o. AUC [mmhꢀ1
]
F [%]
rat[a]
24ꢂ8.0
8.4ꢂ2
4.2ꢂ1.0
7.7ꢂ2.0
16ꢂ4.0
0.36ꢂ0.3
0.29ꢂ0.02
0.1ꢂ0.04
2.3ꢂ1.0
1.7ꢂ0.3
1.2ꢂ0.4
~9
~35
~17
dog[b]
monkey[b]
5.2ꢂ0.3
[a] 5 mgkgꢀ1 i.v. (3% DMA in 40% HPbCD; 30 mgkgꢀ1 p.o. (0.4% HPMC in water). [b] 1 mgkgꢀ1 i.v. (20%
HPbCD; 2 mgkgꢀ1 p.o. (10% T80/90% PEG400).
a
component of an all-oral
direct-acting antiviral treatment
for the cure of HCV. Results from
early clinical studies in both
healthy volunteers and HCV-in-
fected patients indicate that the
favorable preclinical profile of
MK-8742 translates into a clinical-
ly efficacious drug with broad
activity and pharmacokinetics
suggestive of moderate once-
daily dosing.
Scheme 3. Synthesis of MK-8742 (31). Reagents and conditions: a) 1. p-bromophenylhydrazine, AcOH, EtOH, reflux,
2. PPA, 1108C; b) a,a-dibromotoluene, K2CO3, DMF, 1008C; c) bis(pinacolato)diboron, KOAc, Pd(dppf)Cl2, dioxane,
reflux; d) 1. 22b, Na2CO3, Pd(dppf)Cl2, THF, H2O, reflux, 2. HCl, MeOH; e) N-Moc-l-valine, BOP, DIPEA, DMF, then
chiral SFC.
groups provided the desired compound 31d in 57% yield.
Experimental Section
Amide coupling with N-methoxycarbonyl-l-valine afforded
compound 31 as a mixture of diastereomers which were easily
separated by SFC chromatography. MK-8742 was the second
diastereomer to elute and was determined to be the S,S,S,S,S
diastereomer by single-compound X-ray analysis. An enantiose-
lective synthesis of the tetracyclic scaffold has since been es-
tablished and will be reported elsewhere.
Biology
All animal studies were performed according to the NIH Guide for
the Care and Use of Laboratory Animals, and experimental proto-
cols were approved by the Merck Institutional Animal Care and
Use Committee. The HCV genotype was determined by a line
probe assay (Versant HCV genotype assay, LiPa, Bayer Diagnostics/
Innogenetics) and confirmed by RT-PCR rescue and sequencing of
HCV genetic material.[12] HCV-infected chimpanzees were dosed
orally at 1 mgkgꢀ1 for a single dose or q.d. for seven days by the
voluntary ingestion of MK-4882 (in a Tang vehicle). Viral load deter-
minations were performed on plasma samples using the HCV
TaqMan assay (Cenetron Diagnostics, Austin, TX, USA). MK-4882
drug concentrations in plasma or liver biopsy specimens were con-
ducted as described above (under pharmacokinetic studies). Viral
resistance analysis of chimpanzee plasma samples was conducted
similarly to a previously published protocol using NS5A-specific pri-
mers.[13] Primary screening assays were conducted against gt1b,
gt2a, or gt1a replicon cell lines using a luciferase-based reporter
assay.[14] EC50 determinations against the panel of genotype or
mutant replicon cell lines were conducted using a TaqMan-based
assay.[15] The NS5A sequences for the genotypes are GT1a (H77),
GT1b (con1), GT2a (JFH), GT2b (AB030907), GT3a (NC009824), and
GT4a (DQ418742).
Conclusions
A concerted research effort that combined components from
both internal and literature compounds led to a series of ben-
zofuran-based NS5A structures which exhibited good potency
versus genotype 1b. A detailed medicinal chemistry effort was
undertaken and included the exploration of various amide
bond isosteres. This work ultimately led to the incorporation of
two imidazole subunits and afforded compounds with sub-
nanomolar EC50 values against genotypes 1a and 2a. Further
optimization of this series using an expanded panel of HCV
genotypes and clinically relevant NS5A resistant mutant strains
led to the discovery of the early lead compound MK-4882
which showed efficacy in a non-human primate model at
a moderate dose. Viral breakthrough with the genotype 1a in-
fected chimpanzee, however, focused attention on developing
analogues that were more potent against resistant variants
while maintaining or improving the broad genotype profile.
Strategic incorporation of a cyclic constraint and further op-
timization led to the discovery of MK-8742, a tetracyclic indole-
based analogue of MK-4882 which showed both a broad geno-
typic potency profile and an increased potency against clinical-
Chemistry
Solvents, reagents, and intermediates that are commercially avail-
able were used as received. Reagents and intermediates that were
not commercially available were prepared in the manner as de-
scribed below. 1H NMR spectra were obtained on a Varian VNMR
System 400 (400 MHz) and are reported as ppm downfield from
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Me4Si with number of protons, multiplicities, and coupling con-
stants in Hz indicated parenthetically. Where LC–MS data are pre-
sented, analyses was performed using an Agilent 6110A MSD or an
Applied Biosystems API-100 mass spectrometer, and the parent ion
is given. Flash column chromatography was performed using pre-
packed normal-phase silica from Biotage, Inc. or bulk silica from
Fisher Scientific. Unless otherwise indicated, column chromatogra-
phy was performed using a gradient elution of hexanes/ethyl ace-
tate, from 100% hexanes to 100% ethyl acetate.
7.42 (d, J=8.6 Hz, 2H), 5.28 (s, 1H), 4.24 (t, J=7.3 Hz, 2H),
1.28 ppm (d, J=7.1 Hz, 3H); 13C NMR (400 MHz, CDCl3): d=168.1,
135.1, 135.1, 132.2, 132.2, 130.5, 123.7, 62.9, 45.9, 14.1 ppm.
5-Bromo-2-(4-bromophenyl)benzofuran (22d): To a solution of
22c (2.0 g, 6.2 mmol) in DMF (20 mL) was added 5-bromosalicylal-
dehyde (1.2 g, 6.0 mmol) and Cs2CO3 (2.0 g, 12.3 mmol) under N2
protection. The resulting suspension was stirred for 5 h at 1608C,
cooled and treated with water. The resulting precipitate was fil-
tered, and the filtrate cake was dried to give the desired com-
pound 22d as a white solid which was used directly in next step.
Rf =0.57 (hexane); 1H NMR: (400 MHz, CDCl3): d=7.69–7.71 (m,
3H), 7.58 (d, J=8.6 Hz, 2H), 7.35 (m, 2H), 6.95 ppm (s, 1H).
Procedure for the synthesis of MK-4882 (22)
(S)-tert-Butyl-2-(1H-imidazol-2-yl)pyrrolidine-1-carboxylate (22a):
To (S)-Boc-prolinal (80.0 g, 0.40 mol) was added a solution of am-
monia in MeOH (prepared from 150 mL of 7n NH3/MeOH and
200 mL MeOH, 1.05 mol, 260 mol%). An exotherm was noted with
the internal temperature rising to ~308C. The solution was allowed
to stir for 0.5 h at ambient temperature, then glyoxal (76 g,
0.52 mol) was added over 5 min in portions, with the internal tem-
perature rising to ~608C and then returning to room temperature
after 1 h. The reaction was allowed to stir for an additional 15 h,
and the reaction mixture was concentrated. The resulting residue
was diluted with CH2Cl2 (1 L) and H2O (0.5 L) was added, and the
organic phase was washed with H2O (0.25 L), dried over MgSO4, fil-
tered and concentrated. The residue obtained was slurried with
warm EtOAc (~100 mL) and hexane (100 mL), then was cooled and
filtered. The solid obtained was washed with 30% EtOAc/hexane
4,4,5,5-Tetramethyl-2-(4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)benzofuran-2-yl)phenyl)-1,3,2-dioxaborolane (22e):
A
suspension of 22d (4.43 g, 12.58 mmol), bis(pincolato)diboron
(8.31 g, 32.72 mmol), KOAc (3.72 g, 37.7 mmol) and Pd(dppf)Cl2
(921 mg, 1.26 mmol) in dioxane (100 mL) was heated at reflux for
4 h under a nitrogen atmosphere. The mixture was cooled and
concentrated and the residue was partitioned between H2O and
CH2Cl2. The aqueous phase was extracted with CH2Cl2 (20 mLꢁ3),
and the combined organic extracts were washed with brine, dried
over Na2SO4 then concentrated. The residue was purified by chro-
matography on silica gel (EtOAc/hexanes 1:4) to afford the desired
1
compound (5.0 g, 85%). H NMR: (400 MHz, CDCl3): d=8.12 (s, 1H),
7.86–7.94 (m, 4H), 7.79 (d, J=8.4 Hz, 1H), 7.55 (d, J=8.4 Hz, 1H),
7.12 (s, 1H), 1.41 (s, 12H), 1.39 ppm (s, 12H).
1
to provide compound 22a (66.2 g, 70% yield). H NMR (400 MHz,
[D6]DMSO): d=11.6 (brs, 1H), 6.86 (s, 2H), 4.76 (m, 1H), 3.48 (m,
1H), 3.35–3.29 (m, 1H), 2.23–1.73 (m, 4H), 1.15 ppm (s, 9H);
13C NMR (400 MHz, [D6]DMSO): d=154.4, 154.0, 150.5, 149.7, 135.8,
79.1, 78.7, 55.7, 55.1, 47.1, 46.8, 33.9, 32.2, 28.8, 28.5, 24.4,
23.6 ppm; MS (ESI) m/z 238 [M+H]+.
2-((S)-Pyrrolidin-2-yl)-5-(4-(5-(2-((S)-pyrrolidin-2-yl)-1H-imidazol-
5-yl)benzofuran-2-yl)phenyl)-1H-imidazole (22 f): A suspension of
bromoimidazole 22b (1.58 g, 5.0 mmol), boronate ester 22e
(892 mg, 2.0 mmol), Pd(dppf)Cl2 (146 mg, 0.20 mmol), and Na2CO3
(636 mg, 6.0 mmol) were held at reflux in THF/H2O (10:1, 33 mL)
overnight under N2 protection. The mixture was cooled and fil-
tered. The filtrate was washed with water (50 mL) then extracted
with EtOAc (100 mL). The organic phase was removed and washed
with brine (20 mL) then dried over anhydrous Na2SO4. The solution
was concentrated, and the resulting residue was purified by
column chromatography (petroleum ether/EtOAc=8:1!5:1) to
afford the desired compound. MS (ESI) m/z 641 [M+H]+. The prod-
uct was added into 3m HCl/CH3OH (20 mL) and the mixture was
stirred at RT for 3 h. The mixture was concentrated and the crude
product was used directly in the next step without further purifica-
tion. 1H NMR (400 MHz, [D6]DMSO): d=10.5 (brs, 1H), 10.1 (brs,
1H), 8.3 (s, 1H), 8.25 (m, 2H), 8.05–8.15 (m, 5H), 7.92 (dd, J=1.8,
6.8 Hz), 7.81 (d, J=6.8 Hz, 7.64 (s, 1H), 5.12 (t, J=8.4 Hz, 1H), 5.09
(t, J=8.3 Hz, 1H), 3.35–3.53 (m, 4H), 2.53–2.61 (m, 6H), 2.19–
2.29 ppm (m, 2H); 13C NMR (400 MHz, [D6]DMSO): d=156.6, 155.0,
143.0, 142.2, 135.0, 130.1, 129.9, 126.4, 126.1, 123.7, 123.4, 118.9,
117.6, 116.4, 112.6, 103.5, 53.4, 53.0, 46.2, 46.1, 30.2, 30.1, 24.8,
24.7 ppm; MS (ESI) m/z 441 [M+H]+.
(S)-tert-Butyl-2-(4-bromo-1H-imidazol-2-yl)pyrrolidine-1-carbox-
ylate (22b): To a suspension of compound 22a (140 g, 0.59 mol) in
THF (2000 mL) was added N-bromosuccinimide (200 g, 1.1 mol).
The mixture was allowed to stir at ambient temperature under N2
gas for ~15 h. The solvent was then removed in vacuo, and the
residue obtained was purified by silica gel chromatography (EtOAc
eluent) to provide 230 g of the desired 4,5-dibromoimidazole ana-
1
logue. Rf =0.58 (EtOAc/hexanes 2:3); H NMR (400 MHz, CDCl3): d=
10.8 (brs, 1H), 4.74 (s, 1H), 3.38 (s, 2H), 2.81 (m, 1H), 2.1–2.2 (m,
2H), 1.92 (m, 1H), 1.49 ppm (s, 9H); MS (ESI) m/z 396 [M+H]+. The
compound (230 g, 0.58 mol) was suspended in EtOH/H2O (1:1 ratio,
3000 mL) and 733 g (5.80 mol). Na2SO3 was added. The resulting
mixture was allowed to stir at mild reflux for ~15 h. After cooling
to room temperature, the mixture was extracted with CH2Cl2 twice,
and the combined organic washings were concentrated to afford
a semi-solid. The residue obtained was purified using chromatogra-
phy on silica gel to provide 22b (131 g, 70%). Rf =0.47 (EtOAc/hex-
anes 2:3); 1H NMR (400 MHz, [D6]DMSO): d=12.12 (brs, 1H), 7.10
(m, 1H), 4.70 (m, 1H), 3.31 (m, 1H; overlapped with water signal),
2.25–1.73 (m, 4H), 1.39/1.17 ppm (s, 3.8H + 5.2H); 13C NMR
(400 MHz, [D6]DMSO): d=154.4, 153.9, 151.0, 150.3, 115.8, 115.4,
113.3, 79.4, 78.9, 55.6, 55.1, 47.1, 46.9, 33.9, 32.3, 28.8, 28.5, 24.4,
23.6 ppm; MS (ESI) m/z 317 [M+H]+.
MK-4882 (22): To a mixture of proline analogue 22 f (440 mg,
1.0 mmol), N-Moc-l-valine (367 mg, 2.1 mmol) and DIPEA (0.4 mL)
in DMF (3 mL) was added BOP reagent (968 mg, 2.2 mmol). The re-
sulting mixture was stirred at RT for 16 h. The solution was subject-
ed directly to reversed-phase HPLC to afford 405 mg (52%) of the
desired compound 22; mp: 130–1318C; 1H NMR (400 MHz,
CD3OD): d=7.7–8.1 (m, 10H), 7.4 (m, 1H), 5.3 (m, 2H), 4.3 (m, 2H),
4.1 (d, J=4.8 Hz, 2H), 3.9 (m, 2H), 3.7 (m, 6H), 2.6 (d, J=4.8 Hz,
2H), 2.0–2.4 (m, 8H), 1.3–1.4 (m, 2H), 0.9–1.0 ppm (m, 12H);
13C NMR (400 MHz, [D6]DMSO): d=170.9, 157.3, 156.1, 153.5, 149.9,
149.4, 139.9, 139.1, 129.7, 127.6, 125.2, 125.0, 116.4, 113.6, 112.1,
Ethyl 2-bromo-2-(4-bromophenyl)acetate (22c): To a stirred solu-
tion of ethyl 4-bromophenylacetate (50.0 g, 205.8 mmol) in CCl4
(500 mL) was added NBS (38.0 g, 214.7 mmol) then 48% aqueous
HBr (4 drops). After the addition, the solution was stirred overnight
at 808C under argon. The reaction was cooled, filtered, and con-
centrated. The resulting oil was directly used the next step without
purification. 1H NMR (400 MHz, CDCl3): d=7.49 (d, J=8.4 Hz, 2H),
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111.2, 101.8, 58.5, 54.7, 51.9, 31.3, 30.3, 24.7, 19.5, 19.0 ppm; MS
N2. The reaction mixture was cooled to room temperature, filtered,
and the filtrate was washed with water (50 mL) then extracted
with EtOAc (100 mL). The organic extract was washed with brine,
dried over anhydrous Na2SO4, filtered and concentrated. The result-
ing residue was purified using column chromatography on silica
gel (EtOAc) to provide 31d (460 mg). MS (ESI) m/z 768 [M+H]+.
The compound was dissolved in 5 mL of MeOH and added to HCl/
CH3OH (5 mL, 3m). The resulting reaction was allowed to stir at
room temperature for 3 h then concentrated to provide the amine
salt (409 mg, 57%), which was used without further purification.
1H NMR (400 MHz, [D6]DMSO): d=11.9 (brs, 1H), 11.1 (brs, 1H), 7.9
(s, 1H), 7.75 (m, 1H), 7.69 (s, 1H), 7.33–7.65 (m, 4H), 7.22–7.30 (m,
4H), 6.93–7.05 (m, 3H), 4.72 (m, 1H), 3.75 (m, 2H), 2.09–2.19 (m,
2H), 1.84–2.02 (m, 4H), 1.0–1.15 ppm (m, 6H); MS (ESI) m/z 568
[M+H]+.
(ESI) m/z 780 [M+H]+.
Procedure for the synthesis of MK-8742
5-Bromo-2-(5-bromo-1H-indol-2-yl)phenol (31a): A mixture of 5’-
bromo-2’-hydroxyacetophenone (4.2 g, 20 mmol) and 4-bromo-
phenyl hydrazine hydrochloride (4.4 g, 20 mmol) in AcOH and
EtOH (1:10, 100 mL) was heated at reflux and allowed to stir at this
temperature for 6 h. The reaction mixture was cooled to room
temperature and concentrated to provide 7.2 g (94%) of the de-
sired hydrazone as a solid. The hydrazone was subsequently cov-
ered in PPA and heated at 808C for 2 h. The reaction mixture was
cooled to room temperature and poured into iced water. The re-
sulting solution was extracted with CH2Cl2 and the organic extract
was washed with brine, dried over Na2SO4, filtered and concentrat-
ed. The residue was purified using column chromatography (2:3
EtOAc/hexanes) to provide 31a (4.8 g, 65%). Rf =0.70 (EtOAc/hex-
anes 2:3); 1H NMR (400 MHz, [D6]DMSO): d=11.3 (s, 1H), 10.7 (s,
1H), 7.70 (m, 2H), 7.42 (d, J=8.6 Hz, 1H), 7.19 (m, 2H), 7.11 (dd,
J=2.0, 8.4 Hz, 1H), 6.98 ppm (d, J=2.0 Hz, 1H); 13C NMR (400 MHz,
[D6]DMSO): d=154.2, 134.5, 133.9, 128.9, 127.9, 122.5, 121.1, 120.7,
119.4, 117.8, 116.7, 112.1, 110.4, 99.8 ppm; MS (ESI) m/z 368
[M+H]+.
MK-8742 (31): To a solution of 31d (300 mg, 0.53 mmol), N-me-
thoxycarbonyl-l-valine (185 mg, 1.05 mmol) and DIPEA (0.7 mL,
4.2 mmol) in CH3CN (5 mL) was added BOP reagent (515 mg,
1.1 mmol). The resulting reaction was allowed to stir at room tem-
perature and monitored by LC–MS. After LC–MS showed the start-
ing material to be consumed, the reaction mixture was filtered and
the filtrate was purified by reversed-phase HPLC to provide crude
compound 31 as a mixture of diastereomers (300 mg). 1H NMR
(400 MHz, CD3OD): d=7.94 (s, 1H), 7.85 (d, J=8.0 Hz, 1H), 7.74 (s,
1H), 7.63 (s, 1H), 7.48 (s, 1H), 7.35–7.37 (m, 2H), 7.31 (s, 1H), 7.17–
7.18 (m, 4H), 7.11 (s, 1H), 6.96–6.98 (d, J=7.6 Hz, 2H), 5.09–5.17
(m, 2H), 4.13 (t, J=8.0 Hz, 2H), 3.99 (brs, 2H), 3.78 (brs, 2H), 3.56
(s, 6H), 2.44–2.47 (m, 2H), 1.92–2.19 (m, 8H), 0.77–0.85 ppm (m,
12H); MS (ESI) m/z 882 [M+H]+. The diastereomers were separated
on a chiral SFC column to afford compound 31 (140 mg, 30%) and
the C6 epimer 32 (145 mg, 31%):
3,10-Dibromo-6-phenyl-6H-benzo[5,6][1,3]oxazino[3,4-a]indole
(31b): A mixture of 31a (1.1 g, 3.0 mmol), a,a-dibromotoluene
(2.25 g, 9.0 mmol) and K2CO3 (1.2 g, 9.0 mmol) in 15 mL of DMF
was heated at 1008C and allowed to stir at this temperature for
3 h. The reaction mixture was cooled to room temperature, con-
centrated, and the residue obtained was dissolved with CH2Cl2 and
water. The aqueous phase was extracted with 3ꢁ20 mL of CH2Cl2.
The combined organic extracts were washed with brine, dried over
Na2SO4, filtered and concentrated. The resulting residue was puri-
fied using flash column chromatography on silica gel to provide
31b (830 mg, 61%) as a white solid. Rf =0.65 (EtOAc/hexanes
5:95); 1H NMR (400 MHz, CDCl3): d=7.72 (brs, 1H), 7.44–7.46 (d,
J=8.4 Hz, 1H), 7.21–7.28 (m, 3H), 7.09–7.12 (m, 3H), 7.04 (s, 1H),
6.99–7.01 (brs, J=6.8 Hz, 2H), 6.78 (s, 1H), 6.63–6.65 ppm (d, J=
8.4 Hz, 1H); 13C NMR (400 MHz, CDCl3): d=150.2, 136.4, 134.1,
132.3, 130.9, 130.1, 129.2, 127.1, 126.6, 125.8, 125.3, 123.6, 122.6,
121.5, 117.2, 114.3, 111.4, 97.0, 84.7 ppm; MS (ESI) m/z 456 [M+H]+.
MK-8742 (31): mp: 160–1618C; Rf =0.46 (MeOH/CH2Cl2 1:9);
1H NMR (400 MHz, CD3OD): d=7.90 (s, 1H), 7.81–7.83 (m, 1H), 7.72
(s, 1H), 7.62 (s, 1H), 7.45 (s, 1H), 7.14–7.33 (m, 6H), 7.09 (s, 1H),
6.93–6.95 (m, 2H), 5.06–5.14 (m, 2H), 3.71–4.11 (m, 6H), 3.52 (s,
6H), 2.41–2.44 (m, 2H), 1.90–2.15 (m, 8H), 0.74–0.86 ppm (m, 12H);
13C NMR (400 MHz, [D6]DMSO): d=171.0, 157.4, 149.6, 138.3, 134.6,
129.8, 129.5, 127.0, 116.2, 83.5, 58.6, 54.9, 52.1, 47.5, 31.6, 30.5,
24.9, 19.6, 19.2 ppm; MS (ESI) m/z 882 [M+H]+.
1
MK-8742 diastereomer (32): Rf =0.46 (MeOH/CH2Cl2 1:9); H NMR
(400 MHz, CD3OD): d=8.08 (s, 1H), 7.91–7.93 (m, 1H), 7.72 (s, 1H),
7.56 (s, 1H), 7.24–7.43 (m, 7H), 7.19 (s, 1H), 7.03–7.05 (m, 2H),
5.16–5.24 (m, 2H), 3.81–4.21 (m, 6H), 3.62 (s, 6H), 2.52–2.54 (m,
2H), 2.00–2.25 (m, 8H), 0.84–0.91 ppm (m, 12H); MS (ESI) m/z 882
[M+H]+.
6-Phenyl-3,10-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
6H-benzo[5,6][1,3]oxazino[3,4-a]indole (31c): To a solution of
compound 31b (456 mg, 1.0 mmol) in 1,4-dioxane was added bis(-
pinacolato)diboron (559 mg, 2.2 mmol), Pd(dppf)Cl2 (0.04 mmol),
and KOAc (392 mg, 4.0 mmol). The reaction mixture was heated at
110 8C under a blanket of N2 for 3 h. The reaction mixture was
cooled to room temperature, concentrated, and the residue ob-
tained was purified using column chromatography on silica gel
(7:3 EtOAc/hexanes) to provide 31c (590 mg, 87% yield). Rf =0.42
(EtOAc/hexanes 1:4); 1H NMR (400 MHz, CDCl3): d=8.13 (s, 1H),
7.60 (d, J=7.6 Hz, 1H), 7.52 (d, J=8.0 Hz, 1H), 7.36–7.39 (m, 1H),
7.14–7.19 (m, 4H), 6.93–6.95 (m, 3H), 6.90 (s, 1H), 1.26–1.29 ppm
(s, 24H); 13C NMR (400 MHz, CDCl3): d=148.7, 137.5, 137.3, 131.7,
129.5, 129.3, 129.0, 128.9, 128.9, 128.8, 126.9, 124.3, 123.4, 121.0,
109.1, 98.4, 84.1, 84.0, 83.8, 83.7, 32.1, 25.2, 25.1, 25.0, 14.3 ppm;
MS (ESI) m/z 550 [M+H]+.
Keywords: antiviral agents · hepatitis C · inhibitors · medicinal
chemistry · structure–activity relationships
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Received: August 22, 2013
Published online on October 14, 2013
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