Discovery of (7 R)-14-Cyclohexyl-7-{[2-(dimethylamino)ethyl](methyl) amino}-7,8-dihydro-6 H -indolo[1,2- e ][1,5]benzoxazocine-11-carboxylic Acid (MK-3281), a potent and orally bioavailable finger-loop inhibitor of the hepatitis C virus NS5B polymerase

Article abstract of DOI:10.1021/jm1013105

Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel therapies are in urgent demand. The polymerase of HCV is responsible for the replication of viral genome and has been a prime target for drug discovery eff

Full text of DOI:10.1021/jm1013105

J. Med. Chem. 2011, 54, 289–301 289
DOI: 10.1021/jm1013105
Discovery of (7R)-14-Cyclohexyl-7-{[2-(dimethylamino)ethyl](methyl) amino}-7,8-dihydro-6H-
indolo[1,2-e][1,5]benzoxazocine-11-carboxylic Acid (MK-3281), a Potent and Orally Bioavailable
Finger-Loop Inhibitor of the Hepatitis C Virus NS5B Polymerase†
‡
‡
‡
‡
Frank Narjes,*,‡ Benedetta Crescenzi, Marco Ferrara, Jorg Habermann, Stefania Colarusso,
€
Maria del Rosario Rico Ferreira,‡ Ian Stansfield, Angela Claire Mackay,‡ Immacolata Conte,‡ Caterina Ercolani,‡
Simone Zaramella,‡ Maria-Cecilia Palumbi,‡ Philip Meuleman,§ Geert Leroux-Roels,§ Claudio Giuliano,‡ Fabrizio Fiore,‡
Stefania Di Marco,‡ Paola Baiocco,‡ Uwe Koch,‡ Giovanni Migliaccio,‡ Sergio Altamura,‡ Ralph Laufer,‡
Raffaele De Francesco,‡ and Michael Rowley‡
‡Istituto Di Ricerche Di Biologia Molecolare, P. Angeletti SpA (Merck Research Laboratories, Rome), Via Pontina Km 30,600,
I-00040 Pomezia, Italy, and Center for Vaccinology, Ghent University and Hospital, De Pintelaan 185, B-9000, Gent, Belgium
§
Received October 12, 2010
Infections caused by hepatitis C virus (HCV) are a significant world health problem for which novel
therapies are in urgent demand. The polymerase of HCV is responsible for the replication of viral
genome and has been a prime target for drug discovery efforts. Here, we report on the further development
of tetracyclic indole inhibitors, binding to an allosteric site on the thumb domain. Structure-activity
relationship (SAR) studies around an indolo-benzoxazocine scaffold led to the identification of compound 33
(MK-3281), an inhibitor with good potency in the HCV subgenomic replication assay and attractive
molecular properties suitable for a clinical candidate. The compound caused a consistent decrease in
viremia in vivo using the chimeric mouse model of HCV infection.
Introduction
patient compliance.7 Moreover, duration of therapy and
response rate are genotype specific. The SOC is efficient in
about 80% of patients infected with the gt 2 and 3 of the virus
but only in about 50% of patients infected with gt 1, which
accounts for approximately 70% of infections in the Western
world.8 To improve treatment outcome for chronic hepatitis C
infection with respect to efficacy, tolerability, and duration,
research in the pharmaceutical industry and in academia has
focused on the characterization of viral enzymes as targets for
small molecule intervention.9 Proof-of-concept in the clinic
has been obtained for small molecule inhibitors which target
viral proteins such as the NS3/4A protease, the NS5B poly-
merase, and most recently also NS5A.10-14 The RNA-depen-
dent RNA polymerase NS5B plays a key role in the life cycle
of the virus since it is responsible for the replication of the viral
genome.15 Classical nucleoside analogues16 as well as allosteric
inhibitors, which were shown to bind to at least four distinct
sites on the polymerase, have been described.17
Recently, we and other groups reported on the discovery of
indole-based inhibitors of NS5B, also called thumb-pocket 1
or finger-loop inhibitors.18-20 This class of molecules was
shown to inhibit the initiation of RNA synthesis by binding to
the upper part of the thumb domain, where they interrupt a
key interaction between the λ1 loop, which extends from the
fingertips, and the thumbdomain, impeding a conformational
change of the enzyme thought to be important for RNA
synthesis.21-23 Successive optimization of our indole series,
exemplified by 1 (Figure 1),24 led to conformationally con-
strainedindoles, where the orthopositionof the C2-aryl group
is tethered to the indole nitrogen,25 an approach that was also
disclosed recently by a group of scientists at Japan Tobacco.26
The tetracyclic benzodiazocine 2had good intrinsic and cell-based
It has been estimated that more than 170 million people
worldwide are infected with the hepatitis C virus (HCVa).1,2
Although the infection is typically slow progressing and
often asymptomatic, it becomes chronic in a majority of
patients and in a significant fraction of these can develop into
chronic hepatitis, livercirrhosis, and eventually hepatocellular
carcinoma. Chronic HCV infection has become the leading
indication for liver transplants in the developed world3 and is
the principal cause of death in HIV coinfected patients.4
Although the number of new infections is diminishing, the
mortality due to HCV infection is expected to increase in
Europe and the USA.5 The genetic heterogeneity of the virus,
which has been classified into six different genotypes (gt),
having as much as 35% nucleotide sequence difference among
them, has been a major obstacle for the development of a
vaccine.6 Currently, the standard of care (SOC) is based upon
a combination of subcutaneous pegylated R-interferon and
oral ribavirin. It is poorly tolerated and associated with severe
flu-like symptoms, depression, and anemia, resulting in poor
†PDB deposition number: 2xwy.
*To whom correspondence should be addressed. Phone: þ46 46 337
373. Fax: þ46 46 337 119. E-mail: Frank.Narjes@astrazeneca.com.
Current address: AstraZeneca R&D Lund, Department of Medicinal
Chemistry, 221 87 Lund, Sweden.
a Abbreviations: 1,2-DCE, 1,2-dichloroethane; DCM, dichloromethane;
DIPEA, diisopropylethylamine; DMP, Dess-Martin periodinane; FCS,
fetal calf serum; gt, genotype; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N0,
N0-tetramethyl uronium hexafluorophosphate; HCV, hepatitis C Virus;
NHS, normal human serum; SAR, structure-activity relationship; SD,
standard deviation; SFC, supercritical fluid chromatography; SOC, stan-
dard of care; TBAT, tetrabutylammonium triphenyldifluorosilicate; TFA,
trifluoroacetic acid.
r
2010 American Chemical Society
Published on Web 12/08/2010
pubs.acs.org/jmc

290 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Narjes et al.
Figure 1
potency and a superior DMPK profile with respect to 1.27 Our
efforts to improve the potency of this inhibitor class focused
on replacement of the indole core and optimization of the
tether moiety. Thieno[3,2-b]pyrroles were identified as the
only viable replacements for the indole core.28 Optimization
led to tetracyclic thienopyrrole 3, which had similar potency
and pharmacokinetic properties with respect to 2.29 Since we
were unable to improve potency of this series further, we
turned our attention to the modification of the tether moiety
in the indole series, which was more successful. SAR around 2
and related series had shown that 7- as well as 8-membered
rings were equally well tolerated.25,26 Starting from the tetra-
cyclic benzoazepine 4, novel pentacyclic compounds such as 5
with superior potency in the cell-based assay with respect to
our previous compounds were discovered.30 However, differ-
ent from 2, pentacycle 5 and related compounds lacked oral
bioavailability in rodents.
Several base-catalyzed conditions, such as sodium hydride
in DMF, LHMDS in THF, or cesium carbonate in DMF,
induced cyclization to the benzoxazocine alcohol (S)-10a,
with the latter giving the highest yield. Reaction of 8 with
(R)-glycidyl nosylate led to epimeric alcohol10b. Oxidation of
either alcohol gaveketone11, which was reacted witha variety
of amines under reducing conditions to yield the racemic
amino acids 12-27 after hydrolysis of the methyl ester. The
compounds exist as mixtures of atropisomers, as was evident
from their NMR spectra (see Experimental Section).
Yields were satisfactory for primary amines, but secondary
amines such as dimethylamine or piperidine gave only low
yields, racemic alcohol 10 being the major product. Better
yields were obtained for compounds 21-27 by using a double
reductive amination procedure, where 11 was first reacted with
the primary amine and subsequently with the corresponding
aldehyde to introduce the R2-substituent.
In our search for alternative tetracyclic scaffolds which even-
tually could combine the potency of 5 with the good pharmaco-
kinetic properties of 2, we also had prepared the simple
benzoxazocine 6, which showed better cell-based activity
compared to tetracyclic benzoazepine 4 and thus was deemed
to provide a better starting point for optimization.26b In this
paper, we describe the synthesis and structure-activity relation-
ship within this class of compounds, leading to the identifica-
tion of compound 33, which combined improved potency
with good oral bioavailability in preclinical animal models
and which was evaluated in the SCID/Alb-uPA mouse model
of HCV infection.31
The N-acylated derivatives 28 and 29 were obtained as
shown in Scheme 2. For the preparation of compound 29, the
methyl ester needed to be hydrolyzed prior to amine acylation
to avoid cleavage of the tertiary amide bond during the final
ester deprotection.
Compound 21 could be separated into the enantiomers 33
and 34. Thesewerealsoprepared independently fromalcohols
10a and 10b. Tosylate 30a was obtained from 10a, and reaction
with TMS-azide and TBAT as the fluoride source33 (Scheme 3)
gave azide 31a with inversion of configuration in 84% yield
and 99.3% enantiomeric excess (ee). This method was super-
ior with respect to yield and enantiomeric purity to reaction of
the tosylatewithsodiumazidein DMF (50%yield, 72% ee) or
classicalMitsunobuconditionsof alcohol10a, which gave low
yields (31% yield, 99.5% ee). The high enantiomeric purity of
31a also showed that epoxide 9a was obtained by nearly
exclusive attack of the phenol on the nosylate moiety of (S)-
glycidyl 3-nitrobenzenesulfonate. This excludes a potential
racemization at this step via epoxide opening and Payne
rearrangement to give the enantiomer of 9a.32 Reduction of
the azide provided the amino ester 32a, which was converted
to 33 in three more steps with an enantiomeric excess of 96%,
Chemistry
Benzoxazocine 6 was readily prepared from the phenol 8,
theSuzuki-coupling product obtained frombromide7 andthe
corresponding boronic acid, by double alkylation with dibromo-
propane (not shown). To obtain a suitable handle for further
modification of the tether, ketone 11 was targeted, but all
attempts of alkylating 8 with dichloroacetone failed (Scheme 1).
As an alternative, 8 was reacted with (S)-glycidyl nosylate
under the influence ofcesium fluoride togive(S)-epoxide 9a.32

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 291
Scheme 1a
a Reagents and conditions: (a) (2-hydroxyphenyl)boronic acid, PdCl2[P(Ph)3]2, Na2CO3, dioxane, 110 °C; (b) (S)-glycidyl 3-nitrobenzenesulfonate,
CsF, DMF, RT; (c) Cs2CO3, dioxane, 100 °C; (d) (i) (R)-glycidyl 3-nitrobenzenesulfonate, CsF, DMF, RT, (ii) Cs2CO3, dioxane, 100 °C; (e) DMP, DCM
(f ) 12-20: R1R2NH, NaBH(OAc)3, AcOH, 1,2-DCE, rt; 21-27: (i) R1NH2, NaBH(OAc)3, AcOH, 1,2-DCE, (ii) R2CHO, NaBH(OAc)3; (g) KOH,
MeOH/THF (1:1), reflux.
Scheme 2a
1b in a Huh-7 hepatoma cell line in the presence of 10% fetal calf
serum (FCS) or 50% normal human serum (NHS) for com-
pounds with replicon EC50s<1 μM in 10% FCS.24
Introduction of a basic amine onto the indole carboxylic
acids had usually proven the method of choice to gain cell-
based potency for this series of compounds.25 This was also
the case for benzoxazocine 6 (Table 1). Methyl amine 12 was
already 2-fold more active in the replicon assay in 10% FCS
compared to 6. Introduction of another methyl group in-
creased cellular activity even further (13, EC50 570 nM),
although intrinsic potency of 12 and 13 did not change much
a Reagents and conditions: (a) NH4OAc or MeNH2 HCl NaOAc;
with respect to 6. Introduction of an ethane-1,2-diamine gave
14, which displayed similar levels of cell-based activity with
respect to 13. Methylation of the distal amine yielded 15, with
a 3-fold improvement in EC50. Elongation of the distance
between the two amines, as in 16, or incorporating the outer
amine into a piperidine ring as in 17, led to a slight loss in cell-
based potency with respect to 15. Testing compounds 13-17
in high serum conditions resulted in a moderate 4-5-fold
shift in replicon potency.
Compound 15 had the same level of potency when compared
to 2 and was characterized further with respect to metabolic
stability and rodent PK. The compound demonstrated an en-
couraging PK profile in Sprague-Dawley rats after single intra-
venous and oral doses. Following intravenous dosing, plasma
clearance and half-lives were significantly improved with respect
to 2, consistent with the increased metabolic stability of 15 in rat
liver microsomes (Table 2). After an oral dose of 3 mg/kg of 15,
bioavailability was determined to be 20%, slightly lower than
that for 1 (31%). Elevated concentrations of 15 were found in rat
liver tissue 6 h after oral dosing, corresponding to a liver tissue to
plasma concentration ratio of 21 at this point in time (16 for
compound 1). The stability in dog and human liver microsome
preparations was similar to rat, with the low intrinsic clearance
rates observed in all three species.
3
NaBH3CN, MeOH, RT; (b) 28: (i) N,N-dimethylglycine, HATU, DIPEA,
DCM, RT, (ii) BBr3, DCM; 29: (i) KOH, 75 °C; (ii) N,N-dimethylglycine,
HATU, DIPEA, DCM, RT.
as determined by separation on a chiral column. The same
route led from 10b to 34 having the (S)-configuration in 99%
enantiomeric excess (Scheme 3).
Alkylation of alcohol 10a with 2-chloro-N,N-dimethylethane-
amine yielded, after hydrolysis of the ester, the aminoether
35 (Scheme 4). Enantiomer 36 was prepared in the same way
starting from the (R)-configurated alcohol 10b.
Spiroazetidines 38 and 39 could be accessed from the reaction
of 8 with 5,5-bis(bromomethyl)-2,2-dimethyl-1,3-dioxane,34
leading to spiroketal 37. Hydrolysis, conversion of the alcohols
into the corresponding triflates, and reaction with isopropyl-
amine or N,N-dimethylethane-1,2-diamine gave the desired
compounds after hydrolysis of the methyl ester (Scheme 5).
Results and Discussion
Optimization of Replicon Activity. Compounds were assayed
in a polymerase enzyme inhibition assay (IC50) on the HCV
genotype 1b NS5B (ΔC21) polymerase.21 The cellular activity
(EC50) was determined in the replicon system expressing genotype

292 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Narjes et al.
Scheme 3a
a Reagents and conditions: (a) TsCl, pyridine; (b) TMSN3, TBAT, THF, 70 °C; (c) H2, Pd/C, MeOH; (d) N-Boc-aminoacetaldehyde, HC(OMe)3;
NaCNBH3, HOAc, MeOH; (e) DCM/TFA (1:1); HCHO, NaOAc, NaCNBH3, DCM; (f) KOH, dioxane/H2O (2:1); (g) separation by SFC.
Scheme 4a
Scheme 5a
a Reagents and conditions: (a) NaH, DMF, 70 °C; 5,5-bis(bromomethyl)-
2,2-dimethyl-1,3-dioxane, 70 °C, 50%; (b) TsOH H2O, MeOH/THF,
3
RT, 89%; (c) Tf2O, MeCN, DIPEA, 0 °C, then iPrNH2 (38) or H2N-
(CH2)2NMe2 (39), 70 °C; (d) KOH, dioxane, 75 °C.
potency, whereas 21 shifted only 4-fold under high serum
conditions, reflecting the higher binding to human plasma
protein of 29 (0.5% free) versus 21 (2.7% free). With respect
to replicon potency under low and high serum conditions, the
methyl substitution on the two amines proved to be optimal,
although several analogues with modifications on the distal
amine such compounds 22-25 displayed similar replicon
potency under standard replicon conditions. For the most potent
of these analogues, N-methylpiperazine 25 (EC50 66 nM),
a low free fraction in the presence of human plasma proteins
(1.1%free), ledtoa 9-foldshift inthe replicon assay. Exchanging
the methyl group on the inner amine for an ethyl- and iso-
propyl group as in compounds 26 and 27 resulted in a 2-fold
loss in cell-based activity and a more pronounced shift (7-fold)
under high serum conditions.
a Reagents and conditions: (a) 2-chloro-N,N-dimethylethanamine,
Bu4NBr, toluene/30% NaOH, 60 °C.
Encouraged by these results, we undertook further SAR
around the ethane-diamine side chain of 15 and key results
are summarized in Table 3. A distal basic amine was required
for cell-based activity. Methylether 18 lost an order of magni-
tude in activity compared to 15, and amide 19 was 4-fold less
potent. In both cases, the loss in intrinsic potency was only
about 2-fold. Conversely, the conversion of the inner amine
to an amide as in 28 was tolerated and cell-based activity was
further improved by methylation of the nitrogen, leading
to tertiary amide 29 (EC50 73 nM). Removal of the amide
oxygen gave the fully methylated bis-amine 21, which was
equipotent to 29 in the standard replicon conditions. Com-
pounds 21 and 29 were the first examples displaying an
EC50 < 100 nM in the tetracyclic indole series. In the presence
of 50% NHS, compound 29 showed a nearly 10-fold shift in
To investigate the impact of the stereochemistry on the
tether, compound 21 was separated into the enantiomers 33
and 34. The activity resided mostly in the (R)-configurated
compound 33, which was 4-fold more potent intrinsically with
respect to the (S)-enantiomer 34 (Table 4). Moreover, 33 was

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 293
about 10-fold more potent than 34 in the cell-based assay,
with an EC50 of 38 nM and a moderate 3-fold shift under
high serum conditions. The absolute configuration of 33 was
established also by synthesis from 10a and later confirmed by
an X-ray structure of 33 bound to NS5B (vide infra).
Further SAR around the side chain established that the
inner methylamine group could be replaced with oxygen,
giving compounds 35 and 36. The more active epimer 35 was
2-fold less potent in the replicon assay with respect to 33, and
the EC50 shifted 6-fold under high serum conditions. Differ-
ent from what had been observed for bis-amines 33 and 34,
the activity in the resulting amino ethers 35 and 36 resided
mostly in (S)-enantiomer 35, having the opposite configuration
at position 7 of the tether. We also briefly investigated the
possibility to eliminate the stereogenic center at this position by
introduction of a spirocycle. Geminal disubstitution was toler-
ated, as spiro-azetidines 38 and 39 show. Monoamine 38 had
similar potency levels with respect to monoamine 13 (seeTable1),
and bisamine 39 showed slightly improved intrinsic potency
with respect to 33 but was 2-fold less active in the replicon assay,
with a 5-6-fold shift in the presence of 50% NHS.
On the basis of its potency in cell-based assay, compound
33 was characterized further.
Against a panel of chimeric replicons containing different
NS5B genotypes, 33 was an equally potent inhibitor of gt 1a
(EC50=28nM), 1bBK(EC50=9nM), and3a(EC50=37 nM)
replicons, whereas it resulted 2 orders of magnitude less active
on 2a (EC50=2018 nM) and 2b (EC50=1833 nM) replicons.35
The lower sensitivity of the finger-loop inhibitors against HCV
genotype 2b is due to differences in the amino acid composition
of the binding pocket, resulting in a different shape between
the two genotypes.36
Table 1. Enzymatic and Cell-Based Activity of Compounds 6 and 12-16
Crystal Structure of 33 Bound to NS5B. Compound 33 was
successfully soaked into crystals of a NS5B ΔC55 genotype
1b construct (Figure 2). As had been previously observed
with the related indole and thienopyrrole inhibitors, the tetra-
cyclic compound binds in the allosteric pocket in the thumb
domain, which in the apoenzyme is occupied by a short R-helix
protruding from the finger domain and displays the same
binding mode.37 The cyclohexyl ring is tightly bound in a
lipophilic pocket, and the indole scaffold itself is accommo-
dated mainly in a lipophilic environment with its carboxylate
forming a salt bridge to Arg503. The dihedral angle between
the C2-phenyl ring and the indole in the complex structure is
46°, as predicted form our docking studies. The amine containing
side chain is pointing out into solvent, preferring an orientation
which brings the basic amine into proximity of the indole
caboxylate. The structure confirms the (R)-configuration on
the 7-position of the benzoxazocine ring. In this configuration,
the 2-(dimethylamino)ethyl]-(methyl)amino moiety occupies
the pseudoequatorial position on the benzoxazocine-ring,
which is calculated to be 13 kJ/mol lower in energy with respect
to the corresponding pseudoaxial conformation which would
be required for binding of the (S)-configurated epimer 34.38
In the enantiomorphic pseudoequatorial conformation of
the S-epimer, the ring would adopt an orientation incompa-
tible with binding because it clashes with the enzyme surface.
This might be the reason for the difference in activity for the
enantiomers. In the oxygen-linked series, the more active
isomer, compound 35, has the side chain in the pseudoaxial
position. We hypothesize that the pseudoaxial arrangement
is favored by a gauche effect between the exocyclic O-atom
and the endocyclic nitrogen and oxygen atoms. Sterically the
ether O-atom is less disfavoring for the pseudoaxial arrange-
ment than the sterically more demanding N-Me group.
Animal Pharmacokinetics and Safety Assessment. The
pharmacokinetic profile of compound 33 was evaluated in rats,
dogs, and rhesus monkeys (Table 5). The compound displayed
moderate plasma clearance in rats, dogs, and rhesus monkeys,
a Values are the mean ( SD of at least three independent measurements.
Values without SD are the average of at two independent measurements.
Table 2. Pharmacokinetic Profile for Compounds 2 and 15 Following Single Dose Administration to Ratsa and Their Stability in Liver Microsome
Preparations
iv, 3 mg/kg
po, 3 mg/kg
AUC ( μM h) F (%)
compd
Cl (mL/min/kg)
t1/2 (h)
Vd (L/kg)
Cmax ( μM)
rat [liver] 6 h ( μM)
Clint r, d, h ( μL/min/mg)
3
2
15
61
21
1.6
3.0
5.1
3.2
0.2
0.3
0.7
0.9
31
20
0.64
1.3
100, 26, 5
7, 7, 5
a Compounds 2 and 15 were dosed as their respective TFA salts to Sprague-Dawley rat, n = 3 (DMSO/PEG400/water 20%/60%/20%); po (PEG400).

294 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Table 3. Enzymatic and Cell-Based Activity of Compounds 18-29
Narjes et al.
Table 4
a Values are the mean ( SD of at least three independent measurements.
Values without SD are the average of at two independent measurements.
Figure 2. Crystal structure of HCV ΔC55 NS5B polymerase from
genotype 1b in complex with compound 3337 (2.53 A resolution,
˚
compound is in stick representation in green, with nitrogen in blue
and oxygen in red). The lipophilic protein surface is in white and
light gray, except for Arg503, shown in stick representation.
a Values are the mean ( SD of at least three independent measurements.
Values without SD are the average of at two independent measurements.
monkeys. Terminal half-lives were 3.4 h in rats, 6.1 h in dogs,
and 5.7 h in monkeys. The oral bioavailability of the amorphous
bis-HCl salt was 49% in rats, 78% in dogs, and 35% in monkeys.
with 22, 7, and 7 mL/min/kg, respectively. The volumes of
distribution were 3.7 in rats, 2.6 in dogs, and 2.2 L/kg in

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 295
Table 5. Pharmacokinetic Profile of Compound 33 Following Single Dose Administrationa
species
Cl (mL/min/kg)
Vdss (L/kg)
t1/2 (h)
Cmax (μM)
AUCpo (μM h)
F (%)
3
rat
dog
22 ( 3
7 ( 2
7 ( 1.9
3.7 ( 0.5
2.6 ( 0.4
2.2 ( 0.6
3.4 ( 0.1
6.1 ( 0.9
5.7 ( 0.2
0.5 ( 0.1
1.0
2.4 ( 0.1
8.6
49 ( 2
78
rhesus
0.8 ( 0.5
7.4 ( 0.5
35 ( 12
a Compound 33 wasdosedasthebis-HClsalt;rat,dog,andrhesusn= 3 (except dog po n= 2); iv 3, 1, and 2 mg/kg (DMSO/PEG400/water 20%/60%/20%);
po 3, 2, and 4 mg/kg (PEG400 rat and dog, PEG400/water 70%/30% rhesus).
Figure 3. Antiviral response induced by treatment with compound 33 of humanized uPAþ/þ-SCID mice (A, B, C) infected with HCV of genotype 1b.
LOQ: lower limit of detection of HCV RNA (= 315 IU/mL). Vehicle = PEG400/water 30%/70%.
After an oral dose of 3 mg/kg to rat, liver concentrations 6 h after
dosing of 33 were determined to be 0.42 ( 0.16 μM, which
corresponds to a liver tissue to plasma concentration ratio of 5.
The levels are 3-fold lower than those observed for compound 15
at the same dose, but they are 4-fold higher than the EC50
in 50% NHS, which compares favorably with compounds
2 and 15, where this ratio is about 1 and 2, respectively.
Compound 33 displayed relatively high binding to rat (97%),
dog (97.3%), monkey (92.6%), and human (96.9%) plasma
proteins. NADPH-dependent metabolism was observed in
liver microsomes, with low intrinsic clearance in dog, human,
and rat, 3, 7, and 7 μL/min/mg, respectively. In liver micro-
somes supplemented with UDPGA, 33 exhibited very low
turnover in all species, < 4 μL/min/mg.
The compound did not inhibit CYP3A4, CYP2D6, or
CYP2C9 at concentrations up to 50 μM and was not a time-
dependent inhibitor of human CYP3A4. In the PXR transacti-
vation assay, 33 showed no potential for inducing CYP3A4.
In the PanLabs screen, only 2 off-target activities were seen
for matrix metalloprotease 12 (IC50 = 8.3 μM) and protein
kinase CR (IC50 = 59 μM). Selectivity versus inhibition of
NS5B is >1000-fold. The human DNA polymerases R, β,
and γ were not inhibited at concentrations up to 50 μM.
Viral Response in HCV-Infected Chimeric Mice. The in vivo
efficacy of 33 was demonstrated preclinically in a humanized
mouse model for the study of viral hepatitis. T- and B-cell defi-
cient mice carrying a tandem array of murine urokinase-type
plasminogen activator transgenes under the control of the
mouse albumin promoter (Alb/uPAþ/þ-SCID) suffer from a
severe liver disease and consequently are excellent recipients for
primary human hepatocyte transplantation. Several weeks after
transplantation, the liver of these mice can harbor up to 90% of
well-organized, functional, and healthy human hepatocytes.39
They can then be inoculated with infectious HCV particles
of different origins and genotypes and support durable replica-
tion of the virus at levels equivalent to those seen in infected
humans.31a,40 This animal model has been validated with IFN-R,
non-nucleoside inhibitors of the polymerase such as HCV-796
and pyrano-indole-based inhibitors, protease inhibitors such as
BILN-2061, and entry inhibitors like anti-CD81 antibodies.41
In preparation of the experiment, SCID mice (n = 3) were
dosed by intraperitoneal injection for five consecutive days twice
daily with 50 mg/kg of compound 33. The compound was well
tolerated, and no gross changes in body or organ weight were
observed. Twelve hours after administration of the last dose,
plasma levels of compound were 3.6 μM. The same dose of
50 mg/kg of 33 was then administered by intraperitoneal injection
to three humanized uPAþ/þ-SCID mice infected with HCV of
genotype 1b twice daily over the course of 4 days. Treatment
caused a consistent decrease in the viremia in the three animals
A-C (A, -2.84 log10; B, -3.94 log10; C, -2.60 log10), with a
mean decrease of HCV plasma RNA levels of 3.13 ( 0.7 log10.
The mean plasma trough concentration of 33 12 h after admin-
istration of the last dose was similar in all three animals, 2.6 (
0.2 μM. Viral titers rebounded after treatment withdrawal as
measured on day 21 (Figure 3). Three mice were also dosed only
with the vehicle (30% PEG400 þ 70% water) for four days, and
no decrease in viremia was observed (range 0-0.3 log10).
At a 5-fold lower dose of 10 mg/kg, the mean C12 h of 33
was determined to be 0.67 ( 0.2 μM, and also at this dose an
antiviral effect with a mean drop in HCV RNA of 1.4 ( 0.25
log10 was observed (data not shown).
Conclusion
In summary, a novel second-generation series of tetracyclic
allosteric finger-loop inhibitors of the HCV NS5B polymerase

296 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Narjes et al.
was identified. Exploration of the SAR aroundthe amine side-
chain on the benzoxazocine tether of compound 6 led to the
identification of compound 33, designated as MK-3281,42
which combined excellent cell-based potency with good phar-
macokinetic properties in preclinical species. The compound
was efficacious in the chimeric mouse model of HCV infection,
where at a twice daily dose of 10 mg/kg and 50 mg/kg bid a
consistent drop in viremia of 1.4 log10 and 3.1 log10, respectively,
was observed. On the basis of this promising profile, the com-
pound was further characterized for efficacy in HCV-infected
chimpanzee and advanced into phase I clinical trials.43
with EtOAc and washed with water and brine. The crude product
was purified by trituation with Et2O to afford 9a as a colorless
foam (5.11 g, 74%); [R]D20 = þ22° (c =0.5, CHCl3). 1H NMR
(400 MHz, DMSO-d6) δ 1.14-1.40 (m, 3H), 1.62-1.80 (m, 5H),
1.81-1.96 (m, 2H), 2.58-2.68 (m, 1H), 2.59 (dd, J=2.5, 5.0 Hz,
1H), 2.76 (t, J = 5.0 Hz, 1H), 3.19-3.25 (m, 1H), 3.85 (s, 3H),
3.91 (dd, J=6.2, 11.6 Hz, 1H), 4.33 (dd, J=2.7, 11.6 Hz, 1H),
7.11 (t, J=7.4 Hz, 1H), 7.20 (d, J=8.3 Hz, 1H), 7.32 (dd, J=
1.6, 7.4 Hz, 1H), 7.44 (dt, J=1.6, 8.3 Hz, 1H), 7.58 (dd, J=1.3,
8.4 Hz, 1H), 7.79 (d, J=8.4 Hz, 1H), 7.98 (d, J=1.2 Hz, 1H),
11.28 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 26.3, 27.07, 27.1,
33.0, 33.1, 36.6, 44.6, 50.3, 51.8, 68.4, 113.2, 113.5, 119.5, 120.0,
120.2, 121.7, 122.1, 122.9, 130.4, 129.6, 131.7, 133.8, 135.4, 156.2,
168.3. MS (ESþ) m/z 406.5 (M þ H)þ.
Experimental Section
Methyl (7S)-14-Cyclohexyl-7-hydroxy-7,8-dihydro-6H-indolo-
[1,2-e][1,5]benzoxazocine-11-carboxylate (10a).44 A solution of
9a (4.36 g, 10.7 mmol) in dioxane (220 mL) was treated with
Cs2CO3 (7.0 g, 21.5 mmol), and the resulting suspension heated
to reflux for 48 h. The reaction was cooled to RT, diluted with
EtOAc, and washed with water and brine. The crude product
Chemistry. Solvents and reagents were obtained from com-
mercial suppliers and were used without further purification.
Organic extracts were dried over sodium sulfate and were concen-
trated (after filtration of the drying agent) on rotary evaporators
operating under reduced pressure. Flash chromatography puri-
fications were performed on Merck silica gel (200-400 mesh) as
the stationary phase or on commercial flash chromatography
systems (Biotage Corporation and Jones Flashmaster) utilizing
prepacked columns. Petroleum ether (PE) refers to the fraction
boiling between 40 and 60 °C. 1H NMR and 13C NMR spectra
were recorded at 300 K (if not stated otherwise) in the indicated
solvent on Bruker AMX spectrometers operating at the re-
ported frequencies. Chemical shifts (δ) for signals correspond-
ing to nonexchangeable protons (and exchangeable protons
where visible) are recorded in parts per million (ppm) relative
to tetramethylsilane or to the residual solvent peak. Signals
are tabulated in the order: multiplicity (s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet; b, broad, and combinations thereof);
coupling constant(s) in hertz; number of protons. HPLC-MS
data were obtained on an Acquity Waters UPLC operating in
negative (ES-) or positive (ESþ) ionization mode, and results
are reported as the ratio of mass over charge (m/z) for the parent
ion only. High resolving power accurate mass measurement
electrospray (ES) mass spectral data were acquired by use of a
Bruker Daltonics apex-Qe Fourier transform ion cyclotron
resonance mass spetrometer (FT-ICR MS). External calibration
was accomplished with oligomers of polypropylene. The purity
of the final compounds was determined by analytical RP-HPLC
with a Waters Alliance separation module 2695, using aceto-
nitrile/water gradients (both modified with 0.1% TFA). Peaks
were detected with a Waters 996 PDA detector using the
Maxplot option (210-400 nM) contained in the PDA program.
Method A: Waters Sunfire C18 column (2.1 mmꢀ50 mm, 3.5 μm),
flow rate 0.5 mL/min; gradient 10% acetonitrile, 0.5 min isocratic,
linear to 100% acetonitrile over 6 min, then isocratic. Method B:
Waters Sunfire C18 column (4.6 mm ꢀ 150 mm, 5 μM), flow
1.0 mL/min; gradient 70% acetonitrile, 1 min isocratic, then linear
to 70% acetonitrile over 10 min, then linear to 100% acetonitrile
over 5 min and then isocratic. All compounds described in this
article showed purities higher than 95% in both analytical
methods, except for compound 14 (method A 99%, method B
91%) and 27 (method A 96.9%, method B 90.5%). A table with
purity data and retention times can be found in the Supporting
Information. Preparative scale HPLC separations were carried
out on a Waters 2525 pump, equipped with a 2487 dual absorbance
detector or on a mass-triggered automated Waters MassLynx
purification system. Compounds were eluted with linear gradi-
ents of water and MeCN with water containing 0.1% TFA.
Supercritical fluid chromatography (SFC) was performed on a
Berger system equipped with a Berger ALS 719.
was purified by flash chromatography on silica gel (DCM/MeOH
20
98:2) to afford 10a as a colorless powder (2.38 g, 55%); [R]D
=
-28° (c = 0.5, CHCl3). 1H NMR (400 MHz, DMSO-d6, 2 dia-
stereomers, 2:1*) δ 1.02-1.40 (m, 3H), 1.48-1.59 (m, 1H), 1.60-
1.78 (m, 2H), 1.79-1.90 (m, 1H), 1.90-2.06 (m, 3H), 2.60-2.78
(m, 1H), 3.63 (dd, J=10.6, 14.7 Hz, 1H), 3.68 (dd, J=8.2, 12.0
Hz, 1H), 3.77* (d, J=12.7 Hz, 1H), 3.80-3.83 (m, 1H), 3.86*,
3.89 (s, 3H), 3.90-4.06* (m, 3 H), 4.32 (dd, J=4.4, 12.0 Hz, 1H),
4.40 (dd, J=1.0, 14.7 Hz, 1H), 4.58* (dd, J=1.3, 12.7 Hz, 1H),
5.24*, 5.66 (d, J = 3.0*, 4.8 Hz, 1H, OH), 7.15-7.33 (m, 3H),
7.47*, 7.52 (dt, J=1.7*, 8.4*, 1.9, 8.5 Hz, 1H), 7.61*, 7.68 (d, J=
8.4*, 8.5 Hz, 1H), 7.84*, 7.89 (d, J = 8.4*, 8.5 Hz, 1H), 8.20,
8.22* (s, 1H). 13C NMR (100 MHz, CDCl3) δ 26.2, 26.9, 27.0,
33.0, 33.1, 33.3*, 33.4*, 36.6, 46.2, 46.5*, 51.9, 68.1*, 68.4, 72.7*,
78.5, 111.3*, 112.9, 119.4*, 119.9, 120.0*, 120.03, 120.3*, 120.4,
121.0, 122.3, 123.0*, 123.3*, 123.7, 130.1*, 130.4, 130.7*, 131.0,
132.2, 133.2*, 135.8, 136.9*, 137.4*, 138.0, 158.6*, 159.0, 168.2.
MS (ESþ) m/z 406.6 (M þ H)þ.
Methyl 14-Cyclohexyl-7-oxo-7,8-dihydro-6H-indolo[1,2-e]-
[1,5]benzoxazocine-11-carboxylate (11). A solution of 10a (3.59 g,
8.85 mmol) in DCM (85 mL) was treated with Dess-Martin
periodinane (4.5 g, 10.6 mmol), and the mixture was stirred for 1 h
at RT. At this point, another 0.45 g of DMP (1.06 mmol) were
added and the reaction left overnight. The reaction mixture was
diluted with EtOAc and washed with an aqueous solution of a 1:1
mixture containing sodium thiosulfate and sodium hydrogen car-
bonate (both saturated solutions) and then with water and brine.
Drying over Na2SO4, filtration, and concentration in vacuo gave
crude 11 (3.4 g, 95%), which was used without further purification.
1H NMR (400 MHz, CDCl3) δ 1.30-1.48 (m, 3H), 1.71-1.85 (m,
3H), 1.86-2.03 (m, 4H), 2.73-2.89 (m, 1H), 3.95 (s, 3H), 4.46 (d,
J=16.7 Hz, 1H), 4.62 (d, J=17.2 Hz, 1H), 4.74 (d, J=17.2 Hz,
1H), 4.84(d, J=16.7 Hz, 1H), 7.30 (d, J=8.1 Hz, 1H), 7.34 (dt, J=
0.9, 7.6 Hz, 1H), 7.43 (dd, J=0.8, 7.6 Hz, 1H), 7.53 (dt, J=1.8, 8.1
Hz, 1H), 7.82 (dd, J= 1.2, 8.6 Hz, 1H), 7.87 (d, J= 8.6 Hz, 1H),
8.07 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 26.2, 26.9, 27.0, 33.3,
33.5, 36.6, 52.0, 53.7, 77.8, 111.4, 120.5, 120.7, 121.1, 121.4, 121.6,
123.8, 125.0, 130.5, 131.3, 131.5, 136.3, 137.0, 157.4, 167.9, 202.6.
MS (ESþ) m/z 404.5 (M þ H)þ.
General Procedure for the Reductive Amination of Ketone 11
and Ester Hydrolysis: 14-Cyclohexyl-7-(methylamino)-7,8-dihydro-
6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylic Acid (12). To
a suspension of 11 (40 mg, 0.1 mmol) in 1,2-dichloroethane (2 mL)
was added acetic acid (0.008 mL, 0.15 mmol) and methylamine
(0.14 mL, 1 M in THF). After stirring for 15 min at RT, sodium
triacetoxyborohydride (32 mg, 0.15 mmol) was added in one
portion. The resulting mixture was stirred at RT for 2 h. Sodium
hydroxide was added (2 mL, 1N) and after stirring for 5 min the
mixture was taken into EtOAc. The phases were separated, and
the organic phase was washed with water and brine. The crude
Methyl 3-Cyclohexyl-2-{2-[(2S)-oxiran-2-ylmethoxy]phenyl}-
1H-indole-6-carboxylate (9a). To a solution of 8 (6.0 g, 17 mmol)
in DMF (200 mL) were added cesium fluoride (7.74 g, 51 mmol)
and (S)-glycidyl 3-nitrobenzenesulfonate (5.79 g, 22 mmol). The
resulting mixture was stirred at RT overnight and then diluted

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 297
methyl ester was used without further purification. The ester
was dissolved in a mixture of THF and methanol (2 mL, 1:1 v/v)
and treated with 1 N KOH (1 mL). The resulting solution was
heated to 60 °C for 4 h. After cooling to RT, the reaction mixture
was brought to pH 2 by the dropwise addition of hydrochloric
acid (1 N) and then diluted with MeCN and purified by RP-HPLC
to afford 12 as its TFA salt (31 mg, 58%). 1H NMR (300 MHz,
DMSO-d6) δ 1.07-1.28 (m, 1H), 1.30-1.46 (m, 2H), 1.56 (bd, J=
11.9 Hz, 1H), 1.65-1.79 (m, 2H), 1.80-2.09, m, 4H), 2.61-2.80
(m, 1H), 2.77 (s, 3H), 3.57-3.71 (m, 1H), 3.91 (bt, J=12.8 Hz,
1H), 4.08-4.27 (m, 2H), 4.85 (d, J = 14.4 Hz, 1H), 7.22-7.35
(m, 3H), 7.53 (dt, J=2.1, 7.9 Hz, 1H), 7.72 (d, J=8.6 Hz, 1H),
7.91 (d, J = 8.6 Hz, 1H), 8.30 (s, 1H), 8.94 (bs, 1H), 12.68 (bs,
1H). 13C NMR (75 MHz, DMSO-d6, 320 K) δ 25.6, 26.6, 30.8,
32.6, 36.4, 41.3, 55.4, 67.8, 11.9, 119.1, 119.3, 120.1, 120.3, 121.3,
123.1, 124.2, 129.7, 131.1, 132.7, 135.4, 136.7, 157.9, 168.2. MS
(ESþ) m/z 405.3 (M þ H)þ. HRMS (M þ H)þ calcd for C25H29-
N2O3 405.2178; found 405.218.
14-Cyclohexyl-7-{[2-(dimethylamino)ethyl]amino}-7,8-dihydro-
6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylic Acid (15).
Compound 15 was prepared following the procedure for the
synthesis of 12 from 11 (100 mg, 0.25 mmol) and N,N-dimethyl-
ethane-1,2-diamine (0.033 mL, 0.30 mmol). HPLC analysis of
the crude methyl ester (120 mg, quantitative) indicated a ratio
of ester to racemic alcohol 10 of about 9:1; MS (ESþ) m/z 476
(M þ H)þ. Hydrolysis and purification by RP-HPLC afforded
15 as its bis-TFA salt (109 mg). 1H NMR (400 MHz, DMSO-d6,
2 atropisomers 7:1*) δ 1.09-1.22 (m, 1H), 1.25-1.46 (m, 2H),
1.50-1.58 (m, 1H), 1.65-1.78 (m, 2H), 1.82-1.88 (m, 1H), 1.89-
2.07 (m, 3H), 2.64-2.75 (m, 1H), 2.79*, 2.82 (s, 6H), 2.98-3.10
(m, 1H), 3.10-3.90 (m, 5H, together with water peak and probably
the three exchangeable protons), 3.96-4.02 (m, 1H), 4.18, 4.32*
(dd, J = 3.0, 12.6 Hz, 1H), 4.74, 4.84* (d, J = 11.9 Hz, 1H),
7.22-7.28 (m, 2H), 7.30 (dd, J=1.9, 7.9 Hz, 1H), 7.52 (dt, J=
1.9, 8.5 Hz, 1H), 7.64*, 7.70 (dd, J=1.0, 8.4 Hz, 1H), 7.86*, 7.99
(d, J=8.5*, 8.4 Hz, 1H), 8.22, 8.35* (s, 1H). 13C NMR (75 MHz,
DMSO-d6, 320 K, only data for the major atropisomer shown)
δ 25.6, 26.7, 32.7, 36.4, 40.4, 42.9, 43.3, 54.4, 55.5, 70.3, 111.6,
118.9, 119.8, 120.06, 120.08, 121.3, 122.9, 124.0, 129.6, 131.1,
132.6, 135.4, 136.9, 158.3, 168.2. MS (ESþ) m/z 462.3 (M þ H)þ.
HRMS (M þ H)þ calcd for C28H36N3O3 462.2757; found
462.2766.
14-Cyclohexyl-7-{[2-(dimethylamino)ethyl](methyl)amino}-
7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylic
Acid (21). To a solution of crude methyl ester of compound 15
(100 mg, 0.21 mmol) in DCM (2 mL) were added aq formalde-
hyde (37%, 0.050 mL, 0.63 mmol) and acetic acid (0.018 mL,
0.32 mmol), followed after 5 min by solid sodium cyanoboro-
hydride (20 mg, 0.32 mmol). After stirring at RT for 2 h, sodium
hydroxide (4 mL, 1 N) was added and the resulting suspension
stirred for another 5 min before being taken into EtOAc and
washed with water and brine. Drying over sodium sulfate and con-
centration in vacuo gave the crude ester, which was used without
any further purification. MS (ESþ) m/z 490 (M þ H)þ. The ester
was dissolved in a mixture of dioxane/water (2 mL, 2:1, v/v), and
aq potassium hydroxide (1 mL, 1N) was added. The mixture was
stirred at 70 °C for 6 h and then brought to pH 2 by the dropwise
addition of hydrochloric acid (1 N) and then diluted with
MeCN. After purification by RP-HPLC, 21 was obtained as its
bis-TFA salt (66 mg, 45%). For proton and carbon spectra, see
compound 33. MS (ESþ) m/z 476.5 (M þ H)þ. HRMS (M þ H)þ
calcd for C29H38N3O3 476.2913; found 476.2924.
acetonitrile (1 mL). Potassium hydroxide (1 N, 0.31 mL, 0.310
mmol) was added, and the mixture was stirred at 45 °C for three
days to destroy the mixed anhydride. The reaction mixture was
concentrated in vacuo, dissolved in DMSO, and purified by RP-
HPLC to afford 29 as its TFA salt (16 mg, 58%). 1H NMR (400
MHz, DMSO-d6, two atropisomers 1:1) δ 1.09-1.44 (m, 3H),
1.47-1.59 (m, 1H), 1.63-2.03 (m, 6H), 2.26 (s, 1.5 H), 2.62-
2.94 (m, 7H), 2.97 (s, 1.5 H), 3.81 (d, J=17.0, 0.5H), 3.90 (d, J=
15.4, 0.5H), 4.13 (d, J=17.0, 0.5H), 4.15-4.39 (m, 3.5H), 4.46
(dd, J = 14.0, 4.3, 0.5H), 4.56 (d, J = 14.6, 0.5H), 4.62-4.72
(m, 1H), 7.31-7.39 (m, 3H), 7.54-7.63 (m, 1H), 7.65-7.70 (m, 1H),
7.76 (bs, 0.5H), 7.87-7.92 (m, 1H), 8.10 (bs, 0.5H), 9.50-9.79
(bm, 1H), 12.51-12.82 (bs, 1H). 13C NMR (100 MHz, CD3OD)
27.3, 28.2, 31.3, 34.5, 34.7, 38.3, 44.2, 45.2, 54.3, 59.6, 78.1, 111.9,
121.1, 121.6, 123.8, 125.9, 127.1, 131.6, 132.7, 132.8, 138.3, 138.4,
161.2, 166.6, 171.2. MS (ESþ) m/z 490.4 (M þ H)þ. HRMS
(M þ H)þ calcd for C29H35N3O4 490.2706; found 490.2717.
Methyl (7S)-14-Cyclohexyl-7-{[(4-methylphenyl)sulfonyl]oxy}-
7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylate
(30a). To a solution of 10a (325 mg, 0.802 mmol) in pyridine
(10 mL), tosyl chloride (229 mg, 1.203 mmol) was added and the
reaction was stirred at room temperature overnight. The reaction
mixture was diluted with EtOAc and washed with hydrochloric
acid (1 N), sodium hydrogen carbonate (saturated solution),
and brine. The crude product was purified by flash chromatog-
raphy on silica gel (PE/EtOAc 9:1) to afford 30a as a pale-yellow
oil (423 mg, 94%). 1H NMR (400 MHz, CDCl3, 2 atropisomers
7:3) δ 1.29-1.44 (m, 3H), 1.58-1.68 (m, 1H) 1.69-1.81 (m, 2H),
1.82-2.09 (m, 4H), 2.43*(s, 3H*), 2.46 (s, 3H), 2.68-2.80 (m,
1H), 3.92 (dd, J = 6.6, 12.8, 1H), 3.97* (s, 3H*), 4.01 (s, 3H),
4.03-4.17 (m, 2H), 4.26* (dd, J=6.5, 12.8, 1H*), 4.51-4.66 (m,
2H), 4.73* (dd, J=2.4, 16.0, 1H*), 4.98-5.55* (bs, 1H*), 7.05-
7.28 (m, 3H, partially overlapped with CHCl3 peak), 7.31* (d,
J=8.1, 2H*), 7.39 (d, J=8.1, 2H), 7.37-7.47 (m, 1H), 7.75* (d,
J=8.3, 1H*), 7.79-7.87 (m, 2H þ 1H*), 7.90 (d, J=8.3, 2H),
8.18 (s, 1H), 8.21* (s, 1H*). MS (ESþ) m/z 560 (M þ H)þ.
Methyl (7R)-7-Azido-14-cyclohexyl-7,8-dihydro-6H-indolo-
[1,2-e][1,5]benzoxazocine-11-carboxylate (31a). To a solution
of 30a (423 mg, 0.757 mmol) in anhyd THF (12 mL), azido-
trimethylsilane (0.26 mL, 2.06 mmol) and tetrabutylammonium
triphenyldifluorosilicate (1.140 g, 2.112 mmol) were added at RT.
The reaction was stirred for 20 h at 65 °C, and then more azido-
trimethylsilane (0.26 mL, 2.06 mmol) and tetrabutylammonium
triphenyldifluorosilicate (1.140 g, 2.112 mmol) were added. The
mixture was stirred at 65 °C for another 36 h. The volatiles were
evaporated in vacuo, and the residue was dissolved in EtOAc
and washed with hydrochloric acid (1 N), sodium hydrogen
carbonate (saturated solution) and brine. The crude product
was purified by flash chromatography on silica gel (PE/EtOAc
95:5) to afford azide 31a as white foam (210 mg, 62%). 1H NMR
(400 MHz, CDCl3, atropisomers 6:4*) δ 1.14-1.31 (m, 1H),
1.32-1.45 (m, 2H), 1.62-1.72(m, 1H), 1.73-1.83 (m, 2H), 1.84-
2.13 (m, 4H), 2.69-2.84 (m, 1H), 3.58-3.66* (m, 1H*), 3.67-3.76
(m, 1H), 3.93-4.03 (m, 2H), 3.99 (s, 3H), 4.06* (d, J = 11.8,
1H*), 4.19-4.27 (m, 1H), 4.58 (dd, J=3.9, 15.1, 1H), 4.63* (dd,
J=3.5, 12.9, 1H*), 4.71* (dd, J=4.6, 15.6, 1H*), 7.13-7.32 (m,
3H), 7.40-7.50 (m, 1H), 7.75-7.84 (m,1H), 7.89 (d, J=8.6,1H),
8.13 (s, 1H), 8.29* (s, 1H*). MS (ESþ) m/z 431 (M þ H)þ. SFC
chromatography indicated an ee of 99.3% (Chiracel OJ-H
250 μm ꢀ 1.0, 5 μm; Tcol 35 °C, Pcol 100 bar, flow 9.99 mL/min,
modifier MeOH, 0.2% diethylamine (A); gradient 0-1 min 20%
A, 1-6.7 min to 60% A, then another 2 min isocratic.
14-Cyclohexyl-7-[(N,N-dimethylglycyl)(methyl)amino]-7,8-
dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylic Acid (29).
To a solution of 12 (23 mg, 0.044 mmol) in DCM (0.5 mL) was
added N,N-diisopropylethylamine (0.015 mL, 0.088 mmol), followed
by a solution of N,N-dimethylglycine (18 mg, 0.18 mmol),
HATU (67 mg, 0.18 mmol), and N,N-diisopropylethylamine
(38 μL, 0.22 mmol) in dichloromethane (1 mL). The reaction
mixture was concentrated in vacuo after 2 h and dissolved in
Methyl (7R)-7-Amino-14-cyclohexyl-7,8-dihydro-6H-indolo-
[1,2-e][1,5]benzoxazocine-11-carboxylate (32a). A solution of 31a
(210 mg, 0.520 mmol) in methanol (10 mL) containing Pd/C (10%,
w/w, 30 mg) was stirred under hydrogen at atmospheric pressure for
4 h. The catalyst was filtered off, and the solution was concentrated
to dryness under reduced pressure to afford 32a (192 mg, 92%). 1H
NMR (400 MHz, CDCl3, 2 atropisomers 6:4*) δ 1.14-1.30
(m,1H), 1.31-1.52 (m, 2H), 1.58-1.70 (m, 1H), 1.71-1.86 (m,

298 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Narjes et al.
2H), 1.87-2.26 (m, 4H), 2.68-2.89 (m, 1H), 3.44* (s, 3H*),
3.68-3.84 (bs, 4H), 3.85-4.02* (bs, 1H*), 4.03-4.25 (m, 3H þ
1H*), 4.31* (dd, J=2.9, 13.6, 1H*), 4.89 (t, J=12.9, 1H þ 1H*),
5.02* (dd, J=3.8, 16.3, 1H*), 7.00-7.12 (m, 1H þ 2H*), 7.19 (dd,
J = 1.4, 7.6, 1H), 7.22-7.31 (m, 2H, partialy overlapped with
CHCl3 peak), 7.34* (t, J=7.5, 1H*), 7.43* (d, J=8.6, 1H*), 7.47-
32.4, 36.1, 37.5, 42.0, 47.6, 50.2, 61.1, 73.2, 111.7, 119.1, 119.3,
119.9, 120.1, 121.4, 123.2, 123.9, 129.4, 131.1, 132.4, 135.2,
136.4, 157.5, 168.4. MS (ESþ) m/z 476 (M þ H)þ. HRMS
(M þ H)þ calcd for C29H38N3O3 476.2913; found 476.2922.
Chromatography on Chiralpak AD column gave an ee of 96%
(250 mm ꢀ 4.6 mm, 5 μm; flow 1.0 mL/min, isocratic 70:30;
eluent A 0.2% TFA/n-hexane; eluent B 0.2% TFA in EtOH
(containing 3% MeOH); 300 nm; Rt 5.37 min. [R]D20 = þ36.8
(c =0.5, CHCl3).
(7S)-14-Cyclohexyl-7-{[2-(dimethylamino)ethyl](methyl)amino}-
7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylic
Acid (34). The compound was obtained as described for its enan-
tiomer 33 starting from alcohol 10b. HRMS (M þ H)þ calcd for
C29H38N3O3 476.2913; found 476.2918. Chromatography on
Chiralpak AD column (conditions see compound 33) gave an ee
of 99%; RT 14.55 min. [R]D20 = -40 (c =0.1, CHCl3).
(7S)-14-Cyclohexyl-7-[2-(dimethylamino)ethoxy]-7,8-dihydro-
6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylic Acid (35). To
a suspension of 10a (49 mg, 0.12 mmol) in toluene (2.4 mL) was
added 30% w/w aq NaOH (0.16 mL, 1.20 mmol), followed by
tetrabutylammonium bromide (10 mg, 0.03 mmol). After stir-
ring for 30 min, 2-chloro-N,N-dimethylethanaminium chloride
(35 mg, 0.24 mmol) was added and the reaction mixture was
stirred at 60 °Cfor 16 h. More 2-chloro-N,N-dimethylethanaminium
chloride (17 mg, 0.12 mmol) was added, and the reaction mixture
was stirred at 80 °C for a further 4 h. The reaction mixture was
concentration in vacuo, dissolved in DMSO, and purified by
RP-HPLC to afford the TFA salt of 35 as a white powder (33 mg,
47%). 1H NMR (400 MHz, DMSO-d6, two atropisomers 1:1) δ
1.08-1.23 (m, 1H), 1.24-1.44 (m, 2H), 1.49-1.61 (m, 1H),
1.64-1.77 (m, 2H), 1.80-1.88 (m, 1H), 1.89-2.07 (m, 3H), 2.56
(s, 1.5 H), 2.63-2.73 (m, 1H), 2.78 (s, 1.5H), 3.07 (m, 1H), 3.27
(m, 1H), 3.68 (dd, J = 10.7, 14.5, 0.5H), 3.75-3.97 (m, 3H),
3.98-4.14 (m, 1.5H), 4.24 (dd, J=4.2, 13.2, 0.5H), 4.77 (dd, J=
2.7, 14.6, 0.5H), 4.93 (dd, J = 3.1, 15.6, 0.5H), 7.16-7.34 (m,
3H), 7.49 (d, J=6.8, 0.5H), 7.50 (d, J=6.8, 0.5H), 7.62 (d, J=
8.1, 0.5H), 7.69 (d, J=8.3, 0.5H), 7.82 (d, J=8.6, 0.5H), 7.89 (d,
J=8.3, 0.5H), 8.20 (s, 0.5H), 8.21 (s, 0.5H). 13C NMR (150 MHz,
CD3OD, 300 K, only signals for major atropisomer reported) δ
26.9, 27.8, 34.1, 38.0, 43.3, 44.6, 57.8, 63.9, 72.8, 78.0, 113.9, 120.0,
120.4, 120.8, 122.5, 123.9, 124.0, 124.5, 130.9, 131.9, 133.4, 138.3,
159.6, 171.4. MS (ESþ) m/z 463 (M þ H)þ. HRMS (M þ H)þ
calcd for C28H35N2O4 463.2597; found 463.2591; [R]D20=-41.2
(c =0.31, CH3OH).
7.55* (m, 1H*), 7.70-7.78 (m, 1H), 7.85 (d, J = 8.6, 1H), 8.08
20
(s, 1H), 8.27* (s, 1H*). MS (ESþ) m/z 405 (M þ H)þ; [R]D
þ46.4 (c=1.0, CHCl3).
=
(7R)-14-Cyclohexyl-7-{[2-(dimethylamino)ethyl](methyl)amino}-
7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylic
Acid (33). To a solution of 32a (868 mg, 2.146 mmol) in anhyd
trimethyl orthoformate (6 mL) was added tert-butyl N-(2-oxoethyl)-
carbamate (359 mg, 2.25 mmol), and the mixture was stirred at
room temperature overnight. The solution was concentrated to
dryness under reduced pressure and the crude dissolved in dry
methanol (9 mL). The resulting solution was treated with acetic
acid (0.37 mL, 6.44 mmol) and sodium cyanoborohydride (202 mg,
3.22 mmol). The mixture was stirred at room temperature for
1 h. Sodium hydroxide (2 mL, 1 N) was added, and after stirring
for 5 min the mixture was taken into EtOAc and washed with
water and brine. The crude product was purified by flash chro-
matography on silica gel (1:4 EtOAc þ 0.1% triethylamine/
petroleum ether þ 0.1%triethylamine; then 6/4) to afford methyl
(7R)-7-({2-[(tert-butoxycarbonyl)amino]ethyl}amino)-14-cyclohexyl-
7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylate as
colorless foam (524 mg, 46%). 1H NMR (300 MHz, DMSO-d6)
δ 1.05-1.50 (m, 12H), 1.51-1.62(m,1H),1.64-1.78 (m, 2H), 1.79-
1.88 (m, 1H), 1.89-2.07 (m, 3H), 2.73-2.77 (m, 1H), 3.08-3.40
(m, 5H), 3.65-3.84 (m, 1H), 3.89 (s, 3H), 3.84-4.03 (m, 1H),
4.11-4.29 (m, 2H), 4.91 (dd, J = 3.0, 14.8, 1H), 7.02-7.12 (bt,
1H), 7.15-7.36 (m, 3H), 7.48-7.58 (m, 1H), 7.72 (dd, J = 1.1,
8.3, 1H), 7.93 (d, J = 8.6, 1H), 8.33 (s, 1H). MS (ESþ) m/z 548
(M þ H)þ. This compound (524 mg, 0.956 mmol) was dissolved
in a mixture of dichloromethane/trifluoroacetic acid (3:1, v/v,
8 mL), and the mixture was stirred at room temperature. The
solution was concentrated to dryness under reduced pressure.
The residue was dissolved in dichloromethane and sodium
acetate (470 mg, 5.736 mmol), and 40% aq solution of form-
aldehyde (0.46 mL, 5.74 mmol) and sodium cyanoborohydride
(356 mg, 5.74 mmol) were added. After stirring at RT for 1 H,
sodium hydroxide (2 mL, 1 N) was added, and after stirring for
5 min, the mixture was taken into EtOAc and washed with brine.
Drying over sodium sulfate and concentration in vacuo gave
methyl (7R)-14-cyclohexyl-7-{[2-(dimethylamino)ethyl](methyl)-
amino}-7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-
carboxylate, which was used without any further purification.
MS (ESþ) m/z 490 (M þ H)þ. To a solution of the crude product
(280 mg, 0.572 mmol) in dioxane (12 mL) 1N KOH (3 mL, 1N in
water) was added and the mixture was stirred at 80 °C for 2 h.
Evaporation of the volatiles gave a residue, which was purified
by RP-HPLC (column Waters Symmetry prep. C18, 7 μm,
19 mm ꢀ 300 mm). Fractions containing the pure compound
were combined and freeze-dried to afford the bis-TFA salt of 33
as a white powder (123 mg, 45% over two steps). 1H NMR (400
MHz, DMSO-d6, 2 atropisomers 94:6*) δ 1.09-1.25 (m, 1H),
1.26-1.43 (m, 2H), 1.54 (d, J=12.2, 1H), 1.62-1.78 (m, 2H), 1.79-
2.06 (m, 4H), 2.39 (s, 3H), 2.61*(s, 6H*), 2.65-2.77 (m, 1H),
2.85 (s, 6H), 2.94 (dt, J = 5.5, 13.6, 1H), 3.04-3.15 (m, 2H),
3.18 (dt, J=5.6, 13.0, 1H), 3.23-3.34 (m, 1H), 3.74-3.80* (m,
1H* partially overlapped with the signal at 3.85 ppm), 3.85 (dd,
J=10.2, 14.4, 1 H), 4.07 (dd, J=9.0, 12.1, 1H), 4.11-4.17* (m,
1H* partially overlapped with the signal at 4.07 ppm), 4.31 (dd,
J = 4.3, 12.1, 1H), 4.64 (d, J = 14.4, 1H), 4.75* (d, J = 13.1,
1H*), 4.79-4.87* (m, 1H*), 7.24-7.37 (m, 3H), 7.50-7.59
(m, 1H), 7.63-7.66* (m, 1H* partially overlapped with the
signal at 7.69), 7.69 (dd, J = 0.8, 8.3, 1H), 7.82-7.85* (m, 1H*
partially overlapped with dd at 7.87 ppm), 7.87 (d, J=8.3, 1H),
8.15, 8.32* (s, 1H). 13C NMR (100 MHz; DMSO-d6, 320 K,
only data for the major atropisomer reported) δ 25.7, 26.4, 30.3,
Methyl 140-Cyclohexyl-2,2-dimethylspiro[1,3-dioxane-5,70-
indolo[1,2-e][1,5]benzoxazocine]-11-carboxylate (37). NaH (151 mg,
6.30 mmol, 60% dispersion in mineral oil) was added to a degassed
solution of 8(440 mg, 1.26 mmol) in DMF (10 mL). The suspension
was allowed to stir at RT for 20 min, and the resulting solution was
placed in an oil bath preheated at 70 °C. A degassed solution of 5,5-
bis(bromomethyl)-2,2-dimethyl-1,3-dioxane (0.57 g, 1.89 mmol),
prepared as described in ref 34 in dry DMF (6 mL), was added and
the mixture was stirred for at 70 °C for 1 h. Additional electrophile
(0.57 g, 1.89 mmol) was added in the same way, and stirring was
continued at 70 °C for another 3 h. After cooling to RT, the reaction
was quenched with an aq satd NH4Cl, acidified with 1N HCl and
extracted with Et2O. The crude material was purified by chroma-
tography (PE/EtOAc 5:1) to afford the 37 (50%) as the first fraction
(second fraction: recovered 8, 44%). 1H NMR (400 MHz, CDCl3)
δ 1.21-1.38 (m, 3H), 1.45 (s, 3H), 1.66 (s, 3H), 1.68-2.11 (m, 7H),
2.72-2.80 (m, 1H), 3.49 (d, J=12.3 Hz, 1H), 3.59 (d, J=11.8 Hz,
1H), 3.69 (d, J=12.3 Hz, 1H), 3.72 (d, J=15.3 Hz, 1H), 3.77 (d,
J=12.5 Hz, 1H), 3.83 (d, J=11.8 Hz, 1H), 3.93 (s, 3H), 4.15 (d, J=
12.5 Hz, 1H), 4.79 (d, J= 15.3 Hz, 1H), 7.13-7.17 (m, 2H), 7.23
(dd, J=7.8, 1.7 Hz, 1H), 7.41 (bt, J=7.8 Hz, 1H), 7.74 (dd, J=8.5,
1.3 Hz, 1H), 7.84 (d, J=8.5 Hz, 1H), 8.44 (s, 1H). MS (ESþ) m/z
490 (M þ H)þ.
140-Cyclohexyl-1-[2-(dimethylamino)ethyl]spiro[azetidine-3,70-
indolo[1,2-e][1,5]benzoxazocine]-110-carboxylic Acid (39). To a
suspension of 37 (135 mg, 0.28 mmol) in a mixture of MeOH and

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 299
THF (1:2, v/v, 12 mL) was added TsOH H2O (6 mg, 0.03 mmol),
patients: role of hepatitis B and C virus and alcohol. J. Hepatol.
2005, 42, 799–805.
3
and the solution was stirred at RT for 3 h. Filtration over a pad
of neutral alumina using EtOAc as eluent afforded, after evapo-
ration of the solvent, methyl 14-cyclohexyl-7,7-bis(hydroxymethyl)-
7,8-dihydro-6H-indolo[1,2-e][1,5]benzoxazocine-11-carboxylate
(110 mg, 89%). MS (ESþ) m/z 450.4 (M þ H)þ. Triflic anhydride
(220 mg, 0.78 mmol) was added at 0 °C to a solution of the diol
(100 mg, 0.22 mmol) in anhyd MeCN (4 mL). DIPEA (0.16 mL,
0.89 mmol) was then added, and the mixture was stirred at 0 °C
for 15 min. Another 0.16 mL of DIPEA were added, followed by
N,N-dimethylethane-1,2-diamine (0.049 mL, 0.445 mmol). The
mixture was stirred at 70 °C for 2 h. After removal of the solvent,
EtOAc was added, and the organic phase was washed with water
and brine. After drying and evaporation, the residue was
dissolved in dioxane (2 mL) and 1N KOH (1 mL) was added.
After heating to 75 °C for 2 h, the reaction was cooled to 0 °C,
acidified with 1N HCl, and purified by RP-HPLC to afford the
bis-TFA salt of 39 as a colorless powder (50 mg, 25%). 1H NMR
(300 MHz, CD3OD) δ 1.18-1.27 (m, 1H), 1.43 (bt, J=9.9 Hz,
2H), 1.67 (bd, J=12.6 Hz, 1H), 1.74-1.85 (m, 2H), 1.86-2.22
(m, 4H), 2.67-2.81 (m, 1H), 2.81 (s, 6H), 3.27 (t, J = 6.0 Hz,
2H), 3.46 (t, J = 6.0 Hz, 2H), 3.57 (d, J=9.8 Hz, 1H), 3.88 (d,
J = 9.5 Hz, 1H), 3.99 (d, J = 9.8 Hz, 1H), 4.03 (d, J = 9.5 Hz,
1H), 4.08 (d, J=15.4 Hz, 1H), 4.23 (d, J=12.7 Hz, 1H), 4.42 (d,
J=12.7 Hz, 1H), 5.10 (d, J=15.4 Hz, 1H), 7.28-7.33 (m, 3H),
7.52 (dt, J=1.8, 7.6 Hz, 1H), 7.81 (d, J=8.5 Hz, 1H), 7.92 (d,
J = 8.5 Hz, 1H), 8.53 (s, 1H). 13C NMR (75 MHz, CD3OD) δ
27.6, 28.45, 28.50, 34.2, 34.6, 38.6, 41.5, 46.0, 48.6, 53.6, 60.5,
62.2, 77.0, 113.5, 121.4, 121.6, 121.9, 122.0, 122.9, 124.4, 125.1,
132.1, 138.4, 139.1, 132.6, 134.5, 160.1, 171.6. MS (ESþ) m/z
488.5 (M þ H)þ. HRMS (M þ H)þ calcd for C30H38N3O3
488.2913; found 488.2916.
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Acknowledgment. We thank Silvia Pesci and Renzo Bazzo
for NMR experiments and Francesca Naimo, Anna Alfieri,
and Fabio Bonelli for analytical chemistry support. We are
indebted to Nadia Gennari, Monica Bisbocci, Sergio Serafini,
and Mauro Cerretani for the inhibition data on the NS5B
enzyme and the replicon, to Giacomo Paonessa and Linda
Bartholomew for the chimeric replicon data, and to Annalisa
Di Marco for CYP inhibition data. We are grateful to Lieven
Verhoye and Petra Premereur for excellent technical assis-
tance with the chimeric mouse experiment. The work was
funded in part by a concerted action grant from UGent (no.
01G00507) and by the Belgian state via the Interuniversity
Attraction Poles Program (P6/36-HEPRO). P.M. is a post-
doctoral fellow supported by The Research Foundation
;
Flanders (FWO-Vlaanderen). This work was supported in part
by a grant from the MIUR.
Supporting Information Available: Synthetic procedures and
spectroscopic data for compounds 6, 8, 10b, 13, 14, 16-20,
22-28, 36, and 38; table with purity data and retention times for
all tested compounds; enzyme and cell-based assay protocols;
soaking experiments and X-ray data collection. This material is
available free of charge via the Internet at http://pubs.acs.org.
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