Discovery of a potent and selective noncovalent linear inhibitor of the hepatitis C virus NS3 protease (BI 201335)

Article abstract of DOI:10.1021/jm100690x

C-Terminal carboxylic acid containing inhibitors of the NS3 protease are reported. A novel series of linear tripeptide inhibitors that are very potent and selective against the NS3 protease are described. A substantial contribution to the potency of these

Full text of DOI:10.1021/jm100690x

6466 J. Med. Chem. 2010, 53, 6466–6476
DOI: 10.1021/jm100690x
Discovery of a Potent and Selective Noncovalent Linear Inhibitor of the Hepatitis C Virus
NS3 Protease (BI 201335)
ꢀ
Montse Llinas-Brunet,* Murray D. Bailey, Nathalie Goudreau, Punit K. Bhardwaj, Josee Bordeleau, Michael Bos,
Yves Bousquet, Michael G. Cordingley, Jiamin Duan, Pat Forgione, Michel Garneau, Elise Ghiro, Vida Gorys,
ꢁ
€
ꢁ
Sylvie Goulet, Ted Halmos, Stephen H. Kawai, Julie Naud, Marc-Andre Poupart, and Peter W. White
Departments of Medicinal Chemistry, and Biological Sciences, Research and Development, Boehringer Ingelheim (Canada) Ltd.,
2100 Cunard Street, Laval, Quebec H7S 2G5, Canada
Received June 8, 2010
C-Terminal carboxylic acid containing inhibitors of the NS3 protease are reported. A novel series of
linear tripeptide inhibitors that are very potent and selective against the NS3 protease are described. A
substantial contribution to the potency of these linear inhibitors arises from the introduction of a C8
substituent on the B-ring of the quinoline moiety found on the P2 of these inhibitors. The introduction of
a C8 methyl group results not only in a modest increase in the cell-based potency of these inhibitors but
more importantly in a much better pharmacokinetic profile in rats as well. Exploration of C8-
substitutions led to the identification of the bromo derivative as the best group at this position, resulting
in a significant increase in the cell-based potency of this class of inhibitors. Structure-activity studies on
the C8-bromo derivatives ultimately led to the discovery of clinical candidate 29 (BI 201335), a very
potent and selective inhibitor of genotype1 NS3 protease with a promising PK profile in rats.
Hepatitis C virus (HCV) infects an estimated 130-170
million people worldwide1 and is one of the major reasons for
liver transplantation.2 Although the virus was discovered over
20 years ago,3 no direct-acting antiviral has yet reached the
market. Current therapy consists of treatment with pegylated
interferon in combination with ribavirin.4 This treatment is
usually accompanied by side effects and, most importantly, is
only effective in ∼40-50% of the patients infected with HCV
genotype 1. The quest for virus-specific therapies to treat HCV
infections began shortly after discovery of the virus and con-
tinues to be major focus of pharmaceutical research.
HCV is a positive strand RNA virus belonging to the
Flaviviridae family. The HCV genome consists of a 9.6 kb
RNA which encodes a polyprotein of ∼3000 amino acids. This
polyprotein is cleaved co- and post-translationally by both host
and viral proteases. The structural proteins are processed by
host peptidases, whereas the nonstructural (NS) proteins are
processed by two virally encoded proteases, the NS2/3 and the
NS3 proteases. The NS2/3 protease is responsible for a single
cleavage between NS2 and NS3, while the NS3 protease is
responsible for the release of the remaining downstream non-
structural proteins.5 Several viral proteins have been shown to
be essential for viral replication in chimpanzees and are thus
considered validated targets for antivirals.6
BILN 2061 (ciluprevir, 1)8,9 To date, no HCV inhibitor has
reached the market, although several compounds are currently
in clinical trials, with the NS3 protease inhibitors telaprevir10
and boceprevir,11 in phase III, being the most advanced.
Inhibitors of serine proteases are typically peptidic com-
pounds containing an electrophilic moiety that reacts with the
active site serine to form a reversible or irreversible covalent
bond.12 For example, telaprevir and boceprevir both feature
R-ketoamides that form a covalent reversible bond with the
active site residue serine 139.13 A distinctive feature of the NS3
protease is that it is also susceptible to product inhibition by
the N-terminal cleavage product of peptide substrates.14,15
The carboxylic acid terminus of cleavage products can estab-
lish crucial interactions with the enzyme active site, which are
unusual among serine proteases and thus also impart selec-
tivity with respect to other serine proteases.16,17 These are
noncovalent inhibitors that have only ionic interactions with
the catalytic site on the NS3 protease
We have reported several series of C-terminus carboxylic
acid containing HCV NS3 protease inhibitors, initially lead-
ing to the discovery of 1,18 a compound belonging to non-
covalent series of inhibitors (Table 1) and containing (1R,2S)-
1-amino-2-vinylcyclopropylcarboxylic acid (vinyl-ACCA)19
as the P120 residue. However, the development of this com-
pound was discontinued because of the observation of cardi-
otoxicity in high-dose monkey toxicology studies.21 These
discoveries fueled an intensive effort to discover novel, non-
covalent NS3 protease inhibitors.22 Several compounds from
these studies have entered clinical development. Clinical
candidates RG-7227/ITMN-191,23 TMC43530,24 MK-7009,25
and BMS-650032 all belong to this class of noncovalent inhibi-
tors containing a derivative of vinyl- or ethyl-ACCA at P1 and a
cyclopropylacylsulfonamide at P10.
The NS3 protease, located at the N-terminal third of the
NS3 protein, is a 180-amino acid chymotrypsin-like serine
protease, and it was one of the first viral proteins investigated
as a target for antiviral development.7 In 2002, the first proof-
of-concept in man for a direct acting anti-HCV drug was
achieved with the Boehringer Ingelheim NS3 protease inhibitor
*To whom correspondence should be addressed. Phone: (450) 682-4640.
Fax: (450) 682-4189. E-mail: montse.llinas-brunet@boehringer-ingelheim.com.
r
pubs.acs.org/jmc
Published on Web 08/17/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6467
Table 2. Capping Group Optimization
Table 1. Early Inhibitors of the HCV NS3 Protease
Synthesis of Inhibitors
The synthesis of the compounds shown in Table 2 was
performed in solution by sequential coupling of each amino
acid derivative using a previously described procedure.18,27
Since most of our SARefforts focused onvariationsof the 4R-
hydroxyproline substituents, represented by the compounds
shownin Tables 3 and 5, we wanted to develop the synthesis of
an advanced intermediate that would allow us to introduce
variations of this group as late as possible in the synthetic
sequence. Tripeptide 7 (Scheme 1) containing a good leaving
group at the 4-oxoproline was chosen as such an intermediate.
The desired compounds shown in Tables 3 and 5 were then
synthesized by introducing, via an SN2 reaction, the required
2-carbomethoxy-4-hydroxyquinoline derivative resulting in
an inversion of configuration at the 4-hydroxyproline. The
synthesis of tripeptide 6 was performed in solution by sequen-
tial coupling of each amino acid derivative, as described
previously.26 The synthesis of (1R,2S)-1-amino-2-vinylcyclo-
propylcarboxylic acid methyl ester used in the preparation of
these compounds and the synthesis of dipeptide 5 have also
both been reported.28
Coupling of dipeptide derivative 5 to the tert-leucine cyclo-
pentyl carbamate 4 yielded tripeptide 6. Next, the p-nitro-
benzoate group in 6 was hydrolyzed and the resulting free
hydroxyl group was converted to the brosylate 7 by treatment
with p-bromobenzensulfonyl chloride. The brosylate group
was displaced with the appropriately 8-substituted 2-carbo-
methoxy4-hydroxy-7-methoxyquinoline derivatives11a-e to
afford tripeptides 12a-e. Next, conversion of the quinoline
2-carbomethoxy moiety of tripeptides 12a-e into the final
aminothiazole derivatives was carried out by a selective
hydrolysis of the methyl ester, followed by conversion of the
resulting carboxylic acid to the various diazoketones and then
to the corresponding bromoketones 13a-e. Conversion to
diazoketones is carried out using diazomethane, and extreme
We decided to continue our work on the C-terminal
carboxylic acid, vinyl-ACCA containing inhibitors, since in
addition to providing very good potency, it also provides
excellent selectivity and better solubility than observed for other
classes of inhibitors. In addition to the macrocyclic inhibitor 1,
we have also published our work on a related series of linear
inhibitors, with compound 2 (Table 1) being representative of
this class.26 One advantage of the linear series of inhibitors lies in
the fact that the synthetic challenges and costs associated with the
production of the drug substance are significantly reduced.
In an attempt to merge both series, we introduced the
quinoline moiety found in 1 into the linear derivative 2. This
resulted in compound 3 (Table 1) which displayed more than a
10-fold improvement in potency in the replicon assay, provid-
ing the first noncovalent linear tripeptide inhibitor with
cellular activity below 100 nM. With this encouraging result,
our goal was to further increase the cell-based potency of this
linear series while evaluating and improving its ADME/PKa
profile. In this paper, we report our optimization studies that
have focused on the N-terminal end of the molecule (capping
group) and the P2 aminothiazolquinoline moiety that have
culminated in the discovery of compound 29 (BI 201335), a
potent and selective noncovalent linear tripeptide inhibitor
currently being investigated in phase IIb clinical trials.
a Abbreviations: ADME, administration, distribution, metabolism, and
excretion; AUC0-¥, area under the curve; DCM, dichloromethane; DIPEA.
diisopropylethylamine; DMAP, dimethylaminopyridine ; DMSO, dimethyl
sulfoxide; EtOAc, ethyl acetate; HPLC, high pressure liquid chromato-
graphy; PK, pharmacokinetic; MES, 2-(N-morpholino)ethanesulfonic acid;
TBTU, O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium tetrafluoro-
borate; TCEP, tris(2-carboxyethyl)phosphine; TFA, trifluoroacetic acid;
THF, tetrahydrofuran; Tris, tris(hydroxymethyl)aminomethane.

6468 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Table 3. Impact of Substitution on the A-Ring
Llinaꢀs-Brunet et al.
key interactions with the catalytic triad and the oxyanion
hole,17 providing not only good potency but also specificity
toward NS3 versus human and other serine proteases.16 In
addition, another advantage over the other classes of reported
NS3 protease inhibitors is the increase in solubility at high pH
for this series. The tert-leucine found at P3 was also main-
tained. One of the main roles of the P3 tert-butyl side chain is
to rigidify the peptidic backbone and to favor the overall
extended conformation, found in the bound state, for the
inhibitor in solution.26 Therefore, the current study focused
essentially on optimizing both the capping group and the P2
aminothiazolquinoline moiety. First, truncating the com-
pound to the hydroxyl dipeptide 15 (Table 2) resulted in a
substantial loss in potency. Urea derivatives were also eval-
uated but, as observed in previous series, did not provide any
significant advantage.18,26 Carbamate substituents, other
than the cyclopentyl, were next investigated. The derivative
16 containing a tert-butyl carbamate was found to be less
potent, while the corresponding four-membered ring (17) and
six-membered ring (18) carbamates displayed similar potency
to the cyclopentyl analogue (Table 2). However, in the cellular
assay the cyclopentyl derivative 3 was found to be the best,
displaying a 2-fold improved activity compared to the other
cyclic carbamates. Ultimately, this group was maintained
throughout our subsequent SAR efforts, since we had shown
previously that it fits well in the S4 binding pocket and also
imparts good properties to the inhibitor including chemical
stability.26
We evaluated the pharmacokinetic properties of reference
compound 3 in rats in order to see if the linear tripeptide series
had the potential to provide good oral bioavailability. Com-
pound 3 which was quite stable (T1/2 > 100 min) upon
incubation in both human and rat livermicrosomes was found
to have a good pharmacokinetic profile in rats, with an oral
bioavailability of 54% (Table 4). Specifically, compound 3
displayed a maximum plasma concentration (Cmax) of
caution should be used since diazomehane gas may explode
violently. Thebromoketones13a-e werethen treatedwiththe
properly substituted thioureas 14a-g to produce the required
aminothiazole derivatives. Finally the C-terminal esters were
hydrolyzed under basic conditions to produce the compounds
shown in Tables 3 and 5.
The synthesis of different 8-substituted 2-carbomethoxy
4-hydroxy-7-methoxyquinoline derivatives was carried out
from the corresponding anilines according to a reported pro-
cedure29 shown in Scheme 2.
Commercially or readily available anisidines (8a-e) were
treated with dimethylacetylene dicarboxylate 9 to form aducts
10a-ewhich, uponheatingat∼250 ꢀC, underwentcyclization
to produce the different 8-substituted 2-carbomethoxy 4-hydroxy-
7-methoxyquinoline derivatives 11a-e.
0.79 μM and an AUC0-¥ of 2.4 μM h after an oral dose of
3
5 mg/kg in rats, as well as a half-life (T1/2) of 2.6 h and a
moderate clearance (Cl) of 24 (mL/min)/kg following an
intravenous (iv) dose of 2 mg/kg. Encouraged by this very
promising profile, we focused on improving the cell-based
potency of this linear tripeptide series, setting a target of
EC50 < 10 nM while maintaining/improving ADME/PK
properties. On the basis of past success with the macrocyclic
series,18 we felt that modifying the P2 aminothiazol-quinoline
moiety might also represent the most attractive way of
achieving this goal.
A quick evaluation of the SAR at the aminothiazol moiety
revealed that an acetyl group as in compound 19 was well
tolerated providing a compound with slightly improved
potency over the reference compound 3 bearing an isopropyl,
as shown in Table 3. Increasing the size of the acyl group to a
tert-butylacetyl moiety as in 20 led to a partial loss in both the
enzymatic and cellular potencies. At this point, the potency of
these inhibitors still had to be improved by at least 10-fold,
and we reasoned that this gain would likely not come solely
through modification of the aminothiazol substituent. We
had previously shown in a tetrapeptide series that substitu-
tions around the B-ring of the quinoline were only tolerated at
the C7 and C8 positions, with the introduction of either a C7-
or a C8-methoxy producing a significant increase in potency
and giving rise to inhibitors with almost equipotent acti-
vities.26 Unfortunately, combining both the C7- and C8-methoxy
substituents into the same analogue was found to be detrimental
Results and Discussion
Compound 3 (Table 2), which resulted from the introduc-
tion of the compound 1 quinoline moiety into our linear, tri-
peptidyl series of inhibitors, was selected as the starting point
for further optimization. From the outset, the peptide back-
bone was retained, as we had already shown that it makes
optimal interactions with the enzyme which mimic the cano-
nical substrate binding mode, with both the NH and CO
groups of the P3 residue and the NH group of the P1 moiety
being involved in key hydrogen bonds with the protein.26 In
addition, both the vinyl-ACCA derivative at P1 and the tert-
butylglycine moiety at P3 were kept constant, as they already
had been subjected to extensive optimization in the past and
found to be optimal. The C-terminal carboxylic acid makes

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6469
Scheme 1. General Synthetic Route for Inhibitors of Tables 3 and 5a
a Reagents and conditions: (a) TBTU, DIPEA, DCM, room temp, 14 h; (b) 1 equiv of LiOH, H2O/THF, 0 ꢀC, 3 h; (c) brosyl chloride, DMAP, DCM,
0 ꢀC to room temp; (d) 11a-11c, Cs2CO3, NMP, 70 ꢀC, 7 h; (e) 1.1 equiv of 1 N NaOH, H2O-THF, 0 ꢀC to room temp; (f) IBCF, TEA, THF, 0 ꢀC to
room temp; (g) CH2N2, Et2O, 0 ꢀC to room temp; (h) HBr (48% aq), THF; (i) 14a-g, i-PrOH, 50 ꢀC, 1 h; (j) LiOH, H2O-MeOH-THF.
Scheme 2. Synthesis of the Different 8-Substituted 2-Carbo-
methoxy-4-hydroxy-7-methoxyquinoline Derivativesa
methoxy moiety at C7. Specifically, the introduction of a
methyl group at C8 was found to be well tolerated in both the
alkyl and acyl aminothiazol series (Table 3). Indeed, the intro-
duction of a C8-methyl to the reference analogue 3 resulted in
21, an equipotent inhibitor. Interestingly, the introduction of
the C8-methylonthe aminothiazoleacylderivative19 resulted
in an increase in the cell-based potency of the corresponding
inhibitor (22, EC50=19 nM). Extending the acetyl chain by
one carbon to the propionyl (compound 23) was also well
tolerated, giving rise to an equipotent analogue. Compounds
22 and 23 represented our first examples of acyclic tripeptide
inhibitors displaying EC50 < 20 nM in the replicon assay.
Encouraged by these results, we then evaluated the phar-
macokinetic parameters of these new analogues in order to see
if we had been able to maintain or improve their overall PK
profile through the simple chemical modifications we had
introduced. Surprisingly, compound 19, which was found to
be stable (T1/2 > 100 min) upon incubation in both human
and rat liver microsomes, did not achieve significant plasma levels
when dosed orally in rats at 5 mg/kg, in contrast to the reference
derivative 3. We generally found throughout our optimization
efforts that the rat oral absorption of these linear compounds was
greatly influenced by the nature of the substitution on the amino
a Reagents and conditions: (a) 8a-e, MeOH, 70 ꢀC, 5 h; (b) Ph2O,
240 ꢀC, 6-8 min.
to activity, not providing the desired additive effect. Never-
theless, we felt that it might be worth revisiting disubstitution
of the quinoline ring at C7 and C8, with the introduction
this time of smaller substituents at C8 while maintaining the

6470 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Llinaꢀs-Brunet et al.
Table 4. Pharmacokinetic Parameters in Rats after Oral and Intravenous Administration
oral, 5 mg/kg
iv, 2 mg/kg
Vss (L/kg)
compd
Cmax (μM)
AUC0-¥ (μM h)
T1/2 (h)
Cl ((mL/min)/kg)
F (%)
3
3
0.79
1.1
2.4
2.8
3.5
2.6
1.2
1.7
3.7
24
14
15
54
39
56
20
23
0.83
1.8
0.82
Table 5. Effect of C8-Substitution on the Potency of the Inhibitors
group of the aminothiazole moiety. Small alkyl (e.g., methyl) or
acyl groups (such as in 19) produced quite potent compounds
that unfortunately displayed very low oral absorption in rats or
higher species (data not shown). Increasing the size of the alkyl or
acyl group led, in most cases, to an increase in rat oral absorption.
For instance, compounds containing either an isopropyl such as
reference compound 3 or a tert-butylacetyl moiety as 20 showed
good oral PK profile when dosed in rats as highlighted in Table 4.
Interestingly, while an oral dose of 5 mg/kg showed a similar
profile for both compounds, an intravenous dose of 2 mg/kg led
to somewhat different parameters, with 20 showing a slower
clearance (14 (mL/min)/kg) and a smaller volume of distribution
(Vss= 0.83 vs 3.7 L/kg).
As mentioned earlier, the introduction of a C8-methyl on
the quinoline ring of the aminothiazol acyl series resulted in an
improvement in the cell-based potency of these inhibitors,
with 22 and 23 both displaying EC50 < 20 nM. However, the
full benefit of introducing the C8-Me became more evident
when we profiled the ADME properties of these inhibitors.
For instance, compound 22, when dosed orally in rats at
4 mg/kg, achieved plasma concentrations of 0.3 and 0.4 μM at
1 h and 2 h postdosing. This represented a clear advantage over
compound 19 which did not show significant plasma levels
(<0.02 μM) at a similar dose. Similarly, when compound 21
was dosed orally in rats (4 mg/kg), respective plasma concen-
trations of 0.9 and 1.2 μM were detected at 1 and 2 h time
points postdosing. Compound 23, containing a propionyl
group instead of the acetyl group, showed the best overall
profile, and its full rat PK evaluation is shown in Table 4. Very
good oral absorption was observed for this compound with a
Cmax of 0.82 μM and an AUC0-¥ of 3.5 μM h achieved after
3
administration of dose of 5 mg/kg. Its iv profile was also quite
interesting with a half-life(T1/2) of 1.7 h and a clearance (Cl) of
15 (mL/min)/kg following a 2 mg/kg dose. In addition to
displaying a good overall PK profile in rat, with a bioavail-
ability of 56%, another advantage of compound 23 over
the other analogues was its potency of <20 nM in the replicon
assay. Analogue 23 therefore became our new reference
compound.
Having observed an increase in potency and rat bioavail-
ability by introducing the C8-Me, we wanted to investigatethe
effect of other substituents such as halogen at this position. As
shown in Table 5, replacement of the C8-methyl moiety of 23
by a fluoro, chloro, or bromo substituent was well tolerated,
providing inhibitors with equipotent activities in the enzy-
matic assay (IC50 = 7 nM). The effect of these substituents on
the cellular potency was slightly different, with the C8-fluoro
analogue 24 being 2-fold less potent while both the C8-chloro
and C8-bromo analogues (25 and 26) showed improved
activities. Ultimately, the introduction of the C8-bromo sub-
stituent was found to be optimal, with analogue 26 exhibiting
an EC50 of 5 nM which represented our first observation of
single-digit nanomolar activity in the replicon assay for a
linear tripeptide inhibitor. The effect of the C8-bromo sub-
stituent on the overall ADME properties was subsequently
evaluated and is shown in Table 6.
Compound 26 was found to have human and rat liver
microsome stablilities (T1/2 > 300 min) comparable to that of
reference compound 23. However, when the bromo derivative
26 was dosed orally in rats, a significantly lower overall
plasma exposure was observed. Indeed, after oral dosing at
5 mg/kg, the Cmax (0.13 μM) and AUC0-¥ (0.34 μM h)
3
achieved for 26 (Table 6) were found to be considerably lower
(7- to 10-fold) than those observed for the C8-methyl analo-
gue 23 (Table 4). Following an iv dose of 2 mg/kg, a similar
half-life and volume of distribution were observed for both
compounds, although 26 showed a much faster clearance
(28 (mL/min)/kg), which may have contributed to the sub-
stantial decrease in overall bioavailability (F = 11%). Having
nevertheless achieved the desired level of potency with deri-
vative 26, we decided to keep the C8-bromo substituent and
revisit the SAR at the amonothiazole moiety with the goal of
finding a substituent that might display a better balance

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6471
Table 6. Pharmacokinetic Parameters in Rats after Oral and Intravenous Administration
oral, 5 mg/kg
iv, 2 mg/kg
Cl ((mL/min)/kg)
compd
Cmax (μM)
AUC0-¥ (μM h)
T1/2 (h)
Vss (L/kg)
F (%)
3
26
28
29
0.13
0.13
0.6
0.34
0.9
1.7
2.9
1.2
1.8
1.2
1.9
28
6
11
29
40
1.7
20
between potency and ADME profile. A wide variety of sub-
stituents were tolerated on the aminothiazole moiety in the
C8-bromo series, which ultimately enabled us to identify
several derivatives that fulfilled the desired potency criteria
(EC50 < 10 nM). Table 5 highlights three of these inhibitors,
suchasthe methylandisopropylcarbamate derivatives27and
28, respectively, and the acyl analogue 29. This last compound
displayed the best overall potency with IC50 and EC50 values
of 3 nM. Moreover, the in vitro ADME profile of these three
compounds revealed that they were all quite stable in human
(T1/2 > 100 min) and rat liver microsomes (T1/2 > 300 min).
Unfortunately, the two aminothiazole carbamate derivatives
did not achieve good plasma exposure when dosed orally in
rats, as was earlier observed for compound 26. For instance,
analogue 27 showed a plasma level of only 0.1 μM at 1 h
postdosing (4mg/kg oraldose). Only amarginalimprovement
was observed for compound 28 over 26 when its full PK
profile was evaluated in rat following an oral dose of 5 mg/kg
different substitutions at the aminothiazole moiety met our
potency criteria. Differentiation of these inhibitors was pos-
sible based on their rat PK profiles, and compound 29 was
ultimately selected for development. Initial four-week anti-
viral results for this promising drug candidate have been
reported in both treatment-naive patients and in patients
who failed previous treatment with current gold-standard
therapy.30 Compound 29 is now being evaluated in phase
IIb clinical trials, and this compound could become the first
noncovalent linear NS3 protease inhibitor available for treat-
ment of HCV patients
Experimental Section
General. All commercially obtained solvents and reagents
were used as received without further purification. All reactions
were carried out under an atmosphere of argon. Temperatures
are given in degrees Celsius. Solution percentages express a
weight to volume relationship, and solution ratios express a
volume to volume relationship, unless stated otherwise. NMR
spectra were recorded on a Bruker AVANCEII (400 MHz for
1H NMR) spectrometer and were referenced to either DMSO-d6
(2.50 ppm) or CDCl3 (7.27 ppm). Data are reported as follows:
chemical shift (ppm), multiplicity (s = singlet, d = doublet, t =
triplet, br = broad, m = multiplet), coupling constant (J, reported
to the nearest 0.5 Hz), and integration. Mass spectra were obtained
from a Micromass AutoSpec instrument using FAB as ionization
mode with NBA as a matrix support. Purification of crude material
was performed either by flash column chromatography or by
using a CombiFlash Companion using RediSep Silica or SilicaSep
columns according to preprogrammed gradient and flow rate
separation conditions in hexane/EtOAc or DCM/MeOH. The
final compounds were purified either by column chromatography
to yield the neutral compound or by preparative HPLC in a Waters
2767 sample manager with pumps 2525, column fluidics organizer
(CFO), PDA detector 2996, and MassLynx 4.1 using either a
Whatman Partisil 10-ODS-3 column, 2.2 cm ꢀ 50 cm, or a YMC
Combi-Prep ODS-AQ column, 50 mm ꢀ 20 mm i.d., S, 5 μm,
(Cmax = 0.13 μM, AUC0-¥ = 0.90 μM h) and a 2 mg/kg iv
3
dose (T1/2 = 2.9 h, Cl = 6 (mL/min)/kg), resulting in an oral
bioavailability of 29% (Table 6). However, the aminothiazole
acyl derivative 29 was demonstrated to possess an improved
PK profile when evaluated at these oral and intravenous doses
(Table 6). Indeed, the oral exposure was significantly im-
proved with respect to the other C8-bromo containing deri-
vatives with a Cmax of 0.60 μM, AUC0-¥ = 1.7 μM h. The
3
T1/2 was found to be 1.2 h with moderate clearance. The
overall oral bioavailability in rats for compound 29 was 40%.
Although the PK profile of 29 was not as good as that of 23
(F = 56%), its superior potency in both the enzymatic and
replicon assays made it a more interesting candidate for
further development. A further beneficial feature of com-
pound 29 lies in the fact that the compound partitions
favorably into the liver in rats with a 40-fold increase in liver
versus plasma concentration after oral dosing. Moreover,
differences in the cross-species PK profile of these compounds
further supported the selection of 29 (manuscript in pre-
paration). For all these reasons 29 was selected for further
advancement in development, and the complete preclinical
profile of this compound will be published elsewhere. Com-
pound 29 is currently being evaluated in phase IIb clinical trials.
˚
120 A, and a linear gradient program from 2% to 100% AcCN/
water (0.06% TFA). Fractions were analyzed by analytical HPLC,
and the pure fractions were combined, concentrated, frozen, and
lyophilized to yield the desired compound as the trifluoroacetate
salt. The lyophilized TFA salt was dissolved in water, and either
2 N or 10 N NaOH was added dropwise until a pH of 14 was
maintained. The pH was readjusted to 7 with 2 N HCl and the
neutral product extracted into EtOAc (3ꢀ). The combined extracts
were washed with water (2ꢀ) and brine (1ꢀ), dried over MgSO4,
filtered, and evaporated to dryness. The obtained solid was
dissolved in acetonitrile/water, frozen, and relyophilized to provide
the neutral product. The neutral inhibitor HPLC purity was
measured by using an Waters Alliance 2695 separation module
with a Waters 2487 dual λ absorbance detector. A YMC Com-
biscreen ODS-AQ HPLC column was used: 50 ꢀ 4.6 mm i.d.; S-5
μm, 12 nm (catalog no. CCAQS05-0546WT, AQ-300-CC). Linear
gradient at 220 nm was as follows: 5-100% B; solvent A, 0.06%
TFA/H2O; sovlent B, 0.06% TFA/CH3CN. Parameters of the
analytical C18 reversed phase column and the eluting system
were as follows: 0.06% TFA in water-0.06% TFA in acetoni-
trile gradient (20-100% acetonitrile over 30 min). All com-
pounds tested in vitro were determined to be of >95% purity
by HPLC.
Conclusion
Very potent and specific linear tripeptide inhibitors of the
NS3 serine protease have been identified. Our initial goal of
obtaining linear inhibitors with cell-based potencies less than
10 nM and with good overall ADME profiles was achieved.
Crucial to these achievements was the discovery of the impact
of the C8-substituent of the P2 quinoline moiety of these
inhibitors on the properties of the present inhibitors. The
introduction of a C8-Me group was beneficial to increasing
the plasma exposure in rats after oral dosing and, in some
cases, also improved the potency of the inhibitors. Scanning
different substituents at the C8-position of the quinoline
revealed that the bromo group was optimal for increasing
the cell-based potency of this series. Several derivatives with

6472 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Llinaꢀs-Brunet et al.
Enzymatic Assays. Biochemical assays were carried out as
described previously.31 In brief, reactions comprised 0.5 nM
genotype 1b NS3-NS4A enzyme,32 5 μM fluorogenic depsipep-
tide substrate anthranilyl-DDIVPAbu[C(O)-O]AMY(3-NO2)-
TW-OH, and inhibitor in a reaction buffer composed of 50 mM
Tris-HCl, pH 8.0, 0.25 M sodium citrate, 0.01% n-dodecyl-β-D-
maltoside, 1 mM TCEP, and 5% DMSO. Mixtures were
incubated at 23 ꢀC for 60-70 min and terminated by the
addition of 1 M MES (pH 5.8). Fluorescence of the N-terminal
product anthranilyl-DDIVP-Abu was measured using a Polar-
Star Galaxy plate reader (BMG). Calculated percent inhibition
at each inhibitor concentration was then used to determine the
median effective concentration (IC50) by the NLIN procedure of
SAS (Statistical Software System; SAS Institute Inc., Cary, NC).
Cell-Based Replicon Assay. The bicistronic luciferase reporter
replicon, encoding the Con1 genotype 1b NS2-NS5B coding
region, and the experimental procedures for measuring EC50
values in the experiments reported above have been described
elsewhere.33 Compounds were incubated with cells for 72 h, and
the relative levels of luciferase present were determined using the
Bright-Glo luciferase substrate (Promega) on a Packard Top-
count instrument EC50 values were determined by the nonlinear
regression routine NLIN procedure of SAS (EC50).
Pharmacokinetics. All protocols involving animal experimen-
tation were reviewed and approved by the local Animal Care
and Use Committee. In-life procedures were in compliance with
the Guide for the Care and Use of Laboratory Animals from the
Canadian Council of Animal Care. All rat PK and distribution
studies were performed at Boehringer Ingelheim (Canada)
Ltd. Rat pharmacokinetic studies were performed using male
Sprague-Dawley rats (275-300 g, Charles River, St-Constant,
Quebec, Canada). Animals were fasted overnight with access to
10% dextrose in water. Standard oral PK with individual
compounds were performed at an oral dose of 5 mg/kg in a dosing
volume of 10 mL/kg of a vehicle consisting of 0.5% Methocel and
0.3% Tween-80. Blood samples were collected from the canulated
right carotid at 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, and 8 h postdosing, and
plasma samples from three rats were pooled at each time point.
Intravenous PK experiments were performed at a dose of 2 mg/kg,
with an additional sample collection time at 5 min. For rapid oral
PK screening studies, a cassette of four compounds (4 mg/
compound) were dosed in two rats and plasma samples collected
only at 1 and 2 h postdosing. Extracts for LC-MS analysis were
obtained from plasma samples by solid-phase extraction. The
temporal profiles of drug concentrations in plasma were analyzed
by noncompartmental methods using WinNonlin (version 3.1;
Scientific Consulting, Inc., Cary, NC).
described above gave 11c. 1H NMR (400 MHz, CDCl3): δ 8.96 (s,
1H), 8.10 (dd, J = 9.0, 1.5 Hz, 1H), 7.09 (dd, J = 9.0, 8.4 Hz, 1H),
6.92 (s, 1H), 4.06 (s, 3H), 4.05 (s, 3H). MS (ESI): 249.9 (M - H)-,
252.0 (M þ H)þ. HPLC homogeneity: 97%.
2-Carbomethoxy-8-chloro-4-hydroxy-7-methoxyquinoline (11d).
Starting with 2-chloro-3-methoxyaniline 8d and using the proce-
dure described above gave 11d. H NMR (400 MHz, CDCl3):
1
δ 9.47 (s, 1H), 8.32 (d, J = 8.8 Hz, 1H), 7.18 (s, 1H), 7.12 (d, J =
8.7 Hz, 1H), 4.06 (s, 3H), 4.05 (s, 3H). MS (ESI): 265.9 (M - H)-,
267.8 (M þ H)þ. HPLC homogeneity: >99%.
2-Carbomethoxy-8-bromo-4-hydroxy-7-methoxyquinoline (11e).
Starting with 2-bromo-3-methoxyaniline 8e and using the proce-
1
dure described above gave 11e. H NMR (400 MHz, CDCl3): δ
9.37 (s, 1H), 8.33 (d, J= 8.9 Hz, 1H), 7.06 (d, J= 8.9 Hz, 1H), 6.95
(s, 1H), 4.07 (s, 6H). MS (ESI): 310.0, 312.0 (M - H)-, 312.0, 314.0
(M þ H)þ. HPLC homogeneity: 98%.
Synthesis of Inhibitors (Compounds from Tables 3 and 5). The
inhibitors from these tables were synthesized as outlined in the
chemistry section of the manuscript. The synthetic sequence
leading to the preparation of compound 29 serves as a repre-
sentative example for the series.
N-[(Cyclopentyloxy)carbonyl]-L-tert-leucine (4). To a suspen-
sion of carbonic acid cyclopentyl ester 2,5-dioxopyrrolidin-1-yl
ester (9.00 g; 39.6 mmol) and tert-leucine (6.24 g; 47.5 mmol) in
THF (350 mL) were added distilled water (100 mL) and triethyl-
amine (16.6 mL, 119 mmol). The homogeneous solution was
allowed to stir at room temperature for 2.5 h. The THF was
evaporated and the aqueous residue further diluted with water
(100 mL). The mixture was rendered basic by the addition of 1 N
NaOH (25 mL, final pH >10) and then extracted with EtOAc
(2 ꢀ 200 mL). The aqueous phase was then acidified with 1 N
HCl (∼70 mL, final pH <2) and the turbid solution extracted
with EtOAc (2 ꢀ 200 mL). The organic phase was dried
(MgSO4), filtered, and concentrated to afford carbamate 3
1
(8.69 g) as a white solid. H NMR (400 MHz DMSO-d6): δ
12.46 (s, 1H), 7.11 (d, J = 8.9 Hz, 1H), 4.94 (bs, 1H), 3.78 (d, J =
8.9 Hz, 1H), 1.85-1.72 (m, 2H), 1.70-1.48 (m, 6H), 0.94 (s, 9H).
MS (ESI): 241.8 (M - H)-, 243.9 (M þ H)þ, 265.9 (M þ Na)þ.
HPLC homogeneity: 95%.
N-[(Cyclopentyloxy)carbonyl]-3-methyl-L-valyl-(4S)-N-[(1R,2S)-
2-ethenyl-1-(methoxycarbonyl)cyclopropyl]-4-hydroxy-L-prolin-
amide (6). Carbamate 4 (6.15 g, 22.5 mmol) and TBTU (7.72 g,
24.7 mmol) were suspended in dichloromethane (100 mL) and
rapidly stirred before the addition of DIPEA (3.92 mL, 22.5
mmol) at room temperature. After 10 min, a solution of (3S,5S)-
5-{[(1R,2S)-2-ethenyl-1-(methoxycarbonyl)cyclopropyl]carbamoyl}-
pyrrolidin-3-yl 4-nitrobenzoate (5) (10.39 g, 23.6 mmol) in
anhydrous dichloromethane (100 mL) containing DIPEA
(4.11 mL, 23.62 mmol) was poured into the reaction mixture.
The resulting yellow solution was stirred for 14 h before being
concentrated in vacuo. The resulting residue was diluted with
EtOAc (450 mL) and washed with 0.05 N HCl (2 ꢀ 200 mL),
saturated Na2CO3 (300 mL), and finally saturated brine (150 mL).
The combined extracts were dried over MgSO4, filtered, and
concentrated to afford tripeptide 6 as a pale yellow foam (15.68 g,
quantitative). 1H NMR (400 MHz, CDCl3): about 90:10
mixture of cis-trans proline rotamers, major rotamer descrip-
tion; δ 8.27 (d, J = 8.4 Hz, 2H), 8.19 (d, J = 8.4 Hz, 2H), 7.96 (s,
1H), 5.77-5.66 (m, 1H), 5.62-5.56 (m, 1H), 5.29-5.15 (m, 2H),
5.15-5.04 (m, 2H), 4.82 (d, J = 9.0 Hz, 1H), 4.32-4.25 (m, 2H),
3.93 (d, J = 11.8 Hz, 1H), 3.58 (s, 3H), 3.04 (br d, J = 14.4 Hz,
1H), 2.34-2.25 (m, 1H), 2.10-2.01 (m, 1H), 1.96-1.54 (m, 9H),
1.41 (dd, J = 9.4, 5.5 Hz, 1H), 1.07 (s, 9H). MS (ESI): 627.4
(M - H)-, 629.4 (M þ H)þ, 652.4 (M þ Na)þ. HPLC homo-
geneity: 96%.
2-Carbomethoxy-4-hydroxy-7-methoxy-8-methylquinoline (11b).
Dimethyl acetylenedicarboxylate 9 (3.6 mL, 29.3 mmol) was added
dropwise to a solution of 2-methyl-3-methoxyaniline 8b (3.95 g,
28.8 mmol) in MeOH (100 mL) (reaction is exothermic). The
mixture was heated at a gentle reflux for 5 h, cooled, and concen-
trated under vacuum. The crude material was purified by column
chromatography (SiO2) to provide diester 10b (6.5 g, 80.8%).
Diester 10b (6.5 g, 23.3 mmol) was then dissolved in diphenyl ether
(12 mL) and the reaction mixture placed into a preheated sand bath
at a bath temperature of 350-400 ꢀC. Once the reaction mixture
attained an internal temperature of 240 ꢀC (MeOH evolution
observed at 230-240 ꢀC), a count of 6 min was begun before the
bath was removed and the reaction mixture allowed to cool to room
temperature. When the mixture cooled, the product precipitated.
The mixture was diluted with diethyl ether, filtered, and dried to
give a tan colored solid (3.48 g crude). The crude material was
purified by silica gel chromatography to provide 11b (2.1 g, 37%) as
a pale yellow solid. 1H NMR (400 MHz, CDCl3): δ 8.81 (s, 1H),
8.25 (d, J = 9.0 Hz, 1H), 7.05 (d, J = 9.0 Hz, 1H), 6.92 (s, 1H), 4.06
(s, 3H), 3.99 (s, 3H), 2.38 (s, 3H). MS (ESI): 246.0 (M - H)-, 248.1
(M þ H)þ. HPLC homogeneity: 95%.
N-[(Cyclopentyloxy)carbonyl]-3-methyl-L-valyl-(4S)-N-[(1R,2S)-2-
ethenyl-1-(methoxycarbonyl)cyclopropyl]-4-hydroxy-L-prolinamide (7).
Tripeptide 6 (15.68 g, 24,97 mmol) was dissolved in THF (200 mL)
and water (30 mL), and the resulting solution was cooled to 0 ꢀC. A
solution of lithium hydroxide monohydrate (1.18 g, 28.12 mmol) was
2-Carbomethoxy-8-fluoro-4-hydroxy-7-methoxyquinoline (11c).
Starting with 2-fluoro-3-methoxyaniline 8c and using the procedure

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6473
added over 3 min during vigorous stirring. After 3 h at 0 ꢀC, the excess
base was neutralized with 1 N HCl (final pH ∼6) before the THF was
evaporated. The resulting aqueous suspension (yellow gum) was
extracted with EtOAc (2 ꢀ 200 mL) and washed with saturated
NaHCO3 (2 ꢀ 300 mL). The combined extracts were dried over
MgSO4 and concentrated to give a yellow foam. Silica gel chroma-
tography using EtOAc as an eluent afforded the free alcohol as a white
amorphous solid (9.77 g, 91%). The alcohol (9.77 g, 20.85 mmol) was
combined with brosyl chloride (11.19 g, 43.79 mmol) and DMAP (254
mg, 2.09 mmol) in dichloromethane (75 mL) at 0 ꢀC before being
treated dropwise with triethylamine (10.2 mL, 72.98 mmol). The
yellow solution was stirred 1 h at 0 ꢀC and then slowly warmed to
room temperature and stirred for 60 h. The reaction mixture was
concentrated to dryness, diluted with EtOAc, and washed with
saturated NaHCO3, water, and saturated brine before being dried
(MgSO4), filtered, and concentrated to dryness. The crude material
was purified by silica gel chromatography with a gradient of EtOAc in
hexanes to provide the tripeptide 6 as a white foam (11.66 g, 74% over
three steps). 1H NMR (400 MHz, DMSO-d6): about 84:16 mixture of
cis-trans proline rotamers, major rotamer description; δ 8.58 (s, 1H),
7.92(d,J= 8.6 Hz, 2H), 7.85 (d, J= 8.6 Hz, 2H), 6.94 (d, J=8.7Hz,
1H), 5.68-5.56 (m, 1H), 5.23 (dd, J = 17.3, 1.6 Hz, 1H), 5.20-5.12
(m,1H),5.09(dd,J= 10.3, 1.8 Hz, 1H), 4.94-4.89 (m, 1H), 4.36 (dd,
J = 9.0, 4.7 Hz, 1H), 4.07-3.95 (m, 2H), 3.59 (dd, J = 11.7, 4.1 Hz,
1H), 3.56 (s, 3H), 2.43-2.27 (m, 1H), 2.09-1.99 (m, 2H), 1.84-1.70
(m, 2H), 1.69-1.46 (m, 7H), 1.25 (dd, J = 9.3, 5.3 Hz, 1H), 0.92
(s, 9H). MS (ESI): 698.1, 699.1 (M - H)-, 698.2, 700.2 (M þ H)þ.
HPLC homogeneity: 99%.
N-[(Cyclopentyloxy)carbonyl]-3-methyl-L-valyl-(4R)-4-{[8-bromo-
7-methoxy-2-(methoxycarbonyl)quinolin-4-yl]oxy}-N-[(1R,2S)-
2-ethenyl-1-(methoxycarbonyl)cyclopropyl]-L-prolinamide (12e).
Tripeptide 7 (0.5 g, 0.71 mmol), bromoquinoline 11e (234 mg,
0.75 mmol), and ground cesium carbonate (56 mg; 0.78 mmol)
were dissolved in 1-methyl-2-pyrrolidinone (7.6 mL) before
being heated to 70 ꢀC (7 h). The solution was cooled to room
temperature and poured into EtOAc before being washed with
H2O, saturated NaHCO3, and brine. The organic phase was
dried (MgSO4), filtered, and concentrated to dryness before
being purification by silica gel chromatography (7:3 EtOAc in
hexanes) to afford tripeptide 12e (0.429 g, 77%) as a white solid.
1H NMR (400 MHz, CDCl3): about 89:11 mixture of cis-trans
proline rotamers, major rotamer description; δ 8.21 (d, J =
9.3 Hz, 1H), 7.58 (s, 1H), 7.51 (s, 1H), 7.30 (d, J = 9.3 Hz, 1H),
5.81-5.70 (m, 1H), 5.42-5.36 (m, 1H), 5.31 (dd, J = 17.3, 1.2
Hz, 1H), 5.25 (d, J = 9.2 Hz, 1H), 5.15 (dd, J = 10.2, 1.2 Hz,
1H), 4.90-4.84 (m, 1H), 4.81 (t, J = 7.6 Hz, 1H), 4.48 (d, J =
12.1 Hz, 1H), 4.36 (d, J = 9.6 Hz, 1H), 4.08 (s, 3H), 4.07 (s, 3H),
3.96 (dd, J = 12.0, 4.4 Hz, 1H), 3.69 (s, 3H), 3.06-2.98 (m, 1H),
2.44 (dd, J = 14.2, 8.2 Hz, 1H), 2.14 (dd, J = 17.2, 8.8 Hz, 1H),
1.88 (dd, J = 8.1, 5.7 Hz, 1H), 1.84-1.49 (m, 8H), 1.44 (dd, J =
9.6, 5.6 Hz, 1H), 1.07 (s, 9H). MS (electrospray): 775.2 (M þ
2H)þ. HPLC homogeneity: 96%.
saturated NaHCO3 (2ꢀ), brine (1ꢀ), and then dried (MgSO4),
filtered, and concentrated to give the corresponding diazoketone
(2.91 g). The diazoketone (2.91 g, 3.7 mmol) was dissolved in THF
(80 mL) and cooled to 0 ꢀC before the dropwise addition of an HBr
solution (48% aq, 2.7 mL) followed by stirring for 1.25 h. The
mixture was quenched with a saturated NaHCO3 solution and the
THF evaporated in vacuo. The residue was diluted with EtOAc,
washed with a saturated NaHCO3 and saturated brine before
being dried (MgSO4), filtered, and concentrated to provide the
crude bromoketone 13e (2.99 g, 96%). 1H NMR (400 MHz,
CDCl3): about 90:10 mixture of cis-trans proline rotamers, major
rotamer description; δ 8.19 (d, J = 9.2 Hz, 1H), 7.58 (s, 1H), 7.41
(s, 1H), 7.31 (d, J = 9.3 Hz, 1H), 5.81-5.70 (m, 1H), 5.43-5.35
(m, 1H), 5.31 (d, J = 17.2 Hz, 1H), 5.25-5.10 (m, 4H), 4.86-4.74
(m, 2H), 4.45 (d, J = 12.0 Hz, 1H), 4.32 (d, J = 9.5 Hz, 1H), 4.08
(s, 3H), 3.94 (dd, J = 12.1, 4.1 Hz, 1H), 3.69 (s, 3H), 3.06-2.98 (m,
1H), 2.43 (dd, J = 13.5, 8.4 Hz, 1H), 2.14 (dd, J = 17.2, 8.8 Hz,
1H), 1.91-1.85 (m, 1H), 1.84-1.37 (m, 9H), 1.05 (s, 9H). MS
(electrospray): 773.3 (MH þ 2)þ 771.3 (M þ H)þ 769 (M - H)-.
HPLC homogeneity: 95%.
N-[(Cyclopentyloxy)carbonyl]-3-methyl-L-valyl-(4R)-4-[(8-bromo-
7-methoxy-2-{2-[(2-methylpropanoyl)amino]-1,3-thiazol-4-yl}quinolin-
4-yl)oxy]-N-[(1R,2S)-1-carboxy-2-ethenylcyclopropyl]-L-prolin-
amide (29). A yellow solution of R-bromoketone 13e (0.80 g, 0.956
mmol) and isobutyrylthiourea 14e (0.159 g, 1.05 mmol) in iso-
propanol (30 mL) was heated at 75 ꢀC for 1 h. The solution was
cooled to room temperature and evaporated to dryness. The crude
material was purified by silica gel chromatography to provide the
methyl ester of compound 29 (0.59 g, 70% yield). To a solution of
the methyl ester (0.59 g, 0.67 mmol) in a 12 mL mixture of THF/
MeOH/H2O (2:1:1) was added solid LiOH monohydrate (0.28 g,
6.66 mmol). The resulting mixture was stirred at room temperature
overnight and then concentrated. The residue was partitioned
between EtOAc (30 mL) and saturated brine (30 mL) before the
pH was adjusted to 6.0 with 1 N HCl. The aqueous phase was re-
extracted several times with EtOAc. The combined organic extract
were washed with water and saturated brine, dried (MgSO4), and
concentrated to yield compound 29 as a pale yellow solid (0.57 g,
92% yield). 1H NMR (400 MHz, DMSO-d6): about 86:14 mixture
of rotamers, major rotamer description; δ 12.48 (br s, 1H) 12.34
(s, 1H), 8.56 (s, 1H), 8.17 (d, J = 9.2 Hz, 1H), 8.03 (s, 1H), 7.46
(s, 1H), 7.34 (d, J = 9.4 Hz, 1H), 7.02 (d, J = 8.6 Hz, 1H), 5.80-
5.65 (m, 1H), 5.48-5.37 (m, 1H), 5.18 (dd, J = 17.0, 1.4 Hz, 1H),
5.05 (dd, J = 10.1, 1.6 Hz, 1H), 4.63-4.55 (m, 1H), 4.43 (t, J =
8.6 Hz, 1H), 4.37 (d, J = 11.7 Hz, 1H), 4.07 (d, J = 8.6 Hz, 1H),
4.01 (s, 3H), 3.94 (br d, J = 9.4 Hz, 1H), 2.85-2.77 (m, 1H),
2.58-2.49 (m, 1H), 2.30-2.20 (m, 1H), 2.06-1.97 (m, 1H),
1.78-1.20 (m, 10H), 1.14 (d, J = 6.7 Hz, 6H), 0.96 (s, 9H). MS
(ESI): 869.1, 871.1 (M - H)-, 867.1, 869.1 (M þ H)þ. HPLC
homogeneity: >99%.
Synthesis of Compounds from Tables 3 and 5. The synthesis
was performed using the same procedure as outlined above for
compound 29 but using the appropriate quinoline and thiourea
derivatives. The overall reaction sequence was performed on a
similar scale and gave comparable yields. The characterization
of these compounds is as follows.
N-[(Cyclopentyloxy)carbonyl]-3-methyl-L-valyl-(4R)-N-[(1R,2S)-
1-carboxy-2-ethenylcyclopropyl]-4-({7-methoxy-2-[2-(propan-
2-ylamino)-1,3-thiazol-4-yl]quinolin-4-yl}oxy)-L-prolinamide (3).
1H NMR (400 MHz, DMSO-d6): about 82:18 mixture of rota-
mers, major rotamer description; δ 12.50 (br s, 1H), 8.54 (s, 1H),
8.02 (d, J = 9.0 Hz, 1H), 7.70 (d, J = 7.2 Hz, 1H), 7.44 (s, 1H),
7.18 (s, 1H), 7.28 (s, 1H), 7.01 (d, J = 8.6 Hz, 1H), 6.98 (d, J =
9.0 Hz, 1H), 5.43-5.35 (m, 1H), 5.19 (d, J = 16.6 Hz, 1H), 5.06
(d, J = 11.1 Hz, 1H), 4.77-4.70 (m, 1H), 4.43 (t, J = 8.3 Hz,
1H), 4.32 (d, J = 11.3 Hz, 1H), 4.12 (d, J = 8.6 Hz, 1H),
4.00-3.93 (m, 1H), 3.90 (s, 3H), 3.86-3.76 (m, 1H), 2.53-2.45
(m, 1H), 2.08-1.98 (m, 1H), 1.81-1.36 (m, 9H), 1.31-1.25 (m,
1H), 1.25 (d, J = 5.7 Hz, 6H), 0.97 (s, 9H). MS (ESI): 761.4 (M -
H)-, 763.5 (M þ H)þ. HPLC homogeneity: 99%.
N-[(Cyclopentyloxy)carbonyl]-3-methyl-L-valyl-(4R)-4-{[8-bromo-
2-(bromoacetyl)-7-methoxyquinolin-4-yl]oxy}-N-[(1R,2S)-2-ethenyl-
1-(methoxycarbonyl)cyclopropyl]-L-prolinamide (13e). Tripeptide
12e (4.00 g, 5.17 mmol) in 75 mL of a 4:1 mixture of THF and
H2O was cooled to 0 ꢀC before the addition of an aqueous solution
of 1 N NaOH (5.7 mL). The resulting solution was stirred for
15 min at 0 ꢀC, followed by 1.5 h at room temperature. The mixture
was quenched with 1 M HCl, evaporated to near dryness, diluted
with water, then frozen and lyophilized to provide the correspond-
ing acid as the sodium salt (3.93 g). The sodium salt was dissolved
in THF (65 mL) followed by triethylamine (0.981 mL, 7.04 mmol)
before being cooled to 0 ꢀC. Isobutyl chloroformate (0.913 mL,
7.04 mmol) was added dropwise, and the resulting white suspen-
sion was stirred at 0 ꢀC for 1 h. This mixture was then treated with a
solution of diazomethane (0.67 M in diethyl ether, 45 mL, 30.19
mmol). The reaction mixture was stirred for 15 min at 0 ꢀC and
then 1 h at room temperature before being concentrated to a thick
suspension. This suspension was dissolved in EtOAc, washed with

6474 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17
Llinaꢀs-Brunet et al.
(1R,2S)-2-Ethenyl-1-{[(4R)-1-[(2S)-2-hydroxy-3,3-dimethyl-
butanoyl]-4-({7-methoxy-2-[2-(propan-2-ylamino)-1,3-thiazol-
4-yl]quinolin-4-yl}oxy)-L-prolyl]amino}cyclopropanecarboxylic
Acid (15). 1H NMR (400 MHz, DMSO-d6): about 82:18 mixture
of rotamers, major rotamer description; δ 10.40 (br s, 1H), 8.60
(s, 1H), 8.18 (br s, 1H), 8.06 (d, J = 9.2 Hz, 1H), 8.02 (d, J =
8.0 Hz, 1H), 7.81 (br s, 1H), 7.71 (s, 1H), 7.39 (br d, J = 8.3 Hz,
1H), 5.79-5.65 (m, 2H), 5.20 (d, J = 16.9 Hz, 1H), 5.07 (d, J =
10.2 Hz, 1H), 4.53 (t, J = 8.6 Hz, 1H), 4.33 (d, J = 12.7 Hz, 1H),
4.21-4.08 (m, 1H), 3.98 (s, 3H), 3.99-3.93 (m, 1H), 3.90 (br s,
1H), 2.57 (dd, J = 12.8, 7.2 Hz, 1H), 2.35-2.27 (m, 1H),
2.08-2.00 (m, 1H), 1.92-1.82 (m, 1H), 1.56 (dd, J = 7.8, 4.9
Hz, 1H), 1.25 (d, J = 6.7 Hz, 6H), 0.90 (s, 9H). MS (ESI): 650.3
(M - H)-, 652.3 (M þ H)þ. HPLC homogeneity: 95%.
amino]-1,3-thiazol-4-yl}-7-methoxyquinolin-4-yl)oxy]-L-prolin-
amide (20). 1H NMR (400 MHz, DMSO-d6): about 85:15
mixture of rotamers, major rotamer description; δ 12.42 (br
s, 1H), 12.29 (s, 1H), 8.54 (s, 1H), 8.07 (d, J = 9.2 Hz, 1H), 8.02
(br s, 1H), 7.46 (s, 1H), 7.35 (s, 1H), 7.06 (d, J = 9.2 Hz, 1H),
7.00 (d, J = 8.6 Hz, 1H), 5.79-5.66 (m, 1H), 5.47-5.38 (m,
1H), 5.19 (d, J = 17.2 Hz, 1H), 5.06 (d, J = 10.3 Hz, 1H),
4.78-4.67 (m, 1H), 4.48-4.33 (m, 2H), 4.12 (d, J = 8.4 Hz,
1H), 4.03-3.93 (m, 1H), 3.92 (s, 3H), 2.58-2.49 (m, 1H), 2.43
(s, 2H), 2.31-2.19 (m, 1H), 2.08-1.98 (m, 1H), 1.83-1.15 (m,
10H),), 1.04 (s, 9H),), 0.97 (s, 9H). MS (ESI): 817.4 (M - H)-,
819.4 (M þ H)þ. HPLC homogeneity: > 99%.
N-[(Cyclopentyloxy)carbonyl]-3-methyl-L-valyl-(4R)-N-[(1R,2S)-
1-carboxy-2-ethenylcyclopropyl]-4-({7-methoxy-8-methyl-
2-[2-(propan-2-ylamino)-1,3-thiazol-4-yl]quinolin-4-yl}oxy)-L-
prolinamide (21). 1H NMR (400 MHz, DMSO-d6): about 88:12
mixture of rotamers, major rotamer description; δ 12.55 (br s,
1H), 8.58 (s, 1H), 8.07 (d, J = 7.1 Hz, 1H), 8.06 (br s, 1H), 7.89
(d, J = 9 Hz, 1H), 7.57 (br d, J = 6.9 Hz, 1H), 7.34 (br s, 1H),
7.02 (d, J = 8.6 Hz, 1H), 5.78-5.66 (m, 1H), 5.61-5.49 (br s,
1H), 5.23 (d, J = 17.0 Hz, 1H), 5.07 (d, J = 11.9 Hz, 1H), 4.55
(br s, 1H), 4.46 (t, J = 8.4 Hz, 1H), 4.38 (d, J = 11.2 Hz, 1H),
4.08 (d, J = 8.6 Hz, 1H), 4.03-3.97 (m, 1H), 3.96 (s, 3H),
3.92-3.83 (m, 1H), 2.56 (s, 3H), 2.53-2.47 (m, 1H), 2.38-2.25
(m, 1H), 2.08-1.98 (m, 1H), 1.71-1.35 (m, 9H), 1.35-1.27 (m,
1H), 1.27 (d, J = 6.3 Hz, 6H), 0.96 (s, 9H). MS (ESI): 775.4 (M -
H)-, 777.5 (M þ H)þ. HPLC homogeneity: 95%.
N-(tert-Butoxycarbonyl)-3-methyl-L-valyl-(4R)-N-[(1R,2S)-
1-carboxy-2-ethenylcyclopropyl]-4-({7-methoxy-2-[2-(propan-
2-ylamino)-1,3-thiazol-4-yl]quinolin-4-yl}oxy)-L-prolinamide (16).
1H NMR (400 MHz, DMSO-d6): about 90:10 mixture of rota-
mers, major rotamer description; δ 12.52 (br s, 1H), 8.60 (s, 1H),
8.27, (br s, 1H), 8.22 (d, J = 9.2 Hz, 1H), 8.06 (d, J = 7.3 Hz, 1H),
7.86 (s, 1H), 7.75 (s, 1H), 7.26 (d, J = 8.6 Hz, 1H), 6.77 (d, J = 8.0
Hz, 1H), 5.81-5.68 (m, 2H), 5.19 (d, J = 17.2 Hz, 1H), 5.06 (d,
J = 10.2 Hz, 1H), 4.54-4.43 (m, 2H), 4.22-4.09 (m, 1H), 4.01 (d,
J = 8.3 Hz, 1H), 4.00-3.90 (m, 1H), 3.97 (s, 3H), 2.59 (dd, J =
13.9, 7.8 Hz, 1H), 2.38-2.29 (m, 1H), 2.01 (dd, J = 17.2, 8.9 Hz,
1H), 1.56 (dd, J = 7.7, 5.0 Hz, 1H), 1.31-1.25 (m, 1H), 1.26 (d,
J = 6.7 Hz, 6H), 1.19 (s, 9H), 0.97 (s, 9H). MS (ESI): 751.2 (M -
H)-, 749.2 (M þ H)þ. HPLC homogeneity: H2O): 97%.
N-[(Cyclopentyloxy)carbonyl]-3-methyl-L-valyl-(4R)-4-({2-[2-
(acetylamino)-1,3-thiazol-4-yl]-7-methoxy-8-methylquinolin-4-yl}-
oxy)-N-[(1R,2S)-1-carboxy-2-ethenylcyclopropyl]-L-prolinamide
(22). 1H NMR (400 MHz, DMSO-d6): about 85:15 mixture of
rotamers, major rotamer description; δ 12.32 (br s, 2H), 8.57 (s,
1H), 8.15-8.03 (m, 1H), 8.04 (d, J = 9.2 Hz, 1H), 7.47-7.37 (m,
1H), 7.29 (d, J = 8.8 Hz, 1H), 7.03 (d, J = 8.6 Hz, 1H),
5.78-5.65 (m, 1H), 5.45-5.38 (m, 1H), 5.19 (d, J = 17.0 Hz,
1H), 5.05 (d, J = 10.7 Hz, 1H), 4.72-4.62 (m, 1H), 4.46-4.32
(m, 2H), 4.16-4.08 (m, 1H), 4.03-3.90 (m, 1H), 3.94 (s, 3H),
2.60 (s, 3H), 2.30-2.19 (m, 1H), 2.20 (s, 3H), 2.06-1.97 (m, 1H),
1.81-1.21 (m, 11H), 0.97 (s, 9H). MS (ESI): 775.4 (M - H)-,
777.5 (M þ H)þ. HPLC homogeneity: 99%.
N-[(Cyclobutyloxy)carbonyl]-3-methyl-L-valyl-(4R)-N-[(1R,2S)-
1-carboxy-2-ethenylcyclopropyl]-4-({7-methoxy-2-[2-(propan-
2-ylamino)-1,3-thiazol-4-yl]quinolin-4-yl}oxy)-L-prolinamide (17).
1H NMR (400 MHz, DMSO-d6): about 87:13 mixture of rota-
mers, major rotamer description; δ 12.47 (br s, 1H), 8.59 (s, 1H),
8.29-8.20(brs, 1H), 8.19 (d, J = 9.2Hz, 1H), 8.04 (brs, 1H), 7.85
(br s, 1H), 7.73 (br s, 1H), 7.32 (br d, J = 8.6 Hz, 1H), 7.17 (d, J =
8.2 Hz, 1H), 5.79-5.67 (m, 2H), 5.20 (dd, J = 17.1, 1.4 Hz, 1H),
5.07 (dd, J = 10.2, 1.8 Hz, 1H), 4.53-4.42 (m, 2H), 4.36-3.96(m,
3H), 3.98 (s, 3H), 3.93 (dd, J = 12.5, 3.1 Hz, 1H), 2.64-2.54 (m,
1H), 2.38-2.28 (m, 1H), 2.06-1.91 (m, 3H), 1.89-1.74 (m, 2H),
1.70-1.52 (m, 2H), 1.47-1.37 (m, 1H), 1.29-1.26 (m, 1H), 1.26
(d, J = 6.7 Hz, 6H), 0.96 (s, 9H). MS (ESI): 747.4 (M - H)-,
749.3 (M þ H)þ. HPLC homogeneity: 97%.
N-[(Cyclopentyloxy)carbonyl]-3-methyl-L-valyl-(4R)-N-[(1R,2S)-
1-carboxy-2-ethenylcyclopropyl]-4-({7-methoxy-8-methyl-
2-[2-(propanoylamino)-1,3-thiazol-4-yl]quinolin-4-yl}oxy)-L-pro-
linamide (23). 1H NMR (400 MHz, DMSO-d6): about 85:15
mixture of rotamers, major rotamer description; δ 12.37 (br s,
1H), 12.33 (br s, 1H), 8.55 (s, 1H), 8.15-8.02 (m, 1H), 8.04 (d,
J = 9.0 Hz, 1H), 7.49-7.38 (m, 1H), 7.29 (d, J = 9.2 Hz, 1H),
6.99 (d, J = 8.8 Hz, 1H), 5.78-5.66 (m, 1H), 5.46-5.39 (m, 1H),
5.18 (d, J = 17.0 Hz, 1H), 5.05 (d, J = 10.0 Hz, 1H), 4.71-4.62
(m, 1H), 4.47-4.32 (m, 2H), 4.15-4.09 (m, 1H), 4.03-3.92 (m,
1H), 3.94 (s, 3H), 2.60 (s, 3H), 2.58-2.40 (m, 2H), 2.30-2.20 (m,
1H), 2.07-1.97 (m, 1H), 1.80-1.21 (m, 11H), 1.13 (t, J = 7.5
Hz, 3H), 0.97 (s, 9H). MS (ESI): 789.4 (M - H)-, 791.4 (M þ
H)þ. HPLC homogeneity: 98%.
N-[(Cyclopentyloxy)carbonyl]-3-methyl-L-valyl-(4R)-N-[(1R,2S)-
1-carboxy-2-ethenylcyclopropyl]-4-({8-fluoro-7-methoxy-
2-[2-(propanoylamino)-1,3-thiazol-4-yl]quinolin-4-yl}oxy)-L-pro-
linamide (24). 1H NMR (400 MHz, DMSO-d6): about 85:15
mixture of rotamers, major rotamer description; δ 12.39 (br s,
1H) 12.35 (s, 1H), 8.54 (s, 1H), 8.03 (s, 1H), 7.92 (d, J = 9.2 Hz,
1H), 7.47 (s, 1H), 7.38 (t, J = 8.3 Hz, 1H), 6.98 (d, J = 8.7 Hz,
1H), 5.78-5.65 (m, 1H), 5.47-5.38 (m, 1H), 5.18 (dd, J = 17.2,
1.4 Hz 1H), 5.05 (dd, J = 10.3, 1.6 Hz, 1H), 4.69-4.60 (m, 1H),
4.48-4.30 (m, 2H), 4.14-3.90 (m, 2H), 3.98 (s, 3H), 2.60-2.39 (m,
3H), 2.30-2.20 (m, 1H), 2.06-1.97 (m, 1H), 1.80-1.37 (m, 8H),
1.37-1.20 (m, 2H), 1.12 (t, J =7.5 Hz, 3H), 0.96 (s, 9H). MS (ESI):
793.3 (M - H)-, 795.3 (M þ H)þ. HPLC homogeneity: 98%.
N-[(Cyclopentyloxy)carbonyl]-3-methyl-L-valyl-(4R)-N-[(1R,2S)-
1-carboxy-2-ethenylcyclopropyl]-4-({8-chloro-7-methoxy-2-[2-
(propanoylamino)-1,3-thiazol-4-yl]quinolin-4-yl}oxy)-L-prolinamide (25).
N-[(Cyclohexyloxy)carbonyl]-3-methyl-L-valyl-(4R)-N-[(1R,2S)-1-
carboxy-2-ethenylcyclopropyl]-4-({7-methoxy-2-[2-(propan-
2-ylamino)-1,3-thiazol-4-yl]quinolin-4-yl}oxy)-L-prolinamide (18).
1H NMR (400 MHz, DMSO-d6): about 88:12 mixture of rota-
mers, major rotamer description; δ 12.51 (br s, 1H), 8.62 (s, 1H),
8.26 (br s, 1H), 8.22 (d, J = 8.2 Hz, 1H), 8.05 (br s, 1H), 7.86 (br s,
1H), 7.72 (br s, 1H), 7.24 (br d, J = 7.9 Hz, 1H), 7.11 (d, J =
8.4 Hz, 1H), 5.82-5.61 (m, 2H), 5.21 (d, J = 17.2 Hz, 1H), 5.08
(d, J = 10.2 Hz, 1H), 4.61-4.37 (m, 2H), 4.25-3.86 (m, 4H), 3.98
(s, 3H), 2.64-2.48 (m, 1H), 2.38-2.28 (m, 1H), 2.01 (dd, J =
17.1, 8.8 Hz, 1H), 1.72-1.41 (m, 5H), 1.32-1.05 (m, 7H), 1.28 (d,
J = 6.8 Hz, 6H), 0.98 (s, 9H). MS (ESI): 775.4 (M - H)-, 777.3
(M þ H)þ. HPLC homogeneity: 98%.
N-[(Cyclopentyloxy)carbonyl]-3-methyl-L-valyl-(4R)-4-({2-[2-
(acetylamino)-1,3-thiazol-4-yl]-7-methoxyquinolin-4-yl}oxy)-
N-[(1R,2S)-1-carboxy-2-ethenylcyclopropyl]-L-prolinamide (19).
1H NMR (400 MHz, DMSO-d6): about 86:14 mixture of rotamers,
major rotamer description; δ 12.38 (br s, 2H), 8.57 (s, 1H), 8.37 (br
s, 1H), 8.18 (d, J = 9.2 Hz, 1H), 7.63 (s, 1H), 7.56 (s, 1H), 7.21 (d,
J = 9.0 Hz, 1H), 7.02 (d, J = 7.9 Hz, 1H), 5.77-5.67 (m, 1H), 5.59
(br m, 1H), 5.19 (d, J = 17.2 Hz, 1H), 5.06 (d, J = 10.2 Hz, 1H),
4.61-4.54 (m, 1H), 4.51-4.41 (m, 2H), 4.07 (d, J = 8.6 Hz, 1H),
4.01-3.95 (m, 1H), 3.95 (s, 3H), 2.59 (dd, J=12.9, 7.2 Hz, 1H),
2.35-2.26 (m, 1H), 2.23 (s, 3H), 2.07-1.97 (m, 1H), 1.71-1.31 (m,
9H), 1.27 (dd, J = 9.4, 4.9 Hz, 1H), 0.97 (s, 9H). MS (ESI): 761.1
(M - H)-, 763.1 (M þ H)þ. HPLC homogeneity: > 99%.
N-[(Cyclopentyloxy)carbonyl]-3-methyl-L-valyl-(4R)-N-[(1R,2S)-
1-carboxy-2-ethenylcyclopropyl]-4-[(2-{2-[(3,3-dimethylbutanoyl)-

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 17 6475
References
1H NMR (400 MHz, DMSO-d6): about 88:12 mixture of rotamers,
major rotamer description; δ12.49 (br s, 1H) 12.34 (s, 1H), 8.55 (s, 1H),
8.12 (d, J= 9.2 Hz, 1H), 8.05 (s, 1H), 7.48 (s, 1H), 7.39 (d, J=9.2Hz,
1H), 6.98 (d, J = 8.6 Hz, 1H), 5.80-5.66 (m, 1H), 5.49-5.37 (m, 1H),
5.19 (d, J = 17.0 Hz, 1H), 5.07 (d, J = 10.4 Hz, 1H), 4.70-4.54 (m,
1H),4.45(t,J= 8.6 Hz, 1H), 4.38 (d, J= 11.5 Hz, 1H), 4.09 (d, J=8.8
Hz,1H),4.01(s,3H),3.96(brd,J= 10.6 Hz, 1H), 2.60-2.43 (m, 3H),
2.32-2.22(m, 1H), 2.06-1.98(m, 1H), 1.80-1.41 (m, 8H), 1.39-1.21
(m, 2H), 1.13 (t, J = 7.5 Hz, 3H), 0.96 (s, 9H). MS (ESI): 809.3 (M -
H)-, 811.3 (M þ H)þ. HPLC homogeneity: 96%.
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(26). 1H NMR (400 MHz, DMSO-d6): about 85:15 mixture of
rotamers, major rotamer description; δ 12.50 (br s, 1H) 12.33
(s, 1H), 8.55 (s, 1H), 8.17 (d, J = 9.2 Hz, 1H), 8.04 (s, 1H), 7.47
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5.80-5.66 (m, 1H), 5.47-5.37 (m, 1H), 5.19 (d, J = 17.1 Hz,
1H), 5.07 (d, J = 10.4 Hz, 1H), 4.67-4.55 (m, 1H), 4.45 (t, J =
8.4 Hz, 1H), 4.38 (d, J = 11.7 Hz, 1H), 4.08 (d, J = 8.8 Hz, 1H),
4.01 (s, 3H), 3.95 (br d, J = 8.6 Hz, 1H), 2.60-2.47 (m, 3H),
2.32-2.21 (m, 1H), 2.06-1.98 (m, 1H), 1.77-1.37 (m, 8H),
1.36-1.23 (m, 2H), 1.13 (t, J = 7.6 Hz, 3H), 0.96 (s, 9H). MS
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N-[(Cyclopentyloxy)carbonyl]-3-methyl-L-valyl-(4R)-4-[(8-bromo-
7-methoxy-2-{2-[(methoxycarbonyl)amino]-1,3-thiazol-4-yl}-
quinolin-4-yl)oxy]-N-[(1R,2S)-1-carboxy-2-ethenylcyclopropyl]-
L-prolinamide (27). 1H NMR (400 MHz, DMSO-d6): about
88:12 mixture of rotamers, major rotamer description; δ 12.49
(br s, 1H) 12.06 (s, 1H), 8.55 (s, 1H), 8.16 (d, J = 9.2 Hz, 1H),
8.04 (s, 1H), 7.45 (s, 1H), 7.35 (d, J = 9.4 Hz, 1H), 6.98 (d, J =
8.6 Hz, 1H), 5.80-5.66 (m, 1H), 5.48-5.37 (m, 1H), 5.19 (d, J =
17.1 Hz, 1H), 5.06 (d, J = 10.2 Hz, 1H), 4.65-4.55 (m, 1H), 4.45
(t, J = 8.5 Hz, 1H), 4.37 (d, J = 11.9 Hz, 1H), 4.08 (d, J = 8.8
Hz, 1H), 4.01 (s, 3H), 3.96 (br d, J = 9.4 Hz, 1H), 3.78 (s, 3H),
2.58-2.48 (m, 1H), 2.32-2.21 (m, 1H), 2.06-1.98 (m, 1H),
1.71-1.37 (m, 8H), 1.36-1.26 (m, 2H), 0.96 (s, 9H). MS (ESI):
855.1, 857.1 (M - H)-, 857.2, 859.2 (M þ H)þ. HPLC homo-
geneity: >99%.
N-[(Cyclopentyloxy)carbonyl]-3-methyl-L-valyl-(4R)-4-{[8-bromo-
7-methoxy-2-(2-{[(propan-2-yloxy)carbonyl]amino}-1,3-thiazol-
4-yl)quinolin-4-yl]oxy}-N-[(1R,2S)-1-carboxy-2-ethenylcyclopropyl]-
L-prolinamide (28). 1H NMR (400 MHz, DMSO-d6): about
85:15 mixture of rotamers, major rotamer description; δ 12.45
(br s, 1H) 11.90 (s, 1H), 8.55 (s, 1H), 8.16 (d, J = 9.2 Hz, 1H),
8.03 (s, 1H), 7.45 (s, 1H), 7.35 (d, J = 9.4 Hz, 1H), 6.98 (d, J =
8.6 Hz, 1H), 5.81-5.66 (m, 1H), 5.48-5.37 (m, 1H), 5.19 (d, J =
17.0 Hz, 1H), 5.07 (d, J = 10.4 Hz, 1H), 4.99 (m, 1H), 4.67-4.55
(m, 1H), 4.45 (t, J = 8.4 Hz, 1H), 4.38 (d, J = 11.9 Hz, 1H), 4.08
(d, J = 8.8 Hz, 1H), 4.01 (s, 3H), 3.96 (br d, J = 9.4 Hz, 1H),
2.58-2.48 (m, 1H), 2.32-2.21 (m, 1H), 2.06-1.98 (m, 1H),
1.71-1.37 (m, 8H), 1.36-1.26 (m, 2H), 1.29 (d, J = 6.3 Hz, 6H),
0.96 (s, 9H). MS (ESI): 883.2, 885.2 (M - H)-, 885.3, 887.3
(M þ H)þ. HPLC homogeneity: >99%.
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ꢁ
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