Discovery of GS-9669, a thumb site II non-nucleoside inhibitor of NS5B for the treatment of genotype 1 chronic hepatitis C infection

Article abstract of DOI:10.1021/jm401420j

Investigation of thiophene-2-carboxylic acid HCV NS5B site II inhibitors, guided by measurement of cell culture medium binding, revealed the structure-activity relationships for intrinsic cellular potency. The pharmacokinetic profile was enhanced through incorporation of heterocyclic ethers on the N-alkyl substituent. Hydroxyl groups were incorporated to modulate protein binding. Intrinsic potency was further improved through enantiospecific introduction of an olefin in the N-acyl motif, resulting in the discovery of the phase 2 clinical candidate GS-9669. The unexpected activity of this compound against the clinically relevant NS5B M423T mutant, relative to the wild type, was shown to arise from both the N-alkyl substituent and the N-acyl group.

Full text of DOI:10.1021/jm401420j

Article
pubs.acs.org/jmc
Discovery of GS-9669, a Thumb Site II Non-Nucleoside Inhibitor of
NS5B for the Treatment of Genotype 1 Chronic Hepatitis C Infection
Scott E. Lazerwith,* Willard Lew, Jennifer Zhang, Philip Morganelli, Qi Liu, Eda Canales,
Michael O. Clarke, Edward Doerffler, Daniel Byun, Michael Mertzman,† Hong Ye, Lee Chong,
Lianhong Xu, Todd Appleby, Xiaowu Chen, Martijn Fenaux,‡ Ahmad Hashash,§ Stephanie A. Leavitt,
Eric Mabery,∥ Mike Matles, Judy W. Mwangi, Yang Tian, Yu-Jen Lee, Jingyu Zhang, Christine Zhu,
Bernard P. Murray, and William J. Watkins*
Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, California 94404, United States
S
* Supporting Information
ABSTRACT: Investigation of thiophene-2-carboxylic acid HCV NS5B site II
inhibitors, guided by measurement of cell culture medium binding, revealed
the structure−activity relationships for intrinsic cellular potency. The
pharmacokinetic profile was enhanced through incorporation of heterocyclic
ethers on the N-alkyl substituent. Hydroxyl groups were incorporated to
modulate protein binding. Intrinsic potency was further improved through
enantiospecific introduction of an olefin in the N-acyl motif, resulting in the
discovery of the phase 2 clinical candidate GS-9669. The unexpected activity
of this compound against the clinically relevant NS5B M423T mutant, relative to the wild type, was shown to arise from both the
N-alkyl substituent and the N-acyl group.
retain useful levels of activity only in genotype (GT) 1 HCV,
although a recent publication has reported compounds with
EC50 ≤ 80 nM against GTs 2−6.20 Characteristic of antiviral
agents with this mechanism of action is the emergence of NS5B
M423T as a resistant mutant, both clinically and in in vitro
studies.4
The attractiveness of NS5B thumb site II inhibitors for use in
combination regimens was increased by early reports of clinical
results with 2, which demonstrated a 2.3 log10 reduction in
HCV RNA in plasma when dosed as monotherapy at 700 mg
twice daily for 8 days in GT 1 infected patients,21 and by 3
(then referred to as VCH-222), which achieved a 3.7 log10
reduction in plasma viral RNA after 3 days of treatment at 750
mg twice daily.22
As part of a broad HCV discovery effort, we initiated a
program to discover a new NS5B thumb site II inhibitor for use
in future single-tablet regimens. Such an agent would be more
potent than previous examples (to minimize dose), would
exhibit improved pharmacokinetics (to enable once-daily
dosing), and would have physicochemical properties compat-
ible with convenient coformulation with other agents. In view
of the clinical potency of 3 and the relatively small molecular
size of the class, we chose to explore the thiophene-2-carboxylic
acids for optimization.
INTRODUCTION
■
In recent years, as clinical data from numerous new small-
molecule hepatitis C virus (HCV) inhibitors with different
modes of action have been reported, it has become increasingly
clear that interferon-based treatment regimens will soon be
supplanted by all-oral combinations of direct-acting antiviral
agents.1,2 In this context, regimens that deliver the maximum
benefit to patients in terms of efficacy and brevity of treatment
duration will be those that consist of multiple agents targeting
different inhibitory mechanisms with complementary resistance
profiles. An ideal combination would include drugs with once
daily dosing schedules that could be coformulated into a single
tablet regimen.3
One of the most well-studied targets for the inhibition of
HCV replication by small molecules is the viral polymerase
NS5B.4 Inhibitors fall into two broad categories: nucleos(t)ide
analogues that become incorporated into the nascent RNA
chain and result in defective elongation, and non-nucleoside
inhibitors whose activity results from binding at diverse
allosteric sites, four of which have been shown to be exploitable
for clinical efficacy. Among these is thumb site II, located ∼30
Å from the active site. Screening hits from various HCV
discovery programs5−10 are now known to act by binding at this
site, and their optimization11−18 has resulted in the
identification of several clinical candidates, three of which
(covering disparate chemotypes) are illustrated in Figure 1. The
medicinal chemistry optimization programs leading to HCV-
371 (1)5 and filibuvir (2)16 have been published, but the
structure of lomibuvir (3) was revealed only upon recent
assignment of a nonproprietary name.19 All of these inhibitors
Special Issue: HCV Therapies
Received: September 13, 2013
© XXXX American Chemical Society
A
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Figure 1. Structures of selected clinical candidates.
a
Scheme 1. Generalized Synthetic Route
a
Reagents and conditions: (a) trifluoroacetic anhydride; (b) LDA, then I2; (c) t-Bu acetylene, Pd(Ph)2Cl2, CuI; (d) K2CO3.
METHODS AND RESULTS
Table 1. CCM Binding and HCV Replicon Activity of Initial
Analogues
■
Potency was determined using subgenomic GT 1a and 1b replicon
assays23−25 in a medium containing 10% fetal bovine serum. To
establish that the observed replicon inhibition was specific, cytotoxicity
was determined in the replicon cell line in parallel with potency, and
all the compounds in this article exhibited CC50 > 20 μM. Cytotoxicity
in the MT4 cell line was measured as previously described.25 In order
to better interpret structure−activity relationships, the unbound
fraction in both cell culture medium (CCM) and human plasma was
measured by dialysis for many compounds. Intrinsic potency was
defined as [replicon EC50 × free fraction in CCM]. Plasma-adjusted
potency was defined as [replicon EC50 × (plasma/CCM free fraction
ratio)] and was used to estimate the potency expected in a clinical
setting.
Most of the new analogues were synthesized using the general
synthetic route shown in Scheme 1, which begins with the amino
protection and selective iodination of commercially available methyl 3-
aminothiophene-2-carboxylate (4) to afford the 5-iodothiophene 5.
Sonagashira coupling with tert-butylacetylene and deprotection give
the versatile 3-aminothiophene 6. This intermediate is then
monoalkylated via reductive amination, forming 7, and acylated with
an acid chloride. The resulting hindered amide may then be subject to
further elaboration of the N-alkyl substituent and is finally hydrolyzed
to the desired carboxylic acid 8.
a
b
fu: fraction unbound. Huh7 CC50 for all the analogues reported
c
herein was ≥20 μM. ND: not determined.
corroborated in the hydroxyl-containing analogues 12 and 13. Neither
of the methyl groups in the N-alkyl substituent of 10 are in van der
Waals contact with the protein; we infer that the increased
conformational rigidity around the amide plays a significant role in
the potency increase. Consistent with this, atropisomers were
observable in the proton NMR spectra for analogues bearing chiral
N-substituents (such as 12).27 A similar phenomenon has recently
been reported in a closely related series of anthranilates.28 The
importance of the amide as a polar and atom-efficient scaffolding
element for positioning of the acyl moiety relative to the thiophene
core has previously been emphasized.29,30
Noting the similar intrinsic potency of compounds (3 and 10−12),
which vary only in the apparently solvent exposed N-alkyl substituent,
we anticipated that a broader investigation in this region of the
structure might enable modulation of other properties. Examples of
further variations at this position are listed in Table 2.
The observed and intrinsic potencies of a concise set of initial
analogues exploring the impact of key substituents upon activity are
reported in Table 1.
In general for compounds in this series, activity against the GT 1a
replicon was weaker than for GT 1b, and in order to maximize activity
in all GT 1 patients, further optimization was therefore directed
toward GT 1a. Comparison of 914 and 1026 illustrates the benefit of
replacement of the 5-phenyl substituent with the more recently
discovered tert-butylacetylene moiety, resulting in a 30-fold improve-
ment in intrinsic potency. The intrinsic potencies of 3, 11, and 12 are
broadly similar to 10, with the differences in observed activity largely
attributable to changes in CCM binding, as might be expected by
inspection of the cocrystal structure of 10 bound in thumb site II
(Figure 2) in which it is evident that the N-alkyl substituent is directed
away from the protein. The requirement for branching of the N-alkyl
substituent in order to confer high levels of potency14 was
The results for compounds 14−17 demonstrate that despite the
apparent lack of direct contact with Δ21 NS5B protein (Figure 2), not
B
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Table 3. Intrinsic Potency, in Vitro Metabolism, and Rat
Pharmacokinetics of 17 and 20−27
Figure 2. Cocrystal structure of 10 bound to GT 1b NS5B Δ21.
Table 2. CCM Binding and HCV Replicon Activity of
Further N-Alkyl Substituents
a
From liver microsomal stability assays with cofactors for glucur-
onidation added.
Table 4. Survey of tert-Butyl Variants
all branched N-alkyl substituents confer the same level of intrinsic
potency in the cellular replicon assay (where NS5B forms part of a
multimeric protein assembly known as the replicase complex).31 The
pyridyl ether 17, however, displayed intrinsic potency similar to the
best analogues in Table 1, and analogues 18 and 19 illustrate the
structure−activity relationships (SAR) of linking units of this motif
with the rest of the molecule. Intrinsic potency was slightly diminished
in the case of the cyclobutane linker but could be maintained through
use of a branched, linear chain.
Compound 17 was profiled in drug metabolism and pharmacoki-
netic studies, and on the basis of the promising stability in microsomal
assays25 and long mean residence time in rat, a variety of related ethers
was explored, as summarized in Table 3.
Both trans- and cis-4-substituted cyclohexyl ethers (and thioethers,
data not shown) provided analogues retaining intrinsic potency across
a range of heterocycles. Curiously, the mean residence times of the cis
analogues in rat were short and inconsistent with predictions from in
vitro metabolism assays (20, 25). By contrast, the trans analogues were
well-behaved in vivo, resulting in long mean residence times. The
predicted human hepatic extraction ratios of several of these analogues,
including the saturated heterocyclic ether 27, were lower than could be
readily measured in in vitro assays.
In order to explore the potential of the tert-butyl motif for further
optimization, variants at this position in the context of the pyridazonyl
ether 24 were briefly explored (vide infra, Table 4). These analogues
were prepared as shown in Scheme 2. The advanced 5-iodothiophene
intermediate 28 was treated with isopropylmagnesium bromide
followed by an appropriate Weinreb amide, giving ketone 29. After
hydroxyl deprotection, Seyferth−Gilbert homologation32 using
Bestmann’s reagent33 generated the alkyne 31. Generation of the
pyridazonyl ether 32 was accomplished by SNAr reaction of the
alkoxide of 31 with 3,6-dichloropyridazine and subsequent hydrolysis.
The utility of the trifluoromethylcyclopropyl group as a
metabolically stable tert-butyl replacement has recently been high-
lighted.34 While both 33 and 34 did indeed exhibit improved stability,
plasma-adjusted potency was compromised, and the cyclobutyl
analogue 35 was worse in both respects.
C
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a
Scheme 2. General Synthetic Route to Alkyne Analogues
a
Reagents and conditions: (a) i-PrMgBr; (b) RCON(Me)OMe; (c) TFA; (d) K2CO3, MeOH; (e) NaH, then 3,6-dichloropyridazine; (f) NaOAc,
AcOH.
It is evident from a comparison of the observed and intrinsic
potencies in Table 3 that the degree of CCM binding varied
considerably across these analogues, and an assessment of the impact
of human plasma binding and its association with log D was
informative. As a preliminary safety screen, we also assessed the
cytotoxicity of the analogues in the rapidly dividing lymphoid MT4 cell
line, as summarized in Table 5.
A trend was noted in which the more lipophilic heteroaromatic
ethers tended to exhibit greater intrinsic cytotoxicity and less favorable
plasma-adjusted potency. Over 140 N-alkyl substituents of this type
were examined in a search to combine potency, low cytoxicity,
metabolic stability, and an in vivo pharmacokinetic profile into a single
compound. Ultimately, the results with the saturated heterocyclic
analogue 27 led to further exploration of this subset of ether-linked
compounds, with an emphasis on reduction in log D to further
optimize plasma-adjusted potency (Table 6).
Variation of the heterocycle ring size gave analogues with similar
intrinsic potency, but the microsomal stability of 36 was reduced
(predicted human hepatic extraction 16%) and the plasma-adjusted
potency of 37 was compromised. The addition of hydroxyl groups
produced diverse outcomes: on an extended alkynyl motif (38)
intrinsic potency was reduced and the mean residence time in rat was
very short (despite predicted rat hepatic extraction of only 8%);
adjacent to the amide carbonyl group (39)11 no significant impact on
log D resulted and the in vivo profile was compromised; and when
incorporated on the amide N-alkyl substituent (40) resulted in an
analogue that exhibited a rat mean residence time similar to that of 27
but with improved plasma-adjusted potency. As in earlier cases, the rat
mean residence time was exquisitely sensitive to the stereochemistry of
the N-alkyl substituent (41 vs 40).
Table 5. log D, Plasma-Adjusted Potency, and Intrinsic
Cytotoxicity for Selected Analogues
The stringent SAR of the amide acyl group, which binds in the
lipophilic pocket lined by L419, R422, M423, and W528,35 was
illustrated in earlier work14 where it was reported that a 4-methyl
substituent was required to be present and equatorial on the
cyclohexyl ring in order to achieve submicromolar levels of activity
in the replicon. We next elected to explore this motif further in an
effort to improve intrinsic potency (Table 7).
Correction for CCM binding reveals that the magnitude of the
increase upon incorporation of the equatorial 4-methyl substituent (10
vs 42) is ∼100-fold, typical of the maximum observed in a survey of all
methyl group effects and consistent with its placement deep in the
hydrophobic pocket.36 The sensitivity of the SAR for the 4-substituent
of the cyclohexyl motif was further confirmed by the weaker activity of
43 and 44. The dramatic loss in activity in 45 likely reflects the known
intolerance for any axial 4-substituent. The intrinsic potency of the
unsubstituted cycloheptyl variant 46 was similar to that of the
D
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Table 6. Profiles of Saturated Heterocyclic Ethers
a
ND: not determined.
into the six-membered ring resulted in remarkably dissimilar
outcomes: although the conjugated cyclohexene 50 was much less
active than 10, the unsaturated isomers 51 and 52 demonstrated a
small but reproducible improvement in both the observed and intrinsic
potency, with the R enantiomer the more active of the two.
Table 7. CCM Binding and HCV Replicon Activity of Acyl
Variants
Finally, when the preferred unsaturated acyl motif ((R)-methyl-
cyclohexenyl from compound 52) was combined with the previously
optimized N-alkyl substituent (from compound 40), the resulting
compound (53) was shown to have a favorable profile including high
potency, high metabolic stability in vitro, and an encouraging mean
residence time in rat (Table 8). A broader summary of its preclinical
Table 8. Profile of 53
predicted
hepatic
extraction
ratio (%)
GT 1a EC50 (nM)
intrinsic
plasma- MT4 CC50
rat mean
residence
time (h)
observed intrinsic adjusted
(nM)
human rat
11 2.9 128
12138
<7
<5
2.7
characteristics has been previously described.25 53 (GS-9669) is
currently being evaluated in phase 2 clinical combination studies for
chronic HCV.
Compound 53 retains a high level of activity against the NS5B
M423T mutation.25 In order to explore the contribution of the
different substituents to this phenomenon, side-by-side transient
transfection GT 1b assays24 with wild type (WT) and M423T NS5B
replicons were performed on a concise set of analogues, with the
results shown in Table 9.
cyclohexane 42, but attempts to increase potency through modulation
of conformation or the addition of an extra carbon at the distal end of
the ring (47−49) were unsuccessful. The introduction of unsaturation
E
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profile was enhanced through incorporation of heterocyclic
ethers on the N-alkyl substituent. Hydroxyl groups were added
to modulate lipophilicity, thereby reducing protein binding.
Intrinsic potency was further improved through enantiospecific
introduction of an olefin in the acyl motif, resulting in the
discovery of 53 (GS-9669). The surprising activity of this
compound against M423T was shown to arise from both the N-
alkyl substituent and the acyl group. In phase 1 clinical studies,
compound 53 exhibited a median half-life of 6−7 h in chronic
HCV patients and produced a 3.6 log10 reduction in plasma
viral RNA after 3 days of treatment at 500 mg once daily.38
This compound is currently being evaluated in phase 2 clinical
studies in combination with other direct acting anti-HCV
agents.
Table 9. Transient Transfection Results with WT and
M423T NS5B Replicons
EXPERIMENTAL SECTION
■
All commercial reagents were used as provided. Flash chromatography
was performed using ISCO Combiflash Companion purification
system with RediSep Rf prepacked silica gel cartridges supplied by
Teledyne Isco. 1H NMR spectra were recorded on a Varian Inova 300
MHz, a Varina Mercury Plus 400 MHz, or a Bruker Advance 400 MHz
spectrometer. Proton chemical shifts are reported in ppm from an
internal standard or residual solvent. Purity of tested compounds was
assessed to be at least 95% by HPLC analysis unless indicated
otherwise. A Gemini C18 110 Å column (50 mm × 4.6 mm, 5 μm
particle size) was used with gradient elution of acetonitrile in water,
with detection at 254 nm wavelength. For all samples 0.1% TFA was
added to both eluents. Mass spectrometry was performed on a
Finnigan LCQ Advantage MAX mass spectrometer with a Thermo
LCQ Advantage HPLC. The samples were run on a Phenomenex
Gemini, C18 column using reverse phase chromatography and
acetonitrile/water containing 0.1% acetic acid as eluent.
5-(3,3-Dimethylbut-1-ynyl)-3-((R)-N-((1R,4S)-4-hydroxy-4-
(((S)-tetrahydrofuran-3-yloxy)methyl)cyclohexyl)-4-methylcy-
clohex-3-enecarboxamido)thiophene-2-carboxylic Acid (53).
Scheme 1, Steps a and b. Trifluoroacetic anhydride (18 mL, 27.3
g, 130 mmol) was added dropwise to a solution of methyl 3-
aminothiophene-2-carboxylate (20 g, 127 mmol) in ethyl ether (140
mL), stirring at −5 to −10 °C over 1 h. The resulting suspension was
slowly warmed to room temperature with vigorous stirring overnight.
The mixture was washed successively with 1 N aqueous HCl (60 mL),
water (50 mL), and brine (50 mL) and dried over anhydrous Na2SO4.
Both the N-alkyl substituent and the acyl motif that are
incorporated in 53 confer greater activity against the mutant than
would be anticipated based on the profile of 10, as demonstrated by
the reduction in fold shift for 40 and 52, respectively. The unexpected
contribution of the N-alkyl substituent further highlights the unknown
aspects of this region of the structure in the context of the replicase
complex in cells. The combination of the two motifs in 53 provides an
additive effect, resulting in a minimal reduction in activity against this
clinically relevant mutation.
The synthetic route to 53 (Scheme 3) utilized intermediate 6
(Scheme 1), which was subjected to a reductive amination with the
monoketal of 1,4-cyclohexanedione. Secondary amine 54 was then
acylated under mild buffered conditions with the acid chloride of (R)-
4-methylcyclohex-3-enecarboxylic acid37 resulting in the fully sub-
stituted amide 55. Ketal deprotection followed by Corey−Chaykovsky
epoxidation gave epoxide 56 stereoselectively. Finally, ester
saponification and epoxide opening with (S)-tetrahydrofuran-3-ol
afforded the desired compound.
CONCLUSION
■
Investigation of thiophene 2-carboxylic acid HCV NS5B site II
inhibitors, guided by measurement of CCM binding, revealed
the SAR for intrinsic cellular potency. The pharmacokinetic
a
Scheme 3. Synthesis of GS-9669
a
Reagents and conditions: (a) Bu2SnCl2, PhSiH3; (b) oxalyl chloride; (c) K3PO4; (d) HCl; (e) Me3SO+I−, NaH; (f) LiOH; (g) (S)-tetrahydrofuran-
3-ol, KOt-Bu.
F
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Concentration in vacuo and trituration with diethyl ether gave a white
solid which was used without purification (32 g, 99% yield). 1H NMR
(400 MHz, DMSO-d6): δ 11.2 (s, 1H), 8.02 (d, J = 5.6 Hz, 1H), 7.71
(d, J = 5.2 Hz, 1H), 3.85 (s, 3H).
Celite, rinsed with dichloromethane, and concentrated to give (1R)-4-
methylcyclohex-3-enecarboxylic acid chloride (4.28 g, 95% yield),
which was used immediately in the next step.
(1R)-4-Methylcyclohex-3-enecarboxylic acid chloride (4.28 g, 27.0
mmol), 54 (3.40 g, 9.0 mmol), and potassium phosphate tribasic (5.73
g, 27.0 mmol) were suspended in dichloroethane (22.5 mL), placed in
an oil bath at 80 °C, and stirred under nitrogen. After 6 h, an
additional 5 mL of dichloroethane was added. After 16 h, the mixture
was cooled and partitioned between ethyl acetate and water. The
organic layer was separated and washed with water and brine and dried
over anhydrous Na2SO4, filtered, and concentrated. Flash chromatog-
raphy (EtOAc/hexanes) afforded the desired methyl 5-(3,3-
dimethylbut-1-ynyl)-3-((1R)-4-methyl-N-(1,4-dioxaspiro[4.5]decan-8-
yl)cyclohex-3-enecarboxamido)thiophene-2-carboxylate (55) as a
n-BuLi (2.5 M in hexane, 123 mL, 308 mmol) was added dropwise
over 30 min to a solution of diisopropylethylamine (47 mL, 355
mmol) in anhydrous THF (300 mL) at −78 °C under argon. The
mixture was stirred for 30 min at −78 °C. To this freshly prepared
LDA solution, methyl 3-(2,2,2-trifluoroacetamido)thiophene-2-carbox-
ylate from the previous step (23.6 g, 93 mmol) was added dropwise as
a solution in THF (110 mL) over 40 min. The resulting mixture was
stirred at −78 °C for 1 h, and then a solution of I2 (35.5 g, 140 mmol)
in THF (110 mL) was added over 1 h. The reaction mixture was
continually stirred for another 1 h at −78 °C, quenched with saturated
aqueous NH4Cl (500 mL), and extracted with EtOAc (800 mL). The
organic layer was separated and washed with 15% aqueous Na2S2O3 (2
× 500 mL), 10% citric acid (500 mL), brine (500 mL), and dried over
anhydrous MgSO4. Concentration in vacuo and purification by silica
gel flash column (EtOAc/hexanes) gave methyl 5-iodo-3-(2,2,2-
trifluoroacetamido)thiophene-2-carboxylate (5) as a gray solid (23.2
1
white foam (3.31 g, 74% yield). H NMR (400 MHz, DMSO-d6): δ
7.29 (d, J = 18.9 Hz, 1H), 5.25 (br d, J = 14.1 Hz, 1H), 4.42 (br t, J =
12.2 Hz, 1H), 3.87−3.65 (m, 7H), 2.30−1.53 (m, 10H), 1.51 (s, 3H),
1.44−1.32 (m, 1H), 1.30 (d, J = 2.7 Hz, 9H), 1.06−0.89 (m, 1H).
Scheme 3, Steps d and e. 55 (3.31 g, 6.63 mmol) was dissolved in
THF (53 mL), and 4 M HCl (26.5 mL) was added. The reaction
mixture was heated to 45 °C and stirred for 2 h. Ethyl acetate was
added, and the organic layer was separated. The aqueous was extracted
again with ethyl acetate, and the combined organics were washed with
saturated aqueous sodium bicarbonate, water, and brine. The organic
layer was dried over anhydrous Na2SO4, filtered, and concentrated to
afford 5-(3,3-dimethylbut-1-ynyl)-3-[(1R)-4-methylcyclohex-3-enecar-
bonyl)-(4-oxocyclohexyl)amino]thiophene-2-carboxylic acid methyl
ester as a white foam (3.03 g, 100% yield).
1
g, 70% yield). H NMR (400 MHz, CDCl3): δ 11.2 (s, 1H), 8.01 (s,
1H), 3.84 (s, 3H).
Scheme 1, Steps c and d. A 500 mL three-neck flask equipped with
a thermometer and a stirrer was charged with 5 (23.2 g, 61 mmol),
CuI (2.33 g, 12.2 mmol), tert-butylacetylene (10.0 g, 122 mmol),
Pd(PPh3)2Cl2 (4.29 g, 6.1 mmol), DMF (300 mL), and Et3N (4.3 g).
The mixture was stirred at 80 °C under nitrogen for 1.5 h, cooled to
room temperature, diluted with EtOAc (1 L), and filtered to remove
solids. The filtrate was washed with water (500 mL), brine (500 mL)
and dried over anhydrous Na2SO4. Concentration in vacuo and
purification by flash chromatography (EtOAc/hexanes) gave methyl 5-
(3,3-dimethylbut-1-ynyl)-3-(2,2,2-trifluoroacetamido)thiophene-2-car-
A solution of trimethylsulfoxonium chloride (1.07 g, 8.31 mmol) in
DMSO (24 mL) was treated with sodium hydride (0.293 g, 60% oil
dispersion, 7.32 mmol) portionwise over 10 min and stirred at ambient
temperature for 20 min. 5-(3,3-Dimethylbut-1-ynyl)-3-[(1R)-4-meth-
ylcyclohex-3-enecarbonyl)-(4-oxocyclohexyl)amino]thiophene-2-car-
boxylic acid methyl ester (3.03 g, 6.65 mmol) in THF (24 mL) was
added dropwise over 5 min, and the reaction mixture was stirred for 45
min. The orange solution was acidified to pH 3 with 5% citric acid and
partitioned between water and ethyl acetate. The organic layer was
separated, and the aqueous layer was extracted again with ethyl acetate.
The combined organic extracts were washed with 5% aqueous LiCl,
water, and brine and dried over anhydrous Na2SO4. After filtration and
concentration, the residue was purified by flash chromatography
(EtOAc/hexanes) to afford the desired methyl 5-(3,3-dimethylbut-1-
ynyl)-3-((R)-4-methyl-N-((3S,6s)-1-oxaspiro[2.5]octan-6-yl)cyclohex-
3-enecarboxamido)thiophene-2-carboxylate 56 as a white solid (2.70 g,
1
boxylate as a pale yellow solid (12.3 g, 60% yield). H NMR (400
MHz, CDCl3): δ 11.1 (s, 1H), 8.00 (s, 1H), 3.92 (s, 3H), 1.32 (s, 9H).
MS (ESI−) m/z: 332 [M − 1]−.
A mixture of methyl 5-(3,3-dimethylbut-1-ynyl)-3-(2,2,2-
trifluoroacetamido)thiophene-2-carboxylate (12.3 g, 36.8 mmol) and
K2CO3 (10.2 g, 73.6 mmol) in methanol (70 mL), THF (70 mL), and
H2O (70 mL) was stirred at room temperature for 2.5 h and
neutralized with 1 N HCl to pH 8. The mixture was concentrated to
remove the volatile solvents, and the aqueous solution was extracted
with EtOAc (500 mL). The organic layer was dried over anhydrous
Na2SO4 and concentrated to dryness. The residue obtained was
triturated with petroleum ether and dried in a vacuum oven to give
methyl 3-amino-5-(3,3-dimethylbut-1-ynyl)thiophene-2-carboxylate
(6) as a white solid (7.9 g, 91% yield). 1H NMR (400 MHz,
DMSO-d6): δ 6.62 (s, 1H), 6.54 (s, broad, 2H), 3.70 (s, 3H), 1.27 (s,
9H). MS (ESI+) m/z: 238 [M + 1]+.
1
86% yield). H NMR (400 MHz, DMSO-d6): δ 7.33 (d, J = 18.9 Hz,
1H), 5.26 (br d, J = 17.2 Hz, 1H), 4.59−4.39 (m, 1H), 3.76 (d, J = 6.1
Hz, 3H), 2.55 (q, J = 4.8 Hz, 2H), 2.28−1.54 (m, 11H), 1.52 (s, 3H),
1.43−1.32 (m, 1H), 1.30 (d, J = 2.7 Hz, 9H), 1.17−0.93 (m, 3H).
Scheme 3, Steps f and g. 56 (2.69 g, 5.73 mmol) in THF (31 mL)
and water (26 mL) was treated with lithium hydroxide (1.92 g, 46
mmol) with stirring at ambient temperature. After 16.5 h, the mixture
was cooled to 0 °C and acidified to pH 4 with 10% citric acid (60 mL).
The white cloudy mixture was extracted with ethyl acetate and washed
with water and brine. After drying over anhydrous Na2SO4, the organic
layer was filtered and concentrated to afford crude 5-(3,3-dimethylbut-
1-ynyl)-3-((R)-4-methyl-N-((3S,6s)-1-oxaspiro[2.5]octan-6-yl)-
cyclohex-3-enecarboxamido)thiophene-2-carboxylic acid as an off-
white foam.
(S)-Tetrahydrofuran-3-ol (2.53 g, 28.7 mmol) in 1-methylpyrroli-
din-2-one (19 mL) at 0 °C was treated portionwise with potassium
tert-butoxide (2.57 g, 22.9 mmol) over 2 min. 5-(3,3-Dimethylbut-1-
ynyl)-3-((R)-4-methyl-N-((3S,6s)-1-oxaspiro[2.5]octan-6-yl)cyclohex-
3-enecarboxamido)thiophene-2-carboxylic acid (5.73 mmol) from the
previous step was added in one portion, washing in the residue with 9
mL of 1-methylpyrrolidin-2-one. The mixture was warmed to 40 °C
and stirred for 16 h. After cooling to 0 °C, the mixture was acidified to
pH 3 with 2 M HCl, partitioned between ethyl acetate and water, and
separated. The organic layer was washed with water and brine and
dried over anhydrous Na2SO4. After filtration and removal of solvent
under vacuum, the residue was purified by flash chromatography
Scheme 3, Step a. Dibutyltin dichloride (3.03 g, 0.1 mmol) was
added to compound 6 (23.7g, 100 mmol) and 1,4-dioxa-spiro[4.5]-
decan-8-one (18.7g, 120 mmol) in THF (20 mL) and DMF (10 mL).
After 5 min, phenylsilane (13.6 mL, 111 mmol) was added and the
resulting mixture was stirred overnight. Ethyl acetate was added, and
the reaction mixture was washed with 5% LiCl solution and brine. The
organic phase was separated and dried over anhydrous Na2SO4. After
concentration, the crude material was purified by flash chromatog-
raphy, eluting with ethyl acetate, to give methyl 3-(1,4-dioxaspiro[4.5]-
decan-8-ylamino)-5-(3,3-dimethylbut-1-ynyl)thiophene-2-carboxylate
1
(54) as a light yellow solid (32.1 g, 89% yield). H NMR (400 MHz,
DMSO-d6): δ 7.036 (s, 1H), 6.671 (d, J = 8.4 Hz, 1H), 3.86 (m, 4H),
3.71 (s, 1H), 3.58 (m, 1H), 1.85 (m, 2H), 1.62 (m, 4H), 1.47 (m, 2H),
1.28 (s, 9H). MS (ESI+) m/z: 378 [M + 1]+.
Scheme 3, Steps b and c. (1R)-4-Methylcyclohex-3-enecarboxylic
acid36 (4.00 g, 28.5 mmol, azeotropically dried by evaporation of a
toluene solution) and freshly ground potassium phosphate tribasic
(7.55 g, 35.6 mmol) were suspended in dichloromethane (81 mL),
treated with DMF (0.120 mL), and placed in a 0 °C bath. After 5 min,
the mixture was treated portionwise over 15 min with oxalyl chloride
(8.10 mL, 85.5 mmol). After the mixture was stirred for 1 h, the
heterogeneous mixture was quickly filtered through a small pad of
G
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Journal of Medicinal Chemistry
Article
(EtOAc/dichloromethane) and recrystallized from warm acetonitrile/
water to afford 5-(3,3-dimethylbut-1-ynyl)-3-((R)-N-((1R,4S)-4-hy-
droxy-4-(((S)-tetrahydrofuran-3-yloxy)methyl)cyclohexyl)-4-methyl-
cyclohex-3-enecarboxamido)thiophene-2-carboxylic acid (53) as a
white powder (1.96 g, 63% yield from 56). MS (ESI+) m/z: 544.0
[M + H]+. 1H NMR (400 MHz, DMSO-d6): δ 13.49 (s, 1H), 7.18 (d,
J = 18.3 Hz, 1H), 5.25 (d, J = 15.8 Hz, 1H), 4.39−4.23 (m, 1H), 4.10−
4.01 (m, 1H), 3.99 (s, 1H), 3.76−3.56 (m, 4H), 3.13−3.00 (m, 2H),
2.25−1.96 (m, 2H), 1.96−1.33 (m, 18H), 1.29 (d, J = 2.7 Hz, 9H),
1.23−1.09 (m, 1H).
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ASSOCIATED CONTENT
* Supporting Information
Experimental details for the syntheses and spectroscopic
characterization of the compounds in this paper and further
assay details. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
Accession Codes
PDB code for HCV NS5B inhibitors: 4eo6.
AUTHOR INFORMATION
Corresponding Authors
*S.E.L.: phone, 650-522-1564; e-mail, scott.lazerwith@gilead.
com.
*W.J.W.: phone, 650-522-5384; e-mail, will.watkins@gilead.
com.
■
(9) Nittoli, T.; Curran, K.; Insaf, S.; DiGrandi, M.; Orlowski, M.;
Chopra, R.; Agarwal, A.; Howe, A. Y. M.; Prashad, A.; Brawner Floyd,
M.; Johnson, B.; Sutherland, A.; Wheless, K.; Feld, B.; O’Connell, J.;
Mansour, T. K.; Bloom, J. Identification of Anthranilic Acid
Derivatives as a Novel Class of Allosteric Inhibitors of Hepatitis C
NS5B Polymerase. J. Med. Chem. 2007, 50, 2108−2116.
(10) May, M. M.; Brohm, D.; Harrenga, A.; Marquardt, T.; Riedl, B.;
Kreuter, J.; Zimmermann, H.; Ruebsamen-Schaeff, H.; Urban, A.
Discovery of Substituted N-Phenylbenzenesulphonamides as a Novel
Class of Non-Nucleoside Hepatitis C Virus Polymerase Inhibitors.
Antiviral Res. 2012, 95, 182−191.
Present Addresses
†M.M.: Bristol-Myers Squibb Research and Development, P.O.
Box 4000, Princeton, NJ 08543.
‡M.F.: Novartis Institutes for BioMedical Research, 4560
Horton Street, Emeryville, CA 94608.
§A.H.: Ironwood Pharmaceuticals, 301 Binney Street, Cam-
bridge, MA 02142.
∥E.M.: Reset Therapeutics, 863A Mitten Road Suite 100C,
Burlingame, CA 94010.
Notes
(11) LaPorte, M. G.; Draper, T. L.; Miller, L. E.; Blackledge, C. W.;
Leister, L. K.; Amparo, E.; Hussey, A. R.; Young, D. C.; Chunduru, S.
K.; Benetatos, C. A.; Rhodes, G.; Gopalsamy, A.; Herbertz, T.; Burns,
C. J.; Condon, S. M. The Discovery and Structure−Activity
Relationships of Pyrano[3,4-b]indole Based Inhibitors of Hepatitis C
Virus NS5B Polymerase. Bioorg. Med. Chem. Lett. 2010, 20, 2968−
2973.
(12) Jackson, R. W.; LaPorte, M. G.; Herbertz, T.; Draper, T. L.;
Gaboury, J. A.; Rippin, S. R.; Patel, R.; Chunduru, S. K.; Benetatos, C.
A.; Young, D. C.; Burns, C .J.; Condon, S. M. The Discovery and
Structure−Activity Relationships of Pyrano[3,4-b]indole-Based Inhib-
itors of Hepatitis C Virus NS5B Polymerase. Bioorg. Med. Chem. Lett.
2011, 22, 3227−3231.
The authors declare the following competing financial
interest(s): All of the authors of this manuscript are current
or recent employees of Gilead Sciences.
ABBREVIATIONS USED
■
ACN, acetonitrile; AcOH, acetic acid; 9-BBN, 9-borabicyclo-
[3.3.1]nonane; CC50, 50% cytostatic concentration; CCM, cell
culture medium; Cmpd, compound; DIAD, diisopropyl
azodicarboxylate; Et3N, triethylamine; EtOAc, ethyl acetate;
fu, fraction unbound; GT, genotype; HCl, hydrochloric acid;
HCV, hepatitis C virus; i-PrMgBr, isopropylmagnesium bro-
mide; KHMDS, potassium hexamethyldisilazide; KOt-Bu,
potassium tert-butoxide; LDA, lithium diisopropylamide;
MeCN, acetonitrile; MeOH, methanol; min, minute; NaOAc,
sodium acetate; NS, nonstructural; SNAr, nucleophilic aromatic
substitution; WT, wild type
(13) Chan, L.; Das, S. K.; Reddy, T. J.; Poisson, C.; Proulx, M.;
Pereira, O.; Courchesne, M.; Roy, C.; Wang, W.; Siddiqui, A.;
Yannopoulos, C. G.; Nguyen-Ba, N.; Labrecque, D.; Bethell, R.;
Hamel, M.; Courtemanche-Asselin, P.; L’Heureux, L.; Maud, D.;
́
Nicolas, O.; Brunette, S.; Bilimoria, D.; Bedard, J. Discovery of
Thiophene-2-carboxylic Acids as Potent Inhibitors of HCV NS5B
Polymerase and HCV Subgenomic RNA Replication. Part 1:
Sulfonamides. Bioorg. Med. Chem. Lett. 2004, 14, 793−796.
(14) Chan, L.; Pereira, O.; Reddy, T. J.; Das, S. K.; Poisson, C.;
Courchesne, M.; Proulx, M.; Siddiqui, A.; Yannopoulos, C. G.;
Nguyen-Ba, N.; Roy, C.; Nasturica, D.; Moinet, C.; Bethell, R.; Hamel,
M.; L’Heureux, L.; Maud, D.; Nicolas, O.; Courtemanche-Asselin, P.;
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