Highly potent HCV NS4B inhibitors with activity against multiple genotypes

Article abstract of DOI:10.1021/jm401646w

The exploration of novel inhibitors of the HCV NS4B protein that are based on a 2-oxadiazoloquinoline scaffold is described. Optimization to incorporate activity across genotypes led to a potent new series with broad activity, of which inhibitor 1 displayed the following EC₅₀ values: 1a, 0.08 nM; 1b, 0.10 nM; 2a, 3 nM; 2b, 0.6 nM, 3a, 3.7 nM; 4a, 0.9 nM; 6a, 3.1 nM.

Full text of DOI:10.1021/jm401646w

Brief Article
pubs.acs.org/jmc
Highly Potent HCV NS4B Inhibitors with Activity against Multiple
Genotypes
Barton Phillips,* Ruby Cai, William Delaney, Zhimin Du, Mingzhe Ji, Haolun Jin, Johnny Lee, Jiayao Li,
Anita Niedziela-Majka, Michael Mish, Hyung-Jung Pyun, Joe Saugier,† Neeraj Tirunagari, Jianhong Wang,
Huiling Yang, Qiaoyin Wu, Chris Sheng,‡ and Catalin Zonte
Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404, United States
S
* Supporting Information
ABSTRACT: The exploration of novel inhibitors of the HCV NS4B protein that are based on
a 2-oxadiazoloquinoline scaffold is described. Optimization to incorporate activity across
genotypes led to a potent new series with broad activity, of which inhibitor 1 displayed the
following EC50 values: 1a, 0.08 nM; 1b, 0.10 nM; 2a, 3 nM; 2b, 0.6 nM, 3a, 3.7 nM; 4a, 0.9 nM;
6a, 3.1 nM.
INTRODUCTION
■
Infection with the hepatitis C virus (HCV) is a global health
concern, with more than 170 million affected people world-
wide, including 4 million in the U.S.1 HCV is a diverse disease,
with six major HCV genotypes (GTs 1−6) and 52 sub-GTs
(1a, 1b, 2a, 2b, 3a, etc.). In North America and Europe, HCV
Figure 1. Representative NS4B inhibitors.
GTs 1a and 1b are most prevalent, but over 25% of patients are
infected with GTs 2 and 3.2 In North Africa and the Middle
these strains have the variants T, L, and L at positions 94, 98,
East, GT 4 is prevalent, while GT 6 predominates in East Asia.3
and 105, respectively, suggesting that these differences
Because of the lack of the proofreading ability in the HCV RNA
contribute to the reduced activity of NS4B inhibitors against
dependent RNA polymerase,4 high genetic variability and
2a strains. In this paper we describe attempts to develop a new
polymorphism occur, even within the subgenotypes. Because of
series of NS4B inhibitors with potent activity across genotypes,
the high variability in the virus, developing direct acting
focusing on GT 2a optimization.
antivirals effective across all genotypes is challenging. The
In the course of our HCV research program we discovered a
recently approved protease inhibitors (telaprevir and bocepre-
new 6,6-heterocyclic based system that provided potent
vir) are highly effective against GT 1 but lack pan-genotype
inhibitors of the NS4B replicon. Compound 4 (Figure 2) is a
activity. Furthermore, viral strains resistant to protease
inhibitors have emerged, demonstrating the need for other
direct-acting antiviral agents with complementary mechanisms
of action.5 A multidrug combination regimen, without
interferon and effective across HCV GTs, remains a major
goal of antiviral research.
Most current clinical compounds and approved drugs
targeting HCV act at and induce mutations in one of three
targets: the NS3, NS5A, and NS5B proteins.6 NS4B, or HCV
Figure 2. Early quinolone scaffold inhibitor.
nonstructural 4B, is a more recent target for HCV inhibitors.
NS4B is a 27 kDa integral membrane protein believed to act
primarily as an endoplasmic reticulum-localized scaffold for the
assembly of the replicase complexes needed for HCV RNA
replication.7 Recently, several chemotypes that target NS4B and
inhibit HCV GT 1 replication have been reported, including
clemizole 1, imidazopyridines (e.g., anguizole 2),8−10 6-(indol-
2-yl)pyridine-3-sulfonamides (e.g., PTC-725 3)11,12 (Figure 1),
and piperazinones.13 Although highly potent GT 1 inhibitors
have emerged, particularly in the imidazopyridine series, to date
no agents with potent GT 2 activity have been described.9 An
analysis of the 19 available GT 2a sequences in the HCV
European database revealed that the NS4B proteins of all of
representative early lead from this series, showing potent
inhibition against the wild-type GT 1a and 1b replicons, with
EC50’s of 0.9 and 1.3 nM, respectively. A 42-fold shift in
potency against the GT 1b-H94N replicon was observed, a
polymorph well-known to be less sensitive to other small
molecule NS4B inhibitors.9,11 Furthermore, 4 showed high
Special Issue: HCV Therapies
Received: October 23, 2013
Published: February 10, 2014
© 2014 American Chemical Society
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Brief Article
binding affinity for the NS4B protein; titration with purified
wild type GT 1b NS4B protein afforded a Kd of 31 nM. The
GT 2a replicon also proved to be much less sensitive to
compounds from the series, with a dramatic (2725-fold) drop
in activity for 4. Potent activity in GT 2a was needed for the
targeted broad genotype inhibitor profile, and thus, activity at
GT 2a became a major focus of optimization efforts.
Early efforts focused on modifying the flat, hydrophobic
phenyl groups at each end of the inhibitor. Alkyl ethers, such as
methylene-1,3-dioxolane, proved to be equipotent with phenyl
as the aminooxadiazole substituent, while O-linked alkyl ethers
were effective replacements for the C6 phenyl. Combining both
changes in 10 gave similar activity to 9 in GT 1b but little
improvement in GT 2a activity. The greatly reduced log D of
10, however, resulted in a substantial increase in LLE. In
contrast, smaller alkyl substituents at C6, such as 11, provided a
sharp drop in potency. Replacing the C6 ether O with the
corresponding N-linker (e.g., aniline 12) produced a drop in
lipophilicity with an improvement in potency. This combina-
tion proved critical in optimizing the final compounds.
CHEMISTRY
■
The synthesis of 4 (Scheme 1) is representative of all the
compounds in the series. Beginning with 4-amino-3-tert-
a
Scheme 1. Synthesis of Oxadiazole 4
The tert-butyl group at C8 in 4 provided a 4-fold increase in
potency over the corresponding trifluoromethyl analogue 10, in
GTs 1b and 2a. The increase in potency was sufficient to
improve LLE despite a small increase in log D. Less lipophilic
side chains such as trifluoromethyl were consistently less active,
and the drop in log D was insufficient to offset this.
A variety of unsubstituted alkyl substituents on the
aminooxadiazole, such as cyclopentyl 14, proved to be similar
in activity to the dioxolane. Unfortunately, the increase in log D
for the unsubstituted alkyls led to a sharp drop in LLE and was
typically accompanied by an increase in the rate of metabolism,
a common but not universal trend in the higher log D
analogues prepared. Adding hydroxyl substituents to the
cycloalkyl proved to be very effective in reducing metabolism,
and in some isomers it increased potency as well. Stereo-
chemistry was critical for such potency increases with a 1,3-cis-
substitution pattern favored for ring sizes 4−6, as illustrated in
the aminocyclobutanol isomers 15 and 16. Other substitution
patterns saw no such improvement, although the trans-isomers
were as stable as the corresponding cis-isomers. Given the
effectiveness of the hydroxy substituents, we combined this
modification with the C6 trifluoroethylanilino group. Example
18 showed an EC50 against 2a-JFH of 25 nM, a >10000-fold
improvement over 9, and a LLE of 3.9. In contrast, the more
lipohilic ether counterpart 17 was >10-fold less active.
Combined with an alcohol substituent in 18, the C6 aniline
functionality offered no liability in microsomal stability
compared with C6 ether 17. A systematic exploration of
cycloalkyl alcohols revealed the (1S,3R)-cis-aminocyclohexanol
side chain as a superior side chain, with analogue 20 showing
inhibition of the GT 2a replicon in the low single digit
nanomolar range, an overall increase in LLE from −0.8 to 4.9
(9−20). The cyclohexanol system was quite stereospecific, with
the (1R,3S)-enantiomer of 20 being 67-fold less potent as an
inhibitor in the 2a replicon system (201 nM). A resistance
selection was performed with 20 using a GT 2a replicon and
yielded mutations in NS4B including K20R, H53Y, L64F, and
I109V.
a
Conditions: (i) BnBr, NaH, DMSO, 64%; (ii) diethylacetylene
dicarboxylate, EtOH, 60 °C, 1 h; (iii) neat, 220 °C, 15 min, 73%; (iv)
Tf2O, lutidine, 0 °C, 1 h, 96%; (v) MeB(OH)2, Pd(dppf)-CH2Cl2,
Cs2CO3, dioxane, 100 °C, 4 h, 100%; (vi) H2, Pd−C, 1 h, 92%; (vii)
2,2,2-trifluoroethyl trifluoromethanesulfonate, K2CO3, DMF, 100 °C,
4 h, 51%; (viii) hydrazine hydrate, EtOH, 70 °C, 1 h, 100%; (ix) O-
phenyl (1,3-dioxolan-2-yl)methylcarbamothioate, TEA, EDCI (3.5
equiv), DMF 16 h, 74%.
butylphenol 5, protection of the phenol as its benzyl ether
was followed by a two-step, one-pot condensation with diethyl
acetylenedicarboxylate and subsequent cyclization under
thermal conditions, providing quinolone 6, which served as a
versatile intermediate.
The C-4 Me functionality was conveniently installed via
Suzuki coupling of the intermediate triflate derivative with
methylboronic acid. To introduce the C-6 2,2,2-trifluoroethyl
ether functionality, debenzylation was followed by alkylation of
the phenol to provide 8. The 2-aminooxadiazole was generated
at the end of the synthesis via a two-step procedure beginning
with formation of the acyl hydrazide and followed by
condensation with the appropriate phenyl thiocarbamate in
the presence of EDCI to selectively remove H2S.
RESULTS AND DISCUSSION
■
Compound 9 (Table 1), the initial hit in the series, showed
promising activity against GT 1b but was clearly limited by high
lipophilicity and low potency against GT 2a. Efforts at
optimization focused on (1) reducing log D to 3.5 or below
and (2) improving potency in GT 2a into the low nanomolar
range, both combining to provide a target LLE of 4.5. New
analogues were screened for inhibition of the HCV GT 1a, 1b,
1b-H94N, and 2-JFH replicon assays. LLEs were calculated
from measured log D and GT 2a activity. Given the
exceptionally low potency seen in the 2a-JFH replicon, it
served as a surrogate for untested genotypes during the
preliminary assessment. Table 1 outlines representative SAR
from these efforts, along with predicted clearance values derived
from stability upon incubation with human liver microsomes
(HLMs).
Given the excellent potency of 20, it was tested in a panel of
replicons along with 18 and 19 to assess potency across a broad
range of genotypes (Table 2). 20 displayed potent (EC50 < 5
nM) activity against all the replicons in this study, including GT
1b resistant polymorphs (a GT 5 replicon was not tested).
Although the GT 1b vs 2a shift remained significant for 20 (30-
fold), the shift was greatly reduced from that observed for 4
(2725-fold). The corresponding C6 ether 19 was much less
active against GTs 2a and 3a, although it did show better
stability in microsomes. Both 18 and 19 also showed a larger
shift between GT 1b and GT 2a (312-fold and 300-fold,
respectively). All three compounds show potential in a
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Journal of Medicinal Chemistry
Brief Article
Table 1. Inhibition of HCV RNA in the Replicon System and in Vitro Metabolic Stability
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Brief Article
a
Table 2. Genotype Profile of 18, 19, and 20
a
The GT 1a, 1b, 2a, 3a, 4a, 6a replicons are subgenomic replicons derived from only the indicated strain of HCV. The GT 2b replicon is chimeric,
expressing full length GT 2b NS4B protein in backbone of a GT 2a replicon.
raphy was conducted under medium pressure on silica (ISCO RediSep
Rf). HPLC purification was performed on Shimadzu LC instruments,
with a 20 min gradient of 0.1% aqueous TFA and 10−97% acetonitrile,
at a flow rate of 20 mL/min, using as the stationary phase a
Phenomenex SnyderSi4 4 μm Polar RP column, 20 mm × 150 mm,
and peak acquisition based on UV detection at 254 nm. NMR spectra
were collected on a Varian 300 Inova. Frequencies for nuclei were 400
multigenotype drug regimen for treating HCV, with 20 holding
a clear advantage due to its flatter activity profile.
DMPK. Encouraged by the results outlined above, we
profiled three compounds from Table 1 in rat PK experiments.
The results are summarized in Table 3, along with in vitro
1
MHz for H and 75 MHz for 13C, using DMSO as solvent unless
Table 3. Rat pK Profile for 9, 18, and 20
indicated otherwise. LCMS was performed on a Dionex MSQ, using
Phenomenex Synergi Hydro RP 80A column: eluent, water/
acetonitrile, formic acid (0.05%); gradient 1% acetonitrile to 100%
acetonitrile; run length, 2 or 4 min. Analytical RP-HPLC was
performed on Agilent 1100 series with Phenomenex Luna C18 and
mobile phase gradual mix of water and acetonitrile containing 0.1%
TFA. Monitoring wavelength was 254 nm. High resolution mass
spectrometry was performed on an Agilent model 6220 time of flight
mass spectrometer with an Agilent 1200 rapid resolution HPLC
instrument. Data processing was via Agilent MassHunter B.02
Qualitative Analysis. The lock masses that were used during the run
were 118.086 255 and 922.009 798.
CL
Vss
T1/2
Fpo
HLM
RLM
compd (L h−1 kg−1
)
(L/kg) (h) (%) (L h−1 kg−1
)
(L h−1 kg−1
)
18
20
9
0.87
0.65
1.05
5.0
2.4
1.8
5.2
11
25
88
0.20
0.22
0.46
0.22
0.24
0.70
2.3
stability in human and rat liver microsomes (HLMs and RLMs,
respectively). Compounds 18 and 20 each showed good
bioavailability and relatively low CL. The high volumes help to
provide a long half-life. The observed CL was slightly higher
than predicted by rat liver microsomes. The initial lead 9
showed similar but somewhat inferior results, demonstrating
that optimization for broader-GT potency had not compro-
mised the in vivo PK profile of the series.
(1S,3R)-3-(5-(8-tert-Butyl-4-methyl-6-(2,2,2-trifluoroethyl-
amino)quinolin-2-yl)-1,3,4-oxadiazol-2-ylamino)cyclohexanol
(20).
SUMMARY
■
We described the development of a novel series of NS4B
inhibitors with broad genotype activity, starting from a lead
largely limited to activity against GT 1. Early lead compounds
showed an excellent pharmacokinetic profile and high potency
against GT 1 but little activity against GT 2a. Subsequent
efforts to optimize LLE and potency led to a 3 log
improvement in activity against GT 2a together with a small
drop in log D. Activity in the low nanomolar range was seen
across the broad range of genotypes tested. Representative 20
showed low nanomolar or better inhibitory activity in the 1a,
1b, 2a, 2b, 3a, 4a, and 6a replicon systems.
A solution of (1S,3R)-3-aminocyclohexanol (29 mg, 0.25 mmol) in 1
mL DCM was cooled to 0 °C and treated with O-phenyl
carbonochloridothioate (447 mg, 0.26 mmol). TEA (77 μL, 0.55
mmol) was added slowly over 15 min with vigorous stirring. After 2 h,
the mixture was diluted with 1 mL of 10% citric acid solution and
stirred for an additional 5 min. The organic layer was separated
(rinsing with additional DCM), dried with sodium sulfate, and
concentrated to provide a tan semisolid. This material was taken up in
2 mL of DMF and treated with 8-tert-butyl-4-methyl-6-(2,2,2-
trifluoroethylamino)quinoline-2-carbohydrazide (67 mg, 0.22 mmol)
and TEA (22 mg, 0.22 mmol) and heated to 50 °C for 3 days. The
mixture was then cooled to rt and treated with EDCI (110 mg, 0.66
mmol). The mixture was heated to 50 °C for 3 h, then cooled and
diluted with EtOAc. The organic solution was washed with water,
dried with sodium sulfate, and concentrated to provide an oily brown
solid. Purification by silica gel chromatography provided the product
EXPERIMENTAL SECTION
■
Chemistry. The purity of the final compounds was determined to
be ≥95% by 1H NMR and LCMS; structural asignments were
consistent with the spectroscopic data. Solvents were purified and
stored according to standard procedures. Flash column chromatog-
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Journal of Medicinal Chemistry
Brief Article
(23 mg, 25% yield) as a white solid. LCMS tR = 2.38 min; [M + H] =
to Dharmaraj Samuel and Nikolai Novikov for their work in
purifying the NS4B protein.
1
478.23; 19F NMR δ −70.60 (t, J = 9.7 Hz); H NMR (400 MHz,
DMSO-d6) δ 7.92 (s, 1H), 7.83 (d, J = 7.6 Hz, 1H), 7.75 (s, 1H), 7.27
(d, J = 2.5 Hz, 1H), 6.83 (t, J = 6.8 Hz, 1H), 4.65 (d, J = 4.6 Hz, 1H),
4.25−3.88 (m, 2H), 3.41 (d, J = 9.0 Hz, 1H), 2.86 (s, 1H), 2.70 (d, J =
0.7 Hz, 1H), 2.57 (d, J = 1.0 Hz, 3H), 2.18 (d, J = 11.9 Hz, 1H), 1.95
(s, 1H), 1.78 (s, 1H), 1.76−1.60 (m, 2H), 1.59 (s, 9H), 1.35−0.83 (m,
2H). HRMS calcd for C24H30F3N5O2, M+ 477.2352; found, 477.2353.
N-((1,3-Dioxolan-2-yl)methyl)-5-(8-tert-butyl-4-methyl-6-
(2,2,2-trifluoroethoxy)quinolin-2-yl)-1,3,4-oxadiazol-2-amine
(4).
ABBREVIATIONS
■
AcOH, acetic acid; DCE, dichloroethane; DCM, dichloro-
methane; DIAD, diisopropyl azodicarboxylate; TEA, triethyl-
amine; EtOAc, ethyl acetate; GT, genotype; Hex, hexanes; HCl,
hydrochloric acid; HCV, hepatitis C virus; KHMDS, potassium
hexamethyldisilazide; KO-t-Bu, potassium tert-butoxide;
LiHMDS, lithium hexamethyldisilazide; MeCN, acetonitrile;
MeOH, methanol; min, minute; NaOAc, sodium acetate; NS,
nonstructural; RP, reverse phase; SNAr, nucleophilic aromatic
substitution; WT, wild type
REFERENCES
A solution of (1,3-dioxolan-2-yl)methanamine (0.311 mL, 3.02 mmol)
and TEA (1.01 mL, 7.24 mmol) in 10 mL DCM was cooled to 0 °C
and treated dropwise with thiophosgene (0.28 mL, 3.62 mmol). After
being stirred at rt for 2.5 h, the mixture was diluted with Et2O and
washed with water. The organic layer was separated and dried with
sodium sulfate before filtering through a plug of silica gel.
Concentration provided a faintly yellow oil, which was taken up in
30 mL of DCE, treated with 8-tert-butyl-4-methyl-6-(2,2,2-
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(s, 1H), 5.04 (t, J = 4.3 Hz, 1H), 4.96 (q, J = 8.9 Hz, 2H), 3.92 (t, J =
6.8 Hz, 2H), 3.80 (t, J = 6.9 Hz, 2H), 3.41−3.35 (m, 2H), 2.69 (s,
3H), 1.61 (s, 9H); 19F NMR (376.1 MHz) δ −72.82, −75.17 (TFA
salt); MS [M + H]+ = 467.2; LCMS tR = 2.58 min. HRMS calcd for
C22H25F3N4O4, M+ 466.1828; found, 466.1815.
■
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S
* Supporting Information
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.
AUTHOR INFORMATION
■
Corresponding Author
(10) Miller, J. F.; Chong, P. Y.; Shotwell, J. B.; Catalano, J. G.; Tai, V.
W. F.; Fang, J.; Banka, A. L.; Roberts, C. D.; Youngman, M.; Zhang,
H.; Xiong, Z.; Mathis, A.; Pouliot, J. J.; Hamatake, R. K.; Price, D. J.;
Seal, J. W.; Stroup, L. L.; Creech, K. L.; Carballo, L. H.; Todd, D.;
Spaltenstein, A.; Furst, S.; Hong, Z.; Peat, A. J. Hepatitis C Replication
Inhibitors That Target the Viral NS4B Protein. J. Med. Chem. [Online
early access]. DOI: 10.1021/jm400125h. Published Online: Apr 1,
2013.
(11) Zhang, X.; Zhang, N.; Chen, G.; Turpoff, A.; Ren, H.; Takasugi,
J.; Morrill, C.; Zhu, J.; Li, C.; Lennox, W.; Paget, S.; Liu, Y.; Almstead,
N.; Njoroge, F.; Gu, Z.; Komatsu, T.; Clausen, V.; Espiritu, C.; Graci,
J.; Colacino, J.; Lahser, F.; Risher, N.; Weetall, M.; Nomeir, A.; Karp,
G. M. Bioorg. Med. Chem. Lett. 2013, 23, 3947−3953.
*Bart Phillips.: Phone: 650-522-5060. E-mail: bart.phillips@
gilead.com.
Present Addresses
†J.S.: 622 Daisyfield Drive, Livermore, CA 94551.
‡C.S.: Accelas Pharmaceutical Inc., No. 9 Weidi Rd., Xianlin
University City, Qixia District, Nanjing, China.
Notes
The authors declare the following financial interests: The
authors are employees of Gilead Sciences except for C. S. and J.
S. who were employed at Gilead Sciences during this research.
All authors are shareholders of Gilead Sciences.
(12) Zhang, N.; Zhang, X.; Zhu, J.; Turpoff, A.; Chen, G.; Morrill, C.;
Huang, S.; Lennox, W.; Kakarla, R.; Liu, R.; Li, C.; Ren, H.; Almstead,
N.; Venkatraman, S.; Njoroge, F. G.; Gu, Z.; Clausen, V.; Graci, J.;
Jung, S. P.; Zheng, Y.; Colacino, J. M.; Lahser, F.; Sheedy, J.; Mollin,
A.; Weetall, M.; Nomeir, A.; Karp, G. M.. Structure−Activity
ACKNOWLEDGMENTS
■
Special thanks are given to Kathy Brendza for her work
obtaining high resolution mass spectral data for the project and
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