Non-Nucleoside Inhibitors of HVC NS5B Polymerase
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 27 6891
8H, major trans-rotamer), 1.09 (s, 1H, minor cis-rotamer); 13
NMR (100 MHz, DMSO-d6) δ 164.9, 157.8, 155.0, 152,3, 143.6,
133.7, 127.5, 124.9114.9, 113.7, 111.5, 110.1, 82.3, 77.7, 52.3,
30.7, 28.2, 27.3, 18.8; HRMS (FAB) m/z C24H32N3O5S (MH+)
calcd 474.2063, found 474.2058. Anal. Calcd for C24H31N3O5S:
C, 60.87; H, 6.60; N, 8.87. Found: C, 60.71; H, 6.57; N, 8.67.
1.17 (s, 1H, minor cis-rotamer); 13C NMR (100 MHz, DMSO-
d6) δ 195.4, 155.2, 152.2, 143.7, 133.8, 127.2, 125.4, 115.1,
111.6, 110.5, 110.1, 82.3, 78.3, 58.2, 55.9, 28.1, 27.3; HRMS
(FAB) m/z C22H29N4O6 (MH+) calcd 445.2087, found 445.2094.
Anal. Calcd for C22H28N4O6: C, 59.45; H, 6.35; N, 12.60.
Found: C, 59.43; H, 6.37; N, 11.49.
C
The diazomethyl ketone from above (3.93 g, 8.84 mmol) was
dissolved in EtOAc (50 mL) and the solution cooled to -45 °C
in a dry ice-acetone bath. A solution of 45% HBr in AcOH
(1.30 mL, 10.04 mmol, 1.14 equiv) was added dropwise over
75 min, the progress of the reaction being monitored by TLC.
After completion, the cold reaction mixture was diluted with
TBME (150 mL) and the solution washed with 5% aqueous
citric acid (2 × 50 mL), saturated NaHCO3 (2 × 50 mL), and
brine (50 mL). After drying (Na2SO4), the solution was
concentrated under reduced pressure and the residue purified
by flash chromatography on silica gel using 30% EtOAc in
hexane as eluent. The desired bromomethyl ketone 44 was
obtained as a yellow foam (3.614 g, 82% yield): mp 82-90 °C
(dec); Rf 0.39 (50% EtOAc - hexane); [R]D +0.4 (c 1.0, CHCl3);
1H NMR (400 MHz, DMSO-d6) indicated the presence of
pseudo-cis- and trans-rotamers of the carbamate bond in a 8:1
ratio, δ 10.97 (br s, 1H), 7.35 (d, J ) 1.6 Hz, 1H), 7.33 (d, J )
7.4 Hz, 1H), 7.32 (d, J ) 8.8 Hz, 1H), 7.21 (d, J ) 1.4 Hz, 1H),
6.85 (dd, J ) 8.8, 2.2 Hz, 1H), 4.47 (s, 1H), 4.46 (s, 1H), 4.39
(m, 1H), 3.13 (dd, J ) 14.7, 4.7 Hz, 1H, part of AB), 2.87 (dd,
J ) 14.5, 9.4 Hz, 1H, part of AB), 1.48 (s, 9H), 1.31 (s, 8H,
major trans-rotamer), 1.18 (s, 1H, minor cis-rotamer); 13C NMR
(100 MHz, DMSO-d6) δ 201.0, 155.4, 152.2, 143.8, 133.8, 127.2,
125.5, 115.1, 111.7, 110.3, 110.1, 82.3, 78.5, 58.7, 35.5, 28.1,
27.3; HRMS (FAB) m/z C22H29BrN2O6 (MH+) calcd 496.1209,
found 496.1209. Anal. Calcd for C22H29BrN2O6: C, 53.13; H,
5.88; N, 5.63. Found: C, 53.26; H, 5.96; N, 5.81.
General Procedure for the Preparation of Thiazolyl
Derivatives of General Formula 45 from Bromomethyl
Ketone 44. Carbonic Acid 3-[(S)-2-(2-Aminothiazol-4-yl)-
2-tert-butoxycarbonylaminoethyl]-1H-indol-5-yl Ester tert-
Butyl Ester (Compound 45, X ) NH2). Bromomethyl ketone
44 (1.180 g, 2.37 mmol) was dissolved in acetonitrile (10 mL),
and thiourea (0.226 g, 2.97 mmol, 1.25 equiv) was added. The
mixture was refluxed for 24 h. The reaction was then diluted
with TBME (150 mL) and the solution washed with saturated
NaHCO3 (25 mL). After drying (Na2SO4), volatiles were
removed under reduced pressure, and the residue was purified
by flash chromatography on silica gel using 70-100% EtOAc
in hexane to give the desired thiazolyl derivative as a white
solid (1.050 g, 93% yield): mp 117-123 °C (dec); Rf 0.27 (70%
EtOAc-hexane); [R]D -14.2 (c 1.0, CHCl3); 1H NMR (400 MHz,
DMSO-d6) indicated the presence of pseudo-cis- and trans-
rotamers of the carbamate bond in a 8:1 ratio, δ 10.87 (br s,
1H), 7.29 (d, J ) 8.6 Hz, 1H), 7.25 (d, J ) 1.6 Hz, 1H), 7.08 (d,
J ) 1.3 Hz, 1H), 6.99 (d, J ) 9.0 Hz, 1H), 6.90 (br s, 2H), 6.83
(dd, J ) 8.6, 2.2 Hz, 1H), 6.20 (s, 1H)4.58 (m, 1H), 3.15 (dd, J
) 14.7, 4.5 Hz, 1H, part of AB), 2.86 (dd, J ) 14.7, 9.2 Hz, 1H,
part of AB)1.49 (s, 9H), 1.29 (s, 8H, major trans-rotamer), 1.07
(s, 1H, minor cis-rotamer); 13C NMR (100 MHz, DMSO-d6) δ
168.3, 155.0, 153.8, 152.3, 143.6, 133.7, 127.6, 124.7, 114.8,
111.9, 111.5, 110.1, 100.3, 82.3, 77.6, 52.5, 30.3, 28.2, 27.3;
HRMS (FAB) m/z C23H31N4O5S (MH+) calcd 475.2015, found
475.2015. Anal. Calcd for C23H30N4O5S: C, 58.21; H, 6.37; N,
11.81. Found: C, 58.41; H, 6.41; N, 11.35.
General Procedure for Coupling Thiazolyl Deriva-
tives to Benzimidazole Carboxylic Acid 2. Preparation
of 1-Cyclohexyl-2-furan-3-yl-1H-benzimidazole-5-carbox-
ylic Acid [(S)-1-(2-Aminothiazol-4-yl)-2-(5-hydroxy-1H-
indol-3-yl)ethyl]amide 30. The bis-Boc-protected aminothi-
azole derivative from above (0.160 g, 0.34 mmol, 1.4 equiv)
was dissolved in CH2Cl2 (1 mL), and TFA (0.5 mL) was added.
The mixture was stirred for 1 h at room temperature, and
volatiles were removed under reduced pressure. The residue
was dissolved in DMSO (2 mL), and carboxylic acid 2 (0.075
g, 0.24 mmol, 1 equiv) was added followed by triethylamine
(0.28 mL, 2 mmol, 8.3 equiv) and TBTU (0.078 g, 0.24 mmol,
1 equiv). The reaction mixture was stirred for 1 h at room
temperature (complete by RP-HPLC analysis) and acidified
with TFA and the product isolated directly from the reaction
mixture by preparative reversed-phase HPLC using 0.1%
TFA-acetonitrile gradients. The TFA salt of inhibitor 30 (30
mg, 18% yield) was isolated as a yellow amorphous solid after
lyophilization of pure fractions: 1H NMR (400 MHz, DMSO-
d6) δ 10.50 (d, J ) 1.6 Hz, 1H), 8.86 (d, J ) 7.8 Hz, 1H), 8.33
(s, 1H), 8.22 (d, J ) 1.0 Hz, 1H), 7.96 (m, 2H), 7.78 (dd, J )
8.6, 1.0 Hz, 1H), 7.08 (d, J ) 11.7 Hz, 1H), 7.02 (s, 1H), 6.97
(dd, J ) 13.9, 2.0 Hz, 1H), 6.70 (s, 1H), 6.59 (dd, J ) 8.6, 2.2
Hz, 1H), 5.27 (dt, J ) 7.8, 6.9 Hz, 1H), 4.45 (tt, J ) 12.3, 3.3
Hz, 1H), 3.24 (dd, J ) 14.7, 6.3 Hz, 1H, part of AB), 3.17 (dd,
J ) 14.5, 8.8 Hz, 1H, part of AB), 2.32-2.19 (m, 2H), 1.97-
1.80 (m, 4H), 1.68 (m, 1H), 1.50-1.35 (m, 3H); 13C NMR (100
MHz, DMSO-d6) δ 169.83, 166.1, 150.3, 147.6, 144.6, 143.9,
135.3, 130.6, 128.2, 127.9, 124.1, 122.2, 117.8, 115.0, 112.9,
111.6, 111.3, 111.1, 109.2, 102.8, 102.4, 56.9, 48.5, 30.4, 29.5,
25.4, 24.3; HRMS (FAB) m/z C31H31N6O3S (MH+) calcd 567.2178,
found 567.2197; HPLC homogeneity 99.6% (system A), 99.6%
(system B).
Biological Assays. IC50 determinations were carried out
as previously described11 using an N-terminally tagged full-
length NS5B construct. Reported values are the average of
duplicate measurements carried out on two separate weight-
ings. Specificity for HCV polymerase was evaluated using the
rdrp of the poliovirus and a DNA-dependent RNA mammalian
polymerase isolated from calf-thymus, also as previously de-
scribed.11,25 EC50 determinations in the cell-based replicon
assay were performed in duplicates on two separate weightings
using RT-PCR for RNA quantification as described elsewhere.25
Acknowledgment. We thank Norman Aubry,
Sylvain Bordeleau, Colette Boucher, and Michael Little
for analytical support. We are also grateful to Guillaume
Arguin, Fre´de´rik Be´langer, Philippe Courtemanche-
Asselin, Laura Feldcamp, Sylvain Lefebvre, Martin Pel-
licelli, and Louise Thauvette for biochemical assays and
Peter Kohlbauer for the synthesis of some inhibitors.
Supporting Information Available: Analytical data (1H
and 13C NMR, HRMS, and HPLC homogeneities in two solvent
systems) are provided for inhibitors 3-29 and 31-34. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Carbonic Acid 3-[(S)-2-tert-Butoxycarbonylamino-2-(2-
methylthiazol-4-yl)ethyl]-1H-indol-5 -yl Ester tert-Butyl
Ester (compound 45, X ) Me). Following a similar procedure
to that described above but using thioacetamide instead of
thiourea, the title compound was obtained as a white solid in
60% yield after purification by flash chromatography: mp 80-
83 °C (dec); Rf 0.64 (70% EtOAc-hexane); [R]D -9.9 (c 1.0,
CHCl3); 1H NMR (400 MHz, DMSO-d6) indicated the presence
of pseudo-cis- and trans-rotamers of the carbamate bond in a
8:1 ratio, δ 10.89 (s, 1H), 7.28 (d, J ) 8.6 Hz, 1H), 7.22 (m,
2H), 7.12 (d, J ) 8.6 Hz, 1H), 7.11 (s, 1H), 6.83 (dd, J ) 8.8,
2.2 Hz, 1H), 4.84 (m, 1H), 3.19 (dd, J ) 14.5, 5.1 Hz, 1H), 2.97
(dd, J ) 14.5, 8.8 Hz, 1H), 2.63 (s, 3H), 1.49 (s, 9H), 1.29 (s,
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