Synthesis and HepatitisC Antiviral Activity of 1-Aminobenzyl-1H-indazole-3-carboxamide Analogues

Full text of DOI:10.1002/cmdc.201300083

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DOI: 10.1002/cmdc.201300083
Synthesis and Hepatitis C Antiviral Activity of 1-
Aminobenzyl-1H-indazole-3-carboxamide Analogues
Jing-Jing Shi,[a] Fei-Hong Ji,[b] Pei-Lan He,[b] Ya-xi Yang,[a] Wei Tang,[b] Jian-Ping Zuo,*[b] and
Yuan-Chao Li*[a]
Hepatitis C virus (HCV) is a small (+)-RNA virus classified in the
genus Hepaciviru of the family Flaviviridae.[1] Nearly 3% of the
global population (approximately 170 million) have been in-
fected with HCV, while 3–4 million are newly infected each
year. Untreated HCV infections can progress to liver fibrosis,
steatosis, cirrhosis, and even hepatocellular carcinoma.[2] Since
the discovery of the virus in 1989,[3] much effort has been
made to find efficient antiviral therapies.[4] Currently, the stan-
dard of care for patients with chronic hepatitis C is a combina-
tion of pegylated interferon alpha (PEG-IFN-a) and ribavirin.[5]
However, this treatment regime is only effective for 40–60% of
people infected with HCV genotype-1, which accounts for the
majority of infections in the US, Europe, and Asia.[6] Further-
more, the low success rate and high cost associated with this
treatment restrict its usage. Viral protease and polymerase in-
hibitors are promising agents currently under development. In
2011, two NS3A/4A protease inhibitors (boceprevir and telapre-
vir) were approved by the US Food and Drug Administration
(FDA) for the treatment of chronic hepatitis C infection. In
combination with PEG-IFN-a and ribavirin, both boceprevir
and telaprevir produce a higher cure rate and shorter period
of treatment.[7] HCV NS5B polymerase is another attractive
target for antiviral therapy.[8] Because of the high mutation and
replication rates of HCV,[9] it is unlikely that a preventive vac-
cine will become available in the coming years, and as such,
more effective anti-HCV drugs are urgently needed.
ture–HCV antiviral activity relationships
of 1-aminobenzyl-1H-indazole-3-carbox-
amide derivatives.
The synthesis of compounds 5–7 is
depicted in Scheme 1. Compounds 2a–
c were obtained from commercially
available 3-carboxylic indazole 1 by pre-
paring the aroyl chloride and then re-
acting it with the appropriate amine. Alkylation at position 1 of
2a with 1-(bromomethyl)-3-nitrobenzene was performed in
In previous studies,[10] we applied a HCV-infected system in
combination with an automated high-throughput system to
screen an available compound library. We identified 1-(3-ami-
nobenzyl)-1H-indazole-3-carboxamide (4a) as a modest inhibi-
tor of the HCV-infected system with an IC50 value of 5–10 mm
and low cytotoxicity against Huh 7.5.1 cells (CC50 >10 mm). Uti-
lizing 4a as a lead, we initiated a study to explore the struc-
Scheme 1. Synthesis of acylamide compounds 5a–5w, 6a–6b and 7. Re-
agents and conditions: a) 1. SOCl2, 808C, 4 h, 2. R1NH2, THF, 08C, 5 h, 52–
57%; b) K2CO3, 1-(bromomethyl)-3-nitrobenzene, DMF, RT, 6 h, 85–87%;
c) SnCl2·2H2O, THF, RT, overnight, 70–79%; d) R2CO2H, HBTU, Et3N, DMF, RT,
overnight, or R2COCl, Et3N, CH2Cl2, 08C, 5 h, 65–91%.
[a] J.-J. Shi,+ Y.-x. Yang, Prof. Y.-C. Li
the presence of potassium carbonate to give nitro compound
3a; compounds 3b–c were synthesized analogously. After re-
duction of the nitro groups with tin(II) chloride dihydrate, key
intermediate amines 4a–c were obtained. Acylamides 5–7
were subsequently accessed via acylation.
Department of Medicinal Chemistry
Shanghai Institute of Materia Medica, Chinese Academy of Sciences
555 Road Zu Chong Zhi, Zhangjiang Hi-Tech Park
Shanghai 201203 (P. R. China)
E-mail: ycli@mail.shcnc.ac.cn
[b] F.-H. Ji,+ P.-L. He, W. Tang, Prof. J.-P. Zuo
Acylamide compounds 10a,b and 13a,b were prepared
from 1H-indazole-3-carboxamide 2a, through benzylation, re-
duction and acylation (Scheme 2).
Laboratory of Immunopharmacology
State Key Laboratory of Drug Research
Shanghai Institute of Materia Medica, Chinese Academy of Sciences
555 Road Zu Chong Zhi, Zhangjiang Hi-Tech Park
Shanghai 201203 (P. R. China)
Initial efforts were focused on evaluating different substitu-
ents at various positions on the benzyl group (Table 1). Acyla-
tion of the 3’-amino group of compound 4a gives 5a,b, and
these compounds exhibit significantly improved inhibitory ac-
tivities, especially compound 5b (IC50 =0.125 mm), which has
E-mail: jpzuo@mail.shcnc.ac.cn
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300083.
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yl analogues 5d,e and h were
devoid of HCV inhibitory activity
at a compound concentration
of 0.5 mm. Additionally, phenyl-
acetic analogue 5c was found
to be a potent inhibitor (IC50 =
0.539 mm). These findings sug-
gested that the presence of an
aryl moiety in the acyl group
could lead to improved bioac-
tivity. Furthermore, a primary
amino group on the 3-carboxa-
mide was found to be critical
for activity, as substitutions
(e.g., 6a, 6b and 7) caused sig-
nificant decrease in activity.
Scheme 2. Synthesis of acylamides A) 10a–b and B) 13a–b. Reagents and conditions: a) K2CO3, 1-(bromomethyl)-2-
nitrobenzene (for 10a–b) or 1-(bromomethyl)-4-nitrobenzene (for 13a–b), DMF, RT, 6 h, 69–72%; b) SnCl2·2H2O,
THF, RT, overnight, 70–79%; c) RCOCl, Et3N, CH2Cl2, 08C, 5 h, 83–90%.
The
results
summarized
above led to a study that tar-
geted further optimization of the inhibitory activity by varying
aroyl substituents at the 3’-amino group (Table 3). As anticipat-
ed, the compounds of this series showed improved bioactivity.
In particular, the analogues with an aroyl group substituted in
Table 1. Structures and anti-HCV activities of 1-aminobenzyl-1H-indazole-
3-carboxamide analogues determined in HCV-infected Huh 7.5.1 cells.
Table 2. Structures and anti-HCV activities of 1-aminobenzyl-1H-indazole-
3-carboxamide analogues determined in HCV-infected Huh 7.5.1 cells.
Compd
R
CC50 [mm]
IC50[a] [mm]
SI[b]
4a
5a
3’-A
3’-B
3’-C
2’-A
2’-B
2’-C
4’-A
4’-B
4’-C
>10
>10
>10
>10
>10
>10
>10
>10
>10
5–10
1.17
0.125
>10
>10
1.70
>10
>10
>10
>1
>8.5
>80
5b
9
10a
10b
12
Compd R1
R2
CC50 [mm] IC50[c] [mm]
SI[d]
–
–
5c
H
>10
92.1[a]
86.0[a]
0.539
>18.5
>5.9
–
–
–
5d
5e
H
H
CH3
0[b]
–
–
13a
13b
10.3[b]
[a] Mycophenolic acid (MPA) was used as reference agent/positive con-
trol; in this assay, the IC50 value was 0.713 mm. [b] Selective index (SI) cal-
culated as CC50/IC50; IC50 values were estimated by inhibition at five con-
centrations; assays were performed in triplicate, and data represent simi-
lar results.
5 f
H
H
>10
0.674
0.267
>14.8
>37.4
5g
>10
89.2[a]
5h
6a
H
8.71[b]
6.05[b]
–
–
approximately a 40-fold higher bioactivity than 4a. Further-
more, 2’-amino (9, 10a,b) and 4’-amino (12, 13a, 13b) ana-
logues were found not to exhibit significant HCV inhibition,
with the exception of compound 10b that has a moderate IC50
value of 1.70 mm although this is significantly poorer than the
similarly substituted analogue 5a (IC50 =1.17 mm). The findings
indicate that an amino group at position 3’ of the 1-aminoben-
zyl-1H-indazole-3-carboxamide core is crucial for HCV inhibitory
activity and that acylation of the amino group influences activi-
ty.
Ph
96.2[a]
94.8[a]
6b
7
Ph
0[b]
–
–
CH2COOMe
>10
>10
[a] Cell viability (%) at 0.5 mm. [b] Percentage inhibition at 0.5 mm. [c] My-
cophenolic acid (MPA) was used as reference agent/positive control; in
this assay, the IC50 value was 0.807 mm. [d] Selective index (SI) calculated
as CC50/IC50; IC50 values were estimated by inhibition at five concentra-
tions; assays were performed in triplicate, and data represent similar re-
sults.
Subsequently, the focus was shifted to different substitutions
around the amino group in position 3’ of the 3-carboxamide
moiety. Aroyl analogues of 4a (e.g., 5 f and 5g) were observed
to exhibit improved antiviral activity (Table 2). However, alkano-
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the para position (e.g., 5n, 5q, 5t, 5u and 5v) exhibited signif-
icantly increased activity compared with unsubstituted deriva-
tive 5b (IC50 =0.125 mm). Specifically, compounds 5n (IC50 =
0.013 mm) and 5t (IC50 =0.007 mm) were found to be the most
potent inhibitors with low cytotoxicity. However, analogues
with substitutions at either the ortho or meta position (e.g., 5j,
5l, 5m, 5o, 5p, 5r and 5s) did not show improved activity,
with the exception of 5i (IC50 =0.035 mm). Furthermore, the an-
tiviral potencies of compounds 5i, 5n, 5q and 5t were validat-
ed by observation of their activities in an HCV replicon assay
(Table 4). Analogues 5n and 5q possess superb anti-HCV activi-
ty; both are efficient HCV replicon inhibitors, with EC50 values
of 0.018 mm and 0.024 mm, respectively, coupled with low toxic-
ity (CC50 =20.67 mm and CC50 >100 mm, respectively).
In conclusion, starting from compound 4a as an early lead
inhibitor of HCV, extensive structure–activity relationships stud-
ies around multiple positions on the core structure were per-
formed. The results showed that the amino group of the 3-car-
boxamide scaffold could not be substituted, and that com-
pounds in which the benzamide with a para orientation at po-
sition 3’ showed the best bioactivity. The results led to the dis-
covery of two potent anti-HCV, compounds 5n (IC50 =0.013 mm
and EC50 =0.018 mm; SI=1150) and 5t (IC50 =0.007 mm and
EC50 =0.024 mm; SI> 4160). Given the potent biological activity
and selectivity of compounds 5n and 5t, studies are planned
aimed at the further optimization of these series. Their acute
toxicities and pharmacokinetic properties will be conducted, as
well as mechanistic studies on this class of compounds against
HCV.
Table 3. Structures and anti-HCV activities of 1-aminobenzyl-1H-indazole-
3-carboxamide analogues determined in HCV-infected Huh 7.5.1 cells.
Compd
R
CC50 [mm]
>10
IC50[a] [mm]
SI[b]
5i
0.035
>286
5j
>10
>10
0.230
0.115
>43
>87
5k
5l
>10
0.115
>87
5m
5n
>10
>10
0.238
0.013
>42
>769
5o
>10
0.603
>16
5p
5q
>10
>10
0.346
0.035
>29
>285
Experimental Section
General: Details of the instruments used are given in the Support-
ing Information, along with characterization data for compounds
2–13 not given below.
5r
>10
0.739
>14
1H-Indazole-3-carboxamide (2a): A mixture of 1H-indazole-3-car-
boxylic acid (1) (3.24 g, 10 mmol) in SOCl2 (30 mL) was stirred at
reflux for 4 h. Volatiles were removed by a stream of nitrogen fol-
lowed by vacuum to give 1H-indazole-3-carbonyl chloride as
a solid. The solid was redissolved in THF (50 mL), and the solution
was cooled to 08C and then treated dropwise with 26% NH3·H2O
(8 mL). The mixture was stirred at RT for 5 h. Water (20 mL) was
added, and the resultant precipitate was isolated by filtration,
washed with water (30 mL) and THF (30 mL), and air dried to give
1H-indazole-3-carboxamide (2a) as a white solid (1.84 g, 57%):
1H NMR (300 MHz, [D6]DMSO): d=13.49 (s, 1H), 8.15 (d, J=8.1 Hz,
1H), 7.69 (s, 1H), 7.58 (d, J=8.1 Hz, 1H), 7.38 (t, J=7.5 Hz, 1H),
7.30 (s, 1H), 7.21 ppm (t, J=7.5 Hz, 1H); LC-MS (ESI): m/z (%):184.1
(100) [M+Na]+.
5s
5t
>10
>10
0.213
0.007
>47
>1430
5u
5v
6.35
0.044
0.040
144
>10
>250
5w
>10
0.110
>91
Compounds 2b,c were prepared by the same procedure as de-
scribed for 2a.
[a] Mycophenolic acid (MPA) was used as reference agent/positive con-
trol; in this assay, the IC50 value was 0.780 mm. [b] Selective index (SI) cal-
culated as CC50/IC50; IC50 values were estimated by inhibition at five con-
centrations; assays were performed in triplicate, and data represent simi-
lar results.
1-(3-Nitrobenzyl)-1H-indazole-3-carboxamide (3a): A mixture of
2a (1.61 g, 10 mmol), K2CO3 (2.76 g, 20 mmol) and 1-(bromometh-
yl)-3-nitrobenzene (2.26 g, 10.5 mmol) in DMF (30 mL) was stirred
at RT for 6 h. Water (50 mL) was then added, and the resultant pre-
cipitate was isolated by filtration, washed with water (15ꢁ3 mL),
and air dried to give 1-(3-nitrobenzyl)-1H-indazole-3-carboxamide
1
(3a) as a white solid (2.58 g, 87%): H NMR (300 MHz, [D6]DMSO):
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Acknowledgements
Table 4. Results of the HCV replicon assays for compounds 5i, 5n, 5q,
and 5t.
This work was supported by a National Science and Technology
Major Project (China): “Chemical Innovative Drug Research and
Development System” (grant no. 2012ZX09301-001).
Compd
CC50 [mm]
EC50 [mm]
SI[a]
322
1150
156
5i
10.31
20.67
28.42
0.032
0.018
0.182
0.024
5n
5q
5t
>100
>4160
Keywords: antiviral agents · carboxamides · hepatitis C ·
organic synthesis · structure–activity relationships
[a] Selective index (SI) calculated as CC50/EC50; EC50 values were estimated
by inhibition at five concentrations; assays were performed in triplicate,
and data represent similar results.
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7.44 (m, 2H), 7.27 (t, J=7.5 Hz, 1H), 5.89 ppm (s, 2H); LC-MS (ESI):
m/z (%): 319.0 (100) [M+Na]+.
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Compounds 3b,c, 8, 11 were prepared by the same procedure as
described for 3a.
1-(3-Aminobenzyl)-1H-indazole-3-carboxamide (4a): A mixture of
3a (400 mg, 1.35 mmol) and SnCl2·2H2O (1.22 g, 5.4 mmol) in THF
(10 mL) was stirred at RT overnight, and then the solution was
treated with 5m aq NaOH to pH 11, extracted with EtOAc (2ꢁ
50 mL), washed with brine (50 mL), dried (Na2SO4), filtered and con-
centrated. Purification by chromatography (EtOAc/Pet. ether, 1:1)
gave 4a as a white solid (252 mg, 70%): 1H NMR (300 MHz,
[D6]DMSO): d=8.17 (d, J=8.1 Hz, 1H), 7.67 (d, J=8.6 Hz, 2H), 7.40
(m, 2H), 7.23 (t, J=7.5 Hz, 1H), 6.93 (t, J=7.5 Hz, 1H), 6.40 (m, 2H),
6.32 (s, 1H), 5.55 (s, 2H), 5.10 ppm (br s, 2H); 13C NMR (100 MHz,
[D6]DMSO): d=164.4, 149.4, 141.1, 137.9, 137.8, 129.6, 127.1, 123.0,
122.8, 122.5, 115.1, 113.8, 112.7, 111.0, 53.2 ppm; LC-MS (ESI): m/z
(%): 289.0 (100) [M+Na]+.
[5] T. Schaller, N. Appel, G. Koutsoudakis, S. Kallis, V. Lohmann, T. Pietsch-
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Compounds 4b,c, 9, 12 were prepared by the same procedure as
described for 4a.
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1-(3-(Cyclopropanecarboxamido)benzyl)-1H-indazole-3-carboxa-
mide (5a): A solution of 4a (27 mg, 0.1 mmol) and Et3N (0.028 mL,
0.2 mmol) in dry CH2Cl2 (1 mL) at 08C under Ar was treated with
cyclopropanecarbonyl chloride (0.010 mL, 0.11 mmol). The mixture
was stirred at 08C for 30 min. The solvent was removed in vacuo
to give the crude product, which was purified by chromatography
(CH2Cl2/MeOH, 50:1) to give 5a as a white solid (29 mg, 88%):
1H NMR (300 MHz, [D6]DMSO): d=10.14 (s, 1H), 8.18 (d, J=8.3 Hz,
1H), 7.69 (m, 2H), 7.51 (d, J=8.4 Hz, 1H), 7.41 (m, 3H), 7.23 (dd,
J=15.6, 7.6 Hz, 2H), 6.90 (d, J=7.2 Hz, 1H), 5.68 (s, 2H), 1.69 (m,
1H), 0.73 ppm (m, 4H); 13C NMR (100 MHz, [D6]DMSO): d=171.7,
163.8, 140.7, 139.6, 137.6, 137.4, 129.0, 126.7, 122.5, 122.4, 122.1,
121.8, 118.3, 117.5, 110.4, 52.4, 14.5, 7.16 ppm (2C); LC-MS (ESI): m/
z (%): 357.0 (100) [M+Na]+.
Received: February 22, 2013
Published online on && &&, 0000
Compounds 5b–w, 6a,b, 7, 10a,b, 13a,b were prepared by the
same procedure as described for 5a.
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Fighting HCV: Two potent antiviral ana-
logues were developed from a previous-
ly identified lead as novel agents
against hepatitis C virus. Their potency
and selectivity (5n: IC50 =0.013 mm and
EC50 =0.018 mm; 5t: IC50 =0.007 mm and
EC50 =0.024 mm) make them good can-
didates for further development as anti-
viral agents.
J.-J. Shi, F.-H. Ji, P.-L. He, Y.-x. Yang,
W. Tang, J.-P. Zuo,* Y.-C. Li*
&& – &&
Synthesis and Hepatitis C Antiviral
Activity of 1-Aminobenzyl-1H-
indazole-3-carboxamide Analogues
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ChemMedChem 0000, 00, 1 – 4
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