N-(3-cyano-1H-indol-5-yl)isonicotinamide and N-(3-cyano-1H-indol-5-yl)-1H-benzo[d]imidazole-5-carboxamide derivatives: Novel amide-based xanthine oxidase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2021.105181

Our previous work demonstrated that amide is an efficient linker to explore chemical space of xanthine oxidase (XO) inhibitors that are entirely different from febuxostat and topiroxostat. In this effort, with 3-cyano-1H-indol-5-yl as a key moiety, two series of amide-based XO inhibitors, N-(3-cyano-1H-indol-5-yl)isonicotinamides (2a-w) and N-(3-cyano-1H-indol-5-yl)-1H-benzo[d]imidazole-5-carboxamides (3a-i), were designed and synthesized. The structure-activity relationship investigation identified N-(3-cyano-1-cyclopentyl-1H-indol-5-yl)-1H-benzo[d]imidazole-5-carboxamide (3i, IC₅₀ = 0.62 μM) as the most promising compound, with 14.4-fold higher in vitro inhibitory potency than allopurinol (IC₅₀ = 8.91 μM). Molecular simulations provided reasonable interaction modes for the representative compounds. Furthermore, in vivo activity evaluation demonstrated that compound 3i (oral dose of 12.8 mg/kg) has obviously hypouricemic effect on a potassium oxonate induced hyperuricemic rat model. Cytotoxicity assay and ADME prediction also supported that 3i is an excellent lead for further exploration of amide-based XO inhibitors.

Full text of DOI:10.1016/j.bioorg.2021.105181

Bioorganic Chemistry 115 (2021) 105181
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
N-(3-cyano-1H-indol-5-yl)isonicotinamide and
N-(3-cyano-1H-indol-5-yl)-1H-benzo[d]imidazole-5-carboxamide
derivatives: Novel amide-based xanthine oxidase inhibitors
1
1
*
Shun Tu , Ting-jian Zhang , Yi Zhang, Xu Zhang, Zhen-hao Zhang, Fan-hao Meng
School of Pharmacy, China Medical University, 77 Puhe Road, North New Area, Shenyang 110122, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Our previous work demonstrated that amide is an efficient linker to explore chemical space of xanthine oxidase
XO) inhibitors that are entirely different from febuxostat and topiroxostat. In this effort, with 3-cyano-1H-indol-
-yl as a key moiety, two series of amide-based XO inhibitors, N-(3-cyano-1H-indol-5-yl)isonicotinamides (2a-w)
Synthesis
(
Xanthine oxidase inhibitor
SAR
5
and N-(3-cyano-1H-indol-5-yl)-1H-benzo[d]imidazole-5-carboxamides (3a-i), were designed and synthesized.
The structure-activity relationship investigation identified N-(3-cyano-1-cyclopentyl-1H-indol-5-yl)-1H-benzo[d]
Hyperuricemia
imidazole-5-carboxamide (3i, IC50 = 0.62
inhibitory potency than allopurinol (IC50 = 8.91
modes for the representative compounds. Furthermore, in vivo activity evaluation demonstrated that compound
i (oral dose of 12.8 mg/kg) has obviously hypouricemic effect on a potassium oxonate induced hyperuricemic
μ
M) as the most promising compound, with 14.4-fold higher in vitro
μ
M). Molecular simulations provided reasonable interaction
3
rat model. Cytotoxicity assay and ADME prediction also supported that 3i is an excellent lead for further
exploration of amide-based XO inhibitors.
1
. Introduction
Hyperuricemia is a chronic metabolic disease with elevated serum
allopurinol and other purine-type XO inhibitors affect purine meta-
bolism and cause serious side effects, including fever, liver damage,
kidney damage, and Stevens-Johnson syndrome [13]. In the past ten
years, many nonpurine-based XO inhibitors with different scaffolds have
been released, including furans [14] pyrano[3,2-d]pyrimidines [15]
flavonoids [16,17] imidazoles [18] 5-aryl-1H-tetrazoles [19] thiazole-5-
carboxylic acid derivatives [20] dihydropyrimidine-5-carboxylic acid
derivatives [21] 5-arylazo-tropolone derivatives [22] caffeic acid phe-
nethyl ester derivatives [23] and chalcone derivatives [24,25]. In clin-
ical application, nonpurine XO inhibitor febuxostat was approved by the
US Food and Drug Administration (FDA) in 2009, and another non-
purine XO inhibitor, topiroxostat, was subsequently approved by the
Japanese Food and Drug Administration in 2013. Both topiroxostat and
febuxostat are novel nonpurine XO inhibitors based on the characteristic
five-membered ring linker. However, after clinical application, it was
found that topiroxostat may increase the incidence of gouty arthritis
[26]. And on February 21, 2019, FDA added a boxed warning for
increased risk for heart-related death with febuxostat. Hence, novel
nonpurine XO inhibitors are still urgently needed to meet the clinical
requirement.
uric acid caused by purine metabolism disorder [1,2]. Serum uric acid is
the final oxidation product of human purine catabolism, and the
continuous increase of serum uric acid will induce the formation of
monosodium urate crystal deposits, eventually leading to gout [3]. Be-
sides, high levels of serum uric acid are associated with many other
chronic diseases, such as cardiovascular disease, hypertension, and
kidney disease [4–6]. Xanthine oxidase (XO) is a multifunctional
molybdoflavin protein and a key rate-limiting enzyme in the purine
metabolic pathway [7]. XO can catalyze the oxidation of hypoxanthine
and xanthine to uric acid and generate reactive oxygen species (ROS)
[
8,9]. Excessive ROS can cause cell damage and participate in many
pathological processes, such as diabetes, atherosclerosis, and chronic
heart failure [10]. Therefore, XO has become an important target, not
only for the treatment of gout and hyperuricemia but also for many
other diseases related to ROS [9,11].
Allopurinol is a hypoxanthine isomer that has been used to treat
hyperuricemia and gout for decades [12]. However, as purine analog,
*
Corresponding author.
E-mail address: fhmeng@cmu.edu.cn (F.-h. Meng).
These two authors contributed equally to this work.
1
https://doi.org/10.1016/j.bioorg.2021.105181
Received 30 November 2020; Received in revised form 5 July 2021; Accepted 12 July 2021
Available online 15 July 2021
0
045-2068/© 2021 Elsevier Inc. All rights reserved.

S. Tu et al.
Bioorganic Chemistry 115 (2021) 105181
In recent years, our team has been engaged in the discovery of novel
2. Results and discussion
XO inhibitors, and numerous promising compounds with diverse che-
motypes have been published [9,27–33]. Especially, through employing
amide fragment to be an opened-ring isostere of five-membered ring
linker of classic XO inhibitors (e.g., thiazole of febuxostat and 1,2,4-tri-
azole of topiroxostat), we successfully identified N-phenyl-
isonicotinamide, as a novel XO inhibitor scaffold for the first time [27].
This finding provides important clues for the discovery of novel XO in-
hibitors bearing an amide linker (amide-based XO inhibitors). In the
present work, amide-based XO inhibitors with stronger structural di-
versity were further explored.
2.1. Chemistry
The synthesis of N-(1-alkyl-3-cyano-1H-indol-5-yl) isonicotinamides
(2a-i), N-(1-alkyl-3-cyano-1H-indol-5-yl)-2-chloroisonicotinamides (2j-
o) and N-(1-alkyl-3-cyano-1H-indol-5-yl)-2-fluoroisonicotinamides (2p-
w) was performed as outlined in Scheme 1. The formyl group was
introduced into commercially available 5-nitro-1H-indole under
Vilsmeier-Haack conditions to provide 5-nitro-1H-indole-3-carbalde-
hyde (4) [40]. The corresponding formyl group was further converted
into a cyano group in the presence of hydroxylamine hydrochloride and
sodium formate in formic acid at reflux to afford 5-nitro-1H-indole-3-
carbonitriles (5) [41]. The reduction of 5 by hydrogen under the catal-
ysis of Pd/C yielded 5-amino-1H-indole-3-carbonitrile (6), which was
acylated with corresponding acyl chloride in the presence of triethyl-
amine to provide N-(1-alkyl-3-cyano-1H-indol-5-yl) heterocyclic amides
(2a, 2j, 2o, and 3a) [42,43]. Alkylation of compounds (2a, 2j, and 2o)
with various alkyl chlorides or alkyl bromides obtained N-(1-alkyl-3-
cyano-1H-indol-5-yl) heterocyclic amides (2a-w) [44].
Guiding by the structure based drug design (CBDD), we speculated
that introducing larger aromatic (such as fused aromatic ring) on one or
both sides of the amide linker may balance two vital H-bond interactions
with residues Asn768 and Glu1261, respectively, and facilitate the
binding affinity and inhibitory potency. Indole, as an endogenous bio-
logically active substance and a popular drug construction fragment, has
been widely used in the design of anti-tumor, anti-viral, anti-
inflammatory, and even anti-hyperuricemia drugs [34–38]. Therefore,
using indole as a key moiety, a series of N-(3-cyano-1H-indol-5-yl)iso-
nicotinamide derivatives (2a-w) was firstly designed and synthesized.
Furthermore, isonicotinamide, as the first discovered amide framework,
despite exhibiting a great contribution to binding affinity (e.g., forming
The synthesis of N-(1-alkyl-3-cyano-1H-indol-5-yl)-1H-benzo[d]
imidazole-6-carboxamide (3b-i) was carried out by a similar procedure
as shown in Scheme 2. 5-nitro-1H-indole-3-carbonitrile (5) was alky-
lated with various alkyl chlorides or alkyl bromides in the presence of
sodium hydride in dimethylformamide (DMF) to give 1- alkyl-5-nitro-
1H-indole-3-carbonitrile (7). The reduction of 7 by tin chloride dehy-
drate in the presence of hydrochloric acid yielded 1-alkyl-5-amino-1H-
indole-3-carbonitrile (8), which was acylated with 1H-benzo[d]imid-
azole-6-carbonyl chloride in the presence of triethylamine to provide N-
(1-alkyl-3-cyano-1H-indol-5-yl)-1H-benzo[d]imidazole-6-carboxamide
(3b-i) [45].
π
-
π
stack with Phe914 and generating hydrogen bonds with Glu1261),
was assumed that it is not irreplaceable. Next, we utilized a bicyclic 5-
H-benzo[d]imidazole-5-carbonyl moiety to replace the monocyclic
isonicotinoyl moiety of 2a-w to provide another series of XO inhibitors,
1
N-(1H-indol-5-yl)-1H-benzo[d]imidazole-5-carboxamide
derivatives
′
(
3a-i) as shown in Fig. 1. Additionally, 3 -cyano was introduced onto the
indole moiety to serve as an H-bond receptor to interact with Asn768
′
residue of XO, and the substituent linked at 1 -position was expected to
form hydrophobic interaction with lipophilic amino acid residues(such
as Leu648, Phe649, and Phe1013).
The synthesis of N-(1H-indol-5-yl)isonicotinamide (10) and N-(3-
formyl-1H-indol-5-yl)isonicotinamide (11) were carried out by a similar
procedure as shown in Scheme 3. Commercially available 5-nitroindole
was reduced by hydrogen under the catalysis of Pd/C to yield 1H-indol-
5-amine (9), which was acylated with isonicotinoyl chloride in the
presence of triethylamine to provide N-(1H-indol-5-yl)isonicotinamide
(10). The formyl group was introduced into compound 10 under
Vilsmeier-Haack conditions to provide N-(3-formyl-1H-indol-5-yl)iso-
nicotinamide (11).
After the structure-activity relationship investigations, the most
potent compounds were submitted to steady-state kinetic analysis and
molecular simulations studies to investigate the inhibition behaviors.
Meanwhile, the in vivo hypouricemic effects, cytotoxicity and ADMET
(
absorption, distribution, metabolism, excretion, and toxicity) predic-
tion were further performed for the optimized compound.
Fig. 1. Design strategy of the target compounds.
2

S. Tu et al.
Bioorganic Chemistry 115 (2021) 105181
◦
◦
Scheme 1. Reagents and conditions: (i) POCl
3
, DMF, 0 C, 30 min, then room temperature overnight; (ii) HONH
2
HCl, HCOONa, AcOH, 110 C for 2 h; (iii) Pd/C, H
2
,
◦
N, THF, ꢀ 5 ◦C, 1 h, then room temperature overnight; (v) RCl or RBr, NaH, KI, DMF, ꢀ 5 ◦C, 30 min, then
EtOH, 25 C, 4 h; (iv) heterocyclic acid chloride, Et
3
◦
6
0
C, overnight.
′
2
.2. Biological activity in vitro
Considering the potential steric effect, the length of the alkyl at 1 -po-
sition of indole scaffold was initially locked within three carbon atoms.
′
The in vitro bovine XO inhibitory activity of compounds 2a-t, 3a-h,
a-d, 11, and 12 was measured spectrophotometrically by determining
Therefore, the influence of some three atom carbon chains at 1 -position
7
(2d, 2e, 2f, and 2 g) was investigated. As indicated in Table 1, branched-
chain alkyl groups may be more suitable for N-(1-alkyl-3-cyano-1H-
indol-5-yl)isonicotinamide than their straight-chain isostere (2e vs 2d;
uric acid production at 294 nm. Topiroxostat and allopurinol were
included as reference compounds. The testing results are shown in
Table 1.
IC50 = 4.43
allyl or propargyl, the potency was maintained (2f vs 2d; IC50 = 7.65
vs 7.34 M vs 7.34
M) or 6.2 times improved (2g vs 2d; IC50 = 1.18
M). This demonstrated that unsaturated alkyl may be more conducive
μM vs 7.34
μ
M). When the propyl group was changed into
As shown in Table 1, in the N-(1-alkyl-3-cyano-1H-indol-5-yl)iso-
μM
nicotinamides series, except compounds 2b (R
1
= methyl, not active, <
μ
μ
6
0% inhibition at 33
μM) and 2h (R
1
= benzyl, IC50 = 12.46
μ
M), other
μ
′
′
compounds with different alkyl groups at the 1 -position of indole
to N-(3-cyan-1H-indol-5-yl)isonicotinamide derivatives. When the 1 -
position was substituted by a benzyl, the potency was weakened, (2h vs
scaffold presented much higher potency than unsubstituted compound
′
2
a (IC50 = 8.59
μ
M). This indicated that the alkyl groups at 1 -position of
2a; IC50 = 12.46
significantly improved the potency (2i vs 2a; IC50 = 0.73
M), suggesting that for N-(3-cyan-1H-indol-5-yl)isonicotinamide, the
introduction of benzyl group may not be desirable while the substitution
μ
M vs 8.59
μ
M), while the introduction of a cyclopentyl
the indole scaffold played a crucial role for these compounds. From
methyl to ethyl, the efficiency varied from inactive to active, while, from
ethyl to propyl, there was no obvious discrepancy in potency.
μ
M vs 8.59
μ
3

S. Tu et al.
Bioorganic Chemistry 115 (2021) 105181
◦
◦
◦
Scheme 2. Reagents and conditions: (i) RCl or RBr, NaH, KI, DMF, ꢀ 5 C, 30 min, then 60 C, overnight; (ii) SnCl
2
, HCl, EtOH, 60 C, overnight; (iii) 1H-benzo[d]
imidazole-5-carbonyl chloride, Et
3
N, THF, ꢀ 5 ◦C, 1 h, then room temperature overnight.
◦
N, THF, ꢀ 5 ◦C, 1 h, then room temperature overnight; (iii) POCl
Scheme 3. Reagents and conditions: (i) Pd/C, H
2
, EtOH, 25 C, 18 h; (ii) isonicotinoyl chloride, Et
3
3
,
◦
DMF, 0 C, 30 min, then room temperature overnight.
of cyclopentyl would bring great benefits. Especially, cyclopentyl de-
rivative 2i displayed the highest inhibitory activity in the N-(1-alkyl-3-
cyano-1H-indol-5-yl)isonicotinamides series, which was 12-fold better
improved as the isonicotinamide series, and even presented an opposite
effect. Except for the slight increase in activity of compound 2u (IC50
5.04 M) relative to compound 2p, the potency of other compounds
=
μ
than compound 2a. This result was also found in our previous studies
reduced compared with compound 2j or 2p, some derivatives (e.g., 2 k,
2 l, 2n, 2o, 2q, 2 s, and 2v) even were inactive. And most of the de-
rivatives with halogens introduced at the 2-position of isonicotinamide
displayed weaker inhibitory potency than their corresponding coun-
terparts in N-(1-alkyl-3-cyano-1H-indol-5-yl)isonicotinamides series (e.
g., 2 l vs 2c, 2q vs 2c, 2n vs 2d, 2 s vs 2d, and 2u vs 2 g), indicating that
the introduction of halogens at the 2-position of isonicotinamide series
were detrimental to the XO inhibitory activity.
′
[
30]. In addition, when the 3 -position substitution of indole was
changed into hydrogen (compound 11, < 60% inhibition at 33
μ
M) or
formyl group (compound 12, < 60% inhibition at 33 M), the potency
μ
′
disappeared, which indicated that maintaining the 3 -position cyano
group was essential for potency.
To investigate the influence of halogen-substituted on iso-
nicotinamide fragment, 2-chloro and 2-fluoro were introduced to pro-
vide N-(1-alkyl-3-cyano-1H-indol-5-yl)-2-chloroisonicotinamides and
Generally speaking, N-(1-alkyl-3-cyano-1H-indol-5-yl)-1H-benzo[d]
imidazole-5-carboxamide exhibited higher XO inhibitory activity than
N-(1-alkyl-3-cyano-1H-indol-5-yl)isonicotinamide counterpart (e.g., 3a
vs 2a, 3b vs 2b, 3c vs 2c, 3d vs 2d, 3f vs 2f, and 3h vs 2h), which
revealed that the benzo[d]imidazole-5-carboxamide may be more
N-(1-alkyl-3-cyano-1H-indol-5-yl)-2-fluoroisonicotinamides.
When
chloro and fluoro were introduced at the 2-position of N-(1-alkyl-3-
cyano-1H-indol-5-yl)isonicotinamide, the XO inhibitory activity was
improvement (2j vs 2a, and 2p vs 2a IC50 = 4.30
μ
M vs 8.59
μ
M, and
IC50 = 6.95
μ
M vs 8.59 M, respectively), which gave us considerable
μ
effective than isonicotinamide moiety. And with the extension of the
′
encouragement. Surprisingly, when alkyl groups were introduced at the
length of the alkyl groups at 1 -position (3b, 3c, 3d IC50 = 4.49
μ
M, 2.16
′
1
-position of the indole scaffold, the potency was not as generally
μ
M, 2.17 M, respectively), the variation pattern of efficacy showed
μ
4

S. Tu et al.
Bioorganic Chemistry 115 (2021) 105181
Table 1
In vitro XO inhibitory potency of compounds.
a
Compounds
R
1
groups
R
2
groups
R
3
groups
IC50( M)
μ
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
1
1
a
b
c
d
e
f
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
F
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
/
8.59 ± 0.19
b
Methyl
Ethyl
n.a.
c
c
7.89 ± 0.16
7.34 ± 0.14
4.43 ± 0.12
7.65 ± 0.24
1.18 ± 0.06
Propyl
iso-Propyl
Allyl
c
c
g
h
i
Prop-2-yn-1-yl
Benzyl
Cyclopentyl
H
12.46 ± 0.66
0.73 ± 0.02
4.30 ± 0.24
n.a.
j
k
l
Methyl
Ethyl
n.a.
m
n
o
p
q
r
Propyl
iso-Propyl
Allyl
16.19 ± 1.32
n.a.
n.a.
c
H
6.95 ± 0.29
Ethyl
F
n.a.
Propyl
iso-Propyl
Allyl
F
12.64 ± 1.10
n.a.
s
F
t
F
21.79 ± 1.21
c
u
v
w
a
b
c
d
e
f
Prop-2-yn-1-yl
Benzyl
Cyclopentyl
H
F
5.04 ± 0.04
F
n.a.
F
12.64 ± 0.48
3.52 ± 0.07
4.49 ± 0.13
2.16 ± 0.22
2.17 ± 0.17
4.25 ± 0.04
2.04 ± 0.07
4.27 ± 0.34
1.52 ± 0.03
/
Methyl
Ethyl
/
/
/
/
Propyl
iso-Propyl
Allyl
/
/
/
/
/
/
g
h
i
Prop-2-yn-1-yl
Benzyl
Cyclopentyl
H
/
/
/
/
#
/
/
0.62 ± 0.02
0
1
H
H
/
H
n.a.
H
CHO
n.a.
Allopurinol
/
/
/
8.9 ± 0.09
0.021 ± 0.003
Topiroxostat
/
/
a
Values are means ± SD of three independent experiments.
b
c
n.a.: not active (<60% inhibition at 33
P < 0.05, versus allopurinol.
μ
M).
#
P < 0.05, versus compound 2i.
similarity to that of N-(1-alkyl-3-cyano-1H-indol-5-yl)isonicotinamides
N-(3-cyano-4-(cyclopentyloxy)phenyl)isonicotinamide that possessed
the same cyclopentyl substitution, the activities of compounds 2i and 3i
have been greatly improved [27]. Particularly, compound 3i exhibited
′
series. Interestingly, the effect of alkyl groups at 1 -position with three
carbon atom lengths on inhibitory potency differed from that of the N-
(
1-alkyl ꢀ 3-cyano-1H-indol-5-yl)isonicotinamides series. Straight chain
14.4-fold higher potency than the positive control allopurinol (IC50
8.9 M), although, it still 30-fold less effective than another positive
M). To investigate the inhibition
=
alkyl may be more suitable for N-(1-alkyl-3-cyano-1H-indol-5-yl)-1H-
benzo[d]imidazole-5-carboxamide than branched alkyl (3d vs 3e; IC50
μ
control topiroxostat (IC50 = 0.021
μ
=
2.17
propargyl group, the potency was remained (3f vs 3d; IC50 = 2.04
.17 M vs 4.27 M), indicating
μ
M vs 4.25
μ
M). When the propyl group was replaced by allyl or
type of compound 3i on XO, enzyme kinetics studies were performed.
The Lineweaver-Burk plot (Fig. 2) revealed that compound 3i acted as a
μM vs
2
μ
M) or decreased (3g vs 3d; IC50 = 1.18
μ
μ
mixed-type inhibitor. Additionally, the K
increased but the Vmax was decreased by increasing the inhibitor con-
centration from 0 to 2.1 M. Specifically, the K and Vmax values of
compound 3i at 0 M, 1 M and 2.1 M were 107.41, 253.56, and
387.00 M and 43.69, 37.26, and 32.96 M/min, respectively.
m m
and slope (K /Vmax) were
that saturated alkyl may be more conducive to N-(3-cyan-1H-indol-5-
yl)ꢀ 1H-benzo[d]imidazole-5-carboxamide derivatives. The introduc-
tion of benzyl and cyclopentyl both improved the XO inhibitory activity
μ
m
μ
μ
μ
(
3h vs 3a, and 3i vs 3a IC50 = 1.52
μ
M vs 3.52
μ
M, and IC50 = 0.62
μ
M vs
μ
μ
3
.52 M, respectively), especially compound 3i presented as the most
μ
potent compound in this work. On the whole, the introduction of a small
2
.3. Molecular docking
′
alkyl group at the 1 -position was advantageous to the XO inhibitory
effect of N-(1-alkyl-3-cyano-1H-indol-5-yl)-1H-benzo[d]imidazole-5-
carboxamide, which was similar to the N-(1-alkyl-3-cyano-1H-indol-5-
yl)isonicotinamides series. Moreover, in these two series, compounds 2i
and 3i with the best XO inhibitory activity both introduced cyclopentyl
To investigate the possible binding modes of these compounds in the
XO catalytic pocket, the representative compounds, 2i and 3i, were
subjected to molecular docking studies using MOE (Molecular Operating
Environment, version 2015.1001, Chemical Computing Group Inc.,
Canada) software [46]. The crystal structure of XO in complex with
′
at the 1 -position. Compared with our previously discovered compound
5

S. Tu et al.
Bioorganic Chemistry 115 (2021) 105181
[
47]. The backbone root means square deviation (RMSD) of the complex
was exhibited in Fig. 4A. It can be seen from Fig. 4A that the RMSD
values of the complexes XO/3i and XO/topiroxostat tended to be
convergent with fluctuations around 2.3 Å and 2.3 Å respectively after 5
ns and 2 ns of simulation, clearly indicating that the whole system has
been equilibrated. The balanced conformation was picked up and
rendered with MOE software (Fig. 4B and 4C). As showed in Fig. 4C,
after MD simulation, the conformational changes of topiroxostat in the
active pocket were not significant, indicating that the MD protocol was
reliable. And compound 3i retained the majority of the interactions
observed in molecular docking after MD simulation (Fig. 4B), such as the
cyano group forming H-bond with Asn768, the benzo[d]imidazole N
atom generating H-bond with Glu1261 residue, and the cyclopentyl tail
interacting with lipophilic amino acid residues (e.g., Phe649, Leu648,
and Phe1013). Interestingly, the H-bond between amide NH and residue
Glu802 disappeared; instead, the carbonyl accepted an H-bond from
residue Thr1010.
2
.5. Hypouricemic effect in vivo
The in vivo hypouricemic effects of compound 3i were further
investigated by measuring serum uric acid levels in an acute hyperuri-
cemia rat model induced by potassium oxazinate. Topiroxostat was used
as a positive control. Four experimental groups were set, which marked
as blank group (normal rat orally administered blank solvent), model
group (model rat orally administered blank solvent), 3i group (model rat
orally administered 12.8 mg/kg of 3i) and topiroxostat group (model rat
orally administered 10.0 mg/kg of topiroxostat), respectively. Oral
doses of compound 3i and topiroxostat had the same amount of sub-
stance. As shown in Fig. 5, compared with the blank group, the serum
uric acid levels of the model group were markedly increased (P < 0.01),
suggesting that the model was successfully established by intraperito-
neal injection of potassium oxonate (300 mg/kg). Moreover, 3i group
showed an apparent reduction of 30.1% in uric acid levels (P < 0.05) at
2 h and a reduction of 20.8% in the AUC (uric acid, 0.5–8 h) (P < 0.05)
compared with the model group. While topiroxostat group displayed a
more obvious reduction of 73.9% in uric acid levels (P < 0.0001) at 2 h
and a reduction of 66.4% in the AUC (uric acid, 0.5–8 h) (P < 0.0001)
compared with the model group, which was 2.46 and 3.19 times more
effective than compound 3i in reducing serum uric acid levels, respec-
tively. Thus, the results of the hypouricemic effect in vivo suggested that
compound 3i could be a promising lead compound for the treatment of
hyperuricemia.
Fig. 2. Lineweaver-Burk plot analysis of XO inhibition by compound 3i.
topiroxostat (PDB code: 1 V97) [39] was processed as a docking mode.
As shown in Fig. 3, two vital H-bond interactions were observed in
both binding modes of 2i (Fig. 3A) and 3i (Fig. 3B), as expected, which
were Asn768 residue forming H-bond with cyano group of both 2i and
3
i, and Glu1261 interacting with pyridine N atom of 2i and benzo[d]
imidazole N atom of 3i, respectively. This result verified our idea of
balancing the interactions with residues Asn768 and Glu1261 by
introducing an indole moiety. Moreover, amide fragments can strongly
interact with amino acid residues Glu802 and Thr1010 in the active
pocket as reported in our previous studies [39]. In addition, the cyclo-
pentyl tail was surrounded by some lipophilic amino acid residues, such
as Phe649, Leu648, and Phe1013, which may also contribute to the
binding affinity. These interactions explained the relatively high activ-
ities of compounds 2i and 3i.
2
.4. Molecular dynamics simulations
To obtain a more integrated and precise view of the binding process,
molecular dynamics (MD) simulations of 3i docking complex and top-
iroxostat/XO co-crystal (PDB code:1V97) were carried out using Gro-
ningen Machine for Chemicals Simulations (GROMACS) 5.0 package
Fig. 3. Docking poses of compound 2i (A, cyan) and 3i (B, bright yellow) within the protein binding pocket and overlaid with the original ligand topiroxostat
(
green). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
6

S. Tu et al.
Bioorganic Chemistry 115 (2021) 105181
Fig. 4. Results of MD simulations. (A) Backbone RMSD of the complex versus time (20 ns); (B) MD conformation of XO in complex with compound 3i; (C) MD
conformation of XO in complex with topiroxostat.
Fig. 5. Effect of compound 3i and topiroxostat on the serum uric acid levels in the potassium oxonate-induced hyperuricemic rat model. (a) Time course changes of
serum uric acid levels after 0.5–8 h of treatment. (b) Serum uric acid levels after 2 h of treatment. (c) The AUC of serum uric acid after 0.5–8 h of treatment. Blank
group: normal rat orally administered blank solvent, model group: model rat orally administered blank solvent. The data are shown as the mean ± SD. (n = 6). *P <
0
.05, **P < 0.01, ***P < 001 and ****P < 0.0001, significantly different from the model group by Student’s t-test.
2
.6. In vitro cytotoxicity assay
and both exhibited non-cytotoxicity on normal cells under the high
concentration of 10 M. It can be concluded that toxicity did not emerge
from 3i even after high-dose incubation.
μ
The MTT assay was used to evaluate the cytotoxicity of the com-
pound 3i in two healthy cell lines: MCF10A (human breast cells) and
6HBE (human bronchial epithelial cells). The cell viability assay data
treated with different concentrations of 3i (0.1 M, 1 M and 10 M)
were quantified (Fig. 6), and the highest incubation concentration was
1
2
.7. ADME/T properties prediction
μ
μ
μ
In order to assess the drug-like properties, pharmacokinetic behav-
1
6 times higher than IC50 value of 3i. As shown in Fig. 6, MCF10A and
6HBE showed 91.06% and 81.14% cell viability at 10 M, respectively,
iors (absorption, distribution, biotransformation and excretion) and
1
μ
toxicity of compound 3i were predicted using pre-ADMET web service
7

S. Tu et al.
Bioorganic Chemistry 115 (2021) 105181
benzo[d]imidazole-5-carboxamide could serve as a novel scaffold for the
discovery and development of amide-based XO inhibitors, and com-
pound 3i could be a promising lead for the further investigation.
4
. Experimental protocols
4
.1. Chemistry
Unless otherwise indicated, reagents and solvents were purchased
from commercial sources and used without further purification. All re-
actions were monitored by TLC using silica gel aluminum cards (0.2 mm
1
13
thickness) with a fluorescent indicator of 254 nm. H NMR and C NMR
spectra were recorded on a Bruker spectrometer. Chemical shifts were
expressed in parts per million using tetramethylsilane as an internal
reference and DMSO‑d
6
as the solvent. ESI-HRMS data were gathered
using a Bruker microTOF-Q instrument.
Fig. 6. Toxic effects of compound 3i on healthy cell lines MCF10A and 16HBE.
4
.1.1. Synthesis of 5-nitro-1H-indole-3-carbaldehyde (4)
(
https://preadmet.bmdrc.kr/), and the results were summarized in
A mixture of anhydrous DMF (3.38 g, 46.25 mmol) and phosphorus
◦
oxychloride (14.18 g, 92.51 mmol) was stirred at 0 C for 30 min. Then a
Table 2 [48]. Low blood–brain barrier (BBB) permeability was found (C.
brain/C.blood < 1), suggesting that 3i could be inactive in the central
nervous system. Importantly, Caco-2 permeability was used to evaluate
the suitability of compounds for oral dosing, and 3i has significantly
better human intestinal permeability than allopurinol. In addition,
human intestinal absorption (HIA) is a key factor that affects oral
bioavailability, compound 3i demonstrated excellent bioavailability.
The high plasma-protein binding (PPB) of 3i means a long half-life and
stable efficacy, which could maintain a durable potency and adequate
stability of the compound. The inhibition of CYP3A4 by a drug consti-
tutes the majority of cases of drug-drug interaction; it was found that 3i
may not be an inhibitor of CYP3A4. Another hand, cardiotoxicity may
not be avoided with the medium risk of hERG inhibition.
solution of 5-nitro-1H-indole (5.00 g, 30.84 mmol) in anhydrous DMF
(
6 mL) was added dropwise to the above mixture. After the addition, the
mixture was stirred at room temperature overnight. Then, the reaction
mixture was poured into ice water, diluted with an aqueous solution of
sodium hydroxide and refluxed for 1 h. After cooling, the precipitate was
collected by filtration, washed with a large quantity of water and dried
in vacuum to give 5-nitro-1H-indole-3-carbaldehyde 5 (5.34 g, yield
1
9
1.1%) as a brown solid. H NMR (500 MHz, DMSO‑d
6
) δ 12.67 (s, 1H),
10.01 (s, 1H), 8.92 (s, 1H), 8.55 (s, 1H), 8.13 (d, J = 6.2 Hz, 1H), 7.70 (d,
1
3
J = 6.6 Hz, 1H). C NMR (125 MHz, DMSO‑d
6
) δ 185.34, 142.71,
141.24, 139.97, 123.36, 118.90, 118.61, 116.89, 113.06.
4
.1.2. Synthesis of 5-nitro-1H-indole-3-carbonitrile (5)
A mixture of compound 4 (2.00 g, 10.52 mmol), hydroxylamine
3
. Conclusions
hydrochloride (3.65 g, 52.59 mmol) and sodium formate (3.93 g, 57.85
mmol) in formic acid (30 mL) was refluxed for 2 h. After that, the
resulted solution was cooled and poured into ice water (400 mL). The
formed precipitate was collected by filtration, washed with a large
quantity of water and dried to provide 5-nitro-1H-indole-3-carbonitrile
5 (1.62 g, yield 82.2%) as a light yellow solid. 1H NMR (500 MHz,
Herein, we designed and synthesized two series of amide-based XO
inhibitors, N-(3-cyano-1H-indol-5-yl)isonicotinamides and N-(3-cyano-
H-indol-5-yl)-1H-benzo[d]imidazole-5-carboxamides. Biological eval-
1
uation indicated that the latter series are more active and more struc-
turally diverse than the former series, indicating that amide is a versatile
linker for designing novel XO inhibitor, and the chemical space of
amide-based XO inhibitors is broad, and not limited in isonicotinamides.
DMSO‑d
6
) δ 12.81 (s, 1H), 8.53 (s, 1H), 8.49 (s, 1H), 8.14 (d, J = 8.8 Hz,
13
1H), 7.74 (d, J = 8.9 Hz, 1H). C NMR (125 MHz, DMSO‑d
6
) δ 142.41,
1
38.31, 138.18, 125.86, 118.45, 114.91, 114.75, 113.65, 86.69.
The structure-activity relationship investigation identified 3i (IC50
=
0
.62 M) as the most potent compound, which exhibited 14.4-fold
μ
4
.1.3. Synthesis of 5-amino-1H-indole-3-carbonitrile (6)
higher potency than the positive control allopurinol. During in vivo
assay, compound 3i showed an effective hypouricemic effect at an oral
dose of 12.8 mg/kg in a rat model of potassium oxonate-induced hy-
peruricemia. MTT results indicated that compound 3i is non-toxic to
A mixture of compound 5 (1.00 g, 5.34 mmol) and Pd/C (5%; 0.10 g)
◦
in ethanol (40 mL) under 40 psi of hydrogen atmosphere, was at 25 C
for 4 h. After the completion of the reaction, the insoluble solid was
filtered out. The filtrate was evaporated under reduced pressure to give
healthy cells at a high concentration of 10 M. And ADME/T prediction
μ
supports the well drug-like properties of compound 3i. In short, this
work presented convincing evidence that N-(3-cyano-1H-indol-5-yl)-1H-
5-amino-1H-indole-3-carbonitrile 6 (0.75 g, yield 89.4%) as an off-white
1
solid. H NMR (500 MHz, DMSO‑d
6
) δ 11.72 (s, 1H), 7.96 (d, J = 2.0 Hz,
1
H), 7.22 (d, J = 8.6 Hz, 1H), 6.73 (s, 1H), 6.63 (d, J = 8.6 Hz, 1H), 4.92
s, 2H). 13C NMR (125 MHz, DMSO‑d
) δ 143.82, 132.73, 128.15,
28.13, 116.96, 113.42, 112.90, 100.44, 82.29.
(
6
1
Table 2
a
Predicted ADMR/T profiles.
4
5
.1.4. General procedure for the synthesis of N-(1-alkyl-3-cyano-1H-indol-
-yl) heterocyclic amides (2a, 2j, 2p and 3a)
ADME/Tox properties
Compound
3
i
Allopurinol
Into a solution of compound 6 (0.50 g, 3.18 mmol) and triethylamine
(0.97 g, 9.54 mmol) in tetrahydrofuran (80 mL) at ꢀ 5 ◦C, was added
aroyl chloride (4.77 mmol). The mixture was maintained at the same
temperature for 1 h and then stirred at room temperature overnight.
After the completion of the reaction, the insoluble materials were
removed by filtration and washed with THF. The filtrate was evaporated
in vacuum to remove about 3/4 of the solvent. The residue was
dispersed into water, filtered under reduced pressure, washed and dried
to provide crude products, which was further purified by
Buffer solubility (SK atomic types, mg/L)
Blood-brain barrier penetration (BBB)
In vivo Caco-2 cell permeability (nm/sec)
Human intestinal absorption (HIA, %)
Plasma protein binding (PPB, %)
CYP_3A4_inhibition
7.23
395.38
0.25
0.83
20.17
16.61
92.97
76.95
94.41
8.96
Non
Inhibitor
Weakly
Low risk
CYP_3A4_substrate
Substrate
Medium risk
hERG inhibition
a
https://preadmet.bmdrc.kr.
8

S. Tu et al.
Bioorganic Chemistry 115 (2021) 105181
recrystallization from 50% ethanol to provide corresponding products
powder, yield 85.5%. 1H NMR (500 MHz, DMSO‑d
8.80 (d, J = 3.5 Hz, 2H), 8.31 (s, 1H), 8.22 (s, 1H), 7.90 (d, J = 4.0 Hz,
6
) δ 10.55 (s, 1H),
2
a, 2j, 2p and 3a.
1
3
2
H), 7.69 (s, 2H), 4.28 (q, J = 7.0 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H).
C
4
.1.4.1. N-(3-cyano-1H-indol-5-yl)isonicotinamide (2a). A gray powder,
NMR (125 MHz, DMSO) δ 163.65, 150.11, 141.81, 136.45, 133.35,
1
yield 78.8%. H NMR (600 MHz, DMSO‑d
6
) δ 12.21 (s, 1H), 10.54 (s,
H), 8.79 (dd, J = 4.5, 1.5 Hz, 2H), 8.24 (s, 1H), 8.22 (d, J = 1.6 Hz, 1H),
.90 (dd, J = 4.5, 1.5 Hz, 2H), 7.65 (dd, J = 8.8, 1.8 Hz, 1H), 7.56 (d, J =
.8 Hz, 1H). 13C NMR (125 MHz, DMSO‑d
) δ 164.26, 150.73, 142.51,
131.82, 127.22, 121.37, 117.44, 115.86, 111.44, 109.92, 83.28, 41.31,
14.89. ESI-HRMS calcd. for C17
289.1077.
14 4
H N
O [Mꢀ H]ꢀ 289.1089, found:
1
7
8
1
1
6
35.42, 133.70, 132.67, 127.35, 122.03, 118.16, 116.84, 113.49,
4.1.5.3. N-(3-cyano-1-propyl-1H-indol-5-yl)isonicotinamide
white powder, yield 89.7%. 1H NMR (600 MHz, DMSO‑d
(2d). A
10.24, 84.78. ESI-HRMS calcd. for C15
H
10
N
4
O [Mꢀ H]ꢀ 261.0776,
6
) δ 10.56 (s,
found:261.0792.
1H), 8.80 (d, J = 5.2 Hz, 2H), 8.30 (s, 1H), 8.22 (d, J = 0.8 Hz, 1H), 7.90
d, J = 5.8 Hz, 2H), 7.71 (d, J = 8.9 Hz, 1H), 7.68 (dd, J = 8.9, 1.5 Hz,
1H), 4.21 (t, J = 7.0 Hz, 2H), 1.84–1.78 (m, 2H), 0.84 (t, J = 7.4 Hz, 3H).
13
(
4
.1.4.2. N-(3-cyano-1H-indol-5-yl)-2-chloroisonicotinamide
(2j). A
1
brown powder, yield 69.7%. H NMR (500 MHz, DMSO‑d
6
) δ 12.23 (s,
6
C NMR (125 MHz, DMSO‑d ) δ 164.29, 150.75, 142.43, 137.73,
1
8
H), 10.61 (s, 1H), 8.62 (d, J = 4.6 Hz, 1H), 8.25 (s, 1H), 8.20 (s, 1H),
133.95, 132.76, 127.75, 122.02, 118.05, 116.54, 112.24, 110.48, 83.81,
48.47, 23.28, 11.38. ESI-HRMS calcd. for
303.1246, found: 303.1259.
C
18
H
16
N
4
O
[Mꢀ H]ꢀ
.03 (s, 1H), 7.91 (d, J = 4.2 Hz, 1H), 7.64 (d, J = 8.7 Hz, 1H), 7.56 (d, J
=
8.7 Hz, 1H). 13C NMR (125 MHz, DMSO‑d
) δ 162.08, 150.69, 150.51,
6
1
1
45.41, 134.82, 132.82, 132.13, 126.71, 122.08, 121.08, 117.46,
16.14, 112.89, 109.68, 84.20. ESI-HRMS calcd. for C15
Mꢀ H]ꢀ 295.0387, found: 295.0408.
9
H ClN
4
O
4.1.5.4. N-(3-cyano-1-isopropyl-1H-indol-5-yl)isonicotinamide
white powder, yield 84.6%. 1H NMR (500 MHz, DMSO‑d
H), 8.80 (d, J = 4.6 Hz, 2H), 8.42 (s, 1H), 8.22 (s, 1H), 7.90 (d, J = 4.5
Hz, 2H), 7.74 (d, J = 8.9 Hz, 1H), 7.69 (d, J = 8.9 Hz, 1H), 4.86–4.81 (m,
(2e). A
[
6
) δ 10.55 (s,
1
4
.1.4.3. N-(3-cyano-1H-indol-5-yl)-2-fluoroisonicotinamide
(2p). A
1
13
brown powder, yield 72.6%. H NMR (500 MHz, DMSO‑d
6
) δ 12.23 (s,
1H), 1.49 (d, J = 6.6 Hz, 6H). C NMR (125 MHz, DMSO‑d
6
) δ 163.65,
1
7
H), 10.61 (s, 1H), 8.46 (d, J = 4.9 Hz, 1H), 8.26 (s, 1H), 8.21 (s, 1H),
150.10, 141.81, 133.93, 133.35, 131.61, 127.21, 121.37, 117.38,
.87 (d, J = 4.2 Hz, 1H), 7.70 (s, 1H), 7.63 (d, J = 8.8 Hz, 1H), 7.57 (d, J
115.93, 111.58, 109.92, 83.65, 48.06, 22.03. ESI-HRMS calcd. for
8.7 Hz, 1H). 13C NMR (125 MHz, DMSO‑d
) δ 163.24 (d, J = 235.8
O [Mꢀ H]ꢀ 303.1246, found: 303.1224.
=
6
18 16 4
C H N
Hz), 162.18 (d, J = 3.7 Hz), 148.29 (d, J = 14.9 Hz), 147.98 (d, J = 7.4
Hz), 134.84, 132.80, 132.13, 126.71, 120.04 (d, J = 4.0 Hz), 117.49,
4.1.5.5. N-(1-allyl-3-cyano-1H-indol-5-yl)isonicotinamide (2f). A light
1
1
16.15, 112.91, 109.69, 107.83 (d, J = 39.5 Hz), 84.19. ESI-HRMS
6
yellow powder, yield 76.6%. H NMR (500 MHz, DMSO‑d ) δ 10.55 (s,
ꢀ
calcd. for C15
H
9
FN
4
O [Mꢀ H] 279.0682, found:279.0691.
1H), 8.80 (d, J = 3.6 Hz, 2H), 8.27 (s, 1H), 8.23 (s, 1H), 7.90 (d, J = 4.2
Hz, 2H), 7.68 (d, J = 8.8 Hz, 1H), 7.63 (d, J = 8.8 Hz, 1H), 6.06–6.02 (m,
4
.1.4.4. N-(3-cyano-1H-indol-5-yl)-1H-benzo[d]imidazole-5-carboxamide
1H), 5.22 (d, J = 10.2 Hz, 1H), 5.10 (d, J = 17.1 Hz, 1H), 4.92 (d, J = 4.6
1
13
(
3a). A gray solid, yield 74.7%. H NMR (500 MHz, DMSO‑d
6
) δ 12.71
6
Hz, 2H). C NMR (125 MHz, DMSO‑d ) δ 163.68, 150.11, 141.80,
(
s, 1H), 12.15 (s, 1H), 10.27 (s, 1H), 8.44 (s, 1H), 8.38 (s, 1H), 8.26 (s,
137.09, 133.45, 133.00, 132.07, 127.19, 121.37, 117.72, 117.57,
1
1
1
1
H), 8.22 (s, 1H), 7.90 (s, 1H), 7.80–7.62 (m, 2H), 7.53 (d, J = 8.7 Hz,
14 4
115.69, 111.75, 109.94, 83.68, 48.74. ESI-HRMS calcd. for C18H N O
1
3
[Mꢀ H]ꢀ 301.1089, found: 301.1084.
H). C NMR (125 MHz, DMSO‑d
6
) δ 165.68, 143.89, 139.60, 136.46,
34.44, 134.00, 131.68, 128.40, 126.78, 121.65, 117.64, 116.32,
15.48, 114.30, 112.61, 109.30, 84.07. ESI-HRMS calcd. for C17
Mꢀ H]ꢀ 300.0885, found:300.0896.
H
11
N
5
O
4.1.5.6. N-(3-cyano-1-(prop-2-yn-1-yl)-1H-indol-5-yl)isonicotinamide
[
(2g). A yellow powder, yield 73.4%. 1H NMR (600 MHz, DMSO‑d
) δ
6
1
0.62 (s, 1H), 8.80 (d, J = 5.6 Hz, 2H), 8.30 (s, 1H), 8.27 (d, J = 1.5 Hz,
1H), 7.90 (dd, J = 7.2, 4.9 Hz, 3H), 7.76–7.71 (m, 2H), 5.89 (d, J = 6.5
Hz, 2H). 13C NMR (150 MHz, DMSO‑d
) δ 202.81, 164.41, 150.75,
142.34, 135.40, 134.78, 131.74, 128.16, 122.03, 118.63, 115.69,
4
5
.1.5. General procedure for the synthesis of N-(1-alkyl-3-cyano-1H-indol-
-yl)isonicotinamides 2b-i, N-(1-alkyl-3-cyano-1H-indol-5-yl)-2-
6
chloroisonicotinamides 2k-o and N-(1-alkyl-3-cyano-1H-indol-5-yl)-2-
fluoroisonicotinamides 2q-w
12 4
112.77, 110.56, 97.43, 89.85, 86.96. ESI-HRMS calcd. for C18H N O
[Mꢀ H]ꢀ 299.0933, found: 299.0952.
To a cooled solution of compound 7 (0.76 mmol) in anhydrous DMF
(
10 mL) was added NaH (1.91 mmol as 60% dispersion in paraffin oil)
and kept at ꢀ 5 ◦C for 30 min. Then, alkyl chloride/bromide (0.92
4.1.5.7. N-(1-benzyl-3-cyano-1H-indol-5-yl)isonicotinamide (2h). A light
1
mmol), and potassium iodide (0.05 mmol) were successively added. The
yellow powder, yield 70.5%. H NMR (500 MHz, DMSO‑d ) δ 10.54 (s,
6
◦
mixture was stirred under nitrogen atmosphere at 60 C overnight. After
1H), 8.79 (d, J = 4.5 Hz, 2H), 8.45 (s, 1H), 8.23 (s, 1H), 7.89 (d, J = 4.4
the completion of the reaction, the insoluble solid was filtered out and
the filtrate was diluted with water (200 mL). The formed precipitate was
collected, washed with water and dried to yield a crude product, which
was further purified by recrystallization from 50% ethanol to provide
corresponding products 2b-i, 2 k-o and 2q-w.
Hz, 2H), 7.65 (s, 2H), 7.33 (d, J = 6.9 Hz, 2H), 7.29 (d, J = 6.5 Hz, 3H),
5.52 (s, 2H). 13C NMR (125 MHz, DMSO‑d ) δ 163.68, 150.10, 141.78,
6
137.42, 136.49, 133.50, 132.03, 128.57, 127.67, 127.32, 127.08,
121.36, 117.67, 115.64, 111.87, 109.98, 83.93, 49.85. ESI-HRMS calcd.
ꢀ
for C22H16N O [Mꢀ H] 351.1246, found: 351.1231.
4
4
.1.5.1. N-(3-cyano-1-methyl-1H-indol-5-yl)isonicotinamide
(2b). A
4.1.5.8. N-(3-cyano-1-cyclopentyl-1H-indol-5-yl)isonicotinamide (2i). A
1
white powder, yield 68.4%. 1H NMR (500 MHz, DMSO‑d ) δ 10.55 (s,
white powder, yield 88.7%. H NMR (600 MHz, DMSO‑d
6
) δ 10.56 (s,
6
1
H), 8.80 (d, J = 5.1 Hz, 2H), 8.23 (s, 1H), 8.22 (d, J = 1.5 Hz, 1H), 7.90
1H), 8.80 (d, J = 4.2 Hz, 2H), 8.38 (s, 1H), 8.21 (s, 1H), 7.90 (d, J = 4.4
(
d, J = 5.9 Hz, 2H), 7.70 (dd, J = 8.9, 1.8 Hz, 1H), 7.64 (d, J = 8.9 Hz,
Hz, 2H), 7.73 (d, J = 8.9 Hz, 1H), 7.69 (d, J = 9.0 Hz, 1H), 4.96–4.92 (m,
H), 3.87 (s, 3H). 13C NMR (125 MHz, DMSO‑d
) δ 164.30, 150.75,
13
1
1
1
2
6
1H), 2.20 (d, J = 7.7 Hz, 2H), 1.91–1.85 (m, 4H), 1.72 (s, 2H). C NMR
42.44, 138.51, 134.01, 133.38, 127.67, 122.03, 118.03, 116.51,
(125 MHz, DMSO‑d ) δ 163.64, 150.09, 141.82, 134.33, 133.41, 132.25,
6
12.10, 110.35, 83.58, 33.94. ESI-HRMS calcd. for C16
H
12
N
4
O [Mꢀ H]ꢀ
127.31, 121.37, 117.38, 115.92, 111.84, 109.86, 83.61, 57.37, 31.85,
ꢀ
75.0933, found: 275.0959.
23.30. ESI-HRMS calcd. for C20H18N O [Mꢀ H] 329.1402, found:
4
3
29.1390.
4
.1.5.2. N-(3-cyano-1-ethyl-1H-indol-5-yl)isonicotinamide (2c). A white
9

S. Tu et al.
Bioorganic Chemistry 115 (2021) 105181
4
.1.5.9. N-(3-cyano-1-methyl
ꢀ 1H-indol-5-yl)-2-chloroisonicotinamide
) δ
0.62 (s, 1H), 8.63 (d, J = 4.8 Hz, 1H), 8.24 (s, 1H), 8.20 (s, 1H), 8.03 (s,
H), 7.91 (d, J = 4.6 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.65 (d, J = 8.8
J = 4.0 Hz), 117.38, 115.82, 111.64, 109.94, 107.83 (d, J = 39.1 Hz),
2k). A white powder, yield 87.7%. 1H NMR (500 MHz, DMSO‑d
83.24, 47.85, 22.64, 10.73. ESI-HRMS calcd. for C18
321.1152, found: 321.1160.
H15FN
O [Mꢀ H]
ꢀ
(
6
4
1
1
1
3
Hz, 1H), 3.87 (s, 3H). C NMR (125 MHz, DMSO‑d
6
) δ 162.12, 150.70,
4.1.5.16. N-(3-cyano-1-isopropyl-1H-indol-5-yl)-2-fluoroisonicotinamide
(2 s). A white powder, yield 78.5%. 1H NMR (500 MHz, DMSO‑d ) δ
1
1
50.53, 145.35, 137.92, 133.14, 132.84, 127.03, 122.07, 121.08,
6
17.33, 115.80, 111.49, 109.79, 83.01, 33.31. ESI-HRMS calcd. for
10.62 (s, 1H), 8.49–8.40 (m, 2H), 8.21 (s, 1H), 7.87 (d, J = 4.1 Hz, 1H),
C
16
H
11ClN
4
O [Mꢀ H]ꢀ 309.0543, found: 309.0577.
7.78–7.65 (m, 3H), 4.84 (dt, J = 12.9, 6.4 Hz, 1H), 1.49 (d, J = 6.5 Hz,
1
3
6
H). C NMR (125 MHz, DMSO‑d
6
) δ 163.25 (d, J = 235.9 Hz), 162.19
4
.1.5.10. N-(3-cyano-1-ethyl
ꢀ 1H-indol-5-yl)-2-chloroisonicotinamide
) δ
0.62 (s, 1H), 8.63 (d, J = 5.0 Hz, 1H), 8.32 (s, 1H), 8.20 (s, 1H), 8.03 (s,
H), 7.91 (d, J = 4.2 Hz, 1H), 7.69 (q, J = 8.9 Hz, 2H), 4.28 (q, J = 7.1
(d, J = 3.8 Hz), 148.31 (d, J = 15.1 Hz), 147.89 (d, J = 7.0 Hz), 134.05,
133.09, 131.67, 127.17, 120.03 (d, J = 4.0 Hz), 117.30, 115.92, 111.66,
(
2l). A white powder, yield 88.2%. 1H NMR (500 MHz, DMSO‑d
6
1
1
109.94, 107.83 (d, J = 39.8 Hz), 83.65, 48.05, 22.04. ESI-HRMS calcd.
ꢀ
for C18
H
15FN
4
O [Mꢀ H] 321.1152, found: 321.1175.
1
3
Hz, 2H), 1.41 (t, J = 7.2 Hz, 3H). C NMR (125 MHz, DMSO‑d
6
) δ
1
1
62.10, 150.70, 150.53, 145.33, 136.53, 133.11, 131.89, 127.20,
4.1.5.17. N-(1-allyl-3-cyano-1H-indol-5-yl)-2-fluoroisonicotinamide
(2
1
22.07, 121.08, 117.34, 115.83, 111.49, 109.94, 83.30, 41.32, 14.90.
t). A light yellow powder, yield 73.4%. H NMR (500 MHz, DMSO‑d ) δ
6
ESI-HRMS calcd. for C17
H
13ClN
4
O [Mꢀ H]ꢀ 323.0700, found: 323.0712.
10.63 (s, 1H), 8.46 (d, J = 4.9 Hz, 1H), 8.28 (s, 1H), 8.23 (s, 1H), 7.87 (d,
J = 4.5 Hz, 1H), 7.70 (s, 1H), 7.66 (q, J = 9.0 Hz, 2H), 6.05–6.01 (m,
4
.1.5.11. N-(3-cyano-1- propyl ꢀ 1H-indol-5-yl)-2-chloroisonicotinamide
1H), 5.22 (d, J = 10.2 Hz, 1H), 5.10 (d, J = 17.1 Hz, 1H), 4.92 (d, J = 5.0
1
13
(
2 m). A white powder, yield 85.1%. H NMR (500 MHz, DMSO‑d
6
) δ
0.61 (s, 1H), 8.63 (d, J = 4.8 Hz, 1H), 8.30 (s, 1H), 8.20 (s, 1H), 8.03 (s,
H), 7.91 (d, J = 4.8 Hz, 1H), 7.71 (d, J = 8.9 Hz, 1H), 7.67 (d, J = 8.9
Hz, 2H). C NMR (125 MHz, DMSO‑d
6
) δ 163.24 (d, J = 236.5 Hz),
1
1
162.23 (d, J = 3.7 Hz), 148.31 (d, J = 14.9 Hz), 147.88 (d, J = 7.8 Hz),
137.20, 133.18, 133.00, 132.14, 127.16, 120.03 (d, J = 4.0 Hz), 117.72,
Hz, 1H), 4.21 (t, J = 6.9 Hz, 2H), 1.86–1.76 (m, 2H), 0.84 (t, J = 7.2 Hz,
117.49, 115.67, 111.83, 109.97, 107.84 (d, J = 39.1 Hz), 83.68, 48.74.
1
3
ꢀ
3
1
1
H). C NMR (125 MHz, DMSO‑d
6
) δ 162.10, 150.70, 150.54, 145.33,
ESI-HRMS calcd. for C18
H
13FN
4
O [Mꢀ H] 319.0995, found: 319.1012.
37.14, 133.07, 132.21, 127.11, 122.07, 121.08, 117.34, 115.84,
11.64, 109.91, 83.23, 47.85, 22.64, 10.74. ESI-HRMS calcd. for
4.1.5.18. N-(3-cyano-1-(prop-2-yn-1-yl)-1H-indol-5-yl)-2-fluo-
roisonicotinamide (2u). A yellow powder, yield 74.5%. 1H NMR (500
C
18
H
15ClN
4
O [Mꢀ H]ꢀ 337.0856, found: 337.0868.
MHz, DMSO‑d
6
) δ 10.68 (s, 1H), 8.46 (d, J = 5.0 Hz, 1H), 8.33 (d, J =
4
.1.5.12. N-(3-cyano-1-
oisonicotinamide (2n). A white powder, yield 87.7%. H NMR (500
MHz, DMSO‑d
isopropyl
ꢀ 1H-indol-5-yl)-2-chlor-
8.2 Hz, 1H), 8.26 (s, 1H), 7.92 (d, J = 9.0 Hz, 1H), 7.87 (d, J = 4.5 Hz,
1
13
1H), 7.77–7.69 (m, 3H), 5.90 (d, J = 6.5 Hz, 2H). C NMR (125 MHz,
6
) δ 10.63 (s, 1H), 8.63 (d, J = 4.9 Hz, 1H), 8.44 (s, 1H),
.21 (s, 1H), 8.04 (s, 1H), 7.91 (d, J = 4.9 Hz, 1H), 7.75 (d, J = 8.9 Hz,
H), 7.67 (d, J = 9.0 Hz, 1H), 4.86–4.81 (m, 1H), 1.49 (d, J = 6.5 Hz,
DMSO‑d
6
) δ 202.20, 163.25 (d, J = 230.2 Hz), 162.30 (d, J = 2.5 Hz),
8
1
6
1
1
148.32 (d, J = 14.8 Hz), 147.80 (d, J = 6.9 Hz), 134.84, 133.88, 131.22,
127.53, 120.04 (d, J = 4.1 Hz), 117.98, 114.98, 112.19, 110.03, 107.85
1
3
H). C NMR (125 MHz, DMSO‑d
6
) δ 162.10, 150.70, 150.54, 145.33,
(d, J = 39.6 Hz), 96.78, 89.21, 86.36. ESI-HRMS calcd. for C18
4
H11FN O
ꢀ
34.04, 133.11, 131.66, 127.16, 122.07, 121.09, 117.27, 115.92,
[Mꢀ H] 317.0839, found: 317.0864.
4
11.65, 109.92, 83.65, 48.05, 22.04. ESI-HRMS calcd. for C18H15ClN O
[
Mꢀ H]ꢀ 337.0856, found: 337.0869.
4.1.5.19. N-(1-benzyl-3-cyano-1H-indol-5-yl)-2-fluoroisonicotinamide
2v). A light yellow powder, yield 68.8%. 1H NMR (500 MHz,
DMSO‑d
) δ 10.69 (s, 1H), 8.50–8.43 (m, 2H), 8.23 (s, 1H), 7.87 (s, 1H),
7.73–7.60 (m, 3H), 7.33 (d, J = 6.9 Hz, 2H), 7.29 (d, J = 6.4 Hz, 3H),
5.52 (s, 2H). 13C NMR (125 MHz, DMSO‑d
(
4
.1.5.13. N-(1-allyl-3-cyano-1H-indol-5-yl)-2-chloroisonicotinamide
6
(
2o). A light yellow powder, yield 81.2%. 1H NMR (500 MHz,
) δ 10.64 (s, 1H), 8.63 (d, J = 4.9 Hz, 1H), 8.28 (s, 1H), 8.22 (s,
H), 8.04 (s, 1H), 7.91 (d, J = 4.8 Hz, 1H), 7.65 (q, J = 8.9 Hz, 2H),
.06–6.00 (m, 1H), 5.22 (d, J = 10.1 Hz, 1H), 5.09 (d, J = 17.3 Hz, 1H),
DMSO‑d
6
6
) δ 163.23 (d, J = 236.6 Hz),
1
6
4
1
1
162.25 (d, J = 3.9 Hz), 148.30 (d, J = 14.8 Hz), 147.88 (d, J = 6.9 Hz),
137.53, 136.49, 133.27, 132.08, 128.58, 127.68, 127.28, 127.09,
1
3
.92 (d, J = 5.0 Hz, 2H). C NMR (125 MHz, DMSO‑d
6
) δ 162.14,
120.05 (d, J = 4.0 Hz), 117.61, 115.64, 111.96, 110.00, 107.85 (d, J =
ꢀ
50.70, 150.54, 145.33, 137.19, 133.20, 133.00, 132.13, 127.16,
39.0 Hz), 83.90, 49.82. ESI-HRMS calcd. for C22
369.1152, found: 369.1174.
H
15FN
4
O [Mꢀ H]
22.08, 121.08, 117.72, 117.46, 115.67, 111.82, 109.95, 83.69, 48.74.
ESI-HRMS calcd. for C18
H
13ClN
4
O [Mꢀ H]ꢀ 335.0700, found: 335.0709.
4
.1.5.20. N-(3-cyano-1-cyclopentyl-1H-indol-5-yl)-2-fluo-
roisonicotinamide (2w). A white powder, yield 65.4%. 1H NMR (500
MHz, DMSO‑d
4
.1.5.14. N-(3-cyano-1-ethyl-1H-indol-5-yl)-2-fluoroisonicotinamide
(
2q). A white powder, yield 86.5%. 1H NMR (500 MHz, DMSO‑d
6
) δ
0.64 (s, 1H), 8.46 (d, J = 3.7 Hz, 1H), 8.32 (s, 1H), 8.21 (s, 1H), 7.87 (s,
6
) δ 10.62 (s, 1H), 8.46 (d, J = 4.8 Hz, 1H), 8.40 (s, 1H),
1
8.21 (s, 1H), 7.87 (d, J = 3.3 Hz, 1H), 7.74 (d, J = 8.9 Hz, 1H), 7.72–7.65
(m, 2H), 4.99–4.92 (m, 1H), 2.19 (d, J = 7.9 Hz, 2H), 1.87 (d, J = 16.1
1
3
1
H), 7.75–7.64 (m, 3H), 4.31–4.25 (m, 2H), 1.41 (t, J = 6.7 Hz, 3H).
C
NMR (125 MHz, DMSO‑d
6
) δ 163.25 (d, J = 236.2 Hz), 162.20 (d, J =
Hz, 4H), 1.71 (s, 2H). 13C NMR (125 MHz, DMSO‑d
6
) δ 163.25 (d, J =
3
1
1
.2 Hz), 148.29 (d, J = 14.8 Hz), 147.89 (d, J = 7.6 Hz), 136.54, 133.09,
235.7 Hz), 162.18 (d, J = 3.8 Hz), 148.30 (d, J = 14.8 Hz), 147.88 (d, J
= 6.9 Hz), 134.44, 133.14, 132.31, 127.28, 120.02 (d, J = 4.0 Hz),
31.89, 127.20, 120.03 (d, J = 4.0 Hz), 117.37, 115.84, 111.51, 109.96,
07.83 (d, J = 39.8 Hz), 83.29, 41.32, 14.90. ESI-HRMS calcd. for
117.30, 115.91, 111.91, 109.89, 107.82 (d, J = 39.9 Hz), 83.61, 57.35,
ꢀ
ꢀ
C
17
H
13FN
4
O [Mꢀ H] 307.0995, found: 307.1023.
31.85, 23.28. ESI-HRMS calcd. for C20
H
17FN
4
O [Mꢀ H] 347.1308,
found: 347.1329.
4
.1.5.15. N-(3-cyano-1-propyl-1H-indol-5-yl)-2-fluoroisonicotinamide
2r). A white powder, yield 76.5%. 1H NMR (500 MHz, DMSO‑d
0.62 (s, 1H), 8.46 (d, J = 4.9 Hz, 1H), 8.31 (s, 1H), 8.21 (s, 1H), 7.87 (d,
J = 4.2 Hz, 1H), 7.74–7.65 (m, 3H), 4.22 (t, J = 6.9 Hz, 2H), 1.85–1.77
(
6
) δ
4.1.6. General procedure for the synthesis of 1-alkyl-5-nitro-1H-indole-3-
carbonitrile (7)
1
To a cooled solution of compound 5 (1.00 g, 5.34 mmol) in anhy-
drous DMF (15 mL) was added NaH (0.53 g, 13.36 mmol as 60%
1
3
(
m, 2H), 0.84 (t, J = 7.3 Hz, 3H). C NMR (125 MHz, DMSO‑d
63.25 (d, J = 236.3 Hz), 162.19 (d, J = 3.9 Hz), 148.30 (d, J = 14.8
Hz), 147.89 (d, J = 7.6 Hz), 137.13, 133.05, 132.23, 127.12, 120.02 (d,
6
) δ
◦
1
dispersion in paraffin oil) and kept at ꢀ 5 C for 30 min. Alkyl chloride/
bromide (6.41 mmol), and potassium iodide (0.05 mmol) was then
1
0

S. Tu et al.
Bioorganic Chemistry 115 (2021) 105181
◦
added. The mixture was stirred under nitrogen atmosphere at 60
C
g, 25.0 mmol) and hydrochloric acid (5 mL) in ethanol (50 mL) and
◦
overnight. After the completion of the reaction, the insoluble solid was
filtered out and the filtrate was diluted with water (250 mL). The formed
precipitate was collected, washed with water and dried to provide cor-
responding key intermediates 1- alkyl-5-nitro-1H-indole-3-carbonitrile
water (5 mL) was heated at 60 C overnight. After the completion of the
reaction, the solution was basified with an aqueous solution of sodium
hydroxide and extracted with ethyl acetate (100 mL*3). The ethyl ace-
tate extracts were combined, dried over sodium sulfate, and evaporated
to dryness to provide corresponding key intermediates 5-amino-1- alkyl
7
a-h.
ꢀ
1H-indole-3-carbonitrile 8a-h, which was directly used in the next step
4
.1.6.1. 1-methyl-5-nitro-1H-indole-3-carbonitrile (7a). A yellow solid,
without further purification. Into a solution of compound 9 (1.17 mmol)
and triethylamine (0.35 g, 3.50 mmol) in tetrahydrofuran (50 mL) at
ꢀ 5 ◦C, was added 1H-benzo[d]imidazole-5-carbonyl chloride (0.32 g,
1.75 mmol). The mixture was maintained at the same temperature for 1
h and then stirred at room temperature overnight. After the completion
of the reaction, the insoluble materials were removed by filtration and
washed with THF. The filtrate was evaporated in vacuum to give crude
products, which were then purified using column chromatography to
obtain the pure corresponding products 3b-i.
1
yield 92.1%. H NMR (500 MHz, DMSO‑d ) δ 8.50 (s, 1H), 8.43 (s, 1H),
13
6
8
.16 (d, J = 9.1 Hz, 1H), 7.84 (d, J = 9.1 Hz, 1H), 3.93 (s, 3H). C NMR
(
125 MHz, DMSO‑d ) δ 142.51, 141.36, 138.53, 126.02, 118.28, 114.98,
6
1
14.38, 112.33, 85.65, 33.79.
4
.1.6.2. 1-ethyl-5-nitro-1H-indole-3-carbonitrile (7b). A yellow solid,
1
yield 89.3%. H NMR (500 MHz, DMSO‑d
6
) δ 8.61 (s, 1H), 8.43 (s, 1H),
8
2
1
1
.15 (d, J = 9.1 Hz, 1H), 7.92 (d, J = 9.1 Hz, 1H), 4.36 (q, J = 7.1 Hz,
13
H), 1.41 (t, J = 7.2 Hz, 3H). C NMR (125 MHz, DMSO‑d
6
) δ 142.47,
40.03, 137.65, 126.17, 118.27, 115.11, 114.41, 112.26, 85.97, 41.85,
4.1.7.1. N-(3-cyano-1-methyl-1H-indol-5-yl)-1H-benzo[d]imidazole-5-
carboxamide (3b). A brown powder, yield 78.7%. 1H NMR (500 MHz,
4.81.
6
DMSO‑d ) δ 12.78 (s, 1H), 10.32 (s, 1H), 8.39 (s, 1H), 8.35 (s, 1H), 8.28
(
s, 1H), 8.21 (s, 1H), 7.90 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 8.4 Hz, 1H),
4
.1.6.3. 1-propyl-5-nitro-1H-indole-3-carbonitrile (7c). A yellow solid,
1
3
1
7
.71 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 8.5 Hz, 1H), 3.87 (s, 3H). C NMR
yield 85.5%. H NMR (500 MHz, DMSO‑d
6
) δ 8.61 (s, 1H), 8.47 (s, 1H),
(
125 MHz, DMSO‑d
6
) δ 165.71, 143.91, 139.85, 138.42, 137.56, 134.34,
8
2
.17 (d, J = 8.7 Hz, 1H), 7.96 (d, J = 9.1 Hz, 1H), 4.31 (t, J = 6.8 Hz,
13
1
1
3
32.40, 128.48, 127.10, 121.69, 117.48, 116.01, 115.63, 114.20,
H), 1.82 (dd, J = 14.2, 7.1 Hz, 2H), 0.84 (t, J = 7.2 Hz, 3H). C NMR
) δ 142.54, 140.57, 138.06, 126.13, 118.33, 115.17,
11.22, 109.37, 82.85, 33.26. ESI-HRMS calcd. for C18
14.1042, found:314.1059.
H
13
N
5
O [Mꢀ H]ꢀ
(
125 MHz, DMSO‑d
6
1
14.41, 112.45, 86.00, 48.25, 22.63, 10.64.
4
.1.7.2. N-(3-cyano-1-ethyl-1H-indol-5-yl)-1H-benzo[d]imidazole-5-car-
4
.1.6.4. 1-isopropyl-5-nitro-1H-indole-3-carbonitrile (7d). A yellow
1
1
boxamide (3c). A gray powder, yield 76.3%. H NMR (500 MHz,
DMSO‑d ) δ 12.77 (s, 1H), 10.34 (s, 1H), 8.40 (s, 1H), 8.35 (s, 1H), 8.30
d, J = 2.6 Hz, 2H), 7.91 (d, J = 8.4 Hz, 1H), 7.75–7.67 (m, 3H), 4.28 (q,
solid, yield 83.4%. H NMR (500 MHz, DMSO‑d
6
) δ 8.75 (s, 1H), 8.43 (s,
1
1
1
2
H), 8.14 (d, J = 9.2 Hz, 1H), 7.96 (d, J = 9.2 Hz, 1H), 5.01–4.92 (m,
6
13
(
H), 1.50 (d, J = 6.6 Hz, 6H). C NMR (125 MHz, DMSO‑d
6
) δ 142.49,
1
3
J = 7.0 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H). C NMR (125 MHz, DMSO‑d
6
)
37.51, 137.42, 126.15, 118.19, 115.09, 114.45, 112.34, 86.45, 48.78,
δ 165.71, 143.91, 139.72, 138.35, 136.18, 134.30, 131.43, 128.48,
1.95.
1
4
27.26, 121.70, 117.49, 116.05, 115.69, 114.36, 111.25, 109.51, 83.12,
1.28, 14.93. ESI-HRMS calcd. for C19
H
15
N
5
O [Mꢀ H]ꢀ 328.1198,
4
.1.6.5. 1-allyl-5-nitro-1H-indole-3-carbonitrile (7e). A yellow solid,
1
found:328.1201.
yield 81.4%. H NMR (500 MHz, DMSO‑d
6
) δ 8.58 (s, 1H), 8.50 (s, 1H),
8
5
2
1
.18 (d, J = 9.4 Hz, 1H), 7.86 (d, J = 9.2 Hz, 1H), 6.11–5.97 (m, 1H),
4
.1.7.3. N-(3-cyano-1-propyl-1H-indol-5-yl)-1H-benzo[d]imidazole-5-
carboxamide (3d). An off-white powder, yield 78.1%. 1H NMR (500
MHz, DMSO‑d
) δ 10.32 (s, 1H), 8.39 (s, 1H), 8.36 (s, 1H), 8.28 (d, J =
.24 (d, J = 10.3 Hz, 1H), 5.09 (d, J = 17.1 Hz, 1H), 5.02 (d, J = 5.1 Hz,
1
3
H). C NMR (125 MHz, DMSO‑d ) δ 142.66, 140.66, 137.93, 132.50,
6
26.23, 118.48, 118.12, 115.23, 114.29, 112.62, 86.32, 49.09.
6
5
.0 Hz, 2H), 7.91 (d, J = 8.3 Hz, 1H), 7.75–7.67 (m, 3H), 4.21 (t, J = 6.5
Hz, 2H), 1.82 (d, J = 7.0 Hz, 1H), 1.80 (d, J = 6.8 Hz, 1H), 0.84 (t, J =
4
.1.6.6. 5-nitro-1-(prop-2-yn-1-yl)-1H-indole-3-carbonitrile (7f). A yel-
7.1 Hz, 3H). 13C NMR (125 MHz, DMSO‑d ) δ 165.72, 143.93, 139.66,
1
6
low solid, yield 76.4%. H NMR (500 MHz, DMSO‑d
6
) δ 8.57 (s, 1H),
1
38.42, 136.77, 134.27, 131.77, 128.47, 127.19, 121.68, 117.52,
8
.46 (s, 1H), 8.21 (d, J = 9.1 Hz, 1H), 7.91 (d, J = 9.1 Hz, 1H), 5.37 (d, J
5.9 Hz, 1H), 5.31 (s, 2H). 1 C NMR (125 MHz, DMSO‑d
3
116.04, 115.66, 114.34, 111.36, 109.51, 83.08, 47.82, 22.64, 10.74.
=
6
) δ 142.91,
ESI-HRMS calcd. for C20
H
17
N
5
O [Mꢀ H]ꢀ 342.1355, found:342.1375.
1
6
40.10, 137.53, 126.31, 118.77, 115.32, 114.03, 112.59, 86.82, 77.26,
6.45, 36.56.
4
.1.7.4. N-(3-cyano-1-isopropyl-1H-indol-5-yl)-1H-benzo[d]imidazole-5-
1
carboxamide (3e). A gray powder, yield 74.2%. H NMR (500 MHz,
4
.1.6.7. 1-benzyl-5-nitro-1H-indole-3-carbonitrile (7 g). A yellow solid,
1
DMSO‑d ) δ 12.80 (s, 1H), 10.31 (s, 1H), 8.41 (s, 1H), 8.39 (s, 1H), 8.34
6
yield 73.8%. H NMR (500 MHz, DMSO‑d
6
) δ 8.77 (s, 1H), 8.51 (s, 1H),
(
s, 1H), 8.28 (s, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.76–7.68 (m, 3H),
8
2
1
1
.17 (d, J = 9.0 Hz, 1H), 7.91 (d, J = 9.1 Hz, 1H), 7.34 (d, J = 5.7 Hz,
13
H), 7.31 (s, 3H), 5.63 (s, 2H). 1 C NMR (125 MHz, DMSO‑d
6
) δ 142.73,
3
4.86–4.81 (m, 1H), 1.50 (d, J = 6.5 Hz, 6H). C NMR (125 MHz,
DMSO‑d ) δ 165.69, 143.94, 139.64, 138.44, 134.30, 133.67, 131.21,
6
40.91, 137.90, 135.88, 128.69, 127.91, 127.18, 126.37, 118.63,
1
1
3
28.45, 127.22, 121.68, 117.39, 116.14, 115.61, 114.28, 111.40,
15.32, 114.28, 112.70, 86.57, 50.17.
09.46, 83.46, 47.97, 22.06. ESI-HRMS calcd. for C20
42.1355, found:342.1361.
H
17
N
5
O [Mꢀ H]ꢀ
4
.1.6.8. 1-cyclopentyl-5-nitro-1H-indole-3-carbonitrile (7 h). A yellow
1
solid, yield 80.5%. H NMR (500 MHz, DMSO‑d
6
) δ 8.71 (s, 1H), 8.45 (s,
4
.1.7.5. N-(1-allyl-3-cyano-1H-indol-5-yl)-1H-benzo[d]imidazole-5-car-
1
1
H), 8.16 (d, J = 9.1 Hz, 1H), 7.96 (d, J = 9.1 Hz, 1H), 5.13–5.02 (m,
1
13
boxamide (3f). A yellow powder, yield 76.1%. H NMR (500 MHz,
DMSO‑d ) δ 12.76 (s, 1H), 10.31 (s, 1H), 8.39 (s, 1H), 8.35 (s, 1H), 8.29
H), 2.22 (d, J = 7.1 Hz, 2H), 1.87 (d, J = 11.7 Hz, 4H), 1.72 (s, 2H).
) δ 142.52, 138.08, 137.92, 126.24, 118.23,
15.07, 114.50, 112.62, 86.37, 57.79, 31.94, 23.28.
C
6
NMR (125 MHz, DMSO‑d
6
(
s, 1H), 8.25 (s, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.74 (d, J = 8.9 Hz, 1H),
1
7
5
2
1
.71 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.9 Hz, 1H), 6.08–6.00 (m, 1H),
.22 (d, J = 10.2 Hz, 1H), 5.10 (d, J = 17.1 Hz, 1H), 4.92 (d, J = 4.8 Hz,
4
5
.1.7. General procedure for the synthesis of N-(1-alkyl-3-cyano-1H-indol-
-yl)-1H-benzo[d]imidazole-5-carboxamides 3b-i
1
3
6
H). C NMR (125 MHz, DMSO‑d ) δ 165.72, 143.90, 139.43, 138.33,
37.85, 136.85, 134.40, 133.09, 131.68, 128.31, 127.22, 121.69,
A solution of compound 7 (2.50 mmol), tin chloride dihydrate (5.63
1
1

S. Tu et al.
Bioorganic Chemistry 115 (2021) 105181
1
17.65, 117.59, 116.50, 114.41, 111.58, 109.51, 83.51, 48.71. ESI-
solution of compound 10 (0.50 g, 2.11 mmol) in anhydrous DMF (2 mL)
was added dropwise to the above mixture. After the addition, the
mixture was stirred overnight at room temperature. Then, the reaction
mixture was poured into ice water, basified with an aqueous solution of
sodium hydroxide and refluxed for 1 h. After cooling, the precipitate was
collected by filtration, washed with a large quantity of water and dried
HRMS calcd. for C20
H
15
N
5
O [Mꢀ H]ꢀ 340.1198, found:340.1216.
4
.1.7.6. N-(3-cyano-1-(prop-2-yn-1-yl)-1H-indol-5-yl)-1H-benzo[d]imid-
1
azole-5-carboxamide (3g). A yellow powder, yield 62.5%. H NMR (500
MHz, DMSO‑d ) δ 12.89 (s, 1H), 10.35 (s, 1H), 8.41 (s, 1H), 8.35 (s, 1H),
6
8
.30 (s, 2H), 7.91 (d, J = 7.9 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.70 (t, J
in vacuum to give N-(3-formyl-1H-indol-5-yl)isonicotinamide 11 (0.43
8.6 Hz, 2H), 5.22 (s, 2H), 3.56 (s, 1H). 1 C NMR (125 MHz, DMSO‑d
3
)
1
=
6
g, yield 76.9%) as a white solid. H NMR (500 MHz, DMSO‑d
6
) δ 12.14
δ 165.72, 143.87, 139.41, 138.22, 136.42, 134.66, 131.32, 128.50,
(s, 1H), 10.51 (s, 1H), 9.93 (s, 1H), 8.79 (d, J = 4.3 Hz, 2H), 8.55 (s, 1H),
1
7
3
27.24, 121.77, 117.80, 115.57, 115.49, 114.44, 111.52, 109.57, 84.15,
8.29 (s, 1H), 7.91 (d, J = 4.4 Hz, 2H), 7.72 (d, J = 8.7 Hz, 1H), 7.51 (d, J
[Mꢀ H]ꢀ
13
7.76, 76.62, 36.01. ESI-HRMS calcd. for
38.1042, found:338.1057.
C
20
H
13
N
5
O
= 8.7 Hz, 1H). C NMR (125 MHz, DMSO‑d
6
) δ 184.62, 163.43, 150.05,
141.96, 138.88, 133.97, 133.37, 123.94, 121.40, 118.15, 117.72,
1
12.93, 112.06.
4
.1.7.7. N-(1-benzyl-3-cyano-1H-indol-5-yl)-1H-benzo[d]imidazole-5-
carboxamide (3h). A gray powder, yield 70.6%. 1H NMR (500 MHz,
4
.2. Assay of in vitro XO inhibitory activity
Bovine XO inhibitory potency in vitro was assayed spectrophoto-
DMSO‑d
6
) δ 10.31 (s, 1H), 8.44 (s, 1H), 8.36 (s, 1H), 8.33 (s, 1H), 8.29
(
s, 1H), 7.87 (d, J = 8.2 Hz, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.62 (d, J =
metrically by measuring the uric acid formation at 294 nm using a UV-
8
2
1
1
4
.6 Hz, 1H), 7.34 (d, J = 6.7 Hz, 2H), 7.29 (d, J = 6.5 Hz, 3H), 5.51 (s,
13
◦
2
100 spectrophotometer (UNICO) at 25 C. The testing method was
H). C NMR (125 MHz, DMSO‑d ) δ 165.78, 144.14, 139.71, 138.63,
6
based on the procedure reported by Matsumoto et al. [49] with modi-
fication. The assay mixture contained 0.1 M sodium pyrophosphate
37.14, 136.58, 134.48, 131.62, 128.57, 128.27, 127.65, 127.35,
27.09, 121.54, 117.71, 115.83, 115.65, 114.34, 111.67, 109.54, 83.76,
2
buffer (pH 8.3), 0.3 mM Na EDTA, 1 mM xanthine, 25 U/L XO (Sigma,
9.80. ESI-HRMS calcd. for
24 17 5
C H N O
[Mꢀ H]ꢀ 390.1355,
X1875), and the test compound. The enzyme was pre-incubated for 10
min with the test compound, and the reaction was started by the addi-
tion of xanthine. The XO inhibition by various compounds was calcu-
lated by the reduction of uric acid production in the first 2 min.
Allopurinol and topiroxostat were used as positive controls. All tests
were performed in triplicate. Compounds presenting inhibitory effects
found:390.1375.
4
5
.1.7.8. N-(3-cyano-1-cyclopentyl-1H-indol-5-yl)-1H-benzo[d]imidazole-
1
-carboxamide (3i). A brown powder, yield 70.6%. H NMR (500 MHz,
DMSO‑d
6
) δ 12.74 (s, 1H), 10.32 (s, 1H), 8.37 (s, 2H), 8.28 (s, 1H), 7.91
(
s, 1H), 7.84–7.62 (m, 3H), 4.94 (d, J = 6.3 Hz, 1H), 2.19 (s, 2H), 1.86 (s,
H), 1.71 (s, 2H). 1 C NMR (125 MHz, DMSO‑d
3
over 60% at a concentration of 33 M were further tested at a wide range
μ
4
1
1
2
6
) δ 165.68, 143.92,
of concentrations to calculate their IC50 values using SPSS 25.0 (SPSS
Inc., Chicago, IL, USA) software.
39.64, 138.55, 134.37, 134.09, 131.85, 128.50, 127.33, 122.28,
17.39, 116.13, 115.87, 114.31, 111.67, 109.40, 83.41, 57.29, 31.84,
3.29. ESI-HRMS calcd. for
C
22
H
19
N
5
O
[Mꢀ H]ꢀ 368.1511,
4
.3. Molecular docking
found:368.1542.
Molecular modeling studies were performed with MOE software
4
.1.8. Synthesis of 1H-indol-5-amine (9)
[
46]. The co-crystal structure of bovine XO in complex with topiroxostat
A mixture of 5-nitro-1H-indole (2.00 g, 12.33 mmol) and Pd/C (5%;
(
PDB code 1V97) [50] was downloaded from RCSB Protein Data Bank
0
.20 g) in ethanol (40 mL) under 40 psi of hydrogen atmosphere, was
◦
(http://www.rcsb.org/), which was optimized by a series of procedures
of Structure Preparation. Protonate 3D and Structure Refine (RMSD
gradient = 0.1 kcal/mol, AMBER99 force field) to provide a docking
receptor [51]. The ligands were generated by Builder protocol in MOE.
The docking procedure was adopted the standard protocol implemented
in MOE and all parameters were maintained as the defaults.
stirred at 25 C for 18 h. After the completion of the reaction, the
insoluble solid was filtered out. The filtrate was evaporated under
reduced pressure to give 1H-indol-5-amine 9 (1.42 g yield 87.1%) as a
1
light red solid. H NMR (600 MHz, DMSO‑d
6
) δ 10.56 (s, 1H), 7.13 (t, J
=
2.7 Hz, 1H), 7.10 (d, J = 8.5 Hz, 1H), 6.70 (d, J = 2.0 Hz, 1H), 6.51
13
(
dd, J = 8.5, 2.1 Hz, 1H), 6.14 (dd, J = 3.4, 1.4 Hz, 1H), 4.42 (s, 2H).
C
NMR (150 MHz, DMSO‑d ) δ 141.45, 130.26, 129.03, 125.20, 112.29,
6
4
.4. Molecular dynamics simulations
1
11.86, 103.71, 100.09.
MD simulations studies were performed using GROMACS 5.0 pack-
4
.1.9. Synthesis of N-(1H-indol-5-yl)isonicotinamide (10)
age [52,53]. The CHARMM36 force field was adopted for the simula-
tions [54]. Ligand topology files were generated using CHARMM
General Force Field [55]. Corresponding counterions were added to
neutralize the solvated TIP3P water model present in the dodecahedron
box. The energy was minimized using the steepest descent algorithm.
The particle mesh Ewald (PME) method was employed for energy
calculation, electrostatic, and Van der Waals interactions. The systems
were equilibrated in the NVT ensemble for 20,000 steps followed by
equilibration in the NPT ensemble for an additional 20,000 steps.
Finally, 20 ns MD simulations were performed at 300 K under periodic
boundary conditions with a 2.0 fs time step [56,57].
Into a solution of compound 9 (1.00 g, 7.57 mmol) and triethylamine
◦
(
2.30 g, 22.70 mmol) in tetrahydrofuran (80 mL) at ꢀ 5 C, was added
isonicotinyl chloride (1.60 g, 11.35 mmol). The mixture was maintained
at the same temperature for 1 h and then stirred at room temperature
overnight. After the completion of the reaction, the insoluble materials
were removed by filtration and washed with THF. The filtrate was
evaporated in vacuum to remove about 3/4 of the solvent. The residue
was dispersed into water, filtered under reduced pressure, washed and
dried to give N-(1H-indol-5-yl)isonicotinamide 10 (1.37 g, yield 76.1%)
1
as a white solid. H NMR (600 MHz, DMSO‑d
6
) δ 11.07 (s, 1H), 10.33 (s,
1
H), 8.78 (dd, J = 4.5, 1.5 Hz, 2H), 8.02 (s, 1H), 7.90 (dd, J = 4.5, 1.5
Hz, 2H), 7.44–7.38 (m, 2H), 7.35 (t, J = 2.7 Hz, 1H), 6.49–6.41 (m, 1H).
1
3
4.5. Hypouricemic effect in vivo
6
C NMR (150 MHz, DMSO‑d ) δ 163.94, 150.66, 142.91, 133.74,
1
30.86, 127.91, 126.54, 122.03, 116.50, 112.91, 111.64, 101.73.
Twenty-four six-week-old Sprague-Dawley rats, half male and half
female, weighing 180–220 g, were provided by Beijing Huafukang
Biotechnology Co., Ltd (Beijing, China). The production license number
of experimental animals was SCXK 2019-0008. Experimental animals
were raised in the SPF Animal Experiment Center of the Laboratory
4
.1.10. Synthesis of N-(3-formyl-1H-indol-5-yl)isonicotinamide (11)
A mixture of anhydrous DMF (0.23 g, 3.16 mmol) and phosphorus
◦
oxychloride (0.97 g, 6.32 mmol) was stirred at 0 C for 30 min. Then a
1
2

S. Tu et al.
Bioorganic Chemistry 115 (2021) 105181
Animal Department of China Medical University (experimental animal
license number: SYXK 2018-0008). The maintenance and treatment of
animals complied with the protocol approved by the Animal Experiment
Ethics Review Committee of China Medical University. This study was
approved by the Laboratory Animal Welfare Ethics Committee of China
Medical University (IACUC: NO CMU2020116).
Acknowledgments
This work was supported by the National Natur-
al Science Foundation of China (Grant No. 81803355) and Young Tal-
ents Program of China Medical University.
The rats had free access to food and water and kept a 12-hour light-
dark cycle in a room with controlled temperature and humidity for one
week. After that, the rats were randomly divided into 4 groups of six
each as follows: blank group, model group, test (3i) group and positive
control (topiroxostat) group, and then fasted and free drinking water for
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105181.
1
2 h. Subsequently, rats in the blank group were intraperitoneally
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