Design, synthesis and biological evaluation of 1-alkyl-5/6-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-indole-3-carbonitriles as novel xanthine oxidase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112077

Xanthine oxidase (XO) has emerged as an important target for the treatment of hyperuricemia and gout. In this study, to obtain novel nonpurine XO inhibitors, a series of 1-alkyl-5/6-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-indole-3-carbonitriles (1a-1u, 2c, 2e, 2h and 2n) were designed using a bioisosteric replacement strategy and were synthesized through a five-step procedure with good yields. Thereafter, the in vitro XO inhibitory potencies of these compounds were evaluated by spectrophotometry, showing inhibitory profiles in the micromolar/submicromolar range. Particularly, compound 1h emerged as the strongest XO inhibitor, with an IC₅₀ value of 0.36 μM, which was approximately 21-fold more potent than the positive control allopurinol. Additionally, the structure-activity relationships revealed that the 5-oxo-4,5-dihydro-1,2,4-oxadiazole moiety linked at the 5-position of the indole scaffold was more preferable than the 6-position for the XO inhibitory potency. Enzyme kinetic studies indicated that compound 1h acted as a mixed-type XO inhibitor. Moreover, molecular modeling studies were performed on compound 1h to gain insights into its binding modes with XO. The results showed that the 5-oxo-4,5-dihydro-1,2,4-oxadiazole moiety could interact with Arg880 and Thr1010 in the innermost part of the active pocket through hydrogen bonds, while the cyano group could form hydrogen bonds with Asn768 and Lys771 in the subpocket. Furthermore, the in vivo hypouricemic effect of compound 1h was further investigated in a hyperuricemia rat model induced by potassium oxonate. The results suggested that compound 1h could effectively reduce serum uric acid levels at an oral dose of 10 mg/kg. Therefore, compound 1h could be a promising lead compound for the treatment of hyperuricemia and gout.

Full text of DOI:10.1016/j.ejmech.2020.112077

European Journal of Medicinal Chemistry 190 (2020) 112077
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and biological evaluation of 1-alkyl-5/6-(5-oxo-4,5-
dihydro-1,2,4-oxadiazol-3-yl)-1H-indole-3-carbonitriles as novel
xanthine oxidase inhibitors
a
b
a
a
a
c
a
Jun Gao , Xuegui Liu , Bing Zhang , Qing Mao , Zhuo Zhang , Qian Zou , Xiwen Dai ,
a, *
Shaojie Wang
a
Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical
University, 103 Wenhua Road, Shenhe District, Shenyang, 110016, PR China
b
Institute of Functional Molecules, Shenyang University of Chemical Technology, Shenyang, Liaoning, 110142, PR China
Wuya College of Innovation, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenhe District, Shenyang, 110016, PR China
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 December 2019
Received in revised form
Xanthine oxidase (XO) has emerged as an important target for the treatment of hyperuricemia and gout.
In this study, to obtain novel nonpurine XO inhibitors, a series of 1-alkyl-5/6-(5-oxo-4,5-dihydro-1,2,4-
oxadiazol-3-yl)-1H-indole-3-carbonitriles (1a-1u, 2c, 2e, 2h and 2n) were designed using a bio-
isosteric replacement strategy and were synthesized through a five-step procedure with good yields.
Thereafter, the in vitro XO inhibitory potencies of these compounds were evaluated by spectropho-
tometry, showing inhibitory profiles in the micromolar/submicromolar range. Particularly, compound 1h
11 January 2020
Accepted 16 January 2020
Available online 21 January 2020
emerged as the strongest XO inhibitor, with an IC50 value of 0.36 mM, which was approximately 21-fold
Keywords:
more potent than the positive control allopurinol. Additionally, the structure-activity relationships
revealed that the 5-oxo-4,5-dihydro-1,2,4-oxadiazole moiety linked at the 5-position of the indole
scaffold was more preferable than the 6-position for the XO inhibitory potency. Enzyme kinetic studies
indicated that compound 1h acted as a mixed-type XO inhibitor. Moreover, molecular modeling studies
were performed on compound 1h to gain insights into its binding modes with XO. The results showed
that the 5-oxo-4,5-dihydro-1,2,4-oxadiazole moiety could interact with Arg880 and Thr1010 in the
innermost part of the active pocket through hydrogen bonds, while the cyano group could form
hydrogen bonds with Asn768 and Lys771 in the subpocket. Furthermore, the in vivo hypouricemic effect
of compound 1h was further investigated in a hyperuricemia rat model induced by potassium oxonate.
The results suggested that compound 1h could effectively reduce serum uric acid levels at an oral dose of
Hyperuricemia
Xanthine oxidase inhibitor
1,2,4-Oxadiazole
Biological evaluation
10 mg/kg. Therefore, compound 1h could be a promising lead compound for the treatment of hyper-
uricemia and gout.
©
2020 Elsevier Masson SAS. All rights reserved.
1
. Introduction
molybdoflavoprotein, is a critical and rate-limiting enzyme that
catalyzes the oxidation of hypoxanthine and xanthine to uric acid in
humans [5e8]. Thus, XO is considered the most promising target
for the treatment of hyperuricemia and gout [6,9].
To date, only one purine-type XO inhibitor, allopurinol (1966),
and two nonpurine-type XO inhibitors, febuxostat (2009) and
topiroxostat (2013) (Fig.1), have been approved for the treatment of
hyperuricemia and gout [5]. Allopurinol, an analog of hypoxan-
thine, was the first marketed XO inhibitor that has been clinically
used as a drug for the treatment of hyperuricemia and gout for
decades [6]. Unfortunately, allopurinol and its active metabolite
oxypurinol (Fig. 1) can be converted by hypoxanthine-guanine
Gout is a common and disabling disease that is associated with
chronic hyperuricemia [1,2]. The reported prevalence of gout
worldwide ranges from 0.1% to 10% and the incidence of gout has
been steadily increasing in many parts of the world in recent years
[
1,3]. Additionally, gout is associated with multiple comorbidities,
such as hypertension, diabetes, cardiovascular disease and chronic
kidney disease [4]. Xanthine oxidase (XO), versatile
a
*
Corresponding author.
E-mail address: Wangshaojie@syphu.edu.cn (S. Wang).
https://doi.org/10.1016/j.ejmech.2020.112077
223-5234/© 2020 Elsevier Masson SAS. All rights reserved.
0

2
J. Gao et al. / European Journal of Medicinal Chemistry 190 (2020) 112077
Fig. 1. Chemical structures of selected XO inhibitors.
phosphoribosyltransferase and orotate phosphoribosyltransferase
to nucleotide analogs such as allopurinol-1-ribonucleotide and
oxypurinol-7-ribonucleotide, which further interfere with normal
purine metabolism and pyrimidine synthesis [5,9], and subse-
quently produce severe side effects such as hypersensitivity, skin
rash, gastrointestinal distress, eosinophilia, and renal toxicity
[
5,6,10]. As a result, various nonpurine-based XO inhibitors with
different scaffolds have been extensively investigated, including
thiazoles [11e14], pyrazoles [15], isoxazoles [16], imidazoles [17,18],
selenazoles [19], furans [20], isocytosines [21e23], pyrimidines
[
24e27], benzimidazoles [28], 1,2,3-triazole derivatives [29,30], 5-
aryl-1H-tetrazoles [31], chalcone derivatives [32,33], coumarin
derivatives [34e36] and flavonoid derivatives [37,38]. However,
febuxostat, a representative nonpurine XO inhibitor, was mandated
to have a black box warning by the FDA in February 2019, consid-
ering that the cardiovascular mortality of febuxostat was higher
than that of allopurinol [39,40]. Therefore, there is an urgent need
to develop new nonpurine XO inhibitors for the treatment of hy-
peruricemia and gout [41].
As a planar acidic heterocycle (pKa ¼ ~6e7), 5-oxo-4,5-dihydro-
Fig. 2. Design strategy.
1
,2,4-oxadiazole has been widely adopted by medicinal chemists to
design new agents with diverse biological activities, including anti-
fungus, anti-hypertension and anti-inflammation [42e45]. More-
over, it is obvious that the 5-oxo-4,5-dihydro-1,2,4-oxadiazole
moiety could be used as a pyrimidone bioisostere [46e48],
considering that allopurinol can be divided into two parts, namely,
a pyrimidone and pyrazole. Thus, it is reasonable to use the 5-oxo-
1
,2,4-oxadiazol-3-yl)-1H-indole-3-carbonitriles (1a-1u, 2c, 2e, 2h
and 2n) are described. In addition, steady-state kinetic analysis and
molecular modeling studies were also performed to investigate the
inhibitory behaviors of representative compound 1h. Meanwhile,
the hypouricemic effects of compound 1h were investigated in a
potassium oxonate-induced hyperuricemia rat model.
4
,5-dihydro-1,2,4-oxadiazole moiety as a pyrimidone bioisostere to
design novel allopurinol analogs to obtain new nonpurine XO in-
hibitors [46e48]. Indole, as a privileged scaffold, could be found in
many synthetic and natural products and these products usually
possess various biological activities including anti-inflammation,
anti-oxidant, anti-hypertension, anti-diabetes, anti-virus and anti-
tumor [49e52]. Thus, it is widely used in drug design. Particu-
larly, Song et al. designed a series of 2-indol thiazole derivatives,
and the results showed that these compounds exhibited excellent
XO inhibitory potency [11,12]. Therefore, it might be a reliable
practice to replace the pyrazole ring of allopurinol with an indole
scaffold through a bioisosteric replacement strategy.
Accordingly, we connected the pyrimidone and pyrazole rings of
allopurinol with a single bond and employed a bioisosteric
replacement strategy to replace these two parts of allopurinol with
a 5-oxo-4,5-dihydro-1,2,4-oxadiazole moiety and an indole scaf-
fold, respectively, to design a series of 1-alkyl-5-(5-oxo-4,5-
dihydro-1,2,4-oxadiazol-3-yl)-1H-indole-3-carbonitriles (1a-1u)
as novel nonpurine XO inhibitors [15]. Meanwhile, to investigate
the influence of the pending 5-oxo-4,5-dihydro-1,2,4-oxadiazole
position on the inhibitory potency, the 6-position-substituted
2
. Results and discussion
2.1. Chemistry
The reaction sequence for the synthesis of the target compounds
is shown in Scheme 1. Commercially available 1H-indole-5-
carbonitrile (3) was alkylated with specific alkyl halides in the
presence of sodium hydride in dimethylformamide (DMF) to give
good yields of 1-alkyl-1H-indole-5-carbonitriles (5b-5u) [53].
Subsequently, the precursor 3 or intermediates 5b-5u were reacted
with hydroxylamine hydrochloride and sodium carbonate in
dimethyl sulfoxide (DMSO) to provide N-hydroxy-1-alkyl-1H-
indole-5-carboximidamides (7a-7u), which were further cyclized
in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and
0
N,N -carbonyldiimidazole (CDI) in dichloromethane, furnishing key
intermediates
3-(1-alkyl-1H-indol-5-yl)-1,2,4-oxadiazol-5(4H)-
ones (9a-9u) with excellent yields [54]. The aldehyde group was
introduced into intermediates 9a-9u under Vilsmeier-Haack con-
ditions to provide 1-alkyl-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-
yl)-1H-indole-3-carbaldehydes (11a-11u) [55]. The corresponding
aldehyde group was further converted into a cyano group in the
presence of hydroxylamine hydrochloride and sodium formate in
formic acid at reflux to afford compounds 1-alkyl-5-(5-oxo-4,5-
dihydro-1,2,4-oxadiazol-3-yl)-1H-indole-3-carbonitriles (1a-1u)
compounds
1-alkyl-6-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-
1
H-indole-3-carbonitriles (2c, 2e, 2h and 2n) were also designed,
as is shown in Fig. 2.
In this study, the synthesis, in vitro XO inhibitory evaluation and
structure-activity relationships of 1-alkyl-5/6-(5-oxo-4,5-dihydro-

J. Gao et al. / European Journal of Medicinal Chemistry 190 (2020) 112077
3
ꢀ
ꢀ
ꢀ
ꢀ
Scheme 1. Reagents and conditions: i. RCl or RBr, NaH, KI, DMF, 25e80 C; ii. H
NOH$HCl, Na CO
2 2 3
, DMSO, 80 C; iii. CDI, DBU, CH
2
Cl
2
, 30 C; iv. POCl
3
, DMF, 0 C; v. H
2
NOH$HCl,
HCOONa, NMP, HCOOH, reflux.
in good yields [24]. The 1-alkyl-6-(5-oxo-4,5-dihydro-1,2,4-
oxadiazol-3-yl)-1H-indole-3-carbonitriles (2c, 2e, 2h and 2n)
were also prepared as previously described using 1H-indole-6-
carbonitrile (4) as a precursor.
1.02 mM, respectively), which indicated that the iso-propyl group
was favorable for the potency. Interestingly, when the cyclopentyl
group was introduced instead of cyclopropylmethyl group in
compound 1g to obtain compound 1h, the inhibitory potency was
increased by approximately 2.4-fold (1h vs 1g, IC50 ¼ 0.36
.88 M, respectively), and further expanding the ring to a cyclo-
hexylmethyl group (1i, IC50 ¼ 2.86 M) resulted in an approxi-
mM and
0
m
2
2
.2. Biological evaluation
m
mately 7.9-fold decrease in potency compared with that of
compound 1h, suggesting that the cyclopentyl group was prefer-
able for these saturated cycloalkyl substituted XO inhibitors, which
was in accordance with the conclusion from the 1,2,3-triazole de-
rivatives [30].
.2.1. Xanthine oxidase inhibitory activity
Evaluation of the in vitro bovine XO inhibitory potency of
compounds 1a-1u, 2c, 2e, 2h, 2n, 9c and 9h was performed with
allopurinol as a reference compound. The results showed that most
of the target compounds displayed IC50 values ranging between
To explore the influence of the benzyl group on inhibitory po-
tency, compounds 1j-1u with a benzyl or para- or meta-substituted
benzyl group at the 1-position of the indole scaffold were prepared,
and the results showed that these compounds also possessed
remarkable XO inhibitory potency with IC50 values ranging from
0
.36
m
M and 6.56
m
M, except 9c and 9h (not active, <50% inhibition
M), with a
at 10
mM). In particular, compound 1h (IC50 ¼ 0.36
m
cyclopentyl group at 1-position and a 5-oxo-4,5-dihydro-1,2,4-
oxadiazole ring at 5-position of the indole scaffold, exhibited the
most active XO inhibitory potency, which was approximately 21-
1.02 to 3.60
1r, 1s, 1t and 1u IC50 ¼ 3.60
1.70 M, respectively) to the corresponding para-counterparts (1k,
1l, 1m, 1n and 1o IC50 ¼ 1.71 M, 2.40 M, 2.07 M, 1.14 M and
1.02 M, respectively), the potencies of the meta-substituted
mM. However, by comparing the meta-derivatives (1q,
fold more potent than that of allopurinol (IC50 ¼ 7.59
over, as exemplified by compounds 9c and 9h (<50% inhibition at
M), replacement of the cyano group with a hydrogen atom at 3-
m
M). More-
m
M, 2.93 M, 2.60 M, 2.16 M and
m
m
m
m
1
0
m
m
m
m
m
position of the indole scaffold resulted in a dramatic loss of potency.
This result demonstrated that the cyano group was indispensable
for this series of compounds to produce XO inhibitory potency,
which was similar to the 2-(indol-5-yl) thiazole derivatives [11].
m
compounds were weaker than the corresponding para-substituted
compounds, confirming that, for the halogen (F, Cl and Br), methyl
and methoxy groups, the para position was preferred for potency.
To investigate the influence of the pending 5-oxo-4,5-dihydro-
1,2,4-oxadiazole moiety position on potency, compounds 2c, 2e, 2h
As shown in Table 1, except for compound 1b (IC50 ¼ 5.41
mM),
M),
compounds 1c-1u (IC50 values ranging from 0.36 M to 3.60
m
m
containing a 5-oxo-4,5-dihydro-1,2,4-oxadiazole moiety at the 5-
position and different alkyl groups at the 1-position of the indole
scaffold, were more active than unsubstituted compound 1a
and 2n with
a
5-oxo-4,5-dihydro-1,2,4-oxadiazole moiety
substituted at the 6-position of the indole scaffold were prepared.
The results showed that the inhibitory potencies of these com-
pounds were largely mitigated (2c vs 1c, 2e vs 1e, 2h vs 1h, and 2n
(
1
IC50 ¼ 4.13
mM). This finding implied that the hydrophobic group at
-position of the indole scaffold played a crucial role for these
vs 1n; IC50 ¼ 1.80
m
m
M vs 0.41 M, 6.56
m
mM vs 1.02 mM, 0.82 mM vs
compounds to exhibit XO inhibitory potency, which was consistent
0.36 M, and 1.84
m
M vs 1.14 mM, respectively), which meant that
with pyrimidones [25]. Specifically, the inhibitory potency of
the 5-oxo-4,5-dihydro-1,2,4-oxadiazole moiety linked at the 5-
position was more preferable for the XO inhibitory potency.
compound 1c (IC50 ¼ 0.41
position, was approximately 13.2-, 2.7- and 2.5-fold higher than
those of compounds 1b, 1d and 1e (IC50 ¼ 5.41 M, 1.09 M and
mM), with an iso-propyl group at the 1-
m
m

4
J. Gao et al. / European Journal of Medicinal Chemistry 190 (2020) 112077
Table 1
In vitro XO inhibitory potency of the designed compounds.
Compd.
R
R
1
IC50
(m
M)a
Compd.
R
R
1
IC50 (m
M)a
1
1
1
1
1
1
1
1
1
1
1
1
1
1
a
b
c
d
e
f
g
h
i
j
k
l
m
n
H
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
CN
4.13 ± 0.15
5.41 ± 0.09
0.41 ± 0.01
1.09 ± 0.03
1.02 ± 0.04
1.24 ± 0.03
0.88 ± 0.03
0.36 ± 0.01
2.86 ± 0.11
1.72 ± 0.07
1.71 ± 0.05
2.40 ± 0.06
2.07 ± 0.04
1.14 ± 0.03
1o
1p
1q
1r
1s
1t
1u
9c
9h
2c
2e
2h
4-methoxybenzyl
4-(tert-butyl) benzyl
3-fluorobenzyl
3-chlorobenzyl
3-bromobenzyl
3-methylbenzyl
3-methoxybenzyl
iso-propyl
cyclopentyl
iso-propyl
iso-pentyl
cyclopentyl
CN
CN
CN
CN
CN
CN
CN
H
1.02 ± 0.01
1.44 ± 0.07
3.60 ± 0.07
2.93 ± 0.07
2.60 ± 0.07
2.16 ± 0.11
1.70 ± 0.05
methyl
iso-propyl
iso-butyl
iso-pentyl
3-methylbut-2-en-1-yl
cyclopropylmethyl
cyclopentyl
cyclohexylmethyl
benzyl
4-fluorobenzyl
4-chlorobenzyl
4-bromobenzyl
4-methylbenzyl
b
n.a.
n.a.
b
H
CN
CN
CN
CN
1.80 ± 0.06
6.56 ± 0.32
0.82 ± 0.03
1.84 ± 0.06
7.59 ± 0.21
2n
4-methylbenzyl
Allopurinol
a
All values are expressed as the mean ± SD of triplicate determinations.
n.a.: Not active (<50% inhibition at 10 mM).
b
2
.2.2. Enzyme inhibitory kinetics
To explore the mode of action of representative compound 1h,
inhibitor preferentially bound to the free enzyme rather than to the
enzyme-substrate complex [57e59]. Moreover, dose-dependent
inhibition of XO by compound 1h is shown in Fig. 4.
enzyme kinetic studies were performed. As shown in Fig. 3,
Lineweaver-Burk plot analysis revealed that compound 1h was a
mixed-type XO inhibitor, which was different from allopurinol with
2.2.3. Molecular modeling
a competitive inhibition mode [56]. Additionally, the K
/Vmax) were increased but the Vmax was decreased by increasing
the inhibitor concentration from 0 to 1.325 M. Specifically, the K
and Vmax values of compound 1h were 171.35, 198.38, 203.46,
43.71, and 319.68 M and 0.189, 0.165, 0.150, 0.107, and 0.041 M/
min, respectively. Moreover, the values of K (equilibrium constant
m
and slope
To further understand the enzyme-inhibitor interactions, mo-
(K
m
lecular docking of compounds 1h, 2h and allopurinol into the
binding pocket of the xanthine dehydrogenase (XDH) crystal
structure (PDB 1N5X) was performed. The molybdenum-pterin
centers of both XDH and XO are identical in terms of binding
modes and substrate catalysis [60,61].
m
m
2
m
m
i
for binding with free enzyme) and Kis (equilibrium constant for
binding with enzyme-substrate complex) were calculated to be
0
As shown in Fig. 5a, compound 1h was accommodated in the
active site through hydrogen bonds and p-p stacking interactions
.138 mM and 0.331 mM, respectively, which confirmed that the
with primary amino acids, including Asn768, Lys771, Arg880,
Phe914, Phe1009 and Thr1010, explaining why compound 1h could
produce its XO inhibitory potency. Specifically, the 5-oxo-4,5-
dihydro-1,2,4-oxadiazole moiety interacted with the innermost
part of the active pocket via 3 hydrogen bonds, in which the
carbonyl group acted as a hydrogen bond acceptor interacting with
Fig. 3. Lineweaver-Burk plots of XO in the presence of compound 1h.
Fig. 4. Dose-dependent inhibition of compound 1h (means ± SD, n ¼ 3).

J. Gao et al. / European Journal of Medicinal Chemistry 190 (2020) 112077
5
Fig. 5. The binding modes of compounds 1h and 2h and allopurinol within the binding pocket of XDH (PDB 1N5X). (a) The interactions of compound 1h (brown) with the amino
acid residues in XDH. (b) The binding mode of compound 2h (purple) with XDH. (c) The docking pose of allopurinol (green) with XDH. The ligands are depicted as a ball-and-stick
model. The amino acid residues in XDH interacting with ligands are shown as cyan lines. The hydrogen bonds and
p-p stacking interactions are shown as green and violet dashed
lines, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
the amino group of Thr1010 and the guanidine group of Arg880,
which was in accordance with 5-aryl-1H-tetrazoles [31]. In addi-
tion, the 5-oxo-4,5-dihydro-1,2,4-oxadiazole ring was sandwiched
between Phe914 and Phe1009 through face-to-face and face-to-
group, a significant hyperuricemia rat model was established by
intraperitoneal injection of potassium oxonate (300 mg/kg) after
1 h of drug administration (P < 0.0001). Obviously, compound 1h
showed a reduction of 20.8% in uric acid levels (P < 0.001) at 3 h and
a reduction of 12.8% in the AUC (uric acid, 1e8 h) (P < 0.0001) compared
with the model group at an oral dose of 10 mg/kg. Therefore, the
results on the hypouricemic effect in vivo suggested that compound
1h could be a promising lead compound for the treatment of hy-
edge
rinol and pyrano[3,2-d] pyrimidine derivatives [27]. Moreover, the
-cyano group formed two hydrogen bonds with Asn768 and
Lys771 in the subpocket of the active cavity, which was similar to 2-
indol-5-yl) thiazole derivatives and pyrimidones [11,24]. Addi-
p-p stacking interactions, which was consistent with allopu-
3
(
peruricemia. However, although compound 1h exhibited a
tionally, several hydrophobic interactions between the cyclopentyl
group and Leu648, Phe649, Val1011 and Phe1013 were also
observed at the entrance of the active pocket. In contrast, the
allopurinol only engaged in one hydrogen bond with Glu802, two
considerable reduction in serum urate levels, it did not show oral
efficacy comparable to that of allopurinol. This result could be due
to its poor oral bioavailability.
p-p stacking interactions with Phe914 and Phe1009 and several
3
. Conclusion
hydrophobic interactions with Leu873, Val1011, Leu1014 and
Ala1078 (Fig. 5c). Therefore, the binding modes mentioned above
could effectively explain the potency difference between com-
pound 1h and allopurinol; it also emphasized the importance of the
In conclusion, using allopurinol as a prototype drug, a novel
class of 1-alkyl-5/6-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-
indole-3-carbonitriles (1a-1v, 2c, 2e, 2h and 2n) were developed as
effective XO inhibitors using a bioisosteric replacement strategy. In
particular, the in vitro biological evaluation results revealed that
compound 1h was the strongest XO inhibitor with an IC50 value of
5
-oxo-4,5-dihydro-1,2,4-oxadiazole moiety and 3-cyano group in
these compounds to display apparent inhibitory potency. In com-
parison to compound 1h, compound 2h presented similar
hydrogen bonds and
acids including Asn768, Lys771, Arg880, Phe914 and Thr1010
Fig. 5b). However, the loss of the stacking interaction with
p-p stacking interactions with the key amino
0.36 mM. In addition, the structure-activity relationship analysis
revealed that the 3-cyano group was indispensable for these
compounds to produce XO inhibitory potency and the 5-oxo-4,5-
dihydro-1,2,4-oxadiazole moiety linked at 5-position rather than
(
p-p
Phe1009 may be the main reason for its reduced potency, which
could also be observed for other compounds (2c, 2e and 2n) with a
6
-position of the indole scaffold was preferable. Additionally, the
5
-oxo-4,5-dihydro-1,2,4-oxadiazole moiety linked at the 6-position
enzyme kinetic studies indicated that compound 1h acted as a
mixed-type XO inhibitor. The molecular docking results suggested
that the 5-oxo-4,5-dihydro-1,2,4-oxadiazole moiety could interact
with Arg880 and Thr1010 in the innermost part of the active pocket
through 3 hydrogen bonds, while the cyano group could form two
hydrogen bonds with Asn768 and Lys771. Moreover, compound 1h
showed an effective hypouricemic effect at an oral dose of 10 mg/kg
in a rat model of potassium oxonate-induced hyperuricemia.
Accordingly, the results described above strongly suggest that
compound 1h could be a promising lead compound for the treat-
ment of hyperuricemia and gout.
of the indole scaffold.
For a deeper understanding of the hydrophobic alkyl groups on
the inhibitory potency, molecular docking studies have been also
performed on compounds 1b-1e, 1g and 1i at the active pocket. The
partial docking scores were calculated by adding the scores of van
der Waals force between hydrophobic alkyl groups and amino acids
including Leu648, Phe649, Val1011 and Phe1013 at the entrance of
the active pocket. As shown in Table 2, the partial docking scores
displayed the same tendency with the in vitro inhibitory potency of
compounds 1b-1e and 1g-1i, respectively. And these results pro-
vided a reasonable explanation for the observed potencies of these
compounds.
4
. Experimental section
2.2.4. Hypouricemic effect in vivo
4.1. Chemistry
The in vivo hypouricemic effect of compound 1h (10 mg/kg) was
further investigated and compared with the positive control allo-
purinol (5 mg/kg, Fig. 6). As expected, compared with the blank
Unless otherwise noted, the solvents and reagents, obtained
from commercial companies, were used directly without further

6
J. Gao et al. / European Journal of Medicinal Chemistry 190 (2020) 112077
Table 2
Partial docking scores of compounds 1b-1e and 1g-1i at the active pocket of XO.
Compounds
R
IC50
(m
M)
Partial docking score (kcal/mol)
1
1
1
1
1
1
1
b
c
methyl
iso-propyl
iso-butyl
iso-pentyl
cyclopropylmethyl
cyclopentyl
cyclohexylmethyl
5.41 ± 0.09
0.41 ± 0.01
1.09 ± 0.03
1.02 ± 0.04
0.88 ± 0.03
0.36 ± 0.01
2.86 ± 0.11
ꢁ1.26
ꢁ8.15
ꢁ5.06
ꢁ4.83
ꢁ9.36
ꢁ11.35
ꢁ6.40
d
e
g
h
i
purification. All reactions were run using a Teflon-coated magnetic
stir bar at the indicated temperature on the benchtop. Reactions
that were run at elevated temperatures were run in an oil bath at
the indicated temperature. Reaction progress was monitored by
thin-layer chromatography with a 254 nm or 365 nm fluorescence
indicator. The purity of the target compounds was determined to be
greater than 95% by HPLC. H NMR and C NMR spectra were
recorded on a Bruker 400 MHz spectrometer or a Bruker 600 MHz
spectrometer. Chemical shifts are reported in parts per million
DMSO‑d
6
)
d
8.07 (d, J ¼ 1.6 Hz, 1H), 7.62 (d, J ¼ 8.5 Hz, 1H), 7.53 (d,
J ¼ 3.2 Hz, 1H), 7.48 (dd, J ¼ 8.5, 1.6 Hz, 1H), 6.59 (d, J ¼ 3.2 Hz, 1H),
þ
3.84 (s, 3H); ESI-MS m/z 157.05 [MþH] .
4.1.1.2. 1-Isopropyl-1H-indole-5-carbonitrile (5c). A yellow oil; yield
1
81.2%; H NMR (400 MHz, DMSO‑d )
6
d 8.09e8.06 (m, 1H), 7.72 (d,
1
13
J ¼ 2.2 Hz, 1H), 7.70 (d, J ¼ 3.3 Hz, 1H), 7.46 (dd, J ¼ 8.6, 1.6 Hz, 1H),
6.63 (d, J ¼ 3.3 Hz, 1H), 4.84 (hept, J ¼ 6.7 Hz, 1H), 1.46 (d, J ¼ 6.7 Hz,
þ
6H); ESI-MS m/z 185.09 [MþH] .
(
ppm) relative to tetramethylsilane (TMS), and spin multiplicities
are given as s (singlet), d (doublet), t (triplet), q (quartet), hept
heptet) or m (multiplet). Low-resolution mass spectrometry (MS)
4
8
1
.1.1.3. 1-Isobutyl-1H-indole-5-carbonitrile (5d). A yellow oil; yield
4.2%; H NMR (400 MHz, Chloroform-d)
H), 7.41 (dd, J ¼ 8.6, 1.5 Hz, 1H), 7.36 (d, J ¼ 8.4 Hz, 1H), 7.19 (d,
(
1
d
7.96 (dd, J ¼ 1.5, 0.7 Hz,
were recorded with an Agilent 1100 ion trap liquid
chromatography-mass spectrometer. High-resolution mass spectra
J ¼ 3.2 Hz, 1H), 6.56 (dd, J ¼ 3.2, 0.8 Hz, 1H), 3.93 (d, J ¼ 7.3 Hz, 2H),
(
HRMS) data were taken with the Agilent Q-TOF system. Melting
2
.24e2.11 (m, 1H), 0.92 (d, J ¼ 6.6 Hz, 6H); ESI-MS m/z 199.13
points were determined on
uncorrected.
a
YRT-3 apparatus and were
þ
[MþH] .
4
.1.1.4. 1-Isopentyl-1H-indole-5-carbonitrile (5e). A white crystal-
ꢀ
ꢀ
1
4
6
.1.1. General procedure for the preparation of 1-alkyl-1H-indole-5/
-carbonitriles (5b-5u, 6c, 6e, 6h and 6n)
A solution of 3 (or 4, 80 mmol) in DMF (80 mL) at 0 C was treat
line powder; yield 87.1%; M.p 47.8 Ce49.4 C; H NMR (400 MHz,
DMSO‑d6)
d
8.08 (d, J ¼ 0.9 Hz, 1H), 7.67 (dt, J ¼ 8.6, 0.9 Hz, 1H), 7.61
ꢀ
(d, J ¼ 3.2 Hz, 1H), 7.48 (dd, J ¼ 8.6, 1.6 Hz, 1H), 6.60 (dd, J ¼ 3.2,
0.8 Hz, 1H), 4.30e4.21 (m, 2H), 1.65 (q, J ¼ 7.0 Hz, 2H), 1.53e1.43 (m,
with sodium hydride (60% dispersion in mineral oil, 90 mmol)
portionwise, and the reaction was stirred until the hydrogen was
completely evolved. The appropriate alkyl halide (100 mmol) and
potassium iodide (8 mmol) were then added to the reaction
þ
1H), 0.91 (d, J ¼ 6.6 Hz, 6H); ESI-MS m/z 213.14 [MþH] .
4
.1.1.5. 1-(3-Methylbut-2-en-1-yl)-1H-indole-5-carbonitrile
(5f).
7.96
ꢀ
1
mixture and the reaction was stirred at 25e80 C. After completion,
A yellow oil; yield 92.4%; H NMR (400 MHz, Chloroform-d)
d
the reaction was quenched with caution by the dropwise addition
of water, treated with saturated sodium chloride, and extracted
with ethyl acetate. The combined organic layer was washed with
brine, dried over anhydrous sodium sulfate and concentrated under
reduced pressure. The crude product was purified by flash column
chromatography to afford the title compound.
(
1
dd, J ¼ 1.6, 0.7 Hz, 1H), 7.41 (dd, J ¼ 8.6, 1.6 Hz, 1H), 7.38e7.33 (m,
H), 7.21 (d, J ¼ 3.2 Hz, 1H), 6.55 (dd, J ¼ 3.3, 0.8 Hz, 1H), 5.35 (tdq,
J ¼ 6.9, 2.9,1.4 Hz,1H), 4.71 (dt, J ¼ 7.0,1.0 Hz, 2H),1.83 (d, J ¼ 1.3 Hz,
þ
3
H), 1.78 (q, J ¼ 1.3 Hz, 3H); ESI-MS m/z 211.12 [MþH] .
4
.1.1.6. 1-(Cyclopropylmethyl)-1H-indole-5-carbonitrile (5g). A yel-
1
low oil; yield 75.3%; H NMR (400 MHz, DMSO‑d
6
)
d
8.08 (dd,
4
.1.1.1. 1-Methyl-1H-indole-5-carbonitrile (5b). A white crystalline
J ¼ 1.6, 0.7 Hz, 1H), 7.72 (dd, J ¼ 8.6, 0.8 Hz, 1H), 7.65 (d, J ¼ 3.2 Hz,
ꢀ
ꢀ
1
powder; yield 93.6%; M.p 77.1 Ce77.8 C; H NMR (400 MHz,
1H), 7.47 (dd, J ¼ 8.6,1.6 Hz,1H), 6.60 (dd, J ¼ 3.2, 0.8 Hz,1H), 4.11 (d,
Fig. 6. Anti-hyperuricemic effects of compound 1h and allopurinol. (a) Time-course changes of serum uric acid levels after 1e8 h of treatment. (b) Serum uric acid levels after 3 h of
treatment. (c) The AUC of serum uric acid after 1e8 h of treatment. The data are shown as the mean ± SD. (n ¼ 6). ***P < 0.001, ****P < 0.0001, significantly different from the model
group by Student’s t-test.

J. Gao et al. / European Journal of Medicinal Chemistry 190 (2020) 112077
7
J ¼ 7.0 Hz, 2H), 1.30e1.16 (m, 1H), 0.55e0.47 (m, 2H), 0.44e0.34 (m,
4.1.1.16. 1-(3-Fluorobenzyl)-1H-indole-5-carbonitrile (5q). A white
þ
ꢀ
ꢀ
1
2
H); ESI-MS m/z 197.10 [MþH] .
crystalline powder; yield 91.6%; M.p 67.5 Ce68.9 C; H NMR
400 MHz, Chloroform-d)
(
d
7.93 (d, J ¼ 1.4 Hz, 1H), 7.33 (dd, J ¼ 8.6,
4
.1.1.7. 1-Cyclopentyl-1H-indole-5-carbonitrile (5h). A yellow oil;
1.5 Hz, 1H), 7.23 (d, J ¼ 2.1 Hz, 1H), 7.19e7.16 (m, 2H), 6.91 (td,
J ¼ 8.4, 2.5 Hz, 1H), 6.79 (d, J ¼ 7.7 Hz, 1H), 6.68 (dt, J ¼ 9.5, 2.1 Hz,
1H), 6.58 (d, J ¼ 3.2 Hz, 1H), 5.27 (s, 2H); ESI-MS m/z 251.09
1
yield 57.6%; H NMR (400 MHz, Chloroform-d)
1
d
7.95 (t, J ¼ 1.1 Hz,
H), 7.44 (d, J ¼ 8.6 Hz, 1H), 7.41 (dd, J ¼ 8.6, 1.5 Hz, 1H), 7.31 (d,
þ
J ¼ 3.3 Hz, 1H), 6.57 (dd, J ¼ 3.3, 0.6 Hz, 1H), 4.85e4.76 (m, 1H),
[MþH] .
þ
2
.29e2.19 (m, 2H), 2.01e1.71 (m, 6H); ESI-MS m/z 211.14 [MþH] .
4
.1.1.17. 1-(3-Chlorobenzyl)-1H-indole-5-carbonitrile (5r). A white
ꢀ
ꢀ
1
4
.1.1.8. 1-(Cyclohexylmethyl)-1H-indole-5-carbonitrile (5i). A white
crystalline powder; yield 91.5%; M.p 108.2 Ce109.5 C; H NMR
400 MHz, Chloroform-d)
ꢀ
ꢀ
1
crystalline powder; yield 78.2%; M.p 116.2 Ce118.1 C; H NMR
400 MHz, Chloroform-d) 7.99 (s, 1H), 7.49e7.37 (m, 2H), 7.20 (d,
J ¼ 3.2 Hz, 1H), 6.59 (d, J ¼ 3.3 Hz, 1H), 3.99 (d, J ¼ 7.2 Hz, 2H),
(
d
8.02 (d, J ¼ 1.4 Hz, 1H), 7.43 (dd, J ¼ 8.6,
(
d
1.6 Hz,1H), 7.34e7.26 (m, 4H), 7.13e7.09 (m,1H), 7.00e6.93 (m,1H),
6
.68 (dd, J ¼ 3.2, 0.8 Hz, 1H), 5.35 (s, 2H); ESI-MS m/z 267.06
þ
1.90e1.80 (m, 1H), 1.77e1.60 (m, 5H), 1.27e1.14 (m, 3H), 1.07e0.97
[MþH] .
þ
(
m, 2H); ESI-MS m/z 239.17 [MþH] .
4
.1.1.18. 1-(3-Bromobenzyl)-1H-indole-5-carbonitrile (5s). A white
4
.1.1.9. 1-Benzyl-1H-indole-5-carbonitrile (5j). A white crystalline
ꢀ
ꢀ
1
crystalline powder; yield 90.7%; M.p 117.8 Ce118.4 C; H NMR
ꢀ
ꢀ
1
powder; yield 97.3%; M.p 104.5 Ce105.6 C; H NMR (400 MHz,
Chloroform-d)
(400 MHz, Chloroform-d)
d
8.00 (dd, J ¼ 1.6, 0.7 Hz, 1H), 7.45e7.39
d
8.02 (d, J ¼ 1.4 Hz, 1H), 7.42 (dd, J ¼ 8.5, 1.5 Hz, 1H),
(
1
m, 2H), 7.32e7.26 (m, 2H), 7.24 (d, J ¼ 3.2 Hz, 1H), 7.18 (t, J ¼ 7.9 Hz,
7
1
.38e7.25 (m, 5H), 7.12 (dd, J ¼ 7.5, 2.0 Hz, 2H), 6.66 (d, J ¼ 3.2 Hz,
H), 6.99e6.96 (m, 1H), 6.65 (dd, J ¼ 3.3, 0.9 Hz, 1H), 5.32 (s, 2H);
þ
H), 5.38 (s, 2H); ESI-MS m/z 233.17 [MþH] .
þ
ESI-MS m/z 310.98, 313.00 [MþH] .
4
.1.1.10. 1-(4-Fluorobenzyl)-1H-indole-5-carbonitrile (5k). A white
ꢀ
ꢀ
1
4.1.1.19. 1-(3-Methylbenzyl)-1H-indole-5-carbonitrile (5t). A yellow
crystalline powder; yield 94.1%; M.p 101.9 Ce102.5 C; H NMR
400 MHz, Chloroform-d)
7.99 (dd, J ¼ 1.6, 0.7 Hz, 1H), 7.45 (d,
1
oil; yield 87.1%; H NMR (400 MHz, Chloroform-d)
d 7.99e7.96 (m,
(
d
1
H), 7.40e7.36 (m, 1H), 7.32 (dt, J ¼ 8.6, 0.8 Hz, 1H), 7.24 (d,
J ¼ 8.4 Hz, 2H), 7.40 (dd, J ¼ 8.6, 1.5 Hz, 1H), 7.28 (dd, J ¼ 8.7, 0.9 Hz,
J ¼ 3.2 Hz, 1H), 7.18 (d, J ¼ 7.6 Hz, 1H), 7.09 (d, J ¼ 7.7 Hz, 1H), 6.92 (s,
1
0
H), 7.23 (d, J ¼ 3.2 Hz, 1H), 6.96 (d, J ¼ 8.4 Hz, 2H), 6.64 (dd, J ¼ 3.3,
þ
1H), 6.88 (d, J ¼ 7.7 Hz,1H), 6.61 (dd, J ¼ 3.2, 0.8 Hz,1H), 5.29 (s, 2H),
.9 Hz, 1H), 5.30 (s, 2H); ESI-MS m/z 251.10 [MþH] .
þ
2
.29 (s, 3H); ESI-MS m/z 247.11 [MþH] .
4
.1.1.11. 1-(4-Chlorobenzyl)-1H-indole-5-carbonitrile (5l). A white
ꢀ
ꢀ
1
crystalline powder; yield 95.2%; M.p 122.3 Ce124.1 C; H NMR
400 MHz, Chloroform-d)
8.02 (d, J ¼ 1.4 Hz, 1H), 7.42 (dd, J ¼ 8.6,
4.1.1.20. 1-(3-Methoxybenzyl)-1H-indole-5-carbonitrile
white crystalline powder; yield 93.4%; M.p 86.3 Ce87.9 C; H NMR
(5u). A
ꢀ
ꢀ
1
(
d
1
.5 Hz, 1H), 7.33e7.30 (m, 3H), 7.26 (d, J ¼ 3.2 Hz, 1H), 7.04 (d,
(400 MHz, Chloroform-d)
d
7.98 (dd, J ¼ 1.6, 0.7 Hz, 1H), 7.39 (dd,
J ¼ 8.4 Hz, 2H), 6.67 (dd, J ¼ 3.3, 0.8 Hz, 1H), 5.34 (s, 2H); ESI-MS m/z
J ¼ 8.6, 1.5 Hz, 1H), 7.32 (dt, J ¼ 8.6, 0.8 Hz, 1H), 7.25e7.20 (m, 2H),
6.82 (ddd, J ¼ 8.3, 2.6, 0.9 Hz, 1H), 6.67 (ddq, J ¼ 7.6, 1.6, 0.8 Hz, 1H),
þ
2
67.02 [MþH] .
6
.64e6.59 (m, 2H), 5.31 (s, 2H), 3.73 (s, 3H); ESI-MS m/hept z 263.10
þ
[MþH] .
4
.1.1.12. 1-(4-Bromobenzyl)-1H-indole-5-carbonitrile (5m). A white
ꢀ
ꢀ
1
crystalline powder; yield 94.4%; M.p 145.4 Ce146.7 C; H NMR
400 MHz, Chloroform-d)
7.99 (dd, J ¼ 1.6, 0.7 Hz, 1H), 7.44 (d,
(
d
4
.1.1.21. 1-Isopropyl-1H-indole-6-carbonitrile (6c). A yellow oil;
J ¼ 8.4 Hz, 2H), 7.40 (dd, J ¼ 8.6, 1.5 Hz, 1H), 7.28 (dd, J ¼ 8.7, 0.9 Hz,
1
yield 86.7%; H NMR (400 MHz, Chloroform-d)
1
d
7.72 (q, J ¼ 1.0 Hz,
1
0
H), 7.23 (d, J ¼ 3.2 Hz, 1H), 6.95 (d, J ¼ 8.4 Hz, 2H), 6.64 (dd, J ¼ 3.3,
H), 7.66 (dd, J ¼ 8.2, 0.7 Hz, 1H), 7.42 (d, J ¼ 3.2 Hz, 1H), 7.32 (dd,
þ
.9 Hz, 1H), 5.30 (s, 2H); ESI-MS m/z 310.98, 312.97 [MþH] .
J ¼ 8.2, 1.3 Hz, 1H), 6.58 (dd, J ¼ 3.2, 0.8 Hz, 1H), 4.68 (hept,
þ
J ¼ 6.7 Hz, 1H), 1.55 (d, J ¼ 6.7 Hz, 6H); ESI-MS m/z 185.11 [MþH] .
4
.1.1.13. 1-(4-Methylbenzyl)-1H-indole-5-carbonitrile (5n). A white
ꢀ
ꢀ
1
crystalline powder; yield 92.6%; M.p 69.4 Ce70.8 C; H NMR
400 MHz, Chloroform-d)
7.98e7.97 (m, 1H), 7.38 (dd, J ¼ 8.6,
4
.1.1.22. 1-Isopentyl-1H-indole-6-carbonitrile (6e). A yellow oil;
(
d
1
yield 88.9%; H NMR (400 MHz, Chloroform-d)
H), 7.32 (dd, J ¼ 8.1, 1.4 Hz, 1H), 7.29 (d, J ¼ 3.1 Hz, 1H), 6.55 (dd,
J ¼ 3.2, 0.8 Hz, 1H), 4.15 (t, J ¼ 7.5 Hz, 2H), 1.74 (q, J ¼ 7.5 Hz, 2H),
d
7.66 (d, J ¼ 8.2 Hz,
1
.5 Hz, 1H), 7.34e7.30 (m, 1H), 7.23 (d, J ¼ 3.2 Hz, 1H), 7.12 (d,
2
J ¼ 7.8 Hz, 2H), 6.99 (d, J ¼ 7.9 Hz, 2H), 6.61 (dd, J ¼ 3.2, 0.8 Hz, 1H),
þ
5
.29 (s, 2H), 2.32 (s, 3H); ESI-MS m/z 247.11 [MþH] .
1
.66e1.53 (m, 1H), 0.98 (d, J ¼ 6.6 Hz, 6H); ESI-MS m/z 213.12
þ
[MþH] .
4
.1.1.14. 1-(4-Methoxybenzyl)-1H-indole-5-carbonitrile
(5o). A
ꢀ
ꢀ
1
white crystalline powder; yield 96.1%; M.p 101.7 Ce103.3 C; H
4
.1.1.23. 1-Cyclopentyl-1H-indole-6-carbonitrile (6h). A yellow oil;
NMR (400 MHz, Chloroform-d)
d
7.97 (dd, J ¼ 1.5, 0.8 Hz, 1H), 7.39
1
yield 61.3%; H NMR (400 MHz, Chloroform-d)
d 7.76e7.73 (m, 1H),
(
dd, J ¼ 8.6, 1.5 Hz, 1H), 7.33 (dt, J ¼ 8.5, 0.8 Hz, 1H), 7.22 (d,
7
.65 (d, J ¼ 8.2 Hz, 1H), 7.39 (d, J ¼ 3.2 Hz, 1H), 7.31 (dd, J ¼ 8.2,
J ¼ 3.2 Hz, 1H), 7.05 (d, J ¼ 8.7 Hz, 2H), 6.84 (d, J ¼ 8.7 Hz, 2H), 6.60
1.4 Hz,1H), 6.56 (d, J ¼ 3.2 Hz,1H), 4.79 (p, J ¼ 7.0 Hz,1H), 2.29e2.21
(
[
dd, J ¼ 3.3, 0.9 Hz, 1H), 5.27 (s, 2H), 3.77 (s, 3H); ESI-MS m/z 263.07
þ
þ
(m, 2H), 1.99e1.78 (m, 6H); ESI-MS m/z 211.15 [MþH] .
MþH] .
4
.1.1.15. 1-(4-Tert-butylbenzyl)-1H-indole-5-carbonitrile
(5p). A
4.1.1.24. 1-(4-Methylbenzyl)-1H-indole-6-carbonitrile (6n). A white
ꢀ
ꢀ
1
ꢀ
ꢀ
1
white crystalline powder; yield 85.2%; M.p 111.8 Ce113.2 C;
H
crystalline powder; yield 92.4%; M.p 81.3 Ce82.9 C; H NMR
(400 MHz, Chloroform-d)
NMR (400 MHz, Chloroform-d)
dd, J ¼ 8.6, 1.5 Hz, 1H), 7.36e7.31 (m, 3H), 7.24 (d, J ¼ 3.2 Hz, 1H),
.03 (d, J ¼ 8.4 Hz, 2H), 6.61 (dd, J ¼ 3.2, 0.8 Hz,1H), 5.31 (s, 2H),1.28
d
7.98 (dd, J ¼ 1.6, 0.8 Hz, 1H), 7.39
d
7.67 (dd, J ¼ 8.3, 0.7 Hz, 1H), 7.60 (dd,
(
7
J ¼ 1.4, 0.8 Hz,1H), 7.34e7.30 (m, 2H), 7.13 (d, J ¼ 7.7 Hz, 2H), 7.00 (d,
J ¼ 8.0 Hz, 2H), 6.60 (dd, J ¼ 3.1, 0.9 Hz,1H), 5.29 (s, 2H), 2.33 (s, 3H);
þ
þ
(
s, 9H); ESI-MS m/z 289.17 [MþH] .
ESI-MS m/z 247.10 [MþH] .

8
J. Gao et al. / European Journal of Medicinal Chemistry 190 (2020) 112077
4
1
.1.2. General procedure for the preparation of N-hydroxy-1-alkyl-
H-indole-5/6-carboximidamides (7a-7u, 8c, 8e, 8h and 8n)
-Alkyl-1H-indole-5/6-carbonitrile (3, 5b-5u, 6c, 6e, 6h and 6n,
0 mmol), sodium carbonate (120 mmol) and hydroxylamine hy-
4.1.2.8. 1-Cyclopentyl-N-hydroxy-1H-indole-5-carboximidamide
(7h). A white crystalline powder; yield 89.3%; M.p
ꢀ ꢀ
1
152.3 Ce154.4 C; 1H NMR (400 MHz, DMSO‑d
6
) d 9.36 (s, 1H),
6
7.85e7.83 (m, 1H), 7.51e7.47 (m, 2H), 7.45 (d, J ¼ 3.2 Hz,1H), 6.46 (d,
J ¼ 3.2 Hz, 1H), 5.68 (s, 2H), 4.88 (tt, J ¼ 12.9, 5.6 Hz, 1H), 2.21e2.09
(m, 2H), 1.91e1.79 (m, 4H), 1.76e1.67 (m, 2H); ESI-MS m/z 244.14
drochloride (72 mmol) were suspended in 60 mL of DMSO. The
reaction mixture was stirred at 40 C for 30 min and then stirred at
8
matography, the solvent was cooled to room temperature and
poured into cold water with vigorous stirring. The resulting pre-
cipitate was filtered, washed with water and dried to obtain the
corresponding title compound, which was analytically pure and
used without further purification.
ꢀ
ꢀ
þ
0 C for 2e3 h. After completion, monitored by thin-layer chro-
[MþH] .
4.1.2.9. 1-(Cyclohexylmethyl)-N-hydroxy-1H-indole-5-
carboximidamide (7i). A white crystalline powder; yield 83.1%; M.p
ꢀ
ꢀ
1
192.4 Ce194.2 C; H NMR (400 MHz, DMSO‑d
(d, J ¼ 1.5 Hz, 1H), 7.48 (dd, J ¼ 8.6, 1.6 Hz, 1H), 7.43 (d, J ¼ 8.7 Hz,
H), 7.33 (d, J ¼ 3.1 Hz, 1H), 6.44 (d, J ¼ 3.1 Hz, 1H), 5.70 (s, 2H), 4.00
6
) d 9.38 (s, 1H), 7.84
1
(
d, J ¼ 7.2 Hz, 2H), 1.83e1.72 (m, 1H), 1.70e1.55 (m, 3H), 1.53e1.40
4.1.2.1. N-Hydroxy-1H-indole-5-carboximidamide (7a). A white
ꢀ
ꢀ
1
(m, 2H), 1.14e1.10 (m, 3H), 1.01e0.92 (m, 2H); ESI-MS m/z 272.15
crystalline powder; yield 85.9%; M.p 143.6 Ce145.3 C; H NMR
400 MHz, DMSO‑d
11.14 (s, 1H), 9.37 (s, 1H), 7.87 (d, J ¼ 1.6 Hz,
H), 7.47 (dd, J ¼ 8.6, 1.7 Hz, 1H), 7.38e7.33 (m, 2H), 6.46 (t,
þ
[
MþH] .
(
1
6
) d
þ
4.1.2.10. 1-Benzyl-N-hydroxy-1H-indole-5-carboximidamide
A white crystalline powder; yield 86.4%; M.p 158.2 Ce159.7 C; H
(7j).
J ¼ 2.7 Hz, 1H), 5.69 (s, 2H); ESI-MS m/z 176.06 [MþH] .
ꢀ
ꢀ
1
NMR (400 MHz, DMSO‑d
6
)
d
9.39 (s,1H), 7.86 (d, J ¼ 1.5 Hz,1H), 7.52
4
.1.2.2. N-Hydroxy-1-methyl-1H-indole-5-carboximidamide
(7b).
(
7
6
d, J ¼ 3.1 Hz,1H), 7.45 (dd, J ¼ 8.7,1.6 Hz,1H), 7.41 (d, J ¼ 8.7 Hz,1H),
ꢀ
ꢀ
1
A white crystalline powder; yield 71.2%; M.p 169.2 Ce171.1 C; H
NMR (400 MHz, DMSO‑d
.30 (dd, J ¼ 8.1, 6.5 Hz, 2H), 7.26e7.21 (m, 1H), 7.21e7.15 (m, 2H),
6
)
d
9.39 (s,1H), 7.85 (d, J ¼ 1.7 Hz,1H), 7.52
.51 (d, J ¼ 3.1 Hz, 1H), 5.70 (s, 2H), 5.42 (s, 2H); ESI-MS m/z 266.09
(
1
dd, J ¼ 8.6, 1.7 Hz, 1H), 7.40 (d, J ¼ 8.6 Hz, 1H), 7.33 (d, J ¼ 3.1 Hz,
þ
[MþH] .
H), 6.44 (dd, J ¼ 3.1, 0.9 Hz, 1H), 5.72 (s, 2H), 3.79 (s, 3H); ESI-MS
þ
m/z 190.08 [MþH] .
4
.1. 2.11. 1-(4-Fluorobenzyl)-N-hydroxy-1H-indole-5-
carboximidamide (7k). A white crystalline powder; yield 92.6%;
ꢀ
ꢀ
1
4
.1.2.3. N-Hydroxy-1-isopropyl-1H-indole-5-carboximidamide (7c).
M.p 184.5 Ce186.4 C; H NMR (400 MHz, DMSO‑d
7.86 (dd, J ¼ 1.6, 0.7 Hz, 1H), 7.52 (d, J ¼ 3.1 Hz, 1H), 7.46 (dd, J ¼ 8.7,
.6 Hz, 1H), 7.44e7.41 (m, 1H), 7.28e7.20 (m, 2H), 7.17e7.09 (m, 2H),
6
) d 9.39 (s, 1H),
ꢀ
ꢀ
1
A white crystalline powder; yield 89.4%; M.p 126.3 Ce129.5 C; H
NMR (400 MHz, DMSO‑d 9.35 (s, 1H), 7.86e7.83 (m, 1H),
.51e7.45 (m, 3H), 6.47 (d, J ¼ 3.2 Hz, 1H), 5.68 (s, 2H), 4.75 (hept,
6
) d
1
7
6.51 (dd, J ¼ 3.2, 0.7 Hz, 1H), 5.70 (s, 2H), 5.41 (s, 2H); ESI-MS m/z
þ
þ
J ¼ 6.6 Hz, 1H), 1.45 (d, J ¼ 6.6 Hz, 6H); ESI-MS m/z 218.10 [MþH] .
2
84.07 [MþH] .
4
.1.2.4. N-Hydroxy-1-isobutyl-1H-indole-5-carboximidamide (7d).
4.1. 2.12. 1-(4-Chlorobenzyl)-N-hydroxy-1H-indole-5-
ꢀ
ꢀ
1
carboximidamide (7l). A white crystalline powder; yield 89.3%; M.p
A white crystalline powder; yield 86.4%; M.p 121.4 Ce122.4 C; H
NMR (400 MHz, DMSO‑d
9.36 (s, 1H), 7.84 (dd, J ¼ 1.6, 0.7 Hz,
ꢀ
ꢀ
1
)
d
170.9 Ce174.4 C; H NMR (400 MHz, DMSO‑d
6
) d 9.40 (s, 1H), 7.87
6
(
d, J ¼ 1.6 Hz, 1H), 7.52 (d, J ¼ 3.2 Hz, 1H), 7.46 (dd, J ¼ 8.6, 1.7 Hz,
1
H), 7.48 (dd, J ¼ 8.7, 1.6 Hz, 1H), 7.44 (dt, J ¼ 8.8, 0.8 Hz,1H), 7.35 (d,
J ¼ 3.1 Hz, 1H), 6.45 (dd, J ¼ 3.1, 0.7 Hz, 1H), 5.69 (s, 2H), 3.97 (d,
1H), 7.41 (s, 1H), 7.36 (d, J ¼ 8.4 Hz, 2H), 7.19 (d, J ¼ 8.5 Hz, 2H), 6.52
(
dd, J ¼ 3.1, 0.7 Hz, 1H), 5.71 (s, 2H), 5.43 (s, 2H); ESI-MS m/z 300.07
J ¼ 7.3 Hz, 2H), 2.20e2.01 (m, 1H), 0.84 (d, J ¼ 6.7 Hz, 6H); ESI-MS
þ
þ
m/z 232.11 [MþH] .
[MþH] .
4
.1. 2.13. 1-(4-Bromobenzyl)-N-hydroxy-1H-indole-5-
4
.1.2.5. N-Hydroxy-1-isopentyl-1H-indole-5-carboximidamide (7e).
ꢀ
ꢀ
1
carboximidamide (7m). A white crystalline powder; yield 89.3%;
M.p 195.5 Ce197.8 C; H NMR (400 MHz, DMSO‑d
A white crystalline powder; yield 91.4%; M.p 139.8 Ce141.2 C; H
NMR (400 MHz, DMSO‑d
ꢀ
ꢀ
1
6
) d 9.42 (s, 1H),
6
)
d
9.37 (s, 1H), 7.84 (d, J ¼ 1.6 Hz, 1H), 7.50
7
.88 (d, J ¼ 1.4 Hz, 1H), 7.54e7.45 (m, 4H), 7.39 (d, J ¼ 8.6 Hz, 1H),
(
1
dd, J ¼ 8.7, 1.7 Hz, 1H), 7.42 (d, J ¼ 8.7 Hz, 1H), 7.39 (d, J ¼ 3.1 Hz,
7.12 (d, J ¼ 8.1 Hz, 2H), 6.53 (d, J ¼ 3.1 Hz, 1H), 5.72 (s, 2H), 5.41 (s,
H), 6.44 (dd, J ¼ 3.1, 0.8 Hz, 1H), 4.18 (t, J ¼ 7.3 Hz, 2H), 1.68e1.60
þ
2
H); ESI-MS m/z 344.05, 346.05 [MþH] .
(
2
m, 2H), 1.55e1.42 (m, 1H), 0.91 (d, J ¼ 6.6 Hz, 6H); ESI-MS m/z
þ
84.5 [MþK] .
4
.1.2.14. N-Hydroxy-1-(4-methylbenzyl)-1H-indole-5-
carboximidamide (7n). A white crystalline powder; yield 89.3%;
4
.1.2.6. N-Hydroxy-1-(3-methylbut-2-en-1-yl)-1H-indole-5-
ꢀ
ꢀ
1
M.p 164.7 Ce166.0 C; H NMR (400 MHz, DMSO‑d
.85 (d, J ¼ 1.5 Hz, 1H), 7.49 (d, J ¼ 3.1 Hz, 1H), 7.45 (dd, J ¼ 8.6,
.6 Hz, 1H), 7.39 (d, J ¼ 8.7 Hz, 1H), 7.09 (s, 4H), 6.49 (dd, J ¼ 3.1,
.7 Hz, 1H), 5.70 (s, 2H), 5.36 (s, 2H), 2.23 (s, 3H); ESI-MS m/z 280.12
6
) d 9.38 (s, 1H),
carboximidamide (7f). A white crystalline powder; yield 79.1%; M.p
7
1
ꢀ ꢀ
1
144.1 Ce145.2 C; H NMR (400 MHz, DMSO‑d ) d 9.36 (s, 1H), 7.85
6
(
0
dd, J ¼ 1.7, 0.6 Hz, 1H), 7.49 (dd, J ¼ 8.6, 1.6 Hz, 1H), 7.37 (dt, J ¼ 8.7,
0
.8 Hz, 1H), 7.33 (d, J ¼ 3.1 Hz, 1H), 6.44 (dd, J ¼ 3.1, 0.8 Hz, 1H), 5.68
þ
[MþH] .
(
3
s, 2H), 5.35e5.30 (m, 1H), 4.79e4.73 (m, 2H), 1.81 (d, J ¼ 1.3 Hz,
þ
H), 1.71 (d, J ¼ 1.3 Hz, 3H); ESI-MS m/z 244.13 [MþH] .
4
.1.2.15. N-Hydroxy-1-(4-methoxybenzyl)-1H-indole-5-
carboximidamide (7o). A white crystalline powder; yield 79.1%; M.p
ꢀ ꢀ
1
4
.1.2.7. 1-(Cyclopropylmethyl)-N-hydroxy-1H-indole-5-
163.9 Ce165.5 C; H NMR (400 MHz, DMSO‑d ) d 9.39 (s, 1H), 7.85
6
carboximidamide (7g). A white crystalline powder; yield 86.7%; M.p
(d, J ¼ 1.5 Hz, 1H), 7.49 (d, J ¼ 3.1 Hz, 1H), 7.48e7.39 (m, 2H), 7.17 (d,
J ¼ 8.6 Hz, 2H), 6.85 (d, J ¼ 8.6 Hz, 2H), 6.48 (d, J ¼ 3.1 Hz, 1H), 5.70
ꢀ
ꢀ
1
1
62.1 Ce163.2 C; H NMR (400 MHz, DMSO‑d
6
) d 9.36 (s, 1H), 7.85
þ
(
t, J ¼ 1.1 Hz, 1H), 7.51e7.45 (m, 2H), 7.44 (d, J ¼ 3.1 Hz, 1H), 6.45 (d,
(s, 2H), 5.33 (s, 2H), 3.69 (s, 3H); ESI-MS m/z 296.11 [MþH] .
J ¼ 3.1 Hz, 1H), 5.69 (s, 2H), 4.04 (d, J ¼ 6.9 Hz, 2H), 1.18e1.28 (m,
1
H), 0.53e0.47 (m, 2H), 0.40e0.34 (m, 2H); ESI-MS m/z 230.12
4.1.2.16. 1-(4-Tert-butylbenzyl)-N-hydroxy-1H-indole-5-
carboximidamide (7p). A white crystalline powder; yield 82.4%;
þ
[MþH] .

J. Gao et al. / European Journal of Medicinal Chemistry 190 (2020) 112077
9
ꢀ
ꢀ
1
M.p 112.4 Ce113.8 C; H NMR (400 MHz, DMSO‑d
7
7
6
)
d
9.39 (s, 1H),
1.4 Hz, 1H), 6.46e6.42 (m, 1H), 5.79 (s, 2H), 4.96e4.82 (m, 1H),
2.23e2.10 (m, 2H), 1.93e1.81 (m, 4H), 1.76e1.69 (m, 2H); ESI-MS m/
z 244.14 [MþH] .
.86 (d, J ¼ 1.5 Hz, 1H), 7.51 (d, J ¼ 3.1 Hz, 1H), 7.49e7.38 (m, 2H),
þ
.31 (d, J ¼ 8.3 Hz, 2H), 7.12 (d, J ¼ 8.0 Hz, 2H), 6.50 (d, J ¼ 3.1 Hz,
1
H), 5.70 (s, 2H), 5.37 (s, 2H), 1.22 (s, 9H); ESI-MS m/z 322.19
þ
[MþH] .
4.1.2.25. N-Hydroxy-1-(4-methylbenzyl)-1H-indole-6-
carboximidamide (8n). A white crystalline powder; yield 93.1%;
ꢀ ꢀ
1
4
.1. 2.17. 1-(3-Fluorobenzyl)-N-hydroxy-1H-indole-5-
M.p 176.1 Ce177.0 C; H NMR (400 MHz, DMSO‑d ) d 9.47 (s, 1H),
6
carboximidamide (7q). A white crystalline powder; yield 95.6%;
7.77 (s, 1H), 7.54e7.48 (m, 2H), 7.39 (dd, J ¼ 8.4, 1.4 Hz, 1H), 7.10 (s,
4H), 6.47 (d, J ¼ 3.1 Hz, 1H), 5.74 (s, 2H), 5.37 (s, 2H), 2.23 (s, 3H);
ꢀ
ꢀ
1
6
M.p 188.7 Ce190.8 C; H NMR (400 MHz, DMSO‑d ) d 9.40 (s, 1H),
þ
7
1
.87 (d, J ¼ 1.5 Hz,1H), 7.54 (d, J ¼ 3.1 Hz,1H), 7.47 (dd, J ¼ 8.7,1.6 Hz,
ESI-MS m/z 280.12 [MþH] .
H), 7.42 (d, J ¼ 8.7 Hz, 1H), 7.34 (td, J ¼ 8.1, 6.1 Hz, 1H), 7.07 (td,
J ¼ 8.5, 2.4 Hz, 1H), 7.02e6.97 (m, 2H), 6.53 (d, J ¼ 3.1 Hz, 1H), 5.71
4
5
.1.3. General procedure for the preparation of 3-(1-alkyl-1H-indol-
/6-yl)-1,2,4-oxadiazol-5(4H)-ones (9a-9u, 10c, 10e, 10h and 10n)
N-Hydroxy-1-alkyl-1H-indole-5/6-carboximidamide (7a-7u, 8c,
þ
(
s, 2H), 5.45 (s, 2H); ESI-MS m/z 284.07 [MþH] .
4
.1. 2.18. 1-(3-Chlorobenzyl)-N-hydroxy-1H-indole-5-
carboximidamide (7r). A white crystalline powder; yield 93.1%; M.p
8
e, 8h and 8n, 50 mmol) and DBU (100 mmol) were dissolved in
ꢀ
dichloromethane (50 mL) at 30 C. Thereafter, CDI (55 mmol) was
added to the reaction mixture, which was stirred at 30 C for 2 h.
ꢀ
ꢀ
1
1
81.3 Ce183.1 C; H NMR (400 MHz, DMSO‑d
d, J ¼ 1.5 Hz,1H), 7.55 (d, J ¼ 3.1 Hz,1H), 7.47 (dd, J ¼ 8.6,1.6 Hz,1H),
.43 (d, J ¼ 8.7 Hz, 1H), 7.36e7.30 (m, 2H), 7.24 (d, J ¼ 2.0 Hz, 1H),
.13 (dt, J ¼ 6.8, 1.9 Hz, 1H), 6.54 (d, J ¼ 3.1 Hz, 1H), 5.72 (s, 2H), 5.45
6
)
d
9.41 (s, 1H), 7.88
ꢀ
(
7
After the completion of the reaction, the solvent was evaporated in
vacuo. Ice water was added to the residue, and the pH was brought
to 4 with 2 M hydrochloric acid. The resulting precipitate was
filtered, dried, washed with water and recrystallized with anhy-
drous methanol to obtain the corresponding title compound.
7
(
þ
s, 2H); ESI-MS m/z 300.06 [MþH] .
4
.1. 2.19. 1-(3-Bromobenzyl)-N-hydroxy-1H-indole-5-
carboximidamide (7s). A white crystalline powder; yield 87.9%; M.p
4
.1.3.1. 3-(1H-Indol-5-yl)-1,2,4-oxadiazol-5(4H)-one (9a). A white
ꢀ
ꢀ
1
186.6 Ce188.2 C; H NMR (400 MHz, DMSO‑d
6
)
d
9.42 (s, 1H), 7.88
ꢀ
1
amorphous powder; yield 80.3%; M.p > 250 C; H NMR (400 MHz,
DMSO‑d 12.78 (s, 1H), 11.53 (s, 1H), 8.06 (s, 1H), 7.55 (d,
(
d, J ¼ 1.5 Hz, 1H), 7.55 (d, J ¼ 3.1 Hz, 1H), 7.50e7.41 (m, 3H), 7.39 (d,
6
) d
J ¼ 1.7 Hz, 1H), 7.26 (t, J ¼ 7.8 Hz, 1H), 7.17 (d, J ¼ 7.7 Hz, 1H), 6.54 (d,
J ¼ 1.4 Hz, 2H), 7.53e7.48 (m, 1H), 6.59 (dd, J ¼ 3.1, 1.9 Hz, 1H); ESI-
J ¼ 3.1 Hz, 1H), 5.73 (s, 2H), 5.44 (s, 2H); ESI-MS m/z 344.05, 346.04
-
MS m/z 200.03 [M ꢁ H] .
þ
[MþH] .
4
.1.3.2. 3-(1-Methyl-1H-indol-5-yl)-1,2,4-oxadiazol-5(4H)-one (9b).
4
.1.2.20. N-Hydroxy-1-(3-methylbenzyl)-1H-indole-5-
ꢀ
ꢀ
A white amorphous powder; yield 86.3%; M.p 223.8 Ce225.6 C;
carboximidamide (7t). A white crystalline powder; yield 91.3%; M.p
1
H NMR (400 MHz, DMSO‑d
6
)
d
12.72 (s, 1H), 8.05 (t, J ¼ 1.1 Hz, 1H),
ꢀ
ꢀ
1
199.5 Ce202.1 C; H NMR (400 MHz, DMSO‑d
6
) d 9.40 (s, 1H), 7.86
7
3
.66e7.57 (m, 2H), 7.47 (d, J ¼ 3.1 Hz, 1H), 6.58 (d, J ¼ 3.1 Hz, 1H),
(
d, J ¼ 1.5 Hz,1H), 7.50 (d, J ¼ 3.1 Hz,1H), 7.46 (dd, J ¼ 8.7,1.6 Hz,1H),
þ
.84 (s, 3H); ESI-MS m/z 216.08 [MþH] .
7
6
2
.40 (d, J ¼ 8.7 Hz, 1H), 7.17 (t, J ¼ 7.5 Hz, 1H), 7.09e7.00 (m, 2H),
.96 (d, J ¼ 7.6 Hz, 1H), 6.51 (d, J ¼ 3.1 Hz, 1H), 5.71 (s, 2H), 5.37 (s,
þ
4.1.3.3. 3-(1-Isopropyl-1H-indol-5-yl)-1,2,4-oxadiazol-5(4H)-one
9c). A white amorphous powder; yield 76.2%; M.p
H), 2.23 (s, 3H); ESI-MS m/z 280.11 [MþH] .
(
ꢀ
ꢀ
1
198.8 Ce201.0 C; H NMR (400 MHz, DMSO‑d
6
)
d
12.75 (s, 1H),
4
.1.2.21. N-Hydroxy-1-(3-methoxybenzyl)-1H-indole-5-
8
.05 (d, J ¼ 1.7 Hz, 1H), 7.70 (d, J ¼ 8.7 Hz, 1H), 7.64 (d, J ¼ 3.3 Hz,
carboximidamide (7u). A white crystalline powder; yield 90.0%;
M.p 147.6 Ce150.2 C; H NMR (400 MHz, DMSO‑d ) d 9.40 (s, 1H),
6
ꢀ
ꢀ
1
1H), 7.57 (dd, J ¼ 8.7, 1.8 Hz, 1H), 6.62 (d, J ¼ 3.3 Hz, 1H), 4.82 (hept,
-
J ¼ 6.7 Hz, 1H), 1.47 (d, J ¼ 6.7 Hz, 6H); ESI-MS m/z 242.08 [M ꢁ H] .
7.90e7.81 (m, 1H), 7.51 (d, J ¼ 3.1 Hz, 1H), 7.47 (dd, J ¼ 8.7, 1.6 Hz,
1
2
H), 7.41 (d, J ¼ 8.7 Hz, 1H), 7.20 (t, J ¼ 7.9 Hz, 1H), 6.80 (dd, J ¼ 8.2,
.6 Hz, 1H), 6.77 (d, J ¼ 1.8 Hz, 1H), 6.72 (dd, J ¼ 7.6, 1.5 Hz, 1H), 6.51
4
.1.3.4. 3-(1-Isobutyl-1H-indol-5-yl)-1,2,4-oxadiazol-5(4H)-one
(9d). A white amorphous powder; yield 93.2%; M.p
(
2
d, J ¼ 3.1 Hz, 1H), 5.71 (s, 2H), 5.39 (s, 2H), 3.68 (s, 3H); ESI-MS m/z
ꢀ
ꢀ
1
þ
200.1 Ce201.6 C; H NMR (400 MHz, DMSO‑d )
8
6
d
12.79 (s, 1H),
96.11 [MþH] .
.06 (d, J ¼ 1.6 Hz, 1H), 7.69 (d, J ¼ 8.7 Hz, 1H), 7.58 (dd, J ¼ 8.7,
1
.7 Hz, 1H), 7.51 (d, J ¼ 3.1 Hz, 1H), 6.60 (dd, J ¼ 3.1, 0.8 Hz, 1H), 4.04
4
.1.2.22. N-Hydroxy-1-isopropyl-1H-indole-6-carboximidamide (8c).
1
(d, J ¼ 7.3 Hz, 2H), 2.22e2.05 (m, 1H), 0.85 (d, J ¼ 6.7 Hz, 6H); ESI-
A yellow oil; yield 92.3%; H NMR (400 MHz, DMSO‑d
1
1
6
)
d
9.47 (s,
-
MS m/z 256.10 [M ꢁ H] .
H), 7.82 (d, J ¼ 1.4 Hz, 1H), 7.51 (d, J ¼ 3.2 Hz, 1H), 7.51e7.46 (m,
H), 7.40 (dd, J ¼ 8.3, 1.4 Hz, 1H), 6.45 (dd, J ¼ 3.2, 0.7 Hz, 1H), 5.80
(
s, 2H), 4.77 (hept, J ¼ 6.7 Hz, 1H), 1.47 (d, J ¼ 6.7 Hz, 6H); ESI-MS m/
4.1.3.5. 3-(1-Isopentyl-1H-indol-5-yl)-1,2,4-oxadiazol-5(4H)-one
(9e). A white amorphous powder; yield 89.2%; M.p
þ
z 218.10 [MþH] .
ꢀ ꢀ
1
180.8 Ce182.1 C; H NMR (400 MHz, DMSO‑d ) d 12.79 (s, 1H),
6
4
.1.2.23. N-Hydroxy-1-isopentyl-1H-indole-6-carboximidamide (8e).
8.06 (s, 1H), 7.66 (d, J ¼ 8.7 Hz, 1H), 7.59 (dd, J ¼ 8.7, 1.7 Hz, 1H), 7.54
(d, J ¼ 3.2 Hz,1H), 6.60 (d, J ¼ 3.1 Hz,1H), 4.24 (t, J ¼ 7.4 Hz, 2H),1.66
(q, J ¼ 7.2 Hz, 2H), 1.49 (hept, J ¼ 6.7 Hz, 1H), 0.92 (d, J ¼ 6.7 Hz, 6H);
ꢀ
ꢀ
1
A white crystalline powder; yield 83.1%; M.p 108.1 Ce109.8 C; H
NMR (400 MHz, DMSO‑d 9.48 (s, 1H), 7.76 (s, 1H), 7.49 (d,
6
) d
-
J ¼ 8.3 Hz, 1H), 7.43e7.36 (m, 2H), 6.41 (d, J ¼ 3.0 Hz, 1H), 5.80 (s,
ESI-MS m/z 270.4 [M ꢁ H] .
2
0
H), 4.19 (t, J ¼ 7.3 Hz, 2H),1.67 (q, J ¼ 7.1 Hz, 2H), 1.56e1.46 (m, 1H),
þ
.93 (d, J ¼ 6.6 Hz, 6H); ESI-MS m/z 246.14 [MþH] .
4.1.3.6. 3-[1-(3-Methylbut-2-en-1-yl)-1H-indol-5-yl]-1,2,4-
oxadiazol-5(4H)-one (9f). A white amorphous powder; yield 79.3%;
ꢀ ꢀ
1
4
.1.2.24. 1-Cyclopentyl-N-hydroxy-1H-indole-6-carboximidamide
M.p 173.2 Ce174.5 C; H NMR (400 MHz, DMSO‑d ) d 12.77 (s, 1H),
6
(
1
(
8h). A white
crystalline
powder;
22.3 Ce123.3 C; H NMR (400 MHz, DMSO‑d
d, J ¼ 1.3 Hz, 1H), 7.48 (dd, J ¼ 5.7, 2.6 Hz, 2H), 7.40 (dd, J ¼ 8.3,
yield
87.3%;
M.p
8.05 (s, 1H), 7.64e7.56 (m, 2H), 7.48 (d, J ¼ 3.2 Hz, 1H), 6.59 (d,
J ¼ 3.2 Hz, 1H), 5.40e5.30 (m, 1H), 4.82 (d, J ¼ 7.0 Hz, 2H), 1.82 (s,
ꢀ
ꢀ
1
6
) d 9.46 (s, 1H), 7.82
-
3H), 1.72 (s, 3H); ESI-MS m/z 268.10 [M ꢁ H] .

10
J. Gao et al. / European Journal of Medicinal Chemistry 190 (2020) 112077
-
4
5
.1.3.7. 3-[1-(Cyclopropylmethyl)-1H-indol-5-yl]-1,2,4-oxadiazol-
3H); ESI-MS m/z 320.15 [M ꢁ H] .
(4H)-one (9g). A white amorphous powder; yield 85.6%; M.p
ꢀ
ꢀ
1
214.0 Ce215.9 C; H NMR (400 MHz, DMSO‑d
6
)
d
12.77 (s, 1H),
4.1.3.16. 3-[1-(4-Tert-butylbenzyl)-1H-indol-5-yl]-1,2,4-oxadiazol-
8
.05 (d, J ¼ 1.6 Hz, 1H), 7.71 (d, J ¼ 8.6 Hz, 1H), 7.58 (dd, J ¼ 8.6,
5(4H)-one (9p). A white amorphous powder; yield 93.7%; M.p
ꢀ ꢀ
1
1
.9 Hz, 2H), 6.60 (d, J ¼ 3.1 Hz, 1H), 4.10 (d, J ¼ 7.0 Hz, 2H), 1.30e1.20
235.4 Ce237.0 C; H NMR (400 MHz, DMSO‑d ) d 12.79 (s, 1H),
6
(
[
m, 1H), 0.53e0.47 (m, 2H), 0.41e0.37 (m, 2H); ESI-MS m/z 254.09
M ꢁ H] .
8.06 (d, J ¼ 1.6 Hz, 1H), 7.68 (dt, J ¼ 8.7, 0.8 Hz, 1H), 7.65 (d,
J ¼ 3.1 Hz, 1H), 7.56 (dd, J ¼ 8.7, 1.7 Hz, 1H), 7.32 (d, J ¼ 8.4 Hz, 2H),
-
7
.14 (d, J ¼ 8.3 Hz, 2H), 6.64 (dd, J ¼ 3.2, 0.8 Hz,1H), 5.44 (s, 2H), 1.22
-
4
.1.3.8. 3-(1-Cyclopentyl-1H-indol-5-yl)-1,2,4-oxadiazol-5(4H)-one
(s, 9H); ESI-MS m/z 346.20 [M ꢁ H] .
(9h). A white amorphous powder; yield 86.8%; M.p
ꢀ
ꢀ
1
192.6 Ce194.1 C; H NMR (400 MHz, DMSO‑d
6
)
d
12.76 (s, 1H),
4.1.3.17. 3-[1-(3-Fluorobenzyl)-1H-indol-5-yl]-1,2,4-oxadiazol-
8
.05 (d, J ¼ 1.6 Hz,1H), 7.71 (d, J ¼ 8.7 Hz,1H), 7.61 (d, J ¼ 3.3 Hz,1H),
5(4H)-one (9q). A white amorphous powder; yield 86.3%; M.p
ꢀ ꢀ
1
7
1
.58 (dd, J ¼ 8.7, 1.7 Hz, 1H), 6.62 (d, J ¼ 3.2 Hz, 1H), 5.01e4.89 (m,
232.2 Ce234.9 C; H NMR (400 MHz, DMSO‑d ) d 12.77 (s, 1H),
6
H), 2.22e2.12 (m, 2H), 1.93e1.78 (m, 4H), 1.78e1.68 (m, 2H); ESI-
8.08 (d, J ¼ 1.7 Hz, 1H), 7.72e7.63 (m, 2H), 7.57 (dd, J ¼ 8.7, 1.7 Hz,
1H), 7.36 (td, J ¼ 8.0, 6.0 Hz, 1H), 7.15e6.99 (m, 3H), 6.68 (d,
-
MS m/z 268.10 [M ꢁ H] .
-
J ¼ 3.2 Hz, 1H), 5.52 (s, 2H); ESI-MS m/z 308.11 [M ꢁ H] .
4
5
.1.3.9. 3-[1-(Cyclohexylmethyl)-1H-indol-5-yl]-1,2,4-oxadiazol-
(4H)-one (9i). A white amorphous powder; yield 89.7%; M.p
4.1.3.18. 3-[1-(3-Chlorobenzyl)-1H-indol-5-yl]-1,2,4-oxadiazol-
ꢀ
ꢀ
1
2
(
1
13.0 Ce217.2 C; H NMR (400 MHz, DMSO‑d
d, J ¼ 1.6 Hz, 1H), 7.67 (d, J ¼ 8.7 Hz, 1H), 7.58 (dd, J ¼ 8.7, 1.7 Hz,
H), 7.48 (d, J ¼ 3.1 Hz, 1H), 6.59 (d, J ¼ 3.1 Hz, 1H), 4.06 (d,
J ¼ 7.2 Hz, 2H), 1.80 (ddh, J ¼ 11.0, 7.3, 3.6 Hz, 1H), 1.71e1.54 (m, 3H),
6
)
d
12.76 (s,1H), 8.05
5(4H)-one (9r). A white amorphous powder; yield 79.6%; M.p
ꢀ ꢀ
1
247.0 Ce249.3 C; H NMR (400 MHz, DMSO‑d ) d 12.77 (s, 1H),
6
8.08 (d, J ¼ 1.6 Hz, 1H), 7.72e7.64 (m, 2H), 7.57 (dd, J ¼ 8.7, 1.7 Hz,
1H), 7.39e7.25 (m, 3H), 7.15 (dt, J ¼ 6.8, 1.9 Hz, 1H), 6.68 (dd, J ¼ 3.2,
-
1
.53e1.43 (m, 2H), 1.18e1.07 (m, 3H), 0.98 (qd, J ¼ 11.7, 3.4 Hz, 2H);
0.8 Hz, 1H), 5.51 (s, 2H); ESI-MS m/z 324.10 [M ꢁ H] .
-
ESI-MS m/z 296.16 [M ꢁ H] .
4
.1.3.19. 3-[1-(3-Bromobenzyl)-1H-indol-5-yl]-1,2,4-oxadiazol-
4
.1.3.10. 3-(1-Benzyl-1H-indol-5-yl)-1,2,4-oxadiazol-5(4H)-one (9j).
5(4H)-one (9s). A white amorphous powder; yield 87.4%; M.p
ꢀ
ꢀ
1
ꢀ ꢀ
1
A white amorphous powder; yield 88.7%; M.p 235.8 Ce237.2 C; H
NMR (400 MHz, DMSO‑d
248.5 Ce250.2 C; H NMR (400 MHz, DMSO‑d ) d 12.77 (s, 1H),
6
6
)
d
12.76 (s, 1H), 8.06 (d, J ¼ 1.6 Hz, 1H),
8.08 (d, J ¼ 1.6 Hz, 1H), 7.71e7.65 (m, 2H), 7.57 (dd, J ¼ 8.7, 1.7 Hz,
1H), 7.48e7.41 (m, 2H), 7.28 (t, J ¼ 7.8 Hz, 1H), 7.19 (dt, J ¼ 7.7, 1.3 Hz,
7
6
.70e7.60 (m, 2H), 7.55 (dd, J ¼ 8.7, 1.7 Hz, 1H), 7.36e7.17 (m, 5H),
-
.66 (d, J ¼ 3.1 Hz, 1H), 5.49 (s, 2H); ESI-MS m/z 290.12 [M ꢁ H] .
1H), 6.68 (dd, J ¼ 3.2, 0.8 Hz, 1H), 5.51 (s, 2H); ESI-MS m/z 368.06,
-
3
70.06 [M ꢁ H] .
4
5
.1.3.11. 3-[1-(4-Fluorobenzyl)-1H-indol-5-yl]-1,2,4-oxadiazol-
(4H)-one (9k). A white amorphous powder; yield 87.4%;
4.1.3.20. 3-[1-(3-Methylbenzyl)-1H-indol-5-yl]-1,2,4-oxadiazol-
ꢀ
1
M.p > 250 C; H NMR (400 MHz, DMSO‑d
6
)
d
12.77 (s, 1H), 8.07 (d,
5(4H)-one (9t). A white amorphous powder; yield 87.8%; M.p
ꢀ
ꢀ
1
J ¼ 1.6 Hz, 1H), 7.71e7.65 (m, 2H), 7.57 (dd, J ¼ 8.7, 1.7 Hz, 1H),
6
225.2 Ce227.9 C; H NMR (400 MHz, DMSO‑d ) d 12.64 (s, 1H),
7
5
.31e7.25 (m, 2H), 7.18e7.11 (m, 2H), 6.66 (dd, J ¼ 3.2, 0.8 Hz, 1H),
8.07 (d, J ¼ 1.6 Hz, 1H), 7.67e7.62 (m, 2H), 7.55 (dd, J ¼ 8.7, 1.6 Hz,
-
.48 (s, 2H); ESI-MS m/z 308.11 [M ꢁ H] .
1H), 7.19 (t, J ¼ 7.9 Hz, 1H), 7.07 (d, J ¼ 7.1 Hz, 2H), 6.98 (d, J ¼ 7.6 Hz,
1
H), 6.65 (d, J ¼ 3.1 Hz, 1H), 5.44 (s, 2H), 2.24 (s, 3H); ESI-MS m/z
-
4
5
2
1
.1.3.12. 3-[1-(4-Chlorobenzyl)-1H-indol-5-yl]-1,2,4-oxadiazol-
304.12 [M ꢁ H] .
(4H)-one (9l). A white amorphous powder; yield 78.7%; M.p
ꢀ
ꢀ
1
04.4 Ce207.6 C; H NMR (400 MHz, DMSO‑d
6
)
d
8.07e8.01 (m,
4.1.3.21. 3-[1-(3-Methoxybenzyl)-1H-indol-5-yl]-1,2,4-oxadiazol-
H), 7.64e7.57 (m, 2H), 7.53 (dd, J ¼ 8.6, 1.7 Hz, 1H), 7.48 (d,
5(4H)-one (9u). A white amorphous powder; yield 78.8%; M.p
ꢀ
ꢀ
1
J ¼ 8.5 Hz, 2H), 7.12 (d, J ¼ 8.5 Hz, 2H), 6.62 (dd, J ¼ 3.2, 0.8 Hz, 1H),
204.6 Ce206.1 C; H NMR (400 MHz, DMSO‑d
6
) d 12.81 (s, 1H),
-
5
.44 (s, 2H); ESI-MS m/z 324.11 [M ꢁ H] .
8.07 (dd, J ¼ 1.8, 0.7 Hz, 1H), 7.68e7.64 (m, 2H), 7.56 (dd, J ¼ 8.7,
1
.7 Hz, 1H), 7.22 (t, J ¼ 7.9 Hz, 1H), 6.85e6.78 (m, 2H), 6.75e6.73 (m,
4
5
2
1
.1.3.13. 3-[1-(4-Bromobenzyl)-1H-indol-5-yl]-1,2,4-oxadiazol-
1H), 6.65 (dd, J ¼ 3.2, 0.8 Hz, 1H), 5.45 (s, 2H), 3.70 (s, 3H); ESI-MS
-
(4H)-one (9m). A white amorphous powder; yield 79.7%; M.p
m/z 320.16 [M ꢁ H] .
ꢀ
ꢀ
1
47.8 Ce249.0 C; H NMR (400 MHz, DMSO‑d
6
) d 8.09e8.06 (m,
H), 7.66e7.61 (m, 2H), 7.56 (dd, J ¼ 8.6, 1.7 Hz, 1H), 7.51 (d,
4.1.3.22. 3-(1-Isopropyl-1H-indol-6-yl)-1,2,4-oxadiazol-5(4H)-one
J ¼ 8.4 Hz, 2H), 7.15 (d, J ¼ 8.4 Hz, 2H), 6.66 (dd, J ¼ 3.2, 0.8 Hz, 1H),
(10c). A white amorphous powder; yield 87.2%; M.p
-
191.0 Ce194.1 C; 1H NMR (600 MHz, DMSO‑d
ꢀ ꢀ
5
.47 (s, 2H); ESI-MS m/z 368.07, 370.06 [M ꢁ H] .
6
) d 12.81 (s, 1H),
8
.03 (s, 1H), 7.73e7.69 (m, 2H), 7.48 (dd, J ¼ 8.2, 1.5 Hz, 1H), 6.58 (d,
4
5
2
8
.1.3.14. 3-[1-(4-Methylbenzyl)-1H-indol-5-yl]-1,2,4-oxadiazol-
J ¼ 3.2 Hz, 1H), 4.78 (hept, J ¼ 6.7 Hz, 1H), 1.51 (d, J ¼ 6.7 Hz, 6H);
-
(4H)-one (9n). A white amorphous powder; yield 89.7%; M.p
ESI-MS m/z 242.09 [M ꢁ H] .
ꢀ
ꢀ
1
20.1 Ce222.5 C; H NMR (400 MHz, DMSO‑d
6
) d 12.76 (s, 1H),
.08e8.05 (m, 1H), 7.67e7.62 (m, 2H), 7.55 (dd, J ¼ 8.7, 1.7 Hz, 1H),
4.1.3.23. 3-(1-Isopentyl-1H-indol-6-yl)-1,2,4-oxadiazol-5(4H)-one
7
.11 (s, 4H), 6.64 (dd, J ¼ 3.2, 0.8 Hz, 1H), 5.42 (s, 2H), 2.24 (s, 3H);
(10e). A white amorphous powder; yield 94.2%; M.p
-
ꢀ ꢀ
1
ESI-MS m/z 304.13 [M ꢁ H] .
179.1 Ce183.0 C; H NMR (600 MHz, DMSO‑d ) d 12.86 (s, 1H),
6
7
.96 (s, 1H), 7.70 (d, J ¼ 8.3 Hz, 1H), 7.61 (d, J ¼ 3.1 Hz, 1H), 7.47 (dd,
4
5
2
8
1
.1.3.15. 3-[1-(4-Methoxybenzyl)-1H-indol-5-yl]-1,2,4-oxadiazol-
J ¼ 8.2, 1.5 Hz, 1H), 6.54 (d, J ¼ 3.1 Hz, 1H), 4.23 (t, J ¼ 7.5 Hz, 2H),
(4H)-one (9o). A white amorphous powder; yield 90.4%; M.p
1.69 (q, J ¼ 7.2 Hz, 2H), 1.55e1.48 (m, 1H), 0.93 (d, J ¼ 6.7 Hz, 6H);
ꢀ
ꢀ
1
-
27.9 Ce229.3 C; H NMR (400 MHz, DMSO‑d
6
)
d
12.76 (s, 1H),
ESI-MS m/z 270.13 [M ꢁ H] .
.07e8.04 (m, 1H), 7.67 (dt, J ¼ 8.7, 0.8 Hz, 1H), 7.63 (d, J ¼ 3.1 Hz,
H), 7.55 (dd, J ¼ 8.7, 1.7 Hz, 1H), 7.20 (d, J ¼ 8.6 Hz, 2H), 6.87 (d,
4.1.3.24. 3-(1-Cyclopentyl-1H-indol-6-yl)-1,2,4-oxadiazol-5(4H)-one
(10h). A white amorphous powder; yield 79.8%; M.p
J ¼ 8.7 Hz, 2H), 6.63 (dd, J ¼ 3.2, 0.8 Hz, 1H), 5.39 (s, 2H), 3.70 (s,

J. Gao et al. / European Journal of Medicinal Chemistry 190 (2020) 112077
11
ꢀ
ꢀ
1
ꢀ
ꢀ
1
1
78.4 Ce180.2 C; H NMR (600 MHz, DMSO‑d
6
)
d
12.81 (s, 1H),
amorphous powder; yield 87.3%; M.p 228.1 Ce230.0 C; H NMR
(400 MHz, DMSO‑d
8
1
2
.02 (d, J ¼ 1.3 Hz, 1H), 7.72e7.67 (m, 2H), 7.47 (dd, J ¼ 8.2, 1.5 Hz,
6
)
d
12.91 (s, 1H), 9.96 (s, 1H), 8.64 (d, J ¼ 1.3 Hz,
H), 6.57 (d, J ¼ 3.2 Hz, 1H), 4.88 (p, J ¼ 7.2 Hz, 1H), 2.23e2.16 (m,
1H), 8.45 (s, 1H), 7.77 (d, J ¼ 1.2 Hz, 2H), 5.42 (ddt, J ¼ 8.5, 7.0, 1.4 Hz,
H), 1.92e1.83 (m, 4H), 1.78e1.69 (m, 2H); ESI-MS m/z 268.08
1H), 4.94 (d, J ¼ 7.1 Hz, 2H),1.85 (d, J ¼ 1.3 Hz, 3H),1.75 (d, J ¼ 1.4 Hz,
-
-
[
M ꢁ H] .
3H); ESI-MS m/z 296.13 [M ꢁ H] .
4
5
.1.3.25. 3-[1-(4-Methylbenzyl)-1H-indol-6-yl]-1,2,4-oxadiazol-
4.1.4.7. 1-(Cyclopropylmethyl)-5-(5-oxo-4,5-dihydro-1,2,4-
(4H)-one (10n). A white amorphous powder; yield 84.7%; M.p
oxadiazol-3-yl)-1H-indole-3-carbaldehyde (11g). A purple amor-
ꢀ
ꢀ
1
ꢀ
1
213.2 Ce214.8 C; H NMR (600 MHz, DMSO‑d
6
)
d
12.84 (s, 1H),
phous powder; yield 85.5%; M.p > 250 C; H NMR (400 MHz,
DMSO‑d6)
7
1
.98 (d, J ¼ 1.3 Hz, 1H), 7.75e7.71 (m, 2H), 7.47 (dd, J ¼ 8.3, 1.5 Hz,
d
9.97 (s, 1H), 8.64 (d, J ¼ 1.6 Hz, 1H), 8.54 (s, 1H), 7.86 (d,
H), 7.16e7.10 (m, 4H), 6.60 (d, J ¼ 3.0 Hz, 1H), 5.42 (s, 2H), 2.24 (s,
J ¼ 8.6 Hz, 1H), 7.76 (dd, J ¼ 8.7, 1.7 Hz, 1H), 4.20 (d, J ¼ 7.2 Hz, 2H),
1.40e1.29 (m, 1H), 0.64e0.54 (m, 2H), 0.50e0.43 (m, 2H); ESI-MS
m/z 282.10 [M ꢁ H] .
-
3
H); ESI-MS m/z 304.13 [M ꢁ H] .
-
4
4
.1.4. General procedure for the preparation of 1-alkyl-5/6-(5-oxo-
,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-indole-3-carbaldehydes (11a-
4
1
.1.4.8. 1-Cyclopentyl-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-
11u, 12c, 12e, 12h and 12n)
H-indole-3-carbaldehyde (11h). A purple amorphous powder;
A 250 mL three-necked round flask was supplemented with 3-
ꢀ
ꢀ
1
yield 80.7%; M.p 209.8 Ce212.5 C; H NMR (400 MHz, DMSO‑d
12.98 (s, 1H), 9.96 (s, 1H), 8.64 (d, J ¼ 1.7 Hz, 1H), 8.60 (s, 1H), 7.89
d, J ¼ 8.7 Hz, 1H), 7.76 (dd, J ¼ 8.7, 1.8 Hz, 1H), 5.03 (p, J ¼ 7.0 Hz,
H), 2.33e2.19 (m, 2H), 2.01e1.82 (m, 4H), 1.82e1.68 (m, 2H); ESI-
6
)
(
1-alkyl-1H-indol-5/6-yl)-1,2,4-oxadiazol-5(4H)-one (9a-9u, 10c,
d
(
1
ꢀ
10e, 10h and 10n, 40 mmol) and anhydrous DMF (50 mL) at 0 C.
3
Phosphorus oxychloride (POCl , 17 mL) was then added dropwise
via an addition funnel maintaining the temperature below 10 C.
The resulting mixture was stirred for 40 min at 0 C and then room
ꢀ
-
MS m/z 296.12 [M ꢁ H] .
ꢀ
temperature for an additional 1 h. The reaction was quenched with
chilled water, followed by the addition of aqueous sodium hy-
droxide solution. The precipitate was filtered and washed with
water. The filter cake was dried in vacuo in the presence of phos-
phorus pentoxide to yield the title compound, which was analyti-
cally pure and used without further purification.
4
3
.1.4.9. 1-(Cyclohexylmethyl)-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
-yl)-1H-indole-3-carbaldehyde (11i). A purple amorphous pow-
ꢀ
1
6
der; yield 80.7%; M.p > 250 C; H NMR (400 MHz, DMSO‑d ) d 9.97
(
7
1
s, 1H), 8.65 (d, J ¼ 1.6 Hz, 1H), 8.47 (s, 1H), 7.88 (d, J ¼ 8.7 Hz, 1H),
.79 (dd, J ¼ 8.7, 1.8 Hz, 1H), 4.20 (d, J ¼ 7.3 Hz, 2H), 1.92e1.80 (m,
H), 1.73e1.47 (m, 5H), 1.24e0.96 (m, 5H); ESI-MS m/z 324.19
-
[M ꢁ H] .
4
.1.4.1. 5-(5-Oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-indole-3-
carbaldehyde (11a). A white amorphous powder; yield 91.3%;
M.p > 250 C; H NMR (400 MHz, DMSO‑d ) d 12.72 (s, 1H), 10.00 (s,
6
4
.1.4.10. 1-Benzyl-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-
ꢀ
1
indole-3-carbaldehyde (11j). A yellow amorphous powder; yield
8
1
2
H), 8.92 (s, 1H), 8.64 (d, J ¼ 1.5 Hz, 1H), 8.47 (s, 1H), 7.78e7.69 (m,
ꢀ
1
1.6%; M.p > 250 C; H NMR (400 MHz, DMSO‑d ) d 12.31 (s, 1H),
6
-
H); ESI-MS m/z 228.04 [M ꢁ H] .
1
0
2
0.00 (s, 1H), 8.65 (d, J ¼ 1.6 Hz, 1H), 8.63 (s, 1H), 7.80 (dd, J ¼ 8.8,
.7 Hz, 1H), 7.75 (dd, J ¼ 8.7, 1.7 Hz, 1H), 7.41e7.25 (m, 5H), 5.61 (s,
4
.1.4.2. 1-Methyl-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-
-
H); ESI-MS m/z 318.14 [M ꢁ H] .
indole-3-carbaldehyde (11b). A purple amorphous powder; yield
ꢀ
1
7
9
2
2.3%; M.p > 250 C; H NMR (400 MHz, DMSO‑d ) d 12.62 (s, 1H),
6
4.1.4.11. 1-(4-Fluorobenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-
.95 (s, 1H), 8.63 (t, J ¼ 1.2 Hz, 1H), 8.44 (s, 1H), 7.79 (d, J ¼ 1.2 Hz,
-
yl)-1H-indole-3-carbaldehyde (11k). A white amorphous powder;
yield 80.4%; M.p > 250 C; H NMR (400 MHz, DMSO‑d
H), 3.95 (s, 3H); ESI-MS m/z 242.06 [M ꢁ H] .
ꢀ
1
6
) d 10.01 (s,
1
H), 8.67 (d, J ¼ 2.3 Hz, 2H), 7.85 (d, J ¼ 8.7 Hz, 1H), 7.78 (dd, J ¼ 8.7,
4
.1.4.3. 1-Isopropyl-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-
1.8 Hz, 1H), 7.43 (dd, J ¼ 8.5, 5.6 Hz, 2H), 7.20 (t, J ¼ 8.8 Hz, 2H), 5.62
indole-3-carbaldehyde (11c). A white amorphous powder; yield
7
-
ꢀ ꢀ
1
(s, 2H); ESI-MS m/z 336.11 [M ꢁ H] .
9.2%; M.p 220.7 Ce222.4 C; H NMR (400 MHz, DMSO‑d ) d 13.02
6
(
(
6
s, 1H), 9.98 (s, 1H), 8.66e8.65 (m, 2H), 7.90 (d, J ¼ 8.7 Hz, 1H), 7.78
4
.1.4.12. 1-(4-Chlorobenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
dd, J ¼ 8.7, 1.8 Hz, 1H), 4.93 (hept, J ¼ 6.6 Hz, 1H), 1.55 (d, J ¼ 6.6 Hz,
-
3-yl)-1H-indole-3-carbaldehyde (11l). A yellow amorphous pow-
der; yield 81.6%; M.p > 250 C; H NMR (400 MHz, DMSO‑d )
6
H); ESI-MS m/z 270.09 [M ꢁ H] .
ꢀ
1
d
10.01 (s, 1H), 8.67 (d, J ¼ 3.3 Hz, 2H), 7.86e7.74 (m, 2H), 7.43 (d,
4
.1.4.4. 1-Isobutyl-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-
J ¼ 8.2 Hz, 2H), 7.37 (d, J ¼ 8.2 Hz, 2H), 5.64 (s, 2H); ESI-MS m/z
indole-3-carbaldehyde (11d). A purple amorphous powder; yield
8
-
ꢀ
ꢀ
1
352.11 [M ꢁ H] .
1.3%; M.p 238.0 Ce240.0 C; H NMR (400 MHz, DMSO‑d
6
) d 9.97
(
7
1
s, 1H), 8.65 (d, J ¼ 1.6 Hz, 1H), 8.48 (s, 1H), 7.88 (d, J ¼ 8.7 Hz, 1H),
.77 (dd, J ¼ 8.7, 1.8 Hz, 1H), 4.17 (d, J ¼ 7.4 Hz, 2H), 2.24e2.13 (m,
4.1.4.13. 1-(4-Bromobenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
3-yl)-1H-indole-3-carbaldehyde (11m). A white amorphous pow-
der; yield 83.3%; M.p > 250 C; H NMR (400 MHz, DMSO‑d )
6
-
H), 0.89 (d, J ¼ 6.6 Hz, 6H); ESI-MS m/z 284.11 [M ꢁ H] .
ꢀ
1
4
.1.4.5. 1-Isopentyl-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-
d
10.00 (s,1H), 8.66 (d, J ¼ 1.7 Hz,1H), 8.65 (s,1H), 7.81 (d, J ¼ 8.7 Hz,
indole-3-carbaldehyde (11e). A white amorphous powder; yield
1H), 7.75 (dd, J ¼ 8.7, 1.7 Hz, 1H), 7.56 (d, J ¼ 8.4 Hz, 2H), 7.29 (d,
1
-
ꢀ
8
9
1
4.6%; M.p > 250 C; H NMR (400 MHz, DMSO‑d
6
)
d
12.99 (s, 1H),
J ¼ 8.2 Hz, 2H), 5.61 (s, 2H); ESI-MS m/z 398.06 [M ꢁ H] .
.95 (s, 1H), 8.63 (d, J ¼ 1.6 Hz, 1H), 8.51 (s, 1H), 7.83 (d, J ¼ 8.7 Hz,
H), 7.75 (dd, J ¼ 8.7, 1.8 Hz, 1H), 4.34 (t, J ¼ 7.4 Hz, 2H), 1.73 (q,
4.1.4.14. 1-(4-Methylbenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
3-yl)-1H-indole-3-carbaldehyde (11n). A yellow amorphous pow-
der; yield 83.8%; M.p > 250 C; H NMR (400 MHz, DMSO‑d ) d 9.99
J ¼ 7.0 Hz, 2H), 1.61e1.47 (m, 1H), 0.94 (d, J ¼ 6.6 Hz, 6H); ESI-MS m/
-
ꢀ
1
z 298.1205 [M ꢁ H] .
6
(
s, 1H), 8.65 (d, J ¼ 1.6 Hz, 1H), 8.61 (s, 1H), 7.79 (d, J ¼ 8.7 Hz, 1H),
4
.1.4.6. 1-(3-Methylbut-2-en-1-yl)-5-(5-oxo-4,5-dihydro-1,2,4-
7.74 (dd, J ¼ 8.7,1.7 Hz,1H), 7.24 (d, J ¼ 8.0 Hz, 2H), 7.16 (d, J ¼ 7.8 Hz,
-
oxadiazol-3-yl)-1H-indole-3-carbaldehyde (11f). A purple
2H), 5.55 (s, 2H), 2.26 (s, 3H); ESI-MS m/z 332.15 [M ꢁ H] .

12
J. Gao et al. / European Journal of Medicinal Chemistry 190 (2020) 112077
4
.1.4.15. 1-(4-Methoxybenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-
J ¼ 7.5 Hz, 2H), 1.76 (q, J ¼ 7.2 Hz, 2H), 1.63e1.53 (m, 1H), 0.96 (d,
-
oxadiazol-3-yl)-1H-indole-3-carbaldehyde (11o). A white amor-
J ¼ 6.6 Hz, 6H); ESI-MS m/z 298.12 [M ꢁ H] .
ꢀ
1
phous powder; yield 83.4%; M.p > 250 C; H NMR (400 MHz,
DMSO‑d
9.98 (s, 1H), 8.64 (d, J ¼ 1.6 Hz, 1H), 8.60 (s, 1H), 7.83 (d,
J ¼ 8.7 Hz, 1H), 7.75 (dd, J ¼ 8.6, 1.7 Hz, 1H), 7.33 (d, J ¼ 8.2 Hz, 2H),
.92 (d, J ¼ 8.3 Hz, 2H), 5.51 (s, 2H), 3.72 (s, 3H); ESI-MS m/z 348.17
6
)
d
4.1.4.24. 1-Cyclopentyl-6-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-
1H-indole-3-carbaldehyde (12h). A white amorphous powder; yield
ꢀ
ꢀ
1
6
87.2%; M.p 180.2 Ce182.4 C; H NMR (400 MHz, DMSO‑d
6
) d 9.96
-
[M ꢁ H] .
(s, 1H), 8.61 (s, 1H), 8.26 (s, 1H), 8.22 (d, J ¼ 8.3 Hz, 1H), 7.77e7.72
(
m, 1H), 4.99 (t, J ¼ 7.0 Hz, 1H), 2.28 (h, J ¼ 6.1, 5.5 Hz, 2H), 2.11e1.82
-
4
.1.4.16. 1-(4-Tert-butylbenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-
(m, 4H), 1.83e1.68 (m, 2H); ESI-MS m/z 296.10 [M ꢁ H] .
oxadiazol-3-yl)-1H-indole-3-carbaldehyde (11p). A white amor-
ꢀ
1
phous powder; yield 83.8%; M.p > 250 C; H NMR (400 MHz,
DMSO‑d
13.02 (s, 1H), 9.99 (s, 1H), 8.65 (d, J ¼ 1.7 Hz,1H), 8.63 (s,
4.1.4.25. 1-(4-Methylbenzyl)-6-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
3-yl)-1H-indole-3-carbaldehyde (12n). A white amorphous pow-
6
)
d
ꢀ
ꢀ
1
1
H), 7.85 (d, J ¼ 8.7 Hz, 1H), 7.73 (dd, J ¼ 8.7, 1.8 Hz, 1H), 7.37 (d,
der; yield 84.3%; M.p 205.3 Ce206.4 C; H NMR (400 MHz,
DMSO‑d
9.99 (s, 1H), 8.65 (s, 1H), 8.25 (d, J ¼ 8.3 Hz, 1H), 8.17 (d,
J ¼ 1.4 Hz, 1H), 7.72 (dd, J ¼ 8.3, 1.4 Hz, 1H), 7.28 (d, J ¼ 7.8 Hz, 2H),
J ¼ 8.4 Hz, 2H), 7.26 (d, J ¼ 8.3 Hz, 2H), 5.56 (s, 2H), 1.23 (s, 9H); ESI-
6
) d
-
MS m/z 374.25 [M ꢁ H] .
7
.17 (d, J ¼ 7.8 Hz, 2H), 5.54 (s, 2H), 2.26 (s, 3H); ESI-MS m/z 332.15
-
4.1.4.17. 1-(3-Fluorobenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-
[M ꢁ H] .
yl)-1H-indole-3-carbaldehyde (11q). A white amorphous powder;
ꢀ
1
yield 87.3%; M.p > 250 C; H NMR (400 MHz, DMSO‑d
H), 8.69e8.66 (m, 2H), 7.84 (d, J ¼ 8.7 Hz, 1H), 7.77 (dd, J ¼ 8.7,
.7 Hz, 1H), 7.41 (td, J ¼ 8.0, 6.1 Hz, 1H), 7.24 (dt, J ¼ 9.9, 2.1 Hz, 1H),
6
)
d
10.01 (s,
4.1.5. General procedure for the preparation of 1-alkyl-5/6-(5-oxo-
4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-indole-3-carbonitriles (1a-
1u, 2c, 2e, 2h and 2n)
1
1
7
.16 (dd, J ¼ 8.4, 2.5 Hz, 2H), 5.65 (s, 2H); ESI-MS m/z 336.12
1-Alkyl-5/6-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-
indole-3-carbaldehyde (11a-11u, 12c, 12e, 12h and 12n, 20 mmol),
sodium formate (70 mmol), hydroxylamine hydrochloride
(40 mmol), formic acid (30 mL) and N-methyl pyrrolidone (NMP,
5 mL) were added to a 250 mL round bottom flask. The reaction
mixture was stirred under reflux for 6 h. After completion, the
mixture was cooled to ambient temperature and then diluted with
water. The precipitate was filtered and washed with water. The
crude product was purified by flash column chromatography to
afford the target compound.
-
[M ꢁ H] .
4
3
.1.4.18. 1-(3-Chlorobenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
-yl)-1H-indole-3-carbaldehyde (11r). A white amorphous powder;
ꢀ
1
yield 81.6%; M.p > 250 C; H NMR (400 MHz, DMSO‑d
1
6
) d 10.01 (s,
H), 8.67 (d, J ¼ 1.6 Hz, 2H), 7.84 (d, J ¼ 8.7 Hz, 1H), 7.77 (dd, J ¼ 8.7,
1
1
.8 Hz, 1H), 7.47 (d, J ¼ 2.1 Hz, 1H), 7.42e7.37 (m, 2H), 7.33e7.24 (m,
-
H), 5.64 (s, 2H); ESI-MS m/z 352.12 [M ꢁ H] .
4
3
.1.4.19. 1-(3-Bromobenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
-yl)-1H-indole-3-carbaldehyde (11s). A white amorphous powder;
4.1.5.1. 5-(5-Oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-indole-3-
ꢀ
1
yield 81.3%; M.p > 250 C; H NMR (400 MHz, DMSO‑d
1
6
)
d
10.01 (s,
carbonitrile (1a). A purple amorphous powder; yield 70.9%;
ꢀ
1
H), 8.71e8.62 (m, 2H), 7.84 (d, J ¼ 8.7 Hz, 1H), 7.77 (dd, J ¼ 8.7,
M.p > 250 C; H NMR (400 MHz, DMSO‑d
1H), 8.40 (d, J ¼ 2.8 Hz, 1H), 8.18 (s, 1H), 7.77e7.69 (m, 2H); C NMR
(100 MHz, DMSO‑d 160.41, 158.22, 137.35, 136.89, 127.16, 121.15,
6
) d 12.90 (s, 1H), 12.55 (s,
13
1
2
.8 Hz, 1H), 7.61 (t, J ¼ 1.4 Hz, 1H), 7.55e7.48 (m, 1H), 7.37e7.28 (m,
-
H), 5.63 (s, 2H); ESI-MS m/z 396.09, 398.07 [M ꢁ H] .
6
) d
1
17.99, 117.23, 116.12, 114.55, 85.90; ESI-HRMS calcd. for C11
H
5
N
4
O
2
-
4
3
.1.4.20. 1-(3-Methylbenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
[M ꢁ H] 225.0418, found 225.0421.
-yl)-1H-indole-3-carbaldehyde (11t). A white amorphous powder;
ꢀ
1
yield 81.1%; M.p > 250 C; H NMR (400 MHz, DMSO‑d
H), 10.00 (s, 1H), 8.65 (d, J ¼ 1.6 Hz, 1H), 8.62 (s, 1H), 7.81 (d,
J ¼ 8.7 Hz, 1H), 7.74 (dd, J ¼ 8.7, 1.8 Hz, 1H), 7.24 (t, J ¼ 7.6 Hz, 1H),
6
)
d
12.84 (s,
4.1.5.2. 1-Methyl-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-
1
indole-3-carbonitrile (1b). A white amorphous powder; yield
ꢀ
1
6
84.3%; M.p > 250 C; H NMR (600 MHz, DMSO‑d ) d 12.91 (s, 1H),
7
.17 (s, 1H), 7.11 (d, J ¼ 7.6 Hz, 2H), 5.56 (s, 2H), 2.27 (s, 3H); ESI-MS
8.39 (s, 1H), 8.17 (d, J ¼ 1.5 Hz, 1H), 7.83 (d, J ¼ 8.7 Hz, 1H), 7.79 (dd,
-
13
m/z 332.16 [M ꢁ H] .
J ¼ 8.7, 1.6 Hz, 1H), 3.92 (s, 3H); C NMR (100 MHz, DMSO‑d
6
)
d
160.39, 158.13, 140.03, 137.89, 127.43, 121.08, 118.07, 117.47, 115.78,
113.20, 84.83, 34.14; ESI-HRMS calcd. for C12
239.0574, found 239.0576.
-
4
.1.4.21. 1-(3-Methoxybenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-
H
7
N
4
O
2
[M ꢁ H]
oxadiazol-3-yl)-1H-indole-3-carbaldehyde (11u). A yellow amor-
ꢀ
ꢀ
1
phous powder; yield 81.2%; M.p 249.3 Ce251.7 C; H NMR
400 MHz, DMSO‑d
10.00 (s, 1H), 8.66 (d, J ¼ 1.6 Hz, 1H), 8.64 (s,
H), 7.81 (d, J ¼ 8.7 Hz, 1H), 7.76 (dd, J ¼ 8.7, 1.7 Hz, 1H), 7.27 (t,
(
1
6
)
d
4.1.5.3. 1-Isopropyl-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-
indole-3-carbonitrile (1c). A white amorphous powder; yield 88.2%;
ꢀ
1
J ¼ 7.9 Hz, 1H), 6.96 (t, J ¼ 2.0 Hz, 1H), 6.87 (td, J ¼ 6.6, 3.3 Hz, 2H),
M.p > 250 C; H NMR (400 MHz, DMSO‑d
6
) d 12.91 (s, 1H), 8.62 (s,
-
5
.57 (s, 2H), 3.73 (s, 3H); ESI-MS m/z 348.15 [M ꢁ H] .
1H), 8.18 (d, J ¼ 1.7 Hz, 1H), 7.94 (d, J ¼ 8.8 Hz, 1H), 7.78 (dd, J ¼ 8.8,
13
1
.7 Hz, 1H), 4.93 (hept, J ¼ 6.6 Hz, 1H), 1.50 (d, J ¼ 6.6 Hz, 6H);
NMR (100 MHz, DMSO‑d 160.36, 158.08, 136.76, 136.22, 127.55,
121.03, 118.21, 117.49, 115.89, 113.30, 85.54, 48.99, 22.66; ESI-HRMS
C
4
1
.1.4.22. 1-Isopropyl-6-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-
6
) d
H-indole-3-carbaldehyde (12c). A yellow amorphous powder;
ꢀ
1
-
yield 86.2%; M.p > 250 C; H NMR (400 MHz, DMSO‑d
1
6
)
d
9.97 (s,
calcd. for C14
H
11
N
4
O
2
[M ꢁ H] 267.0887, found 267.0895.
H), 8.67 (s, 1H), 8.27 (d, J ¼ 1.4 Hz, 1H), 8.24 (d, J ¼ 8.3 Hz, 1H), 7.74
(
6
dd, J ¼ 8.3, 1.4 Hz, 1H), 4.89 (hept, J ¼ 6.7 Hz, 1H), 1.58 (d, J ¼ 6.6 Hz,
4.1.5.4. 1-Isobutyl-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-
-
H); ESI-MS m/z 270.09 [M ꢁ H] .
indole-3-carbonitrile (1d). A white amorphous powder; yield
ꢀ ꢀ
1
7
8.2%; M.p 237.5 Ce239.5 C; H NMR (600 MHz, DMSO‑d ) d 12.93
6
4
.1.4.23. 1-Isopentyl-6-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-
(s, 1H), 8.46 (s, 1H), 8.19 (d, J ¼ 1.7 Hz, 1H), 7.93 (d, J ¼ 8.8 Hz, 1H),
indole-3-carbaldehyde (12e). A yellow amorphous powder; yield
8
7.78 (dd, J ¼ 8.7, 1.7 Hz, 1H), 4.15 (d, J ¼ 7.4 Hz, 2H), 2.20e2.13 (m,
ꢀ
ꢀ
1
13
5.6%; M.p 129.4 Ce131.6 C; H NMR (400 MHz, DMSO‑d
6
)
d
9.96
1H), 0.86 (d, J ¼ 6.6 Hz, 6H); C NMR (100 MHz, DMSO‑d
6
) d 160.38,
(
s, 1H), 8.55 (s, 1H), 8.26e8.19 (m, 2H), 7.80e7.69 (m, 1H), 4.35 (t,
158.09, 139.56, 137.61, 127.40, 121.10, 118.20, 117.43, 115.79, 113.51,

J. Gao et al. / European Journal of Medicinal Chemistry 190 (2020) 112077
13
-
8
2
5.15, 54.06, 29.35, 20.01; ESI-HRMS calcd. for C15
81.1044, found 281.1051.
H
13
N
4
O
2
[M ꢁ H]
1H), 8.62 (s, 1H), 8.19 (d, J ¼ 1.6 Hz, 1H), 7.88 (d, J ¼ 8.7 Hz, 1H), 7.76
(dd, J ¼ 8.7, 1.7 Hz, 1H), 7.38 (dd, J ¼ 8.6, 5.6 Hz, 2H), 7.17 (t,
13
J ¼ 8.9 Hz, 2H), 5.57 (s, 2H); C NMR (100 MHz, DMSO‑d
6
) d 159.88,
4
.1.5.5. 1-Isopentyl-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-
157.54, 139.06, 136.63, 135.30, 132.62, 129.21, 128.77, 127.19, 120.95,
indole-3-carbonitrile (1e). A white amorphous powder; yield 79.2%;
117.84, 117.31, 115.05, 113.01, 85.40, 49.29; ESI-HRMS calcd. for
ꢀ
ꢀ
1
-
M.p 194.5 Ce196.2 C; H NMR (400 MHz, DMSO‑d
6
)
d
12.92 (s,
C
18
H10FN
4
O
2
[M ꢁ H] 333.0793, found 333.0797.
1
H), 8.49 (s, 1H), 8.18 (d, J ¼ 1.6 Hz, 1H), 7.90 (d, J ¼ 8.7 Hz, 1H), 7.78
(
1
dd, J ¼ 8.7,1.6 Hz,1H), 4.32 (t, J ¼ 7.5 Hz, 2H),1.70 (q, J ¼ 7.2 Hz, 2H),
4.1.5.12. 1-(4-Chlorobenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
13
.57e1.44 (m, 1H), 0.92 (d, J ¼ 6.6 Hz, 6H); C NMR (150 MHz,
3-yl)-1H-indole-3-carbonitrile (1l). A white amorphous powder;
ꢀ
1
DMSO‑d
6
)
d
160.39, 158.11, 139.10, 137.18, 127.56, 121.15, 118.24,
yield 75.6%; M.p > 250 C; H NMR (600 MHz, DMSO‑d ) d 12.92 (s,
6
1
17.49, 115.76, 113.23, 85.21, 45.57, 38.56, 25.64, 22.64; ESI-HRMS
1H), 8.62 (s, 1H), 8.20 (d, J ¼ 1.6 Hz, 1H), 7.86 (d, J ¼ 8.7 Hz, 1H), 7.76
(dd, J ¼ 8.8, 1.7 Hz, 1H), 7.41 (d, J ¼ 8.6 Hz, 2H), 7.32 (d, J ¼ 8.6 Hz,
-
calcd. for C16
H
15
N
4
O
2
[M ꢁ H] 295.1200, found 295.1222.
13
2
6
H), 5.59 (s, 2H); C NMR (100 MHz, DMSO‑d ) d 160.39, 158.05,
4
.1.5.6. 1-(3-Methylbut-2-en-1-yl)-5-(5-oxo-4,5-dihydro-1,2,4-
139.57, 137.14, 135.81, 133.13, 129.72, 129.28, 127.70, 121.46, 118.35,
oxadiazol-3-yl)-1H-indole-3-carbonitrile (1f). A white amorphous
117.82, 115.56, 113.52, 85.91, 49.80; ESI-HRMS calcd. for
ꢀ
ꢀ
1
-
powder; yield 81.3%; M.p 215.8 Ce217.5 C; H NMR (400 MHz,
DMSO‑d 12.93 (s, 1H), 8.42 (s, 1H), 8.21e8.17 (m, 1H), 7.84e7.77
m, 2H), 5.38 (tp, J ¼ 6.5, 1.4 Hz, 1H), 4.92 (d, J ¼ 7.0 Hz, 2H), 1.83 (d,
C
18
H10ClN
4
O
2
[M ꢁ H] 349.0497, found 349.0523.
6
) d
(
4.1.5.13. 1-Methyl-1H-indole-5-carbonitrile (5b)1-(4-Bromobenzyl)-
1
3
J ¼ 1.3 Hz, 3H),1.74 (d, J ¼ 1.4 Hz, 3H); C NMR (100 MHz, DMSO‑d
6
)
5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-indole-3-carbonitrile
ꢀ
1
d
160.36, 158.09, 138.82, 138.38, 137.07, 127.70, 121.12, 118.99,118.21,
(1m). A white amorphous powder; yield 86.3%; M.p > 250 C; H
NMR (400 MHz, DMSO‑d 12.91 (s, 1H), 8.60 (s, 1H), 8.19 (d,
J ¼ 1.6 Hz, 1H), 7.84 (d, J ¼ 8.8 Hz, 1H), 7.75 (dd, J ¼ 8.7, 1.7 Hz, 1H),
1
C
17.51, 115.75, 113.35, 85.20, 45.18, 25.79, 18.42; ESI-HRMS calcd. for
6
) d
-
H
16 13
N
4
O
2
[M ꢁ H] 293.1044, found 293.1046.
1
3
7
.54 (d, J ¼ 8.4 Hz, 2H), 7.25 (d, J ¼ 8.5 Hz, 2H), 5.56 (s, 2H); C NMR
(100 MHz, DMSO‑d 160.37, 158.02, 139.55, 137.11, 136.21, 132.19,
130.01, 127.69, 121.67, 121.44, 118.34, 117.79, 115.56, 113.48, 85.92,
4
.1.5.7. 1-(Cyclopropylmethyl)-5-(5-oxo-4,5-dihydro-1,2,4-
6
) d
oxadiazol-3-yl)-1H-indole-3-carbonitrile (1g). A white amorphous
ꢀ
ꢀ
1
-
powder; yield 78.5%; M.p 227.6 Ce229.3 C; H NMR (400 MHz,
DMSO‑d
1
1
49.86; ESI-HRMS calcd. for C18
393.0020.
H10BrN
4
O
2
[M ꢁ H] 392.9992, found
6
)
d
12.91 (s, 1H), 8.52 (s, 1H), 8.19 (s, 1H), 7.94 (d, J ¼ 8.8 Hz,
H), 7.78 (d, J ¼ 8.7 Hz, 1H), 4.19 (d, J ¼ 7.2 Hz, 2H), 1.36e1.26 (m,
13
H), 0.58e0.54 (m, 2H), 0.47e0.43 (m, 2H); C NMR (100 MHz,
160.38, 158.12, 138.94, 137.33, 127.54, 121.12, 118.16,
4.1.5.14. 1-(4-Methylbenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
DMSO‑d
6
)
d
3-yl)-1H-indole-3-carbonitrile (1n). A white amorphous powder;
ꢀ ꢀ
1
1
C
17.48, 115.78, 113.36, 85.22, 51.33, 11.63, 4.25; ESI-HRMS calcd. for
yield 81.5%; M.p 239.2 Ce241.5 C; H NMR (600 MHz, DMSO‑d )
6
þ
H
15 11
N
4
O
2
[MþH] 279.0887, found 279.0891.
d
1
12.90 (s, 1H), 8.59 (s, 1H), 8.18 (d, J ¼ 1.6 Hz, 1H), 7.84 (d, J ¼ 8.7 Hz,
H), 7.74 (dd, J ¼ 8.7, 1.6 Hz, 1H), 7.20 (d, J ¼ 7.9 Hz, 2H), 7.14 (d,
13
4
.1.5.8. 1-Cyclopentyl-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-
J ¼ 7.9 Hz, 2H), 5.51 (s, 2H), 2.24 (s, 3H); C NMR (100 MHz,
DMSO‑d 160.41, 158.07, 139.47, 137.77, 137.13, 133.75, 129.80,
127.84, 127.71, 121.30, 118.28, 117.69, 115.63, 113.55, 85.64, 50.38,
21.10; ESI-HRMS calcd. for C19
329.1069.
1
H-indole-3-carbonitrile (1h). A white amorphous powder; yield
6
) d
ꢀ
1
76.7%; M.p > 250 C; H NMR (600 MHz, DMSO‑d
6
)
d
12.91 (s, 1H),
-
8
.56 (s, 1H), 8.16 (d, J ¼ 1.6 Hz, 1H), 7.92 (d, J ¼ 8.8 Hz, 1H), 7.77 (dd,
13
H N
4
O
2
[M ꢁ H] 329.1044, found
J ¼ 8.8, 1.6 Hz, 1H), 5.02 (p, J ¼ 7.1 Hz, 1H), 2.27e2.16 (m, 2H),
1
3
1.94e1.80 (m, 4H), 1.76e1.67 (m, 2H);
C NMR (100 MHz,
DMSO‑d 160.37, 158.07, 137.40, 136.53, 127.66, 121.02, 118.14,
6
)
d
4.1.5.15. 1-(4-Methoxybenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-
1
C
17.53, 115.87, 113.50, 85.51, 58.15, 32.54, 23.92; ESI-HRMS calcd. for
oxadiazol-3-yl)-1H-indole-3-carbonitrile (1o). A white amorphous
-
ꢀ
1
H
16 13
N
4
O
2
[M ꢁ H] 293.1044, found 293.1053.
powder; yield 76.4%; M.p > 250 C; H NMR (400 MHz, DMSO‑d
12.91 (s, 1H), 8.58 (s, 1H), 8.18 (d, J ¼ 1.6 Hz, 1H), 7.88 (d, J ¼ 8.8 Hz,
1H), 7.75 (dd, J ¼ 8.8, 1.7 Hz, 1H), 7.29 (d, J ¼ 8.7 Hz, 2H), 6.89 (d,
6
)
d
4
3
.1.5.9. 1-(Cyclohexylmethyl)-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
-yl)-1H-indole-3-carbonitrile (1i). A white amorphous powder;
13
J ¼ 8.7 Hz, 2H), 5.48 (s, 2H), 3.70 (s, 3H); C NMR (100 MHz,
DMSO‑d 160.47, 159.48, 158.13, 139.33, 137.06, 129.45, 128.64,
127.74, 121.27, 118.26, 117.70, 115.65, 114.65, 113.56, 85.59, 55.57,
ꢀ
ꢀ
1
yield 79.7%; M.p 240.0 Ce242.0 C; H NMR (400 MHz, DMSO‑d
12.88 (s, 1H), 8.41 (s, 1H), 8.17 (s, 1H), 7.90 (d, J ¼ 8.7 Hz, 1H), 7.76
d, J ¼ 8.7 Hz, 1H), 4.15 (d, J ¼ 7.3 Hz, 2H), 1.81 (m, 1H), 1.72e1.39 (m,
H), 1.05 (m, 5H); 13C NMR (100 MHz, DMSO‑d
160.41, 158.11,
39.56, 137.66, 127.41, 121.11, 118.17, 117.44, 115.75, 113.47, 85.13,
2.93, 38.39, 30.30, 26.21, 25.51; ESI-HRMS calcd. for C18
6
)
6
) d
d
(
5
1
-
50.12; ESI-HRMS calcd. for C19
345.1018.
H
13
N
4
O
3
[M ꢁ H] 345.0993, found
6
) d
5
H
17
N
4
O
2
4.1.5.16. 1-(4-Tert-butylbenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-
-
[M ꢁ H] 321.1357, found 321.1371.
oxadiazol-3-yl)-1H-indole-3-carbonitrile (1p). A white amorphous
ꢀ
1
powder; yield 86.4%; M.p > 250 C; H NMR (400 MHz, DMSO‑d
6
)
4
.1.5.10. 1-Benzyl-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-
d 12.92 (s, 1H), 8.62 (s, 1H), 8.23e8.17 (m, 1H), 7.94e7.86 (m, 1H),
indole-3-carbonitrile (1j). A white amorphous powder; yield 78.6%;
7.76 (dd, J ¼ 8.8, 1.7 Hz, 1H), 7.36 (d, J ¼ 8.4 Hz, 2H), 7.24 (d,
ꢀ
1
13
M.p > 250 C; H NMR (400 MHz, DMSO‑d
6
)
d
12.91 (s, 1H), 8.63 (s,
J ¼ 8.3 Hz, 2H), 5.54 (s, 2H), 1.23 (s, 9H); C NMR (100 MHz,
1
H), 8.20 (d, J ¼ 1.5 Hz, 1H), 7.89e7.84 (m, 1H), 7.75 (dd, J ¼ 8.8,
6
DMSO‑d ) d 160.37, 158.05, 150.92, 139.48, 137.20, 133.86, 127.69,
13
1.7 Hz, 1H), 7.40e7.25 (m, 5H), 5.59 (s, 2H); C NMR (150 MHz,
127.55, 126.02, 121.36, 118.29, 117.68, 115.62, 113.55, 85.71, 50.22,
-
DMSO‑d 160.42, 158.09, 139.57, 137.21, 136.80, 129.28, 128.44,
6
)
d
34.70, 31.48; ESI-HRMS calcd. for C22
found 371.1542.
H
19
N
4
O
2
[M ꢁ H] 371.1513,
1
27.76, 127.71, 121.38, 118.32, 117.75, 115.61, 113.55, 85.76, 50.57;
-
ESI-HRMS calcd. for C18
H
11
N
4
O
2
[M ꢁ H] 315.0887, found 315.0899.
4.1.5.17. 1-(3-Fluorobenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-
4
.1.5.11. 1-(4-Fluorobenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-
yl)-1H-indole-3-carbonitrile (1q). A white amorphous powder;
ꢀ
ꢀ
1
yl)-1H-indole-3-carbonitrile (1k). A white amorphous powder;
yield 83.4%; M.p > 250 C; H NMR (600 MHz, DMSO‑d
yield 87.3%; M.p 219.3 Ce231.7 C; H NMR (600 MHz, DMSO‑d
6
)
ꢀ
1
6
)
d
12.92 (s,
d
12.92 (s, 1H), 8.63 (s,1H), 8.20 (d, J ¼ 1.6 Hz,1H), 7.88 (d, J ¼ 8.7 Hz,

14
J. Gao et al. / European Journal of Medicinal Chemistry 190 (2020) 112077
1
H), 7.76 (dd, J ¼ 8.7,1.7 Hz,1H), 7.39 (td, J ¼ 8.0, 6.0 Hz,1H), 7.19 (dt,
(dd, J ¼ 8.4,1.5 Hz,1H), 4.29 (t, J ¼ 7.6 Hz, 2H),1.72 (q, J ¼ 7.2 Hz, 2H),
13
13
J ¼ 9.9, 2.0 Hz, 1H), 7.16e7.08 (m, 2H), 5.60 (s, 2H); C NMR
100 MHz, DMSO‑d 159.86, 157.51, 139.07, 138.72, 136.63, 133.38,
30.70, 127.97, 127.26, 127.17, 125.96, 121.00, 117.86, 117.33, 115.03,
1.56e1.50 (m, 1H), 1H), 0.93 (d, J ¼ 6.6 Hz, 6H); C NMR (100 MHz,
(
1
6
)
d
6
DMSO‑d ) d 160.39, 158.15, 139.84, 135.08, 130.17, 120.30, 119.59,
118.63, 115.72, 110.59, 84.77, 45.68, 38.38, 25.74, 22.63; ESI-HRMS
-
-
112.94, 85.52, 49.34; ESI-HRMS calcd. for C18
H10FN
4
O
2
[M ꢁ H]
calcd. for C16H
15
N
4
O
2
[M ꢁ H] 295.1200, found 295.1223.
3
33.0793, found 333.0798.
4
.1.5.24. 1-Cyclopentyl-6-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-
4
3
.1.5.18. 1-(3-Chlorobenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
1H-indole-3-carbonitrile (2h). A white amorphous powder; yield
ꢀ
1
-yl)-1H-indole-3-carbonitrile (1r). A white amorphous powder;
67.7%; M.p > 250 C; H NMR (600 MHz, DMSO‑d ) d 12.94 (s, 1H),
6
ꢀ
1
yield 77.6%; M.p > 250 C; H NMR (600 MHz, DMSO‑d
1
6
)
d
12.92 (s,
8.59 (s, 1H), 8.19e8.13 (m, 1H), 7.80 (d, J ¼ 8.4 Hz, 1H), 7.69 (dd,
J ¼ 8.4, 1.4 Hz, 1H), 4.94 (p, J ¼ 7.2 Hz, 1H), 2.27e2.22 (m, 2H),
H), 8.63 (s, 1H), 8.20 (d, J ¼ 1.7 Hz, 1H), 7.88 (d, J ¼ 8.8 Hz, 1H), 7.76
13
(
7
dd, J ¼ 8.7, 1.7 Hz, 1H), 7.44 (d, J ¼ 2.1 Hz, 1H), 7.36 (m, 2H),
1.98e1.80 (m, 4H), 1.79e1.67 (m, 2H);
C NMR (100 MHz,
13
.25e7.20 (m, 1H), 5.59 (s, 2H); C NMR (100 MHz, DMSO‑d
6
)
DMSO‑d
6
) d 160.42, 158.24, 137.33, 135.38, 130.30, 120.24, 119.73,
d
1
4
3
160.36, 158.02, 139.58, 139.23, 137.14, 133.89, 131.20, 128.48,
118.56, 115.84, 111.01, 85.03, 58.34, 32.40, 23.77; ESI-HRMS calcd.
-
27.77, 127.67, 126.47, 121.51, 118.36, 117.83, 115.54, 113.44, 86.03,
for C16H
13
N
4
O
2
[M ꢁ H] 293.1044, found 293.1068.
-
9.85; ESI-HRMS calcd. for C18
49.0521.
H10ClN
4
O
2
[M ꢁ H] 349.0497, found
4.1.5.25. 1-(4-Methylbenzyl)-6-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
3
-yl)-1H-indole-3-carbonitrile (2n). A white amorphous powder;
ꢀ
1
4
3
.1.5.19. 1-(3-Bromobenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
yield 89.5%; M.p > 250 C; H NMR (600 MHz, DMSO‑d ) d 12.99 (s,
6
-yl)-1H-indole-3-carbonitrile (1s). A white amorphous powder;
1H), 8.65 (s, 1H), 8.13 (d, J ¼ 1.4 Hz, 1H), 7.84 (d, J ¼ 8.4 Hz, 1H), 7.69
(dd, J ¼ 8.4, 1.4 Hz, 1H), 7.22 (d, J ¼ 7.9 Hz, 2H), 7.16 (d, J ¼ 7.8 Hz,
ꢀ
1
yield 84.3%; M.p > 250 C; H NMR (600 MHz, DMSO‑d
H), 8.64 (s, 1H), 8.20 (d, J ¼ 1.7 Hz, 1H), 7.89 (d, J ¼ 8.8 Hz, 1H), 7.77
dd, J ¼ 8.7, 1.7 Hz, 1H), 7.58 (t, J ¼ 1.8 Hz, 1H), 7.50 (dt, J ¼ 7.9, 1.5 Hz,
H), 7.30 (t, J ¼ 7.8 Hz, 1H), 7.26 (dt, J ¼ 7.7, 1.3 Hz, 1H), 5.59 (s, 2H);
6
) d 12.92 (s,
13
1
(
6
2H), 5.50 (s, 2H), 2.25 (s, 3H); C NMR (100 MHz, DMSO‑d )
d 160.39, 158.07, 140.28, 137.81, 135.15, 133.69, 130.31, 129.84,
1
127.79, 120.42, 119.93, 118.87, 115.60, 110.69, 85.27, 50.36, 21.11; ESI-
1
3
[MþH]þ 331.1190, found 331.1182.
C NMR (100 MHz, DMSO‑d
6
)
d
160.37, 158.05, 139.60, 139.49,
15 4 2
HRMS calcd. for C19H N O
137.14, 131.49, 131.39, 130.64, 127.67, 126.85, 122.45, 121.53, 118.37,
1
C
17.85, 115.54, 113.48, 86.01, 49.78; ESI-HRMS calcd. for
4.2. Biological evaluation
-
18
H10BrN
4
O
2
[M ꢁ H] 392.9992, found 393.0021.
4.2.1. Assay for the in vitro XO inhibitory potency
4
3
.1.5.20. 1-(3-Methylbenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
In vitro bovine XO inhibitory potency with allopurinol as a
-yl)-1H-indole-3-carbonitrile (1t). A white amorphous powder;
positive control was measured by spectrophotometric determina-
ꢀ
ꢀ
1
ꢀ
yield 87.3%; M.p 230.9 Ce234.1 C; H NMR (600 MHz, DMSO‑d
6
)
tion of uric acid formation at 295 nm at 25 C, with modifications
d
1
12.92 (s, 1H), 8.61 (s, 1H), 8.19 (d, J ¼ 1.6 Hz, 1H), 7.86 (d, J ¼ 8.7 Hz,
[62]. The enzyme solution (75 U/L) was prepared by dissolving XO
(X4875, Sigma-Aldrich Chemical Co.) in phosphate buffer (0.3 mM
Na EDTA and 0.1 M sodium pyrophosphate decahydrate, pH 8.3).
2
The substrate solution was prepared by dissolving xanthine (J&K)
in a buffer. The tested compounds were diluted with buffer to
varied concentrations. The reaction was performed in 96-well
H), 7.75 (dd, J ¼ 8.7, 1.7 Hz, 1H), 7.22 (t, J ¼ 7.6 Hz, 1H), 7.14 (s, 1H),
7
3
.10 (d, J ¼ 7.6 Hz, 1H), 7.07 (d, J ¼ 7.7 Hz, 1H), 5.53 (s, 2H), 2.26 (s,
13
H); C NMR (100 MHz, DMSO‑d
6
)
d
160.37, 158.06, 139.57, 138.55,
1
37.21, 136.70, 129.19, 129.12, 128.36, 127.69, 124.90, 121.36, 118.31,
117.69, 115.64, 113.56, 85.70, 50.56, 21.41; ESI-HRMS calcd. for
-
C
H
19 13
N
4
O
2
[M ꢁ H] 329.1044, found 329.1068.
plates (COSTAR 3599) containing 200
m
L of assay mixture, which
L of varying concentra-
contained 40 L of enzyme solution, 53
m
m
4
.1.5.21. 1-(3-Methoxybenzyl)-5-(5-oxo-4,5-dihydro-1,2,4-
tions of the tested compounds (or buffer for the blank) and 67
buffer. After incubating for 25 min at 25 C, the reaction was
initiated by the addition of a 40 mL xanthine solution (500 mM/l),
and the XO inhibitory potency of the tested compounds was
measured by the reduction of uric acid production in the first 2 min.
The final concentration of DMSO is less than 5%. All the compounds
were tested in triplicate with 5 different concentrations. Statistical
analyses of data were conducted with Microsoft Excel 2016
(Microsoft Inc., Redmond, USA). The IC50 values were calculated
using IBM SPSS Statistics 24.0 (SPSS Inc., Chicago, IL, USA) software.
m
L of
ꢀ
oxadiazol-3-yl)-1H-indole-3-carbonitrile (1u). A white amorphous
ꢀ
ꢀ
1
powder; yield 76.2%; M.p 207.8 Ce209.1 C; H NMR (400 MHz,
DMSO‑d
12.91 (s, 1H), 8.61 (s, 1H), 8.19 (d, J ¼ 1.7 Hz, 1H), 7.87 (d,
6
)
d
J ¼ 8.8 Hz, 1H), 7.75 (dd, J ¼ 8.8, 1.7 Hz, 1H), 7.25 (t, J ¼ 7.9 Hz, 1H),
6
1
d
.92 (t, J ¼ 2.1 Hz, 1H), 6.86 (dd, J ¼ 8.3, 2.4 Hz, 1H), 6.83e6.80 (m,
13
H), 5.54 (s, 2H), 3.72 (s, 3H); C NMR (100 MHz, DMSO‑d
6
)
160.37, 159.98, 158.05, 139.59, 138.27, 137.22, 130.45, 127.67,
121.38, 119.83, 118.31, 117.71, 115.62, 113.85, 113.63, 113.56, 85.73,
-
5
5.57, 50.50; ESI-HRMS calcd. for C19
13
H N
4
O
3
[M ꢁ H] 345.0993,
found 345.1019.
4.2.2. Enzyme inhibitory kinetics
4
1
8
8
.1.5.22. 1-Isopropyl-6-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-
The mode of action of representative compound 1h was further
investigated using enzyme kinetic studies, which were performed
in the same way as the in vitro XO assay but by varying the xanthine
H-indole-3-carbonitrile (2c). A white amorphous powder; yield
ꢀ
1
6.2%; M.p > 250 C; H NMR (400 MHz, DMSO‑d
6
)
d
12.95 (s, 1H),
.65 (s, 1H), 8.20 (d, J ¼ 1.4 Hz, 1H), 7.83 (d, J ¼ 8.4 Hz, 1H), 7.70 (dd,
concentrations to 80, 100, 120 and 140
mM/l. The results were
J ¼ 8.4,1.4 Hz,1H), 4.86 (hept, J ¼ 6.7 Hz,1H),1.54 (d, J ¼ 6.7 Hz, 6H);
analyzed using a Lineweaver-Burk plot, from which we could
13
C NMR (100 MHz, DMSO‑d
6
)
d
160.36, 158.22, 137.04, 134.67,
m i
calculate the value of K , Vmax, K and Kis in the presence and
1
30.23, 120.29, 119.66, 118.53, 115.83, 110.84, 85.08, 49.29, 22.56;
absence of inhibitor.
-
ESI-HRMS calcd. for C14
H
11
N
4
O
2
[M ꢁ H] 267.0887, found 267.0911.
4.2.3. Molecular modeling
4.1.5.23. 1-Isopentyl-6-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-1H-
Molecular modeling studies on the high-resolution crystal
indole-3-carbonitrile (2e). A white amorphous powder; yield 79.7%;
structure of XDH (PDB 1N5X) were carried out with GLIDE (2016,
Schrodinger Suite) [63,64]. The enzyme preparation, grid, ligands
and docking parameter files were in accordance with our previous
ꢀ
ꢀ
1
M.p 238.7 Ce239.9 C; H NMR (600 MHz, DMSO‑d
6
) d 12.98 (s,
1
H), 8.51 (s, 1H), 8.09 (d, J ¼ 1.3 Hz, 1H), 7.80 (d, J ¼ 8.4 Hz, 1H), 7.68

J. Gao et al. / European Journal of Medicinal Chemistry 190 (2020) 112077
M. Coyfish, S. Guillo, T.L. Jansen, H. Janssens, F. Liot
15
eꢀ , C. Mallen, G. Nuki,
report [25]. All ligands were built within Maestro BUILD and pre-
pared using the LIGPREP module (2016, Schrodinger Suite) [63].
The protein was prepared by adding hydrogen atoms, deleting
waters and repairing the missing and terminal residues of poly-
peptide chains using an OPLS force field [63]. The grid size was set
F. Perez-Ruiz, J. Pimentao, L. Punzi, T. Pywell, A. So, A.K. Tausche, T. Uhlig,
J. Zavada, W. Zhang, F. Tubach, T. Bardin, 2016 updated EULAR evidence-based
recommendations for the management of gout, Ann. Rheum. Dis. 76 (2017)
29e42.
[
[
2] W. Grassi, R.D. Angelis, Clinical features of gout, Reumatismo 63 (2011)
238e245.
3
as 25 ꢂ 25 ꢂ 25 Å around the center of the ligand. The confor-
3] C.-F. Kuo, M.J. Grainge, W. Zhang, M. Doherty, Global epidemiology of gout:
prevalence, incidence and risk factors, Nat. Rev. Rheumatol. 11 (2015)
mation with the highest score and rational conformation was
selected for discussion. Additionally, the results of docking were
visualized using Discovery Studio Visualizer 2017 software [65].
649e662.
[
[
4] A.M. Abeles, M.H. Pillinger, Gout and cardiovascular disease: crystallized
confusion, Curr. Opin. Rheumatol. 31 (2019) 118e124.
5] R. Kumar, G. Joshi, H. Kler, S. Kalra, M. Kaur, R. Arya, Toward an understanding
of structural insights of xanthine and aldehyde oxidases: an overview of their
inhibitors and role in various diseases, Med. Res. Rev. 38 (2018) 1073e1125.
4.2.4. Hypouricemic effect in vivo
Six-week-old male Sprague-Dawley rats were obtained from the
ꢁ
ꢁ
ꢁ
ꢁ
[
6] A. Smelcerovi ꢀc , K. Tomovi ꢀc , Z. Smelcerovi ꢀc , Z. Petronijevi ꢀc , G. Koci ꢀc ,
T. Toma ꢁs i ꢁc , Z. Jakopin, M. Anderluh, Xanthine oxidase inhibitors beyond
allopurinol and febuxostat; an overview and selection of potential leads based
on in silico calculated physico-chemical properties, predicted pharmacoki-
netics and toxicity, Eur. J. Med. Chem. 135 (2017) 491e516.
ꢁ
Animal Center of Shenyang Pharmaceutical University (Shenyang,
China). The animals were placed in an animal house for 1 week to
adapt to the new experimental environment. During this period,
the rats were maintained under a 12:12-h light-dark cycle and had
free access to food and water. All the study protocols were approved
by the Ethics Review Committee for Animal Experimentation of
Shenyang Pharmaceutical University (Agreement no. SYPU-IACUC-
C2019-9-25-210).
Overnight-fasted rats were randomly divided into four groups of
six each as follows: blank group, model group, 1h group and allo-
purinol group. Potassium oxonate (300 mg/kg) was intraperitone-
ally injected into rats to induce hyperuricemia, except for the blank
group. One hour after administering potassium oxonate, the rats
were intragastrically administered with an equal volume of 0.5%
CMC-Na, compound 1h (15 mg/kg) and allopurinol (5 mg/kg)
[7] M.G. Battelli, A. Bolognesi, L. Polito, Pathophysiology of circulating xanthine
oxidoreductase: new emerging roles for a multi-tasking enzyme, Biochim.
Biophys. Acta (BBA) - Mol. Basis Dis. 1842 (2014) 1502e1517.
8] C. Vorbach, A. Scriven, M.R. Capecchi, The housekeeping gene xanthine
oxidoreductase is necessary for milk fat droplet enveloping and secretion:
gene sharing in the lactating mammary gland, Genes Dev. 16 (2002)
[
3223e3235.
[
9] G. Luna, A.V. Dolzhenko, R.L. Mancera, Inhibitors of xanthine oxidase: scaffold
diversity and structure-based drug design, ChemMedChem 14 (2019)
714e743.
10] P. Pacher, A. Nivorozhkin, C. Szabo, Therapeutic effects of xanthine oxidase
inhibitors: renaissance half a century after the discovery of allopurinol,
Pharmacol. Rev. 58 (2006) 87e114.
[11] J.U. Song, S.P. Choi, T.H. Kim, C.-K. Jung, J.-Y. Lee, S.-H. Jung, G.T. Kim, Design
and synthesis of novel 2-(indol-5-yl)thiazole derivatives as xanthine oxidase
inhibitors, Bioorg. Med. Chem. Lett 25 (2015) 1254e1258.
12] J.U. Song, J.W. Jang, T.H. Kim, H. Park, W.S. Park, S.-H. Jung, G.T. Kim, Structure-
based design and biological evaluation of novel 2-(indol-2-yl) thiazole de-
rivatives as xanthine oxidase inhibitors, Bioorg. Med. Chem. Lett 26 (2016)
[
[
21,66]. Approximately 0.5 mL of blood sample was collected via
[
orbital vein bleeding at 1e8 h after drug treatment. After coagu-
lation for 2 h at room temperature, the serum sample was obtained
950e954.
ꢀ
by centrifugation at 3500g for 10 min at 4 C. Serum urate levels
[
13] M.R. Ali, S. Kumar, O. Afzal, N. Shalmali, M. Sharma, S. Bawa, Development of
2-(substituted benzylamino)-4-methyl-1, 3-thiazole-5-carboxylic acid de-
rivatives as xanthine oxidase inhibitors and free radical scavengers, Chem.
Biol. Drug Des. 87 (2016) 508e516.
14] X. Xu, L. Deng, L. Nie, Y. Chen, Y. Liu, R. Xie, Z. Li, Discovery of 2-
phenylthiazole-4-carboxylic acid, a novel and potent scaffold as xanthine
oxidase inhibitors, Bioorg. Med. Chem. Lett 29 (2019) 525e528.
15] S. Ishibuchi, H. Morimoto, T. Oe, T. Ikebe, H. Inoue, A. Fukunari, M. Kamezawa,
I. Yamada, Y. Naka, Synthesis and structureeactivity relationships of 1-
phenylpyrazoles as xanthine oxidase inhibitors, Bioorg. Med. Chem. Lett 11
(2001) 879e882.
were determined with a uric acid assay kit (Nanjing Jiancheng
Bioengineering Institute, China) in accordance with the manufac-
turer’s instructions. Statistical analysis was analyzed with Student’s
t-test. The figures were obtained with the GraphPad Prism 7.0
[
(
GraphPad Inc., San Diego, USA) statistical system.
[
4.2.5. Statistical analysis
Data analysis and image processing were performed using
Microsoft Excel 2016 (Microsoft Inc., Redmond, USA), IBM SPSS
Statistics 24.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 7.0
[
16] S. Wang, J. Yan, J. Wang, J. Chen, T. Zhang, Y. Zhao, M. Xue, Synthesis of some
5
-phenylisoxazole-3-carboxylic acid derivatives as potent xanthine oxidase
inhibitors, Eur. J. Med. Chem. 45 (2010) 2663e2670.
[17] S. Chen, T. Zhang, J. Wang, F. Wang, H. Niu, C. Wu, S. Wang, Synthesis and
evaluation of 1-hydroxy/methoxy-4-methyl-2-phenyl-1H-imidazole-5-
(
GraphPad Inc., San Diego, USA). All data were presented as
mean ± standard deviation (SD). The comparisons between two
groups were conducted by Student’s t-test. P < 0.05 was considered
a statistically significant difference.
carboxylic acid derivatives as non-purine xanthine oxidase inhibitors, Eur. J.
Med. Chem. 103 (2015) 343e353.
[18] T. Zhang, Y. Lv, Y. Lei, D. Liu, Y. Feng, J. Zhao, S. Chen, F. Meng, S. Wang, Design,
synthesis and biological evaluation of 1-hydroxy-2-phenyl-4-pyridyl-1H-
imidazole derivatives as xanthine oxidase inhibitors, Eur. J. Med. Chem. 146
Declaration of competing interest
(2018) 668e677.
[
19] Q. Guan, Z. Cheng, X. Ma, L. Wang, D. Feng, Y. Cui, K. Bao, L. Wu, W. Zhang,
Synthesis and bioevaluation of 2-phenyl-4-methyl-1,3-selenazole-5-
carboxylic acids as potent xanthine oxidase inhibitors, Eur. J. Med. Chem. 85
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
(
2014) 508e516.
[20] H.-J. Tang, W. Li, M. Zhou, L.-Y. Peng, J.-X. Wang, J.-H. Li, J. Chen, Design,
synthesis and biological evaluation of novel xanthine oxidase inhibitors
bearing
a 2-arylbenzo[b]furan scaffold, Eur. J. Med. Chem. 151 (2018)
Acknowledgments
849e860.
[
[
[
21] B.R. C, A. Kulkarni-Almeida, K.V. Katkar, S. Khanna, U. Ghosh, A. Keche, P. Shah,
A. Srivastava, V. Korde, K.V. Nemmani, N.J. Deshmukh, A. Dixit, M.K. Brahma,
U. Bahirat, L. Doshi, R. Sharma, H. Sivaramakrishnan, Identification of novel
isocytosine derivatives as xanthine oxidase inhibitors from a set of virtual
screening hits, Bioorg. Med. Chem. 20 (2012) 2930e2939.
22] K. Bajaj, S. Burudkar, P. Shah, A. Keche, U. Ghosh, P. Tannu, S. Khanna,
A. Srivastava, N.J. Deshmukh, A. Dixit, Y. Ahire, A. Damre, K.V. Nemmani,
A. Kulkarni-Almeida, B.R. C, R. Sharma, H. Sivaramakrishnan, Lead optimiza-
tion of isocytosine-derived xanthine oxidase inhibitors, Bioorg. Med. Chem.
Lett 23 (2013) 834e838.
23] S. Khanna, S. Burudkar, K. Bajaj, P. Shah, A. Keche, U. Ghosh, A. Desai,
A. Srivastava, A. Kulkarni-Almeida, N.J. Deshmukh, A. Dixit, M.K. Brahma,
U. Bahirat, L. Doshi, K.V. Nemmani, P. Tannu, A. Damre, B.R. C, R. Sharma,
H. Sivaramakrishnan, Isocytosine-based inhibitors of xanthine oxidase:
design, synthesis, SAR, PK and in vivo efficacy in rat model of hyperuricemia,
This work was financially supported by the National Key
Research and Development Program of China (2018YFD0200102).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112077.
References
[
1] P. Richette, M. Doherty, E. Pascual, V. Barskova, F. Becce, J. Casta n~ eda-Sanabria,

16
J. Gao et al. / European Journal of Medicinal Chemistry 190 (2020) 112077
Bioorg. Med. Chem. Lett 22 (2012) 7543e7546.
24] Q. Mao, X. Dai, G. Xu, Y. Su, B. Zhang, D. Liu, S. Wang, Design, synthesis and
biological evaluation of 2-(4-alkoxy-3-cyano)phenyl-6-oxo-1,6-
dihydropyrimidine-5-carboxylic acid derivatives as novel xanthine oxidase
inhibitors, Eur. J. Med. Chem. 181 (2019) 111558.
25] B. Zhang, X. Dai, Z. Bao, Q. Mao, Y. Duan, Y. Yang, S. Wang, Targeting the
subpocket in xanthine oxidase: design, synthesis, and biological evaluation of
derivatives as novel xanthine oxidase inhibitors, Eur. J. Med. Chem. 183
(2019) 111717.
[
[
[
[42] J.N. Sangshetti, R.R. Nagawade, D.B. Shinde, Synthesis of novel 3-(1-(1-
substituted piperidin-4-yl)-1H-1,2,3-triazol-4-yl)-1,2,4-oxadiazol-5(4H)-one
as antifungal agents, Bioorg. Med. Chem. Lett 19 (2009) 3564e3567.
[43] Y. Kohara, K. Kubo, E. Imamiya, T. Wada, Y. Inada, T. Naka, Synthesis and
angiotensin II receptor antagonistic activities of benzimidazole derivatives
bearing acidic heterocycles as novel tetrazole bioisosteres, J. Med. Chem. 39
(1996) 5228e5235.
[44] C. Ballatore, D.M. Huryn, A.B. Smith 3rd, Carboxylic acid (bio)isosteres in drug
design, ChemMedChem 8 (2013) 385e395.
[45] H. Rakugi, K. Enya, K. Sugiura, Y. Ikeda, Comparison of the efficacy and safety
of azilsartan with that of candesartan cilexetil in Japanese patients with grade
2-[4-alkoxy-3-(1H-tetrazol-1-yl)
phenyl]-6-oxo-1,6-dihydropyrimidine-5-
carboxylic acid derivatives, Eur. J. Med. Chem. 181 (2019) 111559.
26] J. Evenas, F. Edfeldt, M. Lepisto, N. Svitacheva, A. Synnergren, B. Lundquist,
M. Granse, A. Ronnholm, M. Varga, J. Wright, M. Wei, S. Yue, J. Wang, C. Li,
X. Li, G. Chen, Y. Liao, G. Lv, A. Tjornebo, F. Narjes, HTS followed by NMR based
counterscreening. Discovery and optimization of pyrimidones as reversible
and competitive inhibitors of xanthine oxidase, Bioorg. Med. Chem. Lett 24
IeII essential hypertension:
a randomized, double-blind clinical study,
(
2014) 1315e1321.
Hypertens. Res. 35 (2012) 552e558.
[
[
[
[
27] M. Kaur, A. Kaur, S. Mankotia, H. Singh, A. Singh, J.V. Singh, M.K. Gupta,
S. Sharma, K. Nepali, P.M.S. Bedi, Synthesis, screening and docking of fused
pyrano[3,2-d]pyrimidine derivatives as xanthine oxidase inhibitor, Eur. J.
Med. Chem. 131 (2017) 14e28.
28] K. Tomovic, B.S. Ilic, Z. Smelcerovic, M. Miljkovic, D. Yancheva, M. Kojic,
A.T. Mavrova, G. Kocic, A. Smelcerovic, Benzimidazole-based dual dipeptidyl
peptidase-4 and xanthine oxidase inhibitors, Chem. Biol. Interact. 315 (2020)
[46] S.R. Langdon, P. Ertl, N. Brown, Bioisosteric replacement and scaffold hopping
in lead generation and optimization, Mol. Inform. 29 (2010) 366e385.
[47] L.M. Lima, E.J. Barreiro, Bioisosterism: a useful strategy for molecular modi-
fication and drug design, Curr. Med. Chem. 12 (2005) 23e49.
[48] P.H. Olesen, The use of bioisosteric groups in lead optimization, Curr. Opin.
Drug Discov. Dev 4 (2001) 471e478.
[49] N. Chadha, O. Silakari, Indoles as therapeutics of interest in medicinal
chemistry: bird’s eye view, Eur. J. Med. Chem. 134 (2017) 159e184.
[50] V. Garg, R.K. Maurya, P.V. Thanikachalam, G. Bansal, V. Monga, An insight into
the medicinal perspective of synthetic analogs of indole: a review, Eur. J. Med.
Chem. 180 (2019) 562e612.
[51] A. Kumari, R.K. Singh, Medicinal chemistry of indole derivatives: current to
future therapeutic prospectives, Bioorg. Chem. 89 (2019) 103021.
[52] M.-Z. Zhang, Q. Chen, G.-F. Yang, A review on recent developments of indole-
containing antiviral agents, Eur. J. Med. Chem. 89 (2015) 421e441.
[53] R.R. Singh, R.-S. Liu, Gold-catalyzed oxidative couplings of two indoles with
one aryldiazo cyanide under oxidant-free conditions, Chem. Commun. 53
(2017) 4593e4596.
[54] X. Yu, K. Chen, F. Yang, S. Zha, J. Zhu, Oxadiazolone-enabled synthesis of
primary azaaromatic amines, Org. Lett. 18 (2016) 5412e5415.
[55] H.-M. Liu, F.-Z. Suo, X.-B. Li, Y.-H. You, C.-T. Lv, C.-X. Zheng, G.-C. Zhang, Y.-
J. Liu, W.-T. Kang, Y.-C. Zheng, H.-W. Xu, Discovery and synthesis of novel
indole derivatives-containing 3-methylenedihydrofuran-2(3H)-one as irre-
versible LSD1 inhibitors, Eur. J. Med. Chem. 175 (2019) 357e372.
[56] G.B. Elion, Enzymatic and metabolic studies with allopurinol, Ann. Rheum. Dis.
25 (1966) 608e614.
108873.
29] T.J. Zhang, Q.X. Wu, S.Y. Li, L. Wang, Q. Sun, Y. Zhang, F.H. Meng, H. Gao,
Synthesis and evaluation of 1-phenyl-1H-1,2,3-triazole-4-carboxylic acid
derivatives as xanthine oxidase inhibitors, Bioorg. Med. Chem. Lett 27 (2017)
3812e3816.
30] T.-j. Zhang, S.-y. Li, Y. Zhang, Q.-x. Wu, F.-h. Meng, Design, synthesis, and
biological evaluation of 5-(4-(pyridin-4-yl)-1H-1,2,3-triazol-1-yl)benzonitrile
derivatives as xanthine oxidase inhibitors, Chem. Biol. Drug Des. 91 (2018)
526e533.
[
[
31] I. Fatima, H. Zafar, K.M. Khan, S.M. Saad, S. Javaid, S. Perveen, M.I. Choudhary,
Synthesis, molecular docking and xanthine oxidase inhibitory activity of 5-
aryl-1H-tetrazoles, Bioorg. Chem. 79 (2018) 201e211.
32] Z. Xie, X. Luo, Z. Zou, X. Zhang, F. Huang, R. Li, S. Liao, Y. Liu, Synthesis and
evaluation of hydroxychalcones as multifunctional non-purine xanthine oxi-
dase inhibitors for the treatment of hyperuricemia, Bioorg. Med. Chem. Lett 27
(
2017) 3602e3606.
[
33] S. Burmaoglu, S. Ozcan, S. Balcioglu, M. Gencel, S.A.A. Noma, S. Essiz, B. Ates,
O. Algul, Synthesis, biological evaluation and molecular docking studies of bis-
chalcone derivatives as xanthine oxidase inhibitors and anticancer agents,
Bioorg. Chem. 91 (2019) 103149.
[57] H. Singh, S. Sharma, R. Ojha, M.K. Gupta, K. Nepali, P.M. Bedi, Synthesis and
evaluation of naphthoflavones as a new class of non purine xanthine oxidase
inhibitors, Bioorg. Med. Chem. Lett 24 (2014) 4192e4197.
[58] H.S. Virdi, S. Sharma, S. Mehndiratta, P.M. Bedi, K. Nepali, Design, synthesis
and evaluation of 2,4-diarylpyrano[3,2-c]chromen-5(4H)-one as a new class
of non-purine xanthine oxidase inhibitors, J. Enzym. Inhib. Med. Chem. 30
(2015) 730e736.
[
34] A. Fais, B. Era, S. Asthana, V. Sogos, R. Medda, L. Santana, E. Uriarte, M.J. Matos,
F. Delogu, A. Kumar, Coumarin derivatives as promising xanthine oxidase
inhibitors, Int. J. Biol. Macromol. 120 (2018) 1286e1293.
[
35] H.-C. Lin, S.-H. Tsai, C.-S. Chen, Y.-C. Chang, C.-M. Lee, Z.-Y. Lai, C.-M. Lin,
Structureeactivity relationship of coumarin derivatives on xanthine oxidase-
inhibiting and free radical-scavenging activities, Biochem. Pharmacol. 75
(
2008) 1416e1425.
[59] J.V. Singh, G. Mal, G. Kaur, M.K. Gupta, A. Singh, K. Nepali, H. Singh, S. Sharma,
S.B. Pm, Benzoflavone derivatives as potent antihyperuricemic agents, Med-
chemcomm 10 (2019) 128e147.
[60] C. Enroth, B.T. Eger, K. Okamoto, T. Nishino, T. Nishino, E.F. Pai, Crystal
structures of bovine milk xanthine dehydrogenase and xanthine oxidase:
structure-based mechanism of conversion, Proc. Natl. Acad. Sci. 97 (2000)
10723e10728.
[61] R. Hille, T. Nishino, Flavoprotein structure and mechanism. 4. Xanthine oxi-
dase and xanthine dehydrogenase, FASEB J. 9 (1995) 995e1003.
[62] K. Matsumoto, K. Okamoto, N. Ashizawa, T. Nishino, FYX-051: a novel and
potent hybrid-type inhibitor of xanthine oxidoreductase, J. Pharmacol. Exp.
Ther. 336 (2011) 95e103.
[
36] S. Sharma, K. Sharma, R. Ojha, D. Kumar, G. Singh, K. Nepali, P.M.S. Bedi,
Microwave assisted synthesis of naphthopyrans catalysed by silica supported
fluoroboric acid as a new class of non purine xanthine oxidase inhibitors,
Bioorg. Med. Chem. Lett 24 (2014) 495e500.
37] N. Malik, P. Dhiman, A. Khatkar, Mechanistic approach towards interaction of
newly synthesized Hesperidin derivatives against xanthine oxidase, Int. J. Biol.
Macromol. 135 (2019) 864e876.
38] M.D. Santi, M. Paulino Zunini, B. Vera, C. Bouzidi, V. Dumontet, A. Abin-Car-
riquiry, R. Grougnet, M.G. Ortega, Xanthine oxidase inhibitory activity of
natural and hemisynthetic flavonoids from Gardenia oudiepe (Rubiaceae)
in vitro and molecular docking studies, Eur. J. Med. Chem. 143 (2018)
[
[
577e582.
[63] Schrodinger Glide, LLC, New York, NY, 2016.
[
39] F.H. Messerli, M. Burnier, Cardiovascular disease and uric acid: is the not-so-
innocent bystander becoming a true culprit and does the US black box
warning for febuxostat indicate that not all uric acid lowering is beneficial?
Eur. Heart J. 40 (2019) 1787e1789.
[64] K. Okamoto, B.T. Eger, T. Nishino, S. Kondo, E.F. Pai, T. Nishino, An extremely
potent inhibitor of xanthine oxidoreductase: crystal structure of the enzyme-
inhibitor complex and mechanism of inhibition, J. Biol. Chem. 278 (2003)
1848e1855.
[
40] W.B. White, K.G. Saag, M.A. Becker, J.S. Borer, P.B. Gorelick, A. Whelton,
B. Hunt, M. Castillo, L. Gunawardhana, Cardiovascular safety of febuxostat or
allopurinol in patients with gout, N. Engl. J. Med. 378 (2018) 1200e1210.
41] T.J. Zhang, Y. Zhang, S. Tu, Y.H. Wu, Z.H. Zhang, F.H. Meng, Design, synthesis
and biological evaluation of N-(3-(1H-tetrazol-1-yl)phenyl)isonicotinamide
[65] A.D.S, Visualizer, Version 4. 5, Softw. Vis. Anal. Protein Struct. (2017).
[66] Y.-c. Zeng, S. Li, C. Liu, T. Gong, X. Sun, Y. Fu, Z.-r. Zhang, Soluplus micelles for
improving the oral bioavailability of scopoletin and their hypouricemic effect
in vivo, Acta Pharmacol. Sin. 38 (2017) 424e433.
[