Design, synthesis and biological evaluation of indole-2-carboxylic acid derivatives as IDO1/TDO dual inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.111985

Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) are involved in the key steps of tryptophan metabolism and are potential new targets for tumor immunotherapy. In this work, a variety of indole-2-carboxylic acid derivatives were synthesized, and their inhibitory activities against both enzymes along with structure-activity relationships were investigated. As a result, a number of 6-acetamido-indole-2-carboxylic acid derivatives were found to be potent dual inhibitors with IC₅₀ values at low micromolar levels. Among them, compound 9o-1 was the most potent inhibitor with an IC₅₀ value of 1.17 μM for IDO1, and 1.55 μM for TDO, respectively. In addition, a para-benzoquinone derivative 9p-O, resulted from the oxidation of compound 9p, was also identified and it showed strong inhibition against the two enzymes with IC₅₀ values at the double digit nanomolar level. Using molecular docking and molecular dynamic simulations, we predicted the binding modes of this class of compounds within IDO1 and TDO binding pocket. The results provide insights for further structural optimization of this series of IDO1/TDO dual inhibitors.

Full text of DOI:10.1016/j.ejmech.2019.111985

Journal Pre-proof
Design, synthesis and biological evaluation of indole-2-carboxylic acid derivatives as
IDO1/TDO dual inhibitors
Guonan Cui, Fangfang Lai, Xiaoyu Wang, Xiaoguang Chen, Bailing Xu
PII:
S0223-5234(19)31137-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.111985
EJMECH 111985
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 October 2019
Revised Date: 4 December 2019
Accepted Date: 17 December 2019
Please cite this article as: G. Cui, F. Lai, X. Wang, X. Chen, B. Xu, Design, synthesis and biological
evaluation of indole-2-carboxylic acid derivatives as IDO1/TDO dual inhibitors, European Journal of
Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2019.111985.
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Graphical Abstract:
Design, synthesis and biological evaluation of indole-2-carboxylic acid
derivatives as IDO1/TDO dual inhibitors
Guonan Cui, Fangfang Lai, Xiaoyu Wang, Xiaoguang Chen, Bailing Xu
The indole-2-carboxylic acid derivatives were
disclosed as novel IDO1/TDO dual inhibitors.

Design, synthesis and biological evaluation of indole-2-carboxylic acid
derivatives as IDO1/TDO dual inhibitors
a,#
b,#
a
b,*
a,*
Guonan Cui , Fangfang Lai , Xiaoyu Wang , Xiaoguang Chen , Bailing Xu
a
Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of
Materia Medica, Chinese Academy of Medical Sciences Peking Union Medical College,
Beijing, 100050, China
State Key Laboratory of Bioactive Substances and Functions of Natural Medicines,
＆
b
Institute of Materia Medica, Chinese Academy of Medical Sciences＆Peking Union Medical
College, Beijing, 100050, China
Abstract
Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO)
are involved in the key steps of tryptophan metabolism and are potential new targets
for tumor immunotherapy. In this work, a variety of indole-2-carboxylic acid
derivatives were synthesized, and their inhibitory activities against both enzymes
along with structure-activity relationships were investigated. As a result, a number of
6
-acetamido-indole-2-carboxylic acid derivatives were found to be potent dual
inhibitors with IC values at low micromolar levels. Among them, compound 9o-1
5
0
was the most potent inhibitor with an IC value of 1.17 µM for IDO1, and 1.55 µM
5
0
for TDO, respectively. In addition, a para-benzoquinone derivative 9p-O, resulted
from the oxidation of compound 9p, was also identified and it showed strong
inhibition against the two enzymes with IC values at the double digit nanomolar
5
0
level. Using molecular docking and molecular dynamic simulations, we predicted the
binding modes of this class of compounds within IDO1 and TDO binding pocket. The
results provide insights for further structural optimization of this series of IDO1/TDO
dual inhibitors.
Keywords: indoleamine 2,3-dioxygenase 1, tryptophan 2,3-dioxygenase,
indole-2-carboxylic acid, dual inhibitors
#
The first two authors contributed equally.
Corresponding author. Bailing Xu, Tel: +86 10 63166764; Xiaoguang Chen, Tel: +86 10 63166302
*
E-mail address: xubl@imm.ac.cn (B. Xu); chxg@imm.ac.cn (X. Chen).

1
. Introduction
Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO)
are heme-containing enzymes that catalyze the first and rate-limiting step of
tryptophan catabolism along the kynurenine pathway, generating tryptophan dioxide,
which was converted to N-formyl kynurenine and kynurenine derivatives [1]. IDO1
and TDO are found to be overexpressed in various cancer cells with different levels [2,
3
]. In the tumor microenvironment, tryptophan depletion and kynurenine metabolites
accumulation led to the enhancement of the number and function of Tregs cells [4]
and inhibition of effector T-cells [5]. These two mechanisms are synergistic to
immunosuppress, helping cancer cells to escape a potentially effective immune
response [2, 6]. These discoveries have triggered a great deal of new interest in
targeting TDO and IDO for drug discovery.
Although IDO1 and TDO have similar biological roles, their distribution is
different. IDO1 is an extrahepatic cytosolic enzyme that is distributed in various
tissues, including placenta, lung, liver, spleen, kidney, stomach, large intestine, small
intestine and colon, playing a key role in the tryptophan oxidative cleavage reaction [1,
7
]; TDO is mainly expressed in the liver, controlling the concentration of tryptophan
in the blood [1]. There is also a large difference in the structure between IDO1 and
TDO. IDO1 is a monomeric enzyme, and TDO is a homotetrameric enzyme. However,
their active sites are highly similar, though their sequence identity is only 16% [8, 9].
Furthermore, TDO exhibited high substrate specificity and catalyzed the oxidative
cleavage of Trp preferentially. In contrast, IDO1 showed greater substrate promiscuity
than TDO, and a range of indoleamines including Trp, serotonin and tryptamine could
be catabolized by IDO1 [10].
At present, many pharmaceutical companies and research institutes have
conducted extensive studies on IDO1 inhibition [11-31]. And numerous structurally
distinct chemical entities were disclosed, and this inhibitor structure diversity along
with its substrate promiscuity likely resulted from the flexibility of the active site
within IDO1 in part [25]. For the present, several IDO1 inhibitors have advanced into

clinical trials such as Epacadostat [26, 27], BMS986205 [28], Indoximod [29],
PF-0684003 [30] and Navoximod [31]. Compared with inhibitors of IDO1, there are
few studies on TDO inhibitors [32-37], and no inhibitors enter the clinical trial.
However, the current clinical results of IDO1 inhibitors are not very satisfying.
Epacadostat combined with PD-1 checkpoint inhibitor pembrolizumab for the
treatment of unresectable or metastatic melanoma, no significant improvement in
progression-free survival rate (PFS) was observed compared with pembrolizumab
alone [38]. It is speculated that selective inhibition of IDO1 has limited effects on
tumor growth, probably due to inhibition of IDO1 leading to TDO activation in
tumors, which compensates for the increased ratio of Trp/Kyn caused by IDO1
inhibition. Therefore, The IDO1/TDO dual inhibitors may become a new strategy for
tumor immunotherapy [39, 40].
A random screening of our in-house compound library, which featured an indole
scaffold, was conducted. And indole-2-carboxylic acid compounds 9a and 9k were
found to be the dual inhibitors with a novel scaffold, although these two hits displayed
weak inhibitory activity against both IDO1 and TDO. Thus, we took compounds 9a
and 9k as the starting point to embark on extensive structural modifications with an
aim to search for novel IDO1/TDO dual inhibitors with improved potency. Herein, the
chemical synthesis of these derivatives and the investigation on the structure-activity
relationships were reported.
9
a R = H, R = F, IC = 40.7 µM (IDO1), IC = 99.2 µM (TDO)
6 7 50 50
9
k R = CH , R = H, IC = 35.6 µM (IDO1), IC = 22.5 µM (TDO)
6 3 7 50 50
Figure 1. The structures of the hits 9a, 9k and the general structure of designed derivatives
2
. Results and discussion

2
.1 Chemistry
Two methods were used to construct the key intermediates
-bromo-1H-indole-2-carboxylate derivatives as shown in scheme 1. The
4
Hemetsberger's method was utilized to produce compounds 3a, 3c-3f and 3i-3m. In
the presence of strong base sodium ethoxide, the substituted benzaldehydes (1a, 1c-1f，
1i-1m) were condensed with ethyl azidoacetate to form alkenyl azides 2 with yields of
12-42%. Compounds 2 were heated to 180 ℃to afford 4-bromo-indole intermediates
3 in 30-93% yields. Compounds 3g and 3n were prepared using Fisher indole
synthesis method. It was noted that compound 6n was cyclized using PPA as the
1
solvent to give rise to two isomers. The H-NMR spectroscopy confirms that the two
isomers were respectively ethyl 4-nitro-6-bromide-1H-indole- 2-carboxylate (δH-7 7.93
ppm, 49.4%) and the desired ethyl 4-bromo-6-nitro-1H-indole-2-carboxylate (3n, δH-7
8
.38 ppm, 33.1%). The nitro group of 3n was reduced and acetylated to provide
-acetamido-4-bromo-indole intermediate 3o.
6
Scheme 1. Reagents and conditions: (i) NaOEt, ethyl azidoacetate, ethyl trifluoroacetate,
EtOH, 12.0%-42.3%. (ii) 1,2-dichlorobenzene, reflux, 30.0%-93.0%. (iii) NaNO , SnCl , HCl,
2
2
H O, -30 ℃, 89.2%-99.0%. (iv) ethyl 2-oxopropanoate, r.t., 37.0%-82.8%. (v) PPA, 80 ℃,
2
3
0.0%-33.1%. (vi) Fe, NH Cl, EtOH and H O, 80 ℃, 86%. (vii) CH COCl, TEA, DMF, r.t.,
4 2 3
9
6.6%.

As shown in scheme 2, the majority of target compounds 9 were constructed via
two steps starting from the key intermediates 3. 4-Bromo-substituted indole
derivatives 3 were subjected to a variety of aryl amines under the condition of
Buchwald-Hartwig cross coupling reaction to prepare aryl indole amines 8 in the yield
of 26-91%, which were hydrolyzed under basic conditions, providing target
compounds 9a-9g, 9i-9o, 9a-1-9a-17 and 9o-1-9o-24. By using iron powder and
ammonium chloride, the 7-nitro-substituted intermediate 8g was reduced and acylated
to generate compound 8h, which was subjected to hydrolysis to give the target
compound 9h with a 7-acetamido group on the indole ring. The target compound 9p
with a 6-ethylamine was obtained by reduction of 6-acetamido-substituted compound
8
o and hydrolysis. Starting from compounds 8a and 8k, the N-1 methyl substituted
compounds 9q and 9r were readily prepared by methylation and ester hydrolysis in
high yields. Compound 11 was a key substrate for the preparation of benzothiophene
derivative 3s. Starting from 4-acetamidobenzaldehyde 10, 2-aminobenzoic acid was
reacted with an aldehyde group to form an imine as a transient directing group, which
was further subjected to Pd (II)-catalyzed ortho-C-H chlorination in the presence of
Pd(OAc) , AgTFA and NCS to achieve the dichloro substituted aldehyde compound
2
11 in 57.8% yield [41]. Then compound 11 was reacted with ethyl thioglycolate in the
presence of K CO to form the benzothiophene intermediate 3s, which was converted
2
3
into target compound 9s by coupling and hydrolysis reaction.

Scheme 2. Reagents and conditions: (i) aryl amine, Pd (dba) , DavePhos, K PO (aq), toluene,
2
3
3
4
8
0 ℃ ; aryl amine, Pd (dba) , XantPhos, Na CO (aq), toluene, reflux; aryl amine, Pd (dba) ,
2 3 2 3 2 3
X-phos, Cs CO , 1,4-dioxane, 100 ℃ ; aryl amine, Pd (dba) , Xantphos, Cs CO , 1,4-dioxane,
2
3
2
3
2
3
1
00 ℃ , 25.9%-90.7%. (ii) NaOH, THF, EtOH and H O, r.t.; LiOH, THF, EtOH and H O, 40 ℃ ;
2 2
Na CO , EtOH and H O, reflux, 23.8%-99.7%. (iii) Fe, NH Cl, EtOH and H O, 80 ℃ , 54.9%;
2
3
2
4
2
CH COCl, DIEA, DCM, r.t., 58.9%. (iv) BH ·THF, THF, 0 ℃ ~r.t., 39.9%. (v) CH I, K CO ,
3
3
3
2
3
DMF, 97.5%-98.6%. (vi) NCS, Pd(OAc) , Ag(TFA), 2-aminobenzoic acid, TFA, DCE, 60 ℃ ,
2
5
7.8%. (vii) K CO , ethyl thioglycolate, DMF, 60 ℃ , 62.4%.
2 3
The variation of the substituents on the 2-position of indole ring was performed
and the synthesis of the corresponding compounds 14a-14g was depicted in Scheme 3.
-Methyl substituted compound 14a was constructed by two steps reaction. Using the
Bartoli indole synthesis method, 2-fluoro-5-bromonitrobenzene 12 and isopropenyl
magnesium bromide reacted at low temperature to yield
-methyl-4-bromo-7-fluoro-1H-indole 13, which was transformed into the target
2
2
product 14a by a coupling reaction. Upon treatment of 2-carboxyl acid substituted
compound 9a with Cu powder and quinolone as a solvent, compound 14b was
obtained in 46.5% yield through a decarboxylation reaction at a high temperature.

Upon treatment of compound 9a with borane in THF solution,
2
-hydroxymethyl-substituted compound 14c was resulted in 55.7% yield. Compounds
4d and 14e were prepared by condensation reaction of compound 9a with
1
ammonium hydroxide and hydroxylamine, respectively. Utilizing a series of
hydrolysis, condensation and dehydration reaction, compound 3a was converted into
2
-cyano-4-bromo-7-fluoroindole
15,
which
was
then
coupled
with
3
-chloro-4-fluoroaniline to give compound 14f. Subsequently, compound 14f was
transformed into the target compound 14g in the presence of hydroxylamine and TEA.
Scheme 3. Reagents and conditions: (i) isopropenyl magnesium bromide, THF, -40 ℃ , 20.6%.
(
ii) Pd (dba) , DavePhos, K PO (aq), toluene, 100 ℃ , 28.1%. (iii) Cu, quinoline, MW, 240 ℃ ,
2 3 3 4
4
6.5%; BH ·THF, THF, 0 ℃ , 55.7%; EDCI, HOBt, NH H O or NH OH·HCl, TEA, r.t.
3 3· 2 2
3
6.8%-48.7%. (iv) NaOH, THF, EtOH, H O, 40 ℃ , 99.0%; oxalyl chloride, DMF, DCM,
2
NH H O, r.t., 99.0%; POCl , Tol, Ar, reflux, 88.5%. (v) Pd (dba) , X-phos, Cs CO ,
3
·
2
3
2
3
2
3
1
,4-dioxane, MW, 100 ℃ , 40.9%. (vi) NH OH, TEA, EtOH, r.t., 59.0%.
2
The synthesis of compounds 17a-17f with different linker groups between indole
scaffold and aromatic group was outlined in Scheme 4. Compound 16a with an S
atom as the linker group could be constructed by the C-S coupling reaction between
compound 3a and 4-fluoro-3-chlorothiophenol, although the yield is very low.
Compound 3a was coupled with t-butyl carbamate, followed by removing the Boc
protecting group to form the 4-amino substituted intermediate 19. Reductive
amination of compound 19 with 3-chloro-4-fluorobenzaldehyde resulted in compound
1
6b. The reaction of compound 19 with 3-chloro-4-fluorobenzoyl chloride or

3
-chloro-4-fluorophenylacetyl chloride led to compounds 16c and 16e. Compound 19
was subjected to a nucleophilic addition reaction with 3-chloro-4-fluorophenyl
isocyanate to give 16f. Compound 16d was prepared by a Pd-catalyzed
aminocarbonylation reaction. The 4-Br substituted compound 3a was carbonylated
with carbon monoxide, forming in situ by CHCl and CsOH, and then directly reacted
3
with 3-chloro-4-fluoroaniline in a one-pot reaction to produce compound 16d in 16.9%
yield [42]. The target compounds 17a-17f were obtained by ester hydrolysis of
compounds 16a-16f.
Scheme
4.
Reagents and conditions: (i) 3-chloro-4-fluorobenzenethiol, CuI,
N,N-dimethylglycine, Cs CO , MW, 150 ℃ , 9.7%. (ii) LiOH, THF, EtOH and H O, 40 ℃ ,
2
3
2
4
3.4% ~ 97.1%. (iii) NH Boc, X-phos, Cs CO , 1,4-dioxane, 110 ℃ , 70.4%. (iv) CF COOH,
2 2 3 3
DCM, r.t., 94.9%. (v) 3-chloro-4-fluorobenzaldehyde, HOAc, NaBH , DCM, r.t., 66.5%;
4
3
3
3
-chloro-4-fluorobenzoic
acid,
SOCl2,
DMF,
DCM,
TEA,
43.3%;
-chloro-4-fluorophenylacetic acid, oxalyl chloride, DMF, DCM, TEA, 65%;
-chloro-4-fluorophenyl isocyanate, DCM, 50.9%. (vi) DPEPhos, Pd(OAc) , CHCl , CsOH
2
3
H O, 3-chloro-4-fluoroaniline, Tol, 80 ℃ , 16.9%.
2
2
.2 Inhibitory activities of indole derivatives against IDO1 and TDO enzyme
In order to investigate the structure-activity relationship of indole derivatives, we
carried out extensive structure variations on the indole scaffold, including the
,7-position of the indole skeleton, the indole NH group, 2-carboxyl moiety, and the
6

substituents on the 4-position. All compounds were evaluated by using recombinant
enzyme of IDO1 and TDO and the results were shown in Tables 1-4.
2
.2.1 Structural modifications on the indole skeleton
By scrutinizing the hits 9a and 9k, we speculated that the indole aryl amine frame
was a unique structural feature for this class of inhibitors. Therefore, the indole
fragment and the aromatic group on the 4-position of indole ring were the focus of the
modifications. Initially, the 2-carboxyl-indole ring was kept intact, a variety of
substituents were introduced onto the 6- and 7-position of the indole scaffold. As
shown in Table 1, when the 7-fluorine atom of the hit (9a) was removed (9b) or
replaced with an electron-donating group, such as methyl (9d), acetamido (9h), or an
electron withdrawing group, such as Cl (9c), CN (9f), NO (9g), the inhibitory
2
activities were lost. Only 7-methoxy substituted compound (9e, IDO1 IC = 32.4 µM;
5
0
TDO IC50 = 19.5 µM) retained the inhibitory activity against IDO1 and TDO.
Obviously, the modification space of the 7-position is very limited.
The variation of 6-substituents on the inhibitory effects was presented in Table 1.
The hit (9k) with a 6-methyl group had a micromolar level inhibitory activity, and the
IC50 values for IDO1 and TDO were 35.6 µM and 22.5 µM, respectively. The
replacement of methyl group with F (9i), Cl (9j), OCH (9l), CN (9m), or NO (9n)
3
2
substituent led to the loss of potency, while the acetamido (9o) or ethylamino group
9p) was incorporated onto the 6-position, the compounds had strong inhibitory
activities against both enzymes. The IC values for IDO1 of 8.40 µM and 1.75 µM,
(
5
0
respectively, and the inhibitory activity against TDO was comparable to that of IDO1,
showing IC50 values of 8.48 µM and 3.53 µM, respectively. The 6-acetamido
substituent or 6-ethylamine group was favorable for increasing the inhibitory activity.
It was assumed that these substituents might contribute to the binding of IDO1 or
TDO through hydrogen bonds. Among this series of compounds, the 6-ethylamino
substituted compound (9p) showed the strongest inhibitory activity, which was 23
times more active than the hit compound (9a). However, due to the high electron
density nature of this indole ring, compound (9p) was readily oxidized and unstable.

Therefore, in the subsequent structural optimization of the substituents on the
-position of the indole scaffold, we selected the 6-acetamido substituted compound
4
(9o) as a new lead compound for further SAR exploration.
The methylation of indole nitrogen was conducted on the hits 9a and 9k as well.
And none of the two compounds (9q and 9r) had inhibitory activity. Furthermore, we
also tentatively replaced the indole ring with a benzothiophene fragment (9s), which
had no inhibitory activity against IDO1. These results suggested that the NH in the
indole ring was important for the binding activity, presumably because NH was
involved in a hydrogen bonding and/or a steric hindrance was generated by the
introduction of the methyl group.
Table 1. Enzyme inhibitory activities for 6- or 7-substituted or 1-substituted
indole-2-carboxylic acid derivatives 9a-9s
F
HN
Cl
COOH
X
R
IC (µM)±SD
5
0
Compound
R
X
IDO1
TDO
9
a
7-F
H
7-Cl
7-CH
7-OCH
7-CN
7-NO
7-NHCOCH
6-F
6-Cl
6-CH
6-OCH
6-CN
6-NO
6-NHCOCH
6-NHC
7-F
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
40.7±3.03
>100
>100
>100
32.4±4.95
>100
99.2±51.5
N.D.
N.D.
N.D.
19.5±0.47
N.D.
a
9
b
9
c
9
d
3
9
9
9
e
f
g
3
>100
>100
>100
>100
N.D.
N.D.
N.D.
N.D.
2
9
h
3
9
9
i
j
9
k
3
35.6±3.93
>100
22.5±3.30
N.D.
9
l
3
9
m
>100
>100
8.40±1.70
1.75±0.12
>100
>100
>100
8.48±0.40
3.53±0.83
N.D.
9
n
2
9
o
p
q
3
9
9
H
2 5
NCH
3

9
9
r
s
6-CH
6-NHCOCH
Epacadostat -
3
NCH
3
>100
>100
0.058±0.012
0.075±0.017
N.D.
N.D.
-
S
-
-
3
Navoximod
-
0.80±0.051
a
not detected.
The SAR on the 2-position of the indole scaffold was investigated preliminarily
with an attempt to displace carboxyl group to improve the drug-like property.
However, as shown in Table 2, the results demonstrated that the 2-carboxyl group
played a critical role in the binding. The replacement of the carboxyl group with a
methyl (14a), hydroxymethyl (14c) or cyano group (14f), or removal of the carboxyl
group (14b) resulted in loss of inhibition. In particular, the close bioisosteric group of
carboxyl moiety, including the carbamoyl (14d), hydroxamic acid (14e), and
hydroxycarbamimidoyl group (14g), was grafted onto the 2-position, the inhibition
disappeared as well. We conjectured that the carboxyl anion might act as an electron
donor to cooperate with the iron ion in the heme of IDO1. After incubation compound
9
a with IDO1, the UV spectrum was measured and the wavelength did not show the
red-shift [43], suggesting that compound 9a did not cooperate with the iron ion in
IDO1. Therefore, we speculated that the carboxyl group was supposed to bind with
the key amino acids within IDO1/TDO via electrostatic interactions.
Table 2. Enzyme inhibitory activities for 2-substituted indole derivatives 14a-14g
IC (µM)
5
0
Compound
R2
IDO1
TDO
9
a
COOH
40.7±3.03
>100
99.2±51.5
a
1
1
1
4a
4b
4c
CH
H
3
N.D.
>100
>100
N.D.
N.D.
N.D.
CH
OH
2
1
4d
>100

N.D.
1
4e
4f
4g
>100
>100
1
CN
N.D.
N.D.
-
1
>100
Epacadostat
Navoximod
-
-
0.058±0.012
0.075±0.017 0.80±0.051
a
not detected.
The NH group on the 4-position of indole ring could be regarded as a linker
between the indole scaffold and the aromatic group. The influence of this linker on the
inhibition was also evaluated. As shown in Table 3, one-atom linker such as S atom
(17a), two-atom linker such as aminomethylene (17b), carbamoyl (17c), formamido
(
17d), or three-atom linker such as NHCOCH (17e) or urea group (17f) was utilized
2
and all of the resultant compounds showed no inhibition against IDO1. These results
indicated that the linker NH was an important pharmacophore fragment. It is assumed
that the NH group not only provides a suitable distance between the indole ring and
the aromatic moiety, but also might serve as a hydrogen bond donor to contribute to
the binding.
Table 3. Enzyme inhibitory activities for 4-substituted indole-2-carboxylic acid
compounds 17a-17f.
IC (µM)
5
0
Compound
Y
IDO1
TDO
9
a
NH
S
-NHCH
-NHCO-
-CONH-
-NHCOCH
40.7±3.03
>100
99.2±51.5
a
1
7a
7b
7c
7d
7e
7f
N.D.
1
2
-
>100
>100
>100
>100
N.D.
N.D.
N.D.
N.D.
N.D.
1
1
1
2
-
1
-NHCONH-
>100

Epacadostat
Navoximod
-
-
0.058±0.012
0.075±0.017 0.80±0.051
-
a
not detected.
Taken together, the efforts on the modifications of indole ring demonstrated that
-carboxyl indole ring and 4-amino linker were the essential structure moiety for
2
producing inhibition against IDO1 and TDO. Grafting an acetamido or ethylamino
group was favorable for the binding and giving rise to micromolar inhibitors, although
only very limited substituents were allowed to incorporate onto the 6- and 7-position
of the indole ring. Therefore, we selected two representative indole scaffolds, one is
7
-fluoro-2-carboxyl-indole and the other is 6-acetamido-2-carboxyl-indole, as the
templates for further SAR explorations on the aromatic moiety of compounds 9a and
9
2
o.
.2.2 Structural modifications on the aryl ring
The variation of aromatic group of compound 9a was performed and the results
were presented in Table 4. When the 4'-position was a fluorine atom, the 3'-position
was substituted by Cl (9a), Br (9a-2), OCH (9a-3) or OCF (9a-4), respectively, the
3
3
obtained compounds had moderate inhibitory activities, while a fluorine atom (9a-1)
or cyano group (9a-5) was placed, loss of inhibition was observed. When the 3'-Cl in
the hit 9a was maintained, and the 4'-F was replaced by various groups, such as Cl,
OCH , OCF or CF , none of them (9a-6~9a-9) produced inhibitory effects on IDO1.
3
3
3
These results indicated that a steric bulky hydrophobic group was allowed on the
'-position while there is a limited space for the substituents on the 4'-position. The F
3
atom on the 4'-position of the benzene ring was beneficial to the inhibitory activity.
Except for the 3',4'-disubstituted phenyl groups, other substituted benzene rings
were explored as well. The derivatives bearing 3'-methoxy-5'-trifluoromethyl (9a-10),
3
'-methoxy-5'-chloro (9a-11) or 2'-carboxy-5'-methoxy (9a-13) substituent had no
inhibitory activity. Compounds with 2'-fluoro-5'-bromo (9a-12, IDO1 IC = 28.1 µM,
5
0
TDO IC = 41.4 µM) or 3',4',5'-trimethoxyl (9a-14, IDO1 IC = 22.2 µM; TDO IC
50
5
0
50
=
23.3 µM) substituent had moderate inhibitory activity. We also examined the

inhibitory effect of the indoleamine and its derivatives substituted for the
3
'-chloro-4'-fluoro-benzene amine. When the indoleamine (9a-15) or
'-F-indoleamine (9a-16) was utilized, the compounds showed no inhibitory activity.
7
Surprisingly, compound 9a-17 bearing 7'-fluoro-2'-carboxy-indoleamine at the
-position of the indole scaffold showed a pronounced potency against IDO1 and
TDO, the IC values are 2.72 µM and 3.48 µM, respectively, which is 15 and 28.5
4
5
0
times higher than that of hit compound 9a. Compound 9a-17 had the strongest
inhibitory activity against IDO1 among the 7-fluoro-indole-2-carboxylic acid series of
compounds. Overall, both the substitution pattern of the benzene ring and the
properties of the substituents have impacts on the inhibitory activity, and further
modifications on this moiety might provide chance to improve the potency.
Table 4. Enzyme inhibitory activities for 4-arylamino-7-fluoro-indole-2-carboxylic acid
derivatives 9a-1~9a-17
IC (µM)
5
0
Compound
Ar
IDO1
TDO
9
a
40.7±3.03
99.2±51.5
a
9a-1
9a-2
9a-3
9a-4
9a-5
9a-6
9a-7
9a-8
>100
9.93±0.85
54.4±15.7
16.3±2.33
>100
N.D.
10.5±0.06
31.4±1.44
16.5±1.96
N.D.
>100
N.D.
>100
N.D.
>100
N.D.

9
a-9
>100
>100
N.D.
N.D.
9a-10
9a-11
9a-12
9a-13
9a-14
9a-15
9a-16
9a-17
>100
28.1±3.72
>100
N.D.
41.4±4.25
N.D.
22.2±2.34
>100
23.3±0.46
N.D.
>100
N.D.
2.72±0.67
3.48±0.22
-
Epacadostat
Navoximod
a
-
-
0.058±0.012
0.075±0.017 0.80±0.051
not detected.
The SAR investigation based on the template 6-acetamido-indole-2-carboxylic
acid was also carried out and the inhibitory activities were listed in Table 5.
According to the previous SAR results, the fluoro atom was kept on the 4'-position of
the benzene ring, and a variety of substituents were incorporated onto the 3'-position
to explore the SAR of this series of derivatives first. Satisfyingly, grafting a fluoro
atom and various hydrophobic groups of different sizes (9o-1 ~ 9o-13), all compounds
produced noticeable inhibition against IDO1 and TDO, the IC value ranged from
5
0
1
.17 µM to 10.0 µM for IDO1 and 1.44 µM to 9.63 µM for TDO. Compound 9o-1
with a 3', 4'-difluoro phenyl group exhibited marked inhibition against both enzymes
IDO1 IC = 1.17 µM, TDO IC = 1.55 µM), and it is the most potent dual inhibitor
(
5
0
50

in this class of indole inhibitors. Among the alkyloxyl substituted derivatives (9o-2 ~
o-8), compounds with a relatively smaller size group, such as methoxyl (9o-2),
9
ethoxyl (9o-3), propoxyl (9o-4) and trifluoromethoxyl (9o-5), showed strong
inhibition against IDO1 with IC50 values at the low micromolar level, and potent
inhibition against TDO as well. In contrast, the placement of somewhat larger groups,
such as isopropoxyl (9o-6), cyclopropylmethoxyl (9o-7), or trifluoroethoxyl (9o-8)
moiety would result in a slightly decrease in potency. The incorporation of even
more bulky substituents, such as phenoxyl (9o-9) and benzyloxyl (9o-10) moiety led
to more reduction in inhibition. In comparison with that of compound 9o-1, the
inhibition of compound 9o-10 decreased by 8.5-fold against IDO1 and 3.4-fold
against TDO, respectively. These results demonstrated that the inhibitory activity
decreased with increasing the size of the 3'-substituent on the benzene ring.
Table 5. Enzyme inhibitory activities for 4-arylamine-6-acetamido-indole-2-carboxylic
acid derivatives 9o-1 ~ 9o-24
IC (µM)
5
0
Compound
Ar
IDO1
TDO
9
o
8.40±1.70
8.48±0.40
9o-1
9o-2
9o-3
9o-4
9o-5
9o-6
1.17±0.15
1.23±0.33
2.20±0.01
1.48±0.15
1.37±0.18
3.09±0.35
1.55±0.25
2.49±0.16
5.13±0.68
4.46±0.08
3.62±0.21
6.46±1.59
9
o-7
3.11±0.35
4.09±0.98

9
o-8
o-9
4.62±0.83
5.83±1.53
9.63±0.63
1.44±0.13
9
9
9
9
o-10
o-11
o-12
10.0±1.07
1.83±0.18
2.95±0.38
5.24±0.94
1.70±0.49
5.45±0.18
9
o-13
3.89±0.31
2.53±0.40
9
9
9
9
9
9
9
9
9
o-14
o-15
o-16
o-17
o-18
o-19
o-20
o-21
o-22
5.28±1.01
1.71±0.53
3.18±0.27
23.7±6.89
55.5±39.4
38.5±2.65
10.8±0.83
3.41±0.39
4.73±0.49
7.69±0.11
3.01±0.01
5.50±0.31
5.71±0.78
23.2±2.14
46.1±6.24
11.7±0.91
3.62±0.31
5.27±2.55
9
o-23
o-24
3.18±0.56
7.52±2.35
7.12±0.27
6.78±0.05
9
Epacadostat
Navoximod
-
-
0.058±0.012
0.075±0.017
-
0.80±0.051
As to the alkyl substituted derivatives, the similar trend was observed.
Compound 9o-11 (IDO1 IC = 1.83 µM; TDO IC = 1.70 µM) with a methyl group
5
0
50

was more potent than compound 9o-12 (IDO1 IC = 2.95 µM; TDO IC = 5.45 µM)
5
0
50
with a cyclopropyl substituent. The thienyl substituted derivative 9o-13 had similar
potency to compound 9o-12. These results demonstrated that a small hydrophobic
group such as F, OCH and CH on the 3'-position was favorable for the inhibition.
3
3
The compounds with mono-substituted benzene ring or pyridine ring (9o-14 ~
o-22) were also evaluated as shown in Table 5. The 4'-fluoro substituted derivative
9
(9o-14) displayed comparable activity to compound 9o. Compound 9o-15 (IDO1 IC50
=
1.71 µM; TDO IC = 3.01 µM) with a methoxyl group produced an improved
5
0
activity compared with compounds 9o and 9o-14. Especially, when the substituents
OCF H or OCF was brought in, the inhibition against IDO1 would be inflicted
2
3
significantly (IDO1 inhibition, 4'-OCH > 4'-OCF H > 4'-OCF ). These data indicated
3
2
3
that the oxygen atom on the 4'-position played an important role and it might serve as
an H-bond acceptor, as the ability of forming H-bond decreased with the fluoro atom
increasing. In addition, compound 9o-18 (IDO1 IC = 55.5 µM; TDO IC = 23.2 µM)
50
50
with a trifluoromethyl group showed pronounced reduction against both enzymes.
This result further supported that an H-bond acceptor such as F or O atom on the
4
'-position was critical for the binding.
In comparison with compounds 9o-14 and 9o-15, the pyridine derivatives (9o-19
9o-22) showed reduction in potency, partly due to the decreased H-bond forming
~
capability of the fluoro atom and the methoxyl group, presumably.
In this series, we also prepared compound 9o-23 with a trimethoxyl substituted
benzene ring and compound 9o-24 bearing an indazole fragment. Both of them
displayed micromolar inhibition against the two enzymes. This result further
suggested that various aromatic groups could be tolerated at the 4-position of the
indole scaffold.
Generally, 6-acetamido-indole-2-carboxylic acid derivatives are more potent
than the 7-fluoro-substituted series, as exemplified by compounds 9o-1 (IDO1 IC =
5
0
1
.17 µM; TDO IC = 1.55 µM) and 9o-2 (IDO1 IC = 1.23 µM; TDO IC = 2.49
50 50 50

µM), which possess remarkable potency against both enzymes, while the counterpart
of 7-fluoro-substituted derivatives (compounds 9a-1 and 9a-3) showed very weak
-5
inhibition with IC > 10 M. In addition, the aromatic moiety on the 4-position of
5
0
6
-acetamido substituted series was allowed to be varied to a greater extent, and thus
providing more opportunities for further improving potency.
2
.3 The structure confirmation and activity evaluation of the oxidation product
of compound 9p
We accidentally found that compound 9p was unstable and easily oxidized. The
1
oxidation product was isolated and identified by H-NMR and MS, which could be
p-benzoquinone 9p-O. In order to confirm the structure of compound 9p-O, a targeted
synthesis was designed as shown in Figure 2. Compound 8p was oxidized with
mCPBA to yield compound 8p-O, which was confirmed to be a p-benzoquinone by
X-ray single crystal diffraction (Figure 2). The ester hydrolysis of compound 8p-O
gave rise to compound 9p-O. Surprisingly, compound 9p-O exhibited strong
inhibitory activity against IDO1 with IC value of 18 nM and TDO with IC value of
5
0
50
2
5 nM, respectively.
F
N
Cl
O
EtO
N
N
H
H
O
8p-O
F
F
F
Cl
N
Cl
N
Cl
NH
i
ii
COOEt
COOH
COOEt
N
H
N
H
N
H
N
H
N
N
H
H
O
O
8p-O
9p-O
8p
IDO1 IC = 18 nM
5
0
TDO IC = 25 nM
5
0
Reagents and conditions: (i) m-CPBA, 0 ℃ , DCM, 81%. (ii) LiOH, r.t., THF, H O, 59.5%
2
Figure 2. The crystal structure of compound 8p-O and the preparation of oxidation product 9p-O.

2
.4 Predicted binding modes of compound 9o to IDO1 and TDO
In order to explore the binding mode of this class of compounds, compound 9o
which served as a template and lead structure of the 6-acetamido-indole-2-carboxylic
acid series was tentatively selected and docked into the crystal structures of IDO1
(5WMU) [44] and TDO (5TIA) [8], respectively, by using CDOCKER protocol
integrated in Accelrys Discovery Studio [45]. And then molecular dynamics
simulation was performed with Amber16 package [46] to further optimize the binding
pose of compond 9o within the binding pocket of each enzyme (the results of MD
simulation shown in supplementary information S1). As shown in Figure 3,
compound 9o occupies the pocket on the top of heme molecule in both IDO1 and
TDO, the overall orientations are alike. However, the precise binding poses are
somewhat different. In IDO1 (Figure 3A and 3B), the NH and carbonyl oxygen on
6
-acetamido group form hydrogen bonds with key amino acid residues Gly262 and
Cys129, respectively. The 2-carboxylate group on the indole scaffold creates H-bonds
with key amino acid residues Tyr126 and Ser167 as well. These hydrogen bonding
interactions are supposed to make critical contributions to the binding since the
acetamido and carboxyl moieties are essential pharmacorphore groups as SAR
demonstrated. The indole ring forms a T-shape π-π interaction with Phe163, which is
also an important feature for known IDO1 inhibitors [27, 47]. The
4
'-fluoro-3'-chloro-phenyl group on the 4-position extends into a subpocket close to
Arg231, and the 4'-F can build a hydrogen bond with the key amino acid Arg231. This
is also consistant with the SAR that an H-bond acceptor on the 4'-position is benefical
to the inhibitory potency.

Figure 3. Predicted binding modes of compound 9o to IDO1 (A and B) and TDO (C and D) using
CDOCKER and dynamics simulation. (A, C): The binding mode of compound 9o to
IDO1(5WMU, A) and TDO (5TIA, C), the enzyme is shown in gold, compound 9o is shown as
sticks with sky blue carbon atoms. The residues that interact with compound 9o are shown as
sticks with gold carbon atoms, and hydrogen bonds are indicated by red lines. The images were
generated by using Chimera 1.12. (B, D): The schematic 2D diagram of the key interactions in
between compound 9o with IDO1 (5WMU, B) and TDO (5TIA, D), respectively.
Compared with IDO1, one of the key differences within the binding pocket is
that an His76 is situated on the top of heme in TDO and the corresponding amino acid
is Ser167 in IDO1 (Figure 3C and 3D). Due to the presence of a large side chain of
His76, the carboxyl group of compound 9o moves slightly toward the heme molecule
in TDO, resulting in a different binding pose within the cavity of TDO. The 2-COOH
not only constructs an H-bond with His76, but also can build an electrostatic
interaction with the ferrous ion of the heme. The NHCOCH group can interact with
3

Ala150 via a hydrogen bond. The indole ring establishes hydrophobic interactions
with Ala150 and Phe72. These binding features of compound 9o suggested that 6-
acetamido-2-carboxyl-indole scaffold played a crucial role in TDO binding as well. It
is noted that the NH group on the 4-position of the indole ring forms an H-bond with
the carbonyl oxygen of Arg144 in TDO, while it has no interaction in IDO1. The 4'-F
of benzene ring forms a hydrogen bond with the key amino acid Thr336. This also
demonstrated that an appropriate H-bond acceptor such as F or OCH group on the
3
4
'-position could contribute to the binding affinity to some extent. Taken together, the
binding modes indicated that the 6-acetamido-2-carboxyl-indole ring and the
'-fluoro-substituted benzene ring are characteristic moieties of this class of dual
4
inhibitors, which make essential contributions to the binding to both enzymes,
although the binding poses of compound 9o and the involved key amino acids within
the pocket are somewhat different.
2
.5 Cellular IDO1/TDO inhibitory activities of indole-2-carboxylic acid
derivatives
As the series of compounds bearing the 6-acetamido-indole-2-carboxylic acid
scaffold presented potent enzymatic inhibition, most of them were further tested for
their cellular IDO1 and TDO inhibitory activity using A172 cells. IFNγ was used to
induce the overexpression of IDO1 and TDO in A172 cells. The inhibitory activity
was determined by measuring the amount of the kynurenine released from the cells
[
48, 49]. The results (see supplementary information S2) showed that compound
o-23 was the most potent inhibitor, which had an inhibition rate of 77.7% at the
concentration of 100 µM and 55.9% at 50 µM concentration, respectively.
9
2
.6 Effects of indole-2-carboxylic acid derivatives on T cell proliferation
Both IDO1 and TDO could contribute to the tumor progression through its
capability to block T lymphocyte proliferation by consuming Trp locally. Thus, to
determine whether this class of dual inhibitors could enhance the T cell proliferation
which was stimulated by LLC cells, we performed T cell proliferation assay using
compound 9o-23 with the cellular inhibitory activity [17]. As shown in Figure 4,

Compound 9o-23 could increase the proliferation of T cells at a concentration of 10
µM noticeably, although it much less potent than Navoximod. The results
demonstrated that compound 9o-23 could reverse the suppression of T lymphocyte
caused by IDO1 and TDO.
Figure 4. Compound 9o-23 increased the proliferation of T cells stimulated with LLC tumor cells.
Each bar of the graph indicates the mean of three replicate wells with standard error of the mean.
(
*) P < 0.05, (**) P < 0.01, (***) P < 0.001 compare to vehicle control.
2
.7 Antitumor activity evaluation of compound 9o-23
As compound 9o-23 exhibited potent IDO1/TDO inhibition on both enzymatic
and cellular assay, along with its capability to induce T cell proliferation, it was
selected for further in vivo antitumor evaluation. The antitumor activity of compound
9
o-23 against mouse B16F10 melanoma was carried out on a mouse subcutaneous
xenograft model. After consecutive administration of 19 days, the tumor growth
inhibition was observed (Figure 5, A). The inhibition was 43.2% at the dose of 50
mg/kg, and 44.7% at the dose of 100 mg/kg, respectively. In addition, the body weight
of the treated mice had no significant loss compared with the control group. These
data further suggested that this series of novel dual inhibitors could serve as
promising lead structure for developing highly potent and drug-like dual inhibitors to
be useful for the treatment of cancer.

Figure 5. In vivo anti-tumor activity of 9o-23 in B16F10 xenograft mice. (A) Tumors weights of
each group after 19 days of treatment. The control group mice bearing B16F10 xenografts were
dosed orally with vehicle (0.5% Sodium salt of Caboxymethyl Cellulose, CMC-Na); the CTX
(Cyclophosphamide) group were administered CTX intraperitoneally at the dose of 100 mg/kg;
the treated group were administered 9o-23 orally at the dose of 50 mg/kg or 100 mg/kg. * p < 0.05
and *** p < 0.001 versus vehicle. (B) The body weight of each group after the treatment. There is
no obvious body weight difference among all of the groups.
3. Conclusion
Based on the hits 9a and 9k identified by a random screening, a variety of
indole-2-carboxylic acid derivatives were synthesized and their inhibitory activity
against IDO1 and TDO were evaluated. The structure-activity relationships were
investigated extensively and resulted in a new class of dual inhibitors with a
6
-acetamido-indole-2-carboxylic acid scaffold, as exemplified by compounds 9o and
o-1 ~ 9o-24. The binding modes of compound 9o gave some insights for
9
rationalizing the SARs. The scaffold situated in a relatively restricted subpocket on
the top of the heme and the space of structural modifications was very limited. In
contrast, the aromatic moiety on the 4-position extended into an area around a loop
and Arg231 (Arg144 in TDO), which possessed some extent flexibility and provided
an opportunity for structural optimization to improve the potency and diversity, as the
SAR results demonstrated.
In addition, a new para-benzoquinone derivative compound 9p-O was identified,
which displayed strong inhibitory activity with IC50 value at the double digit
nanomolar level against both IDO1 and TDO, respectively.

In summary, novel IDO1 and TDO dual inhibitors were disclosed and the results
obtained in this work will guide further structural optimizations for the development
of diversified new chemical entities with improved potency.
4
4
. Experimental section
.1 Chemical Synthesis General
1
H NMR spectra were recorded with a Varian Mercury 400 or 500 spectrometer
using tetramethylsilane (TMS) as the internal standard in Acetone d , DMSO d or
6
6
CDCl . High preparation mass spectra (HRMS) were recorded on an Agilent
3
Technologies LC/MSD TOF spectrometer. Melting points were measured on a Yanaco
micro melting point apparatus. All chemicals and solvents used were of reagent grade
without further purification or dried before used. All the reactions were monitored by
thin-layer chromatography (TLC) under a UV lamp at 254 nm. Column
chromatography separations were performed with silica gel (200-300 mesh).
4
.2. General procedure for preparation of 1H-indole-2-carboxylic acids (9a-9s,
9
a-1~9a-17, 9o-1~9o-24)
Method 1: To a solution of compound 8 in THF/EtOH (1:1) was added 1.0 M NaOH
solution (5 eq) dropwise and stirred at room temperature overnight. The solvent was
evaporated in vacuo and the residue was acidified with 1N HCl. After filtration, the
title compound was obtained. Compounds 9a-9e, 9g-9o, 9q-9s, 9a-2~9a-4, 9a-6,
9
a-12~9a-14, 9a-17, 9o-2, 9o-3, 9o-5, 9o-11~9o-13, 9o-23 were prepared with
method 1.
Method 2: To a solution of compound 8 in THF/EtOH (1:1) was added 1.0 M LiOH
solution (5 eq) dropwise and stirred at 40 ℃ until the ester group was completely
hydrolyzed. The solvent was evaporated in vacuo and the residue was acidified with
1
9
2
N HCl. After filtration, the title compound was obtained. Compounds 9f, 9a-1, 9a-5,
a-7~9a-11, 9o-1, 9o-4, 9o-6~9o-10, 9o-14~9o-22, 9o-24 were prepared with method
.

Method 3: To a solution of compound 8 in EtOH was added 1.0 M Na CO solution
2
3
(5 eq) and stirred at refluxing temperature until the ester group was completely
hydrolyzed. The solvent was evaporated in vacuo and the residue was acidified with
1
N HCl. After filtration, the title compound was obtained. Compounds 9p, 9a-15,
9
a-16 were prepared with method 3.
4
.2.1. 4-((3-Chloro-4-fluorophenyl)amino)-7-fluoro-1H-indole-2-carboxylic acid
1
(
9a): copper green solid, yield: 99.0%, mp: 135-137 ℃ , H-NMR (400 MHz,
DMSO-d ) δ (ppm): 13.06 (brs, 1H), 12.24 (s, 1H), 8.30 (s, 1H), 7.27-7.24 (m, 2H),
6
7
.17 (dd, J = 6.5 Hz, J = 2.5 Hz, 1H), 7.05 (ddd, J = 9.0 Hz, J = 4.0 Hz, J = 3.0
1 2 1 2 3
Hz, 1H), 6.97 (dd, J = 10.5 Hz, J = 8.0 Hz, 1H), 6.73 (dd, J = 8.5 Hz, J = 3.5 Hz,
1
2
1
2
+
1
H); HRMS (ESI): m/z, Calcd. for C H O N ClF [M+H] : 323.0393, Found
15 10 2 2 2
3
23.0393.
4
.2.2. 4-((3-Chloro-4-fluorophenyl)amino)-1H-indole-2-carboxylic acid (9b):
1
copper green solid, yield: 94.0%, mp: 182-183 ℃ , H-NMR (400 MHz, Acetone-d ) δ
6
(ppm): 10.87 (brs, 1H), 7.34 (s, 1H), 7.31 (d, J = 5.6 Hz, 1H), 7.24-7.14 (m, 4H), 6.92
+
(
d, J = 7.2 Hz, 1H); HRMS (ESI): m/z, Calcd. for C H O N ClF [M+H] : 305.0488,
1
5
11
2
2
Found 305.0495.
4
.2.3. 7-Chloro-4-((3-chloro-4-fluorophenyl)amino)-1H-indole-2-carboxylic acid
1
(
9c): brown solid, yield: 98.0%, mp: 237-239 ℃ , H-NMR (400 MHz, DMSO-d ) δ
6
(ppm): 13.02 (brs, 1H), 11.87 (s, 1H), 8.45 (s, 1H), 7.41 (s, 1H), 7.33-7.27 (m, 2H),
7
.18-7.14 (m, 2H), 6.79 (d, J = 8.4 Hz, 1H); HRMS (ESI): m/z, Calcd. for
+
C H O N Cl F [M+H] : 339.0098, Found 339.0097.
15
10
2
2
2
4
.2.4. 4-((3-Chloro-4-fluorophenyl)amino)-7-methyl-1H-indole-2-carboxylic acid
1
(
9d): white solid, yield: 95.0%, mp: 224-226 ℃ , H-NMR (400 MHz, DMSO-d ) δ
6
(ppm): 12.82 (s, 1H), 11.58 (s, 1H), 8.21 (s, 1H), 7.24 (t, J = 8.8 Hz, 2H), 7.15 (dd, J1
=
6.0 Hz, J = 1.6 Hz, 1H), 7.05-7.03 (m, 1H), 6.92 (d, J = 7.2 Hz, 1H), 6.75 (d, J =
2
+
7
.6 Hz, 1H), 2.44 (s, 3H); HRMS (ESI): m/z, Calcd. for C H O N ClF [M+H] :
1
6
13
2
2

3
19.0644, Found 319.0645.
4
.2.5.
4-((3-Chloro-4-fluorophenyl)amino)-7-methoxy-1H-indole-2-carboxylic
1
acid (9e): brownish green solid, yield: 99.0%, mp: 193-195 ℃ , H-NMR (400 MHz,
DMSO-d ) δ (ppm): 11.71 (s, 1H), 8.05 (s, 1H), 7.20 (t, J = 8.8 Hz, 1H), 7.10 (s, 1H),
6
7
.01 (d, J = 5.2 Hz, 1H), 6.92-6.89 (m, 1H), 6.77 (d, J = 8.0 Hz, 1H), 6.71 (d, J = 8.0
+
Hz, 1H), 3.87 (s, 3H); HRMS (ESI): m/z, Calcd for C H O N ClF [M+H] :
1
6
13
3
2
3
35.0593, Found 335.0595.
4
.2.6. 4-((3-Chloro-4-fluorophenyl)amino)-7-cyano-1H-indole-2-carboxylic acid
1
(
9f): yellow solid, yield: 97.7%, mp: >250 ℃ , H-NMR (400 MHz, DMSO-d ) δ
6
(
ppm): 13.07 (brs, 1H), 12.50 (s, 1H), 9.03 (s, 1H), 7.57-7.55 (m, 2H), 7.47 (dd, J =
1
6
.4 Hz, J = 2.4 Hz, 1H), 7.43 (t, J = 8.8 Hz, 1H), 7.34-7.30 (m, 1H), 6.75 (d, J = 8.0
2
+
Hz, 1H); HRMS (ESI): m/z, Calcd for C H O N ClF [M+H] : 330.0440, Found
1
6
10
2
3
3
30.0426.
4
.2.7. 4-((3-Chloro-4-fluorophenyl)amino)-7-nitro-1H-indole-2-carboxylic acid
1
(
9g): red-brown solid, yield: 97.0%, mp: 242-244 ℃ , H-NMR (400 MHz, DMSO-d )
6
δ (ppm): 10.80 (s, 1H), 9.71 (s, 1H), 8.14 (d, J = 9.2 Hz, 1H), 7.74 (s, 1H), 7.60 (dd,
J = 6.4 Hz, J = 2.4 Hz, 1H), 7.50 (t, J = 8.8 Hz, 1H), 7.43-7.40 (m, 1H), 6.72 (d, J =
1
2
+
9
3
.2 Hz, 1H); HRMS (ESI): m/z, Calcd for C H O N ClF [M+H] : 350.0338, Found
15 10 4 3
50.0341.
4
.2.8. 7-Acetamido-4-((3-chloro-4-fluorophenyl)amino)-1H-indole-2-carboxylic
1
acid (9h): brown solid, yield: 79.1%, mp: 177-179 ℃ , H-NMR (400 MHz, DMSO-d )
6
δ (ppm): 13.07 (brs, 1H), 11.55 (s, 1H), 9.67 (s, 1H), 8.25 (s, 1H), 7.72 (d, J = 8.0 Hz,
1
7
3
3
H), 7.25 (t, J = 9.2 Hz, 1H), 7.23 (s, 1H), 7.15 (dd, J = 6.4 Hz, J = 2.8 Hz, 1H),
1 2
.04 (ddd, J = 8.8 Hz, J = 4.0 Hz, J = 2.8 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 2.13 (s,
1
2
3
+
H); HRMS (ESI): m/z, Calcd for C H O N ClF [M+H] : 362.0702, Found
1
7
14
3
3
62.0694.
.2.9. 4-((3-Chloro-4-fluorophenyl)amino)-6-fluoro-1H-indole-2-carboxylic acid
4

1
(
9i): white solid, yield: 93.0%, mp: 226-229℃ , H-NMR (400 MHz, DMSO-d ) δ
6
(ppm): 12.83 (s, 1H), 11.78 (s, 1H), 8.59 (s, 1H), 7.37-7.33 (m, 3H), 7.25-7.23 (m,
1
H), 6.63 (d, J = 9.2 Hz, 1H), 6.53 (d, J = 12.4 Hz, 1H); HRMS (ESI): m/z, Calcd. for
+
C H O N ClF [M+H] : 323.0393, Found 323.0403.
15
10
2
2
2
4
.2.10. 6-Chloro-4-((3-chloro-4-fluorophenyl)amino)-1H-indole-2-carboxylic acid
1
(
9j): white solid, yield: 94.0%, mp: 205-207 ℃ , H-NMR (400 MHz, DMSO-d ) δ
6
(
ppm): 11.86 (d, J = 1.6 Hz, 1H), 8.63 (brs, 1H), 7.39-7.33 (m, 3H), 7.23 (ddd, J =
1
8
.8 Hz, J = 4.4 Hz, J = 2.8 Hz, 1H), 6.95-6.94 (m, 1H), 6.66 (d, J = 1.6 Hz, 1H);
2 3
+
HRMS (ESI): m/z, Calcd. for C H O N Cl F [M+H] : 339.0098, Found 339.0103.
1
5
10
2
2
2
4
.2.11. 4-((3-Chloro-4-fluorophenyl)amino)-6-methyl-1H-indole-2-carboxylic acid
1
(
9k): brown solid, yield: 94.0%, mp: 155-157 ℃ , H-NMR (400 MHz, DMSO-d ) δ
6
(
ppm): 11.57 (s, 1H), 8.28 (brs, 1H), 7.29 (t, J = 12.0 Hz, 1H), 7.24 (dd, J = 8.4 Hz,
1
J2 = 2.8 Hz, 1H), 7.19 (s, 1H), 7.16-7.12 (m, 1H), 6.79 (s, 1H), 6.63 (s, 1H), 2.33 (s,
+
3
H); HRMS (ESI): m/z, Calcd. for C H O N ClF [M+H] : 319.0644, Found
16
13
2
2
3
19.0655.
4
.2.12.
4-((3-Chloro-4-fluorophenyl)amino)-6-methoxy-1H-indole-2-carboxylic
1
acid (9l): celadon solid, yield: 82.6%, mp: 108-110 ℃ , H-NMR (400 MHz,
DMSO-d ) δ (ppm): 12.63 (s, 1H), 11.56 (s, 1H), 8.37 (s, 1H), 7.32 (t, J = 9.2 Hz, 1H),
6
7
4
3
3
.29 (dd, J = 6.4 Hz, J = 2.8 Hz, 1H), 7.24-7.23 (m, 1H), 7.18 (ddd, J = 8.8 Hz, J =
1 2 1 2
.0 Hz, J = 2.8 Hz, 1H), 6.43 (d, J = 1.2 Hz, 1H), 6.38 (d, J = 2.0 Hz, 1H), 3.74 (s,
3
+
H); HRMS (ESI): m/z, Calcd. for C H O N ClF [M+H] : 335.0593, Found
16
13
3
2
35.0590.
4
.2.13. 4-((3-Chloro-4-fluorophenyl)amino)-6-cyano-1H-indole-2-carboxylic acid
1
(
9m): khaki solid, yield: 97.8%, mp: >250 ℃ , H-NMR (400 MHz, DMSO-d ) δ
6
(ppm): 13.27 (brs, 1H), 12.27 (s, 1H), 8.66 (s, 1H), 7.43-7.35 (m, 4H), 7.29-7.25 (m,
+
1
H), 6.89 (s, 1H); HRMS (ESI): m/z, Calcd. for C H O N ClF [M+H] : 330.0440,
16
10
2
3
Found 330.0437.