Design, synthesis and biological evaluation of 7-nitro-1H-indole-2- carboxylic acid derivatives as allosteric inhibitors of fructose-1,6- bisphosphatase

Article abstract of DOI:10.1016/j.bmc.2014.01.047

A series of novel indole derivatives was synthesized as inhibitors of fructose-1,6-bisphosphatase (FBPase). Extensive structure-activity relationships were conducted and led to a potent FBPase inhibitor 3.9 with an IC₅₀ of 0.99 μM. The binding mode of this series of indoles was predicted using CDOCKER algorithm. The results of this research will shed light on the further design and optimization of novel small molecules as FBPase inhibitors.

Full text of DOI:10.1016/j.bmc.2014.01.047

Accepted Manuscript
Design, synthesis and biological evaluation of 7-nitro-1H-indole-2-carboxylic
acid derivatives as allosteric inhibitors of fructose-1,6-bisphosphatase
Jianbo Bie, Shuainan Liu, Jie Zhou, Bailing Xu, Zhufang Shen
PII:
S0968-0896(14)00075-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.01.047
BMC 11378
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 December 2013
15 January 2014
27 January 2014
Please cite this article as: Bie, J., Liu, S., Zhou, J., Xu, B., Shen, Z., Design, synthesis and biological evaluation of
7-nitro-1H-indole-2-carboxylic acid derivatives as allosteric inhibitors of fructose-1,6-bisphosphatase, Bioorganic
& Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.01.047
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Design, synthesis and biological evaluation of
7-nitro-1H-indole-2-carboxylic acid derivatives as allosteric
inhibitors of fructose-1,6-bisphosphatase
Jianbo Biea, Shuainan Liua, Jie Zhoua, Bailing Xua*, Zhufang Shena*
aBeijing 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
Abstract: A series of novel indole derivatives was synthesized as inhibitors of
Fructose-1,6-bisphosphatase (FBPase). Extensive structure-activity relationships were conducted
and led to a potent FBPase inhibitor 3.9 with an IC50 of 0.99 ꢀM. The binding mode of this series
of indoles was predicted using CDOCKER algorithm. The results of this research will shed light
on the further design and optimization of novel small molecules as FBPase inhibitors.
Keywords: Fructose-1, 6-bisphosphatase inhibitor; Allosteric inhibitor; Indole; Diabetes
1. Introduction
Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease and manifests fasting
hyperglycemia and exaggerated postprandial glucose levels. Excessive hepatic
glucose output is a key factor leading to fasting hyperglycemia1 and it also makes a
significant contribution to postprandial hyperglycemia in type 2 diabetes patients.2,3
Gluconeogenesis is a primary process for liver to produce glucose, where
three-carbon substrates such as pyruvate, lactate and glycerol are converted into
glucose. Fructose-1,6-bisphosphatase (FBPase), acting as a rate-limiting enzyme in
gluconeogenesis pathway, can catalyze the hydrolysis of fructose-1,6-bisphosphate
into fructose-6-phosphate and inorganic phosphate. It has been demonstrated that
FBPase plays an important role in the control of blood glucose since FBPase in liver
is elevated in insulin-resistant and insulin-deficient animal models of diabetes.4,5
Furthermore, FBPase inhibitors have been verified to be capable of reducing hepatic
glucose production and lower blood glucose levels in animal models of diabetes.6-8
Therefore, Inhibition of FBPase may become a new strategy for the development of
novel antidiabetic agents.
X-ray crystal structure of FBPase revealed that it is a homotetramer,9 and each
monomer consists of a substrate (fructose-1,6-bisphosphate) binding domain and an
allosteric AMP binding domain. Probably due to the more druggable AMP binding
site, many efforts have been devoted to the discovery of allosteric inhibitors via both
* Corresponding author. Bailing Xu, Tel: +86 10 63166764; Zhufang Shen, Tel: +86 10 83172669.
E-mail address: xubl@imm.ac.cn (B. Xu); shenzhf@imm.ac.cn (Z. Shen).
1

high-throughput screening of compound libraries10-16 and structure-guided design of
AMP mimetics.17-23 Among several classes of known FBPase inhibitors, phosphonic
acid-containing thiazoles CS-917 and MB07803 have been advanced to human
clinical trials.8, 20, 21
An indole-template FBPase inhibitor 3-(2-carboxy-ethyl)-4,6-dichloro-1H-indole-2-
carboxylic acid10 (MDL-29951, IC50 = 2.5 ꢀM Figure 1) was identified by Pfizer via
screening a compound library. In our efforts to search for FBPase inhibitors without a
phosphonate or phosphate group, compound MDL-29951 was chosen as a lead
structure in view of the fact that it is a small molecule with known drug-like
properties. In this work, a series of novel 7-nitro-1H-indole-2-carboxylic acid
derivatives (A, Figure 1) were designed by scrutinizing both the structure-activity
relationships (SAR) of the known indole inhibitors and the binding features of AMP
in the crystal structure of AMP complexed with FBPase9 . The indole-2-carboxylic
acid essential for the inhibitory activity was retained as a scaffold. A nitro group was
tentatively incorporated on 7-position of the indole ring with an aim to construct
H-bond interactions with residues Thr31 and/or Val17, which were commonly
utilized by many FBPase inhibitors.6-10,12,19,20,22 In addition, a hydrophobic surface
formed by residues Met177, Leu30 and Val160 was explored by several known
inhibitors.6-10,12,19,20 Therefore, variations on 3-, 4- and/or 5-position of
7-nitro-1H-indole-2-carboxylic acid with a wide range of hydrophobic substituents
were conducted in order to improve the hydrophobic interactions. Herein, the
chemical synthesis of these new indole derivatives is described in details. The
inhibitory activities against FBPase of these synthetic compounds are presented along
with their SAR analysis as follows.
2. Results and discussion
2.1 Chemistry
The synthesis of compounds (1.6a-1.6f) was shown in Scheme 1 and their chemical
structures were presented in Table 1. (2-Nitrophenyl)hydrazine hydrochloride was
converted into hydrozone compound 1.2, which was further transformed into indole
1.3 under the catalysis of PPA in 41% yield24. Regioselective bromination on
3-position of compound 1.3 was accomplished in 85% yield in the presence of NBS in
DMF. Compounds 1.5a-1.5f were achieved via Suzuki coupling of compound 1.4
with various boronic acids in moderate to high yields. The hydrolysis of compounds
1.5a-1.5f yielded the corresponding carboxylic acids 1.6a-1.6f in good yields.
The chemical structures of target compounds (2.7a-2.7u, 2.8a-2.8y and 2.10a-2.10b)
were shown in Table 2 and their preparations were illustrated in Scheme 2.
5-Chloro-4-nitroaniline was converted into (5-chloro-4-nitrophenyl)hydrazine
hydrochloride 2.2 via diazotization reaction followed by reduction of diazonium salt
with SnCl2/HCl. The hydrazone derivative 2.3 obtained via the condensation of
2

compound 2.2 with ethyl pyruvate was heated at 80 in PPA to give rise to the key
indole intermediate 2.4 in 30% yield. Utilizing Suzuki-Miyaura reaction or
Buchwald-Hartwig reaction, compound 2.4 was converted into the corresponding C-C
and C-N coupling products 2.5 or 2.6 (32-98% yields), respectively, which were
hydrolyzed to the carboxylic acid derivatives 2.7a-2.7p and 2.8a-2.8l in good yields.
The carboxylic acid compounds 2.10a-2.10b were easily accessed by hydrolysis of
esters 2.9a-2.9b, which were derived from the alkylation of compound 2.5c with alkyl
halides in the yield of 48% and 20%, respectively.
The target compounds (3.6a-3.6c, 3.9) were synthesized according to Scheme 3 and
their structures were presented in Table 3. Starting from 4-bromo-2-nitroaniline 3.1,
the indole intermediate 3.4 was prepared straightforwardly via Fischer indole
synthesis. Suzuki coupling of compound 3.4 with boronic acid derivatives produced
compounds 3.5a-3.5c in moderate to high yields. Upon treatment of compound 3.5a
with NBS in DMF, compound 3.7 was afforded in 93% yield. Suzuki coupling of
compound 3.7 with ethylboronic acid was conducted to generate compound 3.8 in
80% yield. The hydrolysis of compounds 3.5a-3.5c and 3.8 provided the
corresponding carboxylic acids 3.6a-3.6c and 3.9 in reasonable yields.
2.2 Biological results and discussion
Recombinant human FBPase activity was assayed by employing the coupling
enzymes phosphoglucose isomerase and glucose-6-phosphate dehydrogenase. The
concomitant reduction of NADP+ to NADPH was monitored spectrophotometrically.25
All target compounds (1.6a-1.6f, 2.7a-2.7p, 2.8a-2.8l, 2.10a-2.10b, 3.6a-3.6c, 3.9)
were tested for their inhibitory activities against human liver FBPase. The
corresponding results summarized in Tables 1-3 were expressed as IC50 values. AMP
and compound MDL-29951 were used as reference molecules.
Variations at 3-position of the indole scaffold were initially conducted and all of the
resulted derivatives (1.6a-1.6f) displayed inhibitory activities against FBPase as
shown in Table 1. The ethyl or isobutyl substituted compounds (1.6a and 1.6b)
showed inhibition with an IC50 value of 6.2 ꢀM and 10.3 ꢀM, respectively. When
aromatic substituents were incorporated, the inhibitory activities of compounds
1.6c-1.6f against FBPase varied with an IC50 value ranging from 4.5 ꢀM to 35.6 ꢀM.
The 3-fluorophenyl substituted derivative (1.6f, IC50 = 4.5 ꢀM) was the most potent
compound in this series.
The SAR was extensively explored on 4-substituted indoles. As shown in Table 2, a
wide range of aromatic amino groups (2.7a-2.7p, 2.10a-2.10b) and substituted phenyl
groups (2.8a-2.8k) were directly installed at 4-position of the indole scaffold. In the
series of diaryl amines (2.7a-2.7p, 2.10a-2.10b), seven compounds were found to be
active against FBPase (IC50, 3.4-33.9 ꢀM). Among which, the analogs bearing
4-nitrophenylamino
(2.7l),
3-methylphenylamino
(2.7m)
or
3

3,4-dimethoxyphenylamino fragment exhibited potent inhibitory activity, which was
comparable to that of AMP. It was observed that the compound with a
3-methoxyphenylamino or 3-methylphenylamino substituent was more potent than
that with a 4-methoxyphenylamino or 4-methylphenylamino group (compound 2.7c vs
2.7d; 2.7m vs 2.7n). In contrast, the compound bearing a 4-trifluoromethyl or 4-nitro
substituent displayed stronger potency against FBPase than that with a
3-trifluoromethyl or 3-nitro group (compound 2.7g vs 2.7h; 2.7k vs 2.7l). These
results suggested that the placement of a hydrophobic electron-donating group at
3-position and an electron-withdrawing group at 4-position of the phenyl ring would
be favorable for potency against FBPase. In addition, no inhibitory effect was
detected when phenylamino (2.7a), 3-chlorophenylamino (2.7i), 4-chlorophenylamino
(2.7j), 3-acetamidophenylamino (2.7o) or 3-aminophenylamino (2.7p) was chosen as
4-substituent. Furthermore, in comparison with 3-methoxyphenylamino substituted
derivative (2.7c), substitutions with methoxyl group at 2- or 4-position (2.7b and 2.7d)
led to complete loss of activity. Taken together, it was suggested that the substitution
pattern and the electron property and hydrophobicity of the substituents on the
phenylamino ring likely play a key role in the inhibitory activity of this series
compouds. It was noted that alkylation of the nitrogen atom of the diaryl amine 2.7c
resulted in loss of activity completely (2.7c vs 2.10a and 2.10b), indicating that a
hydrogen bond donor (NH) was essential or a bulky group was not tolerated in this
position.
Compared to the biaryl amine derivatives, the biaryl compounds (2.8a-2.8k) generally
produced weaker inhibition. In the series of the biaryl compounds, only two
derivatives with 3-methylphenyl (2.8g) and 3-acetamidophenyl (2.8j) groups as
4-substituents showed inhibitory activity against FBPase with an IC50 value of 25.9
ꢀM and 18.5 ꢀM, respectively. Interestingly, the 4-isobutyl substituted derivative
(2.8l) had an IC50 value of 9.7 ꢀM, which was about 2-fold as potent as that of the
biaryl derivatives (2.8g and 2.8j).
The primary SAR investigation was conducted on 5-substituents (R5). Three distinct
groups such as isobutyl, 3-acetamidophenyl and 3-methoxyphenylamino moieties
were chosen based on the SAR studies on the 3- and 4-substituents. All designed
analogues (3.6a-3.6c) displayed inhibitory activity against FBPase with IC50 values at
low micromolar level, indicating that different substituents at 5-position were
tolerated. And it was also indicated that the hydrophobic binding site formed by
residues Met177, Leu30 and Val160 was more easily accessible by the 5-substituents,
compared to the 3- and 4-substituents. Simultaneous modification on 3- and
5-position led to indole derivative 3.9, which is the most potent FBPase inhibitor in
this work with an IC50 value of 0.99 ꢀM. As compared with the corresponding
monosubstituted analogs 3.6a and 1.6a, the inhibitory activity of compound 3.9 was
5-6 fold as active as that of compound 3.6a and 1.6a. Therefore, dual substitutions on
3- and 5-position were more benefical than monosubstitution to improve the binding
affinity.
4

In order to get some insight for further structure-based modification of indoles as
FBPase inhibitors, molecular modeling was performed using CDOCKER docking
program (Accelrys Discovery Studio 2.5.5).26 The coordinates of the AMP-FBPase
complex (pdb code: 1FTA)9 were employed. The binding modes of most of the
evaluated target compounds were similar to that of AMP and exemplified by the most
potent compound 3.9 as shown in Figure 2. The key hydrogen bonding interactions
were observed between the carboxylate and residues Thr 27, Lys 112 and Arg 140,
which were also recognized by the phosphate group in AMP. It was believed that this
hydrogen bonding network made crucial contributions to the binding affinity. The
indole ring was situated in a hydrophobic pocket involved in residues Leu30 and
Leu34. The 7-nitro group interacted with the hydroxyl group of Thr31 via a hydrogen
bond. Nevertheless, in comparison with the purine ring in AMP, the indole moiety
orientated slightly outward from the purine binding pocket. Therefore, we envisioned
that the indole scaffold could occupied the purine pocket more deeply if the
carboxylate side chain was extended by 2 or 3-bonds length, and this could facilitate
to enhance the hydrophobic contacts. The 3-ethyl and 5-isobutyl groups were
positioned at a large hydrophobic site formed by residues Tyr113, Val160 and
Met177. Compound 3.9 produced more potency probably because of the more
hydrophobic contacts between the alkyl groups and FBPase. It should be addressed
that modification on the 3-, 4- and 5-position of the indole ring simultaneously with a
variety of substituents might provide more chances to improve the binding affinity.
3. Conclusion
In summary, a series of novel indole-based chemical entities were designed and
synthesized as potent FBPase inhibitors. Compound 3.9 was identified as the most
potent inhibitor with an IC50 value of 0.99 ꢀM. The SAR and the binding mode of the
titled compounds were also explored, and that will guide us to develop more potent
FBPase inhibitors, which will be reported in due course.
4. Experimental section
4.1. Chemistry
4.1.1. General
Melting points were measured on a Yanaco micro melting point apparatus and are
1
uncorrected. H NMR (300 MHz or 400 MHz) on a Varian Mercury 300 or 400
spectrometer was recorded in DMSO-d6, acetone-d6 or CDCl3. Chemical shifts are
reported in ꢁ (ppm) units relative to the internal standard tetramethylsilane (TMS).
High resolution mass spectra (HRMS) were obtained on an Agilent Technologies
LC/MSD TOF spectrometer. All chemicals and solvents used were of reagent grade
without purified or dried before use. All the reactions were monitored by thin-layer
chromatography (TLC) on pre-coated silica gel G plates at 254 nm under a UV lamp.
5

Column chromatography separations were performed with silica gel (200-300 mesh).
4.1.2. Synthesis of 3-subsituted-7-nitro-1H-indole-2-carboxylic acid derivatives
4.1.2.1 Ethyl 2-(2-(2-nitrophenyl)hydrazono)propanoate (1.2)
To a solution of (2-nitrophenyl)hydrazine hydrochloride (5.0 g, 26.4 mmol) in
ethanol (30 mL), ethyl pyruvate (3.7 g, 32 mmol) was added dropwise. The reaction
mixture was stirred at room temperature for 10 min, and then poured into ice-water
(60 mL). After filtration, the title compound 1.2 was obtained as yellow solid (5.56 g,
1
82%); mp: 106-107 ; H NMR (CDCl3) ꢁ (ppm): 10.96 (brs, 1H), 8.21 (q, J = 8.4
Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.00 (t, J = 8.0 Hz, 1H),
4.36 (q, J = 7.2 Hz, 2H), 2.25 (s, 3H), 1.40 (t, J = 7.2 Hz, 3H); HRMS (ESI): m/z,
calcd for C11H14N3O4 [M+H+]: 252.0979, found 252.0986.
4.1.2.2. Ethyl 7-nitro-1H-indole-2 -carboxylate (1.3)24
The reaction mixture of compound 1.2 (5.3 g, 21 mmol) in PPA (26.5 g) was heated
at 50-60 for 4 h and then heated at 80 for 3 h. The reactiong mixture was cooled
to room temperature and water (30 mL) was added to the mixture to destroy the PPA.
The resulting solution was extracted with EtOAc (40 mL × 2). The crude product
obtained after concentration was purified with column chromatography to afford the
title compound 1.3 (2.02 g, 41%) as light yellow solid; mp: 94-95 ; 1H NMR (CDCl3)
ꢁ (ppm): 10.34 (brs, 1H), 8.30 (d, J = 8.0 Hz, 1H), 8.05 (d, J = 7.6 Hz, 1H), 7.36 (s,
1H), 7.28 (t, J = 7.6 Hz, 1H), 4.47 (q, J = 7.2 Hz, 2H), 1.45 (t, J = 7.2 Hz, 3H);
HRMS (ESI): m/z, calcd for C11H11N2O4 [M+H+]: 235.0713, found 235.0722.
4.1.2.3. Ethyl 3-bromo-7-nitro-1H-indole-2 -carboxylate (1.4)
A solution of NBS (84 mg, 0.47 mmol) in DMF (2 mL) was added dropwise to a
solution of 1.3 (100 mg, 0.43 mmol) in DMF (2 mL), which was cooled by an
ice-water bath. After addition, the reaction mixture was stirred at room temperature
for 1.5 hours, and then poured into ice-water (15 mL). After filtration, the title
compound 1.4 was afforded as off-white solid (115 mg, 85%); mp: 135-137 ; 1H
NMR (CDCl3) ꢁ (ppm): 10.41 (brs, 1H), 8.37 (d, J = 8.1 Hz, 1H), 8.07 (d, J = 7.8 Hz,
1H), 7.37 (t, J = 7.8 Hz, 1H), 4.52 (q, J = 7.2 Hz, 2H), 1.49 (t, J = 7.2 Hz, 3H);
HRMS (ESI): m/z, calcd for C11H10BrN2O4 [M+H+]: 312.9819, found 312.9815.
4.1.2.4. Synthesis of ethyl 3-substituted-7-nitro-1H-indole-2-carboxylate
(1.5a-1.5f)
4.1.2.4.1. Ethyl 3-ethyl-7-nitro-1H-indole-2 -carboxylate (1.5a)
To a solution of 1.4 (200 mg, 0.64 mmol) in toluene (15 mL), Pd (OAc)2 (7 mg,
0.032 mmol), Josiphos ligand (20 mg, 0.032 mmol), K3PO4 (679 mg, 3.2 mmol, 1mL
H2O) and ethylboronic acid (142 mg, 1.92 mmol) were sequentially added. The
reaction mixture was heated at 80 ℃ for 4 hours under argon atmosphere, and then
concentrated under reduced pressure. The residue was dissolved in EtOAc (40 mL),
and washed with brine (20 mL×3) and water (20 mL×3). The crude product obtained
after concentration was purified with column chromatography to afford the product
1.5a as off-white solid (165 mg, 99%); mp: 94-96 ; 1H NMR (CDCl3) ꢁ (ppm):
10.13 (brs, 1H), 8.29 (d, J = 8.0 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.25 (t, J = 8.0 Hz,
1H), 4.47 (q, J = 7.2 Hz, 2H), 3.16 (q, J = 7.2 Hz, 2H), 1.46 (t, J = 6.8 Hz, 3H), 1.30
(t, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for C13H15N2O4 [M+H+]: 263.1026,
found 263.1024.
6

4.1.2.4.2. Ethyl 3-isobutyl-7-nitro-1H-indole-2-carboxylate (1.5b) bjb-1301
Following the procedure of 4.1.2.4.1, starting from compound 1.4 (200 mg, 0.64
mmol), the title compound 1.5b was obtained as yellow solid (177 mg, 85%); mp:
95-96 ; 1H NMR (CDCl3) δ (ppm): 10.19 (brs, 1H), 8.29 (d, J = 8.1 Hz, 1H), 8.03 (d,
J = 7.8 Hz, 1H), 7.24 (t, J = 8.1 Hz, 1H), 4.46 (q, J = 7.2 Hz, 2H), 3.01 (d, J = 7.5 Hz,
2H), 1.98-2.07 (m, 1H), 1.46 (t, J = 7.2 Hz, 3H), 0.96 (d, J = 6.6 Hz, 3H); HRMS
(ESI): m/z, calcd for C15H19N2O4 [M+H+]: 291.1339, found 291.1335.
4.1.2.4.3. Ethyl 7-nitro-3-phenyl-1H-indole-2 -carboxylate (1.5c)
To a solution of 1.4 (200 mg, 0.64 mmol) in 1,4-dioxane (15 mL), PdCl2(dppf) (47
mg, 0.064 mmol), Na2CO3 (203 mg, 1.92 mmol, 2 mL H2O) and phenylboronic acid
(234 mg, 1.92 mmol) were sequentially added. The reaction mixture was heated at
100 ℃ for 2 hours under argon atmosphere, and then concentrated under reduced
pressure. The residue was dissolved in EtOAc (40 mL), and washed with brine (20
mL×3) and water (20 mL×3). The crude product obtained after concentration was
purified with column chromatography to afford the desired product 1.5c as yellow
solid (250 mg, 84%); mp: 118-120 ; 1H NMR (CDCl3) ꢁ (ppm): 10.42 (brs, 1H),
8.34 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.42-7.54 (m, 5H), 7.26 (t, J = 8.0
Hz, 1H), 4.35 (q, J = 7.2 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd
for C17H15N2O4 [M+H+]: 311.1026, found 311.1019.
4.1.2.4.4. Ethyl 3-methoxyphenyl-7-nitro-1H-indole-2 -carboxylate (1.5d)
Following the procedure of 4.1.2.4.3, starting from compound 1.4 (200 mg, 0.64
mmol), the title compound 1.5d was afforded as light yellow solid (185 mg, 85%); mp:
114-115 ; 1H NMR (CDCl3) ꢁ (ppm): 10.41 (s, 1H), 8.33 (d, J = 8.0 Hz, 1H), 8.00 (d,
J = 8.0 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 7.26 (t, J = 8.0 Hz, 1H), 7.10 (t, J = 7.6 Hz,
1H), 7.07 (s, 1H), 6.99 (d, J = 8.0 Hz, 1H), 4.36 (q, J = 7.2 Hz, 2H), 3.86 (s, 3H), 1.29
(t, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for C18H17N2O5 [M+H+]: 341.1132, found
341.1126.
4.1.2.4.5. Ethyl 3-acetamidophenyl-7-nitro-1H-indole-2-carboxylate (1.5e)
Following the procedure of 4.1.2.4.3, starting from compound 1.4 (170 mg, 0.54
mmol), the title compound 1.5e was afforded as yellow solid (150 mg, 58%), mp:
150-151 ; 1H NMR (CDCl3) ꢁ (ppm): 10.41 (s, 1H), 8.33 (d, J = 8.0 Hz, 1H), 8.01 (d,
J = 7.6 Hz, 1H), 7.74 (s, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H),
7.24-7.31 (m, 2H), 4.36 (q, J = 7.2 Hz, 2H), 2.19 (s, 3H), 1.29 (t, J = 7.2 Hz, 3H);
HRMS (ESI): m/z, calcd for C19H18N3O5 [M+H+]: 368.1236, found 368.1241.
4.1.2.4.6. Ethyl 3-fluorophenyl-7-nitro-1H-indole-2 -carboxylate (1.5f)
Following the procedure of 4.1.2.4.3, starting from compound 1.4 (220 mg, 0.70
mmol), the title compound 1.5f was obtained as light yellow solid (210 mg, 91%); mp:
124-126 ; 1H NMR (CDCl3) ꢁ (ppm): 10.45 (s, 1H), 8.35 (d, J = 8.0 Hz, 1H), 7.97 (d,
J = 8.0 Hz, 1H), 7.10 (dd, J1 = 14.0 Hz, J2 = 7.6 Hz, 1H), 7.24-7.31 (m, 3H), 7.15 (t, J
= 8.4 Hz, 1H), 4.36 (q, J = 7.2 Hz, 2H), 1.29 (t, J = 7.2 Hz, 3H); HRMS (ESI): m/z,
calcd for C17H14FN2O4 [M+H+]: 329.0932, found 329.0926.
4.1.2.5. Synthesis of 3-substituted-7-nitro-1H-indole-2-carboxylic acid (1.6a-1.6f)
4.1.2.5.1. 3-ethyl-7-nitro-1H-indole-2-carboxylic acid (1.6a)
To a solution of 1.5a (100 mg, 0.38 mmol) in THF (4 mL) and ethanol (2 mL), a
solution of NaOH (76 mg, 1.9 mmol) in water (2 mL) was added dropwise. The
7

resulting mixture was stirred at room temperature for overnight, and then concentrated
under reduced pressure. The residue was dissolved in water (10 mL), which was
acidified to pH=3-4 with diluted HCl. After filtration, the title compound 1.6a was
obtained as light yellow solid (60 mg, 67%); mp: 231-233 ; 1H NMR (DMSO-d6) ꢁ
(ppm): 13.58 (brs, 1H), 10.58 (s, 1H), 8.28 (d, J = 8.0 Hz, 2H), 7.34 (t, J = 8.0 Hz,
1H), 3.12 (q, J = 7.2 Hz, 2H), 1.22 (t, J = 7.6 Hz, 3H); HRMS (ESI): m/z, calcd for
C11H11N2O4 [M+H+]: 235.0713, found 235.0710.
4.1.2.5.2. 3-isobutyl-7-nitro-1H-indole-2-carboxylic acid (1.6b)
Following the procedure of 4.1.2.5.1, starting from compound 1.5b (120 mg, 0.41
mmol), the title compound 1.6b was obtained as light yellow solid (60 mg, 56%); mp:
185-187 ; 1H NMR (DMSO-d6) ꢁ (ppm): 13.54 (s, 1H), 10.60 (s, 1H), 8.27 (d, J =
8.8 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H), 7.33 (t, J = 8.0 Hz, 1H), 2.99 (d, J = 6.8 Hz,
2H), 1.93-1.99 (m, 1H), 0.89 (d, J = 6.4 Hz, 3H); HRMS (ESI): m/z, calcd for
C13H15N2O4 [M+H+]: 263.1026, found 263.1023.
4.1.2.5.3. 7-nitro-3-phenyl-1H-indole-2-carboxylic acid (1.6c)
Following the procedure of 4.1.2.5.1, starting from compound 1.5b (127 mg, 0.41
mmol), the title compound 1.6c was obtained as off-white solid (110 mg, 95%); mp:
244-245 ; 1H NMR (DMSO-d6) ꢁ (ppm): 13.49 (brs, 1H), 11.03 (s, 1H), 8.33 (d, J =
8.0 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.41-7.54 (m, 5H), 7.37 (t, J = 8.0 Hz, 1H);
HRMS (ESI): m/z, calcd for C15H11N2O4 [M+H+]: 283.0713, found 283.0708.
4.1.2.5.4. 3-methoxyphenyl-7-nitro-1H-indole-2-carboxylic acid (1.6d)
Following the procedure of 4.1.2.5.1, starting from compound 1.5d (120 mg, 0.35
mmol), the title compound 1.6d was obtained as light yellow solid (110 mg, 100%);
mp: 219-221 ; 1H NMR (DMSO-d6) ꢁ (ppm): 11.04 (s, 1H), 8.33 (d, J = 8.0 Hz, 1H),
8.00 (d, J = 8.0 Hz, 1H), 7.41 (t, J = 8.4 Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 7.09 (d, J =
7.2 Hz, 1H), 7.08 (s, 1H), 7.00 (d, J = 8.8 Hz, 1H), 3.80 (s, 3H); HRMS (ESI): m/z,
calcd for C16H13N2O5 [M+H+]: 313.0819, found 313.0813.
4.1.2.5.5. 3-acetamidophenyl-7-nitro-1H-indole-2-carboxylic acid (1.6e)
Following the procedure of 4.1.2.5.1, starting from compound 1.5e (80 mg, 0.22
mmol), the title compound 1.6e was obtained as light yellow solid (80 mg, 100%);
mp >250 ; 1H NMR (DMSO-d6) ꢁ (ppm): 11.06 (s, 1H), 10.05 (s, 1H), 8.33 (d, J =
8.0 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.74 (s, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.36-7.42
(m, 2H), 7.19 (d, J = 8.0 Hz, 1H), 2.06 (s, 3H); HRMS (ESI): m/z, calcd for
C17H14N3O5 [M+H+]: 340.0928, found 340.0922.
4.1.2.5.6. 3-fluorophenyl-7-nitro-1H-indole-2-carboxylic acid (1.6f)
Following the procedure of 4.1.2.5.1, starting from compound 1.5f (120 mg, 0.37
mmol), the titile compound 1.6f was obtained as light yellow solid (90 mg, 82%), mp:
195-196 ; 1H NMR (DMSO-d6) ꢁ (ppm): 13.59 (s, 1H), 11.11 (s, 1H), 8.33 (d, J =
8.0 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.52 (dd, J1 = 14.8 Hz, J2 = 8.0 Hz, 1H),
7.35-7.39 (m, 3H), 7.26 (t, J = 8.4 Hz, 1H); HRMS (ESI): m/z, calcd for C15H10FN2O4
[M+H+]: 301.0619, found 301.1403.
4.1.3. Synthesis of 4-subsituted-7-nitro-1H-indole-2-carboxylic acid derivatives
4.1.3.1. (5-chloro-2-nitrophenyl)hydrazine hydrochloride (2.2)
A solution of 5-chloro-2-nitroaniline (5.0 g, 2.9 mmol) in concentrated HCl (57.2 g,
8

580 mmol) was heated at 40 for 2 h. The reaction mixture was cooled to -10 , and
then a solution of NaNO2 (2.1 g, 30.5 mmol) in water (20 mL) was added dropwise.
After stirring for 2 h, the mixture was cooled to -30
and added to a solution of
SnCl2 (19.6 g, 87 mmol) in concentrated HCl (30 mL) precooled to -30 . The
reaction mixture was stirred at -20 for 1, and then warmed to room temperature and
filtered. The title compound 2.2 was obtained as light yellow solid (7.0 g, 100%); mp:
178-180 ; 1H NMR (DMSO-d6) ꢁ ( ppm ): 9.39 (brs, 1H), 8.15 (d, J = 8.8 Hz, 1H),
7.45 (s, 1H), 7.06 (d, J = 8.4 Hz, 1H), 3.83 (brs, 3H); HRMS (ESI): m/z, calcd for
C6H7ClN3O2 [M+H+]: 188.0221, found 188.0220.
4.1.3.2. Ethyl 2-(2-(5-chloro-2-nitrophenyl)hydrazono)propanoate (2.3)
To a solution of 2.2 (1.0 g, 4.46 mmol) in ethanol (20 mL), ethyl pyruvate (0.54 g,
4.69 mmol) was added dropwise. The reaction mixture was stirred at room
temperature for 5 min, and then poured into ice-water (60 mL). After filtration, the
title compound 2.3 was obtained as yellow solid (469 mg, 37%); mp: 107-109 ; 1H
NMR (acetone-d6) ꢁ (ppm): 10.82 (br, 1H), 8.24 (d, J = 8.8 Hz, 1H), 7.95 (s, 1H),
7.10 (dd, J1 = 9.2 Hz, J2 = 1.6 Hz, 1H), 4.32 (q, J = 7.2 Hz, 2H), 2.26 (s, 3H), 1.35 (t,
J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for C11H13ClN3O4 [M+H+]: 286.0589, found
286.0582.
4.1.3.3. Ethyl 4-chloro-7-nitro-1H-indole-2-carboxylate (2.4)
The reaction mixture of compound 2.3 (2.84 g, 9.94 mmol) in PPA (57 g) was
heated at 80℃ for 12 h, and then cooled to room temperature. Water (30 mL) was
added to the mixture to destroy the PPA, and the residue was extracted with EtOAc
(40 mL×2). The crude product obtained after concentration was purified with column
chromatography to afford 2.4 (810 mg, 30%) as light yellow solid; mp: 170-171 ;1H
NMR (acetone-d6) ꢁ (ppm): 11.01 (brs, 1H), 8.31 (d, J = 8.7 Hz, 1H), 7.45 (d, J = 8.4
Hz, 1H), 7.40 (s, 1H), 4.45 (q, J = 7.2 Hz, 2H), 1.42 (t, J = 7.2 Hz, 3H); HRMS (ESI):
m/z, calcd for C11H10ClN2O4 [M+H+]: 269.0324, found 269.0318.
4.1.3.4. Synthesis of ethyl 4-substituted-7-nitro-1H-indole-2-carboxylate
4.1.3.4.1. Ethyl 7-nitro-4-phenylamino-1H-indole-2-carboxylate (2.5a)
To a solution of 2.4 (50 mg, 0.19 mmol) in 1,4-dioxane (10 mL), Pd2(dba)3 (17 mg,
0.019 mmol), Xantphos (22 mg, 0.038 mmol) and t-BuONa (55 mg, 0.57 mmol) and
aniline (53 mg, 0.57 mmol) were added in order. The reaction mixture was refluxed
for 3 h under argon atmosphere, and then concentrated under reduced pressure. The
residue was dissolved in EtOAc (20 mL), and washed with brine (10 mL×3) and
water (10 mL×3). The crude product obtained after concentration was purified with
column chromatography to afford product 2.6a as yellow solid (42 mg, 65%); mp:
211-212 ; 1H NMR (acetone-d6) ꢁ (ppm): 10.59 (brs, 1H), 8.85 (brs, 1H), 8.17 (d, J
= 9.0 Hz, 1H), 7.69 (s, 1H), 7.46-7.51 (m, 4H), 7.22-7.27 (m, 1H), 6.84-6.88 (m, 1H),
4.41 (q, J = 7.5 Hz, 2H), 1.40 (t, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for
C17H16N3O4 [M+H+]: 326.1135, found 326.1126.
4.1.3.4.2.
(2.5b)
Ethyl 4-(2-methoxyphenylamino)-7-nitro-1H-indole-2-carboxylate
To a solution of 2.4 (250 mg, 0.93 mmol) in toluene (15 mL), Pd2(dba)3 (85 mg,
0.093 mmol), Xantphos (108 mg, 0.19 mmol) and Na2CO3 (296 mg, 2.79 mmol, 2 mL
H2O) and 2-methoxyaniline (344 mg, 2.79 mmol) were added in order. The reaction
mixture was refluxed for 4 h under argon atmosphere, and then concentrated under
9

reduced pressure. The residue was dissolved in EtOAc (30 mL), and washed with
brine (20 mL×3) and water (20 mL×3). The crude product obtained after
concentration was purified with column chromatography to afford product 2.6b as
yellow solid (270 mg, 82%); mp: 137-139 ; 1H NMR (CDCl3) ꢁ (ppm): 10.46 (brs,
1H), 8.20 (d, J = 9.2 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 1.2 Hz, 1H), 7.16
(t, J = 8.0 Hz, 2H), 7.00-7.04 (m, 3H), 6.92 (d, J = 8.8 Hz, 1H), 4.46 (q, J = 7.2 Hz,
2H), 3.92 (s, 3H), 1.45 (t, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for C18H18N3O5
[M+H+]: 356.1241, found 356.1231.
4.1.3.4.3.
(2.5c)
Ethyl 4-(3-methoxyphenylamino)-7-nitro-1H-indole-2-carboxylate
Following the procedure of 4.1.3.4.2, starting from compound 2.4 (220 mg, 0.82
mmol), the title compound 2.5c was obtained as yellow solid (285 mg, 98%); mp:
188-190 ; 1H NMR (DMSO-d6) ꢁ (ppm): 10.94 (s, 1H), 9.64 (s, 1H), 8.13 (d, J = 9.3
Hz, 1H), 7.84 (s, 1H), 7.35 (t, J = 7.8 Hz, 1H), 6.96 (d, J = 7.8 Hz, 1H), 6.92-6.93 (m,
1H), 6.77-6.82 (m, 2H), 4.37 (q, J = 7.5 Hz, 2H), 3.78 (s, 3H), 1.36 (t, J = 7.2 Hz, 3H);
HRMS (ESI): m/z, calcd for C18H18N3O5 [M+H+]: 356.1241, found 356.1231.
4.1.3.4.4.
(2.5d)
Ethyl 4-(4-methoxyphenylamino)-7-nitro-1H-indole-2-carboxylate
Following the procedure of 4.1.3.4.2, starting from compound 2.4 (350 mg, 1.30
mmol), the title compound 2.5d was obtained as yellow solid (389 mg, 84%); mp:
209-211 ; 1H NMR (CDCl3) ꢁ (ppm): 10.47 (brs, 1H), 8.14 (d, J = 9.3 Hz, 1H), 7.33
(d, J = 2.4 Hz, 1H), 7.25 (d, J = 8.7 Hz, 2H), 6.98 (d, J = 9.0 Hz, 1H), 6.72 (brs, 1H),
6.50 (d, J = 9.0 Hz, 1H), 4.43 (q, J = 7.2 Hz, 2H), 3.86 (s, 1H), 1.43 (t, J = 7.5 Hz,
3H); HRMS (ESI): m/z, calcd for C18H18N3O5 [M+H+]: 356.1231, found 356.1241.
4.1.3.4.5. Ethyl 4-(3,4-dimethoxyphenylamino)-7-nitro-1H-indole-2-carboxylate
(2.5e)
Following the procedure of 4.1.3.4.2, starting from compound 2.4 (300 mg, 1.12
mmol), the title compound 2.5e was obtained as orange-red solid (420 mg, 98%); mp:
205-206 ; 1H NMR (CDCl3) ꢁ (ppm): 10.46 (brs, 1H), 8.15 (d, J = 9.0 Hz, 1H), 7.35
(d, J = 1.8 Hz, 1H), 6.78-6.95 (m, 4H), 6.55 (d, J = 8.7 Hz, 1H), 4.43 (q, J = 7.2 Hz,
2H), 3.93 (s, 1H), 3.88 (s, 3H), 1.42 (t, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for
C19H20N3O6 [M+H+]: 386.1347, found 386.1333.
4.1.3.4.6. Ethyl 4-(3,4,5-trimethoxyphenylamino)-7-nitro-1H-indole-2-carboxy
-late (2.5f)
Following the procedure of 4.1.3.4.2, starting from compound 2.4 (200 mg, 0.74
mmol), the title compound 2.5f was obtained as orange-red solid (292 mg, 94%); mp:
179-181 ; 1H NMR (CDCl3) ꢁ (ppm): 10.46 (brs, 1H), 8.18 (d, J = 9.2 Hz, 1H), 7.35
(d, J = 1.2 Hz, 1H), 6.75 (brs, 1H), 6.70 (d, J = 9.2 Hz, 1H), 6.56 (s, 2H), 4.44 (q, J =
7.2 Hz, 2H), 3.89 (s, 1H), 3.86 (s, 6H), 1.43 (t, J = 7.2 Hz, 3H); HRMS (ESI): m/z,
calcd for C20H22N3O7 [M+H+]: 416.1452, found 416.1439.
4.1.3.4.7. Ethyl 4-(3-(trifluoromethyl)phenylamino)-7-nitro-1H-indole-2-carboxy
-late (2.5g)
Following the procedure of 4.1.3.4.2, starting from compound 2.4 (250 mg, 0.93
mmol), the title compound 2.5g was obtained as light yellow solid (360 mg, 98%);
mp >250 ; 1H NMR (acetone-d6) ꢁ (ppm): 10.62 (s, 1H), 8.96 (s, 1H), 8.21 (d, J =
10

9.2 Hz, 1H), 7.67-7.80 (m, 4H), 7.52 (d, J = 7.6 Hz, 1H), 6.99-7.01 (m, 1H), 4.41 (q, J
= 7.2 Hz, 2H), 1.40 (t, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for C18H15F3N3O4
[M+H+]: 394.1009, found 394.0997.
4.1.3.4.8. Ethyl 4-(4-(trifluoromethyl)phenylamino)-7-nitro-1H-indole-2-carboxy
-late (2.5h)
Following the procedure of 4.1.3.4.2, starting from compound 2.4 (200 mg, 0.74
mmol), the title compound 2.5h was obtained as yellow solid (250 mg, 86%); mp:
239-241 ; 1H NMR (acetone-d6) ꢁ (ppm): 10.67 (brs, 1H), 9.05 (s, 1H), 8.23 (d, J =
8.8 Hz, 1H), 7.77 (d, J = 8.4 Hz, 2H), 7.69 (s, 1H), 7.66 (d, J = 8.4 Hz, 2H), 7.12-7.15
(m, 1H), 4.41 (q, J = 7.2 Hz, 2H), 1.39 (t, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd
for C18H15F3N3O4 [M+H+]: 394.1009, found 394.0997.
4.1.3.4.9. Ethyl 4-(3-chlorophenylamino)-7-nitro-1H-indole-2-carboxylate (2.5i)
Following the procedure of 4.1.3.4.2, starting from compound 2.4 (200 mg, 0.74
mmol), the title compound 2.5i was obtained as light yellow solid (257 mg, 96%); mp:
220-221 ; 1H NMR (DMSO-d6) ꢁ (ppm): 11.01 (s, 1H), 9.67 (s, 1H), 8.16 (d, J = 8.8
Hz, 1H), 7.81 (s, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.42 (s, 1H), 7.37 (d, J = 8.0 Hz, 1H),
7.23 (d, J = 7.6 Hz, 1H), 6.84 (d, J = 8.8 Hz, 1H), 4.37 (q, J = 7.5 Hz, 2H), 1.36 (t, J =
7.2 Hz, 3H); HRMS (ESI): m/z, calcd for C17H15ClN3O4 [M+H+]: 360.0746, found
360.0736.
4.1.3.4.10. Ethyl 4-(4-chlorophenylamino)-7-nitro-1H-indole-2-carboxylate (2.5j)
Following the procedure of 4.1.3.4.2, starting from compound 2.4 (200 mg, 0.74
mmol), the title compound 2.5j was obtained as yellow solid (265 mg, 99%); mp:
244-245 ; 1H NMR (acetone-d6) ꢁ (ppm): 10.63 (brs, 1H), 8.87 (s, 1H), 8.19 (d, J =
9.2 Hz, 1H), 7.69 (s, 1H), 7.48 (m, 4H), 6.89-6.91 (m, 1H), 4.41 (q, J = 7.2 Hz, 2H),
1.40 (t, J = 7.2 Hz, 3H) ; HRMS (ESI): m/z, calcd for C17H15ClN3O4 [M+H+]:
360.0746, found 360.0736.
4.1.3.4.11. Ethyl 4-(3-nitrophenylamino)-7-nitro-1H-indole-2-carboxylate (2.5k)
Following the procedure of 4.1.3.4.2, starting from compound 2.4 (220 mg, 0.82
mmol), the title compound 2.5k was obtained as light yellow solid (265 mg, 87%);
1
mp >250 ; H NMR (DMSO-d6) ꢁ (ppm): 11.05 (s, 1H), 9.84 (s, 1H), 8.17 (d, J =
9.2 Hz, 1H), 8.14 (s, 1H), 7.96 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.79 (s,
1H), 7.69 (t, J = 8.0 Hz, 1H), 6.96 (d, J = 8.8 Hz, 1H), 4.37 (q, J = 7.2 Hz, 2H), 1.36
(t, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for C17H15N4O6 [M+H+]: 371.0986,
found 371.0981.
4.1.3.4.12. Ethyl 4-(4-nitrophenylamino)-7-nitro-1H-indole-2-carboxylate (2.5l)
Following the procedure of 4.1.3.4.2, starting from compound 2.4 (220 mg, 0.82
mmol), the title compound 2.5l was obtained as yellow solid (242 mg, 84%);
1
mp >250 ; H NMR (DMSO-d6) ꢁ (ppm): 11.16 (s, 1H), 10.00 (s, 1H), 8.24 (d, J =
8.8 Hz, 2H), 8.21 (d, J = 9.2 Hz, 1H), 7.78 (s, 1H), 7.53 (d, J = 9.2 Hz, 2H), 7.15 (d, J
= 8.8 Hz, 1H), 4.37 (q, J = 7.2 Hz, 2H), 1.36 (t, J = 7.2 Hz, 3H); HRMS (ESI): m/z,
calcd for C17H14N4O6 [M+H+]: 371.0986, found 371.0973.
4.1.3.4.13.
(2.5m)
Ethyl
4-(3-methylphenylamino)-7-nitro-1H-indole-2-carboxylate
Following the procedure of 4.1.3.4.2, starting from compound 2.4 (220 mg, 0.82
11

mmol), the title compound 2.5m was obtained as yellow solid (260 mg, 93%); mp:
190-191 ; 1H NMR (DMSO-d6) ꢁ (ppm): 10.89 (s, 1H), 9.62 (s, 1H), 8.13 (d, J = 9.2
Hz, 1H), 7.83 (s, 1H), 7.33 (t, J = 8.0 Hz, 1H), 7.16-7.19 (m, 2H), 7.04 (d, J = 7.6 Hz,
1H), 6.72 (d, J = 9.2 Hz, 1H), 4.37 (q, J = 7.2 Hz, 2H), 2.34 (s, 3H), 1.36 (t, J = 7.2
Hz, 3H); HRMS (ESI): m/z, calcd for C18H18N3O4 [M+H+]: 340.1292, found
340.1289.
4.1.3.4.14.
(2.5n)
Ethyl
4-(4-methylphenylamino)-7-nitro-1H-indole-2-carboxylate
Following the procedure of 4.1.3.4.2, starting from compound 2.4 (250 mg, 0.93
mmol), the title compound 2.5m was obtained as yellow solid (256 mg, 81%); mp:
232-234 ; 1H NMR (CDCl3) ꢁ (ppm): 10.47 (brs, 1H), 8.16 (d, J = 9.2 Hz, 1H), 7.33
(s, 1H), 7.25 (d, J = 7.2 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H), 6.71 (brs, 1H), 6.65 (d, J =
8.8 Hz, 2H), 4.44 (q, J = 7.2 Hz, 2H), 2.40 (s, 3H), 1.43 (t, J = 7.2 Hz, 3H); HRMS
(ESI): m/z, calcd for C18H18N3O4 [M+H+]: 340.1292, found 340.1281.
4.1.3.4.15. Ethyl 4-(3-(acetamidophenyl)amino)-7-nitro-1H-indole-2-carboxylate
(2.5o)
Following the procedure of 4.1.3.4.2, starting from compound 2.4 (220 mg, 0.82
mmol), the title compound 2.5o was obtained as brick-red solid (170 mg, 54%);
mp >250 ; 1H NMR (DMSO-d6) ꢁ (ppm): 10.91 (s, 1H), 10.05 (s, 1H), 9.69 (s, 1H),
8.12 (d, J = 9.3 Hz, 1H), 7.85-7.86 (m, 1H), 7.79 (s, 1H), 7.29-7.35 (m, 2H), 7.04 (d,
J = 6.9 Hz, 1H), 6.77 (d, J= 9.3 Hz, 1H), 4.37 (q, J = 7.2 Hz, 2H), 2.05 (s, 3H), 1.35 (t,
J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for C19H19N4O5 [M+H+]: 383.1350, found
383.1336.
4.1.3.5. Synthesis of 4-substituted-7-nitro-1H-indole-2-carboxylic acid (2.7a-2.7p)
4.1.3.5.1. 7-nitro-4-phenylamino-1H-indole-2-carboxylic acid (2.7a)
Following the procedure of 4.1.2.5.1, starting from compound 2.5a (100 mg, 0.31
mmol), the title compound 2.7a was obtained as orange solid (70 mg, 76%); mp:
237-240 ; 1H NMR (DMSO-d6) ꢁ (ppm): 13.37 (brs, 1H), 10.72 (s, 1H), 9.65 (s, 1H),
8.11 (d, J = 9.2 Hz, 1H), 7.74 (s, 1H), 7.46 (t, J = 7.6 Hz, 2H), 7.38 (d, J = 7.6 Hz,
1H), 7.22 (t, J = 6.8 Hz, 1H), 6.71 (q, J = 9.2 Hz, 1H); HRMS (ESI): m/z, calcd for
C15H12N3O4 [M+H+]: 298.0822, found 298.0811.
4.1.3.5.2. 4-(2-methoxyphenylamino)-7-nitro-1H-indole-2-carboxylic acid (2.7b)
Following the procedure of 4.1.2.5.1, starting from compound 2.5b (180 mg, 0.51
mmol), the title compound 2.7b was obtained as orange solid (130 mg, 78%); mp:
245-246 ; 1H NMR (DMSO-d6) ꢁ (ppm): 10.65 (s, 1H), 9.38 (s, 1H), 8.05 (d, J = 9.2
Hz, 1H), 7.70 (s, 1H), 7.33-7.37 (m, 2H), 7.21 (d, J = 8.0 Hz, 1H), 7.06 (t, J = 7.6 Hz,
1H), 6.14 (d, J = 9.2 Hz, 1H), 3.78 (s, 3H); HRMS (ESI): m/z, calcd for C16H14N3O5
[M+H+]: 328.0928, found 328.0923.
4.1.3.5.3. 4-(3-methoxyphenylamino)-7-nitro-1H-indole-2-carboxylic acid (2.7c)
Following the procedure of 4.1.2.5.1, starting from compound 2.5c (140 mg, 0.39
mmol), the title compound 2.7c was obtained as orange solid (102 mg, 79%); mp:
217-219 ; 1H NMR (DMSO-d6) ꢁ (ppm): 10.73 (s, 1H), 9.62 (s, 1H), 8.12 (d, J = 9.2
Hz, 1H), 7.75 (s, 1H), 7.35 (t, J = 8.0 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 6.92 (s, 1H),
6.77-6.80 (m, 2H), 3.77 (s, 3H); HRMS (ESI): m/z, calcd for C16H14N3O5 [M+H+]:
328.0928, found 328.0921.
12

4.1.3.5.4. 4-(4-methoxyphenylamino)-7-nitro-1H-indole-2-carboxylic acid (2.7d)
Following the procedure of 4.1.2.5.1, starting from compound 2.5d (350 mg, 0.99
mmol), the title compound 2.7d was obtained as orange solid (300 mg, 99%); mp:
254-255 ; 1H NMR (DMSO-d6) ꢁ (ppm): 10.59 (s, 1H), 9.55 (s, 1H), 8.05 (d, J = 9.3
Hz, 1H), 7.61 (s, 1H), 7.31 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 8.4 Hz, 2H), 6.48 (d, J =
9.3 Hz, 1H), 3.80 (s, 3H); HRMS (ESI): m/z, calcd for C16H14N3O5 [M+H+]: 328.0928,
found 328.0921.
4.1.3.5.5. 4-(3,4-dimethoxyphenylamino)-7-nitro-1H-indole-2-carboxylic acid
(2.7e)
Following the procedure of 4.1.2.5.1, starting from compound 2.5e (320 mg, 0.83
mmol), the title compound 2.7e was obtained as sorrel solid (291 mg, 98%); mp:
267-269 ; 1H NMR (DMSO-d6) ꢁ (ppm): 13.34 (brs, 1H), 10.69 (s, 1H), 9.61 (s, 1H),
8.09 (d, J = 9.3 Hz, 1H), 7.72 (d, J = 2.1 Hz, 1H), 7.04 (d, J = 8.7 Hz, 1H), 6.91-6.97
(m, 2H), 6.58 (d, J = 9.3 Hz, 1H), 3.79 (s, 3H), 3.77 (s, 3H); HRMS (ESI): m/z, calcd
for C17H16N3O6 [M+H+]: 358.1034, found 358.1029.
4.1.3.5.6. 4-(3,4,5-trimethoxyphenylamino)-7-nitro-1H-indole-2-carboxylic acid
(2.7f)
Following the procedure of 4.1.2.5.1, starting from compound 2.5f (190 mg, 0.46
mmol), the title compound 2.7f was obtained as sorrel solid (145 mg, 82%); mp:
1
278-280 ; H NMR (DMSO-d6) ꢁ (ppm): 10.73 (brs, 1H), 9.66 (s, 1H), 8.12 (d, J =
9.0 Hz, 1H), 7.76 (d, J = 2.1 Hz, 1H), 6.78 (d, J = 9.3 Hz, 1H), 6.70 (s, 2H), 3.80 (s,
6H), 3.69 (s, 3H); HRMS (ESI): m/z, calcd for C18H18N3O7 [M+H+]: 388.1139, found
388.1133.
4.1.3.5.7. 7-nitro-4-(3-(trifluoromethyl)phenylamino)-1H-indole-2-carboxylic acid
(2.7g)
Following the procedure of 4.1.2.5.1, starting from compound 2.5g (124 mg, 0.32
mmol), the title compound 2.7g was obtained as sorrel solid (42 mg, 37%);
1
mp >250 ; H NMR (DMSO-d6) ꢁ (ppm): 10.81 (s, 1H), 9.75 (s, 1H), 8.16 (d, J =
9.2 Hz, 1H), 7.64-7.71 (m, 4H), 7.51 (d, J = 7.2 Hz, 1H), 6.85 (d, J = 9.2 Hz, 1H);
HRMS (ESI): m/z, calcd for C16H11F3N3O4 [M+H+]: 366.0696, found 366.0694.
4.1.3.5.8. 7-nitro-4-(4-(trifluoromethyl)phenylamino)-1H-indole-2-carboxylic acid
(2.7h)
Following the procedure of 4.1.2.5.1, starting from compound 2.5h (140 mg, 0.36
mmol), the title compound 2.7h was obtained as orange solid (90 mg, 69%); mp:
244-246 ; 1H NMR (DMSO-d6) ꢁ (ppm): 13.41 (brs, 1H), 10.84 (s, 1H), 9.79 (s, 1H),
8.15 (d, J = 8.8 Hz, 1H), 7.74-7.76 (m, 3H),7.55 (d, J = 8.0 Hz, 2H), 6.98 (d, J = 8.8
Hz, 1H); HRMS (ESI): m/z, calcd for C16H11F3N3O4 [M+H+]: 366.0696, found
366.0694.
4.1.3.5.9. 4-(3-chlorophenylamino)-7-nitro-1H-indole-2-carboxylic acid (2.7i)
Following the procedure of 4.1.2.5.1, starting from compound 2.5i (190 mg, 0.46
mmol), the title compound 2.7i was obtained as orange solid (103 mg, 81%);
1
mp >250 ; H NMR (acetone-d6) ꢁ (ppm): 13.20 (brs, 1H), 10.78 (s, 1H), 9.64 (s,
1H), 8.14 (d, J = 9.2 Hz, 1H), 7.71 (d, J = 1.2 Hz, 1H), 7.45 (t, J = 8.0 Hz, 1H), 7.41
(s, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.23 (d, J = 7.6 Hz, 1H), 6.82 (d, J = 8.8 Hz, 1H);
13

HRMS (ESI): m/z, calcd for C15H11ClN3O4 [M+H+]: 332.0432, found 332.0427.
4.1.3.5.10. 4-(4-chlorophenylamino)-7-nitro-1H-indole-2-carboxylic acid (2.7j)
Following the procedure of 4.1.2.5.1, starting from compound 2.5j (190 mg, 0.46
mmol), the title compound 2.7j was obtained as orange solid (145 mg, 82%);
mp >250 ; 1H NMR (DMSO-d6) ꢁ (ppm): 13.78 (s, 1H), 10.77 (s, 1H), 9.65 (s, 1H),
8.12 (d, J = 9.2 Hz, 1H), 7.72 (s, 1H), 7.49 (d, J = 8.8 Hz, 2H), 7.40 (d, J = 8.4 Hz,
2H), 6.74 (d, J = 9.2 Hz, 1H); HRMS (ESI): m/z, calcd for C15H11ClN3O4 [M+H+]:
332.0432, found 332.0430.
4.1.3.5.11. 4-(3-nitrophenylamino)-7-nitro-1H-indole-2-carboxylic acid (2.7k)
Following the procedure of 4.1.2.5.1, starting from compound 2.5k (122 mg, 0.33
mmol), the title compound 2.7k was obtained as orange solid (105 mg, 93%);
1
mp >250 ; H NMR (DMSO-d6) ꢁ (ppm): 13.42 (brs, 1H), 10.86 (s, 1H), 9.84 (s,
1H), 8.15-8.17 (m, 2H), 7.96 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.72 (s,
1H), 7.69 (t, J = 8.0 Hz, 1H), 6.95 (d, J = 9.2 Hz, 1H); HRMS (ESI): m/z, calcd for
C15H10N4O6 [M+H+]: 343.0673, found 343.0665.
4.1.3.5.12. 4-(4-nitrophenylamino)-7-nitro-1H-indole-2-carboxylic acid (2.7l)
Following the procedure of 4.1.2.5.1, starting from compound 2.5l (140 mg, 0.38
mmol), the title compound 2.7l was obtained as orange solid (113 mg, 88%);
mp >250 ; 1H NMR (DMSO-d6) ꢁ (ppm): 10.97 (s, 1H), 10.03 (s, 1H), 8.25 (d, J =
8.8 Hz, 2H), 8.21 (d, J = 8.8 Hz, 1H), 7.12 (s, 1H), 7.53 (d, J = 8.8 Hz, 2H), 7.15 (d, J
= 9.2 Hz, 1H); HRMS (ESI): m/z, calcd for C15H11N4O6 [M+H+]: 343.0673, found
343.0672.
4.1.3.5.13. 4-(3-methylphenylamino)-7-nitro-1H-indole-2-carboxylic acid (2.7m)
Following the procedure of 4.1.2.5.1, starting from compound 2.5m (190 mg, 0.46
mmol), the title compound 2.7m was obtained as yellow solid (90 mg, 80%);
mp >250 ; 1H NMR (DMSO-d6) ꢁ (ppm): 10.43 (s, 1H), 9.49 (s, 1H), 8.03 (d, J =
8.8 Hz, 1H), 7.47 (s, 1H), 7.31 (t, J = 8.0 Hz, 1H), 7.16-7.19 (m, 2H), 7.01 (d, J = 7.6
Hz, 1H), 6.69 (d, J = 9.2 Hz, 1H), 2.33 (s, 3H); HRMS (ESI): m/z, calcd for
C16H14N3O4 [M+H+]: 312.0979, found 312.0976.
4.1.3.5.14. 4-(4-methylphenylamino)-7-nitro-1H-indole-2-carboxylic acid (2.7n)
Following the procedure of 4.1.2.5.1, starting from compound 2.5n (190 mg, 0.46
mmol), the title compound 2.7n was obtained as yellow solid (160 mg, 97%); mp:
257-259 ; 1H NMR (DMSO-d6) ꢁ (ppm): 10.70 (s, 1H), 9.61 (s, 1H), 8.09 (d, J = 9.2
Hz, 1H), 7.74 (s, 1H), 7.27 (s, 4H), 6.62 (d, J = 9.2 Hz, 1H), 2.34 (s, 3H); HRMS
(ESI): m/z, calcd for C16H14N3O4 [M+H+]: 312.0979, found 312.0973.
4.1.3.5.15. 4-(3-acetamidophenylamino)-7-nitro-1H-indole-2-carboxylic acid
(2.7o)
Following the procedure of 4.1.2.5.1, starting from compound 2.5o (190 mg, 0.46
mmol), the title compound 2.7o was obtained as sorrel solid (100 mg, 98%);
mp >250 ; 1H NMR (DMSO-d6) ꢁ (ppm): 10.72 (s, 1H), 10.08 (s, 1H), 9.69 (s ,1H),
8.11 (d, J = 9.2 Hz, 1H), 7.78 (m, 2H), 7.33-7.36 (m, 2H), 7.04 (d, J = 5.6 Hz, 1H),
6.75 (d, J = 8.8 Hz, 1H), 2.05 (s, 3H); HRMS (ESI): m/z, calcd for C17H15N4O5
[M+H+]: 355.1037, found 355.1033.
14

4.1.3.5.16. 4-(3-aminophenylamino)-7-nitro-1H-indole-2-carboxylic acid (2.7p)
To a solution of 2.7o (120 mg, 0.339 mmol) in acetic acid (4 mL), concentrated
HCl (2 mL) was added. The resulting mixture was refluxed for 12 hours. After
filtration, the residue was dissolved in NaOH aqueous (2 mL, 2M), and extacted with
EtOAc. The aqueous layer was acidified to PH=3-4 with diluted HCl. After filtration,
1
the title compound 2.7p was obtained as dark solid (60 mg, 57%); mp >250 ; H
NMR (DMSO-d6) ꢁ (ppm): 11.81 (s, 1H), 9.78 (s, 1H), 8.14 (d, J = 9.0 Hz, 1H), 7.79
(d, J = 2.1 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.24 (m, 2H), 7.01 (d, J = 7.5 Hz, 1H),
6.84 (d, J = 9.0 Hz, 1H); HRMS (ESI): m/z, calcd for C15H13N4O4 [M+H+]: 313.0931,
found 313.0927.
4.1.3.6. Synthesis of ethyl 4-substituted-7-nitro-1H-indole-2-carboxylate
(2.6a-2.6j, 2.6l)
4.1.3.6.1. Ethyl 7-nitro-4-phenyl-1H-indole-2-carboxylate (2.6a)
To a solution of 2.4 (50 mg, 0.19 mmol) in DMF (5 mL), Pd(OAc)2 (8.4 mg, 0.037
mmol), dppf (41 mg, 0.074 mmol), Et3N (95 mg, 0.94 mmol) and phenylboronic acid
(68 mg, 0.56 mmol) were added in order. The reaction mixture was heated to 80 for
20 h under argon atmosphere, and concentrated under reduced pressure. The residue
was dissolved in EtOAc (20 mL), and washed with brine (10 mL×3) and water (10
mL×3). The crude product obtained after concentration was purified with column
chromatography to afford product 2.6a as light yellow solid (90 mg, 60%); mp:
180-181 ; 1H NMR (acetone-d6) ꢁ (ppm): 10.86 (brs, 1H), 8.41 (d, J = 8.0 Hz, 1H),
7.77 (d, J = 7.6 Hz, 2H), 7.54-7.64 (m, 3H), 7.47 (d, J = 8.4 Hz, 1H), 7.44 (s, 1H),
4.43 (q, J = 7.2 Hz, 2H), 1.40 (t, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for
C17H15N2O4 [M+H+]: 311.1026, found 311.1017.
4.1.3.6.2. Ethyl 4-(3-methoxyphenyl)-7-nitro-1H-indole-2-carboxylate (2.6b)
To a solution of 2.4 (250 mg, 0.93 mmol) in toluene (15 mL), Pd2(dba)3 (85 mg,
0.093 mmol), Xantphos (108 mg, 0.186 mmol) and Na2CO3 (296 mg, 2.79 mmol, 2
mL H2O) and 3-methoxyphenylboronic acid (424 mg, 2.79 mmol) were sequentially
added. The reaction mixture was refluxed for 3 h under argon atmosphere, and
concentrated under reduced pressure. The residue was dissolved in EtOAc (30 mL),
and washed with brine (20 mL×3) and water (20 mL×3). The crude product obtained
after concentration was purified with column chromatography to afford product 2.6b
1
as light yellow solid (268 mg, 85%); mp: 182-183 ; H NMR (CDCl3) ꢁ (ppm):
10.47 (s, 1H), 8.35 (d, J = 8.4 Hz, 1H), 7.45-7.47 (m, 2H), 7.31 (d, J = 8.4 Hz, 1H),
7.25 (d, J = 9.2 Hz, 1H), 7.18 (s, 1H), 7.04 (d, J = 8.4 Hz, 1H), 4.46 (q, J = 7.2 Hz,
2H), 3.89 (s, 3H), 1.43 (t, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for C18H17N2O5
[M+H+]: 341.1132, found 341.1119.
4.1.3.6.3. Ethyl 4-(3-chlorophenyl)-7-nitro-1H-indole-2-carboxylate (2.6c)
Following the procedure of 4.1.3.6.2, starting from compound 2.4 (220 mg, 0.82
mmol), the title compound 2.6c was obtained as light yellow solid (190 mg, 67%), mp:
148-150 ; 1H NMR (acetone-d6) ꢁ (ppm): 10.90 (brs, 1H), 8.40 (d, J = 8.4 Hz, 1H),
7.75 (s, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.64 (t, J = 8.0 Hz, 1H), 7.58 (d, J = 8.0 Hz,
1H), 7.49 (d, J = 8.0 Hz, 1H), 7.41 (s, 1H), 4.43 (q, J = 7.2 Hz, 2H), 1.40 (t, J = 7.2
Hz, 3H); HRMS (ESI): m/z, calcd for C17H14ClN2O4 [M+H+]: 345.0637, found
345.0624.
4.1.3.6.4. Ethyl 4-(4-chlorophenyl)-7-nitro-1H-indole-2-carboxylate (2.6d)
15

Following the procedure of 4.1.3.6.2, starting from compound 2.4 (220 mg, 0.82
mmol), the title compound 2.6d was obtained as light yellow solid light green solid
(200 mg, 71%); mp: 186-187 ℃; 1H NMR (acetone-d6) ꢁ (ppm): 8.40 (d, J = 8.4 Hz,
1H), 7.78-7.81 (m, 2H), 7.63-7.66 (m, 2H), 7.43-7.49 (m, 2H), 4.43 (q, J = 7.2 Hz,
2H), 1.39 (t, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for C17H14ClN2O4 [M+H+]:
345.0637, found 345.0625.
4.1.3.6.5. Ethyl 4-(3-cyanophenyl)-7-nitro-1H-indole-2-carboxylate (2.6e)
Following the procedure of 4.1.3.6.2, starting from compound 2.4 (250 mg, 0.93
mmol), the title compound 2.6e was obtained as light yellow solid (200 mg, 71%); mp:
195-197 ℃; 1H NMR (acetone-d6) ꢁ (ppm): 10.95 (brs, 1H), 8.44 (d, J = 8.4 Hz, 1H),
8.17 (s, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.85 (t, J = 8.0 Hz,
1H), 7.55 (d, J = 8.4 Hz, 1H), 7.48 (s, 1H), 4.44 (q, J = 7.2 Hz, 2H), 1.39 (t, J = 7.2
Hz, 3H); HRMS (ESI): m/z, calcd for C18H14N3O4 [M+H+]: 336.0979, found
336.0968.
4.1.3.6.6. Ethyl 4-(4-cyanophenyl)-7-nitro-1H-indole-2-carboxylate (2.6f)
Following the procedure of 4.1.3.6.2, starting from compound 2.4 (250 mg, 0.93
mmol), the title compound 2.6f was obtained as light yellow solid light yellow solid
(248 mg, 80%); mp: 213-215 ℃; 1H NMR (CDCl3) ꢁ (ppm): 10.52 (brs, 1H), 8.39 (d,
J = 8.4 Hz, 1H), 7.86 (d, J = 8.7 Hz, 2H), 7.78 (d, J = 8.1 Hz, 2H),7.37-7.38 (m, 1H),
7.32 (d, J = 8.1 Hz, 1H), 4.47 (q, J = 6.9 Hz, 2H), 1.44 (t, J = 6.9 Hz, 3H); HRMS
(ESI): m/z, calcd for C18H14N3O4 [M+H+]: 336.0979, found 336.0968.
4.1.3.6.7. Ethyl 4-(3-methylphenyl)-7-nitro-1H-indole-2-carboxylate (2.6g)
Following the procedure of 4.1.3.6.2, starting from compound 2.4 (220 mg, 0.82
mmol), the title compound 2.6g was obtained as light yellow solid (176 mg, 66%); mp:
1
91-92 ℃; H NMR (acetone-d6) ꢁ (ppm): 10.83 (brs, 1H), 8.39 (d, J = 8.0 Hz, 1H),
7.43-7.57 (m, 5H), 7.37 (d, J = 7.2 Hz, 1H), 4.43 (q, J = 7.2 Hz, 2H), 2.47 (s, 3H),
1.39 (t, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for C18H17N2O4 [M+H+]: 325.1183,
found 325.1172.
4.1.3.6.8. Ethyl 7-nitro-4-(3-(trifluoromethyl)phenyl)-1H-indole-2-carboxylate
(2.6h)
Following the procedure of 4.1.3.6.2, starting from compound 2.4 (220 mg, 0.82
mmol), the title compound 2.6h was obtained as light yellow solid (256 mg, 83%);
mp: 137-139 ℃; 1H NMR (acetone-d6) ꢁ (ppm): 11.02 (brs, 1H), 8.51 (d, J = 8.4 Hz,
1H), 8.16 (d, J = 6.8 Hz, 1H), 7.13 (s, 1H), 7.96-7.98 (m, 2H), 7.63 (d, J = 8.4 Hz,
1H), 7.48 (s, 1H), 4.52 (q, J = 7.2 Hz, 2H), 1.47 (t, J = 7.2 Hz, 3H); HRMS (ESI): m/z,
calcd for C18H14F3N2O4 [M+H+]: 379.0900, found 379.0886.
4.1.3.6.9. Ethyl 7-nitro-4-(3-(trifluoromethyl)phenyl)-1H-indole-2-carboxylate
(2.6i)
Following the procedure of 4.1.3.6.2, starting from compound 2.4 (220 mg, 0.82
mmol), the title compound 2.6i was obtained as light yellow solid (225 mg, 73%); mp:
178-179 ℃; 1H NMR (acetone-d6) ꢁ (ppm): 10.96 (brs, 1H), 8.43 (d, J = 8.4 Hz, 1H),
8.00 (d, J = 8.4 Hz, 2H), 7.96 (d, J = 8.0 Hz, 2H), 7.54 (d, J = 8.4 Hz, 1H), 7.44 (s,
1H), 4.43 (q, J = 7.2 Hz, 2H), 1.39 (t, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for
C18H14F3N2O4 [M+H+]: 379.0900, found 379.0890.
16

4.1.3.6.10. Ethyl 4-(3-acetamidophenyl)-7-nitro-1H-indole-2-carboxylate (2.6j)
Following the procedure of 4.1.3.6.2, starting from compound 2.4 (300 mg, 1.12
mmol), the title compound 2.6j was obtained as light yellow solid (248 mg, 60%); mp:
183-184 ; 1H NMR (CDCl3) ꢁ (ppm): 10.45 (s, 1H), 8.33 (d, J = 8.0 Hz, 1H), 7.84 (s,
1H), 7.63 (d, J = 7.6 Hz, 1H), 7.47-7.51 (m, 3H), 7.39 (d, J= 7.6 Hz, 1H), 7.30 (d, J =
8.4 Hz, 1H), 4.45 (q, J = 7.2 Hz, 2H), 2.23 (s, 3H), 1.43 (t, J = 7.2 Hz, 3H); HRMS
(ESI): m/z, calcd for C19H18N3O5 [M+H+]: 368.1241, found 368.1228.
4.1.3.6.11. Ethyl 4-isobutyl-7-nitro-1H-indole-2-carboxylate (2.6l)
To a solution of compound 2.4 (250 mg, 0.93 mmol) in toluene (12 mL), Pd(OAc)2
(21 mg, 0.093 mmol), t-Bu3P·HBF4 (54 mg, 0.186 mmol) and K3PO4 (987 mg, 4.65
mmol, 1 mL H2O) and isobutylboronic acid (284 mg, 2.79 mmol) were sequentially
added. The reaction mixture was heated to 90 for 2 h under argon atmosphere, and
concentrated under reduced pressure. The residue was dissolved in EtOAc (20 mL),
and washed with brine (20 mL×3) and water (20 mL×3). The crude product obtained
after concentration was purified with column chromatography to afford product 2.6n
1
as off-white solid (216 mg, 80%); mp: 108-109 ; H NMR (CDCl3) ꢁ (ppm): 10.36
(s, 1H), 8.23 (d, J = 8.0 Hz, 1H), 7.38 (s, 1H), 7.05 (d, J = 8.0 Hz, 1H), 4.47 (q, J =
7.2 Hz, 2H), 2.86 (d, J = 7.2 Hz, 2H), 2.04-2.15 (m, 1H), 1.46 (t, J = 7.2 Hz, 2H),
0.97 (d, J = 7.2 Hz, 6H); HRMS (ESI): m/z, calcd for C15H19N2O4 [M+H+]: 291.1339,
found 291.1331.
4.1.3.7. Synthesis of 4-substituted-7-nitro-1H-indole-2-carboxylic acid (2.8a-2.8l)
4.1.3.7.1. 7-nitro-4-phenyl-1H-indole-2-carboxylic acid (2.8a)
Following the procedure of 4.1.2.5.1, starting from compound 2.6a (90 mg, 0.29
mmol), the title compound 2.8a was obtained as light yellow solid (55 mg, 68%);
mp >250 ; 1H NMR (DMSO-d6) ꢁ (ppm): 13.61 (brs, 1H), 11.26 (s, 1H), 8.34 (d, J =
8.4 Hz, 1H), 7.72 (d, J = 6.9 Hz, 2H), 7.53-7.62 (m, 3H), 7.42 (d, J = 8.4 Hz, 1H),
7.31 (s, 1H); HRMS (ESI): m/z, calcd for C15H11N2O4 [M+H+]: 283.0713, found
283.0710.
4.1.3.7.2. 4-(3-methoxyphenyl)-7-nitro-1H-indole-2-carboxylic acid (2.8b)
Following the procedure of 4.1.2.5.1, starting from compound 2.6b (128 mg, 0.38
mmol), the title compound 2.8b was obtained as light yellow solid (106 mg, 91%);
mp: 248-249 ; 1H NMR (DMSO-d6) ꢁ (ppm): 13.59 (brs, 1H), 11.27 (s, 1H), 8.33 (d,
J = 8.4 Hz, 1H), 7.51 (t, J = 8.0 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.31 (s, 1H), 7.30
(d, J = 7.6 Hz, 1H), 7.22 (s, 1H), 7.11 (d, J = 8.4 Hz, 1H), 3.80 (s, 3H); HRMS (ESI):
m/z, calcd for C16H13N2O5 [M+H+]: 313.0819, found 313.0817.
4.1.3.7.3. 4-(3-chlorophenyl)-7-nitro-1H-indole-2-carboxylic acid (2.8c)
Following the procedure of 4.1.2.5.1, starting from compound 2.6c (130 mg, 0.38
mmol), the title compound 2.8c was obtained as light yellow solid (50 mg, 42%);
mp >250 ; 1H NMR (DMSO-d6) ꢁ (ppm): 13.70 (brs, 1H), 11.25 (s, 1H), 8.33 (d, J =
8.0 Hz, 1H), 7.75 (s, 1H), 7.70 (d, J = 6.4 Hz, 1H), 7.61-7.64 (m, 2H), 7.45 (d, J = 8.4
Hz, 1H), 7.24 (s, 1H); HRMS (ESI): m/z, calcd for C15H10ClN2O4 [M+H+]: 317.0324,
found 317.0316.
4.1.3.7.4. 4-(4-chlorophenyl)-7-nitro-1H-indole-2-carboxylic acid (2.8d)
Following the procedure of 4.1.2.5.1, starting from compound 2.6d (100 mg, 0.29
17

mmol), the title compound 2.8d was obtained as light yellow solid (25 mg, 27%);
mp >250 ; 1HNMR (DMSO-d6) ꢁ (ppm): 13.62 (brs, 1H), 11.32 (s, 1H), 8.35 (d, J =
8.0 Hz, 1H), 7.76 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.0 Hz,
1H), 7.32 (s, 1H); HRMS (ESI): m/z, calcd for C15H10ClN2O4 [M+H+]: 317.0324,
found 317.0323.
4.1.3.7.5. 4-(3-cyanophenyl)-7-nitro-1H-indole-2-carboxylic acid (2.8e)
Following the procedure of 4.1.2.5.1, starting from compound 2.6e (110 mg, 0.33
mmol), the title compound 2.8e was obtained as white solid (96 mg, 95%);
mp >250 ; 1H NMR (DMSO-d6) ꢁ ( ppm ): 11.32 (s, 1H), 8.33 (d, J = 8.4 Hz, 1H),
8.17 (s, 1H), 8.05 (d, J = 7.6 Hz, 1H), 7.98 (d, J = 7.2 Hz, 1H), 7.78 (t, J = 7.6 Hz,
1H), 7.47 (d, J = 8.4 Hz, 1H), 7.32 (s, 1H); HRMS (ESI): m/z, calcd for C16H10N3O4
[M+H+]: 308.0666, found 308.0652.
4.1.3.7.6. 4-(4-cyanophenyl)-7-nitro-1H-indole-2-carboxylic acid (2.8f)
Following the procedure of 4.1.2.5.1, starting from compound 2.6f (202 mg, 0.60
mmol), the title compound 2.8f was obtained as light yellow solid (116 mg, 63%);
mp >250 ;1H NMR (DMSO-d6) ꢁ (ppm): 13.64 (brs, 1H), 11.38 (s, 1H), 8.35 (d, J =
8.0 Hz, 1H), 8.04 (d, J = 8.0 Hz, 2H), 7.92 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz,
1H), 7.30 (s, 1H); HRMS (ESI): m/z, calcd for C16H11N3O4 [M+H+]: 308.0666, found
308.0657.
4.1.3.7.7. 4-(3-methylphenyl)-7-nitro-1H-indole-2-carboxylic acid (2.8g)
Following the procedure of 4.1.2.5.1, starting from compound 2.6g (116 mg, 0.36
mmol), the title compound 2.8g was obtained as light yellow solid (80 mg, 75%);
mp >250 ; 1H NMR (DMSO-d6) ꢁ (ppm): 13.61 (brs, 1H), 11.26 (s, 1H), 8.35 (d, J =
8.0 Hz, 1H), 7.47-7.54 (m, 3H), 7.42 (d, J = 8.0 Hz, 1H), 7.36 (d, J = 7.2 Hz, 1H),
7.32 (s, 1H), 2.44 (s, 3H); HRMS (ESI): m/z, calcd for C16H13N2O4 [M+H+]: 297.0870,
found 297.0867.
4.1.3.7.8. 4-(3-(trifluromethyl)phenyl)-7-nitro-1H-indole-2-carboxylic acid (2.8h)
Following the procedure of 4.1.2.5.1, starting from compound 2.6h (141 mg, 0.37
mmol), the title compound 2.8h was obtained as light yellow solid (68 mg, 52%); mp:
217-219 ; 1H NMR (DMSO-d6) ꢁ (ppm): 11.29 (brs, 1H), 8.35 (d, J = 8.4 Hz, 1H),
8.05 (d, J = 7.6 Hz, 1H), 8.00 (s, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.83 (t, J = 8.0 Hz,
1H), 7.50 (d, J = 8.0 Hz, 1H), 7.23 (s, 1H); HRMS (ESI): m/z, calcd for C16H10F3N2O4
[M+H+]: 351.0587, found 351.0580.
4.1.3.7.9. 4-(4-(trifluoromethyl)phenyl)-7-nitro-1H-indole-2-carboxylic acid (2.8i)
Following the procedure of 4.1.2.5.1, starting from compound 2.6i (140 mg, 0.37
mmol), the title compound 2.8i was obtained as light yellow solid (75 mg, 58%); mp:
221-223 ; 1H NMR (DMSO-d6) ꢁ (ppm): 13.63 (brs, 1H), 11.39 (s, 1H), 8.38 (d, J =
8.0 Hz, 1H), 7.95 (m, 4H), 7.50 (d, J = 8.4 Hz, 1H), 7.34 (s, 1H); HRMS (ESI): m/z,
calcd for C16H10F3N2O4 [M+H+]: 351.0587, found 351.0587.
4.1.3.7.10. 4-(3-acetamidophenyl)-7-nitro-1H-indole-2-carboxylic acid (2.8j)
Following the procedure of 4.1.2.5.1, starting from compound 2.6j (90 mg, 0.25
mmol), the title compound 2.8j was obtained as light yellow solid (65 mg, 78%);
1
mp >250 ; H NMR (DMSO-d6) ꢁ (ppm): 13.60 (brs, 1H), 11.27 (s, 1H), 10.17 (s,
1H), 8.35 (d, J = 8.4 Hz, 1H), 8.02 (s, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.50 (t, J = 7.8
18

Hz, 1H), 7.36-7.42 (m, 3H), 2.08 (s, 3H); HRMS (ESI): m/z, calcd for C17H14N3O5
[M+H+]: 340.0928, found 340.0921.
4.1.3.7.11. 4-(3-aminophenyl)-7-nitro-1H-indole-2-carboxylic acid (2.8k)
To a solution of 2.8j (125 mg, 0.37 mmol) in methanol (3 mL), concentrated HCl (3
mL, 6 N) was added. The resulting mixture was refluxed for 3 h. After filtration, the
residue was dissolved in NaOH aqueous (2 mL, 2M), and extracted with EtOAc. The
aqueous layer was acidified to pH=3-4 with diluted HCl. After filtration, the title
compound 2.8k was obtained as light yellow solid (38 mg, 35%); mp >250 ; 1H
NMR (DMSO-d6) ꢁ (ppm): 10.30 (brs, 1H), 8.16 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 8.8
Hz, 1H), 7.20 (t, J = 7.6 Hz, 1H), 6.95 (s, 1H), 6.91 (s, 1H), 6.83 (d, J = 7.6 Hz, 1H),
6.68 (d, J = 7.6 Hz, 1H), 5.33 (s, 2H); HRMS (ESI): m/z, calcd for C15H12N3O4
[M+H+]: 298.0822, found 298.0819.
4.1.3.7.12. 4-isobutyl-7-nitro-1H-indole-2-carboxylic acid (2.8l)
Following the procedure of 4.1.2.5.1, starting from compound 2.6l (156 mg, 0.54
mmol), the title compound 2.8l was obtained as white solid (116 mg, 82%); mp:
1
195-197 ; H NMR (DMSO-d6) ꢁ (ppm): 13.53 (s, 1H), 11.02 (s, 1H), 8.21 (d, J =
8.4 Hz, 1H), 7.49 (s, 1H), 7.15 (d, J = 8.0 Hz, 1H), 2.88 (d, J = 7.2 Hz, 2H), 1.96-2.06
(m, 1H), 0.91 (d, J = 6.4 Hz, 6H); HRMS (ESI): m/z, calcd for C13H15N2O4 [M+H+]:
263.1026, found 263.1024.
4.1.3.8. Synthesis of ethyl 4-substituted-7-nitro-1H-indole-2-carboxylate
(2.9a-2.9b)
4.1.3.8.1. Ethyl
-carboxylate (2.9a)
4-((3-methoxyphenyl)(methyl)amino)-7-nitro-1H-indole-2
To a solution of 2.6c (200 mg, 0.56 mmol) in DMF (10 mL), Cs2CO3 (363 mg, 1.12
mmol) and iodomethane (79 mg, 0.56 mmol) was added. The reaction mixture was
stirred at room temperature for 3 h, and then concentrated under reduced pressure.
The residue was dissolved in EtOAc (20 mL), washed with water (20 m ×3), dried
with anhydrous MgSO4 and concentrated under reduced pressure. The crude product
was purified by silica gel column chromatography using petroleum ether/ethyl acetate
as eluent to provide compound 2.9a as yellow solid (100 mg, 48%); mp: 138-139
;
1H NMR (CDCl3) ꢁ (ppm): 10.44 (brs, 1H), 8.22 (d, J = 9.3 Hz, 1H), 7.36 (t, J = 8.1
Hz, 1H), 6.89-6.92 (dd,?? 1H), 6.83 (d, J = 7.2 Hz, 1H), 6.77 (s, 1H), 6.50 (d, J = 9.0
Hz, 1H), 6.25-6.26 (m, 1H), 4.34 (q, J = 7.2 Hz, 2H), 3.80 (s, 3H), 3.60 (s, 3H), 1.35
(t, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for C19H20N3O5 [M+H+]: 370.1398,
found 370.1386.
4.1.3.8.2. Ethyl
-carboxylate (2.9b)
4-((3-methoxyphenyl)(propyl)amino)-7-nitro-1H-indole-2
Following the procedure of 4.1.3.8.1, starting from compound 2.6c (250 mg, 0.70
mmol), the title compound 2.8l was obtained as light yellow solid (55 mg, 20%), mp:
115-117 ; 1H NMR (CDCl3) ꢁ (ppm): 10.43 (brs, 1H), 8.20 (d, J = 9.0 Hz, 1H), 7.36
(t, J = 8.4 Hz, 1H), 6.91 (d, J = 8.7 Hz, 1H), 6.82 (d, J = 7.8 Hz, 1H), 6.76 (s, 1H),
6.50 (d, J = 9.3 Hz, 1H), 6.05 (d, J = 2.1 Hz, 1H), 4.32 (q, J = 7.2 Hz, 2H), 3.87 (t, J =
8.1 Hz, 2H), 3.81 (s, 3H), 1.76-1.89 (m, 2H), 1.34 (t, J = 7.2 Hz, 3H), 1.00 (t, J = 7.5
Hz, 3H); HRMS (ESI): m/z, calcd for C21H24N3O5 [M+H+]: 398.1711, found
398.1697.
19

4.1.3.9. Synthesis of 4-substituted-7-nitro-1H-indole-2-carboxylic (2.10a-2.10b)
4-((3-methoxyphenyl)(methyl)amino)-7-nitro-1H-indole-2-carboxylic
4.1.3.9.1
acid (2.10a)
Following the procedure of 4.1.2.5.1, starting from compound 2.9a (100 mg, 0.27
mmol), the title compound 2.10a was obtained as orange solid (30 mg, 33%); mp:
223-225 ; 1H NMR (DMSO-d6) ꢁ (ppm): 13.24 (brs, 1H), 10.64 (s, 1H), 8.20 (d, J =
9.2 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H), 7.00 (d, J = 8.0 Hz, 1H), 6.95 (s, 1H), 6.89 (d, J
= 8.0 Hz, 1H), 6.69 (d, J = 9.2 Hz, 1H), 5.94 (s, 1H), 3.76 (s, 3H), 3.55 (s, 3H);
HRMS (ESI): m/z, calcd for C17H16N3O5 [M+H+]: 342.1085, found 342.1083.
4.1.3.9.2.
acid (2.10b)
4-((3-methoxyphenyl)(propyl)amino)-7-nitro-1H-indole-2-carboxylic
Following the procedure of 4.1.2.5.1, starting from compound 2.9b (50 mg, 0.13
mmol), the title compound 2.10b was obtained as yellow solid (40 mg, 86%); mp:
191-192 ; 1HNMR (DMSO-d6) ꢁ (ppm): 13.23 (brs, 1H), 10.60 (brs, 1H), 8.18 (d, J
= 9.3 Hz, 1H), 7.42 (t, J = 7.8 Hz, 1H), 7.01 (d, J = 8.1 Hz, 1H), 6.93 (s, 1H), 6.88 (d,
J = 8.4 Hz, 1H), 6.75 (d, J = 9.3 Hz, 1H), 5.73 (d, J = 1.5 Hz, 1H), 3.89 (t, J = 7.8 Hz,
2H), 3.77 (s, 3H), 1.67-1.75 (m, 2H), 0.94 (t, J = 7.5 Hz, 3H); HRMS (ESI): m/z,
calcd for C19H20N3O5 [M+H+]: 370.1398, found 370.1398.
4.1.4. Synthesis of 5-subsituted-7-nitro-1H-indole-2-carboxylic acid derivatives
4.1.4.1. Ethyl 2-(2-(4-bromo-2-nitrophenyl)hydrazono)propanoate (3.3) bjb-1540
A solution of 4-bromo-2-nitroaniline (20 g, 92 mmol) in concentrated HCl (100 mL)
was heated to 40 for 2 h. The mixture was then cooled to -10 , and a solution of
NaNO2 (6.98 g, 101.2 mmol) in water (50 mL) was added `dropwise. After 2 h, the
mixture was cooled to -30 and added to a solution of SnCl2 (62 g, 276 mmol) in
concentrated HCl (100 mL) precooled to -30 . The reaction mixture was stirred at
-20 for 1 h, then warmed to room temperature and filtered. The title compound 3.2
was obtained as light yellow solid (24 g, 97%);
To a solution of compound 3.2 (24 g, 89.3 mmol) in ethanol (60 mL) ethyl pyruvate
(11.4 g, 98.3 mmol) was added dropwise. The reaction mixture was stirred at room
temperature for 10 min. After filtration, the title compound 3.3 was obtained as yellow
solid (27 g, 92%); mp: 124-125 ; 1H NMR (CDCl3) ꢁ (ppm): 13.91 (brs, 1H), 8.34 (s,
1H), 7.92 (d, J = 9.2 Hz, 1H), 7.62 (d, J = 9.2 Hz, 1H), 4.39 (q, J = 7.2 Hz, 2H), 2.25
(s, 3H), 1.40 (t, J = 7.2 Hz, 3H); HRMS (ESI): m/z, calcd for C11H13BrN3O4 [M+H+]:
330.0084, found 330.0074.
4.1.4.2. Ethyl 5-bromo-7-nitro-1H-indole-2 -carboxylate (3.4)
The reaction mixture of compound 3.3 (27 g, 81.8 mmol) in PPA (270 g) was
heated at 90-100 for 4 h, and then cooled to room temperature. Water (400 mL) was
added to the mixture to destroy the PPA, and the residue was extracted with EtOAc
(100 mL×4). The crude product obtained after concentration was purified with
column chromatography to afford title compound 3.4 (11.7 g, 44%) as light yellow
solid; mp: 135-137 ; 1H NMR (CDCl3) ꢁ (ppm): 10.31 (brs, 1H), 8.40 (s, 1H), 8.17
(s, 1H), 7.30 (s, 1H), 4.47 (q, J = 7.2 Hz, 2H), 1.45 (t, J = 7.2 Hz, 3H); HRMS (ESI):
m/z, calcd for C11H10BrN2O4 [M+H+]: 312.9818, found 312.9807.
4.1.4.3. Synthesis of ethyl 5-substituted-7-nitro-1H-indole-2-carboxylate
20

(3.5a-3.5c)
4.1.4.3.1. Ethyl 5-isobutyl-7-nitro-1H-indole-2 -carboxylate (3.5a)
To a solution of compound 3.4 (750 mg, 2.40 mmol) in toluene (28 mL), Pd(OAc)2
(54 mg, 0.24 mmol), t-Bu3P·HBF4 (139 mg, 0.48 mmol) and K3PO4 (2.04 g, 9.6 mmol,
5.5 mL H2O) and isobutylboronic acid (489 mg, 4.8 mmol) were sequentially added.
The reaction mixture was heated at 90 ℃ for 5 h under argon atmosphere, and then
concentrated under reduced pressure. The residue was dissolved in EtOAc (60 mL),
and washed with brine (20 mL×3) and water (20 mL×3). The crude product obtained
after concentration was purified with column chromatography to afford title product
3.5a as light yellow solid (614 mg, 88%); mp: 99-100 ; 1H NMR (CDCl3) ꢁ (ppm):
10.23 (brs, 1H), 8.11 (s, 1H), 7.81 (s, 1H), 7.29 (s, 1H), 4.46 (q, J = 7.2 Hz, 2H), 2.65
(d, J = 7.2 Hz, 2H), 1.91-1.98 (m, 1H), 1.44 (t, J = 7.2 Hz, 3H), 0.94 (d, J = 6.8 Hz,
6H); HRMS (ESI): m/z, calcd for C15H19N2O4 [M+H+]: 291.1339, found 291.1330.
4.1.4.3.2. Ethyl 5-(3-acetamidophenyl)-7-nitro-1H-indole-2-carboxylate (3.5b)
Following the procedure of 4.1.3.4.2, starting from compound 3.4 (250 mg, 0.64
mmol), the title compound 3.5b was obtained as yellow solid (238 mg, 81%); mp:
124-125 ℃; 1H NMR (CDCl3) ꢁ (ppm): 10.30 (s, 1H), 8.50 (s, 1H), 8.23 (s, 1H), 7.89
(s, 1H), 7.37-7.49 (m, 5H), 4.48 (q, J = 7.2 Hz, 2H), 2.23 (s, 3H), 1.46 (t, J = 7.2 Hz,
3H); HRMS (ESI): m/z, calcd for C19H18N3O5 [M+H+]: 368.1241, found 368.1223.
4.1.4.3.3. Ethyl 5-((3-methoxyphenyl)amino)-7-nitro-1H-indole-2-carboxylate
(3.5c)
To a solution of compound 3.4 (300 mg, 0.96 mmol) in toluene (19 mL), Pd2(dba)3
(88 mg, 0.096 mmol), Davephos (76 mg, 0.19 mmol) and K3PO4 (611 mg, 2.88 mmol,
2.5 mL H2O) and 3-methoxyaniline (489 mg, 4.8 mmol) were sequentially added. The
reaction mixture was heated at 90 for 10 h under argon atmosphere, and then
concentrated under reduced pressure. The residue was dissolved in EtOAc (40 mL),
and washed with brine (20 mL×3) and water (20 mL×3). The crude product obtained
after concentration was purified with column chromatography to afford title product
3.5c as sorrel solid (242 mg, 71%); mp: 99-100 ; 1H NMR (CDCl3) ꢁ (ppm): 10.18
(brs, 1H), 8.11 (d, J = 2.1 Hz, 1H), 7.79 (d, J = 1.5 Hz, 1H), 7.18-7.24 (m, 2H),
6.51-6.63 (m, 3H), 4.46 (q, J = 7.2 Hz, 2H), 3.79 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H);
HRMS (ESI): m/z, calcd for C18H18N3O5 [M+H+]: 356.1241, found 356.1228.
4.1.4.4. Synthesis of 5-substituted-7-nitro-1H-indole-2-carboxylic acid (3.6a-3.6c)
4.1.4.4.1. 5-isobutyl-7-nitro-1H-indole-2-carboxylic acid (3.6a)
Following the procedure of 4.1.2.5.1, starting from compound 3.5a (166 mg, 0.57
mmol), the title compound 3.6a was obtained as light yellow solid (155 mg, 100%);
mp >300 ; 1H NMR (DMSO-d6) ꢁ (ppm): 10.25 (brs, 1H), 7.93 (s, 1H), 7.89 (s, 1H),
6.86 (s, 1H), 2.63 (d, J = 7.2 Hz, 2H),1.86-1.94 (m, 1H), 0.89 (d, J = 6.9 Hz, 6H);
HRMS (ESI): m/z, calcd for C13H15N2O4 [M+H+]: 263.1026, found 263.1026.
4.1.4.4.2. 5-(3-acetamidophenyl)-7-nitro-1H-indole-2-carboxylic acid (3.6b)
Following the procedure of 4.1.2.5.1, starting from compound 3.5b (142 mg, 0.39
21

mmol), the title compound 3.6b was obtained as yellow solid (130 mg, 100%); mp:
282-284 ℃; 1H NMR (DMSO-d6) ꢁ (ppm): 13.60 (brs, 1H), 11.25 (s, 1H), 10.09 (s,
1H), 8.45 (s, 1H), 8.40 (d, J = 1.5 Hz, 1H), 7.98 (s, 1H), 7.60-7.63 (m, 1H), 7.39-7.45
(m, 3H), 2.06 (s, 3H); HRMS (ESI): m/z, calcd for C17H14N3O5 [M+H+]: 340.0928,
found 340.0920.
4.1.4.4.3. 5-((3-methoxyphenyl)amino)-7-nitro-1H-indole-2-carboxylic acid (3.6c)
Following the procedure of 4.1.2.5.1, starting from compound 3.6c (130 mg, 0.37
mmol), the title compound 3.6c was obtained as yellow solid (120 mg, 100%);
1
mp >300 ; H NMR (DMSO-d6) ꢁ (ppm): 10.86 (s, 1H), 8.42 (s, 1H), 8.00 (d, J =
0.9 Hz, 1H), 7.91 (d, J = 1.5 Hz, 1H), 7.27 (d, J = 1.8 Hz, 1H), 7.17 (t, J = 7.8 Hz,
1H), 6.66 (d, J = 7.8 Hz, 1H), 6.61 (s, 1H), 6.45 (dd, J1 = 7.8 Hz, J2 = 1.5 Hz, 1H),
3.72 (s, 3H); HRMS (ESI): m/z, calcd for C16H14N3O5 [M+H+]: 328.0928, found
328.0923.
4.1.4.5. Ethyl 3-bromo-5-isobutyl-7-nitro-1H-indole-2-carboxylate (3.7)
To a solution of 3.5a (250 mg, 0.86 mmol) in DMF (4 mL), a solution of NBS (168
mg, 0.95 mmol) in DMF (2 mL) was added dropwise under the ice-water bath. The
reaction mixture was stirred at room temperature for 2 h, and then poured into
ice-water (35 mL). After filtration, the title compound 1.4 was afforded as light
1
yellow solid (295 mg, 93%); mp: 84-86 ; H NMR (CDCl3) ꢁ (ppm): 10.29 (brs,
1H), 8.17 (s, 1H), 7.80 (s, 1H), 4.50 (q, J = 7.2 Hz, 2H), 2.69 (d, J = 7.2 Hz, 2H),
1.96-2.01 (m, 1H), 1.48 (t, J = 7.2 Hz, 3H), 0.96 (d, J = 6.8 Hz, 6H); HRMS (ESI):
m/z, calcd for C15H18BrN2O4 [M+H+]: 369.0445, found 369.0441.
4.1.4.6. Ethyl 3-ethyl-5-isobutyl-7-nitro-1H-indole-2-carboxylate (3.8)
To a solution of 3.7 (250 mg, 0.68 mmol) in toluene (18 mL), Pd (OAc)2 (7.6 mg,
0.034 mmol), Josiphos (44 mg, 0.068 mmol) and K3PO4 (722 mg, 3.4 mmol, 2.5 mL
H2O) and ethylboronic acid (151 mg, 2.04 mmol) were sequentially added. The
reaction mixture was heated at 80
for 36 h under argon atmosphere, and then
concentrated under reduced pressure. The residue was dissolved in EtOAc (40 mL),
and washed with brine (20 mL×3) and water (20 mL×3). The crude product obtained
after concentration was purified with column chromatography to afford product 3.8 as
1
light yellow solid (171 mg, 80%); mp: 72-73 ; H NMR (CDCl3) ꢁ (ppm): 10.03
(brs, 1H), 8.11 (s, 1H), 7.79 (s, 1H), 4.46 (q, J = 7.2 Hz, 2H), 3.14 (q, J = 7.2 Hz, 2H),
2.66 (d, J = 7.2 Hz, 2H), 1.91-1.98 (m, 1H), 1.45 (t, J = 7.2 Hz, 3H), 1.29 (t, J = 7.2
Hz, 3H), 0.95 (d, J = 6.4 Hz, 6H); HRMS (ESI): m/z, calcd for C17H23N2O4 [M+H+]:
319.1652, found 319.16510.
4.1.4.7. 3-ethyl-5-isobutyl-7-nitro-1H-indole-2-carboxylic acid (3.9)
Following the procedure of 4.1.2.5.1, starting from compound 3.8 (120 mg, 0.38
mmol), the title compound 3.9 was obtained as light yellow solid (97 mg, 89%); mp:
1
168-170 ; H NMR (DMSO-d6) ꢁ (ppm): 10.47 (s, 1H), 8.08 (s, 1H), 8.05 (s, 1H),
3.10 (q, J = 6.9 Hz, 2H), 2.68 (d, J = 6.9 Hz, 2H), 1.90-1.94 (m, 1H), 1.21 (t, J = 7.2
Hz, 3H), 0.90 (d, J = 6.6 Hz, 6H); HRMS (ESI): m/z, calcd for C15H19N2O4 [M+H+]:
22

291.1339, found 291.1337.
4.2 Biological evaluation
4.2.1 Isolation and purification of human liver FBPase.
An expression plasmid (pcDNA3.1-hFBP) for human liver FPBase was constructed
by inserting the human liver FBPase gene from plasmid EX-C0133-B31
(GeneCopoeia) into the vector pcDNA3.1. The FBPase gene from EX-C0133-B31
had a C-terminal 6xHis tag for use in purification.
For expression of the human liver FBPase, chemically competent BL21(DE3)
Rosetta cells (Novagen, Inc.) were transformed with pcDNA3.1-hFBP. A 5 mL
overnight culture grown in LB media with 150 ꢀg/mL of ampicillin was back-diluted
1000-fold into 2 L of 2YT media with 0.4% glycerol and 150 ꢀg/mL ampicillin.
Bacterial growth at 37°C was monitored using the A560 and the doubling-time was
calculated. When the growth reached an A560 of 0.4, 0.1 mM IPTG was added to
induce the cell, following which the cells were allowed to grow for three additional
doubling times. The cells were then harvested by centrifugation (4400 x g),
resuspended and lysed by sonication. The lysate was clarified by centrifugation at
31,000 x g for 15 min. The protein was purified through a Chelating Sepharose Fast
Flow column (GE Healthcare), which had been charged with 0.3 M NiSO4. The
human liver FBPase was eluted with buffer containing 50 mM sodium citrate and 50
mM NaCl, pH 4.0. The protein-containing fractions were pooled and dialyzed against
50 mM Tris-acetate buffer, 150 mM NaCl, pH 7.5 at 4 °C overnight.
Protein purity was confirmed by sodium dodecyl sulfate polyacrylamide gel
electrophoresis. Protein concentration was determined using the BioRad version of
the Bradford dye-binding assay with bovine serum albumin as the standard.28
4.2.2 Measurement of FBPase activity
FBPase activity was measured spectrophotometrically by employing the coupling
enzymes phosphoglucose isomerase and glucose-6-phosphate dehydrogenase. The
reduction of NADP+ to NADPH was monitored directly at 340 nm.29 Specifically,
buffer (100 mM Tris, 2 mM MgCl2, 0.1 mM EDTA, pH 7.5), 10 ꢀM of inhibitor and
0.72 unit of FBPase were mixed in a cuvette and equilibrated at 37°C. 0.2 mM of
NADP+, 0.01 units of phosphoglucose isomerase, 0.01 units of glucose-6-phosphate
dehydrogenase and FBP were then added to initiate the reaction. Reactions were
performed in duplicate. Inhibition curves were obtained for each compound by
plotting the relative activity versus inhibitor concentration.
4.3 Molecular docking
All molecular computation studies were performed on a Dell 2.83 GHz Core 2
running Windows XP. The X-ray crystal structure of FBPase complexed with AMP
was retrieved from protein data bank (PDB code: 1FTA).9 The CDOCKER protocol in
Discovery Studio 2.5.5 (Accelrys Software Inc., San Diego, CA) was used in this
study to investigate the binding mode of compound 3.9 in the crystal structure
FBPase.26 CDOCKER uses molecular dynamics (MD) with CHARMm force field
23

scheme to dock ligands into a binding site of targeted protein. The water molecules in
protein were removed and the protein was prepared by adding hydrogen and
correcting the incomplete residues using Clean Protein tool of DS, then the protein
was refined with CHARMm. The binding site was constructed within 7.6 Å with
AMP set as the center. Compound 3.9 was built and minimized using Prepare Ligands
tool of DS and refined with CHARMm force field. Docking of compound 3.9 into
FBPase with CDOCKER was done using the default parameters except that Pose
Cluster Radius was defined as 0.5 Å for increasing the diversity of the docked poses.
The pose with the top –CDOCKER_INTERACTION_ENERGY was chosen for
analyzing the binding features of compound 3.9 and FBPase.
Acknowledgments
This work is supported by Beijing Natural Science Foundation (No. 7132138).
References and notes
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25

MDL-29951
A
Figure 1. Chemical structures of MDL-29951 and designed compounds (A).
26

Figure 2. CDOCKER-modeled binding mode of compound 3.9 (carbon atoms colored orange) in comparison with
the crystal structure (1FTA in PDB)9 of AMP (carbon atoms colored green). H-Bonding interactions are presented
with light blue line. The chemical structure of reference molecule is presented. Molecular image was generated
with UCSF Chimera.27
27

Scheme 1. Reagents and conditions: (a) ethyl pyruvate, EtOH, r.t. (b) PPA, 80 . (c) NBS, DMF.
(d) R3B(OH)2, Pd(OAc)2, Josiphos, K3PO4(aq), toluene, 80 ; or R3B(OH)2, PdCl2(dppf),
Na2CO3(aq), 1,4-dioxane, 100 . (e) NaOH, THF, EtOH and H2O, r.t.
28

Scheme 2. Reagents and conditions: (a) (i) NaNO2, HCl, -10 ; (ii) SnCl2, HCl, -30 . (b) ethyl
pyruvate, EtOH, r.t. (c) PPA, 80 . (d) R4B(OH)2 or ArNH2, Pd2(dba)3, Xantphos, 1M Na2CO3(aq),
toluene, 80-110 ; or i-BuB(OH)2, Pd(OAc)2, t-Bu3P·HBF4, K3PO4(aq), toluene, 90 . (e) NaOH,
THF, EtOH and H2O, r.t. (f) R6X, Cs2CO3, DMF, 80
.
29