Discovery of novel indole derivatives as allosteric inhibitors of fructose-1,6-bisphosphatase

Article abstract of DOI:10.1016/j.ejmech.2014.11.049

A series of novel indole derivatives was designed and synthesized as inhibitors of fructose-1,6-bisphosphatase (FBPase). The most potent compound 14c was identified with an IC₅₀ value of 0.10 1/4M by testing the inhibitory activity against recombinant human FBPase. The structure-activity relationships were investigated on the substitution at 4- and 5-position of the indole scaffold. The binding interactions of the title compounds at AMP binding site of FBPase were predicted using CDOCKER algorithm.

Full text of DOI:10.1016/j.ejmech.2014.11.049

European Journal of Medicinal Chemistry 90 (2015) 394e405
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery of novel indole derivatives as allosteric inhibitors of
fructose-1,6-bisphosphatase
Jianbo Bie 1, Shuainan Liu 1, Zhanmei Li, Yongzhao Mu, Bailing Xu*, Zhufang Shen*
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
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel indole derivatives was designed and synthesized as inhibitors of fructose-1,6-
Received 9 August 2014
Received in revised form
30 October 2014
Accepted 24 November 2014
Available online 25 November 2014
bisphosphatase (FBPase). The most potent compound 14c was identified with an IC50 value of 0.10 mM
by testing the inhibitory activity against recombinant human FBPase. The structure-activity relationships
were investigated on the substitution at 4- and 5-position of the indole scaffold. The binding interactions
of the title compounds at AMP binding site of FBPase were predicted using CDOCKER algorithm.
© 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Fructose-1,6-bisphosphatase inhibitor
Diabetes
Allosteric inhibitor
Indole derivative
1. Introduction
namely the conversion of fructose-1,6-bisphosphate into fructose-
6-phosphate. FBPase in liver is elevated in insulin-resistant and
Type 2 diabetes mellitus is a metabolic disorder, which is usually
characterized by insulin deficiency, insulin resistance and excess
endogenous glucose production (EGP), leading to high blood
glucose levels in both fasting and postprandial state of patients. At
present, the majority of current anti-diabetic drugs reduce blood
glucose via either augmenting insulin secretion or improving in-
sulin resistance [1,2]. Although excess EGP is a major contributor to
the hyperglycemia [3e5], Metformin is the only available drug
which can effectively lower glucose levels by the indirect influence
on the EGP process [6,7]. Gluconeogenesis (GNG) is a primary EGP
process for liver to produce glucose, where three carbon substrates
such as pyruvate, lactate and glycerol are transformed into glucose.
Direct blocking GNG pathway represents a promising strategy for
the development of anti-diabetics, which can be used for the
treatment of both fasting and postprandial hyperglycemia. So far,
not any drugs have been developed into the market by directly
acting on the GNG process.
insulin-deficient animal models of diabetes [8e10], and this fact
highlights the importance of this enzyme in the control of blood
glucose. In particular, the recent reports of oral FBPase inhibitors
provided proof-of-concept in both T2DM animal models and pa-
tients [11e17]. Therefore, FBPase inhibitors are expected to serve as
a novel class of anti-diabetic agents.
FBPase is a homotetramer which exists in active (R) and inactive
(T) conformational states. It has been demonstrated that FBPase is
subjected to the competitive substrate inhibition of fructose-2,6-
bisphosphate and to the allosteric inhibition of AMP via the
conformational change from R sate toT state or stabilization of the T
state [18,19]. Up to date, a number of pharmaceutical companies
and academic laboratories have been involved in exploring the
allosteric inhibitors of AMP binding site in FBPase and there have
been many types of chemical entities reported in the literature
[11e13,20e24]. Among them, only AMP mimetic MB07803, which
is a prodrug of MB07729, has been advanced into phase II clinical
trial [16,17]. Therefore, there are enormous unmet needs to develop
FBPase inhibitors as potential anti-diabetic agents.
Fructose-1,6-bisphosphatase (FBPase) is a key enzyme in hepatic
GNG pathway, which catalyzes the penultimate rate-limiting step,
In our previous work, we described 7-nitro-1H-indole-2-
carboxylic acid derivatives [25] (A, Fig. 1, IC50: 0.99 mMe35.6 mM)
as novel allosteric inhibitors of FBPase, which were low molecular
weight and devoid of a phosphonate or phosphate group. With a
goal to increase the binding affinity of this series of indoles, we re-
* Corresponding authors.
E-mail addresses: xubl@imm.ac.cn (B. Xu), shenzhf@imm.ac.cn (Z. Shen).
1
The first two authors contributed equally.
http://dx.doi.org/10.1016/j.ejmech.2014.11.049
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

J. Bie et al. / European Journal of Medicinal Chemistry 90 (2015) 394e405
395
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 NADPþ
to NADPH was monitored spectrophotometrically [27]. All target
compounds (5aeg, 6, 8, 10aeb, 12aem and 14aed) were tested for
their inhibitory activities against human liver FBPase. The corre-
sponding results summarized in Tables 1e2 were expressed as IC50
values. AMP and compound MDL-29951 were used as reference
molecules.
According to our design strategy, namely, installation of the
carboxyethyl group at 3-position and nitro group at 7-position on
the indole-2-carboxylic acid scaffold, compound 5d was first syn-
thesized. As shown in Table 1, compound 5d displayed moderate
Fig. 1. Structural design of the target indole derivatives.
inhibitory activity with an IC50 value of 5.17 mM. In order to further
explore the contribution of 7-nitro group to the inhibition, com-
pounds 5aec and 8 were synthesized and tested. When the nitro
group was introduced at 4, 5 or 6-position of the indole ring, the
inhibitory effects of the resulting compounds 5aec decreased
drastically in comparison with compound 5d. The reduction of 7-
nitro group to 7-amino group (compound 8) led to loss of activity
as well. These results demonstrated that 7-nitro group played a key
role in the binding interaction between FBPase and this series of
indole derivatives. In addition, the esterfication of 2-carboxylic acid
or N-alkylation of compound 5d gave rise to compounds 6 and
10aeb, which presented no inhibitory activity. These results were
consistent with the known SARs of MDL-29951 derivatives [26],
and it was indicated that the designed indoles very likely took the
similar binding mode in the AMP binding site of FBPase as MDL-
29951 did. The 2-carboxylic acid formed crucial hydrogen bonds
with FBPase. The NH group on the indole ring might function as a
hydrogen bond donor or a relatively bulky group was not tolerated
due to the steric hindrance.
examined the lead structure of MDL-29951 identified by Pfizer via
screening a compound library [26] and lead structure A discovered
by our group, a series of novel indole derivatives (B, Fig. 1) was
designed by the integration of the characteristic pharmacophore
groups present in the lead structures A and MDL-29951. By keeping
the 7-nitro-1H-indole-2-carboxylic acid scaffold intact, the car-
boxyethyl fragment was introduced as R3 group as it was in MDL-
29951. According to the SARs from series A [25], a wide range of
hydrophobic groups were chosen as R4 and R5 substituents for se-
ries B. Herein, we described the synthesis, enzymatic activities and
structure-activity relationships of these new indole derivatives as
novel FBPase inhibitors.
2. Results and discussion
2.1. Chemistry
The synthesis of compounds 5aeg and 6 was shown in Scheme
1 and their chemical structures were presented in Table 1. Starting
from the substituted anilines 1aef, the JappeKingemann reactions
yielded the corresponding phenylhydrazones 2aef in 15e83%
yields. The subsequent Fischer esterification of phenylhydrazone
2aef delivered compounds 3aef (28e92% yields), which were
converted into the key indole intermediates 4aeg in 11e50% yields
via Fischer indole synthesis in the presence of p-toluenesulfonic
acid in toluene or using PPA as solvent. Under the basic reaction
conditions, compounds 4aeg were hydrolyzed into the corre-
sponding carboxylic acid derivatives 5aeg in high yields. Com-
pound 6 was obtained in 64% yield directly via Fischer indole
synthesis of phenylhydrazone compound 2a under the catalysis of
concentrated H2SO4 in toluene.
The chemical structures of target compounds (8 and 10aeb)
were shown in Table 1 and their preparations were illustrated in
Scheme 2. The nitro group of compound 4d was reduced to amine 7
upon treatment with Fe/CH3COOH in 79% yield. The followed hy-
drolysis of ester 7 produced the carboxylic acid 8 in good yield. The
hydrolysis of esters 9aeb, which were derived from the N-alkyl-
ation of compound 4d with alkyl halides in 90e94% yields, gave rise
to the formation of carboxylic acids 10aeb in acceptable chemical
yields.
With an aim to further examine the design concept, compounds
5eeg were synthesized using a chloro or bromo residue as the R4 or
R5 substituent. Satisfyingly, compared with compound 5d, these
compouds showed stronger inhibition with IC50 values of
0.88e2.07
mM. In view of the above results, it was reasonably
assumed that the structure B was a viable template to discover
more potent FBPase inhibitors by variations of R4 and R5
substituents.
The SARs of substitution at 4-position of the indole ring with
aryl, alkyl or arylamino group were summarized in Table 2. The
analogs substituted with 4-aryl groups (12aeh) had IC50 values
ranging from 1.10 to 5.27 mM. Among which, most of them showed
a 2~5-fold increase in activity in comparison with compound 5d.
Substitution on the benzene ring of R4 group in compound 12a
resulted in marginal effects on the inhibitory activity, except for
compounds 12c and 12e. The 4-ethyl and 4-isobutyl substituted
analogs (12i and 12j) showed inhibition with an IC50 value of
2.76
mM and 0.69 mM, respectively. Introducing arylamino sub-
stituents (12kem) at 4-position of the indole scaffold resulted in a
3~7-fold increase in potency compared with compound 5d. It was
noticed that the arylamino group was somewhat more favorable to
the binding affinity than the aryl substituent, as comparing com-
pounds 12k (IC50, 0.69
mM) and 12l (IC50, 1.31
mM) with compounds
The target compounds 12aem and 14aed were prepared ac-
cording to the method as depicted in Scheme 3 and their chemical
structures were shown in Table 2. Utilizing SuzukieMiyaura reac-
tion or BuchwaldeHartwig reaction, the intermediates 4e and 4g
delivered the corresponding coupling products 11aem and 13aed
(27e87% yields), respectively. The hydrolysis of 11aem and 13aed
yielded the corresponding carboxylic acid derivatives 12aem and
14aed in good yields.
12a (IC50, 1.40 M) and 12e (IC50, 5.22 m
m
M), respectively. Taken
together, the SAR studies on 4-position demonstrated that a variety
of hydrophobic substituents were well tolerated, and the chloro,
isobutyl and phenylamino moiety were preferred.
The influence of R5 substituents on the inhibitory activity was
then explored primarily.
As summarized in Table 2, all synthesized compounds 14aed
exhibited inhibitory activities with IC50 values in a range of

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J. Bie et al. / European Journal of Medicinal Chemistry 90 (2015) 394e405
Scheme 1. Reagents and conditions: (a) (i) NaNO2, HCl, H2O, 0 ꢀC; (ii) NaOAc, ethyl-2-oxocyclopentane-carboxylate, 15 ꢀC; (iii) Et3N, H2O, reflux. (b) conc. H2SO4, EtOH, reflux. (c)
TsOH, toluene, reflux; or PPA, 90 ꢀC. (d) NaOH, THF, EtOH and water, rt. (e) conc. H2SO4, toluene, reflux.
0.10e6.43
mM. The ethyl and isobutyl substituted derivatives (14b
4. Experimental section
and 14c) were more potent than the analogs with a chloro, bromo,
phenyl and phenylamino substituent (5f, 5g, 14a and 14d). Com-
4.1. Chemistry
pound 14c (IC50, 0.10 mM) was found to be the most potent FBPase
inhibitor with the indole scaffold. It produced more than 20-fold
and 50-fold enhancement in inhibition compared with MDL-
29951 and compound 5d, respectively. Therefore, it was indicated
that substitution at 5-position with a small hydrophobic alkyl
moiety would be favorable for binding affinity. Furthermore, it has
been demonstrated that dual substitutions on 3- and 5-position
could achieve more pronounced inhibitory activity as we ex-
pected [25].
4.1.1. General
Melting points were measured on a Yanaco micro melting point
apparatus and are uncorrected. 1H 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
d (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. Col-
umn chromatography separations were performed with silica gel
(200e300 mesh).
Molecular docking was performed using CDOCKER program
(Accelrys Discovery Studio 2.5.5) [28] to gain insights into the
protein-inhibitor interactions within AMP binding site of FBPase.
The coordinates of the AMP-FBPase complex (pdb code: 1FTA) [29]
were employed. The representative binding pose of the synthesized
indoles was illustrated by the most potent compound 14c as shown
in Fig. 2. The indole ring was situated in a hydrophobic pocket
formed by residues Val17, Leu30 and Leu34. Nevertheless, in
comparison with the purine ring in AMP, the indole moiety shifted
slightly outward from the purine binding pocket. The 7-nitro group
interacted with the hydroxyl group on the side chain of Thr31 via a
hydrogen bond. The 2-carboxylate interacted with residues Thr27,
Lys112 and Arg140 through a hydrogen bonding network, which
was also observed between the phosphate group in AMP and
FBPase. It was believed that these hydrogen bonding interactions
played important roles in the binding affinity. This was in accor-
dance with the results that esterfication of 2-carboxylic acid elim-
inated the inhibitory activity. The 3-carboxyethyl side chain
extended into a solvent exposed surface, and that was similar to
that of MDL-29951 [26]. The 5-ethyl group was located in a hy-
drophobic region lined with residues Leu34, Val160 and Met177.
Presumably due to the better shape complementarity of ethyl
group with this hydrophobic subpocket, the hydrophobic in-
teractions made great contributions to the binding affinity of
compound 14c.
4.1.2. General procedure for synthesis of phenylhydrazones (2aef)
Taking 2a as an example: A solution of 2-nitroaniline (6.91 g,
50 mmol) in concentrated hydrochloric acid (12.5 mL) and water
(100 mL) was cooled to 0 ꢀC. A solution of NaNO2 (3.45 g, 50 mmol)
in water (20 mL) was then added to the above solution slowly so as
to the reaction temperature was maintained below 0 ꢀC. After the
addition was complete, a solution of sodium acetate (22.56 g,
275 mmol) in water (61 mL) was prepared and added. Then ethyl 2-
oxocyclopentanecarboxylate (9.37 g, 60 mmol) was added and
stirred vigorously for 15 min at 0 ꢀC and 60 min at room temper-
ature. The mixture was extracted with ethyl acetate (70 mL ꢁ 2).
The combined organic layers were concentrated in vacuo and the
crude material was added to a solution of boiling Et3N (2 mL) in
water (150 mL). The solution was refluxed for 30 min and cooled to
room temperature to precipitate. The yellow solid was obtained
and recrystallized from ethyl acetate to give compound 2a (8.28 g,
51%).
4.1.2.1. 6-Ethoxy-5-(2-(2-nitrophenyl)hydrazono)-6-oxohexanoic
acid (2a). Starting from 2-nitroaniline (6.91 g, 50 mmol), com-
pound 2a was afforded as yellow solid (8.28 g, 51%); mp:
3. Conclusion
157e158 ꢀC; 1H NMR (300 MHz, CDCl3):
d 13.89 (s, 1H), 8.20 (d,
J1 ¼1.5 Hz, J2 ¼ 8.4 Hz, 1H), 7.99 (d, J ¼ 8.7 Hz, 1H), 7.56 (m, 1H), 6.94
(m, 1H), 4.39 (q, J ¼ 6.9 Hz, 2H), 2.68 (t, J ¼ 7.2 Hz, 2H), 2.48 (t,
J ¼ 7.5 Hz, 2H), 1.98e2.08 (m, 2H), 1.40 (t, J ¼ 6.9 Hz, 3H). HRMS
(ESI): m/z, calcd. for C14H18N3O6 [MþH]þ: 324.1190, found 324.1184.
In summary, by integrating the key structure features of MDL-
29951 and indole-based inhibitors identified previously in our
group, we further developed a series of new indole derivatives as
FBPase inhibitors with improved potency. The preliminary SARs
were explored and led to a potent compound 14c with an IC50 value
of 0.10
m
M. This represents a novel lead structure with low mo-
4.1.2.2. 6-Ethoxy-5-(2-(3-nitrophenyl)hydrazono)-6-oxohexanoic
acid (2b). Starting from 3-nitroaniline (5.52 g, 40 mmol), com-
pound 2b was afforded as yellow solid (8.04 g, 59%). mp:
lecular weight. Therefore, it will provide more chances to further
develop druggable FBPase inhibitors.

J. Bie et al. / European Journal of Medicinal Chemistry 90 (2015) 394e405
397
Table 1
The chemical structures and inhibitory activities against FBPase of compounds
5aeg, 6, 8, 10aeb.a
Compd
R1
R2
X
IC50 (m
M)b
5a
5b
5c
5d
5e
5f
5g
6
8
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
ethyl
H
4-NO2
5-NO2
6-NO2
7-NO2
4-Cl, 7-NO2
5-Cl, 7-NO2
5-Br, 7-NO2
7-NO2
7-NH2
7-NO2
NDc
>50
NDc
5.17
0.88
2.07
1.29
>50
>50
>50
>50
Scheme 3. Reagents and conditions: (a) ArB(OH)2 or ArNH2, Pd2(dba)3, Xantphos,
Na2CO3(aq), toluene, 80 ꢀC; or Alkylboronic acid, Pd(OAc)2, tBu3P$HBF4, K3PO4(aq),
toluene, 90 ꢀC; or ArNH2, Pd2(dba)3, Davephos, K3PO4(aq), toluene, 80 ꢀC. (b) NaOH,
THF, EtOH and H2O, rt.
H
10a
10b
cyclo -propylmethyl
benzyl
H
H
Table 2
7-NO2
The chemical structures and inhibitory activities against FBPase of compounds
a
AMP and compound MDL-29951 were used as reference molecules. IC50 for AMP
M IC50 for compound MDL-29951 was 2.0e2.8 M.
Compound dose ( M) required to inhibit FBPase activity by 50%.
The inhibition of compound 5a and 5c were 57.5% and 79.5%, respectively, at
mM concentration.
12aem, 14aed.a
was 3.2
m
m
b
m
c
50
Compd
R4
R5
IC50 (m
M)b
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
14a
14b
14c
14d
phenyl
H
H
H
H
H
H
H
H
H
H
H
H
1.40
2.40
5.27
2.40
5.22
1.80
1.10
2.00
2.76
0.69
0.69
1.31
1.7
2-fluorophenyl
3-fluorophenyl
4-fluorophenyl
3-methoxyphenyl
4-methoxyphenyl
3-nitrophenyl
4-nitrophenyl
ethyl
isobutyl
phenylamino
(3-methoxyphenyl)amino
(4-methoxyphenyl)amino
H
H
H
H
phenyl
isobutyl
ethyl
6.43
0.45
0.10
1.60
Scheme 2. Reagents and conditions: (a) Fe/CH3COOH. (b) NaOH, THF, EtOH and water,
rt. (c) Cs2CO3, RX, DMF, 60 ꢀC.
H
phenylamino
a
AMP and compound MDL-29951 were used as reference molecules. IC50 for AMP
M IC50 for compound MDL-29951 was 2.0e2.8 M.
Compound dose ( M) required to inhibit FBPase activity by 50%.
was 3.2
m
m
164e165 ꢀC; 1H NMR (300 MHz, acetone-d6):
d 10.21 (s, 1H),
b
m
8.07e8.08 (m, 1H), 7.73e7.76 (m, 1H), 7.64e7.67 (m, 1H), 7.55 (t,
J ¼ 8.4 Hz,1H), 4.26 (q, J ¼ 7.2 Hz, 2H), 2.68 (t, J ¼ 8.1 Hz, 2H), 2.51 (t,
J ¼ 6.6 Hz, 2H), 1.73e1.82 (m, 2H), 1.32 (t, J ¼ 7.2 Hz, 3H). HRMS
(ESI): m/z, calcd. for C14H18N3O6 [MþH]þ: 324.1190, found 324.1183.
(1.73 g, 10 mmol), compound 2d was afforded as yellow solid
(1.56 g, 44%). mp: 168e169 ꢀC; 1H NMR (400 MHz, CDCl3):
13.91
d
(s, 1H), 8.15 (d, J ¼ 8.8 Hz, 1H), 7.97 (s, 1H), 6.90 (d, J ¼ 8.8 Hz, 1H),
4.40 (q, J ¼ 7.2 Hz, 2H), 2.69 (t, J ¼ 7.2 Hz, 2H), 2.48 (t, J ¼ 7.2 Hz, 2H),
2.03 (m, 2H), 1.40 (t, J ¼ 7.2 Hz, 3H). HRMS (ESI): m/z, calcd. for
4.1.2.3. 6-Ethoxy-5-(2-(4-nitrophenyl)hydrazono)-6-oxohexanoic
acid (2c). Starting from 4-nitroaniline (3.46 g, 25 mmol), com-
pound 2c was afforded as yellow solid (6.70 g, 83%). mp:
C
14H17N3O6Cl [MþH]þ: 358.0800, found 358.0797.
164e165 ꢀC; 1H NMR (300 MHz, acetone-d6):
d 10.44 (s,1H), 8.20 (d,
J ¼ 9.0 Hz, 2H), 7.40 (d, J ¼ 9.0 Hz, 2H), 4.26 (q, J ¼ 7.2 Hz, 2H), 2.70
(t, J ¼ 7.5 Hz, 2H), 2.52 (t, J ¼ 6.6 Hz, 2H), 1.72e1.82 (m, 2H), 1.32 (t,
J ¼ 7.2 Hz, 3H). HRMS (ESI): m/z, calcd. for C14H18N3O6 [MþH]þ:
324.1190, found 324.1185.
4.1.2.5. 6-Ethoxy-5-(2-(4-chloro-2-nitrophenyl)hydrazono)-6-
oxohexanoic acid (2e). Starting from 4-chloro-2-nitroaniline (5.2 g,
30 mmol), compound 2e was afforded as yellow solid (2.44 g, 23%).
mp: 168e169 ꢀC; 1H NMR (300 MHz, DMSO-d6):
d 13.71 (s, 1H),
12.71 (brs,1H), 8.15 (d, J ¼ 2.4 Hz, 1H), 7.91e7.95 (d, J ¼ 9.0 Hz, 1H),
7.77 (dd, J1 ¼ 2.4 Hz, J2 ¼ 9.0 Hz, 1H), 4.31 (q, J ¼ 7.2 Hz, 2H), 2.57 (t,
J ¼ 7.2 Hz, 2H), 2.30 (t, J ¼ 7.2 Hz, 2H),1.84 (m, 2H), 1.31 (t, J ¼ 7.2 Hz,
4.1.2.4. 6-Ethoxy-5-(2-(5-chloro-2-nitrophenyl)hydrazono)-6-
oxohexanoic acid (2d). Starting from 5-chloro-2-nitroaniline

398
J. Bie et al. / European Journal of Medicinal Chemistry 90 (2015) 394e405
Fig. 2. CDOCKER-modeled binding mode of compound 14c (carbon atoms colored orange). (A) The binding orientation and interactions of compound 14c in comparison with that of
AMP (carbon atoms colored green) in the cocrystal structure (1FTA in PDB) [29]; (B) The molecular shape and volume of compound 14c in the binding site of FBPase (1FTA in PDB).
H-Bonding interactions are presented with purple lines. The surface of compound 14c was presented with blue meshes. Molecular image was generated with UCSF Chimera.[30].
3H). HRMS (ESI): m/z, calcd. for C14H17N3O6Cl [MþH]þ: 358.0800,
J ¼ 9.0 Hz, 2H), 4.27e4.37 (m, 4H), 2.65 (t, J ¼ 7.5 Hz, 2H), 2.50 (m,
2H), 1.77 (m, 2H), 1.33e1.42 (m, 6H). HRMS (ESI): m/z, calcd. for
C
found 358.0793.
16H22N3O6 [MþH]þ: 352.1503, found 352.1498.
4.1.2.6. 6-Ethoxy-5-(2-(4-bromo-2-nitrophenyl)hydrazono)-6-
oxohexanoic acid (2f). Starting from 4-bromo-2-nitroaniline (6.5 g,
30 mmol), compound 2f was afforded as yellow solid (1.8 g, 15%).
4.1.3.4. Diethyl
2-(2-(5-chloro-2-nitrophenyl)hydrazono)hex-
anedioate (3d). Starting from compound 2d (1.56 g, 4.36 mmol),
compound 3d was afforded as yellow solid (1.24 g, 74%). mp:
mp: 159e161 ꢀC; 1H NMR (400 MHz, CDCl3):
d 8.34 (s, 1H), 7.90 (d,
80e81 ꢀC; 1H NMR (400 MHz, CDCl3):
d 13.91 (s, 1H), 8.15 (d,
J ¼ 8.0 Hz,1H), 7.63 (d, J ¼ 8.0 Hz,1H), 4.40 (q, J ¼ 7.2 Hz, 2H), 2.68 (t,
J ¼ 8.0 Hz, 2H), 2.47 (t, J ¼ 8.0 Hz, 2H), 2.02 (m, 2H), 1.40 (t,
J ¼ 7.2 Hz, 3H). HRMS (ESI): m/z, calcd. for C14H17N3O6Br [MþH]þ:
402.0295, found 402.0289.
J ¼ 8.8 Hz,1H), 7.97 (s,1H), 6.90 (d, J ¼ 8.8 Hz,1H), 4.40 (q, J ¼ 7.2 Hz,
2H), 4.15 (q, J ¼ 7.2 Hz, 2H), 2.67 (t, J ¼ 7.2 Hz, 2H), 2.42 (t, J ¼ 7.2 Hz,
2H), 2.01 (quint, J ¼ 7.2 Hz, 2H), 1.40 (t, J ¼ 7.2 Hz, 3H), 1.26 (t,
J ¼ 7.2 Hz, 3H). HRMS (ESI): m/z, calcd. for C16H21O6N3Cl [MþH]þ:
386.1113, found 386.1116.
4.1.3. General procedure for synthesis of diethyl derivatives (3a-f)
Taking 3a as an example: To a solution of 2a (1.00 g, 3.10 mmol)
in ethanol (20 mL), concentrated H2SO4 (0.31 mL) was added
dropwise. The reaction mixture was heated to reflux for 4 h, and
then poured into the ice-water. The mixture was filtered to afford
the crude product, which was dried and recrystallized from ethyl
acetate to give the desired product 3a (856 mg, 79%) as orange
solid.
4.1.3.5. Diethyl
2-(2-(4-chloro-2-nitrophenyl)hydrazono)hex-
anedioate (3e). Starting from compound 2e (2.44 g, 6.82 mmol),
compound 3e was afforded as yellow solid (2.41 g, 92%). mp:
79e80 ꢀC; 1H NMR (300 MHz, CDCl3):
d 13.81 (s, 1H), 8.19 (d,
J ¼ 2.4 Hz, 1H), 7.96 (d, J ¼ 9.0 Hz, 1H), 7.77 (dd, J1 ¼ 2.4 Hz,
J2 ¼ 9.0 Hz, 1H), 4.39 (q, J ¼ 7.2 Hz, 2H), 4.13 (q, J ¼ 7.2 Hz, 2H), 2.65
(t, J ¼ 7.2 Hz, 2H), 2.40 (t, J ¼ 7.2 Hz, 2H), 2.00 (quint, J ¼ 7.2 Hz, 2H),
1.39 (t, J ¼ 7.2 Hz, 3H), 1.25 (t, J ¼ 7.2 Hz, 3H). HRMS (ESI): m/z, calcd.
for C16H21O6N3Cl [MþH]þ: 386.1113, found 386.1108.
4.1.3.1. Diethyl 2-(2-(2-nitrophenyl)hydrazono)hexanedioate (3a).
Starting from compound 2a (1.00 g, 3.1 mmol), compound 3a was
afforded as orange solid (856 mg, 79%). mp: 77e78 ꢀC; 1H NMR
(300 MHz, CDCl3):
d
13.89 (s, 1H), 8.20 (d, J ¼ 8.1 Hz, 1H), 8.00 (d,
4.1.3.6. Diethyl
2-(2-(4-bromo-2-nitrophenyl)hydrazono)hex-
J ¼ 8.4 Hz, 1H), 7.57 (t, J ¼ 8.1 Hz, 1H), 6.96 (t, J ¼ 7.8 Hz, 1H), 4.40 (q,
J ¼ 7.2 Hz, 2H), 4.14 (q, J ¼ 7.2 Hz, 2H), 2.66 (t, J ¼ 7.5 Hz, 2H), 2.42 (t,
J ¼ 7.2 Hz, 2H), 2.02 (quint, J ¼ 7.2 Hz, 2H), 1.40 (t, J ¼ 7.2 Hz, 3H),
1.25 (t, J ¼ 7.2 Hz, 3H). HRMS (ESI): m/z, calcd. for C16H22N3O6
[MþH]þ: 352.1503, found 352.1498.
anedioate (3f). Starting from compound 2f (1.70 g, 3.95 mmol),
compound 3f was afforded as light yellow solid (1.57 g, 92%). mp:
80e82 ꢀC; 1H NMR (300 MHz, CDCl3):
d 13.91 (s, 1H), 8.34 (d,
J ¼ 2.1 Hz, 1H), 7.90 (d, J ¼ 8.7 Hz, 1H), 7.61e7.65 (dd, J1 ¼ 2.1 Hz,
J2 ¼ 8.7 Hz, 1H), 4.39 (q, J ¼ 6.9 Hz, 2H), 4.41 (q, J ¼ 7.2 Hz, 2H), 2.65
(t, J ¼ 7.2 Hz, 2H), 2.41 (t, J ¼ 7.2 Hz, 2H), 2.01 (quint, J ¼ 7.2 Hz, 2H),
1.40 (t, J ¼ 6.9 Hz, 3H),1.25 (t, J ¼ 7.2 Hz, 3H). HRMS (ESI): m/z, calcd.
for C16H21O6N3Br [MþH]þ: 430.0608, found 430.0609.
4.1.3.2. Diethyl 2-(2-(3-nitrophenyl)hydrazono)hexanedioate (3b).
Starting from compound 2b (3.69 g,11.42 mmol), compound 3b was
afforded as yellow solid (1.13 g, 28%). mp: 82e83 ꢀC; 1H NMR
(300 MHz, CDCl3): d 12.12 (s, 1H), 7.86 (s, 1H), 7.61 (m, 1H), 7.25 (m,
4.1.4. General procedure for synthesis of indole-2-carboxylate
derivatives (4aed) and (4eeg)
2H), 4.15 (q, J ¼ 7.2 Hz, 2H), 3.97 (q, J ¼ 6.9 Hz, 2H), 2.43 (t, J ¼ 7.2 Hz,
2H), 2.25 (t, J ¼ 7.2 Hz, 2H), 1.82 (quint, J ¼ 7.2 Hz, 2H), 1.22 (t,
J ¼ 7.2 Hz, 3H), 1.09 (t, J ¼ 6.9 Hz, 3H). HRMS (ESI): m/z, calcd. for
Taking 4a and 4c as an example by which compounds 4aed
were prepared in toluene: To a solution of 3b (0.95 g, 2.71 mmol) in
toluene (20 mL), p-toluenesulfonic acid (0.78 g, 4.07 mmol) was
added. The resulting mixture was heated to reflux for 12 h and then
concentrated under vacuum. The residue was dissolved in EtOAc
(40 mL), washed with saturated NaHCO3 aqueous solution
(30 mL ꢁ 2) and dried over anhydrous MgSO4. The crude product
obtained after concentration was purified by column
C
16H22N3O6 [MþH]þ: 352.1503, found 352.1497.
4.1.3.3. Diethyl 2-(2-(4-nitrophenyl)hydrazono)hexanedioate (3c).
Starting from compound 2c (2.00 g, 6.19 mmol), compound 3c was
afforded as yellow solid (1.94 g, 90%). mp: 80e82 ꢀC; 1H NMR
(300 MHz, CDCl3):
d
10.36 (s, 1H), 8.21 (d, J ¼ 9.0 Hz, 2H), 7.40 (d,

J. Bie et al. / European Journal of Medicinal Chemistry 90 (2015) 394e405
399
chromatography to afford isomers 4a (272 mg, 30%) and 4c
(260 mg, 29%) as yellow solid, respectively.
4.1.4.6. Ethyl 5-bromo-3-(3-ethoxy-3-oxopropyl)-7-nitro-1H-indole-
2-carboxylate (4g). Starting from compound 3f (1.47 g, 3.4 mmol),
compound 4g was afforded as off-white solid (690 mg, 49%). mp:
Taking 4e as an example by which compounds 4eeg were
prepared in PPA: The reaction mixture of compound 3d (1.0 g,
2.60 mmol) in PPA (2 g) was heated at 90e100 ꢀC for 4 h. The re-
action 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 (20 mL ꢁ 2). The crude product obtained
after concentration was purified by column chromatography to
afford the title compound 4e (252 mg, 26%) as light yellow solid.
120e121 ꢀC; 1H NMR (300 MHz, CDCl3):
d 10.18 (s, 1H), 8.40 (s, 1H),
8.24 (s, 1H), 4.48 (q, J ¼ 6.9 Hz, 2H), 4.10 (q, J ¼ 6.9 Hz, 2H), 3.39 (t,
J ¼ 7.2 Hz, 2H), 2.69 (t, J ¼ 7.2 Hz, 2H), 1.46 (t, J ¼ 6.9 Hz, 3H), 1.22 (t,
J ¼ 6.9 Hz, 3H). HRMS (ESI): m/z, calcd. for C16H18O6N2Br [MþH]þ:
413.0343, found 413.0338.
4.1.5. General procedure for synthesis of indole-2-carboxylic acid
derivatives (5a-g)
Taking 5a as an example: A solution of NaOH (140 mg,
3.50 mmol) in water (2 mL) was added dropwise to a solution of
compound 4a (236 mg, 0.70 mmol) in THF (4 mL) and ethanol
(2 mL). The reaction mixture was stirred at room temperature for
overnight and concentrated under vacuum. The residue was dis-
solved in water (10 mL), which was acidified to pH ¼ 3e4 with
diluted HCl. After filtration, the title compound 5a was obtained as
yellow solid (752 mg, 90%).
4.1.4.1. Ethyl 3-(3-ethoxy-3-oxopropyl)-4-nitro-1H-indole-2-
carboxylate (4a) and Ethyl 3-(3-ethoxy-3-oxopropyl)-6-nitro-1H-
indole-2-carboxylate (4c). Starting from compound 3b (0.95 g,
2.71 mmol), compounds 4a (272 mg, 30%) and 4c (260 mg, 29%)
were afforded as yellow solid, respectively.
(4a) mp: 115e117 ꢀC; 1H NMR (300 MHz, CDCl3): 9.39 (s, 1H),
7.77 (d, J ¼ 8.1 Hz, 1H), 7.66 (d, J ¼ 8.4 Hz, 1H), 7.35 (m, 1H), 4.47 (q,
J ¼ 7.2 Hz, 2H), 4.12 (q, J ¼ 6.9 Hz, 2H), 3.45 (t, J ¼ 7.5 Hz, 2H), 2.70 (t,
J ¼ 8.1 Hz, 2H), 1.45 (t, J ¼ 7.2 Hz, 3H), 1.23 (t, J ¼ 7.2 Hz, 3H). HRMS
(ESI): m/z, calcd. for C16H19N2O6 [MþH]þ: 335.1238, found 335.1231.
(4c) mp: 134e135 ꢀC; 1H NMR (300 MHz, CDCl3): 9.29 (s, 1H),
8.33 (s, 1H), 8.02 (d, J ¼ 9.0 Hz, 1H), 7.83 (d, J ¼ 9.3 Hz, 1H), 4.47 (q,
J ¼ 6.9 Hz, 2H), 4.09 (q, J ¼ 6.9 Hz, 2H), 3.42 (t, J ¼ 7.5 Hz, 2H), 2.70 (t,
J ¼ 7.5 Hz, 2H), 1.46 (t, J ¼ 6.9 Hz, 3H), 1.19 (t, J ¼ 6.9 Hz, 3H). HRMS
(ESI): m/z, calcd. for C16H19N2O6 [MþH]þ: 335.1238, found 335.1231.
4.1.5.1. 3-(2-Carboxyethyl)-4-nitro-1H-indole-2-carboxylic acid (5a).
Starting from compound 4a (236 mg, 0.71 mmol), compound 5a
was afforded as light yellow solid (163 mg, 83%). mp: >300 ꢀC; 1H
NMR (300 MHz, DMSO-d6): 12.76 (brs, 2H), 12.37 (s, 1H), 7.79 (d,
J ¼ 8.1 Hz, 1H), 7.74 (d, J ¼ 7.5 Hz, 1H), 7.36e7.41 (m, 1H), 3.25 (t,
J ¼ 8.1 Hz, 2H), 2.43 (t, J ¼ 8.1 Hz, 2H). HRMS (ESI): m/z, calcd. for
C
12H11N2O6 [MþH]þ: 279.0607, found 279.0612.
4.1.4.2. Ethyl 3-(3-ethoxy-3-oxopropyl)-5-nitro-1H-indole-2-
carboxylate (4b). Starting from compound 3c (1.95 g, 5.54 mmol),
compound 4b was afforded as yellow solid (402 mg, 22%). mp:
4.1.5.2. 3-(2-Carboxyethyl)-5-nitro-1H-indole-2-carboxylic acid (5b).
Starting from compound 4b (200 mg, 0.60 mmol), compound 5b
was afforded as light yellow solid (145 mg, 87%). mp: 265e267 ꢀC;
1H NMR (400 MHz, DMSO-d6): 12.23 (s, 1H), 8.74 (s, 1H), 8.09e8.12
(m, 1H), 7.54 (d, J ¼ 9.2 Hz, 1H), 3.30e3.32 (m, 2H), 2.56 (t,
J ¼ 7.6 Hz, 2H). HRMS (ESI): m/z, calcd. for C12H11N2O6 [MþH]þ:
279.0608, found 279.0612.
163.5e164.0 ꢀC; 1H NMR (300 MHz, acetone-d6):
d 11.34 (brs, 1H),
8.78 (d, J ¼ 1.8 Hz, 1H), 8.16 (dd, J1 ¼1.8 Hz, J2 ¼ 9.0 Hz, 1H), 7.65 (d,
J ¼ 9.0 Hz, 1H), 4.42 (q, J ¼ 6.9 Hz, 2H), 4.03 (q, J ¼ 6.9 Hz, 2H), 3.48
(t, J ¼ 7.5 Hz, 2H), 2.72 (t, J ¼ 7.5 Hz, 2H), 1.40 (t, J ¼ 6.9 Hz, 3H), 1.14
(t, J ¼ 6.9 Hz, 3H). HRMS (ESI): m/z, calcd. for C16H19N2O6 [MþH]þ:
335.1238, found 335.1234.
4.1.5.3. 3-(2-Carboxyethyl)-6-nitro-1H-indole-2-carboxylic acid (5c).
Starting from compound 4c (285 mg, 0.85 mmol), compound 5c
was afforded as light yellow solid (173 mg, 73%). mp: 262e264 ꢀC;
4.1.4.3. Ethyl 3-(3-ethoxy-3-oxopropyl)-7-nitro-1H-indole-2-
carboxylate (4d). Starting from compound 3a (0.79 g, 2.25 mmol),
compound 4d was afforded as light yellow solid (372 mg, 50%). mp:
1H NMR (300 MHz, acetone-d6):
d 11.08 (s, 1H), 8.22 (s, 1H),
76e78 ꢀC; 1H NMR (300 MHz, CDCl3):
d 10.21 (s, 1H), 8.30 (d,
7.72e7.80 (m, 2H), 3.23 (t, J ¼ 7.8 Hz, 2H), 2.49 (t, J ¼ 7.8 Hz, 2H).
HRMS (ESI): m/z, calcd. for C12H11N2O6 [MþH]þ: 279.0612, found
279.0609.
J ¼ 8.1 Hz, 1H), 8.14 (d, J ¼ 8.1 Hz, 1H), 7.27 (t, J ¼ 8.1 Hz, 1H), 4.48 (q,
J ¼ 7.2 Hz, 2H), 4.08 (q, J ¼ 7.2 Hz, 2H), 3.44 (t, J ¼ 7.5 Hz, 2H), 2.71 (t,
J ¼ 7.5 Hz, 2H), 1.47 (t, J ¼ 7.2 Hz, 3H), 1.19 (t, J ¼ 7.2 Hz, 3H). HRMS
(ESI): m/z, calcd. for C16H19N2O6 [MþH]þ: 335.1237, found 335.1233.
4.1.5.4. 3-(2-Carboxyethyl)-7-nitro-1H-indole-2-carboxylic
acid
(5d). Starting from compound 4d (1.00 g, 3.00 mmol), compound
5d was afforded as yellow solid (752 mg, 90%). mp: 254e256 ꢀC; 1H
4.1.4.4. Ethyl 4-chloro-3-(3-ethoxy-3-oxopropyl)-7-nitro-1H-indole-
2-carboxylate (4e). Starting from compound 3d (1.00 g, 2.60 mmol),
compound 4e was afforded as light yellow solid (252 mg, 26%). mp:
NMR (400 MHz, DMSO-d6):
d 12.86 (brs, 2H), 10.60 (s, 1H), 8.25 (d,
J ¼ 8.0 Hz,1H), 8.23 (d, J ¼ 8.0 Hz, 1H), 7.30 (t, J ¼ 8.0 Hz,1H), 3.28 (t,
135e137 ꢀC; 1H NMR (400 MHz, CDCl3):
d 10.37 (s, 1H), 8.19 (d,
J ¼ 7.6 Hz, 2H), 2.55 (t, J ¼ 7.6 Hz, 2H); 13C NMR (100 MHz, DMSO-
J ¼ 8.4 Hz, 1H), 7.22 (d, J ¼ 8.4 Hz, 1H), 4.48 (q, J ¼ 7.2 Hz, 2H), 4.15
(q, J ¼ 7.2 Hz, 2H), 3.73 (t, J ¼ 8.4 Hz, 2H), 2.68 (t, J ¼ 8.4 Hz, 2H),1.46
(t, J ¼ 7.2 Hz, 3H), 1.25 (t, J ¼ 7.2 Hz, 3H). HRMS (ESI): m/z, calcd. for
d6):
d 173.71, 162.16, 132.89, 131.18, 129.59, 127.96, 126.92, 122.96,
122.11, 119.55, 34.82, 19.55. HRMS (ESI): m/z, calcd. for C12H11N2O6
[MþH]þ: 279.0612, found 279.0609.
C
16H18O6N2Cl [MþH]þ: 369.0848, found 369.0850.
4.1.5.5. 3-(2-Carboxyethyl)-4-chloro-7-nitro-1H-indole-2-carboxylic
acid (5e). Starting from compound 4e (120 mg, 0.33 mmol), com-
pound 5e was afforded as light yellow solid (90 mg, 44%). mp:
4.1.4.5. Ethyl 5-chloro-3-(3-ethoxy-3-oxopropyl)-7-nitro-1H-indole-
2-carboxylate (4f). Starting from compound 3e (2.25 g, 5.8 mmol),
compound 4f was afforded as off-white solid (706 mg, 11%). mp:
>250 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 13.80 (brs,1H),12.23 (brs,
120e121 ꢀC; 1H NMR (300 MHz, CDCl3):
d
10.17 (s, 1H), 8.27 (s, 1H),
1H), 10.94 (s, 1H), 8.23 (d, J ¼ 8.4 Hz, 1H), 7.39 (d, J ¼ 8.4 Hz, 1H),
8.10 (s, 1H), 4.48 (q, J ¼ 7.2 Hz, 2H), 4.09 (q, J ¼ 7.2 Hz, 2H), 3.39 (t,
J ¼ 7.2 Hz, 2H), 2.69 (t, J ¼ 7.2 Hz, 2H), 1.46 (t, J ¼ 7.2 Hz, 3H), 1.21 (t,
J ¼ 7.2 Hz, 3H). HRMS (ESI): m/z, calcd. for C16H18O6N2Cl [MþH]þ:
369.0848, found 369.0844.
3.56 (t, J ¼ 8.4 Hz, 2H), 2.53 (t, J ¼ 8.8 Hz, 2H); 13C NMR (100 MHz,
DMSO-d6):
d 173.34, 161.82, 135.06, 132.12, 129.19, 128.27, 126.57,
122.78, 122.17, 121.10, 35.67, 19.76. HRMS (ESI): m/z, calcd. for
C
12H10N2O6Cl [MþH]þ: 313.0222, found 313.0221.

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J. Bie et al. / European Journal of Medicinal Chemistry 90 (2015) 394e405
4.1.5.6. 3-(2-Carboxyethyl)-5-chloro-7-nitro-1H-indole-2-carboxylic
acid (5f). Starting from compound 4f (130 mg, 0.35 mmol), com-
pound 5f was afforded as off-white solid (105 mg, 96%). mp:
dried with anhydrous MgSO4 and concentrated under reduced
pressure. The crude product was purified by silica gel column
chromatography to provide compound 9a as yellow oil (283 mg,
97%).
>250 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 10.93 (s, 1H), 8.40 (s, 1H),
8.23 (s, 1H), 3.28 (t, J ¼ 7.6 Hz, 2H), 2.56 (t, J ¼ 7.6 Hz, 2H). 13C NMR
(100 MHz, DMSO-d6):
d
173.71, 161.90, 133.05, 132.18, 128.53,
4.1.9.1. Ethyl 1-(cyclopropyl)methyl-3-(3-ethoxy-3-oxopropyl)-7-
nitro-1H-indole -2-carboxylate (9a). Starting from compound 4d
(250 mg, 0.75 mmol), compound 9a was afforded as yellow oil
128.50, 126.59,123.38,122.45,121.32, 34.69, 19.33. HRMS (ESI): m/z,
calcd. for C12H10N2O6Cl [MþH]þ: 313.0222, found 313.0215.
(283 mg, 97%). 1H NMR (300 MHz, acetone-d6):
d
8.19 (d, J ¼ 8.1 Hz,
4.1.5.7. 3-(2-Carboxyethyl)-5-bromo-7-nitro-1H-indole-2-carboxylic
acid (5g). Starting from compound 4g (130 mg, 0.31 mmol), com-
pound 5g was afforded as off-white solid (102 mg, 91%). mp:
1H), 7.94 (d, J ¼ 7.8 Hz, 1H), 7.32e7.38 (m, 1H), 4.43e4.50 (m, 4H),
4.05 (q, J ¼ 7.2 Hz, 2H), 3.41 (t, J ¼ 7.5 Hz, 2H), 2.67 (t, J ¼ 7.5 Hz, 2H),
1.44 (t, J ¼ 7.2 Hz, 3H), 1.14 (t, J ¼ 7.2 Hz, 3H), 0.72e0.81 (m, 1H),
0.28e0.34 (m, 2H), 0.08e0.13 (m, 2H). HRMS (ESI): m/z, calcd. for
>250 ꢀC; 1H NMR (400 MHz, DMSO-d6):
d 10.93 (s, 1H), 8.51 (s, 1H),
8.30 (s, 1H), 3.27 (t, J ¼ 7.6 Hz, 2H), 2.54 (t, J ¼ 7.6 Hz, 2H). 13C NMR
C
20H25N2O6 [MþH]þ: 389.1699, found 389.1707.
(100 MHz, DMSO-d6):
d 173.71, 161.90, 133.36, 132.73, 131.45,
128.29, 126.85, 123.79, 122.35, 110.41, 34.69, 19.33. HRMS (ESI): m/z,
4.1.9.2. Ethyl 1-benzyl-3-(3-ethoxy-3-oxopropyl)-7-nitro-1H-indole-
2-carboxylate (9b). Starting from compound 4d (260 mg,
0.78 mmol) and benzyl bromide (334 mg, 1.95 mmol), compound
9b was afforded as yellow oil (305 mg, 92%). 1H NMR (300 MHz,
calcd. for C12H10N2O6Br [MþH]þ: 356.9710, found 356.9717.
4.1.6. 3-(2-(Ethoxycarbonyl)-7-nitro-1H-indol-3-yl)propanoic acid
(6)
acetone-d6):
d
8.21 (dd, J1 ¼ 7.8 Hz, J2 ¼ 0.9 Hz, 1H), 7.83 (d,
To a solution of 2a (10.57 g, 33 mmol) in toluene (165 mL),
concentrated H2SO4 (0.31 mL) was dropwise added. The reaction
mixture was heated to reflux for 4 h, and then poured into the ice-
water. The residue was filtered, dried and recrystallized from ethyl
acetate to give product 6 (6.46 g, 64%) as yellow solid. mp:
J1 ¼7.8 Hz, J2 ¼ 0.9 Hz, 1H), 7.32 (t, J ¼ 7.8 Hz, 1H), 7.14e7.16 (m, 3H),
6.69e6.72 (m, 2H), 5.84 (s, 2H), 4.44 (q, J ¼ 7.2 Hz, 2H), 4.05 (q,
J ¼ 7.2 Hz, 2H), 3.45 (t, J ¼ 7.5 Hz, 2H), 2.71 (t, J ¼ 7.5 Hz, 2H), 1.40 (t,
J ¼ 7.2 Hz, 3H), 1.15 (t, J ¼ 7.2 Hz, 3H). HRMS (ESI): m/z, calcd. for
C
23H25N2O6 [MþH]þ: 425.1707, found 425.1697.
186e187 ꢀC; 1H NMR (300 MHz, CDCl3):
d 10.22 (s, 1H), 8.30 (d,
J ¼ 7.8 Hz, 1H), 8.11 (d, J ¼ 8.1 Hz, 1H), 7.24e7.29 (m, 1H), 4.48 (q,
J ¼ 7.2 Hz, 2H), 3.44 (t, J ¼ 7.5 Hz, 2H), 2.76 (t, J ¼ 7.5 Hz, 2H), 1.46 (t,
J ¼ 7.2 Hz, 3H). HRMS (ESI): m/z, calcd. for C14H15N2O6 [MþH]þ:
307.0925, found 307.0918.
4.1.10. Synthesis of N-alkyl indole-2-carboxylic acid derivatives
(10aeb)
4.1.10.1. 1-(Cyclopropyl)methyl-3-(2-carboxyethyl)-7-nitro-1h-
indole-2-carboxy -lic acid (10a). Following the procedure of 4.1.5,
and starting from compound 9a (160 mg, 0.41 mmol), compound
10a was afforded as white solid (112 mg, 82%). mp: 216e218 ꢀC. 1H
4.1.7. Ethyl 7-amino-3-(3-ethoxy-3-oxopropyl)-1H-indole-2-
carboxylate (7)
NMR (300 MHz, acetone-d6):
d
8.23 (d, J ¼ 8.1 Hz, 1H), 7.94 (d,
To a solution of 4d (500 mg, 1.50 mmol) in acetic acid (30 mL), Fe
powder (672 mg, 12.00 mmol) was added. The reaction mixture
was heated to 40 ꢀC for 30 min, diluted with EtOAc (20 mL), filtered
to remove Fe residue and concentrated under vacuum. The residue
was dissolved in EtOAc (30 mL ꢁ 3), washed with saturated NaHCO3
aqueous solution (10 mL ꢁ 3) and water (10 mL ꢁ 3) and dried over
anhydrous MgSO4. The crude product obtained after concentration
was purified by silica gel column chromatography to afford 7 as
light yellow solid (361 mg, 79%). mp: 99e100 ꢀC; 1H NMR
J ¼ 8.1 Hz, 1H), 7.35 (t, J ¼ 8.1 Hz, 1H), 4.50 (d, J ¼ 6.9 Hz, 2H), 3.45 (t,
J ¼ 7.5 Hz, 2H), 2.72 (t, J ¼ 7.5 Hz, 2H), 0.74e0.82 (m, 1H), 0.28e0.34
(m, 2H), 0.09e0.14 (m, 2H). HRMS (ESI): m/z, calcd. for C16H17N2O6
[MþH]þ: 333.1081, found 333.1075.
4.1.10.2. 1-Benzyl-3-(2-carboxyethyl)-7-nitro-1H-indole-2-
carboxylic acid (10b). Following the procedure of 4.1.5, and starting
from compound 9b (170 mg, 0.40 mmol), compound 10b was
afforded as white solid (130 mg, 88%). mp: >250 ꢀC. 1H NMR
(300 MHz, CDCl3):
d
9.34 (s, 1H), 7.22 (d, J ¼ 8.1 Hz, 1H), 6.98e7.03
(300 MHz, acetone-d6):
d
8.25 (d, J ¼ 7.8 Hz, 1H), 7.83 (d, J ¼ 7.8 Hz,
(m, 1H), 6.68 (d, J ¼ 7.5 Hz, 1H), 4.45 (q, J ¼ 7.2 Hz, 2H), 4.14 (q,
J ¼ 7.2 Hz, 2H), 3.97 (brs, 2H), 3.41 (t, J ¼ 8.1 Hz, 2H), 2.68 (t,
J ¼ 8.1 Hz, 2H), 1.46 (t, J ¼ 7.2 Hz, 3H),1.24 (t, J ¼ 7.2 Hz, 3H). HRMS
1H), 7.32 (t, J ¼ 8.1 Hz, 1H), 7.13e7.15 (m, 3H), 6.70e6.73 (m, 2H),
5.90 (s, 2H), 3.50 (t, J ¼ 7.5 Hz, 2H), 2.76 (t, J ¼ 7.5 Hz, 2H). HRMS
(ESI): m/z, calcd. for C19H17N2O6 [MþH]þ: 369.1074, found 369.1081.
(ESI): m/z, calcd. for
305.1491.
C
16H21N2O4 [MþH]þ: 305.1496, found
4.1.11. General procedure for synthesis of 4-substituted indole-2-
carboxylate derivatives (11aem)
4.1.8. 7-Amino-3-(2-carboxyethyl)-1H-indole-2-carboxylic acid (8)
Following the procedure of 4.1.5, starting from compound 7
(604 mg, 1.98 mmol), compound 8 was afforded as off-white solid
Taking 11a as an example: To a solution of 4e (250 mg,
0.68 mmol) in toluene (12 mL), Pd2(dba)3 (62 mg, 0.068 mmol),
Xantphos (79 mg, 0.14 mmol), Na2CO3 (216 mg, 2.03 mmol) and
phenylboronic acid (248 mg, 2.03 mmol) were sequentially added.
The reaction mixture was heated to 100 ꢀC for 4 h under argon
atmosphere, and then 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 by silica gel column chromatog-
raphy to afford product 11a as yellow solid (256 mg, 77%).
(441 mg, 90%). mp: >300 ꢀC; 1H NMR (300 MHz, DMSO-d6):
d 11.03
(s, 2H), 6.84 (d, J ¼ 8.1 Hz, 1H), 6.74e6.79 (m, 1H), 6.38 (d, J ¼ 7.2 Hz,
1H), 3.19 (t, J ¼ 7.8 Hz, 2H), 2.46 (t, J ¼ 7.8 Hz, 2H). HRMS (ESI): m/z,
calcd. for C12H13N2O4 [MþH]þ: 249.0870, found 249.0865.
4.1.9. General procedure for synthesis of N-alkyl indole-2-
carboxylate derivatives (9a-b)
Taking 9a as an example: To a solution of 4d (250 mg,
0.75 mmol) in DMF (8 mL), Cs2CO3 (719 mg, 2.25 mmol), a catalytic
amount of KI and cyclopropylmethyl bromide (253 mg, 1.88 mmol)
was sequentially added. The reaction mixture was heated to 60 ꢀC
for 2 h, and then concentrated under reduced pressure. The residue
was dissolved in EtOAc (30 mL), washed with water (10 mL ꢁ 3),
4.1.11.1. Ethyl
3-(3-ethoxy-3-oxopropyl)-7-nitro-4-phenyl-1H-
indole-2-carboxy -late (11a). Starting from compound 4e (250 mg,
0.68 mmol) and phenylboronic acid (248 mg, 2.03 mmol), com-
pound 11a was afforded as yellow solid (256 mg, 77%). mp:
110e112 ꢀC; 1H NMR (400 MHz, CDCl3):
d 10.46 (s, 1H), 8.31 (d,

J. Bie et al. / European Journal of Medicinal Chemistry 90 (2015) 394e405
401
J ¼ 8.4 Hz, 1H), 7.46e7.47 (m, 3H), 7.40 (m, 2H), 7.08 (d, J ¼ 8.4 Hz,
1H), 4.45 (q, J ¼ 7.2 Hz, 2H), 3.98 (q, J ¼ 7.2 Hz, 2H), 2.97 (t,
J ¼ 8.4 Hz, 2H), 2.19 (t, J ¼ 8.4 Hz, 2H), 1.43 (t, J ¼ 7.2 Hz, 3H), 1.16 (t,
J ¼ 7.2 Hz, 3H). HRMS (ESI): m/z, calcd. for C22H23N2O6 [MþH]þ:
411.1511, found 411.1511.
3.24 mmol), compound 11g was afforded as yellow solid (148 mg,
27%). mp: 148e149 ꢀC; 1H NMR (300 MHz, CDCl3):
10.51 (s, 1H),
d
8.34e8.38 (m, 2H), 8.30 (s, 1H), 7.77 (d, J ¼ 7.8 Hz, 1H), 7.70 (t,
J ¼ 7.8 Hz,1H), 7.10 (d, J ¼ 8.1 Hz,1H), 4.47 (q, J ¼ 6.9 Hz, 2H), 3.95 (q,
J ¼ 6.9 Hz, 2H), 2.81 (m, 2H), 2.25e2.28 (m, 2H), 1.43 (t, J ¼ 6.9 Hz,
3H), 1.13 (t, J ¼ 6.9 Hz, 3H). HRMS (ESI): m/z, calcd. for C22H22N3O8
[MþH]þ: 456.1401, found 456.1400.
4.1.11.2. Ethyl 3-(3-ethoxy-3-oxopropyl)-4-(2-fluorophenyl)-7-nitro-
1H-indole-2 -carboxylate (11b). Starting from compound 4e
(250 mg, 0.68 mmol) and 2-fluorophenylboronic acid (285 mg,
2.03 mmol), compound 11b was afforded as light yellow solid
(248 mg, 86%). mp: 117e119 ꢀC; 1H NMR (300 MHz, DMSO-d6):
4.1.11.8. Ethyl 3-(3-ethoxy-3-oxopropyl)-4-(4-nitrophenyl)-7-nitro-
1H-indole-2 -carboxylate (11h). Starting from compound 4e
(250 mg, 0.68 mmol) and 4-nitrophenylboronic acid (340 mg,
2.03 mmol), compound 11g was afforded as yellow solid (160 mg,
d
10.45 (s, 1H), 8.34 (d, J ¼ 8.1 Hz, 1H), 7.44e7.50 (m, 1H), 7.18e7.38
(m, 3H), 7.11 (d, J ¼ 8.1 Hz, 1H), 4.45 (q, J ¼ 7.2 Hz, 2H), 4.00 (q,
J ¼ 6.9 Hz, 2H), 2.90e3.00 (m, 2H), 2.21e2.28 (m, 2H), 1.43 (t,
J ¼ 7.2 Hz, 3H), 1.18 (t, J ¼ 6.9 Hz, 3H). HRMS (ESI): m/z, calcd. for
52%). mp: 134e135 ꢀC; 1H NMR (300 MHz, CDCl3):
d 10.51 (s, 1H),
8.33e8.37 (m, 3H), 7.61 (d, J ¼ 8.4 Hz, 2H), 7.08 (d, J ¼ 8.4 Hz, 1H),
4.47 (q, J ¼ 7.2 Hz, 2H), 3.95 (q, J ¼ 7.2 Hz, 2H), 2.92 (t, J ¼ 8.1 Hz, 2H),
2.21 (t, J ¼ 8.1 Hz, 2H), 1.44 (t, J ¼ 7.2 Hz, 3H), 1.13 (t, J ¼ 7.2 Hz, 3H).
HRMS (ESI): m/z, calcd. for C22H22N3O8 [MþH]þ: 456.1391, found
456.1401.
C
22H22N2O6F [MþH]þ: 429.1456, found 429.1450.
4.1.11.3. Ethyl 3-(3-ethoxy-3-oxopropyl)-4-(3-fluorophenyl)-7-nitro-
1H-indole-2 -carboxylate (11c). Starting from compound 4e
(200 mg, 0.54 mmol) and 3-fluorophenylboronic acid (227 mg,
1.62 mmol), compound 11c was afforded as light yellow solid
4.1.11.9. Ethyl 3-(3-ethoxy-3-oxopropyl)-4-ethyl-7-nitro-1H-indole-
2-carboxylate (11i). To a solution of 4e (250 mg, 0.68 mmol) in
toluene (15 mL), Pd(OAc)2 (15 mg, 0.068 mmol), (tBu)3P$HBF4
(39 mg, 0.14 mmol), K3PO4 (720 mg, 3.40 mmol, 1 mL H2O) and
ethylboronic acid (451 mg, 2.04 mmol) were added in order. The
reaction mixture was heated to 90 ꢀC for 4 h under argon atmo-
sphere, 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 by column chromatography to afford product 11i as
light yellow solid (162 mg, 55%). mp: 85e87 ꢀC; 1H NMR (400 MHz,
(222 mg, 77%). mp: 136e137 ꢀC; 1H NMR (400 MHz, CDCl3):
d 10.47
(s, 1H), 8.31 (d, J ¼ 8.0 Hz, 1H), 7.45 (dd, J1 ¼7.6 Hz, J2 ¼ 14.0 Hz, 1H),
7.17e7.19 (m, 2H), 7.12 (d, J ¼ 9.2 Hz, 1H), 7.08 (d, J ¼ 8.0 Hz, 1H),
4.46 (q, J ¼ 7.2 Hz, 2H), 4.00 (q, J ¼ 7.2 Hz, 2H), 2.95e2.97 (m, 2H),
2.24 (t, J ¼ 7.2 Hz, 2H), 1.43 (t, J ¼ 7.2 Hz, 3H), 1.18 (t, J ¼ 7.2 Hz, 3H).
HRMS (ESI): m/z, calcd. for C22H22N2O6F [MþH]þ: 429.1456, found
429.1458.
4.1.11.4. Ethyl 3-(3-ethoxy-3-oxopropyl)-4-(4-fluorophenyl)-7-nitro-
1H-indole-2 -carboxylate (11d). Starting from compound 4e
(250 mg, 0.68 mmol) and 4-fluorophenylboronic acid (285 mg,
2.03 mmol), compound 11d was afforded as light yellow solid
CDCl3):
d
10.39 (s, 1H), 8.22 (d, J ¼ 8.0 Hz, 1H), 7.05 (d, J ¼ 8.0 Hz,
1H), 4.47 (q, J ¼ 7.2 Hz, 2H), 4.16 (q, J ¼ 7.2 Hz, 2H), 3.59 (t, J ¼ 8.4 Hz,
2H), 3.18 (q, J ¼ 7.6 Hz, 2H), 2.66 (t, J ¼ 8.4 Hz, 2H), 1.46 (t, J ¼ 7.2 Hz,
3H), 1.39 (t, J ¼ 7.2 Hz, 3H), 1.26 (t, J ¼ 7.6 Hz, 3H). HRMS (ESI): m/z,
calcd. for C18H23N2O6 [MþH]þ: 363.1551, found 363.1542.
(210 mg, 72%). mp: 134e135 ꢀC; 1H NMR (300 MHz, CDCl3):
d 10.46
(s, 1H), 8.31 (d, J ¼ 8.4 Hz, 1H), 7.38 (dd, J1 ¼ 5.4 Hz, J2 ¼ 8.7 Hz, 2H),
7.17 (t, J ¼ 8.7 Hz, 2H), 7.06 (d, J ¼ 8.1 Hz, 1H), 4.46 (q, J ¼ 7.2 Hz, 2H),
4.01 (q, J ¼ 7.2 Hz, 2H), 2.98 (t, J ¼ 8.7 Hz, 2H), 2.19 (t, J ¼ 8.7 Hz, 2H),
1.43 (t, J ¼ 7.2 Hz, 3H), 1.18 (t, J ¼ 7.2 Hz, 3H). HRMS (ESI): m/z, calcd.
for C22H22N2O6F [MþH]þ: 429.1456, found 429.1450.
4.1.11.10. Ethyl
3-(3-ethoxy-3-oxopropyl)-4-isobutyl-7-nitro-1H-
indole-2ecarboxy -late (11j). Following the procedure of 4.1.11.9,
starting from compound 4e (200 mg, 0.54 mmol) and iso-
butylboronic acid (165 mg, 2.62 mmol), compound 11j was afforded
as light yellow solid (150 mg, 71%). mp: 73e75 ꢀC; 1H NMR
4.1.11.5. Ethyl 3-(3-ethoxy-3-oxopropyl)-4-(3-methoxyphenyl)-7-
nitro-1H-indole-2 -carboxylate (11e). Starting from compound 4e
(200 mg, 0.54 mmol) and 3-methoxyphenylboronic acid (246 mg,
1.62 mmol), compound 11e was afforded as yellow solid (245 mg,
(300 MHz, CDCl3):
d
10.40 (s, 1H), 8.20 (d, J ¼ 8.4 Hz, 1H), 6.97 (d,
J ¼ 8.4 Hz,1H), 4.47 (q, J ¼ 6.9 Hz, 2H), 4.17 (q, J ¼ 6.9 Hz, 2H), 3.57 (t,
J ¼ 8.4 Hz, 2H), 2.96 (d, J ¼ 7.2 Hz, 2H), 2.64 (t, J ¼ 8.4 Hz, 2H),
1.91e2.00 (m,1H),1.46 (t, J ¼ 6.9 Hz, 3H),1.26 (t, J ¼ 6.9 Hz, 3H),1.00
(d, J ¼ 6.6 Hz, 6H). HRMS (ESI): m/z, calcd. for C20H27N2O6 [MþH]þ:
391.1864, found 391.1859.
82%). mp: 124e125 ꢀC; 1H NMR (300 MHz, CDCl3):
d 10.45 (s, 1H),
8.31 (d, J ¼ 8.4 Hz,1H), 7.38 (t, J ¼ 7.8 Hz,1H), 7.09 (d, J ¼ 8.4 Hz,1H),
6.95e7.02 (m, 2H), 6.91 (m, 1H), 4.45 (q, J ¼ 6.9 Hz, 2H), 4.00 (q,
J ¼ 7.2 Hz, 2H), 3.85 (s, 3H), 2.99 (t, J ¼ 8.4 Hz, 2H), 2.24 (t, J ¼ 8.4 Hz,
2H), 1.43 (t, J ¼ 6.9 Hz, 3H), 1.17 (t, J ¼ 7.2 Hz, 3H). HRMS (ESI): m/z,
calcd. for C23H25N2O7 [MþH]þ: 441.1656, found 441.1655.
4.1.11.11. Ethyl
3-(3-ethoxy-3-oxopropyl)-7-nitro-4-phenylamino-
1H-indole-2 -carboxylate (11k). Starting from compound 4e
(250 mg, 0.68 mmol) and aniline (189 mg, 2.03 mmol), compound
11k was afforded as orange solid (164 mg, 57%). mp: 144e146 ꢀC;
4.1.11.6. Ethyl 3-(3-ethoxy-3-oxopropyl)-4-(4-methoxyphenyl)-7-
nitro-1H-indole-2-carboxylate (11f). Starting from compound 4e
(250 mg, 0.68 mmol) and 4-methoxyphenylboronic acid (309 mg,
2.03 mmol), compound 11f was afforded as light yellow solid
1H NMR (400 MHz, CDCl3):
d 10.51 (s, 1H), 9.55 (s, 1H), 8.12 (d,
J ¼ 9.2 Hz, 1H), 7.38e7.44 (m, 4H), 7.17 (t, J ¼ 6.8 Hz, 1H), 6.91 (d,
J ¼ 9.2 Hz,1H), 4.46 (q, J ¼ 7.2 Hz, 2H), 4.16 (q, J ¼ 7.2 Hz, 2H), 3.53 (t,
J ¼ 5.6 Hz, 2H), 3.00 (t, J ¼ 5.6 Hz, 2H), 1.46 (t, J ¼ 7.2 Hz, 3H), 1.23 (t,
J ¼ 7.2 Hz, 3H). HRMS (ESI): m/z, calcd. for C22H24N3O6 [MþH]þ:
426.1660, found 426.1658.
(266 mg, 89%). mp: 119e120 ꢀC; 1H NMR (300 MHz, CDCl3):
d 10.44
(s, 1H), 8.29 (d, J ¼ 8.1 Hz, 1H), 7.32 (d, J ¼ 8.7 Hz, 2H), 7.06 (d,
J ¼ 8.1 Hz, 1H), 6.99 (d, J ¼ 8.7 Hz, 2H), 4.45 (q, J ¼ 7.5 Hz, 2H), 3.99
(q, J ¼ 6.9 Hz, 2H), 3.88 (s, 3H), 3.04 (t, J ¼ 8.4 Hz, 2H), 2.19 (t,
J ¼ 8.4 Hz, 2H), 1.43 (t, J ¼ 7.5 Hz, 3H), 1.16 (t, J ¼ 6.9 Hz, 3H). HRMS
(ESI): m/z, calcd. for C23H25N2O7 [MþH]þ: 441.1656, found 441.1649.
4.1.11.12. Ethyl 3-(3-ethoxy-3-oxopropyl)-7-nitro-4-(3-
methoxyphenylamino)-1H -indole-2-carboxylate (11l). Starting
from compound 4e (250 mg, 0.68 mmol) and 3-methoxyaniline
(250 mg, 2.03 mmol), compound 11l was afforded as yellow solid
4.1.11.7. Ethyl 3-(3-ethoxy-3-oxopropyl)-4-(3-nitrophenyl)-7-nitro-
1H-indole-2 -carboxylate (11g). Starting from compound 4e
(400 mg, 1.08 mmol) and 3-nitrophenylboronic acid (541 mg,
(270 mg, 87%). mp: 126e128 ꢀC; 1H NMR (300 MHz, CDCl3):
d
10.50
(brs, 1H), 9.57 (s, 1H), 8.13 (d, J ¼ 9.3 Hz, 1H), 7.32 (t, J ¼ 8.1 Hz, 1H),

402
J. Bie et al. / European Journal of Medicinal Chemistry 90 (2015) 394e405
6.96e7.00 (m, 3H), 6.71 (dd, J1 ¼ 1.8 Hz, J2 ¼ 8.1 Hz, 1H), 4.46 (q,
J ¼ 7.2 Hz, 2H), 4.15 (q, J ¼ 7.2 Hz, 2H), 3.84 (s, 3H), 3.52 (t, J ¼ 6.0 Hz,
2H), 3.00 (t, J ¼ 6.0 Hz, 2H), 1.46 (t, J ¼ 7.2 Hz, 3H), 1.23 (t, J ¼ 7.2 Hz,
3H). HRMS (ESI): m/z, calcd. for C23H26N3O7 [MþH]þ: 456.1765,
found 456.1765.
J ¼ 8.0 Hz, 1H), 2.81 (t, J ¼ 8.4 Hz, 2H), 2.08 (t, J ¼ 8.4 Hz, 2H); 13C
NMR (100 MHz, DMSO-d6):
d
173.03, 162.23 (JCF ¼ 244.4 Hz),
162.06, 144.56, 135.10, 132.22, 130.55 (JCF ¼ 8.4 Hz), 128.58, 127.95,
127.60, 122.96, 121.99, 121.63, 115.12 (JCF ¼ 21.7 Hz), 34.29, 19.78.
HRMS (ESI): m/z, calcd. for C18H14N2O6F [MþH]þ: 373.0830, found
373.0824.
4.1.11.13. Ethyl 3-(3-ethoxy-3-oxopropyl)-7-nitro-4-(4-
methoxyphenylamino)-1H
-indole-2-carboxylate
(11m).
4.1.12.5. 3-(2-Carboxyethyl)-4-(3-methoxyphenyl)-7-nitro-1H-
indole-2-carboxylic acid (12e). Following the procedure of 4.1.5,
and starting from compound 11e (140 mg, 0.32 mmol), compound
12e was afforded as light yellow solid (112 mg, 91%). mp:
Starting from compound 4e (250 mg, 0.68 mmol) and 4-
methoxyaniline (251 mg, 2.04 mmol), compound 11m was affor-
ded as orange solid (252 mg, 82%). mp: 85e87 ꢀC; 1H NMR
220e222 ꢀC; 1H NMR (300 MHz, DMSO-d6):
d 10.76 (s, 1H), 8.29 (d,
(300 MHz, CDCl3):
d
10.51 (s, 1H), 9.30 (s, 1H), 8.09 (d, J ¼ 9.3 Hz,
1H), 7.31 (d, J ¼ 8.7 Hz, 2H), 6.97 (d, J ¼ 8.7 Hz, 2H), 6.66 (d,
J ¼ 9.3 Hz,1H), 4.45 (q, J ¼ 7.2 Hz, 2H), 4.15 (q, J ¼ 7.2 Hz, 2H), 3.85 (s,
3H), 3.53 (t, J ¼ 5.4 Hz, 2H), 2.98 (t, J ¼ 5.4 Hz, 2H), 1.46 (t, J ¼ 7.2 Hz,
3H), 1.22 (t, J ¼ 7.2 Hz, 3H). HRMS (ESI): m/z, calcd. for C23H26N3O7
[MþH]þ: 456.1765, found 456.1758.
J ¼ 8.1 Hz, 1H), 7.40 (t, J ¼ 8.1 Hz, 1H), 7.13 (d, J ¼ 8.1 Hz, 1H),
6.98e7.05 (m, 3H), 3.79 (s, 3H), 2.81 (t, J ¼ 8.4 Hz, 2H), 2.13 (t,
J ¼ 8.4 Hz, 2H); 13C NMR (100 MHz, DMSO-d6):
d 173.03, 162.08,
158.88, 145.51, 140.13, 129.33, 128.59, 127.88, 127.59, 123.15, 121.69,
121.60, 120.68, 114.28, 113.77, 55.20, 34.38, 19.79. HRMS (ESI): m/z,
calcd. for C19H17N2O7 [MþH]þ: 385.1030, found 385.1027.
4.1.12. Synthesis of 4-substituted indole-2-carboxylic acid
derivatives (12aem)
4.1.12.1. 3-(2-Carboxyethyl)-7-nitro-4-phenyl-1H-indole-2-
carboxylic acid (12a). Following the procedure of 4.1.5, and starting
from compound 11a (130 mg, 0.32 mmol), compound 12a was
afforded as light yellow solid (103 mg, 92%). mp: > 250 ꢀC; 1H NMR
4.1.12.6. 3-(2-Carboxyethyl)-4-(4-methoxyphenyl)-7-nitro-1H-
indole-2-carboxylic acid (12f). Following the procedure of 4.1.5, and
starting from compound 11f (125 mg, 0.28 mmol), compound 12f
was afforded as light yellow solid (93 mg, 85%). mp: >250 ꢀC; 1H
NMR (300 MHz, DMSO-d6):
d
10.74 (s, 1H), 8.28 (d, J ¼ 8.1 Hz, 1H),
(400 MHz, DMSO-d6): d 13.70 (brs, 1H), 11.81 (brs, 1H), 10.78 (s, 1H),
7.39 (d, J ¼ 8.4 Hz, 2H), 7.10 (d, J ¼ 8.1 Hz,1H), 7.04 (d, J ¼ 8.4 Hz, 2H),
8.30 (d, J ¼ 8.4 Hz, 1H), 7.47e7.48 (m, 5H), 7.12 (d, J ¼ 8.4 Hz, 1H),
3.82 (s, 3H), 2.86 (t, J ¼ 8.1 Hz, 2H), 2.08 (t, J ¼ 8.1 Hz, 2H); 13C NMR
2.80 (t, J ¼ 8.0 Hz, 2H), 2.06 (t, J ¼ 8.0 Hz, 2H); 13C NMR (100 MHz,
(100 MHz, DMSO-d6): d 173.11, 162.10, 159.47, 145.82, 131.80, 130.93,
DMSO-d6):
d 172.95, 162.07, 145.70, 138.79, 132.11, 128.62, 128.44,
129.69, 128.72, 127.96, 127.41, 123.27, 122.02, 121.70, 113.69, 55.25,
34.24, 19.82. HRMS (ESI): m/z, calcd. for C19H17N2O7 [MþH]þ:
385.1030, found 385.1022.
128.34, 128.15, 127.85, 127.57, 123.11, 121.85, 121.66, 34.29, 19.73.
HRMS (ESI): m/z, calcd. for C18H15N2O6 [MþH]þ: 355.0925, found
355.0923.
4.1.12.7. 3-(2-Carboxyethyl)-7-nitro-4-(3-nitrophenyl)-1H-indole-2-
carboxylic acid (12g). Following the procedure of 4.1.5, and starting
from compound 11g (100 mg, 0.32 mmol), compound 12g was
afforded as yellow solid (75 mg, 85%). mp: 134e136 ꢀC; 1H NMR
4.1.12.2. 3-(2-Carboxyethyl)-4-(2-fluorophenyl)-7-nitro-1H-indole-
2-carboxylic acid (12b). Following the procedure of 4.1.5, and
starting from compound 11b (120 mg, 0.28 mmol), compound 12b
was afforded as light yellow solid (99 mg, 95%). mp: >250 ꢀC; 1H
(400 MHz, DMSO-d6):
d 10.87 (s, 1H), 8.32e8.37 (m, 3H), 7.97 (d,
NMR (400 MHz, DMSO-d6):
d
10.82 (s, 1H), 8.31 (d, J ¼ 8.4 Hz, 1H),
J ¼ 7.6 Hz, 1H), 7.82 (t, J ¼ 8.0 Hz, 1H), 7.21 (d, J ¼ 8.0 Hz, 1H), 3.34 (t,
7.54 (dd, J1 ¼ 6.8 Hz, J2 ¼ 13.2 Hz, 1H), 7.48 (t, J ¼ 7.2 Hz, 1H),
7.32e7.38 (m, 2H), 7.16 (d, J ¼ 8.4 Hz, 1H), 2.80e2.85 (m, 1H),
2.65e2.73 (m, 1H), 2.07 (t, J ¼ 8.0 Hz, 2H); 13C NMR (100 MHz,
J ¼ 7.2 Hz, 2H), 2.13 (t, J ¼ 7.2 Hz, 2H); 13C NMR (125 MHz, DMSO-
d6):
d 172.89, 161.97, 147.34, 142.57, 140.21, 135.14, 132.73, 130.00,
128.54, 128.04, 127.82, 123.27, 123.04, 122.41, 121.97, 121.61, 34.16,
19.90. HRMS (ESI): m/z, calcd. for C18H14N3O8 [MþH]þ: 400.0775,
found 400.0775.
DMSO-d6):
d
172.87, 162.01, 158.67 (JCF ¼ 242.1 Hz), 138.32, 132.76,
131.10 (JCF ¼ 8.0 Hz), 130.93, 128.43, 128.38, 127.80, 126.04
(JCF ¼ 16.0 Hz), 124.56, 122.71, 122.30, 121.61, 115.58 (JCF ¼ 21.0 Hz),
34.40, 19.38. HRMS (ESI): m/z, calcd. for C18H14N2O6F [MþH]þ:
373.0830, found 373.0821.
4.1.12.8. 3-(2-Carboxyethyl)-7-nitro-4-(4-nitrophenyl)-1H-indole-2-
carboxylic acid (12h). Following the procedure of 4.1.5, and starting
from compound 11h (100 mg, 0.32 mmol), compound 12h was
affoarded as yellow solid (75 mg, 85%). mp: >250 ꢀC; 1H NMR
4.1.12.3. 3-(2-Carboxyethyl)-4-(3-fluorophenyl)-7-nitro-1H-indole-
2-carboxylic acid (12c). Following the procedure of 4.1.5, and
starting from compound 11c (110 mg, 0.26 mmol), compound 12c
was afforded as light yellow solid (90 mg, 94%). mp: >250 ꢀC; 1H
(300 MHz, DMSO-d6):
d
10.91 (s, 1H), 8.35 (d, J ¼ 8.1 Hz, 3H), 7.80 (d,
J ¼ 8.4 Hz, 2H), 7.19 (d, J ¼ 8.1 Hz, 1H), 2.77 (t, J ¼ 7.8 Hz, 2H), 2.08 (t,
J ¼ 7.8 Hz, 2H); 13C NMR (100 MHz, DMSO-d6):
d 172.98, 161.99,
NMR (400 MHz, DMSO-d6):
d
10.82 (s, 1H), 8.30 (d, J ¼ 8.0 Hz, 1H),
147.37, 145.57, 142.89, 132.77, 130.10, 128.50, 128.05, 127.59, 123.30,
7.51e7.54 (m, 1H), 7.29e7.38 (m, 3H), 7.14 (d, J ¼ 8.0 Hz, 1H), 2.80 (t,
J ¼ 8.0 Hz, 2H), 2.12 (t, J ¼ 8.0 Hz, 2H); 13C NMR (125 MHz, DMSO-
122.46, 121.59, 121.54, 34.25, 19.89. HRMS (ESI): m/z, calcd. for
C
18H14N3O8 [MþH]þ: 400.0775, found 400.0767.
d6):
d
172.96, 162.04, 161.67 (JCF ¼ 243.8 Hz), 143.90, 141.01
(JCF ¼ 7.9 Hz), 132.35, 130.27 (JCF ¼ 8.5 Hz), 128.50, 127.88, 127.80,
124.75, 122.73, 121.73, 121.51, 115.44 (JCF ¼ 22.1 Hz), 115.27
(JCF ¼ 20.9 Hz), 34.28, 19.76. HRMS (ESI): m/z, calcd. for C18H14N2O6F
[MþH]þ: 373.0830, found 373.0828.
4.1.12.9. 3-(2-Carboxyethyl)-4-ethyl-7-nitro-1H-indole-2-carboxylic
acid (12i). Following the procedure of 4.1.5, and starting from
compound 11i (110 mg, 0.29 mmol), compound 12i was afforded as
light yellow solid (40 mg, 45%). mp: 197e198 ꢀC; 1H NMR
4.1.12.4. 3-(2-Carboxyethyl)-4-(4-fluorophenyl)-7-nitro-1H-indole-
2-carboxylic acid (12d). Following the procedure of 4.1.5, and
starting from compound 11d (120 mg, 0.28 mmol), compound 12d
was afforded as light yellow solid (78 mg, 75%). mp: >250 ꢀC; 1H
(300 MHz, DMSO-d6):
d
10.62 (s, 1H), 8.22 (d, J ¼ 8.4 Hz, 1H), 7.16 (d,
J ¼ 8.4 Hz, 1H), 3.44 (t, J ¼ 7.2 Hz, 2H), 3.15 (q, J ¼ 7.5 Hz, 2H),
2.50e2.56 (m, 2H), 1.31 (t, J ¼ 7.5 Hz, 3H); 13C NMR (100 MHz,
DMSO-d6):
d 173.48, 162.10, 149.30, 131.16, 128.54, 127.75, 126.74,
NMR (400 MHz, DMSO-d6):
7.52 (dd, J1 ¼ 5.6 Hz, J2 ¼ 8.4 Hz, 2H), 7.32 (t, J ¼ 8.4 Hz, 2H), 7.12 (d,
d
10.76 (s, 1H), 8.28 (d, J ¼ 8.0 Hz, 1H),
123.17, 122.40, 120.01, 35.71, 25.73, 20.72, 15.25. HRMS (ESI): m/z,
calcd. for C14H15N2O6 [MþH]þ: 307.0925, found 307.0919.

J. Bie et al. / European Journal of Medicinal Chemistry 90 (2015) 394e405
403
4.1.12.10. 3-(2-Carboxyethyl)-4-isobutyl-7-nitro-1H-indole-2-
carboxylic acid (12j). Following the procedure of 4.1.5, and starting
from compound 11j (110 mg, 0.28 mmol), compound 12j was
afforded as light yellow solid (82 mg, 88%). mp: 199e200 ꢀC; 1H
afforded as light yellow solid (188 mg, 94%). mp: 88e89 ꢀC; 1H NMR
(400 MHz, CDCl3): 10.10 (s, 1H), 8.11 (s, 1H), 7.86 (s, 1H), 4.47 (q,
d
J ¼ 7.2 Hz, 2H), 4.09 (q, J ¼ 7.2 Hz, 2H), 3.42 (t, J ¼ 8.0 Hz, 2H),
2.65e2.70 (m, 4H), 1.92e1.98 (m, 1H), 1.46 (t, J ¼ 7.2 Hz, 3H), 1.19 (t,
J ¼ 7.2 Hz, 3H), 0.95 (d, J ¼ 6.4 Hz, 6H). HRMS (ESI): m/z, calcd. for
NMR (400 MHz, DMSO-d6):
d
10.62 (brs,1H), 8.20 (d, J ¼ 8.4 Hz,1H),
7.09 (d, J ¼ 8.4 Hz, 1H), 3.42 (t, J ¼ 8.0 Hz, 2H), 2.95 (d, J ¼ 6.8 Hz,
C
20H27N2O6 [MþH]þ: 391.1864, found 391.1857.
2H), 2.50e2.54 (m, 2H), 1.89e1.92 (m, 1H), 0.95 (d, J ¼ 6.4 Hz, 6H);
13C NMR (100 MHz, DMSO-d6):
d
173.45, 162.09, 146.65, 131.32,
4.1.13.3. Ethyl 3-(3-ethoxy-3-oxopropyl)-5-ethyl-7-nitro-1H-indole-
2-carboxylate (13c). Following the procedure of 4.1.11.9, and start-
ing from compound 4g (270 mg, 0.65 mmol) and ethylboronic acid
(145 mg, 1.96 mmol), compound 13c was afforded as light yellow
solid (150 mg, 63%). mp: 95e96 ꢀC; 1H NMR (400 MHz, CDCl3):
128.74, 128.11, 126.90, 123.01, 122.19, 121.82, 41.88, 35.67, 30.19,
22.03, 20.62. HRMS (ESI): m/z, calcd. for C16H19N2O6 [MþH]þ:
335.1238, found 335.1231.
4.1.12.11. 3-(2-Carboxyethyl)-7-nitro-4-phenylamino-1H-indole-2-
carboxylic acid (12k). Following the procedure of 4.1.5, and starting
from compound 11k (103 mg, 0.24 mmol), compound 12k was
afforded as yellow solid (85 mg, 95%). mp: 235e237 ꢀC; 1H NMR
d
10.09 (s, 1H), 8.17 (s, 1H), 7.91 (s, 1H), 4.47 (q, J ¼ 7.2 Hz, 2H), 4.09
(q, J ¼ 7.2 Hz, 2H), 3.42 (t, J ¼ 7.6 Hz, 2H), 2.84 (q, J ¼ 7.6 Hz, 2H), 2.69
(t, J ¼ 7.6 Hz, 2H), 1.46 (t, J ¼ 7.2 Hz, 3H), 1.34 (t, J ¼ 7.6 Hz, 3H), 1.20
(t, J ¼ 7.2 Hz, 3H). HRMS (ESI): m/z, calcd. for C18H23N2O6 [MþH]þ:
363.1551, found 363.1543.
(400 MHz, DMSO-d6):
d
10.52 (s, 1H), 9.34 (s, 1H), 8.11 (d, J ¼ 9.2 Hz,
1H), 7.45 (t, J ¼ 7.6 Hz, 2H), 7.36 (d, J ¼ 7.6 Hz, 2H), 7.19 (t, J ¼ 7.2 Hz,
1H), 6.87 (d, J ¼ 9.2 Hz,1H), 3.50 (t, J ¼ 6.8 Hz, 2H), 2.74 (t, J ¼ 6.8 Hz,
4.1.13.4. Ethyl 3-(3-ethoxy-3-oxopropyl)-5-phenylamino-7-nitro-1H-
indole-2-car -boxylate (13d). To a solution of 4g (250 mg,
2H); 13C NMR (100 MHz, DMSO-d6):
d
175.70, 161.93, 149.00, 140.11,
130.89, 129.60, 125.96, 124.68, 124.58, 124.21, 123.99, 122.15, 117.32,
104.56, 35.23, 19.94. HRMS (ESI): m/z, calcd. for C18H16N3O6
[MþH]þ: 370.1034, found 370.1033.
0.56 mmol) in toluene (15 mL), Pd2(dba)3 (51 mg, 0.056 mmol),
Davephos (44 mg, 0.11 mmol), K3PO4 (357 mg, 1.68 mmol, 2 mL
H2O) and aniline (156 mg, 1.68 mmol) were added in order. The
reaction mixture was heated to 80 ꢀC for 6 h under argon atmo-
sphere, 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 by column chromatography to afford product 13d as
sorrel solid (210 mg, 68%). mp: 145e147 ꢀC; 1H NMR (400 MHz,
4.1.12.12. 3-(2-Carboxyethyl)-7-nitro-4-(3-methoxyphenylamino)-
1H-indole-2-carboxylic acid (12l). Following the procedure of 4.1.5,
and starting from compound 11l (160 mg, 0.35 mmol), compound
12l was afforded as yellow solid (130 mg, 93%). mp: 248e250 ꢀC; 1H
NMR (300 MHz, DMSO-d6):
d 10.53 (s, 1H), 9.32 (s, 1H), 8.13 (d,
J ¼ 9.3 Hz, 1H), 7.34 (t, J ¼ 8.1 Hz, 1H), 6.85e6.94 (m, 3H), 6.76 (d,
CDCl3):
d
10.04 (s, 1H), 8.12 (d, J ¼ 1.6 Hz, 1H), 7.84 (s, 1H), 7.30 (t,
J ¼ 7.8 Hz, 1H), 3.77 (s, 3H), 3.49 (t, J ¼ 6.6 Hz, 2H), 2.73 (t, J ¼ 6.6 Hz,
J ¼ 8.0 Hz, 2H), 7.03 (d, J ¼ 8.0 Hz, 2H), 6.97 (t, J ¼ 8.0 Hz, 1H), 4.47
(q, J ¼ 7.2 Hz, 2H), 4.07 (q, J ¼ 7.2 Hz, 2H), 3.36 (t, J ¼ 7.6 Hz, 2H), 2.67
(t, J ¼ 7.6 Hz, 2H),1.46 (t, J ¼ 7.2 Hz, 3H),1.18 (t, J ¼ 7.2 Hz, 3H). HRMS
2H); 13C NMR (125 MHz, DMSO-d6):
d 175.71, 161.93, 160.29, 148.71,
141.43, 130.84, 130.36, 125.86, 124.78, 124.75, 123.91, 117.59, 114.00,
109.55,107.52,105.13, 55.15, 35.22,19.93. HRMS (ESI): m/z, calcd. for
(ESI): m/z, calcd. for
426.1660.
C
22H24N3O6 [MþH]þ: 426.1651, found
C
19H18N3O7 [MþH]þ: 400.1139, found 400.1138.
4.1.12.13. 3-(2-Carboxyethyl)-7-nitro-4-(4-methoxyphenylamino)-
1H-indole-2-carboxylic acid (12m). Following the procedure of
4.1.5, and starting from compound 11m (148 mg, 0.33 mmol),
compound 12m was afforded as yellow solid (90 mg, 69%). mp:
4.1.14. Synthesis of 5-substituted indole-2-carboxylic acid
derivatives (14aed)
4.1.14.1. 3-(2-Carboxyethyl)-7-nitro-5-phenyl-1H-indole-2-
carboxylic acid (14a). Following the procedure of 4.1.5, and starting
from compound 13a (120 mg, 0.29 mmol), compound 14a was
afforded as yellow solid (102 mg, 98%). mp: >250 ꢀC; 1H NMR
>250 ꢀC; 1H NMR (300 MHz, DMSO-d6):
d 10.49 (s, 1H), 9.11 (s, 1H),
8.06 (d, J ¼ 9.3 Hz, 1H), 7.30 (d, J ¼ 8.7 Hz, 2H), 7.05 (d, J ¼ 8.7 Hz,
2H), 6.49 (d, J ¼ 9.3 Hz, 1H), 3.80 (s, 3H), 3.52 (t, J ¼ 6.9 Hz, 2H), 2.72
(400 MHz, DMSO-d6):
d 10.70 (s, 1H), 8.57 (s, 1H), 8.45 (s, 1H), 7.84
(t, J ¼ 6.9 Hz, 2H); 13C NMR (125 MHz, DMSO-d6):
d
175.35, 161.93,
(d, J ¼ 7.6 Hz, 2H), 7.53 (t, J ¼ 7.6 Hz, 2H), 7.42 (t, J ¼ 7.6 Hz, 1H), 3.38
156.86, 150.44, 132.32, 131.00, 126.28, 125.44, 124.14, 123.63, 116.00,
(t, J ¼ 7.6 Hz, 2H), 2.61 (t, J ¼ 7.6 Hz, 2H); 13C NMR (100 MHz, DMSO-
114.86, 103.56, 55.34, 35.26, 19.98. HRMS (ESI): m/z, calcd. for
d6): d 173.90, 162.31, 138.78, 133.10, 132.03, 129.07, 128.34, 127.62,
C
19H18N3O7 [MþH]þ: 400.1139, found 400.1131.
127.28, 127.05, 122.96, 120.19, 39.94, 19.46. HRMS (ESI): m/z, calcd.
for C18H15N2O6 [MþH]þ: 355.0925, found 355.0918.
4.1.13. General procedure for synthesis of 5-substituted indole-2-
carboxylate derivatives (13aed)
4.1.14.2. 3-(2-Carboxyethyl)-5-isobutyl-7-nitro-1H-indole-2-
carboxylic acid (14b). Following the procedure of 4.1.5, and starting
from compound 13b (120 mg, 0.31 mmol), compound 14b was
afforded as yellow solid (75 mg, 74%). mp: >250 ꢀC; 1H NMR
4.1.13.1. Ethyl
3-(3-ethoxy-3-oxopropyl)-7-nitro-5-phenyl-1H-
indole-2-carboxy -late (13a). Following the procedure of 4.1.11.9,
starting from compound 4g (200 mg, 0.48 mmol) and phenyl-
boronic acid (176 mg, 1.44 mmol), compound 13a was afforded as
light yellow solid (222 mg, 90%). mp: 85e87 ꢀC; 1H NMR (400 MHz,
(400 MHz, DMSO-d6):
d 13.19 (brs, 2H), 10.35 (s, 1H), 8.03 (s, 1H),
8.01 (s, 1H), 3.29 (t, J ¼ 7.6 Hz, 2H), 2.65 (d, J ¼ 7.2 Hz, 2H), 2.58 (t,
CDCl3):
d
10.18 (s, 1H), 8.55 (s, 1H), 8.32 (s, 1H), 7.68 (d, J ¼ 7.6 Hz,
J ¼ 7.6 Hz, 2H), 1.88e1.95 (m, 1H), 0.90 (d, J ¼ 6.8 Hz, 6H); 13C NMR
2H), 7.51 (t, J ¼ 7.6 Hz, 2H), 7.42 (t, J ¼ 7.2 Hz, 1H), 4.49 (q, J ¼ 7.2 Hz,
2H), 4.08 (q, J ¼ 7.2 Hz, 2H), 3.48 (t, J ¼ 7.6 Hz, 2H), 2.73 (t, J ¼ 7.6 Hz,
2H), 1.48 (t, J ¼ 7.2 Hz, 3H), 1.17 (t, J ¼ 7.2 Hz, 3H). HRMS (ESI): m/z,
calcd. for C22H23N2O6 [MþH]þ: 411.1551, found 411.1543.
(100 MHz, DMSO-d6): d 173.92, 163.08, 132.43, 132.11, 131.80,
130.36, 129.21, 126.20, 121.71, 119.86, 43.76, 35.12, 29.95, 21.95,
19.40. HRMS (ESI): m/z, calcd. for C16H19N2O6 [MþH]þ: 335.1238,
found 335.1233.
4.1.13.2. Ethyl
3-(3-ethoxy-3-oxopropyl)-5-isobutyl-7-nitro-1H-
4.1.14.3. 3-(2-Carboxyethyl)-5-ethyl-7-nitro-1H-indole-2-carboxylic
acid (14c). Following the procedure of 4.1.5, and starting from
compound 13c (110 mg, 0.30 mmol), compound 14c was afforded as
light yellow solid (72 mg, 77%). mp: >250 ꢀC; 1H NMR (400 MHz,
indole-2-carboxy -late (13b). Following the procedure of 4.1.11.9,
and starting from compound 4g (210 mg, 0.51 mmol) and iso-
butylboronic acid (156 mg, 1.53 mmol), compound 13b was

404
J. Bie et al. / European Journal of Medicinal Chemistry 90 (2015) 394e405
DMSO-d6):
d
10.55 (s, 1H), 8.13 (s, 2H), 3.29 (t, J ¼ 7.6 Hz, 2H), 2.82
4.3. Molecular docking
(q, J ¼ 7.6 Hz, 2H), 2.56 (t, J ¼ 7.6 Hz, 2H), 1.28 (t, J ¼ 7.6 Hz, 3H); 13
C
NMR (100 MHz, DMSO-d6):
d
173.76, 162.21, 135.61, 132.56, 131.34,
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) [29]. 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 14c in the crystal
structure FBPase [28]. CDOCKER uses molecular dynamics (MD)
with CHARMm force field 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 con-
structed within 7.6 Å with AMP set as the center. Compound 14c
was built and minimized using Prepare Ligands tool of DS and
refined with CHARMm force field. Docking of compound 14c 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
eCDOCKER_INTERACTION_ENERGY was chosen for analyzing the
binding features of compound 14c and FBPase.
128.33, 126.99, 126.73, 122.56, 121.92, 34.78, 27.62, 19.51, 15.91.
HRMS (ESI): m/z, calcd. for C14H15N2O6 [MþH]þ: 307.0925, found
307.0918.
4.1.14.4. 3-(2-Carboxyethyl)-5-phenylamino-7-nitro-1H-indole-2-
carboxylic acid (14d). Following the procedure of 4.1.5, and starting
from compound 13d (120 mg, 0.28 mmol), compound 14d was
afforded as sorrel solid (92 mg, 89%). mp: >250 ꢀC; 1H NMR
(400 MHz, DMSO-d6):
d 10.48 (s, 1H), 8.42 (brs, 1H), 8.02 (s, 1H),
7.93 (s, 1H), 7.27 (t, J ¼ 8.0 Hz, 2H), 7.11 (d, J ¼ 8.0 Hz, 2H), 6.86 (t,
J ¼ 8.0 Hz, 1H), 3.24 (t, J ¼ 7.2 Hz, 2H), 2.56 (t, J ¼ 7.2 Hz, 2H); 13C
NMR (100 MHz, DMSO-d6):
d 173.77, 162.17, 143.71, 136.34, 132.90,
131.72, 129.37, 127.33, 123.88, 121.93, 119.85, 116.70, 115.95, 113.54,
109.30, 34.77, 19.60. HRMS (ESI): m/z, calcd. for C18H16N3O6
[MþH]þ: 370.1034, found 370.1028.
4.2. Biological evaluation
4.2.1. Isolation and purification of human liver FBPase
Acknowledgment
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.
This work is supported by Beijing Natural Science Foundation
(No. 7132138) and PUMC Graduate Innovation Fund (No.2013-
1007-24).
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
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.11.049.
150
mg/mL of ampicillin was back-diluted 1000-fold into 2 L of 2 YT
media with 0.4% glycerol and 150
mg/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 ꢁ g), resuspended and
lysed by sonication. The lysate was clarified by centrifugation at
31,000 ꢁ 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 poly-
acrylamide gel electrophoresis. Protein concentration was deter-
mined using the BioRad version of the Bradford dye-binding assay
with bovine serum albumin as the standard [31].
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