Design, synthesis and biological evaluation of a novel series of indole-3-carboxamide derivatives for cancer treatment as EGFR inhibitors

Article abstract of DOI:10.2174/1570180814666170929093258

Background: As reported EGFR is a sialoglycoprotein with tyrosine kinase activity involved in control of cellular survival, multiplication, differentiation and metastasis. Dysregulation or aberrant expression of EGFR has been implicated in cell transformation and having oncogenic roles in a number of human cancers. Therefore EGFR has become a significant target for developing targeted therapy for cancer. Methods: A novel series of indole-3-carboxamide derivatives as EGFR inhibitors were designed, synthesized and evaluated for the anticancer activity in vitro against three EGFR high-expressed cancer cell lines (A549, HeLa, and SW480), one EGFR low-expressed cell line (HepG2) and one human liver normal cell line (HL7702) by MTT assay. Results: The target compounds 6c, 6f, 6i, 6j, 6l, 6r, 6u and 6x exhibited potent anticancer activities against the three tested cancer cell lines and weak cytotoxic effects on HepG2, which imply that the target compounds are probably effective in inhibiting EGFR. And they also did not show measurable activities in HL7702, which imply the target compounds are likely to overcome the nonspecific toxicity against normal cells. Particularly, the target compound 6x indicated equal to the positive control erlotinib. In addition, molecular docking studies demonstrated the target compound 6x may be the potential inhibitor to EGFR. Conclusion: A new EGFR inhibitor scaffold and a preliminary discussion on their SARs provide promising opportunities to guide further research on indole-3-carboxamide derivatives as novel anticancer agents.

Full text of DOI:10.2174/1570180814666170929093258

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Letters in Drug Design & Discovery, 2018, 15, 70-83
70
RESEARCH ARTICLE
ISSN: 1570-1808
eISSN: 1875-628X
Impact
Factor:
1.17
Design, Synthesis and Biological Evaluation of a Novel Series of Indole-3-
Carboxamide Derivatives for Cancer Treatment as EGFR Inhibitors
BENTHAM
S
CIENCE
Lan Zhanga, Xin-Shan Denga, Guang-Peng Menga, Chao Zhanga, Cong-Chong Liua, Gu-Zhou Chena,
Xu-Liang Jianga,*, Qing-Chun Zhaoa,b and Chun Hua,*
aKey Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical
b
University, Shenyang 110016, P.R. China; Department of Pharmacy, General Hospital of Shenyang Military Area
Command, Shenyang 110016, P.R. China
Abstract: Background: As reported EGFR is a sialoglycoprotein with tyrosine kinase activity
involved in control of cellular survival, multiplication, differentiation and metastasis. Dysregulation
or aberrant expression of EGFR has been implicated in cell transformation and having oncogenic
roles in a number of human cancers. Therefore EGFR has become a significant target for develop-
ing targeted therapy for cancer.
Methods: A novel series of indole-3-carboxamide derivatives as EGFR inhibitors were designed,
synthesized and evaluated for the anticancer activity in vitro against three EGFR high-expressed
A R T I C L E H I S T O R Yꢀ
cancer cell lines (A549, HeLa, and SW480), one EGFR low-expressed cell line (HepG2) and one
human liver normal cell line (HL7702) by MTT assay.
Received: February 07, 2017
Revised: September 07, 2017
Accepted: September 13, 2017
Results: The target compounds 6c, 6f, 6i, 6j, 6l, 6r, 6u and 6x exhibited potent anticancer activities
against the three tested cancer cell lines and weak cytotoxic effects on HepG2, which imply that the
DOI:
10.2174/1570180814666170929093258
target compounds are probably effective in inhibiting EGFR. And they also did not show measur-
able activities in HL7702, which imply the target compounds are likely to overcome the nonspecific
toxicity against normal cells. Particularly, the target compound 6x indicated equal to the positive
control erlotinib. In addition, molecular docking studies demonstrated the target compound 6x may
be the potential inhibitor to EGFR.
Conclusion: A new EGFR inhibitor scaffold and a preliminary discussion on their SARs provide
promising opportunities to guide further research on indole-3-carboxamide derivatives as novel
anticancer agents.
Keywords: Synthesis, indole-3-carboxamide derivatives, EGFR inhibitors, anticancer activity, structure-activity relationship,
cancer.
1. INTRODUCTION
oncogenic roles in a number of human cancers as lung,
breast, colon, gastric, prostatic, and head and neck cancers
[8, 9]. Therefore EGFR has become a significant target for
developing targeted therapy for cancer.
The World Cancer Report 2014 reveals that cancer is
becoming a primary cause of worldwide death, and in the
next two decades, cancer related deaths are likely to go up by
almost 70% [1]. Hence the research of novel cancer targets
and associated pathways establishes the era of molecular-
targeted medications [2-4]. Among the several target recep-
tors of cancer, the epidermal growth factor receptor (EGFR)
is a sialoglycoprotein with tyrosine kinase activity involved
in control of cellular survival, multiplication, differentiation
and metastasis [5-7]. Dysregulation or aberrant expression of
EGFR has been implicated in cell transformation and having
EGFR tyrosine kinase inhibitors (TKIs) show excellent
anticancer activity in patients and first generation EGFR
TKIs (gefitinib and erlotinib, Fig. 1) and the second genera-
tion EGFR TKIs (afatinib and lapatinib, Fig. 1) are approved
on a worldwide scale [10]. However, it has since been rec-
ognized that there are clinical problems in a subgroup of
patients who have primary and acquired resistance to the
EGFR TKIs [11-14]. As a consequence, there is an emergent
need to develop novel effective EGFR inhibitors that en-
hance the efficacy and safety for cancer treating.
*Address correspondence to these authors at the Key Laboratory of Structure-
based Drug Design & Discovery, Ministry of Education, Shenyang Pharma-
ceutical University, Shenyang 110016, P.R. China; Tel: + 86-24-43520246;
E-mails: xuliangjiang1975@163.com and chunhu@syphu.edu.cn
Indole is a key structural moiety known to have broad
biological activities. Previous studies by Hollis Showalter
have showed that a series of 4-anilinopyrimidoindole deriva-
1875-628X/18 $58.00+.00
©2018 Bentham Science Publishers

Design, Synthesis and Biological Evaluation of a Novel Series
Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1 71
ꢀ
Fig. (1). Chemical structures of gefitinib, erlotinib, afatinib and lapatinib.
Fig. (2). Design of a novel series of indole-3-carboxamide derivatives ꢀbased on the biologically active compounds
tives especially representative compound N-(3-bromophenyl)-
6-methoxy-9H-pyrimido[4,5-b]indol-4-amine (Fig. 2) exhibited
antiproliferative activity as potent inhibitors of EGFR [15].
Based on these tricyclic systems, the structure of lead com-
pound was simplified and the pyrimidine ring was opened to
get indole-3-carboxamide as the target compounds (Fig. 2).
With the purpose of improving anticancer activities, different
substituted benzylamine and various hydrophilic side chains
which have been frequently utilized in designing EGFR in-
hibitors were introduced into 3-position and 5-position of
indole-3-carboxamide scaffold, respectively. Moreover,
hereinafter docking studies were presented as well.
EGFR levels of A549, HeLa and SW480 cells were highly-
expressed [16-17], while that of HepG2 cells were low [18].
As expected, some target compounds exhibited obvious anti-
cancer activities and potency superior to positive control
drug against the tested cancer cells that expressed high level
of EGFR and did not show measurable activities in the very
low level of EGFR cells and human liver normal cells, which
implies that it is in all probability that the target compounds
are effective in inhibiting EGFR and conquering the toxicity
against normal cells. The structure–activity relationships
(SARs) were preliminary investigated based on the obtained
experimental results.
The synthesis of target compounds is described in
Scheme 1 with the intention of identifying much more con-
tributing anticancer drugs. Herein, the anticancer activity of
all target compounds was evaluated against A549 (human
lung adenocarcinoma), HeLa (Henrietta Lacks strain of can-
cer), SW480 (human colorectal cancer), HepG2 (human liver
cancer) and HL7702 (human liver) cell lines. As known, the
2. MATERIALS AND METHODS
2.1. Synthesis
All the starting materials were commercially available
and directly used without further purification. All the reac-
tion progress was monitored by thin layer chromatography

72 Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1
Zhang et al.
analysis, carried out on the silica gel plates (Qingdao Jiyida
silica reagent factory, Qingdao, China). All compounds'
melting points were determined on a melting-point apparatus
with microscope (Zhengzhou Mingyi Instrument Equipment
Corporation, Zhengzhou, China), and were uncorrected. ESI
mass spectra were performed on a Waters Q-TOF spec-
trometer (Waters Corporation, USA). The IR spectra were
recorded on a Bruker IFS55 spectrometer (KBr pellets
method). 1H NMR and 13C NMR spectra were recorded on a
Bruker 400MHz or 101 MHz NMR spectrometer (Faellan-
den, Switzerland) with TMS as an internal standard and
DMSO-d6 or CDCl3 as the solvent, chemical shifts (d values)
and coupling constants (J values) are respectively given in ppm
and Hz. High-resolution mass spectral (HRMS) analyses
were recorded on Agilent Technologies 6530 Accurate-Mass
Q-TOF Mass Spectrometer (Agilent Technologies, USA).
temperature, the reaction was acidized with hydrochloric
acid to pH 2. Then the reaction mixture was filtered and the
residue was purified by recrystallization with ethanol to af-
ford the product as solid.
2.1.3.1.
Acid (4a)
5-Hydroxy-1,2-dimethyl-1H-indole-3-carboxylic
Yellow solid; yield: 77.0%; M.p.:216-217°C; ESI-MS,
m/z: calcd. 205.07(M+); found 204.1([M-H]-); 1H NMR (400
MHz, DMSO-d6): δ 11.76 (s, 1H), 8.84 (s, 1H), 7.38 (d, J =
2.3 Hz, 1H), 7.25 (d, J = 8.7 Hz, 1H), 6.63 (dd, J = 8.7, 2.4
Hz, 1H), 3.63 (s, 3H), 2.65 (s, 3H).
2.1.3.2. 1-Ethyl-5-hydroxy-2-methyl-1H-indole-3-carboxylic
Acid (4b)
Yellow solid; yield: 68.3%; M.p.: 221-223°C; ESI-MS,
m/z: calcd. 219.09(M+); found 218.1([M-H]-); 1H NMR (400
MHz, DMSO-d6): δ 11.76 (s, 1H), 8.84 (s, 1H), 7.38 (d, J =
2.3 Hz, 1H), 7.26 (d, J = 8.7 Hz, 1H), 6.62 (dd, J = 8.7, 2.4
Hz, 1H), 4.13 (q, J = 7.1 Hz, 2H), 2.66 (s, 3H), 1.21 (t, J =
7.1 Hz, 3H).
2.1.1. General Procedure for the Preparation of Ethyl (Z)-
3-(substituted amino)but-2-enoate
To ethyl acetoacetate (15.01 g, 0.12 mol), methylamine
or ethylamine (0.36 mol, aqueous solution) was added drop-
wise and the reaction mixture was stirred at ambient tem-
perature for 3 h. The organic phase was successively washed
with water and brine and dried over anhydrous magnesium
sulfate to give the corresponding product as a transparent
liquid. The resulting product was used directly in the follow-
ing step without further purification.
2.1.4. General Procedure for the Preparation of 1-Alkyl-
N-(substituent
carboxamide
benzyl)-5-hydroxy-2-methyl-1H-indole-3-
A solution of 4a or 4b (0.020 mol), benzylamine or 4-
fluorobenzylamine (0.060 mol), 1-(3-(dimethylamino)propyl)-
3-ethyl-carbodiimide hydrochloride (EDCI, 5.75g, 0.030
mol), 1-hydroxybenzotriazole (HOBt, 4.05g, 0.030 mol) and
triethylamine (6.07 g, 0.060 mol) in N,N-dimethylformamide
(DMF, 100 mL) and dichloromethane (100 mL) was stirred
at ambient temperature for 24 h. Water (200 mL) was added
and the reaction mixture was stirred for 1 h. Then the mix-
ture was filtered and the residue was purified by recrystalli-
zation with 70% ethanol aqueous solution to afford product
as solid.
2.1.1.1. Ethyl (Z)-3-(methylamino)but-2-enoate (2a)
Yellow liquid; yield: 91.4%.
2.1.1.2. Ethyl (Z)-3-(ethylamino)but-2-enoate (2b)
White liquid; yield: 90.3%.
2.1.2. General Procedure for the Preparation of Ethyl 1-
alkyl-5-hydroxy-2-methyl-1H-indole-3-carboxylate
2.1.4.1.
N-Benzyl-5-hydroxy-1,2-dimethyl-1H-indole-3-
To a solution of benzoquinone (12.40 g, 0.11 mol) in ace-
tone (120 mL), 2a or 2b (0.11 mol) was added dropwise at
30°C, and the reaction mixture was stirred at this temperature
for 2 h. After cooling to room temperature, the reaction mix-
ture was concentrated in vacuo and the residue was purified by
recrystallization with acetone to afford the product as solid.
carboxamide (5a)
White solid; yield: 53.5%; M.p.: 211-213°C; ESI-MS,
m/z: calcd. 294.14(M+); found 295.1([M+H]+), 317.1
([M+Na]+).; 1H NMR (400 MHz, DMSO-d6): δ 8.81 (s, 1H),
7.94 (d, J = 2.3 Hz, 1H), 7.37 (d, J = 8.6 Hz, 1H), 7.36 – 7.22
(m, 5H), 7.16 (dd, J = 8.7, 2.3 Hz, 1H), 4.47 (d, J = 6.0 Hz,
2H), 3.61 (s, 3H), 2.56 (s, 3H).
2.1.2.1. Ethyl 5-hydroxy-1,2-dimethyl-1H-indole-3-carboxylate
(3a)
2.1.4.2.
N-(4-Fluorobenzyl)-5-hydroxy-1,2-dimethyl-1H-
White solid; yield: 75.2%; M.p.:208-210°C (lit.[19]: 208-
209°C). ESI-MS, m/z: calcd. 233.11(M+); found 234.1
([M+H]+), 356.1([M+Na]+).
indole-3-carboxamide (5b)
White solid; yield: 73.0%; M.p.: 217-218°C; ESI-MS,
m/z: calcd. 312.13(M+); found 313.1([M+H]+), 335.1
([M+Na]+).; 1H NMR (400 MHz, DMSO-d6): δ 8.82 (s, 1H),
7.95 (d, J = 2.4 Hz, 1H), 7.39 (dd, J = 8.4, 5.7 Hz, 2H), 7.24
(d, J = 8.7 Hz, 1H), 7.18 – 7.11 (m, 3H), 4.44 (d, J = 6.0 Hz,
2H), 3.62 (s, 3H), 2.56 (s, 3H).
2.1.2.2. Ethyl 1-ethyl-5-hydroxy-2-methyl-1H-indole-3-
carboxylate (3b)
White solid; yield: 76.8%; M.p.:184-186°C (lit.[20]: 184-
185°C). ESI-MS, m/z: calcd. 247.12(M+); found 248.1
([M+H]+), 270.1([M+Na]+).
2.1.4.3. N-Benzyl-1-ethyl-5-hydroxy-2-methyl-1H-indole-3-
carboxamide (5c)
2.1.3. General Procedure for the Preparation of 1-Alkyl -5-
hydroxy-2-methyl-1H-indole-3-carboxylic Acid
Yellow solid; yield: 59.2%; M.p.: 189-190°C; ESI-MS,
m/z: calcd. 308.15(M+); found 309.2([M+H]+); 1H NMR
(400 MHz, DMSO-d6): δ 8.81 (s, 1H), 7.94 (d, J = 2.3 Hz,
1H), 7.35 (d, J = 8.7 Hz, 1H), 7.34 – 7.23 (m, 5H), 7.15 (dd,
A solution of 3a or 3b (0.0043 mol) and sodium hydrox-
ide (17.15 g, 0.043 mol) in ethanol (100 mL) and water (50
mL) was stirred at reflux for 12 h. After cooling to ambient

Design, Synthesis and Biological Evaluation of a Novel Series
Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1 73
J = 8.7, 2.3 Hz, 1H), 4.46 (d, J = 6.0 Hz, 2H), 4.11 (q, J = 7.1
Hz, 2H), 2.56 (s, 3H), 1.20 (t, J = 7.1 Hz, 3H).
160.74(C8), 153.88(C5), 139.34(C2), 139.29(C-Ph), 128.83
(C9), 127.93(CH-Ph), 127.42(CH-Ph), 125.76(CH-Ph), 111.12
(C6), 110.31(C3), 107.52(C7), 103.55(C4), 68.65(C1’’), 49.10
(NCH2), 47.63(NCH2), 43.60(C2’), 29.75(NCH3), 22.30
(CH2), 20.80(CH2), 11.91(indole-CH3), 11.49(CH3), 11.39
(CH3); HRMS (ESI): calcd for C26H33N3O3, [M]+ m/z,
435.2522; found, 436.2581([M+H]+), 458.2391([M+Na]+).
2.1.4.4.
1-Ethyl-N-(4-fluorobenzyl)-5-hydroxy-2-methyl-
1H-indole-3-carboxamide (5d)
Yellow solid; yield: 58.4%; M.p.: 167-168°C; ESI-MS,
m/z: calcd. 326.14(M+); found 327.1([M+H]+), 349.1
([M+Na]+).; 1H NMR (400 MHz, DMSO-d6): δ 8.81 (s, 1H),
7.94 (d, J = 2.3 Hz, 1H), 7.28 (dd, J = 8.5, 5.6 Hz, 2H), 7.16
(d, J = 8.7 Hz, 1H), 7.11 – 6.96 (m, 3H), 4.56 (d, J = 5.8 Hz,
2H), 4.07 (q, J = 7.2 Hz, 2H), 2.67 (s, 3H), 1.30 (t, J = 7.2
Hz, 3H).
2.1.5.3. N-Benzyl-5-[2-(di-sec-butylamino)-2-oxoethoxy]-
1,2-dimethyl-1H-indole-3-carboxamide (6c)
Yellow solid; yield: 70.9%; M.p.: 105-106°C; IR: (KBr,
cm-1): υ 3425.2, 2919.0, 2850.0, 1621.9, 1485.5, 1384.5,
1194.7, 1120.5, 821.2, 778.6, 697.9; ESI-MS, m/z: calcd.
463.28(M+); found 464.3([M+H]+); 1H NMR (400 MHz,
CDCl3): δ 7.43 (d, J = 2.3 Hz, 1H, indole-H7), 7.38 – 7.27
(m, 5H, Ar–H), 7.19 (d, J = 8.9 Hz, 1H, indole-H4), 6.90 (dd,
J = 8.8, 2.4 Hz, 1H, indole-H6), 4.71 (d, J = 5.6 Hz, 2H,
NHCH2), 4.70 (s, 2H, OCH2), 3.65 (s, 3H, NCH3), 3.20 –
3.11(m, 2H, NCH), 2.72 (s, 3H, indole-CH3), 1.29 – 1.25 (m,
4H, CH2), 0.92 (d, J = 6.7 Hz, 6H, CH3), 0.78 (t, J = 6.7 Hz,
6H, CH3); 13C NMR (101 MHz, CDCl3): δ 168.71(C1’),
166.30(C2’’), 160.17(C8), 153.82(C5), 139.28(C2), 137.48(C-
Ph), 128.83(C9), 127.94(CH-Ph), 127.42(CH-Ph), 125.74
(CH-Ph), 111.09(C6), 110.26(C3), 107.49(C7), 103.73(C4),
68.60(C1’’), 54.48(NCH), 52.64(NCH), 43.68(C2’), 29.84
(NCH3), 27.46(CH2), 26.26(CH2), 20.25(CH3), 20.19(CH3),
11.91(indole-CH3), 11.00(CH3), 10.54(CH3); HRMS (ESI):
calcd for C28H37N3O3, [M]+ m/z, 463.2835; found, 464.
2892([M+H]+), 486.2705([M+Na]+).
2.1.5. General Procedure for the Preparation of Indole-3-
carboxamide Derivatives 6a-6x
A solution of 5a, 5b, 5c or 5d (0.0034 mol), 2-
chloroacetamide(0.017 mol), anhydrous potassium carbon-
ate(4.70 g, 0.034 mol), potassium iodide(0.06 g, 0.00034
mol) in N,N-dimethylformamide (DMF, 20 mL) was stirred
at 80oC for 16 h. Water (100 mL) was added and the reaction
mixture was stirred for 4 h. The mixture was filtered and the
residue was purified by column chromatography on silica gel
using petroleum ether/ ethyl acetate(5/1, v/v) as eluent to
afford the corresponding products (6a-6x).
2.1.5.1.
N-Benzyl-5-[2-(diethylamino)-2-oxoethoxy]-1,2-
dimethyl-1H-indole-3-carboxamide (6a)
White solid; yield: 63.0%; M.p.: 119-120°C; IR: (KBr,
cm-1): υ 3444.3, 2927.6, 1660.6, 1620.2, 1431.2, 1383.6,
1257.3, 1190.8, 1085.3, 834.3, 776.2, 745.1, 701.8; ESI-MS,
m/z: calcd. 407.22(M+); found 408.3([M+H]+), 430.2
2.1.5.4.
N-Benzyl-1,2-dimethyl-5-[2-oxo-2-(piperidin-1-
yl)ethoxy]-1H-indole-3-carboxamide (6d)
1
([M+Na]+), 446.2([M+K]+).; H NMR (400 MHz, CDCl3): δ
White solid; yield: 58.5%; M.p.: 78-80°C; IR: (KBr, cm-1):
υ 3429.6, 2923.2, 2853.6, 1641.2, 1618.1, 1488.8, 1440.1,
1212.0, 1163.4, 1082.8, 1017.8, 874.0, 699.6; ESI-MS, m/z:
calcd. 419.22(M+); found 420.4([M+H]+), 442.3([M+Na]+);
1H NMR (400 MHz, CDCl3): δ 7.43 (d, J = 2.4 Hz, 1H, in-
dole-H7), 7.38 – 7.26 (m, 5H, Ar–H), 7.20 (d, J = 8.9 Hz, 1H,
indole-H4), 6.91 (dd, J = 8.9, 2.4 Hz, 1H, indole-H6), 4.71 (d,
J = 5.8 Hz, 2H, NHCH2), 4.66 (s, 2H, OCH2), 3.66 (s, 3H,
NCH3), 3.52 (t, J = 5.3 Hz, 2H, NCH2), 3.45 (t, J = 5.0 Hz,
2H, NCH2), 2.72 (s, 3H, indole-CH3), 1.61-1.53 (m, 6H,
CH2); 13C NMR (101 MHz, CDCl3): δ 166.52(C1’),
166.15(C2’’), 160.43(C8), 153.62(C5), 142.55(C2), 139.16(C-
Ph), 128.69(C9), 127.83(CH-Ph), 127.29(CH-Ph), 125.64
(CH-Ph), 111.03(C6), 110.24(C3), 107.42(C7), 103.31(C4),
68.65(C1’’), 46.44(C2’), 43.56(NCH2), 43.17(NCH2), 29.62
(NCH3), 26.49(CH2), 25.55(CH2), 24.48(CH2), 11.78(indole-
CH3); HRMS (ESI): calcd for C25H29N3O3, [M]+ m/z,
419.2209; found, 442.2077([M+Na]+).
7.43 (d, J = 2.3 Hz, 1H, indole-H7), 7.37 – 7.27 (m, 5H, Ar–
H), 7.21 (d, J = 8.9 Hz, 1H, indole-H4), 6.91 (dd, J = 8.9, 2.4
Hz, 1H, indole-H6), 4.71 (d, J = 5.7 Hz, 2H, NHCH2), 4.66
(s, 2H, OCH2), 3.66 (s, 3H, NCH3), 3.38 (dd, J = 7.1, 2.9 Hz,
2H, NCH2), 3.35 (dd, J = 7.1, 2.9 Hz, 2H, NCH2), 2.72 (s,
3H, indole-CH3), 1.17 (t, J = 7.1 Hz, 3H, CH3), 1.11 (t, J =
7.1 Hz, 3H, CH3); 13C NMR (101 MHz, CDCl3): δ
167.51(C1’), 166.30(C2’’), 158.24(C8), 153.86(C5), 139.28(C2,
C-Ph), 132.28(CH-Ph), 128.83(C9), 127.97(CH-Ph), 127.43
(CH-Ph), 126.95(CH-Ph), 111.24(C6), 110.33(C3), 107.55
(C7), 103.50(C4), 68.71(C1’’), 43.69(C2’), 41.71(NCH2), 40.39
(NCH2), 29.76(NCH3), 12.97(CH3), 12.78(CH3), 11.92
(indole-CH3); HRMS (ESI): calcd for C24H29N3O3, [M]+ m/z,
407.2209; found, 408.2270([M+H]+), 430.2083([M+Na]+).
2.1.5.2. N-Benzyl-5-[2-(dipropylamino)-2-oxoethoxy]-1,2-
dimethyl-1H-indole-3-carboxamide (6b)
White solid; yield: 73.0%; M.p.: 123-125°C; IR: (KBr,
cm-1): υ 3349.0, 2964.9, 2871.1, 1656.2, 1484.3, 1382.6,
1270.8, 1190.0, 1143.3, 1084.6, 926.2, 839.0, 798.2, 741.2,
697.8; ESI-MS, m/z: calcd. 435.25(M+); found 436.2
2.1.5.5.
N-Benzyl-1,2-dimethyl-5-[2-(morpholin-4-yl)-2-
oxoethoxy]-1H-indole-3-carboxamide (6e)
1
([M+H]+), 458.2([M+Na]+); H NMR (400 MHz, CDCl3): δ
White solid; yield: 54.6%; M.p.: 110-112°C; IR: (KBr,
cm-1): υ 3442.6, 2856.0’ 1653.8, 1483.8, 1384.1, 1274.2,
1207.7, 1162.8, 1031.3, 923.2, 850.8, 698.4; ESI-MS, m/z:
calcd. 421.20(M+); found 422.2([M+H]+), 444.2([M+Na]+);
1H NMR (400 MHz, CDCl3): δ 7.44 (d, J = 2.3 Hz, 1H, in-
dole-H7), 7.38 – 7.27 (m, 5H, Ar–H), 7.21 (d, J = 8.9 Hz, 1H,
indole-H4), 6.89 (dd, J = 8.9, 2.4 Hz, 1H, indole-H6), 4.71 (d,
J = 5.7 Hz, 2H, NHCH2), 4.68 (s, 2H, OCH2), 3.67 (s, 3H,
7.43 (d, J = 2.3 Hz, 1H, indole-H7), 7.38 – 7.26 (m, 5H, Ar–
H), 7.20 (d, J = 8.9 Hz, 1H, indole-H4), 6.90 (dd, J = 8.9, 2.4
Hz, 1H, indole-H6), 4.71 (d, J = 5.7 Hz, 2H, NHCH2), 4.67
(s, 2H, OCH2), 3.66 (s, 3H, NCH3), 3.26 (q, J = 7.0 Hz, 4H,
NCH2), 2.72 (s, 3H, indole-CH3), 1.56 – 1.48 (m, 4H, CH2),
0.89 (t, J = 7.4 Hz, 3H, CH3), 0.84 (t, J = 7.4 Hz, 3H, CH3);
13C NMR (101 MHz, CDCl3): δ 167.96(C1’), 166.32(C2’’),

74 Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1
Zhang et al.
NCH3), 3.63 – 3.57 (m, 8H, CH2), 2.72 (s, 3H, indole-CH3);
13C NMR (101 MHz, CDCl3): δ 167.08(C1’), 166.20(C2’’),
160.23(C8), 153.39(C5), 142.69(C2), 139.25(C-Ph), 128.86
(C9), 127.99(CH-Ph), 127.49(CH-Ph), 125.85(CH-Ph),
111.08(C6), 110.46(C3), 107.64(C7), 103.35(C4), 68.67(C1’’),
66.95(OCH2), 66.91(OCH2), 46.07(C2’), 43.73(NCH2), 42.53
(NCH2), 29.79(NCH3), 11.94(indole-CH3); HRMS (ESI):
calcd for C24H27N3O4, [M]+ m/z, 421.2002; found, 422.
2065([M+H]+), 444.1875([M+Na]+).
NMR (400 MHz, CDCl3): δ 7.40 (dd, J = 8.7, 5.4 Hz, 2H,
Ar–H), 7.28 (d, J = 2.3 Hz, 1H, indole-H7), 7.21 (d, J = 8.9
Hz, 1H, indole-H4), 7.03 (t, J = 8.7 Hz, 2H, Ar–H), 6.89 (dd,
J = 8.8, 2.4 Hz, 1H, indole-H6), 4.70 (s, 2H, OCH2), 4.67 (d,
J = 5.6 Hz, 2H, NHCH2), 3.66 (s, 3H, NCH3), 3.29 – 3.24
(m, 4H, NCH2), 2.72 (s, 3H, indole-CH3), 1.54 – 1.47 (m,
4H, CH2), 0.90 (t, J = 7.4 Hz, 3H, CH3), 0.83 (t, J = 7.4 Hz,
3H, CH3); 13C NMR (101 MHz, CDCl3): δ 167.42(C1’),
166.29(C2’’), 160.95(C8), 160.63(C-F), 154.41(C5), 138.45
(C2), 133.69(C-C6H4F), 129.65(CH-C6H4F), 129.57(CH-
C6H4F), 128.06(C9), 115.70(CH-C6H4F), 115.55(CH-C6H4F),
111.05(C6), 110.87(C3), 110.36(C7), 103.47(C4), 68.75(C1’’),
58.41(NCH2), 57.95(NCH2), 49.11(C2’), 29.76(NCH3), 22.30
(CH2), 20.80(CH2), 12.15(indole-CH3), 11.47(CH3), 11.35
(CH3); HRMS (ESI): calcd for C26H32FN3O3, [M]+ m/z,
453.2428; found, 477.2335([M+Na]+).
2.1.5.6. N-Benzyl-5-{2-{[2-(2-methoxyphenoxy)ethyl]amino}-
2-oxoethoxy}-1,2-dimethyl-1H-indole-3-carboxamide (6f)
White solid; yield: 58.8%; M.p.: 180-181°C; IR: (KBr,
cm-1): υ 3426.8, 2923.0, 2852.3, 1628.8, 1384.4, 1253.4,
1124.5, 777.8, 618.2; ESI-MS, m/z: calcd. 501.23(M+);
found 502.4([M+H]+), 524.4([M+Na]+); 1H NMR (400 MHz,
CDCl3): δ 7.40 (d, J = 2.3 Hz, 1H, indole-H7), 7.38 – 7.27
(m, 5H, benzyl-H), 7.22 (d, J = 8.9 Hz, 1H, indole-H4), 6.90
– 6.81 (m, 5H, Ar–H), 4.68 (d, J = 5.6 Hz, 2H, NHCH2),
4.50 (s, 2H, OCH2CO), 4.10 (t, J = 5.1 Hz, 2H, OCH2), 3.76
(s, 3H, NCH3), 3.74 – 3.71 (m, 2H, CH2), 3.66 (s, 3H,
OCH3), 2.72 (s, 3H, indole-CH3); 13C NMR (101 MHz,
CDCl3): δ 169.00(C1’), 168.96(C2’’), 160.50(C8), 153.10(C5),
150.28(O-C-Ph), 150.22(O-C-Ph), 139.13(C2), 137.31(C-
Ph), 128.95(C9), 127.87(CH-Ph), 127.60(CH-Ph), 125.88
(CH-Ph), 122.48(CH-Ph), 121.15(CH-Ph), 120.30(CH-Ph),
115.62(CH-Ph), 112.19(C6), 111.07(C3), 110.46(C7), 103.83
(C4), 69.01(OCH2), 68.84(C1’’), 56.14(OCH3), 43.72(C2’),
38.50(NHCH2), 29.79(NCH3), 11.91(indole-CH3); HRMS
(ESI): calcd for C29H31N3O5, [M]+ m/z, 501.2264; found,
502.2322([M+H]+), 524.2134([M+Na]+).
2.1.5.9.
5-[2-(Di-sec-butylamino)-2-oxoethoxy]-N-(4-
fluorobenzyl)-1,2-dimethyl-1H-indole-3-carboxamide (6i)
White solid; yield: 57.1%; M.p.: 137-139°C; IR: (KBr,
cm-1): υ 3355.2, 2961.8, 1656.6, 1540.3, 1485.8, 1385.1,
1215.7, 1145.0, 1082.5, 821.1, 795.3, 758.4, 712.4, 589.9;
ESI-MS, m/z: calcd. 481.27(M+); found 482.4([M+H]+),
1
504.4([M+Na]+); H NMR (400 MHz, CDCl3): δ 7.41 (dd, J
= 8.7, 5.4 Hz, 2H, Ar–H), 7.30 (d, J = 2.3 Hz, 1H, indole-
H7), 7.20 (d, J = 8.9 Hz, 1H, indole-H4), 7.02 (t, J = 8.7 Hz,
2H, Ar–H), 6.89 (dd, J = 8.8, 2.4 Hz, 1H, indole-H6), 4.73 (s,
2H, OCH2), 4.67 (d, J = 5.6 Hz, 2H, NHCH2), 3.66 (s, 3H,
NCH3), 3.20 – 3.13 (m, 2H, NCH), 2.72 (s, 3H, indole-CH3),
2.01 – 1.87 (m, 4H, CH2), 0.93 (d, J = 6.7 Hz, 6H, CH3),
0.77 (t, J = 6.7 Hz, 6H, CH3); 13C NMR (101 MHz, CDCl3):
δ 168.79(C1’), 166.29(C2’’), 160.65(C8), 160.04(C-F), 153.84
(C5), 139.85(C2), 132.31(C-C6H4F), 129.65(CH-C6H4F), 129.
57(CH-C6H4F), 127.69(C9), 115.71(CH-C6H4F), 115.50(CH-
C6H4F), 111.03(C6), 110.31(C3), 107.29(C7), 103.77(C4),
68.69(C1’’), 54.46(NCH), 54.28(NCH), 52.62(C2’), 29.76
(NCH3), 27.44(CH2), 26.25(CH2), 18.87(CH3), 18.81(CH3),
11.92(indole-CH3), 10.07(CH3), 9.83(CH3); HRMS (ESI):
calcd for C28H36FN3O3, [M]+ m/z, 481.2741; found,
482.2804([M+H]+), 504.2614([M+Na]+).
2.1.5.7. 5-[2-(Diethylamino)-2-oxoethoxy]-N-(4-fluorobenzyl)-
1,2-dimethyl-1H-indole-3-carboxamide (6g)
White solid; yield: 50.4%; M.p.: 133-134°C; IR: (KBr,
cm-1): υ 3442.8, 2972.6, 2931.4, 1658.0, 1619.9, 1487.1,
1383.6, 1218.2, 1143.2, 1086.9, 974.6, 822.1, 757.6, 568.6;
ESI-MS, m/z: calcd. 425.21(M+); found 426.3([M+H]+),
1
448.4([M+Na]+); H NMR (400 MHz, CDCl3): δ 7.41 (dd, J
= 8.6, 5.4 Hz, 2H, Ar–H), 7.29 (d, J = 2.3 Hz, 1H, indole-
H7), 7.21 (d, J = 8.9 Hz, 1H, indole-H4), 7.03 (t, J = 8.7 Hz,
2H, Ar–H), 6.91 (dd, J = 8.8, 2.4 Hz, 1H, indole-H6), 4.68 (s,
2H, OCH2), 4.66 (d, J = 5.6 Hz, 2H, NHCH2), 3.66 (s, 3H,
NCH3), 3.37 (q, J = 6.9 Hz, 4H, NCH2), 2.72 (s, 3H, indole-
CH3), 1.17 (t, J = 7.1 Hz, 3H, CH3), 1.10 (t, J = 7.1 Hz, 3H,
CH3); 13C NMR (101 MHz, CDCl3): δ 167.60(C1’), 166.29
(C2’’), 161.77(C8), 161.46(C-F), 153.89(C5), 143.01(C2),
132.28(C-C6H4F), 129.73(CH-C6H4F), 129.67(CH-C6H4F),
127.61(C9), 115.70(CH-C6H4F), 115.48(CH-C6H4F), 111.19
(C6), 110.38(C3), 109.37(C7), 103.52(C4), 68.75(C1’’), 41.72
(C2’), 40.41(NCH2), 40.33(NCH2), 29.73(NCH3), 14.46
(CH3), 12.95(CH3), 11.92(indole-CH3); HRMS (ESI): calcd
for C24H28FN3O3, [M]+ m/z, 425.2115; found, 426.2178
([M+H]+), 448.1989([M+Na]+).
2.1.5.10.
N-(4-Fluorobenzyl)-1,2-dimethyl-5-[2-oxo-2-
(piperidin-1-yl)ethoxy]-1H-indole-3-carboxamide (6j)
White solid; yield: 40.0%; M.p.: 92-94°C; IR: (KBr, cm-1):
υ 3423.8, 2920.5, 2851.1, 1626.7, 1509.2, 1445.4, 1220.0,
1156.9, 1014.3, 874.3, 803.4, 714.5; ESI-MS, m/z: calcd.
437.21(M+); found 438.4([M+H]+), 560.4([M+Na]+); 1H
NMR (400 MHz, CDCl3): δ 7.41 (dd, J = 8.6, 5.4 Hz, 2H,
Ar–H), 7.29 (d, J = 2.4 Hz, 1H, indole-H7), 7.21 (d, J = 8.9
Hz, 1H, indole-H4), 7.03 (t, J = 8.7 Hz, 2H, Ar–H), 6.90 (dd,
J = 8.8, 2.4 Hz, 1H, indole-H6), 4.69 (s, 2H, OCH2), 4.67 (d,
J = 5.8 Hz, 2H, NHCH2), 3.66 (s, 3H, NCH3), 3.51 (t, J = 5.2
Hz, 2H, NCH2), 3.45 (t, J = 5.1 Hz, 2H, NCH2), 2.72 (s, 3H,
indole-CH3), 1.55 – 1.47 (m, 6H, CH2); 13C NMR (101 MHz,
CDCl3): δ 166.95(C1’), 166.70(C2’’), 159.27(C8), 159.16(C-
F), 153.80(C5), 136.18(C2), 132.27(C-C6H4F), 129.69(CH-
C6H4F), 129.61(CH-C6H4F), 127.99(C9), 115.70(CH-C6H4F),
115.49(CH-C6H4F), 111.15(C6), 110.41(C3), 108.77(C7),
103.44(C4), 68.83(C1’’), 49.25(NCH2), 49.14(NCH2), 43.32
(C2’), 29.77(NCH3), 25.69(CH2), 25.63(CH2), 24.60(CH2),
11.92(indole-CH3); HRMS (ESI): calcd for C25H28FN3O3,
2.1.5.8. 5-[2-(Dipropylamino)-2-oxoethoxy]-N-(4-fluorobenzyl)-
1,2-dimethyl-1H-indole-3-carboxamide (6h)
White solid; yield: 73.1%; M.p.: 145-147°C; IR: (KBr,
cm-1): υ 3446.5, 2930.0, 2360.9, 1655.5, 1508.8, 1484.3,
1383.4, 1271.9, 1190.1, 1095.7, 819.2, 798.4, 777.4; ESI-
MS, m/z: calcd. 453.24(M+); found 454.4([M+H]+); 1H

Design, Synthesis and Biological Evaluation of a Novel Series
Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1 75
[M]+ m/z, 437.2115; found, 438.2178([M+H]+), 460.1988
([M+Na]+).
OCH2), 4.12 (q, J = 7.2 Hz, 2H, indole-NCH2), 3.36 (q, J =
7.0 Hz, 4H, NCH2), 2.72 (s, 3H, indole-CH3), 1.33 (t, J = 7.2
Hz, 3H, CH3), 1.17 (t, J = 7.1 Hz, 3H, CH3), 1.11 (t, J = 7.1
Hz, 3H, CH3); 13C NMR (101 MHz, CDCl3): δ 167.53(C1’),
166.34(C2’’), 153.79(C5), 141.82(C2), 139.29(C-Ph), 128.81
(C8), 128.72(CH-Ph), 128.59(CH-Ph), 127.96(C9), 127.41
(CH-Ph), 126.28(CH-Ph), 126.01(CH-Ph), 111.23(C6),
110.35(C3), 107.65(C7), 103.57(C4), 68.70(C1’’), 43.68(C2’),
41.69(NCH2), 40.38(NCH2), 38.02(NCH2), 15.10(CH3),
14.47(CH3), 12.96(CH3), 11.63(indole-CH3); HRMS (ESI):
calcd for C25H31N3O3, [M]+ m/z, 421.2365; found, 422.
2427([M+H]+), 444.2234([M+Na]+).
2.1.5.11. N-(4-Fluorobenzyl)-1,2-dimethyl-5-[2-(morpholin-
4-yl)-2-oxoethoxy]-1H-indole-3-carboxamide (6k)
Yellow solid; yield: 75.9%; M.p.: 181-182°C; IR: (KBr,
cm-1): υ 3422.9, 2919.7, 2860.2, 1677.1, 1627.6, 1506.2,
1444.1, 1214.8, 1162.4, 1117.0, 1032.2, 845.3, 808.7, 720.8;
ESI-MS, m/z: calcd. 439.19(M+); found 440.3([M+H]+),
1
462.3([M+Na]+); H NMR (400 MHz, CDCl3): δ 7.41 (dd, J
= 8.5, 5.4 Hz, 2H, Ar–H), 7.28 (d, J = 2.4 Hz, 1H, indole-
H7), 7.21 (d, J = 8.9 Hz, 1H, indole-H4), 7.03 (t, J = 8.7 Hz,
2H, Ar–H), 6.88 (dd, J = 8.8, 2.4 Hz, 1H, indole-H6), 4.70 (s,
2H, OCH2), 4.67 (d, J = 5.8 Hz, 2H, NHCH2), 3.66 (s, 3H,
NCH3), 3.63 – 3.58 (m, 8H, CH2), 2.71 (s, 3H, indole-CH3);
13C NMR (101 MHz, CDCl3): δ 167.13(C1’), 166.18(C2’’),
163.42(C8), 161.03(C-F), 153.42(C5), 142.89(C2), 132.35(C-
C6H4F), 129.69(CH-C6H4F), 129.61(CH-C6H4F), 125.77(C9),
115.73(CH-C6H4F), 115.51(CH-C6H4F), 111.06(C6), 110.51
(C3), 107.49(C7), 103.33(C4), 68.73(C1’’), 66.94(OCH2), 66.90
(OCH2), 46.07(C2’), 42.96(NCH2), 42.54(NCH2), 29.79
(NCH3), 11.93(indole-CH3); HRMS (ESI): calcd for C24H26
FN3O4, [M]+ m/z, 439.1907; found, 440.1971([M+H]+),
462.1779([M+Na]+).
2.1.5.14. N-Benzyl-5-[2-(dipropylamino)-2-oxoethoxy]-1-
ethyl-2-methyl-1H-indole-3-carboxamide (6n)
White solid; yield: 74.3%; M.p.: 129-130°C; IR: (KBr,
cm-1): υ 3444.2, 2930.3, 1657.5, 1618.2, 1482.3, 1383.6,
1272.0, 1193.7, 1145.4, 1090.3, 795.4, 698.8; ESI-MS, m/z:
calcd. 449.27(M+); found 450.2([M+H]+), 472.3([M+Na]+);
1H NMR (400 MHz, CDCl3): δ 7.42 (d, J = 2.2 Hz, 1H, in-
dole-H7), 7.37 – 7.27 (m, 5H, Ar–H), 7.20 (d, J = 8.9 Hz, 1H,
indole-H4), 6.89 (dd, J = 8.9, 2.3 Hz, 1H, indole-H6), 4.70 (d,
J = 5.8 Hz, 2H, NHCH2), 4.67 (s, 2H, OCH2), 4.11 (q, J =
7.1 Hz, 2H, indole-NCH2), 3.29 – 3.21 (m, 4H, NCH2), 2.71
(s, 3H, indole-CH3), 1.60 – 1.52 (m, 4H, CH2), 1.34 (t, J =
7.3 Hz, 3H, CH3), 0.89 (t, J = 7.4 Hz, 3H, CH3), 0.84 (t, J =
7.4 Hz, 3H, CH3); 13C NMR (101 MHz, CDCl3): δ
167.95(C1’), 166.34(C2’’), 153.79(C5), 141.87(C2), 139.30(C-
Ph), 128.90(C8), 128.80(CH-Ph), 128.58(CH-Ph), 127.92
(C9), 127.39(CH-Ph), 126.79(CH-Ph), 125.99(CH-Ph),
111.10(C6), 110.32(C3), 107.61(C7), 103.62(C4), 68.61(C1’’),
49.07(C2’), 47.61(NCH2), 43.66(NCH2), 38.00(NCH2),
22.28(CH2), 20.79(CH2), 15.08(CH3), 11.62(indole-CH3),
11.47(CH3), 11.38(CH3); HRMS (ESI): calcd for C27H35
N3O3, [M]+ m/z, 449.2678; found, 450.2740([M+H]+),
472.2550([M+Na]+).
2.1.5.12. N-(4-Fluorobenzyl)-5-{2-{[2-(2-methoxyphenoxy)
ethyl]amino}-2-oxoethoxy}-1,2-dimethyl-1H-indole-3-
carboxamide (6l)
White solid; yield: 43.4%; M.p.: 187-189°C; IR: (KBr,
cm-1): υ 3453.5, 3238.6, 2912.8, 1681.0, 1627.1, 1506.3,
1485.9, 1384.4, 1252.9, 1222.8, 1126.3, 1026.5, 829,.6,
790.9, 736.4; ESI-MS, m/z: calcd. 519.22(M+); found
520.3([M+H]+), 542.2([M+Na]+); 1H NMR (400 MHz,
CDCl3): δ 7.35 (dd, J = 8.6, 5.4 Hz, 2H, 4-fluorobenzyl-H),
7.23 (d, J = 2.3 Hz, 1H, indole-H7), 7.19 (d, J = 8.9 Hz, 1H,
indole-H4), 7.03 (t, J = 8.7 Hz, 2H, 4-fluorobenzyl-H), 6.95
(dd, J = 3.8, 1.2 Hz, 1H, indole-H6), 6.89 – 6.83 (m, 4H, Ar–
H), 4.64 (d, J = 5.8 Hz, 2H, NHCH2), 4.53 (s, 2H, OCH2CO),
4.10 (t, J = 5.1 Hz, 2H, OCH2), 3.75 (s, 3H, NCH3), 3.74 –
3.70 (m, 2H, CH2), 3.66 (s, 3H, OCH3), 2.71 (s, 3H, indole-
CH3); 13C NMR (101 MHz, CDCl3): δ 169.67(C1’), 167.83
(C2’’), 160.94(C8), 160.57(C-F), 153.28(C5), 150.27(O-C-Ph),
150.21(O-C-Ph), 139.09(C2), 133.42(C-C6H4F), 129.45(CH-
C6H4F), 129.26(CH-C6H4F), 127.58(C9), 123.55(CH-Ph),
122.43(CH-Ph), 121.39(CH-Ph), 115.80(CH-C6H4F), 115.52
(CH-C6H4F), 112.20(C6), 111.17(C3), 110.36(C7), 103.81
(C4), 69.07(OCH2), 68.73(C1’’), 56.19(OCH3), 43.62(C2’),
40.21(NHCH2), 29.97(NCH3), 11.93(indole-CH3); HRMS
(ESI): calcd for C29H30FN3O5, [M]+ m/z, 519.2169; found,
520.2231([M+H]+), 542.2037([M+Na]+).
2.1.5.15. N-Benzyl-5-[2-(di-sec-butylamino)-2-oxoethoxy]-
1-ethyl-2-methyl-1H-indole-3-carboxamide (6o)
White solid; yield: 56.0%; M.p.: 101-103°C; IR: (KBr,
cm-1): υ 3423.1, 2959.1, 2920.8, 1656.7, 1621.6, 1543.7,
1484.8, 1422.4, 1384.8, 1209.4, 1143.3, 1091.9, 817.5,
744.8, 698.8; ESI-MS, m/z: calcd. 477.30(M+); found
478.4([M+H]+), 500.3([M+Na]+); 1H NMR (400 MHz,
CDCl3): δ 7.43 (d, J = 2.3 Hz, 1H, indole-H7), 7.38 – 7.25
(m, 5H, Ar–H), 7.20 (d, J = 8.9 Hz, 1H, indole-H4), 6.89 (dd,
J = 8.8, 2.3 Hz, 1H, indole-H6), 4.71 (d, J = 5.8 Hz, 2H,
NHCH2), 4.66 (s, 2H, OCH2), 4.12 (q, J = 7.1 Hz, 2H, in-
dole-NCH2), 3.21 – 3.14 (m, 2H, NCH), 2.72 (s, 3H, indole-
CH3), 2.02 – 1.67 (m, 4H, CH2), 1.33 (t, J = 7.1 Hz, 3H,
CH3), 0.92(d, J = 6.7 Hz, 6H, CH3), 0.79 (t, J = 6.7 Hz, 6H,
CH3); 13C NMR (101 MHz, CDCl3): δ 168.70(C1’), 166.35
(C2’’), 153.73(C5), 141.92(C2), 139.29(C-Ph), 128.82(C8),
128.55(CH-Ph), 128.28(CH-Ph), 127.94(C9), 127.40(CH-Ph),
126.86(CH-Ph), 125.99(CH-Ph), 111.06(C6), 110.28(C3),
107.57(C7), 103.82(C4), 68.58(C1’’), 54.47(NCH), 52.62
(NCH), 43.67(C2’), 38.00(NCH2), 27.45(CH2), 26.25(CH2),
20.24(CH3), 20.17(CH3), 15.07(CH3), 11.63(indole-CH3),
10.29(CH3), 10.21(CH3); HRMS (ESI): calcd for C29H39
N3O3, [M]+ m/z, 477.2991; found, 500.2862([M+Na]+).
2.1.5.13.
N-Benzyl-5-[2-(diethylamino)-2-oxoethoxy]-1-
ethyl-2-methyl-1H-indole-3-carboxamide (6m)
White solid; yield: 45.9%; M.p.: 105-106°C; IR: (KBr,
cm-1): υ 3434.3, 2925.2, 1661.7, 1617.5, 1530.2, 1478.3,
1347.7, 1259.2, 1189.2, 1139.2, 1082.9, 835.4, 756.0, 704.4;
ESI-MS, m/z: calcd. 421.24(M+); found 422.3([M+H]+),
444.3([M+Na]+); 1H NMR (400 MHz, CDCl3): δ 7.43 (d, J =
2.3 Hz, 1H, indole-H7), 7.36 – 7.27 (m, 5H, Ar–H), 7.21 (d, J
= 8.9 Hz, 1H, indole-H4), 6.90 (dd, J = 8.9, 2.4 Hz, 1H, in-
dole-H6), 4.71 (d, J = 5.7 Hz, 2H, NHCH2), 4.65 (s, 2H,

76 Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1
Zhang et al.
2.1.5.16. N-Benzyl-1-ethyl-2-methyl-5-[2-oxo-2-(piperidin-
1-yl)ethoxy]-1H-indole-3-carboxamide (6p)
128.60(CH-Ph), 127.87(C9), 127.58(CH-Ph), 126.62(CH-
Ph), 126.13(CH-Ph), 122.51(CH-Ph), 121.17(CH-Ph), 120.
49(CH-Ph), 112.22(C6), 111.11(C3), 110.48(C7), 103.94(C4),
69.03(OCH2), 68.87(C1’’), 55.93(OCH3), 43.67(C2’), 38.63
(NCH2), 38.08(NCH2), 15.10(CH3), 11.63(indole-CH3);
HRMS (ESI): calcd for C30H33N3O5, [M]+ m/z, 515.2420;
found, 516.2483([M+H]+), 538.2296([M+Na]+).
White solid; yield: 57.4%; M.p.: 89-90°C; IR: (KBr, cm-1):
υ 3551.3, 3404.6, 3336.9, 1646.8, 1613.0, 1546.0, 1488.7,
1419.9, 1384.5, 1214.2, 1164.4, 1977.0, 984.4, 850.0, 806.9,
727.7, 695.2; ESI-MS, m/z: calcd. 433.24(M+); found
1
434.7([M+H]+); H NMR (400 MHz, CDCl3): δ 7.43 (d, J =
2.3 Hz, 1H, indole-H7), 7.38 – 7.25 (m, 5H, Ar–H), 7.21 (d, J
= 8.9 Hz, 1H, indole-H4), 6.90 (dd, J = 8.8, 2.4 Hz, 1H, in-
dole-H6), 4.71 (d, J = 5.7 Hz, 2H, NHCH2), 4.66 (s, 2H,
OCH2), 4.12 (q, J = 7.2 Hz, 2H, indole-NCH2), 3.52 (t, J =
5.3 Hz, 2H, NCH2), 3.44 (t, J = 5.0 Hz, 2H, NCH2), 2.71 (s,
3H, indole-CH3), 1.66 – 1.48 (m, 6H, CH2), 1.33 (t, J = 7.2
Hz, 3H, CH3); 13C NMR (101 MHz, CDCl3): δ 166.70(C1’),
166.33(C2’’), 153.68(C5), 141.81(C2), 139.30(C-Ph), 128.81
(C8), 128.67(CH-Ph), 128.43(CH-Ph), 127.96(C9), 127.41
(CH-Ph), 127.33(CH-Ph), 126.02(CH-Ph), 111.14(C6), 110.36
(C3), 107.65(C7), 103.47(C4), 68.75(C1’’), 46.56(NCH2),
43.69(NCH2), 43.29(C2’), 38.02(NCH2), 26.62(CH2),
25.67(CH2), 24.60(CH2), 15.10(CH3), 11.63(indole-CH3);
HRMS (ESI): calcd for C26H31N3O3, [M]+ m/z, 433.2365;
found, 434.2433([M+H]+), 456.2243([M+Na]+).
2.1.5.19. 5-[2-(Diethylamino)-2-oxoethoxy]-1-ethyl-N-(4-
fluorobenzyl)-2-methyl-1H-indole-3-carboxamide (6s)
White solid; yield: 55.2%; M.p.: 135-137°C; IR: (KBr,
cm-1): υ 3423.8, 3278.3, 2925.4, 1646.1, 1625.0, 1510.1,
1482.6, 1383.4, 1263.2, 1207.6, 1155.5, 1070.6, 822.5,
791.0, 706.7; ESI-MS, m/z: calcd. 439.23(M+); found
440.4([M+H]+); 1H NMR (400 MHz, CDCl3): δ 7.40 (dd, J =
8.5, 5.5 Hz, 2H, Ar–H), 7.29 (d, J = 2.3 Hz, 1H, indole-H7),
7.21 (d, J = 8.9 Hz, 1H, indole-H4), 7.02 (t, J = 8.7 Hz, 2H,
Ar–H), 6.89 (dd, J = 8.8, 2.3 Hz, 1H, indole-H6), 4.67 (s, 2H,
OCH2), 4.65 (d, J = 5.8 Hz, 2H, NHCH2), 4.11 (q, J = 7.2
Hz, 2H, indole-NCH2), 3.38 (q, J = 7.1 Hz, 4H, NCH2), 2.71
(s, 3H, indole-CH3), 1.32 (t, J = 7.2 Hz, 3H, CH3), 1.17 (t, J
= 7.1 Hz, 3H, CH3), 1.10 (t, J = 7.1 Hz, 3H, CH3); 13C NMR
(101 MHz, CDCl3): δ 167.58(C1’), 166.31(C2’’), 160.93(C-F),
153.79(C5), 142.03(C2), 135.16(C-C6H4F), 129.65(C8),
129.57(CH-C6H4F), 129.31(CH-C6H4F), 125.90(C9), 115.65
(CH-C6H4F), 115.43(CH-C6H4F), 111.14(C6), 110.37(C3),
107.47(C7), 103.57(C4), 68.72(C1’’), 42.89(C2’), 41.67(NCH2),
40.38(NCH2), 38.00(NCH2), 15.07(CH3), 14.43(CH3), 12.93
(CH3), 11.61(indole-CH3); HRMS (ESI): calcd for C25H30
FN3O3, [M]+ m/z, 439.2271; found, 440.2333([M+H]+),
462.2142([M+Na]+).
2.1.5.17. N-Benzyl-1-ethyl-2-methyl-5-[2-(morpholin-4-yl)-
2-oxoethoxy]-1H-indole-3-carboxamide (6q)
Yellow solid; yield: 77.7%; M.p.: 161-162°C; IR: (KBr,
cm-1): υ 3338.5, 2969.3, 2931.8, 1663.5, 1546.4, 1481.6,
1440.8, 1279.5, 1196.4, 1161.6, 1112.1, 1035.4, 970.1,
851.3, 758.6, 707.8; ESI-MS, m/z: calcd. 435.22(M+); found
1
436.7([M+H]+); H NMR (400 MHz, CDCl3): δ 7.44 (d, J =
2.3 Hz, 1H, indole-H7), 7.38 – 7.27 (m, 5H, Ar–H), 7.22 (d, J
= 8.8 Hz, 1H, indole-H4), 6.88 (dd, J = 8.8, 2.3 Hz, 1H, in-
dole-H6), 4.71 (d, J = 5.4 Hz, 2H, NHCH2), 4.67 (s, 2H,
OCH2), 4.16 (q, J = 7.1 Hz, 2H, indole-NCH2), 3.64 – 3.52
(m, 8H, CH2), 2.72 (s, 3H, indole-CH3), 1.34 (t, J = 7.2 Hz,
3H, CH3); 13C NMR (101 MHz, CDCl3): δ 167.04(C1’),
166.24(C2’’), 153.34(C5), 141.80(C2), 139.27(C-Ph), 128.85
(C8), 128.70(CH-Ph), 127.99(CH-Ph), 127.47(C9), 127.14
(CH-Ph), 126.97(CH-Ph), 126.11(CH-Ph), 111.05(C6), 110.47
(C3), 107.75(C7), 103.43(C4), 68.67(C1’’), 66.95(OCH2),
66.92(OCH2), 46.07(NCH2), 43.76(NCH2), 42.52(C2’),
38.06(NCH2), 15.11(CH3), 11.65(indole-CH3); HRMS (ESI):
calcd for C25H29N3O4, [M]+ m/z, 435.2158; found,
436.2227([M+H]+), 458.2037([M+Na]+).
2.1.5.20. 5-[2-(Dipropylamino)-2-oxoethoxy]-1-ethyl-N-(4-
fluorobenzyl)-2-methyl-1H-indole-3-carboxamide (6t)
Yellow solid; Yield: 46.9%; M.p.: 120-122°C; IR: (KBr,
cm-1): υ 3304.4, 2963.1, 2931.9, 1658.7, 1619.4, 1510.7,
1483.0, 1423.2, 1272.9, 1207.6, 1158.4, 1091.3, 835.3,
794.0, 706.7; ESI-MS, m/z: calcd. 467.26(M+); found
468.4([M+H]+), 490.4([M+Na]+); 1H NMR (400 MHz,
CDCl3): δ 7.40 (dd, J = 8.5, 5.4 Hz, 2H, Ar–H), 7.29 (d, J =
2.3 Hz, 1H, indole-H7), 7.21 (d, J = 8.9 Hz, 1H, indole-H4),
7.02 (t, J = 8.7 Hz, 2H, Ar–H), 6.88 (dd, J = 8.8, 2.4 Hz, 1H,
indole-H6), 4.69 (s, 2H, OCH2), 4.67 (d, J = 5.8 Hz, 2H,
NHCH2), 4.12 (q, J = 7.2 Hz, 2H, indole-NCH2), 3.32 – 3.21
(m, 4H, NCH2), 2.72 (s, 3H, indole-CH3), 1.59 – 1.49 (m,
4H, CH2), 1.33 (t, J = 7.2 Hz, 3H, CH3), 0.90 (t, J = 7.4 Hz,
3H, CH3), 0.83 (t, J = 7.4 Hz, 3H, CH3); 13C NMR (101
MHz, CDCl3): δ 168.03(C1’), 166.33(C2’’), 160.96(C-F),
153.81(C5), 142.16(C2), 135.18(C-C6H4F), 129.64(C8),
129.56(CH-C6H4F), 129.39(CH-C6H4F), 125.91(C9), 115.68
(CH-C6H4F), 115.47(CH-C6H4F), 111.03(C6), 110.37(C3),
107.43(C7), 103.67(C4), 68.71(C1’’), 49.08(NCH2), 47.63
(NCH2), 42.91(C2’), 38.03(NCH2), 22.29(CH2), 20.83(CH2),
15.09(CH3), 11.63(indole-CH3), 11.46(CH3), 11.38(CH3);
HRMS (ESI): calcd for C27H34FN3O3, [M]+ m/z, 467.2584;
found, 468.2652([M+H]+), 490.2463([M+Na]+).
2.1.5.18.
N-Benzyl-1-ethyl-5-{2-{[2-(2-methoxyphenoxy)
ethyl]amino}-2-oxoethoxy}-2-methyl-1H-indole-3-
carboxamide (6r)
White solid; yield: 86.7%; M.p.: 157-159°C; IR: (KBr,
cm-1): υ 3455.6, 2922.3, 1686.6, 1619.2, 1508.6, 1482.9,
1384.0, 1253.7, 1209.9, 1127.8, 1027.1, 975.5, 745.5, 698.1;
ESI-MS, m/z: calcd. 515.24(M+); found 516.3([M+H]+),
538.2([M+Na]+); 1H NMR (400 MHz, CDCl3): δ 7.40 (d, J =
2.4 Hz, 1H, indole-H7), 7.38 – 7.27 (m, 5H, benzyl-H), 7.23
(d, J = 8.8 Hz, 1H, indole-H4), 6.89 – 6.84 (m, 5H, Ar–H),
4.68 (d, J = 5.7 Hz, 2H, NHCH2), 4.50 (s, 2H, OCH2), 4.14 –
4.09 (m, 4H, CH2), 3.76 (s, 3H, OCH3), 3.73 (t, J = 5.3 Hz,
2H, CH2), 2.72 (s, 3H, indole-CH3), 1.34 (t, J = 7.2 Hz, 3H,
CH3); 13C NMR (101 MHz, CDCl3): δ 168.95(C1’),
166.22(C2’’), 153.06(C5), 150.23(O-C-Ph), 147.92(O-C-Ph),
141.92(C2), 139.14(C-Ph), 128.94(C8), 128.60(CH-Ph),
2.1.5.21. 5-[2-(Di-sec-butylamino)-2-oxoethoxy]-1-ethyl-N-
(4-fluorobenzyl)-2-methyl-1H-indole-3-carboxamide (6u)
Yellow solid; yield: 50.7%; M.p.: 112-114°C; IR: (KBr,
cm-1): υ 3444.7, 3295.3, 2960.1, 2921.4, 1665.1, 1631.3,

Design, Synthesis and Biological Evaluation of a Novel Series
Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1 77
1509.6, 1483.6, 1384.2, 1213.4, 1151.4, 1090.2, 842.8,
821.8, 796.1, 749.7, 691.1; ESI-MS, m/z: calcd. 495.29(M+);
found 496.3([M+H]+), 518.3([M+Na]+); 1H NMR (400 MHz,
CDCl3): δ 7.40 (dd, J = 8.6, 5.4 Hz, 2H, Ar–H), 7.30 (d, J =
2.3 Hz, 1H, indole-H7), 7.20 (d, J = 8.9 Hz, 1H, indole-H4),
7.02 (t, J = 8.7 Hz, 2H, Ar–H), 6.88 (dd, J = 8.8, 2.4 Hz, 1H,
indole-H6), 4.73 (s, 2H, OCH2), 4.66 (d, J = 5.9 Hz, 2H,
NHCH2), 4.12 (q, J = 7.2 Hz, 2H, indole-NCH2), 3.20 – 3.15
(m, 2H, NCH), 2.71 (s, 3H, indole-CH3), 2.08 – 1.99 (m, 4H,
CH2), 1.19 (t, J = 7.1 Hz, 3H, CH3), 0.93 (d, J = 6.8 Hz, 6H,
CH3), 0.76 (t, J = 6.7 Hz, 6H, CH3); 13C NMR (101 MHz,
CDCl3): δ 168.77(C1’), 166.32(C2’’), 160.95(C-F), 153.73
(C5), 142.14(C2), 135.18(C-C6H4F), 129.63(C8), 129.55(CH-
C6H4F), 129.03(CH-C6H4F), 125.91(C9), 115.67(CH-C6H4F),
115.46(CH-C6H4F), 110.98(C6), 110.31(C3), 107.40(C7),
103.86(C4), 68.66(C1’’), 54.43(NCH), 52.60(NCH), 42.89
(C2’), 38.00(NCH2), 27.42(CH2), 26.22(CH2), 20.22(CH3),
20.14(CH3), 15.06(CH3), 11.62(indole-CH3), 10.13(CH3),
9.89(CH3); HRMS (ESI): calcd for C29H38FN3O3, [M]+ m/z,
495.2897; found, 496.2965([M+H]+), 518.2773([M+Na]+).
129.66(CH-C6H4F), 128.64(CH-C6H4F), 126.03(C9), 115.74
(CH-C6H4F), 115.52(CH-C6H4F), 111.06(C6), 110.54(C3),
109.37(C7), 103.42(C4), 68.75(C1’’), 66.95(OCH2), 66.89
(OCH2), 46.48(NCH2), 46.08(NCH2), 42.99(C2’), 42.54
(NCH2), 15.12(CH3), 11.67(indole-CH3); HRMS (ESI):
calcd for C25H28FN3O4, [M]+ m/z, 453.2064; found,
454.2134([M+H]+), 476.1944([M+Na]+).
2.1.5.24. 1-Ethyl-N-(4-fluorobenzyl)-5-{2-{[2-(2-methoxy-
phenoxy)ethyl]amino}-2-oxoethoxy}-2-methyl-1H-indole-3-
carboxamide (6x)
White solid; yield: 64.9%; M.p.: 166-168°C; IR: (KBr,
cm-1): υ 3268.1, 1673.3, 1622.3, 1507.4, 1482.0, 1254.0,
1220.4, 1125.7, 1026.3, 831.0, 739,8, 720.1; ESI-MS, m/z:
calcd. 533.23(M+); found 534.2([M+H]+), 556.2([M+Na]+);
1H NMR (400 MHz, CDCl3): δ 7.35 (dd, J = 8.6, 5.4 Hz, 2H,
4-fluorobenzyl-H), 7.23 (d, J = 2.3 Hz, 1H, indole-H7), 7.21
(d, J = 8.9 Hz, 1H, indole-H4), 7.03 (t, J = 8.7 Hz, 2H, 4-
fluorobenzyl-H), 6.94 (dd, J = 8.9, 2.3 Hz, 1H, NHCH2),
6.89 – 6.82 (m, 4H, Ar–H), 4.64 (d, J = 5.8 Hz, 2H,
NHCH2), 4.53 (s, 2H, OCH2), 4.14 – 4.10 (m, 4H, CH2),
3.76 (s, 3H, OCH3), 3.74 – 3.70 (m, 2H, CH2), 2.71 (s, 3H,
indole-CH3), 1.34 (t, J = 7.2 Hz, 3H, CH3); 13C NMR (101
MHz, CDCl3): δ 169.02(C1’), 166.26(C2’’), 161.04(C-F),
153.13(C5), 150.22(O-C-Ph), 147.86(O-C-Ph), 142.17(C2),
135.00(C-C6H4F), 129.51(C8), 129.43(CH-C6H4F), 128.66
(CH-C6H4F), 126.07(C9), 122.58(CH-Ph), 121.19(CH-Ph),
115.81(CH-C6H4F), 115.71(CH-C6H4F), 112.21(CH-Ph),
111.11(C6), 110.53(C3), 110.28(CH-Ph), 107.70(C7), 104.03
(C4), 69.06(OCH2), 68.99(C1’’), 55.97(OCH3), 42.97(C2’),
42.81(NCH2), 38.63(NHCH2), 15.09(CH3), 11.62(indole-
CH3); HRMS (ESI): calcd for C30H32FN3O5, [M]+ m/z,
533.2326; found, 534.2394([M+H]+), 556.2205([M+Na]+).
2.1.5.22. 1-Ethyl-N-(4-fluorobenzyl)-2-methyl-5-[2-oxo-2-
(piperidin-1-yl)ethoxy]-1H-indole-3-carboxamide (6v)
Yellow solid; yield: 42.9%; M.p.: 126-127°C; IR: (KBr,
cm-1): υ 3513.3, 3308.9, 2918.1, 2849.7, 1655.9, 1608.0,
1544.5, 1510.5, 1439.8, 1231.5, 1210.6, 1160.6, 1087.6,
837.8, 809.2, 771.2, 689.3; ESI-MS, m/z: calcd. 451.23(M+);
found 452.2([M+H]+), 474.3([M+Na]+); 1H NMR (400 MHz,
CDCl3): δ 7.41 (dd, J = 8.4, 5.5 Hz, 2H, Ar–H), 7.28 (d, J =
2.3 Hz, 1H, indole-H7), 7.21 (d, J = 8.8 Hz, 1H, indole-H4),
7.02 (t, J = 8.7 Hz, 2H, Ar–H), 6.89 (dd, J = 8.8, 2.3 Hz, 1H,
indole-H6), 4.68 (s, 2H, OCH2), 4.66 (d, J = 5.9 Hz, 2H,
NHCH2), 4.12 (q, J = 7.2 Hz, 2H, indole-NCH2), 3.52 (t, J =
5.1 Hz, 2H, NCH2), 3.44 (t, J = 5.0 Hz, 2H, NCH2), 2.71 (s,
3H, indole-CH3), 1.67 – 1.52 (m, 6H, CH2), 1.33 (t, J = 7.2
Hz, 3H, CH3); 13C NMR (101 MHz, CDCl3): δ 166.73(C1’),
166.33(C2’’), 161.00(C-F), 153.72(C5), 142.08(C2), 135.16
(C-C6H4F), 129.68(C8), 129.60(CH-C6H4F), 129.13(CH-
C6H4F), 125.94(C9), 115.67(CH-C6H4F), 115.46(CH-C6H4F),
111.12(C6), 110.42(C3), 107.44(C7), 103.53(C4), 68.82(C1’’),
46.56(NCH2), 46.41(NCH2), 43.25(C2’), 42.94(NCH2), 26.61
(CH2), 25.67(CH2), 24.58(CH2), 15.09(CH3), 11.64(indole-
CH3); HRMS (ESI): calcd for C26H30FN3O3, [M]+ m/z,
451.2271; found, 452.2334([M+H]+), 474.2146([M+Na]+).
2.2. Biological Assay
For observing the antitumor activity of the target com-
pounds in vitro, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) assay was used. A549 cells, HeLa
cells, SW480 cells, HepG2 cells and HL7702 cells were har-
vested in the logarithmic growth phase and seeded in 96-well
plates at a density of 8000 cells per well, and cultured at
37oC in humidified atmosphere containing 5% CO2 in Dul-
becco’s modified Eagle’s medium (DMEM or RPMI-1640)
with 10% fetal bovine serum (FBS) for 24 h before any
treatments. Tested compounds were dissolved in DMSO and
diluted in the culture fluid to get various concentrations. The
cells were treated with target compounds subsequently and
incubated overnight. Then 20 μL of MTT (5 mg/mL) was
added in each well and after 4 h incubation, the medium was
removed immediately and MTT formazan was solubilized in
150 μL of DMSO. The optical densities (OD) were measured
with a microplate reader (Bio-Tek instruments, INC. USA)
at 490 nm. Inhibitory effects were determined as IC50 value.
2.1.5.23. 1-Ethyl-N-(4-fluorobenzyl)-2-methyl-5-[2-(morpholin-
4-yl)-2-oxoethoxy]-1H-indole-3-carboxamide (6w)
White solid; yield: 79.3%; M.p.: 151-153°C; IR: (KBr,
cm-1): υ 3369.5, 2966.9, 2851.8, 1669.9, 1620.5, 1510.2,
1481.1, 1352.4, 1188.6, 1112.9, 1028.0, 841.8, 777.3, 707.4;
ESI-MS, m/z: calcd. 453.21(M+); found 454.2([M+H]+),
1
476.3([M+Na]+); H NMR (400 MHz, CDCl3): δ 7.42 (dd, J
= 8.7, 5.4 Hz, 2H, Ar–H), 7.29 (d, J = 2.4 Hz, 1H, indole-
H7), 7.23 (d, J = 8.9 Hz, 1H, indole-H4), 7.04 (t, J = 8.7 Hz,
2H, Ar–H), 6.88 (dd, J = 8.8, 2.4 Hz, 1H, indole-H6), 4.70 (s,
2H, OCH2), 4.67 (d, J = 5.7 Hz, 2H, NHCH2), 4.13 (q, J =
7.2 Hz, 2H, indole-NCH2), 3.63 – 3.59 (m, 8H, CH2), 2.72
(s, 3H, indole-CH3), 1.34 (t, J = 7.2 Hz, 3H, CH3); 13C NMR
(101 MHz, CDCl3): δ 167.14(C1’), 166.24(C2’’), 159.20(C-F),
153.36(C5), 142.03(C2), 135.14(C-C6H4F), 129.68(C8),
3. RESULTS AND DISCUSSION
3.1. Chemistry
As depicted in Scheme 1, a novel series of indole-3-
carboxamide derivatives were synthesized starting from the
commercially available ethyl acetoacetate (1). Reaction of

78 Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1
Zhang et al.
Scheme 1. Synthetic route of the target indole-3-carboxamide derivatives. Reagents and conditions: (a) R1NH2, rt, 3 h; (b) benzoquinone,
acetone, 30oC, 2 h; (c) NaOH, H2O, ethanol, reflux, 12 h; (d) EDCI, HOBt, triethylamine, DMF, CH2Cl2, rt, 24 h; (e) ClCH2CONR2R3,
K2CO3, KI, DMF, 80oC, 16 h.
ethyl acetoacetate with methylamine or ethylamine at ambi-
ent temperature afforded compound 2a or 2b, which was
converted to indole-3-carboxylate 3a or 3b via Nenitzescu
reaction with benzoquinone. Hydrolysis of compound 3a or
3b using sodium hydroxide in ethanol-aqueous solution gave
the acid 4a or 4b. Treatment of 4a or 4b with benzylamine
or 4-fluorobenzylamine in the presence of EDCI and HOBt
provided key intermediates 5a-5d. Reaction of 5a-5d with
corresponding 2-chloroacetamide derivatives by using anhy-
drous K2CO3 and KI gave indole-3-carboxamide derivatives
6a-6x, respectively. The structures of the target compounds
were characterized by IR, ESI-MS, 1H NMR and 13C NMR.
effect on the activity. However, the target compounds con-
taining [2-(2-methoxyphenoxy)ethyl]amino exhibited strong
cytotoxic activities. These results revealed that the introduc-
tion of a large aryl group was advantageous for anticancer
activity. (4) The target compounds 6c, 6i, 6j and 6u showed
excellent selective activity for HeLa cells over the other
tested cancer cells. (5) The target compound 6x (IC50 = 4.99,
6.81 and 13.07 μmol/L, respectively) exhibited strong cyto-
toxic activities against A549, HeLa and SW480 (6). In addi-
tion, all target compounds exhibited weak cytotoxic effects
on human liver normal cells HL7702.
3.3. Molecular Docking Study
3.2. Evaluation of the Bioactivity
Molecular docking studies were carried out to explore the
binding modes of the target compounds with the active site
of EGFR. Based on the in vitro cytotoxicity results, the tar-
get compound 6a, the moderate EGFR inhibitor and the tar-
get compound 6x, the best inhibitor in the study was selected
as the ligand example. The 3D structure of EGFR was built
as the docking model (PDB ID code: 1M17[21]).
Using erlotinib as control, the synthesized target com-
pounds 6a-6x were evaluated for the cytotoxic activities in
vitro against five cell lines (A549, HeLa, SW480, HepG2
and HL7702) by MTT colorimetric assay. The results ex-
pressed as IC50 values are summarized in Table 1.
The SAR analysis revealed as follows: (1) Compared to
the target compound 6f, the target compound 6r exhibited
potent cytotoxic activities against A549 and SW480 cells.
These results suggest that there was no significant difference
between methyl and ethyl in the 1-position of indole-3-
carboxamide core structure; (2) The target compound 6x
exhibited better activity than the target compound 6r against
EGFR high-expressed cancer cells. These results show that
the introduction of 4-fluorobenzylamine in 3-position was
beneficial for anticancer activity than that of benzylamine,
meanwhile also implying that the presence of fluorine atom
on benzyl group was appropriate to increase the activity. (3)
By comparison, the target compounds 6a-6f showed variable
activity. In general, the introduction of 2-(morpholin-4-yl)-2-
oxoethoxy at the 5-position resulted in weak activity. Fur-
thermore, the presence of 2-(dipropylamino)-2-oxoethoxy
and 2-(di-sec-butylamino)-2-oxoethoxy at the 5-position
resulted in a decrease or loss of the activity. In contrast, the
presence of 2-(diethylamino)-2-oxoethoxy and 2-oxo-2-
(piperidin-1-yl)ethoxy played no significant effect or slight
The binding modes of lead compound N-(3-
bromophenyl)-6-methoxy-9H-pyrimido[4,5-b]indol-4-amine
and the target compound 6a are shown in Fig. (3). As de-
picted in Fig. (3), the lead compound and the target com-
pound 6a could pretty nearly overlap in the binding model. It
is demonstrated that the docking mode of the target com-
pound 6a was similar to the lead compound by analysis of
the binding mode of the target compound 6a in the active
binding site.
The most active compound 6x was further docked into
the binding site of EGFR and the theoretical binding mode is
shown in Fig. (4). As shown in Fig. (4), the fluorine atom of
6x formed a hydrogen bond with residue Thr766 and the 5-
acetoxyl group of 6x formed a hydrogen bond with residue
Ala698. The binding modes also showed that 3-carboxamide
and indole part formed weak hydrophobic interactions with
residues Leu768, Ala719, Lys721, Val702 and Leu694. The
molecular docking study and above-mentioned results of

Design, Synthesis and Biological Evaluation of a Novel Series
Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1 79
Table 1. In vitro cytotoxicity of the target compounds.
IC50(μM)
Compd.
A549
HeLa
SW480
HepG2
HL7702
O
O
O
N
H
N
39.18
23.97
62.01
>100
>100
N
6a
6b
6c
6d
O
O
O
N
N
H
>100
>100
38.47
>100
6.34
>100
16.17
22.72
>100
78.22
47.67
>100
1.66
>100
>100
48.29
31.38
10.57
>100
73.35
57.15
>100
>100
>100
>100
>100
86.10
67.83
>100
97.63
>100
>100
>100
>100
>100
>100
N
O
O
O
N
H
N
N
O
O
O
N
N
N
H
N
O
O
O
O
N
H
O
N
6e
6f
O
O
O
N
H
N
H
OCH3
N
O
O
N
O
N
F
H
22.69
>100
>100
N
6g
O
O
N
O
N
F
F
H
N
6h
6i
O
O
N
O
N
H
>100
N
(Table 1) contd….

80 Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1
Zhang et al.
IC50(μM)
Compd.
A549
HeLa
SW480
HepG2
HL7702
O
O
O
O
O
N
H
F
N
N
54.77
9.15
>100
>100
>100
>100
>100
N
6j
O
O
N
H
F
>100
7.40
>100
4.67
>100
27.47
>100
>100
O
N
6k
O
O
O
N
H
F
N
H
OCH3
N
6l
6m
6n
O
O
O
N
H
N
>100
66.75
58.02
>100
62.78
24.32
52.33
57.66
>100
>100
>100
>100
>100
>100
>100
N
O
O
O
N
N
H
N
O
O
O
N
H
N
N
6o
6p
6q
6r
O
O
O
N
N
H
>100
>100
5.35
>100
>100
65.74
>100
>100
11.59
>100
>100
>100
>100
83.27
>100
N
O
O
O
N
H
N
O
N
O
O
O
O
N
N
H
H
OCH3
N
(Table 1) contd….

Design, Synthesis and Biological Evaluation of a Novel Series
Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1 81
IC50(μM)
Compd.
A549
HeLa
SW480
HepG2
HL7702
O
O
O
N
H
F
N
>100
64.43
>100
>100
>100
N
6s
O
O
O
N
F
N
H
>100
54.27
42.29
>100
17.25
9.77
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
N
N
6t
O
O
O
N
H
F
N
6u
O
O
O
N
F
N
N
H
61.47
>100
N
6v
O
O
O
O
N
H
F
O
N
6w
O
O
O
N
H
F
N
H
4.99
6.81
13.07
16.72
>100
>100
OCH3
N
6x
（-）
（-）
Erlotinib
13.41
15.34
cancer cell lines and weak activity against the EGFR low-
expressed cancer cell lines and human liver normal cells,
which was a supporting evidence for the target compounds
as EGFR inhibitors with low toxicity. The target compounds
6c, 6f, 6i, 6j, 6l, 6r, 6u and 6x demonstrated an excellent
strong anticancer activities (6x showed equal activity to er-
lotinib). What’s more, a new EGFR inhibitor scaffold and a
preliminary discussion on their SARs provide promising
opportunities to guide further research on indole-3-
carboxamide derivatives as novel anticancer agents.
SARs analysis could contribute to the rational design of po-
tential EGFR inhibitors.
CONCLUSION
In summary, a novel series of indole-3-carboxamide de-
rivatives were designed and synthesized. All of the target
compounds were evaluated against four cancer cell lines and
one human liver normal cell lines. Some target compounds
showed moderate to strong activity as compared to the posi-
tive control erlotinib against the EGFR highly-expressed

82 Letters in Drug Design & Discovery, 2018, Vol. 15, No. 1
Zhang et al.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENTS
This work was supported by the National Science Foun-
dation of China (NFSC) for the grant No. 21342006. The
authors would like to thank Molegro ApS for kindly provid-
ing a free evaluation copy of their software package.
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