Design, synthesis, and biological evaluation of sorafenib derivatives containing indole (ketone) semicarbazide analogs as antitumor agents

Article abstract of DOI:10.1002/jhet.3972

A series of new sorafenib derivatives was designed and synthesized. The antiproliferative activity of the synthesized compounds against human lung cancer cell (A549), human pancreatic cancer cell (PC-3), human leukemia cell (K562), and human hepatoma cell (SMMC-7721) was evaluated by MTT assay. The results revealed that several compounds displayed more significant antitumor activities than commercial anticancer agent sorafenib against SMMC-7721. In addition, compounds 7a, 7g, 7l, 7m, and 7p represented obvious growth inhibition with IC₅₀ values of 1-9 μM against four cancer cell lines, demonstrating more predominant activities against cancer cells as compared to sorafenib. Furthermore, some structure-activity relationships have also been established. Compounds containing indole and benzene ring substituted by halogen showed better activity than sorafenib. Wound healing assay suggested that cells would be targeted on their migratory capacity by 7g, potentially affecting the migration activity of these tumors. The effects of A549 and PC-3 cell apoptosis induced by compound 7g were significantly increased compared with sorafenib. Importantly, the result of western blot assay showed that 7g inhibited cell growth by suppressing the activity of EGFR, especially the expression of p-EGFR (Tyr1068).

Full text of DOI:10.1002/jhet.3972

Received: 19 December 2019
DOI: 10.1002/jhet.3972
Revised: 2 March 2020
Accepted: 9 March 2020
A R T I C L E
Design, synthesis, and biological evaluation of sorafenib
derivatives containing indole (ketone) semicarbazide
analogs as antitumor agents
Wen Li1 | Ya-Yun Qi1 | Yuan-Yuan Wang1 | Yi-Yuan Gan1 | Li-Hui Shao1
|
Li-Qiong Zhang1 | Zhen-Hua Tang1 | Mei Zhu1 | Si-Yu Tang1
|
Zhen-Chao Wang1
| Gui-Ping Ouyang1,2,3
1School of Pharmaceutical Sciences,
Guizhou University, Guiyang, People's
Republic of China
2State Key Laboratory of Functions and
Applications of Medicinal Plants, Guizhou
Medical University, Guiyang, People's
Republic of China
3Guizhou Engineering Laboratory for
Synthetic Drugs, Guiyang, People's
Republic of China
Abstract
A series of new sorafenib derivatives was designed and synthesized. The antip-
roliferative activity of the synthesized compounds against human lung cancer
cell (A549), human pancreatic cancer cell (PC-3), human leukemia cell (K562),
and human hepatoma cell (SMMC-7721) was evaluated by MTT assay. The
results revealed that several compounds displayed more significant antitumor
activities than commercial anticancer agent sorafenib against SMMC-7721. In
addition, compounds 7a, 7g, 7l, 7m, and 7p represented obvious growth inhi-
bition with IC50 values of 1-9 μM against four cancer cell lines, demonstrating
more predominant activities against cancer cells as compared to sorafenib.
Furthermore, some structure-activity relationships have also been established.
Compounds containing indole and benzene ring substituted by halogen
showed better activity than sorafenib. Wound healing assay suggested that
cells would be targeted on their migratory capacity by 7g, potentially affecting
the migration activity of these tumors. The effects of A549 and PC-3 cell apo-
ptosis induced by compound 7g were significantly increased compared with
sorafenib. Importantly, the result of western blot assay showed that 7g
inhibited cell growth by suppressing the activity of EGFR, especially the
expression of p-EGFR (Tyr1068).
Correspondence
Zhen-Chao Wang and Gui-Ping Ouyang,
School of Pharmaceutical Sciences,
Guizhou University, Huaxi District,
Guiyang, Guizhou Province, 550025,
People's Republic of China.
Email: wzc.4884@163.com (Z.-C. W) and
oygp710@163.com (G.-P. O.)
Funding information
the National Natural Science Foundation
of China, Grant/Award Number:
21867004; the project of State Key
Laboratory of Functions and Applications
of Medicinal Plants, Guizhou Medcial
University, Grant/Award Number:
FAMP201707K; the Science Technology
Program of Guizhou province, Grant/
Award Number: 20185781
1 | INTRODUCTION
Recently, sorafenib was proved to be effective for the treat-
ment of non-small cell lung cancer, breast cancer, and thy-
Sorafenib, a diaryl urea multiple-targeted antitumor agent,
jointly developed by Bayer and Onyx, US, was proved to
inhibit kinases including Raf, VEGFR, PDGFR, and KIT
involved in tumor proliferation and angiogenesis.[1,2]
roid cancer.[3–5] At present, there are three ways by which
sorafenib fights against cancer. First, sorafenib blocks can-
cer cell proliferation by inhibiting BRaf and Raf 1/c-Raf
kinase phosphorylation in the protein kinase pathway.
Second, sorafenib prevents tumor-associated angiogenesis
by inactivating vascular endothelial growth factor
Wen Li and Ya-Yun Qi contributed equally to this work.
J Heterocyclic Chem. 2020;1–13.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals, Inc.
1

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LI ET AL.
receptors (VEGFR), the platelet-derived growth factor
receptor-β (PDGFR-β) and stem cell growth factor receptor
(c-kit). Third, sorafenib induces tumor cell apoptosis by
reducing elF4E phosphorylation and downregulating
Mcl-1 levels.[6–14] Sorafenib has been proven to be of broad
spectrum and well tolerated, but due to the various side
effects of sorafenib, it is necessary to explore for new anti-
tumor drugs with high efficiency and low toxicity.
superior antitumor activity. It is known that indole is versa-
tile and privileged in drug discovery,[15–18] such as the
nintedanib for the treatment of idiopathic pulmonary fibrosis
(IPF),[19] the vinorelbine for the treatment of non-small cell
lung cancer and breast cancer,[20] acemetacin for the treat-
ment of rheumatoid arthritis,[21] and sunitinib for the treat-
ment of renal cancer and gastrointestinal stromal tumors
(Figure 1).[22] A series of indole vinyl sulfones compounds
were reported by Li in 2019; the bioassay showed that com-
pound A presented potent activity against HepG2, A549, and
K562 (Figure 1).[23] Especially, derivatives bearing amino
and ureide moiety based on indole scaffold usually have
noteworthy antitumor activity, for example, compound B
(Figure 1) was reported by Eldehna in 2019, which emerged
as the most potent multikinase inhibitor in the ureido ana-
logues series with VEGFR-2, FGFR-1, and PDGFR-b IC50 of
0.18, 0.23, and 0.10 μM, respectively. And it was also very
effective toward HepG2 cells (IC50 = 2.67 0.14 μM) and
A498 cells (IC50 = 1.00 0.02 μM).[24]
In most studies including our previous work, it was
found that indole constituted various compounds with
On the basis of the previous considerations and our
high interest at the development of sorafenib and indole,
a series of new sorafenib derivatives were designed and
synthesized by substituting semicarbazide indole deriva-
tives for urea (Figure 2).
FIGURE 1 Structures of indole derivatives
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
The synthesis route of the target molecules 6a–c and 7a–q
was outlined in Scheme 1. 1H-indole-3-carbaldehyde (1a)
was prepared via a one-step procedure that involved the
FIGURE 2 Design idea of the target compounds
SCHEME 1 Synthetic routes of the target compounds 6a–c and 7a–q

LI ET AL.
3
Vilsmeier–Hacck reaction of 1H-indole with acylation
agent DMF under the POCl3 catalyzing to give.[25] Simi-
larly, 2-chloro-1H-indole-3-carbaldehyde (1b) was synthe-
sized according to a method previously reported in the
literature.[26] Molecules 5a–i were selected as our key
intermediates for this series of reactions, and were synthe-
sized by means of a two-step procedure that involved the
substitution reaction of substituted aniline 3a–i with phe-
nyl carbonochloridate to give carbonate compounds 4a–i,
which were then reacted with 80% hydrazine.[27] One of
the objectives of this study was the preparation of target
compounds 6a–c and 7a–q, a structure analogue of
sorafenib. Preparation of N-(substituted phenyl)-
2-(2-oxoindolin-3-ylidene)hydrazine-1-carboxamide 6a–
13C NMR spectra of 6a, 6c, 7c, 7i, and 7j disclosed the
PhCF3 quartets at around δ 123.42-123.01 ppm, whose
coupling constant is calculated manually. Furthermore,
the measured value of HRMS [M+H]+, [M+Na]+, or
[M-H]+ of the target compounds were consistent with
the theoretical value, and the errors were within m/z
0.005, which further confirmed the structures of the
target compounds. Finally, the melting point determina-
tion of the target molecules indicated the melting point
range of indolone semicarbazide sorafenib analogues
6a–c at about 250ꢀC-261ꢀC, while that of the indole
semicarbazide sorafenib derivatives 7a–q was around
193ꢀC-237ꢀC.
c
and 2-((substituted 1H-indol-3-yl) methylene)-N-
(substituted phenyl)hydrazine-1-carboxamide 7a–q
was accomplished via the condensation reaction
of substituted isatin or 2a–d with semicarbazide
compounds 5a–i.
2.2 | MTT assay
The antitumor activities against four cell lines of all of the
synthesized compounds were evaluated using sorafenib as
positive control. The results for the inhibitions rate of the
derivatives are listed in Table 1. The IC50 (dose of the com-
pound that caused a 50% reduction of survival values) are
shown in Table 2. From the analysis of Table 1, at the con-
centration of 10 μM, the great majority of compounds
exhibited comparable or even better antitumor activity
against the four cell lines. For example, compounds 6a, 7a,
7g, 7j, 7l, 7m, 7n, and 7q had the inhibition rates of 51.39
The structures of 6a–c and 7a–q were characterized
and confirmed by means of 1H NMR, 13C NMR, and
HRMS spectra. The characteristic hydrogen atoms of the
target compounds were affiliated to the 1H NMR spectrum
as follows. The 1H NMR spectra of 7a–i revealed the pres-
ence of singlet signals at δ 12.40-11.52 ppm corresponding
1
to indole-NH protons; the H NMR spectra of 6a–c and
7a–i manifested the existence of CONHN and PhNHCO
singlet signals at around δ 10.88-10.39 ppm and δ
3.39%, 57.45 2.32%, 67.05 3.82%, 59.71
3.81%,
1
9.96-8.41 ppm, respectively. Whereas the H NMR spectra
55.45 3.62%, 68.69 1.45%, 46.52 4.09%, and 57.63
0.06% against A549, PC-3, SMMC-7721, and K562 respec-
tively, which was more active than the control sorafenib
(35.30 3.45%, 41.95 1.84, 38.34 8.86, and 48.25 1.08
against A549, PC-3, SMMC-7721, and K562 respectively).
Additionally, compounds 7a, 7g, 7l, 7m, and 7p possessed
excellent antitumor activity against PC-3, with the inhibi-
tion rates of 55.21 6.35%, 50.49 6.84%, 58.03 3.06%,
56.32 5.60%, and 58.81 0.71%, respectively, compared
with the control sorafenib. As for the K562, compounds 7a,
7g, 7l, 7m, 7n, and 7p were found to possess better inhibi-
tion activities than positive control. Even better, most of
compounds possessed more potency in antiproliferation
effect against SMMC-7721.
According to the inhibition rate results of the target com-
pounds 6a–c and 7a–q against four cancer cell lines
(Table 1), we picked 16 compounds with high inhibition of
cancer cells and measured their IC50 values. As shown in
Table 2, all of the test compounds had excellent activities with
the IC50 values ranging from 28.76 3.72 to 4.08 1.21 μM
against A549, from 46.91 1.99 to 3.53 0.44 μM against
PC-3, from 13.95 2.89 to 1.73 0.25 μM against SMMC-
7721, from 116.25 1.91 to 1.43 0.14 μM against K562,
which showed better antitumor activities than sorafenib
(IC50 = 23.01 1.33 μM against A549, 16.69 4.44 μM
of compounds 6a–c showed indolone-NH single peaks at
1
around δ 10.56-10.50 ppm, and the H NMR spectra of
compounds 7a–q showed N CH single peaks at
about δ 8.25-8.16 ppm, and the 1H NMR spectra of
compounds 7a–d showed indole-2-H double peaks at
about δ 7.84-7.79 ppm. Additionally, the quartets at δ
4.33-4.31 ppm was assigned to the methylene group on
NCH2CH3 of target molecules 7j–p. The triplet at
δ = 4.26 ppm was designated to the first methylene group
on NCH2CH2CH3 of compounds 7q; the single peaks at
δ = 3.74 ppm came under the jurisdiction of the signal of
three hydrogens on Ph-OCH3 of 7h and 7o; the single
peaks at δ 2.33-2.27 ppm belonged to Ph-CH3 resonances
of 6c and 7d; the multiple peaks at δ 1.83-1.70 ppm were
the two hydrogen signals of the second methylene group
on NCH2CH2CH3 of 7q; and the triplets at
δ
1.30-0.87 ppm were the three hydrogen signals of the
methyl group on CH2CH3 of 7j–q. Likewise, the character-
istic carbon atoms of the target compounds were attached
to the 13C NMR spectrum as follows. The 13C NMR spectra
of compounds 6a–c showed the chemical shift of indole-
2-carbonyl carbon at about δ 165.22-165.06 ppm, the 13C
NMR spectra of 6a–c and 7a–i manifested the existence of
NHCONH signal at around δ 153.69-152.75 ppm, and the

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LI ET AL.
TABLE 1
the tested compounds using MTT assay
Antitumor activities of
Inhibition rate (%)a SD
Compound
A549
PC-3
SMMC-7721
24.65 0.95
57.14 1.87
51.77 6.97
58.72 1.06
22.27 3.77
26.03 6.51
7.61 8.93
K562
on the four cell lines at 10 μM
6a
51.39 3.39
8.01 1.14
17.02 3.99
57.45 2.32
17.11 1.62
21.67 5.84
7.11 3.19
8.15 8.63
38.13 6.76
67.05 3.82
25.29 3.82
8.63 1.28
59.71 3.81
30.08 3.29
55.45 3.62
68.69 1.45
46.52 4.09
33.26 6.75
32.75 2.31
57.63 0.06
35.30 3.45
21.53 3.15
20.27 0.05
19.96 2.72
55.21 6.35
25.45 9.16
15.90 0.96
4.95 2.70
33.63 4.82
32.47 7.59
50.49 6.84
18.34 8.50
24.11 3.08
13.86 7.88
26.09 3.74
58.03 3.06
56.32 5.60
17.08 7.60
35.30 3.92
58.81 0.71
26.27 4.92
41.95 1.84
22.07 6.38
16.95 2.28
15.63 9.54
87.35 0.37
23.43 7.12
24.29 2.62
13.00 5.24
6.84 2.14
31.00 2.94
88.65 1.15
24.72 2.83
19.77 6.12
43.65 3.92
13.81 6.25
75.53 1.97
82.76 1.08
70.00 3.55
14.97 0.66
75.55 3.16
22.76 2.22
48.25 1.08
6b
6c
7a
7b
7c
7d
7e
66.80 3.13
45.27 6.83
67.64 0.67
32.62 3.42
30.12 9.58
63.41 3.77
52.77 1.14
67.12 5.36
69.03 4.95
72.09 1.24
58.24 1.84
66.01 0.81
65.16 1.49
38.34 8.86
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
Sorafenibb
aThe average of three trials.
bCommercial sorafenib was used as positive control.
against PC-3, 13.62 0.52 μM against SMMC-7721 and
12.33 2.52 μM against K562). Interestingly, compounds 7a,
7g, 7l, 7m, and 7p showed superb IC50 values against the
four cell lines in vitro, respectively.
inhibition activity of compounds substituted by chlorine
was the least. From the comparison between compounds
7m, 7n, and 7k, the ortho-substitution performed promi-
nent activity than meta-substitution and para-substitution,
which possessed the least activity. Third, the structural
change at R2 appeared to have a considerable effect
on the anticancer activity. From comparison of
compounds 7g (R2 = -H), 7l (R2 = -CH2CH3) and 7q
(R2 = -CH2CH2CH3), it was obvious that compound 7g
(IC50: 4.93 1.69 μM, 3.53 0.44 μM, 5.48 0.81 μM,
and 1.43 0.14 μM against the four cell lines, respec-
tively) showed better comparable cytotoxicity than 7l
(IC50: 8.99 2.60 μM, 7.72 0.76 μM, 2.97 0.65 μM,
and 2.25 0.31 μM against the four cell lines, respec-
tively) and compound 7q (IC50: 6.89 0.86 μM against
A549, 1.73 0.25 μM against SMMC-7721).
Based on the analysis of the anticancer activity of the
compounds obtained by changing the types and positions
of the substituents, a structure-activity relationship was
preliminarily established. First, it was found that com-
pounds containing indolyl showed more significant anti-
tumor activities than those containing the indole ketone
group. Second, the substitutions on the benzene ring were
important to their inhibition activities against the four cell
lines. For example, compounds substituted by electron-
donating (eg, -CH3 and -OCH3) compounds, such as 7d,
7o, and 7h, and the substitution of trifluoromethyl and
chlorine-containing (eg, 7c, 7i, and 7j) compounds,
exerted weaker inhibition activities than the others. How-
ever, compounds substituted by halogen (eg, 7m con-
taining fluorine, 7p containing chlorine, 7a containing
bromine) showed better cytotoxicity than sorafenib. Fur-
ther, compounds 7a (3-Br), 7g (3-Br), and 7l (3-Br) gave
the marked activity than 7m (1-F), 7n (2-F), and the
2.3 | Wound healing assay
The role of target compounds at 5 μM in A549 cell
migration was assessed using a wound healing assay.

LI ET AL.
5
TABLE 2
Anticancer IC50 values of the tested compounds using MTT assay on the four cell lines
IC50 (μM)a SD
Compound
A549
PC-3
SMMC-7721
8.56 2.61
3.58 1.06
6.74 1.30
4.30 0.99
7.82 0.87
9.33 0.82
7.82 0.88
5.48 0.81
1.83 0.52
12.88 1.15
2.97 0.65
1.86 0.39
3.41 1.60
13.95 2.89
2.26 0.55
1.73 0.25
13.62 0.52
K562
6a
11.52 3.17
10.49 2.49
25.23 9.68
8.03 1.23
12.86 1.27
28.76 3.72
12.86 1.28
4.93 1.69
4.76 0.60
24.01 6.15
8.99 2.60
4.08 1.21
5.72 0.87
27.65 5.00
6.93 0.58
6.89 0.86
23.01 1.32
NT
NT
6b
18.49 9.18
46.91 1.99
4.66 0.28
NT
NT
6c
NT
7a
1.77 0.39
29.72 1.98
116.25 1.91
29.72 1.98
1.43 0.14
15.00 0.77
NT
7b
7e
36.64 1.70
NT
7f
7g
3.53 0.44
31.94 9.19
15.19 8.81
7.72 0.76
3.88 0.66
24.85 3.54
18.70 5.82
4.69 1.18
33.37 8.92
16.69 4.44
7j
7k
7l
2.25 0.31
1.73 0.18
3.82 0.32
NT
7m
7n
7o
7p
2.81 0.31
6.89 0.86
12.33 2.52
7q
Sorafenib
Abbreviations: NT, not tested.
aThe average of three trials.
bCommercial sorafenib was used as positive control.
FIGURE 3 A, The picture of wound healing (10× magnification). B, The migration ratios of A549
Significant closure of the scratch wound was
observed for the control group after 24 hours.
However, scratch wound closure was significantly
inhibited in the 7g group, showing
in cell migration following stimulating with 7g
(Figure 3).
a
defect

6
LI ET AL.
2.4 | Apoptosis evaluation by flow
cytometry
against A549, from 53.1% to 69.9% against PC-3) com-
pared with sorafenib groups (from 12.9% to 33.0% against
A549, from 25.4% to 53.1% against PC-3).
To investigate the function of potential compounds in cell
apoptosis, flow cytometry assay was conducted. As shown
in (Figure 4A,B), all of the hopeful compounds induced
cell apoptosis in A549 and PC-3 cells in different concen-
tration (5, 10, and 20 μM). These data hasted that as the
concentration increasing, the compounds increasingly
induced A549 and PC-3 cells apoptosis, as compared with
the control group (Figure 4C,D). The apoptosis ratios
(including the early and late apoptosis rates) of A549 and
PC-3 were remarkable influenced by 7g in a dose-
dependent manner. The apoptosis ratios of treatment
with 7g were significantly increased (from 50.0% to 55.9%
2.5 | Western blot
At last, the functions of 7g in the expression and activ-
ity of EGFR was estimated through adopting the west-
ern blot assay. In Figure 5, phosphorylated EGFR
(Tyr1068) and EGFR protein levels were hindered by 7g
management at the concentrations of 5 μM. Unlike
sorafenib, 7g inhibited cell growth by suppressing the
activity of EGFR, especially the expression of p-EGFR
(Tyr1068).
FIGURE 4 A, Annexin/PI statin of A549. B, Annexin/PI statin of PC-3. C, The apoptosis rate of A549. D, The apoptosis rate of PC-3

LI ET AL.
7
FIGURE 5 A, Detection of EGFR expression in A549 by western blot. B, The statistical results
3 | CONCLUSIONS
bromopropane (61.25 mmol) were added. After the mix-
ture was tempestuously stirred and refluxed at 60ꢀC for
10 hours. After finish of the reaction, filtered fleetly and
washed twice by acetone, then the filtrate was dried
under reduced pressure. The residue was stirred for
30 minutes after adding water, then ultrasonicated for
2 minutes, filtered, dried, and recrystallized from metha-
nol to gain compounds 2a–d.
In conclusion, a series of sorafenib derivatives containing
indole (ketone) semicarbazide analogues were designed
and synthesized. Further, the biological activities of these
compounds were evaluated according to the MTT method,
wound healing assay, flow cytometry and western blot
analysis. Compound 7g with the excellent activity could
be promising lead candidate for the following research.
4.3 | General procedure for the synthesis
of 4a–i
4 | EXPERIMENTAL SECTION
4.1 | Materials and methods
To a solution of substituted aniline 3a–i (50 mmol) in
anhydrous acetone (50 mL), K2CO3 (13.8 g, 0.100 mol)
was added, and phenyl carbonochloridate (50 mmol) was
distributed droplets in the mixture keeping the tempera-
ture between 0ꢀC and5ꢀC, then the mixture was vigor-
ously stirred at room temperature. After completion of
the reaction, as captured by TLC, the solvent was evapo-
rated to dryness under reduced pressure. The residue was
washed three times by water, filtered, and dried to obtain
the corresponding intermediates 4a–i.
All reagents were purchased from commercial sources as
analytical grade and were used without further purifica-
tion. All reactions were monitored by thin-layer chroma-
tography (TLC). 1H NMR and 13C NMR spectra were
performed on a Bruker Ascend 400 NMR spectrometer
and JEOL-ECX 500 NMR spectrometer (except for the 1H
NMR spectra of 7l was measured on a Bruker Avance
spectrometer at 600 MHz) using deuterated dimethyl
sulfoxide (DMSO-d6) as solvent and TMS as internal stan-
dard. Chemical shifts were expressed as δ ppm and cou-
pling constants were given as J Hz. High-resolution mass
spectra (HRMS) were recorded on Thermo Scientific Q
Extractive series with an ESI source. Melting points (ꢀC)
were obtained on an X-4D Micro melting point apparatus
and were uncorrected.
4.4 | General procedure for the synthesis
of 6a–c and 7a–q
The key intermediates 5a–i (3.05 mmol) and various
indole (ketone) derivatives substituted isatins or 2a–d
(3.36 mmol) were dissolved in CH3CH2OH (10 mL) at
room temperature, then catalytic amount of sulfuric acid
(0.15 mmol) were dropwise added to the solution, and
monitored by TLC (PE: EA = 1:4). After the reaction was
finished, the mixture was cooled to room temperature
and the solid precipitate was filtered, washed with etha-
nol (5 mL × 2), dried, and purified by recrystallization
4.2 | General procedure for the synthesis
of 2a–d
To a solution of 1a–b (55.68 mmol) in anhydrous acetone
(225 mL), K2CO3 (61.25 mmol) and bromoethane or

8
LI ET AL.
using methanol to yield the target compounds 6a–c and
7a–q.
J = 272.7 Hz), 121.67, 118.81 (q, J = 5.4 Hz), 116.03,
110.73, 21.05. HRMS (ESI) m/zcalcd for C17H12ClF3N4O2
[M-H]+ 395.05171, found 395.05276.
4.5 | N-(4-chloro-3-(trifluoromethyl)
phenyl)-2-(2-oxoindolin-3-ylidene)
hydrazine -1-carboxamide (6a)
4.8 | 2-((1H-indol-3-yl)methylene)-N-
(3-bromophenyl)hydrazine-
1-carboxamide(7a)
1
Yellow solid, yield: 69.78%, mp 260ꢀC-261ꢀC; H NMR
(500 MHz, DMSO-d6) δ 10.77 (s, 1H, CONHN), 10.54 (s,
1H, indoloneNH), 9.96 (s, 1H, PhNHCO), 8.15 (d,
J = 2.5 Hz, 1H, HAr), 8.04 (d, J = 7.6 Hz, 1H, HAr), 7.88
(dd, J = 8.8, 2.5 Hz, 1H, HAr), 7.64 (d, J = 8.8 Hz, 1H,
HAr), 7.35 (td, J = 7.7, 0.8 Hz, 1H, HAr), 7.07-7.01 (m,
1H, HAr), 6.88 (d, J = 7.8 Hz, 1H, HAr).13C NMR
(101 MHz, DMSO-d6) δ 165.15, 153.24, 143.76, 138.77,
136.25, 132.72, 132.52 (d, J = 3.6 Hz), 125.99, 124.11,
124.08, 123.56, 123.43 (q, J = 275.73 Hz), 121.89, 118.66
(dd, J = 4.4, 1.9 Hz), 115.95, 111.05. HRMS (ESI)
m/zcalcd for C16H10ClF3N4O2 [M-H]+ 381.03606, found
381.03729.
Off-white powder, yield: 45.36%, mp 214ꢀC-215ꢀC; 1H
NMR (500 MHz, DMSO-d6) δ 11.52 (s, 1H, indoleNH),
10.44 (s, 1H, CONHN), 8.72 (s, 1H, PhNHCO), 8.18 (d,
J = 7.5 Hz, 1H, HAr), 8.16 (s, 1H, CH N, HAr), 8.00 (d,
J = 1.9 Hz, 1H, HAr), 7.79 (d, J = 2.7 Hz, 1H, indole-
2-CH), 7.58-7.54 (m, 1H, HAr), 7.40 (d, J = 7.8 Hz, 1H,
HAr), 7.23 (t, J = 8.1 Hz, 1H, HAr), 7.19-7.13 (m, 3H,
HAr). 13C NMR (101 MHz, DMSO-d6) δ 153.43, 141.53,
139.63, 137.42, 130.93, 130.11, 125.16, 124.65, 122.94,
122.10, 121.99, 121.94, 120.91, 118.78, 112.24, 111.75.
HRMS (ESI) m/zcalcd for C16H13BrN4O [M-H]+
355.01890, found 355.02020.
4.6 | N-(3,4-difluorophenyl)-
2-(2-oxoindolin-3-ylidene)hydrazine-
1-carboxamide(6b)
4.9 | 2-((1H-indol-3-yl)methylene)-N-
(3,4-difluorophenyl)hydrazine-
1-carboxamide(7b)
1
Yellow powder, yield: 69.55%, mp 253ꢀC-254ꢀC; 1H NMR
(500 MHz, DMSO-d6) δ 10.80 (s, 1H, CONHN), 10.50 (s,
1H, indoloneNH), 9.74 (s, 1H, PhNHCO), 8.08 (d,
J = 7.6 Hz, 1H, HAr), 7.83-7.74 (m, 1H, HAr), 7.44-7.33
(m, 3H, HAr), 7.09 (t, J = 7.5 Hz, 1H, HAr), 6.92 (d,
J = 7.8 Hz, 1H, HAr). 13C NMR (101 MHz, DMSO-d6) δ
165.22, 153.10, 146.77, 143.67, 143.63, 136.19 (dd, J = 9.5,
2.4 Hz), 135.80, 132.60, 125.88, 122.14, 117.93 (d,
J = 18.0 Hz), 116.21 (dd, J = 5.5, 3.2 Hz), 115.99, 111.04,
108.90 (d, J = 21.6 Hz). HRMS (ESI) m/zcalcd for
C15H10F2N4O2 [M-H]+ 315.06881, found 315.06952.
White powder, yield: 70.12%, mp 213ꢀC-214ꢀC; H NMR
(500 MHz, DMSO-d6) δ 11.55 (s, 1H, indoleNH), 10.47 (s,
1H, CONHN), 8.79 (s, 1H, PhNHCO), 8.23 (d, J = 7.6 Hz,
1H, HAr), 8.20 (s, 1H, CH N), 7.88-7.83 (m, 1H, HAr),
7.83 (d, J = 2.7 Hz, 1H, indole-2-CH), 7.44 (d, J = 7.9 Hz,
2H, HAr), 7.37 (dt, J = 18.4, 9.1 Hz, 1H, HAr), 7.24-7.14
(m, 2H, HAr). 13C NMR (101 MHz, DMSO-d6) δ153.56,
149.40 (dd, J = 242.1, 13.0 Hz), 145.18 (dd, J = 239.8,
12.5 Hz), 139.69, 137.43, 137.00 (dd, J = 9.4, 2.6 Hz),
130.13, 124.63, 122.95, 122.21, 120.90, 117.51 (d,
J = 17.6 Hz), 116.20 (dd, J = 5.5, 3.1 Hz), 112.22, 111.76,
108.83 (d, J = 21.6 Hz). HRMS (ESI) m/zcalcd for
C16H12F2N4O [M-H]+ 313.08954, found 313.09061.
4.7 | N-(4-chloro-3-(trifluoromethyl)
phenyl)-2-(5-methyl-2-oxoindolin-
3-ylidene)hydrazine-1-carboxamide(6c)
4.10 | 2-((1H-indol-3-yl)methylene)-N-
(4-chloro-3-(trifluoromethyl)phenyl)
hydrazine-1-carboxamide(7c)
Orange yellow powder, yield: 70.00%, mp 250ꢀC-252ꢀC;
1H NMR (500 MHz, DMSO-d6) δ 10.68 (s, 1H, CONHN),
10.56 (s, 1H, indoloneNH), 9.90 (s, 1H, PhNHCO), 8.21
(d, J = 2.6 Hz, 1H, HAr), 7.97-7.92 (m, 2H, HAr), 7.68 (d,
J = 8.8 Hz, 1H, HAr), 7.20 (dd, J = 7.9, 0.7 Hz, 1H, HAr),
6.81 (d, J = 7.9 Hz, 1H, HAr), 2.33 (s, 3H, Ph-CH3). 13C
NMR (101 MHz, DMSO-d6) δ 165.17, 153.38, 141.50,
138.76, 136.34, 133.01, 132.50, 131.13, 127.31 (d,
J = 2.5 Hz), 127.00 (d, J = 2.5 Hz), 126.49, 123.01 (q,
1
White needle solid, yield: 75.00%, mp 224ꢀC-225ꢀC; H
NMR (500 MHz, DMSO-d6) δ 11.59 (s, 1H, indoleNH),
10.59 (s, 1H, CONHN), 9.09 (s, 1H, PhNHCO), 8.34 (d,
J = 2.5 Hz, 1H, HAr), 8.26 (d, J = 7.7 Hz, 1H, HAr), 8.21
(s, 1H, CH N, HAr), 7.95 (dd, J = 8.8, 2.4 Hz, 1H, HAr),
7.84 (d, J = 2.6 Hz, 1H, indole-2-CH), 7.65 (d, J = 8.8 Hz,
1H, HAr), 7.44 (d, J = 7.9 Hz, 1H, HAr), 7.23-7.18 (m,

LI ET AL.
9
1H, HAr), 7.16 (td, J = 7.6, 1.1 Hz, 1H, HAr). 13C NMR
(101 MHz, DMSO-d6) δ 153.53, 139.99, 139.59, 137.41,
132.12, 130.17, 127.05 (d, J = 4.9 Hz), 126.75 (d,
J = 3.3 Hz), 126.47, 124.91, 123.42(q, J = 273.71 Hz)
123.13, 122.95, 120.85, 118.61 (dd, J = 10.8, 4.9 Hz),
112.21, 111.72; HRMS (ESI) m/zcalcd for C17H12ClF3N4O
[M-H]+ 379.05680, found 379.05777.
J = 2.0 Hz, 1H, HAr), 7.55 (d, J = 8.2 Hz, 1H, HAr),
7.38-7.30 (m, 2H, HAr), 7.23 (tdd, J = 8.5, 7.2, 1.3 Hz, 2H,
HAr), 7.07 (dd, J = 7.9, 2.0 Hz, 1H, HAr). 13C NMR
(101 MHz, DMSO-d6) δ 153.32, 141.27, 137.11, 135.41,
133.39, 130.61, 126.88, 124.28, 123.56, 122.40, 121.99, 121.72,
119.35, 118.57, 111.54, 107.45. HRMS (ESI) m/zcalcd for
C16H12Cl2N4O [M-H]+ 345.03044, found 345.03140.
4.11 | 2-((1H-indol-3-yl)methylene)-N-(p-
tolyl)hydrazine-1-carboxamide(7d)
4.14 | N-(3-bromophenyl)-2-((2-chloro-
1H-indol-3-yl)methylene)hydrazine-
1-carboxamide(7g)
1
Yellow needle solid, yield: 45.52%, mp 236ꢀC-237ꢀC; H
NMR (400 MHz, DMSO-d6) δ 11.57 (s, 1H, indoleNH),
10.39 (s, 1H, CONHN), 8.49 (s, 1H, PhNHCO), 8.23 (d,
J = 2.7 Hz, 1H, HAr), 8.20 (s, 1H, CH N), 7.83 (d,
J = 2.7 Hz, 1H, indole-2-CH), 7.54 (d, J = 8.4 Hz, 2H,
HAr), 7.46 (dd, J = 6.4, 1.9 Hz, 1H, HAr), 7.25-7.17 (m,
2H, HAr), 7.13 (d, J = 8.3 Hz, 2H, HAr), 2.27 (s, 3H, Ph-
CH3). 13C NMR (101 MHz, DMSO-d6) δ 153.61, 139.03,
137.45, 137.10, 131.54, 129.99, 129.49 (2C), 124.61, 122.93,
121.96, 120.92, 119.87 (2C), 112.29, 111.85, 20.87. HRMS
(ESI) m/zcalcd for C17H16N4O [M-H]+ 291.12404, found
291.12534.
Yellow powder, yield: 42.35%, mp 214ꢀC-215ꢀC; 1H NMR
(500 MHz, DMSO-d6) δ 11.55 (s, 1H, indoleNH), 10.47 (s,
1H, CONHN), 8.75 (s, 1H, PhNHCO), 8.19 (s, 1H), 8.03
(t, J = 1.9 Hz, 1H, HAr), 7.83 (d, J = 2.7 Hz, 1H, HAr),
7.59 (dd, J = 8.2, 0.9 Hz, 1H, HAr), 7.44 (d, J = 8.0 Hz,
1H, HAr), 7.27 (t, J = 8.0 Hz, 1H, HAr), 7.22-7.15 (m, 3H,
HAr). 13C NMR (101 MHz, DMSO-d6) δ 153.45, 141.54,
139.64, 137.43, 130.92, 130.13, 125.17, 124.65, 122.96,
122.13, 122.02, 121.96, 120.92, 118.79, 112.26, 111.76.
HRMS (ESI) m/z calcd for C16H12BrClN4O [M+Na]+
412.97752, found 412.97845.
4.12 | 2-((2-chloro-1H-indol-3-yl)
methylene)-N-(3,4-difluorophenyl)
hydrazine-1-carboxamide(7e)
4.15 | 2-((2-chloro-1H-indol-3-yl)
methylene)-N-(4-methoxyphenyl)
hydrazine-1-carboxamide(7h)
1
1
Light yellow powder, yield: 70.58%, mp 207ꢀC-208ꢀC; H
Off-white solid, yield: 68.78%, mp 202ꢀC-203ꢀC; H NMR
NMR (400 MHz, DMSO-d6) δ 12.40 (s, 1H, indoleNH),
10.63 (s, 1H, CONHN), 8.83 (s, 1H, PhNHCO), 8.30 (dd,
J = 6.7, 1.9 Hz, 1H, HAr), 8.23 (s, 1H, CH N, HAr), 7.85
(ddd, J = 13.5, 7.5, 2.4 Hz, 1H, HAr), 7.48-7.31 (m, 3H,
HAr), 7.29-7.18 (m, 2H, HAr). 13C NMR (101 MHz,
DMSO-d6) δ 153.43, 149.40 (dd, J = 242.2, 13.2 Hz),
145.29 (dd, J = 240.0, 12.8 Hz), 137.16, 136.87 (dd,
J = 9.2, 2.7 Hz), 135.43, 126.88, 124.29, 123.54, 122.05,
121.69, 117.50 (d, J = 17.6 Hz), 116.41 (dd, J = 5.6,
3.2 Hz), 111.52, 109.04 (d, J = 21.5 Hz), 107.47. HRMS
(ESI) m/zcalcd for C16H11ClF2N4O [M-H]+ 347.05057,
found 347.05157.
(400 MHz, DMSO-d6) δ 12.37 (s, 1H, indoleNH), 10.47 (s,
1H, CONHN), 8.43 (s, 1H, PhNHCO), 8.26 (d, J = 7.1 Hz,
1H, HAr), 8.20 (s, 1H, CH N), 7.52 (d, J = 8.9 Hz,
2H, HAr), 7.37 (d, J = 7.1 Hz, 1H, HAr), 7.30-7.15
(m, 2H, HAr), 6.91 (d, J = 8.9 Hz, 2H, HAr), 3.74 (s, 3H,
PhOCH3). 13C NMR (101 MHz, DMSO-d6) δ 155.43,
153.69, 136.30, 135.44, 132.57, 126.55, 124.32, 123.51,
122.11(2C), 121.82, 121.69, 114.24(2C), 111.56, 107.58,
55.66. HRMS (ESI) m/zcalcd for C17H15ClN4O2 [M-H]+
341.07998, found 341.08112.
4.16 | 2-((2-chloro-1H-indol-3-yl)
methylene)-N-(4-chloro-3-(trifluoromethyl)
phenyl)hydrazine-1-carboxa-mide(7i)
4.13 | 2-((2-chloro-1H-indol-3-yl)
methylene)-N-(3-chlorophenyl)hydrazine-
1-carboxamide(7f)
White solid, yield: 50.00%, mp 232ꢀC-233ꢀC; 1H NMR
(500 MHz, DMSO-d6) δ 12.37 (s, 1H, indoleNH), 10.67 (s,
1H, CONHN), 9.07 (s, 1H, PhNHCO), 8.28 (dd, J = 4.8,
2.2 Hz, 2H, HAr), 8.19 (s, 1H, CH N), 7.89 (dd, J = 8.8,
2.5 Hz, 1H, HAr), 7.61 (d, J = 8.8 Hz, 1H, HAr), 7.37-7.30
(m, 1H, HAr), 7.22-7.19 (m, 1H, HAr), 7.19-7.15 (m, 1H,
1
Light yellow powder, yield: 51.25%,mp 223ꢀC-225ꢀC; H
NMR (500 MHz, DMSO-d6) δ 12.39 (s, 1H, indoleNH),
10.64 (s, 1H, CONHN, HAr), 8.80 (s, 1H, PhNHCO), 8.27
(dd, J = 6.7, 1.7 Hz, 1H, HAr), 8.20 (s, 1H, CH N), 7.87 (t,

10
LI ET AL.
HAr). 13C NMR (101 MHz, DMSO-d6) δ 153.41, 139.47,
137.54, 135.41, 132.11, 127.41 (d, J = 7.2 Hz), 127.05 (d,
J = 3.5 Hz), 126.77, 126.46, 125.12, 123.57, 123.32, 123.30
(q, J = 274.72 Hz), 121.66, 118.80 (q, J = 5.6 Hz), 111.51,
107.44. HRMS (ESI) m/zcalcd for C17H11Cl2F3N4O [M-
H]+ 413.01892, found 413.01907.
1H, PhNHCO), 8.33 (d, J = 7.8 Hz, 1H, HAr), 8.23 (s,
1H, CH N), 8.01 (t, J = 1.9 Hz, 1H, HAr), 7.59 (dd,
J = 12.6, 4.6 Hz, 2H, HAr), 7.33-7.30 (m, 1H, HAr), 7.27
(ddd, J = 7.8, 3.4, 2.0 Hz, 2H, HAr), 7.22-7.18 (m, 1H,
HAr), 4.32 (q, J = 7.1 Hz, 2H, NCH2CH3), 1.29 (t,
J = 7.2 Hz, 3H, NCH2CH3). 13C NMR (101 MHz, DMSO-
d6) δ 153.27, 141.41, 137.16, 135.37, 130.91, 127.92,
125.32, 125.26, 123.69, 123.66, 122.22, 122.07, 121.91,
118.99, 110.49, 107.62, 38.83, 15.13. HRMS (ESI) m/
zcalcd for C18H16BrClN4O [M+H]+ 419.02688, found
419.02551.
4.17 | 2-((2-chloro-1-ethyl-1H-indol-3-yl)
methylene)-N-(4-chloro-3-(trifluoromethyl)
phenyl)hydrazine-1-carboxamide(7j)
1
White powder, yield: 68.74%, mp 230ꢀC-231ꢀC; H NMR
(400 MHz, DMSO-d6) δ 10.75 (s, 1H, CONHN), 9.13 (s,
1H, PhNHCO), 8.37 (d, J = 7.6 Hz, 1H, HAr), 8.32 (d,
J = 2.5 Hz, 1H, HAr), 8.25 (s, 1H, CH N), 7.93 (dd,
J = 8.8, 2.4 Hz, 1H, HAr), 7.65 (d, J = 8.8 Hz, 1H, HAr),
7.60 (d, J = 8.1 Hz, 1H, HAr), 7.35-7.28 (m, 1H, HAr),
7.28-7.23 (m, 1H, HAr), 4.33 (q, J = 7.1 Hz, 2H,
NCH2CH3), 1.30 (t, J = 7.1 Hz, 3H, NCH2CH3). 13C NMR
(101 MHz, DMSO-d6) δ 153.38, 139.46, 137.59, 135.38,
132.13, 128.09 (d, J = 5.1 Hz), 127.10 (d, J = 3.2 Hz),
126.79, 126.67, 125.14, 123.34, 123.32123.10 (q, J =
278.76 Hz) 122.41, 118.82 (dd, J = 4.7, 2.3 Hz), 110.46,
107.59, 38.85, 15.12. HRMS (ESI) m/zcalcd for
C19H15Cl2F3N4O [M-H]+ 441.04913, found 441.05048.
4.20 | 2-((2-chloro-1-ethyl-1H-indol-3-yl)
methylene)-N-(2-fluorophenyl)hydrazine-
1-carboxamide(7m)
Pink solid, yield: 73.58%, mp 205ꢀC-207ꢀC; 1H NMR
(400 MHz, DMSO-d6) δ 10.88 (s, 1H, CONHN), 8.56 (d,
J = 2.1 Hz, 1H, PhNHCO), 8.23 (s, 1H, CH N), 8.22-8.12
(m, 2H, HAr), 7.61 (d, J = 8.2 Hz, 1H, HAr), 7.31 (t,
J = 8.4 Hz, 2H, HAr), 7.21 (dt, J = 15.8, 7.7 Hz, 2H,
HAr), 7.08 (dd, J = 13.2, 6.2 Hz, 1H, HAr), 4.31 (q,
J = 7.0 Hz, 2H, NCH2CH3), 1.28 (t, J = 7.1 Hz, 3H,
NCH2CH3). 13C NMR (101 MHz, DMSO-d6) δ 152.92 (d,
J = 241.0 Hz), 152.75, 136.83, 135.45, 128.05, 127.51 (d,
J = 10.0 Hz), 125.11, 125.08, 123.71, 123.67, 123.56
(d, J = 7.6 Hz), 122.03, 121.12 (d, J = 12.8 Hz), 115.50
(d, J = 19.0 Hz), 110.83, 107.43, 38.84, 15.11. HRMS (ESI)
m/zcalcd for C18H16ClFN4O [M-H]+ 357.09129, found
357.09280.
4.18 | 2-((2-chloro-1-ethyl-1H-indol-3-yl)
methylene)-N-(4-fluorophenyl)hydrazine-
1-carboxamide(7k)
Light pink solid, yield: 67.92%, mp 219ꢀC-221ꢀC; 1H
NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H, CONHN),
8.65 (s, 1H, PhNHCO), 8.32 (d, J = 7.5 Hz, 1H, HAr), 8.22
(s, 1H, CH N, HAr), 7.69-7.56 (m, 3H, HAr), 7.36-7.28
(m, 1H, HAr), 7.28-7.23 (m, 1H, HAr), 7.19-7.11 (m, 2H,
HAr), 4.32 (q, J = 7.1 Hz, 2H, NCH2CH3), 1.29
(t, J = 7.1 Hz, 3H, NCH2CH3). 13C NMR (101 MHz,
DMSO-d6) δ 158.23 (d, J = 239.5 Hz), 153.57, 136.68,
135.94, 135.35, 127.76, 123.65(2C), 122.20, 122.12
(d, J = 7.6 Hz, 2C), 122.04, 115.49 (d, J = 22.2 Hz, 2C),
110.49, 107.64, 38.81, 15.15. HRMS (ESI) m/zcalcd for
C18H16ClFN4O [M-H]+ 357.09129, found 357.09250.
4.21 | 2-((2-chloro-1-ethyl-1H-indol-3-yl)
methylene)-N-(3-fluorophenyl)hydrazine-
1-carboxamide(7n)
Milky white powder, yield: 71.70%, mp 210ꢀC-211ꢀC;
1H NMR (400 MHz, DMSO-d6)
δ 10.68 (s, 1H,
CONHN), 8.82 (s, 1H, PhNHCO), 8.36-8.28 (m, 1H,
HAr), 8.24 (s, 1H, CH N), 7.66 (dt, J = 12.0, 2.2 Hz,
1H, HAr), 7.58 (d, J = 7.6 Hz, 1H, HAr), 7.46-7.40 (m,
1H, HAr), 7.38-7.24 (m, 3H, HAr), 6.83 (td, J = 8.2,
1.9 Hz, 1H, HAr), 4.31 (q, J = 7.1 Hz, 2H, NCH2CH3),
1.28 (t, J = 7.1 Hz, 3H, NCH2CH3). 13C NMR
(101 MHz, DMSO-d6) δ 162.72 (d, J = 240.4 Hz),
153.27, 141.56 (d, J = 11.4 Hz), 137.07, 135.35, 130.53
(d, J = 9.7 Hz), 127.90, 123.68, 123.66, 122.13 (d,
J = 10.0 Hz), 115.74 (d, J = 2.3 Hz), 110.50, 109.08 (d,
J = 21.1 Hz), 107.60, 106.68, 106.42, 38.82, 15.13.
HRMS (ESI) m/zcalcd for C18H16ClFN4O [M-H]+
357.09129, found 357.09271.
4.19 | N-(3-bromophenyl)-2-((2-chloro-
1-ethyl-1H-indol-3-yl)methylene)hydrazine-
1-carboxamide(7l)
Pink solid, yield: 69.47%, mp 216ꢀC-217ꢀC; 1H NMR
(600 MHz, DMSO-d6) δ 10.66 (s, 1H, CONHN), 8.79 (s,

LI ET AL.
11
4.22 | 2-((2-chloro-1-ethyl-1H-indol-3-yl)
methylene)-N-(4-methoxyphenyl)
hydrazine-1-carboxamide(7o)
4.25 | MTT assay
Antitumor activities of compounds 6a–c and 7a–q
against four types of cancer cells named A549, K562,
PC-3, and SMMC-7721 were also tested at 10 μM, and
assessed according to the method of cell growth rate. The
PC-3 was maintained in monolayer culture at 37ꢀC and
5% CO2 in DMEM medium supplemented with 10% fetal
bovine serum and 1% Penicillin Streptomycin. The others
were cultured in RPMI1640 supplemented with the same
mixtures. The cells that grew well observed by an
inverted phase microscope (Nikon Ti-s) were seeded in
96-well plates in 180 μL aliquots at 3 × 104 cells/mL for
MTT assays. For each compound, five concentrations
(0.625, 1.25, 2.5, 5, and 10 μM) were evaluated and each
was done in five copies. The plates were further incu-
bated for 24 hours and the assay was terminated by the
addition of 20 μL of 5% MTT and incubated for 4 hours at
37ꢀC. Finally, the supernatant was discarded. The bound
amount of stain was subsequently eluted with 150 μL of
DMSO and the absorbance was measured with an
enzyme-labeled instrument (TECAN infinite M200 PRO)
at 490 nm wavelength. The growth percentage was calcu-
lated on a plate-by-plate basis for test wells relative to
control wells. The sensitivity of the cancer cells to each
test compound was expressed in terms of IC50, indicated
as mean values SD for three independent experiments.
Statistical analysis was performed using Graphpad prism
version 7 for Student's test. Values of P < .05 were con-
sidered significant.
Light pink solid, yield: 74.22%, mp 193ꢀC-194ꢀC; 1H
NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H, CONHN),
8.41 (s, 1H, PhNHCO), 8.30 (d, J = 7.7 Hz, 1H, HAr), 8.22
(s, 1H, CH N), 7.59 (d, J = 7.9 Hz, 1H, HAr), 7.51 (d,
J = 8.9 Hz, 2H, HAr), 7.34-7.22 (m, 2H, HAr), 6.90 (d,
J = 8.9 Hz, 2H, HAr), 4.32 (q, J = 6.8 Hz, 2H, NCH2CH3),
3.74 (s, 3H, PhOCH3), 1.29 (t, J = 7.1 Hz, 3H, NCH2CH3).
13C NMR (101 MHz, DMSO-d6) δ 155.44, 153.65, 136.32,
135.37, 132.56, 127.60, 123.72, 123.62, 122.12, 122.07,
122.03, 114.37, 114.24(2C), 110.50, 107.73, 55.67, 38.80,
15.14. HRMS (ESI) m/zcalcd for C19H19ClN4O2 [M+H]+
371.12693, found 371.12653.
4.23 | 2-((2-chloro-1-ethyl-1H-indol-3-yl)
methylene)-N-(3-chlorophenyl)hydrazine-
1-carboxamide (7p)
1
Light pink powder, yield: 65.37%, mp 203ꢀC-204ꢀC; H
NMR (400 MHz, DMSO-d6) δ 10.66 (s, 1H, CONHN),
8.79 (s, 1H, PhNHCO), 8.33 (d, J = 7.3 Hz, 1H, HAr), 8.25
(s, 1H, CH N), 7.88 (t, J = 1.9 Hz, 1H, HAr), 7.62-7.53
(m, 2H, HAr), 7.36-7.24 (m, 3H, HAr), 7.07 (dd, J = 7.9,
1.3 Hz, 1H, HAr), 4.32 (q, J = 7.1 Hz, 2H, NCH2CH3),
1.30 (t, J = 7.1 Hz, 3H, NCH2CH3). 13C NMR (101 MHz,
DMSO-d6) δ 153.28, 141.26, 137.15, 135.38, 133.41,
130.59, 127.91, 123.71, 123.66, 122.41, 122.21, 122.07,
119.36, 118.56, 110.49, 107.62, 38.83, 15.13. HRMS (ESI)
m/zcalcd for C18H16Cl2N4O [M-H]+ 373.06174, found
373.06299.
4.26 | Wound healing assay
The cells were seeded in six-well plate, and each hole was
1 mL, at 37ꢀC, 5% CO2 and saturated humidity cell culture
medium 24 hours to cell fusion. The 10-μL micropipette
was vertically scratched in the six-well plates, and the PBS
solution was rinsed two times. The compounds were added
and divided into five solutions, each hole was 1 mL, and
the negative control group was added with the same vol-
ume of cell culture medium. Digital images of the wounded
monolayers were obtained by photomicroscope (Ti-S,
Nikon) at 0, 6, 12, and 24 hours. The unfilled scratched
zones were quantified by Java's Image J software.
4.24 | N-(3-bromophenyl)-2-((2-chloro-
1-propyl-1H-indol-3-yl)methylene)
hydrazine-1-carboxamide(7q)
1
Off-white solid, yield: 66.28%, mp 222ꢀC-223ꢀC; H NMR
(400 MHz, DMSO-d6) δ 10.65 (s, 1H, CONHN), 8.78 (s,
1H, PhNHCO), 8.32 (d, J = 7.4 Hz, 1H, HAr), 8.24 (s, 1H,
CH N), 8.01 (t, J = 1.7 Hz, 1H, HAr), 7.60 (t, J = 6.9 Hz,
2H, HAr), 7.35-7.18 (m, 4H, HAr), 4.26 (t, J = 7.1 Hz, 2H,
NCH2CH2CH3), 1.83-1.70 (m, 2H, NCH2CH2CH3), 0.87
(t, J = 7.4 Hz, 3H, NCH2CH2CH3). 13C NMR (101 MHz,
DMSO-d6) δ 153.28, 141.41, 137.20, 135.94, 130.92,
128.37, 125.33, 123.63, 123.57, 122.21, 122.16, 122.05,
121.90, 119.00, 110.76, 107.53, 45.21, 23.00, 11.40. HRMS
(ESI) m/zcalcd for C19H18BrClN4O [M+H]+ 433.04253,
found 433.04111.
4.27 | Apoptosis evaluation by flow
cytometry
The A549 cells stimulated by potential compounds was
assayed 24 hours later using an Annexin V-fluorescein

12
LI ET AL.
isothiocyanate (FITC)/propidium iodide (PI) apoptosis
assay. Briefly, cells were incubated with 5 μL Annexin
V-FITC (Solabio, Beijing, China) under conditions devoid
of light for 15 minutes at 4ꢀC. Next, 5 μL PI was adopted
for dying cells for 10 minutes at room temperature.
A flow cytometer (C6; BD Biosciences) was used to exam-
ine the rate of apoptosis.
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ACKNOWLEDGMENTS
This study was supported by the project of State Key
Laboratory of Functions and Applications of Medicinal
Plants, Guizhou Medical University (No. FAMP201707K),
and the National Natural Science Foundation of China
(No. 21867004), and the Science Technology Program of
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Additional supporting information including 1H, 13C
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ORCID
Zhen-Chao Wang https://orcid.org/0000-0003-1859-
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How to cite this article: Li W, Qi Y-Y,
Wang Y-Y, et al. Design, synthesis, and biological
evaluation of sorafenib derivatives containing
indole (ketone) semicarbazide analogs as
antitumor agents. J Heterocyclic Chem. 2020;1–13.
https://doi.org/10.1002/jhet.3972
SUPPORTING INFORMATION
Additional supporting information may be found online
in the Supporting Information section at the end of this
article.