Synthesis and Antiproliferative Activity of New N-Acylhydrazone Derivatives Containing Benzothiazole and Indole Based Moiety

Article abstract of DOI:10.1007/s11094-020-02215-w

Through a structure-based molecular hybridization strategy, a series of new N-acylhydrazone derivatives containing the benzothiazole and indole based moiety were designed, synthesized and screened for in vitro antiproliferative activity against Hep G2 cancer cell line. One compound (7a) exhibited excellent antiproliferative activity with IC₅₀ values of 0.78 μM against Hep G2. In addition, C-5 substitutions of the indole ring of target compounds might be crucial for their cytotoxic activities. Additionally, the relative configuration of target compounds was confirmed as the E isomer. Further chemical manipulation of derivative 7a can make it possible to obtain new potential antitumor agents.

Full text of DOI:10.1007/s11094-020-02215-w

DOI 10.1007/s11094-020-02215-w
Pharmaceutical Chemistry Journal, Vol. 54, No. 4, July, 2020 (Russian Original Vol. 54, No. 4, April, 2020)
SYNTHESIS AND ANTIPROLIFERATIVE ACTIVITY
OF NEW N-ACYLHYDRAZONE DERIVATIVES CONTAINING
BENZOTHIAZOLE AND INDOLE BASED MOIETY
1
Kai Liu, Yangyang Ding, and Congmin Kang
1
1,*
Original article submitted January 23, 2020.
Through a structure-based molecular hybridization strategy, a series of new N-acylhydrazone derivatives con-
taining the benzothiazole and indole based moiety were designed, synthesized and screened for in vitro
antiproliferative activity against Hep G2 cancer cell line. One compound (7a) exhibited excellent
antiproliferative activity with IC50 values of 0.78 mM against Hep G2. In addition, C-5 substitutions of the
indole ring of target compounds might be crucial for their cytotoxic activities. Additionally, the relative con-
figuration of target compounds was confirmed as the E isomer. Further chemical manipulation of derivative 7a
can make it possible to obtain new potential antitumor agents.
Keywords: synthesis; N-acylhydrazone; benzothiazole; indole based moiety; antiproliferative activity.
1
. INTRODUCTION
Cancer is one of the most life-threatening diseases
tiviral [6] anti-inflammatory [7], and particularly in discov-
ery of new antitumor agents [8, 9]. In recent years, more new
indole derivatives have been developed as potent antitumor
agents, including cediranib (1) [10], obatoclax (2) [11],
D-24851 (3) and sunitinib (4) [12] (Fig. 1).
worldwide. Despite tremendous efforts to develop effective
treatment strategies, satisfactory results have not yet been
obtained, and the overall survival rate and disease-free sur-
vival rate of most cancers are still very low [1, 2]. Therefore,
discovery of more effective and safer anticancer agents re-
mains urgently needed.
Benzothiazoles are heterocyclic compounds with diverse
range of biological activities. Many studies reported that
benzothiazoles derivatives exhibited various activities, in-
cluding antimicrobial [13], anti-inflammatory [14], antifun-
gal [15] anticancer [16], antidiabetic [17], anticonvulsant
Indole, as a typical N-heterocyclic aromatic compound is
widespread in nature, It has been considered as an important
structural component in many pharmaceutical agents includ-
ing antidepressant [3], anticonvulsant [4], antifungal [5], an-
[
18], antiviral [19], antitubercular [20], antimalarial [21], and
antihelmintic [22]. Particularly, 2-substituted benzothiazoles
are among privileged structures in medicinal chemistry
23, 24]. Recently, some 2-substituted benzothiazole deriva-
tives have been evaluated as potential antitumor agents 5
[25] and 6 [26] (Fig. 2).
[
1
College of Chemical Engineering, Qingdao University of Science and
Technology, Qingdao 266042, P. R. China.
*
e-mail: qustndds@163.com
Fig. 1. Structures of compounds bearing indole based moiety.
345
0
091-150X/20/5404-0345 © 2020 Springer Science+Business Media, LLC

3
46
Kai Liu et al.
Fig. 2. Structures of benzothiazole and N-acylhydrazone derivatives.
According to structure-based molecular hybridization
strategy, researchers have attempted to combine benzothia-
zole and indole through different linkers to obtain new candi-
dates with useful pharmacological activity [27, 28]. The
N-acylhydrazone (NAH) scaffold is usually employed in the
design of heterocyclic compounds, which endow molecules
with good thermal, hydrolytic, and chemical stability, as well
as the feasibility of changing chemical composition by vari-
ous substituents [29, 30]. In recent years, some compounds
containing NAH scaffold have been reported as antitumor
agents, e.g., PAC-1 (7) [31], LASSBio-1586 (8) [32], and
compound 9 [33] (Fig. 2), and it was indicated that introduc-
tion of NAH as a linker was an effective approach for devel-
oping more potent and broad spectrum antitumor agents.
In this study, based on a structure-based molecular hy-
bridization strategy – a common and effective approach to
developing small molecules with multi-targeted molecular
mechanism, we have designed and synthesized a series of
new NAH derivatives containing the benzothiazole and in-
dole-based moiety (Fig. 3). Herein, twelve new NAH deriva-
tives and their antiproliferative activities in vitro against Hep
G2 cancer cell line are reported. Additionally, the configura-
tion of target compounds was confirmed as the E isomer. The
proposed structures of target compounds were confirmed by
1
13
H NMR, C NMR, NOESY NMR, and IR spectroscopy
data.
2. EXPERIMENTAL CHEMICAL PART
2.1. Chemicals and Instruments
All chemicals and solvents were obtained from commer-
cial sources and used without further purification. Reactions
were monitored by thin layer chromatography (TLC) using
glass plates precoated with a layer of silica gel (GF254) con-
taining a fluorescent indicator. TLC plates were visualized
by exposure to ultraviolet light (254 nm). The melting points
of synthesized compounds were determined in an electric
1
melting point apparatus and remained uncorrected. The H
1
NMR and C NMR spectra were measured on 500 MHz
3
Bruker spectrometer in DMSO-d using TMS as internal
6
standard. Infrared (IR) spectra were recorded on Nicolet 370
FT-IR spectrometer.
Fig. 3. The design of target compounds.

Synthesis and Antiproliferative Activity
347
Scheme 1. Synthetic route to compounds 2 and 3. Reagents and conditions: (a) diethyl oxalate, 140°C, 4 h; (b) 80% hydrazine hydrate, ethanol,
0°C, 9 h.
8
2
.2. Synthetic Routes
densation of compound 1 with diethyl oxalate furnished
compound 2, which was reacted with hydrazine to obtain tar-
get hydrazides 3.
An efficient method for the synthesis of linked N-acyl-
hydrazone derivatives containing the benzothiazole and
indole-based moiety (7a – 7e, 8a – 8g) was developed as
shown in Schemes 1 and 2. According to a convergent syn-
thesis approach, we initially synthesized the key intermedi-
ates benzothiazole-2-carbohydrazide (3) and substituted
The synthetic route to compounds 4a – 4e is outlined in
Scheme 2. Under Vilsmeier-Haack (DMF-POCl ) condi-
3
tions, compounds 4a – 4e were transformed into the corre-
sponding 3-carboxaldehyde functionalized indoles 5a – 5e.
The N-ethyl, N-isopropyl and N-phenylsulfonyl derivatives
(6a – 6g) of indole-3-carboxaldehyde were obtained via re-
action of 5a – 5c with bromoethane, 2-bromopropane or ben-
1
H-indole-3-carbaldehydes (5a – 5e, 6a – 6g) to afford the
proposed acylhydrazones. As illustrated in Scheme 1, con-
1
2
Compd.
R
R
4
a,5a,7a
H
5-OCH
5-Br
5-Cl
5-F
4b,5b,7b
3
4
c,5c,7c
4d,4d,7d
4
e,5e,7e
6a,8a
H
Et
6
b,8b
c,8c
d,8d
5-OCH
5-Br
H
3
Et
6
Et
i-Pr
6
6
e,8e
f,8f
g,8g
5-OCH
5-Br
H
3
i-Pr
6
i-Pr
6
Phenylsulfonyl
Scheme 2. Synthetic route to compounds 5a -– 5e, 6a – 6g and target compounds 7a -– 7e and 8a – 8g. Reagents and conditions: (c) (i) POCl ,
3
DMF, 0°C, 20 min; (ii) 35°C, 1 h; (d) (i) 20% NaOH, DMSO, r.t, 15 min; (ii) R Br, 30°C, 5 h; (e) glacial acetic acid, ethanol, reflux 6 h.
2

3
48
Kai Liu et al.
Fig. 4. NOESY of representative compound 7a.
zenesulfonyl chloride in the presence of sodium hydroxide
Due to the presence of an imino bond, all target com-
pounds 7a – 7-e and 8a – 8g could exist in either E or Z iso-
meric form, hence, the next step in this work was to deter-
mine the relative configuration of the imino double bond in
(
NaOH) in DMSO and water. This was the first time that
DMSO and water were used as a mixed solvent for this reac-
tion and such a high yield has not been reported before.
Finally, the condensation of compound 3 with compounds
1
target compounds. By careful analysis of the H-NMR spec-
5
a – 5e or compounds 6a – 6g, produced the target com-
tra of compounds 7a – 7e and 8a – 8g, we only detected the
presence of one imino hydrogen signal (Table 1). According
to the literature [34], this was attributed to the presence of
only (E)-diastereomers in N-acylhydrazone derivatives.
Compound 7a was further selected to identify the stereoche-
mistry by undergoing NOESY NMR. Results (Fig. 4) sho-
wed that an evident NOE signal was observed between the
pounds 7a – 7e and 8a – 8g. In summary, this synthetic route
for a series of N-acylhydrazone derivatives containing the
benzothiazole and indole-based moiety shows significant ad-
vantages, such as mild conditions, high yields, and simple
operation process. The results are summarized in Table 1.
1
H
2
(-CH=N-, d = 7.86 ppm) and
H
(=N-NH-, d =
1
13
TABLE 1. Main H and C NMR Signals for Target Compounds
11.69 ppm) in the E isomer (E-7a), owing to the large intra-
molecular H-H distance (Z-7a), NOE signal should not be
observed in the putative Z isomer. Thus, target compounds
7a – 7e and 8a – 8g were clearly confirmed as E isomers.
7
a – 7e and 8a – 8g
1
13
H NMR (ppm)
Imine protons
C NMR (ppm)
Compd. Acylhydrazone
carbonyl
M.p.
°C)
Yield
(%)
(
(
E)
(Z)
-
2.3. Synthesis of Benzothiazole-2-carboxylate (2)
7
a
164.51
164.58
164.35
164.36
164.40
164.51
164.57
164.35
164.54
164.61
164.37
163.80
7.86
7.80
7.91
7.95
7.94
7.94
7.87
8.01
7.94
7.89
8.10
7.99
278 – 280
270 – 271
311 – 313
303 – 305
294 – 296
201 – 202
172 – 174
197 – 199
251 – 252
220 – 221
185 – 187
210 – 211
76
77
82
72
76
77
83
76
84
82
84
78
A mixture of 2-aminothiophenol (0.05 mol) and ethyl ox-
7
b
-
alate (0.1 mol) was stirred at 140 °C for 4 h. After comple-
tion of reaction as indicated by TLC, the reaction mixture
was cooled to room temperature and poured into a solution
containing 50 mL of hydrochloric acid, 150 mL of water and
70 mL of ethanol with stirring, the oil dissolved and a solid
formed. Then the mixture was separated by filtering under
vacuum and washed with water, dried to afford compound 2:
white solid; yield 95%; m.p. 68 – 69°C (lit.[35] 68 – 70°C);
7
c
-
7
d
-
7
e
-
8
a
-
8
b
-
8
c
-
8
d
-
–
IR (KBr, cm ): 1748(C=O), 1500, 1097(C-O), 1012, 722;
1
8
e
-
1
H NMR (DMSO-d ; d, ppm): 8.29 – 8.19 (m, 2H, ArH),
6
8
f
-
7
.72 – 7.61 (m, 2H, ArH), 4.46 (q, J = 7.0 Hz, 2H,
8
g
-
-
CH -C=O), 1.38 (t, J = 7.1 Hz, 3H, -CH ).
2
3

Synthesis and Antiproliferative Activity
349
2
.4. Synthesis of Benzothiazole-2-carbohydrazide (3)
1-Ethyl-1H-indole-3-carbaldehyde (6a): yellow solid;
–1
m.p. 97–99°C (lit.[40] 98–100°C); IR (KBr, cm ): 3102
1
-CH ), 2978 (-CH -), 1695 (C=O), 1447, 781, 748; H NMR
Compound 2 (0.01 mol) was dissolved in ethanol
(
3 2
(
60 mL). hydrazine hydrate (80%) (0.02 mol) was added to
(
DMSO-d ; d, ppm): 9.90 (s, 1H, CHO), 8.35 (s, 1H, ArH),
the solution dropwise with constant stirring and the reaction
mixture was refluxed for 9 h. The reaction mixture was
cooled to room temperature, filtered, washed with water,
dried overnight and finally recrystallized from absolute etha-
nol, filtered and dried to afford compound 3: light yellow
solid; yield 87%; m.p. 173 – 174°C (lit.[36] 172 – 174°C);
6
8
7
4
3
.11 (d, J = 7.8 Hz, 1H, ArH), 7.64 (d, J = 8.2 Hz, 1H, ArH),
.32 (t, J = 7.5 Hz, 1H, ArH), 7.26 (t, J = 7.4 Hz, 1H, ArH),
.31 (q, J = 7.3 Hz, 2H, -CH -), 1.43 (dd, J = 9.3, 5.2 Hz,
2
H, CH3).
1
-Ethyl-5-methoxy-1H-indole-3-carboxaldehyde (6b):
light pink solid; m.p. 100 – 101°C (lit.[40] 97°C).
-Bromo-1-ethyl-1H-indole-3-carbaldehyde
white solid; m.p. 106 – 107°C (lit.[40] 98°C).
-Isopropyl-1H-indole-3-carbaldehyde (6d): yellow
–
IR (KBr, cm ): 3310, 3205 (N-H), 1680 (C=O), 1501, 1201
1
5
(6c):
1
C-N), 1022 (N-N), 750; H NMR (DMSO-d ; d, ppm):
(
6
1
0.46 (s, 1H, N-H), 8.22 (d, J = 7.9 Hz, 1H, ArH), 8.11 (d,
1
J = 8.1 Hz, 1H, ArH), 7.62 (t, J = 7.6 Hz, 1H, ArH), 7.57 (t,
J = 7.5 Hz, 1H, ArH), 4.72 (s, 2H, NH2).
–
solid; m.p. 81 – 83°C; IR (KBr, cm ): 3120 (-CH ),2984
1
3
1
CH aliphatic), 1655 (CHO), 1340, 790, 751; H NMR
(
2
.5. Synthesis of Substituted 1H-Indole-3-carbaldehydes
(DMSO-d ; d, ppm): 9.92 (s, 1H, CHO), 8.47 (s, 1H, ArH),
8.11 (d, J = 7.8 Hz, 1H, ArH), 7.68 (d, J = 8.2 Hz, 1H, ArH),
6
(
5a – 5e)
7
.33 – 7.29 (m, 1H, ArH), 7.26 (t, J = 7.4 Hz, 1H, ArH), 4.90
General procedure. POCl (5mL) was dropped into
3
–
4.81 (m, 1H, CH aliphatic), 1.52 (d, J = 6.6 Hz, 6H, CH3).
DMF (15mL) in an ice bath and stirred for 30 min. After that,
Indole (4a – 4e, 0.01mol) dissolved in 5ml DMF was added
into the mixture and heated to 40°C for 1 h. The resulting re-
action mixture was poured into 50 mL ice water and then ad-
justed to pH 8 using 20% NaOH aq. The solution was filtered
and the residue was washed with water, dried and finally
recrystallized from absolute ethanol, filtered and dried to af-
ford pure compounds 5a – 5e in 95 – 98% yields.
1
-Isopropyl-5-methoxy-1H-indole-3-carbaldehyde (6e):
yellow solid; m.p. 116 – 117°C.
-Bromo-1-isopropyl-1H-indole-3-carbaldehyde (6f):
light green solid; m.p. 117 – 119°C.
-(Phenylsulfonyl)-1H-indole-3-carbaldehyde
5
1
(6g):
1
white solid; m.p. 157 – 158°C; H NMR (DMSO-d ; d,
6
ppm): 10.09 (s, 1H, CHO), 8.91 (s, 1H, phenylsulfonyl), 8.13
(
t, J = 6.9 Hz, 3H, phenylsulfonyl), 7.98 (d, J = 8.3 Hz, 1H,
phenylsulfonyl), 7.78 (t, J = 7.5 Hz, 1H, ArH), 7.67 (t,
J = 7.9 Hz, 2H, ArH), 7.47 (dd, J = 11.5, 4.2 Hz, 1H, ArH),
1
H-Indole-3-carbaldehyde (5a): orange solid; m.p.
1
94 – 196°C (lit.[37] 196 – 198°C); H NMR (DMSO-d ; d,
1
6
ppm): 12.15 (s, 1H, N-H), 9.92 (s, 1H, CHO), 8.27 (s, 1H,
ArH), 8.07 (d, J = 7.7 Hz, 1H, ArH), 7.50 (d, J = 8.0 Hz, 1H,
ArH), 7.25 (t, J = 7.5 Hz, 1H, ArH), 7.20 (t, J = 7.3 Hz, 1H,
ArH).
7
.41 (t, J = 7.5 Hz, 1H, ArH).
2.7. Preparation of target compounds (7a – 7e and 8a – 8g)
General procedure: A mixture of compound 3
5
-Methoxy-1H-indole-3-carbaldehyde (5b): white
solid; m.p. 180 – 182°C (lit.[38] 181 – 182°C).
-Bromo-1H-indole-3-carbaldehyde (5c): light pink
solid; m.p. 206 – 207°C (lit.[37] 202 – 204°C).
-Chloro-1H-indole-3-carbaldehyde (5d): light yellow
solid; m.p. 217 – 218°C (lit.[37] 215 – 216°C).
-Fluoro-1H-indole-3-carbaldehyde (5e): white solid;
m.p. 164 – 165°C (lit.[39] 162 – 163°C).
(
0.002 mol) and compounds 5a – 5e (6a – 6g) (0.0022 mol)
and two drop of glacial acetic acid in ethanol (20 mL) was
heated at reflux for 6 h and then cooled to room temperature,
the precipitate was filtered and washed with diethyl ether.
Finally recrystallized from absolute ethanol and dried in air
to furnish pure compounds 7a – 7e and 8a – 8g.
5
5
5
(
E)-N¢-[(1H-Indol-3-yl)methylene]benzothiazole-2-car-
bohydrazide (7a): yellow solid; yield 76%; m.p.
–
78 – 280°C; IR (KBr, cm ): 3292 (N-H aromatic), 1621
1
2
.6. Synthesis of Substituted 1-Ethyl-1H-indole-3-
carbaldehydes (6a – 6c), Substituted 1-Isopropyl-1H-
indole-3-carbaldehydes (6d – 6f), and 1-(Phenylsulfonyl)-
2
1
C=O), 1607, 1544 (C=N), 1235 (C-N), 1127 (N-N), 745; H
(
NMR (DMSO-d ; d, ppm): 12.40 (s, 1H, N-H), 11.69 (s, 1H,
6
1
H-indole-3-carbaldehyde (6g)
=
N-NH-), 8.82 (s, 1H, ArH), 8.30 (d, J = 7.9 Hz, 1H, ArH),
.28 (d, J = 7.9 Hz, 1H, ArH), 8.20 (d, J = 8.1 Hz, 1H, ArH),
.86 (d, J = 2.7 Hz, 1H, -CH=N-), 7.67 (t, J = 7.6 Hz, 1H,
8
General procedure. A mixture of compound 5a, 5b or
c (0.02 mol) , DMSO (30 mL) and 20% NaOH solution
7
5
ArH), 7.62 (t, J = 7.5 Hz, 1H, ArH), 7.46 (d, J = 7.9 Hz, 1H,
ArH), 7.23 (t, J = 7.3 Hz, 1H, ArH), 7.18 (t, J = 7.3 Hz, 1H,
(
10 mL) was stirred at r.t. for 15 min. After that, bromo-
ethane, 2-bromopropane or benzenesulfonyl chloride was
dropwise added into the mixture and heated to 35°C for 2 – 5
h. After completion of reaction as indicated by TLC, the re-
action mixture was cooled to room temperature and poured
into water (150 mL), stirring until a solid formed. Filtering
under vacuum and washed with water. Recrystallization af-
forded compound 6a – 6g with yields of 85–94 %.
ArH). 13C NMR (DMSO-d ; d, ppm): 164.51 (C=O),
6
155.40, 152.80, 147.43 (C=N-NH-), 137.05, 135.98, 131.05,
127.14, 126.85, 124.33, 123.94, 122.98, 122.73, 121.96,
120.58, 111.87, 111.56.
(E)-N¢-[(5-Methoxy-1H-indol-3-yl)methylene]benzo-
thiazole-2-carbohydrazide (7b): yellow solid; yield 77%;

3
50
Kai Liu et al.
–
1
–1
201 – 202°C; IR (KBr, cm ): 2998 (-CH ), 1648 (C=O),
m.p. 270 – 271°C; IR (KBr, cm ): 3301 (N-H aromatic),
3
1
1608, 1544 (C=N), 1245 (C-N), 1188 (N-N), 762; H NMR
3
261 (-CH ), 1671 (C=O), 1608, 1544 (C=N), 1215 (C-N),
3
1
022(N-N), 764; H NMR (DMSO-d ; d, ppm): 12.37 (s, 1H,
1
(DMSO-d ; d, ppm): 12.38 (s, 1H, =N-NH-), 8.81 (s, 1H,
6
6
N-H), 11.55 (s, 1H, =N-NH-), 8.80 (s, 1H, ArH), 8.28 (d,
J = 8.0 Hz, 1H, ArH), 8.20 (d, J = 8.1 Hz, 1H, ArH), 7.87 (s,
H, ArH), 7.80 (d, J = 2.6 Hz, 1H, -CH=N-), 7.67 (t, J = 7.5
Hz, 1H, ArH), 7.61 (t, J = 7.6 Hz, 1H, ArH), 7.35 (d, J = 8.8
Hz, 1H, ArH), 6.87 (d, J = 8.8 Hz, 1H, ArH), 3.81 (s, 3H,
ArH), 8.31 (d, J = 7.8 Hz, 1H, ArH), 8.27 (d, J = 7.9 Hz, 1H,
ArH), 8.20 (d, J = 8.1 Hz, 1H, ArH), 7.94 (s, 1H, -CH=N-),
7.67 (t, J = 7.6 Hz, 1H, ArH), 7.61 (t, J = 7.5 Hz, 1H, ArH),
7.57 (d, J = 8.2 Hz, 1H, ArH), 7.28 (t, J = 7.5 Hz, 1H, ArH),
7.22 (t, J = 7.4 Hz, 1H, ArH), 4.27 (q, J = 7.2 Hz, 2H,
1
1
CH3). C NMR (DMSO-d ; d, ppm): 164.58 (C=O), 155.35,
3
13
-CH -), 1.42 (t, J = 7.2 Hz, 3H, -CH ). C NMR (DMSO-d6;
6
2
3
1
54.56, 152.82, 147.44 (C=N-NH-), 135.99, 132.01, 131.33,
27.12, 126.83, 124.99, 123.92, 122.98, 112.50, 112.36,
11.34, 104.20, 55.28 (O-C).
E)-N¢-[(5-Bromo-1H-indol-3-yl)methylene]benzothia-
zole-2-carbohydrazide (7c): yellow solid; yield 82%; m.p.
d, ppm): 164.51 (C=O), 155.38, 152.81, 146.95 (C=N-NH-),
136.60, 135.99, 133.15, 127.14, 126.86, 124.89, 123.93,
122.99, 122.76, 122.21, 120.82, 110.75, 110.28, 40.68,
15.19.
1
1
(
(E)-N¢-[(1-Ethyl-5-methoxy-1H-indol-3-yl)methylene]-
benzothiazole-2-carbohydrazide (8b): yellow solid; yield
–1
3
11 – 313°C; IR (KBr, cm ): 3204 (N-H aromatic), 1671
–
83%; m.p. 172 – 174°C; IR (KBr, cm ): 2997 (-CH ), 1648
1
(C=O), 1608, 1544 (C=N), 1255 (C-N), 1185 (N-N), 758; H
1
(
C=O), 1618, 1534 (C=N), 1290 (C-N), 1131 (N-N), 757,
1
21 (C-Br); H NMR (DMSO-d ; d, ppm): 12.46 (s, 1H,
3
6
6
N-H), 11.85 (s, 1H, =N-NH-), 8.77 (s, 1H, ArH), 8.45 (s, 1H,
ArH), 8.26 (d, J = 7.9 Hz, 1H, ArH), 8.18 (d, J = 8.1 Hz, 1H,
ArH), 7.91 (d, J = 2.4 Hz, 1H, -CH=N-), 7.65 (t, J = 7.3 Hz,
NMR (DMSO-d ; d, ppm): 12.36 (s, 1H, =N-NH-), 8.78 (s,
6
1H, ArH), 8.27 (d, J = 8.0 Hz, 1H, ArH), 8.20 (d, J = 8.1 Hz,
1H, ArH), 7.87 (s, 2H -CH=N-), 7.67 (t, J = 7.6 Hz, 1H,
ArH), 7.61 (t, J = 7.6 Hz, 1H, ArH), 7.48 (d, J = 8.8 Hz, 1H,
ArH), 6.92 (d, J = 8.9 Hz, 1H, ArH), 4.22 (q, J = 7.3 Hz, 2H,
1
H, ArH), 7.60 (t, J = 7.6 Hz, 1H, ArH), 7.42 (d, J = 8.6 Hz,
1
3
1
H, ArH), 7.33 (dd, J = 8.6, 1.8 Hz, 1H, ArH). C NMR
13
(
DMSO-d ; d, ppm): 164.35 (C=O), 155.52, 152.80, 146.92
-CH -), 3.82 (s, 3H, -CH ), 1.40 (t, J = 7.2 Hz, 3H).
C
6
2
3
(
C=N-NH-), 135.99, 135.80, 132.42, 127.16, 126.90, 125.96,
NMR (DMSO-d ; d, ppm): 164.57 (C=O), 155.30, 154.79,
6
1
25.27, 124.15, 123.96, 123.01, 113.97, 113.29, 111.17.
E)-N¢-[(5-Chloro-1H-indol-3-yl)methylene]benzothia-
zole-2-carbohydrazide (7d): yellow solid; yield 72%; m.p.
152.81, 146.94 (C=N-NH-), 135.99, 133.30, 131.71, 127.14,
126.84, 125.54, 123.92, 123.00, 112.22, 111.02, 110.34,
104.48, 55.32 (O-C), 40.83, 15.24.
(
–
1
3
1
7
03 – 305°C; IR (KBr, cm ): 3201 (N-H aromatic),
(E)-N¢-[(5-Bromo-1-ethyl-1H-indol-3-yl)methylene]-
benzothiazole-2-carbohydrazide (8c): yellow solid; yield
–1
671(C=O), 1604, 1544 (C=N), 1291 (C-N), 1131 (N-N),
1
57, 728(C-Cl); H NMR (DMSO-d ; d, ppm): 12.48 (s, 1H,
76%; m.p. 197 – 199°C; IR (KBr, cm ): 3102 (-CH ), 1671
6
3
N-H), 11.87 (s, 1H, =N-NH-), 8.80 (s, 1H, ArH), 8.32 (s, 1H,
ArH), 8.28 (d, J = 7.9 Hz, 1H, ArH), 8.20 (d, J = 8.0 Hz, 1H,
ArH), 7.95 (d, J = 2.5 Hz, 1H, -CH=N-), 7.67 (t, J = 7.3 Hz,
(C=O), 1608, 1544 (C=N), 1201 (C-N), 1022 (N-N), 757,
1
621 (C-Br); H NMR (DMSO-d ; d, ppm): 12.46 (s, 1H,
6
=N-NH-), 8.78 (s, 1H, ArH), 8.48 (s, 1H, ArH), 8.28 (d,
J = 8.0 Hz, 1H, ArH), 8.20 (d, J = 8.1 Hz, 1H, ArH), 8.01 (s,
1H, -CH=N-), 7.67 (t, J = 7.5 Hz, 1H, ArH), 7.62 (t, J = 7.3
Hz, 1H, ArH), 7.59 (d, J = 8.7 Hz, 1H, ArH), 7.41 (d, J = 8.5
1
H, ArH), 7.62 (t, J = 7.5 Hz, 1H, ArH), 7.48 (d, J = 8.6 Hz,
1
3
1
H, ArH), 7.24 (d, J = 8.6 Hz, 1H, ArH). C NMR
(
DMSO-d ; d, ppm): 164.36 (C=O), 155.53, 152.80, 146.91
6
(
C=N-NH-), 135.99, 135.54, 132.57, 127.16, 126.89, 125.33,
Hz, 1H, ArH), 4.26 (q, J = 7.1 Hz, 2H, CH ), 1.40 (t, J = 7.2
2
1
Hz, 3H, CH3). C NMR (DMSO-d ; d, ppm): 164.35 (C=O),
3
1
25.25, 123.95, 123.00, 122.72, 121.12, 113.50, 111.28.
E)-N¢-[(5-Fluoro-1H-indol-3-yl)methylene]benzothia-
zole-2-carbohydrazide (7e): yellow solid; yield 76%; m.p.
6
(
155.51, 152.79, 146.45 (C=N-NH-), 136.00, 135.41, 134.42,
127.17, 126.90, 126.39, 125.25, 124.36, 123.95, 123.01,
113.61, 112.50, 110.35, 40.93, 15.14.
–1
2
94 – 296°C; IR (KBr, cm ): 3189 (N-H aromatic), 1680
(
C=O), 1610, 1544 (C=N), 1317 (C-N), 1288 (C-F), 1044
1
N-N), 764; H NMR (DMSO-d ; d, ppm): 12.44 (s, 1H,
(E)-N¢-[(1-Isopropyl-1H-indol-3-yl)methylene]benzo-
thiazole-2-carbohydrazide (8d): yellow solid; yield 84%;
(
6
–
m.p. 251 – 252°C; IR (KBr, cm ): 3190 (-CH ), 1657
1
N-H), 11.77 (s, 1H, =N-NH-), 8.80 (s, 1H, ArH), 8.28 (d,
J = 7.9 Hz, 1H, ArH), 8.20 (d, J = 8.1 Hz, 1H, ArH), 8.01 (d,
J = 9.7 Hz, 1H, ArH), 7.94 (s, 1H, -CH=N-), 7.67 (t, J = 7.5
Hz, 1H, ArH), 7.62 (t, J = 7.5 Hz, 1H, ArH), 7.47 (dd,
3
(C=O), 1648, 1544 (C=N), 1188 (C-N,), 1021 (N-N), 754;
1
H NMR (DMSO-d ; d, ppm): 12.38 (s, 1H, =N-NH-), 8.81
6
(s, 1H, ArH), 8.31 (d, J = 7.8 Hz, 1H, ArH), 8.27 (d, J = 7.9
Hz, 1H, ArH), 8.20 (d, J = 8.1 Hz, 1H, ArH), 7.94 (s, 1H,
-CH=N-), 7.67 (t, J = 7.6 Hz, 1H, ArH), 7.61 (t, J = 7.5 Hz,
1H, ArH), 7.57 (d, J = 8.2 Hz, 1H, ArH), 7.28 (t, J = 7.5 Hz,
1H, ArH), 7.22 (t, J = 7.4 Hz, 1H, ArH), 4.27 (q, J = 7.2 Hz,
1
3
J = 8.6, 4.5 Hz, 1H, ArH), 7.08 (t, J = 8.2 Hz, 1H, ArH).
C
NMR (DMSO-d ; d, ppm): 164.40 (C=O), 155.52, 152.80,
6
1
1
1
46.98 (C=N-NH-), 135.99, 133.66, 132.69, 127.12, 126.85,
23.93, 122.96, 113.05, 111.70, 110.95, 110.75, 106.85,
06.66.
1
1H, CH), 1.42 (t, J = 7.2 Hz, 6H, CH3). C NMR
3
(
E)-N¢-[(1-Ethyl-1H-indol-3-yl)methylene]benzothia-
(DMSO-d ; d, ppm): 164.54 (C=O), 155.41, 152.81, 147.01
6
zole-2-carbohydrazide (8a): yellow solid; yield 77%; m.p.
(C=N-NH-), 136.45, 135.98, 130.18, 127.14, 126.86, 124.84,

Synthesis and Antiproliferative Activity
351
1
23.94, 122.99, 122.70, 122.24, 120.86, 111.07, 110.40,
7.04, 22.28.
E)-N¢-[(1-Isopropyl-5-methoxy-1H-indol-3-yl)methyl-
ene]benzothiazole-2-carbohydrazide (8e): yellow solid;
mors) was used as positive controls. The results presented as
IC50 values are summarized in Table 2, where the values
show average of at least two independent experiments. As
can be seen from Table 2, the results indicated that part of the
synthesized target compounds showed moderate to excellent
cytotoxic activity against Hep G2 cancer cell line. In general,
when the C-5 position of indole was substituted by elec-
tron-donating group, this was not beneficial to the antiproli-
ferative activity. Notably, compound 7a exhibited the best
antiproliferative activity with IC50 values of 0.78 mM for
Hep G2 cells, and thus was more potent than the positive
control compound sunitinib. Furthermore, electronic and
steric effects of the N-1 position of indole were studied by in-
4
(
–1
yield 82%; m.p. 220 – 221°C; IR (KBr, cm ): 3267 (-CH3),
1
7
8
680 (C=O), 1550, 1544 (C=N), 1203 (C-N), 1023 (N-N),
1
54; H NMR (DMSO-d ; d, ppm): 12.40 (s, 1H, =N-NH-),
6
.79 (s, 1H, ArH), 8.27 (d, J = 7.9 Hz, 1H, ArH), 8.20 (d,
J = 8.1 Hz, 1H, ArH), 7.95 (s, 1H, ArH), 7.89 (d, J = 2.0 Hz,
H, -CH=N-), 7.67 (t, J = 7.5 Hz, 1H, ArH), 7.61 (t, J = 7.5
Hz, 1H, ArH), 7.51 (d, J = 8.9 Hz, 1H, ArH), 6.93 – 6.90 (m,
H, ArH), 4.76 (dd, J = 13.2, 6.5 Hz, 1H, CH), 3.82 (s, 3H,
1
1
13
CH ), 1.49 (d, J = 6.6 Hz, 6H, CH ). C NMR (DMSO-d6;
3
3
2
troduction of various R groups. The results revealed that in-
d, ppm): 164.61 (C=O), 155.34, 154.77, 152.82, 147.00
C=N-NH-), 135.99, 131.54, 130.35, 127.13, 126.84, 125.48,
23.92, 122.99, 112.15, 111.11, 110.64, 104.50, 55.32 (O-C),
7.20, 22.31.
E)-N¢-[(5-Bromo-1-isopropyl-1H-indol-3-yl)methyl-
ene]benzothiazole-2-carbohydrazide (8f): yellow solid;
(
creasing size of substituent at the N-1 position of indole
1
clearly decreased the antiproliferative activity (7a vs. 8a,
4
8d), which suggested that steric hindrance of the group in
2
(
this region of R groups exhibited a negative effect on the
cytotoxic activity. On the whole, it seemed that the C-5 sub-
–1
yield 84%; m.p. 185 – 187°C; IR (KBr, cm ): 3067 (-CH3),
1
648 (C=O), 1610, 1544 (C=N), 1197 (C-N), 1025 (N-N),
1
60, 625 (C-Br); H NMR (DMSO-d ; d, ppm): 12.50 (s, 1H,
7
6
TABLE 2. Antiproliferative Activity of Target compounds 7a – 7e
and 8a – 8g against Hep G2 Cancer Cell Line
=
N-NH-), 8.79 (s, 1H, ArH), 8.49 (s, 1H, ArH), 8.28 (d,
J = 7.9 Hz, 1H, ArH), 8.20 (d, J = 8.0 Hz, 1H, ArH), 8.10 (s,
H, -CH=N-), 7.67 (t, J = 7.5 Hz, 1H, ArH), 7.62 (t, J = 7.1
Hz, 2H, ArH), 7.42 – 7.38 (m, 1H, ArH), 4.81 (dt, J = 13.1,
1
IC50 (mmol/L)
Compd.
R
1
R
2
Hep G2
13
6
.6 Hz, 1H, CH), 1.50 (d, J = 6.6 Hz, 6H, CH3). C NMR
7
a
H
5-OCH
5-Br
5-Cl
5-F
0.78 ± 0.26*
>50
(
DMSO-d ; d, ppm): 164.37 (C=O), 155.54, 152.80, 146.51
6
7
b
3
(
C=N-NH-), 136.00, 135.23, 131.50, 127.15, 126.89, 126.33,
1
25.19, 124.40, 123.95, 123.00, 113.59, 112.53, 110.69,
7.42, 22.22.
E)-N¢-[(1-(Phenylsulfonyl)-1H-indol-3-yl)methylene]-
benzothiazole-2-carbohydrazide (8g): yellow solid; yield
7c
>50
4
7d
>50
(
7
e
35.15 ± 10.28
16.79 ± 4.08*
8
a
H
–1
7
8%; m.p. 210 – 211°C; IR (KBr, cm ): 3292, 3055, 1621
(
C=O), 1607, 1544 (C=N), 1290 (S=O), 1195 (C-N), 1022
1
N-N), 745; H NMR (DMSO-d ; d, ppm): 12.82 (s, 1H,
8b
5-OCH
5-Br
H
3
>50
>50
(
6
=
N-NH-), 8.84 (s, 1H, ArH), 8.42 (d, J = 5.7 Hz, 2H, ArH),
.28 (d, J = 7.9 Hz, 1H, ArH), 8.22 (d, J = 8.1 Hz, 1H, ArH),
.09 (d, J = 7.7 Hz, 2H, ArH), 7.99 (d, J = 8.2 Hz, 1H,
CH=N-), 7.74 (t, J = 7.5 Hz, 1H, ArH), 7.68 (t, J = 7.2 Hz,
H, ArH), 7.63 (td, J = 7.6, 4.3 Hz, 3H, ArH), 7.46 (t, J = 7.4
8
c
8
8
-
8d
20.93 ± 3.47
1
13
Hz, 1H, ArH), 7.41 (t, J = 7.5 Hz, 1H, ArH). C NMR
8
e
5-OCH
5-Br
H
3
>50
>50
>50
(
DMSO-d ; d, ppm): 163.80 (C=O), 156.10, 152.73, 144.79
6
(
C=N-NH-), 136.64, 136.04, 134.96, 134.72, 130.53, 129.98,
1
1
27.24, 127.07, 126.89, 126.84, 125.95, 124.39, 124.06,
23.29, 123.04, 118.02, 113.08.
8
f
3
. EXPERIMENTAL BIOLOGICAL PART
8g
By using MTT assay, all target compounds were evalu-
ated for their in vitro antiproliferative activity against Hep
G2 (human liver cancer) cells, and sunitinib (a multi-targeted
tyrosine kinase inhibitor possessing anti-angiogenic and
antitumor activity against a variety of advanced solid tu-
a
sunitinib
3.22 ± 0.52
a
p < 0.05 compared to the value for sunitinib; positive control.
*

3
52
Kai Liu et al.
1
0. U. A. Matulonis, S. Berlin, P. Ivy, et al., J. Clin. Oncol., 27(33),
stitution of the indole ring of compounds might be crucial for
their cytotoxic activities.
5
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1
In view of above observation, these cytotoxic activity re-
sults provided a valuable leading compound 7a with excel-
lent antiproliferative activity, and highlighted the potential
for further development of new N-acylhydrazone derivatives
as potent antitumor agents.
1
1
1
1
1
1
3. P. P. Prabhu, S. Pande and C. S. Shastry, Int. J. Chem. Tech. Res.,
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4
. RESULTS AND DISCUSSION
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In this study, a series of N-acylhydrazone derivatives
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designed, synthesized and evaluated for the IC50 values
against Hep G2 cancer cell line. The relative configuration of
target compounds was confirmed as the E isomer. Addi-
tionally, we optimized the synthesis of N-substituted indoles
to reach a high yield that has not been reported before.
17. S. R. Pattan, C. H. Suresh, V. D. Pujar, et al., Indian J. Chem. B.,
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1
2
2
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1
Part of the synthesized target compounds showed moder-
ate to excellent cytotoxic activity. In general, it seemed that
the C-5 substitutions of the indole-ring of these compounds
might be crucial for their cytotoxic activity. Notably, com-
pound 7a showed better activity against Hep G2 cancer cell
line than the positive control drug sunitinib. Thus, compound
2
2
7
a emerged as a valuable lead for further structural modifica-
1
tions. Further studies on structure optimization are underway
in our laboratory. Overall, the structurally new N-acylhydra-
zone derivatives provide us promising lead compounds for
future anticancer drug discovery.
2
2
2
2
2
2
2
3
3
3
3
3
3
3
4
ACKNOWLEDGEMENTS
3
The project was funded by the key Laboratory of Marine
Bioactive Substances and Modern Analysis Technology,
SOA (MBSMAT-2017-06)
(
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