Synthesis and antiproliferative evaluation of piperazine-1- carbothiohydrazide derivatives of indolin-2-one

Article abstract of DOI:10.1016/j.bmcl.2013.03.099

By varying the substituents (R¹) at the indolin-2-one scaffold, a series of indolin-2-one derivatives bearing 4-phenylpiperazine-1- carbothiohydrazide moiety at the C3-position were synthesized and evaluated for their antiproliferative activity against three human cancer cell lines. We further selected the 5-chloroindolin-2-one moiety for the extension to another series of compounds by varying the substituents (R²) at the phenyl group connected with the piperazine ring. Among all the compounds synthesized, 6d and 6l were most potent with IC₅₀ values of 3.59 and 5.58 μM, respectively against A549 lung cancer cells, while 5f and 6l possessed IC ₅₀ values of 3.49 and 4.57 μM, respectively against HCT-116 colon cancer cells which were comparable to that of Sunitinib, an indolin-2-one derivative in cancer therapy.

Full text of DOI:10.1016/j.bmcl.2013.03.099

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3304–3307
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antiproliferative evaluation of piperazine-
1-carbothiohydrazide derivatives of indolin-2-one
Hui-Hui Lin a, Wei-Yao Wu a,b, Sheng-Li Cao a,b, , Ji Liao b,c, Li Ma a, Man Gao a,b
,
⇑
Zhong-Feng Li a, Xingzhi Xu b,c,
⇑
a Department of Chemistry, Capital Normal University, Beijing 100048, China
b Beijing Key Laboratory of DNA Damage Response, Capital Normal University, Beijing 100048, China
c College of Life Science, Capital Normal University, Beijing 100048, China
a r t i c l e i n f o
a b s t r a c t
By varying the substituents (R1) at the indolin-2-one scaffold, a series of indolin-2-one derivatives bear-
ing 4-phenylpiperazine-1-carbothiohydrazide moiety at the C3-position were synthesized and evaluated
for their antiproliferative activity against three human cancer cell lines. We further selected the 5-chlor-
oindolin-2-one moiety for the extension to another series of compounds by varying the substituents (R2)
at the phenyl group connected with the piperazine ring. Among all the compounds synthesized, 6d and 6l
Article history:
Received 23 September 2012
Revised 17 March 2013
Accepted 26 March 2013
Available online 4 April 2013
were most potent with IC50 values of 3.59 and 5.58
5f and 6l possessed IC50 values of 3.49 and 4.57
which were comparable to that of Sunitinib, an indolin-2-one derivative in cancer therapy.
Ó 2013 Elsevier Ltd. All rights reserved.
l
M, respectively against A549 lung cancer cells, while
Keywords:
Indolin-2-one
Piperazine-1-carbothiohydrazide
Synthesis
lM, respectively against HCT-116 colon cancer cells
Antiproliferative activity
Indolin-2-one has been shown to be an attractive scaffold of a
class of new antitumor agents,1 most of which appear to act as
inhibitors of various protein kinase families, particularly receptor
tyrosine kinases (RTKs) and serine/threonine-specific protein ki-
nases such as the cyclin-dependent kinases (CDKs).2 For example,
Sunitinib (SU11248, Fig. 1) is the first kinase inhibitor of the indo-
lin-2-one type targeting at multiple kinases, which has been ap-
proved for the treatment of renal cell carcinoma and
gastrointestinal stromal tumor.3 On the other hand, thiosemicarba-
zone derivatives are known to possess a potent antitumor activ-
ity,4–7 which have attracted considerable attention in the
development of antitumor agents. For example, 3-aminopyridine-
2-carboxaldehyde thiosemicarbazone (3-AP, Fig. 1) is a potent
ribonucleotide reductase inhibitor that has entered phase II clinical
trials for the treatment of a number of cancer, including non-small-
cell lung cancer and renal carcinoma.8,9 Recently, the incorporation
of thiosemicarbazone moiety into the C3-position of indolin-2-one
has proven to be an efficient strategy to design novel antitumor
agents.10–14 In additon, we have synthesized a number of 2-
methyl-4-oxoquinazoline or 2,4-diaminoquinazoline derivatives
bearing 4-substituted-piperazine-1-carbodithioate side chains (I,
II, Fig. 1).15,16 Some of the synthesized compounds significantly
inhibited the proliferation of human tumor cells in culture, sug-
gesting that the piperazine-1-carbodithioate moiety might con-
tribute to the cytotoxic activity. In view of these observations,
we integrated the structural features of indolin-2-one, thiosemi-
carbazone and piperazine-1-carbodithioate to design and synthe-
size a new class of piperazine-1-carbothiohydrazide derivatives
of indolin-2-one, aiming at the identification of novel small mole-
cules with potent antiproliferative effects on tumor cells.
As shown in Scheme 1, methyl hydrazinecarbodithioate 1 was
prepared by reaction of 85% hydrazine hydrate, carbon disulfide
and iodomethane in isopropanol/water in the presence of potas-
sium hydroxide, according to the procedure reported by Klayman
et al.17 Reaction of 1 with 1-phenylpiperazine 2a in ethanol at re-
flux afforded 4-phenylpiperazine-1-carbothiohydrazide 3a, which
was condensed with various indoline-2,3-diones 4a–l to give the
first series of target compounds 5a–l. The results of cell prolifera-
tive inhibition assay showed that compound 5f was the most po-
tent among this series (Table 1). Consequently, reaction of 1 with
various 1-substituted piperazines 2b–m gave intermediates 3b–
m, which reacted with 5-chloroindoline-2,3-dione 4f, respectively,
to furnish the second series of target compounds 6a–l. (For syn-
thetic procedures and spectroscopic data of compounds 3a–m,
5a–l and 6a–l, see Supplementary data.)
The target compounds may exist as either the Z or E-isomers
due to the exocyclic C@N double bond at the C3 position of indo-
lin-2-one.18 The 1H NMR spectra of 5a–l and 6a–l displayed the
chemical shifts of the NH protons in carbothiohydrazide moiety
⇑
Corresponding authors. Tel./fax: +86 10 68902974 (S.-L. Cao); tel./fax: +86 10
68902240 (X. Xu).
E-mail addresses: slcao@cnu.edu.cn (S.-L. Cao), xingzhi_xu@mail.cnu.edu.cn
(X. Xu).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.099

H.-H. Lin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3304–3307
3305
Figure 1. Structures of Sunitinib, 3-AP, compounds I, II and target compounds 5, 6.
Scheme 1. Reagents and conditions: (a) CS2, CH3I, KOH, i-PrOH/H2O; 0 °C, 2 h; (b) 1-phenylpiperazine (2a), EtOH, reflux, 10 h; (c) indoline-2,3-diones 4a–l, EtOH, reflux, 10 h;
(d) 1-substituted-piperazines 2b–m, EtOH, reflux, 5–10 h; (e) 5-chloroindoline-2,3-dione (4f), EtOH, reflux, 10 h.
ranging from 13.06 to 13.31 ppm as single singlets, indicating that
these compounds were obtained as pure geometrical isomers.
Compared to the chemical shifts of the NH protons in intermedi-
ates 3a–m (d 9.12–9.21 ppm), the downfield shift of the NH
protons in carbothiohydrazide moiety of 5a–l and 6a–l (d 13.06–
13.31 ppm) was due to the deshielding effect of the adjacent
carbonyl group (Fig. 2). Therefore, we believe that compounds
5a–l and 6a–l exist as the thermodynamically stable Z-isomers
forming a six-membered ring with an intramolecular hydrogen
bond between the NH proton and the C2 carbonyl oxygen atom
of indolin-2-one (Fig. 2), and this configuration is in agreement
with the crystal structure of the similar compounds reported by
Hall et al.14
The MTT [3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl-tetrazo-
lium bromide] cell proliferation assay was used to evaluate the
antiproliferative activity of the synthesized compounds against
three human cancer cell lines including A549 (lung cancer), MCF-
7 (breast adenocarcinoma) and HCT-116 (colorectal cancer) cell
lines (see Supplementary data). The inhibition of cell proliferation
was determined 72 h after cells were exposed to the tested com-
while maintaining the phenyl ring at the N4-position of piperazine
to synthesize a series of target compounds 5a–l (Scheme 1) and
evaluate their antiproliferative activity. As shown in Table 1, the
unsubstituted compound 5a exhibited moderate inhibitory activity
against the proliferation of A549, MCF-7 and HCT-116 cell lines
with IC50 values of 18.56, 24.86 and 11.13 lM, respectively. Intro-
duction of an electron-donating group of methyl (5b) or methoxyl
(5c) into the 5-position of indolin-2-one ring led to an obvious in-
crease in antiproliferative activity against A549 and MCF-7 cells,
and a slight increase against HCT-116 cells, in comparison to the
parent compound 5a. Compounds 5g and 5h bearing a strong elec-
tron-withdrawing group of a fluoro or nitro group, respectively, at
the 5-position of indolin-2-one ring exhibited a decreased or sim-
ilar antiproliferative activity compared to compound 5a. However,
introduction of a weak electron-withdrawing substituent of bromo
(5d) into the 5-position of indolin-2-one ring resulted in an in-
crease in the antiproliferative activity. Further introduction of a
bromo substituent into the 7-position of indolin-2-one ring of 5d
resulted in compound 5e, which possessed higher antiproliferative
activity against three cell lines than 5d. Interestingly, compound 5f
possessing a chloro substituent at the 5-position of indolin-2-one
ring yielded comparable potency to 5d against A549 and MCF-7
cells, but more potent than 5d against HCT-116 cells with an IC50
pounds at a concentration of 100 lM. The compounds with 50%
or more inhibition compared with vehicle-treated cells were con-
sidered active. Inhibition of cell proliferation by these active com-
pounds at various concentrations were further measured, and their
IC50 (the concentration that causes 50% of cell proliferation inhibi-
tion) values were determined and summarized in Table 1. Suniti-
nib was used as a positive control.
To explore the structure–activity relationship and optimize the
structures of piperazine-1-carbothiohydrazide derivatives of indo-
lin-2-one, we first varied the substituents at the indolin-2-one ring
value of 3.49 lM. These results suggest that an electron-donating
group, a weak or medium electron-withdrawing group is more
favorable for enhancing the activity than a strong electron-with-
drawing group.
Compared to 4-substituted compounds 5i (R1 = 4-Br) and 5j
(R1 = 4-Cl), 6-substituted compounds 5k (R1 = 6-Br) and 5l
(R1 = 6-Cl) were more active, suggesting that substituents at the

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H.-H. Lin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3304–3307
Table 1
ring is important for antitumor activity. We thus selected 5-chlor-
oindolin-2-one moiety to serve as a template for further investiga-
tion of structure–activity relationship.
By maintaining the 5-chloro substituent at the indolin-2-one
moiety, we introduced various substituents into the phenyl ring
at the N4-position of piperazine ring to generate the second series
of target compounds 6a–l (Scheme 1) and evaluated for their anti-
proliferative activity. As shown in Table 1, the IC50 values of this
series of compounds against three cell lines were generally lower
Antiproliferative activity of compounds 5a–l and 6a–l against three human cancer cell
lines
S
N
N
N
N
H
R2
R1
O
N
H
5a-l 6a-l
,
than 20 lM, furthermore, these compounds were more active than
the parent compound 5a. In particular, the antiproliferative activity
Compound R1
R2
IC50 (lM) SD
a
A549
MCF-7
HCT-116
of compounds 6a–l against A549 cells were obviously more potent
than the first series of compounds 5a–l (IC50, 3.59–11.77
7.34–55.93 M), with compound 6d being the most potent with
an IC50 value of 3.59 M. In fact, the second series of compounds
were generally more potent against HCT-116 cells (IC50, 4.57–
lM vs
5a
5b
5c
H
5-CH3
5-
H
H
H
18.56 1.70 24.86 0.28 11.13 2.89
9.88 0.82 14.86 1.14
7.52 0.54 14.41 0.17
9.48 0.17
9.53 0.73
l
l
OCH3
5-Br
5,7-
diBr
5-Cl
5-F
5-NO2
4-Br
4-Cl
6-Br
6-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5d
5e
H
H
12.25 1.42 20.50 3.21 10.39 0.99
12.0
42.14
(IC50, 4.91
ity to the positive control Sunitinib (IC50, 4.71
l
l
M) than the first series of compounds (IC50
M) as well, of which compounds 6c (IC50, 4.91
M) and 6l (IC50, 4.57
,
l
3.49–
M), 6f
6.11 1.87 11.55 0.94
6.32 0.56
5f
H
H
H
H
H
H
H
7.34 2.18 10.39 1.84
3.49 1.26
l
l
M) exhibited comparable activ-
5g
5h
5i
5j
5k
5l
6a
6b
6c
6d
20.73 4.25 24.30 5.05 12.35 0.11
17.91 2.97 23.33 5.41 15.09 0.68
53.97 4.95 40.69 0.47 33.60 5.74
55.93 5.26 52.07 2.23 42.14 2.67
15.84 2.82 19.65 2.56 14.51 1.53
27.65 2.78 22.98 0.62 17.86 0.10
lM).
As compared with compound 5f with no substituent at the phe-
nyl ring, the antiproliferative activity of compounds 6a, 6b, 6e, 6h,
and 6k bearing a methoxyl, methyl, chloro, fluoro, or nitro group at
the 40-position of the phenyl ring were in general less active
against three cell lines. However, compounds 6c, 6f and 6l bearing
a methyl, chloro or nitro group at the 20-position of the phenyl ring
were more potent than their 4-substituted counterparts 6b, 6e and
6k against three tumor cell lines and comparable to compound 5f,
whereas 6i (R2 = 20-F) was less potent than 6h (R2 = 40-F). In addi-
tion, introduction of two substituents into both the 20- (or 30- for
6g) and 40-position of the phenyl ring resulted in compounds 6d,
6g and 6j, of which only 6d containing 2,4-dimethylphenyl group
4-OCH3
4-CH3
2-CH3
2,4-
diCH3
4-Cl
2-Cl
2,4-diCl
4-F
7.68 1.28 13.79 1.99
10.13 0.82 17.13 2.30
6.52 1.24 10.90 2.36
3.59 0.77 10.83 1.86
7.74 0.89
6.39 0.81
4.91 0.49
7.18 0.39
6e
6f
6g
6h
6i
6j
6k
6l
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
5-Cl
8.18 0.42 13.10 0.35
7.92 0.59 11.73 1.30
7.95 0.52 10.82 0.18
8.90 0.49 12.32 0.54
10.51 0.83 15.23 1.30
11.38 0.81 20.06 1.56
11.77 0.90 20.86 1.68
5.39 1.09
4.91 0.36
5.82 0.67
6.35 0.10
8.29 1.23
5.89 1.11
12.0 0.86
4.57 0.44
4.71 0.32
2-F
2,4-diF
4-NO2
2-NO2
was more active against A549 cells (IC50, 3.59
lM) than compound
5f (IC50, 7.34 M), whereas 6g (3,4-dichlorophenyl) and 6j (2,4-
l
5.58 0.22
2.44 0.22
7.49 1.06
6.29 0.22
difluorophenyl) exhibited comparable or weaker activity against
three cell lines compared to 5f. These results suggest that the pres-
ence of a bulky group at the 20-position of the phenyl ring is favor-
able for retaining the potency, and the electronic effect of
substituents was not important for the antiproliferative activity.
In order to investigate the effect of the piperazine moiety on
antiproliferative activity, we synthesized compounds 9a–c that
are the analogs having no piperazine moiety of representative
compounds 5a, 5f and 6d. As illustrated in Scheme 2, treatment
of 1-isothiocyanatobenzene 7a or 1-isothiocyanato-2,4-dimethyl-
benzene 7b with 85% hydrazine hydrate to give N-substituted
hydrazinecarbothioamides 8a,b, which were condensed with indo-
line-2,3-dione 4a or 5-chloroindoline-2,3-dione 4f, respectively to
furnish 9a–c (see Supplementary data). Unfortunately, the reaction
of 1-isothiocyanato-2-nitrobenzene with 85% hydrazine hydrate
did not take place at room temperature or even at reflux, so that
we failed to prepare the counterpart of the active compound 6d.
It can be seen from Table 2 that compounds 9a and 9c were
inactive against the proliferation of A549, MCF-7 and HCT-116 cells
Sunitinib
a
The concentration that causes 50% of cell proliferation inhibition. Data are
expressed as means SD of three separate experiments.
Figure 2. Compounds 5a–l and 6a–l adopt the Z-configuration due to the formation
of a hydrogen bond.
6-position of indolin-2-one ring are favorable for the antiprolifera-
tive activity. Nevertheless, both compounds 5i (R1 = 4-Br) and 5k
(R1 = 6-Br) were generally less active than compound 5d (R1 = 5-
Br). Similarly, compounds 5j (R1 = 4-Cl) and 5l (R1 = 6-Cl) were less
potent than compound 5f (R1 = 5-Cl). These results indicate that
the presence of a substituent at the 5-position of indolin-2-one
(IC50 > 100
ate or strong antiproliferative activity against three cell lines (IC50
3.49–24.86 M). Compound 9b showed moderate antiproliferative
lM), whereas their analogs 5a and 6d exhibited moder-
,
l
Scheme 2. Reagents and conditions: (a) 85% hydrazine hydrate, i-PrOH, rt, 1-5 h; (b) indoline-2,3-diones 4a,f, EtOH, acetic acid, reflux, 10 h.

H.-H. Lin et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3304–3307
3307
Table 2
Acknowledgements
Antiproliferative activity of compounds 9a–c against three human cancer cell lines
S
This work was supported by National Natural Science Founda-
tion of China (Project Nos. 20972099 and 31130017), Beijing Muni-
cipal Commission of Education (Project No. KZ201210028035), the
973 project 2013CB911000, and the Funding Project for Academic
Human Resources Development in Institutions of Higher Learning
Under the Jurisdiction of Beijing Municipality (PHR20110508).
N
H
N
N
H
R2
R1
O
N
H
9a-c
Compound
R1
R2
IC50 (lM) SD
Supplementary data
A549
MCF-7
HCT-116
9a
9b
9c
H
5-Cl
5-Cl
H
H
>100a
33.14 1.04
>100
>100
35.00 0.90
>100
>100
30.76 0.21
>100
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.03.
099.
2,4-diCH3
a
IC50 >100
lM indicates that cell proliferation inhibition is lower than 50% at the
concentration of 100
l
M.
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7.34, 10.39 and 3.49 lM against A549, MCF-7 and HCT-116 cells,
respectively. Moreover, introduction of a substituent (R2) into the
20-position, instead of 40-position, of the phenyl group connected
with piperazine ring was favorable for retaining the activity and
the steric effect of substituents was more important for the
potency than the electronic effect. Thus, compounds 6c (R2 = 20-
CH3), 6f (R2 = 20-Cl) and 6l (R2 = 20-NO2) exhibited comparable
antiproliferative activity against HCT-116 cells to Sunitinib, while
compound 6d (R2 = 20,40-diCH3) was the most potent against
A549 cells. Further studies to investigate the possible mechanism
of action of the active compounds are currently in progress.
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