Synthesis, in vitro and in vivo antitumor activity of symmetrical bis-Schiff base derivatives of isatin

Article abstract of DOI:10.1016/j.ejmech.2013.04.040

Eighteen symmetrical bis-Schiff base derivatives of isatin were synthesized by condensation of the natural or synthetic isatins with hydrazine and were evaluated for their in vitro and in vivo antitumor activities. More than half of the obtained compounds showed potent cytotoxicity according to the MTT assay on five different human cancer cell lines (i.e. HeLa, SGC-7901, HepG2, U251, and A549), with compound 3b 3,3′-(hydrazine-1,2-diylidene)bis (5-methylindolin-2-one) being the most potent compound on HepG2 (IC₅₀ ～ 4.23 μM). 3b was also found to be able to inhibit substantially the tumor growth on the HepS-bearing mice at a dose of 40 mg/kg. The real-time live cell imaging and tracking in the H₂B-labeled HeLa cells revealed that 3b could induce mitosis interference and apoptosis-associated cell death. In mechanism study, 3b arrested the cell cycle at the G2/M phase in HepG2 cells by down-regulating the expression of cyclin B1 and cdc 2.

Full text of DOI:10.1016/j.ejmech.2013.04.040

Accepted Manuscript
Synthesis, in vitro and in vivo antitumor activity of symmetrical bis-Schiff base
derivatives of isatin
Chengyuan Liang, Juan Xia, Dong Lei, Xiang Li, Qizheng Yao, Jing Gao
PII:
S0223-5234(13)00272-9
10.1016/j.ejmech.2013.04.040
EJMECH 6150
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 November 2012
Revised Date: 2 March 2013
Accepted Date: 18 April 2013
Please cite this article as: C. Liang, J. Xia, D. Lei, X. Li, Q. Yao, J. Gao, Synthesis, in vitro and in vivo
antitumor activity of symmetrical bis-Schiff base derivatives of isatin, European Journal of Medicinal
Chemistry (2013), doi: 10.1016/j.ejmech.2013.04.040.
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Synthesis, in vitro and in vivo antitumor activity of novel symmetrical
bis-Schiff base derivatives of isatin
Chengyuan Lianga,†, Juan Xiab,†, Dong Leia, Xiang Lib, Qizheng Yaoa,*, Jing Gaob,*
a School of Pharmacy, China Pharmaceutical University, Nanjing 210009, People’s Republic of China;
b School of Pharmacy, Jiangsu University, Zhenjiang 212013, People’s Republic of China;
† These two authors contributed equally to this work
*Correspondence: Q.Z. Yao: E-mail: qz_yao@yahoo.com.cn; Phone/Fax, +86-25-86634730;
J. Gao: E-mail: jinggao@ujs.edu.cn; Phone/Fax,+86-511-88791552;
Novel bis-isatins Schiff base analogues were designed and synthesized. Most of these
derivatives possessed superior in vitro and moderate in vivo antitumor activities based upon the
interference of mitosis.

highlights.docx
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Research Highlights
1. Conformational flexible bis-isatins Schiff base analogs.
2. Superior in vitro and moderate in vivo antitumor activity.
3. 3b induced mitosis interference and cell death related to apoptosis.
4. The long-term real-time live cell imaging was first presented.
5. 3b arrested the cell cycle at the G2/M phase in HepG2 cells.

EJMECH-D-12-01706.docx
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Synthesis, in vitro and in vivo antitumor activity of symmetrical
bis-Schiff base derivatives of isatin
Chengyuan Lianga,†, Juan Xiab,†, Dong Leia, Xiang Lib, Qizheng Yaoa,*, Jing Gaob,*
a School of Pharmacy, China Pharmaceutical University, Nanjing 210009, People’s Republic of China;
b School of Pharmacy, Jiangsu University, Zhenjiang 212013, People’s Republic of China;
† These two authors contributed equally to this work
*Correspondence: Q.Z. Yao: E-mail: qz_yao@yahoo.com.cn Phone/Fax, +86-25-86634730;
J. Gao: E-mail: jinggao@ujs.edu.cn
Phone/Fax, +86-511-88791552;
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Abstract
Eighteen symmetrical bis-Schiff base derivatives of isatin were synthesized by condensation
of the natural or synthetic isatins with hydrazine and were evaluated for their in vitro and in vivo
antitumor activities. More than half of the obtained compounds showed potent cytotoxicity
according to the MTT assay on five different human cancer cell lines (i.e. HeLa, SGC-7901,
HepG2, U251, and A549), with compound 3b 3,3'-(hydrazine-1,2-diylidene)bis
(5-methylindolin-2-one) being the most potent compound on HepG2 (IC50~4.23 μM). 3b was also
found to be able to inhibit substantially the tumor growth on the HepS-bearing mice at a dose of
40 mg/kg. The real-time live cell imaging and tracking in the H2B-labeled HeLa cells revealed that
3b could induce mitosis interference and apoptosis-associated cell death. In mechanism study, 3b
arrested the cell cycle at the G2/M phase in HepG2 cells by down-regulating the expression of
cyclin B1 and cdc 2.
Keywords isatin, Schiff base, cytotoxicity, anti-tumor, mitosis interference, apoptosis
1 Introduction
Isatin (1H-indole-2,3-dione) has been found to be a common structural motif in a variety of
dyes, agrochemicals, and pharmacologically active compounds by virtue of its unique size and
privileged electronic properties [1-3]. Isatin-based chemicals and drug candidates have already
been pursued in the pharmaceutical industry [4]. For example, Sunitinib is an orally administered
tyrosine kinase inhibitor approved for the treatment of the metastatic renal cell carcinoma and the
Imatinib-resistant gastrointestinal stromal tumor in 2006 [5]. Additionally, the triple angiokinase
inhibitor Intedanib (against VEGFR, PDGFR, and FGFR) was developed as a potential anticancer
agent [6]. The multiple tyrosine kinase receptor inhibitors Semaxanib and TSU-68 were
discovered as the lead compounds among a series of small-molecule inhibitors for the purpose of
developing a potential anti-cancer treatment [7]. Structurally, both Semaxanib and TSU-68 are
close analogs of Sunitinib since they all share the isatin motif (Fig. 1).
Fig. 1.
The isatin nucleus is also embedded in various bis-isatin compounds (Fig. 2). Among them,
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indigo is probably the oldest and most famous colorant. Thousand tons of deep blue
synthetic vat dye known as indigo were produceded per annum chiefly as the dye for jeans. [8].
An isomer of indigo, indirubin, seen in small amounts in the organically derived indigo plant, is
the active ingredient in the traditional Chinese remedy called Dang Gui Long Hui Wan which is
used to treat chronic myeloid leukemia [9]. When combined with another bis-isatin, i.e.
meisoindigo, indirubin has further been found to be able to induce a hematologic remission in
patients with chronic phase (CP) CML as effectively as hydroxyurea and busulfan [10]. Indirubin
has also been identified as a potent inhibitor for cyclin-dependent kinases (CDKs) and GSK-3β
with IC50 values of 0.075 μM and 0.190 μM respectively. Specifically, Indirubin inhibits CDK5
and GSK-3β-mediated tau phosphorylation, a process over-active in the Alzheimer’s disease [11].
However, the poor aqueous solubility and bioavailability preclude the extensive clinical
application of indirubin. To solve this problem, our research group previously synthesized the
potentially more water soluble azaindirubin and found that azaindirubin also exhibited favorable
antiproliferative activities against the ovarian adenoma cell lines via its inhibitory effects on
CDK2/cyclinA complex [12].
Fig. 2.
Previous studies on isatin and a myriad of its functionalized derivatives have captured the
imagination of medicinal chemists. Notably, Schiff bases of isatin have been reported to possess
anti-HIV, anticonvulsant, antibacterial, antiprotozoal, antifungal, antiviral, and anthelminthic
activities [13, 14]. Schiff bases of isatin also have been used as ligands for complexation of metals
such as copper [15]. In continuation of our search for pharmacologically active isatin derivatives,
it seemed to be reasonable to prepare and evaluate new Schiff bases of isatin for their biological
properties. We envisioned that if two isatin-containing molecules are to be directly connected via a
bis-Schiff base linker as shown in Fig. 3, the resulting symmetrical compounds would possess
enhanced flexiblity and aqueous solubility as compared to indirubin and meisoindigo, which may
also open an avenue for an enhanced biological activity profile (i.e. enhanced bioavailability and
at least preserved pharmacological activity). Based on this hypothesis, in the current study we
synthesized and evaluated the symmetrical Schiff base compounds shown in Fig. 3 for their in
vitro and in vivo antitumor activity.
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Fig. 3.
2 Chemistry
The synthetic route to target compounds 3 is outlined in Scheme 1. Preparation of the
intermediate N1-substituted isatin 2 was achieved by the reaction of indole-2,3-dione 1 with
sodium hydride in DMF, followed by N1-alkylation with appropriate alkyl halide at room
temperature. Compounds 3 were obtained from the condensation of 1 or 2 with hydrazine hydrate
in ethanol at reflux in the presence of a catalytic amount of acetic acid for 0.5–2 h.
Scheme 1.
3 Biological Activity
3.1 Cytotoxicity evaluation
The synthesized compounds 3a-3r were first evaluated with five human cancer cell lines
(HeLa, SGC-7901, HepG2, U251, and A549) in vitro by the standard
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method adopting 5-FU as
the positive control. The half-inhibitory concentration (IC50) values were calculated and
summarized in Table 1. Most of the evaluated compounds were observed to exhibit favorable
growth inhibitory activities against tested cell lines. In comparison with 5-FU, four compounds
(i.e. 3a, 3b, 3e, 3h) exhibited significant cytotoxicities against HepG2 cells with IC50 values
ranging from 4.23 μM to 15.81 μM. The particularly cytotoxic compound 3b showed a
comparable or a stronger cytotoxicity against all the cancer cells as compared to 5-FU; moreover,
it is about 8-fold more potent against SGC-7901 cell (12.66 μM) and 5-fold more potent against
HepG2 cell (4.23 μM). Thus, compound 3b was regarded as the most promising compound and
was chosen for further evaluation.
Table 1
3.2 Structure-activity relationship (SAR) of the bis-Schiff base derivatives of isatin
From the above cytotoxicity evaluation, a SAR for the test compounds 3a-3r could be
drawn as follows. When R2 were alkyl groups (3j, 3k, and 3l), the IC50 values were dramatically
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increased with no significant activities in tested cell lines. As for the substitution of benzyl groups
(3m, 3n, and 3o), it was found that the cytotoxic activities were more potent when aromatic rings
of isatin were substituted with 5-Me (3n compared with 3m and 3o). It seemed that the
replacement of the 5,5’-bis-halogeno substituents with electron-donating groups such as methyl
(3b) and methoxy (3f) would enhance the antiproliferative activity. Moreover, it was also noted
that among the bis-halogenoisatin derivatives, the bis-fluoro compounds (3d, 3g, and 3i) exhibited
slightly higher activities than the corresponding bis-chloro and bis-bromo compounds (3c, 3e, and
3h).
The above cytotoxicity evaluation also indicated that compound 3h showed stronger
inhibitory activity on HepG2 but weak activity on HeLa, while 3n showed modest inhibition on
HepG2 but a decent inhibition on HeLa. It is difficult at this time to draw a definitive conclusion
why these two compounds behaved differently among the cell lines used in this study.
3.3 Tumor growth inhibition assessment with the HepS xenograft
We investigated the effect of 3b treatment on tumor growth using HepS xenografts. As
indicated in Fig. 4A, there was not a gross growth inhibition toward the 3b treated mice, in fact,
the body weights of the tumor-bearing mice treated with 3b had a fairly profound increase as
compared to the control group, i.e. 6.5 g (20 mg/kg of 3b) and 6.2 g (40 mg/kg of 3b) versus 5.0 g
(control). On the contrary, the body weights of the tumor-bearing mice treated with 5-fluorouracil
(5-FU, 25 mg/kg) increased only by 4.3 g.
However, compound 3b treatment resulted in a significant attenuation in both the tumor
weight (Fig. 4.B) and the tumor size (Fig. 4.C) in a dose-dependent manner. In specific, a 60.09%
reduction in the tumor weight was achieved following 3b treatment (40 mg/kg), whereas 5-FU
treatment (25 mg/kg) only afforded a 47.10% tumor weight reduction (Table 2). The in vivo
antitumor efficacy of 3b was consistent with its in vitro cytotoxicity. Furthermore, as showed in
Fig. 4.D and Fig. 4.E, mice treated with 3b showed improved index of thymus and spleen than
those treated with 5-FU. Therefore, it reveals that 3b possessed stronger antitumor efficacy and
less side-effect than 5-FU in the HepS tumor model.
Table 2
Fig. 4.
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3.4 The pathological sections of tumor tissue
To further evaluate the antitumor efficacy of 3b on the tumor-bearing mice, the tumors were
excised for pathology. Fig.5 presents the tissue sections from different mice groups. The group
intragastrically administrated with the vehicle showed typical pathological characteristics of tumor,
such as compactly arranged tumor cells. Tumor tissue from the group treated with 5-FU or 3b
showed spotty necrosis and intercellular blank. These results further supported the notion that 3b
possesses an effective tumor growth inhibitory effect.
Fig.5
3.5 The mitosis interference and the apoptosis-associated cell death induced by compound 3b
treatment
By using a real-time live cell imaging system (LCS), combined with genetically engineered
chromosome labeling technique, the behavior of the nucleus during cell growth could be observed
and tracked for several days in a high-throughput manner. To examine whether compound 3b
could interfere with mitosis of the neoplasm cells, the morphological changes of the nuclei and the
cells themselves of the H2B-GFP-labeled HeLa cells incubated with 8 μM of 3b for 48 h were
observed with LCS. During the first 24 hours of the 3b treatment, cells were observed to shrink
and to detach from the culture plate and float in the culture medium, which was followed by
chromatin condensation and nuclei fragmentation (Fig.6). With a prolonged 3b incubation,
nuclei were observed to become condensed and to be divided into several parts, and more and
more cells began to exhibit these features in a time-dependent manner (See Supplementary movie
1 and 2). These results suggested that 3b could interfere with mitosis and induce the
apoptosis-associated cell death.
Fig. 6
3.6 Compound 3b arrested the cell cycle at the G2/M phase in HepG2 cells
Indirubin inhibits cyclin dependent kinases (CDKs) by competing with ATP binding at the
catalytic site, resulting in cell cycle arrest in the late G1 and G2/M phases [16-19]. In order to
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examine whether the anti-proliferative effect of compound 3b is associated with cell cycle arrest,
we tested the effect of compound 3b on cell cycle distribution by flow cytometry (Fig. 7). Cell
populations in the G0/G1, S and G2/M phases were 63.58%, 25.96% and 10.46%, respectively, in
the control group. After 24 h of incubation with 8 μM compound 3b, the percentage of G0/G1
phase cells decreased to 46.36%, while the cell population in the G2/M phase increased to 27.32%
(Fig. 3B). However, the populations of S phase cells after compound 3b treatment (26.31%)
remained similar to that of the control. Such changes in the cell cycle distribution upon compound
3b treatment seems to indicate that it induced cell cycle arrest at the G2/M phase. G2 to M phase
progression is regulated by a number of the Cdk/cyclin family members [20-22]. Activation of
Cdk1/cyclin B1 complex is required for transition from G2 to M phase of the cell cycle, so we
further evaluated the relative expression levels of cell cycle related proteins by immunoblotting.
As shown in Figure 7C, compound 3b downregulated the expression of cyclin B1 and cdc 2 in
time- and dose-dependent manner in HepG2 cells.
Fig. 7
4 Conclusion
Starting from the commercially available isatins, a practical synthesis of their bis-Schiff
base derivatives as well as the biological activity evaluation of these derivatives have been
accomplished. The present study demonstrated that symmetrical bis-Schiff base derivatives of
isatin could markedly inhibit the proliferation of cancer cell lines (HeLa, SGC-7901, HepG2,
U251, and A549). Among them, 3,3'-(hydrazine-1,2-diylidene)bis(5-methylindolin-2-one) 3b
exhibited a stronger cytotoxicity than 5-FU in vitro with IC50 values of 4.23 μM on HepG2 cells,
12.66 μM on SGC-7901 cells, 12.78 μM on A549 cells, respectively. The preliminary SAR
showed that the N1-unsubstituted bis-isatin structures with 5,5’-bis electron-donating groups could
improve the anti-tumor activity. Gratifyingly, 3b exhibited an excellent in vivo antitumor profile
(i.e. high efficacy and and low side-effect) in HepS xenograft model as compared to 5-FU.
Mechanistic studies demonstrated that compound 3b arrested the cell cycle at the G2/M phase
with down-regulation of cyclin B1 and cdc 2 expression. By using a real-time live cell imaging
technique, 3b could induce mitosis interference and the apoptosis. Given the anti-tumor potential
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observed for the batch of the bis-Schiff base derivatives of isatin in this study, a more
comprehensive SAR exploration would be warranted. On the other hand, the determination of the
PK(pharmacokinetic) /PD(pharmacodynamic) properties of compound 3b and the further
mechanistic studies of its anti-tumor effect are current underway in our laboratories.
5 Experimental protocols
5.1 General methods
Unless noted, all solvents and reagents were freshly distilled or purified according to
standard procedures. Melting points were taken on XT-4 micro melting point apparatus and were
uncorrected. Mass spectra were recorded on electron impact ionization (EI) techniques.
1
Compounds were visualized under UV lamp (254 nm). H-NMR and 13C-NMR spectra were
obtained on a Bruker AV-300 MHz NMR spectrometer (1H-NMR at 300 MHz, 13C-NMR at 75
1
MHz) at ambient temperature. H-NMR spectra were reported in ppm on the δ scale and
referenced to the internal tetramethylsilane. The data were presented as follows: chemical shift,
multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad, app =
apparent), coupling constant(s) in Hertz (Hz), and integration. Chemical shifts (δ) were recorded
1
relative to residual DMSO-d6 (δ = 2.50 in H-NMR and δ = 35.2 in 13C-NMR). Analytical TLC
was carried out with plates pre-coated with silica gel 60 F254 (0.25 mm thick). Flash column
chromatography was carried out on silica gel 200–300 mesh. The purities of all new compounds
were more than 97%, which was confirmed by HPLC.
5.2 Typical experimental procedure for the synthesis of N1-substituted isatins 2
An N1-substituted isatin 2 was obtained by the reaction of indole-2,3-dione 1(10 mmol) with
sodium hydride(10 mmol) in anhydrous DMF(15 mL), followed by the N1-alkylation by adding an
appropriate alkyl halide (10 mmol) at room temperature. The mixture was then stirred at 60~65 oC
for 1 h. The reaction mixture was cooled down and poured into water (250 mmol). The product
was then purified from the obtained filtrate by column chromatography (200~300 mesh silica gel;
ethyl acetate/ligroin, 1:5).
5.3 General experimental procedure for the synthesis of target compounds 3
Target compounds 3 were prepared by coupling compounds 1 or 2 with hydrazine hydrate
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under a mild condition. A mixture of isatin or an N1-substituted isatin (10 mmol) and hydrazine
hydrate (5 mmol) was refluxed in ethanol (50 mL) in the presence of acetic acid as the catalyst for
0.5–2 h. After completion of the reaction (monitored by TLC), the solvent was removed under
reduced pressure and the crude product was washed with water and recrystallized from ethanol to
give pure products (Table 1). Some compounds synthesized in the current study are known
compounds and their physicochemical characteristics are consistent with those reported in earlier
literature. Characteristic data for all the bis-Schiff base derivatives are as follows:
3,3'-(Hydrazine-1,2-diylidene)diindolin-2-one (3a)
Red powder; 1H-NMR (300MHz, DMSO-d6) : 10.78 (s, 2H), 7.86(d, 2H, J=7.29Hz), 7.81(d, 2H,
J=7.50Hz), 7.50(t, 2H, J=7.29Hz), 7.26(t,2H, J=7.50Hz); 13C-NMR (75MHz, DMSO-d6) : 169.0,
141.2, 138.0, 131.2, 129.4, 124.4, 119.4, 117.7; IR(KBr) ν 3269(2NH),1731(2C=O),1608(2C=N)
cm-1; HRMS (EI) for (M+H)+: calcd 291.0882, found 291.0889.
3,3'-(Hydrazine-1,2-diylidene)bis(5-methylindolin-2-one) (3b)
Red powder; 1H-NMR(300MHz, DMSO-d6) : 10.90 (2H, s), 7.33(2H, s), 7.03(2H, d, J=7.29Hz),
6.84(2H, d, J=7.29Hz), 2.22(6H, s); 13C-NMR(75 MHz, DMSO-d6) : 169.0, 138.0, 134.1, 131.5,
129.6, 121.6, 117.6, 21.3; IR(KBr) ν 3383-3248(2NH),1723(2C=O),1613(2C=N) cm-1; HRMS (EI)
for (M+H)+: calcd 319.1195, found 319.1183.
3,3'-(Hydrazine-1,2-diylidene)bis(5-bromoindolin-2-one) (3c)
Red powder; 1H-NMR (300MHz, DMSO-d6) : 10.99(s, 2H), 8.10(s, 2H), 7.75(d, 2H, J=7.29Hz),
7.56(d, 2H, J=7.29Hz); 13C-NMR(75MHz, DMSO-d6) : 169.0, 141.8, 140.2, 138.0, 136.9, 132.9,
119.9, 118.8, 118.0; IR(KBr) ν 3427-3241(2NH),1718(2C=O),1619(2C=N) cm-1; HRMS (EI) for
(M+H)+: calcd 446.9092, found 446.9096;
3,3'-(Hydrazine-1,2-diylidene)bis(5-fluoroindolin-2-one) (3d)
Red powder; 1H-NMR (300MHz, DMSO-d6) : 10.89(s, 2H), 7.84(d, 2H, J=6.80Hz), 7.78(s, 2H),
7.34(d, 2H, J=6.80Hz); 13C-NMR(75MHz, DMSO-d6) : 169.0, 158.6, 138.0, 136.9, 119.3, 118.0,
112.7, 110.9; IR(KBr) ν 3239(2NH),1735(2C=O),1617(2C=N) cm-1; HRMS (EI) for (M+H)+:
calcd 327.0694, found 327.0690;
3,3'-(Hydrazine-1,2-diylidene)bis(5-chloroindolin-2-one) (3e)
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Red powder; 1H-NMR(300MHz, DMSO-d6) : 11.00 (2H, s), 7.48 (2H, d, J=7.10Hz), 7.42 (2H, d,
J=7.10Hz), 6.93(2H, s); 13C-NMR (75MHz, DMSO-d6) : 163.4, 144.8, 142.9, 134.7, 131.4, 128.5,
115.8, 110.8; IR(KBr) ν 3234(2NH),1734(2C=O),1612(2C=N) cm-1; HRMS (EI) for (M+H)+:
calcd 359.0103, found 359.0107;
3,3'-(Hydrazine-1,2-diylidene)bis(5-methoxyindolin-2-one) (3f)
Red powder; 1H-NMR (300MHz, DMSO-d6) : 11.00(s, 2H), 7.75(d, 2H, J=7.30Hz), 7.48(s, 2H),
7.04(d, 2H, J=7.30Hz), 3.83(s,6H); 13C-NMR (75MHz, DMSO-d6) : 169.0, 156.3, 138.0, 133.5,
122.7, 118.7, 116.8, 113.5, 55.8; IR(KBr) ν 3281(2NH),1725(2C=O),1614(2C=N) cm-1; HRMS
(EI) for (M+H)+: calcd 351.1093, found 351.1096;
3,3'-(Hydrazine-1,2-diylidene)bis(7-fluoroindolin-2-one) (3g)
Red powder; 1H-NMR (300MHz, DMSO-d6) : 11.02(s, 2H), 7.60(d, 2H), 7.35(m, 4H); 13C-NMR
(75MHz, DMSO-d6) : 169.0, 162.9, 138.0, 126.0, 125.7, 125.0, 119.3, 118.0; IR(KBr) ν
3413-3321(2NH),1733(2C=O),1619(2C=N) cm-1; HRMS (EI) for (M+H)+: calcd 327.0694, found
327.0698;
3,3'-(Hydrazine-1,2-diylidene)bis(6-chloroindolin-2-one) (3h)
Yellow powder; 1H-NMR (300MHz, DMSO-d6) :11.00 (s, 2H), 8.10(s, 2H), 7.75(d, 2H,
J=6.90Hz), 7.26(d, 2H, J=6.90Hz); 13C-NMR (75MHz, DMSO-d6) :169.0, 148.2, 140.6, 138.0,
130.8, 124.5, 119.8, 115.8; IR(KBr) ν 3446-3281(2NH),1743(2C=O),1621(2C=N) cm-1; HRMS
(EI) for (M+H)+: calcd 359.0103, found 359.0105;
3,3'-(Hydrazine-1,2-diylidene)bis(6-fluoroindolin-2-one) (3i)
1
Red powder; H-NMR (300MHz, DMSO-d6) : 11.00 (s, 2H), 7.79 (d, 2H, J=7.25Hz), 7.59 (s,
2H), 7.10 (d, 2H, J=7.25Hz); 13C-NMR (75MHz, DMSO-d6) : 169.0, 165.4, 148.4, 138.0, 131.0,
113.3, 111.2, 110.2; IR(KBr) ν 3385-3269(2NH),1741(2C=O),1618(2C=N) cm-1; HRMS (EI) for
(M+H)+: calcd 327.0694, found 327.0699;
3,3'-(Hydrazine-1,2-diylidene)bis(1-ethylindolin-2-one) (3j)
Red powder; 1H-NMR (300MHz, DMSO-d6) : 7.86(d, 2H, J=7.29Hz), 7.81(d, 2H, J=7.62Hz),
7.50(t, 2H, J=7.29Hz), 7.26(t, 2H, J=7.62Hz), 4.28(q, 4H, J=10Hz), 1.31(t, 6H, J=10Hz);
13C-NMR (75MHz, DMSO-d6) :163.2, 147.4, 138.0, 131.2, 129.4, 124.4, 117.7, 115.8, 42.2, 13.7;
IR(KBr) ν 1741(2C=O),1627(2C=N) cm-1; HRMS (EI) for (M+H)+: calcd 347.1508, found
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347.1504;
3,3'-(Hydrazine-1,2-diylidene)bis(1-ethyl-5-methylindolin-2-one) (3k)
Red powder; 1H-NMR (300MHz, DMSO-d6) : 8.01 (s, 2H), 7.74 (d, 2H, J=6.90Hz), 7.16 (d, 2H,
J=6.90Hz), 3.88 (q, 4H, J=8.89Hz), 2.34 (s, 6H), 1.51 (t, 6H, J=8.89Hz); 13C-NMR (75MHz,
DMSO-d6) :163.2, 144.4, 142.3, 134.1, 129.6, 120.6, 117.6, 116.1, 42.2, 21.3, 13.7; IR(KBr) ν
1738(2C=O),1613(2C=N) cm-1; HRMS (EI) for (M+H)+: calcd 375.1821, found 375.1825
3,3'-(Hydrazine-1,2-diylidene)bis(1-ethyl-5-fluoroindolin-2-one) (3l)
Red powder; 1H-NMR (300MHz, DMSO-d6) :7.84 (d, 2H, J=7.29Hz ), 7.78 (s, 2H ), 7.34 (d, 2H,
J=7.29Hz ) ,3.88 (q, 4H, J=10Hz), 1.50 (t, 6H, J=10Hz ); 13C-NMR (75MHz, DMSO-d6) : 163.2,
158.6, 143.0, 138.0, 125.0, 119.3, 118.0, 110.9, 42.2, 13.7; IR(KBr) ν 1731(2C=O),1618(2C=N)
cm-1; HRMS (EI) for (M+H)+: calcd 383.1320, found 383.1325;
3,3'-(Hydrazine-1,2-diylidene)bis(1-benzylindolin-2-one) (3m)
1
Yellow powder; H-NMR (300MHz, DMSO-d6) : 7.86-7.81 (m, 4H), 7.50 (t, 2H, J=6.56Hz ),
7.33 (t, 4H, J=6.29Hz ), 7.26-7.23 (m, 8H), 4.94 (s, 4H); 13C-NMR (75MHz, DMSO-d6) : 163.5,
147.8, 139.0, 136.3, 131.2, 129.9, 128.4, 126.9, 124.1, 122.6, 117.7, 115.8, 47.3; IR(KBr) ν
1730(2C=O),1614(2C=N) cm-1; HRMS (EI) for (M+H)+: calcd 471.1821, found 471.1827;
3,3'-(Hydrazine-1,2-diylidene)bis(1-benzyl-5-methylindolin-2-one) (3n)
1
Yellow powder; H-NMR (300MHz, DMSO-d6) : 8.13 (s, 2H), 7.74 (t, 2H, J=6.95Hz ), 7.33 (t,
4H), 7.25 (m, 6H), 7.16 (d, 2H, J=6.95Hz), 4.94(s, 4H), 3.13(s, 6H); 13C-NMR (75MHz,
DMSO-d6) :163.4, 143.1, 140.9, 137.8, 136.1, 134.3, 128.8, 126.7, 120.6, 117.6, 115.8, 47.3,
21.3; IR(KBr) ν 1735(2C=O),1623(2C=N) cm-1; HRMS (EI) for (M+H)+: calcd 499.2134, found
499.2137
3,3'-(Hydrazine-1,2-diylidene)bis(1-benzyl-5-fluoroindolin-2-one) (3o)
Pink powder; 1H-NMR (300MHz, DMSO-d6) : 7.90-7.70 (m, 4H), 7.34 (t, 2H, J=7.05Hz ), 7.33
(t, 4H, J=7.05Hz), 7.26-7.23 (m, 6H), 4.94 (s, 4H); 13C-NMR (75MHz, DMSO-d6) : 163.0, 158.1,
142.7, 138.0, 136.1, 128.5, 127.0, 126.8, 124.8, 119.3, 118.0, 110.9, 47.3; IR(KBr) ν
1735(2C=O),1623(2C=N) cm-1; HRMS (EI) for (M+H)+: calcd 507.1633, found 507.1637;
3,3'-(Hydrazine-1,2-diylidene)bis(1-isopropylindolin-2-one) (3p)
Pale powder; 1H-NMR (300MHz, DMSO-d6) : 7.91-7.82 (m, 4H), 7.52 (t, 2H, J=7.09Hz), 7.21 (t,
11

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2H, J=7.09Hz), 3.84 (m, 2H), 1.20 (m, 12H); 13C-NMR (75MHz, DMSO-d6) : 163.1, 147.4,
138.0, 131.0, 128.6, 124.7, 118.4, 115.3, 58.4, 20.6; IR(KBr) ν 1740(2C=O),1609(2C=N) cm-1;
HRMS (EI) for (M+H)+: calcd 375.1821, found 375.1826;
3,3'-(Hydrazine-1,2-diylidene)bis(1-isopropyl-5-methylindolin-2-one) (3q)
Pink powder; 1H-NMR (300MHz, DMSO-d6) : 8.16 (s, 2H), 7.74 (d, 2H, J=7.29Hz), 7.16 (d, 2H,
J=7.29Hz), 3.84 (m, 2H), 2.34 (s, 6H), 1.20 (m, 12H) ; 13C-NMR (75MHz, DMSO-d6) :162.9,
144.4, 138.0, 134.1, 129.6, 120.6, 117.6, 116.1, 58.4, 21.3, 20.6; IR(KBr)
1747(2C=O),1604(2C=N) cm-1; HRMS (EI) for (M+H)+: calcd 403.2134, found 403.2139;
3,3'-(Hydrazine-1,2-diylidene)bis(5-fluoro-1-isopropylindolin-2-one) (3r)
ν
Pale powder; 1H-NMR (300MHz, DMSO-d6) : 7.84 (d, 2H, J=7.29Hz), 7.78 (s, 2H), 7.34 (d, 2H,
J=7.29Hz), 3.94 (m, 2H), 1.20 (m, 12H); 13C-NMR (75MHz, DMSO-d6) : 162.9, 158.6, 143.0,
138.0, 125.0, 119.3, 118.0, 110.9, 58.4, 20.6; IR(KBr) ν 1730(2C=O),1614(2C=N) cm-1; HRMS
(EI) for (M+H)+: calcd 411.1633, found 411.1637;
5.4 Cytotoxicity
5.4.1 Cell culture
Five human cancer cell lines including HeLa, SGC-7901, HepG2, U251, and A549 were
obtained from Cancer Cell Repository (Shanghai cell bank). Cells were maintained in DMEM
medium or RPMI-1640 medium (Gibco, USA) supplemented with 10% (v/v) heat-inactivated fetal
bovine serum and antibiotics (100 U/ml penicillin and 100 U/ml streptomycin) at 37°C in a
humidified atmosphere of 5% CO2.
5.4.2 In vitro anti-proliferation assay with compounds 3
Cells were plated in 96-well culture plates at an initial density of 4×103 viable cells per well.
After 24 h of cell growth, a test compound at various concentrations was incubated with the
cells. Cell viability was estimated by measuring the metabolism of MTT. Briefly, 100 μL of MTT
solution (1 mg/mL) was added into each well of a 96-well plate, and cells were maintained for 4 h
at 37°C. The medium was aspirated and the formazan contained in cells was solubilized by 100 μL
of DMSO for 1 h. The absorbance was measured at 570 nm by a plate reader (Spectra MAX 190,
Molecular Devices Corporation). The rate of inhibition was calculated as follows [23]:
12

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Rate of Inhibition =(1-OD570 drug treated/OD570 control)×100
IC50 values were determined graphically from the growth inhibition curves obtained after a 48 h
exposure to 3b, using the software from China Pharmaceutical University.
5.5 In Vivo antitumor activity assay with 3b
Female ICR mice, purchased from The Experimental Animal Center of Jiangsu University,
were maintained on a standard diet and water made freely available in a conventional animal
colony. The mice were 6-8 weeks old at the beginning of the experiment. The tumor used was
HepS that forms solid tumors when injected subcutaneously. HepS cells for initiation of
subcutaneous tumors were obtained from the ascitic form of the tumors in mice, which were
serially transplanted once per week. Subcutaneous tumors were implanted by injecting 0.2 mL of
NS containing 1×107 viable tumor cells under the skin on the right oxter. Twenty-four hours after
implantation, the tumor-bearing mice were randomly assigned into four experimental groups (6
per group). All the mice were given a daily intraperitoneal injection of 5-FU (positive control) and
intragastric administration of 3b (20 mg/kg or 40 mg/kg) pre-dissolved in 4% Tween 80 in NS, for
nine consecutive days; and the vehicle alone was used as the negative control. Twenty-four hours
after the last administration, animal welfare and experimental procedures were carried out strictly
in accordance with the Guide for the Care and Use of Laboratory Animals (The Ministry of
Science and Technology of China, 2006) and the related ethical regulations of our university.
Every effort was made to minimize animals' suffering and to reduce the number of animals used.
The tumor wet weights of the treated (Tw) and control (Cw) groups were measured on the last day
of each experiment and the percentage of tumor growth inhibition was calculated as follows [24]:
Inhibition (%) =[1-(TW/CW)]×100
Observations were also made to assess the toxicity of 3b on thymus and spleen.
5.6 Live cell imaging
Live cell imaging was performed as described [25, 26]. Briefly, HeLa cells expressing
H2B-GFP were grown on 24-well culture plates at a density of 4×104 cells per well for 24 h. Then,
with the incubation of 3b at 8 μM, the morphological changes of the H2B-GFP-labeled Hela cells
were observed using Nikon Ti-E microscope with LCS which can provide CO2 and temperature
13

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control as well as position fixing. Images were automatically acquired at multiple locations on the
24-well culture plates. Fluorescence and differential interference contrast images were obtained
every 10 min for a period of 48 h. The bright and fluorescence images of cells were recorded and
analyzed. Time-lapse records of live cell imaging experiments were exported as image series.
They were further converted into movie stacks using NIS-Elements AR software (Universal
Imaging Corporation).
5.7 Cell cycle analysis
HepG2 cells (8×104 cells/well) were seeded in a 6-well plate for 24 h at 37 °C. Cells were
washed with 1×PBS, replaced with fresh media, and then treated with the varying concentrations
of compound 14 for different times. Then, the cells were dissociated with trypsin, washed in cold
PBS and fixed with 70% cold ethanol at -20 °C for 24 h. The suspensions were centrifuged at
1500 rpm for 5 min. The pellets were resuspended in a solution containing 50 μg/ml propidium
iodide, 50 μg/ml RNase A and 0.1% Triton-X-100 and stayed at 37 °C for 20 min. Then the pellets
were analyzed by a flow cytometer.
5.8 Western blot analysis
Cells were lysed in 0.5% TritonX100, 100 mM Tris–HCl, 150 mM NaCl and 0.1 U ml-1
aprotinin for 30 min on ice and centrifuged at 12 000 g for 6 min. Equal amounts of total protein
lysates were separated on 10% polyacrylamide gels, transferred onto PVDF membranes and
probed with the following primary antibodies: anti-cyclin B1 (1:1000), anti-cdc2 (1:1000),
andanti-GAPDH (1:2000). All antibodies were obtained from SantaCruz Biotechnology (Santa
Cruz, CA). Immunoblotting was detected by enhanced chemiluminescence reagent (Beyotime
Institute of Biotechnology, China). Western blot bands were quantified using the NIH ImageJ
software.
Acknowledgment
This work was supported by National Science and Technology Major Project of the Ministry
of Science and Technology of China (No. 2009ZX09301-006). We would like to thank Dr.
14

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Weiping Zheng for his assistance in the preparation of this manuscript. We appreciate the support
from Center for Instrumental Analysis, China Pharmaceutical University, for their contribution in
the structural confirmation.
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tables.docx
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Table 1. In vitro antiproliferative activity of the synthesized compounds against various cancer cell
lines.
Compd.
IC50(μM)a
A549
HeLa
HepG2
U251
>100b
SGC-7901
7.20±0.86
3a
3b
3c
21.80±3.54 13.44±5.26 12.48±1.78
12.78±0.44 8.6±0.13
81.75±2.82 14.09±0.17
4.23±0.68 29.00±0.21 12.66±0.21
>100 >100 37.17±0.39
9.45±0.81 25.29±1.28 22.76±1.03 47.85±2.56
3d
3e
8.57±0.48
>100
>100
>100
15.81±0.54 21.65±1.28 84.26±4.52
>100 11.72±0.45 18.42±1.71
3f
23.91±1.02
3g
3h
3i
28.37±2.09 30.82±2.14 19.88±0.11 7.09±0.02 47.64±2.49
42.89±2.83 24.58±1.09 8.74±0.31
>100
11.87±0.44
>100
5.38±0.42
>100
4.71±0.21 58.39±1.82 23.71±1.50
3j
>100
>100
>100
>100
>100
>100
>100
>100
>100
3k
3l
>100
43.76±1.54
>100
87.69±0.80 13.90±0.29 88.11±1.73
3m
3n
3o
3p
3q
3r
34.78±1.29
49.33±0.87
>100
>100
74.98±2.74
>100
35.59±0.25 3.37±0.10 47.49±0.24
>100
19.40±0.13 65.10±0.81
48.65±0.90 87.34±0.69 26.39±1.82 79.13±4.09
>100
>100
>100
23.41±01 64.93±0.29
>100
>100
>100
>100
>100
>100
>100
>100
>100
5-FU
17.31±0.87 7.32±0.19 19.76±0.83 28.45±1.34
Data are presented as mean±S.D. (n = 3).
a cancer cell lines used: A549, lung adenocarcinoma epithelial cell; Hela, epithelial carcinoma
cell; HepG2, liver hepatocellular cell; U251, glioma cell; SGC-7901, human gastric carcinoma
cells.
bThe concentration of a compound that inhibits cells’ growth by 50%.
c When a 50% inhibition could not be reached at the highest compound concentration, >100
μM was given.

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Table 2. The inhibitory effect of 3b on HepS tumor xenograft.
Compd.
Rate of inhibition
47.10%
5-FU (25 mg/kg)
3b (20 mg/kg)
3b (40 mg/kg)
17.09%
60.09%

Schemes.docx
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Scheme 1. The synthetic route to the bis-Schiff base derivatives of isatin (3a-3r).
Isolated
Entry
R1
R2
Product
Mp(oC)
Yield(%)
81
1
2
H
5-Me
5-Br
5-F
H
H
3a
3b
3c
3d
3e
3f
171-173
188-190
183-185
186-188
184-186
177-179
199-201
183-185
190-192
195-197
189-191
193-196
187-189
205-207
199-201
201-202
203-205
192-194
89
3
H
83
4
H
77
5
5-Cl
5-MeO
7-F
H
82
6
H
71
7
H
3g
3h
3i
78
8
6-Cl
6-F
H
83
9
H
65
10
11
12
13
14
15
16
17
18
H
Et
Et
Et
Bn
Bn
Bn
i-Pr
i-Pr
i-Pr
3j
79
5-Me
5-F
3k
3l
58
87
H
3m
3n
3o
3p
3q
3r
73
5-Me
5-F
64
81
H
72
5-Me
5-F
64
83

figures.doc
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Figure Legends
(Note: please use the revised legends under figures.)
Fig. 1. The chemical structures of some reported drugs containing the isatin nucleus.
Fig. 2. The chemical structures of some known bis-isatins.
Fig. 3. A structural comparison of the designed symmetrical bis-Schiff base derivatives of isatin
with indirubin and meisoindigo.

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A
28
26
24
22
20
18
#
#
Control
5-Fu
3b(20 mg/kg)
3b(40 mg/kg)
#
*
0
1
2
3
4
5
6
7
8
9
10 11
days
B1.5
1.0
C
Control
5-FU(25 mg/kg)
*
*
0.5
3b(20 mg/kg)
3b(40 mg/kg)
0.0
Control
5-FU
20
40
3b(mg/kg)
E 8
D 5
4
3
2
1
0
6
4
2
0
*
Control
5-FU
20
40
Control
5-FU
20
40
3b(mg/kg)
3b(mg/kg)
Fig.4. In vivo antitumor efficacy assay. The body weight changes of tumor-bearing mice as a
function of time (A); the weight (B) and size (C) of tumor mass of each treatment group at the time
of sacrifice; effects of 3b against thymus (D) and spleen index (E) of HepS xenografts bearing
mice. Thymus and Spleen indexes were calculated by using the formulas: thymus index = (thymus
weight/final body weight); spleen index = (spleen weight/final body weight). Data represent
#
mean±S.E.M(n=6), P<0.05,vs 5-FU; *P <0.05, vs control.⁄⁄P <0.01 (Student’s t-test). (n = 7).*p﹤
0.05

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A
B
D
C
Fig.5. Tissue sections of the tumors isolated 24 h after the last treatment
administration and stained with hematoxylin and eosin (H&E) for histopathological
analysis. (A) Control, (B) 5-FU (25 mg/kg), (C) 3b (20 mg/kg), and (D) 3b (40
mg/kg) .

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A
B
DIC
GFP
DIC
GFP
6 h
6 h
12 h
12 h
24 h
48 h
24 h
48 h
Fig. 6. Compound 3b induced mitosis interference and apoptosis-associated cell death in the
H2B-GFP-labeled HeLa cells, revealed by a real-time LCS. The cells were exposed to 8 μM of
3b, and then observed for the morphological changes of the cells and their nuclei. (A) Control
(B) 8 μM 3b. DIC: the bright imaging of cells; GFP: the fluorescence imaging of nuclei.

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A
G0/G1:46.36%
S:26.31%
G0/G1: 63.58 %
S: 25.96 %
G0/G1:55.21%
S:29.68%
G2/M: 27.32 %
G2/M: 10.46 %
G2/M:15.11%
0
50
100
150
0
50
100
150
0
50
100
150
DNA Content
DNA Content
DNA Content
Control
12 h
24 h
B
C
Compound 3b (μmol/L)
16
Compound 3b (8 μmol/L)
Control 12 h 24 h 48 h
Control
4
8
cyclin B1
cdc 2
cyclin B1
cdc 2
GAPDH
GAPDH
Fig.7. compound 3b induced G2/M phase arrest in HepG2 cells. (A) Cell cycle analysis. Cells were
treated with 8 μM of compound 3b for 12 and 24 h, harvested and stained with propidium iodide (PI) for
20 min. Cells were then subjected to flow cytometric analysis for cell distributions at each phase of cell
cycle. (B, C) Western blot analysis. Cells treated with compound 3b were lysed for subjection to western
blotting analysis with antibodies to cdc2 and cyclin B1,GAPDH was used as a loading control.