European Journal of Medicinal Chemistry 74 (2014) 742e750
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, in vitro and in vivo antitumor activity of symmetrical bis-Schiff base derivatives of isatin Chengyuan Liang a, y, Juan Xia b, y, Dong Lei a, Xiang Li b, Qizheng Yao a, *, Jing Gao b, ** a b
School of Pharmacy, China Pharmaceutical University, Nanjing 210009, PR China School of Pharmacy, Jiangsu University, Zhenjiang 212013, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 November 2012 Received in revised form 2 March 2013 Accepted 18 April 2013 Available online 13 June 2013
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 3,30 -(hydrazine-1,2-diylidene)bis (5-methylindolin-2-one) being the most potent compound on HepG2 (IC50 w 4.23 mM). 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 H2Blabeled 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. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: Isatin Schiff base Cytotoxicity Antitumor 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 [1e3]. 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 anticancer treatment [7]. Structurally, both Semaxanib and TSU-68 are close analogs of Sunitinib since they all share the isatin motif (Fig. 1). The isatin nucleus is also embedded in various bis-isatin compounds (Fig. 2). Among them, indigo is probably the oldest and * Corresponding author. Tel./fax: þ86 25 86634730. ** Corresponding author. Tel./fax: þ86 511 88791552. E-mail addresses: [email protected] (Q. Yao), [email protected] (J. Gao). y These two authors contributed equally to this work. 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.04.040
most famous colorant. Thousand tons of deep blue synthetic vat dye known as indigo were produced 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-3b with IC50 values of 0.075 mM and 0.190 mM respectively. Specifically, Indirubin inhibits CDK5 and GSK-3b-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 [12a]. 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 antiHIV, anticonvulsant, antibacterial, antiprotozoal, antifungal, antiviral, and anthelmintic activities [13,14]. Schiff bases of isatin also
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Fig. 1. The chemical structures of some reported drugs containing the isatin nucleus.
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 flexibility 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.
cell (4.23 mM). Thus, compound 3b was regarded as the most promising compound and was chosen for further evaluation. 3.2. Structureeactivity relationship (SAR) of the bis-Schiff base derivatives of isatin
3. Biological activity
From the above cytotoxicity evaluation, a SAR for the test compounds 3ae3r could be drawn as follows. When R2 were alkyl groups (3j, 3k, and 3l), the IC50 values were dramatically 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 5,50 -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-halogenisation derivatives, the bisfluoro 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.
3.1. Cytotoxicity evaluation
3.3. Tumor growth inhibition assessment with the HepS xenograft
The synthesized compounds 3ae3r 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)-2,5diphenyl 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 mM to 15.81 mM. 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 mM) and 5-fold more potent against HepG2
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. 4B) and the tumor size (Fig. 4C) 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
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 0.5e2 h.
Fig. 2. The chemical structures of some known bis-isatins.
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Fig. 3. A structural comparison of the designed symmetrical bis-Schiff base derivatives of isatin with indirubin and meisoindigo.
was consistent with its in vitro cytotoxicity. Furthermore, as showed in Fig. 4D and Fig. 4E, 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 sideeffect than 5-FU in the HepS tumor model. 3.4. The pathological sections of tumor tissue To further evaluate the antitumor efficacy of 3b on the tumorbearing 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.
Entry
Product
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 mM of 3b for 48 h were observed with LCS. During the first 24 h 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
Isolated
Mp(oC)
R1
R2
1
H
H
3a
81
171-173
2
5-Me
H
3b
89
188-190
3
5-Br
H
3c
83
183-185
4
5-F
H
3d
77
186-188
5
5-Cl
H
3e
82
184-186
6
5-MeO
H
3f
71
177-179
7
7-F
H
3g
78
199-201
8
6-Cl
H
3h
83
183-185
9
6-F
H
3i
65
190-192
10
H
Et
3j
79
195-197
11
5-Me
Et
3k
58
189-191
Yield(%)
12
5-F
Et
3l
87
193-196
13
H
Bn
3m
73
187-189
14
5-Me
Bn
3n
64
205-207
15
5-F
Bn
3o
81
199-201
16
H
i-Pr
3p
72
201-202
17
5-Me
i-Pr
3q
64
203-205
18
5-F
i-Pr
3r
83
192-194
Scheme 1. The synthetic route to the bis-Schiff base derivatives of isatin (3ae3r).
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Table 1 In vitro antiproliferative activity of the synthesized compounds against various cancer cell lines. Compd.
IC50 (mM)a A549
3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l 3m 3n 3o 3p 3q 3r 5-FU
21.80 3.54 12.78 0.44 81.75 2.82 8.57 0.48 >100c 23.91 1.02 28.37 2.09 42.89 2.83 5.38 0.42 >100 >100 >100 34.78 1.29 35.59 0.25 >100 19.40 0.13 >100 >100 17.31 0.87
HeLa 13.44 5.26 8.6 0.13 14.09 0.17 9.45 0.81 >100 >100 30.82 2.14 24.58 1.09 4.71 0.21 >100 >100 >100 >100 3.37 0.10 48.65 0.90 65.10 0.81 >100 >100 7.32 0.19
HepG2 12.48 1.78 4.23 0.68 >100 25.29 1.28 15.81 0.54 >100 19.88 0.11 8.74 0.31 58.39 1.82 >100 >100 87.69 0.80 49.33 0.87 47.49 0.24 87.34 0.69 >100 >100 >100 19.76 0.83
U251
SGC-7901 b
>100 29.00 0.21 >100 22.76 1.03 21.65 1.28 11.72 0.45 7.09 0.02 >100 23.71 1.50 >100 >100 13.90 0.29 >100 >100 26.39 1.82 23.41 01 >100 >100 28.45 1.34
7.20 0.86 12.66 0.21 37.17 0.39 47.85 2.56 84.26 4.52 18.42 1.71 47.64 2.49 11.87 0.44 >100 >100 43.76 1.54 88.11 1.73 74.98 2.74 >100 79.13 4.09 64.93 0.29 >100 >100 >100
Data are presented as mean S.D. (n ¼ 3). Bold value signifies P < 0.05. 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. b The 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 mM was given.
manner (See Supplementary movie 1 and 2). These results suggested that 3b could interfere with mitosis and induce the apoptosis-associated cell death. 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 [9,16e18]. In order to examine whether the antiproliferative 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 mM 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 [19e21]. 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 Fig. 7C, compound 3b downregulated the expression of cyclin B1 and cdc 2 in time- and dosedependent manner in HepG2 cells. 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, 3,30 -(hydrazine-1,2-diylidene)bis(5-methylindolin2-one) 3b exhibited a stronger cytotoxicity than 5-FU in vitro with IC50 values of 4.23 mM on HepG2 cells, 12.66 mM on SGC-7901 cells, 12.78 mM on A549 cells, respectively. The preliminary SAR showed that the N1-unsubstituted bis-isatin structures with 5,50 -bis electron-donating groups could improve the antitumor activity. Gratifyingly, 3b exhibited an excellent in vivo antitumor profile (i.e. high efficacy 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 downregulation 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 antitumor potential 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 antitumor 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. Compounds were visualized under UV lamp (254 nm). 1 H-NMR and 13C-NMR spectra were obtained on a Bruker AV300 MHz NMR spectrometer (1H-NMR at 300 MHz, 13C-NMR at 75 MHz) at ambient temperature. 1H-NMR spectra were reported in ppm on the d scale and referenced to the internal tetramethylsilane. The data were presented as follows: chemical shift, multiplicity (s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet, br ¼ broad, app ¼ apparent), coupling constant(s) in Hertz (Hz), and integration. Chemical shifts (d) were recorded relative to residual
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A
28
average body weight(g)
#
Control 5-Fu 3b(20 mg/kg) 3b(40 mg/kg)
#
26
#
*
6
7
24 22 20 18 0
1
2
3
4
8
9
10
11
days
B1.5 Average tumor weight(g)
5
C Control
1.0
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)
D 4 3 2
* 1 0 Control
E8 index of spleen(mg/g)
index of thymus(mg/g)
5
5-FU
20
40
3b(mg/kg)
6 4 2 0 Control
5-FU
20
40
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 ¼ (thymus weight/final body weight); spleen index ¼ (spleen weight/final body weight). Data represent mean S.E.M. (n ¼ 6), #P < 0.05, vs 5-FU; *P < 0.05, vs control. ⁄⁄P < 0.01 (Student’s t-test); n ¼ 7, *P < 0.05.
DMSO-d6 (d ¼ 2.50 in 1H-NMR and d ¼ 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 200e300 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 N1substituted 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 Table 2 The inhibitory effect of 3b on HepS tumor xenograft. Compd.
Rate of inhibition (%)
5-FU (25 mg/kg) 3b (20 mg/kg) 3b (40 mg/kg)
47.10 17.09 60.09
an appropriate alkyl halide (10 mmol) at room temperature. The mixture was then stirred at 60e65 C 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 (200e300 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 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 0.5e2 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
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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).
with those reported in earlier literature. Characteristic data for all the bis-Schiff base derivatives are as follows:
110.9; IR (KBr) n 3239 (2NH), 1735 (2C¼O), 1617 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 327.0694, found 327.0690.
5.3.1. 3,30 -(Hydrazine-1,2-diylidene)diindolin-2-one (3a) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 10.78 (s, 2H), 7.86 (d, 2H, J ¼ 7.29 Hz), 7.81 (d, 2H, J ¼ 7.50 Hz), 7.50 (t, 2H, J ¼ 7.29 Hz), 7.26 (t, 2H, J ¼ 7.50 Hz); 13C-NMR (75 MHz, DMSO-d6) d: 169.0, 141.2, 138.0, 131.2, 129.4, 124.4, 119.4, 117.7; IR (KBr) n 3269 (2NH), 1731 (2C¼O), 1608 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 291.0882, found 291.0889.
5.3.5. 3,30 -(Hydrazine-1,2-diylidene)bis(5-chloroindolin-2-one) (3e) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 11.00 (2H, s), 7.48 (2H, d, J ¼ 7.10 Hz), 7.42 (2H, d, J ¼ 7.10 Hz), 6.93 (2H, s); 13C-NMR (75 MHz, DMSO-d6) d: 163.4, 144.8, 142.9, 134.7, 131.4, 128.5, 115.8, 110.8; IR (KBr) n 3234 (2NH), 1734 (2C¼O), 1612 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 359.0103, found 359.0107.
5.3.2. 3,30 -(Hydrazine-1,2-diylidene)bis(5-methylindolin-2-one) (3b) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 10.90 (2H, s), 7.33 (2H, s), 7.03 (2H, d, J ¼ 7.29 Hz), 6.84 (2H, d, J ¼ 7.29 Hz), 2.22 (6H, s); 13 C-NMR (75 MHz, DMSO-d6) d: 169.0, 138.0, 134.1, 131.5, 129.6, 121.6, 117.6, 21.3; IR (KBr) n 3383-3248 (2NH), 1723 (2C¼O), 1613 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 319.1195, found 319.1183. 5.3.3. 3,30 -(Hydrazine-1,2-diylidene)bis(5-bromoindolin-2-one) (3c) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 10.99 (s, 2H), 8.10 (s, 2H), 7.75 (d, 2H, J ¼ 7.29 Hz), 7.56 (d, 2H, J ¼ 7.29 Hz); 13C-NMR (75 MHz, DMSO-d6) d: 169.0, 141.8, 140.2, 138.0, 136.9, 132.9, 119.9, 118.8, 118.0; IR (KBr) n 3427-3241 (2NH), 1718 (2C¼O), 1619 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 446.9092, found 446.9096. 5.3.4. 3,30 -(Hydrazine-1,2-diylidene)bis(5-fluoroindolin-2-one) (3d) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 10.89 (s, 2H), 7.84 (d, 2H, J ¼ 6.80 Hz), 7.78 (s, 2H), 7.34 (d, 2H, J ¼ 6.80 Hz); 13C-NMR (75 MHz, DMSO-d6) d: 169.0, 158.6, 138.0, 136.9, 119.3, 118.0, 112.7,
5.3.6. 3,30 -(Hydrazine-1,2-diylidene)bis(5-methoxyindolin-2-one) (3f) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 11.00 (s, 2H), 7.75 (d, 2H, J ¼ 7.30 Hz), 7.48 (s, 2H), 7.04 (d, 2H, J ¼ 7.30 Hz), 3.83 (s, 6H); 13CNMR (75 MHz, DMSO-d6) d: 169.0, 156.3, 138.0, 133.5, 122.7, 118.7, 116.8, 113.5, 55.8; IR (KBr) n 3281 (2NH), 1725 (2C¼O), 1614 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 351.1093, found 351.1096. 5.3.7. 3,30 -(Hydrazine-1,2-diylidene)bis(7-fluoroindolin-2-one) (3g) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 11.02 (s, 2H), 7.60 (d, 2H), 7.35 (m, 4H); 13C-NMR (75 MHz, DMSO-d6) d: 169.0, 162.9, 138.0, 126.0, 125.7, 125.0, 119.3, 118.0; IR (KBr) n 3413-3321 (2NH), 1733 (2C¼O), 1619 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 327.0694, found 327.0698. 5.3.8. 3,30 -(Hydrazine-1,2-diylidene)bis(6-chloroindolin-2-one) (3h) Yellow powder; 1H-NMR (300 MHz, DMSO-d6) d: 11.00 (s, 2H), 8.10 (s, 2H), 7.75 (d, 2H, J ¼ 6.90 Hz), 7.26 (d, 2H, J ¼ 6.90 Hz); 13CNMR (75 MHz, DMSO-d6) d: 169.0, 148.2, 140.6, 138.0, 130.8, 124.5, 119.8, 115.8; IR (KBr) n 3446-3281 (2NH), 1743 (2C¼O), 1621 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 359.0103, found 359.0105.
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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 mM of 3b, and then observed for the morphological changes of the cells and their nuclei. (A) Control (B) 8 mM 3b. DIC: the bright imaging of cells; GFP: the fluorescence imaging of nuclei.
5.3.9. 3,30 -(Hydrazine-1,2-diylidene)bis(6-fluoroindolin-2-one) (3i) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 11.00 (s, 2H), 7.79 (d, 2H, J ¼ 7.25 Hz), 7.59 (s, 2H), 7.10 (d, 2H, J ¼ 7.25 Hz); 13C-NMR (75 MHz, DMSO-d6) d: 169.0, 165.4, 148.4, 138.0, 131.0, 113.3, 111.2, 110.2; IR (KBr) n 3385-3269 (2NH), 1741 (2C¼O), 1618 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 327.0694, found 327.0699. 5.3.10. 3,30 -(Hydrazine-1,2-diylidene)bis(1-ethylindolin-2-one) (3j) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 7.86 (d, 2H, J ¼ 7.29 Hz), 7.81 (d, 2H, J ¼ 7.62 Hz), 7.50 (t, 2H, J ¼ 7.29 Hz), 7.26 (t, 2H, J ¼ 7.62 Hz), 4.28 (q, 4H, J ¼ 10 Hz), 1.31 (t, 6H, J ¼ 10 Hz); 13CNMR (75 MHz, DMSO-d6) d: 163.2, 147.4, 138.0, 131.2, 129.4, 124.4, 117.7, 115.8, 42.2, 13.7; IR (KBr) n 1741 (2C¼O), 1627 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 347.1508, found 347.1504. 5.3.11. 3,30 -(Hydrazine-1,2-diylidene)bis(1-ethyl-5-methylindolin2-one) (3k) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 8.01 (s, 2H), 7.74 (d, 2H, J ¼ 6.90 Hz), 7.16 (d, 2H, J ¼ 6.90 Hz), 3.88 (q, 4H, J ¼ 8.89 Hz), 2.34 (s, 6H), 1.51 (t, 6H, J ¼ 8.89 Hz); 13C-NMR (75 MHz, DMSO-d6) d: 163.2, 144.4, 142.3, 134.1, 129.6, 120.6, 117.6, 116.1, 42.2, 21.3, 13.7; IR (KBr) n 1738 (2C¼O), 1613 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 375.1821, found 375.1825.
5.3.12. 3,30 -(Hydrazine-1,2-diylidene)bis(1-ethyl-5-fluoroindolin2-one) (3l) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 7.84 (d, 2H, J ¼ 7.29 Hz), 7.78 (s, 2H), 7.34 (d, 2H, J ¼ 7.29 Hz), 3.88 (q, 4H, J ¼ 10 Hz), 1.50 (t, 6H, J ¼ 10 Hz); 13C-NMR (75 MHz, DMSO-d6) d: 163.2, 158.6, 143.0, 138.0, 125.0, 119.3, 118.0, 110.9, 42.2, 13.7; IR (KBr) n 1731 (2C¼O), 1618 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 383.1320, found 383.1325. 5.3.13. 3,30 -(Hydrazine-1,2-diylidene)bis(1-benzylindolin-2-one) (3m) Yellow powder; 1H-NMR (300 MHz, DMSO-d6) d: 7.86e7.81 (m, 4H), 7.50 (t, 2H, J ¼ 6.56 Hz), 7.33 (t, 4H, J ¼ 6.29 Hz), 7.26-7.23 (m, 8H), 4.94 (s, 4H); 13C-NMR (75 MHz, DMSO-d6) d: 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) n 1730 (2C¼O), 1614 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 471.1821, found 471.1827. 5.3.14. 3,30 -(Hydrazine-1,2-diylidene)bis(1-benzyl-5-methylindolin2-one) (3n) Yellow powder; 1H-NMR (300 MHz, DMSO-d6) d: 8.13 (s, 2H), 7.74 (t, 2H, J ¼ 6.95 Hz), 7.33 (t, 4H), 7.25 (m, 6H), 7.16 (d, 2H, J ¼ 6.95 Hz), 4.94 (s, 4H), 3.13 (s, 6H); 13C-NMR (75 MHz, DMSO-d6)
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Fig. 7. Compound 3b induced G2/M phase arrest in HepG2 cells. (A) Cell cycle analysis. Cells were treated with 8 mM 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.
d: 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) n 1735 (2C¼O), 1623 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 499.2134, found 499.2137. 5.3.15. 3,30 -(Hydrazine-1,2-diylidene)bis(1-benzyl-5-fluoroindolin2-one) (3o) Pink powder; 1H-NMR (300 MHz, DMSO-d6) d: 7.90-7.70 (m, 4H), 7.34 (t, 2H, J ¼ 7.05 Hz), 7.33 (t, 4H, J ¼ 7.05 Hz), 7.26e7.23 (m, 6H), 4.94 (s, 4H); 13C-NMR (75 MHz, DMSO-d6) d: 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) n 1735 (2C¼O), 1623 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 507.1633, found 507.1637. 5.3.16. 3,30 -(Hydrazine-1,2-diylidene)bis(1-isopropylindolin-2-one) (3p) Pale powder; 1H-NMR (300 MHz, DMSO-d6) d: 7.91-7.82 (m, 4H), 7.52 (t, 2H, J ¼ 7.09 Hz), 7.21 (t, 2H, J ¼ 7.09 Hz), 3.84 (m, 2H), 1.20 (m, 12H); 13C-NMR (75 MHz, DMSO-d6) d: 163.1, 147.4, 138.0, 131.0, 128.6, 124.7, 118.4, 115.3, 58.4, 20.6; IR (KBr) n 1740 (2C¼O), 1609 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 375.1821, found 375.1826. 5.3.17. 3,30 -(Hydrazine-1,2-diylidene)bis(1-isopropyl-5methylindolin-2-one) (3q) Pink powder; 1H-NMR (300 MHz, DMSO-d6) d: 8.16 (s, 2H), 7.74 (d, 2H, J ¼ 7.29 Hz), 7.16 (d, 2H, J ¼ 7.29 Hz), 3.84 (m, 2H), 2.34 (s, 6H), 1.20 (m, 12H) ; 13C-NMR (75 MHz, DMSO-d6) d: 162.9, 144.4, 138.0, 134.1, 129.6, 120.6, 117.6, 116.1, 58.4, 21.3, 20.6; IR (KBr) n 1747 (2C¼O), 1604 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 403.2134, found 403.2139. 5.3.18. 3,30 -(Hydrazine-1,2-diylidene)bis(5-fluoro-1isopropylindolin-2-one) (3r) Pale powder; 1H-NMR (300 MHz, DMSO-d6) d: 7.84 (d, 2H, J ¼ 7.29 Hz), 7.78 (s, 2H), 7.34 (d, 2H, J ¼ 7.29 Hz), 3.94 (m, 2H), 1.20
(m, 12H); 13C-NMR (75 MHz, DMSO-d6) d: 162.9, 158.6, 143.0, 138.0, 125.0, 119.3, 118.0, 110.9, 58.4, 20.6; IR (KBr) n 1730 (2C¼O), 1614 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: 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 mL 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 mL 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 [22]:
Rate of Inhibition ¼ ð1 OD570 drug treated=OD570 controlÞ 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.
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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 6e8 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 [23]:
Inhibition ð%Þ ¼ ½1 ðTw=CwÞ 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 [24,25]. 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 mM, 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 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. Timelapse 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 mg/ml propidium iodide, 50 mg/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% Triton-X-100, 100 mM TriseHCl, 150 mM NaCl and 0.1 U ml1 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), and antiGAPDH (1:2000). All antibodies were obtained from Santa Cruz 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. 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. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2013.04.040. References [1] L. Zhou, Y. Liu, W. Zhang, P. Wei, C. Huang, J. Pei, Y. Yuan, L. Lai, J. Med. Chem. 49 (2006) 3440e3443. [2] R. Ghahremanzadeh, M. Sayyafi, S. Ahadi, A. Bazgir, J. Comb. Chem. 11 (2009) 393e396. [3] S.K. Sridhar, S.N. Pandeya, J.P. Stables, A. Ramesh, Eur. J. Pharm. Sci. 16 (2002) 129e132. [4] Z.H. Chohan, H. Pervez, A. Rauf, K.M. Khan, C.T. Supuran, J. Enzyme Inhib. Med. Chem. 19 (2004) 417e423. [5] R.J. Motzer, T.E. Hutson, P. Tomczak, M.D. Michaelson, R.M. Bukowski, O. Rixe, S. Oudard, S. Negrier, C. Szczylik, S.T. Kim, N. Engl. J. Med. 356 (2007) 115e124. [6] Q. Xiang, F. Wang, X. Su, Y. Liang, L. Zheng, Y. Mi, W. Chen, L. Fu, Cell. Oncol. 34 (2011) 33e44. [7] M. Krug, A. Hilgeroth, Mini-Rev. Med. Chem. 8 (2008) 1312e1327. [8] J. Adachi, Y. Mori, S. Matsui, H. Takigami, J. Fujino, H. Kitagawa, C.A. Miller Iii, T. Kato, K. Saeki, T. Matsuda, J. Biol. Chem. 276 (2001) 31475e31478. [9] R. Hoessel, S. Leclerc, J.A. Endicott, M. Nobel, A. Lawrie, P. Tunnah, M. Leost, E. Damiens, D. Marie, D. Marko, Nat. Cell. Biol. 1 (1999) 60e67. [10] Z. Xiao, L. Qian, B. Liu, Y. Hao, Br. J. Haematol. 111 (2000) 711e712. [11] S. Leclerc, M. Garnier, R. Hoessel, D. Marko, J.A. Bibb, G.L. Snyder, P. Greengard, J. Biernat, Y.Z. Wu, E.M. Mandelkow, J. Biol. Chem. 276 (2001) 251e260. [12] (a) Z.H. Wang, W.Y. Li, F.L. Li, L. Zhang, W.Y. Hua, J.C. Cheng, Q.Z. Yao, Chin. Chem. Lett. 20 (2009) 542e544; (b) Z.H. Wang, Y. Dong, T. Wang, M.H. Shang, W.Y. Hua, Q.Z. Yao, Chin. Chem. Lett. 21 (2010) 297e300. [13] S. Pandeya, D. Sriram, G. Nath, E. DeClercq, Eur. J. Pharm. Sci. 9 (1999) 25e31. [14] A. Jarrahpour, M. Zarei, Molbank (2004) M377. [15] V.C. da Silveira, J.S. Luz, C.C. Oliveira, I. Graziani, M.R. Ciriolo, A.M.C. Ferreira, J. Inorg. Biochem. 102 (2008) 1090e1103. [16] J-j Xu, X-m Dai, H-l Liu, W-j Guo, J. Gao, C-h Wang, W-b Li, Q-z Yao, J. Appl. Toxicol. 31 (2011) 164e172. [17] G. Eisenbrand, F. Hippe, S. Jakobs, S. Muehlbeyer, J. Cancer. Res. Clin. 130 (2004) 627e635. [18] F. Canduri, W.F. Azevedo Jr., Curr. Comput-Aid. Drug 1 (2005) 53e64. [19] B. Feng, Y.-W. Guo, C.-G. Huang, L. Li, R.-H. Chen, B.-H. Jiao, Chem-Biol. Interact. 183 (2010) 142e153. [20] B. Lewin, Cell 61 (1990) 743e752. [21] M. Dorée, S. Galas, FASEB J. 8 (1994) 1114e1121. [22] H. Yamaue, H. Tanimura, K. Noguchi, T. Hotta, M. Tani, T. Tsunoda, M. Iwahashi, M. Tamai, S. Iwakura, Br. J. Cancer 66 (1992) 794e799. [23] J. Wu, C. Li, M. Zhao, W. Wang, Y. Wang, S. Peng, Bioorg. Med. Chem. 18 (2010) 6220e6229. [24] X. Rao, Y. Zhang, Q. Yi, H. Hou, B. Xu, L. Chu, Y. Huang, W. Zhang, M. Fenech, Q. Shi, Mutat. Res-Fundam. Mol. Mech. Mut. 646 (2008) 41e49. [25] H. Hisamichi, R. Naito, A. Toyoshima, N. Kawano, A. Ichikawa, A. Orita, M. Orita, N. Hamada, M. Takeuchi, M. Ohta, S.-I. Tsukamoto, Bioorg. Med. Chem. 13 (2005) 6277e6279.
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, in vitro and in vivo antitumor activity of symmetrical bis-Schiff base derivatives of isatin Chengyuan Liang a, y, Juan Xia b, y, Dong Lei a, Xiang Li b, Qizheng Yao a, *, Jing Gao b, ** a b
School of Pharmacy, China Pharmaceutical University, Nanjing 210009, PR China School of Pharmacy, Jiangsu University, Zhenjiang 212013, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 November 2012 Received in revised form 2 March 2013 Accepted 18 April 2013 Available online 13 June 2013
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 3,30 -(hydrazine-1,2-diylidene)bis (5-methylindolin-2-one) being the most potent compound on HepG2 (IC50 w 4.23 mM). 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 H2Blabeled 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. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: Isatin Schiff base Cytotoxicity Antitumor 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 [1e3]. 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 anticancer treatment [7]. Structurally, both Semaxanib and TSU-68 are close analogs of Sunitinib since they all share the isatin motif (Fig. 1). The isatin nucleus is also embedded in various bis-isatin compounds (Fig. 2). Among them, indigo is probably the oldest and * Corresponding author. Tel./fax: þ86 25 86634730. ** Corresponding author. Tel./fax: þ86 511 88791552. E-mail addresses: [email protected] (Q. Yao), [email protected] (J. Gao). y These two authors contributed equally to this work. 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.04.040
most famous colorant. Thousand tons of deep blue synthetic vat dye known as indigo were produced 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-3b with IC50 values of 0.075 mM and 0.190 mM respectively. Specifically, Indirubin inhibits CDK5 and GSK-3b-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 [12a]. 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 antiHIV, anticonvulsant, antibacterial, antiprotozoal, antifungal, antiviral, and anthelmintic activities [13,14]. Schiff bases of isatin also
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Fig. 1. The chemical structures of some reported drugs containing the isatin nucleus.
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 flexibility 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.
cell (4.23 mM). Thus, compound 3b was regarded as the most promising compound and was chosen for further evaluation. 3.2. Structureeactivity relationship (SAR) of the bis-Schiff base derivatives of isatin
3. Biological activity
From the above cytotoxicity evaluation, a SAR for the test compounds 3ae3r could be drawn as follows. When R2 were alkyl groups (3j, 3k, and 3l), the IC50 values were dramatically 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 5,50 -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-halogenisation derivatives, the bisfluoro 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.
3.1. Cytotoxicity evaluation
3.3. Tumor growth inhibition assessment with the HepS xenograft
The synthesized compounds 3ae3r 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)-2,5diphenyl 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 mM to 15.81 mM. 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 mM) and 5-fold more potent against HepG2
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. 4B) and the tumor size (Fig. 4C) 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
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 0.5e2 h.
Fig. 2. The chemical structures of some known bis-isatins.
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Fig. 3. A structural comparison of the designed symmetrical bis-Schiff base derivatives of isatin with indirubin and meisoindigo.
was consistent with its in vitro cytotoxicity. Furthermore, as showed in Fig. 4D and Fig. 4E, 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 sideeffect than 5-FU in the HepS tumor model. 3.4. The pathological sections of tumor tissue To further evaluate the antitumor efficacy of 3b on the tumorbearing 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.
Entry
Product
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 mM of 3b for 48 h were observed with LCS. During the first 24 h 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
Isolated
Mp(oC)
R1
R2
1
H
H
3a
81
171-173
2
5-Me
H
3b
89
188-190
3
5-Br
H
3c
83
183-185
4
5-F
H
3d
77
186-188
5
5-Cl
H
3e
82
184-186
6
5-MeO
H
3f
71
177-179
7
7-F
H
3g
78
199-201
8
6-Cl
H
3h
83
183-185
9
6-F
H
3i
65
190-192
10
H
Et
3j
79
195-197
11
5-Me
Et
3k
58
189-191
Yield(%)
12
5-F
Et
3l
87
193-196
13
H
Bn
3m
73
187-189
14
5-Me
Bn
3n
64
205-207
15
5-F
Bn
3o
81
199-201
16
H
i-Pr
3p
72
201-202
17
5-Me
i-Pr
3q
64
203-205
18
5-F
i-Pr
3r
83
192-194
Scheme 1. The synthetic route to the bis-Schiff base derivatives of isatin (3ae3r).
C. Liang et al. / European Journal of Medicinal Chemistry 74 (2014) 742e750
745
Table 1 In vitro antiproliferative activity of the synthesized compounds against various cancer cell lines. Compd.
IC50 (mM)a A549
3a 3b 3c 3d 3e 3f 3g 3h 3i 3j 3k 3l 3m 3n 3o 3p 3q 3r 5-FU
21.80 3.54 12.78 0.44 81.75 2.82 8.57 0.48 >100c 23.91 1.02 28.37 2.09 42.89 2.83 5.38 0.42 >100 >100 >100 34.78 1.29 35.59 0.25 >100 19.40 0.13 >100 >100 17.31 0.87
HeLa 13.44 5.26 8.6 0.13 14.09 0.17 9.45 0.81 >100 >100 30.82 2.14 24.58 1.09 4.71 0.21 >100 >100 >100 >100 3.37 0.10 48.65 0.90 65.10 0.81 >100 >100 7.32 0.19
HepG2 12.48 1.78 4.23 0.68 >100 25.29 1.28 15.81 0.54 >100 19.88 0.11 8.74 0.31 58.39 1.82 >100 >100 87.69 0.80 49.33 0.87 47.49 0.24 87.34 0.69 >100 >100 >100 19.76 0.83
U251
SGC-7901 b
>100 29.00 0.21 >100 22.76 1.03 21.65 1.28 11.72 0.45 7.09 0.02 >100 23.71 1.50 >100 >100 13.90 0.29 >100 >100 26.39 1.82 23.41 01 >100 >100 28.45 1.34
7.20 0.86 12.66 0.21 37.17 0.39 47.85 2.56 84.26 4.52 18.42 1.71 47.64 2.49 11.87 0.44 >100 >100 43.76 1.54 88.11 1.73 74.98 2.74 >100 79.13 4.09 64.93 0.29 >100 >100 >100
Data are presented as mean S.D. (n ¼ 3). Bold value signifies P < 0.05. 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. b The 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 mM was given.
manner (See Supplementary movie 1 and 2). These results suggested that 3b could interfere with mitosis and induce the apoptosis-associated cell death. 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 [9,16e18]. In order to examine whether the antiproliferative 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 mM 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 [19e21]. 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 Fig. 7C, compound 3b downregulated the expression of cyclin B1 and cdc 2 in time- and dosedependent manner in HepG2 cells. 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, 3,30 -(hydrazine-1,2-diylidene)bis(5-methylindolin2-one) 3b exhibited a stronger cytotoxicity than 5-FU in vitro with IC50 values of 4.23 mM on HepG2 cells, 12.66 mM on SGC-7901 cells, 12.78 mM on A549 cells, respectively. The preliminary SAR showed that the N1-unsubstituted bis-isatin structures with 5,50 -bis electron-donating groups could improve the antitumor activity. Gratifyingly, 3b exhibited an excellent in vivo antitumor profile (i.e. high efficacy 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 downregulation 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 antitumor potential 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 antitumor 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. Compounds were visualized under UV lamp (254 nm). 1 H-NMR and 13C-NMR spectra were obtained on a Bruker AV300 MHz NMR spectrometer (1H-NMR at 300 MHz, 13C-NMR at 75 MHz) at ambient temperature. 1H-NMR spectra were reported in ppm on the d scale and referenced to the internal tetramethylsilane. The data were presented as follows: chemical shift, multiplicity (s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet, br ¼ broad, app ¼ apparent), coupling constant(s) in Hertz (Hz), and integration. Chemical shifts (d) were recorded relative to residual
746
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A
28
average body weight(g)
#
Control 5-Fu 3b(20 mg/kg) 3b(40 mg/kg)
#
26
#
*
6
7
24 22 20 18 0
1
2
3
4
8
9
10
11
days
B1.5 Average tumor weight(g)
5
C Control
1.0
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)
D 4 3 2
* 1 0 Control
E8 index of spleen(mg/g)
index of thymus(mg/g)
5
5-FU
20
40
3b(mg/kg)
6 4 2 0 Control
5-FU
20
40
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 ¼ (thymus weight/final body weight); spleen index ¼ (spleen weight/final body weight). Data represent mean S.E.M. (n ¼ 6), #P < 0.05, vs 5-FU; *P < 0.05, vs control. ⁄⁄P < 0.01 (Student’s t-test); n ¼ 7, *P < 0.05.
DMSO-d6 (d ¼ 2.50 in 1H-NMR and d ¼ 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 200e300 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 N1substituted 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 Table 2 The inhibitory effect of 3b on HepS tumor xenograft. Compd.
Rate of inhibition (%)
5-FU (25 mg/kg) 3b (20 mg/kg) 3b (40 mg/kg)
47.10 17.09 60.09
an appropriate alkyl halide (10 mmol) at room temperature. The mixture was then stirred at 60e65 C 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 (200e300 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 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 0.5e2 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
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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).
with those reported in earlier literature. Characteristic data for all the bis-Schiff base derivatives are as follows:
110.9; IR (KBr) n 3239 (2NH), 1735 (2C¼O), 1617 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 327.0694, found 327.0690.
5.3.1. 3,30 -(Hydrazine-1,2-diylidene)diindolin-2-one (3a) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 10.78 (s, 2H), 7.86 (d, 2H, J ¼ 7.29 Hz), 7.81 (d, 2H, J ¼ 7.50 Hz), 7.50 (t, 2H, J ¼ 7.29 Hz), 7.26 (t, 2H, J ¼ 7.50 Hz); 13C-NMR (75 MHz, DMSO-d6) d: 169.0, 141.2, 138.0, 131.2, 129.4, 124.4, 119.4, 117.7; IR (KBr) n 3269 (2NH), 1731 (2C¼O), 1608 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 291.0882, found 291.0889.
5.3.5. 3,30 -(Hydrazine-1,2-diylidene)bis(5-chloroindolin-2-one) (3e) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 11.00 (2H, s), 7.48 (2H, d, J ¼ 7.10 Hz), 7.42 (2H, d, J ¼ 7.10 Hz), 6.93 (2H, s); 13C-NMR (75 MHz, DMSO-d6) d: 163.4, 144.8, 142.9, 134.7, 131.4, 128.5, 115.8, 110.8; IR (KBr) n 3234 (2NH), 1734 (2C¼O), 1612 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 359.0103, found 359.0107.
5.3.2. 3,30 -(Hydrazine-1,2-diylidene)bis(5-methylindolin-2-one) (3b) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 10.90 (2H, s), 7.33 (2H, s), 7.03 (2H, d, J ¼ 7.29 Hz), 6.84 (2H, d, J ¼ 7.29 Hz), 2.22 (6H, s); 13 C-NMR (75 MHz, DMSO-d6) d: 169.0, 138.0, 134.1, 131.5, 129.6, 121.6, 117.6, 21.3; IR (KBr) n 3383-3248 (2NH), 1723 (2C¼O), 1613 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 319.1195, found 319.1183. 5.3.3. 3,30 -(Hydrazine-1,2-diylidene)bis(5-bromoindolin-2-one) (3c) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 10.99 (s, 2H), 8.10 (s, 2H), 7.75 (d, 2H, J ¼ 7.29 Hz), 7.56 (d, 2H, J ¼ 7.29 Hz); 13C-NMR (75 MHz, DMSO-d6) d: 169.0, 141.8, 140.2, 138.0, 136.9, 132.9, 119.9, 118.8, 118.0; IR (KBr) n 3427-3241 (2NH), 1718 (2C¼O), 1619 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 446.9092, found 446.9096. 5.3.4. 3,30 -(Hydrazine-1,2-diylidene)bis(5-fluoroindolin-2-one) (3d) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 10.89 (s, 2H), 7.84 (d, 2H, J ¼ 6.80 Hz), 7.78 (s, 2H), 7.34 (d, 2H, J ¼ 6.80 Hz); 13C-NMR (75 MHz, DMSO-d6) d: 169.0, 158.6, 138.0, 136.9, 119.3, 118.0, 112.7,
5.3.6. 3,30 -(Hydrazine-1,2-diylidene)bis(5-methoxyindolin-2-one) (3f) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 11.00 (s, 2H), 7.75 (d, 2H, J ¼ 7.30 Hz), 7.48 (s, 2H), 7.04 (d, 2H, J ¼ 7.30 Hz), 3.83 (s, 6H); 13CNMR (75 MHz, DMSO-d6) d: 169.0, 156.3, 138.0, 133.5, 122.7, 118.7, 116.8, 113.5, 55.8; IR (KBr) n 3281 (2NH), 1725 (2C¼O), 1614 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 351.1093, found 351.1096. 5.3.7. 3,30 -(Hydrazine-1,2-diylidene)bis(7-fluoroindolin-2-one) (3g) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 11.02 (s, 2H), 7.60 (d, 2H), 7.35 (m, 4H); 13C-NMR (75 MHz, DMSO-d6) d: 169.0, 162.9, 138.0, 126.0, 125.7, 125.0, 119.3, 118.0; IR (KBr) n 3413-3321 (2NH), 1733 (2C¼O), 1619 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 327.0694, found 327.0698. 5.3.8. 3,30 -(Hydrazine-1,2-diylidene)bis(6-chloroindolin-2-one) (3h) Yellow powder; 1H-NMR (300 MHz, DMSO-d6) d: 11.00 (s, 2H), 8.10 (s, 2H), 7.75 (d, 2H, J ¼ 6.90 Hz), 7.26 (d, 2H, J ¼ 6.90 Hz); 13CNMR (75 MHz, DMSO-d6) d: 169.0, 148.2, 140.6, 138.0, 130.8, 124.5, 119.8, 115.8; IR (KBr) n 3446-3281 (2NH), 1743 (2C¼O), 1621 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 359.0103, found 359.0105.
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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 mM of 3b, and then observed for the morphological changes of the cells and their nuclei. (A) Control (B) 8 mM 3b. DIC: the bright imaging of cells; GFP: the fluorescence imaging of nuclei.
5.3.9. 3,30 -(Hydrazine-1,2-diylidene)bis(6-fluoroindolin-2-one) (3i) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 11.00 (s, 2H), 7.79 (d, 2H, J ¼ 7.25 Hz), 7.59 (s, 2H), 7.10 (d, 2H, J ¼ 7.25 Hz); 13C-NMR (75 MHz, DMSO-d6) d: 169.0, 165.4, 148.4, 138.0, 131.0, 113.3, 111.2, 110.2; IR (KBr) n 3385-3269 (2NH), 1741 (2C¼O), 1618 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 327.0694, found 327.0699. 5.3.10. 3,30 -(Hydrazine-1,2-diylidene)bis(1-ethylindolin-2-one) (3j) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 7.86 (d, 2H, J ¼ 7.29 Hz), 7.81 (d, 2H, J ¼ 7.62 Hz), 7.50 (t, 2H, J ¼ 7.29 Hz), 7.26 (t, 2H, J ¼ 7.62 Hz), 4.28 (q, 4H, J ¼ 10 Hz), 1.31 (t, 6H, J ¼ 10 Hz); 13CNMR (75 MHz, DMSO-d6) d: 163.2, 147.4, 138.0, 131.2, 129.4, 124.4, 117.7, 115.8, 42.2, 13.7; IR (KBr) n 1741 (2C¼O), 1627 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 347.1508, found 347.1504. 5.3.11. 3,30 -(Hydrazine-1,2-diylidene)bis(1-ethyl-5-methylindolin2-one) (3k) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 8.01 (s, 2H), 7.74 (d, 2H, J ¼ 6.90 Hz), 7.16 (d, 2H, J ¼ 6.90 Hz), 3.88 (q, 4H, J ¼ 8.89 Hz), 2.34 (s, 6H), 1.51 (t, 6H, J ¼ 8.89 Hz); 13C-NMR (75 MHz, DMSO-d6) d: 163.2, 144.4, 142.3, 134.1, 129.6, 120.6, 117.6, 116.1, 42.2, 21.3, 13.7; IR (KBr) n 1738 (2C¼O), 1613 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 375.1821, found 375.1825.
5.3.12. 3,30 -(Hydrazine-1,2-diylidene)bis(1-ethyl-5-fluoroindolin2-one) (3l) Red powder; 1H-NMR (300 MHz, DMSO-d6) d: 7.84 (d, 2H, J ¼ 7.29 Hz), 7.78 (s, 2H), 7.34 (d, 2H, J ¼ 7.29 Hz), 3.88 (q, 4H, J ¼ 10 Hz), 1.50 (t, 6H, J ¼ 10 Hz); 13C-NMR (75 MHz, DMSO-d6) d: 163.2, 158.6, 143.0, 138.0, 125.0, 119.3, 118.0, 110.9, 42.2, 13.7; IR (KBr) n 1731 (2C¼O), 1618 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 383.1320, found 383.1325. 5.3.13. 3,30 -(Hydrazine-1,2-diylidene)bis(1-benzylindolin-2-one) (3m) Yellow powder; 1H-NMR (300 MHz, DMSO-d6) d: 7.86e7.81 (m, 4H), 7.50 (t, 2H, J ¼ 6.56 Hz), 7.33 (t, 4H, J ¼ 6.29 Hz), 7.26-7.23 (m, 8H), 4.94 (s, 4H); 13C-NMR (75 MHz, DMSO-d6) d: 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) n 1730 (2C¼O), 1614 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 471.1821, found 471.1827. 5.3.14. 3,30 -(Hydrazine-1,2-diylidene)bis(1-benzyl-5-methylindolin2-one) (3n) Yellow powder; 1H-NMR (300 MHz, DMSO-d6) d: 8.13 (s, 2H), 7.74 (t, 2H, J ¼ 6.95 Hz), 7.33 (t, 4H), 7.25 (m, 6H), 7.16 (d, 2H, J ¼ 6.95 Hz), 4.94 (s, 4H), 3.13 (s, 6H); 13C-NMR (75 MHz, DMSO-d6)
C. Liang et al. / European Journal of Medicinal Chemistry 74 (2014) 742e750
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Number of cells
G0/G1:46.36% S:26.31% G2/M: 27.32 %
0
50
100
150
0
0
0
200
200
800 600
G0/G1:55.21% S:29.68% G2/M:15.11%
400
Number of cells
400
600
G0/G1: 63.58 % S: 25.96 % G2/M: 10.46 %
200
Number of cells
800
600
1000
1000
A
0
50
100
DNA Content
DNA Content
Control
12 h
B
150
0
50
100
150
DNA Content
24 h
C Compound 3b (µmol/L) Control
4
8
Compound 3b (8 µmol/L) Control 12 h
16
cyclin B1
24 h
48 h
cyclin B1
cdc 2
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 mM 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.
d: 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) n 1735 (2C¼O), 1623 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 499.2134, found 499.2137. 5.3.15. 3,30 -(Hydrazine-1,2-diylidene)bis(1-benzyl-5-fluoroindolin2-one) (3o) Pink powder; 1H-NMR (300 MHz, DMSO-d6) d: 7.90-7.70 (m, 4H), 7.34 (t, 2H, J ¼ 7.05 Hz), 7.33 (t, 4H, J ¼ 7.05 Hz), 7.26e7.23 (m, 6H), 4.94 (s, 4H); 13C-NMR (75 MHz, DMSO-d6) d: 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) n 1735 (2C¼O), 1623 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 507.1633, found 507.1637. 5.3.16. 3,30 -(Hydrazine-1,2-diylidene)bis(1-isopropylindolin-2-one) (3p) Pale powder; 1H-NMR (300 MHz, DMSO-d6) d: 7.91-7.82 (m, 4H), 7.52 (t, 2H, J ¼ 7.09 Hz), 7.21 (t, 2H, J ¼ 7.09 Hz), 3.84 (m, 2H), 1.20 (m, 12H); 13C-NMR (75 MHz, DMSO-d6) d: 163.1, 147.4, 138.0, 131.0, 128.6, 124.7, 118.4, 115.3, 58.4, 20.6; IR (KBr) n 1740 (2C¼O), 1609 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 375.1821, found 375.1826. 5.3.17. 3,30 -(Hydrazine-1,2-diylidene)bis(1-isopropyl-5methylindolin-2-one) (3q) Pink powder; 1H-NMR (300 MHz, DMSO-d6) d: 8.16 (s, 2H), 7.74 (d, 2H, J ¼ 7.29 Hz), 7.16 (d, 2H, J ¼ 7.29 Hz), 3.84 (m, 2H), 2.34 (s, 6H), 1.20 (m, 12H) ; 13C-NMR (75 MHz, DMSO-d6) d: 162.9, 144.4, 138.0, 134.1, 129.6, 120.6, 117.6, 116.1, 58.4, 21.3, 20.6; IR (KBr) n 1747 (2C¼O), 1604 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: calcd 403.2134, found 403.2139. 5.3.18. 3,30 -(Hydrazine-1,2-diylidene)bis(5-fluoro-1isopropylindolin-2-one) (3r) Pale powder; 1H-NMR (300 MHz, DMSO-d6) d: 7.84 (d, 2H, J ¼ 7.29 Hz), 7.78 (s, 2H), 7.34 (d, 2H, J ¼ 7.29 Hz), 3.94 (m, 2H), 1.20
(m, 12H); 13C-NMR (75 MHz, DMSO-d6) d: 162.9, 158.6, 143.0, 138.0, 125.0, 119.3, 118.0, 110.9, 58.4, 20.6; IR (KBr) n 1730 (2C¼O), 1614 (2C¼N) cm1; HRMS (EI) for (M þ H)þ: 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 mL 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 mL 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 [22]:
Rate of Inhibition ¼ ð1 OD570 drug treated=OD570 controlÞ 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.
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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 6e8 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 [23]:
Inhibition ð%Þ ¼ ½1 ðTw=CwÞ 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 [24,25]. 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 mM, 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 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. Timelapse 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 mg/ml propidium iodide, 50 mg/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% Triton-X-100, 100 mM TriseHCl, 150 mM NaCl and 0.1 U ml1 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), and antiGAPDH (1:2000). All antibodies were obtained from Santa Cruz 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. 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. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2013.04.040. References [1] L. Zhou, Y. Liu, W. Zhang, P. Wei, C. Huang, J. Pei, Y. Yuan, L. Lai, J. Med. Chem. 49 (2006) 3440e3443. [2] R. Ghahremanzadeh, M. Sayyafi, S. Ahadi, A. Bazgir, J. Comb. Chem. 11 (2009) 393e396. [3] S.K. Sridhar, S.N. Pandeya, J.P. Stables, A. Ramesh, Eur. J. Pharm. Sci. 16 (2002) 129e132. [4] Z.H. Chohan, H. Pervez, A. Rauf, K.M. Khan, C.T. Supuran, J. Enzyme Inhib. Med. Chem. 19 (2004) 417e423. [5] R.J. Motzer, T.E. Hutson, P. Tomczak, M.D. Michaelson, R.M. Bukowski, O. Rixe, S. Oudard, S. Negrier, C. Szczylik, S.T. Kim, N. Engl. J. Med. 356 (2007) 115e124. [6] Q. Xiang, F. Wang, X. Su, Y. Liang, L. Zheng, Y. Mi, W. Chen, L. Fu, Cell. Oncol. 34 (2011) 33e44. [7] M. Krug, A. Hilgeroth, Mini-Rev. Med. Chem. 8 (2008) 1312e1327. [8] J. Adachi, Y. Mori, S. Matsui, H. Takigami, J. Fujino, H. Kitagawa, C.A. Miller Iii, T. Kato, K. Saeki, T. Matsuda, J. Biol. Chem. 276 (2001) 31475e31478. [9] R. Hoessel, S. Leclerc, J.A. Endicott, M. Nobel, A. Lawrie, P. Tunnah, M. Leost, E. Damiens, D. Marie, D. Marko, Nat. Cell. Biol. 1 (1999) 60e67. [10] Z. Xiao, L. Qian, B. Liu, Y. Hao, Br. J. Haematol. 111 (2000) 711e712. [11] S. Leclerc, M. Garnier, R. Hoessel, D. Marko, J.A. Bibb, G.L. Snyder, P. Greengard, J. Biernat, Y.Z. Wu, E.M. Mandelkow, J. Biol. Chem. 276 (2001) 251e260. [12] (a) Z.H. Wang, W.Y. Li, F.L. Li, L. Zhang, W.Y. Hua, J.C. Cheng, Q.Z. Yao, Chin. Chem. Lett. 20 (2009) 542e544; (b) Z.H. Wang, Y. Dong, T. Wang, M.H. Shang, W.Y. Hua, Q.Z. Yao, Chin. Chem. Lett. 21 (2010) 297e300. [13] S. Pandeya, D. Sriram, G. Nath, E. DeClercq, Eur. J. Pharm. Sci. 9 (1999) 25e31. [14] A. Jarrahpour, M. Zarei, Molbank (2004) M377. [15] V.C. da Silveira, J.S. Luz, C.C. Oliveira, I. Graziani, M.R. Ciriolo, A.M.C. Ferreira, J. Inorg. Biochem. 102 (2008) 1090e1103. [16] J-j Xu, X-m Dai, H-l Liu, W-j Guo, J. Gao, C-h Wang, W-b Li, Q-z Yao, J. Appl. Toxicol. 31 (2011) 164e172. [17] G. Eisenbrand, F. Hippe, S. Jakobs, S. Muehlbeyer, J. Cancer. Res. Clin. 130 (2004) 627e635. [18] F. Canduri, W.F. Azevedo Jr., Curr. Comput-Aid. Drug 1 (2005) 53e64. [19] B. Feng, Y.-W. Guo, C.-G. Huang, L. Li, R.-H. Chen, B.-H. Jiao, Chem-Biol. Interact. 183 (2010) 142e153. [20] B. Lewin, Cell 61 (1990) 743e752. [21] M. Dorée, S. Galas, FASEB J. 8 (1994) 1114e1121. [22] H. Yamaue, H. Tanimura, K. Noguchi, T. Hotta, M. Tani, T. Tsunoda, M. Iwahashi, M. Tamai, S. Iwakura, Br. J. Cancer 66 (1992) 794e799. [23] J. Wu, C. Li, M. Zhao, W. Wang, Y. Wang, S. Peng, Bioorg. Med. Chem. 18 (2010) 6220e6229. [24] X. Rao, Y. Zhang, Q. Yi, H. Hou, B. Xu, L. Chu, Y. Huang, W. Zhang, M. Fenech, Q. Shi, Mutat. Res-Fundam. Mol. Mech. Mut. 646 (2008) 41e49. [25] H. Hisamichi, R. Naito, A. Toyoshima, N. Kawano, A. Ichikawa, A. Orita, M. Orita, N. Hamada, M. Takeuchi, M. Ohta, S.-I. Tsukamoto, Bioorg. Med. Chem. 13 (2005) 6277e6279.
