Design, synthesis and anti-proliferative studies of a novel series of indirubin derivatives

Article abstract of DOI:10.1016/j.cclet.2010.05.026

A series of novel derivatives of indirubin were synthesized and evaluated for their anti-proliferative activity against human cancer cell lines of SGC7901, A549, HL-60, SK-BR-3 and HCT116. Most of the compounds displayed more potent activity than Sunitinib. In addition, the derivatives showed improved water solubility, which may be favorable to their pharmacokinetic performances.

Full text of DOI:10.1016/j.cclet.2010.05.026

Available online at www.sciencedirect.com
Chinese Chemical Letters 21 (2010) 1407–1410
www.elsevier.com/locate/cclet
Design, synthesis and anti-proliferative studies of a novel series
of indirubin derivatives
b,
Tian Cai Wang a, Ju Zhi Wei b, Chuan Sheng Guo b, Hui Bin Zhang a, , Hou Xing Fan
*
*
a Center of Drug Discovery, College of Pharmacy, China Pharmaceutical University, Nanjing 210009, China
b Shanghai Sun-sail Pharmaceutical Science & Technology Co., Ltd., Shanghai 201203, China
Received 15 April 2010
Abstract
A series of novel derivatives of indirubin were synthesized and evaluated for their anti-proliferative activity against human
cancer cell lines of SGC7901, A549, HL-60, SK-BR-3 and HCT116. Most of the compounds displayed more potent activity than
Sunitinib. In addition, the derivatives showed improved water solubility, which may be favorable to their pharmacokinetic
performances.
# 2010 Hui Bin Zhang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Anti-proliferative activity; Indirubin; Sunitinib
Indirubin (Fig. 1), a minor component of Qing Dai (Indigo naturalis), is the biological active component of Qing
Dai [1,2]. Indirubin is the active ingredient of a traditional Chinese medicine recipe, ‘‘Danggui Longhui Wan’’, used to
treat various diseases, including chronic myelocytic leukemia [3]. It is revealed that indirubin and some of its
analogues are potent inhibitors of CDKs, which play an important role in cell cycles. Thus, indirubin and its analogues
have a great potential for the treatment of cancer [4–6]. Further studies show that the mechanism of action of indirubins
is complex. Additional targets include GSK-3, c-Src, AhR, GP, JNK, VEGFR, FLT3, Auroa’s, LCK, etc. [7].
Sunitinib(Fig.1) isamultitargetedreceptortyrosinekinaseinhibitor, withbothdirectanti-proliferativeeffectsandanti-
angiogenic properties, targeting the VEGFRs, PDGFR-b, and c-Kit [8]. The excellent pharmacokinetic profiles of
Sunitinib may attribute to the water-soluble amide chain. Indirubin was not considered a good candidate for drug
developmentbecauseitspoorwatersolubilitywouldadverselyaffectoralbioavailability[9].Thusvariousofwater-soluble
amide side chains were introduced into indirubin to find desired anti-proliferative agents, which may inhibit multiple
tyrosinekinasesincludingVEGFRbasedonthestructuralpropertiesandthetargetmechanismsofindirubinandSunitinib.
1. Experimental
Compound 3 was synthesized from 5-Br-indole according to the known method [10,11]. As shown in Scheme 1,
condensation of compound 3 with N1,N1-diethylethane-1,2-diamine afforded compound 4 in yield of 80%, which
* Corresponding authors.
E-mail addresses: zhanghb80@163.com (H.B. Zhang), hxfan@sun-sail.cn (H.X. Fan).
1001-8417/$ – see front matter # 2010 Hui Bin Zhang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2010.05.026

T.C. Wang et al. / Chinese Chemical Letters 21 (2010) 1407–1410
1[(Fig._1)TD$FIG] 408
Fig. 1. Indirubin, Sunitinib and our designed compounds.
[(Scheme_1)TD$FIG]
Scheme 1. Reagents and conditions: (a) CuCN, NMP, 230 8C, 5 h; (b) (1) 10% KOH, reflux, 15 h; (2) 10% HCl; (c) HOBt, EDCÁHCl, DMF, Et3N,
r.t., overnight; (d) I2, NaOH, MeOH, r.t., 1 h; (e) HgOAc, HOAc, 80 8C, overnight; (f) indoline-2,3-dione, Na2CO3, MeOH, r.t., 3 h.
underwent iodination reaction to obtain compound 5. Treatment of compound 5 with HgOAc in acetic acid gave
compound 6 in yield of 56% with two steps. Finally, condensation of compound 6 with indoline-2,3-dione [12]
afforded compound 7 in good yield. Compound 11 was synthesized according to the similar method in Scheme 1.
Hydrolysis of compound 11 gave key intermediate 12, which condensed with various amines to afford the target
1
compounds 13–20 (Scheme 2). All the target compounds were confirmed by H NMR and MS (ESI) data [14].
2. Results and discussion
The result of in vitro inhibition ratio of compounds 7 and 13–20 against VEGFR-2 high-expressed HMEC at a
concentration of 10 mmol/L is summarized in Table 1. All tested compounds except compound 20 displayed more
potent anti-proliferative activity than the positive control Sunitinib. Fluorine can be introduced to the indirubin without
the decrease in activity indicated by the result of compounds 7 and 14. Compound 16 exhibited more potent activity
than compound 15, the activity increased with the increment of length of side chain. It may be explained that
compound 16 has a stronger binding affinity with the receptor than that of compound 15.
The target compounds were further subjected to in vitro anti-proliferative activity assay. As shown in Table 1, most
of the compounds displayed more potent anti-proliferative activity than Sunitinib against cell lines of SGC7901
(gastric), A549 (lung), HL-60 (leukemia) and SK-BR-3 (breast), and are inferior to Sunitinib against HCT116 (colon).
[(Scheme_2)TD$FIG]
Scheme 2. Reagents and conditions: (a) H2SO4, MeOH, 70 8C, 3 h; (b) I2, NaOH, MeOH, r.t., 1 h; (c) HgOAc, HOAc, 80 8C, overnight; (d) 5-
fluoroindoline-2,3-dione [13], Na2CO3, MeOH, r.t., 3 h; (e) LiOH, THF/MeOH, r.t., 16 h; (f) HoBt, EDCÁHCl, DMF, Et3N, r.t., overnight.

T.C. Wang et al. / Chinese Chemical Letters 21 (2010) 1407–1410
1409
Table 1
Anti-proliferative effects of indirubin derivatives.
Compound
R1
R2
Inhibition ratioa (%)
IC50 (mmol/L)
SGC7901
7.8
A549
4.7
HL-60
SK-BR-3
0.0008
HCT116
3.49
[TD$INLINE]
7
H
88.1 Æ 4.8
8.6
9.0
4.6
1.7
[TD$INLINE]
13
14
15
F
F
F
68.3 Æ 4.4
88.5 Æ 5.9
69.0 Æ 5.5
1.42
9.12
1.22
4.9
4.3
2.17
3.63
1.13
[TD$INLINE]
3.3
0.06
2.2
[TD$INLINE]
3.18
[TD$INLINE]
16
17
18
F
F
F
80.9 Æ 10.2
86.9 Æ 6.4
74.6 Æ 3.1
4.33
ndb
0.76
3.2
7.6
3.2
0.3
0.1
0.4
0.5
3.46
2.58
1.16
[TD$INLINE]
[TD$INLINE]
1.54
1.17
[TD$INLINE]
19
20
F
60.9 Æ 2.6
1.51
0.76
0.03
0.6
2.57
[TD$INLINE]
F
–
59.2 Æ 11.5
59.9 Æ 13.9
1.16
2.28
2.77
4.48
1.3
5.5
7.4
3.81
2.2
Sunitinib
–
3.55
a
VEGFR-2 high-expressed HMEC.
Not determined.
b
In addition, Introduce of amide side chain greatly improved the water solubility, which exemplified by the HCl salt of
compound 17 with solubility of 45 mg/mL in water (data not shown).
In summary, a series of novel derivatives of indirubin were synthesized and evaluated for their anti-proliferative
activity against human cancer cell lines of SGC7901, A549, HL-60, SK-BR-3 and HCT116. Most of the compounds
displayed more potent activity than Sunitinib, especially compound 7 exhibited the most potent activity against SK-
BR-3 cell line with IC50 of 0.8 nmol/L. In addition, the derivatives showed improved water solubility, which may be
favorable to their pharmacokinetic performances. Further pharmacokinetic profiles and in vivo anti-proliferative
studies of this series of compounds are ongoing in our laboratory.
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[13] M.K. Uddin, S.G. Reignier, T. Coulter, et al. Bioorg. Med. Chem. Lett. 17 (2007) 2854.
[14] Analytical data for compounds 7, 11, 13–20. Compound 7: mp: 165–166 8C. 1H NMR (300 MHz, DMSO-d6): d 11.24 (s, 1H), 10.96 (s, 1H),
8.78 (d, 1H, J = 7.6 Hz), 8.57 (s, 1H), 8.18 (d, 1H, J = 1.4 Hz), 8.08 (dd, 1H, J1 = 1.6 Hz, J2 = 8.4 Hz), 7.47 (d, 1H, J = 8.1 Hz), 7.28 (m, 1H),
7.04 (m, 1H), 6.91 (d, 1H, J = 7.6 Hz), 3.45-3.52 (m, 2H), 2.49–2.51 (m, 6H), 1.11 (t, 6H, J = 5.9 Hz). ESI-MS (m/z): 405 (MH+). Compound
11: mp: >290 8C. 1H NMR (300 MHz, DMSO-d6): d 11.15 (br s, 2H), 8.54 (d, 1H, J = 9.7 Hz), 8.11 (d, 2H, J = 7.8 Hz), 7.48 (d, 1H,
J = 8.0 Hz), 7.09 (d, 1H, J = 7.7 Hz), 6.83–6.87 (m, 1H), 3.80 (s, 3H). ESI–MS (m/z): 339 (MH+). Compound 13: mp: 267–269 8C. 1H NMR
(300 MHz, DMSO-d6): d 11.29 (s, 1H), 10.96 (s, 1H), 8.51–8.58 (m, 2H), 8.18 (s, 1H), 8.07 (d, 1H, J = 7.7 Hz), 7.45 (d, 1H, J = 8.4 Hz), 7.08–
7.12 (m, 1H), 6.88 (m, 1H), 3.42–3.54 (m, 2H), 2.23 (s, 6H), 2.10–2.16 (m, 2H). ESI–MS (m/z): 395 (MH+). Compound 14: mp: 233–235 8C.
1H NMR (300 MHz, CDCl3): d 10.54 (s, 1H), 8.64 (dd, 1H, J1 = 2.4 Hz, J2 = 9.9 Hz), 8.51 (br s, 1H), 8.05 (s, 1H), 7.91 (d, 1H, J = 9.0 Hz),
6.95–7.04 (m, 3H), 6.78–6.82 (m, 1H), 3.50–3.57 (m, 2H), 2.62–2.74 (m, 6H), 1.07 (t, 6H, J = 7.3 Hz). ESI–MS (m/z): 423 (MH+). Compound
15: mp: 238–240 8C. 1H NMR (300 MHz, DMSO-d6): d 11.31 (s, 1H), 10.97 (s, 1H), 8.54–8.60 (m, 2H), 8.20 (d, 1H, J = 1.5 Hz), 8.09 (dd, 1H,
J1 = 1.8 Hz, J2 = 8.4 Hz), 7.47 (d, 1H, J = 8.4 Hz), 7.10–7.15 (m, 1H), 6.87-6.91 (m, 1H), 3.45 (m, 2H), 2.45–2.65 (m, 6H), 1.64–1.68 (m, 4H).
ESI-MS (m/z): 421 (MH+). Compound 16: mp: 159–161 8C. 1H NMR (300 MHz, DMSO-d6): d 11.27 (s, 1H), 10.94 (s, 1H), 8.54–8.60 (m, 2H),
8.15 (d, 1H, J = 1.2 Hz), 8.06 (dd, 1H, J1 = 1.8 Hz, J2 = 8.4 Hz), 7.45 (d, 1H, J = 8.4 Hz), 7.08–7.10 (m, 1H), 6.87 (dd, 1H, J1 = 4.8 Hz,
J2 = 8.4 Hz), 3.21–3.27 (m, 2H), 2.38–2.45 (m, 6H), 1.64–1.75 (m, 6H). ESI–MS (m/z): 435 (MH+). Compound 17: mp: >290 8C. 1H NMR
(300 MHz, DMSO-d6): d 11.28 (s, 1H), 10.93 (s, 1H), 8.56 (dd, 1H, J1 = 2.5 Hz, J2 = 10.0 Hz), 8.30 (d, 1H, J = 8.1 Hz), 8.21 (d, 1H,
J = 1.5 Hz), 8.07 (dd, 1H, J1 = 2.0 Hz, J2 = 8.6 Hz), 7.44 (d, 1H, J = 8.1 Hz), 7.07–7.14 (m, 1H), 6.87 (dd, 1H, J1 = 4.6 Hz, J2 = 8.7 Hz), 3.68–
3.72 (m, 1H), 2.70–2.74 (m, 2H), 2.15 (s, 3H), 1.93 (t, 2H, J = 11.2 Hz), 1.76 (m, 2H), 1.54–1.63 (m, 2H). ESI–MS (m/z): 421 (MH+).
Compound 18: mp: 250–252 8C. 1H NMR (DMSO-d6): d 11.28 (s, 1H), 10.93 (s, 1H), 8.57 (dd, 1H, J1 = 2.7 Hz, J2 = 10.5 Hz), 8.31 (d, 1H,
J = 7.2 Hz), 8.21 (d, 1H, J = 1.2 Hz), 8.08 (dd, 1H, J1 = 1.6 Hz, J2 = 8.8 Hz), 7.45 (d, 1H, J = 8.4 Hz), 7.23–7.36 (m, 5H), 7.08–7.15 (m, 1H),
6.88 (dd, 1H, J1 = 4.9 Hz, J2 = 8.7 Hz), 3.74–3.79 (m, 1H), 3.47(s, 2H), 2.82 (m, 2H), 2.02 (m, 2H), 1.78 (m, 2H), 1.56–1.65 (m, 2H). ESI–MS
(m/z): 497.3 (MH+). Compound 19: mp: >290 8C. 1H NMR (DMSO-d6): d 11.28 (s, 1H), 10.94 (s, 1H), 8.57 (dd, 1H, J1 = 3.3 Hz,
J2 = 10.5 Hz), 8.47–8.52 (m, 2H), 8.32 (d, 1H, J = 7.2 Hz), 8.21 (d, 1H, J = 1.2 Hz), 8.08 (dd, 1H, J1 = 1.6 Hz, J2 = 8.8 Hz), 7.72 (m, 1H), 7.46
(d, 1H, J = 8.5 Hz), 7.34–7.37 (m, 1H), 7.08-7.15 (m, 1H), 6.88 (dd, 1H, J1 = 5.2 Hz, J2 = 8.5 Hz), 3.74–3.79 (m, 1H), 3.51 (s, 2H), 2.81 (m,
2H), 2.05 (m, 2H), 1.79 (m, 2H), 1.56–1.63 (m, 2H). ESI–MS (m/z): 498.3 (MH+). Compound 20: mp: 277–278 8C. 1H NMR (DMSO-d6): d
10.92 (s, 1H), 10.89 (s, 1H), 8.56 (dd, 1H, J1 = 2.7 Hz, J2 = 10.3 Hz), 8.50 (d, 2H, J = 5.0 Hz), 8.32 (d, 1H, J = 7.4 Hz), 8.21 (m, 1H), 8.08 (d,
1H, J = 9.1 Hz), 7.46 (d, 1H, J = 8.5 Hz), 7.31 (d, 2H, J = 4.9 Hz), 7.08–7.15 (m, 1H), 6.88 (dd, 1H, J1 = 4.6 Hz, J2 = 8.1 Hz), 3.74–3.79 (m,
1H), 3.51 (s, 2H), 2.79 (m, 2H), 2.05 (m, 2H), 1.78 (m, 2H), 1.58–1.65 (m, 2H), ESI–MS (m/z): 498.3 (MH+).