Synthesis and cytotoxic activities of a series of novel N-methylbisindolylmaleimide amino acid ester conjugates

Article abstract of DOI:10.1016/j.cclet.2012.01.033

A series of novel N-methylbisindolylmaleimides and natural amino acid conjugates were synthesized and evaluated for their inhibitory activity against six tumor cell lines. Most of the compounds exhibited moderate in vitro cytotoxic activities in the range

Full text of DOI:10.1016/j.cclet.2012.01.033

Available online at www.sciencedirect.com
Chinese Chemical Letters 23 (2012) 462–465
www.elsevier.com/locate/cclet
Synthesis and cytotoxic activities of a series of novel
N-methylbisindolylmaleimide amino acid ester conjugates
Ke Wang b, Zheng Yan b, Nan Wang b, Zhan Zhu Liu a,b,
*
a State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica,
Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, China
b Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of Materia Medica,
Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, China
Received 7 December 2011
Available online 3 March 2012
Abstract
A series of novel N-methylbisindolylmaleimides and natural amino acid conjugates were synthesized and evaluated for their
inhibitory activity against six tumor cell lines. Most of the compounds exhibited moderate in vitro cytotoxic activities in the range of
10–100 mmol/L.
# 2012 Zhan Zhu Liu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Keywords: Bisindolylmaleimide; Amino acid; Synthesis; Cytotoxicity
Staurosporine (Fig. 1) was isolated from Streptomyces staurosporeus in 1977. It has been found to possess a wide
range of biological activities, including antifungal, antimicrobial, hypotensive, platelet aggregation activities and cell
cytotoxicity. The mechanism of its cytotoxic activity against tumor cells was the inhibition of protein kinase C, which
is the most important aspect of its biological profile [1–4]. However, the poor selectivity for kinase family renders it a
blunt tool for searching for new drugs. Nonetheless, staurosporine has been a useful lead for the discovery of antitumor
drugs. For example, a number of bisindolylmaleimides structurally related to staurosporine has been synthesized,
some of which are being in clinical trials [5–7].
As part of our ongoing effort to discover potent anti-tumor compounds through the structural modification of
natural products, we recently designed and synthesized a novel series of bisindolylmaleimide amino acid ester
conjugates. Amino acids have been introduced into different bioactive molecules to improve their pharmacological
profiles including both potency and bioavailability. The rich side chains as well as the carboxylic or amino
functionality render amino acids an ideal moiety in the structural modification of bioactive natural products [8]. In our
previous paper, a series of N-methylbisindolylmaleimide amino acids conjugates featuring an acetyl linker were
synthesized and evaluated for their in vitro anti-tumor activity [9,10].
* Corresponding author at: State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Peking
Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, China.
E-mail address: liuzhanzhu@imm.ac.cn (Z.Z. Liu).
1001-8417/$ – see front matter # 2012 Zhan Zhu Liu. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
doi:10.1016/j.cclet.2012.01.033

K. Wang et al. / Chinese Chemical Letters 23 (2012) 462–465
463
H
N
O
N
N
O
MeO
HN
Fig. 1. The structure of Staurosporine.
In this study, a new series of derivatives of N-methylmaleimide conjugated with a number of natural amino acids by
the linker of ethylamine were designed and synthesized. In addition, the in vitro inhibition activities of these
derivatives were tested against a panel of human tumor cell lines including human intestinal adenocarcinoma (HCT-8),
human hepatoma cell (BEL-7402), human ovarian carcinoma (A2780), human breast carcinoma (MCF-7), non-small
cell lung carcinoma (A549), human gastric carcinoma (BGC-823). Furthermore, a preliminary structure–activity
relationship was discussed.
Bisindolylmaleimide nucleus was synthesized from N-methylpyrrole 1. Bromination and oxidation of N-
methylpyrrole gave N-methyldibromomaleimide 2 [11]. Indolyllithium was coupled with N-methyldibromomalei-
mide 2 to afford the bisindolylmaleimide 3 [12]. The indole nitrogen was alkylated with 2-bromoethyl amine in the
presence of NaH to afford amine 4. Then 4 was coupled with various N-Boc-L-amino acids in the presence of 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 4-(dimethylamino)pyridine (DMAP) to give 5.
Finally, deprotection of 5 afforded target compound 6 in a reasonable yield (Scheme 1) [13].
N
O
O
N
O
O
N
d
O
O
O
c
a
N
N
N
N
N
b
H
Br
Br
H
H
1
3
4
2
H N
2
N
O
N
O
O
e
f
N
N
H
N
N
H
HN
R
O
HN
R
O
Boc
N
5
6
H
NH
2
R-(6a-6j) :
6a=
6f=
6b=
H C
3
6c=
6d=
6e=
S
H N
6g=
2
N
N
N
O
O
O
O
O
O
6h=
6i=
6j=
N
H
N
N
N
N
H
N
H
H
N
HN
HN
HN
NH
O
NH
2
O
O
2
Scheme 1. Reagents and conditions: (a) NBS, THF, (b) HNO3, 46%; (c) indole, LiHMDS, toluene, 73%; (d) BrCH2CH2NH2, NaH, DMF, 80 8C,
48%; (e) EDC, DMAP, CH2Cl2, 60–90%; (f) CF3COOH, CH2Cl2, 50–85%.

464
K. Wang et al. / Chinese Chemical Letters 23 (2012) 462–465
Table 1
Cytotoxicity of compounds 6a–j.
Compound
In vitro cytotoxicity (IC50, mmol/L)
BCT-8
BEL-7402
A2780
21.85
MCF-7
20.10
A549
24.77
BGC-823
6a
6b
20.11
20.78
24.35
23.63
22.62
33.79
28.97
18.09
24.17
>100
36.11
7.39
26.99
36.59
71.87
52.12
62.67
59.29
35.02
38.02
>100
50.24
1.02
15.97
19.02
26.50
27.00
40.32
14.63
24.97
25.86
27.19
30.25
59.45
50.24
25.96
25.23
19.69
22.52
18.56
40.40
76.21
23.24
45.25
30.38
23.72
85.17
35.55
6c
6d
6e
6f
6g
>100
26.19
78.75
6h
6i
>100
>100
>100
>100
6j
Cisplatin
33.28
3.08
42.71
4.83
58.49
3.36
59.89
1.75
The IC50 values represent the compound concentration (mmol/L) required to inhibit tumor cell proliferation by 50%.
By the MTT assay, cytotoxicity of these derivatives was evaluated against six human cancer cell lines: human
intestinal adenocarcinoma (HCT-8), human hepatoma cell (BEL-7402), human ovarian carcinoma (A2780), human
breast carcinoma (MCF-7), non-small cell lung carcinoma (A549), human gastric carcinoma (BGC-823). The results
of cytotoxicity studies were summarized in Table 1.
As for the ten derivatives of amino acids, compound 6i, which was derived from b-aminopropionic acid, were the
only one that was inactive to all tumor cell lines. Compound 6b, the only aromatic amino acid derivative, which was
derived from L-phenylalanine, showed moderate cytotoxic activities against all tested human tumor cell lines with IC50
values of 15–40 mmol/L. Compound 6a, which was derived from L-leucine, exhibited inhibitory activities against the
tested cell lines, with IC50 values of 20–30 mmol/L. Interestingly, compound 6d, which was derived from L-isoleucine,
exhibited similar inhibitory potency against the tested cell lines, with IC50 values of 20–100 mmol/L. Ala (6e) and Val
(6c) conjugates, which share a similar hydrophobic residue, exhibited similar antiproliferative profile. Lys conjugate
6f, which has two amino residues, exhibited inhibitory activities against the tested five cell lines with IC50 values of
28–80 mmol/L, but was inactive against A549. Compound 6g, which was derived from L-methionine, exhibited
inhibitory activities against the tested cell lines, with IC50 values of 14–35 mmol/L. In the cases of the Gly (6h) and Pro
(6j) conjugates, both exhibited inhibitory activities against the tested cell lines, with IC50 values of 24–100 mmol/L
and 33–60 mmol/L respectively. In addition, it could be seen that all of these derivatives are less active against the
human gastric carcinoma (BGC-823).
In conclusion, a series of novel N-methylbisindolylmaleimide amino acid conjugates were prepared. All of a-
amino acid conjugates except the b-aminopropionic acid conjugate 6i showed moderate antiproliferative activity
against human cancer cell lines including HCT-8, BEL-7402, A2780, MCF-7, A549 and BGC-823, which means that
the a-amino acid moiety is essential for the cytotoxicity of this type of N-methylbisindolylmaleimide conjugates. This
study demonstrated that this type of bisindolylmaleimide derivatives could be promising lead compounds for the
discovery of novel antitumor drugs. Further studies of the antiproliferative mechanism of this series of compounds are
in progress.
References
[1] S. Omura, Y. Iwai, A. Hirano, et al. J. Antibiot. 30 (1977) 275.
[2] T. Tamaoki, H. Nomoto, I. Takahishi, et al. Biochem. Biophys. Res. Commun. 135 (1986) 397.
[3] U. Pindur, Y.S. Kim, F. Mehrabani, Curr. Med. Chem. 6 (1999) 29.
[4] G.W. Gribble, S.J. Berthel, in: Atta-ur-Rahman (Ed.), Studies in Natural Product Chemistry, vol. 12, Elsevier, Amsterdam, 1993, p. 365.
[5] P.D. Davis, C.H. Hill, E. Keech, et al. FEBS Lett. 259 (1989) 61.
[6] L.P. Arello, M.R. Jirousek, G.L. King, et al., PCT Int. Appl. WO 9740831 (1997).
[7] P.D. Davis, C.H. Hill, G. Lawton, Eur. Patent Appl. EP 328026 (1989).
[8] M. Zhao, L. Bi, W. Wang, et al. Bioorg. Med. Chem. Lett. 14 (2006) 6998.
[9] K. Wang, X.Y. Li, X.G. Chen, et al. J. Asian Nat. Prod. Res. 1 (2010) 36.
[10] K. Wang, Z.Z. Liu, Eur. J. Med. Chem. 45 (2010) 4175.

K. Wang et al. / Chinese Chemical Letters 23 (2012) 462–465
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[11] D.S. Choi, S. Huang, M. Huang, et al. J. Org. Chem. 63 (1998) 2646.
[12] M. Brenner, H. Rexhausen, B. Steffan, et al. Tetrahedron 10 (1988) 2887.
[13] General procedure and data for the preparation of compounds 6a–j: To a solution of 5 (0.18 mmol) in anhydrous dichloromethane (2 mL) was
added trifluoroacetic acid (2 mL) and the reaction mixture was stirred at 0 8C under argon for 2 h. The reaction mixture was concentrated in
vacuo, and then diluted with H2O (2 mL). Saturated aqueous NaHCO3 was added dropwise until neutralization. After extraction with ethyl
acetate, the combined organic phase was dried over Na2SO4. The solvent was removed, and the residue was purified on a column of silica gel
D
using MeOH–CHCl3 (1/50) as eluent to give the product 6. Yield: 50–88%. Compound 6a: Yield 86.2%, red solid, mp: 142–144 8C; [a]20
+61.3 (c 0.006, CH3OH); HRMS (ESI) calcd. for C29H31N5O3Na [M+Na]+: 520.2319, found 520.2318. 1H NMR (300 MHz, DMSO-d6): d
11.73 (s, 1H, N–H), 8.67 (t, 1H, J = 4.8 Hz), 8.10 (br, 1H), 7.76 (s, 1H), 7.70 (d, 1H, J = 2.7 Hz), 7.52 (d, 1H, J = 8.1 Hz), 7.37 (d, 1H,
J = 8.1 Hz), 7.01 (m, 2H), 6.70 (m, 1H), 6.65 (m, 2H), 4.33 (m, 2H), 3.61 (m, 2H), 3.07 (m, 4H), 1.51 (m, 1H), 1.42 (m, 1H), 0.87 (m, 1H), 0.77
(d, 6H, J = 5.4 Hz). 13C NMR (100 MHz, DMSO-d6): d 171.68, 169.44, 136.00, 135.72, 132.44, 129.26, 127.06, 126.23, 125.84, 125.19,
121.84, 121.64, 121.48, 121.11, 119.68, 119.44, 111.79, 110.06, 105.54, 105.15, 50.93, 45.66, 44.78, 38.86, 23.90, 23.44, 22.42, 21.48.
D
Compound 6b: Yield 83.3%, red solid, mp: 151–153 8C; [a]20 À13.0 (c 0.002, CH2Cl2); HRMS (ESI) calcd. for C32H30N5O3 [M+H]+:
532.2348, found 532.2347. 1H NMR (300 MHz, DMSO-d6): d 11.68 (s, 1H), 8.05 (t, 1H), 7.74 (s, 1H), 7.72 (s, 1H), 7.49 (d, 1H, J = 8.4 Hz),
7.35 (d, 1H, J = 8.1 Hz), 7.21 (m, 4H), 7.00 (m, 3H), 6.80 (t, 2H, J = 8.1 Hz), 6.64 (m, 2H), 4.26 (t, 2H, J = 6.0 Hz), 3.40 (m, 3H), 3.02 (s, 3H),
2.87 (dd, 1H, J = 13.2, 4.5 Hz), 2.55 (m, 1H), 1.98 (br, 2H). 13C NMR (100 MHz, DMSO-d6): d 174.49, 171.70, 138.64, 135.97, 135.84, 132.31,
129.23, 128.09, 127.16, 126.35, 126.09, 125.84, 125.26, 121.73, 121.64, 121.29, 121.09, 119.58, 119.44, 111.75, 110.14, 105.59, 105.05,
56.18, 45.14, 40.62, 38.51, 23.92. Compound 6c: Yield 45%, red solid, mp: 169–171 8C; [a]20D +24.0 (c 0.007, CH3OH); HRMS (ESI) calcd
for C28H30N5O3 [M+H]+: 484.2349, found 484.2325. 1H NMR (300 MHz, CDCl3): d 9.08 (s, 1H), 7.70 (d, 1H, J = 2.7 Hz), 7.57 (s, 1H), 7.39 (t,
1H, J = 6.0 Hz), 7.30 (d, 2H, J = 8.4 Hz), 7.01 (m, 4H), 6.75 (t, 2H, J = 7.5 Hz), 4.26 (m, 2H), 3.64 (m, 2H), 3.15 (m, 4H), 2.26 (m, 1H), 0.95 (d,
3H, J = 6.9 Hz), 0.80 (d, 3H, J = 7.2 Hz). 13C NMR (75 MHz, CDCl3): d 175.14, 172.45, 136.01, 135.88, 131.68, 128.39, 127.35, 126.34,
125.37, 122.45, 122.30, 121.77, 120.27, 120.08, 111.34, 109.36, 107.03, 106.22, 60.03, 45.91, 38.97, 30.66, 24.16, 19.64, 16.09. Compound
6d: Yield 34%, red solid, mp: 147–149 8C; [a]20 À42.0 (c 0.007, CH2Cl2); HRMS (ESI) calcd. for C29H32N5O3 [M+H]+: 498.2505, found
D
498.2530. 1H NMR (300 MHz, CDCl3): d 9.19 (s, 1H, N–H), 7.69 (d, 1H, J = 2.7 Hz), 7.57 (s, 1H), 7.41 (t, 1H, 6.3 Hz), 7.29 (m, 2H), 7.03 (m,
3H), 6.74 (t, 2H, 7.2 Hz), 4.22 (m, 2H), 3.49 (m, 2H), 3.17 (m, 4H), 1.94 (m, 2H), 1.61 (br, 2H), 1.08 (m, 1H), 0.90 (m, 6H). 13C NMR (75 MHz,
CDCl3): d 174.71, 172.03, 135.60, 135.49, 131.24, 128.03, 126.85, 125.92, 124.94, 121.86, 121.33, 119.84, 119.62, 110.95, 108.93, 106.54,
105.80, 59.37, 45.43, 38.54, 37.37, 23.71, 23.25, 15.62, 11.47. Compound 6e: Yield 73.2%, red solid, mp: 182–184 8C; [a]20D: +10.0 (c 0.008,
CH3OH); HRMS (ESI) calcd. for C26H26N5O3 [M+H]+: 456.2030, found 456.2034. 1H NMR (300 MHz, CDCl3): d 9.22 (s, 1H), 7.70 (d, 1H,
J = 2.4 Hz), 7.56 (s, 1H), 7.29 (m, 3H), 7.01 (m, 4H), 6.74 (t, 2H, J = 7.8 Hz), 4.28 (m, 2H), 3.51 (m, 3H), 3.17 (s, 3H), 1.28 (d, 3H, J = 7.2 Hz),
0.85 (m, 2H). 13C NMR (75 MHz, CDCl3): d 176.44, 172.47, 136.09, 135.93, 131.68, 128.47, 127.43, 126.31, 125.37, 122.42, 122.29, 121.75,
120.26, 120.05, 111.38, 109.36, 106.97, 106.25, 50.57, 45.60, 39.14, 24.14, 21.39. Compound 6f: Yield 63.6%, red solid, mp: 155–157 8C;
[a]20D +45.0 (c 0.009, CH3OH); HRMS (ESI) calcd. for C29H33N6O3 [M+H]+: 513.2614, found 513.2595. 1H NMR (300 MHz, CD3OD-d4): d
7.69 (s, 1H), 7.60 (s, 1H), 7.37 (d, 1H, J = 8.1 Hz), 7.29 (d, 1H, J = 8.1 Hz), 6.95 (m, 2H), 6.76 (m, 2H), 6.58 (m, 2H), 4.27 (t, 2H, J = 6.0 Hz),
3.52 (m, 2H), 3.11 (t, 1H, J = 6.0 Hz), 3.04 (s, 3H), 2.68 (t, 2H, J = 7.2 Hz), 1.45 (m, 6H). 13C NMR (125 MHz, CD3OD-d4): d 177.84, 174.01,
137.76, 137.59, 133.39, 133.31, 130.25, 129.27, 129.21, 127.82, 127.79, 127.06, 123.34, 123.10, 123.06, 121.06, 120.74, 112.50, 110.60,
107.62, 58.78, 49.63, 41.92, 40.80, 31.07, 30.07, 24.28, 20.84. Compound 6g: Yield 71.3%, red solid, mp: 168–171 8C; [a]20D +34.0 (c 0.005,
CH2Cl2); HRMS (ESI) calcd. for C28H30N5O3S [M+H]+: 516.2069, found 516.2058. 1H NMR (300 MHz, CDCl3): d 9.10 (s, 1H), 7.68 (d, 1H,
J = 2.7 Hz), 7.57 (s, 1H), 7.38 (t, 1H, J = 6.0 Hz), 7.31 (m, 2H), 7.04 (m, 2H), 6.95 (dd, 2H, J = 7.8, 3.0 Hz), 6.74 (m, 2H), 4.30 (t, 2H,
J = 5.1 Hz), 3.55 (m, 2H), 3.41 (dd, 1H, J = 8.1, 4.2 Hz), 3.17 (s, 3H), 2.54 (t, 2H, J = 6.9 Hz), 2.12 (m, 1H), 2.08 (s, 1H), 1.72 (m, 1H). 13
C
NMR (75 MHz, CDCl3): d 175.26, 172.49, 172.42, 136.03, 135.86, 131.75, 128.41, 127.37, 127.28, 126.25, 125.38, 122.45, 122.31, 121.74,
120.26, 120.10, 111.34, 109.33, 106.98, 106.18, 54.06, 45.61, 39.14, 33.65, 30.59, 24.16, 15.23. Compound 6h: Yield 73.5%, red solid, mp:
123–126 8C; HRMS (ESI) calcd. for C25H24N5O3 [M+H]+: 442.1879, found 442.1874. 1H NMR (300 MHz, CDCl3): d 9.47 (s, 1H), 7.69 (d,
1H, J = 2.1 Hz), 7.54 (s, 1H), 7.28 (m, 3H), 7.05 (m, 3H), 6.90 (d, 1H, J = 8.1 Hz), 6.73 (m, 2H), 4.28 (t, 2H, J = 5.4 Hz), 3.53 (q, 2H,
J = 5.7 Hz), 3.25 (s, 2H). 13C NMR (75 MHz, CDCl3): d 173.16, 172.02, 135.52, 131.26, 128.16, 127.10, 126.69, 125.88, 124.86, 121.84,
121.26, 119.81, 119.54, 111.03, 108.83, 106.36, 105.75, 45.32, 44.09, 38.56, 23.71. Compound 6i: Yield 73.5%, red solid, mp: 152–154 8C;
HRMS (ESI) calcd. for C26H26N5O3 [M+H]+: 456.2035, found 456.2031. 1H NMR (300 MHz, CD3OD-d4): d 7.71 (s, 1H), 7.63 (s, 1H), 7.35 (d,
1H, J = 8.4 Hz), 7.27 (d, 1H, J = 7.8 Hz), 6.99 (t, 1H, J = 7.5 Hz), 6.92 (t, 1H, J = 7.8 Hz), 6.77 (t, 2H, J = 8.7 Hz,), 6.55 (m, 2H), 4.31 (t, 2H,
J = 6.3 Hz), 3.51 (t, 2H), 3.25 (d, 3H, J = 0.9 Hz), 2.95 (t, 2H, J = 6.9 Hz), 2.40 (t, 2H, J = 6.3 Hz). 13C NMR (125 MHz, CD3OD-d4): d 172.84,
172.62, 136.40, 136.28, 131.74, 128.84, 127.89, 126.58, 126.52, 125.62, 121.82, 121.67, 121.64, 121.15, 119.60, 119.30, 111.10, 109.22,
106.20, 105.98, 44.92, 38.95, 36.06, 32.70, 22.85. Compound 6j: Yield 72.5%, red solid, mp: 171–174 8C; [a]20D +75.0 (c 0.008, CH2Cl2);
HRMS (ESI) calcd. for C28H28N5O3 [M+H]+: 482.2192, found 482.2178. 1H NMR (300 MHz, CDCl3): d 9.38 (s, 1H), 7.71 (s, 2H), 7.55 (s,
1H), 7.32 (t, 2H, J = 8.1 Hz), 7.04 (m, 3H), 6.92 (d, 1H, J = 8.1 Hz), 6.75 (t, 2H, J = 7.2 Hz), 4.27 (m, 2H), 3.72 (q, 1H), 3.57 (m, 1H), 3.17 (s,
3H), 2.91 (m, 1H), 2.72 (m, 1H), 2.09 (m, 1H), 1.84 (m, 1H), 1.62 (m, 2H), 0.86 (m, 1H). 13C NMR (125 MHz, CDCl3): d 174.49, 172.47,
136.28, 136.04, 131.64, 128.60, 127.60, 127.43, 126.31, 125.40, 122.46, 122.36, 122.34, 121.86, 120.32, 120.11, 111.50, 109.43, 107.00,
106.36, 60.30, 46.97, 45.68, 39.33, 30.45, 24.16, 22.68.