Synthesis and cytotoxicity of novel 3-amino-4-indolylmaleimide derivatives

Article abstract of DOI:10.1007/s12272-011-0401-z

In an attempt to develop potent and selective antitumor agents, a series of novel 3-amino-4- indolylmaleimides were designed and synthesized. The reaction showed high regioselectivity. The structure of compound 7a was determined by an X-ray single crystal

Full text of DOI:10.1007/s12272-011-0401-z

Arch Pharm Res Vol 34, No 4, 519-526, 2011
DOI 10.1007/s12272-011-0401-z
Synthesis and Cytotoxicity of Novel 3-Amino-4-indolylmaleimide
Derivatives
Sheng-Yin Zhao1, Yan-Wu Yang1, Hai-Quan Zhang1, Yun Yue2, and Mei Fan1
2
1Department of Chemistry, Donghua University, Shanghai 201620, China and Department of Pharmacology, Foshan
University, Foshan 528000, China
(Received April 29, 2010/Revised September 1, 2010/Accepted September 2, 2010)
In an attempt to develop potent and selective antitumor agents, a series of novel 3-amino-4-
indolylmaleimides were designed and synthesized. The reaction showed high regioselectivity.
The structure of compound 7a was determined by an X-ray single crystal diffraction method.
The cytotoxicities of the title compounds were evaluated against HeLa, SMMC 7721 and HL
60 cancer cell lines by a standard MTT assay in vitro. The pharmacological results showed
that some of the title compounds displayed moderate or high cytotoxic activity against the
tested cell lines. Compound 7d was the most promising compound against the tested cancer
cell lines. Structure-activity relationships are discussed based on the experimental data
obtained. A hydroxyethylamino group at the 3-position in the side chain of indolylmaleimide
is associated with an increase in cytotoxicity.
Key words: 3-Amino-4-indolylmaleimide, Synthesis, Crystal structure, Cytotoxicity
growth factor (VEGF)-induced tumor development
and angiogenesis, and in the apoptosis-regulating
INTRODUCTION
Cancer is the second leading cause of death in the phosphatidylinositol 3-kinase (PI3K)/Akt pathway.
world. Angiogenesis is the process of generating new Recently, protein kinase C was shown to be an
capillary blood vessels, and it plays an important role important target in cancer chemotherapy (Serova et al.,
in the proliferation, invasion and metastasis of 2006). The development of PKC inhibitors as cancer
malignant tumors (Argyriou et al., 2009). Blocking chemotherapy agents is an active area of research
tumor-induced angiogenesis continues to be an at- (Jirousek et al., 1996; Pajak et al., 2008; Rucci et al.,
tractive strategy for cancer therapy (Fujita et al., 2008; Zhao et al., 2009). The natural product,
2008; Ali et al., 2009).
staurosporine, although a potent inhibitor of PKC, has
Protein kinase C (PKC), a family of serine/threonine limited selectivity in vitro for both ATP-dependent
specific kinases, composed of at least 12 isozymes, are kinases and individual PKC isozymes (Tamaoki et al.,
involved in signal transduction pathways that govern 1986). A series of novel indolylmaleimides were
a wide range of physiological processes including developed for clinical trials (Zhao et al., 2008), such as
differentiation, proliferation, gene expression, brain Enzastaurin (Chen and LaCasce, 2008) and So-
function, membrane transport and the organization of trastaurin (Wagner et al., 2009). Enzastaurin is an
cytoskeletal and extracellular matrix proteins that acyclic bisindolylmaleimide derivative that potently
regulate vascular function (Serova et al., 2006; Martiny and selectively inhibits PKC isoforms. Enzastaurin
-Baron and Fabbro, 2007). PKC overexpression has displayed anticancer efficacy in several preclinical
been linked to several types of cancer. A PKC isoform cancer models and in clinical trials in patients with
was suspected to be involved in vascular endothelial advanced cancers. It is currently undergoing phase III
clinical trials for relapsed glioblastoma multiforme
and diffuse B-cell lymphoma (Chen and LaCasce,
2008).
Indolylmaleimides (e.g., JTT 010), were reported to
Correspondence to: Sheng-Yin Zhao, Department of Chemistry,
Donghua University, Shanghai, P.O. Box 201620, China
Tel: 86-21-67792776, Fax: 86-21-67792608
E-mail: syzhao8@dhu.edu.cn
519

520
S.-Y. Zhao et al.
exhibit remarkable PKC inhibition (Tanaka et al., thoroughly in DMF (10 mL). To this solution, an
2006). We reasoned that by introducing nitrogen amine (6 mmol) was added. The reaction mixture was
atoms into the indolylmaleimide ring, one might be stirred at 100oC for 16 h. Water (20 mL) was added.
able to enhance the binding of the compounds to PKC. The mixture was extracted with ethyl acetate (40 mL
On the other hand, SAR studies on indolylmaleimides × 2), and washed with saturated NaHCO3 (40 mL × 2)
indicated that attaching hydrophilic substituents on and water (40 mL × 2). The solvent was removed and
the appropriate position of the compounds could purified by flash column chromatography (dichloro-
enhanced their inhibitory potency toward PKC (Tanaka methane/methanol = 10:1, v/v) to give red yellow solids
et al., 2004; Dodo et al., 2005). This result prompted us
to design a series of novel 3-amino-4-indolylmaleimide procedure given above. Compound
analogues in an attempt to improve potency against from
or using ethanolamine as solvent. The
PKC and to find more potent anticancer compounds.
physical and spectral data of compounds 5a-5h 7a-7d
are as follows.
5
. Compound
7
was synthesized from
6 following the
8
was synthesiz- ed
4
6
,
Recently, our research group has been interested in and
the synthesis and development of indolylmaleimide
8
derivatives as anticancer agents (Zhao et al., 2008,
2009; Jiang et al., 2010). In our continuing research
3-n-propylamino-4-(1H-indol-3-yl)-1-methyl-1H
-pyrrole-2,5-dione (5a)
program directed toward the synthesis of novel in- Red crystalline solid; Yield 73%, m.p. 199~202ºC; IR
dolylmaleimide derivatives in this area, we describe
ν
max (KBr r, cm-1): 3332, 1757, 1698, 1534, 1457, 1384,
1
herein the synthesis and preliminary biological 745; H-NMR (DMSO-d6):
δ 0.84 (t, 3H, J = 8.4 Hz,
evaluation of novel 4-amino-3-indolylmaleimide deriva- CH3), 1.15-1.24 (m, 2H, CH2), 2.90 (s, 3H, CH3), 2.95-
tives against various human cancer cell lines in vitro
2.99 (m, 2H, CH2), 4.02 (s, 1H, NH), 6.99 (t, 1H, J =
.
8.0 Hz, Ar-H), 7.09 (t, 1H,
(m, 2H, Ar-H), 7.39 (t, 1H,
J = 8.0 Hz, Ar-H), 7.27-7.34
J
= 8.0 Hz, Ar-H), 11.22 (s,
MATERIALS AND METHODS
Chemicals
1H, NH); MS (ESI) m/e [M+H]+ 284.2, [M+Na]+ 306.3;
Anal. Calcd. for C16H17N3O2: C, 67.83; H, 6.05; N,
Melting points were determined with an RY-1 14.83. Found: C, 67.65; H, 6.08; N, 14.40%.
apparatus, and are uncorrected. IR spectra were
determined as KBr pellets on a Shimadzu model 470
3-isopropylamino-4-(1H-indol-3-yl)-1-methyl-1H
-pyrrole-2,5-dione (5b)
spectrophotometer. 1H-NMR spectra were recorded
using a Bruker AV 400 MHz spectrometer in DMSO- Red crystalline solid; Yield 67%, m.p. 203~206ºC; IR
d6, CDCl3 and acetone-d6 with tetramethylsilane as an νmax (KBr, cm-1): 3448, 3319, 1700, 1647, 1615, 1549,
internal standard. EI mass spectra were recorded on a 1385, 745; 1H-NMR (CDCl3):
δ 0.97 (d, 6H, J = 8.4 Hz,
Shimadzu QP-2010 GC-MS system and a Waters 2CH3), 3.07 (s, 3H, CH3), 3.68-3.77 (m, 1H, CH), 5.05
Micromass GCT system. ESI mass spectra were (d, 1H, = 8.4 Hz, NH), 7.14 (t, 1H, = 7.4 Hz, Ar-H),
obtained on a Shimadzu 2010A LC-MS instrument. 7.21-7.26 (m, 2H, Ar-H), 7.39 (d, 1H, = 8.0 Hz, Ar-
= 8.0 Hz, Ar-H), 8.34 (s, 1H, NH);
J
J
J
Elemental analyses were performed using a Vario EL H), 7.48 (d, 1H,
J
III elemental analyser. Crystal structures were MS (ESI) m/e [M+H]+ 284.2, [M+Na]+ 306.1; Anal.
determined using a Bruker SMART CCD area detector Calcd. for C16H17N3O2: C, 67.83; H, 6.05; N, 14.83.
diffractometer. All chemicals and reagents were Found: C, 67.50; H, 6.08; N, 14.51%.
purchased from known commercial suppliers and
were used without further purification. 3,4-Dibro-
momaleimide, 3,4-dibromo-N-methylmaleimide, 2-bromo
3-n-butylamino-4-(1H-indol-3-yl)-1-methyl-1H-
pyrrole-2,5-dione (5c)
-3-(1
dolylmaleimide were synthesized according to pu- νmax (KBr, cm-1): 3401, 2861, 1700, 1653, 1541, 1456,
blished procedures with minor changes (Scharf et al., 1350; 1H-NMR (acetone-d6):
0.58 (t, 3H, = 8.2 Hz,
1965; Brenner et al., 1988; Zhao et al., 2009). Target CH3), 0.97-1.01 (m, 2H, CH2), 1.29-1.32 (m, 2H, CH2),
compounds were prepared according to the synthetic 2.96 (s, 3H, CH3), 3.17 (t, 2H, = 8.4 Hz, CH2), 7.02-
H-indol-3-yl)-N-methylmaleimide and bisin- Red crystalline solid; Yield 77%, m.p. 183~186ºC; IR
δ
J
J
routes shown in Scheme 1 and Scheme 2.
7.06 (m, 1H, Ar-H), 7.10-7.14 (m, 1H, Ar-H), 7.33 (s,
1H, Ar-H), 7.42-7.47 (m, 2H, Ar-H), 10.55 (brs, 1H,
NH); MS (ESI) m/e: [M+H]+ 298.2, [M+Na]+ 320.2;
Anal. Calcd. for C17H19N3O2: C, 68.67; H, 6.44; N,
General procedure for preparation of com-
pound 5
4
(5 mmol) and triethylamine (10 mmol) were mixed 14.13. Found: C, 68.44; H, 6.50; N, 13.90%.

3-Amino-4-indolylmaleimide Derivatives as Antitumor Agents
521
νmax (KBr, cm-1): 3461, 3316, 3218, 3027, 2930, 1699,
3-benzylamino-4-(1H-indol-3-yl)-1-methyl-1H-
pyrrole-2,5-dione (5d)
1644, 1550, 1358, 1056; 1H-NMR (DMSO-d6):
δ
2.91 (s,
= 5.8
νmax (KBr, cm-1): 3409, 1692, 1653, 1537, 1456, 1339; Hz, 2H, CH2), 4.58 (s, 1H, OH), 6.90 (s, 1H, NH), 7.00-
1H-NMR (DMSO-d6):
2.90 (s, 3H, CH3), 2.99 (s, 2H, 7.03 (m, 1H, Ar-H), 7.09-9.12 (m, 1H, Ar-H), 7.29-7.31
CH2), 4.23 (s, 1H, NH), 6.83 (d, 1H, = 8.4 Hz, Ar-H), (m, 1H, Ar-H), 7.37-7.40 (m, 2H, Ar-H), 11.17 (s, 1H,
6.87 (d, 1H,
= 8.4 Hz, Ar-H), 7.10-7.15 (m, 4H, Ar- NH); MS (ESI, m/z): 286.2 [M+H]+, 308.3 [M+Na]+.
H), 7.28 (d, 1H, = 8.4 Hz, Ar-H), 7.38 (d, 2H, = 8.0 Anal. Calcd. for C15H15N3O3: C, 63.15; H, 5.30; N,
Hz, Ar-H), 7.72 (d, 1H, = 8.0 Hz, Ar-H), 11.22 (d, 1H, 14.73. Found: C, 63.08; H, 5.01; N, 14.42%.
Red crystalline solid; Yield 78%, m.p. 193~195ºC; IR 3H, CH3), 3.11 (t,
J = 5.8 Hz, 2H, CH2), 3.24 (t, J
δ
J
J
J
J
J
NH); MS (ESI, m/z)
m/e: [M+H]+ 332.3, [M+Na]+
354.3, [M-H]− 330.1. This compound was reported in
the literature (Schultz et al., 1991).
3-(dimethylamino)-4-(1H-indol-3-yl)-1H-pyrrole-
2,5-dione (7a)
Red crystalline solid; Yield 62%, m.p. 161~163oC; IR
νmax (KBr, cm-1): 3370, 3290, 2945, 1650, 1544, 1422,
3-(di-n-butylamino)-4-(1H-indol-3-yl)-1-methyl-
1H-pyrrole-2,5-dione (5e)
1
1367; H-NMR (DMSO-d6):
δ 2.96 (s, 6H, CH3), 7.00-
Red crystalline solid; Yield 84%, m.p. 197~199ºC; IR 7.04 (m, 1H, Ar-H), 7.09-7.12 (m, 1H, Ar-H), 7.26-7.29
max (KBr, cm-1): 3365, 3278, 1695, 1646, 1542, 1350, (m, 1H, Ar-H), 7.36-7.39 (m, 2H, Ar-H), 10.32 (s, 1H,
1238; 1H-NMR (acetone-d6): = 8.4 Hz, NH), 11.22 (s, 1H, NH); MS (EI, m/z): 255 (M+), 240,
0.70-0.74 (t,
6H, 2CH3), 1.00 -1.05 (m, 4H, 2CH2), 1.44-1.48 (m, 4H, 207, 169, 128, 113, 92, 77. HRMS (EI): Calcd for
2CH2), 2.94 (s, 3H, CH3), 3.50 (t, 4H,
= 8.4 Hz, C14H13N3O2 255.1008 (M+). Found 255.1011.
ν
δ
J
J
2CH2), 7.03-7.05 (m, 1H, Ar-H), 7.09-7.14 (m, 1H, Ar-
H), 7.31 (s, 1H, Ar-H), 7.40-7.44 (m, 2H, Ar-H), 10.55
(s, 1H, NH); MS (ESI, m/z): 354.3 [M+H]+, 376.4
3-(pyrrolidin-1-yl)-4-(1H-indol-3-yl)-1H-pyrrole-
2,5-dione (7b)
[M+Na]+. Anal. Calcd. for C21H27N3O2: C, 71.36; H, Red crystalline solid; Yield 79%, m.p. 147~149ºC; IR
7.70; N, 11.89. Found: C, 71.02; H, 7.41; N, 11.59%.
νmax (KBr, cm-1): 3434, 3287, 2972, 1743, 1687, 1622,
1534, 1430, 1236; 1H-NMR (DMSO-d6):
1.62-1.66 (m,
4H, 2CH2), 3.37 (t, 4H, = 6.4 Hz, 2CH2), 6.92-6.96
(m, 1H, Ar-H), 7.02-7.05 (m, 1H, Ar-H), 7.18 (d, 1H,
δ
J
3-(pyrrolidin-1-yl)-4-(1H-indol-3-yl)-1-methyl-1H
-pyrrole-2,5-dione (5f)
J
Red crystalline solid; Yield 88%, m.p. 209~212ºC; IR = 2.4 Hz, Ar-H), 7.28-7.33 (m, 2H, Ar-H), 10.24 (s, 1H),
νmax (KBr, cm-1): 3384, 3269, 1683, 1630, 1542, 1358, 11.09 (s, 1H); MS (EI, m/e): [M]+ 281, 252, 238, 168,
1
1243; H-NMR (CDCl3):
δ
1.73-1.76 (m, 4H, 2CH2), 127. HRMS (EI): Calcd. for C16H15N3O2 281.1164 (M+).
3.06 (s, 3H, CH3), 3.53-3.58 (m, 4H, 2CH2), 7.09-7.13 Found 281.1166.
(m, 2H, Ar-H), 7.16-7.20 (m, 1H, Ar-H), 7.35 (d, 1H,
= 8.4 Hz, Ar-H), 7.46 (d, 1H, = 7.8 Hz, Ar-H), 8.28 (s,
1H, NH); MS (ESI, m/e): [M+H]+ 296.3, [M+Na]+
J
J
3-(morpholin-4-yl)-4-(1H-indol-3-yl)-1H-pyrrole-
2,5-dione (7c)
318.1. Anal. Calcd. for C17H17N3O2: C, 69.14; H, 5.80; Red crystalline solid; Yield 79%, m.p. 147~149ºC; IR
N, 14.23. Found: C, 69.04; H, 5.57; N, 14.12%.
(KBr, cm-1): 3386, 2981, 1701, 1624, 1531, 1431, 1110;
1H-NMR (400 MHz, DMSO-d6):
3.44 (d, 4H, = 3.6
Hz, 2CH2), 3.50 (d, = 3.6 Hz, 4H, 2CH2), 7.04 (t, 1H,
= 7.4 Hz, Ar-H), 7.12 (t, 1H,
δ
J
J
3-(morpholin-4-yl)-4-(1H-indol-3-yl)-1-methyl-
1H-pyrrole-2,5-dione (5g)
J
J
J
= 7.4 Hz, Ar-H),
= 7.8 Hz, 1H, Ar-
Red crystalline solid; Yield 87%, m.p. 209~213ºC; IR 7.375-7.40 (m, 2H, Ar-H), 7.46 (d,
νmax (KBr, cm-1): 3391, 3282, 1701, 1651, 1539, 1452, H), 10.46 (s, 1H, NH), 11.33 (s, 1H, NH); MS (EI, m/
1
1386; H-NMR (CDCl3):
δ
3.09 (s, 3H, CH3), 3.67 (m,
= 8.4 Hz, Ar-H), 7.24 (t, 1H, Calcd. for C16H15N3O3: C, 64.64; H, 5.09; N, 14.13.
= 8.4 Hz, Ar-H), 7.37-7.40 (m, 1H, Ar-H), 7.46 (d, Found: C, 64.38; H, 5.31; N, 14.40%.
e
): [M]+ 297, 252, 238, 226, 168, 140, 127, 84. Anal.
8H, 4CH2), 7.18 (t, 1H,
J
J
1H,
J = 8.4 Hz, Ar-H), 7.55 (d, 1H, J = 8.4 Hz, Ar-H),
8.38 (s, 1H, NH); MS (ESI) m/e: [M++H] 312.3, [M++Na]
334.2; Anal. Calcd. for C17H17N3O3: C, 65.58; H, 5.50;
N, 13.50. Found: C, 65.58; H, 5.45; N, 13.32%.
3-(2-hydroxyethylamino)-4-(1H-indol-3-yl)-1H-
pyrrole-2,5-dione (7d)
Red crystalline solid; Yield 65%, m.p. 179~182ºC; IR
νmax (KBr, cm-1): 3488, 3294, 3157, 1697, 1648, 1554,
1
1421, 1350, 1059; H-NMR (400 MHz, DMSO-d6):
δ
3-(2-hydroxyethylamino)-4-(1H-indol-3-yl)-1-
methyl-1H-pyrrole-2,5-dione (5h)
3.10 (t, 2H,
J
= 5.8 Hz, CH2), 3.22 (q, 2H,
J = 5.7 Hz,
Red crystalline solid; Yield 78%, m.p. 219~222oC; IR CH2), 4.62 (t, 1H,
J
= 5.3 Hz, OH), 6.72 (s, 1H, NH),

522
S.-Y. Zhao et al.
7.00 (d, 1H,
J
= 7.2 Hz, Ar-H), 7.08 (t, 1H,
J
= 7.7 Hz, and 5% CO2, cells were incubated with various con-
Ar-H), 7.29 (t,
J
= 2.4 Hz, 1H, Ar-H), 7.37 (t, 2H,
J
=
centrations of test drugs for 48 h. MTT was added at
7.2 Hz, Ar-H), 10.35 (s, 1H, NH), 11.22 (s, 1H, NH); a final concentration of 0.5 mg/mL and incubated with
MS (EI, m/e): [M]+ 271, 240, 238, 197, 129. HRMS cells for 4 h. The formazan crystals that formed in
(EI): Calcd. for C14H14N3O3 271.0957 (M+). Found each well were dissolved in 100
µL DMSO, and the
271.0955.
optical density was measured at 492 nm (for
absorbance of MTT formazan) and 630 nm (for the
reference wavelength). Antitumor potency of the
target compounds were expressed as IC50 values that
3-(2-hydroxyethylamino)-1-(2-hydroxyethyl)-4-
(1H-indol-3-yl)-1H-pyrrole-2,5-dione (8)
Red crystalline solid; Yield 78%, m.p. 232~235ºC; IR were calculated by linear regression analysis of the
νmax (KBr, cm-1): 3488, 3294, 1759, 1697, 1647, 1554, concentration-response curves obtained for the target
1421, 1350; 1H-NMR (DMSO-d6):
δ
3.12 (t, 2H,
J = 5.8 compounds. Assays were performed in triplicate in
Hz, CH2), 3.22 (t, 2H,
J = 5.8 Hz, CH2), 3.47-3.50 (m, each of three independent experiments.
4H, 2CH2), 4.60 (brs, 1H, OH), 4.84 (brs, 1H, OH),
6.92-6.95 (m, 1H, Ar-H), 7.01-7.03 (m, 1H, Ar-H), 7.07-
7.10 (m, 1H, Ar-H), 7.30 (s, 1H, NH), 7.39-7.42 (m, 2H,
Ar-H), 11.23 (s, 1H, NH); MS (EI, m/z): 315 [M]+, 296,
284, 238, 197, 169, 155, 128. Anal. Calcd. for C16H17N3O4
RESULTS AND DISCUSSION
Chemistry
The target compounds were synthesized according
: C, 60.94; H, 5.43; N, 13.33. Found: C, 60.58; H, 5.76; to the route shown in Scheme 1, and the yields were
N, 12.99%.
not optimized. 2-Bromo-3-(1
maleimide as the key intermediate was easily
synthesized from succinimide by bromination with
H-indol-3-yl)-N-methyl-
4
X-ray crystallography
Crystals for 3-(dimethylamino)-4-(1H-indol-3-yl)- bromine, methylation with methyl iodide and by the
1H-pyrrole-2,5-dione 7a were obtained by dissolving reaction of indole with 2,3-dibromo-N-methylmaleimide
compound 7a (0.3 g, 1.7 mmol) in ethyl acetate (5 mL) in the presence of magnesium and ethyl bromide in
and ethanol (5 mL) and evaporating the solvent slowly 35% yield for the three steps (Zhao et al., 2009).
at room temperature for about 2 days.
The following were the procedures and the programs toluene was used as the solvent (Brenner et al., 1988).
we used to do these procedures: Data collection: 2-Bromo-3-(1 -indol-3-yl)maleimide was obtained
SMART; cell refinement: SAINT; data reduction: from compound by the reaction of indole with 2,3-
SAINT; programs used to solve structure: SHELXS97 dibromomaleimide in the presence of magnesium and
Bisindolylmaleimide, a side-product, was formed if
H
6
2
;
programs used to refine structure: SHELXL97; mole-
cular graphics: SHELXTL; software used to prepare
material for publication: SHELXTL
.
Crystallographic data (excluding structural factors)
for the structure of compound 7a in this paper have
been deposited with the Cambridge Crystallographic
Data Centre as supplementary publication number
CCDC 759817. These data can be obtained free of
charge via www.ccdc.cam.ac.uk/data_request/cif, by e-
mailing data_request@ccdc.cam.ac.uk, or by contacting
The Cambridge Crystallographic Data Cntre, 12 Union
Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223-
336033.
Cytotoxicity assay
In vitro cytotoxicity of novel 3-amino-4-indolyl-
maleimide derivatives against HeLa, SMMC 7721
and HL 60 cells were evaluated by a standard MTT
assay using bisindolylmaleimide as a positive control
(Zhao et al.,2009). Cells were seeded in 96-well plates
at a concentration of 40,000 cells per well in 100 mL
RPMI 1640 medium. After culture for 24 h at 37oC
Scheme 1. Synthetic route for compounds 5a
-5h and 7a-
7d
.

3-Amino-4-indolylmaleimide Derivatives as Antitumor Agents
523
ethyl bromide in 49% yield for the two steps. Com- the structure of 1-(2-hydroxylethyl)-3-(2-hydroxyethyla-
pounds and were then treated with different amines mino)-4-indolylmaleimide . Using the same procedure,
in DMF to give 3-amino-4-indolyl-N-methylmaleimides compound was treated with ethanolamine (1.2 equiv.
and 3-amino-4-indolylmaleimides as red crystals. and 2.0 equiv.), and compounds 7d and were obtained
We found that when compounds and were in 65% and 80% yield, respectively. When treating
treated with primary amines, besides the main compound 5h or 7d with ethanolamine, compound
products and
the reaction mixture. For example, first, we treated (Scheme 2).
compound (1.0 equiv.) with ethanolamine (1.2 equiv.) However, when compounds
4
6
8
6
5
7
8
4
6
8
5
7, a side-product also was isolated from was obtained in 72% and 78% yield, respectively
4
4
and
6
were treated
in the presence of triethylamine (1.5 equiv.) as a with secondary amines, such as piperidine or di-n-
base, in DMF at 100°C to give 3-(2-hydroxyethylamino)- butylamine, a main product of 3-aminoindolylmalei-
4-indolyl-N-methylmaleimide 5h as the main product mides were obtained in 70~85% yield.
with a 78% yield. When compound
4
(1.0 equiv.) was
Crystals of compound 7a suitable for X-ray analysis
treated with ethanolamine (2.0 equiv.), a new com- were obtained by slow evaporation of a solution of this
pound was detected by TLC analysis besides compound compound in a mixture of ethyl acetate and ethanol
5h. With increasing amounts of ethanolamine, the (1:1). A single red crystal with dimensions 0.470
yield of the new compound increased. It turned out to 0.392 0.351 mm was chosen for X-ray diffraction
be a main product when ethanolamine was used as a study. The data were collected on a CAD-4 diffracto-
solvent. After purification by flash column chroma- meter equipped with graphite-monochromatic MoK
×
×
α
tography, a red crystal was isolated using spectral radiation (
detection. The H-NMR spectrum showed that the
λ
= 0.71037 Å) using an
ω
scan mode at 293
1
proton signal of the methyl group at
disappeared, and one new signal was exhibited in the
range of 2 ~ 4 ppm. Further, the EI mass spectrum
of this compound showed it to be a molecular ion at
315 (M+). On the basis of spectral data, we assumed
that ring opening and closing reactions happened in
this reaction and assigned to the obtained compound
δ 3.0 ppm had
Table I. Crystal data and structure refinement for compound
7a
δ
CCDDC deposit no.
Molecular formula
Molecular weight
Temperature (K)
CCDC759817
C16H17N3O3
299.33
293 (2)
Radiation
λ
0.71073 A
Monoclinic
P2 (1)/n
13.5807 (13)
8.5960 (8)
26.395 (3)
3010.2 (5)
8
Crystal system
Space group
a / Å
b / Å
c / Å
V / Å3
Z, calculated density
(g cm-3)
Dcalc (g cm-3)
Crystal size (mm)
Crystal colour
1.321
0.470 × 0.392 × 0.351
Red crystal
Absorption coefficient (cm-1) 0.093
Absorption correction
Tmin and Tmax
F(000)
Range for data collection
Limiting indices
Empirical
1.0000 and 0.0441
1264
1.87-25.50o
−
12
≤
≤
h
≤
16,
31
−10≤k≤10,
−29
l
≤
Reflections collected/unique 15441/5622 [R(int)=0.0519]
Completeness to
Refinement method
θ
= 25.50
99.9%
Full-matrix least-squares
on F2
Goodness-of fit on F2
1.031
Final R indices [I > 2
σ
(I)]
R1 = 0.0683, wR2 = 0.2035
R1 = 0.0921, wR2 = 0.2235
0.0012 (11)
R
indices (all data)
Extinction coefficient
Largest diff. Peak and hole 0.466 and
−0.34
(eÅ-3)
Scheme 2. Synthetic route for compound 8.

524
S.-Y. Zhao et al.
The asymmetric unit contains two 3-dimethylamine-
4-indolylmaleimide molecules and one ethyl acetate
molecule. The average molecular formula is C16H17N3O3.
The average molecular weight is 299.33. Two 3-
dimethylamine-4-indolylmaleimide molecules show
different conformations. In one of them, the indole
ring and maleimide are nearly coplanar, whereas in
the other one, they are highly twisted with respect to
each other. In the crystal packing, indolylmaleimide
molecules are linked by intermolecular N-H··· O
hydrogen bonds. The layers are further connected into
a three-dimensional network by N-H··· O hydrogen
bonds involving the maleimide molecules as H-donors
and by weak π−π stacking interactions involving
neighbouring indole and maleimide rings. Atoms N1,
N3, N4 and N6 act as hydrogen bond donors,
respectively, via O3, O2, O1 and O4 atoms to generate
the supramolecular structures. The ethyl acetate
molecule was located in the middle of the cave of the
crystal cell.
Fig. 1. ORTEP structure of the compound 7a, showing 50%
probability ellipsoids.
Cytotoxicity assay
The cytotoxicity of the novel 3-aminoindolylmaleimide
derivatives 5a-5h, 7a-7d and
8 was evaluated with
HeLa, SMMC 7721 and HL 60 in vitro by a standard
MTT assay using bisindolylmaleimide as a positive
control. Antitumor potencies of the title compounds
were expressed as IC50 values that were calculated by
linear regression analysis of the concentration-response
curves obtained for each compound. The results are
summarized in Table II.
As shown in Table II, while most of the prepared
compounds showed potent inhibitory effects on SMMC
7721 cell lines, and the potencies of some compounds
were better or comparative to the lead compound
bisindolylmaleimide, some compounds showed mo-
derate cytotoxicity against tested cancer cell lines,
although they were less potent than bisindolymalei-
mide. It can be concluded that the cytotoxicity of the
tested compounds against SMMC 7721 is higher than
against HeLa and HL 60 cell lines, reflecting the
excellent selectivity for a particular human hepatocel-
lular cancer cell type.
Fig. 2. Packing view of compound 7a in the unit cell,
showing N-H··· O hydrogen-bonding interaction.
The cytotoxicities of the resulting derivatives
appeared to be related to the nature of the leaving
(2) K. In the range of 1.87o <
θ
< 25.50o, a total of group at 1-position in the maleimide ring, which was
15441 reflections were collected, of which 5622 were in agreement with the cytotoxic mechanism reported.
independent (Rint = 0.0519) and 3856 were observed It has been observed from Table II that most of the
with I > 2
σ
(I).
derivatives with hydroxyethyl moieties have higher
The crystal data and refinement details are listed in cytotoxicity than the derivatives with an alkyl group.
Table I. A view of the title structure is illustrated in For example, cytotoxicities of compound 7d were 22.7,
Fig. 1. A perspective view of the crystal packing in the 7.5 and 19.2
unit cell is shown in Fig. 2. 60, respectively. A hydroxyethylamino group at the 3-
µM against HeLa, SMMC 7721 and HL

3-Amino-4-indolylmaleimide Derivatives as Antitumor Agents
525
Table II. 3-aminoindolylmaleimide derivatives and their cytotoxicity
IC50 (µM)
R1 R2
Compound
HeLa
SMMC 7721
HL 60
5a
5b
5c
5d
5e
5f
5g
5h
7a
7b
7c
7d
-H -CH(CH3)2
-H -(CH2)2CH3
-H -(CH2)3CH3
30.5
39.1
30.5
>100
62.7
>100
59.7
31.5
29.6
87.6
41.8
22.7
>100
56.8
21.6
27.2
21.6
45.6
18.1
56.5
39.8
18.4
28.9
30.1
25.5
7.5
33.6
46.2
33.6
>100
43.6
>100
78.7
18.9
25.3
46.5
34.2
19.2
>100
33.4
-H -PhCH2
-(CH2)3CH3 -CH2)3CH3
-CH2CH2CH2CH2-
-CH2CH2OCH2CH2-
-H -CH2CH2OH
-CH3 -CH3
-CH2CH2CH2CH2-
-CH2CH2OCH2CH2-
-H -CH2CH2OH
-CH2CH2OH -CH2CH2OH
8
33.5
20.2
bisindolylmaleimide
position in the side chain of indolylmaleimide is data obtained. A hydroxyethylamino group at the 3-
associated with an increase in cytotoxicity. We suppose position in the side chain of indolylmaleimide is
specific hydrogen bonds interactions with PKC may associated with an increase in cytotoxicity. This study
play an important role in cytotoxicity (He et al., 1994; may provide valuable information for further designing
Levy et al., 2008).
On the other hand, the cytotoxicity of compounds 7a evaluation and structural optimization of indolylmalei-
7d is superior to compound 5a 5h against tested mide derivatives are currently underway in our
more potent anticancer drugs. Further biological
-
-
cancer cell lines. The results indicate that the series laboratories.
bearing N-methylmaleimide displays lower activity
than the series 7a-7d. This study may provide
valuable information for further designing more
potent anticancer agents. Structural optimizing of
ACKNOWLEDGEMENTS
We thank the Scientific Research Foundation for the
indolylmaleimide derivatives are currently underway Returned Overseas Chinese Scholars, State Education
in our laboratories. Ministry of China (No.[2008]890) and Donghua
In summary, we have synthesized a series of novel Univesity for financial support for funding the
3-amino-4-indolylmaleimides from succinimide. The outstanding young faculty (S.Y. Zhao) and Foshan
structure of compound 7a was determined by X-ray University for initial funding for the investigator (Y.
single crystal diffraction method. Cytotoxicity was Yun).
evaluated for the synthesized compounds against
cervical cancer HeLa cells, human hepatocellular
cancer SMMC 7721 cells, and HL 60 cell lines in vitro
by a standard MTT assay. Some of the target com-
pounds exhibited more potent cytotoxic effects against
HeLa and SMMC 7721 cell lines. Structure-activity
relationships were discussed based on the experimental
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