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

Article abstract of DOI:10.3184/030823409X430202

A series of novel 3-amido-4-indolylmaleimides have been synthesised from succinimide in five steps sequence consisting of bromination, indole addition, azide substitution, reduction and selective acylation. Cytotoxicity was evaluated for the products agai

Full text of DOI:10.3184/030823409X430202

198 RESEARCH PAPER
MARCH, 198-200
JOURNAL OF CHEMICAL RESEARCH 2009
Synthesis and cytotoxicity of noveI3-amido-4-indolylmaleimide
derivatives
Sheng Yin Zhaoa*, Shi Wei Moua, Zhi Long Chenb, Yun Yuec and Yun Sunb
Departments
ofaChemistry
and bBiological Sciences, Donghua University, Shanghai 201620, P.R. China
CDepartment of Pharmacology,
Foshan University, Foshan 528000, P.R. China
A
series of novel 3-amido-4-indolylmaleimides
have been synthesised from succinimide in five steps sequence
consisting of bromination, indole addition, azide substitution, reduction and selective acylation. Cytotoxicity was
evaluated for the products against cervical cancer Hela cell lines and human hepatocellular cancer SMMC 7721 cell
line by standard MTT assay in vitro. Some ofthese compounds showed moderate cytotoxic potencies.
Keywords: 3-amido-4-indolylmaleimide, synthesis, cytotoxicity
Angiogenesis is the process of generating new capillary blood
vessels, which plays an important role in the proliferation,
invasion and metastasis of malignant tumours. Blocking
tumour-induced angiogenesis continues to be an attractive
strategy for cancer therapy. 1
inhibitor of PKC, has limited selectivity in vitro for both ATP-
dependent kinases and individual PKC isozymes. A series of
novel bisindolylmaleimides have been developed for clinical
trail,S including Enzastaurin9 and Sotrastaurin.lO Enzastaurin
is an acyclic bisindolylmaleimide that potently and selectively
inhibits the PKC isoform. Enzastaurin displayed anticancer
efficacy in several preclinical cancer models and in clinical
trials in patients with advanced cancers. It is currently
undergoing phase III development for relapsed glioblastoma
multiforme and diffuse B-celllymphoma.9
Recently, our research group has been interested in the
synthesis and development of indolylmaleimide derivatives
as anticancer agents.s In continuation of this research, we
describe the synthesis and preliminary biological evaluation of
some novel 3-amido-4-indolylmaleimide derivatives against
cervical cancer Hela and human hepatocellular cancer SMMC
7721 cell line in vitro.
Protein kinase C(PKC) is a family of serine/threonine specific
kinases, composed of at least 12 isozymes, which are involved
in signal transduction pathways that govern a wide range of
physiological processes including differentiation, proliferation,
gene expression, brain function, membrane transport and the
organisation of cytoskeletal and extracellular matrix proteins
that regulate vascular function.2-4 PKC overexpression has
been linked to several types of cancer, with the PKC isoform
suspected to be involved in vascular endothelial growth
factor(VEGF)-induced tumor development and angiogenesis
and in the apoptosis-regulating phosphatidyl inositol
3-kinase(PI3K)/Akt pathway. Targeting PKC therefore
represents a potentially effective strategy for the treatment of
cancer.5-? The natural product staurosporine, although a potent
The title compounds were synthesised according to the
route shown in Fig. 1, and the yields were not optimised.
H
H
H
C2H5Br, Mg
°
N
°
NaN3
QUO
•
Br2 0;::(
..
Br
Br
-
01<
00
N
H
N
H
3
2
H
N
H
N
H
o
0
o
0
N
0
01<-~
RCOCI
~
1
V~H2
1
1
N3
0 :
1
o 0-H N ~1O
THF, NEt3
~
N
H
~
R
W
N
N
H
H
5
4
6
H
N
T7
o
0
rt.
7a
~I
V~H2
W
N
~HF
H
50°C
~
5
7b
Fig.1 Synthetic route for compounds
6a-f and 7a-b.
* Correspondent.E-mail:syzhao8@dhu.edu.cn

JOURNAL OF CHEMICAL RESEARCH 2009
199
General procedure for preparation of6a-f and7a-b
The key intermediate 2-bromo-3-(lH-indol-3-yl)maleimide
was easily synthesised from succinimide by bromination with
bromine.l l Indole was added to 2,3-dibromomaleimide in the
presence of magnesium and ethyl bromide in 45% yield for the
two steps. Bisindolylmaleimide, a side-product, was formed
3
Compound 5 (5 mmol) and triethylamine (10 mmol) were mixed
thoroughly in THF (10 ml). The acyl cWoride in THF (5 ml) was
added dropwise at room temperature to this solution. The reaction
mixture was stirred for 6 h. Water (5 ml) was added, extracted with
ethyl acetate and washed with saturated NaHC03 and water. The
solvent was removed and purified by flash column chromatography
to give a red-yellow solid. The physical and spectra data of the
compounds 6a-f and 7a-b are as follows.
if toluene were used as solvent.12 Compound 3 was treated
with sodium azide in DMF to give 3-azideindolylmaleimide
4
which was reduced by PPh3 in the presence of TsOH to give
3-aminoindolylmaleimides 5.13 There are three nitrogen atoms
in the compound 5, NH2 group, N atom in the indole and
atom in the maleimide. Schultz and co-workers14 reported
that 3-amido-4-indolyl-N-methylmaleimide derivatives were
synthesised from N-Boc-protected
starting material, and removed the Boc-protected
N-( 4-(1 H-indol-3-yl)-2.
5-dioxo-2. 5-dihydro-l H-pyrrol-3-
yl)acetamide (6a): Yellow crystals 70% yield; m.p. 248-250°C;
IR (cm"): l704(C=O), 1642(C=O). 'H NMR: 8 1.98(s 3H), 7.07(t,
lH,J= 7.2 Hz), 7.l5(t, lH, J= 7.2 Hz), 7.44-7.50 (m, 2H), 7.85(s,
lH), 9.99(s, lH), 10.86(s, lH), l1.8l(s, lH). MS(ESI, m/z): 270.1 [(M
+ HW, 292.2 [(M +NaW. Anal. Calcd for C'4HllN303: C, 62.45; H,
4.12; N, 15.61. Found: C, 62.84; H, 4.31; N, 15.29%.
N
indolylmaleimides
as
group at the
N-( 4-(1 H-indol-3-yl)-2.
5-dioxo-2. 5-dihydro-l H-pyrrol-3-
final step. Here, we developed a selective acylation procedure.
Without purification compound 5 was treated with an acyl
chloride in THF at the room temperature to provide the target
yl)butyramide (6b):Yellow crystals 65% yield; m.p. 240-243°C. IR
(cm"): l70l(C=O), l638(C=O). 'H NMR: 8 0.8l(t, 3H, J= 7.6 Hz),
1.44(q, 2H, J= 7.2 Hz), 2.28(t, 2H, J= 7.6 Hz), 7.04(t, lH, J= 7.2 Hz),
7.l4(t, lH, J= 7.2 Hz), 7.43-7.48(m, 2H), 7.84(s, lH), 9.90(s, lH),
10.87(s, lH), l1.79(s, lH). MS(ESI, m/z): 298.2[(M + HW, 320.2
[(M + NaW. Anal. Calcd for C'6H,sN303: C, 64.64; H, 5.09; N,
14.13. Found: C, 64.39; H, 5.01; N, 13.92%.
N-( 4-(1 H-indol-3-yl)-2. 5-dioxo-2. 5-dihydro-l H-pyrrol-3-yl)
benzamide (6c): Red crystals 59% yield; m.p. 232-235°C.
IR(cm"): l697(C=O), l648(C=O). 'HNMR: 86.87(t, lH,J= 7.2 Hz),
compound
6 with 60% yield. The enamine reacted more
readily with the acyl choride compared to the other groups.
When the compound 5 reacted with chloroacetyl chloride,
the compounds 7a and 7b were obtained in THF at room
temperature
in the absence of NEt3, respectively. A reasonable explanation
is that the formation of amine hydrochloride salt on NH2
in the presence of NEt3 and in THF at 50°C
7.08(t, lH,
J = 7.2 Hz), 7.42-7.58(m, 5H), 7.88-7.94(m, 3H),
10.30(s, lH), 10.96(s, lH), l1.83(s, lH). MS(ESI, m/z):332.2[(M
HW, 354.1 [(M + NaW. Anal. Calcd for C19H13N303:C, 68.88; H,
3.95; N, 12.68. Found: C, 68.98; H, 3.76; N, 12.95%.
+
group makes it more difficult to obtain the acylation product
7a in high temperature. The product 7b, with chloroacetylation
N-(4-( lH -indol-3 -yl)- 2,5-dioxo- 2,5-dihydro-lH -pyrrol- 3-yl )-4-
chlorobenzamide (6d): Red crystals 56% yield; m.p. 258-26l°C. IR
(cm"): 1703(C=O), l639(C=O). 'H NMR: 8 6.88(t, lH, J= 7.2 Hz),
on the
N atom of maleimide, was shown to be the main
product by the existence of the signal of indole NH proton at
11.48 ppm.
7.08(t, lH, J = 7.2 Hz), 7.43(d, lH,
J = 8.0 Hz), 7.5l(d, lH,
The cytotoxicity
of the novel compounds
(6a-f) was
J = 8.0 Hz), 7.58(d, 2H, J = 8.4 Hz), 7.93-7.95(m, 3H), 10.45(s,
lH), 10.99(s, lH), l1.86(s, lH). MS(ESI, m/z): 364.0 [(M-H)]'.Anal.
Calcd for C19H12ClN303: C, 62.39; H, 3.31; N, 11.49. Found: C,
62.67; H, 3.12; N, 11.18%.
evaluated with Hela and SMMC 7721 cells in vitro by MIT
assay. The results expressed as ICso were summarised
in
Table 1. It was be found that these compounds (6a-f) show
N- (4- (1 H-indol- 3-yl)-2. 5 -dioxo-2. 5-dihydro-l H-pyrrol-3 -yl)-4-
nitrobenzamide (6e): Red crystals 62% yield; m.p. 265-268°C. IR
(cm"): l7l2(C=O), l657(C=O). 'H NMR: 8 6.89(t, lH, J= 7.2 Hz),
7.09(t, lH, J = 7.2 Hz), 7.43-7.52(m, 2H), 7.96(d, lH, J = 7.2 Hz),
8.13(d, 2H, J = 8.8 Hz), 8.33(d, 2H, J = 8.8 Hz), 10.76(s, lH), l1.03(s,
lH), l1.89(s, lH). MS(ESI, m/z): 375.0 [(M-H) ]'. Anal. Calcd for
C19H12N40S:C, 60.64; H, 3.21; N, 14.89. Found: C, 60.42; H, 3.41; N,
14.69%.
moderate cytotoxicity
on Hela and SMMC 7721 cell line.
For example, cytotoxicity of the compound 6f against Hela
and SMMC 7721 was 21.2 and 12.5 /.tM, respectively.
In conclusion, a series of nove13-amido-4- indolylmaleimides
have been synthesised from succinimide.
evaluated for the synthesised compounds
cancer Hela cell lines and human hepatocellular
SMMC 7721 cell line by standard MIT assay. Some of the
compounds exhibited potent cytotoxicity against Hela cell
Cytotoxicity
against cervical
cancer
was
N-( 4-(1 H-indol-3-yl)-2. 5-dioxo-2. 5-dihydro-l H-pyrrol-3-yl)-
4-hydroxy-3-nitrobenzamide
(6f): Red crystals 42% yield; m.p.
260-262°C. IR (cm"): l7l0(C=O), l669(C=O). 'H NMR: 86.89
(m, lH, J= 7.2 Hz), 7.07 (t, lH, J= 7.2 Hz), 7.20(d, lH, J= 8.8 Hz),
7.43(d, lH, J= 8.0 Hz), 7.49(d, lH, J= 8.0 Hz), 7.93(s, lH), 8.06(d,
lH, J = 8.8 Hz), 8.53(s, lH), 10.47(s, lH), 10.98(s, lH), l1.80(s,
lH), l1.85(s,lH). MS(ESI, m/z):391.0 [(M-H)]'. Anal. Calcd for
C19H12N406:C, 58.17; H, 3.08; N, 14.28. Found: C, 58.42; H, 3.11;
N,13.96%.
line and SMMC 7721 cell line. Further biological evaluation
and structure optimisation of indoly lmaleimide derivatives are
currently underway in our laboratories.
Experimental
N- (4- (1 H-indol- 3-yl)-2. 5 -dioxo-2. 5-dihydro-l H-pyrrol-3 -yl)-2-
chloroacetamide (7a): Yellow crystals 82% yield; m.p. 170-172°C.
IR (cm"): l706(C=O), l653(C=O). 'H NMR: 8 4.28(s, 2H), 7.08(t,
lH, J= 7.2 Hz), 7.l6(t, lH, J= 7.2 Hz), 7.45-7.49(m, 2H), 7.88(s,
lH), 10.37(s, lH), 10.97(s, lH), l1.86(s, lH). MS(ESI, m/z): 303.9
[(M + HW. Anal. Calcd for C'4HIOClNP3: C, 55.37; H, 3.32;
N, 13.84. Found: C, 55.13; H, 3.61; N, 13.50%.
Melting points were determined with RY-l apparatus, and were
uncorrected. IR spectra were determined as KEr pellets on
Shimadzu model 470 spectrophotometer. 'H NMR spectra were
recorded using a Broker AV 400 MHz spectrometer in DMSO-d6
with tetramethylsilane as internal standard. Electrospray impact mass
spectra were recorded on Simadzu LCMS-20l0 system. Elemental
analyses were performed on a Vario EL III elemental analyser.
a
3-amino-l-(2-chloroacetyl)-4-(1
dione (7b): Yellow crystals 55% yield; m.p. l60-l62°C. IR (em"):
1695(C=O), l646(C=O).'HNMR:84.88(s,2H), 7.03(t, lH,J=7.2Hz),
H-indol-3-yl)-1 H-pyrrole-2. 5-
Table
1
The cytotoxicity data of indolylmaleimide derivatives
7. 13(t, lH, J= 7.2 Hz), 7.26(s, 2H), 7.42(d, lH, J= 8.0 Hz), 7.53(s,
lH), 7.59(d, lH, J = 8.0 Hz), l1.48(s, lH). MS(ESI, m/z): 304.0
[(M + HW. Anal. Calcd for C,4HIOClN303: C, 55.37; H, 3.32; N,
13.84. Found: C, 55.02; H, 3.41; N, 13.59%.
6a-f in vitro
Compound
IC50 (I-lM)
Hela
SMMC 7721
Bioassay of cytotoxicity testing
6a
6b
6c
6d
6e
6f
35.5
42.5
29.5
38.9
31.2
21.2
23.8
9.2
21.2
26.2
16.3
20.4
12.5
10.3
The cytotoxicity of the novel compounds (6a-f) were evaluated
against Hela(cervical cancer cell lines) and SMMC-772l(human
hepatocellular cancer cell lines) in vitro by MTT assay'S, Ro 31-
6233 as the positive controL'6 The results expressed as ICso were
summarised in Table 1. The cells were seeded in 96-well plate at the
concentration of 4000 cells per well in 100 mL RPM I 1640 medium.
After being cultured for 12 h at 37°C and 5% CO2, the cells were
Ro 31-6233

200 JOURNAL OF CHEMICAL RESEARCH 2009
6
7
H.J. Mackay and C.J. Twelves, Endocr.-Relat. Cancer, 2003, 10, 389.
K. Podar, M.S. Raab, D. Chauhan aod K.C. Anderson, Expert Opin.
Invest. Drug, 2007, 16, 1693.
incubated with various concentrations of samples for 24 h. MTT
was added at a terminal concentration of 5 mg/mL and incubated
with cells for 4 h. The formazan crystals were dissolved in 100 mL
DMSO each well, and the optical density was measured at 492 nm
(for absorbance of MTT formazan) and 630 nm (for the reference
wavelength).
S.Y. Zhao, Z.Y. Shao, W.M. Qin aod D.Q. Zhang, Chin. J Org. Chem.,
2008,28, 1676.
9
L.A. Sorbera, N. Serradell, J. Bolos aod E. Rosa, Drug Future, 2007,
32,297.
10 R. Albert, N.G. Cooke, S. Cottens, C. Ehrhardt, J.P. Evenou,
R. Sedrani, P. Vonmatt, J. Wagner aod G. Zenke, WO 2002038561, 2002.
(CA, 136:386017).
We thank the Donghua University for financial support for
funding the young faculty.
11 H.D. Scharf, F. Korte, H. Seidler aod R. Dittmar, Chem. Ber., 1965,
98,764.
Received 2 November 2008; accepted 17February 2009
Paper 08/0274 doi: 10.3184/030823409X430202
Published online: 6 April 2009
12 M. Brenoer, H. Rexhausen, B. Steffao aod W. Steglieh, Tetrahedron,
1988,44,2887.
13 S. Mahboobi, S. Eluwa, M. Koller, A. Popp aod D. Seholhneyer,
J
Heterocyclic Chem., 2000, 37, 1177.
References
14 M. Schultz, C. Tsaklakidis, R. Haag, W. Scheuer aod E. Russmann,
DE 4005970, 1991.(CA, 115:256019)
1
2
3
4
5
A.C. Newton, J BioI. Chem., 1995, 270, 28495.
K.J. Way, E. Chou aod G.L. King, Trends Pharmacol. Sci., 2000, 21, 181.
J. Hofmano, FASEB J, 1997,11,649.
H. Mellor aod P.J. Parker, Biochem. J , 1998,332,281.
M. Serova, A. Ghoul, K.A. Benhadji, E. Cvitkovie, S. Faivre, F. Calvo,
F. Lokiee aod E. Raymond, Semin. Oncol., 2006, 33, 466.
15 T. Mosmano aod J. Inunuool. Methods, 1983,65,55.
16 M. Taoaka, S. Sagawa, J. Hoshi, F. Shimoma, 1. Matsuda, K. Sakoda,
T. Sasase, M. Shindo aod T. 1uaba, Bioorg. Med.Chem. Lett., 2004,
14,5171.