Rapid synthesis and bioactivities of 3-(nitromethylene)indolin-2-one analogues

Article abstract of DOI:10.2478/s11696-010-0047-1

A new synthesis method of 3-(nitromethylene)indolin-2-one analogues is described, using the Henry reaction of isatin and N-substituted isatins with nitromethane followed by dehydration of the nitroaldol adduct with mesyl chloride. The use of diethylamine (rather than DBU) as the base catalyst in a solvent-free Henry reaction gave the nitroaldol adduct in sufficient purity as to allow its direct dehydration to nitroalkene. Overall yields for this two-step synthesis are satisfactory (typically 50-77 % after chromatographic purification). 3-(Nitromethylene)indolin-2-one analogues are valued protective agents against H₂O₂-induced apoptosis using PC12 cells, and for their cytotoxicity against the A549 and P388 lung cancer cell lines. One compound, (E)-1-benzyl-3-(nitromethylene)indolin-2-one (VIII), exhibited potent activity in the latter assay.

Full text of DOI:10.2478/s11696-010-0047-1

Chemical Papers 64 (5) 673–677 (2010)
DOI: 10.2478/s11696-010-0047-1
SHORT COMMUNICATION
Rapid synthesis and bioactivities of 3-(nitromethylene)indolin-2-one
analogues
c
d
a,bGang Chen, b,cXiao-Jiang Hao*, Qian-Yun Sun, Jian Ding
aCollege of Chemistry & Chemical Engineering, Xi’an Shiyou University, Xi’an, Shaanxi 710065, China
bState Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming, Yunnan 650204, China
cKey Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences,
Guiyang 550002, China
dState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China
Received 28 November 2009; Revised 23 February 2010; Accepted 2 March 2010
A new synthesis method of 3-(nitromethylene)indolin-2-one analogues is described, using the
Henry reaction of isatin and N-substituted isatins with nitromethane followed by dehydration of
the nitroaldol adduct with mesyl chloride. The use of diethylamine (rather than DBU) as the base
catalyst in a solvent-free Henry reaction gave the nitroaldol adduct in sufficient purity as to allow its
direct dehydration to nitroalkene. Overall yields for this two-step synthesis are satisfactory (typically
50–77 % after chromatographic purification). 3-(Nitromethylene)indolin-2-one analogues are valued
protective agents against H2O2-induced apoptosis using PC12 cells, and for their cytotoxicity against
the A549 and P388 lung cancer cell lines. One compound, (E)-1-benzyl-3-(nitromethylene)indolin-
2-one (VIII ), exhibited potent activity in the latter assay.
c
ꢀ 2010 Institute of Chemistry, Slovak Academy of Sciences
Keywords: 3-(nitromethylene)indolin-2-one, diethylamine, antitumor neuroprotection, catalyst
Isatin is an endogenous compound in mammalian
tissues and body fluids (Glover et al., 1988) as well
as in many plants. In addition, isatin is a versatile
intermediate in the synthesis of potential drugs. Re-
cently, 3-substituted indolin-2-ones have attracted at-
tention due to their antitumor therapeutic activities
(Sun et al., 1999, 2000). 3-(Methylene)indolin-2-one
derivatives in particular have received extensive bio-
logical scrutiny (Laird et al., 2000; Fong et al., 1999;
Mendel et al., 2000). One such analogue, SU11248
(product name: Sunitinib malate), was found to be
clinically effective as an antiangiogenesis drug.
2,3ꢀ-oxindole] derivatives (Palmisano et al., 1996; Fe-
jes et al., 2001; Long et al., 1978). For this purpose,
the intermediate 3-hydroxy-3-(nitromethyl)indolin-2-
one (I ) (Fig. 1, R = H) was synthesized by the
Henry nitroaldol reaction between isatin and ni-
tromethane followed by a dehydration reaction to give
3-(nitromethylene)indolin-2-one (II ) (Fig. 1, R = Me).
Conn and Lindwall (1936) reported the synthesis of I
in an ethanol solution using diethylamine as the cat-
alyst; however, the reaction time was long (one day)
and the yield was only moderate (71 %). The use of a
stronger organic base, 1,8-diazabicyclo[5.4.0]undec-7-
ene (DBU), as the catalyst, did not improve this yield
(Palmisano et al., 1996; Fejes et al., 2001; Long et al.,
1978).
3-(Nitromethylene)indolin-2-one derivatives have
been used as intermediates in the synthesis, by 1,3-
dipolar cycloadditon reactions, of spiro[pyrrolidine-
*Corresponding author, e-mail: gangchen@xsyu.edu.cn

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G. Chen et al./Chemical Papers 64 (5) 673–677 (2010)
NO2
HO
O
O
NO2
ii
i
Method A
N
R
N
R
O
N
R
O
I
II–VIII
NO2
HO
O
O
NO2
iii
iv
ii
Method B
Method C
N
R
N
R
O
O
N
R
O
NO2
H3N
O
O
NO2
N
R
N
R
N
R
O
Fig. 1. Synthesis of (E)-1-alkyl-3-(nitromethylene)indolin-2-ones (II, R = H; III, R = methyl; IV, R = ethyl; V, R = propyl; VI, R
= butyl; VII, R = pentyl; VIII, R = benzyl). Reaction conditions: i) CH3NO2, Et2NH; ii) CH3SO2Cl, Et3N; iii) CH3NO2,
DBU; iv) CH3NO2, AcOH, NH4OAc.
In order to increase the overall yield, a solvent-
free synthesis of N-substituted analogues of I us-
ing diethylamine as the catalyst was developed.
Without further purification of the nitroaldol in-
termediate, MsCl-dependent dehydration produced
3-(nitromethylene)indolin-2-one derivatives (Fig. 1,
method A). A small series of these derivatives was
prepared and their protective bioactivities on H2O2-
induced apoptosis of PC12 cells and cytotoxicity
against lung cancer A549 and P388 cell lines of these
compounds were evaluated as a result of our continued
interest in indole alkaloids (Chen et al., 2006, 2009; Di
et al., 2007).
All starting materials and solvents (A.R. grade)
were commercially available and were used without
further purification. NMR spectra were recorded using
a Bruker Drx-400 spectrometer operating at 400 MHz
for 1H and at 100 MHz for 13C. Mass spectra were
recorded on a Micromass Platform spectrometer using
a direct-inlet system operating in the electron impact
(EI) mode at 75 eV. Elemental analyses were obtained
using a Carlo Erba 1106 elemental analyzer.
Spectral and analytical data of known ( )-3-
hydroxy-3-(nitromethyl)indolin-2-one (I ) (Fig. 1,
R = H) and (E)-3-(nitromethylene)indolin-2-one (II )
(Fig. 1, R = H) were in accordance with those al-
ready published (Elinson et al., 2008; Conn & Lind-
wall, 1936; Morales-Ríos et al., 2000).
New (E)-1-alkyl-3-(nitromethylene)indolin-2-ones
(Fig. 1: III, R = methyl; IV, R = ethyl; V, R = propyl;
VI, R = butyl; VII, R = pentyl; VIII, R = benzyl)
were prepared according to the general procedure as
follows: N-substituted isatin (0.294 g, 2 mmol) was
dissolved in nitromethane (15 mL). One drop of di-
ethylamine was added and the mixture was stirred at
room temperature for a few minutes until the orange-
colored solution turned colorless. The solvent was re-
moved under reduced pressure to give the nitroaldol
adduct as a white powder. This powder was dissolved
in CH2Cl2 (20 mL), followed by an addition of triethy-
lamine (3 mL) and methanesulfonyl chloride (0.5 mL).
The reaction mixture was cooled to 0◦ and stirred at
this temperature overnight. The solvent was removed
under reduced pressure and the residue was puri-
fied by column chromatography (silica gel, petroleum
ether/ethyl acetate, gradient of ϕr = 50 : 1 to ϕr = 20
: 1) to give the target products. Analytical samples of
red (or orange) crystals of 3-(nitromethylene)indolin-
2-ones were obtained by crystallization from ethanol.
E-stereochemistry was assigned to these products on
the basis of literature data (Palmisano et al., 1996;
Fejes et al., 2001). Analytical and spectral data are
given in Tables 1 and 2.
The use of diethylamine as the base catalyst in
the solvent-free reaction of isatin and nitromethane
resulted in an effective and fast reaction, proceed-
ing within a few minutes. Removal of the sol-
vent from this reaction mixture gave 3-hydroxy-3-
(nitromethyl)indolin-2-one (I, R = H) in a nearly
quantitative yield. The yield and purity of this inter-
mediate allowed its direct dehydration, without its iso-
lation (Fig. 1, method A), with methanesulfonyl chlo-
ride to give 3-(nitromethylene)indolin-2-one (II, R =
H). The overall yield of these two steps reached 77 %
after chromatographic purification. (E)-1-Methyl-3-
(nitromethylene)indolin-2-one (III ) was obtained by
this method in the 61 % yield. For comparison, previ-
ous syntheses (Fig. 1, method B) of two compounds
(II and III ), as well as classical synthesis of (E)-β-
nitrostyrene (Fig. 1, method C), were repeated. The
results showed that method A consistently provided
improved yields compared to methods B and C. More-
over, method A offers short reaction times for the first
step.

G. Chen et al./Chemical Papers 64 (5) 673–677 (2010)
675
Table 1. Characterization data of newly prepared (E)-1-alkyl-3-(nitromethylene)indolin-2-ones
wi(calc.)/mass %
wi(found)/mass %
Yield
M.p.
Compound
Formula
Mr
C
H
N
%
61
55
53
53
51
62
◦C
III
IV
C10H8N2O3
C11H10N2O3
C12H12N2O3
C13H14N2O3
C14H16N2O3
C16H12N2O3
204.18
218.21
232.24
246.26
260.29
280.29
58.82
58.80
60.55
60.56
62.06
62.01
63.40
63.44
64.60
64.62
68.56
68.59
3.95
3.95
4.62
4.60
5.21
5.22
5.73
5.71
6.20
6.21
4.32
4.33
13.72
13.71
12.84
12.80
12.06
12.07
11.38
11.33
10.76
10.75
9.99
137–138
142–143
147–148
150–151
151–153
165–167
V
VI
VII
VIII
10.02
Table 2. Spectral data of newly prepared (E)-1-alkyl-3-(nitromethylene)indolin-2-ones
Compound
Spectral data
III
1H NMR (CDCl3), δ: 11.40 (s, 1H, HCNO2), 8.10 (d, 1H, J = 7.2 Hz, Haryl), 7.26 (s, 1H, Haryl), 7.09 (t, 1H, J =
7.6 Hz, Haryl), 6.83 (t, 1H, J = 7.2 Hz, Haryl), 3.28 (s, 3H, CH3)
13
—
C NMR (CDCl3), δ: 26.2 (CH3), 121.2, 122.5, 123.9, 126.6, 128.1, 138.2 (CHNO2), 141.5, 144.5, 169.1 (C O)
—
MS, m/z (Ir/%): 204 (24) (M+
)
IV
V
1H NMR (CDCl3), δ: 11.60 (s, 1H, HCNO2), 8.12 (d, 1H, J = 7.6 Hz, Haryl), 7.37 (t, 1H, J = 7.6 Hz, Haryl), 7.07
(t, 1H, J = 7.6 Hz, Haryl), 6.85 (d, 1H, J = 7.6 Hz, Haryl), 3.81 (q, 2H, J = 7.2 Hz, NCH2), 1.29 (t, 3H, J = 7.2
Hz, CH3)
13C NMR (CDCl3), δ: 14.6 (CH3), 31.2 (CH2), 121.1, 123.2, 123.7, 126.7, 128.4, 138.8 (CHNO2), 141.7, 146.6,
—
169.1(C O)
—
MS, m/z (Ir/%): 218 (18) (M+
)
1H NMR (CDCl3), δ: 11.30 (s, 1H, HCNO2), 8.11 (d, 1H, J = 7.2 Hz, Haryl), 7.39 (t, 1H, J = 8.0 Hz, Haryl), 7.08
(t, 1H, J = 8.0 Hz, Haryl), 6.86 (d, 1H, J = 8.0 Hz, Haryl), 3.72 (t, 2H, J = 7.2 Hz, NCH2), 1.73 (m, 2H, CH2),
0.98 (t, 3H, J = 7.2 Hz, CH3)
13C NMR (CDCl3), δ: 22.1 (CH3), 26.8 (CH2), 40.1 (CH2), 121.2, 122.3, 124.0, 125.9, 128.7, 137.9 (CHNO2), 142.0,
—
146.5, 169.1 (C O)
—
MS, m/z (Ir/%): 232 (21) (M+
)
VI
1H NMR (CDCl3), δ: 11.20 (s, 1H, HCNO2), 8.12 (d, 1H, J = 7.6 Hz, Haryl), 7.37 (m, 1H, Haryl), 7.07 (m, 1H,
H
aryl), 6.86 (d, 1H, J = 8.0 Hz, Haryl), 3.76 (t, 2H, J = 7.2 Hz, NCH2), 1.68 (m, 2H, CH2), 1.41 (m, 2H, CH2),
0.96 (t, 3H, J = 7.6 Hz, CH3)
13C NMR (CDCl3), δ: 22.4 (CH3), 26.9 (CH2), 28.8 (CH2), 40.2 (CH2), 121.4, 122.5, 123.5, 126.9, 128.0, 138.7
—
(CHNO2), 141.8, 146.2, 169.0 (C O)
—
MS, m/z (Ir/%): 246 (27) (M+
)
VII
VIII
1H NMR (CDCl3), δ: 11.18 (s, 1H, HCNO2), 8.12 (d, 1H, J = 7.2 Hz, Haryl), 7.39 (t, 1H, J = 8.0 Hz, Haryl), 7.08
(t, 1H, J = 7.6 Hz, Haryl), 6.86 (d, 1H, J = 8.0 Hz, Haryl), 3.75 (t, 2H, J = 7.2 Hz, NCH2), 1.70 (m, 2H, CH2),
1.37 (m, 4H, CH2), 0.90 (m, 3H, CH3)
13C NMR (CDCl3), δ: 14.2 (CH3), 22.5 (CH2), 26.9 (CH2), 28.9 (CH2), 40.2 (CH2), 121.5, 122.6, 123.5, 126.9,
—
128.0, 138.7 (CHNO2), 141.8, 146.2, 169.0 (C O)
—
MS, m/z (Ir/%): 260 (33) (M+
)
1H NMR (CDCl3), δ: 11.40 (s, 1H, HCNO2), 8.13 (d, 1H, J = 7.6 Hz, Haryl), 7.20–7.41 (m, 6H, Haryl), 7.10 (m,
1H, Haryl), 6.76 (d, 1H, J = 7.6 Hz, Haryl), 4.97 (s, 2H, CH2)
13C NMR (CDCl3), δ: 48.7 (CH2), 121.4, 122.5, 123.1, 123.9, 126.6, 127.2, 127.6, 128.1, 128.8, 138.2 (CHNO2),
—
146.5, 169.1 (C O)
—
MS, m/z (Ir/%) 280 (12) (M+
)
Compared with the yield of III (61 %), the yields of
the other N-substituted isatin derivatives were slightly
lower (51–62 %, after chromatographic purification).
Compounds III–VII as well as isatin were screened
for their protective effect on the apoptosis of PC12
cells induced by H2O2 and for their cytotoxicity

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G. Chen et al./Chemical Papers 64 (5) 673–677 (2010)
Table 3. Inhibitory and protective effects of (E)-1-alkyl-3-(nitromethylene)indolin-2-ones
Inhibitory effect/%a
Protective effect/%b
Compound
200 µM
20 µM
2 µM
200 µM
20 µM
2 µM
Isatin
III
IV
V
VI
92
62
78
92
66
83
–
0
14
6
16
27
19
–
3
22
7
17
14
19
–
–
–
–
44
–
–
20
0
28
63
–
40
0
52
66
0
VII
VE
0
–
0
22
–
a) Inhibition of PC12 cell growth; b) protective effect on the apoptosis of PC12 cells induced by H2O2.
J.-X., Ding, J., & Hao, X.-J. (2009). Synthesis and bioac-
tivity evaluation of 3-hydroxy-3-(phenylethynyl)indol-2-one
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(2006). l-Proline-catalyzed asymmetric aldol condensation
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2333. DOI: 10.3987/COM-06-10856.
Conn, W. R., & Lindwall, H. G. (1936). Oxindole amines from
isatin. Journal of the American Chemical Society, 58, 1236–
1239. DOI: 10.1021/ja01298a042.
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niphyllum calycillum. Organic Letters, 9, 1355–1358. DOI:
10.1021/ol070218r.
Elinson, M. N., Ilovaisky, A. I., Merkulova, V. M., Barba, F., &
Batanero, B. (2008). Electrochemically induced Henry reac-
tion of nitromethane and carbonyl compounds. Tetrahedron,
64, 5915–5919. DOI: 10.1016/j.tet.2008.04.039.
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Table 4. In vitro inhibitory activities against P388 and A549
cell line
Inhibitory activity/%
Compound
P388
A549
Isatin
III
IV
V
VI
28
17
53
51
53
88
90
49
63
53
62
58
90
85
VIII
R-XK469
against A549 and P388 cell lines by reported meth-
ods (Chen et al., 2009; Mosmann, 1983). The results
are given in Table 3.
Compounds IV and V as well as isatin showed po-
tent activity, and were more effective than vitamin
E (VE, ( )-α-tocopherol), with percentages of 52 %,
66 %, and 40 % at concentrations of 2 µM, respec-
tively. Besides, the three compounds were weakly cy-
totoxic to PC12 cells at the concentrations of 2–20
µM, while the other compounds were either inactive
or cytotoxic to PC12 cells.
The in vitro cytotoxicity of compounds III–VI,
VIII, and isatin against A549 and P388 cell lines
at the concentration of 100 µM are given in Ta-
ble 4. Almost all compounds inhibited both cancer
cell lines at the concentration of 100 µM (≥ 50 %),
and were more potent than isatin. Only isatin and
III were inactive against P388. Compound VIII was
the most potent of the nitromethylene series show-
ing 88 % inhibition of P388 and 90 % inhibition of
A549, and also more potent than (R)-2-(4-(7-chloro-2-
quinoxalinyloxy)phenoxy)propanoic acid (R-XK469).
Fong, T. A. T., Shawver, L. K., Sun, L., Tang, C., App, H.,
Powell, T. J., Kim, Y. H., Schreck, R., Wang, X., Risau, W.,
Ullrich, A., Hirth, K. P., & McMahon, G. (1999). SU5416 is
a potent and selective inhibitor of the vascular endothelial
growth factor receptor (Flk-1/KDR) that inhibits tyrosine
kinase catalysis, tumor vascularization, and growth of mul-
tiple tumor types. Cancer Research, 59, 99–106.
Glover, V., Halket, J. M., Watkins, P. J., Clow, A., Goodwin,
B. L. & Sandler, M. (1988). Isatin: identity with the puri-
fied endogenous monoamine oxidase inhibitor tribulin. Jour-
nal of Neurochemistry, 51, 656–659. DOI: 10.1111/j.1471-
4159.1988.tb01089.x.
Laird, A. D., Vajkoczy, P., Shawver, L. K., Thurnher, A., Liang,
C., Mohammadi, M., Schlessinger, J., Ullrich, A., Hubbard,
S. R., Blake, R. A., Fong, T. A. T., Strawn, L. M., Sun,
L., Tang, C., Hawtin, R., Tang, F., Shenoy, N., Hirth, K.
P., McMahon, G., & Cherrington, J. M. (2000). SU6668 is
a potent antiangiogenic and antitumor agent that induces
regression of established tumors. Cancer Research, 60, 4152–
4160.
Acknowledgements. This work was financially supported by
the grants from the Chinese Academy of Sciences (29O702441
2P1, O7023112S1) and the Natural Science Research Plan
Projects of the Shaanxi Science and Technology Department
(SJ08B20).
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