Design, synthesis and evaluation of 3-methylene-substituted indolinones as antimalarials

Article abstract of DOI:10.1016/j.ejmech.2011.01.008

The design, synthesis and evaluation of 3-methylene-substituted indolinones as falcipain inhibitors and antiplasmodial agents are described. These compounds react readily with thiols via an addition-elimination mechanism, indicating their potential as cys

Full text of DOI:10.1016/j.ejmech.2011.01.008

European Journal of Medicinal Chemistry 46 (2011) 927e933
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and evaluation of 3-methylene-substituted
indolinones as antimalarials
,
a
S. Praveen Kumar a, Jiri Gut b, Rita C. Guedes a, Philip J. Rosenthal b, Maria M.M. Santos a , Rui Moreira
*
a Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal
b Department of Medicine, San Francisco General Hospital, University of California, San Francisco, CA 94143, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The design, synthesis and evaluation of 3-methylene-substituted indolinones as falcipain inhibitors and
antiplasmodial agents are described. These compounds react readily with thiols via an addition-elimi-
nation mechanism, indicating their potential as cysteine protease inhibitors. Several indolinones con-
taining a Leu-i-amyl recognition moiety were found to be moderate inhibitors of the Plasmodium
falciparum cysteine protease falcipain-2, but not of the related protease falcipain-3, and displayed
antiplasmodial activity against the chloroquine-resistant P. falciparum W2 strain in the low micromolar
range. Coupling a 7-chloroquinoline moiety to the 3-methylene-substituted indolinone scaffold led to
a significant improvement in antiplasmodial activity.
Received 6 August 2010
Received in revised form
4 November 2010
Accepted 9 January 2011
Available online 15 January 2011
Keywords:
Indolin-2-ones
Falcipain inhibitor
Antiplasmodial activity
Malaria
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
2. Results and discussion
Malaria is a serious global health problem, with devastating
social and economic consequences in countries where the disease
is endemic, due in part to the rapid emergence and spread of
parasites that are resistant to well-established antimalarial drugs
[1]. As a result, there is an urgent need for novel drugs, preferably
acting on underexploited parasite targets in order to delay or
overcome the selection of clinical resistance [2]. Cysteine proteases
from malaria parasites are of particular interest as therapeutic
targets due to their key role in parasite development [3]. Plasmo-
dium falciparum expresses four cysteine proteases from the papain
family known as falcipains, of which falcipain-2 (FP-2) and falci-
pain-3 (FP-3) are the most relevant as therapeutic targets [4e6]. As
part of our ongoing studies on the design and synthesis of Michael
acceptors as cysteine protease inhibitors [4,7e9], we became
interested in the study of the 3-methylene-indolinone scaffold
(Fig.1). This scaffold was already used with success for VEGFR, Trk A
and CDK inhibitors [10e14]. In particular, indolin-2-ones
substituted at C-3 with an aminomethylene group bearing different
amino acid moieties were described as antitumor and antibacterial
agents [15,16]. However, the potential of 3-methylene-indolinones
as antimalarial agents remains to be explored.
2.1. Chemistry
To evaluate the 3-methylene-indolinone scaffold as a Michael
acceptor, we first studied the reaction of 1a with a thiol. Compound
1a was synthesized from indolin-2-one and DMA-DMF as described
in the literature [17].
Interestingly, compound 3 was isolated in 36e50% yields from
reaction of 1a with benzyl mercaptate 2 using different conditions
(Scheme 1), consistent with conjugate addition of the thiol or thi-
olate followed by elimination of the amine moiety (Scheme 1).
A series of 3-(N,N-dimethylaminomethylene)indolin-2-ones
1bee was then synthesized in 77e90% yield. The compounds
were obtained as a mixture of Z/E isomers. The Z/E isomeric ratio
(Table 1) was determined from the 1H NMR spectrum on the signal
of the olefinic protons, two singlets around 7.4 and 7.6 ppm,
respectively.
The next stage was the design of selective falcipain inhibitors, by
incorporating the adequate recognition moiety to the double bond.
We started by studying the reaction of amines with compound 1a.
Compound 4 (Fig. 2) was obtained in 60% yield by reaction of 1a
with aniline in the presence of hydrochloric acid [16]. However, no
reaction occurred when L-phenylalanine and glycine ethyl esters
were used. Only when the reaction was performed in the presence
of acetic acid at 70 ꢀC, were we able to obtain compounds 5aeb
(Fig. 2).
* Corresponding author. Tel.: þ351 21 794 6400; fax: þ351 21 794 6470.
E-mail address: mariasantos@ff.ul.pt (M.M.M. Santos).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.008

928
S. Praveen Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 927e933
Table 1
R
O
Yields, Z/E ratio and antiplasmodial activity for compounds 1aee, 3, 4, 5aeb, 7aei
and 10aef.
O
R1
H
N
HO2C
Compds
Yield
(%)
h
Z/E ratio Falcipain-2
IC50
Falcipain-3
P. falciparum W2
IC50
N
R2
N
H
,
m
M
IC50
,
m
Ma
, mM
O
O
1a
1b
1c
1d
1e
3
4
5a
5b
7a
7b
7c
85
82
77
83
90
1:1
1:1
1.25:1
1.5:1
1.1:1
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
ND
ND
ND
ND
ND
ND
ND
ND
ND
>50
>50
>50
>50
>50
>50
>50
>50
>50
ND
>50
>50
>50
>50
>50
>10
›10
E-64-c
3-Methylene indolinone
Fig. 1. Chemical structure of 3-methylene-substituted indolinone and E-64c.
36e50 3:1
60
29
20
26
40
55
52
55
50
35
40
30
40
52
62
56
70
68
e
3:1
10:1
10:1
10:1
20:1
20:1
20:1
20:1
3:1
10.79 ꢁ 1.77
>10
A series of compounds 7aei was designed based on the struc-
30.73 ꢁ 1.71
18.72 ꢁ 1.31
15.94 ꢁ 0.46
15.66 ꢁ 1.08
23.50 ꢁ 3.49
26.66 ꢁ 3.70
18.55 ꢁ 0.40
>50
13.44 ꢁ 1.77
0.42 ꢁ 0.02
0.20 ꢁ 0.01
0.18 ꢁ 0.02
0.17 ꢁ 0.02
0.29 ꢁ 0.02
0.14 ꢁ 0.01
ture of the epoxide cysteine protease inhibitor E-64c (Fig. 1), which
contains a Leu-isoamyl recognition moiety. Phe, homoPhe and Gly
residues were also selected in order to modulate the interaction
with FP-2. The recognition moiety was obtained starting with
different N-Boc amino acids (Leu, Phe, homoPhe and Gly) and iso-
amyl amine in the presence of BOP and THF. Compounds 6aed
were obtained with 62e70% yields after deprotection of the
nitrogen with TFA.
Several conditions were used to couple compounds 6aed to
dimethylaminomethylenes 1aee. However, no reaction occurred
using the Moreau method (HCl in MeOH) [16], or AcOH in CH2Cl2.
Finally, using the method described by Abass [17], compounds 7aei
were obtained in 26e55% yields (Scheme 2) as a mixture of Z/E
isomers, which were inseparable by column chromatography. The
Z/E isomeric ratio (Table 1) was determined from the 1H NMR
spectrum based on the signals of the vinylic protons, which
appeared ca. 7.43 (d, J ¼ 12 Hz) and 7.62 ppm (d, J ¼ 14 Hz). The ratio
of Z/E isomers varied with the amino acid used in the recognition
moiety. Compounds 7a and 7g gave a 10:1 Z/E ratio, while
compounds 7bee and 7i gave a 20:1 mixture of Z/E isomers. In
contrast, compounds 7f and 7h gave a 3:1 mixture of Z/E isomers. In
the case of the homoPhe derivatives, only the Z isomer was
obtained probably because of steric hindrance exerted by the
homoPhe group in the vicinity of the C]C bond. Overall, the
preferred formation of the Z isomer can be ascribed to hydrogen
bonding between the NH and the carbonyl oxygen atom of the
indole ring (Fig. 3).
The reaction of compound 7a with benzyl mercaptate 2 was also
studied using methanol and triethylamine. In this case, the starting
material 7a was consumed at a slower rate than 1a suggesting that
incorporation of the recognition moiety in the indolin-2-one scaf-
fold reduces the reactivity toward the thiol or thiolate.
In order to further study the potential of 3-methylene-indolin-
2-ones as antimalarials we prepared compounds 10aee, in which
the 3-methylene- indolin-2-one scaffold is coupled to a 4-amino-
quinoline moiety. The synthesis of derivatives 10aee involved the
preparation of the quinoline intermediate 9 [18] from reaction of
4,7-dichloro quinoline 8, with 1,3 propane diamine in 94% yield
(Scheme 3). Reaction of 9 with 3-(N,N-dimethylaminomethylene)
indolin-2-ones 1aee, led to 10aee in 40e70% yield (Scheme 3).
In order to study the effect of carbon chain length between
44.1 ꢁ 1.3
48.5 ꢁ 2.0
42.1 ꢁ 2.3
>50
7d
7e
7f
>50
7g
7h
7i
10a
10b
10c
10d
10e
10f
E-64
10:1
3:1
20:1
2:1
49.9 ꢁ 0.3
43.3 ꢁ 0.7
44.0 ꢁ 1.3
>50
2:1
>50
ND
ND
ND
ND
2:1
>50
2:1
>50
10:1
3:1
>50
ND
ND
e
111.7 ꢁ 25.4 481.5 ꢁ 6.36 1.94 ꢁ 0.004
(nM)
ND
(nM)
ND
Chloroquine
e
e
0.14 ꢁ 0.02
a
ND ¼ not done.
indolinone and quinoline scaffolds, compound 10f was also
synthesized using as starting material 1,2 ethylene diamine
(Scheme 3, 68% yield).
2.2. Biological activity
Compounds 1aee, 3, 4, 5aeb, 7aei and 10aef were then
screened against the parasite cysteine proteases FP-2 and FP-3.
Inspection of the data presented in Table 1, shows that the recog-
nition moiety seems to be essential for FP-2 inhibition, as
compounds 1aee, 3, 4, 5aeb and 10aef, lacking the amino acid-
isoamyl moiety were devoid of enzyme inhibitory activity. In
contrast, none of the compounds tested was active for FP-3. Also
worth noting, the presence of a chlorine atom at the indolinone
moiety seems to be beneficial for FP-2 inhibition (e.g. 7a vs 7bed),
but the position of substitution had no major impact on activity
(5-Cl, 7b vs 6-Cl, 7c vs 7-Cl, 7d).
Compounds 1aee, 3, 4, 5aeb, 7aei and 10aef were then tested
against the chloroquine-resistant W2 strain of P. falciparum
(Table 1). Compounds containing the amino acid-isoamyl moiety,
R
NHPh
O
N
H
CO2Et
NMe2
S
Ph
HS
Ph
2
O
O
O
N
H
N
H
N
H
N
H
4
5a R=H
5b R=CH2Ph
1a
3
Scheme 1. Reaction of 1a with benzyl mercaptate 2. Conditions: AcOH or, CH2Cl2/TEA
or MeOH/TEA.
Fig. 2. Chemical structure of compounds 4 and 5aeb.

S. Praveen Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 927e933
929
R3
H
N
NMe2
O
N
H
R3
R1
H
N
O
R1
+
O
H2N
N
R2
N
R2
O
1a R1=H; R2=H
6a R3=H
7a R1=H; R2=H; R3=CH2CH(CH3)2
7b R1=5-Cl; R2=H; R3=CH2CH(CH3)2
7c R1=6-Cl; R2=H; R3=CH2CH(CH3)2
7d R1=7-Cl; R2=H; R3=CH2CH(CH3)2
7e R1=H; R2=Me; R3=CH2CH(CH3)2
7f R1=H; R2=H; R3=H
1b R1=5-Cl; R2=H
1c R1=6-Cl; R2=H
1d R1=7-Cl; R2=H
1e R1=H; R2=Me
6b R3=CH2CH(CH3)2
6c R3=CH2Ph
6d R3=CH2CH2Ph
7g R1=H; R2=H; R3=CH2CH2Ph
7h R1=5-Cl; R2=H; R3=CH2Ph
7i R1=7-Cl; R2=H; R3=CH2CH2Ph
Scheme 2. Synthesis of indolinones 7aei. Conditions: AcOH, 70 ꢀC.
i.e. 7, had moderate antiplasmodial activity, with IC50 values
ranging from 13 to 30 M. In addition, compound 5a, containing
a phenylalanine ethyl ester residue, had an IC50 value of 11 M.
These results are similar to those reported for other 2-oxoindole
based inhibitors, such as thiosemicarbazone isatin derivatives [19].
Data from Table 1 suggests that FP-2 inhibition might contribute to
some extent to the antiplasmodial activity displayed by compounds
7bed and 7hei.
anticipated active site, and hydrophobic interactions between the
inhibitors and non polar regions of the falcipain-2 surface.
m
m
Our calculations demonstrate a clear difference in the scores
obtained for the docking of inhibitors 7a, 7e and 7f (goldscore
between 50 and 52) and the most potent compounds of the 7 series
(goldscore always superior to 54). When 7a was docked to falci-
pain-2, two binding poses were identified. One presents the indo-
linone moiety in the S01 subsite and the Leu-isoamyl in the S2
pocket, while the other binding pose is inverted, presenting the
indolinone moiety well within the S2 pocket, Leu in S1 and Leu-
isoamyl in the S01 subsite. The distances between the reactive
carbon and the Cys42 sulfur atom are in the 3.5e5 Å range. A
similar inversion in the top binding poses is observed in compound
7e, with the indolinone moiety occupying S2 or S1 subsites and the
reactive carbon 3.5 Å distant from the Cys42 sulfur. Another
important feature in the active site binding pose of these
compounds is that the reactive carbon is never well oriented to the
Cys42 sulfur. Interestingly, the less potent compounds (7e and 7f),
with the indolinone ring in the S1 subsite, are the only compounds
occupying the top part of this subsite.
Compounds 10aef did not inhibit FP-2, which can be explained
by the absence of a recognition moiety. In contrast, compounds
10aef displayed potent antiplasmodial activity, with an IC50 value
of 0.14e0.42
mM against the P. falciparum W2 strain (Table 1).
Probably, FP-2 inhibition is not the primary mode by which
derivatives 10a-f exert their antiplasmodial activities. It is also
worth noting, that variation in carbon chain length between
indolinone and quinoline scaffolds had no major impact on activity
(3 carbons, 10d vs 2 carbons, 10f).
2.3. Docking studies
With the exception of 7h, all compounds that demonstrated
inhibition, possess the reactive carbon close to the Cys42 sulfur
(2.9e3.5 Å) and this carbon is always well oriented to a potential
attack. Compounds 7g and 7i have exactly the same binding pose,
To gain some insight to the binding mode of our series of
inhibitors we studied their molecular interactions with falcipain-2
using GOLD software. Compounds containing Gly, Leu, Phe, and
homoPhe (7ae7i) were docked into the enzyme active site and the
results were compared with the crystallized pose of E-64 [5]. The
active site of the enzyme is located in a cleft between the struc-
turally distinct domains of the papain like fold, and E-64 interacts
with the residues located at S1, S2 and S3 [5].
Each potential inhibitor was energy - minimized and subjected
to 2000 docking runs. The top 5 solutions (those presenting the
highest fitness score) were visually analyzed. In order to elucidate
the inhibitor binding poses we gave special attention to the
distances between the potentially reactive carbon and the sulfur
atom of the catalytic Cys42 residue, the subsites occupied in the
Cl
N
HN
N
NH2
Cl
(i)
Cl
8
9
(ii)
R1
N
NH
N
H
N
R2
R3
O
H
Cl
N
Cl
N
H
N
10a R1=H; R2=H
H
N
H
O
10b R1=5-Cl; R2=H
10c R1=6-Cl; R2=H
10d R1=7-Cl; R2=H
10e R1=H; R2=Me
R1
Cl
HN
N
O
O
10f
N
R2
Scheme 3. Synthesis of aminoquinoline derivatives 10aef. Conditions: i) 1,3 propane
diamine, 120 ꢀC; ii) 1, AcOH, 70 ꢀC.
Fig. 3. Hydrogen bond in Z-configuration.

930
S. Praveen Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 927e933
occupying the S2 and S01 subsites, with homoPhe very close to the
S3 subsite. Compound 7h has a different binding pattern with the
distance between the reactive carbon and Cys42 sulfur being
around 4.5. This is the only compound that has the indolinone
moiety in the S1 subsite, and the amino acid in P2 (Phe in this case)
is pointing toward the S3 subsite in a similar way as in the case
of E-64.
J ¼ 8.0 Hz), 7.26 (d,1H, J ¼ 8.0 Hz), 6.93 (dd,1H, J ¼ 8.0 Hz, J ¼ 4.0 Hz),
6.70e6.91 (m, 4H), 6.69 (d,1H, J ¼ 8.0 Hz), 3.63 (s br, 3H), 3.28 (s, 6H),
3.27 (s br, 3H). 13C NMR (100 MHz, DMSO-d6)
d 170.80, 165.94,
148.83, 147.93, 137.92, 135.72, 128.74, 122.94, 120.19, 119.78, 119.32,
114.40, 108.59, 107.76, 94.81, 93.02, 43.90. HRMS-ESI-TOF: m/z calc
C11H13N2O (M þ 1) 189.1028, found 189.1022.
Our most potent inhibitor, 7d, is the one that possesses the
reactive carbon closer and adequately oriented to the sulfur atom of
Cys42 (2.9e3.2 Å), suggesting that this could in principle establish
a covalent bond between the enzyme and the inhibitor.
The difference between the inhibition presented by the active
studied compounds and the FP-2 inhibitor E-64 can be explained by
the different pose that they adopt in the active site. In E-64 the
amino acid in P2 is placed in the S2 pocket, which does not occur in
our compounds.
4.1.1.2. 3-(N,N-Dimethylaminomethylene) indolin-2-one 1b. Obtained
in 82% yield, Z/E mixture 2/1. M.p. 204e206 ꢀC. IR (KBr) 3112, 1682,
1593, 1402, 1223, 1153 cmꢂ1 1H NMR (400 MHz, DMSO-d6)
d 10.16 (s
br, 1H, NH), 10.07 (s br, 1H, NH), 7.66 (s, 1H), 7.46 (s, 1H), 7.36 (d, 1H,
J ¼ 4.0 Hz), 7.30 (d,1H, J ¼ 4.0 Hz), 6.93 (dd,1H, J ¼ 8.0 Hz, J ¼ 4.0 Hz),
6.82 (dd,1H, J ¼ 8.0 Hz, J ¼ 4.0 Hz), 6.74 (d,1H, J ¼ 8.0 Hz), 6.65 (d,1H,
J ¼ 8.0 Hz), 3.65 (s br, 3H), 3.31 (s, 6H), 3.26 (s br, 3H). 13C NMR
(100 MHz, DMSO-d6) d 170.62, 165.80, 149.92, 149.08, 136.47, 134.24,
130.97, 124.80, 123.83, 122.09, 120.75, 119.37, 114.14, 109.50, 108.75,
93.83, 92.26, 46.59, 42.30. HRMS-ESI-TOF: m/z calc C11H12ClN2O
(M þ 1) 223.0638, found 223.0633.
3. Conclusion
In conclusion, 3-methylene-indolinone-2-ones incorporating
a recognition moiety (Leu-iso-amyl) for FP-2 inhibition were shown
to display moderate inhibitory activity against the parasitic
cysteine protease as well as antiplasmodial activity against a chlo-
roquine-resistant strain of P. falciparum. Importantly, no FP-2
inhibition could be observed for compounds lacking the recogni-
tion moiety, consistent with interaction of the Leu-iso-amyl
sequence with the S2 pocket. Furthermore, the antiplasmodial
activity could be significantly improved when a 4-aminoquinoline
was coupled to the 3-methylene moiety of the indolinone-2-one
scaffold, suggesting that these 3-methylene-indolinone-2-ones are
potential lead compounds for the development of new antimalarial
agents.
4.1.1.3. 3-(N,N-Dimethylaminomethylene) indolin-2-one 1c. Obtained
in 77% yield, Z/E mixture 1.25/1. M.p. 224e226 ꢀC. IR (KBr) 2907,
1682, 1580, 1421, 1223, 1121 cmꢂ1 1H NMR (400 MHz, DMSO-d6)
d
10.19 (s, 1H, NH), 10.10 (s, 1H, NH), 7.61 (s, 1H), 7.44 (s, 1H), 7.35 (d,
1H, J ¼ 8.0 Hz), 7.26 (d,1H, J ¼ 8.0 Hz), 6.79e6.83 (m, 2H), 6.76 (d,1H,
J ¼ 2 Hz), 6.69 (d, 1H, J ¼ 2 Hz), 3.63 (s br, 3H), 3.30 (s, 6H), 3.26 (s br,
3H). 13C NMR (100 MHz, DMSO-d6)
d 170.76, 165.78, 149.41, 148.71,
139.01, 136.66, 127.82, 126.98, 125.62, 122.00, 121.02, 119.25, 118.90,
115.46, 108.32, 107.56, 93.79, 92.13, 46.60, 42.34. HRMS-ESI-TOF: m/z
calc C11H12ClN2O (M þ 1) 223.0638, found 223.0633.
4.1.1.4. 3-(N,N-Dimethylaminomethylene) indolin-2-one 1d. Obtained
in 83% yield, Z/E mixture 1.5/1. M.p. 230e232 ꢀC. IR (KBr) 3100,1670,
1587, 1414, 1223, 1127 cmꢂ1 1H NMR (400 MHz, DMSO-d6)
d 10.41 (s,
4. Experimental protocols
1H, NH), 10.34 (s, 1H, NH), 7.65 (s, 1H), 7.51 (s, 1H), 7.34 (d, 1H,
J ¼ 8.0 Hz), 7.24 (d, 1H, J ¼ 8.0 Hz), 6.96 (d, 1H, J ¼ 8.0 Hz), 6.77e6.88
(m, 3H), 3.66 (s br, 3H), 3.32 (s, 6H), 3.29 (s br, 3H). 13C NMR
4.1. Chemistry
(100 MHz, DMSO-d6) d 170.58, 165.68, 150.21, 149.31, 134.80, 132.62,
All reagents and solvents were obtained from commercial
suppliers and were used without further purification. Melting
points were determined using a Kofler camera Bock monoscope M
and are uncorrected. The infrared spectra were collected on
a Nicolet Impact 400 FTIR infrared spectrophotometer. High reso-
lution mass spectra (HMRS) were performed in Unidade de
Espectrometria de Masas, Santiago de Compostela. Low mass
spectra (MS) was performed in Faculdade de Farmácia, Lisbon.
Elemental analyses were carried out on a C. Erba Model 1106
(Elemental Analyzer for C, H and N) and the results are within
ꢁ0.4% of the theoretical values. Merck Silica Gel 60 F254 plates
were used as analytical TLC; flash column chromatography was
performed on Merck Silica Gel (200e400 mesh). 1H and 13C NMR
spectra were recorded on a Bruker 400 Ultra-Shield (400 MHz). 1H
130.77, 125.00, 122.29, 121.03, 120.80, 120.46, 118.52, 113.10, 112.92,
112.30, 94.30, 92.72, 46.70, 42.43. HRMS-ESI-TOF: m/z calc
C11H12ClN2O (M þ 1) 223.0638, found 223.0633.
4.1.1.5. 3-(N,N-Dimethylaminomethylene) indolin-2-one 1e. Obtained
in 90% yield, Z/E mixture 1.1/1. M.p. 58e60 ꢀC. IR (KBr) 3382, 1663,
1574, 1440, 1255, 1159 cmꢂ1 1H NMR (400 MHz, DMSO-d6)
d 7.61 (s,
1H), 7.51 (s, 1H), 7.43 (d, 1H, J ¼ 8 Hz), 7.33 (d, 1H, J ¼ 8 Hz), 7.02 (td,
1H, J ¼ 8.0 Hz, J ¼ 1.2 Hz), 6.94 (td, 1H, J ¼ 8.0 Hz, J ¼ 1.2 Hz),
6.82e6.90 (m, 4H), 3.65 (s br, 3H), 3.31 (s, 6H), 3.28 (s br, 3H), 3.18 (s,
3H), 3.16 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
d 169.40, 164.09,
149.15, 148.06, 139.15, 136.97, 127.48, 123.00, 122.16, 121.29, 120.32,
120.04, 119.94, 114.25, 107.31, 106.76, 93.88, 92.02, 43.99, 25.73,
25.62. HRMS-ESI-TOF: m/z calc C12H15N2O (M þ 1) 203.1184, found
203.1179.
and 13C chemical shifts are expressed in
d (ppm) referenced to the
solvent used and the proton coupling constants (J) in hertz.
4.1.2. Procedures for the preparation of indolin-2-one 3
4.1.1. General procedure for the preparation of 3-(N,N-
dimethylaminomethylene) indolin-2-ones 1aee
Method A: The benzyl mercaptane (0.223 mmol) was added to
a
solution of 3-(N,N-dimethylaminomethylene) indolin-2-one
DMF-DMA (2.66 mL, 20.0 mmol) was added to a solution of the
appropriate indole (0.78 mmol) in toluene (3 mL). The mixture was
stirred for 2e4 h. The precipitate was collected by filtration and
washed with dichloromethane.
(0.223 mmol) in acetic acid (3 mL) and the reaction mixture was
heated at 70 ꢀC for 12 h. After completion of reaction (monitor by
TLC after small workup), the acetic acid was evaporated and the
residue was purified by flash chromatography (hexane/ethyl
acetate 1:1). The final product was recrystallized in EtOAc and
Hexane, yielded 36%.
4.1.1.1. 3-(N,N-Dimethylaminomethylene) indolin-2-one 1a. Obtained
in 85% yield, Z/E mixture 1/1. M.p. 210e212 ꢀC. IR (KBr) 3112, 1670,
1580, 1427, 1229, 1114 cmꢂ1 1H NMR (400 MHz, DMSO-d6)
d
10.03 (s
Method B: The mixture of benzyl mercaptane (0.223 mmol), TEA
(0.397 mmol) in CH2Cl2 was stirred for 15 min at 0 ꢀC before
br, 1H, NH), 9.95 (s br, 1H, NH), 7.54 (s, 1H), 7.40 (s, 1H), 7.38 (d, 1H,

S. Praveen Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 927e933
931
addition of 3-(N,N-dimethylaminomethylene) indolin-2-one
(0.265 mmol), then was stired for 48 h at RT. After completion of
reaction (monitor by TLC after small workup), water was added and
washed with CH2Cl2, the organic layer was washed with brine and
Na2SO4 evaporated, afforded after purification by column chro-
matography (hexane/ethyl acetate 1:1) 50% yields.
Method C: The mixture of benzyl mercaptane (0.223 mmol), TEA
(0.397 mmol) in MeOH was stirred for 15 min at 0 ꢀC before
addition of 3-(N,N-dimethylaminomethylene) indolin-2-one
(0.265 mmol), then was stired for 12 h at RT. After completion of
reaction (monitor by TLC after small workup), evaporation the
methanol, recrystallization from ethyl acetate and hexane was
afforded 56% yields.
(d, 2H, J ¼ 4.0 Hz), 1.59 (t, 3H, J ¼ 8.0 Hz). 13C NMR (100 MHz, CDCl3)
d
169.13, 146.84, 135.39, 124.56, 123.58, 121.16, 115.59, 109.77, 61.91,
49.80, 14.16. HRMS-ESI-TOF (Mþ
247.1083, found 247.1077.
þ
1) calcd for C13H15N2O3
4.1.4.3. Indolin-2-one 5b. Obtained in 20% yield, Z/E mixture 10/1.
M.p. 146e148 ꢀC. IR (KBr) 3205, 3110, 1739, 1678 cmꢂ1. Z-isomer -
1H NMR (400 MHz, CDCl3)
d
9.01 (s br, 1H, J ¼ 8 Hz NH), 8.79 (s, 1H,
NH), 7.24e7.34 (m, 6H), 7.09e7.17(m, 3H), 7.04 (t, 1H, J ¼ 8.0 Hz),
6.94 (t, 2H, J ¼ 8.0 Hz), 4.25 (q, 2H, J ¼ 8.0 Hz), 3.32 (dd, 1H,
J ¼ 12.0 Hz, J ¼ 4.0 Hz), 3.13 (dd, 1H, J ¼ 12.0 Hz, J ¼ 8.0 Hz), 1.29 (t,
3H, J ¼ 8.0 Hz). 13C NMR (100 MHz, CDCl3)
d 170.70, 170.54, 144.90,
135.69, 134.63, 129.63, 128.93, 127.51, 124.73, 123.62, 120.98, 115.54,
109.66, 97.48, 63.07, 62.09, 40.23, 14.24. HRMS-ESI-TOF (Mþ þ 1)
calcd for C20H21N2O3 337.1552, found 337.1547.
4.1.2.1. Indolin-2-one 3. M.p. 171e173 ꢀC. IR (KBr) 3133, 1682, 1590,
670 cmꢂ1 1H NMR (400 MHz, CDCl3)
d 8.07 (s br, 1H, NH), 7.86 (s,
1H), 7.54 (d, 1H, J ¼ 8.0 Hz), 7.28e7.42 (m, 5H), 7.18 (t, 1H,
4.1.4.4. Indolin-2-one 7a. Obtained in 26% yield; Z/E mixture 10ꢂ/11.
Z-isomer - 1H NMR (400 MHz, CDCl3):
d 8.72e8.77 (m, 1H), 8.39 (s,
J ¼ 8.0 Hz), 7.03 (t, 1H, J ¼ 8.0 Hz), 6.85 (d, 1H, J ¼ 8.0 Hz), 4.25 (s.
M.p. 174e176 ꢀC. IR (KBr) 3450, 3024, 2928, 1675, 1606, 1211 cm
.
2H). 13C NMR (100 MHz, CDCl3)
d
167.28, 140.35, 139.81, 136.12,
129.12, 129.05, 128.95, 128.93, 128.24, 128.02, 123.88, 122.04,
1H), 7.43 (d, 1H, J ¼ 12.0 Hz), 7.25 (d, 1H, J ¼ 4.0 Hz), 7.06 (t, 1H,
J ¼ 8.0 Hz), 6.98 (t, 1H, J ¼ 8.0 Hz), 6.92 (d, 1H, J ¼ 8.0 Hz), 6.21 (s br,
1H), 3.89e3.94 (m, 1H), 3.27 (q, 2H, J ¼ 8.0 Hz), 1.86e1.89 (m, 1H),
1.71e1.74 (m, 2H), 1.55e1.59 (m, 1H), 1.37 (q, 2H, J ¼ 8.0 Hz), 0.97
(dd, 6H, J ¼ 8.0 Hz, J ¼ 4.0 Hz), 0.88 (dd, 6H, J ¼ 8.0 Hz, J ¼ 4.0 Hz).
109.52, 40.07. HRMS-ESI-TOF: m/z calc C16H14NOS (M
268.0796, found 268.0791.
þ
1)
4.1.3. General procedure for the preparation of indolin-2-ones
6aed
13C NMR (100 MHz, CDCl3):
d 171.74, 145.29, 135.65, 124.36, 124.16,
The amino acid (0.432 mmol) and BOP (191 mg, 0.432 mmol)
were dissolved in anhydrous THF (4 mL) and stirred with dry TEA
121.39, 115.95, 109.71, 98.36, 61.68, 41.77, 38.42, 38.27, 26.05, 24.84,
23.24, 22.54, 22.53, 21.46. Anal. Cald for C20H29N3O2: C, 69.94; H,
8.51; N, 12.23; O 9.32. Found: C, 69.35; H, 8.60; N, 11.76.
(57
mL, 57.4 mmol) for 30 min at room temperature. Then, a mixture
of isopentyl amine (37.7 mg, 0.432 mmol) and dry TEA (57
mL,
57.4 mmol) in THF was added under nitrogen atmosphere. The
resulting mixture was stirred for 24 h at room temperature. The
solvent was evaporated under reduced pressure. The residue was
dissolved in ethyl acetate (10 mL), washed with water (2 ꢃ 15 mL),
brine (10 mL), dried over Na2SO4, filtered and concentrated. The
product was isolated after column chromatography (hexane/ethyl
acetate 7:3) in yield 60e70%. Then, the product (0.5 mmol) was
dissolved in a 1:1 solution of TFA and DCM (1 mL). The reaction
mixture was stirred for 1 h at RT and evaporated under reduced
pressure. CCl4 (3 ꢃ 3 mL) was added and evaporated under reduced
pressure. This compound was used without further purification.
4.1.4.5. Indolin-2-one 7b. Obtained in 40% yield; Z/E mixture 20/1.
M.p. 173e175 ꢀC. IR (KBr) 3316, 3172, 2956, 1657 cmꢂ1; Z-isomer -
1H NMR (400 MHz, CDCl3):
d 8.82e8.88 (m, 1H), 8.12 (s, 1H), 7.45 (d,
1H, J ¼ 12.0 Hz), 7.24 (s, 1H), 7.02 (dd, 1H, J ¼ 8.0 Hz, J ¼ 4.0 Hz), 6.83
(d, 1H, J ¼ 8.0 Hz), 6.06 (s br, 1H), 3.89e3.93 (m, 1H), 3.28 (q, 2H,
J ¼ 8.0 Hz), 1.86e1.90 (m, 1H), 1.70e1.80 (m, 2H), 1.56e1.60 (m, 1H),
1.38 (q, J ¼ 8.0 Hz, 2H), 0.97 (dd, J ¼ 8.0 Hz, J ¼ 4.0 Hz, 6H), 0.89 (d,
J ¼ 4.0 Hz, 6H). 13C NMR (100 MHz, CDCl3): 171.49, 170.84, 146.08,
134.17, 126.39, 126.11, 123.25, 115.72, 110.52, 97.28, 61.33, 42.00,
38.31, 25.99, 24.71, 23.08, 22.46, 21.62. HRMS-ESI-TOF (Mþ þ 1)
calcd for C20H29ClN3O2 378.1948, found 378.1943.
4.1.4. General procedure for the preparation of indolin-2-ones 4, 5a,
5b, 7aei and 10aef
4.1.4.6. Indolin-2-one 7c. Obtained in 55% yield; Z/E mixture 20/1.
M.p. 193e195 ꢀC. IR (KBr) 3436, 3016, 2932, 1735, 1621 cmꢂ1; Z-
The appropriate amine (0.39 mmol) was added to a solution of
3-(N,N-dimethylaminomethylene) indolin-2-one (0.39 mmol) in
acetic acid (3 mL) and the reaction mixture was heated at 70 ꢀC for
12 h. After completion of reaction (monitor by TLC after small
workup), the acetic acid was evaporated and the residue was
purified by flash chromatography (hexane then hexane/ethyl
acetate 2:8 to 1:1). The final product was recrystallized in EtOAc
and hexane.
isomer- 1H NMR (400 MHz, CDCl3):
d 8.74e8.79 (m,1H, NH), 8.15 (s,
1H), 7.42 (d, 1H, J ¼ 12.0 Hz), 7.14 (d, 1H, J ¼ 8.0 Hz), 6.96 (dd, 1H,
J ¼ 8.0 Hz, J ¼ 1.5 Hz), 6.92 (s, 1H), 6.06 (s br, 1H), 3.88e3.93 (m, 1H),
3.25e3.31 (m, 2H), 1.80e1.90 (m, 1H), 1.69e1.74 (m, 3H), 1.56e1.57
(m, 1H), 1.38 (q, 2H, J ¼ 8.0 Hz), 0.97 (dd, 6H, J ¼ 8.0 Hz, J ¼ 4.0 Hz),
0.89 (d, 6H, J ¼ 8.0 Hz). 13C NMR (100 MHz, CDCl3): 171.43, 170.84,
145.58, 136.24, 129.41, 122.94, 121.40, 116.70, 110.08, 97.63, 61.68,
41.82, 38.45, 38.32, 26.08, 24.85, 23.23, 22.56, 21.52. HRMS-ESI-TOF
(Mþ þ 1) calcd for C20H29ClN3O2 378.1948, found 378.1943.
4.1.4.1. Indolin-2-one 4. Obtained in 60% yield, Z/E mixture 5/1.
M.p. 160e162 ꢀC. IR (KBr) 3230, 3113, 1672, 1572 cmꢂ1 1H NMR
4.1.4.7. Indolin-2-one 7d. Obtained in 52% yield; Z/E mixture 20/1.
M.p. 168e170 ꢀC. IR (KBr) 3285, 2928, 1681, 1613 cmꢂ1; Z-isomer -
(400 MHz, MeOD)
J ¼ 8.0 Hz), 7.38 (m, 2H), 7.26 (d, 2H, J ¼ 8.0 Hz), 6.91e7.10 (m, 4H).
13C NMR (100 MHz, MeOD)
192.52, 141.51, 139.43, 138.23, 131.20,
d 8.34 (s, 1H, NH), 7.79 (s, 1H, NH), 7.49 (d, 1H,
1H NMR (400 MHz, CDCl3):
d 8.87e8.92 (m, 1H, NH), 8.41 (d, 1H,
d
J ¼ 8.0 Hz), 7.48 (d, 1H, J ¼ 12.0 Hz), 7.14 (d, 1H, J ¼ 8.0 Hz), 7.04 (d,
1H, J ¼ 8.0 Hz), 6.92 (t, 1H, J ¼ 8.0 Hz), 6.19 (s br, 0.5H, NH),
5.98e6.00 (m, 0.5H, NH), 3.91e3.93 (m, 1H), 3.74e3.76 (m, 2H),
1.61e1.90 (m, 3H), 1.55e1.60 (m, 1H), 1.32e1.41 (m, 2H), 0.96 (dd,
6H, J ¼ 8.0 Hz, J ¼ 4.0 Hz), 0.88 (d, 6H, J ¼ 8.0 Hz). 13C NMR
(100 MHz, CDCl3): 171.39, 170.47, 146.41, 132.93, 125.86, 123.58,
122.14, 114.98, 114.13, 98.22, 68.11, 61.66, 41.81, 38.42, 38.31, 26.06,
24.83, 23.21, 22.54, 21.51. HRMS-ESI-TOF (Mþ þ 1) calcd for
C20H29ClN3O2 378.1948, found 378.1943.
125.67, 125.02, 122.58, 121.71, 117.94, 117.24, 110.99, 24.69. HRMS-
ESI-TOF (Mþ þ 1) calcd for C15H13N2O 237.1028, found 237.1148.
4.1.4.2. Indolin-2-one 5a. Obtained in 29% yield, Z/E mixture 10/1.
M.p. 162e164 ꢀC. IR (KBr) 3420, 3250, 1710, 1680 cmꢂ1. Z-isomer -
1H NMR (400 MHz, CDCl3)
d 9.15 (s br, 1H, NH), 9.00 (s br, 1H, NH),
7.60 (d, 1H, J ¼ 12.0 Hz), 7.55 (s, 1H), 7.48 (d, 1H, J ¼ 8.0 Hz), 7.33 (t,
1H, J ¼ 8.0 Hz), 7.26 (t, 1H, J ¼ 8.0 Hz), 4.54 (q, 2H, J ¼ 8.0 Hz), 4.40

932
S. Praveen Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 927e933
4.1.4.8. Indolin-2-one 7e. Obtained in 55% yield; Z/E mixture 20/1.
M.p. 183e185 ꢀC. IR (KBr) 3292, 3022, 2971, 1673, 1622 cmꢂ1
Z-isomer - 1H NMR (400 MHz, CDCl3):
8.72e8.78 (m, 1H), 7.43 (d,
NH), 8.39 (d,1H, J ¼ 4 Hz), 8.27 (d,1H, J ¼ 8 Hz), 7.90 (d,1H, J ¼ 12 Hz),
7.79 (s, 1H), 7.45e7.47 (m, 1H), 7.38 (s br, 1H, NH), 7.25 (d, 1H,
J ¼ 8 Hz), 6.75e6.90 (m, 3H), 6.49 (d,1H, J ¼ 4 Hz), 3.48 (m, 2H), 3.33
;
d
1H, J ¼ 12.0 Hz), 7.27 (d, 1H, J ¼ 4.0 Hz), 7.11 (td, 1H, J ¼ 8.0 Hz,
J ¼ 4.0 Hz), 7.04 (t, J ¼ 8.0 Hz, 1H), 6.84 (d, 1H, J ¼ 8.0 Hz), 6.44 (s br,
1H), 3.91e3.96 (m 1H), 3.27 (s, 3H), 3.26e3.32 (m, 2H), 1.88e1.91
(m,1H),1.71e1.74 (m, 2H),1.58e1.60 (m,1H),1.39 (q, 2H, J ¼ 8.0 Hz),
0.95 (dd, 6H, J ¼ 8.0 Hz, J ¼ 4.0 Hz), 0.87 (dd, 6H, J ¼ 8.0 Hz,
J ¼ 2.0 Hz). 13C NMR (100 MHz, CDCl3): 171.89, 169.13, 144.62,
138.23, 124.07, 123.37, 121.31, 115.62, 107.91, 98.26, 61.60, 41.75,
38.42, 38.21, 26.01, 25.59, 24.83, 23.27, 22.53, 22.51, 21.36. HRMS-
ESI-TOF (Mþ þ 1) calcd for C21H32N3O2 358.2495, found 358.2481.
(m, 2H), 1.98 (m, 2H). 13C NMR (100 MHz, DMSO)
d 169.82, 151.73,
150.01, 148.79, 147.80, 135.56, 133.49, 127.11, 125.31, 124.09, 124.04,
122.04, 119.67, 117.90, 114.98, 108.71, 98.73, 94.72, 46.08, 39.08,
29.43. HRMS-EI: calcd for C21H19ClN4O 378.1247, found 378.1248.
4.1.4.14. Indolin-2-one 10b. Obtained in 52% yield; Z/E ꢂm1ixture 2/1.
M.p. 278e280 ꢀC. IR (KBr) 3287, 1668, 1560, 1456 cm
;
1H NMR
(400 MHz, MeOD):
d
8.31 (d, 1H, J ¼ 4 Hz,1H), 8.11 (d, 1H, J ¼ 12 Hz),
7.77 (s, 1H), 7.70 (d, 1H, J ¼ 2 Hz), 7.39 (dd, 1H, J ¼ 12, 4 Hz), 7.11 (d,
1H, J ¼ 2 Hz), 6.89 (dd, 1H, J ¼ 12, 4 Hz), 6.78 (s, 1H), 6.67 (d, 1H,
J ¼ 8 Hz), 3.61 (m, 4H), 2.13 (m, 2H). 13C NMR (100 MHz, MeOD)
4.1.4.9. Indolin-2-one 7f. Obtained in 50% yield; Z/E mixture 3/1.
M.p. 203e205 ꢀC. IR (KBr) 3318, 3022, 2958, 1667, 1603 cmꢂ1
;
d 171.74, 154.67, 150.13, 148.37, 138.29, 135.12, 128.21, 127.16, 127.13,
Z-isomer - 1H NMR (400 MHz, DMSO-d6):
d
10.18 (s, 1H, NH),
124.86, 124.11, 123.19, 119.36, 117.93, 116.03, 111.13, 99.67, 95.97,
48.22, 41.88, 30.14. HRMS-EI: calcd for C21H19Cl2N4O 412.0858,
found 412.0848.
8.76e8.83 (m, 1H), 8.01e8.04 (m, 1H), 7.80 (d, 1H, J ¼ 13.12 Hz), 7.26
(d,1H, J ¼ 8.0 Hz), 6.76e6.97 (m, 3H), 3.99 (d, 2H, J ¼ 8.0 Hz), 3.11 (q,
2H, J ¼ 8.0 Hz), 1.55e1.62 (m, 1H), 1.32 (q, 2H, J ¼ 8.0 Hz), 0.87 (d,
6H, J ¼ 8.0 Hz). 13C NMR (100 MHz, DMSO-d6):
d
169.59, 168.14,
4.1.4.15. Indolin-2-one 10c. Obtained in 62% yield; Z/E mixture 2/1;
mp: 265e267 ꢀC. IR (KBr): Cmꢂ1 3292, 3216, 1679,1586, 1450, 1220;
147.98, 135.91, 125.28, 122.32, 119.77, 115.10, 108.69, 95.40, 50.22,
38.08, 36.90, 25.13, 22.40. Anal. Cald for C16H21N3O2: C, 66.88; H,
7.37; N, 14.62. Found: C, 66.49; H, 7.25; N, 14.18.
1H NMR (400 MHz, DMSO-d6):
d 10.33 (s, 1H, NH), 8.95 (t br, 1H,
J ¼ 8 Hz, NH), 8.39 (d, 1H, J ¼ 4 Hz), 8.26 (d, 1H, J ¼ 8 Hz), 7.97 (d, 1H,
J ¼ 12 Hz), 7.79 (s,1H), 7.44 (d,1H, J ¼ 12 Hz), 7.40 (s br,1H, NH), 7.24
(d, 1H, J ¼ 8 Hz), 6.87 (d, 1H, J ¼ 8 Hz), 6.76 (d, 1H, J ¼ 8 Hz), 6.49 (d,
1H, J ¼ 4 Hz), 3.48 (m, 2H), 3.33 (m, 2H), 1.98 (m, 2H); 13C NMR
4.1.4.10. Indolin-2-one 7g. Obtained in 35% yield; Z/E mixture 10/1.
M.p. 134e136 ꢀC. IR (KBr) 3312, 2941, 1661, 1593 cmꢂ1; Z-isomer -
1HNMR (400 MHz, CDCl3):
d
8.81e8.86 (m, 1H, NH), 8.14 (s br, 1H,
(101 MHz, DMSO-d6) d 169.58, 151.82, 150.18, 149.00, 148.70, 136.67,
NH), 7.36 (d, 1H, J ¼ 12.0 Hz), 7.19e7.31 (m, 6H), 7.08 (t, 1H,
J ¼ 8.0 Hz), 7.01 (t, 1H, J ¼ 8.0 Hz), 6.94 (d, 1H, J ¼ 8.0 Hz), 6.13 (s br,
1H), 3.75e3.80 (m, 1H), 3.26 (q, 2H, J ¼ 8.0 Hz), 2.82e2.85 (m, 1H),
2.70e2.73 (m, 1H), 2.46e2.48 (m, 1H), 2.09e2.13 (m, 1H), 1.50e1.56
(m, 1H), 1.37 (q, 2H, J ¼ 8.0 Hz), 0.88 (d, 6H, J ¼ 8.0 Hz). 13C NMR
(100 MHz, CDCl3): 170.88, 170.59, 145.25, 139.86, 135.52, 128.65,
128.56, 126.40, 124.13, 121.33, 115.87, 109.57, 98.43, 62.13, 38.31,
38.17, 34.07, 31.71, 25.93, 22.42. Anal. Cald for C24H29N3O2: C, 73.63;
H, 7.47; N, 10.73. Found: C, 73.84; H, 7.53; N, 10.45.
133.51, 127.54, 126.01, 124.37, 124.15, 119.43, 119.22, 117.51, 116.14,
108.57, 98.82, 93.79, 46.34, 39.52, 29.37. HRMS-ESI-TOF (Mþ þ 1)
calcd for C21H19Cl2N4O 413.0936, found 413.0930.
4.1.4.16. Indolin-2-one 10d. Obtained in 56% yield; Z/E ꢂm1ixture 2/1.
;
1H NMR
M.p. 123e125 ꢀC. IR (KBr) 3275, 1672, 1568, 1461 cm
(400 MHz, DMSO-d6)
d
10.58 (s,1H, NH), 9.04 (t br,1H, J ¼ 8 Hz, NH),
8.39 (d, 1H, J ¼ 4 Hz), 8.26 (d, 1H, J ¼ 8 Hz), 8.02 (d, 1H, 12 Hz), 7.79
(s,1H), 7.47 (d,1H, J ¼ 8 Hz), 7.39 (s br, 1H, NH), 7.22 (d,1H, J ¼ 8 Hz),
6.91 (d,1H, J ¼ 8 Hz), 6.84 (d,1H, J ¼ 8 Hz), 6.49 (d,1H, J ¼ 4 Hz), 3.51
(m, 2H), 3.35 (m, 2H), 1.99 (m, 2H); 13C NMR (100 MHz, CDCl3)
4.1.4.11. Indolin-2-one 7h. Obtained in 40% yield; Z/E mixture 3/1;
M.p. 242e244 ꢀC. IR (KBr) 3436, 3024, 1729, 1681 cmꢂ1; Z-isomer e
d
168.89, 151.42, 149.55, 148.75, 148.64, 133.00, 132.20, 126.94,
1H NMR (400 MHz, DMSO-d6):
d
10.32 (s, 1H), 9.00e9.05 (m, 1H,
126.91, 123.62, 123.57, 121.01, 120.46, 117.04, 112.97, 112.61, 98.17,
NH), 8.22 (t, 1H, J ¼ 8.0 Hz,), 7.82 (d, 1H, J ¼ 12.0 Hz), 7.18e7.31 (m,
6H), 6.89 (dd, 1H, J ¼ 8.0 Hz, J ¼ 4.0 Hz), 6.74 (d, 1H, J ¼ 8.0 Hz),
4.22e4.27 (m,1H), 2.99e3.15 (m, 4H),1.44e1.49 (m,1H),1.25 (q, 2H,
J ¼ 8.0 Hz), 0.84 (d, 6H, J ¼ 4.0 Hz). 13C NMR (100 MHz, DMSO-d6):
169.44, 169.37, 147.16, 136.70, 134.48, 129.46, 128.30, 127.05, 126.63,
124.21, 121.67, 115.04, 109.82, 94.84, 62.01, 37.86, 36.82, 24.94,
22.36, 22.34. Anal. Cald for C23H26ClN3O2: C, 67.06; H, 6.36; N,10.20.
Found: C, 66.87; H, 5.96; N, 9.81.
93.85, 45.96, 39.40, 28.76. MS-ESI m/z: C21H19Cl2N4O 412.96 (Mþ).
4.1.4.17. Indolin-2-one 10e. Obtained in 70% yield; Z/Eꢂm1ixture 10/
1. M.p. 63e65 ꢀC. IR (KBr) 3275, 1681, 1575, 1445 cm
;
1H NMR
(400 MHz, CDCl3):
d
8.80 (t br, 1H, J ¼ 8 Hz, NH), 8.19 (s, 1H, NH),
8.02 (d, 1H, J ¼ 8 Hz), 7.94 (d, 1H, J ¼ 8 Hz), 7.70 (s, 1H), 7.47 (d, 1H,
J ¼ 12 Hz), 7.18 (d, 1H, J ¼ 8 Hz), 7.16 (d, 1H, J ¼ 8 Hz), 7.08 (t, 1H,
J ¼ 8 Hz), 6.97 (t, 1H, J ¼ 8 Hz), 6.84 (d, 1H, J ¼ 8 Hz), 6.07 (d, 1H,
J ¼ 4 Hz), 3.52 (m, 2H), 3.40 (m br, 2H), 3.29 (s, 3H), 2.05 (m, 2H); 13C
4.1.4.12. Indolin-2-one 7i. Obtained in 30% yield; Z/E mixture 20/1.
NMR (100 MHz, CDCl3) d 168.98, 153.14, 146.47, 145.55, 142.30,
M.p. 66e68 ꢀC. IR (KBr) 3304, 3019, 1681, 1609 cmꢂ1; Z-isomer 1
H
137.66, 137.55, 126.32, 123.93, 123.84, 123.30, 122.69, 121.18, 115.99,
115.13, 107.80, 97.63, 96.01, 46.78, 40.63, 29.39, 25.66. HRMS-EI:
calcd for C22H21ClN4O 392.1404, found 392.1403.
NMR (400 MHz, CDCl3): d 9.29 (s br, 1H, NH), 9.12e9.18 (m, 1H, NH),
7.48 (d, 1H, J ¼ 12.0 Hz), 7.26e7.30 (m, 3H), 7.17e7.21 (m, 3H), 7.13
(d, 1H, J ¼ 8.0 Hz,), 7.04 (d, 1H, J ¼ 8.0 Hz), 6.92 (t, 1H, J ¼ 8.0 Hz),
6.46e6.48 (m, 1H), 3.86e3.92 (m, 1H), 3.25e3.30 (m, 2H),
2.65e2.85 (m, 2H), 2.40e2.50 (m, 1H), 2.12e2.16 (m, 1H), 1.55e1.58
(m, 1H), 1.39 (q, J ¼ 8.0 Hz, 2H), 0.88 (d, 6H, J ¼ 4.0 Hz). 13C NMR
(100 MHz, CDCl3): 170.80, 170.29, 146.64, 139.91, 133.06, 128.70,
128.54, 126.45, 125.95, 123.45, 122.04, 115.03, 114.00, 98.07, 62.09,
38.32, 38.30, 34.35, 31.70, 25.98, 22.46. HRMS-ESI-TOF (Mþ þ 1)
calcd for C24H29ClN3O2 426.1948, found 426.1943.
4.1.4.18. Indolin-2-one 10f. Obtained in 68% yield; Z/Eꢂm1 ixture 3/1.
;
1H NMR
M.p. >300 ꢀC. IR (KBr) 3269, 1682, 1559, 1430 cm
(400 MHz, DMSO-d6):
d
10.55 (s, 1H, NH), 9.09 (t, 1H, J ¼ 8 Hz, NH),
8.41 (d, 1H, J ¼ 8 Hz), 8.25 (d, 1H, J ¼ 8 Hz), 7.94 (d, 1H, J ¼ 12 Hz),
7.79 (s, 1H), 7.47 (s br, 1H, NH), 7.45 (d, 1H, J ¼ 8 Hz), 7.13 (d, 1H,
J ¼ 4 Hz), 6.89 (d, 1H, J ¼ 8 Hz), 6.82 (t, 1H, J ¼ 8 Hz), 6.65 (d, 1H,
J ¼ 4 Hz), 3.65 (m br, 2H), 3.52 (m br, 2H); 13C NMR (100 MHz,
DMSO-d6)
d 169.18, 151.99, 150.01, 149.08, 133.46, 132.67, 127.64,
4.1.4.13. Indolin-2-one 10a. Obtained in 40% yield; Z/E mixtuꢂre1 2/1.
127.42, 124.25, 124.03, 121.51, 120.91, 117.50, 113.38, 113.08, 112.70,
99.16, 94.63, 46.91, 43.51. HRMS-EI: calcd for C20H16Cl2N4O
398.0701, found 398.0704.
mp: 133e135 ꢀC. IR (KBr): 3275, 3207, 1652, 1592, 1449 cm
;
1H
NMR (400 MHz, DMSO-d6):
d
10.18 (s,1H, NH), 8.88 (t br,1H, J ¼ 8 Hz,

S. Praveen Kumar et al. / European Journal of Medicinal Chemistry 46 (2011) 927e933
933
4.2. Pharmacology
minimal deviation of its atomic coordinates (RMSD < 2 Å). Since the
validation study revealed that our model and software protocol
could accurately reproduce the experimentally binding mode of the
E-64 inhibitor, a series of synthesized 3-methylene-substituted
indolinones were covalently docked into the falcipain-2 binding
site using the Goldscore fitness function [22].
4.2.1. Falcipain-2 and -3 assays
Both enzymes were assayed at 25 ꢀC using Z-Leu-Arg-AMC as
substrate at a concentration of 25
6.0, 5 mM DTT, 0.75% DMSO. One microliter of the compound dis-
solved in DMSO was diluted into 100 L of assay buffer. Fifty
mM in 100 mM sodium acetate pH
m
microliters of the enzyme in assay buffer were added and the
resulting mixture was incubated for 10 min. The reaction was
Acknowledgments
initiated by addition of 50
substrate and read immediately in a Fluoroskan Ascent microplate
spectrofluorometer. Every compound was assayed at eight different
mL of assay buffer containing the
This work was supported by Fundação para a Ciência e a Tec-
nologia (Portugal) through project PTDC/SAU-FCF/098734/2008,
project REDE/1518/REM/2005 and postdoctoral fellowship to S.P.K..
R.M. and P.J.R. acknowledge FLAD for financial support through the
Luso-American Collaborative Response Awards. P.J.R. is a Distin-
guished Clinical Scientist of the Doris Duke Charitable Foundation.
concentrations, with 50
mM being the highest inhibitor concen-
tration; all other concentrations were created by exponential five-
fold dilution. The IC50 values were determined by plotting the
percentage of inhibition relative to the control reaction in the
absence of inhibitor over the logarithmic compound concentration
by using the equation v/v0 ¼ 1/(1 þ 10log[I]/IC50). Data analysis was
done with the program GraphPad Prism 4 (GraphPad Software).
References
[1] A. Martinelli, R. Moreira, P.V.L. Cravo, Mini Rev. Med. Chem.
8 (2008)
201e212.
[2] D.A. Fidock, R.T. Eastman, S.A. Ward, S.R. Meshnick, Trends Parasitol. 24 (2008)
537e544.
[3] M. Schlitzer, Arch. Pharm. Chem. Life Sci. 341 (2008) 149e163.
[4] C. Valente, R.C. Guedes, R. Moreira, J. Iley, J. Gut, P.J. Rosenthal, Bioorg. Med.
Chem. Lett. 16 (2006) 4115e4119.
[5] I.D. Kerr, J.H. Lee, K.C. Pandey, A. Harrison, M. Sajid, P.J. Rosenthal, L.S. Brinen,
J. Med. Chem. 52 (2009) 852e857.
[6] R. Ettari, F. Bova, M. Zappalà, S. Grasso, N. Micale, Med. Res. Rev. 30 (2010)
136e167.
[7] M.M.M. Santos, R. Moreira, Mini Rev. Med. Chem. 7 (2007) 1040e1050.
[8] R. Capela, R. Oliveira, L.M. Gonçalves, A. Domingos, J. Gut, P.J. Rosenthal,
F. Lopes, R. Moreira, Bioorg. Med. Chem. Lett. 19 (2009) 3229e3232.
[9] A.S. Newton, L.M. Gonçalves, P.M.C. Glória, D.J.V.A. dos Santos, R. Moreira,
R.C. Guedes, M.M.M. Santos, Eur. J. Med. Chem. 45 (2010) 3858e3863.
[10] A. Arora, E.M. Scholar, J. Pharmacol. Exp. Ther. 315 (2005) 971e979.
[11] E.R. Wood, L. Kuyper, K.G. Petrov, R.N. Hunter, P.A. Harris, K. Lackey, Bioorg.
Med. Chem. Lett. 14 (2004) 953e957.
4.2.2. In vitro antiplasmodial activity in human red blood cells
Human red blood cells infected with 1% ring stage P. falciparum
strains synchronized with 5% sorbitol were incubated with tested
compounds (stock solutions in DMSO) in 96 well plates at 37 ꢀC for
48 h in RPMI-1640 medium, supplemented with 25 mM HEPES pH
7.4, 10% heat inactivated human serum (or 0.5% Albumax, 2%
human serum), and 100 uM Hypoxanthine under an atmosphere of
3% O2, 5% CO2, 91% N2. After 48 h the cells were fixed in 2% HCHO in
PBS, transferred into PBS with 100 mM NH4Cl, 0.1% Triton X-100,
1 nM YOYO-1, and then analyzed in a flow cytometer (FACSort,
Beckton Dickinson; EX 488 nm, EM 520 nm) IC50 were calculated
using GraphPad PRISM software.
[12] H.N. Bramson, J. Corona, S.T. Davis, S.H. Dickerson, M. Edelstein, S.V. Frye,
R.T. Gampe Jr., P.A. Harris, A. Hassell, W.D. Holmes, R.N. Hunter, K.E. Lackey,
B. Lovejoy, M.J. Luzzio, V. Montana, W.J. Rocque, D. Rusnak, L. Shewchuk,
J.M. Veal, D.H. Walker, L.F. Kuyper, J. Med. Chem. 44 (2001) 4339e4353.
[13] K.-C. Luk, M.E. Simcox, A. Schutt, K. Rowan, T. Thompson, Y. Chen,
U. Kammlott, W. DePinto, P. Dunten, A. Dermatakis, Bioorg. Med. Chem. Lett.
14 (2004) 913e917.
[14] C.L. Woodard, Z. Li, A.K. Kathcart, J. Terrell, L. Gerena, M. Lopez-Sanchez,
D.E. Kyle, A.K. Bhattacharjee, D.A. Nichols, W. Ellis, S.T. Prigge, J.A. Geyer,
N.C. Waters, J. Med. Chem. 46 (2003) 3877e3882.
4.3. Computational studies
Molecular docking studies were performed, using the GOLD
software [20] (version 5) to predict the interactions and binding
modes of 7 series of synthesized inhibitors in the falcipain-2 active
site, and to evaluate their relative binding affinities. The 3D struc-
ture coordinates of falcipain-2 were obtained after a search in the
Protein Data Bank and electing the structure with code 3BPF with
a resolution of 2.9 Å [5]. To prepare the enzyme for the docking
studies, the E-64 inhibitor included in the 3BPF structure was
removed, hydrogen atoms were added to falcipain-2, crystallo-
graphic waters were removed and the protonation states were
correctly assigned using the Protonate-3D tool within the Molec-
ular Operating Environment (MOE) 2009.10 software package [21].
To evaluate and validate the effectiveness of our model system and
the performance of the docking protocol, a set of studies was first
performed using the co-crystallized inhibitor to reproduce the
experimental binding pose. The best docking pose obtained was
[15] M. Sassatelli, E. Debiton, B. Aboab, M. Prudhomme, P. Moreau, Eur. J. Med.
Chem. 41 (2006) 709e716.
[16] F. Bouchikhi, E. Rossignol, M. Sancelme, B. Aboab, F. Anizon, D. Fabbro,
M. Prudhomme, P. Moreau, Eur. J. Med. Chem. 43 (2008) 2316e2322.
[17] M. Abass, B.B. Mostafa, Bioorg. Med. Chem. 13 (2005) 6133e6144.
[18] C.C. Musonda, D. Taylor, J. Lehman, J. Gut, P.J. Rosenthal, K. Chibale, Bioorg.
Med. Chem. Lett. 14 (2004) 3901e3905.
[19] I. Chiyanzu, E. Hansell, J. Gut, P.J. Rosenthal, J.H. McKerrow, K. Chibale, Bioorg.
Med. Chem. Lett. 13 (2003) 3527e3530.
[20] (a) G. Jones, P. Willett, R.C. Glen, J. Mol. Biol. 245 (1995) 43e53;
(b) G. Jones, P. Willett, R.C. Glen, A.R. Leach, R. Taylor, J. Mol. Biol. 267 (1997)
727e748.
[21] http://www.chemcomp.com/software-moe2009.htm.
[22] http://www.ccdc.cam.ac.uk/support/documentation/gold/3_2/doc/portable_
html/gold_portable-3-077.html.
overlaid with the experimental crystal structure showing
a