Azide-alkyne cycloaddition en route to novel 1H-1,2,3-triazole tethered isatin conjugates with in vitro cytotoxic evaluation

Article abstract of DOI:10.1016/j.ejmech.2012.06.057

1H-1,2,3-triazole tethered isatin conjugates have been synthesized and evaluated for cytotoxicity on four human cancer cell lines. The results revealed 5a, 5c, 5e and 5n proved to be twice as potent as 5-fluorouracil on THP-1 cell line with 5a and 5c being most active exhibiting IC₅₀ values of <1 against all cell lines except Caco-2. Activity profiles showed dependence on the substituents on isatin rings with a preference for hydrogen while a strong electron withdrawing fluoro as well as nitro substituents on either ring decreased the anticancer activity.

Full text of DOI:10.1016/j.ejmech.2012.06.057

European Journal of Medicinal Chemistry 55 (2012) 455e461
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Azide-alkyne cycloaddition en route to novel 1H-1,2,3-triazole tethered isatin
conjugates with in vitro cytotoxic evaluation
a
b
a
b
c
c
Pardeep Singh , Pooja Sharma , Amit Anand , P.M.S. Bedi , Tandeep Kaur , A.K. Saxena ,
Vipan Kumar
a
a
,
*
Department of Chemistry, Guru Nanak Dev University, Amritsar 143005, India
Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar 143005, India
Cancer Pharmacology Division, Indian Institute of Integrative Medicine, Jammu 180001, J & K, India
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 March 2012
Received in revised form
1H-1,2,3-triazole tethered isatin conjugates have been synthesized and evaluated for cytotoxicity on four
human cancer cell lines. The results revealed 5a, 5c, 5e and 5n proved to be twice as potent as
5-fluorouracil on THP-1 cell line with 5a and 5c being most active exhibiting IC50 values of <1 against all
8
June 2012
cell lines except Caco-2. Activity profiles showed dependence on the substituents on isatin rings with
a preference for hydrogen while a strong electron withdrawing fluoro as well as nitro substituents on
either ring decreased the anticancer activity.
Accepted 28 June 2012
Available online 7 July 2012
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Click chemistry
Isatin conjugates
1
H-1,2,3-Triazole
Anticancer evaluation
Structureeactivity relationship
1. Introduction
Australian mollusk Dicathais orbita has been reported as a cytotoxic
marine compound [6]. 6-bromo isatin, a major decomposition
The isatin (1H-indole-2,3-dione) moiety is a privileged scaffold
with wide possibility for chemical modification and responsible for
a broad spectrum of biological properties in many synthetically
versatile molecules [1]. Among these properties, cytotoxic and
antineoplastic activities of this moiety have been found to be
interesting and mechanistically can be associated with its affinity to
inhibit tyrosine kinase [2] and serine/threonine-specific protein
kinases such as the cyclic-dependent kinases (CDKs) [3] and
carbonic anhydrase isozymes (CAIs) [4]. Literature rationale
reveals that in particular, halogenated isatin derivatives have
been reported to exhibit enhanced anticancer activity compared to
the parent molecule. 5-Bromo-3-o-nitrophenyl isatin hydrazone
product formed through the oxidation of tyriverdin (precursor of
Tyrian purple), has been shown to have a weaker anticancer activity
against a human lymphoma cell line in comparison with 6-bromo-
2-methylthio-3H-indol-3-one [7]. Moreover, N-benzylation of
5,7-dibromoisatin further increased the cytotoxicity against U937
(human monocyte-like histiocytic lymphoma) cells [8] targeting of
microtubules and activity against a range of human cancer cell lines
including a metastatic breast adenocarcinoma cell line (MDA-MB-
231) [9]. Isatin-pyrazoline and isatin-4-thiazolidinone conjugates
have been recently targeted as prospective anticancer agents with
highly selective influence on the leukemia subpanel and moderate
selectivity toward the CNS and renal cancer subpanels [10]. In the
and
a
series of 5-bromo-(2-oxo-3-indolinyl) thiazolidine-
recently approved drugs by FDA a 5-fluoro-3-substituted-2-
2
,4-diones substituted by various Mannich bases were found to
oxoindole, SU11248 (Sutent), is provided for the treatment of
gastrointestinal stromal tumors and advanced renal-cell carcinoma
[11,12].
exhibit anticancer activity against Walker carcinoma-256 and
P388 lymphocytic leukemia in mice, respectively [5]. 6-Bromo-2-
methylthio-3H-indol-3-one, tyrindoleninone,
precursor to Tyrian purple, isolated from the egg masses of the
a
brominated
Over the past few years, 1,2,3-triazoles have attracted the
attention of organic and medicinal chemists because of their varied
biological properties including anti-allergic, antibacterial, anti-
fungal, anti-HIV, anticonvulsant, anti-inflammatory, antitubercular
and anticancer activities [13]. The favorable properties of
1,2,3-triazole ring like moderate dipole character, hydrogen
*
Corresponding author. Tel.: þ91183 2258802 09x3320; fax: þ91183 2258819 20.
E-mail address: vipan_org@yahoo.com (V. Kumar).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.057

456
P. Singh et al. / European Journal of Medicinal Chemistry 55 (2012) 455e461
bonding capability, rigidity and stability under in vivo conditions are
evidently responsible for their enhanced biological activities [14].
Recent revelation from our lab has shown the synthesis and
in vitro cytotoxic evaluation of a series of N-alkyl bromo and
N-alkylphthalimido isatins against four human cancer cell lines
compounds were prepared in DMSO. The stock solutions were
serially diluted with complete growth medium containing 50 g/
mL of gentamycin to obtain working test solutions of required
concentrations.
In vitro cytotoxicity against human cancer cell lines of different
tissues was determined [18,19] using 96-well tissue culture plates.
m
[15]. As an extension to this approach and our interest in the
synthesis of biologically imperative frameworks with medicinal
potential [16], the present manuscript explicates the synthesis of
novel 1H-1,2,3-triazole tethered isatin conjugates via azide-alkyne
cycloaddition as elucidated in Fig. 1 along with their anticancer
evaluation on four human cancer cell lines.
The 100 ml of cell suspension was added to each well of the 96-well
tissue culture plate. The cells were allowed to grow in carbon
ꢀ
dioxide incubator (37 C, 5% CO
2
, 90% RH) for 24 h. Test materials in
l) were added after 24 h of incu-
complete growth medium (100
m
bation to the wells containing cell suspension. The plates were
further incubated for 48 h in a carbon dioxide incubator. The cell
growth was stopped by gently layering trichloroacetic acid (50%,
2
. Synthetic chemistry and pharmacology
5
0
m
l) on top of the medium in all the wells. The plates were
ꢀ
2.1. Synthetic chemistry
incubated at 4 C for 1 h to fix the cells attached to the bottom of the
wells. The liquid of all the wells was gently pipetted out and dis-
carded. The plates were washed five times with distilled water to
remove trichloroacetic acid, growth medium low molecular weight
metabolites, serum proteins etc and air-dried. The plates were
The procedure for the synthesis of N-alkyl azido isatin deriva-
tives 3 was based on a combination of the literature methods [17]
involving an initial base-assisted N-alkylation of substituted
isatins with dibromoethane to yield the corresponding N-alkyl
bromo isatins 2, followed by subsequent reaction with sodium
stained with sulforhodamine B dye (0.4% in 1% acetic acid, 100 ml)
for 30 min. The plates were washed five times with 1% acetic acid
and then air-dried [19]. The adsorbed dye was dissolved in TriseHCl
ꢀ
azide in DMF at 60 C. The second precursor, substituted
N-propargylated isatins
4
were prepared by base assisted
Buffer (100 ml, 0.01 M, pH 10.4) and plates were gently stirred for
propargylation as shown in Scheme 1. The target compounds 5
were synthesized by utilizing azide-alkyne cycloaddition
reaction of appropriate precursors 3 and 4 in the presence of
10 min on a mechanical stirrer. The optical density (OD) was
recorded on ELISA reader at 540 nm. The cell growth was deter-
mined by subtracting mean OD value of respective blank from the
mean OD value of experimental set. Percent growth in presence of
test material was calculated considering the growth in absence of
any test material as 100% and in turn percent growth inhibition in
presence of test material was calculated.
CuSO
4 2
$5H O/sodium ascorbate in ethanol:water mixture (Scheme
1
). The purification of the reaction mixture, after usual work up,
via column chromatography resulted in the isolation of desired
scaffolds, the structures to which were assigned on the basis of
spectral data and analytical evidences.
3. Results and discussion
2
2
.2. Pharmacology
The synthesized 1,2,3-triazole tethered isatin conjugates were
.2.1. Anticancer evaluation
The human cancer cell lines were procured from National
evaluated for their anticancer activity against four human cancer cell
lines viz. A-549(lung), PC-3(prostate), THP-1(leukemia) and Caco-
2(colon) using sulforhodamine B assay. The concentration depen-
dent cytotoxicity of these scaffolds against human cancer cell lines is
shown in Table 1. Paclitexal has been used as standard in case of lung
(A-549), 5-fluorouracil in case of THP-1(leukemia) and Caco-
2(colon) while mitomycin in case of PC-3(prostate). The analysis of
Table 1 revealed interesting structure activity relationship (SAR) and
substituent effect with regard to C-5 position of isatin rings A and B
and considerable growth inhibition at low concentrations. As
evident from Table 1, the test compound 5a has shown almost
Cancer Institute, Frederick, PO Box B, Frederick, MD 21702-1201,
U.S.A. Cells were grown in tissue culture flasks in complete growth
medium (RPMI-1640 medium with 2 mM glutamine, pH 7.4, sup-
plemented with 10% fetal calf serum, 100
m
g/mL streptomycin and
ꢀ
100 units/mL penicillin) in a carbon dioxide incubator (37 C, 5%
2
CO , 90% RH). The cells at subconfluent stage were harvested from
the flask by treatment with trypsin [0.05% in PBS (pH 7.4) con-
taining 0.02% EDTA]. Cells with viability of more than 98% as
determined by trypan blue exclusion, were used for determination
5
of cytotoxicity. The cell suspension of 1 ꢁ10 cells/mL was prepared
complete growth inhibition on all cell lines except Caco-2 at 10 mM
concentration and was 2e3 folds more active than 5-fluorouracil on
ꢂ2
in complete growth medium. Stock solutions (2 ꢁ 10
M) of
O
R
A
O
N
O
O
R
A
N3
N
N
N
N
O
N
R1
B
O
R1
O
B
O
N
Fig. 1. General structure of lead compound and target hybrid compounds.

P. Singh et al. / European Journal of Medicinal Chemistry 55 (2012) 455e461
457
O
O
O
R
R
R
(
i)
(ii)
O
O
O
N
N
N
H
1
2
3
N3
Br
O
R1
R
(
iii)
O
N
4
O
O
A
N N
N
N
N
R1
B
O
O
5
1
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
a. R=H, R =H
1
b. R=H, R =Br
1
c. R=H, R =Cl
1
d. R=H, R =F
1
e. R=H, R =CH
3
1
f. R=H, R =NO
2
1
g. R=F, R =H
1
h. R=F, R =Br
1
i. R=F, R =Cl
1
j. R=F, R =F
1
k. R=F, R =CH
3
1
l. R=F, R =NO
2
1
m. R=Cl, R =Br
1
n. R=Cl, R =CH
3
1
o. R=Cl, R =NO
2
ꢀ
ꢀ
Scheme 1. Synthesis of isatin-conjugates: (i) 1,2-dibromoethane, NaH, DMF, 60 C, 2 h; (ii) sodium azide, DMF, 60 C, 3 h; (iii) sodium ascorbate, CuSO
4
$5H
2
O, ethanol:H
2
O, 8 h.
THP-1 cell line while compounds 5c, 5e and 5n were twice as potent
as 5-flouorouracil.The compound 5c has also shown considerable
growth inhibition on A-549 and PC-3 cell lines while 5b and 5n
proved to be active against PC-3 and A-549 cell lines at low
concentration. These preliminary results at low concentration indi-
cated that the synthesized scaffold 5a proved to be the most active of
the series showing a marked preference for hydrogen substituent on
both the rings. The activity profiles of 5b, 5c and 5e also corroborates
toward the preference of H-substituent on ring A while the poor
activity exhibited by 5d and 5f might be attributed to the presence of
strong electron withdrawing fluoro and nitro substituents on ring B.
Careful scrutinizing of Table 1 proved the attribution of poor activity
to electron withdrawing fluoro and nitro substituents on ring A as
evident by the activity profiles of compounds 5gel. The reduction in
cytotoxic profile with the introduction of electron withdrawing
substituents has further authenticated by the poor activity profiles
5e (A-549 and PC-3), 5f (A-549, PC-3, THP-1) and 5n (A-549 and
THP-1). The compound 5g and 5k showed a significant increase in
cytotoxic activity against A-549 and THP-1 cell lines while 5f
showed the similar enhancements in case of A-549, PC-3 and THP-1
cell lines. The compound 5b showed 96% and 99% growth inhibition
on A-549 and PC-3 cell lines respectively at 50
mM while
compounds 5h and 5m showed a significant increase in cytotoxic
activity against A-549 and THP-1 cell lines. The series of
compounds having fluoro substituent on ring A (5gel) however
does not show any marked improvement of anticancer profile at
50
compound 5h (R ¼ R ¼ F) and 5l (R ¼ F, R ¼ NO
improvement in cytotoxic profiles. A further increase in concen-
tration to 100 M showed complete growth inhibition in most of
mM except in case of 5h (A-549 and THP-1) and 5k (THP-1). The
1
1
2
) showed least
m
the compounds especially 5aee in all cancer cell lines tested except
Caco-2 while 5h, 5i, 5k, 5m and 5n showed almost complete
growth inhibition (97e99%) on A-549 cell lines (Fig. 2). The IC50
values, which is the concentration required to inhibit 50% of the cell
viability by the test compounds after exposure to cells, of the test
compounds were calculated and the results are summarized in
Table 2.
1
1
observed in case of 5j (R ¼ R ¼ F) and 5l (R ¼ F, R ¼ NO
2
); both being
least active in the series.
The evaluation studies at 50 mM further improved the cytotoxic
profiles in case of all the test compounds, the effects being more
pronounced in case 5b (A-549, PC-3), 5d (A-549, PC-3 and THP-1),

458
P. Singh et al. / European Journal of Medicinal Chemistry 55 (2012) 455e461
Table 1
Table 2
In vitro cytotoxicity against human cancer cell lines.
IC50 determination of cytotoxicity of compounds against human cancer cell lines.
Compound Cell lines
Lung
A-549
IC50
<1
Compound
Concentration Cell lines
A-549 PC-3 THP-1 Caco-2 C log Pa [20]
Age growth inhibition
96
Prostate
PC-3
Leukemia
THP-1
Colon
%
Caco-2
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
10
99
100
100
94
99
100
99
100
100
20
98
100
63
100
100
24
97
99
0
32
82
0
2
51
0
4
9
0
10
30
0
31
48
2
11
33
29
43
70
36
55
70
30
44
72
ND
ND
60
100
100
100
10
53
88
80
90
100
10
96
100
89
100
100
11
97
100
8
9
20
36
1
10
25
6
24
38
0
1
22
0
0
17
0
4
18
0
1
10
0
7
13
2
12
30
0
0.148
1.189
1.039
0.469
0.647
0.274
0.469
1.511
1.361
0.791
0.968
0.595
2.081
1.538
1.165
(mM)
5
00
10
0
98
99
41
96
97
94
98
100
11
97
99
35
99
100
13
96
98
6
17
32
41
79
98
64
73
97
5
23
62
8
57
99
7
22
64
10
83
98
81
95
98
11
80
99
69
ND
ND
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
<1
<1
<1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
32.2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
14.56
<1
51.86
<1
<1
50
d
e
f
g
h
i
j
k
l
38.9
18.39
39.2
30.1
3.03
30.7
71.1
>100
>100
>100
85.2
95.2
51.4
32.6
53.2
39.9
<1
00
10
42.8
88.1
27.6
36.3
83.3
38.75
86.2
26.9
<1
50
>100
00
10
16.39
2.65
90.7
49.9
93.2
42.2
<1
d
e
f
50
00
10
50
m
n
o
00
10
44.9
>100
39.3
26.0
50
00
10
5
00
10
5
00
10
5
00
10
5
00
10
5
00
10
5
00
10
5
00
10
5
g
h
i
0
15
90
26
85
94
20
70
92
1
13
92
14
88
89
2
Among the four human cancer cell lines used, most of the
compounds showed activity on all cancer cell lines except Caco-2
with the effect more pronounced against THP-1 followed by
A-549 and PC-3 cell lines respectively. The tested compounds
proved to be inactive against Caco-2 cell lines except 5m and 5n
0
0
with IC50 values of 32.2 and 44.9
analogs in the series proved to be 5a and 5c exhibiting IC50 values of
1 against A-549, PC-3 and THP-1 cell lines followed by 5n with
mM respectively. The most active
j
0
0
10
0
15
44
0
<
IC50 values of 0.03, 0.48, 32.6 and 44.9
PC-3 and Caco-2 cell lines respectively.
mM against A-549, THP-1,
k
l
0
In conclusion, the present manuscript explicates the synthesis
of novel 1H-1,2,3-triazole tethered isatin conjugates and their
evaluation against a panel of four human cancer cell lines. The
preliminary studies have shown that most of these compounds are
active on all cancer cell lines except Caco-2 with activity showing
dependence on C-5 substituent of the synthesized scaffolds. The
compound 5a, 5c, 5e and 5n proved to be twice as potent as
0
14
90
28
82
90
71
91
92
29
82
88
ND
70
ND
0
0
m
n
o
36
57
68
39
52
56
17
21
33
ND
68
ND
0
0
5-fluorouracil on THP-1 cell line. These preliminary results indicate
00
10
5
00
1
a marked dependence of cytotoxicity on the presence of substit-
uent on the isatin ring with a distinct preference for hydrogen
substituent. The presence of a strong electron withdrawing fluoro
as well as nitro substituents markedly decreased the activity
profiles with compound 5j and 5l being the least active of the test
compounds. Based on these observations, the compound 5a may
be considered as a good hit in terms of %age growth inhibition, IC50
and partition coefficient C log P (0.148) values. Further studies in
order to generalize substantiate and improve the activity profiles
of the scaffolds are underway in the lab and will soon be
communicated.
0
Paclitaxel
-Flourouracil
5
20
1
Mitomycin-C
a
Calculated using Chem Draw ultra 10.0; ND ¼ Not Determined.
4. Experimental section
Melting points were determined by open capillary using Veego
Precision Digital Melting Point apparatus (MP-D) and are uncor-
rected. IR spectra were recorded on a Shimadzu D-8001 spectro-
1
photometer. H NMR spectra were recorded in deuterochloroform
6
and dimethylsulfoxide d with Jeol 300 (300 MHz) and BRUKER
AVANCE II (400 MHz) spectrometers using TMS as internal stan-
dard. Chemical shift values are expressed as parts per million
downfield from TMS and J values are in hertz. Splitting patterns are
indicated as s: singlet, d: doublet, t: triplet, m: multiplet, dd: double
doublet, ddd: doublet of a doublet of a doublet, and br: broad peak.
13
Fig. 2. Comparison of activity of test compounds on four cancer cell line at 100
mM.
C NMR spectra were recorded on Jeol 300 (75 MHz)

P. Singh et al. / European Journal of Medicinal Chemistry 55 (2012) 455e461
459
spectrometers in deuterochloroform using TMS as internal stan-
dard. Mass spectra were recorded on Shimadzu GCMS-QP-2000
mass spectrometer. Elemental analyses were performed on Her-
aus CHN-O-Rapid Elemental Analyzer. Column chromatography
was performed on a silica gel (60e120 mesh).
which was recrystallized using chloroform:methanol (10:2)
mixture.
4.4.1. 1-{1-[2-(2,3-Dioxo-2,3-dihydro-indol-1-yl)-ethyl]-1H-[1,2,3]
triazol-4-ylmethyl}-1H-indole-2,3-dione (5a)
ꢀ
1
Orange solid; yield: 71%; m.p: 124e126 C. H NMR (300 MHz,
CDCl ): ), 4.67e4.71
ppm ¼ 4.45e4.48 (t, J ¼ 5.4 Hz, 2H, CH
t, J ¼ 5.4 Hz, 2H, CH ), 4.92 (s, 2H, CH ), 7.10e7.15 (m, 3H, ArH),
.28e7.59 (m, 5H, ArH), 8.22 (s, 1H, H-triazole ring). C NMR
75 MHz, CDCl ):
ppm ¼ 35.4, 41.7, 48.1, 112.9, 115.6, 116.3, 118.5,
21.1, 123.6, 123.8, 125.2, 127.6, 137.7, 139.5, 139.9, 141.2, 144.1, 156.5,
3
d
2
4.1. Typical procedure for the synthesis of substituted N-alkyl
(
2
2
bromo isatins (2)
13
7
d
(
3
d
To a stirred suspension of sodium hydride (1.5 mmol) in dry
DMF (10 mL) was added isatin (1 mmol), resulting in the formation
of purple colored anion. The solution was stirred at room temper-
ature till the evolution of hydrogen ceases. To this reaction mixture
was then added drop wise a solution of dibromoethane (1.1 mmol)
1
1
ꢂ1
58.1, 181.4, 182.0. IR (cm ): 1749, 1732, 1648, 1627, 1512. MS (EI)
þ
m/z: 401 (M ). Calcd for C21
15 5 4
H N O : C, 62.84; H, 3.77; N, 17.45.
Found C, 63.04; H, 3.99; N, 17.23.
ꢀ
in DMF. The reaction mixture was heated to 60 C with constant
4
1
.4.2. 5-Bromo-1-{1-[2-(2,3-dioxo-2,3-dihydro-indol-1-yl)-ethyl]-
stirring for about 2 h. After the completion of reaction, as evident by
TLC, it was quenched by drop wise addition of water (20 mL) and
subsequently extracted with ethyl acetate (3 ꢁ 30 mL). The
combined organic layers were washed with brine solution, dried
H-[1,2,3]triazol-4-ylmethyl}-1H-indole-2,3-dione (5b)
ꢀ
1
Yellow solid; yield: 78.6%; m.p: 112e114 C. H NMR (400 MHz,
CDCl ): ), 4.71e4.73
ppm ¼ 4.55e4.57 (t, J ¼ 5.5 Hz, 2H, CH
), 4.98 (s, 2H, CH ), 7.17e7.49 (m, 4H, ArH),
3
d
2
d
(
7
t, J ¼ 5.5 Hz, 2H, CH
2
2
2 4
over anhydrous Na SO and concentrated under reduced pressure.
.59e7.61 (dd, 1H, J ¼ 3.6, 8.4 Hz, ArH), 7.72e7.74 (d, 1H, J ¼ 8.4 Hz,
Purification of the reaction mixture via column chromatography
using hexane:ethyl acetate (8:2) mixture furnished the desired
N-alkyl bromo derivatives in good yields.
ArH), 7.77e7.78 (d, 1H, J ¼ 3.6 Hz, ArH), 7.83 (s, 1H, H-triazole ring).
1
3
C NMR (75 MHz, CDCl
3
):
d
ppm ¼ 35.5, 42.1, 48.6, 113.9, 115.7,
117.4, 119.5, 122.1, 123.6, 124.8, 127.0, 128.7, 138.2, 140.4, 141.9, 144.7,
ꢂ1
149.6, 157.5, 158.9, 181.6, 182.4. IR (cm ): 1757, 1738, 1633, 1607,
4.2. Typical procedure for the synthesis of substituted N-alkyl azido
þ
1517. MS (EI) m/z: 480 (M ). Calcd for C21
5 4
H14BrN O : C, 52.52; H,
isatins (3)
2.94; N, 14.58. Found C, 52.71; H, 3.05; N, 14.53.
To the stirred suspension of N-alkyl bromo isatin in dry DMF was
4
1
.4.3. 5-Chloro-1-{1-[2-(2,3-dioxo-2,3-dihydro-indol-1-yl)-ethyl]-
added NaN
3
(1.5 mmol) and the reaction mixture was heated at
H-[1,2,3]triazol-4-ylmethyl}-1H-indole-2,3-dione (5c)
ꢀ
6
0
C for about 3 h (monitored by TLC). Upon completion, the
ꢀ
1
Orange solid; yield: 68.7%; m.p: 138e140 C. H NMR (400 MHz,
CDCl ): ), 4.71e4.73 (t,
ppm ¼ 4.55e4.57 (t, J ¼ 5.7 Hz, 2H, CH
J ¼ 5.7 Hz, 2H, CH ), 4.98 (s, 2H, CH ), 7.20e7.44 (m, 4H, ArH),
.53e7.56 (dd, 1H, J ¼ 3.6, 8.0 Hz, ArH), 7.56e7.58 (m, 2H, ArH), 7.72
reaction mixture was extracted with ethyl acetate and the organic
layer was washed with brine, dried over anhydrous Na SO and
3
d
2
2
4
2
d
2
concentrated under reduced pressure to yield N-alkyl azido isatin
derivatives in good yields.
7
13
(
4
1
s, 1H, H-triazole ring). C NMR (75 MHz, CDCl
2.9, 49.1, 114.3, 115.9, 117.7, 120.7, 122.8, 124.3, 125.1, 127.7, 129.4,
38.9, 141.2, 141.7, 142.4, 145.6, 156.8, 157.8, 181.1, 183.5. IR
3
):
d
ppm ¼ 36.1,
4
.3. Typical procedure for the synthesis of substituted N-propargy
lated isatins (4)
ꢂ1
þ
(cm ):1763, 1744,1654,1613,1515. MS (EI) m/z: 436 (M ). Calcd for
C
21
H
14ClN : C, 57.87; H, 3.24; N, 16.07. Found C, 58.02; H, 3.41; N,
5 4
O
To a stirred suspension of sodium hydride (1.5 mmol) in dry
DMF (10 mL) was added isatin (1 mmol), resulting in the formation
of purple colored anion. Solution was stirred at room temperature
till the evolution of hydrogen ceases followed by the drop wise
addition of a solution of propargyl bromide (1.5 mmol) in DMF. The
reaction mixture was stirred at room temperature for about 2 h and
on completion, as evident by TLC, was quenched by drop wise
addition of water (20 mL) and extracted with ethyl acetate
15.87.
4.4.4. 1-{1-[2-(2,3-Dioxo-2,3-dihydro-indol-1-yl)-ethyl]-1H-[1,2,3]
triazol-4-ylmethyl}-5-fluoro-1H-indole-2,3-dione (5d)
Orange solid; yield: 70.56%; m.p: 107e109 C. 1H NMR
300 MHz, CDCl ): ),
ppm ¼ 4.53e4.56 (t, J ¼ 5.7 Hz, 2H, CH
.75e4.77 (t, J ¼ 5.7 Hz, 2H, CH ), 4.94 (s, 2H, CH ), 7.19e7.48
ꢀ
(
4
3
d
2
2
2
(m, 5H, AreH), 7.52e7.53 (d, 1H, J ¼ 3.3 Hz, ArH), 7.71e7.73
(
(
(
3 ꢁ 30 mL). The combined organic layers were washed with brine
13
d, 1H, J ¼ 8.1 Hz, ArH), 7.78 (s, 1H, H-triazole ring). C NMR
solution, dried over anhydrous Na SO and concentrated under
2
4
75 MHz, CDCl
3
):
d
ppm ¼ 37.9, 43.9, 49.8, 116.1, 117.4, 119.3, 121.5,
reduced pressure. Purification of the reaction mixture via column
chromatography using hexane:ethyl acetate (8:2) mixture fur-
nished the desired N-propargylated isatin derivatives in good
yields.
1
1
23.9, 125.9, 127.1, 129.3, 131.4, 140.6, 142.8, 143.3, 145.3, 147.7, 160.1,
ꢂ1
63.6, 182.9, 184.4. IR (cm ): 1788, 1759, 1658, 1622, 1512. MS (EI)
þ
m/z: 420 (M ). Calcd for C21
Found C, 60.01; H, 3.49; N, 16.58.
5 4
H14FN O : C, 60.14; H, 3.36; N, 16.70.
4.4. Typical procedure for the synthesis of triazole tethered
4.4.5. 1-{1-[2-(2,3-Dioxo-2,3-dihydro-indol-1-yl)-ethyl]-1H-[1,2,3]
functionalized isatin-conjugates (5)
triazol-4-ylmethyl}-5-methyl-1H-indole-2,3-dione (5e)
ꢀ
1
Orange solid; yield: 71.3%; m.p: 128e130 C. H NMR (300 MHz,
CDCl ): ), 4.51e4.54 (t, J ¼ 5.7 Hz, 2H, CH ),
ppm ¼ 2.35 (s, 3H, CH
4.70e4.73 (t, J ¼ 5.7 Hz, 2H, CH ), 4.91 (s, 2H, CH ), 7.03e7.06 (d, 1H,
J ¼ 7.8 Hz, ArH), 7.19e7.45 (m, 5H, ArH), 7.65 (s, 1H, ArH), 8.15 (s, 1H,
To the stirred solution of N-propargylated isatin (1 mmol) and
N-alkyl azido isatin (1 mmol) in ethanol:water (10:1) was added in
succession copper sulfate (0.055 mmol) and sodium ascorbate
3
d
3
2
2
2
1
3
(
0.143 mmol) at room temperature. On completion of the reaction,
H-triazole ring); C NMR (75 MHz, CDCl
3
):
d
ppm ¼ 24.5, 40.7, 47.5,
as monitored by TLC, water (15 mL) was added to the reaction
mixture and extracted with chloroform (2 ꢁ 50 mL). Combined
organic layers were dried over anhydrous sodium sulfate and
concentrated under reduce pressure to result in a crude product
50.1, 110.9, 114.8, 115.7, 117.5, 120.5, 123.1, 123.6, 124.6, 127.0, 136.6,
ꢂ1
138.2, 139.5, 140.2, 143.4, 155.3, 157.8, 181.3, 182.0. IR (cm ):1766,
þ
1728, 1639, 1617, 1515. MS (EI) m/z: 416 (M ). Calcd for C22
H
17
N
5
O
4
:
C, 63.61; H, 4.12; N, 16.86. Found C, 63.87; H, 4.21; N, 16.73.

460
P. Singh et al. / European Journal of Medicinal Chemistry 55 (2012) 455e461
4.4.6. 1-{1-[2-(2,3-Dioxo-2,3-dihydro-indol-1-yl)-ethyl]-1H-[1,2,3]
4.4.11. 1-{2-[4-(2,3-Dioxo-2,3-dihydro-5-methyl-indol-1-ylmethyl)-
triazol-4-ylmethyl}-5-nitro-1H-indole-2,3-dione (5f)
[1,2,3]triazol-1-yl]-ethyl}-5-fluoro-1H-indole-2,3-dione (5k)
ꢀ
1
ꢀ
1
Orange solid; yield: 71%; m.p: 126e128 C. H NMR (300 MHz,
CDCl ): ), 4.69e4.72
ppm ¼ 4.19e4.21 (t, J ¼ 5.4 Hz, 2H, CH
t, J ¼ 5.7 Hz, 2H, CH ), 4.91 (s, 2H, CH ), 7.19e7.71 (m, 4H, ArH),
.77 (s, 1H, H-triazole ring); 8.10e8.51 (m, 3H, ArH); C NMR
75 MHz, CDCl ):
ppm ¼ 37.9, 43.7, 49.9, 117.9, 118.7, 119.5, 121.9,
23.7, 126.5, 128.8, 129.5, 131.7, 140.7, 142.9, 143.8, 145.9, 147.5,
Orange solid; yield: 69%; m.p: 119e121 C. H NMR (300 MHz,
3
d
2
CDCl
3
):
d
ppm ¼ 2.31 (s, 3H, CH
3
), 4.44e4.46 (t, J ¼ 5.4 Hz, 2H, CH
2
),
(
2
d
2
4.57e4.59 (t, J ¼ 5.4 Hz, 2H, CH
2
), 4.78 (s, 2H, CH ), 7.21e7.25 (dd,
d
2
13
7
1H, J ¼ 2.8, 8.1 Hz, ArH), 7.32e7.35 (dd, 1H, J ¼ 2.8, 8.4 Hz, ArH),
7.50e7.53 (d, 1H, J ¼ 2.8 Hz, ArH), 7.59e7.81 (m, 3H, ArH), 8.03
(
3
d
13
1
(s, 1H, H-triazole ring). C NMR (75 MHz, CDCl
3
):
d
ppm ¼ 22.3,
ꢂ1
158.3, 160.7, 183.7, 185.3. IR (cm ):1767, 1738, 1643, 1627, 1517. MS
46.0, 49.7, 52.1, 108.7, 112.6, 113.4, 115.8, 118.7, 121.1, 122.6, 123.3,
125.0, 134.9, 137.1, 137.2, 139.0, 142.8, 153.3, 155.2, 181.1, 182.8. IR
þ
(
EI) m/z: 446 (M ). Calcd for C21
14 6 6
H N O : C, 56.51; H, 3.16; N, 18.83;
ꢂ1
þ
Found C, 56.65; H, 3.11; N, 18.91.
(cm ):1744, 1731, 1638, 1627, 1516. MS (EI) m/z: 434 (M ). Calcd for
: C, 60.97; H, 3.72; N, 16.16. Found C, 61.13; H, 3.85; N,
22 5 4
C H16FN O
4.4.7. 1-{2-[4-(2,3-Dioxo-2,3-dihydro-indol-1-ylmethyl)-[1,2,3]
15.98.
triazol-1-yl]-ethyl}-5-fluoro-1H-indole-2,3-dione (5g)
ꢀ
1
Orange solid; yield: 76%; m.p: 108e110 C. H NMR (300 MHz,
CDCl ): ), 4.64e4.67
ppm ¼ 4.16e4.19 (t, J ¼ 5.4 Hz, 2H, CH
t, J ¼ 5.7 Hz, 2H, CH ), 4.90 (s, 2H, CH ), 6.33e6.37 (dd, 1H,
J ¼ 3.6, 8.4 Hz, ArH), 7.09e7.58 (m, 6H, AreH), 7.70 (s, 1H, H-triazole
4.4.12. 1-{2-[4-(2,3-Dioxo-2,3-dihydro-5-nitro-indol-1-ylmethyl)-
[1,2,3]triazol-1-yl]-ethyl}-5-fluoro-1H-indole-2,3-dione (5l)
3
d
2
ꢀ
(
2
d
2
Orange solid; yield: 64%; m.p: 132e134 C.
d
ppm ¼ 4.00e4.02
), 4.85
), 7.25e7.29 (dd, 1H, J ¼ 2.8, 8.1 Hz, ArH), 7.50e7.82
(m, 2H, ArH), 8.00 (s, 1H, H-triazole ring); 8.10e8.45 (m, 3H,
(t, J ¼ 5.1 Hz, 2H, CH
2
), 4.49e4.52 (t, J ¼ 5.1 Hz, 2H, CH
2
d
13
ring); C NMR (75 MHz, CDCl
3
):
d
ppm ¼ 37.6, 42.9, 49.0, 116.5,
(s, 2H, CH
2
1
18.1, 119.6,121.7, 123.2,126.4, 128.6, 129.3, 131.4, 140.6,142.8, 143.3,
ꢂ1
13
1
1
45.3, 147.7, 158.4, 160.3, 183.2, 184.9. IR (cm ):1767, 1738, 1643,
ArH). C NMR (75 MHz, CDCl
3
):
d
ppm ¼ 45.9, 47.0, 49.9,
þ
627, 1517. MS (EI) m/z: 420 (M ). Calcd for C21
H
14FN
5
O
4
: C, 60.14;
109.6, 110.9, 111.1, 117.9, 119.6, 121.5, 123.8, 129.3, 135.7, 137.0,
ꢂ1
H, 3.36; N, 16.70. Found C, 60.27; H, 3.31; N, 16.62.
141.4, 145.5, 146.8, 149.9, 158.0, 159.7, 180.3, 181.2. IR (cm ): 1737,
724, 1635, 1621, 1513. MS (EI) m/z: 464 (M ). Calcd for
þ
1
4.4.8. 1-{2-[4-(2,3-Dioxo-2,3-dihydro-5-bromo-indol-1-ylmethyl)-
21 6 6
C H13FN O : C, 54.32; H, 2.82; N, 18.10; Found C, 54.23; H, 3.01;
[
1,2,3]triazol-1-yl]-ethyl}-5-fluoro-1H-indole-2,3-dione (5h)
N, 18.22.
Yellow-orange solid; yield: 77%; m.p: 105e107 C. 1H NMR
400 MHz, CDCl ): ),
ppm ¼ 4.07e4.09 (t, J ¼ 5.2 Hz, 2H, CH
.60e4.63 (t, J ¼ 5.2 Hz, 2H, CH ), 4.87 (s, 2H, CH ), 6.74e6.77 (dd,
H, J ¼ 2.8, 8.8 Hz, ArH), 7.02e7.04 (d,1H, J ¼ 8.4 Hz, ArH), 7.20e7.25
ꢀ
(
4
1
3
d
2
4.4.13. 1-{2-[4-(2,3-Dioxo-2,3-dihydro-5-bromo-indol-1-ylmethyl)-
[1,2,3]triazol-1-yl]-ethyl}-5-chloro-1H-indole-2,3-dione (5m)
2
d
2
ꢀ
1
Orange solid; yield: 74.7%; m.p: 122e124 C. H NMR (300 MHz,
CDCl ): ), 4.47e4.50
ppm ¼ 4.41e4.44 (t, J ¼ 5.7 Hz, 2H, CH
(t, J ¼ 5.7 Hz, 2H, CH ), 4.61 (s, 2H, CH ), 7.51e7.54 (dd, 1H,
J ¼ 3.6, 8.4 Hz, ArH), 7.72e7.80 (m, 4H, ArH), 7.96 (d, 1H, J ¼ 3.6 Hz,
(
dd, 1H, J ¼ 2.8, 8.8 Hz, ArH), 7.35e7.37 (d, 1H, J ¼ 8.8 Hz, ArH),
3
d
2
7
8
4
.70e7.71 (d, 1H, J ¼ 2.8 Hz, ArH), 7.96e7.97 (d, 1H, J ¼ 2.8 Hz, ArH),
2
2
13
.19 (s, 1H, H-triazole ring). C NMR (75 MHz, CDCl
9.5, 49.7, 111.1, 111.4, 113.2, 115.1, 118.0, 119.0, 123.6, 123.9, 124.6,
26.6, 139.8, 141.2, 146.3, 148.8, 157.1, 158.0, 181.7, 182.1. IR
3
):
d
ppm ¼ 46.1,
13
ArH), 8.13 (s, 1H, H-triazole ring); C NMR (75 MHz, CDCl
ppm ¼ 46.3, 49.2, 52.0, 108.9, 111.7, 112.6, 116.2, 119.4, 120.7, 123.5,
124.3, 125.6, 132.9, 136.8, 137.9, 140.4, 141.6, 153.8, 156.2, 181.6,
3
):
1
d
ꢂ1
þ
(
cm ):1754, 1728, 1720, 1648, 1518. MS (EI) m/z: 498 (M ). Calcd
for C21 13BrFN : C, 50.62; H, 2.63; N, 14.06. Found C, 50.81; H,
.76; N, 13.93.
ꢂ1
þ
H
5
O
4
182.0. IR (cm ):1756, 1744, 1624, 1622, 1513. MS (EI) m/z: 514 (M ).
Calcd for C21 13BrClN : C, 49.00; H, 2.55; N,13.61. Found C, 49.13;
H, 2.71; N, 13.49.
2
H
5 4
O
4.4.9. 1-{2-[4-(2,3-Dioxo-2,3-dihydro-5-chloro-indol-1-ylmethyl)-
[
1,2,3]triazol-1-yl]-ethyl}-5-fluoro-1H-indole-2,3-dione (5i)
4.4.14. 1-{2-[4-(2,3-Dioxo-2,3-dihydro-5-methyl-indol-1-ylmethyl)-
[1,2,3]triazol-1-yl]-ethyl}-5-chloro-1H-indole-2,3-dione (5n)
Orange solid; yield: 70.5%; m.p: 124e126 C. 1H NMR
ꢀ
1
Orange solid; yield: 72%; m.p: 120e122 C. H NMR (300 MHz,
CDCl ): ), 4.66e4.69
ppm ¼ 4.14e4.17 (t, J ¼ 5.4 Hz, 2H, CH
t, J ¼ 5.4 Hz, 2H, CH ), 4.88 (s, 2H, CH ), 6.80e6.83 (dd, 1H,
J ¼ 2.4, 8.4 Hz, ArH), 7.11e7.13 (d, 1H, J ¼ 8.4 Hz, ArH), 7.23e7.27 (dd,
H, J ¼ 2.8, 8.4 Hz, ArH), 7.42e7.44 (d, 1H, J ¼ 8.4 Hz, ArH),
.80e7.84 (m, 2H, ArH), 8.21 (s, 1H, H-triazole ring); 13C NMR
75 MHz, CDCl ):
ppm ¼ 46.9, 49.8, 50.7, 110.9, 112.8, 113.9, 119.8,
21.2, 123.9, 125.2, 131.9, 137.4, 138.2, 143.7, 146.1, 147.9, 148.4, 159.2,
ꢀ
3
d
2
(
2
d
2
(300 MHz, CDCl
J ¼ 5.7 Hz, 2H, CH
CH
), 7.38e7.41 (dd, 1H, J ¼ 3.6, 8.4 Hz, ArH), 7.53e7.56 (dd, 1H,
J ¼ 3.6, 8.4 Hz, ArH), 7.71e7.80 (m, 4H, ArH), 7.99 (s, 1H, H-triazole
3
):
d
ppm ¼ 2.11 (s, 3H, CH
3
), 4.23e4.27 (t,
2
), 4.46e4.49 (t, J ¼ 5.7 Hz, 2H, CH
2
), 4.58 (s, 2H,
1
7
(
1
2
1
3
3
d
ring); C NMR (75 MHz, CDCl
3
):
d
ppm ¼ 21.3, 45.9, 49.2, 52.3,
109.2, 112.3, 112.8, 116.5, 118.9, 121.7, 123.2, 123.8, 125.5, 135.4,
137.8, 138.4, 139.6, 143.1, 153.9, 155.7, 181.9, 182.3. IR (cm ): 1738,
1732, 1616, 1609, 1510. MS (EI) m/z: 450 (M ). Calcd for
ꢂ1
ꢂ1
1
60.9, 181.9, 182.2. IR (cm ): 1748, 1730, 1640, 1630, 1515. MS (EI)
þ
þ
m/z: 454 (M ). Calcd for C21
H13ClFN
5
O
4
: C, 55.58; H, 2.89; N, 15.43.
Found C, 55.71; H, 3.01; N, 15.22.
C
22
H
16ClN
5 4
O : C, 58.74; H, 3.59; N, 15.57. Found C, 58.63; H, 3.71;
N, 15.49.
4.4.10. 1-{2-[4-(2,3-Dioxo-2,3-dihydro-5-flouro-indol-1-ylmethyl)-
[
1,2,3]triazol-1-yl]-ethyl}-5-fluoro-1H-indole-2,3-dione (5j)
4.4.15. 1-{2-[4-(2,3-Dioxo-2,3-dihydro-5-nitro-indol-1-ylmethyl)-
ꢀ
1
Orange solid; yield: 65%; m.p: 110e112 C. H NMR (300 MHz,
CDCl ): ), 4.46e4.49
ppm ¼ 4.01e4.03 (t, J ¼ 5.1 Hz, 2H, CH
t, J ¼ 5.1 Hz, 2H, CH ), 4.83 (s, 2H, CH ), 7.23e7.27 (dd, 1H,
J ¼ 2.8, 8.1 Hz, ArH), 7.32e7.36 (dd, 1H, J ¼ 2.8, 8.1 Hz, ArH),
.50e7.82 (m, 4H, ArH), 8.00 (s, 1H, H-triazole ring). 13C NMR
75 MHz, CDCl ):
ppm ¼ 45.5, 47.6, 49.7, 109.4, 110.8, 111.4, 117.5,
19.8, 121.7, 123.4, 129.2, 135.6, 137.9, 141.3, 145.1, 146.9, 149.8, 158.5,
[1,2,3]triazol-1-yl]-ethyl}-5-chloro-1H-indole-2,3-dione (5o)
ꢀ
1
3
d
2
Orange solid; yield: 63.2%; m.p: 137e139 C. H NMR (300 MHz,
CDCl ): ), 4.48e4.51
ppm ¼ 4.40e4.44 (t, J ¼ 5.7 Hz, 2H, CH
(t, J ¼ 5.7 Hz, 2H, CH ), 4.66 (s, 2H, CH ), 7.53e7.80 (m, 3H, ArH),
8.13 (s, 1H, H-triazole ring); 8.23e8.71 (m, 3H, ArH). C NMR
(75 MHz, CDCl ):
(
2
d
2
3
d
2
2
2
13
7
(
1
1
3
d
3
d
ppm ¼ 46.2, 49.1, 52.7, 108.8, 111.9, 112.5, 116.9,
119.5, 120.8, 123.6, 124.3, 125.1, 133.3, 136.2, 137.9, 140.0, 141.8,
ꢂ1
ꢂ1
59.4, 180.6, 180.9. IR (cm ): 1737, 1724, 1635, 1621, 1513. MS (EI)
153.5, 156.1, 181.9, 182.4. IR (cm ):1756, 1744, 1624, 1622, 1513. MS
þ
þ
m/z: 438 (M ). Calcd for C21
H
13
2
F N
5
O
4
: C, 57.67; H, 3.00; N, 16.01.
(EI) m/z: 480 (M ). Calcd for C21
H13ClN
6
O
6
: C, 52.46; H, 2.73; N,
Found C, 57.85; H, 3.07; N, 15.92.
17.48; Found C, 52.43; H, 2.82; N, 17.58.

P. Singh et al. / European Journal of Medicinal Chemistry 55 (2012) 455e461
461
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