Ultrasound promoted one pot synthesis of novel fluorescent triazolyl spirocyclic oxindoles using DBU based task specific ionic liquids and their antimicrobial activity

Article abstract of DOI:10.1016/j.ejmech.2014.03.016

Spirocyclic oxindoles and triazolyl derivatives posses remarkable biological activities. In present work, we have described an efficient one pot four-component domino reaction of 1-(prop-2-ynyl)indoline-2,3-dione, cyclic 1,3-diketones, malononitrile and v

Full text of DOI:10.1016/j.ejmech.2014.03.016

European Journal of Medicinal Chemistry 77 (2014) 145e154
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Ultrasound promoted one pot synthesis of novel fluorescent triazolyl
spirocyclic oxindoles using DBU based task specific ionic liquids and
their antimicrobial activity
,
b
b
Harjinder Singh a, Jayant Sindhu a, Jitender M. Khurana a , Chetan Sharma , K.R. Aneja
*
a Department of Chemistry, University of Delhi, Delhi 110007, India
b Department of Microbiology, Kurukshetra University, Kurukshetra, Haryana, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Spirocyclic oxindoles and triazolyl derivatives posses remarkable biological activities. In present work,
we have described an efficient one pot four-component domino reaction of 1-(prop-2-ynyl)indoline-2,3-
dione, cyclic 1,3-diketones, malononitrile and various aryl azides in DBU based ionic liquids [DBU-H]OAc
and [DBU-Bu]OH under ultrasonic irradiation for the construction of heterocycles, comprising spiro-
oxindole, 2-amino-4H-pyran, and 1,2,3-triazoles substructures. The antimicrobial activity of all com-
pounds has been investigated against six microbial strains. All compounds showed good antimicrobial
activity. All newly synthesized compounds exhibit fluorescence in methanol with large stoke shift.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Received 2 January 2014
Received in revised form
22 February 2014
Accepted 6 March 2014
Available online 7 March 2014
Keywords:
Multicomponent reactions
Ultrasonic irradiation
Ionic liquids
1,2,3-Triazole
Antimicrobial activity
1. Introduction
elimination of intermediate steps, diversity, high efficiency, selec-
tivity and minimal waste production [4e9]. MCRs using task spe-
Infectious diseases caused by microbes such as bacteria and
fungi are one of the leading causes of morbidity and mortality and
the major reason for the increase in microbial infections is the
resistance developed by these microbial organisms, particularly
Gram-positive bacteria Staphylococcus aureus and species of the
genus Enterococcu towards existing antimicrobial drugs [1].
Therefore development of alternative new more effective antimi-
crobial agents with new modes of action and a broad spectrum of
activities is a one of the major challenges in drug discovery. Mo-
lecular hybridization involves combining two or more heterocyclic
rings in a single molecule wherein combining units are derived
from known bioactive molecules [2]. Pharmacophore hybridization
is believed to be analogous to conventional combination therapy
wherein the two drugs are covalently linked and available as a
single entity [3].
cific ionic liquid (TSIL) with dual role of reaction media and catalyst
as well as alternative sources of energy such as ultrasonic/micro-
wave irradiation have gained much attention as efficient synthetic
tools for synthesis of complex novel molecules/hybrids in the realm
of green chemistry [10e13]. DBU based TSILs exhibit basicity
similar to DBU and are accompanied with the general features of
ILs. However they are less explored in organic synthesis as
compared to other ionic liquids [14].
Spirocyclic oxindoles are privileged structures with diverse
biological activities including antibacterial, antifungal, anticonvul-
sant, antiviral, and antiproliferative [15]. They act as potent in-
hibitors of monoamine oxidase in human urine and rat tissues [16]
and acetylcholinesterase [17], antagonist of in vitro receptor bind-
ing by atrial natriuretic peptide [18] and possess a wide range of
CNS activities [19]. Spiro oxindole ring system is also a part of many
alkaloids with biological activities such as paraherquamide A which
possess antiparasitic activity and antinematodal properties (Fig. 1.)
[20]. Pyrans and fused pyran derivatives are biologically interesting
compounds with activities such as antibacterial [21], antifungal
[22], antitumor [23], hypotensive [24] and analgesic [25]. For
example huajiaosimuline exhibits a selective cytotoxicity profile
showing the greatest activity with estrogen receptor-positive ZR-
Multicomponent reactions (MCRs) offer valuable strategies for
the hybridization of molecules with several advantages e.g.
* Corresponding author.
E-mail addresses: jmkhurana@chemistry.du.ac.in, jmkhurana1@yahoo.co.in (J.
M. Khurana).
http://dx.doi.org/10.1016/j.ejmech.2014.03.016
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

146
H. Singh et al. / European Journal of Medicinal Chemistry 77 (2014) 145e154
Table 1
Optimization of reaction conditions for the proposed four component condensation
O
in [DBU-H]OAc under ultrasonic irradiation.a
O
OH
C8H17
N
H
N
N
S. No.
Solvent
Base catalyst
Time (min)
Yield (%)
OH
NH
N
F
O
N
N
N
O
O
N
1.
2.
3.
4.
5.
6.
7.
8.
Ethanol
Ethanol
Water
Methanol
e
e
e
e
e
120
80
80
120
20
20
eb
N
[DBU-H]OAc (20 mol%)
[DBU-H]OAc (20 mol%)
[DBU-H]OAc (20 mol%)
[DBU-H]OAc (20 mol%)
[DBU-H]OAc (30 mol%)
[DBU-H]OAc (10 mol%)
[DBU-H]OAc (20 mol%)
55c
65c
60c
92
O
F
Fluoconazole
paraherquamide A
Huajiaosimuline
89
Fig. 1. Some representative compounds containing spirocyclic oxindole, pyran and
50
120
75
1,2,3-triazole moiety.
52d
a
Reaction was carried out using 1-(prop-2-ynyl)indoline-2,3-dione (1.0 mmol),
dimedone (1.0 mmol), 4-nitrophenyl azide (1.0 mmol), malononitrile (1.0 mmol) in
presence of CuSO4.5H2O (10 mol %) and sodium ascorbate (20 mol %).
75-1 breast cancer cells (Fig. 1) [26]. 1,2,3-Triazoles are another
privileged structures associated with a plethora of biological ac-
tivities including antiHIV [27], antimicrobial [28], antiviral [29],
antiproliferative [30], antibiotics [31], insecticides [32] and fungi-
cidal [33]. Fluconazole is a well known antifungal drug consisting of
1,2,3-triazole moiety (Fig. 1).
b
No reaction.
Mixture of products.
The reaction was carried out using stirring at room temperature in absence of
c
d
ultrasound irradiation.
Therefore, in continuation of our research interest in the syn-
thesis of potentially bioactive heterocyclic compounds with diverse
applications through hybridization [34], we decided to combine
spirocyclic oxindole, 2-amino-4H-pyran and 1,2,3-triazoles in a
single matrix through MCR approach in ionic liquids under ultra-
sonic irradiation and to study their biological activity and mode of
action against pathogenic microorganisms.
[DBU-H]OAc (20 mol%) also gave poor yields of 2a (Table 1, entries
3, 4). However, when the reaction was attempted in absence of any
solvent using [DBU-H]OAc (20 mol%) with dual role of catalyst and
medium under ultrasonic irradiation at room temperature, the
reaction time reduced dramatically to 20 min and yielded 92% of
the desired product 2a after a simple workup (Scheme 2, Table 1,
entry 5).
Higher amount of [DBU-H]OAc (30 mol%) did not affect the re-
action time and product yield (Table 1, entry 6) while lower amount
of [DBU-H]OAc (10 mol%) resulted in a longer reaction time and
inferior yield (Table 1, entry 7). When the reaction was carried out
in [DBU-H]OAc in absence of ultrasonic irradiation, the reaction was
incomplete even after 120 min and gave a mixture of products with
only 52% of the desired product 2a (Table 1, entry 8).
Thus, condensation of four-components using CuSO4.5H2O
(10 mol%) and sodium ascorbate (20 mol%) in 20 mol% of [DBU-H]
OAc under ultrasonic irradiation at room temperature proved to be
the optimum condition for this reaction. Subsequently reactions
were carried out by changing aryl azides. All the reactions preceded
smoothly for both electron rich and electron deficient aryl azides
which afforded the desired products (2be2g) in high yields
(Table 2, Method A, entries 2e7). Replacement of dimedone with
cyclohexane-1,3-dione under otherwise identical conditions also
afforded corresponding triazolyl spiro-oxindoles (2he2k) in high
yields (Table 2, Method A, entries 8e11). Further we examined the
scope of reaction by replacing dimedone with 1,3-
dimethylbarbituric acid under otherwise identical conditions. Re-
actions proceeded smoothly and yielded corresponding triazolyl
spiro oxindoles (2le2o) in high yields (Table 2, Method A, entries
12e15). These results have been summarized in Table 2 (Method A),
Scheme 3.
2. Results and discussion
2.1. Chemistry
We report herein an efficient multicomponent synthesis of
novel fluorescent triazolyl spirocyclic oxindole derivatives (2ae2o)
comprising spiro-oxindole, 2-amino-4H-pyran, and 1,2,3-triazole
substructures by one pot four-component reaction of 1-(prop-2-
ynyl)indoline-2,3-dione, cyclic 1,3-diketones such as dimedone/
cyclohexan-1,3-dione/1,3-dimethylbarbituric acid, malononitrile
and aryl azides in the presence of 10 mol% CuSO4.5H2O and 20 mol%
sodium ascorbate in ionic liquids [DBU-H]OAc and [DBU-Bu]OH
under ultrasonic irradiation at room temperature. The antimicro-
bial activity and fluorescence properties of all new compounds
were investigated.
Initially, 1-(prop-2-ynyl)indoline-2,3-dione, one of the compo-
nent of MCR, was synthesized by the hitherto unreported proce-
dure as outlined in Scheme 1.
The methodology for the proposed four-component reaction
was established for the synthesis of 2a using a model reaction
comprising 1-(prop-2-ynyl)indoline-2,3-dione (1.0 mmol), malo-
nonitrile (1.0 mmol), dimedone (1.0 mmol) and 4-nitrophenyl azide
(1.0 mmol) in presence of 10 mol% CuSO4.5H2O and 20 mol% sodium
ascorbate under different reaction conditions as shown in Table 1.
There was no reaction when the above components were heated in
ethanol (Table 1, entry 1). The above reaction was then attempted in
presence of [DBU-H]OAc (20 mol%) in ethanol. After workup
(80 min), 55% of the desired product (2a) was obtained (Table 1,
entry 2). Reaction repeated in water and methanol in presence of
After successful synthesis of triazolyl spiro-oxindoles in [DBU-
H]OAc under ultrasonic irradiation, we decided to explore this re-
action in presence of another DBU based ionic liquid [DBU-Bu]OH.
Therefore,
a
reaction of 1-(prop-2-ynyl)indoline-2,3-dione
(1.0 mmol), malononitrile (1.0 mmol), dimedone (1.0 mmol) and
4-nitrophenyl azide (1.0 mmol) was attempted in [DBU-Bu]OH
(20 mol%) in the presence of 10 mol% CuSO4.5H2O and 20 mol%
sodium ascorbate. The reaction was complete in 15 min under ul-
trasonic irradiation at room temperature and yielded 94% of the
desired product 2a. Higher amount of [DBU-Bu]OH did not affect
the reaction time or yield of reaction, while use of 10 mol% of [DBU-
Bu]OH resulted in longer reaction time. The reaction was incom-
plete even after 120 min when performed in absence of ultrasonic
irradiation. Subsequently, reactions of other aryl azides were
attempted which proceeded smoothly to yield corresponding
O
O
K2CO3 , PEG-400
O
Br
+
O
N
)))
N
H
(1)
Scheme 1. Synthesis of 1-(prop-2-ynyl)indoline-2,3-dione.

H. Singh et al. / European Journal of Medicinal Chemistry 77 (2014) 145e154
147
H2N
NC
O
N3
O
CuSO4.5H2O (10 mol %)
Sodium ascorbate (20 mol %)
O
CN
CN
O
+
+
+
[DBU-H]OAc (20 mol%), ))), RT
N
O
O
N
N
O
NO2
N
N
NO2
2a
Scheme 2. Four component condensation for synthesis of spiro oxindole derivatives.
products (2ae2g) in high yields as shown in Table 2 (Method B,
entries 1e7). Also when cyclohexane-1,3-dione and 1,3-
dimethylbarbituric acid were used in the place of dimedone,
promising results were obtained with high yield of products (2he
2o) (Table 1, Method B, entries 8e15) Scheme 3.
The structures of all compounds (2ae2o) have been confirmed
on the basis of their spectral data. IR spectrum of compound 2a
showed characteristic absorption bands at 3367 and 3161 cmꢀ1 due
to NH stretch of amino group. The 1H NMR spectrum of compound
The recovery of reaction medium and catalyst is an important
aspect of green chemistry. Therefore, recyclability of [DBU-Bu]OH
was investigated. After completion of the reaction for the synthe-
sis of 2a in [DBU-Bu]OH, water (5 mL) was added to the reaction
mixture. The ionic liquid dissolved in the water, and the solution
was filtered to isolate the product and washed again with water
(5 mL). Water was evaporated from water-ionic liquid mixture and
dried under reduced pressure. The recovered ionic liquid was
reused to study its catalytic activity in subsequent runs for the
synthesis of 2a. No appreciable loss in the yield of 2a was observed
after three cycles as 2a was obtained in 90%, 88% and 85% yield after
first, second and third cycle, respectively.
2a showed singlet at
another singlet at 7.25 corresponding to two NH2 protons. The
eight aromatic protons of 2a were observed in the range of 8.28e
d 8.62 because of one triazolyl proton and
d
d
6.85. Two methylene protons adjacent to nitrogen atom (eNCHaHb)
of 2a appeared as AB system and were observed as two sets of
The plausible reaction mechanism for the synthesis of triazolyl
spirocyclic oxindole derivatives has been shown in Scheme 4.
Initially, Huiseng 1,3-dipolar cycloaddition takes place between 1-
(prop-2-ynyl)indoline-2,3-dione (1) and aryl azide in presence of
Cu(1) generated in situ leading to formation of a triazole derivative
(1A) as an intermediate. The formation of 1A was confirmed by
comparing with an authentic sample on TLC. This is followed by
Knoevenagel condensation with malononitrile in task specific ionic
liquid to give another intermediate 1B. The unsaturated nitrile
derivative so formed (1B) undergo subsequent reactions with 1,3-
diketone in presence of ionic liquid to give the desired triazolyl
spirocyclic oxindole derivatives (2).
In order to provide evidence for this plausible mechanism,
compound 1A was synthesized using 4-nitrophenyl azide. It was
then reacted with malononitrile in the presence of [DBU-Bu]OH
under ultrasonic irradiation at room temperature to give com-
pound 1B after 5 min. Dimedone was added to the reaction mixture
and the desired product 2a was obtained in 92% yield after 10 min.
doublets at
adjacent to geminal dimethyl group also act as AB system and were
observed as two sets of doublets at 2.51, 2.43 (COCHa.Hb) and at
2.08, 1.99 (CHa.Hb), respectively. The six protons of two geminal
methyl groups were seen as two different singlets at 0.92 and
0.87, respectively. 13C NMR spectra of compound 2a showed
twenty six signals corresponding to twenty six non-equivalent
carbons. In 13C NMR spectra signal at
195.28 accounted for the
carbonyl carbon, while signal at 176.31 accounted for carbonyl
carbon of oxindole ring system. The signal for spiro carbon of 2a
was observed at 49.85, while two methyl groups were observed at
27.63 and
d 5.03 and 4.93. The protons of two methylene groups
d
d
d
d
d
d
d
d
d 26.90, respectively. HRMS of compound 2a showed a
molecular ion peak at 538.1819 [Mþ þ H]. The position of NH2
protons in the 1H NMR spectra of all compounds depends upon the
NMR solvent, when NMR was recorded in CDCl3, NH2 protons
appeared in the region of
d 5.20e4.79, while in DMSO-d6, NH2
protons appeared in the region of
d 7.69e7.25.
2.2. Biological screening of novel triazolyl spirocyclic oxindoles
derivatives
Table 2
2.2.1. In vitro antibacterial activity
Synthesis of triazolyl spirocyclic oxindole derivatives under ultrasonic irradiation.
Entry Ar Product Method A Method B
Time (min) Yield (%) Time (min) Yield (%)
Antibacterial activity of all new compounds (2ae2o) was eval-
uated against four bacterial strains. Two Gram-positive bacteria (S.
aureus MTCC 96 and Bacillus subtilis MTCC 121); two Gram-negative
bacteria (Escherichia coli MTCC 1652 and Pseudomonas aeruginosa
MTCC 741) were used in the present study for evaluation of anti-
bacterial activity of all new compounds. The results of the anti-
bacterial activity evaluation reveal that all compounds possessed
good Gram-positive antibacterial activity against S. aureus and B.
subtilis. However, none of the compounds exhibited activity against
Gram-negative (E. coli and P. aeruginosa) bacteria. The results of
antibacterial activity of all compounds are shown in Table 3 and
Fig. 2. All compounds showed diameter of growth of inhibition
zone in the range of 13.6e20.6 mm against S. aureus bacteria, and in
the range of 15.6e22.3 mm against B. subtilis bacteria.
1
2
3
4
5
6
7
8
4-NO2C6H4
2a
2b
2c
2d
20
40
30
45
50
35
40
20
25
35
40
25
30
35
45
92
87
90
89
85
81
90
88
93
84
81
90
87
92
86
15
20
15
20
20
20
25
15
10
20
25
15
15
20
20
94
88
93
87
89
90
82
92
88
92
90
89
92
91
92
3-ClC6H4
4-BrC6H4
4-MeC6H4
4-MeOC6H4 2e
C6H5 2f
4-F,3-ClC6H3 2g
4-NO2C6H4
4-BrC6H4
4-MeC6H4
4-MeOC6H4 2k
4-NO2C6H4
4-BrC6H4
4-MeC6H4
4-MeOC6H4 2o
2h
2i
2j
9
10
11
12
13
14
15
2l
2m
2n
Minimum inhibitory concentration (MIC) and minimum bacte-
ricidal concentration (MBC) of all compounds (2ae2o) was
measured against Gram-positive bacteria. All compounds showed
Method A: Reactions performed using task specific ionic liquid [DBU-H]OAc.
Method B: Reactions performed using task specific ionic liquid [DBU-Bu]OH.
MIC values in the range of 32e256 mg/mL against S. aureus and in

148
H. Singh et al. / European Journal of Medicinal Chemistry 77 (2014) 145e154
O
H2N
NC
O
R1
R1
R1
O
R1
O
O
N
N
O
N
Ar
N
CN
CN
Method A or Method B
O
+
+
Ar-N3
2a - 2g, R1 = CH3
2h - 2k, R1 = H
N
O
N
H2N
NC
N
O
N
O
O
O
N
O
O
N
N
Method A:- CuSO4.5H2O (10 mol %), sodium ascorbate (20 mol %), [DBU-H]OAc (20 mol%), ))), RT
Method B:- CuSO4.5H2O (10 mol %), sodium ascorbate (20 mol %), [DBU-Bu]OH (20 mol%), ))), RT
N
Ar
N
2l - 2o
Scheme 3. Synthesis of triazolyl spiro-oxindoles in presence of task specific ionic liquids under ultrasonic irradiation at room temperature.
the range of 16e128
compounds showed MBC values in the range of 64e512
m
g/mL against B. subtilis bacteria (Table 4). All
g/mL
g/mL against B.
2.2.2. In vitro antifungal activity
m
Antifungal activity of all compounds (2ae2o) was evaluated by
selecting two yeast strains Candida albicans (MTCC 227) and
Saccharomyces cerevisiae (MTCC 170) on the basis of their clinical
importance. The diameter of growth of inhibition zone (mm) and
against S. aureus and in the range of 64e256
subtilis bacteria (Table 4).
The results of antibacterial activity (Tables 3 and 4) revealed that
among all novel compounds, 2m was found to be most active
against Gram-positive bacteria with a diameter of growth of inhi-
bition zone of 20.6 mm against S. aureus and 22.3 mm against B.
m
MIC
(mg/mL) of all compounds was determined using
amphotericin-B as a standard drug. The results of antifungal ac-
tivity of all compounds (2ae2o) are shown in Table 5, Fig. 3. It can
be seen from Table 5 that all compounds showed good antifungal
activity. The diameter of growth of inhibition zone was observed in
the range of 12.3e16.6 mm for yeast C. albicans and in the range of
13.6e17.6 mm for yeast S. cerevisiae for compounds 2ae2o. All
compounds showed MIC in the range of 128e512
albicans and in the range of 64e512 g/mL against S. cerevisiae.
Compound 2e showed highest activity against yeast C. albicans with
subtilis. Compound 2m showed MIC value of 32
value of 64 g/mL against S. aureus, while it showed MIC value of
16 g/mL and MBC value of 64 g/mL against B. subtilis. The
mg/mL and MBC
m
m
m
structure activity relationship (SAR) study of these compounds
revealed that compounds 2b, 2c, 2g, 2i and 2m having halogen
atom in their moiety showed better activity as compared to other
compounds.
mg/mL for C.
m
O
Ar-N
O
N
O
N
L
Cu
Ar
O
O
O
N
N
N
N
H
L
Cu
N
CuL
Ar
[L Cu]
N
N
O
O
N
(1)
O
R
O
N
CN
CN
N
N
Ar
N
N
N
RN
O
CN
NC
NC
H
(1A )
N
O
NC
N
N
Ar
X
N
N
R
N
N
NH
NC
H
NC
N
OH
O
O
CN
O
NC
O
Ar
O
N
N
N
N
Ar
N
N
N
N
N
N
N
N
Ar
(2)
R
R
=
=
H, X = OAc for [DBU-H]OAc
NC
NC
O
O
n-Bu, X = OH for [DBU-Bu]OH
H
TSIL
Ar
N
N
N
N
(1B )
Scheme 4. Plausible mechanism for synthesis of triazolyl spirocyclic oxindoles.

H. Singh et al. / European Journal of Medicinal Chemistry 77 (2014) 145e154
149
Table 3
Table 4
Antibacterial activity (diameter of growth of inhibition zone) of all compounds.
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration
(MBC) of compounds (2a-2o) against Gram positive bacteria.
Entry Product
Diameter of growth of inhibition zone (mm)a
Entry Product
MIC (
m
g/mL)
MBC (mg/mL)
Staphylococcus Bacillus Escherichia coli Pseudomonas
aureus
subtilis
aeruginosa
Staphylococcus Bacillus Staphylococcus Bacillus subtilis
aureus
subtilis aureus
1
2
3
4
5
6
7
8
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
16.6
19.3
19.3
14.3
15.6
14.6
19.6
17.6
18.6
13.6
14.0
15.3
20.6
14.3
15.3
17.3
20.6
22.2
15.6
17.3
16.3
21.3
18.6
20.3
16.3
15.6
18.6
22.3
16.3
17.6
24.0
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
25.0
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
22.0
1
2
3
4
5
6
7
8
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
128
64
32
128
128
128
64
128
64
256
64
128
32
128
256
6.25
128
32
16
128
128
128
256
256
128
128
256
64
256
128
64
32
256
256
256
128
256
128
128
128
128
64
128
128
32
128
32
9
10
11
12
13
14
15
16
9
10
11
12
13
14
15
16
128
32
64
512
512
512
64
16
128
128
256
512
128
256
6.25
Ciprofloxacin 26.6
Ciprofloxacin
6.25
6.25
e No activity.
a
Values, including diameter of the well (8 mm), are means of three replicates.
satisfactory results were obtained. The binding site for CYP51 in-
hibitors is located at residues Phe22, Tyr76, Met79, Phe83, Lys74,
Phe255, Ala256, His259, Leu321, Ile322, Ile323, His430, Met433,
Val434, Val435, Met253 and the heme [36]. The binding mode of
compound 2e at the active site of CYP51 is shown in Fig. 4. The
nitrogen atom of amino group of 2e coordinates with iron atom of
Hem460, while nitrogen atom of triazole ring formed a hydrogen
bond with Lys74 amino acid residue and is surrounded by Met79,
Ile322, Leu324, Met325, Tyr76, His259, Ala256, Arg96, Met253
amino acid residues. These interactions may be responsible for the
antifungal activity of compound 2e. However, we cannot refute
other mechanisms which are responsible for antifungal activity and
only experiments on isolated CYP51 could confirm above
hypothesis.
diameter of growth of inhibition zone of 16.6 mm and MIC value of
128
against S. cerevisiae with diameter of growth of inhibition zone of
17.6 mm and MIC value of 64 g/mL. Compounds 2e, 2k, 2o showed
MIC value of 64 g/mL against S. cerevisiae lower than the standard
mg/mL. While compound 2o was found to be most active
m
m
drug amphotericin-B. It can be inferred from the results of anti-
fungal activity that the presence of methoxy group in molecules
(2e, 2k, 2o) enhances the antifungal activity of these compounds.
The molecular basis of the antifungal activity of azole antifungal
drugs is the inhibition of ergosterol biosynthesis which is necessary
to maintain fungal cell membrane integrity and permeability.
Indeed, azole antifungals inhibit the cytochrome P-450 lanosterol
14
a
-demethylase (CYP51) which is responsible for the oxidative
-methyl group of lanosterol [35]. In order to
removal of the 14
a
understand the possible mechanism of antifungal activity, the
docking of most potent compound 2e was performed at active site
of CYP51 (PDB ID 1EA1). The docking studies were performed using
autodock 4 programe with default parameters. The crystallographic
2.2.3. Fluoresecne properties
Novel fluorescent molecules are potential candidates as fluo-
rescent probes, for imaging in clinical diagnostics and biomedical
research. We observed that all compounds showed strong fluo-
rescence in methanol when excited at wavelength 266 nm as
shown in Fig. 5. The results of photophysical properties of all tri-
azolyl spirocyclic oxindoles are summarized in Table 6. Moreover,
structure of Cytochrome P450 14a-sterol demethylase (MTCYP51)
with bound inhibitor (fluconazole) was downloaded from protein
data bank (PDB ID 1EA1). Fluconazole was re-docked into the active
site in order to check the validity of docking protocol and
30
Staphylococcus aureus
Escherichia coli
Bacillus subtilis
25
20
15
10
5
Pseudomonas aeruginosa
0
Compounds
Fig. 2. Graphical representation of diameter of growth of inhibition zone (mm) of compounds (2ae2o) against bacterial strains.

150
H. Singh et al. / European Journal of Medicinal Chemistry 77 (2014) 145e154
Table 5
Antifungal activity of all compounds (2ae2o).
Entry Product
Diameter of growth
MIC (mg/mL)
of inhibition zone (mm)
Candida Saccharomyces Candida Saccharomyces
albicans cerevisiae albicans cerevisiae
1
2
3
4
5
6
7
8
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
14.3
12.3
13.3
12.6
16.6
13.3
13.6
12.3
12.6
14.3
16.0
12.6
13.3
12.3
14.6
15.3
13.6
14.6
14.0
15.6
14.3
14.3
13.6
14.3
15.6
16.3
13.6
15.3
14.3
17.6
18.3
256
512
512
512
128
256
256
512
512
128
128
512
256
512
128
100
256
256
128
256
64
256
256
256
256
128
64
9
10
11
12
13
14
15
16
512
128
256
64
Fig. 4. Binding mode of compound 2e at the active site of CYP51 (PDB ID: 1EA1).
Amphotericin-B 17.6
100
4. Experimental
All chemicals were commercial and were purchased from
SigmaeAldrich, Spectrochem and were used as received. F254 Pre-
coated aluminum plates with silica gel 60 from Merck were used to
monitor reaction progress. Ultrasonic bath (54 KHz, 300 W, 3 L,
capacity) of Throughclean ultrasonic Pvt. Ltd. (India) was used for
reactions. Melting points were measured on Buchi M-560 melting
point apparatus and are uncorrected. IR (KBr) spectra were recor-
ded on Perkin Elmer FTIR spectrophotometer and the values are
expressed as nmax cmꢀ1. The NMR (1H and 13C) spectra were
recorded on Jeol JNM ECX-400P at 400 MHz and 100 MHz,
the UVevisible spectra of all compounds contain intense absorp-
tion maxima in the range of 239e287 nm. These compounds
exhibit emission band in the range of 366e417 nm with stokes shift
in the range of 97e156 nm.
3. Conclusion
In conclusion, We have developed an efficient synthesis of novel
structurally diverse fluorescent triazolyl spirocyclic oxindole de-
rivatives by one pot four-component domino reaction in presence
of [DBU-H]OAc and [DBU-Bu]OH under ultrasonic irradiation at
room temperature. The antimicrobial activity of all compounds was
evaluated against six microbial strains. Compound 2m was found to
be most potent antibacterial agent against S. aureus and against B.
subtilis. All compounds showed good antifungal activity. Com-
pounds 2e, 2k, 2o showed MIC values lower than the standard drug
amphotericin-B against S. cerevisiae. The docking studies of com-
respectively. The chemical shift values are recorded on
d scale and
the coupling constants (J) are in Hertz. The mass spectra were
recorded on Agilent 6520-QTOF LCMS having ESI source in positive
mode. Ultravioletevisible (UVeVis) absorption spectra were
recorded on Analytikjena specord 250 spectrophotometer. The
fluorescence spectra were measured at Cary Eclipse Fluorescence
spectrophotometer.
4.1. Preparation of ionic liquid [DBU-H]OAc
pound 2e at the active site of cytochrome P-450 lanosterol 14a-
demethylase (CYP51) were also performed. The compounds exhibit
strong fluorescence in methanol with large stokes shift.
[DBU-H]OAc was prepared by reaction of DBU with acetic acid
by reported procedure [37].
20
Candida albicans
Saccharomyces cerevisiae
18
16
14
12
10
8
6
4
2
0
Compounds
Fig. 3. Graphical representation of diameter of growth of inhibition zone (mm) of compounds (2ae2o) against yeast strains.

H. Singh et al. / European Journal of Medicinal Chemistry 77 (2014) 145e154
151
(10 mL) was added to the reaction mixture. The precipitate formed
was collected by filtration at pump, washed with water to afford
pure 1-(prop-2-ynyl)indoline-2,3-dione (M.p. 156e158 ꢁC) [39] in
92% yield as orange solid.
4.4. General procedure for the domino synthesis of triazolyl
spirocyclic oxindole derivatives (2ae2o) under ultrasonic
irradiation in presence of [DBU-H]OAc (Method A)
A mixture of 1-(prop-2-ynyl)indoline-2,3-dione (1.0 mmol),
dimedone/cyclohexane-1,3-dione/1,3-dimethyl barbituric acid
(1.0 mmol), malononitrile (1.0 mmol), aryl azide (1.0 mmol),
CuSO4.5H2O (10 mol%), sodium ascorbate (20 mol%) and 20 mol%
[DBU-H]OAc was placed in a 50 mL round bottomed flask. The re-
action mixture was sonicated for appropriate time until the com-
plete disappearance of starting materials. The progress of the
reaction was monitored by TLC using ethyl acetate: petroleum ether
(40:60, v/v) as eluent. After completion of the reaction as indicated
by TLC, water (10 mL) was added to the reaction mixture. The
precipitate formed was collected by filtration at pump and washed
with water several times to remove Cu (II) from product. The
product thus obtained was recrystallized from ethanol to yield pure
products (2ae2o) in high yields as mentioned in Table 2 (Method
A). The products were characterized by IR, 1H NMR, 13C NMR and
Mass spectra.
Fig. 5. Fluorescence spectra of compounds (2ae2o) in methanol.
4.2. Preparation of ionic liquid [DBU-Bu]OH
Firstly [DBU-Bu]Br was prepared by reaction of DBU with n-
butyl bromide in cyclohexane by reported procedure [38]. In sec-
ond step, KOH (1.2 eq) was added to a solution of [DBU-Bu]Br in
dichloromethane. After 48 h of stirring at room temperature, the
reaction mixture was filtered through a sintered funnel. The filtrate
was concentrated on a rotary evaporator to yield a viscous liquid.
Dichloromethane was added to the residue to cause precipitation of
any remaining KBr. The precipitate was filtered through a sintered
funnel and the filtrate concentrated again on rotary evaporator to
obtain 92% of [DBU-Bu]OH.
4.5. General procedure for the synthesis of triazolyl spirocyclic
oxindole derivatives (2ae2o) under ultrasonic irradiation in
presence of [DBU-Bu]OH (Method B)
A mixture of 1-(prop-2-ynyl)indoline-2,3-dione (1.0 mmol),
dimedone/cyclohexane-1,3-dione/1,3-dimethyl barbituric acid
(1.0 mmol), malononitrile (1.0 mmol), aryl azide (1.0 mmol),
CuSO4.5H2O (10 mol%), sodium ascorbate (20 mol%) and 20 mol%
[DBU-Bu]OH was placed in a 50 mL round bottomed flask. The re-
action mixture was sonicated for appropriate time until the com-
plete disappearance of starting materials. The progress of the
reaction was monitored by TLC using ethyl acetate: petroleum ether
(40:60, v/v) as eluent. After completion of the reaction as indicated
by TLC, water (10 mL) was added to the reaction mixture. The
precipitate formed was collected by filtration at pump and washed
with water several times to remove Cu (II) from product. The
product thus obtained was recrystallized from ethanol to yield pure
products (2ae2o) (Table 2, Method B). The products were charac-
terized by IR, 1H NMR, 13C NMR and Mass spectra.
1H NMR (400 MHz, CDCl3)
5H), 1.58e1.46 (m, 9H), 1.37e1.07 (m, 5H), 0.75e0.69 (m, 3H); 13C
NMR (100 MHz, CDCl3):
¼ 175.45, 49.33, 45.37, 36.83, 29.59,
28.84, 28.71, 28.30, 27.91, 25.58, 25.24, 20.11, 13.64; IR (nmax cmꢀ1
d
¼ 3.26e3.15 (m, 3H), 2.41e2.19 (m,
d
,
KBr) ¼ 3445, 2928, 1634, 1485, 1326.
4.3. Procedure for synthesis of 1-(prop-2-ynyl)indoline-2,3-dione
A mixture of isatin (1.0 mmol), propargyl bromide (1.2 mmol),
K2CO3 (2.0 mmol) and PEG 400 (5 mL) was placed in a 50 mL round-
bottomed flask. The reaction mixture was sonicated at room tem-
perature for 4 h. After completion of reaction as monitored by TLC
using ethyl acetate: petroleum ether (30:70, v/v) as eluent, water
4.6. Spectral data for compounds (2ae2o)
Table 6
4.6.1. 2-Amino-7,7-dimethyl-10-((1-(4-nitrophenyl)-1H-1,2,3-
triazol-4-yl)methyl)-20,5-dioxo-5,6,7,8-tetrahydrospiro[chromene-
4,30-indoline]-3-carbonitrile (2a)
Photophysical data of all compounds (2ae2o) in methanol.
Product labs (nm) ε ꢂ 105 (L molꢀ1 cmꢀ1
)
lem (nm) Stoke shift (Dn) nm
White Solid, M.pt.: 270 ꢁC (decomp), 1H NMR (400 MHz, DMSO-
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
239
241
261
262
264
270
287
269
269
265
269
270
269
257
270
0.29
0.28
0.43
0.60
0.75
0.71
0.25
0.19
0.78
0.92
0.93
0.61
0.41
0.25
0.55
373
366
417
368
378
367
384
385
398
377
405
369
385
413
377
134
125
156
106
114
97
d6)
d
¼ 8.62 (s, 1H, triazolyl-H), 8.28 (d, 2H, J ¼ 8.8 Hz, ArH), 7.97 (d,
2H, J ¼ 8.8 Hz, ArH), 7.25 (s, 2H, eNH2), 7.12e7.08 (t, 1H, J ¼ 8.8 Hz,
ArH), 6.96e6.85 (m, 3H, ArH), 5.03 and 4.93 (AB system,
J ¼ 16.12 Hz, 2H, eNCHa.Hb), 2.51 and 2.43 (AB system, J ¼ 17.61 Hz,
2H, eCHa.Hb), 2.08 and 1.99 (AB system, J ¼ 16.12 Hz, 2H, eCHa.Hb),
0.92 (s, 3H, CH3), 0.87 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6):
97
116
129
112
136
99
116
156
107
d
¼ 195.28, 176.31, 164.69, 158.71, 146.62, 144.18, 141.64, 140.64,
133.47, 128.43, 125.60, 123.05, 122.88, 121.86, 120.40, 120.08, 117.57,
110.47, 108.77, 56.86, 49.85, 46.35, 35.23, 31.96, 27.63, 26.90; IR
(
nmax cmꢀ1, KBr) ¼ 3367, 3161, 1708, 1669, 1611, 1497; HRMS (ESI)
[Mþ þ H] calcd. for C28H23N7O5: 538.1838, found: 538.1819
[Mþ þ H].

152
H. Singh et al. / European Journal of Medicinal Chemistry 77 (2014) 145e154
4.6.2. 2-Amino-10-((1-(3-chlorophenyl)-1H-1,2,3-triazol-4-yl)
methyl)-7,7-dimethyl-20,5-dioxo-5,6,7,8-tetrahydrospiro[chromene-
4,30-indoline]-3-carbonitrile (2b)
4.6.6. 2-Amino-7,7-dimethyl-20,5-dioxo-10-((1-phenyl-1H-1,2,3-
triazol-4-yl)methyl)-5,6,7,8 tetrahydrospiro[chromene-4,30-
indoline]-3-carbonitrile (2f)
White Solid, M.pt.: 176 ꢁC, 1H NMR (400 MHz, CDCl3)
d
¼ 8.33 (s,
White Solid, M.pt.: 164 ꢁC, 1H NMR (400 MHz, CDCl3)
d
¼ 8.19 (s,
1H, triazolyl-H), 7.73 (s, 1H, ArH), 7.56 (d, 1H, J ¼ 7.36 Hz, ArH),
7.32e7.25 (m, 2H, ArH), 7.15e7.11 (t,1H, J ¼ 7.32 Hz, ArH), 6.98e6.95
(m, 2H, ArH), 6.76 (d, 1H, J ¼ 8.04 Hz, ArH), 5.16 (s, 2H, eNCH2), 4.88
(s, 2H, eNH2), 2.53 and 2.43 (AB system, J ¼ 17.56 Hz, 2H, eCHa.Hb),
2.21 and 2.12 (AB system, J ¼ 16.88 Hz, 2H, eCHa.Hb), 1.08 (s, 3H,
1H, triazolyl-H), 7.66 (d, 2H, J ¼ 7.32 Hz, ArH), 7.39e7.35 (m, 3H,
ArH), 7.30e7.27 (m, 1H, ArH), 7.15e7.11 (m, 1H, ArH), 6.97e6.92 (m,
1H, ArH), 6.78 (d, 1H, J ¼ 7.32 Hz, ArH), 5.18 (s, 2H, eNCH2), 4.83 (s,
2H, eNH2), 3.75 (s, 3H, eCH3), 2.52 and 2.42 (AB system, J ¼ 17.6 Hz,
2H, eCHa.Hb), 2.21 and 2.12 (AB system, J ¼ 16.88 Hz, 2H, eCHa.Hb),
1.08 (s, 3H, CH3), 1.02 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3):
CH3), 1.02 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3):
d
¼ 195.30,
176.36, 164.22, 158.30, 141.61, 140.34, 139.09, 138.96, 137.89, 135.33,
132.49, 131.45, 130.63, 129.26, 128.44, 123.48, 123.10, 120.52, 118.17,
119.21, 109.39, 50.51, 46.70, 40.92, 36.56, 32.28, 28.61, 27.52; IR
d
¼ 195.18, 176.64, 171.84, 168.23, 164.12, 158.30, 141.71, 137.05,
132.50, 130.08, 128.81, 129.24, 128.42, 127.78, 123.72, 121.03, 120.24,
111.60, 109.50, 50.50, 46.34, 40.89, 36.63, 32.36, 28.61, 27.53; IR
(
nmax cmꢀ1, KBr): ¼3371, 3120, 1702, 1661, 1611, 1540; HRMS (ESI)
(
nmax cmꢀ1, KBr): ¼3308, 3153, 1685, 1670, 1610, 1353; HRMS (ESI)
[Mþ þ H] calcd. for C28H23ClN6O3: 527.1598, found: 527.1590
[Mþ þ H] calcd. for C28H24N6O3: 493.1988, found: 493.1992
[Mþ þ H].
[Mþ þ H].
4.6.3. 2-Amino-10-((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)
methyl)-7,7-dimethyl-20,5-dioxo-5,6,7,8-tetrahydrospiro[chromene-
4,30-indoline]-3-carbonitrile (2c)
4.6.7. 2-Amino-10-((1-(3-chloro-4-fluorophenyl)-1H-1,2,3-triazol-
4-yl)methyl)-7,7-dimethyl-20,5-dioxo-5,6,7,8-tetrahydrospiro
[chromene-4,30-indoline]-3-carbonitrile (2g)
White Solid, M.pt.: 240 ꢁC (decomp), 1H NMR (400 MHz, DMSO-
White Solid, M.pt.: 270 ꢁC (decomp), 1H NMR (400 MHz, CDCl3)
d6)
d
¼ 8.42 (s, 1H, triazolyl-H), 7.63 (s, 2H, eNH2), 7.26 (s, 1H, ArH),
d
¼ 8.28 (s, 1H, triazolyl-H), 7.80e7.78 (m, 1H, ArH), 7.57e7.53 (m,
7.11e7.07 (t, 1H, J ¼ 7.32 Hz, ArH), 6.97 (d, 2H, J ¼ 7.32 Hz, ArH),
6.92e6.85 (m, 4H, ArH), 5.01 and 4.89 (AB system, J ¼ 16.12 Hz, 2H,
eNCHa.Hb), 2.51 and 2.44 (AB system, J ¼ 17.6 Hz, 2H, eCHa.Hb),
2.08 and 1.99 (AB system, J ¼ 16.12 Hz, 2H, eCHa.Hb), 0.92 (s, 3H,
1H, ArH), 7.16e7.11 (m, 2H, ArH), 6.98e6.95 (m, 2H, ArH), 6.76 (d,
1H, J ¼ 7.32 Hz, ArH), 5.16 (s, 2H, eNCH2), 4.90 (s, 2H, eNH2), 2.53
and 2.44 (AB system, J ¼ 17.56 Hz, 2H, eCHa.Hb), 2.22 and 2.12 (AB
system, J ¼ 16.12 Hz, 2H, eCHa.Hb), 1.08 (s, 3H, CH3), 1.02 (s, 3H,
CH3), 0.88 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6):
d
¼ 195.80,
CH3); 13C NMR (100 MHz, CDCl3):
d
¼ 195.30, 176.18, 164.20, 158.24,
176.74, 165.24, 159.48, 142.38, 136.21, 134.02, 133.38, 133.25, 128.99,
128.90, 123.60, 123.35, 122.33, 121.91, 118.21, 116.04, 110.28, 109.31,
156.36, 144.09, 141.43, 133.61, 132.46, 123.50, 123.12, 122.73, 122.23,
122.07, 121.18, 119.86, 117.51, 117.28, 116.69, 111.60, 109.35, 50.53,
46.67, 40.92, 36.49, 32.29, 28.61, 27.53; IR (nmax cmꢀ1, KBr): ¼ 3325,
3304, 3113, 1710, 1668, 1612, 1508; HRMS (ESI) [Mþ þ H] calcd. for
57.31, 50.42, 46.92, 35.91, 32.53, 28.18, 27.48; IR (nmax cmꢀ1
,
KBr): ¼3363, 3160, 1707, 1670, 1610, 1497; HRMS (ESI) [Mþ þ H]
calcd. for C28H23BrN6O3: 571.1093, found: 571.1089 [Mþ þ H].
C
28H22ClFN6O3: 545.1504 found: 545.1512 [Mþ þ H].
4.6.4. 2-Amino-7,7-dimethyl-20,5-dioxo-10-((1-p-tolyl-1H-1,2,3-
triazol-4-yl)methyl)-5,6,7,8-tetrahydrospiro[chromene-4,30-
indoline]-3-carbonitrile (2d)
4.6.8. 2-Amino-10-((1-(4-nitrophenyl)-1H-1,2,3-triazol-4-yl)
methyl)-20,5-dioxo-5,6,7,8-tetrahydrospiro[chromene-4,30-
indoline]-3-carbonitrile (2h)
White Solid, M.pt.: 250 ꢁC (decomp), 1H NMR (400 MHz, CDCl3)
White Solid, M.pt.: 267 ꢁC, 1H NMR (400 MHz, DMSO-d6)
d
¼ 8.29 (s, 1H, triazolyl-H), 7.56 (d, 2H, J ¼ 8.24 Hz, ArH), 7.21e7.14
(m, 3H, ArH), 7.01e6.96 (m, 2H, ArH), 6.82 (d, 1H, J ¼ 7.8 Hz, ArH),
5.20 (s, 2H, eNH2), 5.08 (s, 2H, eNCH2), 2.55 and 2.45 (AB system,
J ¼ 17.84 Hz, 2H, eCHa.Hb), 2.33 (s, 3H, CH3), 2.23 and 2.14 (AB
system, J ¼ 16.48 Hz, 2H, eCHa.Hb), 1.10 (s, 3H, CH3), 1.04 (s, 3H,
d
¼ 8.74 (s, 1H, triazolyl-H), 8.39 (d, 2H, J ¼ 8.24 Hz, ArH), 8.08 (d,
2H, J ¼ 8.28 Hz, ArH), 7.34 (s, 2H, eNH2), 7.24e6.98 (m, 4H, ArH),
5.24 and 5. 04 (AB system, J ¼ 15 .56 Hz, 2H, eNCHa.Hb), 2.68e2.61
(m, 2H, eCH2), 2.26e2.18 (m, 2H, eCH2), 1.94e1.92 (m, 2H, eCH2);
CH3); 13C NMR (100 MHz, CDCl3):
d
¼ 195.20, 176.55, 164.18, 158.41,
13C NMR (100 MHz, DMSO-d6):
d
¼ 195.65, 176.55, 166.75, 158.55,
141.70, 138.48, 134.76, 132.55, 131.90, 130.08, 129.16, 128.40, 126.92,
123.37, 123.04, 121.02, 120.15, 111.52, 109.45, 50.46, 46.73, 40.85,
36.61, 32.20, 28.56, 27.46, 20.98; IR (nmax cmꢀ1, KBr): ¼3327, 3210,
1724, 1673, 1611, 1518; HRMS (ESI) [Mþ þ H] calcd. for C29H26N6O3:
507.2144, found: 507.2158 [Mþ þ H].
146.56, 144.38, 141.76, 140.49, 137.89, 133.34, 132.42, 128.06, 125.62,
123.68, 121.86, 120.06, 117.61, 111.76, 108.17, 56.02, 46.44, 39.08,
26.88, 19.86; IR (nmax cmꢀ1, KBr): ¼3365, 3110, 1707, 1661, 1611,
1520; HRMS (ESI) [Mþ þ H] calcd. for C26H19N7O5: 510.1525 found:
510.1532 [Mþ þ H].
4.6.5. 2-Amino-10-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)
methyl)-7,7-dimethyl-20,5-dioxo-5,6,7,8-tetrahydrospiro[chromene-
4,30-indoline]-3-carbonitrile (2e)
4.6.9. 2-Amino-10-((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)
methyl)-20,5-dioxo-5,6,7,8-tetrahydrospiro[chromene-4,30-
indoline]-3-carbonitrile (2i)
White Solid, M.pt.: 214 ꢁC, 1H NMR (400 MHz, CDCl3)
d
¼ 8.19 (s,
1H, triazolyl-H), 7.55 (d, 2H, J ¼ 6.6 Hz, ArH), 7.13e7.11 (m, 1H, ArH),
6.95e6.88 (m, 4H, ArH), 6.79 (d, 1H, J ¼ 8.04 Hz, ArH), 5.17 (s, 2H, e
NCH2), 4.79 (s, 2H, eNH2), 3.75 (s, 3H, eOCH3), 2.52 and 2.42 (AB
system, J ¼ 17.84 Hz, 2H, eCHa.Hb), 2.20 and 2.11 (AB system,
J ¼ 16.08 Hz, 2H, eCHa.Hb), 1.07 (s, 3H, CH3), 1.01 (s, 3H, CH3); 13C
White Solid, M.pt.: 250 ꢁC (decomp), 1H NMR (400 MHz, DMSO-
d6)
d
¼ 8.53 (s, 1H, triazolyl-H), 7.74e7.69 (m, 3H, ArH), 7.35 (s, 2H,
eNH2), 7.22e7.18 (m, 1H, ArH), 7.10 (d, 2H, J ¼ 7.32 Hz, ArH), 7.02e
6.95 (m, 2H, ArH), 5.11 and 5.01 (AB system, J ¼ 16.12 Hz, 2H, e
NCHa.Hb), 2.67e2.66 (m, 2H, eCH2), 2.26e2.20 (m, 2H, eCH2),
1.94e1.91 (m, 2H, eCH2); 13C NMR (100 MHz, DMSO-d6):
NMR (100 MHz, DMSO-d6):
d
¼ 195.23, 176.17, 164.67, 159.24,
158.93, 141.89, 133.48, 129.91, 128.52, 128.48, 123.04, 122.76, 121.47,
121.40, 121.03, 117.66, 114.89, 110.50, 108.74, 56.80, 55.53, 49.89,
46.38, 35.48, 31.97, 27.62, 27.00; IR (nmax cmꢀ1, KBr): ¼3393, 3157,
1702, 1671, 1601, 1520; HRMS (ESI) [Mþ þ H] calcd. for C29H26N6O4:
523.2093, found: 523.2105 [Mþ þ H].
d
¼ 195.21, 176.08, 166.66, 158.76, 141.66, 135.63, 133.56, 132.70,
128.22, 126.42, 125.33, 123.18, 122.68, 121.93, 121.04, 117.44, 116.51,
111.42, 108.50, 56.89, 46.30, 36.24, 25.70, 19.82; IR (nmax cmꢀ1
,
KBr): ¼3345, 3120, 1710, 1652, 1607, 1497 HRMS (ESI) [Mþ þ H]
calcd. for C26H19BrN6O3: 543.0780 found: 543.0786 [Mþ þ H].

H. Singh et al. / European Journal of Medicinal Chemistry 77 (2014) 145e154
153
4.6.10. 2-Amino-20,5-dioxo-10-((1-4-tolyl-1H-1,2,3-triazol-4-yl)
methyl)-5,6,7,8-tetrahydrospiro [chromene-4,30-indoline]-3-
carbonitrile (2j)
J ¼ 16.04 Hz, 2H, eNCHaHb), 3.38 (s, 3H, eNCH3), 3.03 (s, 3H, e
NCH3), 2.33 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6):
d
¼ 181.18,
176.14, 160.72, 158.68, 152.57, 149,89, 142.19, 139.66, 134.64, 133.21,
130.72, 129.14, 124.40, 123.06, 121.60, 120.65, 119.53, 117.83, 109.18,
100.18, 47.66, 36.02, 30.36, 28.47, 21.05; IR (nmax cmꢀ1, KBr): ¼ 3356,
3102, 1709, 1689, 1489; HRMS (ESI) [Mþ þ H] calcd. for C27H22N8O4:
523.1842 found:523.1850 [Mþ þ H].
White Solid, M.pt.: 248 ꢁC, 1H NMR (400 MHz, DMSO-d6)
d
¼ 8.43 (s, 1H, triazolyl-H), 7.63 (s, 2H, eNH2), 7.35e7.19 (m, 4H,
ArH), 7.10e6.99 (m, 4H, ArH), 5.11 and 4.99 (AB system,
J ¼ 12.44 Hz, 2H, eNCHaHb), 2.68e2.66 (m, 2H, eCH2), 2.49e2.23
(m, 5H, eCH2, CH3), 2.07e1.92 (m, 2H, eCH2); 13C NMR (100 MHz,
4.6.15. 70-Amino-10,30-dimethyl-2,20,40-trioxo-1-((1-4-methoxyl-
1H-1,2,3-triazol-4-yl)methyl)-10,20,30,40-tetrahydrospiro[indoline-
3,50-pyrano[2,3-d]pyrimidine]-60-carbonitrile (2o)
DMSO-d6):
d
¼ 195.45, 176.28,166.56, 158.95, 147.38, 143.35,142.02,
138.16, 134.25, 133.61, 130.25, 128.39, 123.08, 122.53, 121.03, 119.69,
117.65, 111.43, 108.10, 56.82, 46.46, 36.29, 35.52, 20.51, 19.72; IR
nmax cmꢀ1, KBr): ¼3325, 3292, 1725, 1667, 1611; HRMS (ESI)
White Solid, M.pt.: 205 ꢁC, 1H NMR (400 MHz, DMSO-d6)
(
[Mþ þ H] calcd. For C27H22N6O3: 479.1831, found: 479.1841
[Mþ þ H].
d
¼ 8.43 (s, 1H, triazolyl-H), 7.68e7.66 (m, 4H, eNH2, ArH), 7.23e
7.19 (m, 2H, ArH), 7.08e6.97 (m, 4H, ArH), 5.13 and 5 .03 (AB system,
J ¼ 15.4 Hz, 2H, eNCHaHb), 3.78 (s, 3H, eOCH3), 3.38 (s, 3H, eNCH3),
4.6.11. 2-Amino-10-((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)
methyl)-20,5-dioxo-5,6,7,8-tetrahydrospiro[chromene-4,30-
indoline]-3-carbonitrile (2k)
3.03 (s, 3H, eNCH3); 13C NMR (100 MHz, DMSO-d6):
d
¼ 175.55,
159.74, 158.92, 158.08, 152.17, 149.61, 142.06, 141.29, 132.41, 129.72,
128.59, 123.85, 122.72, 122.25, 121.55, 121.20, 117.75, 116.80, 114.79,
108.5, 56.87, 55.42, 46.73, 29.25, 27.53; IR (nmax cmꢀ1, KBr): ¼3356,
3109, 1715, 1686, 1519; HRMS (ESI) [Mþ þ H] calcd. for C27H22N8O5:
539.1791 found: 539.1805 [Mþ þ H].
White Solid, M.pt.: 197 ꢁC, 1H NMR (400 MHz, DMSO-d6)
d
¼ 8.38 (s,1H, triazolyl-H), 7.66 (d, J ¼ 8.08 Hz, 2H, ArH), 7.35 (s, 2H,
eNH2), 7.19e6.98 (m, 6H, ArH), 5.15 and 5.01 (AB system,
J ¼ 14.92 Hz, 2H, eNCHaHb), 3.76 (s, 3H, OCH3), 2.68e2.66 (m, 2H, e
CH2), 2.24e2.22 (m, 2H, eCH2), 1.99e1.92 (m, 2H, eCH2); 13C NMR
Acknowledgments
(100 MHz, DMSO-d6):
d
¼ 195.29, 176.20, 166.30, 159.38, 158.62,
141.70, 133.48, 130.04, 128.10, 123.20, 122.85, 121.48, 121.05, 117.58,
115.13, 114.43, 113.67, 113.29, 108.47, 56.98, 55.58, 46.40, 35.45, 26.
74, 19.71; IR (nmax cmꢀ1, KBr): ¼3345, 3192, 1715, 1611, 1597; HRMS
(ESI) [Mþ þ H] calcd. for C27H22N6O4: 495.1780 found: 495.1786
[Mþ þ H].
HS and JS thank UGC, New Delhi, India for the grant of Junior and
senior Research Fellowships.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.016.
4.6.12. 70-Amino-10,30-dimethyl-1-((1-(4-nitrophenyl)-1H-1,2,3-
triazol-4-yl)methyl)-2,20,40-trioxo-10,20,30,40-tetrahydrospiro
[indoline-3,50-pyrano[2,3-d]pyrimidine]-60-carbonitrile (2l)
White Solid, M.pt.: 252 ꢁC, 1H NMR (400 MHz, DMSO-d6)
References
d
¼ 8.48 (s, 1H, triazolyl-H), 7.74 (d, J ¼ 8.18 Hz, 2H, ArH), 7.55 (s, 2H,
[1] M. Grare, M. Mourer, S. Fontanay, J.B. Regnouf-de-Vains, C. Finance, R.E. Duval,
Journal of Antimicrobial Chemotherapy 60 (2007) 575e581.
[2] C. Viegas-Junior, A. Danuello, V.S. Bolzani, E.J. Barreiro, C.A.M. Fraga, Current
Medicinal Chemistry 14 (2007) 1829e1852.
[3] C. Biot, K. Chibale, Infectious Disorders Drug Targets 6 (2006) 173e204.
[4] A. Chanda, V.V. Fokin, Chemical Reviews 109 (2009) 725e748.
[5] C.C.A. Cariou, G.J. Clarkson, M. Shipman, Journal of Organic Chemistry 73
(2008) 9762e9764.
[6] L. Zhou, D.S. Bohle, H.F. Jiang, C.J. Li, Synlett (2009) 937e940.
[7] G. Ren, J. Zhang, Z. Duan, M. Cui, Y. Wu, Australian Journal of Chemistry 62
(2009) 75e81.
[8] C. Mukhopadhyay, P.K. Tapaswi, M.G.B. Drew, Tetrahedron Letters 51 (2010)
3944e3950.
eNH2), 7.29e7.02 (m, 6H, ArH), 5.12 and 5.02 (AB system,
J ¼ 14.90 Hz, 2H, eNCHaHb), 3.26 (s, 3H, eNCH3), 3.05 (s, 3H, e
NCH3); 13C NMR (100 MHz, DMSO-d6):
d
¼ 182.28, 179.12, 160.22,
157.48,150.54, 145.84, 140.29, 129.76,134.44,132.50,130.72,128.34,
124.80, 122.46, 121.45, 120.30, 119.33, 117.65, 108.78, 101.28, 46.56,
34.08, 31.54, 28.67; IR (nmax cmꢀ1, KBr): ¼3370, 3150, 1710, 1696,
1520; HRMS (ESI) [Mþ þ H] calcd. for C26H19N9O6: 554.1536 found:
554.1540 [Mþ þ H].
4.6.13. 70-Amino-1-((1-(4-bromophenyl)-1H-1,2,3-triazol-4-yl)
methyl)-10,30-dimethyl-2,20,40-trioxo-10,20,30,40-tetrahydrospiro
[indoline-3,50-pyrano[2,3-d]pyrimidine]-60-carbonitrile (2m)
White Solid, M.pt.: 215 ꢁC, 1H NMR (400 MHz, DMSO-d6)
[9] N.G. Kozlov, A.P. Kadutskii, Tetrahedron Letters 49 (2008) 4560e4562.
[10] N. Isambert, M.M.S. Duque, J.C. Plaquevent, Y. Enisson, J. Rodriguez,
T. Constantieux, Chemical Society Reviews 40 (2011) 1347e1357.
[11] M. Petkovic, K.R. Seddon, L.P.N. Rebelo, C.S. Pereira, Chemical Society Reviews
40 (2011) 1383e1403.
d
¼ 8.58 (s, 1H, triazolyl-H), 7.82e7.74 (m, 4H, ArH), 7.68 (s, 2H, e
[12] (a) T. Welton, Chemical Reviews 99 (1999) 2071e2084;
(b) C. Gordon, Applied Catalysis A 222 (2001) 101e117;
(c) P. Wasserscheid, W. Keim, Angewandte Chemie International Edition 39
(2000) 3772e3789;
NH2), 7.23e7.20 (m, 2H, ArH), 7.03e6.97 (m, 2H, ArH), 5.13 and 5.04
(AB system, J ¼ 16.12 Hz, 2H, eNCHaHb), 3.36 (s, 3H, eNCH3), 3.03 (s,
3H, eNCH3); 13C NMR (100 MHz, DMSO-d6):
d
¼ 181.47, 176.22,
(d) M.J. Earle, K.R. Seddon, Pure and Applied Chemistry 72 (2000) 1391e1398;
(e) J. Dupont, R.F. Souza, P.A.Z. Suarez, Chemical Reviews 102 (2002) 3667e
3691.
162.43, 156.88, 151.44, 146.87, 140.89, 128.42, 132.33, 131.53, 130.58,
125.87, 123.88, 122.65, 121.55, 120.45, 119.23, 118.55, 107.88, 104.98,
45.56, 33.91, 30.67, 28.40; IR (nmax cmꢀ1, KBr): ¼ 3340, 3112, 1720,
1686, 1501; HRMS (ESI) [Mþ þ H] calcd. for C26H19BrN8O4: 587.0790
found: 587.0801 [Mþ þ H].
[13] (a) C.O. Kappe, Angewandte Chemie International Edition 43 (2004) 6250e
6284;
(b) R.S. Varma, Green Chemistry 1 (1999) 43e55;
(c) A.K. Bose, M.S. Manhas, S.N. Ganguly, A.H. Sharma, B.K. Banik, Synthesis
(2002) 1578e1591;
(d) D.R. Baghurst, D.M.P. Mingos, Chemical Society Reviews 20 (1991) 1e47.
[14] A.G. Ying, L.M. Wang, H.X. Deng, J.H. Chen, X.Z. Chen, W.D. Yeb, ARKIVOC 11
(2009) 288e298.
[15] J.F.M. Silva, S.J. Garden, A.C. Pinto, Journal of the Brazilian Chemical Society 12
(2001) 273e324.
[16] V. Glover, J.M. Halket, P.J. Watkins, A. Clow, B.L. Goodwin, M. Sandler, Journal
of Neurochemsitry 51 (1998) 656e659.
[17] R. Kumar, R.C. Bansal, A. Mahmood, Biogenic Amines 9 (1993) 281e289.
[18] A.E. Medvedev, A. Clow, M. Sandler, V. Glover, Biochemical Pharmacology 52
(1996) 385e391.
4.6.14. 70-Amino-10,30-dimethyl-2,20,40-trioxo-1-((1-4-tolyl-1H-
1,2,3-triazol-4-yl)methyl)-10,20,30,40-tetrahydrospiro[indoline-3,50-
pyrano[2,3-d]pyrimidine]-60-carbonitrile (2n)
White Solid, M.pt.: 215 ꢁC (decomp), 1H NMR (400 MHz, DMSO-
d6)
d
¼ 8.49 (s, 1H, triazolyl-H), 7.69 (s, 2H, eNH2), 7.63 (d,
J ¼ 8.24 Hz, 2H, ArH), 7.33 (d, J ¼ 8.24 Hz, 2H, ArH), 7.23e7.19 (m,
2H, ArH), 7.01e6.97 (m, 2H, ArH), 5.13 and 5. 08 (AB system,

154
H. Singh et al. / European Journal of Medicinal Chemistry 77 (2014) 145e154
[19] (a) S.K. Bhattacharya, S.K. Mitra, S.B. Acharya, Journal of Psychopharmacology
5 (1991) 202e206;
[31] M. Kume, T. Kubota, Y. Kimura, H. Nakashimizu, K. Motokawa, M. Nakano, The
Journal of Antibiotics 46 (1993) 177e192.
(b) S.K. Bhattacharya, V. Glover, I. McIntyre, G. Oxenkrug, M. Sandler,
Neuroscience Letters 92 (1982) 218e221.
[20] M. Yamazaki, E. Okuyama, Tetrahedron Letters 22 (1981) 135e136.
[21] J. Zamocka, E. Misikova, Pharmazie 41 (1991) 610e611.
[22] T. Ohira, M. Yatagai, Journal of the Japan Wood Research Society 39 (1993)
237e242.
[32] I.K. Boddy, G.G. Briggs, R.P. Harrison, T.H. Jones, M.J. O’Mahony, I.D. Marlow,
B.G. Roberts, R.J. Willis, R. Bardsley, Journal of Pesticide Science 48 (1996)
189e196.
[33] K.H. Buechel, H. Gold, P.E. Frohberger, H. Kaspers, German Patent 2407305,
1975; Chemical Abstracts, 83 (1975) 206.
[34] H. Singh, J. Sindhu, J.M. Khurana, Journal of the Iranian Chemical Society 10
(2013) 883e888;
[23] S.J. Mohr, M.A. Chirigos, F.S. Fuhrman, J.W. Pryor, Cancer Research 35 (1975)
3750e3754.
(a) J.Sindhu, H.Singh, J. M.Khurana, C.Sharma, K. R.Aneja, Australian Journal of
[24] V.K. Tandon, M. Vaish, S. Jain, D.S. Bhakuni, R.C. Srimal, Indian Journal of
Pharmaceutical Sciences 53 (1991) 22e23.
Chemistry, 66(2013) 710 e 717; (b) H. Singh, J. Sindhu, J.M. Khurana,
C. Sharma, K.R. Aneja, Australian Journal of Chemistry 66 (2013) 1088e1096;
(c) H. Singh, J. Sindhu, J.M. Khurana, RSC Advances 3 (2013) 22360e22366;
(d) H. Singh, J. Sindhu, J.M. Khurana, C. Sharma, K.R. Aneja, RSC Advances 4
(2014) 5915e5926;
(e) H. Singh, J. Sindhu, J.M. Khurana, Sensors and Actuators B: Chemical 192
(2014) 536e542;
[25] B.L. Bourdonnec, R.T. Windh, L.K. Leister, Q.J. Zhou, C.W. Ajello, M. Gu,
G.H. Chu, P.A. Tuthill, W.M. Barker, M. Koblish, D.D. Wiant, T.M. Graczyk,
S. Belanger, J.A. Cassel, M.S. Feschenko, B.L. Brogdon, S.A. Smith, M. Der-
elanko, S. Kutz, P.J. Little, R.N. Dehaven, D.L. Dehaven-Hudkins, R.E. Dolle,
Journal of Medicinal Chemistry 52 (2009) 5685e5702.
[26] T.S. Chen, S.J. Wu, I.L. Tsai, T.S. Wu, J.M. Pezzuto, M.C. Lu, H. Chai, N. Suh,
C.M. Teng, Journal of Natural Products 57 (1994) 1206e1211.
[27] S. Velazquez, R. Alvarez, C. Perez, F. Gago, M.J. Camarasa, Antiviral Chemistry &
Chemotherapy 9 (1998) 481e489.
(f) J.M. Khurana, A. Chaudhary, A. Lumb, B. Nand, Green Chemistry 14 (2012)
2321e2327.
[35] H.M. Guardiola, L. Foster, D. Mushrush, A.D. Vaz, Biochemical Pharmacology
61 (2001) 1463e1470.
[28] M.J. Genin, D.A. Allwine, D.J. Anderson, M.R. Barbachyn, D.E. Emmert,
S.A. Garmon, D.R. Graber, K.C. Grega, J.B. Hester, D.K. Hutchinson, J. Morris,
R.J. Reischer, C.W. Ford, G.E. Zurenko, J.C. Hamel, R.D. Schaadt, D. Stapert,
B.H. Yagi, Journal of Medicinal Chemistry 43 (2000) 953e970.
[29] A.K. Jordão, V.F. Ferreira, T.M. Souza, G.G. Faria, V. Machado, J.L. Abrantes,
M.C. Souza, A.C. Cunha, Bioorganic and Medicinal Chemistry 19 (2011) 1860e
1865.
[36] W. Samee1, O. Vajragupta, African Journal of Pharmacy and Pharmacology 5
(2011) 477e485.
[37] A.G. Ying, L. Liu, G.F. Wu, G. Chen, X.Z. Chen, W.D. Ye, Tetrahedron Letters 50
(2009) 1653e1657.
ꢀ
[38] J. Nowicki, M. Muszynskia, S. Gryglewiczb, Journal of Chemical Technology
and Biotechnology 89 (2014) 48e55.
[39] R. Bouhfid, Synthetic Communications 41 (2011) 2096e2102.
[30] S.G. Agalave, S.R. Maujan, V.S. Pore, Chemistry e An Asian Journal 6 (2011)
2696e2718.