Click-chemistry-based multicomponent condensation approach for design and synthesis of spirochromene-tethered 1,2,3-triazoles as potential antitubercular agents

Article abstract of DOI:10.1007/s11164-017-2955-y

A series of spirochromene-tethered 1,2,3-triazoles (1,2,3-triazolylspirochromenes) were designed and synthesized via click-chemistry-based one-pot five-component reaction between N-propargyl isatins, malononitrile, dimedone (or 4-hydroxyl-6-methyl-2H-pyran-2-one), arylalkyl halides, and sodium azide using cellulose-supported CuI nanoparticles (Cell-CuI NPs) as heterogeneous catalyst. All synthesized compounds were screened for inhibitory activity against Mycobacterium tuberculosis H37Ra (ATCC 25177) and Mycobacterium bovis BCG (ATCC 35743), in active as well as dormant state. During screening, compounds 6h, j, l were found to exhibit promising antimycobacterial activity against M.?bovis BCG, while compounds 7d, h, l showed promising antimycobacterial activity against M.?tuberculosis H37Ra as well as M.?bovis BCG. The active compounds were found to be noncytotoxic to three human cancer cell lines (MCF-7, HCT116, and A549). The active compounds exhibited selectivity index >10, indicating potential as antitubercular agents. The active compounds were also evaluated for in?vitro antibacterial activity, with five (6h, l, 7b, f, l) showing good antibacterial activity against Gram-positive as well as Gram-negative bacteria. Compounds 6h, j, l exhibiting promising activity against M.?bovis BCG can serve as good leads for further modification and optimization.

Full text of DOI:10.1007/s11164-017-2955-y

Res Chem Intermed
DOI 10.1007/s11164-017-2955-y
Click-chemistry-based multicomponent condensation
approach for design and synthesis of spirochromene-
tethered 1,2,3-triazoles as potential antitubercular
agents
Pramod V. Chavan1 Kapil S. Pandit1
•
•
Uday V. Desai1 Prakash P. Wadgaonkar2
Laxman Nawale3 Sujit Bhansali3 Dhiman Sarkar3
•
•
•
•
Received: 12 November 2016 / Accepted: 8 April 2017
Ó Springer Science+Business Media Dordrecht 2017
Abstract A series of spirochromene-tethered 1,2,3-triazoles (1,2,3-triazolyl-
spirochromenes) were designed and synthesized via click-chemistry-based one-pot
five-component reaction between N-propargyl isatins, malononitrile, dimedone (or
4-hydroxyl-6-methyl-2H-pyran-2-one), arylalkyl halides, and sodium azide using
cellulose-supported CuI nanoparticles (Cell-CuI NPs) as heterogeneous catalyst. All
synthesized compounds were screened for inhibitory activity against Mycobac-
terium tuberculosis H37Ra (ATCC 25177) and Mycobacterium bovis BCG (ATCC
35743), in active as well as dormant state. During screening, compounds 6h, j,
l were found to exhibit promising antimycobacterial activity against M. bovis BCG,
while compounds 7d, h, l showed promising antimycobacterial activity against
M. tuberculosis H37Ra as well as M. bovis BCG. The active compounds were found
to be noncytotoxic to three human cancer cell lines (MCF-7, HCT116, and A549).
The active compounds exhibited selectivity index [10, indicating potential as
antitubercular agents. The active compounds were also evaluated for in vitro
antibacterial activity, with five (6h, l, 7b, f, l) showing good antibacterial activity
against Gram-positive as well as Gram-negative bacteria. Compounds 6h, j, l ex-
hibiting promising activity against M. bovis BCG can serve as good leads for further
modification and optimization.
Electronic supplementary material The online version of this article (doi:10.1007/s11164-017-2955-y)
contains supplementary material, which is available to authorized users.
&
Uday V. Desai
uvdchem2011@gmail.com
1
2
Department of Chemistry, Shivaji University, Kolhapur 416 004, India
Polymer Science and Engineering Division, CSIR National Chemical Laboratory,
Pune 411 008, India
3
Combi Chem–Bio Resource Centre, CSIR National Chemical Laboratory, Pune 411 008, India
123

P. V. Chavan et al.
Keywords Click chemistry Á 1,2,3-Triazoles Á Triazolylspirochromenes Á
Tuberculosis Á Cytotoxicity Á Heterogeneous catalysis
Introduction
Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis,
remaining one of the greatest health problems even in the 21st century [1, 2]. The
main problem associated with spread of tuberculosis is emergence of strains
resistant to one or more drugs and the very long time required for treatment of
multiple-drug-resistant tuberculosis (MDR-TB) as well as extensively drug-resistant
tuberculosis (XDR-TB) [3–6]. For the last few years, the combination of rifampicin,
ethambutol, and isoniazid remains the effective therapy for treatment of TB [7] and
as far as discovery of new, effective drugs to encounter TB is concerned, except
bedaquiline, no remarkable breakthroughs have been achieved in the recent past.
With the possibility that tubercular bacilli will acquire resistance to all these drugs,
there is an urgent need to develop new antitubercular agents [8]. Amongst various
approaches developed for introduction of new antitubercular agents, molecular
hybridization involving design of new chemical entities by covalent fusion of two or
more pharmacophores is an attractive strategy [9–12]. Synthesis of such hybrid
molecules may involve separate synthesis of two or more subunits with known
bioprofiles followed by their combination in a final step, or synthesis in a single step
using a multicomponent synthesis strategy [13–17]. Amongst these approaches, the
multicomponent condensation pathway is certainly advantageous in terms of atom
economy, yield, selectivity, efficiency, and minimum waste generation [18]. With
all these concepts in mind and our continued interest in multicomponent synthesis
[19–25], we undertook synthesis of novel hybrid molecules as possible antituber-
cular agents.
It is well known that, owing to their diverse biological properties, heterocyclic
compounds play a very important role in organic chemistry. Among various
heterocyclic compounds, spiroheterocycles, especially spirooxindoles, represent a
medicinally privileged scaffold present in many naturally occurring bioactive
alkaloids (Fig. 1a) [26–29]. In recent years, spirooxindole derivatives fused with
2-amino-4H-chromene unit, viz. spirochromenes (Fig. 1b), have become immensely
important due to their diuretic, spasmolytic, anticancer, anticoagulant, and
antianaphylactic activities [30–32]. Like spirochromenes, compounds containing
1,2,3-triazole structural motif are also known to possess antitubercular, anticancer,
antifungal, antiallergic, antiviral, antimicrobial, antiepileptic, as well as anti-human
immunodeficiency virus (HIV) properties (Fig. 1c–f) [33–48]. Considering the
biological potential of spirochromenes as well as of 1,2,3-triazoles, we surmised that
hybrid molecules containing both of these structural units (Fig. 2) may possess
enhanced antitubercular activity.
From the retrosynthetic viewpoint, synthesis of such hybrid molecules involves
construction of the spirochromene motif in a first step then construction of the 1,2,3-
triazole unit in a subsequent step, or vice versa. The best known pathway for
synthesis of spirochromenes involves multicomponent condensation between isatin,
123

Click-chemistry-based multicomponent condensation…
Fig. 1 Spirooxindole- and 1,2,3-triazole-containing bioactive compounds
Fig. 2 1,2,3-Triazolyl-
spirochromene hybrid molecule
active methylene compound (malononitrile/alkyl cyanoacetate), and cyclic 1,3-
dicarbonyl compound (dimedone, 4-hydroxyl-6-methyl-2H-pyran-2-one, etc.). Such
condensation is reported to proceed under the influence of base as well as CuI as
catalyst [49, 50]. On the other hand, copper-catalyzed azide–alkyne cycloaddition
(CuAAC, click chemistry) remains the approach of choice for synthesis of 1,2,3-
triazoles [51–55] (Scheme 1a, b).
Scheme 1 Retrosynthetic analysis for synthesis of spirochromenes and 1,2,3-triazoles
123

P. V. Chavan et al.
We previously reported cellulose-supported cuprous iodide nanoparticles (Cell-
CuI NPs) as heterogeneous and reusable catalyst for synthesis of 1,4-disubstituted
1,2,3-triazoles [56] and recently also reported environmentally benign protocols for
synthesis of 2-amino-4H-chromenes [19–21]. Based on these results, we speculated
that, if cuprous iodide nanoparticles (Cell-CuI NPs) are effective for construction of
spirochromene moiety, it would be possible to synthesize the targeted hybrid
molecules using a one-pot five-component condensation approach. As an outcome
of this philosophy, we report herein synthesis of spirochromene-tethered 1,2,3-
triazoles by one-pot five-component condensation between N-propargyl isatins,
malononitrile, dimedone (or 4-hydroxy-6-methyl-2H-pyran-2-one), arylalkyl
halides, and sodium azide using Cell-CuI NPs as reusable catalyst (Scheme 2).
Results and discussion
Chemistry
The synthetic route started with preparation of N-propargyl isatins. Various
substituted N-propargylated isatins were prepared by potassium-carbonate-catalyzed
propargylation of isatins with propargyl bromide using dimethylformamide (DMF)
as solvent and tetra-n-butylammonium bromide (TBAB) as phase-transfer catalyst
[56].
We first focused on exploring the feasibility of using Cell-CuI NPs as catalyst for
synthesis of spirochromenes. Accordingly, a model reaction was performed using N-
propargyl isatin, malononitrile, and dimedone as substrates (Scheme 3). Thus, to an
equimolar mixture of N-propargyl isatin, malononitrile, and dimedone (1 mmol,
each) in ethanol (5 mL) were added Cell-CuI NPs (100 mg, 3.7 mol% CuI)
followed by continuous stirring at ambient temperature for 12 h. We did not observe
any appreciable reaction progress, so the reaction was continued under reflux
condition. Timely analysis of the reaction mixture by thin-layer chromatography
Scheme 2 One-pot five-component synthesis of 1,2,3-triazolylspirochromenes 6, 7
123

Click-chemistry-based multicomponent condensation…
Scheme 3 Cell-CuI NP-catalyzed synthesis of spirochromenes
(TLC) indicated formation of a new product in major quantity. Upon reaction
completion, the resultant product was isolated, purified by column chromatography,
and identified as the expected spirochromene, A (78%, Scheme 3). Our primary aim
with this trial reaction was simply to check the feasibility of using Cell-CuI NPs in
synthesis of spirochromenes; however, as the desired spirochromene A was
obtained in acceptable yield (78%) during the trial reaction, no further attempts
were made towards improving the yield of A (Scheme 3).
Encouraged by this initial success, we next planned to undertake one-pot
synthesis of 1,2,3-triazolylspirochromene 6a (Scheme 2, R1 = R2 = H) as model
compound. Accordingly, to well-stirred solution of N-propargyl isatin, malonon-
itrile, dimedone, benzyl bromide (1 mmol, each), and sodium azide (1.1 mmol) in
ethanol (5 mL) were added Cell-CuI NPs (200 mg, 7.4 mol% CuI). Stirring was
continued under reflux, and reaction progress monitored by TLC. Upon reaction
completion followed by work-up as well as spectral analysis, we were pleased to
note formation of expected 1,2,3-triazolylspirochromene 6a in acceptable yield
(72%). Thereafter, attempts were directed towards improving the yield of desired
6a. In this context, the model reaction was repeated in different reaction media. It
was observed that, in common organic solvents such as toluene, acetonitrile, as well
as tetrahydrofuran, the yield of the desired product 6a, remained nearly the same.
However, with water, water–ethanol, as well as water–acetonitrile as reaction
medium, significantly increased yield of desired 6a was found (Table 1, entries 3, 7,
8). However, with these water-based reaction media, product isolation proved to be
slightly cumbersome. Hence, the model reaction was then carried out in
dimethylformamide–water (2:1, v/v) as mixed solvent system (Table 1, entries
9–11). This modification proved to be useful in terms of both yield as well as
isolation of the desired product 6a (Table 1, entry 9). The results summarized in
Table 1 also reveal that use of less than the optimized amount of catalyst failed to
furnish the desired product 6a in the same yield (Table 1, entries 10, 11). It is
noteworthy that, in absence of added catalyst, the reaction furnished desired product
6a in low yield (35%).
Having established appropriate reaction conditions, we turned our attention to
examine the generality of the reaction conditions. Thus, the benzyl bromide
component of the model reaction was replaced by 4-methyl (4b), 4-fluoro (4c), and
4-chlorobenzyl bromide (4d). Under the optimized reaction conditions, correspond-
ing 1,2,3-triazolylspirochromenes 6b–d were produced in excellent yield (Table 2).
123

P. V. Chavan et al.
Table 1 Optimization of reaction conditions for one-pot synthesis of 1,2,3-triazolylspirochromene 6a
Entry
Solvent
Catalyst (mg)
Temp. (°C)
Time (h)
Yielda (%)
1
Ethanol
200
200
200
200
200
200
200
200
200
150
100
RT
6
5
6
5
5
6
5
5
2
3
4
70
85
84
70
75
60
86
80
92
83
80
2
Ethanol
Reflux
80
3
Water
4
Toluene
100
5
Acetonitrile
Reflux
Reflux
Reflux
80
6
Tetrahydrofuran
Water–ethanol (1:1)
Water–acetonitrile (1:1)
Water 1 DMF (2:1)
Water ? DMF (2:1)
Water ? DMF (2:1)
7
8
9
70
10
11
70
70
Reaction conditions: N-propargyl isatin, malononitrile, dimedone, benzyl bromide (1 mmol, each), NaN3
(1.1 mmol), Cell-CuI NPs, solvent
b
Yields refer to pure isolated product
To examine the scope of the reaction conditions, various N-propargylated isatins
derived from 5-chloroisatin (1b), 5-bromoisatin (1c), and 5-fluoroisatin (1d) were
then used. Once again, the reaction furnished expected 1,2,3-triazolylspirochrome-
nes 6e–p in acceptable yield (Table 2). Finally, the dimedone component in each
successful reaction was replaced with 4-hydroxyl-6-methyl-2H-pyran-2-one (3b)
(Scheme 2). Most gratifyingly, in each case, corresponding 1,2,3-triazolyl-
spirochromenes 7a–p were produced in acceptable yield (Table 2). In this
multicomponent synthesis of 1,2,3-triazolylspirochromenes under the influence of
copper catalyst, formation of the chromene unit precedes construction of the 1,2,3-
triazole unit (TLC). A plausible mechanism for the formation of the desired 1,2,3-
triazolylspirochromene is depicted in Fig. 3.
In heterogeneously catalyzed reactions, reusability as well as stability of the
catalyst is of importance from both economic and environmental points of view.
Hence, upon completion of the model reaction, the catalyst was separated by
filtration, washed repeatedly with ethyl acetate as well as acetone, dried in air, and
used in the next run with the same substrates. The recovered catalyst also furnished
desired 1,2,3-triazolylspirochromene 6a in nearly the same yield (Fig. 4). Subse-
quent studies on reusability of the catalyst revealed that the catalyst could be used
for four consecutive cycles without appreciable change in the yield of the desired
triazole 6a.
In copper-catalyzed click synthesis of biologically active molecules, study of
catalyst leaching is also of prime importance. Therefore, upon reaction completion
followed by catalyst separation, resultant filtrate was analyzed by atomic absorption
spectroscopy, revealing no detectable copper content.
123

Click-chemistry-based multicomponent condensation…
Table 2 Cell-CuI NP-catalyzed synthesis of 1,2,3-triazolylspirochromenes 6, 7
Entry N-Propargyl 1,3-Dicarbonyl
isatin (1)
Arylalkyl
bromide (4)
Product Time Yielda (%) M.p. (°C)
compound (3a, b)
(6, 7)
(h)
1
1a
1a
1a
1a
1b
1b
1b
1b
1c
1c
1c
1c
1d
1d
1d
1d
1a
1a
1a
1a
1b
1b
1b
1b
1c
1c
1c
1c
1d
1d
1d
1d
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
3a
3b
3b
3b
3b
3b
3b
3b
3b
3b
3b
3b
3b
3b
3b
3b
3b
4a
4b
4c
4d
4a
4b
4c
4d
4a
4b
4c
4d
4a
4b
4c
4d
4a
4b
4c
4d
4a
4b
4c
4d
4a
4b
4c
4d
4a
4b
4c
4d
6a
6b
6c
6d
6e
6f
2
3
3
4
2
2
3
4
3
4
5
5
3
4
5
5
3
4
4
5
3
4
3
5
3
5
3
5
3
5
3
5
92
94
91
82
84
76
80
83
84
83
81
78
92
88
85
84
87
83
92
84
87
81
83
86
84
85
86
84
83
92
85
83
142–144
150–152
145–147
155–158
158–160
214–216
148–150
160–162
218–220
178–180
176–178
190–192
178–180
160–162
182–184
205–207
228–230
235–238
223–225
208–210
160–162
182–184
150–152
150–152
180–182
182–184
188–190
185–187
260–262
222–224
243–245
218–220
2
3
4
5
6
7
6g
6h
6i
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
6j
6k
6l
6m
6n
6o
6p
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
a
Reaction conditions: N-propargyl isatin, malononitrile, dimedone or 4-hydroxyl-6-methyl-2H-pyran-2-one,
arylalkyl bromide (1 mmol, each), NaN3 (1.1 mmol), Cell-CuI (7 mol%), water–DMF (2: 1, v/v, 6 mL), 70 °C
b
Yields refer to pure isolated product
123

P. V. Chavan et al.
Fig. 3 Plausible mechanism for synthesis of 1,2,3-triazolylspirochromene
Fig. 4 Reusability of Cell-CuI catalyst
123

Click-chemistry-based multicomponent condensation…
Biological evaluation
Antitubercular studies
As well as designing and synthesizing novel organic molecules, we also aimed to
screen their antitubercular activity. Hence, all synthesized compounds were
screened for in vitro antitubercular activity against M. tuberculosis H37Ra (MTB)
(ATCC 25177) and M. bovis BCG (ATCC 35743) by twofold dilution technique.
The minimum inhibitory concentration (MIC) and 50% inhibition concentration
(IC50) values of the tested compounds are presented in Table 3. Rifampicin, a drug
in regular clinical use, was applied as reference standard. During primary screening,
of 32 synthesized compounds, 20 were found to be active against both species with
MIC in the range of 2.87–10.00 lg/mL (Table 3). In particular, compounds 6h, j,
l were found to be most active against M. bovis BCG (MIC from 2.87 to 6.23 lg/
mL) in active as well as dormant state. On the other hand, compounds 7d, h, l were
found to be promisingly active (MIC \ 7 lg/mL) against both M. tuberculosis
H37Ra as well as M. bovis BCG. From the antimycobacterial activity data
summarized in Table 3, it is evident that the activity depends upon the nature of the
substituents R1 and R2 present on the phenyl rings. Replacement of hydrogen atoms
on phenyl rings at R1 and R2 by halogen resulted in increased antitubercular
activity. Amongst halogens, presence of chlorine substituent on phenyl as well as
isatin ring proved to be useful for enhanced antitubercular activity. Regarding the
choice of the 1,3-diketone, replacement of dimedone with 4-hydroxyl-6-methyl-2H-
pyran-2-one did not cause any significant change in the antimycobacterial activity
against M. bovis BCG, while for M. tuberculosis H37Ra it was increased to
moderate extent.
Cytotoxicity
All synthesized compounds (6a–7p) were then evaluated for their in vitro
cytotoxicity against three human cancer cell lines, viz. MCF-7, HCT116, and
A549, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay with paclitaxel as positive control [57–59]. The results are summarized in
Table 4. The 50% growth inhibition concentration (GI50) values ([100 lg/mL) of
the active compounds 6a–7f, m–p indicate that these compounds are potent and
specific inhibitors of MTB. Especially 7g–l were found to be active antiproliferative
compounds with GI50 in the range of 22.08–71.68 lg/mL against MCF-7 cell line
but not against HCT116 or A549.
GI50 values obtained from cytotoxicity studies allow calculation of the selectivity
index, which reflects the concentration of a compound at which it is active against
mycobacteria but not toxic towards host cells. Higher selectivity index indicates that
the compound can be used as a therapeutic agent. The selectivity of triazolyl-
spirochromenes 6a–7p towards human cell lines and against M. bovis BCG was
therefore described in terms of the selectivity index, and the results are presented in
123

P. V. Chavan et al.
Table 3 Antimycobacterial activity of various 1,2,3-triazolylspirochromenes
Entry
R1
R2
M. tuberculosis H37Ra
Active stage Dormant stage
M. bovis BCG
Active stage
Dormant stage
MIC
IC50
MIC
IC50
MIC
IC50
MIC
IC50
6a
6b
6c
H
H
[30
[30
[30
49.15
[30
[50
39.61
[50
39.79
29.23
46.35
[50
[30
[50
34.30
[30
[30
13.99
27.7
5.21
37.35
[50
[50
5.39
[30
[30
26.27
6.63
30
25.64 [30
27.81
[30
[30
12.4
29.59
11.11
17.36
3.06
7.06
5.1
28.22
[30
28.88
8.98
27.43
8.15
6.60
2.87
7.87
2.87
7.28
4.32
18.09
10.96
[50
12.37
27.54
7.42
7.51
2.97
13.07
5.24
8.04
3.04
5.93
10.39
6.82
4.12
30
7.8
26.56
[30
27.64
8.97
8.43
[30
13.75
4.26
16.93
4.2
H
Me
F
[30
[30
18.96
[30
[30
23.16
[30
16.44
3.6
H
6d
6e
H
Cl
H
Cl
Cl
Cl
Cl
Br
Br
Br
Br
F
29.43 [30
18.49
1.67
3.2
26.82
9.24
6f
Me
F
22.65
17.25
15.45
5.86
21.47
6g
6h
6i
23.96
8.07
9.44
3.45
0.6
Cl
H
1.33
3.47
1.33
3.17
1.8
3.09
13.48
8.89
4.66
2.06
1.33
2.42
1.24
4.73
4.63
3.25
5.34
16.76
1.97
3.25
2.06
1.42
1.26
1.14
1.56
4.21
5.11
2.61
1.14
15.61
9.17
17.39
4.57
0.075
6j
Me
F
3.24
6.23
6k
6l
29.81
22.81
10.33
10.60
5.38
5.89
25.93
14.05
8.33
17.65
15.19
1.71
4.53
1.43
13.93
7.93
6.6
8.09
Cl
H
3.16
6m
6n
6o
6p
7a
7b
7c
27.56 [30
3.78
4.13
13.65
5.34
15.46
1.68
2.91
0.7
19.39
18.69
23.91
13.86
26.48
5.36
F
Me
F
29.23
13.39
26.59
16.17
F
F
Cl
H
15.62 [30
16.53 [30
H
H
Me
F
2.13
4.31
1.63
18.2
17.67
42.06
1.84
7.3
H
16.16
4.39
10.13
4.35
7d
7e
H
Cl
Cl
Cl
Cl
Br
Br
Br
Br
F
Cl
H
23.09
11.59
11.18
4.81
5.53
2.53
2.42
0.45
2.36
5.8
5.74
7f
Me
F
5.23
7g
7h
7i
6.69
Cl
H
2.32
21.31
6.21
7.01
3.07
28.78
6.15
[30
8.88
0.05
7.73
14.63 [30
10.45
9.71
7j
Me
F
6.39
4.72
2.71
28.72
3.56
[30
4.47
0.002
[30
26.77
6.8
7k
7l
2.67
2.11
6.14
Cl
H
3.97
7m
7n
7o
7p
Rifampicin
30
17.53 [30
9.41 [30
18.57 [30
F
Me
F
29.56
[30
8.81
0.51
28.93
[30
4.5
[30
[30
23.12
0.45
F
F
Cl
5.14
17.04
0.81
0.75
0.005
MIC minimum inhibitory concentration, IC50 50% inhibitory concentration
Table 5. It is evident that compounds 6h, j, l, 7b, d, h, l exhibited selectivity index
value [10, indicating potential as antitubercular agents, thus they should be
investigated further as possible anti-TB agents.
123

Click-chemistry-based multicomponent condensation…
Table 4 In vitro cytotoxicity
profile of selected 1,2,3-
Product code
MCF-7
HCT116
GI50 (lg/mL)
A549
GI50 (lg/mL)
GI50 (lg/mL)
triazolylspirochromenes against
three human cancer cell lines
6a–7f
[100
43.22
71.68
22.08
29.12
41.73
48.57
[100
[100
0.0048
[100
[100
[100
[100
[100
[100
[100
[100
[100
0.026
[100
[100
[100
[100
[100
[100
[100
[100
[100
0.0035
7g
7h
7i
7j
7k
7l
7m–p
Rifampicin
Paclitaxel
GI50 50% growth inhibition
concentration
Table 5 Selectivity index (SI) of the most active compounds 6h, j, l, 7d, h, l against active as well as
dormant M. BCG vs. three human cancer cell lines
Product MIC against M. bovis SI against MCF-7
BCG (mg/mL)
SI against HCT116
(colon)
SI against A549
(lung)
(breast)
Active
state
Dormant
state
Active
state
Dormant
state
Active
state
Dormant
state
Active
state
Dormant
state
6h
6j
2.87
2.87
4.32
7.42
2.97
3.04
4.12
0.45
3.09
6.23
3.16
5.36
4.35
7.73
3.97
0.81
35
35
32
16
35
35
32
16
35
35
32
16
6l
23
32
23
32
23
32
7b
7d
7h
7l
13
19
13
19
13
19
34
23
34
23
34
23
24
09
33
13
33
13
12
12
24
25
24
25
Rif
222
123
222
123
222
123
Rif rifampicin
Antibacterial studies
Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus
subtilis are common pathogens that have harmful effects on human health. We
thought it worthwhile to evaluate the antimicrobial activity of 1,2,3-triazolyl-
spirochromenes against these microorganisms [60]. This assay was performed by
well plate method (zone of inhibition) using ampicillin and kanamycin as reference
standards. The results are summarized in Table 6 (ESI, Table S6). Five compounds
(6h, l, 7b, f, l) showed more than 90% inhibition at concentration of 3 lg/mL.
Compounds showing good antibacterial activity were further analyzed to determine
MIC values using dose–response curves. Compounds 6h, l, 7b, f, l showed good
antibacterial activity against E. coli, P. aeruginosa, S. aureus, and B. subtilis, with
MIC values varying from 1.1 to 5.29 lg/mL.
123

P. V. Chavan et al.
Table 6 In vitro antibacterial activity of 1,2,3-triazolylspirochromenes against E. coli, S. aureus,
P. aeruginosa, and B. subtilis
Product code
Gram negative
Gram positive
E. coli
MIC90 (lg/mL)
P. aeruginosa
MIC90 (lg/mL)
S. aureus
MIC90 (lg/mL)
B. subtilis
MIC90 (lg/mL)
6h
1.78
[30
2.86
1.97
29.89
2.65
[30
2.75
1.46
1.62
5.29
[30
1.57
3.08
26.28
4.88
[30
2.33
4.36
0.49
3.55
[30
1.09
4.27
[30
2.41
[30
3.86
1
1.14
[30
3.18
2.85
26.15
7.19
[30
5.27
10.32
1.35
6j
6l
7b
7d
7f
7h
7l
Amp.
Kana.
[30
Amp ampicillin, Kana kanamycin
All the results above clearly indicate that target compound 6h is mycobacteria
specific and can be further explored as a potential antitubercular drug.
Conclusions
Based on the structure of compounds with proven biological activity and our
experience in click chemistry, green chemistry, and multicomponent reactions,
studies were undertaken on the design and one-pot synthesis of novel compounds as
possible antitubercular agents. We designed and synthesized for the first time a
series of 1,2,3-triazolylspirochromenes by one-pot five-component condensation
between N-propargyl isatin, malononitrile, dimedone (or 4-hydroxyl-6-methyl-2H-
pyran-2-one), arylalkyl halide, and sodium azide using cellulose-supported CuI
nanoparticles (Cell-CuI NPs) as heterogeneous catalyst. Simple experimental
procedure, wide scope, and catalyst reusability for five consecutive runs are the
main merits of this ecobenign protocol. The synthesized compounds were screened
for their antimycobacterial activity. Among all the compounds, 6h, j, l showed very
good antimycobacterial activity against M. bovis BCG. Some of the synthesized
1,2,3-triazolylspirochromenes (7d, h, l) showed promising antimycobacterial
activity against Mycobacterium tuberculosis H37Ra as well as M. bovis BCG.
The active compounds were found to be noncytotoxic to three cell cancer lines.
Selectivity studies revealed that these active compounds have potential as possible
antitubercular agents. The specificity of these compounds was checked by screening
their antibacterial activity against four bacterial strains. Compounds 6h, l, 7b, f,
l showed good antibacterial activity. All the above screening results clearly indicate
that 1,2,3-triazolylspirochromenes can be further explored as potential antituber-
cular drugs via chemical modification.
123

Click-chemistry-based multicomponent condensation…
Acknowledgements U. V. D. and P. V. C. thank the University Grants Commission (UGC), New Delhi,
India for financial assistance [F. 43—221/2014 (SR)] and for the award of a teacher fellowship,
respectively. We gratefully acknowledge Dr. K. G. Kanade, Principal, Yashavantrao Chavan Institute of
Science, Satara, for encouragement.
Appendix
Experimental
General
Isatins (Aldrich), benzyl halides (Aldrich), sodium azide (S. D. Fine-Chem.
Limited, Mumbai), dimedone, malononitrile, copper(I) iodide (Spectrochem,
Mumbai), and microcrystalline cellulose (SRL, Mumbai) were used as received.
Cell-CuI NPs as catalyst were prepared according to the procedure developed and
reported by us earlier [56]. All melting points were recorded using Kumar melting
point apparatus. Infrared (IR) spectra were recorded neat using a Thermo Scientific
Nicolet iS10 FT-IR spectrometer. 1H nuclear magnetic resonance (NMR)
(300 MHz) and 13C NMR (75.4 MHz) spectra were recorded using Bruker
Avance II spectrometer. High-resolution mass spectra (HRMS) were recorded
using Thermo Scientific Q-Exactive, Accela 1250 pump, instrument.
Representative procedure for one-pot synthesis of 1,2,3-triazolylspirochromenes
N-Propargyl isatin (1 mmol), malononitrile (1 mmol), dimedone (1 mmol), benzyl
bromide (1 mmol), and sodium azide (1.1 mmol) were placed in a round-bottomed
flask. DMF–water (1:2, 6 mL, v/v) and cellulose-CuI NPs as catalyst (0.2 g) were
added, and the reaction mixture was heated at 70 °C. After reaction completion
(TLC), the reaction mixture was filtered and the filter was washed with ethyl acetate
(4 9 10 mL). The organic extract was washed with water and brine and dried over
Na2SO4. Removal of solvent under vacuum furnished corresponding 1,4-disubsti-
tuted 1,2,3-triazolylspirochromene as solid product. The resultant solid was
successively washed using a mixture of hexane–chloroform (80: 20 v/v), and dried.
None of the resultant products required any further purification. The recovered
catalyst was washed with acetone, dried in air, and reused for five consecutive runs.
Biological methods
Antimycobacterial activity
All synthesized compounds were screened for their in vitro activity against
M. tuberculosis H37Ra (MTB) (ATCC 25177) and M. bovis BCG (ATCC 35743)
using twofold dilution technique to determine the minimum inhibitory concentration
(MIC). Screening against M. tuberculosis H37Ra was carried out by XTT reduction
menadione assay (XRMA) by reading the absorbance at 470 nm, while screening of
M. bovis BCG was carried out by nitrate reductase (NR) assay [61–63]. The optical
123

P. V. Chavan et al.
density was read on a microplate reader using a 470-nm filter for XTT and a 540-nm
filter for NR against blank prepared from cell-free wells. Absorbance given by cells
treated with vehicle alone was taken as 100% cell growth. Initially, primary
screening was done at 30, 10, and 3 lg/ml. Compounds showing 90% inhibition of
bacilli at or lower than 30 lg/ml were selected for further dose–response curve
analysis. All experiments were performed in triplicate, with values expressed as
average standard deviation. MIC and IC50 values of selected compounds were
calculated from dose–response curves using Origin 6 software. Percentage inhibi-
tion was calculated using the formula: % inhibition = [(control - CMP)/(con-
trol - blank)] 9 100, where ‘‘control’’ is the activity of mycobacteria without
compounds, ‘‘CMP’’ is the activity of mycobacteria in presence of compound, and
‘‘blank’’ is the activity of culture medium without mycobacteria.
Cytotoxicity and selectivity index
To check the selectivity, all compounds were assayed for their cytotoxic effects
against three different cell lines (MCF-7, A549, and HCT116) by MTT assay
(Table 4) [64–66]. The cell lines were maintained at 37 °C under 5% CO2/95% air
humidified environment. The concentration range for each compound was selected as
30, 10, and 3 lg/mL. Each concentration was tested in duplicate in a single
experiment. GI50 and MIC values were calculated using OriginPro software. Viability
and growth in presence of test material were calculated using the following formula: %
cytotoxicity = [(average absorbance of control - absorbance of compound)/(ab-
sorbance of control - absorbance of blank)] 9 100, where control is the culture
medium with cells and dimethyl sulfoxide (DMSO), and blank is the culture medium
without cells. The selectivity index (SI) was calculated by dividing the 50% growth
inhibition concentration (GI50) value for each cell line (MCF-7, A549, and HCT116)
by the MIC for in vitro activity against active/dormant MTB and BCG [60].
Antibacterial activity
All bacterial cultures were first grown in Luria-Bertani (LB) medium at 37 °C at
180 RPM. Once the culture reached 1 OD, it was used for antibacterial assay. Bacterial
strains E. coli (NCIM 2688), P. aeruginosa (NCIM 2036) as Gram-negative and
B. subtilis (NCIM 2079), S. aureus (NCIM 2010) as Gram-positive strains were
obtained from NCIM (NCL, Pune) and grown in Luria-Bertani medium. Screening
was carried out by adding 0.1% 1-OD inoculated culture to each well of a 96-well plate
containing the compounds to be tested and measuring the optical density at 620 nm
after 8 h for Gram-negative or 12 h for Gram-positive bacteria.
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