Click Chemistry Approach for Bis-Chromenyl Triazole Hybrids and Their Antitubercular Activity

Article abstract of DOI:10.1111/j.1747-0285.2012.01441.x

1,4-Disubstituted bis-chromenyl triazole hybrids 5a-m have been synthesized in a three-step reaction sequence from 4-(bromomethyl)-2H-chromen-2-ones 3a-m. The intermediate azides 4a-m underwent a regioselective 1,3-dipolar cycloaddition with a 2H-chromen-2-one linked acetylenic dipolarophile in the presence of Cu (II)/ascorbate/water/n-butanol reaction medium. Three compounds 5h-j exhibited 6.25μg/mL MIC against M. tuberculosis. Among the compounds screened for antifungal activity, lowest MIC of 6.25μg/mL was observed for 5c against A. niger that also exhibited DNA cleavage observed by agarose gel electrophoresis. All the compounds were moderately active against both Gram-positive and Gram-negative bacterial strains. The cytotoxic effect of potent compounds on normal cells (V79 and HBL100) was assessed by MTT assay.

Full text of DOI:10.1111/j.1747-0285.2012.01441.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01441.x
Chem Biol Drug Des 2012
Research Article
Click Chemistry Approach for Bis-Chromenyl
Triazole Hybrids and Their Antitubercular Activity
Reshma J. Naik1, Manohar V. Kulkarni1,*,
possessing hydrazone moiety, akin to naturally occurring calanolide
(8), have recently been screened against M. tuberculosis (9). Biodeg-
radation of 2H-chromen-2-ones leads to in situ generation of carbox-
ylic and phenolic -OH groups that might facilitate the penetration
K. Sreedhara Ranganath Pai2 and Pawan G.
Nayak2
1P.G. Department of Chemistry, Karnatak University, Dharwad 580
through the cell wall of the bacterial species (10). 1,2,3-Triazoles have
been the nuclei of choice in recent years because of their excellent
pharmacokinetic characteristics, favourable safety profile, latent abil-
ity for the formation of hydrogen bonds, moderate dipole character,
rigidity and stability under in vivo conditions (11). Further, they have
also been found to be inhibitors of thymidine monophosphate kinase
(TMP Kinase), which catalyses the phosphorylation of deoxythymidine
monophosphate (dTMP) to deoxythymidine diphosphate (dTDP) utiliz-
ing ATP as a phosphoryl donor (12).
003, Karnataka, India
2Department of Pharmacology, Manipal College of Pharmaceutical
Sciences, Manipal University, Manipal-576104, Karnataka, India
*Corresponding author: Manohar V. Kulkarni,
manohar274@gmail.com
1,4-Disubstituted bis-chromenyl triazole hybrids
5a–m have been synthesized in
a
three-step
reaction sequence from 4-(bromomethyl)-2H-chro-
men-2-ones 3a–m. The intermediate azides 4a–m
underwent a regioselective 1,3-dipolar cycloaddi-
tion with a 2H-chromen-2-one linked acetylenic di-
polarophile in the presence of Cu (II)/ascorbate/
water/n-butanol reaction medium. Three com-
pounds 5h–j exhibited 6.25 lg/mL MIC against
M. tuberculosis. Among the compounds screened
for antifungal activity, lowest MIC of 6.25 lg/mL
was observed for 5c against A. niger that also
exhibited DNA cleavage observed by agarose gel
electrophoresis. All the compounds were moder-
ately active against both Gram-positive and Gram-
negative bacterial strains. The cytotoxic effect of
potent compounds on normal cells (V79 and
HBL100) was assessed by MTT assay.
'Click' chemistry has emerged as a new trend for constructing
1,2,3-triazoles through exergonic Huisgen 1,3-dipolar cycloaddition
of alkynes to azides (13). Classical Huisgen 1,3-dipolar cycloaddition
with unsymmetrical dipolarophiles leads to a mixture of 1,4- and
1,5-regioisomers. Innovations in click chemistry have demonstrated
the use of copper and ruthenium salts under aqueous conditions for
the regioselective synthesis of 1,4 and 1,5-disubstituted triazoles,
respectively, which cannot be achieved in the conventional Huisgen
approaches. 1,4-Substituted triazoles were synthesized by copper-
catalyzed azide alkyne cycloaddition reaction using sodium ascor-
bate as reducing agent (14), and synthesis of the same compound
with 1,5-substitution was achieved by using ruthenium catalyst (15).
For 1,4-substituted triazoles, the origin of the observed regioselec-
tivity lies in the inherent ability of Cu(I) to form an in situ covalent
bond with the acetylenic dipolariphile and coordinate exclusively
with the nitrogen that is linked to the R-group in the azide. The
nucleophilic attack of the anionic nitrogen on the other actylenic
carbon is followed by ring closure, which is the rate-determining
step in this stepwise mechanism. This pathway was predicted by
DFT calculations (16) and has been supported by a real-time infra-
red analysis (17). Unusual anisotropic effects observed in the DMAD
adducts of 4-azido methyl 2H-chromen-2-ones have been reported
by our group (18). In the light of the above observations, it was
thought of considerable interest to undertake a regioselective syn-
thesis of 2H-chromen-2-one triazole hybrids as potential antituber-
cular and antimicrobial agents using click chemistry conditions.
Key words: 1,2,3-triazole, 2H-chromen-2-one, antibacterial, antifun-
gal, antitubercular, click reaction, cytotoxic
Received 12 January 2012, revised 7 June 2012 and accepted for publi-
cation 19 June 2012
Designing drugs for chemotherapy of tuberculosis continues to be an
active area of medicinal chemistry in view of the rapid emergence of
multidrug-resistant strains. Molecular and structural diversity associ-
ated with heterocycles have made them the most promising candi-
dates for antitubercular activity (1). Pharmacophoric moieties like
hydrazone, guanidine, b-amino ethanol, b-amino glycoside, etc. have
been incorporated in quinoline (2), triazole (3), 2H-chromen-2-one (4),
benzofuran (5), thiophene (6), etc., which have resulted in potent
antitubercular compounds exhibiting low minimum inhibitory concen-
trations (Figure 1). In view of their extensive natural occurrence and
biocompatibility, 2H-chromen-2-ones have been found to exhibit
variety of biological activities (7). 2H-chromen-2-one derivatives
Experimental Section
Materials and methods
The melting points were determined by open capillary method and
are uncorrected. The IR spectra (KBr disc) were recorded on a Nico-
1

Naik et al.
General procedure for the synthesis of
4-(azidomethyl)-2H-chromen-2-ones (4a–m)
4-(Bromomethyl)-2H-chromen-2-ones 3a–m (0.01 mol equiv) was
taken in 20 mL of acetone in a round-bottom flask. To this, sodium
azide (0.012 mol equiv) in 3 mL of water was added drop wise with
stirring, which was continued for 10 h (reaction was monitored by
TLC). The reaction mixture was then poured in to ice-cold water.
Separated solid was filtered and recrystallized using ethanol
(18,23). Among azides, four azides (4e, 4f, 4l, 4m) are newly syn-
thesized.
4-(Azidomethyl)-5,7-dimethyl-2H-chromen-2-one
(4e)
Colourless solid (ethanol); yield, 72%; mp 156–158 ꢀC. IR (KBr) per
1
cm 1711 (C=O), 2110 (N3); H-NMR (CDCl3, 400 MHz, TMS): d ppm
2.37 (s, 3H, CH3), 2.65 (s, 3H, CH3), 4.65 (s, 2H, –CH2N–), 6.45 (s,
1H, C3-H), 6.91 (s, 1H, C6-H), 7.02 (s, 1H, C8-H). MS m ⁄ z 229. Anal.
Calcd for C12H11N3O2 (%): Calcd. C, 62.87; H, 4.84; N, 18.33; Found:
C, 62.73; H, 4.82; N, 18.24.
Figure 1: Antimycobacterial agents possessing triazole and 2H-
chromen-2-one moieties.
4-(Azidomethyl)-7,8-dimethyl-2H-chromen-2-one
(4f)
let-5700 FT-IR spectrophotometer. 1H-NMR spectra were recorded
on Bruker 300 MHz spectrometer using CDCl3 and DMSO-d6 as
solvents and TMS as an internal standard (Figure S1). The chemical
shifts are expressed in d ppm. The mass spectra were recorded
using Agilent-single Quartz GC-MS. The elemental analysis was car-
ried out using Heraus CHN rapid analyzer. The purity of the com-
pound was checked by T.L.C. All the chemicals purchased were of
analytical grade and were used without further purification unless
otherwise stated.
Colourless solid (ethanol); yield, 70%; mp 130–132 ꢀC. IR (KBr) per
1
cm 1715 (C=O), 2117 (N3); H-NMR (CDCl3, 400 MHz, TMS): d ppm
2.38 (s, 3H, CH3), 2.40 (s, 3H, CH3), 4.54 (s, 2H, –CH2N–), 6.46 (s,
1H, C3-H), 7.12 (d, 1H, C6-H, J = 8Hz), 7.27 (d, 1H, C5-H, J = 8Hz).
MS m ⁄ z 229. Anal. Calcd for C12H11N3O2 (%): Calcd. C, 62.87; H,
4.84; N, 18.33; Found: C, 62.82; H, 4.79; N, 18.29.
4-(Azidomethyl)-6-bromo-2H-chromen-2-one (4l)
Yield solid (ethanol); yield, 74%; mp 120–122 ꢀC. IR (KBr) per cm
1
1719 (C=O), 2132(N3); H- NMR (CDCl3, 400 MHz, TMS): d ppm 4.55
Procedure for the synthesis of 4-methyl-7-(prop-
2-ynyloxy)-2H-chromen-2-one (2)
(s, 2H, –CH2N–), 6.56 (s, 1H, C3-H), 7.25–7.66 (m, 3H, Ar-H). MS
m ⁄ z 279. Anal. Calcd for C10H6BrN3O2 (%): Calcd. C, 42.88; H, 2.16;
N, 15.00; Found: C, 42.78; H, 2.12; N, 14.98.
To a solution of 7-hydroxy-4-methyl-2H-chromen-2-one 1 (0.01 mol
equiv) in dry acetone, anhydrous potassium carbonate (0.01 mol
equiv) and 3-bromoprop-1-yne (0.01 mol equiv) were added. The
resultant mixture was stirred at 50 ꢀC for 18 h, cooled and the
solvent was removed under reduced pressure. The residue was
treated with 50 mL of cold water, and the so-obtained white solid
was filtered and washed with water (19). The crude product was
purified by crystallization from ethyl alcohol. White solid; yield,
95%; mp 132–134 ꢀC, IR (KBr) per cm 1726 (C=O), 3304 (C”C–H);
1H-NMR (CDCl3, 300 MHz, TMS): d ppm 2.71 (s, 3H, CH3), 2.59 (s,
1H, C”C–H), 4.77 (s, 2H, –CH2O–), 6.16 (s, 1H, C3-H), 6.95–6.93
(m, 2H, C8-H & C6-H), 7.55–7.52 (m, 1H, C5-H). MS m ⁄ z 214. Anal.
Calcd for C13H10O3 (%): Calcd. C, 72.89; H, 4.71; Found: C, 72.76;
H, 4.78.
4-(Azidomethyl)-7-methyl-6,8-dinitro-2H-
chromen-2-one (4m)
Yield solid (ethanol); yield, 74%; mp 122–124 ꢀC. IR (KBr) per cm
1
1750 (C=O), 2117 (N3); H-NMR (CDCl3, 400 MHz, TMS): d ppm 2.61
(s, 3H, CH3), 4.65 (s, 2H, –CH2N–), 6.70 (s, 1H, C3-H), 8.32 (s, 1H,
C5-H). MS m ⁄ z 305. Anal. Calcd for C11H7N5O6 (%): Calcd. C, 43.29;
H, 2.31; N, 22.95; Found: C, 43.22; H, 2.30; N, 22.92.
General procedure for the synthesis of
4-methyl-7-((1-((2-oxo-2H-chromen-4-yl)methyl)-
1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-
ones (5a–m)
To a solution of compound 2 (1.0 molar equiv) in tert-BuOH ⁄ H2O
1 ⁄ 1(v ⁄ v), CuSO4. 5H2O (0.15 molar equiv) and sodium ascorbate
(0.30 molar equiv) were added. The mixture was stirred at room
temperature for 15 min. Then azide 4a–m (1.0 molar equiv) were
General procedure for the synthesis of 4-
(bromomethyl)-2H-chromen-2-ones (3a–m)
The required 4-(bromomethyl)-2H-chromen-2-ones (20,21) have been
synthesized by the Pechmann cyclization of substituted phenols with
ethyl 4-bromoacetoacetate (22).
2
Chem Biol Drug Des 2012

Bis-Chromenyl Triazole Hybrids and Their Antitubercular Activity
added, and the resulting reaction mixture was refluxed on water
6.19 (s, 2H, –CH2N), 6.23 (s, 1H, C3-H), 7.07–7.71 (m, 5H, Ar-H), 8.37
(s, 1H, Tri-H). MS m ⁄ z 443. Anal. Calcd for C25H21N3O5 (%): Calcd. C,
67.71; H, 4.77; N, 9.48; Found: C, 67.75; H, 4.73; N, 9.52.
bath until the starting material was consumed as judged by TLC.
Then the reaction mixture was cooled, obtained solid was filtered
and washed with water and recrystallized from DMF.
4-Methyl-7-((1-((7,8-dimethyl-2-oxo-2H-chromen-
4-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (5f)
White solid; yield, 96%; mp 210–212 ꢀC. IR (KBr) per cm 1714 (C=O);
1H-NMR (DMSO-d6, 300 MHz, TMS): d ppm 2.28 (s, 3H, CH3), 2.36
(s, 3H, CH3), 2.39 (s, 3H, CH3), 5.31 (s, 2H, –CH2O–), 5.80 (s, 1H, C3-
H), 5.97 (s, 2H, –CH2N), 6.22 (s, 1H, C3-H), 7.02–7.69 (m, 5H, Ar-H),
8.45 (s, 1H, Tri-H). MS m ⁄ z 443. Anal. Calcd for C25H21N3O5 (%):
Calcd. C, 67.71; H, 4.77; N, 9.48; Found: C, 67.68; H, 4.76; N, 9.50.
4-Methyl-7-((1-((6-methyl-2-oxo-2H-chromen-4-
yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (5a)
White solid; yield, 97%; mp 236–238 ꢀC, IR (KBr) per cm 1741
1
(C=O), 1742 (C=O); H-NMR (DMSO-d6, 300 MHz, TMS): d ppm 2.37
(s, 3H, CH3), 2.40 (s, 3H, CH3), 5.33 (s, 2H, –CH2O–), 5.78 (s, 1H, C3-
H), 6.00 (s, 2H, –CH2N), 6.23 (s, 1H, C3-H), 7.03–7.70 (m, 6H, Ar-H),
8.45 (s, 1H, Tri-H). MS m ⁄ z 429. Anal. Calcd for C24H19N3O5 (%):
Calcd. C, 67.13; H, 4.46; N, 9.79; Found: C, 67.12; H, 4.42; N, 9.82.
4-Methyl-7-((1-((7-hydroxy-2-oxo-2H-chromen-4-
yl)methyl)-1H-1,2,3-triazol-4-yl) methoxy)-2H-
chromen-2-one (5g)
4-Methyl-7-((1-((7-methyl-2-oxo-2H-chromen-4-
yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (5b)
White solid; yield, 96%; mp 118–120 ꢀC. IR (KBr) per cm 1719
(C=O); 1H-NMR (DMSO-d6, 300 MHz, TMS): d ppm 2.42 (s, 6H,
2CH3), 5.32 (s, 2H, –CH2O–), 5.80 (s, 1H, C3-H), 5.98 (s, 2H, –CH2N),
6.23 (s, 1H, C3-H), 7.05–7.71 (m, 6H, Ar-H), 8.44 (s, 1H, Tri-H). MS
m ⁄ z 429. Anal. Calcd for C24H19N3O5 (%): Calcd. C, 67.13; H, 4.46;
N, 9.79; Found: C, 67.15; H, 4.44; N, 9.80.
Yellow solid; yield, 93%; mp 224–226 ꢀC. IR (KBr) per cm 1709
1
(C=O), 3437 (OH); H-NMR (DMSO-d6, 300 MHz, TMS): d ppm 2.40
(s, 3H, CH3), 3.43 (s, 1H, OH), 5.33 (s, 2H, –CH2O–), 5.57 (s, 1H, C3-
H), 5.95 (s, 2H, –CH2N), 6.22 (s, 1H, C3-H), 6.80–7.70 (m, 6H, Ar-H),
8.45 (s, 1H, Tri-H). MS m ⁄ z 431. Anal. Calcd for C23H17N3O6 (%):
Calcd. C, 64.04; H, 3.97; N, 9.74; Found: C, 64.06; H, 3.97; N, 9.79.
4-Methyl-7-((1-((6-chloro-2-oxo-2H-chromen-4-
yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (5h)
4-Methyl-7-((1-((6-methoxy-2-oxo-2H-chromen-4-
yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (5c)
Yellow solid; yield, 95%; mp 190–192 ꢀC. IR (KBr) per cm 1709
1
(C=O), 1724 (C=O); H-NMR (DMSO-d6, 300 MHz, TMS): d ppm 2.39
White solid; yield, 96%; mp 218–220 ꢀC. IR (KBr) per cm 1711
(s, 3H, CH3), 5.33 (s, 2H, –CH2O–), 5.90 (s, 1H, C3-H), 6.02 (s, 2H, –
CH2N), 6.22 (s, 1H, C3-H), 7.03–7.94 (m, 6H, Ar-H), 8.43 (s, 1H, Tri-
H). 13C-NMR (DMSO-d6, 400 MHz, TMS): d ppm 18.08, 48.99,
61.53, 101.67, 111.33, 112.63, 113.42, 115.05, 118.49, 118.78,
124.31, 125.96, 126.47, 128.59, 132.19, 142.72, 149.08, 151.76,
153.32, 154.61, 158.84, 160.06, 160.86. MS m ⁄ z 449. Anal. Calcd
for C23H16ClN3O5 (%): Calcd. C, 61.41; H, 3.59; N, 9.34; Found: C,
61.42; H, 3.57; N, 9.36.
1
(C=O); H-NMR (DMSO-d6, 300 MHz, TMS): d ppm 2.40 (s, 3H, CH3),
3.79 (s, 3H, OCH3), 5.33 (s, 2H, –CH2O–), 5.89 (s, 1H, C3-H), 6.01 (s,
2H, –CH2N), 6.22 (s, 1H, C3-H), 7.02–7.70 (m, 6H, Ar-H), 8.45 (s, 1H,
Tri-H). MS m ⁄ z 445. Anal. Calcd for C24H19N3O6 (%): Calcd. C,
64.72; H, 4.30; N, 9.43; Found: C, 64.74; H, 4.29; N, 9.40.
4-Methyl-7-((1-((7-methoxy-2-oxo-2H-chromen-4-
yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (5d)
White solid; yield, 96%; mp 220–222 ꢀC. IR (KBr) per cm
1711(C=O); 1H-NMR (DMSO-d6, 300 MHz, TMS): d ppm 2.39 (s,
3H, CH3), 3.86 (s, 3H, -OCH3), 5.32 (s, 2H, –CH2O–), 5.68 (s, 1H,
C3-H), 5.97 (s, 2H, –CH2N), 6.22 (s, 1H, C3-H), 6.98–7.95 (m, 6H,
Ar-H), 8.44 (s, 1H, Tri-H). MS m ⁄ z 445. Anal. Calcd for C24H19N3O6
(%): Calcd. C, 64.72; H, 4.30; N, 9.43; Found: C, 64.75; H, 4.32; N,
9.42.
4-Methyl-7-((1-((7-chloro-2-oxo-2H-chromen-4-
yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (5i)
Yellow solid; yield, 94%; mp 232–234 ꢀC IR (KBr) per cm 1713
1
(C=O); H-NMR (DMSO-d6, 300 MHz, TMS): d ppm 2.40 (s, 3H, CH3),
5.32 (s, 2H, –CH2O–), 5.92 (s, 1H, C3-H), 6.00 (s, 2H, –CH2N), 6.22
(s, 1H, C3-H), 7.02–7.87 (m, 6H, Ar-H), 8.43 (s, 1H, Tri-H). MS m ⁄ z
449. Anal. Calcd for C23H16ClN3O5 (%): Calcd. C, 61.41; H, 3.59; N,
9.34; Found: C, 61.43; H, 4.02; N, 9.29.
4-Methyl-7-((1-((5,7-dimethyl-2-oxo-2H-chromen-
4-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (5e)
White solid; yield, 94%; mp 248–250 ꢀC. IR (KBr) per cm 1712 (C=O);
1H-NMR (DMSO-d6, 300 MHz, TMS): d ppm 2.36 (s, 3H, CH3), 2.40 (s,
3H, CH3), 2.70 (s, 3H, CH3), 5.10 (s, 1H, C3-H), 5.37 (s, 2H, –CH2O–),
1-((4-((4-methyl-2-oxo-2H-chromen-7-
yloxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-3H-
benzo[f]chromen-3-one (5j)
Yellow solid; yield, 96%; mp 256–258 ꢀC. IR (KBr) per cm
1719.7(C=O); 1H-NMR (DMSO-d6, 300 MHz, TMS): d ppm 2.40 (s,
Chem Biol Drug Des 2012
3

Naik et al.
3H, CH3), 5.34 (s, 2H, –CH2O–), 5.50 (s, 1H, C3-H), 6.22 (s, 1H, C3-
H), 6.49 (s, 2H, –CH2N), 7.61–8.46 (m, 10H, Ar-H, Tri-H). MS m ⁄ z
465. Anal. Calcd for C27H19N3O5 (%): Calcd. C, 69.67; H, 4.11; N,
9.03; Found: C, 69.69; H, 4.13; N, 9.02.
reagent and 10% tween 80 were added to the plate and incubated
for 24 h. A blue colour in the well was interpreted as no bacterial
growth, and pink colour was scored as growth. The MIC was
defined as lowest drug concentration that prevented the colour
change from blue to pink.
4-((4-((4-Methyl-2-oxo-2H-chromen-7-
yloxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-2H-
benzo[h]chromen-2-one (5k)
Antimicrobial screening
Nine dilutions of each drug have to be performed with BHI for MIC
(25). In the initial tube, 20 lL of drug was added into the 380 lL of
BHI broth. For dilutions, 200 lL of BHI broth was added into the
next nine tubes separately. Then from the initial tube, 200 lL was
transferred to the first tube containing 200 lL of BHI broth. This
was considered as 10)1 dilution. From 10)1 diluted tube, 200 lL
was transferred to second tube to make 10)2 dilution. The serial
dilution was repeated up to 10)9 dilution for each drug. From the
maintained stock cultures of required organisms, 5 lL was taken
and added into 2 mL of BHI (brain heart infusion) broth. In each seri-
ally diluted tube, 200 lL of above culture suspension was added.
The tubes were incubated for 24 h and observed for turbidity.
Yellow solid; yield, 96%; mp 230–232 ꢀC. IR (KBr) per cm 1712
1
(C=O); H-NMR (DMSO-d6, 300 MHz, TMS): d ppm 2.38 (s, 3H, CH3),
5.33 (s, 2H, –CH2O–), 5.97 (s, 1H, C3-H), 6.11 (s, 1H, C3-H), 6.21 (s,
2H, –CH2N), 7.04–8.48 (m, 10H, Ar-H, Tri-H). MS m ⁄ z 465. Anal.
Calcd for C27H19N3O5 (%): Calcd. C, 69.67; H, 4.11; N, 9.03; Found:
C, 69.65; H, 4.14; N, 9.01.
4-Methyl-7-((1-((6-bromo-2-oxo-2H-chromen-4-
yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
chromen-2-one (5l)
Yellow solid; yield, 93%; mp 188–190 ꢀC. IR (KBr) per cm 1723
1
(C=O); H-NMR (DMSO-d6, 300 MHz, TMS): d ppm 2.40 (s, 3H, CH3),
5.34 (s, 2H, –CH2O–), 5.89 (s,1H, C3-H), 6.02 (s, 2H, –CH2N), 6.22 (s,
1H, C3-H), 7.05–8.06 (m, 6H, Ar-H), 8.43 (s, 1H, Tri-H). 13C-NMR
(DMSO-d6, 400 MHz, TMS): d ppm 16.75, 47.67, 60.24, 100.38,
110.02, 111.32, 112.12, 113.68, 115.13, 117.64, 117.73, 124.62,
125.15, 125.90, 133.69, 141.40, 147.71, 150.87, 151.99, 153.31,
157.47, 158.73, 159.56. MS m ⁄ z 493. Anal. Calcd for
C23H16BrN3O5(%): Calcd. C, 55.89; H, 3.26; N, 8.50; Found: C, 55.91;
H, 3.24; N, 8.53.
Cytotoxic study
The cytotoxic effect of drugs on normal cells (V79 and HBL100) was
assessed by MTT assay (26). In brief, exponentially growing cells
(1 · 104 cells ⁄ well) were plated in 96-well plates and allowed to
adhere for 24 h prior to extract addition. The drugs were dissolved
in 0.1% DMSO and then diluted with the medium. The cells were
then exposed to different concentrations of drug (5–200 lg ⁄ mL) for
24 h. The cells in the control wells received medium containing the
same volume of DMSO (0.1%). After the incubation, 100 lL of MTT
reagent (1 mg ⁄ mL in PBS) was added, and cells were incubated for
an additional 4 h. The formazan produced by the viable cells was
solubilized by addition of 100 lL DMSO. The suspension was
placed on a microvibrator for 5 min, and absorbance was recorded
at 540 nm by the plate reader (ELx800; BioTek, Winooski, VT, USA).
The experiment was performed in triplicate. Doxorubicin was used
as positive control. The percentage of growth inhibition was calcu-
lated with respect to vehicle control using the formula:
4-Methyl-7-((1-((7-methyl-6,8-dinitro-2-oxo-2H-
chromen-4-yl)methyl)-1H-1,2,3-triazol-4-
yl)methoxy)-2H-chromen-2-one (5m)
Yellow solid; yield, 93%; mp 206–208 ꢀC. IR (KBr) per cm 1757
1
(C=O), 1714 (C=O); H-NMR (DMSO-d6, 300 MHz, TMS): d ppm 2.40
(s, 3H, CH3), 2.51 (s, 3H, CH3), 5.32 (s, 2H, –CH2O–), 6.07 (s, 2H, –
CH2N), 6.22 (s, 2H, 2C3-H), 7.04–8.41 (m, 4H, Ar-H), 8.72 (s, 1H, Tri-
H). MS m ⁄ z 519. Anal. Calcd for C24H17N5O9 (%): Calcd. C, 61.47;
H, 4.13; N, 11.47; Found: C, 61.48; H, 4.12; N,11.43.
% Inhibition = [{(Control absorbance ) Blank absorbance) ) (Test
absorbance ) Blank absorbance)} ⁄ (Control absorbance ) Blank
absorbance)] · 100.
Biological evaluation
Anti-TB activity using Alamar Blue Dye
DNA cleavage experiment
Antimycobacterial activity of compounds was assessed against
M. tuberculosis using microplate Alamar Blue assay (MABA) (24).
This methodology is non-toxic, uses a thermally stable reagent and
shows good correlation with proportional and BACTEC radiometric
method. Briefly, 200 lL of sterile deionized water was added to all
outer perimeter wells of sterile 96-well plate to minimize evapora-
tion of medium in the test wells during incubation. The 96-well
plate received 100 lL of the Middlebrook 7H9 broth, and serial
dilution of compounds was made directly on plate. The final drug
concentrations tested were 100–0.2 lg ⁄ mL. Plates were covered
and sealed with parafilm and incubated at 37 ꢀC for 5 days. After
this time, 25 lL of freshly prepared 1:1 mixture of Almar Blue
Preparation of culture media
Potato dextrose broth (PDB): 250 g of peeled potato was boiled for
20 min and squeezed and filtered (27). To this filtrate, 20 g of dex-
trose was added, and the volume was made up to 1000 mL by dis-
tilled water. The spores of the culture were inoculated in to the
autoclaved media and grown at ꢀ27 ꢀC for 48 h.
Isolation of DNA
Two gram of sample was grounded using 25 mL of prechilled mortar
and pestle. 1 mL of lysozyme solution was added to the above sus-
4
Chem Biol Drug Des 2012

Bis-Chromenyl Triazole Hybrids and Their Antitubercular Activity
pension and incubated at 37 ꢀC for 30 min, shaking occasionally.
When the gel attains ꢀ55 ꢀC, it was poured into the gel cassette fit-
ted with comb. After the gel was solidified, the comb was carefully
removed, and the gel was placed in the electrophoresis chamber
flooded with TAE buffer. 20 lL of DNA sample (mixed with brom-
ophenol blue dye at 1:1 ratio) was loaded carefully into the wells,
along with standard DNA marker and pass the constant 50 V of elec-
tricity for around 30 min. The gel was removed and carefully stained
with ethidium bromide solution (2 lg ⁄ mL) for 10–15 min, and the
bands were observed under UV transilluminator (UVP, Germany).
After the incubation, the lysis was completed by adding 2 mL of
sodium dodecyl sulphate (SDS) solution, this preparation was heated
for 10 min at a 60 ꢀC water bath, and finally, the solution was cooled
to room temperature. 5 M sodium chloride solution was added to the
lysed preparation. An equal volume of chloroform–ethanol mixture
(24:1) was added to the lysed preparation suspended in 1 M sodium
chloride and slowly shaken (30–60 oscillations ⁄ min) in a tightly stop-
pered flask for 30 min at room temperature. The resulting emulsion
was separated by centrifugation for 5 min at 10 000 · g at room
temperature. After centrifuging, the top aqueous solution was care-
fully pipetted out from the coagulated protein emulsion at the inter-
phase. The pipetted aqueous phase was kept in a beaker. The nucleic
acid solution was gently stirred with a sterilized glass rod while
slowly adding 95% ethanol down the side of a beaker, so that ethanol
is layered over the viscous aqueous phase. The preparation was stir-
red to mix ethanol throughout the entire aqueous phase, and all the
gelatinous thread-like DNA-rich precipitate was spooled off by the
glass rod. Excess fluid from the spooled crude DNA was drained off
by pressing the rod against the wall of the beaker until no further
fluid can be squeezed from the spooled preparation. The crude DNA
was dissolved on stirring it in 9 mL of dilute (1 ⁄ 10 fold) saline citrate
in a test tube or small beaker. 3 M sodium acetate and 1 mM EDTA
were added to the even suspension (pH 7.0). The preparation was
transferred to a 100-mL beaker, and the sample was gently swirled by
dripping in 5.4 mL isopropanol. The pellet was centrifuged at 8 500 g
for 10 min and dried at room temperature. The pellet was dissolved
in minimum volume of Tris–HCl buffer (50 mM, pH 8.2).
Result and Discussion
Chemistry
Acetylenic dipolarophile used in this investigation, 4-methyl-7-(prop-
2-ynyloxy)-2H-chromen-2-one 2, was synthesized by the reaction of 7-
hydroxy 4-methyl 2H-chromen-2-one 1 with 3-bromoprop-1-yne using
potassium carbonate as base in acetone medium. Ethyl
4-bromoacetoacetate obtained from the bromination of ethylacetoac-
etate was treated with substituted phenols under Pechmann cycliza-
tion conditions using neat sulphuric acid as the condensing agent.
The reaction resulted in the formation of 4-(bromomethyl)-2H-chro-
men-2-ones 3a–m. The reactivity of 4-(bromomethyl)-2H-chromen-2-
ones has been explored in a cascaded manner via an allylic displace-
ment. The required dipolar azide intermediates 4a–m were
synthesized by the reaction of sodium azide with various 4-(bromom-
ethyl)-2H-chromen-2-ones 3a–m in aqueous acetone at room
temperature and were quite stable even above 100 ꢀC. Copper cataly-
sed the reaction of various dipolar 2H-chromen-2-one azides with
acetylenic dipolarophile in the presence of sodium ascorbate in aque-
ous medium resulting in the exclusive formation of 1,4-substituted
bis-chromenyl triazole hybrids, which is in accordance with the earlier
observations on click reactions (14) (Scheme1).
Agarose gel electrophoresis
A total of 200 mg of agarose was weighed and dissolved in 25 mL
of TAE buffer (4.84 g Tris base, pH 8.0, 0.5 M EDTA ⁄ L) by boiling.
Scheme 1: Synthesis of 4-
methyl-7-((1-((2-oxo-2H-chromen-4-
yl)methyl)-1H-1,2,3-triazol-4-yl)meth-
oxy)-2H-chromen-2-ones 5a–m.
Chem Biol Drug Des 2012
5

Naik et al.
Formation of acetylenic dipolarophile 2 was supported by the appear-
ance of two singlets in the 1H-NMR at 2.59 and 4.77 ppm because of
C”C–H and C”C–CH2 protons, respectively. In the 1H-NMR spectrum
of the azides (4e, 4f, 4l, 4m), the C4-CH2 protons linked to the azide
group were observed between 4.54 and 4.65 ppm, and the C3-H of
OMe), 5e (C5,C7-diMe) and 5f (C7,C8-diMe) showed weak activity
with MIC > 100 lg ⁄ mL. Compounds 5a (C6-Me), 5g (C7-OH), 5k
(benzo[h] fused chromen), 5l (C6-Br), 5m (C7-Me-C6,C8-diNO2)
showed good activity with MIC of 12.50 lg ⁄ mL. Compounds 5h (C6-
Cl), 5i (C7-Cl) and 5j (benzo[f] fused chromen derivative) were as
highly active as streptomycin with MIC of 6.25 lg ⁄ mL. Above obser-
vations clearly indicate that chloro and benzo substituents on the
coumarin ring have remarkable impact on the antitubercular activity
of the title compounds. Thus, chloro and benzo substituents reinforce
the antitubercular activity of coumarin triazole hybrids.
2H-chromen-2-one appeared as
a singlet between 6.45 and
6.70 ppm. In IR spectrum of azide, band at 2117–2132 per cm was
observed because of the presence of azide group. The IR spectrum of
triazole adduct 5a–m showed absence of azide band at 2117–2132
1
per cm, and the H-NMR spectrum showed two interesting features
viz., (i) C4-CH2 protons showed a downfield shift and were observed
at 6.00 ppm when compared with 4.56 ppm observed in the case of
4-arylamino methyl 2H-chromen-2-one. (ii) The C3-H of 2H-chromen-2-
one in the triazole adduct experienced an upfield shift and was
observed as a singlet at 5.78 ppm, as against 6.60–6.70 ppm in the
case of both 4-arylaminomehtyl and 4-phenoxymethyl 2H-chromen-2-
one (20,28). The observed results are in agreement with our earlier
report (18). This rare type of shielding effect of 2H-chromen-2-one C3-
H and simultaneous pronounced deshielding of the C4-CH2 protons
were because of the triazole on the C4-CH2 group, which has been
consistently observed in spectral data of all the synthesized bis-
chromenyl triazole hybrids.
2H-chromen-2-one derivatives possessing hydrazone moiety (8) and
benzyl triazoles (1) exhibited antimycobacterial activity with MIC of
50–100 lg ⁄ mL and 16 lg ⁄ mL, respectively. Compared with the
above compounds, bis-chromenyl triazole hybrids exhibited higher
antimycobacterial (6.25 lg ⁄ mL) activity. Thus, the attachment of
triazole moiety to the 2H-chromen-2-one dramatically increases the
antimycobacterial activity of the parental 2H-chromen-2-one
compared with chromenes possessing the hydrazone unit. This
comparison clearly supports the existence of a better effect in 2H-
chromen-2-one triazole hybrids.
Antibacterial screening
Biology
All the synthesized titled compounds were evaluated for their antibac-
terial activity against (i) Gram-positive bacteria: Streptococcus faecal-
is (MTCC 3382) and Staphylococcus aureus (MTCC 3160) and (ii)
Gram-negative bacteria: Pseudomonas aeruginosa (MTCC 1034) and
Escherichia coli (MTCC 1089). The antibacterial data (Table 1)
revealed that some of the bis-chromenyl triazole hybrids exhibited
Anti-TB activity using Alamar Blue Dye
All the synthesized titled compounds were evaluated for their anti-
mycobacterial activity against M. tuberculosis using MABA. The data
(Table 1) revealed that compounds 5b (C7-Me), 5c (C6-OMe), 5d (C7-
Table 1: Results of biological evaluation of compounds 5a–m MICs (lg ⁄ mL)
Antibacterial activity
Gram-positive
Gram-negative
Antifungal activity
O
O
O
N
N
N
O
O
R
Streptococcus
faecalis
vStreptococcus
aureus
Pseudomonas
aeruginosa
Escherichia
coli
Candida
albicans
Aspergillus
niger
Compounds
R
Anti tubercular activity
5a
5b
C6-Me
C7-Me
C6-OMe
C7-OMe
C5,C7-diMe
C7,C8-diMe
C7-OH
C6-Cl
C7-Cl
benzo[f]
benzo[h]
C6-Br
>100
50
50
50
100
50
>100
50
50
50
50
50
25
50
50
50
25
25
25
25
25
50
50
25
1
>100
>100
25
>100
>100
>100
>100
25
50
50
>100
>100
>100
1
>100
>100
25
25
25
50
50
50
50
50
50
25
50
1
25
50
50
50
50
25
>100
50
50
50
50
25
50
–
50
25
6.25
25
25
50
50
25
25
50
25
25
50
–
12.5
>100
>100
>100
>100
>100
12.5
6.25
6.25
6.25
12.5
12.5
12.5
–
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
50
>100
1
C7-Me-C6,C8-diNO2
Ciprofloxacin
Streptomycin
Fluconazole
–
–
–
–
–
–
–
–
–
–
–
–
16
–
8
6.25
–
6
Chem Biol Drug Des 2012

Bis-Chromenyl Triazole Hybrids and Their Antitubercular Activity
Table 2: IC50 value of after 24 h drug incubation with V79 and
HBL100 cell lines by MTT assay
IC50 (lg ⁄ mL)
V79
Compounds
HBL100
C6-Cl
C7-Cl
Ben[f]
Doxorubicin
44.24 € 2.15
61.88 € 3.74
48.25 € 5.36
0.19 € 0.02
36.91 € 1.63
54.39 € 2.07
52.03 € 2.55
0.30 € 0.03 lM
Figure 2: Gel electrophoresis picture of compound 5c. Photo-
graph showing the effect of representative compound 5c on DNA
of A. niger. Lane M: DNA marker, Lane C: untreated DNA, Lanes 1,
2, 3, 4, 5 and 6 correspond to 1.562, 3.125, 6.25, 12.5, 25,
50 lg ⁄ mL.
moderate activity. Compounds 5b (C7-Me), 5f (C7,C8-diMe), 5g (C7-
OH), 5h (C6-Cl), 5i (C7-Cl), 5j (benzo[f] fused chromen) and 5m (C7-
Me-C6,C8-diNO2) showed moderate activity with MIC of 25 lg ⁄ mL
against S. aureus. Compounds 5c (C6-OMe) and 5h (C6-Cl) groups
showed moderate activity with MIC of 25 lg ⁄ mL against P. aerugin-
osa. Compounds 5c (C6-OMe), 5d (C7-OMe), 5e (C5,C7-diMe) and 5l
(C6-Br) showed moderate activity with MIC of 25 lg ⁄ mL against
E. coli. All compounds showed weak activity against S. faecalis. The
compounds screened had no significant specificity for Gram-positive
or Gram-negative species. In all the cases, -OCH3 and -Cl groups had
the best effect on the antimicrobial activity of the tested compounds.
chromen-2-one, have been carried out against A. niger by agarose
gel electrophoresis method. Gel electrophoresis technique works on
the migration of DNA under the influence of electric potential. The
photograph (Figure 2) shows the molecular weight differences com-
pared with control and is the differentiating criterion for the DNA
cleaving ability of the tested compounds with A. niger. Control
experiments using DNA alone does not indicate any significant
cleavage of DNA even after long exposure time. After marker M
and control C, the first six lanes correspond to six different concen-
trations viz., 50, 25, 12.5, 6.25, 3.125, 1.562 lg ⁄ mL. Compound 5c
with concentration 50 lg ⁄ mL shows the absence of the control
band, which could be attributed to the DNA cleavage, but later the
extent of cleavage has decreased with the decrease in the concen-
tration of compound 5c.
Antifungal screening
All the synthesized title compounds were screened for their antifun-
gal activity against Candida albicans and Aspergillus niger. The anti-
fungal data (Table 1) revealed that some of the bis-chromenyl triazole
hybrids exhibited moderate activity. Compounds 5a (C6-Me), 5f
(C7,C8-diMe) and 5l (C6-Br) showed moderate activity with MIC of
25 lg ⁄ mL against C. albicans. Compounds 5b (C7-Me), 5d (C7-OMe),
5e (C5, C7-diMe), 5h (C6-Cl), 5i (C7-Cl), 5k (benzo[h] fused chromen)
and 5l (C6-Br) showed moderate activity with MIC of 25 lg ⁄ mL
against A. niger. Compound 5c (C6-OMe) is highly active, which was
higher than the activity of fluconazole, with MIC of 6.25 lg ⁄ mL
against A. niger. The tested compounds possessing electron-donating
groups at C-6 and C-7 in the coumarin moiety enhanced the antifun-
gal activity, which was more significant against A.niger than C. albi-
cans. A fourfold reduction in MIC was observed in the 6-methoxy-
substituted compound 5c against A. niger, which confirms the effect
of electron-donating groups on the antifungal activity.
Conclusions
We have synthesized a series of bis-chromenyl triazole hybrids
under click reaction conditions. Antimycobacterial screening data
reveal that the synthesized compounds showed significant antimyco-
bacterial activity against M. tuberculosis. Compounds 5h (C6-Cl), 5i
(C7-Cl) and 5j (benzo[f] fused chromen) showed activity equivalent
to streptomycin. All the compounds showed moderate activity
against microbials. On the other hand, 5c (C6-OMe) showed excel-
lent fungicidal activity, which was higher than fluconazole, and
DNA cleavage against A. niger. These compounds act as potent
antitubercular agent rather than antimicrobial agent and are active
against Gram-positive bacteria compared with Gram-negative bacte-
ria. Cytotoxicity study reveals that compounds do not inhibit the
growth of normal cell lines (V79 and HBL100) even at higher con-
centrations. Higher activity of these compounds in antitubercular
studies indicates that these compounds may act as good structural
leads in developing new antimycobacterial drugs.
Cytotoxic study
The potent compounds 5h, 5i and 5j (C6-Cl, C7-Cl and benzo[f]) were
tested in normal cells (V79 and HBL100) for toxicity and showed IC50
ranging between 40 and 60 lg ⁄ mL (Table 2). As observed from the
MIC values (6.25 lg ⁄ mL), the compounds were microbicidal at signifi-
cantly lower doses, four to six times lesser than IC50 values. There-
fore, it may be inferred that the compounds do not inhibit the growth
of normal cell lines (V79 and HBL100), at concentrations active for mi-
crobicidal action. These classes of compounds may be taken further
for in vivo testing to confirm their microbicidal activity.
Acknowledgments
Reshma Naik is grateful to DST, New Delhi, for the financial assis-
tance in the form of INSPIRE Fellowship. We thank University Sci-
entific Instrumentation Center (USIC), Karnatak University, Dharwad,
for IR, NMR, MS and analytical data.
DNA cleavage analysis
The DNA cleavage studies of 5c (C6-OMe), 4-methyl-7-((1-((6-meth-
oxy-2-oxo-2H-chromen-4-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-
Chem Biol Drug Des 2012
7

Naik et al.
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Conflicts of Interest
Authors have no conflicts of interest.
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Additional Supporting Information may be found in the online ver-
sion of this article:
1
Figure S1. H and 13C NMR spectra.
Please note: Wiley-Blackwell is not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
corresponding author for the article.
8
Chem Biol Drug Des 2012