Novel 4H-1,2,4-triazol-3-yl cycloalkanols as potent antitubercular agents

Article abstract of DOI:10.1007/s00044-012-0043-9

Enzymes of shikimate pathway, dehydroquinase and shikimate kinase represent comparatively newer targets for antitubercular research. Molecular hybridization approach was implemented by integrating the essential features of inhibitors acting on these enzymes of shikimate pathway. Considering the flexibility of alicyclic ring of reported dehydroquinase (DHQ) inhibitors and triazole ring, key feature of the virtual hits of Mtb shikimate kinase, a series of structurally novel, substituted 4H-1,2,4-triazol-3-yl cycloalkanols were designed as antimycobacterial agents. Docking studies of the molecules was carried out on the enzyme DHQ. All the synthesized compounds exhibited promising activity (MIC 0.59-15.5 μg/ml) against H₃₇Rv strains of Mycobacterium tuberculosis using resazurin microtiter assay. Five of the evaluated compounds exhibit MIC < 1 μg/ml. CC₅₀ values indicate compounds are non-toxic, with selectivity indices >28. These compounds could serve as leads for further optimization to obtain novel antimycobacterial agents.

Full text of DOI:10.1007/s00044-012-0043-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0043-9
ORIGINAL RESEARCH
Novel 4H-1,2,4-triazol-3-yl cycloalkanols as potent antitubercular
agents
•
Nutan H. Palsule Desai Ranjeet Bairwa
•
•
Manoj Kakwani Nilesh Tawari M. K. Ray
•
•
•
M. G. Rajan Mariam Degani
Received: 1 August 2011 / Accepted: 27 March 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Enzymes of shikimate pathway, dehydroquin-
ase and shikimate kinase represent comparatively newer
targets for antitubercular research. Molecular hybridization
approach was implemented by integrating the essential
features of inhibitors acting on these enzymes of shikimate
pathway. Considering the flexibility of alicyclic ring of
reported dehydroquinase (DHQ) inhibitors and triazole
ring, key feature of the virtual hits of Mtb shikimate kinase,
a series of structurally novel, substituted 4H-1,2,4-triazol-
3-yl cycloalkanols were designed as antimycobacterial
agents. Docking studies of the molecules was carried out
on the enzyme DHQ. All the synthesized compounds
exhibited promising activity (MIC 0.59–15.5 lg/ml)
against H37Rv strains of Mycobacterium tuberculosis using
resazurin microtiter assay. Five of the evaluated com-
pounds exhibit MIC \ 1 lg/ml. CC50 values indicate
compounds are non-toxic, with selectivity indices [28.
These compounds could serve as leads for further optimi-
zation to obtain novel antimycobacterial agents.
Introduction
Tuberculosis (TB) is an airborne infectious disease that is
preventable and curable. Up to a quarter of the people with
tuberculosis in some regions, remain untreated with stan-
dard drug regimens. The stark finding of the new World
Health Organization report estimates that 440,000 people
had multidrug-resistant TB (MDR-TB) in 2008, a third of
whom died. In the case of Multidrug and Extensively Drug-
Resistant Tuberculosis (M/XDR-TB), 2010 Global Report
on Surveillance and Response underlines that almost half
of all the cases of MDR-TB are estimated to occur in China
and India. In Africa, an estimated 69,000 cases have
emerged. The spread of HIV epidemic has worsened the
condition further (Global Report on Surveillance and
Report, 2010). Inspite of the pain staking efforts in the
antitubercular research, new drugs, having novel mecha-
nism of action, has remained a dream since 1965. Hence,
there is an urgent need to develop potent new inhibitors,
against latent and resistant tubercle bacilli, acting on new
targets which would have minimal chances of cross resis-
tance with current drugs.
Keywords Tuberculosis ꢀ Triazoles ꢀ Alicyclic ꢀ
Molecular hybridization ꢀ TMSCN
Targeting enzymes of various metabolic pathways of
Mycobacterium tuberculosis is a pivotal strategy for
development of newer antitubercular agents. Enzymes of
shikimate pathway, dehydroquinase and shikimate kinase
represent comparatively newer targets for antitubercular
research (Prazeres et al., 2006). Hence, dual inhibitors
acting on both these enzymes would be valuable in the
mycobacterial resistance scenario. Mutation studies of
genes encoding for the enzymes of shikimate pathway
suggests that mycobacteria cannot survive in the absence of
these enzymes. Evidence that shikimate pathway is
essential for M. tuberculosis, even in the presence of
exogenous supplements, reinforces its attractiveness as a
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-012-0043-9) contains supplementary
material, which is available to authorized users.
N. H. P. Desai ꢀ R. Bairwa ꢀ M. Kakwani ꢀ N. Tawari ꢀ
M. Degani (&)
Institute of Chemical Technology, Nathalal Parekh Marg,
Matunga, Mumbai 400019, India
e-mail: ms.degani@ictmumbai.edu.in; m_degani@yahoo.com
M. K. Ray ꢀ M. G. Rajan
Radiation Medicine Centre, Tata Memorial Hospital,
Parel, Mumbai, India
123

Med Chem Res
drug target (Parish and Stoker, 2002). The active site of
mycobacterial dehydroquinase enzyme is small and
accommodates small, flexible alicyclic molecules. Hence,
the alicyclic scaffold constitutes a backbone of reported
Mycobacterium tuberculosis dehydroquinase inhibitors
(Fig. 1a) (Concepcion and Luis, 2007). Also, a structure
based virtual screening protocol for shikimate kinase, has
been reported, wherein 42 % of the 644 hits obtained,
possess five membered heteroaromatic triazole or tetrazole
ring (Fig. 1b). According to the virtual studies, identified
triazole or tetrazole moieties could exhibit their potential as
antimycobacterial agents by their ability to mimic the key
interactions involved in shikimate kinase -substrate com-
plex formation (Cabrera and Perez, 2008). In addition, the
anti-tubercular activity of various substituted 1,2,4-tria-
zoles like 1,2,4-triazole-3-thione (Ku¨c¸u¨kgu¨zel et al., 2008),
3-pyridyl-1,2,4-triazole-5-thiol (Mali et al., 2009), clubbed
1,2,4-triazole (Shiradkar et al., 2007; Joshi et al., 2008)
1,2,4-triazol-3-yl-benzene-1,2,3-triol (Mandal et al., 2010)
etc. is reported. 1,2,4-Triazoles are also well known for
their antimycobacterial action via inhibiting the cell wall
biosynthesis (Bechet et al., 1972).
Fig. 2 General structure of 4H-1,2,4-triazol-3-yl cycloalkanols
compound 4a in the active site of Mtb type-II dehydro-
quinase and Fig. 3b shows that designed molecule fit well
and occupy the space of active site cavity of Mtb type-II
dehydroquinase. Further, design of 4H-1,2,4-triazol-3-yl
cycloalkanols involved substitution of the triazole ring with
various groups like aliphatic, aromatic, heteroaromatic
substituents etc. QikProp (2008) analysis of these designed
compounds was carried out to determine their drug-likeli-
ness and compounds showing good results were further
considered for synthesis.
Based on these literature reports, molecular hybridiza-
tion approach was implemented, to explore molecular
scaffolds having flexibility of alicyclic ring of reported
DHQ inhibitors and potential enhancement of antimyco-
bacterial activity owing to triazole ring, key feature of the
virtual hits of Mtb shikimate kinase (Fig. 1c).
Experimental
Accordingly, novel 4H-1,2,4-triazol-3-yl cycloalkanols
were designed which would exhibit promising antituber-
cular activity (Fig. 2).
Chemistry
A retrosynthetic analysis of the designed compounds sug-
gested that reaction of various alicyclic cyanohydrins with
aliphatic, aromatic or heteroaromatic hydrazides would
lead to corresponding triazoles. General scheme for syn-
thesis of these triazoles is represented in Scheme 1.
Docking study of the designed molecules was carried
out in the active site of Mtb type-II dehydroquinase (PDB
code: 1HOS) using validated protocol of GLIDE module of
¨
Schrodinger LLC, NY. It was observed that these mole-
cules exhibit favourable binding interactions with the
active site residues (Arg 19, Tyr 24 and Ile 102) of Mtb
type-II dehydroquinase, similar to those of the reported
inhibitors. Figure 3a shows the H-bond interaction of
Various substituted and unsubstituted alicyclic alde-
hydes and ketones 1a–w were subjected to cyanation
reaction using sodium cyanide to fetch corresponding
Fig. 1 Molecular hybridization of alicyclic scaffold and triazole
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Med Chem Res
Table 1 List of synthesized 4H-1,2,4-triazol-3-yl cycloalkanols
Compound R
R0
m n
4a
4b
4c
4d
4e
4f
–H
-3,5,50-triCH3
–Methyl
–Methyl
0
0
1
1
0
0
0
0
0
0
1
1
1
1
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
–
1
1
H, double bond at C3–C4 –Methyl
-3,4-diOH
–Methyl
–H
-4-OH–phenyl
-4-Pyridyl
-Phenyl
–H
4g
4h
4i
–H
–H
-4-OH–phenyl
-4-Pyridyl
-3-Pyridyl
–H
4j
–H
4k
4l
–H
-4-Methyl–thiophene 0
-3,5,50-triCH3
-3,5,50-triCH3
-3,5,50-triCH3
-3,5,50-triCH3
–Phenyl
0
0
0
0
1
1
1
1
1
4m
4n
4o
4p
4q
4r
4s
-4-OH–phenyl
-4-Pyridyl
-3-Pyridyl
–H, double bond at C3–C4 –Phenyl
–H, double bond at C3–C4 -4-Pyridyl
–H, double bond at C3–C4 -3-Pyridyl
-3,4-dihydroxy
-3,4-dihydroxy
-1-adamentyl ring
-4-CH3
–Phenyl
4t
-4-Pyridyl
4u
4v
4w
-4-Methyl–thiophene 0
-4-Pyridyl
–Phenyl
0
0
-4-CH3
Melting points were recorded on Thermomik Compbell
electronics, having oil-heating system and are uncorrected.
The purity of the compounds was routinely checked by thin
layer chromatography (TLC) using silica gel G (Merck).
HPLC analysis of compounds was carried out on Agilent
HPLC instrument. Mobile phase used for the purpose was
MeOH:H2O (90:10). FTIR spectra were recorded on
‘‘Perkin Elmer infrared spectrophotometer’’ using KBr
pellets. The NMR spectra were recorded on FT-NMR
JEOL, 60 MHz or JEOL AL 300 MHz, spectrometer with
DMSO-d6 or CDCl3 as solvent using tetramethylsilane
(TMS) as an internal standard. All chromatographic sol-
vents were distilled before use. Absolute ethanol used in
reaction as solvent for synthesis of intermediates and final
Fig. 3 a Hydrogen bond interaction of compound 4a in the active site
of Mtb type II dehydroquinase. b Designed molecule in the active site
of Mtb type II dehydroquinase
alicyclic cyanohydrins 2a–w (Kondo and Iwatsuki, 1984).
These cyanohydrins were reacted with various acid
hydrazides 3 by refluxing in n-butanol in presence of
K2CO3 as a base to obtain corresponding alicyclic triazoles
4a–w (Yeung et al., 2005). The list of synthesized
4H-1,2,4-triazol-3-yl cycloalkanols is enumerated in Table 1.
Scheme 1 Synthetic scheme for 4H-1,2,4-triazol-3-yl cycloalkanols
123

Med Chem Res
3,3,5-Trimethyl-1-(5-methyl-4H-1,2,
4-triazol-3-yl)cyclohexanol 4b
molecules; was obtained from S. D. Fine Chemicals Ltd.
All reactants were obtained from S. D. Fine Chemicals
Ltd., and Spectrochem/Aldrich. Sodium cyanide was
obtained from Salpra Pharmaceuticals and Chemicals,
Mumbai.
Yield: 66.17 %; Rf: 0.55 [Ethylacetate Hexane (1:1)];
m.p.: 118–120 °C; IR (KBr): Vmax cm-1 3373, 3238, 2845,
1670, 1618, 1588, 1443, 1073; MS m/z: 224 [M?1].
General procedure for synthesis of alicyclic
cyanohydrins
Cyclohex-3-enyl(5-methyl-4H-1,2,4-triazol-3-yl)methanol
4c
Alicyclic aldehyde/ketone (1a–w) (10 mmol) was added
dropwise to 25 ml of aqueous solution of sodium bisulphite
(12 mmol) over a period of 45 min. Temperature of reac-
tion mixture was increased to 40–50 °C. Water (30 ml)
was added and reaction mixture was cooled to 10–15 °C.
Aqueous solution (15 ml) of sodium cyanide (15 mmol)
was added dropwise over 30 min. The reaction mixture
was warmed to 30 °C for 1 h. The reaction mixture was
diluted with 100 ml water and extracted with EtOAc
(2 9 25 ml). The combined organic layers were then
washed with brine (50 ml), dried over anhydrous sodium
sulphate (Na2SO4), and concentrated in vacuum to obtain
cyanohydrins (2a–w) in yields ranging from 62.5 to
90.91 %.
Yield: 66.10 %; Rf: 0.62 [Ethylacetate Hexane (1:1)];
m.p.: 121–123 °C; IR (KBr):Vmax cm-1 3476, 3179, 2849,
1671, 1510, 1384, 1284, 143.9; H NMR:10.09 (bs, 1H, -
1
triazole-NH, D2O exchange), 5.74–5.66 (t, 2H, –CH=CH–,
J = 11.7 Hz), 4.46 (s, 1H, –CH–OH), 2.28 (s, 3H, –CH3),
2.2–1.54 (m, 8H, cyclohexyl-H, J = 21 Hz); 13C NMR:
150.84, 150.83, 126.97, 125.28, 77.48, 36.54, 28.34, 25.99,
24.31, 20.28;MS: 193.1 [M?].
4-(Hydroxy(5-methyl-4H-1,2,4-triazol-3-
yl)methyl)cyclohexane-1,2-diol 4d
Yield: 68.17 %; Rf: 0.25 [Ethylacetate (100 %)]; m.p.:
148–150 °C; IR (KBr) Vmax cm-1 3258, 2916, 1684, 1633,
1555, 484, 1176, 1015; MS: 228 [M?1].
General procedure for synthesis of alicyclic triazoles
A mixture of a-hydroxynitrile (2a–w) (10 mmol), acid
hydrazide (3) (12 mmol) and K2CO3 (15 mmol) was sus-
pended in n-butanol (20–30 ml) and the mixture was sub-
jected to microwave irradiation at a power of 100 W, for
10–20 min, 115 °C. The reaction mixture was then cooled
to room temperature; concentrated under reduced pressure
and partitioned between EtOAc (50 ml) and water (50 ml).
The organic layer was separated, and the aqueous layer was
extracted further with EtOAc (2 9 25 ml). The combined
organic layers were washed with brine (50 ml), dried over
sodium sulphate (Na2SO4), and concentrated under vac-
uum. Purification of compound was carried out by column
chromatography (Silica gel, 6G, 20 % EtOAc/hexane) to
obtain pure 1,2,4-triazole derivative (4a–4w) in yields
ranging from 34 to 75 %.
4-(5-(1-Hydroxycyclopentyl)-4H-1,2,4-triazol-3-yl)phenol
4e
Yield: 44.88 %; Rf: 0.39 [Ethylacetate: Hexane (2:3)];
m.p.: 161–163 °C; IR (KBr) Vmax cm-1 3434, 3196, 3050,
2926, 1633, 1552, 1287, 1058; MS: 246 [M?1].
1-(5-(Pyridin-4-yl)-4H-1,2,4-triazol-3-yl)cyclopentanol 4f
Yield: 57.91 %; Rf: 0.36 [Ethylacetate: Hexane (2:3)];
m.p: 170–172 °C; IR (KBr) Vmax cm-1: 3257, 3064, 2933,
2849, 1645, 1518, 1112, 946; 1H NMR (CDCl3, 300 MHz):
10.19 (bs, 1H, -triazole-NH, D2O exchange), 7.86–7.84 (d,
2H, pyridyl-H, J = 6 Hz), 7.53–7.499 (d, 2H, pyridyl-H,
J = 12 Hz), 6.1 (bs, 1H, OH), 2.39–1.39 (m, 8H, cyclo-
pentyl-H, 11.5 Hz); 13C NMR : 166.41, 166.32, 42.50,
41.74, 128.38, 127.50, 121.76, 120.21, 58.90. 38.94, 38.76,
24.87, 22.53; MS: 231 [M?1].
1-(5-Methyl-4H-1,2,4-triazol-3-yl)cyclohexanol 4a
Yield: 75.97 %; Rf: 0.5 [Ethylacetate: Hexane (1:1)]; m.p.:
110–112 °C; IR (KBr): Vmax cm-1 3459.4, 3231.4, 2911,
2849, 1662, 1549, 1251, 1070; 1H NMR (CDCl3,
300 MHz)-9.33 (bs, 1H, -triazole-NH, D2O exchange),
5.21 (bs, 1H, –OH), 3.14 (s, 3H, –CH3), 2.047–1.24 (m,
10H, cyclohexyl-H, J = 14 Hz); 13C NMR: 169.58,
169.44, 99.28, 34.05, 33.57, 24.73, 22.12, 21.78, 20.67; MS
m/z: 181[M?].
1-(5-Phenyl-4H-1,2,4-triazol-3-yl)cyclohexanol 4g
Yield: 44.81 %; Rf: 0.42 [Ethylacetate: Hexane (3:7)];
m.p.: 125–127 °C; IR (KBr) Vmax cm-1: 3414, 3185.8,
3071, 2843, 1670, 1384, 1290, 1010; H NMR:10.21(bs,
1
1H, -triazole-NH, D2O exchange), 7.87–7.85 (m, 2H, Ar–H,
123

Med Chem Res
4-(5-(1-Hydroxy-3,3,5-trimethylcyclohexyl)-4H-1,2,
4-triazol-3-yl)phenol 4m
J = 7 Hz), 7.56–7.453 (m, 3H, Ar–H, J = 7.6 Hz), 5.96
(bs, 1H, OH), 2.3–1.20 (m, 10H, cyclohexyl-H, J =
12 Hz); 13C NMR: 166.35, 166.31, 42.65, 41.65, 128.53,
128.36, 127.47, 121.49, 59.48, 33.89, 33.84, 24.46, 21.88,
21.88; MS: 244.22 [M?1].
Yield: 60.47 %; Rf: 0.35 [Ethylacetate: Hexane (1:4)];
m.p.: 153–155 °C; 1H NMR (CDCl3, 300 MHz): 10.04 (bs,
1H, -triazole-NH, D2O exchange), 7.73–7.70 (d, 2H, Ar–H,
J = 9 Hz), 6.82–6.79 (d, 2H, Ar–H, J = 9 Hz), 5.83 (bs,
1H, OH), 2.4–1.27 (m, 8H, cyclohexyl-H, J = 14 Hz),
0.94–0.80 (m, 9H, –CH3, J = 22 Hz); 13C NMR : 166.05,
160.55, 129.45, 123.07, 122.69, 114.89, 57.70, 46.99,
45.00, 42.65, 33.65, 31.07, 25.85, 25.21, 21.68; MS
:302.125 [M?1].
4-(5-(1-Hydroxycyclohexyl)-4H-1,2,4-triazol-3-yl)phenol
4h
Yield: 53.08 %; Rf: 0.40 [Ethylacetate: Hexane (3:7)];
m.p.: 148–150 °C; IR (KBr) Vmax cm-1: 3405, 3243, 3071,
2963, 1601, 154, 1400, 1193; MS: 260 [M?1].
3,3,5-Trimethyl-1-(5-(pyridin-4-yl)-4H-1,2,
4-triazol-3-yl)cyclohexanol 4n
1-(5-(Pyridin-4-yl)-4H-1,2,4-triazol-3-yl)cyclohexanol 4i
Yield: 67.62 %; Rf: 0.35 [Ethylacetate: Hexane (3:7)];
m.p.: 158–160 °C; IR (KBr) Vmax cm-1: 3389, 3247, 3050,
2859, 1661, 1579, 1455, 403; 1H NMR (CDCl3, 300 MHz):
9.75 (bs, 1H, -triazole-NH, D2O exchange), 8.27–8.22 (d,
2H, pyridyl-H, J = 15 Hz), 7.80–7.79 (m, 2H, pyridyl-H,
J = 3 Hz), 3.94 (bs, 1H, OH), 2.18–1.26 (m, 10H, cyclo-
hexyl-H, J = 12 Hz); 13C NMR: 165.6, 165.50, 150.18,
49.69, 49.64, 121.54, 121.05, 60.25, 34.48, 34.39, 24.81,
22.25, 22.17; MS: 244 [M?1].
Yield: 61.33 %; Rf: 0.38 [Ethylacetate: Hexane (1:4)];
m.p.: 182–184 °C; 1H NMR (CDCl3, 300 MHz): 10.94 (bs,
1H, -triazole-NH, D2O exchange), 8.71–8.68 (d, 2H, pyr-
idyl-H, J = 2 Hz), 7.73–7.69 (d, 2H, pyridyl-H, J =
12 Hz), 2.88–1.16 (m, 8H, cyclohexyl-H, J = 13.5 Hz),
0.99–0.76 (m, 9H, –CH3, J = 31.2); 13C NMR: 167.96,
166.47, 150.53, 141.59, 122.18, 122.10, 67.62, 43.29,
40.52, 36.10, 34.18, 32.21, 29.75, 28.54, 25.59, 22.69.
3,3,5-Trimethyl-1-(5-(pyridin-3-yl)-4H-1,2,
4-triazol-3-yl)cyclohexanol 4o
1-(5-(Pyridin-3-yl)-4H-1,2,4-triazol-3-yl)cyclohexanol 4j
Yield: 56.35 %; Rf: 0.33 [Ethylacetate: Hexane (3:7)];
m.p.: 169–171 °C; IR (KBr) Vmax cm-1: 3362, 3196.2,
3019, 2926, 2843, 1648, 1539, 1062.
Yield: 58.41 %; Rf: 0.36m [Ethylacetate: Hexane (1:4)];
m.p.: 191–193 °C; IR (KBr) Vmax cm-1: 3433, 3320, 2951,
1610, 1581, 1479, 484, 1185.6; MS: 287.18[M?1].
1-(5-(5-Methylthiophen-2-yl)-4H-1,2,4-triazol-3-
yl)cyclohexanol 4k
Cyclohex-3-enyl(5-phenyl-4H-1,2,4-triazol-3-yl)
methanol 4p
Yield: 48.47 %; Rf: 0.48 [Ethylacetate: Hexane (3:7)];
m.p.: 105–107 °C; IR (KBr) Vmax cm-1: 3416, 3273, 3009,
2833, 1602, 1583, 426, 1292; MS: 264.1 [M?1].
Yield: 58.46 %; Rf: 0.44 [Ethylacetate: Hexane (3:7)];
m.p.: 126–128 °C; IR (KBr) Vmax cm-1: 3311, 3267, 3050,
2869, 1635, 1560, 467, 1159; 1H NMR (CDCl3, 300 MHz):
9.95 (bs, 1H, -triazole-NH, D2O exchange), 7.92–7.26 (m,
52H, Ar–H, J = 7.5 Hz), 5.71–5.68 (s, 2H, –CH=CH–,
J = 9 Hz), 5.08 (s, 1H, –CH–OH), 4.02 (bs, 1H, OH),
2.36–1.55 (m, 10H, cyclohexyl-H, J = 14.1); 13C
NMR:158.45, 158.34, 42.50, 41.78, 129.09, 128.85,
127.23, 127.12, 124.74, 124.61, 77.45, 35.29, 28.12, 24.76,
24.46.
3,3,5-Trimethyl-1-(5-phenyl-4H-1,2,4-triazol-3-
yl)cyclohexanol 4l
Yield: 52.75 %; Rf: 0.40 [Ethylacetate: Hexane (1:4)];
m.p.: 146–148 °C; IR (KBr) Vmax cm-1: 3456, 3254, 3082,
2916, 1674, 1529, 1446, 1212; 1H NMR (CDCl3,
300 MHz):10.58 (bs, 1H, -triazole-NH, D2O exchange),
8.2 (m, 2H, Ar–H, J = 8.1), 7.45 (m,3H,Ar–H, J = 6 Hz),
3.3 (bs, 1H, OH), 2.89–1.07 (m, 8H, cyclohexyl-H,
J = 13.5 Hz), 0.99–0.76 (m, 9H, 3-CH3, J = 22 Hz; 13C
NMR :164.38, 163.18, 44.11, 41.17, 128.50, 128.23,
127.58, 127.44, 65.37, 42.85, 35.53, 33.93, 33.69, 31.78,
22.27; MS: 286 [M?1].
Cyclohex-3-enyl(5-(pyridin-4-yl)-4H-1,2,
4-triazol-3-yl)methanol 4q
Yield: 61.00 %; Rf: 0.34 [Ethylacetate: Hexane (3:7)];
m.p.: 143–145 °C; IR (KBr) Vmax cm-1: 3403, 3011, 2884,
1643, 1465, 1280, 1181, 1027; 1H NMR (CDCl3,
123

Med Chem Res
4-Methyl-1-(5-phenyl-4H-1,2,4-triazol-3-yl)cyclohexanol
4w
300 MHz): 8.76–8.75 (d, 2H, pyridyl-H, J = 5.1 Hz),
7.68–7.66 (d, 2H, pyridyl-H, J = 6 Hz), 5.72–5.69 (d, 2H,
–CH=CH–, J = 9.9 Hz), 5.4 (s, 1H, –CH–OH), 4.025 (bs,
1H, OH), 2.36–1.54 (m, 7H, cyclohexenyl-H, J = 8.1 Hz);
13C NMR: 166.22, 166.21, 150.92, 150.57, 49.30, 127.24,
127.08, 124.57, 124.47, 77.48, 35.26, 27.59, 24.80, 24.36.
Yield: 64.65 %; Rf: 0.45 [Ethylacetate: Hexane (1:4)];
m.p.: 129–41 °C; IR (KBr) Vmax cm-1: 3416, 3285, 2953,
1653, 1610, 1581, 403, 1246; 1H NMR (CDCl3,
300 MHz):10.22 (bs, 1H, -triazole-NH, D2O exchange),
7.85–7.48 (m, 5H, Ar–H, J = 7 Hz), 3.99 (bs, 1H, OH),
2.14–1.02 (m, 9H, cyclohexyl-H, J = 13.5 Hz), 0.89–0.88
(d, 3H, –CH3, J = 3 Hz); 13C NMR: 166.30, 166.26,
42.60, 41.67, 128.39, 127.59, 127.46, 59.92, 33.98, 31.02,
30.76, 28.14, 21.75; MS: 258.06 [M?1].
Cyclohex-3-enyl(5-(pyridin-3-yl)-4H-1,2,
4-triazol-3-yl)methanol 4r
Yield: 56.72 %; Rf: 0.39 [Ethylacetate: Hexane (3:7)];
m.p.: 155–157 °C; IR (KBr) Vmax cm-1: 3414.9, 3241,
3009, 2864, 1631, 1528, 1283, 1129; MS: 257.10 [M?1].
Microbiology
4-(Hydroxy(5-phenyl-4H-1,2,
4-triazol-3-yl)methyl)cyclohexane-1,2-diol 4s
The synthesized, structurally novel 4H-1,2,4-triazol-3-yl
cycloalkanols were tested by Resazurin Microtitre Assay
(REMA) plate method against H37Rv strains of Mycobac-
terium tuberculosis. Isoniazid was used as a reference drug.
MIC is defined as the minimum concentration of com-
pound required for complete inhibition of bacterial growth,
as indicated by the colour change of resazurin (from blue to
pink in case of growth) inspected visually (Palomino et al.,
2002). The Minimum Inhibitory Concentration (MIC),
CC50 values and selectivity indices of the synthesized tri-
azole derivatives (4a–4w) are listed in Table 2.
Yield: 34.50 %; Rf: 0.32 [Ethylacetate: Methanol (9:1]);
m.p.: 200–202 °C; IR (KBr) Vmax cm-1: 3412, 3333, 3061,
1635, 1544, 1424, 428, 1188; MS: 290.39 [M?1].
4-(Hydroxy(5-(pyridin-4-yl)-4H-1,2,
4-triazol-3-yl)methyl)cyclohexane-1,2-diol 4t
Yield: 37.86 %; Rf: 0.25 [Ethylacetate: Methanol (9:1)];
m.p.: 220–222 °C; IR (KBr) Vmax cm-1: 3457, 3276, 3069,
2838, 1632, 1581, 1252, 1182; MS: 291 [M?1].
Antitubercular activity
1-(5-(5-Methylthiophen-2-yl)-4H-1,2,
4-triazol-3-yl)admentanol 4u
All synthesized compounds (4a–4w) were dissolved in
dimethyl sulfoxide (DMSO) at a concentration of 5 mg/ml
and were screened against M. tuberculosis H37Rv.
Yield: 44.95 %; Rf: 0.58 [Ethylacetate: Hexane (1:4)];
m.p.: 105–107 °C; IR (KBr) Vmax cm-1: 3458, 3245, 2909,
2840, 1657, 1512, 478, 1068; 1H NMR (CDCl3,
300 MHz):8.49 (bs, 1H, -triazole-NH, D2O exchange),
7.62–7.58 (d, 1H, thiophene-H, J = 12 Hz), 7.01–6.96 (d,
1H, thiophene-H, 15 Hz), 2.28 (s, 3H, –CH3), 2.07–1.76
(m, 14H, admentyl-H, J = 16 Hz).
Preparation of mycobacterial cell culture Mycobacte-
rium tuberculosis (H37Rv) was grown in Youman’s media
till mid log phase. The pellicle was homogenized in Mid-
dlebrook 7H9 broth by vortexing along with glass beads in
a sealed tube. The suspension was allowed to stand for
15 min and then the supernatant from the top was removed
to a fresh tube and OD540 adjusted to 0.1 (&107 cells/ml).
4-Methyl-1-(5-(pyridin-4-yl)-4H-1,2,4-triazol-3-
yl)cyclohexanol 4v
Resazurin microplate assay (REMA) Compounds were
tested against M. tuberculosis H37Rv to determine the
actual minimum inhibitory concentration (MIC) in the
broth microdilution assay with REMA method. Homoge-
nous mycobacterial (H37Rv) culture suspension was seeded
in microtitre plates at density of 105 cells per well along
with serially diluted compound to a final volume of 200 ll.
The control received equivalent amount of DMSO. The
plates were incubated at 37 °C for 7 days in CO2 incubator.
Resazurin dye (0.02 %) was added after 7 days and plates
were again incubated for 48 h. MIC was determined by
Yield: 58.72 %; Rf: 0.41 [Ethylacetate: Hexane (2:3)];
m.p.: 144–146 °C; IR (KBr) Vmax cm-1: 3341, 3261, 3019,
2843, 1654, 1627, 1578, 433; 1H NMR (CDCl3, 300 MHz):
9.35 (bs, 1H, -triazole-NH, D2O exchange), 8.84–8.82 (d,
2H, pyridyl-H, J = 6 Hz), 8.38–8.36 (d, 2H, pyridyl-H,
J = 6 Hz), 3.95 (bs, 1H, OH), 1.98–1.6 (m, 9H, cyclo-
hexyl-H, J = 12 Hz), 1.06–1.03 (d, 3H, –CH3); 13C NMR:
161.22, 160.64, 156.69, 150.69, 142.30, 121.32,
121.28,69.40, 49.72, 38.44, 30.41, 29.94, 29.50, 22.60; MS
: 259.25 [M?1].
123

Med Chem Res
optical density values (OD570) with respect to non-treated
cells by 50 %.
Table 2 Biological evaluation data of synthesized 4H-1,2,4-triazol-
3-yl cycloalkanols
Compound
MIC lg/ml*
CC50 lg/ml**
SI***
4a
6.25
4.50
5.15
3.25
3.2
392
380
318
378
347
382
422
340
412
397
373
383
352
414
368
358
388
371
429
429
356
403
437
199
63
84
Results and discussion
4b
4c
62
In the present study, five compounds 4f, 4i, 4n, 4t and 4v
exhibited good activity with MIC in the range of 0.59–
0.95 lg/ml. Also, MIC for rest of the compounds was
found to be in the range of 1.25–12.5 lg/ml. All the
compounds exhibit CC50 values in the range of 318–
437 lg/ml indicating that the compounds are non-toxic,
with selectivity indices [28.
4d
4e
116
108
490
34
4f
0.78
12.36
7.5
4g
4h
4i
45
0.59
4.5
698
88
4j
Structure activity relationship reveals that compounds
with methyl substituent on triazole ring of 4H-1,2,4-tria-
zol-3-yl cycloalkanols, 4a–4d, showed good activity.
Further, replacement of methyl group by phenyl ring on
the triazole ring, 4g, 4l, 4p, and 4s, results in detrimental
effect on the antitubercular activity. However, 4-hydroxy
phenyl substituent on the triazole ring, 4h and 4m, leads
to reasonable improvement in activity as compared to 4g
and 4l, with unsubstituted phenyl ring. Heteroaryl sub-
stituents like 3-pyridyl, 4-pyridyl and 4-methyl thiophen-
2yl on triazole ring result in significant improvement in
activity. Amongst the heteroaryl substituents, 4-pyridyl
was better than 3-pyridyl and 4-methylthiophen-2-yl sub-
stituents. Compounds 4f, 4i, 4n, 4t and 4v with 4-pyridyl
substituent on the alicyclic triazole ring exhibit very good
antitubercular activity. Amongst these five compounds,
unsubstituted five and six membered alicyclic ring con-
taining compounds, 4f and 4i, showed almost equivalent
activity. However, compounds with unsubstituted six
membered ring showed slightly better activity than com-
pound with trimethyl substituted six membered rings 4n
and compound with dihydroxyl substituted six membered
ring 4t.
4k
4l
1.65
15.36
10.5
0.65
12.5
12.5
1.25
8.5
226
25
4m
4n
4o
33
637
29
4p
4q
4r
29
310
44
4s
7.75
0.90
4.25
0.95
15.5
0.4
55
4t
477
84
4u
4v
424
28
4w
Isoniazid
499
* Compounds were tested against Mtb H37Rv strains using REMA
method
** Cytotoxicity studies were done on Vero Cell line C1008 using
MTT assay
*** Selectivity index = CC50/MIC
visual inspection of the dye colour (blue to pink). Isoniazid
was used as the reference drug.
Conclusion
Cytotoxicity
In summary, a series of novel, alicyclic triazoles was
designed based on the molecular hybridization of alicyclic
scaffold and triazole. Synthesized compounds were eval-
uated for activity against Mycobacterium tuberculosis
H37Rv and cytotoxicity on VERO C1008 cell line. Few of
these compounds exhibit promising antitubercular activity
and could serve as leads for further development of anti-
tubercular agents.
Cytotoxicity evaluation of these compounds was done on
Mammalian VERO cell line (C1008) by MTT assay
(Kasugai et al., 1990). Cells were seeded in 96-well
microtitre plates (Falcon Becton–Dickinson, Franklin
Lakes, NJ, USA) at a density of 1 9 105 cells/well and
incubated at 37 °C in 5 % CO2 and observed for mor-
phological changes at 24, 48 and 72 h. Following 72 h of
incubation at 37 °C in 5 % CO2, viability was assessed on
the basis of cellular conversion of (3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) into a for-
mazan product. The concentration required for 50 %
inhibition (CC50 value) was determined by reducing the
Acknowledgments The authors, N.H.P.D and R.B. are thankful to
University Grants Commission, India and authors, M.K. and N.R.T.
are thankful to Department of Biotechnology, India for the financial
assistance.
123

Med Chem Res
Conflict of interest The authors report no conflict of interest. The
authors alone are responsible for the content and the writing of the
paper.
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