Chemical synthesis and biological evaluation of triazole derivatives as inhibitors of InhA and antituberculosis agents

Article abstract of DOI:10.1016/j.ejmech.2012.03.029

A series of triazoles have been prepared and evaluated as inhibitors of InhA as well as inhibitors of Mycobacterium tuberculosis H₃₇R _v. Several of these new compounds possess a good activity against InhA, particularly compounds 17 and 18 for which molecular docking has been performed. Concerning their activities against M. tuberculosis H ₃₇R_V strain, two of them, 3 and 12, were found to be good inhibitors with MIC values of 0.50 and 0.25 μg/mL, respectively. Particularly, compound 12 presenting the best MIC value of all compounds tested (0.6 μM) is totally inactive against InhA.

Full text of DOI:10.1016/j.ejmech.2012.03.029

European Journal of Medicinal Chemistry 52 (2012) 275e283
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Chemical synthesis and biological evaluation of triazole derivatives as inhibitors
of InhA and antituberculosis agents
,
,
,
Christophe Menendez a b, Aurélien Chollet a b, Frédéric Rodriguez a b, Cyril Inard c, Maria Rosalia Pasca d,
Christian Lherbet a , Michel Baltas
,
b
,
a,b,
*
*
a CNRS, Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique, LSPCMIB, UMR-5068, 118 Route de Narbonne, F-31062 Toulouse Cedex 9, France
b Université de Toulouse, UPS, Laboratoire de Synthèse et Physico-Chimie de Molécules d’Intérêt Biologique, LSPCMIB, 118 route de Narbonne, F-31062 Toulouse Cedex 9, France
c Inserm U 563, Institut Claudius Regaud, 20-24 rue du pont Saint-Pierre, 31052 Toulouse Cedex, France
d University of Pavia, Dipartimento di Biologia e Biotecnologie «Lazzaro Spallanzani», via Ferrata 1, 27100 Pavia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of triazoles have been prepared and evaluated as inhibitors of InhA as well as inhibitors of
Mycobacterium tuberculosis H37Rv. Several of these new compounds possess a good activity against InhA,
particularly compounds 17 and 18 for which molecular docking has been performed. Concerning their
activities against M. tuberculosis H37RV strain, two of them, 3 and 12, were found to be good inhibitors
Received 6 February 2012
Received in revised form
15 March 2012
Accepted 15 March 2012
Available online 23 March 2012
with MIC values of 0.50 and 0.25
m
g/mL, respectively. Particularly, compound 12 presenting the best MIC
M) is totally inactive against InhA.
Ó 2012 Elsevier Masson SAS. All rights reserved.
value of all compounds tested (0.6
m
Keywords:
Inhibitor
Triazole
Enoyl ACP reductase
InhA
Tuberculosis
1. Introduction
inhibitors of the 2-trans-enoyl-ACP (acyl carrier protein) reductase
enzyme (InhA or ENR, EC number: 1.3.1.9) from M. tuberculosis have
focused on the synthesis of triazole derivatives to mimic the phenol
central core of triclosan analogues as shown in Fig. 1 [10].
Among the different molecules conceived, synthetized and
tested on InhA and M. tuberculosis H37Rv strain, compound 3 (Fig. 1)
has been proven to be a good inhibitor of M. tuberculosis strain but
a weak inhibitor of InhA [10].
In this study, we report the synthesis and the evaluation of
a series of potential InhA inhibitors bearing different substituents
around the triazole core as shown in Fig.1. We assessed: 1) the effect
of the nature of the substituent on the phenyl group; 2) the effect of
the alkyl chain length and 3) the effect of the linking positions on
the triazole fragment on InhA and M. tuberculosis activities.
Tuberculosis caused by Mycobacterium tuberculosis is respon-
sible for more than two million deaths worldwide every year. The
World Health Organization (WHO) has estimated than one-third of
the world population is infected by M. tuberculosis [1]. Multidrug-
resistant M. tuberculosis (MDR-MT) [2,3] and extensively drug-
resistant MT (XDR-MT) [4] have become resistant at an alarming
rate [5] to existing antibiotics: isoniazid, rifampicin (for MDR-MT)
and fluoroquinolone kanamicyn, amikacin, and capreomycin (for
XDR-MT). Currently there is an urgent need of new chemothera-
peutics to combat the spread of this disease. Different families of
compounds have been reported with antitubercular activities such
as cinnamic derivatives [6], hydrazones [7], nitroimidazoles [8]. In
recent years, triazoles have received much attention as central core
in medicinal chemistry and especially in the synthesis of antitu-
bercular agents [9]. Our own efforts on the development of potential
2. Results and discussion
2.1. Chemistry
* Corresponding authors. CNRS, Laboratoire de Synthèse et Physico-Chimie
de Molécules d’Intérêt Biologique, LSPCMIB, UMR-5068, 118 Route de Narbonne,
F-31062 Toulouse Cedex 9, France. Tel.: þ33 (0)5 61556289; fax: þ33 (0)5 61556011.
E-mail addresses: lherbet@chimie.ups-tlse.f (C. Lherbet), baltas@chimie.ups-
tlse.fr (M. Baltas).
The triazole moiety was synthesized by copper catalyzed Huis-
gen 1,3 dipolar cycloaddition reaction between azides and alkynes.
This cycloaddition remains very attractive to generate numerous
applications and especially in medicinal chemistry. Also, they are
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.03.029

276
C. Menendez et al. / European Journal of Medicinal Chemistry 52 (2012) 275e283
R
OH
N
N
N
X
triazole
OH
Cl
K2CO3
m
N
N
10
R
N
N
N
N
O
n
10
Br
Compound 3
10
acetone
R
Cl
O
µ
40% inhibition at 50
M
5
µ
6 Cl
MIC (H37Rv) < 2 g/mL
7
OMe
8PP
Scheme 2. Synthesis of triazoles 6 and 7.
Fig. 1. Analogies between triclosan analogue 8PP (also called 8PS) [11] and
compounds in this publication (left); inhibitor 3 [10] (right).
Concerning the first family, when two carbon atoms separate the
aromatic ring from the triazole core, fair inhibition activities were
obtained for compounds 1 and 3 and a much better one for
compound 2 possessing 9-carbonalkyl chain. It is interesting to point
out that when oxygen atom replaces the methylene group, a better
activity was observed (54% vs 40% for compounds 4 and 3 respec-
tively). 2,4-Dichloro substitutions on the aromatic ring are delete-
rious for theactivity which is restored by replacementof the 2-chloro
by the 2-OMe group (5% vs 35% for compounds 6 and 7 respectively).
While compounds 31 and 32, bearing an aromatic ring directly
attached to the triazole core are weak or no inhibitors at all,
compounds with one methylene group between these two frames
present much better activities. In fact, triazole derivatives 17, 18 and
characterized by mild reaction conditions, high regioselectivities
and high yields [12,13].
A series of 1,2,3-triazoles bearing alkyl and arylalkyl chains
attached at either the carbon or the nitrogen atom of the triazole
frame, were synthesized by click chemistry from the corresponding
azide and commercially available alkynes.
Aryl ether derivatives were obtained through two different
manners. First, the ether analogue 4 of compound 3 (Fig. 1) was
synthesized in 79% from the corresponding azide and alkyne
(R ¼ PhO, n ¼ 1), easily prepared from phenol and propargyl
bromide [14] (Scheme 1).
Halogenated compound 5 was used for the elaboration of the
other aryl ether derivatives. Compounds 6 and 7 were synthesized
in 94% and 66% yield respectively, by condensing the bromide
derivative 5, synthetized through click chemistry between prop-
argyl bromide and dodecyl azide, with the corresponding phenols
in the presence of potassium carbonate (Scheme 2).
Finally, two other molecules, 33 and 34, were synthesized as
isomers of compounds 3 and 9. To gain access to these 1,5-
disubstituted triazoles, the corresponding azides and alkynes
were combined and heated under reflux in toluene for 35 h. A small
amount was purified enough to perform the biological test. Struc-
tural identification of compounds 33 and 34 was based upon
comparing their spectroscopic (1H and 13C NMR) and thin layer
chromatography data with those of 1,4-regioisomers 3, 9 respec-
tively, obtained through classical copper-induced click reaction
(Scheme 3).
19 showed 73%, 75% and 58% inhibition activities at 50
mM in
comparison to 46%, 58% and 40% for compounds 1, 2 and 3
respectively. These inhibitors, even though they are relatively
similar by their structure to triclosan analogue 8PS [11], still
present
a much lower activity than the latter compound
(IC50 ¼ 5 nM). A hypothetical placement of compound 18 obtained
by molecular docking calculations in the active site of InhA (PDB
code 2B37) shown in Fig. 2. Both compounds 8PS and 18 can easily
be superimposed. However, the nanomolar activity of compound
8PS could be attributed to the hydroxyl-substituted ring of triclosan
that forms a hydrogen bond to Tyr158 and the 20-hydroxy group of
NADþ, thus inducing a most favourable
between the phenol and the NADþ rings [11].
p-stacking interaction
Considering the second family of compounds, when two carbon
atoms separate the aromatic and triazole frame, fair inhibition
activities were obtained for compounds 9 and 10. No major changes
were observed when introducing a 2-OMe or a 2-Me substituent on
the aromatic ring (compounds 13, 14 and 15, 16 respectively).
An eOMe substitution at another positions of the aromatic ring are
deleterious for the activities (compounds 11 and 12). When one
methylenic group separates the two frames, the compounds tested
showed comparable (9/22; 20/23; 13/26) or less (14/26; 13/25)
activities against InhA.
2.2. Biology
2.2.1. InhA inhibition assay
Recombinant M. tuberculosis InhA was expressed in E. coli and
subsequently purified according to a previous reported procedure
[10]. The synthetic compounds were evaluated in vitro for the
inhibition of InhA from M. tuberculosis at 50
a commonly used method [10]. Triclosan showed a complete
inhibition of InhA at 10 M. The results are shown in Table 2.
mM by applying
Finally, compounds 33 and 34 a-substituted on the triazole core
do not present any worth considering activity.
m
In analysing the results more closely, two families of
compounds may be considered. The first one when the aromatic
system is connected to the carbon atom of the triazole core; the
second one when it is connected to the nitrogen atom of the tri-
azole frame.
N
N
N
10
N
N
toluene, reflux
N3
35 h
33
10
R
CuSO4 (0.2 eq)
NaAsc (0.4 eq)
N
n
R
8
N
N3
n
N
N
R1
N3
tBuOH/H2O 1/1
R1
toluene, reflux
rt, 24h
8
35 h
4-5,8-32
34
Scheme 1. Synthesis of triazoles 4,5, 8e32.
Scheme 3. Synthesis of 1,5-triazoles 33 and 34.

C. Menendez et al. / European Journal of Medicinal Chemistry 52 (2012) 275e283
277
Table 1
Synthesis of triazole derivatives.
Product
1,2,3-Triazole
Yield (%)
79
Product
1,2,3-triazole
Yield(%)
N
N
N
N
N
N
O
N
4
20
76
10
5
N
N
N
N
N
5
30
94
21
22
80
79
Br
7
10
N
N
Cl
N
N
N
N
N
O
O
6
7
10
10
9
Cl
Cl
N
N
OMe
N
N
N
66
23
82
11
N
OMe
OMe
OMe
N
N
5
8
99
89
76
90
24
25
26
27
78
76
90
70
N
N
N
5
9
N
N
N
N
9
9
N
N
N
N
N
N
11
N
10
11
N
N
11
N
OMe
N
N
MeO
MeO
9
N
N
9
5
MeO
N
9
Me
N
N
MeO
MeO
11
N
12
83
28
N
N
81
Me
Cl
N
N
N
OMe
13
14
15
89
86
78
29
30
31
58
61
68
N
N
N
N
5
9
N
N
Cl
OMe
Me
N
N
11
N
N
OH
N
N
7
9
N
7
N
N
Me
N
Me
N
N
16
17
69
78
32
33
98
N
N
7
N
N
N
N
N
N
10
NDa
N
7
(continued on next page)

278
C. Menendez et al. / European Journal of Medicinal Chemistry 52 (2012) 275e283
Table 1 (continued )
Product
1,2,3-Triazole
Yield (%)
60
Product
1,2,3-triazole
Yield(%)
NDa
N
N
N
8
N
18
19
34
N
N
8
N
N
N
73
11
a
ND for Not determined.
2.2.2. Bacterial growth inhibition experiments
respectively 9 and 10-carbon chain. It is also noteworthy to point
out that compound 12 showing the best MIC value among all
compounds tested does not inhibit at all the InhA enzyme.
Compound 11, possessing the same substitution pattern on the
aromatic ring as compound 12, presents also a good MIC value
(5.3 mM) while it is a very weak InhA inhibitor. In contrast, the other
six active compounds of the series (3, 4, 13e16) showed also fairly
good inhibitions of InhA.
All of thesynthesized compounds were evaluated by determining
the minimal inhibitory concentration (MIC) on M. tuberculosis H37Rv
strain (Table 3). Triclosan was used for comparison.
Among all compounds tested, those possessing on methylenic
group between the aromatic group and the triazole frame do not
present any notable activities against growth of M. tuberculosis
H37Rv strain. In fact, all values are superior or equal to 4
better results were obtained for compounds 26, 29 and 30 with MIC
values of 4 g/mL (11.2, 14.4 and 12.0 M respectively). It has to be
mg/mL. The
Finally, the 1,5-disubstitued triazole regioisomers are not well
recognized by comparison with the 1,4-disubstituted isomers (3
versus 33 and 9 versus 34).
m
m
noted that the length of the alkyl chain attached to the triazole core
does not influence sharply the activities.
The most active compounds are to be found among those pos-
sessing two atoms between the two frames (aromatic and triazolo).
3. Conclusion
Thus
8
compounds present activities against growth of
g/mL
To increase the chance of effective compounds coming from the
research that can become available for TB care, we feel that the
number and the structural variety of the compounds that might be
conceived to enter the pipeline should be sufficiently large. In that
respect, among the families of compounds developed in our group
(cinnamic, triazolophtalazine ...) the triazolo ones become part as
valuable scaffolds for that purpose.
M. tuberculosis H37Rv strain with values inferior or equal to 4
m
(compounds 3, 4, 11e16) and this independently of the linking
positions of the aromatic or alkyl chain on the triazole core.
In general, the length of the alkyl chain is of importance. In fact
the best results are obtained for the 12-carbon chain derivatives 3
and 12 that possess MIC values of 1.6 mM and 0.6 mM respectively
and are 8e10 times more active than compounds 2 and 11 bearing
Concerning the triazolo derivatives presented here, a series of
alkyl triazoles were synthesized in which the A ring has been
substituted with a variety of groups. Several of these new
compounds possess a good activity against InhA. In particular,
compounds 17 and 18, possessing one methylene group between
the triazolo and the aromatic rings present the best values among
all compounds tested. Concerning their activities against
M. tuberculosis H37RV strain, two of them, 3 and 12, were found to
Table 2
Enzyme inhibition values. Results are expressed as a percentage of InhA inhbition.
Compd.
Triclosan
% Inhibition
Compd.
% Inhibition
at 50
m
M
at 50
mM
>99
18
75
N
be good inhibitors with MIC values of 0.50 and 0.25
respectively. Particularly, compound 12 presenting the best MIC
mg/mL,
N
N
N
N
a
7
8
46
58
40
19
20
21
58
21
34
1
value of all compounds tested (0.6
InhA.
mM) is totally inactive against
N
N
In conclusion, the results presented here may highlight not only
compounds active against well established target (InhA), but also
potentially active compounds with unknown target. In addition,
these molecules bearing a relatively simple scaffold, could be
structurally improved to afford better inhibitors. Concerning
compound 12, it should be interesting to search for its biological
target. Studies are underway to improve their efficiency against
InhA and M. tuberculosis.
a
2
N
N
a
11
3
4
6
7
8
54
< 5
35
10
43
67
21
NIb
48
61
37
58
73
22
23
24
25
26
27
28
29
30
31
32
33
34
44
52
NIb
20
46
24
19
NIb
42
NIb
33
9
4. Experimental
9
10
11
12
13
14
15
16
17
4.1. Material
Kinetic studies were perfomed on a Cary Bio 100. All chemicals
were obtained from Aldrich or Acros Organics and used without
further purification. Nuclear magnetic resonance spectra
were recorded on a Bruker AC 300 spectrometer (1H and 13C NMR),
and mass spectra were measured on a LCT Premier Waters
spectrometer.
33
a
See reference [10].
NI for no inhibition at the given concentration.
b

C. Menendez et al. / European Journal of Medicinal Chemistry 52 (2012) 275e283
279
Fig. 2. Hypothetical binding mode of compounds 8PS (up, cyan) and 18 (down, magenta) in the active site of InhA enzyme (PDB ID 2B37) obtained by molecular docking. Coc-
rystallised cofactor NADþ and 8PS are coloured in green and white respectively; the molecular surface of protein is coloured using a hydrophobic scale and rendered using UCSF
Chimera [15]. Tyr158 residue is coloured (white for 2B37 native state) in a final conformation obtained after docking. The same calculation protocol was used in both cases and the
docking of 8PS shows an agreement in reproducing crystallography (RMSD ¼ 0.94 Å) without displacing Tyr158. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)
4.2. Chemistry
AscNa (0.4 eq) at room temperature. The reaction mixture was
stirred at rt for 24 h, then H2O was added and extracted with
EtOAc. The organic layer was washed with brine, dried over
MgSO4, filtered, concentrated under reduced pressure. The desired
product was purified by flash chromatography (EtOAc/petroleum
ether).
4.2.1. Synthesis of azide derivatives
To a DMF solution (3 mL) of bromide derivative, were added
sodium iodide (0.05 eq) and sodium azide (1.2 eq) at 40 ꢀC. The
reaction mixture was stirred for overnight. Et2O (20 mL) was added
to the mixture then the organic layer was washed with water
(3 ꢁ 20 mL), dried over MgSO4 and concentrated under reduced
pressure to give the azide derivative. Spectroscopic data of
compounds are identical to those previously reported [10].
4.2.2.1. 1-Dodecyl-4-(phenoxymethyl)-1H-1,2,3-triazole
(4). (The
alkyne, (prop-2-ynyloxy)benzene, was synthesized following
a known procedure [14].). White powder. Mp 75 ꢀC. Yield 79%. 1H
NMR (CDCl3) d 7.58 (s, 1H); 7.27 (m, 2H); 6.96 (m, 3H); 5.19 (s, 2H);
4.2.2. Preparation of compounds 4,8e32
4.32 (t, J ¼ 7.2 Hz, 2H); 1.88 (m, 2H); 1.25 (m, 18H); 0.87 (t,
A typical experimental procedure for the preparation of these
compounds from the corresponding commercially available
alkynes is described below. To a solution of alkyne (1.0 eq) with
azide (1.2 eq) in tBuOH/H2O (1/1), were added CuSO4 (0.2 eq),
J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d 158.1; 144.1; 129.4; 122.4; 121.1;
114.6; 61.9; 50.3; 31.8; 30.1; 29.5; 29.4; 29.2; 29.2; 28.9; 26.3; 22.6;
14.0; HRMS: (DCI/CH4, m/z) calc. for C21H34N3O: 344.2702. Found:
344.2711.

280
C. Menendez et al. / European Journal of Medicinal Chemistry 52 (2012) 275e283
Table 3
4.2.2.7. 1-(2-Methoxyphenethyl)-4-decyl-1H-1,2,3-triazole
(13). White powder. Mp 56 ꢀC. Yield 89%. 1H NMR (CDCl3)
d
7.20 (td,
Compounds tested as inhibitory agents of M. tuberculosis growth.
J ¼ 7.8 Hz,1.8 Hz,1H); 7.00 (s,1H); 6.92 (dd, J ¼ 7.4 Hz,1.7 Hz,1H);6.83
(t, J ¼ 8.5 Hz, 1H); 6.79 (dd, J ¼ 8.4 Hz, 0.9 Hz, 1H); 4.51 (t, J ¼ 7.1 Hz,
2H); 3.81 (s, 3H); 3.16 (t, J ¼ 7.3 Hz, 2H); 2.63 (t, J ¼ 7.5 Hz, 2H); 1.58
(m, 2H); 1.24 (m, 14H); 0.86 (t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
1,2,3-triazoles
MIC (
m
g/mL)/(
m
M)
1,2,3-triazoles
MIC (mg/mL)/(mM)
Triclosan
1
2
3
4
6
7
8
10/34.5
10/35
5/16.7
0.5/1.6
2/5.8
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
25/87.6
25/76.3
25/102.8
> 4/ > 14.7
> 4/ > 13.4
> 4/ > 12.2
> 4/ > 14.6
> 4/ > 12.1
4/11.2
> 4/ > 11.1
ND
4/14.4
4/12.0
ND
> 4/ > 14.7
> 4/ > 11.7
> 4/ > 12.8
d
157.3; 147.9; 130.5; 128.3; 125.3; 120.7; 120.4; 110.1; 55.1; 49.6;
32.0; 31.8; 29.5; 29.4; 29.4; 29.3; 29.2; 29.0; 25.5; 22.5; 14.0; HRMS:
(DCI/CH4, m/z) calc. for C21H34N3O: 344.2702. Found: 344.2701.
4/9.7
> 4/ > 9.8
10/38.9
4/12.8
4/11.7
2/5.3
0.25/0.6
2/5.8
2/5.4
9
4.2.2.8. 1-(2-Methoxyphenethyl)-4-dodecyl-1H-1,2,3-triazole (14).
10
11
12
13
14
15
16
17
White powder. Mp 60 ꢀC. Yield 86%. 1H NMR (CDCl3)
d 7.22 (td,
J ¼ 7.8 Hz, 1.8 Hz, 1H); 7.00 (s, 1H); 6.93 (dd, J ¼ 7.4 Hz, 1.7 Hz, 1H);
6.85 (t, J ¼ 7.8 Hz, 1H); 6.81 (dd, J ¼ 7.4 Hz, 1.0 Hz, 1H); 4.52 (t,
J ¼ 7.1 Hz, 2H); 3.83 (s, 3H); 3.16 (t, J ¼ 7.2 Hz, 2H); 2.64 (t, J ¼ 7.6 Hz,
2H); 1.59 (m, 2H); 1.25 (m, 18H); 0.87 (t, J ¼ 6.7 Hz, 3H); 13C NMR
2/6.7
2/6.1
25/92.1
(CDCl3) d 157.4; 147.9; 130.6; 128.3; 125.4; 120.7; 120.5; 110.2; 55.2;
49.7; 32.1; 31.8; 29.6; 29.6; 29.5; 29.5; 29.4; 29.3; 29.3; 29.1; 25.6;
22.6; 14.0; HRMS: (DCI/CH4, m/z) calc. for C23H38N3O: 372.3015.
Found: 372.3026.
4.2.2.2. 4-Hexyl-1-phenethyl-1H-1,2,3-triazole (8). White powder.
Mp 40 ꢀC. Yield 99%. 1H NMR (CDCl3)
7.14 (m, 3H); 6.97 (dd,
d
4.2.2.9. 1-(2-Methylphenethyl)-4-octyl-1H-1,2,3-triazole (15). White
J ¼ 7.9 Hz, 1.8 Hz, 2H); 6.96 (s, 1H); 4.42 (t, J ¼ 7.1 Hz, 2H); 3.07 (t,
powder. Mp 58 ꢀC. Yield 98%. 1H NMR (CDCl3)
d 7.14 (m, 3H); 7.01 (d,
J ¼ 7.3 Hz, 2H); 2.54 (t, J ¼ 7.4 Hz, 2H); 1.50 (m, 2H); 1.18 (m, 6H);
J ¼ 7.2 Hz, 1H); 6.95 (s, 1H); 4.50 (t, J ¼ 7.2 Hz, 2H); 3.18 (t, J ¼ 7.5 Hz,
0.77 (t, J ¼ 6.6 Hz, 3H); 13C NMR (CDCl3)
d 147.5; 136.9; 128.3; 128.3;
126.5; 120.7; 51.0; 36.4; 31.2; 29.0; 28.4; 25.2; 22.1; 13.7; HRMS:
2H); 2.66 (t, J ¼ 7.5 Hz, 2H); 2.22 (s, 3H); 1.60 (m, 2H); 1.27 (m, 10H);
0.88 (t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d 148.2; 136.2; 135.4; 130.5;
(DCI/CH4, m/z) calc. for C16H24N3: 258.1970. Found: 258.1982.
129.3; 127.1; 126.3; 120.8; 50.4; 34.1; 31.8; 29.4; 29.3; 29.2; 29.1;
25.5; 22.6; 19.1; 14.0; HRMS: (DCI/CH4, m/z) calc. for C19H30N3:
300.2440. Found: 300.2442.
4.2.2.3. 4-Decyl-1-phenethyl-1H-1,2,3-triazole (9). White powder.
Mp 70 ꢀC. Yield 90%. 1H NMR (CDCl3)
d
7.25 (m, 3H); 7.08 (dd, J ¼ 7.9,
1.7 Hz, 2H); 6.98 (s, 1H); 4.52 (t, J ¼ 7.2 Hz, 2H); 3.17 (t, J ¼ 7.3 Hz,
4.2.2.10. 1-(2-Methylphenethyl)-4-decyl-1H-1,2,3-triazole (16). White
2H); 2.64 (t, J ¼ 7.5 Hz, 2H); 1.59 (m, 2H); 1.25 (m, 14H); 0.87 (t,
powder. Mp 64 ꢀC Yield 69%. 1H NMR (CDCl3)
d 7.14 (m, 3H); 7.01 (d,
J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d
148.0; 137.2; 128.6; 128.6; 126.9;
J ¼ 7.3 Hz, 1H); 6.96 (s, 1H); 4.50 (t, J ¼ 7.2 Hz, 2H); 3.18 (t, J ¼ 7.5 Hz,
120.9; 51.4; 36.8; 31.8; 29.5; 29.4; 29.3; 29.2; 29.1; 25.5; 22.6; 14.0;
HRMS: (DCI/CH4, m/z) calc. for C20H32N3: 314.2596. Found:
314.2610.
2H); 2.65 (t, J ¼ 7.5 Hz, 2H); 2.22 (s, 3H); 1.60 (m, 2H); 1.26 (m, 14H);
0.87 (t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d 148.2; 136.2; 135.4; 129.3;
127.1; 126.3; 120.8; 50.4; 34.1; 31.8; 29.5; 29.5; 29.4; 29.3; 29.3; 29.1;
25.5; 22.6; 19.1; 14.1; HRMS: (DCI/CH4, m/z) calc. for C21H34N3:
328.2753. Found: 328.2753.
4.2.2.4. 4-Dodecyl-1-phenethyl-1H-1,2,3-triazole
powder. Mp 76 ꢀC. Yield: 76%. 1H NMR (CDCl3)
(10). White
7.24 (m, 3H); 7.07
d
(dd, J ¼ 7.9 Hz, 1.8 Hz, 2H); 6.98 (s, 1H); 4.52 (t, J ¼ 7.2 Hz, 2H); 3.16
4.2.2.11. 4-Benzyl-1-octyl-1H-1,2,3-triazole (17). White powder. Mp
(t, J ¼ 7.3 Hz, 2H); 2.64 (t, J ¼ 7.5 Hz, 2H); 1.59 (m, 2H); 1.24 (m,
53 ꢀC. Yield 78%. 1H NMR (CDCl3)
d 7.15e7.29 (m, 5H); 7.13 (s, 1H);
18H); 0.86 (t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d
148.0; 137.1; 128.6;
4.20 (t, J ¼ 7.3 Hz, 2H); 4.03 (s, 2H); 1.80 (m, 2H); 1.23 (m, 10H); 0.84
128.6; 126.9; 120.9; 51.4; 36.7; 31.8; 29.6; 29.5; 29.5; 29.4; 29.3;
29.2; 29.0; 25.5; 22.6; 14.0; HRMS: (DCI/CH4, m/z) calc. for
C22H36N3: 342.2909. Found: 342.2914.
(t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d 147.1; 138.9; 128.4; 128.3;
126.1; 121.0; 49.9; 32.0; 31.4; 30.0; 28.7; 28.6; 26.2; 22.3; 13.8;
HRMS: (DCI/CH4, m/z) calc. for C17H26N3: 272.2127. Found: 272.2134.
4.2.2.5. 1-(3,4-Dimethoxyphenethyl)-4-decyl-1H-1,2,3-triazole (11).
4.2.2.12. 4-Benzyl-1-nonyl-1H-1,2,3-triazole (18). White powder.
White powder. Mp 89 ꢀC. Yield 90%. 1H NMR (CDCl3)
d
6.98 (s, 1H);
Mp 58 ꢀC. Yield 60%. 1H NMR (CDCl3)
d 7.27 (m, 5H); 7.13 (s, 1H);
6.76 (d, J ¼ 8.2 Hz, 1H); 6.63 (dd, J ¼ 8.1 Hz, 2.0 Hz, 1H); 6.51 (d,
J ¼ 1.9 Hz, 1H); 4.49 (t, J ¼ 7.1 Hz, 2H); 3.84 (s, 3H); 3.78 (s, 3H); 3.11
(t, J ¼ 7.1 Hz, 2H); 2.64 (t, J ¼ 7.6 Hz, 2H); 1.59 (m, 2H); 1.24 (m,14H);
4.26 (t, J ¼ 7.3 Hz, 2H); 4.09 (s, 2H); 1.85 (m, 2H); 1.24 (m, 12 H);
0.87 (t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d 147.5; 139.1; 128.6; 128.5;
126.4; 121.1; 50.2; 32.2; 31.7; 30.2; 29.2; 29.0; 28.9; 26.4; 22.5;
14.0; HRMS: (DCI/CH4, m/z) calc. for C18H28N3: 286.2283. Found:
286.2292.
0.86 (t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d 148.9; 148.0; 147.9; 129.7;
121.0; 120.6; 111.7; 111.3; 55.8; 55.7; 51.7; 36.4; 31.8; 29.5; 29.5;
29.3; 29.2; 29.1; 25.5; 22.6; 14.0; HRMS: (DCI/CH4, m/z) calc. for
C22H36N3O2: 374.2808. Found: 374.2805.
4.2.2.13. 4-Benzyl-1-dodecyl-1H-1,2,3-triazole (19). White powder.
Mp 61 ꢀC. Yield 73%. 1H NMR (CDCl3)
d 7.26 (m, 5H); 7.15 (s, 1H);
4.2.2.6. 1-(3,4-Dimethoxyphenethyl)-4-dodecyl-1H-1,2,3-triazole(12).
4.28 (t, J ¼ 7.3 Hz, 2H); 4.1 (s, 2H); 1.86 (m, 2H); 1.25 (m, 18H); 0.89
White powder. Mp 94 ꢀC. Yield 83%. 1H NMR (CDCl3)
d
6.97 (s, 1H);
(t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d 147.6; 139.1; 128.6; 128.5;
6.74 (d, J ¼ 8.2 Hz, 1H); 6.60 (dd, J ¼ 8.1 Hz, 1.9 Hz, 1H); 6.49 (d,
J ¼ 1.9 Hz, 1H); 4.47 (t, J ¼ 7.1 Hz, 2H); 3.81 (s, 3H); 3.76 (s, 3H); 3.08 (t,
J ¼ 7.1 Hz, 2H); 2.61 (t, J ¼ 7.5 Hz, 2H); 1.57 (m, 2H); 1.21 (m,18H); 0.84
126.3; 121.1; 50.2; 32.2; 31.8; 30.2; 29.5; 29.4; 29.3; 29.2; 28.9;
26.4; 22.6; 14.0; HRMS: (DCI/CH4, m/z) calc. for C21H34N3: 328.2753.
Found: 328.2757.
(t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d 148.9; 147.8; 129.6; 121.0; 120.5;
111.7; 111.2; 55.7; 55.6; 51.6; 36.3; 31.8; 29.5; 29.5; 29.4; 29.4; 29.2;
29.2; 29.1; 25.5; 22.5; 14.0; HRMS: (DCI/CH4, m/z) calc. for
C24H40N3O2: 402.3121. Found: 402.3139.
4.2.2.14. 1-Benzyl-4-hexyl-1H-1,2,3-triazole (20). White powder.
Mp 57 ꢀC. Yield 76%. 1H NMR (CDCl3)
d
7.22e7.35 (m, 6H); 5.56 (s,
2H); 2.64 (t, J ¼ 7.7 Hz, 2H); 1.67 (m, 2H); 1.31 (m, 6H); 0.87 (t,

C. Menendez et al. / European Journal of Medicinal Chemistry 52 (2012) 275e283
281
J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d
148.9; 135.1; 129.0; 128.6; 127.9;
1H); 6.85 (s, 2H); 5.39 (s, 2H); 2.67 (t, J ¼ 7.6 Hz, 2H); 2.28 (s, 6H); 1.63
120.6; 53.9; 31.5; 29.4; 28.9; 25.7; 22.5; 14.1; HRMS: (DCI/CH4, m/z)
(m, 2H); 1.29 (m, 6H); 0.85 (t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d 148.8;
calc. for C15H22N3: 244.1814. Found: 244.1818.
138.6; 134.8; 130.1; 125.7; 120.4; 53.9; 31.5; 29.3; 28.8; 25.7; 22.5;
21.1; 14.0; HRMS: (DCI/CH4, m/z) calc. for C17H26N3: 272.2127. Found:
272.2139.
4.2.2.15. 1-Benzyl-4-octyl-1H-1,2,3-triazole (21). White powder. Mp
72 ꢀC. Yield 80%. 1H NMR (CDCl3)
d 7.31 (m, 3H); 7.24 (m, 2H); 7.20
(s, 1H); 5.46 (s, 2H); 2.66 (t, J ¼ 7.5 Hz, 2H); 1.61 (m, 2H); 1.23 (m,
4.2.2.23. 1-(3-Chlorobenzyl)-4-hexyl-1H-1,2,3-triazole (29). White
10H); 0.85 (t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d
148.8; 134.9; 128.9;
powder. Mp 63 ꢀC. Yield 58%. 1H NMR (CDCl3)
d 7.28 (m, 4H); 7.11
128.4; 127.8; 120.4; 53.8; 31.7; 29.3; 29.2; 29.1; 29.1; 25.6; 22.5;
14.0; HRMS: (DCI/CH4, m/z) calc. for C17H26N3: 272.2127. Found:
272.2127.
(m, 1H); 5.44 (s, 2H); 2.67 (t, J ¼ 7.6 Hz, 2H); 1.62 (m, 2H); 1.27 (m,
6H); 0.84 (t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d 149.1; 136.9; 134.8;
130.2; 128.7; 127.8; 125.8; 120.5; 53.1; 31.4; 29.2; 28.8; 25.6; 22.4;
13.9; HRMS: (DCI/CH4, m/z) calc. for C15H21N3Cl: 278.1424. Found:
278.1435.
4.2.2.16. 1-Benzyl-4-decyl-1H-1,2,3-triazole (22). White powder.
Mp 81 ꢀC. Yield 79%. White powder. Mp ꢀC. Yield %. 1H NMR (CDCl3)
d
7.35 (m, 3H); 7.26 (m, 2H); 7.18 (s, 1H); 5.48 (s, 2H); 2.67 (t,
J ¼ 7.5 Hz, 2H); 1.63 (m, 2H); 1.24 (m, 14H); 0.87 (t, J ¼ 6.7 Hz, 3H);
13C NMR (CDCl3)
148.9; 135.0; 129.0; 128.5; 127.9; 120.4; 53.9;
4.2.2.24. 1-(3-Chlorobenzyl)-4-decyl-1H-1,2,3-triazole (30). White
powder. Mp 67 ꢀC. Yield 61%. 1H NMR (CDCl3)
d 7.29 (m, 2H); 7.21
d
(m, 2H); 7.11 (m, 1H); 5.45 (s, 2H); 2.68 (t, J ¼ 7.5 Hz, 2H); 1.63 (m,
31.8; 29.5; 29.5; 29.4; 29.3; 29.3; 29.2; 25.7; 22.6; 14.1; HRMS: (DCI/
CH4, m/z) calc. for C19H30N3: 300.2440. Found: 300.2443.
2H); 1.23 (m, 14H); 0.86 (t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d 149.1;
136.9; 134.9; 130.3; 128.7; 127.9; 125.9; 120.5; 53.2; 31.8; 29.5;
29.5; 29.3; 29.3; 29.2; 29.2; 25.6; 22.6; 14.0; HRMS: (DCI/CH4, m/z)
calc. for C19H29N3Cl: 334.2050. Found: 334.2047.
4.2.2.17. 1-Benzyl-4-dodecyl-1H-1,2,3-triazole (23). White powder.
Mp 85 ꢀC. Yield 82%. 1H NMR (CDCl3)
d 7.39 (m, 3H); 7.23 (m, 2H);
7.18 (s, 1H); 5.48 (s, 2H); 2.67 (t, J ¼ 7.7 Hz, 2H); 1.62 (m, 2H); 1.24
4.2.2.25. 3-Octyl-1H-1,2,3-triazol-4-yl)phenol (31). White powder.
(m, 18H); 0.87 (t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d
148.9; 135.0;
Mp 69 ꢀC. Yield 68%. 1H NMR (CDCl3)
d 7.68 (s, 1H); 7.64 (m, 1H);
129.0; 128.5; 127.9; 120.4; 55.9; 31.8; 29.6; 29.6; 29.5; 29.5; 29.3;
29.2; 25.7; 22.6; 14.1; HRMS: (DCI/CH4, m/z) calc. for C21H34N3:
328.2753. Found: 328.2756.
7.24 (m, 2H); 6.90 (m, 1H); 4.36 (t, J ¼ 7.2 Hz, 2H); 1.91 (m, 2H); 1.28
(m, 10H); 0.86 (t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d 157.0; 147.5;
131.4; 130.1; 119.9; 117.5; 115.8; 113.0; 50.6; 31.7; 30.2; 29.0; 28.9;
26.4; 22.6; 14.0; HRMS : (DCI/CH4, m/z) calc. for C16H24N3O:
274.1919. Found: 274.1917.
4.2.2.18. 1-(3-Methoxybenzyl)-4-hexyl-1H-1,2,3-triazole (24). White
powder. Mp 44 ꢀC. Yield 78%. 1H NMR (CDCl3)
d
7.28 (t, J ¼ 7.9 Hz,
1H); 7.18 (s, 1H); 6.87 (dd, J ¼ 8.3 Hz, 2.6 Hz, 1H); 6.83 (d, J ¼ 7.5 Hz,
1H); 6.77 (t, J ¼ 1.9 Hz, 1H); 5.46 (s, 2H); 3.78 (s, 3H); 2.68 t,
J ¼ 7.7 Hz, 2H; 1.63 (m, 2H); 1.29 (m, 6H); 0.86 (t, J ¼ 7.7 Hz, 3H); 13C
4.2.2.26. 1-Octyl-4-m-tolyl-1H-1,2,3-triazole (32). White powder.
Mp 75 ꢀC. Yield 98%. 1H NMR (CDCl3)
d 7.73 (s, 1H); 7.68 (s, 1H); 7.58
(d, J ¼ 7.7 Hz,1H); 7.27 (t, J ¼ 7.6 Hz,1H); 7.10 (d, J ¼ 7.6 Hz,1H); 4.32
NMR (CDCl3)
d 160.1; 149.0; 136.4; 130.1; 120.5; 120.1; 114.1; 113.5;
(t, J ¼ 7.2 Hz, 2H); 2.36 (s, 3H); 1.88 (m, 2H); 1.27 (m, 10H); 0.85 (t,
55.3; 53.9; 31.5; 29.3; 29.0; 25.7; 22.5; 14.0; HRMS: (DCI/CH4, m/z)
calc. for C16H24N3O: 274.1919. Found: 274.1927.
J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d 147.5; 138.2; 130.5; 128.6; 128.5;
126.1; 122.6; 119.3; 50.2; 31.5; 30.1; 28.9; 28.8; 26.3; 22.4; 21.2;
13.9; HRMS : (DCI/CH4, m/z) calc. for C17H26N3: 272.2127. Found:
272.2126.
4.2.2.19. 1-(3-Methoxybenzyl)-4-decyl-1H-1,2,3-triazole (25). White
powder. Mp 59 ꢀC. Yield 76%. 1H NMR (CDCl3)
d
7.26 (t, J ¼ 7.9 Hz,
1H); 7.18 (s, 1H); 6.83 (m, 2H); 6.75 (t, J ¼ 1.9 Hz, 1H); 5.44 (s, 2H);
3.76 (s, 3H); 2.66 (t, J ¼ 7.6 Hz, 2H); 1.62 (m, 2H); 1.23 (m, 14H); 0.86
4.2.3. Synthesis of compounds 6 and 7
Compound 5 was synthesized as described for compounds
8e32. Compounds 6 and 7 were synthesized according to the
protocol bellow. To a solution of triazole 5 (1.0 eq) and phenol (1.0)
in acetone, was added K2CO3 at room temperature. The reaction
was stirred at reflux for overnight. Then the solution was concen-
trated, diluted with AcOEt. The organic layer was washed with
water and brine, dried over MgSO4 and concentrated under
reduced pressure. The desired compound was purified by flash
chromatography (petroleum ether/EtOAc : 9/1).
(t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d 160.0; 148.9; 136.4; 130.0;
120.4; 120.0; 114.0; 113.4; 55.2; 53.8; 31.8; 29.5; 29.5; 29.3; 29.3;
29.2; 29.2; 25.7; 22.6; 14.0; HRMS: (DCI/CH4, m/z) calc. for
C20H32N3O: 330.2545. Found: 330.2560.
4.2.2.20. 1-(3-Methoxybenzyl)-4-dodecyl-1H-1,2,3-triazole
White powder. Mp 65 ꢀC. Yield 90%. 1H NMR (CDCl3)
(26).
7.28 (t,
d
J ¼ 7.9 Hz, 1H); 7.19 (s, 1H); 6.87 (dd, J ¼ 8.3 Hz, 2.5 Hz, 1H); 6.83 (d,
J ¼ 7.5 Hz, 1H); 6.76 (t, J ¼ 1.9 Hz,1H); 5.46 (s, 2H); 3.77 (s, 3H); 2.68
(t, J ¼ 7.5 Hz, 2H); 1.63 (m, 2H); 1.24 (m, 18H); 0.87 (t, J ¼ 6.7 Hz,
4.2.3.1. 4-((2,4-Dichlorophenoxy)methyl)-1-dodecyl-1H-1,2,3-triazole
3H); 13C NMR (CDCl3)
d
160.0; 136.4; 130.1; 120.1; 114.1; 113.5;
(6). White powder. Mp 50 ꢀC. Yield 94%. 1H NMR (CDCl3)
d 7.62 (m,
55.3; 31.9; 29.6; 29.6; 29.5; 29.3; 29.2; 25.7; 22.7; 14.1; HRMS:
(DCI/CH4, m/z) calc. for C22H36N3O: 358.2858. Found: 358.2868.
1H); 7.35 (d, J ¼ 2.5 Hz, 1H); 7.17 (dd, J ¼ 8.8 Hz, 2.5 Hz, 1H); 7.05 (d,
J ¼ 8.8 Hz,1H); 5.27 (s, 2H); 4.34 (t, J ¼ 7.2 Hz, 2H); 1.89 (m, 2H); 1.24
(m, 18H); 0.87 (t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d 152.5; 143.4;
4.2.2.21. 1-(3,5-Dimethoxybenzyl)-4-decyl-1H-1,2,3-triazole (27).
130.0; 127.6; 126.4; 123.9; 122.6; 115.1; 63.6; 50.5; 31.8; 30.2; 29.5;
29.5; 29.4; 29.3; 29.3; 28.9; 26.4; 22.6; 14.1; HRMS: (DCI/CH4, m/z)
calc. for C21H32N3OCl2: 412.1922. Found: 412.1918.
White powder. Mp 77 ꢀC. Yield 70%. 1H NMR (CDCl3)
d 7.19 (s, 1H);
6.37 (m, 3H); 5.38 (s, 2H); 3.73 (s, 6H); 2.66 (t, J ¼ 7.6 Hz, 2H); 1.62
(m, 2H); 1.23 (m, 14H); 0.85 (t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d
161.2; 148.9; 137.1; 120.5; 105.8; 100.2; 55.3; 53.9; 31.8; 29.5;
4.2.3.2. 4-((4-Chloro-2-methoxyphenoxy)methyl)-1-dodecyl-1H-1,2,
29.5; 29.3; 29.3; 29.2; 29.2; 25.6; 22.6; 14.0; HRMS: (DCI/CH4, m/z)
3-triazole (7). White powder. Mp 61 ꢀC. Yield 66%. 1H NMR (CDCl3)
calc. for C21H34N3O2: 360.2651. Found: 360.2663.
d
7.59 (s, 1H); 6.95 (d, J ¼ 8.7 Hz, 1H); 6.84 (s, 1H); 6.82 (dd,
J ¼ 8.6 Hz, 2.4 Hz, 1H); 5.24 (s, 2H); 4.31 (t, J ¼ 7.3 Hz, 2H); 3.93 (s,
4.2.2.22. 1-(3,5-Dimethylbenzyl)-4-hexyl-1H-1,2,3-triazole (28). White
3H); 1.87 (m, 2H); 1.23 (m, 18H); 0.86 (t, J ¼ 6.7 Hz, 3H); 13C NMR
powder. Mp 75 ꢀC. Yield 81%. 1H NMR (CDCl3)
d
7.17 (s, 1H); 6.96 (s,
(CDCl3) d 150.2; 146.4; 143.9; 126.6; 122.7; 120.4; 115.2; 112.5;

282
C. Menendez et al. / European Journal of Medicinal Chemistry 52 (2012) 275e283
63.4; 56.1; 50.5; 30.3; 29.6; 29.5; 29.4; 29.4; 29.3; 29.3; 29.0; 26.5;
22.7; 14.1; HRMS: (DCI/CH4, m/z) calc. for C22H35N3O2Cl: 408.2418.
Found: 408.2424.
serial dilutions of appropriate compound. MIC values were scored
as the lowest drug concentrations inhibiting bacterial growth. All
assays were repeated three times.
4.2.4. Synthesis of compounds 33 and 34
4.3.5. Molecular docking studies
A solution of azide and alkyne was heated to reflux in toluene
for 35 h. After cooling to room temperature, the reaction mixture
was diluted with EtOAc, washed with water, dried over MgSO4,
filtered and concentrated over reduced pressure. The mixture was
purified by flash chromatography using a linear gradient 100%
petroleum ether (solvent A) to 5/5 solvent A/solvent B (CH2Cl2/
AcOEt 5/5) over 15 min.
The conformation active site of InhA is known to be flexible with
states corresponding to a major or a minor portal opening and loop
reordering (residues 195e210) [16e18]. The best active ligand
(compound18)andsomeproductsof thelibrary(cf. Table 1)areclosed
in shape by comparison with 5-octyl-2-phenoxy-phenol known as
8PS hetero compound ID in Protein Data Bank (PDB) [19]. Conse-
quently the corresponding structure of InhA cocrystallized with 8PS
and NADþ (2B37 PDB ID, chain C) was used for docking studies [11].
Molegro Virtual Docker (MVD) 5.0 software (Molegro ApS,
Aarhus, Denmark) was used [10,20]. The structures (protein, NADþ,
ligand) were imported in MVD. The cavity detection algorithm
implemented in MVD was used to optimize the definition of a 15 Å
(radius) potential binding site. The corresponding crystallographic
NADþ molecule was used as cofactor using the MVD features. The
water molecules were not taken in account. The side chains around
the cofactor (30 residues around and upper the NADþ) were set as
flexible with a tolerance of 0.99 and strength of 0.91 (MVD units). A
calculation scheme combining Moldock Score [GRID] and Moldock
optimizer was used with a population size of 50, maximum itera-
tion of 2000, scaling factor of 0.50, crossover rate of 0.90 and
a variation-based termination scheme. After calculation minimi-
zation steps (global, lateral chain, ligands), optimization of
hydrogen bonds was performed using MVD default features. For
each ligand and a given calculation scheme, 20 runs (a set) were
carried out using Tabu clustering. If the best values of Moldock
Score and Rerank Score were found for the same pose, the corre-
sponding conformation was retained as the best pose.
4.2.4.1. 1-Dodecyl-5-phenethyl-1H-1,2,3-triazole
(33). White
powder. Mp 50 ꢀC. 1H NMR (CDCl3)
d
7.48 (s, 1H); 7.28 (m, 3H); 7.15
(d, J ¼ 7.8 Hz, 2H); 4.10 (t, J ¼ 7.4 Hz, 2H); 2.95 (m, 4H); 1.78 (m, 2H);
1.24 (m, 18H); 0.87 (t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d 139.8;
128.7; 128.3; 126.6; 47.7; 34.6; 31.8; 29.9; 29.5; 29.4; 29.3; 29.3;
29.0; 26.5; 25.3; 22.6; 14.1; HRMS: (DCI/CH4, m/z) calc. for
C22H36N3: 342.2909. Found: 342.2916.
4.2.4.2. 5-Decyl-1-phenethyl-1H-1,2,3-triazole (34). Colourless oil.
1H NMR (CDCl3)
d 7.36 (s, 1H); 7.23 (m, 3H); 7.02 (m, 2H); 4.41 (t,
J ¼ 7.2 Hz, 2H); 3.19 (t, J ¼ 7.2 Hz, 2H); 2.20 (t, J ¼ 7.5 Hz, 2H); 1.43
(m, 2H); 1.24 (m, 14H); 0.87 (t, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3)
d
137.7; 137.4; 131.7; 128.8; 128.7; 127.0; 49.0; 36.9; 31.9; 29.5;
29.5; 29.3; 29.2; 29.1; 27.9; 22.8; 22.7; 14.1; HRMS: (DCI/CH4, m/z)
calc. for C20H32N3: 314.2596. Found: 314.2595.
4.3. Biology
4.3.1. InhA expression and purification
The production and purification were performed as described in
reference 10.
Acknowledgements
4.3.2. Inhibition Kinetics
We thank the CNRS and the “ UniversitéPaul Sabatier” for
financial support.
Stock solutions of all compounds were prepared in DMSO such
that the final concentration of this co-solvent was constant at 5% v/
v in a final volume 1 mL for all kinetic reactions. Kinetic assays using
trans-2-dodecenoyl-Coenzyme A (DD-CoA) and wild-type InhA
were performed as described in reference 10. Reactions were
initiated by addition of InhA (100 nM final) to solutions containing
Appendix A. Supplementary material
Supporting information related to this article can be found
online at doi:10.1016/j.ejmech.2012.03.029.
DD-CoA (50 mM final), inhibitor, and NADH (250 mM final) in 30 mM
PIPES, 150 mM NaCl, pH 6.8, buffer. Control reactions were carried
out with the same conditions as described above but without
inhibitor. The inhibitory activity of each derivative was expressed as
the percentage inhibition of InhA activity (initial velocity of the
reaction) with respect to the control reaction without inhibitor. All
activity assays were performed in triplicate.
References
[1] World Health Organization: http://www.who.int/tb/publications/global_
report/2010/en/index.html.
[2] M.H. Cynamon, Y. Zhang, T. Harpster, S. Cheng, M.S. DeStefano, High-dose
isoniazid therapy for isoniazid-resistant murine Mycobacterium tuberculosis
infection, Antimicrob. Agents Chemother. 43 (1999) 2922e2924.
[3] P. Bemer-Melchior, A. Bryskier, H.B. Drugeon, Comparison of the in vitro
activities of rifapentine and rifampicin against Mycobacterium tuberculosis
complex, J. Antimicrob. Chemother. 46 (2000) 571e576.
4.3.3. Growth conditions
M. tuberculosis H37Rv strain was grown either in Middlebrook
7H9 broth (Difco) supplemented with 0.05% Tween 80, or on
Middlebrook 7H11 agar (Difco) supplemented with 0.5% glycerol,
both supplemented with 10% (vol/vol) OADC. All compounds were
dissolved in dimethyl sulfoxide (DMSO). Mycobacterial cultures
were usually grown at 37 ꢀC without shaking.
[4] A. Jain, R. Mondal, Extensively drug-resistant tuberculosis: current challenges
and threats, FEMS Immunol. Med. Microbiol. 53 (2008) 145e150.
[5] A.S. Fauci, Multidrug-resistant and extensively drug-resistant tuberculosis:
the National Institute of Allergy and Infectious Diseases Research agenda and
recommendations for priority research, J. Infect. Dis. 197 (2008) 1493e1498.
[6] (a) P. De, G. Koumba Yoya, P. Constant, F. Bedos-Belval, H. Duran, N. Saffon,
M. Daffé, M. Baltas, Design, synthesis, and biological evaluation of new cin-
namic derivatives as antituberculosis agents, J. Med. Chem. 54 (2011)
1449e1461;
(b) M.D. Kakwani, P. Suryavanshi, M. Ray, M.G. Rajan, S. Majee, A. Samad,
P. Devarajan, M.S. Degani, Design, synthesis and antimycobacterial activity of
4.3.4. MIC determinations
A single colony of M. tuberculosis strain was used to inoculate
complete Middlebrook 7H9. The cultures were incubated at 37 ꢀC
until exponential growth phase (w108 CFU/mL) was reached, cor-
responding to an OD600nm ranging from 0.8 to 1.0. Cultures were
cinnamide derivatives:
a molecular hybridization approach, Bioorg. Med.
Chem. Lett. 21 (2011) 1997e1999.
ꢀ
ꢀ
ꢀ
ꢀ
}
[7] (a) E. Vavríková, S. Polanc, M. Kocevar, J. Kosmrlj, K. Horváti, S. Bosze,
ꢀ
J. Stolaríková, A. Imramovský, J. Vinsová, New series of isoniazid hydrazones
linked with electron-withdrawing substituents, Eur. J. Med. Chem. 46 (2011)
5902e5909;
diluted to the final concentration of about 107 CFU/mL; 1
m
l of the
diluted cultures was then streaked onto plates containing two-fold
(b) F.R. Pavan, P.I. da S Maia, S.R. Leite, V.M. Deflon, A.A. Batista, D.N. Sato,

C. Menendez et al. / European Journal of Medicinal Chemistry 52 (2012) 275e283
283
S.G. Franzblau, C.Q. Leite, Thiosemicarbazones, semicarbazones, dithiocarba-
zates and hydrazide/hydrazones: anti-Mycobacterium tuberculosis activity and
cytotoxicity, Eur. J. Med. Chem. 45 (2010) 1898e1905.
affinity InhA inhibitors with activity against drug-resistant strains of Myco-
bacterium tuberculosis, ACS Chem. Biol. 1 (2006) 43e53.
[12] V.V. Rostovtsev, L.G. Green, V.V. Fokin, K.B. Sharpless, A stepwise huisgen
cycloaddition process: copper(I)-catalyzed regioselective "ligation" of azides
and terminal alkynes, Angew. Chem. Int. Ed. 41 (2002) 2596e2599.
[13] C.M. Tornoe, C. Christensen, M. Meldal, Peptidotriazoles on solid phase:
[1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloaddi-
tions of terminal alkynes to azides, J. Org. Chem. 67 (2002) 3057e3064.
[14] M. van Scherpenzeel, E.E. Moret, L. Ballell, R.M.J. Liskamp, U.J. Nilsson,
H. Leffler, R.J. Pieters, Synthesis and evaluation of new thiodigalactoside-based
chemical probes to label galectin-3, Chem. Bio. Chem. 10 (2009) 1724e1733.
[15] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt,
E.C. Meng, T.E. Ferrin, UCSF Chimera - a visualization system for exploratory
research and analysis, J. Comput. Chem. 13 (2004) 1605e1612.
[16] D.A. Rozwarski, C. Vilcheze, M. Sugantino, R. Bittman, C. Sacchettini, Crystal
structure of the Mycobacterium tuberculosis Enoyl-ACP reductase, InhA, in
complex with NADþ and a C16 Fatty acyl Substrate, J. Biol. Chem. 274 (1999)
15582e15589.
[17] J.S. Freundlich, F. Wang, C. Vilcheze, G. Gulten, R. Langley, G.A. Schiehser,
D.P. Jacobus, W.R. Jacobs Jr., J.C. Sacchettini, Triclosan derivatives: towards
potent inhibitors of drug-sensitive and drug-resistant Mycobacterium tuber-
culosis, Chem. Med. Chem. 4 (2009) 241e248.
[8] (a) J. Cherian, I. Choi, A. Nayyar, U.H. Manjunatha, T. Mukherjee, Y.S. Lee,
H.I. Boshoff, R. Singh, Y.H. Ha, M. Goodwin, S.B. Lakshminarayana,
P. Niyomrattanakit, J. Jiricek, S. Ravindran, T. Dick, T.H. Keller, V. Dartois,
C.E. Barry III, Structure-activity relationships of antitubercular nitro-
imidazoles. 3. Exploration of the linker and lipophilic tail of ((s)-2-nitro-6,7-
dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl)-(4-trifluoromethoxybenzyl)
amine (6-amino PA-824), J. Med. Chem. 54 (2011) 5639e5659;
(b) S.H. Lee, S. Kim, M.H. Yun, Y.S. Lee, S.N. Cho, T. Oh, P. Kim, Synthesis and
antitubercular activity of monocyclic nitroimidazoles: insights from econa-
zole, Bioorg. Med. Chem. Lett. 21 (2011) 1515e1518.
[9] (a) P. Shanmugavelan, S. Nagarajan, M. Sathishkumar, A. Ponnuswamy,
P. Yogeeswari, D. Sriram, Efficient synthesis and in vitro antitubercular activity
of 1,2,3-triazoles as inhibitors of Mycobacterium tuberculosis, Bioorg. Med.
Chem. Lett. 21 (2011) 7273e7275;
(b) A.K. Jordão, P.C. Sathler, V.F. Ferreira, V.R. Campos, M.C. de Souza,
H.C. Castro, A. Lannes, A. Lourenco, C.R. Rodrigues, M.L. Bello, M.C. Lourenco,
G.S. Carvalho, M.C. Almeida, A.C. Cunha, Synthesis, antitubercular activity, and
SAR study of N-substituted-phenylamino-5-methyl-1H-1,2,3-triazole-4-
carbohydrazides, Bioorg. Med. Chem. 19 (2011) 5605e5611;
(c) G.R. Labadie, A. de la Iglesia, H.R. Morbidoni, Targeting tuberculosis
through a small focused library of 1,2,3-triazoles, Mol. Divers. 15 (2011)
1017e1024.
[18] S.R. Luckner, N. Liu, C.W. am Ende, P.J. Tonge, C. Kisker, A Slow, Tight binding
inhibitor of InhA, the enoyl-acyl carrier protein reductase from Mycobacterium
tuberculosis, J. Biol. Chem. 285 (2010) 14330e14337.
[10] C. Menendez, S. Gau, C. Lherbet, F. Rodriguez, C. Inard, M.R. Pasca, M. Baltas,
Synthesis and biological activities of triazole derivatives as inhibitors of InhA
and antituberculosis agents, Eur. J. Med. Chem. 46 (2011) 5524e5531.
[11] T.J. Sullivan, J.J. Truglio, M.E. Boyne, P. Novichenok, X. Zhang, C.F. Stratton,
H.J. Li, T. Kaur, A. Amin, F. Johnson, R.A. Slayden, C. Kisker, P.J. Tonge, High
[19] H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig,
I.N. Shindyalov, P.E. Bourne, The protein data bank, Nucleic Acids Res. 28
(2000) 235e242.www.pdb.org.
[20] R. Thomsen, M.H. Christensen, MolDock: a new technique for high-accuracy
molecular docking, J. Med. Chem. 49 (2006) 3315e3321.