Synthesis and biological activities of triazole derivatives as inhibitors of InhA and antituberculosis agents

Article abstract of DOI:10.1016/j.ejmech.2011.09.013

InhA, the enoyl reductase from the mycobacterial type II fatty acid biosynthesis pathway, is a target for the development of novel drugs against tuberculosis. We exploited copper-catalyzed [3+2] cycloaddition between alkynes and different azides to afford 1,4-disubstituted triazole or α-ketotriazole derivatives. Several compounds bearing a lipophilic chain mimicking the substrate were able to inhibit InhA. Among them, 1-dodecyl-4-phenethyl-1H-1,2,3-triazole displayed a minimum inhibitory concentration inferior to 2 μg/mL against Mycobacterium tuberculosis H37Rv.

Full text of DOI:10.1016/j.ejmech.2011.09.013

European Journal of Medicinal Chemistry 46 (2011) 5524e5531
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological activities of triazole derivatives as inhibitors of InhA
and antituberculosis agents
,
,
,
b
,
a,
Christophe Menendeza b, Sylvain Gaua b, Christian Lherbeta , Frédéric Rodriguez b, Cyril Inardc,
*
Maria Rosalia Pascad, Michel Baltasa
,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 Dipartimento di Genetica e Microbiologia, University of Pavia, 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:
InhA, the enoyl reductase from the mycobacterial type II fatty acid biosynthesis pathway, is a target for
Received 22 April 2011
Received in revised form
2 September 2011
Accepted 10 September 2011
Available online 16 September 2011
the development of novel drugs against tuberculosis. We exploited copper-catalyzed [3þ2] cycloaddition
between alkynes and different azides to afford 1,4-disubstituted triazole or a-ketotriazole derivatives.
Several compounds bearing a lipophilic chain mimicking the substrate were able to inhibit InhA. Among
them, 1-dodecyl-4-phenethyl-1H-1,2,3-triazole displayed a minimum inhibitory concentration inferior to
2 mg/mL against Mycobacterium tuberculosis H37Rv.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Inhibitor
Triazole
Enoyl ACP reductase
InhA
Tuberculosis
1. Introduction
activation by KatG, the mycobacterial catalase-peroxidase, forms an
adduct with NADH called NAD-INH [8,9]. The X-ray structure of the
complex InhA-isonicotinoyl moiety of this adduct shows that it
occupies a hydrophobic pocket [10]. Different groups validated this
cavity as a suitable site to increase inhibitors potency [11]. Different
approaches exist toward the design of new inhibitors for InhA.
High-throughput screen of a bank of compounds is one of them. By
this way, He et al. found that pyrrolidine carboxamides is a class of
inhibitors of InhA [12]. Other methods involve modifying existing
structures of triclosan [13] (5-chloro-2-(2,4-dichloro-phenoxy)
phenol ether, (Fig. 1)), one of the lead compounds. Indeed, several
research groups have focused their attention on the synthesis of 5-
substituted triclosan derivatives and of different molecules bearing
this diphenyl ether skeleton (Fig. 1) [14e16].
In this study, we report the design, synthesis and evaluation of
potential InhA inhibitors by replacing the phenolic moiety (ring B)
in triclosan derivatives with 1,4-disubstituted triazole using click
chemistry. Changes in the ring B have already been reported in the
literature especially with the use of piperidine [17] or pyrrolidine
carboxamide [12] as the core structure. This modification to triazole
could permit the synthesis of various inhibitors bearing a wide
range of substituents. 1,2,3-Triazoles have received increased
attention for their biological applications [18]. For example,
Tuberculosis (TB) is a threat to worldwide public health [1].
Despite the availability of effective treatments, tuberculosis is
responsible for more than three million deaths annually world-
wide. The high susceptibility of human immunodeficiency virus-
infected persons to the disease [2], the emergence of multi-drug-
resistant (MDR-TB) strains [3,4] and extensively drug-resistant
(XDR-TB) ones [5] which are mainly resistant to the most effec-
tive drugs (isoniazid; rifampicin) have brought this infectious
disease back into scientific focus. For this reason, there is a growing
need and urgency to discover new classes of chemical compounds
acting with different mechanisms from those currently used. The
design of new TB drugs has been an important challenge for
chemists and biochemists in recent years.
It is believed that isoniazid (INH) inhibits InhA, an essential
enzyme in the FASII system involved in the synthesis of the
mycobacterial mycolic acids [6,7]. INH is a prodrug that upon
* 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 address: baltas@chimie.ups-tlse.fr (M. Baltas).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.013

C. Menendez et al. / European Journal of Medicinal Chemistry 46 (2011) 5524e5531
5525
Cl
A
OH
B
O
Cl
OH
O
O
R
H
R
Cl
Cl
Cl
Triclosan
O
O
Fig. 1. Representative structure of enoyl reductase inhibitors (Triclosan and
N
?
derivatives).
R
R
N
TMS
N
R1
OH
N
N
N
N
N
O
Scheme 2. Routes to
a-ketotriazoles.
N
O
as described in Scheme 1. The desired products (1 and 2) were
obtained in excellent yields. Azides were easily obtained from their
corresponding bromide or chloride derivatives by nucleophilic
substitutions (See experimental section).
Class II
Class I
Fig. 2. Representative cores used in the synthesis of our molecules.
Then, we attempted to get
a-ketotriazoles in a “one pot”
cycloaddition between (TMS)ethynyl ketones and azides mediated
by Cu(II) without prior TMS deprotection.
Colombano et al. exploited copper-catalyzed [3þ2] cycloaddition
reaction between different azides and alkynes in order to synthe-
size a series of inhibitors and generate structureeactivity rela-
tionships against nicotinamide phosphoryltransferase [19].
Moreover, this reaction was applied to the synthesis of dihy-
drobenzofuran derivatives with antitubercular activities [20]. Two
different classes of 1,2,3-triazoles have been explored as shown in
1-Trimethylsilyl-1-alkynyl ketones are useful intermediates for
the synthesis of various molecules through copper-dependent
cycloadditions [22,23]. For example, Karpov and Müller used
them in a one-pot three component reaction to provide 2,4-
disubtituted pyrimidines [24]. Usually a prior step is necessary to
furnish the terminal alkyne (Scheme 2). Different reagents such as
fluoride sources, silver salts or base are frequently used to deprotect
sequentially or in “one pot” TMS [25,26]. Because ynones by
comparison with terminal alkynes are very reactive, we wondered
if smooth deprotection conditions could be found. We chose as
model reaction partners, commercially available benzyl azide and
4-(trimethylsilyl)-1-phenylbut-3-yn-2-one. The reaction was first
conducted at room temperature in the presence of Cu(II) sources
(e.g. CuCl2) and sodium ascorbate in CH3CN/H2O. The reaction is
very slow and the yield is too low after 1h of reaction (entry 1,
Table 1). However, increasing temperature to reflux allowed the
starting materials to be consumed after 1 h as checked by TLC and
the isolation of 1,2,3-triazole with a good yield (78%, entry 2). When
the reaction was performed with Cu(OAc)2, a 62% yield was ob-
tained (entry 3).
To evaluate the role of water present as co-solvent, the reaction
was carried out under anhydrous conditions (dry acetonitrile only)
and reflux affording 1,2,3-triazole 3 in a very poor yield 7% (entry
4). In view of these results it seems likely that water might facilitate
the deprotection of 1-trimethylsilyl-ynone in situ.
Finally, for comparison with TMS-ynone, treatment of 1-phenyl-
2-(trimethylsilyl)acetylene with benzyl azide led to the corre-
sponding 1,2,3-triazole with a very poor yield (entry 5, Table 1).
This result indicates that the starting material is not hydrolyzed as
quickly as the TMSethynyl ketone under our conditions.
Fig. 2. Class I is a a-ketoheterocycle-based inhibitor bearing a a-
ketotriazole moiety as a central fragment. A second one, class II,
was investigated as a 1,4-dialkyl-substituted triazole to verify the
contribution of the carbonyl group in the binding site of InhA. All
the molecules were evaluated as inhibitors against InhA and for
their antitubercular activity against Mycobacterium tuberculosis
H37Rv strain.
2. Results and discussion
2.1. Chemistry
The catalyzed Huisgen 1,3 dipolar cycloaddition reaction
between azides and alkynes has generated numerous applications.
These cycloadditions are characterized by mild reaction conditions
and high yields [18,21,22].
Class I compounds. In the literature, the use of ynones in cyclo-
addition reactions to obtain triazoles is rare [23]. (Trimethylsilyl)
ethynyl ketones are significantly more stable than the parent
ynones and act usually as a precursor for the terminal alkyne before
cycloaddition. (TMS)ethynyl ketones can be easily generated by
different ways (1) addition of bis(trimethylsilyl)acetylene in the
presence of aluminum trichloride in a FriedeleCrafts acylation; (2)
addition of lithium (trimethylsilyl)-acetylide to Weinreb amides;
(3) Stille coupling of acyl chlorides with (TMS)-ethynyl tributyl-
stannane or (4) Sonogashira cross coupling of TMS acetylene and
acyl chlorides [24]. Finally we decided to prepare our trimethylsilyl
ynone from phenyl acetylchloride derivatives, bis(trimethylsilyl)
acetylene and aluminum trichloride in a Friedel and Crafts reaction
After exploring different reaction conditions for a variety of
azide reagents, we obtained the optimized results in all cases when
introducing the reactants at room temperature then heating
progressively to reflux conditions for 1 h.
R
R
R
TMS
AlCl3
Cl
TMS TMS
OH
SOCl2
O
O
CH2Cl2
0°C
O
CH2Cl2, reflux
R
R
R
two steps R = H 94 % 1
Cl 92 % 2
Scheme 1. Synthesis of trimethylsilyl ynones 1 and 2.

5526
C. Menendez et al. / European Journal of Medicinal Chemistry 46 (2011) 5524e5531
Table 1
Table 2
Screening of the conditions for click chemistry with TMS-ynones.
Synthesis of inhibitors for InhA.
N
N
TMS
R1
R
R
TMS
CuCl2 0.1 eq
NaAsc 0.2 eq
N
N
N
1
1-2
O
O
N
O
conditions
R
AcCN/H2O 4/1
rt to reflux
R
3-15
N3
O
R1
N3
3
Compounds
R
Azides
Yield
(%)
Entry
Conditions
Time
(h)
Yield
(%)
1
2
3
4
5
6
CuCl2 (0.1 equiv), NaAsc (0.2 equiv),
AcCN/H2O (4/1), rt
CuCl2 (0.1 equiv), NaAsc (0.2 equiv),
AcCN/H2O (4/1), 80 ꢀC
Cu(OAc)2 (0.1 equiv), NaAsc (0.2 equiv),
AcCN/H2O (4/1), 80 ꢀC
CuCl2 (0.1 equiv), NaAsc (0.2 equiv),
AcCN, 80 ꢀC
CuCl2 (0.1 equiv), NaAsc (0.2 equiv),
AcCN/H2O (4/1), 80 ꢀC
CuCl2 (0.1 equiv), NaAsc (0.2 equiv),
AcCN/H2O (4/1), rt to 80 ꢀC
1
1
1
1
2
1
14
78
62
7
N3
N3
3
H
90
82
4
5
H
H
OMe
MeO
7a
90
N3
63
80
a
OMe
Reaction performed with 1-phenyl-2-(trimethylsilyl)acetylene.
Me
N3
6
H
In order to verify the generality of our methodology, we
extended it to different (trimethylsilyl)ynones and azides (Table 2).
The yields remained good regardless of the different benzyl groups
used, bearing various electron-donating or electron-withdrawing
substituents. In the case of cyclohexylmethyl azide, a lower yield
was observed under our standard reaction conditions and a higher
when performing the reaction directly at 80 ꢀC (54% vs. 32%). The
reaction with 3,5-dimethoxybenzyl azide confirmed that higher
yields are obtained when the reaction starts at room temperature
to reflux.
Me
N3
7
8
9
H
H
H
84
O2N
Octyl azide
N3
69
32(54)a
In order to verify the biological contribution of the
a-keto
N3
functionality we also synthesized two -hydroxy triazole deriva-
a
10
11
Cl
Cl
72
67
tives as racemates, in good yield, by reducing the keto-functionality
of 15 using sodium borohydride in methanol-dichloromethane
(Scheme 3).
Class II compounds. A series of 1,2,3-triazoles were synthesized
in order to evaluate the impact of the carbonyl and the chain length
in our molecules. The reaction was performed with Cu(OAc)2 and
sodium ascorbate in tBuOH/H2O. The results are reported in Table 3.
The desired products were obtained in good yields (60% to
quantitative).
N3
OMe
MeO
N3
12
13
14
Cl
Cl
66
61
OMe
Me
N3
2.2. Biology
2.2.1. InhA inhibition assay
Me
Recombinant M. tuberculosis InhA was expressed in Escherichia
coli and subsequently purified with slight modifications according
to previously reported procedure [12]. The synthetic compounds
were evaluated in vitro for the inhibition of InhA from
N3
Cl
Cl
86
66
O2N
M. tuberculosis at 10
mM (and/or at 50 mM) by applying a commonly
15
Octyl azide
used method [27]. Triclosan was tested first at the same concen-
a
In brackets, yield for the reaction performed directly under reflux.
tration and showed a complete inhibition of InhA at 10
results are shown in Table 4.
mM. The
The
aromatic ring A are very weak (or not) inhibitors of InhA. In fact the
best of them present a 10% inhibition at 50 M. The 2,4-
dichlorinated analogues of these compounds (10e14) are not
recognized at 10 M. Beyond 10 M, they are not fully soluble even
at 5% DMSO final in the cuvette. In addition, compounds bearing no
a-ketotriazoles (3e7) bearing no chlorine atoms on the
carbonyl functionality (18e23) also did not display any inhibitory
abilities at 10 M (Table 4).
Based on previous reported research’s, we hypothesized that
substituents with alkyl chains mimicking the InhA substrate might
provide compounds with higher affinity. The introduction of
m
m
m
m

C. Menendez et al. / European Journal of Medicinal Chemistry 46 (2011) 5524e5531
5527
Table 4
N
N
N
N
R
N
R
NaBH4 2 eq
Enzyme inhibition values. Results are expressed as a percentage of InhA
inhibition.
N
7
7
CH3OH/CH2Cl2 3/1
rt
OH
O
R
Compounds
% Inhibition at
10 M (50 M)
>99
R
m
m
R = H 77% 16
Cl 90% 17
Triclosan
3
4
5
ND(13)
ND(10)
NI
Scheme 3. Synthesis of the corresponding alcohols.
6
7
9
ND(10)
NI
ND(12)
NI
NI
NI
NI
NI
NI
NI
lipophilic chains might facilitate more hydrophobic interactions in
the pocket [16]. The results show an increase in the inhibitory
activity (Table 5). Compound 8 by comparison with 3 presents 25%
inhibition at 50 mM. In the same way as the benzyl analogue 10,
dichloro-substituted molecule 15 was not recognized in the
10
11
12
13
14
18
19
20
21
22
23
Table 3
NI(15)
ND(11)
NI
Synthesis of class II triazole inhibitors.
Cu(OAc)2 0.1 eq
NaAsc 0.2 eq
N
N
R1
N
NI
n
ND for not determined, NI for no inhibition at the given concentration.
t-BuOH/H2O 1/1
n
18-26
R1
Compounds
18
N3
rt
binding site of InhA. These results show that chloro substitution on
ring A is detrimental to the recognition of the enzyme for this
family of compounds.
1,2,3-Triazole
Yield
(%)
Compounds bearing no carbonyl group and different alkyl
chains (24e26) were also evaluated. The results are presented in
Table 5. Highest inhibitory potencies were observed with
compounds 24 and 25 bearing respectively C8- and C9-chain
lengths. When the chain becomes longer (C12; compound 26), we
observed a decrease in affinity. This is in agreement with the results
reported by Sullivan et al. on 5-alkyl substituted des-chloro tri-
closan derivatives. They found that the activity of these molecules
was optimal when a C8 chain was introduced in the case of B ring
para-substituted triclosan analogues [14].
N
N
N
86
N
N
N
19
82
N
N
N
Both compounds (16 and 17) showed no significant activity at
20
21
60
10
m
M (Table 5). Generally, these in vitro studies showed that our
-hydroxy- or -ketotriazoles are
molecules bearing either
a
a
Me
poorly recognized or not recognized at all, in the binding site of
InhA.
Me
63a
N
N
N
Triazole derivative 24 (class II core and 1-alkyl-substituted) is
one of the most active compounds in vitro. Fig. 3 shows a hypothetic
placement of 24 obtained by molecular docking calculations in the
active site of InhA (PDB code 2X22 (chain A)). The conformation of
compound 24 shows the conservation of the hydrogen bonding
network. Its placement may be compared to that of a triclosan
derivative, TCU (5-hexyl-2-(2-methylphenoxy)phenol), which
shares some structure analogy and binds the active site of InhA in
the structure PDB code 2X22.
OMe
N
N
22
23
61a
85
N
N
N
N
Table 5
N
N
N
N
Influence of the alkyl chain. Results are expressed as a percentage of InhA
inhibition.
N
24
25
99
90
93
7
Compounds
Inhibition (%) at
10 M (50 M)
m
m
N
N
8
14(25)
NI
NI
8
15
16
17
24
25
26
NI
N
N
23(46)
ND(58)
ND(40)
26
11
a
ND for not determined, NI for no inhibition at the given concentration.
0.5 equiv Cu(OAc)2 and 1 equiv AscNa used.

5528
C. Menendez et al. / European Journal of Medicinal Chemistry 46 (2011) 5524e5531
Among these compounds tested (Table 6), two different series
could be compared. Concerning the first family of compounds
bearing a benzyl group in position 4 on the triazole (compounds
3e7), compound 4 bearing meta-methoxy substitution gave the
best MIC. For compounds 10e13 bearing chloro-substituents on the
other aryl moiety, the similar trend of biological activity was
observed. Clearly, the benzyl group bearing meta-methoxy
substituent is important for activity. The presence of chloro-
substituents in position 2 and 4 on the aryl ring allows for a 5-
fold lower MIC (11 compared to 4). The lack of inhibition for this
compound on InhA in contrast with the low MIC suggests that the
target is clearly not InhA. Therefore, we believe that these new
triazole derivatives might have biological target other than InhA
and a mode of action different than triclosan thereby making them
relevant candidates for further drug design in MDR-TB research.
Significantly, introduction of an alkyl chain in position 4 on
triazole (compounds 8, 15, 24e26) resulted in low MIC values.
Again, compound 15 bearing chloro-substituents exhibits 2.5-fold
lower MIC by comparison with compound 8, which is a non-
chloro derivative, inspite of the fact that 8 is a better inhibitor of
InhA than 15. Compound 24 bearing no carbonyl group is more
active than compound 8. If the carbonyl group is reduced or
removed for a methylene group, the MIC values are found to be
higher and the best result was obtained for compound 26 with a 12-
carbon chain with a MIC inferior to 2
mg/mL. Activity was increased
more than 5-fold by comparison with 24 (C8-chain) and more than
2-fold by comparison with 25 (C9-chain). In general, these
compounds are not only active on InhA but also on M. tuberculosis
strain H37Rv.
Fig. 3. Hypothetical binding mode of an alkyl triazole derivative (compound 24, cyan)
in the active site of InhA enzyme obtained by molecular docking. The cofactor NADþ
found in the crystallographic structure is shown in gray. The TCU conformation
(yellow) was obtained using the same calculation protocol as compound 24 and shows
good agreement with crystallography (RMSD ¼ 0.63 Å). Clipped model of 2X22 (chain
A) PDB structure with hydrophobicity scale coloring of molecular surface [28] and
outlined Tyr158 (green). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
3. Conclusion
The one pot synthesis of
a-ketotriazoles by Cu(II)-catalyzed
[3þ2] cycloaddition has been successfully accomplished using
a mild synthetic protocol. 1,4-Dialkyl substituted-triazoles were
also synthesized as another class of triazoles. Inhibition studies
showed that a-ketotriazoles are poor inhibitors of InhA compared
2.2.2. Bacterial growth inhibition experiments
to class II compounds, particularly those bearing an C8- or C9-alkyl
chain (24e26), which were found to be good inhibitors of InhA.
They showed the best inhibitory activity as well as an MIC below
All compounds were evaluated by determining the minimal
inhibitory concentration (MIC) on M. tuberculosis H37Rv strain
(Table 6). Triclosan was used as a reference drug.
2 mg/mL for 26. The development of this family of compounds and
more detailed inhibition studies in order to get insights into the
mechanism of action are underway. Finally, clear correlation
between InhA inhibition and MIC-value were not observed with
compound 11. But we feel that this difference in activity could
indicate that compound 11 may have a different mode of action,
other than InhA inhibition. This different mode of action could be
relevant to the MDR-TB drug research in the future.
Table 6
Compounds tested as inhibitory agents of M. tuberculosis growth.
1,2,3-Triazoles
MIC (mg/mL)/(mM)
Triclosan
3
4
5
6
10/34.5
100/360.6
25/81.3
>100/>296.4
>100/>327.4
100/310.3
25/83.5
7
8
4. Experimental
9
25/88.2
100/288.9
5/13.3
100/246.1
100/267.2
100/255.6
10/27.2
10/33.2
25/67.5
>100/>425
50/200.6
25/94.9
10/34.3
50/185.6
10/35
5/16.7
ꢁ2/ꢁ5.9
4.1. Material
10
11
12
13
14
15
16
17
18
19
20
21
23
24
25
26
Kinetic studies were performed 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 an LCT Premier Waters
spectrometer.
4.2. Chemistry
4.2.1. Synthesis of azide derivatives
Sodium azide (1.2 equiv) was added at room temperature to
a solution of bromide derivative (1 equiv, mmol) in DMF (3 mL). The

C. Menendez et al. / European Journal of Medicinal Chemistry 46 (2011) 5524e5531
5529
reaction mixture was stirred overnight. Et2O (20 mL) was added to
the mixture then the organic layer was washed with water
(3 ꢂ 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: azidooctane
[23], 4-nitrobenzylazide [23], (azidomethyl)cyclohexane [23], 1-
2H); 8.09 (s, 1H); 8.23 (d, J ¼ 8.7 Hz, 2H); 13C NMR (CDCl3)
d
46.0;
53.3; 124.4; 126.3; 127.0; 128.5; 128.9; 129.8; 133.7; 140.5; 148.0;
148.2; 192.1; HRMS: (DCI/CH4, m/z) calc. for C17H22N4O3: 323.1144.
Found: 323.1142.
4.2.3.6. 1-(1-(Octyl)-1H-1,2,3-triazol-4-yl)-2-phenylethanone
(azidomethyl)-3-methoxybenzene
[29],
1-(azidomethyl)-3,5-
(8). White powder. Mp 105 ꢀC. Yield 99%. 1H NMR (CDCl3)
d
0.88 (t,
J ¼ 6.9 Hz, 3H); 1.29 (m, 10H); 1.92 (m, 2H); 4.38 (t, J ¼ 7.2 Hz, 2H);
4.43 (s, 2H); 7.31 (m, 5H); 8.05 (s, 1H); 13C NMR (CDCl3)
14.0; 22.5;
dimethoxybenzene [30], 1-(azidomethyl)-3,5-dimethylbenzene [31].
d
4.2.2. Preparation of trimethylsilylethynyl ketones
These compounds were prepared according to a known proce-
dure [32].
26.3; 28.8; 28.9; 30.0; 31.6; 45.9; 50.7; 125.8; 126.8; 128.5; 129.9;
134.0; 147.4; 192.3; HRMS: (DCI/CH4, m/z) calc. for C18H26N3O:
300.2076. Found: 300.2089.
4.2.2.1. 1-(2,4-Dichlorophenyl)-4-(trimethylsilyl)but-3-yn-2-one
4.2.3.7. 1-(1-(Cyclohexylmethyl)-1H-1,2,3-triazol-4-yl)-2-
(2). Yellow oil. 1H NMR (CDCl3)
d
0.19 (s, 9H); 3.95 (s, 2H); 7.17 (d,
J ¼ 8.2 Hz, 1H); 7.24 (dd, J ¼ 8.2, 2.1 Hz, 1H); 7.42 (d, J ¼ 2.1 Hz, 1H);
13C NMR (CDCl3)
phenylethanone (9). White solid. Mp 122 ꢀC. Yield 32%. 1H NMR
(CDCl3)
d
1.12 (m, 5H); 1.75 (m, 6H); 4.23 (d, J ¼ 7.1 Hz, 2H); 4.46 (s,
d
ꢃ0.9; 48.2; 100.5; 101.1; 127.2; 129.4; 130.2;
2H); 7.34 (m, 5H); 8.05 (s, 1H); 13C NMR (CDCl3)
d
25.4; 25.9; 30.3;
132.6; 134.1; 135.6; 182.9; HRMS: (DCI/CH4, m/z) calc. for
38.6; 45.9; 56.7; 126.4; 126.8; 128.5; 129.9; 134.0; 147.3; 192.4;
HRMS: (DCI/CH4, m/z) calc. for C17H22N3O: 284.1763. Found:
284.1765.
C13H15Cl2OSi: 285.0269. Found: 285.0272.
4.2.3. Preparation of the a-ketotriazole products
A typical experimental procedure for the preparation of these
compounds from the corresponding trimethylsilylethynyl ketones is
described below. CuCl2 (0.1 equiv), AscNa (0.2 equiv) were added at
room temperature to a solution of 1-trimethylsilyl-1-alkynyl ketone
(1 equiv) with azide (1.2 equiv) in CH3CN/H2O (4/1). Then the reac-
tion mixture was warmed to reflux for 45e60 min. After cooling the
reaction to room temperature, 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).
4.2.3.8. 1-(1-Benzyl-1H-1,2,3-triazol-4-yl)-2-(2,4-dichlorophenyl)
ethanone (10). White powder. Mp 128 ꢀC. Yield 72%. 1H NMR
(CDCl3)
d
4.57 (s, 2H); 5.57 (s, 2H); 7.22 (m, 2H); 7.31 (m, 2H); 7.39
43.5; 54.5; 125.7; 127.06;
(s, 4H); 8.02 (s, 1H); 13C NMR (CDCl3)
d
128.3; 129.2; 129.2; 129.3; 131.1; 132.7; 133.5; 133.6; 135.4; 147.5;
190.2; HRMS: (DCI/CH4, m/z) calc. for C17H14Cl2N3O: 346.0514.
Found: 346.0515.
4.2.3.9. 1-(1-(3-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)-2-(2,4-
dichlorophenyl)ethanone (11). Lightly yellow powder. Mp 98 ꢀC.
Yield 67%. 1H NMR (CDCl3)
d 3.79 (s, 3H); 4.57 (s, 2H); 5.53 (s, 2H);
4.2.3.1. 1-(1-Benzyl-1H-1,2,3-triazol-4-yl)-2-phenylethanone
6.82 (t, J ¼ 1.9 Hz, 1H); 6.90 (td, J ¼ 8.4, 2.5 Hz, 2H); 7.21 (d,
(3). White crystal. Mp 102 ꢀC. Yield 90%. 1H NMR (CDCl3)
d
4.42 (s,
J ¼ 1.2 Hz, 2H); 7.31 (t, J ¼ 8.0 Hz, 1H); 7.40 (t, J ¼ 1.1 Hz, 1H); 8.02 (s,
2H); 5.54 (s, 2H); 7.37 (m,10H); 7.97 (s,1H); 13C NMR (CDCl3)
d
45.9;
1H); 13C NMR (CDCl3)
d 43.5; 54.5; 55.3; 114.1; 114.5; 120.5; 127.1;
54.5; 126.0; 126.9; 128.3; 128.5; 129.2; 129.3; 129.9; 133.5; 133.9;
192.2; HRMS: (DCI/CH4, m/z) calc. for C17H16N3O: 278.1293. Found:
278.1301 (M þ H).
129.2; 130.4; 131.1; 132.7; 133.6; 134.8; 135.4; 160.2; 190.2; HRMS:
(DCI/CH4, m/z) calc. for C18H15Cl2N3O2: 376.0620. Found: 376.0621.
4.2.3.10. 1-(1-(3,5-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)-2-(2,4-
4.2.3.2. 1-(1-(3-Methoxybenzyl)-1H-1,2,3-triazol-4-yl)-2-
dichlorophenyl)ethanone (12). White powder. Mp 105 ꢀC. Yield 66%.
phenylethanone (4). Lightly yellow oil. Yield 82%. 1H NMR (CDCl3)
1H NMR (CDCl3)
d 3.77 (s, 6H); 4.58 (s, 2H); 5.49 (s, 2H); 6.42 (d,
d
3.78 (s, 3H); 4.41 (s, 2H); 5.49 (s, 2H); 6.83 (m, 1Hþ2H); 7.30 (m,
J ¼ 2.1 Hz, 2H); 6,45 (t, J ¼ 2.1 Hz,1H); 7.22 (d, J ¼ 1.2 Hz, 2H); 7.41 (t,
6H); 7.98 (s, 1H); 13C NMR (CDCl3)
d
45.9; 54.3; 55.2; 114.0; 114.4;
J ¼ 1.2 Hz, 1H); 8,02 (s, 1H); 13C NMR (CDCl3)
d 43.5; 54.6; 55.4;
120.4; 126.0; 126.8; 128.5; 129.8; 130.3; 133.9; 134.9; 147.6; 160.1;
192.2; HRMS: (DCI/CH4, m/z) calc. for C18H18N3O2: 308.1399. Found:
308.1396 (M þ H).
100.6; 106.4; 125.8; 127.1; 129.3; 131.1; 132.7; 133.6; 135.4; 147.5;
161.4; 190.2; HRMS: (DCI/CH4, m/z) calc. for C19H18Cl2N3O3:
406.0725. Found: 406.0722.
4.2.3.3. 1-(1-(3,5-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)-2-
4.2.3.11. 1-(1-(3,5-Dimethylbenzyl)-1H-1,2,3-triazol-4-yl)-2-(2,4-
phenylethanone (5). Yellow gummy solid. Yield 63%. 1H NMR
dichlorophenyl)ethanone (13). Yellow powder. Mp 153 ꢀC. Yield
(CDCl3)
d
3.76 (s, 6H); 4.41 (s, 2H); 5.45 (s, 2H); 6.40 (d, J ¼ 2.2 Hz,
61%. 1H NMR (CDCl3)
d
2.31 (s, 6H); 4.58 (s, 2H); 5.49 (s, 2H); 6.92 (s,
2H); 7.02 (s, 1H); 7.22 (s, 2H); 7.41 (s, 1H); 7.98 (s, 1H); 13C NMR
(CDCl3) 21.2; 43.5; 54.6; 125.7; 126.2; 127.1; 129.3; 130.8; 131.1;
2H); 6.44 (t, J ¼ 2.2 Hz, 1H); 7.30 (m, 5H); 7.98 (s, 1H); 13C NMR
(CDCl3)
d
45.9; 54.5; 55.4; 100.6; 106.4; 126.0; 126.9; 128.5; 129.9;
d
133.9; 135.5; 147.7; 161.4; 192.2; HRMS: (DCI/CH4, m/z) calc. for
C19H19N3O3: 338.1505. Found: 338.1499 (M þ H).
132.8; 133.2; 133.6; 135.5; 139.1; 147.4; 190.3; HRMS: (DCI/CH4, m/
z) calc. for C19H18Cl2N3O: 374.0827. Found: 374.0827.
4.2.3.4. 1-(1-(3,5-Dimethylbenzyl)-1H-1,2,3-triazol-4-yl)-2-
4.2.3.12. 1-(1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-yl)-2-(2,4-
phenylethanone (6). White crystal. Mp 104 ꢀC. Yield 80%. 1H NMR
dichlorophenyl)ethanone (14). Yellow powder. Mp 97 ꢀC. Yield 86%.
(CDCl3)
d
2.30(s,6H);4.42(s,2H);5.45(s,2H);6.90(s,2H);7.01(s,1H);
21.1; 45.8; 54.4; 125.9;
1H NMR (CDCl3)
d
4.58 (s, 2H); 5.70 (s, 2H); 7.23 (d, J ¼ 1.1 Hz, 2H);
7.39 (s, 1H); 7.45 (d, J ¼ 8.6 Hz, 2H); 8.18 (s, 1H); 8.23 (d, J ¼ 8.6 Hz,
2H); 13C NMR (CDCl3)
43.6; 54.5; 124.5; 126.0; 127.2; 128.9; 129.3;
7.25 (m, 5H); 7.97 (s, 1H); 13C NMR (CDCl3)
d
126.1; 126.8; 128.4; 129.8; 130.7; 133.3; 133.9; 139.0; 147.5; 192.2;
d
HRMS: (DCI/CH4, m/z) calc. for C19H20N3O: 306.1606. Found: 306.1610.
130.9; 132.8; 133.8; 135.4; 140.4; 147.8; 190.1; HRMS: (DCI/CH4, m/
z) calc. for C17H13Cl2N4O3: 391.0365. Found: 391.0371.
4.2.3.5. 1-(1-(4-Nitrobenzyl)-1H-1,2,3-triazol-4-yl)-2-
phenylethanone (7). Yellow powder. Mp 84 ꢀC. Yield 84%. 1H NMR
4.2.3.13. 2-(2,4-Dichlorophenyl)-1-(1-octyl-1H-1,2,3-triazol-4-yl)
(CDCl3)
d
4.42 (s, 2H); 5.66 (s, 2H); 7.31 (m, 5H); 7.44 (d, J ¼ 8.7 Hz,
ethanone (15). Yellow powder. Mp 77 ꢀC. Yield 66%. 1H NMR

5530
C. Menendez et al. / European Journal of Medicinal Chemistry 46 (2011) 5524e5531
(CDCl3)
d
0.87 (t, J ¼ 6.9 Hz, 3H); 1.29 (m, 10H); 1.94 (t, J ¼ 7.1 Hz,
4.2.5.3. 1-(Cyclohexylmethyl)-4-phenyl-1H-1,2,3-triazole
2H); 4.41 (t, J ¼ 7.2 Hz, 2H); 4.59 (s, 2H); 7.23 (d, J ¼ 0.9 Hz, 2H); 7.42
(23). White powder. Mp 54 ꢀC. Yield 85%. 1H NMR (CDCl3)
(s, 1H); 8.09 (s, 1H); 13C NMR (CDCl3)
d
14.0; 22.6; 26.4; 28.9; 29.0;
d
0.90e1.86 (m, 11H); 3.03 (m, 4H); 4.10 (d, J ¼ 7.2 Hz, 2H); 7.04 (s,
30.1; 31.6; 43.5; 50.8; 125.6; 127.1; 129.3; 131.2; 132.8; 133.6; 135.5;
147.2; 190.4; HRMS: (DCI/CH4, m/z) calc. for C18H24Cl2N3O:
368.1296. Found: 374.1307.
1H); 7.26 (m, 5H); 13C NMR (CDCl3)
d
25.4; 25.9; 27.4; 30.3; 35.5;
38.6; 56.1; 121.4; 125.9; 128.2; 128.4; 141.1; 146.7; HRMS: (DCI/CH4,
m/z) calc. for C17H24N3: 270.1970. Found: 270.1975.
4.2.4. Preparation of products 16 and 17
4.2.5.4. 1-(Octyl)-4-phenethyl-1H-1,2,3-triazole (24). White pow-
NaBH4 was added slowly to a solution of
a
-ketotriazole in
der.Mp 47 ꢀC. Yield 99%.1H NMR (CDCl3)
d
0.91 (t, J ¼ 6.9 Hz, 3H); 1.30
(m,10H);1.86 (m, 2H);3.05(m, 4H);4.30(t, J¼ 7.1 Hz, 2H);7.11 (s,1H);
7.41 (m, 5H); 13C NMR (CDCl3)
14.0; 22.5; 26.3; 27.4; 28.9; 29.0; 30.2;
CH2Cl2/MeOH 1/3 at room temperature. The reaction was followed
by TLC. After complete consumption of the starting material, water
was added followed by CH2Cl2. The organic phases were dried over
MgSO4, filtered, and concentrated under reduced pressure. The
desired product was purified by flash chromatography.
d
31.6; 35.5; 50.1; 120.7; 126.0; 128.3; 128.4; 141.1; 147.0; HRMS: (DCI/
CH4, m/z) calc. for C18H28N3: 286.2283. Found: 286.2281.
4.2.5.5. 1-Nonyl-4-phenethyl-1H-1,2,3-triazole (25). White solid.
4.2.4.1. 1-(1-Octyl)-1H-1,2,3-triazol-4-yl-2-phenylethanol
Mp 48 ꢀC. Yield 92%.1H NMR (CDCl3)
d
0.90 (t, J ¼ 7.1 Hz, 3H); 1.28 (m,
12H); 1.85 (m, 2H); 3.03 (m, 4H); 4.29 (t, J ¼ 7.1 Hz, 2H); 7.11 (s, 1H);
7.24 (m, 5H); 13C NMR (CDCl3)
14.0; 22.6; 16.4; 27.4; 28.9; 29.1; 29.3;
(16). White powder. Mp 46 ꢀC. Yield 77%. 1H NMR (CDCl3)
d 0.87 (t,
J ¼ 6.7 Hz, 3H); 3.09 (dd, J ¼ 13.7, 8.1 Hz, 1H); 3.27 (dd, J ¼ 13.7,
d
5.1 Hz, 1H); 4.28 (t, J ¼ 7.2 Hz, 2H); 5.11 (dd, J ¼ 8.1, 5.1 Hz, 1H); 7.25
30.2; 31.7; 35.5; 50.1; 120.7; 126.0; 128.3; 128.4; 141.1; 147.0; HRMS:
(DCI/CH4, m/z) calc. for C19H30N3: 300.2440. Found: 300.2454.
(m, 6H); 13C NMR (CDCl3)
d
14.0; 22.5; 26.4; 28.9; 29.0; 30.2; 31.6;
43.9; 50.3; 68.1; 120.6; 126.6; 128.4; 129.5; 137.4; 150.3; HRMS:
(DCI/CH4, m/z) calc. for C18H28N3O: 302.2232. Found: 302.2239.
4.2.5.6. 1-Dodecyl-4-phenethyl-1H-1,2,3-triazole (26). White pow-
der. Mp 51 ꢀC. Yield 93%. 1H NMR (CDCl3)
d
0.90 (t, J ¼ 6.9 Hz, 3H);
1.28 (m,17H); 1.85 (m, 2H); 3.03 (m, 4H); 4.27 (t, J ¼ 7.2 Hz, 2H); 7.11
(s, 1H); 7.23 (m, 5H); 13C NMR (CDCl3)
14.0; 22.5; 26.3; 27.4; 28.8;
4.2.4.2. 2-(2,4-Dichlorophenyl)-1-(1-octyl-1H-1,2,3-triazoyl-4-yl)
ethanol (17). White powder. Mp 49 ꢀC. Yield 90%. 1H NMR (CDCl3)
d
d
0.87 (t, J ¼ 6.8 Hz, 3H); 1.26 (m, 10 H); 1.85 (m, 2H); 2.79 (broad s,
29.2; 29.2; 29.3; 29.4; 30.1; 30.7; 35.4; 50.0; 120.7; 125.9; 128.2;
128.3; 141.0; 146.9; HRMS: (DCI/CH4, m/z) calc. for C22H36N3:
342.2909. Found: 342.2908.
1H); 3.22 (dd, J ¼ 13.9, 8.2 Hz, 1H); 3.37 (dd, J ¼ 13.9, 5.2 Hz, 1H);
4.30 (t, J ¼ 7.2 Hz, 2H); 5.17 (dd, J ¼ 8.1, 5.2 Hz, 1H); 7.13 (dd, J ¼ 8.2,
2.0 Hz, 1H); 7.18 (d, J ¼ 8.2 Hz, 1H); 7.35 (d, J ¼ 2.0 Hz, 1H); 7.37 (s,
1H); 13C NMR (CDCl3)
d
14.0; 22.6; 26.4; 28.9; 29.0; 30.2; 31.7; 40.7;
4.3. Biology
50.4; 66.2; 120.6; 126.9; 132.8; 133.1; 134.0; 135.0; 149.8; HRMS:
(DCI/CH4, m/z) calc. for C18H26Cl2N3O: 370.1453. Found: 370.1444.
4.3.1. InhA expression and purification
The production and purification of the InHA-6xHis protein from
a protease-deficient strain of E. coli (BL21) transformed with the
pHAT5/InhA plasmid were performed as followed. 1 mL of the
bacteria was grown in 100 mL of LB medium containing ampicillin
4.2.5. Preparation of the triazole products
A typical experimental procedure for the preparation of these
compounds from the corresponding commercially available
alkynes is described below. Cu(OAc)2 (0.1 equiv), AscNa (0.2 equiv)
were added at room temperature to a solution of alkyne (1.0 equiv)
with azide (1.2 equiv) in tBuOH/H2O (1/1),. The reaction mixture
was stirred at room temperature 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).
(100 m
g/mL) and 2% glucose at 37 ꢀC. After 4 h, the solution was re-
diluted in 1 L of the same medium and re-grown at 37 ꢀC. When the
proper concentration (OD595 ¼ 0.6e0.8) was reached, the culture
was centrifuged at 3300 g for 10 min at 4 ꢀC and bacteria were
suspended in LB medium containing ampicillin (100
Protein expression was induced for overnight incubation in 1 mM
isopropyl-
-galactopyranoside (IPTG) at 20 ꢀC. Cells were har-
mg/mL).
b-D
vested by centrifugation at 6000 g for 30 min at 4 ꢀC. The dry pellet
was kept at ꢃ80 ꢀC for several months. The purification was per-
formed with Ni-NTA Agarose from QIAGEN as described by the
manufacturer’s protocol. The purified recombinant protein was
applied to PD-10 desalting columns (GE Healthcare, Piscataway, NJ)
equilibrated with PIPES 30 mM pH 6.8, 150 mM NaCl to remove
imidazole.
The compounds reported could be compared with those of the
previously reported spectroscopic data: 1-benzyl-4-phenyl-1H-
1,2,3-triazole (18) [33], 1,4-dibenzyl-1H-1,2,3-triazole (19) [34], 1-
benzyl-4-phenethyl-1H-1,2,3-triazole (20) [35].
4.2.5.1. 1-(3,5-Dimethylbenzyl)-4-phenethyl-1H-1,2,3-triazole
(21). White powder. Mp 95 ꢀC. Yield 63%. 1H NMR (CDCl3)
d
2.28 (s,
6H); 2.98 (t, J ¼ 7.5 Hz, 2H); 5.36 (s, 2H); 6.82 (s, 2H); 6.95 (s, 1H);
7.01 (s, 1H); 7.15 (m, 5H); 13C NMR (CDCl3)
21.1; 27.5; 35.5; 54.0;
Samples were analyzed using SDS-PAGE and Coomassie blue
staining and then stored at 4 ꢀC for short term storage or ꢃ80 ꢀC
with 20% glycerin for long-term storage.
d
121.0; 125.7; 126.0; 128.3; 128.4; 130.2; 134.6; 138.7; 141.1; 147.5;
HRMS: (DCI/CH4, m/z) calc. for C19H22N3: 292.1814. Found:
292.1813.
4.3.2. Inhibition kinetics
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 previously. Reactions were initiated
by addition of InhA (100 nM final) to solutions containing DD-CoA
4.2.5.2. 1-(3-Methoxybenzyl)-4-phenethyl-1H-1,2,3-triazole
(22). Lightly yellow oil. 1H NMR (CDCl3)
d
3.02 (t, J ¼ 7.5 Hz, 2H);
3.03 (t, J ¼ 7.5 Hz, 2H); 3.82 (s, 3H); 5.46 (s, 2H); 6.77 (t, J ¼ 1.9 Hz,
1H); 6.82 (dt, J ¼ 0.5, 7.5 Hz, 1H); 6.91 (ddd, J ¼ 0.5, 2.5 Hz,
J ¼ 8.3 Hz, 1H); 7.07 (s, 1H); 7.16 (m, 6H); 13C NMR (CDCl3)
d
27.5;
(50 mM final), inhibitor, and NADH (250 mM final) in 30 mM PIPES,
35.4; 53.8; 55.2; 113.4; 113.9; 120.0; 121.0; 126.0; 128.3; 128.4;
130.0; 136.3; 141.0; 147.6; 160.0; HRMS: (DCI/CH4, m/z) calc. for
C18H20N3O: 294.1606. Found: 294.1604.
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

C. Menendez et al. / European Journal of Medicinal Chemistry 46 (2011) 5524e5531
5531
percentage of InhA activity inhibition (initial velocity of the reac-
Appendix. Supplementary material
tion) with respect to the control reaction without inhibitor. All
activity assays were performed in triplicate.
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2011.09.013.
4.3.3. Molecular docking studies
The structures (protein, NADþ, ligand) were imported in Mole-
gro Virtual Docker (MVD) 4.2 software (Molegro ApS, Aarhus,
Denmark). The cavity detection algorithm implemented in MVD
was used to optimize the definition of a 15 Å (radius) potential
binding site but not for constraining results to the cavity. In the
present paper 2X22a PDB structures was used for molecular
studies. The corresponding crystallographic NADþ molecules were
used as cofactor using the MVD features. The water molecules were
taken in account with an entropy reward of 0.5 (MVD units). The
side chains around ligand (61 residues for 2X22a) were set as
flexible with a tolerance of 1.0 and strength of 0.9 (MVD units).
Three calculation schemes combining Moldock Score [GRID] [36]
and Plants Score [GRID] [37] and optimizers (Moldock SE and
Moldock optimizer) were used. For each ligand and a given calcu-
lation scheme, 20 runs were carried out using Tabu clustering. The
poses were checked visually and plotted against different param-
eters such as Total Energy vs. Total interaction Energy or Moldock
Score (or Rerank Score) vs. Plants Score (when Plants Score [GRID]
was used in calculation scheme). The best pose corresponding to
these three plots was selected. If the 3 best poses were not the same
(RMSD < 1.2 Å for alkyl TCU like molecules) a new calculation
process was done. After calculation minimization steps (global,
lateral chain, ligands) were done using MVD default features. This
protocol ensures us a good accuracy (RMSD < 0.7 Å) in the repro-
duction of crystallography results.
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complete Middlebrook 7H9. The cultures were incubated at 37 ꢀC
until the exponential growth phase (w108 CFU/mL) was reached,
corresponding to an OD600nm ranging from 0.8 to 1.0. Cultures were
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Acknowledgments
We are grateful to Professor Christopher Abell and Doctor Sachin
Surade (Christ’s College, University of Cambridge) for the generous
gift of pHAT5/InhA plasmid. The authors are grateful to Dr.
Prithwiraj De for his help in preparing this manuscript. We would
like to thank the CNRS and the “ Université Paul Sabatier” for their
financial support.