Synthesis and evaluation of α-ketotriazoles and α,β- diketotriazoles as inhibitors of Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2013.06.042

Two series of α-ketotriazole and α,β-diketotriazole derivatives were synthesized and evaluated for antitubercular and cytotoxic activities. Among them, two α,β-diketotriazole compounds, 6b and 9b, exhibited good activities (minimum inhibitory concentratio

Full text of DOI:10.1016/j.ejmech.2013.06.042

Accepted Manuscript
Synthesis and Evaluation of α-ketotriazoles and α,β-diketotriazoles as inhibitors of
Mycobacterium tuberculosis
Christophe Menendez, Frédéric Rodriguez, Ana Luisa de Jesus Lopes Ribeiro,
Francesca Zara, Céline Frongia, Valérie Lobjois, Nathalie Saffon, Maria Rosalia
Pasca, Christian Lherbet, Michel Baltas
PII:
S0223-5234(13)00410-8
10.1016/j.ejmech.2013.06.042
EJMECH 6270
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 February 2013
Revised Date: 14 June 2013
Accepted Date: 18 June 2013
Please cite this article as: C. Menendez, F. Rodriguez, A.L.d.J.L. Ribeiro, F. Zara, C. Frongia, V. Lobjois,
N. Saffon, M.R. Pasca, C. Lherbet, M. Baltas, Synthesis and Evaluation of α-ketotriazoles and α,β-
diketotriazoles as inhibitors of Mycobacterium tuberculosis, European Journal of Medicinal Chemistry
(2013), doi: 10.1016/j.ejmech.2013.06.042.
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ACCEPTED MANUSCRIPT
Synthesis and Evaluation of -ketotriazoles and ,-diketotriazoles as
inhibitors of Mycobacterium tuberculosis
Christophe Menendez, Frédéric Rodriguezb Ana Luisa de Jesus Lopes Ribeiro, Francesca Zara,
Céline Frongia, Valérie Lobjois, Nathalie Saffon, Maria Rosalia Pasca, Christian Lherbet,
Michel Baltas

*Highlights (for review)
ACCEPTED MANUSCRIPT
 A series of ,-diketotriazoles were synthesized.
 Two compounds displayed inhibitory activity of 7 M against M. tuberculosis H37Rv
and multi-drug resistant M. tuberculosis strains
 ,-Diketotriazoles are not cytotoxic on two human cell lines

*Revised Manuscript
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Synthesis and Evaluation of -ketotriazoles and ,-diketotriazoles as inhibitors of
Mycobacterium tuberculosis
Christophe Menendez,a,b Frédéric Rodriguez,a,b Ana Luisa de Jesus Lopes Ribeiro,c Francesca
Zara,d Céline Frongia,e Valérie Lobjois,e Nathalie Saffon,f Maria Rosalia Pasca,c Christian
Lherbet,*a,b Michel Baltas*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.
Dipartimento di Biologia e Biotecnologie « Lazarro Spallanzani, University di Pavia, via Ferrata 1, 27100 Pavia, Italy.
d. Dipartimento di Scienze Clinico-chirurgiche, diagnostiche e pediatriche, University of Pavia, via Brambilla 74, 27100 Pavia, Italy.
e. Université de Toulouse; ITAV-UMS3039, F-31106 Toulouse, France.
f. Université de Toulouse, UPS and CNRS, ICT FR2599, 118 route de Narbonne, 31062 Toulouse, France
Fax: 33 (0)5 61556011; Tel: 33 (0)5 61556289; E-mail: lherbet@chimie.ups-tlse.fr; baltas@chimie.ups-tlse.fr
Keywords: inhibitor, ,-diketotriazole, tuberculosis, oxidation, cytotoxicity
Abstract
Two series of -ketotriazole and ,-diketotriazole derivatives were synthesized and
evaluated for antitubercular and cytotoxic activities. Among them, two ,-diketotriazole
compounds, 6b and 9b, exhibited good activities (minimum inhibitory concentration = 7.6
μM and 6.9 μM, respectively) on Mycobacterium tuberculosis and multi-drug resistant M.
tuberculosis strains and presented no cytotoxicity (IC50 > 50 M) on colorectal cancer
HCT116 and normal fibroblast GM637H cell lines. These two compounds represent
promising leads for further optimization.

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1. Introduction
Tuberculosis (TB) caused by Mycobacterium tuberculosis (M.tb) remains a major cause of
mortality worldwide infecting 8 million and killing two million people annually [1].The
mortality and the spread of this disease have been aggravated mainly by its co-infection with
Human Immunodeficiency Virus (HIV) [2]. The worsening situation has prompted the World
Health Organization (WHO) to declare tuberculosis a global public health threat [1]. Most of
the drugs which are classified as a first-line TB treatment were discovered in the 1950’s and
1960’s. Streptomycin [3] was the first drug used to treat tuberculosis, followed by isoniazid
(isonicotinylhydrazine, INH) in the early 1950’s [4]. Pyrazinamide (PZA) appeared as a
potential drug against TB in 1952 [5]. The two most recent first-line antitubercular drugs are
ethambutol (EMB) [6,7] and rifampicin (RIF) [8,9] which were discovered in the 1960’s.
Today, an improvement of the treatment has been observed by combining these drugs. Strains
of M.tb resistant to both INH and RIF have been called multidrug resistant (MDR) [10,11]. To
treat MDR-TB, the WHO have recommended using second-line drugs such as
fluoroquinolones [12], ethionamide [13,14] and cycloserin [15,16,17]. These agents besides
being unsuitable for short-course treatment could also be less effective and more toxic.
Recently, the emergence of extensively drug-resistant (XDR) strains has been observed [18].
M.tb XDR strains are MDR isolates resistant to a fluoriquinolone or a second-line injectable
drug [18].
More than ever, there is an urgent need to develop new, potent and fast acting anti-
tuberculosis drugs to combat the spread of TB. Different classes of compounds have been
reported with antitubercular activities such as cinnamic [19,20,21], phenolic [22], but also
five-membered ring heterocycle derivatives. Indeed, over the past ten years, a variety of
imidazole [23,24,25], furan [26], 1H-1,2,4-triazole [27,28] but also 1H-1,2,3-triazole [29-35]
derivatives have been reported to have antitubercular activities (Figure 1).

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In an ongoing effort to develop novel scaffolds against tuberculosis, we reported a series of -
ketotriazoles and triazoles with good antitubercular activities [36,37]. During our first
synthesis of -ketotriazoles as potential inhibitors of InhA (an essential enzyme involved in
the synthesis of the mycobacterial mycolic acids), a side product identified as ,-
diketotriazole was observed. From this observation, a general method for obtaining ,-
diketotriazoles by oxidation of -ketotriazoles in the presence of catalytic amounts of CuI and
2,9-dimethyl-1,10-phenanthroline as ligand, using air as oxidant, was established [38].
Herein, we report the synthesis of two classes of compounds, namely -ketotriazoles and ,-
diketotriazoles, their evaluation as inhibitors of M.tb strain H37Rv and their cytotoxicity on
two human cell lines.
2. Results and discussion
2.1. Chemistry
Two different methods were employed in the synthesis of ,-diketotriazoles. The first (A,
Table 1) consisted of a two-step synthesis as previously described and provides access to the
first and second classes of compounds, namely -ketotriazoles and ,-diketotriazoles,
respectively [36,38]. -Ketotriazoles were readily available in one-step procedure with TMS
deprotection followed by Huisgen 1,3-dipolar cycloaddition (Scheme 1). ,-Diketotriazoles
can then be synthesized from -ketotriazole in the presence of CuI or CuCl2 and 2,9-
dimethyl-1,10-phenanthroline in refluxing acetonitrile.
In an alternative method of preparation (B, Table 2), ,-diketones were synthesized in a one-
pot three-step reaction: TMS-deprotection of the trimethylsilyl ynones / Huisgen 1,3-dipolar

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cycloaddition between the azides and the deprotected ynones / oxidation of the benzylic
moiety (Scheme 1). The yields ranged from 28 to 43% as summarized in Table 1. The two-
step procedure afforded higher yields, except for entry 8. The main difference between the
two protocols is the absence of water in the oxidation step, when starting directly from -
ketotriazoles (method A). The structure of 9b was confirmed by X-ray crystallography
(Figure 2) [39].
2.2. Biology
2.2.1. Antimycobacterial activity
All the -ketotriazoles synthesized and their corresponding ,-diketotriazoles (herein and
ref. 38) were evaluated by determining the minimum inhibitory concentration (MIC) on M.
tuberculosis H37Rv strain (Table 2). Triclosan and INH were used for comparison.
Most of the compounds of the first class, corresponding to -ketotriazoles, exhibited weak in
vitro activities against M. tuberculosis with MIC values ranging from 20.9 to 360 M.
Interestingly, compounds 5a and 14a possessing an octyl chain attached to the triazole core
presented the best activities with MIC values of 83.5 and 20.9 M, respectively. The latter
one displayed an activity equivalent to triclosan.
As shown in Table 2, the second class of compounds, corresponding to ,-diketotriazole
derivatives, generally displayed higher activities than -ketotriazoles, with MIC ranging
from 6.9 to 171 M. This was the case for all compounds possessing a remote aromatic group
at the N-1 position of the triazole frame. The only exceptions concern compounds possessing
an octyl chain at the N-1 position (5a/5b and 14a/14b), for which MIC activities were either
comparable (5a ≈ 5b) or reversed (14a > 14b). For all ,-diketotriazole compounds, the

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most potent were found to be 6b and 9b with MICs of 2.5g/mL (7.6 and 6.9 M,
respectively). These compounds present different substituents (p-methoxy or 2,4-dichloro) on
the aromatic moiety. Among all 2,4-dichloro derivatives 9b-12b evaluated, those possessing a
remote aromatic ring at the N-1 position of the triazole ring exhibited comparable in vitro
activities against M. tuberculosis (MIC values: 9b, 6.9 M; 10b, 9.5 M; 12b, 13.4 M),
except for the p-NO2 derivative 11b (MIC = 79.0 M) .
Furthermore, the cytotoxicity of different -ketotriazoles and ,-diketotriazoles was
evaluated on two human cell lines, the human colon cancer cell line HCT116 and the human
fibroblast cell line GM637 (Table 2). The data showed that the IC50 of the different
compounds tested were above 50 M, indicating that these compounds are not cytotoxic
against human cell lines.
2.2.2. Bacterial growth inhibition experiments
On the basis of antituberculous activities, compounds 6b and 9b both exhibiting MICs of 2.5
g/mL were selected and evaluated for testing against multi-drug-resistant M. tuberculosis
strains. The results are reported in Table 3 and show that 6b and 9b were active in all of the
resistant strains with the same level of efficiency.
3. Conclusion
Two series of -ketotriazole and ,-diketotriazole derivatives were synthesized. The three-
step one-pot synthesis of ,-diketotriazoles was successfully accomplished with yields
ranging from 28 to 43%. The latter approach will be further improved in order to prepare,
through automated parallel synthesis, a variety of ,-diketotriazoles. Among the compounds
synthesized and evaluated, two compounds 6b and 9b exhibit interesting antituberculosis

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activities with MIC values of 2.5 g/mL and no apparent cytotoxicities toward HCT116 or
GM637 human cells. Furthermore, compounds 6b and 9b present the same in vitro efficiency
against MDR-TB strains. In conclusion, it was shown that the potency and low
cytotoxicity of these compounds make them good leads for synthesizing new derivatives
with enhanced activities. Further studies will focus on the identification of the protein
targets of these molecules.
4. Experimental Section
4.1. Material
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). 1H NMR spectra were recorded at 300 MHz by using CDCl3
(7.26 ppm) as an internal standard. 13C NMR spectra were recorded at 75.0 MHz and are
referenced against the central line of the CDCl3 triplet at  = 77.0 ppm. Mass spectrometry
(MS) data were obtained on a ThermoQuest TSQ 7000 spectrometer, high-resolution mass
spectra (HRMS) were recorded on a ThermoFinnigan MAT 95 XL spectrometer using
electrospray ionization (ESI) methods. Melting points were measured on a Mettler Toledo
MP50 melting point system. IR spectra were recorded on a Perkin Elmer 1725.
Crystallographic data for compound 9b [39] were collected on a Bruker-AXS Quazar APEX
II diffractometer using a 30 W air-cooled microfocus source (ImS) with focusing multilayer
optics at a temperature of 193(2)K, with MoK radiation (wavelength = 0.71073 Å) using
phi- and omega-scans. The data were integrated with SAINT [40], and an empirical
absorption correction with SADABS [41] was applied. The structures were solved by direct
methods, using SHELXS-97 and refined using the least–squares method on F2. [42] All non-

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H atoms were treated anisotropically. The H atoms were fixed geometrically and treated as a
riding model.
4.2. Chemistry
4.2.1. Synthesis of -ketotriazoles and ,-diketotriazoles in two steps.
Compounds 1a-6a, 1b-6b, 9a-11a, 9b-11b, 13a, 13b, 15a-19a, 15b-19b were synthesized
according to the general procedure and exhibited identical spectral data as indicated in a
previous report [38].
4.2.1.1. 2-(4-Methoxyphenyl)-1-(1-phenethyl-1H-1,2,3-triazol-4-yl)ethanone (7a). White
1
powder. M.p. 119.2°C. Yield 60%. IR (/cm-1): 3141; 1688; 1616; 1516; 1456; H NMR
(CDCl3) 7.82 (s, 1H); 7.27 (m, 5H); 7.07 (m, 2H); 6.85 (d, J = 8.7 Hz, 2H); 4.60 (t, J = 7.2
Hz, 2H); 4.34 (s, 2H); 3.77 (s, 3H); 3.21 (t, J = 7.3 Hz, 2H); 13C NMR (CDCl3)  192.4;
158.5; 147.0; 136.3; 130.8; 128.8; 128.5; 127.2; 126.2; 125.9; 113.9; 55.1; 51.7; 45.0; 36.3;
HRMS: (DCI/CH4, m/z) calc. for C19H20N3O2: 322.1556. Found: 322.1550.
4.2.1.2. 1-(4-Methoxyphenyl)-2-(1-phenethyl-1H-1,2,3-triazol-4-yl)ethane-1,2-dione (7b).
1
Yellow gummy solid. Yield 49%. IR (/cm-1): 3116; 1672; 1653; 1600; 1517; H NMR
(CDCl3) 8.00 (s, 1H); 7.99 (d, J = 8.9 Hz, 2H); 7.27 (m, 3H); 7.10 (m, 2H); 6.96 (d, J = 8.9
Hz, 2H); 4.67 (t, J = 7.2 Hz, 2H); 3.88 (s, 3H); 3.24 (t, J = 7.2 Hz, 2H); 13C NMR (CDCl3) 
190.4; 185.7; 165.0; 143.9; 136.2; 132.7; 128.9; 128.7; 128.6; 127.4; 125.3; 114.3; 55.6; 52.0;
36.4; HRMS: (DCI/CH4, m/z) calc. for C19H18N3O3: 336.1348. Found: 336.1355.
4.2.1.3. 1-(1-(4-Methoxyphenethyl)-1H-1,2,3-triazol-4-yl)-2-(4-methoxyphenyl)ethanone (8a).
1
White powder. M.p. 124.7°C. Yield 54%. IR (/cm-1): 3121; 1693; 1613; 1515; 1460; H
NMR (CDCl3) 7.80 (s, 1H); 7.26 (d, J = 8.7 Hz, 2H); 6.96 (d, J = 8.7 Hz, 2H); 6.84 (d, J =
8.7 Hz, 2H); 6.78 (d, J = 8.7 Hz, 2H); 4.56 (t, J = 7.1 Hz, 2H); 4.33 (s, 2H); 3.763 (s, 3H);
3.757 (s, 3H); 3.14 (t, J = 7.1 Hz, 2H); 13C NMR (CDCl3)  192.4; 158.7; 158.5; 147.0; 130.8;

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129.5; 128.2; 126.3; 125.9; 114.2; 113.9; 55.1; 52.0; 45.0; 36.0; HRMS: (DCI/CH4, m/z) calc.
for C20H22N3O3: 352.1661. Found: 352.1656.
4.2.1.4.
1-(1-(4-Methoxyphenethyl)-1H-1,2,3-triazol-4-yl)-2-(4-methoxyphenyl)ethane-1,2-
dione (8b). Yellow gummy solid. Yield 37%. IR (/cm-1): 3132; 1683; 1597; 1573;
1
1514;1463; H NMR (CDCl3) 7.99 (m, 3H); 6.98 (t, J = 8.9 Hz, 2H); 6.96 (d, J = 9.1 Hz,
2H); 6.80 (d, J = 8.7 Hz, 2H); 4.62 (t, J = 7.1 Hz, 2H); 3.88 (s, 3H); 3.76 (s, 3H); 3.17 (t, J =
7.1 Hz, 2H); 13C NMR (CDCl3)  190.4; 185.8;165.0; 158.8; 143.9; 132.7; 129.6; 128.8;
128.1; 125.3; 114.31; 114.25; 55.6; 55.2; 52.2; 35.5; HRMS: (DCI/CH4, m/z) calc. for
C20H20N3O4: 366.1454. Found: 366.1458.
4.2.1.5. 2-(2,4-Dichlorophenyl)-1-(1-phenethyl-1H-1,2,3-triazol-4-yl)ethanone (12a). White
1
powder. M.p. 137.2°C. Yield 68%. IR (/cm-1): 3135; 1685; 1532; 1474; H NMR (CDCl3)
7.91 (s, 1H); 7.43 (s, 1H); 7.30 (m, 3H); 7.25 (br s, 2H); 7.12 (m, 2H); 4.68 (t, J = 7.2 Hz,
13
2H); 4.60 (s, 2H); 3.27 (t, J = 7.2 Hz, 2H); C NMR (CDCl3)  190.2; 146.9; 136.2; 135.3;
133.5; 132.7; 131.2; 129.2; 128.9; 128.5; 127.3; 127.0; 126.1; 51.9; 43.4; 36.3; HRMS:
(DCI/CH4, m/z) calc. for C18H16N3OCl2: 360.0670. Found: 360.0681.
4.2.1.6. 1-(2,4-Dichlorophenyl)-2-(1-phenethyl-1H-1,2,3-triazol-4-yl)ethane-1,2-dione (12b).
1
White powder. M.p. 116.5°C. Yield 51%. IR (/cm-1): 3133; 1670; 1580; 1533; H NMR
(CDCl3) 8.00 (s, 1H); 7.82 (d, J = 8.3 Hz, 1H); 7.45 (d, J = 1.8 Hz, 1H); 7.42 (dd, J = 8.3
Hz, 1.9 Hz, 1H); 7.27 (m, 3H); 7.08 (m, 2H); 4.71 (t, J = 7.1 Hz, 2H); 3.27 (t, J = 7.1 Hz, 2H);
13C NMR (CDCl3)  191.0; 183.3; 143.0; 140.5; 136.1; 134.8; 132.9; 131.6; 130.3; 128.9;
128.8; 128.6; 127.8; 127.3; 52.1; 36.4; HRMS: (DCI/CH4, m/z) calc. for C18H14N3O2Cl2:
374.0463. Found: 374.0468.
4.2.1.7. 2-(2,4-Dichlorophenyl)-1-(1-nonyl-1H-1,2,3-triazol-4-yl)ethanone (14a). Yellow
1
gummy solid. Yield 63%. IR (/cm-1): 2924; 2854; 1684; 1582; 1553; 1437 ; H NMR
(CDCl3) 8.12 (s, 1H); 7.38 (d, J = 1.6 Hz, 1H); 7.20 (m, 2H); 4.56 (s, 2H); 4.38 (t, J = 7.2
13
Hz, 2H); 1.90 (m, 2H); 1.23 (m, 12H); 0.85 (t, J = 6.7 Hz, 3H); C NMR (CDCl3)  190.2;

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147.1; 135.4; 133.5; 132.7; 131.2; 129.1; 127.0; 125.7; 50.6; 43.3; 31.6; 30.0; 29.2; 29.0;
28.8; 26.3; 22.5; 14.0; HRMS: (DCI/CH4, m/z) calc. for C19H26N3OCl2: 382.1453. Found:
382.1447.
4.2.1.8. 1-(2,4-Dichlorophenyl)-2-(1-nonyl-1H-1,2,3-triazol-4-yl)ethane-1,2-dione (14b).
White powder. M.p. 60.4°C. Yield 51%. IR (/cm-1): 2921; 2852; 1689; 1588; 1471; 1H NMR
(CDCl3) 8.31 (s, 1H); 7.84 (d, J = 8.3 Hz, 1H); 7.46 (d, J = 1.9 Hz, 1H); 7.42 (dd, J = 8.3
Hz, 2.0 Hz, 1H); 4.46 (t, J = 7.3 Hz, 2H); 1.97 (m, 2H); 1.26 (m, 12H); 0.88 (t, J = 6.7 Hz,
3H); 13C NMR (CDCl3)  191.1; 183.5; 143.5; 140.5; 135.0; 133.0; 131.6; 130.5; 128.3;
127.9; 50.8; 31.7; 30.1; 29.2; 29.1; 28.9; 26.3; 22.6; 14.0; HRMS: (DCI/CH4, m/z) calc. for
C19H24N3O2Cl2: 396.1259. Found: 396.1247.
4.2.2. Representative procedure for the one-pot synthesis of ,-diketotriazoles from TMS-
ynones and azides.
A typical experimental procedure for the preparation of these compounds from the
corresponding trimethylsilylethynyl ketones is described below. CuCl2 (0.1 mol equiv)
and sodium ascorbate (0.2 mol equiv) were added at room temperature to a solution of 1-
trimethylsilyl-1-alkynyl ketone (1 mol equiv) with azide (1.2 mol equiv) in CH3CN/H2O
(4/1). Then the reaction mixture was warmed to reflux for 60 min and 2,9-dimethyl-1,10-
phenanthroline was added and the reaction was stirred for 20 h. After cooling the reaction
mixture to room temperature, H2O was added and extracted with EtOAc. The organic
layer was washed with brine, dried over MgSO4, filtered, and concentrated under reduced
pressure. The desired product was obtained by purification by flash chromatography
(petroleum ether/EtOAc).
4.3. Biology
4.3.1. Growth conditions and minimum inhibitory concentration (MIC) determination. M.
tuberculosis H37Rv was used as the reference strain. The strains were grown at 37 °C in

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Middlebrook 7H9 broth (Difco), supplemented with 0.05% Tween 80, or on solid
Middlebrook 7H11 medium (Difco) supplemented with oleic acid-albumin-dextrose-catalase
(OADC). MICs for the new compounds were determined by means of the micro-broth
dilution method. Dilutions of M. tuberculosis wild-type and clinical isolate cultures (about
105–106 cfu/ml) were streaked onto 7H11 solid medium containing a range of drug
concentrations (0.25 g/mL to 40 g/mL). Plates were incubated at 37 °C for about 21 days
and the growth was visually evaluated. The lowest drug dilution at which visible growth
failed to occur was taken as the MIC value. Results were expressed as the average of at least
three independent determinations.
4.3.2. M. tuberculosis clinical isolates and drug susceptibility testing. Three M. tuberculosis
MDR isolates were collected at the Sondalo Division of the Valtellina and Valchiavenna,
Italy, hospital authority in the 2012. Their resistance profile is shown in Table 3. All clinical
isolates were grown in BACTEC™ MGIT™ 960 and Lowenstein–Jensen slants. Drug
susceptibility testing for all first-line antitubercular drugs was performed with the BACTEC™
MGIT™ 960 System (Becton-Dickinson Diagnostic Systems, Sparks, Maryland) for isoniazid
(0.1 μg/ml; 0.4 μg/ml), rifampicin (1 μg/ml), streptomycin (1 μg/ml; 4 μg/ml), ethambutol (5
μg/ml) and pyrazinamide (100 μg/ml), in accordance with the manufacturer's instructions..
MIC determination to second-line drugs (cycloserine, 50 μg/ml; amikacin, 1-5 μg/ml;
ciprofloxacin, 2 μg/ml; ethionamide, 5 or 10 μg/ml; para-aminosalicylic acid, 4 or 8 μg/ml;
ofloxacin, 10 μg/ml) was also performed by the MGIT™ 960 System.
4.3.3.Cytotoxicity
Human colon cancer cell line HCT116 (ATCC) and human fibroblasts (GM637 cell line)
were cultured in DMEM supplemented with 10% fetal calf serum. For cytotoxicity
evaluation, 3000 cells were seeded per well in 96-wells plates and, 24 h later, were treated

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with concentrations ranging from 100 nM to 50 µM (8 replicates for each). After 4 days of
treatment, the cytotoxicity of each compound was measured by using the WST-1 kit (Roche).
Acknowledgments
We thank the CNRS and the “Université Paul Sabatier” for financial support.
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[39] Crystal data for 9b: C17H11Cl2N3O2, M=360.19, Orthorhombic, space group Pca21,
a=13.2479(11)Å, b=7.8752(7)Å, c=30.784(3)Å, V=3211.7(5) Å3, Z=8, crystal size 0.20 x 0.12 x 0.12
mm3, 45337 reflections collected (5423 independent, Rint=0.0669), 434 parameters, R1 [I>2σ(I)]=
0.0466, wR2 [all data]= 0.1217, largest diff. peak and hole: 0.401 and -0.328 e.Å-3. CCDCC 923093
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Table(s)
ACCEPTED MANUSCRIPT
Table 1. One-pot reaction to obtain ,-diketotriazoles from TMS-ynones and azides.
CuCl2 (0.1 mol equiv),
Sodium ascorbate (0.2 mol equiv)
CH3CN / H2O (4/1), 80 °C, 1 h
Then 2,9-diMe-1,10-Phen (0.2 mol equiv)
80 °C, 20 h
TMS
1) CuCl2 (0.1 mol equiv)
Sodium ascorbate (0.2 mol equiv)
CH3CN/H2O (4/1)
N
N
R2
R
O
TMS
N
R
R
O
R1
O
O
Consecutive steps
Method A
2) CuI (0.1 mol equiv)
2,9-diMe-1,10-Phen (0.2 mol equiv)
CH3CN, reflux
R1
R1
R2
N3
R2
N3
Two steps
Method B
Entry
Product
Yielda (%)
Method A
Overall yielda (%)
Method B
(step 1/step 2)
49
N
N
O
N
1
35
1b
6b
7b
8b
9b
O
N
N
O
2
3
4
5
28
33
N
O
N
MeO
NDb
NDb
30
30 (60 / 49)
20 (54 / 37)
52
N
O
N
O
N
MeO
N
O
OMe
N
O
MeO
N
N
Cl
O
N
O
Cl
N
N
Cl
O
6
43
61
11b
N
O
Cl
NO2
NDb
41
35 (68 / 51)
N
N
7
8
12b
13b
Cl
O
N
O
Cl
33
N
N
Cl
O
N
O
Cl
NDb
32 (63 / 51)
N
N
Cl
O
9
14b
N
7
O
Cl
a.
Yields for isolated compounds.
Not determined.
b.

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Table 2. Compounds tested as inhibitors of M. tuberculosis growth.
R1
N
R1
O
N
N
N
R3
R3
N
N
O
O
R2
R2
Compounds Xa
Compounds Xb
Compound
R1
R2
R3
MIC
Cytotoxicity
(M)
ND
(g/ml)/ (M)
10/34.5
---
---
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
---
---
H
H
H
H
H
H
H
H
H
H
---
---
Triclosan
INH
1a
0.05/0.4
100/360.6
50/171.6
100/310.3
4/11.89
ND
Bn
>50
>50
>50
>50
ND
Bn
1b
4-NO2-Bn
4-NO2-Bn
3,5-diMeO-Bn
3,5-diMeO-Bn
3,5-diMe-Bn
3,5-diMe-Bn
octyl
2a
2b
>100/>296.4
4/11.38
3a
>50
>50
>50
>50
ND
3b
>100/>327.4
16/50
4a
4b
25/83.5
5a
octyl
16/51
5b
OMe
OMe
OMe
OMe
OMe
OMe
Cl
Bn
ND
ND
6a
Bn
>50
ND
6b
2.5/7.6
Ph(CH2)2
Ph(CH2)2
4-MeOPh(CH2)2
4-MeOPh(CH2)2
Bn
>32/>112.0
>32/>95.4
>32/>91.1
>32/>87.6
100/288.9
2.5/6.9
7a
>50
ND
7b
8a
ND
8b
>50
>50
ND
9a
Cl
Bn
9b
Cl
3,5-di-MeO-Bn
3,5-di-MeO-Bn
100/246.1
4/9.51
10a
10b
Cl
>50

ACCEPTED MANUSCRIPT
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
4-NO2-Bn
4-NO2-Bn
Ph(CH2)2
Ph(CH2)2
cyclohexyl
cyclohexyl
Octyl
100/255.6
32/79.0
>32/>88.8
5/13.4
ND
>50
ND
>50
ND
ND
ND
>50
ND
11a
11b
12a
12b
13a
13b
14a
14b
15a
ND
16/43.7
8/20.9
Octyl
32/80.7
ND
COOMe
O
Cl
Cl
16/39.16
ND
15b
COOMe
O
H
H
4-NO2
4-NO2
4-NO2
4-NO2
H
Bn
ND
32/95.1
ND
ND
>50
ND
>50
ND
>50
ND
>50
16a
16b
17a
17b
18a
18b
19a
19b
Bn
H
3,5-di-MeO-Bn
3,5-di-MeO-Bn
Bn
H
64/161.4
ND
MeO
MeO
MeO
MeO
H
Bn
4/12.44
ND
H
3,5-di-MeO-Bn
3,5-di-MeO-Bn
H
8/21.0

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Table 3. Activities against multi-drug-resistant M. tuberculosis strains.
MIC (g/ml)
M. tuberculosis
H37Rv
M. tuberculosis clinical isolates
IC1a
IC2a
IC3a
Compound
g/mL / M
g/mL / M
g/mL / M
g/mL / M
2.5 / 6.9
2.5 / 7.6
2.5 / 6.9
2.5 / 7.6
2.5 / 6.9
2.5 / 7.6
2.5 / 6.9
2.5 / 7.6
6b
9b
aMtb clinical isolate: IC1 drug resistance profile: resistant to streptomycin, isoniazid, rifampicin, ethambutol; IC2
drug resistance profile: resistant to streptomycin, isoniazid, rifampicin, ethambutol, pyrazinamide,
ethionammide, capreomicin; IC3 drug resistance profile: resistant to streptomycin, isoniazid, rifampicin,
ethambutol, pyrazinamide, ethionammide.

Figure(s)
ACCEPTED MANUSCRIPT
Figure 1. Different triazole-based anti-TB drugs.

ACCEPTED MANUSCRIPT
N
N
N
R1
R
O
ref. 38
ref. 36
TMS
N
N
O
N
R1
R
R
O
O
this work
R1
N3
Scheme 1. Two methods to synthesize ,-diketotriazole derivatives.

ACCEPTED MANUSCRIPT
Figure 2. Molecular view of 9b. Only one of the two independent molecules in the asymmetric unit is shown.
Hydrogen atoms are omitted for clarity (crystallographic data for 9b are provided in the Supporting Information)