RSC Medicinal Chemistry
Research Article
(C), 120.3 (CH), 116.9 (CH), 49.8 (CH2), 31.4 (CH2), 29.0
(CH2), 25.5 (CH2), 22.3 (CH2), 13.9 (CH3). ESI-HRMS calcd.
for (M + H+) C14H19N3O 246.1606, found 246.1601.
needed, solid medium Middlebrook 7H11 supplemented with
ADS (1/10 v/v) and glycerol (1% v/v) was used.
In vitro activity against M. tuberculosis H37Rv. Stock
solutions for all the tested compounds were made in DMSO
at 40 mM. Working solutions were made by dilution in the
above described 7H9-ADS-G medium at a final concentration
of 400 μM. Antimycobacterial activity was determined by two-
fold dilution of the compounds in Middlebrook 7H9-ADS-Gly
medium as described previously.16 For this purpose, 96-well
plates (Falcon, Cat number 3072, Becton Dickinson, Lincoln
Park, NJ) were used. The 96 well-plates received 100 μL of
Middlebrook 7H9 broth and a serial two-fold dilution of the
compounds was made directly on the plate. The initial and
final drug concentrations tested were 20 μM and 1.25 μM,
respectively. The compounds were tested in three biological
repetitions each one in technical duplicate. Rifampicin (final
concentrations ranging from 2 mg mL−1 to 0.16 mg mL−1;
stock solution prepared as a 10 mg mL−1 solution in
methanol) was used as a control drug. The inoculum was
prepared as a 1/25 dilution of a fresh mid-log M. tuberculosis
H37Rv suspension (O.D equivalent to Mc Farland 1.0 scale
value) made in Middlebrook 7H9-ADS-G. A 100 μL aliquot
(containing approximately 106 colony forming units) was
used to inoculate the wells. The plates were sealed with
Parafilm and incubated at 37 °C for five days. After visual
inspection of the plates, the turbidity was recorded and the
final value for the MIC was assessed by addition of 30 μL of a
stock MTT solution as described elsewhere.35 The minimum
inhibitory concentration (MIC) was defined as the lowest
drug concentration preventing mycobacterial growth (yellow
color = growth inhibition).
InhA inhibition assay. The InhA inhibition activity was
tested using trans-2-dodecenoyl-coenzyme A (DD-CoA) and
wild-type InhA as described previously.36 Reactions were
initiated by the addition of 100 nM InhA to solutions
containing 25 μM DD-CoA, 40–50 μM inhibitor, and 250 μM
NADH in 30 mM PIPES and 150 mM NaCl, pH 6.8 buffer.
Control reactions were carried out under the same conditions
as described above but without an 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 an inhibitor.
In vitro antileishmanial assay. Leishmania donovani
promastigotes of the S1 Sudan strain (2 × 106 cells per mL)
were cultured at 26 °C in plastic flasks (25 cm2) containing
RPMI-1640 medium (without sodium bicarbonate and
sodium pyruvate) with 10% FBS. L. donovani promastigotes
General procedure for the synthesis of 1,2,3-triazoles by
thermal cycloaddition. Alkyne (1 eq.) and azide (1 eq.) were
suspended in 2 mL per eq. of toluene. The mixture was
stirred and heated under reflux for 35 hours. Then, it was
cooled down to room temperature it was cooled down to
room temperature, brine was added, and the solution was
extracted with dichloromethane. Combined organic extracts
were dried with sodium sulphate and evaporated. The
resulting residue was purified by column chromatography
over silica gel using an increasing AcOEt/hexane gradient to
afford desired pure products.
Synthesis of (1-benzyl-4-pentyl-1H-1,2,3-triazol-5-yl)methanol
(11a). White solid. MP: 59.3–60.0 °C. 1H NMR (300 MHz,
CDCl3) δ: 7.29–7.30 (m, 3H); 7.22–7.19 (m, 2H); 5.59 (s, 2H);
4.53 (d, J = 4.7 Hz, 2H); 2.56 (t, J = 7.7 Hz, 2H); 1.60 (p, J = 7.0
Hz, 2H); 1.27 (m, 4H); 0.85 (t, J = 6.5 Hz, 3H). 13C NMR (75
MHz, CDCl3) δ: 146.5 (C); 135.2 (C); 131.9 (C); 128.9 (CH);
128.3 (CH); 127.5 (CH); 52.3 (CH2); 52.1 (CH2); 31.5 (CH2);
29.6 (CH2); 26.8 (CH2); 22.4 (CH2); 14.0 (CH3). ESI-HRMS
calcd. for (M + H+) C15H21N3O 260.1763; found 260.1757.
Synthesis of (4-pentyl-1-(3-phenylpropyl)-1H-1,2,3-triazol-5-yl)
methanol (13a). Colorless oil. 1H NMR (300 MHz, CDCl3) δ:
7.31–7.17 (m, 5H); 4.62 (d, J = 4.3 Hz, 2H); 4.36 (t, J = 7.3 Hz,
2H); 2.69 (t, J = 7.3 Hz, 2H); 2.61 (t, J = 7.6 Hz, 2H); 2.29 (q, J
= 7.1 Hz, 2H); 1.68–1.62 (m, 2H); 1.31 (m, 4H, C7–H); 0.88 (t,
J = 7.0 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ: 146.1 (C); 140.6
(C); 131.3 (C); 128.5 (CH); 128.4 (CH); 126.2 (CH); 52.3 (CH2);
47.9 (CH2); 32.7 (CH2); 31.5 (CH2); 31.4 (CH2); 29.7 (CH2);
24.9 (CH2); 22.4 (CH2); 14.0 (CH3). ESI-HRMS calcd. for (M +
H+) C17H26N3O 288.2076; found 288.2070.
Synthesis of (1-(naphthalen-2-ylmethyl)-5-pentyl-1H-1,2,3-
1
triazol-4-yl)methanol (15b). White solid. MP: 79.2–80.2 °C. H
NMR (300 MHz, CDCl3) δ: 7.80 (m, 3H); 7.76 (s, 1H); 7.50–
7.47 (m, 2H); 7.27 (m, 1H); 5.64 (s, 2H); 4.72 (d, J = 5.4 Hz,
2H); 2.59 (t, J = 7.5 Hz, 2H); 1.34 (p, J = 7.5 Hz, 2H); 1.14–1.12
(m, 4H); 0.72 (t, J = 7.0 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ:
144.9 (C); 135.2 (C); 133.2 (C); 133.0 (C); 132.4 (C); 129.0
(CH); 127.8 (CH); 127.8 (CH); 126.6 (CH); 126.5 (CH); 126.2
(CH); 124.7 (CH); 55.9 (CH2); 52.2 (CH2); 31.4 (CH2); 28.5
(CH2); 22.6 (CH2); 22.1 (CH2); 13.7 (CH3). ESI-HRMS calcd.
for (M + H+) C19H24N3O 310.1841; found 310.1904.
3.2. Biology
Bacterial strain M. tuberculosis H37Rv. M. tuberculosis
strain H37Rv (kindly provided by Dr. L. Barrera, Instituto
Nacional de Microbiología “C. G. Malbrán”, Argentina) was
routinely grown at 37 °C under gentle agitation in
Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI, USA)
supplemented with 1/10 v/v of ADS (50 g L−1 BSA fraction V,
20 g L−1 dextrose and 8.1 g L−1 NaCl) and glycerol (1% w/v).
Tween 80 was added to prevent clumping (0.05% w/v). This
medium was designated as 7H9-ADS-Gly for short. When
were subcultured twice a week, with the highest cell
concentration in the range of 20–25 × 106 promastigotes per
mL. Compounds with appropriate dilution were added to a
96 well microplate with promastigotes (2 × 106 cells per mL)
reaching final concentrations of 40, 8 and 1.6 μg mL−1. The
plates were incubated at 26 °C for 72 h and the growth was
determined by the Alamar blue assay.37 pentamidine and
amphotericin
B were used as standard antileishmanial
agents. IC50 values were obtained by non-linear regression of
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RSC Med. Chem.