N. Glanzmann et al.
Biomedicine & Pharmacotherapy 141 (2021) 111857
13
azidodecane, previously synthesized according to Ren et al. (2011) [31],
were dissolved in 15 mL of an ethanol and water mixture (4:1). In
sequence, 0.044 mmol Copper (II) sulfate pentahydrate and 0.36 mmol
sodium ascorbate were slowly added under stirring. The reactant
mixture was kept at room temperature for 7 days and, after that, water
1,2,3-triazolic signals in the 1H (δ 7.42 ppm) and C NMR spectra (δ
121.4 and 146.2 ppm) is the main evidence of product formation, it is
also important to note that the NOESY correlation spectra (Fig. 1) can
endorse the connectivity between the 1,2,3-triazole nucleus and the
remaining molecular moieties. For the main synthetic route used to
obtain the derivative 4, the correlation peaks between the 1,2,3-tria-
was added and the mixture was extracted with CH
were dried (anhydrous sodium sulfate), the CH Cl
under reduced pressure and thin layer chromatography (TLC) analysis,
using the eluent CH Cl /CH OH (9:1), determined the presence of the
2
Cl
2
. The organics
′
′
′
2
2
was evaporated
zolic hydrogen (H-7 ) and the hydrogens H-5 and H-9 , highlighted in
Fig. 1, provides solid proof that the 1,2,3-triazolic ring is connected both
to the alkyl side chain and the quinoline containing remnant of the
molecule. Nonetheless, in the specific case of the derivative 4 obtained
as a side product in the synthesis of derivative 5, the correlation peak
2
2
3
product (Retention factor = 0.4). The desired product was isolated from
the starting material residues through column chromatography
′
′
(
CH
2
Cl
2
3
/CH OH gradient). After removing the solvent from the pure
between hydrogens H-5 and H-3 confirms the connection between the
1,2,3-triazolic and quinolinic parts of the hybrid.
fractions, derivative 4 was obtained with a 15.5% yield as a light yellow
1
solid. Melting range = 90–93 ºC. H NMR (500 MHz, CDCl
Hz): 0.87 (t, J = 6.9, 3H); 1.15 – 1.37 (m, 14H); 1.87 (quint, J = 7.6,
H); 2.05 (bs, 2H, NH
); 3.10 (t, J = 5.6, 2H); 3.38 (t, J = 5.7, 2H); 3.99
s, 2H); 4.31 (t, J = 7.3, 2H); 6.25 (bs, 1H, NH); 6.36 (d, J = 5.5, 1H);
.38 (dd, J = 8.9 e 2.2, 1H); 7.42 (s, 1H); 7.89 (d, J = 8.9, 1H); 7.95 (d,
3
) δ (ppm), J
(
2
2
2.2. Ethics, animals and parasites
(
7
Female BALB/c mice (4–6 weeks old) were purchased from the
Institute of Reproduction Biology of UFJF and maintained under
1
3
J = 2.2, 1H); 8.49 (d, J = 5.4, 1H). C NMR (126 MHz, CDCl
3
) δ (ppm):
◦
1
5
1
4
4.2; 22.8; 26.6; 29.1; 29.4; 29.5; 29.6; 30.4; 32.0; 42.5; 44.0; 46.9;
controlled conditions: temperature at 22 C, cycle of 12/12 h light/dark,
0.5; 99.1; 117.5; 121.4; 122.0; 125.5; 128.3; 135.2; 146.2; 148.7;
water and food ad libitum. The study was approved by the Committee
for the Ethical Handling of Research Animals (protocol number 007/
2018 and 008/2018) from Federal University of Juiz de Fora (UFJF, Juiz
de Fora, Minas Gerais, Brazil). Leishmania amazonensis (IFLA/BR/1967/
50.4; 151.6. HR-ESI-MS: m/z calculated for C24
H
36ClN
6
[M+H]+
43.26845, found 443.26837.
◦
2
.1.3. Synthesis of the intermediate 1,2,3-triazole 3
PH8) strain was cultivated at 25 C in Warren’s medium [brain heart
The same conditions as the previous procedure were used,
substituting the alkyne to propargyl chloride. Thin layer chromatog-
raphy (TLC) analysis, using the eluent CH Cl /CH OH (95:5), deter-
mined the presence of the product (Retention factor = 0.8). Derivative
was obtained with an 18.7% yield as a white solid. Melting
infusion (BHI) containing hemin and folic acid] at pH 7.2, supplemented
with 10% Fetal Bovine Serum (FBS), and 0.1% of the antibiotic solution
composed by 100 UI/mL penicillin G and 0.1 mg/mL of streptomycin
[24].
2
2
3
3
1
range = 39–41 ºC. H NMR (500 MHz, CDCl
J = 6.9, 3H); 1.11 – 1.54 (m, 14H); 1.89 (quint, J = 7.2, 2H); 4.33 (t,
3
) δ (ppm), J (Hz): 0.86 (t,
2.3. In vitro antipromastigote activity
1
3
J = 7.3, 2H); 4.70 (s, 2H); 7.57 (s, 1H). C NMR (126 MHz, CDCl
3
) δ
6
(
ppm): 14.2; 22.8; 26.6; 29.1; 29.3; 29.5; 29.6; 30.4; 32.0; 36.4; 50.7;
Leishmania amazonensis promastigotes (2 × 10 ), which were
1
2
22.6; 144.8. HR-ESI-MS: m/z calculated for C13
H25ClN
3
[M+H]+
collected in an exponential growth phase, were incubated in the pres-
58.17315, found 258.17307.
ence of derivatives 1–5 (1.6–100.0
μ
M) in 96-well culture plates (Nunc,
◦
Nunclon, Roskilde, Denmark) for 72 h at 25 C. Miltefosine at
2
.1.4. Synthesis of the quinoline/1,2,3-triazole hybrid 5
To a round-bottom flask, 0,58 mmol of derivative 3 and 0,86 mmol
3.1–100.0
μ
M (Cayman Chemical Company, Michigan, USA) was used
as drug control. The parasite viability was assessed by measuring of the
MTT (3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide,
Sigma-Aldrich, USA) colorimetric method. The optical density (OD)
values were read in a microplate spectrophotometer reader (Multiskan
MS microplate reader, LabSystems Oy, Helsink, Finland) at 570 nm
[32]. The results were expressed as the inhibitory concentration of 50%
of parasite growth (IC50) and were calculated from three independent
experiments performed in duplicate.
of derivative 1 were added and dissolved in 5 mL of ethanol. The
reactant mixture was submitted to constant stirring at 60 ºC for 3 days,
when thin layer chromatography (TLC) analysis, using the eluent
CH
Retention factor = 0.6) and derivative 4. At this point, the reaction was
stopped, the ethanol was removed under reduced pressure and the
products were isolated through column chromatography (CH Cl
CH OH gradient). After removing the solvent from the pure fractions,
2 2 3
Cl /CH OH (9:1), showed two products, identified as derivative 5
(
2
2
/
3
the previously described derivative 4 was obtained with a 5.6% yield
and the reaction also yielded the major product derivative 5 (21.6%) as
2.4. Treatment of Leishmania-infected macrophages
1
a light brown solid. Melting range = 79–81 ºC. H NMR (500 MHz,
5
CDCl
3
) δ (ppm), J (Hz): 0.87 (t, J = 7.0, 6H); 1.20 – 1.32 (m, 28H); 1.83
Murine macrophages (6 × 10 cells) were platted in 24-well plates
(
quint, J = 7.0, 4H); 1.96 (bs, 2H, NH); 2.96 (t, J = 5.4, 2H); 3.36 (q,
J = 4.7, 2H); 3.88 (s, 4H); 4.28 (t, J = 7.3, 4H); 6.30 (d, J = 5.4, 1H);
.87 (bs, 1H, NH); 7.45 (dd, J = 8.9 e 2.2, 1H); 7.51 (s, 2H); 7.94 (d,
containing sterile glass coverslips in RPMI 1640 medium supplemented
◦
with 10% FBS, and incubated for 24 h at 37 C in 5% CO . After that,
2
6
cultures were infected with stationary promastigotes of L. amazonensis
1
3
6
J = 2.1, 1H); 8.25 (d, J = 9.0, 1H); 8.48 (d, J = 5.3, 1H). C NMR
126 MHz, CDCl ) δ (ppm): 14.2; 22.8; 26.6; 29.1; 29.4; 29.5; 29.6; 30.4;
2.0; 41.0; 48.3; 50.5; 50.8; 98.9; 117.8; 122.7; 122.9; 125.4; 128.4;
(6 × 10 cells, at a ratio of 10 parasites per one macrophage) for 4 h, at
◦
(
3
33 C in 5% CO . In sequence, free parasites were removed by extensive
2
3
1
washing with PBS 1x, and infected macrophages were treated with
35.0; 144.4; 149.1; 150.5; 152.0. HR-ESI-MS: m/z calculated for
increasing concentrations (0.2–100.0 µM) of derivatives 1–5, for 72 h
[M+H]+ 664.45765, found 664.45789.
◦
C
37
H
59ClN
9
at 33 C in 5% CO . Miltefosine (0.8–25.0 µM) was used as control drug
2
in this assay. After incubation, cells were fixed with absolute ethanol and
stained with Giemsa, A total of 200 non-parasited or parasited macro-
phages were counted in a light microscope [32]. The results were
calculated as a percentage of reduction of the global burden of the
amastigotes per infected cell in relation to the non-treated control. From
these results, the IC50 of each molecule was determined using the Probit
program.
2
.1.5. Further NMR characterization of derivative 4
The main compound used for the mechanism of action assays in this
work was fully characterized via unidimensional and bidimensional
NMR techniques which can be found in the supplementary material. The
1
13
H and C NMR described above were fully attributed based on COSY,
HSQC and HMBC correlation spectroscopy. Although the presence of the
3