E.T. da Silva, et al.
European Journal of Pharmaceutical Sciences xxx (xxxx) xxxx
synthesis by inhibition of aranbinosyltransferase. Due to this Arabinan,
a component of arabinogalactan that constitue the cell wall, is not
generated (Villamizar-Mogotocoro et al., 2020).
were the more actives. However, the exception is 6d with activity of 93
μM. The azide derivatives 10 (a - d) presented reasonable activity
(44 – 143 μΜ) with low log P values. Compounds substituted with butyl
groups at the position 4 of the quinoline ring 4 (a, c, d) (9 – 41 μM)
were more actives than the compounds 2 (a – d), 3 (a – d), 5 (a -d), 6
(a – d), 7 (a, b, d), 8b, 9 (a – d), 10 (a – d), and 11 (a – d) (≥ 44 μM).
The amino alcohol group present in Ethambutol at the 4-position, 9
(a – d), as well as of similar group, 11 (a – c), and simplified groups, 2
(a, c, d), 3c, 5d, and 6b, displayed moderated activity (150 – 392 μM).
The compounds 2b, 3a, 3b, 3d, 5 (a – c), 6a, 6c, 7b and 11d were
inactive.
The pharmacophore amino alcohol group of the Ethambutol was
introduced at 4-position of the quinoline ring (group in red, Figure 1,
n
=
1,
R
=
NHCH
2
CH
2
OH), as well as of similar (n
=
2,
R = NHCH
2
CH
2
OH) and simplified groups (R = NH
2
, OH), including
groups with lipophilic characteristics (R = CH
2
CH
3
, Cl, N ). Mod-
3
ifications on the quinoline ring were promoted, with the insertion of
1
lipophilic substituents (R = Cl, Br) and the exchange position of the
1
trifluoromethyl group (Figure 1, R = CF
3
). The absence of substituents
on the quinoline ring, as well as the induction effect of electron-do-
nating and electron-withdrawing groups, were also evaluated. The use
of bromo at 6-position of the quinoline ring was motivated by Be-
daquiline, which present this atom in this position (Hards et al., 2015).
Analyzing only the substituents at 6-position, the CF group seems
3
to impair the activity concerning to others evaluated substituents de-
spite it confer the highest lipophilicity according to values log P
(Table 1). It assumes that property electron-withdrawing of the CF
3
group in this position affect the activity. In general, the bromo sub-
stituent resulted in more actives compounds. However, unsubstituted
compounds showed interesting similar activity and, in some cases, were
more actives (6d vs. 6c, 8d vs. 8c, 10d vs. 10c). The majority of the
compounds substituted with chloro group not were more actives than
the corresponding unsubstituted.
2
. Results and discussion
2.1. Chemistry
For the synthesis of the new analogues 2 (a – d) - 11 (a – d,
Scheme 1), we used the synthetic methodology previously described by
our group (Araújo et al., 2019). The first step reaction is the formation
of the quinoline nucleus, based on the condensation reaction with dif-
ferent anilines with PPA at 150°C for 2-3 hours. The formed nucleus
possessed at 6-position, chloro, trifluoromethyl, bromo and hydrogen,
and also a free hydroxyl group at 4-position. This hydroxyl group was
then alkylated using methyl iodide to form a leaving group, which will
react with nucleophiles. Methylation was made by using methyl iodide
As done in the previous work (Araújo et al., 2019), the more active
compounds against sensitive strain (13 compounds) were evaluated
against resistant strains (SR2571/0215; T113/09 strains resistant to
Rifampicin and Isoniazid) and are demonstrated in Table 2. Nine
compounds displayed higher activity to resistant strains (lower MIC
value) than to sensitive strain. The compound 4a with activity at 41 μM
showed excellent activity against resistant strain at 4 μΜ. Compound 4d
with activity at 12 μM displayed activity against resistant strain at 5
μΜ. The compound 10d was also much more active to resistant strain
(11 μΜ) than to sensitive strain (44 μΜ; Table 2). A hypothesis raised
by us is that activity of these new analogues was believed to be through
a different mechanism of action, or more than one, perhaps similar to
that presented by Mefloquine, which is like that of Bedaquiline
(Hards et al., 2015). This latter presents its antimycobacterial action by
inhibiting the enzyme F1Fo-ATP synthase, causing the death of even
non-replicating cells.
in acetone at room temperature in the presence of Na
2
CO , for 4 – 20
3
hours, to furnish the compounds 1 (a – d) in 50 – 70% yield. These
compounds reacted with diamines, butylamine, and amino alcohols at
9
0 – 120°C to furnish the derivatives 2 (a – d), 3 (a – d), 4 (a – d), 5
(
a – d), and 6 (a – d) in 15-95% yield after 1-3 hours (4d: 48 h). The
compounds 5 (a – d) and 6 (a – d) were converted to chloro derivatives
(a – d) and 8 (a – d) by treatment with SOCl in CH Cl or CHCl at
7
2
2
2
3
reflux in 40 – 95% yield after 1-3 hours of reaction (Scheme 1).
The compounds 9 (a – d) were obtained after reaction of 7 (a – d)
with ethanolamine under heating at 110°C for 1-2 hours in 67 – 91%
yield. The azido compounds 10 (a – d) were obtained after reaction of 7
The more active compounds in sensitive and resistant strains were
assayed to cellular viability in the presence and absence of test com-
pounds by Mosmans's MTT (3-(4,5-demethylthylthiazol-2-yl)-2,5-di-
methyl tetrazolium bromide; Merck) microcultured tetrazolium assay,
utilizing human macrophages cells (Mosmann, 1983). The results were
expressed as percentage cell viability in different concentrations: 1, 10,
100, and 1000 μM (Table 3). The more active compounds to resistant
strains 4a, 4d, 7c, 8d, and 10d were not cytotoxic to the host cells at
concentrations near the MIC. The more active compounds to sensitive
strain 4d, 6d, 8d, and 10d also were not cytotoxic to the host cells at
concentrations near the MIC.
(
a – d) with sodium azide in DMF at 130°C for 2 – 3.5 hours in 79 – 95%
yield. The compounds 11 (a – d) were obtained after reaction of 8
(
a – d) with ethanolamine at 110°C for 1-2 hours in 59 – 93% yield.
All these new compounds were identified by detailed spectral data,
1
13
including H NMR, C NMR, and high resolution mass spectra. In
1
general, the H NMR spectrum showed four or five quinoline protons at
1
3
8
.90 – 6.20 ppm and the aliphatic protons at 4.00 – 0.95 ppm. The
C
NMR spectrum showed the quinoline carbons signals at the region of
9
1
2
3
2.84 – 164.52 ppm and the aliphatic carbons at the regions of
3.64 – 60.49 ppm. The CF group showed a quartet with J about 270-
74 Hz. In the IR spectra, characteristic signals of NH were observed at
3
3. Material and methods
−
1
−1
200 – 3380 cm , OH were observed at 3500 – 4000 cm , and N
3
All experimental procedures for synthesis and characterization of all
substances described are in supporting information.
−
1
were observed at 2090 – 2100 cm . In all compounds were observed
-
1
for the C-F axial deformation signals at 1080 – 1300 cm .
3.1. Biological Evaluation against Mycobacterium tuberculosis
2
.2. Antimycobacterial activity against M. tuberculosis H37Rv (ATCC
7294)
2
The antimycobacterial activities of all tested compounds were as-
sessed against sensitive M. tuberculosis strain H37Rv (ATCC 27294) and
resistant M. tuberculosis strain SR571/0215 and T113/09 (resistant to
Isoniazid and Rifampicin, no clinical strains), using the microplate
Alamar Blue assay (MABA, Tables 1 and 2) (Franzblau et al., 1998) and
performed in duplicate. Briefly, 200 microliters of sterile deionized
water were added to all outer-perimeter wells of sterile 96-well plates
(Falcon, 3072: Becton Dickinson, Lincoln Park, NJ, USA) to minimize
evaporation of the medium in the test wells during incubation. The 96
plates received 100 μL of the Middlebrook 7H9 broth (Difco
The antimycobacterial activities of derivatives 2 (a – d) – 11 (a – d),
showed in Table 1, were assessed against M. tuberculosis H37Rv (ATCC
2
7294) using the microplate Alamar Blue assay (MABA)
(
Franzblau et al., 1998).
Of these forty planned and synthesized compounds, twenty-nine
exhibited activity with MIC values of 9 to 392 μM (Table 1). In general,
compounds possessing lipophilic groups at the 4-position of the qui-
noline ring 4 (a – d), 7 (a, c, d), 8 (a – d), and 10 (a – d) (9 – 182 μM),
3
da Silva, Emerson Teixeira
