Design, synthesis, and evaluation of new 2-(quinoline-4-yloxy)acetamide-based antituberculosis agents

Article abstract of DOI:10.1016/j.ejmech.2020.112179

Using a classical molecular simplification approach, a series of 36 quinolines were synthesized and evaluated as in vitro inhibitors of Mycobacterium tuberculosis (M. tuberculosis) growth. Structure–activity relationship (SAR) studies leaded to potent antitubercular agents, with minimum inhibitory concentration (MIC) values as low as 0.3 μM against M. tuberculosis H37Rv reference strain. Furthermore, the lead compounds were active against multidrug-resistant strains, without cross-resistance with some first- and second-line drugs. Testing the molecules against a spontaneous mutant strain containing a single mutation in the qcrB gene (T313A) indicated that the synthesized quinolines targeted the cytochrome bc₁ complex. In addition, leading compounds were devoid of apparent toxicity to HepG2 and Vero cells and showed moderate elimination rates in human liver S9 fractions. Finally, the selected structures inhibited M. tuberculosis growth in a macrophage model of tuberculosis infection. Taken together, these data indicate that this class of compounds may furnish candidates for the future development of antituberculosis drugs.

Full text of DOI:10.1016/j.ejmech.2020.112179

Journal Pre-proof
Design, synthesis, and evaluation of new 2-(quinoline-4-yloxy)acetamide-based
antituberculosis agents
Ana Flávia Borsoi, Josiane Delgado Paz, Bruno Lopes Abbadi, Fernanda Souza
Macchi, Nathalia Sperotto, Kenia Pissinate, Raoní S. Rambo, Alessandro Silva
Ramos, Diana Machado, Miguel Viveiros, Cristiano Valim Bizarro, Luiz Augusto
Basso, Pablo Machado
PII:
S0223-5234(20)30146-X
DOI:
https://doi.org/10.1016/j.ejmech.2020.112179
EJMECH 112179
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 December 2019
Revised Date: 12 February 2020
Accepted Date: 20 February 2020
Please cite this article as: Ana.Flá. Borsoi, J.D. Paz, B.L. Abbadi, F.S. Macchi, N. Sperotto, K. Pissinate,
Raoní.S. Rambo, A.S. Ramos, D. Machado, M. Viveiros, C.V. Bizarro, L.A. Basso, P. Machado, Design,
synthesis, and evaluation of new 2-(quinoline-4-yloxy)acetamide-based antituberculosis agents,
European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112179.
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Design, synthesis, and evaluation of new 2-(quinoline-4-yloxy)acetamide-based
antituberculosis agents
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Ana Flávia Borsoia,b, Josiane Delgado Paza,c, Bruno Lopes Abbadia, Fernanda Souza Macchia,c,
Nathalia Sperottoa,b, Kenia Pissinatea, Raoní S. Ramboa, Alessandro Silva Ramosa, Diana
Machadod, Miguel Viveirosd, Cristiano Valim Bizarroa,c, Luiz Augusto Bassoa,b,c,*, Pablo
Machadoa,c,*
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aInstituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia
Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul, 90616-900,
Porto Alegre, Rio Grande do Sul, Brazil
bPrograma de Pós-Graduação em Medicina e Ciências da Saúde, Pontifícia Universidade
Católica do Rio Grande do Sul, 90616-900, Porto Alegre, Rio Grande do Sul, Brazil
cPrograma de Pós-Graduação em Biologia Celular e Molecular, Pontifícia Universidade Católica
do Rio Grande do Sul, 90616-900, Porto Alegre, Rio Grande do Sul, Brazil
dGlobal Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT,
Universidade NOVA de Lisboa, UNL, Lisbon, Portugal
*Corresponding author
E-mail addresses:
luiz.basso@pucrs.br (Luiz Augusto Basso); pablo.machado@pucrs.br (Pablo Machado)

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Abstract
Using a classical molecular simplification approach, a series of 36 quinolines were synthesized
and evaluated as in vitro inhibitors of Mycobacterium tuberculosis (M. tuberculosis) growth.
Structure–activity relationship (SAR) studies leaded to potent antitubercular agents, with
minimum inhibitory concentration (MIC) values as low as 0.3 µM against M. tuberculosis
H37Rv reference strain. Furthermore, the lead compounds were active against multidrug-
resistant strains, without cross-resistance with some first- and second-line drugs. Testing the
molecules against a spontaneous mutant strain containing a single mutation in the qcrB gene
(T313A) indicated that the synthesized quinolines targeted the cytochrome bc1 complex. In
addition, leading compounds were devoid of apparent toxicity to HepG2 and Vero cells and
showed moderate elimination rates in human liver S9 fractions. Finally, the selected structures
inhibited M. tuberculosis growth in a macrophage model of tuberculosis infection. Taken
together, these data indicate that this class of compounds may furnish candidates for the future
development of antituberculosis drugs.
Keywords: Mycobacterium tuberculosis; molecular simplification; multidrug-resistant strains;
SAR; intracellular activity; cytochrome bc1 complex.

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1. Introduction
Tuberculosis (TB) is an airborne infectious disease, described among the top 10 causes of
death worldwide. The disease has Mycobacterium tuberculosis (M. tuberculosis) as its main
etiological agent, and it was responsible for claiming 1.5 million lives in 2018 [1]. In the same
year, about 10.0 million people developed TB, according to the World Health Organization [1].
This public health problem has been aggravated by the emergence of rifampicin-resistant TB
(RR-TB), multidrug-resistant TB (MDR-TB), HIV coinfection, and the large number of
individuals infected with latent or dormant bacilli [1,2]. The recommended treatment includes a
set of four drugs administered in two different combinations for six months. Although it has a
high cure rate, the current therapeutic regimen suffers from low adherence of the patients, with
increased drug resistance, adverse effects, and the impossibility of co-administration with some
antiretroviral drugs [3,4]. In the early 2010s, the approval of bedaquiline [5] and delamanid [6]
for the treatment of adult patients with pulmonary MDR-TB ended a period of more than four
decades without approval of a new anti-TB drug. This addition to the therapeutic arsenal has
been met with caution since these drugs have resulted in certain toxicity events, including drug-
induced QT interval prolongation. In addition, the adaptive capacity of M. tuberculosis has
already led to the emergence of bedaquiline- and delamanid-resistant strains [7], indicating the
need for continuous effort to obtain new therapeutic alternatives to TB treatment.
Within this context and as part of our ongoing research, we evaluated the antimycobacterial
activity of 2-(quinolin-4-yloxy)acetamides 1 (Figure 1) and their derivatives [8,9]. The
compounds demonstrated submicromolar activity against resistant and non-resistant M.
tuberculosis strains by targeting the QcrB subunit of menaquinol cytochrome c oxidoreductase

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(bc1 complex) [10]. Despite their high capacity to inhibit M. tuberculosis growth in vitro, their
good membrane permeability, and their synergistic in vitro effect with rifampin, this chemical
class has shown moderate metabolic stability [9]. The microsomal instability of 2-(quinolin-4-
yloxy)acetamides has been attributed to the amide group lability, which is a probable point of
esterase-mediated hydrolysis [11]. Such instability could reduce the pharmacokinetic exposure of
these molecules when evaluated in in vivo models of tuberculosis. Thus, a change in this
chemical group with a reduction in electrophilicity could lead to compounds containing better
metabolic profiles. Our hypothesis was that molecular simplification of the attached acetamide
group could provide novel compounds with optimized properties (Figure 1). The major
challenge would be to maintain antimycobacterial activity while altering the amide since this
group has been described as an important pharmacophoric point of compounds with potent
activity against M. tuberculosis growth [8,9,12]. Furthermore, it has been described that
quinolines containing ether groups at the 4-position are devoid of antitubercular activity [13].
Therefore, in an attempt to obtain new compounds with activity against drug-susceptible and,
mostly, drug-resistant M. tuberculosis strains, a new series of simplified 2-methyl-6-methoxy-2-
quinolines was synthesized. First, the structural requirements for potency of molecules (SAR)
were evaluated using minimal inhibitory concentration (MIC) values. Subsequently, the most
active structures against M. tuberculosis H37Rv were tested against a panel of well characterized
multidrug-resistant strains, while the viability of HepG2 and Vero cells was used as an indicator
of the toxicity and selectivity of the compounds. Finally, the in vitro metabolic stability and
intracellular activity in a macrophage model of M. tuberculosis infection were also evaluated.

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Figure 1. Molecular simplification of the amide group from 2-(quinolin-4-yloxy)acetamides as a
strategy for the discovery and optimization of new antimycobacterial compounds with better
metabolic properties.
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2. Results and discussion
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The designed compounds were obtained in two synthetic steps. First, 4-hydroxyquinoline (2)
was synthesized in a classical Conrad-Limpach cyclocondensation reaction between ethyl 3-
oxobutanoate and 4-methoxyaniline, according to an already-reported protocol [9]. The second
step was accomplished through O-alkylation in a second-order nucleophilic substitution reaction
(SN2). Importantly, the substituents of the alkylating agents were chosen from different electron-
donating and electron-withdrawing groups, including bulky alkyl and aryl groups. The
quinolines 3a–m were obtained from the reaction of 4-hydroxyquinoline (2) and 2-bromo-1-
arylethanones using potassium carbonate (K2CO3) as a base and dimethylformamide (DMF) as
the solvent. The reactants were stirred for 18 h at 25°C, leading to products 3a–m with 13–90%
yields (Scheme 1). Using the same procedure, the quinolines 4a–w were synthesized by the
reaction of 4-hydroxyquinoline (2) and benzyl bromides, with 20−97% yields (Scheme 1). In
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general, the presence of a carbonyl group in the alkylating agent provided products in lower
yields when compared to alkylation reactions using benzyl bromides. Spectroscopic and
spectrometric data were found to be in agreement with the proposed structures (Supporting
Information).
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Scheme 1. Reagents and conditions. (i) = Alkyl halide, K2CO3, DMF, 25°C, 18 h.
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The synthesized compounds 3 and 4 were evaluated in a whole-cell assay against the M.
tuberculosis H37Rv strain using isoniazid as positive control [14,15]. The quinolines 3a–m
presented good activity against the bacillus, with MIC values ranging from 1.3 to 31.1 µM under
the tested conditions (Table 1). The antimycobacterial activity results showed that carbonyl-
containing compounds 3a and 3h yielded lower MICs than the first line drug, isoniazid.
Positioning the methoxy group at the 4-position of the benzene ring led to a compound with
reduced activity against M. tuberculosis, whereas the unsubstituted compound 3a exhibited a
MIC of 1.3 µM; the presence of the methoxy group at the 4-position of 3b (MIC = 7.4 µM)
reduced the activity more than 5-fold. Additionally, the presence of the methoxy group at the 3-
(3c) and 2-positions (3d) reduced the activity of the molecules to a greater extent, with MIC
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values of 14.8 and 29.6 µM, respectively. By changing the electron-donating group to 4-methyl,
the potency was also reduced since quinoline 3e showed a MIC of 15.6 µM. The same pattern
was observed with the use of electron-withdrawing groups as substituents. The 4-fluor- (3f) and
4-chloro-substituted (3g) compounds showed MICs of 30.7 and 12.9 µM, respectively. Notably,
changing the chloro atom from the 4- to 3-position significantly altered the antimycobacterial
activity because molecule 3h exhibited a MIC value of 1.8 µM. This value differed only slightly
from 3a (1.3 µM) and showed increased potency of nearly 7-fold compared to the 4-substituted
derivative 3g. Substitution with chlorine atoms at position 3 and 4 of the benzyl ring reduced the
inhibitory activity against M. tuberculosis. 3,4-Dichlorophenyl-substituted 3i showed a MIC of
6.6 µM, which was more than 3.5-fold lower than its monosubstituted analog, 3h. Additionally,
the 4-bromophenyl-substituted 3j exhibited a MIC of 14.6 µM, denoting that the classic
bioisosteric replacement between the chlorine and bromine was able to maintain similar and
reduced potencies. The significant increase in the lipophilicity when using 4-iso-butyl and 4-
phenyl groups did not increase the activity of the molecules against the M. tuberculosis H37Rv
strain since structures 3k and 3l presented MICs of 6.9 and 13.0 µM, respectively. Finally, the
presence of an α-methyl group in quinoline 3m reduced the activity nearly 24-fold, suggesting
that changes in this position do not sustain the antimycobacterial activity of this chemical class.

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Table 1. ClogP values and in vitro activity of quinolines 3 and 4 against the M. tuberculosis
H37Rv strain.
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Entry
3a
3b
3c
R1
H
H
H
H
H
H
H
H
H
H
H
H
Me
–
R2
Ph
R3
ClogPa
4.18
4.40
4.40
4.40
4.67
4.39
4.96
4.96
5.58
5.11
6.13
6.06
4.48
4.99
4.90
MIC (µM)
1.3
–
4-MeO-Ph
3-MeO-Ph
2-MeO-Ph
4-Me-Ph
4-F-Ph
4-Cl-Ph
3-Cl-Ph
3,4-(Cl)2-Ph
4-Br-Ph
4-i-Bu-Ph
4-Ph-Ph
Ph
–
7.4
–
14.8
29.6
15.6
30.7
12.9
1.8
–
3d
3e
–
–
3f
–
3g
3h
3i
–
–
6.6
–
14.6
6.9
3j
–
3k
3l
–
13.0
31.1
5.6
–
Ph
3m
4a
4b
–
–
–
3-MeO-Ph
32.3

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4c
4d
4e
4f
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
3,5-(MeO)2-Ph
4-F-Ph
4.99
5.13
5.13
5.13
5.20
5.67
5.67
5.70
6.29
6.29
6.42
5.85
5.85
5.87
5.87
4.73
6.41
6.81
5.31
6.16
29.5
16.8
16.8
19.2
3.9
3-F-Ph
2-F-Ph
4g
4h
4i
3,4-(F)2-Ph
4-Cl-Ph
15.9
7.9
3-Cl-Ph
2-Cl-Ph
18.8
0.3
4j
3,4-(Cl)2-Ph
2,3-(Cl)2-Ph
3-Cl-4-Br-Ph
4-Br-Ph
4k
4l
28.7
1.6
4m
4n
4o
4p
4q
4r
4s
13.9
13.9
7.2
3-Br-Ph
4-F3C-Ph
3-F3C-Ph
4-O2N-Ph
4-i-Pr-Ph
4-t-Bu-Ph
Bn
7.2
30.8
1.9
3.7
4t
34.1
7.6
4u
4v
2-Naphthyl
–
–
–
–
4.95
–
30.9
2.3
4w
–
INH

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aClogP calculated with ChemBioDraw Ultra, version 13.0.0.3015. INH, Isoniazid.
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In the second round of obtaining new anti-TB drug candidates, the antimycobacterial activity
of 2-quinolin-4-yloxy derivatives 4a-w against the M. tuberculosis H37Rv strain was determined
(Table 1). In general, dihalogenated- or alkyl-substituted compounds showed the best activities
under the tested conditions. The unsubstituted quinoline 4a showed a MIC of 5.6 µM. The
presence of the methoxy group attached at the 3-position of the benzene ring yielded compound
4b, which exhibited a MIC of 32.3 µM. This result demonstrates that this electron-donating
group reduced the activity by more than 5-fold compared to the unsubstituted analogue, 4a.
Similarly, the use of another methoxy group attached at the 5-position of the benzene ring led to
molecule 4c, which presented a MIC of 25.5 µM. Fluorine attached at the 4- (4d) and 3-positions
(4e) generated equipotent compounds, with a MIC of 16.8 µM. Quinoline 4f, containing a
fluorine atom at the 2-position, showed a MIC of 19.2 µM. When two fluorine atoms were
positioned at the 3- and 4-positions of the benzene ring, the capacity to inhibit the bacillus
growth increased. The MIC presented by difluorinated derivative 4g was 3.9 µM. This MIC
value indicated a slightly superior activity compared to that presented by compound 4a (MIC =
5.6 µM). The chlorine atom attached at the 4- and 3-positions of 4h and 4i yielded compounds
with MIC values of 15.9 µM and 7.9 µM, respectively. Once more, the presence of a halogen at
the 2-position reduced the potency against M. tuberculosis since molecule 4j exhibited a MIC of
18.8 µM. On the other hand, 3,4-dichlorobenzyl derivative 4k showed the highest
antimycobacterial activity of the series of synthesized quinolines. The presence of chlorine atoms
at the 3- and 4-positions in structure 4k led to a MIC value of 0.3 µM. This finding indicated that
compound 4k was approximately 7.6-fold more effective than isoniazid (MIC = 2.3 µM). The
importance of this molecular arrangement is evident by the comparison with the activity of

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structure 4l. Changing the chlorine atom from 4- to 2-position greatly reduced antimycobacterial
activity as quinoline 4l showed MIC of 28.7 µM. In addition, steric properties seem to be directly
related to the activity presented by the compounds rather than the physicochemical parameters as
CLogP of 4k and 4l are identical (CLogP = 6.19). Another finding related to the specificity of
the activity showed by molecule 4k was that exchanging the 4-chlorine with the 4-bromine in
quinoline 4m reduced the activity more than 5-fold, leading to a MIC value of 1.6 µM. Notably,
independent of the position of the bromine atom attached to the benzene ring in molecules 4n
and 4o, the MIC values were 13.9 µM. The same pattern was observed with trifluoromethylated
compounds 4p and 4q, which exhibited MIC values of 7.2 µM. Additionally, by increasing the
polarization of quinoline 4r with the 4-nitro substituent attached to the aryl group, the activity
was greatly reduced. This electron-withdrawing group yielded a compound that inhibited M.
tuberculosis H37Rv growth, with a MIC of 30.8 µM. By contrast, replacement of the hydrogen
of the benzene ring with alkyl groups at the 4-position was well tolerated. However, the 4-iso-
propyl group in molecule 4s was able to inhibit M. tuberculosis, with a MIC of 1.9 µM. The 4-
tert-butyl group in 4t presented a MIC value of 3.7 µM. In particular, these results showed that
structure 4s has a slightly higher potency than isoniazid under the experimental conditions used.
Moreover, the use of a methylene group as a spacer greatly reduced the activity of quinoline 4u.
The MIC of this structure was 34.1 µM, which was approximately 6-fold less effective than
compound 4a. Finally, the 2-naphthyl group in 4v presented a MIC value of 7.6 µM, while
benzo[d][1,3]dioxole derivative 4w was able to inhibit M. tuberculosis, with a MIC of 30.9 µM.
This result reveal that increasing the polarity in the substituent reduced the inhibitory capacity of
the molecule nearly 4-fold.

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Using the MIC value of 2.3 µM, presented by isoniazid, as a threshold, quinolines 3a, 3h, 4k,
and 4s were selected for inhibitory activity against a panel of multidrug-resistant M. tuberculosis
strains (Table 2). Due to the antimycobacterial activity of compounds 4k and 4m and their
structural similarity, only 3,4-dichlorobenzyl derivative 4k was selected. M. tuberculosis strains
PT2, PT12, and PT20 have been described as resistant to drugs such as isoniazid, rifampin,
streptomycin, ethionamide, and rifabutine. Additionally, PT12 and PT20 are also resistant to
drugs such as pyrazinamide and ethambutol and PT12 present additional resistance to amikacin
and capreomycin. The genome of these strains has been sequenced, and the genotypic alterations
responsible for the resistant phenotypes have been already reported [16]. Notably, the evaluated
quinolines exhibited similar activities, and they were even more potent against PT2, PT12, and
PT20 strains than the M. tuberculosis H37Rv strain (Table 2). Compounds 3a and 4k exhibited
similar activities between the different drug-susceptible and drug-resistant M. tuberculosis
strains. Molecules 3h and 4s were 1.9- to 9.5-fold more potent against MDR strains than the
drug-susceptible M. tuberculosis H37Rv strain. From the results obtained, one can conclude that
the synthesized quinolines do not share in vitro cross-resistance with some important and
clinically useful anti-TB drugs. These data suggest promising potential of these structures against
drug-susceptible and MDR M. tuberculosis strains, probably involving different molecular
targets from known drugs.
In an attempt to shed light on the mechanism of action of synthesized quinolines 3a, 3h, 4k,
and 4s, the MICs of the compounds were determined against a 2-(quinolin-4-yloxy)acetamide-
resistant M. tuberculosis strain (Table 2). Whole genome sequencing of this spontaneous
resistant strain has revealed a mutation in the qcrB gene which substituted an adenine nucleotide
at 937 position by a guanine resulting in the T313A amino acid exchange [10]. The four

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evaluated compounds showed MIC values that were at least 4-fold higher than those displayed
against the M. tuberculosis H37Rv strain. These data suggested the involvement of the qcrB gene
product in the antimycobacterial activity elicited by these molecules. The qcrB gene encodes the
b-subunit of the cytochrome bc1 complex, which is part of the respiratory electron transport chain
and required for ATP biosynthesis. Therefore, the molecular simplification from 2-(quinolin-4-
yloxy)acetamides, which culminates in the withdrawal of amide function in compounds 3 and 4,
maintained the cytochrome bc1 complex as a possible molecular target for this series of
molecules.
Table 2. In vitro activity of the selected quinolines against M. tuberculosis H37Rv, MDR strains,
and the 2-(quinolin-4-yloxy)acetamide-resistant strain. Evaluation of the viability of HepG2 and
Vero cells. Metabolic stability evaluated in human liver S9 fractions.
b
b
c
d
Entry MIC
MIC MIC MIC MICa
CC50
CC50
Clint
t1/2
H37Rv PT2 PT12 PT20 qcrB-
(µM) (µM) (µM) (µM) T313A
(µM)
HepG2
Vero (mL/min/kg) (min)
(µM)
(µM)
1.3
1.8
0.3
1.9
2.3
0.05
2.0
0.5
0.2
0.5
2.0
0.9
0.5
1.0
1.0
0.2
0.2
0.2
32.5
7.3
14.4
7.8
2.3
–
>20
>20
>20
>20
>20
–
7.1
7.7
12.6
17.4
–
87.5
78.2
30.6
10.0
–
3a
3h
>20
14.4e;14.9f
4k
13.1e;13.0f
4s
291.7 72.9 145.8
>48.6 >48.6 12.2
–
–
INH
RIF
–
–
–

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a2-(Quinolin-4-yloxy)acetamide-resistant spontaneous mutant containing a unique alteration in
b
the qcrB gene (ACC to GCC at nucleotide 937 position or T313A amino substitution). The
toxicity and selectivity of the compounds was studied on HepG2 and Vero cells. The 50%
c
cytotoxic concentration determined by MTT and Neutral Red assays. Human S9 intrinsic
clearance. dHalf-live. eDetermined by the MTT method. fDetermined by the Neutral Red method.
INH, Isoniazid. RIF, Rifampin.
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In addition, quinolines 3a, 3h, 4k, and 4s were evaluated for their cytotoxicity using HepG2
and Vero cells (Table 2). Cellular viability was determined using MTT and Neutral Red uptake
assays [9,17] after exposing the cell lineages to the quinolines for 72 h [18]. While MTT
determines mitochondrial activity, neutral red assesses the lysosomal viability of the cells.
Incubation of carbonyl-containing compounds 3a and 3h at a concentration of 20 µM did not
significantly affect the viability of either cell lineage. Furthermore, the viability of the Vero cells
exposed to 20 µM of compounds 4k and 4s was not affected. By contrast, molecules 4k and 4s
exhibited CC50 (50% cytotoxic concentration) values of 14.4/14.9 µM and 13.1/13.0 µM,
respectively, when incubated with HepG2 cells. These findings point out that there is a
difference of 6.8–6.9-fold between the antimycobacterial activity elicited by structure 4s against
M. tuberculosis H37Rv and its cytotoxic concentration. Taking this data alone, these values
could be considered indicative of toxicity and, therefore, motivation for exclusion of 4s from
subsequent trials. However, when considering the MIC values of molecule 4s against MDR
strains, this difference increases to at least 13-fold. Therefore, the viability results suggested that
structures 3a, 3h, 4k, and 4s present a reasonable to high degree of selectivity for M.
tuberculosis, propelling us to continue our research efforts.

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Afterward, the metabolic stability of quinolines 3a, 3h, 4k, and 4s was determined using
human liver S9 fractions (Table 2). Compounds 3a, 3h, and 4k showed moderate elimination
rates based on human S9 intrinsic clearance values (15< Clint >5 mL/min/kg) [19]. By contrast,
molecule 4s exhibited a high elimination rate under the evaluated conditions (Clint >15
mL/min/kg) [19]. Interestingly, carbonyl-containing structures 3a and 3h demonstrated half-lives
that were at least 2.5-fold longer than ether derivatives 4k and 4s. Another finding was that the
presence of the 4-iso-propylbenzyl group reduced the half-life of quinoline 4s 3-fold when
compared to 3,4-dichlorobenzyl derivative 4k. This may be related to the activation of the
benzene ring by the alkyl group, which is prone to oxidation reactions from microsomal
enzymes. However, further studies are needed to confirm such hypothesis. For purposes of
comparison, the metabolic stability of 2-(quinolin-4-yloxy)acetamides, determined under the
same experimental conditions, showed half-lives with mean values of 18.7 min [8,9]. This data
suggests, once more [11], that the presence of an amide function generates a hydrolysis soft spot
that can be circumvented by the molecular simplification strategy.
In order to evaluate the ability of the compounds to pass cell membranes and to inhibit the
intracellular growth of the bacilli, quinolines 3a, 3h, and 4k were evaluated in a macrophage
model of M. tuberculosis infection. It is important to mention that these molecules were chosen
based on cytotoxicity and metabolic stability studies. Macrophages from the untreated group
(0.5% DMSO) showed an increase of around 1.21 log10 CFU, within five days, compared to the
early control group. This data denoted the bacteria's ability to multiply intracellularly (Table 3).
Treatments with molecules 3h and 4k prevented bacterial growth and kept the bacterial loads
stable inside the macrophages. By contrast, phenyl-substituted compound 3a was ineffective
when statically compared to the early control and the untreated group. The data obtained

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suggested that structures 3h and 4k were able to have a bacteriostatic effect on intracellular M.
tuberculosis growth (P<0.05). It is noteworthy that these simplified quinolines also exhibited the
best activities against MDR M. tuberculosis strains, making these lead compounds highly
attractive.
Table 3. Intracellular activity of compounds 3a, 3h, and 4k in murine macrophages infected with
the virulent M. tuberculosis H37Rv strain.
Log10 CFU/well (Mean ± SD)
Entry
Early Control
Untreated
3a (5 µM)
3h (5 µM)
4k (5 µM)
3.45 ± 0.07*
4.66 ± 0.23**
4.06 ± 0.11*
3.80 ± 0.43*
3.80 ± 0.17*
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SD, standard deviation; *P < 0.05 compared to untreated group (0.5% DMSO); **P < 0.01
compared to early control (EC) group.
3. Conclusion
In summary, herein the design and synthesis of a new series of simplified quinolines was
shown, and we demonstrated their in vitro antimycobacterial activity. The synthetic procedures
were performed using readily accessible reagents and reactants under mild reaction conditions.
In addition, the compounds showed selective activity against drug-sensitive and MDR M.
tuberculosis strains, with MIC values in the low micromolar or submicromolar range.
Interestingly, the lead compounds carry out their antitubercular activity by targeting the

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cytochrome bc1 complex. This possible mechanism of action/resistance expands the potential use
of these structures for non-replicating forms of M. tuberculosis. Furthermore, the design strategy
provided molecules that were metabolically more stable than their counterparts, which were able
to inhibit the intracellular M. tuberculosis growth with a bacteriostatic effect. Finally, the
submicromolar activity against MDR M. tuberculosis strains elicited by leading quinolines
coupled to the metabolic stability and intracellular activity suggests that this class of compounds
may yield candidates for the development of new anti-TB drugs. New structural modifications of
the compounds, as well as bioavailability studies, are currently underway.
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4. Experimental section
4.1 Synthesis and structure: apparatus and analysis
The commercially available reactants and solvents were obtained from commercial suppliers
and were used without additional purification. The melting points were measured using a
Microquímica MQAPF-302 apparatus. 1H and 13C NMR spectra were acquired on an Avance III
HD Bruker spectrometer (Pontifical Catholic University of Rio Grande do Sul). Chemical shifts
(δ) were expressed in parts per million (ppm) relative to DMSO-d6, which was used as the
solvent, and to TMS, which was used as the internal standard. High-resolution mass spectra
(HRMS) were obtained on an LTQ Orbitrap Discovery mass spectrometer (Thermo Fisher
Scientific, Bremer, Germany). This system combines an LTQ XL linear ion-trap mass
spectrometer and an Orbitrap mass analyzer. The analyses were performed through the direct
infusion of the sample in MeOH/CH3CN (1:1) with 0.1% formic acid (flow rate of 10 µL/min) in
positive-ion mode using electrospray ionization (ESI). For the elemental composition, the
calculations used the specific tool included in the Qual Browser module of Xcalibur (Thermo

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Fisher Scientific, release 2.0.7) software. The compound purity was determined using an Äkta
HPLC system (GE Healthcare® Life Sciences) equipped with a binary pump, manual injector,
and UV detector. Unicorn 5.31 software (Build 743) was used for data acquisition and
processing. The HPLC conditions were as follows: RP column, 5 µm Nucleodur C-18 (250 × 4.6
mm); flow rate, 1.5ꢀmL/min; UV detection, 254 nm; 100% water (0.1% acetic acid) was
maintained from 0 to 7 min, followed by a linear gradient from 100% water (0.1% acetic acid) to
90% acetonitrile/methanol (1:1, v/v) from 7 to 15 min (15–30 min) and subsequently returned to
100% water (0.1% acetic acid) in 5 min (30–35 min) and maintained for an additional 10 min
(35–45 min). All the evaluated compounds were ≥90% pure.
4.2 General procedure for the synthesis of quinolines 3 and 4
The synthesis of 4-hydroxyquinoline (2) was performed in accordance to an already reported
procedure [9]. The appropriate alkyl halide (1.2 mmol) was added to a mixture of 4-
hydroxyquinoline (1 mmol) and potassium carbonate (K2CO3, 3.12 mmol) in DMF (6 mL). The
reaction mixture was stirred at 25°C for 18 h. Afterwards, the reaction mixture was diluted in
water (10 mL) with concomitant precipitation of the product. The solid was separated using a
centrifuge (18,000 RPM, 4°C, 10 min), washed with water (3 × 15 mL), and dried under reduced
pressure to afford the products in good purity, which was measured by HPLC experiments. In
some cases where the purity of the products was not satisfactory, the solids were washed with
ethyl ether or purified by flash chromatography using ethyl acetate and hexane in a ratio of 3:7;
1:1, and, finally, 7:3, respectively.
4.2.1 2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-1-phenylethan-1-one (3a)

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Yield 38%; m.p.: 155-156 °C; HPLC 90% (tR = 14.53 min); H NMR (400 MHz, DMSO-d6) δ
2.52 (s, 3H), 3.89 (s, 3H), 5.89 (s, 2H), 6.93 (s, 1H), 7.35 (dd, J = 9.1, 2.9 Hz, 1H), 7.48 (d, J =
2.9 Hz, 1H), 7.61 (t, J = 7.6 Hz, 2H), 7.68 – 7.84 (m, 2H), 8.04 – 8.13 (m, 2H). 13C NMR (101
MHz, DMSO-d6) δ 24.7, 55.1, 70.2, 99.5, 119.5, 121.2, 127.7 (2C), 128.6 (4C), 129.3, 133.6,
134.0, 156.0, 156.7, 159.1, 193.2; HRMS (FTMS + pESI) m/z calcd. for C19H17NO3 (M)+:
308.1281; found: 308.1273.
4.2.2 2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-1-(4-methoxyphenyl)ethan-1-one (3b)
1
Yield 58%; m.p.: 145-146 °C; HPLC 97% (tR = 14.72 min); H NMR (400 MHz, DMSO-d6) δ
2.52 (s, 3H), 3.88 (s, 6H), 5.81 (s, 2H), 6.88 (s, 1H), 7.12 (d, J = 8.7 Hz, 2H), 7.35 (dd, J = 9.1,
2.9 Hz, 1H), 7.48 (d, J = 2.8 Hz, 1H), 7.79 (d, J = 9.1 Hz, 1H), 8.06 (d, J = 8.8 Hz, 2H). 13C
NMR (101 MHz, DMSO-d6) δ 25.0, 55.8, 70.5, 99.8, 102.4, 114.0 (2C), 119.8, 121.4, 127.4,
129.4, 130.3 (3C), 144.1, 156.5, 156.9, 159.4, 163.6, 191.6; HRMS (FTMS + pESI) m/z calcd.
for C20H19NO4 (M)+: 338.1387; found: 338.1401.
4.2.3 2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-1-(3-methoxyphenyl)ethan-1-one (3c)
Yield 78%; m.p.: 93-95 °C; HPLC 90% (tR = 15.80 min); 1H NMR (400 MHz, DMSO-d6) δ 2.53
(s, 3H), 3.86 (s, 3H), 3.89 (s, 3H), 5.88 (s, 2H), 6.93 (s, 1H), 7.29 – 7.31 (m, 1H), 7.35 (dd, J =
9.1, 2.9 Hz, 1H), 7.48 (d, J = 2.9 Hz, 1H), 7.53 (t, J = 7.9 Hz, 1H), 7.57 – 7.59 (m, 1H), 7.68 (dd,
13
J = 6.7, 1.2 Hz, 2H), 7.79 (d, J = 9.1 Hz, 1H). C NMR (101 MHz, DMSO-d6) δ 25.5, 55.8,
55.9, 71.0, 100.2, 103.0, 113.2, 120.2, 120.3, 120.8, 122.0, 130.0, 130.3, 136.1, 144.7, 156.7,
157.4, 159.8, 160.0, 193.8; HRMS (FTMS + pESI) m/z calcd. for C20H19NO4 (M)+: 338.1387;
found: 338.1379.

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4.2.4 2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-1-(2-methoxyphenyl)ethan-1-one (3d)
1
Yield 57%; m.p.: 140-142 °C; HPLC 90% (tR = 14.93 min); H NMR (400 MHz, DMSO-d6) δ
2.51 (s, 3H), 3.86 (s, 3H), 3.96 (s, 3H), 5.58 (s, 2H), 6.75 (s, 1H), 7.11 (t, J = 7.5 Hz, 1H), 7.26
(d, J = 8.4 Hz, 1H), 7.34 (dd, J = 9.1, 2.9 Hz, 1H), 7.41 (d, J = 2.8 Hz, 1H), 7.65 (t, J = 7.8 Hz,
1H), 7.78 (d, J = 8.9 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 24.9, 55.3, 56.0, 73.4, 99.8,
102.2, 112.6, 119.8, 120.7, 121.4, 124.6, 129.5, 129.7, 134.8, 144.2, 156.2, 156.8, 159.1, 159.5,
194.5; HRMS (FTMS + pESI) m/z calcd. for C20H19NO4 (M)+: 338.1387; found: 338.1385.
4.2.5 2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-1-(p-tolyl)ethan-1-one (3e)
1
Yield 25%; m.p.: 138-139 °C; HPLC 90% (tR = 15.01 min); H NMR (400 MHz, DMSO-d6) δ
2.52 (s, 3H), 3.88 (s, 3H), 5.84 (s, 2H), 6.91 (s, 1H), 7.35 (dd, J = 9.1, 2.9 Hz, 1H), 7.41 (d, J =
8.0 Hz, 2H), 7.48 (d, J = 2.8 Hz, 1H), 7.79 (d, J = 9.1 Hz, 1H), 7.98 (d, J = 8.2 Hz, 2H). 13C
NMR (101 MHz, DMSO-d6) δ 21.2, 25.1, 55.3, 70.3, 100.1, 102.5, 119.8, 121.4, 127.8 (2C),
129.3 (2C), 129.4, 131.8, 144.1, 144.4, 156.3, 156.8, 159.4, 192.7; HRMS (FTMS + pESI) m/z
calcd. for C20H19NO3 (M)+: 322.1438; found: 322.1451.
4.2.6 1-(4-Fluorophenyl)-2-((6-methoxy-2-methylquinolin-4-yl)oxy)ethan-1-one (3f)
1
Yield 31%; m.p.: 128-130 °C; HPLC 92% (tR = 14.69 min); H NMR (400 MHz, DMSO-d6) δ
2.53 (s, 3H), 3.89 (s, 3H), 5.88 (s, 2H), 6.95 (s, 1H), 7.36 (dd, J = 9.2, 2.7 Hz, 1H), 7.42 – 7.49
(m, 3H), 7.80 (d, J = 9.1 Hz, 1H), 8.18 (dd, J = 8.3, 5.8 Hz, 2H). 13C NMR (101 MHz, DMSO-
d6) δ 25.0, 55.3, 70.3, 99.7, 102.5, 115.9 (d, J = 22.0 Hz, 2C), 119.8, 121.5, 126.9, 129.6, 131.0

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(d, J = 5.4 Hz, 2C), 144.2, 156.3, 156.9, 159.3, 165.4 (d, J = 252.2 Hz), 192.2. HRMS (FTMS +
pESI) m/z calcd. for C19H16FNO3 (M)+: 326.1187; found: 326.1188.
4.2.7 1-(4-Chlorophenyl)-2-((6-methoxy-2-methylquinolin-4-yl)oxy)ethan-1-one (3g)
1
Yield 13%; m.p.: 134-136 °C; HPLC 93% (tR = 15.26 min); H NMR (400 MHz, DMSO-d6) δ
2.53 (s, 3H), 3.88 (s, 3H), 5.87 (s, 2H), 6.95 (s, 1H), 7.35 (dd, J = 9.1, 2.9 Hz, 1H), 7.47 (d, J =
2.9 Hz, 1H), 7.69 (d, J = 8.6 Hz, 2H), 7.79 (d, J = 9.2 Hz, 1H), 8.09 (d, J = 8.6 Hz, 2H). 13C
NMR (101 MHz, DMSO-d6) δ 25.0, 55.3, 70.4, 99.7, 102.5, 119.7, 121.5, 128.0, 129.5, 129.9
(2C), 131.9 (2C), 138.8, 144.2, 156.3, 156.9, 159.3, 192.8. HRMS (FTMS + pESI) m/z calcd. for
C19H16ClNO3 (M)+: 342.0891; found: 342.0897.
4.2.8 1-(3-Chlorophenyl)-2-((6-methoxy-2-methylquinolin-4-yl)oxy)ethan-1-one (3h)
1
Yield 63%; m.p.: 141-142 °C; HPLC 99% (tR = 14.76 min); H NMR (400 MHz, DMSO-d6) δ
2.53 (s, 3H), 3.89 (s, 3H), 5.90 (s, 2H), 6.99 (s, 1H), 7.36 (dd, J = 9.1, 2.9 Hz, 1H), 1H), 7.47 (d,
J = 2.9 Hz, 1H), 7.65 (t, J = 7.9 Hz, 1H), 7.76 – 7.84 (m, 2H), 8.10 – 8.15 (m, 1H), 8.02 (d, J =
7.7 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 25.4, 55.8, 71.0, 100.2, 103.1, 120.2, 122.1,
127.0, 128.3, 129.9, 131.3, 134.03 134.2, 136.6, 144.5, 156.8, 157.4, 159.8, 193.1; HRMS
(FTMS + pESI) m/z calcd. for C19H16ClNO3 (M)+: 342.0891; found: 342.0893.
4.2.9 1-(3,4-Dichlorophenyl)-2-((6-methoxy-2-methylquinolin-4-yl)oxy)ethan-1-one (3i)
1
Yield 53%; m.p.: 149-151 °C; HPLC 95% (tR = 15.80 min); H NMR (400 MHz, DMSO-d6) δ
2.53 (s, 3H), 3.89 (s, 3H), 5.88 (s, 2H), 6.99 (s, 1H), 7.35 (dd, J = 9.1, 2.9 Hz, 1H), 7.46 (d, J =
2.9 Hz, 1H), 7.79 (d, J = 9.1 Hz, 1H), 7.89 (d, J = 8.3 Hz, 1H), 8.01 (dd, J = 8.4, 2.0 Hz, 1H),

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8.31 (d, J = 2.0 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 24.9, 55.3, 70.5, 99.7, 102.5, 119.7,
121.6, 129.7, 129.4, 130.0, 131.2, 131.9, 136.6, 144.0, 156.3, 156.9, 159.2, 191.8; HRMS
(FTMS + pESI) m/z calcd. for C19H15Cl2NO3 (M)+: 376.0502; found: 376.0508.
4.2.10 1-(4-Bromophenyl)-2-((6-methoxy-2-methylquinolin-4-yl)oxy)ethan-1-one (3j)
1
Yield 55%; m.p.: 134-136 °C; HPLC 91% (tR = 15.37 min); H NMR (400 MHz, DMSO-d6) δ
2.53 (s, 3H), 3.89 (s, 3H), 5.86 (s, 2H), 6.95 (s, 1H), 7.35 (dd, J = 9.1, 2.9 Hz, 1H), 7.47 (d, J =
13
2.9 Hz, 1H), 7.77 – 7.85 (m, 3H), 8.01 (d, J = 8.6 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ
25.0, 55.3, 70.4, 99.7, 102.5, 119.7, 121.5, 128.0, 129.5, 129.9 (2C), 131.9 (2C), 133.2, 144.2,
156.3, 156.9, 159.3, 192.8; HRMS (FTMS + pESI) m/z calcd. for C19H16BrNO3 (M)+: 386.0386;
found: 386.0391.
4.2.11 1-(4-Isobutylphenyl)-2-((6-methoxy-2-methylquinolin-4-yl)oxy)ethan-1-one (3k)
1
Yield 90%; m.p.: 130-132 °C; HPLC 96% (tR = 15.99 min); H NMR (400 MHz, DMSO-d6) δ
0.88 (s, 3H), 0.89 (s, 3H), 1.85 – 1.93 (m, 1H), 2.52 (s, 3H), 2.56 (d, J = 7.2 Hz, 2H), 3.88 (s,
3H), 5.86 (s, 2H), 6.92 (s, 2H), 7.35 (dd, J = 9.1, 2.9 Hz, 1H), 7.35 – 7.42 (m, 2H), 7.48 (d, J =
13
2.9 Hz, 1H), 7.79 (d, J = 9.1 Hz, 1H), 7.97 – 8.04 (m, 2H). C NMR (101 MHz, DMSO-d6) δ
22.6 (2C), 25.5, 30.0, 45.0, 55.8, 70.8, 100.2, 102.9, 120.3, 122.0, 128.4 (2C), 129.8 (2C), 130.0,
132.6, 144.7, 148.4, 156.7, 157.4, 159.9, 193.5; HRMS (FTMS + pESI) m/z calcd. for
C23H25NO3 (M)+: 364.1907; found: 364.1894.
4.2.12 1-([1,1'-Biphenyl]-4-yl)-2-((6-methoxy-2-methylquinolin-4-yl)oxy)ethan-1-one (3l)

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Yield 38%; m.p.: 160-162 °C; HPLC 97% (tR = 16.25 min); H NMR (400 MHz, DMSO-d6) δ
2.54 (s, 3H), 3.89 (s, 3H), 5.93 (s, 2H), 6.97 (s, 1H), 7.36 (dd, J = 9.2, 2.9 Hz, 1H), 7.44 – 7.50
(m, 4H), 7.48 – 7.58 (m, 3H), 7.75 – 7.84 (m, 3H), 7.87 – 7.94 (m, 2H), 8.14 – 8.21 (m, 2H). 13C
NMR (101 MHz, DMSO-d6) δ 25.0, 55.3, 70.4, 99.7, 102.5, 119.8, 121.5, 126.9 (2C), 127.0
(2C), 128.5, 128.7 (2C), 129.1 (2C), 129.5, 133.0, 138.8, 144.2, 145.3, 156.3, 156.9, 159.4,
193.0. HRMS (FTMS + pESI) m/z calcd. for C25H21NO3 (M)+: 384.1594; found: 384.1596.
4.2.13 2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-1-phenylpropan-1-one (3m)
1
Yield 71%; m.p.: 142-144 °C; HPLC 94% (tR = 14.58min); H NMR (400 MHz, DMSO-d6) δ
1.71 (d, J = 6.8 Hz, 3H), 2.48 (s, 3H), 3.87 (s, 3H), 6.30 (q, J = 6.7 Hz, 1H), 6.75 (d, J = 1.2 Hz,
1H), 7.35 (ddd, J = 9.2, 2.9, 1.1 Hz, 1H), 7.43 (dd, J = 2.9, 1.1 Hz, 1H), 7.57 – 7.63 (m, 2H),
7.69 – 7.74 (m, 1H), 7.77 (dd, J = 9.1, 1.1 Hz, 1H), 8.12 (dd, J = 8.4, 1.2 Hz, 2H). 13C NMR (101
MHz, DMSO-d6) δ 18.1, 25.0, 55.3, 75.1, 99.9, 102.5, 119.9, 121.5, 128.5, 129.0 (2C), 129.5
(2C), 133.95, 134.01, 144.2, 156.3, 156.7, 158.6, 197.2; HRMS (FTMS + pESI) m/z calcd. for
C20H19NO3 (M)+: 322.1438; found: 322.1433.
4.2.14 4-(Benzyloxy)-6-methoxy-2-methylquinoline (4a)
1
Yield 49%; m.p.: 140-142 °C; HPLC 90% (tR = 14.59 min); H NMR (400 MHz, DMSO-d6) δ
2.56 (s, 3H), 3.85 (s, 3H), 5.38 (s, 2H), 7.00 (s, 1H), 7.34 (dd, J = 9.1, 2.9 Hz, 1H), 7.41 – 7.49
(m, 2H), 7.57 (dt, J = 6.3, 1.4 Hz, 2H), 7.78 (d, J = 9.1 Hz, 1H); 13C NMR (101 MHz, DMSO-d6)
δ 25.0, 55.3, 69.5, 99.7, 102.4, 119.9, 121.2, 127.5 (2C), 128.0 (2C), 128.5, 129.6, 136.3, 144.1,
156.2, 156.9, 159.5; HRMS (FTMS + pESI) m/z calcd. for C18H17NO2 (M)+: 280.1332; found:
280.1324.

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4.2.15 6-Methoxy-4-((3-methoxybenzyl)oxy)-2-methylquinoline (4b)
1
Yield 86%; m.p.: 103-104 °C; HPLC 98% (tR = 14.63 min); H NMR (400 MHz, DMSO-d6) δ
2.56 (s, 3H), 3.79 (s, 3H), 3.86 (s, 3H), 5.35 (s, 2H), 6.91 – 6.97 (m, 1H), 6.99 (m, 1H), 7.10 –
13
7.16 (m, 2H), 7.33 – 7.43 (m, 3H), 7.79 (d, J = 9.1 Hz, 1H). C NMR (101 MHz, DMSO-d6) δ
25.6, 55.6, 55.8, 69.9, 100.2, 103.0, 113.5, 114.0, 120.1, 120.4, 121.9, 130.1, 130.3, 138.4, 144.6,
156.8, 157.6, 159.9, 160.1; HRMS (FTMS + pESI) m/z calcd. for C19H19NO3 (M)+: 310.1438;
found: 310.1452.
4.2.16 4-((3,5-Dimethoxybenzyl)oxy)-6-methoxy-2-methylquinoline (4c)
1
Yield 88%; m.p.: 106-108 °C; HPLC 93% (tR = 15.04 min); H NMR (400 MHz, DMSO-d6) δ
2.57 (s, 3H), 3.77 (s, 6H), 3.87 (s, 3H), 5.32 (s, 2H), 6.50 (d, J = 2.2 Hz, 1H), 6.72 (d, J = 2.2 Hz,
2H), 7.36 (dd, J = 9.1, 2.9 Hz, 1H), 7.00 (s, 1H), 7.41 (d, J = 2.8 Hz, 1H), 7.79 (d, J = 9.1 Hz,
13
1H). C NMR (101 MHz, DMSO-d6) δ 25.3, 55.6 (3C), 70.0, 100.1, 100.2, 103.0, 105.7 (2C),
120.4, 122.0, 129.7, 139.0, 144.1, 156.8, 157.4, 160.2, 161.1 (2C); HRMS (FTMS + pESI) m/z
calcd. for C20H21NO4 (M)+: 340.1543; found: 340.1534.
4.2.17 4-((4-Fluorobenzyl)oxy)-6-methoxy-2-methylquinoline (4d)
1
Yield 67%; m.p.: 113-114 °C; HPLC 96% (tR = 15.01 min); H NMR (400 MHz, DMSO-d6) δ
2.58 (s, 3H), 3.85 (s, 3H), 5.35 (s, 2H), 6.98 (s, 1H), 7.28 (t, J = 8.9 Hz, 2H), 7.34 (dd, J = 9.0,
2.9 Hz, 1H), 7.38 (d, J = 2.7 Hz, 1H), 7.63 (dd, J = 8.6, 5.6 Hz, 2H), 7.80 (d, J = 9.0 Hz, 1H);
13C NMR (101 MHz, DMSO-d6) δ 25.1, 55.3 (d, J = 1.8 Hz), 68.9, 99.8, 102.5, 115.5 (d, J =
21.4 Hz, 2C), 119.9, 121.3, 129.6, 129.9 (d, J = 8.3 Hz, 2C), 132.6 (d, J = 3.0 Hz), 144.1,

25
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495
496
497
498
499
156.31, 157.1, 159.5, 162.0 (d, J = 243.9 Hz); HRMS (FTMS + pESI) m/z calcd. for
C18H16FNO2 (M)+: 298.1238; found: 298.1251.
4.2.18 4-((3-Fluorobenzyl)oxy)-6-methoxy-2-methylquinoline (4e)
Yield 84%; m.p.: 79-80 °C; HPLC 98% (tR = 15.80 min); 1H NMR (400 MHz, DMSO-d6) δ 2.57
(s, 3H), 3.87 (s, 3H), 5.40 (s, 2H), 6.98 (s, 1H), 7.22 (td, J = 8.4, 2.0 Hz, 1H), 7.33 – 7.44 (m,
4H), 7.46 – 7.54 (m, 1H), 7.80 (d, J = 9.1 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 25.5,
55.8, 69.2 (d, J = 2.1 Hz), 100.2, 103.0, 114.7 (d, J = 22.1 Hz), 115.3 (d, J = 20.9 Hz), 120.3,
121.9, 123.9 (d, J = 2.8 Hz), 130.0, 131.2 (d, J = 8.4 Hz), 139.7 (d, J = 7.5 Hz), 144.6, 156.8,
157.5, 159.8, 162.7 (d, J = 243.7 Hz), 163.9; HRMS (FTMS + pESI) m/z calcd. for C18H16FNO2
(M)+: 298.1238; found: 298.1251.
4.2.19 4-((2-Fluorobenzyl)oxy)-6-methoxy-2-methylquinoline (4f)
1
Yield 52%; m.p.: 139-141 °C; HPLC 90% (tR = 14.59 min); H NMR (400 MHz, DMSO-d6) δ
2.58 (s, 3H), 3.83 (s, 3H), 5.42 (s, 2H), 7.07 (s, 1H),7.26 – 7.38 (m, 4H), 7.43 – 7.52 (m, 1H),
7.68 (td, J = 7.5, 1.7 Hz, 1H), 7.75 – 7.83 (m, 1H); 13C NMR (101 MHz, DMSO-d6) δ 24.9, 55.2,
64.0 (d, J = 3.8 Hz), 99.7, 102.3, 115.4 (d, J = 20.9 Hz), 119.7, 121.1, 123.0 (d, J = 14.3 Hz),
124.6 (d, J = 3.5 Hz), 129.5, 130.3 (d, J = 4.0 Hz), 130.5 (d, J = 8.3 Hz), 144.1, 156.2, 156.9,
159.3, 160.3 (d, J = 246.4 Hz); HRMS (FTMS + pESI) m/z calcd. for C18H16FNO2 (M)+:
298.1238; found: 298.1229.
4.2.20 4-((3,4-Difluorobenzyl)oxy)-6-methoxy-2-methylquinoline (4g)

26
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500
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502
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504
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513
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517
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521
Yield 90%; m.p.: 119-121 °C; HPLC 95% (tR = 15.33 min); H NMR (400 MHz, DMSO-d6) δ
2.57 (s, 3H), 3.86 (s, 3H), 5.36 (s, 2H), 6.99 (s, 1H), 7.34 (dd, J = 9.1, 2.9 Hz, 1H), 7.39 (d, J =
2.8 Hz, 1H), 7.39 – 7.47 (m, 1H), 7.46 – 7.55 (m, 1H), 7.65 (ddd, J = 11.4, 7.8, 2.0 Hz, 1H), 7.78
(d, J = 9.1 Hz, 1H); 13C NMR (101 MHz, DMSO-d6) δ 25.0, 55.3, 68.3, 99.8, 102.5, 116.8 (dd, J
= 17.5, 3.9 Hz), 117.7 (d, J = 17.2 Hz), 119.8, 121.4, 124.6 (dd, J = 6.8, 3.5 Hz), 129.55, 134.06
(dd, J = 6.1, 3.8 Hz), 144.09, 148.07 (dd, J = 24.7, 12.6 Hz), 150.52 (dd, J = 24.6, 12.5 Hz),
156.96, 157.80 (d, J = 301.4 Hz, 2C); HRMS (FTMS + pESI) m/z calcd. for C18H15F2NO2 (M)+:
316.1144; found: 316.1144.
4.2.21 4-((4-Chlorobenzyl)oxy)-6-methoxy-2-methylquinoline (4h)
1
Yield 73%; m.p.: 159-160 °C; HPLC 92% (tR = 15.61 min); H NMR (400 MHz, DMSO-d6) δ
2.57 (s, 3H), 3.86 (s, 3H), 5.35 (s, 2H), 6.95 (s, 1H), 7.34 (dd, J = 9.1, 2.5 Hz, 1H), 7.39 (s, 1H),
7.50 (d, J = 8.3 Hz, 2H), 7.59 (d, J = 8.2 Hz, 2H), 7.80 (d, J = 9.0 Hz, 1H); 13C NMR (101 MHz,
DMSO-d6) δ 25.1, 55.4, 68.8, 99.8, 102.6, 119.9, 121.4, 128.7 (2C), 129.5, 129.6 (2C), 132.7,
135.4, 144.1, 156.3, 157.1, 159.4; HRMS (FTMS + pESI) m/z calcd. for C18H16ClNO2 (M)+:
314.0942; found: 314.0956.
4.2.22 4-((3-Chlorobenzyl)oxy)-6-methoxy-2-methylquinoline (4i)
1
Yield 90%; m.p.: 101-102 °C; HPLC 93% (tR = 15.65 min); H NMR (400 MHz, DMSO-d6) δ
2.57 (s, 3H), 3.87 (s, 3H), 5.40 (s, 2H), 6.99 (s, 1H), 7.35 (dd, J = 9.1, 2.9 Hz, 1H), 7.40 (d, J =
2.8 Hz, 1H), 7.41 – 7.51 (m, 2H), 7.54 (d, J = 7.2 Hz, 1H), 7.62 (s, 1H), 7.79 (d, J = 9.1 Hz, 1H);
13C NMR (101 MHz, DMSO-d6) δ 25.0, 55.3, 68.7, 99.7, 102.5, 119.8, 121.4, 126.1, 127.3,

27
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523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
128.0, 129.4, 130.6, 133.2, 138.8, 143.9, 156.3, 157.0, 159.4; HRMS (FTMS + pESI) m/z calcd.
for C18H16ClNO2 (M)+: 314.0942; found: 319.0945.
4.2.23 4-((2-Chlorobenzyl)oxy)-6-methoxy-2-methylquinoline (4j)
Yield 86%; m.p.: 86-87 °C; HPLC 94% (tR = 14.92 min); 1H NMR (400 MHz, DMSO-d6) δ 2.56
(s, 3H), 3.85 (s, 3H), 5.38 (s, 2H, CH2), 7.00 (s, 1H), 7.34 (dd, J = 9.1, 2.9 Hz, 1H), 7.37 – 7.40
(m, 2H), 7.41 – 7.49 (m, 2H), 7.57 (d, J = 6.8 Hz, 1H), 7.78 (d, J = 9.1 Hz, 1H); 13C NMR (101
MHz, DMSO-d6) δ 25.0, 55.2, 67.2, 99.7, 102.4, 119.8, 121.2, 127.4, 129.5, 129.6, 130.0, 130.1,
132.7, 133.5, 144.1, 156.2, 157.0, 159.3; HRMS (FTMS + pESI) m/z calcd. for C18H16ClNO2
(M)+: 314.0942; found: 314.0935.
4.2.24 4-((3,4-Dichlorobenzyl)oxy)-6-methoxy-2-methylquinoline (4k)
1
Yield 63%; m.p.: 141-143 °C; HPLC 97% (tR = 16.21 min); H NMR (400 MHz, DMSO-d6) δ
2.56 (s, 3H), 3.85 (s, 3H), 5.38 (s, 2H), 7.00 (s, 1H), 7.34 (dd, J = 9.1, 2.9 Hz, 1H), 7.39 (d, J =
13
2.9 Hz, 1H), 7.45 (t, J = 7.3 Hz, 2H), 7.53 – 7.59 (m, 1H), 7.78 (d, J = 9.1 Hz, 1H); C NMR
(101 MHz, DMSO-d6) δ 24.8, 55.1, 67.8, 99.5, 102.2, 119.5, 121.1, 127.5, 129.2, 129.4, 130.4,
130.6, 131.0, 137.3, 143.9, 156.1, 156.7, 159.0; HRMS (FTMS + pESI) m/z calcd. for
C18H15Cl2NO2 (M)+: 348.0553; found: 348.0546.
4.2.25 4-((2,3-Dichlorobenzyl)oxy)-6-methoxy-2-methylquinoline (4l)
1
Yield 93%; m.p.: 127-129 °C; HPLC 99% (tR = 15.73 min); H NMR (400 MHz, DMSO-d6) δ
2.59 (s, 3H), 3.86 (s, 3H), 5.47 (s, 2H), 7.06 (s, 1H), 7.34 – 7.42 (m, 2H), 7.46 (t, J = 7.9 Hz,
1H), 7.66 – 7.71 (m, 2H), 7.77 – 7.84 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 25.3, 55.9,

28
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567
68.1, 100.3, 103.1, 120.2, 122.1, 128.7, 129.0, 129.7, 130.8, 131.0, 132.6, 136.6, 144.1, 156.9,
157.5, 159.9; HRMS (FTMS + pESI) m/z calcd. for C18H15Cl2NO2 (M)+: 348.0553; found:
348.0541.
4.2.26 4-((4-Bromo-3-chlorobenzyl)oxy)-6-methoxy-2-methylquinoline (4m)
1
Yield 96%; m.p.: 150-152 °C; HPLC 99% (tR = 15.99 min); H NMR (400 MHz, DMSO-d6) δ
2.57 (s, 3H), 3.87 (s, 3H), 5.37 (s, 2H), 6.97 (s, 1H), 7.35 (dd, J = 9.0, 2.8 Hz, 1H), 7.39 (d, J =
13
2.9 Hz, 1H), 7.47 (dd, J = 8.2, 2.1 Hz, 2H), 7.76 – 7.86 (m, 3H). C NMR (101 MHz, DMSO-
d6) δ 25.4, 55.8, 68.5, 100.2, 102.9, 120.2, 121.4, 121.9, 128.3, 129.8, 129.9, 133.7, 134.5, 138.5,
144.4, 156.8, 157.4, 159.8; HRMS (FTMS + pESI) m/z calcd. for C18H15BrClNO2 (M)+:
392.0047; found: 392.0035.
4.2.27 4-((4-Bromobenzyl)oxy)-6-methoxy-2-methylquinoline (4n)
1
Yield 78%; m.p.: 173-174 °C; HPLC 98% (tR = 15.72 min); H NMR (400 MHz, DMSO-d6) δ
2.55 (s, 3H), 3.86 (s, 3H), 5.36 (s, 2H), 6.98 (s, 1H), 7.34 (dd, J = 9.1, 2.9 Hz, 1H), 7.38 (d, J =
13
2.8 Hz, 1H), 7.52 (d, J = 8.3 Hz, 2H), 7.60 – 7.67 (m, 2H), 7.78 (d, J = 9.1 Hz, 1H). C NMR
(101 MHz, DMSO-d6) δ 24.9, 55.2, 68.7, 99.9, 102.4, 119.7, 121.0, 121.1, 129.4, 129.5 (2C),
131.3 (2C), 135.7, 144.0, 156.2, 156.8, 159.3; HRMS (FTMS + pESI) m/z calcd. for
C18H16BrNO2 (M)+: 358.0437; found: 358.0455.
4.2.28 4-((3-Bromobenzyl)oxy)-6-methoxy-2-methylquinoline (4o)
Yield 95%; m.p.: 111-112 °C; HPLC 97% (tR = 15.68 min); ); 1H NMR (400 MHz, DMSO-d6) δ
2.56 (s, 3H), 3.87 (s, 3H), 5.39 (s, 2H), 6.98 (s, 1H), 7.35 (dd, J = 9.1, 2.9 Hz, 1H), 7.39 (d, J =