New insights into the SAR and drug combination synergy of 2-(quinolin-4-yloxy)acetamides against Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.ejmech.2016.11.048

2-(Quinolin-4-yloxy)acetamides have been described as potent and selective in vitro inhibitors of Mycobacterium tuberculosis (Mtb) growth. Herein, a new series of optimized compounds were found to demonstrate highly potent antitubercular activity, with minimum inhibitory concentration (MIC) values against drug-susceptible and drug-resistant Mycobacterium tuberculosis strains in the submicromolar range. Furthermore, the most active compounds had no apparent toxicity to mammalian cells, and they showed intracellular activities similar to those of isoniazid and rifampin in a macrophage model of Mtb infection. Use of the checkerboard method to investigate the association profiles of lead compounds with first- and second-line antituberculosis drugs showed that 2-(quinolin-4-yloxy)acetamides have a synergistic effect with rifampin. Ultimately, the good permeability, moderate rates of metabolism and low risk of drug-drug interactions displayed by some of the synthesized compounds indicate that 2-(quinolin-4-yloxy)acetamides may yield candidates to use in the development of novel alternative therapeutics for tuberculosis treatment.

Full text of DOI:10.1016/j.ejmech.2016.11.048

Accepted Manuscript
New insights into the SAR and drug combination synergy of 2-(quinolin-4-
yloxy)acetamides against Mycobacterium tuberculosis
Bruno Couto Giacobbo, Kenia Pissinate, Valnês Rodrigues-Junior, Anne Drumond
Villela, Estêvão Silveira Grams, Bruno Lopes Abbadi, Fernanda Teixeira Subtil,
Nathalia Sperotto, Rogério Valim Trindade, Davi Fernando Back, Maria Martha
Campos, Luiz Augusto Basso, Pablo Machado, Diógenes Santiago Santos
PII:
S0223-5234(16)30986-2
10.1016/j.ejmech.2016.11.048
EJMECH 9081
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 October 2016
Revised Date: 20 November 2016
Accepted Date: 21 November 2016
Please cite this article as: B.C. Giacobbo, K. Pissinate, V. Rodrigues-Junior, A.D. Villela, E.S. Grams,
B.L. Abbadi, F.T. Subtil, N. Sperotto, R.V. Trindade, D.F. Back, M.M. Campos, L.A. Basso, P. Machado,
D.S. Santos, New insights into the SAR and drug combination synergy of 2-(quinolin-4-yloxy)acetamides
against Mycobacterium tuberculosis, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.11.048.
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ACCEPTED MANUSCRIPT

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New insights into the SAR and drug combination synergy of 2-(quinolin-4-
yloxy)acetamides against Mycobacterium tuberculosis
Bruno Couto Giacobboa,b, Kenia Pissinatea, Valnês Rodrigues-Juniora, Anne Drumond Villelaa,
Estêvão Silveira Gramsa, Bruno Lopes Abbadia,b, Fernanda Teixeira Subtila,b, Nathalia
Sperottoa,c, Rogério Valim Trindadea,b, Davi Fernando Backd, Maria Martha Camposa-c, Luiz
Augusto Bassoa-c, Pablo Machado*,a,b and Diógenes Santiago Santos*,a,b
aInstituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia
Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul, 90619-900,
Porto Alegre, Rio Grande do Sul, Brazil
bPrograma de Pós-graduação em Biologia Celular e Molecular, Pontifícia Universidade Católica
do Rio Grande do Sul, 90619-900, Porto Alegre, Rio Grande do Sul, Brazil
cPrograma de Pós-Graduação em Medicina e Ciências da Saúde, Pontifícia Universidade
Católica do Rio Grande do Sul, 90619-900, Porto Alegre, Rio Grande do Sul, Brazil
dDepartamento de Química, Laboratório de Materiais Inorgânicos, Universidade Federal de
Santa Maria, 97105-900, Santa Maria, RS, Brazil
*Corresponding authors. Phone/Fax: +55 51 3320 3629
E-mail addresses: pablo.machado@pucrs.br (P. Machado); diogenes@pucrs.br (D.S. Santos)

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Abstract
2-(Quinolin-4-yloxy)acetamides have been described as potent and selective in vitro inhibitors of
Mycobacterium tuberculosis (Mtb) growth. Herein, a new series of optimized compounds were
found to demonstrate highly potent antitubercular activity, with minimum inhibitory
concentration (MIC) values against drug-susceptible and drug-resistant Mycobacterium
tuberculosis strains in the submicromolar range. Furthermore, the most active compounds had no
apparent toxicity to mammalian cells, and they showed intracellular activities similar to those of
isoniazid and rifampin in a macrophage model of Mtb infection. Use of the checkerboard method
to investigate the association profiles of lead compounds with first- and second-line
antituberculosis drugs showed that 2-(quinolin-4-yloxy)acetamides have a synergistic effect with
rifampin. Ultimately, the good permeability, moderate rates of metabolism and low risk of drug-
drug interactions displayed by some of the synthesized compounds indicate that 2-(quinolin-4-
yloxy)acetamides may yield candidates to use in the development of novel alternative
therapeutics for tuberculosis treatment.
KEYWORDS: Tuberculosis, intracellular activity, synergism, drug-resistant strains

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1. Introduction
Although it has been regarded by the World Health Organization (WHO) as a global public
health emergency since the early 1990s, human tuberculosis (TB) still claims thousands of lives
annually [1]. This infectious disease is caused mainly by Mycobacterium tuberculosis (Mtb), and
it has been reported as being among the top current causes of death resulting from a single
pathogen. According to the WHO, 9.6 million new cases of the disease and 1.5 million deaths
were reported worldwide in 2014 [1]. Multidrug-resistant TB (MDR-TB) and extensively drug-
resistant TB (XDR-TB) [2], HIV coinfection [3], the limited effectiveness of the Mycobacterium
bovis bacillus Calmette-Guérin (BCG) vaccine [4], and the high number of individuals infected
with latent or dormant bacilli [5] have strongly contributed to elevated incidences and mortality
from TB. Further complicating this scenario, an increasing number of cases of infections are
caused by drug-resistant strains for which treatment options are restricted and suboptimal [1,6].
Together, these points highlight the paramount need for novel drugs, ideally with innovative
mechanisms of action that would be effective against drug-resistant strains. Within this context,
quinoline-containing compounds have been used in several drug discovery campaigns with the
aim of uncovering novel antimycobacterial compounds [7]. In addition, the quinoline scaffold is
found in clinically useful anti-TB drugs such as bedaquiline [8] and some fluoroquinolones [9].
In line with research into new antitubercular candidates bearing a quinoline ring, we have
recently described the structural optimization of a previously reported 2-(quinolin-4-
yloxy)acetamide 1 [10] (Figure 1), with some encouraging results [11]. Compound 1 was
obtained from phenotypic screening together with a set of 177 lead anti-TB compounds [10]. Our
optimization efforts yielded hit 2 (Figure 1), which was 8.8 times more potent than 1, with a
minimum inhibitory concentration (MIC) of 0.05 µM against M. tuberculosis H37Rv [11]. In

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addition, compound 2 exhibited inhibitory activity against drug-resistant Mtb strains. Recently,
and independently, two research groups have described their findings about this chemical class
[12-13]. In one of these studies, the antimycobacterial activity of 2-(quinolin-4-yloxy)acetamide
derivatives against Mtb strains carrying cytochrome bd oxidase deletion and single-nucleotide
polymorphism at the qcrB gene have suggested the menaquinol cytochrome c oxidoreductase
(bc1 complex) as a possible molecular target [12].
Therefore, as part of our ongoing project to obtain compounds with improved activity against
drug-sensitive and drug-resistant Mtb strains, a new series of 2-(quinolin-4-yloxy)acetamides
and some of their analogs was synthesized and assayed against M. tuberculosis H37Rv. First, the
structural requirements for the potency of compounds 1 and 2 were evaluated by modifying the
substituents at the 2-, 3-, 4- and 6-positions of the quinoline ring. Additionally, analogs of
compound 2 were designed by molecular simplification. Thereafter, the most active structures
against M. tuberculosis H37Rv were tested against clinically isolated drug-resistant strains, and
the viability of mammalian cells after exposure to the compounds was determined. Finally, the
intracellular activity of the compounds in a macrophage model of Mtb infection, drug
combination studies, and some in vitro ADME parameters were also shown.
2. Results and Discussion
The synthesis of 2-(quinolin-4-yloxy)acetamides 6a-i was performed using three steps. First, 4-
hydroxyquinolines 5a-j were prepared using classical Conrad-Limpach reactions between β-
ketoesters 3a-f and anilines 4a-f (Scheme 1) through one-pot procedures [14]. These reactions
were performed in the presence of magnesium sulfate and acetic acid using refluxing ethanol as a

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solvent for 16 h to yield β-anilinoacrylate intermediates, which were not isolated or
characterized. The thermal cyclization of the intermediates occurred after the crude products
were heated using Dowtherm A® at 230–250 °C for 15 min to afford 4-hydroxyquinolines 5a-f
with yields of 29–75%. By contrast, halogenated 4-hydroxyquinolines 5g-j were synthesized
through cyclocondensation reactions between β-ketoester 3a and anilines 4c-f in the presence of
polyphosphoric acid (PPA) at 80–120 °C for 6 hours with yields of 17–56% (Scheme 1).
Notably, even after several attempts, compounds 5a-f were not obtained using polyphosphoric
acid-mediated reactions. Moreover, according to spectroscopic data, no signs of Knorr products
(2-hydroxyquinolines) were observed when either cyclocondensation method was used. In the
second reaction step, 2-bromo-N-(2-methoxyphenyl)acetamide was obtained by the acylation of
2-methoxyaniline, using bromoacetyl chloride, according to a previously reported method [15-
16]. Finally, the O-alkylation of 4-hydroxyquinolines 5b-j with 2-bromo-N-(2-
methoxyphenyl)acetamide using potassium carbonate and N,N-dimethylformamide (DMF) as the
solvent afforded 2-(quinolin-4-yloxy)acetamides 6a-i with 25–85% yields.
To evaluate the effect of exchanging the oxygen at the 4-position of the quinoline ring with
nitrogen, the 2-(quinolin-4-ylamino)acetamides 9a-b were synthesized (Scheme 2). The products
were obtained after the alkylation reaction of 4-aminoquinoline 7 with 2-bromo-N-
arylacetamides 8a-b, which were prepared as described previously [15-16]. The reaction was
performed in the presence of sodium hydride that had been dissolved in dry DMSO under an
argon atmosphere. The amino derivative compounds 9a-b were isolated at yields of 28–31%.

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Structural analogs of compound 2 were synthesized using the 4-hydroxyquinoline 5a (Scheme
3). The alkylation reaction of 5a using 2-bromo-1-(naphthalen-2-yl)ethan-1-one in the presence
of an excess of potassium carbonate with DMF as the solvent afforded quinoline 10 at a 61%
yield. The same reaction utilizing 2-(bromomethyl)naphthalene as the alkylating agent yielded
compound 11 at a 76% yield. Finally, 2-(quinolin-4-yloxy)acetamides 12a-n were prepared by
the O-alkylation of 4-hydroxyquinoline 5a (Scheme 3). The synthetic protocol was the same as
that described previously, and it used potassium carbonate as a base and DMF as the solvent to
obtain the targeted molecules with 31–99% yields. All of the synthesized compounds showed the
expected structures, on the basis of their analytical data (Supporting Information). In addition,
the structure of 2-(quinolin-4-yloxy)acetamide 12n in its solid state was determined by single-
crystal diffraction (Figure 2) [17]. The crystal data and details of the data collection and
structure refinement are shown in Table S2 (Supporting Information).
The synthesized quinoline-based compounds were evaluated in a whole-cell assay against M.
tuberculosis H37Rv using the anti-TB drug isoniazid (INH) as a positive control [10,18]. In
general, alkyl lipophilic substituents that were directly attached to the N-arylacetamide group
yielded compounds with potent antimycobacterial action, whereas increasing the alkyl chain
length at the 2-position or changing the methoxy group attached at the 6-position of the quinoline
ring to yield compounds with decreased activity (Table 1). In fact, exchanging the methyl group
at the 2-position of the quinoline ring (R1) with an ethyl group decreased the potency by almost
two-fold. The inhibitory activity of compound 1 (R1 = Me) in our experimental conditions was
0.44 µM whereas 2-(quinolin-4-yloxy)acetamide 6a (R1 = Et) exhibited an MIC of 0.86 µM. In
addition, the replacement of the alkyl side chain R1 with propyl or isopropyl groups in

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compounds 6b and 6c, respectively, greatly decreased the activity, and both molecules exhibited
MIC values > 26.3 µM.
Similarly, the presence of the bulky lipophilic benzyl group at the 3-position of the quinoline
ring (R2) in structure 6d decreased the antitubercular potency in vitro (MIC > 22.6 µM). 2-
(Quinolin-4-yloxy)acetamides 6e-i were synthesized to evaluate whether increasing the alkyl
chain length as well as the presence of a halogen at the 6-position of the quinoline ring (R3)
would improve the activity. As previously reported, the presence of the methoxy group at this
position is important for potency because its replacement by hydrogen significantly decreased
the activity [11-13]. The ethoxy-substituted compound 6e showed an MIC value of 1.75 µM,
which was nearly four-fold less active than 1 (R3 = MeO). Among the halogenated compounds,
bromo-substituted 6h was the most effective structure with an MIC of 0.78 µM, while 2-
(quinolin-4-yloxy)acetamides 6f (R3 = F) and 6g (R3 = Cl) exhibited MICs of 14.7 µM and 1.75
µM, respectively. Notably, the order of potency followed increases in both the lipophilicity
(Table 1) and atomic radii of the evaluated halogen atoms. Compound 6i, bearing a
trifluoromethyl group at R3, also showed decreased activity, with an MIC > 25.6 µM, thus once
again showing the necessity of the methoxy group at the 6-position of the heterocycle for potent
activity. Similar trends have been obtained for structurally related compounds [12-13].
In the next round of our SAR study, the substituents that were attached at the 2-, 3-, and 6-
positions of the quinoline rings of compounds 1 and 2 were maintained while the oxygen at the
4-position was replaced by a nitrogen. This modification produced molecules with increased
polarity because of the presence of the NH group, which can act as a hydrogen bonding donor-
acceptor pair at a putative target. 2-(Quinolin-4-ylamino)acetamides 9a and 9b presented MIC

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values > 28.5 µM and 6.73 µM, respectively, thus indicating the importance of the oxygen at the
4-position for the anti-Tb activity of the synthesized compounds.
To assess the importance of the amide group in the antimycobacterial activity, analogs 10 and 11
were synthesized, and their inhibitory action against Mtb growth was determined. Quinoline 10,
which retained the ketone group, exhibited an MIC of 1.75 µM, which was 35-fold less active
than compound 2. A similar MIC value of 1.90 µM was observed for molecule 11, which does
not encompass the entire amide group. In evaluating the importance of the hydrogen attached to
the amide nitrogen, the activity elicited by 2-(quinolin-4-yloxy)acetamides 12a-c indicated that
secondary amides can be detrimental for the potent anti-Tb activity of this chemical class, given
that all three compounds were devoid of activity at the tested concentrations (MIC > 25.1 µM). It
is worth mentioning that similar findings have corroborated the importance of the amide group in
obtaining potent in vitro antimycobacterial activity [13]. Among other data, the bioisosteric
replacement of the amide group targeting structures endowed with increased metabolic stability
yielded less active compounds [13].
In focusing our SAR study on hit compound 2, structures 12d-h were proposed and synthesized
for further evaluation against the M. tuberculosis H37Rv strain. First, the 2-tetrahydronaphthyl
derivative 12d that attached directly at the arylamide group was proposed to evaluate the
necessity of an aromatic and planar 2-naphthyl group at this position for the activity of 2-
(quinolin-4-yloxy)acetamide 2. Notably, when the 2-naphthyl group was replaced with a 2-
tetrahydronaphthyl substituent, the potency was maintained, and the MIC value of both
molecules (2 and 12d) was 0.05 µM. This finding indicated that the potency of compound 2 does
not appear to be dependent on the aromaticity and planarity of the 2-naphthyl ring. By reducing
the hydrophobicity of structure 12d by using a 2,3-dihydro-1H-inden-5-yl group, the activity of

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compound 12e was also maintained in the submicromolar range, with an MIC of 0.05 µM. By
contrast, the 1-tetrahydronaphthyl derivative 12f was almost 17-fold less active than its analog
12d (0.05 µM), which had an MIC of 0.83 µM. Chiral substituents attached at the N-arylamide
group of compounds 12g and 12h yielded molecules with decreased activity compared with that
of compound 2. Interestingly, the S stereoisomer was eight times more potent against the bacilli
than the R stereoisomer, with observed MIC values of 1.66 µM for 12g and 13.28 µM for
compound 12h.
Finally, structures 12i-n were proposed, with the goals of molecular simplification of 2-
(quinolin-4-yloxy)acetamide 12d and mimicking the hydrophobic six-membered ring attached at
the benzene group. Similar MIC values were obtained for the 3,4-dimethyl-substituted
compound 12i and the 4-methyl-substituted derivative 12j. Compound 12i had an MIC of 0.11
µM, whereas 2-(quinolin-4-yloxy)acetamide 12j inhibited Mtb growth at a minimal
concentration of 0.12 µM. Increasing the alkyl chain length with 4-ethyl and 4-propyl groups
attached to the benzene ring of compounds 12k and 12l, respectively, afforded molecules with a
potency of a similar order to that obtained for compound 2. Structures 12k and 12l exhibited
MICs of 0.06 and 0.05 µM, respectively. By contrast, the in vitro antimycobacterial activity of
butyl-substituted 12m was only half as effective as its counterparts (0.10 µM). Notably, when a
pentyl substituent was attached at the 4-position of the benzene ring, it yielded a highly potent
antitubercular structure with an MIC value as low as 0.005 µM. Compound 12n was tenfold
more potent than structure 2 and 292 times more potent than first-line drug INH (Table 1). This
finding places this molecule in a select group of structures with an MIC in the nanomolar range
against Mtb. Notably, the increase of the lipophilicity parameter alone during the lead

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optimization process does not explain the improved activity, because several molecules with
similar ClogP values displayed different MIC activities (Table 1).
2-(Quinolin-4-yloxy)acetamides with MIC values lower than 0.2 µM (12d-e and 12i-n) were
selected to study their inhibitory activity against clinically isolated strains (PE-003 and CDCT-4)
(Table 1). The PE-003 [19] and CDCT-4 strains have been described as multidrug-resistant
clinical isolates with resistance to drugs such as isoniazid, rifampin, ethambutol, and
streptomycin. Targeted sequencing of the PE-003 strain has revealed a mutation in the inhA
regulatory region C(-15)T [19], whereas CDCT-4 has been described as possessing mutations in
its rpoB (531 TCG-TAG) and katG (315 AGC-ACC) genes. Interestingly, in most cases, the
evaluated compounds were even more potent against the PE-003 and CDCT-4 strains than
against the M. tuberculosis H37Rv strain (Table 1). 2-(Quinolin-4-yloxy)acetamide 12n
exhibited an unexpectedly low MIC value of 1.0 nM and was almost 100 times more potent than
the fluoroquinolone-based drug moxifloxacin was against the PE-003 strain. Exposing the
HepG2, Vero, RAW 264.7, HaCat, and N9 cell lineages to a 10 µM concentration of compounds
12d-e and 12i-n for 72 hours did not significantly affect cell viability (Supporting Information,
Table S3) [20]. The tested concentration (10 µM) was at least 85 times higher than the MICs of
the synthesized compounds against M. tuberculosis H37Rv. These findings suggest that these
compounds have low toxicity in mammalian cells and a likely high degree of selectivity for Mtb.
The promising and selective activity elicited by the new compounds prompted us to investigate
their intracellular activity in a macrophage model of Mtb infection [21]. RIF- and INH-treated
groups showed decreases of 1.7 log10 (P < 0.001) and 2.3 log10 (P < 0.001) in the CFU counts
compared with the untreated early control and late control groups, respectively (Table 1). In this
model, an early control was defined as a control group of infected macrophages without any

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previous treatment that was lysed on the day of treatment onset, whereas the late control group
was lysed, for further cell counting, after 5 days of incubation. Treatments with the compounds
12d-e and 12i-n resulted in statistically significant decreases in CFU units compared with those
for both the early and late control groups. This finding suggests that the compounds present
intracellular bactericidal activity in this infection model at a concentration of 5 µM (Table 1).
Interestingly, as compared with the late control, all of the evaluated compounds decreased the
CFU counts at an elevated significance level (P < 0.001). In particular, treatments with 2-
(quinolin-4-yloxy)acetamides 12k and 12l showed higher activities that were not significantly
different from those of the positive control groups INH and RIF.
Moreover, the possibility of synergism between 2-(quinolin-4-yloxy)acetamides 12l and 12n in
combination with the anti-TB drugs isoniazid, rifampin, ethambutol, ethionamide, moxifloxacin
and streptomycin was evaluated using the checkerboard assay [22]. As shown in Table 2, there
was no change in the MIC of 12l in combination with ethambutol or moxifloxacin. The same
trend was observed in the association of 12n with isoniazid or moxifloxacin (Table 2). Even
though the combination of 12l with isoniazid, ethionamide, and streptomycin and 12n with
streptomycin and ethionamide showed improved activity on the basis of the MIC values, the
analysis of fractional inhibitory concentration indexes (FICI) suggested that the evaluated
structures act independently of one another (Table 2). In addition, the MIC of 12n with
ethambutol was 2-fold higher, but the FICI value indicated no interaction between these
compounds (Table 1). By contrast, the MIC values of 12l and 12n in the presence of rifampin
were decreased 8-fold and 4-fold, respectively, whereas the MIC of rifampin in the presence of
either 12l or 12n was decreased 4-fold. The FICI values were 0.375 and 0.5 for compounds 12l
and 12n, respectively (Table 2), thus suggesting a synergistic effect. The interaction profiles of

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2-(quinolin-4-yloxy)acetamides 12l and 12n with rifampin against the M. tuberculosis H37Rv
strain may provide a basis for alternative therapeutic regimens targeting drug-resistant Mtb
strains. Notably, no combination tested here demonstrated antagonist interactions under our
experimental conditions. Checkerboard experiments were performed in three independent assays
with concordant results.
Some in vitro ADME parameters such as aqueous solubility, permeability, intrinsic clearance,
and drug-drug interaction were determined for 2-(quinolin-4-yloxy)acetamides 12d-e and 12i-n
(Table 3). As previously reported, this class of compounds exhibited better aqueous solubility at
acid pH [11]. When tested at pH 5.8, structures 12d-e and 12i-n showed kinetic solubility
ranging from 2.2 to 17.0 µM, whereas at pH 7.4, the solubility was determined in a range from
1.2 to 17.9 µM. Notwithstanding the low solubilities observed at physiological pH, 2-(quinolin-
4-yloxy)acetamides 12e and 12i-m exhibited the aqueous solubilities at pH 1.5 or pH 4.0
(Supporting Information, Table S5) above the threshold value of 100 µM, which has been used
in early drug discovery programs [23]. Furthermore, the passive permeability features of 2-
(quinolin-4-yloxy)acetamides 12d-e and 12i-n were studied using the parallel artificial
membrane permeability assay (PAMPA). Overall, the investigation revealed good membrane
permeability for compounds 12e, 12i-j, 12l and 12n with values ranging from 1.3 to 13.1 × 10-6
cm/s. 2-(Quinolin-4-yloxy)acetamides 12d, 12k, 12m, and hit compound 2 were not quantified
in the receiver buffer solutions under the experimental conditions used here, thus indicating their
very low permeability. Nevertheless, an in vitro intrinsic clearance analysis revealed that the
tested compounds 12d-e, 12i, 12k, and 12m exhibited high metabolic rates. However,
compounds 12j, 12l, and 12n were metabolically more stable than their counterparts, with
increased in vitro half-lives. In particular, the half-life of 2-(quinolin-4-yloxy)acetamide 2l was

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increased nearly two-fold compared with those of molecules 1 and 2. Similar structures
evaluated in metabolic stability experiments have indicated that the amide bond is a possible
point of hydrolysis that is mediated by esterases [13]. Finally, the possibility of drug-drug
interactions was evaluated by assaying compound 12n against a panel of human CYP450
isoforms (3A4, 1A2, 2C9, 2C19, 2D6, and 2E1). The results indicated the low risk of drug-drug
interactions because the IC50 of the compound was > 30 µM for all of the tested isoforms.
3. Conclusions
In summary, here, we showed the synthesis of a new series of 2-(quinolin-4-yloxy)acetamides
and their in vitro antitubercular activities. The compounds were obtained with well-established
synthetic protocols using accessible reactants and reagents that produced the molecules in
reasonable yields. In addition, the synthesized compounds showed potent and selective activity
against drug-sensitive and drug-resistant Mtb strains with no apparent cytotoxicity to mammalian
cells, and exhibited intracellular activities similar to those of the first-line drugs isoniazid and
rifampin. The combined effectiveness of the evaluated 2-(quinolin-4-yloxy)acetamides (12l and
12n) and rifampin may be useful for further novel anti-TB regimens, especially when patients
cannot use the current available treatments because of drug-resistant infection, toxicity events or
drug-drug interactions. Finally, the submicromolar antitubercular activity elicited by 2-(quinolin-
4-yloxy)acetamides coupled with some drug-like parameters suggests that this class of
compounds may yield candidates for the future development of novel drugs for TB treatment.
Studies evaluating the in vivo oral bioavailability of the lead compounds (a possible attrition
point for this chemical class) and to assess their efficacy in a murine model of Mtb infection are
in progress.

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4. Experimental Section
4.1. Synthesis and structure: apparatus and analysis
All the commercially available solvents and reagents were obtained from commercial suppliers
and were used without further purification. All the reactions involving reactants, reagents or
intermediates that were sensitive to air or moisture were performed under an inert argon
atmosphere. The reactions were monitored by thin layer chromatography (TLC) with Macherey-
Nagel ALUGRAM® Xtra SIL G/UV254 (0.2 mm) plates. The melting points were measured using
1
a Microquímica MQAPF-302 apparatus. H NMR spectra were acquired on a Varian 400 or
Agilent DD2 spectrometer (Federal University of Rio Grande do Sul, UFRGS/Brazil) (1H at
400.13 MHz or 500.13 MHz, respectively) at 25 °C in 5 mm sample tubes. 13C NMR spectra
were acquired on a Varian 400 or Agilent DD2 spectrometer (13C at 100.63 MHz or 125.63
MHz, respectively) at 25 °C. Chemical shifts (δ) were expressed in parts per million (ppm)
1
relative to DMSO-d6 (δ 2.49 for H and δ 39.5 for 13C), which was used as the solvent and to
TMS, which was used as the internal standard. Splitting patterns were designated as follows: s,
singlet; d, doublet; dd, doublet of doublets; and m, multiplet. High-resolution mass spectra
(HRMS) were obtained for all the compounds on an LTQ Orbitrap Discovery mass spectrometer
(Thermo Fisher Scientific, Bremen, Germany). This hybrid system combines an LTQ XL linear
ion trap mass spectrometer and an Orbitrap mass analyzer. The experiments were performed
through the direct infusion of the sample in MeOH:H2O (1:1) with 0.1% formic acid (flow rate
10 µL/min) in positive-ion mode using electrospray ionization (ESI). Elemental composition
calculations were executed using the specific tool included in the Qual Browser module of
Xcalibur (Thermo Fisher Scientific, release 2.0.7) software. Crystal data were collected using a
Bruker APEX II CCD area-detector diffractometer using graphite-monochromatized MoKα

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radiation. Compound purity was determined by HPLC using an Äkta HPLC system (GE
Healthcare® Life Sciences) equipped with a binary pump, manual injector, and UV detector.
Unicorn 5.31 (Build 743) software 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 1.5
mL/min; UV detection at 254 nm; 100% water (0.1% acetic acid) was maintained from 0 to 7
min and was 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; and the last partition was maintained for an
additional 15 min (15 to 30 min) and subsequently returned to 100% water (0.1% acetic acid) in
5 min (30 to 35 min) and maintained for an additional 10 min (35 to 45 min). All the evaluated
compounds were ≥ 95% pure.
4.2. General procedure for the synthesis of 2-(quinolin-4-yloxy)acetamides 6a-i and 12a-n
4-Hydroxyquinolines 5 (1.1 mmol) were added to a stirred solution containing 2-bromo-N-aryl-
acetamide (1.0 mmol) and K2CO3 (3.12 mmol) in 6 mL of N,N-dimethylformamide (DMF) under
an argon atmosphere. After being stirred for 16 h, the reaction mixture was dissolved in 20 mL
of distilled water. The precipitated product was filtered off, washed with water and dried under a
vacuum. Purification of the compounds was performed by flash chromatography on silica gel
(Macherey-Nagel, 35-70 mesh) using hexane:ethyl acetate (1:1) as the eluent.
4.2.1. 2-((6-Methoxy-2-ethylquinolin-4-yl)oxy)-N-(2-methoxyphenyl)acetamide (6a)

ACCEPTED MANUSCRIPT
1
Yield 25%; m.p.: 157-158 ºC; HPLC 98% (tR = 15.01 min); H NMR (500 MHz, DMSO-d6)
δ
ppm 1.3 (t, J = 6.8 Hz, 3 H), 2.8 (m, 2 H), 3.8 (s, 3 H), 3.9 (s, 3 H), 5.1 (s, 2 H), 6.9 (s, 2 H), 7.1
(m, 2 H), 7.4 (d, J = 8.8 Hz, 1 H), 7.5 (s, 1 H), 7.8 (d, J = 8.8 Hz, 1 H), 8.1 (d, J = 6.8 Hz, 1 H),
13
9.4 (s, 1 H); C NMR (126 MHz, DMSO-d6)
δ ppm 13.5, 31.6, 55.4, 55.8, 67.0, 100.1, 101.4,
111.2, 119.8, 120.4, 120.9, 121.2, 124.7, 126.4, 129.8, 144.1, 149.1, 156.4, 158.9, 161.8, 165.5;
FTMS (ESI) m/z 367.1644 [M + H]+; calcd for C21H22N2O4: 367.1652.
4.2.2. 2-((6-Methoxy-2-propylquinolin-4-yl)oxy)-N-(2-methoxyphenyl)acetamide (6b)
Yield 51%, m.p.: 155-156 ºC; HPLC 98% (tR = 15.45 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 0.9 (s, 3 H), 1.7 (s, 2 H), 2.8 (s, 2 H), 3.8 (s, 3 H), 3.9 (s, 3 H), 5.1 (s, 2 H), 6.9 (s, 2 H), 7.1
(s, 2 H), 7.4 (d, J = 8.3 Hz, 1 H), 7.5 (s, 1 H), 7.8 (d, J = 8.3 Hz, 1 H), 8.1 (s, 1 H), 9.4 (s, 1 H);
13C NMR (126 MHz, DMSO-d6)
δ ppm 13.8, 22.3, 40.5, 55.4, 55.8, 67.0, 100.1, 101.9, 111.2,
119.8, 120.4, 120.9, 121.2, 124.7, 126.4, 129.8, 144.2, 149.2, 156.4, 158.9, 160.7, 165.5; FTMS
(ESI) m/z 381.1802 [M + H]+; calcd for C22H24N2O4: 381.1809.
4.2.3. 2-((6-Methoxy-2-isopropylquinolin-4-yl)oxy)-N-(2-methoxyphenyl)acetamide (6c)
Yield 47%; m.p.: 145-146 ºC; HPLC 96% (tR = 14.62 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 1.3 (s, 3 H), 1,2 (s, 3 H), 3.1 (m, 1 H), 3.8 (s, 3 H), 3.9 (s, 3 H), 5.08 (s, 2 H), 6.9 (m, 1 H),
6,95 (s, 1H), 7.1 (m, 2 H), 7.4 (dd, J = 9.0, 2.7 Hz, 1 H), 7.5 (d, J = 2.4 Hz, 1 H), 7.8 (d, J = 9.3
Hz, 1 H), 8.1 (d, J=7.8 Hz, 1 H), 9.4 (s, 1 H); 13C NMR (126 MHz, DMSO-d6)
δ ppm 22.3, 36.6,
55.4, 55.8, 67.1, 100.1, 100.3, 111.2, 120.0, 120.5, 121.0, 121.2, 124.8, 126.4, 130.0, 144.0,

ACCEPTED MANUSCRIPT
149.2, 156.4, 159.0, 165.6, 165.6; FTMS (ESI) m/z 381.1802 [M + H]+; calcd for C22H24N2O4:
381.1809.
4.2.4. 2-((6-Methoxy-3-benzyl-2-methylquinolin-4-yl)oxy)-N-(2-methoxyphenyl)acetamide (6d)
Yield 75%; m.p.: 162-163 ºC; HPLC 95% (tR = 17.86 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 2.47 (s, 3 H), 3.73 (s, 3 H), 3.90 (s, 3 H), 4.29 (s, 2 H), 4.77 (s, 2 H), 7.0 (t, J = 7.6 Hz, 1
H), 7.1 (d, J = 7.8 Hz, 1 H), 7.1 (d, J = 8.3 Hz, 1 H), 7.2 (m, 3 H), 7.3 (t, J = 7.6 Hz, 2 H), 7.4
(dd, J = 9.0, 2.7 Hz, 1 H), 7.5 (d, J = 2.4 Hz, 1 H), 7.9 (d, J = 9.3 Hz, 1 H), 8.1 (d, J = 7.8 Hz, 1
H), 9.4 (s, 1 H); 13C NMR (126 MHz, DMSO-d6)
δ ppm 23.2, 31.3, 55.4, 55.6, 72.7, 100.1,
111.0, 120.3, 120.7, 121.3, 122.4, 124.2, 124.6, 126.0, 126.3, 127.9, 128.4, 130.1, 139.2, 143.7,
149.0, 157.0, 157.3, 158.0, 162.4, 165.8; FTMS (ESI) m/z 443.1960 [M + H]+; calcd for
C27H26N2O4: 443.1965.
4.2.5. 2-((6-Ethoxy-2-methylquinolin-4-yl)oxy)-N-(2-methoxyphenyl)acetamide (6e)
Yield 58%; m.p.: 196-197 ºC; HPLC 97% (tR = 15.26 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 1.4 (s, 3 H), 2.5 (s, 3 H), 3.9 (s, 3 H), 4.2 (q, J = 5.9 Hz, 2 H), 5.0 (s, 2 H), 6.9 (m, 2 H), 7.1
(s, 2 H), 7.4 (d, J = 8.3 Hz, 1 H), 7.5 (s, 1 H), 7.8 (d, J = 8.8 Hz, 1 H), 8.1 (d, J = 6.4 Hz, 1 H),
13
9.4 (s, 1 H); C NMR (126 MHz, DMSO-d6)
δ ppm 14.6, 25.0, 55.8, 63.5, 67.0, 101.0, 102.4,
111.2, 119.6, 120.5, 120.7, 121.3, 124.7, 126.4, 129.6, 144.1, 149.0, 155.6, 156.9, 158.7, 165.4;
FTMS (ESI) m/z 367.1642 [M + H]+; calcd for C21H22N2O4: 367.1652.

ACCEPTED MANUSCRIPT
4.2.6. 2-((6-Fluoro-2-methylquinolin-4-yl)oxy)-N-(2-methoxyphenyl)acetamide (6f)
Yield 85%; m.p.: 188-189 ºC; HPLC 98% (tR = 16.86 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 2.6 (s, 3 H), 3.9 (s, 3 H), 5.0 (s, 2 H), 7.0 (t, J = 7.3 Hz, 1 H), 7.0 (s, 1 H), 7.1 (m, 2 H), 7.6
(td, J = 8.8, 2.9 Hz, 1 H), 7.9 (dd, J = 9.5, 1.7 Hz, 1 H), 8.0 (dd, J = 9.3, 5.4 Hz, 1 H), 8.1 (d, J =
7.3 Hz, 1 H), 9.4 (s, 1 H); 13C NMR (126 MHz, DMSO-d6)
δ ppm 25.1, 55.7, 67.1, 103.0, 104.8,
105.0, 109.5, 111.1, 119.3, 119.4, 119.4, 119.5, 120.5, 120.7, 124.7, 126.3, 130.8, 130.9, 145.4,
149.0, 158.1, 159.0, 159.0, 159.3, 159.4, 160.0, 165.0; FTMS (ESI) m/z 341.1290 [M + H]+;
calcd for C19H17FN2O3: 341.1296.
4.2.7. 2-((6-Chloro-2-methylquinolin-4-yl)oxy)-N-(2-methoxyphenyl)acetamide (6g)
Yield 70%; m.p.: 201-202 ºC; HPLC 95% (tR = 18.51 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 2.60 (s, 3 H), 3.91 (s, 3 H), 5.00 (s, 2 H), 6.95 (s, 1 H), 7.04-7.11 (m, 2 H), 7.73 (s, 1 H),
7.89 (s, 1 H), 8.09 (s, 1 H), 8.18 (s, 1 H), 8.3 (s, 1 H), 9.3 (s, 1 H); 13C NMR (126 MHz, DMSO-
d6)
δ ppm 25.1, 55.8, 67.2, 103.1, 109.3, 111.1, 119.7, 120.0, 120.4, 120.5, 124.6, 126.3, 129.4,
130.0, 130.1, 146.7, 158.5, 160.4, 164.8; FTMS (ESI) m/z 357.0994 [M + H]+; calcd for
C19H17ClN2O3: 357.1000.
4.2.8. 2-((6-Bromo-2-methylquinolin-4-yl)oxy)-N-(2-methoxyphenyl)acetamide (6h)
Yield 32%; m.p.: 196-197 ºC; HPLC 97% (tR = 19.46 min); 1H NMR (400 MHz, DMSO-d6)
δ
ppm 2.6 (s, 3 H), 3.9 (s, 3 H), 5.0 (s, 2 H), 7.0 (m, 1 H), 7.0 (s, 1 H), 7.1 (m, 2 H), 7.8 (m, 2 H),
13
8.1 (d, J=8.2 Hz, 1 H), 8.3 (d, J=1.2 Hz, 1 H), 9.4 (s, 1 H); C NMR (101 MHz, DMSO-d6)
δ

ACCEPTED MANUSCRIPT
ppm 25.4; 56.0; 67.2; 103.3; 111.2; 118.0; 120.3; 120.5; 120.8; 123.4; 124.8; 126.4;
130.4; 132.9; 146.9; 149.1; 158.6; 160.8; 165.1; FTMS (ESI) m/z 401.0490 [M + H]+; calcd
for C19H17BrN2O3: 401.0495.
4.2.9. 2-((6-Trifluoromethyl-2-methylquinolin-4-yl)oxy)-N-(2-methoxyphenyl)acetamide (6i)
Yield 56%; m.p.: 203-204 ºC; HPLC 95% (tR = 18.66 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 2.6 (s, 3 H), 3.8 (s, 3 H), 5.1 (s, 2 H), 6.9 (t, J = 7.1 Hz, 1 H), 7.1 (m, 3 H), 8.0 (d, J = 8.8
13
Hz, 1 H), 8.1 (d, J = 7.8 Hz, 2 H), 8.5 (s, 1 H), 9.3 (s, 1 H); C NMR (126 MHz, DMSO-d6)
δ
ppm 25.3, 55.6, 67.3, 103.5, 111.2, 118.2, 119.2, 119.3, 120.3, 120.9, 123.0, 124.6, 124.9, 125.0,
125.2, 126.3, 129.5, 149.2, 149.2, 149.4, 160.0, 162.9, 164.8; FTMS (ESI) m/z 391.1251 [M +
H]+; calcd for C20H17F3N2O3: 391.1264.
4.2.10.
2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-N-(2-methoxyphenyl)-N-methylacetamide
(12a)
Yield 72%; m.p.: 171-172 ºC; HPLC 96% (tR = 14.05 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 2.5 (s, 3 H), 3.1 (s, 3 H), 3.8 (s, 3 H), 3.9 (s, 3 H), 4.5 (d, J = 15.7 Hz, 1 H), 4.7 (d, J = 15.2
Hz, 1 H), 6.5 (s, 1 H), 7.0 (t, J = 6.8 Hz, 1 H), 7.2 (d, J = 8.3 Hz, 1 H), 7.3 (s, 1 H), 7.3 (d, J =
8.8 Hz, 1 H), 7.4 (m, 2 H), 7.7 (d, J = 8.8 Hz, 1 H); 13C NMR (126 MHz, DMSO-d6)
δ ppm 25.1,
35.9, 55.3, 55.8, 65.5, 99.6, 101.6, 112.8, 119.6, 121.2, 121.5, 129.1, 129.4, 129.6, 130.1, 144.1,
154.6, 156.1, 156.5, 159.4, 166.2; FTMS (ESI) m/z 367.1643 [M + H]+; calcd for C21H22N2O4:
367.1652.

ACCEPTED MANUSCRIPT
4.2.11. 2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-1-(piperidin-1-yl)ethan-1-one (12b)
Yield 90%; m.p.: 105-105 ºC; HPLC 96% (tR = 13.70 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 1.5 (m, 2 H), 1.6 (m, 4 H), 2.5 (s, 3 H), 3.4 (s, 4 H), 3.9 (s, 3 H), 5.1 (s, 2 H), 6.8 (s, 1 H),
7.3 (d, J = 9.3 Hz, 1 H), 7.4 (s, 1 H), 7.8 (d, J = 9.3 Hz, 1 H); 13C NMR (126 MHz, DMSO-d6)
δ
ppm 23.9, 25.1, 25.3, 26.0, 42.2, 45.1, 55.3, 66.0, 99.7, 102.3, 119.8, 121.4, 129.5, 144.1, 156.2,
156.8, 159.5, 164.5; FTMS (ESI) m/z 315.1694 [M + H]+; calcd for C18H22N2O3: 315.1703.
4.2.12. 2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-N,N-diphenylacetamide (12c)
Yield 31%; m.p.: 173-174 ºC; HPLC 98% (tR = 14.61 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 2.6 (s, 3 H), 3.8 (s, 3 H), 4.9 (s, 2 H), 6.7 (s, 1 H), 7.3 (m, 12 H), 7.7 (d, J = 9.3 Hz, 1 H);
13C NMR (126 MHz, DMSO-d6)
δ
ppm 25.0, 55.2, 66.0, 99.5, 101.8, 119.6, 121.5, 129.4, 144.0,
156.0, 156.7, 159.2, 166.0; FTMS (ESI) m/z 399.1698 [M + H]+; calcd for C25H22N2O3:
399.1703.
4.2.13.
2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-N-(5,6,7,8-tetrahydronaphthalen-2-
yl)acetamide (12d)
Yield 99%; m.p.: 163-164 ºC; HPLC 96% (tR = 15.57 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 1.7 (m, 4 H), 2.5 (s, 3 H), 2.7 (m, 4 H), 3.9 (s, 3 H), 5.0 (s, 2 H), 6.8 (s, 1 H), 7.0 (d, J = 8.3
Hz, 1 H), 7.3 (d, J = 7.8 Hz, 1 H), 7.4 (m, 2 H), 7.5 (s, 1 H), 7.8 (d, J = 8.8 Hz, 1 H), 10.1 (s, 1
H); 13C NMR (126 MHz, DMSO-d6)
δ ppm 22.6, 22.7, 25.0, 28.2, 28.9, 55.3, 67.0, 100.0, 102.1,

ACCEPTED MANUSCRIPT
117.2, 119.7, 119.9, 121.5, 129.0, 129.4, 132.0, 135.6, 136.7, 144.1, 156.1, 156.7, 159.4, 165.2;
FTMS (ESI) m/z 377.1850 [M + H]+; calcd for C23H24N2O3: 377.1860.
4.2.14.
2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-N-(2,3-dihydro-1H-inden-5-yl)acetamide
(12e)
Yield 73%; m.p.: 192-193 ºC; HPLC 96% (tR = 15.33 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 2.0 (q, J = 7.3 Hz, 2 H), 2.5 (s, 3 H) 2.8 (m, 4 H), 3.9 (s, 3 H), 5.0 (s, 2 H), 6.8 (s, 1 H), 7.2
(d, J = 7.8 Hz, 1 H), 7.3 (m, 2 H), 7.5 (d, J = 2.9 Hz, 1 H), 7.6 (s, 1 H), 7.8 (d, J = 9.3 Hz, 1 H),
13
10.1 (s, 1 H); C NMR (126 MHz, DMSO-d6)
δ ppm 25.0, 25.1, 31.8, 32.5, 55.4, 67.1, 100.1,
102.2, 115.9, 117.8, 119.7, 121.5, 124.2, 129.5, 136.5, 139.0, 144.1, 144.2, 156.2, 156.8, 159.4,
165.3; FTMS (ESI) m/z 363.1695 [M + H]+; calcd for C22H22N2O3: 363.1703.
4.2.15.
2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-N-(5,6,7,8-tetrahydronaphthalen-1-
yl)acetamide (12f)
Yield 66%; m.p.: 218-219 ºC; HPLC 96% (tR = 15.39 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 1.7 (m, 4 H), 2.6 (s, 3 H), 2.6 (m, 2 H), 2.7 (m, 2 H), 3.9 (s, 3 H), 5.0 (s, 2 H), 6.9 (s, 1 H),
6.9 (d, J = 7.3 Hz, 1 H), 7.1 (t, J = 7.6 Hz, 1 H), 7.3 (d, J = 7.8 Hz, 1 H), 7.3 (dd, J = 9.0, 2.7 Hz,
1 H), 7.6 (s, 1 H), 7.8 (d, J = 8.8 Hz, 1 H), 9.4 (s, 1 H); 13C NMR (126 MHz, DMSO-d6)
δ ppm
22.2, 22.3, 24.1, 25.0, 29.1, 55.4, 67.3, 100.3, 102.3, 119.7, 121.4, 122.6, 125.1, 126.4, 129.4,
135.1, 137.5, 144.2, 156.3, 156.7, 159.2, 165.6; FTMS (ESI) m/z 377.1847 [M + H]+; calcd for
C23H24N2O3: 377.1860.

ACCEPTED MANUSCRIPT
4.2.16.
(S)-2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-N-(1,2,3,4-tetrahydronaphthalen-1-
yl)acetamide (12g)
Yield 52%; m.p.: 190-191ºC; HPLC 97% (tR = 15.04 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 1.8 (m, 2 H), 1.9 (m, 2 H), 2.5 (s, 3 H), 2.7 (m, 2 H), 3.8 (s, 3 H), 4.8 (s, 2 H), 5.1 (m, 1 H),
6.8 (s, 1 H), 7.1 (m, 4 H), 7.3 (dd, J = 9.0, 2.7 Hz, 1 H), 7.4 (d, J = 2.4 Hz, 1 H), 7.8 (d, J = 8.8
Hz, 1 H), 8.6 (d, J = 8.8 Hz, 1 H); 13C NMR (126 MHz, DMSO-d6)
δ ppm 20.1, 25.0, 28.7, 29.6,
46.4, 55.3, 67.1, 100.2, 102.1, 119.7, 121.5, 125.7, 126.8, 127.9, 128.8, 129.4, 137.1, 137.1,
144.1, 156.1, 156.7, 159.4, 166.3; FTMS (ESI) m/z 377.1848 [M + H]+; calcd for C23H24N2O3:
377.1860.
4.2.17.
(R)-2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-N-(1,2,3,4-tetrahydronaphthalen-1-
yl)acetamide (12h)
Yield 79%; m.p.: 190-191 ºC; HPLC 98% (tR = 15.13 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 1.7 (m, 2 H), 1.9 (m, 2 H), 2.5 (s, 3 H), 2.6-2.7 (m, 2 H), 3.8 (s, 3 H), 4.9 (s, 2 H), 5.1 (m, 1
H), 6.8 (s, 1 H), 7.1 (m, 4 H), 7.3 (d, J = 9.3 Hz, 1 H), 7.4 (s, 1 H), 7.8 (d, J = 8.8 Hz, 1 H), 8.6
13
(d, J = 8.3 Hz, 1 H); C NMR (126 MHz, DMSO-d6)
δ ppm 20.1, 25.0, 28.7, 29.6, 46.4, 55.3,
67.1, 100.2, 102.1, 119.7, 121.5, 125.7, 126.8, 127.9, 128.7, 129.4, 137.1, 137.1, 144.1, 156.1,
156.7, 159.4, 166.3; FTMS (ESI) m/z 377.1860 [M + H]+; calcd for C23H24N2O3: 377.1860.
4.2.18. 2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-N-(3,4-dimethylphenyl)acetamide (12i)

ACCEPTED MANUSCRIPT
Yield 88%; m.p.: 169-170 ºC; HPLC 98% (tR = 14.94 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 2.1 (s, 3 H), 2.2 (s, 3 H), 2.5 (s, 3 H), 3.9 (s, 3 H), 5.0 (s, 2 H), 6.8 (s, 1 H), 7.1 (d, J = 8.3
Hz, 1 H), 7.4 (m, 1 H), 7.4 (s, 1 H), 7.5 (d, J = 2.4 Hz, 1 H), 7.8 (d, J = 8.8 Hz, 1 H), 8.3 (s, 1 H),
10.1 (s, 1 H); 13C NMR (126 MHz, DMSO-D6)
δ ppm 18.8, 19.6, 25.1, 55.4, 67.1, 100.1, 102.2,
117.2, 119.7, 120.8, 121.6, 129.5, 129.6, 131.5, 136.1, 136.4, 144.2, 156.2, 156.8, 159.5, 165.3;
FTMS (ESI) m/z 351.1696 [M + H]+; calcd for C21H22N2O3: 351.1703.
4.2.19. 2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-N-(p-tolyl)acetamide (12j)
Yield 56%; m.p.: 188-189 ºC; HPLC 96% (tR = 14.78 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 2.2 (s, 3 H) 2.5 (s, 3 H) 3.9 (s, 3 H) 5.0 (s, 2 H) 6.8 (s, 1 H) 7.1 (d, J = 7.8 Hz, 2 H) 7.3 (d, J
= 9.3 Hz, 1 H) 7.5 (m, 3 H) 7.8 (d, J = 8.8 Hz, 1 H) 10.2 (s, 1 H); 13C NMR (126 MHz, DMSO-
d6)
δ ppm 20.4, 25.1, 55.4, 67.1, 100.1, 102.2, 119.7, 119.7, 121.5, 129.1, 129.5, 132.7, 135.8,
144.2, 156.2, 156.8, 159.4, 165.4; FTMS (ESI) m/z 337.1540 [M + H]+; calcd for C20H20N2O3:
337.1547.
4.2.20. 2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-N-(4-ethylphenyl)acetamide (12k)
Yield 68%; m.p.: 198-199 ºC; HPLC 95% (tR = 14.52 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 1.1 (s, 3 H), 2.5 (s, 5 H), 3.9 (s, 3 H), 5.0 (s, 2 H), 6.8 (s, 1 H), 7.2 (d, J = 4.4 Hz, 2 H), 7.3
13
(d, J = 5.9 Hz, 1 H), 7.5 (d, J = 12.7 Hz, 3 H), 7.8 (s, 1 H), 10.2 (s, 1 H); C NMR (126 MHz,
DMSO-d6)
δ ppm 15.6, 25.0, 27.5, 55.3, 67.1, 100.1, 102.1, 119.7, 121.5, 127.9, 129.4, 136.0,

ACCEPTED MANUSCRIPT
139.1, 144.1, 156.2, 156.8, 159.4, 165.3; FTMS (ESI) m/z 351.1696 [M + H]+; calcd for
C21H22N2O3: 351.1703.
4.2.21. 2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-N-(4-propylphenyl)acetamide (12l)
Yield 83%; m.p.: 194-195 ºC; HPLC 96% (tR = 15.68 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 0.9 (t, J = 7.3 Hz, 3 H), 1.6 (s, J = 7.4 Hz, 2 H), 2.5 (m, 2 H), 2.5 (s, 3 H), 3.9 (s, 3 H), 5.0
(s, 2 H), 6.9 (s, 1 H), 7.1 (d, J = 8.8 Hz, 2 H), 7.4 (dd, J = 8.8, 2.9 Hz, 1 H), 7.5 (d, J = 2.4 Hz, 1
H), 7.5 (d, J = 8.8 Hz, 2 H), 7.8 (d, J = 9.3 Hz, 1 H), 10.2 (s, 1 H); 13C NMR (126 MHz, DMSO-
d6)
δ ppm 13.6, 24.1, 25.1, 36.7, 55.4, 67.1, 100.1, 102.2, 119.7, 121.6, 128.6, 129.5, 136.1,
137.5, 144.2, 156.2, 156.9, 159.5, 165.4; FTMS (ESI) m/z 365.1851 [M + H]+; calcd for
C22H24N2O3: 365.1860.
4.2.22. 2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-N-(4-butylphenyl)acetamide (12m)
Yield 84%; m.p.: 143-174 ºC; HPLC 95% (tR = 15.34 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 0.9 (t, J = 7.3 Hz, 3 H), 1.3 (s, J = 7.3 Hz, 2 H), 1.5 (q, J = 7.5 Hz, 2 H), 2.5 (m, 2 H), 2.5
(s, 3 H), 3.9 (s, 3 H), 5.0 (s, 2 H), 6.9 (s, 1 H), 7.1 (d, J = 8.3 Hz, 2 H), 7.4 (dd, J = 9.0, 2.7 Hz, 1
13
H), 7.5 (d, J = 2.9 Hz, 1 H), 7.5 (d, J = 8.3 Hz, 2 H), 7.8 (d, J = 9.3 Hz, 1 H); C NMR (126
MHz, DMSO-d6)
δ ppm 13.7, 21.6, 25.0, 33.1, 34.1, 55.3, 67.0, 100.0, 102.1, 119.6, 121.5,
128.4, 129.4, 136.0, 137.6, 144.1, 156.1, 156.7, 159.4, 165.3; FTMS (ESI)m/z 379.2023 [M +
H]+; calcd for C23H26N2O3: 379.2016.

ACCEPTED MANUSCRIPT
4.2.23. 2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-N-(4-pentylphenyl)acetamide (12n)
Yield 95%; m.p.: 118-119 ºC; HPLC 98% (tR = 16.77 min); 1H NMR (400 MHz, DMSO-d6)
δ
ppm 0.8 (t, J = 7.3 Hz, 3 H), 1.2 (m, 4 H), 1.5 (q, J = 7.5 Hz, 2 H), 2.5 (m, 2H), 2.5 (s, 3 H), 3.9
(s, 3 H), 5.0 (s, 2 H), 6.8 (s, 1H), 7.1 (d, J=8.3 Hz, 2 H), 7.3 (dd, J = 9.0, 2.7 Hz, 1 H), 7.5 (d, J
13
= 2.9 Hz, 1 H), 7.5 (d, J = 8.3 Hz, 2 H), 7.7 (d, J = 9.3 Hz, 1 H), 10.1 (s, 1 H); C NMR (101
MHz, DMSO-d6)
δ ppm 13.8, 21.9, 25.1, 30.8, 30.8, 34.5, 55.4, 67.1, 100.1, 102.1, 119.6,
121.5, 128.5, 129.5, 136.0, 137.7, 144.2, 156.2, 156.8, 159.4, 165.4; FTMS (ESI) m/z 393.2147
[M + H]+; calcd for C24H28N2O3: 393.2173.
4.3. General procedure for the synthesis of 2-(quinolin-4-ylamino)acetamides 9a-b
Under an argon atmosphere, sodium hydride (25 mg) that was dispersed in oil was washed with
dry hexane and then stirred in dry DMSO (4 mL) at 45 °C for 20 min. The solution containing
the 2-methyl-6-methoxy-4-aminoquinoline 7 (0.5 mmol) that was dissolved in 4 mL of dry
DMSO was added dropwise to the mixture, which was stirred for 20 min at 75 °C. 2-Bromo-N-
acetamide 8a or 8b (0.5 mmol) in DMSO (2 mL) was kept under an argon atmosphere and added
to the reaction mixture with subsequent stirring for 3 h at 80 °C. After the completion of the
reaction (as evaluated by TLC), the reaction mixture was allowed to cool, poured into crushed
ice, and then extracted with ethyl acetate (3 x 50 mL). The organic layer was washed with water,
dried using magnesium sulfate, evaporated, and dried under vacuum. The purification of the
compound was performed by flash chromatography on silica gel (Macherey-Nagel, 35-70 mesh)

ACCEPTED MANUSCRIPT
with elution using hexane and ethyl acetate (7:3, 1:1, 3:7, 0:1), and, finally, ethyl acetate and
methanol (9:1).
4.3.1. 2-((6-Methoxy-2-methylquinolin-4-yl)amino)-N-(2-methoxyphenyl)acetamide (9a)
1
Yield 31%; m.p.: 174-175 ºC; HPLC 95% (tR = 14.25 min); H NMR (500 MHz, DMSO-d6)
δ
ppm 2.4 (s, 3 H), 3.7 (s, 3 H), 3.9 (s, 3 H), 4.2 (s, 2 H), 6.4 (s, 1 H), 6.9 (t, J = 7.1 Hz, 1 H), 7.0
(d, J = 7.8 Hz, 1 H), 7.0 (d, J = 7.3 Hz, 1 H), 7.3 (d, J = 8.8 Hz, 1 H), 7.6 (s, 1 H), 7.7 (m, 2 H),
13
8.0 (d, J = 7.3 Hz, 1 H), 9.3 (s, 1 H); C NMR (126 MHz, DMSO-d6)
δ ppm 24.3, 46.4, 55.6,
55.7, 99.0, 100.9, 111.2, 117.8, 120.4, 120.8, 120.9, 124.4, 126.9, 128.7, 149.1, 149.8, 155.4,
155.8, 168.0; FTMS (ESI) m/z 352.1669 [M + H]+; calcd for C20H21N3O3: 352.1656.
4.3.2. 2-((6-Methoxy-2-methylquinolin-4-yl)amino)-N-(naphthalen-2-yl)acetamide (9b)
Yield 28%; m.p.: 243-244 ºC; HPLC 96% (tR = 14.94 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 2.39 (s, 3H), 3.89 (s, 3H), 4.19 (s, 2H), 6.28 (s, 1H), 7.23 (d, J = 9.3 Hz, 1H), 7.3 (m, 2 H),
7.4 (m, 1 H), 7.5 (s, 1 H), 7.6 (d, J = 7.8 Hz, 2H), 7.7-7,8 (m, 3 H), 8.29 (s, 1H), 10.3 (s, 1H); 13C
NMR (126 MHz, DMSO-d6)
δ ppm 25.0, 46.4, 55.5, 98.7, 100.9, 115.4, 117.93, 120.0, 120.2,
124.6, 126.4, 127.2, 127.4, 128.4, 129.8, 133.3, 136.4, 143.6, 149.4, 155.5, 155.9, 168.6; FTMS
(ESI) m/z 372.1707 [M + H]+; calcd for C23H21N3O2: 372.1707.
4.4. General procedure for the synthesis of 4-hydroxyquinolines 10 and 11

ACCEPTED MANUSCRIPT
To a solution containing 4-hydroxyquinoline 5a (1.1 mmol) and an excess of K2CO3 (3.12
mmol) dissolved in 6 mL of dimethylformamide (DMF), 2-bromo-1-(naphthalen-2-yl)ethan-1-
one or 2-(2-bromoethyl)naphthalene (1 mmol) was added. The resulting solution was kept under
an argon atmosphere and stirred for 8 h at 25 °C. Afterwards, the reaction mixture was dissolved
in 20 mL of distilled water. The precipitated product was filtered off, washed with water and
dried under vacuum. The purification of the compounds was performed by flash chromatography
on silica gel (Macherey-Nagel, 35-70 mesh) using hexane: ethyl acetate (1:1) as the eluent.
4.4.1. 2-((6-Methoxy-2-methylquinolin-4-yl)oxy)-1-(naphthalen-2-yl)ethan-1-one (10)
o
Yield 61%; m.p.: 176-177 C; HPLC 97% (tR = 15.69 min); 1H NMR (500 MHz, DMSO-d6)
δ
ppm 2.5 (s, 3 H), 3.9 (s, 3 H), 6.0 (s, 2 H), 7.0 (s, 1 H), 7.3 (d, J = 9.3 Hz, 1 H), 7.5 (s, 1 H), 7.7
(m, 2 H), 7.8 (dd, J = 9.3, 2.0 Hz, 1 H), 8.0 (d, J = 8.8 Hz, 2 H), 8.1 (m, 1 H), 8.2 (d, J = 8.3 Hz,
13
1 H), 8.8 (s, 1 H); C NMR (126 MHz, DMSO-d6)
δ ppm 25.0, 55.3, 70.5, 99.7, 102.5, 119.8,
121.5, 123.3, 127.1, 127.8, 128.5, 128.9, 129.5, 129.6, 130.0, 131.5, 132.1, 135.3, 144.2, 156.3,
156.9, 159.4, 193.4; FTMS (ESI) m/z 358.1432 [M + H]+; calcd for C23H19NO3: 358.1438.
4.4.2. 6-Methoxy-2-methyl-4-(naphthalen-2-ylmethoxy)quinoline (11)
o
Yield 76% (0.245 g); m.p.: 158-159 C; HPLC 96% (tR = 16.11 min); 1H NMR (500 MHz,
DMSO-d6)
δ ppm 2.5 (s, 3 H), 3.8 (s, 3 H), 5.5 (s, 2 H), 7,0 (s, 1 H), 7.3 (d, J = 8.8 Hz, 1 H), 7.4
(s, 1 H), 7.5 (s, 2 H), 7.7 (d, J = 7.8 Hz, 1 H), 7.8 (d, J=9.3 Hz, 1 H), 8.0 (m, 3 H), 8.1 (s, 1 H);
13C NMR (126 MHz, DMSO-d6)
δ
ppm 25.1, 55.3, 69.7, 99.7, 102.5, 119.9, 121.3, 125.5, 126.3,

ACCEPTED MANUSCRIPT
126.3, 126.4, 127.6, 127.9, 128.3, 129.6, 132.6, 132.8, 133.9, 144.1, 156.3, 157.0, 159.6; FTMS
(ESI) m/z 330.1467 [M + H]+; calcd for C22H19NO2: 330.1489.
4.5. Mycobacterium tuberculosis inhibition assay
The measurement of the minimum inhibitory concentration (MIC) for each tested compound was
performed in 96-well U-bottom polystyrene microplates. Isoniazid (INH, control drug) and
compound solutions were prepared at a 1 mg/mL concentration in DMSO. They were diluted in
Middlebrook 7H9 medium containing 10% ADC (albumin, dextrose, and catalase) to a
concentration of 20 µg/mL of each compound containing 5% DMSO. Serial two-fold dilutions of
each drug in 100 µL of Middlebrook 7H9 medium containing 10% ADC were prepared directly
in 96-well plates at concentration ranges of 10.0 to 0.02 µg/mL, or 0.2 to 0.0004 µg/mL. Growth
controls containing no antibiotic and sterile controls without inoculation were included. The MIC
was determined for M. tuberculosis H37Rv and for the clinical isolates PE-003 and CDCT-4
(1105/09). Mycobacterial strains were grown in Middlebrook 7H9 containing 10% OADC (oleic
acid, albumin, dextrose, and catalase) and 0.05% Tween 80. The cells were vortexed with sterile
glass beads (4 mm) for 5 min to disrupt any clumps and were allowed to settle for 20 min. The
supernatant was measured spectrophotometrically at an absorbance of 600 nm. The Mtb
suspensions were divided into aliquots and stored at -20 °C. Each suspension was appropriately
diluted in Middlebrook 7H9 broth containing 10% ADC to achieve an optical density at 600 nm
of 0.006, and 100 µL was added to each well of the plate except the sterile controls. A final
concentration of 2.5% DMSO was maintained in each well. The plates were covered, sealed with
parafilm, and incubated at 37 °C. After 7 days of incubation, 60 µL of 0.01% resazurin solution
was added to each well, and the samples were incubated for an additional 48 hours at 37 °C [18].