Copia de Novel 4-Aminoquinolines - Synthesis, Inhibition of The Mycobacterium Tuberculosis Enoyl-Acyl Carrier

European Journal of Medicinal Chemistry 245 (2023) 114908
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry

journal homepage: www.elsevier.com/locate/ejmech
Research paper
Novel 4-aminoquinolines: Synthesis, inhibition of the Mycobacterium

tuberculosis enoyl-acyl carrier protein reductase, antitubercular activity,
SAR, and preclinical evaluation
Josiane Delgado Paz a, b, Nathalia Denise de Moura Sperotto a, Alessandro Silva Ramos a,
Kenia Pissinate a, Valnês da Silva Rodrigues Junior a, Bruno Lopes Abbadi a, Ana Flávia Borsoi a, c,
Raoní Scheibler Rambo a, Ana Carolina Corso Minotto a, Adilio da Silva Dadda a, Luiza Galina a, b,
Fernanda Souza Macchi Hopf a, b, Mauro Neves Muniz a, Leonardo Kras Borges Martinelli a,
Candida Deves Roth a, Rodrigo Braccini Madeira Silva a, Marcia Alberton Perelló a,
Alexia de Matos Czeczot a, b, Christiano Ev Neves a, b, Lovaine Silva Duarte a, Mariana Leyser d,
Sílvia Dias de Oliveira b, d, Cristiano Valim Bizarro a, b, Pablo Machado a, b, c, **,
Luiz Augusto Basso a, b, c, *
a
Instituto 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
b
Programa 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
c
Programa 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
d
Laboratório de Imunologia e Microbiologia, Pontifícia Universidade Católica do Rio Grande do Sul, 90616-900, Porto Alegre, Rio Grande do Sul, Brazil
A R T I C L E I N F O A B S T R A C T
Keywords: Herein a series of 4-aminoquinolines were synthesized in an attempt to optimize and study the structural features
Tuberculosis related to LABIO-17 biological activity, a Mycobacterium tuberculosis NADH-dependent enoyl-acyl carrier protein
Multidrug-resistant strains reductase (MtInhA) inhibitor previously identified by a virtual-ligand-screening approach. Structure-activity
InhA
relationships led to novel submicromolar inhibitors of MtInhA and potent antitubercular agents. The lead
Hit optimization
In vivo activity
compound is 87-fold more potent as enzymatic inhibitors and 32-fold more potent against M. tuberculosis H37Rv
Antitubercular drug candidate strain in comparison with LABIO-17. These molecules were also active against multidrug-resistant strains, devoid
of apparent toxicity to mammalian cells and showed favorable in vitro ADME profiles. Additionally, these
compounds were active in an intracellular model of tuberculosis (TB) infection, showed no genotoxicity signals,
satisfactory absorption parameters and absence of in vivo acute toxicity. Finally, treatment with selected 4-amino
quinoline for two weeks produced bacteriostatic effect in a murine model of TB. Taken together, these findings
indicate that this chemical class may furnish candidates for the future development of drug-sensitive and drug-
resistant tuberculosis treatments.
1. Introduction disease has been impacted by COVID-19 pandemic raising to levels seen
in 2017 and reversing years of progress and achievements in reducing
Tuberculosis (TB) is a human infectious disease caused mainly by TB disease burden [1]. The number of people newly diagnosed with TB
Mycobacterium tuberculosis (Mtb). The number of deaths from this declined from 7.1 millions in 2019 to 5.8 millions in 2020 falling short
* Corresponding author. Av. Ipiranga 6681, TECNOPUC, Prédio 92A, 90619-900, Porto Alegre, RS, Brazil.
** Corresponding author. Instituto Nacional de Ciência e Tecnologia em Tuberculose, Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Uni
versidade Católica do Rio Grande do Sul, 90616-900, Porto Alegre, Rio Grande do Sul, Brazil.
E-mail addresses: [email protected] (P. Machado), [email protected] (L.A. Basso).
https://doi.org/10.1016/j.ejmech.2022.114908
Received 23 August 2022; Received in revised form 25 October 2022; Accepted 3 November 2022
Available online 18 November 2022
0223-5234/© 2022 Elsevier Masson SAS. All rights reserved.
J.D. Paz et al. European Journal of Medicinal Chemistry 245 (2023) 114908
from the estimated 10 million people affected by the disease in 2020 [1]. cell wall, leading to cell lysis [18]. MtInhA is a component of the
Owing to reduced access to diagnosis and treatment, estimates for 2020 Mycobacterial type II dissociated fatty acid biosynthesis system (FAS-II)
include 1.3 million deaths from TB among HIV-negative (1.2 million in [19]. The FAS-II system elongates acyl fatty acid precursors yielding the
2019) and 214,000 among HIV-positive (209,000 in 2019) patients [1]. long carbon chain of meromycolate branch of mycolic acids, the hall
The World Health Organization (WHO) recommended treatment for mark of M. tuberculosis. We have previously described a virtual-ligand
patients with drug-susceptible TB disease is a 6-month regimen of four screening approach of a 3D-pharmacophore model of four points built
first-line drugs (isoniazid, rifampicin, ethambutol and pyrazinamide) based on X-ray crystal structures of MtInhA, which was employed to
[1]. Resistance to both isoniazid (INH) and rifampicin (the two most select protein ligands from the ZINC database [20]. The 4-aminoquino
effective first-line agents), defined as multidrug resistant TB (MDR-TB), line compound (LABIO-17, Fig. 1) was amongst these ligands and was
and rifampicin-resistant TB (RR-TB) continues to be a public health subsequently shown to inhibit the MtInhA-catalyzed chemical reaction
threat. Treatment of MDR/RR-TB requires a course of second-line drugs in a time-independent manner with an IC50 value of 20 (±3) μM [21].
for at least 9 months and up to 20 months, and it is estimated that a cure Steady-state kinetics results showed a non-competitive type of inhibition
rate of only 56% is achieved for MDR-TB patients [2]. WHO currently towards NADH with inhibition constant values (Kii and Kis) of, respec
recommends expanded access to all-oral regimens [3]. Even though the tively, 8 (±1) μM and 8 (±3) μM. The LABIO-17 displayed a competitive
approval of bedaquiline (2012) [4], delamanid (2014) [5], and pre mode of inhibition with respect to 2-trans-dodecenoyl-CoA (DD-CoA)
tomanid (2019) [6] (Fig. 1) ended decades of insufficient scientific substrate with Kis value of 6.3 (±0.7) μM [21]. Equilibrium dissociation
innovation in the treatment of drug-resistant TB, genotypic changes constants (KD) assessed by the quench in protein fluorescence titration of
associated with resistance have already been described for the first two LABIO-17 binding to MtInhA:NADH binary complex at various tem
of these drugs [7]. Accordingly, there is a continuous need to develop peratures yielded a linear van’T Hoff relationship with changes in
new anti-TB agents that should ideally be more active than the existing enthalpy (ΔH◦ ), entropy (ΔS◦ ), and Gibbs Free energy (ΔG◦ ) values of,
drugs to reduce the treatment time course, be effective against respectively, − 23.3 (±1.9) kcal/mol, − 54.5 (±6.5) cal/mol K, and − 7.1
drug-resistant strains, be compatible with current anti-retroviral ther (±0.8) kcal/mol [21]. The minimum inhibitory concentration (MIC)
apy, and have diminished side effects. values to arrest in vitro growth of M. tuberculosis H37Rv (virulent
The M. tuberculosis NADH-dependent enoyl-acyl carrier protein pan-sensitive ATCC 27294) and PE-003 (multidrug-resistant clinical
(ACP) reductase (MtInhA; EC 1.3.1.9) protein catalyzes the hydride isolate) [22] strains were, respectively, 5 μg/ml (12.8 μM) and 2.5 μg/ml
transfer of the 4S hydrogen of NADH to carbon-3 of long chain enoyl (6.4 μM) [21]. The LABIO-17 compound was also shown to not signifi
thioester substrates to yield NAD+ and acyl-ACP(CoA) products [8], and cantly affect cell viability of the HaCat (human keratinocytes), Vero
has been shown to be the major target of INH [9]. INH is a prodrug that (African green monkey kidney) and RAW 264.7 (murine macrophages)
is activated by the mycobacterial katG-encoded catalase-peroxidase cell lines [21]. Importantly, the LABIO-17 compound (5 μM) resulted in
enzyme (KatG) in the presence of manganese ions, NAD(H) and oxy statistically significant reductions in Colony-Forming Unit (CFU) counts
gen [10–13]. The KatG-produced acylpyridine fragment of INH is in M. tuberculosis-infected macrophages and bactericidal activity [21].
covalently attached to the C4 position of NAD(H), and forms a binary These results suggested that further efforts on this class of compounds
complex with the wild-type MtInhA [14] with a dissociation constant were warranted.
value lower than 0.4 nM [15]. The INH-NAD adduct has been shown to To try to elucidate the structural requirements for biological activity
be a slow, tight-binding competitive inhibitor of wild-type (WT) MtInhA, of this class of compounds to obtain optimized chemical candidates, a
in which the initial rapidly reversible weak binding (Ki = 16 nM) is series of 4-aminoquinolines were synthesized and structure-activity
followed by a slow isomerization leading to a tighter enzyme-inhibitor relationship (SAR) studies were pursued. The compounds were evalu
complex with an overall dissociation constant (Ki*) value of 0.75 nM ated for their ability to inhibit both the reaction catalyzed by MtInhA
[16]. Experimental pieces of evidence suggesting MtInhA as the primary and Mtb growth in vitro. The most effective molecules were further
target of INH include, but are not limited to, genetic data in which a tested against drug-resistant Mtb strains, evaluated for cytotoxicity
point mutation (Ser94Ala) in inhA WT gene conferred INH resistance in (selectivity), and in vitro ADME properties determined. Finally, the ac
M. tuberculosis [17] and thermal inactivation of InhA in Mycobacterium tivity in an in vitro macrophage TB infection model, pharmacokinetics,
smegmatis that resulted in morphological changes to the mycobacterial acute toxicity, and murine TB model were also investigated.
Fig. 1. Chemical structures of bedaquiline, delamanid, preteomanid and LABIO-17. The latter is a 4-aminoquinoline that was discovered by virtual screening using a
pharmacophoric model from the crystallographic structures of the Mycobacterium tuberculosis NADH-dependent enoyl-acyl carrier protein reductase (MtInhA).
2
2. Results and discussion step of chemical synthesis.

The second round of synthetic procedures involved modification at
2.1. Synthesis and characterization of chemical compounds the 4-position of the heterocyclic ring (Scheme 2). For this purpose,
different substituted anilines (14a-l) were used in an aromatic nucleo
Mastering the synthesis route is of paramount importance to allow philic substitution reaction with the ethyl 4-chloroquinoline-6-carbox
multimilligram scale compound production, thereby enabling SAR ylate (12), which was synthesized as described in Scheme 1. The
studies and pre-clinical assays to be carried out. The synthesis of LABIO- chemical reactions of anilines (14a-l) and 12 were carried out in the
17 was designed using 7 conversion steps (Scheme 1). The synthesis presence of pyridine and HCl using isopropanol as solvent. The products
started with the acylation reaction of 4-methylaniline (5) with acetic (15a-l) were obtained after stirring for 16 h at 85 ◦ C with yields ranging
anhydride. The product was obtained with 97% yield after 30 min of from 30 to 99%.
stirring at 50 ◦ C. The second step was the oxidation reaction of N-acetyl- To assess the role, if any, of the C-6 ethyl ester group of LABIO-17 in
4-methylaniline (6) in the presence of potassium permanganate. The biological activity, analogs were synthesized by varying the substituents
reaction mixture was heated at up to 80 ◦ C for 1 h to provide 4-acetyla attached to 6-position of the quinolinic ring (Scheme 3), while the N-(4-
minobenzoic acid (7) with 67% yield. The deprotection reaction was aminophenyl)piperidine substituent was kept at 4-position. The cyclo
carried out under acidic conditions using concentrated HCl. 4-Amino condensation reaction was carried out between the substituted anilines
benzoic acid (PABA) was obtained in 86% yield (8). Esterification re (16a-k) and the β-ketoester ethyl 3-oxobutanoate (10) to obtain the
action of 8 in the presence of sulfuric acid and ethanol after 2 h of reflux desired 4-hydroxyquinolines (17a-k). The reactions were performed in
resulted in ethyl 4-aminobenzoate (9) synthesis with 30% yield. To the presence of magnesium sulfate and acetic acid using ethanol under
obtain the quinolinic ring, the classic Conrad-Limpach reaction between reflux for 16 h. The thermal cyclization of the β-anilinoacrylate in
β-ketoesters (10) and anilines (9) was used. This cyclocondensation re termediates were obtained using Dowtherm® A as the heating transfer
action was performed under experimental conditions as described fluid at 230–250 ◦ C for 15 min to produce the 4-hydroxyquinolines
elsewhere by our research group [23] and the resulting 4-hydroxyquino (17a-k) with yields of 17–48%. These molecules in the presence of
line (11) was isolated with 42% yield. The next step was to obtain ethyl phosphoryl chloride provided the 4-chlorosubstituted compounds (18a-
4-chloroquinoline-6-carboxylate (12) using phosphoryl chloride. The k) with 26–98% yields that were used in subsequent nucleophilic aro
substitution reaction was carried out in toluene under reflux for 2 h matic substitution reactions. The reaction between 4-chloroquinolines
leading to the product with 81% yield. Finally, nucleophilic aromatic (18a-k) and N-(4-aminophenyl)piperidine (13) was performed using a
substitution reaction using a commercially available N-(4-aminophenyl) mixture of pyridine, hydrochloric acid in isopropanol. The products
piperidine (13) led to LABIO-17 (4) synthesis. The reaction was carried (19a-k) were obtained after 16 h of stirring at 85 ◦ C with 33–92% yields
out using isopropanol as solvent under reflux for 2 h and provided this (Scheme 3).
4-aminoquinoline with 54% yield (Scheme 1). It is noteworthy that the As the biological data emerged showing that the halogenated sub
yields here reported were obtained from the isolated products. The stituents (19j and 19k) provided the most effective compounds
yields were calculated using masses of products found in the previous (Table 1), which is characteristic of function-guided chemical changes of
Scheme 1. Reagents and conditions: i = HCl, Ac2O, NaOAc, 30 min, 50 ◦ C; ii = MgSO₄, KMnO4, 1 h, 55–80 ◦ C; iii = HCl, H2O, 1 h, 100 ◦ C; iv = EtOH, H2SO4, 2 h,
80 ◦ C; v = MgSO4, AcOH, EtOH, 90 ◦ C, 16 h; vi = Dowtherm® A, 230–250 ◦ C, 15 min; vii = POCl3, Toluene, 110 ◦ C, 2 h; viii = Pyridine, HCl, (CH3)2CHOH, 85 ◦ C,
16 h.
3
Scheme 2. Reagents and conditions: i = Pyridine, HCl, (CH3)2CHOH, 85 ◦ C, 16 h.
structure-activity relationship efforts, a round of synthesis was pursued example, HPLC chromatogram and UV–Vis spectrum of compound 19k
keeping bromine or chlorine atoms attached to the 6-position of the is given in Supporting Information (Fig. S66).
heterocyclic rings in 18k and 20a-b. Analogs containing ethyl groups
attached to the 2-position of the quinolinic ring were also synthesized.
2.2. Determination of in vitro biological activity
The not yet mentioned chemical compounds in Scheme 4 are the
following: 6-bromo-4-chloro-2-ethylquinoline (20a); 4,6-dichloro-2-
The 4-aminoquinoline derivatives of LABIO-17 were initially evalu
ethylquinoline (20b); 1-(4-aminophenyl pyrrolidin-2-yl)methanol
ated for their ability to inhibit the MtInhA-catalyzed chemical reaction
(21a); and 4-(4-phenylpiperidin-1-yl)aniline (21b). These changes
(IC50) using a protocol described elsewhere [21]. These compounds
were focused on 6-, 4- and 2-positions using different substituents
were also tested as inhibitors in in vitro M. tuberculosis H37Rv growth
employing the synthetic approach described here with yields of products
assay (MIC) by the resazurin reduction method [24] using the anti-TB
(22a-f) in the 40–96% range. Analytical data for all compounds syn
drugs isoniazid (INH) and rifampicin (RIF) as positive controls (Table 1).
thesized were consistent with the chemical structures shown. NMR
Overall, the results described in Table 1 show that there is correla
spectra are given in Supporting Information (Figs. S1–S58). As an
tion between inhibition, or lack, of MtInhA activity and MIC values for
4
Scheme 3. Reagents and conditions: i = MgSO4, AcOH, EtOH, 90 ◦ C, 16 h; ii = Dowtherm® A, 230–250 ◦ C, 15 min; iii = POCl3, Toluene, 110 ◦ C, 2 h; iv = Pyridine,
HCl, (CH3)2CHOH, 85 ◦ C, 16 h.
the 4-aminoquinolines derivatives (15a-l, 19a-k, 22a, c, e-f), with few the MIC value (6.2 μM), showing a 2-fold better activity in comparison
exceptions (22b and 22d). Steric factors and lipophilicity (molecular with the parent compound (4). Interestingly, the presence of a methy
volumes) seem to have more influence on the activities than electronic lene spacer (15d) was detrimental to the inhibitory activity on the
effects of the substituents. The increase in polarity with the insertion of enzyme (>50 μM) and in vitro Mtb growth (49.6 μM). It is tempting to
morpholine (15a) replacing the piperidine group in 4 (LABIO-17) suggest that greater planarity and lower mobility are needed for
maintained the ability to inhibit MtInhA activity (19.5 μM) although improved activity at 4-position of the heterocyclic ring. Alkyl groups
with reduced inhibition of Mtb growth. On the other hand, the thio directly attached to benzene ring (15e-g) demonstrated that the increase
morpholine ring (15b) resulted in reduced inhibitory activity of MtInhA in molecular volume and lipophilicity in this region of the molecule
enzyme activity (>50 μM). The MIC value observed was 51.1 μM for 15a increases enzymatic inhibition and antimycobacterial activities
while 15b was inactive at the highest concentration that could be (Table 1). Compounds 15e-g inhibited MtInhA activity with IC50 values
evaluated (>12.3 μM). Increasing liposolubility and molecular volume of, respectively, 41.3 μM, 19.6 μM and 11.2 μM. The MICs reduced from
with the presence of a methyl group attached to the piperidine ring 29.9 μM (15e) to 6.9 μM (15g). Interestingly, methylene group addition
(15c) improved the enzyme inhibitory activity (10.9 μM) and reduced to the alkyl chain resulted in increased inhibitory activity by a factor of
5
Table 1
ClogP, IC50 and MIC values.
Comps. R1 R2 R3 ClogP IC50 MtInhA (μM) MIC H37Rv (μM)
4 (LABIO17) CO2Et Me 6.34 20 ± 3 12.8
15a CO2Et Me 4.96 19.5 ± 2.3 51.1
15b CO2Et Me 5.79 >50 >12.3
15c CO2Et Me 6.86 10.9 ± 1.3 6.2
15d CO2Et Me 6.53 >50 49.6
15e CO2Et Me Et 6.53 41.3 ± 3.6 29.9

15f CO2Et Me Pr 7.06 19.6 ± 1.2 14.3
15g CO2Et Me But 7.59 11.2 ± 0.9 6.9
15h CO2Et Me 7.24 26.2 ± 1.6 10.4
15i CO2Et Me 7.02 42.1 ± 2.5 >47.3
15j CO2Et Me 5.62 8.0 ± 0.9 >6.7
15k CO2Et Me 5.78 21.7 ± 1.2 13.3
15l CO2Et Me 4.70 44.5 ± 2.8 >49.6
19a MeO Me 5.98 4.7 ± 0.6 3.6
19b EtO Me 6.51 9.5 ± 1.0 6.9
19c MeS Me 6.36 1.0 ± 0.2 0.40
19d CN Me 5.33 19.6 ± 2.0 14.6
19e N(Me)2 Me 6.24 20.0 ± 2.9 6.9
19f NO2 Me 5.62 18.6 ± 1.8 6.9
19g Me Me 6.16 17.2 ± 0.9 7.5
19h iPr Me 7.09 8.2 ± 1.2 6.9
19i F Me 5.91 38.3 ± 3.2 14.9
(continued on next page)
6
Table 1 (continued )
Comps. R1 R2 R3 ClogP IC50 MtInhA (μM) MIC H37Rv (μM)
19j Cl Me 6.48 1.1 ± 0.1 0.9
19k Br Me 6.63 0.23 ± 0.02 0.4
22a Br Me 7.15 1.4 ± 0.2 0.7
22b Br Me 6.69 >50 1.33
22c Br Me 5.94 4.85 ± 0.2 3.3
22d Br Me 5.35 >50 12.1
22e Br Et 7.16 1.54 ± 0.2 0.75
22f Cl Et 7.01 2.90 ± 0.4 1.7
Scheme 4. Reagents and conditions: i = Pyridine, HCl, (CH3)2CHOH, 85 ◦ C, 16 h.
7
approximately 2-fold each. Increases in degrees of freedom may tenta for the chlorine (19j) and 0.49 μM for the bromine (19k) substituents.
tively be invoked to explain this trend. The presence of 1-benzylpipera Compared to LABIO-17 compound (4), changing the C-6 ethyl ester
zine in 4-aminoquinoline (15h) replacing piperidine did not alter the group to bromine (19k) resulted in approximately 87-fold better MtInhA
levels of both enzyme and bacilli inhibition in comparison to 4. The inhibitory activity and 32-fold lower MIC value for in vitro growth of
presence of bulky 1H-benzo[d]imidazole substituent was detrimental to bacilli. The MIC values for the chlorine (19j) and bromine (19k)
both inhibitory activities (15i). Reduction of six-membered ring substituted 4-aminoquinolines exhibited MIC values of, respectively,
(piperidine) to five-membered rings improved inhibitory enzyme ac ~2.6- and ~5.8-fold better (Table 1) than isoniazid (2.3 μM), under the
tivity for pyrazole (15j) and had no apparent positive effect for pyrro experimental conditions here described (Table 4).
lidine (15k). These results suggest that 1,2-diazole and azacyclopentane As the C-6 bromine compound (19k) displayed the best inhibitory
rings are tolerated in this region of the molecule as compared to com properties, a next round of chemical modifications focused on trying to
pound 4. The MIC values were >6.7 μM for 15j and 13.3 μM for 15k, assess the effects, if any, of substitutions of the 4-position of the het
which are similar to 4, though larger concentrations of 15j could not be erocyclic ring (R3, Scheme 4). Methyl piperidine (22a) and pyrazole
tested due to limited solubility in the growth medium. The presence of a (22c), and exploratory benzopiperidine (22b) and C2-methylene hy
carbonyl group between the benzene ring of 4-aminoquinoline and five- droxyl pyrrolidine (22d) modifications were thus pursued (Table 1).
membered pyrrolidine (15l) resulted in approximately 2-fold decrease There was no improvement in inhibitory activity for 22a and 22c as IC50
in enzyme inhibitory activity (44.5 μM) and larger MIC value (>49.6 values were, respectively, 1.4 μM and 4.85 μM, and MIC values were,
μM) as compared to 15k and the parent compound (4). Interestingly, the respectively, 0.7 μM and 3.3 μM (as compared to IC50 = 0.23 μM and
piperidine and the shorter pyrrolidine could be interchangeable with no MIC = 0.4 μM for 19k). Although the IC50 values for the benzopiperidine
decrease in inhibitory activity as borne out by comparing LABIO-17 (4) (22b) and C2 methyl hydroxyl pyrrolidine (22d) were rather large (>50
with 15k, suggesting that the 6- piperidine and 5-atom pyrrolidine rings μM), these compounds showed inhibitory activity of in vitro Mtb growth
may be regarded as bioisosteres for this biological system. (MIC = 1.33 μM and 12.1 μM, respectively). These results suggest that
In the second round of proposed modifications, the roles, if any, of 22b and 22d may engage a yet unknown intracellular target in
substituents at 6-position of the quinolinic ring were evaluated whereas M. tuberculosis. To assess the role of the methyl group attached to C2 (R2)
the N-(4-aminophenyl)piperidine group was retained at the 4-position of of 19k, ethyl (22e) substitution was performed. The ethyl substitution
the chemical derivatives (19a-k). The simplification of the C-6 ethyl (22e) resulted in no better inhibitory activity as compared to 19k
ester group of LABIO-17 (4) to ether and shorter carbon chains (19a and (Table 1). The IC50 (1.54 μM) and MIC (0.75 μM) values for 22e were,
19b) improved inhibitory activity. Interestingly, the shorter 6-methoxyl respectively, 7- and 2-fold larger than 19k (0.23 μM and 0.4 μM). As the
electron-donating group of 19a resulted in approximately 2-fold better C6-chlorine compound (19j) showed favorable biological activity, ethyl
inhibitory activity (IC50 = 4.7 μM and MIC = 3.6 μM) as compared to 6- substitution at C2 (22f) was also evaluated. The IC50 (2.90 μM) and MIC
ethoxyl chemical function of 19b (IC50 = 9.5 μM and MIC = 6.9 μM). It is (1.7 μM) values for 22f were approximately 2-fold larger than 19j (1.1
noteworthy that 6-methoxyquinoline derivatives have been obtained in μM and 0.9 μM).
potent and selective molecules against drug-sensitive and drug-resistant
Mtb strains [23,25]. Notably, bioisosteric replacement of methoxyl
2.3. Steady-state kinetics of MtInhA enzyme inhibition
(19a) by the methylthio group led to one of the most potent molecule in
this series (19c) with IC50 and MIC values of, respectively, 1.0 μM and
Steady-state kinetics measurements were carried out to determine
0.40 μM. These results suggest that steric factors play a role in inhibitory
the mode of MtInhA enzyme inhibition of the 4-aminoquinolines 19j-k,
activity (protein binding) at this particular position. Generally, polar
19c and 22a as they were amongst the compounds that showed micro
izability increases as the volume occupied by electrons increases and it is
and sub-micromolar IC50 values. Before embarking on efforts to deter
thus tempting to suggest that the larger polarizability of the methylthio
mine the mode of inhibition of these compounds, initial velocity mea
as compared to the methoxyl group may result in increased induced
surements as a function of time were evaluated to ascertain whether or
dipole moment with concomitant improvement of van der Waals inter
not they displayed time-dependent inhibition of MtInhA activity. None
molecular interactions. In comparison to LABIO-17 (4), the 19c com
of them showed time-dependent enzyme inhibition (data not shown).
pound was 20-fold more potent as inhibitor of MtInhA enzyme activity
Steady-state kinetics revealed that at varying NADH concentrations
and 32-fold more effective against growth of bacilli (Table 1). Nitrile
(10–160 μM) with fixed-non-saturating concentration of 2-trans-dode
(19d), dimethylamino (19e), nitro (19f), and methyl (19g) groups
cenoyl-CoA (DD-CoA; 45 μM) the inhibition constants ranged from 0.14
attached at 6-position of the heterocyclic ring yielded fairly similar
μM to 0.72 μM (Table 2). For varying DD-CoA concentrations (15–135
inhibitory effects on MtInhA activity as the ethyl ester-substituted
μM) in the presence of fixed-non-saturating NADH concentration (60
LABIO-17 compound (4). Thus, regardless of the electron-donating
μM) the inhibition constant values ranged from 0.14 μM to 1.33 μM
(dimethylamino and methyl) or electron-withdrawing (nitrile and
(Table 2).
nitro) characteristics of the substituents at this position, the enzyme
For all four compounds (19j-k, 19c and 22a), the double reciprocal
inhibitory activity remained similar with IC50 ranging from 17.2 (19g)
plots showed a pattern of lines intersecting to the left of the y-axis which
to 20.0 μM (19e). The 4-aminoquinolines 19e and 19f showed identical
is consistent with non-competitive mode of inhibition towards NADH
MIC values of 6.9 μM whereas 19d and 19g inhibited Mtb growth with
(Fig. 2a for 19k and Fig. S59a for all four compounds), which was also
MIC values of, respectively, 14.6 μM and 7.5 μM. Replacing the C-6 ethyl
displayed by the LABIO-17 (4) compound as reported elsewhere (Kis = 8
ester (4) with isopropyl group (19h) yielded a molecule with more
μM and Kii = 8 μM) [21]. This type of inhibitor displays binding affinity
potent inhibitory MtInhA activity (IC50 = 8.2 μM) and improved anti
mycobacterial killing (MIC = 6.9 μM). Finally, halogens (F, Cl, and Br)
Table 2
were employed as univalent isosteres at 6-position of quinolic ring
Inhibition constants (Kii, Kis) of 19j-k, 19c and 22a compounds of MtInhA
(19i-19k). Notably, the increase in volume and polarizability of halogen
enzyme activity.
atom correlated with the increase in both enzymatic inhibitory and
Comps. NADH DD-CoA
antimycobacterial activities. These results suggest that van der Waals
forces may play a role in MtInhA ligand binding as the value of 38.3 μM Kii (μM) Kis (μM) Kis (μM)
for IC50 for the low polarizable fluorine-containing compound (19i) 19j 0.46 ± 0,02 0.43 ± 0.05 1.33 ± 0.30
decreased to 1.1 μM for the intermediate polarizable chlorine (19j) and 19k 0.15 ± 0.05 0.14 ± 0.01 0.14 ± 0.02
even further to 0.23 μM for the more polarizable bromine (19k) halogen 19c 0.41 ± 0.05 0.43 ± 0.05 0.62 ± 0.07
22a 0.69 ± 0.08 0.72 ± 0.19 0.79 ± 0.13
substituent. The MIC values were 14.9 μM for the fluorine (19i), 0.9 μM
8
Fig. 2. Determination of inhibition mode of 19k (0–0.5 μM). (a) Lineweaver-Burk plot displaying a pattern of lines intersecting at the left of the y-axis, which are
diagnostic of non-competitive inhibition with respect to NADH. The data were thus fitted to Eq. (2). (b) Lineweaver-Burk plot displaying a pattern of lines intersecting
at the y-axis, consistent with competitive mode of inhibition with respect to DD-CoA. These data were fitted to Eq. (3).
for both the free enzyme and the enzyme-substrate complex or subse which provided mesurements of the individual contributions of the
quent species [17], yielding two dissociation constants, one for standard enthalpy (ΔH◦ ) and entropy (ΔS◦ ) to the Gibbs Free energy
enzyme-inhibitor (EI) binary complex (Kis) and one for the ternary change (ΔG◦ ) for the binding processes. This type of analysis provided
enzyme-substrate-inhibitor (ESI) ternary complex (Kii) [26]. The assessments of the thermodynamic signatures of non-covalent in
non-competitive mode of inhibition is likely due to the ability of 19j-k, teractions to each binding process. The van’t Hoff predicts a linear
19c and 22a chemical compounds to make intermolecular interactions relationship between the association constant at equilibrium (KA =
with the large enoyl thioester binding site of MtInhA even in the absence 1/KD) and ΔH◦ (slope) and ΔS◦ (intercept on the ordinate) for a binding
of NADH. The fairly similar Kii and Kis values (Table 2) suggest that process, in which the two latter thermodynamic parameters are inde
NADH binding to MtInhA has no effect on the overall inhibition con pendent of temperature (as long as change in heat capacity at constant
stants of 19j-k, 19c and 22a compounds. The Lineweaver-Burk plots for pressure, ΔCp, is independent of temperature). Table 3 gives the KD,
DD-CoA as the variable substrate reveal a family of lines intersecting at ΔH◦ , ΔS◦ and ΔG◦ values for ligand binding to MtInhA:NADH binary
the y-axis (Fig. 2b for 19k and Fig. S59b for all four compounds) indi complex.
cating competitive type of inhibition for all tested compounds (Table 2), Titration of MtInhA:NADH complex with 19k displayed hyperbolic
in agreement with data reported for the LABIO-17 (4) (Kis = 6.3 μM) curves at all tested temperatures (Fig. 3), yielding KD values of 0.28
[21]. (±0.02) μM, 0.47 (±0.03) μM, 0.81 (±0.04) μM and 1.34 (±0.13 μM) at,
This type of inhibition predicts that the 19j-k, 19c and 22a com respectively, 15 ◦ C, 20 ◦ C, 25 ◦ C and 30 ◦ C. The data for 19c (Fig. S60),
pounds and the DD-CoA substrate compete for binding to the active site 19j (Fig. S61) and 22a (Fig. S62) are given in Supporting Information. A
of MtInhA enzyme. The results indicate that the modifications made to linear relationship was found between the natural logarithm of KD and
LABIO-17 have not changed the steady-state kinetics mode of inhibition the inverse of Kelvin temperature for 19k (Fig. 4), which suggests that
of both substrates, likely due to the fact that the modifications are rather the ΔH◦ and ΔS◦ are independent of the temperature and isobaric heat
small in relation to the cavity of the enzyme active site. Replacement of capacity of the system (ΔCp) remained constant. The van’t Hoff linear
the C-6 ethyl ester group (4) by either Cl- (19j) or Br- (19k) halogens or relationships for 19c (Fig. S63), 19j (Fig. S64) and 22a (Fig. S65) are
methylthio group (19c) improved the overall inhibition constants to given in Supporting Information, and thereby the same conclusions
wards both substrates. Towards NADH (non-competitive), decreases of arrived to 19k apply to these compounds. The hyperbolic saturation
17- and ~19-fold for 19j, 53- and 57-fold for 19k, ~19- and ~18-fold for curves for ligand binding at all temperatures (Fig. 3) suggest that there is
19c, and ~12- and 11-fold for 22a, were determined for, respectively, no cooperativity upon 19k binding to MtInhA:NADH binary complex, as
Kii and Kis values (Table 2). Towards DD-CoA (competitive), decreases of also shown for 19c, 19j and 22a (Fig. S60, S61 and S62, respectively). It
~5-fold for 19j, 45-fold for 19k, ~10-fold for 19c, and ~8-fold for 22a, is interesting to note that the lower ΔH◦ for 22a is compensated by
were determined for Kis values (Table 2). Interestingly, the most increased ΔS◦ resulting in rather similar overall dissociation constant
noticeable effect was on the overall inhibition constants towards NADH (KD) value as compared to 19j, 19k and 19c compounds (Table 3). The
and DD-CoA for 19k compound, in which the C-6 ethyl ester group was methyl group attached to the 4-position of the piperidine heterocyclic
replaced by Br-halogen. The C-6 thiomethyl substitution (19c) has also ring (R3, Scheme 4) was detrimental to interatomic interactions between
resulted in improved inhibitory activity, though to a lower extent. As the MtInhA:NADH binary complex and 22a (lower though favorable
compared to 19k, replacing piperidine (19k) with methyl piperidine ΔH◦ ) that was compensated by positive ΔS◦ (favorable) value, which
(22a) was slightly detrimental to enzyme inhibition constants (Table 2),
even though the latter showed improved activity as compared to the Table 3
parent compound (4). Dissociation constant and thermodynamics parameters for binding of 19j-k, 19c
and 22a chemical compounds to MtInhA:NADH binary complex determined by
fluorescence spectroscopya.
2.4. Thermodynamics of ligand binding by fluorescence spectroscopy
1
KD (μM) ΔH◦ (kcal ΔS◦ (cal mol− ΔG◦ (kcal
mol− 1) K− 1) mol− 1)
The thermodynamics parameters of binding of compounds 19j, 19k,
19c and 22a were determined by monitoring the quench in intrinsic 19j 0.90 ± − 23.2 ± 1.9 − 49.8 ± 6.4 − 8.3 ± 1.1
protein fluorescence upon ligand binding at various temperatures. A 0.07
19k 0.81 ± − 18.3 ± 0.3 − 33.4 ± 1.0 − 8.3 ± 0.3
direct analysis of ligand interactions is most suitably carried out by 0.04
isothermal titration calorimetry (ITC) [27–29]. Nonetheless, owing to 19c 0.87 ± − 25.8 ± 1.6 − 55.7 ± 5.3 − 8.3 ± 0.7
solubility issues that prevented reliable ITC data collection, fluorescence 0.07
spectroscopy and van’t Hoff analysis were employed to evaluate the 22a 1.25 ± − 5.6 ± 0.3 8.3 ± 1.1 − 8.0 ± 1.0
0.06
thermodynamics of ligand binding. Accordingly, dissociation constant
values (KD) were determined as a function of variable temperature, a
Dissociation constant values determined at 298.15 K (25 ◦ C).
9
Table 4
In vitro activity of the selected 4-aminoquinolines against M. tuberculosis H37Rv, MDR strains, and evaluation of the viability of HepG2 and Vero cells.
Entry MIC H37Rv (μM) MIC PT2 (μM) MIC PT12 (μM) MIC PT20 (μM) CC50a HepG2 (μM) CC50a Vero (μM) SId HepG2 SId Vero
b c b c b c
19c 0.4 0.1 0.2 0.1 5.9 ; 12.2 11.1 ; 8.5 14.7 ; 30.5 27.7b; 21.2c
19j 0.9 0.4 0.9 0.2 >20b; 16.4c 19.2b; 11.7c >22.2b; 18.2c 21.3b; 13c
19k 0.4 0.1 0.2 0.1 >20b; 18.3c >20b; 12.2c >50b; 45.7c >50b; 30.5c
22a 0.7 0.2 0.2 0.1 5.3b; 12.2c 9.7b; 6.2c 7.6b; 17.4c 13.8b; 8.9c
INH 2.3 291.7 145.8 291.7 – – – –
RIF 0.05 >48.6 >48.6 >48.6 – – – –
a
The toxicity and selectivity of the compounds was studied on HepG2 and Vero cells. The results were expressed as the concentration capable of reducing cell
viability by 50% (CC50) by MTT and Neutral Red assays.
b
Determined by the MTT method.
c
Determined by the Neutral Red method.
d
Selectivity index (SI = CC50/MIC H37Rv). INH, Isoniazid. RIF, Rifampin.
Fig. 3. Fluorescence spectroscopy of the equilibrium binding of compound 19k. Changes in intrinsic protein fluorescence upon 19k binding to MtInhA:NADH binary
complex were plotted as the relative fluorescence change as a function of increasing chemical compound concentration at 15 ◦ C (a), 20 ◦ C (b), 25 ◦ C (c), and
30 ◦ C (d).
may suggest that release of “bound” water molecules to the bulk solvent
increased the degree of disorder of the bound as compared to free system
even though a reduction in conformational states upon ternary complex
formation conceivably made an unfavorable entropic contribution.
2.5. Susceptibility against in vitro growth of drug-resistant M. tuberculosis

strains
The 19c, 19j-k and 22a chemical compounds were evaluated for
inhibitory activity against a panel of multidrug-resistant M. tuberculosis
strains (Table 4). M. tuberculosis strains PT2, PT12, and PT20 have been
described as resistant to drugs such as isoniazid, rifampin, streptomycin,
ethionamide, and rifabutine [30]. The PT12 and PT20 strains are also
resistant to pyrazinamide and ethambutol, and PT12 present additional
resistance to amikacin and capreomycin. Whole genome sequencing has
revealed the genotypic alterations related with resistant phenotypes of
these strains [30]. The 4-aminoquinolines 19c, 19j–k, and 22a exhibited
similar or even more potent inhibition of in vitro growth of drug-resistant
PT2, PT12, and PT20 as compared to drug-susceptible M. tuberculosis
Fig. 4. Dissociation constant as a function of temperature for 19k. The curve H37Rv strain (Table 4). For the PT2 strain, 19c was 4-fold more potent
was fitted to the van’t Hoff equation (Eq. (5)) allowing determination of ΔHo whereas 19j, 19k and 22a were, respectively, 2.25-, 4-, and 3.5-fold
and ΔSo values. Data are expressed as the means ± SD. more effective in vitro growth inhibitors as compared to
10
drug-susceptible M. tuberculosis H37Rv strain (Table 4). The genome of 2.8. Chemical and metabolic stability, solubility and permeability of 19c,
PT2 strain harbors an S94A mutation in the inhA coding sequence of 19j and 19k compounds
M. tuberculosis InhA [30], which is associated with isoniazid resistance.
It is noteworthy that this mutation did not reverse the antimycobacterial Chemical stability was determined upon incubations at 37 ◦ C for 24 h
activity of 19c, 19j–k, and 22a 4-aminoquinoline derivatives (Table 4). at different pH values for 19c, 19j and 19k 4-aminoquinolines (Table 5).
The activity of the structures against PT12 and PT20 strains showed a Notably, these molecules were fairly stable at pH 1.2. The 19j and 19k
similar profile. 4-Aminoquinolines 19c, 19j–k, and 22a exhibited either compounds showed no change in concentration at pH 7.4, whereas
similar or better inhibitory activity against growth of drug-resistant reduction of more than 30% in 19c concentration indicated reduced
PT12 and PT20 strains (Table 4). The genome of M. tuberculosis PT12 stability at neutral pH (Table 5). On the other hand, 19j and 19k showed
and PT20 strains harbour S315T mutation in the katG gene that encodes reduced stability at basic pH, whereas no concentration change was
the catalase-peroxidase enzyme, which has been implicated in isoniazid detected for the 6-thiomethyl-substituted 19c compound at pH 9.1
resistance [30]. These results suggest that, unlike isoniazid, the mole (Table 5). The 6-chlorine-substituted 19j molecule lost more than 75%
cules 19c, 19j–k, and 22a appear not to require the catalase-peroxidase of the initial concentration after 24 h of incubation at pH 9.1. This effect
activation mechanism. Moreover, taken together, these findings was not so drastic on 6-brominated-derivative 19k which had its con
demonstrate that the 19c, 19j–k, and 22a 4-aminoquinolines do not centration reduced by approximately 50% under the same experimental
share in vitro cross-resistance with important and clinically useful conditions. Larger solubility at acidic pH (1.2) compared with a neutral
anti-TB drugs as they are shown to be active against drug-susceptible one (7.4) was observed for 19c, 19j and 19k (Table 5). At pH 1.2 the
and MDR M. tuberculosis strains (Table 4). solubility of the molecules was greater than 2.5 mM at 24 ◦ C after 4 h of
stirring. The 4-aminoquiline 19c was the most soluble structure at pH
2.6. Cytotoxicity evaluation 7.4 with equilibrium concentration of 38 μM. The chlorinated (19j) and
brominated (19k) analogs showed soluble concentrations at pH 7.4 of
Building on these promising results, toxicity was evaluated in HepG2 18.6 μM and 3.9 μM, respectively (Table 5). Passive permeability was
and Vero cells that allowed to determine selectivity of 19c, 19j–k, and thereafter assessed using the parallel artificial membrane permeability
22a compounds (Table 4). These 4-aminoquinolines were incubated for assay (PAMPA) [34,35] for 19c, 19j and 19k compounds (Table 5).
72 h and viability was determined by using MTT [31] and Neutral Red Compound 19c exhibited a value of 0.03 × 10− 6 cm/s, which has been
[32] protocols, which assess, respectively, mitochondrial viability attributed to low permeability structures (≤1 × 10− 6 cm/s) [36]. By
(reflecting the number of viable cells present) and lysosomal homeo contrast, 19j and 19k presented values consistent with high perme
stasis (loss of neutral red uptake by lysosomes corresponds to loss of cell ability structures (≥1 × 10− 6 cm/s) [36]. The metabolic stabilities of
viability). Overall, molecules 19c and 22a showed the lowest CC50 19c, 19j and 19k were determined using rat liver microsomes fractions
(concentration capable of reducing cell viability by 50%) in HepG2 and (Table 5). Compounds 19c and 19j showed high elimination rates based
Vero cell lines, whereas these values were larger for 19j and 19k on rat liver intrinsic clearance values (Clint > 47 mL/min/kg) [36]. By
(Table 4). The lowest selectivity index values (SI = CC50/MIC) were contrast, molecule 19k exhibited a moderate elimination rate under the
determined for 22a (Table 4). As suggested by the Tuberculosis Anti evaluated conditions (47< Clint >16 mL/min/kg) [36]. Interestingly,
microbial Acquisition & Coordinating Facility of USA, for a compound to the brominated structure 19k demonstrated a half-life that was more
move forward through screening programs SI should be larger than 10 than 3.7-fold longer than thiomethyl- and chlorinated-derivatives,
[33]. Accordingly, as 19c, 19j and 19k showed SI values consistently respectively, 19c and 19j.
larger than 13 (Table 4), further efforts were deemed warranted.
2.7. Evaluation of possible broad spectrum activity 2.9. Intracellular activity of 19j and 19k in macrophage model of
M. tuberculosis infection
The compounds 19c, 19j and 19k were evaluated against Staphylo
coccus aureus ATCC 25923, Escherichia coli ATCC 25922, and the As the 4-aminoquinoline 19c compound showed low permeability
multidrug-resistant clinical isolates Acinetobacter baumannii and Klebsi and low metabolic stability, it was deemed appropriate to proceed with
ella pneumoniae. Using a concentration of 20 μM, none of the structures the halogenated derivatives 19j–k in further efforts. In order to evaluate
were able to inhibit bacterial growth, denoting specificity of these the ability of these compounds to cross cell membranes and inhibit
compounds for M. tuberculosis in vitro growth inhibition (data not intracellular growth of bacilli, molecules 19j–k were evaluated in a
shown). It should, however, be pointed out that a limited panel of mi macrophage model of M. tuberculosis infection [37]. Macrophages from
croorganisms has been evaluated. the untreated group showed an increase of approximately 1.2 log10
Colony Forming Units (log10 CFU), within five days, compared to the
early control group (Table 6), thereby showing intracellular growth of
bacilli in macrophages. Treatments with 19j–k prevented bacterial
Table 5
Chemical stability, solubility, permeability, and metabolic stability of the 4-aminoquinolines 19c, 19j–k.
Entry Chemical Stabilitya Solubilityb Permeability Metabolic Stability
pH 1.2c (%) pH 7.4d (%) pH 9.1e (%) pH 1.2c (mM) pH 7.4d (μM) PAMPA (10− 6
cm/s) Clintf (mL/min/kg) t1/2g (min)
19c 100 68 100 >2.7 38.0 0.03 60 5.9

19j 98.6 100 24.7 >2.8 18.6 11.5 60.5 5.9
19k 94.6 100 47 >2.5 3.9 3.16 36.2 22
a
Percentage of remaining compound after incubation at 37 ◦ C for 24 h.
b
Solubility determined after incubation and stirring of compound suspensions at 25 ◦ C for 4 h.
c
0.1 M HCl solution.
d
PBS.
e
0.1 M NH4HCO3 solution.
f
Rat liver microsomes intrinsic clearance.
g
Half-life.
11
Table 6 safety profile (Fig. 5). Although 19k showed lower solubility than 19j at
Intracellular activity of compounds 19j–k in murine macrophages pH 7.4 (Table 5), the larger metabolic stability and longer half-life may
infected with the virulent M. tuberculosis H37Rv strain. be translated into greater pharmacokinetic exposure. The possibility of
Entry log10 CFU/well (Mean ± SD) the 19k producing acute toxicity when orally administered (gavage) in
Early Control 3.02 ± 0.26
mice was studied first. This experiment was carried out with two ob
Untreated 4.19 ± 0.41** jectives: 1) to evaluate the acute toxicity of compound 19k and/or of its
19j (5 μM) 2.39 ± 0.04### possible metabolites; 2) to rationalize the use of animals as high toxicity
19k (5 μM) 2.74 ± 0.19## would require a larger number of mice to assess its effectiveness. The
SD, standard deviation; **P < 0.05 compared to early control group; administration of a single dose of 300 mg/kg (≈757 μM/kg) was not able
##
P < 0.01 compared to untreated (0.5% DMSO) group; ###P < to generate any indicative of toxicity in the animals (n = 6) observed for
0.001 compared to untreated (0.5% DMSO) group. 14 days (Supporting Information, Table S1). Furthermore, no deaths
were observed and there was no significant change in weight of treated
growth and kept the bacterial loads stable inside the macrophages at animals versus the group that received only the vehicle (Table S1). After
values statistically comparable to the early control group (Table 6). 14 days from administration, the animals were euthanized and kidneys,
These data show bacteriostatic effect of 4-aminoquinolines 19j–k on liver and spleen were dissected and evaluated in relation to weight and
intracellular growth of M. tuberculosis (P < 0.01). macroscopic properties. No significant differences were observed
compared to the vehicle group (Table S1).
A preliminary experiment was carried out to measure and quantify
2.10. Genotoxicity evaluation of 19j-k
the ability of compound 19k to reach the systemic circulation after oral
administration in mice (data not shown). After gavage administration of
In order to shed light on the possible toxicity of the 4-aminoquino
300 μM/kg of 19k, a maximum plasma concentration (Cmax) value of 55
lines under study, genotoxicity evaluation using the alkaline comet
μM was obtained after 30 min. These data demonstrated that 4-amino
assay was performed [38]. The results demonstrated that compounds
quinoline 19k can reach the systemic circulation at concentration
19j–k did not produce DNA damage at the evaluated experimental
value 137-fold larger than the MIC (0.4 μM) for the H37Rv strain
conditions (Fig. 5), indicating favorable safety profile for the 4-amino
(Table 1). Accordingly, acute toxicity data and preliminary absorption
quinolines 19j–k.
results provided further pieces of evidence to qualify 19k for evaluation
of antituberculosis activity in a TB mice model. After 7 days of infection,
2.11. In vivo model of tuberculosis infection the experimental groups were treated by 14 days with a daily dose of
300 μM/kg and 450 μM/kg of 19k [39]. As positive controls, isoniazid
These data prompted further studies including the evaluation of the (INH) and ethambutol (EMB) were used. After treatment the animals
in vivo effectiveness of 19k as a representative of this 4-aminoquinoline were euthanized, and their lungs and spleens dissected. The lung and
class of chemical compounds. Choosing 19k for in vivo studies was based spleen were placed in saline solution and disrupted in a glass tissue
on a number of reasons: 1) low IC50 and MIC values (Table 1), 2) low in homogenizer, and the resulting suspension seeded in culture medium to
vitro enzyme inhibition constant values (Table 2), 3) not inferior ΔG◦ determine CFU counts. Unlike INH, no molecules tested (EMB or 19k)
value in comparison to 19j and 19c (Table 3), 4) fairly good activity reversed mycobacterial growth in the spleen and lungs of mice (Sup
against drug-resistant strains (Table 4), 5) amongst the best selectivity porting Information, Table S2). Both evaluated concentrations of 19k
index values (Table 4), 6) favorable chemical stability at pH 7.4 (300 μM/kg and 450 μM/kg) were able to reduce the growth of bacilli in
(Table 5), 7) high passive permeability (Table 5), 8) moderate elimi the lungs of mice when compared to the group that received only the
nation rate and longer half-life (Table 5), 9) intracellular growth inhi vehicle (p < 0.05 and p < 0.01) (Fig. 6). Compound 19k showed no
bition activity (Table 6), and 10) no genotoxicity indicating favorable difference when compared to the early control, defined as the initial
bacterial load, denoting a bacteriostatic action under the experimental
conditions employed. Notably, the action of 19k molecule was not
different from that presented by first line drug EMB administered at 300
μM/kg. This drug administered by gavage at 300 μM/kg presented an
adjusted p value of 0.11 and 0.18 when compared with treated groups
with 300 μM/kg and 450 μM/kg of 19k, respectively. Among the mol
ecules evaluated, only INH at 150 μM/kg was able to reduce bacterial
load compared to early control and vehicle (Fig. 6). Thus, as expected,
INH showed bactericidal activity against M. tuberculosis under the
experimental conditions evaluated. It is also noteworthy that there were
no significant changes in body weight between the groups of animals
during the experiment (Table S2). A trend is observed for weight gain in
the groups treated with INH, EMB and 19k (Table S2). These results may
be indicative of reduced infection as the vehicle showed the opposite
trend, but further studies are needed to clarify this point.
3. Conclusions
Herein, potent inhibitors of MtInhA enzyme activity were synthe

sized. The synthetic process involved simple and easily accessible re
agents and reactants employing well-established protocols. The
Fig. 5. DNA damage index measured by alkaline comet assay in HepG2 cells
incubated for 24 h with 19j-k. NC = control vehicle group (0.5% DMSO), MMS compounds were designed stepwise from LABIO-17 (4), an MtInhA in
= methyl methanesulfonate, positive control group. Data were expressed as the hibitor identified by in silico pharmacophore-based screening [20,21].
mean ± standard error from two independent experiments. Statistical analysis The most potent 4-aminoquinoline compounds were able to inhibit the
was performed by one-way analysis of variance followed by Dunnett’s post-test. enzyme with IC50 and Ki in the submicromolar range with a
***p < 0.001. non-competitive inhibition mode with respect to NADH and competitive
12
values of, respectively, 6.3 μM 0.14 μM. Fluorescence spectroscopy and

van’t Hoff analysis of ligand binding to MtInhA:NADH binary complex
showed approximately 9-fold lower overall dissociation constant for
19k (KD = 0.81 μM) in comparison with LABIO-17 (KD = 7.2 μM) [21].
Although it is tempting to suggest that 19k is more efficient than
LABIO-17 for killing drug-resistant strains of M. tuberculosis, with lower
MIC values for the former (0.1 μM for PT2, 0.12 μM for PT12, and 0.1 μM
for PT20) than the latter (6.4 μM for PE-003), it shoul be considered with
caution as these strains present different genetic backgrounds [22,30].
Interestingly, intracellular activity of compounds 19k and LABIO-17
[21] in murine macrophages infected with the virulent M. tuberculosis
H37Rv strain showed the former as bacteriostatic and the latter bacte
ricidal. However, it should be pointed out that the concentration
employed in these infected Mtb-macrophage murine cell line (RAW
264.7) was 4-fold larger for LABIO-17 (20 μM) [21] than 19k (5 μM).
Selectivity index, chemical and metabolic stability, solubility and
permeability, and in vivo data are not yet available for LABIO-17, hence
comparisons will have to await further experimental efforts. Notwith
standing, 19k compares favorably well with direct MtInhA inhibitors
that have been reported. Recent examples of direct MtInhA inhibitors
with antimycobacterial activity include thiourea-based compounds
[40], thiadiazolylhidrazones [41], and diazoborines [42], to name a
Fig. 6. CFU counts in lungs from M. tuberculosis-infected mice after treatment few. Comprehensive reviews on direct MtInhA inhibitors are given
with 19k (300 μM/kg and 450 μM/kg) for 14 days. Data representing the mean
elsewhere [43–45]. The results here presented suggest that 19k repre
± standard deviation. Statistical analysis was determined by one-way analysis
sents a lead compound for the development of novel oral drugs to treat
of variance (ANOVA), followed by Tukey’s multiple comparisons test using
GraphPad Prism 9.0.0 (San Diego, CA, USA). ###p < 0.001 significantly from drug-sensitive and drug-resistant tuberculosis.
early control group. *p < 0.05, **p < 0.01, and ***p < 0.001 significantly from
vehicle group. N = 4–11 animals per group. 4. Experimental section
inhibition mode considering DD-CoA as variable substrate. The inhibi 4.1. Synthesis and structure: apparatus and analysis
tion of MtInhA, a validated target for anti-TB drug development, seems
to be part of the mode of action of these compounds, which showed The commercially available reactants and solvents were obtained
minimal inhibitory concentration (MIC) as low as 0.40 μM (19k) against from commercial suppliers and were used without additional purifica
pan-sensitive M. tuberculosis H37Rv strain. This finding represented an tion. The melting points were measured using a Microquímica MQAPF-
increment of 32-fold in inhibitory potency compared to that presented 302 apparatus. 1H and 13C NMR spectra were acquired on an Avance III
by the hit compound LABIO17 (4). In addition, the most potent 4-amino HD Bruker spectrometer (Fällanden, Switzerland). Chemical shifts (δ)
quinolines were able to inhibit M. tuberculosis drug-resistant strains with were expressed in parts per million (ppm) relative to DMSO‑d6, which
reduced MIC values in comparison to INH and RIF. These results showed was used as the solvent, and to TMS, which was used as the internal
that this class of compounds does not appear to share the same mech standard. High-resolution mass spectra (HRMS) were obtained on an
anisms of action of the main drugs in clinical use, including those that LTQ Orbitrap Discovery mass spectrometer (Thermo Fisher Scientific,
have the MtInhA as molecular target. The viability of HepG2 and Vero Bremer, Germany). This system combines an LTQ XL linear ion-trap
cells were used to assess the selectivity of the most potent structures mass spectrometer and an Orbitrap mass analyzer. The analyses were
obtained in the mycobacterial growth inhibition assays. Selectivity performed through the direct infusion of the sample in MeOH/CH3CN
index values indicated that some molecules (19c, 19j-k) have cytotox (1:1) with 0.1% formic acid (flow rate of 10 μL/min) in positive-ion
icity consistent with drug-like properties. Furthermore, the most effec mode using electrospray ionization (ESI). For the elemental composi
tive and selective compounds exhibited good chemical stability at tion, the calculations used the specific tool included in the Qual Browser
different pH conditions, high solubility in acidic medium, high perme module of Xcalibur (Thermo Fisher Scientific, release 2.0.7) software.
ability determined by PAMPA assay, and high to moderate elimination The compounds’ purities were determined using a Dionex Ultimate 3000
rates in rat liver microsomes. The 19j-k compounds were able to inhibit UHPLC chromatograph (Dionex Corporation, Germering, Germany).
intracellular growth of bacilli in a macrophage model of M. tuberculosis Chromeleon software (version 6.80 SR11 Build 3160 (183147)) was
infection, and showed no signs of genotoxicity in the alkaline comet used for data acquisition and processing. The liquid chromatography
assay. Compound 19k was selected for studies in M. tuberculosis mouse conditions were as follows: RP column, 5 mm Nucleodur C-18ec (250 ×
infection model, showing absence of in vivo acute toxicity and bacte 4.6 mm); flow rate, 1.5 mL min− 1; UV detection, 260–280 nm; 100%
riostatic effect similar to ethambutol (a first-line anti-TB drug). The water (0.1% acetic acid) was maintained from 0 to 7 min, followed by a
structure-activity data here described resulted in 19k as the best linear gradient from 100% water (0.1% acetic acid) to 90% acetonitrile/
candidate of this class of compounds for further evaluation aiming at the methanol (1:1, v/v) from 7 to 15 min (15–30 min), and subsequently
development of a chemotherapeutic agent to treat TB. In short, 19k returned to 100% water (0.1% acetic acid) in 5 min (30–35 min) and
chemical compound showed lower MIC (0.40 μM) than LABIO-17 (12.8 maintained for an additional 10 min (35–45 min). All the evaluated
μM) against pan-sensitive H37Rv strain of M. tuberculosis, lower IC50 for compounds were ≥95% pure.
MtInhA enzyme inhibition (0.23 μM for 19k, 20 μM for LABIO-17).
Steady-state kinetics data showed that LABIO-17 and 19k were 4.2. Procedure for the synthesis of N-(4-methylphenyl)-acetamide (6)
non-competitive inhibitors towards NADH with the latter displaying
lower inhibition constants (Kii = 0.15 μM and Kis = 0.14 μM) in com p-Toluidine (5, 4 g, 37.3 mmol) was added to a solution of water
parison to the former (Kii = 8 μM and Kis = 8 μM) [21]. Towards DD-CoA (100 mL) and HCl 37% (4 mL). The reaction mixture was stirred at 50 ◦ C
substrate, LABIO-17 [21] and 19k are competitive inhibitors with Kis for 20 min. Afterwards, to the resulting solution was added acetic an
hydride (4.2 mL, 44.4 mmol) and thereafter a solution of sodium acetate
13
(6.5 g, 79.2 mmol) in water (10 mL). The reaction mixture was stirred at 4.7. Procedure for the synthesis of ethyl 4-chloro-2-methylquinoline-6-
50 ◦ C for additional 10 min. After cooling to room temperature, a white carboxylate (12)
solid was obtained. This solid was filtered off, washed with cold water
and subsequently dried under reduced pressure to afford the product in To a solution of 4-hydroxy-2-methyl-6-quinolinecarboxylate (11,
good purity. White solid; Yield 5.4 g (97%); m.p.: 148–150 ◦ C; UHPLC: 0.65 g, 2.8 mmol) and toluene (10 mL) was added phosphorus(V) oxy
97% (tR = 15.74 min); FTMS (ESI) m/z: 150.0925 [M+H]+; calcd. for chloride (0.65 mL, 6.9 mmol). The reaction mixture was stirred at
C9H11NO: 150.0913. 110 ◦ C for 2 h. Thereafter, excess phosphorus(V) oxychloride was
removed under reduced pressure and the resulting residue was cooled in
4.3. Procedure for the synthesis of 4-(acetylamino)-benzoic acid (7) an ice bath and neutralized with saturated sodium bicarbonate solution.
The product was extracted with ethyl acetate (3 × 50 mL), the organic
To a mixture containing N-(4-methylphenyl)-acetamide (6, 4 g, 26.8 phases were combined and treated with anhydrous sodium sulfate, and
mmol) and magnesium sulfate in water (50 mL) at 55 ◦ C was added the solvent evaporated under reduced pressure. Finally, the solid
potassium permanganate (KMnO4) (15 g, 94.9 mmol). The resulting resulting was dried under reduced pressure to afford the product in good
reaction mixture was stirred at 80 ◦ C for 1 h. Afterwards, ethanol was purity. Pale red solid; Yield 0.57 g (81%); m.p.: 109–111 ◦ C; UHPLC:
added to mixture kept stirring for another 5 min. The resulting mixture 99% (tR = 18.96 min); FTMS (ESI) m/z: 250.0630 [M+H]+; calcd. for
was filtered off using a Celite cartridge and the solid formed was washed C13H12ClNO2: 250.0629.
with hot water. Finally, the aqueous phase (colorless) was cooled in an
ice bath and slowly acidified with a solution of sulfuric acid (20%) until 4.8. Procedure for the synthesis of ethyl 2-methyl-4-((4-(piperidin-1-yl)
complete precipitation of the product. This solid was filtered off, washed phenyl)amino)quinoline-6-carboxylate (4, LABIO-17)
with cold water and subsequently dried under reduced pressure to afford
the product in good purity. White solid; Yield 3.2 g (67%); m.p.: The mixture of ethyl 4-chloro-2-methylquinoline-6-carboxylate (12,
259–261 ◦ C; UHPLC: 93% (tR = 14.08 min); FTMS (ESI) m/z: 180.0669 0.53 g, 2 mmol), 4-(piperidin-1-yl)aniline (0.57 g, 3 mmol), pyridine
[M+H]+; calcd. for C9H9NO3: 180.0655. (0.24 mL) and HCl 37% (0.2 mL) in isopropanol (15 mL) was stirred at
85 ◦ C for 16 h. After this time, the solvent was removed under reduced
4.4. Procedure for the synthesis of 4-aminobenzoic acid (8) pressure and the residue was treated with saturated sodium bicarbonate
solution. The solid obtained was filtered off and dissolved in ethyl ace
4-(Acetylamino)-benzoic acid (7, 1 g, 5.6 mmol) diluted in a solution tate (50 mL). Finally, the ethyl acetate was washed with water (3 × 15
(30 mL) of water and HCl 37% (1:1; v/v) was stirred at 100 ◦ C for 1 h. mL), the organic phase was dried with anhydrous magnesium sulfate,
Thereafter, the solution was cooled on an ice bath and alkalinized to pH and the solvent evaporated under reduced pressure. The product iso
8 with ammonia hydroxide. Under continuous stirring, the product was lated as green solid was purified by successive washes with hot hexane
precipitated after glacial acetic acid (3 mL) addition. Finally, the solid (4 × 10 mL). Pale green solid; Yield 0.42 g (54%); m.p.: 215–216 ◦ C;
was filtered off and dried under reduced pressure to afford the product in UHPLC: 96% (tR = 18.04 min); 1H NMR (400 MHz, DMSO‑d6) δ ppm
good purity. Yellow solid; Yield 0.66 g (86%); m.p.: 185–187 ◦ C; UHPLC: 1.36 (t, J = 7.2 Hz, 3 H), 1.51–1.65 (m, 6 H), 2.39 (s, 3 H), 3.13 (m, 4 H),
98% (tR = 12.36 min); FTMS (ESI) m/z: 138.0561 [M+H]+; calcd. for 4.4 (q, J = 7.0 Hz, 2 H), 6.54 (s, 1 H), 6.99 (d, J = 9.0 Hz, 2 H), 7.18 (d, J
C7H7NO2: 138.0550. = 9.0 Hz, 2 H), 7.77 (d, J = 8.6 Hz, 1 H), 8.07 (dd, J = 8.6, 1.6 Hz, 1 H),
9.04 (d, J = 1.6 Hz, 1 H), 9.10 (s, 1 H, NH). 13C NMR (101 MHz,
4.5. Procedure for the synthesis of ethyl 4-aminobenzoate (9) DMSO‑d6) δ ppm 14.3, 23.9, 25.3, 25.3, 49.7, 60.7, 100.8, 109.5, 116.6,
117.2, 124.6, 125.2, 125.3, 128.3, 128.7, 130.6, 149.1, 150.4, 150.8,
The solution of 4-aminobenzoic acid (8, 1.85 g, 13.5 mmol) in 161.2, 165.8; FTMS (ESI) m/z: 390.2176 [M+H]+; calcd. for
ethanol (10 mL) was cooled using ice bath for 20 min. To this solution C24H27N3O2: 390.2176.
was slowly added concentrated sulfuric acid (0.6 mL). Afterwards, the
reaction mixture was stirred at 80 ◦ C for 2 h. After returning to room 4.9. General procedure for the synthesis of 4-aminoquinolines 15a–l
temperature, the reaction mixture was treated with sodium bicarbonate
until pH 7.4. Finally, the solid obtained was filtered off and dried under The compounds 15a-l were synthesized from 4-chloro-2-methylqui
reduced pressure to afford the product in good purity. White solid; Yield noline-6-carboxylate (12) using the same reported procedure for
0.67 g (30%); m.p.: 89–91 ◦ C; UHPLC: 96% (tr = 15.94 min); FTMS (ESI) obtaining the 4-aminoquinoline 4 (LABIO-17). The anilines 14-l were
m/z: 166.0874 [M+H]+; calcd. for C9H11NO2: 166.0863. commercially available.
4.6. Procedure for the synthesis of ethyl 4-hydroxy-2-methyl-6-quinoli 4.9.1. Ethyl 2-methyl-4-((4-morpholinophenyl)amino)quinoline-6-
necarboxylate (11) carboxylate (15a)
Yield: 0.36 g (46%); mp.: 226–228 ◦ C; UHPLC: 98% (tR = 14.73 min);
1
The synthesis of 11 was performed in accordance to an already re H NMR (400 MHz, DMSO‑d6) δ ppm 1.38 (dt, J = 7.1, 0.7 Hz, 3H), 2.41
ported procedure [14]. To a solution containing ethyl 4-aminobenzoate (s, 3H), 3.14 (t, J = 4.9 Hz, 4H), 3.76 (t, J = 4.8 Hz, 4H), 4.39 (q, J = 7.4,
(9, 1.2 g, 7.3 mmol) and anhydrous magnesium sulfate (1.08 g, 9.0 6.8 Hz, 2H), 6.57 (s, 1H), 7.03 (d, J = 8.8 Hz, 2H), 7.24 (d, J = 8.6 Hz,
mmol) in ethanol (15 mL) was added acetic acid (0.33 mL) and ethyl 2H), 7.79 (d, J = 8.8 Hz, 1H), 8.09 (dd, J = 8.5 Hz, 1,6 Hz 1H), 9.06 (s,
3-oxobutanoate (1.8 mL, 14.1 mmol). The reaction mixture was stirred 1H), 9.16 (s, 1H); 13C NMR (101 MHz, DMSO‑d6) δ ppm 13.97, 24.78,
at 90 ◦ C for 16 h. Afterwards, the suspended magnesium sulfate was 48.46 (2C), 60.38, 65.88 (2C), 100.75, 115.66 (2C), 117.06, 124.63,
removed by filtration and the ethanol was evaporated under reduced 124.86, 124.98 (2C), 128.09, 131.21, 148.21, 150.24, 150.30, 150.32,
pressure to afford acrylate intermediate. To complete the reaction the 160.65, 165.55; FTMS (ESI) m/z: 392.1983 [M+H]+; calc for
intermediate was heated in the presence of Dowtherm® A (15 mL) at C23H26N3O3: 392.1969.
230–250 ◦ C for 15 min. The reaction mixture was cooled to room tem
perature, the precipitate was filtered and washed with hexane, diethyl 4.9.2. Ethyl 2-methyl-4-((4-thiomorpholinophenyl)amino)quinoline-6-
ether and ethyl acetate. Finally, the product was dried under reduced carboxylate (15b)
pressure. Pale yellow solid; Yield 0.71 g (42%); m.p.: 252–254 ◦ C; Yield: 0.77 g (94%); mp.: 217–219 ◦ C; UHPLC: 95% (tR = 14.40 min);
1
UHPLC: 96% (tR = 15.78 min); FTMS (ESI) m/z: 232.0977 [M+H]+; H NMR (400 MHz, DMSO‑d6) δ ppm 1.37 (t, J = 7.1 Hz, 3H), 2.41 (s,
calcd. for C13H13NO3: 232.0968. 3H), 2.66–2.73 (m, 4H), 3.48–3.55 (m, 4H), 4.38 (q, J = 7.1 Hz, 2H),
14
6.59 (s, 1H), 7.00 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 8.3 Hz, 2H), 7.79 (d, J 34.31, 60.78, 101.54, 117.51, 123.20 (2C), 124.90, 125.27, 128.36,
= 8.8 Hz, 1H), 8.09 (d, J = 8.8 Hz, 1H), 9.05 (s, 1H), 9.14 (s, 1H); 13C 128.51, 129.18 (2C), 137.58, 138.42, 149.68, 150.39, 161.06, 165.74;
NMR (101 MHz, DMSO‑d6) δ ppm 14.25, 25.12, 25.73 (2C), 51.27 (2C), FTMS (ESI) m/z: 363.2061[M+H]+; calc for C23H27N2O2: 363.2067.
60.71, 100.88, 117.17 (2C), 117.23, 124.68, 125.14, 125.24 (2C),
128.34, 128.46, 131.05, 148.04, 150.33, 150.57, 161.07, 165.78; FTMS 4.9.8. Ethyl 4-((4-(4-benzylpiperazin-1-yl)phenyl)amino)-2-
(ESI) m/z: 408.1720 [M+H]+; calc for C23H26N3O2S: 408.1740. methylquinoline-6-carboxylate (15h)
1
4.9.3. Ethyl 2-methyl-4-((4-(4-methylpiperidin-1-yl)phenyl)amino) H NMR (400 MHz, DMSO‑d6) δ ppm 1.38 (t, J = 7.1 Hz, 3H), 2.43 (s,
quinoline-6-carboxylate (15c) 3H), 2.54 (t, J = 5.0 Hz, 4H), 3.18 (t, J = 5.0 Hz, 4H), 3.55 (s, 2H), 4.39
Yield: 0.60 g (75%); mp.: 114–117 ◦ C; UHPLC: 99% (tR = 16.70 min); (q, J = 7.1 Hz, 2H), 6.57 (s, 1H), 7.01 (d, J = 8.5 Hz, 2H), 7.22 (d, J =
1
H NMR (400 MHz, DMSO‑d6) δ ppm 0.95 (d, J = 6.4 Hz, 3H), 1.16–1.32 8.5 Hz, 2H), 7.24–7.32 (m, 1H), 7.35 (d, J = 4.4 Hz, 4H), 7.81 (d, J = 8.8
(m, 2H), 1.38 (t, J = 7.1 Hz, 3H), 1.46–1.55 (m, 1H), 1.65–1.77 (m, 2H), Hz, 1H), 8.13 (dd, J = 8.8, 1.8 Hz, 1H), 9.09 (s, 1H), 9.39 (s, 1H); 13C
2.40 (s, 3H), 2.66 (dt, J = 12.1, 2.6 Hz, 2H), 3.66 (dt, J = 12.5, 3.4 Hz, NMR (101 MHz, DMSO‑d6) δ ppm 14.27, 24.32, 48.23 (2C), 52.54 (2C),
2H), 4.38 (q, J = 7.0 Hz, 2H), 6.56 (s, 1H), 7.00 (d, J = 8.9 Hz, 2H), 7.19 60.87, 62.03, 100.77, 116.10 (2C), 116.96, 125.14, 125.29, 125.41
(d, J = 8.5 Hz, 2H), 7.78 (d, J = 8.8 Hz, 1H), 8.08 (dd, J = 8.7, 1.9 Hz, (2C), 127.03, 127.16, 128.21 (2C), 128.97, 129.11 (2C), 130.37,
1H), 9.08 (d, J = 16.7 Hz, 2H); 13C NMR (101 MHz, DMSO‑d6) δ ppm 137.90, 148.66, 149.02, 151.24, 160.19, 165.65; FTMS (ESI) m/z:
14.25, 21.72, 25.25, 30.08, 33.58 (2C), 48.98 (2C), 60.67, 100.75, 481.2595[M+H]+; calc for C30H33N4O2: 481.2598.
116.50 (2C), 117.20, 124.56, 125.13, 125.22 (2C), 128.21, 128.65,
130.50, 148.80, 150.36, 150.80, 161.17, 165.80; FTMS (ESI) m/z: 4.9.9. Ethyl 4-((4-(1H-benzo[d]imidazole-1-yl)phenyl)amino)-2-
404.2333 [M+H]+; calc for C25H30N3O2: 404.2333. methylquinoline-6-carboxylate (15i)
1
4.9.4. Ethyl 2-methyl-4-((4-(piperidin-1-ylmethyl)phenyl)amino) H NMR (400 MHz, DMSO‑d6) δ ppm 1.40 (t, J = 7.1 Hz, 3H), 2.54 (s,
quinoline-6-carboxylate (15d) 3H), 4.41 (q, J = 7.1 Hz, 2H), 7.03 (s, 1H), 7.28–7.41 (m, 2H), 7.63–7.68
Yield: 0.62 g (77%); mp.: 151–153 ◦ C; UHPLC: 96% (tR = 18.90 min); (m, 3H), 7.71–7.76 (m, 2H), 7.78–7.82 (m, 1H), 7.92 (d, J = 8.8 Hz, 1H),
1
H NMR (400 MHz, DMSO‑d6) δ ppm 1.38 (t, J = 7.1 Hz, 5H), 1.47–1.54 8.17 (dd, J = 8.8, 1.8 Hz, 1H), 8.57 (s, 1H), 9.15 (d, J = 1.8 Hz, 1H), 9.75
(m, 4H), 2.30–2.36 (m, 4H), 2.46 (s, 3H), 3.41 (s, 2H), 4.39 (q, J = 7.1 (s, 1H); 13C NMR (101 MHz, DMSO‑d6) δ ppm 14.28, 24.64, 60.93,
Hz, 2H), 6.85 (s, 1H), 7.32 (s, 4H), 7.83 (d, J = 8.8 Hz, 1H), 8.11 (dd, J = 102.69, 110.69, 117.86, 119.93, 122.41, 123.42, 123.58 (2C), 124.87
8.8, 1.8 Hz, 1H), 9.08 (d, J = 1.8 Hz, 1H), 9.27 (s, 1H); 13C NMR (101 (2C), 125.40, 125.49, 128.99, 131.51, 133.27, 139.90, 143.29 (2C),
MHz, DMSO‑d6) δ ppm 14.22, 24.00, 25.23, 25.54 (2C), 53.85 (2C), 143.71, 149.33, 149.68, 160.81, 165.68; FTMS (ESI) m/z: 423.1818
60.71, 62.40, 101.90, 117.70, 122.44 (2C), 124.83, 125.25, 128.30, [M+H]+; calc for C26H23N4O2: 423.1816.
128.73, 129.66 (2C), 134.28, 138.85, 149.16, 150.83, 161.32, 165.75;
FTMS (ESI) m/z: 404.2335 [M+H]+; calc for C25H30N3O2: 404.2333. 4.9.10. Ethyl 4-((4-(1H-pyrazol-1-yl)phenyl)amino)-2-methylquinoline-6-
carboxylate (15j)
4.9.5. Ethyl 4-((4-ethylphenyl)amino)-2-methylquinoline-6-carboxylate Yield: 0.71 g (96%); mp.: 115–118 ◦ C; UHPLC: 99% (tR = 13.70 min);
1
(15e) H NMR (400 MHz, DMSO‑d6) δ ppm 1.39 (t, J = 7.1 Hz, 3H), 2.49 (s,
Yield: 0.56 g (84%); mp.: 144–145 ◦ C; UHPLC: 98% (tR = 17.14 min); 3H), 4.41 (q, J = 7.1 Hz, 2H), 6.57 (t, J = 2.1 Hz, 1H), 6.89 (s, 1H),
1
H NMR (400 MHz, DMSO‑d6) δ ppm 1.22 (t, J = 7.6 Hz, 3H), 1.38 (t, J 7.47–7.56 (m, 2H), 7.77 (d, J = 1.8 Hz, 1H), 7.86 (d, J = 8.8 Hz, 1H),
= 7.1 Hz, 3H), 2.44 (s, 3H), 2.63 (q, J = 7.6 Hz, 2H), 4.39 (q, J = 7.1 Hz, 7.89–7.94 (m, 2H), 8.15 (dd, J = 8.8, 1.8 Hz, 1H), 8.51 (d, J = 2.5 Hz,
2H), 6.77 (s, 1H), 7.24–7.33 (m, 4H), 7.82 (d, J = 8.8 Hz, 1H), 8.11 (dd, 1H), 9.09 (d, J = 1.8 Hz, 1H), 9.46 (s, 1H); 13C NMR (101 MHz,
J = 8.8, 1.8 Hz, 1H), 9.07 (d, J = 1.9 Hz, 1H), 9.26 (s, 1H); 13C NMR DMSO‑d6) δ ppm 14.28, 25.02, 60.85, 102.23, 107.71, 117.72, 119.51
(101 MHz, DMSO‑d6) δ ppm 14.27, 15.58, 25.21, 27.65, 60.76, 101.57, (2C), 123.74 (2C), 125.10, 125.26, 127.52, 128.40, 128.63, 135.95,
117.58, 123.24 (2C), 124.81, 125.25, 128.36, 128.65 (3C), 137.71, 138.36, 140.76, 149.31, 150.37, 161.16, 165.74; FTMS (ESI) m/z:
139.73, 149.54, 150.74, 161.28, 165.79; FTMS (ESI) m/z: 335.1747 373.1651 [M+H]+; calc for C22H21N4O2: 373.1659.
[M+H]+; calc for C21H23N2O2: 335.1754.
4.9.11. Ethyl 2-methyl-4-((4-(pyrrolidin-1-yl)phenyl)amino)quinoline-6-
4.9.6. Ethyl 2-methyl-4-((4-propylphenyl)amino)quinoline-6-carboxylate carboxylate (15k)
(15f) Yield: 0.65 g (87%); mp.: 214–216 ◦ C; UHPLC: 96% (tR = 18.15 min);
1
Yield: 0.60 g (87%); mp.: 154–155 ◦ C; UHPLC: 95% (tR = 18.19 min); H NMR (400 MHz, DMSO‑d6) δ ppm 1.38 (t, J = 7.1, 1.6 Hz, 3H),
1
H NMR (400 MHz, DMSO‑d6) δ ppm 0.93 (t, J = 7.3 Hz, 3H), 1.38 (t, J 1.90–2.06 (m, 4H), 2.38 (s, 3H), 3.22–3.34 (m, 4H), 4.38 (q, J = 7.1, 6.6
= 7.1 Hz, 3H), 1.55–1.69 (m, 2H), 2.44 (s, 3H), 2.57 (t, J = 8.5, 6.7 Hz, Hz, 2H), 6.43 (s, 1H), 6.63 (d, J = 7.4 Hz, 2H), 7.15 (d, J = 9.0 Hz, 2H),
2H), 4.39 (q, J = 7.1 Hz, 2H), 6.77 (s, 1H), 7.22–7.33 (m, 4H), 7.82 (d, J 7.77 (d, J = 8.8 Hz, 1H), 8.08 (d, J = 8.6 Hz, 1H), 9.03 (s, 1H), 9.05 (s,
= 8.8 Hz, 1H), 8.11 (dd, J = 8.8, 1.8 Hz, 1H), 9.07 (d, J = 1.9 Hz, 1H), 1H); 13C NMR (101 MHz, DMSO‑d6) δ ppm 14.06, 24.76 (2C), 24.84,
9.25 (s, 1H); 13C NMR (101 MHz, DMSO‑d6) δ ppm 13.64, 14.26, 24.07, 47.26 (2C), 60.44, 100.32, 112.02 (2C), 116.87, 124.49, 124.94, 126.16
25.23, 36.73, 60.75, 101.59, 117.58, 123.10 (2C), 124.80, 125.25, (2C), 127.20, 128.07, 128.12, 145.65, 150.25, 151.16, 160.60, 165.63;
128.35, 128.68, 129.22 (2C), 137.73, 138.09, 149.48, 150.76, 161.29, FTMS (ESI) m/z: 376.2027 [M+H]+; calc for C23H26N3O2: 376.2020.
165.78; FTMS (ESI) m/z: 349.1904 [M+H]+; calc for C22H25N2O2:
349.1911. 4.9.12. Ethyl 2-methyl-4-((4-(pyrrolidine-1-carbonyl)phenyl)amino)
quinoline-6-carboxylate (15l)
4.9.7. Ethyl 4-((4-butylphenyl)amino)-2-methylquinoline-6-carboxylate Yield: 0.59 g (73%); mp.: 213–216 ◦ C; UHPLC: 98% (tR = 14.93 min);
1
(15g) H NMR (400 MHz, DMSO‑d6) δ ppm 1.38 (t, J = 7.1 Hz, 3H), 1.86 (s,
Yield: 0.51 g (70%); mp.: 152–155 ◦ C; UHPLC: 95% (tR = 19.05 min); 4H), 2.51 (s, 3H), 3.47–3.51 (m, 4H), 4.40 (q, J = 7.1 Hz, 2H), 7.04 (s,
1
H NMR (400 MHz, DMSO‑d6) δ ppm 0.92 (t, J = 7.3 Hz, 3H), 1.29–1.41 1H), 7.40–7.44 (m, 2H), 7.57–7.61 (m, 2H), 7.86 (d, J = 8.8 Hz, 1H),
(m, 5H), 1.53–1.63 (m, 2H), 2.45 (s, 3H), 2.60 (t, J = 7.7 Hz, 2H), 4.39 8.13 (dd, J = 8.8, 1.9 Hz, 1H), 9.04 (d, J = 1.9 Hz, 1H), 9.41 (s, 1H); 13C
(q, J = 7.1 Hz, 2H), 6.77 (s, 1H), 7.31–7.23 (m, 4H), 7.83 (d, J = 8.8 Hz, NMR (101 MHz, DMSO‑d6) δ ppm 14.28, 23.91, 25.13, 26.08, 46.06,
1H), 8.12 (dd, J = 8.8, 1.8 Hz, 1H), 9.08 (d, J = 1.9 Hz, 1H), 9.32 (s, 1H); 49.06, 60.89, 103.47, 118.17, 120.71 (2C), 125.22, 125.34, 128.61,
13
C NMR (101 MHz, DMSO‑d6) δ ppm 13.78, 14.27, 21.76, 25.04, 33.16, 128.72 (2C), 131.65, 142.18, 148.34, 150.61, 161.37, 161.39, 165.75,
15
167.82; FTMS (ESI) m/z: 404.1970 [M+H]+; calc for C24H26N3O3: 118.16, 118.56, 124.58 (2C), 128.69, 131.70, 141.88, 146.92, 147.39,
404.1969. 148.33, 153.66. FTMS (ESI) m/z: 361.2386 [M+H]+; calc for C23H29N4:
361.2387.
4.10. General procedure for the synthesis of 4-aminoquinolines 19a–k
4.10.6. 2-Methyl-6-nitro-N-(4-(piperidin-1-yl)phenyl)quinolin-4-amine
The three synthetic steps were performed as described for obtaining (19f)
the 4-aminoquinoline 4 (LABIO-17). In addition, the cyclocondensation Yield: 0.64 g (88%); mp.: 216–219 ◦ C; UHPLC: 97% (tR = 15.28 min);
1
reactions between anilines and ethyl 3-oxobutanoate to afford 4-hy H NMR (400 MHz, DMSO‑d6) δ ppm 1.50–1.58 (m, 2H), 1.59–1.69 (m,
droxy-2-methyl-quinolines were accomplished as already reported [14]. 4H), 2.43 (s, 3H), 3.12–3.19 (m, 4H), 6.62 (s, 1H), 7.01 (d, J = 8.4 Hz,
2H), 7.20 (d, J = 8.3 Hz, 2H), 7.86 (d, J = 9.3 Hz, 1H), 8.32 (d, J = 8.8
4.10.1. 6-Methoxy-2-methyl-N-(4-(piperidin-1-yl)phenyl)quinolin-4- Hz, 1H), 9.36 (s, 1H), 9.45 (s, 1H); 13C NMR (101 MHz, DMSO‑d6) δ ppm
amine (19a) 23.81, 25.28 (2C), 25.32, 49.59 (2C), 101.11, 116.51 (2C), 116.61,
Yield: 0.60 g (87%); mp.: 243–246 ◦ C; UHPLC: 99% (tR = 13.25 min); 120.00, 122.54, 125.19 (2C), 129.78, 129.88, 142.67, 149.29, 150.94,
1 151.38, 162.86; FTMS (ESI) m/z: 363.1813 [M+H]+; calc for
H NMR (400 MHz, DMSO‑d6) δ ppm 1.53–1.60 (m, 2H), 1.63–1.70 (m,
4H), 2.38 (s, 3H), 3.13–3.18 (m, 4H), 3.91 (s, 3H), 6.52 (s, 1H), C21H23N4O2: 363.1816.
6.94–7.03 (m, 2H), 7.16–7.21 (m, 2H), 7.27 (dd, J = 9.1, 2.8 Hz, 1H),
7.65–7.72 (m, 2H), 8.30 (s, 1H); 13C NMR (101 MHz, DMSO‑d6) δ ppm 4.10.7. 2,6-Dimethyl-N-(4-(piperidin-1-yl)phenyl)quinolin-4-amine (19g)
23.46, 24.29 (2C), 24.94, 49.52 (2C), 55.34, 100.15, 101.14, 116.22 Yield: 0.22 g (33%); m.p.: 218–220 ◦ C; UHPLC: 99% (tR = 13.31
(2C), 118.05, 119.98, 124.64 (2C), 129.27, 131.14, 143.68, 148.22, min); 1H NMR (400 MHz, DMSO‑d6) δ ppm 1.51–1.60 (m, 2H),
148.51, 155.38, 155.48; FTMS (ESI) m/z: 348.2065 [M+H]+; calc for 1.62–1.69 (m, 4H), 2.37 (s, 3H), 2.49 (s, 3H), 3.10–3.19 (m, 4H), 6.52 (s,
C22H26N3O: 348.2070. 1H), 6.96–7.01 (m, 2H), 7.15–7.20 (m, 2H), 7.45 (dd, J = 8.5, 1.8 Hz,
1H), 7.65 (d, J = 8.5 Hz, 1H), 8.10 (d, J = 1.6 Hz, 1H), 8.43 (s,1H); 13C
4.10.2. 6-Ethoxy-2-methyl-N-(4-(piperidin-1-yl)phenyl)quinolin-4-amine NMR (101 MHz, DMSO‑d6) δ ppm 21.26, 23.88, 24.99, 25.38 (2C),
(19b) 49.91 (2C), 100.05, 116.72 (2C), 117.75, 120.70, 124.89 (2C), 128.08,
Yield: 0.42 g (58%); mp.: 251–254 ◦ C; UHPLC: 96% (tR = 14.30 min); 130.93, 131.31, 132.71, 146.85, 148.67, 148.83, 157.55; FTMS (ESI) m/
1 z: 332.2122 [M+H]+; calc for C22H26N3: 332.2121.
2H), 1.61–1.69 (m, 4H), 2.34–2.36 (m, 3H), 3.14 (t, J = 5.3 Hz, 4H),
4.17 (q, J = 6.9 Hz, 2H), 6.50 (s, 1H), 6.99 (d, J = 8.0 Hz, 2H), 7.16 (d, J 4.10.8. 6-Isopropyl-2-methyl-N-(4-(piperidin-1-yl)phenyl)quinolin-4-
= 8.3 Hz, 2H), 7.24 (dt, J = 9.2, 1.8 Hz, 1H), 7.61–7.69 (m, 2H), 8.37 (s, amine (19h)
1H); 13C NMR (101 MHz, DMSO‑d6) δ ppm 14.53, 23.73, 24.74, 25.24 Yield: 0.66 g (92%); mp.: 255–257 ◦ C; UHPLC: 99% (tR = 15.76 min);
1
(2C), 49.76 (2C), 63.42, 100.08, 101.65, 116.54 (2C), 118.20, 120.54, H NMR (400 MHz, DMSO‑d6) δ ppm 1.38 (d, J = 7.1 Hz, 6H); 1.58–1.65
124.85 (2C), 129.72, 131.27, 144.01, 148.14, 148.67, 154.80, 155.72; (m, 2H), 1.66–1.75 (m, 4H), 2.43 (s, 3H), 3.06–3.26 (m, 5H), 6.55 (s,
FTMS (ESI) m/z: 362.2227 [M+H]+; calc for C23H28N3O: 362.2227. 1H), 7.03 (d, J = 8.2 Hz, 2H), 7.23 (d, J = 8.3 Hz, 2H), 7.57 (d, J = 8.7
Hz, 1H), 7.73 (d, J = 8.7 Hz, 1H), 8.18 (s, 1H), 8.55 (s, 1H); 13C NMR
4.10.3. 2-Methyl-6-(methylthio)-N-(4-(piperidin-1-yl)phenyl)quinolin-4- (101 MHz, DMSO‑d6) δ ppm 23.51, 23.54 (2C), 24.37, 24.99 (2C),
amine (19c) 33.32, 49.53 (2C), 99.86, 116.23 (2C), 117.37, 117.71, 124.76 (2C),
Yield: 0.62 g (86%); mp.: 233–236 ◦ C; UHPLC: 99% (tR = 13.80 min); 127.49, 128.07, 130.96, 143.51, 146.46, 148.61, 148.97, 156.91; FTMS
1 (ESI) m/z: 360.2421 [M+H]+; calc for C24H30N3: 360.2434.
4H), 2.38 (s, 3H), 2.60 (s, 3H), 3.05–3.24 (m, 4H), 6.51 (s, 1H),
6.93–7.02 (m, 2H), 7.13–7.21 (m, 2H), 7.53 (dd, J = 8.8, 2.0 Hz, 1H), 4.10.9. 6-Fluoro-2-methyl-N-(4-(piperidin-1-yl)phenyl)quinolin-4-amine
7.68 (d, J = 8.8 Hz, 1H), 8.13 (d, J = 2.1 Hz, 1H), 8.66 (s, 1H); 13C NMR (19i)
(101 MHz, DMSO‑d6) δ ppm 15.39, 23.58, 24.29, 25.05 (2C), 49.52 Yield: 0.51 g (76%); mp.: 195–197 ◦ C; UHPLC: 98% (tR = 12.32 min);
1
(2C), 100.40, 116.28 (2C), 118.01, 118.05, 125.00 (2C), 127.94, H NMR (400 MHz, DMSO‑d6) δ ppm 1.53–1.60 (m, 2H), 1.62–1.70 (m,
128.50, 130.59, 133.24, 145.65, 148.57, 148.84, 157.14; FTMS (ESI) m/ 4H), 2.40 (s, 3H), 3.13–3.19 (m, 4H), 6.57 (s, 1H), 6.95–7.01 (m, 2H),
z: 364.1842 [M+H]+; calc for C22H26N3O: 364.1842. 7.15–7.20 (m, 2H), 7.47 (ddd, J = 9.2, 8.2, 2.8 Hz, 1H), 7.80 (dd, J =
9.2, 5.8 Hz, 1H), 8.11 (dd, J = 11.0, 2.8 Hz, 1H), 8.35 (s, 1H); 13C NMR
4.10.4. 2-Methyl-4-((4-(piperidin-1-yl)phenyl)amino)quinoline-6- (101 MHz, DMSO‑d6) δ ppm 23.44, 24.51, 24.92, 49.46, 100.23, 105.18,
carbonitrile (19d) 105.42, 116.19, 117.69, 117.94, 118.04, 124.59, 130.47, 130.56,
Yield: 0.61 g (89%); mp.: 255–257 ◦ C; UHPLC: 95% (tR = 12.41 min); 130.70, 145.35, 148.62, 148.67, 156.91, 157.61, 159.31; FTMS (ESI) m/
1 z: 336.1870 [M+H]+; calc for C21H23FN3: 336.1871.
4H), 2.40 (s, 3H), 3.10–3.14 (m, 4H), 6.59 (s, 1H), 6.94–7.02 (m, 2H),
7.12–7.21 (m, 2H), 7.81 (d, J = 8.7 Hz, 1H), 7.87 (dd, J = 8.7, 1.7 Hz, 4.10.10. 6-Chloro-2-methyl-N-(4-(piperidin-1-yl)phenyl)quinolin-4-amine
1H), 8.92 (d, J = 1.8 Hz, 1H), 8.97 (s, 1H); 13C NMR (101 MHz, (19j)
DMSO‑d6) δ ppm 23.80, 25.27 (2C), 25.30, 49.61 (2C), 100.93, 105.37, Yield: 0.42 g (60%); mp.: 205–207 ◦ C; UHPLC: 98% (tR = 13.60 min);
1
116.54 (2C), 117.46, 119.34, 125.11 (2C), 128.81, 129.69, 129.84, H NMR (400 MHz, DMSO‑d6) δ ppm 1.50–1.58 (m, 2H), 1.60–1.67 (m,
129.95, 149.23, 149.38, 149.99, 162.13; FTMS (ESI) m/z: 343.1910 4H), 2.39 (s, 3H), 3.09–3.17 (m, 4H), 6.57 (s, 1H), 6.94–6.99 (m, 2H),
[M+H]+; calc for C22H23N4: 343.1917. 7.14–7.19 (m, 2H), 7.58 (dd, J = 9.0, 2.3 Hz, 1H), 7.75 (d, J = 8.9 Hz,
1H), 8.44 (d, J = 2.3 Hz, 1H), 8.59 (s, 1H); 13C NMR (101 MHz, DMSO) δ
4.10.5. 6-dimethylamine-2-methyl-N-(4-(piperidin-1-yl)phenyl)quinolin- ppm 23.57, 24.78, 25.05 (2C), 49.53 (2C), 100.49, 116.31 (2C), 118.54,
4-amine (19e) 120.72, 124.70 (2C), 127.76, 128.93, 130.19, 130.59, 146.89, 148.37,
Yield: 0.31 g (43%); mp.: 268–270 ◦ C; UHPLC: 98% (tR = 18.20 min); 148.75, 158.88; FTMS (ESI) m/z: 352.1568 [M+H]+; calc for
1 C21H23ClN3: 352.1575.
4H), 2.32 (s, 3H), 3.00 (s, 6H), 3.09–3.17 (m, 4H), 6.45 (s, 1H), 6.96 (d,
J = 8.3 Hz, 2H), 7.15 (d, J = 8.3 Hz, 2H), 7.20–7.30 (m, 2H), 7.60 (d, J = 4.10.11. 6-Bromo-2-methyl-N-(4-(piperidin-1-yl)phenyl)quinolin-4-amine
9.1 Hz, 1H), 8.12 (s, 1H); 13C NMR (101 MHz, DMSO‑d6) δ ppm 23.52, (19k)
24.40, 25.02 (2C), 40.41 (2C), 49.67 (2C), 100.13, 100.42, 116.31 (2C), Yield: 0.49 g (62%); mp.: 227–229 ◦ C; UHPLC: 99% (tR = 14.11 min);
16
1
H NMR (400 MHz, DMSO‑d6) δ ppm 1.50–1.58 (m, 2H), 1.59–1.68 (m, 4.11.5. 6-Bromo-2-ethyl-N-(4-(piperidin-1-yl)phenyl)quinolin-4-amine
4H), 2,38 (s, 3H), 3.13 (t, J = 5.4 Hz, 4H), 6.56 (s, 1H), 6.97 (d, J = 8.9 (22e)
Hz, 2H), 7.17 (d, J = 8.9 Hz, 2H), 7.63–7.75 (m, 2H), 8.59 (d, J = 2.0 Hz, Yield: 0.79 g (96%); mp.: 218–220 ◦ C; UHPLC: 99% (tR = 14.52 min);
1H), 8.66 (s, 1H); 13C NMR (101 MHz, DMSO‑d6) δ ppm 23.81, 24.85, 1
H NMR (400 MHz, DMSO‑d6) δ ppm 1.19 (t, J = 7.6 Hz, 3H), 1.50–1.58
25.29 (2C), 49.70 (2C), 100.52, 116.42, 116.58 (2C), 119.17, 124.21, (m, 2H), 1.60–1.69 (m, 4H), 2.65 (q, J = 7.6 Hz, 2H), 3.10–3.18 (m, 4H),
124.98 (2C), 130.16, 130.46, 132.10, 146.75, 148.65, 149.03, 159.06; 6.60 (s, 1H), 6.94–7.02 (m, 2H), 7.13–7.22 (m, 2H), 7.70 (d, J = 1.2 Hz,
FTMS (ESI) m/z: 396.1063 [M+H]+; calc for C21H23BrN3: 396.1070. 2H), 8.59 (t, J = 1.4 Hz, 1H), 8.66 (s, 1H); 13C NMR (101 MHz, DMSO) δ
ppm 13.19, 23.64, 25.13 (2C), 31.31, 49.59 (2C), 99.60, 116.12, 116.38
4.11. General procedure for the synthesis of 4-aminoquinolines 22a-f (2C), 119.39, 123.97, 124.56 (2C), 130.58, 130.66, 131.62, 147.10,
148.36, 148.73, 163.90; FTMS (ESI) m/z: 410.1231 [M+H]+; calc for
The compounds 22a-f were synthesized from 4-chloro-2-methylqui C22H25BrN3: 410.1226.
nolines 18k, 20a-b using the same described procedure for obtaining the
4-aminoquinoline 4 (LABIO-17). The anilines 13, 14c, 14j, 21a-b were 4.11.6. 6-chloro-2-ethyl-N-(4-(piperidin-1-yl)phenyl)quinolin-4-amine
commercially available. (22f)
Yield: 0.33 g (45%); mp.: 208–210 ◦ C; HPLC: 97% (tR = 14.30 min);
1
4.11.1. 6-Bromo-2-methyl-N-(4-(4-methylpiperidin-1-yl)phenyl)quinolin- H NMR (400 MHz, DMSO‑d6) δ ppm 1.18 (t, J = 7.6 Hz, 3H), 1.49–1.57
4-amine (22a) (m, 2H), 1.59–1.67 (m, 4H), 2.65 (q, J = 7.6 Hz, 2H), 3.04–3.22 (m, 4H),
Yield: 0.58 g (71%); mp.: 228–230 ◦ C; UHPLC: 99% (tR = 19.17 min); 6.60 (s, 1H), 6.98 (d, J = 8.5 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H), 7.61 (dd,
1
H NMR (400 MHz, DMSO‑d6) δ ppm 0.95 (d, J = 6.5 Hz, 3H), 1.18–1.32 J = 9.0, 2.3 Hz, 1H), 7.78 (d, J = 8.9 Hz, 1H), 8.46 (d, J = 2.3 Hz, 1H),
(m, 2H), 1.41–1.56 (m, 1H), 1.66–1.75 (m, 2H), 2.37 (s, 3H), 2.64 (dt, J 8.71 (s, 1H); 13C NMR (101 MHz, DMSO‑d6) δ ppm 13.57, 23.81, 25.31
= 12.2, 2.6 Hz, 2H), 3.65 (dt, J = 12.8, 3.4 Hz, 2H), 6.56 (s, 1H), (2C), 31.51, 49.73 (2C), 99.63, 116.60 (2C), 118.89, 120.97, 124.76
6.96–7.03 (m, 2H), 7.13–7.21 (m, 2H), 7.64–7.76 (m, 2H), 8.59 (d, J = (2C), 128.01, 129.28, 130.65, 130.74, 147.08, 148.55, 148.88, 164.02;
2.1 Hz, 1H), 8.73 (s, 1H); 13C NMR (101 MHz, DMSO) δ ppm 21.72, FTMS (ESI) m/z: 366.1740 [M+H]+; calc for C22H25ClN3: 366.1732.
25.09, 30.08, 33.58 (2C), 49.01 (2C), 100.53, 116.27, 116.54 (2C),
119.24, 124.12, 124.93 (2C), 130.56, 130.58, 131.88, 147.22, 148.38,
4.12. MtInhA expression and purification
148.67, 159.28; FTMS (ESI) m/z: 410.1221 [M+H]+; calc for
C22H25BrN3: 410.1226.
The recombinant MtInhA was expressed and purified as previously
described [21,46]. The substrate DD-CoA was synthetized [46] and
4.11.2. 6-Bromo-2-methyl-N-(4-(4-phenylpiperidin-1-yl)phenyl)quinolin-
purified [47] from 2-trans-dodecenoic acid and coenzyme A (CoA) via
4-amine (22b)
anhydride formation following acylation.
1
H NMR (400 MHz, DMSO‑d6) δ ppm 1.71–1.92 (m, 4H), 2.38 (s, 3H),
2.60–2.70 (m, 1H), 2.71–2.80 (m, 2H), 3.79 (dt, J = 12.6, 3.1 Hz, 2H), 4.13. In vitro inhibition studies by steady-state kinetics
6.60 (s, 1H), 7.00–7.06 (m, 2H), 7.17–7.24 (m, 3H), 7.25–7.36 (m, 4H),
7.67–7.75 (m, 2H), 8.62 (d, J = 2.0 Hz, 1H), 8.77 (s, 1H); 13C NMR (101 The IC50 value, which defines the concentration of inhibitor required
MHz, DMSO) δ ppm 25.11, 32.78 (2C), 41.43, 49.53 (2C), 100.59, to half-saturate the enzyme population, was used in order to assess the
116.32, 116.71 (2C), 119.29, 124.16, 124.90 (2C), 126.04, 126.64 (2C), relative potency of the compounds. Experiments were performed at
128.33 (2C), 130.61, 130.86, 131.89, 145.97, 147.26, 148.34, 148.58, 25 ◦ C, in 100 mM Pipes pH 7.0 and were started with the addition of the
159.30; FTMS (ESI) m/z: 472.1352 [M+H]+; calc for C27H27BrN3: 2.2 μM MtInhA to a total reaction volume of 500 μL for a 1 min, using an
472.1383. UV-2550 UV/Visible spectrophotometer (Shimadzu©), monitoring the
NADH oxidation at 340 nm (εβ-NADH = 6.22 M− 1 cm− 1), in the forward
4.11.3. N-(4-(1H-pyrazol-1-yl)phenyl)-6-bromo-2-methylquinolin-4- direction. The maximal rate for the enzyme reaction was determined in
amine (22c) the absence of inhibitor, in the presence of fixed non-saturating con
Yield: 0.45 g (59%); mp.: 211–213 ◦ C; HPLC: 99% (tR = 13.67 min); centration of NADH (60 μM ≅ Km) and DD-CoA (45 μM ≅ Km) [21]. The
1
H NMR (400 MHz, DMSO‑d6) δ ppm 2.46 (s, 3H), 6.56 (t, J = 2.4, 1.8 reaction velocity was analyzed as the percentage of inhibition as a
Hz, 1H), 6.91 (s, 1H), 7.46–7.51 (m, 2H), 7.72–7.80 (m, 3H), 7.87–7.92 function of inhibitor concentration and the data were fitted to Eq (1).
(m, 2H), 8.50 (d, J = 2.5 Hz, 1H), 8.62 (d, J = 2.1 Hz, 1H), 9.05 (s, 1H); vi 1
13
C NMR (101 MHz, DMSO‑d6) δ ppm 25.12, 102.15, 107.65, 116.79, = ( ) Eq. 1
v0 1 + [1]
119.52 (2C), 119.76, 123.17 (2C), 124.27, 127.49, 130.72, 132.16, IC50
135.63, 138.42, 140.70, 146.93, 147.33, 159.57; FTMS (ESI) m/z:

The compounds that have presented the lowest IC50 value were
379.0569 [M+H]+; calc for C19H16BrN4: 379.0553.
further evaluated. In order to assess the mode of inhibition and to
determine the inhibition constant (Ki), initial rates were measured as a
4.11.4. (1-(4-((6-Bromo-2-methylquinolin-4-yl)amino)phenyl)pyrrolidin-
function of NADH concentration (10–160 μM) at fixed non-saturating
2-yl)methanol (22d)
DD-CoA concentration (45 μM) and fixed-varied inhibitor concentra
1 tions (0.05–2 μM) of the molecules. The Ki values towards NADH for
H NMR (400 MHz, DMSO‑d6) δ ppm 1.82–2.08 (m, 3H), 2.36 (s, 3H),
compounds 19c, 19j, 19k, and 22a were calculated using the using Eq.
3.00–3.11 (m, 1H), 3.22 (t, J = 9.7 Hz, 1H), 3.30–3.43 (m, 2H),
(2), which describes a non-competitive inhibition, where [I] is the in
3.50–3.61 (m, 1H), 3.65–3.74 (m, 1H), 4.78 (s, 1H), 6.44 (s, 1H),
hibitor concentration, [S] is the substrate concentration, Km and Vmax
6.64–6.72 (m, 2H), 7.09–7.17 (m, 2H), 7.63–7.75 (m, 2H), 8.61 (d, J =
are the Michaelis-Menten constants, Kii is the overall inhibition constant
2.1 Hz, 1H), 8.68 (s, 1H); 13C NMR (101 MHz, DMSO‑d6) δ ppm 22.68,
for the ESI complex and Kis is the overall inhibition constant for the EI
24.84, 27.94, 48.22, 60.22, 61.29, 100.13, 112.13 (2C), 115.98, 119.00,
complex [48].
123.97, 125.88 (2C), 127.46, 130.27, 131.66, 145.20, 146.96, 149.06,
158.99; FTMS (ESI) m/z: 412.1006 [M+H]+; calc for C21H23BrN3O: Vmax [S]
v0 = ( ) ( ) Eq. 2
412.1019. km 1 + K[I]is + [S] K[I]ii
Inhibition studies were also carried out in the presence of fixed non-
17
saturating concentration of NADH (60 μM) and fixed-varied inhibitor reference strain (ATCC 27294) by the resazurin reduction microplate
concentrations (0.2–4 μM), when DD-CoA was the variable substrate assay (REMA), as described elsewhere [24]. Stock solutions (2 mg mL− 1)
(15–135 μM). For compounds 19c, 19j, 19k, and 22a, the inhibition of the test compounds were made in neat DMSO (Sigma-Aldrich), and
constant for the DD-CoA substrate were determined using the competi aliquots were stored at − 20 ◦ C. The compounds were further diluted in
tive equation, Eq. (3), in which [I] is the inhibitor concentration, [S] is 1 mL of Difco™ Middlebrook 7H9 broth (Becton Dickinson, BD), sup
the substrate concentration, Km and Vmax are the Michaelis-Menten plemented with 10% (v/v) BBL™ Middlebrook ADC enrichment (albu
constants, and Kis is the overall inhibition constant for the EI complex min, dextrose, and catalase; BD) and 5% (v/v) DMSO. The maximum
[48]. concentration tested for each compound ranged from 0.08 to 40 μg mL− 1
due to differences in solubility. The compounds were prepared in 2-fold
V [S]
v0 = ( max ) Eq. 3 serial dilutions directly in 96-well plates. Three independent experi
km 1 + K[I]is + [S] ments were performed, and the Minimumm Inhibitory Concentration
(MIC) was considered to be the lowest compound concentration that
4.14. Binding experiments and thermodynamics analysis prevented resazurin (Sigma-Aldrich) reduction, which, otherwise, is
indicated by a color conversion from blue to pink. The MIC value re
Binding interactions between the enzyme and ligands were evalu ported for each compound was the most frequent value among the three
ated by monitoring the change in protein intrinsic fluorescence upon assays or the highest value obtained, and it was expressed in molar
binding in a RF-5301PC Spectrofluorophotometer (Shimadzu©). The concentration (μM). Additionally, 4-aminoquinolines 19c, 19j-K, and
excitation wavelength employed with MtInhA was 295 nm and the 22a were further tested by the REMA assay for their inhibitory potential
emission wavelength range was 310 nm–500 nm, the temperature used against three MDR clinical isolates of M. tuberculosis [30]. The clinical
was 25 ◦ C, and the excitation and emission slits were 5 nm and 10 nm, isolates (named PT2, PT12, and PT20) were obtained from patients in
respectively [21]. Fluorescence titration of pre-formed MtInhA:NADH the Lisbon Health Region, Lisbon, Portugal. INH and RIF were used as
binary complex was carried out by making microliter additions of the control drugs to demonstrate the MDR phenotype of these isolates.
250 μM 19c (0.032–3087 μM final concentration), 250 μM 19j
(0.032–3208 μM final concentration), 250 μM 19k (0.032–2598 μM 4.16. Cytotoxicity evaluation
final concentration), 250 μM 22a (0,124–3696 μM final concentration),
to 2 mL of 3 μM MtInhA in the presence of 20 μM NADH, keeping the Cellular viability determination after incubation with the test com
dilution to a maximum of 1%. This binding experiment was performed pounds was performed using two different methods: the MTT method
with the compounds and the pre-formed MtInhA:NADH binary complex [31] and neutral red [32] uptake assay. First, HepG2 and Vero cells were
due to the non-competitive mode of inhibition of the molecules, because grown in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supple
their affinities are the same for the free enzyme and the binary complex, mented with 10% heat-inactivated fetal bovine serum (FBS), 1% anti
and in the experiments that led to the identification of the compounds, biotic (penicillin-streptomycin) and 0.01% antifungal (amphotericin B).
the NADH coenzyme was always treated as part of the protein in the Cells were seeded at 4 × 103 (HepG2) or 2 × 103 cells/well (Vero) in a
docking simulations [20]. Control experiments were employed to both 96-well microtiter plate and incubated for 24 h. Test compounds were
determine the maximum ligand concentrations to be used with no inner diluted in three different concentrations (1, 5 and 20 μM) using 1%
filter effect and dilution effect on protein fluorescence. Data from DMSO and were incubated with the cell lines for 72 h at 37 ◦ C under 5%
equilibrium fluorescence spectroscopy were fitted to Eq. (4) for hyper CO2. For the MTT assay, the cultures were incubated with MTT reagent
bolic binding isotherms, in which F0 is the observed fluorescence signal; (5 mg/mL) for 4 h. The absorbance was measured at 595 nm (Ez Read
Fmax is the maximal fluorescence intensity; F∞ is the maximum change in 400 microplate reader, Biochrom, UK). The precipitated purple for
fluorescence at saturating ligand (L) concentration and KD represents the mazan crystals were directly proportional to the number of live cells
dissociation constant for binding of compounds to MtInhA. with active mitochondria. For the neutral red assay, after 72 h of incu
bation with the compounds, the cells were washed with PBS before the
F0 − F
=
[L]
Eq. 4 addition of 200 μL of neutral red dye solution (25 μg/mL, Sigma) pre
F0 − Fmax KD + [L] pared in serum-free medium. The plate was incubated for an additional
The determination of the thermodynamics binding parameters were 3 h at 37 ◦ C under 5% CO2. After incubation, cells were washed with
assessed by the relationship between the equilibrium dissociation con PBS, followed by incubation with 100 μL of a desorb solution
stant (KD), determined by spectrofluorimetry, and the experimental (CH3COOH/EtOH/H2O, 1:50:49) for 30 min, with gentle shaking to
temperature. This relationship states that a change in the KD at different extract neutral red dye from the viable cells. The absorbance was
temperatures yields values for changes in the enthalpy (ΔHo), in entropy measured at 562 nm using a microtiter plate reader. The percentage of
(ΔSo) and in the Gibbs Free energy (ΔGo). In short, the KD was deter cell viability for the treated groups was reported by considering the
mined by fluorescence titration in a range of various temperatures control wells (1% DMSO) as 100% of cell viability: cell viability (%) =
(15 ◦ C–30 ◦ C) to assess whether or not there is a linear relationship (absorbance of treated wells/absorbance of control wells) × 100. Sta
between the KD and the tested temperatures. Data from the experiment tistical analysis was performed using GraphPad Prism 5.0 software (San
were fitted to the van’t Hoff Equation (Eq. (5)), in which KD is the Diego, CA, USA).
dissociation constant, R is the ideal gas constant 1.987 cal mol− 1 K− 1,
and T is temperature in Kelvin, yielding ΔHo and ΔSo. Eq. (6) provides 4.17. Evaluation of possible broad spectrum activity
ΔGo from the relation between ΔHo and ΔSo.
( o) The microorganisms evaluated were Staphylococcus aureus ATCC
ΔH 1 ΔSo 25923, Escherichia coli ATCC 25922, and the multidrug-resistant clinical
ln KD = − Eq. 5
R T R isolates Acinetobacter baumannii and Klebsiella pneumoniae. The mini
mum inhibitory concentrations (MIC) were determined by broth
ΔGo = ΔH o − TΔSo Eq. 6 microdilution method, in triplicate, for 19c, 19j and 19k compounds as
described elsewhere [25].
4.15. Susceptibility testing against M. tuberculosis strains
4.18. Chemical stability evaluation
The synthesized compounds were tested for their potential inhibitory
activity of in vitro growth of pan-senstitive M. tuberculosis H37Rv The measurement of chemical stability at different pHs provides
18
information on the amount or concentration of the test drug that re sterile PBS (pH 7.4) to remove non-adherent cells, and the infection
mains constant or stable and, consequently, available for absorption. 4- occurred as follows. One isolated colony of the M. tuberculosis H37Rv
aminoquinolines 19c, 19j, and 19k (1–10 μM) were incubated at 37 ◦ C reference strain (ATCC 27294) was cultured in 5 mL of 7H9-ADC broth,
for 24 h in the presence of a buffer solution at controlled pH 1.2 supplemented with 0.05% (v/v) Tween 80 (Sigma-Aldrich) and 0.2%
(simulating the pH of the stomach; 0.1 sol. M HCl), pH 7.4 (simulating (v/v) glycerol (MERCK) until the mid-log phase (OD600 ≅ 0.8). The
the pH of the plasma; PBS) and pH 9.1 (simulating the pH of the intes culture was diluted in pre-heated RPMI medium, and approximately 5 ×
tine; 0.1 M NH4HCO3). After this period, the test compounds were 104 CFU was added to each well. The infection was allowed to continue
quantified by HPLC-EM/MS. Alprenolol was used as an analytical con for 3 h at 37 ◦ C with 5% CO2. Afterwards, the infected cells were washed
trol. This experiment was carried out by CEMSA (São Paulo, SP, Brazil). twice with sterile PBS to remove non-internalized mycobacteria. Cells of
the early control (EC) group were lysed on the day of treatment onset
4.19. Solubility assay with 1 mL of 0.025% SDS (dissolved in sterile 0.9% saline). Lysates were
serially diluted in sterile 0.9% saline and plated on Difco™ Middlebrook
The solubility tests were performed according to a previously pub 7H10 Agar (BD), supplemented with 10% BBL™ Middlebrook OADC
lished protocol with slight modifications [49]. Accordingly, 1 mL of a enrichment (oleic acid, albumin, dextrose, and catalase; BD) and 0.5%
prepared solution (1 × PBS, pH 7.4 or 0.1 M HCl, pH 1.0) was added to 1 (v/v) glycerol. Thereafter, the infected cells were treated with 2.5 μM of
mg of compound (in triplicate). The final solutions were vortexed (1 each test compound in triplicate. Compounds 19j and 19k were first
min) and the resulting suspensions were shaken for 4 h at 25 ◦ C. Then, solubilized (4 mM) in neat DMSO, and then diluted in 2 mL of RPMI
the suspensions were centrifuged (13000 rpm for 15 min at 25 ◦ C) medium to a final concentration of 5 μM. The final DMSO concentration
obtaining a pellet and the remaining solutions were quantified by liquid was maintained at 0.5% in each well. After 5 days of treatment, the
chromatography (as per the conditions already described for UHPLC RPMI medium was removed, and each well was gently washed with PBS.
experiments) using single-point calibration of a known concentration of The treated macrophages were lysed with 0.025% SDS, serially diluted
the compounds in DMSO. in 0.9% saline, and plated on 7H10 agar. After an incubation period of
2–3 weeks at 37 ◦ C, the CFUs were counted, setting a limit of detection
4.20. Permeability assay (LOD) between 20 and 200 CFU per plate. The calculated CFU values
were converted into logarithms of CFU before statistical analysis, and
The Parallel Artificial Membrane Permeability Assay (PAMPA) the result was expressed as the mean of the log10 CFU values per well ±
comprises the use of an artificial membrane as an alternative, efficient the standard deviation (mean log10 CFU/well ± SD). Groups were
and rapid model for the study and evaluation of the drug candidates compared by one-way analysis of variance (ANOVA), followed by the
regarding its (passive) permeation potential across the intestinal mem Tukey post-test, using GraphPad Prism 9.0 (GraphPad, San Diego, CA,
brane. 4-Aminoquinolines 19c, 19j, and 19k were quantified (HPLC- USA). Significance between groups was determined using p < 0.05.
EM/EM), after incubation, in two solutions separated by an artificial and
porous lipid membrane. The result of this test was expressed in unit of 4.23. Genotoxicity
diffusion rate (permeation). First, compounds at concentration of 10 μM
(in 2% DMSO) were added to donor aqueous phase (pH 7.4). After 5 h at The alkaline comet assay [38] was performed using HepG2 cells. The
room temperature, one aliquot of the receptor solutions (pH 7.4), in cells were seeded at 8 × 104 cells/well in a 24-well culture plate for 24 h
which the compounds were, in theory, transported by passive diffusion, before treatment. After this time, compounds 19j and 19k were added at
were withdrawn and the concentration of test compounds were mens concentrations of 1 μM and 5 μM, and the plate was incubated for an
urated. With the concentration results, the permeation velocity value additional 24 h. Cells were mixed with low-melting point agarose and
was determined. Alprenolol was used as analytical control. The placed on a microscope slide precovered with normal agarose and
permeability has been described as low (<1.0 × 10− 6 (cm/s)) and high placed at 4 ◦ C for 10 min for total agarose solidification. The microscope
(>1.0 × 10− 6 (cm/s)) [36]. This experiment was carried out by CEMSA slides were then exposed to a lysis solution (2.5 M NaCl, 100 mM
(São Paulo, SP, Brazil). Na2EDTA, 10 mM Tris with 1% Triton X-100, and 10% DMSO) for 48 h.
The slides were washed with PBS and then exposed to alkali conditions
4.21. Metabolic stability (300 mM NaOH, 1 mM ethylenedinitrilotetraacetic acid, pH > 13) at
4 ◦ C for 20 min in order to allow the DNA to unfold and the expression of
In brief, test compounds at a concentration of 2 μM were incubated alkali-labile sites to occur. Electrophoresis was then performed for 20
with 1 mg/mL of the rat microsomes preparation containing NADPH at min at 25 V and 300 mA. Subsequently, the slides were neutralized,
37 ◦ C. Consumption of the compounds from the incubation mixture was fixed, and stained using silver nitrate. After they were completely dry,
measured at 0, 5, 15, and 30 min using the HPLC-MS/MS technique to the cells were observed on a microscope. Incubation with methyl
determine the in vitro disappearance half-life. Verapamil was used as the methanesulfonate (MMS) at 100 μM was used as positive control group.
positive control. The intrinsic clearance has been described as low (<16 Overall, 100 cells from each slide, and two slides per treatment, were
mL/min/kg), moderate (16–47 mL/min/kg), and high (>47 mL/min/ selected randomly for the analysis and for quantifying DNA damage.
kg) [36]. This experiment was carried out by CEMSA (São Paulo, SP, Slides were visually scored according to the size and amount of DNA
Brazil). present in the tail. Separately, each cell was given an arbitrary value of
0 (undamaged) to 4 (maximum damage). The damage score was thus
4.22. Intracellular activity in a macrophage model of M. tuberculosis attributed to each slide and ranged from 0 (undamaged: 100 cells × 0) to
infection 400 (maximum damage: 100 cells × 4). Finally, damage score was
expressed as the mean with standard error from two independent
Compounds 19j and 19k were evaluated in a macrophage model of experiments.
M. tuberculosis infection, as previously described [37], with slight
modifications. Murine macrophage RAW 264.7 cells were cultured in 4.24. In vivo model of tuberculosis infection
RPMI 1640 medium (Gibco), supplemented with 10% FBS, without
penicillin–streptomycin, and about 5 × 104 cells were seeded in each The evaluation of 4-aminoquinline 19k was performed in accordance
well of a sterile flat-bottom 24-well plate. After an incubation period of to an already reported procedure with some modifications [39]. One
24 h in a bacteriological chamber (at 37 ◦ C with 5% CO2 and a humid isolated colony of the M. tuberculosis H37Rv was cultured in 5 mL of
atmosphere), the adhered cells were washed once with pre-heated 7H9-ADC broth, supplemented with 0.05% (v/v) Tween 80 and 0.2%
19
(v/v) glycerol until the mid-log phase (OD600 ≅ 0.8). Male CF-1 mice Desenvolvimento Econômico e Social (BNDES/FUNTEC) [grant number
were (25–30 g) obtained from the Center of Experimental Biological 14.2.0914.1] and FAPERGS [grant numbers 17/1265-8 INCT-TB and
Models from Pontifícia Universidade Católica do Rio Grande do Sul 19/1724-3 PQG]. L.A.B. (CNPq, grant 303499/2021-4), C.V.B (CNPq,
(CeMBE/PUCRS). The animals were maintained at controlled humidity grant 311949/2019-3) and P.M. (CNPq, grant 305203/2018-5) are
(40–60%) and temperature room (24 ± 2 ◦ C), under a 12/12 h light/ Research Career Awardees of CNPq. This study was financed in part by
dark cycle. Food and water were provided ad libitum. All experimental the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—
protocols were approved by the Animal Ethics Committee from Pontif Brasil (CAPES), Finance Code 001. We thank Dr. Miguel Viveiros for his
ícia Universidade Católica do Rio Grande do Sul (CEUA-PUCRS) (pro help with MDR strains.
tocol number:8637). Mice were anaesthetized by intraperitoneal
injection of a mixture containing ketamine (100 mg/kg) and xylazine Appendix A. Supplementary data
(10 mg/kg), for subsequently infection with 106 viable M. tuberculosis
H37Rv cells suspended in 200 μL of saline solution through the Supplementary data to this article can be found online at https://doi.
retro-orbital venous plexus. Following infection, mice were randomly org/10.1016/j.ejmech.2022.114908.
divided into treatment groups. Treatment was started 7 days post
infection and compound 19k or control drugs (INH and EMB) were Abbreviations
administered daily by oral gavage. The chemotherapeutic experiment
consisted of a 15-day schedule of treatment. Treatment groups received ADME absorption, distribution, metabolism and excretion
300 μM/kg or 450 μM/kg of 19k prepared with propylene glycol (PG): ANOVA one-way analysis of variance
Tween 80 (4:1 vol/vol) vehicle, INH (150 μM/kg) and EMB (300 μM/kg) CC50 concentration capable of reducing cell viability by 50%
in saline solution were employed as positive treatment controls. Vehicle CFU colony-forming unit
group (infected and not treated) received treatments vehicle (PG:Tween Clint intrinsic clearance
80) during the treatment period. To assess lung and spleen CFU counts DD-CoA 2-trans-dodecenoyl-CoA
and splenomegaly, mice were euthanized by isoflurane inhalation 2 and DMEM Dulbecco’s Modified Eagle Medium
3 days after the last dose of drugs. The lung and spleen were placed in 3 EMB ethambutol
mL of saline solution and were disrupted in a glass tissue homogenizer ESI electrospray ionization
(Góes Vidros Especiais, Porto Alegre, RS, Brazil). The number of viable ESI enzyme-substrate-inhibitor complex
organisms was determined by plating serial dilutions of homogenates on FTMS fourier transform mass spectrometry
Middlebrook 7H10 agar (Difco, Sparks, MD) plates containing 10% HIV human immunodeficiency virus
Middlebrook OADC (oleic acid–albumin–dextrose–catalase) enrichment IC50 concentration of inhibitor that reduces enzyme velocity by
(Becton Dickinson, Franklin Lakes, NJ) and 0.2% (v/v) glycerol. Plates half
were incubated at 37 ◦ C for approximately 21 days prior to counting of INH isoniazid
viable M. tuberculosis cells. To investigate the bactericidal activity of ITC isothermal titration calorimetry
19k, a group of infected mice was sacrificed at the start of treatment as KD dissociation constant
pre-treatment controls (early control group - EC) and the numbers of Kii dissociation constants for the ternary ESI complex
viable organisms in the spleen and lungs were determined. Finally, for Kis dissociation constants for the binary enzyme-inhibitor
M. tuberculosis cell counts, the numbers were converted into logarithms complex
of CFU (log10 CFU). Data were evaluated by one-way analysis of vari m.p. melting point
ance (ANOVA) followed by Tukey’s post-test using GraphPad Prism 9.0 MDR multidrug-resistant
(GraphPad Software Inc., San Diego, CA). Significance between groups MIC minimum inhibitory concentration
was determined using p < 0.05. Animals with a variation above of two MMS methyl methanesulfonate
standard deviations, compared within your own group, were considered Mtb Mycobacterium tuberculosis
outliers, and were excluded from analysis. Using this criterion two an MtInhA Mycobacterium tuberculosis NADH-dependent enoyl-acyl
imals were excluded from lung evaluation (vehicle and 300 μM/kg carrier protein reductase
groups). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide)
Author contributions PABA 4-aminobenzoic acid
PAMPA parallel artificial membrane permeability assay
All authors have materially participated in the research and the PBS phosphate buffered saline;
manuscript was written through contributions of all authors. All authors ppm parts per million
have given approval to the final version of the manuscript. PT12 Portugal 12
PT2 Portugal 2
Declaration of competing interest PT20 Portugal 20
RIF rifampicin
The authors declare that they have no known competing financial SAR structure-activity relationship;
interests or personal relationships that could have appeared to influence SI selectivity index
the work reported in this paper. t1/2 half-live;
TB tuberculosis
Data availability TMS tetramethylsilane
tR retention time;
Data will be made available on request. UHPLC ultra-high-performance liquid chromatography
WHO World Health Organization
Acknowledgment ΔG◦ Gibbs free energy change
ΔH◦ enthalpy change
This work was supported by National Institute of Science and ΔS◦ entropy change
Technology on Tuberculosis (Decit/SCTIE/MS-MCT-CNPq-FNDTC-
CAPES-FAPERGS) [grant number 421703/2017-2], Banco Nacional de
20
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Copia de Novel 4-Aminoquinolines - Synthesis, Inhibition of The Mycobacterium Tuberculosis Enoyl-Acyl Carrier

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European Journal of Medicinal Chemistry 245 (2023) 114908

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

Novel 4-aminoquinolines: Synthesis, inhibition of the Mycobacterium

2. Results and discussion step of chemical synthesis.

Scheme 2. Reagents and conditions: i = Pyridine, HCl, (CH3)2CHOH, 85 ◦ C, 16 h.

Comps. R1 R2 R3 ClogP IC50 MtInhA (μM) MIC H37Rv (μM)

4 (LABIO17) CO2Et Me 6.34 20 ± 3 12.8

15a CO2Et Me 4.96 19.5 ± 2.3 51.1

15b CO2Et Me 5.79 >50 >12.3

15c CO2Et Me 6.86 10.9 ± 1.3 6.2

15d CO2Et Me 6.53 >50 49.6

15e CO2Et Me Et 6.53 41.3 ± 3.6 29.9

15i CO2Et Me 7.02 42.1 ± 2.5 >47.3

15j CO2Et Me 5.62 8.0 ± 0.9 >6.7

15k CO2Et Me 5.78 21.7 ± 1.2 13.3

15l CO2Et Me 4.70 44.5 ± 2.8 >49.6

19a MeO Me 5.98 4.7 ± 0.6 3.6

19b EtO Me 6.51 9.5 ± 1.0 6.9

19c MeS Me 6.36 1.0 ± 0.2 0.40

19d CN Me 5.33 19.6 ± 2.0 14.6

19e N(Me)2 Me 6.24 20.0 ± 2.9 6.9

19f NO2 Me 5.62 18.6 ± 1.8 6.9

19g Me Me 6.16 17.2 ± 0.9 7.5

19h iPr Me 7.09 8.2 ± 1.2 6.9

19i F Me 5.91 38.3 ± 3.2 14.9

(continued on next page)

19j Cl Me 6.48 1.1 ± 0.1 0.9

19k Br Me 6.63 0.23 ± 0.02 0.4

22a Br Me 7.15 1.4 ± 0.2 0.7

22b Br Me 6.69 >50 1.33

22c Br Me 5.94 4.85 ± 0.2 3.3

22d Br Me 5.35 >50 12.1

22e Br Et 7.16 1.54 ± 0.2 0.75

22f Cl Et 7.01 2.90 ± 0.4 1.7

Scheme 4. Reagents and conditions: i = Pyridine, HCl, (CH3)2CHOH, 85 ◦ C, 16 h.

2.5. Susceptibility against in vitro growth of drug-resistant M. tuberculosis

19c 100 68 100 >2.7 38.0 0.03 60 5.9

Herein, potent inhibitors of MtInhA enzyme activity were synthe­

values of, respectively, 6.3 μM 0.14 μM. Fluorescence spectroscopy and

135.63, 138.42, 140.70, 146.93, 147.33, 159.57; FTMS (ESI) m/z:

References resistance in Mycobacterium tuberculosis. Antimicrob, Agents Chemother. 46

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Herein, potent inhibitors of MtInhA enzyme activity were synthe