Design, synthesis and antitubercular potency of 4-hydroxyquinolin-2(1H)-ones

Article abstract of DOI:10.1016/j.ejmech.2017.06.061

In this study, a 50-membered library of substituted 4-hydroxyquinolin-2(1H)-ones and two closely related analogues was designed, scored in-silico for drug likeness and subsequently synthesized. Thirteen derivatives, all sharing a common 3-phenyl substituent showed minimal inhibitory concentrations against Mycobacterium tuberculosis H37Ra below 10 μM and against Mycobacterium bovis AN5A below 15 μM but were inactive against faster growing mycobacterial species. None of these selected derivatives showed significant acute toxicity against MRC-5 cells or early signs of genotoxicity in the Vitotox assay at the active concentration range. The structure activity study relation provided some insight in the further favourable substitution pattern at the 4-hydroxyquinolin-2(1H)-one scaffold and finally 6-fluoro-4-hydroxy-3-phenylquinolin-2(1H)-one (38) was selected as the most promising member of the library with a MIC of 3.2 μM and a CC₅₀ against MRC-5 of 67.4 μM.

Full text of DOI:10.1016/j.ejmech.2017.06.061

Accepted Manuscript
Design, synthesis and antitubercular potency of 4-hydroxyquinolin-2(1H)-ones
Maíra Bidart de Macedo, Roman Kimmel, Damijana Urankar, Martin Gazvoda,
Antonio Peixoto, Freya Cools, Eveline Torfs, Luc Verschaeve, Emerson Silva Lima,
Antonín Lyčka, David Milićević, Antonín Klásek, Paul Cos, Stanislav Kafka, Janez
Košmrlj, Davie Cappoen
PII:
S0223-5234(17)30515-9
10.1016/j.ejmech.2017.06.061
EJMECH 9556
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 April 2017
Revised Date: 16 June 2017
Accepted Date: 28 June 2017
Please cite this article as: Maí. Bidart de Macedo, R. Kimmel, D. Urankar, M. Gazvoda, A. Peixoto, F.
Cools, E. Torfs, L. Verschaeve, E.S. Lima, Antoní. Lyčka, D. Milićević, Antoní. Klásek, P. Cos, S. Kafka,
J. Košmrlj, D. Cappoen, Design, synthesis and antitubercular potency of 4-hydroxyquinolin-2(1H)-ones,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.061.
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Graphical abstract

ACCEPTED MANUSCRIPT
Design, synthesis and antitubercular potency of
4-hydroxyquinolin-2(1H)-ones
Maíra Bidart de Macedoa, Roman Kimmelb, Damijana Urankarc, Martin Gazvodac, Antonio
Peixotod, Freya Coolsa, Eveline Torfsa, Luc Verschaevee, Emerson Silva Limaf, Antonín Lyčkag,
David Milićevićb, Antonín Klásekb, Paul Cosa, Stanislav Kafkab,*, Janez Košmrljc,*, Davie
Cappoena,*
a Laboratory of Microbiology, Parasitology and Hygiene (LMPH), S7, Faculty of Pharmaceutical,
Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk,
Belgium
b Faculty of Technology, Tomas Bata University, Vavre
čkova 275, CZ-760 01 Zlín, Czech Republic
c Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ve
Ljubljana, Slovenia
čna pot 113, SI-1000
d Centre National de la Recherche Scientifique, IPBS, UMR 5089, F-31077 Toulouse, France, 2 Univ.
Toulouse, UPS, F-31000 Toulouse, France
e Department of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk,
Belgium
f Faculty of Pharmaceutical Sciences, Federal Univeristy of Amazonas, Avenida General Rodrigo Otávio
Campos de Jordão, 6200, Coroado, Manaus, 69077-000, Amazonas, Brazil
g Faculty of Science, University of Hradec Králové, Rokitanského 62, CZ-500 03 Hradec Králové III,
Czech Republic

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Abstract
In this study, a 50-membered library of substituted 4-hydroxyquinolin-2(1H)-ones and two
closely related analogues was designed, scored in-silico for drug likeness and subsequently
synthesized. Thirteen derivatives, all sharing a common 3-phenyl substituent showed minimal
inhibitory concentrations against Mycobacterium tuberculosis H37Ra below 10 µM and against
Mycobacterium bovis AN5A below 15 µM but were inactive against faster growing
mycobacterial species. None of these selected derivatives showed significant acute toxicity
against MRC-5 cells or early signs of genotoxicity in the VitotoxTM assay at the active
concentration range. The structure activity study relation provided some insight in the further
favourable substitution pattern at the 4-hydroxyquinolin-2(1H)-one scaffold and finally 6-fluoro-
4-hydroxy-3-phenylquinolin-2(1H)-one (38) was selected as the most promising member of the
library with a MIC of 3.2 µM and a CC50 against MRC-5 of 67.4 µM.
Keywords: Mycobacterium tuberculosis, tuberculosis, 4-hydroxyquinolin-2(1H)-ones,
antibiotic, cytotoxicity, genotoxicity
1. Introduction
Since 2015, tuberculosis (TB) has been declared by the WHO as the most common cause of
death from a single infectious agent, on par with HIV. Although a highly effective
chemotherapeutic treatment exists, in 2015 an estimated 1.4 million people died from TB related
complications [1]. Apart from the socio-economic reasons, why TB is still a threat, the
increasing incidence of multi- and extensively-drug resistant Mycobacterium tuberculosis (Mtb),
fuelled by the HIV pandemic has reinforced the urgent demand for novel antibiotics for TB

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treatment. Finding compounds with activity towards TB has proven to be less trivial mainly
because of the high antibiotic and xenobiotic tolerance of Mtb which results from its elaborate
adaptation for survival within its host [2]. Repurposing existing drugs or known pharmacophores
is a strategy that has gained tremendous interest in recent years in the search of novel
antitubercular compounds. A major advantage being that both the in vivo kinetics as well as the
toxicity of the analogues can be more easily anticipated. Such a pharmacophore can be found in
the group of antibacterial fluoroquinolones norfloxacin, ciprofloxacin, ofloxacin, enoxacin,
pefloxacin and grepafloxacin, which are worldwide patented drugs. In addition, the synthesis of
4-hydroxyquinolin-2(1H)-ones has received much attention from medicinal chemists due to a
wide variety of pharmacological properties attributed to this compound class itself. 4-
Hydroxyquinolin-2(1H)-ones have been reported to show antivirotical activity [3,4,5,6,7],
inclusive of anti-HIV [8], antibacterial activity against both Gram-negative and Gram-positive
bacteria [9], antifungal activity against Aspergillus niger, A. fumigatus and A. flavus [9,10], and
antimycobacterial activity [11]. To that extent, Arya and Agarwal [11] reported on the potency of
series of 3-unsubstituted fluorinated 4-hydroxyquinolin-2(1H)-ones against the Mtb strain
H37Rv. From their findings, it was concluded that compounds bearing 7-trifluoromethyl and 8-
trifluoromethyl substituents showed elevated potency.
2. RESULTS AND DISCUSSION
2.1. Chemistry
The construction of the 4-hydroxyquinolin-2(1H)-one heterocyclic skeleton in 1–38 was
accomplished by the condensation of aniline derivatives with diethyl esters of substituted
malonic acids by using previously established [12] procedures with modifications from our

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laboratories (Scheme 1) [13]. Derivatives having hydroxyl group at the fused benzene ring 25,
33–35 were prepared from the corresponding methoxy anilines via the intermediacy of methoxy
derivatives 20, 30–32 that were subjected to HBr mediated demethylation in the last step of the
reaction sequence (Scheme 1) [14].
Scheme 1. Synthesis of 4-hydroxyquinolin-2(1H)-ones 1–38 and 4-O-acetyl derivatives 39–50:
(i) 220–230 °C, 1h, then 260–270 °C, 3–6 h; (ii) 48% HBr, reflux 4–5 h; (iii) acetic acid/acetic
anhydride 4:1 (v/v), AcONa, reflux 2.5 h; (iv) Ac2O, pyridine, rt.
4-O-Acetyl derivative 39 was prepared by reaction of unsubstituted 4-hydroxyquinolin-2(1H)-
one with acetic anhydride in boiling acetic acid in the presence of catalytic sodium acetate
(Scheme 1). Acetates 40–50 were obtained by treating of the appropriate 4-hydroxyquinolin-
2(1H)-ones (1, 2, 4, 19–22, 30–32, 38) with acetic anhydride in pyridine at ambient temperature
[15].

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The synthesis of 4-hydroxypyridin-2(1H)-ones 51 and 52 was achieved by thermal
condensation of racemic camphoranil with bis-2,4,6-trichlorophenyl malonate as established
earlier (Scheme 2) [16].
Scheme 2. Synthesis of 4-hydroxypyridin-2(1H)-ones: (i) 240–250 °C, 10–90 min.
13
The 4-O-acetyl derivatives 41–50 were fully characterized by 1H and C NMR spectroscopy
and the corresponding resonances were assigned on the basis of 2D NMR experiments including
1H–1H gs-COSY, NOESY, 1H–13C gs-HMQC and 1H–13C gs-HMBC. The spectra were recorded
for DMSO-d6 solutions and the results are collected in Tables S3 and S4 (Supporting
Information). Broad singlets found in the range of δ 11.1–12.3 ppm were characteristic for the
NH protons, and the resonances of the acetate methyl protons appeared in the expected range of
13
δ 2.12–2.50 ppm. Characteristic for the downfield regions of the C NMR spectra were three
resonances belonging to the acetate carbonyl (δ 167.8–168.5 ppm), quinolin-2(1H)-one C-2 (δ
161.1–162.7 ppm) and C-4 (δ 151.0–152.4 ppm) atoms. The other carbon resonances of the
quinolin-2(1H)-one ring were found in the aromatic region of the spectra and were highly
dependent on the type of substituent R3–R6. The acetate methyl carbon resonated at δ 20.3–20.5
ppm.
2.2. Biological evaluation
2.2.1. In-silico prediction of drug likeness properties

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The values of in-silico predicted physicochemical parameters of the designed 4-
hydroxyquinolin-2(1H)-ones were predicted using the FAF-Drugs3 tool and are shown in Table
S1 of the Supporting Information. Their optimum range followed by 95% of clinically approved
drugs of synthetic origin is also shown in the footnotes of Table S1 [17]. The results of the
predicted drug-likeness parameters revealed that values of descriptors like MW, HBA, HBD and
log P followed Lipinski rule of five and their values lied within the prescribed optimum range.
Furthermore, all compounds comply to both the VEBER (≤10 rotatable bonds and tPSA ≤ 140
Å2 (or ≤12 HBA+HBD) and the EGAN (−1 ≤ log P ≤ 5.8 and tPSA ≤ 130 Å2) rules which
predict a good oral bioavailability. Furthermore, all compounds comply to the GSK 4/400 rule
for drug safety profiling but poorly to the Pfizer 3/75 rules. Using admetSAR, Predicted acute
oral toxicity values of all the compounds lie in “class III” which revealed that, compounds
possessed fairly high lethal dose and can be considered suitable for the druggable point of view
[18]. All designed compounds exhibited good predicted values of percentage human oral
absorption and none of the compound possessed mutagenicity (predicted in qualitative terms). So
overall, based upon the values of predicted drug likeness parameters, all envisaged derivatives
possessed the drug-likeness behaviour.
2.2.2. Evaluation of antitubercular activity
Initially, the 4-hydroxyquinolin-2(1H)-ones and the structurally related compounds from this
study were evaluated for antitubercular activity against the Mtb H37Ra strain. The screening was
performed with the luminescent strain reporter strain H37Ralux that harbours the pSMT1 plasmid
carrying the luxAB genes from Vibrio harveyi as previously described. Briefly, the activity of the
investigated compounds was deduced from the reduction in light emission by the reporter strain

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when exposed to the compounds compared to the non-exposed control culture. The compounds
were solubilized in DMSO and further diluted in 7H9 + OADC to a final concentration of 10
µM, 1 µM and 0.1 µM. Growth Inhibition (GI) in % as displayed in Table 1 [19]. Compounds
with the potency to reduce the bacterial growth of the reporter strain were selected for further
study and their structures were used as templates to guide the further synthesis of 4-
hydroxyquinolin-2(1H)-one analogues.
In a first attempt to improve the antitubercular potency of the compounds 1–18, focus was put
in creating derivatives that varied in the substituents at N1 and C3, i.e., R1 and R2. Results of this
initial screening are shown in Table 1. Three compounds could be selected from the first round,
3-ethyl-4-hydroxyquinolin-2(1H)-one (2) (GI 98.6%), 4-hydroxy-3-phenylquinolin-2(1H)-one
(4) (GI 92.4%) and 4-hydroxy-1,3-diphenylquinolin-2(1H)-one (14) (GI 98.3%). With the
exception of 4-hydroxy-1,3-diphenylquinolin-2(1H)-one (14), adding substituents at N1 (R1)
(compounds 6–18) did not improve the antitubercular activity of the derivatives compared with
the unsubstituted compound 1. Interestingly, derivative 14 shared the same phenyl substituent R2
as one of the two other active derivatives 4. From these observations, attention was drawn on the
potential importance of substituent R2 for the antitubercular potency of the derivatives and the
potency was higher for compounds that carried a phenyl or an ethyl group. In the second round
the library was further expanded with compounds 19–38 to study the potency of the derivatives
in function of the R4, R5, R6 and R7 substitution pattern when combined with the favourable R2
substituents from the previous screening. The synthesis was focused on 3-ethyl-4-
hydroxyquinolin-2(1H)-ones (R2 = ethyl; compounds 19–25) and 4-hydroxy-3-phenylquinolin-
2(1H)-ones (R2 = phenyl; compounds 30–38) as they were selected from the first attempt.
However, it was observed that derivatives 19–25, did not show the anticipated potency to reduce

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the mycobacterial growth of the reporter strain H37Ralux by 90% at 10 µM. In contrast to this
discouraging observation, derivatives 33–38, sharing the R2 phenyl substituent, all showed the
predicted potency. The three most active compounds of the series were 4-hydroxy-7-methoxy-3-
phenylquinolin-2(1H)-one (31) (GI= 98.2%), 4,7-dihydroxy-3-phenylquinolin-2(1H)-one (34)
(GI= 96.3%) and the fluorinated derivative 6-fluoro-4-hydroxy-3-phenylquinolin-2(1H)-one (38)
(GI= 99.5%). As acetylation of pharmacophores to construct prodrugs is a strategy often applied
to improve the biochemical properties or reduce toxicity, the synthesis of a series of acetylated
analogues (39–50) was envisaged, synthesized and screened. The design of the 4-O-acetyl
derivative analogues was based on the selected derivatives from the preceding attempts. Despite
the antitubercular potency displayed by the previous analogues, only one 4-O-acetyl derivative
(50) meets the criteria of GI>90 at 10 µM with a GI of 99.2%. A possible explanation for the
lack of observable potency in the other derivatives might be due to the essential role of 4-
hydroxyl moiety. In a final effort the synthesis and screening of a less planar scaffold was
executed. With the generation of a more out-of-plane 4-hydroxypyridin-2(1H)-one derivatives
51–52 possible negative side effects such as DNA intercalation, potentially leading to
genotoxicity, can be prevented. However, the activity was completely abrogated in both
derivatives, discouraging further attempts.
Table 1. Growth inhibition at 10 µM, 1 µM and 0.1 µM
Cmpd.
Growth inhibition (Mtb H37Ralux) %
10 µM
55.5
1 µM
46.6
78.2
47.4
0.1 µM
43.6
1
2
3
98.6
59.2
69.3
42.5

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92.4
59.2
40.2
32.9
10.5
49.5
0.0
56.0
7.5
4
53.5
83.8
23.9
87.2
8.2
5
4.0
6
0.0
7
29.8
0.0
8
9
74.5
25.6
56.6
47.3
98.3
56.3
33.2
71.5
49.5
68.2
60.8
71.0
57.3
86.2
55.1
49.4
78.2
64.8
59.1
62.6
90.8
98.2
95.0
95.1
96.3
48.5
34.8
46.3
46.3
53.7
54.7
25.3
42.4
38.8
56.3
36.0
66.4
51.3
46.4
48.8
34.3
38.5
35.4
38.7
10.7
24.5
74.4
47.5
52.1
61.7
36.0
22.5
10.4
34.4
30.1
20.3
20.9
31.2
24.8
0.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
23.7
65.8
40.0
16.9
33.3
25.6
13.2
21.6
30.0
0.0
9.4
56.5
42.5
32.3
53.7

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95.3
67.1
51.2
48.3
42.6
21.1
31.8
9.4
60.0
36.2
32.5
35.1
14.5
19.2
3.5
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
92.3
90.7
99.5
35.4
45.2
29.4
27.8
28.6
31.2
44.5
72.3
79.6
81.2
76.3
99.2
12.5
7.5
10.1
15.3
13.3
14.0
35.4
43.2
39.7
32.5
38.0
8.1
0.0
0.0
0.0
0.0
10.9
22.5
20.9
8.9
31.0
0.0
1.2
0.0
2.2.3. Evaluation of cytotoxicity and genotoxicity
The in vitro cytotoxicity of the selected 4-hydroxyquinolin-2(1H)-ones was evaluated against
MRC-5 human lung fibroblast cell (ATCC® CCL-171™) that serve as a suitable in vitro model
for the host cells. The acute cytotoxicity was investigated by the neutral red assay, a test in which
the ability of living cells to bind and incorporate toluene red in their lysosomal structures is a
measure of their viability. The critical concentration CC50 of a compound is the concentration at
which the retention of the neutral red dye has been reduced by 50% [20]. In addition, the
Minimal Inhibitory Concentration (MIC) at which the growth of the H37Ralux reporter strain was
reduced by 90% of the selected compounds was investigated using serial dilutions (128 µM–0.5

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µM) of the selected derivatives in order to derive the in vitro therapeutic index (TI). The TI =
(CC50/MIC) was used as an in vitro model to predict the degree of selectivity of the derivatives
against the Mtb over the host cells. Genotoxicity is often overlooked in the early phase of drug
discovery. Compounds sometimes undergo an expensive time consuming and expensive in vivo
evaluation during drug development only to be discarded if genotoxic properties are discovered
in later stages. For this reason, the VitotoxTM assay from Gentaur was included in the biological
evaluation of the 4-hydroxyquinolin-2(1H)-ones to exclude the possibility of early signs of
genotoxicity. Briefly, the VitotoxTM assay makes use of a recombinant luminescent Salmonella
typhimurim T104 (RecN2-4) reporter strain and closely correlates with the Ames test (91%),
with a high specificity of 94%. The S. typhimurim reporter strain carries an integrated lux operon
under transcriptional control of the pr1 promotor allowing a rapid and sensitive detection of early
signs of genotoxicity. DNA damage is reported as an increase in luminescence elicited by
activation of the bacterial SOS DNA repair mechanism. By adding S9 liver fraction, the
genotoxicity of the investigated compounds can be distinguished from that of its metabolites.
For most of the selected compounds we observed a comparable MIC ranging from 3.2 µM to
9.1 µM, indicating that substitution pattern at the R4, R5, R6 and R7 positions did not greatly
improve the activity against the Mtb strain H37Ra. A MIC of 3.2 µM and 3.8 µM was observed
for the most potent derivatives, 6-fluoro-4-hydroxy-3-phenylquinolin-2(1H)-one (38) and 6-
fluoro-2-oxo-1,2-dihydroquinolin-4-yl acetate (50) respectively. Low cytotoxicity of 4-
hydroxyquinolin-2(1H)-one derivatives against the MRC-5 was observed with CC50
concentrations ten-times higher than the MIC. From the calculated TI, 6-fluoro-4-hydroxy-3-
phenylquinolin-2(1H)-one (38), 4-hydroxy-1,3-diphenylquinolin-2(1H)-one (14), and 6-fluoro-2-
oxo-1,2-dihydroquinolin-4-yl acetate 50 were selected as the most promising derivatives. As

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shown in Table 2, no signs of early genotoxicity were observed for any of the derivatives or their
metabolites with the VitotoxTM assay (data plus controls presented in Table S2).
Table 2. Growth inhibition of selected compounds on Mtb and acute toxicity against MRC-5
cells.
Cmpd.
MICa (µM)
CC50b (µM)
TIc CC50/MIC
Genox
Cytox
-S9
+S9
-S9
+S9
6.8
8.5
6.4
8.7
9.1
7.9
8.3
8.2
8.3
8.0
9.2
3.2
3.8
0.1
0.3
84.7
96.5
12.5
11.4
>20.0
8.7
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
4
-
-
-
>128.0
75.4
-
-
-
14
-
-
-
30
59.0
6.5
-
-
-
31
104.2
>128.0
92.1
13.2
15.4
11.2
10.1
>16.0
6.7
-
-
-
32
33
-
-
-
-
-
-
34
84.1
-
-
-
35
>128.0
61.8
-
-
-
36
-
-
-
-
-
-
37
67.4
21.1
23.3
N.D.
N.D.
38
88.4
N.D.f
-
-
-
50
RIFd
INHe
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
a The concentration of the analogues at which a growth inhibition 90% was observed of the Mtb
H37Ra lab strain compared to the negative control. b The concentration of the analogues at which
viability of the MRC-5 cells was reduced by 50%. The therapeutic index was calculated by
d,e
dividing the CC50 by the MIC.
RIF and INH, the first line Mtb antibiotics Rifampycin and
isoniazid used as a positive control. f N.D. = Not Determined.
2.2.4. Activity against clinical relevant mycobacterial species
In order to investigate if the potency of the selected compounds is limited to Mtb within the
mycobacteria, the derivatives were screened against several clinically relevant mycobacterial

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species. Mycobacterium bovis, together with Mycobacterium africanum and Mycobacterium
microti, constitutes the M. tuberculosis complex. Mycobacterium avium ssp. Avium is
pathogenic for birds but can cause opportunistic infections in AIDS patient, whereas M. avium
ssp. Paratuberculosis is the etiological agent of John’s disease or paratuberculosis (in ruminants)
which may be linked to Chron’s disease in humans. Mycobacterium smegmatis is generally
considered a non-pathogenic microorganism and rarely causes opportunistic infections but is
considered a good tool to study the activity of compounds against mycobacteria in general.
The selected compounds showed potency against M. bovis AN5lux at the same size order as
with Mtb (Table 3). The potency against both M. avium ssps and against M. smegmatis was
significantly lower. Without the precise mechanism of action, there is currently no explanation
for the increase in MIC. Possibly, the shorter generation time of these species enables them to
overcome the antibacterial properties of the quinolin-2(1H)-ones.
Table 3 The MIC of the derivatives against other mycobacteria.
Cmpd.
MIC (µM)
M. avium ssp. paratuberculosis
M. bovis
AN5lux
9.5
M. avium ssp. avium
M. smegmatis
MC2155lux
23.2
ATCC19698lux
ATCC15769lux
14.5
18.4
1
7.2
21.2
19.8
23.5
4
7.8
23.8
35.4
10.3
14
30
31
32
33
34
35
36
37
38
9.3
18.6
17.5
15.3
9.5
16.5
13.8
19.5
8.6
18.0
13.0
15.1
10.4
11.5
9.9
13.2
15.6
16.8
15.7
16.3
18.5
13.7
17.0
20.0
8.7
13.2
12.6
19.2
13.2
5.3
20.3
15.7
17.3
11.5
8.5
8.4

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4.7
7.5
8.9
< 0.1
9.5
50
INHa
< 0.1
< 0.1
< 0.1
a First line Mtb drug Isoniazid used as a positive control.
3. Conclusions
In this investigation we have observed the antimycobacterial potency of a small library of 4-
hydroxyquinolin-2(1H)-ones and structurally related compounds. Derivatives carrying a 3-
phenyl substituent were favoured. The most active derivatives carried a fluoro substitution,
ultimately leading to the most active derivative 6-fluoro-4-hydroxy-3-phenylquinolin-2(1H)-one
(38), with a MIC of 3.2 µM and a CC50 of 67.4 µM and no observable early signs of
genotoxicity. Although the activity was not in the same order as the first line drugs for TB
treatment, Isoniazid and Rifampycin, this work proves that the 4-hydroxyquinolin-2(1H)-one
library can be altered by structural modification and change in substituents of the heterocyclic
scaffold. When screened against other mycobacterial species it was observed that the activity
was severely reduced against mycobacteria with shorter generation time. Knowing the enzymatic
target would also contribute to a more directed structural optimization of the compounds. For
that reason the future perspectives will be to elucidate the mechanism of action. One promising
observation that can be derived from Table 1 is that hit compounds 30–38 (defined as derivatives
with MIC < 10 µM) can be structurally clustered. In contrast to singletons this highlights the
plasticity of the compound class and the degree of freedom to adjust the physiochemical
properties of the derivatives without decreasing their potency. This is an extremely useful trait
when further hit expansion studies are focused on the metabolic stability, solubility and bio-
distribution.

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4. Materials and Methods
4.1. Chemistry
4.1.1. Materials-instrumentation-physical measurements
The reagents and solvents were used as obtained from the commercial sources. 4-
Hydroxyquinolin-2(1H)-one was purchased from Sigma-Aldrich. TLC was carried out on pre-
coated TLC sheets ALUGRAM® SIL G/UV254 for TLC, MACHEREY-NAGEL. The melting
points were determined on the microscope hot stage, Kofler, PolyTherm, manufacturer Helmut
Hund GmbH, Wetzlar or Gallenkamp apparatus and are uncorrected. NMR data were collected
with a Bruker Avance DPX 300 spectrometer operating at 300 MHz (1H) and 75 MHz (13C),
Bruker Avance 500 spectrometer operating at 500 MHz (1H), 126 MHz (13C) and 471 MHz (19F)
at 300 K. Proton spectra were referenced to TMS as internal standard, in some cases to the
13
residual DMSO-d5 (at δ 2.50 ppm). Carbon chemical shifts were determined relative to the C
signal of DMSO-d6 (39.5 ppm). 19F NMR spectra were referenced to external CCl3F in a
capillary. Chemical shifts are given on the δ scale (ppm). Coupling constants (J) are given in Hz
and are reported a read from the spectra. Multiplicities are indicated as follows: s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet) or br (broadened). Some compounds were
1
1
1
analyzed by two-dimensional H–1H gs-COSY, NOESY, H–13C gs-HMQC and H–13C gs-
HMBC techniques.
The IR spectra were recorded with a Perkin–Elmer 421 or 1310, Mattson 3000, or Bruker
Alpha spectrophotometer using samples in potassium bromide disks. Electron impact mass
spectra were performed with a Shimadzu QP-2010 instrument via direct impact technique.
Samples were prepared by drying of 10 µL of solution (0.03 mg mL–1) in dichloromethane in DI
cuvette at 50 °C. DI method: 80 °C/1 min, 40 °C/min to 350 °C, hold for required time, IS 200

ACCEPTED MANUSCRIPT
°C/70 eV. Only peaks of relative abundance exceeding 5% are listed. Data are reported as m/z
(relative intensity). High-resolution mass spectra were obtained with Q-TOF Premier instrument
or Agilent 6224 Accurate Mass TOF LC/MS system with electrospray ionization (ESI).
Elemental analyses (C, H, N) were performed with FlashEA1112 Automatic Elemental Analyzer
(Thermo Fisher Scientific Inc.).
4.1.2. General procedures for the synthesis of 4-hydroxyquinolin-2(1H)-ones 1–24, 26–32 and
36–38.
Compounds 1–24, 26–32 and 36–38 were synthesized by a modified literature procedure [13].
A mixture of the appropriate aniline (100 mmol) and substituted diethyl malonate (102–105
mmol) was heated on a metal bath at 220–230 °C for 1 h and then at 260–270 °C for 3–6 h (the
reaction was complete when the distillation of evolving ethanol stopped). After cooling, the solid
product was crushed, dissolved in aqueous sodium hydroxide solution (0.5 M, 250 mL) and after
filtration the filtrate was washed three times with toluene (40 mL). The aqueous layer was
filtered and the product was precipitated from alkaline water layer with concentrated
hydrochloric acid. The resulting pure 4-hydroxyquinolin-2(1H)-one product was collected by
filtration and washed with water. For the analytical purposes and biological screenings, the crude
products were recrystallized from the solvent indicated below.
Spectral and analytical for 4-hydroxy-3-methylquinolin-2(1H)-one (1) [21], 3-ethyl-4-
hydroxyquinolin-2(1H)-one (2) [21], 4-hydroxy-3-isopropylquinolin-2(1H)-one (3) [22], 4-
hydroxy-3-phenylquinolin-2(1H)-one (4) [21], 3-benzyl-4-hydroxyquinolin-2(1H)-one (5) [23],
4-hydroxy-1,3-dimethylquinolin-2(1H)-one (6) [24], 3-butyl-1-methyl-4-hydroxyquinolin-
2(1H)-one (7) [21], 4-hydroxy-1-methyl-3-phenylquinolin-2(1H)-one (8) [21], 1-ethyl-4-

ACCEPTED MANUSCRIPT
hydroxy-3-phenylquinolin-2(1H)-one (9) [25], 4-hydroxy-3-methyl-1-phenylquinolin-2(1H)-one
(10) [13], 3-ethyl-4-hydroxy-1-phenylquinolin-2(1H)-one (11) [13], 4-hydroxy-1-phenyl-3-
propylquinolin-2(1H)-one (12) [13], 3-butyl-4-hydroxy-1-phenylquinolin-2(1H)-one (13) [26],
4-hydroxy-1,3-diphenylquinolin-2(1H)-one (14) [26], 3-benzyl-4-hydroxy-1-phenylquinolin-
2(1H)-one (15) [27], 1-benzyl-3-butyl-4-hydroxyquinolin-2(1H)-one (16) [28], 1-benzyl-4-
hydroxy-3-phenylquinolin-2(1H)-one (17) [29], 1,3-dibenzyl-4-hydroxyquinolin-2(1H)-one (18)
[25], 3-ethyl-4-hydroxy-6-methoxyquinolin-2(1H)-one (19) [21], 3-ethyl-4-hydroxy-7-
methoxyquinolin-2(1H)-one (20) [21], 3-ethyl-4-hydroxy-8-methoxyquinolin-2(1H)-one (21)
[21], 3-ethyl-6-fluoro-4-hydroxyquinolin-2(1H)-one (22) [30], 5-chloro-3-ethyl-4-hydroxy-8-
methylquinolin-2(1H)-one (24) [21], 6-fluoro-4-hydroxy-3-propylquinolin-2(1H)-one (26) [30],
3-butyl-6-chloro-4-hydroxyquinolin-2(1H)-one (27) [31], 3-butyl-5-chloro-4-hydroxy-8-
methylquinolin-2(1H)-one (28) [31], 3-butyl-4-hydroxy-6,8-dimethoxyquinolin-2(1H)-one (29)
[21], 4-hydroxy-6-methoxy-3-phenylquinolin-2(1H)-one (30) [32], 4-hydroxy-7-methoxy-3-
phenylquinolin-2(1H)-one (31) [32], 4-hydroxy-8-methoxy-3-phenylquinolin-2(1H)-one (32)
[32], 4-hydroxy-6-methyl-3-phenylquinolin-2(1H)-one (36) [33], and 6-fluoro-4-hydroxy-3-
phenylquinolin-2(1H)-one (38) [33], were in agreement with the literature reports.
4.1.2.1. 3-Ethyl-4-hydroxy-5,8-dimethylquinolin-2(1H)-one hemihydrate (23·½H2O).
Synthesized from 3,5-dimethylaniline (12.12 g) and diethyl ethylmalonate (19.61 g, 104.2
mmol), and isolated as white solid (13.86 g, 63.79 mmol, 64%); mp 205–211 °C (EtOH); IR:
3429, 2966, 2930, 2870, 1633, 1604, 1586, 1449, 1388, 1302, 1260, 1201, 1159, 1082, 635, 608,
1
786, 604 cm–1; H NMR (500 MHz, DMSO-d6) δ (ppm) 1.00 (t, 3H, J = 7.3 Hz), 2.34 (s, 3H),
2.59 (q, 2H, J = 7.3 Hz), 2.69 (s, 3H), 4.10 (br s, 1H), 6.80 (d, 1H, J = 7.5 Hz), 7.12 (d, 1H, J =

ACCEPTED MANUSCRIPT
13
7.5 Hz), 10.14 (s, 1H); C NMR (126 MHz, DMSO-d6) δ (ppm) 13.2, 16.1, 17.4, 24.3, 112.3,
+
114.4, 120.9, 124.6, 130.4, 133.4, 136.7, 160.1, 162.8; HRMS (ESI+) m/z for C13H16NO2 [M +
H]+: calcd 218.1176, found 218.1186. Anal. Calcd. for C13H15NO2·½H2O (225.90): C, 69.01; H,
7.13; N, 6.19. Found: C, 68.97; H, 7.15; N, 5.90.
4.1.2.2. 4-Hydroxy-6,7-dimethyl-3-phenylquinolin-2(1H)-one (37).
Synthesized from 3,4-dimethylaniline (12.12 g) and diethyl phenylmalonate (24.80 g, 105.0
mmol), and isolated as colorless crystals (22.82 g, 86.01 mmol, 86%); mp 344–346 °C (AcOH).
1
IR: 2966, 1646, 1599, 1429, 1365, 1246, 1194, 1158, 886, 688, 539 cm–1; H NMR (500 MHz,
DMSO-d6) δ (ppm) 2.27 (s, 3H), 2.29 (s, 3H), 7.06 (s, 1H), 7.26–7.43 (m, 5H), 7.71 (s, 1H), 9.87
(br s, 1H), 11.30 (s, 1H); 13C NMR (126 MHz, DMSO-d6) δ (ppm) 19.1, 19.8, 111.8, 113.4,
115.3, 123.1, 126.7, 127.6, 129.4, 131.3, 133.6, 136.5, 139.8, 157.3, 162.6; HRMS (ESI+) m/z
+
for C17H16NO2 [M + H]+: calcd 266.1176, found 266.1175. Anal. Calcd. for C17H15NO2
(265.31): C, 76.96; H, 5.70; N, 5.28. Found: C, 76.87; H, 5.99; N, 5.24.
4.1.3. General procedures for the preparation of 4-hydroxyquinolin-2(1H)-ones 25, 33–35.
Compounds 25, 33–35 were synthesized by a modified literature procedure [14]. The mixture
of the appropriate methoxy substituted 4-hydroxyquinolin-2(1H)-one (20, 30, 31 or 32) (10.0
mmol) and 48% hydrobromic acid (0.88–1.15 mol) was refluxed until the starting material could
no longer be detected by TLC. During the course of the reaction, the reaction mixture turned
from a white suspension into a yellow solution. After completion, the reaction mixture was
concentrated in vacuo at 80 °C. Water was added to the residue and the resulting precipitate was

ACCEPTED MANUSCRIPT
filtered. The filtrate consisted of pure product 25, 33, 34 or 35 that was pure according to TLC
analysis.
Spectral and analytical data for 4,6-dihydroxy-3-phenylquinolin-2(1H)-one (33) [34] and 4,7-
dihydroxy-3-phenylquinolin-2(1H)-one (34) [34] were in agreement with those reported in the
literature.
4.1.3.1. 3-Ethyl-4,7-dihydroxyquinolin-2(1H)-one hydrate (25·H2O).
Synthesized from 3-ethyl-4-hydroxy-7-methoxyquinolin-2(1H)-one (20) (2.19 g) and 48%
hydrobromic acid (100 mL, 0.88 mol); reaction time was 5 h. Isolated as white solid (2.04 g,
9.14 mmol, 91%) pure by TLC analysis and IR spectrum. Recrystallization from EtOH afforded
1.38 g (0.618 mmol, 62%) of white solid; mp 247–252 °C (EtOH); IR 3539, 3360, 2989, 1635,
1
1616, 1555, 1478, 1462, 1400, 1261, 1241, 1221, 856, 780, 497 cm–1; H NMR (300 MHz,
DMSO-d6) δ (ppm) 0.98 (t, 3H, J = 7.2 Hz), 2.50 (q, 2H, J = 7.2 Hz), 6.58 (dd, 1H, J = 8.7 Hz,
2.0 Hz), 6.62 (d, 1H, J = 2.0 Hz), 7.67 (d, 1H, J = 8.7 Hz), 9.74 (br s, 1H), 9.87 (br s, 1H), 11.01
13
(br s, 1H); C NMR (75 MHz, DMSO-d6) δ (ppm) 13.4, 16.2, 99.6, 108.3, 109.7, 110.6, 124.0,
139.1, 157.1, 159.0, 163. 9. Anal. Calcd. for C11H11NO3·H2O (223.23): C, 59.19; H, 5.87; N,
6.27. Found: C, 59.36; H, 5.90; N, 6.33.
4.1.3.2. 4,6-Dihydroxy-3-phenylquinolin-2(1H)-one (33).
Prepared from 4-hydroxy-6-methoxy-3-phenylquinolin-2(1H)-one (30) (2.68 g) and 48%
hydrobromic acid (130 mL, 1.15 mol); reaction time was 4.5 h. Isolated as off-white solid (4.94
g, 19.5 mmol, 98%) pure according to TLC. Recrystallization from EtOH afforded 4.50 g (17.8
mmol, 89%) of white solid; 330–332 °C (EtOH); IR 3178, 3020, 1644, 1621, 1507, 1498, 1476,

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1446, 1310, 1282, 1200, 1185, 821, 707, 701 cm–1; 1H NMR (500 MHz, DMSO-d6) δ (ppm) 7.01
(dd, 1H, J = 8.8, 2.4 Hz), 7.15 (d, 1H, J = 8.8 Hz), 7.25–7.33 (m, 2H), 7.33–7.43 (m, 4H), 9.36
13
(s, 1H), 9.83 (br s, 1H), 11.25 (s, 1H); C NMR (126 MHz, DMSO-d6) δ (ppm) 107.1, 112.8,
116.1, 116.3, 120.0, 126.8, 127.7, 131.2, 131.4, 133.6, 151.7, 156.8, 162.1; HRMS (ESI+) m/z
+
for C15H12NO3 [M + H]+: calcd 254.0812, found 254.0815. Anal. Calcd. for C15H11NO3
(253.25): C, 71.14; H, 4.38; N, 5.53. Found: C, 71.23; H, 4.57; N, 5.23.
4.1.3.3. 4,7-Dihydroxy-3-phenylquinolin-2(1H)-one hemihydrate (34·½H2O).
Prepared from 4-hydroxy-7-methoxy-3-phenylquinolin-2(1H)-one (31) (9.36 g, 35.0 mmol)
and 48% hydrobromic acid (350 mL, 3.09 mol); reaction time was 9 h. Isolated as colourless
powder (7.54 g, 29.8 mmol, 85%) after recrystallization from EtOH; mp 325–329 °C (EtOH);
mp [34] 315–318 °C. IR 3537, 1644, 1621, 1509, 1445, 1408, 1293, 1189, 877, 704, 548 cm–1;
1H NMR (500 MHz, DMSO-d6) δ (ppm) 6.63 (dd, 1H, J = 8.8, 2.3 Hz), 6.68 (d, 1H, J = 2.3 Hz),
7.24–7.30 (m, 1H), 7.32–7.41 (m, 4H), 7.76 (d, 1H, J = 8.8 Hz), 9.82 (s, 1H), 10.07 (s, 1H),
11.23 (s, 1H); 13C NMR (126 MHz, DMSO-d6) δ (ppm) 99.6, 108.1, 109.5, 110.9, 124.7, 126.5,
+
127.6, 131.3, 133.7, 139.9, 157.7, 159.7, 163.0; HRMS (ESI+) m/z for C15H12NO3 [M + H]+:
calcd 254.0812, found 254.0818. Anal. Calcd. for C15H11NO3·½H2O (262.26): C, 68.70; H, 4.61;
N, 5.34. Found: C, 68.74; H, 4.79; N, 5.30.
4.1.3.4. 4,8-Dihydroxy-3-phenylquinolin-2(1H)-one (35).
Prepared from 4-hydroxy-8-methoxy-3-phenylquinolin-2(1H)-one (32) (2.67 g, 10 mmol) and
48% hydrobromic acid (100 mL, 0.88 mmol); reaction time was 17 h. Isolated as colourless
powder (2.10 g, 8.29 mmol, 83%) after recrystallization from EtOH; mp 215–219 °C (EtOH). IR

ACCEPTED MANUSCRIPT
3237, 1644, 1592, 1514, 1431, 1352, 1243, 1199, 1140, 1067, 822, 702, 542 cm–1; 1H NMR (500
MHz, DMSO-d6) δ (ppm) 6.95–7.05(m, 2H), 7.26–7.33 (m, 1H), 7.33–7.46 (m, 6H), 9.99 (s,
1H), 10.27 (br s, 1H). 13C NMR (126 MHz, DMSO-d6) δ (ppm) 112.6, 113.5, 114.8, 116.5,
+
121.5, 127.0, 127.3, 127.8, 131.2, 133.3, 143.6, 158.2, 161.9; HRMS (ESI+) m/z for C15H12NO3
[M + H]+: calcd 254.0812, found 254.0811.
4.1.4.1. 2-Oxo-1,2-dihydroquinolin-4-yl acetate (39).
A solution of acetic anhydride (98%, 17.0 mL, 18.0 g, 179 mmol), acetic acid (68 mL) and
sodium acetate (25 mg, 0.30 mmol) was added to 4-hydroxyquinolin-2(1H)-one (483 mg, 3
mmol) and the reaction mixture was refluxed for 2.5 h. After cooling, the reaction mixture was
filtered. The filtrate was evaporated to dryness in vacuo and the residue was crystallized from
EtOH yielding 39 as colourless needles (515 mg, 2.53 mmol, 85%); mp 219–223 °C (EtOH), mp
[35] 214–217 °C (EtOH). IR 2847, 1761, 1505, 1439, 1382, 1264, 1201, 1077, 914, 881, 767
1
cm–1; H NMR (500 MHz, DMSO-d6) δ (ppm) 2.43 (s, 3H), 6.40 (s, 1H), 7.19–7.25 (m, 1H),
7.37 (d, 1H), 7.55–7.60 (m, 1H), 7.66 (dd, 1H), 11.91 (s, 1H); 13C NMR (126 MHz, DMSO-d6) δ
(ppm) 20.8, 112.4, 114.8, 115.5, 122.0, 122.4, 131.5, 138.9, 156.1, 162.3, 168.1; MS (EI) m/z
(%): 204 (3, [M + 1]+), 203 (27, [M]+), 162 (10), 161 (100), 133 (26), 132 (13), 120 (30), 119
(58), 105 (18), 104 (12), 92 (21), 77 (26), 65 (10), 51 (12), 43 (88); HRMS (ESI+) m/z for
+
C11H10NO3 [M + H]+: calcd 204.0655, found 204.0642. Anal. Calcd. for C11H9NO3 (203.19): C,
65.02; H, 4.46; N, 6.89. Found: C, 64.89; H, 4.40; N, 6.78.
4.1.5. General procedures for the preparation of 3-alkyl/aryl-2-oxo-1,2-dihydroquinolin-4-yl
acetates 40–50.

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Acetic anhydride (30 mL, 0.31 mol) was added dropwise to a solution of an appropriate 4-
hydroxyquinolin-2(1H)-one (15.0 mmol) in pyridine (45 mL, 0.55 mol) within 10 minutes. The
reaction mixture was stirred at room temperature until TLC indicated complete consumption of
the starting material (30–120 min). The resulting suspension was evaporated in vacuo to dryness.
The residue was triturated with water (50 mL), and the resulting precipitate was collected by
filtration. The filter cake was washed with water until the filtrate was neutral and crystallized
from a suitable solvent affording pure product (40–50).
Spectral and analytical for 3-methyl-2-oxo-1,2-dihydroquinolin-4-yl acetate (40) [36], 3-
ethyl-2-oxo-1,2-dihydroquinolin-4-yl acetate (41) [37], 3-ethyl-7-methoxy-2-oxo-1,2-
dihydroquinolin-4-yl acetate (43) [38], 3-ethyl-8-methoxy-2-oxo-1,2-dihydroquinolin-4-yl
acetate (44) [38], 3-phenyl-2-oxo-1,2-dihydroquinolin-4-yl acetate (46) [34], and 3-phenyl-8-
methoxy-2-oxo-1,2-dihydroquinolin-4-yl acetate (49) [32] were in agreement with those reported
in the literature.
4.1.5.1. 3-Methyl-2-oxo-1,2-dihydroquinolin-4-yl acetate (40).
Synthesized from 4-hydroxy-3-methylquinolin-2(1H)-one (1) (2.63 g) and isolated as white
solid (3.04 g, 14.0 mmol, 93%), pure according to TLC and IR spectrum, which was crystallized
from benzene affording final 40 of the same appearance (2.86 g, 13.2 mmol, 88%); mp 255–256
°C (benzene), mp [36] 244–245 °C (EtOH); IR 3011, 2963, 2906, 2861, 1758, 1664, 1583, 1443,
1
1364, 1276, 1212, 1183, 1136, 1094, 899, 756, 695 cm–1; H NMR (500 MHz, DMSO-d6) δ
(ppm) 1.91 (s, 3H), 2.49 (s, 3H), 7.20 (ddd, 1H, J = 7.6, 7.6, 0.9 Hz), 7.35 (d, 1H, J = 8.0 Hz),
7.52 (ddd, 1H, J =7.7, 7.7, 1.2 Hz), 7.57 (d, 1H, J = 8.0 Hz), 11.98 (s, 1H); 13C NMR (126 MHz,

ACCEPTED MANUSCRIPT
DMSO-d6) δ (ppm) 10.1, 20.3, 115.2, 115.2, 120.3, 122.0, 122.0, 130.3, 136.9, 151.9, 162.7,
+
167.9; HRMS (ESI+) m/z for C12H12NO3 [M + H]+: calcd 218.0812, found 218.0809.
4.1.5.2. 3-Ethyl-2-oxo-1,2-dihydroquinolin-4-yl acetate (41).
Synthesized from 3-ethyl-4-hydroxyquinolin-2(1H)-one (2) (2.84 g, mmol) and isolated as
colourless needles (3.29 g, 14.2 mmol, 95%) after recrystallization from EtOH; mp 255–259 °C
(EtOH); mp [37] 247–248 °C (EtOH); IR 2967, 1766, 1661, 1577, 1501, 1435, 1368, 1198,
1137, 1099, 760, 684, 658 cm–1; MS (EI) m/z (%): 232 (5, [M + 1]+), 231 (34, [M]+), 190 (13),
189 (99), 188 (43), 175 (11), 174 (100), 120 (15), 92 (19), 69 (11), 65 (12), 55 (10). Anal. Calcd.
for C13H13NO3 (231.25): C, 67.52; H, 5.67; N, 6.06. Found: C, 67.28; H, 5.62; N, 5.94. 1H NMR
(500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data are collected in Table S3.
4.1.5.3. 3-Ethyl-6-methoxy-2-oxo-1,2-dihydroquinolin-4-yl acetate (42).
Synthesized from 3-ethyl-4-hydroxy-6-methoxyquinolin-2(1H)-one (19) (3.29 g) and isolated
as colourless crystals (2.92 g, 11.2 mmol, 75%) after recrystallization from EtOH; mp 232–237
°C (EtOH); IR 2935, 1769, 1651, 1624, 1502, 1423, 1366, 1273, 1223, 1195, 1137, 1097, 1037,
853, 741, 645, 615 cm–1; MS (EI) m/z (%): 262 (6, [M + 1]+), 261 (34, [M]+), 220 (13), 219
(100), 218 (39), 205 (10), 204 (77), 106 (14), 69 (11), 55 (11). Anal. Calcd. for C14H15NO4
1
(261.27): C, 64.36; H, 5.79; N, 5.36. Found: C, 64.16; H, 5.76; N, 5.23. H NMR (500 MHz,
DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data are collected in Table S3.
4.1.5.4. 3-Ethyl-7-methoxy-2-oxo-1,2-dihydroquinolin-4-yl acetate (43).

ACCEPTED MANUSCRIPT
Synthesized from 3-ethyl-4-hydroxy-7-methoxyquinolin-2(1H)-one (20) (3.29 g) and isolated
as colourless needles (3.45 g, 13.2 mmol, 88%) after recrystallization from EtOH; mp 225–227
°C (EtOH); mp [38] 223 °C (aq. EtOH); IR 2975, 1763, 1642, 1621, 1491, 1383, 1270, 1222,
1200, 1132, 1084, 1042, 735, 680 cm–1; MS (EI) m/z (%): 262 (4, [M + 1]+), 261 (27, [M]+), 219
(64), 218 (16), 205 (12), 204 (100). Anal. Calcd. for C14H15NO4 (261.27): C, 64.36; H, 5.79; N,
1
13
5.36. Found: C, 64.31; H, 5.75; N, 5.20. H NMR (500 MHz, DMSO-d6) and C NMR (125
MHz, DMSO-d6) data are collected in Table S3.
4.1.5.5. 3-Ethyl-8-methoxy-2-oxo-1,2-dihydroquinolin-4-yl acetate (44).
Synthesized from 3-ethyl-4-hydroxy-8-methoxyquinolin-2(1H)-one (21) (3.29 g) and isolated
as colourless crystals (3.34 g, 12.8 mmol, 85%) after recrystallization from EtOH; mp 174–181
°C (EtOH), mp [38] 180–182 °C (EtOH); IR 2975, 1763, 1642, 1621, 1491, 1383, 1270, 1222,
1200, 1132, 1084, 1042, 735, 680 cm–1; MS (EI) m/z (%): 262 (9, [M + 1]+), 261 (59, [M]+), 220
(14), 219 (100), 218 (83), 204 (48), 190 (24), 167 (11), 150 (11), 149 (66), 141 (16), 111 (11), 99
(14), 97 (16), 95 (10), 85 (36), 83 (16), 71 (52), 70 (15), 69 (38), 57 (69), 56 (13), 55 (32). Anal.
Calcd. for C14H15NO4 (261.27): C, 64.36; H, 5.79; N, 5.36. Found: C, 64.16; H, 5.74; N, 5.16. 1H
NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data are collected in Table S3.
4.1.5.6. 3-Ethyl-6-fluoro-2-oxo-1,2-dihydroquinolin-4-yl acetate (45).
Synthesized from 3-ethyl-6-fluoro-4-hydroxyquinolin-2(1H)-one (22) (3.11 g) and isolated as
colourless needles (3.46 g, 13.9 mmol, 93%) after recrystallization from EtOH; mp 271–275 °C
(EtOH); IR 2941, 1769, 1664, 1503, 1427, 1370, 1259, 1198, 1183, 1095, 1039, 859, 817, 645
cm–1; MS (EI) m/z (%): 250 (5, [M + 1]+), 249 (32, [M]+), 208 (13), 207 (100), 206 (40), 192

ACCEPTED MANUSCRIPT
(76), 149 (25), 138 (10), 110 (11), 85 (13), 83 (11), 71 (17), 69 (28), 57 (27), 55 (17). Anal.
Calcd. for C13H12FNO3 (249.24): C, 62.65; H, 4.85; N, 5.62. Found: C, 62.53; H, 4.85; N, 5.48.
1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data are collected in Table
S3.
4.1.5.7. 3-Phenyl-2-oxo-1,2-dihydroquinolin-4-yl acetate (46).
Synthesized from 4-hydroxy-3-phenylquinolin-2(1H)-one (4) (3.56 g) and isolated as
colourless needles (4.04 g, 14.5 mmol, 96%) after recrystallization from EtOH; mp 265–269 °C
(EtOH), mp [34] 259–262 °C (MeOH); IR 2897, 1767, 1657, 1497, 1431, 1361, 1190, 1077, 880,
752, 705, 648, 556 cm–1; MS (EI) m/z (%): 280 (4, [M + 1]+), 279 (23, [M]+), 238 (16), 237
(100), 236 (88), 120 (36), 92 (19). Anal. Calcd. for C17H13NO3 (279.29): C, 73.11; H, 4.69; N,
1
13
5.02. Found: C, 72.82; H, 4.64; N, 4.91. H NMR (500 MHz, DMSO-d6) and C NMR (125
MHz, DMSO-d6) data are collected in Table S4.
4.1.5.8. 6-Methoxy-3-phenyl-2-oxo-1,2-dihydroquinolin-4-yl acetate (47).
Synthesized from 4-hydroxy-6-methoxy-3-phenylquinolin-2(1H)-one (30) (4.01 g) and
isolated as colourless needles (4.18 g, 13.5 mmol, 90%) after recrystallization from EtOH; mp
274–278 °C (EtOH); IR 2854, 1774, 1656, 1626, 1496, 1355, 1282, 1219, 1177, 1063, 1034,
846, 756, 703, 649 cm–1; MS (EI) m/z (%): 310 (7, [M + 1]+), 309 (34, [M]+), 268 (17), 267
(100), 266 (64), 150 (14), 122 (10), 106 (12). Anal. Calcd. for C18H15NO4 (309.32): C, 69.89; H,
4.89; N, 4.53. Found: C, 69.62; H, 4.81; N, 4.43. 1H NMR (500 MHz, DMSO-d6) and 13C NMR
(125 MHz, DMSO-d6) data are collected in Table S4.

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4.1.5.9. 7-Methoxy-3-phenyl-2-oxo-1,2-dihydroquinolin-4-yl acetate (48).
Synthesized from 4-hydroxy-7-methoxy-3-phenylquinolin-2(1H)-one (31) (4.01 g) and
isolated as colourless crystals (3.60 g, 11.6 mmol, 78%) after recrystallization from EtOH; mp
236–239 °C (EtOH); IR 2942, 1770, 1660, 1625, 1575, 1514, 1445, 1361, 1287, 1263, 1195,
1134, 1070, 882, 815, 701, 685 cm–1; MS (EI) m/z (%): 310 (4, [M + 1]+), 309 (18, [M]+), 268
(17), 267 (100), 266 (71), 150 (52), 43 (14). Anal. Calcd. for C18H15NO4 (309.32): C, 69.89; H,
4.89; N, 4.53. Found: C, 69.67; H, 4.86; N, 4.39. 1H NMR (500 MHz, DMSO-d6) and 13C NMR
(125 MHz, DMSO-d6) data are collected in Table S4.
4.1.5.10. 8-Methoxy-3-phenyl-2-oxo-1,2-dihydroquinolin-4-yl acetate (49).
Synthesized from 4-hydroxy-8-methoxy-3-phenylquinolin-2(1H)-one (32) (4.01 g) and
isolated as colourless crystals (4.18 g, 13.5 mmol, 90%) after recrystallization from EtOH; mp
195–197 °C (EtOH); IR 3004, 1775, 1644, 1503, 1362, 1279, 1265, 1191, 1140, 1061, 875, 775,
734, 698 cm–1; MS (EI) m/z (%): 310 (5, [M + 1]+), 309 (25, [M]+), 268 (17), 267 (100), 266
(88), 251 (12), 238 (31), 237 (15), 150 (16), 92 (11), 43 (28). Anal. Calcd. for C18H15NO4
1
(309.32): C, 69.89; H, 4.89; N, 4.53. Found: C, 69.61; H, 4.84; N, 4.36. H NMR (500 MHz,
DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data are collected in Table S4.
4.1.5.11. 6-Fluoro-3-phenyl-2-oxo-1,2-dihydroquinolin-4-yl acetate (50).
Synthesized from 6-fluoro-4-hydroxy-3-phenylquinolin-2(1H)-one (38) (3.83 g) and isolated
as colourless crystals (3.81 g, 12.8 mmol, 85%) after recrystallization from EtOH; mp 261–268
°C (EtOH); IR 2834, 1773, 1651, 1498, 1446, 1423, 1268, 1172, 1063, 866, 754, 701, 651, 605
cm–1; MS (EI) m/z (%): 298 (4, [M + 1]+), 297 (19, [M]+), 256 (16), 255 (100), 254 (90), 138

ACCEPTED MANUSCRIPT
(27), 110 (12). Anal. calcd. for C17H12FNO3 (297.28): C, 68.68; H, 4.07; N, 4.71. Found: C,
1
13
68.54; H, 4.12; N, 4.58. H NMR (500 MHz, DMSO-d6) and C NMR (125 MHz, DMSO-d6)
data are collected in Table S4.
4.1.6. General procedures for the synthesis of rac-4-hydroxy-8,9,9-trimethyl-1-phenyl-5,6,7,8-
tetrahydro-5,8-methanoquinolin-2(1H)-ones 51 and 52.
Compounds were synthesized by a modified literature procedure [16]. A mixture of rac-1,7,7-
trimethyl-N-phenylbicyclo[2.2.1]heptan-2-imine (3.41 g, 15.0 mmol) and the bis(2,4,6-
trichlorophenyl) butylmalonate (7.40 g, 14.3 mmol) or bis(2,4,6-trichlorophenyl)
phenylmalonate (7.98 g, 14.2 mmol) was heated to 240–250 °C (metal bath) for 1.5 h or 10 min,
respectively. Cooled reaction mixture was triturated with petroleum ether (30 mL). Solid phase
was collected by filtration, washed gradually with petroleum ether (2 × 20 mL) and diethyl ether
(2 × 10 mL) and recrystallized from ethanol.
Data for rac-4-hydroxy-8,9,9-trimethyl-1,3-diphenyl-5,6,7,8-tetrahydro-5,8-methanoquinolin-
2(1H)-one were have been reported previously [16].
4.1.6.1.
rac-3-Butyl-4-hydroxy-8,9,9-trimethyl-1-phenyl-5,6,7,8-tetrahydro-5,8-
methanoquinolin-2(1H)-one (52).
Colourless needles (2.46 g, 7.00 mmol, 49%); mp 296–299 °C (EtOH); IR 2955, 1619, 1535,
1
1434, 1388, 1209, 1114, 768, 693, 622 cm–1; H NMR (500 MHz, DMSO-d6) δ (ppm) 0.21 (s,
3H), 0.74 (s, 6H), 0.86 (t, 3H, J = 7.2 Hz), 1.01–1.10 (m, 1H), 1.22–1.39 (m, 5H), 1.54 (ddd, 1H,
J = 12.4, 9.1, 3.6 Hz), 1.88–1.98 (m, 1H), 2.36 (tt, 2H, J = 8.2, 4.2 Hz), 3.06 (d, 1H, J = 3.3 Hz),
7.13–7.22 (m, 2H), 7.40–7.51 (m, 3H), 9.52 (br s, 1H); 13C NMR (126 MHz, DMSO-d6) δ (ppm)

ACCEPTED MANUSCRIPT
12.0, 14.0, 19.0, 19.7, 22.4, 23.5, 26.2, 30.4, 32.1, 46.9, 56.0, 56.2, 108.0, 114.0, 128.2, 128.6,
+
128.6, 128.7, 129.9, 138.9, 150.7, 158.7, 163.5; HRMS (ESI+) m/z for C23H30NO2 [M + H]+:
calcd 352.2271, found 352.2271. Anal. calcd. for C23H29NO2 (351.48): C, 78.59; H, 8.32; N,
3.99. Found: C, 78.38; H, 8.64; N, 3.72.
4.2. Biology
4.2.1. Materials
7H9 Growth medium was purchased as a powder from BD Science (Franklin Lakes, NJ,
USA). OADC, mycobactin J, penicillin, fungizone and hygromycin were from Roche (Basel,
CH) and Triton X-100, glycerol, Tween 80 and n-decanal were purchased from Sigma Aldrich
(St. Louis, MO, USA). M. tuberculosis H37Ra (American Type Culture Collection (ATCC)
25177) is known to be sensitive to the five first line anti-tuberculosis drugs (streptomycin,
isoniazid, rifampin, ethambutol and pyrazinamide). Four other strains used for screening were M.
bovis strain AN5, M. avium ssp. avium ATCC 15769, M. avium ssp. paratuberculosis
ATCC19698. All strains were cultivated in 7H9 medium supplemented with 10% oleic
acidalbumin- dextrose-catalase (OADC) and 0.2% glycerol. For M. bovis, the glycerol was
replaced with 0.05% Tween 80. The growth medium of M. avium ssp. paratuberculosis was
further supplemented with mycobactin J (2 µg/mL) required for optimal in vitro growth. Both M.
avium strains and the M. bovis strain were grown at 39 °C, M. ulcerans was grown at 32 °C and
M. tuberculosis was grown at 37 °C.
4.2.2. Biological evaluation
4.2.2.1. Evaluation of antitubercular activity