Ring-substituted 8-hydroxyquinoline-2-carboxanilides as potential antimycobacterial agents

Article abstract of DOI:10.1016/j.bmc.2015.06.047

In this study, a series of twenty-two ring-substituted 8-hydroxyquinoline-2-carboxanilides was prepared and characterized. Primary in vitro screening of the synthesized compounds was performed against Mycobacterium tuberculosis H37Ra, Mycobacterium avium complex and M. avium subsp. paratuberculosis. Some of the tested compounds showed the antimycobacterial activity against M. avium subsp. paratuberculosis comparable with or higher than that of rifampicin. 8-Hydroxy-N-[3-(trifluoromethyl)phenyl]- and 8-hydroxy-N-[4-(trifluoromethyl)phenyl]quinoline-2-carboxamide showed MIC = 24 μM against all tested mycobacterial strains. 3-Methoxyphenyl- and 3-methylphenyl derivatives expressed MIC = 27 or 29 μM also against all the tested strains. Their activity against M. avium subsp. paratuberculosis was 4-fold higher than that of rifampicin. 2-Bromophenyl- and 2-(trifluoromethyl)phenyl derivatives had MIC = 23 or 24 μM against M. tuberculosis. A significant decrease of mycobacterial cell metabolism (viability of M. tuberculosis H37Ra) was observed using MTT assay. Screening of cytotoxicity of the compounds was performed using the THP-1 cells, and no significant lethal effect was observed up to tested concentration 30 μM. The structure-activity relationships are discussed.

Full text of DOI:10.1016/j.bmc.2015.06.047

Accepted Manuscript
Ring-substituted 8-hydroxyquinoline-2-carboxanilides as potential antimyco-
bacterial agents
Jiri Kos, Iveta Zadrazilova, Eoghan Nevin, Michal Soral, Tomas Gonec, Peter
Kollar, Michal Oravec, Aidan Coffey, Jim O’Mahony, Tibor Liptaj, Katarina
Kralova, Josef Jampilek
PII:
S0968-0896(15)00541-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.06.047
BMC 12403
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
16 April 2015
13 June 2015
18 June 2015
Please cite this article as: Kos, J., Zadrazilova, I., Nevin, E., Soral, M., Gonec, T., Kollar, P., Oravec, M., Coffey,
A., O’Mahony, J., Liptaj, T., Kralova, K., Jampilek, J., Ring-substituted 8-hydroxyquinoline-2-carboxanilides as
potential antimycobacterial agents, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.
2
015.06.047
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Ring-substituted 8-hydroxyquinoline-2-carboxanilides as
potential antimycobacterial agents
a
Jiri Kos , Iveta Zadrazilova , Eoghan Nevin , Michal Soral , Tomas Gonec , Peter Kollar ,
a
b
c
a
d
e
b
b
c
f
Michal Oravec , Aidan Coffey , Jim O'Mahony , Tibor Liptaj , Katarina Kralova , Josef
a,
Jampilek *
a
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and
Pharmaceutical Sciences, Palackeho 1/3, 612 42 Brno, Czech Republic
b
Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland
c
Department of NMR Spectroscopy and Mass Spectrometry, Faculty of Chemical and Food
Technology, Slovak University of Technology in Bratislava, Radlinskeho 9, 812 37 Bratislava,
Slovakia
d
Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of
Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 612 42 Brno, Czech Republic
e
Global Change Research Centre AS CR, Belidla 986/4a, 603 00 Brno, Czech Republic
f
Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina
Ch-2, 842 15 Bratislava, Slovakia
*
Corresponding author: E-mail: josef.jampilek@gmail.com
1

Abstract:
In this study, a series of twenty-two ring-substituted 8-hydroxyquinoline-2-carboxanilides
was prepared and characterized. Primary in vitro screening of the synthesized compounds was
performed against Mycobacterium tuberculosis H37Ra, M. avium complex and M. avium
subsp. paratuberculosis. Some of the tested compounds showed the antimycobacterial activity
against M. avium subsp. paratuberculosis comparable with or higher than that of rifampicin.
8
-Hydroxy-N-[3-(trifluoromethyl)phenyl]-
and
8-hydroxy-N-[4-
(
trifluoromethyl)phenyl]quinoline-2-carboxamide showed MIC = 24 µM against all tested
mycobacterial strains. 3-Methoxyphenyl- and 3-methylphenyl derivatives expressed
MIC = 27 or 29 µM also against all the tested strains. Their activity against M. avium subsp.
paratuberculosis was 4-fold higher than that of rifampicin. 2-Bromophenyl- and 2-
(
trifluoromethyl)phenyl derivatives had MIC = 23 or 24 µM against M. tuberculosis.
A significant decrease of mycobacterial cell metabolism (viability of M. tuberculosis H37Ra)
was observed using MTT assay. Screening of cytotoxicity of the compounds was performed
using the THP-1 cells, and no significant lethal effect was observed up to tested concentration
3
0 µM. The structure-activity relationships are discussed.
Keywords: 8-hydroxyquinolines; in vitro antimycobacterial activity; MTT assay; in vitro
cytotoxicity; structure-activity relationships.
2

1
. Introduction
Tuberculosis (TB) remains one of the world’s deadliest communicable diseases.
Mycobacterium tuberculosis, the pathogen responsible for TB, uses diverse strategies to
survive in a variety of host lesions and to evade immune surveillance. In 2013, an estimated
9
.0 million people developed TB and 1.5 million died from the disease, 360 000 of whom
were HIV-positive. Multidrug-resistant TB strains (MDR-TB) are a great worldwide problem.
Drug resistance surveillance data indicate that in 2013 approximately 480 000 people
developed MDR-TB worldwide. Among TB patients reported by national TB programmes in
2
013, there were an estimated 300 000 cases of MDR-TB. Extensively drug-resistant TB
(
XDR-TB, defined as MDR-TB plus resistance to any fluoroquinolone and any second-line
injectable drugs) has been identified in 100 countries globally in 2013, and the average
proportion of MDR-TB cases with XDR-TB was 9.0%. Also non-tuberculous mycobacteria
(
NTM), for example, M. avium, M. kansasii, M. abscessus, M. fortuitum, M. chelonae,
M. smegmatis, which become the main human pathogens and cause difficult-to-treat or
incurable diseases ending in death of mainly HIV-positive patients, are a threat. NTM can
cause a broad spectrum of diseases, such as pulmonary disease, lymphadenitis, skin and soft
tissue disease, gastrointestinal and skeletal infections. The number of infections that can be
associated with specific species as well as the number of new species as etiological agents has
exploded in the last few years due to the increase in the number of immunocompromised
patients. The emergence of MDR-TB and NTM makes the discovery of new molecular
1–4
scaffolds a priority to achieve effective control of both TB and NTM.
5
The quinoline nucleus can be considered as a privileged structure; quinoline-based
compounds exhibit various activities such as anti-inflammatory, cardiovascular,
6
–12
anticonvulsant, antiproliferative, antiprotozoan, antifungal and antibacterial.
Quinoline
6
–8,13,14
analogues
antimycobacterial agents.
Sirturo™) belonging to diarylquinolines, a new class of antitubercular drugs, was approved
and especially 8-hydroxyquinolines have been also recognized as promising
1
5–18
The best known quinoline anti-TB derivative, bedaquiline
(
1
by FDA for treatment of MDR-TB. Structural modifications of quinolines by various groups
9
have led to the identification of potent anti-TB compounds exhibiting significant activity
13,14,20-29
especially against strains of M. tuberculosis.
It was found that these anti-TB
quinolines exhibit various mechanisms of action; for example, they inhibit mycobacterial
2
3,30
31 32
mycobacterial FtsZ protein, glutathione S-transferase, enoyl-ACP
ATP synthase,
2
0,21,33
24
or decaprenylphosphoryl-β-D-ribose-2′-epimerase; also other sites of actions
reductase
3

of 8-hydroxyquinolines can be found due to the bidentate metal-chelating property of
1
5–18
8
-hydroxyquinolines.
The aim of this contribution is synthesis and antimycobacterial screening of
-hydroxyquinoline-2-carboxanilides with following analysis of the structure-activity
8
relationships as a continuation of the investigation of recently designed ring-substituted
9
-11,34-37
quinoline analogues.
Based on the above mentioned facts all the presented compounds
were designed to bear an 8-hydroxyquinoline fragment and an amide moiety. Both structural
features are important functional groups that are able to interact with a number
of enzymes/receptors via hydrogen bonds and in this manner to affect the biological
3
8-40
response.
2
. Results and Discussion
2
.1 Chemistry and physicochemical properties
All studied compounds 1–8c were prepared according to Scheme 1 by modified
4
1,42
microwave-assisted (MW) synthesis;
thus the synthesis of the target compounds was
carried out in only one step with yields 66–76%. At first the carboxyl group was activated
with phosphorus trichloride. The final amide was immediately formed by aminolysis of the
acyl chloride by ring-substituted aniline in dry chlorobenzene. All the crude target compounds
were recrystallized from ethanol.
Scheme 1. Synthesis of ring-substituted 8-hydroxyquinoline-2-carboxanilides 1–8c
Within structure-activity relationship studies various parameters describing
physicochemical properties are investigated. Lipophilicity is a property that has a major effect
on solubility, absorption, distribution and biotransformation as well as pharmacological
activity, because drugs cross biological membranes through passive transport, which strongly
depends on their lipophilicity. Lipophilicity of the studied compounds was determined
4

by RP-HPLC as capacity factor logarithm (log k) and calculated as log P using ACD/Percepta
ver. 2012 (Advanced Chemistry Development, Inc., Toronto, ON, Canada, 2012). The
corresponding values of ring-substituted 8-hydroxyquinoline-2-carboxanilides 1–8c are shown
in Table 1.
Table 1. Structure of discussed 8-hydroxyquinoline-2-carboxanilides 1–8c, experimentally
determined values of lipophilicity log k, predicted values of compound lipophilicity log P and
electronic Hammett’s σ parameters of R substituents, in vitro antimycobacterial activity
(
MIC) of compounds in comparison with rifampicin (RIF) standard and in vitro cytotoxicity
assay (IC50) of selected compounds.
MIC [µM]
MAC
30
IC50
a
a
Comp.
1
R
log k log P
σ
MT
30
27
27
849
29
29
29
28
53
53
27
50
837
23
44
729
24
24
24
97
26
26
10
MAP [µM]
H
0.7600 2.55
0.7935 2.67
0.8164 2.61
0.7129 2.51
0.6944 2.90
0.9686 2.90
0.9521 2.90
0.6806 2.59
0.9420 2.76
0.8598 2.59
0.9566 3.07
1.1718 3.28
1.1543 3.05
1.0536 3.16
1.2357 3.31
1.2347 3.19
0.9147 3.36
1.3206 3.44
1.3653 3.27
1.1277 2.73
0.9845 2.56
1.0495 2.45
0
30
51
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
–
2a
2-OCH
3-OCH
4-OCH
2-CH
3-CH
4-CH
3
-0.28
0.12
-0.27
-0.17
-0.07
-0.17
0.06
0.34
0.06
0.22
0.37
0.23
0.22
0.39
0.23
0.51
0.43
0.51
0.77
0.71
0.78
–
51
2
b
3
27
27
2
c
3
425
54
849
29
3a
3
3
b
3
29
29
3
c
3
54
29
4a
2-F
3-F
53
28
4
b
53
53
4
c
4-F
53
53
5a
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
837
50
27
5
b
50
5
c
50
837
23
6a
729
44
6
b
44
6
c
44
729
24
7a
2-CF
3
752
24
7
b
3-CF
3
24
7
c
4-CF
2-NO
3-NO
4-NO
–
3
24
24
8a
2
2
2
808
808
808
10
97
8
b
808
404
109
8
c
RIF
–
−
a
calculated using ACD/Percepta ver. 2012 (Advanced Chemistry Development, Inc., Toronto, ON,
Canada, 2012); MT = M. tuberculosis H37Ra ATCC 25177, MAC = M. avium complex CIT19/06
(
clinical isolate) and MAP = M. avium subsp. paratuberculosis CIT03 (clinical isolate).
5

The highest experimental lipophilicity was found for 8-hydroxy-N-(4-
trifluoromethylphenyl)quinoline-2-carboxamide (7c), while N-(2-fluorophenyl)-8-
hydroxyquinoline-2-carboxamide (4a) showed the lowest log k value. The results obtained
with the discussed compounds show that the experimentally-determined lipophilicities (log k)
of the ortho-substituted derivatives showed poor match, while the meta- and para-substituted
compounds showed higher match with the predicted log P values. In this case the most
significant deviations can be observed for nitro substituted derivatives; when 2-NO
eliminated, correlation coefficient of the dependence of log k on log P values improves to
r = 0.7502 (n = 6) and after elimination of 3-NO (8b) and 4-NO (8c) correlation coefficient
2
(8a) is
2
2
of the dependence of log k on log P values improves to r = 0.9759 (n = 13), which reflects
intra/intermolecular interactions of the nitro moiety. The influence of R substituents on
lipophilicity of ortho-substituted derivatives is as follows: F < CH
Br < NO ; for meta- and para-substituted derivatives it is as follows: OCH
NO < Cl < Br < CF . Within the individual series the lipophilicity determined by log k values
increases for halogens, methyl and trifluoromethyl substituents as follows: ortho < para <
meta; for methoxy substituent as follows: para < ortho < meta, and for NO as follows: meta
3
3 3
< H < OCH < CF < Cl <
2
3
/ H < F < CH <
3
2
3
2
<
para < ortho.
Similarly poor correlation between experimental and calculated lipophilicity values
9
-11,35,36,43
was found also for different derivatives of 8-hydroquinolines.
This is probably
caused by the fact that between the hydroxyl moiety in C(8) of the quinoline nucleus and
nitrogen of quinoline intramolecular H-bond affecting lipophilicity, solubility and
44
basicity/acidity is formed. Based on the presented results, it can be stated that log k values
specify lipophilicity within individual series of the studied compounds better than log P
values.
The presence of different substituents in the anilide part of the discussed compounds
results in a wide range of electronic properties. Electronic parameters (expressed as
Hammett’s σ parameters) of compounds 1
see Table 1; they ranged from -0.28/-0.27 (compound 2a,c 2-OCH
compound 8a,c 2-NO , 4-NO ).
–
8c were predicted using ACD/Percepta software,
3
, 4-OCH ) to 0.77/0.78
3
(
2
2
2
.2 In vitro antimycobacterial evaluation
6

The evaluation of the in vitro antimycobacterial activity of the compounds was
performed against Mycobacterium tuberculosis H37Ra ATCC 25177 (MT) and clinical
isolates of M. avium complex CIT19/06 (MAC) and M. avium subsp. paratuberculosis CIT03
(
MAP), see Table 1. To reduce risk and make manipulation in the laboratory easier, surrogate
model pathogens for M. tuberculosis can be used in laboratory studies. Avirulent
M. tuberculosis strain H37Ra is very closely related to human-infecting M. tuberculosis
strains (e.g., H37Rv), making it a good model for study especially because of the lower risk
4
5
for laboratory workers. Because of M. tuberculosis, the pathogenic role of NTM in humans
was overshadowed for a long time. Mycobacterium avium complex includes ubiquitous
atypical mycobacteria found in the environment that can easily infect immunosuppressed
4
6
patients and cause disseminated disease, producing fever, sweats, weight loss and anaemia.
M. avium subsp. paratuberculosis is suspected to be a causative agent in gastrointestinal
diseases; it is resistant to standard antimycobacterial therapy, but may be susceptible to some
4
7
standard antibiotics, however, resistance to these antibiotics develops quickly. The activity
of compounds was expressed as the minimum inhibitory concentration (MIC) that is defined
for mycobacteria as a 90% or greater (IC90) reduction of growth in comparison with the
control. The MIC/IC90 value is routinely and widely used in bacterial assays and is a standard
detection limit according to the Clinical and Laboratory Standards Institute (CLSI).
Most of the target compounds showed activity against all three tested mycobacterial
strains. With respect to M. tuberculosis a greater number of more efficient compounds
showing higher effect than para-substituted compounds can be found among ortho- and meta-
substituted derivatives, except for C'(4) substituted derivatives 3c (R = 4-CH
and 8c (R = 4-NO ), which activity was comparable with that of ortho- and meta-substituted
compounds. Compounds 6a (R = 2-Br, MIC = 23 µM), 7a (R = 2-CF , MIC = 24 µM), 7b
3-CF , MIC = 24 µM) and 7c (4-CF , MIC = 24 µM) exhibited the highest activity. The
3 3
), 7c (R = 4-CF )
2
3
(
3
3
antimycobacterial activity of the compounds against M. tuberculosis was expressed as
log(1/MIC [M]), and the dependences of the activity on lipophilicity expressed as log k and
electronic properties expressed as Hammett’s σ constants are illustrated in Figures 1A and 1B.
3
When really inactive para-substituted compounds 2c, 5c and 6c (R = 4-OCH , 4-Cl, 4-Br) are
eliminated (marked by empty symbols), it is evident that neither lipophilicity nor electronic
properties possess any significant effect on the antitubercular activity. Compounds 4b,c
(
R = 3-, 4-F), 5b (R = 3-Cl) and 6b (R = 3-Br) had slightly lower activity than other
4 illustrated compounds. Similar trends can be found for the dependences of the activity
1
7

against M. avium subsp. paratuberculosis on physicochemical parameters, therefore these
dependences are not illustrated. It is important to note that nitro substituted derivatives 8a–c
expressed different activity than it could be expected. As mentioned in section 2.1, all nitro
derivatives 8a–c showed poor correlation between experimental and calculated lipophilicity
values. Especially C'(2) substituted derivative 8a has the highest experimentally determined
lipophilicity (log k = 1.1277) along with the strongest electron-withdrawing properties but the
lowest antitubercular activity within the series. Probably the above mentioned interactions of
the nitro moiety with the amide bond limited binding of the nitro substituted derivatives to
target sites in mycobacterial cells. These interactions are most evident for compound 8a,
therefore this compound is also eliminated (marked by empty symbols in Figures 1A and 1B)
from SAR discussion. Similar observations for compound 8a were made in respect to
M. avium subsp. paratuberculosis.
Figure 1. Dependence of antitubercular activity against M. tuberculosis expressed as
log 1/MIC [M] of tested compounds on lipophilicity expressed as log k (A) and on Hammett’s
σ constants of R substituent (B). (Inactive compounds are marked by empty symbols.)
A 4.8
B 4.8
4.5
4.5
4.2
3.9
3.6
3.3
3.0
2.7
4.2
8a
8a
3.9
3.6
3.3
6c
2c
5c
6c
2c
3.0
5c
2.7
0.5
0.7
0.9
1.1
1.3
1.5
-0.4
-0.2
0.0
0.2
0.4
0.6
para
0.8
1.0
log k
σ
ortho meta
H
ortho meta para
H
3
Compounds 6a (R = 2-Br, MIC = 23 µM), 7b (R = 3-CF , MIC = 24 µM) and
7
c (R = 4-CF , MIC = 24 µM) showed the highest activity against M. avium subsp.
3
paratuberculosis. Based on the results it can be stated that ortho- and especially meta-
substituted derivatives expressed higher activity than para-substituted ones. Only 7c showed
high effect within the C'(4) substituted compounds. Similarly as for the previous strain,
electronic properties show that the character of individual substituents R does not influence
the antimycobacterial activity significantly.
8

Compounds 7b (3-CF
highest activity against M. avium complex. The nitro moiety in all three positions (compounds
a-c) is the least favoured substituent, therefore these compounds are not considered for SAR
3 3
, MIC = 24 µM) and 7c (4-CF , MIC = 24 µM) showed the
8
discussion. Most effective compounds can be found among meta-substituted derivatives. The
antimycobacterial activity of the compounds against M. avium complex was expressed as
log(1/MIC [M]), and the dependences of the activity on lipophilicity expressed as log k and
electronic properties expressed as Hammett’s σ constants are illustrated in Figures 2A and 2B.
Generally, it seems that within the whole discussed series of compounds dependences of
antimycobacterial activity against M. avium complex on lipophilicity and electronic properties
increase slightly with increasing of lipophilicity values and electron-withdrawing effect of
substituents; although for the meta-substituted compounds this dependence is very slight, see
Figures 2A and 2B. The significant dependence of antimycobacterial activity on lipophilicity
and electronic properties of the ortho- and para-substituents can be observed. The ortho-
substituted derivatives are completely ineffective when lipophilicity (log k) is >0.8 along with
Hammett’s σ constants of R substituents >0.1 (see compounds 5a, 6a, 7a), contrary to the
properties of the para-substituted compounds that are ineffective when log k <0.8 and σ < -0.2
(
compound 2c), see Figures 2A and 2B.
Figure 2. Dependence of antimycobacterial activity against M. avium complex expressed as
log 1/MIC [M] of tested compounds on lipophilicity expressed as log k (A) and on Hammett’s
σ constants of R substituent (B). (Compounds 8a–c not included in SAR discussion are
marked by empty symbols.)
A 4.8
.5
.2
.9
.6
.3
.0
.7
B 4.8
4.5
4
4
4.2
3
3.9
3
3.6
2c
3.3
2c
3
8a
6
a
6a
7a
8b
7a
8b
5a
8c
3
3.0
5a
8c 8a
2
2.7
0.5
0.7
0.9
1.1
1.3
1.5
-0.4
-0.2
0.0
0.2
0.4
0.6
para
0.8
1.0
log k
σ
H
ortho meta para
H
ortho
meta
Generally all the prepared compounds showed relatively high lipophilicity
(
log P = 2.5–3.4 or log k = 0.68–1.36) that enables the compounds to permeate through
9

hydrophobic mycobacterial cell wall. Compound 2c (R = 4-OCH
against all three mycobacterial strains; compounds 5c, 6c, 8b and 8c (R = 4-Cl, 4-Br, 3-NO
-NO ) were ineffective against two of the tested mycobacterial strains. It can be stated that
3
) was completely inactive
2
,
4
2
there is no any significant difference between antimycobacterial activities against
M. tuberculosis and M. avium subsp. paratuberculosis, nevertheless ortho-substituted
derivatives seem to be favourable, see 4a>4b=4c, 5a>5b>>>5c, 6a>6b>>>6c, except
7
3 2
a (2-CF ) and 8a (2-NO ) substituted derivatives. On the other hand, the meta-substituted
derivatives showed the highest potency against M. avium complex. When the compounds
showed potency against M. avium complex, they were effective also against M. tuberculosis
and M. avium subsp. paratuberculosis. Thus, it can be postulated that compounds with
potency against all three strains were received by the substitution of C'(3) position of aniline.
On the other hand also the hydroxyl moiety in C(8) of quinoline seems to play a significant
role for activity, because its absence in quinoline-2-carboxanilides led to a decrease of
3
antimycobacterial effect. Similar essential contribution of the hydroxyl moiety amplifying
7
4
1
antimycobacterial potency was also observed for 1-hydroxynaphthalene-2-carboxanilides
4
2
and 6-hydroxynaphthalene-2-carboxanilides versus naphthalene-2-carboxanilides.
37
2
.3 MTT assay
Additionally, a standard MTT assay was performed for the selected most effective
compounds, the MICs of which were previously determined through alamarBlue assays, see
Table 1. The MTT assay is a well-characterized method of assessing cell growth through
measurement of respiration. For the purpose of this assay, a measured viability of
M. tuberculosis H37Ra of less than 70%, as determined by MTT, after exposure to the MIC of
each test compound (as determined by the alamarBlue assay) was considered a positive result.
As such, a low level of cell viability may suggest inhibition of cell growth through respiratory
4
8
inhibition. Model compounds selected from 14 derivatives with the highest antitubercular
activity, namely 3-methoxy (2b, 30.9 %), 3-methyl (3b, 66.4 %), 2-fluoro (4a, 26.9 %), 2-
chloro (5a, 21.4 %), 2-bromo (6a, 26.9 %), 2-trifluoromethyl (7a, 53.4 %),
3
-trifluoromethyl (7b, 18.8 %), 4-trifluoromethyl (7c, 33.1 %), 3-nitro (8b, 7.0 %) and 4-nitro
8c, 17.3 %) derivatives, showed less than 70% viability of M. tuberculosis H37Ra at the
lowest tested concentration (8 µg/mL, i.e. ca. 26 µM). Similar effect was observed previously
(
4
9
with ring-substituted naphthalene-1-carboxanilides
where 3-methoxy and 4-methyl
derivatives showed more than 80% reduction in activity at the lowest tested concentration
1
0

(
8 µg/mL, i.e., ca. 30 µM) after four hours of incubation, similar to the reduction observed in
the rifampicin and ciprofloxacin standards and 2-methoxy and 3-fluoro derivatives achieved
2
0,33
similar levels of inhibition at 16 µg/mL (ca. 60 µM) concentration.
Based on the fact that the change in colour of alamarBlue is caused by a decrease of
mycobacterial cell metabolism, it may be hypothesized that the mechanism of action of these
ring-substituted 8-hydroxyquinoline-2-carboxanilides could be connected with affection of
3
mycobacterial energy metabolism; nevertheless, another possible site of action of the
8
15-18
studied compounds in the mycobacteria cannot be excluded.
2
.4 In vitro cytotoxicity assay
The preliminary in vitro screening of the cytotoxicity of the compounds was
performed using the human monocytic leukemia THP-1 cell line. The cytotoxicity was
evaluated as the IC50 value (compound concentration causing 50% inhibition of cell
population proliferation), see Table 1. A compound is considered as cytotoxic when it
5
0
demonstrates a toxic effect on cells at the concentration up to 10 µM, and the highest tested
concentration that was used for the toxicity assay was three times this value. Treatment with
3
0 µM of the discussed compounds did not lead to significant lethal effect on THP-1 cells.
Based on these observations it can be concluded that the discussed amides can be considered
as non-toxic agents for subsequent design of novel antimycobacterial agents.
3
. Conclusions
A series of twenty-two ring-substituted 8-hydroxyquinoline-2-carboxanilides were
prepared by means of microwave-assisted synthesis and subsequently characterized. All the
compounds were tested for their in vitro antimycobacterial activity against M. tuberculosis
and clinical isolates of M. avium complex and M. avium subsp. paratuberculosis. Some of the
tested compounds showed the antimycobacterial activity against M. avium subsp.
paratuberculosis comparable with or higher than that of rifampicin. 8-Hydroxy-N-[3-
(
trifluoromethyl)phenyl]- (7b) and 8-hydroxy-N-[4-(trifluoromethyl)phenyl]quinoline-2-
carboxamide (7c) showed MIC = 24 µM against all three tested mycobacterial strains. 3-
Methoxyphenyl- (2b) and 3-methylphenyl (3b) derivatives expressed MIC = 27 or 29 µM
1
1

also against all the tested strains. 2-Bromophenyl- (6a) and 2-(trifluoromethyl)phenyl (7a)
derivatives showed MIC = 23 or 24 µM against M. tuberculosis. All the compounds possess
high lipophilicity; they can permeate through hydrophobic mycobacterial cell wall. Based on
the results it can be postulated that compounds with potency against all three strains were
received by the substitution of C'(3) position of aniline. The performed MTT assay of the
selected compounds shows that they cause a decrease of mycobacterial cell metabolism;
probably they influence the mycobacterial respiratory chain. All the compounds were tested for
their in vitro cytotoxicity against the THP-1 cells and within this preliminary screening they
demonstrated insignificant toxicity up to tested concentration 30 µM, therefore it can be
concluded that the discussed anilides can be considered as promising agents for subsequent
design of novel antimycobacterial agents.
4
. Experimental
4
.1. Chemistry
All reagents were purchased from Aldrich (Sigma-Aldrich, St. Louis, MO, USA). TLC
experiments were performed on alumina-backed silica gel 40 F254 plates (Merck, Darmstadt,
Germany). The plates were illuminated under UV (254 nm) and evaluated in iodine vapour. The
melting points were determined on Kofler hot-plate apparatus HMK (Franz Kustner Nacht
KG, Dresden, Germany) and are uncorrected. Infrared (IR) spectra were recorded on a Smart
MIRacle™ ATR ZnSe for Nicolet™ Impact 410 FT-IR spectrometer (Thermo Electron
Corporation, West Palm Beach, FL, USA). The spectra were obtained by accumulation of
−
1
−1
1
13
56 scans with 2 cm resolution in the region of 4000–600 cm . All H- and C-NMR
2
spectra were recorded on an Agilent VNMRS 600 MHz system (Agilent Technologies, Santa
1
. H and
Clara, CA, USA) equipped with a triple resonance HCN probe at 25 °C in DMSO-d
6
1
3
13
19
1
C chemical shifts and C- F coupling constants were determined from the standard H and
1
3
C spectra with digital resolution 0.3 Hz or better. Chemical shifts (δ) are reported in ppm.
1
When necessary, additional experiments were done: C-APT (Attached Proton Test) for
3
discrimination between CH and quaternary carbons; DQF COSY, HSQC and HMBC for
1
1
1
13
through-bond H- H and one- and multiple-bond H- C correlations. Mass spectra were
measured using a LTQ Orbitrap Hybrid Mass Spectrometer (Thermo Electron Corporation)
with direct injection into an APCI source (400 °C) in the negative mode.
1
2

General procedure for synthesis of 8-hydroxy-N-(substituted-phenyl)quinoline-2-
carboxamides 1–8c. 8-Hydroxyquinoline-2-carboxylic acid (5.3 mM) was suspended in dry
chlorobenzene (30 mL) at ambient temperature and phosphorus trichloride (2.7 mM, 0.5 eq.),
and the corresponding substituted aniline (5.3 mM, 1 eq.) was added dropwise. The reaction
mixture was transferred to the microwave reactor, where the synthesis was performed (1st
phase: 10 min, 100 °C, 100 W; 2nd phase: 15 min, 120 °C, 500 W; 3rd phase: 20 min,
1
30 °C, 500 W). Then the mixture was cooled to 50 °C, and then the solvent was removed to
dryness under reduced pressure. The residue was washed with hydrochloric acid and water.
The crude product was recrystallized from isopropanol. Studied compounds 1–8c are
presented in Table 1.
−
1
8
3
1
-Hydroxy-N-phenylquinoline-2-carboxamide (1). Yield 78%; mp 197 °C; IR (cm ):
336, 3046, 1679, 1600, 1575, 1527, 1505, 1461, 1444, 1429, 1389, 1366, 1310, 1287, 1235,
1
197, 1129, 1117, 1082, 931, 906, 879, 847, 814, 802, 761, 752, 722, 692; H-NMR
(
DMSO-d
6
), δ: 7.183 (tt, 1H, J=7.4, 1.1 Hz), 7.228 (dd, 1H, J=7.6, 1.2 Hz), 7.447 (m, 2H),
7
.524 (dd, 1H, J=8.1, 1.2 Hz), 7.608 (t, 1H, J=7.9 Hz), 7.901 (m, 2H), 8.272 (d, 1H,
1
J=8.5 Hz), 8.571 (d, 1H, J=8.5 Hz), 10.404 (s, 1H), 11.171 (s, 1H); C-NMR (DMSO-d
3
6
), δ:
1
1
2
12.03, 117.64, 118.95, 120.70 (2C), 124.13, 128.77 (2C), 129.69, 129.72, 136.36, 138.10,
-
38.25, 147.28, 153.70, 162.09; HR-MS: C16
63.0822 m/z.
11 2 2
H N O [M-H] calculated 263.0826 m/z, found
8
-Hydroxy-N-(2-methoxyphenyl)quinoline-2-carboxamide
(2a).
Yield
71%;
−1
mp 187–189 °C; IR (cm ): 3446, 3372, 1679, 1601, 1538, 1505, 1485, 1456, 1432, 1326,
1
289, 1236, 1198, 1135, 1105, 1083, 1046, 1030, 929, 888, 848, 813, 744, 722, 680; H-NMR
1
(
DMSO-d
6
), δ: 3.349 (s, 3H), 7.027 (td, 1H, J=7.7, 1.3 Hz), 7.151 (dd, 1H, J=8.3, 1.3 Hz),
7
.211 (dd, 1H, J=7.6, 1.2 Hz), 7.243(ddd, 1H, J=9.1, 7.5, 1.6 Hz), 7.510 (dd, 1H,
J=8.2, 1.2 Hz), 7.587 (t, 1H, J=7.9 Hz), 7.800 (dd, 1H, J=7.8, 1.6 Hz), 8.221 (d, 1H,
1
J=8.5 Hz), 8.544 (d, 1H, J=8.5 Hz), 10.374 (s, 1H), 10.885 (s, 1H); C-NMR (DMSO-d
3
6
), δ:
5
1
2
5.66, 111.50, 112.13, 117.65, 118.84, 120.26, 124.95, 126.14, 126.22, 129.56, 129.77,
-
36.64, 137.95, 147.44, 152.05, 153.78, 162.28; HR-MS: C17
93.0932 m/z, found 293.0928 m/z.
13 2 3
H N O [M-H] calculated
1
3

8
-Hydroxy-N-(3-methoxyphenyl)quinoline-2-carboxamide
(2b).
Yield
69%;
−1
mp 199–201 °C; IR (cm ): 3331, 3307, 1684, 1657, 1605, 1592, 1525, 1504, 1456, 1416,
1
7
391, 1358, 1328, 1286, 1265, 1247, 1224, 1201, 1159, 1081, 1053, 1033, 994, 874, 843, 822,
1
74, 765, 758, 736, 723, 702, 688; H-NMR (DMSO-d
6
), δ: 3.813(s, 3H), 6.768 (dd, 1H,
J=8.3, 2.5 Hz), 7.230 (dd, 1H, J=7.6, 1.3 Hz), 7.349 (t, 1H, J=8.2 Hz), 7.499 (dm, 1H,
J=8.1 Hz), 7.524 (d, 1H, J=8.2 Hz), 7.577 (t, 1H, J=2.2 Hz), 7.609 (t, 1H, J=7.9 Hz), 8.269
1
3
(
(
d, 1H, J=8.5 Hz), 8.572 (d, 1H, J=8.5 Hz), 10.413 (s, 1H), 11.133 (s, 1H); C-NMR
DMSO-d
6
), δ: 55.12, 106.38, 109.60, 112.08, 112.91, 117.66, 118.96, 129.59, 129.71,
-
1
29.76, 136.36, 138.13, 139.44, 147.24, 153.71, 159.58, 162.13; HR-MS: C17
13 2 3
H N O [M-H]
calculated 293.0932 m/z, found 293.0927 m/z.
8
-Hydroxy-N-(4-methoxyphenyl)quinoline-2-carboxamide (2c). Yield 66%, mp 209 °C;
−1
IR (cm ): 3334w, 3208w, 1675m, 1627m, 1593m, 1528s, 1510s, 1464s, 1410w, 1302w,
1
7
287w, 1230s, 1174m, 1163m, 1110m, 1086m, 1036s, 937w, 855m, 828s, 815s, 762s, 748s,
1
23s, 677w; H-NMR (DMSO-d
6
), δ: 3.779(s, 3H), 7.017 (m, 2H), 7.217 (dd, 1H,
J=7.6, 1.2 Hz), 7.511 (dd, 1H, J=8.2, 1.2 Hz), 7.594 (t, 1H, J=7.9 Hz), 7.795 (m, 2H), 8.253
1
3
(
(
d, 1H, J=8.5 Hz), 8.553 (d, 1H, J=8.5 Hz), 10.357 (s, 1H), 11.605 (s, 1H); C-NMR
DMSO-d
6
), δ: 55.22, 111.94, 113.93(2C), 117.63, 118.92, 122.26(2C), 129.61(2C), 131.30,
-
[M-H] calculated
1
2
36.36, 138.04, 147.46, 153.66, 155.89, 161.74; HR-MS: C17
93.0932 m/z; found 293.0925 m/z.
H
13
N
2
O
3
8
1
1
d
1
7
1
1
-Hydroxy-N-(2-methylphenyl)quinoline-2-carboxamide (3a). Yield 73%; mp 139–
−1
40 °C; IR (cm ): 3177, 3043, 2960, 1661, 1615, 1587, 1538, 1523, 1456, 1428, 1367, 1325,
1
247, 1208, 1144, 1106, 1083, 1067, 1050, 933, 882, 846, 814, 758, 710; H-NMR (DMSO-
6
), δ: 2.303 (s, 3H), 7.211 (dd, 1H, J=7.6, 1.0 Hz), 7.232 (td, 1H, J=7.3, 1.2 Hz), 7.291 (t,
H, J=7.5 Hz), 7.347(d, 1H, J=7.5 Hz), 7.451 (d, 1H, J=7.7 Hz), 7.525(d, 1H, J=8.3 Hz ),
.602 (t, 1H, J=7.9 Hz), 8.230 (d, 1H, J=8.5 Hz), 8.562 (d, 1H, J=8.5 Hz), 10.240 (s, 1H),
1
3
1.055 (s, 1H); C-NMR (DMSO-d ), δ: 17.96, 111.84, 117.61, 118.91, 126.16, 126.25,
6
26.49, 129.63, 129.65, 130.43, 133.72, 135.81, 136.44, 137.99, 147.28, 153.64, 162.27; HR-
-
[M-H] calculated 277.0983 m/z, found 277.0977 m/z.
MS: C17
H
13
N
2
O
2
8
-Hydroxy-N-(3-methylphenyl)quinoline-2-carboxamide (3b). Yield 75%; mp 203–204 °C;
−1
IR (cm ): 3342, 3312, 3049, 1686, 1656, 1612, 1538, 1505, 1489, 1462, 1392, 1358, 1327,
1
308, 1289, 1224, 1181, 1163, 1117, 1084, 1046, 940, 897, 881, 853, 779, 758, 741, 724, 692;
1
H-NMR (DMSO-d ), δ: 2.368 (s, 3H), 7.000 (dm, 1H, J=7.5 Hz), 7.226 (dd, 1H,
6
1
4

J=7.6, 1.2 Hz), 7.321 (t, 1H, J=7.7 Hz), 7.520 (dd, 1H, J=8.1, 1.2 Hz), 7.606 (t, 1H,
J=7.9 Hz), 7.720 (s, 1H), 7.729 (d, 1H, J=7.7 Hz), 8.265 (d, 1H, J=8.5 Hz), 8.568 (d, 1H,
1
J=8.5 Hz), 10.407 (s, 1H), 11.107 (s, 1H); C-NMR (DMSO-d
3
6
), δ: 21.17, 112.04, 117.64,
1
1
2
17.81, 118.94, 121.17, 124.81, 128.62, 129.68, 129.71, 136.37, 137.99, 138.10, 138.18,
-
47.33, 153.72, 162.02; HR-MS: C17
77.0980 m/z.
13 2 2
H N O [M-H] calculated 277.0983 m/z, found
8
-Hydroxy-N-(4-methylphenyl)quinoline-2-carboxamide (3c). Yield 68%; mp 208 °C; IR
−
1
(
cm ): 3457, 3204, 2917, 1683, 1630, 1590, 1527, 1501, 1464, 1404, 1319, 1297, 1260,
1
230, 1185, 1163, 1086, 1047, 1019, 980, 931, 889, 851, 809, 761, 749, 724; H-NMR
1
(
DMSO-d
6
), δ: 2.321(s, 3H), 7.221 (dd, 1H, J=7.6, 1.3 Hz), 7.248 (m, 2H), 7.521 (dd, 1H,
J=8.2, 1.2 Hz), 7.604 (t, 1H, J=7.9 Hz), 7.779 (m, 2H), 8.259 (d, 1H, J=8.5 Hz), 8.556 (d, 1H,
1
J=8.5 Hz), 10.382 (s, 1H), 11.110 (s, 1H); C-NMR (DMSO-d
3
6
), δ: 20.530, 112.00, 117.64,
1
1
18.94, 120.67(2C), 129.18(2C), 129.66, 129.67, 133.16, 135.73, 136.36, 138.08, 147.38,
-
13 2 2
53.69, 162.92; HR-MS: C17H N O [M-H] calculated 277.0983m/z; found 277.0988 m/z.
N-(2-fluorophenyl)-8-hydroxyquinoline-2-carboxamide (4a). Yield 68%; mp 145 °C; IR
−
1
(
cm ): 3489, 3376, 1689, 1618, 1594, 1537, 1505, 1455, 1359, 1327, 1290, 1252, 1232,
1
194, 1169, 1121, 1083, 1031, 887, 848, 757, 722; H-NMR (DMSO-d
1
1
6
), δ: 11.168 (s, 1H),
0.352 (s, 1H), 8.576 (d, 1H, J=8.5 Hz), 8.230 (d, 1H, J=8.5 Hz), 7.745 (td, 1H,
J=7.8, 1.8 Hz), 7.608 (dd, 1H, J=7.7, 8.1 Hz), 7.523 (dd, 1H, J=8.2, 1.2 Hz), 7.40-7.28 (m,
1
H), 7.219 (dd, 1H, J=7.7, 1.2 Hz); C-NMR (DMSO-d
3
3
1
6
), δ: 162.54, 155.81 (J=247.4 Hz),
53.74, 146.76, 138.06, 136.46, 129.82, 129.73, 127.22 (J=9.2 Hz), 127.20, 125.05
(
J=12.2 Hz), 124.45 (J=3.6 Hz), 118.92, 115.95(J=19.8 Hz), 117.60, 111.93; HR-MS:
-
[M-H] calculated 281.0732 m/z, found 281.0737 m/z.
C
16
H10FN
2
O
2
N-(3-fluorophenyl)-8-hydroxyquinoline-2-carboxamide (4b). Yield 72%; mp 197 °C; IR
−
1
(
cm ): 3333, 1686, 1612, 1599, 1528, 1504, 1463, 1443, 1391, 1365, 1325, 1295, 1276,
1
237, 1200, 1174, 1145, 1111, 1083, 965, 872, 846, 775, 760, 748, 722, 684; H-NMR
1
(
6
DMSO-d ), δ: 11.265 (s, 1H), 10.376 (s, 1H), 8.572 (d, 1H, J=8.5 Hz), 8.263 (d, 1H,
J=8.5 Hz), 7.866 (dt, 1H, J=11.6, 2.2 Hz), 7.698 (ddd, 1H, J=8.1, 1.9, 0.9 Hz), 7.611 (t, 1H,
J=7.9 Hz), 7.522 (dd, 1H, J=8.2, 1.2 Hz), 7.484 (dt, 1H, J=6.8, 8.2 Hz), 7.233 (dd, 1H,
1
J=7.6, 1.2 Hz), 7.012 (td, 1H, J=8.4, 2.5 Hz); C-NMR (DMSO-d
3
6
), δ: 162.35, 162.33
(
J=241.6 Hz), 153.67, 146.87, 140.01(J=11.0 Hz), 138.20, 136.32, 130.43 (J=9.5 Hz), 129.87,
1
5

1
29.77, 118.95, 117.68, 116.33 (J=2.6 Hz), 112.10, 110.61 (J=20.9 Hz), 107.30 (J=26.1 Hz);
-
[M-H] calculated 281.0732 m/z, found 281.0736 m/z.
HR-MS: C16
H10FN
2
O
2
N-(4-fluorophenyl)-8-hydroxyquinoline-2-carboxamide (4c). Yield 71%; mp 200 °C; IR
−
1
(
cm ): 3318, 1677, 1655, 1609, 1528, 1528, 1504, 1464, 1405, 1359, 1309, 1286, 1225,
1
189, 1156, 1133, 1089, 1083, 932, 886, 854, 827, 760, 723, 678; H-NMR (DMSO-d
1
1
2
1
1
1
6
), δ:
1.206 (s, 1H), 10.349 (s, 1H), 8.565 (d, 1H, J=8.5 Hz), 8.260 (d, 1H, J=8.5 Hz), 7.909 (m,
H), 7.604 (dd, 1H, J=8.1, 7.7 Hz), 7.519 (dd, 1H, J=8.2, 1.2 Hz), 7.290 (m, 2H), 7.226 (dd,
1
3
H, J=7.6, 1.2 Hz); C-NMR (DMSO-d ), δ: 162.05, 158.57 (J=240.9 Hz), 153.64, 147.12,
6
38.12, 136.33, 134.60 (J=2.7 Hz), 129.74, 129.68, 122.56 (2C, J=8.0 Hz), 118.93, 117.65,
-
[M-H] calculated 281.0732 m/z,
15.41(2C, J=22.4 Hz), 111.99; HR-MS: C16
H
10FN
2
O
2
found 281.0728 m/z.
N-(2-chlorophenyl)-8-hydroxyquinoline-2-carboxamide (5a). Yield 71%; mp 151–152 °C;
−1
IR (cm ): 3355, 1695, 1595, 1581, 1535, 1505, 1461, 1433, 1362, 1328, 1310, 1290, 1237,
1
198, 1136, 1115, 1088, 1052, 1032, 885, 848, 743, 724, 668; H-NMR (DMSO-d
1
1
1
6
), δ:
1.224 (s, 1H), 10.322 (s, 1H), 8.571 (d, 1H, J=8.5 Hz), 8.229 (d, 1H, J=8.5 Hz), 7.746 (dd,
H, J=7.9, 1.6 Hz), 7.630 (dd, 1H, J=8.0, 1.4 Hz), 7.610 (t, 1H, J=7.9 Hz), 7.528 (dd, 1H,
J=8.2, 1.1 Hz), 7.458 (td, 1H, J=7.6, 1.5 Hz), 7.352 (td, 1H, J=7.8, 1.6 Hz), 7.233 (dd, 1H,
1
J=7.6, 1.2 Hz); C-NMR (DMSO-d
3
6
), δ: 162.57, 153.72, 146.77, 138.07, 136.50, 134.58,
1
29.80, 129.79, 129.67, 129.14, 128.09, 127.63, 127.59, 118.86, 117.62, 111.97; HR-MS:
-
C
16 2 2
H10ClN O [M-H] calculated 297.0436 m/z, found 297.0437 m/z.
N-(3-chlorophenyl)-8-hydroxyquinoline-2-carboxamide (5b). Yield 75%; mp 227 °C; IR
−
1
(
cm ): 3342, 1689, 1589, 1524, 1502, 1481, 1464, 1405, 1324, 1296, 1222, 1191, 1169,
1
118, 1097, 1078, 977, 881, 855, 817, 786, 765, 722, 685; H-NMR (DMSO-d
1
1
6
), δ: 11.243 (s,
H), 10.354 (s, 1H), 8.573 (d, 1H, J=8.5 Hz), 8.261 (d, 1H, J=8.5 Hz), 8.060 (t, 1H,
J=2.1 Hz), 7.874 (ddd, 1H, J=8.2, 2.0, 0.9 Hz), 7.614 (dd, 1H, J=7.7, 8.1 Hz), 7.524 (dd, 1H,
J=8.2, 1.1 Hz), 7.478 (t, 1H, J=8.1 Hz), 7.240 (ddd, 1H, J=8.0, 2.1, 0.9 Hz), 7.236 (dd, 1H,
1
J=7.6, 1.2 Hz); C-NMR (DMSO-d
3
6
), δ: 162.33, 153.65, 146.82, 139.74, 138.20, 136.30,
1
33.11, 130.50, 129.88, 129.76, 123.80, 119.98, 118.93, 118.90, 117.67, 112.07; HR-MS:
-
C
16 2 2
H10ClN O [M-H] calculated 297.0436 m/z, found 297.0432 m/z.
1
6

N-(4-chlorophenyl)-8-hydroxyquinoline-2-carboxamide (5c). Yield 75%; mp 272–273 °C;
−1
IR (cm ): 3390, 3316, 1686, 1651, 1589, 1528, 1502, 1466, 1399, 1362, 1307, 1283, 1226,
1
187, 1090, 1008, 889, 839, 804, 748, 722, 671; H-NMR (DMSO-d
1
1
1
6
), δ: 11.229 (s, 1H),
0.356 (s, 1H), 8.563 (d, 1H, J=8.5 Hz), 8.255 (d, 1H, J=8.5 Hz), 7.938 (m, 2H), 7.605 (dd,
H, J=8.1, 7.7 Hz), 7.515 (dd, 1H, J=8.1, 1.2 Hz), 7.502 (m, 2H, J=7.9 Hz), 7.226 (dd, 1H,
1
J=7.6, 1.2 Hz); C-NMR (DMSO-d
3
6
), δ: 162.19, 153.66, 146.98, 138.16, 137.22, 136.32,
1
29.81, 129.72, 128.71 (2C), 127.79, 122.17 (2C), 118.93, 117.66, 112.04; HR-MS:
-
C
16 2 2
H10ClN O [M-H] calculated 297.0436 m/z, found 297.0433 m/z.
N-(2-bromophenyl)-8-hydroxyquinoline-2-carboxamide (6a). Yield 76%; mp 176–177 °C;
−1
IR (cm ): 3452, 3336, 1696, 1592, 1575, 1532, 1504, 1460, 1431, 1362, 1328, 1305, 1235,
1
196, 1134, 1109, 1087, 1045, 1021, 848, 814, 758, 723, 668; H-NMR (DMSO-d
1
1
1
6
), δ:
1.208 (s, 1H), 10.306 (s, 1H), 8.571 (d, 1H, J=8.5 Hz), 8.223 (d, 1H, J=8.5 Hz), 7.786 (dd,
H, J=8.0, 1.4 Hz), 7.700 (dd, 1H, J=7.9, 1.6 Hz), 7.610 (t, 1H, J=7.9 Hz), 7.528 (dd, 1H,
J=8.2, 1.1 Hz), 7.499 (td, 1H, J=7.7, 1.4 Hz), 7.283 (td, 1H, J=7.7, 1.7 Hz), 7.219 (dd, 1H,
1
J=7.7, 1.2 Hz); C-NMR (DMSO-d
3
6
), δ: 162.56, 153.72, 146.80, 138.07, 136.50, 136.10,
1
32.80, 129.80, 129.78, 128.50, 128.24, 128.05, 120.10, 118.86, 117.62, 111.95; HR-MS:
-
C
16 2 2
H10BrN O [M-H] calculated 340.9931 m/z, found 340.9935 m/z.
N-(3-bromophenyl)-8-hydroxyquinoline-2-carboxamide (6b). Yield 75%; mp 235 °C; IR
−
1
(
cm ): 3445, 3279, 3057, 1626, 1584, 1536, 1504, 1504, 1478, 1409, 1329, 1306, 1230,
1
190, 1163, 1123, 1072, 994, 881, 850, 722, 747, 725, 677; H-NMR (DMSO-d
1
6
), δ: 11.228
(
s, 1H), 10.347 (s, 1H), 8.572 (d, 1H, J=8.5 Hz), 8.257(d, 1H, J=8.5 Hz), 8.183 (t, 1H,
J=2.0 Hz), 7.924 (ddd, 1H, J=8.1, 1.9, 1.0 Hz), 7.612 (dd, 1H, J=7.8, 8.1 Hz), 7.522 (dd, 1H,
J=8.3, 1.2 Hz), 7.415 (t, 1H, J=8.0 Hz), 7.368 (ddd, 1H, J=7.9, 1.9, 1.1 Hz), 7.233 (dd, 1H,
1
J=7.7, 1.2 Hz); C-NMR (DMSO-d
3
6
), δ: 162.31, 153.65, 146.82, 139.88, 138.21, 136.30,
1
30.81, 129.88, 129.76, 126.70, 122.81, 121.54, 119.28, 118.94, 117.68, 112.08; HR-MS: for
-
C
16 2 2
H10BrN O [M-H] calculated 340.9931 m/z, found 340.9937 m/z.
N-(4-bromophenyl)-8-hydroxyquinoline-2-carboxamide (6c). Yield 75%; mp 283–284 °C;
−1
IR (cm ): 3386, 3316, 1686, 1650, 1585, 1524, 1500, 1393, 1305, 1280, 1224, 1179, 1161,
1
111, 1072, 1005, 933, 887, 837, 822, 811, 801, 720; H-NMR (DMSO-d
1
1
2
6
), δ: 11.221 (s, 1H),
0.356 (s, 1H), 8.565 (d, 1H, J=8.5 Hz), 8.255 (d, 1H, J=8.5 Hz), 7.888 (m, 2H), 7.631 (m,
H), 7.607 (t, 1H, J=7.9 Hz), 7.519 (dd, 1H, J=8.2, 1.2 Hz), 7.227 (dd, 1H, J=7.6, 1.2 Hz);
1
7

1
3
C-NMR (DMSO-d
6
), δ: 162.19, 153.65, 146.96, 138.16, 137.63, 136.32, 131.62 (2C),
-
1
29.82, 129.72, 122.51 (2C), 118.93, 117.66, 115.87, 112.04; HR-MS: C16
2 2
H10BrN O [M-H]
calculated 340.9931 m/z, found 343.9932 m/z.
8
-Hydroxy-N-(2-trifluoromethylphenyl)quinoline-2-carboxamide (7a). Yield 71%;
−1
mp 142–143 °C; IR (cm ): 3312, 1666, 1590, 1524, 1505, 1483, 1459, 1365, 1315, 1280,
1
240, 1202, 1170, 1117, 1056, 1032, 935, 894, 857, 817, 754, 733, 668; H-NMR
1
(
DMSO-d
J=8.5 Hz), 7.867 (dm, 1H, J=7.9 Hz), 7.807 (t, 1H, J=7.8 Hz), 7.706 (dm, 1H, J=7.8 Hz),
.612 (dd, 1H, J=7.7, 8.2 Hz), 7.592 (tm, 1H, J=7.7 Hz), 7.529 (dd, 1H, J=8.2, 1.2 Hz), 7.218
6
), δ: 11.221 (s, 1H), 10.269 (s, 1H), 8.569 (d, 1H, J=8.5 Hz), 8.208 (d, 1H,
7
1
dd, 1H, J=7.6, 1.2 Hz); C-NMR (DMSO-d
3
(
6
), δ: 163.49, 153.69, 146.62, 138.07, 136.49,
1
35.36 (q, J=2.9 Hz), 133.32, 130.53, 129.83, 129.78, 127.49, 126.61 (q, J=4.9 Hz), 126.03
(
[
10 3 2 2
q, J=29.4 Hz), 123.60 (q, J=273.5 Hz), 118.80, 117.60, 111.91; HR-MS: C17H F N O
-
M-H] calculated 331.0699 m/z, found 331.0703 m/z.
8
-Hydroxy-N-(3-trifluoromethylphenyl)quinoline-2-carboxamide (7b). Yield 68%;
−1
mp 213–214 °C; IR (cm ): 3293, 1691, 1657, 1614, 1541, 1493, 1393, 1329, 1224, 1161,
1
111, 1096, 880, 854, 794, 754, 724, 694; H-NMR (DMSO-d
1
1
1
7
1
6
), δ: 11.373 (s, 1H), 10.343 (s,
H), 8.575 (d, 1H, J=8.5 Hz), 8.267 (d, 1H, J=8.5 Hz) 8.321 (t, 1H, J=2.2 Hz), 8.199 (dm,
H, J=8.1 Hz), 7.689 (t, 1H, J=8.0 Hz), 7.613 (t, 1H, J=7.9 Hz), 7.527 (dm, 1H, J=7.6 Hz),
1
.522 (dd, 1H, J=8.3, 1.2 Hz), 7.237 (dd, 1H, J=7.6, 1.1 Hz); C-NMR (DMSO-d
3
6
), δ:
62.54, 153.66, 146.75, 139.08, 138.22, 136.31, 130.05, 129.91, 129.79, 129.54 (q,
J=31.5 Hz), 124.11 (q, J=272.2 Hz), 124.08, 120.40 (q, J=3.9 Hz), 118.94, 117.69, 116.57 (q,
-
[M-H] calculated 331.0699 m/z, found
J=4.0 Hz), 112.08; HR-MS: C17
H
10
F
3
N
2
O
2
3
31.0698 m/z.
8
-Hydroxy-N-(4-trifluoromethylphenyl)quinoline-2-carboxamide (7c). Yield 72%;
−1
mp 253 °C; IR (cm ): 3315, 1692, 1662, 1614, 1595, 1530, 1501, 1464, 1409, 1360, 1318,
1
228, 1183, 1108, 1063, 1016, 933, 852, 837, 755, 722; H-NMR (DMSO-d
1
1
6
), δ: 11.378 (s,
H), 10.398 (s, 1H), 8.577 (d, 1H, J=8.5 Hz), 8.273 (d, 1H, J=8.5 Hz), 8.146 (m, 2H), 7.818
(
m, 2H), 7.617 (t, 1H, J=7.9 Hz), 7.526 (dd, 1H, J=8.2, 1.3 Hz), 7.238 (dd, 1H,
1
J=7.6, 1.3 Hz); C-NMR (DMSO-d
3
6
), δ: 162.57, 153.69, 146.75, 141.89, 138.23, 136.32,
1
29.95, 129.81, 126.07 (q, 2C, J=3.8 Hz), 124.34 (q, J=271.3 Hz), 124.05 (q, J=32.2 Hz),
1
8

-
1
20.45 (2C), 118.98, 117.69, 112.13; HR-MS: C17
10 3 2 2
H F N O [M-H] calculated 331.0699 m/z,
found 331.0704 m/z.
8
-Hydroxy-N-(2-nitrophenyl)quinoline-2-carboxamide (8a). Yield 79%; mp 239–241 °C;
−1
IR (cm ): 3462, 3287, 1697, 1606, 1581, 1548, 1499, 1464, 1447, 1427, 1363, 1338, 1292,
1
271, 1234, 1198, 1143, 1132, 1115, 1088, 935, 862, 847, 809, 783, 753, 736, 724, 668;
1
H-NMR (DMSO-d ), δ: 7.247 (d, 1H, J=7.7 Hz), 7.502 (t, 1H, J=7.7 Hz), 7.545 (d, 1H,
6
J=8.2 Hz), 7.632(t, 1H, J=7.8 Hz), 7.845 (t, 1H, J=7.7 Hz), 7.934 (d, 1H, J=8.1 Hz), 8.101 (d,
1
1
1
H, J=8.1 Hz), 8.206 (d, 1H, J=8.5 Hz), 8.590 (d, 1H, J=8.5 Hz), 10.219 (s, 1H), 11.826 (s,
1
3
H); C-NMR (DMSO-d ), δ: 112.08, 117.73, 118.92, 125.23, 126.01, 126.21, 129.92,
6
30.07, 130.84, 134.21, 136.45, 138.30, 142.99, 146.20, 153.69, 162.46; HR-MS:
-
[M-H] calculated 308.0677 m/z, found 308.0673 m/z.
C
16
H
10
N
3
O
4
8
-Hydroxy-N-(3-nitrophenyl)quinoline-2-carboxamide (8b). Yield 73%; mp 273–276 °C;
−1
IR (cm ): 3463, 3276, 1680, 1594, 1525, 1502, 1456, 1429, 1347, 1295, 1275, 1230, 1186,
1
164, 1124, 1076, 946, 894, 882, 833, 803, 755, 735, 724, 668; H-NMR (DMSO-d
1
7
6
), δ:
.244 (dd, 1H, J=7.6, 1.2 Hz), 7.538 (dd, 1H, J=8.1, 1.0 Hz), 7.627 (t, 1H, J=8.0 Hz), 7.746
(
t, 1H, J=8.1 Hz), 8.030 (dd, 1H, J=8.2, 2.1 Hz), 8.278 (d, 1H, J=8.5 Hz), 8.350 (dd, 1H,
J=8.2, 1.8 Hz), 8.594 (d, 1H, J=8.5 Hz), 8.854 (t, 1H, J=2.1 Hz), 10.360 (s, 1H), 11.469 (s,
1
3
1
1
6
H); C-NMR (DMSO-d ), δ: 112.10, 114.55, 117.70, 118.53, 118.95, 126.37, 129.82,
30.00, 130.25, 136.29, 138.26, 139.47, 146.53, 147.99, 153.66, 162.66; HR-MS:
-
[M-H] calculated 308.0677 m/z, found 308.0672 m/z.
C
16
H
10
N
3
O
4
8
-Hydroxy-N-(4-nitrophenyl)quinoline-2-carboxamide (8c). Yield 75%; mp 297–300 °C;
−1
IR (cm ): 3253, 3116, 1690, 1595, 1567, 1535, 1501, 1464, 1409, 1328, 1303, 1244, 1177,
1
113, 1083, 977, 942, 914, 847, 802, 748, 690; H-NMR (DMSO-d
1
6
), δ: 7.242 (dd, 1H,
J=7.7, 1.2 Hz), 7.537 (dd, 1H, J=8.2, 1.2 Hz), 7.631 (t, 1H, J=7.9 Hz), 8.189 (m, 2H), 8.273
(
d, 1H, J=8.5 Hz), 8.347 (m, 2H), 8.593 (d, 1H, J=8.5 Hz), 10.442 (s, 1H), 11.531 (s, 1H);
1
3
C-NMR (DMSO-d
6
), δ: 112.51, 117.99, 119.23, 120.48(2C), 125.08(2C), 130.11, 130.37,
-
1
10 3 4
36.55, 138.55, 143.03, 144.62, 146.65, 153.85, 163.05; HR-MS: C16H N O [M-H]
calculated 308.0677 m/z, found 308.0675 m/z.
4
.2. Lipophilicity determination by HPLC (Capacity factor k/calculated log k)
1
9

A HPLC system Agilent 1200 equipped with DAD detector (Agilent, Santa Clara, CA,
®
USA) was used. A chromatographic column Symmetry C18 5 µm, 4.6×250 mm, Part No.
WAT054275, (Waters Corp., Milford, MA, USA) was used. The HPLC separation process
was monitored and evaluated by EZChrom Elite software ver. 3.3.2 (Agilent). Isocratic
2
elution by a mixture of MeOH p.a. (60%) and H O-HPLC Mili-Q grade (40%) as a mobile
phase was used. The total flow of the column was 1.0 mL/min, injection 20 µL, column
temperature 40 °C and sample temperature 10 °C. The detection wavelength 210 nm was
chosen. The KI methanolic solution was used for the dead time (t
times (t ) were measured in minutes. The capacity factors k were calculated according to
formula k = (t − t )/t , where t is the retention time of the solute, whereas t denotes the
D
) determination. Retention
R
R
D
D
R
D
dead time obtained using an unretained analyte. Log k, calculated from the capacity factor k,
is used as the lipophilicity index converted to log P scale. The log k values of the individual
compounds are shown in Table 1.
4
.3. In vitro antimycobacterial evaluation
Mycobacterium tuberculosis H37Ra ATCC 25177 and well characterised clinical
isolates of M. avium complex CIT19/06, and M. avium subsp. paratuberculosis CIT03 were
grown in Middlebrook broth (MB), supplemented with Oleic-Albumin-Dextrose-Catalase
supplement (OADC, Becton Dickinson, UK) and mycobactin J (2 µg/mL). Identification of
these isolates was performed using biochemical and molecular protocols. At log phase
growth, a culture sample (10 mL) was centrifuged at 15,000 rpm/20 min using a bench top
centrifuge (Model CR 4-12, Jouan Inc., UK). Following removal of the supernatant, the pellet
was washed in fresh Middlebrook 7H9GC broth and re-suspended in fresh supplemented MB
8
10 mL). The turbidity was adjusted to match McFarland standard No. 1 (3×10 cfu) with MB
(
broth. A further 1:20 dilution of the culture was then performed in MB broth. The
antimicrobial susceptibility of all three mycobacterial species was investigated in a 96-well
plate format. In these experiments, sterile deionised water (300 µL) was added to all outer-
perimeter wells of the plates to minimize evaporation of the medium in the test wells during
incubation. Each evaluated compound (100 µL) was incubated with each of the mycobacterial
species (100 µL). Dilutions of each compound were prepared in duplicate. For all synthesized
compounds, final concentrations ranged from 1,000 µg/mL to 8 µg/mL. All compounds were
prepared in DMSO and subsequent dilutions were made in supplemented MB. The plates
were sealed with parafilm and incubated at 37 °C, for 5 days in the case of M. avium complex,
2
0

7
days in the case of M. tuberculosis and 11 days in the case of M. avium paratuberculosis.
Following incubation, a 10% addition of alamarBlue (AbD Serotec, Kidlington, UK) was
mixed into each well and readings at 570 nm and 600 nm were taken, initially for background
subtraction and subsequently after 24 h re-incubation. The background subtraction is
necessary for strongly coloured compounds, where the colour may interfere with the
interpretation of any colour change. For non-interfering compounds, a blue colour in the well
was interpreted as an absence of growth and a pink colour was scored as growth. The
minimum inhibitory concentration (MIC) was defined as the lowest concentration of the
compound at which no visible bacterial growth was observed, i.e. the MIC is the lowest
concentration that prevented a visual colour change from blue to pink. The MIC value is
routinely and widely used in bacterial assays and is a standard detection limit according to the
Clinical and Laboratory Standards Institute (CLSI, www.clsi.org). Rifampicin (Sigma-
Aldrich) was used as the standard, as it is a clinically used antimycobacterial drug. The results
are summarized in Table 1.
4
.4. MTT assay
For the MTT assay, the outer wells of a 96-well plate were filled with 200 µL of sterile
water, and the inner wells were filled with 100 µL of the tested compound at the
concentration to be examined. Compounds were prepared as previously stated and diluted in
Middlebrook media to achieve the desired concentration. Mycobacterium tuberculosis H37Ra
ATCC 25177 was suspended in ODAC supplemented Middlebrook broth at a MacFarland
standard of 1.0 and then diluted through a 1:20 dilution using Middlebrook broth as a diluent.
The diluted mycobacteria (100 µL) were added to each well containing the compound to be
tested. A negative growth control was composed of 100 µL of DMSO and 100 µL of media,
and the diluted mycobacteria in broth absent of inhibiting compounds were used as a positive
growth control. All compounds and controls were prepared in triplicate. Plates were incubated
at 37°C for 7 days. After the incubation period, 10% well volume of MTT reagent was mixed
into each well and incubated at 37 °C for 24 hours. The reagent and media were then aspirated
from the wells, to which 50 µL 99% propan-2-ol was then added, and plates were read at
5
70 nm. The absorbance readings from the cells, grown in the presence of the tested
compounds, were compared with uninhibited cell growth (using DMSO as the blank) to
determine the relative percent viability. The percent viability was determined through the
MTT assay. The percent viability is calculated through comparison of a measured value
2
1

against that of the uninhibited control: %viability = OD570E/OD570P×100, where OD570E is
the reading from the compound-exposed cells, while OD570P is the reading from the
uninhibited cells (positive control). Cytotoxic potential is determined by a percent viability of
<
70%.
4
.5. In vitro cytotoxicity assay
Human monocytic leukemia THP-1 cells were used for in vitro toxicity assay. Cells
were obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK) and
routinely cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2%
2
L-glutamine, 1% penicillin and streptomycin at 37 °C with 5% CO . Cells were passaged at
approximately 1 week intervals. Cytotoxicity of the compounds was determined using a
WST-1 assay kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s
instructions. The tested compounds were dissolved in DMSO and added in five increasing
concentrations (0.37, 1.1, 3.3, 10, and 30 µM) to the cell suspension in the culture RPMI 1640
medium. The maximum concentration of DMSO in the assays never exceeded 0.1%.
2
Subsequently, the cells were incubated for 24 h at 37 °C with 5% CO . For WST-1 assays,
4
cells were seeded into 96-well plates (5×10 cells/well in 100 µL culture medium) in triplicate
in serum-free RPMI 1640 medium and measurements were taken 24 h after the treatment with
the compounds. The median inhibition concentration values, IC50, were deduced through the
production of a dose-response curve. All data were evaluated using GraphPad Prism 5.00
software (GraphPad Software, San Diego, CA, USA). The results are summarized in Table 1.
Acknowledgments
This study was supported by IGA VFU Brno 37/2014/FaF, 304/2015/FaF, 320/2015/FaF,
3
22/2015/FaF, Slovak Grant Agency VEGA 1/0770/15, by Project APVV-0061-11 and by the
Irish Department of Agriculture Fisheries and Food (FIRM): Refs. 08RDCIT601 &
8RDCIT617. The system of high-performance liquid chromatography coupled to an LTQ
0
Orbitrap XL high-resolution mass spectrometer forms a part of the National Infrastructure
CzeCos/ICOS (LM2010007).
References
2
2

1. World Health Organization. Global tuberculosis report 2014. WHO Press, Geneva,
Switzerland, 2014.
2
3
4
. Wagner, D.; Young, L. S. Infection 2004, 32, 257.
. Koul, A.; Arnoult, E.; Lounis, N.; Guillemont, J.; Andries, K. Nature 2011, 469, 483.
. Baquero, F.; Coque, T.M.; de la Cruz, F. Antimicrob. Agents Chemother. 2011, 55,
3
649.
5
6
7
8
. Polanski, J.; Kurczyk, A.; Bak, A.; Musiol, R. Curr. Med. Chem. 2012, 19, 1921.
. Kumar, S.; Bawa, S.; Gupta, H. Mini-Rev. Med. Chem. 2009, 9, 1648.
. Pati, B.; Banerjee, S. J. PharmaSciTech 2014, 3, 59.
. Marella, A.; Tanwar, O.P.; Saha, R.; Ali, M.R.; Srivastava, S.; Akhter, M.;
Shaquiquzzaman, M.; Alam, M.M. Saudi. Pharm. J. 2013, 21, 1.
9
. Musiol, R.; Jampilek, J.; Buchta, V.; Niedbala, H.; Podeszwa, B.; Palka, A.; Majerz-
Maniecka, K.; Oleksyn, B.; Polanski, J. Bioorg. Med. Chem. 2006, 14, 3592.
0. Musiol, R.; Serda, M.; Hensel-Bielowka, S.; Polanski, J. Curr. Med. Chem. 2010, 17,
1
1
960.
1
1
1. Cieslik, W.; Musiol, R.; Nycz, J.; Jampilek, J.; Vejsova, M.; Wolff, M.; Machura, B.;
Polanski, J. Bioorg. Med. Chem. 2012, 20, 6960.
2. Sahu, K.B.; Ghosh, S.; Banerjee, M.; Maity, A.; Mondal, S.; Paira, R.; Saha, P.; Naskar,
S.; Hazra, A.; Banerjee, S.; Samanta, A.; Mondal N.B. Med. Chem. Res. 2013, 22, 94.
3. Beena; Rawat, D.S. Med. Res. Rev. 2013, 33, 693.
1
1
1
1
4. Keri, R.S.; Patil, S.A. Biomed. Pharmacother. 2014, 68, 1161.
5. Fraser, S.S.R.; Creanor, J. Biochem. J. 1975, 147, 401.
6. Hongmanee, P.; Rukseree, K.; Buabut, B.; Somsri, B.; Palittapongarnpim, P.
Antimicrob. Agents Chemother. 2007, 51, 1105.
1
1
7. Darby, C.M.; Nathan, C.F. J. Antimicrob. Chemother. 2010, 65, 1424.
8. Prachayasittikul, V.; Prachayasittikul, S.; Ruchirawat, S.; Prachayasittikul, V. Drug
Des. Devel. Ther. 2013, 7, 1157.
1
2
9. Sirturo. http://www.sirturo.com/. (April 9, 2015)
0. Gratraud, P.; Surolia, N.; Besra, G.S.; Surolia, A.; Kremer, L. Antimicrob. Agents
Chemother. 2008, 52, 1162.
2
2
1. Thomas, K.D.; Adhikari, A.V.; Chowdhury, I.H.; Sumesh, E.; Pal, N.K. Eur. J. Med.
Chem. 2011, 46, 2503.
2. Thomas, K.D.; Adhikari, A.V.; Telkar, S.; Chowdhury, I.H.; Mahmood, R.; Pal, N.K.;
Row, G.; Sumesh E. Eur. J. Med. Chem. 2011, 46, 5283.
2
3

2
2
3. Khan, S.R.; Singh, S.; Roy, K.K.; Akhtar, M.S.; Saxena, A.K.; Krishnan, M.Y. Int. J.
Antimicrob. Agents 2013, 41, 41.
4. Naik, M.; Humnabadkar, V.; Tantry, S.J.; Panda, M.; Narayan, A.; Guptha, S.; Panduga,
V.; Manjrekar, P.; Jena, L.K.; Koushik, K.; Shanbhag, G.; Jatheendranath, S.;
Manjunatha, M.R.; Gorai, G.; Bathula, C.; Rudrapatna, S.; Achar, V.; Sharma, S.;
Ambady, A.; Hegde, N.; Mahadevaswamy, J.; Kaur, P.; Sambandamurthy, V.K.;
Awasthy, D.; Narayan, C.; Ravishankar, S.; Madhavapeddi, P.; Reddy, J.; Prabhakar,
K.; Saralaya, R.; Chatterji, M.; Whiteaker, J.; McLaughlin, B.; Chiarelli, L.R.; Riccardi,
G.; Pasca, M.R.; Binda, C.; Neres, J.; Dhar, N.; Signorino-Gelo, F.; McKinney, J.D.;
Ramachandran, V.; Shandil, R.; Tommasi, R.; Iyer, P.S.; Narayanan, S.; Hosagrahara,
V.; Kavanagh, S.; Dinesh, N.; Ghorpade, S.R. J. Med. Chem. 2014, 57, 5419.
5. Marriner, G.A.; Nayyar, A.; Uh, E.; Wong, S.Y.; Mukherjee, T.; Via, L.E.; Carroll, M.;
Edwards, R.L.; Grubber, T.D.; Choi, I.; Lee, J.; Arora, K.; England, K.D.; Boshoff,
H.I.M.; Barry, C.E. Top. Med. Chem. 2011, 7, 47.
2
2
6. Ballell, L.; Bates, R.H.; Young, R.J.; Alvarez-Gomez, D.; Alvarez-Ruiz, E.; Barroso,
V.; Blanco, D.; Crespo, B.; Escribano, J.; Gonzalez, R.; Lozano, S.; Huss, S.; Santos-
Villarejo, A.; Martin-Plaza, J.J.; Mendoza, A.; Rebollo-Lopez, M.J.; Remuinan-Blanco,
M.; Lavandera, J.L.; Perez-Herran, E.; Gamo-Benito, F.J.; Garcia-Bustos, J.F.; Barros,
D.; Castro, J.P.; Cammack, N. ChemMedChem 2013, 8, 313.
2
2
7. Upadhayaya, R.S.; Vandavasi, J.K.; Kardile, R.A.; Lahore, S.V.; Dixit, S.S.; Deokar,
H.S.; Shinde, P.D.; Sarmah, M.P.; Chattopadhyaya, J. Eur. J. Med. Chem. 2010, 45,
1
854.
8. Nayyar, A.; Monga, V.; Malde, A.; Coutinho, E.; Jain, R. Bioorg. Med. Chem. 2007, 15,
26.
6
2
3
9. Nayyar, A.; Jain R. Ind. J. Chem. 2008, 47B, 117.
0. Bueno, R. V.; Braga, R. C.; Segretti, N. D.; Ferreir, E. I.; Trossini, G. H.; Andrade, C.
H. Curr. Pharm. Des. 2014, 20, 4474.
3
3
3
1. Mathew, B.; Ross, L.; Reynolds, R.C. Tuberculosis 2013, 93, 398.
2. Paritala, H.; Carroll, K.S. Infect. Disord. Drug Targets 2013, 13, 85.
3. Joshi, S.D.; More, U.A.; Dixit, S.R.; Dubey, D.; Tripathi, A.; Kulkarni, V.H. Ind. Am. J.
Pharm. Res. 2014, 4, 864–877.
3
4. Jampilek, J.; Dolezal, M.; Kunes, J.; Buchta, V.; Silva, L.; Kralova, K. Med. Chem.
2
005, 1, 591.
2
4

3
3
3
5. Musiol, R.; Jampilek, J.; Kralova, K.; Richardson, D.R.; Kalinowski, D.; Podeszwa, B.;
Finster, J.; Niedbala, H.; Palka, A.; Polanski, J. Bioorg. Med. Chem. 2007, 15, 1280.
6. Musiol, R.; Tabak, D.; Niedbala, H.; Podeszwa, B.; Jampilek, J.; Kralova, K.; Dohnal, J.;
Finster, J.; Mencel, A.; Polanski, J. Bioorg. Med. Chem. 2008, 16, 4490.
7. Gonec, T.; Bobal, P.; Pesko, M.; Guo, J.H.; Kralova, K.; Kos, J.; Coffey, A.; Kollar, P.;
Imramovsky, A.; Jampilek, J. Molecules 2012, 17, 613.
3
3
4
4
8. Zumla, A.; Nahid, P.; Cole, S. T. Nat. Rev. Drug Discov. 2013, 12, 388.
9. Pattabiraman, V. R.; Bode, J. W. Nature 2011, 480, 471.
0. Lavecchia, A.; Di Giovanni, C. Curr. Med. Chem. 2013, 20, 2839.
1. Gonec, T.; Kos, J.; Zadrazilova, I.; Pesko, M.; Keltosova, S.; Tengler, J.; Bobal, P.;
Kollar, P.; Cizek, A.; Kralova, K.; Jampilek, J. Bioorg. Med. Chem. 2013, 21, 6531.
2. Kos, J.; Nevin, E.; Soral, M.; Kushkevych, I.; Gonec, T.; Bobal, P.; Kollar, P.; Coffey,
A.; O´Mahony, J.; Liptaj, T.; Kralova, K.; Jampilek, J. Bioorg. Med. Chem. 2015, 23,
4
2
035.
4
4
4
4
4
3. Musiol, R.; Jampilek, J.; Podeszwa, B.; Finster, J.;Tabak, D.; Dohnal, J.; Polanski, J.
Cent. Eur. J. Chem. 2009, 7, 586.
4. Palmer, M.H. The Structure and Reactions of Heterocyclic Compounds. Edward Arnold
Publisher, London, UK, 1967; pp. 105–144.
5. Zheng, H.; Lu, L.; Wang, B.; Pu, S.; Zhang, X.; Zhu, G.; Shi, W.; Zhang, L.; Wang, H.;
Wang, S.; Zhao, G.; Zhang, Y. PLoS One 2008, 3, e2375.
6. Havlik, J. A.; Horsburgh, C. R.; Metchock, B.; Williams, P.; Fann, S. A.; Thompson, S.
E. J. Infect. Dis. 1992, 165, 577.
7. Rath, T.; Roderfeld, M.; Blocher, S.; Rhode, A.; Basler, T.; Akineden, O.;
Abdulmawjood, A.; Halwe, J. M.; Goethe, R.; Bulte, M.; Roeb, E. BMC
Gastroenterology 2011, 11, 34.
4
4
5
8. Bueno, J. Antitubercular in vitro drug discovery: Tools for begin the search. In:
Understanding Tuberculosis-New Approaches to Fighting against Drug Resistance,
Cardona, P.J. (Ed.). InTech, Rijeka, Croatia, 2012; pp. 147–168.
9. Gonec, T.; Kos, J.; Nevin, E.; Govender, R.; Pesko, M.; Kushkevych, I.; Oravec, M.;
Kollar, P.; O´Mahony, J.; Kralova, K.; Coffey, A.; Jampilek, J. Molecules 2014, 19,
1
0386.
0. Suffness, M.; Douros, J. J. Nat. Prod. 1982, 45, 1.
2
5

2
6