Discovery of novel enhancers of isoniazid toxicity in mycobacterium tuberculosis

Article abstract of DOI:10.3390/molecules23040825

Abstract: The number of effective first-line antibiotics for the treatment of Mycobacterium tuberculosis infection is strongly limited to a few drugs. Due to emerging resistance against those drugs, second- and third-line antibiotics have been established in therapy with certain problems and also increasing mycobacterial resistance. An alternative to such novel drugs or combined therapeutic regimes which may reduce resistance development is finding enhancers of mycobacterial drug effectiveness, especially enhancers that counteract causative resistance mechanisms. Such enhancers may reduce the extracellular drug efflux mediated by bacterial efflux pumps and thus enhance the intracellular drug toxicity. We developed novel 1,4-dihydropyridines (DHPs) as potential efflux pump inhibitors with some determined P-gp affinities. The influence on the antituberculotic drug toxicity has been investigated for three prominent antituberculotic drugs. Exclusive and selective toxicity enhancing effects have been detected for isoniazid (INH) which could be related to certain substituent effects of the 1,4-DHPs. So, structure-dependent activities have been found. Thus, promising enhancers could be identified and a suggested efflux pump inhibition is discussed.

Full text of DOI:10.3390/molecules23040825

molecules
Communication
Discovery of Novel Enhancers of Isoniazid Toxicity in
Mycobacterium tuberculosis
Fabian Lentz 1, Norbert Reiling 2,3, Ana Martins 4, Joseph Molnár 4 and Andreas Hilgeroth 1,
*
1
Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, 06120 Halle, Germany;
fabian.lentz@pharmazie.uni-halle.de
Research Center of Borstel, Leibniz Lung Center, 23845 Borstel, Germany; nreiling@fz-borstel.de
German Center for Infection Research (DZIF), Partner Site Hamburg-Lübeck-Borstel, 23845 Borstel, Germany
Department of Medical Microbiology, University of Szeged, 6720 Szeged, Hungary;
anasfmartins@gmail.com (A.M.); molnar.jozsef@med.u-szeged.hu (J.M.)
2
3
4
*
Correspondence: andreas.hilgeroth@pharmazie.uni-halle.de; Tel.: +49-345-55-25168
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 23 February 2018; Accepted: 1 April 2018; Published: 4 April 2018
Abstract: The number of effective first-line antibiotics for the treatment of Mycobacterium tuberculosis
infection is strongly limited to a few drugs. Due to emerging resistance against those drugs, second-
and third-line antibiotics have been established in therapy with certain problems and also increasing
mycobacterial resistance. An alternative to such novel drugs or combined therapeutic regimes
which may reduce resistance development is finding enhancers of mycobacterial drug effectiveness,
especially enhancers that counteract causative resistance mechanisms. Such enhancers may reduce
the extracellular drug efflux mediated by bacterial efflux pumps and thus enhance the intracellular
drug toxicity. We developed novel 1,4-dihydropyridines (DHPs) as potential efflux pump inhibitors
with some determined P-gp affinities. The influence on the antituberculotic drug toxicity has been
investigated for three prominent antituberculotic drugs. Exclusive and selective toxicity enhancing
effects have been detected for isoniazid (INH) which could be related to certain substituent effects of
the 1,4-DHPs. So, structure-dependent activities have been found. Thus, promising enhancers could
be identified and a suggested efflux pump inhibition is discussed.
Keywords: antibacterial enhancing activity; synthesis; derivatives; structure-activity; lead structure
1. Introduction
The most recent tuberculosis (Tb) report of the World Health Organization (WHO) states that TB
is the ninth leading cause of death worldwide and the leading cause from a single infectious agent,
ranking above HIV/AIDS. In 2016, there were an estimated 1.3 million TB deaths among HIV-negative
people (down from 1.7 million in 2000) and additional 374,000 deaths among HIV-positive people.
In addition, an estimated 10.4 million people fell ill with TB [1]. A major concern is the steady increase
of resistant and multidrug-resistant Mycobacterium tuberculosis (Mtb) complex strains. A total of
490,000 cases of multidrug-resistant TB (MDR-TB) have been reported for 2016. Almost half (47%) of
these cases were in India, China, and the Russian Federation [1].
Isoniazid (INH), rifampicin (RIF), ethambutol (EMB), and pyrazinamide (PZA) are considered as
first-line anti-TB drugs and form the core of standard treatment regimens [2]. If a given Tb isolate is
found susceptible to the used drug regime, EMB or PZA are usually discontinued after two months.
The ongoing therapy concentrates on the use of INH and RIF, which documents them as most important
antituberculotic drugs [3]. INH has the strongest early bactericidal action and significantly contributes
to rapidly making patients non-infectious. RIF has been shown to act on tubercle bacilli even in the
state of nonreplicating persistence (NRP) [3].
Molecules 2018, 23, 825; doi:10.3390/molecules23040825
www.mdpi.com/journal/molecules

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Resistance against at least INH and RIF, which represent the fundamental components of
any regimen for the treatment of drug-susceptible Tb, has been defined using the acronym
MDR-TB (multidrug-resistant tuberculosis) [3]. In such a case, second-line antibiotics are used in a
combined regime of four or five drugs including fluoroquinolone and aminoglycoside compounds [
3].
An MDR-TB isolate which in addition also shows resistance against a fluoroquinolone and an
aminoglycoside is termed extensively drug resistant (XDR-Tb). Only very limited therapy options do
currently exist for an effective treatment of patients infected with XDR-Tb strains.
Novel antituberculotic drugs in development are strongly limited in number and their usage with
partly insufficient knowledge about their mode of action and their effectiveness will be critical [
The costs of the therapy with such novel drugs will be too high for the developing countries which are
mainly affected by Tb [ ]. Moreover, the use of the established PZA becomes more doubtful due to
4].
5
observed initial resistances in Mtb strains as monoresistance or polyresistance [6,7].
One strategy to strengthen the effectiveness of the used antituberculotic drugs and to additionally
combat the resistance developments may be the use of enhancers of the antituberculotic drug effects
which are counteracted by an extracellular drug efflux. Only very few established drugs have been
investigated to increase the intracellular toxicity of antituberculotic drugs. All of those used drugs have
pharmacological activities like phenothiazines, verapamil, or reserpine which make them toxic for the
use as potential enhancers of the antituberculotic drug activity by the inhibition of the extracellular
efflux [8]. For most of those drugs, inhibitory activities of the human P-glycoprotein (P-gp) have
been reported in earlier studies. P-gp is a transmembrane efflux pump in mammalian cells which
is found overexpressed in cancer cells to export various anticancer drugs out of the cells, mostly
independent of the compound structure [9]. Thus, P-gp mainly contributes to the MDR phenomenon
in cancer treatment.
We developed a novel class of 1,4-dihydropyridines (DHP) with substituted phenyl residues both
at the 4- and the nitrogen position of the DHP scaffold and investigated the P-gp inhibitory activities
in a P-gp overexpressing cell assay system. The determined activities encouraged us to characterize
potential compound effects on the toxicity of antituberculotic drugs in Mycobacterium tuberculosis.
However, the compounds’ P-gp activities cannot be related to any activity data determined in Mtb
because P-gp as an efflux pump is not present in Mtb. The novel compound class could be profiled
in the use of the three most prominent antituberculotic drugs INH, RIF, and EMB with exclusive and
selective effects on the INH toxicity which is discussed in relation to substituent effects.
2. Results and Discussion
2.1. Synthesis of the 1,4-Dihydropyridines
For the synthesis of our 1,4-dihydropyridines
1–10 (Scheme 1) two equivalents of ethyl
acetoacetate, one equivalent of the respective aromatic aldehyde, and one equivalent of the substituted
aniline compound were heated under reflux conditions in alcohol, either methanol or ethanol.
The reaction was monitored by TLC until no more of the starting compounds aromatic aldehyde
or aniline derivatives were detectable. After removal of the solvent, the remaining oil was purified by
column chromatography using a mixture of cyclohexane and ethyl acetate in a ratio of 75:25. From the
unified compound-containing fractions, the target compounds were crystallized from methanol or
ethanol or, alternatively, from a mixture of diethyl ether and methanol in a ratio of 1:5. The isolated
compound yields varied from merely 2% to 21%. The lower yields may be caused by a reduced
nucleophilic activity of the aniline nitrogen compared to that of ammonia used for the synthesis of the
clinically used 1,4-dihydropyridines without a nitrogen substituent and higher isolated yields varying
from the reaction conditions [10
ester carbonyl vibrational bond at about 1690 cm−1 in the IR spectra and the shift of the 4-proton of the
1,4-dihydropyridine scaffold to about 5 ppm in the 1H NMR spectra. In compounds
, and with a
20-anilino substituent, we found a double set of proton signals in the compound spectra which could
,11]. The spectroscopic compound characteristics were the conjugated
2,
3
7

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be dedicated to two forms of isomers A and B, one with the 2-substituent at the N-phenyl residue on
the left and the other with that substituent on the right site of the molecule due to a sterically hindered
rotability of the nitrogen aniline bond. That hindered0rotabality is caused by the close location of the
0
2- and 6-methyl substituent, respectively, to the near 2 -anilino substituent. In the case of the 3 -anilino
substituted compounds 4, 5, and 8–10, we found one set of proton signals, accordingly. Also the
unsubstituted aniline compounds 1 and 6 resulted in just one set of proton signals.
R1
R1
O
H
O
O
O
O
O
O
O
O
a
+
O
O
N
NH2
R2
R2
1 - 10
Scheme 1. Synthesis of the 1,4-dihydropyridines 1–10, (a) MeOH, EtOH, 8–10 h reflux.
2.2. P-glycoprotein Inhibitory Activity of the 1,4-Dihydropyridines
The P-gp inhibitory activity of the compounds has been determined in a mouse T-lymphoma cell
model of two cell lines, one of which lacked P-gp expression and the other one expressing human
P-gp after retroviral gene transfection with the human MDR1 gene encoding for P-gp. The latter
cell line was cultured under colchicine conditions to provide a stable P-gp expression in the tested
cells. Rhodamine 123 has been used as fluorescent P-gp substrate and the uptake of the substrate was
measured in both cell lines without and with the use of our potential P-gp inhibitors. The fluorescence
uptake was measured by flow cytometry. A fluorescence ratio (FAR) value was determined from
the fluorescence amounts determined in the P-gp expressing cell line and the non-expressing cell
line under inhibitor application after the values were corrected by those of the non-inhibitor-treated
cells. FAR values > 1.1 prove a P-gp inhibitory activity of our compounds because the efflux of the
fluorescent substrate in the P-gp expressing cell line will be decreased under inhibitor application.
The N-(2-tolyl) substituent of compound
value of 1.45 (Table 1, right column). If the 2-methyl function moved to the 3-position of the N-phenyl
residue the activity was almost unchanged with a FAR value of 1.57 for compound . If replaced
with a chloro substitution, the activities of both compounds and slightly increased to 2.59 for the
N-(2-chlorophenyl) substitution and to 3.61 for the N-(3-chlorophenyl) substitution. If combined with
a 3-methoxy function at the 4-phenyl residue of compounds and both the N-2- and the N-3-tolyl
derivatives showed the lowest activities again, with FAR values of 1.53 and 1.48, respectively, similar
to the 4-phenyl unsubstituted compounds and . A N-(3-chlorophenyl) substitution combined with
that 3-methoxyphenyl function in compound was also not favourable with a FAR value of 1.57.
2 showed the lowest P-gp inhibitory activity with a FAR
4
3
5
7
8
2
4
9
If the 3-methoxy function at the 4-phenyl residue was replaced with a nitro group the activity of the
N-(3-tolyl) compound 10 moderately increased with a FAR value of 4.05.
It can be stated that overall our compounds showed some P-gp inhibitory activities almost
similar to the used standard verapamil with a FAR value of 1.40. If compared to the clinically
used 1,4-dihydropyridine nifedipine with a FAR value < 1.1, the novel compounds show some

Molecules 2018, 23, 825
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improved activities, whereas nifedipine was not active. Those clinically used 1,4-dihydropyridines
are unsubstituted at the nitrogen atom. Such an unsubstituted nitrogen is reported as an essential
feature for their use as a calcium antagonist [10]. So, our novel compounds with their N-phenyl
substitution will show no calcium antagonistic activity, but a preferred efflux pump inhibition due to
their N-aryl substitution.
Table 1. Isoniazid (INH) toxicity enhancing activity and P-gp-inhibiting properties of target compounds 1–10.
Increased Growth Inhibition (%) a,b
Cpd.
R1
R2
FAR Value a
c
1
2
3
4
5
6
7
8
9
H
H
H
H
H
3-MeO
3-MeO
3-MeO
3-MeO
3-NO2
H
35 ± 1.6
49 ± 0.7
61 ± 6.3
64 ± 2.5
80 ± 0.8
12 ± 0.7
52 ± 1.0
86 ± 1.3
79 ± 1.3
76 ± 1.3
n.d.
2-Me
2-Cl
3-Me
3-Cl
H
2-Me
3-Me
3-Cl
3-Me
b
1.45 ± 0.09
2.59 ± 0.14
1.57 ± 0.09
3.61 ± 0.22
2.21 ± 0.31
1.53 ± 0.09
1.48 ± 0.08
1.57 ± 0.09
4.05 ± 0.71
10
a
c
Mean of three determinations; percent growth inhibition, each related to INH growth inhibition; n.d.,
not determined.
2.3. Antituberculotic Drug Toxicity Enhancing Properties of the 1,4-Dihydropyridines
With the determined P-gp inhibitory activities of our compounds we decided to determine a
potential effect on the extracellular efflux of antituberculotic drugs similar to the reported drugs with
P-gp inhibitory activities which could not be further developed due to their own pharmacological
properties and resulting severe side-effects.
We started our studies with the most prominent antituberculotic drug, INH, that is also used
prophylactically in latently infected patients [12]. The bacterial growth of the Mtb strain H37Rv
expressing the green fluorescence protein (GFP) was characterized. We measured the GFP fluorescence
related to the bacterial growth under the use of subinhibitory concentrations of INH alone and INH
combined with our 1,4-DHPs to determine a potential drug toxicity enhancing effect. We observed
increasing effects on the INH toxicity after compound application. The resulting decreased growth
data were related to the INH growth inhibition alone. The growth inhibition data have been calculated
accordingly and are shown in Table 1 as percent-increased growth inhibition data.
The N-phenyl and 4-phenyl substituted derivative
of 35% compared to the inhibition of the sole INH. The introduction of a 2-methyl function in the
N-phenyl residue of compound increased the growth inhibition to 49%. A 2-chloro function of the
N-phenyl residue led to a further increase to 61% for compound . If both N-phenyl functions move to
1 resulted in an increased growth inhibition
2
3
the 3-position of the N-phenyl residue the growth inhibition further increased to 64% for the 3-tolyl
residue in compound 4 and to 80% for the 3-chlorophenyl substituent in compound 5.
If combined with a 3-methoxy function at the 4-phenyl residue, we found a lowered growth
inhibition for the unsubstituted N-phenyl residue in compound
N-(2-tolyl) compound again showed an increased growth inhibition of 52%. If the methyl group
moved to the 3-position of the N-phenyl residue the corresponding compound showed an increased
growth inhibition to 86% and the N-(3-chlorophenyl) compound showed a growth inhibition of 79%,
similar to the 4-phenyl substituted derivative . If the 3-methoxy function of the 4-phenyl residue
6 of just 12%. The corresponding
7
8
9
5
was replaced with a nitro function and combined with the most favourable 3-methyl function of the
N-phenyl residue in compound 10, the growth inhibition was slightly reduced to 76% compared
to compound 8. The results demonstrate that N-3-phenyl substituted inhibitors with a 3-methoxy
function at the 4-phenyl residue showed the best activities as enhancers of INH toxicity, as they increase
the INH mediated growth inhibition up to 86% for compound 8.

Molecules 2018, 23, 825
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Next, we tested four of our best compounds identified in the growth inhibition assay with INH,
namely compounds 10, and , to potentially enhance the growth inhibitory capacity of RIF under
8
,
5
,
4
the same used conditions. However, only compound 10 slightly increased the antimycobacterial
growth activity of RIF by 15%. All the other compounds showed almost unchanged rifampicin
activity data. Finally, we investigated a potential inhibitory effect of our compounds on the growth
inhibition of EMB in our assay system. We again used our four derivatives
practically no effects on the growth inhibition of EMB under compound application with calculated
data of 5% for compound , 9% for compound , 8% for compound 10, and finally 2% for compound 4.
8, 5, 10, and 4. We observed
8
5
Taken together, our data show that our novel compound class selectively enhances the
antituberculotic activity of INH if compared to the other studied antituberculotic drugs. Those
compounds with a 3-substitution of the N-phenyl residue preferably combined with a substituent at
the 3-position of the 4-phenyl residue have been identified as lead compounds. Although the fact that
a combination of our compounds with RIF and EMB showed no changes in the antituberculotic drug
activity already suggested that our compounds themselves show no antituberculotic drug activity,
we tested all compounds alone to inhibit the antituberculotic growth. None of the compounds showed
any relevant activities, as expected. So, possible toxic effects of the compounds themselves can be
excluded because such effects would reduce the growth of Mtb.
2.4. Efflux Pumps as a Potential Target of the 1,4-Dihydropyridines
In Mtb, efflux pumps of three families have been described; the major facilitator superfamily (MFS),
the ATP-binding cassette (ABC) superfamily, and the resistance nodulation division family (RND) [8].
Several efflux pumps of these families have been related to antituberculotic drug resistance. All efflux
pumps reported show no specificity for one single antituberculotic drug under normal cell culture
conditions, rather they transport various drugs of the established first-line therapeutics [8].
As we observed a specific drug toxicity enhancing effect for INH when combined with our novel
compounds, we wondered whether the used subinhibitory concentration of INH might have induced
an efflux pump sensitive to our compounds as potential inhibitors. In a recent study, induction of efflux
pumps was described for subinhibitory concentrations of the first-line antituberculotic drugs INH, RIF,
and EMB [13].
For INH, the induction of the efflux pump encoding genes Rv1273c, Rv1819c, and Rv1250 has been
described under conditions closely related to our cell culture conditions. Similarly, for RIF, induction
of the Rv1273c and the Rv1819c genes has been described under the used subinhibitory conditions.
For the used subinhibitory concentrations of EMB, efflux pump gene inductions have been observed
for Rv1273c and Rv0194. As our compounds showed only effects on INH toxicity, but not on the RIF
and EMB toxicity, we wondered whether Rv1250 might be the efflux pump which was selectively
affected by our novel compounds. As no specific detectable substrates of one single efflux pump exist,
it will be not possible to prove such a specify towards the respective efflux pump. However, it may be
investigated in future studies.
3. Material and Methods
3.1. Chemical Reagents and Instruments
Commercial reagents were used without further purification. The 1H-NMR spectra (400 MHz)
were measured using tetramethylsilane as internal standard. TLC was performed on E. Merck
5554 silica gel plates. The high-resolution mass spectra were recorded on a Finnigan LCQ Classic
mass spectrometer.
3.2. General Procedure for the Synthesis of Compounds 1–10
One equivalent of the aniline compound and the aromatic aldehyde and two equivalents of
ethyl acetoacetate were heated in a small volume of methanol or ethanol under reflux conditions.

Molecules 2018, 23, 825
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The reaction proceeding was followed by thin layer chromatography until no more starting compounds
were detectable. Then, the solution was evaporated to dryness and the remaining oil was purified by
column chromatography using silica gel and an eluent mixture of cyclohexane and ethyl acetate (3:1).
The received compound-containing fractions were unified and evaporated. The compounds were
finally crystallized either from the methanol or ethanol or from mixtures of diethyl ether and methanol
in a ratio of 1: 5.
Diethyl 2,6-Dimethyl-1,4-diphenyl-1,4-dihydropyridine-3,5-dicarboxylate (
IR (ATR) = 7.53–7.44 (m, 3H, 3-, 4-, 5-H of
= 1684 (C=O), 1198 (C–O–C) cm−1; 1H NMR (DMSO-d6)
N-phenyl), 7.32–7.26 (m, 6H, 2-, 6-H of N-phenyl and 2-, 3-, 5-, 6-H of 4-phenyl), 7.19–7.14 (m, 1H, 4-H
1 ;
). Yield 16%; mp 153–157 ◦C
ν
δ
of 4-phenyl), 5.02 (s, 1H, 4-H), 4.08–4.01 (m, 4H, COOCH2CH3), 1.95 (s, 6H, C-2, -6-CH3), 1.16–1.12
(m, 6H, COOCH2CH3); m/z (ESI) 406.63 (M + H+).
Diethyl 2,6-Dimethyl-4-phenyl-1-(o-tolyl)-1,4-dihydropyridine-3,5-dicarboxylate (
2
). Yield 10%;
1
mp 90–91 ◦C; IR (ATR)
ν
= 1691 (C=O), 1212 (C–O–C) cm−1; H NMR (DMSO-d6)
δ
= 7.44–7.11
(m, 18H, aromatic H of isomer A and B), 5.11 and 5.01 (2 x s, 2H, 4-H of isomer A and B), 4.08–3.98
(m, 8H, COOC 2CH3 of isomer A and B), 2.15 and 1.93 (2 x s, 6H, 2-CH3 of N-phenyl of isomer A
H
and B), 1.88 (s, 12H, C-2-, -6-CH3 of isomer A and B), 1.14–1.11 (m, 12H, COOCH2CH3 of isomer A
and B); m/z (ESI) 420.73 (M + H+).
Diethyl 1-(2-Chlorophenyl)-2,6-Dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (
3
). Yield 2%
;
,
◦
mp 87–89 C; IR (ATR)
ν δ = 7.74–7.35 (m, 10H
= 1693 (C=O), 1203 (C–O–C) cm−1; 1H NMR (DMSO-d6)
aromatic H of isomer A or B), 7.31–7.11 (m, 8H, aromatic H of isomer A or B), 5.04 and 4.99 (2 x s, 2H,
4-H of isomer A and B), 4.05–3.96 (m, 8H, COOC 2CH3 of isomer A and B), 1.93 and 1.87 (2 x s, 12H,
H
C-2-, -6-CH3 of isomer A and B), 1.14–1.08 (m, 12H, COOCH2C
H3 of isomer A and B); m/z (ESI) 440.71
(M + H+).
Diethyl 2,6-Dimethyl-4-phenyl-1-(m-tolyl)-1,4-dihydropyridine-3,5-dicarboxylate (
4
). Yield 8%;
1
mp 99–103 ◦C; IR (ATR)
ν
= 1692 (C=O), 1205 (C–O–C) cm−1; H NMR (DMSO-d6)
δ
= 7.41–7.37
(m, 1H, 5-H of N-phenyl), 7.32–7.26 (m, 5H, 4-H of N-phenyl and 2-, 3-, 5-, 6-H of 4-phenyl), 7.19–7.14
(m, 1H, 4-H of 4-phenyl), 5.02 (s, 1H, 4-H), 4.07–4.01 (m, 4H, COOC 2CH3), 2.34 (s, 3H, 3-CH3- of
N-phenyl), 1.96 (s, 6H, C-2-, -6-CH3), 1.16–1.12 (m, 6H, COOCH2CH3); m/z (ESI) 420.86 (M + H+).
H
Diethyl 1-(3-Chlorophenyl)-2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate ( ). Yield 3%
5
;
◦
1
mp 124–125 C; IR (ATR)
ν δ
= 1687 (C=O), 1201 (C–O–C) cm−1; H NMR (DMSO-d6)
= 7.58–7.51
(m, 2H, 5-, 6-H of N-phenyl), 7.31–7.24 (s, 1H, 2-H of N-phenyl), 7.31–7.24 (m, 5H, 4-H of N-phenyl
and 2-, 3-, 5-, 6-H of 4-phenyl), 7.18–7.14 (m, 1H, 4-H of 4-phenyl), 5.02 (s, 1H, 4-H), 4.07–4.02 (m, 4H,
COOC
H2CH3), 1.96 (s, 6H, C-2-, -6-CH3), 1.15–1.12 (m, 6H, COOCH2CH
3); m/z (ESI) 440.71 (M + H+).
Diethyl 4-(3-Methoxyphenyl)-2,6-dimethyl-1-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (
6
). Yield 2%
;
◦
1
mp 116–117 C; IR (ATR)
ν δ = 7.54–7.44
= 1690 (C=O), 1199 (C–O–C) cm−1; H NMR (DMSO-d6)
(m, 3H, 3-, 4-, 5-H of N-phenyl), 7.25–7.23 (m, 2H, 2-, 6-H of N-phenyl), 7.22 (t, J = 7.8 Hz, 1H, 5-H of
4-phenyl), 6.87 (dt, J = 7.8, 1.2 Hz, 1H, 6-H of 4-phenyl), 6.80–6.79 (m, 1H, 1H of 4-phenyl), 6.75 (ddd,
J = 7.8, 2.6, 1.2 Hz, 1H, 4-H of 4-phenyl), 5.01 (s, 1H, 4-H), 4.11–4.00 (m, 4H, COOC
H2CH3), 3.71 (s, 3H,
CH3O), 1.95 (s, 6H, C-2, -6-CH3), 1.17–1.13 (m, 6H, COOCH2CH3); m/z (ESI) 436.64 (M + H+).
Diethyl 4-(◦3-Methoxyphenyl)-2,6-dimethyl-1-(o-tolyl)-1,4-dihydropyridine-3,5-dicarboxylate (
7
). Yield 10%
;
mp 98–99 C; IR (ATR)
ν δ = 7.43–7.29 (m, 7H
= 1691 (C=O), 1211 (C–O–C) cm−1; 1H NMR (DMSO-d6)
,
aromatic H of N-phenyl of A and B), 7.24–7.20 and 7.19–7.15 (m, 2H, 5-H of 4-phenyl of A and B),
7.07–7.05 (m, 1H, aromatic H of N-phenyl of A or B), 6.89–6.87 and 6.84–6.82 (m, 2H, 6-H of 4-phenyl
of A and B), 6.79–6.71 (m, 4H, 2-, 4-H of 4-phenyl of A and B), 5.09 and 4.99 (2 x s, 2H, 4-H of A and B),
4.08–3.99 (m, 8H, COOCH2CH3 of A and B), 3.72 and 3.69 (2 x s, 6H, CH3O of A and B), 2.15 and 1.97
(2 x s, 6H, 2-CH3 of N-phenyl of A and B), 1.88 and 1.87 (2 x s, 12H, C-2, -6-CH3 of A and B), 1.16–1.12
(m, 12H, COOCH2CH3 of A and B); m/z (ESI) 450.66 (M + H+).

Molecules 2018, 23, 825
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Diethyl 4-(◦3-Methoxyphenyl)-2,6-dimethyl-1-(m-tolyl)-1,4-dihydropyridine-3,5-dicarboxylate (
8). Yield 4%;
mp 96–98 C; IR (ATR)
ν δ = 7.41–7.37 (m, 1H,
= 1691 (C=O), 1204 (C–O–C) cm−1; 1H NMR (DMSO-d6)
5-H of N-phenyl), 7.29–7.26 (m, 1H, 4-H of N-phenyl), 7.22 (t, J = 7.9 Hz, 1H, 5-H of 4-phenyl), 7.04–7.03
(m, 2H, 2-, 6-H of N-phenyl), 6.86 (dt, J = 7.9, 1.2 Hz, 1H, 6-H of 4-phenyl), 6.79–6.78 (m, 1H, 2-H of
4-phenyl), 6.75 (ddd, J = 7.9, 2.6, 0.9 Hz, 1H, 4-H of 4-phenyl), 5.01 (s, 1H, 4-H), 4.09–4.02 (m, 4H,
COOCH2CH3), 3.72 (s, 3H, CH3O), 2.33 (s, 3H, 3-CH3 of N-phenyl), 1.96 (s, 6H, C-2, -6-CH3), 1.17–1.14
(m, 6H, COOCH2CH3); m/z (ESI) 472.72 (M + Na+).
Diethyl 1-(3-Chlorophenyl)-4-(3-Methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicar-boxylate (9).
◦
1
Yield 3%; mp 108–110 C; IR (ATR)
ν
= 1692 (C=O), 1203 (C–O–C) cm−1; H NMR (DMSO-d6)
δ = 7.59–7.54 (m, 2H, 5-, 6-H of N-phenyl), 7.42 (s, 1H, 2-H of N-phenyl), 7.25–7.24 (m, 1H, 4-H of
N-phenyl), 7.23 (t, J = 7.8 Hz, 1H, 5-H of 4-phenyl), 6.87 (dt, J = 7.8, 1.2 Hz, 1H, 6-H of 4-phenyl),
6.78 (t, J = 1.2 Hz, 1H, 2-H of 4-phenyl), 6.77 (dt, J = 7.8 Hz, 1.2 Hz, 1H, 4-H of 4-phenyl), 5.02 (s, 1H
4-H), 4.08 (A X3, J = 10.9, 7.1 Hz, 2H, COOCHA HBCH3), 4.07 (A X3, J = 10.9, 7.0 Hz, 2H,
COOCHA BCH3), 3.73 (s, 3H, CH3O), 1.98 (s, 6H, C-2, -6-CH3), 1.16 (AB 3, J = 7.1, 7.0 Hz, 6H,
COOCH2CH3); m/z (ESI) 492.89 (M + Na+).
,
B
B
H
X
Diethyl 2,6-Dimethyl-4-(3-nitrophenyl)-1-(m-tolyl)-1,4-dihydropyridine-3,5-dicarboxylate (10). Yield 21%
;
◦
mp 114–115 C; IR (ATR)
t, J = 2.0 Hz, 1H, 2-H of 4-phenyl), 8.07 (dt, J = 7.8, 2.0 Hz, 1H, 4-H of 4-phenyl), 7.76 (dt, J = 7.8, 2.0 Hz,
ν ; δ = 8.16
= 1694 (C=O), 1206 (C–O–C) cm−1 1H NMR (DMSO-d6)
(
1H, 6-H of 4-phenyl), 7.63 (t, J = 7.8 Hz, 1H, 5-H of 4-phenyl), 7.44–7.40 (m, 1H, 5-H of N-phenyl),
7.32–7.29 (m, 1H, 4-H of N-phenyl), 7.17–7.14 (m, 2H, 2-, 6-H of N-phenyl), 5.09 (s, 1H, 4-H), 4.10–3.99
(m, 4H, COOCH2CH3), 2.36 (s, 3H, 3-CH3 of N-phenyl), 2.00 (s, 6H, C-2, -6-CH3), 1.16–1.13 (m, 6H,
COOCH2CH3); m/z (ESI) 465.72 (M + H+).
3.3. P-gp Inhibitory Activity
Two cell lines, a mouse T-lymphoma parental cell line L5178Y and a P-gp expressing subline
◦
L5178Y mdr, which resulted from retrovirus-mediated gene transfection, were cultured at 37 C under
carbon dioxide containing atmosphere (5%) in McCoys (San Marcos, TX, USA) 5A medium containing
10% of fetal calf serum, L-glutamine (2 mM) and 5 mL of a gentamicin solution (5 mg/mL). The medium
used for culturing the P-gp expressing subline L5178Y mdr has been additionally supplemented with
colchicine (60 ng/mL) to ensure a stable P-gp expression.
Cells of the mouse T-lymphoma parental cell line and the P-gp expressing subline were taken in
an adjusted concentration of 1
medium. 0.5 mL Aliquots were filled into Eppendorf (Hamburg, Germany) centrifuge tubes. The test
compounds 10 taken from prepared stock solutions in dimethylsulfoxide (1.0 mg/mL) were added
in a volume of 5 L. After 10 min of inhibitor preincubation at rt the P-gp substrate rhodamine 123 was
added using 5 L of a 0.5 mM solution in water reaching a final inhibitor concentration of 1 M.
×
106 cells per mL medium and resuspended in serum free McCoys 5A
1–
µ
µ
µ
◦
Incubation was continued for 40 min at 37 C. After that, the cells were centrifuged and washed
twice with phosphate-buffered saline (PBS). Then, they were resuspended in PBS for measurement.
The non-inhibitor containing cells were treated in the same way as the inhibitor preincubated cells.
The fluorescence uptake of rhodamine 123 within a number of 1 × 104 counted cells was determined
by flow cytometry using a Becton Dickinson FACScan (Franklin Lakes, NJ, USA) flow cytometer.
The fluorescence activity ratio (FAR) value was calculated from the quotient of the determined
fluorescence uptake ratios of the P-gp expressing cell (MDR) line and the non-P-gp expressing parental
cell line (parental). Both ratios have been corrected by division with the fluorescence determined in
the inhibitor untreated control cell lines following the equation for the FAR:
MDR treated/MDR control
Parental treated/Parental control

Molecules 2018, 23, 825
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3.4. Mtb Growth Inhibition Assay
Green fluorescence-expressing Mtb H37Rv bacteria were employed for growth analysis as
previously described [14]. In brief, 2
106 bacteria were cultured in 7H9 medium supplemented
with 10% oleic acid-albumin-dextrose-catalase (OADC), 0.05% Tween 80, and 0.2% glycerol in a total
volume of 100 L in a black 96-well plate with a clear bottom (Corning Inc., Corning, NY, USA) sealed
with an air-permeable membrane (Porvair Sciences, Wrexham, UK). Preincubation was carried out
for seven days with subinhibitory concentrations of INH (0.08 g/mL), RIF (0.01 g/mL), and EMB
3 µg/mL) as described [11]. Bacterial growth was measured under 1,4-DHP compound concentrations
×
µ
µ
µ
(
of 10 µg/mL in triplicates as relative light units at 528 nm after excitation at 485 nm in a fluorescence
microplate reader after seven days of culture (Synergy 2, Biotek, Winooski, VT, USA). The fluorescence
values determined for the combined antituberculotic drug and compound application were each
related to those of the determined values for the antituberculotic drug activity alone that were set
to 100%. So, reduced growth values have been given for the combination of INH and the used
compounds. The growth inhibition has been calculated accordingly as the difference between the
determined percent growth values and 100%.
4. Conclusions
Antituberculotic therapy is limited to a small number of drugs. Resistance against those drugs are
critical due to a lack of alternatives. One promising strategy is to increase the cellular antituberculotic
drug effectiveness, which may also decrease the risk of resistance development caused by efflux
pump activity. We developed novel 1,4-dihydropyridines in a simple one-pot reaction that were
demonstrated to selectively enhance INH toxicity in Mtb. The observed selectivity may be explained by
efflux pump inhibition by the compounds under the efflux pump inducing conditions of subinhibitory
concentrations of antituberculotic drugs. Such efflux pump inhibiting properties have not been
reported for 1,4-dihydropyridines before. In the case of the clinically used 1,4-dihydropyridines,
such effects are not expected because they show almost no effect towards P-gp, as demonstrated for
nifedipine. Structure-dependent activities have been found and the first lead compounds have been
identified, representing the first selective enhancers of antituberculotic drug activity.
Acknowledgments: We acknowledge the support of the Szeged Foundation for Cancer Research and the financial
support within the funding programme Open Access Publishing by the German Research Foundation (DFG).
Author Contributions: Fabian Lentz performed the synthesis; Norbert Reiling was responsible for the antibacterial
testing; Ana Martins and Joseph Molnár managed the determination of the P-gp inhibiting activities; Andreas
Hilgeroth designed the synthetic experiments and wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds not are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).