Dually acting nonclassical 1,4-dihydropyridines promote the anti-tuberculosis (Tb) activities of clofazimine

Article abstract of DOI:10.3390/molecules24162873

The number of effective antituberculotic drugs is strongly limited to four first-line drugs in standard therapy. In case of resistances second-line antibiotics are used with a poor efficacy and tolerability. Therefore, novel antituberculotic drugs are urgently needed. We synthesized novel nonclassical 1,4-dihydropyridines and evaluated their antituberculotic properties depending on substituent effects. Preferred substituents could be identified. As related classical 1,4-dihydropyridines are known as inhibitors of the transmembrane efflux pump ABCB1 in cancer cells, we wondered whether a use of our compounds may be of favour to enhance the antituberculotic drug efficacy of the second-line antituberculotic drug clofazimine, which is a known substrate of ABCB1 by a suggested inhibition of a corresponding efflux pump in Mycobacterium tuberculosis (Mtb). For this, we determined the ABCB1 inhibiting properties of our compounds in a mouse T-lymphoma cell line model and then evaluated the drug-enhancing properties of selected compounds in a co-application with clofazimine in our Mtb strain. We identified novel enhancers of clofazimine toxicity which could prevent clofazimine resistance development mediated by an efflux pump activity.

Full text of DOI:10.3390/molecules24162873

molecules
Communication
Dually Acting Nonclassical 1,4-Dihydropyridines
Promote the Anti-Tuberculosis (Tb) Activities
of Clofazimine
Fabian Lentz 1, Norbert Reiling 2, Gabriella Spengler 3, Annamária Kincses 3 , Andrea Csonka 3,
Joseph Molnár 3 and Andreas Hilgeroth 1,
*
1
Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, 06120 Halle, Germany
Research Center of Borstel, Leibniz Lung Center, 23845 Borstel, Germany
Department of Medical Microbiology, University of Szeged, 6720 Szeged, Hungary
Correspondence: andreas.hilgeroth@pharmazie.uni-halle.de; Tel.: +49-345-55-25168
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Peter J. Rutledge
Received: 21 June 2019; Accepted: 1 August 2019; Published: 8 August 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The number of effective antituberculotic drugs is strongly limited to four first-line drugs
in standard therapy. In case of resistances second-line antibiotics are used with a poor efficacy and
tolerability. Therefore, novel antituberculotic drugs are urgently needed. We synthesized novel
nonclassical 1,4-dihydropyridines and evaluated their antituberculotic properties depending on
substituent effects. Preferred substituents could be identified. As related classical 1,4-dihydropyridines
are known as inhibitors of the transmembrane efflux pump ABCB1 in cancer cells, we wondered
whether a use of our compounds may be of favour to enhance the antituberculotic drug efficacy
of the second-line antituberculotic drug clofazimine, which is a known substrate of ABCB1 by a
suggested inhibition of a corresponding efflux pump in Mycobacterium tuberculosis (Mtb). For this, we
determined the ABCB1 inhibiting properties of our compounds in a mouse T-lymphoma cell line
model and then evaluated the drug-enhancing properties of selected compounds in a co-application
with clofazimine in our Mtb strain. We identified novel enhancers of clofazimine toxicity which could
prevent clofazimine resistance development mediated by an efflux pump activity.
Keywords: antibacterial activity; synthesis; substituent; structure–activity; inhibition
1. Introduction
Tuberculosis (Tb) is the ninth cause of global death and the leading one from a single infectious
agent, thus ranking above human immunodeficiency virus (HIV) [1,2]. The present treatment
regimen of Tb with the four drugs isoniazid, rifampicin, ethambutol and pyrazinamide as first-line
antituberculostatic agents lasts for six months with a high efficacy of about 90% in the cases of the
so-called drug-susceptible Tb [
or tolerability lead to resistances [
a drug-resistant Tb results which currently contributes about 50% of the global resistances [
resistance against both isoniazid and rifampicin results in a multidrug-resistant Tb (MDR-Tb) [
cases of such a resistance, second line antibiotics of the fluoroquinolone group or the injectable agents
kanamycin, amikacin or capreomycin are used [ ]. The therapy with those drugs is characterized
by low efficacy, high toxicity, a long duration and a poor outcome of 54% [ ]. A treatment of the
1,3
,
1
4
]. Occurring problems with compliance, sub-optimal drug levels
]. In the case of a resistance against either isoniazid or rifampicin
]. A
]. In
1
1
5
5,6
so-called extensively drug-resistant Tb (MDR/XDR-Tb) which includes additional resistances against
one fluoroquinolone and one-injectable agent is very difficult with an outcome of < 30% and thus
MDR/XDR-Tb mainly contributes to the Tb mortality rates [
1,5
,6]. Therefore, there is a strong need for
novel antituberculostatic drugs.
Molecules 2019, 24, 2873; doi:10.3390/molecules24162873
www.mdpi.com/journal/molecules

Molecules 2019, 24, 2873
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Classical 1,4-dihydropyridines have been investigated to act as antituberculostatic agents [7].
They showed percentual inhibitions of the mycobacterial growth at a given concentration. Their
mode of action was suggested to be similar to that of isoniazide (INH). However, for isoniazide
several modes of actions are discussed to block the synthesis of mycolic acid as an essential part of the
mycobacterial cell wall. Moreover, those classical 1,4-dihydropyridines own an unsubstituted nitrogen
and electron-withdrawing substituents at the 2-position of the 4-phenyl residue. Both structural
properties are a prerequisite for the compounds’ abilities to act as antihypertensive agents. So those
compounds are excluded for a use as antituberculostatic agents.
We developed novel nonclassical 1,4-dihydropyridines with a substituted nitrogen and without
2-substituents at the 4-phenyl residue to provide compounds which may exclusively act as
antituberculostatic agents. Those classical 1,4-dihydropyridines are known to act as inhibitors
of the transmembrane efflux pump ABCB1 in cancer cells where they enhance the toxicity of applicated
cytostatic agents [8–10]. Therefore, we wondered whether our nonclassical 1,4-dihydropyridines may
act in a similar way towards ABCB1 and whether they may increase the toxicity of antituberculostatic
drugs due to an inhibition of a mycobacterial efflux pump similar to ABCB1. Furthermore drug-toxicity
enhancing effects of promising compounds have been investigated.
2. Results and Discussion
2.1. Synthesis of the 1,4-Dihydropyridines
Contrasting the formation of the classical 1,4-dihydropyridines which follows the principles of
the Hantzsch synthesis in ethanol or methanol depending on the nature of the used ethyl or methyl
acetoacetate [11], the nonclassical 1,4-dihydropyridines without a dimethyl substitution in both the 2-
and the 6-position of the 1,4-dihydropyridine scaffold are formed alternatively in a one-pot reaction by
the use of an aromatic aldehyde, ethyl propiolate and an amine compound (Scheme 1).
The mechanism followd a primary formation of the amino acrylate
molecule ethyl propiolate and the used substituted aniline compound 12]. In a second reaction the
aromatic aldehyde condenses with the nucleophilic C-3 of amino acrylate to give intermediate
12 13]. In a reaction of another compound and , that both have been detectable in the reaction
mixtures by mass spectrometry, compound is formed which cyclizes under aniline elimination to
give the target structure of 18 13]. The product formation was carried out in ethanol under acetic
acid catalysis using one equivalent of the aromatic aldehyde and of the aniline and two equivalents
of ethyl propiolate . The reaction proceeding was followed by thin layer chromatography (TLC) and
mass spectrometry.
A by the reaction of one
1
2 [
3
A
B
[
,
A
B
C
4–
[
3
2
1
After finishing the reaction, the acetic acid was removed by extraction with ethyl acetate versus
brine solution (10%). After drying of the organic layer, filtration and evaporation in vacuum the
remaining oil was partly purified by column chromatography over silica gel and mixtures of ethyl
acetate and cyclohexane to give the pure compound fractions from which the respective target structures
crystallized. Spectroscopically the compounds were characterized by high-field shifts of the 4-protons
of the 1,4-dihydropyridine scaffold to about 4.8 ppm and those of the 2- and 6-protons appeared as a
singlet at about 7.4 ppm.

Molecules 2019, 24, 2873
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R2
O
O
O
O
NH2
O
R1
NH
H
R1
1
2
A
3
R2
R2
O
O
O
O
O
A
O
- H2O
HN
NH
N
R1
R1
R1
B
C
R2
O
O
O
O
NH2
N
R1
R1
4 - 13
Scheme 1. Formation of the 1,4-dihydropyridines 4–13 in EtOH, 3–73 h, 80 ◦C.
2.2. Antituberculostatic Activity of the 1,4-Dihydropyridines
The antituberculostatic activity was determined using a green-fluorescent protein (GFP)
expressing virulent Mycobacterium tuberculosis (Mtb) strain H37Rv. This allowed the monitoring
of mycobacterial growth by measuring GFP-fluorescence at 528 nm. The potentially growth-inhibiting
1,4-dihydropyridines 4–18 were incubated with the mycobacteria at a concentration of 1 µg per
mL and isoniazid was used as the control. The reduced measured fluorescence amounts under
1,4-dihydropyridine application as result of a growth inhibition were each related to that of the
untreated control cells and the resulting percentage inhibitions of growth are given in Table 1. We
investigated the effect of methyl- and chloro-substituents in the aniline residue which contribute to
the lipophilic properties of the compounds. Additionally, alkoxy functions were introduced in the
4-phenyl residue to evaluate a lipophilic influence of methoxy and benzyloxy. All these substituents
showed favourable effects in recent studies of the classical 1,4-dihydropyridines [7]. Amino functions
for comparison were excluded as they would have interfered with the synthesis of the compounds in
which the aniline nitrogen function builds the core of the 1,4-dihydropyridines.
Compound
showed a residual antituberculostatic activity of 9%. If the N 2-chloro function was replaced with
a 2-methyl function in compound we found main increases in activity up to 34%. If the 4-phenyl
residue was replaced with a 3-methoxyphenyl residue we found increases in the activity of the N
4 [14] with an unsubstituted 4-phenyl residue and the N 2-chlorophenyl substitution
5
2-chlorophenyl derivative
3-methoxy phenyl function
6
5
and also the N 2-tolyl derivative
reaching an activity of 40%. That activity was almost half of the activity
7 was better than that without the
of the used isoniazid (INH) control. If the 3-methoxy function of the 4-phenyl residue moved to the
4-position in compounds and we found decreases in activity for both either the N 2-chlorophenyl
8
9

Molecules 2019, 24, 2873
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or the 2-tolyl substituted compounds. If two methoxy functions in the 3- and the 4-position of the
4-phenyl residue were combined in derivatives 10 and 11 we found only a residual activity for the
N 2-chlorophenyl derivative 10 whereas the N 2-tolyl compound 11 was no more active. Therefore,
the 3-methoxy substitution was the most favourable 4-phenyl substitution. Next, we replaced that
only 3-methoxy function with a 3-benzyloxy function in compounds 12 and 13. We found a loss
of activity for the N 2-chlorophenyl compound
9 and a main reduction of activity for the N 2-tolyl
compound 13 with 17%. From the first discussed data the following structure–activity relationships can
be concluded: The 2-methyl group with its inductive effect was the most favourable aniline substitution
contrasting the electron-withdrawing chloro-substitution. An additional 3-methoxy phenyl function
with electron-pushing effects strengthened the observed activity also for the less favourable 2-chloro
aniline substitution. The favourable effect of the introduced methoxy function in the 4-phenyl residue
encouraged us to place it alternatively in the aniline residue. While the synthesis with the 3-methoxy
aniline failed, the use of the 4-methoxy aniline was successful. We also tried to synthesize 4-phenyl
derivatives with the chloro-electron withdrawing function in both the 2- and 3-position for comparison,
but failed due to an insufficient aldehyde reactivity and steric reasons, also in the case of an alternative
2-methyl substitution. The only 4-methoxy aniline substituted compound 14 resulted in a main
increase in bacterial growth inhibition of 60%. If that 4-methoxy aniline substitution was combined
with a 3-methoxy function in the 4-phenyl residue in derivative 15 a slight decrease to 41% in the
mycobacterial growth inhibition was found. If the 3-methoxy function moved to the 4-position of
the 4-phenyl residue in compound 16 we found a further reduction of the mycobacterial growth
inhibition to 35%. However, a combination of a 3- and 4-methoxy phenyl substitution in derivative 17
increased the growth inhibition up to 59%. A combination of the 4-methoxy aniline substitution with
the 3-benzyloxy phenyl substitution in compound 18 led to a main reduction of the growth inhibition to
7%. Finally, it can be stated that the 4-methoxy function in the aniline residue with its electron-pushing
effects was the best 1,4-dihydropyridine substitution. Methoxy functions placed in the 4-phenyl residue
showed favourable effects with the 3-substituion being better than the 4-substitution.
Table 1. Mycobacterium tuberculosis (Mtb) cell growth inhibition and ABCB1-inhibiting properties of
target compounds 4–18 expressed as fluorescence activity ratio (FAR) values.
Cpd.
R1
2-Cl
R2
Growth Inhibition (%) (a)
FAR Value (a)
11.20
8.19
4
H
H
9
5
2-Me
2-Cl
34
37
40
24
2
6
3-OMe
3-OMe
4-OMe
4-OMe
3-, 4-OMe
3-, 4-OMe
3-OBn
32.62
33.59
52.49
30.80
59.76
68.30
31.46
42.29
3.9
7
2-Me
2-Cl
8
9
2-Me
2-Cl
10
11
12
13
14
15
16
17
18
9
(b)
(b)
2-Me
2-Cl
n.a.
n.a.
17
60
41
35
59
7
2-Me
4-OMe
4-OMe
4-OMe
4-OMe
4-OMe
3-OBn
H
3-OMe
4-OMe
3-, 4-OMe
3-OBn
3.4
1.2
5.2
(c)
n.d.
(c)
INH
97
n.d.
n.d.
n.d.
(c)
(c)
Verapamil
Tariquidar
4.10
15.3
(a)
Mean of three determinations; (b) not active; (c) not determined.

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2.3. ABCB1 Inhibitory Activity of the 1,4-Dihydropyridines
We used a mouse T-lymphoma cell line model to evaluate the potential ABCB1 inhibitory
properties of our 1,4-dihydropyridines. Within the studies we determined the amount of the ABCB1
fluorescent substrate rhodamine 123 in two types of cells: first in cells which did not express the human
ABCB1 as control cells and then in cells which expressed ABCB1 after ABCB1 retrovirus gene transfer.
Due to the ABCB1 expression the uptake of rhodamine was reduced in those cells. Then both cells
were pre-incubated with our potential inhibitors and the fluorescence amounts of added rhodamine
123 was determined again. In the case of an ABCB1 inhibition the amounts in the ABCB1 expressing
cells mainly increased due to the inhibition. Finally, a FAR (fluorescence activity ratio) value was
calculated by a relation of the amounts of fluorescence uptake into the ABCB1 expressing cells under
inhibitor treatment versus untreated cells and the amounts of fluorescence in the control cells under
inhibitor treatment versus the untreated control cells. Therefore, compounds with FAR values > 1.1
were found active. The FAR values are shown in Table 1.
The determined ABCB1 inhibitory activity of the only 4-phenyl substituted compounds
were moderate with FAR values of 11.20 and 8.19, respectively, similar to that of the used standards
verapamil and tariquidar. The 3-methoxy function in compounds and increased the activity to
similar values of about 33 for both compounds. If the 4-methoxy function moved to the 4-position
of the 4-phenyl residue in derivatives and the activity of the N 2-chlorophenyl compound
increased whereas that of the N 2-tolyl derivative remained almost unchanged. The 3-, 4-dimethoxy
4 and 5
6
7
8
9
8
9
substitution of compounds 10 and 11 increased the activity of the N 2-tolyl compound 11. However,
slight increases were also observed for the N 2-chlorophenyl derivative 10. If the 3-methoxy functions
in derivatives
6 and 7 were replaced with 3-benzyloxy functions in compounds 12 and 13 the activity
of the N 2-chlorophenyl 12 compound remained almost unchanged and slight increases were observed
for the N 2-tolyl derivative 13. The 4-methoxy function in the aniline residue of compound 14 led
to a reduced inhibitory activity and the combination with the 3-methoxy or the 4-methoxy function
in derivatives 15 and 16 led to similar results. However, all three methoxy functions in compound
18 again increased the ABCB1 inhibitory activity. Therefore, it can be stated that methoxy functions
placed in the 4-phenyl residue were favoured for the ABCB1 inhibitory activities which is in accordance
with earlier publications that suggest such favourable methoxy functions act as hydrogen acceptor
functions for a potential inhibitor binding to ABCB1 [15,16].
2.4. Antituberculostatic Drug Toxicity Enhancing Properties of 1,4-Dihydropyridines
The observed ABCB1 inhibiting properties of our 1,4-dihydropyridines encouraged us to
investigate a possible antituberculostatic drug toxicity enhancing effect of them in co-application with
respective antituberculotic drugs by an inhibition of a mycobacterial efflux pump similar to ABCB1.
Such drug toxicity enhancing effects may be a strategy to prevent drug resistance development of
that drug. In the case of being a substrate of an efflux pump the intracellular level of a respective
drug is reduced so that it becomes ineffective which means a drug resistance [17]. Furthermore, it has
recently been demonstrated that such lowered drug levels induce the expression of respective efflux
pumps in Mtb to enforce such a resistance development [18]. So far drug toxicity enhancing effects
have only been described for a strongly limited number of used drugs which generally have their own
pharmacological properties as antihypertensive or antidepressive drugs [19]. These properties do not
allow their use as enhancers.
We investigated drug toxicity enhancing effects of the antituberculotic drug clofazimine.
Clofazimine has originally been used as leprosy drug for the treatment of infections with Mycobacterium
leprae [20]. Due to its potent activity against Mtb, clofazimine has been recommended by the World
Health Organization in 2016 as a core second line agent against multidrug-resistant tuberculosis with
favourable properties in tissue distribution, intracellular activity and prolonged half-life [1,21,22].
Thus, clofazimine became important for the design of second line regimens for the treatment of
tuberculosis [1].

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Clofazimine has been reported as an ABCB1 substrate in a cellular assay system of MDCK cells
with the expression ABCB1 similar to our mouse T-lymphoma assay system although the cells have
been different [23]. However, with the suggested ABCB1 substrate properties we expected a benefit of
our compounds as inhibitors of ABCB1 in a co-application with clofazimine in Mtb cells to increase the
clofazimine toxicity due to an inhibition of an antituberculotic efflux pump similar to ABCB1.
In our studies we used a concentration of clofazimine of 0.3
of the mycobacterial growth of 56%. Then we co-applicated clofazimine with each 1
compounds of our 1,4-dihydropyridines. We chose compounds 11 and 12. All three compounds
µg/mL to result in a mean reduction
µg/mL of selected
4,
have not been practically active as antituberculotic drugs. Therefore, if an increased toxicity would
result under co-application of those drugs with clofazimine the effect could be related to an inhibition
of a mycobacterial efflux pump related to ABCB1.
For a co-application of compound
4 we found a further reduction of the bacterial growth to
28% which meant an increase of the growth inhibition of 44% compared to that of clofazimine alone
(Table 2). Co-application of compound 11 resulted in a further reduction of growth to 23% which
meant a growth inhibition increase of 55% compared to clofazimine. The use of compound 12 led to
a mycobacterial growth reduction to 34% similar to compound 4. Finally, we tested compound 13
which itself showed an antimycobacterial activity with a growth reduction of 19%. In combination
with the clofazimine dose a total growth inhibition of 28% was observed which meant an increase of
the inhibition of clofazimine by 54%.
Table 2. Mtb cell effects of selected target compounds 4 and 11–13 under clofazimine co-application.
Growth (%) (a)
Growth (%)
Target Compound Only
Percentual Growth
Clofazimine + Target Compound Inhibition Increase (%)
Cpd.
4
96
100
100
81
28
23
34
44
55
44
54
11
12
13
28
(a)
Mean of three determinations.
A comparison of the increase of growth inhibition by the use of the four selected compounds with
their ABCB1 inhibiting properties showed that the compounds with the lower ABCB1 affinities and
4
12 resulted in the lower increase of growth inhibition. Compound 11 with highest ABCB1 affinities
resulted in an enhanced growth inhibition activity. Compound 13 with a mean ABCB1 inhibiting
activity compared to the other used compounds was found to cause an increase of growth inhibition
similar to compound 11 due to its own activity in mycobacterial growth inhibition.
Therefore, our results suggested a compound activity on drug toxicity enhancing effects by an
inhibition of a mycobacterial efflux pump similar to ABCB1 which is presently under discussion.
3. Material and Methods
3.1. Chemical Reagents and Instruments
Commercial reagents were used without further purification. The 1H-NMR spectra (500 MHz)
were measured on an Inova Unity 500 (Varian) spectrometer using tetramethylsilane as internal standard.
The 1H NMR spectra are shown in the Supplementary Materials. Thin layer chromatography (TLC)
was performed on E. Merck 5554 silica gel plates. The high-resolution mass spectra were recorded on a
Finnigan LCQ Classic mass spectrometer. Elemental analysis indicated by the symbols of the elements
was within ±0.4% of the theoretical values and was performed using a Leco CHNS-932 apparatus.

Molecules 2019, 24, 2873
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3.2. General Procedure for the Synthesis of Compounds 4–18
Each 1 equivalent (4.2 mmol) of the aniline compound and of the aromatic aldehyde and 2
equivalents (8.2 mmol) of ethyl propiolate were heated in a small volume of ethanol under addition
of acetic acid (10% v/v) under reflux conditions. The reaction proceeding was followed by TLC and
mass spectrometry until no more starting compounds were detectable. Then acetic acid was removed
from the solution by extraction with a brine solution (10% m/v) versus ethyl acetate three times. The
unified ethyl acetate layer was dried over sodium sulphate and filtered. The filtrate was evaporated in
vacuum and the remaining oil was purified by column chromatography using silica gel and an eluent
mixture of cyclohexane and ethyl acetate (10%–20% v/v) if a direct isolation via crystallization failed.
The compounds were finally recrystallized from mixtures of methanol and diethyl ether.
Diethyl N-(2-chlorophenyl)-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (
4). Yield 19%, white powder;
mp 139–140 ◦C; 1H NMR (DMSO-d6)
δ
= 7.68–7.67 (m, 1H, 3- or 6-H of 2-Cl-Ph), 7.64 (m, 1H, 3- or
6-H of 2-Cl-Ph), 7.52–7.45 (m, 2H, 4-H, 5-H of 2-Cl-Ph), 7.36–7.34 (m, 2H, 2-, 6-H of Ph); 7.34 (s, 2H,
2-, 6-H), 7.29–7.26 (m, 2H, 3-, 5-H of Ph), 7.18–7.14 (m, 1H, 4-H of Ph), 4.79 (s, 1H, 4-H), 4.05–3.94 (m,
4H, COOCH2CH3), 1.11–1.08 (m, 6H, COOCH2CH3); 13C NMR (100 MHz, DMSO-d6) δ = 166.4 (CO),
144.4, 144.2, 139.3, 132.2, 130.7, 128.6, 127.7, 127.6, 125.7, 125.4, 123.8, 108.0 (aromat. C, 2-, 6-, 3-, 5-C),
60.9 (OCH2CH3), 37.4 (4-C), 14.2 (OCH2C
H3); m/z (ESI) 412.55 (M + H+); anal. (C23H22ClNO4) calc. C
67.05, H 5.38, N 3.40; found C 66.75, H 5.18, N 3.22.
Diethyl 4-phenyl-N-(o-tolyl)-1,4-dihydropyridine-3,5-dicarboxylate (5). Yield 16%, white powder; mp
105–106 ◦C; 1H NMR (DMSO-d6)
δ
= 7.40–7.27 (m, 8H, 3-, 4-, 5-, 6-H of 2-CH3Ph and 2-, 3-, 5-, 6-H of
Ph), 7.28 (s, 2H, 2-, 6-H), 7.18–7.14 (m, 1H, 4-H of Ph), 4.81 (s, 1H, 4-H), 4.05–3.93 (m, 4H, COOC 2CH3),
2.28 (s, 3H, 2-CH3Ph), 1.11–1.98 (m, 6H, COOCH2C = 166.8
3); 13C NMR (100 MHz, DMSO-d6)
(CO), 144.6, 144.0, 139.1, 131.3, 120.0, 128.6, 127.7, 126.5, 125.8, 123.7, 122.7, 109.0 (aromat. C, 2-, 6-,
3-, 5-C), 60.8 (O H2CH3), 37.5 (4-C), 18.1 (CH3), 14.2 (OCH2
H3); m/z (ESI) 392.58 (M + H+); anal.
H
δ
H
C
C
(C24H25NO4) calc. C 73.64, H 6.44, N 3.58; found C 73.64, H 6.43, N 3.74.
Diethyl N-(2-chlorophenyl)-4-(3-methoxyphenyl)-1,4-dihydropyridine-3,5-dicarboxylate (6). Yield 20%,
◦
1
yellow-greenish powder; mp 127–128 C; H NMR (DMSO-d6)
δ = 7.68–7.66 (m, 1H, 3- or 6-H
of 2-Cl-Ph), 7.63–7.61 (m, 1H, 3- or 6-H of 2-Cl-Ph), 7.51–7.45 (m, 2H, 4-, 5-H of 2-Cl-Ph), 7.34 (s, 2H, 2-,
6-H), 7.19 (t, J = 7.9 Hz, 1H, 5-H of 3-CH3OPh), 6.93 (dt, J = 7.9, 1.5 Hz, 1H, 6-H of 3-CH3OPh), 6.87
(dd, J = 2.6, 1.5 Hz, 1H, 2-H of 3-CH3OPh), 6.74 (ddd, J = 7.9, 2.6, 1.5 Hz, 1H, 4-H of 3-CH3OPh), 4.77 (s,
1H, 4-H), 4.07–3.95 (m, 4H, COOC
NMR (100 MHz, DMSO-d6) = 166.5 (CO), 160.5, 144.6, 141.1, 138.7, 132.3, 130.3, 129.6, 127.3, 125.1,
123.4, 120.0, 113.1, 111.3, 107.3 (aromat. C, 2-, 6-, 3-, 5-C), 60.7 (O H2CH3), 55.8 (OCH3), 37.5 (4-C), 14.2
(OCH2
H3; m/z (ESI) 442.51 (M + H+); anal. (C24H24ClNO5) calc. C 65.23, H 5.47, N 3.17; found C
65.11, H 5.55, N 2.90.
H2CH3), 3.70 (s, 3H, 3-CH3O), 1.12–1.09 (m, 6H, COOCH2CH C
3); 13
δ
C
C
Diethyl 4-(3-methoxyphenyl)-N-(o-tolyl)-1,4-dihydropyridine-3,5-dicarboxylate (
7). Yield 40%, white powder;
mp 110–111 ◦C; 1H NMR (DMSO-d6)
δ
= 7.40–7.33 (m, 4H, 3-, 4-, 5-, 6-H of 2-CH3Ph), 7.28 (s, 2H, 2-,
6-H), 7.20 (t, J = 7.9 Hz, 1H, 5-H of 3-CH3OPh), 6.90 (dt, J = 7.9, 1.5 Hz, 1H, 6-H of 3-CH3OPh), 6.83
(dd, J = 2.6, 1.5 Hz, 1H, 2-H of 3-CH3OPh), 6.75 (ddd, J = 7.9, 2.6, 1.5 Hz, 1H, 4-H of 3-CH3OPh), 4.80 (s,
1H, 4-H), 4.06–3.95 (m, 4H, COOC
6H, COOCH2C
3); 13C NMR (100 MHz, DMSO-d6)
129.6, 129.0, 126.3, 123.5, 122,4, 120.0, 113.1, 111.3, 108.0 (aromat. C, 2-, 6-, 3-, 5-C), 60.4 (O
55.8 (OCH3), 37.8 (4-C), 14.1 (OCH2
H3); m/z (ESI) 422.28 (M + H+); anal. (C25H27NO5) calc. C 71.24,
H
2CH3), 3.71 (s, 3H, 3-CH3OPh), 2.28 (s, 3H, 2-CH3Ph), 1.12–1.09 (m,
= 166.6 (CO), 160.2, 143.9, 143.1, 138.1, 131.0,
H2CH3),
H
δ
C
C
H 6.46, N 3.32; found C 71.27, H 6.46, N 3.49.
Diethyl N-(2-chlorophenyl)-4-(4-methoxyphenyl)-1,4-dihydropyridine-3,5-dicarboxylate (8). Yield 43%, yellow
powder; mp 134–135 ◦C; 1H NMR (DMSO-d6)
δ = 7.68–7.66 (m, 1H, 3- or 6-H of 2-Cl-Ph), 7.64–7.62
(m, 1H, 3- or 6-H of 2-Cl-Ph), 7.51–7.45 (m, 2H, 4-, 5-H of 2-Cl-Ph), 7.31 (s, 2H, 2-, 6-H), 7.26–7.23 (m,
2H, 2-, 6-H of 4-CH3OPh), 6.84–6.81 (m, 2H, 3-, 5-H of 4-CH3OPh), 4.72 (s, 1H, 4-H), 4.06–3.94 (m, 4H,

Molecules 2019, 24, 2873
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COOC
H
2CH3), 3.69 (s, 3H, 4-CH3O), 1.12–1.09 (m, 6H, COOCH2C
H
3); 13C NMR (100 MHz, DMSO-d6)
δ
= 166.8 (CO), 157.6, 144.1, 138.7, 136.7, 132.1, 130.2, 130.0, 127.2, 125.2, 123.4, 114.2, 107.4 (aromat. C,
2-, 6-, 3-, 5-C), 60.3 (O H2CH3), 55.4 (OCH3), 37.3 (4-C), 14.2 (OCH2
H3); m/z (ESI) 442.54 (M + H+);
anal. (C24H24ClNO5) calc. C 65.23, H 5.47, N 3.17; found C 65.32, H 5.46, N 3.17.
C
C
Diethyl 4-(4-methoxyphenyl)-N-(o-tolyl)-1,4-dihydropyridine-3,5-dicarboxylate ( ). Yield 21%, yellow
= 7.40–7.31 (m, 4H, 3-, 4-, 5-, 6-H of 2-CH3Ph),
7.25 (s, 2H, 2-, 6-H), 7.23–7.20 (m, 2H, 2-, 6-H of 4-CH3OPh), 6.85–6.82 (m, 2H, 3-, 5-H of 4-CH3OPh),
4.75 (s, 1H, 4-H), 4.05–3.93 (m, 4H, COOC 2CH3), 3.70 (s, 3H, 4-CH3OPh), 2.28 (s, 3H, 2-CH3Ph),
1.12–1.09 (m, 6H, COOCH2C = 166.6 (CO), 157.2, 144.3, 138.4,
3); 13C NMR (100 MHz, DMSO-d6)
136.8, 131.6, 130.4, 126.9, 123.6, 122.7, 114.1, 108.2 (aromat. C, 2-, 6-, 3-, 5-C), 60.7 (O H2CH3), 55.4
(OCH3), 37.8 (4-C), 18.0 (CH3), 14.1 (OCH2
H3); m/z (ESI) 422.88 (M + H+); anal. (C25H27NO5) calc. C
9
◦
1
powder; mp 87–89 C; H NMR (DMSO-d6)
δ
H
H
δ
C
C
71.24, H 6.46, N 3.32; found C 70.95, H 6.41, N 3.31.
Diethyl N-(2-chlorophenyl)-4-(3,4-dimethoxyphenyl)-1,4-dihydropyridine-3,5-dicarboxylate (10). Yield 20%,
◦
1
white powder; mp 106–108 C; H NMR (DMSO-d6)
7.63–7.61 (m, 1H, 3- or 6-H of 2-Cl-Ph), 7.51–7.45 (m, 2H, 4-, 5-H of 2-Cl-Ph), 7.32 (s, 2H, 2-, 6-H),
6.88–6.85 (m, 3H, 2-, 5-, 6-H of 3,4-(CH3O)2Ph), 4.73 (s, 1H, 4-H), 4.07–3.96 (m, 4H, COOC 2CH3), 3.70,
3.69 (2 s, 6H, 3,4-(CH3O)2), 1.13–1.10 (m, 6H, COOCH2C
3); 13C NMR (100 MHz, DMSO-d6)
166.4 (CO), 149.7, 146.8, 144.1, 138.7, 135.5, 132.2, 130.7, 127.6, 125.4, 123.8, 122.3, 114.1, 112.3, 108.1
δ = 7.68–7.66 (m, 1H, 3- or 6-H of 2-Cl-Ph),
H
×
H
δ =
(aromat. C, 2-, 6-, 3-, 5-C), 60.4 (OCH2CH3), 55.9, 55.8 (OCH3), 37.8 (4-C), 18.0 (CH3), 14.1 (OCH2CH3);
m/z (ESI) 472.88 (M + H+); anal. (C25H26ClNO6) calc. C 63.63, H 5.55, N 2.97; found C 63.53, H 5.49, N
2.91.
Diethyl 4-(3,4-dimethoxyphenyl)-N-(o-tolyl)-1,4-dihydropyridine-3,5-dicarboxylate (11). Yield 35%, yellow
◦
1
powder; mp 110–111 C; H NMR (DMSO-d6)
7.26 (s, 2H, 2-, 6-H), 6.88–6.81 (m, 3H, 2-, 5-, 6-H of 3,4-(CH3O)2Ph), 4.76 (s, 1H, 4-H), 4.07–3.95 (m,
4H, COOC 2CH3), 3.71, 3.69 (2 s, 6H, 3,4-(CH3O)2), 2.29 (s, 3 H, 2-CH3-Ph), 1.14–1.10 (m, 6H,
COOCH2C = 166.4 (CO), 149.3, 146.9, 144.3, 138.2, 135.83, 131.4,
3); 13C NMR (100 MHz, DMSO-d6)
129.8, 126.1, 123.4, 122.4, 122.0, 113.9, 112.1, 107.8 (aromat. C, 2-, 6-, 3-, 5-C), 60.6 (O H2CH3), 55.9, 56.1
(OCH3), 37.9 (4-C), 18.3 (CH3), 14.2 (OCH2
H3); m/z (ESI) 453.05 (M + H+); anal. (C26H29NO6) calc. C
δ = 7.41–7.34 (m, 4H, 3-, 4-, 5-, 6-H of 2-CH3Ph),
H
H
×
δ
C
C
69.16, H 6.47, N 3.10; found C 68.88, H 6.42, N 3.26.
Diethyl 4-(3-(benzyloxy)phenyl)-N-(2-chlorophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (12). Yield 10%,
◦
1
yellow powder; mp 106–110 C; H NMR (DMSO-d6)
δ = 7.68–7.66 (m, 1H, 3- or 6-H of 2-Cl-Ph),
7.63–7.62 (m, 1H, 3- or 6-H of 2-Cl-Ph), 7.51–7.45 (m, 2H, 4-, 5-H of 2-Cl-Ph), 7.42–7.40 (m, 2H, 2-, 6-H
of BnO), 7.38–7.34 (m, 2H, 3-, 5-H of BnO), 7.34 (s, 2H, 2-, 6-H), 7.32–7.29 (m, 1H, 4-H of BnO), 7.19
(t, J = 7.8 Hz, 1H, 5-H of 3-BnOPh), 6.96–8.94 (m, 2H, 2-, 6-H of 3-BnOPh), 6.83 (ddd, J = 7.8, 2.5, 1.1
Hz, 4-H of 3-BnOPh), 5.03 (s, 2H, Ph-O-C
1.12–1.10 (m, 6H, COOCH2C
3); 13C NMR (100 MHz, DMSO-d6)
138.4, 136.7, 132.1, 130.7, 129.6, 128.9, 127.7, 127.6, 127.1, 125.4, 123.8, 120.0, 113.1, 111.3, 107.8 (aromat.
H
2-Ph), 4.77 (s, 1H, 4-H), 4.06–3.95 (m, 4H, COOC
H2CH3),
H
δ
= 166.8 (CO), 160.5, 143.9, 143.8,
C, 2-, 6-, 3-, 5-C), 70.8 (OCH2), 60.9 (OCH2CH3), 55.9, 56.1 (OCH3), 38.1 (4-C), 14.2 (OCH2CH3); m/z
(ESI) 519.32 (M + H+); anal. (C30H28ClNO5) calc. C 69.56, H 5.45, N 2.70; found C 69.60, H 5.43, N 2.66.
Diethyl 4-(3-(benzyloxy)yphenyl)-N-(o-tolyl)-1,4-dihydropyridine-3,5-dicarboxylate (13). Yield 49%, yellow
powder; mp 117–118 ◦C; 1H NMR (DMSO-d6)
δ
= 7.43–7.28 (m, 9H, aromatic H of N-tolyl and OBn),
7.28 (s, 2H, 2-, 6-H), 7.20 (t, J = 8.1 Hz, 1H, 5-H of 3-BnO-Ph), 6.92–6.91 (m, 2H, 2-, 6-H of 3-BnO-Ph),
6.83 (ddd, J = 8.1, 2.4, 1.0 Hz, 1H, 4-H of 3-BnOPh), 5.04 (s, 2H, Ph-O-C 2-Ph), 4.80 (s, 1H, 4-H),
4.06–3.94 (m, 4H, COOC 2CH3), 2.27 (s, 3H, 2-CH3Ph), 1.12–1.09 (m, 6H, COOCH2C
3); 13C NMR
(100 MHz, DMSO-d6) = 166.7 (CO), 160.9, 144.2, 142.9, 138.3, 136.4, 130.9, 129.8, 129.3, 128.5, 127.3,
127.0, 126.1, 123.4, 122.6, 120.2, 112.8, 108.2 (aromat. C, 2-, 6-, 3-, 5-C), 71.2 (OCH2), 60.5 (O H2CH3),
37.6 (4-C), 18.0 (CH3), 14.2 (OCH2
H3); m/z (ESI) 497.71 (M + H+); anal. (C31H31NO5) calc. C 74.83, H
6.28, N 2.81; found C 74.86, H 6.36, N 2.90.
H
H
H
δ
C
C

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Diethyl N-(4-methoxyphenyl)-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate (14). Yield 63%, yellow
powder; mp 116–117 ◦C; 1H NMR (DMSO-d6)
= 7.50 (s, 2H, 2-, 6-H), 7.42–7.38 (m, 2H, 3-, 5-H of
4-CH3O-Ph), 7.27–7.23 (m, 4H, 2-, 3-, 5-, 6-H of 4-Ph), 7.16–7.12 (m, 1H, 4-H of 4-Ph), 7.04–7.00 (m, 2H,
2-, 6-H of 4-MeO-Ph), 4.78 (s, 1H, 4-H), 4.07–3.93 (m, 4H, COOC 2CH3), 3.77 (s, 3H, 4-MeO), 1.12–1.09
(m, 6H, COOCH2C = 167.2 (CO), 153.3, 144.4, 139.3, 133.5, 128.5,
3); 13C NMR (100 MHz, DMSO-d6)
δ
H
H
δ
127.7, 125.7, 123.5, 115.1, 108.5 (aromat. C, 2-, 6-, 3-, 5-C), 61.3 (OCH2CH3), 55.8 (OCH3), 38.3 (4-C), 14.3
(OCH2CH3); m/z (ESI) 408.64 (M + H+); anal. (C24H25NO5) calc. C 70.75, H 6.18, N 3.44; found C 70.39,
H 6.27, N 3.39.
Diethyl 4-(3-methoxyphenyl)-N-(4-methoxyphenyl)-1,4-dihydropyridine-3,5-dicarboxylate (15). Yield 53%,
yellow powder; mp 120–121 ◦C; 1H NMR (DMSO-d6)
δ = 7.50 (s, 2H, 2-, 6-H), 7.41–7.38 (m, 2H, 3-, 5-H
of 4-CH3O-Ph), 7.18 (t, J = 7.9 Hz, 1H, 5-H of 3-CH3O-Ph), 7.04–7.01 (m, 2H, 2-, 6-H of 4-CH3O-Ph),
6.84 (dt, J = 7.9, 1.2 Hz, 1H, 6-H of 3-CH3O-Ph), 6.77 (dd, J = 2.6, 1.2 Hz, 1H, 2-H of 3-CH3O-Ph), 6.73
(ddd, J = 7.9, 2.6, 1.7 Hz, 1H, 4-H of 3-CH3O-Ph), 4.76 (s, 1H, 4-H), 4.08–3.96 (m, 4H, COOC
3.77 (s, 3H, 4-CH3O), 3.69 (s, 3H, 3-CH3O), 1.14–1.11 (m, 6H, COOCH2C
3); 13C NMR (100 MHz,
DMSO-d6) = 167.2 (CO), 160.5, 153.2, 143.2, 138.9, 133.5, 129.6, 123.3, 120.0, 115.3, 113.1, 111.3, 108.1
H2CH3),
H
δ
(aromat. C, 2-, 6-, 3-, 5-C), 61.7 (OCH2CH3), 55.8 (OCH3), 38.2 (4-C), 14.2 (OCH2CH3); m/z (ESI) 438.44
(M + H+); anal. (C25H27NO6) calc. C 68.64, H 6.22, N 3.20; found C 68.14, H 6.17, N 3.17.
Diethyl 1,4-bis(4-methoxyphenyl)-1,4-dihydropyridine-3,5-dicarboxylate (16). Yield 56%, yellow powder; mp
120–121 ◦C; 1H NMR (DMSO-d6)
δ
= 7.48 (s, 2H, 2-, 6-H), 7.41–7.38 (m, 2H, 3-, 5-H of 4- CH3O-N-Ph),
7.17-7.14 (m, 2H, 2-, 6-H of 4-CH3O-4-Ph), 7.04–7.01 (m, 2H, 2-, 6-H of 4-CH3O-N-Ph), 6.82–6.79 (m, 2H,
3-, 5-H of 4-CH3O-4-Ph), 4.71 (s, 1H, 4-H), 4.07–3.95 (m, 4H, COOC 2CH3), 3.77 (s, 3H, 4-CH3O-N-Ph),
3.68 (s, 3H, 4-CH3O-4-Ph), 1.13–1.11 (m, 6H, COOCH2C = 167.0
3); 13C NMR (100 MHz, DMSO-d6)
(CO), 157.6, 153.3, 136.7, 139.4, 133.2, 130.0, 123.5, 115.1, 114.2, 108.1 (aromat. C, 2-, 6-, 3-, 5-C), 61.4
(O
H2CH3), 55.8 (OCH3), 38.3 (4-C), 14.1 (OCH2CH3); m/z (ESI) 438.15 (M + H+); anal. (C25H27NO6)
H
H
δ
C
calc. C 68.64, H 6.22, N 3.20; found C 68.35, H 6.04, N 3.13.
Diethyl 4-(3,4-methoxyphenyl)-N-(4-methoxyphenyl)-1,4-dihydropyridine-3,5-dicarboxylate (17). Yield 51%,
yellow powder; mp 138–139 ◦C; 1H NMR (DMSO-d6)
δ = 7.49 (s, 2H, 2-, 6-H), 7.41–7.38 (m, 2H, 3-, 5-H
of 4-CH3O-N-Ph), 7.04–7.01 (m, 2H, 2-, 6-H of 4-CH3O-N-Ph), 6.83 (d, J = 1H, 5-H of 3,4-CH3O-Ph),
6.81 (d, J = 2.1 Hz, 1H, 2-H of 3,4-CH3O-Ph), 6.75 (dd, J = 8.3, 2.1 Hz, 1H, 6-H of 3,4-CH3O-Ph), 4.71 (s,
1H, 4-H), 4.08–3.97 (m, 4H, COOC
1.15–1.12 (m, 6H, COOCH2C
3); 13C NMR (100 MHz, DMSO-d6)
138.5, 135.5, 133.5, 123.5, 122.3, 115.3, 114.1, 112.3, 108.3 (aromat. C, 2-, 6-, 3-, 5-C), 61.4 (O
55.8 (OCH3), 38.5 (4-C), 14.2 (OCH2
H3); m/z (ESI) 468.52 (M + H+); anal. (C26H29NO7) calc. C 66.80,
H 6.25, N 3.00; found C 66.40, H 6.14, N 2.75.
H
2CH3), 3.77 (s, 3H, 4-CH3O-N-Ph), 3.68 (s, 6H, 3,4-CH3O-4-Ph),
= 167.1 (CO), 153.4, 149.7, 146.8,
H2CH3),
H
δ
C
C
Diethyl 4-(3-(benzyloxy)yphenyl)-N-(4-methoxyphenyl)-1,4-dihydropyridine-3,5-dicarboxylate (18). Yield 55%,
yellow powder; mp 127–128 ◦C; 1H NMR (DMSO-d6)
= 7.49 (s, 2H, 2-, 6-H), 7.41–7.37 (m, 4H, 3-, 5-H
δ
of 4-CH3O-Ph and 2-, 6-H of OBn), 7.36–7.32 (m, 2H, 3-, 5-H of OBn), 7.31–7.27 (m, 1H, 4-H of OBn),
7.19–7.16 (m, 1H, 5-H of 3-BnOPh), 7.04–7.01 (m, 2H, 2-, 6-H of 4-CH3O-Ph), 6.87–6.80 (m, 3H, 2-, 4-,
6-H of 3-BnOPh), 5.03 (s, 2H, Ph-O-CH2-Ph), 4.76 (s, 1H, 4-H), 4.07–3.96 (m, 4H, COOC
3H, CH3O), 1.13–1.11 (m, 6H, COOCH2C = 167.2 (CO), 160.5,
3); 13C NMR (100 MHz, DMSO-d6)
153.1, 143.2, 139.3, 136.7, 133.5, 129.6, 128.9, 127.6, 127.0, 123.3, 120.0, 115.1, 113.1, 111.2, 107.9 (aromat.
H2CH3), 3.77 (s,
H
δ
C, 2-, 6-, 3-, 5-C), 70.6 (OCH2Ph), 61.7 (OCH2CH3), 55.8 (OCH3), 38.5 (4-C), 14.2 (OCH2CH3); m/z (ESI)
514.51 (M + H+); anal. (C31H31NO6) calc. C 72.50, H 6.08, N 2.73; found C 72.32, H 6.08, N 2.65.
3.3. ABCB1 Inhibitory Activity
Two cell lines, a mouse T-lymphoma parental cell line L5178Y and an ABCB1 expressing subline
L5178Y mdr, which resulted from retrovirus-mediated gene transfection, were cultured in McCoy’s
5A medium which was supplemented with 10% calf serum and l-glutamine (2 mM) at 37 ◦C under

Molecules 2019, 24, 2873
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a carbon dioxide atmosphere (5%). The ABCB1 expressing subline was cultured under addition of
colchicine in a concentration of 60 ng/mL to ensure a survival of only ABCB1-overexpressing cells. The
cell suspensions were diluted three times a week with fresh medium in a relation of 1 to 20.
Both cell lines were adjusted to a number of one million cells per mL. After centrifugation at 2000
rpm the upper layer was removed and the remaining cells were resuspended in medium. Each 0.5 mL
of the cell suspensions were placed in an Eppendorf tube and supplemented with the inhibitor from
stock solutions to reach a final concentration of 1
µM for testing. Incubation followed for 20 min. Then
0.5 L of a rhodamine 123 stock solution (0.5 mM) was added and incubation was continued for an
µ
additional 40 min. Then centrifugation followed again at 2000 rpm. The medium was removed and
the cell suspension was washed with phosphate-buffered saline (PBS)buffer twice at a pH of 7.4. Then
the fluorescence was measured in both cell lines and the fluorescence values were each related to the
fluorescence of the untreated control to give the final FAR value by relating both corrected fluorescence
values of the ABCB1-overexpressing cell line and the parental cell line. The determination of the FAR
value followed the equation:
MDR treated/MDR untreated control
FAR = Parental treated/Parental untreated control
3.4. Mtb Growth Inhibition Assay
For growth analyses green fluorescence-expressing Mtb H37Rv bacteria were used as described [24].
In short details 2
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.) sealed with an air-permeable
membrane (Porvair Sciences). Direct bacterial growth was measured under compound concentrations
of 1 g/mL similar to earlier cited studies where higher concentrations have been used [ ]. IC50
values could not be determined due to occurring problems with the compound solubility at the higher
concentrations. In the drug-toxicity enhancing studies a clofazimine concentration of 0.3 g/mL and a
combination of clofazimine (0.3 g/mL) and the respective 1,4-dihydropyridine (1 g/mL) were used.
×
106 bacteria were cultured in 7H9 medium supplemented with 10% oleic
µ
µ
7
µ
µ
µ
The bacterial growth was measured 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).
4. Conclusions
The number of drugs used in antituberculotic therapies is strongly limited. Moreover, drugs used
in case of an MDR treatment of Mtb suffer from low efficacy, part toxicity and a long duration with
a limited successful outcome. Therefore, there is a need for novel antituberculotic drugs and also
drugs that may enhance the activity of drugs currently in use. This is a challenge because presently
drugs that have been investigated cannot be used in therapy because of their own pharmacological
effects. We identified nonclassical 1,4-dihydropyridines as novel dually acting compounds with
antituberculotic activities and drug toxicity enhancing activities. Favourable substitution patterns of
the 1,4-dihydropyridine scaffold have been 2-toly substituents at the nitrogen phenyl substituent and a
methoxy function placed in both aryl substituents. However, for a more detailed structure–activity
discussion the target structure has to be identified in future studies. In case of antibacterial drug
development, the target structures are mostly unclear in the early state as in the case of the recently
discovered lipolanthines [25]. The determined ABCB1 inhibitory activities promised effects on a
mycobacterial efflux pump similar to ABCB1. Therefore, compounds with varying ABCB1 activities
were evaluated to enhance the antituberculotic activities of clofazimine as ABCB1 substrate that may
be transported by a mycobacterial efflux pump related to ABCB1. The best results as enhancers were
observed for those compounds with the best ABCB1 affinities and with combined antituberculotic

Molecules 2019, 24, 2873
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and ABCB1 inhibiting properties. Perspective lead compounds have been identified which prove the
principle of an effective dual activity to combat Mtb and Mtb drug resistance.
Supplementary Materials: The supplementary materials are available online, Spectra S1: 1H NMR spectra.
Author Contributions: F.L. performed the synthesis; N.R. was responsible for the antibacterial testing; G.S.,
A.K., A.C. and J.M. managed the determination of the ABCB1 inhibiting activities; A.H. designed the synthetic
experiments and wrote the paper.
Funding: We acknowledge the support of the Szeged Foundation for Cancer Research and the financial support
within the funding program Open Access Publishing by the German Research Foundation (DFG).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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