Discovery of novel N-phenyl 1,4-dihydropyridines with a dual mode of antimycobacterial activity

Article abstract of DOI:10.1016/j.bmcl.2016.11.010

There is an urgent need for novel drugs for the treatment of tuberculosis (TB) due to the increasing prevalence of antibiotic resistance among Mycobacterium tuberculosis (Mtb) strains against first-line and second-line therapeutics. We developed novel N-phenyl 1,4-dihydropyridines as potential antituberculotic agents. The observed activity depends on the substitution patterns of the aromatic residues. N-unsubstituted 1,4-dihydropyridines are known inhibitors of the cancer-relevant transmembrane efflux pump ABCB1. Based on the similarity of ABCB1 amino acids sequences relevant to 1,4-dihydropyridine binding and the MTb efflux pump Rv0194, we determined ABCB1-inhibitory properties of our compounds in a cell line model. We identified one compound, which substantially increased the activity of two antituberculotic drugs which are substrates of ABCB1. The data indicate that our N-phenyl 1,4-dihydropyridines represent a novel compound class which improves the efficacy of anti-TB drugs by interfering with transmembrane efflux pumps in Mtb.

Full text of DOI:10.1016/j.bmcl.2016.11.010

Accepted Manuscript
Discovery of novel N-phenyl 1,4-dihydropyridines with a dual mode of anti-
mycobacterial activity
Fabian Lentz, Marc Hemmer, Norbert Reiling, Andreas Hilgeroth
PII:
S0960-894X(16)31150-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.11.010
BMCL 24405
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
6 September 2016
3 November 2016
4 November 2016
Please cite this article as: Lentz, F., Hemmer, M., Reiling, N., Hilgeroth, A., Discovery of novel N-phenyl 1,4-
dihydropyridines with a dual mode of antimycobacterial activity, Bioorganic & Medicinal Chemistry Letters (2016),
doi: http://dx.doi.org/10.1016/j.bmcl.2016.11.010
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1
Discovery of novel N-phenyl 1,4-dihydropyridines with a dual mode of
antimycobacterial activity
a
a
b
a,
Fabian Lentz , Marc Hemmer , Norbert Reiling , Andreas Hilgeroth *
a
Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-
Str. 4, 06120 Halle, Germany
b
Research Center Borstel, Leibniz Center for Medicine and Biosciences, Parkallee 22, 23845
Borstel, Germany
Abstract – There is an urgent need for novel drugs for the treatment of tuberculosis (TB)
due to the increasing prevalence of antibiotic resistance among Mycobacterium tuberculosis
(
Mtb) strains against first-line and second-line therapeutics. We developed novel N-phenyl
,4-dihydropyridines as potential antituberculotic agents. The observed activity depends on
1
the substitution patterns of the aromatic residues. N-unsubstituted 1,4-dihydropyridines are
known inhibitors of the cancer-relevant transmembrane efflux pump ABCB1. Based on the
similarity of ABCB1 amino acids sequences relevant to 1,4-dihydropyridine binding and the
MTb efflux pump Rv0194, we determined ABCB1-inhibitory properties of our compounds in
a cell line model. We identified one compound, which substantially increased the activity of
two antituberculotic drugs which are substrates of ABCB1. The data indicate that our N-
phenyl 1,4-dihydropyridines represent a novel compound class which improves the efficacy
of anti-TB drugs by interfering with transmembrane efflux pumps in Mtb.
_
______________
Keywords: 1,4-dihydropyridines, ABCB1 inhibitor, biological activity data, antituberculotic
drugs

2
*
Corresponding author. Tel. +49 345 55 25168; fax +49 345 55 27207; E-mail address:
andreas.hilgeroth@pharmazie.uni-halle.de (A. Hilgeroth).
1
In 2015 Tb ranked alongside HIV as a leading cause of death worldwide. More than 95%
of TB deaths occur in low- and middle-income countries. TB is a leading killer of HIV-
infected individuals. In 2015, 1 in HIV deaths was due to TB. The risk of progressing from
latent to active TB is estimated to be between 12 and 20 times greater in people living with
2
-4
HIV than among those without HIV infection.
The standard therapy of Tb usually consists of a six-month drug regimen using the four
antibiotics isoniazid, rifampicin, ethambutol and pyrazinamide, all of them considered as first-
line anti-TB drugs. Strains resistant against one of the used drugs are treated with second-line
5
-8
antibiotics like fluoroquinolones. However, these compounds are more expensive, less
9
,10
effective and are often known to have toxic side-effects.
Therapy failure of a second-line
drug therapy is often critical. Although third-line therapeutic drugs like clofazimine are
known, their common use is limited due to insufficient experiences in their application and
5
with their mode of action in the Tb progression.
Antibiotic resistance of Mtb strains mediated through chromosomal mutations under
selective pressure of antibiotic use, lead to structural changes in antituberculotic drug target
11
structures. In addition also efflux pumps have been shown to be involved in the natural
resistance against anti-TB drugs. Sixteen Mtb efflux pumps have been identified to contribute
1
2
to such resistance developments. Specific inhibitors of TB efflux pumps are not known to
date. However, non-specific inhibitors of the cancer-relevant transmembrane efflux pump
ABCB1 have been investigated as potential inhibitors of drug resistance-relevant efflux
1
3
pumps in bacteria like Staphylococcus aureus.
We developed novel N-phenyl 1,4-dihydropyridines as potential antituberculotic agents
with ABCB1-inhibiting properties. Structure-activity properties of the novel compounds will

3
be discussed. The effect of ABCB1-related transmembrane efflux pump inhibition in Mtb will
be demonstrated with an increased activity of antituberculotic second- and third-line drugs.
Our N-phenyl-1,4-dihydropyridine target compounds 1a-h resulted from a simple one-pot
reaction in 1 mL of freshly distilled acetic acid at 100 °C for 1-2 hours using five equivalents
of a structurally varied aromatic aldehyde and an aniline compound with changed substitution
1
4
patterns and ten equivalents of ethyl propiolate.
R1
R2
R1
R2
O
O
H
O
O
O
O
O
O
O
a
+
N
NH2
R3
R
1a - h
Scheme 1. Reagents and conditions for compounds 1a-h: (a) HAc, 1-2 h, 100 °C; yields: 3-57%.
The antituberculotic activity of the compounds was measured using Mtb strain H37Rv
1
5
expressing green fluorescent protein (GFP). The extent of measured fluorescence correlates
with the bacterial growth.
The results of the MTb growth inhibition of the compounds 1a-h are shown in table 1. The
4
-phenyl and N-phenyl substituted compound 1a showed a growth inhibition of 57%. When a
methoxy group is introduced into the 4-phenyl residue the activity is lowered to 25% for the
respective compound 1b. The introduction of a methoxy function into the 4-position of the N-

4
phenyl residue slightly increases the activity of derivative 1c. A methoxy function in the 3-
position of the 4-phenyl residue leads to an improved growth inhibition of 61% for compound
1
d that makes about 63% of the isoniazid activity determined at the given concentration.
Two methoxy functions both in the 4-position of the phenyl residues may combine the
favourable effect of the N-4-methoxyphenyl function with the less favourable effect of the 4-
4-methoxyphenyl) residue reaching an inhibition growth of 35% for compound 1e.
(
Table 1. Antituberculotic activity and ABCB1-inhibiting properties of target compounds 1a-
h.
_
__________________________________________________________________________
[a]
[a]
grow inhibition [%]
1 µg/mL
FAR value
1
2
3
Cpd.
R
R
R
1 µM
_
1
1
1
1
1
1
1
1
__________________________________________________________________________
a
b
c
d
e
f
H
H
H
57
25
60
61
35
41
30
60
97
n.d.
4.3
5.8
3.9
1.1
1.2
3.4
7.3
5.2
n.d.
1.4
OMe
H
H
H
H
OMe
H
H
OMe
H
OMe
H
OMe
OMe
H
OMe
g
h
OMe OMe
OMe OMe
OMe
[b]
isoniazid
[b]
verapamil
_
__________________________________________________________________________
Mean of three determinations.
[a]
[b]
n.d., not determined.

5
When the methoxy function of the 4-phenyl residue moves to the 3-position the activity of
the respective compound 1f increases to give a growth inhibition of 41%. That result reveals
the favourable effect of the 3-methoxy function as shown for compound 1d.
If both methoxy function are concentration on the 4-phenyl residue we found a growth
inhibition of 30% for compound 1g. This may be explained with the observed unfavourable
effect of the 4-methoxyphenyl function of derivative 1b and the favourable effect of the 3-
methoxyphenyl function of compound 1d.
If we introduce a third methoxy function into the favourable 4-position of the N-phenyl
residue we found a growth inhibition of 60% for compound 1h.
Early 1,4-dihydropyridines have been identified as modulators of the cancer-relevant
1
7,18
transmembrane efflux pump ABCB1.
However, ABCB1 has not been found in bacteria,
but transmembrane efflux pumps have been identified to contribute to antibacterial drug
resistance. We used the BLASTp suite to identify similar amino acid sequences in ABCB1
1
9
and known efflux pump proteins of Mtb. Highest homology of 46 and 43% between
ABCB1 and Mtb encoded transmembrane efflux pump Rv1272c were found in the amino
acid regions of the nucleotide binding domains of ABCB1. Next hit of close amino acid
sequences was found for the transmembrane efflux pump Rv0194 with a 30% homology in
the region of the first transmembrane segments of ABCB1 and the following first nucleotide
binding domain and with 46% of the amino acid region of the second binding domain.
Early cross-linking studies with ABCB1 in HEK 293 cells suggested a binding of 1,4-
dihydropyridines to amino acids of the transmembrane segment TM6 of ABCB1 with the
2
0
protein amino acids 331 to 351. Recent studies based on the mouse ABCB1 structure
confirmed a binding of 1,4-dihydropyridines to amino acid residues of TM6 and to TM12 of
2
1
amino acids 974 to 994 both in the binding belt region.
As no 1,4-dihydropyridine binding to the nucleotide binding region has been found, we
may suggest Rv0194 as potential target structure from our protein alignment study with the

6
homology of the amino acid sequence that includes the amino acid sequence of the
transmembrane domain TM6 of ABCB1 as 1,4-dihydropyridine binding region.
Next we characterized our N-phenyl 1,4-dihydropyridines as ABCB1 inhibitors in a
ABCB1 expressing cell line model. We used a mouse T-lymphoma cell line and an ABCB1-
expressing subline as model to investigate the ABCB1-modulating activities of our N-phenyl
1
,4-dihydropyridines. The ABCB1 subline resulted from a retroviral gene transfection with
the human MDR1 gene so that both cell lines just differ in the expression of the human
ABCB1 efflux pump. Rhodamine 123 was used as fluorescent substrate of ABCB1. The
ABCB1-expressing cells accumulate less of this ABCB1 substrate than the non-expressing
cells because the substrate undergoes a cellular efflux by the efflux pump activity. The
fluorescence amounts of both cell lines were determined by flow cytometry. The amounts
were expected to be increased in the ABCB1-expressing cells upon addition of a putative
ABCB1 inhibitory compound due to a reduced transport of the fluorescent rhodamin 123 out
of the cells. The measured fluorescence intensity amounts in both cell lines under modulator
application were each related to those of the untreated control cells and a fluorescence activity
ratio (FAR) was calculated by relating the control-corrected fluorescences of the modulator-
2
2
treated ABCB1-expressing cells to that of the non-expressing cells. Thus, only compounds
with a FAR value > 1.1 can be regarded as active ABCB1 modulators. The resulting FAR
values are displayed in table 1.
Compound 1a with a FAR value of 4.3 was more active than the used verapamil standard
with a FAR value of 1.4. The 4-(4-methoxyphenyl) derivative 1b showed an increased activity
with a FAR value of 5.8. When the 4-methoxy function was placed in the N-phenyl residue of
compound 1c the activity decreased. The 4-(3-methoxyphenyl) compound 1d was practically
not active with a determined value of 1.1. If we combine the unfavourable 3-methoxyphenyl
function of derivative 1d with the more favourable 4-methoxy function of the N-phenyl
residue in resulting compound 1f we found a mixed activity with a FAR value of 3.4. The

7
both 4-methoxyphenyl substituted derivative 1e showed mainly decreased activity. If both
methoxy functions are concentrated on the 4-phenyl residue in derivative 1g the activity
mainly increased to a FAR value of 7.3. A third methoxy function introduced in derivative 1h
lowered the activity with a FAR value of 5.2.
With the determined ABCB1 inhibiting activities and the discovered homology of ABCB1
and the respective transmembrane efflux pump of Rv0194 as possible target structure, we
next investigated a possible Mtb efflux pump-inhibiting effect of our novel compounds on the
antibacterial activity of antituberculotic drugs as discussed substrates of ABCB1 und, thus, of
a related Mtb efflux pump. Such drugs should show a reduced activity against Mtb because
they are transported out of the bacteria by the respective Mtb efflux pump activity.
We selected ciprofloxacin, a second-line anti-Mtb drug, since recent studies in cancer cells
2
3
suggested ciprofloxacin as a substrate of ABCB1. In addition we choose clofazimine, a
third-line therapeutic agent with has been used with success in a short, 9-month combined
2
4, 25
drug regimen for the treatment of multidrug resistance (MDR)-TB.
Having determined the growth inhibitory activitiy of ciprofloxacin and clofazimine against
Mtb we chose suboptimal concentrations of 0.4 µg/mL for ciprofloxacin resulting in a grow
inhibition of 19%, and 0.3 µg/ml for clofazimine observing a growth inhibition of 39% (table
2
). We chose compound 1g from our novel compound class to investigate possible effects as
ABCB1 and suggested MTb efflux pump inhibitor that may increase toxic effects of the used
antituberculotic drugs. Compound 1g showed lowest antituberculotic activity at the measured
concentration, but best ABCB1-inhibiting properties in our cellular ABCB1 assay system
(
table 1). We combined compound 1g with the respective concentrations of the chosen
antituberculotic drugs to investigate possible effects of a MTb efflux pump inhibition. We
used a further lowered concentration of only 0.3 µg/mL of compound 1g to minimize the
residual antituberculotic activity which could be determined with a growth inhibition of less
than 10% at 0.5 µg/mL.

8
Table 2. Antituberculotic activity of used antituberculotic drug concentrations without and
with used compound 1g.
_
__________________________________________________________________________
growth inhibition [%]
Drug
drug alone
combination with 1g (0.3 µg/mL)
_
__________________________________________________________________________
ciprofloxacin (0.4 µg/mL)
clofazimine (0.3 µg/mL)
19
39
46
60
_
__________________________________________________________________________
The combination of ciprofloxacin with compound 1g led to a Mtb growth inhibition of
4
6%. That meant an almost two and a halffold increase in the growth inhibition of 27%. A
combination of clofazimine with compound 1g resulted in a Mtb growth inhibion of 60%.
Thus, the antituberculotic activity of both antituberculotic drugs if combined with compound
1
g was substantially increased, when compound 1g was used in a concentration that could not
contribute to the growth inhibition activity as demonstrated.
Taken together N-phenyl 1,4-dihydropyridines represent a novel class of antituberculotic
compounds that lead to Mtb growth inhibition effects depending on the aromatic substitution
pattern. In addition, with demonstrated ABCB1 and suggested Mtb efflux pump inhibiting
effects a substantial increase of the Mtb growth inhibition was demonstrated for ciprofloxacin
and clofazimine as efflux pump substrates when combined with one of our compounds.
It remains a challenge to further increase both compound activities to provide a
perspective drug for combination therapy regimes that may lower the observed resistance
developments partly due to relevant efflux pumps in Mtb.

9
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1
0
1
4. Spectroscopical data of substituted N-phenyl 4-phenyl-1,4-dihydropyridine compound
1
class representing derivative 1g: Yield 23%; mp 59-65 °C; H NMR (400, MHz,
DMSO-D
.11- 3.97 (m, ABX
Hz, 6H, COOCH CH
6
)  = 7.61 (s, 2H, 2-, 6-H), 7.49-6.73 (m, 8H, aromatic H), 4.73 (s, 1H, 4-H),
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, 4H, COOCH
2
CH
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+
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); m/z (ESI) 460 (100, M+Na ). Elemental Anal. Calcd. (%) for
C
25
H27NO
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1
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1

1
2
R1
R2
O
O
O
O
N
R3
1
a - h