Novel thiazolidinedione-hydroxamates as inhibitors of Mycobacterium tuberculosis virulence factor Zmp1

Article abstract of DOI:10.1016/j.ejmech.2019.111812

Zinc metalloprotease 1 (Zmp1) is an extracellular enzyme, which has been found essential for the intracellular survival and pathogenesis of Mycobacterium tuberculosis. In this work, we designed and synthesized a series of novel thiazolidinedione-hydroxamates and evaluated in silico their drug-likeness behavior. Then, their inhibitory properties towards a recombinant Zmp1 from Mycobacterium tuberculosis were analyzed by MALDI-TOF MS. Nine of the tested compounds were found to inhibit the enzymatic reaction more effectively than the generic metalloprotease inhibitor phosphoramidon. Furthermore, the synthesized thiazolidinedione-hydroxamate hybrids were evaluated for their in vitro antimycobacterial activity and acute cytotoxicity using whole-cell assays. Results showed that none of the hybrids exhibited acute cytotoxicity against RAW264.7 macrophages. Whereas extracellular antimycobacterial activity was limited, RAW264.7 macrophage infection results showed that a majority of the hybrids inhibited the intracellular growth of Mycobacterium tuberculosis at a concentration of 100 and 10 μM. The thiazolidinedione-hydroxamate compound 2n was considered to be the best candidate of the evaluated library.

Full text of DOI:10.1016/j.ejmech.2019.111812

European Journal of Medicinal Chemistry xxx (xxxx) xxx
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel thiazolidinedione-hydroxamates as inhibitors of Mycobacterium
tuberculosis virulence factor Zmp1
b
c
c
c
ꢁ a
ꢀ
ꢀ
d
Veronika Slachtova , Marek Sebela , Eveline Torfs , Lauren Oorts , Davie Cappoen ,
d
, *
ꢁ a
ꢁ
Karel Berka , Vaclav Bazgier , Lucie Brulíkova
a Department of Organic Chemistry, Faculty of Science, Palacký University, 17. listopadu 12, 771 46, Olomouc, Czech Republic
b
ꢁ
Department of Protein Biochemistry and Proteomics, Centre of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science, Palacký
ꢀ
ꢂ
University, Slechtitelu 27, 783 71, Olomouc, Czech Republic
c Laboratory of Microbiology, Parasitology and Hygiene (LMPH), S7, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp,
Wilrijk, Belgium
d Department of Physical Chemistry, Regional Centre of Advanced Technologies and Materials, Faculty of Science, Palacky University, 17. listopadu 12, 771
46, Olomouc, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
Zinc metalloprotease 1 (Zmp1) is an extracellular enzyme, which has been found essential for the
intracellular survival and pathogenesis of Mycobacterium tuberculosis. In this work, we designed and
synthesized a series of novel thiazolidinedione-hydroxamates and evaluated in silico their drug-likeness
behavior. Then, their inhibitory properties towards a recombinant Zmp1 from Mycobacterium tuberculosis
were analyzed by MALDI-TOF MS. Nine of the tested compounds were found to inhibit the enzymatic
reaction more effectively than the generic metalloprotease inhibitor phosphoramidon. Furthermore, the
synthesized thiazolidinedione-hydroxamate hybrids were evaluated for their in vitro antimycobacterial
activity and acute cytotoxicity using whole-cell assays. Results showed that none of the hybrids exhibited
acute cytotoxicity against RAW264.7 macrophages. Whereas extracellular antimycobacterial activity was
limited, RAW264.7 macrophage infection results showed that a majority of the hybrids inhibited the
Received 2 May 2019
Received in revised form
7 August 2019
Accepted 23 October 2019
Available online xxx
Keywords:
Zmp1
Mycobacterium tuberculosis
Thiazolidinediones
Hydroxamates
MALDI-TOF
intracellular growth of Mycobacterium tuberculosis at
a concentration of 100 and 10 mM. The
thiazolidinedione-hydroxamate compound 2n was considered to be the best candidate of the evaluated
library.
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction
resistant (XDR) TB. Additionally, treatment is less efficient and
associated with serious side effects [3]. Without doubt, the dis-
Tuberculosis (TB), an infectious disease predominantly caused
by the bacillus Mycobacterium tuberculosis (Mtb) still poses a major
and enduring global health threat [1]. The infectious disease re-
mains one of the top 10 causes of death and is, additionally, the
leading cause of death due to a single infectious agent. In 2017,
approximately 10 million people developed active TB disease and
over 1.6 million persons died as a result [2]. Furthermore, the
epidemic is fueled by the current HIV/AIDS pandemic and ever-
increasing emergence of anti-TB drug resistance [2]. Moreover,
there is only a limited number of drugs available on the market for
the treatment of multidrug-resistant (MDR) and extensively drug-
covery of entirely new compounds with an alternative mechanism
of action to the existing therapeutics and directed towards un-
known targets of Mtb becomes more and more desirable [4,5].
One attractive mycobacterial target is represented by the
promising and validated 20S proteasome protein, which is neces-
sary for persistence in mice and vitality of the bacterium in path-
ogenesis as it protects Mtb against nitrosative stress from the host
[6]. More recently, another interesting mycobacterial enzyme with
protease activity was identified, i.e. zinc metalloprotease 1 (Zmp1).
The enzyme is found to be essential for the intracellular survival
and pathogenesis of Mtb [7e11]. In 2008, Master et al. illustrated
that Zmp1 affects the macrophage phagosome maturation via
suppression of inflammasome activation and subsequent phag-
olysosome formation, desired for the full clearance of the invalid
pathogens (Fig. 1). The exact action mechanism of Zmp1 in the
* Corresponding author.
ꢁ
E-mail address: lucie.brulikova@upol.cz (L. Brulíkova).
https://doi.org/10.1016/j.ejmech.2019.111812
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.
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Please cite this article as: V. Slachtova et al., Novel thiazolidinedione-hydroxamates as inhibitors of Mycobacterium tuberculosis virulence factor
Zmp1, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2019.111812

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V. Slachtova et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
2
the N-terminal threonine residue of each
b-subunit [13]. The
arrangement of the substrate binding pocket in M. tuberculosis 20S
proteasome allows versatility in degrading of hydrophobic, basic as
well as acidic peptide substrates [12]. The enzyme shows not only a
different mechanism but also substrate and cleavage specificity
compared to Zmp1. Thus, it is clear that the designing of new in-
hibitors of these two enzyme targets has to follow unique princi-
ples and approaches.
To date, only a few examples of Zmp1 inhibitors are known
[14e18]. In 2014, the first selective Mtb Zmp1 inhibitors emerged,
when the Botta group identified active structures by combining an
in silico structure-based inhibitor design and biochemical studies
(Fig. 2) [14]. Most of the defined structures in their study comprised
the rhodanine skeleton that further served as a basic structural
motif for the development of other Zmp1 inhibitors [15e18]. The
most potent Zmp1 inhibitor to date was identified in the latest
study from 2018 [16] when Paolino et al. described a series of 8-
hydroxyquinolines modified with a hydroxamate substitution as
the zinc-binding group.
Fig. 1. Inhibition of the phagosome maturation process by mycobacterial Zmp1 (this
figure was adopted from Ferraris and Rizzi) [11].
In the present study, a protocol for synthetic preparation of
thiazolidinedione-hydroxamates was developed. All synthesized
compounds were evaluated for their antimycobacterial activity. In
addition, the inhibitory properties of the studied compounds to-
wards a recombinant Zmp1 from Mycobacterium tuberculosis were
analyzed by MALDI-TOF MS.
pathogenesis, however, has not been fully elucidated. Though,
there is evidence that the zinc metalloprotease, a type of enzyme
which can carry out diverse functions in pathogenic microorgan-
isms, may be released from Mtb and act on the activation of the
caspase-1/interleukin-1
b inflammasome, ultimately disrupting
phagolysosome formation and Mtb clearance [8]. Therefore, Zmp1
and its inhibition leading to virulence attenuation may also
represent a potentially useful drug target and are worthy of further
investigation.
The molecular structures of the mycobacterial 20S proteasome
and Zmp1 are markedly different. Unlike the monomeric Zmp1 [7],
2. Results and discussion
Based on the biological significance of both thiazolidinedione
scaffold [19e21] and known hydroxamate-based zinc-binding
20S proteasome is a large barrel-shaped oligomer: 7
a-subunits are
arranged into an -ring and 7 -subunits form a -ring. These rings
a
b
b
group (ZBG) [22,23],
a new library of thiazolidinedione-
are stacked to a symmetric complex of a 1-7 b 1-7 b 1-7 a 1-7 [12]. The
active site of Zmp1 appears between the two structural domains
hydroxamate hybrids was designed and synthesized (Fig. 3). Two
lipophilic hydroxamates as privileged scaffolds in a multitude of
medicinally useful agents were selected for our initial studies
[23e25]. Thiazolidinedione heterocycle may further be modified by
various benzylidenes or alkylidenes according to various known
biologically active structures [19]. Furthermore, the thiazolidine-
dione and hydroxamate scaffolds are connected via an appropriate
linker R2, where two simple variants were selected for the initial
study.
rich in
a-helices and contains a catalytic zinc ion in tetrahedral
coordination. Because of the presence of hydrophobic residues,
peptide substrates with a large hydrophobic P1’ side chain (e.g. Phe,
Leu, Ile) are well accommodated. There is also a secondary binding
pocket available at the active site, with arginine residues, which has
been suggested
a promising docking site for inhibitors [7].
Conversely, the proteolytic mechanism of 20S proteasome involves
Fig. 2. Examples of known Zmp1 inhibitors and design of novel compounds.
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Please cite this article as: V. Slachtova et al., Novel thiazolidinedione-hydroxamates as inhibitors of Mycobacterium tuberculosis virulence factor
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V. Slachtova et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
3
NMR data confirmed the formation of Z isomers for all our products.
Moreover, the final compounds containing a stereogenic centre
were analyzed by supercritical fluid chromatography (SFC). All
analyses confirmed the racemic form of both mentioned derivatives
(See Supplementary).
3.1. In silico prediction of drug-likeness properties
To select those compounds suitable for further screening, in
silico physicochemical and pharmacokinetic parameters of the
thiazolidinedione-hydroxamates were predicted using the
knowledge-based FAFDrugs4 [29] and admetSAR [30] software
tools [6,31]. Narrowing the library to a series of compounds with a
favourable drug-likeness and no toxicity alerts avoids futile in-
vestment in compounds with possible undesirable effects in later
stages of the drug discovery and development cascade. For each
hybrid, the predicted drug-likeness parameters are shown in
Table 3, whereas ranges of such parameters followed by 95% of
known drugs are also added in the footnotes [32,33]. The results
revealed that the synthesized thiazolidinedione-hydroxamates
meet the Lipinski’s rule of five as the predicted values of the mo-
lecular weight, n-octanol, and water partition coefficient, hydrogen
bond acceptors and hydrogen-bond donors parameters fall within
the prescribed ranges. Furthermore, the number of atoms was also
found within the range, improving drug-likeness [29]. Aqueous
solubility was forecasted to be good for all thiazolidinedione-
hydroxamates, except for 2g, for which it was predicted to be
Fig. 3. Structures selected for the study.
3. Chemistry
First, simple thiazolidinedione-unsubstituted hydroxamates 1
were generated (Scheme 1). The initial thiazolidinedione 3 was
synthesized according to a previously described procedure [26] and
reacted with methyl bromoacetate or methyl 2-bromopropionate
giving compounds 4. This step was followed by the acid-
catalyzed hydrolysis resulting in carboxylic acids 5. The desired
compounds 1 were obtained after a coupling reaction of the car-
boxylic acids 5 with either O-allylhydroxylamine hydrochloride or
O-benzylhydroxylamine
hydrochloride
carbodiimide
using
hydrochloride
N-(3-
reduced with
a
logSw of ꢀ5.22. The thiazolidinedione-
dimethylaminopropyl)-N0-ethyl
hydroxamates were also considered to have a good oral bioavail-
ability, based on Verber’s rule [34]. All hybrids were designated to
class III considering acute oral toxicity, which reveals that the
compounds should possess fairly high lethal doses (i.e.
LD50 ¼ 0.5e5 g/kg) and can be considered druggable. As for muta-
genicity (Ames test), there were no alerts indicated. In contrast,
another rule of thumb such as the GSK 4/400 rule, which antici-
pates higher risks of toxicity, interactions with off-targets or diffi-
culties during development if the logP and the molecular weight is
larger than 4 and 400, respectively, suggested that 2e and 2g could
be less druggable [29]. Though, the overall results indicate that the
synthesized thiazolidinedione-hydroxamates generally do possess
drug-likeness behavior. Therefore, all derivatives are selected for
further investigation, except the compounds 2e and 2g.
(EDC.HCl) under aqueous conditions in moderate yields (see
Table 1).
Next, hydroxamates 2 were synthesized (Scheme 1). The syn-
thetic procedure was initiated with Knoevenagel condensation of
thiazolidinedione 4 with various aldehydes to reach compounds 6.
Then a substitution with methyl bromoacetate or methyl 2-
bromopropionate gave compounds 7. After the acid-mediated hy-
drolysis of 7, the resulting carboxylic acids 8 were coupled with
either O-allylhydroxylamine hydrochloride or O-benzylhydroxyl-
amine hydrochloride using EDC.HCl to give hydroxamates 2 in
yields ranging from 31 to 75% (see Table 2). The lower yields were
caused by losses during column chromatography.
Finally, we assessed the stereochemical outcome of our syn-
thetic protocol. The formation of two geometrical isomers E or Z
after Knoevenagel condensation is possible. These two isomers can
easily be distinguished by their 1H NMR spectral characteristics. It is
well known that the benzylidine proton appears above 7.90 ppm
for Z isomer and below 7.42 ppm for E isomer [27,28]. The measured
3.2. Inhibitory activity towards Zmp1
The inhibitory properties of the studied compounds towards a
recombinant zinc metallopeptidase (Zmp1) from Mtb were
Scheme 1. Synthesis of model compounds 1 and 2.
Reagents and conditions: (i) methyl bromoacetate or methyl 2-bromopropionate, NaH, DMF dry, rt, on; (ii) HBr (40%), reflux, 5 h; (iii) O-allylhydroxylamine hydrochloride or O-
benzylhydroxylamine hydrochloride, EDC.HCl, H2O, rt, 2 h; (iv) aldehyde, piperidine, EtOH, reflux, 5 h.
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Please cite this article as: V. Slachtova et al., Novel thiazolidinedione-hydroxamates as inhibitors of Mycobacterium tuberculosis virulence factor
Zmp1, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2019.111812

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4
V. Slachtova et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
Table 1
Table 2
Overview of synthesized products 1.
Overview of synthesized products 2.
Entry
R1
R2
Yielda [%]
49
Entry
R1
R2
R3
Yielda [%]
58
1a
2a
1b
1c
55
61
2b
2c
51
64
1d
52
2d
2e
62
49
a
Isolated yield after purification.
analyzed by matrix-assisted laser desorption/ionization-time of
flight mass spectrometry (MALDI-TOF MS). The reaction mixture
contained human angiotensin II as a substrate and the respective
inhibitor. The proteolytic digestion of angiotensin II (m/z 1046)
produced two peptide fragments providing characteristic signals in
the mass spectrum, namely DRVY (m/z 552) and IHPF (m/z 513), in
accordance with the literature [35] and as confirmed by tandem MS
(MS/MS) analyses (not shown). The production of the fragments
was accompanied by a simultaneous decrease in the angiotensin II
signal (Supplementary Figs. S1e2).
MALDI is generally not considered a robust ionization technique
for quantitative measurements, but a growing number of reports
have documented its applicability for quantifying peptides [36].
This stems from reduced experimental difficulties because of the
ongoing technological improvement of the instruments [37]. In this
work, we eliminated both sample-to-sample and shot-to-shot
variability of peak intensities by reading intensity ratios of the re-
action product at m/z 552 and substrate at m/z 1046 in mass spectra
averaged from 1000 laser shots. Finally, the inhibition rate was
calculated from the ratio of the determined time-dependence
slopes for inhibited and control reaction (Supplementary
Table S1). Fig. 4 shows the obtained results. All compounds were
found to inhibit the enzymatic reaction but the existing structural
differences were reflected in their inhibitory strength. The most
potent inhibitors were 2c, 2k-m, and 1b (which inhibited almost
completely), whereas 2d and 2g inhibited only by less than 20%.
Using the measured inhibition percentages, IC50 (half-maximal
inhibitory concentration) values of the inhibitors can be estimated
2f
50
48
2g
2h
31
2i
35
40
60
2j
2k
2l
56
75
2m
2n
2o
75
45
2p
45
(100% activity for [I] ¼ 0
20e160 M. Experimental IC50 values were obtained with three
different inhibitor concentrations for 2n (18 M) and 2e (38 M),
mM) and they appear in the range of
m
a
Isolated yield after purification.
m
m
which represent highly efficient and medium-efficient inhibitors
from the studied group, respectively. These numbers correspond to
inhibition constants described for some other Zmp1 inhibitors
[6,14,38].
Supplementary table (Table S2) shows a graphic interpretation
of the influence of chemical functional groups on the inhibitory
properties. The analyzed hybrid compounds can be divided into
five clusters according to the substituent at the thiazolidinedione
moiety (R3): without any substitution, with a benzylidene, fluo-
robenzylidene, trifluorobenzylidene, and pentylidene substituting
group. In each cluster, the presence of either a benzyl or allyl
substitution at the hydroxamate moiety (R1) was considered, plus a
possible methyl group in the linker (R2). As can be seen, the benzyl
group at the hydroxamate function resulted predominantly in a
lower inhibition potency compared to the allyl group at this posi-
tion for the hybrid compounds with both non-substituted and
trifluorbenzylidene-substituted thiazolidinedione ring. An oppo-
site trend was found for those containing the benzylidene-
substituted thiazolidinedione ring, whereas no clear trend was
observed for the other two clusters. The methyl group presence in
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V. Slachtova et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
5
Table 3
In silico predicted physicochemical and pharmacokinetic parameters of the thiazolidinedione-hydroxamates.
Compound MWa LogPb HBDc HBAd No. of
atomse
Rotatable
bondsf
logSwg Solubility Forecast
Index
Oral bioavailability (Veber’s 4/
Ames test
Acute oral
toxicity
rule]
400 toxicity
1a
1b
1c
1d
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
280.3 1.14
230.24 0.29
294.33 1.54
244.27 0.69
368.41 3.26
318.35 2.41
382.43 3.67
332.37 2.82
436.41 4.15
386.35 3.30
450.43 4.55
400.37 3.70
386.4 3.36
336.34 2.51
400.42 3.77
350.36 2.92
348.42 3.52
298.36 2.67
362.44 3.92
312.38 3.07
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
19
15
20
16
26
22
27
23
30
26
31
27
27
23
28
24
24
20
25
21
5
5
5
5
6
6
6
6
7
7
7
7
6
6
6
6
8
8
8
8
ꢀ2.11 Good
ꢀ1.12 Good
ꢀ2.44 Good
ꢀ1.46 Good
ꢀ3.99 Good
ꢀ3.06 Good
ꢀ4.32 Good
ꢀ3.4 Good
ꢀ4.9 Good
ꢀ3.97 Good
ꢀ5.22 Reduced
ꢀ4.3 Good
ꢀ4.17 Good
ꢀ3.23 Good
ꢀ4.5 Good
ꢀ3.57 Good
ꢀ3.79 Good
ꢀ2.84 Good
ꢀ4.12 Good
ꢀ3.18 Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good Non
Good Non
Good Non
Good Non
Good Non
Good Non
Good Non
Good Non
Bad Non
Good Non
Bad Non
Good Non
Good Non
Good Non
Good Non
Good Non
Good Non
Good Non
Good Non
Good Non
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
2m
2n
2o
2p
a
Molecular Weight (MW; 130 to 725).
b
c
d
e
f
Log partition coefficient between n-octanol and water (ꢀ2 to 6.5).
Number of hydrogen bond donors (0e6).
Number of hydrogen acceptors (2e20).
Number of atoms (20e70).
Number of rotatable bonds (0e15).
Log aqueous solubility (ꢀ6.5 to 0.5).
g
in vitro activity against Mycobacterium tuberculosis H37Ra using a
whole-cell assay. Antimycobacterial potency was assessed using
resazurin, as previously reported [39]. The results were expressed
as the IC50, and also minimal inhibitory concentration (MIC) at
which the mycobacterial growth is reduced by 90% (Table 4). In
parallel, the acute cytotoxicity of the thiazolidinedione-
hydroxamates against the eukaryotic RAW264.7 macrophage cell
line was studied using a previously reported neutral red uptake
(NRU) assay [40]. The cytotoxic concentration (CC50) of a compound
is defined as the concentration at which the NRU by the cells is
reduced by 50% (Table 4). The selectivity index (SI) of the synthe-
sized compounds was calculated by dividing the CC50 with the MIC
(Table 4). Results showed that most of the thiazolidinedione-
hydroxamates showed no significant antimycobacterial activity
against extracellular Mtb H37Ra, i.e. IC50 and MIC values > 64 mM
(Table 4). The hybrids 2f, 2k, 2l, 2m and 2n showed a low anti-
mycobacterial potency with IC50 values of 51.7, 44.4, 60.0, 55.0 and
42.2 respectively. For 2n, a MIC value of 61.8 could be calculated.
Except for 2o, none of the compounds exhibited acute cytotoxicity
Fig. 4. Inhibitory activity toward Zmp1. Inhibition rate at a given inhibitor concen-
tration of 40 M was calculated by inverting the ratio of slopes for inhibited and
control reaction. Phosphoramidon (RDF) was used as a reference at a final concen-
tration of 40 M [16].
against RAW264.7 macrophages, i.e. CC50 values > 128
Only 2o showed a low cytotoxic effect with a CC50 value of 43.9
Though, this effect did not exceed the cytotoxic effect of the
M). As a result, a SI of at
mM (Table 4).
m
mM.
m
reference drug tamoxifen (CC50 ¼ 11.1
m
least 2.1 and 0.7, respectively, could be calculated for 2n and 2o.
Although only a low antimycobacterial activity against extracellular
Mtb H37Ra was present, 2n showed to be the most potent of the
evaluated hybrids. Interestingly, this compound belonged to those
providing the strongest inhibitory properties towards the myco-
bacterial enzyme Zmp1.
the linker resulted in lower inhibitory properties when compared
to the counterparts without this group, for the compounds with
pentylidene and trifluorbenzylidene substitutions at the thiazoli-
dinedione ring. It was the opposite for those with the fluo-
robenzylidene substitution. Generally, this interpretation indicates
that the measured inhibition data reflect a structure-based inter-
action with the enzyme molecule as they are not fully random.
As the thiazolidinedione-hydroxamate hybrids were designed
as Zmp1-inhibitors, the in vitro antimycobacterial activity of the
compounds against intracellular residing Mtb H37Ra bacilli was
assessed as well. The intracellular activity was studied using a
luminometric macrophage infection assay [41]. Based on the
luminescent signal of the synthesized compounds and untreated
control, the obtained results were expressed as inhibition
3.3. In vitro biological activity
Following the synthesis and enzymatic assays, the
thiazolidinedione-hydroxamate hybrids were evaluated for their
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V. Slachtova et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
6
Table 4
contrast, 2e excels in antimycobacterial activity against intracel-
lular residing Mtb H37Ra, even at the lower concentration of 10 M,
In vitro antimycobacterial activity and acute cytotoxicity results.
m
Compound
IC50
(
m
M)a
MIC (
m
M)b
CC50
(
m
M)c
SId
whereas it lacks a significant extracellular activity. Structure-
activity relationship deductions indicate that the R2 linker, con-
necting the thiazolidinedione and hydroxamate moieties, did not
influence in vitro intracellular activity too much. In contrast, the R1
substitution appeared largely influential. For example, for the
simple thiazolidinedione-unsubstituted hydroxamates 1, the
benzyl-substitution (1a and 1c) reduced intracellular anti-
mycobacterial activity at lower concentrations. In contrast, the R1
1a
1b
1c
1d
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
>64
>64
>64
>64
>64
>64
>64
>64
59.5
51.7
>64
>64
>64
>64
>64
60.0
55.0
42.2
44.4
>64
0.1
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
61.8
>64
>64
0.3
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
43.9
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
>2.1
>0.7
ND
ND
ND
benzyl-substitution of the thiazolidinedione-hydroxamates
2
increased their in vitro potency against intracellular residing Mtb
H37Ra. For the latter group, the R3 substitution also showed to be
important: the presence of an alkyl reduced the antimycobacterial
activity at low concentrations, whereas substitution with either an
unsubstituted benzyl group or fluorinated benzyl group increased
the inhibition percentage in the intracellular test. Hybrid 2e was
selected as the most potent inhibitor of intracellular residing Mtb
H37Ra, with an inhibition percentage of 70.4 and 48.8% at 100 and
2m
2n
2o
2p
Isoniazid
Tamoxifen
>128
ND
11.1
10 mM, respectively. Hybrid 2n, however, was selected as the most
optimal compound considering the overall results, including the in
silico drug-likeness predictions and target-based enzymatic assays.
ND
ND
ND, not done.
a
Fifty percent inhibitory concentration.
Minimum inhibitory concentration.
Fifty percent cytotoxicity concentration.
Selectivity index (CC50/MIC).
3.4. Pose identification with in silico molecular docking
b
c
d
In order to explain the inhibitory effect of the thiazolidinedione-
hydroxamates, we have employed a molecular docking of 2n into
the internal central cavity of Mycobacterium tuberculosis zinc met-
alloprotease Zmp1 structure (PDB ID: 3ZUK) [7]. The compound
mimics the interactions of the indole part of phosphoramidon in-
hibitor crystal pose as shown in Fig. 6A. Both aromatic moieties, the
indole of phosphoramidon and thiazolidinedione of compound 2n,
percentages. As depicted in Fig. 5, sixteen of the thiazolidinedione-
hydroxamates were found antimycobacterial at a final test con-
centration of 100
intracellular activity. Considering a final concentration of 10
mM. Only 2f, 2h, 2m and 2o completely lacked
m
M,
eight of the thiazolidinedione-hydroxamates showed an anti-
mycobacterial activity in this experimental arrangement, namely
1b, 1d, 2a, 2c, 2e, 2g, 2i, and 2k. The percentage inhibition was 30.2,
21.6, 38.3, 46.9, 48.8, 19.4, 20.6 and 25.1%, respectively. This in-
dicates that the existing structural differences not only influence
the enzymatic inhibitory strength towards Zmp1 but also affect less
straightforward biological properties such as cell membrane
penetration. The hybrid 2n, which showed only a low anti-
mycobacterial activity against extracellular Mtb H37Ra, exhibited
interact via a p-p interaction with the phenyl ring of F48. They also
share the H-bond with R628 and the hydrophobic interactions with
the aliphatic chain in the cleft formed by F48 and W604. Phos-
phoramidon, however, shows a direct interaction with the zinc
atom and its surroundings leading towards much stronger inhibi-
tion (Ki ¼ 35 nM according to PDBBIND) [42]. Compound 2n shows
additional polar contacts with the cavity entrance at R616. This is in
agreement with the previous suggestion of designing such prom-
ising inhibitors, which would target the secondary binding pocket
at the active site of Zmp1 [7].
intracellular activity at the relatively high 100-mM concentration. In
The reasons for a good performance of the compound 2n seem
to be i) nonpolar aliphatic chain interaction with the hydrophobic
cleft, where larger moieties do not fit, ii) unbranched linker joining
the polar moiety of 2n to the thiazolidinedione ring, which allows
more flexibility towards the polar part of the entrance into the
cavity and iii) polar interactions with highly populated charged
residues within the entrance.
The studied thiazolidinedione-hydroxamates are thus expected
to occupy the entrance into the central cavity, approximately 7 Å
apart of the zinc-binding catalytic site (Fig. 6B). This is probably the
reason for their lower efficiency than it could have been expected
from their rational design (combining structural features of previ-
ously described potent inhibitors) as they are only gate-keeping the
entry/exit of the peptidic substrate and do not further interfere
with the reaction on the zinc atom. In contrast, as shown in crystal
structures, phosphoramidon interacts directly with the zinc cata-
lytic site and this allows the compound to inhibit also human zinc
metallopeptidase neprilysin (NEP) structure (PDB ID: 1DMT) [43] as
shown in Fig. 6C. NEP is able to cleave peptide bonds between
hydrophobic residues in a variety of peptides such as opioid Met-
and Leu-enkephalins [44] or angiotensins (e.g. angiotensin II).
However, while the cleft surrounding the zinc-binding catalytic site
is structurally identical, the entrance into the cavity differs
Fig. 5. Intracellular antimycobacterial activity of the thiazolidinedione-hydrox-
amates. The growth inhibition of Mtb H37Ralux inside RAW264.7 macrophages upon
24 h of exposure to 100 and 10
inhibition. Isoniazid (INH) was used as a reference at a final test concentration of
0.1 M. Results are presented as the mean SD.
mM of the hybrids was expressed as the percentage
m
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V. Slachtova et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
7
dramatically between the Mycobacterium and human zinc
metallopeptidases.
4. Conclusion
In conclusion, we have developed an efficient synthetic protocol
for the preparation of a series of thiazolidinedione-hydroxamates
and assessed their inhibitory properties towards a recombinant
Zmp1 from Mycobacterium tuberculosis by MALDI-TOF MS. This
characterization was completed by whole-cell biological activity
and cytotoxicity tests. Results showed that none of the synthesized
thiazolidinedione-hydroxamates possessed acute cytotoxic effects
against RAW264.7 macrophages. The extracellular anti-
mycobacterial activity was rather limited, whereas anti-
mycobacterial activity against intracellular residing bacilli was
present for the majority of the tested hybrid library. The existing
structural differences were reflected in the variability of the in silico
predicted parameters and biologically determined responses.
When looking for a synergic behavior of the studied synthetic
compounds in all performed experimental tests, the compound 2n,
which does not contain aromatic R1 and R3 substituents as well as a
branched R2 linker, was found the most optimal.
5. Experimental section
5.1. Materials and methods
Solvents and chemicals were purchased from Sigma-Aldrich
(www.sigmaaldrich.com) and Fluorochem (www.fluorochem.co.
uk). All reactions were carried out at ambient temperature (21 ꢁC)
unless stated otherwise. Analytical thin-layer chromatography
(TLC) was performed using aluminum plates precoated with silica
gel (silica gel 60 F254).
The LC-MS analyses were carried out on UHPLC-MS system
consisting of UHPLC chromatograph Accela with photodiode array
detector and triple quadrupole mass spectrometer TSQ Quantum
Access (Thermo Scientific, CA, USA), using Nucleodur Gravity C18
column (dimensions 1.8
800
m
m, 2.1 ꢂ 50 mm at 30 ꢁC and a flow rate of
m
l/min (Macherey-Nagel, Germany). The mobile phase was (A)
0.1% ammonium acetate in water, and (B) 0.1% ammonium acetate
in acetonitrile, linearly programmed from 10% to 80% B over
2.5 min, kept for 1.5 min. The column was re-equilibrated with 10%
of solution B for 1 min. The APCI source operated at a discharge
current of 5 m
A, vaporizer temperature of 400 ꢁC and a capillary
temperature of 200 ꢁC.
NMR 1H/13C spectra were recorded on JEOL ECX-500SS
(500 MHz) or JEOL ECA400II (400 MHz) spectrometer at magnetic
field strengths of 11.75 T (with operating frequencies 500.16 MHz
for 1H and 125.77 MHz for 13C) and 9.39 T (with operating fre-
quencies 399.78 MHz for 1H and 100.53 MHz for 13C) at ambient
temperature (~21 ꢁC). Chemical shifts (
d) are reported in parts per
million (ppm), and coupling constants (J) are reported in Hertz (Hz).
NMR spectra were recorded at ambient temperature (21 ꢁC) in
DMSO‑d6 and referenced to the resonance signal of the solvent.
HRMS analysis was performed with LC-MS and an Orbitrap
high-resolution mass spectrometer (Dionex, Ultimate 3000,
Thermo Exactive plus, MA, USA) operating in positive full scan
mode in the range of 80e1200 m/z. The settings for electrospray
ionization were as follows: 150 ꢁC oven temperature and 3.6 kV
source voltage. The acquired data were internally calibrated with
phthalate as a contaminant in methanol (m/z 297.15909). Samples
were diluted to a final concentration of 0.1 mg/ml in a solution of
water and acetonitrile (50:50, v/v). The samples were injected into
the mass spectrometer following HPLC separation on a Kinetex C18
Fig. 6. Pose identification with molecular docking. A e a comparison of the docked
pose of 2n with that of phosphoramidon (from the crystal structure, PDB ID: 3ZUK) at
the catalytic site of Zmp1; B e the docked poses of thiazolidinedione-hydroxamates in
the entrance compared to the crystal structure pose of phosphoramidon blocking the
entrance and also occupying the catalytic site of Zmp1; C e a comparison of the
binding of phosphoramidon in the cavity of Zmp1 (PDB ID: 3ZUK) and neprilysin (NEP;
PDB ID: 1DMT) [43]. Notice the conserved amino acids around the zinc-binding cat-
alytic site; on the contrary, the entrance shows a variability in the constituting amino
acids.
column (2.6
mm, 100 A, 50 ꢂ 3.0 mm) using an isocratic mobile
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phase of 0.01M acetonitrile/ammonium acetate (80/20) at a flow
rate of 0.3 ml minꢀ1
SFC chiral analyses were performed using an Acquity UPC sys-
tem (Waters) consisting of a binary solvent manager, sample
manager, column manager, column heater, convergence manager,
PDA detector 2998, QDA mass detector and chiral analytical col-
5.2.5. Ester hydrolysis
.
Ester 4/7 was dissolved in HBr 40% (8 ml/5 mmol) and refluxed
for 5 h. The mixture was cooled down to rt and diluted with water
(50 ml) and worked up according to method A or B.
Method A: The resulting suspension was filtered and washed
with water.
umns CHIRALPAK IA3, IB3, IC3 and ID3 (4,6 ꢂ 100 mm, 3
m
m par-
Method B: The product was extracted with EtOAc (3 ꢂ 50 ml),
ticle size). The chromatographic runs were performed at a flow rate
washed with brine, dried over Na2SO4 and concentrated in vacuum.
of 2.2 ml minꢀ1, column temperature of 38 ꢁC and ABPR 2000 psi.
5.2.6. 2-(2,4-Dioxo-5-(4-trifluoromethyl)benzylidene)thiazolidin-
5.2. Chemistry
3-yl)propanoic acid 8a
5.2.1. General procedure for Knoevenagel condensation
A mixture of thiazolidinedione 3, aldehyde (1 eq), piperidine
(0.8 eq) and EtOH (130 ml/17 mmol) was refluxed on (16e20 h) and
worked up according to method A or B.
Method A: The reaction mixture was poured into H2O (200 ml),
acidified with AcOH (10 ml) and filtered affording compounds 6.
Method B: The product was concentrated in vacuum and purified
by column chromatography (mobile phase: hexane/EtOAc 6:2)
affording compounds 6.
Method A. Brown solid. Yield: 55% (734 mg). 1H NMR (500 MHz,
5.2.2. General procedure for reaction with bromoester
DMSO‑d6):
d
13.28 (s, 1H), 8.06 (s, 1H), 7.91 (d, J ¼ 8.4 Hz, 2H), 7.86
Thiazolidinedione 3 or 6 was dissolved in dry DMF (10 ml/
8 mmol). NaH (1 eq) was slowly added to the reaction mixture
followed by stirring and stirred for 10 min. Bromoester (1 eq) was
added dropwise. After stirring overnight (24 h), the reaction
mixture was diluted with water (50 ml). The product was extracted
with DCM (5 ꢂ 50 ml), organic layers were washed with 5% HCl,
brine, dried over Na2SO4 and concentrated in vacuum.
(d, J ¼ 8.1 Hz, 2H), 5.03 (q, J ¼ 7.2 Hz, 1H), 1.52 (d, J ¼ 7.2 Hz, 3H). 13
C
NMR (126 MHz, DMSO‑d6):
d 169.89, 166.32, 164.63, 136.83, 131.89,
130.63, 130.01 (q, J ¼ 32.7 Hz), 126.12, 123.79 (q, J ¼ 272.1 Hz),
123.68, 50.49, 13.87. HRMS: m/z: calcd for C14H10F3NO4S: 346.0355
[MþH]þ; found: 346.0358.
5.2.7. 2-(2,4-Dioxo-5-pentylidenethiazolidin-3-yl)propanoic acid
8b
5.2.3. Methyl 2-(2,4-dioxo-5-pentylidenethiazolidin-3-yl)acetate
7a
Method B. Brown solid. Yield: 83% (415 mg). 1H NMR (500 MHz,
Orange oil. Yield: 81% (822 mg). 1H NMR (500 MHz, DMSO‑d6):
DMSO‑d6):
d
13.16 (s, 1H), 7.08 (t, J ¼ 7.7 Hz, 1H), 4.93 (q, J ¼ 7.2 Hz,
d
7.12 (t, J ¼ 7.7 Hz, 1H), 4.44 (s, 2H), 3.70 (s, 3H), 2.28e2.23 (m, 2H),
1.53e1.47 (m, 2H), 1.32 (dd, J ¼ 14.9, 7.4 Hz, 2H), 0.89 (t, J ¼ 7.3 Hz,
3H). 13C NMR (126 MHz, DMSO‑d6):
167.17, 166.63, 163.57, 140.10,
1H), 2.24 (dd, J ¼ 14.8, 7.5 Hz, 2H), 1.51e1.45 (m, 5H), 1.32 (dd,
J ¼ 14.9, 7.4 Hz, 2H), 0.89 (t, J ¼ 7.3 Hz, 3H). 13C NMR (126 MHz,
d
DMSO‑d6): d 169.97, 166.48, 163.58, 139.59, 123.94, 50.06, 30.96,
123.92, 52.58, 41.83, 31.01, 29.23, 21.74, 13.56. HRMS: m/z: calcd for
C
29.26, 21.76, 13.91, 13.57. HRMS: m/z: calcd for C11H15NO4S:
11H15NO4S: 258.0795 [MþH]þ; found: 258.0796.
258.0795 [MþH]þ; found: 258.0795.
5.2.4. Methyl 2-(2,4-dioxo-5-pentylidenethiazolidin-3-yl)
propanoate 7b
5.2.8. Reaction with hydroxylamine.HCl
Carboxylic acid (1 eq) 5/8 was suspended in water (20 ml/
1 mmol). Hydroxylamine.HCl (1.5 eq) was dissolved in water (20
ml/1 mmol). The amine mixture was added to the acid mixture and
the pH was adjusted to 4.5 with 1M NaOH. THF was added until a
homogeneous solution was obtained. EDC.HCl (3 eq) was dissolved
in water (10 ml/1 mmol) and added in aliquots (4 ml/1 min) to the
reaction mixture. The reaction was stirred for 2 h and worked up
according to the method A or B.
Method A: The reaction was filtered and the solid was dried
overnight.
Method B: The product was extracted with EtOAc (3 ꢂ 40 ml),
washed with saturated NaHCO3, brine, dried over Na2SO4 and
concentrated in vacuum.
Mobile phase: DCM/MeOH 98:2. Brown solid. Yield: 44%
(467 mg). 1H NMR (500 MHz, DMSO‑d6):
d
7.10 (t, J ¼ 7.7 Hz, 1H),
5.07 (q, J ¼ 7.1 Hz, 1H), 3.65 (s, 3H), 2.24 (dd, J ¼ 14.8, 7.5 Hz, 2H),
1.51e1.46 (m, 5H), 1.32 (dd, J ¼ 14.9, 7.4 Hz, 2H), 0.89 (t, J ¼ 7.3 Hz,
3H). 13C NMR (126 MHz, DMSO‑d6):
d 169.02, 166.42, 163.42, 140.12,
123.77, 52.57, 49.98, 30.99, 29.24, 21.76, 13.95, 13.57. HRMS: m/z:
5.2.9. N-(benzyloxy)-2-(2,4-dioxothiazolidin-3-yl)acetamide 1a
calcd for C12H17NO4S: 272.0951 [MþH]þ; found: 272.0952.
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Method A. Mobile phase: DCM/MeOH 90:10. White solid. Yield:
49% (277 mg). 1H NMR (500 MHz, DMSO‑d6):
11.22 (s, 1H),
7.42e7.35 (m, 5H), 4.82 (s, 2H), 4.25 (s, 2H), 4.10 (s, 2H). 13C NMR
(126 MHz, DMSO‑d6): 171.84, 171.46, 162.66, 135.65, 128.87,
Method A. White solid. Yield: 58% (451 mg). 1H NMR (500 MHz,
DMSO‑d6):
11.34 (s, 1H), 7.96 (s, 1H), 7.65 (d, J ¼ 7.3 Hz, 2H), 7.56 (t,
J ¼ 7.3 Hz, 2H), 7.52 (d, J ¼ 7.1 Hz, 1H), 7.42e7.37 (m, 5H), 4.84 (s,
2H), 4.28 (s, 2H). 13C NMR (126 MHz, DMSO‑d6):
166.96, 165.15,
162.54, 135.64, 133.50, 132.83, 130.77, 130.15, 129.39, 128.91, 128.33,
121.00, 77.10, 41.50. HRMS: m/z: calcd for C19H16N2O4S: 369.0904
[MþH]þ; found: 369.0907.
d
d
d
d
128.32, 77.05, 41.14, 33.97. HRMS: m/z: calcd for C12H12N2O4S:
281.0591 [MþH]þ; found: 281.0592.
5.2.10. N-(allyloxy)-2-(2,4-dioxothiazolidin-3-yl)acetamide 1b
5.2.14. N-(allyloxy)-2-(5-benzylidene-2,4-dioxothiazolidin-3-yl)
acetamide 2b
Method B. Mobile phase: CHCl3/MeOH 90:10. Yellow oil. Yield:
55% (257 mg). 1H NMR (500 MHz, DMSO‑d6):
d
11.35 (s, 1H),
5.95e5.87 (m, 1H), 5.31 (d, J ¼ 17.3 Hz, 1H), 5.26 (d, J ¼ 10.4 Hz, 1H),
4.27 (s, 4H), 4.03 (s, 2H). 13C NMR (126 MHz, DMSO‑d6):
171.82,
d
Method A. White solid. Yield: 51% (339 mg). 1H NMR (500 MHz,
171.44, 162.49, 132.70, 119.48, 76.05, 41.11, 33.96. HRMS: m/z: calcd
DMSO‑d6):
d
11.26 (s, 1H), 7.96 (s, 1H), 7.64 (d, J ¼ 6.0 Hz, 2H),
for C8H10N2O4S: 231.0434 [MþH]þ; found: 231.0435.
7.58e7.51 (m, 3H), 5.96 (s, 1H), 5.35 (d, J ¼ 17.2 Hz, 1H), 5.28 (d,
J ¼ 9.2 Hz, 1H), 4.30 (d, J ¼ 28.8 Hz, 4H). 13C NMR (126 MHz,
5.2.11. N-(benzyloxy)-2-(2,4-dioxothiazolidin-3-yl)propanamide 1c
DMSO‑d6): d 166.94, 165.13, 162.38, 133.48, 132.82, 132.69, 130.77,
130.14, 129.39, 120.99, 119.54, 76.09, 41.46. HRMS: m/z: calcd for
C
15H14N2O4S: 319.0747 [MþH]þ; found: 319.0749.
5.2.15. 2-(5-benzylidene-2,4-dioxothiazolidin-3-yl)-N-(benzyloxy)
propanamide 2c
Method B. Mobile phase: DCM/MeOH 90:10. Yellow oil. Yield:
61% (342 mg). 1H NMR (500 MHz, DMSO‑d6):
d 11.35 (s, 1H),
7.40e7.38 (m, 4H), 7.37e7.36 (m, 1H), 4.75 (d, J ¼ 7.1 Hz, 2H), 4.68
(q, J ¼ 7.1 Hz, 1H), 4.16 (s, 2H), 1.40 (d, J ¼ 7.2 Hz, 3H). 13C NMR
(126 MHz, DMSO‑d6): d 171.71, 171.31, 165.08, 135.67, 128.94, 128.28,
76.90, 49.39, 34.03, 13.61. HRMS: m/z: calcd for C13H14N2O4S:
Method B. Mobile phase: DCM/MeOH 98:2. Yellow solid. Yield:
295.0747 [MþH]þ; found: 295.0749.
64% (373 mg). 1H NMR (500 MHz, DMSO‑d6):
d 11.46 (s, 1H), 7.94 (s,
1H), 7.65 (d, J ¼ 7.4 Hz, 2H), 7.58e7.50 (m, 3H), 7.40e7.34 (m, 5H),
5.2.12. N-(allyloxy)-2-(2,4-dioxothiazolidin-3-yl)propanamide 1d
4.87 (q, J ¼ 7.1 Hz, 1H), 4.77 (q, J ¼ 10.7 Hz, 2H), 1.50 (d, J ¼ 7.2 Hz,
3H). 13C NMR (126 MHz, DMSO‑d6):
d 166.75, 164.99, 164.90, 135.69,
132.96, 130.63, 130.05, 129.40, 128.97, 128.27, 121.36, 76.92, 49.85,
13.86. HRMS: m/z: calcd for C20H18N2O4S: 383.1060 [MþH]þ;
found: 383.1058.
5.2.16. N-(allyloxy)-2-(5-benzylidene-2,4-dioxothiazolidin-3-yl)
propanamide 2d
Method B. Mobile phase: CHCl3/MeOH 90:10. Yellow oil. Yield:
52% (240 mg). 1H NMR (500 MHz, DMSO‑d6):
d 11.24 (s, 1H),
5.94e5.86 (m, 1H), 5.30 (dd, J ¼ 17.3, 1.6 Hz, 1H), 5.24 (d, J ¼ 10.5 Hz,
1H), 4.65 (q, J ¼ 7.1 Hz, 1H), 4.25e4.23 (m, 2H), 4.16 (s, 2H), 1.38 (d,
J ¼ 7.2 Hz, 3H). 13C NMR (126 MHz, DMSO‑d6):
d 171.66, 171.29,
164.87, 132.80, 119.24, 75.83, 49.33, 34.02, 13.59. HRMS: m/z: calcd
for C9H12N2O4S: 245.0591 [MþH]þ; found: 245.0593.
5.2.13. 2-(5-benzylidene-2,4-dioxothiazolidin-3-yl)-N-(benzyloxy)
acetamide 2a
Method B. Mobile phase: DCM/MeOH 98:2. Yellow solid. Yield:
62% (314 mg). 1H NMR (500 MHz, DMSO‑d6):
d
11.36 (s, 1H), 7.94 (s,
1H), 7.64 (d, J ¼ 7.4 Hz, 2H), 7.58e7.51 (m, 3H), 5.91 (dq, J ¼ 10.7,
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6.1 Hz, 1H), 5.31 (d, J ¼ 17.3 Hz, 1H), 5.24 (d, J ¼ 11.7 Hz, 1H), 4.85 (q,
J ¼ 7.1 Hz, 1H), 4.26 (t, J ¼ 5.2 Hz, 2H), 1.48 (d, J ¼ 7.2 Hz, 3H). 13C
5.2.20. N-(allyloxy)-2-(2,4-dioxo-5-(4-trifluoromethyl)
benzylidene)thiazolidin-3-yl)propanamide 2h
NMR (126 MHz, DMSO‑d6): d 166.72, 164.98, 164.70, 132.95, 132.82,
130.63, 130.05, 129.39, 121.36, 119.30, 75.87, 49.81, 13.85. HRMS: m/
z: calcd for C16H16N2O4S: 333.0904 [MþH]þ; found: 333.0905.
5.2.17. N-(benzyloxy)-2-(2,4-dioxo-5-(4-trifluoromethyl)
benzylidene)thiazolidin-3-yl)acetamide 2e
Method B. Mobile phase: DCM/MeOH 98:2. White solid. Yield:
31% (127 mg). 1H NMR (500 MHz, DMSO‑d6):
d 11.37 (s, 1H), 8.02 (s,
1H), 7.91 (d, J ¼ 8.3 Hz, 2H), 7.85 (d, J ¼ 8.2 Hz, 2H), 5.91 (qd, J ¼ 11.8,
6.1 Hz, 1H), 5.31 (d, J ¼ 17.3 Hz, 1H), 5.24 (d, J ¼ 10.4 Hz, 1H), 4.86 (q,
J ¼ 6.9 Hz, 1H), 4.26 (t, J ¼ 5.1 Hz, 2H), 1.49 (d, J ¼ 7.1 Hz, 3H). 13C
Method A. White solid. Yield: 49% (298 mg). 1H NMR (500 MHz,
NMR (126 MHz, DMSO‑d6):
d 166.38, 164.76, 164.62, 136.94, 132.81,
131.04, 130.54, 129.85 (q, J ¼ 31.5 Hz), 126.18, 124.41, 123.80 (q,
DMSO‑d6):
7.43e7.37 (m, 5H), 4.85 (s, 2H), 4.29 (s, 2H). 13C NMR (126 MHz,
DMSO‑d6): 166.62, 164.93,162.45, 136.81, 135.63, 131.67, 129.97 (q,
d
11.35 (s, 1H), 8.04 (s, 1H), 7.87 (dd, J ¼ 19.3, 8.2 Hz, 4H),
J ¼ 272.1 Hz), 119.34, 75.89, 49.92, 13.79. HRMS: m/z: calcd for
C
17H15F3N2O4S: 401.0777 [MþH]þ; found: 401.0775.
d
J ¼ 31.5 Hz), 128.92, 128.33, 126.13, 124.00, 123.79 (q, J ¼ 273.4 Hz),
5.2.21. N-(benzyloxy)-2-(5-(4-fluorobenzylidene)-2,4-
dioxothiazolidin-3-yl)acetamide 2i
77.11, 41.63. HRMS: m/z: calcd for
[MþH]þ; found: 437.0775.
C20H15F3N2O4S: 437.0777
5.2.18. N-(allyloxy)-2-(2,4-dioxo-5-(4-trifluoromethyl)
benzylidene)thiazolidin-3-yl)acetamide 2f
Method A. Yellow solid. Yield: 75% (617 mg). 1H NMR (500 MHz,
DMSO‑d6):
6.7 Hz, 2H), 7.42e7.36 (m, 7H), 4.84 (s, 2H), 4.27 (s, 2H). 13C NMR
(126 MHz, DMSO‑d6): 166.85, 165.11, 164.06, 162.53, 162.06,
d
11.33 (s, 1H), 7.97 (d, J ¼ 7.0 Hz, 1H), 7.71 (dd, J ¼ 12.5,
Method A. Yellow solid. Yield: 50% (289 mg). 1H NMR (500 MHz,
d
DMSO‑d6):
d
11.27 (s, 1H), 8.03 (s, 1H), 7.89 (d, J ¼ 8.4 Hz, 2H), 7.85
135.64, 132.66, 132.47, 129.51, 128.99, 128.82, 128,41, 128.26, 120.70,
(d, J ¼ 8.4 Hz, 2H), 5.96 (dd, J ¼ 16.1, 9.9 Hz, 1H), 5.35 (d, J ¼ 17.2 Hz,
116.74, 116.67, 116.43, 77.10, 41.51. HRMS: m/z: calcd for
1H), 5.28 (d, J ¼ 10.1 Hz, 1H), 4.33 (s, 4H). 13C NMR (126 MHz,
C
19H15FN2O4S: 387.0809 [MþH]þ; found: 387.0813.
DMSO‑d6):
d 166.60, 164.91, 162.29, 136.81, 132.69, 131.66, 130.63,
129.97 (q, J ¼ 31.5 Hz), 126.16, 124.00, 123.81 (q, J ¼ 272.1 Hz),
119.56, 76.11, 41.59. HRMS: m/z: calcd for C16H13F3N2O4S: 387.0621
[MþH]þ; found: 387.0621.
5.2.22. N-(allyloxy)-2-(5-(4-fluorobenzylidene)-2,4-
dioxothiazolidin-3-yl)acetamide 2j
5.2.19. N-(benzyloxy)-2-(2,4-dioxo-5-(4-trifluoromethyl)
benzylidene)thiazolidin-3-yl)propanamide 2g
Method A. Yellow solid. Yield: 75% (432 mg). 1H NMR (500 MHz,
DMSO‑d6):
d 11.25 (s, 1H), 7.97 (s, 1H), 7.74e7.69 (m, 2H), 7.38 (t,
J ¼ 8.4 Hz, 2H), 5.95 (s, 1H), 5.35 (d, J ¼ 17.1 Hz,2H), 5.28 (d,
Method B. Mobile phase: DCM/MeOH 95:5. White solid. Yield:
J ¼ 9.5 Hz, 2H), 4.32 (br s, 4H). 13C NMR (126 MHz, DMSO‑d6):
48% (126 mg). 1H NMR (500 MHz, DMSO‑d6):
d
11.47 (s, 1H), 8.03 (s,
d 166.82, 165.10, 162.37, 162.06, 132.70, 132.62, 132.43, 129.50,
1H), 7.91 (d, J ¼ 8.3 Hz, 2H), 7.86 (d, J ¼ 8.1 Hz, 2H), 7.41e7.34 (m,
119.54, 116.72, 116.61, 116.55, 116.44, 76.09, 41.47. HRMS: m/z: calcd
5H), 4.88 (q, J ¼ 7.0 Hz,1H), 4.77 (q, J ¼ 8.4 Hz, 2H),1.50 (d, J ¼ 7.1 Hz,
for C15H13FN2O4S: 337.0653 [MþH]þ; found: 337.0653.
3H). 13C NMR (126 MHz, DMSO‑d6):
d 166.41, 164.82, 164.78, 136.94,
135.67, 131.05, 130.53, 129.85 (q, J ¼ 32.7), 128.98, 128.28, 126.16,
5.2.23. N-(benzyloxy)-2-(5-(4-fluorobenzylidene)-2,4-
dioxothiazolidin-3-yl)propanamide 2k
124.41, 123.80 (q, J ¼ 273.4 Hz), 49.95, 13.82. HRMS: m/z: calcd for
C
21H17F3N2O4S: 451.0934 [MþH]þ; found: 451.0934.
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Please cite this article as: V. Slachtova et al., Novel thiazolidinedione-hydroxamates as inhibitors of Mycobacterium tuberculosis virulence factor
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V. Slachtova et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
11
Method B. Mobile phase: hex/EtOAc 60:40. Yellow solid. Yield:
40% (178 mg). 1H NMR (500 MHz, DMSO‑d6):
11.40 (s, 1H), 7.08 (t,
d
J ¼ 7.7 Hz, 1H), 5.91 (ddd, J ¼ 16.5, 11.3, 6.0 Hz, 1H), 5.32 (d,
J ¼ 17.3 Hz,1H), 5.26 (d, J ¼ 10.4 Hz,1H), 4.35e4.13 (m, 4H), 2.24 (dd,
J ¼ 14.7, 7.4 Hz, 2H), 1.50 (dt, J ¼ 15.0, 7.4 Hz, 2H), 1.32 (dd, J ¼ 14.9,
7.4 Hz, 2H), 0.89 (t, J ¼ 7.3 Hz, 3H). 13C NMR (126 MHz, DMSO‑d6):
d
166.76, 163.85, 162.43, 139.28, 132.70, 124.31, 119.48, 76.06, 41.16,
30.94, 29.27, 21.74, 13.57. HRMS: m/z: calcd for C13H18N2O4S:
299.1060 [MþH]þ; found: 299.1060.
Method B. Mobile phase: DCM/MeOH 98:2. Green solid. Yield:
45% (62 mg). 1H NMR (500 MHz, DMSO‑d6):
11.46 (s, 1H), 7.96 (s,
d
1H), 7.72 (dd, J ¼ 8.5, 5.5 Hz, 2H), 7.43e7.34 (m, 7H), 4.87 (q,
5.2.27. N-(benzyloxy)-2-(2,4-dioxo-5-pentylidenethiazolidin-3-yl)
propanamide 2o
J ¼ 7.1 Hz, 1H), 4.77 (d, J ¼ 8.2 Hz, 2H), 1.49 (d, J ¼ 7.2 Hz, 3H). 13C
NMR (126 MHz, DMSO‑d6):
d 166.63, 164.95, 164.89, 163.97, 161.97,
135.69, 132.59, 131.89, 129.62, 128.97, 128.27, 121.07, 116.68, 116.50,
76.92, 49.88, 13.86. HRMS: m/z: calcd for C20H17FN2O4S: 401.0966
[MþH]þ; found: 401.0968.
5.2.24. N-(allyloxy)-2-(5-(4-fluorobenzylidene)-2,4-
dioxothiazolidin-3-yl)propanamide 2l
Method B. Mobile phase: DCM/MeOH 95:5. Yellow oil. Yield: 60%
(160 mg). 1H NMR (500 MHz, DMSO‑d6):
d 11.41 (s, 1H), 7.39e7.34
(m, 5H), 7.04 (t, J ¼ 7.7 Hz, 1H), 4.76 (t, J ¼ 8.8 Hz, 3H), 2.23 (q,
J ¼ 7.4 Hz, 2H), 1.51e1.48 (m, 2H), 1.45 (d, J ¼ 7.2 Hz, 3H), 1.33 (dd,
J ¼ 14.9, 7.4 Hz, 2H), 0.89 (t, J ¼ 7.3 Hz, 3H). 13C NMR (126 MHz,
DMSO‑d6):
d 166.59, 164.94, 163.73, 138.64, 135.68, 128.95, 128.26,
124.51, 76.89, 49.64, 30.87, 29.32, 21.75, 13.85, 13.59. HRMS: m/z:
calcd for C18H22N2O4S: 363.1373 [MþH]þ; found: 363.1372.
Method B. Mobile phase: DCM/MeOH 95:5. Green solid. Yield:
5.2.28. N-(allyloxy)-2-(2,4-dioxo-5-pentylidenethiazolidin-3-yl)
propanamide 2p
45% (54 mg). 1H NMR (500 MHz, DMSO‑d6):
d 11.35 (s, 1H), 7.95 (s,
1H), 7.72 (dd, J ¼ 8.7, 5.4 Hz, 2H), 7.41 (t, J ¼ 8.8 Hz, 2H), 5.95e5.87
(m, 1H), 5.30 (d, J ¼ 16.0 Hz, 1H), 5.24 (d, J ¼ 10.4 Hz, 1H), 4.84 (q,
J ¼ 7.0 Hz, 1H), 4.26 (t, J ¼ 5.2 Hz, 2H), 1.48 (d, J ¼ 7.2 Hz, 3H). 13C
NMR (126 MHz, DMSO‑d6):
d 166.59, 164.94, 164.69, 161.97, 132.81,
132.59, 132.52, 131.88, 129.64, 121.07, 119.31, 116.67, 116.50, 75.88,
49.84, 13.84. HRMS: m/z: calcd for
C16H15FN2O4S: 351.0809
[MþH]þ; found: 351.0811.
Method B. DCM/MeOH 85:15. Yellow oil. Yield: 56% (129 mg). 1H
5.2.25. N-(benzyloxy)-2-(2,4-dioxo-5-pentylidenethiazolidin-3-yl)
acetamide 2m
NMR (500 MHz, DMSO‑d6):
d 11.31 (s, 1H), 7.04 (s, 1H), 5.91 (ddd,
J ¼ 17.0, 11.2, 5.9 Hz, 1H), 5.29 (d, J ¼ 15.8 Hz, 1H), 5.23 (d, J ¼ 8.8 Hz,
1H), 4.76 (q, J ¼ 7.1 Hz, 1H), 4.24 (s, 2H), 2.23 (dd, J ¼ 14.8, 7.4 Hz,
2H), 1.51e1.47 (m, 2H), 1.43 (d, J ¼ 7.2 Hz, 3H), 1.33 (dd, J ¼ 14.9,
7.4 Hz, 2H), 0.89 (t, J ¼ 7.3 Hz, 3H). 13C NMR (126 MHz, DMSO‑d6):
d
166.54, 164.74, 163.71, 138.63, 132.81, 119.25, 75.84, 49.59, 30.87,
29.32, 21.75, 13.84, 13.58. HRMS: m/z: calcd for C14H20N2O4S:
313.1217 [MþH]þ; found: 313.1215.
Method B. Brown solid. Yield: 35% (174 mg). 1H NMR (500 MHz,
5.3. In silico prediction of drug-likeness properties
DMSO‑d6):
d
11.29 (s, 1H), 7.43e7.37 (m, 5H), 7.09 (dt, J ¼ 11.2,
7.6 Hz, 1H), 4.83 (d, J ¼ 8.5 Hz, 2H), 4.21 (s, 2H), 2.27 (dq, J ¼ 11.2,
7.4 Hz, 2H), 1.53 (dd, J ¼ 14.5, 10.9 Hz, 2H), 1.36 (ddd, J ¼ 14.5, 11.1,
7.5 Hz, 2H), 0.92 (t, J ¼ 11.3, 7.3 Hz, 3H). 13C NMR (126 MHz,
Physicochemical and pharmacokinetic parameters of the
thiazolidinedione-hydroxamates were in silico predicted using the
FAFDrugs4 [29] and admetSAR tools [30]. The different parameters
predicted were as follows: molecular weight, octanol/water parti-
tion coefficient, number of hydrogen donors, number of hydrogen
acceptors, number of atoms, number of rotatable bonds, aqueous
solubility, Solubility Forecast Index, oral bioavailability via Veber’s
rule, 4/400 GSK rule, Ames toxicity and acute oral toxicity.
DMSO‑d6):
d 166.79, 163.86, 162.60, 139.33, 135.64, 128.86, 128.33,
124.32, 77.07, 41.19, 30.95, 29.28, 21.75, 13.57. HRMS: m/z: calcd for
C
17H20N2O4S: 349.1217 [MþH]þ; found: 349.1217.
5.2.26. N-(allyloxy)-2-(2,4-dioxo-5-pentylidenethiazolidin-3-yl)
acetamide 2n
5.4. Zmp1 inhibition assay
Human angiotensin II (Sigma-Aldrich, Cat. No. A9525) was dis-
solved in LC-MS-quality water to a concentration of 4 nmol/ml.
Aliquots (24
ml) of the stock solution were mixed with 1 ml of 2M
NH4HCO3 in 0.5-ml test tubes. This was followed by pipetting either
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V. Slachtova et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
12
1
m
l of neat acetone (control) or 1
acetone (sample). After pre-incubation in a thermostat at 37 ꢁC for
3 min, the reaction was started by adding 1 l of a recombinant
m
l of 1 mM inhibitor solution in
5.6. Assessment of acute in vitro cytotoxicity
m
The 50% cytotoxic concentration towards the RAW264.7 murine
macrophage cell line (ATCC® TIB-71™) was determined by a
neutral red uptake (NRU) assay. The RAW264.7 cells were cultured
in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher)
supplemented with 10% (v/v) heat-inactivated fetal calf serum
(iFCS; Thermo Fisher) in a 5% CO2 atmosphere at 37 ꢁC until a semi-
confluent layer of cells was obtained. Next, the cells were harvested
and seeded into transparent, flat-bottomed 96-well plates at a
density of 40.000 cells per well and left for recovery at 37 ꢁC and 5%
CO2. The following day, twofold serial dilutions of the tested com-
pounds were made in DMEM þ10% iFCS with a final starting con-
Zmp1 metallopeptidase from Mycobacterium tuberculosis (ProSci,
Poway, CA, USA; Cat. No. 90e361; diluted 1:4, v/v, with cold 50 mM
NH4HCO3 and kept on ice during the measurements) and pro-
ceeded at 37 ꢁC for 20 min. Aliquots (0.5
ml) of the reaction mixture
were aspirated by a pipette each 2 min, spotted onto the MALDI
target (an MSP BigAnchor 96 BC; Bruker Daltonik, Bremen, Ger-
many), immediately overlaid with matrix solution (a-cyano-4-
hydroxycinnamic acid, 5 mg/ml in acetonitrile: 2.5% (v/v) tri-
fluoroacetic acid, 7:3, v/v), and left to dry and crystallize.
MALDI-TOF MS measurements were carried out on a Microflex
LRF 20 instrument equipped with a 60-Hz nitrogen laser operating
at lmax ¼ 337 nm (Bruker Daltonik). Mass spectra were accumu-
lated from 1000 laser shots in the reflectron positive ion mode
using an acceleration voltage (IS1) of 18.0 kV, extraction voltage
(IS2) of 15.5 kV, lens voltage of 9.3 kV, reflectron voltage of 19.0 kV,
detector voltage of 1590 V and pulsed ion extraction delay time of
350 ns The instrument was calibrated externally with a peptide
mixture (Peptide Calibration Standard II; Bruker Daltonik).
The enzymatic reaction was monitored by the hydrolysis of
angiotensin II yielding a peptide DRVY (m/z 552.3), which is
accompanied by a simultaneous decrease in the angiotensin II
signal (DRVYIHPF; m/z 1046.5). The ratio of m/z 552 versus m/z 1046
signal intensities was plotted against the reaction time to achieve
an increasing linear dependence. The inhibition rate at the given
centration of 128
mM. As a positive control, tamoxifen (Sigma-
Aldrich) was included. The RAW264.7 cells were washed with
sterile phosphate-buffered saline (PBS; Thermo Fisher) and
exposed to the compounds by adding volumes of 100 ml of the serial
dilutions. Tamoxifen was used as a reference drug. Test plates were
left for 24 h at 37 ꢁC and 5% CO2. After 24 h exposure to the com-
pounds, the cells were washed two times with sterile PBS and
100
well. Subsequently, the test plates were incubated for 3 h at 37 ꢁC
and 5% CO2. The cells were washed again with sterile PBS and 150
ml neutral red (Sigma-Aldrich) working solution was added per
ml
of a 1:1 ethanol/acetic acid (Merck) mixture was added in each
well. The plates were left shaking until the color became homog-
enous purple, and the optical density was measured at 530 nm and
620 nm (reference wavelength) using a plate reader (Promega
Discover). Results were presented as the mean of triplicate values.
inhibitor concentration of 40 mM was finally calculated by inverting
the ratio of slopes for inhibited and control reaction. Phosphor-
amidone (RDF) was used as a reference inhibitor [16]. Three
5.7. Macrophage infection assay
different inhibitor concentrations in the range of 20e80 mM were
used to determine the corresponding IC50 value. The measured
responses (percentages of inhibition) were plotted against the
respective concentrations. All data points obtained for a single in-
hibitor were fitted by a straight line (linear regression equation:
The intracellular activity of the thiazolidinedione-hydroxamates
was tested by infecting the murine RAW264.7 macrophage cell line
with Mtb H37Ralux, a laboratory Mtb H37Ra strain transformed with
a pSMT1 luciferase reporter plasmid. The RAW264.7 cells were
cultured, harvested and seeded into transparent, flat-bottomed 96-
well plates as described above. Upon recovery, the cells were
washed with sterile PBS and infected with H37Ralux at a multi-
plicity of infection (MOI) of 10 for 2 h at 37 ꢁC. RAW264.7 cells were
y ¼ a*x þ b) and then IC50 (in
mM) was obtained as a result of
calculating formula (50 - b)/a.
5.5. In vitro antimycobacterial activity
washed two times with sterile PBS, incubated with 100 mg/ml
gentamicin (Sigma-Aldrich) for 1 h to kill the residual extracellular
bacteria and, again, washed with sterile PBS. Then, the infected
RAW264.7 cells were treated with the thiazolidinedione-
In vitro activity of the synthesized hybrids against Mtb H37Ra
(ATCC® 25177™) was evaluated by
a resazurin assay. The
thiazolidinedione-hydroxamates were solubilized in DMSO
(Sigma-Aldrich) at stock concentration of 10 mM. A two-fold serial
dilution of each compound was made in liquid Middlebrook 7H9
broth (Sigma-Aldrich) with 10% oleic acid, albumin, dextrose,
catalase (OADC) enrichment (BD Biosciences; complete 7H9 broth)
hydroxamates at a final test concentration of 10 and 100
Isoniazid was included as a reference drug at 0.1 M. Uninfected
cells were used as control. At 24 h post-exposure, the infected
RAW264.7 cells were washed and lysed with 200 l of 1% Triton X-
100 (Sigma-Aldrich). To assess the intracellular mycobacterial
replication, 25 l of 1% (v/v) n-decanal in ethanol was added to
100 l of the lysate and luminescence was measured using a
mM.
m
m
with final concentrations ranging from 64 to 0.25
m
M. Volumes of
m
100 l of the serial dilutions were added in triplicate to flat-
m
m
bottomed 96-well plates. A mycobacterial suspension was pre-
pared by thawing and dissolving a frozen glycerol-stock of Mtb
H37Ra and, subsequently, diluting it in complete 7H9 broth to
obtain a suspension with an appropriate inoculum size. A volume of
luminometer (Promega Discover). Results were presented the
mean of triplicate values.
5.8. In silico molecular docking
100 ml of the mycobacterial suspension was added to each well of
the test plates. Isoniazid was used as a reference drug. Positive
(100% growth) and negative (0% growth) controls were included as
well. Test plates were incubated at 37 ꢁC for 7 days. After 7 days of
exposure, extracellular mycobacterial replication was assessed by
All 3D structures of the designed library of ligands were ob-
tained with Marvin 15.1.5, software which can be used for drawing,
displaying and characterization of chemical structure, sub-
structures and reactions. Polar hydrogens were added to all ligands
and proteins with the AutoDock Tools program [45]. Docking of the
library of the structures into Mycobacterium tuberculosis zinc met-
alloprotease Zmp1 (PDB ID: 3ZUK) was carried out using AutoDock
Vina 1.1.2 [46]. A grid box with the edge of 21 Å was centred on the
active site of Zmp1 in the crystal structure (grid x: 95.8, y: 93.2, z:
33.0). The exhaustiveness parameter was set to 80 (default: 8).
resazurin. To each test well, 20 ml of a 0.02% resazurin (Sigma-
Aldrich) solution was added. Test plates were incubated at 37 ꢁC
until a color change from blue to pink occurred. Fluorescence was
measured at lex ¼ 550 nm and lem ¼ 590 nm using a spectropho-
tometer (Promega Discover). Results were presented as the mean of
triplicate values.
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Please cite this article as: V. Slachtova et al., Novel thiazolidinedione-hydroxamates as inhibitors of Mycobacterium tuberculosis virulence factor
Zmp1, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2019.111812

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V. Slachtova et al. / European Journal of Medicinal Chemistry xxx (xxxx) xxx
13
Pharm. Biotechnol. 1 (2013) 496e503.
After docking, we compared the docked poses in bulk and that of
the compound 2n was further analyzed and interpreted.
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bind zinc: an examination of innovative approaches to improved metal-
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[23] M.W. Majewski, S. Cho, P.A. Miller, S.G. Franzblau, M.J. Miller, Syntheses and
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[24] M. Flipo, T. Beghyn, J. Charton, V.A. Leroux, B.P. Deprez, R.F. Deprez-Poulain,
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Acknowledgements
This work was supported by grant no. JG_2019_002 from Pal-
acký University in Olomouc.
Appendix A. Supplementary data
components by an indirect oxidation method and syntheses of
siderophore conjugate and a biotin-siderophore conjugate, J. Org. Chem. 64
(1999) 7451e7458.
a DIG-
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Please cite this article as: V. Slachtova et al., Novel thiazolidinedione-hydroxamates as inhibitors of Mycobacterium tuberculosis virulence factor
Zmp1, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2019.111812