Bioorganic Chemistry 118 (2022) 105489

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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg

Design, synthesis and biological evaluation of substituted

3-amino-N-(thiazol-2-yl)pyrazine-2-carboxamides as inhibitors of
mycobacterial methionine aminopeptidase 1
Martin Juhás a, *, Vinod S.K. Pallabothula a, Katarina Grabrijan b, Martina Šimovičová a,
Ondřej Janďourek a, Klára Konečná a, Pavel Bárta a, Pavla Paterová c, Stanislav Gobec b,
Izidor Sosič b, Jan Zitko a, *
a
Charles University, Faculty of Pharmacy in Hradec Králové, Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
b
University of Ljubljana, Faculty of Pharmacy, Aškerčeva cesta 7, SI-1000 Ljubljana, Slovenia
c
University Hospital Hradec Králové, Department of Clinical Microbiology, Sokolská 581, 500 05 Hradec Králové, Czech Republic

A R T I C L E I N F O A B S T R A C T

Keywords: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is the number one cause of deaths due to a single
Aminothiazole infectious agent worldwide. The treatment of TB is lengthy and often complicated by the increasing drug
Antimycobacterial resistance. New compounds with new mechanisms of action are therefore needed. We present the design, syn­
Enzyme inhibition
thesis, and biological evaluation of pyrazine-based inhibitors of a prominent antimycobacterial drug target -
Methionine aminopeptidase 1
Pyrazine
mycobacterial methionine aminopeptidase 1 (MtMetAP1). The inhibitory activities of the presented compounds
were evaluated against the MtMetAP1a isoform, and all derivatives were tested against a broad spectrum of myco
(bacteria) and fungi. The cytotoxicity of the compounds was also investigated using Hep G2 cell lines.
Overall, high inhibition of the isolated enzyme was observed for 3-substituted N-(thiazol-2-yl)pyrazine-2-
carboxamides, particularly when the substituent was represented by 2-substituted benzamide. The extent of
inhibition was strongly dependent on the used metal cofactor. The highest inhibition was seen in the presence of
Ni2+. Several compounds also showed mediocre in vitro potency against Mtb (both Mtb H37Ra and H37Rv).
Despite the structural similarities of bacterial and fungal MetAP1 to mycobacterial MtMetAP1, title compounds
did not exert antibacterial nor antifungal activity. The reasons behind the higher activity of 2-substituted ben­
zamido derivatives, as well as the correlation of enzyme inhibition with the in vitro growth inhibition activity is
discussed.

1. Introduction only mycobacterium of importance. The other so-called non-tuberculous

Tuberculosis (TB) is a highly contagious disease caused by Myco­
bacterium tuberculosis (Mtb). Based on the annual surveillance report by
the World Health Organization (WHO), TB was responsible for more
than 1.2 million deaths in 2019 [1]. This ranks it in the top ten causes of
deaths worldwide and the leading cause of death due to a single infec­
tious agent. Despite a gradual decline in the number of casualties over
the past ten years, TB still represents a significant health threat. Pres­
ently, approximately two billion people are latently infected, potentially
at risk of fully developing this hard-to-treat infection [1]. Mtb is not the
(or atypical) mycobacteria are responsible for both pulmonary and
extra-pulmonary infections [2,3]. However, their impact on the popu­
lation is not quantifiable as easily as for the Mtb since they are not
surveyed by the authorities in the same manner. From all the atypical
mycobacteria, M. avium complex is of the greatest importance as it is
often seen in patients with impaired immune systems like the HIV-
positives. At the same time, some atypical mycobacteria serve as non-
infectious, safe-to-handle surrogates in the TB research, as discussed
later in the text.
Mycobacteria possess a highly lipophilic membrane responsible for

* Corresponding authors.
E-mail addresses: juhasm@faf.cuni.cz (M. Juhás), pallabov@faf.cuni.cz (V.S.K. Pallabothula), katarina.grabrijan@ffa.uni-lj.si (K. Grabrijan), simovicm@faf.cuni.
cz (M. Šimovičová), jando6aa@faf.cuni.cz (O. Janďourek), konecna@faf.cuni.cz (K. Konečná), pavel.barta@faf.cuni.cz (P. Bárta), pavla.paterova@fnhk.cz
(P. Paterová), stanislav.gobec@ffa.uni-lj.si (S. Gobec), izidor.sosic@ffa.uni-lj.si (I. Sosič), jan.zitko@faf.cuni.cz (J. Zitko).

https://doi.org/10.1016/j.bioorg.2021.105489
Received 24 June 2021; Received in revised form 2 November 2021; Accepted 10 November 2021
Available online 14 November 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.
M. Juhás et al.
low drug penetration and natural Mtb resistance to most drugs [4].
Additionally, colonies of Mtb usually encapsulate in the lung tissue
forming granulomas (hence the name tuberculosis; derived from Latin
tuberculum meaning “small lump”), making the already poor drug de­
livery even more challenging [5,6]. To achieve complete eradication,
six-month treatment is generally required [7].
To slow down the onset of drug-resistance (DR) spontaneously
occurring during such a lengthy regimen, a combination of at least three
drugs with different mechanisms of action (MoA) is used [8]. Combi­
nation therapy has proven effective for non-complicated cases, with the
treatment success reaching 85% in previously untreated patients. In
contrast, in cases of DR strains, it is below 57% [1]. Unfortunately,
especially in low-income regions, new DR cases are rising.
Mycobacterial methionine aminopeptidase 1 (MtMetAP1) is a
promising anti-TB targets. MetAPs are highly conserved metalloenzymes
present in all living organisms. Prokaryotes express only type 1 MetAP
(MetAP1), while in the eukaryotes, two types exist, MetAP1 and MetAP2
[9]. MetAPs are responsible for the cleavage of the initiator N-terminal
methionine and are thus implicated in several regulatory processes [9].
Knockout experiments in bacteria have shown that insufficient activity
of MetAP1 is fatal [10], rendering it a promising antimicrobial target
[11–13]. The overall structure of MetAP1 among organisms is closely
related. However, changes in their active site residues have been noted,
allowing potential direct targeting of inhibitors [14,15]. Mycobacterial
MtMetAP1 exists in two isoforms, 1a and 1c. Both have been proved
important for the viability of Mtb [11,13], but only a small focus was
directed towards the isoform 1a.
As a metalloenzyme, MetAP1 is known to work in the presence of
various divalent cations; most studied are cobalt, nickel, iron, zinc, and
manganese (all as M2+). The catalytic activity of MtMetAP1 with
different metals differs greatly, the differences in MtMetAP1a were
studied previously [12]. The identity of physiological metal is unknown.
Nowadays, iron (Fe2+) is considered the most probable cofactor as only
the inhibitors that showed strong enzyme inhibition with Fe2+ were able
to substantially decrease cell growth in the whole-cell assays [16,17].
An important issue with MetAP is the number of metal cofactors. In
the beginning, MetAPs were considered dinuclear metalloenzymes,
supported by a number of crystal structures containing two cocrystal­
lized metal ions in the active site [18]. In all structures, both metals are
always coordinated in the same way by the same five residues
Fig. 1. Representative structures of published MetAP1 inhibitors and the derivatives active in vitro against Mtb used for the design of the title compounds. N/A – data
not available. References: A [27], B [28], C [30] and D [31]
2
Bioorganic Chemistry 118 (2022) 105489
corresponding to His171, Glu204, Glu235, Asp97 and Asp108 in
EcMetAP1. Along the way, several groups have demonstrated that the
activity of the enzyme is seen already in the presence of one metal (M1,
metal coordinated by His171) [19,20] and that the affinity of the
enzyme to the second metal M2 (coordinated by Asp97) is also much
lower [21]. These findings were supported by specific crystallographic
experiments producing monometalated MetAP1 (with Mn2+) [22]. The
two metal ions inside the active site of MetAPs (majority of RCSB PDB
structures) were thus considered as an artefact due to high concentra­
tions of metal ions used in crystallization. This view was rechallenged by
Mitra and coworkers [22], demonstrating that the mutation of the Asp97
(necessary for coordination of the M2) leads to a decrease of enzyme
activity but not a complete loss. This means that the role of M1 is indeed
catalytic, but the presence of M2 might still be physiologically relevant.
1.1. Design of the compounds
Several potent inhibitors of MetAP1 have already been published,
with some also possessing desired antimicrobial activity [16,17]. From
relatively large molecules (e.g. bengamides [23]) to smaller catechol-
based inhibitors [17], virtually all have apparent metal-chelating
properties [24]. For more information, the reader is referred to the re­
view by Helgren and coworkers [18].
Compounds presented in this study are pyrazine isosteres of previ­
ously reported derivatives of thiazole-4-carboxamide (Fig. 1, A) and
pyridine-2-carboxamide (B) [25–28]. These derivatives showed high
potency against EcMetAP1 (submicromolar), some with good selectivity
concerning human MetAP2 isoforms, which are desired characteristics
for an antimicrobial drug. However, their activity against bacterial cells
was weak, and none of them was tested against mycobacteria. Other
structurally related derivatives were shown to inhibit human MetAP1
[29], raising the issue of selectivity and potentially unwanted toxicity of
the N-(thiazol-2-yl)pyridine-2-carboxamide scaffold.
We have previously published numerous compounds with anti­
mycobacterial activity containing pyrazine fragment that proved
particularly effective against Mtb [32–36]. A moderate anti­
mycobacterial activity (65% inhibition at 6.25 µg/mL) was observed for
the derivative C in Fig. 1 by Doležal and coworkers [30], strikingly
related to the pyridine hit compound presented by Luo and coworkers
[25]. Based on the already mentioned similarity of MetAP1 among
M. Juhás et al.
microorganisms and the structural similarity of the described com­
pounds, we supposed that the observed antimycobacterial activity of C
might have been due to the inhibition of MtMetAP1. Similar structural
features as presented in this article were seen in salicylanilides inhibit­
ing isoform MtMetAP1c by Krátký and coworkers [31] (compound D).
These compounds also possessed high antimycobacterial potency
against the multidrug-resistant strains.
As a proof of concept, the best pyridine-based inhibitor of EcMetAP1
[28] (compound 21 in this article) was synthesized and tested to
compare the effect of the isosteric replacement of pyridine by pyrazine.
With the aim of retaining the MetAP1 inhibition and at the same time
preserving the whole-cell antimycobacterial activity (presumably linked
to the pyrazine fragment), we designed and synthesized a series of
pyrazine-2-carboxamide derivatives as presented in Fig. 1. Compounds
were evaluated for the inhibition of MtMetAP1a and screened against
several mycobacterial, bacterial and fungal strains of clinical impor­
tance. Additionally, binding to MtMetAP1c was assessed by molecular
docking in silico.
2. Materials and methods
2.1. Chemistry
For the full description of the synthetic procedures and the analytical
characterization of compounds see the Supplementary material.
2.2. MtMetAP1a inhibition assay
All compounds were pre-screened in 96-well plates at final concen­
tration 12.5 µM obtained by diluting 10 mM DMSO stock solutions as
described previously [22]. Total reaction volume was 80 µL. Each re­
action contained 40 mM HEPES (pH 7.5), 100 mM NaCl, 100 µM Met-
AMC (L-methionine 7-amido-4-methylcoumarin, substrate), 200 nM of
respective ion (Co2+, Mn2+, Ni2+ or Fe2+), 200 nM MtMetAP1a and the
tested compound. Final concentration of DMSO was 1.25% (v/v). In case
of Fe2+, sodium ascorbate was added to prevent oxidation (2:1 in respect
to Fe2+, i.e., final concentration 400 nM). All metal ions were used as
chlorides. Unless stated otherwise, the stock solutions used for further
dilutions were prepared using bi-distilled water in indicated concen­
trations. The buffer was made by mixing solutions of HEPES pH 7.5 and
NaCl. The pH of HEPES solution was adjusted with 3 M NaOH or 3 M
HCl. The substrate-buffer solution was prepared by diluting Met-AMC
solution (c = 20 mM) with the buffer. Enzyme-metal-buffer solution
was prepared by diluting enzyme stock solution (c = 7.64 mg/ml in
polyethylene glycol) with the buffer, followed by addition of the
respective ion solution (c = 1 mM) and ascorbate (c = 0.1 mM), if
necessary.
Firstly, 1 µL of the compound DMSO stock solution was pipetted into
the well, then 10 µL of the enzyme-metal-buffer solution was added.
Lastly, 69 µL of the substrate-buffer solution was added. For Fe2+, the
substrate-buffer solution was left for 2 min after the addition of ascor­
bate to make a reducing environment and then used as usual. The
mixture was slightly shaken, and the measurement of fluorescence
started immediately; no preincubation was done.
Residual activity (RA) was calculated as the slope of the starting
linear part of the detected signal for the measured compound divided by
the corresponding signal of the blank (1 µL DMSO). The measurement
was done on Synergy H4 Hybrid Reader (BioTek Instruments, USA)
using kinetic measurement (15 min, at 25 ◦ C, fluorescent detection, λex
= 360 nm, λem = 460 nm). Results were processed using Gen5 software
(BioTek). The best active compounds (residual activity at or below
approx. 30%) were used for the determination of IC50. RA were plotted
against log(c) using non-linear regression (four-parameter model) as
implemented in GraphPad Prism 9.0.2 (GraphPad Inc, USA). Measure­
ments were done in triplicates. As a positive control, we used clioquinol
(CQ), a well-known inhibitor of MtMetAP1a [37].
3
Bioorganic Chemistry 118 (2022) 105489
2.3. Antimicrobial activity and cytotoxicity screening
Antimicrobial activity and cytotoxicity screenings were performed as
published previously [32]. For the full description see the Supplemen­
tary material.
2.4. In silico studies
For the full description of the in silico studies, see the Supplementary
material.
3. Results and discussion
3.1. Synthesis
The nucleophilicity of the amino group in 3-aminopyrazinoic acid is
low. Thus, different protocols were investigated to increase the feasi­
bility of the acylation reaction. We started with conducting the reaction
at the laboratory temperature in dry acetone or acetonitrile. Yet, the
yields were very low until the reaction was heated up with a significant
excess of the acyl chloride (3–4 equivalents).
Two different synthetic routes were followed (see Fig. 2). Primarily,
we used straightforward Route A. Compound 1 was prepared in bulk and
used as a starting point for other derivatives. Route B generally showed
better yields and easier purification but was more laborious. Therefore,
it was used to prepare compounds that were difficult to purify or of
insufficient quantity for testing. After the first step of Route B, a diacy­
lated product was often observed (isolated and confirmed by NMR and
MS analyses), which was cleaved to intended monoacylated derivative
(Fig. 2, c) using previously published methodology [38]. Pyridine de­
rivative 21 was prepared analogously starting from 3-aminopicolinic
acid. The isolated yields of different steps are presented in Fig. 2.
The data for all synthesized compounds were in agreement with the
proposed structures based on 1H- and 13C-NMR, as well as IR and HR-MS
analyses (see Supplementary material). Purity of final compounds was
analyzed by HPLC-MS.
Spectroscopic characteristics of final compounds included two
distinguishable 1H-NMR signals indicating the presence of amides
around 12.6 and 11.5 ppm. The hydrogens of pyrazine were observed as
two doublets around 8.7 and 8.6 ppm (J = 2.5 Hz). Thiazole hydrogens
were also seen as two doublets at 7.5 and 7.3 ppm (J = 3.6 Hz). Signals
of phenyl hydrogens were observed at the usual ppm range. In some
compounds, not all carbons were clearly observable in 13C-NMR spectra
due to signals merging, e.g. in the case of 14 (R = 4-Br). In all fluorinated
derivatives, the C-F signal splitting was observed.
Strong amide and C– – C vibrations were observed in the IR spectra.
Distinct signals for each amide were observed. The stretching of the
amidic carbonyl at C-3 of the pyrazine ring (further denoted as υNHCO)
had a higher wavenumber, approx. 1700 cm− 1, while the second amide
stretching (υCONH), originating from the pyrazine-2-carboxamide
moiety, was found around 1650 cm− 1. The vibrations were assigned
based on a representative theoretical IR spectrum obtained from the
quantum mechanical calculation of compound 12 used for further
studies in the section In silico studies (for spectrum see Figure S3 in the
Supplementary material). Additional strong signals were observed in the
range of 1450–1600 cm− 1, typical for (hetero)aromatic amides (CONH
bending and C– – C stretching vibrations). Vibrations are indicated in the
Analytical section in the Supplementary material. As determined by the
HPLC, the purities of the final derivatives used for the biological eval­
uations were > 95.0%, except for derivative 3 (R = 2-Me), with 94.2%
purity. Errors in the HR-MS did not exceed 1.39 ppm.
3.2. MtMetAP1a inhibition
Based on the structural characteristics and the presumed MoA, all
compounds were screened for inhibitory activity against MtMetAP1a.
M. Juhás et al. Bioorganic Chemistry 118 (2022) 105489

Fig. 2. Synthesis of the final compounds. Reagents and conditions: (a) 2-aminothiazole, CDI, DMSO(anh), 70 ◦ C, 1 h; (b) substituted acyl chloride, pyridine(anh)/
MeCN, 70 ◦ C, 24 h; (c) iPrOH, hydrazine, THF(anh), reflux, 30 min.

The inhibition was measured as a fluorescence change due to the con­
version of Met-AMC (L-methionine 7-amido-4-methylcoumarin) to AMC
in the presence of the enzyme [39]. Inhibitory activity was expressed as
a percentage of the initial velocity of the reaction in the presence of the
compound relative to the control reaction (DMSO), i.e., residual activity
(RA). Clioquinol (CQ), a well-known inhibitor of MetAPs was used as a
reference [37]. Measurements were done in triplicates; average values
are indicated in Table 1, the SD values of measurements were within
10% if not indicated otherwise. The most active compounds, observed
residual activity at or below approx. 30%, were investigated further to
determine their IC50, obtained values are presented in Table 2. For the
measured single-point RA values and the IC50 curves see Figure S1 and
Figure S2 in the Supplementary material.

Table 1 3.2.1. Role of the metal ion

Results of the MtMetAP1a enzyme inhibition assay with different ions, expressed Metallo-selective inhibitors of MetAP1 have already been described
as residual activity RA (%) at 12.5 µM in the literature [12,18,40]. We thus investigated inhibition properties
using the most studied metal cofactors Ni2+, Co2+, Fe2+ and Mn2+. In
our case, a preference towards Ni2+ and Fe2+ ions was observed. The
activity of the title compounds varied greatly depending on the used
metal. However, the same trends were followed; active compounds were
active with all metals to some extent, except for Mn2+, as discussed later.
Compound X R / structure Co2+ Ni2+ Fe2+
As seen in Table 1, all compounds were the least active in the pres­
1 – – 67% 35% 56% ence of Co2+. The RA measured for the compounds was generally high.
2 N H 51%* 24% 37%
This means the inhibition was weaker compared to the other metals. The
3 N 2-Me 56% 19% 38%
4 N 3-Me 85% 50% 60%
strongest inhibition was seen for the compounds 2 (R = H, RA = 51%), 3
5 N 4-Me 96% 44% 53% (R = 2-CH3, RA = 56%), and for the compound 12 (R = 2-Br, RA =
6 N 2-F 70%* 28% 52% 51%). Thanks to higher solubility in water, we were able to test com­
7 N 3-F 91% 50% 61%* pound 1 at 125 µM, obtaining RA value of 49%.
8 N 4-F 93%* 35% 52%
9 N 2-Cl 61%* 18% 36%
10 N 3-Cl 88% 39% 66%
11 N 4-Cl 92% 40% 80%
Table 2
12 N 2-Br 51%* 21% 33% Inhibition of MtMetAP1a in the presence of Ni2+
13 N 3-Br 81% 46% 66%* cofactor, expressed as IC50 (µM).
14 N 4-Br 91%* 55% 80% Compound IC50 (µM)
15 N 2-CF3 83% 25% 39%
16 N 3-CF3 95%* 61% 109% 2 2.3
17 N 4-CF3 99% 62% 99% 6 3.0
18 N 4-tBu 83% 75% 100% 8 3.5
19 N – 106%* 36% 57% 9 1.1
20 N – 113% 55% 100% 12 0.7
21 C 2-F 70% 48% 54% CQ 11.01
CQ – – 41 %1 49 %1 59 %1 1
literature value IC50 = 9.25 [37]; CQ – clio­
1
RA determined at 125 µM; *SD above 10% - for the error bars see Figure S1 in quinol; for the concentration–response curves see
the Supplementary material; CQ – clioquinol Figure S2 in the Supplementary material

4
M. Juhás et al. Bioorganic Chemistry 118 (2022) 105489

The highest inhibition of MtMetAP1a was observed with Ni2+ (see a sterically demanding substituent) is favorable for the inhibitors of
Table 1, Table 2, and Figure S1 and S2 in the Supplementary material). MetAP1 and should be preferred in further development of optimized
Activity difference was high. In the derivative 12 the RA decreased from inhibitors.
51% to 21% for Co2+ and Ni2+, respectively. Similar decrease of activity The last point is the effect of the isosteric replacement of pyridine
was noticed also in several other compounds. with pyrazine. An improvement in inhibition was seen in the pyrazine
Most active compounds inhibiting MtMetAP1a with Ni2+ exerted containing inhibitor (6) over the pyridine isostere (21), RA = 28% (IC50
some activity also in the presence of Fe2+, which is considered the = 5.0 µM) to RA = 48%, respectively. However, this statement is based
physiologic ion. Compounds 2, 3, 9, 12 and 15 showed the highest only on one representative pair of compounds and needs to be further
inhibitory activity with the Fe2+ with RA ranging from 33% to 40%. verified. We believe pyrazine is underestimated in the current research
The reason of the observed metal-dependent activity is probably the even though there are several successful pyrazine-containing drugs [42].
strength of the metal–ligand interaction as described elsewhere by
Huang and coworkers [41]. 3.3. In vitro determination of antimycobacterial activity
None of the compounds showed inhibition at the tested concentra­
tions with the Mn2+ (values not presented in Table 1). Presumably, this In vitro antimycobacterial activity was determined using modified
is due to the overall low catalytic activity of MtMetAP1a in the presence Microplate Alamar Blue Assay (MABA [43]) on Mycobacterium tubercu­
of Mn2+ as described by Lu and coworkers [12]. According to their re­ losis H37Ra ITM-M006710 (ATCC 9431), M. aurum DSM 43999 (ATCC
sults, MtMetAP1a has the lowest catalytic activity with Mn2+ of all 23366) and M. smegmatis DSM 43465 (ATCC 607). Compounds with the
tested metals, while it is the most active with Ni2+, followed by Co2+ and highest enzyme inhibition activity and/or activity against H37Ra were
Fe2+. also tested against virulent Mtb H37Rv CNCTC My 331/88 (ATCC
We observed a clear correlation of water solubility and enzyme in­ 27294) and M. avium CNCTC My 80/72 (ATCC 15769). The minimum
hibition. Compound 1, with the highest solubility in water medium inhibitory concentration (MIC) values were measured in µg/mL. To ac­
among the tested derivatives was able to distinctly inhibit the enzyme count for molecular weight, the inhibitory activities (expressed as MIC)
with all three metal cofactors, although higher concentration was were recalculated to µM for a clearer comparison. For the complete
needed for Co2+. We propose that improving the water-solubility of our methodology, please see the Supplementary material.
Ni2+-based strong inhibitors could thus lead to higher inhibition also Thanks to its comparable drug susceptibility to Mtb and its fast-
with other metals. growing character [44], M. smegmatis and other fast-growing myco­
bacteria (e.g. M. aurum) are commonly used as surrogates, non-
3.2.2. Structure-activity relationships for MtMetAP1a inhibition pathogenic models for antimycobacterial research. Mtb H37Ra, a non-
The structure–activity relationships described in the following sec­ virulent strain of Mtb H37Rv has also proven an accessible and valid
tion are general. However, specific values are valid for the inhibition in alternative for development purposes [45,46].
the presence of Ni2+, if not stated otherwise. It is worth noticing that the In Mtb H37Ra (all compounds were tested), the best anti­
majority of compounds were more active (in terms of RA) against the mycobacterial activity was observed for derivative 20 containing
MtMetAP1a than the reference, clioquinol, at ten times lower 1–adamantoyl fragment with MIC = 15.625 µg/mL. Moderate activity
concentration. was also seen for compound 1 (non-substituted amino group), and
In most cases, the substitution of the phenyl ring increased the compound 4 (R = 3-CH3), both with MIC = 31.25 µg/mL.
inhibitory activity. Compounds containing the substituent in position 2 All compounds possessing activity against Mtb H37Ra were also
of the phenyl ring were the most active. The inhibition increased with active against M. smegmatis and M. aurum, suggesting a non-Mtb specific
the size of the substituent. While the overall best activity (RA = 21%, mechanism of action. The best results are presented in Table 3; the rest
IC50 = 0.7 µM) was observed for the derivative containing 2-Br substi­ of the compounds were inactive at tested concentrations (MIC > 31.25
tution (12), for much smaller fluorine (compound 6) the inhibition µg/mL). Possible reasons for such a low number of strong enzyme in­
decreased almost 5-fold to 3.0 µM. This value is close to the unsub­ hibitors to inhibit cell growth are discussed later.
stituted phenyl derivative 2 (IC50 = 2.3 µM). Compounds 1–4, 6, 9, 12, 15, 19 and 20 were further investigated
Moving the substituent from position 2 to position 3 had an against virulent strain of Mtb H37Rv and against M. avium. Results
inhibition-decreasing effect, e.g. the difference between 12 (R = 2-Br) against H37Rv agreed with the activity against H37Ra. Highest growth
and 13 (R = 3-Br) is RA = 21% against RA = 46%, respectively. A similar inhibition was seen for compounds 1, 4 and 20, for the MIC values see
trend was also observed for other positional-isomer pairs. A special case
involves fluorinated compounds (6–8). The differences in inhibition
Table 3
activity among the positional isomers exist, although they are small, and Results of antimycobacterial screening represented as MIC values in comparison
the compounds are overall only somewhat better inhibitors than the with standardsa
non-substituted phenyl. Therefore, we propose that the ability of the
MIC in µg/mL (µM)
title compounds to inhibit MtMetAP1a is positively correlated with the
non-coplanarity between the pyrazine and phenyl ring, introduced by Compound Mtb M. aurum M. smegmatis Mtb M. avium
H37Ra H37Rv
large substituents in the ortho position of the latter ring. This conclusion
is consistent also with the fluorine derivatives, as fluorine is roughly the 1 31.25 15.625 125 (565.0) 50 >100
(141.3) (70.6) (226.0)
same size as hydrogen and therefore, the rotational hindrance is weak.
4 31.25 3.91 (11.5) 62.5 (184.2) 12.5 >100
Moving the substituent further from position 3 on the phenyl ring to (92.1) (36.8)
position 4 had a much smaller effect on inhibition than switching from 20 15.625 3.91 (10.2) 62.5 (163.0) 25 25 (62.5)
position 2 to 3. The effects of phenyl substitution on the conformation of (40.7) (62.5)
the compounds were further studied in silico and are discussed in the In INH 0.25 3.91 7.81–15.625 0.2 (1.5) 25
(1.8) (28.5) (56.9–113.9) (182.3)
silico studies section.
RIF 0.003 0.39 1.56–3.125 n.t. n.t.
Our results suggest that 2-substitution of the phenyl ring in the 3- (0.004) (0.5) (1.9–3.8)
benzamido part of the molecule is more favorable for inhibition of CPX 0.25 0.016–0.031 0.063–0.125 n.t. n.t.
MtMetAP1a than 3- or 4-substitution. The same activity trend within (0.8) (0.05–0.09) (0.2–0.4)
structurally related EcMetAP1 inhibitors was also seen in the original a
Compounds not listed in the table were inactive (MICH37Ra > 31.25 µg/mL and
publications by Luo and Cui and colleagues [26–28]. This further con­ MICH37Rv > 100 µg/mL); INH – isoniazid; RIF – rifampicin; CPX – ciprofloxacin;
firms and broadens our conclusions that 2-substitution (preferably with n.t. – not tested.

5
M. Juhás et al. Bioorganic Chemistry 118 (2022) 105489

Table 3. Importantly, compound 20 also showed potency against cytotoxic of all the tested compounds with IC50 > 250 µM. Distinct in
M. avium, being three times more active than INH when molecular vitro cytotoxicity (Hep G2) was seen for the compounds that were also
weight is taken into account. Other tested compounds were inactive at the most active against Mtb, compounds 4 (IC50 (Hep G2) = 66.7,
tested concentrations (MIC > 100 µg/mL). Since M. avium is generally MICH37Ra = 92 µM) and 20 (IC50 (Hep G2) = 68.0, MICH37Ra = 41 µM).
highly resistant to common antimycobacterial drugs [47], we consider Based on similarities of MetAPs among organisms, we assume the
derivative 20 the most promising antimycobacterial agent of the pre­ toxicity might be caused by inhibition of respective human MetAPs. This
sented series. is also supported by high similarity with the already mentioned pyridine
A limited yet discernible correlation of enzyme inhibition and whole- derivatives inhibiting HsMetAP1 [29]. On the other hand, our title
cell Mtb activity was seen in three compounds, namely 1, 4 and 20. It is compounds did not exert general non-specific cytotoxicity as most of the
important to underline that in 1 and 4 we also observed some activity bacterial and fungal strains were unaffected.
against MtMetAP1a with the Fe2+ cofactor, considered physiologic (see No Hep G2 cytotoxicity was observed for compound 1, which is
the discussion above). Translation of in vitro enzyme inhibition to structurally distinct from the above-mentioned compounds, lacking the
growth inhibition in a whole-cell assay is a general problem not only in substituent on the 3-amino moiety. It is not known whether the decrease
antimicrobial research. Thus, even though iron-based MetAP1 inhibitors in cytotoxicity is structure-specific or rather connected to significantly
have already been successful in inhibiting cell growth [16,17], this is better solubility of compound 1.
only true if the compound is able to penetrate through the cell mem­
brane and reach the target. In our title compounds, problems with
3.6. In silico studies
penetration might have been encountered and we deem it to be another
reason (besides low solubility in water) why only some of the enzyme-
Due to the striking difference in the inhibition of MtMetAP1a be­
inhibiting compounds showed whole-cell antimycobacterial activity.
tween compounds substituted at position 2, 3 or 4 of the phenyl ring, we
Membrane transport requires a specific balance of many factors, such as
decided to investigate possible bioactive conformations using compound
the size of the molecule, sufficient water and lipid solubility, polar
12 (R = 2-Br) and 13 (R = 3-Br) as representative examples. We pre­
surface area, and conformational flexibility. Unfortunately, models
sumed that the non-coplanar conformation between the pyrazine and
summarizing the effects of these factors are limited, so the estimation of
the phenyl ring, caused by sterically demanding ortho substituent, might
drug penetration to mycobacteria is virtually impossible without in vitro
be convenient for binding to MtMetAP1a.
testing. More detailed investigations are thus needed to fully understand
the phenomenon of limited activity on whole cells.
3.6.1. Investigations of the lowest energy conformation
For both model compounds 12 and 13, the initial low-energy
3.4. In vitro screening of antibacterial and antifungal activity
conformation was generated by force-field minimization in
MOE2020.09. This conformation was further optimized through quan­
As a complementary test, all compounds were submitted to anti­
tum mechanics (QM) in Gaussian 09 Rev D.01 at B3LYP/cc-pVTZ level
bacterial and antifungal screening based on a microdilution broth
of theory. The plane of the 3-carbonylamino moiety lies in the plane of
method according to EUCAST [48–50]. For methodology and the com­
the pyrazine ring. Therefore, the dihedral angle θ defined as
plete list of tested strains, see the Supplementary material. MIC values
O–– Ccarbonyl-C1′ phenyl-C2′ phenyl (see Fig. 3) effectively represents the
were expressed in µM.
angle between the planes of pyrazine and phenyl ring. In the QM opti­
Compounds 5, 10, 16, 18, 20 were insoluble in the medium and were
mized structure, we measured θ = 125.4◦ for the 2-Br derivative (12)
not tested. Derivatives 11, 14, 19 were tested up to 250 µM; derivatives
and θ = − 8.0◦ for the 3-Br derivative (13), fulfilling the expectation of
6, 7, 13, 21 were tested up to 125 µM, the rest were tested up to 500 µM.
strong non-coplanarity of 12 due to the ortho substitution.
None of the compounds showed any noticeable activity against the
Afterwards, a dihedral scan of θ was performed to uncover the
testing strains at the tested concentrations. Previously published com­
relationship of energy and dihedral angle; the energy was evaluated by
pound 21, which is an inhibitor of EcMetAP1, showed low activity
QM (B3LYP/cc-pVTZ). For the 2-Br derivative 12, the lowest energy was
against Staphylococcus aureus with MIC = 15.6 µg/ml (approx. 46 µM) as
seen at around 120◦ , and the second, energetically close minimum was
described by Luo and coworkers [28]. In our screening, the compound
around 40◦ . The highest energies were observed for 180◦ and 0◦ . For the
was inactive (MIC > 125 µM). This discrepancy may be due to different
3-Br derivative, the lowest energy was seen at around 20◦ , with the
experimental settings and/or different strain.
second minimum being at 160◦ . The highest energy was observed at 90◦ .
For graphical representations of the dihedral scan, see Figure S4 and
3.5. In vitro cytotoxicity determination
Figure S5 in the Supplementary material.
Our in silico calculations for isolated model molecules confirmed that
Due to the similarity of the title compounds with the already pub­
the 2-Br substituted derivative 12 strongly prefers the non-coplanar
lished inhibitors of human MetAP1 [29], cytotoxicity of all compounds
was tested in human hepatocellular carcinoma cell line Hep G2, using a
commercial colorimetric assay based on the reduction of tetrazolium
dye MTS in living cells to formazan. The parameter IC50 was used as a
measure of cytotoxicity, which allows the quantitative comparison of
the toxicity among tested compounds. The complete methodology is
described in the Supplementary material.
The majority of compounds were non-cytotoxic against the Hep G2
at the tested concentration range. However, evaluation of cytotoxicity
was highly impacted by low solubility in the testing water-based me­
dium. As a consequence, compound 6 was tested up to 100 µM, com­
pounds 2, 16, 18, 19 up to 50 µM, compounds 3, 5, 7–9, 12, 15, 21 up to
25 µM and compounds 10, 11, 13, 14 and 17 up to 10 µM. The results
are presented in the Supplementary material in Table S1. The distinct
value could not be determined due to insufficient solubility at higher
concentrations. Fig. 3. Definition of angle θ used for the torsion scan exemplified on the low-
Compound 1, which showed activity against Mtb, was the least energy conformation of compound 12.

6
M. Juhás et al.
conformation. The interactions inside the binding pocket could
dramatically influence the conformation of the bound ligand. Yet, the
binding modes close to the low energy conformations are energetically
more favorable than those requiring the ligand to attain a “forbidden”
geometry.
3.6.2. Molecular docking to MtMetAP1c
No crystallographic structures of the MtMetAP1a are available to
date, but various high-quality experimental structures for the isoform 1c
are available in the RCSB PDB database. We decided to perform the
molecular docking of the most active compound (12, R = 2-Br) into
MtMetAP1c to evaluate the potential of the title compounds to inhibit
this isoform. This is of particular interest as differences in inhibition of
1a vs 1c isoforms have already been observed [12]. For comparison,
compound 12 was also docked to EcMetAP1 (PDB ID: 2Q96) and the
obtained poses were compared to the previously described docking pose
of pyridine-2-carboxamides in EcMetAP1 [28].
Two different crystallographic structures of MtMetAP1c were used to
cover the conformation flexibility of the protein binding site. The pro­
tein conformation in PDB ID: 3IU7 (Fig. 4, green) is similar to EcMetAP1
(orange) in terms of the conformation seen for the Phe211 side chain
(corresponds to Phe177) and the size of the binding cavity governed by
the position of Trp255 (Trp221), i.e. “open” conformation. On the
contrary, the “closed” conformation of MtMetAP1c in PDB ID: 3IU9
(Fig. 4, blue) has a spatially restricted binding cavity due to the shift of
Trp255 deeper into the active site. Furthermore, a new subpocket
(Fig. 5D) formed by Phe211 being in a different conformation in
comparison to EcMetAP1 is observed.
Overall, our docking experiments for compound 12 to EcMetAP1 and
to the open conformation of MtMetAP1c (PDB ID: 3IU7) predicted the
same metal-binding pattern as described previously [28]. The thiazole
nitrogen of 12 was positioned directly in front of metal M2 (2.6 Å in both
enzymes), and the carbonyl oxygen of the pyrazine-2-carboxamide
fragment directly interacted with M1 (2.5 Å in both). The second
interaction of the said carbonyl oxygen formed either an H-bond to
His212 (MtMetAP1c) or another interaction to metal (EcMetAP1), see
Fig. 5 A and B, respectively. In both enzymes, the phenyl ring was in
non-coplanar conformation, stabilized by His114/His79 (MtMetAP1c/
EcMetAP1).
Docking to the closed conformation of MtMetAP1c (PDB ID: 3IU9)
resulted in a significantly different pose of 12. The new subpocket
formed by Phe211 was occupied in majority of poses of 12. We observed
both π-π sandwich (comparably to the 3IU9 cocrystallized ligand) as
well as T-shaped interactions with Phe211. The carbonyl oxygen of the
Fig. 4. Superposition of EcMetAP1 (PDB ID: 2Q96 – orange) and two confor­
mations of MtMetAP1c (PDB IDs: 3IU7 – green, 3IU9 – blue) with emphasis on
the positions of Phe211 and Trp255.
7
Bioorganic Chemistry 118 (2022) 105489
pyrazin-2-carboxamide fragment was interacting with the catalytic M1,
similarly to heteroaromatic nitrogen in the original ligand of 3IU9
(Fig. 5 C). To investigate the effect of the position of Trp255 on possible
binding mode, the pose of compound 12 resulting from the docking to
EcMetAP1 was superposed with the closed conformation of MtMetAP1c
(PDB ID: 3IU9). Strong van der Waals clashes were observed between
the ligand and the closed conformation receptor (Fig. 5 D), originating
particularly from Trp255. This adds to the explanation why distinct
binding modes were observed after docking to the open and closed
conformation of MtMetAP1c.
In all presented docked poses (to EcMetAP1 and both open and
closed conformations of MtMetAP1c), the 2-Br substituted phenyl ring
was non-coplanar to the pyrazine, in agreement with our QM calcula­
tions of the most stable conformation of isolated compound 12.
The isoforms MtMetAP1a and 1c are homologous to great extent.
Still, significant differences in inhibition of the two isoforms by the same
compounds were observed [12]. These can be explained, for example, by
different active site residues (as discussed earlier in the Introduction) or
possibly by the presence of a longer N-terminal sequence implicated in
the regulation of the active site entrance [14]. The most significant
change in the active site residues is the replacement of Met192 in iso­
form 1a with Phe211 in isoform 1c. Since the majority of our compounds
are aromatic derivatives, they could profit from this exchange by being
better stabilized in the binding pocket. This assumption was rationalized
for title compounds through the docking experiments to the unique
crystal structure of the 1c isoform, PDB ID: 3IU9. We thus propose that
the presented derivatives, especially those with the aroyl substituent on
3-amino group of the pyrazine ring, would also be able to inhibit the
isoform 1c of mycobacterial MetAP1. With an already experimentally
observed inhibition of MtMetAP1a, the ability to inhibit both isoforms
would be highly desirable for a potential drug.
4. Conclusion
We have presented 21 derivatives, out of which 20 have not been
previously described in the literature. Several compounds proved to be
low micromolar inhibitors of MtMetAP1a (while using the Ni2+ as
cofactor), best inhibitory activity was observed for compound 12. The
docking studies also suggested probable inhibition of MtMetAP1c. Three
compounds (1, 4 and 20) exerted moderate antimycobacterial activity
and low-to-moderate cytotoxicity. The level of Hep G2 cytotoxicity of
compounds 4 and 20 was close to their antimycobacterial MIC values.
This is caused by low in vitro antimycobacterial activity rather than high
cytotoxicity. The reason why inhibitors of isolated MtMetAP1a are
relatively weak inhibitors of whole-cell growth were discussed in Sec­
tion 3.3, (low water solubility, low membrane penetration). The
observed cytotoxicity/whole-cell activity ratio is not feasible for a final
antimycobacterial drug candidate, but the compounds might be useful
starting points for further optimizations. No activity against bacteria and
fungi was detected. Our results imply that the compounds active in the
antimycobacterial whole-cell assay could indeed exert their activity
through inhibition of MtMetAP1a.
As a direct continuation of this work we propose isosteric replace­
ment of the 2-aminothiazole fragment e.g. for the 2-aminooxazole. This
structural modification could improve the overall polarity of discussed
compounds, and as a consequence increase their water solubility [51],
drug penetration and the whole-cell activity. Increased water solubility
would allow testing the inhibitory activity at higher concentrations that
could result in more detailed structure–activity relationships. Oxazole as
an isostere of thiazole is also stripped of the undesirable metabolic
instability (S-oxidation [51]), which could have also been responsible
for the overall low whole-cell activity of our compounds.
Furthermore, studies on the inhibition of various isoforms of human
MetAPs could open a new direction of research on the title compounds.
Inhibitors of HsMetAP2 have previously been studied as potential anti-
cancer or anti-obesity drugs [52], and compounds 4 and 20 present
M. Juhás et al.
Fig. 5. Docking pose obtained for 12 in A: MtMetAP1c (PDB ID: 3IU7), B: EcMetAP1 (PDB ID: 2Q96). C: Two binding poses of 12 occupying the Phe211-formed
subpocket (PDB ID: 3IU9). D: Pose obtained in EcMetAP1 superposed onto MtMetAP1c (PDB: 3IU9). Dark red cylinders near Trp255 represent strong van der
Waals clashes. (Color annotations: EcMetAP1, PDB ID: 2Q96 - orange; MtMetAP1c, PDB IDs: 3IU7 - green, 3IU9 - blue).
good candidates in these indications.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
Computational resources were supplied by the project “e-Infra­
struktura CZ” (e-INFRA LM2018140) provided within the program
Projects of Large Research, Development and Innovations In­
frastructures. The authors would also like to sincerely thank Dr. Court­
ney Aldrich for kindly providing MtMetAP1a and Dr. Ghada Bouz for
reviewing the manuscript.
Funding
This research was supported by the Czech Science Foundation proj­
ect No. 20-19638Y, Grant Agency of Charles University (Project No.
349721), SVV 260 547, and also by the Slovenian Research Agency
(research core funding No. P1-0208 and a Young Researcher Grant to K.
G.).
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105489.
8
Bioorganic Chemistry 118 (2022) 105489
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