N-Pyrazinoyl substituted amino acids as potential antimycobacterial agents-the synthesis and biological evaluation of enantiomers

molecules

Article

N-Pyrazinoyl Substituted Amino Acids as Potential

Antimycobacterial Agents—the Synthesis and

Biological Evaluation of Enantiomers

Martin Juhás ^1,

* ,

, Lucie Kucˇerová ¹, Ondrˇej Horácˇek ¹, Ondrˇej Jand’ourek ¹

Vladimír Kubícˇek ¹, Klára Konecˇná ¹, Radim Kucˇera ¹, Pavel Bárta ¹, Jirˇí Janoušek ¹

,

Pavla Paterová ², Jirˇí Kuneš ¹, Martin Doležal ¹and Jan Zitko ^1,

*

1

Charles University, Faculty of Pharmacy in Hradec Kr

á

lové

, Akademika Heyrovsk

é

ho 1203, Hradec Králové,

Czech Republic; kuceroval@faf.cuni.cz (L.K.); horaceko@faf.cuni.cz (O.H.); JANDO6AA@faf.cuni.cz (O.J.);

kubicek@faf.cuni.cz (V.K.); konecna@faf.cuni.cz (K.K.); kucerar@faf.cuni.cz (R.K.);

bartp7aa@faf.cuni.cz (P.B.); janousj2@faf.cuni.cz (J.J.); kunes@faf.cuni.cz (J.K.); dolezalm@faf.cuni.cz (M.D.)

University Hospital Hradec Králové, Department of Clinical Microbiology, Sokolská 581,

500 05 Hradec Králové, Czech Republic; pavla.paterova@fnhk.cz

2

*

Correspondence: juhasm@faf.cuni.cz (M.J.); jan.zitko@faf.cuni.cz (J.Z.);

Tel.: +420-495-067-272 (M.J.); +420-495-067-409 (J.Z.)

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

Academic Editor: Maria João Queiroz

Received: 12 March 2020; Accepted: 25 March 2020; Published: 27 March 2020

ꢀꢁꢂꢃꢄꢅꢆ

Abstract: Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (Mtb), each year

causing millions of deaths. In this article, we present the synthesis and biological evaluations of new

potential antimycobacterial compounds containing a fragment of the ﬁrst-line antitubercular drug

pyrazinamide (PZA), coupled with methyl or ethyl esters of selected amino acids. The antimicrobial

activity was evaluated on a variety of (myco)bacterial strains, including Mtb H37Ra, M. smegmatis,

M. aurum, Staphylococcus aureus, Pseudomonas aeruginosa, and fungal strains, including Candida albicans

and Aspergillus ﬂavus. Emphasis was placed on the comparison of enantiomer activities. None of the

synthesized compounds showed any signiﬁcant activity against fungal strains, and their antibacterial

activities were also low, the best minimum inhibitory concentration (MIC) value was 31.25 µM.

However, several compounds presented high activity against Mtb. Overall, higher activity was seen

in derivatives containing l-amino acids. Similarly, the activity seems tied to the more lipophilic

compounds. The most active derivative contained phenylglycine moiety (PC-d/l-Pgl-Me, MIC <

1.95 µg/mL). All active compounds possessed low cytotoxicity and good selectivity towards Mtb.

To the best of our knowledge, this is the ﬁrst study comparing the activities of the d- and l-amino

acid derivatives of pyrazinamide as potential antimycobacterial compounds.

Keywords: amino acids; antibacterial; antimycobacterial; cytotoxicity; pyrazinamide; tuberculosis

1. Introduction

Tuberculosis (TB) is a highly contagious infection caused by Mycobacterium tuberculosis (Mtb) and

was responsible for over 1.5 million deaths worldwide in 2018, which ranks it in the top 10 causes of

deaths and the leading cause of dying due to a single infectious agent according to the World Health

Organization (WHO) [1]. Most often, TB aﬀects the lungs of the infected individuals, although the

cases of so called extra-pulmonary TB are well documented as well. Despite great advances and many

clinically successful drugs, a desired cure still eludes the reaches of modern medicine [1].

The treatment of TB, as well as other bacterial infections nowadays, is hindered by growing

drug resistance (DR). For TB, the WHO diﬀerentiate multidrug-resistant tuberculosis (MDR-TB) and

extensively drug-resistant tuberculosis (XDR-TB). Infections with MDR or XDR strains constitute an

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Molecules 2020, 25, 1518

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enormous ﬁnancial burden for health-care systems (

for non-DR TB) and pose signiﬁcant therapeutic problems compared to non-DR TB. The most recent

global data reports the success rate for the treatment of MDR-TB at only 56%, for XDR-TB lowered

≥

6000 $ per person for DR TB vs. 1000 $ per person

down to 36%, in comparison with >85% for non-DR TB [1]. The drug-resistant cases require the use of

more toxic second-line antituberculars, often in further combinations with other drugs. In some cases,

even this regimen is insuﬃcient. Thus, the search for new antimycobacterial agents and improvements

of the current ones is still a standing research area.

Mtb is not the only mycobacterium responsible for severe infections. The importance of other

so called nontuberculous or atypical mycobacteria (NTM) increases rapidly [

important NTMs are slow-growing M. avium complex and M. kansasii and rapid-growing M. abscessus

and M. fortuitum, with many others following [ ]. The overall colonization of NTM is enormous. They

2]. Nowadays, the most

3

likely reside in soil, pipes and water supplies, causing problems, e.g., in hospital care. As opposed

to Mtb, the NTM infections do not require reporting to the authorities. Therefore, the incidence of

infections caused solely by NTM is diﬃcult to estimate. Infections involving NTM are often diﬃcult to

handle, employing a combination therapy of standard antituberculars and other antibacterial drugs [

Pyrazinamide (PZA) has been used for the treatment of TB since 1952 [ ]. So far, several

potential mechanisms of action (MoA) have been proposed [ ]; however, none seem to be the deﬁnite

one. Firstly, PZA is activated (hydrolysed) by the enzyme pyrazinamidase to pyrazine-2-carboxylic

acid (pyrazinoic acid, POA), which then acts on several targets [ ]. Previously, the activity of PZA

(after hydrolysis to POA) was attributed only to non-speciﬁc eﬀects linked to the acidiﬁcation of

cytoplasm and the disruption of the membrane potentials [ ]. However, nowadays several speciﬁc

enzymatic targets are being studied. Notable is the inhibition of fatty acid synthase I (FAS-I) [ 10],

4].

9,

the disruption of trans-translation via interaction with ribosomal protein S1 (RpsA) [11], the inhibition

of aspartate decarboxylase (PanD) [12], and the inhibition of quinolinic acid phosphoribosyltransferase

(QAPRTase) [13]. Several other proteins have been suggested as targets of PZA/POA [14–17]; however,

PanD has recently been suggested by some authors as the likely prevalent target [18].

PZA represents one of the most important drugs in modern TB treatment. Its biggest advantage

over other antitubercular drugs is in vivo eﬃciency (after metabolization) in penetrating into lung

granulomas and thus its ability to act on dormant mycobacteria [19]. This eﬀect is lacking in most of

the ﬁrst-line antituberculars.

In search for enhancing the activity of PZA, several derivatives were prepared [20]. Most promising

results were noted for the ester prodrugs of POA [21]. The inhibition of Mtb growth exerted by

POA esters in vitro, also observed in PZA-resistant strains, was considerable [22]. Nevertheless, only

sterically protected or long carbon chain esters were stable enough in plasma and could thus preserve

their antitubercular activity in vivo

were proposed. These proved to be signiﬁcantly more stable in plasma and mycobacterial homogenate,

although they were consequently inactive against Mtb even in vitro 24]. Our research group had also

contributed towards the preparation of many active compounds containing the PZA core, focusing

mainly on N-aryl substituted compounds presenting micromolar activities in vitro 25 26]. For more

[23,24]. As a solution, N-alkyl carboxamide derivatives of PZA

[

,

information about the current state of the pyrazinamide-derivatives research, the reader is encouraged

to read the recent review by Correa and Fernandes [20].

Derivatives combining the PZA core with amino acid (aa), as presented in Figure 1, have

caught very little attention so far, despite the fact that amino acid derivatization is very common in

antimicrobial research [27–29]. The reason for this is probably the undesired high stability seen in

N-alkyl derivatives of PZA [24]. Up to this point, the only data on the aa derivatives of PZA and

their antimycobacterial activity are those found in older research by Kushner et al. [30], Badie and

Azab [31], and Pinheiro et al. [32]. In a very recent study by Panda et al. [33], more derivatives of

structure presented in Figure 1 were tested on several (myco)bacterial strains showing mild activity

at neutral pH. Nevertheless, molecules of the mentioned structure have been tested for their other

applications, e.g., as antihistamines [34], insecticides, anthelmintics [35], or proteasome inhibitors [36].

Molecules 2020, 25, 1518

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Figure 1. General structure of the proposed compounds. Asterisk symbol (*) denotes position of the

stereo center.

As a potential drug, PZA core substituted with amino acid (aa) may present numerous advantages

in comparison with simple aliphatic or aromatic amides and esters. Compared to amides, small

PZA-derived peptides could be more easily handled by Mtb peptidases due to their natural similarity

to the peptides (compared to amides) while being stable enough in plasma medium to reach the site of

infection (compared to esters). As for any other amidic-type drugs, the hydrolysis by host peptidases

(amidases) diminishing the activity can occur. However, at this stage of the drug design, this issue,

although important, is very troublesome to address without relevant experimental data concerning

the exact MoA and the plasma stability of the proposed compounds. In aa prodrugs of antimicrobial

drug dapson [29], the half-life in human plasma was shown to be in order of tens to hundreds of

minutes, based on the used aa. If such stability was also observed for our title compounds, it would

rank them—at least from the stability perspective—among aliphatic long carbon chain esters of POA

(half-life in order of tens of minutes [24]) which are stable and active in macrophage model of Mtb

infection. Similarly, they would be hydrolysed considerably quicker than aliphatic amides (half-life in

order of thousands of minutes [24]). Therefore, we believe that the proposed derivatives could act

as both eﬃcient and suﬃciently stable prodrugs of POA but at the same time we do not rule out the

possibility that the presented derivatives can be active in their amidic, non-hydrolysed form.

To test these hypotheses, we have decided to synthesize a series of N-pyrazinoyl substituted

amino acid derivatives and test their activity against a range of mycobacterial, bacterial, and fungal

strains available to our research group. Furthermore, to our knowledge, we are the ﬁrst to investigate

the diﬀerences in the biological activities of compounds derived from l- and d-aa in both mildly acidic

(pH 6) and commonly used pH 6.6.

2. Results and Discussion

2.1. Chemistry

Thirty-nine derivatives of PZA coupled with pure l- or d-aa or with aa enantiomeric mixtures

were synthesized (for structure, see Table 1, for SMILES, calculated molecular descriptors, and selected

biological data, see the Supplementary Material). In compound codes, dl signiﬁes racemic mixtures

and d/l denotes the mixture of enantiomers. Several coupling strategies were evaluated concurrently.

Herein, the unsuccessful strategies (Procedures 1–4) are describe only in brief outlines.

Procedure 1: Reacting pyrazinoyl chloride, (prepared as previously described in Zitko et al. [26]),

with free aa and their further esteriﬁcation was found unsuitable. Multiple products (not identiﬁed)

were observed, as indicated by thin-layer chromatography (TLC), and the desired product was very

diﬃcult to separate. Furthermore, due to the very low solubility of aa in organic solvents, water

solutions had to be employed, resulting in the undesirable hydrolysis of the pyrazinoyl chloride,

further diminishing the yield.

Procedure 2: Reacting pyrazinoyl chloride with aa esters (or their hydrochlorides) partially solved

the problems with solubility in organic solvents, although, side-products remained a problem.

Procedure 3: The aminolysis of methyl pyrazinoate with aa ester (hydrochlorides) in ethanol did

not work, even when heated with addition of catalytic amount of 4-(dimethylamino)pyridine (DMAP).

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Table 1. Structures of the synthesized compounds.

Code

Structure

PC-d-Ala

PC-l-Ala

PC-dl-Ala-Me

PC-d-Ala-Et

PC-l-Ala-Et

PC-d-Asp-diMe

PC-l-Asp-diEt

PC-l-SBn-Cys

PC-d-Glu-diMe

PC-l-Glu-diEt

PC-Gly

PC-Gly-Et

PC-d-Ile-Me

PC-l-Ile-Me

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Table 1. Cont.

Code

Structure

PC-dl-Leu-Et

PC-d-Leu-Me

PC-l-Leu-Me

PC-MeAcr

PC-l-Met-Me

PC-d/l-Pgl-Me

PC-dl-Phe-Et

PC-d-Ser

PC-d-Ser-Me

PC-l-Ser-Me

PC-l-OBn-Ser

PC-l-OBn-Ser-Me

PC-d-OtBu-Ser-Me

PC-l-OtBu-Ser-Me

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Table 1. Cont.

Code

Structure

PC-d-Thr-Me

PC-l-Thr-Me

PC-d-OtBu-Thr-Me

PC-l-OtBu-Thr-Me

PC-d-Trp-Et

PC-l-Trp-Et

PC-d-Tyr-Me

PC-l-Tyr-Et

PC-dl-Val-Et

PC-d-Val-Me

PC-l-Val-Me

Procedure 4: The use of standard coupling reagents as 1,1⁰-carbonyldiimidazole (CDI) in

anhydrous tetrahydrofurane (THF) with aa showed good results, although in some cases starting

pyrazinoic acid was still detected by TLC as a small impurity diﬃcult to separate.

Procedure 5: At last, general procedure, as described in Section 3.1.1. (and shown in Figure 2),

was achieved by adjusting Procedure 4. The method employs esters of aa instead of aa with the free

carboxylic group. The increased solubility of the reactants and products in used organic solvents (THF)

greatly simpliﬁed the syntheses and subsequent puriﬁcations. The products were created in good

yields (positively related to their lipophilicity) and without epimerization.

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Figure 2. Schematic representation of Procedure 5. Asterisk symbol (*) denotes position of the stereo

center. TEA—triethylamine.

Based on the extensive knowledge on mycobacterial esterases [37], the use of esters over free

acids was also favoured to potentially increase the bioavailability of our mostly polar title compounds,

which proved to be a correct assumption based on the activities discussed further below.

We tried to synthesize Pro and His ester derivatives. However, these proved to be readily soluble

in water even as esters. Therefore, we were unable to suﬃciently purify them for the biological

screening. PC-MeAcr derivative was obtained as a by-product by the elimination of the hydroxyl of

serine methyl ester, as previously described by Pinheiro et al. [32].

All compounds were characterized by ¹H- and ¹³C-NMR spectroscopy in hexadeuterodimethyl

sulfoxide (DMSO-d₆), IR spectroscopy using the attenuated total reﬂectance (ATR) method, and mass

spectrometry. All analytical results were in agreements with the presumed structures.

1

In H-NMR, the amidic hydrogen was always observed as a clearly distinguishable doublet,

usually around 8.0–9.5 ppm, and the usual J coupling constant ranged between 8.4–8.5 Hz. Pyrazine

hydrogens were observed as two doublets and one multiplet at 9.1–9.4, 8.8–9.1, and 8.6–8.9 ppm,

respectively. C-α hydrogen was observed as a distinguishable multiplet at 4.4–4.6 ppm in all chiral

compounds. The acidic hydrogen in free carboxylic moiety was sometimes not observed due to

rapid exchange with solvent, which is common in DMSO-d₆¹H-NMR. Alcoholic OH hydrogen in

Ser ester derivatives was observed as a clear doublet of doublets or a triplet at 5.3 ppm. In the free

acid Ser derivative, as well as in Thr esters, the OH hydrogen was sometimes indistinguishable. The

representative spectra of free aliphatic aa derivative (PC-l-Ala), ester derivative (PC-l-Val-Me), and

aromatic and phenylglycine (Pgl) aa ester derivatives (PC-d/l-Pgl-Me, PC-dl-Phe-Et) are enclosed in

the Supplementary Material.

In the IR spectra of all the title compounds, the amidic N-H stretching was observed as a

distinguishable absorption band at 3200–3300 cm⁻¹, amidic C=O stretching was observed at 1640–1670

cm⁻¹, and ester C=O stretching at 1730–1750 cm⁻¹. Free acids presented characteristically broad O-H

stretches around 3000 cm⁻¹. In Ser and Thr derivatives, an aliphatic O-H stretch was visible above

3300 cm⁻¹

Amino acid esteriﬁcation [38] and other common peptide synthesis procedures (e.g., tert-butyl

group cleavage) [39 40] used to obtain the title compounds are considered enantioselective. Acidic

.

,

hydrolysis was previously described to potentially cause epimerization or even racemization of aa

derivatives [41]. CDI-mediated coupling in THF had been shown to cause a low degree of epimerization

(5%) on one model peptidic compound [42]. Generally, no epimerization occurred in our conditions.

Epimerization was seen only in the case of Pgl derivative (PC-d/l-Pgl-Me) synthesized using the d-Pgl

methyl ester hydrochloride. The composition of the mixture of enantiomers was further investigated

by optical rotation measurements and chiral HPLC. Comparing the literature value of the speciﬁc

rotation of l-enantiomer ([

α

]

= +88.8^◦[43]) with the measured value of our sample [

α

]

=

47.3^◦, it

−

D

is clear that d-enantiomer dominates the mixture of enantiomers as judged from the sense of rotation.

According to the chiral HPLC, the sample consists of two enantiomers represented by peaks in a ratio

of approx. 2.1:1, based on the area under the curve (AUC). We thus conclude that the compound

PC-d/l-Pgl-Me is a mixture of d- and l-enantiomer in ratio of 2.1:1 in favour for d-enantiomer. The

epimerization of Pgl is known to occur very easily using standard coupling conditions and thus

stereoselective synthesis would require speciﬁc reagents not employed in this work [44]. Chiral HPLC

Molecules 2020, 25, 1518

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analysis was performed for enantiomeric mixture PC-d/l-Pgl-Me, racemic mixtures (PC-dl-Ala-Me,

PC-dl-Leu-Et, PC-dl-Phe-Et, PC-dl-Val-Et) and for the samples of which we had the complete couple

of antipodes. The chromatograms are presented in the Supplementary Material.

2.2. Biological Evaluation

2.2.1. Antimycobacterial Activity Screening

Mycobacterial screening of the synthesized compounds was done using microdilution assay

(Microplate Alamar Blue Assay, MABA [45]) on M. smegmatis DSM 43465 (ATCC 607), Mycobacterium

aurum DSM 43999 (ATCC 23366), and the avirulent strain of Mtb H37Ra ITM-M006710 (ATCC 9431)—for

results, see Tables 2 and 3. The minimum inhibitory concentration (MIC) values were measure in

µ

g/mL. To account for molecular weight, the inhibitory activity (expressed as MIC) of the discussed

compounds were recalculated to M concentration. Fast-growing M. smegmatis and M. aurum are used

as surrogate model organisms in antimycobacterial research [46 47]. Mtb H37Ra is commonly used

µ

,

as an accessible valid alternative for the virulent strain [48]. The selected title compounds were also

tested in pre-screening against a virulent strain of Mtb H37Rv CNCTC My 331/88 (ATCC 27294), M.

kansasii CNCTC My 235/80 (ATCC 12478), and M. avium ssp. avium CNCTC My 80/72 (ATCC 15769).

The activity of PC-MeAcr was in accordance with previously published results (MIC on Mtb H37Rv

was 50 µg/mL in our study as well as in the study by Pinheiro et al. [32]). Although PC-MeAcr might

have been considered cytotoxic due to the acrylate group, no signiﬁcant cytotoxicity was noted on the

human HepG2 cell line, as presented in Table 4. For the full results of this pre-screening, see Table S2

in the Supplementary Material.

For the purposes of this article, only compounds with MIC values lower than 31.25 µg/mL (roughly

corresponding to 100 µM based on molecular weight) were considered as active.

Notable activity was detected only against Mtb H37Ra at pH 6. No signiﬁcant activity was

detected against M. smegmatis at neither pH 6 nor pH 6.6, low activity was noted only for PC-MeAcr

(MIC = 31.25 µg/mL, pH 6.6). Only one compound (PC-d/l-Pgl-Me) showed notable activity against M.

aurum (MIC = 31.25 µg/mL, pH 6). If not stated otherwise, the following discussion concerns activity

against Mtb H37Ra at pH 6.

Limited data on similar aa derivatives of PZA are available in the literature. In the work of

Panda et al. [33], the l-derivatives of non-esteriﬁed aa (Leu, Val, allo-Ile, Met, Phe, Trp) were tested.

These compounds were more or similarly active at pH 7 compared to isoniazid (INH) (> 70% growth

inhibition at 30 µg/mL). However, as a diﬀerent Mtb strain and methodology was used in the study of

Panda et al. [33], direct comparison to our study is not applicable. PC-Asp-diEt and some atypical aa

derivatives of the structure presented in Figure 1 were tested previously by Kushner et al. [30] with no

activity, which is in accordance with our results presented in Tables 2 and 3.

pH Dependence

Based on one of the proposed MoA of our compounds, that is POA-releasing prodrugs, all

derivatives were tested at mildly acidic pH 6 as required for the in vitro activity of PZA/POA, and

the results were compared to the activities at the commonly used pH 6.6 [22]. The speciﬁc pH value

for the acidic measurement was selected to balance the activity of tested compounds with the growth

rate of cultivated mycobacteria [49]; furthermore, a similar pH is also found in macrophages, where

the intracellular pathogen of Mtb resides [50], making these conditions closer to reality. The necessity

of acidic conditions for the in vitro activity of PZA and POA is a well-known paradigm. A similar

pH-activity dependence is known also for POA esters [22]. However, Pinheiro et al. [32] and den

Hertog et al. [51] have shown that the activity of PZA and POA may also be observed under non-acidic

conditions. According to den Hertog et al. [51], the in vitro activity of PZA is probably tied to speciﬁc

metabolic and energetic eﬀects occurring in acidic medium, although they concluded that the same

eﬀects could also be induced by other stressors, e.g., low temperature or starvation. In general, these

Molecules 2020, 25, 1518

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implications do not aﬀect the correctness of the in vitro measurements presented in this manuscript or

elsewhere but rather propose new ways to investigate the MoA of PZA.

Based on the results presented in Tables 2 and 3 , the impact of the pH value on the activity of our

compounds against Mtb was substantial. At least an 8-fold decrease in MIC was observed for all active

compounds, corresponding to the change from hundreds of µg/mL to tens of µg/mL (or lower). The

eﬀect of pH on the activity against other mycobacterial strains was less profound, similar to PZA itself,

which could be suggesting PZA-linked MoA.

Table 2. Antimycobacterial activities of the tested compounds at pH 6.6 presented as the minimum

inhibitory concentration (MIC) (µg/mL), the most active compound is in bold. The chemical structures

of the derivatives are presented in Table 1.

Code

Mtb H37Ra

M. smegmatis

M. aurum

PC-d-Ala

PC-l-Ala

PC-dl-Ala-Me

PC-d-Ala-Et

≥ 500

≥ 250

≥ 500

≥ 125

≥ 500

≥ 250

≥ 500

250

≥ 500

≥ 250

≥ 500

≥ 125

≥ 500

≥ 250

≥ 500

250

≥ 500

250

≥ 500

≥ 250

≥ 500

≥ 125

≥ 500

≥ 250

≥ 500

250

PC-l-Ala-Et

PC-d-Asp-diMe

PC-l-Asp-diEt

PC-l-SBn-Cys

PC-d-Glu-diMe

PC-l-Glu-diEt

PC-Gly

PC-Gly-Et

PC-d-Ile-Me

PC-l-Ile-Me

PC-dl-Leu-Et

PC-d-Leu-Me

PC-l-Leu-Me

PC-MeAcr

PC-l-Met-Me

PC-d/l-Pgl-Me

PC-dl-Phe-Et

PC-d-Ser

PC-d-Ser-Me

PC-l-Ser-Me

PC-l-OBn-Ser

PC-l-OBn-Ser-Me

PC-d-OtBu-Ser-Me

PC-l-OtBu-Ser-Me

PC-d-Thr-Me

PC-l-Thr-Me

PC-d-OtBu-Thr-Me

PC-l-OtBu-Thr-Me

PC-d-Trp-Et

≥ 500

62.5

250

15.625

≥ 500

250

≥ 500

250

≥ 500

125

31.25

≥ 500

250

≥ 500

250

≥ 500

250

PC-l-Trp-Et

125

250

125

PC-d-Tyr-Me

PC-l-Tyr-Et

PC-dl-Val-Et

PC-d-Val-Me

PC-l-Val-Me

≥ 500

250

≥ 500

500

≥ 500

250

PZA

INH

RIF

≥ 500

0.125–0.25

0.0039–0.0078

0.125–0.25

7.81–15.625

12.5–25

1.95–3.91

0.39–0.78

0.0078–0.0156

CPX

0.0625–0.125

PZA—pyrazinamide, INH—isoniazid, RIF—rifampicin, CPX—ciproﬂoxacin.

Molecules 2020, 25, 1518

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Table 3. Antimycobacterial activities of the tested compounds at pH 6 presented as MIC values, most

active compounds are in bold. The chemical structures of the derivatives are presented in Table 1.

Code

Mtb H37Ra

M. smegmatis

M. aurum

logP **

µg/mL

µM *

µg/mL

PC-d-Ala

PC-l-Ala

PC-dl-Ala-Me

PC-d-Ala-Et

≥ 125

≥ 250

≥ 125

3.91

≥ 125

62.5

≥ 500

31.25

≥ 125

250

≥ 640.4

≥ 1280.9

≥ 597.5

≥ 560.0

17.5

≥ 467.7

≥ 423.3

196.9

≥ 125

62.5

−1.0155

−0.8725

−0.5315

−1.3565

−0.6745

0.9855

−0.9145

−0.2325

−1.4775

−0.9935

0.5415

125

≥ 125

≥ 250

≥ 500

≥ 125

250

≥ 125

125

≥ 500

≥ 125

250

PC-l-Ala-Et

PC-d-Asp-diMe

PC-l-Asp-diEt

PC-l-SBn-Cys

PC-d-Glu-diMe

PC-l-Glu-diEt

PC-Gly

≥ 1777.7

101.0

≥ 690.0

≥ 597.5

994.9

PC-Gly-Et

PC-d-Ile-Me

PC-l-Ile-Me

250

994.9

58.9

≥ 500

≥ 125

≥ 500

≥ 250

≥ 125

125

≥ 500

250

≥ 500

250

≥ 500

≥ 125

62.5

31.25

62.5

0.5415

0.8825

0.5415

−0.5555

−0.2175

0.5745

PC-dl-Leu-Et

PC-d-Leu-Me

PC-l-Leu-Me

PC-MeAcr

PC-l-Met-Me

PC-d/l-Pgl-Me

PC-dl-Phe-Et

PC-d-Ser

PC-d-Ser-Me

PC-l-Ser-Me

PC-l-OBn-Ser

PC-l-OBn-Ser-Me

PC-d-OtBu-Ser-Me

PC-l-OtBu-Ser-Me

PC-d-Thr-Me

PC-l-Thr-Me

PC-d-OtBu-Thr-Me

PC-l-OtBu-Thr-Me

PC-d-Trp-Et

15.625

≥ 125

62.5

< 3.91

< 1.95

31.25

≥ 250

250

≥ 500

7.81

≥ 500

62.5

≥ 497.4

301.7

< 14.5

< 7.3

104.4

1.0035

≥ 1183.8

1110.1

≥ 2220.2

≥ 1659.5

24.8

≥ 1777.4

≥ 2090.0

211.6

62.5

250

−2.0505

−1.9075

0.3495

≥ 500

62.5

0.4925

≥ 500

−0.0855

−1.4455

0.3765

62.5

≥ 500

250

≥ 500

62.5

≥ 500

≥ 1693.0

0.3765

1.3695

62.5

184.7

PC-l-Trp-Et

125

369.4

250

62.5

1.3695

PC-d-Tyr-Me

PC-l-Tyr-Et

PC-dl-Val-Et

PC-d-Val-Me

PC-l-Val-Me

125

7.81

≥ 125

414.9

24.8

≥ 497.4

≥ 526.9

≥ 500

≥ 250

≥ 125

≥ 250

≥ 125

0.3545

0.6955

0.4405

0.0995

PZA

INH

RIF

< 3.91

0.125–0.25

0.0016–0.0062

0.25

< 31.8

0.91–1.82

0.0019–0.0075

0.75

≥ 500

250

−1.4595

−0.7970

4.5560

7.81–15.625

6.25–12.5

0.0625–0.25

3.91–7.81

0.39–1.56

0.0625

CPX

1.0370

* calculated from µg/mL, ** logP—calculated in MOE 2019.0101 (CCG, Quebec, Canada), PZA—pyrazinamide,

INH—isoniazid, RIF—rifampicin, CPX—ciproﬂoxacin.

Lipophilicity and Sidechain Eﬀects

Mycobacterial membrane is very lipophilic. Compared to other bacteria, it contains unusually

high concentrations of speciﬁc lipidic molecules, e.g., mycolic acids [52]. Consequently, highly

lipophilic low molecular weight compounds are expected to penetrate the membrane through a simple

passive diﬀusion more easily. Nevertheless, the exact physico-chemical requirements relevant to the

Mtb membrane are still unknown [53]. To assess the impact of the lipophilicity of the synthesized

compounds on their activities, we investigated the relationship between calculated logP and respective

MIC values at pH 6 (as no signiﬁcant activity was recorded pH 6.6). All compounds were categorized

considering their chemical nature (free carboxylic acid/ester) and enantiomer aspects (as discussed

later). The logP (o/w) chemical descriptor, hereafter referred to as logP, was calculated by Molecular

Operating Environment (MOE) 2019.0101 (Chemical Computing Group (CCG), Quebec, Canada) [54].

The resulting scatter plot of logP and antimycobacterial activity presented as log(1/MIC [M]) (a higher

Molecules 2020, 25, 1518

11 of 29

value means better activity) is shown in Figure 3. The calculated logP and MIC (

µM) values at pH 6

are contained in Table 3.

Figure 3. Scatter plot representing antimycobacterial activity and calculated lipophilicity of the

compounds. The colours denote stereochemistry; the shape of the symbols distinguishes between

esters and free acids. Gly and MeAcr derivatives are presented as green triangles. The MIC values of

the majority of the derivatives in the non-active region were not determined precisely (see Table 3).

Clearly, for the same aa derivatives, the least active compounds in categories free acid–methyl

ester–ethyl ester are embodied by derivatives with free carboxylic moiety (represented by gems in

Figure 3). Furthermore, ethyl esters (represented by squares) were generally more active than methyl

esters in our study. This general observation can be attributed to their higher lipophilicity (as indicated

by increased logP in Table 3 ) compared to the free acids. As an illustrative example, in l-Ala derivatives,

the MIC value of the ethyl ester is at least 64-times lower compared to that of the free acid (PC-l-Ala-Et

MIC = 3.91

for ethyl ester vs.

µ

g/mL vs. PC-l-Ala MIC

≥

250

µ

g/mL), with the logP diﬀerence of approximately 0.5 (

−

0.53

−

1.02 for free acid)—line 1 in Figure 3. In Leu and Glu esters, high activity was noted

only for more lipophilic ethyl esters, while the less lipophilic methyl esters were inactive (PC-dl-Leu-Et

vs. PC-d-Leu-Me—line 2 and PC-l-Glu-diEt). In fact, the majority of methyl esters (represented

by circles) are located below the activity cut-oﬀ in Figure 3, with the exception of highly lipophilic

compounds, e.g., PC-l-OBn-Ser-Me, PC-d/l-Pgl-Me. The eﬀect of growing activity with the lengthening

of the ester carbon chain had previously also been observed in derivatives of POA [21,23] or other

antimycobacterial compounds [55] and it has been attributed to the above-mentioned better penetration

inside the mycobacteria. However, as discussed later, other eﬀects—as stereochemistry—seem to play

an important role in structure–activity relationships in our compounds. Therefore, the eﬀect of ester

moiety on activity stays inconclusive.

When comparing the activities of our title compounds, higher activity is seen mostly in derivatives

of aa with hydrophobic aliphatic or aromatic side chains.

In a homologous series of Gly, Ala, Val, Leu, and Ile derivatives, high activity was noted only

for derivatives of Ala and Leu. Interestingly, no activity was seen in the similar lipophilic ester of

Val or in the derivatives of mutual chain isomers Leu and Ile. The highest activity in aliphatic aa

derivatives was seen in Met derivative PC-l-Met-Me, having comparable activity with PC-l-Ala-Et

(MIC = 3.91 µg/mL). The activity of tested polar aliphatic aa derivatives (Cys, Ser, Thr) seemed tied

Molecules 2020, 25, 1518

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mainly to the chemically protected form (active PC-l-OBn-Ser-Me vs. inactive PC-l-Ser-Me with

unsubstituted hydroxyl), supposedly due to a higher lipophilicity of the protected alternative.

High activities were noted for the majority of aromatic aa derivatives. The highest activity of all

tested compounds was seen in PC-d/l-Pgl-Me (MIC < 1.95 µg/mL), and derivatives of Tyr were active

as well. All observed results may be tied to the generally higher lipophilicity of aliphatic and aromatic

compounds; however, enzymatic preferences towards the hydrolysis of speciﬁc amidic bonds may also

play a signiﬁcant role, as discussed later.

Stereochemical Aspects of Activity

Enzymatic substrate speciﬁcity is the cornerstone of catalysis, and enzymes with one-enantiomer

speciﬁcity are known in microorganisms [56,57]. We were therefore curious to investigate the diﬀerences

in activities of the l- and d-enantiomers of the title compounds, as they may suggest either a speciﬁc

MoA of the unhydrolyzed amide, or a more facile hydrolysis of one enantiomer.

As may be seen from Figure 3 , d-enantiomers (shown in red) were in general inactive. However, as

discussed above, caution is needed when comparing enantiomers, as other factors, such as the overall

lipophilicity, may also inﬂuence the activity. Therefore, it would be safe to compare the activities of the

same esters, while comparing activity of diﬀerent esters of the same aa derivative should be judicious.

Most illustrative examples following the above-mentioned rule (d-isomers are inactive) are once

again represented by derivatives of Ala. A comparison of PC-l-Ala-Et with PC-d-Ala-Et (line 3) clearly

shows the preference of the l-enantiomer (MIC = 3.91

l-derivatives being more active were noted as well, although the activities might be biased by the

ester diﬀerences, as in the case of PC-l-Glu-diEt vs. PC-d-Glu-diMe (MIC = 31.25 g/mL vs. MIC =

500 g/mL, line 4) or PC-l-Tyr-Et vs. PC-d-Tyr-Me (MIC = 7.81 g/mL vs. MIC = 125 g/mL). The

µg/mL vs.

≥

125 µg/mL, respectively). Other

µ

≥

µ

activity of racemic or enantiomeric mixtures (painted in grey), as for PC-d/l-Pgl-Me and PC-dl-Leu-Et,

is not possible to attribute to the exact enantiomer; although, based on the overall trends, we propose

l-Leu derivative to be more active. As Pgl is not a natural amino acid, the activity could by tied

to both enantiomers. We were unable to test this hypothesis as the pure l- or d-enantiomers of Pgl

derivative were not synthesized. Finally, both l- and d-Trp derivatives showed similar, although very

low activity against Mtb H37Ra (one or two dilution steps below the cut-oﬀ value). However, based on

the observed cytotoxicity of PC-l-Trp-Et (l-enantiomer of Trp) on HepG2 cell lines (IC₅₀= 146.6 µM,

Table 4), and the unchanged MIC value depending on the pH, we attributed the antimycobacterial

activity of both Trp derivatives to their non-speciﬁc toxicity of unknown nature.

The only case of d-enantiomer being more active than l-enantiomer (in the case of the Pgl

derivative, we are unable to distinguish the active and inactive enantiomer) was in O-tert-butyl-protected

derivatives of Thr (PC-d-OtBu-Thr-Me, MIC = 62.5

µ

g/mL vs. PC-l-OtBu-Thr-Me, MIC

≥

500). The

activity was low, however. The reason for this preference is unknown. Nevertheless, ether-protected

derivatives are in fact non-natural aa. Observed activity is still very interesting, as we noted that

in bacteria the same d-enantiomer showed no activity while the l-enantiomer was active against

Staphylococcus aureus strains (discussed further in Section 2.2.2.).

2.2.2. Antibacterial Activity Screening

All compounds were tested by the microdilution broth method [58] against Staphylococcus aureus

subsp. aureus CCM 4223 (ATCC 29213), Staphylococcus aureus subsp. aureus methicillin-resistant (MRSA)

CCM 4750 (ATCC 43300), Staphylococcus epidermidis CCM 4418 (ATCC 12228), Enterococcus faecalis

CCM 4224 (ATCC 29212), Escherichia coli CCM 3954 (ATCC 25922), Klebsiella pneumoniae CCM 4415

(ATCC 10031), Acinetobacter baumannii DSM 30007, ATCC 19606, and Pseudomonas aeruginosa CCM 3955

(ATCC 27853). The majority of the compounds did not show any activity against bacteria under testing

conditions (the highest concentrations tested were 500

PC-l-SBn-Cys, and 125 M for PC-l-OBn-Ser-Me. Three compounds (PC-d-Asp-diMe, PC-d-Ile-Me

and PC-l-Ile-Me) were insoluble in the cultivation medium and therefore were not tested. PC-l-Ala-Et

µM for the majority of compounds, 250 µM for

µ

Molecules 2020, 25, 1518

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derivative had low activity against some G- bacteria (MIC = 125

µM, see Supplementary Material),

while the d-enantiomer and dl-racemate were both inactive. The PC-l-OtBu-Thr-Me derivative was

active against S. aureus, including MRSA with MIC = 31.25 M, suggesting that the MoA of the

µ

compound is diﬀerent from the beta-lactams. No diﬀerences in MIC values read after 24 h and 48 h of

incubation were noted.

In previous studies, the activities of the methyl esters of PC-Gly, PC-l-Ala, PC-l-Ser, and PC-l-Tyr

against some bacterial strains were evaluated, yet no activity was noted [31], in agreement with the

observations in this article. The activities of some free acid compounds reported in the study of Panda

et al. [33] could be tied to the methodology diﬀerences and strain susceptibility, as no activity was seen

when corresponding esters were used in our studies. For the full results (Table S4) and methodology,

see the Supplementary Material.

2.2.3. Antifungal Activity Screening

All compounds were tested by the microdilution broth method [59,60] against Candida albicans

CCM 8320 (ATCC 24433), Candida krusei CCM 8271 (ATCC 6258), Candida parapsilosis CCM 8260 (ATCC

22019), Candida tropicalis CCM 8264 (ATCC 750), Aspergillus fumigatus ATCC 204305, Aspergillus ﬂavus

CCM 8363, Lichtheimia corymbifera CCM 8077, and Trichophyton interdigitale CCM 8377 (ATCC 9533).

No compound showed any signiﬁcant activity against tested fungal strains up to the highest tested

concentration, which was 500

µM for the majority of compounds, 250 µM for PC-l-SBn-Cys, and 125

µM for PC-l-OBn-Ser-Me. Three compounds (PC-d-Asp-diMe, PC-d-Ile-Me and PC-l-Ile-Me; the same

as for antibacterial testing) were insoluble in the medium and therefore were not tested. No diﬀerences

in MIC values read after 24 and 48 h (72 h) of incubation were noted. For the full results (Table S6) and

methodology, see the Supplementary Material.

2.2.4. Cytotoxicity Screening Results

The cytotoxicity of selected active compounds was measured using the standard human

hepatocellular carcinoma cell line HepG2. The used CellTiter 96 assay is based on the reduction of

tetrazolium dye MTS in living cells to formazan, which is then determined colorimetrically. The

reduction of the reagent is related to availability of NADH and NADPH. The decline in levels of these

metabolically important compounds in the cell leads to the decreased production of formazan.

The parameter IC₅₀was used as a measure of cytotoxicity, which allows for the quantitative

comparison of the toxicity among tested compounds (Table 4). Overall, the toxicity of the compounds

was very low, some compounds had the IC₅₀values above the tested range (PC-l-Ala-Et). The highest

cytotoxicity illustrated by its lowest IC₅₀value was noted for the aromatic compound PC-l-Trp-Et

(IC₅₀= 146.6 µM).

The selectivity index (SI) for Mtb was calculated for the most active compounds according to the

equation SI = IC₅₀(µM)/MIC(µM), as used by Bagla et al. [61], and the results are presented in Table 4.

The tested compounds were more active against Mtb (SI > 1) than their cytotoxic eﬀect against human

cells, represented by the HepG2 cell line cytotoxicity experiment. Two of the most active compounds

(PC-l-Ala-Et and PC-d/l-Pgl-Me) had SI > 40, which suggests a good cytotoxicity proﬁle for potential

further development.

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Table 4. Calculated selectivity index (SI) of the most active compounds against Mtb at pH 6 and pH 6.6.

The compounds with a good cytotoxicity proﬁle (SI > 10) are in bold.

Tested compound

Tested range (µM)

HepG2 IC₅₀(µM)

Mtb pH 6 MIC (µM)

SI pH 6

PC-l-Ala

PC-l-Ala-Et

PC-dl-Leu-Et

PC-MeAcr

PC-l-Met-Me

PC-d/l-Pgl-Me

PC-l-OBn-Ser-Me

PC-l-OtBu-Thr-Me

PC-l-Trp-Et

1–1000

1–500

1–1000

1–500

> 250*

> 1000

788.2

704.2 **

> 100 *

> 1000

742.3

> 500

146.6

177.1

≥ 1281

≥ 0.2

> 57.1

13.4

1.7

18

59

302

15

< 7

25

1693

125

25

6.9

> 68.5

30.0

0.3

1.2

7.2

1–500

1–1000

PC-l-Tyr-Et

PZA

9619.1 ***

31.8

302.9

* The determination of IC₅₀was impossible due to irrelevant values of absorbance at higher concentrations of

compounds caused by their precipitation in cell culture medium, ** the estimated value based on the curve, ***

converted to µM based on the literature value 1184.3 ± 120.2 µg/mL [62].

3. Materials and Methods

3.1. Chemistry

All chemicals were of reagent or higher grade of purity. l- and d-aa were purchased from

Fluorochem (Derbyshire, UK), TCI (Tokyo, Japan), Alfa Aesar (Kendal, Germany) or Sigma-Aldrich

(Steinheim, Germany). The 1,1⁰-carbonyldiimidazole (CDI) reagent was bought from Sigma-Aldrich,

the solvents were from Sigma-Aldrich or Penta (Prague, Czech Republic). Thin-layer chromatography

(TLC) was performed on Silica 60 F₂₅₄TLC plates (Merck, Darmstadt, Germany).

3.1.1. General Procedure of Coupling Reaction

To a round bottom ﬂask, 248 mg (2 mmol) of pyrazinoic acid (in the codes of ﬁnal compounds

denoted as PC) was suspended in anhydrous THF (3 mL), 324 mg (2 mmol) of CDI was added and the

mixture was heated to 60 ^◦C and stirred for 1 h. Afterwards, the ﬂask was cooled down to laboratory

temperature and 2 mmol of amino acid or amino acid ester (mostly in hydrochloride form) was added

to the mixture with 300 µL (2.14 mmol) of triethylamine. After the addition, the mixture reacted

for 2 h at laboratory temperature and was evaporated on a rotary evaporator. The solid residue

was suspended in water (10 mL) and extracted to ethyl acetate (EtOAc) using repetitive extractions

(typically twice with 15 mL) in separatory funnel. After each separation, the presence of the product

in the phases was monitored on a TLC (silica, methanol-EtOAc 1:1). Finally, the organic layers were

combined and washed with 10 mL of 0.01 M HCl and 10 mL of saturated NaCl solution. At the end, the

organic phases were mixed with approximately 50 mg of activated carbon, stirred for 10 min, ﬁltered

and evaporated to dryness. The ﬁnal product was left in the desiccator overnight and puriﬁed further

using ﬂash chromatography if necessary.

3.1.2. General Procedure for Esteriﬁcation of Amino Acids

Methyl esters of d-Ile and O-benzyl-Ser derivatives were prepared as described in Li and Sha [38

]

using 2 mmol of amino acid, 0.5 mL of trimethylsilyl chloride (TMSCl), and 10 mL of methanol. The

reaction mixture was evaporated to dryness and stored in a desiccator.

3.1.3. Deprotection of O-Tert-Butyl-Protected Hydroxy Amino Acids

Deprotection followed a common deprotection protocol.

A total of 0.5 mmol of

O-tert-butyl-protected derivative was suspended in 1 mL of deprotection solution (0.95 mL triﬂuoracetic

acid, 0.025 mL triisopropyl silane, 0.025 mL distilled water) and stirred for 1 h. Subsequently, the

mixture was evaporated to dryness and the product was puriﬁed using ﬂash chromatography.

Molecules 2020, 25, 1518

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3.1.4. General Procedure for Hydrolysis of Esters

The preparation of l-Ala derivative was achieved by acidic hydrolysis of the parent ester. A total

of 0.5 mmol (approx. 100 mg) of ester was heated in 10 mL of 0.02 M solution of hydrochloric acid at

45 ^◦C until the spot of the ester disappeared on the TLC plate. Then, the mixture was left to evaporate,

and crude product was recrystallized from water.

3.1.5. Flash Chromatography

The compounds were adsorbed on silica gel and puriﬁed on a PuriFlash XS420 chromatographic

system (Interchim, Montluçon, France) using a gradient elution. We used a combination of silica gel

and DIOL-silica gel columns (Interchim) to achieve better separation of the compounds. As the mobile

phase, a mixture of hexane and EtOAc was used.

3.1.6. HPLC Analysis and Optical Rotation Measurements

The chemical and enantiomeric purity of the compounds were tested on a Shimadzu

chromatograph composed of a DGU-20A3 degasser, an LC-20AD binary gradient pump, an SIL-20AC

autosampler with a 100-µL sample loop, a CTO-20AC thermostated column oven, and an SPD-M20A

photodiode array detector (UV-PDA) controlled by a CBM-20A system controller from computer

software LabSolutions (Shimadzu, Kyoto, Japan). The chiral separation was done on a Lux Amylose-1

(amylose tris(3,5-dimethylphenyl)carbamate) (250

×

4.6 mm) column, particle size 5 µm (Phenomenex,

Germany). The mobile phase consisted of methanol or acetonitrile–50 mM aqueous NaClO₄or

methanol–0.2% (v/v) acetic acid, in speciﬁed volumetric ratios. The exact conditions are summarized in

Table S1 of the Supplementary Material. The ﬂow rate was 1 mL/min and the injection volume was 1

µL. UV detection was performed in single wavelength mode (270 nm). For the collection of the UV

spectra, a photodiode array scan mode (in the range 190–400 nm with a 1 nm step) was used.

Optical rotation measurements were used for the investigation of PC-d/l-Pgl-Me derivative and

for further investigations of compounds without exact antipodes (diﬀerent esters of d- and l-aa).

Polarimeter Krüss P3000 (A. Krüss Optronic GmbH, Hamburg, Germany) was used. Compounds

PC-d/l-Pgl-Me and PC-l-Met-Me were dissolved in chloroform (to compare the obtained speciﬁc

rotation with literature), and other compounds were dissolved in methanol to ﬁnal concentrations c =

5 to 20 mg/mL (based on observed rotation and/or available amount of samples). The optical rotation

was measured in a 5 cm long glass cuvette using a 589 nm wavelength at laboratory temperature (25

1 ^◦C). All measurements were done in triplicates and the average values are reported.

±

3.1.7. NMR, IR Analysis, and Melting Point Measurements

The ¹H– and ¹³C–NMR spectra of the compounds were recorded on Varian VNMR S500 (Varian,

Palo Alto, CA, USA) at 500 MHz for ¹H and 126 MHz for ¹³C. Chemical shifts were reported in ppm

1

(δ

) and were referred indirectly to tetramethylsilane via the signal of the solvent (2.49 for H and

39.7 for ¹³C in DMSO-d₆). The infrared spectra were recorded with spectrometer FT-IR Nicolet 6700

(Thermo Scientiﬁc, Waltham, MA, USA) using the attenuated total reﬂectance (ATR) methodology on

germanium crystal. Melting points (mp) were measured on Stuart SMP30 (Bibby Scientiﬁc Limited,

Staﬀordshire, UK) using the capillary method. For compounds gained in small amounts, mp was

measured using Koﬂer melting point apparatus with a microscope.

3.1.8. LC-MS and GC-MS Analysis

Advantage Max ion-trap LCQ mass spectrometer with ESI ion source (Thermo Finnigan, San Jose,

CA, USA) was used for conﬁrmation of the molecular weight of compounds containing free carboxylic

group in negative mode (direct injection). All spectra were processed with Thermo Finnigan Xcalibur

software (Thermo Finnigan, San Jose, CA, USA).

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The GC-MS analysis was performed on a GCMS-QP2010 Plus system operating in EI mode at 70 eV

(Shimadzu, Tokyo, Japan). The chromatographic separation was carried out on a Rtx-5MS column

(30 m

×

0.25 mm

×

0.25 µ

m, Restek, USA). The column temperature was increased by 20 ^◦C/min, from

130 to 320 ^◦C, and this temperature was maintained for 2 min. The injector temperature was 240 ^◦C

and the carrier gas ﬂow rate (helium) was set to 1 mL/min. The detection range was m/z 110–700,

and the ion source temperature was held at 220 ^◦C. The sample (1

µL) was introduced into injector in

split mode (ratio 1:1). The data were recorded and analysed by the GCMS Solution Software ver. 4.45

(Shimadzu, Tokyo, Japan).

3.2. Analytical Results

All the analytical results for the synthesized compounds are presented in this section. If available,

the CAS number is indicated. The results were in accordance with the presumed structure.

PC-d-Ala ((pyrazine-2-carbonyl)-d-alanine)

CAS 1308937-79-2. Yield: 26%. m.p. = 175–178 ^◦C. ¹³C-NMR (126 MHz, DMSO-d₆):

δ 173.74, 162.70,

1

147.95, 144.52, 143.70, 143.61, 47.97, 17.31. H-NMR (500 MHz, DMSO-d₆):

δ 12.80 (bs, 1H), 9.18 (d, J

= 1.5 Hz, 1H), 8.94 (d, J = 7.6 Hz, 1H), 8.89 (d, J = 2.5 Hz, 1H), 8.76–8.74 (m, 1H), 4.54–4.44 (m, 1H),

1.43 (d, J = 7.3 Hz, 3H). IR (ATR-Ge, cm⁻¹): 3370 CONH N-H stretching, 1641 CONH C=O stretching,

1549 CONH C-N stretching, 2923 COOH O-H stretching, 1736 COOH C=O stretching. MS (ESI-):

[M−H]⁻= 194.

PC-l-Ala ((pyrazine-2-carbonyl)-l-alanine)

CAS 117904-05-9. Yellowish solid. Yield: 95%. m.p. = 170–174 ^◦C (lit. from ethanol 170–173 ^◦C [63]).

1

¹³C-NMR (126 MHz, DMSO-d₆):

(500 MHz, DMSO-d₆):

δ

173.72, 162.69, 147.95, 144.52, 143.69, 143.62, 47.98, 17.32. H-NMR

δ

9.18 (d, J = 1.5 Hz, 1H), 8.93 (d, J = 7.5 Hz, 1H), 8.89 (d, J = 2.5 Hz, 1H), 8.75

(dd, J = 2.5, J = 1.5 Hz, 1H), 4.54–4.44 (m, 1H), 1.43 (d, J = 7.2 Hz, 3H). IR (ATR-Ge, cm⁻¹): 3370 CONH

N-H stretching, 1641 CONH C=O stretching, 1549 CONH C-N stretching, 2872 COOH O-H stretching,

1736 COOH C=O stretching. MS (ESI-): [M−H]⁻= 194.

PC-dl-Ala-Me (methyl (pyrazine-2-carbonyl)alaninate)

CAS 955039-15-3. Yellowish solid. Yield: 17%. m.p. = 53–56 ^◦C. ¹³C-NMR (126 MHz, DMSO-d₆):

δ

1

172.63, 162.94, 147.97, 144.48, 143.78, 143.62, 52.24, 48.00, 17.00. H-NMR (500 MHz, DMSO-d₆):

δ 9.18

(d, J = 1.5 Hz, 1H), 9.16 (d, J = 7.1 Hz, 1H), 8.90 (d, J = 2.5 Hz, 1H), 8.77–8.74 (m, 1H), 4.62–4.54 (m, 1H),

3.65 (s, 3H), 1.43 (d, J = 7.3 Hz, 3H). IR (ATR-Ge, cm⁻¹): 3258 CONH N-H stretching, 1671 CONH C=O

stretching, 1537 CONH C-N stretching, 1747 COOR C=O stretching, 2993, 2951 C-H stretching. MS

(EI): M^+·= 209.

PC-d-Ala-Et (ethyl (pyrazine-2-carbonyl)-d-alaninate)

Oily liquid. Yield: 45%. ¹³C-NMR (126 MHz, DMSO-d₆):

60.85, 48.12, 16.99, 14.20. H-NMR (500 MHz, DMSO-d₆):

δ

172.13, 162.94, 147.95, 144.50, 143.74, 143.61,

1

δ 9.18 (d, J = 1.5 Hz, 1H), 9.11 (d, J = 7.4 Hz,

1H), 8.90 (d, J = 2.5 Hz, 1H), 8.77–8.75 (m, 1H), 4.58–4.50 (m, 1H), 4.15–4.08 (m, 2H), 1.44 (d, J = 7.4

Hz, 3H), 1.18 (t, J = 7.1 Hz, 3H). IR (ATR-Ge, cm⁻¹): 3392 CONH N-H stretching, 1675 CONH C=O

stretching, 1522 CONH C-N stretching, 1736 COOR C=O stretching, 2984 C-H stretching. MS (EI):

M^+·= 223.

PC-l-Ala-Et (ethyl (pyrazine-2-carbonyl)-l-alaninate)

Oily yellow liquid. Yield: 32%. ¹³C-NMR (126 MHz, DMSO-d₆):

143.62, 60.86, 48.13, 16.99, 14.22. H-NMR (500 MHz, DMSO-d₆):

δ

172.14, 162.94, 147.96, 144.50, 143.75,

9.19–9.17 (m, 1H), 9.12 (d, J = 7.3

1

δ

Hz, 1H), 8.90 (d, J = 2.5 Hz, 1H), 8.77–8.75 (m, 1H), 4.58–4.50 (m, 1H), 4.15–4.08 (m, 2H), 1.44 (d, J = 7.3

Hz, 3H), 1.19 (t, J = 7.1 Hz, 3H). IR (ATR-Ge, cm⁻¹): 3389 CONH N-H stretching, 1674 CONH C=O

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stretching, 1525 CONH C-N stretching, 1738 COOR C=O stretching, 2984 C-H stretching. MS (EI):

M^+·= 223.

PC-d-Asp-diMe (dimethyl (pyrazine-2-carbonyl)-d-aspartate)

Oily yellow liquid. Yield: 66%. ¹³C-NMR (126 MHz, DMSO-d₆):

143.82, 143.71, 52.64, 51.91, 48.75, 35.46. H-NMR (500 MHz, DMSO-d₆):

δ

171.03, 170.96, 162.94, 148.20, 144.10,

9.27 (d, J = 8.4 Hz, 1H), 9.20

1

δ

(d, J = 1.5 Hz, 1H), 8.91 (d, J = 2.5 Hz, 1H), 8.78–8.69 (m, 1H), 5.10–4.86 (m, 1H), 3.65 (s, 3H), 3.61 (s,

3H), 3.01 (dd, J = 16.6 Hz, J = 6.4 Hz, 1H), 2.94 (dd, J = 16.6 Hz, J = 6.4 Hz, 1H). IR (ATR-Ge, cm⁻¹):

3382 CONH N-H stretching, 1679 CONH C=O stretching, 1519 CONH C-N stretching, 1737 COOR

C=O stretching, 2954C-H stretching. [α]_D= −4.0^◦(methanol). MS (EI): M^+·= 267.

PC-l-Asp-diEt (diethyl (pyrazine-2-carbonyl)-l-aspartate)

CAS 882740-96-7. Yellowish solid. Yield: 25%. m.p. = 54–57 ^◦C (lit. aqueous ethanol 64–65 ^◦C [30]).

¹³C-NMR (126 MHz, DMSO-d₆):

δ

170.47, 170.35, 162.88, 148.18, 144.12, 143.78, 143.69, 61.30, 60.51,

48.87, 35.72, 14.15, 14.11.

¹H-NMR (500 MHz, DMSO-d₆):

δ 9.21 (d, J = 8.2 Hz, 1H), 9.20 (d, J = 1.5 Hz, 1H), 8.91 (d, J = 2.4 Hz,

1H), 8.79–8.74 (m, 1H), 5.01–4.90 (m, 1H), 4.16–4.00 (m, 4H), 3.03–2.86 (m, 2H), 1.16 (t, J = 7.1 Hz, 6H). IR

(ATR-Ge, cm⁻¹): 3311 CONH N-H stretching, 1660 CONH C=O stretching, 1538 CONH C-N stretching,

1732 COOR C=O stretching, 2978 C-H stretching. [α]_D= 12.0^◦(methanol). MS (EI): M^+·= 295.

PC-l-SBn-Cys (S-benzyl-N-(pyrazine-2-carbonyl)-l-cysteine)

Yellowish solid. Yield: 84%. m.p. = 84–87 ^◦C. ¹³C-NMR (126 MHz, DMSO-d₆):

144.20, 143.72, 143.68, 138.36, 129.00, 128.54, 127.03, 51.77, 35.41, 32.31. H-NMR (500 MHz, DMSO-d₆):

δ 171.77, 162.88, 148.12,

1

δ

9.21 (d, J = 1.5 Hz, 1H), 8.99 (d, J = 8.2 Hz, 1H), 8.92 (d, J = 2.5 Hz, 1H), 8.80–8.76 (m, 1H), 7.31–7.24

(m, 4H), 7.24–7.17 (m, 1H), 4.75–4.67 (m, 1H), 3.76 (s, 2H), 3.03–2.97 (m, 2H). IR (ATR-Ge, cm⁻¹): 3357

CONH N-H stretching, 1660 CONH C=O stretching, 1519 CONH C-N stretching, 1732 COOH C=O

stretching, 2916 C-H stretching. [α]_D= −49.3^◦(methanol). MS (ESI-): [M−H]⁻= 316.

PC-d-Glu-diMe (dimethyl (pyrazine-2-carbonyl)-d-glutamate)

Oily yellow liquid. Yield: 57%. ¹³C-NMR (126 MHz, DMSO-d₆):

143.82, 143.63, 52.32, 51.62, 51.53, 30.01, 25.80. H-NMR (500 MHz, DMSO-d₆):

δ

172.87, 171.74, 163.44, 148.01, 144.40,

9.21–9.15 (m, 2H), 8.90

1

δ

(d, J = 2.5 Hz, 1H), 8.79–8.74 (m, 1H), 4.62–4.53 (m, 1H), 3.65 (s, 3H), 3.56 (s, 3H), 2.41 (t, J = 7.4 Hz, 2H),

2.24–2.15 (m, 1H), 2.14–2.02 (m, 1H). IR (ATR-Ge, cm⁻¹): 3377 CONH N-H stretching, 1677 CONH

C=O stretching, 1520 CONH C-N stretching, 1737 COOR C=O stretching, 2954 C-H stretching. [

16.0^◦(methanol). MS (EI): M^+·= 281.

α] =

D

PC-l-Glu-diEt (diethyl (pyrazine-2-carbonyl)-l-glutamate)

Oily yellow liquid. Yield: 72%. ¹³C-NMR (126 MHz, DMSO-d₆):

δ 172.38, 171.25, 163.41, 147.99, 144.43,

1

143.79, 143.62, 61.00, 60.08, 51.74, 30.24, 25.82, 14.20. H-NMR (500 MHz, DMSO-d₆): δ 9.18 (d, J = 1.4

Hz, 1H), 9.13 (d, J = 8.0 Hz, 1H), 8.90 (d, J = 2.5 Hz, 1H), 8.79–8.74 (m, 1H), 4.59–4.51 (m, 1H), 4.17–4.07

(m, 2H), 4.02 (q, J = 7.1 Hz, 2H), 2.43–2.36 (m, 2H), 2.23–2.04 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H), 1.14 (t,

J = 7.1 Hz, 3H). IR (ATR-Ge, cm⁻¹): 3359 CONH N-H stretching, 1677 CONH C=O stretching, 1520

CONH C-N stretching, 1732 COOR C=O stretching, 2982 C-H stretching. [

MS (EI): M^+·= 309.

α

]_D=

12.0^◦(methanol).

−

PC-Gly ((pyrazine-2-carbonyl)glycine)

CAS 57229-37-5. White solid. Yield: 10%. m.p. not measured due to insuﬃcient quantity of

the compound, structure conﬁrmed by MS and NMR, (lit. from dimethylformamide, methanol

233–235^◦C [63], lit. from water, 229 ^◦C [64]). ¹³C-NMR (126 MHz, DMSO-d₆):

δ 171.39, 162.70, 147.88,

1

144.59, 143.69, 143.55, 42.33. H-NMR (500 MHz, DMSO-d₆):

δ 9.18 (s, 1H), 8.93–8.80 (m, 2H), 8.74

(s, 1H), 3.87 (d, J = 5.0 Hz, 2H). IR (ATR-Ge, cm⁻¹): 3349 CONH N-H and COOH O-H stretching

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broad band, 1652 CONH C=O stretching, 1717 COOH C=O stretching, 2932 C-H stretching. MS (ESI-):

[M−H]⁻= 180.

PC-Gly-Et (ethyl (pyrazine-2-carbonyl)glycinate)

CAS 544702-05-8. White solid. Yield: 22%. m.p. = 92–95 ^◦C (lit. 89–93 [65]). ¹³C-NMR (126 MHz,

DMSO-d₆):

δ

169.60, 163.54, 148.07, 144.36, 143.74, 143.70, 60.81, 41.23, 14.27. ¹H-NMR (500 MHz,

δ

9.23 (t, J = 6.1 Hz, 1H), 9.19 (d, J = 1.5 Hz, 1H), 8.90 (d, J = 2.5 Hz, 1H), 8.77–8.75 (m, 1H),

4.12 (q, J = 7.1 Hz, 2H), 4.05 (d, J = 6.1 Hz, 2H), 1.20 (t, J = 7.1 Hz, 3H). IR (ATR-Ge, cm⁻¹): 3371 CONH

N-H stretching, 1666 CONH C=O stretching, 1530 CONH C-N stretching, 1741 COOR C=O stretching,

2986 C-H stretching. MS (EI): M^+·= 209.

PC-d-Ile-Me (methyl (pyrazine-2-carbonyl)-d-isoleucinate)

Oily yellow liquid. Yield: 38%. ¹³C-NMR (126 MHz, DMSO-d₆):

143.66, 55.58, 52.29, 36.71, 25.83, 14.99, 11.55. H-NMR (500 MHz, DMSO-d₆):

δ

171.82, 163.11, 148.15, 144.13, 143.72,

9.19 (d, J = 1.5 Hz, 1H),

1

δ

8.91 (d, J = 2.4 Hz, 1H), 8.81–8.74 (m, 1H), 8.56 (d, J = 8.8 Hz, 1H), 4.64 (dd, J = 8.8, J = 5.1 Hz, 1H), 3.69

(s, 3H), 2.13–1.99 (m, 1H), 1.46–1.30 (m, 1H), 1.23–1.09 (m, 1H), 0.93–0.85 (m, 6H). IR (ATR-Ge, cm⁻¹):

3390 CONH N-H stretching, 1682 CONH C=O stretching, 1518 CONH C-N stretching, 1741 COOR

C=O stretching, 2960 C-H stretching. MS (EI): M^+·= 251.

PC-l-Ile-Me (methyl (pyrazine-2-carbonyl)-l-isoleucinate)

Oily yellow liquid. Yield: 78%. ¹³C-NMR (126 MHz, DMSO-d₆):

143.67, 56.74, 52.13, 36.38, 25.17, 15.58, 11.18. H-NMR (500 MHz, DMSO-d₆):

δ

171.67, 163.06, 148.12, 144.23, 143.75,

9.18 (d, J = 1.4 Hz, 1H),

1

δ

8.91 (d, J = 2.5 Hz, 1H), 8.79–8.74 (m, 1H), 8.71 (d, J = 8.3 Hz, 1H), 4.48 (dd, J = 8.3, J = 6.5 Hz, 1H), 3.68

(s, 3H), 2.07–1.98 (m, 1H), 1.53–1.42 (m, 1H), 1.27–1.17 (m, 1H), 0.93–0.83 (m, 6H). IR (ATR-Ge, cm⁻¹):

3395 CONH N-H stretching, 1681 CONH C=O stretching, 1519 CONH C-N stretching, 1741 COOR

C=O stretching, 2965 C-H stretching. MS (EI): M^+·= 251.

PC-dl-Leu-Et ethyl (pyrazine-2-carbonyl)leucinate)

CAS 2319608-35-8. Oily yellow liquid. Yield: 83%. ¹³C-NMR (126 MHz, DMSO-d₆):

147.95, 144.49, 143.80, 143.61, 60.85, 50.74, 24.57, 22.99, 21.34, 14.21. H-NMR (500 MHz, DMSO-d₆):

δ 172.05, 163.28,

1

δ

9.18 (d, J = 1.3 Hz, 1H), 9.04 (d, J = 8.2 Hz, 1H), 8.89 (d, J = 2.5 Hz, 1H), 8.79–8.73 (m, 1H), 4.61–4.51 (m,

1H), 4.18–4.05 (m, 2H), 1.95–1.82 (m, 1H), 1.69–1.56 (m, 2H), 1.18 (t, J = 7.1 Hz, 3H), 0.89 (d, J = 6.1

Hz, 3H), 0.87 (d, J = 6.1 Hz, 3H). IR (ATR-Ge, cm⁻¹): 3386 CONH N-H stretching, 1675 CONH C=O

stretching, 1520 CONH C-N stretching, 1739 COOR C=O stretching, 2959 C-H stretching. MS (EI):

M^+·= 265.

PC-d-Leu-Me (methyl (pyrazine-2-carbonyl)-d-leucinate)

Oily yellow liquid. Yield: 85%. ¹³C-NMR (126 MHz, DMSO-d₆):

143.62, 52.21, 50.62, 24.55, 23.01, 21.31. H-NMR(500 MHz, DMSO-d₆):

δ

172.55, 163.30, 147.97, 144.47, 143.83,

9.18 (d, J = 1.5 Hz, 1H), 9.09 (d,

1

δ

J = 8.3 Hz, 1H), 8.89 (d, J = 2.7 Hz, 1H), 8.78–8.73 (m, 1H), 4.63–4.55 (m, 1H), 3.65 (s, 3H), 1.95–1.84 (m,

1H), 1.68–1.56 (m, 2H), 0.89 (d, J = 6.0 Hz, 3H), 0.87 (d, J = 6.0 Hz, 3H). IR (ATR-Ge, cm⁻¹): 3325CONH

N-H stretching, 1673 CONH C=O stretching, 1519 CONH C-N stretching, 1743 COOR C=O stretching,

2956 C-H stretching. MS (EI): M^+·= 251.

PC-l-Leu-Me (methyl (pyrazine-2-carbonyl)-l-leucinate)

CAS 1190303-01-5. Oily yellow liquid. Yield: 66%. ¹³C-NMR (126 MHz, DMSO-d₆):

147.96, 144.47, 143.83, 143.62, 52.21, 50.62, 24.55, 23.01, 21.31. H-NMR (500 MHz, DMSO-d₆):

δ

172.54, 163.30,

9.18 (d,

1

δ

J = 1.5 Hz, 1H), 9.09 (d, J = 8.3 Hz, 1H), 8.89 (d, J = 2.5 Hz, 1H), 8.78–8.74 (m, 1H), 4.66–4.53 (m, 1H),

3.65 (s, 3H), 1.95–1.84 (m, 1H), 1.68–1.56 (m, 2H), 0.89 (d, J = 6.0 Hz, 3H), 0.87 (d, J = 6.0 Hz, 3H). IR

(ATR-Ge, cm⁻¹): 3325 CONH N-H stretching, 1674 CONH C=O stretching, 1520 CONH C-N stretching,

1742 COOR C=O stretching, 2955 C-H stretching. MS (EI): M^+·= 251.

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PC-MeAcr (methyl 2-(pyrazine-2-carboxamido)acrylate)

CAS 1027723-22-3. White solid. Yield: 11%. m.p. = 102–104 ^◦C (lit. 95.5 ^◦C [32]). ¹³C-NMR (126 MHz,

1

DMSO-d₆):

δ

163.80, 161.52, 148.64, 143.79, 143.65, 143.48, 131.57, 109.67, 53.31. H-NMR (500 MHz,

δ

10.10 (bs, 1H), 9.27 (d, J = 1.4 Hz, 1H), 8.96 (d, J = 2.5 Hz, 1H), 8.82–8.77 (m, 1H), 6.56

(s, 1H), 5.91 (s, 1H), 3.83 (s, 3H). IR (ATR-Ge, cm⁻¹): 3349 CONH N-H stretching, 1689 CONH C=O

stretching, 1519 CONH C-N stretching, 1712 COOR C=O stretching, 3158, 2957 C-H stretching. MS

(ESI-): [M−H]⁻= 206.

PC-l-Met-Me (methyl (pyrazine-2-carbonyl)-l-methioninate)

CAS 1454692-96-6. Oily yellow liquid. Yield: 66%. ¹³C-NMR (126 MHz, DMSO-d₆):

147.97, 144.47, 143.84, 143.61, 52.32, 51.34, 30.10, 29.97, 14.69. H-NMR (500 MHz, DMSO-d₆):

δ

171.95, 163.48,

9.21

1

δ

(d, J = 8.1 Hz, 1H), 9.19–9.17 (m, 1H), 8.90 (d, J = 2.6 Hz, 1H), 8.78–8.75 (m, 1H), 4.74–4.66 (m, 1H),

3.66 (s, 3H), 2.60–2.42 (m, 2H), 2.23–2.06 (m, 2H), 2.04 (s, 3H). IR (ATR-Ge, cm⁻¹): 3376 CONH N-H

stretching, 1675 CONH C=O stretching, 1519 CONH C-N stretching, 1741 COOR C=O stretching, 2952

C-H stretching. [

M^+·= 269.

α

]

=

22.7^◦(methanol), +33.0^◦(chloroform) (lit. chloroform 27.8^◦[65]). MS (EI):

−

D

PC-d/l-Pgl-Me (methyl 2-phenyl-2-(pyrazine-2-carboxamido)acetate)-enantiomeric mixture

White solid. m.p. = 92–97 ^◦C (lit. racemic mixture from chloroform and light petroleum 101 ^◦C [43],

pure l-enantiomer 53 ^◦C). Yield: 55%. ¹³C-NMR (126 MHz, DMSO-d₆):

143.75, 143.73, 136.62, 128.85, 128.48, 128.04, 56.35, 52.82. H-NMR (500 MHz, DMSO-d₆):

δ

170.66, 162.68, 148.20, 144.17,

9.26 (d, J

1

δ

= 7.5 Hz, 1H), 9.18 (d, J = 1.5 Hz, 1H), 8.91 (d, J = 2.5 Hz, 1H), 8.79–8.75 (m, 1H), 7.51–7.45 (m, 2H),

7.43–7.29 (m, 3H), 5.73 (d, J = 7.5 Hz, 1H), 3.68 (s, 3H). IR (ATR-Ge, cm⁻¹): 3376 CONH N-H stretching,

1665 CONH C=O stretching, 1516 CONH C-N stretching, 1747 COOR C=O stretching, 3090, 2953 C-H

stretching. [α]_D= –47.3^◦(chloroform). MS (EI): M^+·= 271.

PC-dl-Phe-Et (ethyl (pyrazine-2-carbonyl)phenylalaninate)

Brownish solid. Yield: 94%. m.p. = 58–61 ^◦C. ¹³C-NMR (126 MHz, DMSO-d₆):

144.21, 143.68, 143.65, 137.37, 129.26, 128.43, 126.72, 61.02, 53.81, 36.33, 14.15. ¹H-NMR (500 MHz,

DMSO-d₆): 9.13 (d, J = 1.5 Hz, 1H), 9.02 (d, J = 8.1 Hz, 1H), 8.88 (d, J = 2.5 Hz, 1H), 8.75–8.73 (m, 1H),

δ 171.08, 163.01, 148.05,

δ

7.29–7.21 (m, 4H), 7.20–7.15 (m, 1H), 4.81–4.74 (m, 1H), 4.11 (q, J = 7.1 Hz, 2H), 3.27–3.16 (m, 2H), 1.15

(t, J = 7.1 Hz, 3H). IR (ATR-Ge, cm⁻¹): 3334 CONH N-H stretching, 1660 CONH C=O stretching, 1523

CONH C-N stretching, 1741 COOR C=O stretching, 3064, 2977 C-H stretching. MS (EI): M^+·= 299.

PC-d-Ser ((pyrazine-2-carbonyl)-d-serine)

CAS 193957-37-8. White solid. Yield: 20%. m.p. = 185–189 ^◦C (carbonized). ¹³C-NMR (126 MHz,

1

DMSO-d₆):

δ

171.69, 162.54, 148.11, 144.21, 143.66, 143.56, 61.29, 54.93. H-NMR (500 MHz, DMSO-d₆):

δ

9.23–9.19 (m, 1H), 8.91 (d, J = 2.5 Hz, 1H), 8.80–8.75 (m, 1H), 8.68 (d, J = 7.9 Hz, 1H), 4.52–4.45 (m, 1H),

3.89 (dd, J = 11.1, J = 4.0 Hz, 1H), 3.78 (dd, J = 11.1, J = 3.4 Hz, 1H). IR (ATR-Ge, cm⁻¹): 3365 CONH

N-H stretching, 1651 CONH C=O stretching, 1537 CONH C-N stretching, 1708 COOH C=O stretching,

3335 CH₂OH O-H stretching, 2935 C-H stretching. [α]_Dnot measured due to insolubility. MS (ESI-):

[M−H]⁻= 210.

PC-d-Ser-Me (methyl (pyrazine-2-carbonyl)-d-serinate)

White solid. Yield: 45%. m.p. = 54–56 ^◦C (Koﬂer). ¹³C-NMR (126 MHz, DMSO-d₆):

δ

170.95, 163.15,

1

148.52, 144.33, 143.98, 143.95, 61.49, 55.19, 52.64. H-NMR (500 MHz, DMSO-d₆):

δ 9.21 (d, J = 1.5 Hz,

1H), 8.93 (d, J = 2.5 Hz, 1H), 8.81 (d, J = 8.1 Hz, 1H), 8.79 (dd, J = 2.5, J = 1.5 Hz, 1H), 5.27 (bs, 1H),

4.70–4.56 (m, 1H), 3.93–3.85 (m, 1H), 3.83–3.75 (m, 1H), 3.67 (s, 3H). IR (ATR-Ge, cm⁻¹): 1656 CONH

C=O stretching, 1542 CONH C-N stretching, 1737 COOR C=O stretching, 3343 CH₂OH O-H stretching

broad band, 2956 C-H stretching. MS (EI): M^+·= 225.

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PC-l-Ser-Me (methyl (pyrazine-2-carbonyl)-l-serinate)

CAS 139934-07-9. White solid. Yield: 59%. m.p. = 56–58 ^◦C (Koﬂer). ¹³C-NMR (126 MHz, DMSO-d₆):

1

δ

170.66, 162.85, 148.23, 144.04, 143.68, 143.66, 61.20, 54.91, 52.35. H-NMR (500 MHz, DMSO-d₆):

δ

9.21 (d, J = 1.5 Hz, 1H), 8.93 (d, J = 2.7 Hz, 1H), 8.81 (d, J = 8.1 Hz, 1H), 8.79–8.77 (m, 1H), 5.27 (t, J = 5.9

Hz, 1H), 4.66–4.59 (m, 1H), 3.93–3.86 (m, 1H), 3.84–3.76 (m, 1H), 3.68 (s, 3H). IR (ATR-Ge, cm⁻¹): 1657

CONH C=O stretching, 1543 CONH C-N stretching, 1737 COOR C=O stretching, 3340 CH₂OH O-H

stretching broad band, 2956 C-H stretching. MS (EI): M^+·= 225.

PC-l-OBn-Ser (O-benzyl-N-(pyrazine-2-carbonyl)-l-serine)

White solid. Yield: 50%. m.p. = 139–142 ^◦C. ¹³C-NMR (126 MHz, DMSO-d₆): δ 171.20, 162.58, 148.21,

1

144.00, 143.71, 143.60, 138.12, 128.41, 127.70, 127.66, 72.30, 69.20, 52.61. H-NMR (500 MHz, DMSO-d₆):

δ

13.14 (s, 1H), 9.21 (s, 1H), 8.93 (s, 1H), 8.78 (s, 1H), 8.73 (d, J = 8.2 Hz, 1H), 7.37–7.22 (m, 5H), 4.80–4.70

(m, 1H), 4.53 (s, 2H), 4.05–3.91 (m, 1H), 3.88–3.79 (m, 1H). IR (ATR-Ge, cm⁻¹): 3400 CONH N-H

stretching, 1650 CONH C=O stretching, 1542 CONH C-N stretching, 1743 COOH C=O stretching,

2943 COOH O-H stretching broad band, 2935 C-H stretching. [α]

[M−H]⁻= 300.

= 45.3^◦(methanol). MS (ESI-):

D

PC-l-OBn-Ser-Me (methyl O-benzyl-N-(pyrazine-2-carbonyl)-l-serinate)

Oily yellowish liquid. Yield: 95%. ¹³C-NMR (126 MHz, DMSO-d₆):

δ

1

170.53, 163.14, 148.56, 144.23,

144.02, 143.97, 138.32, 128.74, 128.04, 127.99, 72.51, 69.12, 52.83, 52.79. H-NMR (500 MHz, DMSO-d₆):

9.20 (d, J = 1.5 Hz, 1H), 8.92 (d, J = 2.5 Hz, 1H), 8.88 (d, J = 8.2 Hz, 1H), 8.81–8.75 (m, 1H), 7.35–7.28

δ

(m, 2H), 7.31–7.23 (m, 3H), 4.88–4.81 (m, 1H), 4.55 (d, J = 12.2 Hz, 1H), 4.50 (d, J = 12.2 Hz, 1H), 3.93

(dd, J = 9.9, 5.5 Hz, 1H), 3.84 (dd, J = 10.0, 4.0 Hz, 1H), 3.67 (s, 3H). IR (ATR-Ge, cm⁻¹): 3398 CONH

N-H stretching, 1679 CONH C=O stretching, 1519CONH C-N stretching, 1746 COOR C=O stretching,

2921 C-H stretching. [α]_D= 10.7^◦(methanol). MS (EI): M^+·= 315.

PC-d-OtBu-Ser-Me (methyl O-(tert-butyl)-N-(pyrazine-2-carbonyl)-d-serinate)

Oily yellow liquid. Yield: 100%. ¹³C-NMR (126 MHz, DMSO-d₆):

143.78, 143.63, 73.33, 61.54, 53.00, 52.40, 27.28. H-NMR (500 MHz, DMSO-d₆):

δ

170.43, 162.62, 148.32, 143.83,

9.21 (d, J = 1.5 Hz,

1

δ

1H), 8.92 (d, J = 2.5 Hz, 1H), 8.81–8.76 (m, 1H), 8.62 (d, J = 8.3 Hz, 1H), 4.76–4.69 (m, 1H), 3.83 (dd, J =

9.6, 4.4 Hz, 1H), 3.70 (dd, J = 9.6, 3.9 Hz, 1H), 3.68 (s, 3H), 1.11 (s, 9H). IR (ATR-Ge, cm⁻¹): 3404 CONH

N-H stretching, 1681 CONH C=O stretching, 1519 CONH C-N stretching, 1748 COOR C=O stretching,

2973 C-H stretching. MS (EI): M^+·= 281.

PC-l-OtBu-Ser-Me (methyl O-(tert-butyl)-N-(pyrazine-2-carbonyl)-l-serinate)

Oily yellow liquid. Yield: 61%. ¹³C-NMR (126 MHz, DMSO-d₆) 170.43, 162.62, 148.32, 143.84, 143.78,

1

143.64, 73.33, 61.55, 53.01, 52.39, 27.28. H-NMR (500 MHz, DMSO-d₆): δ 9.21 (d, J = 1.5 Hz, 1H), 8.92

(d, J = 2.5 Hz, 1H), 8.80–8.76 (m, 1H), 8.62 (d, J = 8.3 Hz, 1H), 4.76–4.69 (m, 1H), 3.83 (dd, J = 9.6, 4.3

Hz, 1H), 3.70 (dd, J = 9.6, 3.9 Hz, 1H), 3.68 (s, 3H), 1.10 (s, 9H). IR (ATR-Ge, cm⁻¹): 3404 CONH N-H

stretching, 1681 CONH C=O stretching, 1518 CONH C-N stretching, 1747 COOR C=O stretching, 2973

C-H stretching. MS (EI): M^+·= 281.

PC-d-Thr-Me (methyl (pyrazine-2-carbonyl)-d-threoninate)

Oily yellow liquid. Yield: 95%. ¹³C-NMR (126 MHz, DMSO-d₆):

143.66, 66.46, 57.99, 52.37, 20.57. H-NMR (500 MHz, DMSO-d₆):

δ

170.84, 163.12, 148.37, 143.80, 143.75,

1

δ 9.22 (d, J = 1.5 Hz, 1H), 8.94 (d, J =

2.5 Hz, 1H), 8.82–8.78 (m, 1H), 8.54 (d, J = 8.9 Hz, 1H), 4.53 (dd, J = 8.9, J = 2.9 Hz, 1H), 4.27 (qd, J = 6.4,

J = 2.9 Hz, 1H), 3.79 (bs, 1H), 3.68 (s, 3H), 1.12 (d, J = 6.4 Hz, 3H). IR (ATR-Ge, cm⁻¹): 1671 CONH

C=O stretching, 1525 CONH C-N stretching, 1741 COOR C=O stretching, 3387 CH₂OH O-H stretching

broad band, 2955 C-H stretching. MS (EI): M^+·= 239 not visible in GC-MS spectrum, conﬁrmed by MS

(ESI-) [M−H]⁻= 240.

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PC-l-Thr-Me (methyl (pyrazine-2-carbonyl)-l-threoninate)

Brownish solid. Yield: 90%. m.p. = 69–72 ^◦C. ¹³C-NMR (126 MHz, DMSO-d₆):

143.79, 143.74, 143.65, 66.44, 57.98, 52.36, 20.56. H-NMR (500 MHz, DMSO-d₆):

δ

170.83, 163.10, 148.36,

δ 9.22 (d, J = 1.5 Hz,

1

1H), 8.94 (d, J = 2.5 Hz, 1H), 8.82–8.78 (m, 1H), 8.55 (d, J = 8.9 Hz, 1H), 5.40 (bs, 1H), 4.54 (dd, J = 8.9, J

= 2.8 Hz, 1H), 4.27 (qd, J = 6.4, J = 2.9 Hz, 1H), 3.68 (s, 3H), 1.12 (d, J = 6.4 Hz, 3H). IR (ATR-Ge, cm⁻¹):

1664 CONH C=O stretching, 1527 CONH C-N stretching, 1744 COOR C=O stretching, 3380 CH₂OH

O-H stretching broad band, 2981 C-H stretching. MS (EI): M^+·= 239 not visible, same GC-MS spectrum

as PC-d-Thr-Me.

PC-d-OtBu-Thr-Me (methyl O-(tert-butyl)-N-(pyrazine-2-carbonyl)-d-threoninate)

White solid. Yield: 89%. m.p. = 81–84 ^◦C. ¹³C-NMR (126 MHz, DMSO-d₆):

143.84, 143.61, 143.52, 74.02, 67.22, 57.70, 52.37, 28.22, 21.01. H-NMR (500 MHz, DMSO-d₆):

δ

170.61, 162.90, 148.48,

9.23 (d, J

1

δ

= 1.5 Hz, 1H), 8.95 (d, J = 2.5 Hz, 1H), 8.81–8.80 (m, 1H), 8.29 (d, J = 9.2 Hz, 1H), 4.58 (dd, J = 9.2, J

= 2.2 Hz, 1H), 4.32 (qd, J = 6.2, J = 2.2 Hz, 1H), 3.68 (s, 3H), 1.14 (d, J = 6.2 Hz, 3H), 1.11 (s, 9H). IR

(ATR-Ge, cm⁻¹): 3411 CONH N-H stretching, 1685 CONH C=O stretching, 1519 CONH C-N stretching,

1743 COOR C=O stretching, 2977 C-H stretching. MS (EI): M^+·= 295 not visible in GC-MS spectrum,

conﬁrmed by MS (ESI-) [M−H]⁻= 294.

PC-l-OtBu-Thr-Me (methyl O-(tert-butyl)-N-(pyrazine-2-carbonyl)-l-threoninate)

White solid. Yield: 99%. m.p. = 83–86 ^◦C. ¹³C-NMR (126 MHz, DMSO-d₆):

143.83, 143.60, 143.51, 74.01, 67.22, 57.69, 52.36, 28.21, 21.00. H-NMR (500 MHz, DMSO-d₆):

δ

170.60, 162.90, 148.47,

9.23 (d, J

1

δ

= 1.5 Hz, 1H), 8.95 (d, J = 2.4 Hz, 1H), 8.83–8.78 (m, 1H), 8.29 (d, J = 9.2 Hz, 1H), 4.58 (dd, J = 9.2, J = 2.2

Hz, 1H), 4.32 (qd, J = 6.2, J = 2.2 Hz, 1H), 3.68 (s, 3H), 1.14 (d, J = 6.2 Hz, 3H), 1.11 (s, 9H). IR (ATR-Ge,

cm⁻¹): 3410 CONH N-H stretching, 1685 CONH C=O stretching, 1518 CONH C-N stretching, 1743

COOR C=O stretching, 2977 C-H stretching. MS (EI): M^+·= 295 not visible, same GC-MS spectrum as

PC-d-OtBu-Thr-Me.

PC-d-Trp-Et (ethyl (pyrazine-2-carbonyl)-d-tryptophanate)

White solid. Yield: 99%. m.p. = 90–93 ^◦C. ¹³C-NMR (126 MHz, DMSO-d₆):

144.20, 143.67, 143.60, 136.30, 127.31, 123.97, 121.20, 118.57, 118.25, 111.62, 109.41, 61.03, 53.36, 26.84,

14.14. ¹H-NMR (500 MHz, DMSO-d₆):

10.85 (s, 1H), 9.15 (d, J = 1.4 Hz, 1H), 8.91–8.85 (m, 2H),

δ 171.42, 162.90, 148.05,

δ

8.73–8.69 (m, 1H), 7.50 (d, J = 8.1 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H), 7.17 (d, J = 2.4 Hz, 1H), 7.08–7.01 (m,

1H), 6.98–6.90 (m, 1H), 4.85–4.77 (m, 1H), 4.10 (q, J = 7.1 Hz, 2H), 3.41–3.29 (m, 2H), 1.14 (t, J = 7.1 Hz,

3H). IR (ATR-Ge, cm⁻¹): 3366 CONH N-H stretching, 1673 CONH C=O stretching, 1520 CONH C-N

stretching, 1732 COOR C=O stretching, 2980 C-H stretching. MS (EI): M^+·= 338.

PC-l-Trp-Et (ethyl (pyrazine-2-carbonyl)-l-tryptophanate)

Oily yellow liquid. Yield: 96%. ¹³C-NMR (126 MHz, DMSO-d₆):

143.60, 136.30, 127.31, 123.97, 121.19, 118.57, 118.25, 111.62, 109.40, 61.03, 53.36, 26.84, 14.13. H-NMR

δ

171.42, 162.90, 148.05, 144.20, 143.67,

1

(500 MHz, DMSO-d₆):

δ 10.86 (s, 1H), 9.16 (d, J = 1.5 Hz, 1H), 8.89 (d, J = 7.7 Hz, 1H), 8.88 (d, J = 2.5

Hz, 1H), 8.73–8.69 (m, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.18 (d, J = 2.3 Hz, 1H),

7.10–6.97 (m, 1H), 6.97–6.91 (m, 1H), 4.85–4.78 (m, 1H), 4.10 (q, J = 7.1 Hz, 2H), 3.40–3.30 (m, 2H), 1.14

(t, J = 7.1 Hz, 3H). IR (ATR-Ge, cm⁻¹): 3368 CONH N-H stretching, 1672 CONH C=O stretching, 1520

CONH C-N stretching, 1732 COOR C=O stretching, 2981 C-H stretching. MS (EI): M^+·= 338.

PC-d-Tyr-Me (methyl (pyrazine-2-carbonyl)-d-tyrosinate)

Oily yellow liquid. Yield: 99%. ¹³C-NMR (126 MHz, DMSO-d₆):

143.70, 143.68, 130.20, 127.26, 115.30, 54.02, 52.27, 35.58. H-NMR (500 MHz, DMSO-d₆):

δ

171.70, 162.95, 156.18, 148.08, 144.19,

9.21 (s, 1H),

1

δ

9.13 (d, J = 1.5 Hz, 1H), 8.94 (d, J = 8.1 Hz, 1H), 8.88 (d, J = 2.5 Hz, 1H), 8.75–8.73 (m, 1H), 7.03–6.98 (m,

AA’, BB’, 2H), 6.66–6.60 (m, AA’, BB’, 2H), 4.81–4.66 (m, 1H), 3.65 (s, 3H), 3.09 (d, J = 7.0 Hz, 2H). IR

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(ATR-Ge, cm⁻¹): 3351 CONH N-H stretching, 1669 CONH C=O stretching, 1515 CONH C-N stretching,

1736 COOR C=O stretching, 2953 C-H stretching. [α]_D= 12.0^◦(methanol). MS (ESI-): [M−H]⁻= 300.

PC-l-Tyr-Et (ethyl (pyrazine-2-carbonyl)-l-tyrosinate)

White solid. Yield: 65%. m.p. = 119–123 ^◦C. ¹³C-NMR (126 MHz, DMSO-d₆): δ 171.21, 162.92, 156.20,

148.07, 144.21, 143.68, 143.67, 130.23, 127.19, 115.27, 60.99, 54.09, 35.66, 14.19. ¹H-NMR (500 MHz,

DMSO-d₆):

δ 9.21 (s, 1H), 9.14 (d, J = 1.3 Hz, 1H), 8.92–8.86 (m, 2H), 8.76–8.73 (m, 1H), 7.03–6.98 (m,

AA’, BB’, 2H), 6.66–6.60 (m, AA’, BB’, 2H), 4.73–4.65 (m, 1H), 4.11 (q, J = 7.1 Hz, 2H), 3.09 (dd, J = 7.0,

2.7 Hz, 2H), 1.16 (t, J = 7.1 Hz, 3H). IR (ATR-Ge, cm⁻¹): 3367 CONH N-H stretching, 1663 CONH C=O

stretching, 1519 CONH C-N stretching, 1720 COOR C=O stretching, 3440 Ar-OH O-H broad band,

2986 C-H stretching. [α]_D= -5.0^◦(methanol). MS (ESI-): [M−H]⁻= 314.

PC-dl-Val-Et (ethyl (pyrazine-2-carbonyl)valinate)

Oily yellow liquid. Yield: 31%. ¹³C-NMR (126 MHz, DMSO-d₆):

143.67, 60.94, 57.77, 30.21, 19.10, 18.49, 14.25. H-NMR (500 MHz, DMSO-d₆):

δ

171.12, 163.11, 148.12, 144.23, 143.73,

9.18 (d, J = 1.3 Hz, 1H),

1

δ

8.91 (d, J = 2.6 Hz, 1H), 8.80–8.75 (m, 1H), 8.64 (d, J = 8.4 Hz, 1H), 4.41 (dd, J = 8.4, J = 6.3 Hz, 1H),

4.21–4.10 (m, 2H), 2.32–2.19 (m, 1H), 1.20 (t, J = 7.1 Hz, 3H), 0.94 (d, J = 6.8 Hz, 3H), 0.93 (d, J = 6.8 Hz,

3H). IR (ATR-Ge, cm⁻¹): 3395 CONH N-H stretching, 1682 CONH C=O stretching, 1519 CONH C-N

stretching, 1737 COOR C=O stretching, 2966 C-H stretching. MS (EI): M^+·= 251.

PC-d-Val-Me (methyl (pyrazine-2-carbonyl)-d-valinate)

Oily yellow liquid. Yield: 34%. ¹³C-NMR (126 MHz, DMSO-d₆):

143.67, 57.79, 52.16, 30.13, 19.15, 18.53. H-NMR (500 MHz, DMSO-d₆):

δ

171.64, 163.15, 148.11, 144.24, 143.75,

9.18 (d, J = 1.5 Hz, 1H), 8.91

1

δ

(d, J = 2.5 Hz, 1H), 8.78–8.76 (m, 1H), 8.69 (d, J = 8.4 Hz, 1H), 4.43 (dd, J = 8.4, 6.4 Hz, 1H), 3.68 (s, 3H),

2.36–2.16 (m, 1H), 0.93 (dd, J = 6.8 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H). IR (ATR-Ge, cm⁻¹): 3394 CONH

N-H stretching, 1681 CONH C=O stretching, 1519 CONH C-N stretching, 1740 COOR C=O stretching,

2964 C-H stretching. MS (EI): M^+·= 237.

PC-l-Val-Me (methyl (pyrazine-2-carbonyl)-l-valinate)

CAS 73058-33-0. Oily yellow liquid. Yield: 76%. ¹³C-NMR (126 MHz, DMSO-d₆):

148.11, 144.24, 143.76, 143.66, 57.80, 52.16, 30.14, 19.15, 18.54. H-NMR(500 MHz, DMSO-d₆):

δ

171.64, 163.15,

9.18 (d,

1

δ

J = 1.5 Hz, 1H), 8.91 (d, J = 2.6 Hz, 1H), 8.79–8.75 (m, 1H), 8.69 (d, J = 8.5 Hz, 1H), 4.43 (dd, J = 8.5, J

= 6.4 Hz, 1H), 3.68 (s, 3H), 2.32–2.19 (m, J = 6.8 Hz, 1H), 0.93 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.8 Hz,

3H). IR (ATR-Ge, cm⁻¹): 3395 CONH N-H stretching, 1681 CONH C=O stretching, 1519 CONH C-N

stretching, 1741 COOR C=O stretching, 2965 C-H stretching. MS (EI): M^+·= 237.

3.3. Biological Evaluation

3.3.1. Antimycobacterial Activity Screening Against Mtb H37Ra, M. smegmatis, M. aurum

Microdilution assay (Microplate Alamar Blue Assay, MABA) [45]. The antimycobacterial assay

was performed with fast-growing M. smegmatis DSM 43465 (ATCC 607) and M. aurum DSM 43999

(ATCC 23366) from the German Collection of Microorganisms and Cell Cultures (Braunschweig,

Germany) and with the avirulent strain of Mtb H37Ra ITM-M006710 (ATCC 9431) from Belgian

Co-ordinated Collections of Micro-organisms (Antwerp, Belgium). The technique used for activity

determination was microdilution broth panel method using 96-well microtitration plates. The culturing

medium was Middlebrook 7H9 broth (Sigma-Aldrich) with a deﬁned pH of 6.6

±

0.2 enriched with 0.4%

of glycerol (Sigma-Aldrich) and 10% of Middlebrook OADC growth supplement (Himedia, Mumbai,

India). In the case of screening in pH 6, the pH was achieved by addition of 1M HCl (Penta, Prague,

Czech Republic).

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The mycobacterial strains were cultured on Middlebrook 7H9 agar and suspensions were prepared

in Middlebrook 7H9 broth. The ﬁnal density was adjusted to value 1.0 according to McFarland scale

and diluted in the ratio 1:20 (for fast growing mycobacteria) or 1:10 (for Mtb H37Ra) with broth.

The tested compounds were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich), then

Middlebrook 7H9 broth was added to obtain a concentration of 2000 µg/mL. The standards used for

activity determination were isoniazid (INH), rifampicin (RIF), and ciproﬂoxacin (CPX) (Sigma-Aldrich).

The ﬁnal concentrations were reached by binary dilution and the addition of mycobacterial suspension

and were set as 500, 250, 125, 62.5, 31.25, 15.625, 7.81, and 3.91 µg/mL. The ﬁnal concentration of DMSO

in any well did not exceeded 2.5% (v/v) and did not aﬀect the growth of mycobacteria. Positive (broth,

DMSO, bacteria) and negative (broth, DMSO) controls were included.

◦

The plates were sealed with polyester adhesive ﬁlm and incubated in dark at 37 C without

agitation. The addition of 0.01% solution of resazurin sodium salt followed after 48 h of incubation

for M. smegmatis, after 72 h of incubation for M. aurum, and after 120 h of incubation for Mtb H37Ra,

respectively. Stain was prepared by dissolving resazurin sodium salt (Sigma-Aldrich) in deionised

water to get 0.02% solution. Then, 10% aqueous solution of Tween 80 (Sigma-Aldrich) was prepared.

Both liquids were mixed up making use of the same volumes and ﬁltered through a 0.2 µm syringe

membrane ﬁlter. The microtitration panels were then incubated for further 2.5 h for the determination

of activity against M. smegmatis, 4 h for M. aurum, and 18 h for Mtb H37Ra, respectively.

Antimycobacterial activity was expressed as the minimum inhibitory concentration (MIC). The

MIC (in µg/mL) was determined on the basis of stain colour change (blue colour—active; pink

colour—not active). The MIC values for standards are presented in the respective tables. All

experiments were conducted in duplicate.

3.3.2. Antibacterial and Antifungal Activity Screening

For details, refer to the Supplementary Material.

3.3.3. Cytotoxicity Screening

The human hepatocellular liver carcinoma cell line HepG2 purchased from Health Protection

Agency Culture Collections (ECACC, Salisbury, UK) was cultured in MEM (Minimum Essentials Eagle

Medium) (Sigma-Aldrich) supplemented with 10% foetal bovine serum (PAA Laboratories GmbH,

Pasching, Austria), 1% l-glutamine solution (Sigma-Aldrich), and non-essential amino acid solution

(Sigma-Aldrich) in a humidiﬁed atmosphere containing 5% CO₂at 37 ^◦C. For subculturing, the cells

were harvested after trypsin/EDTA (Sigma-Aldrich) treatment at 37 ^◦C. To evaluate cytotoxicity, the

cells treated with the tested substances were used as experimental groups, whereas untreated HepG2

cells served as controls.

The cells were seeded in a density of 10,000 cells per well in a 96-well plate. During the next day,

the cells were treated with each of the tested substances dissolved in DMSO. The tested substances

were prepared at diﬀerent incubation concentrations (1–1000 µM) in triplicates according to their

solubility. Simultaneously, the controls representing 100% cell viability, 0% cell viability (the cells

treated with 10% DMSO), no cell control, and vehiculum controls were also prepared in triplicates.

After 24 h of incubation in a humidiﬁed atmosphere containing 5% CO₂at 37 ^◦C, the reagent from the

kit CellTiter 96 AQueous One Solution Cell Proliferation Assay (CellTiter 96; PROMEGA, Fitchburg,

WI, USA) was added. After 2 h of incubation at 37 ^◦C, the absorbance of the samples was recorded at

490 nm (TECAN, Inﬁnita M200, Austria). A standard toxicological parameter IC₅₀was calculated by

nonlinear regression from a semilogarithmic plot of incubation concentration versus the percentage

of absorbance relative to untreated controls using GraphPad Prism 8 software (GraphPad Software,

San Diego, CA, USA). The compounds were tested in two separate experiments in cell line passage 3

and 23.

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4. Conclusions

To conclude, thirty-nine N-pyrazinoyl aa derivatives were synthesized in this study. Twenty-four

of these compounds (mostly d-enantiomers) had not been previously described in the literature (to date

10th February 2020; racemic mixtures and enantiomeric mixtures were considered unique compounds).

The in vitro antimicrobial activity of all compounds was investigated, focused on antimycobacterial,

antifungal, and antibacterial activity. No activity was detected against any fungal strains. For

antibacterial activities, low activity against Staphylococcus aureus was noted for PC-l-OtBu-Thr-Me

(MIC = 31.25 µM). The main biological evaluation was thus focused on antimycobacterial testing.

The high activity of some compounds was mainly observed against Mtb H37Ra. Signiﬁcant activity

was noted only in the mildly acidic medium of pH 6, whereas, in the commonly utilized pH 6.6,

the compounds were generally inactive. The mixture of enantiomers PC-d/l-Pgl-Me presented the

best activity (MIC < 1.95 µg/mL, < 7.3 µM, pH 6), which—in molar concentrations—represents four

times higher activity than the ﬁrst-line antitubercular drug PZA. Esters PC-l-Ala-Et, PC-l-Met-Me,

PC-l-OBn-Ser-Me and PC-l-Tyr-Et were up to two times more active than PZA (in molar concentrations

at pH 6). The derivatives with the free carboxylic group were generally inactive. Based on the observed

structure–activity relationships (SAR), we conclude that the activity is tightly linked to the lipophilicity

of the compounds (more lipophilic preferred) as well as to the stereochemistry (l-isomers preferred).

For the ﬁrst time, PZA derivatives containing d-aa were investigated. However, their activity was

signiﬁcantly lower compared to the l-enantiomers.

The diﬀerence in antimycobacterial activity between l- and d-isomers might indicate the presence

of a speciﬁc subcellular target for unhydrolyzed product (pharmacodynamic aspect) or simply the

faster hydrolysis of the amidic bond of l-isomers by mycobacterial amidases to release the active

POA from its prodrug transport form (pharmacokinetics aspect). The exact active species (parent

compounds vs. hydrolysed POA) and its MoA on the molecular level are to be elucidated. However,

based on the recent biochemical and crystallographic investigations of PanD [12], the most prominent

target of PZA, we conclude that our compounds do not act in their non-hydrolysed form against this

target, as the binding cavity at the active site of PanD is too small to accommodate anything bigger

than POA or 6-Cl-POA.

The work presented in this article opens the door for further medicinal chemistry

optimizations of the most active compounds (PC-l-Ala-Et, PC-dl-Leu-Et, PC-l-Met-Me, PC-d/l-Pgl-Me,

PC-l-OBn-Ser-Me) and encourages future research. For the next step, we propose the assessment

of derivatives combining aa with antimycobacterially active 5-chloropyrazinamide (5-Cl-PZA) as a

simplest PZA derivative with known MoA [66]. Our preliminary results show that the l-Leu methyl

ester derivative of 5-Cl-PZA is active against Mtb H37Ra (MIC at pH 6.6 was 62.5

µg/mL, at pH

6 it was 3.91 g/mL). As 5-Cl-PZA is more lipophilic than PZA, even the methyl ester is probably

µ

suﬃcient to eﬀectively penetrate mycobacterial membrane. We thus concluded that the lipophilicity

of the aa part of our compounds indeed plays a role in their activity. For a conclusive description of

the structure–activity relationships (SAR) of the proposed compounds, more derivatives need to be

synthesized focusing mainly on the diﬀerences between the activity of diﬀerent enantiomers with the

same ester. This could bring new interesting knowledge of Mtb enzymatic patterns usable also in

other applications.

The design of aa-derived small molecules—notably as hydrolysable prodrugs—could be an

interesting therapeutic approach for mycobacterial infections. Mtb as any other bacterium is dependent

on nitrogen uptake to synthesize vital biomolecules. However, Mtb has been found to prefer

proteinogenic aa as the source of nitrogen over ammonium chloride when cultured in vitro [67,68].

Therefore, if the appropriate mechanisms and enzymes involved in these metabolic pathways were

identiﬁed, the use of aa derived prodrugs could ‘trick’ Mtb to cleave oﬀ the aa and at the same time

release the active form of the drug inside the cell. The plausibility of this strategy can be rationalized

based on the recent discoveries of several selective Mtb proteases essential either for the survival

or virulence of Mtb [69,70], which are proposed as good druggable targets. The ability to uptake

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all proteinogenic aa has been demonstrated, which could help smaller aa-containing (pro)drugs to

pass through the mycobacterial membrane barrier [68]. Penetration through the Mtb membrane

barrier could perhaps also be managed by one of the known Mtb ATP-binding cassette (ABC) peptide

importers, e.g., dipeptide importer DppABCD or oligopeptide importer OppABCD [71]. Notably, the

speciﬁcity of the latter has been shown to be very broad [72], which could support our speculations.

Supplementary Materials: The following are available online. Supplementary material (PDF) containing

representative spectra, methodologies and results of antibacterial and antifungal screening, chiral HPLC

chromatograms, description of calculated properties. SMILES, calculated properties and MIC against Mtb

H37Ra of the title compounds (CSV).

Author Contributions: Conceptualization, M.J., M.D. and J.Z. Investigation, M.J., L.K., O.H., O.J., V.K., K.K., R.K.,

P.B., J.J., P.P. and J.K.; Methodology, O.J., V.K., K.K., R.K., P.B., J.J., P.P., J.K. and J.Z.; Project administration, M.D.

and J.Z.; Supervision, M.D. and J.Z.; Validation, O.J., V.K., K.K., R.K., P.B., J.J., P.P. and J.K.; Visualization, M.J.,

and J.Z.; Writing—original draft, M.J.; Writing—review & editing, M.J., M.D. and J.Z. All authors have read and

agreed to the published version of the manuscript.

Funding: This research was funded by the Ministry of Education, Youth and Sports of the Czech

Republic (SVV 260 547) and by the Czech Science Foundation project No.

20-19638Y; EFSA-CDN

(No. CZ.02.1.01/0.0/0.0/16_019/0000841) co-funded by ERDF, and by CELSA-Project title: Structure-based design of

new antitubercular medicines-KU Leuven (Arthur Van Aerschot)-Charles University in Prague (Martin Doležal).

Acknowledgments: Computational resources were supplied by the Ministry of Education, Youth and Sports of

the Czech Republic under the Projects CESNET (Project No. LM2015042) and CERIT-Scientiﬁc Cloud (Project

No. LM2015085) provided within the program Projects of Large Research, Development and Innovations

Infrastructures. Great thanks go to Ghada Bouz, for language corrections.

Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the

study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to

publish the results.

Abbreviations

aa—amino acid, CDI—1,1⁰-carbonyldiimidazole, DMAP—4-(dimethylamino)pyridine, EtOAc—ethyl

acetate, MeAcr—methyl acrylate, MIC—minimum inhibitory concentration, MoA—mechanism of

action, mp—melting point, Mtb—Mycobacterium tuberculosis, PC—pyrazinecarbonyl, Pgl—phenylglycine,

POA—pyrazinoic acid, PZA—pyrazinamide, THF—tetrahydrofurane, TLC—thin-layer chromatography,

TMSCl—trimethylsilyl chloride.

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Sample Availability: Samples of the compounds are available from the corresponding authors.

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N-Pyrazinoyl substituted amino acids as potential antimycobacterial agents-the synthesis and biological evaluation of enantiomers

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