Design, synthesis and evaluation of N-pyrazinylbenzamides as potential antimycobacterial agents

molecules

Article

Design, Synthesis and Evaluation of

N-pyrazinylbenzamides as Potential

Antimycobacterial Agents

Jan Zitko ^1,

*

, Alžbeˇta Mindlová ¹, Ondrˇej Valášek ¹, Ondrˇej Jand’ourek ¹, Pavla Paterová ²,

Jirˇí Janoušek ¹, Klára Konecˇná ¹and Martin Doležal ¹

1

Faculty of Pharmacy in Hradec Králové, Charles University, Heyrovského 1203, 500 05 Hradec Králové,

Czech Republic; alzbeta.mindlova@gmail.com (A.M.); valaseko@faf.cuni.cz (O.V.);

jando6aa@faf.cuni.cz (O.J.); janousj2@faf.cuni.cz (J.J.); konecna@faf.cuni.cz (K.K.);

dolezalm@faf.cuni.cz (M.D.)

2

Department of Clinical Microbiology, University Hospital, Sokolská 581, 500 05

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

*

Correspondence: jan.zitko@faf.cuni.cz; Tel.: +420-495-067-272

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

ꢀꢁꢂꢃꢄꢅꢆ

Received: 29 August 2018; Accepted: 17 September 2018; Published: 18 September 2018

Abstract: Three series of N-(pyrazin-2-yl)benzamides were designed as retro-amide analogues of

previously published N-phenylpyrazine-2-carboxamides with in vitro antimycobacterial activity.

The synthesized retro-amides were evaluated for in vitro growth inhibiting activity against

Mycobacterium tuberculosis H37Rv (Mtb), three non-tuberculous mycobacterial strains (M. avium,

M. kansasii, M. smegmatis) and selected bacterial and fungal strains of clinical importance. Regarding

activity against Mtb, most N-pyrazinylbenzamides (retro-amides) possessed lower or no activity

compared to the corresponding N-phenylpyrazine-2-carboxamides with the same substitution

pattern. However, the active retro-amides tended to have lower HepG2 cytotoxicity and better

selectivity. Derivatives with 5-chloro substitution on the pyrazine ring were generally more active

compared to their 6-cloro positional isomers or non-chlorinated analogues. The best antimycobacterial

activity against Mtb was found in N-(5-chloropyrazin-2-yl)benzamides with short alkyl (2h: R²= Me

2i: R²= Et) in position 4 of the benzene ring (MIC = 6.25 and 3.13

g/mL, respectively, with SI > 10).

;

µ

N-(5-Chloropyrazin-2-ylbenzamides with hydroxy substitution (2b: R²= 2-OH; 2d: R²= 4-OH)

on the benzene ring or their acetylated synthetic precursors possessed the broadest spectrum

of activity, being active in all three groups of mycobacterial, bacterial and fungal strains.

The substantial differences in in silico calculated properties (hydrogen-bond pattern analysis,

molecular electrostatic potential, HOMO and LUMO) can justify the differences in biological activities

between N-pyrazinylbenzamides and N-phenylpyrazine-2-carboxamides.

Keywords: antibacterial; antifungal; antimycobacterial; cytotoxicity; linker; pyrazinamide;

retro-amide; tuberculosis

1. Introduction

According to the latest Global Tuberculosis Report published by the WHO [1], an estimated

10.4 million people worldwide developed active tuberculosis (TB) in 2016 and TB caused 1.7 million

deaths, including 0.4 million deaths among HIV-positive people. This mortality rate ranks TB

the leading cause of death from infectious diseases and in the top 10 causes of death generally,

following cardiovascular and respiratory diseases [

population (1.7 billion people) is latently infected with TB [

epidemiologic issue connected with TB. As estimated for 2016, there were 490,000 new cases of

1]. A recent study estimated that 23% of global

2

]. Multidrug-resistance is a serious

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Molecules 2018, 23, 2390

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multidrug-resistant TB (MDR-TB; resistant to rifampicin and isoniazid) and additionally 110,000

cases of rifampicin-resistant TB (RR-TB) [ ]. Even the basic regimen for non-resistant TB consists

1

of at least four ﬁrst-line antitubercular drugs (rifampicin, isoniazid, ethambutol and pyrazinamide)

administered for six months and it is challenging for patients’ compliance. The compliance problem

is further deepened in MDR-TB, as the simplest MDR-TB regimen recommended by the WHO takes

6–9 months and requires parenteral application [3]. There is therefore an urgent clinical requirement

for the development of new anti-TB drugs, leading to more effective and shorter therapeutic regimens.

Pyrazinamide (PZA; pyrazine-2-carboxamide) as a ﬁrst-line antitubercular agent is an important

component of the initial phase of all basic TB treatment regimen [

pyrazine-2-carboxylic acid (POA), or their simple structure derivatives were shown to act as

inhibitors of mycobacterial fatty acid synthase I (FAS I) [ ], aspartate decarboxylase (PanD) [ 11],

1]. PZA, its metabolite

4–8

9–

and quinolinic acid phosphoribosyltransferase (QAPRTase) [12]. Based on the results of protein binding

assays and X-ray crystallography, POA was suggested to act as an inhibitor of trans-translation, the

process of rescuing ribosomes stalled during translation [13,14]. However, this mechanism of action

(MoA) was recently disputed [15]. The perception of PZA and its metabolite POA has changed

from a non-speciﬁc cytosol acidiﬁer to a multi-target inhibitor of speciﬁc enzymes and processes of

mycobacteria [16].

Previously published N-phenylpyrazine-2-carboxamides (in other words anilides of POA) with

different simple substituents on the pyrazine ring (R¹is H, methyl, tert-butyl, chloro or combination

thereof) and simple substituents on the benzene ring (R²is short alkyl, hydroxy, halogen, nitro or

combination thereof, Figure 1) possessed in vitro growth inhibiting activity against Mycobacterium

tuberculosis H37Rv (Mtb) and other non-tuberculous mycobacterial strains. Best compounds possessed

activity with MIC at micromolar level (2–20

µM). Structure-activity relationships (SAR) in this class

were reviewed elsewhere [17 19]. Direct inspiration for compounds presented in this paper was our

–

previous series of 5-chloro-N-phenylpyrazine-2-carboxamides [18], which were generally more active

(best compounds with MIC at low micromolar level) than previously studied 6-Cl isomers. The benzene

ring in the series tolerated different substituents, both electron-donating and electron-withdrawing,

and hydrophilic and lipophilic. The drawback was signiﬁcant in vitro cytotoxicity (HepG2) with IC₅₀

for most compounds at units or tens of µM, leading to insufﬁcient selectivity in most of the compounds.

As a continued effort to extend the knowledge of SAR of N-phenylpyrazine-2-carboxamides as

antimycobacterial agents, we designed a series of N-(pyrazin-2-yl)benzamides, which can be regarded

retro-amide analogues (Figure 1).

Figure 1. Design of title compounds inspired by previously published N-phenylpyrazine-2-

carboxamides with antimycobacterial activity.

Exchanging the amide moiety for a retro-amide is a strategy successfully used in medicinal

chemistry and drug design. The obligatory textbook example is the group of local anesthetics.

Cinchocaine is a substituted amide of a heteroaromatic acid, therefore having the -CONH- connecting

bridge between the aromatic core and the basic amino moiety in the side chain. However, signiﬁcantly

more populated group of local anesthetics are anilides having the retro-amide -NHCO- linker

(e.g., lidocaine, trimecaine, bupivacaine). In the ﬁeld of antimicrobial research, the retro-amide

modiﬁcation led recently to derivatives with increased activity against Candida albicans [20].

Molecules 2018, 23, 2390

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The retro-amides of classical

β-lactam antibiotics and their analogues were studied as substrates

and/or inhibitors of beta-lactamases [21–24].

Thus, the main aim of our study was to evaluate the effect of amide (-CONH-) vs retro-amide

(-NHCO-) linker between the pyrazine and benzene core on in vitro antimycobacterial activity. Along

with the linker exchange, we kept the variability both in the pyrazine core (R¹is H, 5-Cl, or 6-Cl) and

in the benzene core (R²). Synthetic work was performed by two undergraduate students (co-authors of

this paper, A.M. and O.V.) as a part of their diploma theses, therefore we kept the chemistry complexity

at a suitable level.

2. Results and Discussion

2.1. Chemistry

Final compounds were prepared by simple benzoylation (Scheme 1,

a) of aminopyrazine (series 1),

5-chloropyrazin-2-amine (series ), or 6-chloropyrazin-2-amine (series ). The source of the substituted

2

3

benzoyls were commercially available benzoyl chlorides. In the case of hydroxy substituted derivatives,

we used acetoxybenzoyl chlorides and consequently cleaved off the protecting acetyl in the product by

mild alkaline hydrolysis of the ester bond (Scheme 1, b).

Scheme 1. Synthesis of ﬁnal compounds: (a) Benzoylation of aminopyrazine; (b) Hydrolysis of

acetylated derivatives.

In the benzoylation step, we were concerned about possible diacylation, which would produce

undesired side products by substitution of both hydrogens of the amino moiety of aminopyrazine.

Such N,N-diacylation was described for aminopyridine, 2-aminopyrimidine, aminopyrazine and

similar heterocyclic amines in a recent study [25]. In that study, the tendency for diacylation increased

with the strength of used base, more speciﬁcally, employment of pyridine yielded dominantly

monoacylated products, whereas triethylamine (Et₃N) yielded dominantly diacylated products

or mixtures [25]. Indeed, this was conﬁrmed by our experiments, when method A described in

Section 3.2.1 with 3 molar equivalents (equiv) of Et₃N instead of pyridine led to diacylated product

1l-SP (Figure 2 ). Using Et₃N as a base, only diacylated products were obtained. Therefore, in all further

reactions we used dry pyridine (3 or 4 equiv) as a base. To further decrease the probability of formation

of diacylated products, the mixture of the benzoyl chloride with pyridine in appropriate solvent was

pre-cooled in the freezer and subsequently cooled in an ice bath during addition of the aminopyrazine

component to prevent spontaneous heating. We expected that increased reaction temperature could

have preferred diacylation.

Molecules 2018, 23, 2390

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Cl

O

N

O

Cl

Figure 2. Structure of N,N-diacylated product 1l-SP.

Final compounds were isolated as white or very pale yellow colored solids. The isolated yields of

chromatographically pure product after all puriﬁcation steps ranged from 8–67% for Method A and

43–85% for Method B. Method B is a modiﬁcation of Method A and was performed under nitrogen

atmosphere and with extended work-up. Method A performed without the inert atmosphere gave

lower, but still acceptable yields.

The deprotection of acetoxy derivatives was achieved by mild base catalyzed hydrolysis of the

acetate bond by dissolving in EtOH and heating with excess of K₂CO₃. However, in some cases,

we observed spontaneous acetate hydrolysis during workup and/or puriﬁcation. For example, in the

attempt to prepare 1b-Ac, the compound hydrolyzed during ﬂash chromatography (this could have

been catalyzed by the acidic properties of silica) or during recrystallization from hot aqueous ethanol.

All ﬁnal products were fully characterized by melting point, ¹H- and ¹³C-NMR spectra,

IR spectra and elementary analysis. The acquired data were fully consistent with proposed structures.

The summary or prepared compounds and their codes are presented in Table 1.

Table 1.

Green—MIC = 3.13–6.25 µg/mL; Yellow—MIC = 12.5–25

White—MIC > 100 µg/mL (not active); n.a. = compound not available (not prepared).

Summary of prepared derivatives—code assignment and activity against Mtb.

µ

g/mL; Red—MIC = 50–100 g/mL;

µ

R²

H

N

R¹

O

R¹

5-Cl

2a

R²

H

1a

6-Cl

3a

n.a.

3e

3f

3g

3h

3i

2-OH

3-OH

4-OH

2-OCH₃

3-OCH₃

4-OCH₃

4-CH₃

4-Et

1b

1c

n.a.

1e

2b

2c

2d

2e

2f

2g

2h

2i

1f

1g

1h

1i

2-Cl

1j

2j

3j

3-Cl

4-Cl

1k

1l

2k

2l

3k

3l

4-Br

n.a.

1n

n.a.

1c-Ac

1d-Ac

2m

2n

2b-Ac

2c-Ac

2d-Ac

n.a.

3n

n.a.

3-CF₃

2-OAc

3-OAc

4-OAc

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2.2. Antimycobacterial Activity

Title compounds were screened for in vitro growth inhibiting activity against Mycobacterium

tuberculosis H37Rv (Mtb), two non-tubercular slow growing mycobacterial strains (M. kansasii and

M. avium) and a strain of fast-growing M. smegmatis. The assays were Microplate Alamar Blue Assays

(MABA) [26] with modiﬁcations. See Table 2 for active compounds and the Supplementary Material

for full results and detailed experimental procedures.

Table 2. Antimycobacterial activity of prepared compounds expressed as MIC (µg/mL) ^a.

Mtb H37Rv

ATCC 27294

MIC (µg/mL)

M. kans.

ATCC 12478

MIC (µg/mL)

M. avium

ATCC 15769

MIC (µg/mL)

M. smeg.

ATCC 607

MIC (µg/mL)

Cpd

R¹

R²

MW

1a

1e

1g

1j

1k

H

5-Cl

6-Cl

-

H

2-OCH₃

4-OCH₃

2-Cl

3-Cl

4-Cl

199.21

229.24

233.66

372.21

291.69

249.65

291.69

247.68

261.71

301.65

263.68

301.65

137.14

822.95

331.35

100

>100

25

>100

50

12.5

>100

6.25

3.13

25

>100

100

>100

50

>100

50

≥500

125

≥500

250

≥500

31.25

15.625

250

1l

50

1l-SP ^b

2b-Ac

2b

4-Cl ^b

2-OAc

2-OH

3-OAc

4-OAc

4-CH₃

4-Et

3-CF₃

3-OCH₃

3-CF₃

-

>100

12.5

50

>100

100 ^c

25

50

2c-Ac

2d-Ac

2h

2i

2n

3f

3n

>100

100 ^c

50

250

≥500

≥500 ^c

125

15.625

1.56

0.195

50 ^c

25

0.1–0.39

-

INH

RFM

CPX

a

6.25–12.5

-

6.25–12.5

-

Compounds not listed in the table were inactive against all tested strains. Complete version of the table is

b

c

located in the Supplementary Material. Diacylated side-product. Precipitate formed in the testing medium.

INH—isoniazid; RFM—rifampicin; CPX—ciproﬂoxacin.

Regarding the antimycobacterial activity against Mtb, series

was the most successful, containing ﬁve active compounds with MIC ranging 3.13–25

The activity in series derived from non-chlorinated aminopyrazine as well as in series

derived from 6-Cl-pyrazin-2-amine was lower (MIC = 25–100 g/mL) and less abundant,

yielding four moderately or low active compounds in series and two moderately or low

active compounds in series (see graphical representation in Table 1 ). The superiority of

2

derived from 5-Cl-pyrazin-2-amine

µg/mL.

1

3

µ

1

3

5-chloropyrazine substituted derivatives is in concordance with relationships observed in previous

series, where 5-chloro-N-phenylpyrazine-2-carboxamides [18] were more active than their 6-Cl

positional isomers [17] or non-chlorinated derivatives [27].

Discussing the substituent R²on the benzene ring, 2-OH derivative (2b) and its acetylated

precursor (2b-Ac) showed MIC of 12.5 µg/mL against Mtb, and importantly showed similar activity

on the non-tubercular mycobacterial strains. As it will be mentioned in Section 2.4, the activity

spectrum of these two compounds expands also to tested fungal strains. Another successful

substituent R²in series

MIC = 3.13–6.25

chlorinated series (compounds 2n and 3n). In the case of compound 3n, the activity spectrum was

broad and covered all tested mycobacterial strains. The most active derivative in series was 1g with

R²= 4-OCH₃. Generally, the most active derivatives in all series

and follow the substitution

2

was short alkyl in position 4 (2h: R²= 4-CH₃; 2i: R²= 4-Et) with

µ

g/mL against Mtb. Substitution R²= 3-CF₃yielded active compounds in both

1

,

2

3

pattern that was proposed for N-phenylpyrazine-2-carboxamides [19], that is, the benzene ring

substituted with electron-withdrawing substituent in position 3 and/or electron-donating substituent

in position 4.

Molecules 2018, 23, 2390

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2.3. Antibacterial Activity

As a complementary test, ﬁnal compounds were screened for in vitro antibacterial activity against

eight G+ and G bacterial strains of clinical importance. The method was a microplate dilution assay

with MICs determined by naked eye. For experimental details, list of tested strains and full results

see Supplementary Material. Table 3 lists compounds with antibacterial MIC 125 M (and some of

their inactive positional isomers for comparison). It is evident that title compounds exerted mainly

anti-staphylococcal activity and that (with the exception of 1n) they belonged to series derived

from 5-Cl-pyrazin-2-amine. Signiﬁcant activity was exerted by hydroxy derivatives 2b 2d and their

acetylated precursors 2b-Ac 2d-Ac. We assume that acetylated derivatives might act as transport

−

≤

µ

2

,

form and undergo hydrolysis by various esterases either in plasma or after penetration to a bacterial

cell. Interestingly, the hydroxy or acetoxy substituent could be in position 2 or 4 of the benzene ring,

but not in position 3—compounds 2c and 2c-Ac were inactive. 2-Substituted derivatives 2b and 2b-Ac

inhibited all three tested Staphylococcus strains (including MRSA), whereas 4-substituted derivatives 2d

and 2d-Ac were active only against the reference strain of S. aureus. Positional isomerism on benzene

ring effected the activity also in other examples—the 2-Cl derivative 2j and 3-OCH₃derivative 2f

showed moderate activity against S. aureus, but their positional isomers 2k

, 2l and 2e, 2g were inactive

(MIC > 125 M). Most compounds of series (derived from 6-Cl-pyrazin-2-amine) could have not

µ

3

been tested due to excessive precipitation in the testing medium.

Table 3. Antibacterial activity of selected compounds expressed as MIC (µM) in comparison with

standard neomycin.

Compound (Code, R²)

1n

2b-Ac

2b

2c-Ac

2c

2d-Ac

2d

2f

2j

Neom

-

Strain Time

3-CF₃2-OAc 2-OH 3-OAc 3-OH 4-OAc 4-OH 3-OCH₃

2-Cl

SA

24 h

48 h

>125

62.5

125

15.62

>125

500

0.98

31.25

62.50

3.90

MRSA 24 h

48 h

>125

125

250

62.50

>125

>500

>125

>500

0.98

SE

24 h

48 h

62.50

125

250

62.50

>125

>500

>125

500

>500

3.90

7.81

SA–Staphylococcus aureus CCM 4516/08; MRSA–S. aureus H 5996/08 methicillin resistant, clinical isolate;

S. epidermidis H 6966/08, clinical isolate. Neom–neomycin. For more standard drugs, see Supplementary Material.

2.4. Antifungal Activity

As a complementary test, ﬁnal compounds were tested for in vitro antifungal activity against

eight species of clinical importance. The testing was based on microdilution method; results are

expressed as MIC (µM) read by naked eye. For experimental details, list of tested strains and full

results see Supplementary Material. The following paragraph concerns the MIC values read after 24 h

of incubation (with the exception of Trichophyton interdigitale, where the ﬁrst reading was after 72 h).

In series

antifungal activity, inhibiting all tested strains with MIC = 62.5 or 125

the acetylated precursor of 2b, showed similarly broad spectrum of activity of moderate-low level

2

(R¹= 5-Cl), compound 2b (R²= 2-OH) exerted moderate and strain non-speciﬁc

µM. Compound 2b-Ac,

(MIC = 125–250

µ

M). It is probable that 2b is generated by hydrolysis of acetate 2b-Ac spontaneously

◦

(cultivation at 35 C in water-based medium) and/or by the pathogen metabolism. Interestingly,

the activity seems to be strongly dependent on the position of the hydroxyl substituent, as 3-OH (2c

and 4-OH (2d) positional isomers were inactive (MIC 500

M). Compound 2f (R²= 3-OCH₃) showed

moderate activity against Candida albicans (MIC = 125 M) but no activity against any other tested

were inactive up to the highest tested

M or 500 M. The antifungal activity

)

≥

µ

fungal species. The rest of the tested compounds from series

concentration, which (dependent on the solubility) were 125

2

µ

Molecules 2018, 23, 2390

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determined for the title compounds must be considered insigniﬁcant in comparison with standard

nystatin, which expressed MIC ranging from 0.98 to 15.62 M for all tested strains (see Supplementary

Material for MIC values of additional standards). No antifungal activity was observed in series and

Most of the compounds of series (derived from 6-Cl-pyrazin-2-amine) could not be tested due

µ

1

3.

3

to excessive precipitation in the testing medium. See Supplementary Material for full results in

tabular form.

2.5. In Vitro Cytotoxicity

Selected active compounds were tested for their in vitro cytotoxic effect in human hepatocellular

carcinoma cell line (HepG2), using a commercial colorimetric assay (CellTiter 96^®AQueous One

Solution Cell Proliferation Assay, Promega, Madison, WI, USA). The cells were incubated with different

concentrations of tested compounds and after 24 h, the viability was compared to untreated cells.

All experiments were performed in triplicates. Viability curves were constructed, and the cytotoxicity

was expressed as IC₅₀, that is, the concentration required to decrease the viability of cell population

to 50% compared to the control of 100% cell viability. See Table 4 for results. The limited solubility

of compounds 2d-Ac

sufﬁciently high concentrations, therefore the viability curve could not be constructed. In other words,

the IC₅₀of these compounds is signiﬁcantly higher than the highest measured concentration (250

or 500 M). To assess the selectivity to (myco)bacterial cells over human cells, we calculated the

, 2h, 2i and 3n in the culture medium did not allow for the measurement at

µM

µ

corresponding selectivity indexes (Table 4). Compounds with SI > 10 are considered to have potential

for further development.

Table 4. In vitro HepG2 cytotoxicity of selected N-pyrazinylbenzamides expressed as IC₅₀

their selectivity indexes (SI) for Mtb and S. aureus.

(µM) and

HepG2

Mtb

MIC (µM)

SA

MIC (µM)

Cpd

IC₅₀(µM)

SI

2b-Ac

2b

2d-Ac

2h

2i

3n

95.37

155.30

>500 *

>250 *

42.85

50.07

inactive

25.23

11.96

82.88

2

3

n.a.

>10

>21

>3

65.20

15.62

0.98

>125

n.t.

1

10

>510

n.a.

* Measurement at higher concentrations was not possible due to the precipitation of the tested compound in

testing medium. SI—calculated as IC₅₀/MIC in molar concentrations rounded to units. SA—Staphylococcus aureus

CCM 4516/08, MIC after 24 h.

As a conﬁrmatory test, 2b and 2i were further tested for prolonged cytotoxic effects on HepG2

cells using commercial ﬂuorescence kit CellTox

values were determined after 24 h and 48 h of incubation with tested compounds—see Table 5 for

results. The results conﬁrmed low cytotoxicity with IC₅₀values at hundreds of M. Importantly,

™ Green Cytotoxicity Assay (Promega). The IC₅₀

µ

the cytotoxicity of 2i did not increase with time. Experimental details for both cytotoxicity tests can be

found in the Supplementary Material.

Table 5. Prolonged cytotoxicity in HepG2 cells.

IC₅₀(µM)

after 24 h Exposure

IC₅₀(µM)

after 48 h Exposure

Range of Tested

Concentrations (µM)

Compound

2b

2i

545.3

264.3

187.6

250.0

10–2000

1–1000

Molecules 2018, 23, 2390

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2.6. Comparison of the Antimycobacterial Activity and HepG2 Cytotoxicity of N-Pyrazinylbenzamides

(Retro-Amides) with the Corresponding N-Phenylpyrazine-2-carboxamides (Amides)

The central question of our study was how the inversion of the amide linker in the

general structure of previously published N-phenylpyrazine-2-carboxamides will inﬂuence their

antimycobacterial activity. To assess this issue, we compared the antimycobacterial activity of title

N-pyrazinylbenzamides (retro-amides, -NHCO-, left side of Table 6) of this study to the activity of

previously published counterparts (-CONH-, right side of Table 6 ) with the same substitution both

on the pyrazine (R¹) and benzene (R²) ring. As the activity on other strains was rather sporadic,

we focused on the activity against Mtb.

As indicated in Table 6, for most of the pairs, the activity of N-pyrazinylbenzamides (retro-amides)

was substantially lower or none in comparison with original N-phenylpyrazine-2-carboxamides.

A signiﬁcant exception was 2i (R¹= 5-Cl, R²= 4-Et), the most active derivative of this study,

whose activity (MIC = 3.13

-carboxamide counterpart (MIC = 0.78 or 1.56

in series

(R¹= H) and (R¹= 6-Cl) is complicated by the fact that for many of the corresponding

amides, the MIC values were not determined and the only value we have for comparison is the

percentage of growth inhibition at ﬁxed concentration 6.25 g/mL. (These results were produced in the

Tuberculosis Antimicrobial Acquisition and Coordinating Facility (TAACF) screening campaign [28]).

In case that the retro-amide is completely inactive (MIC > 100 g/mL), the direct comparison is

µg/mL) against Mtb was comparable with its N-phenylpyrazine-2

µg/mL). The evaluation of linker inversion effect

1

3

µ

possible. In other cases, where there is some activity for the retro-amide and some percentage of

inhibition for the amide, the comparison should be taken as a rough guess, as indicated with the

question mark in Table 6.

Generally, it can be concluded that inversion of the amide linker in N-phenylpyrazine-2-

carboxamides leads to decrease or loss of antimycobacterial activity in most cases.

N.B.: The methodologies used for MIC determination of historically published compounds

(amides) were similar, but not identical, to conditions used in the up-to-date testing used in this paper.

The differences are stated as footnotes to Table 6 and details can be found in the original literature as

referenced. It is well known that the growth of mycobacteria is highly inﬂuenced by the conditions of

cultivation, for example pH [29,30]. Therefore, the comparisons presented in the preceding paragraphs

and in Table 6 are to be taken as an assessment of general trends and not as comparison of precise

MIC values.

Molecules 2018, 23, 2390

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Table 6. Comparison of antimycobacterial activity (Mtb) of prepared N-pyrazinylbenzamides

(retro-amides) with corresponding N-phenylpyrazine-2-carboxamides (amides).

O

R²

H

N

H

R¹

O

R¹

R²

Relation

Retro-Amides

MIC ^a

Amides

Inhibition (%) at

6.25 µg/mL (TAACF)

Code

MIC (µg/mL)

(µg/mL)

>100 ^b[27]

H

1a

1e

1f

1g

1h

1j

100

>100

25

>100

50

=

<

0 [31]

2-OCH₃

3-OCH₃

4-OCH₃

4-CH₃

2-Cl

>100 ^b[27]

100 ^b[27]

86 [32]

<

50 ^c[19]

>100 ^b[27]

3-Cl

4-Cl

1k

1l

=?

>=?

<

=

<

<?

>?

<

14 [33]

4 [33]

99 [32]

50 ^b[27]

50

6.25 [32], 50 ^b[27]

3.13 (1.56) ^c[18]

3.13 (0.78) ^c[18]

6.25 ^c[18]

H

3-CF₃

H

2-OH

3-OH

4-OH

4-Et

2-Cl

3-Cl

4-Br

3-CF₃

1n

2a

2b

2c

2d

2i

>100

12.5

>100

3.13

>100

25

>100

50

>100

25

5-Cl

6-Cl

3.13 (12.5) ^c[18]

1.56 (0.78) ^c[18]

3.13 (0.78) ^c

6.25 (3.13) ^c[18]

3.13 ^c[18]

2j

2k

2m

2n

3a

3e

3f

3h

3j

3k

3l

3n

3.13 (6.25) ^c[18]

25 ^d[34]

H

32 [31]

6 [35]

2 [31]

2-OCH₃

3-OCH₃

4-CH₃

2-Cl

3-Cl

4-Cl

71 [32]

100 ^d[34]

14 [33]

65 [33]

77 [32]

<

3-CF₃

a

Conditions for determination of MIC: Middlebrook 7H9 with OADC growth supplement, pH = 6.6, MABA;

b

c

Middlebrook 7H9, pH = 6.6, MABA; Šula’s semisynthetic medium, pH = 5.6, growth indicated by

d

turbidity; Šula’s semisynthetic medium, pH = 5.6, MABA. TAACF—Tuberculosis Antimicrobial Acquisition

and Coordinating Facility screening campaign. In Relation column—‘?’ indicates a rough guess.

Focusing on the in vitro HepG2 cytotoxicity, the template ‘amides’ exerted signiﬁcant toxicity

with IC₅₀for most compounds at units or tens of

µM, leading to insufﬁcient selectivity in most

of the compounds [18]. The IC₅₀values for here presented retro-amides were in hundreds of

µM.

Direct comparison of identically substituted pairs of amide vs retro-amide analogues are presented

in Table 7. Although based on limited data, it can be implicated that the exchange of amide for

retro-amide linker decreases the HepG2 cytotoxicity 10 times.

Table 7. Comparison of in vitro HepG2 cytotoxicity of prepared N-pyrazinylbenzamides (retro-amides)

with corresponding N-phenylpyrazine-2-carboxamides (amides).

Retro-Amide

-NHCO-

Amide

-CONH-

Substituents

R¹

R²

Code

IC₅₀(µM)

5-Cl

2-OH

4-Et

2b

2i

155.3

>250

30.0 [18]

7.2 [18]

Molecules 2018, 23, 2390

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2.7. In Silico Prediction of Molecular Structure and Properties

As we showed in the previous section, it is obvious that exchange of an amide linker for a

retro-amide linker has profound effects on the biological activity of the pyrazine-linker-benzene-like

compounds. In an attempt to rationalize the differences, we selected a pair constituted by the inactive

retro-amide 2a and its active amide counterpart for an in silico study of their molecular structure

and properties, which could help explain possible differences in binding to a hypothetical molecular

receptor. Molecular mechanics (AMBER10:EHT forceﬁeld) and quantum mechanics (B3LYP/cc-pVTZ

level of theory) were used to predict the energetically minimized conformations. Molecular electrostatic

potential (MEP) and frontier orbitals (HOMO, LUMO) were calculated at the B3LYP/cc-pVTZ level

of theory.

2.7.1. Geometry Optimization

In the structure optimization efforts, we realized that the amide counterpart has two sterically

signiﬁcantly different conformers of similar energy (see Figures 3 and 4)—conf 1 with E_h=

−

1123.499

hartree and conf 2 with E_h= 1123.483 hartree as predicted on B3LYP/cc-pVTZ level of theory.

−

The two conformers both have the plane of the amide bond in the plane of the pyrazine ring

but differ by rotation of 180 degrees. The presence of two energetically close conformers of

N-phenylpyrazine-2-carboxamides was predicted by QM calculations before [36]. With such close

energy values, it is presumable that both of the presented conformers are biologically relevant.

Figure 3.

Analysis of selected pharmacophore features of the minimized conformations.

Turquoise dot—HBA, violet dot—HBD. Spheres represent the respective projections.

Figure 4. Total electron density isosurface colored by molecular electrostatic potential (MEP).

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2.7.2. Analysis of Pharmacophore Features

We analyzed the hydrogen-bonding pattern that the compounds are able to produce in their

minimized conformations. Pharmacophore features (HBD, HBA and their projections) are presented in

Figure 3. Projections are depicted as spheres, which represent the space where the heavy atom of the

bonding partner of the protein should be present. There were signiﬁcant differences among the studied

compounds/conformers. Both retro-amide 2a and amide in conf 1 have three HBAs (which produce in

total four HBA projections) and one HBD. In 2a, the projection sphere of the HBA arising from the

N1 of the pyrazine ring is close to the HBD projection coming from the -NH- moiety of the linker.

It might be difﬁcult for the protein to satisfy both of these demands due to steric hindrance. In conf 1,

the situation is similar, but the projection spheres of the HBA and HBD are overlapping. This means

that one functional group (let us say a hydroxyl) of a protein could satisfy both the HBA and HBD

features of the ligand at the same time. Amide in conf 2 has only three HBAs and no HBD projection.

Of course the potential HBD in the form of -NH- of the linker is still present, but its projection sphere

is occupied by H-3 of the pyrazine core, so in this conformation the HBD cannot interact with protein.

We can conclude that N-phenylpyrazine-2-carboxamide prototype (conf 1) should have the greatest

potential for interaction with a theoretical protein receptor.

2.7.3. Molecular Electrostatic Potential (MEP)

Total electron density isosurface corresponding to the van der Waals size of the molecule

(isosurface contour 0.002 au) was calculated and mapped with molecular electrostatic potential (MEP,

Figure 4). Red color represents the most negative potential (places with relative abundance of electrons),

whereas the positive potential (relative lack of electrons) is represented by blue color. In between values

are colored according to the RGB scale (green representing neutral values with MEP close to zero).

From the MEP, it is evident that the negative charge covers the carbonyl group and the positive region

is over the -NH- group of the linkers. High electronegativity of the carbonyl group makes it the most

reactive part of the molecule in all studied compounds/conformers. Direct comparison of retro-amide

2a with amide in conf 1 reveals the differences in the electron density in the pyrazine nucleus. In 2a

,

both N-1 and N-4 of the pyrazine ring are associated with negative MEP potential and therefore are

expected to be strong HBAs. The amide in conf 1, however, lacks signiﬁcantly negative MEP at N-1.

This can be explained by close vicinity of the carbonyl with electron-withdrawing properties. We an

conclude that the ring N-1 in amide in conf 1 is expected to be weaker HBA compared to N-1 of

the retro-amide. Amide in conf 2 is different to conf 1 and creates a large area of negative potential

around carbonyl and N-1, and a large area of positive potential around -NH- of the linker and H-3 of

the pyrazine.

2.7.4. HOMO and LUMO Orbitals

The HOMO and LUMO orbitals were calculated at the B3LYP/cc-pVTZ level of theory (Figure 5).

The spatial distributions of both HOMO and LUMO were very similar between conf 1 and conf 2 of the

amide, so they will be discussed jointly. HOMO of 2a (retro-amide) was localized on the pyrazine core

and on the linker, whereas HOMO of the amide was on the phenyl and the linker. LUMO of 2a was

localized over the whole molecule, whereas in amide the LUMO was restrained to the pyrazine and

the linker. The HOMO-LUMO gap was 4.789 eV for 2a, 3.891 eV for amide in conf 1, and 4.027 eV for

amide in conf 2. Without further detailed analysis, it can be stated that the exchange of amide linker for

retro-amide linker has profound effects on the localization of the frontier orbitals (HOMO and LUMO)

and is therefore probable to inﬂuence compounds reactivity and interactions to a hypothetic receptor.

Molecules 2018, 23, 2390

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Figure 5. Predicted HOMO and LUMO.

3. Materials and Methods

3.1. General

All reagents and solvents (unless stated otherwise) were purchased from Sigma-Aldrich

(Schnelldorf, Germany). Aminopyrazine (98%, Sigma-Aldrich), and 6-chloropyrazin-2-amine (97%,

Synthonix, Wake Forest, NC, USA) were used without any puriﬁcation. 5-Chloropyrazin-2-amine

(>95%, Synthonix) as purchased was contaminated by a signiﬁcant amount of another compound,

probably a positional isomer as indicated by NMR. Therefore, a routine ﬂash chromatography (silica,

EtOAc in hexane gradient elution) puriﬁcation was applied before usage. Substituted benzoyl chlorides

were purchased from Sigma-Aldrich.

The reaction process and the purity of ﬁnal compounds were checked using Silica 60 F₂₅₄

TLC plates (Merck, Darmstadt, Germany). Flash chromatography of the ﬁnal compounds was

performed on a CombiFlash Rf 200 automated chromatograph (Teledyne Isco, Lincoln, NE, USA)

using columns ﬁlled with Kieselgel 60, 0.040–0.063 mm (Merck) and a detection wavelength of 280 nm.

NMR spectra were recorded on Varian VNMR S500 or at Varian Mercury VX-BB 300 spectrometers

(Varian, Palo Alto, CA, USA). The spectra were recorded in DMSO-d₆or CDCl₃at ambient temperature.

The chemical shifts as

δ values in ppm are indirectly referenced to tetramethylsilane (TMS) via the

solvent signal. IR spectra were recorded on a Nicolet Impact 400 spectrophotometer (Nicolet, Madison,

WI, USA) using ATR-Ge method. Elemental analysis was performed on a Vario MICRO cube Element

Analyzer (Elementar Analysensysteme, Hanau, Germany). All values regarding elemental analyses

are given as percentages. Melting points were determined in open capillary on Stuart SMP30 melting

point apparatus (Bibby Scientiﬁc Limited, Staffordshire, UK) and are uncorrected. Yields are given as

percentages and refer to the amount of chromatographically pure product after all puriﬁcation steps.

3.2. Chemistry

3.2.1. Method A (Used in the Synthesis of Final Products from Series 1 and 3)

A mixture of dry dichloromethane (DCM, 2 mL) and dry pyridine (475 mg, 6 mmol, 3 molar

equiv) was put into 25 mL round-bottom ﬂask, closed with a stopper and cooled in a freezer for

approximately 15 min. A selected benzoyl chloride (2.4 mmol, 1.2 equiv) was diluted with dry DCM

(5 mL) and added dropwise to the cooled (ice bath) pyridine/DCM mixture under stirring, and the

Molecules 2018, 23, 2390

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mixture was stirred for additional 5 min in the closed ﬂask. 2-Aminopyrazine (190 mg, 2 mmol,

1 equiv) or 6-chloropyrazin-2-amine (259 mg, 2 mmol, 1 equiv) was dissolved in DCM (2 mL) and

added dropwise to the cooled reaction mixture over 10 min upon stirring. After additional 15 min,

the reaction was removed from the ice bath and stirred at laboratory temperature. The progress of

reaction was monitored by TLC (silica plates, 33% EtOAc in hexane). After 2 h, no signiﬁcant further

increase in the spot of the product was observed, so the reaction was ended and worked-up.

The reaction mixture was adsorbed on silica (4 g) by evaporating the solvents under

reduced pressure. The mixture on silica was used for solid loading the ﬂash chromatography

pre-column. The separation used the following conditions: manually ﬁlled silica column (30 g),

continuous gradient elution 0–50% EtOAc in hexane, ﬂow rate 35 mL/min, detection wavelength

280 nm, monitoring wavelength 260 nm. Fractions containing pure product were combined and

solvents were evaporated under reduced pressure to yield solid product. If needed, the products

were recrystallized from hot EtOH, the crystallization was induced by cooling and addition of water.

The products were isolated as white solids. In several cases, the ﬁnal products were still contaminated

with non-speciﬁed impurity of brown color. This impurity was easily removed by dispersing the

product in small amount of hexane and immersion of a vertical piece of ﬁltration paper into this

dispersion. The impurity was soluble in hexane and rose by capillary action to the ﬁltration paper.

3.2.2. Method B (Used in the Synthesis of Final Products from Series 2)

Method B is a modiﬁcation of Method A and was performed under nitrogen atmosphere and

with extended work-up. Substituted benzoyl chloride (1.5 mmol, 1.2 equiv) was placed into the ﬂask

under nitrogen, diluted with dry DCM (5 mL) and dry pyridine (400 mg, 5 mmol, 4 equiv) was added.

The mixture was mixed for 5 min under nitrogen. Then, 5-chloropyrazin-2-amine (162 mg, 1.25 mmol,

1 equiv) dissolved in DCM (10 mL) was added dropwise over 10 min under nitrogen ﬂow. The ﬂask

was closed by septum and stirred for additional 6 h. After reaction, the mixture was diluted with DCM

to the ﬁnal volume of 40 mL and washed with water (1

×

30 mL), 5% (m/m) aqueous NaHCO₃solution

(1 30 mL), and brine (1 30 mL). The organic layer was dried over anhydrous Na₂SO₄and adsorbed

×

on silica (4 g) by evaporating the solvents under reduced pressure. Automated ﬂash chromatography

was run using same conditions as described in Method A. If needed, the products were recrystallized

from hot EtOH (crystallization initiated by cooling and dropwise addition of cold water).

3.2.3. Hydrolysis of Acetates

The acetylated product (compounds 1c-Ac, 2b-Ac, 2c-Ac, or 2d-Ac) was placed in a beaker and

dissolved in hot EtOH. Excess (5 equiv) of potassium carbonate (K₂CO₃) was added and the reaction

mixture was stirred at 50 ^◦C for 2 h under TLC control. Reaction mixture was adsorbed on silica and

puriﬁed using ﬂash chromatography (silica, gradient elution EtOAc in hexane 10–60%).

3.3. Analytical Data of Prepared Compounds

Analytical data of prepared compounds, including the references to previously published

compounds, are located in the Supplementary Material.

3.4. Biological Methods

For tested strains and detailed description of the biological methods, refer to the

Supplementary Material.

3.5. In Silico Calculations

Energetically minimized 3D structures of 2a and its amide counterpart were predicted by

MOE v2018.1001 (Molecular Operating Environment, Chemical Computing Group Inc., Montreal,

QC, Canada). All calculations were performed under AMBER10:EHT forceﬁeld (implicit R-ﬁeld

Molecules 2018, 23, 2390

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solvent model). The structures were drawn manually in MOE Builder and initially minimized

(the built-in minimization method combines both steepest gradient and conjugate gradient methods)

to 0.1 kcal

·

mol^{−1 −2}. These minimized conformations were subjected to conformational search

A

·

using low mode molecular dynamics with default settings to determine the global minimum for

each compound. The resulting conformer with lowest energy for each compound was considered

a representative structure. The obtained coordinates were used to prepare the input for DFT

calculations, which were performed in GAMESS (version 2016-pgi-linux-mkl, Mark Gordon’s Quantum

Theory Group, Ames Laboratory/Iowa State University, Ames, IA, USA). In GAMESS, the geometry

of the structures was ﬁrst optimized (RUNTYP = OPTIMIZE, SCF gradient convergence criteria

0.001 Hartree/Bohr) and subsequently the single point energy of the minimized structures was

calculated (RUNTYP = ENERGY). All QM calculations were performed at B3LYP/cc-pVTZ level

of theory, which was previously used for N-phenylpyrazine-2-carboxamides and gave predictions

consistent with experimentally determined properties [36]. Figure 3 was generated in MOE, using the

Pharmacophore Editor and Uniﬁed annotation scheme of pharmacophore features. Figures 4 and 5

were generated in wxMacMolPlt v7.7 (freeware by Brett Bode, NCSA, Urbana, IL, USA) [37].

4. Conclusions

In this study, we have designed and prepared 41 N-(pyrazin-2-yl)benzamides,

which can be regarded as retro-amide analogues of previously studied antimycobacterial

N-phenylpyrazine-2-carboxamides. To our knowledge, 35 of the reported compounds are new,

not previously described in literature (as checked in CAS SciFinder on 27 July 2018). Compounds

1d-Ac, 1g, 1h, 1j, and 1n were previously described in literature (see the Analytical Data section in

Supplementary Material), but nothing had been published on their antimycobacterial or generally

anti-infective activity. Compound 1b was studied for its antimycobacterial properties in vitro [38] and

the previously reported results are in concordance with our data.

Prepared retro-amides were tested for in vitro growth inhibiting activity on Mtb and three

other mycobacterial strains. Our results clearly indicate that inversion to retro-amide linker

usually leads to decrease or loss of activity, but the remaining active retro-amides are more

selective and probably also less toxic to mammalian cells (HepG2) in comparison with the

pattern amides (N-phenylpyrazine-2-carboxamides). The antimycobacterial activity appeared

dominantly in series

2 derived from 5-chloropyrazin-2-amine (in concordance with previous

observations that 5-Cl-N-phenylpyrazine-2-carboxamides were generally more active than their

6-Cl positional isomers or non-chlorinated analogues [18]). Among the prepared retro-amides of

series 2, signiﬁcant antimycobacterial activity was found only for several compounds with speciﬁc

substitution pattern on the benzene ring. This is in sharp contrast with the previously published

5-chloro-N-phenylpyrazine-2-carboxamides (amides), in which the benzene core tolerated many

substituents, both hydrophilic and lipophilic and electron-donating and withdrawing [18].

Considering their MIC values, in vitro cytotoxicity (HepG2) and the resulting selectivity for

mycobacterial cells over mammalian cells, the most promising N-pyrazinylbenzamides were 2h and 2i,

that is, derivatives with short alkyl in position 4 of the benzene ring (see Table 3).

N-Pyrazinylbenzamides with hydroxy substitution on the benzene ring (such as 2b) or their

acetylated precursors (such as 2d-Ac) exerted signiﬁcant activity against S. aureus and sufﬁcient

selectivity over mammalian cells (Table 3). Compound 2b (R²= 2-OH) and its acetylated precursor

2b-Ac (R²= 2-OAc) had the broadest spectrum of activity, covering Mtb, non-tubercular mycobacterial

strains, S. aureus and all the tested fungal strains. To explain the signiﬁcant and broad-spectrum

antimicrobial properties of 2b and 2b-Ac, one should consider that structurally they are salicylanilides,

which are a class of compounds with well-known and repeatedly reported antimicrobial activity [39,40].

In silico calculations were performed to predict the hydrogen-bonding pattern,

molecular electrostatic potential (MEP), and HOMO and LUMO orbitals for 2a (as a prototype of the

Molecules 2018, 23, 2390

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retro-amide) and compared to its amide counterpart. The observed differences can explain different

levels of biological activity.

It can be concluded that the inversion of the amide linker (-CONH-) in N-phenylpyrazine-2-

carboxamides to the retro-amide linker (-NHCO-) usually leads to a loss or decrease of in vitro

antimycobacterial activity, but the resulting active retro-amides are more selective and have tighter

SAR relationships regarding the benzene ring substitution. Some of the presented compounds with

feasible activity and selectivity (2h, 2i for Mtb; 2b, 2d-Ac for S. aureus) may become useful starting

points for further development of antimicrobial compounds.

Supplementary Materials: Supplementary Material associated with this article can be found, in the online

version, at doi: insert doi. Contents: Full procedures of the biological assays. Full results of biological assays.

Analytical data or prepared compounds.

Author Contributions: J.Z. set the topic, designed the study, interpreted the results and wrote the paper, A.M.

and O.V. synthesized the compounds, O.J. performed testing against M. smegmatis, P.P. performed testing against

M. tuberculosis, kansasii and avium, J.J. measured cytotoxicity, K.K. designed and interpreted the testing against

bacteria and fungi, M.D. set up the topic and designed the study.

Funding: This work was supported by the Czech Science Foundation project No. 17-27514Y. This work was

supported by the project EFSA-CDN (No. CZ.02.1.01/0.0/0.0/16_019/0000841) co-funded by ERDF.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Sample Availability: Samples of the compounds from series 1, 2, and 3 are available from the authors.

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