N-pyridinylbenzamides: an isosteric approach towards new antimycobacterial compounds

Article abstract of DOI:10.1111/cbdd.13804

A series of N-pyridinylbenzamides was designed and prepared to investigate the influence of isosterism and positional isomerism on antimycobacterial activity. Comparison to previously published isosteric N-pyrazinylbenzamides was made as an attempt to draw structure–activity relationships in such type of compounds. In total, we prepared 44 different compounds, out of which fourteen had minimum inhibitory concentration (MIC) values against Mycobacterium tuberculosis H37Ra below 31.25?μg/ml, most promising being N-(5-chloropyridin-2-yl)-3-(trifluoromethyl)benzamide (23) and N-(6-chloropyridin-2-yl)-3-(trifluoromethyl)benzamide (24) with MIC?=?7.81?μg/ml (26?μm). Five compounds showed broad-spectrum antimycobacterial activity against M. tuberculosis H37Ra, M. smegmatis and M. aurum. N-(pyridin-2-yl)benzamides were generally more active than N-(pyridin-3-yl)benzamides, indicating that N-1 in the parental structure of N-pyrazinylbenzamides might be more important for antimycobacterial activity than N-4. Marginal antibacterial and antifungal activity was observed for title compounds. The hepatotoxicity of title compounds was assessed in vitro on hepatocellular carcinoma cell line HepG2, and they may be considered non-toxic (22 compounds with IC₅₀ over 200?μm).

Full text of DOI:10.1111/cbdd.13804

PROFESSOR MARTIN DOLEZAL (Orcid ID : 0000-0003-1558-6625)
Article type : Research Article
N-pyridinylbenzamides
–
an isosteric approach towards new antimycobacterial
compounds
Daria Nawrot, Eliška Suchánková, Ondřej Janďourek, Klára Konečná, Pavel Bárta, Martin
Doležal, and Jan Zitko*
Faculty of Pharmacy in Hradec Králové, Charles University, Heyrovského 1203, 500 05 Hradec
Králové,
Czech
Republic;
nawrotd@faf.cuni.cz
(DN);
vaskovae@faf.cuni.cz
(ES);
JANDO6AA@faf.cuni.cz (OJ); konecna@faf.cuni.cz (KK); bartp7aa@faf.cuni.cz (PB);
dolezalm@faf.cuni.cz (MD).
* Correspondence: jan.zitko@faf.cuni.cz (JZ); +420-495-067-409
ORCID-ID
Name
Surname
0000-0002-8858-1529
Daria
Nawrot
not registered
Eliška
Ondřej
Klára
Pavel
Martin
Jan
Suchánková
Janďourek
Konečná
Bárta
0000-0003-4633-2062
0000-0001-5670-7767
0000-0001-5445-5658
0000-0003-1558-6625
0000-0003-0104-9925
Doležal
Zitko
Abstract
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/CBDD.13804
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A series of N-pyridinylbenzamides was designed and prepared to investigate the influence of
isosterism and positional isomerism on antimycobacterial activity. Comparison to previously
published isosteric N-pyrazinylbenzamides was made as an attempt to draw structure activity
relationships in such type of compounds. In total, we prepared 44 different compounds, out of
which fourteen had minimum inhibitory concentration (MIC) values against Mycobacterium
tuberculosis H37Ra below 31.25 µg/mL, most promising being N-(5-chloropyridin-2-yl)- (23) and
N-(6-chloropyridin-2-yl)-3-(trifluoromethyl)benzamide (24) with MIC = 7.81 µg/mL (26 µM). Five
compounds showed broad spectrum antimycobacterial activity against M. tuberculosis H37Ra, M.
smegmatis, and M. aurum. N-(pyridin-2-yl)benzamides were generally more active than N-
(pyridin-3-yl)benzamides, indicating that N-1 in the parental structure of N-pyrazinylbenzamides
might be more important for antimycobacterial activity than N-4. Marginal antibacterial and
antifungal activity was observed for title compounds. The hepatotoxicity of title compounds was
assessed in vitro on hepatocellular carcinoma cell line HepG2, and they may be considered non-
toxic (22 compounds with IC50 over 200 µM).
Keywords: antibacterial; antimycobacterial; drug design; isosterism, N-pyridinylbenzamide
1. Introduction
Tuberculosis (TB) caused by the bacillus Mycobacterium tuberculosis (Mtb) is the leading cause
of death from a single infectious agent worldwide(World_Health_Organisation, 2020). Several
attempts have been made to eradicate this infection, yet the raising issue of antimycobacterial
resistance is proposing a real challenge(World_Health_Organisation, 2020). Since the
introduction of modern therapy, the mortality rate due to drug-sensitive TB has decreased
drastically. First line anti-TB drugs are isoniazid (INH), ethambutol (ETB), pyrazinamide (PZA)
and rifampicin (RIF). The basic treatment regimen for drug-sensitive TB is composed of the
mentioned four agents for two months, then only INH and RIF for additional four to six months.
This lengthy treatment has in return reduced patient adherence and further contributed to
treatment failure. Annually, the number of TB incident cases decreases at a rate of 2% globally.
However, this rate is not fast enough to achieve the goal of the End TB Strategy implemented by
WHO, that is to reduce the number of TB-induced deaths by 95% till 2035 (in comparison to year
2015)(World_Health_Organisation, 2019) . Here we report the design and synthesis of a series of
N-pyridinylbenzamides as potential novel antituberculars.
The design of title compounds was based on structurally related N-pyrazinylbenzamides
(retro-amides) published by Zitko et al(Zitko, Mindlová, 2018). In the current series, we kept the
retro-amide moiety while replacing the pyrazine core with substituted pyridine (Figure 1). In our
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previous studies, retro-amides exerted lower in vitro hepatotoxicity than their corresponding
amides (inversed linker) with the same substitution pattern(Zitko, Mindlová, 2018). In this study,
we investigate the isosteric exchange of one nitrogen atom in the pyrazine heterocycle (N in
position 1 or 4) for carbon and its influence on antimycobacterial activity of resulting pyridinyl
isosteres.
In general, isosterism (in our case exchanging nitrogen to carbon) is a common structural
modification approach for lead optimization in medicinal chemistry. Several examples are
reported in the literature, including guanidine to 2-aminopyrimidine isosteres and their influence
on antibacterial activity(Russell, Stevens, 2019), pyrazine isosteres binding to DNA minor
groove(Miletin, Kopecký, 2015) and hydroxypyridinethiones scaffolds as promiscuous, non-
selective metalloenzyme inhibitors(Adamek, Credille, 2018).
Nitrogen atoms in aromatic heterocycles can act as hydrogen bond acceptors (HBA), contributing
significantly to the affinity of small molecular weight molecules to their macromolecular targets
(receptors)(Juhás and Zitko, 2020). Specifically for pyrazine, there are several examples of
crystallographic complexes (such as pyrazine based inhibitor in human prolyl-tRNA synthetase,
PDB ID: 5VAD)(Adachi, Okada, 2017), where both nitrogen atoms (N-1 and N-4) of the pyrazine-
based ligand form hydrogen bonds to the receptor. On contrary, in pyrazine derivatives with
antimycobacterial effect, we often observe that N-1 nitrogen atom is more important for
ligand-protein interactions than N-4. The most significant example is pyrazinoic acid (POA, the
active metabolite of PZA) or 6-chloropyrazinoic acid (6-Cl-POA) in complex with mycobacterial
aspartate decarboxylase (PanD) – PDB IDs: 6OYY and 6P02, respectively. These POA ligands
are stabilised by three hydrogen bonds – two to the oxygen atoms of the carboxylate moiety and
one to the N-1 of the pyrazine nucleus, whereas the N-4 is not involved in any significant
interaction(Sun, Li, 2020). For this setup it can be concluded that N-1 (that is the nitrogen atom
adjacent to the hydrogen-bond-forming functional group) is more important for activity than N-4.
Indeed, it has been observed that POA and 6-Cl-POA are competitive inhibitors of PanD, but
nicotinic acid, which lacks the ‘N-1’ atom, is inactive(Sun, Li, 2020) (affinity of nicotinic acid to
PanD is about 5 times lower compared to POA)(Gopal, Nartey, 2017). Similarly, POA inhibits the
process of trans-translation by binding to mycobacterial ribosomal protein S1 (RpsA), but nicotinic
acid is devoid of such activity under the same conditions (Shi, Zhang, 2011). As a part of our
study, we will probe the importance of N-1/N-4 for the antimycobacterial activity (in our case, in
the compounds with retro-amide linker). Firstly, we prepared derivatives of the 2-aminopyridine
and 3-aminopyridine series unsubstituted on the pyrazine core (R1 = H). Based on the obtained
activity results, the 2-aminopyridine series was further extended by derivatives chlorinated on the
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pyridine (R1 = 5-Cl, 6-Cl). The position of the chloro substituent was corresponding to the
template series of N-pyrazinylbenzamides(Zitko, Mindlová, 2018). The influence of positional
isosterism (2-amino vs 3-amino; 5-Cl vs 6-Cl) was also investigated.
The designed N-pyridinylbenzamides have been studied for a broad spectrum of biological
activities, for example as inhibitors of human aldosterone synthase(Zimmer, Hafner, 2011),
negative allosteric modulators of glutamate receptor mGlu5(Bates, Rodriguez, 2014), inhibitors of
human nicotinamide phosphoribosyltransferase(Dragovich, Zhao, 2014), modulators of human
vanilloid receptor 1(Jetter, McNally, 2008), cardiovascular agents(Von der Saal, Mertens, 1989),
antiulcer agents(Irikura and Kasuga, 1971), anticonvulsants(Szirtes, Palosi, 1973), and
neuroprotective agents(Luan, Cordeiro, 2013). However, to our best knowledge, they were not
evaluated for antimicrobial activity (except for compounds 1 and 19 as mentioned in the
Conclusion).
1
R2
H
N
N
R1 = H, 5-Cl, 6-Cl
2
3
R1
O
1
R2
2-aminopyridine derivatives
1 33
compounds –
H
4
N
N
2
3
R1
isosteric exchange
O
N
R2
4
4
H
N
N-pyrazinylbenzamides
Zitko et al. 2018
Best Mtb H37Rv
3
R1
R1 = H
3-aminopyridine derivatives
34 44
O
2
N
1
MIC = 3.13–6.25 µM/mL (12–25 µM)
compounds
–
Figure 1. Design of title compounds
2. Results
2.1. Synthesis
Presented series of compounds were prepared by one step acylation of chosen aminopyridine
derivative (2-aminopyridine, 3-aminopyridine, 5-chloropyridin-2-amine or 6-chloropyridin-2-amine)
with substituted benzoyl chlorides in the presence of dry pyridine as a base. Pyridine was
employed to prevent the formation of diacetylated products, which are the major products when a
stronger base (such as triethylamine) is used(Theodorou, Gogou, 2014). Since the reaction is
exothermic, reaction mixture was cooled down in the fridge before the benzoyl chloride was
added. Upon used conditions, we did not observe the formation of the diacylated product (neither
on the TLC nor during the flash-chromatography). Two compounds (34, 43) were isolated as
hydrochloride salts (as confirmed by elemental analysis). In such cases, the hydrochlorides were
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tested for biological activity and subsequently converted into free bases following the procedure
described in section 4.2.2.
Pure products were obtained after flash column chromatography on silica, using hexane and ethyl
acetate as mobile phase. Final compounds were isolated as white to yellow solids and
1
characterized by H NMR and 13C NMR spectra, elemental analysis, IR spectra and melting
1
points. Obtained data were consistent with proposed structures. In the H NMR spectra amidic
hydrogen signal appeared as a singlet at 7.96–10.91 ppm (in CDCl3-d5) or at 10.35–11.58 ppm
(in DMSO-d6), in the 13C NMR spectra the amidic carbon signal appeared at 163.52–169.18 ppm
(in CDCl3-d5) or 165.45–165.90 ppm (in DMSO-d6). Final compounds were isolated as solids in
moderate yields, usually ranging from 21 to 96% (after all purification steps).
2.2.
Biological activity
2.2.1. Antimycobacterial study
Presented compounds were tested for in vitro cell growth inhibitory activity against three different
mycobacterial strains: M. tuberculosis H37Ra, M. aurum, M. smegmatis. The two latter strains
belong to fast growing strains that do not cause tuberculosis infection in humans. However, they
are much safer to handle, and their phenotype and genotype are close to Mtb which allows us to
use them as Mtb surrogate model(Phelan, Maitra, 2015). Antimycobacterial activity expressed as
minimum inhibitory concentration (MIC) is presented in Table 1. Compounds 34 and 43 were
tested also as hydrochlorides. In both cases, the resulting MIC values were equal to free bases
with tolerance of one step on the dilution scale.
Table 1. Prepared compounds, antimycobacterial activity expressed as Minimum Inhibitory
Concentration (MIC), HepG2 cytotoxicity expressed as IC50, and calculated partition coefficient
logP.
Mtb
H37Ra
[µg/mL]
250
M.
smegmatis
[µg/mL]
250
M.
aurum
[µg/mL]
250
HepG2
IC50
No
.
Mtb H37Ra
[µM]a
Structure
R1
R2
H
log P b
[µM]
1
2
3
4
5
6
7
8
H
1261.2
≥2148.9
537.2
2.22
2.78
2.78
2.78
3.33
3.33
2.78
3.33
>1000**
>250*
504.1
>50*
5-Cl
6-Cl
H
≥500
125
250
≥250
≥500
≥500
≥500
250
125
R2
H
N
≥500
≥500
62.5
≥2148.9
≥1871.9
233.9
≥500
N
5-Cl
6-Cl
H
2-Cl
3-Cl
≥500
>50*
R1
O
62.5
362.6
118.1
161.6
62.5
268.6
≥500
≥500
≥250
5-Cl
31.25
116.9
≥250
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9
6-Cl
H
15.625
62.5
≥500
≥500
31.25
31.25
≥500
62.5
62.5
31.25
62.5
62.5
15.625
31.25
7.81
7.81
125
58.5
268.6
≥1871.9
1871.9
136.9
118.9
≥1903.4
273.8
237.9
118.9
273.8
237.9
59.4
≥500
≥500
250
≥500
62.5
≥500
≥500
250
3.33
2.78
3.33
3.33
2.09
2.65
2.65
2.09
2.65
2.65
2.09
2.65
2.65
3.14
3.70
3.70
3.92
4.48
4.48
2.71
3.26
3.26
3.12
3.68
3.68
2.22
2.78
2.78
2.78
2.09
2.09
2.09
3.14
3.92
2.71
3.12
119.5
>250*
199.1*
>50*
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
5-Cl
6-Cl
H
4-Cl
2-OMe
3-OMe
4-OMe
3-CF3
4-tBu
4-Me
≥500
7.81
7.81
31.25
62.5
62.5
62.5
125
>250*
n.a.
5-Cl
6-Cl
H
250
≥500
250
n.a.
>1000**
>100*
>250*
>1000**
>250*
>250*
223
5-Cl
6-Cl
H
250
62.5
250
5-Cl
6-Cl
H
125
250
≥500
500
≥500
500
117.3
25.9
5-Cl
6-Cl
H
15.625
15.625
62.5
62.5
3.91
62,5
≥500
31.25
62.5
31.25
15.625
125
31.25
31.25
250
149.9
137.7
66.2
25.9
491.5
432.9
27.0
5-Cl
6-Cl
H
125
250
>250*
130.7
620,7
>50*
7.81
125
≥500
125
588.9
≥2026.8
63.3
5-Cl
6-Cl
H
≥500
15.625
62.5
62.5
7.81
250
≥500
≥500
31.25
62.5
250
375.7
836.1
95.2
276.2
239.7
29.9
5-Cl
6-Cl
H
4-Et
116.8
>1000**
>500*
>1000**
>500*
>1000**
>1000**
>1000**
406.5
99.8
H
1261.2
≥2148.9
268.6
1074.5
1095.3
547.6
1095.3
117.4
245.7
588.9
138.1
250
H
2-Cl
≥500
62.5
250
250
≥500
62.5
≥500
250
H
3-Cl
62.5
≥500
500
H
4-Cl
N
O
R1
H
2-OMe
3-OMe
4-OMe
3-CF3
4-tBu
4-Me
4-Et
250
H
125
62.5
250
500
N
H
R2
H
250
≥500
250
H
31.25
62.5
125
125
H
62.5
250
62.5
250
H
>1000**
452.6
H
31.25
125
125
INH
RFM
CPX
7.81–
15.625
3.91–
7.81
0.39–
0.78
0.0156
-
-
-
-
-
-
-
0.125–0.25
0.003125
0.91–1.82
0.0038
-0.64
2.70
n.a.
n.a.
1.56–6.25
0.125–
0.0625
-
-
-
0.125–0.25
0.38–0.75
1.32
n.a.
0.0312
a Calculated from MIC [µg/mL]. b Calculated in ChemDraw v18.1. INH – isoniazid, RFM – rifampicin, CPX –
ciprofloxacin. n.a. – not available.
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*determination of IC50 was not possible due to irrelevant values of absorbance at higher concentrations of
compounds caused by their precipitation in cell culture medium; the presented values correspond to the
highest tested concentration. Therefore, selectivity index estimation does not reflect true selectivity of those
compounds. ** IC50 values were over the limit of the tested concentrations.
2.2.2. Antibacterial and antifungal study
Compounds were screened for activity against four Gram-positive bacteria [Staphylococcus
epidermidis (SE), Enterococcus faecalis (EF), Staphylococcus aureus (SA), methicillin resistant
Staphylococcus aureus (MRSA)] and four Gram-negative bacteria [Pseudomonas aeruginosa
(PA), Escherichia coli (EC), Klebsiella pneumoniae (KP) and Serratia marcescens (SM)].
Antibacterial activity is expressed as MIC in µM.
Compounds presented in this paper were tested against eight different fungal pathogens of
clinical importance [Absidia corymbifera (AC), Aspergillus fumigatus (AF), Aspergillus flavus
(AFla), Candida albicans (CA), Candida krusei (CK), Candida parapsilosis (CP), Candida
tropicalis (CT) and Trichophyton interdigitale (TI)]. Antifungal activity is expressed as MIC in µM.
Due to marginal activity, the obtained results are not further discussed and are presented only in
Supplementary Material (Table S1 and S2).
2.2.3 Cytotoxicity
In vitro model of human hepatocellular carcinoma cell line (HepG2) was used to assess
cytotoxicity of presented compounds. This model was chosen since anti-TB drugs are known to
carry the risk of hepatotoxicity (Yew and Leung, 2006). Results are presented as inhibitory
concentration which reduces viability of the cell population to 50% from the maximum viability
(IC50) and are calculated by non-linear regression from a semilogarithmic plot of incubation
concentration versus percentage of the absorbance relative to untreated controls. Results are
presented in Table 1.
3. Discussion of results
Structure-activity relationships are discussed based on antimycobacterial activity against Mtb
H37Ra (if not stated otherwise).
3.1. Influence of isosterism and positional isomerism on the activity
Comparing derivatives of N-(pyridin-2-yl)benzamide to N-(pyridin-3-yl)benzamide with
non-substituted (R1 = H) pyridine ring, we found that compounds from the pyridin-2-yl series
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generally exert better activity than pyridin-3-yl derivatives. Out of 11 matching pairs bearing the
same substituent R2, four of 2-amino derivatives (R2 = 4-Cl, 2/3/4-OMe) had better activities than
corresponding 3-amino derivatives. Exceptions were 3-trifluoromethyl (22, 41), 4-methyl (28, 43),
3-Cl (7, 36) and non-substituted derivatives (1, 34), where both positional isomers exerted the
same activity (MIC 31.25 µg/mL for R2 = 3-CF3, 125 µg/mL for R2 = 4-Me, MIC 62.5 µg/mL for R2
= 3-Cl and 250 µg/mL for R2 = H). Only 4-Et (44) and 4-tBu (42) derivatives of N-(pyridin-3-
yl)benzamide showed better activity than corresponding pyridin-2-yl isomers and the effect was
only seen when pyridinyl ring was not chlorinated (R1 = H). 2-Cl derivatives were inactive
(MIC ≥ 500 µg/mL) in both positional isomers series (4, 35).
In order to further evaluate the importance of individual nitrogen atoms (depicted as N-1 and N-4
in the template N-pyrazinylbenzamides in Figure 1), selected N-(pyrazin-2-yl)benzamides
previously published by our group were re-evaluated for their for in vitro antimycobacterial activity
against Mtb H37Ra and compared to structurally related title compounds of this study (C
isosteres with identical R2) - refer to the Table 2. It must be noted that comparison shown in Table
2 is valid as compounds were evaluated following the same methodology, at the same
department and by the same person (O.J.).
Based on obtained results it was concluded that title 2-aminopyridine based compounds possess
better activity than previously published 2-aminopyrazine derivatives (out of 10 comparable pairs
with identical R2, 8 had better activity and 2 had the same activity).
Table 2. Comparison between antimycobacterial activity against Mtb H37Ra of title compounds
and their pyrazine isosters published in(Zitko, Mindlová, 2018).
R2
R2
H
H
N
N
N
N
N
R1
R1
O
O
Title compounds:
Previously published
2-aminopyridine derivatives
MICMtb H37Ra [µg/mL]
2-aminopyrazine derivatives
R1
H
R2
H
Rel
>
MICMtb H37Ra [µg/mL]
>500 (1a)
250 (1)
62.5 (6)
62.5 (7)
31.25 (8)
6-Cl
H
2-Cl
3-Cl
3-Cl
>
>500 (3j)
=
62.5 (1k)
5-Cl
>
>500 (2k)
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H
H
4-Cl
4-OMe
3-CF3
3-CF3
4-Et
62.5 (10)
62.5 (19)
7.81 (23)
7.81 (24)
62.5 (31)
62.5 (33)
=
>
>
>
>
>
62.5 (1l)
500 (1g)
>500 (2n)
31.25 (3n)
125 (1i)
5-Cl
6-Cl
H
6-Cl
4-Et
>500 (3i)
Rel – relative comparison of antimycobacterial activity. Codes in parentheses are the
corresponding codes in this paper or in the original publication.
3.2. Influence of substituents R1, R2 on the activity
As for the substitution pattern, it can be concluded that substituting both rings is favourable for
antimycobacterial activity (compounds with R1 and/or R2 = H had inferior activity). Substituting the
pyridine ring with chlorine (R1 = Cl) in position 6 contributed to better activities when compared to
chlorine at position 5 (out of 11 matching pairs with identical R2 substituent, eight 6-chloro
derivatives had better activity, in 2 pairs activity was equal for both isomers, and only in 2 pairs
the 5-chloro derivative was having better activity).
Regarding substitution on the benzene ring, higher activities were found for derivatives bearing
trifluoromethyl or short alkyl as R2 [23 (R2 = 3-CF3), 24 (R2 = 3-CF3), 27 (R2 = 4-tBu), 30 (R2 = 4-
Me), 33 (R2 = 4-Et)]. Interestingly, the latter finding is consistent with our previous findings in the
previously published series of N-pyrazinylbenzamide isosters, where the most active compounds
had trifluoromethyl or short alkyl chain as substituent on the benzene ring (Zitko, Mindlová, 2018).
Among all, compounds 23 and 24 (R1 = 5-Cl or 6-Cl and R2 = 3-CF3), 31 and 32 (R1 = H or 5-Cl
and R2 = 4-Et) and 36 (R1 = H and R2 = 3-Cl) had the broadest spectrum of antimycobacterial
activity, inhibiting the growth of all three tested mycobacterial strains.
3.3. Lipophilicity vs activity
Mycobacterial cell-wall is rich in mycolic acids which efficiently prevents penetration of drugs and
thus drugs with higher lipophilicity may exert better action against mycobacterium(Piccaro, Poce,
2015). In some cases, logP might be an important factor for the antimycobacterial activity of title
compounds. Best antimycobacterial activity was exerted by the most lipophilic compounds (23,
24, 27, 33) with calculated logP 3.68–4.48. However, logP is not the sole determinant of activity,
as other derivatives within this range of logP values had only moderate activity (such as 6 and
32). Among 6-chloro derivatives (R1 = 6-Cl), there is a weak positive correlation between activity
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and logP (Figure 2), while the plot of antimycobacterial activity on logP of the other derivatives (R1
= H, 5-Cl) does not show any correlation (Figure S1 in Supplementary Materials).
Figure 2. Plot of antimycobacterial activity of 6-chloropyridin-2-yl derivatives (Mtb H37Ra) on
calculated lipophilicity logP
3.4. Cytotoxicity
Title compounds were tested for their in vitro cytotoxicity on hepatocellular carcinoma cell line
HepG2, for results refer to Table 1.
Out of 42 tested compounds, 22 had IC50 higher than 200 µM (10, 11, 13, 16, 18, 19, 21, 22, 26,
28, 30, 31, 34–41, 43, 44) and can be considered relatively non-toxic. All compounds had IC50
values above 50 µM. Yet it must be noted that some compounds precipitated in the testing
medium (refer to Table 1 - compounds noted with *) and measurements at higher concentrations
were not possible, therefore the corresponding IC50 values do not accurately reflect the cytotoxic
behaviour of the compounds. Since not all compounds in our previous publications were
assessed for their cytotoxicity, we cannot conclude whether title compounds are of less or higher
cytotoxicity.
It can be concluded that the N-(pyridin-3-yl) derivatives are of lower toxicity than their
N-(pyridin-2-yl) isomers (out of 11 compared pairs, six times the N-(pyridine-3-yl) structure was of
lower toxicity, two times of higher, and in three pairs the toxicity was the same – above the tested
range). Between R1 substituted and non-substituted compounds (R1 = 5-Cl, 6-Cl vs R1 = H) the
non-substituted (R1 = H) were of lower cytotoxicity (seven out of 10 compared pairs had lower
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toxicity). No clear statement can be made on the comparison of the cytotoxicity of the positional
isomers (R1 = 5-Cl vs 6-Cl); out of 10 compared pairs, 5-chloro isomers had lower toxicity four
times, 6-chloro isomers five times and in one pair their cytotoxicity was equal.
Obtained IC50 values were used to calculate the selectivity index based on antimycobacterial
activity against Mtb H37Ra (SI = MIC/IC50). Three compounds had SI close to or higher than 5
(23, 24, 25 – having 3-trifluoromethyl or 4-tBu substituent). A plot of antimycobacterial activity on
cytotoxicity is presented in the Supplementary material as Figure S2.
4. Experimental
4.1.
General Information
All reagents and solvents (unless stated otherwise) were purchased from Sigma-Aldrich
(Schnelldorf, Germany) and used without any further purification. Reaction progress and reaction
products purity was monitored using Silica 60 F254 TLC plates (Merck, Darmstadt, Germany).
Flash chromatography of the final compounds was performed on a puriFlash XS420+ (Interchim,
Montluçon, France) with usage of original columns (non-spherical silica, 30 µm) provided by the
same company. Mobile phase was ethyl acetate in hexane, elution gradient 0–100% ethyl acetate
(EtOAc) in hexane (Hex), detection was performed by UV-VIS detector at wavelength of 254 nm
and 280 nm.
The NMR spectra were recorded on a Varian VNMR S500 (Varian, Palo Alto, CA, USA) at 500
1
13
MHz for H and 126 MHz for C. The spectra of compounds 32 and 36 were measured on Jeol
JNM-ECZ600R at 600 MHz for 1H and 151 MHz for 13C. The spectra were recorded in DMSO-
d6 or CDCl3- d5 at ambient temperature. The chemical shifts are reported as δ values in ppm are
1
indirectly referenced to tetramethylsilane (TMS) via the solvent signal (2.49 for H and 39.7 for
13C in DMSO-d6; 7.27 for 1H and 77.0 for 13C in CDCl3-d5). IR spectra were recorded on a
NICOLET 6700 FT-IR spectrophotometer (Nicolet, Madison, WI, USA) using ATR-Ge method.
Elemental analysis was done on a Vario MICRO cube Element Analyzer (Elementar
Analysensysteme, Hanau, Germany) with values given as a percentage. Yields are given in
percent and refer to the amount of pure product after all purification steps. LogP values were
calculated using ChemDraw v18.1. (PerkinElmer Informatics, Waltham, Massachusetts, U.S.).
4.2.
Chemistry
4.2.1. General procedure
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Selected pyridine derivative (2 mmol) was placed in a 50 mL round-bottom flask, 10 mL of
anhydrous dichloromethane (DCM) and anhydrous pyridine (3 mmol, 237.3 mg) were added. The
flask was closed with a stopper and cooled down in a fridge for 20 minutes. Selected benzoyl
chloride (2.4 mmol) was diluted in 3 mL of DCM and added dropwise upon stirring to the mixture,
the flask was closed with a stopper and stirred at room temperature (RT). The progress of
reaction was monitored by TLC (silica gel plates, Hex:EtOAc, 1:1). After 2 h, the reaction was
complete (no further changes on TLC plate were visible after that time), it was stopped and
purified. The reaction mixture was adsorbed on silica by evaporation of the solvent under reduced
pressure. Pre-column was filled with the mixture on silica and submitted to flash chromatography
(silica column 30 g, gradient elution 0–100% EtOAc in Hex, flow rate 20 mL/min, detection
wavelength 254 nm and 280nm). Fractions containing pure product were collected and solvents
were evaporated under reduced pressure to yield oily liquids that after some time crystalized to
give solids.
4.2.2. Conversion of hydrochloride salts into free bases
Compounds which were obtained in the form of hydrochloride salts were dissolved in 30 mL of
water and ethanol mixture (1:1 v/v). Potassium carbonate decahydrate (s) was added in small
portions to the stirred mixture until basic pH was achieved (pH around 8–9 as indicated by
universal indicator paper strips). The mixture was then stirred for 15 minutes at RT. The
undissolved potassium carbonate was filtered off and the filtrate was evaporated under reduced
pressure to obtain solid, that was dissolved in EtOAc and washed with distilled water (2–3 times,
30 mL). Combined organic layers were dried over anhydrous sodium sulphate, filtered, and the
filtrate was evaporated under reduced pressure to obtain solid free base compounds.
4.3.
Analytical Data
N-(pyridin-2-yl)benzamide (1) mp. 75.6–78.4 °C [lit. 76–78 °C (Katritzky, El-Gendy Bel, 2008)],
1
Yield: 26%; white solid; H NMR (500 MHz, CDCl3-d5) δ 8.90 (s, 1H, CONH), 8.43–8.40 (m, 1H,
ArH), 8.25–8.22 (m, 1H, ArH), 7.97–7.92 (m, 2H, ArH), 7.80–7.73 (m, 1H, ArH), 7.60–7.55 (m,
1H, ArH), 7.53–7.47 (m, 2H, ArH), 7.09–7.04 (m, 1H, ArH); 13C NMR (126 MHz, CDCl3-d5) δ
165.81, 151.61, 147.82, 138.48, 134.30, 132.20, 128.79, 127.23, 119.89, 114.23; IR (ATR-Ge,
cm-1): 3170 (ν, NH); 1670 (ν, C=O, amide); 1579; 1527; 1469; Calculated for C12H10N2O (198.23):
72.71% C; 5.09% H; 14.13% N, found: 72.78% C; 4.90% H; 13.59% N.
N-(5-chloropyridin-2-yl)benzamide (2) mp. 120.5–123.7 °C; Yield: 42%; white solid; 1H NMR (500
MHz, CDCl3-d5) δ 8.78 (s, 1H, CONH), 8.39 (d, J = 8.9 Hz, 1H, ArH), 8.17 (d, J = 2.5 Hz, 1H,
ArH), 7.94–7.88 (m, 2H, ArH), 7.72 (dd, J = 8.9, 2.5 Hz, 1H, ArH), 7.61–7.56 (m, 1H, ArH), 7.53–
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7.47 (m, 2H, ArH); 13C NMR (126 MHz, CDCl3-d5) δ 165.66, 149.89, 146.52, 138.05, 133.96,
132.40, 128.86, 127.20, 126.85, 114.81; IR (ATR-Ge, cm-1): 3231 (ν, NH); 1675 (ν, C=O, amide);
1577; 1522; 1489; Calculated for C12H9ClN2O (232.67): 61.95% C; 3.90% H; 12.04% N; found:
61.77% C; 3.66% H; 12.01% N.
1
N-(6-chloropyridin-2-yl)benzamide (3) mp. 46.4–48.2 °C, Yield: 35%; white solid; H NMR (500
MHz, CDCl3-d5) δ 8.64 (s, 1H, CONH), 8.32 (d, J = 8.2 Hz, 1H, ArH), 7.92–7.88 (m, 2H), 7.70 (t, J
= 8.0 Hz, 1H, ArH), 7.60–7.55 (m, 1H, ArH), 7.52–7.45 (m, 2H, ArH), 7.09 (d, J = 7.8 Hz, 1H,
13
ArH); C NMR (126 MHz, CDCl3-d5) δ 165.55, 151.32, 148.93, 140.98, 133.64, 132.46, 128.83,
127.16, 119.80, 112.13; IR (ATR-Ge, cm-1): 3300 (ν, NH); 1664 (ν, C=O, amide); 1570; 1528;
1492; Calculated for C12H9ClN2O (232.67) 61.95% C; 3.90% H; 12.04% N; found: 62.18% C;
3.79% H; 11.85% N.
2-chloro-N-(pyridin-2-yl)benzamide (4) mp. 133.2–139.1 °C [lit. 160–161 °C (Timari, Hajos,
1990)]; white solid; Yield: 29%; 1H NMR (500 MHz, CDCl3-d5) δ 9.66 (s, 1H, CONH), 8.40 (d, J =
8.4 Hz, 1H, ArH), 7.89–7.84 (m, 1H, ArH), 7.78–7.71 (m, 1H, ArH), 7.71–7.66 (m, 1H, ArH), 7.45–
7.39 (m, 2H, ArH), 7.38–7.33 (m, 1H, ArH), 7.01–6.95 (m, 1H, ArH); 13C NMR (126 MHz, CDCl3-
d5) δ 165.25, 151.41, 147.49, 138.56, 135.31, 131.65, 131.02, 130.38, 129.75, 127.12, 120.02,
114.53; IR (ATR-Ge, cm-1): 3118 (ν, NH); 1679 (ν, C=O, amide); 1592; 1578; 1536; Calculated for
C12H9ClN2O (232.67): 61.95% C; 3.90% H; 12.04% N; found: 61.63% C; 3.53% H; 11.58% N.
1
2-chloro-N-(5-chloropyridin-2-yl)benzamide (5) mp. 152.5–154.1 °C; Yield: 37%; white solid; H
NMR (500 MHz, CDCl3-d5) δ 9.57 (s, 1H, CONH), 8.39 (d, J = 8.8 Hz, 1H, ArH), 7.74–7.65 (m,
3H, ArH), 7.49–7.35 (m, 3H, ArH); 13C NMR (126 MHz, CDCl3-d5) δ 165.12, 149.71, 146.20,
138.22, 134.97, 131.96, 130.98, 130.41, 129.77, 127.21, 127.02, 115.12; IR (ATR-Ge, cm-1):
3227 (ν, NH); 1679 (ν, C=O, amide); 1579; 1521; 1471; Calculated for C12H8Cl2N2O (267.11):
53.96% C; 3.02% H; 10.49% N; found: 53.99% C; 2.96% H; 10.32% N.
1
2-chloro-N-(6-chloropyridin-2-yl)benzamide (6) mp. 89.3–95.8 °C; Yield: 64%; white solid; H
NMR (500 MHz, CDCl3-d5) δ 8.64 (s, 1H, CONH), 8.31 (d, J = 8.1 Hz, 1H, ArH), 7.76–7.68 (m,
2H, ArH), 7.48–7.40 (m, 2H, ArH), 7.39–7.35 (m, 1H, ArH), 7.11 (d, J = 7.8 Hz, 1H, ArH); 13C
NMR (126 MHz, CDCl3-d5) δ 164.80, 150.85, 149.12, 141.04, 134.33, 132.12, 130.90, 130.55,
129.93, 127.22, 120.23, 112.29; IR (ATR-Ge, cm-1): 3173 (ν, NH); 1684 (ν, C=O, amide); 1592;
1573; 1525; Calculated for C12H8Cl2N2O (267.11): 53.96% C; 3.02% H; 10.49% N; found: 54.11%
C; 2.72% H; 10.41% N.
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3-chloro-N-(pyridin-2-yl)benzamide (7) mp. 103.0–104.0 °C; Yield 53%; white solid; 1H NMR (500
MHz, CDCl3-d5) δ 9.00 (s, 1H, CONH), 8.38 (d, J = 8.4 Hz, 1H, ArH), 8.27–8.21 (m, 1H, ArH),
7.95 (t, J = 1.9 Hz, 1H, ArH), 7.83–7.78 (m, 1H, ArH), 7.76 (d, J = 1.9 Hz, 1H, ArH), 7.59–7.51 (m,
13
1H, ArH), 7.43 (t, J = 7.9 Hz, 1H, ArH), 7.12–7.06 (m, 1H, ArH); C NMR (126 MHz, CDCl3-d5)
164.48, 151.36, 147.78, 138.63, 136.06, 135.07, 132.21, 130.06, 127.75, 125.24, 120.16, 114.38;
IR (ATR-Ge, cm-1): 3258 (ν, NH ), 1681 (ν, C=O, amide), 1580, 1534, 1472; Calculated for
C12H9ClN2O (232.04): 61.95% C; 3.9% H; 12.04% N; found 60.97% C; 3.46% H; 11.57% N.
1
3-chloro-N-(5-chloropyridin-2-yl)benzamide (8) mp. 112.1–114 °C; Yield 34%; white solid; H
NMR (500 MHz, CDCl3-d5) δ 8.71 (s, 1H, CONH), 8.36 (d, J = 8.9 Hz, 1H, ArH), 8.21 (d, J = 2.6
Hz, 1H, ArH), 7.91 (t, J = 1.9 Hz, 1H, ArH), 7.80–7.69 (m, 2H, ArH), 7.58–7.54 (m, 1H, ArH), 7.45
(t, J = 7.9 Hz, 1H, ArH); 13C NMR (126 MHz, CDCl3-d5) δ 164.25, 149.55, 146.58, 138.18, 135.67,
135.17, 132.45, 130.17, 127.66, 127.21, 125.14, 114.88; IR (ATR-Ge, cm-1): 3271 (ν, NH ), 1695
(ν, C=O, amide), 1573, 1543, 1514; Calculated for C12H8Cl2N2O (266.0): 53.96% C; 3.02% H;
10.49% N; found 54.17% C; 2.62% H; 9.26% N.
1
3-chloro-N-(6-chloropyridin-2-yl)benzamide (9) mp. 119.0–121.0 °C; Yield 40%; white solid; H
NMR (500 MHz, CDCl3-d5) δ 8.52 (s, 1H, CONH), 8.30 (d, J = 8.2 Hz, 1H, ArH), 7.91 (t, J = 1.9
Hz, 1H, ArH), 7.79–7.70 (m, 2H, ArH), 7.50–7.52 (m, 1H, ArH), 7.45 (t, J = 7.9 Hz, 1H, ArH), 7.13
(d, J = 7.7 Hz, 1H, ArH); 13C NMR (126 MHz, CDCl3-d5) δ 164.18, 151.00, 149.10, 141.11,
135.45, 135.24, 132.55, 130.19, 127.66, 125.09, 120.18, 112.17; IR (ATR-Ge, cm-1): 3252 (ν, NH
), 1697 (ν, C=O, amide), 1559, 1520, 1490; Calculated for C12H8Cl2N2O (266.0): 53.96% C;
3.02% H; 10.49% N; found 54.00% C; 2.65% H; 10.42% N.
4-chloro-N-(pyridin-2-yl)benzamide (10) mp. 130.5–132.5 °C [lit. 130–131 °C (Katritzky, El-Gendy
1
Bel, 2008)]; white solid; Yield 77%; white solid; H NMR (500 MHz, CDCl3-d5) δ 9.11 (s, 1H,
CONH), 8.40 (d, J = 8.4 Hz, 1H, ArH), 8.24–8.19 (m, 1H, ArH), 7.93–7.86 (m, 2H, ArH), 7.81–
7.74 (m, 1H, ArH), 7.50–7.43 (m, 2H, ArH), 7.11–7.05 (m, 1H, ArH); 13C NMR (126 MHz, CDCl3-
d5) δ 164.80, 151.43, 147.54, 138.72, 138.57, 132.60, 130.58, 128.90, 120.04, 114.44; IR (ATR-
Ge, cm-1): 3183 (ν, NH), 1673 (ν, C=O, amide), 1583, 1539, 1484; Calculated for C12H9ClN2O
(232.04): 61.95% C; 3.9% H; 12.04% N; found 61.52% C; 3.64% H; 11.53% N.
1
4-chloro-N-(5-chloropyridin-2-yl)benzamide (11) mp. 156.0–159.0 °C; Yield 96%; white solid; H
NMR (500 MHz, CDCl3-d5) δ 9.28 (s, 1H, CONH), 8.44 (d, J = 8.8 Hz, 1H, ArH), 8.26 (s, 1H, ArH),
7.94 (d, J = 8.1 Hz, 2H, ArH), 7.78 (d, J = 9.0 Hz, 1H, ArH), 7.53–7.45 (m, 2H, ArH); 13C NMR
(126 MHZ, CDCl3-d5) 168.83, 149.32, 146.35, 138.04, 133.78, 132.09, 130.73, 129.31, 127.02,
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114.63; IR (ATR-Ge, cm-1): 3244 (ν, NH), 1689 (ν, C=O, amide), 1606, 1593, 1583; Calculated for
C12H8Cl2N2O (266.0): 53.96% C; 3.02% H; 10.49% N; found 53.79% C; 2.64% H; 8.51% N.
1
4-chloro-N-(6-chloropyridin-2-yl)benzamide (12) mp. 139.0–142.0 °C; Yield 90%; white solid; H
NMR (500 MHz, CDCl3-d5) δ 8.58 (s, 1H, CONH), 8.31 (d, J = 8.1 Hz, 1H, ArH), 8.11–8.01 (m,
1H, ArH), 7.89–7.81 (m, 2H, ArH), 7.78–7.67 (m, 1H, ArH), 7.56–7.46 (m, 2H, ArH), 7.50–7.39
(m, 1H, ArH), 7.12 (dt, J = 7.8, 0.6 Hz, 1H, ArH); 13C NMR (126 MHz, CDCl3-d5) δ 168.95, 150.83,
148.89, 140.97, 133.80, 131.92, 130.72, 129.31, 119.98, 112.00; IR (ATR-Ge, cm-1): 3423 (ν,
NH), 1677 (ν, C=O, amide), 1595, 1571, 1526; Calculated for C12H8Cl2N2O (266.0): 53.96% C;
3.02% H; 10.49% N; found 54.17% C; 2.62% H; 9.26% N.
1
2-methoxy-N-(pyridin-2-yl)benzamide (13) mp. 155.1–157.5 °C; Yield 48%; white solid; H NMR
(500 MHz, DMSO-d6) δ 10.45 (s, 1H, CONH), 8.40–8.32 (m, 1H, ArH), 8.26 (d, J = 8.3 Hz, 1H,
ArH), 7.95–7.89 (m, 1H, ArH), 7.88–7.80 (m, 1H, ArH), 7.61–7.49 (m, 1H, ArH), 7.25 (d, J = 8.5
Hz, 1H, ArH), 7.18–7.09 (m, 2H, ArH), 3.99 (s, 3H, OCH3); 13C NMR (126 MHz, CDCl3-d5) 163.56,
157.50, 151.95, 147.90, 138.21, 133.61, 132.43, 121.44, 119.58, 114.74, 111.50, 56.16; IR (ATR-
Ge, cm-1): 3314 (ν, NH), 1669 (ν, C=O, amide), 1603, 1575, 1537; Calculated for C13H12N2O2 (MW
228.09): 68.41% C; 5.30% H; 12.27% N; found 63.81% C; 5.62% H; 10.62% N.
1
N-(5-chloropyridin-2-yl)-2-methoxybenzamide (14) mp. 116.5–118 °C; Yield 62%; white solid; H
NMR (500 MHz, CDCl3-d5) δ 10.40 (s, 1H, CONH), 8.46–8.41 (m, 1H, ArH), 8.31–8.23 (m, 2H,
ArH), 7.73–7.67 (m, 1H, ArH), 7.57–7.50 (m, 1H, ArH), 7.18–7.11 (m, 1H, ArH), 7.05 (d, J = 8.4
Hz, 1H, ArH), 4.09 (s, 3H, OCH3); 13C NMR (126 MHz, CDCl3-d5) δ 163.52, 157.53, 150.32,
146.56, 137.84, 133.88, 132.52, 126.47, 121.58, 121.02, 115.39, 111.57, 56.25; IR (ATR-Ge, cm-
1): 3344 (ν, NH), 1669 (ν, C=O, amide), 1601, 1572, 1528; Calculated for C13H11ClN2O2 (MW
262.05): 59.44% C; 4.22% H; 10.66% N; found 59.78% C; 4.11% H; 10.62% N.
N-(6-chloropyridin-2-yl)-2-methoxybenzamide (15) mp. 142.8–143.8 °C; Yield 48%; white solid;
1H NMR (500 MHz, CDCl3-d5) δ 10.31 (s, 1H, CONH), 8.38 (d, J = 8.2 Hz, 1H, ArH), 8.25 (dd, J =
7.8, 1.8 Hz, 1H, ArH), 7.69 (t, J = 7.9 Hz, 1H, ArH), 7.58–7.50 (m, 1H, ArH), 7.17–7.13 (m, 1H,
ArH), 7.13–7.01 (m, 2H, ArH), 4.09 (s, 3H, OCH3); 13C NMR (126 MHz, CDCl3-d5) δ 163.71,
157.55, 151.86, 148.98, 140.75, 133.96, 132.52, 121.53, 120.95, 119.55, 112.83, 111.55, 56.30;
IR (ATR-Ge, cm-1): 3339 (ν, NH), 1677 (ν, C=O, amide), 1602, 1568, 1538; Calculated for
C13H11ClN2O2 (MW 262.05): 59.44% C; 4.22% H; 10.66% N; found 59.49% C; 4.10% H; 10.46%
N.
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1
3-methoxy-N-(pyridin-2-yl)benzamide (16) mp. 66.3–66.9 °C; Yield 55%; white solid; H NMR
(500 MHz, CDCl3-d5) δ 9.12 (s, 1H, CONH), 8.45–8.40 (m, 1H, ArH), 8.28–8.23 (m, 1H, ArH),
7.81–7.74 (m, 1H, ArH), 7.54–7.47 (m, 2H, ArH), 7.43–7.34 (m, 1H, ArH), 7.17–7.05 (m, 2H,
ArH), 3.88 (s, 3H, OCH3); 13C NMR (126 MHz, CDCl3-d5) 165.75, 159.94, 151.62, 147.54 138.65,
135.66, 129.75, 129.33, 119.88, 119.22, 114.27, 112.36, 55.35; IR (ATR-Ge, cm-1): 3242 (ν, NH),
1683 (ν, C=O, amide), 1596, 1580, 1520; Calculated for C13H12N2O2 (MW 228.09): 68.41% C;
5.3% H; 12.27% N; found 68.14% C; 5.3% H; 11.11% N.
1
N-(5-chloropyridin-2-yl)-3-methoxybenzamide (17) mp. 96.3–97.1 °C; Yield 60%; white solid; H
NMR (500 MHz, CDCl3-d5) δ 8.74 (s, 1H, CONH), 8.38 (d, J = 8.9 Hz, 1H, ArH), 8.20 (d, J = 2.6
Hz, 1H, ArH), 7.75–7.69 (m, 1H, ArH), 7.48–7.37 (m, 3H, ArH), 7.15–7.09 (m, 1H, ArH), 3.88 (d, J
13
= 0.7 Hz, 3H, OCH3); C NMR (126 MHz, CDCl3-d5) δ 165.53, 160.04, 149.86, 146.57, 138.10,
135.39, 129.90, 126.91, 118.96, 118.70, 114.83, 112.50, 55.51; IR (ATR-Ge, cm-1): 3232 (ν, NH),
1672 (ν, C=O, amide), 1577, 1515, 1487; Calculated for C13H11ClN2O2 (MW 262.05): 59.44% C;
4.22% H; 10.66% N; found 59.76% C; 4.27% H; 10.36% N.
1
N-(6-chloropyridin-2-yl)-3-methoxybenzamide (18) mp. 66.3–66.9 °C; Yield 55%; white solid; H
NMR (500 MHz, CDCl3-d5) δ 8.57 (s, 1H, CONH), 8.32 (d, J = 8.2 Hz, 1H, ArH), 7.76–7.68 (m,
1H, ArH), 7.47–7.41 (m, 1H, ArH), 7.39 (t, J = 7.8 Hz, 1H, ArH), 7.14–7.07 (m, 1H, ArH), 3.87 (s,
3H, OCH3); 13C NMR (126 MHz, CDCl3-d5) δ 165.39, 160.02, 151.28, 148.97, 140.99, 135.10,
129.86, 119.84, 118.93, 118.88, 112.32, 112.11, 55.47; IR (ATR-Ge, cm-1): 3396 (ν, NH), 1642 (ν,
C=O, amide), 1591, 1571, 1542; Calculated for C13H11ClN2O2 (MW 262.05): 59.44% C; 4.22% H;
10.66% N; found 56.81% C; 4.43% H; 10.20% N.
4-methoxy-N-(pyridin-2-yl)benzamide (19) mp. 103.5–104 °C [lit. 106–107 °C (Begouin and
Queiroz, 2009)]; white solid; Yield 53%; pale yellow solid; 1H NMR (500 MHz, DMSO-d6) δ 10.59
(s, 1H, CONH), 8.40–8.34 (m, 1H, ArH), 8.18 (d, J = 8.4 Hz, 1H, ArH), 8.09–8.01 (m, 2H, ArH),
7.85–7.78 (m, 1H, ArH), 7.17–7.11 (m, 1H, ArH), 7.08–6.98 (m, 2H, ArH), 3.83 (s, 3H, OCH3); 13C
NMR (126 MHz, DMSO-d6) δ 165.45, 162.37, 152.55, 148.02, 138.18, 130.17, 126.27, 119.73,
114.83, 113.77, 55.61; IR (ATR-Ge, cm-1): 3232 (ν, NH), 1687 (ν, C=O, amide), 1609, 1594,
1579; Calculated for C13H12N2O2 (MW 228.09): 68.41% C; 5.3% H; 12.27% N; found 66.91% C;
5.00% H; 11.83% N.
1
N-(5-chloropyridin-2-yl)-4-methoxybenzamide (20) mp. 138.5–139 °C; Yield 25%; white solid; H
NMR (500 MHz, CDCl3-d5) δ 8.59 (s, 1H, CONH), 8.38 (d, J = 8.9 Hz, 1H, ArH), 8.22 (d, J = 2.6
Hz, 1H, ArH), 7.92–7.84 (m, 2H, ArH), 7.75–7.68 (m, 1H, ArH), 7.02–6.94 (m, 2H, ArH), 3.89 (s,
3H, OCH3); 13C NMR (126 MHz, CDCl3-d5) δ 165.04, 162.97, 150.07, 146.47, 138.00, 129.15,
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126.62, 126.04, 114.73, 114.10, 55.49; IR (ATR-Ge, cm-1): 3238 (ν, NH), 1673 (ν, C=O, amide),
1608, 1589, 1575; Calculated for C13H11ClN2O2 (MW 262.05): 59.44% C; 4.22% H; 10.66% N;
found 59.26% C; 3.99% H; 10.62% N.
1
N-(6-chloropyridin-2-yl)-4-methoxybenzamide (21) mp. 102.5–104 °C; Yield 75%; white solid; H
NMR (500 MHz, CDCl3-d5) δ 8.58 (s, 1H, CONH), 8.32 (d, J = 8.2 Hz, 1H, ArH), 7.89 (d, J = 8.9
Hz, 2H, ArH), 7.71 (t, J = 7.9 Hz, 1H, ArH), 7.09 (d, J = 7.5 Hz, 1H, ArH), 6.99 (d, J = 8.9 Hz, 2H,
ArH), 3.88 (s, 3H, OCH3); 13C NMR (126 MHz, CDCl3-d5) δ 165.06, 163.03, 151.60, 148.88,
140.98, 129.20, 125.81, 119.55, 114.09, 112.10, 55.48; IR (ATR-Ge, cm-1): 3416 (ν, NH), 1689 (ν,
C=O, amide), 1608, 1590, 1574; Calculated for C13H11ClN2O2 (MW 262.05): 59.44% C; 4.22% H;
10.66% N; found 57.30% C; 4.22% H; 9.82% N.
N-(pyridin-2-yl)-3-(trifluoromethyl)benzamide (22) mp. 128.5–129.5 °C; Yield 30%; white solid; 1H
NMR (500 MHz, CDCl3-d5) δ 9.01 (s, 1H, CONH), 8.39 (d, J = 8.4 Hz, 1H, ArH), 8.26–8.19 (m,
2H, ArH), 8.12 (d, J = 7.9 Hz, 1H, ArH), 7.86–7.75 (m, 2H, ArH), 7.68–7.61 (m, 1H, ArH), 7.12–
7.05 (m, 1H, ArH); 13C NMR (126 MHz, CDCl3-d5) δ 164.36, 151.28, 147.90, 138.62, 135.18,
131.46 (q, J = 32.9 Hz), 130.43, 129.46, 128.76 (q, J = 3.7 Hz), 124.45 (q, J = 3.8 Hz), 123. 56 (q,
J = 274.4 Hz), 114.39; IR (ATR-Ge, cm-1): 3252 (ν, NH), 1680 (ν, C=O, amide), 1596, 1583, 1538.
N-(5-chloropyridin-2-yl)-3-(trifluoromethyl)benzamide (23) mp.109.2–111 °C; Yield 78 %; white
solid; 1H NMR (500 MHz, CDCl3-d5) δ 8.74 (s, 1H, CONH), 8.38 (d, J = 8.9 Hz, 1H, ArH), 8.20 (d,
J = 2.6 Hz, 1H, ArH), 7.75–7.69 (m, 1H, ArH), 7.48–7.37 (m, 3H, ArH), 7.15–7.09 (m, 1H, ArH);
13C NMR (126 MHz, CDCl3-d5) 164.16, 149.48, 146.62, 138.24, 134.78, 131.54 (q, J = 33.0 Hz),
129.57, 128.98 (q, J = 3.6 Hz), 127.37, 124.39 (q, J = 3.8 Hz), 123.53 (q, J = 272.0 Hz), 114.94;
IR (ATR-Ge, cm-1): 3153 (ν, NH), 1677 (ν, C=O, amide), 1592, 1580, 1525.
N-(6-chloropyridin-2-yl)-3-(trifluoromethyl)benzamide (24) mp.78.1–80.4 °C; Yield 72 %; white
1
solid; H NMR (500 MHz, CDCl3-d5) 8.63 (s, 1H, CONH), 8.35–8.30 (m, 1H, ArH), 8.21–8.16 (m,
1H, ArH), 8.10 (d, J = 7.7 Hz, 1H, ArH), 7.88–7.82 (m, 1H, ArH), 7.75 (t, J = 8.0 Hz, 1H, ArH),
7.70–7.63 (m, 1H, ArH), 7.17–7.11 (m, 1H, ArH); 13C NMR (126 MHz, CDCl3-d5) δ 164.11,
150.95, 149.134, 141.18, 134.57, 131.60 (q, J = 33.0 Hz), 129.80 (q, J = 3.6 Hz), 124.32 (q, J =
3.8 Hz), 123.49 (q, J = 272.6 Hz), 120.32, 112.27, 29.68; IR (ATR-Ge, cm-1): 3334 (ν, NH), 1660
(ν, C=O, amide), 1589, 1573, 1525.
4-(tert-butyl)-N-(pyridin-2-yl)benzamide (25) mp. 106.0–108.0 °C; Yield 76%; white solid; 1H NMR
(500 MHz, CDCl3-d5) δ 8.77 (s, 1H, CONH), 8.45–8.39 (m, 1H, ArH), 8.29–8.24 (m, 1H, ArH),
7.91–7.85 (m, 1H, ArH), 7.80–7.76 (m, 1H, ArH), 7.76–7.69 (m, 1H, ArH), 7.55–7.48 (m, 1H,
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ArH), 7.38–7.32 (m, 1H, ArH), 7.09–7.03 (m, 1H, ArH), 1.37 (d, J = 0.8 Hz, 9H, -(CH3)3); 13C NMR
(126 MHz, CDCl3-d5) 165.629, 155.879, 151.691, 147.821, 138.421, 131.319, 129.338, 127.077,
125.757, 125.476, 119.755, 114.148, 35.011, 31.111, 30.997; IR (ATR-Ge, cm-1): 3248 (ν, NH),
1674 (ν, C=O, amide), 1609, 1593, 1578; Calculated for C16H18N2O (MW 254.14): 75.56% C;
7.13% H; 11.01% N; found 75.05% C; 7.02% H; 10.36% N.
4-(tert-butyl)-N-(5-chloropyridin-2-yl)benzamide (26) mp. 153.0–154.0 °C; Yield 23%; white solid;
1H NMR (500 MHz, CDCl3-d5) δ 10.83 (s, 1H, CONH), 8.41–8.37 (m, 1H, ArH), 8.22–8.18 (m, 2H,
ArH), 7.95–7.92 (m, 2H, ArH), 7.92–7.88 (m, 1H, ArH), 7.51–7.46 (m, 2H, ArH), 1.27 (s, 9H, -
(CH3)3); 13C NMR (126 MHz, CDCl3-d5) 165.432, 156.198, 149.961, 146.532, 138.034, 130.962,
127.047, 126.759, 125.878, 114.740, 35.072, 31.111; IR (ATR-Ge, cm-1): 2962 (ν, NH), 1680 (ν,
C=O, amide), 1610, 1588, 1574; Calculated for C16H17ClN2O (MW 288.10): 66.5% C; 5.93% H;
9.70% N; found 65.48% C; 5.60% H; 9.36% N.
4-(tert-butyl)-N-(6-chloropyridin-2-yl)benzamide (27) mp. 111.0–112.0 °C; Yield 17%; white solid;
1H NMR (500 MHz, CDCl3-d5) δ 8.58 (s, 1H, CONH), 8.35 (d, J = 8.2 Hz, 1H, ArH), 7.88–7.83 (m,
2H, ArH), 7.72 (t, J = 7.9 Hz, 1H, ArH), 7.58–7.49 (m, 2H, ArH), 7.10 (d, J = 7.7 Hz, 1H, ArH),
1.36 (s, 9H, -(CH3)3); 13C NMR (126 MHz, CDCl3-d5) δ 165.40, 156.28, 151.46, 148.95, 140.98,
130.71, 127.07, 125.85, 119.68, 112.10, 35.05, 31.08; IR (ATR-Ge, cm-1): 3297 (ν, NH), 1683 (ν,
C=O, amide), 1587, 1570, 1531; Calculated for C16H17ClN2O (MW 288.10): 66.5% C; 5.93% H;
9.70% N; found 66.07% C; 5.73% H; 9.43% N.
4-methyl-N-(pyridin-2-yl)benzamide (28) mp. 99.5–103.4 °C; Yield 19%; white solid; 1H NMR (500
MHz, CDCl3-d5) δ 8.78 (s, 1H, CONH), 8.43–8.38 (m, 1H, ArH), 8.28–8.25 (m, 1H, ArH), 7.84 (d,
J = 8.2 Hz, 2H, ArH), 7.78–7.73 (m, 1H, ArH), 7.30 (d, J = 8.2 Hz, 2H, ArH), 7.08–7.04 (m, 1H,
ArH), 2.43 (s, 3H, CH3); 13C NMR (126 MHz, CDCl3-d5) δ 165.67, 151.68, 147.80, 142.83, 138.44,
131.39, 129.46, 127.24, 119.77, 114.17, 21.49; (ATR-Ge, cm-1): 3234 (ν, NH); 1670 (ν, C=O,
amide); 1578; 1537; 1508; Calculated for C13H12N2O (MW 212.25): 73.56% C; 5.70% H;13.20%
N; found 73.78% C; 5.33% H; 12.88% N.
1
N-(5-chloropyridin-2-yl)-4-methylbenzamide (29) mp. 127.3–132.3 °C; Yield 47%; white solid; H
NMR (500 MHz, CDCl3-d5) δ 8.69 (s, 1H, CONH), 8.39 (d, J = 8.9 Hz, 1H, ArH), 8.18 (d, J = 2.6
Hz, 1H, ArH), 7.81 (d, J = 8.1 Hz, 2H, ArH), 7.71 (dd, J = 8.9, 2.6 Hz, 1H, ArH), 7.30 (d, J = 8.1
Hz, 2H, ArH), 2.43 (s, 3H, CH3); 13C NMR (126 MHz, CDCl3-d5) δ 165.56, 149.99, 146.49, 143.10,
138.01, 131.07, 129.53, 127.21, 126.71, 114.76, 21.51; (ATR-Ge, cm-1): 3244 (ν, NH); 1679 (ν,
C=O, amide); 1575; 1530; 1507; Calculated for C13H11ClN2O (MW 246.69): 63.29% C; 4.49% H;
11.36% N; found 63.42% C; 4.10% H; 11.25% N.
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1
N-(6-chloropyridin-2-yl)-4-methylbenzamide (30) mp. 106.0–107.9 °C; Yield 49%; white solid; H
NMR (500 MHz, CDCl3-d5) δ 8.53 (s, 1H, CONH), 8.33 (d, J = 8.1 Hz, 1H, ArH), 7.82–7.78 (m,
2H, ArH), 7.71 (t, J = 8.0 Hz, 1H, ArH), 7.30 (d, J = 8.0 Hz, 2H, ArH), 7.10–7.08 (m, 1H, ArH),
2.43 (s, 3H, CH3); 13C NMR (126 MHz, CDCl3-d5) δ 165.45, 151.44, 148.94, 143.22, 140.96,
130.81, 129.54, 127.19, 119.67, 112.07, 21.49; (ATR-Ge, cm-1): 3292 (ν, NH); 1655 (ν, C=O,
amide); 1570; 1535; 1506; Calculated for C13H11ClN2O (MW 246.69): 63.29% C; 4.49% H;
11.36% N; found 63.25% C; H: 4.30% H; 11.35% N.
1
4-ethyl-N-(pyridin-2-yl)benzamide (31) mp. 59.2–60.7 °C; Yield 15%; white solid; H NMR (500
MHz, CDCl3-d5) δ 8.83 (s, 1H, CONH), 8.44–8.38 (m, 1H, ArH), 8.27–8.21 (m, 1H, ArH), 7.90–
7.83 (m, 2H, ArH), 7.79–7.72 (m, 1H, ArH), 7.35–7.28 (m, 2H, ArH), 7.09–7.02 (m, 1H, ArH), 2.73
13
(q, J = 7.6 Hz, 2H, CH2), 1.28 (t, J = 7.7 Hz, 3H, CH3); C NMR (126 MHz, CDCl3-d5) δ 165.72,
151.70, 149.02, 147.80, 138.42, 131.61, 128.27, 127.34, 119.74, 114.17, 28.80, 15.21; IR (ATR-
Ge, cm-1): 3170 (ν, NH), 1699 (ν, C=O, amide), 1597, 1579, 1528; Calculated for C14H14N2O (MW
226.11): 74.31% C; 6.24% H; 12.38% N; found 73.91% C; 6.03% H; 12.20% N.
1
N-(5-chloropyridin-2-yl)-4-ethylbenzamide (32) mp. 103.0–104.8 °C; Yield 49%; white solid; H
NMR (600 MHz, CDCl3-d5) δ 8.69 (s, 1H, CONH), 8.37 (d, J = 9.0 Hz, 1H, ArH), 8.16 (d, J = 2.5
Hz, 1H, ArH), 7.84–7.79 (m, 2H, ArH), 7.69 (dd, J = 8.9, 2.6 Hz, 1H, ArH), 7.33–7.28 (m, 2H,
ArH), 2.71 (q, J = 7.7 Hz, 2H, CH2), 1.26 (t, J = 7.6 Hz, 3H, CH3); 13C NMR (151 MHz, CDCl3-d5) δ
165.71, 150.13, 149.42, 146.61, 138.13, 131.41, 128.48, 127.44, 126.82, 114.89, 28.93, 15.29;
IR (ATR-Ge, cm-1): 3258 (ν, NH), 1678 (ν, C=O, amide), 1589, 1574, 1522; Calculated for
C14H13ClN2O (MW 260.07): 64.50% C; 5.03% H; 10.74% N; found 64.50% C; 4.93% H; 10.63%
N.
N-(6-chloropyridin-2-yl)-4-ethylbenzamide (33) mp. 91.5–92.7 °C; Yield 63%; white solid; 1H NMR
(500 MHz, CDCl3-d5) δ 8.54 (s, 1H, CONH), 8.36–8.30 (m, 1H, ArH), 7.86–7.80 (m, 2H, ArH),
7.74–7.66 (m, 1H, ArH), 7.36–7.29 (m, 2H, ArH), 7.11–7.06 (m, 1H, ArH), 2.73 (q, J = 7.6 Hz, 2H,
CH2), 1.27 (t, J = 7.6 Hz, 3H, CH3); 13C NMR (126 MHz, CDCl3-d5) δ 165.45, 151.44, 149.39,
148.93, 140.95, 131.00, 128.36, 127.29, 119.66, 112.07, 28.79, 15.12; IR (ATR-Ge, cm-1): 3423
(ν, NH), 1672 (ν, C=O, amide), 1587, 1568, 1532; Calculated for C14H13ClN2O (MW 260.07):
64.50% C; 5.03% H; 10.74% N; found 64.35% C; 4.94% H; 10.61% N.
N-(pyridin-3-yl)benzamide (34) mp. 109.1–114.6 °C [lit. 113–114 °C (Ueda and Nagasawa,
2009)]; Yield 63%; white solid; 1H NMR (500 MHz, CDCl3-d5) δ 10.45 (s, 1H, CONH), 8.94 (d, J =
2.6 Hz, 1H, ArH), 8.31 (dd, J = 4.7, 1.5 Hz, 1H, ArH), 8.22–8.18 (m, 1H, ArH), 8.00–7.96 (m, 2H,
ArH), 7.64–7.58 (m, 1H, ArH), 7.57–7.52 (m, 2H, ArH), 7.41–7.37 (m, 1H, ArH); 13C NMR (126
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MHz, CDCl3-d5) δ 166.12, 144.74, 142.19, 136.01, 134.55, 132.04, 128.63, 127.90, 127.48,
123.68; IR (ATR-Ge, cm-1): 3325 (ν, NH); 1647 (ν, C=O, amide); 1591; 1522; 1479; Calculated for
C12H10N2O (MW 198.23): 72.71% C; 5.09% H; 14.13% N; found 72.25% C; 4.93% H; 13.95% N.
N-(pyridin-3-yl)benzamide hydrochloride mp. 222.2–225.3 °C; Yield 78%; Calculated for
C12H11N2O (MW 234.68): 61.42% C; 4.72% H; 11.94% N; found 61.67% C; 4.54% H; 12.01% N.
2-chloro-N-(pyridin-3-yl)benzamide (35) mp. 124.1–127.6 °C; Yield 7%; white solid; 1H NMR (500
MHz, DMSO-d6) δ 10.91 (s, 1H, CONH), 8.96 (d, J = 2.5 Hz, 1H, ArH), 8.40 (dd, J = 4.9, 1.5 Hz,
1H, ArH), 8.28–8.24 (m, 1H, ArH), 7.79–7.76 (m, 1H, ArH), 7.67–7.38 (m, 4H, ArH); 13C NMR
(126 MHz, CDCl3-d5) δ 169.18, 149.44, 149.33, 136.06, 135.00, 134.92, 131.72, 130.75, 129.89,
129.30, 126.94, 123.81; IR (ATR-Ge, cm-1): 2928 (ν, NH); 1667 (ν, C=O, amide); 1344; 1276;
1237; Calculated for C12H9ClN2O (MW 232.67): 61.95% C; 3.90% H; 12.04% N; found: 61.24% C;
3.02% H; 7.25% N.
1
3-chloro-N-(pyridin-3-yl)benzamide (36) mp. 124.1–127.6 °C; Yield 78%; white solid; H NMR
(500 MHz, DMSO-d6) 1H NMR (600 MHz, DMSO-d6) δ 11.57 (s, 1H, CONH), 9.38 (d, J = 2.4 Hz,
1H, ArH), 8.89–8.84 (m, 1H, ArH), 8.65–8.61 (m, 1H, ArH), 8.13 (t, J = 1.9 Hz, 1H, ArH), 8.07–
8.03 (m, 1H, ArH), 8.03–7.98 (m, 1H, ArH), 7.70–7.65 (m, 1H, ArH), 7.56 (t, J = 7.9 Hz, 1H, ArH);
13C NMR (126 MHz, CDCl3-d5) δ 166.10, 138.64, 137.30, 135.29, 133.62, 132.55, 130.79,
128.05, 127.30, 127.16; IR (ATR-Ge, cm-1): 3110 (ν, NH); 1698 (ν, C=O, amide); 1595; 1547;
1517; Calculated for C12H9ClN2O (MW 232.67): 61.95% C; 3.90% H; 12.04% N; 61.24%; found:
61.01% C; 3.78% H; 11.48% N.
1
4-chloro-N-(pyridin-3-yl)benzamide (37) mp. 145–147.2 °C; Yield 47%; white solid; H NMR (500
MHz, CDCl3-d5) δ 8.82 (s, 1H, CONH), 8.70 (d, J = 2.6 Hz, 1H, ArH), 8.35–8.26 (m, 2H, ArH),
7.85 (d, J = 8.5 Hz, 2H, ArH), 7.42 (d, J = 8.5 Hz, 2H, ArH), 7.35–7.28 (m, 1H, ArH); 13C NMR
(126 MHz, CDCl3-d5) δ 165.44, 145.08, 141.38, 138.56, 135.11, 132.44, 129.01, 128.73, 128.24,
123.92; IR (ATR-Ge, cm-1): 3243 (ν, NH), 1679 (ν, C=O, amide), 1603, 1586, 1546; Calculated for
C12H9ClN2O (MW 232.04): 61.40% C; 3.54% H; 12.00% N; found 61.52% C; 3.64% H; 11.53% N.
1
2-methoxy-N-(pyridin-3-yl)benzamide (38) mp. 112.0–113.0 °C; Yield 80%; pale yellow solid; H
NMR (500 MHz, CDCl3-d5) δ 9.90 (s, 1H, CONH), 8.65 (d, J = 2.5 Hz, 1H, ArH), 8.40–8.34 (m,
2H, ArH), 8.31–8.25 (m, 1H, ArH), 7.56–7.49 (m, 1H, ArH), 7.34–7.28 (m, 1H, ArH), 7.18–7.10
13
(m, 1H, ArH), 7.06 (d, J = 8.3 Hz, 1H, ArH), 4.08 (s, 3H, OCH3); C NMR (126 MHz, CDCl3-d5)
163.70, 157,22, 144.99, 141.59, 135.21, 133.69, 132.50, 127.61, 123.70, 121.74, 121.05, 111.56,
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56.27; IR (ATR-Ge, cm-1): 3315 (ν, NH), 1699 (ν, C=O, amide), 1600, 1545, 1484; Calculated for
C13H12N2O2 (MW 228.09): 68.41% C; 5.3% H; 12.27% N; found 68.08% C; 5.21% H; 12.10% N.
1
3-methoxy-N-(pyridin-3-yl)benzamide (39) mp. 155.1–157.5 °C; Yield 67%; white solid; H NMR
(500 MHz, CDCl3-d5) δ 8.70–8.65 (m, 1H, ArH), 8.45 (s, 1H, CONH), 8.34 (d, J = 4.7 Hz, 1H,
ArH), 8.32–8.26 (m, 1H, ArH), 7.46–7.39 (m, 2H, ArH), 7.40–7.33 (m, 1H, ArH), 7.33–7.25 (m,
1H, ArH), 7.12–7.05 (m, 1H, ArH), 3.84 (s, 3H, OCH3).; 13C NMR (126 MHz, CDCl3-d5) 166.20,
159.95, 145.37, 141.55, 135.63, 134.98, 129.79, 127.737, 123.74, 118.91, 118.34, 112.59, 55.44;
IR (ATR-Ge, cm-1): 3236 (ν, NH), 1678 (ν, C=O, amide), 1653, 1586, 1545; Calculated for
C13H12N2O2 (MW 228.09): 68.41% C; 5.3% H; 12.27% N; found 67.68% C; 5.26% H; 11.94% N.
1
4-methoxy-N-(pyridin-3-yl)benzamide (40) mp. 166.5–168 °C; Yield 83%; white solid; H NMR
(500 MHz, CDCl3-d5) δ 8.69 (d, J = 2.6 Hz, 1H, ArH), 8.51 (s, 1H, CONH), 8.35–8.27 (m, 2H,
ArH), 7.88 (d, J = 8.8 Hz, 2H, ArH), 7.32–7.26 (m, 1H, ArH), 6.94 (d, J = 8.8 Hz, 2H, ArH), 3.86
(s, 3H, OCH3); 13C NMR (126 MHz, CDCl3-d5) δ 165.87, 162.74, 144.86, 141.39, 135.34, 129.18,
127.90, 126.26, 123.76, 113.96, 55.44; IR (ATR-Ge, cm-1): 3242 (ν, NH), 1697 (ν, C=O, amide),
1559, 15544, 1511; Calculated for C13H12N2O2 (MW 228.09): 68.41% C; 5.3% H; 12.27% N;
found 67.97% C; 5.18% H; 12.02% N.
1
N-(pyridin-3-yl)-3-(trifluoromethyl)benzamide (41) mp. 57.3–60.4 °C; Yield 25%; white solid; H
NMR (500 MHz, CDCl3-d5) δ 9.23 (s, 1H, CONH), 8.70 (d, J = 2.5 Hz, 1H, ArH), 8.32–8.26 (m,
2H, ArH), 8.16 (s, 1H, ArH), 8.09 (d, J = 7.8 Hz, 1H, ArH), 7.78 (d, J = 7.8 Hz, 1H, ArH), 7.58 (t, J
= 7.9 Hz, 1H, ArH), 7.33–7.25 (m, 1H, ArH); 13C NMR (126 MHz, CDCl3-d5) δ 165.23, 145.31,
141.56, 135.03, 131.19 (q, J = 33.0 Hz), 129.39, 128.66 (q, J = 3.6 Hz), 128.35, 126.20 (q, J =
271.8 Hz), 124.29 (q, J = 3.9 Hz), 123.93, 50.54; IR (ATR-Ge, cm-1): 3300 (ν, NH), 1678 (ν, C=O,
amide), 1614, 1543, 1484.
4-(tert-butyl)-N-(pyridin-3-yl)benzamide (42) mp. 135.0–138.0 °C [lit. 137–139 °C (Von der Saal,
1
Mertens, 1989)]; Yield 74%; white solid; H NMR (500 MHz, CDCl3-d5) δ 8.73 (s, 1H, CONH),
8.42 (s, 1H, ArH), 8.39–8.33 (m, 2H, ArH), 7.86 (d, J = 8.5 Hz, 2H, ArH), 7.49 (d, J = 8.5 Hz, 2H,
ArH), 7.35–7.29 (m, 1H, ArH), 1.35 (s, 9H, -(CH3)3); 13C NMR (126 MHz, CDCl3-d5) 166.21,
155.93, 144.78, 141.18, 135.29, 127.97, 127.08, 125.76, 123.83, 35.01, 31.10; IR (ATR-Ge, cm-
1): 2967 (ν, NH), 1671 (ν, C=O, amide), 1587, 1541, 1512; Calculated for C16H18N2O (MW
254.14): 75.56% C; 7.13% H; 11.01% N; found 75.38% C; 7.17% H; 10.70% N.
4-methyl-N-(pyridin-3-yl)benzamide (43) mp. 126.9–129.0 °C [lit. 126.5 °C (Elemans, Bijsterveld,
2007)]; Yield 57.68%; white solid; 1H NMR (500 MHz, DMSO-d6) δ 10.35 (s, 1H, CONH), 8.94 (d,
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J = 2.6 Hz, 1H, ArH), 8.30 (dd, J = 4.7, 1.5 Hz, 1H, ArH), 8.19 (ddd, J = 8.3, 2.6, 1.5 Hz, 1H, ArH),
7.93–7.87 (m, 2H, ArH), 7.41–7.32 (m, 3H, ArH), 2.38 (s, 3H, CH3); 13C NMR (126 MHz, DMSO-
d6) δ 165.90, 144.61, 142.17, 142.13, 136.08, 131.64, 129.15, 127.94, 127.44, 123.65, 21.19
(ATR-Ge, cm-1): 3323 (ν, NH); 1652 (ν, C=O, amide); 1529; 1508; 1481; Calculated for C13H12N2O
(MW 212.25): 73.56% C; 5.70% H; 13.20% N; found 73.51% C; 5.49% H; 13.21% N.
4-methyl-N-(pyridin-3-yl)benzamide hydrochloride mp. 270.2–276.5 °C; Yield 84%; Calculated for
C13H13N2O (MW 248.71): 62.78% C; 5.27% H; 11.26% N; found 62.76% C; 4.95% H; 11.28% N;
1
4-ethyl-N-(pyridin-3-yl)benzamide (44) mp. 90.2–91.4 °C; Yield 21%; white solid; H NMR (500
MHz, CDCl3-d5) δ 8.68 (d, J = 2.5 Hz, 1H, ArH), 8.60 (s, 1H, CONH), 8.33–8.27 (m, 2H, ArH),
7.82 (d, J = 8.1 Hz, 2H, ArH), 7.27 (d, J = 8.0 Hz, 3H, ArH), 2.70 (q, J = 7.6 Hz, 2H, CH2), 1.25 (t,
13
J = 7.6 Hz, 3H, CH3). C NMR (126 MHz, CDCl3-d5) δ 166.38, 148.98, 145.03, 141.47, 135.22,
131.53, 128.22, 127.83, 127.33, 123.72, 28.76, 15.20; IR (ATR-Ge, cm-1): 3320 (ν, NH), 1651 (ν,
C=O, amide), 1572, 1525, 1506; Calculated for C14H14N2O (MW 226.11): 74.31% C; 6.24% H;
12.38% N; found 73.81% C; 6.12% H; 12.17% N.
4.4.
Evaluation of biological activity
The methodology is consistent with our previous publications (Ambrozkiewicz, Kucerova-
Chlupacova, 2020). Full description of the methods is enclosed in Supplementary Material.
4.4.1. In Vitro Antimycobacterial Activity
Testing was performed by Microplate Alamar Blue Assay (MABA) (Franzblau, Witzig, 1998),
where results were expressed as MIC in µg/mL in comparison to INH, RFM and ciprofloxacin
(CPX) as standards.
4.4.2. In Vitro Antibacterial Activity
Microdilution broth method according to EUCAST with minor modifications. Results were
expressed as MIC compared to standards of gentamicin (GNT) and CPX as standards.
4.4.3. In Vitro Antifungal Activity
Microdilution broth method performed according to EUCAST protocols with minor modifications.
Results were expressed as MIC compared to standards amphotericin B (AmB) and voriconazole
(VRC).
5. Conclusions
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We prepared 44 different N-pyridinylbenzamides as C isosters of previously published N-
pyrazinylbenzamides (Zitko, Mindlová, 2018) with in vitro antimycobacterial activity. Some N-
pyrazinylbenzamides have been previously reported in literature, exerting wide spectrum of
biological activities (as briefly mentioned in the Introduction). However, none of them was tested
for antibacterial or antimycobacterial activity except for compounds 1 (Joseph, Dixit, 2019) and 19
(Mndzhoyan and Afrikyan, 1957, Mndzhoyan, Apoyan, 1962) for which only limited activity data
are available.
In our study, some of the title compounds exerted good antimycobacterial activity against Mtb
H37Ra (in total fourteen compounds were below the activity cut-off of MIC = 31.25 µg/mL, with
the best activity of MIC = 7.81 µg/mL exerted by compounds 23, 24, 27 and 33), M. smegmatis (9
compounds active, with best activity of 3.91 µg/mL - 27) and M. aurum (3 active compounds, all
with MIC 31.25 µg/mL – 23, 24, 31). Five compounds (23, 24, 31, 32, 36) inhibited the growth of
all three tested mycobacterial strains and therefore are notable for the broadness of their
antimycobacterial affect, at the same time being inactive against tested bacterial and fungal
strains. Consistently with our template series of N-pyrazinylbenzamides (Zitko, Mindlová, 2018),
we found that the most favourable substituent on the benzene ring (R2) was short alkyl (4-Me, 4-
Et) or 3-CF3.
Additionally, we performed supplementary testing against eight bacterial strains and eight fungal
pathogens of clinical importance, yet none of prepared compounds exerted remarkable activity,
suggesting selective antimycobacterial activity. The primary aim of this research was to
investigate the influence of isosteric exchange of nitrogen atoms in the pyrazine ring of parent N-
pyrazinylbenzamides on antimycobacterial activity of the resulting N-pyridinylbenzamides. Based
on obtained results, we can conclude that N-1 nitrogen of N-pyrazinylbenzamides is more
important than N-4, as removal of N-1 led to less active N-pyridin-3-yl derivatives (series 34–44).
On contrary, removal of N-4 in most cases did not have negative effect on antimycobacterial
activity and resulting N-pyridin-2-yl derivatives (1–33) were often more active than parent N-
pyrazinylbenzamides. The higher importance of N-1 is in concordance with molecular interactions
to protein targets of pyrazinamide and/or its metabolite pyrazinoic acid as discussed in the
Introduction. This finding rationalizes future attempts to prepare new antimycobacterial
compounds by isosteric replacement in active pyrazine derivatives.
Acknowledgements
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The authors wish to thank Ida Dufková for performing the antibacterial and antifungal assessment
and MSc. Ghada Bouz, Ph.D. for language corrections.
Financial Support
This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic
(Grant SVV 260 547) and by the Czech Science Foundation project No. 20-19638Y.
Author contributions
Daria Nawrot: Methodology, Investigation, Writing - Original Draft. Eliška Suchánková:
Investigation. Ondřej Janďourek: Investigation. Klára Konečná: Investigation, Writing - Review &
Editing. Pavel Bárta: Investigation. Martin Doležal: Writing - Review & Editing, Supervision. Jan
Zitko: Conceptualization, Writing - Original Draft, Writing - Review & Editing.
Supplementary material
Supplementary material contains methodology of antimycobacterial, antibacterial, and antifungal
testing and cytotoxicity determination, results of antibacterial (Table S1) and antifungal testing
(Table S2), plots of antimycobacterial activity (Mtb H37Ra) on calculated lipophilicity logP for
individual series (Figure S1), plot of antimycobacterial activity on HepG2 cytotoxicity with
selectivity index indication (Figure S2), and representative NMR spectra in graphic form.
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