Synthesis of pyrazinamide analogues and their antitubercular bioactivity

Article abstract of DOI:10.1007/s00044-020-02626-0

The Yamaguchi reaction is widely and generally applied to synthesize esters and lactones. It involves 2,4,6-trichlorobenzoyl chloride as the Yamaguchi reagent, 4-dimethylaminopyridine, and triethylamine. Pyrazinamide is a crucial first-line drug for tuberculosis treatment, therefore their analogues are interesting in organic synthesis. In general, the synthesis pyrazinamide analogues involve reaction of pyrazine-2-carboxylic acids with thionyl chloride to yield the corresponding acyl chlorides, which on treatment with amines gave pyrazine-2-carboxamides. However, thionyl chloride is listed under the Chemical Weapons Convention and releases toxic sulfur oxide when react with carboxylic acid. We successfully synthesized series of pyrazinamide analogues using the Yamaguchi reaction. The synthesis involved reaction of pyrazine-2-carboxylic acid with various aliphatic and aromatic amines in the presence of Yamaguchi reagent and 4-dimethylaminopyridine. The yield of the pyrazine-2-carboxamides and the reaction time depended on the type of the amine (aliphatic vs aromatic), substitution pattern, and number of substituents on the aromatic amines. N-(4-chlorophenyl)pyrazine-2-carboxamides can be prepared by this method in 81% yield; N-(2-ethylhexyl)pyrazine-2-carboxamide and N-(4-fluorobenzyl)pyrazine-2-carboxamide showed the best activity against Mycobacterium tuberculosis H37Rv (<6.25 μg/mL). This result could lead to find more active pyrazinamide analogues.

Full text of DOI:10.1007/s00044-020-02626-0

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02626-0
ORIGINAL RESEARCH
Synthesis of pyrazinamide analogues and their antitubercular
bioactivity
1
First A. Wati1 Prisna U. Adyarini1 Sri Fatmawati1 Mardi Santoso
●
●
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Received: 18 June 2020 / Accepted: 29 August 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
The Yamaguchi reaction is widely and generally applied to synthesize esters and lactones. It involves 2,4,6-trichlorobenzoyl
chloride as the Yamaguchi reagent, 4-dimethylaminopyridine, and triethylamine. Pyrazinamide is a crucial first-line drug for
tuberculosis treatment, therefore their analogues are interesting in organic synthesis. In general, the synthesis pyrazinamide
analogues involve reaction of pyrazine-2-carboxylic acids with thionyl chloride to yield the corresponding acyl chlorides,
which on treatment with amines gave pyrazine-2-carboxamides. However, thionyl chloride is listed under the Chemical
Weapons Convention and releases toxic sulfur oxide when react with carboxylic acid. We successfully synthesized series of
pyrazinamide analogues using the Yamaguchi reaction. The synthesis involved reaction of pyrazine-2-carboxylic acid with
various aliphatic and aromatic amines in the presence of Yamaguchi reagent and 4-dimethylaminopyridine. The yield of the
pyrazine-2-carboxamides and the reaction time depended on the type of the amine (aliphatic vs aromatic), substitution
pattern, and number of substituents on the aromatic amines. N-(4-chlorophenyl)pyrazine-2-carboxamides can be prepared by
this method in 81% yield; N-(2-ethylhexyl)pyrazine-2-carboxamide and N-(4-fluorobenzyl)pyrazine-2-carboxamide showed
the best activity against Mycobacterium tuberculosis H37Rv (<6.25 μg/mL). This result could lead to find more active
pyrazinamide analogues.
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●
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Keywords Synthesis Pyrazinamide analogues Yamaguchi reaction Antitubercular activity
Introduction
drug resistance, which is regarded as a serious health pro-
blem [3]. Besides, second-line TB drugs are used to treat the
resistance TB are more toxic, less effective effect, and lead
to the higher mortality rate [4]. Hence, there is an urge to
develop and find safe and promising candidate anti-
tubercular compounds.
Tuberculosis (TB), an infectious disease caused by Myco-
bacterium tuberculosis, is a major global health problem
[1]. World Health Organization [2] reported that there were
about 10 million people infected by the bacteria, and
Indonesia accounted for around 770,000–923,000 TB cases
in 2018. First-line TB drugs that consist of isoniazid,
rifampicin, pyrazinamide (PZA), streptomycin, and etham-
butol are active against Mycobacterium tuberculosis [2].
However, the effectiveness of these drugs decreased due to
PZA (1) (Fig. 1) is one of the first-line antitubercular
drugs, which is an essential component in short-term intensive
phase of treatment for TB patients [5]. Servusová et al. [6]
have successfully synthesized PZA analogues (2a–f) (Fig. 1)
that showed antitubercular activities. Phenylpyrazine-2-
carboxamides with electron withdrawing group substituents
(2a, 2b, 2d–2f) (Fig. 1) gave excellent antitubercular activity
with minimum inhibitory concentration (MIC) of
0.78–6.25 µg/mL. The compounds (2a–f) were synthesized by
two-step reactions, including transformation of carboxylic acid
into acyl chloride using thionyl chloride, and then carbox-
amide formation [6]. The drawback of this method is the use
of thionyl chloride, which is toxic releasing sulfur dioxide
(SO2) and listed under the chemical weapons convention.
Halogen-substituted N-phenylpyrazine-2-carboxamides were
also prepared by reacting the same equivalent of aniline,
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02626-0) contains supplementary
material, which is available to authorized users.
* Mardi Santoso
tsv09@chem.its.ac.id
1
Department of Chemistry, Faculty of Science, Institut Teknologi
Sepuluh Nopember, Kampus ITS Sukolilo, Surabaya 60111,
Indonesia

Medicinal Chemistry Research
PF254 containing gypsum as stationary phase. Melting points
were assessed by Fisher John apparatus without correction.
Infrared spectra were recorded on FTIR–8400S Shimadzu
using KBr disc. NMR spectra were recorded on NMR Bruker
(300 MHz for 1H NMR and 75 MHz for 13C NMR) or JEOL
1
Resonance (400 MHz for H NMR and 100 MHz for 13C
NMR) or Agilent (125 MHz for 13C NMR) with CDCl3 as
solvent. High-resolution mass spectra were obtained from a
Waters Q-TOF Xevo mass spectrometer.
Chemistry
Fig. 1 Structures of pyrazinamide (1) and analogues (2)
General procedure for synthesis of PZA analogues (3a–t)
pyrazine-2-carboxylic acid, and triphenyl phosphite in pyr-
idine to yield 65–90%. However, the reaction required high
temperature of 100 °C and long time of >24 h [7, 8]. Another
synthetic method involves decarboxylation reaction of 2,3-
pyrazinedicarboxylic anhydride with anilines and amines in
refluxing toluene. However, secondary amines give pyrazine-
2,3-dicarboximides as a by-product [9]. Cerium oxide (CeO2)
is also used to catalyze pyrazine-2-carboxamide synthesis, but
needs high temperature of 160 °C [10, 11]. Therefore, there is
a need to develop environmentally friendly, mild, and effec-
tive method to synthesize PZA series.
Yamaguchi reaction is commonly utilized to prepare
esters and thioesters using 2,4,6-trichlorobenzoyl chloride.
The reaction is carried out by two steps: anhydride forma-
tion followed by reaction with alcohols or thiols in the
presence of 4-dimethylaminopyridine (DMAP). The reac-
tion is considerably mild, fast, and gives good yields [12–
14]. To our knowledge, the reaction has not been well
explored for the synthesis of amides in general [15] and
certainly not for the synthesis of pyrazine-2-carboxamides.
Herein, we developed one-pot Yamaguchi reaction and
synthesized a series of PZA analogues. Furthermore, the
antimycobacterial activity of these compounds was studied.
To the solution of pyrazine-2-carboxylic acid (1.05 eq) in
tetrahydrofuran (20 mL) was added triethylamine (TEA)
(1.05 eq), 2,4,6-trichlorobenzoyl chloride (1.05 eq), 4-DMAP
(1.05 eq), and the corresponding amines (1 eq). The mixture
was refluxed for appropriate time. After reaction completion
(TLC), the reaction mixture was cooled down then filtered.
The filtrate was diluted with cold water (25 mL) and
extracted with dichloromethane (2 × 25 mL). The obtained
extracts were washed subsequently with hydrogen chloride
5% (25 mL), sodium hydroxide 5% (25 mL), sodium bicar-
bonate 5% (25 mL), and water (25 mL). The organic layer
was dried over anhydrous magnesium sulfate, filtered and
concentrated under reduced pressure to give the product,
which was then purified by column chromatography and/or
chromatotron (ethyl acetate:n-hexane).
N-(2-ethylhexyl)pyrazine-2-carboxamide (3a) Off white
solid; yield: 57%, mp: 50–51 °C; IR (KBr) υmax 3379
(N–H), 3092 (C–H aromatic), 2959 and 2932 (C–H ali-
phatic), 1663 (C=O), 1533 (C=N) cm−1; 1H NMR (CDCl3,
300 MHz): δ = 0.91–0.98 (6H, m, CH3), 1.26–1.46 (8H, m,
CH2), 1.57–1.64 (1H, m, CH), 3.38–3,52 (2H, m, CH2),
7.83 (1H, bs, NH), 8.54 (1H, d, J = 0.8 Hz, ArH), 8.76 (1H,
d, J = 2.1, ArH), 9.42 (1H, s, ArH); 13C NMR (CDCl3,
125 MHz): δ = 11.05, 14.17, 23.10, 24.46, 29.04, 31.21,
39.65, 42.40, 142.58 144.55, 144.76, 147.24, 163.08 (C,
C=O); HRESIMS m/z (pos): 258.1523 C13H21N3ONa
(calcd. 258.1582).
Materials and method
Materials
All the chemicals were purchased from Merck, Sigma-
Aldrich, and Alfa Aesar, and used directly without further
purification. The solvents used for chromatography and
chromatotron were technical grade and were distilled before
use. The reaction progress was monitored by thin layer
chromatography (TLC) (aluminium plates coated with silica
gel 60 F254) at 254 and 365 nm wavelength UV detection and
iodine stained. The obtained product was purified by column
chromatography using silica gel Merck 60 (0.063–0.200 mm)
as stationary phase and/or chromatotron with silica gel 60
N-(4-fluorobenzyl)pyrazine-2-carboxamide (3b) Yellowish
solid; yield: 58%, mp 122–123 °C; IR (KBr) υmax 3376 (N–H),
3084 (C–H aromatic), 2934 (C–H aliphatic), 1674 (C=O),
1
1526 (C=N) cm−1; H NMR (CDCl3, 300 MHz): δ = 4.66
(2H, d, J = 6.1 Hz, CH2), 7.05 (2H, t, J = 8.6 Hz, ArH), 7.36
(2H, dd, J = 5.4 Hz, 8.6 Hz, ArH), 8.15 (1H, bs, NH), 8.52
(1H, dd, J = 1.1 Hz, 2.4 Hz, ArH), 8.77 (1H, d, J = 2.4 Hz,
ArH), 9.46 (1H, d, J = 1.1 Hz, ArH); 13C NMR (CDCl3,
2
125 MHz): δ = 42.88 (CH2), 115.74 (d, Jc–f = 21.36 Hz),
3
4
129.68 (d, Jc–f = 8.13 Hz), 133.72 (d, Jc–f = 3.31 Hz),

Medicinal Chemistry Research
142.66, 144.43, 144.65, 147.51, 162.39 (d, 1Jc–f = 244.70 Hz),
163.15 (C, C=O); HRESIMS m/z (pos): 254.0649
C12H10N3OFNa (calcd. 254.0706).
9.65 (1H, bs, NH); 13C NMR (CDCl3, 125 MHz): δ = 31.48
(CH3), 34.59, 119.72, 126.13, 134.74, 142.46, 144.66,
144.80, 147.55, 148.00, 160.64 (C, C=O); HRESIMS m/z
(pos): 278.1231 C15H17N3ONa (calcd. 278.1269).
N,N-dibenzylpyrazine-2-carboxamide (3c) White solid;
yield 44%, mp 87 °C (Lit. 90 °C [16]); IR (KBr) υmax 3059
and 3028 (C–H aromatic), 2928 (C–H aliphatic), 1638
(C=O) cm−1. 1H NMR (CDCl3, 300 MHz): δ = 4.63 (2H, s,
CH2), 4.73 (2H, s, CH2), 7.24–7.42 (10H, m, ArH), 8.55
(1H, dd, J = 1.4, 2.5 Hz, ArH), 8.63 (1H, d, J = 2.5 Hz,
ArH), 9.03 (1H, d, J = 1.4 Hz, ArH).
N-(3-bromophenyl)pyrazine-2-carboxamide (3i) Yellowish
crystalline solid; yield 27%, mp 133–134 °C (Lit.
129–131 °C [7]). IR (KBr) υmax 3283 (N–H), 3067 (C–H
aromatic), 1672 (C=O), 1516 (C=N), 694 (C–Br) cm−1; 1H
NMR (CDCl3, 300 MHz): δ = 7.16–7.27 (2H, m, ArH),
7.61 (1H, dt, J = 1.7, 7.7 Hz, ArH), 7.95 (1H, t, J = 1.9 Hz,
ArH), 8.53 (1H, t, J = 2.0 Hz, ArH), 8.76 (1H, d, J =
2.4 Hz), 9.44 (1H, d, J = 1.4 Hz, ArH), 9.61 (1H, bs, ArH).
N-phenylpyrazine-2-carboxamide (3d) Yellowish solid;
yield 50%, mp. 122–123 °C (Lit. 123–125 °C [17]); IR
(KBr) υmax 3350 (N–H), 1678 (C=O), 1533 (C=N) cm−1
;
N-(2-nitrophenyl)pyrazine-2-carboxamide
(3j) Yellow
1H NMR (CDCl3, 300 MHz): δ = 7.20 (1H, t, J = 7.4 Hz,
ArH), 7.43 (2H, t, J = 7.4 Hz, ArH), 7.78 (2H, dd, J = 0.9,
8.7 Hz, ArH), 8.62 (1H, dd, J = 1.5, 2.4 Hz, ArH), 8.83
(1H, d, J = 2.4 Hz, ArH), 9.54 (1H, d, J = 1.1 Hz, ArH),
9.69 (1H, bs, NH).
solid; yield 28%, mp 163–165 °C (Lit. 150 °C [19]); IR
(KBr) υmax 3297 (N–H), 3125 (C–H aromatic), 1701
(C=O), 1504 (C=N) cm−1; 1H NMR (CDCl3, 300 MHz): δ
= 7.21 (1H, dt, J = 1.5 Hz, 7.8 Hz, ArH), 7.67 (1H, dt, J =
1.5, 7.9 Hz, ArH), 8.23 (1H, dd, J = 1.5, 7.8 Hz, ArH), 8.65
(1H, t, J = 1.5 Hz, ArH), 8.78 (1H, d, J = 2.4 Hz, ArH),
8.95 (1H, dd, J = 1.0, 8.5 Hz, ArH), 9.44 (1H, d, J =
1.0 Hz, ArH), 12.49 (1H, bs, NH).
N-(4-chlorophenyl)pyrazine-2-carboxamide
(3e) Light
brownish crystalline solid; yield 81%, mp. 160–161 °C (Lit.
182–184 °C [18]); IR (KBr) υmax 3351 (N–H), 3057 (C–H
aromatic), 1678 (C=O), 1530 (C=N), 824 (C–Cl) cm−1; 1H
NMR (CDCl3, 300 MHz): δ = 7.29 (2H, d, J = 8.9 Hz,
ArH), 7.65 (2H, d, J = 8.9 Hz, ArH), 8.53 (1H, dd, J =
1.6 Hz, 2.4 Hz, ArH), 8.76 (1H, d, J = 2.4 Hz, ArH), 9.44
(1H, d, J = 1.0 Hz, ArH), 9.61 (1H, bs, NH).
N-(1-naphtalen-1-yl)pyrazine-2-carboxamide
(3k) Light
yellow solid; yield 66%, mp. 146–147 °C (Lit. 135 °C [20]);
IR (KBr) υmax 3370 (N–H), 3057 (C–H aromatic), 1690
1
(C=O), 1547 (C=N) cm−1. H NMR (CDCl3, 300 MHz,):
δ = 7.54–7.65 (3H, m, ArH), 7.76 (1H, d, J = 8.3 Hz, ArH),
7.94 (1H, d, J = 7.4 Hz, ArH), 8.05 (1H, d, J = 8.3 Hz,
ArH), 8.38 (1H, d, J = 7.4 Hz, ArH), 8.70 (1H, dd, J = 1.1,
2.4 Hz, ArH), 8.88 (1H, d, J = 2.4 Hz, ArH), 9.60 (1H, d, J
= 1.1 Hz, ArH), 10.37 (1H, bs, NH).
N-(4-bromophenyl)pyrazine-2-carboxamide (3f) Yellowish
solid; yield 68%, mp 163–165 °C (Lit. 200 °C [19]); IR
(KBr) υmax 3345(N–H), 2922 (C–H aromatic), 1678 (C=O),
;
1531 (C=N), 671 (C–Br) cm−1 1H NMR (CDCl3,
300 MHz): δ = 7.44 (2H, d, J = 8.7 Hz, ArH), 7.60 (2H, d,
J = 8.7 Hz, ArH), 8.53 (1H, s, ArH), 8.76 (1H, d, J =
2.3 Hz, ArH), 9.44 (1H, s, ArH), 9.61 (1H, bs, NH).
N-(3,5-dimethylphenyl)pyrazine-2-carboxamide (3l) Light
orange solid; yield 68%, mp. 128–129 °C; IR (KBr) υmax
3362 (N–H), 3017 (C–H aromatic), 2963 and 2916 (C–H
aliphatic), 1674 (C=O), 1547 (C=N) cm−1 1H NMR
;
N-(4-iodophenyl)pyrazine-2-carboxamide
(3g) Greyish
(CDCl3, 300 MHz): δ = 2.36 (6H, s, CH3), 6.84 (1H, s,
ArH), 7.42 (2H, s, ArH), 8.59 (1H, t, J = 2.4 Hz, ArH), 8.81
(1H, d, J = 2.4 Hz, ArH), 9.52 (1H, d, J = 1.3 Hz, ArH),
9.60 (1H, bs, NH); 13C NMR (CDCl3, 100 MHz): δ = 21.51
(CH3), 117.67, 126.70, 137.16, 139.02, 142.42, 144.64,
144.76, 147.53, 160.62 (C, C=O); HRESIMS m/z (pos):
250.0993 C13H13N3ONa (calcd. 250.0956).
solid; yield 72%, mp 182–184 °C (Lit. 227 °C [8]); IR
(KBr) υmax 3341 (N–H), 1680 (C=O), 1524 (C=N), 507
1
(C–I) cm−1; H NMR (CDCl3, 300 MHz): δ = 7.49 (2H, d,
J = 8.8 Hz, ArH), 7.64 (2H, d, J = 8.8 Hz, ArH), 8.53 (1H,
dd, J = 1.4, 2.5 Hz, ArH), 8.76 (1H, d, J = 2.5 Hz, ArH),
9.44 (1H, d, J = 1.4 Hz, ArH), 9.59 (1H, bs, NH).
N-(4-tert-butylphenyl)pyrazine-2-carboxamide (3h) Yel-
lowish solid; yield 60%, mp 151–152 °C. IR (KBr) υmax
3352 (N–H), 3059 (C–H aromatic), 2963 and 2926 (C–H
N-(3,5-dimethoxyphenyl)pyrazine-2-carboxamide
(3m)
Grey solid; yield 77%, mp. 130–131 °C; IR (KBr) υmax
3354 (N–H), 3071 (C–H aromatic), 2920 and 2843 (C–H
aliphatic), 1688 (C=O), 1545 (C=N), 1213 and 1022
(C–O) cm−1; 1H NMR (CDCl3, 300 MHz): δ = 3.84 (6H, s,
OCH3), 6.32 (1H, s, ArH), 7.02 (2H, d, J = 2.0 Hz, ArH),
8.61 (1H, s, ArH), 8.83 (1H, d, J = 2.3 Hz, ArH), 9.52 (1H,
aliphatic), 1670 (C=O), 1528 (C=N) cm−1 1H NMR
;
(CDCl3, 300 MHz): δ = 1.36 (9H, s, CH3), 7.44 (2H, d, J =
8.6 Hz, ArH), 7.70 (2H, d, J = 8.6 Hz, ArH), 8.61 (1H, s,
ArH), 8.82 (1H, d, J = 2.3 Hz, ArH), 9.54 (1H, s, ArH),

Medicinal Chemistry Research
s, ArH), 9.65 (1H, bs, NH); 13C NMR (CDCl3, 100 MHz):
δ = 55.54 (OCH3), 97.45, 98.09, 138.98, 142.46, 144.36,
144.71, 147.67, 160.73 (C, C=O), 161.24; HRESIMS m/z
(pos): 260.1084 C13H15N3O3 (calcd. 260.1035).
9.35 (1H, bs, NH), 9.45 (1H, d, J = 1.3 Hz, ArH); 13C NMR
(CDCl3, 75 Hz): δ = 123.61, 129.88, 132.47, 134.23,
142.77, 143.75, 144.90, 147.90, 160.88 (C, C=O); HRE-
SIMS m/z (pos): 355.9052 C11H8N3OBr2 (calcd. 355.9034).
N-(3,5-bis(trifluoromethyl)phenyl)pyrazine-2-carboxamide
(3n) White solid; yield 70%, mp. 118–119 °C; IR (KBr)
υmax 3318 (N–H), 3119 and 3096 (C–H aromatic), 1690
N-(2,4,6-trimethylphenyl)pyrazine-2-carboxamide
(3r)
Beige solid; yield 48%, mp. 119–120 °C (Lit. 124–125 °C
[16]); IR (KBr) υmax 3366 (N–H), 2918 and 2857 (C–H
(C=O), 1541 (C=N), 1123 (C–F) cm−1; H NMR (CDCl3,
aliphatic), 1684 (C=O), 1528 (C=N) cm−1 1H NMR
;
1
300 MHz): δ = 7.61 (1H, s, ArH), 8.22 (2H, s, ArH), 8.56
(1H, s, ArH), 8.81 (1H, d, J = 1.4 Hz, ArH), 9.46 (1H, s,
ArH), 9.89 (1H, bs, NH); 13C NMR (CDCl3, 75 MHz): δ =
(CDCl3, 300 MHz): δ = 2.26 (6H, s, CH3), 2.32 (3H, s,
CH3), 6.97 (2H, s, ArH), 8.63 (1H, s, ArH), 8.83 (1H, d,
J = 2.3 Hz, ArH), 9.13 (1H, bs, NH), 9.51 (1H, s, ArH).
3
3
118.08 (m, Jc–f = 3.75 Hz), 119.51 (d, Jc–f = 3.00 Hz),
1
2
123.04 (q, Jc–f = 270.75 Hz, CF3), 132.63 (q, Jc–f
=
N-(4-bromo-2,6-difluorophenyl)pyrazine-2-carboxamide
(3s) Light orange solid; yield 22%, mp. 134–135 °C; IR
(KBr) υmax 3339 (N–H), 3071 and 3048 (C–H aromatic),
33.50 Hz), 138.66, 142.53, 143.36, 144.83, 148.28, 161.20
(C, C=O); HRESIMS m/z (pos): 336.0515 C13H8N3OF6
(calcd. 336.0572).
1694 (C=O), 1531 (C=N), 1047 (C–F), 604 (C–Br) cm−1
;
1H NMR (CDCl3, 300 MHz): δ = 7.14–7.19 (2H, m, ArH),
8.56 (1H, dd, J = 1.5 Hz, 2.4 Hz, ArH), 8.78 (1H, d, J =
N-(3-fluoro-4-methylphenyl)pyrazine-2-carboxamide (3o)
Yellowish solid; yield 26%, mp 146 °C; IR (KBr) υmax 3351
(N–H), 3115 (C–H aromatic), 2926 (C–H aliphatic), 1676
2.4 Hz, ArH), 9.08 (1H, s, NH), 9.42 (1H, d, J = 1.5 Hz,
2
ArH); 13C NMR (CDCl3, 125 MHz): δ = 113.0 (t, Jc–f
=
=
1
2
3
(C=O), 1530 (C=N), 1130 (C–F) cm−1; H NMR (CDCl3,
16.19 Hz), 116.12 (t, Jc–f = 27.01 Hz), 120.06 (t, Jc–f
300 MHz): δ = 2.20 (3H, s, CH3), 7.10 (1H, t, J = 8.3 Hz,
ArH), 7.19–7.24 (1H, m, ArH), 7.58 (1H, dd, J = 1.7 Hz,
11.4 Hz, ArH), 8.52 (1H, s, ArH), 8.74 (1H, d, J = 2.2 Hz,
ArH), 9.43 (1H, s, ArH), 9.58 (1H, bs, NH); 13C NMR
11.48 Hz), 142.83, 143.54, 145.00, 148.15, 157.8 (dd, 1Jc–f
= 254.09 Hz, 3Jc–f = 5.65), 161.26(C, C=O); HRESIMS m/
z (pos): 335.9475 C11H679BrF2N3ONa (calcd. 335.9560),
337.9505 C11H681BrF2N3ONa (calcd. 335.9540).
3
(CDCl3, 125 MHz): δ = 14.28 (d, Jc–f = 3.16 Hz, CH3),
2
3
107.24 (d, Jc–f = 27.26 Hz), 115.11 (d, Jc–f = 3.35 Hz),
N-(3,4,5-trimethoxyphenyl)pyrazine-2-carboxamide (3t)
Off white solid; yield 41%, mp 163 °C; IR (KBr) υmax 3281
(N–H), 3057 (C–H aromatic), 2945 (C–H aliphatic), 1680
2
4
121.28 (d, Jc–f = 17.31 Hz), 131.65 (d, Jc–f = 6.33 Hz),
136.39 (d, 3Jc–f = 10.74 Hz), 142.48, 144.30, 144.80, 147.75,
160.70 (C, C=O), 161.32 (d, 1Jc–f = 242.990 Hz); HRESIMS
m/z (pos): 232.0799 C12H11N3OF (calcd. 232.0886).
(C=O), 1547 (C=N), 1232 and 1018 (C–O) cm−1 1H
;
NMR (CDCl3, 400 MHz): δ = 3.80 (3H, s, OCH3), 3.88
(6H, s, OCH3), 7.06 (2H, s, ArH), 8.56 (1H, s, ArH), 8.79
(1H, d, J = 2.4 Hz, ArH), 9.48 (1H, s, ArH), 9.59 (1H, bs,
NH); 13C NMR (CDCl3, 100 MHz): δ = 56.25 (OCH3),
61.08 (OCH3), 97.48, 133.43, 135.17, 142.44, 144.38,
144.63, 147.67, 153.56, 160.62 (C, C=O); HRESIMS m/z
(pos): 290.1196 C14H16N3O4 (calcd. 290.1141).
N-(3,4-dimethoxyphenyl)pyrazine-2-carboxamide
(3p)
Blackish solid, yield 31%, mp 131–132 °C; IR (KBr) υmax
3360 (N–H), 3009 (C–H aromatic), 2920 and 2837 (C–H
aliphatic), 1680 (C=O), 1545 (C=N), 1230 and 1022
(C–O) cm−1; 1H NMR (CDCl3, 300 MHz): δ = 3.90 (3H, s,
OCH3), 3.95 (3H, s, OCH3), 6.88 (1H, d, J = 8.6 Hz, ArH),
7.15 (1H, dd, J = 2.2 Hz, 8.6 Hz, ArH), 7.60 (1H, d, J =
2.2 Hz, ArH), 8.59 (1H, s, ArH), 8.81 (1H, s, J = 2.3 Hz,
ArH), 9.51 (1H, s, ArH), 9.62 (1H, bs, NH); 13C NMR
(CDCl3, 100 MHz): δ = 56.04 (OCH3), 56.17 (OCH3),
104.43, 111.37, 111.83, 130.95, 142.46, 144.53, 144.58,
146.25, 147.53, 149.19, 160.47 (C, C=O); HRESIMS m/z
(pos): 260.1026 C13H14N3O3 (calcd. 260.1035).
Antitubercular assay
Antitubercular assay was conducted by resazurin microtiter
assay (REMA) method [21]. One milligram of each tested
compounds was dissolved in 1 mL DMSO 20%, then
diluted to the final following concentrations 25, 12.5, 6.25,
3.13, 1.56, and 0.78 µg/mL. Each solution was placed at
microtiter 96-well plate, and 100 µL suspension of Myco-
bacterium tuberculosis H37Rv were added. The plates were
covered, sealed with plastic bags, and incubated at 37 °C for
7 days. After that, 30 µL of 0.01% resazurin solution was
added to each well, incubated further at 37 °C for a day, and
observed for the colour change. Resazurin reduction is
characterized by a colour change from blue to pink, and
N-(2,6-dibromophenyl)pyrazine-2-carboxamide (3q) Yel-
lowish solid; mp. 142–144 °C. IR (KBr) υmax 3356 (N–H),
3072 (C–H aromatic), 1690 (C=O), 1493 (C=N), 689
(C–Br) cm−1; 1H NMR (CDCl3, 300 MHz): δ = 7.04 (1H, t,
J = 8.1 Hz, ArH), 7.59 (2H, d, J = 8.1 Hz, ArH), 8.58 (1H,
dd, J = 1.3 Hz, 2.5 Hz, ArH), 8.79 (1H, d, J = 2.7 Hz, ArH),

Medicinal Chemistry Research
Table 1 Yamaguchi reactions for synthesis of monosubstituted pyrazine-2-carboxamides (3a–t)
Reaction
Time
Isolated
Yield
Reaction
Time
Isolated
Yield
Entry
1
R
R’
H
Product
Entry
12
R
R’
H
Product
3a
40 minutes
44 hours
44 hours
27 hours
1 hour
57%
58%
44%
50%a
81%
3l
17 hours
6 hours
68%
2
3
4
5
H
3b
3c
3d
3e
OMe
CF3
F
13
14
3m
3n
77%b
H
H
OMe
CF3
H
H
2 hours
70%
Cl
F
Br
I
15
16
3o
3p
1 hour
26%
H
H
6
7
3f
3g
3h
1.5 hours
1 hour
68%
72%
60%
H
H
H
OMe
OMe
10 hours
58%b
Br
8
17 hours
17
18
19
3q
3r
3s
17 hours
17 hours
17 hours
20%
48%
22%
H
H
H
Br
Br
9
3i
3j
5 hours
27%
28%
H
H
O2N
F
Br
10
23 hours
F
OMe
11
3k
1 hour
66%
H
OMe
OMe
20
3t
10 hours
41% b
H
aReaction conditions: pyrazine-2-carboxylic acid (1 eq), DMAP (1.5 eq), Yamaguchi (1.5 eq), TEA (1.5 eq), and amine (1 eq)
bReaction conditions: pyrazine-2-carboxylic acid (1.5 eq), DMAP (1.5 eq), Yamaguchi (1.5 eq), TEA (1.5 eq), and amine (1 eq)
thus bacteria grow. The total volume of DMSO in solutions
in each well is less than 2%.
amines in the presence of the Yamaguchi reagent (Table 1)
[22, 23]. The preparation was carried out in one-pot reaction
under reflux conditions in the presence of 4-DMAP and
TEA. 4-DMAP was used as the preferred catalyst since it
has exceptionally high catalytic activity in acyl transfer
reactions, and can easily be removed using chromatography
[24]. Tetrahydrofuran was used as the preferred solvent due
to the insolubility of pyrazine-2-carboxylic acid in dichlor-
omethane. Alcohol solvents, such as methanol and ethanol,
were avoided since they can undergo esterification under
Results and discussion
Chemistry
Pyrazine-2-carboxamides (3) were prepared by reacting
pyrazine-2-carboxylic acid (4) with primary and secondary

Medicinal Chemistry Research
Table 2 MIC values of pyrazine-2-carboxamide analogues (5)
these conditions. The reaction with primary and secondary
aliphatic amines and anilines substituted with electron
donating and withdrawing groups successfully gave the
pyrazine-2-carboxamide 3a–t in 20–81% yields (Table 1).
Based on the yield and reaction times in Table 1, some
general conclusions can be made: (i) primary amines give
better yields and required lesser reaction times to proceed
than anilines. For instance, preparation of 3a required
almost an hour in 57% yield, while unsubstituted N-phe-
nylpyrazine-2-carboxamide 3d need more than 24 h and
gave yield of 50%. (ii) para monosubstituted anilines pro-
ceed to give the products in higher yield and lower reaction
times than disubstituted or trisubstituted anilines. Com-
pound (3e–g), phenylpyrazine-2-carboxamides with 4-
halogen substituent on phenyl moiety, showed only
around an hour reaction, and with satisfied yields (68–81%).
Nevertheless, bromo substituent at meta position (3i) led to
dropped yield of 27%, which might be caused by bromo’s
meta position enhancing the steric effect. Steric effect took
part in the yield was also supported when ortho position of
nitro in aniline (3j) produced low yield (28%) for longer
reaction. On other side, disubstituted anilines (3p, 3q)
exhibited 10–17 h of reaction time with yields less than
60% (except for 3,5-disubstituted anilines), and reaction of
trisubstituted anilines, which were 2,4,6-trimethylaniline
(3r) and 2,6-difluoro-4-bromoaniline (3s), showed same
reaction time (17 h) with yields of 22% and 48%, respec-
tively. (iii) 3,5-Disubstituted anilines showed yields
approaching those of monosubstituted anilines, but with
longer reaction times. To illustrate, N-(3,5-dimethox-
yphenyl)pyrazine-2-carboxamide (3m) displayed excellent
yield (77%), but it took reaction time of 6 h. However, when
we used electron withdrawing group substituent (3,5-CF3)
aniline, it showed shorter reaction time (2 h) but lower yield
(70%). (iv) 3,4- and 2,6-disubstituted anilines gave poor
yields and required longer reaction times, such as 3,4-
methoxyphenyl (3p) that needed 10 h of reaction to yield
58%. (v) 2,6-Disubstituted anilines were poor nucleophiles
and gave the lowest yields as well as required the longest
reaction times. Among all disubstituted anilines, 2,6-diiso-
propylaniline (3q) performed poor reactivity with 17-h
reaction, and just obtained 20% yield.
Compound
MIC (µg/mL)
Compound
MIC (µg/mL)
3a
3b
3c
3d
3e
3f
<6.25
<6.25
>25
>25
>25
25
3l
25
3m
>25
>25
>25
25
3n
3o
3p
3q
>25
>25
>25
>25
0.25
3g
3h
3i
>25
25
3r
3s
12.5
>25
>25
3t
3j
Rifampicin
3k
their first 6-month therapy. The compounds in Table 1 were
designed in such a way to give a good representation for
structure activity studies with secondary and tertiary amides
having aliphatic and aromatic moieties with electron donating
or electron withdrawing substituents on the aromatic rings.
As seen from Table 2, majority of the compounds showed
MIC > 25 with the exception of PZA analogues are 3a and
3b with MIC of 6.25 µg/mL as well as 3i with MIC of
12.5 µg/mL. There are no general trends that can be seen
from Table 2, which can give guidance for further mod-
ifications. However, it seems that secondary amides with
benzyl substituents that have electron withdrawing groups on
the para position are useful leads for further modifications.
N-phenyl-5-chloropyrazine-2-carboxamides exhibited lower
MIC values than N-benzyl-5-chloropyrazine-2-carboxamides
[6]. However, our work showed the opposite trend where N-
phenyl-2-carboxamides exhibited higher MIC values in
comparison to N-benzyl-2-carboxamides.
Conclusions
Series PZA analogues (3a–t) were successfully synthesized
using Yamaguchi reaction. Primary non-aromatic amines
were more reactive than aromatic amines, and the presence of
substituents especially at ortho position of the anilines
reduced reactivity and yields. The antimycobacterial activity
of the synthesized pyrazine-2-carboxamides was evaluated
using in vitro against Mycobacterium tuberculosis H37Rv
strains. The best activity was showed by N-benzylpyrazine-2-
carboxamides and N-alkylpyrazine-2-carboxamide with MIC
less than 6.25 µg/mL. The compound has potentials to be
candidates as antitubercular agents.
Based on the above data, the position, type, and number
of substituents greatly affect the yield and reaction time of
the products. This can be explained by steric effects as well
as the nucleophilicity of the NH2 group of the amines.
Antitubercular assay
All the synthesized compounds were assayed in vitro against
Mycobacterium tuberculosis H37Rv with rifampicin as
reference using REMA method (Table 2) [21]. Rifampicin is
one of first-line TB drugs that are used to treat patients in
Acknowledgements FAW thanks Indonesian government for the
PMDSU Scholarship and Research Grants, and MS thanks Indonesian
government for the WCP program fund and the PDUPT Research Grant.

Medicinal Chemistry Research
Compliance with ethical standards
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Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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