Design, synthesis and biological activity of pyrazinamide derivatives for anti-Mycobacterium tuberculosis

Article abstract of DOI:10.1080/14756366.2017.1367774

A total of 11 pyrazinamide derivatives were designed and synthesised using pyrazinamide as the lead compound, which was optimised by structural modification with alkyl chains, six-membered rings, and bioisosterism, respectively. The target compounds were synthesised using pyrazinecarboxylic acid as the starting material by acylation, amidation, and alkylation, respectively. Their structures were confirmed by ¹H NMR, ¹³C NMR, HRESIMS, and elemental analysis, respectively. The bioactivities of derivatives were assayed using bacteriostatic experiment and minimum inhibitory concentration experiment. It was showed that the derivatives had good inhibitory effect on Mycobacterium tuberculosis. The biological activity of derivative 1f was the best among all compounds, its antibacterial activity was 99.6%, and the minimum inhibitory concentration was 8.0 μg/mL.

Full text of DOI:10.1080/14756366.2017.1367774

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Design, synthesis and biological activity of
pyrazinamide derivatives for anti-Mycobacterium
tuberculosis
Shiyang Zhou, Shanbin Yang & Gangliang Huang
To cite this article: Shiyang Zhou, Shanbin Yang & Gangliang Huang (2017) Design,
synthesis and biological activity of pyrazinamide derivatives for anti-Mycobacterium
tuberculosis, Journal of Enzyme Inhibition and Medicinal Chemistry, 32:1, 1183-1186, DOI:
10.1080/14756366.2017.1367774
To link to this article: http://dx.doi.org/10.1080/14756366.2017.1367774
©
2017 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
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Date: 18 September 2017, At: 03:19

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 32, NO. 1, 1183–1186
https://doi.org/10.1080/14756366.2017.1367774
SHORT COMMUNICATION
Design, synthesis and biological activity of pyrazinamide derivatives for
anti-Mycobacterium tuberculosis
a,b
a,b
a,b
Shiyang Zhou , Shanbin Yang and Gangliang Huang
a
b
College of Chemistry, Chongqing Normal University, Chongqing, China; University Bioactive Substance Engineering Research Center in
Chongqing, Chongqing Normal University, Chongqing, China
ABSTRACT
ARTICLE HISTORY
A total of 11 pyrazinamide derivatives were designed and synthesised using pyrazinamide as the lead
compound, which was optimised by structural modification with alkyl chains, six-membered rings, and bio-
isosterism, respectively. The target compounds were synthesised using pyrazinecarboxylic acid as the start-
Received 3 June 2017
Revised 25 July 2017
Accepted 1 August 2017
1
ing material by acylation, amidation, and alkylation, respectively. Their structures were confirmed by H
1
3
KEYWORDS
NMR, C NMR, HRESIMS, and elemental analysis, respectively. The bioactivities of derivatives were assayed
using bacteriostatic experiment and minimum inhibitory concentration experiment. It was showed that the
derivatives had good inhibitory effect on Mycobacterium tuberculosis. The biological activity of derivative 1f
was the best among all compounds, its antibacterial activity was 99.6%, and the minimum inhibitory con-
centration was 8.0 mg/mL.
Pyrazinamide; derivatives;
design; synthesis; biological
activity
Introduction
in the human body is 12.5 mg/mL, and it can directly kill M. tuber-
culosis in vivo when the concentration reaches 50 mg/mL. The
metabolite of pyrazinamide during antibacterial mechanism is pyr-
azinoic acid. When pyrazinamide gets into M. tuberculosis bacteria
in vivo, the pyrazinoic acid metabolite achieves the bacteriostatic
effect. In addition, it can also replace the nicotinamide to prevent
dehydrogenase interference and dehydrogenation, interfere with
M. tuberculosis on oxygen utilisation, affect the normal metabol-
ism, and result in the death of bacteria M. tuberculosis. The pyrazi-
namide has good antibacterial effect, but there are side effects,
and the main side effect is to cause liver damage. In addition, the
water/fat-solubility of pyrazinamide is not good. Our research
group designed and synthesised a new class of pyrazinamide com-
pounds that achieved better bacteriostatic effect. The structure of
Tuberculosis (TB) is a common and fatal chronic infectious disease,
and it is easy to occur in young people. Its clinical manifestations
include low fever, night sweats, weakness or emaciation, and
respiratory symptoms, such as cough and hemoptysis. The disease
is mainly caused by Mycobacterium tuberculosis infection, also from
M. bovis, M. africanum, M. canetti, M. microti, and other species of
M. tuberculosis infection, but the mycobacteria do not usually
infect healthy adults . TB usually infects the lungs, brain, lymph
system, central nervous system, urinary system, circulatory system,
bones, joints, and even some skin can also be infected. At present,
there are about 600 million people worldwide to have the Bacillus
M. tuberculosis infection, and most infected people are in the
developing world. The infection rate is the highest in Africa’s Per
1
–3
Capita, and most infected people have no corresponding symp- pyrazinamide was optimised by structure modification with alkyl
4
–6
toms for latent tuberculosis infection . With the impact of envir- chains/six membered rings and bioisosterism. The design
onmental pollution and the spread of AIDS, the incidence of TB is (Figure 1) of pyrazinamide derivatives was as follows.
increasing. The WHO announced in 1993 that TB was a global
health emergency, while the termination of TB partners proposed
the “global TB prevention program”. In 1882, scientists found that
Experimental
the pathogenic bacterium of tuberculosis for M. tuberculosis. Until
the 50s of the last century, the researchers found drugs which
could inhibit M. tuberculosis, and the TB infection control was
effective. At present, the drugs commonly used in clinical trials for
anti-tuberculosis include isoniazid, rifampicin, pyrazinamide, and
Synthetic experiments
Synthesis of pyrazine-2-carboxylic acid chloride (3)
The pyrazine carboxylic acid (12.41 g, 0.10 mol) was put into
250 mL round bottom flask, then 100 mL methylene chloride as
7
solvent and 5 drops of N,N-dimethyl formamide (DMF) used as
ethambutol . Latent TB was usually used as a single drug, while
active TB was most appropriate to take several drugs at the same catalyst for the reaction were added. The flask was placed in the
8
,9
time for reducing the risk of drug-resistant bacteria . In view of ice water bath, and a magnetic mixer was used to stir until the
the great harm of TB, it is of great significance to study new anti- reaction liquid became clarified. The thionyl chloride (29 mL,
disease drugs. Pyrazinamide has good inhibitory effect on 0.40 mol) was constantly dropped into the flask under stirring, and
M. tuberculosis at pH 5–5.5, and the inhibition of bacteria is the the drop speed rate and reaction temperature were controlled.
strongest. The minimum inhibitory concentration of pyrazinamide After the reactants were added, the reaction lasted 8 h under
CONTACT Gangliang Huang
01331, China
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
huangdoctor226@163.com; Shanbin Yang
shanbiny@126.com
College of Chemistry, Chongqing Normal University, Chongqing
4
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

1
184
S. ZHOU ET AL.
d ¼ 9.96 (1H, s, pyrazine-H), 9.38 (1H, s, –NH–), 9.10 (1H, dd,
J ¼ 7.8 Hz, pyrazine-H), 8.89 (1H, dd, J ¼ 7.8 Hz, pyrazine-H), 4.03
(2H, s, J ¼ 6.0 Hz, N–CH –), 2.39 (4H, m, –CH –), 1.43 (4H, m,
O
O
N
N
N
N
NH2
N
H
n
X1
X2
2
2
13
–
CH –), 1.30 (2H, m, –CH –); C NMR (CDCl ,75 MHz): d ¼ 161.0
2
2
3
(C¼O), 146.6, 145.0, 144.7, 144.6 (pyrazine-C), 66.1 (CH
4.2, 25.6, 24.5 (CH , CH –CH ); HRESIMS m/z (pos): 220.2760
C H N O (calcd. 220.1324); Anal. Calcd. for C H N O: C, 59.98;
2 2
, N–CH ),
5
2
2
2
Pyrazinamide
Target compounds
1
1
16
4
11 16 4
Figure 1. Design of pyrazinamide derivatives.
H, 7.32; N, 25.44. Found: C, 59.99; H, 7.31; N, 25.43.
N-(piperazin-1-ylmethyl)pyrazine-2-carboxamide (1b): white
ꢀ
1
crystal; yield 88.2%; m.p. 190–192 C; H NMR (CDCl , 300 MHz):
refluxing. The methylene chloride and excessive thionyl chloride
were removed under vacuum. The mixture was dried to get the
crude product of pyrazine formyl chloride. The crude product was
recrystallised with toluene, filtered, and dried, respectively. The
pure pyrazine-2-carboxylic acid chloride (3) was obtained as the
3
d ¼ 9.96 (1H, s, pyrazine-H), 9.38 (1H, s, –NH–), 9.10 (1H, dd,
J ¼ 7.6 Hz, pyrazine-H), 8.89 (1H, dd, J ¼ 7.6 Hz, pyrazine-H), 4.03
(
–
2H, s, J ¼ 5.9 Hz, N–CH –), 2.65 (4H, m, –CH –), 2.34 (4H, m,
2
2
1
3
CH
2
–), 1.07 (1H, s, –NH–); C NMR(CDCl
3
, 75 MHz): d ¼ 161.0
1
0
(C¼O), 146.6, 145.0, 144.7, 144.6 (pyrazine-C), 66.1 (CH , N–CH ),
white crystal .
2
2
5
4.7, 45.9 (CH
2
, CH
2
–CH
2
15 5
); HRESIMS m/z (pos): 221.2640 C10H N O
(
calcd. 221.1277); Anal. Calcd. for C H N O: C, 54.28; H, 6.83; N,
10 15 5
A general method for synthesis of compounds 4a to 4c
The pyrazine formyl chloride (14.25 g, 0.10 mol) was put into
2
31.65. Found: C, 54.29; H, 6.83; N, 31.64.
N-(thiomorpholinomethyl)pyrazine-2-carboxamide (1c): white
ꢀ
1
50 mL round bottom flask, and 16 mL (0.20 mol) bromide methyl- crystal; yield 86.3%; m.p. 201–203 C; H NMR (CDCl , 300 MHz):
3
amine and 100 mL toluene solvent were added. The reaction liquid d ¼ 9.96 (1H, s, pyrazine-H), 9.38 (1H, s, –NH–), 9.10 (1H, dd,
was magnetically stirred, and the reaction lasted 8 h under reflux- J ¼ 7.7 Hz, pyrazine-H), 8.89 (1H, dd, J ¼ 7.7 Hz, pyrazine-H),
ing. The reaction mixture was thermally filtrated and cooled. The 4.03(2H, s, J ¼ 6.0 Hz, N–CH
–), 2.73 (4H, m, –CH –), 2.54 (4H, m,
2 2
1
3
crude product was recrystallised with toluene, filtered, and dried, –CH
respectively. The pure N-(1-bromine methyl) pyrazine formamide 144.7, 144.6 (pyrazine-C), 65.4 (CH
4a) as white crystal was obtained. The general method was used CH –CH ); HRESIMS m/z (pos): 238.3090
238.0888); Anal. Calcd. for C10 OS: C, 50.40; H, 5.92; N, 23.51;
2
–); C NMR (CDCl
3
, 75 MHz): d ¼ 161.0 (C¼O), 146.6, 145.0,
, N–CH ), 57.7, 28.0 (CH
OS (calcd.
2
2
2
,
(
2
2
C H N
10 14 4
to synthesise compounds 4b–4c.
H N
14 4
1
N-(bromomethyl)pyrazine-2-carboxamide (4a): white crystal;
H
S, 13.45. Found: C, 50.41; H, 5.91; N, 23.50; S, 13.46%.
NMR (CDCl
3
, 300 MHz): d ¼ 9.96 (1H, s, pyrazine-H), 9.38 (1H, s,
N-(2-(piperidin-1-yl)ethyl)pyrazine-2-carboxamide (1d): white
ꢀ
1
–
NH–), 9.10 (1H, dd, J ¼ 8.0 Hz, pyrazine-H), 8.89 (1H, dd, J ¼ 8.0 Hz, crystal; yield 84.0%; m.p. 185–187 C; H NMR (CDCl
3
, 300 MHz):
1
3
pyrazine-H), 4.97 (2H, s, J ¼ 6.4 Hz, –CH
2
–);
3
C NMR (CDCl ,
d ¼ 9.96 (1H, s, pyrazine-H), 9.10 (1H, dd, J ¼ 7.6 Hz, pyrazine-H),
7
5 MHz): d ¼ 161.0 (C¼O), 146.0, 145.0, 144.7, 144.6 (pyrazine-C), 8.89 (1H, dd, J ¼ 7.6 Hz, pyrazine-H), 8.81 (1H, s, –NH–), 3.36 (2H, s,
4
2
5.4 (CH ); HRESIMS m/z (pos): 216.0376 C H N OBr (calcd. J ¼ 5.8 Hz, N–CH
–), 2.46 (2H, s, –CH
–), 1.37 (2H, m, –CH –); C NMR (CDCl
d ¼ 160.7 (C¼O), 146.6, 145.0, 144.7, 144.6 (pyrazine-C), 57.2 (CH
N–CH ), 56.8, 54.0, 25.9, 24.5 (CH , CH –CH ); HRESIMS m/z (pos):
O (calcd. 234.3030); Anal. Calcd. for C12 O:
–), 2.42 (4H, m, –CH
–), 1.49
2
6 6 3
2
2
2
1
3
15.7230); Anal. Calcd. for C
6
H
6
N
3
OBr: C, 33.36; H, 2.80; N, 19.45; (4H, m, –CH
2
2
3
, 75 MHz):
Br, 36.99. Found: C, 33.37; H, 2.81; N, 19.43; Br, 36.98%.
2
,
1
N-(2-bromoethyl)pyrazine-2-carboxamide (4b): white crystal;
H
2
2
2
2
NMR (CDCl
J ¼7.9 Hz, pyrazine-H), 8.98 (1H, s, –NH–), 8.89 (1H, dd, J ¼ 7.9 Hz, C, 61.52; H, 7.74; N, 23.91. Found: C, 61.53; H, 7.75; N, 23.90.
C NMR (CDCl ,
N-(2-(piperazin-1-yl)ethyl)pyrazine-2-carboxamide (1e): white
3
, 300 MHz): d ¼ 9.96 (1H, s, pyrazine-H), 9.10 (1H, dd, 234.1481 C12
H
18
N
4
18 4
H N
1
3
pyrazine-H), 3.74 (4H, s, J ¼ 6.3 Hz, –CH –);
2
3
ꢀ
1
7
4
(
5 MHz): d ¼ 160.7 (C¼O), 146.0, 145.0, 144.7, 144.6 (pyrazine-C), crystal; yield 90.4%; m.p. 192–193 C; H NMR (CDCl
3
, 300 MHz):
6.6, 30.9 (CH CH ); HRESIMS m/z (pos): 230.0653 C H N OBr d ¼ 9.96 (1H, s, pyrazine-H), 9.10 (1H, dd, J ¼ 7.7 Hz, pyrazine-H),
2
2
7 8 3
calcd. 229.3426); Anal. Calcd. for C
8.26; Br, 34.73. Found: C, 36.55; H, 3.52; N, 18.26; Br, 34.72%.
N-(3-bromopropyl)pyrazine-2-carboxamide (4c): white crystal; H (4H, m, –CH
NMR (CDCl
, 300 MHz): d ¼ 9.96 (1H, s, pyrazine-H), 9.10 (1H, dd, d ¼ 160.7 (C¼O), 146.6, 145.0, 144.7, 144.6 (pyrazine-C), 57.3 (CH
), 57.2, 56.8, 53.7, 46.2 (CH , CH –CH ); HRESIMS m/z (pos):
O (calcd. 235.2910); Anal. Calcd. for C11 O:
7
H
8
N
3
OBr: C, 36.54; H, 3.51; N, 8.89 (1H, dd, J ¼ 7.7 Hz, pyrazine-H), 8.81 (1H, s, –NH–), 3.36 (2H, s,
J ¼ 5.9 Hz, N–CH –), 2.65 (4H, m, –CH –), 2.46 (2H, s, –CH –), 2.34
–), 1.07 (1H, s, –NH , 75 MHz):
1
2
2
2
1
13
2
2 3
–); C NMR (CDCl
2
,
3
J ¼7.8 Hz, pyrazine-H), 8.98 (1H, s, –NH–), 8.89 (1H, dd, J ¼7.8 Hz, N–CH
2
2
2
2
pyrazine-H), 3.51 (2H, m, J ¼ 6.0 Hz, –CH –), 3.18 (2H, m, J ¼ 6.0 Hz, 235.1433 C11
H
17
N
5
17 5
H N
2
1
3
–
CH
2
–), 2.05 (2H, m, J ¼ 6.0 Hz, –CH
2 3
–); C NMR (CDCl
, 75 MHz): C, 56.15; H, 7.28; N, 29.77. Found: C, 56.16; H,7.27; N, 29.78.
d ¼ 160.7 (C¼O, 146.0, 145.0, 144.7, 144.6 (pyrazine-C), 38.4, 32.6,
N-(2-morpholinoethyl)pyrazine-2-carboxamide (1f): white crystal;
ꢀ
1
3
0.8 (CH CH CH ); HR-ESI-MS m/z (pos): 244.0921 C H N OBr yield 91.2%; m.p. 202–204 C; H NMR (CDCl
3
, 300 MHz): d ¼ 9.96
OBr: C, 39.37; H, 4.13; N, (1H, s, pyrazine-H), 9.10 (1H, dd, J ¼ 7.8 Hz, pyrazine-H), 8.89 (1H,
–),
.36 (2H, s, J ¼ 6.0 Hz, N–CH –), 2.46 (2H, s, –CH –), 2.40 (4H, m,
2
2
2
8 10 3
(calcd. 243.5438); Anal. Calcd. for C
8 10 3
H N
1
7.22; Br, 32.74. Found: C, 39.38; H, 4.14; N, 17.21; Br, 32.73%.
dd, J ¼ 7.8 Hz, pyrazine-H), 8.81 (1H, s, –NH–), 3.50 (4H, m, –CH
2
3
2
2
1
3
–CH
2
–); C NMR (CDCl
3
, 75 MHz): d ¼ 160.7 (C¼O), 146.6, 145.0,
A general method for all titled derivatives 1a–1k
2 2 2
144.7, 144.6 (pyrazine-C), 66.7 (CH , N–CH ), 57.2, 55.8, 54.0 (CH ,
N-(1-bromine methyl) pyrazine formamide (21.60 g, 0.10 mol) was CH –CH ); HRESIMS m/z (pos): 236.2750 C H N O (calcd.
2
2
11 16 4 2
put into 250 mL round-bottomed flask, and 21.5mL (0.20 mol) six- 236.1273); Anal. calcd for C H N O C, 55.92; H, 6.83; N, 23.71.
1
1 16 4 2:
membered ring compound and 100 mL toluene were added. The Found: C, 55.91; H, 6.84; N, 23.70.
reaction liquid was magnetically stirred, and the reaction lasted 12 h N-(2-thiomorpholinoethyl)pyrazine-2-carboxamide (1g): white
under refluxing. The reaction mixture was filtrated and cooled. The crystal; yield 90.4%; m.p. 192–193 C; H NMR (CDCl
crude product was recrystallised with toluene, filtered, and dried, d ¼ 9.96 (1H, s, pyrazine-H), 9.10 (1H, dd, J ¼ 7.7 Hz, pyrazine-H),
respectively. The target (1) as white crystal was obtained.
8.89 (1H, dd, J ¼ 7.7 Hz, pyrazine-H), 8.81 (1H, s, –NH–), 3.36 (2H, s,
N-(piperidin-1-ylmethyl)pyrazine-2-carboxamide (1a): white crys- J ¼ 5.9 Hz, N–CH –), 2.73 (4H, m, –CH –), 2.54 (4H, m, –CH –), 2.46
, 75 MHz): d ¼ 160.7 (C¼O), 146.6,
ꢀ
1
3
, 300 MHz):
2
2
2
ꢀ
1
13
3 2 3
tal; yield 86.2%; m.p. 183–185 C; H NMR (CDCl , 300 MHz): (2H, s, –CH –); C NMR (CDCl

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1185
1
45.0, 144.7, 144.6 (pyrazine-C), 60.3 (CH
2
, N–CH
2
), 57.2, 53.3, 28.3 d ¼ 160.7 (C¼O), 146.6, 145.0, 144.7, 144.6 (pyrazine-C), 60.6 (CH
2
,
(CH , CH –CH ); HRESIMS m/z (pos): 252.3360 C H N OS (calcd. N–CH ), 51.2, 39.7, 28.3, 27.0 (CH , CH –CH ); HRESIMS m/z (pos):
2
2
2
11 16
4
2
2
2
2
2
52.1045); Anal. Calcd. for C H N OS: C, 52.36; H, 6.39; N, 22.20; 266.3630 C H N OS (calcd. 266.1201); Anal. Calcd. for
11 16 4
12 18 4
S, 12.71. Found: C, 52.35; H, 6.40; N, 22.18; S, 12.72%.
12 18 4
C H N OS: C, 54.11; H, 6.81; N, 21.03; S, 12.04. Found: C, 54.10;
N-(3-(piperidin-1-yl)propyl)pyrazine-2-carboxamide (1h): white H, 6.82; N, 21.04; S, 12.04%.
ꢀ
1
crystal; yield 81.0%; m.p. 204–205 C; H NMR (CDCl
d ¼ 9.96 (1H, s, pyrazine-H), 9.10 (1H, dd, J ¼ 7.5 Hz, pyrazine-H),
.98 (1H, s, –NH–), 8.89 (1H, dd, J ¼ 7.5 Hz, pyrazine-H), 3.18 (2H, s,
3
, 300 MHz):
Biological activity experiments
8
J ¼ 5.8 Hz, N–CH –), 2.42 (4H, m, –CH –), 2.36 (2H, s, –CH –), 1.73
2
2
2
TB bacillus strain was the standard strain H Rv. The modified
3
7
1
3
(
2H, s, –CH –), 1.49 (4H, m, –CH –), 1.37 (2H, m, –CH –);
C
2
2
2
roche medium was used for bacteriostatic experiment, and the
MH agar culture medium was used for the experiment of minimal
inhibitory concentration (MIC). The standard strains were dissolved
in sterile distilled water, and the bacteria liquid concentration was
NMR(CDCl
3
, 75 MHz): d ¼ 160.7 (C¼O), 146.6, 145.0, 144.7, 144.6
, N–CH ), 40.4, 39.7, 27.0, 25.9, 24.5 (CH
); HRESIMS m/z (pos): 248.3300 (calcd.
48.1637); Anal. Calcd. for C13 O: C, 62.88; H, 8.12; N, 22.56.
Found: C, 62.89; H, 8.13; N, 22.55.
N-(3-(piperazin-1-yl)propyl)pyrazine-2-carboxamide (1i): white
(pyrazine-C), 57.1 (CH
2
2
2
,
CH –CH
2
2
13 20 4
C H N O
2
H
20
N
4
3
adjusted to 10 CFU/mL. The 0.1 mL bacteria were accurately ino-
culated by pipetting gun on modified roche medium, then pyrazi-
namide derivative (0.1 mL, 25 mg/mL) was put into culture
medium. Each compound was inoculated in five culture tubes,
ꢀ
1
crystal; yield 84.6%; m.p. 210–211 C; H NMR (CDCl
d ¼ 9.96 (1H, s, pyrazine-H), 9.10 (1H, dd, J ¼ 7.6 Hz, pyrazine-H),
.98 (1H, s, –NH–), 8.89 (1H, dd, J ¼ 7.6 Hz, pyrazine-H), 3.18 (2H, s,
–), 2.65 (4H, m, –CH –), 2.36 (2H, s, –CH –), 2.34
–), 1.73 (2H, s, –CH –), 1.07 (2H, s, –CH
, 75 MHz): d ¼ 160.7 (C¼O), 146.6, 145.0, 144.7, 144.6 (pyra-
3
, 300 MHz):
respectively. The culture tubes were put into an incubator with
8
ꢀ
5% CO
2
at 36 C for 20 days, and the wedding difference of coli
J ¼ 5.7 Hz, N–CH
2
2
2
growth was observed. Blank control experiment did not need to
take drug, and directly inoculated with the microorganisms. For
minimum bacteriostatic concentration experiment, various deriva-
tives were mixed into different concentration solutions firstly, then
they were moved into MH agar medium, respectively. After steril-
isation, 0.1 mL standard H37Rv bacteria liquid with multi-point
1
3
(
(
4H, m, –CH
CDCl
2
2
2
–); C NMR
3
zine-C), 57.6 (CH , N–CH ), 51.6, 46.2, 39.7, 27.0 (CH , CH –CH );
2
2
2
2
2
HRESIMS m/z (pos): 249.3180 C H N O (calcd. 249.1590); Anal.
Calcd. for C H N O: C,57.81; H, 7.68; N, 28.09. Found: C, 57.80; H,
1
2 19 5
1
2 19 5
7
.69; N, 28.10.
N-(3-morpholinopropyl)pyrazine-2-carboxamide (1j): white crys-
inoculator was inoculated in the agar plate surface. Vaccination
ꢀ
was added into the incubator with 5% CO
2
at 36 C for 24 h, and
ꢀ
1
tal; yield 87.3%; m.p. 221–223 C; H NMR (CDCl , 300 MHz):
3
the growth differences of n/med Tuberculosis bacili were observed.
d ¼ 9.96 (1H, s, pyrazine-H), 9.10 (1H, dd, J ¼ 7.7 Hz, pyrazine-H),
8
.98 (1H, s, –NH–), 8.89 (1H, dd, J ¼ 7.7 Hz, pyrazine-H), 3.55 (4H,
m, –CH –), 3.18 (2H, s, J ¼ 5.7 Hz, N–CH –), 2.37 (2H, s, –CH –), 2.33 Results and discussion
2
2
2
1
3
(4H, m, –CH
2
–), 1.73 (2H, s, –CH
2
–); C NMR (CDCl
3
, 75 MHz):
The design and synthesis of pyrazinamide derivatives
d ¼ 160.7 (C¼O), 146.6, 145.0, 144.7, 144.6 (pyrazine-C), 62.9 (CH
N–CH ), 59.4, 39.7, 27.0 (CH , CH –CH
); HRESIMS m/z (pos): The pyrazinamide derivatives for anti-Mycobacterium tuberculosis
50.3020
(calcd. 250.1430); Anal. Calcd. for bacilli were designed using the pyrazinamide as lead compound,
2
,
2
2
2
2
2
12 18 4 2
C H N O
C
12
H
18
N
4
O
2
: C, 57.58; H, 7.25; N, 22.38. Found: C, 57.59; H, 7.24; N, which was modified with the alkyl chains/six-member rings and
bioisosterism. In the structure transformation with alkyl chains, the
2
2.37.
N-(3-thiomorpholinopropyl)pyrazine-2-carboxamide (1k): white number of carbon atom was 1, 2, and 3, respectively. In the struc-
ꢀ
1
crystal; yield 84.3%; m.p. 230–231 C; H NMR (CDCl
d ¼ 9.96 (1H, s, pyrazine-H), 9.10 (1H, dd, J ¼ 7.6 Hz, pyrazine-H), NH, O, S were selected, respectively. Through the optimisation of
.98 (1H, s, –NH–), 8.89 (1H, dd, J ¼ 7.6 Hz, pyrazine-H), 3.18 (2H, s, lead compound structure, 20 compounds were designed. Using
J ¼ 5.6 Hz, N–CH –), 2.73 (4H, m, –CH –), 2.54 (4H, m, –CH
–), 2.36 pyrazinecarboxylic acid as the starting material, the pyrazinamide
2H, s, –CH –), 1.73 (2H, s, –CH –); C NMR (CDCl , 75 MHz): derivatives were synthesised by acylation, amidation, and
3
, 300 MHz): ture transformation with six-member rings, the X
1
¼ N; X
2
¼ CH
2
,
8
2
2
2
1
3
(
2
2
3
Figure 2. Synthetic route of pyrazinamide derivatives.

1
186
S. ZHOU ET AL.
Table 1. Biological activities of pyrazinamide derivatives (0.1 mL, 25 lg/mL).
Disclosure statement
Bacteriostatic
rate (%)
No potential conflict of interest was reported by the authors.
Compounds
n X
1
X
2
MIC (lg/mL)
1
1
1
1
1
1
1
1
1
1
1
a
b
c
d
e
f
g
h
i
1 N CH
1 N NH
1 N S
2
80.1
86.7
88.4
85.9
87.8
99.6
93.6
89.8
90.7
92.0
91.3
99.2
27.3
23.4
20.2
15.4
12.2
8.0
10.2
21.5
18.3
16.2
17.1
12.5
Funding
2 N CH
2 N NH
2 N O
2 N S
3 N CH
3 N NH
3 N O
3 N S
–
2
The project was sponsored by the Scientific Research Foundation
for the Returned Overseas Chinese Scholars, State Education
Ministry. The work was also supported by Chongqing Scientific &
Technological Innovation Special Project of Social Undertakings
and People's Livelihood Guarantee [No. cstc2015shmszx80060],
Chongqing Key Research Project of Basic Science & Frontier
Technology [No. cstc2017jcyjBX0012], Scientific and Technological
Research Program of Chongqing Municipal Education Commission
2
j
k
Pyrazinamide
[
Grant No. KJ1400523], Foundation Project of Chongqing Normal
alkylation, respectively. This synthetic route (Figure 2) was simple,
and the yield was higher (79.6%–91.2%). It indicated that the
length of alkyl chain did not obviously affect the reaction yield,
but the six-member ring type more obviously influenced the yield.
University [No. 14XYY020], Chongqing General Research Project of
Basic Science & Frontier Technology [No. cstc2015jcyjA10054],
Chongqing Normal University Postgraduate’s Research and
Innovation Project [No. YKC17004], Open Foundation Project of
University Engineering Research Center for Bioactive Substances in
In general, the production rate with six-member ring (X
1
¼ N and
X
2
¼ O) was obviously higher than other combinations. In addition,
it was showed that the chemical shift of –NH(CH ) – changed Chongqing [No. AS201614], Chongqing University Students’
2
n
from compound 4 to target product 1. So, the synthesis was Training Project of Innovation & Undertaking [No. 201610637076],
successful.
China.
Biological activities
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Conclusion
A total of 11 pyrazinamide derivatives were designed and synthes-
ised using pyrazinamide as the lead compound, which was modi-
fied with the alkyl chains/six-member rings and bioisosterism,
respectively. The chemical structures of these derivatives were
characterised by H NMR, C NMR, MS, and elemental analysis,
respectively. These derivatives had been carried out the assay of
biological activity. It showed that some derivatives had good func-
tion of resisting M. tuberculosis, high bacteriostatic rates, and low
minimum bacteriostatic concentrations. In addition, the derivative
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13
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