Synthesis, antibacterial evaluation and molecular docking study of nitazoxanide analogues

Article abstract of DOI:10.14233/ajchem.2014.16038

A series of nitazoxanide analogues were synthesized and characterized by means of 1H NMR, ¹³C NMR and HR-MS. The antibacterial activities of synthesized compounds against Clostridium difficile were evaluated and 1d was the most promising compound. The preliminary results showed that compounds containing nitro-substituted thiazole ring displayed notable activities. Compounds that thiazole moiety replaced by a pyrimidine ring exhibited moderate activities. Molecular docking study of nitazoxanide and 1d with pyruvate ferredoxin oxidoreductase suggested that the nitro group interacts with thiamine pyrophosphate and surrounding amino acids, which were almost same compared with pyruvate. The results revealed that nitazoxanide may be a competitive inhibitor of pyruvate and nitro-group is necessary for thiazolide's activities against anaerobic organisms containing pyruvate ferredoxin oxidoreductase enzyme.

Full text of DOI:10.14233/ajchem.2014.16038

Asian Journal of Chemistry; Vol. 26, No. 10 (2014), 2921-2926
ASIAN JOURNAL OF CHEMISTRY
http://dx.doi.org/10.14233/ajchem.2014.16038
Synthesis, Antibacterial Evaluation and Molecular Docking Study of Nitazoxanide Analogues
*
X.W. LIU, H. ZHANG, Y.J. YANG, J.Y. LI , B. LI, X.Z. ZHOU and J.Y. ZHANG
Key Laboratory of New Animal Drug Project, Gansu Province; Key Laboratory of Veterinary Pharmaceutical Development, Ministry of
Agriculture; Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS, Lanzhou 730050, P. R. China
*Corresponding author: Tel/Fax: +86 931 2115290; E-mail: lijy1971@163.com; xiwangliu@126.com
Received: 11 July 2013;
Accepted: 21 August 2013;
Published online: 10 May 2014;
AJC-15149
A series of nitazoxanide analogues were synthesized and characterized by means of 1H NMR, 13C NMR and HR-MS. The antibacterial
activities of synthesized compounds against Clostridium difficile were evaluated and 1d was the most promising compound. The preliminary
results showed that compounds containing nitro-substituted thiazole ring displayed notable activities. Compounds that thiazole moiety
replaced by a pyrimidine ring exhibited moderate activities. Molecular docking study of nitazoxanide and 1d with pyruvate ferredoxin
oxidoreductase suggested that the nitro group interacts with thiamine pyrophosphate and surrounding amino acids, which were almost
same compared with pyruvate. The results revealed that nitazoxanide may be a competitive inhibitor of pyruvate and nitro-group is
necessary for thiazolide's activities against anaerobic organisms containing pyruvate ferredoxin oxidoreductase enzyme.
Keywords: Nitazoxanide analogues, Synthesis, Antibacterial activity, Molecular docking.
INTRODUCTION
N
NO2
OH
OAc O
N
Parasitic and bacterial infections are the major cause of
morbidity and mortality in the world especially in developing
countries. Diseases caused by intestinal parasites affect billions
of people every year and there have been few drug innovations
for treating these infections in the past three decades. Fortu-
nately, nitazoxanide (Fig. 1) belongs to nitrothiazole analogue
was developed as a promising compound to treat these diseases.
Nitazoxanide, 2-acetyloloxy-N-(5-nitro-2-thiazolyl)benzamide
was first synthesized by Rossignol and Cavier1 and has shown
activities against Taenia saginata and Hymenolepis nana2.
Further studies reported that nitazoxanide exhibited an
unusually broad spectrum of activities against various parasites
in human being and animals3-5. In 1996, nitazoxanide has been
marketed in Latin America and then approved by the US food
and drug administration as an agent treating infections caused
by parasites6,7. Nitazoxanide also showed broad anti-microbial
action against anaerobic bacteria (Clostridium difficile,
Helicobacter pylori) as well as notable activities against
influenza virus and hepatitis virus8-11. For highly pathogenic
virus rotavirus and mycobacterium tuberculosis, studies have
found that nitazoxanide can kill the virus or cure relative
N
NO2
H3C
S
N
H
Nitazoxanide (
1
)
Metronidazole (2)
Nitrofurantioin (
3)
Benznidazole (4)
Fig. 1. Nitro heterocyclic drugs
reveal that nitazoxanide inhibits pyruvate ferredoxin oxido-
reductase, a key enzyme of central intermediary metabolism
in anaerobic organisms. Nitazoxanide appears to interact with
pyruvate ferredoxin oxidoreductase and the nitro group is not
metabolically reduced. The mechanism was supposed that
the amide anion (nitazoxanide ions) may directly couple with
thiamine pyrophosphate, which is a cofactor of pyruvate
ferredoxin oxidoreductase (Fig. 2)15. Recently, a number of
nitazoxanide derivatives have been synthesized and their
activities were also investigated16-17. The results demonstrated
that the nitro group may be essential for the antimicrobial
efficacy with a minimum inhibitory concentration of organisms
diseases12,13
.
In contrast to other nitro-drugs like metronidazole,
nitazoxanide has been shown to be non-mutagenic, which
suggested that the two class compounds have fundamental
differences with the mode of action14. In fact, recent studies

2922 Liu et al.
Asian J. Chem.
(MIC90) of 0.06-4 mg/L18. Meanwhile, several analogues dis-
the reactions was monitored by TLC and column chromato-
graphy purification was carried out using silica gel. Melting
points were determined by XT-4 melting point apparatus. 1H
and 13C NMR spectrum was recorded on a Varian 400 NMR
spectrometer at room temperature operating at 400 MHz for
1H NMR and 100 MHz for 13C NMR by using DMSO-d6 as
solvent. Mass spectra were obtained at Bruker Daltonics
APEXTM 4.7T ESI/MS. The C. difficile (ATCC 43593) and
culture medium were purchased from China Center of
Industrial Culture Collection.
General procedure of preparation of nitazoxanide
analogues: To an ice cool solution of amino-heterocycle in
distilled tetrahydrofuran (with 1.1 eq triethylamine) or pyridine
was added an equimolar amount of benzoyl chloride with
stirring.After the addition was complete, the reaction mixture
was allowed to stand at room temperature and stirred overnight.
The reaction was judged complete by TLC analysis. The crude
product that separated on dilution was filtered, washed with
10 % NaHCO3 solution, then several times with water. The
pure compound was obtained by gradient column chromato-
graphy. While for circumstances which dilution does not give
precipitate, then the mixture was extracted by EtOAc and
purified by chromatography. Spectral data of novel compounds
were listed as following.
played more potent antiprotozoal and antivirus activities19-22
.
But notably, specific replacements of the nitro group shown
significant activities against intracellular parasites while
invalidity for intestinal parasite Giardia lamblia23. On the other
hand, modifications of the salicylate moiety may reduce the
activity even abrogate it24. This indicated that other enzymes
beside pyruvate ferredoxin oxidoreductase might also be
relevant.
Glucose
A
Pyruvate
Fd or FId
CoA
CO2
Hydrogenase
(H2) or
(Reduced)
PFOR
Reductases
(NADPH)
Fd or FId
(Oxidized)
Acetyl-CoA
B
N-(5-Chlorothiazol-2-yl)-2,4-difluorobenzamide (2a):
Obtained in 56 % yield as white solid; m.p. 155-156 °C; 1H-
NMR (400 MHz, DMSO-d6): δ 12.91(s, 1H), 7.85 (td, 1H, J =
8.3, 6.5 Hz), 7.62 (s, 1H), 7.48 (ddd, 1H, J = 10.9, 9.5, 2.4
Hz), 7.32-7.22 (m, 1H); 13C NMR (100 MHz, DMSO-d6): δ
164.2 (dd, JCF = 252, 12.3 Hz), 162.2, 160.2 (dd, JCF = 255,
13.1 Hz),155.7, 135.9, 132.3 (dd, JCF = 10.7, 3.4 Hz), 118.7,
118.4 (dd, JCF = 13.3, 3.5 Hz), 112 (dd, JCF = 21.8, 3.5 Hz), 105
(t, JCF = 26.1 Hz). HR-MS (ESI): Calcd. for C10H5N2OSClF2
(M + H): 274.9852; Found: 274.9846.
N-(5-Chlorothiazol-2-yl)-3,4-difluorobenzamide (2b):
Obtained in 51 % yield as white solid; m.p. 196-197 °C; 1H
NMR (400 MHz, DMSO-d6): δ 12.99 (s, 1H), 8.18 (ddd, 1H,
J = 11.5, 7.7, 2.1 Hz), 8.04-7.94 (m, 1H), 7.71-7.52 (m, 2H);
13C NMR (100 MHz, DMSO-d6): δ 163.3, 156.4, 152.2 (dd,
JCF = 253, 12.6 Hz), 149.2 (dd, JCF = 247, 13 Hz), 135.8, 128.8,
126 (dd, JCF = 7.5, 3.3 Hz), 118.7, 117.8 (t, JCF = 19.3 Hz).
HR-MS (ESI): Calcd. for C10H5N2OSClF2 (M + H): 274.9852;
Found: 274.9849.
N-(5-Chlorothiazol-2-yl)-2-(trifluoromethyl)benzamide
(2c): Obtained in 54 % yield as white solid; m.p. 207-208 °C;
1H NMR (400 MHz, DMSO-d6): δ 13.10 (s, 1H), 7.89 (d, 1H,
J = 7.5 Hz), 7.85-7.73 (m, 3H), 7.61 (s, 1H); 13C NMR (100
MHz, DMSO-d6): δ 165.8, 155.6, 135.9, 132.9, 132.6, 131.1,
129, 126.5 (d, JCF = 4.6 Hz), 126.2 (q, JCF = 31.7 Hz), 123.5 (q,
JCF = 274 Hz ), 118.8. HR-MS (ESI): Calcd. for C11H6N2OSClF3
(M + H): 306.9914; Found: 306.9920
N-(5-Chlorothiazol-2-yl)-4-(trifluoromethyl)benzamide
(2d): Obtained in 36 % yield as white solid; m.p. 189-190 °C;
1H NMR (400 MHz, DMSO-d6): δ 13.18 (s, 1H), 8.27 (d, 2H,
J = 8.1 Hz), 7.94 (d, 2H, J = 8.3 Hz), 7.66 (s, 1H); 13C NMR
(100 MHz, DMSO-d6): δ 164.4, 156.4, 135.9, 135.3, 132.3
(q, JCF = 32.1 Hz), 129.2, 125.6 (d, JCF = 3.7 Hz), 123.7 (q, JCF
= 273 Hz), 118.8. HR-MS (ESI): Calcd. for C11H6N2OSClF3
(M + H): 306.9914; Found: 306.9923
Two 1e– transfer
Acetyl-CoA
Fig. 2. Mechanism of pyruvate ferredoxin oxidoreductase enzymatic
reaction
Owing to the structural specificity and notably efficacy,
we hypothesized that modifications of the benzene ring and
thiazole moiety may result increased bioactivities. In this work,
the benzene ring hydrogen and nitro-group were substituted
by halides, part of the compounds contained a pyrimidine ring
instead of thiazole moiety. Actually, all the electron-withdra-
wing substituent can lead easily to the formation of nitazo-
xanide ions which can act on thiamine pyrophosphate causing
the inhibition of pyruvate ferredoxin oxidoreductase. The
antibacterial activities of these compounds are evaluated as a
preliminary screening to provide some information about their
antiparasitic efficiency, which is the final purpose of this
project. In addition, a molecular docking study of nitazoxanide
with pyruvate ferredoxin oxidoreductase was firstly established
using discovery studio software package. The results of mole-
cular docking supported the previous mechanism that Nitro
group interacts with the 4-amino group of thiamine pyrophos-
phate and surrounding amino acids.
EXPERIMENTAL
All chemicals employed in this study were analytical
reagents and solvents were distilled before use. The course of

Vol. 26, No. 10 (2014)
Synthesis, Antibacterial Evaluation and Molecular Docking Study of Nitazoxanide Analogues 2923
N-(5-Bromothiazol-2-yl)-2,4-difluorobenzamide (3a):
Obtained in 68 % yield as white solid; m.p. 188-190 °C; 1H
NMR (400 MHz, DMSO-d6): δ 12.93 (s, 1H), 7.86 (td, 1H, J
= 8.5, 6.5 Hz), 7.67 (s, 1H), 7.47 (ddd, 1H, J = 10.9, 9.5, 2.4
Hz), 7.30-7.23 (m, 1H); 13C NMR (100 MHz, DMSO-d6): δ
164.2 (dd, JCF = 252, 12.4 Hz), 162.2, 160.2 (dd, JCF = 255,
13.2 Hz), 158.1, 138.9, 132.3 (d, JCF = 10 Hz), 118.4 (dd, JCF
= 13.4, 3.6 Hz), 112 (d, JCF = 21.8 Hz), 105 (t, JCF = 26.2 Hz),
102.3. HR-MS (ESI): Calcd. for C10H5N2OSBrF2 (M + H):
318.9347; Found: 318.9352.
N-(5-Chloropyrimidin-2-yl)-3,4-difluorobenzamide
(5b): Obtained in 61 % yield as white solid; m.p. 149-150 °C;
1H NMR (400 MHz, DMSO-d6): δ 11.34 (s, 1H), 8.87 (m,
2H), 8.04 (ddd,1H, J = 11.4, 7.8, 2.2 Hz), 7.91-7.82 (m, 1H),
7.60 (dt, 1H, J = 10.4, 8.4 Hz); 13C NMR (100 MHz, DMSO-
d6): δ 163.2, 156.7, 156.2, 151.9 (dd, JCF = 252, 12.7 Hz), 149
(dd, JCF = 247, 12.6 Hz), 131.3 (dd, JCF = 4.8, 3.6 Hz), 126
(dd, JCF = 7.6, 3.4 Hz), 125.3, 117.7 (dd, JCF = 17.9, 14.9 Hz).
HR-MS (ESI): Calcd. for C11H6N3OClF2 (M + H): 270.0240;
Found: 270.0241.
N-(5-Bromothiazol-2-yl)-3,4-difluorobenzamide (3b):
Obtained in 60 % yield as white solid; m.p. 196-197 °C; 1H
NMR (400 MHz, DMSO-d6): δ 13.03 (s, 1H), 8.25-8.10 (m,
1H), 8.05-7.95 (m, 1H), 7.70-7.60 (m, 2H); 13C NMR (100
MHz, DMSO-d6): δ 163.3, 158.8, 152.3 (dd, JCF = 253, 12.4
Hz), 149.2 (dd, JCF = 247, 13.1 Hz), 138.9, 128.8, 126.1, 117.8
(t, JCF = 18.5 Hz), 102.4. HR-MS (ESI): Calcd. for
C10H5N2OSBrF2 (M + H): 318.9347; Found: 318.9348.
N-(5-Bromothiazol-2-yl)-2-(trifluoromethyl)benzamide
(3c): Obtained in 33 % yield as white solid; m.p. 211-213 °C;
1H NMR (400 MHz, DMSO-d6): δ 13.11 (s, 1H), 7.89 (d, 1H,
J = 7.5 Hz), 7.85-7.73 (m, 3H), 7.66 (s, 1H); 13C NMR (100
MHz, DMSO-d6): δ 165.8, 158.0, 138.9, 132.8, 132.6, 131,
129, 126.5 (q, JCF = 4.7 Hz), 126.2 (q, JCF = 31.6 Hz), 123.5 (q,
JCF = 274 Hz), 102.4. HR-MS (ESI): Calcd. for C11H6N2OSBrF3
(M + H): 350.9409; Found: 350.9419.
N-(5-Bromothiazol-2-yl)-4-(trifluoromethyl)benzamide
(3d): Obtained in 49 % yield as white solid, m.p. 202-205 °C; 1H
NMR (400 MHz, DMSO-d6): δ 13.18 (s, 1H), 8.27 (d, 2H, J = 8.2
Hz), 7.94 (d, 2H, J = 8.2 Hz), 7.70 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): δ 164.4, 158.8, 138.9, 135.3, 132.3 (q, JCF = 32.1 Hz),
129.2, 125.5, 123.7 (q, JCF = 271 Hz), 102.5. HR-MS (ESI): Calcd.
for C11H6N2OSBrF3 (M + H): 350.9409; Found: 350.9412.
N-(5-Nitropyrimidin-2-yl)-2-(trifluoromethyl)benzamide
(4c): Obtained in 54 % yield as white solid; m.p. 158-160 °C;
1H NMR (400 MHz, DMSO-d6): δ 12.09 (s, 1H), 9.40 (s, 2H),
7.86 (d, 1H, J = 7.8 Hz), 7.80-7.65 (m, 3H); 13C NMR (100
MHz, DMSO-d6): δ 166.1, 159.7, 154.8, 138.7, 135.2, 132.5,
N-(5-Chloropyrimidin-2-yl)-2-(trifluoromethyl)
benzamide (5c): Obtained in 22 % yield as white solid; m.p.
216-218 °C; 1H NMR (400 MHz, DMSO-d6): δ 11.50 (s, 1H),
8.76 (s, 2H), 7.83 (d, 1H, J = 7.7 Hz), 7.72 (dt, 2H, J = 14.6,
7 Hz), 7.63 (d, 1H, J = 7.5 Hz); 13C NMR (100 MHz, DMSO-
d6): δ 165.8, 156.6, 155.7, 135.6, 132.4, 130.0, 128.2, 126.3
(q, JCF = 4.6 Hz), 125.7 (q, JCF = 31.4 Hz), 123.7, (q, JCF
=
274 Hz). HR-MS (ESI): Calcd. for C12H7N3OClF3 (M + H):
302.0303; Found: 302.0309.
N-(5-Chloropyrimidin-2-yl)-4-(trifluoromethyl)benzamide
(5d): Obtained in 60 % yield as white solid; m.p. 170-171 °C;
1H NMR (400 MHz, DMSO-d6): δ 11.49 (s, 1H), 8.87 (s, 2H),
8.14 (d, 2H, J = 8.6 Hz), 7.90 (d, 2H, J = 8.5 Hz); 13C NMR
(100 MHz, DMSO-d6): δ 164.5, 156.7, 156.2, 137.9, 131.8
(q, JCF = 31.9 Hz), 129.2, 125.3 (d, JCF = 3.9 Hz), 123.8 (q, JCF
= 273 Hz). HR-MS (ESI): Calcd. for C12H7N3OClF3 (M + H):
302.0303; Found: 302.0296.
N-(5-Bromopyrimidin-2-yl)-2,4-difluorobenzamide
(6a): Obtained in 19 % yield as white solid; m.p. 161-163 °C;
1H NMR (400 MHz, DMSO-d6): δ 11.32 (s, 1H), 8.85 (s, 2H),
7.74 (td, 1H, J = 8.4, 6.7 Hz), 7.38 (ddd, 1H, J = 10.8, 9.5, 2.4
Hz), 7.21 (td, 1H, J = 8.5, 2.2 Hz); 13C NMR (100 MHz,
DMSO-d6): δ 163.7 (dd, JCF = 251, 12.4 Hz), 162.2, 159.8
(dd, JCF = 252, 13 Hz), 158.8, 156.1, 132 (dd, JCF = 10.5, 4.2
Hz), 121 (dd, JCF = 14.4, 3.7 Hz), 113.8, 111.8 (dd, JCF = 21.7,
3.5 Hz), 104.5 (t, JCF = 26.3 Hz). HR-MS (ESI): Calcd. for
C11H6N3OBrF2 (M + H): 313.9735; Found: 313.9737.
N-(5-Bromopyrimidin-2-yl)-3,4-difluorobenzamide
(6b): Obtained in 30 % yield as white solid; m.p. 173-174 °C;
1H NMR (400 MHz, DMSO-d6): δ 11.30 (s, 1H), 8.91 (s, 1H),
8.04 (ddd, 1H, J = 11.5, 7.8, 2.2 Hz), 7.91-7.82 (m, 1H), 7.60
(dt, 1H, J = 10.5, 8.4 Hz); 13C NMR (100 MHz, DMSO-d6): δ
163.2, 158.7, 156.5, 151.9 (dd, JCF = 252, 12.7 Hz), 149 (dd,
JCF = 247, 13 Hz), 131.3 (dd, JCF = 4.9, 3.5 Hz), 125.9, 117.6,
114. HR-MS (ESI): Calcd. for C11H6N3OBrF2 (M + H):
313.9735; Found: 313.9739.
130.4, 128.2, 126.4, 125.8 (q, JCF = 31.5 Hz), 123.7 (q, JCF
=
274 Hz). HR-MS (ESI): Calcd. for C12H7N4O3F3 (M + H):
313.0543; Found: 313.0551.
N-(5-Nitropyrimidin-2-yl)-4-(trifluoromethyl)benzamide
(4d): Obtained in 49 % yield as white solid; m.p. 140-143 °C;
1H NMR (400 MHz, DMSO-d6): δ 12.04 (s, 1H), 9.49 (s, 2H),
8.16 (d, 2H, J = 8.1 Hz), 7.92 (d, 2H, J = 8.5 Hz); 13C NMR
(100 MHz, DMSO-d6), δ: 164.8, 160.5, 154.8, 138.7, 137.5,
132.1 (q, JCF = 31.9 Hz), 129.5, 125.4, 123.8 (q, JCF = 273 Hz).
HR-MS (ESI): Calcd. for C12H7N4O3F3 (M + H): 313.0543;
Found: 313.0553.
N-(5-Chloropyrimidin-2-yl)-2,4-difluorobenzamide
(5a): Obtained in 31 % yield as white solid; m.p. 170-171 °C;
1H NMR (400 MHz, DMSO-d6): δ 11.34 (s, 1H), 8.80 (s, 2H),
7.74 (td, 1H, J = 8.4, 6.6 Hz), 7.38 (ddd, 1H, J = 10.8, 9.5, 2.4
Hz), 7.24-7.17 (m, 1H); 13C NMR (100 MHz, DMSO-d6): δ
163.7 (dd, JCF = 251, 12.4 Hz), 162.2, 159.8 (dd, JCF = 252,
13.0 Hz), 156.7, 155.9, 132.0 (d, JCF = 10.2 Hz), 125.1, 121
(dd, JCF = 14.5, 3.6 Hz), 111.8 (d, JCF = 21.6 Hz), 104.5 (t, JCF
= 26.0 Hz). HR-MS (ESI): Calcd. for C11H6N3OClF2 (M + H):
270.0240; Found: 270.0238.
N-(5-Bromopyrimidin-2-yl)-2-(trifluoromethyl)benzamide
(6c): Obtained in 23 % yield as white solid; m.p. 192-193 °C;
1H NMR (400 MHz, DMSO-d6): δ 11.47 (s, 1H), 8.81 (s, 2H),
7.82 (d, 1H, J = 7.5 Hz), 7.72 (dt, 2H, J = 15.2, 7.5 Hz), 7.63
(d, 1H, J = 7.5 Hz); 13C NMR (100 MHz, DMSO-d6): δ 165.8,
158.7, 156, 135.6, 132.4, 130, 128.2, 126.2, 125.7 (q, JCF
=
31.4 Hz), 123.7 (q, JCF = 274 Hz), 113.8. HR-MS (ESI): Calcd.
for C12H7N3OBrF3 (M + H): 345.9797; Found: 345.9792.
N-(5-Bromopyrimidin-2-yl)-4-(trifluoromethyl)benzamide
(6d): Obtained in 62 % yield as white solid; m.p. 180-182 °C;
1H NMR (400 MHz, DMSO-d6): δ 11.45 (s, 1H), 8.93 (s, 2H),
8.14 (d, 2H, J = 8.2 Hz), 7.89 (d, 2H, J = 8.3 Hz); 13C NMR
(100 MHz, DMSO-d6): δ 164.5, 158.8, 156.5, 137.9, 131.8

2924 Liu et al.
Asian J. Chem.
(q, JCF = 31.9 Hz), 129.2, 125.3 (d, JCF = 3.7 Hz), 123.8 (q, JCF
= 273 Hz), 114.1. HR-MS (ESI): Calcd. for C12H7N3OBrF3
(M + H): 345.9797; Found: 345.9801.
prepared and optimized according to the default procedures.
As described in literatures, pyruvate was thought to be binding
to thiamine pyrophosphate and trasformed into CO2, the
interaction between pyruvate and pyruvate ferredoxin oxido-
reductase was analyzed without any additional treatment. The
binding site of molecular docking was settled around pyruvate
with a radius of 10 Å. After the end of molecular docking, the
pose with highest CDOCKER interaction energy (ECD) was
chosen as the most suitable pose and the types of interactions
were analyzed. Finally, the interactions between ligands and
pyruvate ferredoxin oxidoreductase were shown in 2D pictures.
Determination of MIC values against Clostridium
difficile: The antibacterial activities of synthesized compounds
were tested by agar dilution method. C. Difficile (ATCC 43593)
was incubated anaerobically overnight in chopped meat medium
(anaerobe system) from stock and it was subcultured to a new
chopped meat medium for 5 h at 37 °C. It was standardized to
an optical density of 0.1 at OD600. Analogues were then
diluted into the agar media at concentrations ranging from
0.125-16 µg/mL. Ten microliter volumes of the standardized
inoculums were delivered to the surface of the agar plates.
The plates were incubated for 18 h in an anaerobic chamber
and were read visually for growth or no growth. Anaerobic
plates containing no compound were used as controls and all
experiments were performed 3 times in triplicate.
Molecular docking study of ligands with pyruvate
ferredoxin oxidoreductase: The pdb file about the crystal
structure of pyruvate ferredoxin oxidoreductase complex with
thiamine pyrophosphate and pyruvate (2PDA) was obtained
from the RCSB protein data bank (http://www.pdb.org). The
molecular docking procedure was performed using CDOCKER
protocol for receptor-ligand interactions section of discovery
studio 3.5 (Accelrys Software Inc., San Diego, CA). In complex
2PDA, there were two same protein chains, one was deleted
as well as inorganic ions. The ligands (nitazoxanide, 1d, 2d)
and the enzyme pyruvate ferredoxin oxidoreductase were
RESULTS AND DISCUSSION
The synthesis of nitazoxanide analogues was accomplished
according to Fig. 3. The starting compounds amino-heterocycles
(head group analogues) and benzene ring derivatives (tail groups
analogues) were commercially available or prepared by using
common methods. The tail group and head group analogues
were linked in anhydrous tetrahydrofuran with triethylamine
acted as a base or in anhydrous pyridine. The final products
were purified by gradient column chromatography. The yields
of reactions were moderate since the strong electron-with-
drawing effect of halogen substituents and nitro group. The
1
structures of synthesized compounds were confirmed by H
NMR, 13C NMR and HR-MS spectroscopy.
Antibacterial activities:As shown in Table-1, the antibac-
terial efficacy of synthesized compounds against C. difficile
are expressed as minimum inhibitory concentration (MIC in
TABLE-1
MIC VALUES OF COMPOUNDS AGAINST C. difficile
O
N
X
O
N
X
S
N
H
R
N
H
N
R
1a-d, 2a-d, 3a-d
4c-d, 5a-d, 6a-d
Compound
Nitazoxanide
R[a]
X
NO2
NO2
NO2
NO2
NO2
Cl
Cl
Cl
Cl
Br
MIC[b]
cLog P[c]
2.02
2.22
2.22
2.81
2.86
2.94
2.94
3.53
3.58
3.07
3.07
3.66
3.71
Compound
R[a]
X
NO2
NO2
Cl
Cl
Cl
Cl
Br
Br
Br
MIC[b]
4
cLog P[c]
2.60
2.64
2.72
2.72
3.32
3.36
2.86
2.86
3.45
3.50
2-OAc
2,4-2F
3,4-2F
2-CF3
4-CF3
2,4-2F
3,4-2F
2-CF3
4-CF3
2,4-2F
3,4-2F
2-CF3
4-CF3
2-CF3
2-CF3
2,4-2F
3,4-2F
2-CF3
4-CF3
2,4-2F
3,4-2F
2-CF3
4-CF3
0.25
0.5
4c
4d
5a
5b
5c
5d
6a
6b
6c
6d
2
1a
1b
1c
1d
2a
2b
2c
2d
3a
3b
3c
3d
>16
>16
>16
>16
>16
>16
>16
>16
1
0.5
0.125
>16
>16
>16
>16
>16
> 16
>16
>16
Br
Br
Br
Br
Compounds with high activities are shown in bold; [a]R=H, unless otherwise noted; [b]The MIC (µg/ml) values represent the mean of three
experiments performed in triplicate, and the errors were within acceptable limits; [c]Calculated from http://www. molinspiration.com
N
X
O
N
X
X
O
N
O
X
S
H2N
H2N
THF, Et3N
or Py
S
N
N
H
N
,
R
+
R
H
Cl
+
N
R
1a-d, 2a-d, 3a-d
Fig. 3 Synthesis of nitazoxanide analogues
4c-d, 5a-d, 6a-d
N

Vol. 26, No. 10 (2014)
Synthesis, Antibacterial Evaluation and Molecular Docking Study of Nitazoxanide Analogues 2925
µg/mL). The MIC value of compound 1d is 0.125 µg/mL, which
is more potent than nitazoxanide (0.25 µg/mL). Other four
compounds (1b, 1c, 4d, 4e) containing nitro group exhibited
moderate activities. The MIC values of compounds without
nitro-group rank above 16 µg/mL. These data suggested that
the nitro group may be necessary for their outstanding anti-
bacterial effects although amino-heterocycles can partially
exert some influence.As for benzene ring, it is clear that comp-
ounds with para trifluoromethyl possess higher efficacy when
compared with ortho substitution.
Molecular docking: In most anaerobes, the oxidative
decarboxylation of pyruvate thereby form acetyl-coenzyme
A is usually catalyzed by pyruvate ferredoxin oxidoreductase.
Thus, it is a potential target for drug design against certain
anaerobic pathogens. Besides, aiming to a better understanding
of nitazoxanide's antibacterial activity and antiparasitic
activity, the molecular docking study was attempted to prove
the previous mechanism related to pyruvate ferredoxin oxido-
reductase enzyme.
The complex 2PDA was obtained from RSCB protein data
bank, which contains thiamine pyrophosphate, pyruvate and
inorganic ions. It is a crystal strucutre of homodimeric desulfo-
vibrio africanus pyruvate ferredoxin oxidoreductase and shows
the noncovalent fixation of the substrate before the catalytic
reaction25. Without any processing, the interaction between
pyruvate and pyruvate ferredoxin oxidoreductase was ana-
lyzed. The results displayed that thiamine pyrophosphate and
surrounding amino acid residues (ARG114, THR31) interacted
with pyruvate. Then the pyruvate was removed and a binding
site was set around pyruvate followed by docking study about
different ligands nitazoxanide, 1d and 2d. The results gave by
CDOCKER were analyzed and poses with the highest-ECD
were chosen as the most suitable cases. The docking results of
nitazoxanide and 1d suggested that the nitro group was inter-
acted with thiamine pyrophosphate and the surrounding amino
acids (ARG114, THR31, ASN996). Meanwhile, there was
strong π-π interaction between the benzene ring and amino
acid LYS459. While for compound 2d, which the nitro group
replaced by chlorine, the pose with highest ECD show weak
interactions with related amino acids, no interaction with
thiamine pyrophosphate. Actually, the amino acids interacted
with pyruvate were THR31, ARG114 and ASN996, which
were same when nitazoxanide and 1d set as ligand. The
interactions between pyruvate ferredoxin oxidoreductase
enzyme and lignds were shown in Figs. 4 and 5.
The interactions between nitazoxanide and pyruvate
ferredoxin oxidoreductase were almost same compared with
interactions between pyruvate and pyruvate ferredoxin oxido-
reductase, which means that nitazoxanide may be a competitive
inhibitor thus affect the energy metabolism in anaerobic
organisms. Secondly, besides π-π interaction between benzene
ring and amino acid LYS459, the interactions between pyruvate
ferredoxin oxidoreductase and ligands were all related to nitro-
group. This gives a reasonable explanation why compounds
Fig. 4. 2D pictures of interactions between pyruvate ferredoxin oxidoreductase and different ligands. A: Pyruvate; B: Nitazoxanide; C: 1d; D: 2d

2926 Liu et al.
Asian J. Chem.
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ACKNOWLEDGEMENTS
The authors thank Northwest Normal University and
Lanzhou University for the determination of NMR data and
MS spectroscopy, respectively. This study was supported by
the earmarked fund for China Agriculture Research System
(CARS-38) and Program funded by Basic Scientific Research
Funds in Central Agricultural Scientific Research Institutions
(1610322013005).