Synthesis of oxadiazolyl, pyrazolyl and thiazolyl derivatives of thiophene-2-carboxamide as antimicrobial and anti-HCV agents

Article abstract of DOI:10.2174/1874104501711010038

Introduction: Three series of pyrazole, thiazole and 1,3,4-oxadiazole, derivatives were synthesized starting from 5-amino-4-(hydrazinocarbonyl)-3- methylthiophene-2-carboxamide (2). Methods: All compounds were investigated for their preliminary antimicrob

Full text of DOI:10.2174/1874104501711010038

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The Open Medicinal Chemistry Journal, 2017, 11, 38-53
The Open Medicinal Chemistry Journal
Content list available at: www.benthamopen.com/TOMCJ/
DOI: 10.2174/1874104501711010038
RESEARCH ARTICLE
Synthesis of Oxadiazolyl, Pyrazolyl and Thiazolyl Derivatives of
Thiophene-2-Carboxamide as Antimicrobial and Anti-HCV Agents
Ola H. Rizk1,2,*, Omaima G. Shaaban1,2 and Abeer E. Abdel Wahab3
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt
2Department of Analytical and Pharmaceutical Chemistry, Faculty of Pharmacy & Drug Manufacturing, Pharos
University, Alexandria, Egypt
3Genetic Engineering and Biotechnology Research Institute (GEBRI), Mubarak City for Scientific Research and
Technology Application, Borg El-Arab, Alexandria, Egypt
Received: October 19, 2016
Revised: January 06, 2017
Accepted: January 11, 2017
Abstract:
Introduction:
Three series of pyrazole, thiazole and 1,3,4-oxadiazole, derivatives were synthesized starting from 5-amino-4-(hydrazinocarbonyl)-3-
methylthiophene-2-carboxamide (2).
Methods:
All compounds were investigated for their preliminary antimicrobial activity. They were proved to exhibit remarkable antimicrobial
activity against Pseudomonas aeruginosa with insignificant activity towards Gram positive bacterial strains and fungi.
Results:
In-vitro testing of the new compounds on hepatitis-C virus (HCV) replication in hepatocellular carcinoma cell line HepG2 infected
with the virus utilizing the reverse transcription polymerase chain reaction technique (RT-PCR) generally showed inhibition of the
replication of HCV RNA (–) strands at low concentration, while, eight compounds; 3a, 6, 7a, 7b, 9a, 9b, 10a and 11b proved to
inhibit the replication of HCV RNA (+) and (–) strands at very low concentration range 0.08-0.36 μg/mL.
Conclusion:
Compounds 7b and 11b displayed the highest anti-HCV and antimicrobial activities in this study.
Keywords: Synthesis, Thiophene-2-carboxamide, 1,3,4-Oxadiazole, Pyrazole, Antimicrobial activity, Antiviral activity.
INTRODUCTION
Hepatitis C virus (HCV) infection is a great global health issue with recent total population infection estimates of
1-3% [1, 2]. Egypt has the highest prevalence of Hepatitis C in the world, ranging from 6% to more than 40% among
different regions [3]. HCV is believed to act as a carcinogen due to prevalence of hepatocellular carcinoma in patients
infected with chronic active hepatitis. HCV causes chronic hepatic inflammation resulting in cellular disrupt which
contribute to hepatocellular carcinoma [4]. Current therapies for HCV involve a combination of the nucleoside analog
*
Address correspondence to this author at the Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria
21521, Egypt, / Department of Analytical and Pharmaceutical Chemistry, & Drug Manufacturing, Pharos University, Alexandria, 21311, Egypt, Fax:
+(203)4873273 Telephone: +(203)4871317 E-mails: olarizk@yahoo.com, omimagaber@yahoo.com
This work was presented in The XIV European Symposium on Organic Reactivity (ESOR 2013). Prague, Czech Republic, 1-6 September 2013,
poster p. 38.
1874-1045/17
2017 Bentham Open

Substituted Thiophene-2-Carboxamides as Anti-HCV and Antimicrobial Agents
The Open Medicinal Chemistry Journal, 2017, Volume 11 39
ribavirin and interferon-α [5]. This treatment regimen causes unfavorable side effects which often results in poor patient
compliance [6]. Therefore, discovery of new anti HCV agents is urgently needed.
A literature survey revealed that thiophene nucleus was proved to be a core skeleton of several biologically active
compounds [7 - 14]. Moreover, pyrazole, 1,3-thiazole and 1,3,4-oxadiazole moieties have been acknowledged to
display various biological activities as antimicrobial [15 - 18], antitumor [19 - 22] in addition to antiviral [23 - 27]
activities. Recently many research groups have paid attention for the synthesis of pyrazole, thiazole and oxadiazole due
to their interesting pharmaceutical activities [28 - 31].
Encouraged by these findings, A new series of thiophene derivatives comprising the bioactive pyrazoles, thiazoles
and 1,3,4-oxadiazoles at position 4 were synthesized and biologically evaluated.
MATERIALS AND METHODS
Chemistry
All reagents were purchased from commercial suppliers and purified by standard techniques. Melting points were
determined in open glass capillaries using Thomas-Hoover melting point apparatus. Infrared spectra (IR) were recorded
for KBr discs on Nicolet-800 FT-IR infrared spectrophotometer. NMR spectra were scanned on a Bruker 300
ultrashield spectrometer using tetramethylsilane (TMS) as internal standard and DMSO-d6 as solvent (chemical shifts
are given in d/ppm). Splitting patterns were designated as follows: s: singlet, d: doublet, t: triplet, and m: multiplet.
Mass spectra were run on a gas chromatograph/mass spectrometer Schimadzu GCMS-QP 2010 Plus (70 eV).
Microanalyses were performed at the regional Center for Mycology and Biotechnology, El Azhar University and the
found values were within ± 0.3% of the theoretical values. The reaction completion and purity of the compounds was
followed by thin layer chromatography (TLC) on silica gel-precoated aluminium sheets (Type 60 GF254; Merck;
Germany) and the spots were visualized by UV lamp at λ 254 nm for few seconds. The key intermediate 1 was prepared
by Gütschow et al. [32]
5-Amino-4-(hydrazinocarbonyl)-3-Methylthiophene-2-Carboxamide (2)
A mixture of 1 (2.28 g, 10 mmol) and hydrazine hydrate 99% (4 mL, 80 mmol) in ethanol (10 mL) was heated
under reflux for 24 h. The reaction mixture was cooled and the separated product was filtered, washed with ethanol,
dried and crystallized from ethanol.
Yield: 62%, m.p.: 228-30oC. IR (KBr, cm-1): 3387, 3296, 3222, 3115 (NH2, NH), 1657 (C=O), 1624 (C=N), 1271,
1
1017 (C-S-C). H-NMR (DMSO-d6, ppm): 2.43 (s, 3H, CH3), 4.36 (s, 2H, NH2, D2O exchangeable), 7.61 (s, 2H,
thiophene-C5-NH2, D2O exchangeable), 8.07 (s, 2H, CONH2, D2O exchangeable), 8.90 (s, 1H, NH, D2O exchangeable).
13C-NMR (DMSO-d6, ppm): 14.76 (CH3), 105.68 (thiophene C4), 111.44 (thiophene C2), 139.78 (thiophene C3), 163.62,
165.26 (2 C=O), 165.40 (thiophene C5). Anal. Calcd. (%) for C7H10N4O2S (214.24): C, 39.24; H, 4.70; N, 26.15. Found:
C, 39.52; H, 5.12; N, 26.37.
5-Amino-3-Methyl-4-[(3-Methyl-5-Substituted-1H-pyrazol-1-yl)Carbonyl]Thiophene-2-Carboxamide (3a,b)
A mixture of 2 (0.214 g, 1 mmol) and acetylacetone or benzoylacetone (1 mmol) in absolute EtOH (10 mL)
containing 3 drops glacial acetic acid was heated under reflux for 12 h. The reaction mixture was allowed to attain room
temperature and the separated product was filtered, dried and crystallized from DMF.
5-Amino-3-Methyl-4-[(3,5-Dimethyl-1H-Pyrazol-1-yl)Carbonyl]Thiophene-2-Carboxamide (3a)
Yield: 64%, m.p.: 146-8oC. IR (KBr, cm-1): 3377, 3258 (NH2), 1653 (C=O), 1587 (C=N), 1218, 1022 (C-S-C). 1H-
NMR (DMSO-d6, ppm): 2.15 (s, 3H, pyrazole-C3-CH3), 2.59 (s, 3H, CH3), 2.85 (s, 3H, pyrazole-C5-CH3), 6.12 (s, 1H,
pyrazole-C4-H), 7.93 (s, 2H, thiophene-C5-NH2, D2O exchangeable), 8.24 (s, 2H, CONH2, D2O exchangeable). Anal.
Calcd. (%) for C12H14N4O2S (278.33): C, 51.78; H, 5.07; N, 20.13. Found: C, 51.72; H, 5.47; N, 20.22.
5-Amino-3-Methyl-4-[(3-Methyl-5-Phenyl-1H-Pyrazol-1-yl) Carbonyl]Thiophene-2-Carboxamide (3b)
Yield: 68%, m.p.: 108-10oC. IR (KBr, cm-1): 3368, 3234 (NH2), 1652 (C=O), 1577 (C=N), 1218, 1021 (C-S-C). 1H-
NMR (DMSO-d6, ppm): 2.25 (s, 3H, pyrazole-C3-CH3), 2.60(s, 3H, CH3), 6.42 (s, 1H, pyrazole-C4-H), 7.26 (t, 1H, J =
7.5 Hz, phenyl-C4-H), 7.38 (t, 2H, J = 7.5 Hz, phenyl-C3&5-H), 7.73 (d, J = 7.5 Hz, 2H, phenyl-C2,6-H),7.90 (s, 2H,

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Rizk et al.
thiophene-C5-NH2, D2O exchangeable), 8.14 (s, 2H, CONH2, D2O exchangeable). Anal. Calcd. (%) for C17H16N4O2S
(340.4): C, 59.98; H, 4.74; N, 16.46. Found: C, 59.97; H, 5.07; N, 16.58.
Methyl 1-[(2-Amino-5-Carbamoyl-4-Methylthiophen-3-yl) Carbonyl]-5-Phenyl-4,5-Dihydro-1H-Pyrazole-3-
Carboxylate (4)
A mixture of 2 (0.214 g, 1 mmol) and methyl pyruvate (1 mmol) in absolute EtOH (10 mL) containing 3 drops
glacial acetic acid was refluxed for 10 h, and then cooled. The separated solid product was filtered, dried and
crystallized from DMF.
Yield: 64%, m.p.: 114-6°C. IR (KBr, cm-1): 3413, 3314 (NH2), 1662 (C=O), 1582 (C=N), 1255, 1020 (C-S-C),
1175, 1036 (C-O-C). 1H-NMR (DMSO-d6, ppm): 2.62 (s, 3H, CH3), 3.87(s, 3H, COOCH3), 6.42 (s, 1H, pyrazole-C4-H),
7.19 (t, 1H, J = 7.5 Hz, phenyl-C4-H), 7.21 (t, 2H, J = 7.5 Hz, phenyl-C3&5-H), 7.63 (d, J = 7.5 Hz, 2H, phenyl-C2,6-
H),7.82 (s, 2H, thiophene-C5-NH2, D2O exchangeable), 8.04 (s, 2H, CONH2, D2O exchangeable). Anal. Calcd. (%) for
C18H16N4O4S (384.41): C, 56.24; H, 4.20; N, 14.57. Found: C, 56.31; H, 4.52; N, 14.71.
5-Amino-4-[(3,5-Dioxopyrazolidin-1-yl) Carbonyl]-3-Methylthiophene-2-Carboxamide (5)
A mixture of 2 (0.214 g, 2.0 mmol) and diethylmalonate (3.27 g, 20 mmol, 3.1 mL) was heated under reflux for 4 h.
then left to cool. The obtained precipitate was filtered, dried and crystallized from ethanol / water (4:1).
Yield: 70%, m.p.: 210-12oC. IR (KBr, cm-1): 3386, 3239 (NH2, NH), 1658, 1675(C=O), 1607 (C=N), 1249, 1025 (C-
S-C). 1H-NMR (DMSO-d6, ppm): 1.86 (s, 2H, pyrazolidinyl-C4-H), 2.48 (s, 3H, CH3), 7.70 (s, 2H, thiophene-C5-NH2,
D2O exchangeable), 9.41(s, 2H, CONH2, D2O exchangeable), 9.71 (s, 1H, pyrazolidinyl-C2-NH, D2O exchangeable).
13C-NMR (DMSO-d6, ppm): 14.74 (CH3), 59.72 (pyrazole C4), 105.88 (thiophene C4), 110.24 (thiophene C2), 141.87
(thiophene C3), 163.50, 165.32, 165.55, 165.58 (4 C=O), 168.83 (thiophene C5). EI-MS m/z: 282 (M+ /1.03%), 57
(100%). Anal. Calcd. (%) for C10H10N4O4S (282.28): C, 42.55; H, 3.57; N, 19.85. Found: C, 42.63; H, 3.55; N, 20.08.
5-Amino-3-Methyl-4-[(3-Methyl-5-oxo-4,5-Dihydro-1H-Pyrazol-1-yl) Carbonyl]Thiophene-2-Carboxamide (6)
A mixture of 2 (0.214 g, 1 mmol) and ethylacetoacetate (1 mmol) in absolute EtOH (10 mL) containing 3 drops of
glacial acetic acid was refluxed for 10 h, and the reaction mixture was left to attain room temperature. The separated
product was filtered, dried and crystallized from ethanol.
Yield: 65%, m.p.: 208-10oC. IR (KBr, cm-1): 3378, 3272 (NH2), 1694, 1658 (C=O), 1588 (C=N), 1248, 1034 (C-S-
C). 1H-NMR (DMSO-d6, ppm): 1.85 (s, 2H, pyrazol-C4-H), 2.22 (s, 3H, pyrazole-C3-CH3), 2.48 (s, 3H, CH3), 9.85 (s,
2H, thiophene-C5-NH2, D2O exchangeable), 11.05 (s, 2H, CONH2, D2O exchangeable). Anal. Calcd. (%) for
C11H12N4O3S (280.3): C, 47.13; H, 4.32; N, 19.99. Found: C, 47.32; H, 4.37; N, 20.14.
6-Methyl-2-Substituted-5-oxo-4,5-Dihydro-1H-Thieno [2,3-e][1, 2, 4]Triazepine-7-Carboxamide (7a-c)
Compounds 7a-c were prepared by refluxing a mixture of 2 (0.214 g, 1 mmol) and the appropriate aromatic
aldehyde (1 mmol) in absolute ethanol (20 mL) for 4 h. The reaction mixture was left to attain room temperature and
the obtained products were filtered, dried and crystallized from the appropriate solvent.
6-Methyl-2-(4-Nitrophenyl)-5-oxo-4,5-Dihydro-1H-Thieno[2,3-e][1, 2, 4] Triazepine-7-Carboxamide (7a)
Yield: 74%, m.p.: 278-80oC (DMF). IR (KBr, cm-1): 3434, 3316, 3152 (NH2, NH), 1666 (C=O), 1632, 1579 (C=N),
1509, 1337 (NO2), 1267, 1025 (C-S-C). 1H-NMR (DMSO-d6, ppm): 2.61 (s, 3H, CH3), 7.78 (s, 2H, thiophene-C5-NH2,
D2O exchangeable), 7.95 (d, J = 8.9 Hz, 2H, phenyl-C2,6-H), 8.15 (s, 2H, CONH2, D2O exchangeable), 8.20 (s, 1H, NH,
D2O exchangeable), 8.26 (d, J = 8.9 Hz, 2H, phenyl-C3,5-H), 11.12 (s, 1H, NH, D2O exchangeable). 13C-NMR (DMSO-
d6, ppm): 14.75 (CH3), 106.05 (thienotriazepine C3e), 110.16 (thienotriazepine C7), 124.46 (4-nitrophenyl C3,5), 128.31
(4-nitrophenyl C2,6), 132.14 (4-nitrophenyl C1), 141.29 (thienotriazepine C6), 147.50 (thienotriazepine C2), 147.92 (4-
nitrophenyl C4), 165.06, 165.34 (2 C=O), 170.65 (thienotriazepine C2e). Anal. Calcd. (%) for C14H11N5O4S (345.33): C,
48.69; H, 3.21; N, 20.28. Found: C, 48.82; H, 3.18; N, 20.45.

Substituted Thiophene-2-Carboxamides as Anti-HCV and Antimicrobial Agents
The Open Medicinal Chemistry Journal, 2017, Volume 11 41
6-Methyl-2-(4-Methoxyphenyl)-5-Oxo-4,5-Dihydro-1H-Thieno[2,3-e][1, 2, 4] Triazepine-7-Carboxamide (7b)
Yield: 76%, m.p.: 160-2oC (ethanol). IR (KBr, cm-1): 3416, 3296, 3226 (NH2, NH), 1675 (C=O), 1603 (C=N), 1250,
1021 (C-S-C), 1167, 1117 (C-O-C). 1H-NMR (DMSO-d6, ppm): 2.59 (s, 3H, CH3), 3.79 (s, 3H, OCH3), 6.98 (d, J = 8.7
Hz, 2H, phenyl-C2,6-H), 7.65 (d, J = 8.7 Hz, 2H, phenyl-C3,5-H), 7.72 (s, 2H, thiophene-C5-NH2, D2O exchangeable),
7.98 (s, 2H, CONH2, D2O exchangeable), 8.05 (s, 1H, NH, D2O exchangeable), 11.16 (s, 1H, NH, D2O exchangeable).
13C-NMR (DMSO-d6, ppm): 14.77 (CH3), 55.73 (OCH3), 105.60 (thienotriazepine C3e), 109.16 (thienotriazepine C7),
117.34 (4-methoxyphenyl C3,5), 122.14 (4-methoxyphenyl C1), 129.49 (4-methoxyphenyl C2,6), 138.05 (thienotriazepine
C6), 146.04 (thienotriazepine C2), 158.77 (4-methoxyphenyl C4), 165.02, 165.59 (2 C=O), 170.91 (thienotriazepine C2e).
EI-MS m/z: 329 (M+ /3.81%),330 (0.06), 139 (100%). Anal. Calcd. (%) for C15H14N4O3S (330.36): C, 54.53; H, 4.27; N,
16.96. Found: C, 54.72; H, 4.46; N, 17.13.
6-Methyl-2-(3,4-Dimethoxy)-5-Oxo-4,5-Dihydro-1H-Thieno [2,3-e][1, 2, 4] Triazepine-7-Carboxamide (7c)
Yield: 76%, m.p.: 180-2oC (ethanol/water, 4:1). IR (KBr, cm-1): 3383, 3279, 3214 (NH2, NH), 1658 (C=O), 1598
1
(C=N), 1269, 1024 (C-S-C), 1166, 1055 (C-O-C). H-NMR (DMSO-d6, ppm): 2.70 (s, 3H, CH3), 3.79,3.82 (2s, each
3H, 2OCH3), 7.0 (d, J = 8.35 Hz, 1H, phenyl-C6-H), 7.18 (d, J = 8.35 Hz, 1H, phenyl-C5-H), 7.33 (s, 1H, phenyl-C2-H),
7.71(s, 2H, thiophene-C5-NH2, D2O exchangeable), 7.98 (s, 2H, CONH2, D2O exchangeable), 8.03 (s, 1H, NH, D2O
exchangeable), 11.03 (s, 1H, NH, D2O exchangeable). Anal. Calcd. (%) for C16H16N4O4S (360.39): C, 53.32; H, 4.47; N,
15.55. Found: C, 53.41; H, 4.50; N, 15.71.
5-Amino-3-Methyl-4-{[2-(Substitute Dcarbamothioyl) Hydrazino]Carbonyl}Thiophene-2-Carboxamide (9a, b)
To a suspension of 2 (0.214 g, 1 mmol) in absolute ethanol (20 ml), the appropriate isothiocyanate (1.1 mmol) was
drop wisely added over a period of 15 minutes. The reaction mixture was heated under reflux for 7 h. and left to attain
room temperature. The obtained precipitate was filtered, dried and crystallized from DMF/H2O (5:2).
5-Amino-3-Methyl-4-{[2-(Phenylcarbamothioyl) Hydrazino] Carbonyl} Thiophene-2-Carboxamide (9a)
Yield: 72%, m.p.: 205-7oC. IR (KBr, cm-1): 3380, 3268, 3170 (NH2, NH), 1653 (C=O), 1588 (C=N), 1529, 1331,
1122, 932 (NCS), 1242, 1021 (C-S-C). 1H-NMR (DMSO-d6, ppm): 2.59 (s, 3H, CH3), 6.99 (t, 1H, J = 7.6 Hz, phenyl-
C4-H), 7.34 (t, 2H, J = 7.6 Hz, phenyl-C3&5-H), 7.56 (d, J = 7.6 Hz, 2H, phenyl-C2,6-H), 7.74 (s, 1H, CSNH, D2O
exchangeable), 7.80 (s, 2H, thiophene-C5-NH2, D2O exchangeable), 7.84 (s, 1H, CONHNH, D2O exchangeable), 7.95 (s,
2H, CONH2, D2O exchangeable), 10.48 (s, 1H, CONHNH, D2O exchangeable). Anal. Calcd. (%) for C14H15N5O2S2
(349.43): C, 48.12; H, 4.33; N, 20.04. Found: C, 48.33; H, 4.61; N, 20.21.
5-Amino-3-Methyl-4-({2-[(4-Methylphenyl) Carbamothioyl] Hydrazino} Carbonyl)Thiophene-2-Carboxamide
(9b)
Yield: 76%, m.p.: 202-4oC. IR (KBr, cm-1): 3377, 3258, 3170 (NH2, NH), 1652 (C=O), 1587 (C=N), 1529, 1331,
1123, 931 (NCS), 1218, 1022 (C-S-C). 1H-NMR (DMSO-d6, ppm): 2.25 (s, 3H, phenyl-C4-CH3), 2.58 (s, 3H, CH3), 6.99
(t, 1H, J = 7.6 Hz, phenyl-C4-H), 7.34 (t, 2H, J = 7.6 Hz, phenyl-C3&5-H), 7.56 (d, J = 7.6 Hz, 2H, phenyl-C2,6-H), 7.63
(s, 1H, CSNH, D2O exchangeable), 7.83 (s, 2H, thiophene-C5-NH2, D2O exchangeable), 7.87 (s, 1H, CONHNH, D2O
exchangeable), 8.10 (s, 2H, CONH2, D2O exchangeable), 10.36 (s, 1H, CONHNH, D2O exchangeable). EI-MS m/z: 363
(M+ /1.90%), 64 (100%). Anal. Calcd. (%) for C15H17N5O2S2 (363.46): C, 49.57; H, 4.71; N, 19.27. Found: C, 49.87; H,
4.97; N, 19.53.
5-Amino-4-{[(2Z)-2-(3,4-Disubstituted-1,3-Thiazol-2(3H)-Ylidene)Hydrazino]Carbonyl}-3-Methylthiophene-2-
Carboxamide (10a-f)
A mixture of 9a or 9b (1 mmol) and the appropriate phenacyl bromide (1 mmol) in absolute ethanol (20 ml) was
heated under reflux for 20 h. Anhydrous sodium acetate (0.1 g) was then added and the reaction mixture was heated for
further 30 minutes. The reaction mixture was cooled and poured onto ice-cold water. The separated product was
filtered, washed with water and crystallized from the proper solvent.

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5-Amino-4-{[(2Z)-2-(3,4-Diphenyl-1,3-Thiazol-2(3H)-Ylidene)Hydrazino]Carbonyl}-3-Methylthiophene-2-
Carboxamide (10a)
Yield: 73%, m.p.: 195-7oC (ethanol). IR (KBr, cm-1): 3378, 3276, 3170 (NH2, NH), 1654 (C=O), 1583 (C=N), 1524,
1372, 1098, 868 (NCS), 1231, 1039 (C-S-C). 1H-NMR (DMSO-d6, ppm): 2.43 (s, 3H, CH3), 6.37 (s, 1H, thiazoline-C3-
H), 6.95-7.53 (m, 10H, 2 phenyl-H), 7.76 (s, 2H, thiophene-C5-NH2, D2O exchangeable), 7.96 (s, 2H, CONH2, D2O
exchangeable), 10.53 (s, 1H, CONH, D2O exchangeable). Anal. Calcd. (%) for C22H19N5O2S2 (449.55): C, 58.78; H,
4.26; N, 15.58. Found: C, 58.87; H, 4.30; N, 15.73.
5-Amino-4-({(2Z)-2-[4-(4-Bromophenyl)-3-Phenyl-1,3-Thiazol-2(3H)-Ylidene]Hydrazino}Carbonyl)-3-
Methylthiophene-2-Carboxamide (10b)
Yield: 76%, m.p.: 142-4oC (ethanol/water, 3:2). IR (KBr, cm-1): 3404, 3286, 3169 (NH2, NH), 1658 (C=O), 1582
(C=N), 1523, 1325, 1097, 868 (NCS), 1229, 1009 (C-S-C). 1H-NMR (DMSO-d6, ppm): 2.44 (s, 3H, CH3), 6.45 (s, 1H,
thiazoline-C3-H), 6.94 (d, J = 8.4 Hz, 2H, phenyl-C2,6-H), 7.03 (t, 1H, J = 8.4 Hz, phenyl-C4-H), 7.34 (t, 2H, J = 8.4 Hz,
phenyl-C3&5-H), 7.45 (d, J = 8.4 Hz, 2H, 4-bromophenyl-C2,6-H), 7.63 (d, J = 8.4 Hz, 2H, 4-bromophenyl-C2,6-H), 7.78
(s, 2H, thiophene-C5-NH2, D2O exchangeable), 7.97 (s, 2H, CONH2, D2O exchangeable), 10.53 (s, 1H, CONH, D2O
exchangeable). 13C-NMR (DMSO-d6, ppm): 14.71 (CH3), 105.62 (thiophene C4), 106.17 (thiazolidine C3), 108.69
(thiophene C2), 121.29 (phenyl C2,6), 122.76 (4-bromophenyl C4), 123.58 (phenyl C4), 129.58 (4-bromophenyl C1),
129.96 (4-bromophenyl C2,6), 130.11 (phenyl C3,5), 131.83 (4-bromophenyl C3,5), 138.73 (phenyl C1), 143.70 (thiophene
C3), 150.63 (thiazolidine C4), 157.05 (thiazolidine C1), 162.37, 165.18 (2 C=O), 165.93 (thiophene C5). EI-MS m/z: 528
(M+ /2.25%), 125 (100%). Anal. Calcd. (%) for C22H18BrN5O2S2 (528.44): C, 50.00; H, 3.43; N, 13.25. Found: C, 50.13;
H, 3.41; N, 13.37.
5-Amino-4-({(2Z)-2-[4-(4-Methoxyphenyl)-3-Phenyl-1,3-Thiazol-2(3H)-Ylidene]Hydrazino}Carbonyl)-3-
Methylthiophene-2-Carboxamide (10c)
Yield: 72%, m.p.: 148-50oC (ethanol). IR (KBr, cm-1): 3404, 3286, 3169 (NH2, NH), 1658 (C=O), 1582 (C=N),
1523, 1325, 1097, 868 (NCS), 1229, 1009 (C-S-C) 1167, 1117 (C-O-C). 1H-NMR (DMSO-d6, ppm): 2.45 (s, 3H, CH3),
3.81 (s, 3H, OCH3), 6.25 (s, 1H, thiazoline-C3-H), 6.94-7.45 (m, 9H, phenyl & Ar-H), 7.75 (s, 2H, thiophene-C5-NH2,
D2O exchangeable), 7.89 (s, 2H, CONH2, D2O exchangeable), 10.46 (s, 1H, CONH, D2O exchangeable). Anal. Calcd.
(%) for C23H21N5O3S2 (479.57): C, 57.60; H, 4.41; N, 14.60. Found: C, 57.78; H, 4.46; N, 14.87.
5-Amino-4-({(2Z)-2-[3-(4-Methylphenyl)-4-Phenyl-1,3-Thiazol-2(3H)-Ylidene]Hydrazino}Carbonyl)-3-
Methylthiophene-2-Carboxamide (10d)
Yield: 71%, m.p.: 203-205oC (ethanol). IR (KBr, cm-1): 3389, 3277, 3166 (NH2, NH), 1660 (C=O), 1588 (C=N),
1525, 1371, 1104, 872 (NCS), 1231, 1043 (C-S-C). 1H-NMR (DMSO-d6, ppm): 2.26 (s, 3H, phenyl CH3), 2.43 (s, 3H,
CH3), 6.42 (s, 1H, thiazoline-C3-H), 6.91-7.55 (m, 9H, phenyl & Ar-H), 7.73 (s, 2H, thiophene-C5-NH2, D2O
exchangeable), 7.86 (s, 2H, CONH2, D2O exchangeable), 10.50 (s, 1H, CONH, D2O exchangeable). Anal. Calcd. (%) for
C23H21N5O2S2 (463.58): C, 59.59; H, 4.57; N, 15.11. Found: C, 59.72; H, 4.55; N, 15.32.
5-Amino-4-({(2Z)-2-[4-(4-Bromophenyl)-3-(4-Methylphenyl)-1,3-Thiazol-2(3H)-Ylidene]Hydrazino}
Carbonyl)-3-Methylthiophene-2-Carboxamide (10e)
Yield: 73%, m.p.: 190-2oC (ethanol/water, 4:1). IR (KBr, cm-1): 3392, 3285, 3158 (NH2, NH), 1649 (C=O), 1586
(C=N), 1519, 1327, 1101, 869 (NCS), 1233, 1041 (C-S-C). 1H-NMR (DMSO-d6, ppm): 2.27 (s, 3H, phenyl CH3), 2.43
(s, 3H, CH3), 6.43 (s, 1H, thiazoline-C3-H), 6.83 (d, J = 8.2 Hz, 2H, 4-bromophenyl-C2,6-H), 7.14 (d, 2H, J = 8.2 Hz, 4-
bromophenyl-C3,5-H), 7.44 (d, 2H, J = 8.4 Hz, 4-methylphenyl-C3,5-H), 7.63 (d, J = 8.4 Hz, 2H, 4-methylphenyl-C2,6-H),
7.77 (s, 2H, thiophene-C5-NH2, D2O exchangeable), 7.96 (s, 2H, CONH2, D2O exchangeable), 10.49 (s, 1H, CONH, D2O
exchangeable). Anal. Calcd. (%) for C23H20BrN5O2S2 (542.47): C, 50.92; H, 3.72; N, 12.91. Found: C, 50.97; H, 3.74; N,
13.12.

Substituted Thiophene-2-Carboxamides as Anti-HCV and Antimicrobial Agents
The Open Medicinal Chemistry Journal, 2017, Volume 11 43
5-Amino-4-({(2Z)-2-[4-(4-Methoxyphenyl)-3-(4-Methylphenyl)-1,3-Thiazol-2(3H)-Ylidene]Hydrazino}
Carbonyl)-3 Methylthiophene-2-Carboxamide (10f)
Yield: 69%, m.p.: 184-6oC (ethanol). IR (KBr, cm-1): 3388, 3265, 3190 (NH2, NH), 1658 (C=O), 1590 (C=N), 1525,
1
1325, 1105, 888 (NCS), 1235, 1021 (C-S-C) 1170, 1112 (C-O-C). H-NMR (DMSO-d6, ppm): 2.24 (s, 3H, phenyl
CH3), 2.47 (s, 3H, CH3), 3.90 (s, 3H, OCH3), 6.34 (s, 1H, thiazoline-C3-H), 6.97-7.35 (m, 8H, 2 Ar-H), 7.76 (s, 2H,
thiophene-C5-NH2, D2O exchangeable), 7.89 (s, 2H, CONH2, D2O exchangeable), 10.49 (s, 1H, CONH, D2O
exchangeable). Anal. Calcd. (%) for C24H23N5O3S2 (493.6): C, 58.40; H, 4.70; N, 14.19. Found: C, 58.53; H, 4.68; N,
14.32.
5-Amino-3-Methyl-4-{[(2Z)-2-(4-Oxo-3-Substituted-1,3-Thiazolidin-2-ylidene)Hydrazino]Carbonyl}
Thiophene-2-Carboxamide(11a, b)
A mixture of 9a or b (1 mmol) and ethyl bromoacetate (0.2 g, 0.13 mL, 1.1 mmol) in absolute ethanol (20 mL) was
heated under reflux for 24 h. Anhydrous sodium acetate (0.1 g) was added and the reaction mixture was heated for
further 30 minutes. The reaction mixture was cooled and poured onto ice-cold water. The separated product was
filtered, washed with water and crystallized from dioxane.
5-Amino-3-Methyl-4-{[(2Z)-2-(4-Oxo-3-Phenyl-1,3-Thiazolidin-2-Ylidene)Hydrazino]Carbonyl}Thiophene-2-
Carboxamide(11a)
Yield: 58%, m.p.: 194-6 oC. IR (KBr, cm-1): 3392, 3255, 3170 (NH2, NH), 1658, 1640 (C=O), 1587 (C=N), 1535,
1326, 1105, 912 (NCS), 1227, 1059 (C-S-C). 1H-NMR (DMSO-d6, ppm): 2.60 (s, 3H, CH3), 4.78 (s, 2H, thiazolidine-
C3-H), 7.01-7.63 (m, 5H, phenyl-H), 7.68 (s, 2H, thiophene-C5-NH2, D2O exchangeable), 7.82 (s, 2H, CONH2, D2O
exchangeable), 10.36 (s, 1H, CONH, D2O exchangeable). Anal. Calcd. (%) for C16H15N5O3S2 (389.45): C, 49.34; H,
3.88; N, 17.98. Found: C, 49.68; H, 4.01; N, 18.21.
5-Amino-3-Methyl-4-({(2Z)-2-[3-(4-Methylphenyl)-4-Oxo-1,3-Thiazolidin-2-Ylidene]Hydrazino}Carbonyl)
Thiophene-2-Carboxamide (11b)
Yield: 61%, m.p.: 202-4 oC. IR (KBr, cm-1): 3386, 3245, 3175 (NH2, NH), 1670, 1642 (C=O), 1590 (C=N), 1537,
1326, 1109, 918 (NCS), 1225, 1075 (C-S-C). 1H-NMR (DMSO-d6, ppm): 2.26 (s, 3H, phenyl CH3), 2.58 (s, 3H, CH3)
4.88 (s, 2H, thiazolidine-C3-H), 7.12 (d, 2H, J = 8.4 Hz, 4-methylphenyl-C3,5-H), 7.51 (d, J = 8.4 Hz, 2H, 4-
methylphenyl-C2,6-H),7.70 (s, 2H, thiophene-C5-NH2, D2O exchangeable), 7.79 (s, 2H, CONH2, D2O exchangeable),
10.32 (s, 1H, CONH, D2O exchangeable). Anal. Calcd. (%) for C17H17N5O3S2 (403.48): C, 50.61; H, 4.25; N, 17.36;
Found: C, 50.94; H, 4.43; N, 17.63.
5-Amino-3-Methyl-4-[5-Substituted-1,3,4-Oxadiazol-2-yl] Thiophene-2-Carboxamide (12a,b)
A solution of 9a or 9b (1 mmol) in dry dioxane (10 ml) was treated with freshly prepared yellow HgO (0.26 g, 1.2
mmol). The reaction mixture was refluxed for 5 h. and filtered while hot to remove the precipitated black HgS. The
filtrate was evaporated and the remaining residue was crystallized from dioxane.
5-Amino-3-Methyl-4-[5-(Phenylamino)-1,3,4-Oxadiazol-2-yl] Thiophene-2-Carboxamide (12a)
Yield: 70%, m.p.: 232-4oC. IR (KBr, cm-1): 3396, 3252, 3167 (NH2, NH), 1683 (C=O), 1571 (C=N), 1264, 1022 (C-
1
S-C), 1124, 1073 (C-O-C). H-NMR (DMSO-d6, ppm): 2.56 (s, 3H, CH3), 7.12-7.48 (m, 5H, phenyl-H), 7.65 (s, 2H,
thiophene-C5-NH2, D2O exchangeable), 7.87 (s, 2H, CONH2, D2O exchangeable), 10.42 (s, 1H, NH, D2O exchangeable).
Anal. Calcd. (%) for C14H13N5O2S (315.35): C, 53.32; H, 4.16; N, 22.21. Found: C, 53.45; H, 4.14; N, 22.35.
5-Amino-3-Methyl-4-{5-[(4-Methylphenyl) Amino]-1,3,4-Oxadiazol-2-yl}Thiophene-2-Carboxamide(12b)
Yield: 72%, m.p.: 226-8oC. IR (KBr, cm-1): 3395, 3254, 3155 (NH2, NH), 1660 (C=O), 1586 (C=N), 1250, 1032 (C-
S-C), 1118, 1072 (C-O-C). 1H-NMR (DMSO-d6, ppm): 2.26 (s, 3H, phenyl CH3), 2.59 (s, 3H, CH3), 7.15 (d, 2H, J = 8.4
Hz, 4-methylphenyl-C2&6-H), 7.46 (d, J = 8.4 Hz, 2H, 4-methylphenyl-C3,5-H), 7.82 (s, 2H, thiophene-C5-NH2, D2O
exchangeable), 7.98 (s, 2H, CONH2, D2O exchangeable), 10.35 (s, 1H, NH, D2O exchangeable). Anal. Calcd. (%) for
C15H15N5O2S (329.38): C, 54.70; H, 4.59; N, 21.26. Found: C, 54.84; H, 4.55; N, 21.39.

44 The Open Medicinal Chemistry Journal, 2017, Volume 11
Rizk et al.
BIOLOGICAL EVALUATION
Anti-hepatitis C Virus Activity
Evaluation of Cytotoxicity Using Neutral Red Uptake Assay
Isolation of lymphocytes from whole human blood was done using gradient separation by Ficoll-PagueTM Plus
(MP Biomedicals, France), ˜10 × 104 cells were seeded per well in 96 well plates and the plates were incubated in RPMI
(Roswell Park Memorial Institute) media containing different concentrations of the tested compounds for 24, 48, 72 and
96 h. The fraction of viable lymphocyte cells was measured by the Neutral red assay [33]. The neutral red assay
determines the accumulation of the neutral red dye in the lysosomes of viable cells [34]. Following exposure to different
concentrations of tested compounds, cells were incubated for 3 h with neutral red dye (40μg/ml) dissolved in culture
media RPMI. Cells were then washed with phosphate buffered saline (PBS) then 1 ml of elution medium
(ethanol/glacial acetic acid /water 50/1/ 49%) was added followed by gentle shaking for 10 min so that complete
dissolution was achieved. Aliquots of the resulting solutions were transferred to 96-well plates and absorbance at 490
nm was recorded using microliter plate reader spectrophotometer (Biotek, U.S.A). The assay was conducted three
times. The IC50 (the concentration resulting in 50% death of cells) and EC99 (concentration of the compound that
maintained 99% viability of cells) were calculated using GraphPad Prism (GraphPad Software Inc., San Diego, CA)
[35] (Table 1).
Cell Culture
In the last few years, a number of cell culture systems have been developed that support reliable and efficient
progression of this virus. Among several human hepatocyte cell lines analyzed, the hepatocellular carcinoma HepG2
cell line was found to be the most susceptible to HCV infection [36]. HepG2 cells were washed twice with RPM11640
media supplemented with 200μM L-glutamine and 25μM HEPES buffer; N-[2-hydroxyethyl]piperazine-N`-[2-
ethanesulphonic acid] (Lonza). The cells were suspended at 2 X 105 cells/ mL in RPM1 culture media (RPM1
supplemented media, 10% fetal bovine serum (FBS); Gibco-BRL). The cells were left to adhere on the polystyrene 6-
well plates for 4 h in an incubator (37°C, 5% CO2, 95% humidity). The cells were washed twice from debris and dead
cells by using RPM1 supplemented media. HepG2 cell culture was prepared as discussed, then infected with HCV-
infected serum in RPMI culture medium. Each of the tested compounds was added at the specified EC99 Table (1).
Positive and negative control cultures were included. The positive strand and its replicating form (negative strand) were
detected by reverse transcription-polymerase chain reaction (RT-PCR) using HCV specific primers to the 5'-
untranslated region of the virus.
Qualitative In-vitro Anti-HCV Screening
RNA Extraction and RT-PCR of HCV RNA
Total RNA was extracted from HepG2 HCV-infected cells as well as from HepG2 infected cells that are treated
with the test compounds using the method described by El-Awady et al. [37]. Briefly, culture cells were mixed with
200µLof 4M guanidinium isothiocyanate containing 25 mM sodium citrate, 0.5% sarcosyl, 0.1 Mβ- mercaptoethanol,
and 100 µL sodium acetate. The lysed cells were mixed with an equal volume mixture of water-saturated phenol,
chloroform, and isoamyl alcohol. After vortexing of the sample, the mixture was centrifuged at 14 K rpm for 10 min at
4°C. The aqueous layer was collected and mixed with an equal volume of isopropanol. After incubation at –20°C
overnight, RNA was precipitated by centrifugation at14 K rpm for 30 min at 4oC and the precipitated RNA was washed
twice with 70% ethanol. The complimentary DNA (cDNA) and the first PCR reaction of the nested PCR detection
system for the HCV RNA was performed in a 50 µL volume single-step reaction using the Ready-To-Go RT-PCR
beads (Healthcare Life Sciences, USA), 10 µM from each of the RT downstream primer, PCR forward primer and
reverse primer P2. The thermal cycling protocol was manipulated as follows: 30 min at 42oC for cDNA synthesis
followed by 5 min at 95oC and 30 cycles of 1 min at 94°C, 1 min at 55oC and 1 min at 72oC. The nested PCR
amplification was performed in 50 µL reaction mixture containing 0.2 mM from each dNTP, 10 µM from each of the
reverse nested primer and the forward nested primer, two units of taq DNA polymerase (Promega, Madison, WI, USA),
10 µL from the RT-PCR reaction in a 1X buffer supplied by the Vendor. A fragment of 174bp length was identified in
positive samples.

Substituted Thiophene-2-Carboxamides as Anti-HCV and Antimicrobial Agents
The Open Medicinal Chemistry Journal, 2017, Volume 11 45
Antimicrobial Screening
Inhibition-zone Measurements
All the synthesized compounds were evaluated by the agar cup diffusion technique [38 - 40] using a 1 mg/mL
solution in DMSO. The test organisms were Staphylococcus aureus (DSM 1104) and Bacillus subtilis (ATCC 6633) as
Gram-positive bacteria; Escherichia coli (ATCC 11775) and Pseudomonas aeruginosa (ATCC 10145) as Gram-
negative bacteria. Candida albicans (DSM 70014) was also used as a representative for fungi. Each 100 mL of sterile
molten agar (at 45oC) received 1 mL of 6 h-broth culture and then the seeded agar was poured into sterile Petri dishes.
Cups (8 mm in diameter) were cut in the agar. Each cup received 0.1 mL of the 1 mg/mL solution of the test
compounds. The plates were then incubated at 37°C for 24 h or, in case of C. albicans, for 48 h. A control using DMSO
without the test compound was included for each organism. Ampicillin was used as standard antibacterial, while
clotrimazole was used as antifungal reference. The resulting inhibition zones are recorded in Table (2).
Minimal Inhibitory Concentration (MIC) Measurement
The minimal inhibitory concentrations (MIC) of the most active compounds were measured using the twofold serial
broth dilution method [39, 40]. The test organisms were grown in their suitable broth: 24 h for bacteria and 48 h for
fungi at 37oC. Twofold serial dilutions of solutions of the test compounds were prepared using 200, 100, 50, 25, and
12.5 µg/mL. The tubes were then inoculated with the test organisms; each 5 mL received 0.1 mL of the above inoculum
and were incubated at 37°C for 48 h. Then, the tubes were observed for the presence or absence of microbial growth.
The MIC values of the prepared compounds are listed in Table 3.
Minimal Bactericidal Concentration (MBC) Measurement
MIC tests were always extended to measure the MBC as follows: A loop-full from the tube not showing visible
growth (MIC) was spread over a quarter of Müller-Hinton agar plate. After 18 h of incubation, the plates were
examined for growth. Again, the tube containing the lowest concentration of the test compound that failed to yield
growth on subculture plates was judged to contain the MBC of that compound for the respective test organism (Table
3).
RESULTS AND DISCUSSION
Chemistry
Synthesis of the intermediate and target compounds are illustrated in schemes (1 and 2).
In scheme (1), the starting ester (1) [32] was synthesized by heating a mixture of acetoacetamide, ethyl cyanoacetate
and sulfur in the presence of morpholine. Heating the ester 1 with hydrazine hydrate in ethanol gave rise to the
hydrazide 2. Condensation of the hydrazide 2 with acetyl acetone or benzoylacetone in ethanol containing glacial acetic
acid afforded the corresponding 3,5-dimethylpyrazole 3a and 3-methyl-5-phenylpyrazole 3b. The pyrazole derivative 4
was prepared by heating the acid hydrazide 2 with ethyl pyruvate in ethanol containing glacial acetic acid. The 3,5-
dioxopyrazolidine derivative 5 was successfully achieved by heating 2 with diethylmalonate. On the other hand,
refluxing of 2 with ethyl acetoacetate in ethanol containing glacial acetic acid gave the required 5-oxo-4,5-dihydro-1H-
pyrazole derivative 6. The chemical structure of compounds 3a, b, 4, 5 and 6 were verified by analytical and spectral
data.
Surprisingly, heating of the hydrazide 2 with aromatic aldehydes in ethanol containing glacial acetic acid yielded the
1,2,4 triazepine derivatives 7a-c instead of the expected hydrazones 8a-c. 1H-NMR spectra of these compounds lacked
the D2O exchangeable singlets characteristic for NH2 and N=CH.
In scheme 2, the N-substituted thiocarbamoyl hydrazino derivatives 9a, b was prepared by refluxing the acid
hydrazide 2 with aryl isothiocyanate in absolute ethanol. Cyclocondensation of 9a, b with the selected phenacyl
bromide or ethyl bromoacetate in ethanol containing anhydrous sodium acetate produced the respective thiazoline
derivatives 10a-f, and thiazolidinone derivatives 11a, b respectively. Cyclodesulphurization of 9a, b using mercuric
oxide in dry dioxane furnished the target 1,3,4-oxadiazole derivatives 12a, b in good yields.
The structure of the new compounds was elucidated by spectral analysis in addition to microanalysis.

46 The Open Medicinal Chemistry Journal, 2017, Volume 11
Rizk et al.
NH2
S
O
O
O
OC2H5
i
NC
N
+
O
NH2
O
NH2
S
O
H2N
N
1
O
N
H2N
NH2
S
O
O
3a,b
R
H
iii
N
ii
R'
H2N
O
O
8a-c
NH2
O
NH2
S
O
O
S
vii
iv
H
N
N
NH2
N
H2N
H2N
2
O
O
C6H5
4
vi
v
O
H
S
N
R'
NH2
O
H2N
NH2
HN
O
S
O
S
N
N
N
N
H2N
N
H2N
H
O
O
7a-c
O
O
O
6
5
For 3: a, R' = CH3; b, R' = C6H5
7 and 8:a, R' = 4-NO2C6H4; b, R' = 4-OCH3C6H4; c, R' = 3,4-OCH3C6H3
Reagents and conditions: i ) S/morpholine/EtOH/60oC; ii) NH2NH2.H2O/ EtOH/ reflux
iii)C6H5COCH2COCH3 or CH3COCH2COCH3 /EtOH/CH3COOH/reflux; iv) ethylpyruvate/EtOH/CH3COOH/reflux
v) CH2(COOC2H5)2/reflux; vi) CH2COCH2COOC2H5/EtOH/CH3COOH/reflux; vii)R1CHO/EtOH/reflux
Scheme 1. Synthetic pathways for compounds (1-7).
NH2
S
O
H
N
NH2
H2N
2 O
i
NH2
NH2
NH2
S
O
S
S
S
O
S
O
ii
iv
H
N
H
R1
R
N
N
N
N
H
N
R
H2N
N
H2N
H2N
H
N
O
9a,b
O
O
NHR
10a-f
12a,b
iii
NH2
S
O
S
H
O
N
N
N
H2N
R
O
11a,b
R = C6H5, 4-CH3C6H4 R1= C6H5, 4-BrC6H4, 4-OCH3C6H4
Reagents and conditions: i) RNCS/EtOH/reflux; ii) R1COCH2Br/EtOH/NaOAc/reflux; iii) BrCH2COOC2H5/EtOH/NaOAc/reflux
iv) HgO/dioxane/reflux
Scheme 2. Synthetic pathways for compounds (9-12).

Substituted Thiophene-2-Carboxamides as Anti-HCV and Antimicrobial Agents
The Open Medicinal Chemistry Journal, 2017, Volume 11 47
BIOLOGICAL EVALUATION
Anti-hepatitis C virus activity
Evaluation of Cytotoxicity
All synthesized compounds were investigated for their effect on the proliferative activity of HepG2 cancer cell line,
and the IC50 and EC99 in µg/ mL (Table 1) were determined for each test compound using GraphPad Prism.
Table 1. Preliminary anti-HCV activity of the synthesized compounds 3-12 on HepG 2.
a)
b)
EC99
IC50
Anti-HCV Activityc)
Compound No.
(µg/mL)
(µg/mL)
0.10
0.12
0.11
0.17
0.27
0.12
0.08
0.20
0.16
0.36
0.13
0.14
0.08
0.11
0.08
0.18
0.10
0.13
0.15
0.23
3a
3b
5.64
positive
negative
negative
negative
positive
positive
positive
negative
positive
positive
positive
negative
negative
negative
negative
negative
negative
positive
negative
negative
6.60
4
5.50
5
8.77
6
14.33
6.50
7a
7b
4.37
7c
10.40
8.46
9a
9b
18.16
6.80
10a
10b
10c
10d
10e
10f
11a
11b
12a
12b
7.80
4.20
5.75
4.23
9.45
5.30
7.04
8.10
12.16
a) IC50: concentration of the compound that caused 50% death of cells.
b) EC99: concentration of the compound that maintained 90-99% viability of cells.
c) Ability to inhibit the replication of both HCV RNA (+) and (–) strands.
Fig. (1). Agarose gel electrophoresis showing PCR amplified product of HCV RNA (+) and (-) strands in presence of compounds
11a, 7a, 7c3a. Lane 1: molecular weight marker (M) 100bp ladder, lanes 2 and 4 = positive control and lane 3 = negative control.
Lanes 5, 7, 9 and 11 show the effect of compounds 11a, 7a, 7c and 3a on HCV RNA (-) strands and lanes 6, 8, 10 and 12 show the
effect of compounds 11a, 7a, 7c and 3a on HCV RNA (+) strands isolated from HepG2 cells at the EC99 in µg/mL after 3 days of
treatment.

48 The Open Medicinal Chemistry Journal, 2017, Volume 11
Rizk et al.
Qualitative In-vitro Anti-HCV Screening
All synthesized compounds were evaluated for their in-vitro anti-HCV activity utilizing the hepatocellular
carcinoma HepG2 cell line infected with hepatitis-C virus. Detection of viral (+) and/or (–) RNA strands was performed
using qualitative RT-PCR. Detection of the (–) strand HCV-RNA using RT-PCR has been recently reported as a very
important tool for understanding the life cycle of HCV. It is the most reliable biological marker for the diagnosis of
HCV and monitoring the viral response to antiviral therapy [37].
According to these facts, simultaneous detection of the (+) and/or (–) HCV-RNA strands in HepG2 hepatoma cells
infected with HCV was performed. Inhibition of viral replication was detected by amplification of viral RNA segments
using the RT-PCR technique, both in the cultivated infected cells alone (as a positive control) and in presence of the
specified doses of the test compounds at optimal temperature. An active compound is capable of inhibiting viral
replication inside the HepG2 cells, as evidenced by the disappearance of the (+) and/or (–) strands viral RNA-amplified
products detected by the RT-PCR (compared to positive control).
Fig. (2). Agarose gel electrophoresis showing PCR amplified product of HCV RNA (+) and (-) strands in presence of compounds 5,
6, 9a and 7b. Lane 1: molecular weight marker (M) 100bp ladder, lanes 2 and 4 = positive control and lane 3 = negative control.
Lanes 5, 7, 9 and 11 show the effect of compounds 5, 6, 9a and 7b on HCV RNA (-) strands and lanes 6, 8, 10 and 12 show the effect
of compounds 5, 6, 9a and 7b on HCV RNA (+) strands isolated from HepG2 cells at the EC99 in µg/mL after 3 days of treatment.
Fig. (3). Agarose gel electrophoresis showing PCR amplified product of HCV RNA (+) and (-) strands in the presence of compounds
10b, 9b, 10e, 10d, 10a and 11b. Lane 1: molecular weight marker (M) 100bp ladder, lanes 2 and 4 = positive control and lane 3 =
negative control. Lanes 5, 7, 9, 11, 13 and 15 show the effect of compounds 10b, 9b, 10e, 10d, 10a and 11b on HCV RNA (-) strands
and lanes 6, 8, 10, 12, 14 and 16 show the effect of compounds 10b, 9b, 10e, 10d, 10a and 11b on HCV RNA (+) strands isolated
from HepG2 cells at the EC99 in µg/mL after 3 days of treatment.
Inspection of (Figs. (1-4) showed that all the synthesized compounds inhibited the replication of HCV RNA (–)
strands at the EC99. In addition, eight compounds 3a, 6, 7a, 7b, 9a, 9b, 10a and 11b were proved to inhibit replication of

Substituted Thiophene-2-Carboxamides as Anti-HCV and Antimicrobial Agents
The Open Medicinal Chemistry Journal, 2017, Volume 11 49
both (+) and (–) strands at very low concentration range (EC99 = 0.08- 0.36 µg/mL) as shown in Table 1. The obtained
data revealed that the pyrazolyl derivatives 3a and 6 inhibited the replication of both (+) and (–) strands of HCV RNA
at 0.10 and 0.27 µg/mL. Conversion of the acid hydrazide 2 into the dihydro-1H-thieno[2,3-e] [1, 2, 4] triazepine-7-
carboxamides 7a and 7b enhanced the activity against both (+) and (–) strands. Transformation of the hydrazide 2 into
the N-substituted thiocarbamoylhydrazino derivatives 9a, b yielded compounds that are able to inhibit replication of
both (+) and (–) strands at EC99 = 0.16 and 0.36 µg/mL. Finally, cyclization of 2 into the 1,3-thiazolidin-2-
ylidene)hydrazino]carbonyl}thiophene-2-carboxamide derivatives 10a and 11b yielded compounds active against both
(+) and (–) strands.
Fig. (4). Agarose gel electrophoresis showing PCR amplified product of HCV RNA (+) and (-) strands in the presence of compounds
3b, 12a, 12b, 4, 10c and 10f. Lane 1: molecular weight marker (M) 100bp ladder, lanes 2 and 4 = positive control and lane 3 =
negative control. Lanes 5, 7, 9, 11, 13 and 15 show the effect of compounds 3b, 12a, 12b, 4, 10c and 10f on HCV RNA (-) strands
and lanes 6, 8, 10, 12, 14 and 16 show the effect of compounds 3b, 12a, 12b, 4, 10c and 10f on HCV RNA (+) strands isolated from
HepG2 cells at the EC99 in µg/mL after 3 days of treatment.
Antimicrobial Screening
The new compounds were investigated for their in vitro antibacterial activity against Gram-positive bacteria as
Staphylococcus aureus and Bacillus subtilis, and Gram-negative bacteria as Escherichia coli and Pseudomonas
aeruginosa . They were also studied for their in vitro antifungal potential against Candida albicans. The inhibition
zones were measured utilizing the cup-diffusion technique [38, 39] and compounds showing reasonable inhibition zones
(A ≥13 mm) were further evaluated to determine their minimal inhibitory concentration (MIC) and minimum bacterial
concentration (MBC) applying the twofold serial dilution method [39, 40]. Ampicillin was used as antibacterial
reference while clotrimazole was used as antifungal standard. Dimethylsulfoxide (DMSO) was used as blank and
showed no antimicrobial activity.
Table 2. The inhibition zones (IZ) in mm diameter of the synthesized compounds 3-12.
Cpd No.
3a
S. aureus
16
B. subtilis
P. aeroginosa
E. coli
17
C. albicans
14
14
16
16
18
14
14
18
12
16
18
14
12
12
14
12
17
15
16
12
18
18
14
16
18
17
18
14
16
14
14
16
18
12
14
14
3b
4
17
15
16
12
5
18
18
6
14
16
7a
18
15
7b
7c
18
15
14
14
9a
12
16
9b
10a
10b
14
16
12
16
14
12

50 The Open Medicinal Chemistry Journal, 2017, Volume 11
Rizk et al.
(Table 2) contd.....
Cpd No.
10c
10d
10e
10f
S. aureus
B. subtilis
P. aeroginosa
E. coli
16
14
16
12
14
16
14
14
10
-
C. albicans
14
18
18
16
18
16
18
14
9
14
16
18
14
12
14
14
16
12
-
18
12
12
15
14
12
12
15
7
14
14
14
18
16
14
12
14
-
11a
11b
12a
12b
Aa
Cb
c
-
-
10
a A: Ampicillin trihydrate (standard broad spectrum antibiotic).
bC: Clotrimazole (standard broad spectrum antifungal agent).
c (–): Totally inactive.
Results from (Tables 2 and 3) revealed that twenty two compounds showed promising inhibitory effects on the
growth of the tested Gram-positive and Gram-negative microorganisms with high activity against P. aeruginosa.
Regarding the antibacterial activity of the active compounds against S. aureus, compounds 6 and 10a (MIC = 25
µg/mL) exhibited moderate activity, whereas the other compounds displayed weak activity. Concerning the activity
against B. subtilis, compounds 5, 7c and 10a (MIC = 25 µg/mL) displayed half the activity of ampicillin while
compounds 3a, 6, 10f and 11a were four times less active than ampicillin against the same organism. While, P.
aeruginosa was proved to be the most sensitive microorganism to most of the tested compounds. Compounds 3b, 5, 7b,
10b, 11a and 11b (MIC = 25 µg /mL) showed double the activity of ampicillin, whereas compounds 4, 7a, 9a, 9b, 10c,
10d, 10e, 11b, 12a and 12b were equipotent to ampicillin. Furthermore, compounds 7c, 9a, 10d, 10f, 11a, 11b and 12b
(MIC = 25 µg /mL) showed nearly half the activity of ampicillin against E. coli, meanwhile other compounds showed
moderate to mild activity. On the other hand, only three compounds 3b, 4 and 10b produced moderate growth inhibition
(MIC = 25 µg /mL) against C. Albicans. According to the MIC and MBC limits derived from the latest National
Committee on Clinical Laboratory Standards (NCCLS), it can be decided whether the compound is bactericidal or
bacteriostatic. Consequently, data from (Table 3) revealed that only compounds 10d, 11a and 12a were bactericidal
against P. aeruginosa while the other compounds were bacteriostatic against the test organisms.
Table 3. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the synthesized
compounds 3-12 in µg/mL.
Cpd No.
S. aureus
MIC
B.subtilis
MIC
P.aeruginosa
E.coli
C.albicans
MBC
100
100
100
100
50
MBC
100
200
100
50
MIC
MBC
100
50
MIC
100
50
MBC
200
50
MIC
MBC
100
50
3a
3b
100
50
50
100
100
25
100
25
50
25
4
100
100
25
50
100
50
50
100
100
100
100
100
50
25
25
5
25
50
50
50
6
50
50
100
50
200
100
50
50
100
50
100
100
100
100
100
50
7a
100
50
200
50
100
100
25
100
100
50
100
50
7b
25
100
100
50
7c
100
100
50
200
100
50
100
50
100
100
100
100
50
25
9a
100
100
25
200
100
100
200
200
100
200
100
50
25
50
9b
50
50
100
100
100
200
50
50
10a
10b
10c
10d
10e
10f
11a
11b
12a
25
50
100
25
50
100
25
100
50
50
100
100
100
100
200
200
100
100
100
100
100
100
50
100
100
25
100
100
50
50
100
50
100
50
100
100
100
200
100
100
100
50
50
100
100
25
100
25
100
50
50
100
100
100
50
100
25
100
50
50
25
50
100
100
100
100
25
100
50
25
25
50
50
100
200
50

Substituted Thiophene-2-Carboxamides as Anti-HCV and Antimicrobial Agents
The Open Medicinal Chemistry Journal, 2017, Volume 11 51
(Table 3) contd.....
Cpd No.
S. aureus
B.subtilis
P.aeruginosa
E.coli
C.albicans
MIC
MBC
MIC
MBC
MIC
MBC
100
-
MIC
25
10
-
MBC
MIC
MBC
12b
Aa
Cb
100
100
100
12.5
-
100
50
50
25
-
50
-
100
5
-
-
-
-
c
-
-
-
-
5
a A: Ampicillin trihydrate (standard broad spectrum antibiotic).
bC: Clotrimazole (standard broad spectrum antifungal agent).
c (–): Totally inactive (MIC > 200 µg/mL).
The activity of the test compounds could be tentatively correlated to the structural variations and modification. The
pyrazolyl derivatives 3b and 5. showed potent activity against P. aeroginosa (MIC = 25 µg/mL), while compound 5
showed good activity against B. subtilis (MIC = 25 µg/mL). Whereas, the triazepine derivatives 7a and 7b exhibited
substantial activity against P. aeroginosa (MIC = 50 and 25µg/mL respectively). Meanwhile, the triazepine derivative
7c displayed moderate activity against B. Subtilis and E. Coli (MIC = 25 µg/mL). Cyclization of N-substituted
thiocarbamoyl hydrazino derivatives 9a, b into the corresponding thiazoline derivatives 10a-f, and thiazolidinone
derivatives 11a,b didn't significantly affect the antimicrobial potential except for compounds 10b, 10d, 10f, 11a and
11b that showed activity against Gram-negative microorganisms while compound 10a showed activity against Gram-
positive microorganisms.
CONCLUSION
In the present study, twenty new 5-amino-4-substituted-3-methylthiophene-2-carboxamide derivatives were
prepared and their chemical structures were confirmed by spectral analyses in addition to elemental analyses. All
compounds were investigated for their preliminary in-vitro effect on the replication of hepatitis-C virus (HCV) in
HepG2 hepatocellular carcinoma cell line infected with the virus using the reverse transcription polymerase chain
reaction technique. All compounds were found to inhibit the replication of HCV RNA (–) strands at the EC99,
meanwhile, eight compounds (3a, 6, 7a, 7b, 9a, 9b, 10a and 11b) were proved to inhibit replication of both (+) and (–)
strands at very low concentration range (EC99 = 0.08- 0.36 µg/mL).
Moreover, all compounds exhibited remarkable activity against the tested microorganisms with high activity against
the gram-negative organism P. aeruginosa. Compounds 3b, 5, 7b, 10b and 11a and 11b emerged with the highest
potential against P. aeruginosa as they displayed double the activity of ampicillin against this organism. Collectively,
the triazepine derivative 7b and the thiazolidinone derivative 11b could be considered as the most active compounds in
this study with an interesting dual anti-HCV and antimicrobial profile.
Finally, the obtained anti-HCV and antimicrobial data suggest that the 3-methylthiophene-2-carboxamide might be
a potential skeleton for further development of more active and selective compounds with antihepatitis-C and/ or
antimicrobial activities.
DECLARATION/CONFLICT OF INTEREST
The authors declared no conflicts of interest. The authors alone are responsible for the content and writing of the
paper.
ACKNOWLEDGEMENTS
Declared none
REFERENCES
[1]
[2]
Shepard, C.W.; Finelli, L.; Alter, M.J. Global epidemiology of hepatitis C virus infection. Lancet Infect. Dis., 2005, 5(9), 558-567.
[http://dx.doi.org/10.1016/S1473-3099(05)70216-4] [PMID: 16122679]
Alter, M.J.; Kruszon-Moran, D.; Nainan, O.V.; McQuillan, G.M.; Gao, F.; Moyer, L.A.; Kaslow, R.A.; Margolis, H.S. The prevalence of
hepatitis C virus infection in the United States, 1988 through 1994. N. Engl. J. Med., 1999, 341(8), 556-562.
[http://dx.doi.org/10.1056/NEJM199908193410802] [PMID: 10451460]
[3]
[4]
Lehman, E.M.; Wilson, M.L. Epidemic hepatitis C virus infection in Egypt: estimates of past incidence and future morbidity and mortality. J.
Viral Hepat., 2009, 16(9), 650-658.
[http://dx.doi.org/10.1111/j.1365-2893.2009.01115.x] [PMID: 19413698]
Goins, W.F. Microbial Pathogenesis: A Principles-Oriented Approach; McClane, B.A.; Mietzner, T.A., Eds.; Fence Creek Publishing:
Madison, 1999, p. 395.

52 The Open Medicinal Chemistry Journal, 2017, Volume 11
Rizk et al.
[5]
[6]
Hoofnagle, J.H. A step forward in therapy for hepatitis C. N. Engl. J. Med., 2009, 360(18), 1899-1901.
[http://dx.doi.org/10.1056/NEJMe0901869] [PMID: 19403908]
Jackson, R.W.; LaPorte, M.G.; Herbertz, T.; Draper, T.L.; Gaboury, J.A.; Rippin, S.R.; Patel, R.; Chunduru, S.K.; Benetatos, C.A.; Young,
D.C.; Burns, C.J.; Condon, S.M. The discovery and structure-activity relationships of pyrano[3,4-b]indole-based inhibitors of hepatitis C virus
NS5B polymerase. Bioorg. Med. Chem. Lett., 2011, 21(11), 3227-3231.
[http://dx.doi.org/10.1016/j.bmcl.2011.04.052] [PMID: 21550237]
[7]
[8]
Masunari, A.; Tavares, L.C. A new class of nifuroxazide analogues: synthesis of 5-nitrothiophene derivatives with antimicrobial activity
against multidrug-resistant Staphylococcus aureus. Bioorg. Med. Chem., 2007, 15(12), 4229-4236.
[http://dx.doi.org/10.1016/j.bmc.2007.03.068] [PMID: 17419064]
Foroumadi, A.; Oboudiat, M.; Emami, S.; Karimollah, A.; Saghaee, L.; Moshafi, M.H.; Shafiee, A. Synthesis and antibacterial activity of N-
[2-[5-(methylthio)thiophen-2-yl]-2-oxoethyl] and N-[2-[5-(methylthio)thiophen-2-yl]-2-(oxyimino)ethyl] piperazinylquinolone derivatives.
Bioorg. Med. Chem., 2006, 14(10), 3421-3427.
[http://dx.doi.org/10.1016/j.bmc.2005.12.058] [PMID: 16427290]
[9]
Romagnoli, R.; Baraldi, P.G.; Carrion, M.D.; Cara, C.L.; Cruz-Lopez, O.; Preti, D.; Tolomeo, M.; Grimaudo, S.; Di Cristina, A.; Zonta, N.;
Balzarini, J.; Brancale, A.; Sarkar, T.; Hamel, E. Design, synthesis, and biological evaluation of thiophene analogues of chalcones. Bioorg.
Med. Chem., 2008, 16(10), 5367-5376.
[http://dx.doi.org/10.1016/j.bmc.2008.04.026] [PMID: 18440234]
[10] Shiradkar, M.R.; Padhalingappa, M.B.; Bhetalabhotala, S.; Akula, K.C.; Tupe, D.A.; Pinninti, R.R.; Thummanagoti, S. A novel approach to
cyclin-dependent kinase 5/p25 inhibitors: A potential treatment for Alzheimers disease. Bioorg. Med. Chem., 2007, 15(19), 6397-6406.
[http://dx.doi.org/10.1016/j.bmc.2007.06.053] [PMID: 17643991]
[11] Chabert, J.F.; Marquez, B.; Neville, L.; Joucla, L.; Broussous, S.; Bouhours, P.; David, E. Synthesis and evaluation of new
arylbenzo[b]thiophene and diarylthiophene derivatives as inhibitors of the Nor A multidrug transporter of Staphylococcus aureus. Bioorg.
Med. Chem., 2007, 15, 4482-4497.
[http://dx.doi.org/10.1016/j.bmc.2007.04.023] [PMID: 17498961]
[12] Hossan, A.S.; Abu-Melha, H.M.; Al-Omar, M.A.; Amr, Ael-G. Synthesis and antimicrobial activity of some new pyrimidinone and oxazinone
derivatives fused with thiophene rings using 2-chloro-6-ethoxy-4-acetylpyridine as starting material. Molecules, 2012, 17(11), 13642-13655.
[http://dx.doi.org/10.3390/molecules171113642] [PMID: 23165308]
[13] Al-Omar, M.A. Synthesis and antimicrobial activity of new 5-(2-thienyl)-1,2,4-triazoles and 5-(2-thienyl)-1,3,4-oxadiazoles and related
derivatives. Molecules, 2010, 15(1), 502-514.
[http://dx.doi.org/10.3390/molecules15010502] [PMID: 20110905]
[14] Moreau, B.; O’Meara, J. A.; Bordeleau, J.; Garneau, M.; Godbout, C.; Moreau, B.; OMeara, J.A.; Bordeleau, J.; Garneau, M.; Godbout, C.;
Gorys, V.; Leblanc, M.; Villemure, E.; White, P.W.; Llinàs-Brunet, M. Discovery of hepatitis C virus NS34A protease inhibitors with
improved barrier to resistance and favorable liver distribution. J. Med. Chem., 2014, 57(5), 1770-1776.
[http://dx.doi.org/10.1021/jm400121t] [PMID: 23506530]
[15] Arora, P.; Narang, R.; Bhatia, S.; Nayak, S. K.; Singh, S. K.; Narasimhan, B. Synthesis, molecular docking and QSAR studies of 2, 4-
disubstituted thiazoles as antimicrobial agents. J. Appl. Pharm. Sci., 2015, 5(02), 028-042.
[16] Sharma, P.K.; Chandak, N.; Kumar, P.; Sharma, C.; Aneja, K.R. Synthesis and biological evaluation of some 4-functionalized-pyrazoles as
antimicrobial agents. Eur. J. Med. Chem., 2011, 46(4), 1425-1432.
[http://dx.doi.org/10.1016/j.ejmech.2011.01.060] [PMID: 21342734]
[17] Padmavathi, V.; Sudhakar Reddy, G.; Padmaja, A.; Kondaiah, P.; Ali-Shazia, Synthesis, antimicrobial and cytotoxic activities of 1,3,4-
oxadiazoles, 1,3,4-thiadiazoles and 1,2,4-triazoles. Eur. J. Med. Chem., 2009, 44(5), 2106-2112.
[http://dx.doi.org/10.1016/j.ejmech.2008.10.012] [PMID: 19036476]
[18] El-Emam, A.A.; Al-Deeb, O.A.; Al-Omar, M.; Lehmann, J. Synthesis, antimicrobial, and anti-HIV-1 activity of certain 5-(1-adamantyl)-2-
substituted thio-1,3,4-oxadiazoles and 5-(1-adamantyl)-3-substituted aminomethyl-1,3,4-oxadiazoline-2-thiones. Bioorg. Med. Chem., 2004,
12(19), 5107-5113.
[http://dx.doi.org/10.1016/j.bmc.2004.07.033] [PMID: 15351394]
[19] Al-Saadi, M.S.; Rostom, S.A.; Faidallah, H.M. In vitro antitumor screening of some polysubstituted pyrazole analogs. Saudi Pharm. J., 2005,
13, 88-96.
[20] Qian, X.; Li, Z.; Yang, Q. Highly efficient antitumor agents of heterocycles containing sulfur atom: linear and angular thiazonaphthalimides
against human lung cancer cell in vitro. Bioorg. Med. Chem., 2007, 15(21), 6846-6851.
[http://dx.doi.org/10.1016/j.bmc.2007.07.008] [PMID: 17707644]
[21] Aboraia, A.S.; Abdel-Rahman, H.M.; Mahfouz, N.M.; El-Gendy, M.A. Novel 5-(2-hydroxyphenyl)-3-substituted-2,3-dihydro-1,3,4-
oxadiazole-2-thione derivatives: promising anticancer agents. Bioorg. Med. Chem., 2006, 14(4), 1236-1246.
[http://dx.doi.org/10.1016/j.bmc.2005.09.053] [PMID: 16242340]
[22] Desai, N.C.; Bhatt, N.; Somani, H.; Trivedi, A. Synthesis, antimicrobial and cytotoxic activities of some novel thiazole clubbed 1,3,4-
oxadiazoles. Eur. J. Med. Chem., 2013, 67, 54-59.
[http://dx.doi.org/10.1016/j.ejmech.2013.06.029] [PMID: 23835482]

Substituted Thiophene-2-Carboxamides as Anti-HCV and Antimicrobial Agents
The Open Medicinal Chemistry Journal, 2017, Volume 11 53
[23] Hamdy, N.A.; El-Senousy, W.M. Synthesis and antiviral evalution of some novel pyrazoles and pyrazolo[3,4-d]pyridazines bearing 5,6,7,8-
tetrahydronaphthalene. Acta Pol. Pharm., 2013, 70(1), 99-110.
[PMID: 23610964]
[24] Li, Z.; Zhan, P.; Liu, X. 1,3,4-oxadiazole: a privileged structure in antiviral agents. Mini Rev. Med. Chem., 2011, 11(13), 1130-1142.
[http://dx.doi.org/10.2174/138955711797655407] [PMID: 22353222]
[25] Riyadh, S.M.; Farghaly, T.A.; Abdallah, M.A.; Abdalla, M.M.; Abd El-Aziz, M.R. New pyrazoles incorporating pyrazolylpyrazole moiety:
synthesis, anti-HCV and antitumor activity. Eur. J. Med. Chem., 2010, 45(3), 1042-1050.
[http://dx.doi.org/10.1016/j.ejmech.2009.11.050] [PMID: 20022411]
[26] El-Sayed, W.A.; El-Essawy, F.A.; Ali, O.M.; Nasr, B.S.; Abdalla, M.M.; Abdel-Rahman, A.A Synthesis and antiviral evaluation of new 2,5-
disubstituted 1,3,4-oxadiazole derivatives and their acyclic nucleoside analogues. Monatsh. Chem., 2010, 141, 1021-1028.
[http://dx.doi.org/10.1007/s00706-010-0360-y]
[27] Srivastava, P.C.; Pickering, M.V.; Allen, L.B.; Streeter, D.G.; Campbell, M.T.; Witkowski, J.T.; Sidwell, R.W.; Robins, R.K. Synthesis and
antiviral activity of certain thiazole C-nucleosides. J. Med. Chem., 1977, 20(2), 256-262.
[http://dx.doi.org/10.1021/jm00212a014] [PMID: 189032]
[28] Yan, L.; Wu, J.; Chen, H.; Zhang, S.; Wang, Z.; Wang, H.; Wu, F. Synthesis and in vitro antibacterial activity of novel fluoroalkyl-substituted
pyrazolyl oxazolidinones. RSC Advances, 2015, 5, 73660-73669.
[http://dx.doi.org/10.1039/C5RA11782H]
[29] Kesornpun, C.; Aree, T.; Mahidol, C.; Ruchirawat, S.; Kittakoop, P. water-assisted nitrile oxide cycloadditions: synthesis of isoxazoles and
stereoselective syntheses of isoxazolines and 1,2,4-oxadiazoles. Angew. Chem. Int. Ed. Engl., 2016, 55(12), 3997-4001.
[http://dx.doi.org/10.1002/anie.201511730] [PMID: 26914177]
[30] Li, Z.; Qiu, Q.; Xu, X.; Wang, X.; Jiao, L.; Su, X.; Pan, M.; Huang, W.; Qian, H. Design, synthesis and structure-activity relationship studies
of new thiazole-based free fatty acid receptor 1 agonists for the treatment of type 2 diabetes. Eur. J. Med. Chem., 2016, 113, 246-257.
[http://dx.doi.org/10.1016/j.ejmech.2016.02.040] [PMID: 26945112]
[31] Bueno, J.M.; Carda, M.; Crespo, B.; Cuñat, A.C.; de Cozar, C.; León, M.L.; Marco, J.A.; Roda, N.; Sanz-Cervera, J.F. Design, synthesis and
antimalarial evaluation of novel thiazole derivatives. Bioorg. Med. Chem. Lett., 2016, 26(16), 3938-3944.
[http://dx.doi.org/10.1016/j.bmcl.2016.07.010] [PMID: 27432764]
[32] Gütschow, M.; Kuerschner, L.; Neumann, U.; Pietsch, M.; Löser, R.; Koglin, N.; Eger, K. 2-(diethylamino)thieno1,3ŏxazin-4-ones as stable
inhibitors of human leukocyte elastase. J. Med. Chem., 1999, 42(26), 5437-5447.
[http://dx.doi.org/10.1021/jm991108w] [PMID: 10639285]
[33] Repetto, G.; del Peso, A.; Zurita, J.L. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat. Protoc., 2008, 3(7),
1125-1131.
[http://dx.doi.org/10.1038/nprot.2008.75] [PMID: 18600217]
[34] Fotakis, G.; Timbrell, J.A. In vitro cytotoxicity assays: Comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines
following exposure to cadmium chloride. Toxicol. Lett., 2006, 160(2), 171-177.
[http://dx.doi.org/10.1016/j.toxlet.2005.07.001] [PMID: 16111842]
[35] de Oliveira-Pierce, A.N.; Zhang, R.; Machu, T.K. Colchicine: A novel positive allosteric modulator of the human 5-hydroxytryptamine 3A
receptor. J. Pharmacol. Exp. Ther., 2009, 329(2), 838-847.
[http://dx.doi.org/10.1124/jpet.108.146522] [PMID: 19188483]
[36] Kato, N.; Ikeda, M.; Mizutani, T.; Sugiyama, K.; Noguchi, M.; Hirohashi, S.; Shimotohno, K. Replication of hepatitis C virus in cultured non-
neoplastic human hepatocytes. Jpn. J. Cancer Res., 1996, 87(8), 787-792.
[http://dx.doi.org/10.1111/j.1349-7006.1996.tb02101.x] [PMID: 8797883]
[37] El-Awady, M.K.; Ismail, S.M.; El-Sagheer, M.; Sabour, Y.A.; Amr, K.S.; Zaki, E.A. Assay for hepatitis C virus in peripheral blood
mononuclear cells enhances sensitivity of diagnosis and monitoring of HCV-associated hepatitis. Clin. Chim. Acta, 1999, 283(1-2), 1-14.
[http://dx.doi.org/10.1016/S0009-8981(99)00007-8] [PMID: 10404726]
[38] Jain, S.R.; Kar, A. The antibacterial activity of some essential oils and their combinations. Planta Med., 1971, 20(2), 118-123.
[http://dx.doi.org/10.1055/s-0028-1099675] [PMID: 5119010]
[39] Rostom, S.A.; Badr, M.H.; Abd El Razik, H.A.; Ashour, H.M.; Abdel Wahab, A.E. Synthesis of some pyrazolines and pyrimidines derived
from polymethoxy chalcones as anticancer and antimicrobial agents. Arch. Pharm. (Weinheim), 2011, 344(9), 572-587.
[http://dx.doi.org/10.1002/ardp.201100077] [PMID: 21755528]
Scott, A.C. Laboratory control of antimicrobial therapy. In: Mackie and McCartney Practical Medical Microbiology, 13th ed.; Colle, J.G.;
[40]
Duguid, J.P.; Fraser, A.G.; Marmion, B.P., Eds.; Churchill Livingstone: Edinburgh, 1989; Vol. 2, pp. 161-181.
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