New 1,3,4-thiadiazoles based on thiophene-2-carboxylic acid: Synthesis, characterization, and antimicrobial activities

Article abstract of DOI:10.1016/j.molstruc.2019.127470

Novel 1,3,4-thiadiazole derivatives were prepared by cyclization reaction of thiophene-2-carboxylic acid with N-arylthiosemicarbazides and POCl₃. The antibacterial activities of the synthesized compounds were tested against Gram-negative bacter

Full text of DOI:10.1016/j.molstruc.2019.127470

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New 1,3,4-thiadiazoles based on thiophene-2-carboxylic acid: Synthesis,
characterization, and antimicrobial activities
Halit Muğlu, Hasan Yakan, Hanan Almabrok Shouaib
PII:
S0022-2860(19)31579-0
DOI:
https://doi.org/10.1016/j.molstruc.2019.127470
MOLSTR 127470
Reference:
To appear in: Journal of Molecular Structure
Received Date: 5 February 2019
Revised Date: 14 November 2019
Accepted Date: 21 November 2019
Please cite this article as: H. Muğlu, H. Yakan, H.A. Shouaib, New 1,3,4-thiadiazoles based on
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New 1,3,4-Thiadiazoles Based on Thiophene-2-Carboxylic Acid: Synthesis,
Characterization, and Antimicrobial Activities
Halit Muğlua,*, Hasan Yakanb,*, Hanan Almabrok Shouaibc
a
Department of Chemistry, Faculty of Art and Science, Kastamonu University, Kastamonu, Turkey
b Chemistry Education, Faculty of Education, Ondokuz Mayis University, Atakum, Samsun, Turkey
c Department of Biology, Faculty of Science and Arts, Kastamonu University, Kastamonu, Turkey
* Corresponding author’s e-mail address: hmuglu@kastamonu.edu.tr; hasany@omu.edu.tr
Abstract
Novel 1,3,4-thiadiazole derivatives were prepared by cyclization reaction of thiophene-2-
carboxylic acid with N-arylthiosemicarbazides and POCl3. The antibacterial activities of the
synthesized compounds were tested against Gram-negative bacteria (Salmonella enteritidis,
Salmonella typhimurium, Enterobacter aerogenes, Salmonella infantis, Salmonella kentucky,
and Escherichia coli), Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, and
Enterococcus durans), and the fungus Candida albicans using the disk diffusion method.
Selected 1,3,4-thiadiazole derivatives exhibited effective antimicrobial activity against
Staphylococcus aureus, C. albicans, S. typhimurium, Enterobacter aerogenes, and S.
kentucky. Therefore, 1,3,4-thiadiazole derivatives can be considered as bioactive agents for
pharmacological and medicinal applications. The synthesized compounds were characterized
by using IR, 1H NMR, and 13C NMR spectroscopies and elemental analysis.
Keywords: thiophene-2-carboxylic acid, 1,3,4-thiadiazoles, N-arylthiosemicarbazides,
antimicrobial activity, spectroscopic methods.
1

1. Introduction
Heterocyclic chemistry is one of the most active fields of synthetic organic chemistry
research. Thiazoles, thiadiazoles, indoles, oxadiazoles, and pyrroles are important classes of
heterocyclic compounds due to their interesting biological and synthetic applications [1-3].
Thiadiazole is a heterocyclic compound containing both nitrogen and sulfur atoms as part of
the five-membered aromatic ring. There are four isomeric forms of thiadiazole: 1,2,3-
thiadiazole; 1,2,5-thiadiazole; 1,2,4-thiadiazole; and 1,3,4-thiadiazole. The thermally most
stable among these four isomers is 1,3,4-thiadiazole [4].
1,3,4-Thiadiazoles have been widely studied for their biological and therapeutic activities
with the following reported properties; antileishmanial [5], antimicrobial [6,7], antifungal
[8,9], antibacterial [8,10], analgesic [11], anticonvulsant [12,13], antioxidant [14], anti-
inflammatory [15], antipsychotic [16], antitumor [17,18], antidepressive [19],
antihypertensive [20], antiviral [21], antihistamine [22], and antitubercular [23]. Additionally,
1,3,4-thiadiazoles are broadly used in agricultural applications, such as in pesticides,
herbicides, fungicides, insecticides, and bactericides [23-25].
In this study, N-aryl-1,3,4-thiadiazole derivatives were prepared through the reaction of
thiophene-2-carboxylic acid, N-arylthiosemicarbazides, and phosphorous oxychloride
(POCl3). The synthesized thiadiazoles were characterized (IR, NMR, and elemental analysis)
and studied as antimicrobial agents against 10 microorganisms.
2. Experimental
2.1. Measurement and reagents
All starting materials and solvents were purchased from Aldrich, Carlo Erba, Acrõs
Organics, or Merck Chemical Company and used without further purification. Melting points
2

(uncorrected) were measured on a Stuart SMP 30 electrothermal apparatus. The elemental
analysis was performed on CHNS-932 (LECO). IR spectra were recorded on Bruker Alpha
1
13
FT-IR spectrophotometer. H NMR and C NMR spectra were recorded in DMSO-d6 on a
JEOL-ECS 400 MHz spectrometer using tetramethylsilane as an internal standard. The
splitting patterns are indicated as s (singlet), d (doublet), dd (doublet of doublet), t (triplet), td
(triplet of doublet) and m (multiplied).
2.2. Synthesis
The general procedure for thiadiazole synthesis was as follows: The mixture of 2-
thiophenecarboxylic acid (n mol) and N-arylthiosemicarbazide derivatives (n mol) was chilled
in a refrigerator and phosphorous oxychloride (3n mol) was added drop-wise by stirring.
Then, refluxing was continued 90oC for 4 h. After completion of the reaction, the mixture was
cooled to room temperature, poured into ice-cold water with stirring, and then neutralized
with ammonia. The precipitated product was filtered, washed with water, and crystallized in a
suitable solvent. Novel thiadiazole derivatives were synthesized according to the method [6]
illustrated in Scheme 1.
3

S
O
N
S
POCl3
Ar
N
+
H2N
Ar
N
H
N
S
OH
S
N Ar
H
H
Ar
I : C6H5
VI : 4-NO2-C6H5
VII : C6H5CH2
VIII : 2-OCH3-C6H5
IX : 3-OCH3-C6H5
X : 4-OCH3-C6H5
II : 3-Cl-C6H5
III : 4-Cl-C6H5
IV : 2-F-C6H5
V : 4-F-C6H5
N
N
N
N
N
S
H
S
S
S
N
Cl
S
S
H
H
II
I
Cl
N
N
N
N
S
N
S
N
H
H
F
III
IV
F
NO2
N
N
N
N
S
N
S
N
H
S
S
H
VI
V
N
N
N
N
S
S
N
H
N
S
S
H
OCH3
OCH3
VII
VIII
N
N
N
N
S
N
OCH3
S
S
N
H
H
S
IX
X
Scheme 1. Synthetic overview of 1,3,4-thiadiazole formation.
Synthesis of N-phenyl-5-(thiophen-2-yl)-1,3,4-thiadiazol-2-amine (I): Brown solid.
Yield: 88%. m.p. 70–72 °C. IR (KBr) υmax/cm−1: 3360.4 (stretching, –NH), 3099.9 (Arbenzene
,
1
C–H), 2981.6 (Arthiophene, C–H), 1600.1 (thiadiazole, C=N), 709.2 (C–S–C). H NMR (400
MHz, DMSO-d6) δ/ppm: 10.48 (s, 1H, –NH), 7.68-7.66 (1H, d, J = 4.9 Hz, thiophene), 7.60-
7.58 (2H, d, J = 7.9 Hz, ArH), 7.55-7.50 (1H, d, J = 4.0 Hz, thiophene), 7.34-7.30 (2H, t, J =
7.9 Hz, ArH), 7.14-7.12 (1H, t, J = 4.3 Hz, thiophene), 7.00-6.96 (1H, t, J = 7.3 Hz, ArH) 13C
NMR (100 MHz, DMSO-d6) δ/ppm: 164.1, 152.4, 140.9, 132.9, 129.7, 129.1, 128.7, 122.7,
4

118.1, 117.3. Anal. Calcd. for C12H9N3S2 (%): C, 55.58; H, 3.50; N, 16.20; found: C, 55.33;
H, 3.45; N, 15.99.
Synthesis of N-(3'-chlorophenyl-5-(thiophen-2-yl)-1,3,4-thiadiazol-2-amine (II):
White solid. Yield: 84%. m.p. 166–168 °C. IR (KBr) υmax/cm−1 : 3185.0 (stretching, –NH),
3052.3 (Arbenzene, C–H), 2878.8 (Arthiophene, C–H), 1593.8 (thiadiazole, C=N), 716.0 (C–S–C).
1H NMR (DMSO-d6) δ/ppm: 10.69 (s, 1H, –NH), 7.87 (s, 1H, ArH), 7.70-7.68 (1H, d, J = 4.9
Hz, thiophene), 7.57-7.56 (1H, d, J = 3.7 Hz, thiophene), 7.41-7.39 (1H, dd, J = 7.3, 1.2 Hz,
ArH), 7.35-7.31 (1H, t, J = 8.2 Hz, ArH), 7.15-7.13 (1H, t, J = 4.0 Hz, thiophene), 7.04-7.01
(1H, dd, J = 7.3, 1.8 Hz, ArH). 13C NMR (DMSO-d6) δ/ppm: 163.59, 153.15, 142.19, 134.07,
132.70, 131.26, 129.69, 129.41, 128.78, 122.19, 117.46, 116.51. Anal. Calcd. for
C12H8N3S2Cl (%): C, 49.06; H, 2.74; N, 14.30; found: C, 48.74; H, 2.88; N, 14.19.
Synthesis of N-(4'-chlorophenyl)-5-(thiophen-2-yl)-1,3,4-thiadiazol-2-amine (III):
White solid. Yield: 86%. m.p. 223 °C. IR (KBr) υmax/cm−1 : 3250.3 (stretching, –NH), 3036.8
1
(Arbenzene, C–H), 2981.3 (Arthiophene, C–H), 1596.3 (thiadiazole, C=N), 690.2 (C–S–C). H
NMR (DMSO-d6) δ/ppm: 8.24 (1H, s, –NH), 7.67-7.66 (1H, d, J = 5.0 Hz, thiophene), 7.63-
7.61 (2H, d, J = 8.7 Hz, ArH), 7.53-7.52 (1H, d, J = 3.7 Hz, thiophene), 7.37-7.35 (2H, d, J =
13
8.7 Hz, ArH), 7.14-7.12 (1H, t, J = 4.7 Hz, thiophene). C NMR (DMSO-d6) δ/ppm: 163.8,
152.9, 152.6, 139.8, 132.6, 129.5, 129.3, 128.8, 126.1, 119.6. Anal. Calcd. for C12H8N3S2Cl
(%): C, 49.06; H, 2.74; N, 14.30; found: C, 48.95; H, 2.69; N, 14.17.
Synthesis of N-(2'-fluorophenyl)-5-(thiophen-2-yl)-1,3,4-thiadiazol-2-amine (IV):
Yellow solid. Yield: 78%. m.p. 108 °C. IR (KBr) υmax/cm−1: 3434.6 (stretching, –NH), 3124.3
1
(Arbenzene, C–H), 2981.9 (Arthiophene, C–H), 1622.1 (thiadiazole, C=N), 613.2 (C–S–C). H
NMR (DMSO-d6) δ/ppm: 10.29 (1H, s, –NH), 8.33-8.29 (1H, t, J = 8.2 Hz, JHF = 8.1 Hz,
ArH), 7.67-7.66 (1H, d, J = 5.0 Hz, thiophene), 7.53-7.52 (1H, d, J = 3.2 Hz, thiophene),
5

7.27-7.16 (2H, m, ArH), 7.14-7.12 (1H, t, J = 4.2 Hz, thiophene), 7.06-7.00 (1H, m, ArH). 13C
NMR (DMSO-d6) δ/ppm: 164.2, 153.7, 153.6, 151.3, 132.9, 132.2, 130.6, 129.4, 129.2,
129.1, 128.8, 125.4, 125.3, 123.9, 123.8, 121.2, 115.9, 115.7. Anal. Calcd. for C12H8N3S2F
(%): C, 51.97; H, 2.91; N, 15.15; found: C, 51.88; H, 2.88; N, 14.99.
Synthesis of N-(4'-fluorophenyl)-5-(thiophen-2-yl)-1,3,4-thiadiazol-2-amine (V):
White solid. Yield: 82%, mp: 175–178 °C. IR (KBr) υmax/cm−1 : 3259.7 (stretching, –NH),
3164.6 (Arbenzene, C–H), 2876.3 (Arthiophene, C–H), 1587.5 (thiadiazole, C=N), 631.7 (C–S–C).
1H NMR (DMSO-d6) δ/ppm: 10.49 (s, 1H, NH), 7.68-7.66 (1H, d, J = 4.9 Hz, thiophene),
7.63-7.59 (2H, d, J = 4.3 Hz, JHF = 3.4 Hz, ArH), 7.53-7.51 (1H, d, J = 3.7 Hz, thiophene),
7.12-7.19 (3H, m, thiophene(1H) and ArH (2H, J = 9.2 Hz, JHF = 9.1 Hz)). 13C NMR
(DMSO-d6) δ/ppm: 164.20, 159.11, 157.08, 156.74, 152.41, 137.45, 132.89, 129.38, 129.15,
128.73, 119.86, 119.77, 116.35, 116.12. Anal. Calcd. for C12H8N3S2F (%): C, 51.97; H, 2.91;
N, 15.15; found: C, 51.73; H, 2.87; N, 14.93.
Synthesis of N-(4'-nitrophenyl)-5-(thiophen-2-yl)-1,3,4-thiadiazol-2-amine (VI):
Yellow solid. Yield: 95%. m.p. 237–240 °C. IR (KBr) υmax/cm−1 : 3168.9 (stretching, –NH),
3044.4 (Arbenzene, C–H), 2913.4 (Arthiophene, C–H), 1583.3 (thiadiazole, C=N), 687.3 (C–S–C).
1H NMR (DMSO-d6) δ/ppm: 11.21 (s, 1H, -NH), 8.22-8.20 (2H, d, J = 8.5 Hz, ArH), 7.80-
7.78 (2H, d, J = 9.2 Hz, ArH), 7.71-7.70 (1H, d, J = 4.9 Hz, thiophene), 7.59-7.58 (1H, d, J =
13
3.1 Hz, thiophene), 7.15-7.13 (1H, t, J = 4.0 Hz, thiophene). C NMR (DMSO-d6) δ/ppm:
162.9, 154.5, 146.6, 141.5, 132.5, 130.1, 129.8, 128.8, 126.0, 117.6. Anal. Calcd. for
C12H8N4O2S2 (%): C, 47.36; H, 2.65; N, 18.41; found: C, 46.98; H, 2.76; N, 18.44.
Synthesis of N-benzyl-5-(thiophen-2-yl)-1,3,4-thiadiazol-2-amine (VII): Yellow solid.
Yield: 90%. m.p. 95 °C; IR (KBr) υmax/cm−1 : 3188.0 (stretching, –NH), 3080.0 (Arbenzene, C–
1
H), 2981.7 (Arthiophene, C–H), 1571.7 (thiadiazole, C=N), 693.1 (C–S–C). H NMR (DMSO-
6

d6) δ/ppm: 8.42-8.39 (1H, t, J = 5.5 Hz, -NH), 7.59-7.58 (1H, d, J = 4.9 Hz, thiophene), 7.38-
7.37 (1H, d, J = 4.2 Hz, thiophene), 7.35-7.29 (4H, m, ArH), 7.25-7.22 (1H, m, ArH), 7.09-
7.07 (1H, t, J = 4.3 Hz, thiophene), 4.49-4.48 (2H, d, J = 5.5 Hz, -CH2). 13C NMR (DMSO-d6)
δ/ppm: 168.50, 151.04, 138.95, 133.52, 128.95, 128.55, 128.31, 128.28, 128.10, 127.74,
48.61. Anal. Calcd. for C13H11N3S2 (%): C, 57.12; H, 4.06; N, 15.37; found: C, 56.97; H,
3.98; N, 15.16.
Synthesis of N-(2'-methoxyhenyl)-5-(thiophen-2-yl)-1,3,4-thiadiazol-2-amine (VIII):
Yellow solid. Yield: 87%. m.p. 68–70 °C. IR (KBr) υmax/cm−1 : 3187.1 (stretching, –NH),
3082.5 (Arbenzene, C–H), 2928.3 (Arthiophene, C–H), 1569.5 (thiadiazole, C=N), 694.8 (C–S–C).
1H NMR (DMSO-d6) δ/ppm: 9.85 (1H, s, –NH), 8.24-8.22 (1H, d, J = 8.5 Hz, ArH), 7.64-
7.62 (1H, d, J = 4.9 Hz, thiophene), 7.48-7.46 (1H, d, J = 3.7 Hz, thiophene), 7.13-7.10 (1H, t,
13
J = 4.3 Hz, thiophene), 7.03-6.91 (3H, m, ArH), 3.83 (3H, –OCH3). C NMR (DMSO-d6)
δ/ppm: 164.7, 153.0, 148.9, 133.1, 129.9, 129.0, 128.9, 128.7, 123.6, 121.2, 119.7, 111.6,
56.3. Anal. Calcd. for C13H11N3OS2 (%): C, 53.96; H, 3.83; N, 14.52; found: C, 53.88; H,
3.75; N, 14.28.
Synthesis of N-(3'-methoxyhenyl)-5-(thiophen-2-yl)-1,3,4-thiadiazol-2-amine (IX):
White–yellow solid. Yield: 89%. m.p. 201–204 °C. IR (KBr) υmax/cm−1 : 3267.0 (stretching, –
NH), 3062.8 (Arbenzene, C–H), 2960.7 (Arthiophene, C–H), 1578.3 (thiadiazole, C=N), 699.7 (C–
1
S–C). H NMR (DMSO-d6) δ/ppm: 10.49 (s, 1H, -NH), 7.68-7.66 (1H, d, J = 4.9 Hz,
thiophene), 7.53-7.52 (1H, d, J = 3.1 Hz, thiophene), 7.31 (1H, s, ArH), 7.24-7.20 (1H, t, J =
8.2 Hz, ArH), 7.15-7.12 (1H, t, J = 4.2, thiophene), 7.07-7.05 (1H, dd, J = 7.9, 1.8 Hz, ArH),
6.58-6.56 (1H, dd, J = 8.2, 2.1 Hz, ArH), 3.72 (3H, s, -OCH3). 13C NMR (DMSO-d6) δ/ppm:
164.0, 160.5, 152.5, 142.1, 132.9, 130.5, 129.4, 129.2, 128.7, 110.7, 108.1, 104.1, 55.6. Anal.
Calcd. for C13H11N3OS2 (%): C, 53.96; H, 3.83; N, 14.52; found: C, 53.74; H, 3.78; N, 14.60.
7

Synthesis of N-(4'-methoxyhenyl)-5-(thiophen-2-yl)-1,3,4-thiadiazol-2-amine (X):
Yellow solid. Yield: 93%. m.p. 181–184 °C. IR (KBr) υmax/cm−1 : 3241.3 (stretching, –NH),
3111.3 (Arbenzene, C–H), 2981.9 (Arthiophene, C–H), 1588.2 (thiadiazole, C=N), 698.5 (C–S–C).
1H NMR (DMSO-d6) δ/ppm: 10.27 (1H, s, –NH), 7.64-7.62 (1H, dd, J = 5.0, 0.9 Hz,
thiophene), 7.51-7.47 (2H, d, J = 8.2 Hz, ArH), 7.47-7.45 (1H, d, J = 3.7 Hz, thiophene),
7.13-7.10 (1H, t, J = 4.3 Hz, thiophene), 6.92-6.88 (2H, d, J = 8.2 Hz, ArH), 3.69 (3H, –
OCH3). 13C NMR (DMSO-d6) δ/ppm: 164.9, 155.3, 151.4, 134.4, 133.1, 129.0, 128.8, 128.7,
120.1, 114.9, 55.8. Anal. Calcd. for C13H11N3OS2 (%): C, 53.96; H, 3.83; N, 14.52; found: C,
53.90; H, 3.75; N, 14.73.
2.3. Antimicrobial Activity Testing
2.3.1. Microorganism Strains
Gram negative (S. enteritidis, S. typhimurium, Enterobacter aerogenes, S. infantis, S.
kentucky, and E. coli) and Gram positive (Staphylococcus aureus (ATCC 25923), B. subtilis,
and Enterococcus durans) bacteria were selected for antibacterial activity testing. The fungus
C. albicans (ATCC 26555) was selected for antifungal activity testing. Streptomycin and
ketoconazole were used as reference clinical standards for the antibacterial and antifungal
activities, respectively. Most strains were ATCC, DSMZ, and SL type standard ones. Other
strains, which have no standard ID information, were isolated from food samples and
identified in the Department of Biology at Ankara University.
2.3.2. Disc Diffusion Method
The disk diffusion method published by Andrews was used for antimicrobial activity
testing [26]. Petri dishes (90 mm diameter) containing 25 mL of Mueller–Hinton Agar were
used [27,28]. The newly synthesized 1,3,4-thiadiazole derivatives were dissolved in ethanol to
8

make a final concentration of 5 mg/mL. Thereafter, 30 µg/mL, 50 µg/mL, and 80 µg/mL
samples were loaded on empty sterile antibiotic disks. The disks were maintained at room
temperature for 24 h in aseptic conditions. Microorganism suspensions (0.5 McFarland) were
inoculated on the surfaces of the Mueller–Hinton Agar plates and kept in aseptic conditions
for 2–3 min before application to the disks [29]. After 24 h, the zone of inhibition, including
the disk, was measured. The zone diameters were measured in millimeters. The disc diffusion
assay was performed in triplicate. Compounds III, VI, and IX were not tested because they
could not be dissolved in solvent.
3. Results and Discussion
3.1. Spectral characterization
Physical data, IR, 1H NMR, and 13C NMR of the synthesized compounds are presented in
Tables 1–4, respectively.
Table 1. Physical characterization data for synthesized 1,3,4-thiadiazoles.
Melting
Point
(°C)
Yield
%
Elemental Analysis
(Theoretical/Found)
Compounds
-Ar
IUPAC name
N-(phenyl)-5-(thiophen-2-yl)-
1,3,4-thiadiazol-2-amine
N-(3'-chlorophenyl)-5
(thiophen-2-yl)-1,3,4 thiadiazol-
2-amine
(%): C, 55.58; H, 3.50; N,16.20
(%): C, 55.33; H, 3.45; N, 15.99
-C6H5
88
84
70–72
I
3-Cl-
C6H5
(%): C, 49.06; H, 2.74; N,14.30
(%): C, 48.74; H, 2.88; N, 14.19
166–168
II
N-(4'-chlorophenyl)-5-
(thiophen-2-yl)-1,3,4-thiadiazol-
2-amine
4-Cl-
C6H5
(%): C, 49.06; H, 2.74; N,14.30
(%): C, 48.95; H, 2.69; N, 14.17
86
78
82
223
108
III
IV
V
N-(2'-fluorophenyl)-5-
(thiophen-2-yl)-1,3,4-thiadiazol-
2-amine
2-F-
C6H5
(%): C, 51.97; H, 2.91; N,15.15
(%): C, 51.88; H, 2.88; N, 14.99
N-(4'-fluorophenyl)-5-
(thiophen-2-yl)-1,3,4-thiadiazol-
2-amine
4-F-
C6H5
(%): C, 51.97; H, 2.91; N,15.15
(%): C, 51.73; H, 2.87; N, 14.93
175–178
4-NO2-
C6H5
N-(4'-nitrophenyl)-5-(thiophen-
2-yl)-1,3,4-thiadiazol-2-amine
N-( benzyl)-5-(thiophen-2-yl)-
1,3,4-thiadiazol-2-amine
N-(2'-methoxyhenyl)-5-
(thiophen-2-yl)-1,3,4-thiadiazol-
2-amine
(%): C, 47.36; H, 2.65; N,18.41
(%): C, 46.98; H, 2.76; N, 18.44
(%): C, 57.12; H, 4.06; N,15.37
(%): C, 56.97; H, 3.98; N, 15.16
95
90
237–240
95
VI
C6H5CH2
VII
2-OCH3-
C6H5
(%): C, 53.96; H, 3.83; N,14.52
(%): C, 53.88; H, 3.75; N, 14.28
87
89
68–70
VIII
IX
3-OCH3-
N-(3'-methoxyhenyl)-5-
201–204
(%): C, 53.96; H, 3.83; N,14.52
9

C6H5
(thiophen-2-yl)-1,3,4-thiadiazol-
2-amine
(%): C, 53.74; H, 3.78; N, 14.60
N-(4'-methoxyhenyl)-5-
(thiophen-2-yl)-1,3,4-thiadiazol-
2-amine
4-OCH3-
C6H5
(%): C, 53.96; H, 3.83; N,14.52
(%): C, 53.90; H, 3.75; N, 14.73
93
181–184
X
3.2. Interpretation of the IR Spectra
IR spectra were obtained between 4000 and 200 cm−1 on a Bruker Alpha FT-IR
spectrophotometer. At the end of the reaction, the carbonyl group absorption on the starting
material (carboxylic acid) was neither observed near 1700 cm−1 nor the OH stretching band
observed at 3500–2700 cm−1. Furthermore, the asymmetric and symmetric stretching bands of
the N-arylthiosemicarbazides amino group (–NH2) were not observed at 3500–3200 cm−1.
Instead, new absorptions for the amine group (–NH) stretching vibrations were observed at
3434.6–3168.9 cm−1, absorptions for –C=N group on the thiadiazole ring were observed at
1622.1–1569.5 cm−1, the –C-N group absorptions were observed at 1292.3-1194.7cm−1, and
C–S–C vibrations from the thiadiazole ring were observed at 716.0–613.2 cm−1 for the
synthesized compounds (I–X). The other remarkable very strong vibrational bands were in the
spectrum of compounds II-X resulting from the Ar–Cl, Ar-F, -NO2, -CH2, -C-O functions,
respectively. Ar-Cl absorption bands of compounds II and III were observed at 866.2 and
871.3 cm-1. Ar-F absorption bands were observed at 845.9 and 827.3 cm-1 for compounds IV
and V. The asymmetric and symmetric stretching bands of the nitro group (–NO2) were
observed at around 1431.9 and 1304.5 cm−1 for compound VI. The -CH2 absorption band of
compound VII was observed at 2888.7 cm-1. The –C-O group absorption bands of compounds
VIII-X were observed at 1245.1, 1234.7, and 1226.6 cm-1, respectively. These observations
provided key evidence for thiadiazole formation. These data are consistent with the values of
earlier reported for similar compounds [1,6,12,30]. Table 2 lists the key IR absorptions of the
synthesized thiadiazole, and a representative IR spectrum of N-(4'-chlorophenyl)-5-(thiophen-
2-yl)-1,3,4-thiadiazol-2-amine (III) is presented in Figure 1.
10

Table 2. Key IR absorption frequencies of the synthesized compounds (I–X).
Compounds
υ(-NH)-stretching υC-H (Aromatic)
υC-H (Ar-thiophene)
2981.6
υC=N (thiadiazole)
1600.1
1593.8
1596.3
1546.4
1587.5
1583.3
1569.5
1599.9
1578.3
1588.2
υC-N
υC-S-C
709.2
716.0
690.2
613.2
631.7
687.3
694.8
699.3
699.7
698.5
I
II
3360.4
3185.0
3250.3
3434.6
3259.7
3168.9
3187.1
3207.8
3267.0
3241.3
3099.9
3052.3
3036.8
3124.3
3164.6
3044.4
3082.5
3102.3
3062.8
3111.3
1255.3
1259.0
1241.2
1249.9
1258.8
1194.7
1259.5
1292.3
1250.4
1278.3
2878.8
III
IV
V
2981.3
2981.9
2876.3
VI
VII
VIII
IX
X
2913.4
2928.3
2981.8
2960.7
2981.9
Figure 1. IR spectrum of N-(4'-chlorophenyl)-5-(thiophen-2-yl)-1,3,4-thiadiazol-2-amine
(III).
3.3. Interpretation of the 1H NMR Spectra
1
The H NMR spectra of the synthesized compounds were recorded in DMSO-d6 and the
chemical shifts are presented in Table 3. For compounds I-X, aromatic proton signals of the
11

benzene ring were observed between 8.33 and 6.56 ppm, and aromatic proton signals of the
thiophene region were observed between 7.71 and 7.07 ppm. In compounds (VIII, IX, and
X), –OCH3 proton signals were observed as a singlet in the range of 3.83–3.69 ppm. The –NH
proton signals were observed as singlets in the range of 11.21-8.24 ppm for all the
compounds. For compound VII, the methylene group proton signal was detected as a doublet
at 4.49-4.48 (2H, d, J = 5.5 Hz) ppm; the –NH proton signals were observed as a triplet 8.42-
1
8.39 (1H, t, J = 5.5 Hz) A representative H NMR spectrum of N-(4'-chlorophenyl)-5-
(thiophen-2-yl)-1,3,4-thiadiazol-2-amine (III) is presented in Figure 2. As seen in compound
III, while aromatic protons (H4-H8) of benzene ring were observed at 7.63-7.35 ppm,
aromatic protons (H1-H3) of thiophene ring were observed at 7.67-7.12 ppm. The H4 and H8
proton coupled to the H5 and H7 proton and detected doublet peaks at 7.63-7.61 (J = 8.7 Hz)
ppm, respectively. The H5 and H7 proton coupled to the H4 and H8 proton and detected
doublet peaks at 7.37-7.35 (J = 8.7 Hz) ppm, respectively. The H1 proton coupled to the H2
proton and detected doublet peaks at 7.67-7.66 (J = 5.0 Hz) ppm. The H2 proton coupled to
the H1 and H3 proton and detected triplet peaks at 7.14-7.12 (J = 4.7 Hz) ppm. The H3 proton
coupled to the H2 proton and detected doublet peaks at 7.53-7.52 (J = 3.7 Hz) ppm. The
amino group (–NH) proton signal showed as a singlet in the range of 8.24 ppm. DMSO-d6
and water in DMSO (HOD, H2O) signals were seen around at 2.50 (quintet) and 3.30
(variable, based on the solvent and its concentration) ppm, respectively [31]. These results are
consistent with the values of earlier reported for similar compounds [1,6,12, 30]. Moreover,
1
selected compounds’ IR, H NMR, and 13C NMR spectra were given as supplementary
material.
12

Table 3. 1H NMR data of compounds for I–X, (δ/ppm).
H5
H3
H4
H6
H7
N
N
H2
R
S
N
H
S
H1
H8
Aromatic protons (benzene)
H5; H7
Aromatic protons (thiophene)
H2
-OCH3
or
-CH2
Comp.
I
-NH
H4;H8
H6
H1
H3
7.55-7.50
(1H, d, J =
4.0 Hz)
7.60-7.58 (2H,
7.34-7.30 (2H,
7.00-6.96 (1H,
7.68-7.66 (1H,
d, J = 4.9 Hz)
7.14-7.12 (1H, t,
J = 4.3 Hz)
-
-
10.48
d, J = 7.9 Hz)
t, J = 7.9 Hz)
t, J = 7.3 Hz)
7.87 (1H, s)
7.04-7.01 (1H,
dd, J = 7.3, 1.8
Hz)
7.41-7.39 (1H,
dd, J = 7.3, 1.2
Hz)
7.57-7.56
(1H, d, J =
3.7 Hz)
7.35-7.31 (1H
t, J = 8.2 Hz)
7.70-7.68 (1H,
7.15-7.13 (1H, t,
II
10.69
d, J = 4.9 Hz)
J = 4.0 Hz)
7.53-7.52
(1H, d, J =
3.7 Hz)
7.63-7.61 (2H,
d, J = 8.7 Hz)
7.37-7.35 (2H,
d, J = 8.7 Hz)
7.67-7.66 (1H,
d, J = 5.0 Hz)
7.14-7.12 (1H, t,
J = 4.7 Hz)
III
IV
-
-
-
-
-
8.24
10.29
10.49
11.21
8.33-8.29 (1H, t, J = 8.2 Hz, JHF = 8.1 Hz),
7.53-7.52
(1H, d, J =
3.2 Hz)
7.67-7.66 (1H,
d, J = 5.0 Hz)
7.14-7.12 (1H, t,
J = 4.2 Hz)
7.06-7.00 (1H, m), 7.27-7.16 (2H, m)
7.63-7.59 (2H,
d, J = 4.3 Hz,
JHF = 3.4 Hz)
7.13-7.19 (2H,
m, J = 9.2 Hz,
7.53-7.51
(1H, d, J =
3.7 Hz)
7.68-7.66 (1H,
d, J = 4.9 Hz)
7.12-7.13 (1H,
m)
V
-
-
J
HF = 9.1 Hz)
7.80-7.78 (2H,
d, J = 9.2 Hz,
ArH)
8.22-8.20 (2H,
d, J = 8.5 Hz,
ArH),
7.59-7.58
(1H, d, J =
3.1 Hz)
7.71-7.70 (1H,
d, J = 4.9 Hz)
7.15-7.13 (1H, t,
J = 4.0 Hz)
VI
4.49-
4.48
7.38-7.37
(1H, d, J =
4.2 Hz)
7.25-7.22(1H,
m)
7.59-7.58 (1H,
d, J = 4.9 Hz)
7.09-7.07 (1H, t,
J = 4.3 Hz )
8.42-
8.39
VII
VIII
IX
7.35-7.29 (4H, m)
8.24-8.22 (1H, d, J = 8.5 Hz),
7.48-7.46
(1H, d, J =
3.7 Hz)
7.64-7.62 (1H,
d, J = 4.9 Hz)
7.13-7.10 (1H, t,
J = 4.3 Hz)
3.83
3.72
9.85
7.03-6.91 (3H, m, ArH),
7.31 (1H, s)
6.58-6.56 (1H,
dd, J = 8.2, 2.1
Hz)
7.07-7.05 (1H,
dd, J = 7.9, 1.8
7.53-7.52
(1H, d, J =
3.1 Hz)
7.68-7.66 (1H,
d, J = 4.9 Hz)
7.15-7.12 (1H, t,
J = 4.2 Hz)
7.24-7.20 (1H,
10.49
t, J = 8.2 Hz)
Hz)
7.64-7.62
7.47-7.51 (2H,
d, J = 8.2 Hz)
6.88-6.92 (2H,
d, J = 8.2 Hz)
7.13-7.10 (1H, t,
J = 4.3 Hz)
7.45-7.47
(1H, d, J =
3.7 Hz)
X
-
3.69
10.27
(1H, dd, J = 5.0,
0.9 Hz)
13

Figure 2. 1H NMR spectrum of N-(4'-chlorophenyl)-5-(thiophen-2-yl)-1,3,4-thiadiazol-2-amine (III).
3.4. Interpretation of the 13C NMR Spectra
The 13C NMR spectra of the synthesized compounds were recorded in DMSO-d6 and the
chemical shifts are presented in Table 4. The aromatic C signals from the benzene ring (C7–
C12) were observed between 157.1 and 104.1 ppm for all compounds (I–X), those from the
thiophene ring (C1–C4) were observed between 152.4 and 121.2 ppm, and from the 1,3,4-
thiadiazole ring (C5 and C6) were observed between 168.5 and 151.0 ppm.
In the presence of electronegative elements (–N, -O, –Cl, and –F), the resonances of the
carbon atoms shifted down-field (high values of δ). Conversely, the carbon atoms resonated
up-field (low values of δ) due to the increase in electron density in the presence of
electropositive atoms (Li, Si). In compounds III, V (two carbons), VI, and X, the C10
carbons resonated at 152.6; 157.1, 156.7; 151.4; and 146.6 ppm, respectively. As for, the
14

effect of different groups are seen in the compounds III, V, VI, and X, where the signals of
the para carbons are shifted upfield by the 4-Cl, 4-F, 4-NO2, and 4-OCH3 groups, relative to
the signals of benzene (I, 128.5 ppm). Additionally, in compounds IV and V, the C atoms (for
C7-C12) were also split into doublets due to interacting with the atomic nucleus of F.
The C6 signal was down-field compared to the C5 due to the decrease in the electron
density from multiple inductive effects (surrounded by heteroatoms). For all compounds (I–
X) the C6 was observed between 168.5 and 162.9ꢀppm, whereas C5 was observed between
159.1 and151.0 ppm.
In compounds (VIII, IX, and X), the methoxy carbon atoms (–OCH3) resonated at 56.3,
55.6, and 55.8 ppm, respectively. In compounds (VIII, IX, and X), the C7-C12 carbon atoms
were affected by both methoxy (–OCH3) and amino (-NH, C7) groups. Therefore, the shifts
induced by these substituents were added, wherein the signals of the carbons (C7-C12) were
observed up-field or down-field shifts relative to ortho, meta,and para position of these
groups.
For compound VII, the methylene C13 was detected at 48.6 ppm. These data consistent
with the values of earlier reported for similar compounds [1,6,12,30,32]. A representative 13C
NMR spectrum of N-(4'-chlorophenyl)-5-(thiophen-2-yl)-1,3,4-thiadiazol-2-amine (III) is
presented in Figure 3.
Table 4. 13C NMR data of compounds I–X, (δ/ppm)
9
N
3
N
N
8
3
10
N
2
4
13
8
6
5
R
2
9
7
7
5
6
S
N
4
11
S
N
H
S
1
R
S
H
1
12
Compund VII
12
Compounds I-X except for VII
10
11
R
H
I
3-Cl
II
4-Cl
III
2-F
4-F
4-NO2
VI
H
VII
2-OCH3
VIII
3-OCH3
IX
4-OCH3
X
IVa
Va
C1
C2
C3
C4
C5
C6
132.9 131.3 132.6 132.9 128.7 128.8
129.1 128.8 128.8 130.6 137.5 130.1
122.7 122.2 126.1 121.2 132.9 129.8
140.9 132.7 129.3 132.2 152.4 132.5
152.4 153.1 152.9 151.3 159.1 154.5
164.1 163.6 163.8 164.2 164.2 162.9
133.5 129.9
128.6 128.9
127.7 128.7
138.9 129.0
151.0 153.0
168.5 164.7
130.5
128.7
129.2
129.4
152.5
164.0
133.1
128.8
128.7
129.0
155.3
164.9
15

123.9
123.8
J = 4.5 J =
129.4
129.2
C7
C8
129.7 134.1 139.8
117.3 129.4 129.5
141.5
117.6
128.1 133.1
128.2 123.6
142.1
110.7
134.4
114.9
Hz
12.0
Hz
116.4
116.1
J =
129.4
129.2
J = 8
Hz
23.0
Hz
125.4
125.3
J = 16
Hz
129.1
128.8
J = 26
Hz
119.9
119.8
J =
8.6 Hz
157.1
156.7
J =
C9
118.1 129.7 119.6
128.7 117.5 152.6
126.0
146.6
128.9 119.7
128.3 121.2
132.9
108.1
120.1
151.4
C10
234.5
Hz
115.9
115.7
J = 34
Hz
153.7
153.6
J =
119.9
119.8
J =
8.6 Hz
116.4
116.1
J =
C11
C12
118.1 142.2 119.6
117.3 116.5 129.5
126.0
117.6
128.9 111.6
128.2 148.9
160.5
104.1
120.1
114.9
227.5
Hz
23.0
Hz
C13
R
-
-
-
-
-
-
-
-
-
-
-
-
48.6
-
-
-
-
56.3
55.6
55.8
a In compounds IV (different six C) and V (different four C), C7-C12 atoms were observed split into doublets by
interacting with the atomic nucleus of F.
Figure 3. 13C NMR spectrum of N-(4'-chlorophenyl)-5-(thiophen-2-yl)-1,3,4-thiadiazol-2-amine (III).
16

3.5. Antimicrobial Activity
In vitro antimicrobial activity tests of the synthesized 1,3,4-thiadiazoles were performed
against nine different bacteria (Salmonella enteritidis, Staphylococcus aureus, Salmonella
typhimurium, Enterobacter aerogenes, Salmonella infantis, Bacillus subtilis, Salmonella
kentucky, Escherichia coli, and Enterococcus durans) and one fungus (Candida albicans)
using the disk diffusion method. The antimicrobial activities were measured with zone
inhibition in mm and the results were presented in Table 5. Compounds V and VIII revealed
antifungal activity against C. albicans, with inhibition zones of 7 and 8 mm, respectively.
Compared to standard of Ketoconazole (13 mm), compounds V and VIII were shown
moderate antifungal activity. All the tested compounds (compounds III, VI, and IX could not
be tested due to solubility issues) revealed antibacterial activity against Staphylococcus
aureus. Compound II has the best antibacterial activity against Staphylococcus aureus with
inhibition zones of 14 mm at 80 µg/mL. Compared to standard of Streptomycin (18 mm),
compounds V, VII and X have shown good antibacterial activities, while compounds I, II,
IV, and VIII have exhibited moderate antibacterial activities. Compound V demonstrated
good inhibition against Staphylococcus aureus at 50 and 80 µg/mL. The order of the
antimicrobial activity against Staphylococcus aureus is as follows: V=VII>X>I>II>IV>VIII
(30 µg/mL), V>VII>X>I>II>VIII=IV (50 µg/mL), and II>V>VII>X>I>IV>VIII (80
µg/mL).
Compound II reported antibacterial activity against S. typhimurium with an inhibition
zone of 7 mm. Compound VII revealed antibacterial activity against Enterobacter aerogenes
with inhibition zones of 8, 9, and 9 mm using 30, 50, and 80 µg/mL, respectively. Compounds
VIII and X indicated antibacterial activity against S. kentucky with inhibition zones from 7 to
9 mm. Nevertheless, none of the tested compounds demonstrated antibacterial activity against
17

the remaining five bacterial strains (S. enteritidis, S. infantis, B. subtilis, E. coli, and
Enterococcus durans).
The synthesized 1,3,4-thiadiazoles presented differing degrees of antimicrobial activity.
All of the testable compounds presented significant antibacterial activity against
Staphylococcus aureus. Selected compounds presented antimicrobial activity against C.
albicans, S. typhimurium, Enterobacter aerogenes, and S. kentucky; however, none of the
compounds presented antimicrobial activity against S. enteritidis, S. infantis, B. subtilis, E.
coli, and Enterococcus durans. These results corroborate those of Hussain et al [33]. They
synthesized novel 4-amino-2-{5-[(4-substitutedphenyl)amino]-1,3,4-thiadiazole-2-yl} phenol
derivatives and found them to display antibacterial activity against E. coli (Gram negative)
and Staphylococcus aureus (Gram-positive) bacteria. Moreover, Gür and co-workers studied
1,3,4 thiadiazole moiety and their antimicrobial activities [34]. They found antifungal
activity against C. albicans, antibacterial activities against Staphylococcus aureus (Gram-
positive), and Enterobacter aerogenes (Gram negative). Bhat and co-workers studied
antimicrobial activities of 3-(1,3,4-thiadiazole-2-yl)quinoline derivatives [35]. They found
antibacterial activities against S. typhimurium (Gram negative).
Table 5. Antimicrobial activity results of compounds I, II, IV, V, VII, VIII, and X.
Microorganisms, zone of inhibition (mm)
S.
µg/
mL
C.
Stap.
S.
E.
S.
B.
S.
E.
E.
Comp.
enterit
albicans aureus Typhimur. aerogenes
infants
subtilis kentucky coli durans
idis
30
50
80
30
50
80
30
50
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
10
10
10
8
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
8
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
I
-
7
7
7
-
II
9
14
7
IV
8
-
18

80
30
50
80
30
50
80
30
50
80
30
50
80
80
80
-
-
7
9
12
13
13
12
12
12
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
V
VII
VIII
X
-
7
-
-
-
-
-
-
-
-
8
-
-
-
-
-
-
-
-
-
-
8
9
9
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
7
-
-
-
-
9
-
-
-
7
8
-
-
-
9
-
-
-
-
7
8
-
-
-
-
-
-
9
-
-
-
-
11
11
11
18
NA
-
-
7
-
-
-
-
-
-
-
7
-
-
-
-
-
-
-
-
-
7
-
Strept.
5
NA
13
12
NA
15
NA
6
2
14
NA
-
5
NA
NA
NA
NA
Ketoco.
NA
Strep. : Streptomycin Ketoco.: Ketoconazole NA = not applicable
Note: Compounds III, VI, and IX could not be studied due to solubility issues
4. Conclusions
In this study, novel 1,3,4-thiadiazole compounds were synthesized from the reaction of
thiophene-2-carboxylic acid and N-arylthiosemicarbazides in the presence of phosphorous
oxychloride at reflux. The synthesized compounds were characterized by using IR, 1H NMR,
and 13C NMR spectroscopies and elemental analysis. The resulting 1,3,4-thiadiazole
derivatives were prepared in excellent yields (78–95%) and tested for the antimicrobial
activity. Selected compounds demonstrated activity against Staphylococcus aureus, C.
albicans, S. typhimurium, Enterobacter aerogenes, and S. kentucky. It was seen that the 1,3,4-
thiadiazole compounds have antibacterial activity against Staphylococcus aureus, S.
typhimurium, Enterobacter aerogenes, and S. Kentucky, along with antifungal activity against
C. albicans. Compound II is the best antibacterial activity against Staphylococcus aureus
(gram-positive). Compounds V, VII and X were shown good antibacterial activities against
Staphylococcus aureus.
19

Acknowledgements: We would like to thank Kastamonu University (Grant No. KÜBAP-
01/2014-26) for its financial support of this work. We are grateful to the Scientific Research
Center for Industrial and Technological Applications and Research Centre (BETUM) and Dr.
Hatice Karadeniz for taking the NMR spectra.
Supplementary Material
1
IR, H NMR, and 13C NMR, spectra of the compounds are given in the supporting
information.
Conflict of Interest: The authors declare that they have no conflict of interest.
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Highlights
•
•
•
New 1,3,4-thiadiazoles based on thiophene-2-carboxylic acid were synthesized.
The structures were determined using spectroscopic methods.
The synthesized compounds were examined for antimicrobial activities.
•
Some 1,3,4-thiadiazoles had effective antimicrobial activity against S. aureus, S.
typhimurium, E. aerogenes, S. kentucky, and C. albicans.

Author Contribution Statement
Halit Muğlu: Supervision, Methodology, Project administration, Resources.
Hasan Yakan: Conceptualization, Methodology, Writing- Original draft preparation,
Writing-Review & Editing.
Hanan Almabrok Shouaib: Investigation, Methodology, Formal analysis.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: