Preparation, structure elucidation, and antioxidant activity of new bis(thiosemicarbazone) derivatives

Article abstract of DOI:10.3906/KIM-2002-76

Schiff-base–bearing new bis(thiosemicarbazone) derivatives were prepared from terephthalaldehyde and various thiosemicarbazides. FT–IR, ¹ H NMR, ¹³ C NMR, and UV–Vis spectroscopic methods and elemental analysis were used to elucidate

Full text of DOI:10.3906/KIM-2002-76

Turkish Journal of Chemistry
Turk J Chem
2020) 44: 1085 – 1099
TÜBİTAK
doi:10.3906/kim-2002-76
(
©
http://journals.tubitak.gov.tr/chem/
Research Article
Preparation, structure elucidation, and antioxidant activity of new
bis(thiosemicarbazone) derivatives
∗
Hasan YAKAN 
Department of Mathematics and Science Education, Faculty of Education, Ondokuz Mayis University,
Samsun, Turkey
Received: 24.02.2020
•
Accepted/Published Online: 05.06.2020
•
Final Version: 18.08.2020
Abstract: Schiff-base–bearing new bis(thiosemicarbazone) derivatives were prepared from terephthalaldehyde and var-
1
13
ious thiosemicarbazides. FT–IR, H NMR, C NMR, and UV–Vis spectroscopic methods and elemental analysis were
used to elucidate the identification of the synthesized molecules. The in vitro antioxidant activity of the synthesized com-
pounds was analysed with the 1,1-diphenyl-2-picryl hydrazyl free-radical–trapping process. The synthesized compounds
exhibited lower antioxidant activity than the standard ascorbic acid. IC50 values of the synthesized molecules measured
from 3.81 ± 0.01 to 29.05 ± 0.11 µM. Among the synthesized compounds, compound 3 had the best antioxidant
activity. Moreover, this study explained the structure–activity relationship of the synthesized molecules with different
substituents in radical trapping reactions.
Key words: Thiosemicarbazides, Schiff bases, DPPH, structure–activity relationship, spectroscopic methods
1. Introduction
Thiosemicarbazones are an important class of organic chemicals which have an -NH-C(=S)NH-N= bond. They
are used as versatile intermediates for synthesizing numerous moieties as well. Thiosemicarbazones have shown
numerous medicinal properties and biological activities such as anticonvulsant [1], antituberculosis [2], urease
inhibitory [3], anticancer [4], antiviral [5], antimicrobial [1], antibacterial [6], and antioxidant activity [7–11].
Schiff bases have an extensive range of biological and medicinal properties, such as antimicrobial [12],
antiinflammatory [13], antibacterial [14], antifungal [15], insecticidal [16], anticancer [17] antituberculosis [18],
anticonvulsant [19], and antioxidant [20] activity. They are also used as pigments, catalysts, polymer stabilisers,
and intermediates in organic synthesis [21].
The significance of free radicals and reactive oxygen species (ROS) in the pathogenicity of various diseases
such as metabolic disorders, reperfusion damage, inflammatory diseases, cellular aging, and cancer has attracted
substantial consideration [22–24]. Many of these illnesses occur with the bulking of free radicals in humans.
Hence, antioxidants have been noted to play a major role in preserving humans from many potentially fatal
illnesses.
In view of this evidence, Schiff bases are important not only in possessing biological activities, but
also in pharmacological chemistry. In this paper, a series of novel bis(thiosemicarbazones) based on Schiff
bases were prepared by condensation reaction with terephthalaldehyde and various thiosemicarbazides under
∗Correspondence: hasany@omu.edu.tr
1
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This work is licensed under a Creative Commons Attribution 4.0 International License.

YAKAN/Turk J Chem
reflux in ethanol. The structures of the new compounds were characterized by Fourier-transform infrared
1
13
(
FT–IR), proton–carbon nuclear magnetic resonance ( H NMR– C NMR), and ultraviolet–visible (UV–Vis)
spectroscopic techniques, as well as elemental analysis. Antioxidants are very important reagents because they
can trap free radicals and reduce their damaging effects on our bodies. Therefore, the in vitro antioxidant
activity of all of the obtained molecules were evaluated by the 1,1-diphenyl-2-picryl hydrazyl (DPPH) free-
radical trapping process. This paper investigated the effects of diverse structural features on the antioxidative
activity of all the compounds. Moreover, I studied the electronic effect of electron-donating and electron-
withdrawing groups as the substituents on antioxidant activity, as well as their positions. The aim of this study
was to explain the structure–activity relationship of the products with various substituents in radical scavenging
reactions.
2
2
. Experimental
.1. Measurement and reagents
All chemicals and solvents were purchased from Merck & Co., Inc. (Kenilworth, NJ, USA) or Sigma-Aldrich
Chemical Company (St. Louis, MO, USA). They were used without further purification. A Stuart SMP30
melting point apparatus (Cole-Parmer Instruments Co., Vernon Hills, IL, USA) was used to determine melting
points. Infrared (IR) spectra were recorded with a Bruker Alpha Fourier transform IR (FT–IR) spectrometer
(Bruker BioSpin Corp., Billerica, MA, USA). NMR spectra were taken on a Bruker Avance DPX–400 (400
MHz) spectrophotometer using dimethyl sulfoxide-d6 (DMSO–d6) as solvent. A Eurovector EA3000 elemental
analyser was used for elemental analysis (Eurovector S.r.l., Pavia, Italy). UV–Vis spectra and absorption data
were obtained with a Shimadzu UVM-1240 UV–Visible spectrophotometer (Shimadzu Corporation, Kyoto,
Japan).
2.2. Synthesis of bis(thiosemicarbazones) based on Schiff base (1–10)
To a solution of hydrazine monohydrate (5.0 mmol) in ethanol (10 mL), a suspension of an appropriate isoth-
iocyanate (5.0 mmol) in ethanol (10 mL) was added dropwise with vigorous stirring and cooling in an ice bath.
The mixture was allowed to stand overnight. The solid molecule so formed was filtered and dried, producing
thiosemicarbazides as an intermediate product. Terephthalaldehyde (2.0 mmol) and thiosemicarbazide deriva-
tives (4.0 mmol) were stirred into 50 mL of absolute ethanol in the presence of 3 drops of glacial CH3 COOH.
The reaction mixture was refluxed for 4 h. When the reaction was finished, the reaction mixture was cooled
to room temperature. The solid products so formed were filtered and dried (Scheme 1). They were obtained
according to an earlier procedure, with slight changes [25].
2.3. Antioxidant activity
The antioxidant activities of the newly synthesized compounds were evaluated based on the studies of Brand-
Williams (with slight modification) using DPPH radical scavenging activity analysis [26]. Stock solutions of the
compounds in DMSO at a concentration of 250 µM were prepared. The previously prepared DPPH solution was
used in 3 mL with a concentration of 100 µM in ethanol. DPPH solution was added to different concentrations
of the compound solutions (0.5–10.0 µM) and enough ethanol to bring the volume up to a total of 5 mL. This
mixture was allowed to stand at room temperature for 30 min in the dark. The measurement against the gap
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YAKAN/Turk J Chem
Scheme 1. Synthetic route for bis(thiosemicarbazones) based on Schiff base (1–10).
was taken at 517 nm, and the radical scavenging activity was expressed as a percentage of inhibition. Percentage
of inhibition was calculated as follows [27]:
Inhibition (%) = [(A0 – A1) / A0 × 100];
where A0 is the absorbance of the control and A1 is the absorbance of the sample. The inhibition
curves were prepared, and the half-maximal inhibitory concentration (IC50) values were calculated using linear
regression analysis [27].
3
3
. Results and discussion
.1. Physical properties
The results for the physical properties, yields, melting points, and elemental analyses are given in Tables 1 and
2
.
3.2. Vibrational frequencies
In the FT-IR spectra, the signal of the aldehyde group (-CHO, 2 bands) of the starting material was not
−
1
observed at 2780–2660 cm . Moreover, the symmetric and asymmetric stretching peaks of the amino group
−
1
(
-NH2) did not show at 3520–3250 cm . Instead, new vibrations for the –C=N stretching peaks of imine
−
1
group were observed at 1599–1522 cm . These results indicated a successful reaction, as expected. For the
−
1
synthesized compounds (1–10), the amine group (–NH) vibration peaks were observed at 3368–3269 cm , the
C=S signals of the thiosemicarbazone region were observed at 1462–1409 cm , and the -C–N group vibrations
were observed at 1222–1152 cm . The other considerable vibrations were in the spectrum of compounds 1–
0 resulting from the –C-O, –NO2 , Ar–Cl, Ar–F, and –CH2 , functions, respectively. The -C–O stretching
vibrations of compounds 1 and 2 were observed at 1045 and 1048 cm . The nitro group (–NO2) stretching
peaks were observed at around 1508 and 1317 cm
was observed at 767 cm . For compounds 5–7, Ar–F vibration signals were observed at 940, 932, and 936
cm , respectively. In the literature, Ar–Cl and Ar–F vibration peaks were observed at around 811–856 cm
−
1
–
−
1
1
−
1
−
1
for compound 3. The Ar–Cl vibration signal of compound
−
1
4
−
1
−1
,
−
1
9
00–1038 cm , respectively [10,28].
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YAKAN/Turk J Chem
Table 1. Physical properties of the synthesized compounds.
Comp. Compound
name
-R
Yield % Melting
Colour
Yellow
point
◦
(
C)
1
2
3
4
5
6
7
8
9
1
(2,2')-1,4-
2-OCH3-C6H5
86
95
86
90
72
97
91
81
>300
Benzenedicarboxaldehyde-1-
methanylylidene-bis(4-(2-methoxy-
phenyl)thiosemicarbazone)
(2,2')-1,4-
3-OCH3-C6H5
4-NO2-C6H5
3-Cl-C6H5
2-F-C6H5
222–224
270–273
>300
Yellow
Benzenedicarboxaldehyde-
1
-methanylylidene-bis(4-(3-
methoxyphenyl)thiosemicarbazone)
(2,2')-1,4-
Light orange
Dark yellow
Yellow
Benzenedicarboxaldehyde-
1
-methanylylidene-bis(4-(4-
nitrophenyl)thiosemicarbazone)
(2,2')-1,4-
Benzenedicarboxaldehyde-
1
-methanylylidene-bis(4-(3-
chlorophenyl)thiosemicarbazone)
(2,2')-1,4-
>300
Benzenedicarboxaldehyde-
1
-methanylylidene-bis(4-(2-
fluorophenyl)thiosemicarbazone)
(2,2')-1,4-
3-F-C6H5
>300
Dark yellow
Yellow
Benzenedicarboxaldehyde-
1
-methanylylidene-bis(4-(3-
fluorophenyl)thiosemicarbazone)
(2,2')-1,4-
4-F-C6H5
268–270
>300
Benzenedicarboxaldehyde-
1
-methanylylidene-bis(4-(4-
fluorophenyl)thiosemicarbazone)
(2,2')-1,4-
C6H5CH2
Yellow
Benzenedicarboxaldehyde-
1
-methanylylidene-bis(4-
(
benzyl)thiosemicarbazone)
(2,2')-1,4-
C6H5 CH2CH2 80
280–283
>300
Yellow
Benzenedicarboxaldehyde-1-
methanylylidene-bis(4-(2-phenyl
ethane)thiosemicarbazone)
0
(2,2')-1,4-
C6H11
90
Light orange
Benzenedicarboxaldehyde-
1-methanylylidene-bis(4-
(
cyclohexzyl)thiosemicarbazone)
−
1
In compound 4, the –NH stretching vibration was observed at 3298 cm . The –C=N vibration signal
−
1
−1
appeared at 1544 cm , the –C=S signal of thiosemicarbazone region was observed at 1409 cm , and the
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YAKAN/Turk J Chem
Table 2. Elemental analysis results of the compounds.
Comp. Molecular mass (g/mol) Molecular formula Calculated
Experimental
C%
H% N%
(C)% (H)% (N)%
1
2
3
4
5
6
7
8
9
1
492.14
492.14
522.09
500.04
468.10
468.10
468.10
460.15
488.18
444.21
C24H24N6O2S2
C24H24N6O2S2
C22H18N8O4S2
C22H18Cl2N6S2
C22H18F2N6S2
C22H18F2N6S2
C22H18F2N6S2
C24H24N6S2
58.52 4.91 17.06 58.69 4.87
58.52 4.91 17.06 58.32 4.95
50.57 3.47 21.44 50.80 3.38
52.69 3.62 16.76 52.55 3.66
56.39 3.87 17.94 56.21 3.83
56.39 3.87 17.94 56.24 3.84
56.39 3.87 17.94 56.55 3.79
62.58 5.25 18.25 62.33 5.17
63.90 5.78 17.20 63.72 5.92
59.42 7.25 18.90 59.65 7.11
16.98
17.16
21.35
16.59
17.91
17.90
17.86
18.37
17.11
19.01
C26H28N6S2
C22H32N6S2
0
−
1
-
C–N stretching vibration appeared at 1195 cm , as shown in Figure 1. The IR peaks of the compounds
are presented in Table 3 (see Supplementary information). The frequency data of all of the molecules were
consistent with those reported for similar compounds [8,10,28,29].
Figure 1. FT-IR spectrum of compound 4.
1
3
.3. H NMR interpretations
1
The H NMR spectra of all of the compounds were taken in DMSO-d6 ; the chemical shifts are given in Table 4.
Signals of DMSO-d6 and water in DMSO (HOD, H2 O) were seen around 2.00, 2.55 (quintet), and 3.40 (variable,
depend on the solvent and its concentration) ppm, respectively [30,31].
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YAKAN/Turk J Chem
Table 3. Experimental FT-IR values of the compounds (cm− ).
1
Comp. -NH Ar.CH Aliph.CH C=N C=S C-N Spec. vib.
1
2
3
4
5
6
7
8
9
1
3269 3122
3307 3122
3306 3157
3298 3142
3331 3132
3335 3144
3306 3134
3308 3163
3368 3158
3292 3164
2969
2940
-
-
-
-
-
2933
2923
2928
1599 1462 1192 C–O:1045
1545 1455 1217 C–O:1048
1537 1415 1186 NO2:1508–1317
1544 1409 1195 C–Cl:767
1555 1456 1190 C–F:940
1593 1414 1161 C–F:932
1538 1409 1152 C–F:936
1540 1415 1222
1522 1451 1182
1525 1446 1153
-
-
-
0
1
Table 4. H NMR (δ, ppm, in DMSO-d
6
) values of synthesized compounds.
Comp. H1
H2
H3
H4
H5 H6 Ar-NH NH-N
CH=N Spec.
peaks
1
2
-
8.22–8.21 d
-
7.23–7.19 td 7.00–6.96 td 7.13–7.10 d
7.89 s 10.04 s
6.76–6.73 dd 7.91 s 10.07 s
6.61–6.59 d 8.07 s 10.50 s
12.02 s 8.19 s
-OCH3:
3
.90, s
7.16-7.14 d
7.25–7.21
t
11.86 s 8.14 s
-OCH3:
3.72, s
-
-
-
3
4
5
6.61–6.59 d
7.79 s
-
8.28–8.25 d
-
7.56–7.52 m 7.35–7.22
-
8.28–8.25 d
12.30 s 8.00 s
12.05 s 8.21 s
12.06 s 8.18 s
7.62–7.60 dd 7.43–7.39 t
7.29–7.26 dd 7.97 s 10.24 s
7.94 s 11.04 s
m
6
7.58–7.54 d
7.22–7.17 d
-
7.43–7.33
m
-
7.02–6.98 t
7.92 s 10.18 s
7.96 s 10.18 s
11.97 s 8.15 s
11.94 s 8.18 s
-
7
8
7.60-7.55 d
7.60–7.55 d
7.22–7.17 d
7.21–7.17, t
-
7.21–7.17, t 7.32–7.26
7.80 s 9.10–9.07 t 11.63 s 8.05 s
-CH2:
4.82–4.80
d
m
9
1
7.26–7.22 m 7.36–7.29
m
7.26–7.22 m
7.82 s 8.63–8.60 t 11.62 s 8.08 s
Ar-CH2:
2.97–
2
t
.93,
-
CH2-N:
3
3
.82–
.76,
q
0
-
-
-
-
-
7.82 s 8.07 s
11.50 s 8.05 s
-CH:
4
4
.24–
.18,
p
-
CH2:
.91–
.14,
1
1
m
1
2
In compound 4, the signal of imine (-CH=N) was observed as a singlet at 8.21 ppm. The -N H and -N H
proton signals of the thiosemicarbazone region were observed as a singlet at 10.24 and 12.05 ppm, respectively.
The aromatic proton signals of the isatin ring (H1–H5) were detected between 7.79 and 7.26 ppm (Figure 2).
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YAKAN/Turk J Chem
The H1 proton showed as a singlet peak at 7.79 ppm. The H3 proton coupled to the H4 and H5 protons and
observed doublet of doublets peakes at 7.62–7.60 (J = 7.4, 1.8 Hz) ppm. The H4 proton coupled to the H3
and H5 protons and showed triplet peaks at 7.43–7.39 (J = 8.1 Hz) ppm. The H5 proton coupled to the H3
and H4 protons and showed doublet of doublets peaks at 7.29–7.26 (J = 8.0, 3.0 Hz) ppm. The H6 proton
was detected as a singlet peak at 7.97 (s, 2H) ppm. For compounds 1 and 2, the proton signals of the methoxy
group (–OCH3) showed as a singlet at 3.90 and 3.72 ppm. For compound 8, the proton signal of methylene
group (–CH2) was observed as a doublet at 4.82–4.80 (2H, d, J = 6.1 Hz) ppm; the –NH proton signal was
detected as a triplet at 9.10–9.07 (1H, t, J = 6.1 Hz). Similarly, the methylene group (-CH2 –N) proton signal
was observed as a quartet at 3.82–3.76 (2H, q, J = 5.8, 2.4 Hz) ppm; the methylene group (Ar–CH2) proton
signal showed as a triplet at 2.97–2.93 (2H, t, J = 2.4 Hz) ppm. The –NH proton signal was detected as a
triplet at 8.63–8.60 (1H, t, J = 5.8 Hz) for compound 9 (see Supplementary information). These results are
highly consistent with values for similar compounds. [7,10,25,28].
1
Figure 2. H NMR spectrum of compound 4.
1
3
3
.4. C NMR interpretations
1
3
The C NMR spectra of the compounds were taken in DMSO-d6 ; the chemical shifts are summarized in Table
. In the compounds (1–9), the aromatic C signals of the aryl ring (C1–C6) were observed between 163.4
5
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YAKAN/Turk J Chem
and 111.4 ppm and those from the terephthalaldehyde ring (C9–C10) were observed between 141.1 and 128.2
ppm. The characteristic -C=N (C8) peaks of imine were observed at 144.1 and 141.0 ppm for all compounds
(
1–10). The other remarkable characteristic, -C=S (C7) peaks of the thiosemicarbazone region, were observed
at 178.1 and 175.6 ppm for all compounds (1–10). In compounds 1 and 2, the methoxy carbon atoms (–OCH3)
resonated at 56.4 and 56.1 ppm. For compound 8, the methylene carbon signal was detected at 47.1 ppm.
In compound 9, the methylene carbon signals (Ar-CH2 and -CH2 –N) were detected at 35.3 and 45.5 ppm.
For compound 10, the aliphatic C signals of the cyclic ring (C1–C6) were observed between 53.1 and 25.4
ppm. Furthermore, in compounds 5, 6, and 7, the C atoms (for C1–C6) were also split into doublets caused
by interacting with the atomic nucleus of F. Additionally, in compounds 1–7, the signals of the carbon were
shifted downfield (high values of δ) relative to the signal of benzene (128.5 ppm) due to the presence of the
2
–OCH3 (152.1 ppm), 3–OCH3 (159.8 ppm), 4–NO2 (145.9 ppm), 3–Cl (143.2 ppm), 2–F (159.0 ppm), 3–F
(163.4 ppm), and 4–F (161.4 ppm) atoms/groups (see Supplementary information).
1
3
Table 5.
C3 C4
C NMR data of compounds 1–10 (δ/ppm).
Comp. C1
C2
C5
C6
C7
C8
C9
C10
R
1
2
3
4
5
152.1
111.8
159.8
124.2
143.2
116.3
116.1
163.4
160.7
115.3
115.1
124.6
112.3
145.9
125.5
127.7
127.6
112.5
112.3
161.4
158.9
120.4
129.4
124.2
130.1
124.5
124.4
130.1
130.0
115.3
115.1
128.1
118.6
125.1
124.8
130.7
130.2
122.0
121.9
128.8
128.7
126.4
136.0
127.0
132.6
128.7
128.6
141.4
141.2
135.9
135.8
175.6 142.2 135.9 128.2 -OCH3: 56.4
176.3 142.9 140.7 128.5 -OCH3: 56.1
111.4
125.1
125.7
159.0
176.0 144.1 135.9 128.5
176.4 141.0 135.9 128.4
177.8 142.9 135.9 128.3
-
-
-
-
1
56.6
113.0
12.8
128.8
28.7
6
7
176.5 143.2 135.9 128.4
176.9 142.8 141.1 128.3
1
1
8
9
1
127.3
127.9
32.2
128.0
128.9
25.4
127.3
126.7
25.6
128.0
128.9
25.4
127.3
127.9
32.2
139.9
139.7
53.1
178.1 142.1 135.9 128.7 -CH2: 47.1
177.5 141.8 135.9 129.1 Ar-CH2: 35.3 -CH2-N: 45.5
0
176.2 141.9 135.8 127.9
-
1
3
The C NMR spectrum of compound 4 had 10 different resonances, consistent with the target structure
shown in Figure 3.
In compound 4, the -C=S (C7) signal of the thiosemicarbazone region was observed at 176.4 ppm. The
characteristic -C=N (C8) peak of imine was observed at 141.0 ppm. The carbons (C1–C6) of aryl the ring
were detected at 143.2, 132.6, 130.1, 125.7, 125.5, and 124.8 ppm, respectively. The aromatic C signals of the
terephthalaldehyde region (C9–C10) were observed at 135.9 and 128.4 ppm. The C2 (143.2 ppm) carbon atoms
shifted downfield (high values of δ), which was caused by the presence of the chlorine group. These results are
in agreement with the data reported for similar compounds [8–10,28,29].
3.5. UV–Vis interpretations
The electronic absorption spectra of the new bis(thiosemicarbazones) based on Schiff base derivatives (1–10)
show that in all obtained products absorption bands appeared in the range of 256–406 nm in dimethylsulfoxide
(
see Figures S28–S36). The electronic spectra of these compounds show remarkable absorption bands of the
aromatic ring (C=C) and thiosemicarbazone (C=S)–imine (CH=N) region, owing to transitions of a π π*
and an n → π*. The π π*-type transitions of C=C on the aromatic ring are seen at 256 nm, except
→
→
1
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YAKAN/Turk J Chem
1
3
Figure 3.
C NMR spectrum of compound 4.
in compound 1 (258 nm). This transition is a characteristic benzenoid (B) band of the aromatic structure [32].
While the n → π* transitions of the imine C=N double bond were seen at 282–296 nm for all compounds,
the bands at 352–406 nm are assigned to the electronic transition n → π* of the thiosemicarbazone moiety
(
C=S).
In compound 5, the 3 n → π* transitions of the thiosemicarbazone moiety (C=S) are at 352 (shoulder),
70, and 390 nm, respectively, as shown in Figure 4. While the bands in the spectral region 286 nm (shoulder)
π* transitions
3
can be assigned to the azomethine C=N double bond to the n → π* transitions, the π
→
of C=C on the aromatic structure are seen at 256 nm, as shown in Figure 4. The electronic spectral data of
all of the compounds are summarized in Table 6. These values are in agreement with the results that were
reported for similar compounds [11,33–35].
3.6. Evaluation of antioxidant activity
Antioxidant activity of all of the molecules was determined according to a reported procedure [26,27] as
described in the experimental literature, with minor modifications. It can be assumed that the hydrogen losing
capacities/abilities or structurally stable radical creation after the interaction of the compounds with DPPH free
radicals is related to the antioxidant activity of the molecule being studied [36]. Percentage inhibition changes
of ascorbic acid and synthesized molecules are given in Figure 5. All compounds followed a steadily increasing
sequence depending on the concentration. The synthesized molecules produced lower free radical scavenging at
all concentration ranges compared to ascorbic acid.
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YAKAN/Turk J Chem
Figure 4. UV–Vis spectrum of compound 5 in DMSO.
Table 6. Electronic spectral data (nm) of all the compounds (1–10) in DMSO.
Compound
π
→
π*, B band n → π*, C=N n → π*, C=S
1
2
3
4
5
6
7
8
9
258
256
256
256
256
256
256
256
256
256
296
296
290
284
286
282
288
292
294
286
360, 380, 396
358, 378, 396
356, 372, 392
364, 384, 406
352, 370, 390
356, 372, 394
354, 370, 388
358, 378, 396
358, 376, 396
352, 370, 388
1
0
Figure 5. Percentage of inhibition calculated by the DPPH method for ascorbic acid and compounds 1–10.
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YAKAN/Turk J Chem
The antioxidant activity of all sample molecules is expressed with IC50 values in the results given in Table
. IC50 values of the molecules were found to be between 3.81 ± 0.01 and 29.05 ± 0.11 µM. The DPPH test
7
is thought to cause hydrogen atom transfer reaction, which causes formation of the novel radical [37]. Hence,
antioxidant compounds can react with DPPH free radicals by giving hydrogen atoms or by electron donation via
•
a free radical attack on these compounds [38,39]. According to this reaction (DPPH
+
+ R-NH → DPPH-H
•
R-N ), weaker hydrogen bonds are necessary for higher antioxidant activity in a compound. The strength
of these hydrogen bonds (–NH group in the thiosemicarbazone moiety) is proportional to electron density.
Consequently, antioxidant activity depends on two things: the first being the capacity/skill of compounds to
lose hydrogen atoms, and the second being the stability of the formed radical [36,39,40]. Therefore, the structure
of the tested compounds and electronic effects of groups/substituents in structures plays a significant role in
antioxidant activity [36,37,41]. It is generally known that there are 3 types of electron effects: mesomeric (or
resonance), inductive, and steric hindrance. Substituents on the compounds, which are known as electron-
donating groups (OH, NH2 , OCH3 , CH3 , etc.) and electron-withdrawing groups (NO2 , CN, CF3 , F, Cl, Br,
etc.), significantly affect antioxidant activity [42]. On the other hand, the position of these groups/substituents
in the molecules plays a major role in antioxidant activity as well.
Table 7. IC50 values for the synthesized compounds 1–10.
Compounds
DPPH scavenging activity, IC50 (µM)*
9.29 ± 0.01
1
2
3
4
5
6
7
8
9
7.72 ± 0.04
3.81 ± 0.01
7.48 ± 0.04
8.69 ± 0.04
9.92 ± 0.05
9.28 ± 0.05
29.05 ± 0.11
8.26 ± 0.04
1
0
16.96 ± 0.09
Ascorbic acid 2.99 ± 0.01
*
IC50 = Concentration (µM) exhibiting 50% inhibition of DPPH radical.
Values are expressed as means (n = 3).
In view of this information, compound 3, possessing a nitro group (p–NO2) which had strong electron-
withdrawing through inductive effect, showed the strongest antioxidant activity against the DPPH radical
amongst all test molecules. The reason is that the electron-withdrawing groups reduce the bond strength
between nitrogen and hydrogen atoms, thus decreasing electron density in the structure, which causes easier
loss of the hydrogen atom. In a previous study, compounds containing nitro groups, which draw electrons on
the ring, showed much higher antioxidant activity than various substituted structures [10]. At the same time,
experimental results on halogens showed different activities depending on their position according to the –NH.
Antioxidant activity order was 4 (m–Cl) > 5 (o–F) > 7 (p–F) > 6 (m–F). In similar studies, antioxidant
activity among the halogen-containing compounds was –Cl > –F [8,43,44]. As for the effect of each position
of the fluorine atom, o–F and p–F substituents have a strong inductive electron-attracting effect which causes
easier hydrogen atom loss, whereas m–F substituent has poor inductive effect.
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Compound 2 (m–OCH3) had higher antioxidant activity than compound 1 (o–OCH3). While the m–
OCH3 group is an electron-withdrawing substituent with negative inductive effect, the o–OCH3 group is an
electron-donating substituent with a positive resonance effect. The m–OCH3 substituent provides easier loss of
the hydrogen atom via decreased strength of the N-H bond, whereas the o–OCH3 group has the opposite effect
[
8
45]. Compound 10 (cyclic ring) has higher antioxidant activity than compound 8 (benzyl group). Compound
had difficulty losing the hydrogen atom because the benzyl group is a more electron-donating substituent
than that of the cyclic ring. Consequently, IC50 values for the products followed the order of 3 > 4 > 2 >
9
> 5 > 7 > 1 > 6 > 10 > 8.
4. Conclusion
In this study, Schiff-base–bearing new bis(thiosemicarbazones) have been obtained, with excellent yields of 72%–
1
9
7%. The structure of all of the compounds was elucidated using spectroscopic methods such as FT–IR,
H
1
3
NMR, C NMR, and UV–Vis, as well as elemental analysis. The in vitro antioxidant activity of the synthesized
molecules was determined using the DPPH free-radical trapping process. IC50 values of the newly obtained
molecules ranged from 3.81 ± 0.01 to 29.05 ± 0.11 µM. Among the synthesized compounds, compound 3
had the best satisfactory antioxidant activity against the DPPH radical. Additionally, the structure–activity
relationships were studied in relation to the presence, types, and position of substituents. Among all of the
test molecules, a compound which possesses electron-attracting groups has higher antioxidant activity against
DPPH free radicals than electron-donating groups.
Conflict of interest
The author declares that he has no conflict of interest.
Acknowledgments
I would like to thank the Scientific and Technological Research Applications and Center (Gübitam) and Dr
Ömer Faruk Ensari for taking the NMR spectra. I am also grateful to Dr Mohammad Maher Jesry for checking
and proofreading the article.
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Supplementary materials
Figure S1. IR spectrum of compound 1.
Figure S2. IR spectrum of compound 2.
1

Figure S3. IR spectrum of compound 3.
Figure S4. IR spectrum of compound 5.
2

Figure S5. IR spectrum of compound 6.
Figure S6. IR spectrum of compound 7.
3

Figure S7. IR spectrum of compound 8.
Figure S8. IR spectrum of compound 9.
4

Figure S9. IR spectrum of compound 10.
1
Figure S10. H NMR spectrum of compound 1.
5

1
Figure S11. H NMR spectrum of compound 2.
1
Figure S12. H NMR spectrum of compound 3.
6

1
Figure S13. H NMR spectrum of compound 5.
1
Figure S14. H NMR spectrum of compound 6.
7

1
Figure S15. H NMR spectrum of compound 7.
1
Figure S16. H NMR spectrum of compound 8.
8

1
Figure S17. H NMR spectrum of compound 9.
1
Figure S18. H NMR spectrum of compound 10.
9

1
3
Figure S19. C NMR spectrum of compound 1.
1
3
Figure S20. C NMR spectrum of compound 2.
10

1
3
Figure S21. C NMR spectrum of compound 3.
1
3
Figure S22. C NMR spectrum of compound 5.
11

1
3
Figure S23. C NMR spectrum of compound 6.
1
3
Figure S24. C NMR spectrum of compound 7.
12

1
3
Figure S25. C NMR spectrum of compound 8.
1
3
Figure S26. C NMR spectrum of compound 9.
13

1
3
Figure S27. C NMR spectrum of compound 10.
Compound 1
1,8
1,6
1,4
1,2
1
0,8
0,6
0,4
0,2
0
200
250
300
350
400
450
500
550
600
-
0,2
Wavelenght (nm)
Figure S28. UV-Vis spectrum of compound 1 in DMSO.
14

Compound 2
1,6
1,4
1,2
1
0,8
0,6
0,4
0,2
0
200
250
300
350
400
450
500
550
600
-
0,2
Wavelenght (nm)
Figure S29. UV-Vis spectrum of compound 2 in DMSO.
Compound 3
1,8
1,6
1,4
1,2
1
0,8
0,6
0,4
0,2
0
-
0,2 200
250
300
350
400
450
500
550
600
Wavelenght (nm)
Figure S30. UV-Vis spectrum of compound 3 in DMSO.
15