Synthesis, characterization, and antioxidant activity of some new N 4-arylsubstituted-5-methoxyisatin-β-thiosemicarbazone derivatives

Article abstract of DOI:10.1007/s11164-020-04079-x

Abstract: Firstly, thiosemicarbazides were prepared by the reaction of hydrazine monohydrate with isothiocyanates in cold dry ethanol at 0?°C for 1?h. After that, new isatin-β-thiosemicarbazones were synthesized by treatment of 5-methoxyisatin with thiose

Full text of DOI:10.1007/s11164-020-04079-x

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-020-04079-x
Synthesis, characterization, and antioxidant activity
of some new N4‑arylsubstituted‑5‑methoxyisatin‑β‑
thiosemicarbazone derivatives
Halit Muğlu1
Received: 9 October 2019 / Accepted: 2 January 2020
© Springer Nature B.V. 2020
Abstract
Firstly, thiosemicarbazides were prepared by the reaction of hydrazine monohydrate
with isothiocyanates in cold dry ethanol at 0 °C for 1 h. After that, new isatin-β-
thiosemicarbazones were synthesized by treatment of 5-methoxyisatin with thiosem-
icarbazides in aqueous ethanol containing one drop of hydrochloric acid at refux for
3 h. The chemical structures of products were confrmed by means of IR, 1H NMR,
and 13C NMR data and by elemental analysis. All the synthesized compounds were
evaluated for their antioxidant activity by 1,1-diphenyl-2-picryl hydrazyl (DPPH)
radical scavenging method. The synthesized molecules showed lower antioxidant
activity than the standard Trolox (8.757 μM). IC50 values of the newly synthesized
isatin-β-thiosemicarbazone derivatives ranged from 12.455 to 73.471 μM.
Graphic abstract
Keywords 5-Methoxyisatin · Thiosemicarbazones · Antioxidant activity ·
Spectroscopic techniques
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s1116
4-020-04079-x) contains supplementary material, which is available to authorized users.
*
Halit Muğlu
hmuglu@kastamonu.edu.tr
1
Department of Chemistry, Faculty of Science, Kastamonu University, Kastamonu, Turkey
Vol.:(0123456789)
1 3

H. Muğlu
Introduction
Thiosemicarbazones are a signifcant class of synthetic compounds and have a
variety of pharmacological and biological applications [1–6]. They have revealed
wide spectrum of activities which includes anticancer [5, 7], anti-HIV [8, 9],
anticonvulsant [10, 11], antimalarial [12, 13] anti-infammatory [14], enzymatic
inhibition [3, 4, 15], anti-viral [16], antioxidant [17], antifungal [18], and anti-
bacterial [18, 19].
Isatin derivatives have shown an extensive spectrum of biological activities
such as anti-bacterial [20], anti-viral [21], antifungal [20], antimicrobial [22, 23],
antioxidant [24, 25], anti-tubercular [26], anti-HIV [8, 27], and anticonvulsant
[28].
Free radicals are produced under certain environmental conditions and during
normal cellular functions in the body. Many articles have reported potential benef-
cial efects of diferent free radical scavenging antioxidant polyphenols against neo-
plastic diseases in human biology [24]. Schif bases have been investigated as anti-
oxidants by many researchers because of their ability to scavenge free radicals [29].
In this work, isatin-β-thiosemicarbazones including halogen, methoxy, methyl,
and nitro groups were synthesized and characterized by using methods such as
1
IR, H NMR, and 13C NMR spectroscopy and by elemental analysis. The synthe-
sized compounds were determined antioxidant activity in vitro by using DPPH
free radical scavenging method.
Experimental section
Measurement and reagents
All starting materials and solvent were purchased from Aldrich, Sigma, or Merck
Chemical Company and were used without further purifcation. The solvent was
spectroscopic grade. The elemental analysis was performed on a CHNS-932
(LECO). Infrared spectra were recorded on a Bruker Alpha FT-IR spectrometer.
1H NMR and 13C NMR spectra were taken on a JEOL ECX-400 (400 MHz) in
DMSO-d6 spectrophotometer. The splitting patterns are indicated as s (singlet),
d (doublet), dd (doublet of doublets), t (triplet), q (quartet), and m (multiplied).
Melting points were measured using a Gallenkamp melting point apparatus and
were uncorrected. Absorption measurements were recorded with a SHIMADZU
UVM-1240 UV–visible spectrophotometer.
Synthesis of substituted thiosemicarbazides (1a–j)
To a solution of hydrazine monohydrate (5 mmol) in ethanol (10 mL), a suspen-
sion of an appropriate isothiocyanate (5 mmol) in ethanol (10 mL) was added
1 3

Synthesis, characterization, and antioxidant activity of…
dropwise with vigorous stirring and cooling in an ice bath. The mixture was
allowed to stand overnight. The solid product so formed was fltered, dried, and
crystallized from ethanol to give product.
Synthesis of N4‑substituted 5‑methoxyisatin‑β‑thiosemicarbazones (3a–j) N4-sub-
stituted thiosemicarbazide (1a–j) (0.8 mmol), 5-methoxyisatin (0.8 mmol), and one
drop of HCl were added to aqueous EtOH (20 mL), and the mixture was refuxed at
78 °C for 3 h. The obtained precipitate was fltered and washed with EtOH to aford
pure desired products or recrystallized from ethanol. Then the formed color solid
was isolated and dried in air. The products were obtained good yields (68–97%) as
shown in Scheme 1.
N4‑2‑methoxyphenyl‑5‑methoxyisatin‑β‑thiosemicarbazone (3a) Yield: 87%, m.p.:
218–220 °C, IR (KBr, cm−1): ʋ(–NH ist) 3207, ʋ(–C=N–NH and –NH–Ar car-
bazide) 3137, 3092, ʋ(Ar C–H) 3043–2995, ʋ(C=O) 1687, ʋ(C=N) 1544, ʋ(C=S)
1
1487, ʋ(C–N) 1182; H NMR (400 MHz, DMSO-d6) δ/ppm: 12.71 (s, NH ist),
11.01 (s, NH), 10.39 (s, NH), 7.67–7.65 (d, 1H, J=7.9 Hz), 7.28–7.23 (m, 2H,
ArH), 7.10–7.08 (d, 1H, J=7.9 Hz), 6.98–6.94 (t, 1H, J=7.6 Hz), 6.92–6.89 (dd,
1H, J=8.5, 2.4 Hz, ArH), 6.83–6.81 (d, 1H, J=8.5 Hz, ArH), 3.79 (s, 3H, OCH3),
3.71 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO-d6) δ/ppm: 177.07 (C=S), 163.24
(C=O), 155.84 (C–O), 153.90 (C–O), 136.79 (C=N), 132.97, 128.35, 127.55,
127.51, 121.13, 120.65, 118.15, 112.47, 112.34, 106.65, 56.28 (OCH3), 56.08
(OCH3); Elemental Analysis Calcd: C, 57.29; H, 4.53; N, 15.72. Found: C, 56.84;
H, 4.61; N, 15.34.
N4‑2‑fuorophenyl‑5‑methoxyisatin‑β‑thiosemicarbazone (3b) Yield: 82%, m.p.:
234–235 °C, IR (KBr, cm−1): ʋ(–NH ist) 3309, ʋ(–C=N–NH and –NH–Ar car-
bazide) 3147, 3096, ʋ(Ar C–H) 3040–3005, ʋ(C=O) 1686, ʋ(C=N) 1536, ʋ(C=S)
Scheme 1 Synthetic route for the 5-methoxyisatin-β-thiosemicarbazone derivatives (3a–3j)
1 3

H. Muğlu
1
1479, ʋ(C–N) 1193, ʋ(C–F) 906; H NMR (400 MHz, DMSO-d6) δ/ppm: 12.81 (s,
NH ist), 11.02 (s, NH), 10.64 (s, NH), 7.47–7.43 (t, 1H, J=7.3 Hz), 7.39–7.34 (m,
1H, ArH), 7.32–7.30 (d, 2H, J=7.9 Hz), 7.27–7.21 (m, 1H, J=7.7 Hz, ArH), 6.92–
6.89 (dd, 1H, J=8.5, 2.4 Hz, ArH), 6.83–6.81 (d, 1H, J=8.5 Hz, ArH), 3.71 (s, 3H,
OCH3); 13C NMR (100 MHz, DMSO-d6) δ/ppm: 178.54, 163.29, 159.11, 156.64,
155.86, 136.82, 133.51, 130.80, 129.74, 129.66, 126.98, 126.86, 124.99, 124.96,
121.27, 121.16, 118.23, 116.65, 116.45, 112.47, 106.91, 56.11; Elemental Analysis
Calcd: C, 55.81; H, 3.81; N, 16.27. Found: C, 55.44; H, 3.79; N, 16.05.
N4‑4‑methoxyphenyl‑5‑methoxyisatin‑β‑thiosemicarbazone (3c) Yield: 85%, m.p.:
214–215 °C, IR (KBr, cm−1): ʋ(–NH ist) 3318, ʋ(–C=N–NH and –NH–Ar car-
bazide) 3188, 3132, ʋ(Ar C–H) 3070–3032, ʋ(C=O) 1662, ʋ(C=N) 1576, ʋ(C=S)
1
1475, ʋ(C–N) 1174; H NMR (400 MHz, DMSO-d6) δ/ppm: 12.69 (s, NH ist),
11.00 (s, NH), 10.64 (s, NH), 7.42–7.39 (d, 2H, J=9.2 Hz, ArH), 7.36–7.35 (d, 1H,
J=1.8 Hz,), 6.94–6.92 (d, 2H, J=9.2 Hz, ArH), 6.91–6.88 (dd, 1H, J=8.5, 2.4 Hz),
6.82–6.80 (d, 1H, J=8.5 Hz), 3.73 (s, 3H, OCH3), 3.71 (s, 3H, OCH3); 13C NMR
(100 MHz, DMSO-d6) δ/ppm: 177.14, 163.34, 158.00, 155.85, 136.61, 132.61,
131.58, 127.95, 121.01, 117.74, 114.13, 112.36, 106.91, 56.14, 55.81; Elemental
Analysis Calcd: C, 57.29; H, 4.53; N, 15.72. Found: C, 56.88; H, 4.39; N, 15.60.
N4‑3‑fuorophenyl‑5‑methoxyisatin‑β‑thiosemicarbazone (3d) Yield: 80%, m.p.:
226–227 °C, IR (KBr, cm−1): ʋ(–NH ist) 3339, ʋ(–C=N–NH and –NH–Ar car-
bazide) 3184, 3127, ʋ(Ar C–H) 3072–3035, ʋ(C=O) 1680, ʋ(C=N) 1514, ʋ(C=S)
1474, ʋ(C–N) 1178, ʋ(C–F) 900; 1H NMR (400 MHz, DMSO-d6) δ/ppm: 12.81 (s,
NH ist), 11.03 (s, NH), 10.79(s, NH), 7.58–7.54 (dd, 1H, J=11.0, 2.4 Hz, ArH),
7.47–7.38 (m, 2H, ArH), 7.36–7.35 (d, 1H, J=2.4 Hz,), 7.10–7.05 (m, 1H, ArH),
6.92–6.90 (dd, 1H, J=8.5, 2.4 Hz), 6.82–6.80 (d, 1H, J=8.5 Hz), 3.72 (s, 3H,
OCH3); 13C NMR (100 MHz, DMSO-d6) δ/ppm: 176.78 (C=S), 163.35, 160.94,
155.88, 155.79, 140.66, 140.54, 136.80, 133.46, 130.52, 130.43, 122.01, 121.98,
121.11, 118.13, 113.40, 113.19, 113.06, 112.82, 112.43, 107.29, 56.18; Elemental
Analysis Calcd: C, 55.81; H, 3.81; N, 16.27. Found: C, 55.33; H, 3.81; N, 16.15.
N4‑4‑nitrophenyl‑5‑methoxyisatin‑β‑thiosemicarbazone (3e) Yield: 87%, m.p.:
247–248 °C, IR (KBr, cm−1): ʋ(–NH ist) 3307, ʋ(–C=N–NH and –NH–Ar car-
bazide) 3246, 3218, ʋ(Ar C–H) 3079–3014, ʋ(C=O) 1697, ʋ(C=N) 1542, ʋ(C=S)
1466, ʋ(C–N) 1154, ʋ(NO2): 1438–1326 (as, ss); 1H NMR (400 MHz, DMSO-d6) δ/
ppm: 12.96 (s, NH ist), 11.06 (s, NH), 11.01 (s, NH), 8.25–8.23 (d, 2H, J=8.5 Hz,
ArH), 8.04–8.01 (d, 2H, J=9.2 Hz, ArH), 7.35–7.34 (d, 1H, J=2.4 Hz), 6.93–6.91
(dd, 1H, J=8.5, 2.4 Hz), 6.83–6.81 (d, 1H, J=8.5 Hz), 3.72 (s, 3H, OCH3); 13C
NMR (100 MHz, DMSO-d6) δ/ppm: δ 176.89, 163.06, 156.02, 145.18, 144.66,
136.97, 134.07, 125.42, 124.49, 120.51, 118.31, 112.50, 107.44, 56.07; Elemental
Analysis Calcd: C, 51.75; H, 3.53; N, 18.86. Found: C, 50.90; H, 3.42; N, 18.65.
N4‑2,4‑dichlorophenyl‑5‑methoxyisatin‑β‑thiosemicarbazone
(3f) Yield:
97%, m.p.: 249–250 °C, IR (KBr, cm−1): ʋ(–NH ist) 3265, ʋ(–C=N–NH and
1 3

Synthesis, characterization, and antioxidant activity of…
–NH–Ar carbazide) 3172, 3074, ʋ(Ar C–H) 3032–2994, ʋ(C=O) 1689, ʋ(C=N)
1535, ʋ(C=S) 1453, ʋ(C–N) 1162, ʋ(C–Cl) 856; 1H NMR (400 MHz, DMSO-d6) δ/
ppm: 12.84 (s, NH ist), 11.03 (s, NH), 10.73 (s, NH), 7.74–7.73 (d, 1H, J=1.8 Hz,
ArH), 7.55–7.53 (d, 1H, J=8.5 Hz, ArH), 7.49–7.46 (dd, 1H, J=8.5, 2.4 Hz, ArH),
7.29–7.28 (d, 1H, J=2.4 Hz), 6.93–6.90 (dd, 1H, J=8.5, 2.4 Hz), 6.83–6.81 (d, 1H,
J=8.5 Hz), 3.72 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO-d6) δ/ppm: 178.29,
163.31, 155.77, 136.83, 135.96, 133.68, 133.43, 133.03, 132.48, 129.70, 128.10,
121.02, 118.32, 112.62, 106.74, 56.13; Elemental Analysis Calcd: C, 48.62; H, 3.06;
N, 14.18. Found: C, 47.91; H, 2.99; N, 14.03.
N4‑2‑phenylethyl‑5‑methoxyisatin‑β‑thiosemicarbazone (3g) Yield: 86%, m.p.:
179–180 °C, IR (KBr, cm−1): ʋ(–NH ist) 3369, ʋ(–C=N–NH and –NH–Ar car-
bazide) 3235, 3207, ʋ(Ar C–H) 3062–3022, ʋ(C=O) 1680, ʋ(C=N) 1515, ʋ(C=S)
1
1475, ʋ(C–N) 1137; H NMR (400 MHz, DMSO-d6) δ/ppm: 12.58 (s, NH ist),
10.97 (s, NH), 9.28 (t, J=5.8 Hz, 1H, NH–CH2), 7.30–7.23 (m, 4H, ArH), 7.20–
7.19 (d, 1H, J=2.4 Hz), 7.18–7.16 (t, 1H, J=6.4 Hz, ArH), 6.91–6.88 (dd, 1H,
J=8.2, 2.7 Hz), 6.81–6.79 (d, 1H, J=8.5 Hz), 3.78 (dd, 2H, J=15.6, 5.8 Hz, N–
CH2), 3.73 (s, 3H, OCH3), 2.93 (t, 2H, J=7.9 Hz, –CH2); 13C NMR (100 MHz,
DMSO-d6) δ/ppm: 177.56, 163.29, 155.82, 139.14, 136.39, 132.63, 129.13, 129.02,
126.55, 121.53, 117.48, 112.47, 106.19, 56.17, 45.96, 34.43; Elemental Analysis
Calcd: C,61.00; H, 5.12; N, 15.81. Found: C, 59.80; H, 5.01; N, 15.64.
N4‑2‑methylphenyl‑5‑methoxyisatin‑β‑thiosemicarbazone (3h) Yield: 84%, m.p.:
223–224 °C, IR (KBr, cm−1): ʋ(–NH ist) 3311, ʋ(–C=N–NH and –NH–Ar car-
bazide) 3229, 3173, ʋ(Ar C–H) 3065–2992, ʋ(C=O) 1736, ʋ(C=N) 1586, ʋ(C=S)
1
1480, ʋ(C–N) 1165; H NMR (400 MHz, DMSO-d6) δ/ppm: 12.70 (s, NH ist),
11.01 (s, NH), 10.64 (s, NH), 7.35–7.34 (d, 1H, J=1.8 Hz), 7.30–7.28 (m, 1H,
J=2.4 Hz, ArH), 7.24–7.22 (d, 3H, J=9.2 Hz, ArH), 6.91–6.88 (dd, 1H, J=8.5,
2.4 Hz), 6.83–6.80 (d, 1H, J=8.5 Hz), 3.71 (s, 3H, OCH3), 2.21 (s, 3H, CH3);
13C NMR (100 MHz, DMSO-d6) δ/ppm: 177.78, 163.30, 155.72, 137.91, 136.71,
136.13, 132.94, 130.89, 129.07, 128.00, 126.83, 121.31, 118.11, 112.48, 106.84,
56.33, 18.20; Elemental Analysis Calcd: C, 59.98; H, 4.74; N, 16.46. Found: C,
59.43; H, 4.67; N, 16.38.
N4‑4‑fuorophenyl‑5‑methoxyisatin‑β‑thiosemicarbazone (3i) Yield: 68%, m.p.:
235–236 °C, IR (KBr, cm−1): ʋ(–NH ist) 3334, ʋ(–C=N–NH and –NH–Ar car-
bazide) 3194, 3103, ʋ(Ar C–H) 3069–2995, ʋ(C=O) 1693, ʋ(C=N) 1527, ʋ(C=S)
1450, ʋ(C–N) 1151, ʋ(C–F) 936; 1H NMR (400 MHz, DMSO-d6) δ/ppm: 12.76 (s,
NH ist), 11.02 (s, NH), 10.74 (s, NH), 7.57–7.54 (q, 2H, J=4.5 Hz, ArH), 7.35–7.34
(d, 1H, J=2.4 Hz), 7.24–7.20 (t, 2H, J=8.9 Hz, ArH), 6.92–6.89 (dd, 1H, J=8.2,
2.7 Hz), 6.82–6.80 (d, 1H, J=8.5 Hz), 3.72 (s, 3H, OCH3); 13C NMR (100 MHz,
DMSO-d6) δ/ppm: 177.33, 163.35, 161.83, 159.41, 155.86, 136.75, 135.32, 135.29,
133.20, 128.74, 128.65, 121.21, 118.06, 115.80, 115.57, 112.39, 107.13, 56.16; Ele-
mental Analysis Calcd: C, 55.81; H, 3.81; N, 16.27. Found: C, 55.37; H, 3.67; N,
16.13.
1 3

H. Muğlu
N4‑3‑chlorophenyl‑5‑methoxyisatin‑β‑thiosemicarbazone (3j) Yield: 73%, m.p.:
211–212 °C, IR (KBr, cm−1): ʋ(–NH ist) 3332, ʋ(–C=N–NH and –NH–Ar car-
bazide) 3299, 3220, ʋ(Ar C–H) 3032–2992, ʋ(C=O) 1688, ʋ(C=N) 1537, ʋ(C=S)
1476, ʋ(C–N) 1146, ʋ(C–Cl) 856; 1H NMR (400 MHz, DMSO-d6) δ/ppm: 12.82 (s,
NH), 11.04 (s, NH), 10.79 (s, NH), 7.74 (s, 1H, ArH), 7.61–7.59 (d, 1H, J=7.9 Hz,
ArH), 7.43–7.39 (t, 1H, J=7.9 Hz, ArH), 7.36–7.35 (d, 1H, J=1.8 Hz), 7.31–7.29
(dd, 1H, J=7.9, 1.2 Hz, ArH), 6.93–6.91 (dd, 1H, J=8.2, 2.1 Hz), 6.83–6.81 (d, 1H,
J=8.5 Hz), 3.72 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO-d6) δ/ppm: 176.88,
163.24, 155.77, 140.32, 136.84, 133.50, 133.01, 130.53, 126.45, 125.76, 124.77,
121.13, 118.15, 112.44, 107.27, 56.20; Elemental Analysis Calcd: C, 53.26; H, 3.63;
N, 15.53. Found: C, 53.15; H, 3.54; N, 15.35.
Antioxidant activity measurements by using DPPH method
Antioxidant activity of newly synthesized compounds was determined by the
DPPH radical scavenging activity analysis. For this, free radical scavenging abil-
ity was evaluated according to the method of Brand-Williams et al. [30] with
slight modifcation.
In order to evaluate the antioxidant activity of the products, stock solutions of
the compounds were prepared in DMSO to 250 µM. Compounds were dissolved
in DMSO and diluted to fve diferent concentrations. We used DPPH in ethanol
at a concentration of 50 µM. To the previously prepared (5 mL), DPPH solu-
tion were added compound solutions of diferent concentrations (5 µM, 10 µM,
20 µM, 50 µM, and 100 µM) and sufcient ethanol to total 6 mL. This mixture
was allowed to stand in a dark room at room temperature for 30 min and then was
read at 517 nm against a blank. Radical scavenging activity was expressed as a
percentage of inhibition and calculated using the formula:
[(
)
]
Inhibition(%) = A0 − A1 ∕A0 × 100
where A0 is the absorbance of the control (blank, without compound) and A1 is the
absorbance of the compound. The results were expressed as IC50 values (the test
concentration required to sweep 50% free radicals) [31].
Results and discussion
Physical data
Ten of the synthesized compounds were new. The current experimental results for
the physical data, melting points, yields, and elemental analyses are summarized
in Tables 1 and 2.
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Synthesis, characterization, and antioxidant activity of…
Table 1 Physical data of the 5-methoxyisatin-β-thiosemicarbazone derivatives (3a–3j)
Entry Com-
pound
R
Molecular formula Molecular weight m.p. (°C) Yield %
code
1
2
3
4
5
6
7
8
9
10
3a
3b
3c
3d
3e
3f
2-OCH3C6H4
2-FC6H4
4-OCH3C6H4
3-FC6H4
4-NO2C6H4
2,4-CIC6H3
–CH2CH2C6H5 C18H18N4O2S
2-CH3C6H4
4-FC6H4
3-CIC6H4
C17H16N4O3S
C16H13FN4O2S
C17H16N4O3S
C16H13FN4O2S
C16H13N5O4S
C16H12Cl2N4O2S
356.41
344.37
356.41
344.37
371.38
395.27
354.43
340.41
344.37
360.82
218–220
234–235
214–215
226–227
247–248
249–250
179–180
223–224
235–236
211–212
87
82
85
80
87
97
86
84
68
73
3g
3h
3i
C17H16N4O2S
C16H13FN4O2S
C16H13ClN4O2S
3j
Table 2 Results of elemental
Compounds
Calculated
Experimental
analysis for the compounds
C%
H%
N%
(C)%
(H)%
(N)%
3a
3b
3c
3d
3e
3f
57.29
55.81
57.29
55.81
51.75
48.62
61.00
59.98
55.81
53.26
4.53
3.81
4.53
3.81
3.53
3.06
5.12
4.74
3.81
3.63
15.72
16.27
15.72
16.27
18.86
14.18
15.81
16.46
16.27
15.53
56.84
55.44
56.88
55.33
50.90
47.91
59.80
59.43
55.37
53.15
4.61
3.79
4.39
3.81
3.42
2.99
5.01
4.67
3.67
3.54
15.34
16.05
15.60
16.15
18.65
14.03
15.64
16.38
16.13
15.35
3g
3h
3i
3j
Vibrational frequencies
In the FT-IR spectrum of the synthesized compounds, the asymmetric and
symmetric stretching bands of the amino group (–NH2) did not observe at
3600–3200 cm−1, as expected. In compound 3e, while the –NH (isatin) stretch-
ing vibration was observed at 3307 cm−1, the –NH (semicarbazone) stretching
vibrations were observed at 3246 and 3218 cm−1. The C=O signal of isatin ring
was observed at 1697 cm−1, and the –C=N stretching vibration was observed at
1542 cm−1; the C=S signal of isatin ring was observed at 1466 cm−1, and the
–C–N stretching vibration was appeared at 1154 cm−1; the asymmetric and sym-
metric stretching bands of the nitro group (–NO2) were observed at around 1438
and 1326 cm−1, as shown in Fig. 1. These observations provided key evidence for
the products formation. These data are consistent with values reported previously
1 3

H. Muğlu
Fig. 1 IR spectrum of compound 3e
Table 3 FT-IR values of the compounds (cm−1
)
Comp. –NH (ist) –NH (chz) –NH–Ar
(chz)
Ar CH
C=O C=N C=S C–N Spec. vib.
3a
3b
3c
3d
3e
3207
3309
3318
3339
3307
3137
3147
3188
3184
3246
3092
3096
3132
3127
3218
3043–2995 1687 1544 1478 1182
3040–3005 1686 1536 1479 1193 C–F: 906
3070–3032 1662 1576 1475 1174
–
–
3072–3035 1680 1514 1474 1178 C–F: 900
3079–3014 1697 1542 1466 1154 NO2: 1438–
1326(as, ss)
3f
3g
3h
3i
3265
3369
3311
3334
3332
3172
3235
3229
3194
3299
3074
3207
3173
3103
3220
3032–2994 1689 1535 1453 1162 C–Cl: 856
3062–3022 1680 1515 1475 1137
3065–2992 1736 1586 1480 1165
–
–
3069–2995 1693 1527 1450 1151 C–F: 936
3032–2992 1688 1537 1476 1146 C–Cl: 856
3j
chz carbohydrazide, ist isatin, as asymmetric stretching, ss symmetric stretching
1 3

Synthesis, characterization, and antioxidant activity of…
Scheme 2 General scheme for 1H NMR spectral interpretations
for similar compounds [2, 6, 32]. The IR peaks of the compounds are given in
Table 3.
Interpretation of the 1H NMR spectra
1
The H NMR spectra of the synthesized compounds were detected in DMSO-d6
1
as solvent. The general scheme for H NMR spectral interpretations is shown in
Scheme 2.
For compound 3e, while the –NH peak of isatin was observed as a singlet at
12.96 ppm, the –NH signals of semicarbazone were detected as singlets at 11.06
and 11.01 ppm. The proton signals (H1–H2–H3) of isatin ring were observed
Fig. 2 1H NMR spectrum of compound 3e
1 3

H. Muğlu
between 7.35 and 6.81 ppm (Fig. 2). The H1 proton was observed as doublet peak
at 7.35–7.34 (J=2.4 Hz) ppm. The H2 proton coupled to the H3 and H1 proton
showed doublet of doublets peaks at 6.93–6.91 (J=8.5, 2.4 Hz) ppm. The H3 proton
coupled to the H2 proton and detected doublet peaks at 6.83–6.81 (J=8.5 Hz) ppm.
The aromatic protons (H4–H8) of benzene ring were observed at 8.25–8.01 ppm.
The H4 and H8 proton coupled to the H5 and H7 proton and detected doublet peaks
at 8.04–8.01 (J=8.5 Hz) ppm, respectively. The H5 and H7 proton coupled to the
H4 and H8 proton and detected doublet peaks at 8.25–8.23 (J=9.2 Hz) ppm, respec-
tively. The methoxy group (–OCH3) proton signal was observed as a singlet in the
range of 3.72 ppm. DMSO-d6 and water in DMSO (HOD, H2O) signals were shown
around at 2.00, 2.50 (quintet), and 3.30 (variable, based on the solvent and its con-
centration) ppm, respectively [33]. These results are consistent with the values of
formerly reported for similar compounds [2, 6, 32]. Proton chemical shift values
1
of the compounds are presented in Table 4. Moreover, selected compounds’ IR, H
NMR, and 13C NMR spectra were given as supplementary material.
Interpretation of the 13C NMR spectra
The 13C NMR spectra of all compounds were obtained in DMSO-d6 and are shown
in the general scheme for 13C NMR spectral interpretations in Scheme 3.
The 13C NMR spectrum of compound 3e showed 14 diferent resonances in good
agreement with the proposed structure as shown in Fig. 3. In compound 3e, the
–C=S (C9) signal of the carbohydrazide region was detected at 176.89 ppm. The
characteristic –C=N (C8) and –C=O (C7) peaks of the isatin ring were observed at
136.97 and 163.06 ppm, respectively. The carbons (C1–C6) of the isatin ring were
observed at 156.02, 112.50, 120.51, 134.07, 118.31, and 107.44 ppm, respectively.
The C1 carbons atom shifted downfeld (high values of δ) due to the presence of
methoxy group. The aromatic carbons (C10–C15) of aryl ring were observed at
between 145.18 and 124.49 ppm. The C13 (145.18 ppm) carbon atom was shifted
downfeld (high values of δ) due to the presence of nitro group. The methoxy
(–OCH3) peak were detected at 56.07 ppm. Additionally, the C atoms (for C10–C15)
were also split into doublets due to interacting with the atomic nucleus of F for com-
pounds 3b, 3d, and 3i. These data are consistent with the values reported earlier for
similar compounds [2, 6, 32]. The carbon chemical shift values of the synthesized
compounds are given in Table 5 (see Supplementary Material).
Evaluation of antioxidant activity
Due to concentration of Trolox and synthesized compounds, percent inhibition
changes are given in Fig. 4. Accordingly, all the compounds produced a free radi-
cal scavenging which increased with concentration. Others compounds except for 3e
showed a lower inhibition increase depending on concentration.
An antioxidant is defned as any substance that delays or prevents oxidation of a
substrate, even at low concentrations as compared to an oxidizable substrate [34].
Antioxidant activity of all synthesized compounds performed using DPPH method
1 3

Synthesis, characterization, and antioxidant activity of…
1 3

H. Muğlu
Scheme 3 General scheme for 13C NMR spectral interpretations
Fig. 3 13C NMR spectrum of compound 3e
was expressed in IC50 values in Table 6. IC50 values of all the compounds were
between 12.455 and 73.471 μM. Of all the test compounds, compound 3e had the
most potential antioxidant activity against the DPPH radical. In particular, it has
been found that the antioxidant activities of the test compounds are provided by the
presence of electron-withdrawing groups on the aromatic ring [35].
As in the structure of the substituents in the carbohydrazone, the structure of
the substituents in the thiocarbohydrazone plays an important role for antioxidant
activity [36]. As previously reported by Kiran et al. [37] with novel bis-isatin
carbohydrazone derivatives, halogen-bonded aromatic structures were found
to be responsible for antioxidant activity. In addition, researchers have reported
that the compound containing the nitro group (electron-withdrawing group) in
1 3

Synthesis, characterization, and antioxidant activity of…
1 3

H. Muğlu
30
25
20
15
10
5
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
Trolox
0
0.208
0.416
0.832
2.08
4.16
Concentration (µM)
Fig. 4 Percent of inhibition calculated by the DPPH method for Trolox and compounds 3a–3j at diferent
concentrations
Table 6 IC50 values of Trolox
Compounds
IC50 (µM)a
and compounds 3a–3j
Trolox
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
8.757 0.07
66.178 0.11
79.927 0.13
32.626 0.09
36.258 0.09
12.455 0.06
73.471 0.12
64.704 0.10
57.545 0.09
42.269 0.06
64.478 0.06
aIC50 =the concentration (μM) values are expressed in mean SD
(n=3)
the synthesized compounds shows more antioxidant activity than those contain-
ing substituted bromine, fuorine, and chlorine [31, 38–40]. In parallel with these
studies, 3e molecule showed more antioxidant activity than other electron-with-
drawing substituted halogen groups. In this study, it was seen that the substituted
nitro group structures of thiosemicarbazone molecules attached to methoxy isatin
1 3

Synthesis, characterization, and antioxidant activity of…
exhibited active antioxidant properties, unlike the molecules synthesized with thi-
osemicarbazone-free isatin group by Khan et al. [41].
Conclusions
In this study, new 5-methoxyisatin-β-thiosemicarbazone derivatives have been
obtained with excellent yields of 68–97%. All the synthesized compounds were elu-
cidated by 1H NMR, 13C NMR, and IR and by elemental analyses. The in vitro anti-
oxidant activity of the compounds was determined by the DPPH free radical scav-
enging method. IC50 values of the newly synthesized isatin-β-thiosemicarbazone
derivatives ranged from 12.455 to 73.471 μM. Compound 3e of among the test com-
pounds showed the most acceptable antioxidant activity against the DPPH radical.
Acknowledgements I would like to thank the Scientifc Research Center for Industrial and Technological
Applications and Research Centre (BETUM) and Dr. Hatice Karadeniz for taking the NMR spectra.
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