New 5-methylisatin-thiosemicarbazones: preparation, spectroscopic elucidation and antioxidant properties

doi:10.21203/rs.3.rs-4913499/v1

New isatin-thiosemicarbazone (1–6) were obtained from various 5-methylisatin and various isocyanates with good yields and efficient methods. The structures of the synthesized compounds were clarified by FT-IR, ¹H-NMR, and ¹³C-NMR spectroscopic techniques and elemental analysis. In the study using butylated hydroxytoluene (BHT) as a reference antioxidant, 2,2-diphenyl-1-picrylhydrazyl (DPPH) antiradical quenching capacities of the synthesized compounds were determined. The free radical scavenging effects of the synthesized compounds were compared with the IC₅₀ values obtained from the concentration equations and were found in the order BHT > 6 > 5 > 3 > 2 > 1 > 4. Structural and electronic properties of the compounds were examined by density functional theory (DFT) calculations and their relationship with the antioxidant properties of the compounds was discussed. DFT calculations played an important role in determining how the electronic properties of the synthesized compounds, especially in the presence of different substituents and in the ortho and meta positions, and accordingly the antioxidant properties of the compounds, changed. Experimental and theoretical results confirmed that methoxy-containing structures in the synthesized compounds exhibit higher antioxidant effects compared to halogen-containing structures, regardless of their position.

Thiosemicarbazones

5-Methylisatin

Antioxidant activity

Spectroscopic techniques

Isatin (1H-indole-2,3-dione) composes a substantial class of heterocyclic structures found in plant and human tissue, is the indole-based derivatives. They have various medicinal and biological properties such as antibacterial [1], antioxidant [2–4], antifungal [5], antitubercular [6], anticancer [7], and anti-HIV [8].

Thiosemicarbazones have -NH-C(= S) NH-N = bond, are a substantial class of synthetic organic chemistry. They are also used as versatile intermediates for synthesizing a great number of moieties. Thiosemicarbazones have several biological and medicinal properties such as anticancer [9], anticonvulsant [10], antituberculosis [11], urease inhibitory activity [12], antimicrobial [13], antioxidant agents [14–16], antibacterial [17], and antiviral [18].

Thiosemicarbazone-containing compounds are a well-known class of ligands with a wide range of biological activities [19]. It has been reported that these molecules, which have been synthesized and tested in large numbers so far, show good antioxidant properties [20]. Previous results have shown that thiosemicarbazones and isatin-formed derivatives have not been sufficiently studied so far. Therefore, in this study, it was aimed to elucidate their antioxidant properties by synthesizing new thiosemicarbazones and isatin compounds. For this purpose, condensation reaction of 5-methylisatin and various isocyanates was carried out under reflux in ethanol medium and a series of new 5-methylisatin-thiosemicarbazones were obtained. New molecular structures were elucidated by Fourier transform infrared (FT-IR), proton and carbon nuclear magnetic resonance (¹H NMR & ¹³C NMR) and elemental analysis methods. Additionally, the spectral and electronic properties of the compounds were analyzed by DFT calculations. In particular, the effects of substituents on the antioxidant properties of the compounds were discussed.

Synthesis of 5-methylisatin-thiosemicarbazone compounds (1–6)

6.0 mmol of hydrazine monohydrate solution and 6.0 mmol of isothiocyanate molecule species were added dropwise into 20 mL of ethanol while cooling in an ice bath and stirred vigorously. After this mixture was kept in the refrigerator for 24 h, the resulting precipitate was filtered. The filtrate was dried and washed with ethanol to obtain thiosemicarbohydrazide. The formed thiosemicarbohydrazide (3.0 mmol) was added to 20 mL of ethanol with 3.0 mmol of 5-methylisatin and one drop of dilute HCl solution and stirred at 78°C under reflux for 3–5 h. The solid obtained at the end of the reaction was filtered, washed and dried in an oven. Thus, the synthesized compounds were prepared with a yield of (55–85%) via the reaction shown in Scheme 1 according to the previously reported procedure [21] with minor modifications. The detailed knowledge about measurement and reagents is given in supplemental material.

Antioxidant Activity Measurements

One of the widely used and accepted models to evaluate the free radical scavenging capacity is the DPPH (2,2-diphenyl-1-picrylhydrazil) method [22, 23]. In order to achieve this method in this study, firstly solutions of the synthesized compounds in the concentration range of 1.25–12.5 µM were prepared by dissolving them in dimethyl sulfoxide (DMSO). Then, antioxidant activity measurements were performed by incubating the solutions prepared with 1,1-diphenyl-2-picrylhydrazil (DPPH) stock solution in ethanol for 30 minutes at room temperature. The absorbance measurements of the prepared solutions were measured at 517 nm and the percentage of radical scavenging activity of the samples was calculated by the following formula [24]:

Inhibition (%) = [(A₀ - A₁) / A₀] x100

Accordingly; A₀ represents the absorbance of the solution without the compound, and A₁ represents the absorbance of the sample solution containing the synthesized compounds [25]. Then, the IC₅₀ parameter, which helps us rank the antioxidant activities of the compounds and is a measure of the 50% inhibition capacity, was determined from the y = mx + c formula curve obtained using the inhibition (%)-concentration graph [26].

Theoretical Computational Measurements

The calculations were performed using the ORCA 5.0.3 software [27] with density functional theory (DFT) [28, 29]. The computations utilized Becke's three-parameter exchange functional (B3) combined with the Lee-Yang-Parr correlation functional (LYP), as the B3LYP functional. Additionally, the DEF2-TZVPP basis set was employed along with D3BJ dispersion correction. These calculations were conducted without any symmetry constraints on the compounds. The optimized structures of the compounds represent the true minimum energy. This was demonstrated by the absence of imaginary frequencies in the IR vibrational calculations conducted in the gas phase.

Electronic parameters were obtained with the help of same level gas phase calculations. Frontier molecular orbital (FMO) energy eigenvalue data were then used to calculate global chemical reactivity parameters such as chemical hardness, electronegativity and electrophilic index. Further investigation into the relationship between antioxidant activity and molecular structure involved calculating different global reactivity parameters, such as HOMO-LUMO gap (E_g), electronegativity (χ), global hardness (η), electrophilicity (ω), electrodonating power (ω⁻), and electroaccepting power (ω⁺).

Relative chemical shift values were determined by deducting the absolute chemical shielding of tetramethylsilane (TMS) calculated at the B3LYP/DEF2-TZVPP level (31.698 ppm for ¹H NMR and 183.951 ppm for ¹³C NMR), after re-optimizing the compounds in the DMSO phase and employing the CPCM-SCRF procedure to model solvent effects.

The physical properties, yields, melting points and elemental analysis results of the synthesized compounds are given in Tables 1 and 2.

Table 1

Physical properties of the synthesized compounds
Comp.	Structure Name	M. P. (ºC)	Yield %	Colour
1	(Z)-N-(2-chlorophenyl)-2-(5-methyl-2-oxoindolin-3-ylidene)hydrazine-1-carbothioamide	249–250	85	Rose-light purple
2	(Z)-N-(2-fluorophenyl)-2-(5-methyl-2-oxoindolin-3-ylidene)hydrazine-1-carbothioamide	246–247	58	Rose-light purple
3	(Z)-N-(2-methoxyphenyl)-2-(5-methyl-2-oxoindolin-3-ylidene)hydrazine-1-carbothioamide	239–241	83	Rose-light purple
4	(Z)-N-(3-chlorophenyl)-2-(5-methyl-2-oxoindolin-3-ylidene)hydrazine-1-carbothioamide	233–235	62	Rose-light purple
5	(Z)-N-(3-fluorophenyl)-2-(5-methyl-2-oxoindolin-3-ylidene)hydrazine-1-carbothioamide	225–227	55	Light reddish- brick
6	(Z)-N-(3-methoxyphenyl)-2-(5-methyl-2-oxoindolin-3-ylidene)hydrazine-1-carbothioamide	194–195	74	Light reddish- brick

Table 2

Elemental analysis results of the compounds
Comp.	Molecular mass (g / mol)	Molecular formula	Calculated			Experimental
Comp.	Molecular mass (g / mol)	Molecular formula	C%	H%	N%	(C)%	(H)%	(N)%
1	344.8	C₁₆H₁₃ClN₄OS	55.73	3.80	16.25	55.66	3.89	16.32
2	328.4	C₁₆H₁₃FN₄OS	58.53	3.99	17.06	58.41	4.04	17.13
3	340.4	C₁₇H₁₆N₄O₂S	59.98	4.74	16.46	60.09	4.78	16.37
4	344.8	C₁₆H₁₃ClN₄OS	55.73	3.80	16.25	55.65	3.85	16.22
5	328.4	C₁₆H₁₃FN₄OS	58.53	3.99	17.06	58.41	3.87	17.12
6	340.4	C₁₇H₁₆N₄O₂S	59.98	4.74	16.46	59.91	4.73	16.50

FTIR Vibration frequencies

The FTIR spectrum results of the molecules confirmed that the symmetric and unsymmetric stretching bands belonging to amino groups (NH2) were not observed at 3500–3350 cm-1. Also, the -C = N stretching bands belonging to imine group were observed at 1605–1532 cm-1, indicating that a successful reaction took place as expected. As an example of FTIR spectra of compounds, the spectrum of Compound 3 is presented in Fig. 1. For compound 3, C = S vibration was characteristically given at 1476 cm^− 1 and S = C-N vibration at 1314 cm^− 1.

For all compounds, while the -NH stretching vibrations of isatin ring were observed at 3302 − 3230 cm^− 1, the–NH stretching vibrations of thiosemicarbazone region were detected at 3218 − 3126 cm^− 1, respectively (see Figs. S1-S6). Stretching vibrations of the -C = O structure were observed at 1693 − 1680 cm^− 1, the vibration signal of the -C = S structure at 1483 − 1434 cm^− 1 and the stretching vibrations of the -C–N structure at 1316 − 1308 cm^− 1.

For compounds 1 and 4, stretching vibrations of -C–Cl structure were observed at 939 and 1027 cm^− 1, respectively. For compounds 2 and 5, vibrations of -C–F structure were detected at 1202 and 1205 cm^− 1, respectively. For compounds 3 and 6, -C–O structure showed stretching vibrations at 1124 and 1154 cm^− 1, respectively. These FTIR spectra showed significant evidence that the molecules were synthesized correctly, and FTIR data for all molecules are presented in Table 3. These observations are agreement with values published formerly for similar compounds [14, 16, 30, 31].

Table 3

Experimental and calculated IR vibration frequencies of the compounds (cm^− 1)
	Comp.	ν _NH (ist)	ν _NH (tcrb)	ν _C=O (ist)	ν _C=N	ν _C=S	ν _S=C−N ν _ArC−N	Subs.
Experimental	1	3267	3201	1688	1586	1482	1310	C-Cl:939
	2	3230	3174	1685	1587	1473	1308	C-F:1202
	3	3297	3145	1686	1532	1476	1314	C-O:1124
	4	3287	3218	1680	1605	1434	1313	C-Cl:1027
	5	3301	3126	1683	1603	1478	1314	C-F:1205
	6	3302	3176	1693	1599	1483	1316	C-O:1154
Calculated	1	3640.26	3472.81, 3425.02	1757.27	1617.57	1413.17	1382.65, 1263.62	C-Cl: 1052.32
	2	3640.21	3505.13, 3427.73	1757.59	1618.65	1414.10	1390.54, 1274.11	C-F: 1217.31
	3	3640.73	3498.98, 3428.56	1756.01	1612.99	1415.64	1398.10, 1272.55	O-CH₃: 1057.68
	4	3640.23	3507.01, 3425.38	1757.83	1617.05	1412.72	1385.63, 1258.52	C-Cl: 1122.70
	5	3640.08	3509.31, 3425.93	1757.49	1617.22	1413.61	1385.89, 1289.12	C-F: 1285.76
	6	3640.60	3510.62, 3429.46	1756.86	1615.06	1414.59	1391.22, 1309.64	C-O: 1081.44
Subs.: Substituents, Comp.: Compounds

Examination of ¹H NMR results

The synthesized compounds were dissolved in DMSO-d6 and ¹H NMR spectra were obtained. The chemical shift values for the compounds are shown in Table 4. DMSO-d6 signals were seen around 2.00, 2.55 ppm (quintet) and the signals of water impurity (HOD, H₂O) in DMSO were seen around 3.40 ppm [32]. For the synthesized compounds, the -NH proton signals of the isatin ring were detected as a singlet at 11.18–11.11 ppm. The -N¹H and -N²H proton signals of the thiosemicarbazone region in the compounds appeared as a singlet at 10.86–10.42 and 12.85–12.76 ppm, respectively (see Figs. S7-S12).

Table 4

Experimental and calculated ¹H NMR values of the compounds (δ, ppm)

	Comp.	=N–N²H	isatin NH	–N¹H-Ar	isatin H	ArH	-CH₃/OCH₃
Experimental	1	12.84 (s, 1H)	11.11 (s, 1H)	10.74 (s, 1H)	7.55 (s, 1H), 7.20–7.18 (d, J = 7.9 Hz, 1H), 6.85–6.83 (d, J = 7.8 Hz, 1H)	7.61–7.59 (d, J = 7.6 Hz, 2H), 7.46–7.37 (dt, J = 19.6, 7.5 Hz), 2H)	2.31 (s, 3H)
	2	12.85 (s, 1H)	11.15 (s, 1H)	10.69 (s, 1H)	7.55 (s, 1H) 7.19–7.17 (d, J = 7.9 Hz, 1H), 6.84–6.82 (d, J = 7.9 Hz, 1H)	7.53–7.51 (m, 1H), 7.42–7.26 (m, 3H)	2.30 (s, 3H)
	3	12.76 (s, 1H)	11.15 (s, 1H)	10.42 (s, 1H)	7.49 (s, 1H), 7.83–7.81 (d, J = 7.6 Hz, 1H), 6.84–6.82 (d, J = 7.9 Hz, 1H)	7.31–7.27 (t, J = 7.8 Hz, 1H), 7.18–7.13 (dd, J = 12.7, 8.1 Hz, 2H), 7.03–6.99 (t, J = 7.6 Hz, 1H)	3.87 (s, 3H, OCH₃) 2.31 (s, 3H)
	4	12.85 (s, 1H)	11.13 (s, 1H)	10.80 (s, 1H)	7.60 (s, 1H), 7.19–7.17 (d, J = 7.8 Hz, 1H), 6.84–6.82 (d, J = 7.9 Hz, 1H)	7.82 (s, 1H), 7.69–6.67 (d, J = 7.8 Hz, 1H), 7.47–7.43 (t, J = 8.0 Hz, 1H), 7.34–7.32 (d, J = 7.9 Hz, 1H)	2.32 (s, 3H).
	5	12.85 (s, 1H)	11.18 (s, 1H)	10.86 (s, 1H)	7.61 (s, 1H), 7.20–7.18 (d, J = 7.8 Hz, 1H), 6.85–6.83 (d, J = 7.9 Hz, 1H)	7.65 (s, 1H), 7.53–7.51 (d, J = 8.0 Hz, 1H), 7.49–7.44 (dd, 1H), 7.14–7.10 (t, J = 8.3 Hz, 1H)	2.31 (s, 3H).
	6	12.79 (s, 1H)	11.16 (s, 1H)	10.74 (s, 1H)	7.61 (s, 1H), 7.18–7.16 (d, J = 7.9 Hz, 1H), 6.83–6.81 (d, J = 7.9 Hz, 1H)	7.36–7.31 (m, 2H), 7.26–7.24 (d, J = 8.2 Hz, 1H), 6.87–6.85 (m, 1H)	3.79 (s, 3H, OCH₃), 2.31 (s, 3H)
Calculated	1	13.22	7.66	11.23	8.06 (H1), 7.64 (H2), 7.19 (H3)	8.04 (H5), 7.66 (H6), 7.83 (H7), 10.39 (H8)	2.22–2.67 (3H)
	2	13.06	7.65	11.00	8.06 (H1), 7.64 (H2), 7.19 (H3)	7.77 (H5), 7.67 (H6), 7.71 (H7), 10.08 (H8)	2.23–2.69 (3H)
	3	13.06	7.65	11.27	8.11 (H1), 7.64 (H2), 7.19 (H3)	7.48 (H5), 7.67 (H6), 7.44 (H7), 10.04 (H8)	4.14–4.67 (3H, OCH₃) 2.25–2.72 (3H, CH₃)
	4	13.16	7.65	10.53	8.08 (H1), 7.64 (H2), 7.19 (H3)	7.67 (H4), 7.63 (H6), 7.87 (H7), 9.97 (H8)	2.25–2.71 (3H)
	5	13.14	7.65	10.57	8.09 (H1), 7.64 (H2), 7.19 (H3)	7.38 (H4), 7.38 (H6), 7.91 (H7), 9.86 (H8)	2.24–2.70 (3H)
	6	13.05	7.64	10.48	8.07 (H1), 7.63 (H2), 7.19 (H3)	7.14 (H4), 7.18 (H6), 7.85 (H7), 9.52 (H8)	3.94–4.23 (3H, OCH₃) 2.26–2.71 (3H, CH₃)

For all compounds, the aromatic proton signals of the phenyl ring (H5–H8) were observed at 7.82–6.85 ppm. The ¹H NMR spectrum results obtained for compound 3 are shown in Fig. 2. In compound 3, the H5 and H8 protons coupled to the H6 and H7 proton and showed a doublets of doublet peak at 7.18–7.13 ppm; proton H6 bound to protons H5 and H7 showed a triplet peak at 7.31–7.27 (J = 7.8 Hz) ppm. Proton H7 bound to protons H6 and H8 showed a triplet peak at 7.03–6.99 (J = 7.6 Hz) ppm. For the whole compound, aromatic proton signals of isatin ring (H1–H3) were detected between 7.61 and 6.81 ppm. For compound 3, proton H1 appeared as a singlet peak at 7.49 ppm. Proton H2 bound to proton H3 and a doublet peak was observed at 7.83–7.81 (J = 7.6 Hz) ppm. The H3 proton bound to the H2 proton and a doublet peak was detected at 6.84–6.82 (J = 7.9 Hz) ppm. For compound 3, the proton signals of methoxy (–OCH₃) and methyl (–CH₃) groups were observed as a singlet at 3.87 and 2.31 ppm, respectively. These consequences are consistent with values those for similar compounds in the literature [3, 14, 16, 31].

Examination of ¹³C NMR results

The synthesized compounds were dissolved in DMSO-d6 and ¹³C NMR spectra were obtained. The chemical shift values of the compounds are summarized in Table 5. In all compounds (1–6), the characteristic -C = O (C7) peaks belonging to the isatin ring were observed at 163.2–160.9 ppm. The characteristic -C = N (C8) peaks belonging to the imine structure resonated between 140.9 and 140.7 ppm. The characteristic -C = S (C9) peaks belonging to the thiosemicarbazone moiety in the compounds were observed at 178.4–176.5 ppm (see Figs. S13-S18). The aromatic carbon signals attached to the isatin ring (C1–C6) were detected between 133.3 and 111.4 ppm. The aromatic carbon signals of the phenyl ring (C10–C15) were found at 163.3–111.3 ppm. When the ¹³C NMR spectrum for compound 3 is examined, it is seen that there are 15 different resonance carbon atoms compatible with the target structure (Fig. 3). The methoxy (–OCH3) and methyl (–CH3) carbon atoms of compound 3 showed resonance at 56.3 and 21.1 ppm, respectively. In addition, the C10 and C15 carbon atoms of compounds 2 and 5 were separated into doublets by interaction with the F atom nucleus.

Table 5

Experimental and calculated ¹³C NMR data of compounds **1–8**, (δ/ppm)

		C1	C2	C3	C4	C5	C6	C7	C8	C9	C10	C11	C12	C13	C14	C15	Subs.
Experimental	1	131.9	130.8	120.3	132.4	111.4	129.3	163.2	140.9	178.0	133.2	136.6	128.0	130.1	122.1	131.8	21.1^(a)
	2	133.2	131.9	120.4	132.4	122.1	130.5	163.2	140.9	178.4	127.0126.9	159.0156.5	116.5116.3	129.5 129.4	111.4 111.3	124.9 124.8	21.1^(a)
	3	131.9	132.8	120.3	132.4	111.4	127.9	163.2	140.8	176.5	127.5	153.3	112.2	120.5	126.6	121.8	21.1^(a)56.3^(b)
	4	131.9	130.4	120.3	132.5	111.4	126.2	163.2	140.9	176.7	133.3	124.4	140.4	125.4	132.9	122.3	21.1^(a)
	5	133.3	131.9	120.3	132.5	111.4	122.3	160.9	140.9	176.7	140.7140.6	130.4130.3	163.3163.2	121.7 121.6	112.8 112.5	113.2 112.9	21.1^(a)
	6	131.9	132.9	120.4	132.3	111.5	122.3	163.2	140.7	176.5	140.0	111.9	159.7	118.0	129.6	111.3	21.1^(a)55.7^(b)
Calculated	1	142.3	139.5	115.6	148.1	127.7	128.1	169.3	137.6	183.1	143.4	136.9	136.8	131.5	133.9	123.6	22.5^(a)
	2	142.3	139.4	115.6	148.1	127.7	127.9	169.3	137.3	183.5	134.7	163.9	120.9	131.4	130.7	123.5	21.1^(a)
	3	142.2	139.1	115.5	147.8	127.9	127.7	169.3	136.8	181.7	134.9	157.9	114.7	131.4	124.9	122.1	22.6^(a)58.5^(b)
	4	142.3	139.4	115.6	148.1	127.7	127.8	169.3	137.4	182.9	148.4	126.2	149.0	130.9	136.9	120.3	22.6^(a)
	5	142.4	139.4	115.6	148.0	127.7	127.9	169.3	137.3	182.9	148.9	112.3	174.6	116.9	137.6	118.0	22.6^(a)
	6	142.2	139.1	115.6	147.9	127.9	127.6	169.3	136.9	182.5	148.2	113.2	169.9	111.7	136.7	113.5	22.6^(a)58.1^(b)
Subs.: Substituents, a: CH₃, b: OCH₃

In addition, the presence of 2-Cl (136.6 ppm), 2-F (159.0 ppm), 2-OCH₃ (153.3 ppm), 3-Cl (140.4 ppm), 3-F (163.3 ppm) and 3-OCH₃ (159.7 ppm) atoms/groups caused the carbon signals to appear in lower ranges compared to the benzene signal (128.5 ppm), i.e., high δ values in all compounds. These datya are in agreement with the data that reported similar compounds [3, 14, 16, 31, 33].

Evaluation of antioxidant activity

In this study, percent inhibition changes depending on the concentration of butylated hydroxytoluene (BHT) and synthesized compounds, which we used to evaluate antioxidant properties, are given in Fig. 4.

When the DPPH free radical scavenging activity changes were examined, the synthesized compounds 3, 5, and 6 showed a more regular increase depending on the concentration. Compounds 1, 2, and 4 showed a very low percentage of inhibition at the first two concentrations compared to the standard. These inhibition changes, which differ from molecule to molecule, vary according to the position and structure of the thiosemicarbazone-bound substituents rather than the isatin groups. In order to reveal these differences more clearly, IC50 values obtained from the concentration equations for BHT and molecule samples were calculated and the values are given in Table 6. Accordingly, it was found that the free radical scavenging effects of the molecules changed in the order BHT > 6 > 3 > 5 > 1 > 2 > 4.

Table 6

IC₅₀ values for BHT and the synthesized compounds **1–6**
Compound	DPPH scavenging activity IC₅₀ (µM)*	R²
1	23.91 ± 0.06	0.976
2	23.96 ± 0.06	0.967
3	22.31 ± 0.04	0.985
4	27.35 ± 0.06	0.966
5	22.52 ± 0.04	0.888
6	21.74 ± 0.04	0.920
BHT	10.12 ± 0.02	0.956
*IC₅₀ = the concentration (µM) exhibiting 50% inhibition of DPPH radical.
Values are expressed as means (n = 3).

Naik et al. reported that, similar to our study, the antioxidant activity of substituted anilines rather than the isatin structure increased significantly. They found that molecules containing electron-donating methoxy substituents showed greater antioxidant activity. especially in comparison with butylated hydroxyanisole (BHA) [34]. In this study, it was also observed that methoxy substituted structures showed higher antioxidant activity than the others, but showed much lower antioxidant activity than the standard BHT. Groups attached to thiocarbohydrazone structures have important roles on antioxidant activity, as Pakravan et al. summarized earlier and Božić et al. mentioned in their work on synthesizing carbohydrazones compounds [15, 35]. It was reported by G. Kiran et al. that especially halogen-containing systems significantly increased antioxidant activity [3]. Similarly, halogen substituted groups were used in this study and their antioxidant effects were observed. At the same time, methoxy substituted structures were used in this study. As in the study of Bakır and Lawag, it was found that compounds with –OMe structures exhibited better antioxidant activity in this study [36]. When the different substituents attached to the thiosemicarbazide structures in the ortho position in compounds 1, 2, and 3 were examined, it was observed that the antioxidant activity changed in the order –OMe > F > Cl. This order was also found for compounds 4, 5, and 6 attached to the ring in the thiosemicarbazide structure at the meta position.

DFT analysis

The compounds can be examined under two groups according to their ortho- and meta- Cl, F, and MeO substituents, respectively. Although the effects of substituents on the antioxidant properties of compounds depend on many internal and external factors such as intermolecular-intramolecular interactions and solvent effects, it is also obvious that the electronic properties of the compounds will affect their chemical behavior. HOMO-LUMO surfaces of compounds vary depending on the positions of the substituents (Fig. 5; see Supplementary Fig. S19 for all). As seen from both inhibition percentage data and IC₅₀ values, ortho/meta-MeO substituted compounds 3 and 6 exhibited high antioxidant activity. While the LUMO distributions in these compounds do not differ much from each other, it has been observed that in the ortho position of MeO, HOMOs are concentrated on the benzene ring and thiourea region, whereas in the meta position, they are generally concentrated on the thiourea region. ESP surfaces also indicate that the ortho-MeO substituent contributes more to intramolecular interactions. The effects of chlorine and fluorine atoms on HOMO are more obvious than their effects on LUMO. Although the meta-F substituent had a similar effect to meta-MeO in terms of the distribution of HOMOs, it enhanced a wide HOMO distribution in the isatin-thiourea region when in the ortho position. It has been observed that the chlorine atom has an effect on HOMO-LUMOs similar to that of the fluorine atom in the ortho and meta positions. It is quite difficult to compare the effects of electron-withdrawing fluorine and chlorine substituents on the HOMO-LUMO surfaces of compounds (ESP-HOMO-LUMO surface maps are very similar). Essentially, MeO is considered an electron donor group, while fluorine and chlorine are members of the electron-withdrawing group. While methoxy has a strong electron-donating and reactivity-increasing effect in the ortho position, these effects are weak in the meta position, but it is clear that MeO increases the antioxidant effect of the compounds more than fluorine and chlorine, both in the ortho and meta positions.

The electronic properties of compounds 1–3 and 4–6, according to the ortho and meta positions of the substituents, revealed similar data within their groups (Fig. 6). While the E_g values of ortho substituted compounds 1–3 were calculated as 3.349, 3.351, and 3.215 eV, respectively (the effect of MeO is pronounced), the E_g values of meta substituted compounds 4–6 were similarly calculated as 3.321, 3.312, and 3.279 eV, respectively. A low E_g value indicates high reaction ability, but their evaluation is a little more difficult due to the contribution of ortho substituents to intramolecular interactions. For example, for meta-(Cl, F, MeO) substituted compounds, the lowest E_g value was calculated for MeO substituted compound 6 and the highest for Cl substituted compound 4 (see Supplementary Table S1). These results are also consistent with the IC₅₀ values of compounds 4–6. However, for compounds 1–3, while the lowest E_g value of MeO substituted compound 3 is compatible with the experimental antioxidant effect data, the same is not true for the effects of fluorine and chlorine substitutes, but it can be said that they cause effects close to each other. Moreover, meta-substituted compounds exhibit similar inhibition effects across all concentrations, while the influence of ortho substituents on the compound's inhibition varies with concentration, which can be attributed in part to intramolecular effects.

In this paper, new 5-methhylisatin-thiosemicarbazone compounds have been achieved with good yields of 55– 85%. The molecular structures of the newly synthesized compounds were characterized using spectroscopic techniques such as FT–IR, ¹H NMR, ¹³C NMR and elemental analysis methods. In vitro antioxidant activities of these molecules were observed using DPPH free radical scavenging process. IC₅₀ values of the samples were found to vary between 8.44 and 10.66 𝜇M. The results in this study clearly revealed that different substituents were effective on the antioxidant activity in carbothioamide compounds formed with thiosemicarbazone structures. In the synthesized compounds, the methoxy-containing structures exhibited the highest antioxidant effect compared to the halogen-containing structures, regardless of which position they were attached to. DFT calculations reveal some findings by evaluating the effects of the electronic properties of compounds on antioxidant activity. It was observed that compounds with MeO substituted generally had lower E_g values, indicating higher reactivity. Intramolecular effects, especially at ortho substituents, affected the inhibition effects of compounds at different concentrations. While meta-substituted compounds showed more consistent inhibition effects depending on concentrations, the inhibition effect varied in ortho-substituted compounds. These conclusions highlight that electronic properties, especially the presence and position of substituents, play an important role in determining the antioxidant properties of compounds.

Supplementary Files

IR, ¹H NMR, and ¹³C NMR spectra of all compounds are given in the supporting information.

Supplementary Files

IR, ¹H NMR, and¹³C NMR spectra of all compounds are given in the supporting information.

Acknowledgements

I would like to thank Assoc. Prof. M. Serdar Çavuş for the DFT calculation processes, and Assoc. Prof. Dr. Halit Muğlu and Assoc. Prof. Dr. Hasan Yakan for their contributions to the synthesis studies. I would also like to thank the Scientific and Technological Research and Application Center (Tokat Gaziosmanpaşa University, Gübitam) and Dr. Ömer Faruk Ensari for obtaining the NMR spectra.

Author Contribution Statement

Bakır T: Conceptualization, methodology, formal analysis, writing-original draft preparation, investigation, resources, software, writing-review&editing.

Conflict of Interest

The author declares that he has no conflict of interest.

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No competing interests reported.

Scheme1300dpi.jpg
Scheme 1 Synthetic route for the obtained molecules (1–6)
GraphicalAbstract300dpi.jpg
SuplementalFilesRCI.docx

New 5-methylisatin-thiosemicarbazones: preparation, spectroscopic elucidation and antioxidant properties

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Experimental

Synthesis of 5-methylisatin-thiosemicarbazone compounds (1–6)

Antioxidant Activity Measurements

Theoretical Computational Measurements

Results and discussion

FTIR Vibration frequencies

Examination of ¹H NMR results

Examination of ¹³C NMR results

Evaluation of antioxidant activity

DFT analysis

Conclusions

Supplementary Files

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

New 5-methylisatin-thiosemicarbazones: preparation, spectroscopic elucidation and antioxidant properties

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Experimental

Synthesis of 5-methylisatin-thiosemicarbazone compounds (1–6)

Antioxidant Activity Measurements

Theoretical Computational Measurements

Results and discussion

FTIR Vibration frequencies

Examination of 1H NMR results

Examination of 13C NMR results

Evaluation of antioxidant activity

DFT analysis

Conclusions

Supplementary Files

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

Examination of ¹H NMR results

Examination of ¹³C NMR results