Synthesis and characterisation of novel 4,6-dimethoxyindole-7- and -2-thiosemicarbazone derivatives: Biological evaluation as antioxidant and anticholinesterase candidates

Abstract

Two sets of novel indole-based thiosemicarbazone systems 8a–d and 9a–d are prepared by the Schiff base condensation reaction of indole carbaldehydes 4 and 6 with a range of thiosemicarbazides 7a–d in high yields and purity. The antioxidant properties of the synthesised compounds 8a–d and 9a–d are determined by employing three different assays, namely 2,2-diphenyl-1-picrylhydrazyl hydrate–free radical scavenging, ABTS [2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] cationic radical decolarization and cupric ion reducing antioxidant capacity. The anticholinesterase properties of the products are investigated by acetylcholinesterase and butyrylcholinesterase enzyme inhibition assays. The methyl-substituted compounds 8b and 9b display the highest inhibition for the ABTS assay and absorbance values for the cupric ion reducing antioxidant capacity assay, while compound 9c shows the best activity for 2,2-diphenyl-1-picrylhydrazyl hydrate assay. Moderate inhibition of acetylcholinesterase and butyrylcholinesterase is determined in the case of compound 8b.

Introduction

Indoles are important heterocyclic compounds which have been the subject of many synthetic studies.^1–3 Due to the presence of the indole core in many natural products, studies have been directed towards investigations of the biological properties of natural indolic compounds, and a range of medical uses has been identified.^4–7 One of the most important amino acids for the human diet, ⁸ L-tryptophan contains an indole nucleus. Moreover, vinblastine and vincristine are examples of indole-containing systems that show very potent anticancer activity. ⁹ Apart from the biological importance of indole systems, most importantly, activated indole compounds have been used in many synthetic studies as valuable intermediates.^10–12 A range of reactions such as formylation or halogenation can be performed at different locations on indole systems, while the attachment of important functional groups generates valuable building blocks. These intermediates can be readily converted into other functional groups allowing the production of indole-based macrocyclic compounds.^13–15

Thiosemicarbazones are important classes of Schiff base ligands due to the presence of a R¹R²C=N-NH-(C=S)-NR³R⁴ moiety generated by the reaction between an aldehyde and the thiosemicarbazides.^16–18 The conjugated N-N-S system provides an important therapeutic potential to these thiosemicarbazone-based systems, which can interact with biomolecules.^19–21 Due to their biological activity, thiosemicarbazones appear in a large number of medically valuable compounds and considerable effort has been devoted towards the synthesis of these compounds. Thiosemicarbazones have also attracted the attention of researchers due to their high affinity for metal ions such as iron, zinc and copper to produce metal complexes, which are also important biological targets for interactions with nucleic acids.^22–24

The design and synthesis of analogues that are structurally derived from the combination of a biologically active indole and a thiosemicarbazide has yielded new indole thiosemicarbazone systems as potential therapeutic agents.^25,26 An intense literature search revealed that indole thiosemicarbazone scaffolds have been identified as promising compounds for antioxidant studies due to their ability to donate hydrogen or an electron to acceptors (i.e. 2,2-diphenyl-1-picrylhydrazyl hydrate [DPPH]) and to reduce the production of free radicals.^27,28 It has also been suggested that the accumulation of the bioavailable transition metals Fe, Cu and Zn is responsible for Alzheimer’s disease (AD), since the self-aggregation of amyloid-β (Aβ) via metal peptide chelation forms senile plaques, which are deposited outside of neurons. ²⁹ The presence of conjugated N-N-S tridentate systems on thiosemicarbazones would reduce the accumulation of these metals, and their chelation ability would be a potential therapy for the treatment of AD. ²⁹

In the present work, we aimed to prepare two sets of new thiosemicarbazones with different substituents on the 4,6-dimethoxyindole nucleus. The Hemetsberger reaction was chosen as the synthetic preparation method for the designated indole compounds since it readily forms derivatives of 2-carboxyindoles from aromatic or heterocyclic aldehydes.^30–32 The position 2 was chosen as the first location on the indole ring for the preparation of the first set of compounds. 4,6-Dimethoxyindole-2-carbaldehyde ³³ was prepared by reduction and oxidation of methyl 4,6-dimethoxyindole-2-carboxylate, respectively. The dimethoxyindole backbone was chosen since the synthetic strategy can easily accommodate electron-donating substituents^34–36 and there has been significant interest in heterocyclic aromatic systems derived from dimethoxyindoles due to their possible biological and pharmacological activities.^37–39 The nucleophilic reactivity of 4,6-dimethoxyindole was reported in our previous work ⁴⁰ by utilising the Vilsmeier–Haack formylation. ⁴¹ The formylation was found to occur selectively at the 7-position and the products were subsequently converted into 7-thiosemicarbazones as the second set of compounds.

Eight new 4,6-dimethoxyindole thiosemicarbazones 8a–d and 9a–d have been prepared, and full chemical characterization is reported in this work. The targeted compounds have been subjected to the three different assays, namely, DPPH, ABTS and cupric ion reducing antioxidant capacity (CUPRAC), to investigate the antioxidant properties. Moreover, their acetylcholinesterase (ACh) and butyrylcholinesterase (BCh) enzyme inhibition properties have also been evaluated. To the best of our knowledge, our study is the first report on the preparation of designated dimethoxyindole thiosemicarbazone systems and provides a complementary comparison of antioxidant capacities of differently located thiosemicarbazone moieties.

Results and discussion

Chemistry

Methyl 4,6-dimethoxyindole-2-carboxylate 3 (Scheme 1) was chosen as the starting material for the preparation of the desired indole carbaldehydes 4 and 6 (Scheme 2). The final products 8a–d and 9a–d were synthesised by the Schiff base condensation reaction of the corresponding indole carbaldehydes with the appropriate thiosemicarbazides 7a–d using acetic acid in ethanol at room temperature (Scheme 3).

Scheme 1.

Reagents and conditions: i: N₃CH₂CO₂Me, Anhyd. MeOH <–10 °C, 4 h, 78%, ii: Xylene reflux, 6 h.

Scheme 2.

Reagents and conditions: i: POCl₃, DMF, −10 °C overnight. a: LiAlH₄/THF, −10 °C 1 h, rt 3 h b: MnO₂, CH₂Cl₂, reflux, 24 h.

Scheme 3.

Reagents and conditions: i: EtOH, a few drops of AcOH, overnight, rt.

Preparation of methyl 4,6-dimethoxyindole-2-carboxylate 3

According to the literature, the preparation of activated indole scaffolds has been achieved by numerous methods. The Hemetsberger reaction is potentially one of the most important methods for the synthesis of indoles. In our work, methyl 4,6-dimethoxyindole-2-carboxylate was afforded via a vinyl azide, which is generated by the condensation of 2,4-dimethoxybenzaldehyde (1) with methyl azidoacetate in methanolic sodium methoxide (Scheme 1). The thermal decomposition of vinyl azide 2 followed by intramolecular cyclisation gave the desired indole 3.

Preparation of indole carbaldehydes

Methyl 7-formyl-4,6-dimethoxyindole 4

The Vilsmeier–Haack formulation reaction is a very useful process for installing an aldehyde, which can be used for further synthetic transformations. In the current work, the desired formylated methyl 4,6-dimethoxyindole-2-carboxylate was synthesised by treatment of the methyl indole ester 3 with the Vilsmeier reagent at low temperature. The formylation occurred at the C7 position due to the methyl ester group at C2 and the location of the methoxy groups on the benzene ring (Scheme 2).

4,6-Dimethoxyindole-2-carbaldehyde 6

The compound 6 was synthesised by reduction of the methyl ester at C2 on compound 3 and subsequent oxidation of the hydroxymethyl indole 5 by the procedures reported in the previous study ⁴⁰ (Scheme 2).

The preparation of desired dimethoxyindole thiosemicarbazones

Following preparation of the targeted 4,6-dimethoxyindole 2-carbaldehyde and methyl 7-formyl-4,6-dimethoxyindole-2-carboxylate, a range of thiosemicarbazides 7a–d was treated with the prepared indole carbaldehydes to afford the desired thiosemicarbazones (Scheme 3). The reactions were carried on in EtOH and acetic acid was used as the catalyst. The reaction mixtures were stirred at room temperature overnight and the resulting crude products were purified by recrystallization from EtOH/hexane to give the title compounds 8a–d and 9a–d. All the ¹H NMR, ¹³C NMR and high resolution mass spectroscopy (HRMS) spectras are given as supplemental material.

Indole carbaldehyde	Thiosemicarbazide	R1	R2	Product	Yields %
4	7a	H	H	8a	67
4	7b	H	Me	8b	74
4	7c	Me	Me	8c	62
4	7d	H	Et	8d	71
6	7a	H	H	9a	78
6	7b	H	Me	9b	74
6	7c	Me	Me	9c	62
6	7d	H	Et	9d	78

The ¹H NMR spectrum of compound 8a in dimethyl sulfoxide (DMSO) showed three singlets at 3.83, 3.95 and 4.00 ppm corresponding to the methyl ester and the methoxy protons located at the 4- and 6-positions on the indole moiety. The two singlets at 6.53 and 7.12 ppm were assigned to the H5 and H3 protons, respectively. A broad singlet at 8.10 ppm corresponded to the NH₂ protons, while another broad singlet at 11.27 ppm corresponded to the NH proton on the thiosemicarbazone moiety. Furthermore, the characteristic CH proton appeared as a singlet at 8.59 ppm and the NH indole proton appeared as a broad singlet at 10.68 ppm.

The ¹H NMR spectrum of compound 9a in DMSO confirmed the structure of the molecule by displaying the loss of the aldehyde peak at the 2-position, as a result of condensation with the NH₂ group of thiosemicarbazide, and by the appearance of an azomethine CH proton at 7.91 ppm. The presence of two broad singlets at 8.08 and 8.25 ppm corresponding to the NH₂ protons and the singlet at 11.48 ppm showing the NH proton proved the formation of the desired dimethoxyindole-2-thiosemicarbazone compound 9a. The characteristic indole protons signals were assigned at the expected areas along with the appearance of the two methyl ester protons at 3.78 and 3.83 ppm. Three singlets at 6.16, 6.40 and 6.65 ppm correspond to the protons H7, H3 and H5, respectively. The indole NH proton appeared at 10.27 ppm as a broad singlet.

The synthetic strategy was extended to the synthesis of 4,6-dimethoxyindole-2- and -7-thiosemicarbazone systems containing methyl and ethyl substituents at R² (Scheme 3). Hence, 7-formyl-dimethoxyindole 4 and dimethoxyindole-2-carbaldehyde 6 were reacted with 4-methyl and 4-ethyl thiosemicarbazides 7b and 7d in the presence of acetic acid to give substituted thiosemicarbazones 8b and 8d and 9b and 9d.

The ¹H NMR spectrum of compound 8b in DMSO showed two broad singlets at 8.49 and 11.32 ppm corresponding to the NHMe and NH protons and a singlet at 3.03 ppm was assigned to the CH₃ protons on the N4 end. In the case of compound 8d, the only difference was the presence of triplet and quartet signals at 1.22 and 3.60 ppm (J = 7.0 Hz and 6.4 Hz) corresponding to the CH₃ and CH₂ protons.

In the ¹H NMR spectrum of compound 9b, the NH proton appeared as broad singlet at 11.54 ppm, while the NHMe on the thiosemicarbazone moiety appeared as a broad doublet at 8.54 ppm due to coupling (J = 4.4 Hz) with the neighbouring CH₃ groups. The characteristic CH₃ protons resonated as a doublet at 3.07 ppm with the same coupling constant. In the case of compound 9d, the chemical shifts for the indole moiety were similarly assigned. The ¹H NMR spectrum displayed the CH₂ and CH₃ protons for the ethyl substituent at 3.63 and 1.19 ppm as triplet and quartet signals with coupling constants of 6.4 and 6.8 Hz, respectively.

The same synthetic route was applied to the carbaldehydes 4 and 6 with the 4,4-dimethyl thiosemicarbazide 7c in order to generate two analogues with dimethyl substitution on the N4 end. Thus, indole-2-carbaldehyde 4 and indole-7-carbaldehyde 6 were reacted with 4,4-dimethyl thiosemicarbazide (7c) in the presence of acetic acid to generate the target compounds (Scheme 3).

The formation of the desired methyl 4,6-dimethoxyindole-7-thiosemicarbazone 8c was confirmed by ¹H NMR spectroscopy with the appearance of a singlet at 3.31 ppm corresponding to the two methyl groups on the N4 end. In the case of compound 9c, the spectrum displayed two methyl protons at 3.29 ppm as a sharp singlet.

The formation of the targeted compounds was also confirmed by ¹³C NMR spectroscopy, displaying the C=S carbon signals between 175.0 to 180.0 ppm. In addition, the azomethine carbon (C=N) atom resonated around 130–140 ppm. Infrared (IR) spectroscopy showed the expected absorption bands for identification of the designated compounds. The C=S stretching bands appeared around 1650–1700 cm⁻¹, while the NH and NH₂ stretches were found around 3300–3450 ppm as broad bands. The azomethine C=N stretching occurred around 1500–1550, confirming the Schiff base condensation reactions.

Biological studies

The novel methyl 4,6-dimethoxyindole-7-thiosemicarbazones 8a–d and 4,6-dimethoxyindole-2-thiosemicarbazones 9a–d were screened to determine their anticholinesterase activity using acetylcholine and butyrylcholine enzyme inhibition assays. The inhibition of ACh and BCh by the compounds is given as percentage values at 200 µg/mL concentrations in Table 1 and compared with the galanthamine as a standard. The compounds showing higher percentage of inhibitions were evaluated as potent candidates for the treatment of AD. Their antioxidant profiles against free radicals, cationic radicals and reductive efficiencies for Cu metal were also investigated using DPPH, ABTS and CUPRAC antioxidant assays. The DPPH assay was used to measure the hydrogen atom (or one-electron) donation ability of the scavenge molecule to DPPH, resulting in reduction of DPPH to DPPH₂. The ABTS•+ [2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] assay was carried out to determine the cationic radical reducing ability of the synthesised compounds. The CUPRAC method is an antioxidant capacity assay to identify the reductive efficiencies of compounds for Cu metal. It involves reaction of the antioxidant compound with CuCl₂, neocuproine and ammonium acetate at pH 7. The mean and standard deviation (SD) values for each compound were calculated from at least three replicate experiments.

Table 1.

Acetylcholinesterase and butyrylcholinesterase enzyme inhibitory activities of 4,6-dimethoxyindole-7-thiosemicarbazones 8a–d and 2-thiosemicarbazones 9a–d.^{a, b}

Sample	AChE (% inhibition)	BChE (% inhibition)
8a	NA	31.90 ± 0.37
8b	22.59 ± 0.63	26.68 ± 0.99
8c	NA	24.37 ± 0.56
8d	NA	NA
9a	NA	14.93 ± 0.28
9b	NA	3.18 ± 0.05
9c	NA	NA
9d	12.09 ± 0.88	25.58 ± 0.43
Galanthamineb,c	82.37 ± 0.37	84.06 ± 0.51

NA: not active.

a

Values expressed are means ± S.D. of three parallel measurements, (p< 0.05).

b

200 µg/mL.

c

Positive control.

DPPH free radical scavenging assay

Figure 1 shows the DPPH radical scavenging activity of the eight 4,6-dimethoxyindole thiosemicarbazone compounds, 8a–d and 9a–d, and the positive controls, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and alpha-tocopherol (α-TOC). It was observed that the synthesised compounds had a range of impact on the scavenging activity of free radicals with treatments from 10 to 100 µM. Out of the 4,6-dimethoxyindole-2-thiosemicarbazone derivatives 9a–d, compound 9c had the strongest inhibition activities for all the tested concentrations. The inhibition value of 85% at 100 µM was found to be the highest reduction compared with the all standards at the same concentrations. Moreover, compound 9c showed much better inhibition at a concentration of 50 µM, with the value of 80% inhibition being more than 20% additional inhibition compared with the standards BHA and α-TOC. The level of inhibition efficiency of compound 8c, as an example of indole-7-thiosemicarbazone derivatives, was found to be similar to the standards BHA and α-TOC. The best inhibition was achieved by treatment of 50 µM of compound 8c, showing at least 15% and 5% additional inhibition compared with BHA and α-TOC, respectively.

Figure 1.

Inhibition (in %) of the DPPH free radical scavenging activity of compounds 8a–d, 9a–d and controls BHA, BHT and α-Toc. Values are means, ±SD, n = 3, p< 0.05, significantly different from each other with Student’s t test.

ABTS cation radical decolorization assay

The initial cationic radical scavenging activity assay was carried out on concentrations from 10 to 100 µM and the novel 4,6-dimethoxyindole-2- and -7-thiosemicarbazones displayed promising potency and further investigations were carried out using lower concentrations (1–10 µM) and the Figure 2 shows the concentration-dependent behaviour of compounds 8a–d and 9a–d and the controls BHT, BHA and α-TOC (1, 2.5, 5, 10 μM). Generally, the inhibition values for 4,6-dimethoxyindole-2-thiosemicarbazone analogues 9a–d were found to be greater than those of analogues 8a–d with the 7-thiosemicarbazone functionality. Compound 9b exhibited the greatest cationic radical scavenging activity at all the concentrations and the highest inhibition was determined at a 10 µM concentration with an inhibition value of more than 85%, which was better than all the standards. The inhibition values for compounds 9c and 9d at the concentrations of 1, 2.5 and 5 µM were found to be more efficient than all the standards and were similar to the best standard BHA at a 10 µM concentration. The results also revealed that the methyl 4,6-dimethoxyindole-7-thiosemicarbazone derivatives 8a–d were moderately effective for cationic radical scavenging activity. The best results were obtained with compounds 8b and 8d with the highest inhibition value being almost 60%.

Figure 2.

Inhibition (in %) of the ABTS cation radical scavenging activity of compounds 8a–d, 9a–d and controls BHT, BHA α-Toc. Values are means, ±SD, n = 3, p< 0.05, significantly different from each other with Student’s t test.

CUPRAC

Figure 3 shows the absorbance values obtained from the CUPRAC assay for the synthesised compounds 8a–d and 9a–d and the standards BHT, BHA and α-TOC. A higher absorbance value was identified as better reducing capacity. As in the cationic radical scavenging activity, the reducing potency of 4,6-dimethoxyindole-2-thiosemicarbazone compounds 9a–d was found to be more promising than the analogues of methyl 4,6-dimethoxyindole-7-thiosemicarbazones 8a–d. Compounds 9b and 9d were determined as the most potent derivatives with the highest absorbance values at all concentrations and were identified as much better candidates than all the standards. The absorbance values for compound 9a show that it displayed better reducing capacity compared with the best standard BHA at concentrations from 10 to 50 µM. Methyl 4,6-dimethoxyindole-7-thiosemicarbazone derivatives 8a–d showed lower absorbance values indicating lower inhibition. The best absorbance value was displayed in the presence of methyl-substituted compound 8b, which was found to be better than the two standards BHT and TOC.

Figure 3.

Absorbance values for CUPRAC of compounds 8a–d, 9a–d and controls BHT, BHA α-Toc. Values are means, ±SD, n = 3, p< 0.05, significantly different from each other with Student’s t test.

Determination of anticholinesterase activity

The ACh and BCh enzyme inhibitory activities were evaluated for determination of the therapeutic potential of the 4,6-dimethoxyindole-2- and -7-thiosemicarbazones for the treatment of AD. As expected, the positive control galanthamine showed very potent inhibition both on ACh and BCh with values of 82.37% and 84.06%, respectively. In contrast, most of the synthesised compounds demonstrated much less inhibition and no enzyme inhibitory activity for ACh. In the case of compounds 8b and 9d, a moderate increase for ACh enzyme inhibition was determined with values of 22.59% and 12.09%. In the case of the BCh enzyme inhibition assay, improved values were obtained compared with the ACh enzyme inhibition assay. Compound 8a showed the highest BCh enzyme inhibition with a value of 31.90%.

Conclusion

Indole thiosemicarbazone systems were identified as valuable synthetic intermediates for the preparation of complex structures and important ligand systems for metal complexes. Moreover, they were determined as promising targets for antioxidant and anticholinesterase studies due to their ability to donate hydrogen or electron. This work describes the synthesis of two series of indole thiosemicarbazone systems via Schiff base reactions between the corresponding carbaldehydes and thiosemicarbazides. The method involves the preparation of methyl 4,6-dimethoxyindole-2-carboxylate 3 via Hemetsberger reaction followed by Vilsmeier–Haack reaction to afford the carbaldehyde 4. Reduction and oxidation of the 2-methyl ester on the main structure 3 generated the other desired carbaldehyde 6. Biological data revealed that the indole-2-thiosemicarbazone systems 9a–d were more promising structures than indole-7-thiosemicarbazones 8a–d for investigations of novel potent antioxidant scaffolds. The best antioxidant properties were demonstrated by compound 9b, as an example of an indole-2-thiosemicarbazone with methyl substitution on N4. Compound 9b showed better ABTS and CUPRAC antioxidant inhibitions values than all the controls at all concentrations. Similarly, the methyl-substituted indole-7-thiosemicarbazone derivative 8b was also determined as the most potent compound among the indole-7-thiosemicarbazones for the same assays. The DPPH results revealed that compounds 8c and 9c provided better inhibitions in radical scavenging activity assays. The synthesised compounds were found to be less effective for in anticholinesterase activity assays and compound 8b showed moderate activity for both ACh and BCh enzyme inhibition. From a synthetic aspect, our work has provided eight new indole thiosemicarbazone ligands suitable for future studies in the area of metal complexes synthesis and the biological evaluation.

Materials and methods

Chemicals and physical measurements

All commercially available reagents and the standards used for the biological assays were purchased from Sigma Aldrich and used without further purification. The synthetic procedures have been reported for the final compounds as general methods and other procedures have been given individually with appropriate references. All reactions were monitored by thin layer chromatography using Merck TLC Silica gel 60 F₂₅₄. Silica gel 60 (particle size: 0.040–0.063 mm, 230–400 mesh ASTM) for column chromatography was obtained from Merck. ¹H and ¹³C NMR spectra were recorded for all compounds either in DMSO or CDCl₃ solutions on a Bruker DPX 400 MHz spectrometer at 300 K. Chemical shifts are reported in parts per million and referenced to the residual solvent peak (DMSO-d₆: ¹H 2.50 ppm, ¹³C 39.52 ppm and CDCl₃: ¹H 7.24 ppm, ¹³C 77.23 ppm). Coupling constants (J) are reported in Hertz (Hz). Standard conventions indicating multiplicity were used: m = multiplet, t = triplet, d = doublet, s = singlet, dd = doublet of doublets. Infrared spectra were recorded using a Thermo Scientific Nicolet IS10 FT-IR spectrometer between 600 and 4000 cm⁻¹. Melting points were measured using a Mel-Temp melting point apparatus and are uncorrected high-resolution mass spectra [ESI] were recorded on an Orbitrap LTQ XL ion trap mass spectrometer (Thermo Scientific, Waltham, MA, USA) using a nanospray (nano-electrospray) ionisation source.

Biological studies

Antioxidant assays

DPPH free radical scavenging activity

The assay was performed according to the methods^42,43 with minor modifications. BHT, BHA and α-TOC were used as standards for comparison.

ABTS cation radical decolorization activity

The method was followed as in the literatures^43,44 with the minor changes. BHA, BHT and α-TOC were used as standards.

CUPRAC

The procedure was followed as in the literature^45,46 with minor modifications. BHA, BHT and α-TOC were used as standards.

Anticholinesterase activity determination method

ACh and BCh enzyme inhibitory activities were measured by the slightly modified spectrophotometric method reported in the literature.^46,47 Galanthamine was used as the standard. DMSO was used as the solvent to dissolve the samples and controls.

$$\%\mathrm{Inhibition}=({\mathrm{A}}_0-{\mathrm{A}}_1)/{\mathrm{A}}_0\times 100$$

where A₀ is the absorbance of the control and A₁ is the absorbance in the presence of the sample.

Statistical analysis

The results were mean ± SD of three parallel measurements. All statistical comparisons were made by means of Student’s t test; p values< 0.05 were regarded as significant.

Chemical synthesis

Preparation of compounds 3, 4, 5 and 6

The synthetic procedures reported in the previous study ³³ was followed for the preparation of methyl 4,6-dimethoxyindole-2-carboxylate 3, methyl 7-formyl-4,6-dimethoxyindole-2-carboxylate 4, 2-hydroxymethyl-4,6-dimethoxyindole 5 and 4,6-dimethoxyindole-2-carbaldehyde 6.

General procedure for the preparation of thiosemicarbazones

The carbaldehyde (4 or 6) (1 equiv) was reacted with the appropriate thiosemicarbazide 7a–d (1 equiv) in ethanol. Acetic acid (5 drops) was added as catalyst, and the solution was stirred overnight at room temperature. The resulting dark yellow solution was concentrated under vacuum, and the resulting yellow solid was crystallised from ethanol to give the title compounds 8a–d and 9a–d.

Methyl 7-[(E)-(2-carbamothioylhydrazinylidene)methyl]-4,6-dimethoxy-1H-indole-2-carboxylate (8a)

The title compound was synthesised following the general procedure using methyl 7-formyl-4,6-dimethoxyindole-2-carboxylate (4) (300 mg, 1.13 mmol) and thiosemicarbazide (7a) (84.7 mg, 1.13 mmol) in EtOH (25 mL) with 5 drops of acetic acid. The product 8a was obtained as a dark yellow solid powder; yield 255 mg (67%); 262 °C; IR: v_max 3444, 3328, 3142, 2937, 1688, 1595, 1513, 1438, 1371, 1312, 1252, 1211, 1162 987, 831, 750; ¹H NMR (400 MHz, DMSO-d₆): δ 3.83, 3.95, 4.00 (each 3H, s, OMe), 6.53 (1H, s, H5), 7.12 (1H, s, H3), 8.10 (2H, brs, NH₂), 8.59 (1H, s, CH), 10.68 (1H, brs, NH indole), 11.27 (1H, s, NH); ¹³C NMR (100 MHz DMSO-d₆): δ 52.2, 56.3 and 57.4 (OMe), 89.7 (C5), 99.6 (C3), 106.6, 113,4, 126.3, 135.3 (aryl C), 141.1 (C=N), 157.2, 160.3 (aryl C), 161.5 (CO₂Me), 177.5 (C=S); HRMS (ESI⁺): found m/z 337.0959, [M + H]⁺, C₁₄H₁₇N₄O₄S requires 337.0965.

Methyl 4,6-dimethoxy-7-{(E)-[2-(methylcarbamothioyl)hydrazinylidene]H-indole-2-carboxylate (8b)

The title compound was synthesised following the general procedure using methyl 7-formyl-4,6-dimethoxyindole-2-carboxylate (4) (300 mg, 1.13 mmol) and 4-methylthiosemicarbazide (7b) (102 mg, 1.13 mmol) in EtOH (25 mL) with 5 drops of acetic acid. The product 8b was obtained as a pale yellow solid; yield 293 mg (74%); 253 °C; IR: v_max 3403, 3265, 3093, 2996, 2940, 1699, 1597, 1539, 1505, 1431, 1367, 1308, 1252, 1200, 1084, 987, 801, 720; ¹H NMR (400 MHz, DMSO-d₆): δ 3.03 (3H, s, CH₃) 3.84, 3.95, 3.99 (each 3H, s, OMe), 5.74 (CH₂Cl₂), 6.53 (1H, s, H5), 7.14 (1H, s, H3), 8.49 (1H, brs, NHMe), 8.58 (1H, s, CH), 10.72 (1H, brs, NH indole), 11.32 (1H, s, NH); ¹³C NMR (100 MHz DMSO-d₆): δ 31.4 (CH₃), 52.2, 56.3 and 57.4 (OMe), 89.7 (C5), 99.8 (C3), 106.6, 113.4, 126.3, 135.3 (aryl C), 140.4 (C=N), 157.0, 160.0 (aryl C), 161.4 (CO₂Me), 177.9 (C=S); HRMS (ESI⁺): found m/z 351.1120, [M + H]⁺, C₁₅H₁₉N₄O₄S requires 351.1122.

Methyl 7-{(E)-[2-(dimethylcarbamothioyl)hydrazinylidene]H-indole-2-carboxylate (8c)

The title compound was synthesised following the general procedure using methyl 7-formyl-4,6-dimethoxyindole-2-carboxylate (4) (300 mg, 1.13 mmol) and 4,4-dimethylthiosemicarbazide (7c) (124.3 mg, 1.13 mmol) in EtOH (25 mL) with 5 drops of acetic acid. The product 8c was obtained as a bright yellow solid; yield 255 mg (62%); 247 °C; IR: v_max 3436, 3306, 3213, 2944, 2844, 1699, 1591, 1517, 1435, 1367, 1334, 1301, 1252, 1211, 1162, 1088, 987, 812, 710; ¹H NMR (400 MHz, DMSO-d₆): δ 3.31 (3H, s, 2 × CH₃), 3.82, 3.97, 4,00 (each 3H, s, OMe), 6.53 (1H, d J = 4.2 Hz, H5), 7.13 (1H, t, J = 3,6 Hz H3), 8.84 (1H, s, CH), 11.16 (1H, brs, NH indole), 11.97 (1H, s, NH); ¹³C NMR (100 MHz DMSO-d₆): δ 31.4 (CH₃), 52.4, 56.5 and 57.2 (OMe), 89.5 (C5), 100.9 (C3), 106.5, 113.4, 126.3, 135.3 (aryl C), 140.1 (C=N), 156.7, 158.9 (aryl C), 161.3 (CO₂Me), 180.2 (C=S); HRMS (ESI⁺): found m/z 365.1285, [M + H]⁺, C₁₆H₂₁N₄O₄S requires 365.1278.

Methyl 7-{(E)-[2-(ethylcarbamothioyl)hydrazinylidene]H-indole-2-carboxylate (8d)

The title compound was synthesised following the general procedure using methyl 7-formyl-4,6-dimethoxyindole-2-carboxylate (4) (300 mg, 1.13 mmol) and 4-ethylthiosemicarbazide (7d) (124.3 mg, 1.13 mmol) in EtOH (25 mL) with 5 drops of acetic acid. The product 8d was obtained as a pale yellow solid; yield 292 mg (71%); 259 °C; IR: v_max 3459, 3399, 3153, 2990, 1699, 1621, 1599, 1539, 1505, 1435, 1371, 1304, 1244, 1162, 997, 803, 730; ¹H-NMR (400 MHz, DMSO-d₆): δ 1.22 (3H, t, J = 7.0 Hz, CH₃), 3.60 (2H, t, J = 6.4 Hz, CH₂), 3.84, 3.95, 4.00 (each 3H, s, OMe), 6.53 (1H, s, H5), 7.14 (1H, d, J = 2.4 Hz, H3), 8.44 (1H, brs, NHEt), 8.58 (1H, s, CH), 10.66 (1H, brs, NH indole), 11.31 (1H, s, NH); ¹³C NMR (100 MHz DMSO-d₆): δ 15.0 (CH₃), 39.0 (CH₂), 52.2, 56.3 and 57.4 (OMe), 89.7 (C5), 99.7 (C3), 106.6, 113.4, 126.2, 135.3 (aryl C), 140.1 (C=N), 157.1, 160.0 (aryl C), 161.3 (CO₂Me), 176.8 (C=S); HRMS (ESI⁺): found m/z 365.1290, [M + H]⁺, C₁₆H₂₁N₄O₄S requires 365.1278.

(2Z)-2-[(4,6-dimethoxy-1H-indol-2-yl)methylidene]hydrazine-1-carbothioamide (9a)

The title compound was synthesised following the general procedure using 4,6-dimethoxyindole-2-carbaldehyde (6) (300 mg, 1.46 mmol) and thiosemicarbazide (7a) (109.6 mg, 1.46 mmol) in EtOH (25 mL) with 5 drops of acetic acid. The product 9a was obtained as a pale yellow solid; yield 317 mg (78%); 259 °C; IR: v_max 3440, 3354, 3261, 3142, 2922, 1695, 1580, 1546, 1525, 1453, 1334, 1259, 1215, 1140, 1043, 987, 924, 805 ¹H NMR (400 MHz, DMSO-d₆): δ 3.78 and 3.83 (each 3H, s, OMe), 6.16 (1H, s, H7), 6.40 (1H, s, H3), 6.65 (1H, s, H5), 7.91 (1H, s, CH), 8.08 and 8.25 (2H, brs, NH₂), 10.27 (1H, brs, NH indole), 11.48 (1H, s, NH) ¹³C NMR (100 MHz DMSO-d₆): δ 55.6 and 55.7 (OMe), 86.9 (C7), 91.9 (C5), 104.3 (C3), 113.8, 131.6, 133.7 139.2 (C=N), 154.0 159.0 (aryl C), 178.0 (C=S); HRMS (ESI⁺): found m/z 279.0912, [M + H]⁺, C₁₂H₁₅N₄O₂S requires 279.0910.

(2Z)-2-[(4,6-dimethoxy-1H-indol-2-yl)methylidene]-N-methylhydrazine-1-carbothioamide (9b)

The title compound was synthesised following the general procedure using 4,6-dimethoxyindole-2-carbaldehyde (6) (300 mg, 1.46 mmol) and 4-methylthiosemicarbazide (7b) (132.8 mg, 1.46 mmol) in EtOH (25 mL) with 5 drops of acetic acid. The product 9b was obtained as a bright yellow solid; yield 316 mg (74%); 253 °C; IR: v_max 3317, 3235, 3168, 2952, 2926, 1595, 1531, 1498, 1461, 1367, 1597, 1256, 1215, 1140, 1088, 1036, 991, 924, 798; ¹H NMR (400 MHz, DMSO-d₆): δ 3.07 (3H, d, J = 4.8 Hz, CH₃), 3.79 and 3.83 (each 3H, s, OMe), 6.18 (1H, d, J = 1.6 Hz, H7), 6.44 (1H, s, H3), 6.67 (1H, d, J = 1.6 Hz, H5), 7.91 (1H, s, CH), 8.54 (1H, brd, J = 4.4 Hz, NHMe), 11.19 (1H, brs, NH indole), 11.54 (1H, s, NH); ¹³C NMR (100 MHz DMSO-d₆): δ 30.9 (CH₃), 55.6 and 55.7 (OMe), 87.0 (C7), 91.9 (C5), 104.0 (C3), 113.9, 131.7, 133.4 139.1 (C=N), 154.0 159.0 (aryl C), 178.0 (C=S); HRMS (ESI⁺): found m/z 293.1067, [M + H]⁺, C₁₃H₁₇N₄O₂S requires 293.1067.

(2Z)-2-[(4,6-dimethoxy-1H-indol-2-yl)methylidene]-N, N-dimethylhydrazine-1-carbothioamide (9c)

The title compound was synthesised following the general procedure using 4,6-dimethoxyindole-2-carbaldehyde (6) (300 mg, 1.46 mmol) and 4,4-dimethylthiosemicarbazide (7c) (157.8 mg, 1.46 mmol) in EtOH (25 mL) with 5 drops of acetic acid. The product 9c was obtained as a bright yellow solid; yield 277 mg (62%); 247 °C; IR: v_max 3254, 2937, 2836, 1595, 1565, 1517, 1453, 1420, 1371, 1338, 1308, 1274, 1203, 1144, 1039, 987, 931, 787; ¹H NMR (400 MHz, DMSO-d₆): δ 3.29 (6H, s, 2 × CH₃), 3.76 and 3.83 (each 3H, s, OMe), 6.15 (1H, s, H7), 6.52 (1H, s, H3), 6.63 (1H, s, H5), 8.11 (1H, s, CH), 10.81 (1H, brs, NH indole), 11.17 (1H, s, NH); ¹³C NMR (100 MHz DMSO-d₆): δ 42.6 (2 × CH₃), 55.5 and 55.7 (OMe), 87.5 (C7), 92.0 (C5), 104.4 (C3), 113.7, 131.1, 137.4 139.6 (C=N), 153.8 158.7 (aryl C), 180.5 (C=S); HRMS (ESI⁺): found m/z 307.1249, [M + H]⁺, C₁₄H₁₉N₄O₂S requires 307.1223.

(2Z)-2-[(4,6-dimethoxy-1H-indol-2-yl)methylidene]-N-ethylhydrazine-1-carbothioamide (9d)

The title compound was synthesised following the general procedure using 4,6-dimethoxyindole-2-carbaldehyde (6) (300 mg, 1.46 mmol) and 4-ethylthiosemicarbazide (7d) (157.8 mg, 1.46 mmol) in EtOH (25 mL) with 5 drops of acetic acid. The product 9d was obtained as a pale yellow solid; yield 349 mg (78%); 259 °C; IR: v_max 3354, 3287, 3115, 2929, 2952, 1632, 1584, 1531, 1482, 1438, 1353, 1304, 1256, 1215, 1148, 1095, 1043, 987, 928; ¹H NMR (400 MHz, DMSO-d₆): δ 1.19 (3H, t, J = 6.8 Hz, CH₃), 3.63 (2H, t, J = 6.4 Hz, CH₂), 3.78 and 3.83 (each 3H, s, OMe), 6.18 (1H, s, H7), 6.47 (1H, s, H3), 6.67 (1H, s, H5), 7.92 (1H, s, CH), 8.51 (1H, s, NHEt), 11.21 (1H, brs, NH indole), 11.48 (1H, s, NH); ¹³C NMR (100 MHz DMSO-d₆): δ 15.2 (CH₃), 38.5 (CH₂), 55.6 and 55.7 (OMe), 87.0 (C7), 91.9 (C5), 104.3 (C3), 113.9, 131.6, 133.6 139.1 (C=N), 154.0 159.0 (aryl C), 176.8 (C=S); HRMS (ESI⁺): found m/z 307.1252, [M + H]⁺, C₁₄H₁₉N₄O₂S requires 307.1223.

Supplemental Material

supplemental_material – Supplemental material for Synthesis and characterisation of novel 4,6-dimethoxyindole-7- and -2-thiosemicarbazone derivatives: Biological evaluation as antioxidant and anticholinesterase candidates

Supplemental material, supplemental_material for Synthesis and characterisation of novel 4,6-dimethoxyindole-7- and -2-thiosemicarbazone derivatives: Biological evaluation as antioxidant and anticholinesterase candidates by Murat Bingül in Journal of Chemical Research