The synthesis of disubstituted 4-methyl-N,N-dipropylbenzenamine derivatives bearing identical azolethione(thiol) moieties as antioxidant agents

A series of novel 3,3'-(p-tolylazanediyl)di(propanehydrazide) derivatives bearing double sets of thiosemicarbazide, oxadiazolethione, variously N- and S-substituted triazolethione, pyrrole, and hydrazone moieties were synthesized and their molecular structures were confirmed by IR, 1H, 13C NMR spectroscopy and mass spectrometry data. X-ray analysis of 2,2'-[[[(p-tolylazanediyl)bis(ethane-2,1-diyl)]bis(4-phenyl-4H-1,2,4-triazole-5,3-diyl)]bis(sulfanediyl)]bis[1-(p-tolyl)ethan-1-one] crystal has revealed, that the molecules in the crystal are associated by means of intermolecular hydrogen bonds of OH···N type, forming centrosymmetric dimers. The antioxidant activity of the synthesized compounds was screened by a DPPH radical scavenging assay, reducing power assay, and ferric reducing antioxidant power assay. Among the synthesized compounds, 2,2'-[3,3'-(p-tolylazanediyl)bis(propanoyl)]bis(N-phenylhydrazine-1-carbothioamide) has been identified as possessing the highest antioxidant activity, which is 1.25–2 times higher than that of the commercial antioxidant butylated hydroxytoluene.


Introduction
Oxidative stress is caused by a significantly disturbed balance between the production and accumulation of highly oxidizing free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), in cells and tissues and the ability of an endogenous antioxidant system to neutralize them [1,2].ROS can attack and damage the DNA, lipids, and proteins of an organism, as well as cause cellular damage [3].ROS and RNS are unstable chemicals produced under normal physiological and pathophysiological conditions in biological systems.ROS contain free radical intermediates such as superoxide anion (O 2 *-), and hydroxyl radical (OH*) as well as nonradical molecules such as hydrogen peroxide and hypochlorous acid, while RNS consist mainly of nitric oxide (NO*), peroxynitrite, and other nitrates [2].Oxidative stress plays an important role in the normal ageing process, and in the pathogenesis of numerous chronic diseases, including diabetes, atherosclerosis, cancer, cardiovascular diseases, liver injury, immune dysfunction, and neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's diseases, as well as amyotrophic lateral sclerosis [4].
In the evolution, organisms have developed various defence mechanisms to counterbalance and neutralize harmful effects of ROS to maintain their levels within the physiological threshold required for cell survival and redox homeostasis.Antioxidant systems such as endogenous antioxidant enzymes, including superoxide dismutase, glutathione peroxidase, thioredoxin reductase, and catalase, are present in the organisms, which can neutralize the harmful cellular damage caused by ROS [4].However, the efficacy of organism antioxidant systems is limited and environmental factors such as pollution, UV irradiation, and imbalanced diets can produce an excess of ROS, which in turn can add to causing the oxidative stress.Antioxidants represent a group of compounds that are involved in the defense mechanism of the organism against pathologies associated with the attack of free radicals by slowing or inhibiting completely the oxidation processes caused by these radicals.
Antioxidants may be chemical compounds that can neutralize free radicals by accepting or donating electron(s) to eliminate the unpaired condition of the radical.The antioxidant molecules may directly react with the reactive radicals and destroy them, while they may become new free radicals which are less active, longer-lived and less dangerous than those radicals they have neutralized.Numerous antioxidants may directly react with ROS or free radical intermediates induced by ROS and terminate the chain reaction, thereby stopping the ROS-induced damage [5].Protection against free radicals can be enhanced by the intake of antioxidants in the diet [6].Only a few antioxidants, in the form of supplements, have been approved by the responsible authorities for use in human therapy.There is a great interest in synthesizing more potent derivatives or isolating them as natural products with improved absorption/bioavailability/ antioxidant properties [7].
Common examples of NH-based antioxidants include hydrazones [8].These compounds, bearing azomethine -NH-N=CH-group, make another important class of biologically active compounds in medicinal chemistry [38].Hydrazone derivatives, especially those that contain various heterocyclic scaffolds, have been identified to have diverse biological activity, including antioxidant one [39][40][41].This group has also been widely exploited as a convenient linker in synthetic chemistry because of various biological activities.
Combining two or more bioactive moieties into a single molecule is a commonly used strategy in drug design to improve pharmacological properties [11].The potency of the antioxidant agent can be enhanced by combining two or even more structural and functional fragments with proven antioxidant activity.Additionally, or as another approach for the enhancement of the desirable pharmacological properties, including antioxidant activity, a double number of the moieties imparting the desirable activity are introduced.
Based on the antioxidant properties of the scaffolds discussed above and as a continuation of our search for biologically active compounds bearing double sets of identically substituted heterocyclic rings [42][43][44], we report herein the synthesis of a series of 3,3'-(p-tolylazanediyl)di(propanehydrazide) derivatives with double thiosemicarbazide, oxadiazolethione, oxadiazolethiol, variously N-and S-substituted triazolethione, pyrrole, and hydrazone moieties and screening of their antioxidant activity by DPPH radical scavenging, reducing power, and ferric reducing antioxidant power assays.

Chemistry
The compounds 2-27 were synthesized according to the synthesis route as shown in Schemes 1, 2, 3, 4. The structures of the synthesized compounds have been confirmed by 1 H and 13 C NMR, IR, and HRMS or elemental analysis data.
The character of biological activity of 1,2,4-triazolethiones depends on the nature of the substituents, therefore, the chemical functionalisation of the triazole cycle is a promising and facile method for search/synthesis of novel compounds possessing useful properties.Thione-thiol tautomerism is characteristic of triazolethiones; therefore, they easily form S-substituted derivatives, which elicit a broad spectrum of biological activities as well.S-alkylation or S-acylation reactions of triazolethiones take place under basic conditions when thione-thiol equilibrium of 1,2,4-triazolethiones is shifted towards the formation of thioles.The alkylation reactions of 7 were carried out using two methods (A and B) [48] (Scheme 3).The S-alkylated derivative diethyl 2,2'-[[[(p-tolylazanediyl)bis(ethane-2,1-diyl)]bis(4phenyl-4H-1,2,4-triazole-5,3-diyl)]bis(sulfanediyl)]diacetate (13) was synthesized using ethyl chloroacetate according to Method A in DMF in the presence of K 2 CO 3 at 40 °C.Method B was used for alkylation of 7 with chloroacetamide and phenacyl bromides in the presence of triethylamine.The reaction products were isolated in 71-88% yield.
In the 1 H NMR spectra of the S-substituted derivatives 12-16, the singlet of the NH group proton in the heterocyclic moiety is absent in comparison with the spectra of the precursor triazolethione 7 (~ 13.7 ppm) [47], while signals of the H 11,11' protons at the α-position of S-substituent are observed in the range of 3.87-4.86ppm.The additional proton signals in the aromatic region of the 1 H NMR spectra have proven the formation of target compounds 14-16.
The second 1,2,4-triazolethione ring in each side arm was introduced by reaction of 17 with methyl thiocyanate or phenyl isothiocyanate to form thiosemicarbazides 19 and 20, respectively, followed by the cyclization of the latter under alkaline conditions at the boiling point of the reaction mixtures to give 21 and 22.

Crystal structure of compound 15
Compound 15 bearing three 4-methylphenyl moieties was selected as a representative structure to be examined in more detail.Figure 1 shows a perspective view of molecules 15 with thermal ellipsoids and the atom-numbering scheme followed in the text.In the crystal structure, leastsquares planes of molecules 15 are located almost parallel to the crystallographic plane (2 1 1 ).By means of inter- molecular hydrogen bonds of OH•••N type, molecules in the crystal are associated, forming centrosymmetric dimers (Fig. 2).The parameters of these hydrogen bonds are the It should be noted that the occupancy g-factor of water atoms O1w, H1wa, and H1wb is equal 0.5.This means that there is one molecule of water for every 2 molecules of compound 15.Thus, this substance is a semisolvate.Among other shortened intermolecular contacts, the C31•••O1w contact with a length of 3.147(5) Å should be singled out, which can be considered as a weak hydrogen bond of CH

Evaluation of antioxidant activity
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay is one of the most commonly used methods to measure antioxidant activity.The DPPH method involves the addition of prospective antioxidants to the DPPH solution.The DPPH assay is based on either a hydrogen atom transfer or a single electron transfer mechanism.The unpaired valence electron from the nitrogen atom in the DPPH radical is reduced and, as a result, the DPPH-H hydrazine is formed [52].Scavenging of this stable DPPH radical is determined by spectrophotometry at 517 nm.
The antioxidant activity of thiosemicarbazide 4, which contains six NH groups in its structure, is 1.25 times higher than that of BHT.Thiosemicarbazides form a system of electron donating group C=S and several NH groups as hydrogen binding domains that contribute considerably to their antioxidant potential [53].The DPPH radical scavenging ability of the 1,2,4-triazolethione derivative 6 is lower than that of its open-chain precursor 4. Thiosemicarbazide with terminal methyl substituent 3 (77.22%)has lower antioxidant activity than 4. The activity comparison in the pair of thiosemicarbazide 3 -triazolethione 5 follows the same pattern as in the case of 4 and 6, respectively.This structure-activity relationship corresponds to the reported significant decrease of activity of triazole in comparison with the one of thiosemicarbazide [11].One of the reasonable explanations for the higher antioxidant activity of thiosemicarbazides is the existence of the thiourea fragment, which stabilizes free radicals of nitrogen atoms by double conjugation, mainly with the C=S group.The stability of the radical system is further increased by the conjugation between free radicals of the nitrogen atom and π electrons of the aromatic ring.The probable mechanism for the reaction of thiosemicarbazide 4 as a representative compound with DPPH radical is presented in Scheme 5 [54].
In analogy, in 1,2,4-triazolethione there is only the possibility to conjugate free radicals on the nitrogen atom with the thione group as shown in Scheme 6 using compound 6 as an example [54].It can be assumed, that the lower stability of such a radical is responsible for a weaker antioxidant activity.Another reasonable explanation may be based on the decreased number of NH bonds upon the formation of the 1,2,4-triazole ring and more limited electron delocalisation across the aromatic ring [11].
The   [35] who found that conversion of a thiosemicarbazide derivative to the corresponding triazole led to a slight improvement in free radical scavenging capacity.Thus, other substituents and their arrangement in the molecule influence the activity as well as shown in another study of thiosemicarbazide/triazolethione systems with enhanced and decreased activities, depending on the substituent [55].
Another interesting observation can be made while comparing the performance of 4-substituted 1,2,4-triazolethiones 10 and 11 (76.75%), which molecules differ only by a substituent of benzene rings in the side arm.Compound 10 bearing 4-(dimethylamino)phenyl moieties has been identified as one of the compounds possessing a very high DPPH radical scavenging ability, whereas its analogue 11 bearing 4-methoxyphenyl fragments is just slightly more active than BHT.Compounds 15, 7, 14, 1, 24, 12, 25, 8, 13, 23, and 16 demonstrated lower inhibition of DPPH than that of the control.
Another method for determination of antioxidant activity, reducing power assay, is associated with the electron donating capacity.The reducing capacity of a compound can be evaluated by direct reduction of Fe[(CN) 6 ] 3 to Fe[(CN) 6 ] 2 .The addition of free Fe 3+ to the reduced product forms the intense Perl's Prussian blue complex, Fe 4 [Fe(CN − ) 6 ] 3 , which has a strong absorbance at 700 nm.Increasing absorbance at 700 nm indicates an increase in reductive capacity [56].
As seen from the data presented in Fig. 4, the same as in the case of the DPPH assay, thiosemicarbazide 4 (1.89A) has been identified as possessing the highest ferric ion (Fe 3+ ) reducing power in comparison with BHT.Its activity is 1.69  and 6 (1.09A) is higher as well than that of the control.It is interesting to note, that the introduction of the 4-phenyl-1,2,4-triazolylsulfanyl moieties into the structure of thiosemicarbazide 19 decreased the antioxidant activity of the molecule in comparison with that of thiosemicarbazide 3. The reducing power of ferric ion (Fe 3+ ) of thiosemicarbazide 20 (0.84 A), which contains terminal phenyl in the side arms instead of the methyl group in 19, is lower than that of the latter.The presence of two 4-amino-1,2,4-triazolethione moieties in 9 has a positive influence on the reducing capacity of this compound, whereas the replacement of the amino group in 9 with the 4-methoxybenzylidene moiety slightly reduced the activity of hydrazone 11.Contrary to the results obtained by the other two antioxidant activity evaluation assays, the ferric ion (Fe 3+ ) reducing power of hydrazone 10 is lower than those of its dimethylamino analogue 11 and BHT.
The FRAP assay is an essential antioxidant assay, which evaluates the reduction of the ferric ions (Fe 3+ )-ligand complex to the intensely blue-coloured ferrous ions (Fe 2+ ) complex by antioxidants at low pH values.Measuring the increasing absorbance at 593 nm spectrophotometrically estimates this reduction.The obtained absorbance of antioxidants is compared to the absorbance of the standard FeSO 4 ‧7H 2 O calibration curve and the results are expressed as Fe 2+ µmol/dm 3 concentration [6].The FRAP assay takes place via the electron transfer mechanism.

Conclusion
In summary, 26 compounds, bearing 4-methyl-N,Ndipropylbenzenamino fragment and double sets of identical thiosemicarbazide, oxadiazolethione, oxadiazolethiol, variously N-and S-substituted triazolethione, pyrrole, and hydrazone moieties were synthesized.The crystallographic analysis of compound 15 has revealed, that the molecules in the crystal are associated by means of intermolecular hydrogen bonds of OH•••N type, forming centrosymmetric dimers.It has been shown that there is one molecule of water for every two molecules of compound 15, indicating that the investigated compound is a semisolvate.The antioxidant activity of synthesized compounds 1 and 3-27 was screened by DPPH radical scavenging, reducing power, and FRAP assays to identify thiosemicarbazide 4 as possessing the highest antioxidant activity, which is 1.25, 1.7, and 2 times higher, respectively, than that of the commercial antioxidant butylated hydroxytoluene.It could be noted that in the case of smaller molecules, thiosemicarbazides were more powerful DPPH radical scavengers than their heterocyclisation derivatives 1,2,4-triazolethiones, while the lengthening of the side arms resulted in inconsistent pattern of activity.
In the reducing power and FRAP assays, reducing power capacity of several S-substituted 1,2,4-triazole derivatives was on par with those of 1,2,4-triazolethiones.However, further studies would be needed for development of the reported antioxidants.
The course and purity of the synthesized compounds was monitored by TLC using aluminium plates precoated with silica gel 60 F254 (MerckKGaA, Darmstadt, Germany).Melting points were determined on a MEL-TEMP (Electrothermal, A Bibby Scientific Company, Burlington, NJ, USA) melting point apparatus.FT-IR spectra (cm −1 ) were recorded on a Perkin-Elmer Spectrum BX FT-IR spectrometer (Perkin-Elmer Inc., Waltham, MA, USA) using KBr pellets.The 1 H and 13 C NMR spectra were recorded in DMSO-d 6 on a Bruker Avance III (400 MHz, 101 MHz) spectrometer (Bruker BioSpin AG, Fällanden, Switzerland) operating in the Fourier transform mode.Chemical shifts (δ) are reported in parts per million (ppm) calibrated from TMS (0 ppm) as an internal standard for 1 H NMR, and DMSO-d 6 (39.43 ppm) for 13 C NMR. Mass spectra were obtained on a Bruker maXis UHR-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) with positive-ion mode ESI ionization.Elemental analyses (C, H, N) for compounds 10 and 11 were conducted using the CE-440 Elemental Analyzer (Exeter Analytical, Inc., North Chelmsford, MA, USA), their results were found to be in good agreement (± 0.3%) with the calculated values.
The 1 H, 13 C NMR spectra as well as IR spectra were found to be identical with the ones described in [47].

General procedure for the synthesis of compounds 10 and 11
A mixture of 9 (0.15 mmol), the corresponding aromatic aldehyde (0.31 mmol), 20 cm 3 methanol, and 1 drop of concentrated hydrochloric acid was heated at reflux for 8 h.Subsequently, it was cooled to room temperature.The precipitate was filtered, washed methanol, and recrystallized from DMF/H 2 O mixture.

General procedure for the synthesis of compounds 19 and 20
To 0.49 g 17 (0.75 mmol) dissolved in 15 cm 3 methanol, the corresponding isothiocyanate (1.5 mmol) was added dropwise and the mixture was stirred at 50 °C for 2 h.The precipitate was filtered off and recrystallized from the DMF/H 2 O mixture.

General procedure for the synthesis of compounds 21 and 22
A mixture of the corresponding thiosemicarbazide 19 or 20 (0.54 mmol) and 10 cm 3 10% aqueous KOH solution was heated at reflux for 6 h.The reaction mixture was cooled to room temperature and acidified with concentrated hydrochloric acid to pH 4. The precipitate was filtered off, washed with distilled water to pH 7, and recrystallized from the DMF/H 2 O mixture.

General procedure for the synthesis of compounds 23-27
Method A: A mixture of 0.49 g 17 (0.75 mmol), the corresponding aromatic ketone (1.5 mmol), and 15 cm 3 methanol was stirred at 65 °C for 15 h.The precipitate was filtered off, washed with propan-2-ol, and recrystallized from the DMF/ H 2 O mixture.

XRD study of compound 15
Compound 15 was crystallised from methanol at room temperature and produced crystals suitable for X-ray analysis.A suitable crystal with sizes 0.17 × 0.11 × 0.04 mm 3 was selected and mounted on a suitable support on the diffractometer.The crystal was kept at a steady (150.0(1)K) during data collection.
For further details, see crystallographic data for 15 deposited at the Cambridge Crystallographic Data Centre as Supplementary Publications Number CCDC 2254270.Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK.

DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay
The free radical scavenging activity of compounds 1 and 3-27 was measured by the DPPH radical scavenging assay.At first, a solution (20 mM) of each compound was prepared in DMSO.Then, a 0.1 mM solution of DPPH in ethanol was prepared and 1 cm 3 of this solution was added to the solutions of the selected compounds.The mixture was vigorously stirred and allowed to stand at room temperature.After 20 min, the absorbance of the reaction mixture was measured at 517 nm with a UV-1280 spectrophotometer (Shmidazu).Each experiment was repeated three times [52].

Reducing power assay
The solutions of the tested compounds 1 and 3-27 were prepared in DMSO (20 mM).To each of them, 1.25 cm 3 of sodium phosphate buffer (200 mM, pH 6.6) and 1.25 cm 3 of potassium ferricyanide (1%) were added and the solution mixtures were incubated at 50 °C for 20 min.Then 1.25 cm 3 of trichloroacetic acid (10%) was added and the solution mixture was centrifuged at 9000 rpm for 10 min.Subsequently, 0.5 cm 3 of sterilized water and 0.5 cm 3 of ferric chloride (0.1%) were mixed with the supernatant (0.5 cm 3 ).Finally, the absorbance was measured at 700 nm with a UV-1280 spectrophotometer (Shmidazu) against a blank.Butylated hydroxytoluene (2,6-di-tert-butyl-4-methylphenol, BHT) was used as a positive control [56].

Ferric reducing antioxidant power assay (FRAP)
Reducing properties of the synthesized compounds 1 and 3-27 were investigated using the FRAP method, which is based on the reduction of a ferric-tripyridyl triazine complex to its ferrous coloured form in the presence of antioxidants.The FRAP reagent contained 2.5 cm 3 of a 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in 40 mM HCl, also 2.5 cm 3 of FeCl 3 (20 mM) and 25 cm 3 of acetate buffer (0.3 M, pH 3.6).100 mm 3 of the solution of the tested compound (5 mM) were mixed with 3 cm 3 of the FRAP reagent.The absorbance of the reaction mixture was measured at 593 nm with a UV-1280 spectrophotometer (Shmidazu).For comprising of the calibration curve, five concentrations of FeSO 4 •7H 2 O (5, 10, 15, 20, and 25 μM) were used and the absorbances were measured as a sample solution.Each experiment was repeated three times [6].

Fig. 1
Fig. 1 ORTEP diagram for molecule 15 showing atomic labels and 50% probability displacement ellipsoids.Hydrogen atoms are shown as small spheres of arbitrary radii introduction of the 4-phenyl-1,2,4-triazolylsulfanyl moieties into the structure of 19 (83.41%) slightly increased the antioxidant activity of the molecule in comparison with that of thiosemicarbazide 3, whereas the same moiety in thiosemicarbazide 20 had a negative influence on the activity of the molecule in comparison with that of 4 although the number of NH groups did not change in the molecule.Introduction of the second set of 1,2,4-triazolethione moieties in 22 somewhat increased the antioxidant activity compared to that of compound 20.The same tendency has been observed for the analogous pair 21 (84.46%) and 19, respectively.However, a comparison of the DPPH radical scavenging activity of DPPH between thiosemicarbazides 19, 20 and their heterocyclization derivatives 1,2,4-triazolethiones 21, 22 has revealed the opposite trend than in the