Electrochemical generation of phenothiazin-5-ium. A sustainable strategy for the synthesis of new bis(phenylsulfonyl)-10H-phenothiazine derivatives

In this work, the electrochemical generation of phenothiazin-5-ium (PTZox) from the direct oxidation of phenothiazine (PTZ) in a water/acetonitrile mixture using a commercial carbon anode and conventional stainless steel cathode is reported. PTZox is a reactive intermediate with high potential synthetic applications, which is used in this paper for the synthesis of new phenothiazine derivatives. In this work a novel and simple electrochemical methodology for the synthesis of some bis(phenylsulfonyl)-10H-phenothiazine derivatives was established. In this paper, a mechanism for PTZ oxidation in the presence of arylsulfinic acids has been proposed based on the results obtained from voltammetric and coulometric experiments as well as spectroscopic data of the products. These syntheses are performed in a simple cell by applying constant current under mild conditions and at room temperature with high atom economy.


General procedure for synthesis of bis(phenylsulfonyl)-10H-phenothiazine derivatives (2a-2e)
The electroorganic synthesis of bis(phenylsulfonyl)-10H-phenothiazine derivatives (2a-2e) has been carried out under controlled potential as well as constant current conditions at room temperature.In controlled potential electrolysis PTZ (0.25 mmol) and arylsulfinic acid (0.5 mmol) were electrolyzed at 0.55 V versus Ag/AgCl (3M KCl) in the mixture of water (phosphate buffer, pH 2.0, c = 0.2 M)/acetonitrile (50/50, v/v) (80 ml).The progress of the electrolysis was monitored by periodically recording the decrease of the oxidation peak current in cyclic voltammetry and also by using TLC on silica gel (ethyl acetate/n-hexane: 40/60).The samples on the TLC were visualized with a UV lamp (254 nm).To activate the electrode surface, the electrolysis is stopped for a while and the carbon anodes are washed with acetone.Electrolysis was terminated when the oxidation peak current reached 5% of the initial value.In this condition, the amount of electricity consumed is equal to 100 C. At the end of the electrolysis, the contents were placed at room temperature to reduce the volume of the solution (evaporation) to half and lead to the precipitation of the products.The crude precipitate was filtered and washed several times with water.After drying the product was purified by thin layer chromatography on silica gel GF250-60 with ethyl acetate/n-hexane (40/60 V/V).Product yield is calculated by weighing the pure product.Electrolysis under constant current conditions has also been performed by applying a current density of 1.25 mA/cm 2 (30 mA) for 78 min (consumption of 140 C electricity) for oxidation of PTZ in the presence of BSA as well as other nucleophiles (TSA and CSA) under similar conditions as reported for electrolysis in controlled potential method.(35), 77 (45), 158 (40), 266 (50), 407 (100), 547 (M, 10).

Electrochemical study of PTZ in the presence of arylsulfinic acids
The cyclic voltammogram of PTZ (1.0 mM) in a solution of phosphate buffer 0.2 M, pH 2.0)/acetonitrile (50/50 v/v) in scan rate of 100 mV/s is shown in Fig. 3, part I, curve a.It reveals a quasi-reversible two-electron process involving oxidation of PTZ to phenothiazine-5-ime (PTZ ox ) (peak A 1 at 0.55 V vs. Ag/AgCl) and reduction of PTZ ox to PTZ (peak C 1 at 0.45 V vs. Ag/AgCl) 10 .The peak current ratio (I PC1 /I PA1 ) close to one which illustrates no side reaction is accrued in the time scale of voltammetry 10 .Figure 3 ) to be eliminated.Finally, the anodic and cathodic peaks A 3 and C 3 are related to the oxidation and reduction of final products (2b, 2d, 2e), respectively.The occurrence of oxidation of the final products at more positive potentials than the potential of peak A 2 confirms the presence of two BSA groups in the structure of the final products.The third change is the shift of E pA1 towards less positive potentials.This is another confirmation of the reaction between electrochemically generated PTZ ox and BSA 32 .In Fig. 3, part I, curve c is related to BSA in the absence of the PTZ.The peak observed in this cyclic voltammogram is attributed to the one electron oxidation of BSA to the corresponding radical 33 .An important point regarding the peak current ratio (I pC1 /I pA1 ) is the dependence on the nucleophile (BSA) concentration and on the potential scan rate.As shown in the Fig. 3, part I, inset, the peak current ratio (I pC1 /I pA1 ) increases with decreasing BSA concentration.Comparison of this voltammogram with voltammogram b shows that decreasing the concentration of BSA slows down the reaction rate of BSA with PTZ ox and leads to more PTZ ox remaining on the electrode surface, which leads to an increase in the cathodic peak current (I pC1 ).The effect of scan rate on the cyclic voltammograms of PTZ/BSA mixture is shown in Fig. 3, parts II and III.As can be seen, at low scan rates such as 10 mV/s (part II, curve a), peaks A 1 , A 2 , and A 3 are clearly observed in the anodic cycle, and peaks C 1 and C 3 are also observed in the cathodic scan.Increasing the potential scan rate causes remarkable two changes in the current of the peaks.The first change is related to the gradual decrease of the anodic peaks A 2 and A 3 with the increase of the scan rate and the second change is related to the increase of I pC1 with increasing scan rate.When the scan rate increases, there is not enough time for the BSA to react with PTZ ox .In such a situation, most of the PTZ ox molecules participate in the cathodic reaction, which results in an increase in I pC1 .On the one hand, the current of anodic peaks A 2 and A 3 decrease, because these peaks are related to the oxidation of mono-substituted and di-substituted PTZs, respectively, which are not formed under these conditions.
Based on the obtained electrochemical data, the synthesis of bis(phenylsulfonyl)-10H-phenothiazine derivatives (2a-e) was carried out.The products after separation and purification were identified by various spectroscopic methods such as IR, NMR and MS.Based on spectroscopic data as well as voltammetric results, the following mechanism is proposed for the oxidation of PTZ in the presence of BSA (Fig. 4).This mechanism can be extended to other nucleophiles (4-toluenesulfinic acid, TSA and 4-chlorobenzenesulfinic acid, CSA).According to the proposed mechanism, initially PTZ is converted into its oxidized form (phenothiazin-5-ium, PTZ OX ) by losing two electrons and one proton.In the next step, PTZ OX is attacked by anion resulting from deprotonation of arylsulfinic acids (RSO 2 − ) and forms the first intermediate (INT1) [3-(arylsulfonyl)-10H-phenothiazine] after aromatization.It should be noted that the formation of other isomers of INT1 is also possible.However, due to the presence of sulfonium atom in the structure of PTZ OX , it is suggested that C3 atom is the most favorable site for nucleophilic attack of arylsulfinic acids.
The oxidation of INT1 in the next step causes the formation of INT1 OX .There are a few important points regarding this step.First, unlike PTZ, whose two rings A and B are similar, INT1 rings A and B are not the same, and due to the presence of an electron-withdrawing arylsulfinic group in ring A, oxidation takes place on the B-ring.Like PTZ OX , INT1 OX is attacked by another arylsulfinic anion, leading to the final product after aromatization (Fig. 4).As seen in Fig. 4, two types of products are formed in the reaction of arylsulfinic anion with INT1 OX .One of these products is formed by the attack of arylsulfinic anion on the nitrogen atom of INT1 OX (path I).This compound is a sulfone-sulfonamide product.The second product results from an attack similar to the first arylsulfinic attack (path II), which leads to the formation of the sulfone-sulfone product.These products were separated from each other by thin layer chromatography.
To further investigate the oxidation of PTZ in the presence of BSA, the double potential step chronoamperometry method was also used (Fig. 5).To achieve this goal, based on PTZ cyclic voltammogram (Fig. 3, part I, curve a), the electrode potential is changed from an initial value of 0.20 V to a potential of 0.55 V in the forward step (oxidation step) and then to 0.40 V in the reverse step (reduction step).The generated current was recorded for 12 s in both step.Figure 5 curve a shows the chronoamperometry of PTZ (1.0 mM) in phosphate buffer (pH 2.0, c = 0.2 M/acetonitrile mixture solution (50/50 v/v).The forward current corresponds to the oxidation of PTZ to PTZ ox and the reverse current corresponds to the reduction of PTZ ox to PTZ.In this method, the theoretical current ratio for a reversible system at 2τ and τ (I r (2τ) /I f (τ) ) is 0.293 32,[34][35][36] .
The experimental current ratio (I r (2τ) /I f (τ) ) obtained for PTZ is equal to 0.25, which is slightly less than the theoretical value but close to it.This result confirms the relative stability of oxidized PTZ (PTZ ox ) in the time scale of the experiment.Repeating this experiment for the oxidation of PTZ in the presence of BSA and measuring the current at 2τ and τ (I r (2τ) /I f (τ) ) shows two important facts.First, the forward currents in these two experiments are exactly the same, and second, the reverse current (I r (2τ) ) in the presence of BSA is zero.These results confirm that PTZ ox is removed from the electrode surface due to its reaction with BSA on the time scale of the experiment.shows that the oxidation potential of 2b is about 140 mV more anodic than the PTZ oxidation potential.The peak shift can be expected due to the difference in the structure of 2b (the presence of two electron withdrawing sulfonyl groups) with PTZ.In addition, the presence of two sulfonyl groups in the structure of 2b has caused its oxidized form (2b ox ) to become more unstable than the oxidized form of PTZ (PTZ ox ) and thus participate in subsequent chemical reactions.As a result, the current of its cathodic peak (C 3 ) becomes lower than the current of cathodic peak of PTZ (C 1 ). Figure 6 curve d, shows the cyclic voltammogram of 2a.In similar conditions, unlike the cyclic voltammogram of 2b, the cyclic voltammogram of 2a shows the presence of an irreversible process.The main reason for this difference is the attachment of a sulfonyl group to the nitrogen atom.This binding causes the two-electron oxidation of the 2a to form a highly unstable 2a ox compound (Fig. 4).
According to the proposed mechanism in Fig. 4, the structure of the product formed via pathway II (for example 2b) is similar to that of PTZ and therefore, its general electrochemical behavior should also be similar to PTZ.In Fig. 6, we studied and compared the cyclic voltammogram of product 2b with the cyclic voltammogram of PTZ.Based on this, the redox reactions producing the anodic and cathodic peaks A 3 and C 3 is shown in Fig. 7.The electrochemical oxidation of 2a was also studied (Fig. 6, curve d).Replacing the hydrogen atom in the PTZ molecule with a sulfinic group has a great effect on the stability of the oxidized product (PTZ OX ).The removal of the hydrogen atom attached to the nitrogen atom as a proton and giving its electron to the mother molecule causes the relative stability of the oxidized forms of molecules PTZ and 2b (PTZ OX and 2b ox ).In contrast to these molecules, the replacement of the hydrogen atom with a sulfinic group in molecule 2a has caused the oxidized molecule (2a ox ) to be in the dication form.This compound is very unstable and quickly participates in subsequent chemical processes such as ring cleavage 37,38 , dimerization reactions 39,40 , hydroxylation reactions 41,42 ,  hydrolysis 43,44 and/or sulphoxide formation 45 .The occurrence of these reactions makes the cyclic voltammogram of 2a shows the behavior of an irreversible system.
In this part, it is necessary to consider the possibility of formation of different isomers in the oxidation of PTZ in the presence of arylsulfinic acids.Figure 8 shows the structures that may be formed in the oxidation of PTZ in the presence of arylsulfinic acids.The steric energy calculations show that due to the steric hindrance caused by the presence of two arylsulfinic groups in the ortho position to each other, it is not possible to form molecules such as I, X, XIV and XV.
As discussed earlier, it seems that due to the presence of sulfonium atom in PTZ OX structure, beside nitrogen atom, C1 and C3 atoms are the most favorable sites for nucleophilic attack.Accordingly, the probability of formation of molecules IX, XII and XVII is very low and therefore they are excluded from the set of possible structures.It seems that the first nucleophilic attack leads to the formation of one of the following intermediates (INTA, INTB and INTC) shown in Fig. 9.
As discussed, the second oxidation step of the INTA leads to the corresponding dication, which is a very unstable compound and does not appear to be capable of conversion to our desired product (molecule III, 2a).Therefore, we do not consider this pathway possible in the oxidation of PTZ in the presence of arylsulfinyl acids.However, the second oxidation step of the intermediates INTB and INTC leads to the compounds I, III, VI, VII, XI and XVI.By removing compound I for the reasons mentioned earlier, the only remaining compound with the presence of a sulfinic group on the nitrogen atom is compound III (2a).By excluding, compounds I and III, the 1 H NMR spectrum of the product was compared with the simulated 1 H NMR spectra of compounds VI, VII, XI and XVI.This comparison shows that the experimental spectrum is the most consistent with the simulated spectrum for compound VI (Fig. 10).

Controlled potential coulometry
Controlled potential coulometry was carried out in a solution containing 0.25 mmol of PTZ and 0.5 mmol BSA in phosphate buffer (pH 2.0, c = 0.2 M/acetonitrile solution mixture (50/50, v/v) at 0.55 V versus Ag/AgCl.In order to better understand what happens during coulometry, cyclic voltammograms of the electrolyzed solution were recorded during coulometry (Fig. 11).These voltammograms at different time intervals show that the peak A 1 current decreases in the progress of electrolysis.The second change observed in cyclic voltammograms is the appearance of peak A 2 and its relative increase with the progress of coulometry.Plotting the values of peak A 1 current versus the amount of electricity consumed in order to determine the number of electrons consumed in this process shows that peak A 1 disappears with the consumption of about 104 coulombs of electricity (Fig. 11, inset).
According to the electricity consumption and performing the necessary calculations, 4.1 electrons are assigned to each molecule of PTZ.This number is slightly higher than 4 due to electrolysis in an undivided cell.The number of electrons obtained in this experiment (n = 4) is consistent with that reported in Fig. 4. According to Fig. 4, two electrons are used to oxidize PTZ to PTZ ox (peak A 1 ) and the other two electrons are used to oxidize intermediate INT1 to INT1 ox (peak A 2 ).It should be noted that, the lack of increase in I pA3 and I pC3 during electrolysis is due to the insolubility of final products in electrolysis solution (see experimental section).

Constant current synthesis and optimization of effective parameters
In order to facilitate the synthesis of these compounds in a way that can be used by all researchers, in this section, the synthesis of these compounds in constant current mode has been examined and the effective parameters in improving the efficiency and purity of the products, such as applied current density, amount of electricity, solution pH, electrode material and solvent mixture are optimized by one factor at a time method.The current density is one of the most important parameters in electrosynthesis of organic and inorganic compounds, which affects the yield and purity.In this section, the electrochemical synthesis of products (2a and 2b) was investigated at different current densities from 0.41 to 2.08 mA/cm 2 (Fig. 12, part I) while other parameters are kept constant (see the caption of Fig. 12).The results showed that the highest product yield (89%) was obtained at a current www.nature.com/scientificreports/density of 1.25 mA/cm 2 .At current densities less than 1.25 mA/cm 2 , the amount of overvoltage is not sufficient to oxidize PTZ and/or intermediates.On the other hand, at current densities higher than the optimal current density (1.25 mA/cm 2 ), oxidation of the solvent, supporting electrolyte and/or over-oxidation of the product will reduce the production yield (Fig. 12, part I).To optimize the amount of electricity, the electrochemical synthesis of 2a and 2b was investigated at different amount of electricity and at current density of 1.25 mA/cm 2 while other parameters are kept constant.The results showed that the highest product yield was obtained at Q = 140 C.The higher amount of electricity consumed in the constant current method compared to the controlled potential method is due to the inherent difference of the two methods in the selective consumption of electricity.
The effect of solution pH on the yield and purity of products was also investigated (Fig. 12, part II).For this purpose water with different pH values of 1.0, 2.0, 6.0, 8.0 and 10.0/acetonitrile (50/50 v/v) solution mixtures are prepared.Phosphate buffer (0.2 M) was used to prepare solutions with pH values 2.0, 6.0 and 8.0.For pH = 1.0, perchloric acid solution (0.1 M) and for pH = 10.0, carbonate buffer (0.2 M) was used.Other conditions such as current density (1.25 mA/cm 2 ) and electricity consumption (140 C) were constant in all experiments.Figure 12, part II shows that the optimum pH is 2.0 for highest product yield.The instability of PTZ ox in alkaline solutions and participation in side reactions such as dimerization 10,39,40 and/or hydroxylation 10,41,42 is the main reason for the low yield of the product in alkaline solutions.On the other hand, the one-electron oxidation of PTZ in highly acidic solutions 10 and the instability of cation radicals along with nucleophile protonation, reduce the product yield in highly acidic solutions.
In this section, the effect of electrode materials on product efficiency is investigated and the results are given in Table 1.As can be seen, the highest product yield was achieved with carbon anode and stainless steel cathode www.nature.com/scientificreports/method, the synthesis of these compounds by performing electrolysis in constant current mode has also been successful.In this research, a mechanism for the oxidation of PTZ in the presence of arylsulfinic acids was also proposed based on the data obtained from cyclic voltammetric, chronoamperometric and controlled-potential coulometric studies along with the structure of the synthesized products.This mechanism is depicted in Fig. 4. According to Fig. 4, PTZ is converted to the final product (bis(phenylsulfonyl)-10H-phenothiazine derivatives) through the ECEC mechanism.The insolubility of the final product in the electrolysis solution is one of the factors that prevent the re-oxidation of the final product.Finally, we hope that the synergistic effect of the groups added to the phenothiazine molecule will intensify the medicinal properties and/or reduce the side effects of the synthesized molecules.

Figure 1 .
Figure 1.Examples of some biologically active phenothiazine, sulfone and sulfonamide drugs.
, part I, curve b is the cyclic voltammogram of PTZ in the presence of 1.0 mM benzenesulfinic acid (BSA).Compared to cyclic voltammogram Vol.:(0123456789) Scientific Reports | (2024) 14:4276 | https://doi.org/10.1038/s41598-024-53620-0www.nature.com/scientificreports/a, the following changes have occurred in the cyclic voltammogram of b.The first change is the removal of the cathode peak C 1 , which confirms the reaction between PTZ ox and BSA.The second change is the appearance of an ill-defined irreversible peak A 2 and a new reversible redox peak (A 3 and C 3 ) at more positive potentials.The peak A 2 is related to the oxidation of the adduct formed from the reaction of PTZ ox with BSA (PTZ-BSA) (INT1) to INT1 ox (Fig. 3, part I, curve b).The positive shift of peak A 2 compared to peak A 1 can be justified based on the electron withdrawing property of BSA bound to PTZ.After the electrochemical generation of INT1 ox , another rapid chemical reaction occurs and a BSA binds to INT1 ox .This chemical reaction causes the cathodic peak corresponding to the reduction of INT1 ox (C 2

Figure 4 .
Figure 4. Suggested mechanism for electrochemical oxidation of PTZ in the presence of arylsulfinic acids.

Figure 7 .
Figure 7. Suggested mechanism for electrochemical behavior of 2a and 2b.

Figure 8 .
Figure 8. Possible structures in oxidation of PTZ in the presence of arylsulfinic acids.

Figure 10 .
Figure 10.Experimental 1 H NMR spectrum of 2b and simulated 1 H NMR spectra of different compounds.

Figure 12 .
Figure 12.Part I: The effect of current density on the product yield (both 2a and 2b).The amounts of PTZ and BSA 0.25 and 0.5 mmol, respectively.Q = 140 C. Solvent: phosphate buffer (pH = 2.0, c = 0.2 M)/acetonitrile mixture (50/50 v/v).Anode and cathode material: carbon and stainless steel, respectively.Part II: The effect of solution pH on the yield of product.Applied current density: 1.25 mA/cm 2 .Other conditions are the same as part I.All experiments were performed at room temperature.

Table 1 .
46timization of electrode material for the synthesis of 2a and 2b.aThe yield reported is the sum of yields of products 2a and 2b.b Applied current density: 1.25 mA/cm 2 .Electricity consumption: 140 C. Solvent: phosphate buffer (pH = 2.0, c = 0.2 M)/acetonitrile mixture (50/50 v/v).cTheelectrode was fabricated according to the procedure reported in reference46.