Comprehensive electrochemical study of clonazepam an anticonvulsant benzodiazepine drug and its main metabolite. Green late-stage functionalization of clonazepam

Comprehensive electrochemical study of clonazepam an anticonvulsant benzodiazepine drug and its main metabolite. Green late-stage functionalization of clonazepam | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Comprehensive electrochemical study of clonazepam an anticonvulsant benzodiazepine drug and its main metabolite. Green late-stage functionalization of clonazepam Zahra Godini, Davood Nematollahi, Niloofar Mohamadighader, Pegah Mashhadi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6311438/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract The electrochemical synthesis of new benzodiazepine derivatives, via late-stage modification of clonazepam ( CZP ), a benzodiazepine anticonvulsant drug, is one of the two main objectives of this research. To achieve this goal, electrochemical oxidation of this drug was carried out in the presence of arylsulfinic acid derivatives ( ASA ) in an undivided cell equipped with a graphite anode and cathode, in a water/ethanol mixture, under controlled potential conditions. In this method, new benzodiazepines were synthesized under easy workup and environmentally friendly conditions without the need for catalysts, reagents, or toxic solvents, at room temperature and pressure. In another part of this research, the electrochemical behavior of CZP was thoroughly investigated and new information was reported on the electrochemical reaction mechanism, the major metabolite of CZP , and the relationship between potential and pH. The proposed mechanism for the late-stage modification of CZP involves the sequential reduction and oxidation of CZP at the cathode and anode, respectively, followed by reaction with the nucleophile ( ASA ). In addition, docking studies were performed on the synthesized compounds and it was found that the synthesized derivatives can interact well with the gamma-aminobutyric acid agonist ( GABA ). Physical sciences/Chemistry/Organic chemistry/Reaction mechanisms Physical sciences/Chemistry/Electrochemistry Physical sciences/Chemistry/Green chemistry/Sustainability Physical sciences/Chemistry/Synthesis Electrochemical synthesis Clonazepam Cyclic voltammetry Late-stage modification Molecular docking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Introduction In 1848, scientists were able to incorporate electricity into chemical reactions, performing the first organic electrosynthesis. Electrosynthesis uses electrons instead of chemical reagents, so the method is green, waste-free, and sustainable. The time required for electrochemical processes is usually shorter than for chemical processes, thereby minimizing the associated costs. Some other important features of electrochemical methods include: synthesis under mild conditions without using high temperature or pressure, no use of catalyst, reducing waste, higher yield, and easier workup and scalability. Electrochemistry has high selectivity by controlling the applied potential. This unique capability distinguishes electrosynthesis from other methods. 1 – 7 The synthesis of new pharmaceutical derivatives via late-stage modification (LSM) or late-stage functionalization (LSF) has attracted our attention in this work. This method has the ability to purposefully synthesize derivatives of the original drug that have greater activity than the original drug. In addition, this method makes drug discovery, production, and synthesis fast and efficient and allows for changing the physical and chemical properties of the drug by adding new groups to it. 8 – 10 Nobel Prize winner James Black highlighted, “the most fruitful basis for the discovery of a new drug is to start with an old drug”. Raju believes that by modifying the late stage, new drug derivatives with a broader spectrum of activity can be synthesized. 11 Late-stage modification of drug can directly and selectively lead to the synthesis of compounds with high potential in medicine, even in large and complex molecules. The advantages of the late-stage modification of drugs include avoiding lengthy synthesis, enabling rapid drug discovery, and modifying the physical and chemical properties of the drug. The late-stage modification drug is drug functionalization. Such modifications may have the potential to alter the properties, affinity, drug metabolism, or pharmacokinetic properties of the drug, generally without loss or even with an increase in the drug's biological activity. 12 – 14 Finally, the use of late-stage modification of drugs, is growing and is being used as an efficient method for developing new drugs. Benzodiazepines refer to a family of psychotropic drugs whose main structure consists of a benzene ring and a diazepine ring. 15 Clonazepam ( CZP ), 5-(2-chlorphenyl)-1,3-dihydro-7-nitro-2 H -1,4-benzodiazepin-2-one is one of the long-acting and popular benzodiazepines, which is used as an anticonvulsant, anti-anxiety, sedative and muscle relaxant. 16 – 18 The synthesis of this drug is carried out in four steps as shown in Fig. 1. 19 Benzodiazepines are extensively metabolized by cytochrome P-450 enzymes, particularly by CYP3A4 and CYP2C19. 20 In humans, CZP is metabolized primarily to 7-aminoclonazepam ( ACZP ) by nitro-reduction via hepatic cytochrome P450 (Fig. 2 ). ACZP is then N -acetylated to form 7-acetamidoclonazepam, which is excreted in the urine and feces following extensive biotransformation. 21 Many drugs are based on sulfonamides, the most important and main members of this group of antibiotics being sulfacetamide, sulfamethoxazole, and silversulfadazine. Sulfonamides inhibit the bacterial enzyme dihydropteroate synthetase, which is responsible for converting para -aminobenzoic acid into dihydrofolic acid, preventing the formation of folic acid which is vital for bacterial growth. 22–25 On the other hand, drug resistance resulting from continuous use of drugs is an undeniable fact that poses serious problems to human health and, as a result, necessitates the need for new drugs. In summary, in this study, we intend to add a sulfonamide moiety to the clonazepam molecule using an electrochemical method. Given the pharmacological properties of sulfonamides, we hope that these changes in the structure of CZP will create new properties in this molecule that will lead to an increase in its pharmacological activity. On the other hand, a literature review shows that although the electrochemical behavior of CZP has been investigated in a number of papers, these studies are limited and in some cases contain misleading statements. 19,26–29 Therefore, in this research, we decided to complete the electrochemical studies conducted on this drug and correct many of the published data, in addition to synthesizing new CZP derivatives. Experimental Apparatus and reagents. A Behpajooh model BHP-2051 potentiostat/galvanostat was also used for electrosynthesis and controlled potential coulometry. All experiments including cyclic voltammetry and controlled potential coulometry were carried out by a Autolab model PGSTAT 302N potentiostat/galvanostat (Metrohm-Autolab, Netherland) and nova2.1 software. For electrochemical studies, a three-electrode system in an undivided cell has been used. All electrodes are from Azar Electrode, which includes: Ag/AgCl (3.0 M) as the reference electrode, a glassy carbon (GC) disk as the working electrode (diameter 2.0 mm) and a platinum wire as the counter electrode. Before each experiment, the GC electrode was polished with alumina slurry and rinsed with acetone. The solution was purged for 15 minutes with N 2 before the voltammetric experiments to remove oxygen. For macro-scale electrolysis, five graphite rods (length 9 cm and diameter 8 mm) were used as anode (one numbers) and cathode (four number) electrodes. The FTIR spectra of products were recorded in the range 500–4000 cm − 1 using a Perkin-Elmer 1760. The 1 H and 13 C NMR spectra were recorded in DMSO on a Bruker Avance 400 MHz spectrometer. Mass spectrometry analysis was performed in electron impact mode at an ionization potential of 70 eV using an Agilent-5973 mass spectrometer. The melting point of the products was determined using a Barnstead Electro thermal 9100 instrument. Perchloric acid (60%), acetic acid (100%), phosphoric acid (85%), sodium carbonate (98%) and sodium bicarbonate (98%) were used to prepare the buffer solutions were of analytical grade and were obtained from Sigma-Aldrich and used without further purification. Acetone, ethanol, benzenesulfonic acid sodium salt (98%), sodium4-toluenesulfinate (95%) and 4-fluorobenzenesulfinate (95%) were purchased from Sigma-Aldrich and used without further purification. The active ingredient clonazepam was received from Tehrandaro Pharmaceutical Company. Electrochemical synthesis of CZP derivatives. Electrochemical synthesis was performed under controlled potential condition (at -0.82 V vs. Ag/AgCl) in an undivided cell at room temperature. For this purpose, a mixture of water (phosphate buffer pH = 3.0, c = 0.2 M)/ethanol )30/50, v/v) (80 ml) containing CZP (0.5 mmol) and arylsulfinic acid ( ASA ) (1.0 mmol) was electrolyzed in an undivided cell equipped with graphite as both anode and cathode. At the end of the electrolysis, the solution was allowed to evaporate slowly at room temperature to one-third of the initial volume. Then the solution was extracted with ethyl acetate (30 mL). After the evaporation of ethyl acetate, the precipitate solid was collected by filtration and washed several times with distilled water (100 mL). After washing and drying, the product was identified by FT-IR, 1 H NMR, 13 C NMR and MS spectra. Characteristics of products. N-(5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-7-yl)benzenesulfonamide (SUL1), C 21 H 16 ClN 3 O 3 S . Pale orange solid, Yield. 87%, Mp. 180–183°C (Dec.), 1 H NMR, δ ppm (400 MHz, DMSO- d 6 ): 4.10 (s, 2H, CH 2 ), 6.47 (d, 1H, J = 4 Hz, aromatic), 7.10 (d, 1H, J = 12 Hz, aromatic), 7.21 (dd, 1H, J 1 = 12, J 2 = 4 Hz, aromatic), 7.21–7.44 (m, 8H, aromatic), 7.62 (t, 1H, aromatic), 10.67 (s, 1H, NH), 11.1 (broad, NH). 13 C NMR, δ ppm (100 MHz, DMSO- d 6 ): 57.4, 121.5, 122.8, 125.9, 126.8, 127.3, 127.7, 128.1, 129.2, 129.4, 131.3, 132.0, 132.2, 134.6, 137.7, 138.0, 138.8, 169.1, 170.0. IR (KBr) (cm − 1 ): 3430, 3288, 3172, 2919, 2850, 1675, 1640, 1559, 1496, 1412, 1363, 1171, 1068, 1020, 809, 753, 687, 576, 470. MS (m/z) (EI, 70 EV) (relative intensity): 57 (100), 83 (50), 111 (21), 137 (7), 167 (7), 198 (14), 236 (25), 264 (3), 316 (5), 343 (6), 396 (M-CO, 23), 424 (M-H, 1). N-(5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e] [1,4] diazepin-7-yl)-4-methylbenzene sulfonamide (SUL2), C 22 H 18 ClN 3 O 3 S. Orange solid, Yield. 95%, Mp. 190–193°C (Dec), 1 H NMR, δ ppm (400 MHz, DMSO- d 6 ): 2.24 (s, 3H, CH 3 ), 3.33 (s, 2H, CH 2 ), 7.26 (m, 3H, aromatic), 7.68 (dd, 2H, J 1 = 8, J 2 = 4 Hz, aromatic), 7.80 (m, 4H, aromatic), 8.10 (d, 2H, J = 8 Hz, aromatic), 12.23 (s, 1H, NH). 13 C NMR, δ ppm (100 MHz, DMSO- d 6 ): 57.3, 116.5, 116.8, 121.6, 122.8, 127.1, 127.4, 127.7, 128.2, 130.0, 131.3, 132.2, 132.5, 132.7, 137.6, 138.2, 139.8, 169.0, 170.1. IR (KBr) (cm − 1 ): 3350, 3205, 3065, 2922, 2823, 1687, 1625, 1493, 1359, 1164, 1031, 976, 765, 673, 588, 556, 480. MS (m/z) (EI, 70 EV) (relative intensity): 43 (100), 69 (62), 109 (60), 159 (55), 236 (11), 368 (15) 439 (M, 1). N-(5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-7-yl)-4-fluorobenzene sulfonamide (SUL3), C 21 H 15 ClFN 3 O 3 S. Orange solid, Yield. 83%, Mp. 202–205°C (Dec), 1 H NMR, δ ppm (400 MHz, DMSO- d 6 ): 4.20 (s, 2H, CH 2 ), 6.55 (d, 1H, J = 2.8 Hz, aromatic), 7.18 (d, 1H, J = 12 Hz, aromatic), 7.31 (m, 9H, aromatic), 11.25 (s, 1H, NH), 12.23 (s, 1H, NH). 13 C NMR, δ ppm (100 MHz, DMSO- d 6 ): 57.3, 116.4, 116.7, 121.5, 122.7, 127.0, 127.4, 127.6, 128.2, 130.0, 131.2, 132.1, 137.6, 138.1, 138.9, 164.0, 167.3, 169.0, 170.0. IR (KBr) (cm − 1 ): 3404, 3189, 3027, 2923, 2850, 1739, 1678, 1623, 1593, 1493, 1371, 1177, 1154, 1062, 965, 912, 781, 698, 583, 555, 503. MS (m/z) (EI, 70 EV) (relative intensity): 42 (84), 97 (38), 139 (60), 156 (100), 182 (43), 235 (31), 391 (18), 443 (M, 1). Molecular docking studies. The steps and software used for molecular docking in this research are as follows: Initially, we used the www.rcsb.org database (commonly known as the Protein Data Bank) to extract the 3D structure of, gamma-aminobutyric acid receptor subunit alpha-5 (GABA A α5) (ID-PDB: 8BHK) in PDB format. Then, using ViewerLite software (version 4.2), which is a 3D molecular modeling software, we visualized and processed the 3D structure of a protein. In the next step B, C, D and E chains and water molecules were removed from the protein structure using ViewerLite 4.2 software, and hydrogen atoms were added to its structure. The active site of the protein was then determined based on its specific reference and grid box (a region of the protein where docking occurs). 30 The structure of SUL1 was then drawn with the ChemDraw 23.1.1.3 software and optimized with the HyperChem 8.0 software. In the final step, the results of the docking process were checked using AutoDocktools software (1.5.7) and Molegro virtual Docker software (V6.0.1). Result and Discussion General voltammetric studies. The cyclic voltammograms of CZP (1.0 mM) at glassy carbon electrode, in aqueous phosphate buffer ( c = 0.2 M, pH = 3) solution at scan rate, 100 mV s − 1 are shown in Fig. 3 , part I, curve a. When the potential scan is initially performed towards cathodic potentials, the voltammogram contains a well-defined irreversible cathodic peak (C N1 ) at -0.55 V vs. Ag/AgCl, which is attributed to the six-electron reduction of the nitro group present in the CZP structure to the corresponding amine. By changing the potential sweep towards positive potentials, an anodic peak (A 1 ) appears at a potential of 0.14 V. After the anodic scan, if the cathodic scan is performed again, a new cathodic peak (C 1 ) with a potential of 0.06 V appears in the voltammogram. This peak is the counterpart of peak A 1 . In these conditions, Fig. 3 , curve b, shows that if the anodic scan is performed first, no traces of A 1 .and C 1 peaks are seen in the voltammogram, confirming that the peaks are dependent on the reduction of the nitro group. In our previous electrochemical studies on aromatic para diamino compounds such as, N,N -dimethyl- p -phenylenediamine, 4-aminoacetanilide or 4-morpholinoaniline, we showed that these compounds are readily oxidized by loss of two electrons (and often two protons) to the corresponding quinonediimin. 31 – 37 Accordingly, and considering the structure of CZP and the presence of the nitro group in the para position of the NH-amide group, the following mechanism has been proposed for the electrochemical behavior of CZP (Fig. 4 ). As shown in Fig. 4 , in the cathodic scan, the nitro group is reduced to the amino group by gaining six electrons, producing cathodic peak C RP1 . In the structure of 7-amino-5-(2-chlorophenyl)-1 H -benzo[ e ][1,4]diazepin-2(3 H )-one ( ACZP ), the formed amine group is present in the para position of the NH-amide group. This structure provides favorable conditions for the two-electron oxidation of ACZP and the formation of the corresponding p -quinonediimine ( ACZPQ ) (generation of peak A 1 ). 31 – 37 Accordingly, cathodic peak C 1 is related to the reduction of ACZPQ to ACZP . In this experiment, if we increase the cathodic potential sweep to -1.4 V (Fig. 3 , part II), the voltammogram shows another cathodic peak (C RP2 ) at a potential of -1.10 V. Considering the structure of ACZP , this peak seems to be related to the two-electron reduction of ACZP to A2CZP . 38 Under these conditions, when the anodic scan is performed from − 1.4 V, two overlapped anodic peaks (A 1 and A’ 1 ) are observed, corresponding to the oxidation of ACZP and A2CZP , respectively. Since Δ E p for peaks A 1 and A’ 1 is about 30 mV, their cathodic peaks are indistinguishable from each other. For better visualization of peaks A 1 and A’ 1 , the cyclic voltammogram of CZP was recorded at a scan rate of 1000 mV/s and its anodic part is shown as an inset in Fig. 3 , part II. As can be seen, at a scan rate of 1000 mV/s, Δ E p ( E pA’1 – E pA1 ) has increased. This behavior is a typical example of the electrochemical behavior of “ a reversible and a quasi-reversible redox system ” or “ two quasi-reversible redox systems with different degree of quasi-reversibility ” (different k 0 or different Λ at similar scan rate). When the quasi-reversibility of the second redox system is greater than the quasi-reversibility of the first redox system ( \(\:k\genfrac{}{}{0pt}{}{0}{2}\) < \(\:k\genfrac{}{}{0pt}{}{0}{1}\) ), the Δ E p of the forward peaks (here the anodic peaks) increases with increasing scan rate, while the Δ E p of the reverse peaks (here the cathodic peaks) decreases and becomes closer together. These results confirm that the quasi-reversibility of the ACZP / ACZPQ system is higher than that of A2CZP / A2CZPQ ( k 0 ACZP / ACZPQ < k 0 A2CZP/A2CZPQ ). Effects of solution pH. In this part, the effect of solution pH on the redox couple ACZP / ACZPQ was investigated in detail. For this purpose, cyclic voltammograms of CZP in water (with different pH values)/ethanol mixture (30/50 v/v) were recorded and their anodic part is shown in Fig. 5 , part I. These studies, conducted over a wide pH range of 1–13, confirm the relative stability of the drug itself and the ACZP produced from its reduction on the time scale of the recorded voltammograms. As can be seen, in this wide pH range, the shape of the voltammograms remains unchanged and only the peak potentials shift. The results showed that the half-wave potential ( E 1/2 ) of the redox couple ACZP / ACZPQ is dependent on pH and changes to less positive potentials with increasing pH. This potential shift confirms the participation of protons in the redox process of ACZP / ACZPQ . The potential-pH (Pourbaix) diagram of the ACZP / ACZPQ redox couple is shown in Fig. 5 , part II. It should be noted that the half-wave potential ( E 1/2 ) is obtained from the average of the anodic and cathodic peak potentials. The diagram has three lines (A, B and C) with different slopes, indicating that the number of protons involved in the redox reaction of ACZP / ACZPQ changes in each pH range. Line A is located at pH values less than 3.6 and its equation is as follows: E 1/2 = -0.0912pH + 0.4179 (R² = 0.9686). The slope of the line is 91.2 mV/pH, which is close to the theoretical value (88.8 mV/pH) for the two-electron/three-proton process. In this pH range, the dominant electrochemical process appears to be the oxidation of protonated ACZP ( ACZPH + ) (the dominant species) to ACZPQ (Fig. 6 ). Line B is located at pH values more than 3.6 and less than 6.3 (3.6 < pH < 6.3) and its equation is as follows: E 1/2 = -0.0582pH + 0.2986 (R² = 0.9991). The slope of the line (58.2 mV/pH), is close to the theoretical value (59.2 mV/pH) for the two-electron/two-proton process. In this pH range, the dominant ACZP species is ACZP itself, and the electrochemical process is the oxidation of ACZP to ACZPQ (Fig. 6 ). Line C is located at pH values more than 6.3 and its equation is as follows: E 1/2 = -0.0694pH + 0.3697 (R² = 0.9982). In this region, the slope of the line is 69.4, which is greater than the theoretical value of 59.2 (two-electron/two-proton process) and smaller than the value of 88.8 (two-electron/three-proton process). In alkaline solutions, ACZPQ (a ketone in equilibrium with its enol form) appears to be in equilibrium with its enolate form by loss of a proton. In this pH range, the electrochemical process is the oxidation of ACZP to enolate form of ACZPQ (Fig. 6 ). However, since the deprotonation of ACZPQ (formation of enolate anion) is incomplete and occurs partially, the slope of the line C is less than 88.8. By equating the equation of line A with the equation of line B and solving the resulting equation, the p K a of ACZPH + was calculated as 3.62 (line D). Also, by equating the equation of line B with the equation of line C and solving the resulting equation, the p K a of ACZPQH + is obtained as 6.35 (line E). Our studies show that in addition to peaks A 1 and C 1 , the potentials of peaks RP 1 and RP 2 also shift towards more negative potentials with increasing pH. In addition to this predictable behaviour, voltammograms recorded in the alkaline solutions (pH > 9) show a new reversible redox couple (C 0 and A 0 ) at potentials less negative than peak RP 1 (Fig. 5 , part III). In this condition, the cathodic peak C 0 corresponds to the one-electron reduction of CZP to CZP ⁻ • . The anodic peak A 0 is the counterpart of peak C 0 and is related to the oxidation of CZP ⁻ • to CZP. 39 , 40 Fig. 7 , Eq. 1, shows the electrochemical process corresponding to peaks C 0 and A 0 . The absence of this redox couple (C 0 /A 0 ) is due to the participation of the radical anion formed ( CZP ⁻ • ) in the disproportionation reaction (Fig. 7 , Eq. 2). As can be seen, the rate of this reaction is pH dependent and increases with decreasing pH. The increase in disproportionation rate causes the instability of the radical anion on the cyclic voltammetry time scale and the absence of peaks C 0 and A 0 in acidic and neutral solutions. Effects of potential scan rate. Here, the effect of the potential scan rate on the cyclic voltammogram of CZP is investigated (Fig. 8 ). The first result is the dependence of the peak current ratio ( I pC1 / I pA1 ) on the scan rate, such that this ratio increases with increasing scan rate. This indicates the relative instability of ACZPQ and its participation in the chemical reactions such as dimerization or hydroxylation. 2 , 31 – 37 The second result is the shift of the cathodic peak potential of C 1 (and also RP 1 ) to more negative values and the anodic peak potential of A 1 to more positive potentials with increasing potential scan rate, which confirms the quasi-reversible nature of the redox process ACZPQ / ACZP . The third result is that the ACZP oxidation process is diffusion control in water (phosphate buffer, pH 3.0, c = 0.2 M)/ ethanol (30/50 v/v), mixture. To obtain this result, the log I pA1 was plotted against the log ν (potential scan rate) (Fig. 8 , part IX). In this type of diagram, when the slope of the line is 0.5, the process is completely diffusion-controlled, on the other hand, if the slope of the line is 1.0, the redox process is completely adsorption-controlled. Figure 8 , part IX shows that the slope of the line is 0.55 and it can be considered that the ACZP oxidation process under the tested conditions is a diffusion-controlled process. Effects arylsulfinic acids. Here, the effect of p -toluenesulfonic acid ( TSA ) (a nucleophile) on the redox behavior of CZP was studied. Figure 9 , part I shows the cyclic voltammogram of CZP (1.0 mM) in the presence of TSA (2.0 mM) in water (phosphate buffer, pH 3.0, c = 0.2 M)/ethanol mixture (30/50 v/v), at scan rate of 100 mV/s. Comparing the cyclic voltammogram of CZP in the absence of the nucleophile (CV a) with that in its presence (CV b), shows that the presence of the nucleophile has eliminated cathodic peak C 1 . This observation indicates the rapid reaction between ACZPQ and TSA . To confirm this reaction, the effect of scan rate on the cyclic voltammogram of CZP in the presence of TSA was investigated (Fig. 9 , part II). These experiments show that while peak C 1 is absent when scan rate is 10 mV/s, it appears as the scan rate increases and its current gradually increases so that when the scan rate is 500 mV/s, the peak current ratio ( I pC1 / I pA1 ) becomes approximately 1. These results confirm the reaction between ACZPQ and TSA under the mentioned conditions. 2 Controlled potential coulometry, a method with a large time window, was also used to supplement the data needed for mechanistic studies as well as in synthesis planning. For this purpose, a solution containing 0.5 mM CZP and 1.0 mM TSA was electrolyzed in an undivided (simple) cell at the potential required for the reduction of the nitro group (-0.72 V vs. Ag/AgCl) (see Fig. 10 ). In this experiment, cyclic voltammograms of the solution during electrolysis were also recorded to obtain complementary information about the changes made to the drug molecule (Fig. 10 ). The results show that over time, due to the reduction of the nitro group, its current ( \(\:{I}_{p}^{RP1}\) ) decreases. The plot of \(\:{I}_{p}^{RP1}\) versus charge passed is shown in Fig. 10 , inset and confirms that \(\:{I}_{p}^{RP1}\) decrease with the charge consumption. The total amount of charges consumed to terminate the reaction was calculated to be 362 coulombs from extrapolation to the X axis. Accordingly, since six electrons are required to reduce nitro group to the corresponding amine group, theoretically 290 coulombs should be consumed for 0.5 mmol of CZP , but since this electrolysis is carried out in an undivided cell, a process similar to the back reaction occurs. In this reaction, the intermediates generated at the anode are reduced again at the cathode, and the electricity consumed increases. According to theoretical (290 C) and experimental (362 C) values of electricity, the current efficiency in this experiment is 80%. At the end of this section, it is important to note that the anodic peak current ( I pA1 ) also decreases proportionally to the decrease in peak RP 1 . Another point in this experiment is the absence of peak C 1 (described earlier) and the decrease in A 1 peak current proportional to the charge consumed. From this result, it can be concluded that the reaction product of ACZPQ and p -toluenesulfinic acid is not soluble in the electrolysis medium. Considering the chemical structure of the synthesized products as well as the results of electrochemical experiments, the following mechanism is proposed for the electrolysis of CZP in the presence of arylsulfinic acids ( ASA ) (Fig. 11 ). Figure 11 shows that electrolysis of CZP in the presence of arylsulfonic acids ( ASA ) via successive paired strategy leads to the synthesis of new clonazepam derivatives ( SUL ) containing a sulfonamide moiety. The addition of this moiety to the CZP certainly affects the pharmacological properties of this drug, making it a molecule with high potential for pharmaceutical and medical research. According to the proposed mechanism, at a potential of -0.72 V, CZP is initially converted at the cathode to its amine derivative ( ACZP ), which is also one of its metabolites. In the next step, ACZP is oxidized at the anode surface and generates corresponding quinonediimine ( ACZPQ ). Quinonediimines are reactive compounds that act as electrophiles and readily react with nucleophiles. 31 – 37 This quinonediimine ( ACZPQ ) also has the same properties and reacts with arylsulfinic acids (as nucleophiles) to form the corresponding sulfonamide or clonazepam derivatives ( SUL ). One of the key points of this method is the use of an electrochemical successive paired strategy. As seen in Fig. 11 , in this method, successive reduction and oxidation of CZP is necessary. Such conditions can only be achieved by electrolysis in an undivided electrochemical cell. It is these features that distinguish electrochemical synthesis methods from conventional methods and allows these syntheses to be carried out in a one-pot process without the use of reducing and oxidizing reagents. On the other hand, in this strategy (in the syntheses reported in this study), since both cathodic (six-electron reduction) and anodic (two-electron oxidation) reactions contribute to the formation of the final product, it can lead to a 25% reduction in energy consumption compared to conventional electroorganic syntheses. 2 Docking studies. The purpose of the docking study is to investigate the interaction of SUl1 with Gamma-aminobutyric acid ( GABA ). Meanwhile, the structure of the GABA protein has been determined by electron microscopy. The results show that SUL1 has a promising binding pattern with GABA . This synthesized compound can hydrogen bond with the nitrogen of THR208 and also interact with other amino acids shown in Fig. 12 . The two parameters investigated in this study are inhibition constant and binding energy. The lower inhibition constant indicates that the compound has a higher affinity for binding at the active site, which was calculated to be 13.25 µM in this study. Furthermore, the binding energy, which represents the binding energy to the active site, was calculated to be 6.65 kcal/mol, indicating that SUL1 interacts well with GABA . Optimization of effective parameters. In this section, in order to achieve higher yield and purity, optimization of effective parameters has been performed using the " one factor at a time " optimization strategy. In this strategy, one of the factors that affects the synthesis process is changed while the other factors are kept constant. Here, the effect of electrode materials on the yield and purity of SUL2 has been investigated, while other factors such as CZP amount (0.5 mmol), sodium 4-toluenesulfinate ( TSA ) amount (1.0 mmol), solvent (aqueous phosphate buffer, pH 3.0, c = 0.2 M)/ethanol (30/50 v/v), applied potential ( E app , − 0.72 V versus Ag/AgCl), stirring speed and temperature have been kept constant. The results of these experiments are shown in Table 1 . As can be seen, the best yield (95%) was obtained when both electrodes were graphite (entry 3). The results also show that changing the cathode material from graphite to metal electrodes (stainless steel, Cu or Pb) causes a decrease in yield (entries 2, 4 and 5). It seems that the porosity of the graphite surface and the greater adsorption of the drug and its incomplete reduction intermediates, and consequently the complete reduction of the nitro group (six-electron reduction) on it, is the main factor for the higher efficiency on the graphite surface. On the other hand, when the anode is not inert, such as iron (sacrificial metal) (entry 6), the cathodically generated ACZP is not oxidized at the anode and as a result the desired product is not formed. In entry 6, since the anodic process is the oxidation of the electrode itself (rather than the oxidation of ACZP ), this experiment also confirms the paired strategy in the synthesis of the introduced products ( SUL ). Table 1 The effect of electrode material on the yield of SUL2 . a Entry Cathode Anode Yield [%] 1 Graphite Stainless steel 75 2 Stainless steel Graphite 23 3 Graphite Graphite 95 4 Cu Graphite less than 10 5 Lead Graphite 50 6 Graphite Fe no reaction a CZP (0.5 mmol), sodium 4-toluenesulfinate ( TSA ) (1.0 mmol), solvent (aqueous phosphate buffer, pH 3.0, c = 0.2 M)/ethanol (30/50 v/v), applied potential ( E app , − 0.82 V versus Ag/AgCl) at room temperature. The pH of the solution also has a great impact on the yield and purity of the products. Figure 13 shows the effect of solution pH on the yield of SUL2 . It should be noted that other parameters were kept constant as in the previous section. The results show that the optimal pH in the synthesis of SUL2 is 3. As shown in the equation below, the complete reduction of the nitro group to the amine requires six protons in addition to six electrons. Therefore, acidic pH is expected to be more favorable for this reduction. On the other hand, the p K a of 4-toluenesulfinic acid in water is 1.99. 41 This means that in strongly acidic solutions the 4-toluenesulfinate becomes protonated and its nucleophilicity decreases. CZP has low solubility in water, and hence in this study an organic solvent was used as a co-solvent to improve the solubility of CZP . In this section, the effect of co-solvent type on the yield of SUL2 is investigated. In these experiments, water (phosphate buffer, pH 3.0, c = 0.2 M) (without co-solvent), water (phosphate buffer, pH 3.0, c = 0.2 M)/ethanol (30/50 v/v) mixture, and water (phosphate buffer, pH 3.0, c = 0.2 M)/acetonitrile (30/50 v/v mixture were used as solvents. Figure 13 shows that the highest yield was achieved when ethanol, a valuable solvent in terms of green chemistry, was used as the co-solvent. Here, the effect of the ratio of organic to aqueous solvent on the yield of SUL2 synthesis was also investigated. For this purpose, the synthesis of SUL2 in water (phosphate buffer, pH 3.0, c = 0.2 M)/ethanol mixtures with ratios of 10/70 v/v, 50/30 v/v and 30/50 v/v was investigated. Figure 13 shows that the highest yield was achieved when the water to ethanol ratio was 30/50 v/v. Conclusion This paper reports a multidimensional study on CZP . The main objective of this study is to synthesize new CZP derivatives via a green and one-pot process by electrolysis of CZP in the presence of arylsulfinic acids. The most important features of the method are: (1) Successive reduction and oxidation of CZP in a one-pot process without the use of reducing and oxidizing reagents. (2) Energy savings that can lead to a 25% reduction in energy consumption compared to conventional electroorganic syntheses. (3) Selective reduction of nitro group. (4) Using a water/ethanol mixture as a solvent. (5) Using a simple cell and ordinary graphite electrodes. (6) Simple workup and synthesis at room temperature and pressure and (7) Synthesis of unique molecules with high potential in pharmaceutical and medical research. On the other hand, we performed detailed electrochemical studies on CZP and its metabolite ( ACZP ) and reported information that was not reported, incomplete, or incorrectly reported for the drug and its metabolite. The results showed that CZP can be reduced in two steps, in the first step the nitro group is reduced to an amine group and in the second step the C = N bond is reduced. To achieve the desired goal, we selectively reduced the nitro group using controlled potential electrolysis, to afford SUL in excellent yield. Also, the effect of solution pH on the electrochemical behavior of the drug, especially its metabolite, was studied, and the electrochemical and acid/base characteristic of these compounds were evaluated. Using electrochemical data, we investigated the adsorption/diffusion properties of the metabolite and found that under our experimental conditions, this metabolite has no adsorption activity. The properties discovered in these experiments could be useful in designing newer derivatives of this drug and also lead to deeper insight into the evolution of drug and its metabolites in different parts of the patient's bod. Also, based on molecular docking calculations and studies, SUL1 has a high affinity to interact with the GABA receptor. Declarations Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability The datasets used and/or analyzed during the current study available from the corresponding author on request. Acknowledgments The authors acknowledge the Bu-Ali Sina University Research Council and Center of Excellence in Development of Environmentally Friendly Methods for Chemical Synthesis (CEDEFMCS) for their support of this work. Author contributions DN: supervision, project administration, resources, writing-review & editing. ZG: Project administration, investigation & writing-original draft. NM: investigation, PM: investigation. References Kolbe, H. Untersuchungen über die Elektrolyse organischer Verbindungen. 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M. et al. Probing the architecture of the Mycobacterium marinum arylamine N -acetyltransferase active site. Protein Cell 1 , 384-392 (2010). Maleki, A. & Nematollahi, D. An efficient electrochemical method for the synthesis of methylene blue. Electrochem. Commun . 11 , 2261-2264 (2009). Jamshidi, M. & Nematollahi, D. Green electrochemical synthesis of N-phenylquinoneimine derivatives: dual action of 4-morpholinoaniline and N-(4-aminophenyl) acetamide. ACS Sustain. Chem. Eng. 5 , 9423-9430 (2017). Jamshidi, M., Nematollahi, D., Taheri, F. & Alizadeh, H. Paired electrochemical method for synthesis of new phenylcarbonimidoyl dicyanide dyes. ACS Sustain. Chem. Eng. 7 , 1956-1962 (2018). Talebi, M. R., Nematollahi, D. & Massah, A. R. Comparative electrochemical study of N -(4-aminophenyl) and N -(4-hydroxyphenyl) benzenesulfonamide derivatives. Electrochim. Acta 457 , 142499 (2023). Esmaili, R. & Nematollahi, D. Electrochemical oxidation of 4-morpholinoaniline in aqueous solutions: Synthesis of a new trimer of 4-morpholinoaniline. Electrochim. Acta 56 , 3899-3904 (2011). Maleki, A. & Nematollahi, D. Electrochemical synthesis and mechanestic study of quinone imines exploiting the dual character of N,N -dialkyl- p -phenylenediamines. Org. Lett . 13 , 1928-1931 (2011). Esmaili, R. & Nematollahi, D. Electrochemical Method for the synthesis of disulfides of 2-(benzo[d]thiazol(or oxazol)-2-ylamino)-5-morpholinobenzenethiol. J. Org. Chem . 78 , 5018-5021 (2013). Honeychurch, K. C., Brooks, J. & Hart, J. P. Development of a voltammetric assay, using screen-printed electrodes, for clonazepam and its application to beverage and serum samples. Talanta 147 , 510-515 (2016). Amatore, C. et al. Kinetics and mechanism of self-protonation reactions in organic electrochemical processes. J. Am. Chem. Soc . 107 , 1815-1824 (1985). Hosseinpour, M., Nematollahi, D., Ansari, A., Roshani, M. & Mohamadighader, N. 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Cite Share Download PDF Status: Published Journal Publication published 22 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 22 May, 2025 Reviews received at journal 19 May, 2025 Reviews received at journal 17 May, 2025 Reviewers agreed at journal 12 May, 2025 Reviewers agreed at journal 07 May, 2025 Reviewers agreed at journal 02 May, 2025 Reviewers invited by journal 02 May, 2025 Editor assigned by journal 07 Apr, 2025 Editor invited by journal 07 Apr, 2025 Submission checks completed at journal 04 Apr, 2025 First submitted to journal 26 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6311438","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":451989739,"identity":"875429b0-4ba2-415b-8d4b-a2b6f64fba3f","order_by":0,"name":"Zahra Godini","email":"","orcid":"","institution":"Bu-Ali Sina University","correspondingAuthor":false,"prefix":"","firstName":"Zahra","middleName":"","lastName":"Godini","suffix":""},{"id":451989740,"identity":"d75eff5a-fc5e-41a2-bfe2-dad75d618101","order_by":1,"name":"Davood Nematollahi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYDCCAxBKho+Bh+HABwaGBCK1JDDwsAG1HJxBshZmHmK08N1uPrrh5w8bHjb2swcP27bZ5fGzNzB++JiDW4vknWNpN3sS0njYePISDue2JRdL9hxglpy5DbcWgxs5Zjd4Eg4DHZZjANTCnLjhRgIbMy9eLfnfbv4BaeF/Y3DYsq2eGC05bLfBtkgAbWFsO0xYi+SNNLPbMmlAv0i8MTjYc+544syeg814/cJ3I/nZzTc2NnL8/DnGH36UVSf2szcf/PARjxZUwMgGJhuIVQ8Cf0hRPApGwSgYBSMFAADlBlZ7FRdzggAAAABJRU5ErkJggg==","orcid":"","institution":"Bu-Ali Sina University","correspondingAuthor":true,"prefix":"","firstName":"Davood","middleName":"","lastName":"Nematollahi","suffix":""},{"id":451989741,"identity":"9299bdc9-ab58-45ec-ac76-eba5a8171cd8","order_by":2,"name":"Niloofar Mohamadighader","email":"","orcid":"","institution":"Bu-Ali Sina University","correspondingAuthor":false,"prefix":"","firstName":"Niloofar","middleName":"","lastName":"Mohamadighader","suffix":""},{"id":451989745,"identity":"80c8eb86-dcd4-4f8b-95ac-b9897b60ea1a","order_by":3,"name":"Pegah Mashhadi","email":"","orcid":"","institution":"Bu-Ali Sina University","correspondingAuthor":false,"prefix":"","firstName":"Pegah","middleName":"","lastName":"Mashhadi","suffix":""}],"badges":[],"createdAt":"2025-03-26 10:38:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6311438/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6311438/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-12528-z","type":"published","date":"2025-07-22T15:58:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82202781,"identity":"516d9e65-0bc5-4bcb-ba44-2d1e4f1f8124","added_by":"auto","created_at":"2025-05-07 16:37:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":29615,"visible":true,"origin":"","legend":"\u003cp\u003eClonazepam synthesis steps.\u003csup\u003e17\u003c/sup\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6311438/v1/1c7f4b95a1ff186a5a69d05e.png"},{"id":82202780,"identity":"ec4b5e4e-cd6e-4ce8-a112-4f06a26f8199","added_by":"auto","created_at":"2025-05-07 16:37:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17790,"visible":true,"origin":"","legend":"\u003cp\u003eClonazepam metabolite\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6311438/v1/cc92d564f38f0183a42d2028.png"},{"id":82202783,"identity":"e00d524e-c821-47be-8bfa-6e77ffd657d6","added_by":"auto","created_at":"2025-05-07 16:37:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":92164,"visible":true,"origin":"","legend":"\u003cp\u003ePart I: (a) Cyclic voltammogram of \u003cstrong\u003eCZP\u003c/strong\u003e(1.0 mM) at glassy carbon electrode, at scan rate of 100 mV/s in water (phosphate buffer, pH 3.0, \u003cem\u003ec\u003c/em\u003e = 0.2 M)/ethanol (30/50 v/v). Starting potential: -0.20 V and scanning towards cathodic potentials. (b) (Dashed line) Starting potential: -0.2 0 V and scanning towards anodic potentials. Part II: Similar to Part I CVa, but with more negative switching potential. Inset: Similar to Part II at scan rate of 1000 mV/s (cathodic scan is not shown in the inset). All voltammograms were recorded at room temperature.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6311438/v1/ef426809481a4cea8353db6a.png"},{"id":82202786,"identity":"7635ea15-9743-4cc1-a69b-b3be21bd507d","added_by":"auto","created_at":"2025-05-07 16:37:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":65242,"visible":true,"origin":"","legend":"\u003cp\u003eElectrochemical reaction mechanism of \u003cstrong\u003eCZP\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6311438/v1/8d008e38ffb5de6b46d2240b.png"},{"id":82203384,"identity":"1debc000-7240-4611-81fe-94e2caa1ca47","added_by":"auto","created_at":"2025-05-07 16:45:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":141750,"visible":true,"origin":"","legend":"\u003cp\u003ePart I: Cyclic voltammograms of the anodic part of \u003cstrong\u003eCZP\u003c/strong\u003e (1.0 mM) (\u003cstrong\u003eACZP\u003c/strong\u003e/\u003cstrong\u003eACZPQ\u003c/strong\u003e redox couple) in aqueous buffer with different pH/ethanol (30/50 v/v) mixture with different pH values and same ion strength at scan rate of 100 mV/s. The pH values are: 1.50, 2.50, 3.53, 4.0, 4.99, 6.18, 7.0, 8.09, 9.01, 10.01, 11.47, 12.05 and 12.88 respectively. Part II: Pourbaix diagram of \u003cstrong\u003eACZP\u003c/strong\u003e/\u003cstrong\u003eACZPQ\u003c/strong\u003e redox couple. Part III: Cyclic voltammogram of the cathodic part of \u003cstrong\u003eCZP\u003c/strong\u003e (1.0 mM) in water (carbonate buffer, pH 11.0, \u003cem\u003ec\u003c/em\u003e = 0.2 M)/ethanol (30/50 v/v) at scan rate of 100 mV/s. All voltammograms were recorded at room temperature.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6311438/v1/32ab1976c1d1d803ff866ef4.png"},{"id":82203380,"identity":"a5af405c-b5bf-4b62-89bb-c0a33b8c6909","added_by":"auto","created_at":"2025-05-07 16:45:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":66678,"visible":true,"origin":"","legend":"\u003cp\u003eReactions related to lines A to E of Figure 5.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6311438/v1/224cc71e97f14c967f970d87.png"},{"id":82202784,"identity":"4fa5d590-b497-4e75-b1f1-a1af2389a84c","added_by":"auto","created_at":"2025-05-07 16:37:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":49151,"visible":true,"origin":"","legend":"\u003cp\u003eRedox reaction related to C\u003csub\u003e0\u003c/sub\u003e/A\u003csub\u003e0\u003c/sub\u003e peaks and possible disproportionation reaction.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6311438/v1/99535eb0435a89c50b65f93d.png"},{"id":82202796,"identity":"c8652024-c64b-41c5-b632-4608a108059c","added_by":"auto","created_at":"2025-05-07 16:37:16","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":151792,"visible":true,"origin":"","legend":"\u003cp\u003eParts I-IIX: Cyclic voltammograms of \u003cstrong\u003eCZP\u003c/strong\u003e (1.0 mM) in water (phosphate buffer, pH 3.0, \u003cem\u003ec\u003c/em\u003e = 0.2 M)/ ethanol (30/50 v/v), mixture at different scan rates. Working electrode: glassy carbon. Part IX: The plot of log \u003cem\u003eI\u003c/em\u003e\u003csub\u003epA1\u003c/sub\u003e versus log n. All experiments were performed at room temperature.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6311438/v1/1bde1607d873f20105c5d5cb.png"},{"id":82203383,"identity":"a64476db-e18a-4bd8-94b6-380af02eb9ee","added_by":"auto","created_at":"2025-05-07 16:45:16","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":101719,"visible":true,"origin":"","legend":"\u003cp\u003ePart I, Cyclic voltammograms of 1.0 mM \u003cstrong\u003eCZP\u003c/strong\u003e in the absence (—) and presence of (---) \u003cstrong\u003eTSA\u003c/strong\u003e (2.0 mM) at scan rate of 100 mV/s. Part II, Cyclic voltammograms of \u003cstrong\u003eCZP \u003c/strong\u003e(1.0 mM) in the presence of\u003cstrong\u003e TSA\u003c/strong\u003e (1.0 mM) at different scan rates. Scan rates from a to e are: 10, 25, 50,100 and 500 mV/s, respectively. Solvent: water (buffer solution with different pH values)/ethanol (30/50 v/v). Working electrode: glassy carbon. All experiments were performed at room temperature.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6311438/v1/ee371753df33950484855f19.png"},{"id":82202790,"identity":"b2b94d3d-3d34-4ef2-b304-5f160409f467","added_by":"auto","created_at":"2025-05-07 16:37:16","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":82027,"visible":true,"origin":"","legend":"\u003cp\u003eCyclic voltammograms of \u003cstrong\u003eCZP\u003c/strong\u003e (0.5 mmol) in the presence of \u003cem\u003ep\u003c/em\u003e-toluenesulfinic acid (1.0\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6311438/v1/32a909b607c8c90692d9019a.png"},{"id":82204304,"identity":"382892e4-15c4-4069-a7e1-a9cce2680e96","added_by":"auto","created_at":"2025-05-07 16:53:16","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":57953,"visible":true,"origin":"","legend":"\u003cp\u003eElectrochemical reaction mechanism for the synthesis of clonazepam derivatives (\u003cstrong\u003eSUL\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6311438/v1/22d2171891b2c063bfd8d48f.png"},{"id":82202793,"identity":"f949076c-b74d-406b-8067-6acfcc640765","added_by":"auto","created_at":"2025-05-07 16:37:16","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":255320,"visible":true,"origin":"","legend":"\u003cp\u003ea) Gamma-aminobutyric acid receptor subunit alpha-5 protein (ID-PDB: 8BHK), b) \u003cstrong\u003eSUL1\u003c/strong\u003e and c) Interaction of \u003cstrong\u003eSUL1\u003c/strong\u003ewith \u003cstrong\u003eGABA\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-6311438/v1/41d08b1a94c08865b530e7c6.png"},{"id":82202810,"identity":"d5623830-d6aa-49c6-8f24-203ddb17b225","added_by":"auto","created_at":"2025-05-07 16:37:16","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":55806,"visible":true,"origin":"","legend":"\u003cp\u003eOptimization of effective parameters in the synthesis of \u003cstrong\u003eSUL2\u003c/strong\u003e. pH\u003csub\u003e: \u003c/sub\u003e1) pH = 5.0, 2) pH = 1.5, 3) pH = 3.0, when water/ethanol (30/50 v/v). Solvent: 1) water, 2) water/acetonitrile mixture, 3) water/ethanol mixture, when water (phosphate buffer, pH 3.0, \u003cem\u003ec\u003c/em\u003e = 0.2 M)/solvent. Water to organic solvent ratio: 1) 10/70 v/v, 2) 50/30 v/v, 3) 30/50 v/v, when water (phosphate buffer, pH 3.0, \u003cem\u003ec\u003c/em\u003e = 0.2 M)/ethanol (different v/v). \u003cstrong\u003eCZP\u003c/strong\u003e (0.5 mmol), sodium 4-toluenesulfinate\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eTSA\u003c/strong\u003e) (1.0 mmol), applied potential (\u003cem\u003eE\u003c/em\u003e\u003csub\u003eapp\u003c/sub\u003e, − 0.72 V versus Ag/AgCl), cathode: graphite, anode: graphite at room temperature.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-6311438/v1/c61a4b05182074a3784033ea.png"},{"id":82202792,"identity":"ba7bb15c-766f-420d-97bf-07136c6821ed","added_by":"auto","created_at":"2025-05-07 16:37:16","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":14459,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the Experimental section.\u003c/p\u003e","description":"","filename":"Uf1.png","url":"https://assets-eu.researchsquare.com/files/rs-6311438/v1/cd639e88bcb786dfd2dd5540.png"},{"id":82204830,"identity":"bc271be1-b253-4839-9be3-01c1d14089e1","added_by":"auto","created_at":"2025-05-07 17:01:16","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":15208,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the Experimental section.\u003c/p\u003e","description":"","filename":"Uf2.png","url":"https://assets-eu.researchsquare.com/files/rs-6311438/v1/6bae6659950721c2aff0a158.png"},{"id":82202812,"identity":"e39b9745-1491-4b8a-9105-3abd4873fe60","added_by":"auto","created_at":"2025-05-07 16:37:16","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":15255,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the Experimental section.\u003c/p\u003e","description":"","filename":"Uf3.png","url":"https://assets-eu.researchsquare.com/files/rs-6311438/v1/0a319740c17e42bb313c9e90.png"},{"id":87757401,"identity":"b16d8f0f-1ee6-4e8a-9b98-3241e17476b0","added_by":"auto","created_at":"2025-07-28 16:10:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2156047,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6311438/v1/611da479-d965-4c96-8506-6e8e14660307.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comprehensive electrochemical study of clonazepam an anticonvulsant benzodiazepine drug and its main metabolite. Green late-stage functionalization of clonazepam","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn 1848, scientists were able to incorporate electricity into chemical reactions, performing the first organic electrosynthesis. Electrosynthesis uses electrons instead of chemical reagents, so the method is green, waste-free, and sustainable. The time required for electrochemical processes is usually shorter than for chemical processes, thereby minimizing the associated costs. Some other important features of electrochemical methods include: synthesis under mild conditions without using high temperature or pressure, no use of catalyst, reducing waste, higher yield, and easier workup and scalability. Electrochemistry has high selectivity by controlling the applied potential. This unique capability distinguishes electrosynthesis from other methods.\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe synthesis of new pharmaceutical derivatives via late-stage modification (LSM) or late-stage functionalization (LSF) has attracted our attention in this work. This method has the ability to purposefully synthesize derivatives of the original drug that have greater activity than the original drug. In addition, this method makes drug discovery, production, and synthesis fast and efficient and allows for changing the physical and chemical properties of the drug by adding new groups to it.\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Nobel Prize winner James Black highlighted, \u0026ldquo;the most fruitful basis for the discovery of a new drug is to start with an old drug\u0026rdquo;. Raju believes that by modifying the late stage, new drug derivatives with a broader spectrum of activity can be synthesized.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Late-stage modification of drug can directly and selectively lead to the synthesis of compounds with high potential in medicine, even in large and complex molecules. The advantages of the late-stage modification of drugs include avoiding lengthy synthesis, enabling rapid drug discovery, and modifying the physical and chemical properties of the drug. The late-stage modification drug is drug functionalization. Such modifications may have the potential to alter the properties, affinity, drug metabolism, or pharmacokinetic properties of the drug, generally without loss or even with an increase in the drug's biological activity.\u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Finally, the use of late-stage modification of drugs, is growing and is being used as an efficient method for developing new drugs.\u003c/p\u003e \u003cp\u003eBenzodiazepines refer to a family of psychotropic drugs whose main structure consists of a benzene ring and a diazepine ring.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Clonazepam (\u003cb\u003eCZP\u003c/b\u003e), 5-(2-chlorphenyl)-1,3-dihydro-7-nitro-2\u003cem\u003eH\u003c/em\u003e-1,4-benzodiazepin-2-one is one of the long-acting and popular benzodiazepines, which is used as an anticonvulsant, anti-anxiety, sedative and muscle relaxant.\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e The synthesis of this drug is carried out in four steps as shown in Fig.\u0026nbsp;1.\u003csup\u003e19\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBenzodiazepines are extensively metabolized by cytochrome P-450 enzymes, particularly by CYP3A4 and CYP2C19.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e In humans, \u003cb\u003eCZP\u003c/b\u003e is metabolized primarily to 7-aminoclonazepam (\u003cb\u003eACZP\u003c/b\u003e) by nitro-reduction via hepatic cytochrome P450 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003cb\u003eACZP\u003c/b\u003e is then \u003cem\u003eN\u003c/em\u003e-acetylated to form 7-acetamidoclonazepam, which is excreted in the urine and feces following extensive biotransformation.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMany drugs are based on sulfonamides, the most important and main members of this group of antibiotics being sulfacetamide, sulfamethoxazole, and silversulfadazine. Sulfonamides inhibit the bacterial enzyme dihydropteroate synthetase, which is responsible for converting \u003cem\u003epara\u003c/em\u003e-aminobenzoic acid into dihydrofolic acid, preventing the formation of folic acid which is vital for bacterial growth.\u003csup\u003e22\u0026ndash;25\u003c/sup\u003e On the other hand, drug resistance resulting from continuous use of drugs is an undeniable fact that poses serious problems to human health and, as a result, necessitates the need for new drugs. In summary, in this study, we intend to add a sulfonamide moiety to the clonazepam molecule using an electrochemical method. Given the pharmacological properties of sulfonamides, we hope that these changes in the structure of \u003cb\u003eCZP\u003c/b\u003e will create new properties in this molecule that will lead to an increase in its pharmacological activity.\u003c/p\u003e \u003cp\u003eOn the other hand, a literature review shows that although the electrochemical behavior of \u003cb\u003eCZP\u003c/b\u003e has been investigated in a number of papers, these studies are limited and in some cases contain misleading statements.\u003csup\u003e19,26\u0026ndash;29\u003c/sup\u003e Therefore, in this research, we decided to complete the electrochemical studies conducted on this drug and correct many of the published data, in addition to synthesizing new \u003cb\u003eCZP\u003c/b\u003e derivatives.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cp\u003e \u003cb\u003eApparatus and reagents.\u003c/b\u003e A Behpajooh model BHP-2051 potentiostat/galvanostat was also used for electrosynthesis and controlled potential coulometry. All experiments including cyclic voltammetry and controlled potential coulometry were carried out by a Autolab model PGSTAT 302N potentiostat/galvanostat (Metrohm-Autolab, Netherland) and nova2.1 software. For electrochemical studies, a three-electrode system in an undivided cell has been used. All electrodes are from Azar Electrode, which includes: Ag/AgCl (3.0 M) as the reference electrode, a glassy carbon (GC) disk as the working electrode (diameter 2.0 mm) and a platinum wire as the counter electrode. Before each experiment, the GC electrode was polished with alumina slurry and rinsed with acetone. The solution was purged for 15 minutes with N\u003csub\u003e2\u003c/sub\u003e before the voltammetric experiments to remove oxygen. For macro-scale electrolysis, five graphite rods (length 9 cm and diameter 8 mm) were used as anode (one numbers) and cathode (four number) electrodes. The FTIR spectra of products were recorded in the range 500\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e using a Perkin-Elmer 1760. The \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR spectra were recorded in DMSO on a Bruker Avance 400 MHz spectrometer. Mass spectrometry analysis was performed in electron impact mode at an ionization potential of 70 eV using an Agilent-5973 mass spectrometer. The melting point of the products was determined using a Barnstead Electro thermal 9100 instrument.\u003c/p\u003e \u003cp\u003ePerchloric acid (60%), acetic acid (100%), phosphoric acid (85%), sodium carbonate (98%) and sodium bicarbonate (98%) were used to prepare the buffer solutions were of analytical grade and were obtained from Sigma-Aldrich and used without further purification. Acetone, ethanol, benzenesulfonic acid sodium salt (98%), sodium4-toluenesulfinate (95%) and 4-fluorobenzenesulfinate (95%) were purchased from Sigma-Aldrich and used without further purification. The active ingredient clonazepam was received from Tehrandaro Pharmaceutical Company.\u003c/p\u003e \u003cp\u003e \u003cb\u003eElectrochemical synthesis of CZP derivatives.\u003c/b\u003e Electrochemical synthesis was performed under controlled potential condition (at -0.82 V vs. Ag/AgCl) in an undivided cell at room temperature. For this purpose, a mixture of water (phosphate buffer pH\u0026thinsp;=\u0026thinsp;3.0, \u003cem\u003ec\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2 M)/ethanol )30/50, v/v) (80 ml) containing \u003cb\u003eCZP\u003c/b\u003e (0.5 mmol) and arylsulfinic acid (\u003cb\u003eASA\u003c/b\u003e) (1.0 mmol) was electrolyzed in an undivided cell equipped with graphite as both anode and cathode. At the end of the electrolysis, the solution was allowed to evaporate slowly at room temperature to one-third of the initial volume. Then the solution was extracted with ethyl acetate (30 mL). After the evaporation of ethyl acetate, the precipitate solid was collected by filtration and washed several times with distilled water (100 mL). After washing and drying, the product was identified by FT-IR, \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR, \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR and MS spectra.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCharacteristics of products.\u003c/b\u003e \u003cem\u003eN-(5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-7-yl)benzenesulfonamide\u003c/em\u003e (SUL1), \u003cem\u003eC\u003c/em\u003e\u003csub\u003e\u003cem\u003e21\u003c/em\u003e\u003c/sub\u003e\u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003e16\u003c/em\u003e\u003c/sub\u003e\u003cem\u003eClN\u003c/em\u003e\u003csub\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sub\u003e\u003cem\u003eO\u003c/em\u003e\u003csub\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sub\u003e\u003cem\u003eS\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePale orange solid, Yield. 87%, Mp. 180\u0026ndash;183\u0026deg;C (Dec.), \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR, \u003cem\u003eδ\u003c/em\u003e ppm (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e): 4.10 (s, 2H, CH\u003csub\u003e2\u003c/sub\u003e), 6.47 (d, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 Hz, aromatic), 7.10 (d, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12 Hz, aromatic), 7.21 (dd, 1H, \u003cem\u003eJ\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;12, \u003cem\u003eJ\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4 Hz, aromatic), 7.21\u0026ndash;7.44 (m, 8H, aromatic), 7.62 (t, 1H, aromatic), 10.67 (s, 1H, NH), 11.1 (broad, NH). \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR, \u003cem\u003eδ\u003c/em\u003e ppm (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e): 57.4, 121.5, 122.8, 125.9, 126.8, 127.3, 127.7, 128.1, 129.2, 129.4, 131.3, 132.0, 132.2, 134.6, 137.7, 138.0, 138.8, 169.1, 170.0. IR (KBr) (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): 3430, 3288, 3172, 2919, 2850, 1675, 1640, 1559, 1496, 1412, 1363, 1171, 1068, 1020, 809, 753, 687, 576, 470. MS (m/z) (EI, 70 EV) (relative intensity): 57 (100), 83 (50), 111 (21), 137 (7), 167 (7), 198 (14), 236 (25), 264 (3), 316 (5), 343 (6), 396 (M-CO, 23), 424 (M-H, 1).\u003c/p\u003e \u003cp\u003e \u003cem\u003eN-(5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e] [1,4] diazepin-7-yl)-4-methylbenzene sulfonamide\u003c/em\u003e (SUL2), C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eClN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOrange solid, Yield. 95%, Mp. 190\u0026ndash;193\u0026deg;C (Dec), \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR, \u003cem\u003eδ\u003c/em\u003e ppm (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e): 2.24 (s, 3H, CH\u003csub\u003e3\u003c/sub\u003e), 3.33 (s, 2H, CH\u003csub\u003e2\u003c/sub\u003e), 7.26 (m, 3H, aromatic), 7.68 (dd, 2H, \u003cem\u003eJ\u003c/em\u003e\u003csub\u003e\u003cem\u003e1\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;8, \u003cem\u003eJ\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4 Hz, aromatic), 7.80 (m, 4H, aromatic), 8.10 (d, 2H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, aromatic), 12.23 (s, 1H, NH). \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR, \u003cem\u003eδ\u003c/em\u003e ppm (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e): 57.3, 116.5, 116.8, 121.6, 122.8, 127.1, 127.4, 127.7, 128.2, 130.0, 131.3, 132.2, 132.5, 132.7, 137.6, 138.2, 139.8, 169.0, 170.1. IR (KBr) (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): 3350, 3205, 3065, 2922, 2823, 1687, 1625, 1493, 1359, 1164, 1031, 976, 765, 673, 588, 556, 480. MS (m/z) (EI, 70 EV) (relative intensity): 43 (100), 69 (62), 109 (60), 159 (55), 236 (11), 368 (15) 439 (M, 1).\u003c/p\u003e \u003cp\u003e \u003cem\u003eN-(5-(2-chlorophenyl)-2-oxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-7-yl)-4-fluorobenzene sulfonamide\u003c/em\u003e (SUL3), C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eClFN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOrange solid, Yield. 83%, Mp. 202\u0026ndash;205\u0026deg;C (Dec), \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR, \u003cem\u003eδ\u003c/em\u003e ppm (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e): 4.20 (s, 2H, CH\u003csub\u003e2\u003c/sub\u003e), 6.55 (d, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.8 Hz, aromatic), 7.18 (d, 1H, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12 Hz, aromatic), 7.31 (m, 9H, aromatic), 11.25 (s, 1H, NH), 12.23 (s, 1H, NH). \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR, \u003cem\u003eδ\u003c/em\u003e ppm (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e): 57.3, 116.4, 116.7, 121.5, 122.7, 127.0, 127.4, 127.6, 128.2, 130.0, 131.2, 132.1, 137.6, 138.1, 138.9, 164.0, 167.3, 169.0, 170.0. IR (KBr) (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): 3404, 3189, 3027, 2923, 2850, 1739, 1678, 1623, 1593, 1493, 1371, 1177, 1154, 1062, 965, 912, 781, 698, 583, 555, 503. MS (m/z) (EI, 70 EV) (relative intensity): 42 (84), 97 (38), 139 (60), 156 (100), 182 (43), 235 (31), 391 (18), 443 (M, 1).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMolecular docking studies.\u003c/b\u003e The steps and software used for molecular docking in this research are as follows: Initially, we used the \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.rcsb.org\u003c/span\u003e\u003cspan address=\"http://www.rcsb.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e database (commonly known as the Protein Data Bank) to extract the 3D structure of, gamma-aminobutyric acid receptor subunit alpha-5 (GABA A α5) (ID-PDB: 8BHK) in PDB format. Then, using ViewerLite software (version 4.2), which is a 3D molecular modeling software, we visualized and processed the 3D structure of a protein. In the next step B, C, D and E chains and water molecules were removed from the protein structure using ViewerLite 4.2 software, and hydrogen atoms were added to its structure. The active site of the protein was then determined based on its specific reference and grid box (a region of the protein where docking occurs).\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e The structure of \u003cb\u003eSUL1\u003c/b\u003e was then drawn with the ChemDraw 23.1.1.3 software and optimized with the HyperChem 8.0 software. In the final step, the results of the docking process were checked using AutoDocktools software (1.5.7) and Molegro virtual Docker software (V6.0.1).\u003c/p\u003e "},{"header":"Result and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003cp\u003e \u003cb\u003eGeneral voltammetric studies.\u003c/b\u003e The cyclic voltammograms of \u003cb\u003eCZP\u003c/b\u003e (1.0 mM) at glassy carbon electrode, in aqueous phosphate buffer (\u003cem\u003ec\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2 M, pH\u0026thinsp;=\u0026thinsp;3) solution at scan rate, 100 mV s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, part I, curve a. When the potential scan is initially performed towards cathodic potentials, the voltammogram contains a well-defined irreversible cathodic peak (C\u003csub\u003eN1\u003c/sub\u003e) at -0.55 V vs. Ag/AgCl, which is attributed to the six-electron reduction of the nitro group present in the \u003cb\u003eCZP\u003c/b\u003e structure to the corresponding amine. By changing the potential sweep towards positive potentials, an anodic peak (A\u003csub\u003e1\u003c/sub\u003e) appears at a potential of 0.14 V. After the anodic scan, if the cathodic scan is performed again, a new cathodic peak (C\u003csub\u003e1\u003c/sub\u003e) with a potential of 0.06 V appears in the voltammogram. This peak is the counterpart of peak A\u003csub\u003e1\u003c/sub\u003e. In these conditions, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, curve b, shows that if the anodic scan is performed first, no traces of A\u003csub\u003e1\u003c/sub\u003e.and C\u003csub\u003e1\u003c/sub\u003e peaks are seen in the voltammogram, confirming that the peaks are dependent on the reduction of the nitro group. In our previous electrochemical studies on aromatic \u003cem\u003epara\u003c/em\u003e diamino compounds such as, \u003cem\u003eN,N\u003c/em\u003e-dimethyl-\u003cem\u003ep\u003c/em\u003e-phenylenediamine, 4-aminoacetanilide or 4-morpholinoaniline, we showed that these compounds are readily oxidized by loss of two electrons (and often two protons) to the corresponding quinonediimin.\u003csup\u003e\u003cspan additionalcitationids=\"CR32 CR33 CR34 CR35 CR36\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Accordingly, and considering the structure of \u003cb\u003eCZP\u003c/b\u003e and the presence of the nitro group in the para position of the NH-amide group, the following mechanism has been proposed for the electrochemical behavior of \u003cb\u003eCZP\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, in the cathodic scan, the nitro group is reduced to the amino group by gaining six electrons, producing cathodic peak C\u003csub\u003eRP1\u003c/sub\u003e. In the structure of 7-amino-5-(2-chlorophenyl)-1\u003cem\u003eH\u003c/em\u003e-benzo[\u003cem\u003ee\u003c/em\u003e][1,4]diazepin-2(3\u003cem\u003eH\u003c/em\u003e)-one (\u003cb\u003eACZP\u003c/b\u003e), the formed amine group is present in the para position of the NH-amide group. This structure provides favorable conditions for the two-electron oxidation of \u003cb\u003eACZP\u003c/b\u003e and the formation of the corresponding \u003cem\u003ep\u003c/em\u003e-quinonediimine (\u003cb\u003eACZPQ\u003c/b\u003e) (generation of peak A\u003csub\u003e1\u003c/sub\u003e).\u003csup\u003e\u003cspan additionalcitationids=\"CR32 CR33 CR34 CR35 CR36\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Accordingly, cathodic peak C\u003csub\u003e1\u003c/sub\u003e is related to the reduction of \u003cb\u003eACZPQ\u003c/b\u003e to \u003cb\u003eACZP\u003c/b\u003e. In this experiment, if we increase the cathodic potential sweep to -1.4 V (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, part II), the voltammogram shows another cathodic peak (C\u003csub\u003eRP2\u003c/sub\u003e) at a potential of -1.10 V. Considering the structure of \u003cb\u003eACZP\u003c/b\u003e, this peak seems to be related to the two-electron reduction of \u003cb\u003eACZP\u003c/b\u003e to \u003cb\u003eA2CZP\u003c/b\u003e.\u003csup\u003e38\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnder these conditions, when the anodic scan is performed from \u0026minus;\u0026thinsp;1.4 V, two overlapped anodic peaks (A\u003csub\u003e1\u003c/sub\u003e and A\u0026rsquo;\u003csub\u003e1\u003c/sub\u003e) are observed, corresponding to the oxidation of \u003cb\u003eACZP\u003c/b\u003e and \u003cb\u003eA2CZP\u003c/b\u003e, respectively. Since Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e for peaks A\u003csub\u003e1\u003c/sub\u003e and A\u0026rsquo;\u003csub\u003e1\u003c/sub\u003e is about 30 mV, their cathodic peaks are indistinguishable from each other. For better visualization of peaks A\u003csub\u003e1\u003c/sub\u003e and A\u0026rsquo;\u003csub\u003e1\u003c/sub\u003e, the cyclic voltammogram of \u003cb\u003eCZP\u003c/b\u003e was recorded at a scan rate of 1000 mV/s and its anodic part is shown as an inset in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, part II. As can be seen, at a scan rate of 1000 mV/s, Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e (\u003cem\u003eE\u003c/em\u003e\u003csub\u003epA\u0026rsquo;1\u003c/sub\u003e \u0026ndash; \u003cem\u003eE\u003c/em\u003e\u003csub\u003epA1\u003c/sub\u003e) has increased. This behavior is a typical example of the electrochemical behavior of \u0026ldquo;\u003cem\u003ea reversible and a quasi-reversible redox system\u003c/em\u003e\u0026rdquo; or \u0026ldquo;\u003cem\u003etwo quasi-reversible redox systems with different degree of quasi-reversibility\u003c/em\u003e\u0026rdquo; (different \u003cem\u003ek\u003c/em\u003e\u003csup\u003e0\u003c/sup\u003e or different Λ at similar scan rate). When the quasi-reversibility of the second redox system is greater than the quasi-reversibility of the first redox system (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:k\\genfrac{}{}{0pt}{}{0}{2}\\)\u003c/span\u003e\u003c/span\u003e \u0026lt; \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:k\\genfrac{}{}{0pt}{}{0}{1}\\)\u003c/span\u003e\u003c/span\u003e), the Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e of the forward peaks (here the anodic peaks) increases with increasing scan rate, while the Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e of the reverse peaks (here the cathodic peaks) decreases and becomes closer together. These results confirm that the quasi-reversibility of the \u003cb\u003eACZP\u003c/b\u003e/\u003cb\u003eACZPQ\u003c/b\u003e system is higher than that of \u003cb\u003eA2CZP\u003c/b\u003e/\u003cb\u003eA2CZPQ\u003c/b\u003e (\u003cem\u003ek\u003c/em\u003e\u003csup\u003e0\u003c/sup\u003e\u003csub\u003e\u003cb\u003eACZP\u003c/b\u003e/\u003cb\u003eACZPQ\u003c/b\u003e\u003c/sub\u003e \u0026lt; \u003cem\u003ek\u003c/em\u003e\u003csup\u003e0\u003c/sup\u003e\u003csub\u003e\u003cb\u003eA2CZP/A2CZPQ\u003c/b\u003e\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of solution pH.\u003c/b\u003e In this part, the effect of solution pH on the redox couple \u003cb\u003eACZP\u003c/b\u003e/\u003cb\u003eACZPQ\u003c/b\u003e was investigated in detail. For this purpose, cyclic voltammograms of \u003cb\u003eCZP\u003c/b\u003e in water (with different pH values)/ethanol mixture (30/50 v/v) were recorded and their anodic part is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, part I. These studies, conducted over a wide pH range of 1\u0026ndash;13, confirm the relative stability of the drug itself and the \u003cb\u003eACZP\u003c/b\u003e produced from its reduction on the time scale of the recorded voltammograms. As can be seen, in this wide pH range, the shape of the voltammograms remains unchanged and only the peak potentials shift. The results showed that the half-wave potential (\u003cem\u003eE\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e) of the redox couple \u003cb\u003eACZP\u003c/b\u003e/\u003cb\u003eACZPQ\u003c/b\u003e is dependent on pH and changes to less positive potentials with increasing pH. This potential shift confirms the participation of protons in the redox process of \u003cb\u003eACZP\u003c/b\u003e/\u003cb\u003eACZPQ\u003c/b\u003e. The potential-pH (Pourbaix) diagram of the \u003cb\u003eACZP\u003c/b\u003e/\u003cb\u003eACZPQ\u003c/b\u003e redox couple is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, part II. It should be noted that the half-wave potential (\u003cem\u003eE\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e) is obtained from the average of the anodic and cathodic peak potentials. The diagram has three lines (A, B and C) with different slopes, indicating that the number of protons involved in the redox reaction of \u003cb\u003eACZP\u003c/b\u003e/\u003cb\u003eACZPQ\u003c/b\u003e changes in each pH range. Line A is located at pH values less than 3.6 and its equation is as follows: \u003cem\u003eE\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e = -0.0912pH\u0026thinsp;+\u0026thinsp;0.4179 (R\u0026sup2; = 0.9686). The slope of the line is 91.2 mV/pH, which is close to the theoretical value (88.8 mV/pH) for the two-electron/three-proton process. In this pH range, the dominant electrochemical process appears to be the oxidation of protonated \u003cb\u003eACZP\u003c/b\u003e (\u003cb\u003eACZPH\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e) (the dominant species) to \u003cb\u003eACZPQ\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLine B is located at pH values more than 3.6 and less than 6.3 (3.6\u0026thinsp;\u0026lt;\u0026thinsp;pH\u0026thinsp;\u0026lt;\u0026thinsp;6.3) and its equation is as follows: \u003cem\u003eE\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e = -0.0582pH\u0026thinsp;+\u0026thinsp;0.2986 (R\u0026sup2; = 0.9991). The slope of the line (58.2 mV/pH), is close to the theoretical value (59.2 mV/pH) for the two-electron/two-proton process. In this pH range, the dominant \u003cb\u003eACZP\u003c/b\u003e species is \u003cb\u003eACZP\u003c/b\u003e itself, and the electrochemical process is the oxidation of \u003cb\u003eACZP\u003c/b\u003e to \u003cb\u003eACZPQ\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Line C is located at pH values more than 6.3 and its equation is as follows: \u003cem\u003eE\u003c/em\u003e\u003csub\u003e1/2\u003c/sub\u003e = -0.0694pH\u0026thinsp;+\u0026thinsp;0.3697 (R\u0026sup2; = 0.9982). In this region, the slope of the line is 69.4, which is greater than the theoretical value of 59.2 (two-electron/two-proton process) and smaller than the value of 88.8 (two-electron/three-proton process). In alkaline solutions, \u003cb\u003eACZPQ\u003c/b\u003e (a ketone in equilibrium with its enol form) appears to be in equilibrium with its enolate form by loss of a proton. In this pH range, the electrochemical process is the oxidation of \u003cb\u003eACZP\u003c/b\u003e to enolate form of \u003cb\u003eACZPQ\u003c/b\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). However, since the deprotonation of \u003cb\u003eACZPQ\u003c/b\u003e (formation of enolate anion) is incomplete and occurs partially, the slope of the line C is less than 88.8. By equating the equation of line A with the equation of line B and solving the resulting equation, the p\u003cem\u003eK\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e of \u003cb\u003eACZPH\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e was calculated as 3.62 (line D). Also, by equating the equation of line B with the equation of line C and solving the resulting equation, the p\u003cem\u003eK\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e of \u003cb\u003eACZPQH\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e is obtained as 6.35 (line E).\u003c/p\u003e \u003cp\u003eOur studies show that in addition to peaks A\u003csub\u003e1\u003c/sub\u003e and C\u003csub\u003e1\u003c/sub\u003e, the potentials of peaks RP\u003csub\u003e1\u003c/sub\u003e and RP\u003csub\u003e2\u003c/sub\u003e also shift towards more negative potentials with increasing pH. In addition to this predictable behaviour, voltammograms recorded in the alkaline solutions (pH\u0026thinsp;\u0026gt;\u0026thinsp;9) show a new reversible redox couple (C\u003csub\u003e0\u003c/sub\u003e and A\u003csub\u003e0\u003c/sub\u003e) at potentials less negative than peak RP\u003csub\u003e1\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, part III). In this condition, the cathodic peak C\u003csub\u003e0\u003c/sub\u003e corresponds to the one-electron reduction of \u003cb\u003eCZP\u003c/b\u003e to \u003cb\u003eCZP\u003c/b\u003e⁻\u003csup\u003e\u0026bull;\u003c/sup\u003e. The anodic peak A\u003csub\u003e0\u003c/sub\u003e is the counterpart of peak C\u003csub\u003e0\u003c/sub\u003e and is related to the oxidation of \u003cb\u003eCZP\u003c/b\u003e⁻\u003csup\u003e\u0026bull;\u003c/sup\u003e to \u003cb\u003eCZP.\u003c/b\u003e \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Eq.\u0026nbsp;1, shows the electrochemical process corresponding to peaks C\u003csub\u003e0\u003c/sub\u003e and A\u003csub\u003e0\u003c/sub\u003e. The absence of this redox couple (C\u003csub\u003e0\u003c/sub\u003e/A\u003csub\u003e0\u003c/sub\u003e) is due to the participation of the radical anion formed (\u003cb\u003eCZP\u003c/b\u003e⁻\u003csup\u003e\u0026bull;\u003c/sup\u003e) in the disproportionation reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, Eq.\u0026nbsp;2). As can be seen, the rate of this reaction is pH dependent and increases with decreasing pH. The increase in disproportionation rate causes the instability of the radical anion on the cyclic voltammetry time scale and the absence of peaks C\u003csub\u003e0\u003c/sub\u003e and A\u003csub\u003e0\u003c/sub\u003e in acidic and neutral solutions.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects of potential scan rate.\u003c/b\u003e Here, the effect of the potential scan rate on the cyclic voltammogram of \u003cb\u003eCZP\u003c/b\u003e is investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). The first result is the dependence of the peak current ratio (\u003cem\u003eI\u003c/em\u003e\u003csub\u003epC1\u003c/sub\u003e/\u003cem\u003eI\u003c/em\u003e\u003csub\u003epA1\u003c/sub\u003e) on the scan rate, such that this ratio increases with increasing scan rate. This indicates the relative instability of \u003cb\u003eACZPQ\u003c/b\u003e and its participation in the chemical reactions such as dimerization or hydroxylation.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan additionalcitationids=\"CR32 CR33 CR34 CR35 CR36\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe second result is the shift of the cathodic peak potential of C\u003csub\u003e1\u003c/sub\u003e (and also RP\u003csub\u003e1\u003c/sub\u003e) to more negative values and the anodic peak potential of A\u003csub\u003e1\u003c/sub\u003e to more positive potentials with increasing potential scan rate, which confirms the quasi-reversible nature of the redox process \u003cb\u003eACZPQ\u003c/b\u003e/\u003cb\u003eACZP\u003c/b\u003e. The third result is that the \u003cb\u003eACZP\u003c/b\u003e oxidation process is diffusion control in water (phosphate buffer, pH 3.0, \u003cem\u003ec\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2 M)/ ethanol (30/50 v/v), mixture. To obtain this result, the log \u003cem\u003eI\u003c/em\u003e\u003csub\u003epA1\u003c/sub\u003e was plotted against the log ν (potential scan rate) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, part IX). In this type of diagram, when the slope of the line is 0.5, the process is completely diffusion-controlled, on the other hand, if the slope of the line is 1.0, the redox process is completely adsorption-controlled. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, part IX shows that the slope of the line is 0.55 and it can be considered that the \u003cb\u003eACZP\u003c/b\u003e oxidation process under the tested conditions is a diffusion-controlled process.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffects arylsulfinic acids.\u003c/b\u003e Here, the effect of \u003cem\u003ep\u003c/em\u003e-toluenesulfonic acid (\u003cb\u003eTSA\u003c/b\u003e) (a nucleophile) on the redox behavior of \u003cb\u003eCZP\u003c/b\u003e was studied. Figure\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, part I shows the cyclic voltammogram of \u003cb\u003eCZP\u003c/b\u003e (1.0 mM) in the presence of \u003cb\u003eTSA\u003c/b\u003e (2.0 mM) in water (phosphate buffer, pH 3.0, \u003cem\u003ec\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2 M)/ethanol mixture (30/50 v/v), at scan rate of 100 mV/s. Comparing the cyclic voltammogram of \u003cb\u003eCZP\u003c/b\u003e in the absence of the nucleophile (CV a) with that in its presence (CV b), shows that the presence of the nucleophile has eliminated cathodic peak C\u003csub\u003e1\u003c/sub\u003e. This observation indicates the rapid reaction between \u003cb\u003eACZPQ\u003c/b\u003e and \u003cb\u003eTSA\u003c/b\u003e. To confirm this reaction, the effect of scan rate on the cyclic voltammogram of \u003cb\u003eCZP\u003c/b\u003e in the presence of \u003cb\u003eTSA\u003c/b\u003e was investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, part II). These experiments show that while peak C\u003csub\u003e1\u003c/sub\u003e is absent when scan rate is 10 mV/s, it appears as the scan rate increases and its current gradually increases so that when the scan rate is 500 mV/s, the peak current ratio (\u003cem\u003eI\u003c/em\u003e\u003csub\u003epC1\u003c/sub\u003e/\u003cem\u003eI\u003c/em\u003e\u003csub\u003epA1\u003c/sub\u003e) becomes approximately 1. These results confirm the reaction between \u003cb\u003eACZPQ\u003c/b\u003e and \u003cb\u003eTSA\u003c/b\u003e under the mentioned conditions.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eControlled potential coulometry, a method with a large time window, was also used to supplement the data needed for mechanistic studies as well as in synthesis planning. For this purpose, a solution containing 0.5 mM \u003cb\u003eCZP\u003c/b\u003e and 1.0 mM \u003cb\u003eTSA\u003c/b\u003e was electrolyzed in an undivided (simple) cell at the potential required for the reduction of the nitro group (-0.72 V vs. Ag/AgCl) (see Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this experiment, cyclic voltammograms of the solution during electrolysis were also recorded to obtain complementary information about the changes made to the drug molecule (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). The results show that over time, due to the reduction of the nitro group, its current (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{I}_{p}^{RP1}\\)\u003c/span\u003e\u003c/span\u003e) decreases. The plot of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{I}_{p}^{RP1}\\)\u003c/span\u003e\u003c/span\u003e versus charge passed is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e, inset and confirms that \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{I}_{p}^{RP1}\\)\u003c/span\u003e\u003c/span\u003edecrease with the charge consumption. The total amount of charges consumed to terminate the reaction was calculated to be 362 coulombs from extrapolation to the X axis. Accordingly, since six electrons are required to reduce nitro group to the corresponding amine group, theoretically 290 coulombs should be consumed for 0.5 mmol of \u003cb\u003eCZP\u003c/b\u003e, but since this electrolysis is carried out in an undivided cell, a process similar to the back reaction occurs. In this reaction, the intermediates generated at the anode are reduced again at the cathode, and the electricity consumed increases. According to theoretical (290 C) and experimental (362 C) values of electricity, the current efficiency in this experiment is 80%. At the end of this section, it is important to note that the anodic peak current (\u003cem\u003eI\u003c/em\u003e\u003csub\u003epA1\u003c/sub\u003e) also decreases proportionally to the decrease in peak RP\u003csub\u003e1\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAnother point in this experiment is the absence of peak C\u003csub\u003e1\u003c/sub\u003e (described earlier) and the decrease in A\u003csub\u003e1\u003c/sub\u003e peak current proportional to the charge consumed. From this result, it can be concluded that the reaction product of \u003cb\u003eACZPQ\u003c/b\u003e and \u003cem\u003ep\u003c/em\u003e-toluenesulfinic acid is not soluble in the electrolysis medium. Considering the chemical structure of the synthesized products as well as the results of electrochemical experiments, the following mechanism is proposed for the electrolysis of \u003cb\u003eCZP\u003c/b\u003e in the presence of arylsulfinic acids (\u003cb\u003eASA\u003c/b\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e shows that electrolysis of \u003cb\u003eCZP\u003c/b\u003e in the presence of arylsulfonic acids (\u003cb\u003eASA\u003c/b\u003e) via successive paired strategy leads to the synthesis of new clonazepam derivatives (\u003cb\u003eSUL\u003c/b\u003e) containing a sulfonamide moiety. The addition of this moiety to the \u003cb\u003eCZP\u003c/b\u003e certainly affects the pharmacological properties of this drug, making it a molecule with high potential for pharmaceutical and medical research. According to the proposed mechanism, at a potential of -0.72 V, \u003cb\u003eCZP\u003c/b\u003e is initially converted at the cathode to its amine derivative (\u003cb\u003eACZP\u003c/b\u003e), which is also one of its metabolites. In the next step, \u003cb\u003eACZP\u003c/b\u003e is oxidized at the anode surface and generates corresponding quinonediimine (\u003cb\u003eACZPQ\u003c/b\u003e). Quinonediimines are reactive compounds that act as electrophiles and readily react with nucleophiles.\u003csup\u003e\u003cspan additionalcitationids=\"CR32 CR33 CR34 CR35 CR36\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e This quinonediimine (\u003cb\u003eACZPQ\u003c/b\u003e) also has the same properties and reacts with arylsulfinic acids (as nucleophiles) to form the corresponding sulfonamide or clonazepam derivatives (\u003cb\u003eSUL\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eOne of the key points of this method is the use of an electrochemical successive paired strategy. As seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e, in this method, successive reduction and oxidation of \u003cb\u003eCZP\u003c/b\u003e is necessary. Such conditions can only be achieved by electrolysis in an undivided electrochemical cell. It is these features that distinguish electrochemical synthesis methods from conventional methods and allows these syntheses to be carried out in a one-pot process without the use of reducing and oxidizing reagents. On the other hand, in this strategy (in the syntheses reported in this study), since both cathodic (six-electron reduction) and anodic (two-electron oxidation) reactions contribute to the formation of the final product, it can lead to a 25% reduction in energy consumption compared to conventional electroorganic syntheses.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eDocking studies.\u003c/b\u003e The purpose of the docking study is to investigate the interaction of \u003cb\u003eSUl1\u003c/b\u003e with Gamma-aminobutyric acid (\u003cb\u003eGABA\u003c/b\u003e). Meanwhile, the structure of the \u003cb\u003eGABA\u003c/b\u003e protein has been determined by electron microscopy. The results show that \u003cb\u003eSUL1\u003c/b\u003e has a promising binding pattern with \u003cb\u003eGABA\u003c/b\u003e. This synthesized compound can hydrogen bond with the nitrogen of THR208 and also interact with other amino acids shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e. The two parameters investigated in this study are inhibition constant and binding energy. The lower inhibition constant indicates that the compound has a higher affinity for binding at the active site, which was calculated to be 13.25 \u0026micro;M in this study. Furthermore, the binding energy, which represents the binding energy to the active site, was calculated to be 6.65 kcal/mol, indicating that \u003cb\u003eSUL1\u003c/b\u003e interacts well with \u003cb\u003eGABA\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOptimization of effective parameters.\u003c/b\u003e In this section, in order to achieve higher yield and purity, optimization of effective parameters has been performed using the \"\u003cem\u003eone factor at a time\u003c/em\u003e\" optimization strategy. In this strategy, one of the factors that affects the synthesis process is changed while the other factors are kept constant. Here, the effect of electrode materials on the yield and purity of \u003cb\u003eSUL2\u003c/b\u003e has been investigated, while other factors such as \u003cb\u003eCZP\u003c/b\u003e amount (0.5 mmol), sodium 4-toluenesulfinate (\u003cb\u003eTSA\u003c/b\u003e) amount (1.0 mmol), solvent (aqueous phosphate buffer, pH 3.0, \u003cem\u003ec\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2 M)/ethanol (30/50 v/v), applied potential (\u003cem\u003eE\u003c/em\u003e\u003csub\u003eapp\u003c/sub\u003e, \u0026minus; 0.72 V versus Ag/AgCl), stirring speed and temperature have been kept constant. The results of these experiments are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. As can be seen, the best yield (95%) was obtained when both electrodes were graphite (entry 3). The results also show that changing the cathode material from graphite to metal electrodes (stainless steel, Cu or Pb) causes a decrease in yield (entries 2, 4 and 5). It seems that the porosity of the graphite surface and the greater adsorption of the drug and its incomplete reduction intermediates, and consequently the complete reduction of the nitro group (six-electron reduction) on it, is the main factor for the higher efficiency on the graphite surface. On the other hand, when the anode is not inert, such as iron (sacrificial metal) (entry 6), the cathodically generated \u003cb\u003eACZP\u003c/b\u003e is not oxidized at the anode and as a result the desired product is not formed. In entry 6, since the anodic process is the oxidation of the electrode itself (rather than the oxidation of \u003cb\u003eACZP\u003c/b\u003e), this experiment also confirms the paired strategy in the synthesis of the introduced products (\u003cb\u003eSUL\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe effect of electrode material on the yield of \u003cb\u003eSUL2\u003c/b\u003e.\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEntry\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCathode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnode\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYield [%]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGraphite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStainless steel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eStainless steel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGraphite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGraphite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGraphite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGraphite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eless than 10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLead\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGraphite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGraphite\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eno reaction\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003e \u003cb\u003eCZP\u003c/b\u003e (0.5 mmol), sodium 4-toluenesulfinate (\u003cb\u003eTSA\u003c/b\u003e) (1.0 mmol), solvent (aqueous phosphate buffer, pH 3.0, \u003cem\u003ec\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2 M)/ethanol (30/50 v/v), applied potential (\u003cem\u003eE\u003c/em\u003e\u003csub\u003eapp\u003c/sub\u003e, \u0026minus; 0.82 V versus Ag/AgCl) at room temperature.\u003c/p\u003e \u003cp\u003eThe pH of the solution also has a great impact on the yield and purity of the products. Figure\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e shows the effect of solution pH on the yield of \u003cb\u003eSUL2\u003c/b\u003e. It should be noted that other parameters were kept constant as in the previous section. The results show that the optimal pH in the synthesis of \u003cb\u003eSUL2\u003c/b\u003e is 3. As shown in the equation below, the complete reduction of the nitro group to the amine requires six protons in addition to six electrons. Therefore, acidic pH is expected to be more favorable for this reduction. On the other hand, the p\u003cem\u003eK\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e of 4-toluenesulfinic acid in water is 1.99.\u003csup\u003e41\u003c/sup\u003e This means that in strongly acidic solutions the 4-toluenesulfinate becomes protonated and its nucleophilicity decreases.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCZP\u003c/b\u003e has low solubility in water, and hence in this study an organic solvent was used as a co-solvent to improve the solubility of \u003cb\u003eCZP\u003c/b\u003e. In this section, the effect of co-solvent type on the yield of \u003cb\u003eSUL2\u003c/b\u003e is investigated. In these experiments, water (phosphate buffer, pH 3.0, \u003cem\u003ec\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2 M) (without co-solvent), water (phosphate buffer, pH 3.0, \u003cem\u003ec\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2 M)/ethanol (30/50 v/v) mixture, and water (phosphate buffer, pH 3.0, \u003cem\u003ec\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2 M)/acetonitrile (30/50 v/v mixture were used as solvents. Figure\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e shows that the highest yield was achieved when ethanol, a valuable solvent in terms of green chemistry, was used as the co-solvent.\u003c/p\u003e \u003cp\u003eHere, the effect of the ratio of organic to aqueous solvent on the yield of \u003cb\u003eSUL2\u003c/b\u003e synthesis was also investigated. For this purpose, the synthesis of \u003cb\u003eSUL2\u003c/b\u003e in water (phosphate buffer, pH 3.0, \u003cem\u003ec\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.2 M)/ethanol mixtures with ratios of 10/70 v/v, 50/30 v/v and 30/50 v/v was investigated. Figure\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e shows that the highest yield was achieved when the water to ethanol ratio was 30/50 v/v.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis paper reports a multidimensional study on \u003cb\u003eCZP\u003c/b\u003e. The main objective of this study is to synthesize new \u003cb\u003eCZP\u003c/b\u003e derivatives via a green and one-pot process by electrolysis of \u003cb\u003eCZP\u003c/b\u003e in the presence of arylsulfinic acids. The most important features of the method are: (1) Successive reduction and oxidation of \u003cb\u003eCZP\u003c/b\u003e in a one-pot process without the use of reducing and oxidizing reagents. (2) Energy savings that can lead to a 25% reduction in energy consumption compared to conventional electroorganic syntheses. (3) Selective reduction of nitro group. (4) Using a water/ethanol mixture as a solvent. (5) Using a simple cell and ordinary graphite electrodes. (6) Simple workup and synthesis at room temperature and pressure and (7) Synthesis of unique molecules with high potential in pharmaceutical and medical research.\u003c/p\u003e \u003cp\u003eOn the other hand, we performed detailed electrochemical studies on \u003cb\u003eCZP\u003c/b\u003e and its metabolite (\u003cb\u003eACZP\u003c/b\u003e) and reported information that was not reported, incomplete, or incorrectly reported for the drug and its metabolite. The results showed that \u003cb\u003eCZP\u003c/b\u003e can be reduced in two steps, in the first step the nitro group is reduced to an amine group and in the second step the C\u0026thinsp;=\u0026thinsp;N bond is reduced. To achieve the desired goal, we selectively reduced the nitro group using controlled potential electrolysis, to afford \u003cb\u003eSUL\u003c/b\u003e in excellent yield. Also, the effect of solution pH on the electrochemical behavior of the drug, especially its metabolite, was studied, and the electrochemical and acid/base characteristic of these compounds were evaluated. Using electrochemical data, we investigated the adsorption/diffusion properties of the metabolite and found that under our experimental conditions, this metabolite has no adsorption activity. The properties discovered in these experiments could be useful in designing newer derivatives of this drug and also lead to deeper insight into the evolution of drug and its metabolites in different parts of the patient's bod. Also, based on molecular docking calculations and studies, \u003cb\u003eSUL1\u003c/b\u003e has a high affinity to interact with the \u003cb\u003eGABA\u003c/b\u003e receptor.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the Bu-Ali Sina University Research Council and Center of Excellence in Development of Environmentally Friendly Methods for Chemical Synthesis (CEDEFMCS) for their support of this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDN: supervision, project administration, resources, writing-review \u0026amp; editing. ZG: Project administration, investigation \u0026amp; writing-original draft. NM: investigation, PM: investigation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKolbe, H.\u003cspan dir=\"RTL\"\u003e \u003c/span\u003eUntersuchungen \u0026uuml;ber die Elektrolyse organischer Verbindungen. \u003cem\u003eJustus Liebigs Ann. Chem\u003c/em\u003e. \u003cstrong\u003e69\u003c/strong\u003e, 257-294 (1849).\u003c/li\u003e\n\u003cli\u003eNematollahi, D., Alizadeh, S., Amani, A. \u0026amp; Khazalpour. S. \u003cem\u003ePractical Aspects of Electroorganic Synthesis\u003c/em\u003e. Elsevier, Cambridge, MA 02139, United States, 2024.\u003c/li\u003e\n\u003cli\u003eTalebi, M. R. \u0026amp; Nematollahi, D. Electrochemical synthesis of sulfonamide derivatives: electrosynthesis conditions and reaction pathways. \u003cem\u003eChemElectroChem\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e202300728 (2024).\u003c/li\u003e\n\u003cli\u003eZhu, C. Ang, N. W. 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To achieve this goal, electrochemical oxidation of this drug was carried out in the presence of arylsulfinic acid derivatives (\u003cb\u003eASA\u003c/b\u003e) in an undivided cell equipped with a graphite anode and cathode, in a water/ethanol mixture, under controlled potential conditions. In this method, new benzodiazepines were synthesized under easy workup and environmentally friendly conditions without the need for catalysts, reagents, or toxic solvents, at room temperature and pressure. In another part of this research, the electrochemical behavior of \u003cb\u003eCZP\u003c/b\u003e was thoroughly investigated and new information was reported on the electrochemical reaction mechanism, the major metabolite of \u003cb\u003eCZP\u003c/b\u003e, and the relationship between potential and pH. The proposed mechanism for the late-stage modification of \u003cb\u003eCZP\u003c/b\u003e involves the sequential reduction and oxidation of \u003cb\u003eCZP\u003c/b\u003e at the cathode and anode, respectively, followed by reaction with the nucleophile (\u003cb\u003eASA\u003c/b\u003e). In addition, docking studies were performed on the synthesized compounds and it was found that the synthesized derivatives can interact well with the gamma-aminobutyric acid agonist (\u003cb\u003eGABA\u003c/b\u003e).\u003c/p\u003e","manuscriptTitle":"Comprehensive electrochemical study of clonazepam an anticonvulsant benzodiazepine drug and its main metabolite. 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