Synthesis, crystal structure, characterization, and electrochemical properties of 5-benzoyl-6-phenyl-pyrimidin-4-one-2-thione compound; voltammetric, spectrophotometric, and molecular docking studies of its interaction with DNA

Synthesis, crystal structure, characterization, and electrochemical properties of 5-benzoyl-6-phenyl-pyrimidin-4-one-2-thione compound; voltammetric, spectrophotometric, and molecular docking studies of its interaction with DNA | 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 Research Article Synthesis, crystal structure, characterization, and electrochemical properties of 5-benzoyl-6-phenyl-pyrimidin-4-one-2-thione compound; voltammetric, spectrophotometric, and molecular docking studies of its interaction with DNA Nida Nur Adiyan, Abdulkadir LEVENT, Şerife Pınar Yalçın, Mehmet Sönmez This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6683404/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 Research on Chemical Intermediates → Version 1 posted 10 You are reading this latest preprint version Abstract A novel heterocyclic compound, 5-benzoyl-6-phenyl-pyrimidin-4-one-2-thione (PT), was synthesized from the cyclocondensation reaction of dibenzoyl acetic acid-N-carboxymethylamide with thiourea. Microanalysis, FT-IR, NMR, and API-ES mass techniques were used to characterize the structure of the pyrimidinethione compound. Cyclic voltammetric studies performed using a glassy carbon electrode in anhydrous medium revealed that the PT compound gave an irreversible reduction peak at -0.83 V, indicating electrochemical stability and irreversible redox behavior of the compound. The interaction of PT compound with DNA was investigated by spectrophotometric, voltammetric, and molecular docking methods. The interaction of PT with DNA in acetate as supporting electrolyte (pH 4.8) on a glassy carbon electrode was evaluated by the change in the anodic signal for guanine. The voltammetric analysis of the PT-DNA interaction calculated the binding free energy as -6.5 kcal/mol, while spectrophotometric studies supported the binding interactions with DNA. Molecular docking studies showed a stronger binding interaction with DNA and revealed a binding free energy of -8.12 kcal/mol. These results show that the compound performs a thermodynamically favorable binding process with DNA, and molecular docking provides important information about the binding mode and energy profile. The data obtained with all three techniques about the PT-DNA interaction supported each other, and the interaction occurred in the form of electrostatic and minor binding groove. Pyrimidine thione DNA Voltammetry Spectrophotometric Molecular docking Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Heterocyclic compounds, known to have various biological activities, have an important place in medicinal chemistry due to their therapeutic and pharmacological properties[1]. These compounds are present in many drugs, antibiotics, vitamins, natural products, and many biomolecules. Nitrogenous bases form the framework for many basic biomolecules. Pyrimidine and purine pharmacophores are the basic structural elements in molecules such as DNA and RNA, which contain genetic information and have basic components of nucleic acids, and are involved in various biological activities such as cell signaling[1]. They are widely found in nature as N-substituted sugar derivatives (nucleosides) and in vitamin B1[2]. Many researchers have published about the synthesis and properties of pyrimidine and related compounds[3–7]. When these studies are examined, pyrimidine derivatives can be synthesized by many different methods. Pyrimidine thiones, which are heterocyclic compounds, also have biological importance. The structural interactions of thioxopyrimidine derivatives with purine bases such as adenine and guanine are frequently discussed in the literature. Various studies reported that pyrimidine thione derivatives exhibit many biological activities, including antimicrobial, anti-inflammatory, antihypertensive, antidiabetic, antibacterial, antifungal, antitumor, antioxidant, and antiviral activities[8–12]. In particular, 5-fluorouracil and its modified analogue, 5-fluorothiouracil, are widely used anticancer agents. These drugs can insert into RNA and/or DNA and interfere with the maturation of nuclear RNA and/or DNA[13]. In addition, a study reported that some pyrimidine-2-thione derivatives might be used as an attractive antitumor agent with future clinical applications for the treatment of breast cancer due to their antineoplastic activity via inhibition of p-JNK protein in cancer cells[14]. As mentioned above, pyrimidine derivatives were proven to be an important structure in medicinal chemistry due to their wide biological and pharmacological activities[1,3,9,13,14]. In this study, a new thiouracil derivative, 5-benzoyl-6-phenyl-pyrimidin-4-one-2-thione (PT), was synthesized for the first time by a simple synthesis technique. The structural diversity of pyrimidine derivatives allows their interaction with biological macromolecules, and this feature makes them promising candidates for deeper investigation of their molecular mechanisms. Understanding the mechanisms by which small molecules interact with DNA has vital importance, particularly for the development of anticancer drugs[15–17]. Interactions with DNA can cause molecules to alter the structure and function of DNA through various mechanisms, such as intercalation, groove binding, or electrostatic interactions[13,16,18–21]. These interactions often affect cellular processes such as replication and transcription, forming the basis of biological activity. Molecular docking studies provide an atomic-level understanding of where and how a molecule binds to DNA. This method supports experimental data by analyzing both binding energies and specific interaction sites, providing important guidance for the design of new therapeutic agents[16,22–24]. Electrochemical techniques are the most powerful techniques for studying interactions with DNA [16,25–27]. Voltammetric studies allow evaluation of whether a molecule interacts with DNA by examining its redox behavior and of how these interactions change the redox properties of the molecule[16,28,29]. Spectrophotometric methods complete this process by revealing the binding mode of the molecule with DNA and its binding constants[30–33]. In this study, the synthesis, characterization, and electrochemical properties of a new PT compound were investigated. In addition, the interaction of the compound with DNA was evaluated using voltammetric and spectrophotometric techniques, and the binding behavior was modelled in detail at the atomic level by molecular docking studies. Molecular docking analyses provide critical information to evaluate the potential of the compound as a therapeutic agent by elucidating the specific sites where the compound binds to DNA and the binding mechanism. 2. Experimental 2.1 Apparatus and c hemicals All chemicals used in this study were obtained commercially and used without purification. Deoxyribonucleic acid from fish sperm (DNA) was purchased from Sigma-Aldrich (74782). Dibenzoyl acetic acid-N-carboxymethylamide[34] was prepared according to the method given in the literature. Elemental analysis values were recorded with the Thermo Scientific Flash 2000 brand and model elemental analyzer. 1 H and 13 C NMR spectra were recorded with the Bruker High Performance Digital FT-NMR (600 MHz) spectrometer by dissolving the compound in d6-DMSO and using TMS as an internal standard (Figure SI1 and Figure SI2). The Perkin Elmer Spectrum 100 FT-IR Spectrometer (ATR) model was used to examine the FT-IR spectrum of the compound in the range of 4000-400 cm⁻¹. The mass spectrum of 5-benzoyl-6-phenyl-pyrimidin-4-one-2-thione was obtained by atmospheric pressure ionization electrospray mass spectra (API-ES) on an LC-MS/MS ABSciex 3200 Q-trap spectrophotometer. 2.2 Synthesis of 5-benzoyl-6-phenyl-pyrimidin-4-one-2-thione For this, 1 mmol DBANMA and 1 mmol thiourea were mixed well in a 50 mL reaction flask and then subjected to a direct reaction without solvent in an oil bath at 160 °C for 25 min. The oily product obtained was first treated with dry diethyl ether to obtain a solid product and then crystallized from n-butanol and saved in a desiccator (Figure SI 3). Yield: 64%; Mp: 215-216°C. C 17 H 12 O 2 N 2 S 308.13 g/mol. Theoretical: C: 66.23; H: 3.89; N: 9.09; S: 10.40. Found: C: 66.22; H: 4.00; N: 8.97; S: 10.35%. FT-IR(ATR) v, cm -1 : 3061 (NH), 2930 (C-HAr), 1678, 1637(C=O) 1232,741 (C=S). 1 H NMR (600 MHz, DMSO) δ, ppm. 12.85 (s, 1H, NH), 12.81 (s, 1H, NH), 7.86 (d, J = 7.3 Hz, 2H, aromatic proton), 7.57 (t, J = 7.4 Hz, 1H, aromatic proton), 7.42 (t, J = 7.7 Hz, 2H, aromatic proton), 7.34 (tt, J = 14.8, 7.4 Hz, 5H, aromatic proton). 13 C NMR (151 MHz, DMSO) δ ppm. 192.40 (C=O) benzoyl), 176.45 (C=S), 159.97 (C=O) pyrimidine, 152.74, 137.29, 134.19, 131.09, 130.91, 129.63, 129.15, 128.93, 128.67, 116.31 (10 aromatic carbons). Mass: Calculated Mass = 308.13, Observed m/z = 306.85 [M] + mode. 2.3 Voltammetric method Electrochemical properties of PT were investigated using the Autolab PGSTAT 128N potentiostat (Netherlands) and a triple cell stand (BASi). Glassy carbon (GC, Φ: 3 mm, BASi), Ag/AgCl (BASi), and Pt (BASi) wires were used as the working electrode, reference electrode, and auxiliary electrode in voltammetric experiments, respectively. For experiments, 1 mM PT was prepared in DMSO (supporting electrolyte solution containing 0.1 M tetrabutylammonium perchlorate (TBAP)). Before each voltammetric analysis, the surface of the GC electrode was cleaned manually. In studies in an anhydrous environment, pure nitrogen gas was passed through the solution for 10 min before the analyses. Voltammetric measurements of PT in aqueous medium: Cyclic voltammograms were recorded in the potential range of -1.2 V to 1.0 V in DMSO medium containing 1 mM PT and 0.1 M TBAP on the GC electrode. DNA immobilization on the GC electrode surface: For this, 25 mg/L DNA was immobilized on the GC electrode in acetate buffer (ABS, pH 4.80, containing 0.02 M NaCl) medium at a voltage of +0.50 V for 120 seconds. Interaction with PT-DNA: After DNA was immobilized on the GC electrode surface, 1 mg/L PT was left in ABS (pH 4.80 containing 0.02 M NaCl) medium for periods ranging from 5 to 150 seconds. Voltammetric measurement: After the interaction of PT with DNA, voltammograms were recorded in the voltage range of +0.5 V to +1.40 V using the differential pulse technique. 2.4 Spectrophotometric technique The spectra for PT, DNA, and PT-DNA interaction were recorded in the wavelength range of 200 nm to 800 nm with a Shimadzu Pharma Spec UV-1900 spectrophotometer. Spectral measurements of the compounds were carried out in ABS (pH 4.8) medium. 2.5. Docking measurements The two-dimensional structure of PT was drawn with the ChemDraw Ultra program and then converted to pdb files. The 3-dimensional crystal structure of DNA (PDB ID: 1BNA) was obtained from the RCSB database (http://www.rcsb.org/pdb). DNA and grid preparations were made with the help of the AutoDock_vina_1.1.2 program. In this process, water molecules in the DNA file were deleted. After the polar hydrogen atoms were added, Gustier charging was performed. The docking file for the PT compound was prepared in the same way, and molecular docking operations were performed with the AutoDock_vina_1.1.2 program[22]. DNA was placed in AutoDock Tool 4.2 by creating a PDBQT file containing a protein with hydrogens in all polar groups. The grid area of 14.74, 20.98, and 8.80 dimensions was created for the docking site on the target DNA. The Discovery Studio 4.0 program processor was used to create hydrogen bonds and hydrophobic interactions in the PT-DNA interaction. 3. Results and Discussion 3.1 Synthesis and characterization of 5-benzoyl-6-phenyl-pyrimidin-4-one-2-thione compound In this study, for the first time, PT compound was obtained by the reaction of dibenzoyl acetic acid-N-carboxymethyl amide and thiourea in a solvent-free environment at 160 °C in an oil bath for 25 min, as stated in the synthesis section. The synthesized PT compound was purified by crystallization from n-BuOH after washing several times in methanol. Elemental analysis, FT-IR, 1H/13C NMR (Figure SI 2 and 3) and LC-MS/MS spectral values were used for the characterization of the compound. When the FT-IR spectrum of the PT compound was examined, as expected, bands belonging to NH vibrations at 3060 cm⁻¹ and C=O vibrations at 1678 and 1637 cm⁻¹ were observed [14]. Again, vibrations belonging to the thione group were observed at 1237 and 741 cm⁻¹, supporting the synthesized structure [15]. As shown in Figures SI 2 and 3, the 1 H NMR and 13 C NMR spectra of the compound were examined in d6-DMSO at room temperature, respectively. The 1 H NMR spectrum of the pyrimidine compound showed signals as singlets at δ = 12.81 and 12.85 ppm, corresponding to NH signals. Signals corresponding to aromatic protons appeared between δ = 7.34–7.82 ppm. The 13 C NMR spectrum showed characteristic signals at 192.40, 176.45, and 159.97 ppm due to O=C–Ar, C=S, pyrimidine, and C=O, pyrimidine ring of the compound, respectively [16]. It showed 10 peaks in the region of 152.74–116.31 ppm due to other aromatic carbon atoms. All analytical and spectroscopic data obtained support the proposed structure. 3.2 Cyclic voltammetry results of 5-benzoyl-6-phenyl-pyrimidin-4-one-2-thione compound on GCE Cyclic voltammetry results of the PT compound on GCE in anhydrous medium (0.1 M TBAP) are given in Figure 1. In Figure 1a, an irreversible cathodic peak was observed around -0.83 V in three-cycle CV curves at a 100 mV/s voltage scan rate. A higher current density is observed in the first cycle, which indicates that there is adsorption or chemical transformation on the electrode surface. The decrease in current density after the first cycle can be attributed to reasons such as the binding of the compound to the electrode surface or the formation of a surface film. How the reaction kinetics change with increasing scan rates is clearly observed. At low rates, the reaction is more balanced, but at high rates, kinetic and diffusion effects become dominant. In addition, the cathodic peak voltage value shifted to more negative values as the voltage scan rate increased (Figure 1b). The linear relationship between the cathodic peak current value, the voltage scan rate and the slope value between the logarithm of the cathodic peak current value and the voltage scan rate logarithm (0.678) were calculated between 0.5 and 1. These results show that PT has a diffusion-assisted adsorption feature on GCE under these study conditions. 3.3 Voltammetric results for the interaction of PT compound with DNA Voltammetric studies of the PT-DNA interaction were carried out as stated in section 2.3. In these studies, firstly, the conditions were identified under which the anodic signal intensity of the guanine base on GCE of DNA was most sensitive. For this purpose, researchers generally use the DPV technique as a voltammetric technique in drug-DNA interaction studies[16,28]. Thus, optimization experiments were carried out for the guanine anodic signal. Deposition voltage (0 V to +0.6 V) and deposition time (0 seconds to 210 seconds) for the guanine signal in ABS supporting electrolyte (pH 4.8, containing 0.02 M NaCl) were studied. As a result of the experiments, the most sensitive signals were obtained at 120 seconds deposition time and +0.5 V deposition voltage values on GCE (Figure 2, black line). Under these study conditions, a 65% increase in guanine anodic signal intensity and a positive change in anodic voltage value were observed in the interaction of the PT compound with DNA (Figure 2, dashed red line). In this part of the study, DPV curves were recorded between 5 seconds and 150 seconds in order to determine the maximum interaction time for the interaction of DNA with PT (Figure 3). The anodic signal intensity in the guanine base of DNA decreases as the interaction time increases. In this process, a decrease in the anodic signal intensity was detected due to the complex structure formed between DNA and PT. There was no significant change in the anodic signal intensity after 90 s due to the interaction between DNA and PT. Thus, the interaction time was accepted as 90 s unless otherwise stated in the following parts of the analytical study. In the optimum conditions of this study, DPV curves were recorded by adding PT between 0.25 mg/L and 1.25 mg/L while keeping the DNA concentration constant at 25 mg/L in ABS (pH 4.8, containing 0.02 M NaCl) medium (Figure 4). As can be seen from the recorded DPV curves, a decrease in the anodic signal intensity was observed due to the PT-DNA complex compound formed after each PT addition. Analytically, there was no significant change after the addition of 1.25 mg/L PT. The anodic peak voltage value shifted to a more positive region due to the PT-DNA interaction. The interaction of PT with DNA has great importance in terms of determining the binding constant. This binding constant shows how effectively PT enters and binds to the target cells of DNA. As a result of this interaction, the formation of a PT + DNA ↔ PT-DNA complex structure was evaluated as a simple model. The binding constant was calculated from the recorded PT-DNA interaction curves according to the following equation[35]. In the equation, K represents the binding constant, I DNA guanine peak current and I (PT–DNA) represents the guanine peak current measured after the interaction of PT with DNA. The binding constant K of this complex was found using the value where the y-axis intersects the curve obtained by plotting log(1/[PT]) against log(I PT–DNA /I DNA −I( PT–DNA) ) values. The Kb constant between PT and DNA was found to be 1.05×10 +5 . The free energy of binding was calculated as ΔG = -RTInKb, where ΔG=-6.50 kcal/mol. The fact that AG<0 indicates that the PT-DNA complex structure interaction can occur spontaneously on the GCE surface under these working conditions. As can be seen from this interaction, the PT compound probably interacted with DNA via electrostatic interaction or intercalation[36]. In this process, the charge transfer force of the PT compound can intervene between the helical structures of DNA and deform the DNA. 3.4 Spectrophotometric results for PT-DNA interaction Firstly, the spectra were recorded for the PT compound in the wavelength range of 200 nm to 800 nm (Figure 5, red dashed line). PT gave a well-defined maximum absorption band at 274 nm and a very broad-spectrum wave at approximately 325 nm. In the interaction experiments of PT with DNA, the spectra for the complex compound formed after adding 0.5 mg/L to 4 mg/L DNA to the PT solution were recorded in the wavelength range of 200 nm to 800 nm in ABS (pH 4.8) medium (Figure 5). In these experiments, no analytical change was observed in the spectral band observed at approximately 325 nm wavelength after each successive addition of DNA solution. However, in the absorption band observed at 274 nm wavelength, there was an increase in absorbance intensity (hyperchromic shift) and a shift in wavelength towards 269 nm (hypochromic shift) due to the complex compound structure formed after each DNA addition. The increase in absorption during the interaction of PT with DNA indicates that the local structure of DNA is disrupted or base pairs are exposed. This is usually associated with denaturation of DNA or binding of PT to major/minor grooves of DNA. At the same time, hypochromism is caused by the overlapping of π electrons due to the insertion of functional groups from PT between DNA base pairs (intercalation), and these data are also consistent with molecular docking data[37,38]. In addition, the shift in wavelength, the change in absorption intensity, and the isobestic point observed at 325 nm wavelength indicate that a new complex structure formed as a result of the interaction of the PT compound with DNA. The binding constant (K) of this complex compound was calculated using the Benesi-Hildebrand equation [39]. Here A 0 is the absorbance of PT, A is the absorbance of PT-DNA complex, ℰ is the PT absorption coefficient, and ℰ H-G is the absorption coefficient of the PT-DNA complex. When the necessary calculations were made, K=1.19x105 M 1- was found. By using the PT-DNA complex formation constant, the binding free energy (ΔG) was calculated as ∆G=-6.35 kcal/mol. These results show that the interaction with PT-DNA causes the hypochromism effect in the absorption spectrum of DNA[19,37]. These interactions show that the compound binds to DNA via intercalation and groove binding, which is consistent with the molecular docking results. 3.5 Molecular docking results for PT-DNA interaction Molecular docking studies are a powerful computational technique used to understand and model interactions between biomolecules. This technique provides useful preliminary information, especially for studies in the field of drug design. Molecular docking is used to model how potential drug candidates will bind to biomolecules such as target proteins, DNA, or RNA. The evaluation of the obtained data contributes to the understanding of target-drug interactions in the drug development process. As a result of docking studies, whether biomolecular targets are suitable for drug development can be checked. The molecular docking results for the PT-DNA interaction are given in Figure 6. In this computational simulation, two strong interactions were observed. DNA DG B:14 nucleotides made conventional hydrogen bonds with the oxygen in the pyrimidine carbon at an average distance of 2.56 Å and with the oxygen at the benzoyl carbon at an average distance of 3.28 Å. At the same time, DC A:11 nucleotide interacted with the benzoyl phenyl ring and formed a Pi-donor hydrogen bond[40]. These interactions were connected with a binding affinity of -8.12 kcal/mol. The molecular docking binding affinity is compatible with both voltammetric and spectrophotometric results. The PT-DNA interaction appears to involve electrostatic and minor groove binding. 4. Conclusion In this study, we synthesized a new heterocyclic compound PT, and its structural characterization was successfully carried out by methods such as microanalysis, FT-IR, NMR, and API-ES. Voltammetric studies using GCE showed that PT gave an irreversible reduction peak at -0.83 V, which revealed the electrochemical stability of the compound and its irreversible redox behavior. Interaction studies with DNA showed that PT had a strong and thermodynamically favorable binding process with DNA. Voltammetric analysis results calculated the binding free energy as -6.5 kcal/mol, and the results obtained from spectrophotometric and molecular docking studies supported these findings. Molecular docking data determined the binding free energy as -8.12 kcal/mol and revealed that PT interacted with DNA electrostatically and via the minor binding groove. The findings demonstrate the potential of PT to interact with biomolecules and particularly its ability to bind with DNA, suggesting that compounds synthesized based on PT may be candidates for therapeutic and biomedical applications in the future. Declarations All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. Author contributions Nida Nur Adiyan: Conceptualization, Software, Writing - original draft. Abdulkadir Levent: Methodology, Validation, Writing - original draft, Writing - review & editing, Supervision. Mehmet Sönmez: Data curation, Methodology, Investigation, Writing - original draft. All authors read and approved the final version of the manuscript. Data availability The data that support the findings of this study will not be made publicly available. Ethical approval Safety data retrieved from the spontaneous reporting process are anonymous and concur with ethical standards. Therefore, there was no further requirement for the ethical measure. Conflict of interest The authors declare no conflict of interests. References F.O. Sefrji, A.F. 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Gupta, A new epirubicin biosensor based on amplifying DNA interactions with polypyrrole and nitrogen-doped reduced graphene: Experimental and docking theoretical investigations, Sensors and Actuators, B: Chemical. 284 (2019) 568–574. https://doi.org/10.1016/j.snb.2018.12.164. C. Erkmen, B. Bozal-Palabiyik, H. Tayyab, M.Z. Kabir, S.B. Mohamad, B. Uslu, Exploring molecular interaction of cefpirome with human serum albumin: In vitro and in silico approaches, Journal of Molecular Structure. 1275 (2023) 134723. https://doi.org/10.1016/j.molstruc.2022.134723. S. Rauf, J.J. Gooding, K. Akhtar, M.A. Ghauri, M. Rahman, M.A. Anwar, A.M. Khalid, Electrochemical approach of anticancer drugs-DNA interaction, Journal of Pharmaceutical and Biomedical Analysis. 37 (2005) 205–217. https://doi.org/10.1016/j.jpba.2004.10.037. R. Nimal, D. Nur Unal, C. Erkmen, B. Bozal-Palabiyik, M. Siddiq, G. Eren, A. Shah, B. 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Belyakova, Electrochemical DNA sensors for drug determination, Journal of Pharmaceutical and Biomedical Analysis. 221 (2022) 115058. https://doi.org/10.1016/j.jpba.2022.115058. B. Duman, C. Erkmen, M. Zahirul Kabir, L. Ching Yi, S.B. Mohamad, B. Uslu, In vitro interactions of two pesticides, propazine and quinoxyfen with bovine serum albumin: Spectrofluorometric and molecular docking investigations, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 300 (2023) 122907. https://doi.org/10.1016/j.saa.2023.122907. G. Rabbani, M.H. Baig, E.J. Lee, W.-K. Cho, J.Y. Ma, I. Choi, Biophysical Study on the Interaction between Eperisone Hydrochloride and Human Serum Albumin Using Spectroscopic, Calorimetric, and Molecular Docking Analyses, Molecular Pharmaceutics. 14 (2017) 1656–1665. https://doi.org/10.1021/acs.molpharmaceut.6b01124. N. Shahabadi, N. Fatahi, M. Mahdavi, Z.K. Nejad, M. Pourfoulad, Multispectroscopic studies of the interaction of calf thymus DNA with the anti-viral drug, valacyclovir, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 83 (2011) 420–424. https://doi.org/10.1016/j.saa.2011.08.056. L. Wang, Y. Wu, T. Chen, C. Wei, The interactions of phenanthroline compounds with DNAs: Preferential binding to telomeric quadruplex over duplex, International Journal of Biological Macromolecules. 52 (2013) 1–8. https://doi.org/10.1016/j.ijbiomac.2012.08.015. W.M.F. Fabian, G. Kollenz, Y. Akcamur, T.R. K�k, M. Teczan, M. Akkurt, W. Hiller, Reaktionen cyclischer Oxalylverbindungen, 34. Mitt.: Synthese von Dibenzoylacet-N-carboxyalkylamiden und semiempirische Rechnungen zur Keto-Enol Tautomerie, Monatshefte F�r Chemie Chemical Monthly. 123 (1992) 265–275. https://doi.org/10.1007/BF00810475. D.E. Bayraktepe, A voltammetric study on drug-DNA interactions: Kinetic and thermodynamic aspects of the relations between the anticancer agent dasatinib and ds-DNA using a pencil lead graphite electrode, Microchemical Journal. 157 (2020) 104946. https://doi.org/10.1016/j.microc.2020.104946. M. Aslan, F. Aydın, A. Levent, Voltammetric studies and spectroscopic investigations of the interaction of an anticancer drug bevacizumab-DNA and analytical applications of disposable pencil graphite sensor, Talanta. 265 (2023) 124893. https://doi.org/10.1016/j.talanta.2023.124893. Y. Song, D. Zhong, J. Luo, H. Tan, S. Chen, P. Li, L. Wang, T. Wang, Binding characteristics and interactive region of 2‐phenylpyrazolo[1,5‐ c ]quinazoline with DNA, Luminescence. 29 (2014) 1141–1147. https://doi.org/10.1002/bio.2674. P. Singla, V. Luxami, R. Singh, V. Tandon, K. Paul, Novel pyrazolo[3,4-d]pyrimidine with 4-(1H-benzimidazol-2-yl)-phenylamine as broad spectrum anticancer agents: Synthesis, cell based assay, topoisomerase inhibition, DNA intercalation and bovine serum albumin studies, European Journal of Medicinal Chemistry. 126 (2017) 24–35. https://doi.org/10.1016/j.ejmech.2016.09.093. A. Wolfe, G.H. Shimer, T. Meehan, Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA, Biochemistry. 26 (1987) 6392–6396. https://doi.org/10.1021/bi00394a013. A.R. Nekoei, M. Vatanparast, π-Hydrogen bonding and aromaticity: A systematic interplay study, Physical Chemistry Chemical Physics. 21 (2019) 623–630. https://doi.org/10.1039/c8cp07003b. Additional Declarations No competing interests reported. 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Dashed line: 0.1 M TBAP supporting electrolyte\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6683404/v1/aa3691adaba8995cbb078d6e.png"},{"id":83830954,"identity":"a1623e28-23cc-4572-a18c-91ffe7086f57","added_by":"auto","created_at":"2025-06-03 11:50:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":88726,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential pulse voltammograms recorded in ABS (pH 4.8, containing 0.02 M NaCl) supporting electrolyte medium for PT-DNA interaction\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6683404/v1/61b4111ddd68070e416359d9.png"},{"id":83830956,"identity":"ed6f5d8b-28a4-4f6d-8297-c1d4fbfe4a3b","added_by":"auto","created_at":"2025-06-03 11:50:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":169973,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential pulse voltammograms recorded for PT-DNA interaction time\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6683404/v1/eef4f0458616ec0bae4c7530.png"},{"id":83830935,"identity":"cf35daaf-c11d-4587-95e3-d9bc0dcdc8f9","added_by":"auto","created_at":"2025-06-03 11:42:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":132522,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential pulse voltammograms recorded in ABS (pH 4.8, containing 0.02 M NaCl) supporting electrolyte medium using GCE for PT-DNA interaction\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6683404/v1/4aa756a7c8909a90042fba0f.png"},{"id":83830940,"identity":"056bdf01-837b-4d33-8340-8df56412fbaa","added_by":"auto","created_at":"2025-06-03 11:42:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":143784,"visible":true,"origin":"","legend":"\u003cp\u003eSpectrophotometric spectra recorded for the interaction of PT with DNA.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6683404/v1/31261acf39a0b5cb41710184.png"},{"id":83830942,"identity":"14eb9666-ec9d-41ee-8d1f-0b0ed1730074","added_by":"auto","created_at":"2025-06-03 11:42:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":343034,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking images and results recorded for PT-DNA interaction\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6683404/v1/a486d7383a19d3ce9ea4bfe5.png"},{"id":87756779,"identity":"58609a37-904c-45e3-aa9a-868795fc7e71","added_by":"auto","created_at":"2025-07-28 16:09:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1588241,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6683404/v1/8fcdcc65-b8db-43b9-b657-938d92a836cb.pdf"},{"id":83830939,"identity":"c902bb36-df56-480c-86bf-8dffeca1f43b","added_by":"auto","created_at":"2025-06-03 11:42:26","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":97253,"visible":true,"origin":"","legend":"","description":"","filename":"SI17.05.2025.docx","url":"https://assets-eu.researchsquare.com/files/rs-6683404/v1/b706343b2e9c9dff9a2a49b4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis, crystal structure, characterization, and electrochemical properties of 5-benzoyl-6-phenyl-pyrimidin-4-one-2-thione compound; voltammetric, spectrophotometric, and molecular docking studies of its interaction with DNA","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHeterocyclic compounds, known to have various biological activities, have an important place in medicinal chemistry due to their therapeutic and pharmacological properties[1]. These compounds are present in many drugs, antibiotics, vitamins, natural products, and many biomolecules. Nitrogenous bases form the framework for many basic biomolecules. Pyrimidine and purine pharmacophores are the basic structural elements in molecules such as DNA and RNA, which contain genetic information and have basic components of nucleic acids, and are involved in various biological activities such as cell signaling[1]. They are widely found in nature as N-substituted sugar derivatives (nucleosides) and in vitamin B1[2]. Many researchers have published about the synthesis and properties of pyrimidine and related compounds[3\u0026ndash;7]. When these studies are examined, pyrimidine derivatives can be synthesized by many different methods. Pyrimidine thiones, which are heterocyclic compounds, also have biological importance. The structural interactions of thioxopyrimidine derivatives with purine bases such as adenine and guanine are frequently discussed in the literature. Various studies reported that pyrimidine thione derivatives exhibit many biological activities, including antimicrobial, anti-inflammatory, antihypertensive, antidiabetic, antibacterial, antifungal, antitumor, antioxidant, and antiviral activities[8\u0026ndash;12]. In particular, 5-fluorouracil and its modified analogue, 5-fluorothiouracil, are widely used anticancer agents. These drugs can insert into RNA and/or DNA and interfere with the maturation of nuclear RNA and/or DNA[13]. In addition, a study reported that some pyrimidine-2-thione derivatives might be used as an attractive antitumor agent with future clinical applications for the treatment of breast cancer due to their antineoplastic activity via inhibition of p-JNK protein in cancer cells[14].\u003c/p\u003e\n\u003cp\u003eAs mentioned above, pyrimidine derivatives were proven to be an important structure in medicinal chemistry due to their wide biological and pharmacological activities[1,3,9,13,14]. In this study, a new thiouracil derivative, 5-benzoyl-6-phenyl-pyrimidin-4-one-2-thione (PT), was synthesized for the first time by a simple synthesis technique. The structural diversity of pyrimidine derivatives allows their interaction with biological macromolecules, and this feature makes them promising candidates for deeper investigation of their molecular mechanisms.\u003c/p\u003e\n\u003cp\u003eUnderstanding the mechanisms by which small molecules interact with DNA has vital importance, particularly for the development of anticancer drugs[15\u0026ndash;17]. Interactions with DNA can cause molecules to alter the structure and function of DNA through various mechanisms, such as intercalation, groove binding, or electrostatic interactions[13,16,18\u0026ndash;21]. These interactions often affect cellular processes such as replication and transcription, forming the basis of biological activity. Molecular docking studies provide an atomic-level understanding of where and how a molecule binds to DNA. This method supports experimental data by analyzing both binding energies and specific interaction sites, providing important guidance for the design of new therapeutic agents[16,22\u0026ndash;24].\u003c/p\u003e\n\u003cp\u003eElectrochemical techniques are the most powerful techniques for studying interactions with DNA [16,25\u0026ndash;27]. Voltammetric studies allow evaluation of whether a molecule interacts with DNA by examining its redox behavior and of how these interactions change the redox properties of the molecule[16,28,29]. Spectrophotometric methods complete this process by revealing the binding mode of the molecule with DNA and its binding constants[30\u0026ndash;33]. In this study, the synthesis, characterization, and electrochemical properties of a new PT compound were investigated. In addition, the interaction of the compound with DNA was evaluated using voltammetric and spectrophotometric techniques, and the binding behavior was modelled in detail at the atomic level by molecular docking studies. Molecular docking analyses provide critical information to evaluate the potential of the compound as a therapeutic agent by elucidating the specific sites where the compound binds to DNA and the binding mechanism.\u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cp\u003e\u003cstrong\u003e2.1 Apparatus and c\u003c/strong\u003e\u003cstrong\u003ehemicals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll chemicals used in this study were obtained commercially and used without purification. Deoxyribonucleic acid from fish sperm (DNA) was purchased from Sigma-Aldrich (74782). Dibenzoyl acetic acid-N-carboxymethylamide[34] was prepared according to the method given in the literature. Elemental analysis values were recorded with the Thermo Scientific Flash 2000 brand and model elemental analyzer. \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectra were recorded with the Bruker High Performance Digital FT-NMR (600 MHz) spectrometer by dissolving the compound in d6-DMSO and using TMS as an internal standard (Figure SI1 and Figure SI2). The Perkin Elmer Spectrum 100 FT-IR Spectrometer (ATR) model was used to examine the FT-IR spectrum of the compound in the range of 4000-400 cm⁻¹. The mass spectrum of 5-benzoyl-6-phenyl-pyrimidin-4-one-2-thione was obtained by atmospheric pressure ionization electrospray mass spectra (API-ES) on an LC-MS/MS ABSciex 3200 Q-trap spectrophotometer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Synthesis of 5-benzoyl-6-phenyl-pyrimidin-4-one-2-thione\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor this, 1 mmol DBANMA and 1 mmol thiourea were mixed well in a 50 mL reaction flask and then subjected to a direct reaction without solvent in an oil bath at 160 °C for 25 min. The oily product obtained was first treated with dry diethyl ether to obtain a solid product and then crystallized from n-butanol and saved in a desiccator (Figure SI 3). Yield: 64%; Mp: 215-216°C. C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eS 308.13 g/mol. Theoretical: C: 66.23; H: 3.89; N: 9.09; S: 10.40. Found: C: 66.22; H: 4.00; N: 8.97; S: 10.35%. FT-IR(ATR) v, cm\u003csup\u003e-1\u003c/sup\u003e: 3061 (NH), 2930 (C-HAr), 1678, 1637(C=O) 1232,741 (C=S). \u003csup\u003e1\u003c/sup\u003eH NMR (600 MHz, DMSO) δ, ppm. 12.85 (s, 1H, NH), 12.81 (s, 1H, NH), 7.86 (d, J = 7.3 Hz, 2H, aromatic proton), 7.57 (t, J = 7.4 Hz, 1H, aromatic proton), 7.42 (t, J = 7.7 Hz, 2H, aromatic proton), 7.34 (tt, J = 14.8, 7.4 Hz, 5H, aromatic proton). \u003csup\u003e13\u003c/sup\u003eC NMR (151 MHz, DMSO) δ ppm. 192.40 (C=O) benzoyl), 176.45 (C=S), 159.97 (C=O) pyrimidine, 152.74, 137.29, 134.19, 131.09, 130.91, 129.63, 129.15, 128.93, 128.67, 116.31 (10 aromatic carbons). Mass: Calculated Mass = 308.13, Observed m/z = 306.85 [M]\u003csup\u003e+\u003c/sup\u003e mode.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Voltammetric method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eElectrochemical properties of PT were investigated using the Autolab PGSTAT 128N potentiostat (Netherlands) and a triple cell stand (BASi). Glassy carbon (GC, Φ: 3 mm, BASi), Ag/AgCl (BASi), and Pt (BASi) wires were used as the working electrode, reference electrode, and auxiliary electrode in voltammetric experiments, respectively. For experiments, 1 mM PT was prepared in DMSO (supporting electrolyte solution containing 0.1 M tetrabutylammonium perchlorate (TBAP)). Before each voltammetric analysis, the surface of the GC electrode was cleaned manually. In studies in an anhydrous environment, pure nitrogen gas was passed through the solution for 10 min before the analyses.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eVoltammetric measurements of PT in aqueous medium:\u003c/em\u003e Cyclic voltammograms were recorded in the potential range of -1.2 V to 1.0 V in DMSO medium containing 1 mM PT and 0.1 M TBAP on the GC electrode.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDNA immobilization on the GC electrode surface:\u003c/em\u003e For this, 25 mg/L DNA was immobilized on the GC electrode in acetate buffer (ABS, pH 4.80, containing 0.02 M NaCl) medium at a voltage of +0.50 V for 120 seconds.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eInteraction with PT-DNA:\u003c/em\u003e After DNA was immobilized on the GC electrode surface, 1 mg/L PT was left in ABS (pH 4.80 containing 0.02 M NaCl) medium for periods ranging from 5 to 150 seconds.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eVoltammetric measurement:\u003c/em\u003e After the interaction of PT with DNA, voltammograms were recorded in the voltage range of +0.5 V to +1.40 V using the differential pulse technique.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Spectrophotometric technique\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe spectra for PT, DNA, and PT-DNA interaction were recorded in the wavelength range of 200 nm to 800 nm with a Shimadzu Pharma Spec UV-1900 spectrophotometer. Spectral measurements of the compounds were carried out in ABS (pH 4.8) medium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Docking measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe two-dimensional structure of PT was drawn with the ChemDraw Ultra program and then converted to pdb files. The 3-dimensional crystal structure of DNA (PDB ID: 1BNA) was obtained from the RCSB database (http://www.rcsb.org/pdb). DNA and grid preparations were made with the help of the AutoDock_vina_1.1.2 program. In this process, water molecules in the DNA file were deleted. After the polar hydrogen atoms were added, Gustier charging was performed. The docking file for the PT compound was prepared in the same way, and molecular docking operations were performed with the AutoDock_vina_1.1.2 program[22]. DNA was placed in AutoDock Tool 4.2 by creating a PDBQT file containing a protein with hydrogens in all polar groups. The grid area of 14.74, 20.98, and 8.80 dimensions was created for the docking site on the target DNA. The Discovery Studio 4.0 program processor was used to create hydrogen bonds and hydrophobic interactions in the PT-DNA interaction.\u003c/p\u003e"},{"header":"3. Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1 Synthesis and characterization of 5-benzoyl-6-phenyl-pyrimidin-4-one-2-thione compound\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, for the first time, PT compound was obtained by the reaction of dibenzoyl acetic acid-N-carboxymethyl amide and thiourea in a solvent-free environment at 160 °C in an oil bath for 25 min, as stated in the synthesis section. The synthesized PT compound was purified by crystallization from n-BuOH after washing several times in methanol. Elemental analysis, FT-IR, 1H/13C NMR (Figure SI 2 and 3) and LC-MS/MS spectral values were used for the characterization of the compound. When the FT-IR spectrum of the PT compound was examined, as expected, bands belonging to NH vibrations at 3060 cm⁻¹ and C=O vibrations at 1678 and 1637 cm⁻¹ were observed [14]. Again, vibrations belonging to the thione group were observed at 1237 and 741 cm⁻¹, supporting the synthesized structure [15].\u003c/p\u003e\n\u003cp\u003eAs shown in Figures SI 2 and 3, the \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR spectra of the compound were examined in d6-DMSO at room temperature, respectively. The \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of the pyrimidine compound showed signals as singlets at δ = 12.81 and 12.85 ppm, corresponding to NH signals. Signals corresponding to aromatic protons appeared between δ = 7.34–7.82 ppm. The \u003csup\u003e13\u003c/sup\u003eC NMR spectrum showed characteristic signals at 192.40, 176.45, and 159.97 ppm due to O=C–Ar, C=S, pyrimidine, and C=O, pyrimidine ring of the compound, respectively [16]. It showed 10 peaks in the region of 152.74–116.31 ppm due to other aromatic carbon atoms. All analytical and spectroscopic data obtained support the proposed structure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Cyclic voltammetry results of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e5-benzoyl-6-phenyl-pyrimidin-4-one-2-thione compound\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;on GCE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCyclic voltammetry results of the PT compound on GCE in anhydrous medium (0.1 M TBAP) are given in Figure 1. In Figure 1a, an irreversible cathodic peak was observed around -0.83 V in three-cycle CV curves at a 100 mV/s voltage scan rate. A higher current density is observed in the first cycle, which indicates that there is adsorption or chemical transformation on the electrode surface. The decrease in current density after the first cycle can be attributed to reasons such as the binding of the compound to the electrode surface or the formation of a surface film. How the reaction kinetics change with increasing scan rates is clearly observed. At low rates, the reaction is more balanced, but at high rates, kinetic and diffusion effects become dominant. In addition, the cathodic peak voltage value shifted to more negative values as the voltage scan rate increased (Figure 1b). The linear relationship between the cathodic peak current value, the voltage scan rate and the slope value between the logarithm of the cathodic peak current value and the voltage scan rate logarithm (0.678) were calculated between 0.5 and 1. These results show that PT has a diffusion-assisted adsorption feature on GCE under these study conditions.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e3.3 Voltammetric results for the interaction of PT compound with DNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVoltammetric studies of the PT-DNA interaction were carried out as stated in section 2.3. In these studies, firstly, the conditions were identified under which the anodic signal intensity of the guanine base on GCE of DNA was most sensitive. For this purpose, researchers generally use the DPV technique as a voltammetric technique in drug-DNA interaction studies[16,28]. Thus, optimization experiments were carried out for the guanine anodic signal. Deposition voltage (0 V to +0.6 V) and deposition time (0 seconds to 210 seconds) for the guanine signal in ABS supporting electrolyte (pH 4.8, containing 0.02 M NaCl) were studied. As a result of the experiments, the most sensitive signals were obtained at 120 seconds deposition time and +0.5 V deposition voltage values on GCE (Figure 2, black line). Under these study conditions, a 65% increase in guanine anodic signal intensity and a positive change in anodic voltage value were observed in the interaction of the PT compound with DNA (Figure 2, dashed red line).\u003c/p\u003e\n\u003cp\u003eIn this part of the study, DPV curves were recorded between 5 seconds and 150 seconds in order to determine the maximum interaction time for the interaction of DNA with PT (Figure 3). The anodic signal intensity in the guanine base of DNA decreases as the interaction time increases. In this process, a decrease in the anodic signal intensity was detected due to the complex structure formed between DNA and PT. There was no significant change in the anodic signal intensity after 90 s due to the interaction between DNA and PT. Thus, the interaction time was accepted as 90 s unless otherwise stated in the following parts of the analytical study.\u003c/p\u003e\n\u003cp\u003eIn the optimum conditions of this study, DPV curves were recorded by adding PT between 0.25 mg/L and 1.25 mg/L while keeping the DNA concentration constant at 25 mg/L in ABS (pH 4.8, containing 0.02 M NaCl) medium (Figure 4). As can be seen from the recorded DPV curves, a decrease in the anodic signal intensity was observed due to the PT-DNA complex compound formed after each PT addition. Analytically, there was no significant change after the addition of 1.25 mg/L PT. The anodic peak voltage value shifted to a more positive region due to the PT-DNA interaction. The interaction of PT with DNA has great importance in terms of determining the binding constant. This binding constant shows how effectively PT enters and binds to the target cells of DNA. As a result of this interaction, the formation of a PT + DNA \u0026harr; PT-DNA complex structure was evaluated as a simple model. The binding constant was calculated from the recorded PT-DNA interaction curves according to the following equation[35].\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" style=\"width: 395px; height: 59.7936px;\" width=\"395\" height=\"59.7936\"\u003e\u003c/p\u003e\n\u003cp\u003eIn the equation, K represents the binding constant, I\u003cem\u003e\u003csub\u003eDNA\u003c/sub\u003e\u003c/em\u003e guanine peak current and I\u003cem\u003e\u003csub\u003e(PT\u0026ndash;DNA)\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003erepresents the guanine peak current measured after the interaction of PT with DNA. The binding constant K of this complex was found using the value where the y-axis intersects the curve obtained by plotting log(1/[PT]) against log(I\u003csub\u003ePT\u0026ndash;DNA\u003c/sub\u003e/I\u003csub\u003eDNA\u003c/sub\u003e\u0026minus;I(\u003csub\u003ePT\u0026ndash;DNA)\u003c/sub\u003e) values. The Kb constant between PT and DNA was found to be 1.05\u0026times;10\u003csup\u003e+5\u003c/sup\u003e. The free energy of binding was calculated as \u0026Delta;G = -RTInKb, where \u0026Delta;G=-6.50 kcal/mol. The fact that AG\u0026lt;0 indicates that the PT-DNA complex structure interaction can occur spontaneously on the GCE surface under these working conditions. As can be seen from this interaction, the PT compound probably interacted with DNA via electrostatic interaction or intercalation[36]. In this process, the charge transfer force of the PT compound can intervene between the helical structures of DNA and deform the DNA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Spectrophotometric results for PT-DNA interaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFirstly, the spectra were recorded for the PT compound in the wavelength range of 200 nm to 800 nm (Figure 5, red dashed line). PT gave a well-defined maximum absorption band at 274 nm and a very broad-spectrum wave at approximately 325 nm. In the interaction experiments of PT with DNA, the spectra for the complex compound formed after adding 0.5 mg/L to 4 mg/L DNA to the PT solution were recorded in the wavelength range of 200 nm to 800 nm in ABS (pH 4.8) medium (Figure 5). In these experiments, no analytical change was observed in the spectral band observed at approximately 325 nm wavelength after each successive addition of DNA solution. However, in the absorption band observed at 274 nm wavelength, there was an increase in absorbance intensity (hyperchromic shift) and a shift in wavelength towards 269 nm (hypochromic shift) due to the complex compound structure formed after each DNA addition.\u003c/p\u003e\n\u003cp\u003eThe increase in absorption during the interaction of PT with DNA indicates that the local structure of DNA is disrupted or base pairs are exposed. This is usually associated with denaturation of DNA or binding of PT to major/minor grooves of DNA. At the same time, hypochromism is caused by the overlapping of \u0026pi; electrons due to the insertion of functional groups from PT between DNA base pairs (intercalation), and these data are also consistent with molecular docking data[37,38]. In addition, the shift in wavelength, the change in absorption intensity, and the isobestic point observed at 325 nm wavelength indicate that a new complex structure formed as a result of the interaction of the PT compound with DNA. The binding constant (K) of this complex compound was calculated using the Benesi-Hildebrand equation \u003cimg src=\"data:image/png;base64,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\" style=\"width: 243px; height: 38.4876px;\" width=\"243\" height=\"38.4876\"\u003e [39]. Here A\u003csub\u003e0\u003c/sub\u003e is the absorbance of PT, A is the absorbance of PT-DNA complex, ℰ is the PT absorption coefficient, and ℰ\u003csub\u003eH-G\u003c/sub\u003e is the absorption coefficient of the PT-DNA complex. When the necessary calculations were made, K=1.19x105 M\u003csup\u003e1-\u003c/sup\u003e was found. By using the PT-DNA complex formation constant, the binding free energy (\u0026Delta;G) was calculated as ∆G=-6.35 kcal/mol. These results show that the interaction with PT-DNA causes the hypochromism effect in the absorption spectrum of DNA[19,37]. These interactions show that the compound binds to DNA via intercalation and groove binding, which is consistent with the molecular docking results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Molecular docking results for PT-DNA interaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular docking studies are a powerful computational technique used to understand and model interactions between biomolecules. This technique provides useful preliminary information, especially for studies in the field of drug design. Molecular docking is used to model how potential drug candidates will bind to biomolecules such as target proteins, DNA, or RNA. The evaluation of the obtained data contributes to the understanding of target-drug interactions in the drug development process. As a result of docking studies, whether biomolecular targets are suitable for drug development can be checked. The molecular docking results for the PT-DNA interaction are given in Figure 6. In this computational simulation, two strong interactions were observed. DNA DG B:14 nucleotides made conventional hydrogen bonds with the oxygen in the pyrimidine carbon at an average distance of 2.56 \u0026Aring; and with the oxygen at the benzoyl carbon at an average distance of 3.28 \u0026Aring;. At the same time, DC A:11 nucleotide interacted with the benzoyl phenyl ring and formed a Pi-donor hydrogen bond[40]. These interactions were connected with a binding affinity of -8.12 kcal/mol. The molecular docking binding affinity is compatible with both voltammetric and spectrophotometric results. The PT-DNA interaction appears to involve electrostatic and minor groove binding.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, we synthesized a new heterocyclic compound PT, and its structural characterization was successfully carried out by methods such as microanalysis, FT-IR, NMR, and API-ES. Voltammetric studies using GCE showed that PT gave an irreversible reduction peak at -0.83 V, which revealed the electrochemical stability of the compound and its irreversible redox behavior.\u003c/p\u003e\n\u003cp\u003eInteraction studies with DNA showed that PT had a strong and thermodynamically favorable binding process with DNA. Voltammetric analysis results calculated the binding free energy as -6.5 kcal/mol, and the results obtained from spectrophotometric and molecular docking studies supported these findings. Molecular docking data determined the binding free energy as -8.12 kcal/mol and revealed that PT interacted with DNA electrostatically and via the minor binding groove. The findings demonstrate the potential of PT to interact with biomolecules and particularly its ability to bind with DNA, suggesting that compounds synthesized based on PT may be candidates for therapeutic and biomedical applications in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAll authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNida Nur Adiyan:\u0026nbsp;\u003c/strong\u003eConceptualization, Software, Writing - original draft. \u003cstrong\u003eAbdulkadir Levent:\u0026nbsp;\u003c/strong\u003eMethodology, Validation, Writing - original draft, Writing - review \u0026amp; editing, Supervision. \u003cstrong\u003eMehmet S\u0026ouml;nmez:\u003c/strong\u003e Data curation, Methodology, Investigation, Writing - original draft. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study will not be made publicly available.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSafety data retrieved from the spontaneous reporting process are anonymous and concur with ethical standards. Therefore, there was no further requirement for the ethical measure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eF.O. Sefrji, A.F. Alrefaei, M.A. Imam, G.R.S. Ashour, M.M. Abualnaja, R.M.S. Attar, A.A.A. Darwish, N.M. 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Uslu, Investigation of the molecular interaction between apraclonidine, an \u0026alpha;2-adrenergic receptor agonist, and bovine serum albumin using fluorescence and molecular docking techniques, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 326 (2025) 125246. https://doi.org/10.1016/j.saa.2024.125246.\u003c/li\u003e\n\u003cli\u003eM. Baginski, F. Fogolari, J.M. Briggs, Electrostatic and non-electrostatic contributions to the binding free energies of anthracycline antibiotics to DNA, Journal of Molecular Biology. 274 (1997) 253\u0026ndash;267. https://doi.org/10.1006/jmbi.1997.1399.\u003c/li\u003e\n\u003cli\u003eY. Gilad, H. Senderowitz, Docking studies on DNA intercalators, Journal of Chemical Information and Modeling. 54 (2014) 96\u0026ndash;107. https://doi.org/10.1021/ci400352t.\u003c/li\u003e\n\u003cli\u003eA. Khodadadi, E. Faghih-Mirzaei, H. Karimi-Maleh, A. Abbaspourrad, S. Agarwal, V.K. 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Paul, Novel pyrazolo[3,4-d]pyrimidine with 4-(1H-benzimidazol-2-yl)-phenylamine as broad spectrum anticancer agents: Synthesis, cell based assay, topoisomerase inhibition, DNA intercalation and bovine serum albumin studies, European Journal of Medicinal Chemistry. 126 (2017) 24\u0026ndash;35. https://doi.org/10.1016/j.ejmech.2016.09.093.\u003c/li\u003e\n\u003cli\u003eA. Wolfe, G.H. Shimer, T. Meehan, Polycyclic aromatic hydrocarbons physically intercalate into duplex regions of denatured DNA, Biochemistry. 26 (1987) 6392\u0026ndash;6396. https://doi.org/10.1021/bi00394a013.\u003c/li\u003e\n\u003cli\u003eA.R. Nekoei, M. Vatanparast, \u0026pi;-Hydrogen bonding and aromaticity: A systematic interplay study, Physical Chemistry Chemical Physics. 21 (2019) 623\u0026ndash;630. https://doi.org/10.1039/c8cp07003b.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"research-on-chemical-intermediates","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rint","sideBox":"Learn more about [Research on Chemical Intermediates](http://link.springer.com/journal/11164)","snPcode":"11164","submissionUrl":"https://submission.nature.com/new-submission/11164/3","title":"Research on Chemical Intermediates","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pyrimidine thione, DNA, Voltammetry, Spectrophotometric, Molecular docking","lastPublishedDoi":"10.21203/rs.3.rs-6683404/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6683404/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA novel heterocyclic compound, 5-benzoyl-6-phenyl-pyrimidin-4-one-2-thione (PT), was synthesized from the cyclocondensation reaction of dibenzoyl acetic acid-N-carboxymethylamide with thiourea. Microanalysis, FT-IR, NMR, and API-ES mass techniques were used to characterize the structure of the pyrimidinethione compound. Cyclic voltammetric studies performed using a glassy carbon electrode in anhydrous medium revealed that the PT compound gave an irreversible reduction peak at -0.83 V, indicating electrochemical stability and irreversible redox behavior of the compound.\u003c/p\u003e\n\u003cp\u003eThe interaction of PT compound with DNA was investigated by spectrophotometric, voltammetric, and molecular docking methods. The interaction of PT with DNA in acetate as supporting electrolyte (pH 4.8) on a glassy carbon electrode was evaluated by the change in the anodic signal for guanine. The voltammetric analysis of the PT-DNA interaction calculated the binding free energy as -6.5 kcal/mol, while spectrophotometric studies supported the binding interactions with DNA.\u003c/p\u003e\n\u003cp\u003eMolecular docking studies showed a stronger binding interaction with DNA and revealed a binding free energy of -8.12 kcal/mol. These results show that the compound performs a thermodynamically favorable binding process with DNA, and molecular docking provides important information about the binding mode and energy profile. The data obtained with all three techniques about the PT-DNA interaction supported each other, and the interaction occurred in the form of electrostatic and minor binding groove.\u003c/p\u003e","manuscriptTitle":"Synthesis, crystal structure, characterization, and electrochemical properties of 5-benzoyl-6-phenyl-pyrimidin-4-one-2-thione compound; voltammetric, spectrophotometric, and molecular docking studies of its interaction with DNA","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-03 11:42:21","doi":"10.21203/rs.3.rs-6683404/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-28T08:11:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-26T22:34:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-23T10:37:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"113395560608104677956664873665976707949","date":"2025-06-14T21:58:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62338512335316559774201269688933339995","date":"2025-06-14T12:11:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"284569680637599150568595514283527352443","date":"2025-06-14T11:56:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-14T11:18:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-19T02:56:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-19T02:56:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Research on Chemical Intermediates","date":"2025-05-16T20:46:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"research-on-chemical-intermediates","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rint","sideBox":"Learn more about [Research on Chemical Intermediates](http://link.springer.com/journal/11164)","snPcode":"11164","submissionUrl":"https://submission.nature.com/new-submission/11164/3","title":"Research on Chemical Intermediates","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5c278bf1-fd4a-4fef-b5fd-174ed0f42026","owner":[],"postedDate":"June 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-28T16:03:47+00:00","versionOfRecord":{"articleIdentity":"rs-6683404","link":"https://doi.org/10.1007/s11164-025-05694-2","journal":{"identity":"research-on-chemical-intermediates","isVorOnly":false,"title":"Research on Chemical Intermediates"},"publishedOn":"2025-07-22 15:58:05","publishedOnDateReadable":"July 22nd, 2025"},"versionCreatedAt":"2025-06-03 11:42:21","video":"","vorDoi":"10.1007/s11164-025-05694-2","vorDoiUrl":"https://doi.org/10.1007/s11164-025-05694-2","workflowStages":[]},"version":"v1","identity":"rs-6683404","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6683404","identity":"rs-6683404","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}