Design and Discovery of Phthalazinone-Based Potential PARP Inhibitors: Synthesis, Molecular Docking, ADMET Profiling, and In Vitro Evaluation

Design and Discovery of Phthalazinone-Based Potential PARP Inhibitors: Synthesis, Molecular Docking, ADMET Profiling, and In Vitro Evaluation | 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 Design and Discovery of Phthalazinone-Based Potential PARP Inhibitors: Synthesis, Molecular Docking, ADMET Profiling, and In Vitro Evaluation Olga Mikolaichuk, Andrey Khramchikhin, Mariia Skryl’nikova, Dmitrii Antonenko, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8688192/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract This study reports the design, synthesis, and biological evaluation of two novel phthalazinone-based carboxamides, N -(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide and N -(4-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide, as potential poly(ADP-ribose) polymerase-1 (PARP1) inhibitors. Structure-based molecular docking indicated favorable binding of both compounds within the PARP1 active site, with binding energies of − 8.5 and − 7.5 kcal·mol⁻¹, respectively. The stability and key interaction patterns of the more potent meta-substituted analogue were further validated by 250 ns molecular dynamics simulations. In silico ADMET profiling suggested acceptable drug-like properties for the synthesized compounds. Biochemical evaluation revealed enhanced inhibitory activity of the meta-substituted derivative against recombinant PARP1. Furthermore, both compounds exhibited cytotoxic effects in BRCA-mutant breast cancer cell lines, Capan-1 (BRCA2 c.5946delT) and MDA-MB-436 (BRCA1 c.5396 + 1G > A). Comparative analysis highlights the critical influence of the substituent position on the phenyl ring in modulating PARP1 binding stability and inhibitory activity. These findings identify the phthalazinone carboxamide scaffold as a promising platform for further structural optimization toward potent PARP1 inhibitors. PARP-1 phthalazinone-based compounds DFT ADMET count-assay Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction First-generation poly(ADP-ribose) polymerase inhibitors (PARPi), including olaparib, talazoparib, niraparib, and rucaparib, have been approved for the treatment of certain ovarian, breast, prostate, and pancreatic cancers. 1 – 5 These currently approved PARPi inhibit both PARP1 and PARP2 (PARP1/2), which function as critical DNA damage sensors that bind to sites of damage and recruit repair factors through the formation of branched poly(ADP-ribose) (PAR) chains via PARylation. 6 , 7 The therapeutic efficacy of PARP inhibitors is particularly pronounced in tumors harboring BRCA mutations. 8 Tumor cells with defective BRCA1/2 exhibit impaired homologous recombination repair mechanisms, and inhibition of PARP1 activity further exacerbates DNA damage accumulation. 9 This combination results in synthetic lethality, whereby the simultaneous loss of both PARP-mediated base excision repair and BRCA-dependent homologous recombination leads to catastrophic DNA damage and subsequent apoptosis in cancer cells. 10 This mechanism renders BRCA1/2-mutated tumors exceptionally vulnerable to PARP inhibition. However, hematological toxicities associated with the simultaneous inhibition of PARP1 and PARP2 have limited the clinical utility of these agents. 11 Consequently, the development of selective PARP1 inhibitors has emerged as a promising strategy to mitigate these adverse effects while maintaining therapeutic efficacy. 12 The phthalazinone scaffold represents an attractive pharmacophore for the design of selective PARP1 inhibitors based on its well-established binding profile within the PARP1 active site. 13 This planar bicyclic core features multiple sites for hydrogen bonding and π-π stacking interactions, enabling optimal engagement with key amino acid residues including Lys903 , Ser904 , Gly888 , and Tyr907 , which are crucial for potent enzyme inhibition (Fig. 1 ). The nitrogen atoms within the phthalazinone ring serve as anchoring points for hydrogen bonds with polar residues, thereby enhancing binding affinity, while the aromatic system facilitates favorable hydrophobic interactions. Furthermore, this scaffold offers considerable synthetic flexibility, permitting the introduction of diverse substituents to fine-tune selectivity, potency, and pharmacokinetic properties. 14 Given its proven track record as the structural backbone in clinically successful PARP inhibitors, the phthalazinone scaffold provides a rational starting point for structure-based drug design guided by computational approaches and structure–activity relationship (SAR) studies. In this study, we present the design, synthesis, and thorough evaluation of novel N -(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide and N -(4-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide as potential PARP1 inhibitors. Our integrated approach combines molecular docking, ADME profiling, molecular dynamics, biochemical screening of the inhibiting properties and in vitro cytotoxicity assessments against breast carcinoma cells with BRCA mutations Capan-1 (BRCA-2 mutation c.5946 delT), MDA-MB-436 (BRCA-1 mutation c.5396 + 1G > A). Notably, molecular docking studies revealed binding scores of -8.5 kcal·mol - 1 for N -(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide ( 4 ) and − 7.5 kcal·mol - 1 for N -(4-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide ( 5 ) against PARP1, indicating the potential for further optimization of their structures. Additionally, molecular dynamics simulations of compound 4 in water revealed no signs of molecular aggregation at either 50 or 100 ns. This observation is further supported by the solvent-accessible surface area (SASA) plot, which indicated only minor fluctuations in values throughout the simulation period, underscoring the compound's stability in aqueous environments. Moreover, the compound 4 demonstrated higher binding energy than for talazoparib with the appropriate binding pattern during 250 ns MD simulation. Results and discussion Chemistry The synthetic route for the preparation of phthalazinone derivatives 4 and 5 is illustrated in Scheme 1 . 4-oxo-3,4-dihydrophthalazine-1-carboxylic acid 3 was synthesized used previously observed method from naphthalene 1 oxidizing with KMnO 4 to afford intermediate 2 that was stirred in EtOH with hydrazine hydrate at RT for 18h. 15 The structure of compound 1 was confirmed by 1 H NMR spectroscopy, displaying a singlet at δ 13.10 ppm (1H, OH), multiplets between δ 8.61–8.26 ppm (3H, aromatic), and characteristic aromatic signals at δ 7.97 and 7.88 ppm. The 13 C NMR spectrum supported the proposed structure, with resonances at δ 165.3 ppm (COOH), 160.0 ppm (C = O), and several signals between δ 126.3–136.9 ppm (C Ar ). The chloroanhydride of the 4-oxo-3,4-dihydrophthalazine-1-carboxylic acid was obtained by reacting it with thionyl chloride. Subsequently, triethylamine was added to a mixture of intermediate and amines in dichloromethane. This solution was stirred at room temperature overnight, resulting in the formation of the target compounds 4 and 5 . The structure of compounds 4 and 5 were confirmed by 1 H and 13 C NMR spectroscopy. The comparison of the 1 H NMR spectra for N -(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide and N -(4-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide reveals differences in the chemical environments of the aromatic protons, reflecting the influence of the amido substituent's position on the phenyl ring. In both compounds, the characteristic downfield signals at δ 10.57 and δ 10.63 correspond to the NH groups, indicating their similar electronic environments. However, variations are evident in the aromatic region; for instance, the peaks at δ 8.52–8.26 and δ 8.17 in the NMR spectra shift for compound 4 to δ 8.41 and δ 8.32 in the compound 5 , suggesting a change in coupling patterns due to the different substitution on the phenyl ring. Additionally, the aromatic multiplets exhibit slight shifts and changes in multiplicity, indicative of the spatial arrangement and electronic interactions influenced by the amide group positioning. Molecular dynamic studies To gain deeper insights into the aggregation behavior and self-assembly properties of compounds 4 and 5 in aqueous solution, molecular dynamics simulations were performed. Understanding the clustering tendencies of these compounds is crucial for predicting their bioavailability, formulation stability, and potential therapeutic efficacy. 16 Molecular dynamics simulations of compound 5 in aqueous solution revealed that molecular aggregation occurred by 50 ns and 100ns, leading to cluster formation ( Fig. 2 ). At the end of the simulation, stable molecular clusters were observed in the system. The high clustering potential in water was further confirmed by the solvent-accessible surface area (SASA) plot, which showed a continuous decrease throughout the dynamics ( Fig. 2 ). In contrast, molecular dynamics simulations of compound 4 in water showed no molecular aggregation at either 50 or 100 ns ( Fig. 2 ). The absence of significant clustering potential for compound 5 in aqueous solution was also supported by the SASA plot, which demonstrated only minor changes in values throughout the dynamics. The contrasting aggregation profiles observed in MD simulations—pronounced clustering for compound 5 versus stable dispersion for compound 4 . Figure 2. Molecular dynamics simulations of compound 4 (Fig. 2a and 2b) and 5 in aqueous solution (2 c and 2d) In vitro cytotoxicity studies To assess the anticancer potential of the synthesized PARP inhibitors, in vitro cytotoxicity studies were performed against breast carcinoma cell lines (Capan-1 and MDA-MB-436) with BRCA mutations. These cell lines were selected due to their documented sensitivity to PARP inhibition and relevance in cancer research 17 , 18 . Furthermore, an investigation was conducted into the possible cell death mechanisms mediated by hydrogen peroxide (H 2 O 2 ). The effect of compound 4 was stronger than that of compound 5 ( C = 4.5–450 µM) on both cell lines. Molecular docking studies The affinity of ligands to the catalytic domain of PARP1 at the docking stage was evaluated using two FDA aproved PARP inhibitors namely Olaparib and Talazoparib as benchmarks. The docking grid was determined and centered by the ligand structure originally located in the receptor. As a result of Induced Fit docking, the poses with the lowest binding energy calculated by MM-GBSA method were selected for each ligand which were subjected for further molecular dynamics (MD) simulation (Table 1 ). It can be seen that compound 4 having a more stable and favorable interaction with the target compared to compound 5 . Table 1 Results of IFD for compounds 4 and 5 compared to known inhibitors. The top-poses are presented. Inhibitor XP GScore ΔG bind (kcal/mol) 4 -8.505 -76.28 5 -7.555 -73.96 Olaparib -11.524 -47.79 Talazoparib -8.055 -48.4 The pharmacokinetic properties and suitability of the compounds as drugs were evaluated using predictive descriptors, including molecular weight, the number of hydrogen bond donors and acceptors, the percentage of estimated absorption in humans, and the number of violations of the Lipinski five rule (Table S1 and S2). The characteristics of the compound 4 and 5 were compared with those of FDA approved inhibitors. The synthesized compounds, characterized by their structural similarities, exhibited favorable drug-likeness properties. Both compounds possess a molecular weight of 364.403 g/mol, with two hydrogen bond donors and six hydrogen bond acceptors; additionally, they display a logP value of 3.2. The estimated oral absorption is 86%, which indicates a potentially high oral bioavailability. 19 ,20 During 250 ns MD simulation, all known inhibitors demonstrated acceptable stability within the active sites of PARP1 catalytic domain (Fig. 3 ). As regards compound 5 , it showed great stability within the active site of the receptor. Nonetheless, 5 demonstrated several visible fluctuations temporarily while being shifted within the binding sites tending to find a more favorable location. Nevertheless, the compound 5 was still inside the binding site and the average range of RMSD value did not extend 2Å. The main part of the stabilization has been achieved by phthalazin-1(2 H )-one moiety being connected to Tyr907 by π-π stacking and to Gly863 and Ser904 by H-Bonds. In addition, the NH of 3-methylbutanamide fragment was stabilized by H-bond with Asp766. As for compound 4 , it showed flat RMSD graph except one fluctuation on 100 ns which could be explained by the fact that ligand lost connection with Tyr889 and compensated that with Ser904. Results demonstrated almost the same stabilization profile except the 4-methylbutanamide moiety was stabilized by H-bond with Gln759 connected to oxygen atom. In sum, the interaction interface of the compound 4 is comparable to known inhibitors demonstrating H-bonds with Gly863 and Ser904 and π-π stacking with Tyr907 (Fig. 3 ). The key interacting residues of PARP-1 with known inhibitors were also observed fort he compounds 4 and 5 (Table 2 ). The calculated ΔG binding of compound 4 is comparable for that of Talazoparib The compound 4 showed much greater binding affinity than the compound 5 and Talazoparib (73,5 kcal/mol vs . -66, 8 and 59,6 kcal/mol, respectively). Both compounds 4 and 5 were characterized with lower binding strength than that of Olaparib due to the larger interaction surface of the known inhibitor (Table 2 ). These findings align with cytotoxicity data, which indicate that the substitution at position 4( para ) of N -(R-aminophenyl)-3-methylbutanamide enhances its affinity for PARP. Table 2 MM/GBSA free energy of binding (ΔG bind ) of ligands with PARP1 calculated over 250 ns MD simulation. Common residues of PARP-1 interacting with ligands and Olaparib are underlined; common residues with Talazoparib presented in bold. PARP1-ligand complex MM-GBSA mean ΔG bind over 250 ns MD (kcal/mol) Interacting residue positions of PARP-1 4 -73.5 Val762, Asp766 , Gly863 , Tyr889 , Tyr896 , Ser904 , Tyr907 5 -66.8 Gln759, Asp766 , Gly863 , Tyr889 , Tyr896 , Lys 903 , Ser904 , Tyr907 Olaparib -82.1 Asp766 , Asp770, His862, Gly863 , Ser864, Ile872, Tyr896 , Lys903 , Ser904 , Tyr907 Talazoparib -59.6 Glu763, Asp766 , Asn767, Asp770, His862, Gly863 , Ser864, Gly888, Tyr889 , Tyr896 , Ser904 , Tyr907 , Glu988 PARP1 enzymatic activity Ultimately, the inhibitory potential of compounds 4 and 5 against PARP1 enzymatic activity was evaluated. For this purpose, we employed a previously developed fluorescence anisotropy-based assay system, which measures the binding of PARP1 to a fluorescently labeled DNA duplex and its dissociation from complex with DNA under authoPARylation 21 . The results are presented in Fig. 4 . While compound 5 exhibited no discernible effect on PARP1 activity, compound 4 demonstrated a significant inhibitory effect. Specifically, compound 4 suppressed the dissociation of PARP1 from its complex with DNA during the PARylation process, with a half-maximal inhibitory concentration ( IC₅₀ ) of 6.5 µM. Under analogous experimental conditions, the IC₅₀ value for the reference inhibitor olaparib was determined to be 13 nM. In the presence of 200 µM compound 4 , the residual PARP1 activity was approximately 20%. This data aligns consistently with the cell-based cytotoxicity assays. Conclusions In conclusion, this study successfully delineates the design, synthesis, and comprehensive evaluation of two novel compounds, N -(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide and N -(4-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide, as promising inhibitors of PARP1. The molecular docking, ADME profiling, and in vitro cytotoxicity assessments has provided a robust framework for evaluating their potential therapeutic efficacy. The molecular docking results, with binding affinities of -8.5 kcal/mol and − 7.5 kcal/mol, respectively, suggest that both compounds exhibit significant interaction with PARP1, warranting further structural optimization. Both N -(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide and N -(4-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide exhibited a growth-inhibitory effect on native cells, with the effect being more pronounced after pre-incubation with hydrogen peroxide. Notably, the effect of N -(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide was more significant than that of N -(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide, as it was evident across the entire dose range (4.5–450 µM) on both cell lines. The biochemical data obtained from experiments with recombinant PARP1 align consistently with the cell-based cytotoxicity assays: N -(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide ( 4 ) exhibited PARP1 inhibitory effect with micromolar IC 50 value. This correlation supports the proposed mechanism of action, whereby the growth-inhibitory effects observed for compound 4 in cell lines are mediated, at least in part, through the direct inhibition of PARP1 enzymatic function. Experimental Section General Information (Synthesis) 1 Н and 13 С{ 1 Н} NMR spectra were obtained on a Bruker Avance III HD 400 NanoBay spectrometer (Germany) (400.13 MHz for 1 Н and 100.61 MHz for 13 С) in DMSO- d 6 at 298.15 K. The melting point was determined on a Kofler hot stage equipped with a microscope at a heating rate of 1°C/min. The reaction progress was monitored by TLC on Merck Kieselgel 60 F254 plates. Mass spectral analysis was performed on a Shimadzu Nexera X2 LCMS-9030 chromatography-mass spectrometer (Japan). N -(3-aminophenyl)-3-methylbutanamide (CAS 926225-62-9) and N -(4-aminophenyl)-3-methylbutanamide (CAS 189576-50-9) are commercially available. Synthesis of 2-(carboxycarbonyl)benzoic acid (2) Naphthalene (39.2 mmol) was added to 150 mL of 0.5 N NaOH and subjected to reflux. A solution of potassium permanganate (196.2 mmol) in 600 mL of water was added dropwise to the refluxing solution over a period of 1.5 hours. Once the dropwise addition was complete, the reaction was allowed to proceed for an additional 45 minutes to ensure complete oxidation. The reaction was then quenched by the addition of 120 mL of ethanol, after which the mixture was cooled to room temperature and subjected to suction filtration. The resulting filter cake was acidified with 30% hydrochloric acid to a pH of 5 and subsequently extracted with ethyl acetate (100 mL × 3). The organic layers were combined and dried over anhydrous sodium sulfate. The solvent was then evaporated under reduced pressure, yielding compound 2 as a white solid with a yield of 33.0% (2.51 g). The product was characterized as a colorless powder with a melting point (T m ) of 143–145°C. The obtained data were consistent with previously published results 1 . Synthesis of 4-oxo-3,4-dihydrophthalazine-1-carboxylic acid (3) Hydrazine hydrate (15.0 mmol) was dissolved in a solution of ethanol and water in a 1:2 ratio. Subsequently, compound 2 (12.5 mmol) was added to this solution. The resulting mixture was stirred at room temperature for 18 hours. Afterward, the precipitate was filtered, washed twice with dichloromethane, and dried under vacuum to obtain 4-oxo-3,4-dihydrophthalazine-1-carboxylic acid ( 3 ). The yield was 68% (1.6 g), resulting in white crystals with a melting point (Tm) of 230–232°C. 1 H NMR (400 MHz, DMSO- d 6 ), δ: 13.10 (s, 1H, O H ), 8.61–8.52 (m, 1H, Ar), 8.31–8.26 (m, 1H, Ar), 7.97 (ddd, J = 8.4, 7.2, 1.5 Hz, 1H, Ar), 7.88 (td, J = 7.6, 1.2 Hz, 1H, Ar). 13 C NMR (101 MHz, DMSO- d 6 ), δ: 165.3 (COOH), 160.0 (C = O), 136.9 (Ar), 134.4 (Ar), 132.3 (Ar), 128.3 (Ar), 127.9 (Ar), 126.8 (Ar), 126.3 (Ar). The obtained data fully agree with previously published results 2 . Synthesis of compounds 4 and 5 6 mol of thionyl chloride was added to compound 3 (5.5 mmol) in a 20 mL pyridine, and the mixture was heated to reflux for 6 hours. Following this, any excess liquid was evaporated under reduced pressure to yield 4-oxo-3,4-dihydrophthalazine-1-carbonyl chloride. A solution containing 1.0 mmol of the corresponding amine ( N -(3-aminophenyl)-3-methylbutanamide for compound 4 and N -(4-aminophenyl)-3-methylbutanamide for compound 5 ) and 1.2 mmol of triethylamine (Et₃N) in 30 mL of dichloromethane (DCM) was prepared while cooling in an ice bath. A solution of compound 4 (1.1 mmol) in 30 mL of DCM was then added dropwise to the reaction mixture. Subsequently, the mixture was allowed to warm to room temperature and stirred for 10 hours. Upon completion of the reaction, the organic layer was washed with a 10% aqueous solution of potassium carbonate (10 mL × 3). The organic phase was then dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure by rotary evaporation to afford the crude product. Purification was carried out by column chromatography on silica gel using a chloroform/methanol (CHCl₃/MeOH, 9:1, w/w ) eluent system to obtain compounds 4 and 5 as pure fractions. N-(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide (4) Yield: 70.3% (0.559 g). White powder. T m = 251–253°C. 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.08 (s, 1H), 10.63 (s, 1H), 9.95 (s, 1H), 8.52–8.26 (m, 2H), 8.17 (t, J = 2.0 Hz, 1H), 7.95 (dtd, J = 26.1, 7.3, 1.4 Hz, 2H), 7.54–7.34 (m, 2H), 7.28 (t, J = 8.1 Hz, 1H), 2.20 (d, J = 7.1 Hz, 2H), 0.94 (d, J = 6.6 Hz, 6H). 13 C NMR (101 MHz, DMSO) δ 171.23, 162.56, 160.01, 140.56, 140.11, 139.15, 134.42, 132.59, 129.37, 128.16, 127.98, 126.58, 126.44, 115.56, 115.39, 111.53, 46.08, 26.11, 22.76. Found, m/z : 365.1. [M + H] + . C 20 H 20 N 4 O 3 . Calculated, m/z : 365.4. N-(4-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide (5) Yield: 74.4% (0.592 g). Pink powder. T m = 242–244°C. 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.08 (s, 1H), 10.57 (s, 1H), 9.88 (s, 1H), 8.41 (d, J = 8.5 Hz, 1H), 8.32 (dd, J = 8.1, 1.3 Hz, 1H), 8.03–7.87 (m, 2H), 7.80–7.65 (m, 2H), 7.65–7.47 (m, 2H), 2.19 (d, J = 7.1 Hz, 2H), 0.94 (d, J = 6.5 Hz, 6H). 13 C NMR (101 MHz, DMSO) δ 170.95, 162.26, 160.03, 140.46, 136.03, 134.40, 134.09, 132.58, 128.16, 128.01, 126.74, 126.41, 120.96, 119.91, 46.04, 26.07, 22.78. Found, m/z : 365.5. [M + H] + . C 20 H 20 N 4 O 3 . Calculated, m/z : 365.4. Information on Molecular Dynamics Simulation, in vitro cytotoxicity, determination of IC₅₀ by fluorescence anisotropy, and molecular modeling and in silico prediction can be found in the Supplementary Materials Declarations Declaration of competing interest 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. Fundings This work was supported by Ministry of Health of the Russian Federation 1024021500062-3-3.2.21;3.1.5;1.6.4 (Creation of a selective poly(ADP-ribose) polymerase inhibitor of the first type (PARP1) based on condensed heterocyclic systems). Acknowledgements The authors acknowledge the Russian Science Foundation for supporting the recombinant PARP1 purification research project (Grant №25-74-10025), which facilitated the acquisition of PARP1 for this study. Author Contribution 1-4 wrote the main manuscript text 5-7 prepared figures, 1-3, 8,10-11 prepared SI. All authors reviewed the manuscript. 9,12-13 supervised the research. References Jinge Zh., Bo T, Hao PS, Qiang Z (2025) Empowering PARP inhibition through rational combination: Mechanisms of PARP inhibitors and combinations with a focus on the treatment of metastatic castration-resistant prostate cancer. 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Anal Biochem 545:91–97. https://doi.org/10.1016/j.ab.2017.12.033 Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SIPARP2212.docx Onlinefloatimage2.png Scheme 1. Reagents and conditions: ( i ) 0.5 mol/L NaOH ( aq ), KMnO₄, H₂O, reflux, 2 h; ( ii ) hydrazine hydrate (hydrazine, 35%), EtOH, rt, 15 h; ( iii ) SOCl₂, pyridine, reflux, 6 h; N -(3-aminophenyl)-3-methylbutanamide (for 4) or N -(4-aminophenyl)-3-methylbutanamide (for 5), Et₃N, DCM, rt, overnight Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 27 Apr, 2026 Reviews received at journal 27 Apr, 2026 Reviews received at journal 23 Apr, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers agreed at journal 21 Apr, 2026 Reviewers agreed at journal 20 Apr, 2026 Reviews received at journal 04 Apr, 2026 Reviewers agreed at journal 29 Mar, 2026 Reviewers invited by journal 02 Feb, 2026 Editor assigned by journal 02 Feb, 2026 Submission checks completed at journal 27 Jan, 2026 First submitted to journal 24 Jan, 2026 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. 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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-8688192","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":584674805,"identity":"09f48b23-2d7c-4ef8-9481-9df71db5c699","order_by":0,"name":"Olga Mikolaichuk","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIiWNgGAWjYDACdjDJzGDAwMD4AMji4SOohRmhhdkApIWNFC1sEiAmQS38zOwXP92osJY3Z+9Oq/yaYyfDxsD88NENPFokm3mKpXPOpBvu7Dm77bbstmSgw9iMjXPwaDE4zJMgndt2OMHgRu6225LbmIFaeNikCWhJ/p37D6jl/tttxZLb6onRwn5MOrcBZAvvNsaP2w4T1gL0C5t1zrF0ww1ncjdLM247zsPGTMAv/Oztj2/n1FjLGxw/u/Hjz23V9vzszQ8f49MCjDsDOJOZB0ziVQ4C7A/gTMYfBFWPglEwCkbBSAQApVVFgaqsYK4AAAAASUVORK5CYII=","orcid":"","institution":"A.M. Granov Russian Research Centre for Radiology and Surgical Technologies","correspondingAuthor":true,"prefix":"","firstName":"Olga","middleName":"","lastName":"Mikolaichuk","suffix":""},{"id":584674806,"identity":"ccc8b8a4-7bfb-426d-a375-92261f364b44","order_by":1,"name":"Andrey Khramchikhin","email":"","orcid":"","institution":"A.M. Granov Russian Research Centre for Radiology and Surgical Technologies","correspondingAuthor":false,"prefix":"","firstName":"Andrey","middleName":"","lastName":"Khramchikhin","suffix":""},{"id":584674807,"identity":"86120f39-8dff-4b7a-bb86-07794690a087","order_by":2,"name":"Mariia Skryl’nikova","email":"","orcid":"","institution":"A.M. Granov Russian Research Centre for Radiology and Surgical Technologies","correspondingAuthor":false,"prefix":"","firstName":"Mariia","middleName":"","lastName":"Skryl’nikova","suffix":""},{"id":584674808,"identity":"4f287e2e-d606-4a57-a10a-ec809b3d7567","order_by":3,"name":"Dmitrii Antonenko","email":"","orcid":"","institution":"St. Petersburg State Technological Institute","correspondingAuthor":false,"prefix":"","firstName":"Dmitrii","middleName":"","lastName":"Antonenko","suffix":""},{"id":584674809,"identity":"d0265e5d-6a68-4922-a496-fc0d54fb9b04","order_by":4,"name":"Aleksei Chutko","email":"","orcid":"","institution":"First Pavlov State Medical University of St. Petersburg","correspondingAuthor":false,"prefix":"","firstName":"Aleksei","middleName":"","lastName":"Chutko","suffix":""},{"id":584674810,"identity":"51033dad-d80f-4785-b09e-f440625fd70f","order_by":5,"name":"Tatyana Kurgina","email":"","orcid":"","institution":"Institute of Chemical Biology and Fundamental Medicine","correspondingAuthor":false,"prefix":"","firstName":"Tatyana","middleName":"","lastName":"Kurgina","suffix":""},{"id":584674811,"identity":"9481b3ae-3bc9-4258-82b6-b11125b1e061","order_by":6,"name":"Mikhail Kutuzov","email":"","orcid":"","institution":"Institute of Chemical Biology and Fundamental Medicine","correspondingAuthor":false,"prefix":"","firstName":"Mikhail","middleName":"","lastName":"Kutuzov","suffix":""},{"id":584674812,"identity":"efad5d3d-291d-40da-8bb6-6651cc5f4023","order_by":7,"name":"Kirill Timoshchuk","email":"","orcid":"","institution":"A.M. Granov Russian Research Centre for Radiology and Surgical Technologies","correspondingAuthor":false,"prefix":"","firstName":"Kirill","middleName":"","lastName":"Timoshchuk","suffix":""},{"id":584674813,"identity":"5d1f7df0-d47c-44db-8bf2-88932721913c","order_by":8,"name":"Olga Lavrik","email":"","orcid":"","institution":"Institute of Chemical Biology and Fundamental Medicine","correspondingAuthor":false,"prefix":"","firstName":"Olga","middleName":"","lastName":"Lavrik","suffix":""},{"id":584674814,"identity":"7502a36f-e2cb-4ab5-9709-59255e3090ab","order_by":9,"name":"Natalia Petukhova","email":"","orcid":"","institution":"First Pavlov State Medical University of St. Petersburg","correspondingAuthor":false,"prefix":"","firstName":"Natalia","middleName":"","lastName":"Petukhova","suffix":""},{"id":584674815,"identity":"04667765-bd20-479e-ad3c-40a9821aaba0","order_by":10,"name":"Marina Samoylovich","email":"","orcid":"","institution":"A.M. Granov Russian Research Centre for Radiology and Surgical Technologies","correspondingAuthor":false,"prefix":"","firstName":"Marina","middleName":"","lastName":"Samoylovich","suffix":""},{"id":584674816,"identity":"a33ab59b-7a6f-41e2-a593-3e9371174318","order_by":11,"name":"Oleg Molchanov","email":"","orcid":"","institution":"A.M. Granov Russian Research Centre for Radiology and Surgical Technologies","correspondingAuthor":false,"prefix":"","firstName":"Oleg","middleName":"","lastName":"Molchanov","suffix":""},{"id":584674817,"identity":"fb362609-5bc2-4285-93ea-863364dad37b","order_by":12,"name":"Dmitrii Granov","email":"","orcid":"","institution":"A.M. Granov Russian Research Centre for Radiology and Surgical Technologies","correspondingAuthor":false,"prefix":"","firstName":"Dmitrii","middleName":"","lastName":"Granov","suffix":""}],"badges":[],"createdAt":"2026-01-24 16:40:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8688192/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8688192/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101943169,"identity":"6c9010e4-d14f-45a8-b7e0-6018a6f34ac9","added_by":"auto","created_at":"2026-02-05 09:40:53","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8938,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular scaffold interacting with binding sites of the PARP enzyme. Specific amino acid residues—Gly888, Lys903, Ser904, and Tyr907—are highlighted with different colored dashed ovals, indicating their roles as critical interaction points. Additionally, water (H₂O) is shown near Gly888, suggesting its possible involvement in the hydrogen-bonding network at the binding site.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8688192/v1/0525195ae7aff79803664166.jpg"},{"id":101901205,"identity":"b661b40b-e1fd-40f5-92b1-120fcd456f1b","added_by":"auto","created_at":"2026-02-04 19:17:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":76822,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular dynamics simulations of compound 4 (Figure \u003cem\u003e2a and 2b\u003c/em\u003e) and 5 in aqueous solution (2\u003cem\u003ec and 2d)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8688192/v1/44bcf55cbae5f00834e40254.jpg"},{"id":101901206,"identity":"b6ed3ca0-d99f-4985-8266-71f8af6a4fe3","added_by":"auto","created_at":"2026-02-04 19:17:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":487911,"visible":true,"origin":"","legend":"\u003cp\u003eResults of 250 ns MD run of ligands complexes with PARP1 compared to those of known inhibitors.\u003cstrong\u003e \u003c/strong\u003eRMSD plots (upper left) show the RMSD evolution of a protein (left Y-axis). All protein frames are first aligned on the reference frame backbone, and then the RMSD is calculated based on the atom selection. Corresponding protein selection (C-alpha atoms) are indicated by the green color. Ligand RMSD (right Y-axis) indicates how stable the ligand is with respect to the protein and its binding pocket. In the above plot, 'Lig fit Prot' shows the RMSD of a ligand when the protein-ligand complex is first aligned on the protein backbone of the reference and then the RMSD of the ligand heavy atoms is measured. 'Lig fit Lig' shows the RMSD of a ligand that is aligned and measured just on its reference conformation. A timeline representation of the interactions and contacts (H-bonds, Hydrophobic, Ionic, Water bridges) (upper right): the top right panel shows the total number of specific contacts that the protein makes with the ligand throughout the entire trajectory;the panel shows which residues interact with the ligand in each section of the trajectory. Some residues come into more than one specific contact with the ligand, which is represented by a darker shade of orange according to the scale to the right of the graph. A detailed diagram of the interaction of ligand atoms with protein residues (lower left). The scheme demonstrates interactions that occur more than 10.0% of the simulation time on the selected trajectory (from 0.00 to 250.00 nsec). Simulation Interactions Diagram panel (bottom right) of protein-ligand interactions (or 'contacts') categorized into four types: Hydrogen Bonds, Hydrophobic, Ionic and Water Bridges (the stacked bar charts are normalized over the course of the trajectory showing the fraction of the simulation time that each specific interaction was maintained throughout the MD run).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8688192/v1/07913e1c8e7cd64331848945.png"},{"id":101901204,"identity":"c019d66b-6947-4f76-a9f0-837cfd8c307b","added_by":"auto","created_at":"2026-02-04 19:17:14","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":61821,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of PARP1 enzymatic activity by compounds 4 and 5: dose-response curve for compound 4 (а) and 5 (b), PARP1 activity (expressed as a percentage of the activity in the absence of any compound) is plotted against the inhibitor concentration. The IC₅₀ value and the residual PARP1 activity at 200 µM of compound are indicated.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8688192/v1/33015cb99eb2ec7dcd3566b7.jpg"},{"id":102397219,"identity":"64684209-b167-4b56-b63f-8f132207600f","added_by":"auto","created_at":"2026-02-11 10:11:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1447568,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8688192/v1/6db609a5-d454-4ded-bb66-817719be490e.pdf"},{"id":101901208,"identity":"1b3a44c0-a952-4837-80e7-596b5839b3e6","added_by":"auto","created_at":"2026-02-04 19:17:15","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3106674,"visible":true,"origin":"","legend":"","description":"","filename":"SIPARP2212.docx","url":"https://assets-eu.researchsquare.com/files/rs-8688192/v1/6af86b0f293f88202fad5215.docx"},{"id":101901209,"identity":"2d5dcf60-4751-4f99-839a-ae5d8317379c","added_by":"auto","created_at":"2026-02-04 19:17:15","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5183,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1.\u003c/strong\u003e Reagents and conditions: (\u003cem\u003ei\u003c/em\u003e) 0.5 mol/L NaOH (\u003cem\u003eaq\u003c/em\u003e), KMnO₄, H₂O, reflux, 2 h; (\u003cem\u003eii\u003c/em\u003e) hydrazine hydrate (hydrazine, 35%), EtOH, rt, 15 h; (\u003cem\u003eiii\u003c/em\u003e) SOCl₂, pyridine, reflux, 6 h; \u003cem\u003eN\u003c/em\u003e-(3-aminophenyl)-3-methylbutanamide (for \u003cstrong\u003e4\u003c/strong\u003e) or \u003cem\u003eN\u003c/em\u003e-(4-aminophenyl)-3-methylbutanamide (for \u003cstrong\u003e5\u003c/strong\u003e), Et₃N, DCM, rt, overnight\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8688192/v1/1607e21821ade3a1c18a02db.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Design and Discovery of Phthalazinone-Based Potential PARP Inhibitors: Synthesis, Molecular Docking, ADMET Profiling, and In Vitro Evaluation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFirst-generation poly(ADP-ribose) polymerase inhibitors (PARPi), including olaparib, talazoparib, niraparib, and rucaparib, have been approved for the treatment of certain ovarian, breast, prostate, and pancreatic cancers.\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e These currently approved PARPi inhibit both PARP1 and PARP2 (PARP1/2), which function as critical DNA damage sensors that bind to sites of damage and recruit repair factors through the formation of branched poly(ADP-ribose) (PAR) chains via PARylation.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e The therapeutic efficacy of PARP inhibitors is particularly pronounced in tumors harboring BRCA mutations.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Tumor cells with defective BRCA1/2 exhibit impaired homologous recombination repair mechanisms, and inhibition of PARP1 activity further exacerbates DNA damage accumulation.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e This combination results in synthetic lethality, whereby the simultaneous loss of both PARP-mediated base excision repair and BRCA-dependent homologous recombination leads to catastrophic DNA damage and subsequent apoptosis in cancer cells.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e This mechanism renders BRCA1/2-mutated tumors exceptionally vulnerable to PARP inhibition. However, hematological toxicities associated with the simultaneous inhibition of PARP1 and PARP2 have limited the clinical utility of these agents.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Consequently, the development of selective PARP1 inhibitors has emerged as a promising strategy to mitigate these adverse effects while maintaining therapeutic efficacy.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe phthalazinone scaffold represents an attractive pharmacophore for the design of selective PARP1 inhibitors based on its well-established binding profile within the PARP1 active site.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e This planar bicyclic core features multiple sites for hydrogen bonding and π-π stacking interactions, enabling optimal engagement with key amino acid residues including \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eLys903\u003c/span\u003e, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSer904\u003c/span\u003e, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGly888\u003c/span\u003e, and \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eTyr907\u003c/span\u003e, which are crucial for potent enzyme inhibition (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The nitrogen atoms within the phthalazinone ring serve as anchoring points for hydrogen bonds with polar residues, thereby enhancing binding affinity, while the aromatic system facilitates favorable hydrophobic interactions. Furthermore, this scaffold offers considerable synthetic flexibility, permitting the introduction of diverse substituents to fine-tune selectivity, potency, and pharmacokinetic properties.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Given its proven track record as the structural backbone in clinically successful PARP inhibitors, the phthalazinone scaffold provides a rational starting point for structure-based drug design guided by computational approaches and structure\u0026ndash;activity relationship (SAR) studies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this study, we present the design, synthesis, and thorough evaluation of novel \u003cem\u003eN\u003c/em\u003e-(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide and \u003cem\u003eN\u003c/em\u003e-(4-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide as potential PARP1 inhibitors. Our integrated approach combines molecular docking, ADME profiling, molecular dynamics, biochemical screening of the inhibiting properties and \u003cem\u003ein vitro\u003c/em\u003e cytotoxicity assessments against breast carcinoma cells with BRCA mutations Capan-1 (BRCA-2 mutation c.5946 delT), MDA-MB-436 (BRCA-1 mutation c.5396\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;A). Notably, molecular docking studies revealed binding scores of -8.5 kcal\u0026middot;mol\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e for \u003cem\u003eN\u003c/em\u003e-(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide (\u003cb\u003e4\u003c/b\u003e) and \u0026minus;\u0026thinsp;7.5 kcal\u0026middot;mol\u003csup\u003e-\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e for \u003cem\u003eN\u003c/em\u003e-(4-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide (\u003cb\u003e5\u003c/b\u003e) against PARP1, indicating the potential for further optimization of their structures. Additionally, molecular dynamics simulations of compound \u003cb\u003e4\u003c/b\u003e in water revealed no signs of molecular aggregation at either 50 or 100 ns. This observation is further supported by the solvent-accessible surface area (SASA) plot, which indicated only minor fluctuations in values throughout the simulation period, underscoring the compound's stability in aqueous environments. Moreover, the compound \u003cb\u003e4\u003c/b\u003e demonstrated higher binding energy than for talazoparib with the appropriate binding pattern during 250 ns MD simulation.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemistry\u003c/h2\u003e \u003cp\u003eThe synthetic route for the preparation of phthalazinone derivatives \u003cb\u003e4\u003c/b\u003e and \u003cb\u003e5\u003c/b\u003e is illustrated in Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. 4-oxo-3,4-dihydrophthalazine-1-carboxylic acid \u003cb\u003e3\u003c/b\u003e was synthesized used previously observed method from naphthalene \u003cb\u003e1\u003c/b\u003e oxidizing with KMnO\u003csub\u003e4\u003c/sub\u003e to afford intermediate \u003cb\u003e2\u003c/b\u003e that was stirred in EtOH with hydrazine hydrate at RT for 18h.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e The structure of compound 1 was confirmed by \u003csup\u003e1\u003c/sup\u003eH NMR spectroscopy, displaying a singlet at δ 13.10 ppm (1H, OH), multiplets between δ 8.61\u0026ndash;8.26 ppm (3H, aromatic), and characteristic aromatic signals at δ 7.97 and 7.88 ppm. The \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR spectrum supported the proposed structure, with resonances at δ 165.3 ppm (COOH), 160.0 ppm (C\u0026thinsp;=\u0026thinsp;O), and several signals between δ 126.3\u0026ndash;136.9 ppm (C\u003csub\u003eAr\u003c/sub\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe chloroanhydride of the 4-oxo-3,4-dihydrophthalazine-1-carboxylic acid was obtained by reacting it with thionyl chloride. Subsequently, triethylamine was added to a mixture of intermediate and amines in dichloromethane. This solution was stirred at room temperature overnight, resulting in the formation of the target compounds \u003cb\u003e4\u003c/b\u003e and \u003cb\u003e5\u003c/b\u003e. The structure of compounds \u003cb\u003e4\u003c/b\u003e and \u003cb\u003e5\u003c/b\u003e were confirmed by \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eC NMR spectroscopy. The comparison of the \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR spectra for \u003cem\u003eN\u003c/em\u003e-(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide and \u003cem\u003eN\u003c/em\u003e-(4-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide reveals differences in the chemical environments of the aromatic protons, reflecting the influence of the amido substituent's position on the phenyl ring. In both compounds, the characteristic downfield signals at δ 10.57 and δ 10.63 correspond to the NH groups, indicating their similar electronic environments. However, variations are evident in the aromatic region; for instance, the peaks at δ 8.52\u0026ndash;8.26 and δ 8.17 in the NMR spectra shift for compound \u003cb\u003e4\u003c/b\u003e to δ 8.41 and δ 8.32 in the compound \u003cb\u003e5\u003c/b\u003e, suggesting a change in coupling patterns due to the different substitution on the phenyl ring. Additionally, the aromatic multiplets exhibit slight shifts and changes in multiplicity, indicative of the spatial arrangement and electronic interactions influenced by the amide group positioning.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular dynamic studies\u003c/h3\u003e\n\u003cp\u003eTo gain deeper insights into the aggregation behavior and self-assembly properties of compounds \u003cb\u003e4\u003c/b\u003e and \u003cb\u003e5\u003c/b\u003e in aqueous solution, molecular dynamics simulations were performed. Understanding the clustering tendencies of these compounds is crucial for predicting their bioavailability, formulation stability, and potential therapeutic efficacy.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Molecular dynamics simulations of compound \u003cb\u003e5\u003c/b\u003e in aqueous solution revealed that molecular aggregation occurred by 50 ns and 100ns, leading to cluster formation (\u003cb\u003eFig.\u0026nbsp;2\u003c/b\u003e). At the end of the simulation, stable molecular clusters were observed in the system. The high clustering potential in water was further confirmed by the solvent-accessible surface area (SASA) plot, which showed a continuous decrease throughout the dynamics (\u003cb\u003eFig.\u0026nbsp;2\u003c/b\u003e). In contrast, molecular dynamics simulations of compound \u003cb\u003e4\u003c/b\u003e in water showed no molecular aggregation at either 50 or 100 ns (\u003cb\u003eFig.\u0026nbsp;2\u003c/b\u003e). The absence of significant clustering potential for compound \u003cb\u003e5\u003c/b\u003e in aqueous solution was also supported by the SASA plot, which demonstrated only minor changes in values throughout the dynamics.\u003c/p\u003e \u003cp\u003eThe contrasting aggregation profiles observed in MD simulations\u0026mdash;pronounced clustering for compound \u003cb\u003e5\u003c/b\u003e versus stable dispersion for compound \u003cb\u003e4\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;2.\u003c/b\u003e Molecular dynamics simulations of compound \u003cb\u003e4\u003c/b\u003e (Fig.\u0026nbsp;2a and 2b) and \u003cb\u003e5\u003c/b\u003e in aqueous solution (2\u003cem\u003ec and 2d)\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ecytotoxicity studies\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo assess the anticancer potential of the synthesized PARP inhibitors, \u003cem\u003ein vitro\u003c/em\u003e cytotoxicity studies were performed against breast carcinoma cell lines (Capan-1 and MDA-MB-436) with BRCA mutations. These cell lines were selected due to their documented sensitivity to PARP inhibition and relevance in cancer research\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Furthermore, an investigation was conducted into the possible cell death mechanisms mediated by hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). The effect of compound \u003cb\u003e4\u003c/b\u003e was stronger than that of compound \u003cb\u003e5\u003c/b\u003e (\u003cem\u003eC\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.5\u0026ndash;450 \u0026micro;M) on both cell lines.\u003c/p\u003e\n\u003ch3\u003eMolecular docking studies\u003c/h3\u003e\n\u003cp\u003eThe affinity of ligands to the catalytic domain of PARP1 at the docking stage was evaluated using two FDA aproved PARP inhibitors namely Olaparib and Talazoparib as benchmarks. The docking grid was determined and centered by the ligand structure originally located in the receptor. As a result of Induced Fit docking, the poses with the lowest binding energy calculated by MM-GBSA method were selected for each ligand which were subjected for further molecular dynamics (MD) simulation (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). It can be seen that compound \u003cb\u003e4\u003c/b\u003e having a more stable and favorable interaction with the target compared to compound \u003cb\u003e5\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\u003eResults of IFD for compounds \u003cb\u003e4\u003c/b\u003e and \u003cb\u003e5\u003c/b\u003e compared to known inhibitors. The top-poses are presented.\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\u003eInhibitor\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXP GScore\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eΔG\u003csub\u003ebind\u003c/sub\u003e (kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-8.505\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-76.28\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-7.555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-73.96\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOlaparib\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-11.524\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-47.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTalazoparib\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e-8.055\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-48.4\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\u003eThe pharmacokinetic properties and suitability of the compounds as drugs were evaluated using predictive descriptors, including molecular weight, the number of hydrogen bond donors and acceptors, the percentage of estimated absorption in humans, and the number of violations of the Lipinski five rule (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2). The characteristics of the compound \u003cb\u003e4\u003c/b\u003e and \u003cb\u003e5\u003c/b\u003e were compared with those of FDA approved inhibitors. The synthesized compounds, characterized by their structural similarities, exhibited favorable drug-likeness properties. Both compounds possess a molecular weight of 364.403 g/mol, with two hydrogen bond donors and six hydrogen bond acceptors; additionally, they display a logP value of 3.2. The estimated oral absorption is 86%, which indicates a potentially high oral bioavailability.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,20\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eDuring 250 ns MD simulation, all known inhibitors demonstrated acceptable stability within the active sites of PARP1 catalytic domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). As regards compound \u003cb\u003e5\u003c/b\u003e, it showed great stability within the active site of the receptor. Nonetheless, \u003cb\u003e5\u003c/b\u003e demonstrated several visible fluctuations temporarily while being shifted within the binding sites tending to find a more favorable location. Nevertheless, the compound \u003cb\u003e5\u003c/b\u003e was still inside the binding site and the average range of RMSD value did not extend 2\u0026Aring;. The main part of the stabilization has been achieved by phthalazin-1(2\u003cem\u003eH\u003c/em\u003e)-one moiety being connected to Tyr907 by π-π stacking and to Gly863 and Ser904 by H-Bonds. In addition, the NH of 3-methylbutanamide fragment was stabilized by H-bond with Asp766. As for compound \u003cb\u003e4\u003c/b\u003e, it showed flat RMSD graph except one fluctuation on 100 ns which could be explained by the fact that ligand lost connection with Tyr889 and compensated that with Ser904. Results demonstrated almost the same stabilization profile except the 4-methylbutanamide moiety was stabilized by H-bond with Gln759 connected to oxygen atom. In sum, the interaction interface of the compound \u003cb\u003e4\u003c/b\u003e is comparable to known inhibitors demonstrating H-bonds with Gly863 and Ser904 and π-π stacking with Tyr907 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The key interacting residues of PARP-1 with known inhibitors were also observed fort he compounds \u003cb\u003e4\u003c/b\u003e and \u003cb\u003e5\u003c/b\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The calculated ΔG\u003csub\u003ebinding\u003c/sub\u003e of compound \u003cb\u003e4\u003c/b\u003e is comparable for that of Talazoparib The compound \u003cb\u003e4\u003c/b\u003e showed much greater binding affinity than the compound \u003cb\u003e5\u003c/b\u003e and Talazoparib (73,5 kcal/mol \u003cem\u003evs\u003c/em\u003e. -66, 8 and 59,6 kcal/mol, respectively). Both compounds \u003cb\u003e4\u003c/b\u003e and \u003cb\u003e5\u003c/b\u003e were characterized with lower binding strength than that of Olaparib due to the larger interaction surface of the known inhibitor (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These findings align with cytotoxicity data, which indicate that the substitution at position 4(\u003cem\u003epara\u003c/em\u003e) of \u003cem\u003eN\u003c/em\u003e-(R-aminophenyl)-3-methylbutanamide enhances its affinity for PARP.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMM/GBSA free energy of binding (ΔG\u003csub\u003e\u003cem\u003ebind\u003c/em\u003e\u003c/sub\u003e) of ligands with PARP1 calculated over 250 ns MD simulation. Common residues of PARP-1 interacting with ligands and Olaparib are underlined; common residues with Talazoparib presented in bold.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePARP1-ligand\u003c/p\u003e \u003cp\u003ecomplex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMM-GBSA mean ΔG\u003csub\u003ebind\u003c/sub\u003e over 250 ns MD (kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInteracting residue positions of PARP-1\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e-73.5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVal762, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAsp766\u003c/span\u003e, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eGly863\u003c/span\u003e, \u003cb\u003eTyr889\u003c/b\u003e, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eTyr896\u003c/span\u003e, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eSer904\u003c/span\u003e, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eTyr907\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e-66.8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGln759, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAsp766\u003c/span\u003e, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eGly863\u003c/span\u003e, \u003cb\u003eTyr889\u003c/b\u003e, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eTyr896\u003c/span\u003e, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eLys 903\u003c/span\u003e, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eSer904\u003c/span\u003e, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eTyr907\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOlaparib\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-82.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAsp766\u003c/span\u003e, Asp770, His862, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eGly863\u003c/span\u003e, Ser864, Ile872, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eTyr896\u003c/span\u003e, \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eLys903\u003c/span\u003e, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eSer904\u003c/span\u003e, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eTyr907\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTalazoparib\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-59.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGlu763, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eAsp766\u003c/span\u003e, Asn767, Asp770, His862, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eGly863\u003c/span\u003e, Ser864, Gly888, \u003cb\u003eTyr889\u003c/b\u003e, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eTyr896\u003c/span\u003e, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eSer904\u003c/span\u003e, \u003cspan type=\"BoldUnderline\" class=\"BoldUnderline\" name=\"Emphasis\"\u003eTyr907\u003c/span\u003e, Glu988\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003ePARP1 enzymatic activity\u003c/h3\u003e\n\u003cp\u003eUltimately, the inhibitory potential of compounds \u003cb\u003e4\u003c/b\u003e and \u003cb\u003e5\u003c/b\u003e against PARP1 enzymatic activity was evaluated. For this purpose, we employed a previously developed fluorescence anisotropy-based assay system, which measures the binding of PARP1 to a fluorescently labeled DNA duplex and its dissociation from complex with DNA under authoPARylation \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. The results are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhile compound \u003cb\u003e5\u003c/b\u003e exhibited no discernible effect on PARP1 activity, compound \u003cb\u003e4\u003c/b\u003e demonstrated a significant inhibitory effect. Specifically, compound 4 suppressed the dissociation of PARP1 from its complex with DNA during the PARylation process, with a half-maximal inhibitory concentration (\u003cem\u003eIC₅₀\u003c/em\u003e) of 6.5 \u0026micro;M. Under analogous experimental conditions, the \u003cem\u003eIC₅₀\u003c/em\u003e value for the reference inhibitor olaparib was determined to be 13 nM. In the presence of 200 \u0026micro;M compound \u003cb\u003e4\u003c/b\u003e, the residual PARP1 activity was approximately 20%. This data aligns consistently with the cell-based cytotoxicity assays.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, this study successfully delineates the design, synthesis, and comprehensive evaluation of two novel compounds, \u003cem\u003eN\u003c/em\u003e-(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide and \u003cem\u003eN\u003c/em\u003e-(4-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide, as promising inhibitors of PARP1. The molecular docking, ADME profiling, and in vitro cytotoxicity assessments has provided a robust framework for evaluating their potential therapeutic efficacy. The molecular docking results, with binding affinities of -8.5 kcal/mol and \u0026minus;\u0026thinsp;7.5 kcal/mol, respectively, suggest that both compounds exhibit significant interaction with PARP1, warranting further structural optimization. Both \u003cem\u003eN\u003c/em\u003e-(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide and \u003cem\u003eN\u003c/em\u003e-(4-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide exhibited a growth-inhibitory effect on native cells, with the effect being more pronounced after pre-incubation with hydrogen peroxide. Notably, the effect of \u003cem\u003eN\u003c/em\u003e-(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide was more significant than that of \u003cem\u003eN\u003c/em\u003e-(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide, as it was evident across the entire dose range (4.5\u0026ndash;450 \u0026micro;M) on both cell lines. The biochemical data obtained from experiments with recombinant PARP1 align consistently with the cell-based cytotoxicity assays: \u003cem\u003eN\u003c/em\u003e-(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide (\u003cb\u003e4\u003c/b\u003e) exhibited PARP1 inhibitory effect with micromolar \u003cem\u003eIC\u003c/em\u003e\u003csub\u003e\u003cem\u003e50\u003c/em\u003e\u003c/sub\u003e value. This correlation supports the proposed mechanism of action, whereby the growth-inhibitory effects observed for compound \u003cb\u003e4\u003c/b\u003e in cell lines are mediated, at least in part, through the direct inhibition of PARP1 enzymatic function.\u003c/p\u003e"},{"header":"Experimental Section","content":" \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eGeneral Information (Synthesis)\u003c/h2\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eН and \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eС{\u003csup\u003e1\u003c/sup\u003eН} NMR spectra were obtained on a Bruker Avance III HD 400 NanoBay spectrometer (Germany) (400.13 MHz for \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eН and 100.61 MHz for \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eС) in DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e at 298.15 K. The melting point was determined on a Kofler hot stage equipped with a microscope at a heating rate of 1\u0026deg;C/min. The reaction progress was monitored by TLC on Merck Kieselgel 60 F254 plates. Mass spectral analysis was performed on a Shimadzu Nexera X2 LCMS-9030 chromatography-mass spectrometer (Japan).\u003c/p\u003e \u003cp\u003e \u003cem\u003eN\u003c/em\u003e-(3-aminophenyl)-3-methylbutanamide (CAS 926225-62-9) and \u003cem\u003eN\u003c/em\u003e-(4-aminophenyl)-3-methylbutanamide (CAS 189576-50-9) are commercially available.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eSynthesis of 2-(carboxycarbonyl)benzoic acid (2)\u003c/h3\u003e\n\u003cp\u003eNaphthalene (39.2 mmol) was added to 150 mL of 0.5 N NaOH and subjected to reflux. A solution of potassium permanganate (196.2 mmol) in 600 mL of water was added dropwise to the refluxing solution over a period of 1.5 hours. Once the dropwise addition was complete, the reaction was allowed to proceed for an additional 45 minutes to ensure complete oxidation. The reaction was then quenched by the addition of 120 mL of ethanol, after which the mixture was cooled to room temperature and subjected to suction filtration. The resulting filter cake was acidified with 30% hydrochloric acid to a pH of 5 and subsequently extracted with ethyl acetate (100 mL \u0026times; 3). The organic layers were combined and dried over anhydrous sodium sulfate. The solvent was then evaporated under reduced pressure, yielding compound \u003cb\u003e2\u003c/b\u003e as a white solid with a yield of 33.0% (2.51 g). The product was characterized as a colorless powder with a melting point (T\u003csub\u003em\u003c/sub\u003e) of 143\u0026ndash;145\u0026deg;C. The obtained data were consistent with previously published results\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of 4-oxo-3,4-dihydrophthalazine-1-carboxylic acid (3)\u003c/h2\u003e \u003cp\u003eHydrazine hydrate (15.0 mmol) was dissolved in a solution of ethanol and water in a 1:2 ratio. Subsequently, compound 2 (12.5 mmol) was added to this solution. The resulting mixture was stirred at room temperature for 18 hours. Afterward, the precipitate was filtered, washed twice with dichloromethane, and dried under vacuum to obtain 4-oxo-3,4-dihydrophthalazine-1-carboxylic acid (\u003cb\u003e3\u003c/b\u003e). The yield was 68% (1.6 g), resulting in white crystals with a melting point (Tm) of 230\u0026ndash;232\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e), δ: 13.10 (s, 1H, O\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e), 8.61\u0026ndash;8.52 (m, 1H, Ar), 8.31\u0026ndash;8.26 (m, 1H, Ar), 7.97 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4, 7.2, 1.5 Hz, 1H, Ar), 7.88 (td, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6, 1.2 Hz, 1H, Ar). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e), δ: 165.3 (COOH), 160.0 (C\u0026thinsp;=\u0026thinsp;O), 136.9 (Ar), 134.4 (Ar), 132.3 (Ar), 128.3 (Ar), 127.9 (Ar), 126.8 (Ar), 126.3 (Ar). The obtained data fully agree with previously published results\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of compounds 4 and 5\u003c/h2\u003e \u003cp\u003e6 mol of thionyl chloride was added to compound \u003cb\u003e3\u003c/b\u003e (5.5 mmol) in a 20 mL pyridine, and the mixture was heated to reflux for 6 hours. Following this, any excess liquid was evaporated under reduced pressure to yield 4-oxo-3,4-dihydrophthalazine-1-carbonyl chloride. A solution containing 1.0 mmol of the corresponding amine (\u003cem\u003eN\u003c/em\u003e-(3-aminophenyl)-3-methylbutanamide for compound \u003cb\u003e4\u003c/b\u003e and \u003cem\u003eN\u003c/em\u003e-(4-aminophenyl)-3-methylbutanamide for compound \u003cb\u003e5\u003c/b\u003e) and 1.2 mmol of triethylamine (Et₃N) in 30 mL of dichloromethane (DCM) was prepared while cooling in an ice bath. A solution of compound \u003cb\u003e4\u003c/b\u003e (1.1 mmol) in 30 mL of DCM was then added dropwise to the reaction mixture. Subsequently, the mixture was allowed to warm to room temperature and stirred for 10 hours. Upon completion of the reaction, the organic layer was washed with a 10% aqueous solution of potassium carbonate (10 mL \u0026times; 3). The organic phase was then dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure by rotary evaporation to afford the crude product. Purification was carried out by column chromatography on silica gel using a chloroform/methanol (CHCl₃/MeOH, 9:1, \u003cem\u003ew/w\u003c/em\u003e) eluent system to obtain compounds \u003cb\u003e4\u003c/b\u003e and \u003cb\u003e5\u003c/b\u003e as pure fractions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eN-(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide (4)\u003c/h2\u003e \u003cp\u003eYield: 70.3% (0.559 g). White powder. T\u003csub\u003em\u003c/sub\u003e = 251\u0026ndash;253\u0026deg;C. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 13.08 (s, 1H), 10.63 (s, 1H), 9.95 (s, 1H), 8.52\u0026ndash;8.26 (m, 2H), 8.17 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H), 7.95 (dtd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;26.1, 7.3, 1.4 Hz, 2H), 7.54\u0026ndash;7.34 (m, 2H), 7.28 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1 Hz, 1H), 2.20 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1 Hz, 2H), 0.94 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.6 Hz, 6H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO) δ 171.23, 162.56, 160.01, 140.56, 140.11, 139.15, 134.42, 132.59, 129.37, 128.16, 127.98, 126.58, 126.44, 115.56, 115.39, 111.53, 46.08, 26.11, 22.76. Found, \u003cem\u003em/z\u003c/em\u003e: 365.1. [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e. C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e. Calculated, \u003cem\u003em/z\u003c/em\u003e: 365.4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eN-(4-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide (5)\u003c/h2\u003e \u003cp\u003eYield: 74.4% (0.592 g). Pink powder. T\u003csub\u003em\u003c/sub\u003e = 242\u0026ndash;244\u0026deg;C. \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) δ 13.08 (s, 1H), 10.57 (s, 1H), 9.88 (s, 1H), 8.41 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5 Hz, 1H), 8.32 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.1, 1.3 Hz, 1H), 8.03\u0026ndash;7.87 (m, 2H), 7.80\u0026ndash;7.65 (m, 2H), 7.65\u0026ndash;7.47 (m, 2H), 2.19 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1 Hz, 2H), 0.94 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.5 Hz, 6H). \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO) δ 170.95, 162.26, 160.03, 140.46, 136.03, 134.40, 134.09, 132.58, 128.16, 128.01, 126.74, 126.41, 120.96, 119.91, 46.04, 26.07, 22.78. Found, \u003cem\u003em/z\u003c/em\u003e: 365.5. [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e. C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e. Calculated, \u003cem\u003em/z\u003c/em\u003e: 365.4.\u003c/p\u003e \u003cp\u003eInformation on Molecular Dynamics Simulation, in vitro cytotoxicity, determination of IC₅₀ by fluorescence anisotropy, and molecular modeling and in silico prediction can be found in the Supplementary Materials\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eDeclaration of competing interest\u0026nbsp;\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\u003eFundings\u003c/p\u003e\n\u003cp\u003eThis work was supported by Ministry of Health of the Russian Federation 1024021500062-3-3.2.21;3.1.5;1.6.4 (Creation of a selective poly(ADP-ribose) polymerase inhibitor of the first type (PARP1) based on condensed heterocyclic systems).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors acknowledge the Russian Science Foundation for supporting the recombinant PARP1 purification research project (Grant №25-74-10025), which facilitated the acquisition of PARP1 for this study.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003e1-4 wrote the main manuscript text 5-7 prepared figures, 1-3, 8,10-11 prepared SI. 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Anal Biochem 545:91\u0026ndash;97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ab.2017.12.033\u003c/span\u003e\u003cspan address=\"10.1016/j.ab.2017.12.033\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Scheme ","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-computer-aided-molecular-design","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jcam","sideBox":"Learn more about [Journal of Computer-Aided Molecular Design](http://link.springer.com/journal/10822)","snPcode":"10822","submissionUrl":"https://submission.nature.com/new-submission/10822/3","title":"Journal of Computer-Aided Molecular Design","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"PARP-1, phthalazinone-based compounds, DFT, ADMET, count-assay","lastPublishedDoi":"10.21203/rs.3.rs-8688192/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8688192/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study reports the design, synthesis, and biological evaluation of two novel phthalazinone-based carboxamides, \u003cem\u003eN\u003c/em\u003e-(3-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide and \u003cem\u003eN\u003c/em\u003e-(4-(3-methylbutanamido)phenyl)-4-oxo-3,4-dihydrophthalazine-1-carboxamide, as potential poly(ADP-ribose) polymerase-1 (PARP1) inhibitors. Structure-based molecular docking indicated favorable binding of both compounds within the PARP1 active site, with binding energies of \u0026minus;\u0026thinsp;8.5 and \u0026minus;\u0026thinsp;7.5 kcal\u0026middot;mol⁻\u0026sup1;, respectively. The stability and key interaction patterns of the more potent meta-substituted analogue were further validated by 250 ns molecular dynamics simulations. \u003cem\u003eIn silico\u003c/em\u003e ADMET profiling suggested acceptable drug-like properties for the synthesized compounds. Biochemical evaluation revealed enhanced inhibitory activity of the meta-substituted derivative against recombinant PARP1. Furthermore, both compounds exhibited cytotoxic effects in BRCA-mutant breast cancer cell lines, Capan-1 (BRCA2 c.5946delT) and MDA-MB-436 (BRCA1 c.5396\u0026thinsp;+\u0026thinsp;1G\u0026thinsp;\u0026gt;\u0026thinsp;A). Comparative analysis highlights the critical influence of the substituent position on the phenyl ring in modulating PARP1 binding stability and inhibitory activity. These findings identify the phthalazinone carboxamide scaffold as a promising platform for further structural optimization toward potent PARP1 inhibitors.\u003c/p\u003e","manuscriptTitle":"Design and Discovery of Phthalazinone-Based Potential PARP Inhibitors: Synthesis, Molecular Docking, ADMET Profiling, and In Vitro Evaluation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-04 19:17:09","doi":"10.21203/rs.3.rs-8688192/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-27T21:45:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-27T11:06:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-23T15:40:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"100228934032664091927102436389402971830","date":"2026-04-23T14:44:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"120643341379546544763087503224007478863","date":"2026-04-23T04:07:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"290436540658168713227268253153892611404","date":"2026-04-22T04:55:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"216860427967809051361430758740323099117","date":"2026-04-21T07:41:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81862786202667181316119748649707490906","date":"2026-04-21T02:34:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-04T16:54:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"24336984608449533821439895207890877478","date":"2026-03-29T10:47:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-03T01:07:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-03T01:02:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-28T04:51:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Computer-Aided Molecular Design","date":"2026-01-24T16:29:33+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-computer-aided-molecular-design","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jcam","sideBox":"Learn more about [Journal of Computer-Aided Molecular Design](http://link.springer.com/journal/10822)","snPcode":"10822","submissionUrl":"https://submission.nature.com/new-submission/10822/3","title":"Journal of Computer-Aided Molecular Design","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"246177f1-ddd7-4dc8-84f1-c10bd45b4c35","owner":[],"postedDate":"February 4th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T17:38:20+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-04 19:17:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8688192","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8688192","identity":"rs-8688192","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}