Quinoline-6-Carboxylic Acid Derivatives: A New Class of Potent Ectonucleotidase Inhibitors

Quinoline-6-Carboxylic Acid Derivatives: A New Class of Potent Ectonucleotidase Inhibitors | 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 Quinoline-6-Carboxylic Acid Derivatives: A New Class of Potent Ectonucleotidase Inhibitors Aqsa Ishaq, Ismat Nawaz, Javeria Qadir, Salman Alrokayan, Tajamul Hussain, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6824809/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ectonucleotidases, including h -NTPDases, h -ENPP, and h -e5′NT, play a crucial role in regulating extracellular nucleotide levels by converting ATP into immunosuppressive adenosine, thereby facilitating tumor immune evasion. Inhibiting these enzymes can restore antitumor immunity by preventing adenosine accumulation within the tumor microenvironment. Herein, we report the design and synthesis of quinoline-6-carboxylic acid derivatives (4a–4l), a biologically relevant scaffold, and evaluate their potential to inhibit recombinant h-ENPP1, h -e5′NT, and h -NTPDases. This study led to the identification of new and effective inhibitors, such as compound 4d , which showed good inhibition against h -NTPDase1 and -2, as reflected by IC 50 value of 0.28±0.03 µM against h-NTPDase1 and 0.92±0.17 µM against h -NTPDase2. Similarly, compound 4g inhibited h- NTPDase3 with an IC 50 value of 0.32±0.05 µM, and compound 4b inhibited h -NTPDase8 with an IC 50 value of 0.44±0.08 µM. Additionally, compound 4k demonstrated good inhibition against h -ENPP1 as reflected by IC 50 value 0.11±0.02 µM. Finally, compound 4a inhibited e5’NT enzyme with an IC 50 value of 0.092±0.02 µM. Molecular docking studies were performed to complement the in vitro analysis, revealing that the tested compounds show favorable interaction with the amino acid of the target enzymes h -NTPDase1, -2, -3, and -8, h -NPP1, and h -e5′NT enzymes. These interactions involve residues such as Asp201, Asp213, Asp218, Asp326, Ala412, Tyr340, Tyr371, Thr90, Trp408, Trp450, Cys502, Gly178, Phe360, Phe257, Arg392, Ala347, Leu202, Leu290, His50, His360, His380, Ser49, Ser100, Gln148, Glu266, Lys50, Lys295, Gln523, Pro323, and Ile90. Density Functional Theory (DFT) analysis revealed that compound 4f exhibits the lowest energy gap, which correlates well with its strong ectonucleotidase inhibitory activity against NPP1 and NTPDase3. Finally, fluorescence microscopy was conducted to investigate the interaction of the compound 4d with plasma membrane in A549 lung cancer cells. Fluorescence imaging of compound 4d exhibited strong cellular emission, confirming its effective interaction with membrane-bound enzymes in these cancer cells. MTT and apoptosis studies also revealed compound 4i , 4j , and 4k to be potentially cytotoxic in the cancer cells, endorsing the potential of these quinoline derivatives to be used for drug development for cancer management. Chemical Biology h-NTPDase h-NPP1 and h-e5′NT Quinoline-6-Carboxylic Acid derivatives Inhibitors In silico studies and In vitro assessment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. INTRODUCTION Tumor progression is intricately linked to immune cell infiltration and the resulting inflammatory milieu, which collectively promote angiogenesis, immune escape, and metastasis [ 1 ]. Tumors also evolve mechanisms to subvert immune detection by manipulating molecular cues within the tumor microenvironment (TME). A central component of this immune evasion is the accumulation of adenosine, a small molecule with potent immunosuppressive activity that dampens the function of tumor-infiltrating leukocytes through the activation of A2A and A2B adenosine receptors [ 2 ] [ 3 ]. Adenosine levels in the TME are tightly regulated by ectonucleotidases—cell surface enzymes that sequentially hydrolyze extracellular ATP into AMP and subsequently into adenosine microenvironment [ 4 ]. These enzymes consist of several types such as nucleoside triphosphate diphosphohydrolases (NTPDases), alkaline phosphatases (ALPs), nucleotide pyrophosphatases /phosphodiesterases (NPPs), and ecto-5′-nucleotidase (e5′NT) [ 5 , 6 ]. NTPDases hydrolyze ATP to ADP and AMP, while e5′NT converts AMP to adenosine as illustrated in Fig. 1 . NPPs, such as NPP1, also participate by hydrolyzing nucleotides to monophosphates and pyrophosphate [ 7 ]. Overexpression of these ectonucleotidases has been reported in multiple types of cancer, such as melanoma [ 8 ] breast [ 9 ][ 10 ], and prostate cancers[ 11 ]. Their overactivity is associated with tumor growth, suppression of immune responses, and poor clinical outcomes [ 12 – 14 ]. Increased enzymatic activity leads to adenosine accumulation in the TME, which promotes tumor tolerance by inhibiting T and B cell function, natural killer (NK) cell cytotoxicity, and dendritic cell (DC) activation [ 15 , 16 ]. Beyond cancer, aberrant ectonucleotidase expression is implicated in conditions such as insulin resistance [ 17 ], cardiovascular disease [ 18 ], and pathological mineralization, reinforcing their therapeutic relevance[ 19 ]. Inhibiting these enzymes offers a promising strategy to reduce adenosine levels, reverse immunosuppression, and enhance anti-tumor immunity [ 20 – 22 ]. A diverse array of heterocyclic compounds has been explored for the targeted treatment of various cancers, whereby, both nucleotide- and non-nucleotide-based structures, have been investigated as ectonucleotidase inhibitors. Among them, quemliclustat (AB680), a nucleotide-based inhibitor, has shown promising preclinical results and is currently undergoing Phase 1 clinical trials for cancer therapy [ 23 ]. In addition, non-nucleotide ectonucleotidase inhibitors such as sulfonate derivatives have also been reported for their anticancer potential [ 24 – 28 ]. Likewise, several pyrazole-based ectonucleotidase inhibitors have demonstrated remarkable cytotoxic activity against various cancer cell lines, highlighting the therapeutic promise of such scaffolds [ 29 ]. Representative examples of standard ectonucleotidase inhibitors are illustrated in Fig. 2 . The design of quinoline-6-carboxylic acid amides as potential ectonucleotidase inhibitors is based on both structural features and evidence from literature. Quinoline is a well-known heterocyclic scaffold that has been widely studied for its anticancer potential [ 30 , 31 ]. Its flat, aromatic structure and nitrogen atom make it structurally similar to purine-based molecules like ATP, allowing it to fit into nucleotide-binding pockets of the enzymes such as CD39 and CD73 [ 32 – 35 ]. Moreover, quinoline derivatives have shown activity against several cancer-related targets, including topoisomerases[ 30 ] and protein kinases[ 36 ]. Introducing a carboxylic acid group at the 6-position of the quinoline ring allows the molecule to mimic phosphate groups found in natural nucleotides, promoting key polar interactions at the enzyme’s active site. Converting this acid to an amide improves the molecule’s stability, solubility, and membrane permeability—important traits for inhibiting extracellular enzymes like ectonucleotidases [ 37 – 42 ]. Contextual to this, recent studies have reported quinoline-based carboxamides to be effective inhibitors of ATP-hydrolyzing enzymes, suggesting their potential interaction with the catalytic regions of the enzymes involved in purine metabolism [ 37 , 38 ]. Given their known anticancer properties and favorable drug-like features, we were inspired to design, synthesize and explore quinoline-6-carboxylic acid amides as inhibitors of ectonucleotidases—another promising target involved in cancer immune evasion. In addition, molecular docking studies were conducted to predict their binding affinities and interactions with the target proteins in-silico . The biological activity of the synthesized compounds was, then, evaluated through fluorescence assay to assess cellular interaction, MTT assay for measuring cytotoxicity and apoptosis assay to determine the cell-death inducing potential of these compounds. 2. EXPERIMENTAL SECTION 2.1 General Chemistry Methods: All the solvents used in this study were of analytical grade and used without further purification. Chemicals were purchased from local suppliers and used directly in the reactions. Melting points of the synthesized compounds were measured using a Gallenkamp melting point apparatus. Reaction progress was monitored by Thin Layer Chromatography (TLC) using Merck pre-coated silica gel plates (60 F254), with spot detection under UV light at 254 nm to confirm complete consumption of the starting materials. Structural analysis of the compounds was performed using a Bruker Avance III HD NMR spectrometer operating at 300 and 400 MHz. Both proton ( 1 H) and carbon ( 13 C) NMR spectra were recorded, with chemical shifts reported in parts per million (ppm) and coupling constants (J) in Hertz (Hz). Signal multiplicities were reported as follows: s for singlet, d for doublet, t for triplet, and q for quartet. General synthetic procedure (A) for Quinoline-Based Amide Derivatives (4a-4l) Quinoline-6-carboxylic acid (1.15 mmol, 200 mg, 1 equiv) was reacted with thionyl chloride (3.46 mmol, 412 mg, 3 equiv) in the presence of DCM (20 mL) as a solvent under reflux at 50°C for 8 hours. The conversion of the acid into the corresponding acid chloride was monitored by TLC. Once the conversion was complete, the acid chloride was treated with various amines and stirred overnight, with the reaction progress monitored through TLC. Upon completion, the reaction mixture was concentrated using a rotary evaporator to remove the solvent. The resulting residue was added to a beaker containing distilled water and stirred until a precipitate formed. The precipitate was filtered and dried in an oven at 37°C, yielding quinoline-based amide derivatives (4a–4l) in good to excellent yields. N -(4-chlorobenzyl)quinoline-6-carboxamide (4a) . Using the general synthetic protocol, A , (4-Chlorophenyl)methanamine (1.15 mmol, 164 mg, 140 µL, 1 equiv) was employed as the starting amine, yielding the target compound 4a as a sand brown solid (67%); m.p: 185–187°C; R f = 0.3 (hexane/ EtOAc = 3:2); 1 H NMR (400 MHz, DMSO) ppm δ 9.32 (t, J = 6.0 Hz, 1H, N12-H), 8.99 (dd, J = 4.2, 1.7 Hz, 1H, C2-H), 8.55 (d, J = 2.0 Hz, 1H, C5-H), 8.48 (dd, J = 8.4, 1.7 Hz, 1H, C4-H), 8.21 (dd, J = 8.7, 2.0 Hz, 1H, C7-H), 8.09 (d, J = 8.8 Hz, 1H, C8-H), 7.61 (dd, J = 8.3, 4.2 Hz, 1H, C3-H), 7.40 (s, 4H, C15, 16, 18, 19-H), 4.53 (d, J = 5.9 Hz, 2H, C13-H); 13 C NMR (101 MHz, DMSO) δ 166.0 (s, C-11), 152.1 (s, C-2), 148.7 (s, C-9), 138.6 (s, C-14), 137.1 (s, C-4), 132.0 (s, C-17), 131.4 (s, C-10), 129.2 (s, C-15, 19), 129.1 (s, C-10), 128.3 (s, C-5), 128.1(s, C-8), 127.7 (s, C-16, C18), 127.1 (s, C-7), 122.2 (s, C-3), 42.2 (s, C-13) ; FT-IR (neat) ῡ (cm –1 ) = 3453cm − 1 (N-H stretching), 1654cm − 1 (C = O stretching). N -(4-fluorobenzyl)quinoline-6-carboxamide (4b). According to general procedure, A , (4-fluorophenyl)methanamine (1.15 mmol, 145 mg, 141µL, 1 equiv) was employed as the starting amine, yielding the target compound 4b as a chocolate brown solid (64%); m.p: 150–152°C; R f = 0.3 (hexane/ EtOAc = 4.6); 1 H NMR (300 MHz, DMSO) ppm δ 9.31 (t, J = 6.0 Hz, 1H, N12-H), 8.99 (dd, J = 4.2, 1.8 Hz, 1H, C2-H), 8.55 (d, J = 2.0 Hz, 1H, C5-H), 8.48 (dd, J = 8.2, 2.3 Hz, 1H, C4-H), 8.21 (dd, J = 8.8, 2.0 Hz, 1H, C7-H), 8.09 (d, J = 8.8 Hz, 1H, C8-H), 7.61 (dd, J = 8.3, 4.2 Hz, 1H, C3-H), 7.46–7.33 (m, 2H, C16,18-H), 7.17 (t, J = 8.9 Hz, 2H C15,19-H), 4.53 (d, J = 5.9 Hz, 2H, C13-H); 13 C NMR (75 MHz, DMSO) ppm δ 166.3 (s, C-11), 161.7 (d, 1 J C−F = 240.8 Hz, s, C-17), 152.6 (s, C-2), 149.1 (s, C-9), 137.9 (s, C-4), 136.2 (d, 4 J C−F = 2.0 Hz, C-14), 132.6 (s, C-10), 129.8 (d, 3 J C−F = 8.1 Hz, C-15), 129.5 (s, C-5), 128.5 (s, C-8), 128.2 (s, C-7), 127.6 (s, C-6) 122.7 (s, C-3), 115.5 (d, 2 J C−F = 21.1 Hz, C-16), 42.6 (s, C-13); FT-IR (neat) ῡ (cm –1 ) = 3279cm − 1 (N-H stretching), 1635cm − 1 (C = O stretching). N -(4-methylbenzyl)quinoline-6-carboxamide (4c). Using the general synthetic protocol, A , p-tolymethanamine (1.15 mmol, 139 mg, 147 µL, 1 equiv) was employed as the starting amine, yielding the target compound 4c as a light brown solid (70%); m.p: 190–192°C; R f = 0.4 (hexane/ EtOAc = 4:1); 1 H NMR (300 MHz, DMSO) ppm δ 9.33 (t, J = 6.0 Hz, 1H, N12-H), 8.98 (dd, J = 4.2, 1.7 Hz, 1H, C2-H), 8.57 (d, J = 2.0 Hz, 1H, C5-H), 8.47 (d, J = 8.3 Hz, 1H, C4-H), 8.22 (dd, J = 8.8, 2.0 Hz, 1H, C7-H), 8.08 (d, J = 8.8 Hz, 1H, C8-H), 7.61 (dd, J = 8.3, 4.2 Hz, 1H, C3-H), 7.25 (d, J = 7.8 Hz, 2H, C19-H), 7.14 (d, J = 7.8 Hz, 2H, C16-H), 4.49 (d, J = 5.9 Hz, 2H, C13-H), 2.27 (s, 3H, C18-H); 13 C NMR (75 MHz, DMSO) ppm δ 166.1 (s, C-11), 152.2 (s, C-2), 149.1 (s, C-9), 137.6 (s, C-4), 137.0 (s, C-17), 136.3 (s, C-6), 132.7 (s, C-14), 129.4 (s, C-10), 129.3 (s, C-5), 128.5 (s, C-8), 128.2 (s, C-16), 127.8 (s, C-15), 127.6 (s, C-7), 122.6 (s, C-3), 43.0 (s, C-13), 21.1 (s, C18); FT-IR (neat) ῡ (cm –1 ) = 3296cm − 1 (N-H stretching), 1635cm − 1 (C = O stretching). N -(2,6 fluorophenyl)quinoline-6-carboxamide (4d) . Using the general synthetic protocol, A , 2,6 difluoroaniline (1.15 mmol, 149 mg, 124 µL, 1 equiv) was employed as the starting amine, yielding the target compound 4d as a light brown solid (70%); m.p: 243–245°C; R f = 0.2 (hexane/ EtOAc = 7:3); 1 H NMR (300 MHz, DMSO) ppm δ 10.45 (s, 1H, N12-H), 9.04 (dd, J = 4.2, 1.8 Hz, 1H, C2-H), 8.70 (d, J = 2.0 Hz, 1H, C5-H), 8.56 (dd, J = 8.3, 1.8 Hz, 1H, C4-H), 8.29 (dd, J = 8.8, 2.0 Hz, 1H, C7-H), 8.16 (d, J = 8.8 Hz, 1H, C8-H), 7.66 (dd, J = 8.3, 4.2 Hz, 1H, C3-H), 7.44 (tt, J = 8.8, 6.4 Hz, 1H, C16-H), 7.32–7.18 (m, 2H, C17-H); 13 C NMR (75 MHz, DMSO) ppm δ 165.5 (s, C-11), 158.21 (d, 1 J C−F = 247.4 Hz, C-14), 158.18 (d, 1 J C−F = 247.3 Hz, C-18), 152.6 (s, C-2), 149.0 (s, C-9), 137.3 (s, C-4), 131.0 (s, C-6), 129.4 (s, C-10), 129.1 (s, C-5), 129.05 (s, C-8), 128.6 (t, 3 J C−F = 9.8 Hz, C-16), 127.6 (s, C-7), 122.5 (s, C-3), 114.7 (t, 3 J C−F = 9.3 Hz, C-15,17), 112.0 (d, 2 J C−F = 22.3 Hz, C-13); FT-IR (neat) ῡ (cm –1 ) = 3165cm − 1 (N-H stretching), 1665cm − 1 (C = O stretching). N -(2,4 difluorophenyl)quinoline-6-carboxamide (4e ). Using the general synthetic protocol, A , 2,4-difluoroaniline (1.15 mmol, 149 mg, 117 µL, 1 equiv) was employed as the starting amine, yielding the target compound 4e as a brown (60%); m.p: 186–188°C; R f = 0.5 (hexane/ EtOAc = 3:2); 1 H NMR (400 MHz, DMSO) ppm δ 10.40 (s, 1H, N12-H), 9.02 (dd, J = 4.2, 1.8 Hz, 1H, C2-H), 8.66 (d, J = 2.1 Hz, 1H, C5-H, ), 8.54 (dd, J = 8.4, 1.7 Hz, 1H, C4-H), 8.26 (dd, J = 8.8, 2.0 Hz, 1H, C7-H), 8.14 (d, J = 8.8 Hz, 1H, C8-H), 7.70–7.59 (m, 2H, C3, C18-H), 7.39 (ddd, J = 10.6, 9.1, 2.9 Hz, 1H, C17-H), 7.15 (tdd, J = 8.5, 2.8, 1.4 Hz, 1H, C15-H); 13 C NMR (100 MHz, DMSO) (a) ppm δ 165.1 (s, C-11), 152.4 (s, C-2), 148.9 (s, C-9), 137.3 (s, C-4), 131.6 (s, C-6), 129.2 (s, C-10), 128.8 (s, C-5), 128.4 (dd, J = 10.3, 2.8 Hz, C-18), 128.0(s, C-8), 127.1 (s, C-7), 122.4 (s, C-3), 111.3 (d, 2 J C−F = 25.5 Hz, C-13), 104.7–104.0 (m, 2C, C-15,17); FT-IR (neat) ῡ (cm –1 ) = 3279cm − 1 (N-H stretching), 1635cm − 1 (C = O stretching). (a) Due to the dilute nature of the sample, the signals for the two carbons directly attached to fluorine, expected in the range of 159–160 ppm, are missing. N-[5-fluoro-2-(trifluoromethyl) phenyl]quinoline-6-carboxamide (4f). Using the general synthetic protocol, A , 4-fluoro-2-(trifluoromethyl) aniline (1.15 mmol, 206 mg, 150 µL, 1 equiv) was employed as the starting amine, yielding the target compound 4f as a desert brown (65%); m.p: 200–202°C; R f = 0.3 (hexane/ EtOAc = 3:2); 1 H NMR (300 MHz, DMSO ) ppm δ 10.46 (s, 1H, N12-H), 9.03 (dd, J = 4.2, 1.7 Hz, 1H, C2-H), 8.64 (d, J = 2.0 Hz, 1H, C5-H), 8.60–8.50 (m, 1H, C4-H), 8.26 (dd, J = 8.8, 2.0 Hz, 1H, C7-H), 8.16 (d, J = 8.8 Hz, 1H, C8-H), 7.75 (dt, J = 8.9, 1.8 Hz, 1H, C17-H), 7.70–7.60 (m, 3H, C3, 8, 15-H); 13 C NMR (75 MHz, DMSO ) ppm δ 166.2 (s, C-11), 160.3 (d, J = 244.6 Hz, C-17), 152.8 (s, C-2), 148.9 (s, C-9), 137.3 (s, C-4), 134.0 (s, C-6), 133.9 (s, C-5), 132.1 (s, C-13), 131.6 (s, C-10), 129.3 (s, C-5), 128.7 (s, C-8), 127.7 (s, C-7), 127.2 (s, C-19), 122.4 (s, C-3), 120.4 (s, C-16), 120.1 (s, C-18), 114.0 (s, C-14); FT-IR (neat) ῡ (cm –1 ) = 3345cm − 1 (N-H stretching), 1650cm − 1 (C = O stretching). N -(pyridin-2-yl)quinoline-6-carboxamide (4g). Using the general synthetic protocol, A , pyridine-2-amine (1.15 mmol, 109 mg, 1 equiv) was employed as a the starting amine, yielding the target compound 4g as a cream yellow (68%); m.p: 97–99°C; R f = 0.4 (hexane/ EtOAc = 3:7); 1 H NMR (300 MHz, DMSO) ppm δ 11.03 (s, 1H, N12-H), 9.02 (dd, J = 4.2, 1.8 Hz, 1H, C2-H), 8.76 (d, J = 2.0 Hz, 1H, C5-H), 8.51 (ddd, J = 8.4, 1.8, 0.8 Hz, 1H, C4-H), 8.42 (ddd, J = 4.9, 2.0, 0.9 Hz, 1H, C7-H), 8.30 (dd, J = 8.8, 2.1 Hz, 1H, C8-H), 8.26–8.22 (m, 1H, C14-H), 8.11 (d, J = 8.8 Hz, 1H, C3-H), 7.88 (ddd, J = 8.4, 7.3, 2.0 Hz, 1H, C16-H), 7.64 (dd, J = 8.3, 4.2 Hz, 1H, C17-H), 7.19 (ddd, J = 7.3, 4.9, 1.0 Hz, 1H, C15-H); 13 C NMR (75 MHz, DMSO) ppm δ 165.6 (s, C-11), 152.4 (s, C-2), 152.2 (s, C-13), 148.9 (s, C-15), 148.1 (s, C-9), 138.3 (s, C-17), 137.4 (s, C-4), 131.8 (s, C-6), 129.2 (s, C-10), 129.0 (s, C-5), 128.2 (s, C-8), 127.0 (s, C-7), 122.3 (s, C-3), 120.0 (s, C-16), 114.7 (s, C-18); FT-IR (neat) ῡ (cm –1 ) = 3294cm − 1 (N-H stretching), 1683cm − 1 (C = O stretching). N -(2-iodophenyl)quinoline-6-carboxamide (4h). Using the general synthetic protocol, A , 2-Iodoaniline (1.15 mmol, 253 mg, 1 equiv) was employed as the starting amine, yielding the target compound 4h as a Coffee brown solid (65%); m.p: 182–184°C; R f = 0.3 (hexane/ EtOAc = 4:6); 1 H NMR (300 MHz, DMSO ) ppm δ: 10.36 (s, 1H, N12-H), 9.03 (dd, J = 4.2, 1.7 Hz, 1H, C2-H), 8.70 (d, J = 2.0 Hz, 1H, C5-H), 8.58–8.52 (m, 1H, C4-H), 8.32 (dd, J = 8.8, 2.0 Hz, 1H, C7-H), 8.16 (d, J = 8.8 Hz, 1H, C8-H), 7.97 (dd, J = 7.7, 1.2 Hz, 1H, C15-H), 7.65 (dd, J = 8.3, 4.2 Hz, 1H, C3-H), 7.49 (dd, J = 6.7, 1.6 Hz, 2H, C16, 18-H), 7.10 (ddd, J = 7.9, 6.5, 2.4 Hz, 1H, C17-H); 13 C NMR (75 MHz, DMSO) (a) ppm δ: 164.9 (s, C-11), 152.4 (s, C-2), 148.9 (s, C-13), 139.8 (s, C-9), 139.0 (s, C-15), 137.3 (s, C-4), 132.0 (s, C-6), 129.3 (s, C-10), 128.9 (s, C-5), 128.7 (2C, C8,17), 128.5 (s, C-7), 127.9 (s, C-16), 127.2 (s, C-18), 122.4 (s, C-3), 99.1 (s, C-14); FT-IR (neat) ῡ (cm –1 ) = 3392cm − 1 (N-H stretching), 1652cm − 1 (C = O stretching). N -(2, 4-dimethoxybenzyl)quinoline-6-carboxamide (4i). Using the general synthetic protocol, A , (2,4 dimethoxyphenyl)methanamine (1.15 mmol, 193 mg, 174 µL, 1 equiv) was employed as the starting amine, yielding the target compound 4i as a Dark brown solid (65%); m.p: 120–123°C; R f = 0.6 (hexane/ EtOAc = 2:8); 1 H NMR (300 MHz, DMSO- d6) ppm δ 9.05–8.96 (m, 2H, C2-H, N12-H), 8.56 (d, J = 2.0 Hz, 1H, C5-H), 8.48 (dd, J = 8.4, 1.8 Hz, 1H, C4-H), 8.22 (dd, J = 8.8, 2.0 Hz, 1H, C7-H), 8.08 (d, J = 8.8 Hz, 1H, C8-H), 7.62 (dd, J = 8.3, 4.2 Hz, 1H, C3-H), 7.16 (d, J = 8.3 Hz, 1H, C18-H), 6.58 (d, J = 2.4 Hz, 1H, C15-H), 6.49 (dd, J = 8.3, 2.4 Hz, 1H, C17-H), 4.44 (d, J = 5.6 Hz, 2H, C11-H), 3.82 (s, 3H, C19-H), 3.74 (s, 3H, C20-H); 13 C NMR (75 MHz, DMSO) ppm δ 165.8 (s, C-11),, 159.7 (s, C-17), 157.6 (s, C-15), 151.9 (s, C-2), 148.5 (s, C-9), 137.3 (s, C-4), 132.4 (s, C-6), 128.8 (s, C-10), 128.6 (s, C-19), 128.0 (s, C-5), 127.9 (s, C-8), 127.1 (s, C-7), 122.2 (s, C-3), 118.9 (s, C-14), 104.4 (s, C-18), 98.2 (s, C-16), 55.5 (s, C-20), 55.2 (s, C-21), 37.6 (s, C-13); FT-IR (neat) ῡ (cm –1 ) = 3289cm − 1 (N-H stretching), 1638cm − 1 (C = O stretching). N -(2,4 -dichlorobenzyl)quinoline-6-carboxamide (4j) . Using the general synthetic protocol, A , (2,4 dichlorophenyl)methanamine (1.15 mmol, 203 mg, 155 µL,1 equiv) was employed as the starting amine, yielding the target compound 4j as a Light brown solid (67%); m.p: 165–167°C; R f = 0.4 (hexane/ EtOAc = 6:4); 1 H NMR (300 MHz, DMSO- d6 ) δ: 9.33 (t, J = 5.7 Hz, 1H, N12-H), 9.03–8.94 (m, 1H, C2-H), 8.58 (d, J = 2.0 Hz, 1H, C5-H), 8.52–8.43 (m, 1H, C4-H), 8.22 (dd, J = 8.8, 2.0 Hz, 1H, C7-H), 8.10 (d, J = 8.8 Hz, 1H, C8-H), 7.64 (d, J = 0.8 Hz, 1H, C15-H), 7.63–7.57 (m, 1H, C3-H), 7.43 (d, J = 1.2 Hz, 2H, C17,C18-H), 4.57 (d, J = 5.7 Hz, 2H, C11-H); 13 C NMR (75 MHz, DMSO- d6 ) δ: 166.1 (s, C-11), 152.2 (s, C-2), 148.9 (s, C-9), 137.2 (s, C-4), 135.5 (s, C-6), 133.0 (s, C-14), 132.3 (s, C-15), 131.8 (s, C-17), 130.3 (s, C-16), 129.2 (s, C-10), 128.7 (s, C-19), 128.2 (s, C-8), 127.8 (s, C-5), 127.4 (s, C-7), 127.2 (s, C-18), 122.3 (s, C-3), 40.5 (s, C-13); FT-IR (neat) ῡ (cm –1 ) = 3289cm − 1 (N-H stretching), 1638cm − 1 (C = O stretching). N -(4-Iodophenyl)quinoline-6-carboxamide (4k). Using the general synthetic protocol, A , 4-iodoaniline (1.15 mmol, 253 mg, 1 equiv) was employed as the starting amine, yielding the target compound 4k as a Dark brown (63%); m.p: 182–184°C; R f = 0.4 (hexane/ EtOAc = 4:1); 1 H NMR (300 MHz, DMSO- d6 ) δ: 10.72 (s, 1H, N12-H), 9.09 (dd, J = 4.5, 1.7 Hz, 1H, C2-H), 8.75–8.68 (m, 1H, C5-H), 8.69 (d, J = 8.0 Hz, 1H, C4-H), 8.32 (dd, J = 8.8, 2.0 Hz, 1H, C7-H), 8.20 (d, J = 8.9 Hz, 1H, C8-H), 7.90–7.58 (m, 5H, C3, 14, 15, 17, 18-H); 13 C NMR (75 MHz, DMSO- d6 ) δ: 165.0 (s, C-11), 151.2 (s, C-2), 146.8 (s, C-9), 139.2 (s, C-13), 139.0 (s, 2C, C-15,17), 137.4 (s, C-4), 133.0 (s, C-6), 129.0 (s, C-10), 128.8 (s, C-5), 127.6 (s, C-8), 127.2 (s, C-7), 122.5 (s, C-3), 122.5 (s, 2C, C-13,18), 87.7 (s, C-16); FT-IR (neat) ῡ (cm –1 ) = 3288cm − 1 (N-H stretching), 1678cm − 1 (C = O stretching). N-(5-fluoro 2-methylphenyl)quinoline-6-carboxamide (4l). Using the general synthetic protocol, A , 5-fluoro 2-methylaniline (1.15 mmol, 144 mg, 145 µL,1 equiv) was employed as the starting amine, yielding the target compound 4l as a brown solid (122 mg, 66%); m.p: 164–166°C; R f = 0.4 (hexane/ EtOAc = 3:7); 1 H NMR (300 MHz, DMSO) ppm δ: 10.19 (s, 1H, N12-H), 9.02 (dd, J = 4.2, 1.7 Hz, 1H, C2-H), 8.66 (d, J = 2.0 Hz, 1H, C5-H), 8.55 (dd, J = 8.5, 1.8 Hz, 1H, C4-H), 8.28 (dd, J = 8.8, 2.0 Hz, 1H, C7-H), 8.15 (d, J = 8.8 Hz, 1H, C8-H), 7.65 (dd, J = 8.3, 4.2 Hz, 1H, C3-H), 7.34 (ddd, J = 10.3, 8.4, 4.6 Hz, 2H, C18, C16-H), 7.04 (td, J = 8.5, 2.8 Hz, 1H, C15-H), 2.28 (s, 3H, C19-H); 13 C NMR (75 MHz, DMSO) ppm δ: 165.1 (s, C-11), 160.2 (d, 1 J C−F = 239.1 Hz, C-17), 152.4 (s, C-2), 148.9 (s, C-9), 137.6 (d, 3 J C−F = 10.2 Hz, C-15), 137.3 (s, C-4), 132.2 (s, C-6), 131.51 (d, 3 J C−F = 8.8 Hz, C-13), 129.2 (s, C-10), 129.2 (s, C-8), 128.7 (s, C-5), 128.1 (s, C-14), 127.2 (s, C-7), 122.4 (s, C-3), 112.69 (d, 2 J C−F = 45.0 Hz, C-16), 112.68 (s, C-18), 17.76 (s, C-19) ; FT-IR (neat) ῡ (cm –1 ) = 3352cm − 1 (N-H stretching), 1649cm − 1 (C = O stretching). 2.2 Density functional theory Geometry optimizations of the selected molecules were carried out using Density Functional Theory (DFT) at the B3LYP/3-21G level, consistent with methodologies reported in previous literature [ 43 – 45 ]. The chemical reactivity of the synthesized compounds was determined by analyzing the frontier molecular orbitals. The compounds' softness and chemical hardness were measured. Gaussian 09 software was used to conduct quantum mechanical research on the multi-core machine, and Gauss View 6 was used to view the optimized structures. 2.3 Enzymes preparation To produce the enzymes, COS-7 cells were transfected with plasmids containing the h -NTPDase1 gene (GenBank accession number U87967) [ 46 ]. The h -NTPDase2 construct (GenBank accession number NM_203468) was generously provided by Aileen F. Knowles [ 47 ]. We received the h -NTPDase3 plasmid (GenBank accession no AF034840) as a generous gift from Terence L. Kirley. [ 47 ] h -NTPDase8 (GenBank accession no AY430414) [ 48 ] h -NPP1 (GenBank accession no NM006208) a kind gift of James W. Goding [ 49 ] and h -e5’NT (GenBank accession no DQ186653) [ 50 ] as previously described [ 51 ]. Transfection of COS-7 cells was carried out in 10 cm culture plates by incubating them at 37°C for 5 hours using serum-free DMEM/F-12 medium mixed with 6 µg of plasmid DNA and 24 µL Lipofectamine. Following this, the plate received an equal volume of DMEM/F-12 medium containing 20% fetal bovine serum after 6-8hrs. After 44–48 hours of incubation, the cells were washed 3 times with a harvesting buffer containing 95 mM NaCl, 0.1 mM PMSF, and 45 mM Tris at pH 7.5. After scraping, the cells were centrifuged twice (300 g, 5 minutes at 4°C), washed, and resuspended in harvesting buffer with aprotinin (10 µg/mL) before undergoing sonication. Centrifugation at 850 g for 5 minutes at 4°C was performed to remove the nuclei and cellular debris. The supernatant obtained was supplemented with 7.5% glycerol and preserved at − 80°C. Protein concentration was determined using the Bradford assay in a microplate format, with bovine serum albumin (BSA) serving as the standard [ 52 ]. 2.4 Enzyme inhibition assays h -NTPDase Activity Assay A significantly modified version of a previously published spectrophotometric technique was used to study the effect of all the synthesized compounds on h -NTPDase1, -2, -3, and − 8 [ 53 ]. The compounds were screened at a concentration of 100 µM. The assay was performed in an incubation medium containing 50 mM of Tris-HCl and 5 mM of CaCl 2 maintained at pH 7.4. Reaction medium containing buffer (55 µL), test compound (10 µL), and 10 µL of enzyme solution of h -NTPDase1 (12 ng/well) or h -NTPDase2 (37 ng/well) or h -NTPDase3 (43 ng/well) or h -NTPDase8 (63 ng/well) was pre-incubated at 37℃. After 10 min, the reaction absorbance was measured (630 nm) with a microplate reader (FLUOStar Omega. BMG Labtech, Germany). 10 µL of substrate i.e. ATP (100 µM) was added to begin the reaction followed by 15mins incubation. The enzymatic reaction was eventually stopped by adding 15 µL of malachite green reagent. By measuring the absorbance at 630 nm, released inorganic phosphate was identified and the percent inhibition was computed. Compounds with > 50% inhibition of any isoform of h -NTPDase, were further diluted and their IC 50 values were calculated by generating and fitting the dose-response curves with GraphPad Prism 5.0 (San Diego, CA, USA). h -NPP1 Activity Assay With slight modifications to the previously described procedure, an inhibition assay on h -NPP1 was conducted to verify these synthesized derivatives' inhibitory effect [ 54 ]. The test buffer (pH = 9.5) included 50 mM Tris-HCl, 5 mM MgCl 2, and 0.1 mM ZnCl 2 . The synthetic compounds used for h-ENPP screening were prepared in 10% DMSO and subjected to a test concentration of 0.1 mM. The assay, which contained assay buffer, 100 µM of the test drug, the enzyme (27 ng/well), and substrate p-nitrophenyl 5’-thymidine monophosphate, was carried out in a 96 well plate with a total volume of 100 µL/well. A microplate reader (BioTek FL*800TM, Instruments, Inc. USA) was used to measure absorbance at 405 nm following 25 minutes in an incubator at 37℃. Compounds exhibiting more than 50% h -ENPP1 inhibition of were subjected to serial dilution to calculate their IC 50 values. The data was analyzed using GraphPad PRISM 5.0 (San Diego, CA, USA). h- e5’NT Activity Assay Inhibition assay for human ecto-5'-nucleotidase was carried out according to the previously reported method [ 55 ]. The assay buffer was composed of Tris HCL, MgCl 2, and CaCl 2 and its pH was adjusted to 7.4 before use. The assay was first conducted in triplicates to see whether the compounds exhibited any action. The total volume per well was 100 µL. In triplicates, 55 µL of the assay buffer and 10 µL of the e5’NT were added to each well. Each well received 10 µL of assay compound, followed by 10 minutes’ incubation at 37℃. Once 10 µL of AMP (2 mM) was added as a substrate, the enzymatic activity started and continued for 20 minutes at 37℃. After that, 15 µL of malachite green reagent was added to assess the quantity of phosphate released during the enzyme-mediated breakdown of the substrate. The absorbance change at 630 nm was then recorded. The same methodology was used to calculate the IC 50 values of the assay compounds at various concentrations. Non-linear regression analysis was utilized to further examine the IC 50 of only those compounds that exhibited inhibition values higher than 50% using GraphPad Prism 5.0 software (San Diego, CA, USA). 2.5 Fluorescence Spectroscopy and Microscopic Analysis Fluorescence spectroscopy of compound 4d was conducted using a Horiba Fluoromax-4 spectrofluorometer, with excitation wavelength between 400–600 nm, and emission spectra was recorded to characterize its optical properties. To evaluate cellular uptake, A549 lung cancer cells were incubated with the compound and imaged on inverted fluorescence microscope (Nikon ECLIPSE Ni–U). To evaluate the effect of compound 4d on enzyme function, a real-time fluorescent-based assay was used to monitor changes in fluorescent intensity, wavelength shifts, and emission patterns to infer binding events, conformational changes, and enzyme activity. Combined with cell imaging, the compound d was revealed to enter the A549 lung cancer cells and reach its target enzyme, signifying the druggability of the synthesized compound 4d. Fluorescence signals were normalized via min-max normalization and analyzed using the microscope’s built-in software. These assays collectively aimed to quantify intracellular distribution and assess binding affinity to cancer cell membrane enzymes, providing insights into the compound’s localization and potential interactions. 2.6 Molecular Docking Studies Quinoline-6-carboxylic acid derivatives 4a, 4b, 4d, 4g , and 4k were docked with respective enzymes. Molecular docking experiments were conducted on the synthesized derivatives (4a-4l) using Autodock Vina® 1.5.4 software [ 56 ]. The crystal structures of human ectonucleotide pyrophosphatase/phosphodiesterase 1 ( h -ENPP1) and h -e5′NT were retrieved from the Protein Data Bank https://www.rcsb.org/ using PDB IDs 6WEW and 4H2G, respectively. For h -ENTPDase isoenzymes, homology models were utilized due to the unavailability of their experimental structures. Ligand structures were drawn using ChemDraw software, and were then subjected to energy minimization and saved in PDBQT format using Autodock Vina 1.5.4 software. Best pose with minimum binding energy was selected and re-docking was carried out for validation of the docking results. Visualization of the 2D and 3D protein ligand interactions was done using Discovery studio 2021. 2.7 In Silico ADME analysis The SwissADME web server was employed to assess the pharmacokinetics properties of all the compounds (4a-4l). ChemDraw was used to generate the SMILES representation of the synthesized derivatives, which were then uploaded to an online platform for pharmacokinetic profile prediction. 2.8 MTT Assay MTT assay was conducted to check the safety profile of the synthesized derivatives. The assay was conducted in MCF-7 breast cancer cells. Briefly, the cells were plated in a sterile 96-well plate and incubated for 24 h in CO 2 incubator at 37°C. The media was, then, discarded and the cells were washed with PBS. Tested compounds were loaded at a concentration of 100 µM/well in triplicates. 1% DMSO and 100 µM cisplatin were used as negative control and positive control, respectively. A blank well that contained only serum free media served as a reference. The 96 well plate was further incubated for 24 h in 5% CO 2 incubator at 37°C. Following incubation, 100 µL of MTT reagent was loaded/well to achieve a final concentration of 0.2 mg/mL. The cells were then placed in an incubator for further 2 to 4 h and monitored for formation of formazan crystals. Next, the media was aspirated, and 100 µL/well solubilizing agent (SDS solution) was added. Subsequently, the plates were shaken for 30 min to ensure complete dissolution of the formazan crystals. The absorbance was measured using a microplate reader, with readings taken at 570 nm for the formazan and at 630 nm for the background signal. The conversion of MTT reagent to formazan crystals by mitochondrial dehydrogenases was indicative of cell viability. 2.9 Apoptosis Assay and Fluorescent Microscopy FITC Annexin V was used to quantitatively determine the percentage of MCF-7 cells within a population that were actively undergoing apoptosis. It relies on the property of cells to lose membrane asymmetry in the early phases of apoptosis. Annexin V is a calcium-dependent phospholipid-binding protein with high affinity for phospholipid phosphatidylserine (PS), and was used to identify apoptotic cells with exposed PS. Propidium Iodide (PI) served as a viability probe to distinguish viable cells from nonviable ones. Viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeable to PI. Cells that stained positive for FITC Annexin V were undergoing apoptosis, whereas the cells that stained negative for FITC Annexin V were alive and did not undergo measurable apoptosis upon treatment with the selected compounds. 3. RESULTS & DISCUSSION 3.1 Synthesis The synthesis of quinoline-6-carboxylic acid derivatives was carried out in accordance with the reported procedure [57].First step involved the conversion of quinoline-6-carboxylic acid (1) into quinoline-6-acid chloride (2) using thionyl chloride. The reaction was refluxed for 8 hours and monitored by TLC to confirm the consumption of the starting acid and the formation of quinoline-6-acid chloride (2) . Subsequent addition of the respective amine led to the formation of the corresponding amides (4a–4l) in good yields, ranging from 60% to 70%. The amidation step was carried out at 50 °C, and the reaction progress was monitored by TLC, which indicated complete conversion of the acid chloride into its respective amide. The resulting amides were fully characterized using FTIR and 1 H and 13 C NMR spectroscopy. To assess the versatility of the reaction and introduce structural diversity, a diverse set of amine substrates was explored, as depicted in Scheme 1 . This set comprised primary amines, variously substituted benzylamines, functionalized anilines, and heterocyclic aniline derivatives. Among the benzylamines, substituted benzylamines with 4-chloro, 4-fluoro, 4-methyl, and 2,4-dichloro substituents (3a–3c, 3j) produced good to excellent yields of the corresponding amides (4a–4c, 4j) , with yields of 67%, 64%, 70%, and 67%, respectively. In a similar fashion, a range of aniline derivatives were tested, including monosubstituted anilines with electron-donating groups, such as chloro (Cl) at the ortho (3h) and para (3k) positions. Additionally, disubstituted anilines with a mix of electron-donating groups (F, OCH 3 ; 3d, 3e, 3i) and both electron-donating and electron-withdrawing groups (F, CF 3 , CH 3 ; 3f, 3l) were also compatible, yielding the desired products in good to excellent yields. Additionally, heterocyclic pyridyl amine (3g) was also successfully utilized to synthesize the desired compounds (4g) , furnishing the products in good yields. For all the synthesized compounds, characteristic peaks were observed in the FTIR spectra. A stretching band appeared in the range of 3165–3462 cm⁻¹, indicating the presence of the NH group, while the absorption band at 1628–1682 cm⁻¹ confirmed the existence of the C=O group. The structures were further confirmed using 1 H, and 13 C spectroscopy. The 1 H-NMR data of compounds in the δ 9.00-11.06 ppm range confirmed the presence of NH which further confirmed the presence of amide functionality. In 13 C-NMR data, the appearance of a signal at δ 164.9-166.3 ppm for C=O further confirmed the formation of quinoline-6-carboxylic acid amide derivatives (4a-4l) . 3.2 Enzymatic assays The ability of the synthetic compounds to inhibit the activity of the h -NTPDase1, -2, -3, and -8 iso enzymes, h -NPP1, and h -e5′NT was evaluated. The newly synthesized compounds showed pronounced inhibition of these enzymes, as listed in Table 1. The enzymatic tests of the compounds ( 4a–4l ) revealed their strong inhibitory effects, with IC 50 values ranging from 0.092 to 1.00 µM, as shown in Table 2. Table 1 Percentage inhibition of all the compounds against Ectonucleotidases Compound codes % Inhibition of the studied compounds h -NTPDase1 h- NTPDase2 h -NTPDase3 h- NTPDase8 h -NPP1 h -e5’NT 4a 37.02% 23.30% 30.60% 39.46% 66.97% 67.87% 4b 68.92% 26.18% 67.82% 82.98% 44.80% 37% 4c 57.70% 68.38% 78.38% 70.86% 45.25% 50.70% 4d 76.06% 80.13% 58.42% 33.02% 54.52% 35.64% 4e 32.56% 36.36% 40.21% 39.46% 34.62% 66.87% 4f 44.06% 35.41% 66.67% 39.91% 65.61% 48.49% 4g 58.53% 33.91% 81.68% 20.35% 43.67% 81.98% 4h 18.45% 28.29% 84.98% 65.61% 33.94% 87.05% 4i 19.48% 39.64% 25.08% 29.82%% 29.19% 54.86% 4j 33.06% 26.20% 35.64% 28.60% 85.52% 72.39% 4k 33.79% 18.56% 38.45% 27.02% 67.65% 28.43% 4l 32.37% 41.16% 36.14% 26.12% 29.42% 60.98% * Inhibition percentages at the concentration of 100 µM Table 2 Inhibitory activity of the studied compounds against Ectonucleotidases Compound codes IC 50 ± SEM (µM) h -NTPDase1 h- NTPDase2 h -NTPDase3 h- NTPDase8 h -NPP1 h -e5’NT 4a - - - - 0.23±0.02 0.092±0.02 4b 3.46±0.49 - 0.35±0.17 0.44±0.08 - - 4c 6.13±0.64 6.39±1.07 0.67±0.27 0.99±0.13 - 0.19±0.01 4d 0.28±0.03 0.92±0.17 0.82±0.07 - 0.73±0.09 - 4e - - - - - 0.54±0.09 4f - - 0.99±0.29 - 0.87±0.11 - 4g 0.61±0.09 - 0.32±0.05 - - 0.34±0.07 4h - - 0.88±0.29 3.29±0.36 - 0.39±0.1 4i - - - - - 0.99±0.13 4j - - - - 13.5±1.24 0.44±0.07 4k - - - - 0.11±0.02 - 4l - - - - - 0.47±0.26 Reactive Blue 2 16.1±1.02 24.1±3.01 4.31±0.41 101.1±2.34 - - Suramin - - - - 18.5±1.14 42.1±7.8 *Data presented as IC 50 ± SEM 3.3. Fluorescence characterization of the druggable compound 4d and its cellular interaction Fluorescence spectroscopy of compound 4d revealed distinct excitation-dependent emission profiles between 410–500 nm. The emission spectra displayed strong, well-defined fluorescence peaks across the visible region (400–500 nm), demonstrating excellent photostability and consistent emissive behavior under near-UV to blue light irradiation. This robust optical performance, characterized by minimal photobleaching, positions 4d as potential fluorophore for bioimaging applications. Fluorescence microscopy of A549 lung cancer cells treated with 4d provided detailed spatial information about its cellular distribution. Bright-field images showed preserved cell morphology with intact membranes and no signs of cytotoxicity at the tested concentration (10 μM, 24 h incubation). In the red fluorescence channel, intense fluorescence was observed predominantly at the plasma membrane, with some vesicular staining suggesting possible endocytic uptake. Spectral shifts in emission suggest binding interactions between the compound d and the enzyme. The high signal-to-noise ratio (SNR >15:1) and clear membrane integrity indicated viable colocalization. The merged image in Figure 3B highlights this exclusive membrane targeting, with the red fluorescence of 4d forming a continuous rim around cells while remaining distinctly separated from cytoplasmic and nuclear compartments. Microscopic analysis further confirmed compound uptake, intracellular localization, and biological relevance, integrating in vitro findings and cellular behavior. 3.4 Structure-activity relationships (SARs) The structure-activity relationship (SAR) analysis demonstrated a substantial influence of the substituents on the in vitro activity of the synthesized compounds (4a–4l) against h -NTPDases, h -NPP1, and h -e5′NT enzymes. The structures of the most effective compounds are displayed in Figure 4 . It was observed that compound 4a , with a 4-chloro benzyl substitution on the quinoline carboxamide scaffold, exhibited the highest potency against h -e5′NT enzyme, highlighting the importance of chlorine in enhancing activity. Compound 4j , with additional 2-chloro substitution, showed slightly reduced but notable potency, suggesting steric effects. Electron-donating groups (e.g., methyl) at the 4-position (4c) enhanced activity, whereas introduction of electron withdrawing group i.e., fluorine substitution reduced it, emphasizing chlorine’s optimal electronic properties. Pyridyl rings and 2-iodo substitutions maintained good activity, but bulky 4-iodo groups significantly decreased 4k inhibition potential. We also observed that activity varied with substitution positions. Compound 4e , with fluoro groups at 2- and 4-positions, retained potency, however compound 4d , having fluoro groups at 2-, 6- positions exhibited decreased potential, demonstrating the role of precise substituent placement. For h -NPPI, the tested series of compounds exhibited an opposite trend in activity compared to the h -e5′NT enzyme, with the exception of compound 4a , which showed good potency against both enzymes. Notably, the 4-iodo aromatic moiety displayed exceptional efficacy, with an IC 50 value of 0.11 ± 0.02 µM. A structural comparison of the inhibitors against h -NPPI and h -e5′NT revealed that electronegative atoms (F, Cl, I, and CF 3 ) at the ortho or para positions of the phenyl ring, either as single or double substitutions, significantly enhanced inhibitory potential against h -NPPI. The IC 50 values for these inhibitors ranged from 0.1 to 14 µM, highlighting the importance of both substitution patterns and electronic effects in optimizing enzyme inhibition. All the compounds (4a–4l) displayed good to moderate inhibitory activities against h -NTPDases, with IC 50 values ranging from 0.28 ± 0.03 µM to 6.39 ± 1.07 µM. However, compounds 4a, 4e, 4i, 4j, 4k, and 4l , which include halogen-substituted derivatives and anilines with electron-donating groups, showed poor activity against all four enzymes. Compound 4c , featuring a 4-methylbenzyl substitution, was active across all the enzyme targets, while compound 4d , with 2,6-difluoro substitutions, demonstrated the highest potency specifically against h -NTPDase1 and -2. In contrast, compound 4e , containing fluoro groups at the 2- and 4-positions, was inactive against all four isoenzymes. This inactivity could be attributed to steric hindrance or an unfavorable electronic interaction at the active site. Compound 4b , with a 4-fluorobenzyl moiety, exhibited good potency against h -NTPDase1 and -3 and was the most potent inhibitor of h -NTPDase8. Against h -NTPDase3, compounds 4b, 4c, 4d, 4f, 4g , and 4h showed activity, with compound 4g , a pyridyl-substituted derivative, emerging as the most potent inhibitor. Compound 4i , which contains a methoxy substitution on the phenyl ring, showed less than 50% inhibition for all enzymes except h -e5′NT, where it demonstrated good inhibitory activity. 3.5 Molecular docking studies for validation h -ENTPDase1 Amino acid components involved in the binding interactions of h -NTPDase1 are: Asp213, Tyr450, Gly178, Ser361, Phe360, and Cys502. Moleculer docking study of the compound 4d with h -NTPDase1 revealed conventional hydrogen bonding between oxygen of the carbonyl group and the amino acid Trp450. Other interaction shown by the compound are pi-Sigma interaction of Phe360 with phenyl ring. Cys502 refers to the amino acid Cystein (CYS), whereby, cystein contains thiol (-SH) group that can interact with benzene ring and show π-sigma interaction with the aromatic (benzene) ring. Another amino acid, Gly178, shows that carbon-hydrogen bond interaction with NH group can act as a hydrogen donor in hydrogen bonding. Asp213 with its carboxyl side chain can act as a hydrogen bond acceptor. Asp213 shows hydrogen bond interaction with NH group. Gly178 also shows halogen interaction with the fluorine group (Figure 5A) . h -ENTPDase2 Amino acid components engaged in the binding interactions of h -NTPDase2 are: Asp201, Leu202, Tyr350, His50, Ala347, Arg392, Gly435, and Cys502. Compound 4d , the most active compound against h- NTPDase2 was docked using a homology model to analyze the interactions. Molecular docking of the compound 4d with h -NTPDase2 revealed conventional hydrogen bonding of the oxygen group with Ala347, and Arg392 forms conventional hydrogen bonding with the parent quinoline moiety when the guanidinium groups NH or NH 2 act as a hydrogen donor, and the quinoline nitrogen act as the acceptor. His(50) and Tyr350 show π - π stacked and π - π -T shaped with benzene ring. Another amino acid Cys502 shows Pi alkyl interaction with the aromatic ring. Another amino acid Gly435 shows carbon-hydrogen bond interaction with carbon atom attached to the fluorine group. Leu202 and Asp201 show halogen bond interaction with the fluorine group (Figure 5B) . h -ENTPDase3 Amino acid components engaged in the binding interactions of h -NTPDase3 are: Leu144, Leu145, Gln148, Gly141, Ser100, and Glu266. Molecular docking of the compound 4g with h -NTPDase3 revealed conventional hydrogen bonding of hydrogen group with Gln148, whereas, parent moiety quinoline ring was observed to have Pi-alkyl interaction with Leu144 and Leu145. Furthermore, phenyl ring formed Pi anion interaction with Glu266 and the nitrogen atom of quinoline ring show carbon-hydrogen interaction with Gly141 also. Ser100 amino acid show carbon hydrogen bond interaction with π-electronic cloud of the quinoline ring (Figure 5C) . h -ENTPDase8 Amino acid components involved in the binding interactions of h -NTPDase8 are: Tyr402, Tyr357, Trp398, Pro393, and His360. Molecular docking of the compound 4b with h -NTPDase8 revealed conventional hydrogen bonding of oxygen group with Tyr402, and phenyl ring formed π - π -T shaped interaction with Tyr357 and His360. Similarly, Phenyl ring formed Pi alkyl interaction with Pro393, and the quinoline moiety formed π - π-T-shaped interaction with Trp398 amino acid (Figure 5D) . h- ENPP1 Amino acid sites contributing to the binding interactions of h -NPP1 are: Tyr371, Tyr340, Lys295, Phe257, His380, and Pro323. Molecular docking of the compound 4k with h -NPP1 revealed conventional hydrogen bonding between the nitrogen of the quinoline ring and the amino acid lys295. Tyr340 and phe257 are both aromatic amino acids. They show π - π stacked interactions with ligands containing aromatic moiety such as the quinoline group. Similarly, Tyr371 can engage in π - π T-shaped interactions with quinoline ring due to perpendicular orientation of the aromatic ring (Figure 5E) . h -e 5′NT Amino acid residues implicated in the bonding interaction of h -e5′NT are Ile91, Trp408, Gln523, Thr90, Lys50, Ala412, and Ser49. Molecular docking of the compound 4a with h -e5′NT revealed conventional hydrogen bonding between Gln523 and the oxygen atom of the carbonyl group. Similarly, Thr90 shows pi-sigma interaction with the quinoline moiety. The quinoline ring interacts with the sigma bond of atom like oxygen threonine’s hydroxyl group. While Ile90 and Ala412 amino acids interact with the benzene ring through π -alkyl interaction, where the hydrophobic alkyl side chains of Isoleucine (isopropyl group) and Alanine (methyl group) align with and interact with the pi-electrons of the aromatic benzene ring and Lys50 show pi-alkyl interaction with quinoline moiety. Also, π - π T-shaped interaction occurs between Trp408 and benzene ring. Additionally,amide- π stacked interaction occurs between Ser49 and the quinoline moiety where the amide group from Ser49 can stack with quinoline aromatic ring via the NH and carbonyl group, stabilizing the interaction (Figure 5F) . 3.6 In - silico Pharmacokinetics studies Prediction of Pharmacokinetics Properties and Drug-Likeness of the quinoline-6-carboxylic acid derivatives (4a-4l) Pharmacokinetics properties and drug-likeness of the quinoline-6-carboxylic acid derivatives were calculated using SwissADME. The pharmacokinetics properties were determined based on gastrointestinal (GI) absorption, permeation to the blood-brain barrier (BBB), and ability to act as a P-glycoprotein (P-gp) substrate. The drug-likeness of these compounds was determined based on Lipinski’s rule of five numbers of rotatable bonds and topological polar surface area (TPSA). The calculated parameters related to pharmacokinetics properties and drug-likeness are presented in Table 3 and Table 4, respectively. All the studied compounds showed high GI absorption and were permeable to the BBB, however, none of the the compounds was identified as a P-glycoprotein (P-gp) substrate. In addition, these compounds also possessed suitable drug-like properties. There was no violation of Lipinski's rule of five, and the partition co-efficient (MlogP) values were less than 4.15. Moreover, TPSAof these compounds was within the range 20 to 65 Ų, and the number of rotatable bonds was also less than 7. Table 3 Pharmacokinetics Properties of the quinoline-6-carboxylic acid derivatives (4a-4l) Compounds GI-Absorption a BBB-permeant b P-gp substrate c 4a High Yes No 4b High Yes No 4c High Yes No 4d High Yes No 4e High Yes No 4f High Yes No 4g High Yes No 4h High Yes No 4i High Yes No 4j High Yes No 4k High Yes No 4l High Yes No a GI-Absorption; b BBB-permeant; c P-gp substrate Table 4 Drug-Likeness of the quinoline-6-carboxylic acid derivatives (4a-4l) Compounds HBD a HBA b MlogP c No. of violations d TPSA( Ų) e RBN f 4a 1 2 3.07 0 41.99 4 4b 1 3 2.95 0 41.99 4 4c 1 2 2.80 0 41.99 4 4d 1 4 3.37 0 41.99 3 4e 1 4 3.37 0 41.99 3 4f 1 6 3.84 0 41.99 4 4g 1 3 1.89 0 54.88 3 4h 1 2 3.33 0 41.99 3 4i 1 4 1.89 0 60.45 6 4j 1 2 3.57 0 41.99 4 4k 1 2 3.33 0 41.99 3 4l 1 3 3.22 0 41.99 3 a Hydrogen bond donor; b Hydrogen bond acceptor; c M logP; d No of violations; e Topological polar surface area; f Number of rotatable bond The results indicated that the synthesized compounds met all the criteria. SwissADME employs a visual method known as the boiled egg plot, which graphically represents GI absorption and BBB penetration. The analysis revealed that all the synthesized compounds are in the yolk region, except for 4f (Figure 6) . All compounds except 4f exhibit efficient blood–brain barrier permeability and may be considered for further investigation as therapeutic agents against brain tumors. 3.7 Density Functional Theory In this study, density functional theory (DFT) calculations were employed to analyze the HOMO-LUMO characteristics of quinoline derivatives 4a–4l . The energy gap between the frontier orbitals provided valuable insights into the compounds’ electronic properties, including their chemical reactivity, molecular stability, and optical behavior. The optimized geometries of these derivatives are depicted in Figure 7 . Table 5 summarizes the calculated physicochemical properties of the studied compounds, including optimized energies, dipole moments, polarizability, frontier molecular orbital (FMO) energies (E HOMO and E LUMO ), and the corresponding energy gaps (∆E). A higher HOMO energy (E HOMO ) reflects a greater tendency to donate electrons, whereas higher LUMO energy (E LUMO ) corresponds to a reduced ability to accept electrons. The HOMO-LUMO energy gap plays a critical role in determining the kinetic stability as well as the chemical reactivity of the molecules. Compounds with a larger ∆E are typically more kinetically stable, less polarizable, and exhibit lower chemical reactivity, thus classified as hard species. In contrast, a smaller ∆E indicates higher chemical reactivity, greater polarizability, and lower kinetic stability, signifying a soft molecular nature. Among the derivatives, compound 4j exhibited the highest energy gap (∆E = 0.17488 eV), indicating its superior stability and lowest reactivity. In contrast, compound 4f showed the lowest energy gap (∆E = 0.129 eV), classifying it as the most reactive and chemically softest derivative. This electronic behavior is consistent with its strong inhibitory activity against NPP1 and NTPDase3. Moreover, 4f displayed the highest HOMO energy (E HOMO = -0.206 eV), indicating its strong electron-donating ability. This characteristic enhances its binding affinity through hydrogen bonding and facilitates nucleophilic interactions with active site residues. Table 5 Energetic parameters of the quinoline derivatives ( 4a-4l) Codes Optimization Energy Dipole Moment Polarizability (α) HOMO (eV) LUMO (eV) HOMO-LUMO E ( eV) 4a -1293.75 1.95 188.23 -0.2429 -0.0686 0.17432 4b -935.05 1.51 178.47 -0.23664 -0.0639 0.17274 4c -875.46 2.78 191.75 -0.23209 -0.0606 0.17149 4d -994.66 1.26 177.79 -0.23305 -0.06724 0.16581 4e -994.66 2.66 176.36 -0.2324 -0.0676 0.16481 4f -895.28 3.12 184.63 -0.206 -0.076 0.13641 4g -813.20 2.77 174.11 -0.23002 -0.06582 0.16421 4h -7687.22 2.35 192.18 -0.23471 -0.06399 0.17072 4i -1064.15 4.27 211.89 -0.21255 -0.05539 0.15716 4j -1751.14 1.12 197.61 -0.24571 -0.07083 0.17488 4k -7687.23 3.00 210.49 -0.22028 -0.07262 0.14766 4l -935.07 3.28 221.28 -0.22365 -0.07118 0.15247 Global chemical reactivity descriptors: The global reactivity parameters for the selected compounds (4a–4l) were calculated from their HOMO and LUMO energy values using standard formulas. These parameters, which provide insights into the chemical stability and reactivity of the molecules, are shown in Table 6 . Table 6 Global reactivity descriptors of the quinoline derivatives ( 4a-4l ) Codes Chemical Potential µ (eV) Electronegativity X (eV) Hardness ƞ (eV) Softness S (eV-1) Electrophilicity index ω (eV) 4a -0.156 0.156 0.087 -0.3115 0.139 4b -0.150 0.150 0.086 -0.30054 0.131 4c -0.146 0.146 0.086 -0.29269 0.125 4d -0.150 0.150 0.083 -0.30029 0.136 4e -0.150 0.150 0.082 -0.3342 0.137 4f -0.141 0.141 0.065 -0.2821 0.153 4g -0.148 0.148 0.082 -0.29584 0.133 4h -0.149 0.149 0.085 -0.2987 0.131 4i -0.134 0.134 0.079 -0.26794 0.114 4j -0.158 0.158 0.087 -0.31654 0.143 4k -0.146 0.146 0.074 -0.2929 0.145 4l -0.147 0.147 0.076 -0.29483 0.143 Hardness: η = 1/2(ELUMO - EHOMO); Softness: S = 1/2η; Electrophilicity index: ω = µ/2η; Electronegativity: χ = -1/2(E LUMO + E HOMO ); Chemical potential: µ = - χ 3.8 Cytotoxicity Assessment Using MTT Assay The MTT assay was performed to observe the cytotoxic effects of various compounds (coded 4a–4l) on the MCF-7 cells, compared to cisplatin (positive control) and serum-free media (negative control). Among the test compounds, 4k and 4j exhibited the highest cytotoxicity (56.9% and 55.0%, respectively), with corresponding viability values around 43–45%, suggesting strong anti-proliferative effects. In contrast, compound 4e showed the lowest cytotoxicity (14.6%) and highest cell viability (85.4%), indicating minimal toxicity. Notably, cisplatin displayed very high cytotoxicity (86.29%), validating the assay, while the negative control showed 0% cytotoxicity, confirming the specificity of the assay. Some compounds like 4g, 4h, and 4d also showed substantial cytotoxicity (around 47–49%), suggesting potential for further investigation. Overall, compounds 4j and 4k appear the most promising for anti-cancer activity, though additional validation (e.g., apoptosis assays. Table 7: % Cytotoxicity of the synthesized quinoline-6-carboxylic acid derivatives in MCF-7 cancer cells Compound Code % Cytotoxicity % Viability 4a 37.3±1.80 62.7 4b 25.1±6.08 74.9 4c 42.2±0.81 57.8 4d 48.0±12.72 52.0 4e 14.6±2.48 85.4 4f 31.5±2.54 68.5 4g 47.4±1.80 52.6 4h 49.7±2.17 50.3 4i 41.5±0.31 58.5 4j 55.0±1.36 45.0 4k 56.9±16.87 43.1 4l 39.5±15.51 60.5 Cisplatin (Positive Control) 86.29±2.54 13.71 Serum Free Media (Negative Control) 0 100 3.9 Apoptosis Assessment Using Annexin V Assay and Fluorescent Microscopy For additional validation of the cytotoxicity results obtained through MTT assay, apoptosis studies were carried out using FITC Annexin V assay. It was found that the most cytotoxic compounds 4j and 4k showed significant apoptotic induction in the treated MCF-7 cells. Majority of the cells displayed strong Annexin V positive membrane staining, indicative of early apoptosis. Compound 4i revealed late apoptotic events as visible through co-staining with propidium iodide. These findings are supportive of the anti-cancer potential of these quinoline-6-carboxylic acid derivatives as pro-apoptotic agents in cancer management. 4. Conclusion In this study, a series of quinoline-6-carboxylic acid derivatives was synthesized and evaluated for their inhibitory potential against key ectonucleotidases, including h -NTPDase isoenzymes, h -NPP1, and h -e5’NT. The compounds were obtained in good yields (60–70%) and showed compatibility with a variety of substituents (F, Cl, I, CH 3 , CF 3 , OCH 3 ). Their structures were fully confirmed through NMR analysis. Biological screening revealed that compound 4d was most potent inhibitor of h -NTPDase1 and − 2, while compounds 4g, 4b, 4a , and 4k showed selective inhibition against h -NTPDase3, h -NTPDase8, h -e5’NT, and h -NPP1, respectively. Molecular docking indicated that these interactions are stabilized by hydrogen bonding, π-anion, π-alkyl, and π–π stacking forces. Complementary DFT analysis suggested a correlation between LUMO stabilization and inhibitory strength, reinforcing the structure–activity relationship. Moreover, fluorescence imaging of compound 4d demonstrated strong emission at the cellular level, supporting its effective binding to membrane-bound enzymes in the cancer cells. MTT and apoptosis studies also revealed compound 4i , 4j , and 4k to be potentially cytotoxic in the cancer cells, endorsing the potential of these quinoline derivatives to be used for drug development for cancer management. Together, these findings position quinoline-6-carboxylic acid derivatives as promising lead compounds for the development of effective ectonucleotidase inhibitors with potential applications in cancer immunotherapy. Declarations Author Contributions Aqsa Ishaq: conceptualization, synthesis, methodology, biological activities, docking studies, writing. Ismat Nawaz: Synthesis, editing. Salman Alrokayan: Bioassays, editing, fund acquisition. Tajamul Hussain: Bioassays, editing, fund acquisition. Nicolly Espindola Gelsleichter and Julie Pelletier: preparation of recombinant enzymes. Jean Sévigny: providing recombinant enzymes, review & editing. Jamshed Iqbal: supervision, resources (synthesis, bioactivity and docking). Acknowledgment Authors extend their appreciation to the Deanship of Scientific Research, King Saud University for funding through Vice Deanship of Scientific Research Chairs; Research Chair for Biomedical Application of Nanomaterials. JI received support from the Higher Education Commission China Pakistan Economic Corridor-Collaborative Research Grant # P2-345. 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Trott, O., Olson, A.J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. Journal of computational chemistry. 2010, 31, 455-61. Boyarshinov, V., Mikhalev, A., Yushkova, T., Ukhov, S., Kon’Shina, T. Synthesis and biological activity of quinoline-2-carboxylic acid aryl esters and amides. Pharmaceutical Chemistry Journal. 2017, 51, 351-4. Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations The authors declare no competing interests. Supplementary Files AqsaQuinolineMNSSIScRep662025.docx GraphicalAbstract.docx scheme1.png Scheme 1 Synthesis of quinoline-6-carboxylic acid derivatives (4a-4l). Cite Share Download PDF Status: Posted Version 1 posted 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-6824809","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":467337009,"identity":"0512cbfe-9b71-429e-91ee-6e1935d6f15e","order_by":0,"name":"Aqsa Ishaq","email":"","orcid":"","institution":"COMSATS University Islamabad","correspondingAuthor":false,"prefix":"","firstName":"Aqsa","middleName":"","lastName":"Ishaq","suffix":""},{"id":467338791,"identity":"0e0f7eca-8701-4e24-b518-267cb39c09f9","order_by":1,"name":"Ismat Nawaz","email":"","orcid":"","institution":"COMSATS University Islamabad","correspondingAuthor":false,"prefix":"","firstName":"Ismat","middleName":"","lastName":"Nawaz","suffix":""},{"id":467338792,"identity":"55e8f80f-7a7e-4233-a2c6-b9a322945255","order_by":2,"name":"Javeria Qadir","email":"","orcid":"","institution":"COMSATS University Islamabad","correspondingAuthor":false,"prefix":"","firstName":"Javeria","middleName":"","lastName":"Qadir","suffix":""},{"id":467338793,"identity":"02c5076d-90dc-43e2-b3da-75b08486d60d","order_by":3,"name":"Salman Alrokayan","email":"","orcid":"","institution":"King Saud University","correspondingAuthor":false,"prefix":"","firstName":"Salman","middleName":"","lastName":"Alrokayan","suffix":""},{"id":467338794,"identity":"17625d45-5834-4a7f-8cd3-bd0cefa24c38","order_by":4,"name":"Tajamul Hussain","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIiWNgGAWjYDACCSjJzwNmMBPWwQPTItlDohYGBoMzxGqxl+4x/vijxiLP+MzxZxIMFdaJDfyHH+C3ReaMmTTPMYlis7M9ZhIMZ9ITGyTSDAg4LMeMmYFNInHbeR42Cca2w0AtDAS1AB32TyJxcz/7MwnGf0At/Mc/ENJiIMHbJpG4gbfBTIKxAaiFIYeALTfSyqR5+yQSZ5w5Y2yRcCzduE0ipwCvFvYZyZs//vhWl9jfk/7wxocaa9l+/uMb8GpBBQlAzEaC+lEwCkbBKBgFOAAAIJg/46So2aEAAAAASUVORK5CYII=","orcid":"","institution":"King Saud University","correspondingAuthor":true,"prefix":"","firstName":"Tajamul","middleName":"","lastName":"Hussain","suffix":""},{"id":467338795,"identity":"65e07c09-a040-4c2f-af16-190b17e01557","order_by":5,"name":"Espindola Gelsleichter","email":"","orcid":"","institution":"Université Laval","correspondingAuthor":false,"prefix":"","firstName":"Espindola","middleName":"","lastName":"Gelsleichter","suffix":""},{"id":467338796,"identity":"354884c0-916d-4741-b356-81d0206c0e35","order_by":6,"name":"Julie Pelletier","email":"","orcid":"","institution":"Université Laval","correspondingAuthor":false,"prefix":"","firstName":"Julie","middleName":"","lastName":"Pelletier","suffix":""},{"id":467338797,"identity":"e04df45d-ac00-4ae7-a8e5-00bc3aabfac2","order_by":7,"name":"Jean Sévigny","email":"","orcid":"","institution":"Université Laval","correspondingAuthor":false,"prefix":"","firstName":"Jean","middleName":"","lastName":"Sévigny","suffix":""},{"id":467338798,"identity":"c8143509-120b-47ec-a7ad-ec2049636386","order_by":8,"name":"Muhammad Muhammad","email":"","orcid":"","institution":"University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"","lastName":"Muhammad","suffix":""},{"id":467338799,"identity":"11b4dac8-095f-4875-93fe-f833a928e3ea","order_by":9,"name":"Qing Huang","email":"","orcid":"","institution":"University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Huang","suffix":""},{"id":467338800,"identity":"8ec34c6c-e39b-4269-97f5-d1b1b4df86cc","order_by":10,"name":"Jamshed Iqbal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIiWNgGAWjYDCCAwwJYJLheAMDiMkIpAwYwEyCWs4cAKkzIEoLlLwBVkSEFr7bBx5+uvHnjhzfzcfPJB7U/JFtYG/eJsG4Iw2nFslzCcnSuW3PjCVvp5lJJBwzMG7gOVYmwXgmB6cWgzMMCdK5DYcTN9zOYZNIYDNIbJDIMZNgbKvApyX5d86fw/Ubbp4BavkH1CL/hqCWNOkctsMJBjd42CQS20C28IC04HaYJFCLdW7bYcOZZ9KMLRL7jI3beNKKLRLP4PY+3xme5NtAh8nzHT/88OaPb3Ky/eyHN974uCMZpxYGBp4EVD4biEhswKODgf0AFkFGvFpGwSgYBaNghAEAsgZenI6iWxIAAAAASUVORK5CYII=","orcid":"","institution":"COMSATS University Islamabad","correspondingAuthor":true,"prefix":"","firstName":"Jamshed","middleName":"","lastName":"Iqbal","suffix":""}],"badges":[],"createdAt":"2025-06-05 03:34:55","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6824809/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6824809/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84392313,"identity":"c64f5c73-4da2-4602-a100-79f7463678de","added_by":"auto","created_at":"2025-06-11 11:47:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":155550,"visible":true,"origin":"","legend":"\u003cp\u003eAdenosine triphosphate (ATP) is catalytically transformed into adenosine monophosphate (AMP) and adenosine (Ado) by the Ectonucleotidase Family.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6824809/v1/ba2e40c9bc6afdf0b28325aa.png"},{"id":84391922,"identity":"f988d78d-da09-4926-8beb-7d6a35369423","added_by":"auto","created_at":"2025-06-11 11:39:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":50074,"visible":true,"origin":"","legend":"\u003cp\u003eReported inhibitors of Ectonucleotidases retrieved from literature (a) e5′NT inhibitors [23], (b, c) ENNP inhibitors [24], and (d, e, f) standard reported inhibitors of NTPDases [25]\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6824809/v1/db4b6dc9081e7b54d2c9fc48.png"},{"id":84392315,"identity":"e81a739a-831f-4946-abd3-162fb1137f65","added_by":"auto","created_at":"2025-06-11 11:47:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":372887,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectroscopy and microscopy analysis of the antagonist drug molecule 4d. (A) Fluorescence spectroscopy profiles of 4d, excited between 410 nm and 500 nm, with corresponding emissions between 400 nm and 600 nm. (B) The bright-field image of A549 cells; Fluorescence microscopy image (red channel) of A549 cells treated with compound 4d; Merged image combining bright-field and fluorescence channels, highlighting the localization of the compound \u003cstrong\u003e4d\u003c/strong\u003e on the cell membrane.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6824809/v1/e2e7ef72ee5e1c679a5fdbf9.png"},{"id":84391926,"identity":"96d1961f-ff21-41b3-926b-fca00a82842e","added_by":"auto","created_at":"2025-06-11 11:39:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33310,"visible":true,"origin":"","legend":"\u003cp\u003eMost potent compounds among quinoline-6-carboxylic acid derivatives based on IC50 measurements\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6824809/v1/8e32814a32aa1257d6284429.png"},{"id":84392330,"identity":"3def6c58-6023-4ea8-bbf0-a34190af4784","added_by":"auto","created_at":"2025-06-11 11:47:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":233365,"visible":true,"origin":"","legend":"\u003cp\u003eIllustration of the 2D \u0026amp; 3D ligand-protein conformation of \u003cstrong\u003e(A)\u003c/strong\u003e Compound 4d with \u003cem\u003eh\u003c/em\u003e-ENTPDase1 \u003cstrong\u003e(B)\u003c/strong\u003e Compound \u003cstrong\u003e4d\u003c/strong\u003e with \u003cem\u003eh\u003c/em\u003e-ENTPDase2 \u003cstrong\u003e(C)\u003c/strong\u003e Compound \u003cstrong\u003e4g\u003c/strong\u003e with \u003cem\u003eh\u003c/em\u003e-ENTPDase3 (D) Compound \u003cstrong\u003e4b\u003c/strong\u003e with \u003cem\u003eh\u003c/em\u003e-ENTPDase8 \u003cstrong\u003e(E)\u003c/strong\u003e Compound \u003cstrong\u003e4k\u003c/strong\u003e with \u003cem\u003eh\u003c/em\u003e-ENPP1 \u003cstrong\u003e(F)\u003c/strong\u003e Compound \u003cstrong\u003e4a\u003c/strong\u003e with \u003cem\u003eh\u003c/em\u003e-e5′NT.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6824809/v1/8f61c7b455252e82e6758c86.png"},{"id":84391941,"identity":"cfdf8677-2569-48a2-8e52-f87d06876412","added_by":"auto","created_at":"2025-06-11 11:39:57","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":44876,"visible":true,"origin":"","legend":"\u003cp\u003eBOILED-Egg plot of quinoline-6-carboxylic acid derivatives. The X-axis represents WLOGP (Wildman-Crippen logP), indicating lipophilicity, while the Y-axis represents TPSA, a descriptor related to molecular size and hydrogen bonding capacity.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6824809/v1/ce5f3c91fc98d8d14478ca35.png"},{"id":84392324,"identity":"a362592e-4d12-4592-a6ed-da4456d369a8","added_by":"auto","created_at":"2025-06-11 11:47:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":225346,"visible":true,"origin":"","legend":"\u003cp\u003eOptimized structures of the quinoline derivatives (\u003cstrong\u003e4a-4l\u003c/strong\u003e)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6824809/v1/439c7259127ebcd2be029706.png"},{"id":84391944,"identity":"88707c8c-d56e-4010-813e-e5df0d4a93c6","added_by":"auto","created_at":"2025-06-11 11:39:58","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":309695,"visible":true,"origin":"","legend":"\u003cp\u003eHOMO-LUMO structures of the quinoline derivatives (\u003cstrong\u003e4a-4l)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6824809/v1/e8bdc596bda5cdc6c658ad38.png"},{"id":84391929,"identity":"9a58405b-0f41-4034-8007-e8292d3dd8e6","added_by":"auto","created_at":"2025-06-11 11:39:57","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":8921,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Representation of the % Cytotoxicity and % Viability of the Studied Compounds. (PC=Positive Control, Cisplatin; and NC=Negative Control, Serum Free Media)\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6824809/v1/aacefe92272e6e6777dc2c14.png"},{"id":84391945,"identity":"115117a7-2547-46da-aec3-a41992c53343","added_by":"auto","created_at":"2025-06-11 11:39:58","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":440828,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of FITC-Annexin V and PI fluorescence staining showing MCF-7 cell apoptosis after treatment with 100 nM of the compound \u003cstrong\u003e4j\u003c/strong\u003e, \u003cstrong\u003e4k\u003c/strong\u003e, and \u003cstrong\u003e4i\u003c/strong\u003e each. Cell nucleus was visualized by a red signal for PI and Annexin V was visualized by a green signal.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6824809/v1/553c69b190779cb93318d6d7.png"},{"id":84393422,"identity":"a26cd652-f8ef-46dc-a1f7-430651d3adaf","added_by":"auto","created_at":"2025-06-11 12:03:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3718614,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6824809/v1/38173728-cf12-4e52-b1c8-c189ec8ea56a.pdf"},{"id":84392319,"identity":"7cc3dd25-9e19-4656-8359-b05156a2920f","added_by":"auto","created_at":"2025-06-11 11:47:57","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2981838,"visible":true,"origin":"","legend":"","description":"","filename":"AqsaQuinolineMNSSIScRep662025.docx","url":"https://assets-eu.researchsquare.com/files/rs-6824809/v1/6c23e50dcbdcac06241c0ef3.docx"},{"id":84392316,"identity":"8e9ec179-96ba-4210-a39f-3bb3e8055b17","added_by":"auto","created_at":"2025-06-11 11:47:57","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":220883,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-6824809/v1/4d7063f60102898596713af1.docx"},{"id":84392314,"identity":"6f8c3051-3720-4201-9d6c-4d37d6cb1399","added_by":"auto","created_at":"2025-06-11 11:47:57","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":61788,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1 \u003c/strong\u003eSynthesis of quinoline-6-carboxylic acid derivatives (\u003cstrong\u003e4a-4l\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-6824809/v1/6f1402a4c1cbcdb5f09affc2.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eQuinoline-6-Carboxylic Acid Derivatives: A New Class of Potent Ectonucleotidase Inhibitors\u003c/p\u003e","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eTumor progression is intricately linked to immune cell infiltration and the resulting inflammatory milieu, which collectively promote angiogenesis, immune escape, and metastasis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Tumors also evolve mechanisms to subvert immune detection by manipulating molecular cues within the tumor microenvironment (TME). A central component of this immune evasion is the accumulation of adenosine, a small molecule with potent immunosuppressive activity that dampens the function of tumor-infiltrating leukocytes through the activation of A2A and A2B adenosine receptors [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Adenosine levels in the TME are tightly regulated by ectonucleotidases\u0026mdash;cell surface enzymes that sequentially hydrolyze extracellular ATP into AMP and subsequently into adenosine microenvironment [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. These enzymes consist of several types such as nucleoside triphosphate diphosphohydrolases (NTPDases), alkaline phosphatases (ALPs), nucleotide pyrophosphatases /phosphodiesterases (NPPs), and ecto-5\u0026prime;-nucleotidase (e5\u0026prime;NT) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. NTPDases hydrolyze ATP to ADP and AMP, while e5\u0026prime;NT converts AMP to adenosine as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. NPPs, such as NPP1, also participate by hydrolyzing nucleotides to monophosphates and pyrophosphate [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Overexpression of these ectonucleotidases has been reported in multiple types of cancer, such as melanoma [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] breast [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e][\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and prostate cancers[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Their overactivity is associated with tumor growth, suppression of immune responses, and poor clinical outcomes [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIncreased enzymatic activity leads to adenosine accumulation in the TME, which promotes tumor tolerance by inhibiting T and B cell function, natural killer (NK) cell cytotoxicity, and dendritic cell (DC) activation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Beyond cancer, aberrant ectonucleotidase expression is implicated in conditions such as insulin resistance [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], cardiovascular disease [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and pathological mineralization, reinforcing their therapeutic relevance[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Inhibiting these enzymes offers a promising strategy to reduce adenosine levels, reverse immunosuppression, and enhance anti-tumor immunity [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA diverse array of heterocyclic compounds has been explored for the targeted treatment of various cancers, whereby, both nucleotide- and non-nucleotide-based structures, have been investigated as ectonucleotidase inhibitors. Among them, quemliclustat (AB680), a nucleotide-based inhibitor, has shown promising preclinical results and is currently undergoing Phase 1 clinical trials for cancer therapy [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In addition, non-nucleotide ectonucleotidase inhibitors such as sulfonate derivatives have also been reported for their anticancer potential [\u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Likewise, several pyrazole-based ectonucleotidase inhibitors have demonstrated remarkable cytotoxic activity against various cancer cell lines, highlighting the therapeutic promise of such scaffolds [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Representative examples of standard ectonucleotidase inhibitors are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe design of quinoline-6-carboxylic acid amides as potential ectonucleotidase inhibitors is based on both structural features and evidence from literature. Quinoline is a well-known heterocyclic scaffold that has been widely studied for its anticancer potential [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Its flat, aromatic structure and nitrogen atom make it structurally similar to purine-based molecules like ATP, allowing it to fit into nucleotide-binding pockets of the enzymes such as CD39 and CD73 [\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Moreover, quinoline derivatives have shown activity against several cancer-related targets, including topoisomerases[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and protein kinases[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Introducing a carboxylic acid group at the 6-position of the quinoline ring allows the molecule to mimic phosphate groups found in natural nucleotides, promoting key polar interactions at the enzyme\u0026rsquo;s active site. Converting this acid to an amide improves the molecule\u0026rsquo;s stability, solubility, and membrane permeability\u0026mdash;important traits for inhibiting extracellular enzymes like ectonucleotidases [\u003cspan additionalcitationids=\"CR38 CR39 CR40 CR41\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eContextual to this, recent studies have reported quinoline-based carboxamides to be effective inhibitors of ATP-hydrolyzing enzymes, suggesting their potential interaction with the catalytic regions of the enzymes involved in purine metabolism [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Given their known anticancer properties and favorable drug-like features, we were inspired to design, synthesize and explore quinoline-6-carboxylic acid amides as inhibitors of ectonucleotidases\u0026mdash;another promising target involved in cancer immune evasion. In addition, molecular docking studies were conducted to predict their binding affinities and interactions with the target proteins \u003cem\u003ein-silico\u003c/em\u003e. The biological activity of the synthesized compounds was, then, evaluated through fluorescence assay to assess cellular interaction, MTT assay for measuring cytotoxicity and apoptosis assay to determine the cell-death inducing potential of these compounds.\u003c/p\u003e"},{"header":"2. EXPERIMENTAL SECTION","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 General Chemistry Methods:\u003c/h2\u003e \u003cp\u003eAll the solvents used in this study were of analytical grade and used without further purification. Chemicals were purchased from local suppliers and used directly in the reactions. Melting points of the synthesized compounds were measured using a Gallenkamp melting point apparatus. Reaction progress was monitored by Thin Layer Chromatography (TLC) using Merck pre-coated silica gel plates (60 F254), with spot detection under UV light at 254 nm to confirm complete consumption of the starting materials. Structural analysis of the compounds was performed using a Bruker Avance III HD NMR spectrometer operating at 300 and 400 MHz. Both proton (\u003csup\u003e1\u003c/sup\u003eH) and carbon (\u003csup\u003e13\u003c/sup\u003eC) NMR spectra were recorded, with chemical shifts reported in parts per million (ppm) and coupling constants (J) in Hertz (Hz). Signal multiplicities were reported as follows: s for singlet, d for doublet, t for triplet, and q for quartet.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eGeneral synthetic procedure \u003cb\u003e(A)\u003c/b\u003e for Quinoline-Based Amide Derivatives \u003cb\u003e(4a-4l)\u003c/b\u003e\u003c/strong\u003e \u003cp\u003eQuinoline-6-carboxylic acid (1.15 mmol, 200 mg, 1 equiv) was reacted with thionyl chloride (3.46 mmol, 412 mg, 3 equiv) in the presence of DCM (20 mL) as a solvent under reflux at 50\u0026deg;C for 8 hours. The conversion of the acid into the corresponding acid chloride was monitored by TLC. Once the conversion was complete, the acid chloride was treated with various amines and stirred overnight, with the reaction progress monitored through TLC. Upon completion, the reaction mixture was concentrated using a rotary evaporator to remove the solvent. The resulting residue was added to a beaker containing distilled water and stirred until a precipitate formed. The precipitate was filtered and dried in an oven at 37\u0026deg;C, yielding quinoline-based amide derivatives (4a\u0026ndash;4l) in good to excellent yields.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eN\u003c/em\u003e-(4-chlorobenzyl)quinoline-6-carboxamide \u003cb\u003e(4a)\u003c/b\u003e. Using the general synthetic protocol, \u003cb\u003eA\u003c/b\u003e, (4-Chlorophenyl)methanamine (1.15 mmol, 164 mg, 140 \u0026micro;L, 1 equiv) was employed as the starting amine, yielding the target compound \u003cb\u003e4a\u003c/b\u003e as a sand brown solid (67%); m.p: 185\u0026ndash;187\u0026deg;C; R\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e = 0.3 (hexane/ EtOAc\u0026thinsp;=\u0026thinsp;3:2); \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) ppm δ 9.32 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 1H, N12-H), 8.99 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.2, 1.7 Hz, 1H, C2-H), 8.55 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H, C5-H), 8.48 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4, 1.7 Hz, 1H, C4-H), 8.21 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.7, 2.0 Hz, 1H, C7-H), 8.09 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, C8-H), 7.61 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3, 4.2 Hz, 1H, C3-H), 7.40 (s, 4H, C15, 16, 18, 19-H), 4.53 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 2H, C13-H); \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, DMSO) δ 166.0 (s, C-11), 152.1 (s, C-2), 148.7 (s, C-9), 138.6 (s, C-14), 137.1 (s, C-4), 132.0 (s, C-17), 131.4 (s, C-10), 129.2 (s, C-15, 19), 129.1 (s, C-10), 128.3 (s, C-5), 128.1(s, C-8), 127.7 (s, C-16, C18), 127.1 (s, C-7), 122.2 (s, C-3), 42.2 (s, C-13) ; FT-IR (neat) ῡ (cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;3453cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N-H stretching), 1654cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O stretching).\u003c/p\u003e \u003cp\u003e \u003cem\u003eN\u003c/em\u003e-(4-fluorobenzyl)quinoline-6-carboxamide \u003cb\u003e(4b).\u003c/b\u003e According to general procedure, \u003cb\u003eA\u003c/b\u003e, (4-fluorophenyl)methanamine (1.15 mmol, 145 mg, 141\u0026micro;L, 1 equiv) was employed as the starting amine, yielding the target compound \u003cb\u003e4b\u003c/b\u003e as a chocolate brown solid (64%); m.p: 150\u0026ndash;152\u0026deg;C; R\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e = 0.3 (hexane/ EtOAc\u0026thinsp;=\u0026thinsp;4.6); \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO) ppm δ 9.31 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 1H, N12-H), 8.99 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.2, 1.8 Hz, 1H, C2-H), 8.55 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H, C5-H), 8.48 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2, 2.3 Hz, 1H, C4-H), 8.21 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8, 2.0 Hz, 1H, C7-H), 8.09 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, C8-H), 7.61 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3, 4.2 Hz, 1H, C3-H), 7.46\u0026ndash;7.33 (m, 2H, C16,18-H), 7.17 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.9 Hz, 2H C15,19-H), 4.53 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 2H, C13-H); \u003csup\u003e13\u003c/sup\u003eC NMR (75 MHz, DMSO) ppm δ 166.3 (s, C-11), 161.7 (d, \u003csup\u003e1\u003c/sup\u003e\u003cem\u003eJ\u003c/em\u003e\u003csub\u003eC\u0026minus;F\u003c/sub\u003e = 240.8 Hz, s, C-17), 152.6 (s, C-2), 149.1 (s, C-9), 137.9 (s, C-4), 136.2 (d, \u003csup\u003e4\u003c/sup\u003e\u003cem\u003eJ\u003c/em\u003e\u003csub\u003eC\u0026minus;F\u003c/sub\u003e = 2.0 Hz, C-14), 132.6 (s, C-10), 129.8 (d, \u003csup\u003e3\u003c/sup\u003e\u003cem\u003eJ\u003c/em\u003e\u003csub\u003eC\u0026minus;F\u003c/sub\u003e = 8.1 Hz, C-15), 129.5 (s, C-5), 128.5 (s, C-8), 128.2 (s, C-7), 127.6 (s, C-6) 122.7 (s, C-3), 115.5 (d, \u003csup\u003e2\u003c/sup\u003e\u003cem\u003eJ\u003c/em\u003e\u003csub\u003eC\u0026minus;F\u003c/sub\u003e = 21.1 Hz, C-16), 42.6 (s, C-13); FT-IR (neat) ῡ (cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;3279cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N-H stretching), 1635cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O stretching).\u003c/p\u003e \u003cp\u003e \u003cem\u003eN\u003c/em\u003e-(4-methylbenzyl)quinoline-6-carboxamide \u003cb\u003e(4c).\u003c/b\u003e Using the general synthetic protocol, \u003cb\u003eA\u003c/b\u003e, p-tolymethanamine (1.15 mmol, 139 mg, 147 \u0026micro;L, 1 equiv) was employed as the starting amine, yielding the target compound \u003cb\u003e4c\u003c/b\u003e as a light brown solid (70%); m.p: 190\u0026ndash;192\u0026deg;C; R\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e = 0.4 (hexane/ EtOAc\u0026thinsp;=\u0026thinsp;4:1); \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO) ppm δ 9.33 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.0 Hz, 1H, N12-H), 8.98 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.2, 1.7 Hz, 1H, C2-H), 8.57 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H, C5-H), 8.47 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 1H, C4-H), 8.22 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8, 2.0 Hz, 1H, C7-H), 8.08 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, C8-H), 7.61 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3, 4.2 Hz, 1H, C3-H), 7.25 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz, 2H, C19-H), 7.14 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz, 2H, C16-H), 4.49 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.9 Hz, 2H, C13-H), 2.27 (s, 3H, C18-H); \u003csup\u003e13\u003c/sup\u003eC NMR (75 MHz, DMSO) ppm δ 166.1 (s, C-11), 152.2 (s, C-2), 149.1 (s, C-9), 137.6 (s, C-4), 137.0 (s, C-17), 136.3 (s, C-6), 132.7 (s, C-14), 129.4 (s, C-10), 129.3 (s, C-5), 128.5 (s, C-8), 128.2 (s, C-16), 127.8 (s, C-15), 127.6 (s, C-7), 122.6 (s, C-3), 43.0 (s, C-13), 21.1 (s, C18); FT-IR (neat) ῡ (cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;3296cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N-H stretching), 1635cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O stretching).\u003c/p\u003e \u003cp\u003e \u003cem\u003eN\u003c/em\u003e-(2,6 fluorophenyl)quinoline-6-carboxamide \u003cb\u003e(4d)\u003c/b\u003e. Using the general synthetic protocol, \u003cb\u003eA\u003c/b\u003e, 2,6 difluoroaniline (1.15 mmol, 149 mg, 124 \u0026micro;L, 1 equiv) was employed as the starting amine, yielding the target compound \u003cb\u003e4d\u003c/b\u003e as a light brown solid (70%); m.p: 243\u0026ndash;245\u0026deg;C; R\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e = 0.2 (hexane/ EtOAc\u0026thinsp;=\u0026thinsp;7:3); \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO) ppm δ 10.45 (s, 1H, N12-H), 9.04 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.2, 1.8 Hz, 1H, C2-H), 8.70 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H, C5-H), 8.56 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3, 1.8 Hz, 1H, C4-H), 8.29 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8, 2.0 Hz, 1H, C7-H), 8.16 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, C8-H), 7.66 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3, 4.2 Hz, 1H, C3-H), 7.44 (tt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8, 6.4 Hz, 1H, C16-H), 7.32\u0026ndash;7.18 (m, 2H, C17-H); \u003csup\u003e13\u003c/sup\u003eC NMR (75 MHz, DMSO) ppm δ 165.5 (s, C-11), 158.21 (d, \u003csup\u003e1\u003c/sup\u003e\u003cem\u003eJ\u003c/em\u003e\u003csub\u003eC\u0026minus;F\u003c/sub\u003e = 247.4 Hz, C-14), 158.18 (d, \u003csup\u003e1\u003c/sup\u003e\u003cem\u003eJ\u003c/em\u003e\u003csub\u003eC\u0026minus;F\u003c/sub\u003e = 247.3 Hz, C-18), 152.6 (s, C-2), 149.0 (s, C-9), 137.3 (s, C-4), 131.0 (s, C-6), 129.4 (s, C-10), 129.1 (s, C-5), 129.05 (s, C-8), 128.6 (t, \u003csup\u003e3\u003c/sup\u003e\u003cem\u003eJ\u003c/em\u003e\u003csub\u003eC\u0026minus;F\u003c/sub\u003e = 9.8 Hz, C-16), 127.6 (s, C-7), 122.5 (s, C-3), 114.7 (t, \u003csup\u003e3\u003c/sup\u003e\u003cem\u003eJ\u003c/em\u003e\u003csub\u003eC\u0026minus;F\u003c/sub\u003e = 9.3 Hz, C-15,17), 112.0 (d, \u003csup\u003e2\u003c/sup\u003e\u003cem\u003eJ\u003c/em\u003e\u003csub\u003eC\u0026minus;F\u003c/sub\u003e = 22.3 Hz, C-13); FT-IR (neat) ῡ (cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;3165cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N-H stretching), 1665cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O stretching).\u003c/p\u003e \u003cp\u003e \u003cem\u003eN\u003c/em\u003e-(2,4 difluorophenyl)quinoline-6-carboxamide \u003cb\u003e(4e\u003c/b\u003e). Using the general synthetic protocol, \u003cb\u003eA\u003c/b\u003e, 2,4-difluoroaniline (1.15 mmol, 149 mg, 117 \u0026micro;L, 1 equiv) was employed as the starting amine, yielding the target compound \u003cb\u003e4e\u003c/b\u003e as a brown (60%); m.p: 186\u0026ndash;188\u0026deg;C; R\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e = 0.5 (hexane/ EtOAc\u0026thinsp;=\u0026thinsp;3:2); \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO) ppm δ 10.40 (s, 1H, N12-H), 9.02 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.2, 1.8 Hz, 1H, C2-H), 8.66 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.1 Hz, 1H, C5-H, ), 8.54 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4, 1.7 Hz, 1H, C4-H), 8.26 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8, 2.0 Hz, 1H, C7-H), 8.14 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, C8-H), 7.70\u0026ndash;7.59 (m, 2H, C3, C18-H), 7.39 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.6, 9.1, 2.9 Hz, 1H, C17-H), 7.15 (tdd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5, 2.8, 1.4 Hz, 1H, C15-H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO)\u003csup\u003e\u003cem\u003e(a)\u003c/em\u003e\u003c/sup\u003e ppm δ 165.1 (s, C-11), 152.4 (s, C-2), 148.9 (s, C-9), 137.3 (s, C-4), 131.6 (s, C-6), 129.2 (s, C-10), 128.8 (s, C-5), 128.4 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.3, 2.8 Hz, C-18), 128.0(s, C-8), 127.1 (s, C-7), 122.4 (s, C-3), 111.3 (d, \u003csup\u003e2\u003c/sup\u003e\u003cem\u003eJ\u003c/em\u003e\u003csub\u003eC\u0026minus;F\u003c/sub\u003e = 25.5 Hz, C-13), 104.7\u0026ndash;104.0 (m, 2C, C-15,17); FT-IR (neat) ῡ (cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;3279cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N-H stretching), 1635cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O stretching).\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cem\u003e(a)\u003c/em\u003e \u003c/sup\u003eDue to the dilute nature of the sample, the signals for the two carbons directly attached to fluorine, expected in the range of 159\u0026ndash;160 ppm, are missing.\u003c/p\u003e \u003cp\u003eN-[5-fluoro-2-(trifluoromethyl) phenyl]quinoline-6-carboxamide \u003cb\u003e(4f).\u003c/b\u003e Using the general synthetic protocol, \u003cb\u003eA\u003c/b\u003e, 4-fluoro-2-(trifluoromethyl) aniline (1.15 mmol, 206 mg, 150 \u0026micro;L, 1 equiv) was employed as the starting amine, yielding the target compound \u003cb\u003e4f\u003c/b\u003e as a desert brown (65%); m.p: 200\u0026ndash;202\u0026deg;C; R\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e = 0.3 (hexane/ EtOAc\u0026thinsp;=\u0026thinsp;3:2); \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO\u003cem\u003e) ppm\u003c/em\u003e δ 10.46 (s, 1H, N12-H), 9.03 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.2, 1.7 Hz, 1H, C2-H), 8.64 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H, C5-H), 8.60\u0026ndash;8.50 (m, 1H, C4-H), 8.26 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8, 2.0 Hz, 1H, C7-H), 8.16 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, C8-H), 7.75 (dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.9, 1.8 Hz, 1H, C17-H), 7.70\u0026ndash;7.60 (m, 3H, C3, 8, 15-H); \u003csup\u003e13\u003c/sup\u003eC NMR (75 MHz, DMSO\u003cem\u003e)\u003c/em\u003e ppm δ 166.2 (s, C-11), 160.3 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;244.6 Hz, C-17), 152.8 (s, C-2), 148.9 (s, C-9), 137.3 (s, C-4), 134.0 (s, C-6), 133.9 (s, C-5), 132.1 (s, C-13), 131.6 (s, C-10), 129.3 (s, C-5), 128.7 (s, C-8), 127.7 (s, C-7), 127.2 (s, C-19), 122.4 (s, C-3), 120.4 (s, C-16), 120.1 (s, C-18), 114.0 (s, C-14); FT-IR (neat) ῡ (cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;3345cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N-H stretching), 1650cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O stretching).\u003c/p\u003e \u003cp\u003e \u003cem\u003eN\u003c/em\u003e-(pyridin-2-yl)quinoline-6-carboxamide \u003cb\u003e(4g).\u003c/b\u003e Using the general synthetic protocol, \u003cb\u003eA\u003c/b\u003e, pyridine-2-amine (1.15 mmol, 109 mg, 1 equiv) was employed as a the starting amine, yielding the target compound \u003cb\u003e4g\u003c/b\u003e as a cream yellow (68%); m.p: 97\u0026ndash;99\u0026deg;C; R\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e = 0.4 (hexane/ EtOAc\u0026thinsp;=\u0026thinsp;3:7); \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO) ppm δ 11.03 (s, 1H, N12-H), 9.02 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.2, 1.8 Hz, 1H, C2-H), 8.76 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H, C5-H), 8.51 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4, 1.8, 0.8 Hz, 1H, C4-H), 8.42 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.9, 2.0, 0.9 Hz, 1H, C7-H), 8.30 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8, 2.1 Hz, 1H, C8-H), 8.26\u0026ndash;8.22 (m, 1H, C14-H), 8.11 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, C3-H), 7.88 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4, 7.3, 2.0 Hz, 1H, C16-H), 7.64 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3, 4.2 Hz, 1H, C17-H), 7.19 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3, 4.9, 1.0 Hz, 1H, C15-H); \u003csup\u003e13\u003c/sup\u003eC NMR (75 MHz, DMSO) ppm δ 165.6 (s, C-11), 152.4 (s, C-2), 152.2 (s, C-13), 148.9 (s, C-15), 148.1 (s, C-9), 138.3 (s, C-17), 137.4 (s, C-4), 131.8 (s, C-6), 129.2 (s, C-10), 129.0 (s, C-5), 128.2 (s, C-8), 127.0 (s, C-7), 122.3 (s, C-3), 120.0 (s, C-16), 114.7 (s, C-18); FT-IR (neat) ῡ (cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;3294cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N-H stretching), 1683cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O stretching).\u003c/p\u003e \u003cp\u003e \u003cem\u003eN\u003c/em\u003e-(2-iodophenyl)quinoline-6-carboxamide \u003cb\u003e(4h).\u003c/b\u003e Using the general synthetic protocol, \u003cb\u003eA\u003c/b\u003e, 2-Iodoaniline (1.15 mmol, 253 mg, 1 equiv) was employed as the starting amine, yielding the target compound \u003cb\u003e4h\u003c/b\u003e as a Coffee brown solid (65%); m.p: 182\u0026ndash;184\u0026deg;C; R\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e = 0.3 (hexane/ EtOAc\u0026thinsp;=\u0026thinsp;4:6); \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO\u003cem\u003e)\u003c/em\u003e ppm δ: 10.36 (s, 1H, N12-H), 9.03 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.2, 1.7 Hz, 1H, C2-H), 8.70 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H, C5-H), 8.58\u0026ndash;8.52 (m, 1H, C4-H), 8.32 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8, 2.0 Hz, 1H, C7-H), 8.16 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, C8-H), 7.97 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7, 1.2 Hz, 1H, C15-H), 7.65 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3, 4.2 Hz, 1H, C3-H), 7.49 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.7, 1.6 Hz, 2H, C16, 18-H), 7.10 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 6.5, 2.4 Hz, 1H, C17-H); \u003csup\u003e13\u003c/sup\u003eC NMR (75 MHz, DMSO)\u003csup\u003e(a)\u003c/sup\u003e ppm δ: 164.9 (s, C-11), 152.4 (s, C-2), 148.9 (s, C-13), 139.8 (s, C-9), 139.0 (s, C-15), 137.3 (s, C-4), 132.0 (s, C-6), 129.3 (s, C-10), 128.9 (s, C-5), 128.7 (2C, C8,17), 128.5 (s, C-7), 127.9 (s, C-16), 127.2 (s, C-18), 122.4 (s, C-3), 99.1 (s, C-14); FT-IR (neat) ῡ (cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;3392cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N-H stretching), 1652cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O stretching).\u003c/p\u003e \u003cp\u003e \u003cem\u003eN\u003c/em\u003e-(2, 4-dimethoxybenzyl)quinoline-6-carboxamide \u003cb\u003e(4i).\u003c/b\u003e Using the general synthetic protocol, \u003cb\u003eA\u003c/b\u003e, (2,4 dimethoxyphenyl)methanamine (1.15 mmol, 193 mg, 174 \u0026micro;L, 1 equiv) was employed as the starting amine, yielding the target compound \u003cb\u003e4i\u003c/b\u003e as a Dark brown solid (65%); m.p: 120\u0026ndash;123\u0026deg;C; R\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e = 0.6 (hexane/ EtOAc\u0026thinsp;=\u0026thinsp;2:8); \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed6)\u003c/em\u003e ppm δ 9.05\u0026ndash;8.96 (m, 2H, C2-H, N12-H), 8.56 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H, C5-H), 8.48 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4, 1.8 Hz, 1H, C4-H), 8.22 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8, 2.0 Hz, 1H, C7-H), 8.08 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, C8-H), 7.62 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3, 4.2 Hz, 1H, C3-H), 7.16 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 1H, C18-H), 6.58 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.4 Hz, 1H, C15-H), 6.49 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3, 2.4 Hz, 1H, C17-H), 4.44 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.6 Hz, 2H, C11-H), 3.82 (s, 3H, C19-H), 3.74 (s, 3H, C20-H); \u003csup\u003e13\u003c/sup\u003eC NMR (75 MHz, DMSO) ppm δ 165.8 (s, C-11),, 159.7 (s, C-17), 157.6 (s, C-15), 151.9 (s, C-2), 148.5 (s, C-9), 137.3 (s, C-4), 132.4 (s, C-6), 128.8 (s, C-10), 128.6 (s, C-19), 128.0 (s, C-5), 127.9 (s, C-8), 127.1 (s, C-7), 122.2 (s, C-3), 118.9 (s, C-14), 104.4 (s, C-18), 98.2 (s, C-16), 55.5 (s, C-20), 55.2 (s, C-21), 37.6 (s, C-13); FT-IR (neat) ῡ (cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;3289cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N-H stretching), 1638cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O stretching).\u003c/p\u003e \u003cp\u003e \u003cem\u003eN\u003c/em\u003e-(2,4 -dichlorobenzyl)quinoline-6-carboxamide \u003cb\u003e(4j)\u003c/b\u003e. Using the general synthetic protocol, \u003cb\u003eA\u003c/b\u003e, (2,4 dichlorophenyl)methanamine (1.15 mmol, 203 mg, 155 \u0026micro;L,1 equiv) was employed as the starting amine, yielding the target compound \u003cb\u003e4j\u003c/b\u003e as a Light brown solid (67%); m.p: 165\u0026ndash;167\u0026deg;C; R\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e = 0.4 (hexane/ EtOAc\u0026thinsp;=\u0026thinsp;6:4); \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) δ: 9.33 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 1H, N12-H), 9.03\u0026ndash;8.94 (m, 1H, C2-H), 8.58 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H, C5-H), 8.52\u0026ndash;8.43 (m, 1H, C4-H), 8.22 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8, 2.0 Hz, 1H, C7-H), 8.10 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, C8-H), 7.64 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.8 Hz, 1H, C15-H), 7.63\u0026ndash;7.57 (m, 1H, C3-H), 7.43 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.2 Hz, 2H, C17,C18-H), 4.57 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7 Hz, 2H, C11-H); \u003csup\u003e13\u003c/sup\u003eC NMR (75 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) δ: 166.1 (s, C-11), 152.2 (s, C-2), 148.9 (s, C-9), 137.2 (s, C-4), 135.5 (s, C-6), 133.0 (s, C-14), 132.3 (s, C-15), 131.8 (s, C-17), 130.3 (s, C-16), 129.2 (s, C-10), 128.7 (s, C-19), 128.2 (s, C-8), 127.8 (s, C-5), 127.4 (s, C-7), 127.2 (s, C-18), 122.3 (s, C-3), 40.5 (s, C-13); FT-IR (neat) ῡ (cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;3289cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N-H stretching), 1638cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O stretching).\u003c/p\u003e \u003cp\u003e \u003cem\u003eN\u003c/em\u003e-(4-Iodophenyl)quinoline-6-carboxamide \u003cb\u003e(4k).\u003c/b\u003e Using the general synthetic protocol, \u003cb\u003eA\u003c/b\u003e,\u003c/p\u003e \u003cp\u003e4-iodoaniline (1.15 mmol, 253 mg, 1 equiv) was employed as the starting amine, yielding the target compound 4k as a Dark brown (63%); m.p: 182\u0026ndash;184\u0026deg;C; R\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e = 0.4 (hexane/ EtOAc\u0026thinsp;=\u0026thinsp;4:1); \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) δ: 10.72 (s, 1H, N12-H), 9.09 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.5, 1.7 Hz, 1H, C2-H), 8.75\u0026ndash;8.68 (m, 1H, C5-H), 8.69 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H, C4-H), 8.32 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8, 2.0 Hz, 1H, C7-H), 8.20 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.9 Hz, 1H, C8-H), 7.90\u0026ndash;7.58 (m, 5H, C3, 14, 15, 17, 18-H); \u003csup\u003e13\u003c/sup\u003eC NMR (75 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e) δ: 165.0 (s, C-11), 151.2 (s, C-2), 146.8 (s, C-9), 139.2 (s, C-13), 139.0 (s, 2C, C-15,17), 137.4 (s, C-4), 133.0 (s, C-6), 129.0 (s, C-10), 128.8 (s, C-5), 127.6 (s, C-8), 127.2 (s, C-7), 122.5 (s, C-3), 122.5 (s, 2C, C-13,18), 87.7 (s, C-16); FT-IR (neat) ῡ (cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;3288cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N-H stretching), 1678cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O stretching).\u003c/p\u003e \u003cp\u003e \u003cem\u003eN-(5-fluoro 2-methylphenyl)quinoline-6-carboxamide\u003c/em\u003e \u003cb\u003e(4l).\u003c/b\u003e Using the general synthetic protocol, \u003cb\u003eA\u003c/b\u003e, 5-fluoro 2-methylaniline (1.15 mmol, 144 mg, 145 \u0026micro;L,1 equiv) was employed as the starting amine, yielding the target compound 4l as a brown solid (122 mg, 66%); m.p: 164\u0026ndash;166\u0026deg;C; R\u003csub\u003e\u003cem\u003ef\u003c/em\u003e\u003c/sub\u003e = 0.4 (hexane/ EtOAc\u0026thinsp;=\u0026thinsp;3:7); \u003csup\u003e1\u003c/sup\u003eH NMR (300 MHz, DMSO) ppm δ: 10.19 (s, 1H, N12-H), 9.02 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.2, 1.7 Hz, 1H, C2-H), 8.66 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H, C5-H), 8.55 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5, 1.8 Hz, 1H, C4-H), 8.28 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8, 2.0 Hz, 1H, C7-H), 8.15 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, C8-H), 7.65 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3, 4.2 Hz, 1H, C3-H), 7.34 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.3, 8.4, 4.6 Hz, 2H, C18, C16-H), 7.04 (td, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5, 2.8 Hz, 1H, C15-H), 2.28 (s, 3H, C19-H); \u003csup\u003e13\u003c/sup\u003eC NMR (75 MHz, DMSO) ppm δ: 165.1 (s, C-11), 160.2 (d, \u003csup\u003e1\u003c/sup\u003e\u003cem\u003eJ\u003c/em\u003e\u003csub\u003eC\u0026minus;F\u003c/sub\u003e = 239.1 Hz, C-17), 152.4 (s, C-2), 148.9 (s, C-9), 137.6 (d, \u003csup\u003e3\u003c/sup\u003e\u003cem\u003eJ\u003c/em\u003e\u003csub\u003eC\u0026minus;F\u003c/sub\u003e = 10.2 Hz, C-15), 137.3 (s, C-4), 132.2 (s, C-6), 131.51 (d, \u003csup\u003e3\u003c/sup\u003e\u003cem\u003eJ\u003c/em\u003e\u003csub\u003eC\u0026minus;F\u003c/sub\u003e = 8.8 Hz, C-13), 129.2 (s, C-10), 129.2 (s, C-8), 128.7 (s, C-5), 128.1 (s, C-14), 127.2 (s, C-7), 122.4 (s, C-3), 112.69 (d, \u003csup\u003e2\u003c/sup\u003e\u003cem\u003eJ\u003c/em\u003e\u003csub\u003eC\u0026minus;F\u003c/sub\u003e = 45.0 Hz, C-16), 112.68 (s, C-18), 17.76 (s, C-19) ; FT-IR (neat) ῡ (cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u0026thinsp;=\u0026thinsp;3352cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (N-H stretching), 1649cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (C\u0026thinsp;=\u0026thinsp;O stretching).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Density functional theory\u003c/h2\u003e \u003cp\u003eGeometry optimizations of the selected molecules were carried out using Density Functional Theory (DFT) at the B3LYP/3-21G level, consistent with methodologies reported in previous literature [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The chemical reactivity of the synthesized compounds was determined by analyzing the frontier molecular orbitals. The compounds' softness and chemical hardness were measured. Gaussian 09 software was used to conduct quantum mechanical research on the multi-core machine, and Gauss View 6 was used to view the optimized structures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Enzymes preparation\u003c/h2\u003e \u003cp\u003eTo produce the enzymes, COS-7 cells were transfected with plasmids containing the \u003cem\u003eh\u003c/em\u003e-NTPDase1 gene (GenBank accession number U87967) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. The \u003cem\u003eh\u003c/em\u003e-NTPDase2 construct (GenBank accession number NM_203468) was generously provided by Aileen F. Knowles [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. We received the \u003cem\u003eh\u003c/em\u003e-NTPDase3 plasmid (GenBank accession no AF034840) as a generous gift from Terence L. Kirley. [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] \u003cem\u003eh\u003c/em\u003e-NTPDase8 (GenBank accession no AY430414) [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] \u003cem\u003eh\u003c/em\u003e-NPP1 (GenBank accession no NM006208) a kind gift of James W. Goding [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and \u003cem\u003eh\u003c/em\u003e-e5\u0026rsquo;NT (GenBank accession no DQ186653) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] as previously described [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Transfection of COS-7 cells was carried out in 10 cm culture plates by incubating them at 37\u0026deg;C for 5 hours using serum-free DMEM/F-12 medium mixed with 6 \u0026micro;g of plasmid DNA and 24 \u0026micro;L Lipofectamine. Following this, the plate received an equal volume of DMEM/F-12 medium containing 20% fetal bovine serum after 6-8hrs. After 44\u0026ndash;48 hours of incubation, the cells were washed 3 times with a harvesting buffer containing 95 mM NaCl, 0.1 mM PMSF, and 45 mM Tris at pH 7.5. After scraping, the cells were centrifuged twice (300 g, 5 minutes at 4\u0026deg;C), washed, and resuspended in harvesting buffer with aprotinin (10 \u0026micro;g/mL) before undergoing sonication. Centrifugation at 850 g for 5 minutes at 4\u0026deg;C was performed to remove the nuclei and cellular debris. The supernatant obtained was supplemented with 7.5% glycerol and preserved at \u0026minus;\u0026thinsp;80\u0026deg;C. Protein concentration was determined using the Bradford assay in a microplate format, with bovine serum albumin (BSA) serving as the standard [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Enzyme inhibition assays\u003c/h2\u003e \u003cp\u003e \u003cb\u003eh\u003c/b\u003e \u003cb\u003e-NTPDase Activity Assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA significantly modified version of a previously published spectrophotometric technique was used to study the effect of all the synthesized compounds on \u003cem\u003eh\u003c/em\u003e-NTPDase1, -2, -3, and \u0026minus;\u0026thinsp;8 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The compounds were screened at a concentration of 100 \u0026micro;M. The assay was performed in an incubation medium containing 50 mM of Tris-HCl and 5 mM of CaCl\u003csub\u003e2\u003c/sub\u003e maintained at pH 7.4. Reaction medium containing buffer (55 \u0026micro;L), test compound (10 \u0026micro;L), and 10 \u0026micro;L of enzyme solution of \u003cem\u003eh\u003c/em\u003e-NTPDase1 (12 ng/well) or \u003cem\u003eh\u003c/em\u003e-NTPDase2 (37 ng/well) or \u003cem\u003eh\u003c/em\u003e-NTPDase3 (43 ng/well) or \u003cem\u003eh\u003c/em\u003e-NTPDase8 (63 ng/well) was pre-incubated at 37℃. After 10 min, the reaction absorbance was measured (630 nm) with a microplate reader (FLUOStar Omega. BMG Labtech, Germany). 10 \u0026micro;L of substrate i.e. ATP (100 \u0026micro;M) was added to begin the reaction followed by 15mins incubation. The enzymatic reaction was eventually stopped by adding 15 \u0026micro;L of malachite green reagent. By measuring the absorbance at 630 nm, released inorganic phosphate was identified and the percent inhibition was computed. Compounds with \u0026gt;\u0026thinsp;50% inhibition of any isoform of \u003cem\u003eh\u003c/em\u003e-NTPDase, were further diluted and their IC\u003csub\u003e50\u003c/sub\u003e values were calculated by generating and fitting the dose-response curves with GraphPad Prism 5.0 (San Diego, CA, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eh\u003c/b\u003e \u003cb\u003e-NPP1 Activity Assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWith slight modifications to the previously described procedure, an inhibition assay on \u003cem\u003eh\u003c/em\u003e-NPP1 was conducted to verify these synthesized derivatives' inhibitory effect [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The test buffer (pH\u0026thinsp;=\u0026thinsp;9.5) included 50 mM Tris-HCl, 5 mM MgCl\u003csub\u003e2,\u003c/sub\u003e and 0.1 mM ZnCl\u003csub\u003e2\u003c/sub\u003e. The synthetic compounds used for h-ENPP screening were prepared in 10% DMSO and subjected to a test concentration of 0.1 mM. The assay, which contained assay buffer, 100 \u0026micro;M of the test drug, the enzyme (27 ng/well), and substrate p-nitrophenyl 5\u0026rsquo;-thymidine monophosphate, was carried out in a 96 well plate with a total volume of 100 \u0026micro;L/well. A microplate reader (BioTek FL*800TM, Instruments, Inc. USA) was used to measure absorbance at 405 nm following 25 minutes in an incubator at 37℃. Compounds exhibiting more than 50% \u003cem\u003eh\u003c/em\u003e-ENPP1 inhibition of were subjected to serial dilution to calculate their IC\u003csub\u003e50\u003c/sub\u003e values. The data was analyzed using GraphPad PRISM 5.0 (San Diego, CA, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eh-\u003c/b\u003e \u003cb\u003ee5\u0026rsquo;NT Activity Assay\u003c/b\u003e \u003c/p\u003e \u003cp\u003eInhibition assay for human ecto-5'-nucleotidase was carried out according to the previously reported method [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The assay buffer was composed of Tris HCL, MgCl\u003csub\u003e2,\u003c/sub\u003e and CaCl\u003csub\u003e2\u003c/sub\u003e and its pH was adjusted to 7.4 before use. The assay was first conducted in triplicates to see whether the compounds exhibited any action. The total volume per well was 100 \u0026micro;L. In triplicates, 55 \u0026micro;L of the assay buffer and 10 \u0026micro;L of the e5\u0026rsquo;NT were added to each well. Each well received 10 \u0026micro;L of assay compound, followed by 10 minutes\u0026rsquo; incubation at 37℃. Once 10 \u0026micro;L of AMP (2 mM) was added as a substrate, the enzymatic activity started and continued for 20 minutes at 37℃. After that, 15 \u0026micro;L of malachite green reagent was added to assess the quantity of phosphate released during the enzyme-mediated breakdown of the substrate. The absorbance change at 630 nm was then recorded. The same methodology was used to calculate the IC\u003csub\u003e50\u003c/sub\u003e values of the assay compounds at various concentrations. Non-linear regression analysis was utilized to further examine the IC\u003csub\u003e50\u003c/sub\u003e of only those compounds that exhibited inhibition values higher than 50% using GraphPad Prism 5.0 software (San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Fluorescence Spectroscopy and Microscopic Analysis\u003c/h2\u003e \u003cp\u003eFluorescence spectroscopy of compound 4d was conducted using a Horiba Fluoromax-4 spectrofluorometer, with excitation wavelength between 400\u0026ndash;600 nm, and emission spectra was recorded to characterize its optical properties. To evaluate cellular uptake, A549 lung cancer cells were incubated with the compound and imaged on inverted fluorescence microscope (Nikon ECLIPSE Ni\u0026ndash;U). To evaluate the effect of compound \u003cb\u003e4d\u003c/b\u003e on enzyme function, a real-time fluorescent-based assay was used to monitor changes in fluorescent intensity, wavelength shifts, and emission patterns to infer binding events, conformational changes, and enzyme activity. Combined with cell imaging, the compound d was revealed to enter the A549 lung cancer cells and reach its target enzyme, signifying the druggability of the synthesized compound 4d. Fluorescence signals were normalized via min-max normalization and analyzed using the microscope\u0026rsquo;s built-in software. These assays collectively aimed to quantify intracellular distribution and assess binding affinity to cancer cell membrane enzymes, providing insights into the compound\u0026rsquo;s localization and potential interactions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Molecular Docking Studies\u003c/h2\u003e \u003cp\u003eQuinoline-6-carboxylic acid derivatives \u003cb\u003e4a, 4b, 4d, 4g\u003c/b\u003e, and \u003cb\u003e4k\u003c/b\u003e were docked with respective enzymes. Molecular docking experiments were conducted on the synthesized derivatives \u003cb\u003e(4a-4l)\u003c/b\u003e using Autodock Vina\u0026reg; 1.5.4 software [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The crystal structures of human ectonucleotide pyrophosphatase/phosphodiesterase 1 (\u003cem\u003eh\u003c/em\u003e-ENPP1) and \u003cem\u003eh\u003c/em\u003e-e5\u0026prime;NT were retrieved from the Protein Data Bank \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e using PDB IDs 6WEW and 4H2G, respectively. For \u003cem\u003eh\u003c/em\u003e-ENTPDase isoenzymes, homology models were utilized due to the unavailability of their experimental structures. Ligand structures were drawn using ChemDraw software, and were then subjected to energy minimization and saved in PDBQT format using Autodock Vina 1.5.4 software. Best pose with minimum binding energy was selected and re-docking was carried out for validation of the docking results. Visualization of the 2D and 3D protein ligand interactions was done using Discovery studio 2021.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 \u003cem\u003eIn Silico\u003c/em\u003e ADME analysis\u003c/h2\u003e \u003cp\u003eThe SwissADME web server was employed to assess the pharmacokinetics properties of all the compounds \u003cb\u003e(4a-4l).\u003c/b\u003e ChemDraw was used to generate the SMILES representation of the synthesized derivatives, which were then uploaded to an online platform for pharmacokinetic profile prediction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 MTT Assay\u003c/h2\u003e \u003cp\u003eMTT assay was conducted to check the safety profile of the synthesized derivatives. The assay was conducted in MCF-7 breast cancer cells. Briefly, the cells were plated in a sterile 96-well plate and incubated for 24 h in CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C. The media was, then, discarded and the cells were washed with PBS. Tested compounds were loaded at a concentration of 100 \u0026micro;M/well in triplicates. 1% DMSO and 100 \u0026micro;M cisplatin were used as negative control and positive control, respectively. A blank well that contained only serum free media served as a reference. The 96 well plate was further incubated for 24 h in 5% CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C. Following incubation, 100 \u0026micro;L of MTT reagent was loaded/well to achieve a final concentration of 0.2 mg/mL. The cells were then placed in an incubator for further 2 to 4 h and monitored for formation of formazan crystals. Next, the media was aspirated, and 100 \u0026micro;L/well solubilizing agent (SDS solution) was added. Subsequently, the plates were shaken for 30 min to ensure complete dissolution of the formazan crystals. The absorbance was measured using a microplate reader, with readings taken at 570 nm for the formazan and at 630 nm for the background signal. The conversion of MTT reagent to formazan crystals by mitochondrial dehydrogenases was indicative of cell viability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Apoptosis Assay and Fluorescent Microscopy\u003c/h2\u003e \u003cp\u003eFITC Annexin V was used to quantitatively determine the percentage of MCF-7 cells within a population that were actively undergoing apoptosis. It relies on the property of cells to lose membrane asymmetry in the early phases of apoptosis. Annexin V is a calcium-dependent phospholipid-binding protein with high affinity for phospholipid phosphatidylserine (PS), and was used to identify apoptotic cells with exposed PS. Propidium Iodide (PI) served as a viability probe to distinguish viable cells from nonviable ones. Viable cells with intact membranes exclude PI, whereas the membranes of dead and damaged cells are permeable to PI. Cells that stained positive for FITC Annexin V were undergoing apoptosis, whereas the cells that stained negative for FITC Annexin V were alive and did not undergo measurable apoptosis upon treatment with the selected compounds.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS \u0026 DISCUSSION","content":"\u003cp\u003e\u003cstrong\u003e3.1 Synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe synthesis of quinoline-6-carboxylic acid derivatives was carried out in accordance with the reported procedure [57].First step involved the conversion of quinoline-6-carboxylic acid\u003cstrong\u003e\u0026nbsp;(1)\u003c/strong\u003e into quinoline-6-acid chloride\u003cstrong\u003e\u0026nbsp;(2)\u003c/strong\u003e using thionyl chloride. The reaction was refluxed for 8 hours and monitored by TLC to confirm the consumption of the starting acid and the formation of quinoline-6-acid chloride \u003cstrong\u003e(2)\u003c/strong\u003e. Subsequent addition of the respective amine led to the formation of the corresponding amides \u003cstrong\u003e(4a\u0026ndash;4l)\u003c/strong\u003e in good yields, ranging from 60% to 70%. The amidation step was carried out at 50 \u0026deg;C, and the reaction progress was monitored by TLC, which indicated complete conversion of the acid chloride into its respective amide. The resulting amides were fully characterized using FTIR and \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectroscopy.\u003c/p\u003e\n\u003cp\u003eTo assess the versatility of the reaction and introduce structural diversity, a diverse set of amine substrates was explored, as depicted in \u003cstrong\u003eScheme 1\u003c/strong\u003e. This set comprised primary amines, variously substituted benzylamines, functionalized anilines, and heterocyclic aniline derivatives. Among the benzylamines, substituted benzylamines with 4-chloro, 4-fluoro, 4-methyl, and 2,4-dichloro substituents \u003cstrong\u003e(3a\u0026ndash;3c, 3j)\u003c/strong\u003e produced good to excellent yields of the corresponding amides \u003cstrong\u003e(4a\u0026ndash;4c, 4j)\u003c/strong\u003e, with yields of 67%, 64%, 70%, and 67%, respectively. In a similar fashion, a range of aniline derivatives were tested, including monosubstituted anilines with electron-donating groups, such as chloro (Cl) at the ortho (3h) and para (3k) positions. Additionally, disubstituted anilines with a mix of electron-donating groups (F, OCH\u003csub\u003e3\u003c/sub\u003e; 3d, 3e, 3i) and both electron-donating and electron-withdrawing groups (F, CF\u003csub\u003e3\u003c/sub\u003e, CH\u003csub\u003e3\u003c/sub\u003e; 3f, 3l) were also compatible, yielding the desired products in good to excellent yields. Additionally, heterocyclic pyridyl amine \u003cstrong\u003e(3g)\u003c/strong\u003e was also successfully utilized to synthesize the desired compounds \u003cstrong\u003e(4g)\u003c/strong\u003e, furnishing the products in good yields. For all the synthesized compounds, characteristic peaks were observed in the FTIR spectra. A stretching band appeared in the range of 3165\u0026ndash;3462 cm⁻\u0026sup1;, indicating the presence of the NH group, while the absorption band at 1628\u0026ndash;1682 cm⁻\u0026sup1; confirmed the existence of the C=O group. The structures were further confirmed using \u003csup\u003e1\u003c/sup\u003eH, and \u003csup\u003e13\u003c/sup\u003eC spectroscopy. The \u003csup\u003e1\u003c/sup\u003eH-NMR data of compounds in the \u0026delta; 9.00-11.06 ppm range confirmed the presence of NH which further confirmed the presence of amide functionality. In \u003csup\u003e13\u003c/sup\u003eC-NMR data, the appearance of a signal at \u0026delta; 164.9-166.3 ppm for C=O further confirmed the formation of quinoline-6-carboxylic acid amide derivatives \u003cstrong\u003e(4a-4l)\u003c/strong\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Enzymatic assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ability of the synthetic compounds to inhibit the activity of the \u003cem\u003eh\u003c/em\u003e-NTPDase1, -2, -3, and -8 \u003cem\u003eiso\u003c/em\u003eenzymes, \u003cem\u003eh\u003c/em\u003e-NPP1, and \u003cem\u003eh\u003c/em\u003e-e5\u0026prime;NT was evaluated. The newly synthesized compounds showed pronounced inhibition of these enzymes, as listed in Table 1. The enzymatic tests of the compounds (\u003cstrong\u003e4a\u0026ndash;4l\u003c/strong\u003e) revealed their strong inhibitory effects, with IC\u003csub\u003e50\u003c/sub\u003e values ranging from 0.092 to 1.00 \u0026micro;M, as shown in Table 2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Percentage inhibition of all the compounds against Ectonucleotidases\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"647\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003eCompound codes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"6\" valign=\"top\" style=\"width: 554px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Inhibition\u0026nbsp;of the studied compounds\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cem\u003eh\u003c/em\u003e-NTPDase1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cem\u003eh-\u003c/em\u003eNTPDase2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cem\u003eh\u003c/em\u003e-NTPDase3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cem\u003eh-\u003c/em\u003eNTPDase8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cem\u003eh\u003c/em\u003e-NPP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cem\u003eh\u003c/em\u003e-e5\u0026rsquo;NT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e4a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e37.02%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e23.30%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e30.60%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e39.46%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e66.97%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e67.87%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e4b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e68.92%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e26.18%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e67.82%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e82.98%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e44.80%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e37%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e4c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp;57.70%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e68.38%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e78.38%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e70.86%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e45.25%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e50.70%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e4d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e76.06%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e80.13%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e58.42%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e33.02%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e54.52%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e35.64%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e4e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e32.56%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e36.36%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e40.21%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e39.46%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e34.62%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e66.87%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e4f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e44.06%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e35.41%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e66.67%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e39.91%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e65.61%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e48.49%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e4g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e58.53%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e33.91%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp;\u003cstrong\u003e81.68%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e20.35%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e43.67%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e81.98%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e4h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e18.45%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e28.29%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e84.98%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e65.61%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e33.94%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e87.05%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e4i\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e19.48%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e39.64%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e25.08%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e29.82%%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e29.19%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e54.86%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e4j\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e33.06%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e26.20%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e35.64%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e28.60%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e85.52%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e72.39%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e4k\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e33.79%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e18.56%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e38.45%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e27.02%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e67.65%\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; 28.43%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 93px;\"\u003e\n \u003cp\u003e4l\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e32.37%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e41.16%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 92px;\"\u003e\n \u003cp\u003e36.14%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e26.12%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e29.42%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e60.98%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*\u0026nbsp;Inhibition percentages at the concentration of 100 \u0026micro;M\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e Inhibitory activity of the studied compounds against Ectonucleotidases\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 11.4229%;\"\u003e\n \u003cp\u003eCompound codes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"6\" valign=\"top\" style=\"width: 67.4873%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIC\u003csub\u003e50 \u0026plusmn;\u0026nbsp;\u003c/sub\u003eSEM (\u0026micro;M)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e\u003cem\u003eh\u003c/em\u003e-NTPDase1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e\u003cem\u003eh-\u003c/em\u003eNTPDase2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e\u003cem\u003eh\u003c/em\u003e-NTPDase3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e\u003cem\u003eh-\u003c/em\u003eNTPDase8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5039%;\"\u003e\n \u003cp\u003e\u003cem\u003eh\u003c/em\u003e-NPP1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e\u003cem\u003eh\u003c/em\u003e-e5\u0026rsquo;NT\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.4229%;\"\u003e\n \u003cp\u003e4a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; -\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; -\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5039%;\"\u003e\n \u003cp\u003e0.23\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e0.092\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.4229%;\"\u003e\n \u003cp\u003e4b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e3.46\u0026plusmn;0.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e0.35\u0026plusmn;0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e0.44\u0026plusmn;0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5039%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.4229%;\"\u003e\n \u003cp\u003e4c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e6.13\u0026plusmn;0.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e6.39\u0026plusmn;1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e0.67\u0026plusmn;0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e0.99\u0026plusmn;0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5039%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e0.19\u0026plusmn;0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.4229%;\"\u003e\n \u003cp\u003e4d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e0.28\u0026plusmn;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e0.92\u0026plusmn;0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e0.82\u0026plusmn;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5039%;\"\u003e\n \u003cp\u003e0.73\u0026plusmn;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.4229%;\"\u003e\n \u003cp\u003e4e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; -\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5039%;\"\u003e\n \u003cp\u003e\u0026nbsp;-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e0.54\u0026plusmn;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.4229%;\"\u003e\n \u003cp\u003e4f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; -\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e0.99\u0026plusmn;0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5039%;\"\u003e\n \u003cp\u003e0.87\u0026plusmn;0.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.4229%;\"\u003e\n \u003cp\u003e4g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e0.61\u0026plusmn;0.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e0.32\u0026plusmn;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5039%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e0.34\u0026plusmn;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.4229%;\"\u003e\n \u003cp\u003e4h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e0.88\u0026plusmn;0.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e3.29\u0026plusmn;0.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5039%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e0.39\u0026plusmn;0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.4229%;\"\u003e\n \u003cp\u003e4i\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; -\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5039%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e0.99\u0026plusmn;0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.4229%;\"\u003e\n \u003cp\u003e4j\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; -\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5039%;\"\u003e\n \u003cp\u003e13.5\u0026plusmn;1.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e0.44\u0026plusmn;0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.4229%;\"\u003e\n \u003cp\u003e4k\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; -\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5039%;\"\u003e\n \u003cp\u003e0.11\u0026plusmn;0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.4229%;\"\u003e\n \u003cp\u003e4l\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; -\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5039%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e0.47\u0026plusmn;0.26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.4229%;\"\u003e\n \u003cp\u003eReactive Blue 2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e16.1\u0026plusmn;1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e24.1\u0026plusmn;3.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e4.31\u0026plusmn;0.41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e101.1\u0026plusmn;2.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5039%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 11.4229%;\"\u003e\n \u003cp\u003eSuramin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.2916%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 10.5039%;\"\u003e\n \u003cp\u003e18.5\u0026plusmn;1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 11.5542%;\"\u003e\n \u003cp\u003e42.1\u0026plusmn;7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e*Data presented as IC\u003csub\u003e50\u003c/sub\u003e \u0026plusmn; SEM\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Fluorescence characterization of the druggable compound 4d and its cellular interaction\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFluorescence spectroscopy of compound 4d revealed distinct excitation-dependent emission profiles between 410\u0026ndash;500 nm. The emission spectra displayed strong, well-defined fluorescence peaks across the visible region (400\u0026ndash;500 nm), demonstrating excellent photostability and consistent emissive behavior under near-UV to blue light irradiation. This robust optical performance, characterized by minimal photobleaching, positions 4d as potential fluorophore for bioimaging applications. Fluorescence microscopy of A549 lung cancer cells treated with 4d provided detailed spatial information about its cellular distribution. Bright-field images showed preserved cell morphology with intact membranes and no signs of cytotoxicity at the tested concentration (10 \u0026mu;M, 24 h incubation). In the red fluorescence channel, intense fluorescence was observed predominantly at the plasma membrane, with some vesicular staining suggesting possible endocytic uptake. Spectral shifts in emission suggest binding interactions between the compound d and the enzyme. The high signal-to-noise ratio (SNR \u0026gt;15:1) and clear membrane integrity indicated viable colocalization. The merged image in Figure 3B highlights this exclusive membrane targeting, with the red fluorescence of 4d forming a continuous rim around cells while remaining distinctly separated from cytoplasmic and nuclear compartments. Microscopic analysis further confirmed compound uptake, intracellular localization, and biological relevance, integrating in vitro findings and cellular behavior.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Structure-activity relationships (SARs)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe structure-activity relationship (SAR) analysis demonstrated a substantial influence of the substituents on the \u003cem\u003ein vitro\u003c/em\u003e activity of the synthesized compounds (4a\u0026ndash;4l) against \u003cem\u003eh\u003c/em\u003e-NTPDases, \u003cem\u003eh\u003c/em\u003e-NPP1, and\u003cem\u003e\u0026nbsp;h\u003c/em\u003e-e5\u0026prime;NT enzymes. The structures of the most effective compounds are displayed in \u003cstrong\u003eFigure 4\u003c/strong\u003e. It was observed that compound \u003cstrong\u003e4a\u003c/strong\u003e, with a 4-chloro benzyl substitution on the quinoline carboxamide scaffold, exhibited the highest potency against \u003cem\u003eh\u003c/em\u003e-e5\u0026prime;NT enzyme, highlighting the importance of chlorine in enhancing activity. Compound \u003cstrong\u003e4j\u003c/strong\u003e, with additional 2-chloro substitution, showed slightly reduced but notable potency, suggesting steric effects. Electron-donating groups (e.g., methyl) at the 4-position \u003cstrong\u003e(4c)\u003c/strong\u003e enhanced activity, whereas introduction of electron withdrawing group i.e., fluorine substitution reduced it, emphasizing chlorine\u0026rsquo;s optimal electronic properties. Pyridyl rings and 2-iodo substitutions maintained good activity, but bulky 4-iodo groups significantly decreased \u003cstrong\u003e4k\u003c/strong\u003e inhibition potential. We also observed that activity varied with substitution positions. Compound \u003cstrong\u003e4e\u003c/strong\u003e, with fluoro groups at 2- and 4-positions, retained potency, however compound \u003cstrong\u003e4d\u003c/strong\u003e, having fluoro groups at 2-, 6- positions exhibited decreased potential, demonstrating the role of precise substituent placement.\u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eh\u003c/em\u003e-NPPI, the tested series of compounds exhibited an opposite trend in activity compared to the \u003cem\u003eh\u003c/em\u003e-e5\u0026prime;NT enzyme, with the exception of compound \u003cstrong\u003e4a\u003c/strong\u003e, which showed good potency against both enzymes. Notably, the 4-iodo aromatic moiety displayed exceptional efficacy, with an IC\u003csub\u003e50\u003c/sub\u003e value of 0.11 \u0026plusmn; 0.02 \u0026micro;M. A structural comparison of the inhibitors against \u003cem\u003eh\u003c/em\u003e-NPPI and \u003cem\u003eh\u003c/em\u003e-e5\u0026prime;NT revealed that electronegative atoms (F, Cl, I, and CF\u003csub\u003e3\u003c/sub\u003e) at the ortho or para positions of the phenyl ring, either as single or double substitutions, significantly enhanced inhibitory potential against \u003cem\u003eh\u003c/em\u003e-NPPI. The IC\u003csub\u003e50\u003c/sub\u003e values for these inhibitors ranged from 0.1 to 14 \u0026micro;M, highlighting the importance of both substitution patterns and electronic effects in optimizing enzyme inhibition.\u003c/p\u003e\n\u003cp\u003eAll the compounds \u003cstrong\u003e(4a\u0026ndash;4l)\u003c/strong\u003e displayed good to moderate inhibitory activities against \u003cem\u003eh\u003c/em\u003e-NTPDases, with IC\u003csub\u003e50\u003c/sub\u003e values ranging from 0.28 \u0026plusmn; 0.03 \u0026micro;M to 6.39 \u0026plusmn; 1.07 \u0026micro;M. However, compounds \u003cstrong\u003e4a, 4e,\u003c/strong\u003e \u003cstrong\u003e4i, 4j, 4k, and 4l\u003c/strong\u003e, which include halogen-substituted derivatives and anilines with electron-donating groups, showed poor activity against all four enzymes. Compound \u003cstrong\u003e4c\u003c/strong\u003e, featuring a 4-methylbenzyl substitution, was active across all the enzyme targets, while compound \u003cstrong\u003e4d\u003c/strong\u003e, with 2,6-difluoro substitutions, demonstrated the highest potency specifically against \u003cem\u003eh\u003c/em\u003e-NTPDase1 and -2. In contrast, compound \u003cstrong\u003e4e\u003c/strong\u003e, containing fluoro groups at the 2- and 4-positions, was inactive against all four isoenzymes. This inactivity could be attributed to steric hindrance or an unfavorable electronic interaction at the active site. Compound \u003cstrong\u003e4b\u003c/strong\u003e, with a 4-fluorobenzyl moiety, exhibited good potency against \u003cem\u003eh\u003c/em\u003e-NTPDase1 and -3 and was the most potent inhibitor of \u003cem\u003eh\u003c/em\u003e-NTPDase8. Against \u003cem\u003eh\u003c/em\u003e-NTPDase3, compounds \u003cstrong\u003e4b, 4c, 4d, 4f, 4g\u003c/strong\u003e, and \u003cstrong\u003e4h\u003c/strong\u003e showed activity, with compound \u003cstrong\u003e4g\u003c/strong\u003e, a pyridyl-substituted derivative, emerging as the most potent inhibitor. Compound \u003cstrong\u003e4i\u003c/strong\u003e, which contains a methoxy substitution on the phenyl ring, showed less than 50% inhibition for all enzymes except \u003cem\u003eh\u003c/em\u003e-e5\u0026prime;NT, where it demonstrated good inhibitory activity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Molecular docking studies for validation\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eh\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-ENTPDase1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmino acid components involved in the binding interactions of\u003cem\u003e\u0026nbsp;h\u003c/em\u003e-NTPDase1 are: Asp213, Tyr450, Gly178, Ser361, Phe360, and Cys502. Moleculer docking study of the compound \u003cstrong\u003e4d\u0026nbsp;\u003c/strong\u003ewith \u003cem\u003eh\u003c/em\u003e-NTPDase1 revealed conventional hydrogen bonding between oxygen of the carbonyl group and the amino acid Trp450. Other interaction shown by the compound are pi-Sigma interaction of Phe360 with phenyl ring. \u0026nbsp;Cys502 refers to the amino acid Cystein (CYS), whereby, cystein contains thiol (-SH) group that can interact with benzene ring and show \u0026pi;-sigma interaction with the aromatic (benzene) ring. Another amino acid, Gly178, shows that carbon-hydrogen bond interaction with NH group can act as a hydrogen donor in hydrogen bonding. Asp213 with its carboxyl side chain can act as a hydrogen bond acceptor. Asp213 shows hydrogen bond interaction with NH group. Gly178 also shows halogen interaction with the fluorine group \u003cstrong\u003e(Figure 5A)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eh\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-ENTPDase2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmino acid components engaged in the binding interactions of\u003cem\u003e\u0026nbsp;h\u003c/em\u003e-NTPDase2 are: Asp201, Leu202, Tyr350, His50, Ala347, Arg392, Gly435, and Cys502. Compound \u003cstrong\u003e4d\u003c/strong\u003e, the most active compound against \u003cem\u003eh-\u003c/em\u003eNTPDase2 was docked using a homology model to analyze the interactions. Molecular docking of the compound \u003cstrong\u003e4d\u003c/strong\u003e with \u003cem\u003eh\u003c/em\u003e-NTPDase2 revealed conventional hydrogen bonding of the oxygen group with Ala347, and Arg392 forms conventional hydrogen bonding with the parent quinoline moiety when the guanidinium groups NH or NH\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eact as a hydrogen donor, and the quinoline nitrogen act as the acceptor. His(50) and Tyr350 show \u0026pi; - \u0026pi; stacked and \u0026pi; - \u0026pi; -T shaped with benzene ring. Another amino acid Cys502 shows Pi alkyl interaction with the aromatic ring. Another amino acid Gly435 shows carbon-hydrogen bond interaction with carbon atom attached to the fluorine group. Leu202 and Asp201 show halogen bond interaction with the fluorine group \u003cstrong\u003e(Figure 5B)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eh\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-ENTPDase3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmino acid components engaged in the binding interactions of\u003cem\u003e\u0026nbsp;h\u003c/em\u003e-NTPDase3 are: Leu144, Leu145, Gln148, Gly141, Ser100, and Glu266. Molecular docking of the compound 4g with \u003cem\u003eh\u003c/em\u003e-NTPDase3 revealed conventional hydrogen bonding of hydrogen group with Gln148, whereas, parent moiety quinoline ring was observed to have Pi-alkyl interaction with Leu144 and Leu145. Furthermore, phenyl ring formed Pi anion interaction with Glu266 and the nitrogen atom of quinoline ring show carbon-hydrogen interaction with Gly141 also. Ser100 amino acid show carbon hydrogen bond interaction with \u0026pi;-electronic cloud of the quinoline ring \u003cstrong\u003e(Figure 5C)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eh\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-ENTPDase8\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmino acid components involved in the binding interactions of\u003cem\u003e\u0026nbsp;h\u003c/em\u003e-NTPDase8 are: Tyr402, Tyr357, Trp398, Pro393, and His360. Molecular docking of the compound 4b with \u003cem\u003eh\u003c/em\u003e-NTPDase8 revealed conventional hydrogen bonding of oxygen group with Tyr402, and phenyl ring formed \u0026pi; - \u0026pi; -T shaped interaction with Tyr357 and His360. Similarly, Phenyl ring formed Pi alkyl interaction with Pro393, and the quinoline moiety formed \u0026pi; - \u0026pi;-T-shaped interaction with Trp398 amino acid \u003cstrong\u003e(Figure 5D)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eh-\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eENPP1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmino acid sites contributing to the binding interactions of\u003cem\u003e\u0026nbsp;h\u003c/em\u003e-NPP1 are: \u0026nbsp;Tyr371, Tyr340, Lys295, Phe257, His380, and Pro323. Molecular docking of the compound \u003cstrong\u003e4k\u003c/strong\u003e with \u003cem\u003eh\u003c/em\u003e-NPP1 revealed conventional hydrogen bonding between the nitrogen of the quinoline ring and the amino acid lys295. Tyr340 and phe257 are both aromatic amino acids. They show \u0026pi; - \u0026pi; stacked interactions with ligands containing aromatic moiety such as the quinoline group. Similarly, Tyr371 can engage in \u0026pi; - \u0026pi; T-shaped interactions with quinoline ring due to perpendicular orientation of the aromatic ring \u003cstrong\u003e(Figure 5E)\u003c/strong\u003e.\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003e\u003cem\u003eh\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-e\u003c/strong\u003e\u003cstrong\u003e5\u0026prime;NT\u003c/strong\u003e\u003c/h3\u003e\n\u003cp\u003eAmino acid residues implicated in the bonding interaction of \u003cem\u003eh\u003c/em\u003e-e5\u0026prime;NT are Ile91, Trp408, Gln523, Thr90, Lys50, Ala412, and Ser49. Molecular docking of the compound \u003cstrong\u003e4a\u003c/strong\u003e with \u003cem\u003eh\u003c/em\u003e-e5\u0026prime;NT revealed conventional hydrogen bonding between Gln523 and the oxygen atom of the carbonyl group. Similarly, Thr90 shows pi-sigma interaction with the quinoline moiety. The quinoline ring interacts with the sigma bond of atom like oxygen threonine\u0026rsquo;s hydroxyl group. While Ile90 and Ala412 amino acids interact with the benzene ring through \u0026pi; -alkyl interaction, where the hydrophobic alkyl side chains of Isoleucine (isopropyl group) and Alanine (methyl group) align with and interact with the pi-electrons of the aromatic benzene ring and Lys50 show pi-alkyl interaction with quinoline moiety. Also, \u0026pi; - \u0026pi; T-shaped interaction occurs between Trp408 and benzene ring. Additionally,amide- \u0026pi; stacked interaction occurs between Ser49 and the quinoline moiety where the amide group from Ser49 can stack with quinoline aromatic ring via the NH and carbonyl group, stabilizing the interaction \u003cstrong\u003e(Figure 5F)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eIn\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e-\u003cem\u003esilico\u003c/em\u003e Pharmacokinetics studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrediction of Pharmacokinetics Properties and Drug-Likeness of the quinoline-6-carboxylic acid derivatives\u003c/strong\u003e \u003cstrong\u003e(4a-4l)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePharmacokinetics properties and drug-likeness of the quinoline-6-carboxylic acid derivatives were calculated using SwissADME. The pharmacokinetics properties were determined based on gastrointestinal (GI) absorption, permeation to the blood-brain barrier (BBB), and ability to act as a P-glycoprotein (P-gp) substrate. The drug-likeness of these compounds was determined based on Lipinski\u0026rsquo;s rule of five numbers of rotatable bonds and topological polar surface area (TPSA). The calculated parameters related to pharmacokinetics properties and drug-likeness are presented in Table 3 and Table 4, respectively.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll the studied compounds showed high GI absorption and were permeable to the BBB, however, none of the the compounds was identified as a P-glycoprotein (P-gp) substrate. In addition, these compounds also possessed suitable drug-like properties. There was no violation of Lipinski\u0026apos;s rule of five, and the partition co-efficient (MlogP) values were less than 4.15. Moreover, TPSAof these compounds was within the range 20 to 65\u0026nbsp;\u0026Aring;\u0026sup2;, and the number of rotatable bonds was also less than 7.\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3\u0026nbsp;\u003c/strong\u003ePharmacokinetics Properties of the quinoline-6-carboxylic acid derivatives \u003cstrong\u003e(4a-4l)\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompounds\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGI-Absorption \u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBBB-permeant \u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eP-gp substrate \u003csup\u003ec\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4b\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4c\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4d\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4f\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4g\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4i\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4j\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4k\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4l\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eHigh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 131px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003eGI-Absorption; \u003csup\u003eb\u003c/sup\u003eBBB-permeant; \u003csup\u003ec\u003c/sup\u003eP-gp substrate\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4\u003c/strong\u003e Drug-Likeness of the quinoline-6-carboxylic acid derivatives \u003cstrong\u003e(4a-4l)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"633\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompounds\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHBD \u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHBA \u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMlogP \u003csup\u003ec\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo. of \u0026nbsp;violations \u003csup\u003ed\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTPSA(\u003c/strong\u003e\u003cstrong\u003e\u0026Aring;\u0026sup2;) \u003csup\u003ee\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRBN \u003csup\u003ef\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e3.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e41.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4b\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e2.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e41.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4c\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e2.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e41.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4d\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e3.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e41.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e3.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e41.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4f\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e3.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e41.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4g\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e1.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e54.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4h\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e3.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e41.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4i\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e1.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e60.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4j\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e3.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e41.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4k\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e3.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e41.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4l\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e3.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 95px;\"\u003e\n \u003cp\u003e41.99\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e Hydrogen bond donor; \u003cstrong\u003e\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003eHydrogen bond acceptor; \u003cstrong\u003e\u003csup\u003ec\u003c/sup\u003e\u003c/strong\u003eM logP; \u003cstrong\u003e\u003csup\u003ed\u003c/sup\u003e\u003c/strong\u003e No of violations; \u003cstrong\u003e\u003csup\u003ee\u003c/sup\u003e\u003c/strong\u003e Topological polar surface area; \u003cstrong\u003e\u003csup\u003ef\u003c/sup\u003e\u003c/strong\u003e Number of rotatable bond\u003c/p\u003e\n\u003cp\u003eThe results indicated that the synthesized compounds met all the criteria. SwissADME employs a visual method known as the boiled egg plot, which graphically represents GI absorption and BBB penetration. The analysis revealed that all the synthesized compounds are in the yolk region, except for 4f \u003cstrong\u003e(Figure 6)\u003c/strong\u003e. All compounds except 4f exhibit efficient blood\u0026ndash;brain barrier permeability and may be considered for further investigation as therapeutic agents against brain tumors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.7 Density Functional Theory\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, density functional theory (DFT) calculations were employed to analyze the HOMO-LUMO characteristics of quinoline derivatives \u003cstrong\u003e4a\u0026ndash;4l\u003c/strong\u003e. The energy gap between the frontier orbitals provided valuable insights into the compounds\u0026rsquo; electronic properties, including their chemical reactivity, molecular stability, and optical behavior. The optimized geometries of these derivatives are depicted in \u003cstrong\u003eFigure 7\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5\u003c/strong\u003e summarizes the calculated physicochemical properties of the studied compounds, including optimized energies, dipole moments, polarizability, frontier molecular orbital (FMO) energies (E\u003csub\u003eHOMO\u003c/sub\u003e and E\u003csub\u003eLUMO\u003c/sub\u003e), and the corresponding energy gaps (∆E). A higher HOMO energy (E\u003csub\u003eHOMO\u003c/sub\u003e) reflects a greater tendency to donate electrons, whereas higher LUMO energy (E\u003csub\u003eLUMO\u003c/sub\u003e) corresponds to a reduced ability to accept electrons. The HOMO-LUMO energy gap plays a critical role in determining the kinetic stability as well as the chemical reactivity of the molecules. Compounds with a larger ∆E are typically more kinetically stable, less polarizable, and exhibit lower chemical reactivity, thus classified as hard species. In contrast, a smaller ∆E indicates higher chemical reactivity, greater polarizability, and lower kinetic stability, signifying a soft molecular nature. Among the derivatives, compound \u003cstrong\u003e4j\u003c/strong\u003e exhibited the highest energy gap (∆E = 0.17488 eV), indicating its superior stability and lowest reactivity. In contrast, compound \u003cstrong\u003e4f\u003c/strong\u003e showed the lowest energy gap (∆E = 0.129 eV), classifying it as the most reactive and chemically softest derivative. This electronic behavior is consistent with its strong inhibitory activity against NPP1 and NTPDase3. Moreover, \u003cstrong\u003e4f\u003c/strong\u003e displayed the highest HOMO energy (E\u003csub\u003eHOMO\u003c/sub\u003e = -0.206 eV), indicating its strong electron-donating ability. This characteristic enhances its binding affinity through hydrogen bonding and facilitates nucleophilic interactions with active site residues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 5\u003c/strong\u003e Energetic parameters of the quinoline derivatives (\u003cstrong\u003e4a-4l)\u003c/strong\u003e\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"583\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 57px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCodes\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOptimization Energy\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDipole Moment\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePolarizability (\u0026alpha;)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHOMO (eV)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLUMO (eV)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eHOMO-LUMO\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e \u003cstrong\u003eE (\u003c/strong\u003e\u003cstrong\u003eeV)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e-1293.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e1.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e188.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-0.2429\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.0686\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.17432\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e-935.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e1.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e178.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-0.23664\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.0639\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.17274\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e-875.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e2.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e191.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-0.23209\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.0606\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.17149\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e-994.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e1.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e177.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-0.23305\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.06724\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.16581\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e-994.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e2.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e176.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-0.2324\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.0676\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.16481\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e-895.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e3.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e184.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-0.206\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.076\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.13641\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e-813.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e2.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e174.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-0.23002\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.06582\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.16421\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e-7687.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e2.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e192.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e-0.23471\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.06399\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.17072\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4i\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 105px;\"\u003e\n \u003cp\u003e-1064.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e4.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 107px;\"\u003e\n \u003cp\u003e211.89\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e-0.21255\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.05539\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.15716\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4j\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e-1751.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e197.61\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-0.24571\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.07083\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.17488\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4k\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e-7687.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e3.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e210.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-0.22028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.07262\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.14766\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 57px;\"\u003e\n \u003cp\u003e4l\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 105px;\"\u003e\n \u003cp\u003e-935.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e3.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 107px;\"\u003e\n \u003cp\u003e221.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e-0.22365\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.07118\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.15247\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eGlobal chemical reactivity descriptors:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe global reactivity parameters for the selected compounds (4a\u0026ndash;4l) were calculated from their HOMO and LUMO energy values using standard formulas. These parameters, which provide insights into the chemical stability and reactivity of the molecules, are shown in \u003cstrong\u003eTable 6\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 6\u003c/strong\u003e Global reactivity descriptors of the quinoline derivatives (\u003cstrong\u003e4a-4l\u003c/strong\u003e)\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"583\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eCodes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003eChemical Potential \u0026micro; (eV)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003eElectronegativity X (eV)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003eHardness ƞ (eV)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003eSoftness S (eV-1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003eElectrophilicity index \u0026omega; (eV)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e-0.156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.156\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.087\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.3115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.139\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e-0.150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.086\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.30054\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.131\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e-0.146\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.146\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.086\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.29269\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.125\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e-0.150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.083\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.30029\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.136\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e-0.150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.082\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.3342\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.137\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e-0.141\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.141\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.065\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.2821\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.153\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e-0.148\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.148\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.082\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.29584\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.133\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e-0.149\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.149\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.085\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.2987\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.131\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4i\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e-0.134\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.134\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.079\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.26794\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.114\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4j\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e-0.158\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.158\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.087\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.31654\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.143\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4k\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e-0.146\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.146\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.074\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.2929\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.145\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003e4l\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e-0.147\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0.147\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 78px;\"\u003e\n \u003cp\u003e0.076\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e-0.29483\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 115px;\"\u003e\n \u003cp\u003e0.143\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eHardness: \u0026eta; = 1/2(ELUMO - EHOMO); Softness: S = 1/2\u0026eta;; Electrophilicity index: \u0026omega; = \u0026micro;/2\u0026eta;; Electronegativity: \u0026chi; = -1/2(E\u003csub\u003eLUMO\u003c/sub\u003e + E\u003csub\u003eHOMO\u003c/sub\u003e); Chemical potential: \u0026micro; = - \u0026chi;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.8 Cytotoxicity Assessment Using MTT Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe MTT assay was performed to observe the cytotoxic effects of various compounds (coded 4a\u0026ndash;4l) on the MCF-7 cells, compared to cisplatin (positive control) and serum-free media (negative control). Among the test compounds, 4k and 4j exhibited the highest cytotoxicity (56.9% and 55.0%, respectively), with corresponding viability values around 43\u0026ndash;45%, suggesting strong anti-proliferative effects. In contrast, compound 4e showed the lowest cytotoxicity (14.6%) and highest cell viability (85.4%), indicating minimal toxicity. Notably, cisplatin displayed very high cytotoxicity (86.29%), validating the assay, while the negative control showed 0% cytotoxicity, confirming the specificity of the assay. Some compounds like 4g, 4h, and 4d also showed substantial cytotoxicity (around 47\u0026ndash;49%), suggesting potential for further investigation. Overall, compounds 4j and 4k appear the most promising for anti-cancer activity, though additional validation (e.g., apoptosis assays.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 7: % Cytotoxicity of the synthesized quinoline-6-carboxylic acid derivatives in MCF-7 cancer cells\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCompound Code\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Cytotoxicity\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e% Viability\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e4a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e37.3\u0026plusmn;1.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e62.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e4b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e25.1\u0026plusmn;6.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e74.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e4c\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e42.2\u0026plusmn;0.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e57.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e4d\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e48.0\u0026plusmn;12.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e52.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e4e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e14.6\u0026plusmn;2.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e85.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e4f\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e31.5\u0026plusmn;2.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e68.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e4g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e47.4\u0026plusmn;1.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e52.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e4h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e49.7\u0026plusmn;2.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e50.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e4i\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e41.5\u0026plusmn;0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e58.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e4j\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e55.0\u0026plusmn;1.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e45.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e4k\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e56.9\u0026plusmn;16.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e43.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003e4l\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e39.5\u0026plusmn;15.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e60.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eCisplatin (Positive Control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e86.29\u0026plusmn;2.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e13.71\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 192px;\"\u003e\n \u003cp\u003eSerum Free Media (Negative Control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 126px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e3.9 Apoptosis Assessment Using Annexin V Assay and Fluorescent Microscopy\u003c/p\u003e\n\u003cp\u003eFor additional validation of the cytotoxicity results obtained through MTT assay, apoptosis studies were carried out using FITC Annexin V assay. It was found that the most cytotoxic compounds 4j and 4k showed significant apoptotic induction in the treated MCF-7 cells. Majority of the cells displayed strong Annexin V positive membrane staining, indicative of early apoptosis. Compound 4i revealed late apoptotic events as visible through co-staining with propidium iodide. These findings are supportive of the anti-cancer potential of these quinoline-6-carboxylic acid derivatives as pro-apoptotic agents in cancer management.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, a series of quinoline-6-carboxylic acid derivatives was synthesized and evaluated for their inhibitory potential against key ectonucleotidases, including \u003cem\u003eh\u003c/em\u003e-NTPDase isoenzymes, \u003cem\u003eh\u003c/em\u003e-NPP1, and \u003cem\u003eh\u003c/em\u003e-e5\u0026rsquo;NT. The compounds were obtained in good yields (60\u0026ndash;70%) and showed compatibility with a variety of substituents (F, Cl, I, CH\u003csub\u003e3\u003c/sub\u003e, CF\u003csub\u003e3\u003c/sub\u003e, OCH\u003csub\u003e3\u003c/sub\u003e). Their structures were fully confirmed through NMR analysis. Biological screening revealed that compound \u003cb\u003e4d\u003c/b\u003e was most potent inhibitor of \u003cem\u003eh\u003c/em\u003e-NTPDase1 and \u0026minus;\u0026thinsp;2, while compounds \u003cb\u003e4g, 4b, 4a\u003c/b\u003e, and \u003cb\u003e4k\u003c/b\u003e showed selective inhibition against \u003cem\u003eh\u003c/em\u003e-NTPDase3, \u003cem\u003eh\u003c/em\u003e-NTPDase8, \u003cem\u003eh\u003c/em\u003e-e5\u0026rsquo;NT, and \u003cem\u003eh\u003c/em\u003e-NPP1, respectively. Molecular docking indicated that these interactions are stabilized by hydrogen bonding, π-anion, π-alkyl, and π\u0026ndash;π stacking forces. Complementary DFT analysis suggested a correlation between LUMO stabilization and inhibitory strength, reinforcing the structure\u0026ndash;activity relationship. Moreover, fluorescence imaging of compound \u003cb\u003e4d\u003c/b\u003e demonstrated strong emission at the cellular level, supporting its effective binding to membrane-bound enzymes in the cancer cells. MTT and apoptosis studies also revealed compound \u003cb\u003e4i\u003c/b\u003e, \u003cb\u003e4j\u003c/b\u003e, and \u003cb\u003e4k\u003c/b\u003e to be potentially cytotoxic in the cancer cells, endorsing the potential of these quinoline derivatives to be used for drug development for cancer management. Together, these findings position quinoline-6-carboxylic acid derivatives as promising lead compounds for the development of effective ectonucleotidase inhibitors with potential applications in cancer immunotherapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eAqsa Ishaq: conceptualization, synthesis, methodology, biological activities, docking studies, writing. Ismat Nawaz: Synthesis, editing. Salman Alrokayan: Bioassays, editing, fund acquisition. Tajamul Hussain: Bioassays, editing, fund acquisition. Nicolly Espindola Gelsleichter and Julie Pelletier: preparation of recombinant enzymes. Jean S\u0026eacute;vigny: providing recombinant enzymes, review \u0026amp; editing. Jamshed Iqbal: supervision, resources (synthesis, bioactivity and docking).\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e \u003cp\u003eAuthors extend their appreciation to the Deanship of Scientific Research, King Saud University for funding through Vice Deanship of Scientific Research Chairs; Research Chair for Biomedical Application of Nanomaterials. JI received support from the Higher Education Commission China Pakistan Economic Corridor-Collaborative Research Grant # P2-345. J.S. received support from the Natural Sciences and Engineering Research Council of Canada (RGPIN-2023-05498), as well as institutional support from the \u003cem\u003eFonds de recherche du Qu\u0026eacute;bec\u003c/em\u003e (reference: 30641).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBalkwill, F., Mantovani, A. Inflammation and cancer: back to Virchow? The lancet. 2001, 357, 539-45.\u003c/li\u003e\n\u003cli\u003eSharma, P., Allison, J.P. 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Pharmaceutical Chemistry Journal. 2017, 51, 351-4.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"c30c757f-f65d-49d3-b028-0da0407cb0eb","identifier":"10.13039/501100011665","name":"Deanship of Scientific Research, King Saud University","awardNumber":"0012","order_by":0},{"identity":"fbfa8481-feb1-4da1-9943-a90a8a4368f8","identifier":"10.13039/501100010221","name":"Higher Education Commision, Pakistan","awardNumber":"P2-345.","order_by":1},{"identity":"53a98ebe-d169-4b01-a575-24bf2642fecb","identifier":"10.13039/501100000038","name":"Natural Sciences and Engineering Research Council of Canada","awardNumber":"RGPIN-2023-05498","order_by":2}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"COMSATS University Islamabad","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"h-NTPDase, h-NPP1, and h-e5′NT, Quinoline-6-Carboxylic Acid derivatives, Inhibitors, In silico studies and In vitro assessment","lastPublishedDoi":"10.21203/rs.3.rs-6824809/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6824809/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEctonucleotidases, including \u003cem\u003eh\u003c/em\u003e-NTPDases, \u003cem\u003eh\u003c/em\u003e-ENPP, and \u003cem\u003eh\u003c/em\u003e-e5′NT, play a crucial role in regulating extracellular nucleotide levels by converting ATP into immunosuppressive adenosine, thereby facilitating tumor immune evasion. Inhibiting these enzymes can restore antitumor immunity by preventing adenosine accumulation within the tumor microenvironment. Herein, we report the design and synthesis of quinoline-6-carboxylic acid derivatives (4a–4l), a biologically relevant scaffold, and evaluate their potential to inhibit recombinant h-ENPP1, \u003cem\u003eh\u003c/em\u003e-e5′NT, and \u003cem\u003eh\u003c/em\u003e-NTPDases. This study led to the identification of new and effective inhibitors, such as compound \u003cstrong\u003e4d\u003c/strong\u003e, which showed good inhibition against \u003cem\u003eh\u003c/em\u003e-NTPDase1 and -2, as reflected by IC\u003csub\u003e50 \u003c/sub\u003evalue of 0.28±0.03 µM against h-NTPDase1 and 0.92±0.17 µM against \u003cem\u003eh\u003c/em\u003e-NTPDase2. Similarly, compound \u003cstrong\u003e4g\u003c/strong\u003e inhibited \u003cem\u003eh-\u003c/em\u003eNTPDase3 with an IC\u003csub\u003e50\u003c/sub\u003e value of 0.32±0.05 µM, and compound \u003cstrong\u003e4b \u003c/strong\u003einhibited \u003cem\u003eh\u003c/em\u003e-NTPDase8 with an IC\u003csub\u003e50\u003c/sub\u003e value of\u003csub\u003e \u003c/sub\u003e0.44±0.08 µM. Additionally, compound \u003cstrong\u003e4k \u003c/strong\u003edemonstrated good inhibition against \u003cem\u003eh\u003c/em\u003e-ENPP1 as reflected by IC\u003csub\u003e50\u003c/sub\u003e value 0.11±0.02 µM. Finally, compound \u003cstrong\u003e4a \u003c/strong\u003einhibited e5’NT enzyme with an IC\u003csub\u003e50\u003c/sub\u003e value of 0.092±0.02 µM. Molecular docking studies were performed to complement the \u003cem\u003ein vitro\u003c/em\u003e analysis, revealing that the tested compounds show favorable interaction with the amino acid of the target enzymes \u003cem\u003eh\u003c/em\u003e-NTPDase1, -2, -3, and -8, \u003cem\u003eh\u003c/em\u003e-NPP1, and \u003cem\u003eh\u003c/em\u003e-e5′NT enzymes. These interactions involve residues such as Asp201, Asp213, Asp218, Asp326, Ala412, Tyr340, Tyr371, Thr90, Trp408, Trp450, Cys502, Gly178, Phe360, Phe257, Arg392, Ala347, Leu202, Leu290, His50, His360, His380, Ser49, Ser100, Gln148, Glu266, Lys50, Lys295, Gln523, Pro323, and Ile90. Density Functional Theory (DFT) analysis revealed that compound \u003cstrong\u003e4f\u003c/strong\u003e exhibits the lowest energy gap, which correlates well with its strong ectonucleotidase inhibitory activity against\u003cstrong\u003e \u003c/strong\u003eNPP1 and NTPDase3. Finally, fluorescence microscopy was conducted to investigate the interaction of the compound \u003cstrong\u003e4d\u003c/strong\u003e with plasma membrane in A549 lung cancer cells. Fluorescence imaging of compound 4d exhibited strong cellular emission, confirming its effective interaction with membrane-bound enzymes in these cancer cells. MTT and apoptosis studies also revealed compound \u003cstrong\u003e4i\u003c/strong\u003e,\u003cstrong\u003e 4j\u003c/strong\u003e, and \u003cstrong\u003e4k\u003c/strong\u003e to be potentially cytotoxic in the cancer cells, endorsing the potential of these quinoline derivatives to be used for drug development for cancer management.\u003c/p\u003e","manuscriptTitle":"Quinoline-6-Carboxylic Acid Derivatives: A New Class of Potent Ectonucleotidase Inhibitors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-11 11:39:52","doi":"10.21203/rs.3.rs-6824809/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8faa0c81-dc65-42ab-b5fc-d303d9db5657","owner":[],"postedDate":"June 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49861315,"name":"Chemical Biology"}],"tags":[],"updatedAt":"2025-06-11T11:39:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-11 11:39:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6824809","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6824809","identity":"rs-6824809","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}