Novel phthalimide-pyrimidine hybrids as potent anti-tubercular agents

Novel phthalimide-pyrimidine hybrids as potent anti-tubercular agents | 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 Novel phthalimide-pyrimidine hybrids as potent anti-tubercular agents Wael Shehta, Norah A. Alsaiari, Basant Farag, Marwa M. Abdel-Aziz, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4397392/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 Condensation reaction of aromatic aldehydes with 2-(6-amino-2-chloropyrimidin-4-yl)isoindoline-1,3-dione and 2-(6-amino-2-hydrazineylpyrimidin-4-yl)isoindoline-1,3-dione afforded 2-(2-chloro-6-((3 alkylbenzylidene)amino) pyrimidin-4-yl)isoindoline-1,3-dione (6a-f) and 2-(6-amino-2-(2-(arylidene) hydrazineyl)pyrimidin-4-yl)isoindoline-1,3-dione (8a-f) , respectively, as phthalimide-aminopyrimidine hybrids. Compounds showed a wide range of anti-tubercular activity against sensitive MDR and XDR M. tuberculosis strains, with 8f and 6a showing the highest activity. 8f and 6a inhibited sensitive M. tuberculosis with MIC =0.48 μg/ml and 0.98 μg/ml, respectively, comparable to isonizide (INH) (MIC =0.12 μg/ml). Both 8f and 6a inhibited MDR strain with MIC=1.95 μg/ml and 7.81 μg/ml, respectively, and XRD with MIC=7.81 μg/ml and 15.63 μg/ml, respectively. Both 8f and 6a could inhibit mycobacterial InhA enzyme in-vitro (IC 50 =0.717±0.033µM and 1.646±0.069µM, respectively). Molecular docking simulation revealed that 8f and 6a were also capable of interacting at the catalytic site of the InhA enzyme in a manner similar to the native ligand, via binding with NAD + and Tyr158. Compounds 6a and 8f showed physicochemical properties of oral bioavailable drug-like compounds with high gastrointestinal absorption. Predictions showed that compounds have no side effects on the CNS and no anticipated hepatotoxicity, mutagenicity, or acute oral toxicity in models. Mycobacterium tuberculosis ADMET studies Docking simulation Enzyme inhibition Phthalimide-pyrimidine hybrids Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Tuberculosis is a serious lower respiratory tract infection caused by the bacteria Mycobacterium tuberculosis . The infections can be accompanied by other extra-pulmonary ones in skin, brain, and lymph nodes [ 1 ]. The standard four-drug regimen (first-line drugs) for drug-sensitive TB includes isoniazid, rifampicin, ethambutol, and pyrazinamide, which must be taken for at least six months [ 2 ]. However, due to the emergence of both MDR-TB (multidrug-resistant tuberculosis) and XDR-TB (extensively drug-resistant tuberculosis) strains, tuberculosis is still regarded by the World Health Organization (WHO) as a dangerous re-emerging illness with a high fatality rate and remains a major cause of death for patients with autoimmune disease [ 3 ]. Discovering potent and efficient anti-tubercular medications that can eradicate mycobacteria, prevent treatment resistance, and minimise illness recurrence is essential [ 4 ]. Due to its ability to interfere with lipid biosynthesis, Mycobacterium tuberculosis' s unique lipid cell wall—which is necessary for its survival—also renders it susceptible to several treatment drugs [ 5 ]. Mycobacterium tuberculosis enolyl acyl carrier protein reductase (InhA) is a key enzyme that anti-tubercular medications target because it is essential to the biosynthesis of type II fatty acids (FASII), which is only produced by bacteria and not by humans [ 6 , 7 ]. By reducing the trans double bond, InhA covalently links an intermediate to an acyl carrier protein through its carbonyl group. A number of anti-tubercular medications, such as isoniazid, target InhA. (INH). The creation of novel therapeutic drugs continues to be interested in targeting InhA [ 8 ]. Isoniazid (INH)- a first-line anti-TB drug- targets inhA through its activated metabolites. Despite its activity, INH shows high toxicity and side effects due to its nitrogen-centered free radicals (hydrazine metabolites) that can generate highly lipid peroxidase-causing causing-oxygen species with consequent cell death and hepatic necrosis. There are numerous kinds of InhA inhibitors, including diphenyl ether [ 9 ], pyrrolidine carboxamide [ 10 ], hydroxyl pyridines [ 11 ], tetrahydropyran [ 12 ], and 1,3,4-oxadiazole derivatives [ 13 ]. Recently, thiazolidin-4-one-thiazole hybrids showed potent anti-tubercular activity comparable to INH [ 14 ]. Phthalimide is considered a scaffold for anti-tubercular activity [ 15 ]. Compounds I and II demonstrated antitubercular action against sensitive M. TB strains with MICs in the micromolar range and might interact with the mycobacterial InhA active site [ 16 ]. Pyrimidine is considered a core scaffold with anti-tubercular activity as well [ 17 , 18 ]. As shown in Fig. 1 , many pyrimidine-containing compounds are being investigated in clinical trials (GSK286, TBA7371). Ceritinib is a pyrimidine core with confirmed anti-mycobacterial activity. Iclaprim and trimethoprim are examples of antibacterial drugs that are being considered for the treatment of mycobacterial infections [ 19 ]. Arylamides constitute a general class of InhA inhibitors with features that accommodate the structural requirements for interaction at InhA binding site, as shown in Fig. 2 . Arylamides have a broad range of anti-tubercular activity, with MIC in micro- to nonamolar range. Structurally, polyamides are composed of ring A with the amide linker, which plays a role in the interaction at the catalytic site, forming an H -bond interaction at Tyr158, and other amino acids together with an H -bond interaction with the 2'OH of the NAD cofactor at InhA catalytic site (site I). Ring B plays a role in the hydrophobic interaction at the hydrophobic site of the enzyme (Site II), while ring C is important for interaction at the site exposed (site III), being surrounded by polar and non-polar groups at the active site of the enzyme [ 20 ]. In the present investigation, structural modification of arylamide InhA inhibitors will be performed to provide phthalimide –pyrimidine hybrids where phthalimide will take place of ring A together with amide interaction via H -bonding at the catalytic site (Site I), and pyrimidine directly attached to phthalimide will take place of ring C to maintain the proper distances. Ring C will accommodate small groups at position 2, such as a non-polar chlorine group, or at position 6, such as a polar amino group, to allow for interaction at the solvent-exposed site (site II). Different substituted benzene rings will take place in ring B to interact at the hydrophobic site (Site II). They will be introduced at the pyrimidine core either at position 6 through the methylene amino linker or at position 2 through the methylenehydrazinyl linker to afford 2-(2-chloro-6-((3-alkylbenzylidene)amino)pyrimidin-4-yl)isoindoline-1,3-dione ( 6a-f ) and 2-(6-amino-2-(2-(arylidene)hydrazineyl)pyrimidin-4-yl)isoindoline-1,3-dione ( 8a-f ), respectively, as shown in Fig. 2 . The designed compounds will be screened for their anti-tubercular activity against sensitive, multi-drug-resistant (MDR), and extra-drug-resistant (XDR) M. tuberculosis strains compared to INH. Compounds will also be screened for their in-vitro inhibition of mycobacterial InhA enzyme compared to IHN. Molecular docking simulation will be used to study the binding mode of synthesized compounds at the binding site of InhA enzyme. 2. Materials and methods 2.1. Chemistry: All melting points (°C) were measured on Stuart melting point apparatus (SMP 30) and are uncorrected. All the synthesized compounds were monitored by TLC using precoated plastic sheets silica gel (Merck 60 F254) and spots were visualized by irradiation with UV light (254 nm). The used solvent system was chloroform: methanol (9:1) & ethyl acetate: methanol (10:1). IR spectra were recorded on Pye-Uniearn using KEr wafer technique and Beckman spectrophotometers in Zagazig. 1 H-NMR spectra were recorded on Bruker 400 MHz Spectrometer and 13 C-NMR spectra were run at 125 MHz in dimethylsulfoxide (DMSO-d6) and TMS as an internal standard, at applied Nucleic Acid Research Center, Zagazig University, Egypt. Mass spectra were recorded on Direct Inlet part to mass analyzer in Thermo Scientific GCMS model ISQ at the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Nasr City, Cairo. Microanalysis was carried out in microanalytical center, Mansoura University and results were within ± 0.4% from the theoretical values. All the chemicals and reagents used were purchased from Aldrich Chemicals Co, USA, and commercial sources. 6-Aminouracil (l): Compound 1 was commercially available from Aldrich Chemical Company Inc. and was also synthesised in our lab. As described procedure, ethyl cyanoacetate and urea were heated under reflux in sodium ethoxide and ethanol [ 21 – 24 ]. 4-Amino-2,6-dichloropyrimidine (2): Compound 2 was available commercially from Aldrich Chemical Company Inc. and was also synthesised in our laboratory by refluxing 6-aminouracil ( 1 ) with phosphorous oxychloride, as a published method [ 25 , 26 ]. 2-(6-amino-2-chloropyrimidin-4-yl)isoindoline-1,3-dione (4): A mixture of compound ( 2 ) (0.01 mol) and phthalimide [ 27 ] ( 3 ) (0.01 mol) in absolute ethanol (35 mL) in the presence of few drops of piperidine was refluxed for 6h. Once the reaction was finished (TLC), the reaction mixture was cooled and poured onto ice-cold water (50 ml). The resulting solid was filtrated, dried, and recrystallized from methanol. Color: yellow; yield: 58.3%; m.p.= 117–120 o C; IR (KBr) υ max cm − 1 : 3465, 3211 (NH 2 ), 3102 (CH aromatic), 1716 (C = O); 1 H-NMR (400 MHz, DMSO- d 6 , δ, ppm): 5.51 (s, 1H, CH), 6.39 (s, 2 H, NH 2 , exchangeable by D 2 O), 7.82 (s, 4H, Ar-H); 13 C-NMR (125 MHz, DMSO- d 6 , δ, ppm): 74.08, 122.94, 132.65, 134.34, 151.02, 155.21, 164.26, 171.45; MS: m/z (intensity 100%): 276 (2) [M ⁺ +2], 274 (14) [M + ], 239 (17), 213 (21), 197 (13), 185 (100); Anal. calcd. for C 12 H 7 ClN 4 O 2 : C, 52.48; H, 2.57; N, 20.40. Found: C, 52.58; H, 2.59; N, 20.45. 2-(2-chloro-6-((3-alkylbenzylidene)amino)pyrimidin-4-yl)isoindoline-1,3-dione (6a-f): General method Compound ( 4) (0.01 mol) and the appropriate aromatic aldehyde (0.01 mol) were combined with 50 mL of abs. ethanol and two drops of triethylamine (Et 3 N), and the mixture was stirred for 6 to 8h at room temperature. When the reaction (TLC) was complete, the mixture was cooled to room temperature. The solid product was filtered, dried, and then recrystallized from ethanol. 2-(2-chloro-6-((3-nitrobenzylidene)amino)pyrimidin-4-yl)isoindoline-1,3-dione (6a): Stirred time: 8h; color: light yellow; yield: 75%; m.p.= 200–202 o C; IR (KBr) υ max cm − 1 : 3079 (CH aromatic), 2922 (CH aliphatic), 1705 (C = O), 1635 (C = N), 1401, 1337 (NO 2 ); 1 H-NMR (400 MHz, DMSO- d 6 , δ, ppm) 7.60–7.63 (t, J = 8.4 Hz, 1H, Ar-H), 7.82 (s, 4H, Ar- H phthalimide ), 7.86 (s, 1H, CH), 8.01–8.03 (d, J = 7.2 Hz, 1H, Ar-H), 8.32–8.33 (d, J = 7.2 Hz, 1H, Ar-H), 8.51–8.53 (d, J = 7.2 Hz, 1H, Ar-H), 8.68 (s, 1H, CH azomethine ); 13 C-NMR (125 MHz, DMSO- d 6 , δ, ppm): 99.13, 122.94, 128.55, 130.96, 132.61, 134.33, 137.03, 140.95, 142.62, 147.82, 158.08, 163.74, 169.25, 172.83, 191.83; MS: m/z (intensity %): 409 (24) [M ⁺ +2], 407 (36) [M + ], 373 (28), 353 (99), 322 (22), 246 (25), 173 (23), 147 (84), 107 (11), 79 (19), 43 (100); Anal. calcd. for C 19 H 10 ClN 5 O 4 : C, 55.97; H, 2.47; N, 17.18. Found: C, 55.99; H, 2.50, N, 17.25. 2-(2-chloro-6-((4-nitrobenzylidene)amino)pyrimidin-4-yl)isoindoline-1,3-dione (6b): Stirred time: 6h; color: light yellow; yield: 85.4%; m.p.= 180–182 o C; IR (KBr) υ max cm − 1 : 3078 (CH aromatic), 2869 (CH aliphatic), 1679 (C = O), 1624 (C = N), 1513, 1345 (NO 2 ); 1 H-NMR (400 MHz, DMSO- d 6 , δ, ppm): 5.45 (s, 1H, CH pyrimidine ), 7.51–7.53 (d, J = 8.8 Hz, 1H, Ar-H), 7.82 (s, 4H, Ar- H phthalimide ), 8.15–8.17 (d, J = 8.8 Hz, 1H, Ar-H), 8.22–8.24 (d, J = 8.8 Hz, 1H, Ar-H), 8.40–8.43 (d, J = 8.8 Hz, 1H, Ar-H), 9.46 (s, 1H, CH azomethine ); 13 C-NMR (125 MHz, DMSO- d 6 , δ, ppm): 91.14, 122.95, 124.29, 130.66, 132.62, 134.34, 142.84, 147.82, 155.08, 164.73, 168.26, 169.26, 192.36; MS: m/z (intensity %): 409 (17) [M ⁺ +2], 407 (28) [M ⁺ ], 372 (41), 325 (7), 297 (14), 280 (100), 259 (98), 117 (96); Anal. calcd. for C 19 H 10 ClN 5 O 4 : C, 55.97; H, 2.47; N, 17.18. Found: C, 55.98; H, 2.48; N, 17.23. 2-(2-chloro-6-((4-chlorobenzylidene)amino)pyrimidin-4-yl)isoindoline-1,3-dione (6c): Stirred time: 6h; color: yellow; yield: 63.3%; m.p.= 175–177 o C; IR (KBr) υ max cm − 1 : 3090 (CH aromatic), 2849 (CH aliphatic), 1752, 1704 ( 2 C = O), 1676 ( C = N azomethine ) 1631 (C = N); 1 H-NMR (400 MHz, DMSO- d 6 , δ, ppm): 5.23 (s, 1H, CH pyrimidine ), 7.20–7.22 (d, J = 8.8 Hz, 1H, Ar-H), 7.41–7.43 (d, J = 8.8 Hz, 1H, Ar-H), 7.51–7.53 (d, J = 8.8 Hz, 1H, Ar-H), 7.82 (s, 4H, Ar-H phthalimide ), 8.06–8.09 (d, J = 8.8 Hz, 1H, Ar-H), 8.24 (s, 1H, CH azomethine ); 13 C-NMR (125 MHz, DMSO- d6 , δ, ppm): 99.47, 122.98, 128.14, 131.62, 132.64, 134.37, 134.73, 136.79, 153.06, 161.64, 169.30, 169.53, 192.18; MS: m/z (intensity %): 400 (9) [M⁺+4], 398 (9) [M⁺+2], 396 (27) [M ⁺ ], 363 (25), 324 (48), 296 (8), 280 (21), 249 (100); Anal. calcd. for C 19 H 10 Cl 2 N 4 O 2 : C, 57.45; H, 2.54; N, 14.11. Found: C, 57.50; H, 2.56; N, 14.18. 2-(2-chloro-6-((3,4-dimethoxybenzylidene)amino)pyrimidin-4-yl)isoindoline-1,3-dione (6d): Stirred time: 8h; color: yellow; yield: 92%; m.p.= 208–210 o C; IR (KBr) υ max cm − 1 : 3061 (CH aromatic), 2924 (CH aliphatic), 1709 (C = O), 1631 (C = N); 1 H-NMR (400 MHz, DMSO- d 6 , δ, ppm): 3.16 (s, 6H, 2OCH 3 ), 5.43 (s, 1H, CH pyrimidine ), 7.12–7.14 (d, J = 8.4 Hz, 1H, Ar-H), 7.52–7.54 (d, J = 8.4 Hz, 2H, Ar-H), 7.81 (s, 4H, Ar-H phthalimide ), 9.29 (s, 1H, CH azomethine ); MS: m/z (intensity %): 424 (19) [M ⁺ +2], 422 (19) [M ⁺ ], 387 (43), 243 (86), 224 (100); Anal. calcd. for C 21 H 15 ClN 4 O 4 : C, 59.65; H, 3.58; N, 13.25. Found: C, 59.70; H, 3.60; N, 13.30. 2-(2-chloro-6-((2-hydroxybenzylidene)amino)pyrimidin-4-yl)isoindoline-1,3-dione (6e): Stirred time: 6h; color: yellow; yield: 86.8%; m.p.= 188–191 o C; IR (KBr) υ max cm − 1 : 3656 (OH), 3061 (CH aromatic), 2925 (CH aliphatic), 1710 (C = O), 1632 (C = N); 1 H-NMR (400 MHz, DMSO- d 6 , δ, ppm): 6.32 (s, 1H, CH pyrimidine ), 7.07–7.09 (d, J = 7.6 Hz, 1H, Ar-H), 7.21–7.24 (t, J = 7.6 Hz, 1H, Ar-H), 7.29–7.33 (t, J = 7.6 Hz, 1H, Ar-H), 7.53–7.55 (d, J = 7.6 Hz, 1H, Ar-H), 7.81 (s, 4H, Ar-H), 9.25 (s, 1H, CH azomethine ), 11.32 (s, 1H, OH); 13 C-NMR (125 MHz, DMSO- d 6 , δ, ppm): 99.05, 119.47, 122.95, 123.31, 125.96, 130.56, 132.63, 133.86, 134.34, 156.18, 162.84, 168.47, 169.27, 191.83; MS: m/z (intensity %): 380 (40) [M ⁺ +2], 378 (29) [M ⁺ ], 306 (49), 174 (49), 146 (30), 121(100); Anal. calcd. for C 19 H 11 ClN 4 O 3 : C, 60.25; H, 2.93; N, 14.79. Found: C, 60.30; H, 2.95; N, 14.89. 2-(2-chloro-6-((4-hydroxybenzylidene)amino)pyrimidin-4-yl)isoindoline-1,3-dione (6f): Stirred time: 6h; color: yellow; yield: 82.6%; m.p.= 198–200 o C; IR (KBr) υ max cm − 1 : 3631 (OH), 3063 (CH aromatic), 2991 (CH aliphatic), 1710 (C = O), 1633 (C = N); 1 H-NMR (400 MHz, DMSO- d 6 , δ, ppm): 6.33 (s, 1H, CH pyrimidine ), 7.07–7.09 (d, J = 8.4 Hz, 2H, Ar-H), 7.39–7.41 (d, J = 8.4 Hz, 2H, Ar-H), 7.82 (s, 4H, Ar-H), 9.89 (s, 1H, CH azomethine ), 11.32 (s, 1H, OH); 13 C-NMR (125 MHz, DMSO- d 6 , δ, ppm): 99.22, 119.24, 122.94, 126.94, 129.64, 132.61, 134.33, 158.08, 166.30, 166.53, 169.24, 175.24, 192.18; MS: m/z (intensity %): 380 (24) [M + +2], 378 (18) [M + ], 248 (33), 173 (52), 117 (3), 90 (100); Anal. caled. For C 19 H 11 ClN 4 O 3 : C, 60.25; H, 2.93; N, 14.79. Found: C, 60.28; H, 2.98; N, 14.84. 2-(6-amino-2-hydrazineylpyrimidin-4-yl)isoindoline-1,3-dione (7): Compound 4 (0.01 mol) and hydrazine hydrate (0.03 mol) were combined with 20 mL of ethyl alcohol and two drops of triethylamine (Et 3 N), and the mixture was stirred for 9h at room temperature. The mixture was refrigerated once the reaction (TLC) was completed. The solid product was filtrated, dried, and recrystallized from ethanol; color: yellowish-white; yield: 81%; m.p.= 290–292 o C; IR (KBr) υ max cm − 1 : 3320, 3290, 3165 (NH 2 & NH), 3019 (CH aromatic), 1661 (C = O); 1 H-NMR (400 MHz, DMSO- d 6 , δ, ppm): 5.49 (s, 1H, CH pyrimidine ), 7.84–7.86 (d, J = 8.8 Hz, 2H, Ar-H), 7.88 (s, 2H, NH 2 ), 8.03–8.07 (t, J = 8.8 Hz, 2H, Ar-H), 8.08 (s, 2H, NH 2 pyrimidine ), 10.51 (bs, 1H, NH); 13 C-NMR (125 MHz, DMSO- d 6 , δ, ppm): 77.07, 125.18, 127.23, 132.62, 154.71, 164.59, 166.83, 168.71; MS: m/z (intensity %): 271 (42) [M ⁺ ], 255 (33), 237 (19), 199 (29), 184 (100); Anal. calcd. For C 12 H 10 N 6 O 2 : C, 53.33; H, 3.73; N, 31.10. Found: C, 53.36; H, 3.75; N, 31.17. 2-(6-amino-2-(2-(arylidene)hydrazineyl)pyrimidin-4-yl)isoindoline-1,3-dione (8a-f): General method Compound 7 (0.001 mol) and aromatic aldehyde (0.001 mol) were combined with 50 mL of ethanol and two drops of triethylamine (Et 3 N), and the mixture was stirred for 2-11h at room temperature. Following the completion of the reaction (TLC), the solid product that had been separated from the mixture was filtered out, dried, and then recrystallized from ethanol. 2-(6-amino-2-(2-(3-nitrobenzylidene)hydrazineyl)pyrimidin-4-yl)isoindoline-1,3-dione (8a): Stirred time: 6h; color: yellow; yield: 88.9%; m.p.= 140–142 o C; IR (KBr) υ max cm − 1 : 3509, 3453, 3401 (NH 2 &NH), 3081 (CH aromatic), 2922 (CH aliphatic), 1661 (C = O), 1626 (C = N), 1528, 1351 (NO 2 ); 1 H-NMR (400 MHz, DMSO- d 6 , δ, ppm): 4.13 (s, 1H, CH pyrimidine ), 7.22 (s, 2H, NH 2 ), 7.81–7.85 (t, J = 8 Hz, 1H, Ar-H), 8.36–8.39 (t, J = 2.4 Hz, 2H, Ar-H), 8.39–8.41 (d, J = 8 Hz, 1H, Ar-H), 8.72 (s, 1H, CH hydrazone ), 8.93 (s, 4H, Ar-H), 11.50 (bs, 1H, NH); 13 C-NMR (125 MHz, DMSO- d 6 , δ, ppm): 73.07, 118.78, 121.35, 122.67, 125.84, 130.02, 130.67, 131.05, 134.43, 143.14, 148.23, 160.49, 166.43, 167.80, 169.15; MS: m/z (intensity %): 404 (22) [M ⁺ ], 388 (25), 242 (8), 165 (12), 163 (100); Anal. calcd. For C 19 H 13 N 7 O 4 : C, 56.58; H, 3.25; N, 24.31. Found: C, 56.60; H, 3.30; N, 24.40. 2-(6-amino-2-(2-(4-nitrobenzylidene)hydrazineyl)pyrimidin-4-yl)isoindoline-1,3-dione (8b): Stirred time: 3h; color: yellow; yield: 80%; m.p.= 160–162 o C; IR (KBr) υ max cm − 1 : 3452, 3424 (NH 2 &NH), 2922 (CH aliphatic), 1661 (C = O), 1629 (C = N), 1522, 1345 (NO 2 ); 1 H-NMR (400 MHz, DMSO- d 6 , δ, ppm): 4.02 (s, 1H, CH pyrimidine ), 7.55 (s, 2H, NH 2 ), 7.66–7.68 (d, J = 8 Hz, 2H, Ar-H), 7.87 (s, 4H, Ar-H phthalimide ), 8.07 (s, 1H, CH hydrazone ), 8.15–8.17 (d, J = 8.8 Hz, 2H, Ar-H), 11.50 (s, 1H, NH); 13 C-NMR (125 MHz, DMSO- d 6 , δ, ppm): 71.14, 123.99, 125.29, 128.69, 129.59, 131.58, 132.61, 134.12, 143.49, 145.62, 159.20, 166.29, 167.93, 169.58; MS: m/z (intensity %): 403 (59) [M ·⁺ ], 387 (26), 341 (46), 265 (22), 119 (70), 42 (100); Anal. calcd. For C 19 H 13 N 7 O 4 : C, 56.58; H, 3.25; N, 24.31. Found: C, 56.68; H, 3.29; N, 24.38. 2-(6-amino-2-(2-(4-chlorobenzylidene)hydrazineyl)pyrimidin-4-yl)isoindoline-1,3-dione (8c): Stirred time: 4h; color: white; yield: 88.9%; m.p.= 200–202 o C; IR (KBr) υ max cm − 1 : 3456, 3165, 3125 (NH 2 & NH), 3019 (CH aromatic), 2918 (CH aliphatic), 1660 (C = O), 1623 (C = N); 1 H-NMR (400 MHz, DMSO- d 6 , δ, ppm): 4.33 (s, H, CH pyrimidine ), 7.35–7.37 (d, J = 8 Hz, 1H, Ar-H), 7.56 (s, 2H, NH 2 ), 7.58–7.88 (m, J = 8 Hz, 3H, Ar-H), 8.07 (s, 4H, Ar-H phthalimide ), 8.71 (s, 1H, CH hydrazone ), 11.47 (s, 1H, NH); 13 C-NMR (125 MHz, DMSO- d 6 , δ, ppm): 76.07, 125.16, 127.17, 128.25, 129.11, 129.72, 130.05, 132.60, 136.05, 160.62, 164.64, 167.66, 170.08; MS: m/z (intensity 100%): 394 (5) [M ⁺ +2], 392 (12) [M + ], 357 (15), 270 (6), 133 (13), 104 (100); Anal. calcd. For C 19 H 13 ClN 6 O 2 : C, 58.10; H, 3.34; N, 21.40. Found: C, 58.15; H, 3.36; N, 21.49. 2-(6-amino-2-(2-(2-hydroxybenzylidene)hydrazineyl)-pyrimidin-4-yl)isoindoline-1,3-dione (8d): Stirred time: 6h; color: yellow; yield: 95.5%; m.p.= 176–178 o C; IR (KBr) υ max cm − 1 : 3655 (OH), 3450, 3447, 3368 (NH 2 & NH), 3042 (CH aromatic), 2920 (CH aliphatic), 1688 (C = O), 1623 (C = N); 1 H-NMR (400 MHz, DMSO- d 6 , δ, ppm): 3.16 (s, 1H, CH pyrimidine ), 6.95 (s, 2H, NH 2 ), 6.96–6.98 (d, J = 7.2 Hz, 1H, Ar-H), 7.38–7.42 (t, J = 7.2 Hz, 2H, Ar-H), 7.67–7.69 (d, J = 7.2 Hz, 1H, Ar-H), 8.77 (s, 1H, CH hydrazone ), 8.98 (s, 4H, Ar-H phthalimide ), 11.16 (bs, 2H, NH & OH); 13 C-NMR (125 MHz, DMSO- d 6 , δ, ppm): 72.83, 110.61, 116.99, 118.56, 120.16, 131.40, 131.86, 132.43, 132.64, 133.80, 154.23, 159.05, 163.34, 168.90, 174.42; MS: m/z (intensity %): 374 (19) [M ⁺ ], 280 (26), 206 (93), 176 (18), 150 (100); Anal. calcd. For C 19 H 14 N 6 O 3 : C, 60.96; H, 3.77; N, 22.45. Found: C, 60.98; H, 3.80; N, 22.50. 2-(6-amino-2-(2-(4-hydroxybenzylidene)hydrazineyl)pyrimidin-4-yl) isoindoline-1,3-dione (8e) Stirred for 2h; color: yellow; yield: 90.91%; m.p.= 210–212 o C; IR (KBr) υ max cm − 1 : 3656 (OH), 3510, 3452, 3400 (NH 2 & NH), 3042 (CH aromatic), 2919 (CH aliphatic), 1688 (C = O), 1623 (C = N); 1 H-NMR (400 MHz, DMSO- d 6 , δ, ppm): 4.35 (s, 1H, CH pyrimidine ), 6.95 (s, 2H, NH 2 ), 6.97–6.99 (d, J = 9.2, 2H, Ar-H), 7.36–7.38 (d, J = 9.2 Hz, 1H, Ar-H), 7.40 (s, 1H, CH hydrazone ), 7.42–7.44 (d, J = 9.2 Hz, 1H, Ar-H), 7.66–7.68 (d, J = 9.2 Hz, 2H, Ar-H), 7.70–7.72 (d, J = 9.2 Hz, 2H, Ar-H), 9.00 (s, 1H, OH), 11.13 (s, 1H, NH); 13 C-NMR (125 MHz, DMSO- d 6 , δ, ppm): 76.07, 116.57, 118.22, 119.65, 130.88, 133.28, 138.28, 140.10, 151.66, 153.66, 158.67, 162.83, 170.11; MS: m/z (intensity %): 374 (25) [M ⁺ ], 372 (22), 348 (100), 320 (12); Anal. calcd. For C 19 H 14 N 6 O 3 : C, 60.96; H, 3.77; N, 22.45. Found: C, 60.99; H, 3.79; N, 22.50. 2-(6-amino-2-(2-(4-(dimethylamino)benzylidene)hydrazineyl)pyrimidin-4-yl)isoindoline-1,3-dione (8f): Stirred time: 11h; color: yellow; yield: 92.9%; m.p.= 242–244 o C; IR (KBr) υ max cm − 1 : 3509 3452, 3167 (NH 2 & NH), 3018 (CH aromatic), 2918 (CH aliphatic), 1661 (C = O), 1621 (C = N); 1 H-NMR (400 MHz, DMSO- d 6 , δ, ppm): 2.99 (s, 6H, 2CH 3 ), 4.79 (s, 1H, CH pyrimidine ), 6.75–6.77 (d, J = 7.2 Hz, 2H, Ar-H), 7.33 (s, 2H, NH 2 ), 7.63–7.65 (d, J = 7.2 Hz, 2H, Ar-H), 8.33 (s, 4H, Ar-H phthalimide ), 8.49 (s, 1H, CH hydrazone ), 10.81 (s, 1H, NH); 13 C-NMR (125 MHz, DMSO- d 6 , δ, ppm): 48.97, 72.36, 112.15, 124.49, 126.45, 128.07, 130.07, 132.42, 141.13, 155.03, 160.44, 165.69, 169.18, 171.91; MS: m/z (intensity %): 401 (46) [M ⁺ ], 252 (54), 245 (100); Anal. calcd. for C 21 H 19 N 7 O 2 : C, 62.83; H, 4.77; N, 24.42. Found: C, 62.88; H, 4.79; N, 24.48. 2.2. Biological activity: 2.2.1. Anti-tubercular activity: The American Type Culture Collection (ATCC) in the United States provided M. tuberculosis (ATCC 25177/H37Ra) as a drug-sensitive strain (DS) and M. tuberculosis (ATCC 35822) as a multidrug-resistant strain (MDR). Apart from the moxifloxacin-resistant M. tuberculosis (RCMB 2674), pyrazinamide, ethionamide, ethambutol, isoniazid, and rifampicin-resistant strains, an extensively drug-resistant strain (XDR) was obtained from the Culture Collection Unit of the Regional Center for Mycology and Biotechnology (RCMB). Dubos medium mixed with 50 mM sodium nitrate was used to cultivate all strains of M. tuberculosis . The cultures were cultivated aerobically at 37°C and 150 rpm until they reached log-phase optical density (OD595 = 1). Mycobacteria develop as aggregated clumps; thus, they were subjected to ultrasound irradiation and sonication for two minutes using a water bath (Ultrasonic, Freeport, IL, USA). Microplate Alamar blue test was used to assess the minimum inhibitory concentrations (MICs) of drugs against DS, MDR, and XDR M. tuberculosis strains (MABA) [ 28 , 29 ]. Rifampicin and isoniazid were used as positive controls. The chemical stock solutions and final testing concentrations ranged from 1000 to 0.003 µg/mL. In Difco Middlebrook 7H9 Broth (Seebio) supplemented with 0.2% (vol/vol) glycerol, 0.05% Tween 80, and 10% (vol/vol) albumin-dextrose-catalase, M. tuberculosis was raised to late log phase (70 to 100 Klett units) (7H9-ADC-TG). Compounds were produced as two-fold dilutions in 100 µL volumes in 7H9-ADC-TG clear-bottom microplates (BD). A final testing volume of 200 µL was obtained by adding M. tuberculosis (100 µL containing 2 × 105 CFU). The plates were incubated at 37°C, and on the seventh day, each well received the addition of 20 µL of Alamar blue and 12.5 µL of 20% Tween 80. The fluorescence was measured at an excitation of 530 nm and an emission of 590 nm during an incubation period of 16 to 24h at 37°C. The minimum inhibitory concentration (MIC) was identified as the concentration at which a 90% decrease in fluorescence was seen in comparison to duplicate bacterium-only controls. 2.2.2. In-vitro Mycobacterium tuberculosis InhA enzyme inhibition activity: Enzymatic Assay. InhA activity was followed by a colorimetric assay that measured the oxidation of NADH at 340 nm in the presence of 2-trans-octanoyl-CoA in a buffer that contained 30 mM PIPES, pH 7.5, 50 mM NaCl, 0.1 mM EDTA, and 100 nM InhA. This was preincubated for 10 min at room temperature with 0.25 mM NADH and varying concentrations of the compounds with 1% (v/v) DMSO in a 150 µL reaction volume. The reaction was started by the addition of 2-trans-octanoyl-CoA at a final concentration of 1.5 mM. The reactions were followed for 20 min using a plate reader (CLARIOstar, BMG LABTECH) [ 30 ]. 2.3. Molecular docking simulation: Docking was done according to the literature [ 31 ]. The crystallographic structure of the InhA enzyme (PDB ID: 3FNE) was acquired from PDB and prepared for molecular docking by removing ligands, introducing hydrogens, and decreasing energy with MOE 2009. The structure with the lowest energy was then employed as a docking receptor. MOE's site finder method was utilised to identify InhA's catalytic site. Chem Bio Office was used to create the two-dimensional structures of the synthesised compounds, which were subsequently built from fragment libraries in MOE 2009 and reduced energy with the MMFF94x force field in MOE. To discover and analyze the interaction between ligands and the catalytic site of InhA, docking was done with chosen parameters (rescoring function 1 and rescoring function 2: London). 3. Results and discussion 4.1. Chemistry: In continuation of our research interest concerning the synthesis of nitrogen heterocycles and their potential application as anti-cancer agents [ 21 – 24 ] the novelly designed phthalimide-pyrimidine hybrids 6a-f and 8a-f were prepared from 2-(6-amino-2-chloropyrimidin-4-yl)isoindoline-1,3-dione ( 4 ) as precursor. The pyrimidinylisoindolinedione 4 was obtained starting from the readily available 6-aminouracil ( 1) , according to our recent study [ 25 , 26 ] Initially, chlorination of 1 with phosphorus oxychloride (POCl 3 ) yielded 6-amino-2,4-dichlorouracil ( 2 ), which on refluxing in piperidine as basic medium with phthalimide ( 3 ) produced 4 , as shown in Scheme 1 . Compound 4 was proven by 1 H-NMR spectrum, which revealed singlet signals at δ 5.51 and 6.39 ppm, assigned for CH-5 and NH 2 -6 of pyrimidine ring, respectively. The base-mediated condensation of aminopyrimidine derivative 4 with different aromatic aldehydes 5 (namely, 3-, 4-nitro-, 4-chloro-, 3,4-dimethoxy-, 2-, and 4-hydroxybenzaldehydes) in ethanol at room temperature for 6-8h, led to formation of the corresponding Schiff bases 6a-f in good to excellent yields, as shown in Scheme 2 . The chemical structure of 6a-f was confirmed by IR, 1 H-NMR, 13 C-NMR, and mass spectroscopy. IR spectra of 6a-f showed no stretching frequencies for NH 2 , while it showed absorption bands at 2849–2991 cm − 1 assigned for N = CH aliphatic. In addition, 1 H-NMR spectra (DMSO- d 6 ) of 6a-f showed no signals for NH 2 but exhibited singlet signal at δ 8.24–9.89 ppm characteristic for azomethine proton N = C H . On the other hand, chloropyrimidine derivative 4 undergoes a nucleophilic substitution reaction by hydrazine hydrate in ethyl alcohol in the presence of triethylamine (few drops) at room temperature for 9h, giving 2-hydrazino derivative 7 in good yield, as shown in Scheme 3 . IR spectrum of 7 showed three absorption peaks at 3320, 3290, 3165 cm − 1 for (NH 2 & NH). The chemical structure of 7 was further confirmed by 1 H NMR spectroscopy, which showed singlet signal at δ 5.49 characterized for -CH for pyrimidine ring, two singlets at 7.88 & 8.08 for 2 NH 2 , and broad signal at 10.51 ppm for NH proton. Finally, the base-catalyzed condensation of the 2-hydrazinylpyrimidine 7 with aromatic aldehydes in ethanol at room temperature afforded the respective hydrazones 8a-f in good to excellent yield, as shown in Scheme 4 . IR spectra of 8a-f showed absorption bands at 2918–2922 cm − 1 assigned for C-H aliphatic, with the other expected absorption peaks for NH 2 , NH, and imide carbonyl. In addition, 1 H NMR spectra of 8a-f showed singlet signal at δ 7.40–8.77 ppm assigned for CH hydrazone proton, while no hydrazinylamino proton signal was observed. 3.2 . Biological evaluation 3.2.1 . Anti-tubercular activity : All newly synthesized compounds were tested for anti-tubercular activity against M. tuberculosis (ATCC 25177 H37Ra), a drug-sensitive strain, using the microplate Alamar blue assay (MABA) and INH as the reference drug. Table (1) displays the anti-mycobacterial activity of compounds 6a-f and 8a-f given as a minimum inhibitory concentration (MIC). Compounds showed a wide range of activity, where compounds 8d and 8e displayed mild anti-mycobacterial activity against sensitive strains with MIC 125 µg/ml. Compounds 6c , 6d , 8b and 8c showed moderate activity with MIC range of 15.63–62.5 µg/ml. Compounds 6b , 6e , 6f , and 8a were found to be potent anti-tuberculars with MIC range of 3.9–7.81 µg/ml. The highest activity against sensitive TB strains was shown by compounds 8f (MIC = 0.48 µg/ml) and 6a (MI = 0.98 µg/ml). 3.2.2 . Anti-tubercular activity towards isoniazid, cycloserine, kanamycin, and rifampin-resistant M. tuberculosis (ATCC 35822) : Compounds 6a-f and 8a-f were further tested for anti-tubercular activity against the multi-drug resistance strain (MDR) M. tuberculosis ATCC 35822 -isoniazid, cycloserine, kanamycin, and rifampin-resistant strain using the microplate Alamar blue assay. Table 1 displays the anti-mycobacterial activity of compounds 6a-f and 8a-f represented as a minimum inhibitory concentration. Compounds demonstrated a variety of activity from no activity to high potency where compounds 6c , 8c , 8d and 8e were found to be inactive, compounds 6b , 8a and 8b showed mild activity with MIC range of 31.25–62.5 µg/ml while compounds 6a , 6e and 6f were found to be potent with MIC of 7.81 µg/ml. Compound 8f showed the highest anti-mycobacterial activity against MDR strain with MIC of 1.95 µg/ml. 3.2.3. Anti-tubercular activity towards isoniazid, rifampicin, ethambutol, pyrazinamide, ethionamide, and moxifloxacin -resistant M. tuberculosis (RCMB 2674) : The anti-tubercular activity of substances was tested against M. tuberculosis (RCMB 2674), an extensively drug-resistant strain (XDR), using a microplate Alamar blue assay with isoniazid, rifampicin, ethambutol, pyrazinamide, ethionamide, and moxifloxacin. Table (1) shows the anti-mycobacterial activity of compounds 6a-f and 8a-f expressed as a minimum inhibitory concentration. Compounds 6b , 6c , 6d , 8a , 8c , 8d and 8e were found to be inactive while compounds 6a , 6e , 6f , 8b showed moderate activity with MIC range of 15.63–31.25 µg/ml compound 8f was a potent anti-tubercular against XDR strain with MIC of 7.81 µg/ml. Table (1) Anti-tubercular activity of 6a-f and 8a-f : Compds R Sensitive M. tuberculosis MIC(µg/ml) MDR M. tuberculosis MIC(µg/ml) XDR M. tuberculosis MIC(µg/ml) 6a 3-NO 2 0.98 7.81 15.63 6b 4-NO 2 7.81 31.25 NA 6c 4-Cl 31.25 NA NA 6d 3, 4-OCH 3 31.25 125 NA 6e 2-OH 3.9 7.81 31.25 6f 4-OH 3.9 7.81 31.25 8a 3-NO 2 7.81 31.25 NA 8b 4-NO 2 15.63 62.5 31.25 8c 4-Cl 62.5 NA NA 8d 2-OH 125 NA NA 8e 4-OH 125 NA NA 8f 4-N(CH 3 ) 2 0.48 1.95 7.81 Isoniazid 0.12 IA NA MIC = minimal drug concentration required to stop the growth of Mycobacterium tuberculosis Highlighted values represent the most potent derivatives, compared to INH. NA = No activity (MIC > 125 µg/ml) 3.2.4. Mycobacterium tuberculosis InhA enzyme inhibition activity : The InhA enzyme from Mycobacterium TB is necessary for cell wall metabolism and mycolic acid production. Compounds 8f and 6a demonstrated the best anti-tubercular effectiveness while exhibiting the least bacterial resistance. To compare 8f and 6a to isoniazid (INH) at various doses, their in-vitro inhibitory activity of the enzyme InhA was assessed using a previously described methodology [ 30 ]. Table (2) displays the 50% inhibitory concentration (IC 50 ), which is the concentration necessary to inhibit 50% of InhA. 8f and 6a inhibited the inhA enzyme with IC 50 values of 0.717 ± 0.033µM and 1.646 ± 0.069µM, which were equivalent to isoniazid's IC 50 of 0.323 ± 0.014µM. Table (2) : In-vitro Mycobacterium tuberculosis enoyl-acyl carrier protein reductase (InhA) inhibitory activity of 8f,6a and Isoniazid : Compound (InhA IC 50 (mean ± SD) µM 8f 0.717 ± 0.033 6a 1.646 ± 0.069 Isoniazid 0.323 ± 0.014 3.3. Molecular docking simulation : Several anti-tubercular medicines, notably the activated version of INH, target M. tuberculosis's enoyl-acyl carrier protein reductase (InhA). The binding pocket of the InhA enzyme consists of three main sites: the catalytic site (site I), including key amino acids Tyr158, 2`-OH of the nicotinamide ribose of the nicotinamide adenine dinucleotide NAD+, and other amino acids; and the hydrophobic region (site II), which accommodates the substrate binding loop in InhA. It is composed of a number of amino acids, including Met103, Glu104, Phe149, Ala157, Ala198, Met199, Ile202, Ile215, and Leu218; and a size-limiting solvent-exposed site (site III). It has been discovered that site I and parts of site II are occupied by InhA inhibitors. According to the literature[ 31 ], Tyr158 stabilizes substrates during the catalytic reaction of the enzyme, which is important for InhA activity. It was also stated that the interaction of NAD + with inhibitors is important for activity. Tyr158 exists in two conformations: IN-conformation and OUT-conformation.Tyr158 in its IN-form has the ability to bind to inhibitors; the aromatic groups of the inhibitors occupy the hydrophobic site (site II) together with π-π stacking against Phe149, while the hydroxyl group of the inhibitors is directed towards the inhibitors in the catalytic site (site I) [ 32 , 33 ]. With natural ligand 2-(2,4-dichlorophenoxy)-5-(pyridin-2-ylmethyl)phenol (DCPP), the InhA enzyme's X-ray crystal may be seen (PDB ID: 3FNE) [ 34 ]. The native ligand (DCPP) at the InhA binding site showed H -bond interaction with the key amino acids Tyr158 and NAD + at site I that was occupied by phenolic ring, 2,4-dichlorophenyl ring occupied site II and formed hydrophobic interactions at Met199, Ala198, Gly96, and Met161 while the pyridine ring accommodated itself at site III. MOE 2009 was used for docking. To verify the docking procedure, the co-crystallized ligand was re-docked into the InhA enzyme. Between the co-crystallized ligand and docking posture, the computed root means square differences (RMSD) value was less than 2Å. The newly synthesised compounds were docked and docking poses with higher energy scores and a lower root mean square deviation (RMSD) between the docked compound geometry and the co-crystallized native ligand were studied. Compound 8f showed mode of interaction similar to InhA enzyme inhibitors, where ring A occupied site II, where carbonyl group of phthalimide formed H -bond interaction at Tyr158 and NAD. Furthermore, benzene ring of the phthalimide was stacked against Phe 149. Ring B (dimethyl aminobenzene) occupied site II, forming hydrophobic interactions at Ala198, Met199, Phe97, Gly96, and Met161. Ring B showed stacking against Phe97. Ring C (pyrimidine) occupied site III. As shown in Fig. 3 . Compound 6 a acquired an orientation different from 8f , where the interaction at Tyr158 was afforded by ring C (pyrimidine) through N3, while interaction at NAD was afforded via phthalimide carbonyl at Ring A. The hydrophobic interaction at Ala198, Met199 was afforded by ring C . Ring B (3-nitrobenze) was oriented towards site III, as shown by Fig. 4 . 3.4. Drug likeness and ADMET properties: It is critical to study the physicochemical properties as well as the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of drug candidates. Admetlab 2.0 is an online tool that allows systematic evaluation of ADMET properties, as well as some physicochemical properties. Admetlab 2.0 was used to study the physicochemical properties, drug likeness, and ADMET properties of compounds 8f and 6a compared to INH. Figure (1) in supplementary file (S2) shows the generated radar chart for basic physicochemical properties and drug likeness of 8f , 6a and INH. 1-Studied parameters for drug likeness and physicochemical properties: MW = Molecular weight (optimally for a drug-like: 100–600), nHD = Number of hydrogen bond donors, which is Sum of all OHs and NHs (Optimally for a drug-like 0–7), nHA = Number of hydrogen bond acceptors. Sum of all O and N (optimally for a drug-like: 0–12). nRot = Number of rotatable bonds (optimally for a drug-like: 0–11), nRing = Number of rings (Optimally For a drug-like: 0–6), MaxRing = Number of atoms in the biggest ring (optimally for a drug like: 0–18), nHet = Number of heteroatoms. Number of non-carbon atoms (Optimally for a drug-like:1–15), fChar = Formal charge (Optimally for a drug-like: 4 ~ 4), nRig = Number of rigid bonds (Optimally for a drug-like: 0–30), TPSA Topological polar surface area. Sum of tabulated surface contributions of polar fragments (Optimally for a drug-like:0-140) l ogS = The logarithm of aqueous solubility value. Low solubility is unfavorable to good and complete oral absorption Drug-like compounds are in the range from − 4 to 0.5 log mol/. logP = The logarithm of the n-octanol/water distribution coefficient. log is important to determine both membrane permeability and hydrophobic binding to macromolecules. Drug-like compounds are in the range from 0 to 3 log mol/L. logD7.4 = The logarithm of the n-octanol/water distribution coefficients at pH = 7.4. Drug candidates must be able to keep a balance between lipophilicity and hydrophilicity to dissolve as well as be able to penetrate membranes. Drug-like compounds are in the range of 1 to 3 log mol/L. Table (1 ) in supplementary file (S2) shows the predicted physicochemical properties of oral bioavailable drug-like compounds 6a and 8f compared to INH. 2-Studied parameters for ADMET properties: Absorption : HIA (Human intestinal absorption). Poorly absorbed molecules have an absorption of less than 30%. Consequently, molecules having an HIA > 30% were assigned to HIA- (Category 0), whereas molecules having an HIA < 30% were assigned to HIA (Category 1). With a range of 0 to 1, the output result represents the likelihood of being HIA+. Distribution : BBB Drugs that do not need to operate on the central nervous system (CNS) should ideally have minimal or no BBB penetration to prevent adverse effects. Metabolism : Cytochrome P450(CYP2D6) The human cytochrome P450 family (phase I metabolism enzyme) with many isozymes that metabolize drugs they are mainly concentrated in liver. Toxicity : The Ames mutagenicity test : AMES negative (-); AMES positive (+). Rat Oral Acute Toxicity (-ve) low-toxicity, > 500 mg/kg; or (+ ve) high-toxicity; < 500 mg/kg. The output value is the probability of being toxic, within the range of 0 to 1. Table (2 ) in supplementary file (S2) shows the predicted ADMET properties of compounds 6a and 8f compared to INH. Both compounds 6a and 8f demonstrated good oral bioavailability, together with good gastrointestinal absorption. It was anticipated that they would not show hepatotoxicity or CNS toxicity. Compounds showed no mutagenicity as predicted by the Ames test and no acute oral toxicity in the animal model used in Admet Lab 2.0. 4. Conclusion In conclusion, phthalimide-pyrimidine hybrids demonstrated antitubercular efficacy against MDR, XDR, and sensitive strains of tuberculosis. All compounds showed activity against sensitive M. Tuberculosis strain with MIC range of 0.48–62.5 µg/ml. Compounds 6a , 6b , 6e , 6f , 8a , 8b , and 8f showed anti-tubercular activity against MDR M. Tuberculosis strains with MIC range of 1.95–62.5 µg/ml. Compounds 6a , 6e , 6f , 8b , and 8f showed activity against XDR M. Tuberculosi s strains with MIC range of 7.8-31.25 µg/ml. Compounds 8f and 6a had the most potent anti-tubercular activity against senstive, MDR, and XDR M. Tuberculosis strains with MIC of (0.48 µg/ml, 0.98 µg/ml), (1.95 µg/ml, 7.81 µg/ml), and (7.81 µg/ml, 15.63 µg/ml), respectively. Both 8f and 6a inhibited the Mycobacterial InhA enzyme with IC 50 = 0.717 ± 0.033 µM and 1.646 ± 0.069 µM, respectively. Molecular docking simulation revealed that 8f and 6a could interact with the InhA enzyme in an inhibitory mode, forming H -bond interactions at Tyr158 and NAD + at the catalytic site, in agreement with the co-crystalized ligand and other reported inhibitors. 6a and 8f showed physicochemical properties of oral bioavailable drug-like compounds with gastrointestinal absorption. Predictions showed that compounds have no side effects on the CNS and no anticipated hepatotoxicity. Predictions showed no-mutagenicity or acute oral toxicity in models. Declarations Declaration of Competing Interest: The authors have no competing interests to declare Funding sources: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author Contribution: The authors have no competing interests to declare. References Modi, P., S. Patel, and M. Chhabria, Structure-based design, synthesis and biological evaluation of a newer series of pyrazolo [1, 5-a] pyrimidine analogues as potential anti-tubercular agents. Bioorganic Chemistry, 2019. 87 : p. 240-251. Tiberi, S., et al., New drugs and perspectives for new anti-tuberculosis regimens. Pulmonology, 2018. 24 (2): p. 86-98. Augustynowicz-Kopeć, E., et al., Guidelines of Polish Respiratory Society concerning diagnosis, treatment and prevention of tuberculosis in adults and in children. Advances in Respiratory Medicine, 2013. 81 (4): p. 323-379. Mondal, R. and A. Jain, Extensively drug-resistant Mycobacterium tuberculosis, India. Emerging Infectious Diseases, 2007. 13 (9): p. 1429. Vale, N., P. Gomes, and H. A Santos, Metabolism of the antituberculosis drug ethionamide. Current Drug Metabolism, 2013. 14 (1): p. 151-158. Rawat, R., A. Whitty, and P.J. Tonge, The isoniazid-NAD adduct is a slow, tight-binding inhibitor of InhA, the Mycobacterium tuberculosis enoyl reductase: adduct affinity and drug resistance. Proceedings of the National Academy of Sciences, 2003. 100 (24): p. 13881-13886. Takayama, K., C. Wang, and G.S. Besra, Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis. Clinical Microbiology Reviews, 2005. 18 (1): p. 81-101. AlMatar, M., et al., Novel compounds targeting InhA for TB therapy. Pharmacological Reports, 2018. 70 (2): p. 217-226. Wang, L.-Q., C.N. Falany, and M.O. James, Triclosan as a substrate and inhibitor of 3′-phosphoadenosine 5′-phosphosulfate-sulfotransferase and UDP-glucuronosyl transferase in human liver fractions. Drug Metabolism and Disposition, 2004. 32 (10): p. 1162-1169. He, X., et al., Pyrrolidine carboxamides as a novel class of inhibitors of enoyl acyl carrier protein reductase from Mycobacterium tuberculosis. Journal of Medicinal Chemistry, 2006. 49 (21): p. 6308-6323. Ng, P.S., et al., Structure activity relationships of 4-hydroxy-2-pyridones: A novel class of antituberculosis agents. European Journal of Medicinal Chemistry, 2015. 106 : p. 144-156. Pajk, S., et al., New direct inhibitors of InhA with antimycobacterial activity based on a tetrahydropyran scaffold. European Journal of Medicinal Chemistry, 2016. 112 : p. 252-257. Yar, M.S., A.A. Siddiqui, and M. Ashraf Ali, Synthesis and Anti Tuberculostatic Activity of Novel 1, 3, 4‐Oxadiazole Derivatives. Journal of the Chinese Chemical Society, 2007. 54 (1): p. 5-8. Othman, D.I., et al., Novel 2-arylthiazolidin-4-one-thiazole hybrids with potent activity against Mycobacterium tuberculosis. Bioorganic Chemistry, 2022. 124 : p. 105809. Phatak, P.S., et al., Synthesis, antitubercular evaluation and molecular docking studies of phthalimide bearing 1, 2, 3-triazoles. Synthetic communications, 2019. 49 (16): p. 2017-2028. Rani, A., et al., Design and synthesis of 4-Aminoquinoline-isoindoline-dione-isoniazid triads as potential anti-mycobacterials. Bioorganic & Medicinal Chemistry Letters, 2020. 30 (22): p. 127576. Jesumoroti, O.J., et al., Exploration of 4-aminopyrrolo [2, 3-d] pyrimidine as antitubercular agents. Molecular Diversity, 2023. 27 (2): p. 753-765. Al-Soliemy, A.M., R. Sabour, and T.A. Farghaly, Pyrazoles and fused pyrimidines: Synthesis, structure elucidation, antitubercular activity and molecular docking study. Medicinal Chemistry, 2022. 18 (2): p. 181-198. Finger, V., et al., Pyrimidine derivatives with antitubercular activity. European Journal of Medicinal Chemistry, 2023. 246 : p. 114946. Pan, P. and P. J Tonge, Targeting InhA, the FASII enoyl-ACP reductase: SAR studies on novel inhibitor scaffolds. Current topics in medicinal chemistry, 2012. 12 (7): p. 672-693. Srivastava, A., Palladium metal complexes as platforms for anti tumor therapy. J. Xi’an Shiyou Univ. Nat. Sci. Ed, 2021. 17 (6): p. 267-272. Youssif S, El-Bahaie S, Nabih E. A facile one-pot synthesis of pyrido[2,3-d]pyrimidines and pyrido[2,3-d:6,5-d]dipyrimidines. J. Chem. Res. Synop. (2), 112–113 (1999). " 6-Amino-2-thio-, 6-aminouracils as Precursors for the Synthesis of antiviral and antimicrobial methylene bis(2-thiouracil), tricyclic pyrimidines and 6-alkylthio-purine-2-ones. Shaker Youssif and Sahera F. Mohamed, Chemical Monthly , 2008 , 139, 161-168 “Synthesis, Molecular Docking and Anticancer Activity of Some 5-Aryl-5,10-dihydropyrido[2,3- d : 6,5- d' ]dipyrimidine-2,4,6,8-tetraone Derivatives and Pyrido[2,3- d ]pyrimidines”, Basant Farag, Fatimah Agili, Samar El-Kalyoubi, Said A. Said, Shaker Youssif, and Wael Shehta; ChemistrySelect 2022 , 7, e202103834. Wang, H., et al., Large-scale solvent-free chlorination of hydroxy-pyrimidines,-pyridines,-pyrazines and-amides using equimolar POCl3. Molecules, 2012. 17 (4): p. 4533-4544. Shehta W., Agili F., Farag B., J Almehmadi S., Elfeky M.S., Youssif S., & El-Kalyoubi S., Synthesis and in vitro study of pyrimidine-phthalimide hybrids as VEGFR2 inhibitors with antiproliferative activity, Future Med Chem ., 2023, 661-677. doi: 10.4155/fmc-2023-0025. Paradkar, M. V., Gadre, S. Y., Pujari, T. A., Khandekar, P. P., & Kumbhar, V. B. (2005). One‐Pot Synthesis of 3‐Phenacylphthalides. Synthetic communications , 35 (3), 471-474. Lu, Y., et al., Clofazimine analogs with efficacy against experimental tuberculosis and reduced potential for accumulation. Antimicrobial Agents and Chemotherapy, 2011. 55 (11): p. 5185-5193. Elsayed, Z.M., et al., Development of novel isatin–nicotinohydrazide hybrids with potent activity against susceptible/resistant Mycobacterium tuberculosis and bronchitis causing–bacteria. Journal of Enzyme Inhibition and Medicinal Chemistry, 2021. 36 (1): p. 384-393. Sivaraman, S., Structure-activity studies of the inhibition of FabI, the enoyl ACP reductase from Escherichia coli by triclosan . 2002: State University of New York at Stony Brook. Elfeky, S.M., et al., Synthesis, biological screening, and molecular docking of quinazolinone and quinazolinethione as phosphodiesterase 7 inhibitors. Archiv der Pharmazie, 2020. 353 (1): p. 1900211. Sabbah, M., et al., Fragment-based design of Mycobacterium tuberculosis InhA inhibitors. Journal of Medicinal Chemistry, 2020. 63 (9): p. 4749-4761. Kamsri, P., et al., Discovery of new and potent inha inhibitors as antituberculosis agents: structure-based virtual screening validated by biological assays and x-ray crystallography. Journal of Chemical Information and Modeling, 2019. 60 (1): p. 226-234. Freundlich, J.S., et al., Triclosan derivatives: towards potent inhibitors of drug-sensitive and drug-resistant Mycobacterium tuberculosis. ChemMedChem, 2009. 4 (2): p. 241. Schemes Schemes 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Spectrapaper2basant.docx Supplementaryfiles21.docx Schemes1to4.docx Graphicalabstract.jpg 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-4397392","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":301131153,"identity":"7e2f1aa2-805b-46d8-8677-29ff952ce7d8","order_by":0,"name":"Wael Shehta","email":"","orcid":"","institution":"Zagazig University","correspondingAuthor":false,"prefix":"","firstName":"Wael","middleName":"","lastName":"Shehta","suffix":""},{"id":301131154,"identity":"efb59add-ebee-4c30-8133-1d0c495123c3","order_by":1,"name":"Norah A. Alsaiari","email":"","orcid":"","institution":"Najran University","correspondingAuthor":false,"prefix":"","firstName":"Norah","middleName":"A.","lastName":"Alsaiari","suffix":""},{"id":301131155,"identity":"9d56c67f-81f0-4fb6-9263-670832cd42d1","order_by":2,"name":"Basant Farag","email":"","orcid":"","institution":"Zagazig University","correspondingAuthor":false,"prefix":"","firstName":"Basant","middleName":"","lastName":"Farag","suffix":""},{"id":301131156,"identity":"71643563-a85a-4c33-92b8-b8e8c6efc909","order_by":3,"name":"Marwa M. Abdel-Aziz","email":"","orcid":"","institution":"Al-Azhar University","correspondingAuthor":false,"prefix":"","firstName":"Marwa","middleName":"M.","lastName":"Abdel-Aziz","suffix":""},{"id":301131157,"identity":"1cc5b695-bba6-4ce0-b4dd-5fb268606fab","order_by":4,"name":"Shaker Youssif","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYPACCX5+MM1GghbJmQ0kamGQ3HCAWC3yM5KPSf74YyFhfP6MAcOHssMM/PwL8GsxuJGWJs3bJiFhduCMAeOMc4cZJGc8IKBFIsdMmrFBos7sYI8BM2/bYaAhBwg5LMcM6DAJCeNmHgPmv0At9oS0MNzIMZPgYZOQMGADamEE2cLfQMBhZ54lW4P8InGGreBgz7l0HokbBCyRb08+ePPHnzoJ/v7DGx/8KLOW4+8n5DCBBAQbpJaHQSIBu0o44McwE1NkFIyCUTAKRjgAALRPPzjmAf4bAAAAAElFTkSuQmCC","orcid":"","institution":"Zagazig University","correspondingAuthor":true,"prefix":"","firstName":"Shaker","middleName":"","lastName":"Youssif","suffix":""},{"id":301131158,"identity":"8c54f195-e2ca-4580-aae3-3269a595987e","order_by":5,"name":"Sherin M Elfeky","email":"","orcid":"","institution":"Mansoura University","correspondingAuthor":false,"prefix":"","firstName":"Sherin","middleName":"M","lastName":"Elfeky","suffix":""},{"id":301131159,"identity":"93183076-d3a0-4b8c-8b8d-33acf23e4230","order_by":6,"name":"Samar El-Kalyoubi","email":"","orcid":"","institution":"Port Said University","correspondingAuthor":false,"prefix":"","firstName":"Samar","middleName":"","lastName":"El-Kalyoubi","suffix":""},{"id":301131160,"identity":"fe5e4b8a-e3a4-4e65-a09f-73e13e965761","order_by":7,"name":"Nermeen Awni","email":"","orcid":"","institution":"Zagazig University","correspondingAuthor":false,"prefix":"","firstName":"Nermeen","middleName":"","lastName":"Awni","suffix":""}],"badges":[],"createdAt":"2024-05-10 00:23:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4397392/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4397392/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56528938,"identity":"1ca8f6e2-606c-47ed-842b-e853d1bb6122","added_by":"auto","created_at":"2024-05-15 11:20:24","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":246489,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferent reported anti-tubercular compounds\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4397392/v1/00091e93603d239f2bdd3b28.jpg"},{"id":56528939,"identity":"af90d103-41d7-487a-a4a6-218951d6aff7","added_by":"auto","created_at":"2024-05-15 11:20:24","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":230091,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDesigned phthalimide-pyrimidine hybrids.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4397392/v1/df6b577ed066ecd2e462e474.jpg"},{"id":56528943,"identity":"403f45b3-01b3-4ece-88e7-85d9fe5a19d9","added_by":"auto","created_at":"2024-05-15 11:20:24","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":325942,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e2D\u0026amp; 3D representation of 8f at the binding site of Mycobacterial InhA (3FNE)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(8\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ef \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eblue, DCPP Yellow, NAD Green)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4397392/v1/91b3b918dc59780b83439d09.jpg"},{"id":56528941,"identity":"084c0c9e-3fc4-4634-86ee-eee66d5eb106","added_by":"auto","created_at":"2024-05-15 11:20:24","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":273820,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e2D\u0026amp; 3D representation of 6a\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eat the binding site of Mycobacterial InhA (3FNE)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(6a\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eblue, DCPP Yellow, NAD Green)\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4397392/v1/1d7fd0f93f353938342f77e6.jpg"},{"id":56529818,"identity":"1718cc89-c068-4c0b-8c0f-3d54c72b5ed4","added_by":"auto","created_at":"2024-05-15 11:36:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2694080,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4397392/v1/a52af8ce-42af-4324-a4e4-f7b54aeddd8d.pdf"},{"id":56528945,"identity":"deb01c31-c05e-4d44-9210-ae78bdd176fe","added_by":"auto","created_at":"2024-05-15 11:20:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5104835,"visible":true,"origin":"","legend":"","description":"","filename":"Spectrapaper2basant.docx","url":"https://assets-eu.researchsquare.com/files/rs-4397392/v1/99f5efb87fc0be837a12c632.docx"},{"id":56528940,"identity":"d31007d1-fbbd-437d-a7a3-4d69789d161c","added_by":"auto","created_at":"2024-05-15 11:20:24","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":318838,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfiles21.docx","url":"https://assets-eu.researchsquare.com/files/rs-4397392/v1/00d37425ad6aee604c89f6c3.docx"},{"id":56528942,"identity":"b728195d-e7e6-4877-bda8-f218f2e5390b","added_by":"auto","created_at":"2024-05-15 11:20:24","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":81293,"visible":true,"origin":"","legend":"","description":"","filename":"Schemes1to4.docx","url":"https://assets-eu.researchsquare.com/files/rs-4397392/v1/75644772e1b6ebd360e83fd8.docx"},{"id":56528944,"identity":"8fa0fc11-364b-4128-a3e0-b0142eca64ca","added_by":"auto","created_at":"2024-05-15 11:20:24","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":368362,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4397392/v1/2289b7a6fbe946e2a754ff31.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Novel phthalimide-pyrimidine hybrids as potent anti-tubercular agents","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eTuberculosis is a serious lower respiratory tract infection caused by the bacteria \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e. The infections can be accompanied by other extra-pulmonary ones in skin, brain, and lymph nodes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The standard four-drug regimen (first-line drugs) for drug-sensitive TB includes isoniazid, rifampicin, ethambutol, and pyrazinamide, which must be taken for at least six months [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, due to the emergence of both MDR-TB (multidrug-resistant tuberculosis) and XDR-TB (extensively drug-resistant tuberculosis) strains, tuberculosis is still regarded by the World Health Organization (WHO) as a dangerous re-emerging illness with a high fatality rate and remains a major cause of death for patients with autoimmune disease [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Discovering potent and efficient anti-tubercular medications that can eradicate mycobacteria, prevent treatment resistance, and minimise illness recurrence is essential [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Due to its ability to interfere with lipid biosynthesis, \u003cem\u003eMycobacterium tuberculosis'\u003c/em\u003es unique lipid cell wall\u0026mdash;which is necessary for its survival\u0026mdash;also renders it susceptible to several treatment drugs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Mycobacterium tuberculosis enolyl acyl carrier protein reductase (InhA) is a key enzyme that anti-tubercular medications target because it is essential to the biosynthesis of type II fatty acids (FASII), which is only produced by bacteria and not by humans [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. By reducing the trans double bond, InhA covalently links an intermediate to an acyl carrier protein through its carbonyl group. A number of anti-tubercular medications, such as isoniazid, target InhA. (INH). The creation of novel therapeutic drugs continues to be interested in targeting InhA [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Isoniazid (INH)- a first-line anti-TB drug- targets inhA through its activated metabolites. Despite its activity, INH shows high toxicity and side effects due to its nitrogen-centered free radicals (hydrazine metabolites) that can generate highly lipid peroxidase-causing causing-oxygen species with consequent cell death and hepatic necrosis. There are numerous kinds of InhA inhibitors, including diphenyl ether [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], pyrrolidine carboxamide [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], hydroxyl pyridines [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], tetrahydropyran [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and 1,3,4-oxadiazole derivatives [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRecently, thiazolidin-4-one-thiazole hybrids showed potent anti-tubercular activity comparable to INH [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Phthalimide is considered a scaffold for anti-tubercular activity [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Compounds \u003cb\u003eI\u003c/b\u003e and \u003cb\u003eII\u003c/b\u003e demonstrated antitubercular action against sensitive \u003cem\u003eM. TB\u003c/em\u003e strains with MICs in the micromolar range and might interact with the mycobacterial InhA active site [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Pyrimidine is considered a core scaffold with anti-tubercular activity as well [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, many pyrimidine-containing compounds are being investigated in clinical trials (GSK286, TBA7371). Ceritinib is a pyrimidine core with confirmed anti-mycobacterial activity. Iclaprim and trimethoprim are examples of antibacterial drugs that are being considered for the treatment of mycobacterial infections [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eArylamides constitute a general class of InhA inhibitors with features that accommodate the structural requirements for interaction at InhA binding site, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Arylamides have a broad range of anti-tubercular activity, with MIC in micro- to nonamolar range. Structurally, polyamides are composed of ring \u003cb\u003eA\u003c/b\u003e with the amide linker, which plays a role in the interaction at the catalytic site, forming an \u003cem\u003eH\u003c/em\u003e-bond interaction at Tyr158, and other amino acids together with an \u003cem\u003eH\u003c/em\u003e-bond interaction with the 2'OH of the NAD cofactor at InhA catalytic site (site I). Ring \u003cb\u003eB\u003c/b\u003e plays a role in the hydrophobic interaction at the hydrophobic site of the enzyme (Site II), while ring \u003cb\u003eC\u003c/b\u003e is important for interaction at the site exposed (site III), being surrounded by polar and non-polar groups at the active site of the enzyme [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In the present investigation, structural modification of arylamide InhA inhibitors will be performed to provide phthalimide \u0026ndash;pyrimidine hybrids where phthalimide will take place of ring \u003cb\u003eA\u003c/b\u003e together with amide interaction via \u003cem\u003eH\u003c/em\u003e-bonding at the catalytic site (Site I), and pyrimidine directly attached to phthalimide will take place of ring \u003cb\u003eC\u003c/b\u003e to maintain the proper distances. Ring \u003cb\u003eC\u003c/b\u003e will accommodate small groups at position 2, such as a non-polar chlorine group, or at position 6, such as a polar amino group, to allow for interaction at the solvent-exposed site (site II). Different substituted benzene rings will take place in ring \u003cb\u003eB\u003c/b\u003e to interact at the hydrophobic site (Site II). They will be introduced at the pyrimidine core either at position 6 through the methylene amino linker or at position 2 through the methylenehydrazinyl linker to afford 2-(2-chloro-6-((3-alkylbenzylidene)amino)pyrimidin-4-yl)isoindoline-1,3-dione (\u003cb\u003e6a-f\u003c/b\u003e) and 2-(6-amino-2-(2-(arylidene)hydrazineyl)pyrimidin-4-yl)isoindoline-1,3-dione (\u003cb\u003e8a-f\u003c/b\u003e), respectively, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The designed compounds will be screened for their anti-tubercular activity against sensitive, multi-drug-resistant (MDR), and extra-drug-resistant (XDR) \u003cem\u003eM. tuberculosis\u003c/em\u003e strains compared to INH. Compounds will also be screened for their \u003cem\u003ein-vitro\u003c/em\u003e inhibition of mycobacterial InhA enzyme compared to IHN. Molecular docking simulation will be used to study the binding mode of synthesized compounds at the binding site of InhA enzyme.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003e2.1. Chemistry:\u003c/h2\u003e\n\u003cp\u003eAll melting points (\u0026deg;C) were measured on Stuart melting point apparatus (SMP 30) and are uncorrected. All the synthesized compounds were monitored by TLC using precoated plastic sheets silica gel (Merck 60 F254) and spots were visualized by irradiation with UV light (254 nm). The used solvent system was chloroform: methanol (9:1) \u0026amp; ethyl acetate: methanol (10:1). IR spectra were recorded on Pye-Uniearn using KEr wafer technique and Beckman spectrophotometers in Zagazig. \u003csup\u003e1\u003c/sup\u003eH-NMR spectra were recorded on Bruker 400 MHz Spectrometer and \u003csup\u003e13\u003c/sup\u003eC-NMR spectra were run at 125 MHz in dimethylsulfoxide (DMSO-d6) and TMS as an internal standard, at applied Nucleic Acid Research Center, Zagazig University, Egypt. Mass spectra were recorded on Direct Inlet part to mass analyzer in Thermo Scientific GCMS model ISQ at the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Nasr City, Cairo. Microanalysis was carried out in microanalytical center, Mansoura University and results were within \u0026plusmn;\u0026thinsp;0.4% from the theoretical values. All the chemicals and reagents used were purchased from Aldrich Chemicals Co, USA, and commercial sources.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e6-Aminouracil (l):\u003c/h3\u003e\n\u003cp\u003eCompound \u003cstrong\u003e1\u003c/strong\u003e was commercially available from Aldrich Chemical Company Inc. and was also synthesised in our lab. As described procedure, ethyl cyanoacetate and urea were heated under reflux in sodium ethoxide and ethanol [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003e4-Amino-2,6-dichloropyrimidine (2):\u003c/h3\u003e\n\u003cp\u003eCompound \u003cstrong\u003e2\u003c/strong\u003e was available commercially from Aldrich Chemical Company Inc. and was also synthesised in our laboratory by refluxing 6-aminouracil (\u003cstrong\u003e1\u003c/strong\u003e) with phosphorous oxychloride, as a published method [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003e2-(6-amino-2-chloropyrimidin-4-yl)isoindoline-1,3-dione (4):\u003c/h3\u003e\n\u003cp\u003eA mixture of compound (\u003cstrong\u003e2\u003c/strong\u003e) (0.01 mol) and phthalimide [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e] (\u003cstrong\u003e3\u003c/strong\u003e) (0.01 mol) in absolute ethanol (35 mL) in the presence of few drops of piperidine was refluxed for 6h. Once the reaction was finished (TLC), the reaction mixture was cooled and poured onto ice-cold water (50 ml). The resulting solid was filtrated, dried, and recrystallized from methanol. Color: yellow; yield: 58.3%; m.p.= 117\u0026ndash;120 \u003csup\u003eo\u003c/sup\u003eC; IR (KBr) \u0026upsilon;\u003csub\u003emax\u003c/sub\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e: 3465, 3211 (NH\u003csub\u003e2\u003c/sub\u003e), 3102 (CH aromatic), 1716 (C\u0026thinsp;=\u0026thinsp;O); \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 5.51 (s, 1H, CH), 6.39 (s, \u003cem\u003e2\u003c/em\u003eH, NH\u003csub\u003e2\u003c/sub\u003e, exchangeable by D\u003csub\u003e2\u003c/sub\u003eO), 7.82 (s, 4H, Ar-H); \u003csup\u003e13\u003c/sup\u003eC-NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 74.08, 122.94, 132.65, 134.34, 151.02, 155.21, 164.26, 171.45; MS: m/z (intensity 100%): 276 (2) [M\u003csup\u003e⁺\u003c/sup\u003e+2], 274 (14) [M\u003csup\u003e+\u003c/sup\u003e], 239 (17), 213 (21), 197 (13), 185 (100); Anal. calcd. for C\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e7\u003c/sub\u003eClN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e: C, 52.48; H, 2.57; N, 20.40. Found: C, 52.58; H, 2.59; N, 20.45.\u003c/p\u003e\n\u003ch3\u003e2-(2-chloro-6-((3-alkylbenzylidene)amino)pyrimidin-4-yl)isoindoline-1,3-dione (6a-f):\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompound (\u003cstrong\u003e4)\u003c/strong\u003e (0.01 mol) and the appropriate aromatic aldehyde (0.01 mol) were combined with 50 mL of abs. ethanol and two drops of triethylamine (Et\u003csub\u003e3\u003c/sub\u003eN), and the mixture was stirred for 6 to 8h at room temperature. When the reaction (TLC) was complete, the mixture was cooled to room temperature. The solid product was filtered, dried, and then recrystallized from ethanol.\u003c/p\u003e\n\u003ch3\u003e2-(2-chloro-6-((3-nitrobenzylidene)amino)pyrimidin-4-yl)isoindoline-1,3-dione (6a):\u003c/h3\u003e\n\u003cp\u003eStirred time: 8h; color: light yellow; yield: 75%; m.p.= 200\u0026ndash;202 \u003csup\u003eo\u003c/sup\u003eC; IR (KBr) \u0026upsilon;\u003csub\u003emax\u003c/sub\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e: 3079 (CH aromatic), 2922 (CH aliphatic), 1705 (C\u0026thinsp;=\u0026thinsp;O), 1635 (C\u0026thinsp;=\u0026thinsp;N), 1401, 1337 (NO\u003csub\u003e2\u003c/sub\u003e); \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO- \u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm) 7.60\u0026ndash;7.63 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 1H, Ar-H), 7.82 (s, 4H, Ar- H\u003csub\u003ephthalimide\u003c/sub\u003e), 7.86 (s, 1H, CH), 8.01\u0026ndash;8.03 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 1H, Ar-H), 8.32\u0026ndash;8.33 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 1H, Ar-H), 8.51\u0026ndash;8.53 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 1H, Ar-H), 8.68 (s, 1H, CH\u003csub\u003eazomethine\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC-NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 99.13, 122.94, 128.55, 130.96, 132.61, 134.33, 137.03, 140.95, 142.62, 147.82, 158.08, 163.74, 169.25, 172.83, 191.83; MS: m/z (intensity %): 409 (24) [M\u003csup\u003e⁺\u003c/sup\u003e+2], 407 (36) [M\u003csup\u003e+\u003c/sup\u003e], 373 (28), 353 (99), 322 (22), 246 (25), 173 (23), 147 (84), 107 (11), 79 (19), 43 (100); Anal. calcd. for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eClN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: C, 55.97; H, 2.47; N, 17.18. Found: C, 55.99; H, 2.50, N, 17.25.\u003c/p\u003e\n\u003ch3\u003e2-(2-chloro-6-((4-nitrobenzylidene)amino)pyrimidin-4-yl)isoindoline-1,3-dione (6b):\u003c/h3\u003e\n\u003cp\u003eStirred time: 6h; color: light yellow; yield: 85.4%; m.p.= 180\u0026ndash;182 \u003csup\u003eo\u003c/sup\u003eC; IR (KBr) \u0026upsilon;\u003csub\u003emax\u003c/sub\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e: 3078 (CH aromatic), 2869 (CH aliphatic), 1679 (C\u0026thinsp;=\u0026thinsp;O), 1624 (C\u0026thinsp;=\u0026thinsp;N), 1513, 1345 (NO\u003csub\u003e2\u003c/sub\u003e); \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 5.45 (s, 1H, CH\u003csub\u003epyrimidine\u003c/sub\u003e), 7.51\u0026ndash;7.53 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, Ar-H), 7.82 (s, 4H, Ar- H\u003csub\u003ephthalimide\u003c/sub\u003e), 8.15\u0026ndash;8.17 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, Ar-H), 8.22\u0026ndash;8.24 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, Ar-H), 8.40\u0026ndash;8.43 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, Ar-H), 9.46 (s, 1H, CH\u003csub\u003eazomethine\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC-NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e,\u003c/sub\u003e \u0026delta;, ppm): 91.14, 122.95, 124.29, 130.66, 132.62, 134.34, 142.84, 147.82, 155.08, 164.73, 168.26, 169.26, 192.36; MS: m/z (intensity %): 409 (17) [M\u003csup\u003e⁺\u003c/sup\u003e+2], 407 (28) [M\u003csup\u003e⁺\u003c/sup\u003e], 372 (41), 325 (7), 297 (14), 280 (100), 259 (98), 117 (96); Anal. calcd. for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eClN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: C, 55.97; H, 2.47; N, 17.18. Found: C, 55.98; H, 2.48; N, 17.23.\u003c/p\u003e\n\u003ch3\u003e2-(2-chloro-6-((4-chlorobenzylidene)amino)pyrimidin-4-yl)isoindoline-1,3-dione (6c):\u003c/h3\u003e\n\u003cp\u003eStirred time: 6h; color: yellow; yield: 63.3%; m.p.= 175\u0026ndash;177 \u003csup\u003eo\u003c/sup\u003eC; IR (KBr) \u0026upsilon;\u003csub\u003emax\u003c/sub\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e: 3090 (CH aromatic), 2849 (CH aliphatic), 1752, 1704 ( 2 C\u0026thinsp;=\u0026thinsp;O), 1676 ( C\u0026thinsp;=\u0026thinsp;N\u003csub\u003eazomethine\u003c/sub\u003e) 1631 (C\u0026thinsp;=\u0026thinsp;N); \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO- \u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 5.23 (s, 1H, CH\u003csub\u003epyrimidine\u003c/sub\u003e), 7.20\u0026ndash;7.22 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, Ar-H), 7.41\u0026ndash;7.43 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, Ar-H), 7.51\u0026ndash;7.53 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, Ar-H), 7.82 (s, 4H, Ar-H\u003csub\u003ephthalimide\u003c/sub\u003e), 8.06\u0026ndash;8.09 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H, Ar-H), 8.24 (s, 1H, CH\u003csub\u003eazomethine\u003c/sub\u003e); \u003csup\u003e13\u003c/sup\u003eC-NMR (125 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e, \u0026delta;, ppm): 99.47, 122.98, 128.14, 131.62, 132.64, 134.37, 134.73, 136.79, 153.06, 161.64, 169.30, 169.53, 192.18; MS: m/z (intensity %): 400 (9) [M⁺+4], 398 (9) [M⁺+2], 396 (27) [M\u003csup\u003e⁺\u003c/sup\u003e], 363 (25), 324 (48), 296 (8), 280 (21), 249 (100); Anal. calcd. for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e: C, 57.45; H, 2.54; N, 14.11. Found: C, 57.50; H, 2.56; N, 14.18.\u003c/p\u003e\n\u003ch3\u003e2-(2-chloro-6-((3,4-dimethoxybenzylidene)amino)pyrimidin-4-yl)isoindoline-1,3-dione (6d):\u003c/h3\u003e\n\u003cp\u003eStirred time: 8h; color: yellow; yield: 92%; m.p.= 208\u0026ndash;210 \u003csup\u003eo\u003c/sup\u003eC; IR (KBr) \u0026upsilon;\u003csub\u003emax\u003c/sub\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e: 3061 (CH aromatic), 2924 (CH aliphatic), 1709 (C\u0026thinsp;=\u0026thinsp;O), 1631 (C\u0026thinsp;=\u0026thinsp;N); \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 3.16 (s, 6H, 2OCH\u003csub\u003e3\u003c/sub\u003e), 5.43 (s, 1H, CH\u003csub\u003epyrimidine\u003c/sub\u003e), 7.12\u0026ndash;7.14 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 1H, Ar-H), 7.52\u0026ndash;7.54 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 2H, Ar-H), 7.81 (s, 4H, Ar-H\u003csub\u003ephthalimide\u003c/sub\u003e), 9.29 (s, 1H, CH\u003csub\u003eazomethine\u003c/sub\u003e); MS: m/z (intensity %): 424 (19) [M\u003csup\u003e⁺\u003c/sup\u003e+2], 422 (19) [M\u003csup\u003e⁺\u003c/sup\u003e], 387 (43), 243 (86), 224 (100); Anal. calcd. for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eClN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: C, 59.65; H, 3.58; N, 13.25. Found: C, 59.70; H, 3.60; N, 13.30.\u003c/p\u003e\n\u003ch3\u003e2-(2-chloro-6-((2-hydroxybenzylidene)amino)pyrimidin-4-yl)isoindoline-1,3-dione (6e):\u003c/h3\u003e\n\u003cp\u003eStirred time: 6h; color: yellow; yield: 86.8%; m.p.= 188\u0026ndash;191 \u003csup\u003eo\u003c/sup\u003eC; IR (KBr) \u0026upsilon;\u003csub\u003emax\u003c/sub\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e: 3656 (OH), 3061 (CH aromatic), 2925 (CH aliphatic), 1710 (C\u0026thinsp;=\u0026thinsp;O), 1632 (C\u0026thinsp;=\u0026thinsp;N); \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 6.32 (s, 1H, CH\u003csub\u003epyrimidine\u003c/sub\u003e), 7.07\u0026ndash;7.09 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H, Ar-H), 7.21\u0026ndash;7.24 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H, Ar-H), 7.29\u0026ndash;7.33 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H, Ar-H), 7.53\u0026ndash;7.55 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H, Ar-H), 7.81 (s, 4H, Ar-H), 9.25 (s, 1H, CH\u003csub\u003eazomethine\u003c/sub\u003e), 11.32 (s, 1H, OH); \u003csup\u003e13\u003c/sup\u003eC-NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 99.05, 119.47, 122.95, 123.31, 125.96, 130.56, 132.63, 133.86, 134.34, 156.18, 162.84, 168.47, 169.27, 191.83; MS: m/z (intensity %): 380 (40) [M\u003csup\u003e⁺\u003c/sup\u003e+2], 378 (29) [M\u003csup\u003e⁺\u003c/sup\u003e], 306 (49), 174 (49), 146 (30), 121(100); Anal. calcd. for C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e11\u003c/sub\u003eClN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e: C, 60.25; H, 2.93; N, 14.79. Found: C, 60.30; H, 2.95; N, 14.89.\u003c/p\u003e\n\u003ch3\u003e2-(2-chloro-6-((4-hydroxybenzylidene)amino)pyrimidin-4-yl)isoindoline-1,3-dione (6f):\u003c/h3\u003e\n\u003cp\u003eStirred time: 6h; color: yellow; yield: 82.6%; m.p.= 198\u0026ndash;200 \u003csup\u003eo\u003c/sup\u003eC; IR (KBr) \u0026upsilon;\u003csub\u003emax\u003c/sub\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e: 3631 (OH), 3063 (CH aromatic), 2991 (CH aliphatic), 1710 (C\u0026thinsp;=\u0026thinsp;O), 1633 (C\u0026thinsp;=\u0026thinsp;N); \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 6.33 (s, 1H, CH\u003csub\u003epyrimidine\u003c/sub\u003e), 7.07\u0026ndash;7.09 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 2H, Ar-H), 7.39\u0026ndash;7.41 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 2H, Ar-H), 7.82 (s, 4H, Ar-H), 9.89 (s, 1H, CH\u003csub\u003eazomethine\u003c/sub\u003e), 11.32 (s, 1H, OH); \u003csup\u003e13\u003c/sup\u003eC-NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 99.22, 119.24, 122.94, 126.94, 129.64, 132.61, 134.33, 158.08, 166.30, 166.53, 169.24, 175.24, 192.18; MS: m/z (intensity %): 380 (24) [M\u003csup\u003e+\u003c/sup\u003e+2], 378 (18) [M\u003csup\u003e+\u003c/sup\u003e], 248 (33), 173 (52), 117 (3), 90 (100); Anal. caled. For C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e11\u003c/sub\u003eClN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e: C, 60.25; H, 2.93; N, 14.79. Found: C, 60.28; H, 2.98; N, 14.84.\u003c/p\u003e\n\u003ch3\u003e2-(6-amino-2-hydrazineylpyrimidin-4-yl)isoindoline-1,3-dione (7):\u003c/h3\u003e\n\u003cp\u003eCompound \u003cstrong\u003e4\u003c/strong\u003e (0.01 mol) and hydrazine hydrate (0.03 mol) were combined with 20 mL of ethyl alcohol and two drops of triethylamine (Et\u003csub\u003e3\u003c/sub\u003eN), and the mixture was stirred for 9h at room temperature. The mixture was refrigerated once the reaction (TLC) was completed. The solid product was filtrated, dried, and recrystallized from ethanol; color: yellowish-white; yield: 81%; m.p.= 290\u0026ndash;292 \u003csup\u003eo\u003c/sup\u003eC; IR (KBr) \u0026upsilon;\u003csub\u003emax\u003c/sub\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e: 3320, 3290, 3165 (NH\u003csub\u003e2\u003c/sub\u003e\u0026amp; NH), 3019 (CH aromatic), 1661 (C\u0026thinsp;=\u0026thinsp;O); \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 5.49 (s, 1H, CH\u003csub\u003epyrimidine\u003c/sub\u003e), 7.84\u0026ndash;7.86 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 2H, Ar-H), 7.88 (s, 2H, NH\u003csub\u003e2\u003c/sub\u003e), 8.03\u0026ndash;8.07 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 2H, Ar-H), 8.08 (s, 2H, NH\u003csub\u003e2 pyrimidine\u003c/sub\u003e), 10.51 (bs, 1H, NH); \u003csup\u003e13\u003c/sup\u003eC-NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 77.07, 125.18, 127.23, 132.62, 154.71, 164.59, 166.83, 168.71; MS: m/z (intensity %): 271 (42) [M\u003csup\u003e⁺\u003c/sup\u003e], 255 (33), 237 (19), 199 (29), 184 (100); Anal. calcd. For C\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eN\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e: C, 53.33; H, 3.73; N, 31.10. Found: C, 53.36; H, 3.75; N, 31.17.\u003c/p\u003e\n\u003ch3\u003e2-(6-amino-2-(2-(arylidene)hydrazineyl)pyrimidin-4-yl)isoindoline-1,3-dione (8a-f):\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003eGeneral method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e7\u003c/strong\u003e (0.001 mol) and aromatic aldehyde (0.001 mol) were combined with 50 mL of ethanol and two drops of triethylamine (Et\u003csub\u003e3\u003c/sub\u003eN), and the mixture was stirred for 2-11h at room temperature. Following the completion of the reaction (TLC), the solid product that had been separated from the mixture was filtered out, dried, and then recrystallized from ethanol.\u003c/p\u003e\n\u003ch3\u003e2-(6-amino-2-(2-(3-nitrobenzylidene)hydrazineyl)pyrimidin-4-yl)isoindoline-1,3-dione (8a):\u003c/h3\u003e\n\u003cp\u003eStirred time: 6h; color: yellow; yield: 88.9%; m.p.= 140\u0026ndash;142 \u003csup\u003eo\u003c/sup\u003eC; IR (KBr) \u0026upsilon;\u003csub\u003emax\u003c/sub\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e: 3509, 3453, 3401 (NH\u003csub\u003e2\u003c/sub\u003e\u0026amp;NH), 3081 (CH aromatic), 2922 (CH aliphatic), 1661 (C\u0026thinsp;=\u0026thinsp;O), 1626 (C\u0026thinsp;=\u0026thinsp;N), 1528, 1351 (NO\u003csub\u003e2\u003c/sub\u003e); \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 4.13 (s, 1H, CH\u003csub\u003epyrimidine\u003c/sub\u003e), 7.22 (s, 2H, NH\u003csub\u003e2\u003c/sub\u003e), 7.81\u0026ndash;7.85 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 1H, Ar-H), 8.36\u0026ndash;8.39 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.4 Hz, 2H, Ar-H), 8.39\u0026ndash;8.41 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 1H, Ar-H), 8.72 (s, 1H, CH\u003csub\u003ehydrazone\u003c/sub\u003e), 8.93 (s, 4H, Ar-H), 11.50 (bs, 1H, NH); \u003csup\u003e13\u003c/sup\u003eC-NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 73.07, 118.78, 121.35, 122.67, 125.84, 130.02, 130.67, 131.05, 134.43, 143.14, 148.23, 160.49, 166.43, 167.80, 169.15; MS: m/z (intensity %): 404 (22) [M\u003csup\u003e⁺\u003c/sup\u003e], 388 (25), 242 (8), 165 (12), 163 (100); Anal. calcd. For C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eN\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: C, 56.58; H, 3.25; N, 24.31. Found: C, 56.60; H, 3.30; N, 24.40.\u003c/p\u003e\n\u003ch3\u003e2-(6-amino-2-(2-(4-nitrobenzylidene)hydrazineyl)pyrimidin-4-yl)isoindoline-1,3-dione (8b):\u003c/h3\u003e\n\u003cp\u003eStirred time: 3h; color: yellow; yield: 80%; m.p.= 160\u0026ndash;162 \u003csup\u003eo\u003c/sup\u003eC; IR (KBr) \u0026upsilon;\u003csub\u003emax\u003c/sub\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e: 3452, 3424 (NH\u003csub\u003e2\u003c/sub\u003e\u0026amp;NH), 2922 (CH aliphatic), 1661 (C\u0026thinsp;=\u0026thinsp;O), 1629 (C\u0026thinsp;=\u0026thinsp;N), 1522, 1345 (NO\u003csub\u003e2\u003c/sub\u003e); \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 4.02 (s, 1H, CH\u003csub\u003epyrimidine\u003c/sub\u003e), 7.55 (s, 2H, NH\u003csub\u003e2\u003c/sub\u003e), 7.66\u0026ndash;7.68 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 2H, Ar-H), 7.87 (s, 4H, Ar-H\u003csub\u003ephthalimide\u003c/sub\u003e), 8.07 (s, 1H, CH\u003csub\u003ehydrazone\u003c/sub\u003e), 8.15\u0026ndash;8.17 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 2H, Ar-H), 11.50 (s, 1H, NH); \u003csup\u003e13\u003c/sup\u003eC-NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 71.14, 123.99, 125.29, 128.69, 129.59, 131.58, 132.61, 134.12, 143.49, 145.62, 159.20, 166.29, 167.93, 169.58; MS: m/z (intensity %): 403 (59) [M\u003csup\u003e\u0026middot;⁺\u003c/sup\u003e], 387 (26), 341 (46), 265 (22), 119 (70), 42 (100); Anal. calcd. For C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eN\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e: C, 56.58; H, 3.25; N, 24.31. Found: C, 56.68; H, 3.29; N, 24.38.\u003c/p\u003e\n\u003ch3\u003e2-(6-amino-2-(2-(4-chlorobenzylidene)hydrazineyl)pyrimidin-4-yl)isoindoline-1,3-dione (8c):\u003c/h3\u003e\n\u003cp\u003eStirred time: 4h; color: white; yield: 88.9%; m.p.= 200\u0026ndash;202 \u003csup\u003eo\u003c/sup\u003eC; IR (KBr) \u0026upsilon;\u003csub\u003emax\u003c/sub\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e: 3456, 3165, 3125 (NH\u003csub\u003e2\u003c/sub\u003e \u0026amp; NH), 3019 (CH aromatic), 2918 (CH aliphatic), 1660 (C\u0026thinsp;=\u0026thinsp;O), 1623 (C\u0026thinsp;=\u0026thinsp;N); \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 4.33 (s, H, CH\u003csub\u003epyrimidine\u003c/sub\u003e), 7.35\u0026ndash;7.37 (d, \u003cem\u003eJ\u0026thinsp;=\u003c/em\u003e\u0026thinsp;8 Hz, 1H, Ar-H), 7.56 (s, 2H, NH\u003csub\u003e2\u003c/sub\u003e), 7.58\u0026ndash;7.88 (m, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 3H, Ar-H), 8.07 (s, 4H, Ar-H\u003csub\u003ephthalimide\u003c/sub\u003e), 8.71 (s, 1H, CH\u003csub\u003ehydrazone\u003c/sub\u003e), 11.47 (s, 1H, NH); \u003csup\u003e13\u003c/sup\u003eC-NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 76.07, 125.16, 127.17, 128.25, 129.11, 129.72, 130.05, 132.60, 136.05, 160.62, 164.64, 167.66, 170.08; MS: m/z (intensity 100%): 394 (5) [M\u003csup\u003e⁺\u003c/sup\u003e+2], 392 (12) [M\u003csup\u003e+\u003c/sup\u003e], 357 (15), 270 (6), 133 (13), 104 (100); Anal. calcd. For C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eClN\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e: C, 58.10; H, 3.34; N, 21.40. Found: C, 58.15; H, 3.36; N, 21.49.\u003c/p\u003e\n\u003ch3\u003e2-(6-amino-2-(2-(2-hydroxybenzylidene)hydrazineyl)-pyrimidin-4-yl)isoindoline-1,3-dione (8d):\u003c/h3\u003e\n\u003cp\u003eStirred time: 6h; color: yellow; yield: 95.5%; m.p.= 176\u0026ndash;178 \u003csup\u003eo\u003c/sup\u003eC; IR (KBr) \u0026upsilon;\u003csub\u003emax\u003c/sub\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e: 3655 (OH), 3450, 3447, 3368 (NH\u003csub\u003e2\u003c/sub\u003e \u0026amp; NH), 3042 (CH aromatic), 2920 (CH aliphatic), 1688 (C\u0026thinsp;=\u0026thinsp;O), 1623 (C\u0026thinsp;=\u0026thinsp;N); \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 3.16 (s, 1H, CH\u003csub\u003epyrimidine\u003c/sub\u003e), 6.95 (s, 2H, NH\u003csub\u003e2\u003c/sub\u003e), 6.96\u0026ndash;6.98 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 1H, Ar-H), 7.38\u0026ndash;7.42 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 2H, Ar-H), 7.67\u0026ndash;7.69 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 1H, Ar-H), 8.77 (s, 1H, CH\u003csub\u003ehydrazone\u003c/sub\u003e), 8.98 (s, 4H, Ar-H\u003csub\u003ephthalimide\u003c/sub\u003e), 11.16 (bs, 2H, NH \u0026amp; OH); \u003csup\u003e13\u003c/sup\u003eC-NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 72.83, 110.61, 116.99, 118.56, 120.16, 131.40, 131.86, 132.43, 132.64, 133.80, 154.23, 159.05, 163.34, 168.90, 174.42; MS: m/z (intensity %): 374 (19) [M\u003csup\u003e⁺\u003c/sup\u003e], 280 (26), 206 (93), 176 (18), 150 (100); Anal. calcd. For C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eN\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e: C, 60.96; H, 3.77; N, 22.45. Found: C, 60.98; H, 3.80; N, 22.50.\u003c/p\u003e\n\u003ch3\u003e2-(6-amino-2-(2-(4-hydroxybenzylidene)hydrazineyl)pyrimidin-4-yl) isoindoline-1,3-dione (8e)\u003c/h3\u003e\n\u003cp\u003eStirred for 2h; color: yellow; yield: 90.91%; m.p.= 210\u0026ndash;212 \u003csup\u003eo\u003c/sup\u003eC; IR (KBr) \u0026upsilon;\u003csub\u003emax\u003c/sub\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e: 3656 (OH), 3510, 3452, 3400 (NH\u003csub\u003e2\u003c/sub\u003e \u0026amp; NH), 3042 (CH aromatic), 2919 (CH aliphatic), 1688 (C\u0026thinsp;=\u0026thinsp;O), 1623 (C\u0026thinsp;=\u0026thinsp;N); \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 4.35 (s, 1H, CH\u003csub\u003epyrimidine\u003c/sub\u003e), 6.95 (s, 2H, NH\u003csub\u003e2\u003c/sub\u003e), 6.97\u0026ndash;6.99 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.2, 2H, Ar-H), 7.36\u0026ndash;7.38 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.2 Hz, 1H, Ar-H), 7.40 (s, 1H, CH\u003csub\u003ehydrazone\u003c/sub\u003e), 7.42\u0026ndash;7.44 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.2 Hz, 1H, Ar-H), 7.66\u0026ndash;7.68 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.2 Hz, 2H, Ar-H), 7.70\u0026ndash;7.72 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.2 Hz, 2H, Ar-H), 9.00 (s, 1H, OH), 11.13 (s, 1H, NH); \u003csup\u003e13\u003c/sup\u003eC-NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e,\u003c/sub\u003e \u0026delta;, ppm): 76.07, 116.57, 118.22, 119.65, 130.88, 133.28, 138.28, 140.10, 151.66, 153.66, 158.67, 162.83, 170.11; MS: m/z (intensity %): 374 (25) [M\u003csup\u003e⁺\u003c/sup\u003e], 372 (22), 348 (100), 320 (12); Anal. calcd. For C\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eN\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e: C, 60.96; H, 3.77; N, 22.45. Found: C, 60.99; H, 3.79; N, 22.50.\u003c/p\u003e\n\u003ch3\u003e2-(6-amino-2-(2-(4-(dimethylamino)benzylidene)hydrazineyl)pyrimidin-4-yl)isoindoline-1,3-dione (8f):\u003c/h3\u003e\n\u003cp\u003eStirred time: 11h; color: yellow; yield: 92.9%; m.p.= 242\u0026ndash;244 \u003csup\u003eo\u003c/sup\u003eC; IR (KBr) \u0026upsilon;\u003csub\u003emax\u003c/sub\u003e cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e: 3509 3452, 3167 (NH\u003csub\u003e2\u003c/sub\u003e \u0026amp; NH), 3018 (CH aromatic), 2918 (CH aliphatic), 1661 (C\u0026thinsp;=\u0026thinsp;O), 1621 (C\u0026thinsp;=\u0026thinsp;N); \u003csup\u003e1\u003c/sup\u003eH-NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e\u003c/sub\u003e, \u0026delta;, ppm): 2.99 (s, 6H, 2CH\u003csub\u003e3\u003c/sub\u003e), 4.79 (s, 1H, CH\u003csub\u003epyrimidine\u003c/sub\u003e ), 6.75\u0026ndash;6.77 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 2H, Ar-H), 7.33 (s, 2H, NH\u003csub\u003e2\u003c/sub\u003e), 7.63\u0026ndash;7.65 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 2H, Ar-H), 8.33 (s, 4H, Ar-H\u003csub\u003ephthalimide\u003c/sub\u003e), 8.49 (s, 1H, CH\u003csub\u003ehydrazone\u003c/sub\u003e), 10.81 (s, 1H, NH); \u003csup\u003e13\u003c/sup\u003eC-NMR (125 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e\u003cem\u003e6\u003c/em\u003e,\u003c/sub\u003e \u0026delta;, ppm): 48.97, 72.36, 112.15, 124.49, 126.45, 128.07, 130.07, 132.42, 141.13, 155.03, 160.44, 165.69, 169.18, 171.91; MS: m/z (intensity %): 401 (46) [M\u003csup\u003e⁺\u003c/sup\u003e], 252 (54), 245 (100); Anal. calcd. for C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eN\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e: C, 62.83; H, 4.77; N, 24.42. Found: C, 62.88; H, 4.79; N, 24.48.\u003c/p\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003e2.2. Biological activity:\u003c/h2\u003e\n\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n\u003ch2\u003e2.2.1. Anti-tubercular activity:\u003c/h2\u003e\n\u003cp\u003eThe American Type Culture Collection (ATCC) in the United States provided M. tuberculosis (ATCC 25177/H37Ra) as a drug-sensitive strain (DS) and \u003cem\u003eM. tuberculosis\u003c/em\u003e (ATCC 35822) as a multidrug-resistant strain (MDR). Apart from the moxifloxacin-resistant M. tuberculosis (RCMB 2674), pyrazinamide, ethionamide, ethambutol, isoniazid, and rifampicin-resistant strains, an extensively drug-resistant strain (XDR) was obtained from the Culture Collection Unit of the Regional Center for Mycology and Biotechnology (RCMB). Dubos medium mixed with 50 mM sodium nitrate was used to cultivate all strains of \u003cem\u003eM. tuberculosis\u003c/em\u003e. The cultures were cultivated aerobically at 37\u0026deg;C and 150 rpm until they reached log-phase optical density (OD595\u0026thinsp;=\u0026thinsp;1). Mycobacteria develop as aggregated clumps; thus, they were subjected to ultrasound irradiation and sonication for two minutes using a water bath (Ultrasonic, Freeport, IL, USA). Microplate Alamar blue test was used to assess the minimum inhibitory concentrations (MICs) of drugs against DS, MDR, and XDR M. tuberculosis strains (MABA) [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. Rifampicin and isoniazid were used as positive controls. The chemical stock solutions and final testing concentrations ranged from 1000 to 0.003 \u0026micro;g/mL. In Difco Middlebrook 7H9 Broth (Seebio) supplemented with 0.2% (vol/vol) glycerol, 0.05% Tween 80, and 10% (vol/vol) albumin-dextrose-catalase, M. tuberculosis was raised to late log phase (70 to 100 Klett units) (7H9-ADC-TG). Compounds were produced as two-fold dilutions in 100 \u0026micro;L volumes in 7H9-ADC-TG clear-bottom microplates (BD). A final testing volume of 200 \u0026micro;L was obtained by adding \u003cem\u003eM. tuberculosis\u003c/em\u003e (100 \u0026micro;L containing 2 \u0026times; 105 CFU). The plates were incubated at 37\u0026deg;C, and on the seventh day, each well received the addition of 20 \u0026micro;L of Alamar blue and 12.5 \u0026micro;L of 20% Tween 80. The fluorescence was measured at an excitation of 530 nm and an emission of 590 nm during an incubation period of 16 to 24h at 37\u0026deg;C. The minimum inhibitory concentration (MIC) was identified as the concentration at which a 90% decrease in fluorescence was seen in comparison to duplicate bacterium-only controls.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\n\u003ch2\u003e\u003cem\u003e2.2.2. In-vitro Mycobacterium tuberculosis\u003c/em\u003e InhA enzyme inhibition activity:\u003c/h2\u003e\n\u003cp\u003eEnzymatic Assay. InhA activity was followed by a colorimetric assay that measured the oxidation of NADH at 340 nm in the presence of 2-trans-octanoyl-CoA in a buffer that contained 30 mM PIPES, pH 7.5, 50 mM NaCl, 0.1 mM EDTA, and 100 nM InhA. This was preincubated for 10 min at room temperature with 0.25 mM NADH and varying concentrations of the compounds with 1% (v/v) DMSO in a 150 \u0026micro;L reaction volume. The reaction was started by the addition of 2-trans-octanoyl-CoA at a final concentration of 1.5 mM. The reactions were followed for 20 min using a plate reader (CLARIOstar, BMG LABTECH) [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n\u003ch2\u003e2.3. Molecular docking simulation:\u003c/h2\u003e\n\u003cp\u003eDocking was done according to the literature [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e]. The crystallographic structure of the InhA enzyme (PDB ID: 3FNE) was acquired from PDB and prepared for molecular docking by removing ligands, introducing hydrogens, and decreasing energy with MOE 2009. The structure with the lowest energy was then employed as a docking receptor. MOE's site finder method was utilised to identify InhA's catalytic site. Chem Bio Office was used to create the two-dimensional structures of the synthesised compounds, which were subsequently built from fragment libraries in MOE 2009 and reduced energy with the MMFF94x force field in MOE. To discover and analyze the interaction between ligands and the catalytic site of InhA, docking was done with chosen parameters (rescoring function 1 and rescoring function 2: London).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\n \u003ch2\u003e4.1. Chemistry:\u003c/h2\u003e\n \u003cp\u003eIn continuation of our research interest concerning the synthesis of nitrogen heterocycles and their potential application as anti-cancer agents [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e–\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e] the novelly designed phthalimide-pyrimidine hybrids \u003cstrong\u003e6a-f\u003c/strong\u003e and \u003cstrong\u003e8a-f\u003c/strong\u003e were prepared from 2-(6-amino-2-chloropyrimidin-4-yl)isoindoline-1,3-dione (\u003cstrong\u003e4\u003c/strong\u003e) as precursor. The pyrimidinylisoindolinedione \u003cstrong\u003e4\u003c/strong\u003e was obtained starting from the readily available 6-aminouracil (\u003cstrong\u003e1)\u003c/strong\u003e, according to our recent study [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e] Initially, chlorination of \u003cstrong\u003e1\u003c/strong\u003e with phosphorus oxychloride (POCl\u003csub\u003e3\u003c/sub\u003e) yielded 6-amino-2,4-dichlorouracil (\u003cstrong\u003e2\u003c/strong\u003e), which on refluxing in piperidine as basic medium with phthalimide (\u003cstrong\u003e3\u003c/strong\u003e) produced \u003cstrong\u003e4\u003c/strong\u003e, as shown in Scheme \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Compound \u003cstrong\u003e4\u003c/strong\u003e was proven by \u003csup\u003e1\u003c/sup\u003eH-NMR spectrum, which revealed singlet signals at δ 5.51 and 6.39 ppm, assigned for CH-5 and NH\u003csub\u003e2\u003c/sub\u003e-6 of pyrimidine ring, respectively.\u003c/p\u003e\n \u003cp\u003eThe base-mediated condensation of aminopyrimidine derivative \u003cstrong\u003e4\u003c/strong\u003e with different aromatic aldehydes \u003cstrong\u003e5\u003c/strong\u003e (namely, 3-, 4-nitro-, 4-chloro-, 3,4-dimethoxy-, 2-, and 4-hydroxybenzaldehydes) in ethanol at room temperature for 6-8h, led to formation of the corresponding Schiff bases \u003cstrong\u003e6a-f\u003c/strong\u003e in good to excellent yields, as shown in Scheme \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. The chemical structure of \u003cstrong\u003e6a-f\u003c/strong\u003e was confirmed by IR, \u003csup\u003e1\u003c/sup\u003eH-NMR, \u003csup\u003e13\u003c/sup\u003eC-NMR, and mass spectroscopy. IR spectra of \u003cstrong\u003e6a-f\u003c/strong\u003e showed no stretching frequencies for NH\u003csub\u003e2\u003c/sub\u003e, while it showed absorption bands at 2849–2991 cm\u003csup\u003e− 1\u003c/sup\u003e assigned for N = \u003cspan class=\"Underline\"\u003eCH aliphatic.\u003c/span\u003e In addition, \u003csup\u003e1\u003c/sup\u003eH-NMR spectra (DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) of \u003cstrong\u003e6a-f\u003c/strong\u003e showed no signals for NH\u003csub\u003e2\u003c/sub\u003e but exhibited singlet signal at δ 8.24–9.89 ppm characteristic for azomethine proton N = C\u003cspan class=\"Underline\"\u003eH\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eOn the other hand, chloropyrimidine derivative \u003cstrong\u003e4\u003c/strong\u003e undergoes a nucleophilic substitution reaction by hydrazine hydrate in ethyl alcohol in the presence of triethylamine (few drops) at room temperature for 9h, giving 2-hydrazino derivative \u003cstrong\u003e7\u003c/strong\u003e in good yield, as shown in Scheme \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. IR spectrum of \u003cstrong\u003e7\u003c/strong\u003e showed three absorption peaks at 3320, 3290, 3165 cm\u003csup\u003e− 1\u003c/sup\u003e for (NH\u003csub\u003e2\u003c/sub\u003e \u0026amp; NH). The chemical structure of \u003cstrong\u003e7\u003c/strong\u003e was further confirmed by \u003csup\u003e1\u003c/sup\u003eH NMR spectroscopy, which showed singlet signal at δ 5.49 characterized for -CH for pyrimidine ring, two singlets at 7.88 \u0026amp; 8.08 for 2 NH\u003csub\u003e2\u003c/sub\u003e, and broad signal at 10.51 ppm for NH proton.\u003c/p\u003e\n \u003cp\u003eFinally, the base-catalyzed condensation of the 2-hydrazinylpyrimidine \u003cstrong\u003e7\u003c/strong\u003e with aromatic aldehydes in ethanol at room temperature afforded the respective hydrazones \u003cstrong\u003e8a-f\u003c/strong\u003e in good to excellent yield, as shown in Scheme \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. IR spectra of \u003cstrong\u003e8a-f\u003c/strong\u003e showed absorption bands at 2918–2922 cm\u003csup\u003e− 1\u003c/sup\u003e assigned for C-H aliphatic, with the other expected absorption peaks for NH\u003csub\u003e2\u003c/sub\u003e, NH, and imide carbonyl. In addition, \u003csup\u003e1\u003c/sup\u003eH NMR spectra of \u003cstrong\u003e8a-f\u003c/strong\u003e showed singlet signal at δ 7.40–8.77 ppm assigned for CH hydrazone proton, while no hydrazinylamino proton signal was observed.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cstrong\u003e3.2\u003c/strong\u003e. \u003cstrong\u003eBiological evaluation\u003c/strong\u003e\u003c/h2\u003e\n \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e\n \u003ch2\u003e\u003cstrong\u003e3.2.1\u003c/strong\u003e. \u003cstrong\u003eAnti-tubercular activity\u003c/strong\u003e:\u003c/h2\u003e\n \u003cp\u003eAll newly synthesized compounds were tested for anti-tubercular activity against \u003cem\u003eM. tuberculosis\u003c/em\u003e (ATCC 25177 H37Ra), a drug-sensitive strain, using the microplate Alamar blue assay (MABA) and INH as the reference drug. \u003cstrong\u003eTable\u0026nbsp;(1)\u003c/strong\u003e displays the anti-mycobacterial activity of compounds \u003cstrong\u003e6a-f and 8a-f\u003c/strong\u003e given as a minimum inhibitory concentration (MIC). Compounds showed a wide range of activity, where compounds \u003cstrong\u003e8d\u003c/strong\u003e and \u003cstrong\u003e8e\u003c/strong\u003e displayed mild anti-mycobacterial activity against sensitive strains with MIC 125 µg/ml. Compounds \u003cstrong\u003e6c\u003c/strong\u003e,\u003cstrong\u003e6d\u003c/strong\u003e, \u003cstrong\u003e8b\u003c/strong\u003e and \u003cstrong\u003e8c\u003c/strong\u003e showed moderate activity with MIC range of 15.63–62.5 µg/ml. Compounds \u003cstrong\u003e6b\u003c/strong\u003e, \u003cstrong\u003e6e\u003c/strong\u003e, \u003cstrong\u003e6f\u003c/strong\u003e, and \u003cstrong\u003e8a\u003c/strong\u003e were found to be potent anti-tuberculars with MIC range of 3.9–7.81 µg/ml. The highest activity against sensitive TB strains was shown by compounds \u003cstrong\u003e8f\u003c/strong\u003e (MIC = 0.48 µg/ml) and \u003cstrong\u003e6a\u003c/strong\u003e (MI = 0.98 µg/ml).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec30\" class=\"Section3\"\u003e\n \u003ch2\u003e\u003cstrong\u003e3.2.2\u003c/strong\u003e. \u003cstrong\u003eAnti-tubercular activity towards isoniazid, cycloserine, kanamycin, and rifampin-resistant\u003c/strong\u003e \u003cstrong\u003eM. tuberculosis\u003c/strong\u003e \u003cstrong\u003e(ATCC 35822)\u003c/strong\u003e:\u003c/h2\u003e\n \u003cp\u003eCompounds \u003cstrong\u003e6a-f\u003c/strong\u003e and \u003cstrong\u003e8a-f\u003c/strong\u003e were further tested for anti-tubercular activity against the multi-drug resistance strain (MDR) \u003cem\u003eM. tuberculosis\u003c/em\u003e ATCC 35822 -isoniazid, cycloserine, kanamycin, and rifampin-resistant strain using the microplate Alamar blue assay. \u003cstrong\u003eTable\u0026nbsp;1\u003c/strong\u003e displays the anti-mycobacterial activity of compounds \u003cstrong\u003e6a-f\u003c/strong\u003e and \u003cstrong\u003e8a-f\u003c/strong\u003e represented as a minimum inhibitory concentration. Compounds demonstrated a variety of activity from no activity to high potency where compounds \u003cstrong\u003e6c\u003c/strong\u003e, \u003cstrong\u003e8c\u003c/strong\u003e, \u003cstrong\u003e8d\u003c/strong\u003e and \u003cstrong\u003e8e\u003c/strong\u003e were found to be inactive, compounds \u003cstrong\u003e6b\u003c/strong\u003e, \u003cstrong\u003e8a\u003c/strong\u003e and \u003cstrong\u003e8b\u003c/strong\u003e showed mild activity with MIC range of 31.25–62.5 µg/ml while compounds \u003cstrong\u003e6a\u003c/strong\u003e, \u003cstrong\u003e6e\u003c/strong\u003e and \u003cstrong\u003e6f\u003c/strong\u003e were found to be potent with MIC of 7.81 µg/ml. Compound \u003cstrong\u003e8f\u003c/strong\u003e showed the highest anti-mycobacterial activity against MDR strain with MIC of 1.95 µg/ml.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec31\" class=\"Section3\"\u003e\n \u003ch2\u003e\u003cstrong\u003e3.2.3. Anti-tubercular activity towards isoniazid, rifampicin, ethambutol, pyrazinamide, ethionamide, and moxifloxacin -resistant\u003c/strong\u003e \u003cstrong\u003eM. tuberculosis\u003c/strong\u003e \u003cstrong\u003e(RCMB 2674)\u003c/strong\u003e:\u003c/h2\u003e\n \u003cp\u003eThe anti-tubercular activity of substances was tested against \u003cem\u003eM. tuberculosis\u003c/em\u003e (RCMB 2674), an extensively drug-resistant strain (XDR), using a microplate Alamar blue assay with isoniazid, rifampicin, ethambutol, pyrazinamide, ethionamide, and moxifloxacin. \u003cstrong\u003eTable\u0026nbsp;(1)\u003c/strong\u003e shows the anti-mycobacterial activity of compounds \u003cstrong\u003e6a-f and 8a-f\u003c/strong\u003e expressed as a minimum inhibitory concentration. Compounds \u003cstrong\u003e6b\u003c/strong\u003e,\u003cstrong\u003e6c\u003c/strong\u003e, \u003cstrong\u003e6d\u003c/strong\u003e,\u003cstrong\u003e8a\u003c/strong\u003e,\u003cstrong\u003e8c\u003c/strong\u003e,\u003cstrong\u003e8d\u003c/strong\u003e and \u003cstrong\u003e8e\u003c/strong\u003e were found to be inactive while compounds \u003cstrong\u003e6a\u003c/strong\u003e,\u003cstrong\u003e6e\u003c/strong\u003e, \u003cstrong\u003e6f\u003c/strong\u003e,\u003cstrong\u003e8b\u003c/strong\u003e showed moderate activity with MIC range of 15.63–31.25 µg/ml compound \u003cstrong\u003e8f\u003c/strong\u003e was a potent anti-tubercular against XDR strain with MIC of 7.81 µg/ml.\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;(1) Anti-tubercular activity of 6a-f and 8a-f\u003c/strong\u003e:\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Taba\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCompds\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSensitive\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eM. tuberculosis\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eMIC(µg/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMDR\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eM. tuberculosis\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eMIC(µg/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eXDR\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eM. tuberculosis\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eMIC(µg/ml)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e6a\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3-NO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.63\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e6b\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4-NO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e6c\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4-Cl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e6d\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3, 4-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e6e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2-OH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e6f\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4-OH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e8a\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3-NO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e8b\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4-NO\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e62.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.25\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e8c\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4-Cl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e62.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e8d\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2-OH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e8e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4-OH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e8f\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4-N(CH\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.81\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eIsoniazid\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eIA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\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\u003eMIC = minimal drug concentration required to stop the growth of \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eHighlighted values represent the most potent derivatives, compared to INH.\u003c/p\u003e\n \u003cp\u003eNA = No activity (MIC \u0026gt; 125 µg/ml)\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e\n \u003ch2\u003e\u003cstrong\u003e3.2.4.\u003c/strong\u003e \u003cstrong\u003eMycobacterium tuberculosis\u003c/strong\u003e \u003cstrong\u003eInhA enzyme inhibition activity\u003c/strong\u003e:\u003c/h2\u003e\n \u003cp\u003eThe InhA enzyme from Mycobacterium TB is necessary for cell wall metabolism and mycolic acid production. Compounds \u003cstrong\u003e8f\u003c/strong\u003e and \u003cstrong\u003e6a\u003c/strong\u003e demonstrated the best anti-tubercular effectiveness while exhibiting the least bacterial resistance. To compare \u003cstrong\u003e8f\u003c/strong\u003e and \u003cstrong\u003e6a\u003c/strong\u003e to isoniazid (INH) at various doses, their in-vitro inhibitory activity of the enzyme InhA was assessed using a previously described methodology [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e]. \u003cstrong\u003eTable\u0026nbsp;(2)\u003c/strong\u003e displays the 50% inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e), which is the concentration necessary to inhibit 50% of InhA. \u003cstrong\u003e8f\u003c/strong\u003e and \u003cstrong\u003e6a\u003c/strong\u003e inhibited the inhA enzyme with IC\u003csub\u003e50\u003c/sub\u003e values of 0.717 ± 0.033µM and 1.646 ± 0.069µM, which were equivalent to isoniazid's IC\u003csub\u003e50\u003c/sub\u003e of 0.323 ± 0.014µM.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable\u0026nbsp;(2)\u003c/strong\u003e: \u003cstrong\u003eIn-vitro Mycobacterium tuberculosis\u003c/strong\u003e \u003cstrong\u003eenoyl-acyl carrier protein reductase (InhA) inhibitory activity of\u003c/strong\u003e \u003cstrong\u003e8f,6a\u003c/strong\u003e \u003cstrong\u003eand Isoniazid\u003c/strong\u003e:\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tabb\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCompound\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e(InhA\u003c/p\u003e\n \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e (mean ± SD)\u003c/p\u003e\n \u003cp\u003eµM\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e8f\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.717\u003c/strong\u003e ± 0.033\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e6a\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.646\u003c/strong\u003e ± 0.069\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIsoniazid\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.323\u003c/strong\u003e ± 0.014\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec33\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cstrong\u003e3.3. Molecular docking simulation\u003c/strong\u003e:\u003c/h2\u003e\n \u003cp\u003eSeveral anti-tubercular medicines, notably the activated version of INH, target M. tuberculosis's enoyl-acyl carrier protein reductase (InhA). The binding pocket of the InhA enzyme consists of three main sites: the catalytic site (site I), including key amino acids Tyr158, 2`-OH of the nicotinamide ribose of the nicotinamide adenine dinucleotide NAD+, and other amino acids; and the hydrophobic region (site II), which accommodates the substrate binding loop in InhA. It is composed of a number of amino acids, including Met103, Glu104, Phe149, Ala157, Ala198, Met199, Ile202, Ile215, and Leu218; and a size-limiting solvent-exposed site (site III). It has been discovered that site I and parts of site II are occupied by InhA inhibitors. According to the literature[\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e], Tyr158 stabilizes substrates during the catalytic reaction of the enzyme, which is important for InhA activity. It was also stated that the interaction of NAD + with inhibitors is important for activity. Tyr158 exists in two conformations: IN-conformation and OUT-conformation.Tyr158 in its IN-form has the ability to bind to inhibitors; the aromatic groups of the inhibitors occupy the hydrophobic site (site II) together with π-π stacking against Phe149, while the hydroxyl group of the inhibitors is directed towards the inhibitors in the catalytic site (site I) [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. With natural ligand 2-(2,4-dichlorophenoxy)-5-(pyridin-2-ylmethyl)phenol (DCPP), the InhA enzyme's X-ray crystal may be seen (PDB ID: 3FNE) [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. The native ligand (DCPP) at the InhA binding site showed \u003cem\u003eH\u003c/em\u003e-bond interaction with the key amino acids Tyr158 and NAD + at site I that was occupied by phenolic ring, 2,4-dichlorophenyl ring occupied site II and formed hydrophobic interactions at Met199, Ala198, Gly96, and Met161 while the pyridine ring accommodated itself at site III.\u003c/p\u003e\n \u003cp\u003eMOE 2009 was used for docking. To verify the docking procedure, the co-crystallized ligand was re-docked into the InhA enzyme. Between the co-crystallized ligand and docking posture, the computed root means square differences (RMSD) value was less than 2Å. The newly synthesised compounds were docked and docking poses with higher energy scores and a lower root mean square deviation (RMSD) between the docked compound geometry and the co-crystallized native ligand were studied. Compound \u003cstrong\u003e8f\u003c/strong\u003e showed mode of interaction similar to InhA enzyme inhibitors, where ring \u003cstrong\u003eA\u003c/strong\u003e occupied site II, where carbonyl group of phthalimide formed \u003cem\u003eH\u003c/em\u003e-bond interaction at Tyr158 and NAD. Furthermore, benzene ring of the phthalimide was stacked against Phe 149. Ring \u003cstrong\u003eB\u003c/strong\u003e (dimethyl aminobenzene) occupied site II, forming hydrophobic interactions at Ala198, Met199, Phe97, Gly96, and Met161. Ring \u003cstrong\u003eB\u003c/strong\u003e showed stacking against Phe97. Ring \u003cstrong\u003eC\u003c/strong\u003e (pyrimidine) occupied site III. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eCompound 6\u003cstrong\u003ea\u003c/strong\u003e acquired an orientation different from \u003cstrong\u003e8f\u003c/strong\u003e, where the interaction at Tyr158 was afforded by ring \u003cstrong\u003eC\u003c/strong\u003e (pyrimidine) through N3, while interaction at NAD was afforded via phthalimide carbonyl at Ring A. The hydrophobic interaction at Ala198, Met199 was afforded by ring \u003cstrong\u003eC\u003c/strong\u003e. Ring \u003cstrong\u003eB\u003c/strong\u003e (3-nitrobenze) was oriented towards site III, as shown by Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec34\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. Drug likeness and ADMET properties:\u003c/h2\u003e\n \u003cp\u003eIt is critical to study the physicochemical properties as well as the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of drug candidates. Admetlab 2.0 is an online tool that allows systematic evaluation of ADMET properties, as well as some physicochemical properties. Admetlab 2.0 was used to study the physicochemical properties, drug likeness, and ADMET properties of compounds \u003cstrong\u003e8f\u0026nbsp;\u003c/strong\u003eand \u003cstrong\u003e6a\u0026nbsp;\u003c/strong\u003ecompared to INH. \u003cstrong\u003eFigure (1) in supplementary file (S2)\u003c/strong\u003e shows the generated radar chart for basic physicochemical properties and drug likeness of\u003cstrong\u003e\u0026nbsp;8f\u003c/strong\u003e, \u003cstrong\u003e6a\u003c/strong\u003e and INH.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e1-Studied parameters for drug likeness and physicochemical properties:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eMW\u003c/strong\u003e = Molecular weight (optimally for a drug-like: 100–600), \u003cstrong\u003enHD\u003c/strong\u003e = Number of hydrogen bond donors, which is Sum of all OHs and NHs (Optimally for a drug-like 0–7), \u003cstrong\u003enHA\u003c/strong\u003e = Number of hydrogen bond acceptors. Sum of all O and N (optimally for a drug-like: 0–12). \u003cstrong\u003enRot\u003c/strong\u003e = Number of rotatable bonds (optimally for a drug-like: 0–11), \u003cstrong\u003enRing\u003c/strong\u003e = Number of rings (Optimally For a drug-like: 0–6), \u003cstrong\u003eMaxRing\u003c/strong\u003e = Number of atoms in the biggest ring (optimally for a drug like: 0–18), \u003cstrong\u003enHet\u003c/strong\u003e = Number of heteroatoms. Number of non-carbon atoms (Optimally for a drug-like:1–15), \u003cstrong\u003efChar\u003c/strong\u003e = Formal charge (Optimally for\u0026nbsp;\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAACMAAAAcCAYAAADr9QYhAAABOklEQVRYCe2T4Y2GIBBE6cVirMVSrMRCrMNa+DKXe+aJ8Pu+S9jELCzszDBgqV8U5Yu01ClmdBvTmenMyIFRvftm1nWtpZTHd57nY34cx415Xddrre3f971u2/bYlz3G6YoJC43JDkiWZXG5RlAOQVg8teSICkbbn7WhGEgDSuQUJvFaSNp5MNrD4LodAb8rBsJWPcC4Zic8DjikFujrRIBzVwxkkNMAoUEzTniv13HYGRxwyV0xcSTNtjJjzy04dTvAu7DAEOKWcRCS/BLDFUWMI8C4kLr3tSeF1AJ/yH7/UOOY48mo194jcGPGkNoBrqh9b3EjB2xxjfkS07ui2J6vDdyx7VxRS4rwHg64txhA/NBSAyT1HpAdcC8uINhrdhIhybcYF/9qPMWMnJ/OTGdGDozq8838C2c+sh/WLEEddnsAAAAASUVORK5CYII=\" width=\"35\" height=\"28\"\u003e a drug-like: 4 ~ 4), \u003cstrong\u003enRig\u003c/strong\u003e = Number of rigid bonds (Optimally for a drug-like: 0–30), \u003cstrong\u003eTPSA\u003c/strong\u003e Topological polar surface area. Sum of tabulated surface contributions of polar fragments (Optimally for a drug-like:0-140) l\u003cstrong\u003eogS\u003c/strong\u003e = The logarithm of aqueous solubility value. Low solubility is unfavorable to good and complete oral absorption Drug-like compounds are in the range from − 4 to 0.5 log mol/. \u003cstrong\u003elogP\u003c/strong\u003e = The logarithm of the n-octanol/water distribution coefficient. log is important to determine both membrane permeability and hydrophobic binding to macromolecules. Drug-like compounds are in the range from 0 to 3 log mol/L. \u003cstrong\u003elogD7.4\u003c/strong\u003e = The logarithm of the n-octanol/water distribution coefficients at pH = 7.4. Drug candidates must be able to keep a balance between lipophilicity and hydrophilicity to dissolve as well as be able to penetrate membranes. Drug-like compounds are in the range of 1 to 3 log mol/L. \u003cstrong\u003eTable\u0026nbsp;(1\u003c/strong\u003e) \u003cstrong\u003ein supplementary file (S2)\u003c/strong\u003e shows the predicted physicochemical properties of oral bioavailable drug-like compounds \u003cstrong\u003e6a\u003c/strong\u003e \u003cem\u003eand\u003c/em\u003e \u003cstrong\u003e8f\u003c/strong\u003e compared to INH.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e2-Studied parameters for ADMET properties:\u003c/h3\u003e\n\u003cp\u003e\u003cspan class=\"BoldUnderline\"\u003eAbsorption\u003c/span\u003e: \u003cstrong\u003eHIA\u003c/strong\u003e (Human intestinal absorption). Poorly absorbed molecules have an absorption of less than 30%. Consequently, molecules having an HIA \u0026gt; 30% were assigned to HIA- (Category 0), whereas molecules having an HIA \u0026lt; 30% were assigned to HIA (Category 1). With a range of 0 to 1, the output result represents the likelihood of being HIA+.\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldUnderline\"\u003eDistribution\u003c/span\u003e: \u003cstrong\u003eBBB\u003c/strong\u003e Drugs that do not need to operate on the central nervous system (CNS) should ideally have minimal or no BBB penetration to prevent adverse effects.\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldUnderline\"\u003eMetabolism\u003c/span\u003e: \u003cstrong\u003eCytochrome P450(CYP2D6)\u003c/strong\u003e The human cytochrome P450 family (phase I metabolism enzyme) with many isozymes that metabolize drugs they are mainly concentrated in liver.\u003c/p\u003e\n\u003cp\u003e\u003cspan class=\"BoldUnderline\"\u003eToxicity\u003c/span\u003e: \u003cstrong\u003eThe Ames mutagenicity test\u003c/strong\u003e: AMES negative (-); AMES positive (+). \u003cstrong\u003eRat Oral Acute Toxicity\u003c/strong\u003e (-ve) low-toxicity, \u0026gt; 500 mg/kg; or (+ ve) high-toxicity; \u0026lt; 500 mg/kg. The output value is the probability of being toxic, within the range of 0 to 1. \u003cstrong\u003eTable\u0026nbsp;(2\u003c/strong\u003e) \u003cstrong\u003ein supplementary file (S2)\u003c/strong\u003e shows the predicted ADMET properties of compounds \u003cstrong\u003e6a\u003c/strong\u003e \u003cem\u003eand\u003c/em\u003e \u003cstrong\u003e8f\u003c/strong\u003e compared to INH.\u003c/p\u003e\n\u003cp\u003eBoth compounds \u003cstrong\u003e6a\u003c/strong\u003e and \u003cstrong\u003e8f\u003c/strong\u003e demonstrated good oral bioavailability, together with good gastrointestinal absorption. It was anticipated that they would not show hepatotoxicity or CNS toxicity. Compounds showed no mutagenicity as predicted by the Ames test and no acute oral toxicity in the animal model used in Admet Lab 2.0.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn conclusion, phthalimide-pyrimidine hybrids demonstrated antitubercular efficacy against MDR, XDR, and sensitive strains of tuberculosis. All compounds showed activity against sensitive \u003cem\u003eM. Tuberculosis\u003c/em\u003e strain with MIC range of 0.48\u0026ndash;62.5 \u0026micro;g/ml. Compounds \u003cb\u003e6a\u003c/b\u003e, \u003cb\u003e6b\u003c/b\u003e, \u003cb\u003e6e\u003c/b\u003e, \u003cb\u003e6f\u003c/b\u003e, \u003cb\u003e8a\u003c/b\u003e, \u003cb\u003e8b\u003c/b\u003e, and \u003cb\u003e8f\u003c/b\u003e showed anti-tubercular activity against MDR \u003cem\u003eM. Tuberculosis\u003c/em\u003e strains with MIC range of 1.95\u0026ndash;62.5 \u0026micro;g/ml. Compounds \u003cb\u003e6a\u003c/b\u003e, \u003cb\u003e6e\u003c/b\u003e, \u003cb\u003e6f\u003c/b\u003e, \u003cb\u003e8b\u003c/b\u003e, and \u003cb\u003e8f\u003c/b\u003e showed activity against XDR \u003cem\u003eM. Tuberculosi\u003c/em\u003es strains with MIC range of 7.8-31.25 \u0026micro;g/ml. Compounds \u003cb\u003e8f\u003c/b\u003e and \u003cb\u003e6a\u003c/b\u003e had the most potent anti-tubercular activity against senstive, MDR, and XDR \u003cem\u003eM. Tuberculosis\u003c/em\u003e strains with MIC of (0.48 \u0026micro;g/ml, 0.98 \u0026micro;g/ml), (1.95 \u0026micro;g/ml, 7.81 \u0026micro;g/ml), and (7.81 \u0026micro;g/ml, 15.63 \u0026micro;g/ml), respectively. Both \u003cb\u003e8f\u003c/b\u003e and \u003cb\u003e6a\u003c/b\u003e inhibited the Mycobacterial InhA enzyme with IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.717\u0026thinsp;\u0026plusmn;\u0026thinsp;0.033 \u0026micro;M and 1.646\u0026thinsp;\u0026plusmn;\u0026thinsp;0.069 \u0026micro;M, respectively. Molecular docking simulation revealed that \u003cb\u003e8f\u003c/b\u003e \u003cem\u003eand\u003c/em\u003e \u003cb\u003e6a\u003c/b\u003e could interact with the InhA enzyme in an inhibitory mode, forming \u003cem\u003eH\u003c/em\u003e-bond interactions at Tyr158 and NAD\u0026thinsp;+\u0026thinsp;at the catalytic site, in agreement with the co-crystalized ligand and other reported inhibitors. \u003cb\u003e6a\u003c/b\u003e \u003cem\u003eand\u003c/em\u003e \u003cb\u003e8f\u003c/b\u003e showed physicochemical properties of oral bioavailable drug-like compounds with gastrointestinal absorption. Predictions showed that compounds have no side effects on the CNS and no anticipated hepatotoxicity. Predictions showed no-mutagenicity or acute oral toxicity in models.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclaration of Competing Interest:\u003c/h2\u003e\n\u003cp\u003eThe authors have no competing interests to declare\u003c/p\u003e\n\u003ch2\u003eFunding sources:\u003c/h2\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution:\u003c/h2\u003e\n\u003cp\u003eThe authors have no competing interests to declare.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eModi, P., S. Patel, and M. Chhabria, \u003cem\u003eStructure-based design, synthesis and biological evaluation of a newer series of pyrazolo [1, 5-a] pyrimidine analogues as potential anti-tubercular agents.\u003c/em\u003e Bioorganic Chemistry, 2019. \u003cstrong\u003e87\u003c/strong\u003e: p. 240-251.\u003c/li\u003e\n\u003cli\u003eTiberi, S., et al., \u003cem\u003eNew drugs and perspectives for new anti-tuberculosis regimens.\u003c/em\u003e Pulmonology, 2018. \u003cstrong\u003e24\u003c/strong\u003e(2): p. 86-98.\u003c/li\u003e\n\u003cli\u003eAugustynowicz-Kopeć, E., et al., \u003cem\u003eGuidelines of Polish Respiratory Society concerning diagnosis, treatment and prevention of tuberculosis in adults and in children.\u003c/em\u003e Advances in Respiratory Medicine, 2013. \u003cstrong\u003e81\u003c/strong\u003e(4): p. 323-379.\u003c/li\u003e\n\u003cli\u003eMondal, R. and A. Jain, \u003cem\u003eExtensively drug-resistant Mycobacterium tuberculosis, India.\u003c/em\u003e Emerging Infectious Diseases, 2007. \u003cstrong\u003e13\u003c/strong\u003e(9): p. 1429.\u003c/li\u003e\n\u003cli\u003eVale, N., P. Gomes, and H. A Santos, \u003cem\u003eMetabolism of the antituberculosis drug ethionamide.\u003c/em\u003e Current Drug Metabolism, 2013. \u003cstrong\u003e14\u003c/strong\u003e(1): p. 151-158.\u003c/li\u003e\n\u003cli\u003eRawat, R., A. Whitty, and P.J. Tonge, \u003cem\u003eThe isoniazid-NAD adduct is a slow, tight-binding inhibitor of InhA, the Mycobacterium tuberculosis enoyl reductase: adduct affinity and drug resistance.\u003c/em\u003e Proceedings of the National Academy of Sciences, 2003. \u003cstrong\u003e100\u003c/strong\u003e(24): p. 13881-13886.\u003c/li\u003e\n\u003cli\u003eTakayama, K., C. Wang, and G.S. Besra, \u003cem\u003ePathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis.\u003c/em\u003e Clinical Microbiology Reviews, 2005. \u003cstrong\u003e18\u003c/strong\u003e(1): p. 81-101.\u003c/li\u003e\n\u003cli\u003eAlMatar, M., et al., \u003cem\u003eNovel compounds targeting InhA for TB therapy.\u003c/em\u003e Pharmacological Reports, 2018. \u003cstrong\u003e70\u003c/strong\u003e(2): p. 217-226.\u003c/li\u003e\n\u003cli\u003eWang, L.-Q., C.N. Falany, and M.O. James, \u003cem\u003eTriclosan as a substrate and inhibitor of 3\u0026prime;-phosphoadenosine 5\u0026prime;-phosphosulfate-sulfotransferase and UDP-glucuronosyl transferase in human liver fractions.\u003c/em\u003e Drug Metabolism and Disposition, 2004. \u003cstrong\u003e32\u003c/strong\u003e(10): p. 1162-1169.\u003c/li\u003e\n\u003cli\u003eHe, X., et al., \u003cem\u003ePyrrolidine carboxamides as a novel class of inhibitors of enoyl acyl carrier protein reductase from Mycobacterium tuberculosis.\u003c/em\u003e Journal of Medicinal Chemistry, 2006. \u003cstrong\u003e49\u003c/strong\u003e(21): p. 6308-6323.\u003c/li\u003e\n\u003cli\u003eNg, P.S., et al., \u003cem\u003eStructure activity relationships of 4-hydroxy-2-pyridones: A novel class of antituberculosis agents.\u003c/em\u003e European Journal of Medicinal Chemistry, 2015. \u003cstrong\u003e106\u003c/strong\u003e: p. 144-156.\u003c/li\u003e\n\u003cli\u003ePajk, S., et al., \u003cem\u003eNew direct inhibitors of InhA with antimycobacterial activity based on a tetrahydropyran scaffold.\u003c/em\u003e European Journal of Medicinal Chemistry, 2016. \u003cstrong\u003e112\u003c/strong\u003e: p. 252-257.\u003c/li\u003e\n\u003cli\u003eYar, M.S., A.A. Siddiqui, and M. Ashraf Ali, \u003cem\u003eSynthesis and Anti Tuberculostatic Activity of Novel 1, 3, 4‐Oxadiazole Derivatives.\u003c/em\u003e Journal of the Chinese Chemical Society, 2007. \u003cstrong\u003e54\u003c/strong\u003e(1): p. 5-8.\u003c/li\u003e\n\u003cli\u003eOthman, D.I., et al., \u003cem\u003eNovel 2-arylthiazolidin-4-one-thiazole hybrids with potent activity against Mycobacterium tuberculosis.\u003c/em\u003e Bioorganic Chemistry, 2022. \u003cstrong\u003e124\u003c/strong\u003e: p. 105809.\u003c/li\u003e\n\u003cli\u003ePhatak, P.S., et al., \u003cem\u003eSynthesis, antitubercular evaluation and molecular docking studies of phthalimide bearing 1, 2, 3-triazoles.\u003c/em\u003e Synthetic communications, 2019. \u003cstrong\u003e49\u003c/strong\u003e(16): p. 2017-2028.\u003c/li\u003e\n\u003cli\u003eRani, A., et al., \u003cem\u003eDesign and synthesis of 4-Aminoquinoline-isoindoline-dione-isoniazid triads as potential anti-mycobacterials.\u003c/em\u003e Bioorganic \u0026amp; Medicinal Chemistry Letters, 2020. \u003cstrong\u003e30\u003c/strong\u003e(22): p. 127576.\u003c/li\u003e\n\u003cli\u003eJesumoroti, O.J., et al., \u003cem\u003eExploration of 4-aminopyrrolo [2, 3-d] pyrimidine as antitubercular agents.\u003c/em\u003e Molecular Diversity, 2023. \u003cstrong\u003e27\u003c/strong\u003e(2): p. 753-765.\u003c/li\u003e\n\u003cli\u003eAl-Soliemy, A.M., R. Sabour, and T.A. Farghaly, \u003cem\u003ePyrazoles and fused pyrimidines: Synthesis, structure elucidation, antitubercular activity and molecular docking study.\u003c/em\u003e Medicinal Chemistry, 2022. \u003cstrong\u003e18\u003c/strong\u003e(2): p. 181-198.\u003c/li\u003e\n\u003cli\u003eFinger, V., et al., \u003cem\u003ePyrimidine derivatives with antitubercular activity.\u003c/em\u003e European Journal of Medicinal Chemistry, 2023. \u003cstrong\u003e246\u003c/strong\u003e: p. 114946.\u003c/li\u003e\n\u003cli\u003ePan, P. and P. J Tonge, \u003cem\u003eTargeting InhA, the FASII enoyl-ACP reductase: SAR studies on novel inhibitor scaffolds.\u003c/em\u003e Current topics in medicinal chemistry, 2012. \u003cstrong\u003e12\u003c/strong\u003e(7): p. 672-693.\u003c/li\u003e\n\u003cli\u003eSrivastava, A., \u003cem\u003ePalladium metal complexes as platforms for anti tumor therapy.\u003c/em\u003e J. Xi\u0026rsquo;an Shiyou Univ. Nat. Sci. Ed, 2021. \u003cstrong\u003e17\u003c/strong\u003e(6): p. 267-272.\u003c/li\u003e\n\u003cli\u003eYoussif S, El-Bahaie S, Nabih E. A facile one-pot synthesis of pyrido[2,3-d]pyrimidines and pyrido[2,3-d:6,5-d]dipyrimidines. J. Chem. Res. Synop. (2), 112\u0026ndash;113 (1999).\u003c/li\u003e\n\u003cli\u003e\u0026quot; 6-Amino-2-thio-, 6-aminouracils as Precursors for the Synthesis of antiviral and antimicrobial methylene bis(2-thiouracil), tricyclic pyrimidines and 6-alkylthio-purine-2-ones.\u003cstrong\u003e Shaker Youssif\u003c/strong\u003e and Sahera F. Mohamed, \u003cem\u003eChemical Monthly\u003c/em\u003e, \u003cstrong\u003e2008\u003c/strong\u003e, 139, 161-168\u003c/li\u003e\n\u003cli\u003e\u0026ldquo;Synthesis, Molecular Docking and Anticancer Activity of Some 5-Aryl-5,10-dihydropyrido[2,3-\u003cem\u003ed\u003c/em\u003e: 6,5-\u003cem\u003ed\u0026apos;\u003c/em\u003e]dipyrimidine-2,4,6,8-tetraone Derivatives and Pyrido[2,3-\u003cem\u003ed\u003c/em\u003e]pyrimidines\u0026rdquo;, Basant Farag, Fatimah Agili, Samar El-Kalyoubi,\u003csup\u003e \u003c/sup\u003eSaid A. Said, Shaker Youssif, and Wael Shehta; \u003cem\u003eChemistrySelect\u003c/em\u003e \u003cstrong\u003e2022\u003c/strong\u003e, 7, e202103834.\u003c/li\u003e\n\u003cli\u003eWang, H., et al., \u003cem\u003eLarge-scale solvent-free chlorination of hydroxy-pyrimidines,-pyridines,-pyrazines and-amides using equimolar POCl3.\u003c/em\u003e Molecules, 2012. \u003cstrong\u003e17\u003c/strong\u003e(4): p. 4533-4544.\u003c/li\u003e\n\u003cli\u003eShehta W., Agili F., Farag B., J Almehmadi S., Elfeky M.S., Youssif S., \u0026amp; El-Kalyoubi S., Synthesis and in vitro study of pyrimidine-phthalimide hybrids as VEGFR2 inhibitors with antiproliferative activity, Future Med Chem\u003cstrong\u003e., \u003c/strong\u003e2023, 661-677. doi: 10.4155/fmc-2023-0025.\u003c/li\u003e\n\u003cli\u003eParadkar, M. V., Gadre, S. Y., Pujari, T. A., Khandekar, P. P., \u0026amp; Kumbhar, V. B. (2005). One‐Pot Synthesis of 3‐Phenacylphthalides. \u003cem\u003eSynthetic communications\u003c/em\u003e, \u003cem\u003e35\u003c/em\u003e(3), 471-474.\u003c/li\u003e\n\u003cli\u003eLu, Y., et al., \u003cem\u003eClofazimine analogs with efficacy against experimental tuberculosis and reduced potential for accumulation.\u003c/em\u003e Antimicrobial Agents and Chemotherapy, 2011. \u003cstrong\u003e55\u003c/strong\u003e(11): p. 5185-5193.\u003c/li\u003e\n\u003cli\u003eElsayed, Z.M., et al., \u003cem\u003eDevelopment of novel isatin\u0026ndash;nicotinohydrazide hybrids with potent activity against susceptible/resistant Mycobacterium tuberculosis and bronchitis causing\u0026ndash;bacteria.\u003c/em\u003e Journal of Enzyme Inhibition and Medicinal Chemistry, 2021. \u003cstrong\u003e36\u003c/strong\u003e(1): p. 384-393.\u003c/li\u003e\n\u003cli\u003eSivaraman, S., \u003cem\u003eStructure-activity studies of the inhibition of FabI, the enoyl ACP reductase from Escherichia coli by triclosan\u003c/em\u003e. 2002: State University of New York at Stony Brook.\u003c/li\u003e\n\u003cli\u003eElfeky, S.M., et al., \u003cem\u003eSynthesis, biological screening, and molecular docking of quinazolinone and quinazolinethione as phosphodiesterase 7 inhibitors.\u003c/em\u003e Archiv der Pharmazie, 2020. \u003cstrong\u003e353\u003c/strong\u003e(1): p. 1900211.\u003c/li\u003e\n\u003cli\u003eSabbah, M., et al., \u003cem\u003eFragment-based design of Mycobacterium tuberculosis InhA inhibitors.\u003c/em\u003e Journal of Medicinal Chemistry, 2020. \u003cstrong\u003e63\u003c/strong\u003e(9): p. 4749-4761.\u003c/li\u003e\n\u003cli\u003eKamsri, P., et al., \u003cem\u003eDiscovery of new and potent inha inhibitors as antituberculosis agents: structure-based virtual screening validated by biological assays and x-ray crystallography.\u003c/em\u003e Journal of Chemical Information and Modeling, 2019. \u003cstrong\u003e60\u003c/strong\u003e(1): p. 226-234.\u003c/li\u003e\n\u003cli\u003eFreundlich, J.S., et al., \u003cem\u003eTriclosan derivatives: towards potent inhibitors of drug-sensitive and drug-resistant Mycobacterium tuberculosis.\u003c/em\u003e ChemMedChem, 2009. \u003cstrong\u003e4\u003c/strong\u003e(2): p. 241.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eSchemes 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"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":"Mycobacterium tuberculosis, ADMET studies, Docking simulation, Enzyme inhibition, Phthalimide-pyrimidine hybrids","lastPublishedDoi":"10.21203/rs.3.rs-4397392/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4397392/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCondensation reaction of aromatic aldehydes with 2-(6-amino-2-chloropyrimidin-4-yl)isoindoline-1,3-dione and 2-(6-amino-2-hydrazineylpyrimidin-4-yl)isoindoline-1,3-dione afforded\u0026nbsp; 2-(2-chloro-6-((3 alkylbenzylidene)amino) pyrimidin-4-yl)isoindoline-1,3-dione \u003cstrong\u003e(6a-f) \u003c/strong\u003eand 2-(6-amino-2-(2-(arylidene) hydrazineyl)pyrimidin-4-yl)isoindoline-1,3-dione \u003cstrong\u003e(8a-f)\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003erespectively, as phthalimide-aminopyrimidine hybrids. Compounds showed a wide range of anti-tubercular activity against sensitive MDR and XDR \u003cem\u003eM. tuberculosis\u003c/em\u003e strains, with \u003cem\u003e\u003cstrong\u003e8f\u003c/strong\u003e\u003c/em\u003e and \u003cem\u003e\u003cstrong\u003e6a\u003c/strong\u003e\u003c/em\u003e showing the highest activity. \u003cem\u003e\u003cstrong\u003e8f\u003c/strong\u003e\u003c/em\u003e and \u003cem\u003e\u003cstrong\u003e6a\u003c/strong\u003e\u003c/em\u003e inhibited sensitive \u003cem\u003eM. tuberculosis\u003c/em\u003e with MIC =0.48 μg/ml and 0.98 μg/ml, respectively, comparable to isonizide (INH) (MIC =0.12 μg/ml). Both \u003cem\u003e\u003cstrong\u003e8f\u003c/strong\u003e\u003c/em\u003e and \u003cem\u003e\u003cstrong\u003e6a\u003c/strong\u003e\u003c/em\u003e inhibited MDR strain with MIC=1.95 μg/ml and 7.81 μg/ml, respectively, and XRD with MIC=7.81 μg/ml and 15.63 μg/ml, respectively.\u003cem\u003e \u003c/em\u003eBoth\u003cem\u003e \u003c/em\u003e\u003cem\u003e\u003cstrong\u003e8f\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eand \u003cem\u003e\u003cstrong\u003e6a\u003c/strong\u003e\u003c/em\u003e could inhibit mycobacterial InhA enzyme \u003cem\u003ein-vitro\u003c/em\u003e (IC\u003csub\u003e50\u003c/sub\u003e =0.717±0.033µM and 1.646±0.069µM, respectively). Molecular docking simulation revealed that \u003cem\u003e\u003cstrong\u003e8f\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eand \u003cem\u003e\u003cstrong\u003e6a\u003c/strong\u003e\u003c/em\u003e were also capable of interacting at the catalytic site of the InhA enzyme in a manner similar to the native ligand, via binding with NAD\u003csup\u003e+\u003c/sup\u003e and Tyr158. Compounds \u003cem\u003e\u003cstrong\u003e6a \u003c/strong\u003e\u003c/em\u003eand \u003cem\u003e\u003cstrong\u003e8f\u003c/strong\u003e\u003c/em\u003e showed physicochemical properties of oral bioavailable drug-like compounds with high gastrointestinal absorption. Predictions showed that compounds have no side effects on the CNS and no anticipated hepatotoxicity, mutagenicity, or acute oral toxicity in models.\u003c/p\u003e","manuscriptTitle":"Novel phthalimide-pyrimidine hybrids as potent anti-tubercular agents","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-15 11:20:19","doi":"10.21203/rs.3.rs-4397392/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":"c79d60e8-1ca6-4ed6-b3f5-98f7f250540d","owner":[],"postedDate":"May 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-15T11:20:21+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-15 11:20:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4397392","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4397392","identity":"rs-4397392","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}