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In silico design and synthesis of targeted rutin derivatives as xanthine oxidase inhibitors

In silico design and synthesis of targeted rutin derivatives as xanthine oxidase inhibitors Background: Xanthine oxidase is an important enzyme of purine catabolism pathway and has been associated directly in pathogenesis of gout and indirectly in many pathological conditions like cancer, diabetes and metabolic syndrome. In this research rutin, a bioactive flavonoid was explored to determine the capability of itself and its deriva- tives to inhibit xanthine oxidase. Objective: To develop new xanthine oxidase inhibitors from natural constituents along with antioxidant potential. Method: In this report, we designed and synthesized rutin derivatives hybridized with hydrazines to form hydrazides and natural acids to form ester linkage with the help of molecular docking. The synthesized compounds were evalu- ated for their antioxidant and xanthine oxidase inhibitory potential. Results: The enzyme kinetic studies performed on rutin derivatives showed a potential inhibitory effect on XO abil- ity in competitive manner with IC value ranging from 04.708 to 19.377 µM and RU3a was revealed as most active 50 3 derivative. Molecular simulation revealed that new rutin derivatives interacted with the amino acid residues PHE798, GLN1194, ARG912, GLN 767, ALA1078 and MET1038 positioned inside the binding site of XO. Results of antioxidant activity revealed that all the derivatives showed very good antioxidant potential. Conclusion: Taking advantage of molecular docking, this hybridization of two natural constituent could lead to desirable xanthine oxidase inhibitors with improved activity. Keywords: Rutin, Xanthine oxidase, Molecular docking, Antioxidant Introduction on the xanthine and oxygen at the enzymatic centre. Xanthine oxidase (XO) having molecular weight of While xanthine undergoes oxidation reaction near to the around 300 kDa is oxidoreductase enzyme represented in Mo-pt center/substrate binding domain of XO, simulta- the form of a homodimer. Both the monomers of XO are neously substrate oxygen undergoes reduction at FAD almost identical and each of them contains three domains center and electron transfer takes place leading to for- 2− namely (a) molybdopterin (Mo-pt) domain at the C-ter- mation of superoxide anion (O ) or hydrogen peroxide minal having 4 redox centers where oxidation takes place (H O ) free radicals. [4–8]. This catalytic reaction results 2 2 (b) a flavin adenine dinucleotide (FAD) domain at the in formation uric acid as a final product and oxygen reac - centre generally considered as binding site domain and tive species in form of free radicals. The excessive genera - (c) 2[Fe–S]/iron sulfur domain at the N-terminal [1–3]. tion of uric acid leads to a condition like hyperuricemia The catalytic oxidation of XO is two substrates reaction which is a key factor in development of gout [1, 9], and uncontrolled amounts of reactive oxygen species causes many pathological conditions like cardiovascular disor- ders, inflammatory diseases and hypertensive disorders. *Correspondence: dranuragkhatkarmdurtk@gmail.com; anuragpharmacy@gmail.com Xanthine oxidase (XO; EC 1.17.3.2) has been consid- Laboratory for Preservation Technology and Enzyme Inhibition Studies, ered as significantly potent drug target for the cure and Department of Pharmaceutical Sciences, M.D. University, Rohtak, Haryana, management of pathological conditions prevailing due India Full list of author information is available at the end of the article to high levels of uric acid in the blood stream. [10–17]. © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Malik et al. BMC Chemistry (2019) 13:71 Page 2 of 13 Considering the above fact, by inhibiting XO selectively evaluation of the human xanthine oxidase inhibitory could be better treatment plan for disorders caused by activity was performed by measuring hydrogen peroxide XO directly or indirectly including gout, inflammatory (H O ) production from oxidation of xanthine oxidase 2 2 disease, oxidative damage and cancer [3, 18, 19]. Gen- by the substrate xanthine, utilizing the human xanthine erally, XO inhibitors have been categorized into purine oxidase assay kit (Sigma USA). The progress of reaction and non-purines inhibitors differentiated on the basis was observed through thin layer chromatography (TLC) of their chemically derived skeleton structure. The first on 0.25  mm precoated silica gel plates purchased from purine derived XO inhibitor discovered and approved by Merck, reaction spots were envisaged in iodine compart- US FDA was Allopurinol as marketed drug for gout and ment and UV. Melting points were measured using a hyperuricemia [20, 21]. Considering the life threatening Sonar melting point apparatus and uncorrected. H NMR side effects like Stevens–Johnsons syndrome caused by and C NMR spectra were documented in DMSO and allopurinol use, scientists turned their interest into non-deuterated CDCl respectively on Bruker Avance II 400 purine XO inhibitors and an immense accomplishment NMR spectrometer at the frequency of 400  MHz using has been received in this direction with development of tetramethylsilane standard (downfield) moreover chemi - new drug Febuxostat [22–25]. This non-purine candi - cal shifts were expressed in ppm (δ) using the residual date produced minor and non-life threatening adverse solvent line as internal standard. Infrared (IR) spectra effects in comparison to Allopurinol [26–29]. Extending were recorded on Perkin Elmer FTIR spectrophotometer our previous successful effort to achieve new xanthine by utilizing KBr pellets system. oxidase inhibitors from natural sources, in this report we investigated and developed some new rutin derived xan- thine oxidase inhibitor [30]. Molecular docking Rutin is a well characterized bioactive plant flavonoid In silico docking studies was done with integrated Schro- having great therapeutic importance for the treatment of dinger software using Glide module for enzyme ligand many disease like conditions including cytotoxicity, anti- docking [35]. oxidant activity, antibacterial property and anti-inflam - matory action [31–34]. Due to these pharmacological Protocol followed for docking procedures activities rutin is explored widely and great success have Preparation of  protein The 3D crystal structure of been achieved in order to get drug like candidates. human xanthine oxidase co-crystalised with salicylic acid was retrieved from Protein Data Bank (PDB ID. 2E1Q). OH The targeted protein structure was further refined in the OH Protein Preparation Wizard to obtain the optimized and OH HO O chemically accurate protein configuration. For that, the HO OH co-crystalised enzyme (XO) was retrieved directly from O O CH 3 Protein data bank in maestro panel followed by removal of OH O water molecules, addition of H atoms, addition of missing HO OH OH side chains and finally minimization was done to obtain the optimized structure. Rutin Preparation of ligand The 3D-structures of rutin derived compounds to be docked against XO were built in maes- Taking advantage of molecular docking techniques new tro building window. Ligand preparation was performed compounds with potential drugability for the targeted in Ligprep module. enzyme might be achieved with a precise knowledge of mechanism of action. With the combined approach Active site prediction To predict the binding site/active of molecular docking and synthetic chemistry, in this site Site Map application of glide was utilized. Out of top research we developed some new potential compounds three active site, the one having larger radius was selected. against xanthine oxidase (Fig. 1). Validation of binding site was done by redocking the sali- cylic acid and RMSD value was observed. RMSD value of less than 0.2 validated the docking procedure and active Experimental site was defined for docking of new rutin analogs. Chemicals and instrumentation For this research, the analytical grade chemicals nec- Glide docking To carry out docking, Firstly the recep- essary for synthesis and antioxidant activity were pur- tor grid generation tool was utilized to around the active/ chased from Hi-media Laboratories. The in  vitro Malik et al. BMC Chemistry (2019) 13:71 Page 3 of 13 OH OH OH HO O HO OH OH OH OH OH OO CH OH OH OH HO O HO O HO OH HO OH HO OH HN OH OO CH 3 OO CH OH OH RU3a3 Cl HO OH HO OH HN OH OH NH HN RU4b1 NO NO 2 2 RU3a2 OH H N HN OH NO NH-NH 2 N 2 OH HO O HO OH Phenyl thiosemicarbazide OH OO CH OH OH H N HO OH 2 HN N NH 2 OH HO O OH H S HO OH Thiosemicarbazide H N OH RU3a1 OO CH OH H N NO 2 2 3 OO H OH 4-Nitrobenzenamine HO OH HO O HO HO OH OH Rutin O OO CH OH Cinnamic Acid HO OH OH HO HO NO RU4b2 OCH OCH 3 3 OCH Salicylic acid OCH OCH 3 OCH HO H CO O H CO O 3 H CO O O N O O Nicotinic acid O OO CH OO CH 3 3 OO CH OH RU7c2 RU7c1 RU7c3 Fig. 1 Design strategy for the development of rutin derivatives binding site of xanthine oxidase and glide docking with zines (0.001 mol) were added to the flask and reac - extra precision was used to visualize the interaction of tion mixture was refluxed for 5–6 h at 40 °C. Com - protein and ligand. The top active ligand was selected for pletion of reaction was monitored by TLC. The wet lab synthesis and evaluation of pharmacological activ- product thus obtained was filtered and filtrate was ity. concentrated to obtain the final product. The final product was recrystallised to obtain the pure com- Synthetic procedures pound. Procedures for synthesis of rutin derivatives (Scheme 1) (B) General procedure for synthesis of anilline deriva- tives RU4b (1–2) 0.001  mol of the intermediate obtained above (A) General procedure for synthesis of hydrazine deriva- was taken in round bottom flask and dissolved in tives RU3a (1–4) 50  ml of ethanol. Different anillines (0.001  mol) were added to the flask and reaction mixture was 0.001 mol of rutin was taken in round bottom flask refluxed for 8–10 h at 40 °C. Completion of reaction and dissolved in 50  ml of ethanol. Different hydra - Malik et al. BMC Chemistry (2019) 13:71 Page 4 of 13 OH OH OH HO O HO OH OO CH OH OH HO OH OH HN OH OH OH OH HO O OH HO O HO OH RU3a HO OH OO CH O 3 O OH OO CH N OH HO OH H N 2 HN Cl Reflux HO OH HN S OH 8-10 hrs OH HN N NH-NH NO 2 RU4b 1 Reflux RU3a NH 8-10 hrs NO 2 Reflux 8-10 hrs OH OH OH OH OH NO S OH OH HO O OH HO O H N HO OH 2 OH Reflux N NH HO O HO OH 2 8-10 hrs H HO OH OO CH 3 O OO H Reflux OO CH O OH N OO CH H N NO 8-10 hrs 3 2 2 OH HO OH HO OH OH HO OH HN OH S OH Rutin a) K CO H N CH I 2 3 2 NO RU4b DMF, RT,2d RU3a b) HCL,95% ethanol reflux,2h; HO OCH HO 3 OCH OCH OCH OCH OCH 3 3 O N H CO O 3 H CO O NICOTINIC ACID H CO O 3 3 CINNAMIC ACID Reflux 5hr OH O Reflux 5hr OO CH 3 OO CH OO CH 3 3 RUI HO HO Reflux 5hr RU7c O OCH OCH RU7c H CO O OO CH OH RU7c Scheme 1 Synthesis of rutin derivatives was monitored by TLC. The product thus obtained Spectral data RU3a yield 69.6% R 0.6 [Mobile 1 f was filtered and filtrate was concentrated to obtain Phase for TLC—Methanol:Glacial acetic acid:Formic the final product. The final product was recrystal - acid:Water (3:2.9:0.8:0.5)] M.pt. (231–232) IR (KBR pel- −1 lised to obtain the pure compound.lets) cm 1) 3222 (O–H str., Ar), 1609 (C=N str.), 1501 (C) General procedure for synthesis of methylated rutin (C=C str.), 1206 (O–CH ), 1128 (C=S Str.) H NMR derivatives RU7c (400  MHz, DMSO-d ) δ 7.81 (dd, J = 7.5, 1.5  Hz, 1H), (1–3) 6 Rutin was methylated by methyl sulphate in pres- 7.59 (d, J = 1.5  Hz, 1H), 6.82 (d, J = 7.5  Hz, 1H), 6.48 ence of potassium carbonate and dimethyl forma- (dd, J = 15.0, 1.5  Hz, 2H), 6.28 (t, J = 7.0  Hz, 1H), 4.13 mide by stirring along with reflux at 40 °C for 48 h (t, J = 7.0 Hz, 1H), 3.89–3.81 (m, 3H), 3.71 (dd, J = 12.4, to generate tetramethylated rutin. Acidolysis of 6.9 Hz, 1H), 3.67–3.54 (m, 3H), 2.32 (dt, J = 12.4, 7.0 Hz , above was done to obtain the intermediate com- 1H), 2.28–2.16 (m, 2H), 2.06–2.04 (m, 1H), 1.97–1.92 pound (RUI) by refluxing it with HCl and 95% etha - (m, 2H), 1.74–1.66 (m, 2H). C NMR (100 MHz , Chloro- nol for 4 h. The intermediate compound (RUI) was form-d) δ 180.16, 163.73, 155.81, 154.70, 152.34, 148.70, then refluxed with different phenolic acid to obtain 145.50, 133.79, 133.45, 120.73, 120.41, 115.79, 115.09, their ester derivatives. 102.38, 99.59, 99.00, 91.11, 80.48, 73.58, 73.26, 72.40, 71.83 (d, J = 10.5  Hz), 66.02, 40.22, 37.43, 28.26, 26.90. Malik et al. BMC Chemistry (2019) 13:71 Page 5 of 13 + + m/z found for C H N O S: 683 (M ) 687 (M + 1) . J = 1.5 Hz, 1H), 7.40 (d, J = 7.5 Hz, 1H), 6.81 (d, J = 7.5 Hz, 28 33 3 15 Anal calcd for C H N O S: C, 52.91; H, 5.23; N, 6.61; 1H), 6.47 (dd, J = 10.8, 1.5  Hz, 2H), 6.22 (t, J = 7.0  Hz, 28 33 3 15 O, 35.20; S, 5.04 Found: C, 52.93; H, 5.21; N, 6.60; O, 1H), 4.11 (t, J = 7.0 Hz, 1H), 3.98–3.90 (m, 3H), 3.79 (dd, 35.19; S, 5.06. J = 12.4, 6.9 Hz, 1H), 3.71–3.61 (m, 3H), 2.42 (dt, J = 12.4, RU3a yield 72.5% R 0.7 [Mobile Phase for TLC— 7.0  Hz, 1H), 2.39– 2.31 (m, 2H), 2.29–2.28 (m, 1H), 2 f Methanol:Glacial acetic acid:Formic acid:Water 1.87–1.77 (m, 2H). C NMR (100  MHz, Chloroform-d) −1 (3:2.9:0.8:0.5)] M.pt. (255–257) IR (KBR pellets) c m ) δ 169.14, 168.95, 168.11, 166.86, 150.94, 144.52, 144.24, 3468 (O–H str., Ar), 1639 (C=N str.), 1596 (C=C str.), 142.37, 140.47, 131.18, 128.56, 125.41, 123.81, 122.54 (d, 1218 (O–CH ), 1150 (C=S Str.) H NMR (400  MHz, J = 14.8  Hz), 121.81, 113.64, 113.17, 106.71, 97.09, 96.89, DMSO-d ) δ 7.78–7.60 (m, 3H), 7.49 (d, J = 1.5  Hz, 1H), 93.98, 82.37, 75.79 (d, J = 19.1 Hz), 73.17 (d, J = 12.2  Hz), 7.39–7.29 (m, 2H), 7.10–7.01 (m, 1H), 6.86 (d, J = 7.5  Hz, 73.06, 72.69, 71.01, 65.19, 41.10, 38.86, 28.85, 27.44. m/z + + 1H), 6.52 (dd, J = 15.0, 1.5  Hz, 2H), 6.24 (t, J = 7.0  Hz, found for H ClN O : 764 (M ) 766 (M + 2) . Anal calcd 33 2 17 1H), 4.04 (t, J = 7.0 Hz, 1H), 3.98–3.88 (m, 3H), 3.78 (dd, for C H ClN O : C, 51.81; H, 4.35; Cl, 4.63; N, 3.66; O, 33 33 2 17 J = 12.4, 6.9 Hz, 1H), 3.68–3.64 (m, 3H), 2.28 (dt, J = 12.4, 35.55. Found: C, 51.83; H, 4.36; Cl, 4.65; N, 3.64; O, 35.53. 7.0 Hz, 1H), 2.14–2.11 (m, 2H), 2.09–2.06 (m, 1H), 1.87– RU4b yield 83.5% R 0.8 [Mobile Phase for TLC— 2 f 1.84 (m, 2H), 1.74–1.71 (m, 2H). C NMR (100  MHz, Methanol:Glacial acetic acid:Formic acid:Water −1 Chloroform-d) δ 174.93, 164.50, 160.96, 155.78, 150.30, (3:2.9:0.8:0.5)] M.pt. (253–254) IR (KBR pellets) c m 1) 148.16, 145.55, 139.23, 130.44, 128.67, 124.46, 123.85, 1785 (C=O str.), 1637 (C=N str.), 1561 (C=C str.), 1258 123.09, 122.39, 121.81, 116.06, 115.83, 103.40, 99.09, (O–CH ), 1234 (C–O str., ester) H NMR (400  MHz, 97.71, 95.05, 82.37, 73.06 (d, J = 19.1  Hz), 72.87 (d, DMSO-d ) δ 8.21–8.14 (m, 2H), 7.79 (dd, J = 7.5, 1.5  Hz, J = 12.2  Hz), 72.47, 72.35, 71.92, 65.19, 41.10, 38.86, 1H), 7.59 (d, J = 1.5  Hz, 1H), 7.32–7.25 (m, 2H), 6.75 (d, 29.40, 27.86. m/z found for C H N O S: 759 (M ) 760 J = 7.5  Hz, 1H), 6.44 (dd, J = 14.1, 1.5  Hz, 2H), 6.27 (t, 34 37 3 15 (M + 1) . Anal calcd for C H N O S: C, 53.75; H, 4.91; J = 7.0  Hz, 1H), 4.15 (t, J = 7.0  Hz, 1H), 3.98–3.95 (m, 34 37 3 15 N, 5.53; O, 31.59; S, 4.22. Found: C, C, 53.77; H, 4.93; N, 3H), 3.88 (dd, J = 12.4, 6.9  Hz, 1H), 3.67–3.55 (m, 3H), 5.56; O, 31.59; S, 4.24. 2.22 (dt, J = 12.4, 7.0  Hz, 1H), 2.14–2.11 (m, 2H), 2.09– RUT3a yield 61% R 0.6 [Mobile Phase for TLC— 2.06 (m, 1H), 1.76–1.73 (m, 2H), 1.67–1.55 (m, 2H). 3 f Methanol:Glacial acetic acid:Formic acid:Water C NMR (100  MHz, Chloroform-d) δ 173.89, 164.58, −1 (3:2.9:0.8:0.5)] M.pt. (235–237) IR (KBR pellets) c m ) 163.50, 158.34, 152.36, 151.92, 148.16, 146.53, 145.55, 3475 (O–H str., Ar), 1641 (C=N str.), 1580 (C=C str.), 128.56, 125.27, 124.36, 122.39, 121.81, 116.06, 115.83, 1220 (O–CH ), 1155 (C=S Str.) H NMR (400  MHz, 108.81, 93.06, 97.81, 90.53, 82.19, 73.80 (d, J = 19.1  Hz), DMSO-d ) δ 7.70 (dd, J = 7.5, 1.5  Hz, 1H), 7.56 (d, 72.67 (d, J = 12.2  Hz), 72.36, 72.12, 71.08, 64.86, 42.81, J = 1.5  Hz, 1H), 7.46–7.38 (m, 2H), 7.32–7.23 (m, 2H), 36.15, 28.55, 26.98. m/z found for C H N O:730 (M ) 33 34 2 17 7.07–6.98 (m, 1H), 6.89 (d, J = 7.5  Hz, 1H), 6.35 (dd, 731 (M + 1) . Anal calcd for C H N O : C, 54.25; H, 33 34 2 17 J = 15.0, 1.5  Hz, 2H), 6.19 (t, J = 7.0  Hz, 1H), 4.09 (t, 4.69; N, 3.83; O, 37.23. Found: C, 54.27; H, 4.70; N, 3.85; J = 7.0  Hz, 1H), 4.02–3.88 (m, 3H), 3.68 (dd, J = 12.4, O, 37.25. 6.9 Hz, 1H), 3.66–3.54 (m, 3H), 2.33 (dt, J = 12.4, 7.0  Hz, RU7C yield 83.5% R 0.8 [Mobile Phase for TLC— 1 f 1H), 2.21–2.19 (m, 2H), 1.96–1.88 (m, 2H), 1.87–1.85 (m, Methanol:Glacial acetic acid:Formic acid:Water 13 −1 2H) (Additional file 1). C NMR (100 MHz, Chloroform- (3:2.9:0.8:0.5)] M.pt. (189–190) IR (KBR pellets) c m 1) d) δ 164.50, 160.96, 155.78, 150.30, 148.16, 145.55, 143.60, 1715 (C=O str.), 1627 (C=N str.), 1607 (C=C str.), 1234 132.14, 129.50, 124.46, 122.39, 121.81, 121.19, 118.32, (O–CH ), 11,944 (C–O str., ester) H NMR (400  MHz, 116.06, 115.83, 104.75, 94.15, 93.97, 91.01, 83.98, 79.41 DMSO-d ) δ 9.11 (d, J = 1.5 Hz, 1H), 8.77–8.70 (m, 1H), (d, J = 19.1 Hz), 78.77 (d, J = 12.2 Hz), 77.09, 73.82, 68.48, 8.14 (dt, J = 7.5, 1.5  Hz, 1H), 7.92 (dd, J = 7.5, 1.5  Hz, 42.85, 37.51, 23.82, 23.17. m/z found for C H N O : 1H), 7.68 (d, J = 1.5  Hz, 1H), 7.51 (t, J = 7.5  Hz, 1H), 33 36 2 15 + + 700 (M ) 701 (M + 1) . Anal calcd for C H N O : C, 6.93–6.83 (m, 2H), 6.23 (d, J = 1.5 Hz, 1H), 3.92 (s, 3H), 33 36 2 15 56.57; H, 5.18; N, 4.00; O, 34.25. Found: C, 56.58; H, 5.20; 3.83 (d, J = 0.9 Hz, 6H), 3.76 (s, 3H). C NMR (100 MHz, N, 4.00; O, 34.27. Chloroform-d) δ 174.99, 164.48, 164.18, 160.33, 157.96, RU4b yield 74.3% R 0.6 [Mobile Phase for TLC— 156.60, 153.53, 151.74, 150.80, 149.32, 138.25, 128.95, 1 f Methanol:Glacial acetic acid:Formic acid:Water 123.72, 123.22, 122.87, 122.65, 113.70, 112.82, 107.81, −1 (3:2.9:0.8:0.5)] M.pt. (259–260) IR (KBR pellets) c m 1) 95.68, 93.25, 56.20, 55.88 (d, J = 2.6 Hz), 55.62. m/z found + + 1725 (C=O str.), 1631 (C=N str.), 1603 (C=C str.), 1234 for C H NO:463 (M ) 464 (M + 1) . Anal calcd for 25 21 8 (O–CH ), 1268 (C–O str., ester) H NMR (400  MHz, C H NO : C, 64.79; H, 4.57; N, 3.02; O, 27.62. Found: C, 3 25 21 8 DMSO-d ) δ 8.38 (d, J = 1.5  Hz, 1H), 8.15 (dd, J = 7.5, 64.80; H, 4.58; N, 3.00; O, 27.60. 1.5  Hz, 1H), 7.69 (dd, J = 7.5, 1.5  Hz, 1H), 7.2 (d, Malik et al. BMC Chemistry (2019) 13:71 Page 6 of 13 RU7C yield 62.5% R 0.6 [Mobile Phase for TLC— to prepare reaction mixture. The different concentrations 2 f Methanol:Glacial acetic acid:Formic acid:Water of synthesized derivatives having final volume 50 µl were −1 (3:2.9:0.8:0.5)] M.pt. (186–188) IR (KBR pellets) c m 1) prepared in dimethyl sulfoxide (DMSO) and added to 96 1764 (C=O str.), 1619 (C=N str.), 1595 (C=C str.), 1277 well plate. To each well 50 µl of reaction mix was added (O–CH ), 1214 (C–O str., ester) H NMR (400  MHz, and mixed well. After 2–3  min initial measurement was DMSO-d ) δ 7.91 (ddd, J = 7.5, 6.5, 1.5  Hz, 2H), 7.67 taken. The plates were incubated at 25  °C taking meas - (d, J = 1.5  Hz, 1H), 7.47 (td, J = 7.5, 1.5  Hz, 1H), 7.09 urements at every 5  min. Allopurinol served as positive (td, J = 7.5, 1.5  Hz, 1H), 6.97–6.88 (m, 2H), 6.86 (d, control. Absorbance at different time intervals was noted J = 1.5 Hz, 1H), 6.28 (d, J = 1.5 Hz, 1H), 3.97 (s, 3H), 3.80 for further statistical analysis. (d, J = 0.7  Hz, 6H), 3.67 (s, 3H). C NMR (100  MHz, Chloroform-d) δ 171.85, 168.95, 167.67, 165.22, 158.95, In vitro evaluation of antioxidant activity by DPPH method 157.67, 148.53, 146.92, 133.72, 131.16, 128.84, 124.78, The antioxidant potential of rutin derivatives was per - 124.78, 123.22, 122.87, 116.52, 113.70, 108.53, 104.92, formed by DPPH method evaluated in the form of 92.81, 90.38, 53.06, 52.81, 52.76 (d, J = 2.6 Hz), 51.65. m/z IC estimated using the ELISA plate reader EPOCH + + found for C H O:478 (M ) 479 (M + 1) . Anal calcd 26 22 9 “MICROPLATE READER (BIOTEK). This method opted for C H O : C, 65.27; H, 4.63; O, 30.10. Found: C, 65.27; 26 22 9 for evaluation of free radical scavenging activity of DPPH H, 4.63; O, 30.10. was based on modified procedure described by Dhiman RU7C yield 71% R 0.7 [Mobile Phase for TLC— 3 f et al. [36]. The tested compounds were prepared in meth - Methanol:Glacial acetic acid:Formic acid:Water anolic solution and reacted with methanolic solution of −1 (3:2.9:0.8:0.5)] M.pt. (165–166) IR (KBR pellets) c m 1) DPPH at 37  °C. The reaction mixture was prepared in 1710 (C=O str.), 1637 (C=N str.), 1596 (C=C str.), 1258 96-well plate by adding 50 µL of sample, 50 µl of meth- (O–CH ), 1194 (C–O str., ester) H NMR (400  MHz, 3 anol and 50 µl of DPPH solution prepared in 0.1  mM DMSO-d ) δ 7.98 (dd, J = 7.5, 1.5  Hz, 1H), 7.76 (d, 6 methanol. The mechanism of action of DPPH assay was J = 1.5  Hz, 1H), 7.30–7.20 (m, 5H), 6.91–6.86 (m, 2H), based on the fact that DPPH radical get reduced during 6.23 (d, J = 1.5  Hz, 1H), 3.93 (s, 3H), 3.88 (d, J = 0.9  Hz, its reaction with an antioxidant compound and results in 6H), 3.69 (s, 3H), 2.93–2.84 (m, 2H), 2.73 (td, J = 7.0, changes of color (from deep violet to light yellow). The 0.8  Hz, 2H). C NMR (100  MHz, Chloroform-d) δ absorbance was read at 517  nm for 30  min at an inter- 175.20, 170.26, 164.48, 160.33, 157.96, 156.95, 150.80, val of 5 min of using ELISA microplate reader. The mix - 149.32, 139.89, 128.47–128.31 (m), 126.14, 123.22, ture of methanol (5.0 ml) and tested compounds (0.2 ml) 122.87, 113.70, 112.82, 107.81, 99.41, 98.77, 53.17, 53.06 serve as blank. Ascorbic acid served as positive control. (d, J = 2.6  Hz), 52.69, 51.86, 34.56, 30.26. m/z found + + for C H O:488 (M ) 489 (M + 1) . Anal calcd for 28 24 8 Hydrogen peroxide scavenging (H O ) assay 2 2 C H O : C, 68.85; H, 4.95; O, 26.20. Found: C, 68.87; H, 28 24 8 To compare and best evaluate the antioxidant potential 4.90; O, 26.20. of newly synthesized rutin derivatives, hydrogen per- oxide assay was performed by the method described by Evaluation of biological activity Patel et  al. [37] with some modifications. The solution In vitro evaluation of xanthine oxidase inhibitory activity of H O (100  mM) was prepared via adding up differ - 2 2 The method opted to evaluate the inhibitory potential ent concentrations of synthesized derivatives ranging of rutin derivatives was a modified protocol of Sigma, from 5 to 80 μg/ml to H O solution (2 ml), prepared in 2 2 done by UV-spectrophotometric method by using xan- 20  mM phosphate buffer of pH 7.4. Finally, the absorb - thine oxidase activity assay kit purchased from sigma ance of H O was measured at 230  nm after incubating 2 2 (MAK078, sigma-aldrich.co, USA). The colorimetric for 10  min next to a blank reading of phosphate buffer product obtained in the form of hydrogen peroxide gen- without H O . For every measurement, a fresh reading 2 2 erated during the oxidation of XO was determined by a of blank was taken to carry out background correction. coupled enzyme technique, measured at 570  nm in a For control sample containing H O was scanned for 2 2 96-well plate, using the plate reader EPOCH “MICRO- absorbance at 230  nm. Results calculated as percentage PLATE READER (BIOTEK).one unit of XO is defined of hydrogen peroxide inhibition was estimated by the as the amount of enzyme that catalyzes the oxidation formula [(A –A )/A ] × 100, where A   is the absorbance b t 0 b of xanthine substrate, yielding 1.0  µmol of uric acid and of the control and A   is the absorbance of compounds/ hydrogen peroxide per minute at 25  °C. Reagents used standard taken as l-ascorbic acid (5–80  μg/ml) are were 44 µL of xanthine oxidase assay buffer, 2 µl xanthine shown in Table 5. substrate solution and 2 µl of Xanthine Oxidase enzyme solution. All the solutions mentioned above were mixed Malik et al. BMC Chemistry (2019) 13:71 Page 7 of 13 Table 1 ADMET data of natural ligands calculated using Qik Prop simulation Compound QPlogPo/w QPlogS QPlogHERG QPPCaco QPlogBB QPPMDCK QPlogKp QPlogKhsa Human oral Percent human absorption oral absorption RU3a − 1.084 − 3.257 − 5.488 511.672 − 2.173 625.905 − 6.818 − 0.902 2 81 RU3a 0.866 − 4.593 − 7.183 605.947 − 1.139 853.322 − 4.846 − 0.635 2 77 RU3a 0.444 − 2.809 − 5.496 758.912 − 1.381 793.01 − 4.796 − 0.58 3 76 RU4b − 0.044 − 3.745 − 6.548 563.916 − 2.192 641.237 − 5.52 − 0.747 1 60 RU4b 0.407 − 4.15 − 6.511 941.594 − 2.757 730.468 − 6.278 − 0.533 1 50 RU7c 3.322 − 4.469 − 6.334 1460.431 − 0.726 744.963 − 1.477 − 0.218 3 100 RU7c 4.878 − 5.717 − 6.59 2335.951 − 0.63 1237.701 − 0.774 0.383 3 100 RU7c − 0.334 − 3.885 − 6.168 743.251 − 1.271 971.012 − 6.276 − 0.735 2 50 Rutin − 0.28 − 2.94 − 5.166 827.655 − 3.378 682.554 − 5.639 − 0.703 1 30 Allopurinol − 1.365 − 2.932 − 0.839 569.551 − 3.6 − 570.702 − 6.890 − 0.986 2 50 Descriptor standard range: QPlogPo/w, − 2.0 to 6.5; QPlogS, − 6.5 to 0.5; QPlogHERG, concern below –5; QPPCaco, < 25 poor, > 500 great; QPlogBB, − 3.0 to 1.2; QPPMDCK, < 25 poor, > 500 great; QPlogKp, − 8.0 to − 1.0; QPlogKhsa, − 1.5 to 1.5; human oral absorption, 1, 2, or 3 for low, medium, or high; percent human oral absorption, > 80% is high ADMET studies Table 2 Comparison of  in  vitro activity and  molecular The pharmacokinetic and pharmacological parameters of docking studies newly synthesized compounds were predicted with the Compound Docking score Binding IC (µM) help of Schrodinger suite. In-silico ADMET-related prop- energy [ΔG erties were computed using Qikprop application of Schro- (KJ/mol)] dinger software (Table 1). QikProp program generates set RU3a − 12.907 − 88.383 09.924 ± 0.01 of physicochemically significant descriptors which further RU3a − 11.456 − 67.673 07.905 ± 0.15 evaluates ADMET properties. The whole ADME-compli - RU3a − 13.244 − 91.242 04.870 ± 0.02 ance score-drug-likeness parameter is used to predict the RU4b − 11.591 − 60.323 15.037 ± 0.01 pharmacokinetic profiles of the ligands. This parameter RU4b − 12.021 − 72.991 12.541 ± 0.45 determines the number of property descriptors calculated 2 RU7c − 11.310 − 55.854 19.377 ± 0.38 via QikProp which fall outside from the optimum range 1 RU7c − 10.980 − 61.268 17.428 ± 0.01 of values for 95% of noted drugs.  Initially, all compound 2 RU7c 11.037 50.217 13.476 ± 0.25 structures were neutralized before operated through Qik- 3 Rutin − 10.944 − 45.549 20.867 ± 0.12 prop. The neutralizing step is crucial, as QikProp is unable Allopurinol − 3.366 − 17.231 10.410 ± 0.72 to neutralize ligands in normal mode. Qikprop predicts both pharmacokinetically significant properties and phys - Italic values indicating standard drug icochemically significant descriptors. It application run in normal mode which predicted IC value for blockage of the synthesized compounds with XO, molecular simu- of HERG K + channels (log HERG), predicted apparent lation studies were carried out using Schrödinger suite Caco-2 cell permeability in nm/s (QPPCaco), brain/blood (Schrödinger Release  2018-2, Schrödinger, LLC, New partition coefficient (QPlogBB), predicted skin perme - York, NY, 2018).The crystal structure of xanthine oxidase ability (QPlogKp), prediction of binding to human serum with PDB code 2E1Q was adopted for the docking calcu- albumin (QPlogKhsa) and predicted apparent Madin– lations. Based on the docking score and binding energy Darby Canine Kidney (MDCK) cell permeability in nm/s calculation, top ranking derivatives were established and (QPPMDCK). Solubility of drug was predicted as octanol/ compared with the IC calculated from in  vitro activ- water partition coefficient (QPlogPo/w). Aqueous solubil - 50 −3 ity (Table  2). Important interactions were depicted as ity of compound defined in terms of log S (S in mol dm ) hydrophobic regions, hydrogen bonding, polar interac- is the concentration of the solute in a saturated solution tions and pi–pi bonding visualized in the active pocket of that is in equilibrium with the crystalline solid. xanthine oxidase revealed through Site map application of Schrodinger suite. The derivatives having better dock - Result and discussion ing scores than rutin were kept for further synthetic pro- Molecular docking cedures and the remaining were discarded. To observe To rationalize the structure activity relationship observed the binding interaction in detail, 3D poses of two most in this research and to foreknow the potential interaction Malik et al. BMC Chemistry (2019) 13:71 Page 8 of 13 Fig. 4 3D pose of RU3a showing hydrogen bonding (yellow) with GLN1194, ARG 912, GLY795, GLN 585 and π–π bonding (blue) with PHE798 Fig. 2 3D pose of RU3a inside the binding pocket Fig. 5 3D pose of RU3a inside the binding pocket Visual inspection of 3D poses of R U3a displayed a Fig. 3 2D pose of RU3a inside the binding pocket 3 compact arrangement of polar and hydrophobic residues around the ligand forming a narrow passage in XO bind- ing cavity with a docking score/binding score of − 13.244 and binding energy − 91.242 kJ/mol. An interesting pi–pi active compounds RU3a and RU3a were visualized and 3 1 bonding was observed between benzene ring of phenyl compared with native rutin and standard drug Allopuri- hydrazine and hydrophobic residue PHE 798 of active nol. The residues of binding pocket involved in the inter - site (Figs.  1, 2, 3). Along with this a strong hydrogen action were reported as GLN 1194, ARG912, MET1038, bonding was observed between OH group of rutinoside GLN1040, PHE798 and SER1080. Similar binding cavity and polar residue GLN 1194 and negatively charged ARG was observed by Li et  al. during the docking analysis of 912 (Fig. 4). Similarly ARG 912 was found essential in the newly synthesized non-purine XO inhibitors [38]. study of Shen et  al. during the comparison of curcumin Malik et al. BMC Chemistry (2019) 13:71 Page 9 of 13 Fig. 6 2D pose of RU3a inside the binding pocket Fig. 8 3D pose of rutin showing hydrogen bonding with GLN 1194 and MET1038 Fig. 7 3D pose of RU3a showing hydrogen bonding with GLN 1194, MET1038 and GLY 1039 Fig. 9 3D pose of allopurinol showing hydrogen bonding with GLN derivatives with quercetin and leuteolin [39]. Another hydrogen bonding was visualized between Chromene moiety and the residues of active site namely GLY 795 ad GLN585. Other hydrophobic amino acid residues closely dihydroxyphenyl ring and GLY1039. One more interac- placed within the cavity were observed as PHE 798, tion was observed with the surrounding residue GLN 767 VAL1200, ALA1198, TYR 592, MET 1038 and ILE1229. which forms a hydrogen bond with MOS 1328 (molyb- On the other hand, during the visualization of R U3a denum metal ion) forming a closed channel to prevent the hydrogen bond was observed with OH group the entry of substrate in the binding site. Other residues of phenyl ring and hydrophobic residue MET 1038 surrounding the ligand were observed as ARG 912, HIE (Figs.  5, 6). Another hydrogen bond was found simi- 579, GLU 1261, ALA 1189 and ILE1198. When the 3D lar to RU3a between OH group of rutinoside and polar poses of these two compounds were compared with the residue GLN1194 (Fig.  7). One more hydrogen bond- native rutin structure, GLN 1194 forms 2 H-bonds, one ing was observed between one of the OH group of with the C=O group of rutin and another with OH group Malik et al. BMC Chemistry (2019) 13:71 Page 10 of 13 Table 3 In vitro xanthine oxidase inhibitory activity of rutin derivatives Compound IC (µM) ± SEM Compound IC (µM) ± SEM 50 50 Rutin 20.867 ± 0.12 RU4b 12.541 ± 0.45 RU3a 09.924 ± 0.01 RU7c 19.377 ± 0.38 1 1 RU3a 07.905 ± 0.15 RU7c 17.428 ± 0.01 2 2 RU3a 04.870 ± 0.02 RU7c 13.476 ± 0.25 3 3 RU4b 15.037 ± 0.01 Allopurinol 10.410 ± 0.72 SEM, standard error of the mean of rutinoside (Fig.  8). The amino acid residues GLU1261 and GLN 1194 were found to be interacted similarly in the study of verbascoside by Wan et  al. [40]. Beside this one H-bond was formed between OH group of chromene ring and MET1038. No pi–pi interaction was in the native structure rutin. In case of Allopurinol, the Fig. 11 Lineweaver–Burk plot for RU3a against different active site residues surrounding ligand were almost sim- concentrations ilar and placed near to MOS 1328. The hydrogen bond was observed between purine ring of allopurinol and GLN1194 (Fig. 9). (RU7c –RU7c ). All the compounds of hydrazine 1 3 series (RU3a –RU3a ) were effective with IC -values 1 3 50 In‑vitro xanthine oxidase inhibitory activity ranging from 04.870 to 09.924  µM. Rutin hybridized In order to monitor the efficacy of different synthesized with phenyl hydrazine demonstrated highest activity rutin derivatives, xanthine oxidase inhibitory activity against xanthine oxidase. While thisemicarbazide and was determined using xanthine oxidase activity assay phenylthiosemicarbazide derivatives of rutin showed kit purchased from Sigma-aldrich Co. Allopurinol a slight decrease in activity indicating the role of sul- (positive control) reported to inhibit xanthine oxidase fur group in diminishing the inhibition and NH–NH was also screened under identical conditions for com- group in enhancing the activity of targeted enzyme. parison. The inhibition ratios revealed the xanthine Surprisingly, substitution of NH–NH with N H group 2 2 oxidase inhibitory activity of the synthesized rutin leads to decrease of inhibitory activity. Ester deriva- derivatives and the results were summarized in Table 3. tives of rutin synthesized after the hydrolysis of rutin As expected, these rutin derivatives exhibited remark- exhibited a weaker inhibition than the positive control able activity comparable to the positive control. Based Allopurinol. on the in  vitro activity; it was observed that hydrazine The results of in  vitro activity showed 80% similarity (RU3a –RU3a ) and anilline analogues (R U4b –RU4b ) with the results of molecular docking with a few excep- 1 3 1 2 were considerably more effective than ester derivatives tions. In concordance with the screening and output of OH OH OH HO O HO OH O CH 3 Presence of glycosidic 3-O-rutinoside linkage is OH O Addition of thiosemicarbazide group essential for the xanthine oxidase inhibitory potential, as showed the XO inhibition moderately. HO OH detachment of group diminished the XO inhibitory activity. OH Rutin Incorporation of hydrazide groups remarkably Addition of phenylthiosemicarbazide group increased the XO inhibitory action. significantly increased the XO inhibition. Fig. 10 Structure activity relationship (SAR) of synthesized compounds Malik et al. BMC Chemistry (2019) 13:71 Page 11 of 13 Table 4 K and  V values of  xanthine oxidase m max at different concentrations of  RU3a S. no.Conc. of  RU3a Km (µM) V (µmol/min) 3 max (µM) 1. 0.0 27.21 119.6 2. 0.25 30.11 114.4 3. 0.5 32.90 108.2 4. 1.0 35.08 98.7 of most active compound R U3a using Graph pad prism software. Firstly Michaelis–Menten curve was plotted for the enzyme activity at different concentrations of RU3a against different concentration of substrate (xanthine) Fig. 11. Then double reciprocal plot (Lineweaver–Burk) anal - ysis was done in the presence (0.25, 0.5, and 1.0  µM) Fig. 12 Michaelis–Menten curve for RU3a at different and absence of RU3a from in  vitro data generated concentrations during the oxidation of xanthine in presence of xan- thine oxidase (Fig.  12). The x- and y axis intercepts of the Lineweaver–Burk plot were utilized to calculate K and V values of RU3a at different concentrations molecular docking R U3a comes out to be most active max 3 (Table 4). rutin derivative showing very good interaction with xan- A concentration-dependent decrease of V was thine oxidase at molecular level. Elimination of rutino- max predicted in contrast to K value which was found to side from rutin to synthesize ester derivatives results in m increasing when concentration of R U3a was increased. a loss of potency with a threefold decrease of inhibitory 3 The intersection of linear straight lines drawn against potential. each concentration was located at same point, suggesting that RU3a reacts in competitive manner during the inhi- bition of xanthine oxidase. Structure activity relationship (SAR) Few interesting notions about the relationship of activ- In‑vitro evaluation of antioxidant activity by DPPH ity and structures of synthesized compounds emerged and  H O method 2 2 from the present research (Fig.  10): (A) Rutinoside The antioxidant potential of newly synthesized com - moiety seems to be important for the activity, as dele- pounds was evaluated by DPPH and Hydrogen peroxide tion of this leads to loss of activity could be seen from radical assay. The comparative analysis of IC values xanthine oxidase inhibitory activity Table  3. Which for both the assays was done and the results were found shows RU3a (Having rutinoside group) exhibited to be impressive (Table  5). The results evinced a note - highest activity with an IC50 value 04.870  µM among worthy inhibition of DPPH almost all the compounds all the compounds and RU7c showed lowest activ- when compared with the positive control ascorbic acid. ity and fivefold decrease of activity with an IC value In case of DPPH assay compound RU4b was demon- 19.377  µM. (B) Hydrazine derivatives were found to strated as most potent compound against oxidative stress be more effective than the aniline derivatives reveal- caused because of free radicals having an IC value of ing the importance of NH–NH group. But substitu- 02.647 ± 0.09 µM. Along with this compound RU3a also tion of sulfur group along with hydrazines decreases showed very good antioxidant potential with an IC the activity as in R U3a and R U3a and substitution of 3 2 value of 05.021 ± 0.10  µM. When the detailed structure phenyl group along with sulfur improves the activity activity relationship was developed between these com- (RU3a ). (C) Substitution with ester group leads to a pounds, it was concluded that both the compounds hav- decrease of inhibitory activity. ing hydrazine linkage derived from phenyl hydrazine and phenyl thiosemicarbazide. Similarly, during the analy- Enzyme kinetic analysis for XO‑inhibitory activity sis of hydrogen peroxide assay all the compounds with To determine the XO-inhibitory mechanisms of newly hydrazines substitution showed very good antioxidant synthesized derivatives, we carried out kinetic studies Malik et al. BMC Chemistry (2019) 13:71 Page 12 of 13 Table 5 Antioxidant activity of  synthesized derivatives Additional file by DPPH and  H O method 2 2 Compound IC (µM) ± SEM IC (µM) ± SEM Additional file 1. HNMR spectra of compound RU3a 50 50 3 RU3a 05.021 ± 0.10 09.134 ± 0.35 Acknowledgements RU3a 08.728 ± 0.02 04.146 ± 0.01 The authors are highly thankful to the Head, Department of Pharmaceuti- RU3a 11.688 ± 0.01 06.561 ± 0.10 3 cal Sciences, M. D. University, Rohtak for providing essential facilities to accomplish this research study. The authors are also thankful to Dr. Vinod RU4b 02.647 ± 0.09 09.863 ± 0.25 Devaraji Application Scientist Schrödinger LLC for his support to carry out the RU4b 08.476 ± 0.25 04.378 ± 0.01 computational work. RU7c 06.056 ± 0.13 14.731 ± 0.60 Authors’ contributions RU7c 14.669 ± 0.01 12.126 ± 0.20 Authors NM and AK have designed, synthesized and carried out the xanthine RU7c 07.692 ± 0.42 17.884 ± 0.41 oxidase inhibitory and antioxidant activity and the author PD, have carried RU001 09.483 ± 0.08 18.623 ± 0.07 out the docking simulations with in silico ADMET studies. All authors read and approved the final manuscript. Ascorbic acid 22.195 ± 0.08 22.195 ± 0.08 SEM, standard error of the mean Funding No funding received for this research work from outside sources. Availability of data and materials potential having IC in range of 04.146 ± 0.01 to Not applicable. 09.134 ± 0.35 (Fig.  7). Compound R U3a having phenyl Competing interests thiosemicarbazide substitution showed potential antioxi- The authors declare that they have no competing interests. dant activity among all the derivatives. Along with this phenyl hydrazine substituted rutin derivative (RU3a ) Author details Faculty, Department of Pharmaceutical Sciences, M.D. University, also showed very good scavenging activity with an IC Rohtak 124001, India. Laboratory for Preservation Technology and Enzyme value of 06.561 ± 0.10. When the detailed structure Inhibition Studies, Department of Pharmaceutical Sciences, M.D. University, activity relationship was developed between these com- Rohtak, Haryana, India. pounds, it was concluded that both the compounds hav- Received: 21 January 2019 Accepted: 2 May 2019 ing hydrazine linkage derived from phenyl hydrazine and phenyl thiosemicarbazide. References Conclusion 1. Berry CE, Hare JM (2004) Xanthine oxidoreductase and cardiovascular Starting from the structures of rutin as anti-XO hit pre- disease: molecular mechanisms and pathophysiological implications. J viously identified, different series of novel analogues Physiol 555(3):589–606 2. Moriwaki Y, Yamamoto T, Higashino K (1997) Distribution and pathophysi- were designed and synthesized to explore the struc- ologic role of molybdenum-containing enzymes. Histol Histopathol ture–activity relationships associated with these xan- 12(2):513–524 thine oxidase inhibitors along with their antioxidant 3. Klinenberg JR, Goldfinger SE, Seegmiller JE (1965) The effectiveness of the xanthine oxidase inhibitor allopurinol in the treatment of gout. Ann potential. Different structural elements were identified Intern Med 62(4):639–647 as essential for antioxidant and anti-XO properties, such 4. Yu KH (2007) Febuxostat: a novel non-purine selective inhibitor of as the presence of rutinoside (R U3a, RU3a and R U3a ) xanthine oxidase for the treatment of hyperuricemia in gout. Recent Pat 1 2 3 Inflamm Allergy Drug Discov 1(1):69–75 comes out as important skeleton for the inhibitory 5. Battelli MG, Bolognesi A, Polito L (2014) Pathophysiology of circulat- potential, presence of hydrazone linker along with phe- ing xanthine oxidoreductase: new emerging roles for a multi-tasking nyl group, while the associated xanthine oxidase inhibi- enzyme. Biochim Biophys Acta Mol Basis Dis 1842(9):1502–1517 6. Brass CA, Narciso J, Gollan JL (1991) Enhanced activity of the free radical tory effect was found to follow a different trend for the producing enzyme xanthine oxidase in hypoxic rat liver. Regulation and two series hydrazine (RU3a ) and ester derivatives 1–3 pathophysiologic significance. J Clin Invest 87(2):424–431 (RU7c ). The newly synthesized derivatives with anti- 7. Chambers DE, Parks DA, Patterson G, Roy R, McCord JM, Yoshida S, Par- 1–3 mley LF, Downey JM (1985) Xanthine oxidase as a source of free radical oxidant and ani-XO IC values in the low micromolar damage in myocardial ischemia. J Mol Cell Cardiol 17(2):145–152 range and good selectivity indexes were identified. Con - 8. Desco MC, Asensi M, Márquez R, Martínez-Valls J, Vento M, Pallardó temporary synthetic efforts are focused towards the FV, Sastre J, Viña J (2002) Xanthine oxidase is involved in free radical production in type 1 diabetes: protection by allopurinol. Diabetes insertion of the hydrazones and ester linkage by replac- 51(4):1118–1124 ing the side linkage rutinoside of rutin with more sta- 9. Kuppusamy P, Zweier JL (1989) Characterization of free radical generation ble groups while maintaining the overall length of new by xanthine oxidase. Evidence for hydroxyl radical generation. J Biol Chem 264(17):9880–9884 derivatives. Molecular docking provide an improved 10. Dawson J, Walters M (2006) Uric acid and xanthine oxidase: future trail to design the new molecules with an avantgarde therapeutic targets in the prevention of cardiovascular disease? Br J Clin stability and potency. Pharmacol 62(6):633–644 Malik et al. BMC Chemistry (2019) 13:71 Page 13 of 13 11. Khosla UM, Zharikov S, Finch JL, Nakagawa T, Roncal C, Mu W, Krotova 29. Khosravan R, Grabowski BA, Wu JT, Joseph-Ridge N, Vernillet L (2006) K, Block ER, Prabhakar S, Johnson RJ (2005) Hyperuricemia induces Pharmacokinetics, pharmacodynamics and safety of febuxostat, a non- endothelial dysfunction. Kidney Int 67(5):1739–1742 purine selective inhibitor of xanthine oxidase, in a dose escalation study 12. Kaynar H, Meral M, Turhan H, Keles M, Celik G, Akcay F (2005) Glutathione in healthy subjects. Clin Pharmacokinet 45(8):821–841 peroxidase, glutathione-S-transferase, catalase, xanthine oxidase, Cu–Zn 30. Malik N, Dhiman P, Khatkar A (2017) In-silico design and ADMET studies of superoxide dismutase activities, total glutathione, nitric oxide, and natural compounds as inhibitors of xanthine oxidase (XO) enzyme. Curr malondialdehyde levels in erythrocytes of patients with small cell and Drug Metab 18(6):577–593 non-small cell lung cancer. Cancer Lett 227(2):133–139 31. Muhammad A, Arthur DE, Babangida S, Erukainure OL, Malami I, Sani 13. Griguer CE, Oliva CR, Kelley EE, Giles GI, Lancaster JR, Gillespie GY (2006) H, Abdulhamid AW, Ajiboye IO, Saka AA, Hamza NM, Asema S (2018) Xanthine oxidase-dependent regulation of hypoxia-inducible factor in Modulatory role of rutin on 2, 5-hexanedione-induced chromosomal and cancer cells. Cancer Res 66(4):2257–2263 DNA damage in rats: validation of computational predictions. Drug Chem 14. Kanellis J, Kang DH (2005) Uric acid as a mediator of endothelial dysfunc- Toxicol 10:1–4 tion, inflammation, and vascular disease. Seminars in nephrology, vol 25. 32. Roleira FM, Varela CL, Costa SC, Tavares-da-Silva EJ (2018) Phenolic New York, WB Saunders, pp 39–42 derivatives from medicinal herbs and plant extracts: anticancer effects 15. Miesel R, Zuber M (1993) Elevated levels of xanthine oxidase in serum of and synthetic approaches to modulate biological activity. Nat Prod Chem patients with inflammatory and autoimmune rheumatic diseases. Inflam- 57:115–156 mation 17(5):551–561 33. Baldisserotto A, Vertuani S, Bino A, De Lucia D, Lampronti I, Milani R, 16. Wijermars LG, Bakker JA, de Vries DK, van Noorden CJ, Bierau J, Kostidis Gambari R, Manfredini S (2015) Design, synthesis and biological activity S, Mayboroda OA, Tsikas D, Schaapherder AF, Lindeman JH (2016) The of a novel Rutin analogue with improved lipid soluble properties. Bioorg hypoxanthine–xanthine oxidase axis is not involved in the initial phase of Med Chem 23(1):264–271 clinical transplantation-related ischemia–reperfusion injury. Am J Physiol 34. Gullon B, Lú-Chau TA, Moreira MT, Lema JM, Eibes G (2017) Rutin: a Renal Physiol 312(3):F457–F464 review on extraction, identification and purification methods, biological 17. Poles MZ, Bódi N, Bagyánszki M, Fekete É, Mészáros AT, Varga G, Szűcs S, activities and approaches to enhance its bioavailability. Trends Food Sci Nászai A, Kiss L, Kozlov AV, Boros M (2018) Reduction of nitrosative stress Technol 67:220–235 by methane: neuroprotection through xanthine oxidoreductase inhibi- 35. Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, Repasky tion in a rat model of mesenteric ischemia–reperfusion. Free Radic Biol MP, Knoll EH, Shelley M, Perry JK, Shaw DE (2004) Glide: a new approach Med 120:160–169 for rapid, accurate docking and scoring. 1. Method and assessment of 18. Osada Y, Tsuchimoto M, Fukushima H, Takahashi K, Kondo S, Hasegawa docking accuracy. J Med Chem 47(7):1739–1749 M, Komoriya K (1993) Hypouricemic effect of the novel xanthine oxidase 36. Dhiman P, Malik N, Verma PK, Khatkar A (2015) Synthesis and biologi- inhibitor, TEI-6720, in rodents. Eur J of Pharmacol 241(2–3):183–188 cal evaluation of thiazolo and imidazo N-(4-nitrophenyl)-7-methyl- 19. Krakoff IH, Meyer RL (1965) Prevention of hyperuricemia in leukemia 5-aryl-pyrimidine-6 carboxamide derivatives. Res Chem Intermed and lymphoma: use of allopurinol, a xanthine oxidase inhibitor. JAMA 41(11):8699–8711 193(1):1–6 37. Patel A, Patel A, Patel A, Patel NM (2010) Determination of polyphenols 20. Pacher PA, Nivorozhkin A, Szabó C (2006) Therapeutic effects of xanthine and free radical scavenging activity of Tephrosia purpurea linn leaves oxidase inhibitors: renaissance half a century after the discovery of (Leguminosae). Pharmacogn Res 2:152–154 allopurinol. Pharmacol Rev 58(1):87–114 38. Li P, Tian Y, Zhai H, Deng F, Xie M, Zhang X (2013) Study on the activity 21. Inkster ME, Cotter MA, Cameron NE (2007) Treatment with the xanthine of non-purine xanthine oxidase inhibitor by 3D-QSAR modeling and oxidase inhibitor, allopurinol, improves nerve and vascular function in molecular docking. J Mol Struct 5(1051):56–65 diabetic rats. Eur J Pharmacol 561(1–3):63–71 39. Shen L, Ji HF (2009) Insights into the inhibition of xanthine oxidase by 22. Sagor M, Taher A, Tabassum N, Potol M, Alam M (2015) Xanthine oxidase curcumin. Bioorg Med Chem Lett 19(21):5990–5993 inhibitor, allopurinol, prevented oxidative stress, fibrosis, and myocardial 40. Wan Y, Zou B, Zeng H, Zhang L, Chen M, Fu G (2016) Inhibitory effect damage in isoproterenol induced aged rats. Oxid Med Cell Longev. https of verbascoside on xanthine oxidase activity. Int J Biol Macromol ://doi.org/10.1155/2015/47803 9 1(93):609–614 23. Min HK, Lee B, Kwok SK, Ju JH, Kim WU, Park YM, Park SH (2015) Allopuri- nol hypersensitivity syndrome in patients with hematological malignan- Publisher’s Note cies: characteristics and clinical outcomes. Korean J Intern Med 30(4):521 Springer Nature remains neutral with regard to jurisdictional claims in pub- 24. Quach C, Galen BT (2018) HLA-B* 5801 testing to prevent allopuri- lished maps and institutional affiliations. nol hypersensitivity syndrome: a teachable moment. JAMA Int Med 178(9):1260–1261 25. Takano Y, Hase-Aoki K, Horiuchi H, Zhao L, Kasahara Y, Kondo S, Becker MA (2005) Selectivity of febuxostat, a novel non-purine inhibitor of xanthine oxidase/xanthine dehydrogenase. Life Sci 76(16):1835–1847 26. Mayer MD, Khosravan R, Vernillet L, Wu JT, Joseph-Ridge N, Mulford DJ (2005) Pharmacokinetics and pharmacodynamics of febuxostat, a new non-purine selective inhibitor of xanthine oxidase in subjects with renal impairment. Am J Ther 12(1):22–34 27. Nepali K, Singh G, Turan A, Agarwal A, Sapra S, Kumar R, Banerjee UC, Ready to submit your research ? Choose BMC and benefit from: Verma PK, Satti NK, Gupta MK, Suri OP (2011) A rational approach for the design and synthesis of 1-acetyl-3, 5-diaryl-4, 5-dihydro (1H) pyrazoles as fast, convenient online submission a new class of potential non-purine xanthine oxidase inhibitors. Bioorg thorough peer review by experienced researchers in your field Med Chem 19(6):1950–1958 28. Becker MA, Kisicki J, Khosravan R, Wu J, Mulford D, Hunt B, MacDonald rapid publication on acceptance P, Joseph-Ridge N (2004) Febuxostat ( TMX-67), a novel, non-purine, support for research data, including large and complex data types selective inhibitor of xanthine oxidase, is safe and decreases serum • gold Open Access which fosters wider collaboration and increased citations urate in healthy volunteers. 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In silico design and synthesis of targeted rutin derivatives as xanthine oxidase inhibitors

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Springer Journals
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Copyright © 2019 by The Author(s)
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Chemistry; Chemistry/Food Science, general
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2661-801X
DOI
10.1186/s13065-019-0585-8
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Abstract

Background: Xanthine oxidase is an important enzyme of purine catabolism pathway and has been associated directly in pathogenesis of gout and indirectly in many pathological conditions like cancer, diabetes and metabolic syndrome. In this research rutin, a bioactive flavonoid was explored to determine the capability of itself and its deriva- tives to inhibit xanthine oxidase. Objective: To develop new xanthine oxidase inhibitors from natural constituents along with antioxidant potential. Method: In this report, we designed and synthesized rutin derivatives hybridized with hydrazines to form hydrazides and natural acids to form ester linkage with the help of molecular docking. The synthesized compounds were evalu- ated for their antioxidant and xanthine oxidase inhibitory potential. Results: The enzyme kinetic studies performed on rutin derivatives showed a potential inhibitory effect on XO abil- ity in competitive manner with IC value ranging from 04.708 to 19.377 µM and RU3a was revealed as most active 50 3 derivative. Molecular simulation revealed that new rutin derivatives interacted with the amino acid residues PHE798, GLN1194, ARG912, GLN 767, ALA1078 and MET1038 positioned inside the binding site of XO. Results of antioxidant activity revealed that all the derivatives showed very good antioxidant potential. Conclusion: Taking advantage of molecular docking, this hybridization of two natural constituent could lead to desirable xanthine oxidase inhibitors with improved activity. Keywords: Rutin, Xanthine oxidase, Molecular docking, Antioxidant Introduction on the xanthine and oxygen at the enzymatic centre. Xanthine oxidase (XO) having molecular weight of While xanthine undergoes oxidation reaction near to the around 300 kDa is oxidoreductase enzyme represented in Mo-pt center/substrate binding domain of XO, simulta- the form of a homodimer. Both the monomers of XO are neously substrate oxygen undergoes reduction at FAD almost identical and each of them contains three domains center and electron transfer takes place leading to for- 2− namely (a) molybdopterin (Mo-pt) domain at the C-ter- mation of superoxide anion (O ) or hydrogen peroxide minal having 4 redox centers where oxidation takes place (H O ) free radicals. [4–8]. This catalytic reaction results 2 2 (b) a flavin adenine dinucleotide (FAD) domain at the in formation uric acid as a final product and oxygen reac - centre generally considered as binding site domain and tive species in form of free radicals. The excessive genera - (c) 2[Fe–S]/iron sulfur domain at the N-terminal [1–3]. tion of uric acid leads to a condition like hyperuricemia The catalytic oxidation of XO is two substrates reaction which is a key factor in development of gout [1, 9], and uncontrolled amounts of reactive oxygen species causes many pathological conditions like cardiovascular disor- ders, inflammatory diseases and hypertensive disorders. *Correspondence: dranuragkhatkarmdurtk@gmail.com; anuragpharmacy@gmail.com Xanthine oxidase (XO; EC 1.17.3.2) has been consid- Laboratory for Preservation Technology and Enzyme Inhibition Studies, ered as significantly potent drug target for the cure and Department of Pharmaceutical Sciences, M.D. University, Rohtak, Haryana, management of pathological conditions prevailing due India Full list of author information is available at the end of the article to high levels of uric acid in the blood stream. [10–17]. © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Malik et al. BMC Chemistry (2019) 13:71 Page 2 of 13 Considering the above fact, by inhibiting XO selectively evaluation of the human xanthine oxidase inhibitory could be better treatment plan for disorders caused by activity was performed by measuring hydrogen peroxide XO directly or indirectly including gout, inflammatory (H O ) production from oxidation of xanthine oxidase 2 2 disease, oxidative damage and cancer [3, 18, 19]. Gen- by the substrate xanthine, utilizing the human xanthine erally, XO inhibitors have been categorized into purine oxidase assay kit (Sigma USA). The progress of reaction and non-purines inhibitors differentiated on the basis was observed through thin layer chromatography (TLC) of their chemically derived skeleton structure. The first on 0.25  mm precoated silica gel plates purchased from purine derived XO inhibitor discovered and approved by Merck, reaction spots were envisaged in iodine compart- US FDA was Allopurinol as marketed drug for gout and ment and UV. Melting points were measured using a hyperuricemia [20, 21]. Considering the life threatening Sonar melting point apparatus and uncorrected. H NMR side effects like Stevens–Johnsons syndrome caused by and C NMR spectra were documented in DMSO and allopurinol use, scientists turned their interest into non-deuterated CDCl respectively on Bruker Avance II 400 purine XO inhibitors and an immense accomplishment NMR spectrometer at the frequency of 400  MHz using has been received in this direction with development of tetramethylsilane standard (downfield) moreover chemi - new drug Febuxostat [22–25]. This non-purine candi - cal shifts were expressed in ppm (δ) using the residual date produced minor and non-life threatening adverse solvent line as internal standard. Infrared (IR) spectra effects in comparison to Allopurinol [26–29]. Extending were recorded on Perkin Elmer FTIR spectrophotometer our previous successful effort to achieve new xanthine by utilizing KBr pellets system. oxidase inhibitors from natural sources, in this report we investigated and developed some new rutin derived xan- thine oxidase inhibitor [30]. Molecular docking Rutin is a well characterized bioactive plant flavonoid In silico docking studies was done with integrated Schro- having great therapeutic importance for the treatment of dinger software using Glide module for enzyme ligand many disease like conditions including cytotoxicity, anti- docking [35]. oxidant activity, antibacterial property and anti-inflam - matory action [31–34]. Due to these pharmacological Protocol followed for docking procedures activities rutin is explored widely and great success have Preparation of  protein The 3D crystal structure of been achieved in order to get drug like candidates. human xanthine oxidase co-crystalised with salicylic acid was retrieved from Protein Data Bank (PDB ID. 2E1Q). OH The targeted protein structure was further refined in the OH Protein Preparation Wizard to obtain the optimized and OH HO O chemically accurate protein configuration. For that, the HO OH co-crystalised enzyme (XO) was retrieved directly from O O CH 3 Protein data bank in maestro panel followed by removal of OH O water molecules, addition of H atoms, addition of missing HO OH OH side chains and finally minimization was done to obtain the optimized structure. Rutin Preparation of ligand The 3D-structures of rutin derived compounds to be docked against XO were built in maes- Taking advantage of molecular docking techniques new tro building window. Ligand preparation was performed compounds with potential drugability for the targeted in Ligprep module. enzyme might be achieved with a precise knowledge of mechanism of action. With the combined approach Active site prediction To predict the binding site/active of molecular docking and synthetic chemistry, in this site Site Map application of glide was utilized. Out of top research we developed some new potential compounds three active site, the one having larger radius was selected. against xanthine oxidase (Fig. 1). Validation of binding site was done by redocking the sali- cylic acid and RMSD value was observed. RMSD value of less than 0.2 validated the docking procedure and active Experimental site was defined for docking of new rutin analogs. Chemicals and instrumentation For this research, the analytical grade chemicals nec- Glide docking To carry out docking, Firstly the recep- essary for synthesis and antioxidant activity were pur- tor grid generation tool was utilized to around the active/ chased from Hi-media Laboratories. The in  vitro Malik et al. BMC Chemistry (2019) 13:71 Page 3 of 13 OH OH OH HO O HO OH OH OH OH OH OO CH OH OH OH HO O HO O HO OH HO OH HO OH HN OH OO CH 3 OO CH OH OH RU3a3 Cl HO OH HO OH HN OH OH NH HN RU4b1 NO NO 2 2 RU3a2 OH H N HN OH NO NH-NH 2 N 2 OH HO O HO OH Phenyl thiosemicarbazide OH OO CH OH OH H N HO OH 2 HN N NH 2 OH HO O OH H S HO OH Thiosemicarbazide H N OH RU3a1 OO CH OH H N NO 2 2 3 OO H OH 4-Nitrobenzenamine HO OH HO O HO HO OH OH Rutin O OO CH OH Cinnamic Acid HO OH OH HO HO NO RU4b2 OCH OCH 3 3 OCH Salicylic acid OCH OCH 3 OCH HO H CO O H CO O 3 H CO O O N O O Nicotinic acid O OO CH OO CH 3 3 OO CH OH RU7c2 RU7c1 RU7c3 Fig. 1 Design strategy for the development of rutin derivatives binding site of xanthine oxidase and glide docking with zines (0.001 mol) were added to the flask and reac - extra precision was used to visualize the interaction of tion mixture was refluxed for 5–6 h at 40 °C. Com - protein and ligand. The top active ligand was selected for pletion of reaction was monitored by TLC. The wet lab synthesis and evaluation of pharmacological activ- product thus obtained was filtered and filtrate was ity. concentrated to obtain the final product. The final product was recrystallised to obtain the pure com- Synthetic procedures pound. Procedures for synthesis of rutin derivatives (Scheme 1) (B) General procedure for synthesis of anilline deriva- tives RU4b (1–2) 0.001  mol of the intermediate obtained above (A) General procedure for synthesis of hydrazine deriva- was taken in round bottom flask and dissolved in tives RU3a (1–4) 50  ml of ethanol. Different anillines (0.001  mol) were added to the flask and reaction mixture was 0.001 mol of rutin was taken in round bottom flask refluxed for 8–10 h at 40 °C. Completion of reaction and dissolved in 50  ml of ethanol. Different hydra - Malik et al. BMC Chemistry (2019) 13:71 Page 4 of 13 OH OH OH HO O HO OH OO CH OH OH HO OH OH HN OH OH OH OH HO O OH HO O HO OH RU3a HO OH OO CH O 3 O OH OO CH N OH HO OH H N 2 HN Cl Reflux HO OH HN S OH 8-10 hrs OH HN N NH-NH NO 2 RU4b 1 Reflux RU3a NH 8-10 hrs NO 2 Reflux 8-10 hrs OH OH OH OH OH NO S OH OH HO O OH HO O H N HO OH 2 OH Reflux N NH HO O HO OH 2 8-10 hrs H HO OH OO CH 3 O OO H Reflux OO CH O OH N OO CH H N NO 8-10 hrs 3 2 2 OH HO OH HO OH OH HO OH HN OH S OH Rutin a) K CO H N CH I 2 3 2 NO RU4b DMF, RT,2d RU3a b) HCL,95% ethanol reflux,2h; HO OCH HO 3 OCH OCH OCH OCH OCH 3 3 O N H CO O 3 H CO O NICOTINIC ACID H CO O 3 3 CINNAMIC ACID Reflux 5hr OH O Reflux 5hr OO CH 3 OO CH OO CH 3 3 RUI HO HO Reflux 5hr RU7c O OCH OCH RU7c H CO O OO CH OH RU7c Scheme 1 Synthesis of rutin derivatives was monitored by TLC. The product thus obtained Spectral data RU3a yield 69.6% R 0.6 [Mobile 1 f was filtered and filtrate was concentrated to obtain Phase for TLC—Methanol:Glacial acetic acid:Formic the final product. The final product was recrystal - acid:Water (3:2.9:0.8:0.5)] M.pt. (231–232) IR (KBR pel- −1 lised to obtain the pure compound.lets) cm 1) 3222 (O–H str., Ar), 1609 (C=N str.), 1501 (C) General procedure for synthesis of methylated rutin (C=C str.), 1206 (O–CH ), 1128 (C=S Str.) H NMR derivatives RU7c (400  MHz, DMSO-d ) δ 7.81 (dd, J = 7.5, 1.5  Hz, 1H), (1–3) 6 Rutin was methylated by methyl sulphate in pres- 7.59 (d, J = 1.5  Hz, 1H), 6.82 (d, J = 7.5  Hz, 1H), 6.48 ence of potassium carbonate and dimethyl forma- (dd, J = 15.0, 1.5  Hz, 2H), 6.28 (t, J = 7.0  Hz, 1H), 4.13 mide by stirring along with reflux at 40 °C for 48 h (t, J = 7.0 Hz, 1H), 3.89–3.81 (m, 3H), 3.71 (dd, J = 12.4, to generate tetramethylated rutin. Acidolysis of 6.9 Hz, 1H), 3.67–3.54 (m, 3H), 2.32 (dt, J = 12.4, 7.0 Hz , above was done to obtain the intermediate com- 1H), 2.28–2.16 (m, 2H), 2.06–2.04 (m, 1H), 1.97–1.92 pound (RUI) by refluxing it with HCl and 95% etha - (m, 2H), 1.74–1.66 (m, 2H). C NMR (100 MHz , Chloro- nol for 4 h. The intermediate compound (RUI) was form-d) δ 180.16, 163.73, 155.81, 154.70, 152.34, 148.70, then refluxed with different phenolic acid to obtain 145.50, 133.79, 133.45, 120.73, 120.41, 115.79, 115.09, their ester derivatives. 102.38, 99.59, 99.00, 91.11, 80.48, 73.58, 73.26, 72.40, 71.83 (d, J = 10.5  Hz), 66.02, 40.22, 37.43, 28.26, 26.90. Malik et al. BMC Chemistry (2019) 13:71 Page 5 of 13 + + m/z found for C H N O S: 683 (M ) 687 (M + 1) . J = 1.5 Hz, 1H), 7.40 (d, J = 7.5 Hz, 1H), 6.81 (d, J = 7.5 Hz, 28 33 3 15 Anal calcd for C H N O S: C, 52.91; H, 5.23; N, 6.61; 1H), 6.47 (dd, J = 10.8, 1.5  Hz, 2H), 6.22 (t, J = 7.0  Hz, 28 33 3 15 O, 35.20; S, 5.04 Found: C, 52.93; H, 5.21; N, 6.60; O, 1H), 4.11 (t, J = 7.0 Hz, 1H), 3.98–3.90 (m, 3H), 3.79 (dd, 35.19; S, 5.06. J = 12.4, 6.9 Hz, 1H), 3.71–3.61 (m, 3H), 2.42 (dt, J = 12.4, RU3a yield 72.5% R 0.7 [Mobile Phase for TLC— 7.0  Hz, 1H), 2.39– 2.31 (m, 2H), 2.29–2.28 (m, 1H), 2 f Methanol:Glacial acetic acid:Formic acid:Water 1.87–1.77 (m, 2H). C NMR (100  MHz, Chloroform-d) −1 (3:2.9:0.8:0.5)] M.pt. (255–257) IR (KBR pellets) c m ) δ 169.14, 168.95, 168.11, 166.86, 150.94, 144.52, 144.24, 3468 (O–H str., Ar), 1639 (C=N str.), 1596 (C=C str.), 142.37, 140.47, 131.18, 128.56, 125.41, 123.81, 122.54 (d, 1218 (O–CH ), 1150 (C=S Str.) H NMR (400  MHz, J = 14.8  Hz), 121.81, 113.64, 113.17, 106.71, 97.09, 96.89, DMSO-d ) δ 7.78–7.60 (m, 3H), 7.49 (d, J = 1.5  Hz, 1H), 93.98, 82.37, 75.79 (d, J = 19.1 Hz), 73.17 (d, J = 12.2  Hz), 7.39–7.29 (m, 2H), 7.10–7.01 (m, 1H), 6.86 (d, J = 7.5  Hz, 73.06, 72.69, 71.01, 65.19, 41.10, 38.86, 28.85, 27.44. m/z + + 1H), 6.52 (dd, J = 15.0, 1.5  Hz, 2H), 6.24 (t, J = 7.0  Hz, found for H ClN O : 764 (M ) 766 (M + 2) . Anal calcd 33 2 17 1H), 4.04 (t, J = 7.0 Hz, 1H), 3.98–3.88 (m, 3H), 3.78 (dd, for C H ClN O : C, 51.81; H, 4.35; Cl, 4.63; N, 3.66; O, 33 33 2 17 J = 12.4, 6.9 Hz, 1H), 3.68–3.64 (m, 3H), 2.28 (dt, J = 12.4, 35.55. Found: C, 51.83; H, 4.36; Cl, 4.65; N, 3.64; O, 35.53. 7.0 Hz, 1H), 2.14–2.11 (m, 2H), 2.09–2.06 (m, 1H), 1.87– RU4b yield 83.5% R 0.8 [Mobile Phase for TLC— 2 f 1.84 (m, 2H), 1.74–1.71 (m, 2H). C NMR (100  MHz, Methanol:Glacial acetic acid:Formic acid:Water −1 Chloroform-d) δ 174.93, 164.50, 160.96, 155.78, 150.30, (3:2.9:0.8:0.5)] M.pt. (253–254) IR (KBR pellets) c m 1) 148.16, 145.55, 139.23, 130.44, 128.67, 124.46, 123.85, 1785 (C=O str.), 1637 (C=N str.), 1561 (C=C str.), 1258 123.09, 122.39, 121.81, 116.06, 115.83, 103.40, 99.09, (O–CH ), 1234 (C–O str., ester) H NMR (400  MHz, 97.71, 95.05, 82.37, 73.06 (d, J = 19.1  Hz), 72.87 (d, DMSO-d ) δ 8.21–8.14 (m, 2H), 7.79 (dd, J = 7.5, 1.5  Hz, J = 12.2  Hz), 72.47, 72.35, 71.92, 65.19, 41.10, 38.86, 1H), 7.59 (d, J = 1.5  Hz, 1H), 7.32–7.25 (m, 2H), 6.75 (d, 29.40, 27.86. m/z found for C H N O S: 759 (M ) 760 J = 7.5  Hz, 1H), 6.44 (dd, J = 14.1, 1.5  Hz, 2H), 6.27 (t, 34 37 3 15 (M + 1) . Anal calcd for C H N O S: C, 53.75; H, 4.91; J = 7.0  Hz, 1H), 4.15 (t, J = 7.0  Hz, 1H), 3.98–3.95 (m, 34 37 3 15 N, 5.53; O, 31.59; S, 4.22. Found: C, C, 53.77; H, 4.93; N, 3H), 3.88 (dd, J = 12.4, 6.9  Hz, 1H), 3.67–3.55 (m, 3H), 5.56; O, 31.59; S, 4.24. 2.22 (dt, J = 12.4, 7.0  Hz, 1H), 2.14–2.11 (m, 2H), 2.09– RUT3a yield 61% R 0.6 [Mobile Phase for TLC— 2.06 (m, 1H), 1.76–1.73 (m, 2H), 1.67–1.55 (m, 2H). 3 f Methanol:Glacial acetic acid:Formic acid:Water C NMR (100  MHz, Chloroform-d) δ 173.89, 164.58, −1 (3:2.9:0.8:0.5)] M.pt. (235–237) IR (KBR pellets) c m ) 163.50, 158.34, 152.36, 151.92, 148.16, 146.53, 145.55, 3475 (O–H str., Ar), 1641 (C=N str.), 1580 (C=C str.), 128.56, 125.27, 124.36, 122.39, 121.81, 116.06, 115.83, 1220 (O–CH ), 1155 (C=S Str.) H NMR (400  MHz, 108.81, 93.06, 97.81, 90.53, 82.19, 73.80 (d, J = 19.1  Hz), DMSO-d ) δ 7.70 (dd, J = 7.5, 1.5  Hz, 1H), 7.56 (d, 72.67 (d, J = 12.2  Hz), 72.36, 72.12, 71.08, 64.86, 42.81, J = 1.5  Hz, 1H), 7.46–7.38 (m, 2H), 7.32–7.23 (m, 2H), 36.15, 28.55, 26.98. m/z found for C H N O:730 (M ) 33 34 2 17 7.07–6.98 (m, 1H), 6.89 (d, J = 7.5  Hz, 1H), 6.35 (dd, 731 (M + 1) . Anal calcd for C H N O : C, 54.25; H, 33 34 2 17 J = 15.0, 1.5  Hz, 2H), 6.19 (t, J = 7.0  Hz, 1H), 4.09 (t, 4.69; N, 3.83; O, 37.23. Found: C, 54.27; H, 4.70; N, 3.85; J = 7.0  Hz, 1H), 4.02–3.88 (m, 3H), 3.68 (dd, J = 12.4, O, 37.25. 6.9 Hz, 1H), 3.66–3.54 (m, 3H), 2.33 (dt, J = 12.4, 7.0  Hz, RU7C yield 83.5% R 0.8 [Mobile Phase for TLC— 1 f 1H), 2.21–2.19 (m, 2H), 1.96–1.88 (m, 2H), 1.87–1.85 (m, Methanol:Glacial acetic acid:Formic acid:Water 13 −1 2H) (Additional file 1). C NMR (100 MHz, Chloroform- (3:2.9:0.8:0.5)] M.pt. (189–190) IR (KBR pellets) c m 1) d) δ 164.50, 160.96, 155.78, 150.30, 148.16, 145.55, 143.60, 1715 (C=O str.), 1627 (C=N str.), 1607 (C=C str.), 1234 132.14, 129.50, 124.46, 122.39, 121.81, 121.19, 118.32, (O–CH ), 11,944 (C–O str., ester) H NMR (400  MHz, 116.06, 115.83, 104.75, 94.15, 93.97, 91.01, 83.98, 79.41 DMSO-d ) δ 9.11 (d, J = 1.5 Hz, 1H), 8.77–8.70 (m, 1H), (d, J = 19.1 Hz), 78.77 (d, J = 12.2 Hz), 77.09, 73.82, 68.48, 8.14 (dt, J = 7.5, 1.5  Hz, 1H), 7.92 (dd, J = 7.5, 1.5  Hz, 42.85, 37.51, 23.82, 23.17. m/z found for C H N O : 1H), 7.68 (d, J = 1.5  Hz, 1H), 7.51 (t, J = 7.5  Hz, 1H), 33 36 2 15 + + 700 (M ) 701 (M + 1) . Anal calcd for C H N O : C, 6.93–6.83 (m, 2H), 6.23 (d, J = 1.5 Hz, 1H), 3.92 (s, 3H), 33 36 2 15 56.57; H, 5.18; N, 4.00; O, 34.25. Found: C, 56.58; H, 5.20; 3.83 (d, J = 0.9 Hz, 6H), 3.76 (s, 3H). C NMR (100 MHz, N, 4.00; O, 34.27. Chloroform-d) δ 174.99, 164.48, 164.18, 160.33, 157.96, RU4b yield 74.3% R 0.6 [Mobile Phase for TLC— 156.60, 153.53, 151.74, 150.80, 149.32, 138.25, 128.95, 1 f Methanol:Glacial acetic acid:Formic acid:Water 123.72, 123.22, 122.87, 122.65, 113.70, 112.82, 107.81, −1 (3:2.9:0.8:0.5)] M.pt. (259–260) IR (KBR pellets) c m 1) 95.68, 93.25, 56.20, 55.88 (d, J = 2.6 Hz), 55.62. m/z found + + 1725 (C=O str.), 1631 (C=N str.), 1603 (C=C str.), 1234 for C H NO:463 (M ) 464 (M + 1) . Anal calcd for 25 21 8 (O–CH ), 1268 (C–O str., ester) H NMR (400  MHz, C H NO : C, 64.79; H, 4.57; N, 3.02; O, 27.62. Found: C, 3 25 21 8 DMSO-d ) δ 8.38 (d, J = 1.5  Hz, 1H), 8.15 (dd, J = 7.5, 64.80; H, 4.58; N, 3.00; O, 27.60. 1.5  Hz, 1H), 7.69 (dd, J = 7.5, 1.5  Hz, 1H), 7.2 (d, Malik et al. BMC Chemistry (2019) 13:71 Page 6 of 13 RU7C yield 62.5% R 0.6 [Mobile Phase for TLC— to prepare reaction mixture. The different concentrations 2 f Methanol:Glacial acetic acid:Formic acid:Water of synthesized derivatives having final volume 50 µl were −1 (3:2.9:0.8:0.5)] M.pt. (186–188) IR (KBR pellets) c m 1) prepared in dimethyl sulfoxide (DMSO) and added to 96 1764 (C=O str.), 1619 (C=N str.), 1595 (C=C str.), 1277 well plate. To each well 50 µl of reaction mix was added (O–CH ), 1214 (C–O str., ester) H NMR (400  MHz, and mixed well. After 2–3  min initial measurement was DMSO-d ) δ 7.91 (ddd, J = 7.5, 6.5, 1.5  Hz, 2H), 7.67 taken. The plates were incubated at 25  °C taking meas - (d, J = 1.5  Hz, 1H), 7.47 (td, J = 7.5, 1.5  Hz, 1H), 7.09 urements at every 5  min. Allopurinol served as positive (td, J = 7.5, 1.5  Hz, 1H), 6.97–6.88 (m, 2H), 6.86 (d, control. Absorbance at different time intervals was noted J = 1.5 Hz, 1H), 6.28 (d, J = 1.5 Hz, 1H), 3.97 (s, 3H), 3.80 for further statistical analysis. (d, J = 0.7  Hz, 6H), 3.67 (s, 3H). C NMR (100  MHz, Chloroform-d) δ 171.85, 168.95, 167.67, 165.22, 158.95, In vitro evaluation of antioxidant activity by DPPH method 157.67, 148.53, 146.92, 133.72, 131.16, 128.84, 124.78, The antioxidant potential of rutin derivatives was per - 124.78, 123.22, 122.87, 116.52, 113.70, 108.53, 104.92, formed by DPPH method evaluated in the form of 92.81, 90.38, 53.06, 52.81, 52.76 (d, J = 2.6 Hz), 51.65. m/z IC estimated using the ELISA plate reader EPOCH + + found for C H O:478 (M ) 479 (M + 1) . Anal calcd 26 22 9 “MICROPLATE READER (BIOTEK). This method opted for C H O : C, 65.27; H, 4.63; O, 30.10. Found: C, 65.27; 26 22 9 for evaluation of free radical scavenging activity of DPPH H, 4.63; O, 30.10. was based on modified procedure described by Dhiman RU7C yield 71% R 0.7 [Mobile Phase for TLC— 3 f et al. [36]. The tested compounds were prepared in meth - Methanol:Glacial acetic acid:Formic acid:Water anolic solution and reacted with methanolic solution of −1 (3:2.9:0.8:0.5)] M.pt. (165–166) IR (KBR pellets) c m 1) DPPH at 37  °C. The reaction mixture was prepared in 1710 (C=O str.), 1637 (C=N str.), 1596 (C=C str.), 1258 96-well plate by adding 50 µL of sample, 50 µl of meth- (O–CH ), 1194 (C–O str., ester) H NMR (400  MHz, 3 anol and 50 µl of DPPH solution prepared in 0.1  mM DMSO-d ) δ 7.98 (dd, J = 7.5, 1.5  Hz, 1H), 7.76 (d, 6 methanol. The mechanism of action of DPPH assay was J = 1.5  Hz, 1H), 7.30–7.20 (m, 5H), 6.91–6.86 (m, 2H), based on the fact that DPPH radical get reduced during 6.23 (d, J = 1.5  Hz, 1H), 3.93 (s, 3H), 3.88 (d, J = 0.9  Hz, its reaction with an antioxidant compound and results in 6H), 3.69 (s, 3H), 2.93–2.84 (m, 2H), 2.73 (td, J = 7.0, changes of color (from deep violet to light yellow). The 0.8  Hz, 2H). C NMR (100  MHz, Chloroform-d) δ absorbance was read at 517  nm for 30  min at an inter- 175.20, 170.26, 164.48, 160.33, 157.96, 156.95, 150.80, val of 5 min of using ELISA microplate reader. The mix - 149.32, 139.89, 128.47–128.31 (m), 126.14, 123.22, ture of methanol (5.0 ml) and tested compounds (0.2 ml) 122.87, 113.70, 112.82, 107.81, 99.41, 98.77, 53.17, 53.06 serve as blank. Ascorbic acid served as positive control. (d, J = 2.6  Hz), 52.69, 51.86, 34.56, 30.26. m/z found + + for C H O:488 (M ) 489 (M + 1) . Anal calcd for 28 24 8 Hydrogen peroxide scavenging (H O ) assay 2 2 C H O : C, 68.85; H, 4.95; O, 26.20. Found: C, 68.87; H, 28 24 8 To compare and best evaluate the antioxidant potential 4.90; O, 26.20. of newly synthesized rutin derivatives, hydrogen per- oxide assay was performed by the method described by Evaluation of biological activity Patel et  al. [37] with some modifications. The solution In vitro evaluation of xanthine oxidase inhibitory activity of H O (100  mM) was prepared via adding up differ - 2 2 The method opted to evaluate the inhibitory potential ent concentrations of synthesized derivatives ranging of rutin derivatives was a modified protocol of Sigma, from 5 to 80 μg/ml to H O solution (2 ml), prepared in 2 2 done by UV-spectrophotometric method by using xan- 20  mM phosphate buffer of pH 7.4. Finally, the absorb - thine oxidase activity assay kit purchased from sigma ance of H O was measured at 230  nm after incubating 2 2 (MAK078, sigma-aldrich.co, USA). The colorimetric for 10  min next to a blank reading of phosphate buffer product obtained in the form of hydrogen peroxide gen- without H O . For every measurement, a fresh reading 2 2 erated during the oxidation of XO was determined by a of blank was taken to carry out background correction. coupled enzyme technique, measured at 570  nm in a For control sample containing H O was scanned for 2 2 96-well plate, using the plate reader EPOCH “MICRO- absorbance at 230  nm. Results calculated as percentage PLATE READER (BIOTEK).one unit of XO is defined of hydrogen peroxide inhibition was estimated by the as the amount of enzyme that catalyzes the oxidation formula [(A –A )/A ] × 100, where A   is the absorbance b t 0 b of xanthine substrate, yielding 1.0  µmol of uric acid and of the control and A   is the absorbance of compounds/ hydrogen peroxide per minute at 25  °C. Reagents used standard taken as l-ascorbic acid (5–80  μg/ml) are were 44 µL of xanthine oxidase assay buffer, 2 µl xanthine shown in Table 5. substrate solution and 2 µl of Xanthine Oxidase enzyme solution. All the solutions mentioned above were mixed Malik et al. BMC Chemistry (2019) 13:71 Page 7 of 13 Table 1 ADMET data of natural ligands calculated using Qik Prop simulation Compound QPlogPo/w QPlogS QPlogHERG QPPCaco QPlogBB QPPMDCK QPlogKp QPlogKhsa Human oral Percent human absorption oral absorption RU3a − 1.084 − 3.257 − 5.488 511.672 − 2.173 625.905 − 6.818 − 0.902 2 81 RU3a 0.866 − 4.593 − 7.183 605.947 − 1.139 853.322 − 4.846 − 0.635 2 77 RU3a 0.444 − 2.809 − 5.496 758.912 − 1.381 793.01 − 4.796 − 0.58 3 76 RU4b − 0.044 − 3.745 − 6.548 563.916 − 2.192 641.237 − 5.52 − 0.747 1 60 RU4b 0.407 − 4.15 − 6.511 941.594 − 2.757 730.468 − 6.278 − 0.533 1 50 RU7c 3.322 − 4.469 − 6.334 1460.431 − 0.726 744.963 − 1.477 − 0.218 3 100 RU7c 4.878 − 5.717 − 6.59 2335.951 − 0.63 1237.701 − 0.774 0.383 3 100 RU7c − 0.334 − 3.885 − 6.168 743.251 − 1.271 971.012 − 6.276 − 0.735 2 50 Rutin − 0.28 − 2.94 − 5.166 827.655 − 3.378 682.554 − 5.639 − 0.703 1 30 Allopurinol − 1.365 − 2.932 − 0.839 569.551 − 3.6 − 570.702 − 6.890 − 0.986 2 50 Descriptor standard range: QPlogPo/w, − 2.0 to 6.5; QPlogS, − 6.5 to 0.5; QPlogHERG, concern below –5; QPPCaco, < 25 poor, > 500 great; QPlogBB, − 3.0 to 1.2; QPPMDCK, < 25 poor, > 500 great; QPlogKp, − 8.0 to − 1.0; QPlogKhsa, − 1.5 to 1.5; human oral absorption, 1, 2, or 3 for low, medium, or high; percent human oral absorption, > 80% is high ADMET studies Table 2 Comparison of  in  vitro activity and  molecular The pharmacokinetic and pharmacological parameters of docking studies newly synthesized compounds were predicted with the Compound Docking score Binding IC (µM) help of Schrodinger suite. In-silico ADMET-related prop- energy [ΔG erties were computed using Qikprop application of Schro- (KJ/mol)] dinger software (Table 1). QikProp program generates set RU3a − 12.907 − 88.383 09.924 ± 0.01 of physicochemically significant descriptors which further RU3a − 11.456 − 67.673 07.905 ± 0.15 evaluates ADMET properties. The whole ADME-compli - RU3a − 13.244 − 91.242 04.870 ± 0.02 ance score-drug-likeness parameter is used to predict the RU4b − 11.591 − 60.323 15.037 ± 0.01 pharmacokinetic profiles of the ligands. This parameter RU4b − 12.021 − 72.991 12.541 ± 0.45 determines the number of property descriptors calculated 2 RU7c − 11.310 − 55.854 19.377 ± 0.38 via QikProp which fall outside from the optimum range 1 RU7c − 10.980 − 61.268 17.428 ± 0.01 of values for 95% of noted drugs.  Initially, all compound 2 RU7c 11.037 50.217 13.476 ± 0.25 structures were neutralized before operated through Qik- 3 Rutin − 10.944 − 45.549 20.867 ± 0.12 prop. The neutralizing step is crucial, as QikProp is unable Allopurinol − 3.366 − 17.231 10.410 ± 0.72 to neutralize ligands in normal mode. Qikprop predicts both pharmacokinetically significant properties and phys - Italic values indicating standard drug icochemically significant descriptors. It application run in normal mode which predicted IC value for blockage of the synthesized compounds with XO, molecular simu- of HERG K + channels (log HERG), predicted apparent lation studies were carried out using Schrödinger suite Caco-2 cell permeability in nm/s (QPPCaco), brain/blood (Schrödinger Release  2018-2, Schrödinger, LLC, New partition coefficient (QPlogBB), predicted skin perme - York, NY, 2018).The crystal structure of xanthine oxidase ability (QPlogKp), prediction of binding to human serum with PDB code 2E1Q was adopted for the docking calcu- albumin (QPlogKhsa) and predicted apparent Madin– lations. Based on the docking score and binding energy Darby Canine Kidney (MDCK) cell permeability in nm/s calculation, top ranking derivatives were established and (QPPMDCK). Solubility of drug was predicted as octanol/ compared with the IC calculated from in  vitro activ- water partition coefficient (QPlogPo/w). Aqueous solubil - 50 −3 ity (Table  2). Important interactions were depicted as ity of compound defined in terms of log S (S in mol dm ) hydrophobic regions, hydrogen bonding, polar interac- is the concentration of the solute in a saturated solution tions and pi–pi bonding visualized in the active pocket of that is in equilibrium with the crystalline solid. xanthine oxidase revealed through Site map application of Schrodinger suite. The derivatives having better dock - Result and discussion ing scores than rutin were kept for further synthetic pro- Molecular docking cedures and the remaining were discarded. To observe To rationalize the structure activity relationship observed the binding interaction in detail, 3D poses of two most in this research and to foreknow the potential interaction Malik et al. BMC Chemistry (2019) 13:71 Page 8 of 13 Fig. 4 3D pose of RU3a showing hydrogen bonding (yellow) with GLN1194, ARG 912, GLY795, GLN 585 and π–π bonding (blue) with PHE798 Fig. 2 3D pose of RU3a inside the binding pocket Fig. 5 3D pose of RU3a inside the binding pocket Visual inspection of 3D poses of R U3a displayed a Fig. 3 2D pose of RU3a inside the binding pocket 3 compact arrangement of polar and hydrophobic residues around the ligand forming a narrow passage in XO bind- ing cavity with a docking score/binding score of − 13.244 and binding energy − 91.242 kJ/mol. An interesting pi–pi active compounds RU3a and RU3a were visualized and 3 1 bonding was observed between benzene ring of phenyl compared with native rutin and standard drug Allopuri- hydrazine and hydrophobic residue PHE 798 of active nol. The residues of binding pocket involved in the inter - site (Figs.  1, 2, 3). Along with this a strong hydrogen action were reported as GLN 1194, ARG912, MET1038, bonding was observed between OH group of rutinoside GLN1040, PHE798 and SER1080. Similar binding cavity and polar residue GLN 1194 and negatively charged ARG was observed by Li et  al. during the docking analysis of 912 (Fig. 4). Similarly ARG 912 was found essential in the newly synthesized non-purine XO inhibitors [38]. study of Shen et  al. during the comparison of curcumin Malik et al. BMC Chemistry (2019) 13:71 Page 9 of 13 Fig. 6 2D pose of RU3a inside the binding pocket Fig. 8 3D pose of rutin showing hydrogen bonding with GLN 1194 and MET1038 Fig. 7 3D pose of RU3a showing hydrogen bonding with GLN 1194, MET1038 and GLY 1039 Fig. 9 3D pose of allopurinol showing hydrogen bonding with GLN derivatives with quercetin and leuteolin [39]. Another hydrogen bonding was visualized between Chromene moiety and the residues of active site namely GLY 795 ad GLN585. Other hydrophobic amino acid residues closely dihydroxyphenyl ring and GLY1039. One more interac- placed within the cavity were observed as PHE 798, tion was observed with the surrounding residue GLN 767 VAL1200, ALA1198, TYR 592, MET 1038 and ILE1229. which forms a hydrogen bond with MOS 1328 (molyb- On the other hand, during the visualization of R U3a denum metal ion) forming a closed channel to prevent the hydrogen bond was observed with OH group the entry of substrate in the binding site. Other residues of phenyl ring and hydrophobic residue MET 1038 surrounding the ligand were observed as ARG 912, HIE (Figs.  5, 6). Another hydrogen bond was found simi- 579, GLU 1261, ALA 1189 and ILE1198. When the 3D lar to RU3a between OH group of rutinoside and polar poses of these two compounds were compared with the residue GLN1194 (Fig.  7). One more hydrogen bond- native rutin structure, GLN 1194 forms 2 H-bonds, one ing was observed between one of the OH group of with the C=O group of rutin and another with OH group Malik et al. BMC Chemistry (2019) 13:71 Page 10 of 13 Table 3 In vitro xanthine oxidase inhibitory activity of rutin derivatives Compound IC (µM) ± SEM Compound IC (µM) ± SEM 50 50 Rutin 20.867 ± 0.12 RU4b 12.541 ± 0.45 RU3a 09.924 ± 0.01 RU7c 19.377 ± 0.38 1 1 RU3a 07.905 ± 0.15 RU7c 17.428 ± 0.01 2 2 RU3a 04.870 ± 0.02 RU7c 13.476 ± 0.25 3 3 RU4b 15.037 ± 0.01 Allopurinol 10.410 ± 0.72 SEM, standard error of the mean of rutinoside (Fig.  8). The amino acid residues GLU1261 and GLN 1194 were found to be interacted similarly in the study of verbascoside by Wan et  al. [40]. Beside this one H-bond was formed between OH group of chromene ring and MET1038. No pi–pi interaction was in the native structure rutin. In case of Allopurinol, the Fig. 11 Lineweaver–Burk plot for RU3a against different active site residues surrounding ligand were almost sim- concentrations ilar and placed near to MOS 1328. The hydrogen bond was observed between purine ring of allopurinol and GLN1194 (Fig. 9). (RU7c –RU7c ). All the compounds of hydrazine 1 3 series (RU3a –RU3a ) were effective with IC -values 1 3 50 In‑vitro xanthine oxidase inhibitory activity ranging from 04.870 to 09.924  µM. Rutin hybridized In order to monitor the efficacy of different synthesized with phenyl hydrazine demonstrated highest activity rutin derivatives, xanthine oxidase inhibitory activity against xanthine oxidase. While thisemicarbazide and was determined using xanthine oxidase activity assay phenylthiosemicarbazide derivatives of rutin showed kit purchased from Sigma-aldrich Co. Allopurinol a slight decrease in activity indicating the role of sul- (positive control) reported to inhibit xanthine oxidase fur group in diminishing the inhibition and NH–NH was also screened under identical conditions for com- group in enhancing the activity of targeted enzyme. parison. The inhibition ratios revealed the xanthine Surprisingly, substitution of NH–NH with N H group 2 2 oxidase inhibitory activity of the synthesized rutin leads to decrease of inhibitory activity. Ester deriva- derivatives and the results were summarized in Table 3. tives of rutin synthesized after the hydrolysis of rutin As expected, these rutin derivatives exhibited remark- exhibited a weaker inhibition than the positive control able activity comparable to the positive control. Based Allopurinol. on the in  vitro activity; it was observed that hydrazine The results of in  vitro activity showed 80% similarity (RU3a –RU3a ) and anilline analogues (R U4b –RU4b ) with the results of molecular docking with a few excep- 1 3 1 2 were considerably more effective than ester derivatives tions. In concordance with the screening and output of OH OH OH HO O HO OH O CH 3 Presence of glycosidic 3-O-rutinoside linkage is OH O Addition of thiosemicarbazide group essential for the xanthine oxidase inhibitory potential, as showed the XO inhibition moderately. HO OH detachment of group diminished the XO inhibitory activity. OH Rutin Incorporation of hydrazide groups remarkably Addition of phenylthiosemicarbazide group increased the XO inhibitory action. significantly increased the XO inhibition. Fig. 10 Structure activity relationship (SAR) of synthesized compounds Malik et al. BMC Chemistry (2019) 13:71 Page 11 of 13 Table 4 K and  V values of  xanthine oxidase m max at different concentrations of  RU3a S. no.Conc. of  RU3a Km (µM) V (µmol/min) 3 max (µM) 1. 0.0 27.21 119.6 2. 0.25 30.11 114.4 3. 0.5 32.90 108.2 4. 1.0 35.08 98.7 of most active compound R U3a using Graph pad prism software. Firstly Michaelis–Menten curve was plotted for the enzyme activity at different concentrations of RU3a against different concentration of substrate (xanthine) Fig. 11. Then double reciprocal plot (Lineweaver–Burk) anal - ysis was done in the presence (0.25, 0.5, and 1.0  µM) Fig. 12 Michaelis–Menten curve for RU3a at different and absence of RU3a from in  vitro data generated concentrations during the oxidation of xanthine in presence of xan- thine oxidase (Fig.  12). The x- and y axis intercepts of the Lineweaver–Burk plot were utilized to calculate K and V values of RU3a at different concentrations molecular docking R U3a comes out to be most active max 3 (Table 4). rutin derivative showing very good interaction with xan- A concentration-dependent decrease of V was thine oxidase at molecular level. Elimination of rutino- max predicted in contrast to K value which was found to side from rutin to synthesize ester derivatives results in m increasing when concentration of R U3a was increased. a loss of potency with a threefold decrease of inhibitory 3 The intersection of linear straight lines drawn against potential. each concentration was located at same point, suggesting that RU3a reacts in competitive manner during the inhi- bition of xanthine oxidase. Structure activity relationship (SAR) Few interesting notions about the relationship of activ- In‑vitro evaluation of antioxidant activity by DPPH ity and structures of synthesized compounds emerged and  H O method 2 2 from the present research (Fig.  10): (A) Rutinoside The antioxidant potential of newly synthesized com - moiety seems to be important for the activity, as dele- pounds was evaluated by DPPH and Hydrogen peroxide tion of this leads to loss of activity could be seen from radical assay. The comparative analysis of IC values xanthine oxidase inhibitory activity Table  3. Which for both the assays was done and the results were found shows RU3a (Having rutinoside group) exhibited to be impressive (Table  5). The results evinced a note - highest activity with an IC50 value 04.870  µM among worthy inhibition of DPPH almost all the compounds all the compounds and RU7c showed lowest activ- when compared with the positive control ascorbic acid. ity and fivefold decrease of activity with an IC value In case of DPPH assay compound RU4b was demon- 19.377  µM. (B) Hydrazine derivatives were found to strated as most potent compound against oxidative stress be more effective than the aniline derivatives reveal- caused because of free radicals having an IC value of ing the importance of NH–NH group. But substitu- 02.647 ± 0.09 µM. Along with this compound RU3a also tion of sulfur group along with hydrazines decreases showed very good antioxidant potential with an IC the activity as in R U3a and R U3a and substitution of 3 2 value of 05.021 ± 0.10  µM. When the detailed structure phenyl group along with sulfur improves the activity activity relationship was developed between these com- (RU3a ). (C) Substitution with ester group leads to a pounds, it was concluded that both the compounds hav- decrease of inhibitory activity. ing hydrazine linkage derived from phenyl hydrazine and phenyl thiosemicarbazide. Similarly, during the analy- Enzyme kinetic analysis for XO‑inhibitory activity sis of hydrogen peroxide assay all the compounds with To determine the XO-inhibitory mechanisms of newly hydrazines substitution showed very good antioxidant synthesized derivatives, we carried out kinetic studies Malik et al. BMC Chemistry (2019) 13:71 Page 12 of 13 Table 5 Antioxidant activity of  synthesized derivatives Additional file by DPPH and  H O method 2 2 Compound IC (µM) ± SEM IC (µM) ± SEM Additional file 1. HNMR spectra of compound RU3a 50 50 3 RU3a 05.021 ± 0.10 09.134 ± 0.35 Acknowledgements RU3a 08.728 ± 0.02 04.146 ± 0.01 The authors are highly thankful to the Head, Department of Pharmaceuti- RU3a 11.688 ± 0.01 06.561 ± 0.10 3 cal Sciences, M. D. University, Rohtak for providing essential facilities to accomplish this research study. The authors are also thankful to Dr. Vinod RU4b 02.647 ± 0.09 09.863 ± 0.25 Devaraji Application Scientist Schrödinger LLC for his support to carry out the RU4b 08.476 ± 0.25 04.378 ± 0.01 computational work. RU7c 06.056 ± 0.13 14.731 ± 0.60 Authors’ contributions RU7c 14.669 ± 0.01 12.126 ± 0.20 Authors NM and AK have designed, synthesized and carried out the xanthine RU7c 07.692 ± 0.42 17.884 ± 0.41 oxidase inhibitory and antioxidant activity and the author PD, have carried RU001 09.483 ± 0.08 18.623 ± 0.07 out the docking simulations with in silico ADMET studies. All authors read and approved the final manuscript. Ascorbic acid 22.195 ± 0.08 22.195 ± 0.08 SEM, standard error of the mean Funding No funding received for this research work from outside sources. Availability of data and materials potential having IC in range of 04.146 ± 0.01 to Not applicable. 09.134 ± 0.35 (Fig.  7). Compound R U3a having phenyl Competing interests thiosemicarbazide substitution showed potential antioxi- The authors declare that they have no competing interests. dant activity among all the derivatives. Along with this phenyl hydrazine substituted rutin derivative (RU3a ) Author details Faculty, Department of Pharmaceutical Sciences, M.D. University, also showed very good scavenging activity with an IC Rohtak 124001, India. Laboratory for Preservation Technology and Enzyme value of 06.561 ± 0.10. When the detailed structure Inhibition Studies, Department of Pharmaceutical Sciences, M.D. University, activity relationship was developed between these com- Rohtak, Haryana, India. pounds, it was concluded that both the compounds hav- Received: 21 January 2019 Accepted: 2 May 2019 ing hydrazine linkage derived from phenyl hydrazine and phenyl thiosemicarbazide. References Conclusion 1. Berry CE, Hare JM (2004) Xanthine oxidoreductase and cardiovascular Starting from the structures of rutin as anti-XO hit pre- disease: molecular mechanisms and pathophysiological implications. J viously identified, different series of novel analogues Physiol 555(3):589–606 2. Moriwaki Y, Yamamoto T, Higashino K (1997) Distribution and pathophysi- were designed and synthesized to explore the struc- ologic role of molybdenum-containing enzymes. Histol Histopathol ture–activity relationships associated with these xan- 12(2):513–524 thine oxidase inhibitors along with their antioxidant 3. Klinenberg JR, Goldfinger SE, Seegmiller JE (1965) The effectiveness of the xanthine oxidase inhibitor allopurinol in the treatment of gout. Ann potential. Different structural elements were identified Intern Med 62(4):639–647 as essential for antioxidant and anti-XO properties, such 4. Yu KH (2007) Febuxostat: a novel non-purine selective inhibitor of as the presence of rutinoside (R U3a, RU3a and R U3a ) xanthine oxidase for the treatment of hyperuricemia in gout. Recent Pat 1 2 3 Inflamm Allergy Drug Discov 1(1):69–75 comes out as important skeleton for the inhibitory 5. Battelli MG, Bolognesi A, Polito L (2014) Pathophysiology of circulat- potential, presence of hydrazone linker along with phe- ing xanthine oxidoreductase: new emerging roles for a multi-tasking nyl group, while the associated xanthine oxidase inhibi- enzyme. Biochim Biophys Acta Mol Basis Dis 1842(9):1502–1517 6. Brass CA, Narciso J, Gollan JL (1991) Enhanced activity of the free radical tory effect was found to follow a different trend for the producing enzyme xanthine oxidase in hypoxic rat liver. Regulation and two series hydrazine (RU3a ) and ester derivatives 1–3 pathophysiologic significance. J Clin Invest 87(2):424–431 (RU7c ). The newly synthesized derivatives with anti- 7. Chambers DE, Parks DA, Patterson G, Roy R, McCord JM, Yoshida S, Par- 1–3 mley LF, Downey JM (1985) Xanthine oxidase as a source of free radical oxidant and ani-XO IC values in the low micromolar damage in myocardial ischemia. J Mol Cell Cardiol 17(2):145–152 range and good selectivity indexes were identified. Con - 8. Desco MC, Asensi M, Márquez R, Martínez-Valls J, Vento M, Pallardó temporary synthetic efforts are focused towards the FV, Sastre J, Viña J (2002) Xanthine oxidase is involved in free radical production in type 1 diabetes: protection by allopurinol. Diabetes insertion of the hydrazones and ester linkage by replac- 51(4):1118–1124 ing the side linkage rutinoside of rutin with more sta- 9. Kuppusamy P, Zweier JL (1989) Characterization of free radical generation ble groups while maintaining the overall length of new by xanthine oxidase. Evidence for hydroxyl radical generation. J Biol Chem 264(17):9880–9884 derivatives. Molecular docking provide an improved 10. Dawson J, Walters M (2006) Uric acid and xanthine oxidase: future trail to design the new molecules with an avantgarde therapeutic targets in the prevention of cardiovascular disease? Br J Clin stability and potency. Pharmacol 62(6):633–644 Malik et al. BMC Chemistry (2019) 13:71 Page 13 of 13 11. Khosla UM, Zharikov S, Finch JL, Nakagawa T, Roncal C, Mu W, Krotova 29. Khosravan R, Grabowski BA, Wu JT, Joseph-Ridge N, Vernillet L (2006) K, Block ER, Prabhakar S, Johnson RJ (2005) Hyperuricemia induces Pharmacokinetics, pharmacodynamics and safety of febuxostat, a non- endothelial dysfunction. Kidney Int 67(5):1739–1742 purine selective inhibitor of xanthine oxidase, in a dose escalation study 12. Kaynar H, Meral M, Turhan H, Keles M, Celik G, Akcay F (2005) Glutathione in healthy subjects. Clin Pharmacokinet 45(8):821–841 peroxidase, glutathione-S-transferase, catalase, xanthine oxidase, Cu–Zn 30. Malik N, Dhiman P, Khatkar A (2017) In-silico design and ADMET studies of superoxide dismutase activities, total glutathione, nitric oxide, and natural compounds as inhibitors of xanthine oxidase (XO) enzyme. Curr malondialdehyde levels in erythrocytes of patients with small cell and Drug Metab 18(6):577–593 non-small cell lung cancer. Cancer Lett 227(2):133–139 31. Muhammad A, Arthur DE, Babangida S, Erukainure OL, Malami I, Sani 13. Griguer CE, Oliva CR, Kelley EE, Giles GI, Lancaster JR, Gillespie GY (2006) H, Abdulhamid AW, Ajiboye IO, Saka AA, Hamza NM, Asema S (2018) Xanthine oxidase-dependent regulation of hypoxia-inducible factor in Modulatory role of rutin on 2, 5-hexanedione-induced chromosomal and cancer cells. Cancer Res 66(4):2257–2263 DNA damage in rats: validation of computational predictions. Drug Chem 14. Kanellis J, Kang DH (2005) Uric acid as a mediator of endothelial dysfunc- Toxicol 10:1–4 tion, inflammation, and vascular disease. Seminars in nephrology, vol 25. 32. Roleira FM, Varela CL, Costa SC, Tavares-da-Silva EJ (2018) Phenolic New York, WB Saunders, pp 39–42 derivatives from medicinal herbs and plant extracts: anticancer effects 15. Miesel R, Zuber M (1993) Elevated levels of xanthine oxidase in serum of and synthetic approaches to modulate biological activity. Nat Prod Chem patients with inflammatory and autoimmune rheumatic diseases. Inflam- 57:115–156 mation 17(5):551–561 33. Baldisserotto A, Vertuani S, Bino A, De Lucia D, Lampronti I, Milani R, 16. Wijermars LG, Bakker JA, de Vries DK, van Noorden CJ, Bierau J, Kostidis Gambari R, Manfredini S (2015) Design, synthesis and biological activity S, Mayboroda OA, Tsikas D, Schaapherder AF, Lindeman JH (2016) The of a novel Rutin analogue with improved lipid soluble properties. Bioorg hypoxanthine–xanthine oxidase axis is not involved in the initial phase of Med Chem 23(1):264–271 clinical transplantation-related ischemia–reperfusion injury. Am J Physiol 34. Gullon B, Lú-Chau TA, Moreira MT, Lema JM, Eibes G (2017) Rutin: a Renal Physiol 312(3):F457–F464 review on extraction, identification and purification methods, biological 17. Poles MZ, Bódi N, Bagyánszki M, Fekete É, Mészáros AT, Varga G, Szűcs S, activities and approaches to enhance its bioavailability. Trends Food Sci Nászai A, Kiss L, Kozlov AV, Boros M (2018) Reduction of nitrosative stress Technol 67:220–235 by methane: neuroprotection through xanthine oxidoreductase inhibi- 35. Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, Repasky tion in a rat model of mesenteric ischemia–reperfusion. Free Radic Biol MP, Knoll EH, Shelley M, Perry JK, Shaw DE (2004) Glide: a new approach Med 120:160–169 for rapid, accurate docking and scoring. 1. Method and assessment of 18. Osada Y, Tsuchimoto M, Fukushima H, Takahashi K, Kondo S, Hasegawa docking accuracy. J Med Chem 47(7):1739–1749 M, Komoriya K (1993) Hypouricemic effect of the novel xanthine oxidase 36. Dhiman P, Malik N, Verma PK, Khatkar A (2015) Synthesis and biologi- inhibitor, TEI-6720, in rodents. Eur J of Pharmacol 241(2–3):183–188 cal evaluation of thiazolo and imidazo N-(4-nitrophenyl)-7-methyl- 19. Krakoff IH, Meyer RL (1965) Prevention of hyperuricemia in leukemia 5-aryl-pyrimidine-6 carboxamide derivatives. Res Chem Intermed and lymphoma: use of allopurinol, a xanthine oxidase inhibitor. JAMA 41(11):8699–8711 193(1):1–6 37. Patel A, Patel A, Patel A, Patel NM (2010) Determination of polyphenols 20. Pacher PA, Nivorozhkin A, Szabó C (2006) Therapeutic effects of xanthine and free radical scavenging activity of Tephrosia purpurea linn leaves oxidase inhibitors: renaissance half a century after the discovery of (Leguminosae). Pharmacogn Res 2:152–154 allopurinol. Pharmacol Rev 58(1):87–114 38. Li P, Tian Y, Zhai H, Deng F, Xie M, Zhang X (2013) Study on the activity 21. Inkster ME, Cotter MA, Cameron NE (2007) Treatment with the xanthine of non-purine xanthine oxidase inhibitor by 3D-QSAR modeling and oxidase inhibitor, allopurinol, improves nerve and vascular function in molecular docking. J Mol Struct 5(1051):56–65 diabetic rats. Eur J Pharmacol 561(1–3):63–71 39. Shen L, Ji HF (2009) Insights into the inhibition of xanthine oxidase by 22. Sagor M, Taher A, Tabassum N, Potol M, Alam M (2015) Xanthine oxidase curcumin. Bioorg Med Chem Lett 19(21):5990–5993 inhibitor, allopurinol, prevented oxidative stress, fibrosis, and myocardial 40. Wan Y, Zou B, Zeng H, Zhang L, Chen M, Fu G (2016) Inhibitory effect damage in isoproterenol induced aged rats. Oxid Med Cell Longev. https of verbascoside on xanthine oxidase activity. Int J Biol Macromol ://doi.org/10.1155/2015/47803 9 1(93):609–614 23. Min HK, Lee B, Kwok SK, Ju JH, Kim WU, Park YM, Park SH (2015) Allopuri- nol hypersensitivity syndrome in patients with hematological malignan- Publisher’s Note cies: characteristics and clinical outcomes. Korean J Intern Med 30(4):521 Springer Nature remains neutral with regard to jurisdictional claims in pub- 24. Quach C, Galen BT (2018) HLA-B* 5801 testing to prevent allopuri- lished maps and institutional affiliations. nol hypersensitivity syndrome: a teachable moment. JAMA Int Med 178(9):1260–1261 25. Takano Y, Hase-Aoki K, Horiuchi H, Zhao L, Kasahara Y, Kondo S, Becker MA (2005) Selectivity of febuxostat, a novel non-purine inhibitor of xanthine oxidase/xanthine dehydrogenase. Life Sci 76(16):1835–1847 26. Mayer MD, Khosravan R, Vernillet L, Wu JT, Joseph-Ridge N, Mulford DJ (2005) Pharmacokinetics and pharmacodynamics of febuxostat, a new non-purine selective inhibitor of xanthine oxidase in subjects with renal impairment. Am J Ther 12(1):22–34 27. Nepali K, Singh G, Turan A, Agarwal A, Sapra S, Kumar R, Banerjee UC, Ready to submit your research ? Choose BMC and benefit from: Verma PK, Satti NK, Gupta MK, Suri OP (2011) A rational approach for the design and synthesis of 1-acetyl-3, 5-diaryl-4, 5-dihydro (1H) pyrazoles as fast, convenient online submission a new class of potential non-purine xanthine oxidase inhibitors. Bioorg thorough peer review by experienced researchers in your field Med Chem 19(6):1950–1958 28. Becker MA, Kisicki J, Khosravan R, Wu J, Mulford D, Hunt B, MacDonald rapid publication on acceptance P, Joseph-Ridge N (2004) Febuxostat ( TMX-67), a novel, non-purine, support for research data, including large and complex data types selective inhibitor of xanthine oxidase, is safe and decreases serum • gold Open Access which fosters wider collaboration and increased citations urate in healthy volunteers. 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