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Atenolol thiourea hybrid as potent urease inhibitors: Design, biology-oriented drug synthesis, inhibitory activity screening, and molecular docking studies
Journal Pre-proofs Atenolol Thiourea Hybrid as Potent Urease Inhibitors: Design, Biology-Oriented Drug Synthesis, Inhibitory Activity Screening, and Molecular Docking Studies Sana Wahid, Sajid Jahangir, Muhammad Ali Versiani, Khalid Mohammed Khan, Uzma Salar, Muhammad Ashraf, Urva Farzand, Abdul Wadood, Kanwal, Ashfaq-ur-Rehaman, Arshia, Muhammad Taha, Shahnaz Perveen PII: DOI: Reference:
S0045-2068(19)31363-X https://doi.org/10.1016/j.bioorg.2019.103359 YBIOO 103359
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Bioorganic Chemistry
Received Date: Revised Date: Accepted Date:
20 August 2019 25 September 2019 9 October 2019
Please cite this article as: S. Wahid, S. Jahangir, M. Ali Versiani, K. Mohammed Khan, U. Salar, M. Ashraf, U. Farzand, A. Wadood, Kanwal, Ashfaq-ur-Rehaman, Arshia, M. Taha, S. Perveen, Atenolol Thiourea Hybrid as Potent Urease Inhibitors: Design, Biology-Oriented Drug Synthesis, Inhibitory Activity Screening, and Molecular Docking Studies, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103359
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Atenolol Thiourea Hybrid as Potent Urease Inhibitors: Design, Biology-Oriented Drug Synthesis, Inhibitory Activity Screening, and Molecular Docking Studies Sana Wahid,a Sajid Jahangir,a Muhammad Ali Versiani,a Khalid Mohammed Khan,b,c Uzma Salar,d Muhammad Ashraf,e Urva Farzand,e Abdul Wadood,f Kanwal,b Ashfaq-ur-Rehaman,f Arshia,b Muhammad Taha,c Shahnaz Perveeng a
Department of Chemistry, Federal Urdu University of Art, Science and Technology, Karachi, Pakistan b H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan c Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam, Saudi Arabia d Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan e Department of Biochemistry and Biotechnology, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan f Department of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul Wali Khan University Mardan, Khyber Pakhtunkhwa 23200, Pakistan g PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi75280, Pakistan Abstract: Current research deals with the biology-oriented drug synthesis (BIODS) of twenty-three new thiourea analogs of pharmacologically important drug atenolol which is a well-known medicine to treat hypertension as well as cardiovascular diseases (CVDs). Structural characterization of all compounds was done by various spectroscopic techniques. Compounds 1-23 were subjected for urease inhibitory activity in vitro. Screening results revealed that whole library was found to be active having IC50 ranges from 11.73 ± 0.28 to 212.24 ± 0.42 µM. It is noteworthy that several derivatives including 3 (IC50 = 21.65 ± 0.31 µM), 8 (IC50 = 19.26 ± 0.42 µM), 9 (IC50 = 21.27 ± 0.25 µM), 12 (IC50 = 21.52 ± 0.42 µM), 17 (IC50 = 19.26 ± 0.42 µM), 20 (IC50 = 16.78 ± 0.34 µM), and 22 (IC50 = 11.73 ± 0.28 µM) showed excellent inhibitory potential than parent atenolol (IC50 = 64.36 ± 0.19 µM) and standard thiourea (IC50= 21.74±1.76 µM). A most probable structure-activity relationship (SAR) was anticipated by observing varying degree of inhibitory potential given by compounds. However, molecular insights regarding the binding mode of atenolol thiourea analogs within the active pocket of urease enzyme was rationalized by molecular docking studies.
*Corresponding Authors: [email protected], [email protected], Tel.: +922134824910, Fax: +922134819018
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Keywords: Biology-oriented drug synthesis (BIODS); atenolol; thiourea; urease; In vitro; molecular docking study 1.
Introduction
The class of drugs that is used significantly for the treatment of cardiac disorders, hypertension, anxiety, migraine, and other related complications are known as β-adrenoreceptors antagonists or β-blockers [1-3]. Different marketed drugs are being used as β-blockers, among them the most common are acebutolol, atenolol, bisoprolol etc. Nevertheless, these β-adrenoreceptor antagonists are still used in their racemic form. Atenolol, an aryloxypropanol amine derivative bearing one chiral centre selectively blocks β1, is highly polar molecule and is unable to penetrate into the blood-brain barrier [4]. It is used for the cure of hypertension, angina, acute myocardial infarction, and tachycardia. Atenolol is also used in racemic form, however, its (S)-enantiomer is most likely to inhibit β1-adrenoreceptors 50-500 times higher than (R)-enantiomer [5-8]. Urease, a member of the amidohydrolases family, is a nickel containing metalloenzyme (EC 3.5.1.5) having two nickel atoms within its active site. The prominent position of this enzyme in enzymology is mainly due to the fact that it was the first enzyme to be purified and crystallized (by James B. Sumner in 1926). This enzyme is widely distributed in the nature ranging from prokaryotes to eukaryotes with slight variation in the composition, however, the function is same i.e. hydrolysis of urea into carbonic acid and ammonia [9]. Urea being a stable molecule requires an enzyme (catalyst) for hydrolysis to breakdown the molecule around 1014 times greater than the uncatalyzed reaction [10]. Since, ammonia is produced in the hydrolysis of urea which leads to increased pH, thus leading to alkalinity. The increased alkalinity facilitates the growth of a pathogenic bacteria Helicobacter pylori which is responsible of infectious diseases such as urolithiasis, deudonal, peptic, and gastric ulcers [11, 12, 13]. Biology-oriented drug synthesis (BIODS) is the term used when marketed drugs modified via one or two steps chemical transformation and explored for their diversified activities [14-18]. Our research group has modified various drugs using this approach and explored for their diverse biological potentials. In recent past, we have reported the urease inhibition of Snaproxen derivatives [18] (Figure-1). In current study, we incorporated thiourea functionality on drug atenolol. Since, thiourea [19] and its derivatives are well-known as potent urease inhibitors [20], thus we intended to explore these new atenolol thiourea hybrids for urease
2
inhibitory potential. Thiourea moiety also has wide range of medicinal importance including antioxidant [21], antiinflammatory [22], anticonvulsants [23], and antibacterial [24] activities. In current attempt, all twenty-three (23) synthetic atenolol thiourea derivatives and parent atenolol molecule were subjected for evaluation of their urease inhibitory potential in vitro. Enzyme-ligand (synthetic compounds) interactions were accessed by in silico studies and got good results. Till to date, all synthetic compounds never reported before structurally and biologically.
Figure-1: Rationale of Current Study 2.
Results and Discussions
2.1.
Chemistry
In the current study, atenolol was treated with a number of isothiocyanates. In the reaction mechanism, lone pair of secondary aminic nitrogen of atenolol attacks on the electrophilic carbon center of isothiocyanate. Almost in all cases, reaction completed within 5-15 minutes under reflux condition. Products were appeared as solid and crystallized from ethanol (Scheme-1).
Scheme-1: Biology-Oriented Drug Synthesis of Atenolol Thiourea Derivatives 1-23
3
Chemical structures of all compounds were deduced by various spectroscopic techniques including FAB-MS (+ve), HRFAB-MS (+ve), 1H-, 13C-NMR, and FT-IR (see supplementary information). It is worth-mentioning that all synthetic atenolol thiourea derivatives (1-23) were never reported before structurally (Table-1). 2.2.
Urease inhibitory activity In vitro
Newly synthesized atenolol thiourea analogs were subjected for the evaluation of their urease inhibitory activity. It is note-worthy that all thiourea analogs were found to be active. Parent drug atenolol having IC50 = 64.36 ± 0.19 µM, was also screened for its inhibitory potential in order to compare the activity results of derivatives. Thiourea with IC50 = 21.74 ±1.76 µM was used as the standard. Almost all derivatives except 2, 4, 6, 13, 14, and 23 were found to show better activity than parent atenolol (IC50 = 64.36 ± 0.19 µM) whereas analogs 3, 8, 9, 12, 17, 20, and 22 showed even greater inhibitory potential than the standard thiourea (IC50 = 21.74 ± 1.76 µM) (Table-1). Table-1: Urease inhibitory activity of synthetic atenolol thiourea derivatives 1-23.
1-23 IC50 ± SEMa (µM)
Comp.
1
32.42 ± 0.21
14
72.56 ± 0.13
2
91.17 ± 0.13
15
42.37 ± 0.18
3
21.65 ± 0.31
16
56.82 ± 0.54
4
72.56 ± 0.13
17
19.26 ± 0.42
5
58.56 ± 0.59
18
52.20 ± 0.24
Comp.
R
R
IC50 ± SEMa (µM)
4
6
145.25 ± 0.35
19
41.63 ± 0.52
7
41.59 ± 0.52
20
16.78 ± 0.34
8
19.26 ± 0.42
21
52.43 ± 0.27
9
21.27 ± 0.25
22
11.73 ± 0.28
10
29.15 ± 0.12
23
212.24 ± 0.42
11
32.52 ± 0.38
64.36 ± 0.19 Atenolol
21.52 ± 0.42
12
21.74 ± 1.76 87.43 ± 0.54
13
Thioureac
SEMa (standard error mean); Thioureac (Standard inhibitor for urease enzyme)
2.3.
Structure-activity relationship (SAR)
Primarily, different functional groups and their respective positions have great influence on the urease inhibition activity, however, the variations in the inhibitory potential is attributed by varying structural motifs/groups on aryl part (R). Limited SAR was rationalized by looking at diverse range of group “R” and their effect on the inhibitory potential. Amongst all compounds, para-ethoxy substituted derivative 22 (IC50 = 11.73 ± 0.28 µM) was identified as the most potent analog in this series when compared to the standard thiourea (IC50 = 21.74 ± 1.76 µM) as well as parent drug atenolol (IC50 = 64.36 ± 0.19 µM). Effect of ethoxy substitution on urease inhibitory potential can easily be judge by comparing its activity with compound 1 (IC50 = 32.42 ± 0.21 µM) with no substitution on ring R and found
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to be almost three times less active than 22. Compounds 20 (IC50 = 16.78 ± 0.34 µM) and 21 (IC50 = 52.43 ± 0.27 µM) are positional isomers having ortho and para methoxy substitutions, respectively. These two positional isomers showed big difference in the inhibitory potential which indicates that position of methoxy substituent on “R” played a key role in the inhibitory activity. It was also observed that alkyl substituted derivatives 18 (IC50 = 52.20 ± 0.24 µM) and 19 (IC50 = 41.63 ± 0.52 µM) showed moderate activity as compared to alkoxy substituted derivatives (Figure-2).
Figure-2: Comparison of structure-activity relationship of compounds 1 and 18-22 Amongst halogen substituted molecules, para-chloro substituted analog 3 (IC50 = 21.65 ± 0.31 µM) showed inhibition comparable to the standard thiourea. Closely related ortho and para substituted positional isomers 2 (IC50 = 91.17 ± 0.13 µM) and 4 (IC50 = 72.56 ± 0.13 µM) presented steep decline in the activity which suggested that position of chloro moieties have worth importance for inhibitory activity. Dichloro groups containing compounds 5 (IC50 = 58.56 ± 0.59 µM) and 6 (IC50= 145.25 ± 0.35 µM) showed decreased inhibitory activity as compared to monochloro substituted molecules which reveals that increasing the number of chloro didn′t enhance the inhibitory activity. Though, derivative 7 (IC50= 41.59 ± 0.52 µM) having a combination of chloro with nitro group showed relatively good inhibitory potential (Figure-3)
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Figure-3: Comparison of structure-activity relationship of compounds 2-7 Another derivative 12 (IC50 = 21.52 ± 0.42 µM) with fluoro and methoxy substituents para to each another demonstrated good inhibitory potential. Nevertheless, ortho fluoro substituted derivative 10 (IC50 = 29.15 ± 0.12 µM) displayed inhibitory activity comparable to the standard thiourea. Addition of another fluoro substituent at para positon as in derivative 11 (IC50 = 32.52 ± 0.38 µM) slightly decreased the inhibitory potential. Trifluoromethyl substituted analogs 13, 14, and 15 having IC50s = 87.43 ± 0.54 µM, 72.56 ± 0.13 µM, and 42.37 ± 0.18 µM, respectively, showed moderate to weak inhibitory potential (Figure-4).
Figure-4: Comparison of structure activity relationship of compound 5-7 Amongst other halogenated compounds, meta-bromo substituted derivative 17 (IC50 = 19.26 ± 0.42 µM) revealed significant inhibitory potential, compared to thiourea and atenolol. While ortho-bromo substituted isomer 16 (IC50 = 56.82 ± 0.54 µM) exhibited almost three-
7
fold less active which confirmed that position of substituents play a vital role in the inhibitory potential. Amongst the nitro substituted analogs, derivative 8 (IC50 = 21.27 ± 0.25 µM) with meta-nitro group was identified slightly less active towards urease inhibition as compared to its positional isomer 9 (IC50 = 19.27 ± 0.26 µM) having para-nitro group (Figure-5).
Figure-5: Comparison of structure activity relationship of compound 8-9 Limited SAR predicted the effect of different groups such as Me, OMe, OEt, Cl, Br, F, and NO2 in the varying inhibitory potential, however, real picture of protein-ligand interactions were rationalized by the in-silico studies. A plausible demonstration is given below. 2.4.
Molecular docking studies
For the sake to predict the ligand binding interactions with the active site of urease enzyme, molecular docking of atenolol thiourea derivatives 1-23 was performed with urease crystal structure through MOE package. Most favorable docking conformations for each compound were determined with appropriate orientation within the active site. Active site of urease enzyme contained both hydrophobic and hydrophilic amino acid residues. The hydrophobic site comprised of K169, A170, 366, L319, and C322 whereas hydrophilic site contained R339, G166, 223, H 315, 323, 324, D224, 494, and 249. Two Ni ions conjointly play a significant role to link key amino acid residues and ligands. Nevertheless, it was observed from the molecular docking study that each compound’s conformations fit well within the active pocket of urease enzyme. Further, the most promising docked conformation of each
8
analog was assessed for binding mode analysis based on the scores, obtained from the GBVI/WSA binding free energy calculation. Generally, it was observed from the binding mode of all atenolol thiourea analogs that all derivatives possesses varying substituents by “R” including electron withdrawing (EWG) and donating (EDG) groups, and therefore, these substituted groups and their positions gradually altered the enzyme activity. The deep interaction pose analysis for the most potent analog 22 (IC50 = 11.73 ± 0.28 M) shows that this derivative was well fitted into the catalytic cavity and attained most favorable ionic and other interactions (i.e. hydrogen bond, hydrophobic etc.) with the active site residues, such as R338, H249, and with the modified residue KCX220. Additionally, the two embedded Ni198 and Ni799 ions in the active pocket adopt ionic bond with the S8 and O10 of the corresponding compound, and further enhance the activity against urease enzyme. The high potency of analog 22 might be due to the stabilized benzene ring. Benzene ring becomes partial +ve when substituted with EWG, which withdraws electron density from benzene ring through inductive effect. So this ring may adopt π-stacking interaction with other groups to regain stability. Other reason for the potent activity of analog 22 might be due to the ionic interaction with both the Ni ions and as well as with the modified residue which play significant role in enhancing the urease inhibition (Figure-6a). Furthermore, in case of other active compounds which includes 4, 9, 12, 17, and 18 showed best activity against urease enzyme. In case of other active compound in the series, further demonstrate the inhibition pattern in term of adopting various mode of interaction with the active site residues, hence established this ranking in the series of synthetic compounds. All these above-mentioned compounds possess EWG at benzene ring, but at various position, in case of compound 4 possess p-Cl, and further showed favorable interactions with the H315, and K169 active site residues through π-stacking pattern. Additionally, Ni799 adopt ionic interaction with the O9 of the corresponding compound. The high potency of derivative 4 might be due to EWG at para position of ring “R”, which facilitates the benzene ring to established π-interaction with other groups. Compound 17 also showed good interaction pattern with the active site residues (Figure-6c), and same mode of interaction was observed for this compound (Figure-6d). The withdrawing amplitude of the Cl is much more than the Br, so the less potency of compound 17 might be due to the above-mentioned reason, as compared to compounds 4 and 12 which possess Cl at different position but showed better mode of interaction, and inhibitory activity against urease enzyme. The obtained docking results supported well the experimental results grounded on several interactions of ligands (atenolol thiourea derivatives) with key amino
9
acid residues of urease enzyme as well as the docking scores calculated. The docking pose of almost every potent derivative computationally well inhibited the urease catalytic activities by binding firmly via different interactions such as strong polar, hydrophobic, and hydrogen bonding interactions with the key residues of active site. The detail of interaction docking score
embedded
in
(
10
Table-2).
Figure-6: Three dimensional interactions of compounds 4, 12, 17, and 22 with the active site residues of urease enzyme. Different color indicates different ligand, and key residues of the active site were colored to gray, hydrogen bonding are shown in dark color dotted lines, and the both sided arrows indicate the π-stacking interaction. (a) the mode of interaction of compound 22 with the active site residues, (b) for compound 4, (c) for compound 17, and (d) for compound 12.
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Table-2: Docking scores and interactions detail of the compounds (1-23) against urease enzyme. Interactions Detail Comp. 1
2 3 4
5
6 7 8 9 10
11
12 13 14 15 16 17 18 19 20
Ligand
Receptor
Interaction
Distance
E(kcal/mol)
Docking Score (S) -3.1647
O1 S 21 S 21
N ARG 369 (C) CA LEU 365 (C) N ALA 366 (C)
H-acceptor H-acceptor H-acceptor
2.69 4.43 4.43
-0.7 -0.8 -1.3
N 14 O 12 O 12 O 12 6-ring N 14 O 12 O 12 N3 N 14 N 22 O 12 O 12 S 22 N 14 6-ring NI 799 N3 C 10 N 14 O 12 O 12 N 14 O 12 O 12 S21 O12 O1 O 12 O 12 S 21 O1 N 14 O 12 O 12 O1 N14 N 22 O1 O 12 O 12 C 16 O12 O1
O ALA 364 (C) N ALA 366 (C) N MET 367 (C) O Ala 170 (C) CA GLY 280 (C) O ALA 364 (C) N ALA 366 (C) N MET 367 (C) SD MET 367 (C) O ALA 364 (C) SD MET 367 (C) O MET 367 (C) N MET 367 (C) CG ARG 339 (C) O ALA 170(C) CA GLY 280(C) NE2 HIS 139 (C) O THR 362 (C) SD MET 367 (C) O ALA 364 (C) N ALA 366 (C) N MET 367 (C) O ALA 364 (C) N ALA 366 (C) N MET 367 (C)
H-donor H-acceptor H-acceptor H-donor Pi-H H-donor H-acceptor H-acceptor H-donor H-donor H-donor H-donor H-acceptor H-acceptor H-donor Pi-H Metal H-donor H-donor H-donor H-acceptor H-acceptor H-donor H-acceptor H-acceptor
2.81 3.19 2.90 3.27 3.67
-1.0 -0.7 -1.5 -0.7 -0.6
3.36 4.45 3.72
-0.5 -0.4 -0.4
4.40 2.78 3.54 2.79 2.79 3.61 3.22 3.61 2.25 3.02 3.73 2.77 3.08 2.98 2.91 3.18 2.97
-1.5 -0.8 -2.5 -0.5 -2.0 -0.5 -0.5 -0.7 -3.3 -2.8 -0.6 -0.8 -1.0 -2.6 -0.5 -0.8 -1.1
NE2 HIS 323(C) NI NI 799 (C)
H-donor Metal
4.07 2.2
-1.7 -2.4
O ALA 364 (C) N MET 367 (C) CB THR 301 (C) CA LUE 319 (C) O ALA 364 (C) N ALA 366 (C) N MET 367 (C) NH2 ARG 339 (C) OQ2 KCX 220 (C) SD MET 367 (C) CB THR 301 (C) NH2 ARG 339 (C) O ALA 364 (C) 5-RING HIS139 (C) NE2 HIS 315 (C) NI 799 (C)
H-donor H-acceptor H-acceptor H-acceptor H-donor H-acceptor H-acceptor H-acceptor H-donor H-donor H-acceptor H-acceptor H-donor Pi-H H-donor Metal
2.72 3.13 3.19 3.30 2.81 3.13 2.91 2.94 3.02 3.07 3.33 3.27 2.78 3.93 2.92 2.32
-0.6 -1.5 -0.9 -0.7 -0.9 -0.8 -1.2 -3.6 -13.6 -2.7 -0.6 -1.1 -0.6 -0.5 -3.1 -3.2
-6.5793 -7.0997
-5.5778 -7.3714
-6.0903 -6.1940 -5.1088 -7.3044 -8.9114
-6.1996
-7.9425 -7.3565 -5.9060 -5.4293 -8.5344 -7.9859 -4.2697 -6.4847 -8.3034
12
21
N 14 O 12 O 12
22
S 21
23
N 14 O 12 O 12
3.
O ALA 364 (C) N ALA 366 (C) N MET 367 (C) HIS 249 (C) KCX 220 (C) NI 799 (C) NI 798 (C) O ALA 364 (C) N ALA 366 (C) N MET 367 (C)
H-donor H-acceptor H-acceptor H-donor H-donor Metal Metal H-donor H-acceptor H-acceptor
2.78 3.11 3.00 3.71 2.96 2.21 2.7 2.81 3.09 2.94
-0.8 -0.8 -2.5 -1.9 -1.4 -2.3 -1.7 -1.0 -0.8 -2.7
-6.6584
-9.0990
-9.3080
Conclusion
Twenty-three new atenolol thiourea analogs were synthesized and screened for their urease inhibitory potential. All derivatives 1-23 were found excellent to moderately active. Analogs 3, 8, 9, 12, 17, 20, and 22 showed excellent inhibitory potential than parent drug atenolol as well as the standard thiourea. Limited SAR suggested that nature, positions, and number of groups/substituents have considerable effect on inhibitory potential of compounds, however, these interpretations were further confirmed by in silico study. Molecular docking study have identified many important interactions of ligand (synthetic compounds) with the urease active site. This study has identified several drug based molecules which may serve as lead candidates for the development of new potent urease inhibitors. 4.
Experimental
4.1.
Material and Methods
Dried glass wares were used for the reactions. Chemicals including substituted phenyl isothiocyanates were purchased from Merck (Germany) and Aldrich (USA). Evaporation of organic solvent was achieved by rotatory evaporator (Büchi) with a water bath temperature 55 °C. Atenolol was kindly provided by NabiQasim Pharmaceutical Industries Pvt. Merck kieselgel 60F254 Silica gel sheet was used for TLC (Thin layer chromatography). Visualizations were accomplished with UV light and iodine vapours. Melting points were measured by using BUCHI melting point M.560 apparatus in open glass capillaries. FTIR spectra were recorded directly on Bruker FT-IR spectrophotometer (Vector 22) in the range of 4000-400 cm-1 system. FAB and HR-FAB spectra were recorded on JEOL-600H-2 and JMS-HX-110, respectively. 1H-NMR were recorded on Bruker Avance spectrophotometer at 400 MHz and
13
C-NMR were recorded on Bruker Avance spectrophotometer at 100 MHz.
Chemical shifts are represented in ppm and coupling constant J in Hz.
13
4.2.
General procedure for syntheses of atenolol thiourea derivatives 1-23
First atenolol (1 mmol) was completely dissolved in acetonitrile (10 mL) into a dried 100 mL round-bottomed flask. Then, substituted phenyl isothiocayante (1 mmol) was also added into it and refluxed at 80 °C. Product formation as well as purity of compounds were confirmed by thin layer chromatography [TLC system; 100% ethyl acetate EtOAc]. Precipitates were appeared within 5-15 minutes which were filtered, washed with hot water, and crystallized from ethanol. Structures of all synthetic compounds were deduced by various spectroscopic analysis. Spectroscopic data of all compounds is given in supplementary information. 4.3.
Urease inhibition activity
All thiourea derivatives and atenolol were screened for urease inhibition activity by using reported method [25]. In a 96-well plate, urease enzyme 25 µL (Jack bean urease 1 unit/well final concentration) and test sample (1-23 and atenolol) 5 µL (0.5 mM/well final concentration), were incubated for 15 minutes at 30 °C. 55 µL of sodium-phosphate buffer having pH = 6.8 with 100 mM urea substrate was added to each well and incubated at 30 °C for 15 minutes. Determination of ammonia production was done by indophenol method. Concisely, 45 µL of phenol reagent (0.3% w/v phenol and 0.007% w/v sodium nitroprusside) and 70 µL alkali reagent (0.3% w/v NaOH and 0.1% active chloride NaOCl) were added to each well and incubated at 30 °C for 55 minutes. After 55 minutes, absorbance was measured at 630 nm. Triplicate analysis were done for all samples including atenolol. Thiourea was used as the standard inhibitor. The IC50 values for compounds (1-23) were determined by EZFit Enzyme Kinetic Program. The % inhibition was calculated by using following formula. %Inhibition = 100 (100 – OD test well/OD control) x 100 4.4.
Molecular docking study
Molecular Operating Environment (MOE) package [26] was accustomed to perform molecular docking study so as to predict the binding mode of the synthesized compounds (123) within the active site of urease enzyme. First the 3D structures of the synthesized derivatives were generated by using builder tool executed in MOE package. Next, all compounds were protonated, and energy minimized using the default parameters of the MOE (gradient: 0.05, Force Field: MMFF94X), and saved in mdb (Molecular data bank) file format. The 3D structure of the target protein was retrieved from the protein databank (PDB
14
ID 4ubp). The retrieved protein was opened in MOE package, water molecules were removed, and 3D protonation was carried. After 3D protonation the protein was energy minimized to get a stable conformation of the protein using the default parameters of MOE package. For docking studies, the default parameters of MOE package were used i.e., Placement: Triangle Matcher, Rescoring 1: London dG, Refinement: Forcefield, Rescoring 2: GBVI/WSA. For each ligand ten conformations were allowed to be fashioned and the top ranked conformations on the basis of docking score were selected for additional analysis. Acknowledgment: The authors are thankful to the Pakistan Academy of Sciences for providing financial support to Project No. (5-9/PAS/440). 5.
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17
Graphical Abstract
18
Research Highlights
Biology-oriented drug synthesis (BIODS) of new thiourea analogs of drug atenolol.
Structural characterization by various spectroscopic techniques.
Evaluation for their urease inhibitory activity.
Amongst all compounds, compound 22 was the most potent urease inhibitor.
Molecular docking was done to decipher various important mode of interactions.
19
S0045-2068(19)31363-X https://doi.org/10.1016/j.bioorg.2019.103359 YBIOO 103359
To appear in:
Bioorganic Chemistry
Received Date: Revised Date: Accepted Date:
20 August 2019 25 September 2019 9 October 2019
Please cite this article as: S. Wahid, S. Jahangir, M. Ali Versiani, K. Mohammed Khan, U. Salar, M. Ashraf, U. Farzand, A. Wadood, Kanwal, Ashfaq-ur-Rehaman, Arshia, M. Taha, S. Perveen, Atenolol Thiourea Hybrid as Potent Urease Inhibitors: Design, Biology-Oriented Drug Synthesis, Inhibitory Activity Screening, and Molecular Docking Studies, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103359
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© 2019 Published by Elsevier Inc.
Atenolol Thiourea Hybrid as Potent Urease Inhibitors: Design, Biology-Oriented Drug Synthesis, Inhibitory Activity Screening, and Molecular Docking Studies Sana Wahid,a Sajid Jahangir,a Muhammad Ali Versiani,a Khalid Mohammed Khan,b,c Uzma Salar,d Muhammad Ashraf,e Urva Farzand,e Abdul Wadood,f Kanwal,b Ashfaq-ur-Rehaman,f Arshia,b Muhammad Taha,c Shahnaz Perveeng a
Department of Chemistry, Federal Urdu University of Art, Science and Technology, Karachi, Pakistan b H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan c Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam, Saudi Arabia d Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan e Department of Biochemistry and Biotechnology, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan f Department of Biochemistry, Computational Medicinal Chemistry Laboratory, UCSS, Abdul Wali Khan University Mardan, Khyber Pakhtunkhwa 23200, Pakistan g PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi75280, Pakistan Abstract: Current research deals with the biology-oriented drug synthesis (BIODS) of twenty-three new thiourea analogs of pharmacologically important drug atenolol which is a well-known medicine to treat hypertension as well as cardiovascular diseases (CVDs). Structural characterization of all compounds was done by various spectroscopic techniques. Compounds 1-23 were subjected for urease inhibitory activity in vitro. Screening results revealed that whole library was found to be active having IC50 ranges from 11.73 ± 0.28 to 212.24 ± 0.42 µM. It is noteworthy that several derivatives including 3 (IC50 = 21.65 ± 0.31 µM), 8 (IC50 = 19.26 ± 0.42 µM), 9 (IC50 = 21.27 ± 0.25 µM), 12 (IC50 = 21.52 ± 0.42 µM), 17 (IC50 = 19.26 ± 0.42 µM), 20 (IC50 = 16.78 ± 0.34 µM), and 22 (IC50 = 11.73 ± 0.28 µM) showed excellent inhibitory potential than parent atenolol (IC50 = 64.36 ± 0.19 µM) and standard thiourea (IC50= 21.74±1.76 µM). A most probable structure-activity relationship (SAR) was anticipated by observing varying degree of inhibitory potential given by compounds. However, molecular insights regarding the binding mode of atenolol thiourea analogs within the active pocket of urease enzyme was rationalized by molecular docking studies.
*Corresponding Authors: [email protected], [email protected], Tel.: +922134824910, Fax: +922134819018
1
Keywords: Biology-oriented drug synthesis (BIODS); atenolol; thiourea; urease; In vitro; molecular docking study 1.
Introduction
The class of drugs that is used significantly for the treatment of cardiac disorders, hypertension, anxiety, migraine, and other related complications are known as β-adrenoreceptors antagonists or β-blockers [1-3]. Different marketed drugs are being used as β-blockers, among them the most common are acebutolol, atenolol, bisoprolol etc. Nevertheless, these β-adrenoreceptor antagonists are still used in their racemic form. Atenolol, an aryloxypropanol amine derivative bearing one chiral centre selectively blocks β1, is highly polar molecule and is unable to penetrate into the blood-brain barrier [4]. It is used for the cure of hypertension, angina, acute myocardial infarction, and tachycardia. Atenolol is also used in racemic form, however, its (S)-enantiomer is most likely to inhibit β1-adrenoreceptors 50-500 times higher than (R)-enantiomer [5-8]. Urease, a member of the amidohydrolases family, is a nickel containing metalloenzyme (EC 3.5.1.5) having two nickel atoms within its active site. The prominent position of this enzyme in enzymology is mainly due to the fact that it was the first enzyme to be purified and crystallized (by James B. Sumner in 1926). This enzyme is widely distributed in the nature ranging from prokaryotes to eukaryotes with slight variation in the composition, however, the function is same i.e. hydrolysis of urea into carbonic acid and ammonia [9]. Urea being a stable molecule requires an enzyme (catalyst) for hydrolysis to breakdown the molecule around 1014 times greater than the uncatalyzed reaction [10]. Since, ammonia is produced in the hydrolysis of urea which leads to increased pH, thus leading to alkalinity. The increased alkalinity facilitates the growth of a pathogenic bacteria Helicobacter pylori which is responsible of infectious diseases such as urolithiasis, deudonal, peptic, and gastric ulcers [11, 12, 13]. Biology-oriented drug synthesis (BIODS) is the term used when marketed drugs modified via one or two steps chemical transformation and explored for their diversified activities [14-18]. Our research group has modified various drugs using this approach and explored for their diverse biological potentials. In recent past, we have reported the urease inhibition of Snaproxen derivatives [18] (Figure-1). In current study, we incorporated thiourea functionality on drug atenolol. Since, thiourea [19] and its derivatives are well-known as potent urease inhibitors [20], thus we intended to explore these new atenolol thiourea hybrids for urease
2
inhibitory potential. Thiourea moiety also has wide range of medicinal importance including antioxidant [21], antiinflammatory [22], anticonvulsants [23], and antibacterial [24] activities. In current attempt, all twenty-three (23) synthetic atenolol thiourea derivatives and parent atenolol molecule were subjected for evaluation of their urease inhibitory potential in vitro. Enzyme-ligand (synthetic compounds) interactions were accessed by in silico studies and got good results. Till to date, all synthetic compounds never reported before structurally and biologically.
Figure-1: Rationale of Current Study 2.
Results and Discussions
2.1.
Chemistry
In the current study, atenolol was treated with a number of isothiocyanates. In the reaction mechanism, lone pair of secondary aminic nitrogen of atenolol attacks on the electrophilic carbon center of isothiocyanate. Almost in all cases, reaction completed within 5-15 minutes under reflux condition. Products were appeared as solid and crystallized from ethanol (Scheme-1).
Scheme-1: Biology-Oriented Drug Synthesis of Atenolol Thiourea Derivatives 1-23
3
Chemical structures of all compounds were deduced by various spectroscopic techniques including FAB-MS (+ve), HRFAB-MS (+ve), 1H-, 13C-NMR, and FT-IR (see supplementary information). It is worth-mentioning that all synthetic atenolol thiourea derivatives (1-23) were never reported before structurally (Table-1). 2.2.
Urease inhibitory activity In vitro
Newly synthesized atenolol thiourea analogs were subjected for the evaluation of their urease inhibitory activity. It is note-worthy that all thiourea analogs were found to be active. Parent drug atenolol having IC50 = 64.36 ± 0.19 µM, was also screened for its inhibitory potential in order to compare the activity results of derivatives. Thiourea with IC50 = 21.74 ±1.76 µM was used as the standard. Almost all derivatives except 2, 4, 6, 13, 14, and 23 were found to show better activity than parent atenolol (IC50 = 64.36 ± 0.19 µM) whereas analogs 3, 8, 9, 12, 17, 20, and 22 showed even greater inhibitory potential than the standard thiourea (IC50 = 21.74 ± 1.76 µM) (Table-1). Table-1: Urease inhibitory activity of synthetic atenolol thiourea derivatives 1-23.
1-23 IC50 ± SEMa (µM)
Comp.
1
32.42 ± 0.21
14
72.56 ± 0.13
2
91.17 ± 0.13
15
42.37 ± 0.18
3
21.65 ± 0.31
16
56.82 ± 0.54
4
72.56 ± 0.13
17
19.26 ± 0.42
5
58.56 ± 0.59
18
52.20 ± 0.24
Comp.
R
R
IC50 ± SEMa (µM)
4
6
145.25 ± 0.35
19
41.63 ± 0.52
7
41.59 ± 0.52
20
16.78 ± 0.34
8
19.26 ± 0.42
21
52.43 ± 0.27
9
21.27 ± 0.25
22
11.73 ± 0.28
10
29.15 ± 0.12
23
212.24 ± 0.42
11
32.52 ± 0.38
64.36 ± 0.19 Atenolol
21.52 ± 0.42
12
21.74 ± 1.76 87.43 ± 0.54
13
Thioureac
SEMa (standard error mean); Thioureac (Standard inhibitor for urease enzyme)
2.3.
Structure-activity relationship (SAR)
Primarily, different functional groups and their respective positions have great influence on the urease inhibition activity, however, the variations in the inhibitory potential is attributed by varying structural motifs/groups on aryl part (R). Limited SAR was rationalized by looking at diverse range of group “R” and their effect on the inhibitory potential. Amongst all compounds, para-ethoxy substituted derivative 22 (IC50 = 11.73 ± 0.28 µM) was identified as the most potent analog in this series when compared to the standard thiourea (IC50 = 21.74 ± 1.76 µM) as well as parent drug atenolol (IC50 = 64.36 ± 0.19 µM). Effect of ethoxy substitution on urease inhibitory potential can easily be judge by comparing its activity with compound 1 (IC50 = 32.42 ± 0.21 µM) with no substitution on ring R and found
5
to be almost three times less active than 22. Compounds 20 (IC50 = 16.78 ± 0.34 µM) and 21 (IC50 = 52.43 ± 0.27 µM) are positional isomers having ortho and para methoxy substitutions, respectively. These two positional isomers showed big difference in the inhibitory potential which indicates that position of methoxy substituent on “R” played a key role in the inhibitory activity. It was also observed that alkyl substituted derivatives 18 (IC50 = 52.20 ± 0.24 µM) and 19 (IC50 = 41.63 ± 0.52 µM) showed moderate activity as compared to alkoxy substituted derivatives (Figure-2).
Figure-2: Comparison of structure-activity relationship of compounds 1 and 18-22 Amongst halogen substituted molecules, para-chloro substituted analog 3 (IC50 = 21.65 ± 0.31 µM) showed inhibition comparable to the standard thiourea. Closely related ortho and para substituted positional isomers 2 (IC50 = 91.17 ± 0.13 µM) and 4 (IC50 = 72.56 ± 0.13 µM) presented steep decline in the activity which suggested that position of chloro moieties have worth importance for inhibitory activity. Dichloro groups containing compounds 5 (IC50 = 58.56 ± 0.59 µM) and 6 (IC50= 145.25 ± 0.35 µM) showed decreased inhibitory activity as compared to monochloro substituted molecules which reveals that increasing the number of chloro didn′t enhance the inhibitory activity. Though, derivative 7 (IC50= 41.59 ± 0.52 µM) having a combination of chloro with nitro group showed relatively good inhibitory potential (Figure-3)
6
Figure-3: Comparison of structure-activity relationship of compounds 2-7 Another derivative 12 (IC50 = 21.52 ± 0.42 µM) with fluoro and methoxy substituents para to each another demonstrated good inhibitory potential. Nevertheless, ortho fluoro substituted derivative 10 (IC50 = 29.15 ± 0.12 µM) displayed inhibitory activity comparable to the standard thiourea. Addition of another fluoro substituent at para positon as in derivative 11 (IC50 = 32.52 ± 0.38 µM) slightly decreased the inhibitory potential. Trifluoromethyl substituted analogs 13, 14, and 15 having IC50s = 87.43 ± 0.54 µM, 72.56 ± 0.13 µM, and 42.37 ± 0.18 µM, respectively, showed moderate to weak inhibitory potential (Figure-4).
Figure-4: Comparison of structure activity relationship of compound 5-7 Amongst other halogenated compounds, meta-bromo substituted derivative 17 (IC50 = 19.26 ± 0.42 µM) revealed significant inhibitory potential, compared to thiourea and atenolol. While ortho-bromo substituted isomer 16 (IC50 = 56.82 ± 0.54 µM) exhibited almost three-
7
fold less active which confirmed that position of substituents play a vital role in the inhibitory potential. Amongst the nitro substituted analogs, derivative 8 (IC50 = 21.27 ± 0.25 µM) with meta-nitro group was identified slightly less active towards urease inhibition as compared to its positional isomer 9 (IC50 = 19.27 ± 0.26 µM) having para-nitro group (Figure-5).
Figure-5: Comparison of structure activity relationship of compound 8-9 Limited SAR predicted the effect of different groups such as Me, OMe, OEt, Cl, Br, F, and NO2 in the varying inhibitory potential, however, real picture of protein-ligand interactions were rationalized by the in-silico studies. A plausible demonstration is given below. 2.4.
Molecular docking studies
For the sake to predict the ligand binding interactions with the active site of urease enzyme, molecular docking of atenolol thiourea derivatives 1-23 was performed with urease crystal structure through MOE package. Most favorable docking conformations for each compound were determined with appropriate orientation within the active site. Active site of urease enzyme contained both hydrophobic and hydrophilic amino acid residues. The hydrophobic site comprised of K169, A170, 366, L319, and C322 whereas hydrophilic site contained R339, G166, 223, H 315, 323, 324, D224, 494, and 249. Two Ni ions conjointly play a significant role to link key amino acid residues and ligands. Nevertheless, it was observed from the molecular docking study that each compound’s conformations fit well within the active pocket of urease enzyme. Further, the most promising docked conformation of each
8
analog was assessed for binding mode analysis based on the scores, obtained from the GBVI/WSA binding free energy calculation. Generally, it was observed from the binding mode of all atenolol thiourea analogs that all derivatives possesses varying substituents by “R” including electron withdrawing (EWG) and donating (EDG) groups, and therefore, these substituted groups and their positions gradually altered the enzyme activity. The deep interaction pose analysis for the most potent analog 22 (IC50 = 11.73 ± 0.28 M) shows that this derivative was well fitted into the catalytic cavity and attained most favorable ionic and other interactions (i.e. hydrogen bond, hydrophobic etc.) with the active site residues, such as R338, H249, and with the modified residue KCX220. Additionally, the two embedded Ni198 and Ni799 ions in the active pocket adopt ionic bond with the S8 and O10 of the corresponding compound, and further enhance the activity against urease enzyme. The high potency of analog 22 might be due to the stabilized benzene ring. Benzene ring becomes partial +ve when substituted with EWG, which withdraws electron density from benzene ring through inductive effect. So this ring may adopt π-stacking interaction with other groups to regain stability. Other reason for the potent activity of analog 22 might be due to the ionic interaction with both the Ni ions and as well as with the modified residue which play significant role in enhancing the urease inhibition (Figure-6a). Furthermore, in case of other active compounds which includes 4, 9, 12, 17, and 18 showed best activity against urease enzyme. In case of other active compound in the series, further demonstrate the inhibition pattern in term of adopting various mode of interaction with the active site residues, hence established this ranking in the series of synthetic compounds. All these above-mentioned compounds possess EWG at benzene ring, but at various position, in case of compound 4 possess p-Cl, and further showed favorable interactions with the H315, and K169 active site residues through π-stacking pattern. Additionally, Ni799 adopt ionic interaction with the O9 of the corresponding compound. The high potency of derivative 4 might be due to EWG at para position of ring “R”, which facilitates the benzene ring to established π-interaction with other groups. Compound 17 also showed good interaction pattern with the active site residues (Figure-6c), and same mode of interaction was observed for this compound (Figure-6d). The withdrawing amplitude of the Cl is much more than the Br, so the less potency of compound 17 might be due to the above-mentioned reason, as compared to compounds 4 and 12 which possess Cl at different position but showed better mode of interaction, and inhibitory activity against urease enzyme. The obtained docking results supported well the experimental results grounded on several interactions of ligands (atenolol thiourea derivatives) with key amino
9
acid residues of urease enzyme as well as the docking scores calculated. The docking pose of almost every potent derivative computationally well inhibited the urease catalytic activities by binding firmly via different interactions such as strong polar, hydrophobic, and hydrogen bonding interactions with the key residues of active site. The detail of interaction docking score
embedded
in
(
10
Table-2).
Figure-6: Three dimensional interactions of compounds 4, 12, 17, and 22 with the active site residues of urease enzyme. Different color indicates different ligand, and key residues of the active site were colored to gray, hydrogen bonding are shown in dark color dotted lines, and the both sided arrows indicate the π-stacking interaction. (a) the mode of interaction of compound 22 with the active site residues, (b) for compound 4, (c) for compound 17, and (d) for compound 12.
11
Table-2: Docking scores and interactions detail of the compounds (1-23) against urease enzyme. Interactions Detail Comp. 1
2 3 4
5
6 7 8 9 10
11
12 13 14 15 16 17 18 19 20
Ligand
Receptor
Interaction
Distance
E(kcal/mol)
Docking Score (S) -3.1647
O1 S 21 S 21
N ARG 369 (C) CA LEU 365 (C) N ALA 366 (C)
H-acceptor H-acceptor H-acceptor
2.69 4.43 4.43
-0.7 -0.8 -1.3
N 14 O 12 O 12 O 12 6-ring N 14 O 12 O 12 N3 N 14 N 22 O 12 O 12 S 22 N 14 6-ring NI 799 N3 C 10 N 14 O 12 O 12 N 14 O 12 O 12 S21 O12 O1 O 12 O 12 S 21 O1 N 14 O 12 O 12 O1 N14 N 22 O1 O 12 O 12 C 16 O12 O1
O ALA 364 (C) N ALA 366 (C) N MET 367 (C) O Ala 170 (C) CA GLY 280 (C) O ALA 364 (C) N ALA 366 (C) N MET 367 (C) SD MET 367 (C) O ALA 364 (C) SD MET 367 (C) O MET 367 (C) N MET 367 (C) CG ARG 339 (C) O ALA 170(C) CA GLY 280(C) NE2 HIS 139 (C) O THR 362 (C) SD MET 367 (C) O ALA 364 (C) N ALA 366 (C) N MET 367 (C) O ALA 364 (C) N ALA 366 (C) N MET 367 (C)
H-donor H-acceptor H-acceptor H-donor Pi-H H-donor H-acceptor H-acceptor H-donor H-donor H-donor H-donor H-acceptor H-acceptor H-donor Pi-H Metal H-donor H-donor H-donor H-acceptor H-acceptor H-donor H-acceptor H-acceptor
2.81 3.19 2.90 3.27 3.67
-1.0 -0.7 -1.5 -0.7 -0.6
3.36 4.45 3.72
-0.5 -0.4 -0.4
4.40 2.78 3.54 2.79 2.79 3.61 3.22 3.61 2.25 3.02 3.73 2.77 3.08 2.98 2.91 3.18 2.97
-1.5 -0.8 -2.5 -0.5 -2.0 -0.5 -0.5 -0.7 -3.3 -2.8 -0.6 -0.8 -1.0 -2.6 -0.5 -0.8 -1.1
NE2 HIS 323(C) NI NI 799 (C)
H-donor Metal
4.07 2.2
-1.7 -2.4
O ALA 364 (C) N MET 367 (C) CB THR 301 (C) CA LUE 319 (C) O ALA 364 (C) N ALA 366 (C) N MET 367 (C) NH2 ARG 339 (C) OQ2 KCX 220 (C) SD MET 367 (C) CB THR 301 (C) NH2 ARG 339 (C) O ALA 364 (C) 5-RING HIS139 (C) NE2 HIS 315 (C) NI 799 (C)
H-donor H-acceptor H-acceptor H-acceptor H-donor H-acceptor H-acceptor H-acceptor H-donor H-donor H-acceptor H-acceptor H-donor Pi-H H-donor Metal
2.72 3.13 3.19 3.30 2.81 3.13 2.91 2.94 3.02 3.07 3.33 3.27 2.78 3.93 2.92 2.32
-0.6 -1.5 -0.9 -0.7 -0.9 -0.8 -1.2 -3.6 -13.6 -2.7 -0.6 -1.1 -0.6 -0.5 -3.1 -3.2
-6.5793 -7.0997
-5.5778 -7.3714
-6.0903 -6.1940 -5.1088 -7.3044 -8.9114
-6.1996
-7.9425 -7.3565 -5.9060 -5.4293 -8.5344 -7.9859 -4.2697 -6.4847 -8.3034
12
21
N 14 O 12 O 12
22
S 21
23
N 14 O 12 O 12
3.
O ALA 364 (C) N ALA 366 (C) N MET 367 (C) HIS 249 (C) KCX 220 (C) NI 799 (C) NI 798 (C) O ALA 364 (C) N ALA 366 (C) N MET 367 (C)
H-donor H-acceptor H-acceptor H-donor H-donor Metal Metal H-donor H-acceptor H-acceptor
2.78 3.11 3.00 3.71 2.96 2.21 2.7 2.81 3.09 2.94
-0.8 -0.8 -2.5 -1.9 -1.4 -2.3 -1.7 -1.0 -0.8 -2.7
-6.6584
-9.0990
-9.3080
Conclusion
Twenty-three new atenolol thiourea analogs were synthesized and screened for their urease inhibitory potential. All derivatives 1-23 were found excellent to moderately active. Analogs 3, 8, 9, 12, 17, 20, and 22 showed excellent inhibitory potential than parent drug atenolol as well as the standard thiourea. Limited SAR suggested that nature, positions, and number of groups/substituents have considerable effect on inhibitory potential of compounds, however, these interpretations were further confirmed by in silico study. Molecular docking study have identified many important interactions of ligand (synthetic compounds) with the urease active site. This study has identified several drug based molecules which may serve as lead candidates for the development of new potent urease inhibitors. 4.
Experimental
4.1.
Material and Methods
Dried glass wares were used for the reactions. Chemicals including substituted phenyl isothiocyanates were purchased from Merck (Germany) and Aldrich (USA). Evaporation of organic solvent was achieved by rotatory evaporator (Büchi) with a water bath temperature 55 °C. Atenolol was kindly provided by NabiQasim Pharmaceutical Industries Pvt. Merck kieselgel 60F254 Silica gel sheet was used for TLC (Thin layer chromatography). Visualizations were accomplished with UV light and iodine vapours. Melting points were measured by using BUCHI melting point M.560 apparatus in open glass capillaries. FTIR spectra were recorded directly on Bruker FT-IR spectrophotometer (Vector 22) in the range of 4000-400 cm-1 system. FAB and HR-FAB spectra were recorded on JEOL-600H-2 and JMS-HX-110, respectively. 1H-NMR were recorded on Bruker Avance spectrophotometer at 400 MHz and
13
C-NMR were recorded on Bruker Avance spectrophotometer at 100 MHz.
Chemical shifts are represented in ppm and coupling constant J in Hz.
13
4.2.
General procedure for syntheses of atenolol thiourea derivatives 1-23
First atenolol (1 mmol) was completely dissolved in acetonitrile (10 mL) into a dried 100 mL round-bottomed flask. Then, substituted phenyl isothiocayante (1 mmol) was also added into it and refluxed at 80 °C. Product formation as well as purity of compounds were confirmed by thin layer chromatography [TLC system; 100% ethyl acetate EtOAc]. Precipitates were appeared within 5-15 minutes which were filtered, washed with hot water, and crystallized from ethanol. Structures of all synthetic compounds were deduced by various spectroscopic analysis. Spectroscopic data of all compounds is given in supplementary information. 4.3.
Urease inhibition activity
All thiourea derivatives and atenolol were screened for urease inhibition activity by using reported method [25]. In a 96-well plate, urease enzyme 25 µL (Jack bean urease 1 unit/well final concentration) and test sample (1-23 and atenolol) 5 µL (0.5 mM/well final concentration), were incubated for 15 minutes at 30 °C. 55 µL of sodium-phosphate buffer having pH = 6.8 with 100 mM urea substrate was added to each well and incubated at 30 °C for 15 minutes. Determination of ammonia production was done by indophenol method. Concisely, 45 µL of phenol reagent (0.3% w/v phenol and 0.007% w/v sodium nitroprusside) and 70 µL alkali reagent (0.3% w/v NaOH and 0.1% active chloride NaOCl) were added to each well and incubated at 30 °C for 55 minutes. After 55 minutes, absorbance was measured at 630 nm. Triplicate analysis were done for all samples including atenolol. Thiourea was used as the standard inhibitor. The IC50 values for compounds (1-23) were determined by EZFit Enzyme Kinetic Program. The % inhibition was calculated by using following formula. %Inhibition = 100 (100 – OD test well/OD control) x 100 4.4.
Molecular docking study
Molecular Operating Environment (MOE) package [26] was accustomed to perform molecular docking study so as to predict the binding mode of the synthesized compounds (123) within the active site of urease enzyme. First the 3D structures of the synthesized derivatives were generated by using builder tool executed in MOE package. Next, all compounds were protonated, and energy minimized using the default parameters of the MOE (gradient: 0.05, Force Field: MMFF94X), and saved in mdb (Molecular data bank) file format. The 3D structure of the target protein was retrieved from the protein databank (PDB
14
ID 4ubp). The retrieved protein was opened in MOE package, water molecules were removed, and 3D protonation was carried. After 3D protonation the protein was energy minimized to get a stable conformation of the protein using the default parameters of MOE package. For docking studies, the default parameters of MOE package were used i.e., Placement: Triangle Matcher, Rescoring 1: London dG, Refinement: Forcefield, Rescoring 2: GBVI/WSA. For each ligand ten conformations were allowed to be fashioned and the top ranked conformations on the basis of docking score were selected for additional analysis. Acknowledgment: The authors are thankful to the Pakistan Academy of Sciences for providing financial support to Project No. (5-9/PAS/440). 5.
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Graphical Abstract
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Research Highlights
Biology-oriented drug synthesis (BIODS) of new thiourea analogs of drug atenolol.
Structural characterization by various spectroscopic techniques.
Evaluation for their urease inhibitory activity.
Amongst all compounds, compound 22 was the most potent urease inhibitor.
Molecular docking was done to decipher various important mode of interactions.
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