Design, synthesis and biological evaluation of bisabolangelone oxime derivatives as potassium-competitive acid blockers (P-CABs)

Accepted Manuscript Design, synthesis and biological evaluation of bisabolonalone oxime derivatives as potassium-competitive acid blockers (P-CABs) Nian-Yu Huang, Wen-Bin Wang, Lei Chen, Hua-Jun Luo, Jun-Zhi Wang, WeiQiao Deng, Kun Zou PII: DOI: Reference:

S0960-894X(16)30271-2 http://dx.doi.org/10.1016/j.bmcl.2016.03.051 BMCL 23694

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Bioorganic & Medicinal Chemistry Letters

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20 December 2015 29 February 2016 14 March 2016

Please cite this article as: Huang, N-Y., Wang, W-B., Chen, L., Luo, H-J., Wang, J-Z., Deng, W-Q., Zou, K., Design, synthesis and biological evaluation of bisabolonalone oxime derivatives as potassium-competitive acid blockers (PCABs), Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.03.051

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Design, synthesis and biological evaluation of bisabolonalone oxime derivatives as potassium-competitive acid blockers (PCABs)

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Nian-Yu Huang, Wen-Bin Wang, Lei Chen, Hua-Jun Luo, Jun-Zhi Wang, Wei-Qiao Deng, Kun Zou

Bioorganic & Medicinal Chemistry Letters

Design, synthesis and biological evaluation of bisabolonalone oxime derivatives as potassium-competitive acid blockers (P-CABs) Nian-Yu Huang a, Wen-Bin Wang a, Lei Chen a, Hua-Jun Luo a, , Jun-Zhi Wang a, Wei-Qiao Deng b, Kun Zou a a

Hubei Key Laboratory of Natural Products Research and Development, College of Biological and Pharmaceutical Sciences, China Three Gorges University, Yichang 443002 (PR China) b State Key Laboratory of Molecular Reaction Dynamics, Dalian National Laboratory for Clean Energy, Dalian Institute of Chemica l Physics, Chinese Academy of Science, 457 Zhongshan Road, Dalian 116023 (PR China)

A RT I C L E I N F O

A BS T RA C T

Article history: Received Revised Accepted Available online

With the aim of searching novel P-CABs, seven bisabolangelone oxime derivatives were designed, synthesized, characterized and evaluated the H +,K+-ATPase inhibitory activities guided by computer aided drug design methods. The binding free energy calculations were in good agreement with the experiment results with the correlation coefficient R of -0.9104 between ΔGbind and pIC50 of ligands. Compound 5 exhibited the best inhibitory activity (pIC50 = 6.36) and most favorable binding free energy (ΔGbind = -47.67 kcal/mol) than other derivatives. The binding sites of these compounds were found to be the hydrophobic substituted groups with the Cys813 residue by the decomposed binding free energy analysis.

Keywords: P-CABs bisabolangelone H+,K+ -ATPase inhibitory activities drug rational design synthesis

2009 Elsevier Ltd. All rights reserved.

 Corresponding author. Tel.: +86-717-6397980; fax: +86-717-6397980; e-mail: [email protected] (Luo, Hua-Jun)

The gastric H+,K+-ATPase (proton pump) is a P-type ATPdriven cation transporter, which engages in 2K+/2H+/1ATP electroneutral ion exchange to generate a million-fold H+gradient across the parietal cell membrane1. H+,K+-ATPase inhibitors are a well known class of pharmaceutical agents used in the treatment of gastric acid-related diseases such as gastric ulcers, duodenal ulcers, gastro esophageal reflux disease (GERD), and so on2. There are two types of H+,K+-ATPase inhibitors including irreversible and reversible inhibitors. Irreversible H+,K+-ATPase inhibitors such as omeprazole, lansoprazole, rabeprazole, pantoprazole, tenatoprazole and leminoprazole are named proton pump inhibitors (PPIs) and considered as the first-line therapy for acid suppression3. However, PPIs exhibit a delayed onset of acute effect and achieve full effect only incrementally over several dose cycles, because of their chemical structures and irreversible inhibition of H+,K+-ATPase (forming a covalent complex with the protein at specific cysteine residue)4,5. Now reversible inhibitors, also named potassium-competitive acid blockers (P-CABs) including SCH28080, Soraprazan, Revaprazan, and TAK-438, are found to overcome the limitations of conventional PPIs, which reversibly inhibit gastric H+,K+-ATPase by competing with the K+ on the luminal surface6,7. Bisabolangelone (Scheme 1), a bioactive sequiterpene in the roots of Angelica polymorpha, was found to possess potent inhibitory activity against H+,K+-ATPase with the doses of 3.8, 7.6 and 15.3 mg/kg (P < 0.01) in our previous work8. Recently, we found that the bisabolangelone reduction derivative (3R,6R,Z)-3-Hydroxy-3,6-dimethyl-2-(3-methylbutylidene) hexahydrobenzofuran-4(2H)-one (1a) was the potent P-CABs (IC50 = 23.21 µM)9. To enhance the activity of the lead compound, we design the bisabolangelone oxime derivatives (Scheme 1) from 1a and 1b by computer aided drug design methods including homology modeling, induced-fit docking, QM/MM optimization and MM/GBSA binding free energy calculations, and all the bisabolangelone oxime derivatives were synthesized and performed biological evaluation in this paper.

vdW scaling of 0.5 was used for both the protein and ligand nonpolar atoms. The Glide XP mode18 was used for the initial docking, and 20 ligand poses were retained for protein structural refinements. Previous biochemical and mutagenesis studies11,19,20 suggest that Ala335, Tyr799 and Cys813 in pig H+,K+-ATPase are the key amino acid residues in the luminal cavity. Therefore, dimensions for the cubic boundary box centered on the centroid of these three residues were set to 22 Å × 22 Å × 22 Å. Secondly, Prime program was used to generate the induced-fit proteinligand complexes. Each of the 20 structures from the previous step was subjected to side chain and backbone refinements. All residues with at least one atom located within 5.0 Å of each corresponding ligand pose were included in the Prime refinement21. The refined complexes were ranked by Prime energy, and the receptor structures within 30 kcal/mol of the minimum energy structure were passed through for a final round of Glide docking and scoring. Finally, each ligand was redocked into every refined low-energy receptor structure produced in the second step using Glide XP mode at default settings. An IFD score (IFD score = 1.0 Glide_Gscore + 0.05 Prime_Energy) that accounts for both the protein–ligand interaction energy and the total energy of the system was calculated and used to rank the IFD poses. The best pose complex was chosen to run QM/MM optimization. M/MM optimization. The induced-fit docking complexes were energetically optimized by QM/MM method. QM/MM calculations were carried out using the QSite program22 of the Schrödinger suite. The ligands were defined as QM region calculated by the density functional theory DFT/B3LYP (6-31G* basis set). The receptor as MM region was minimized with Truncated Newton algorithm (maximum cycles as 1000; gradient criterion as 0.01). The OPLS 2005 all-atom force field was employed. MM/GBSA calculations. After QM/MM optimization, binding free energy (ΔGbind) calculations were performed for complex using molecular mechanics-generalized Born surface area (MM/GBSA) method. MM/GBSA procedure in Prime program23 was used to calculate ΔGbind of the docked ligands according to the following equations24: Gbind  EMM  Gsolv  TS

Scheme 1. Synthetic routes for the bisabolonalone oxime derivatives.

Homology modeling. Now the structure of gastric H+,K+ATPase is poorly defined, being currently limited to a resolution of 7 Å (PDB code: 3IXZ10, resolution: 6.5 Å; PDB code: 2XZB11, resolution: 7 Å). Therefore the H+,K+-ATPase structure was constructed by homology modeling as previously described12. Molecular docking. The docking simulations were performed using induced-fit docking (IFD) method 13 in the Schrödinger software suite14, which had been reported to be a robust and accurate method to account for both ligand and receptor flexibility13,15. The IFD protocol was carried out in three consecutive steps16,17. Firstly, the ligand was docked into a rigid receptor model with scaled-down van der Waals (vdW) radii. A

Where ΔEMM is the difference of the gas phase MM energy between the complex and the sum of the energies of the protein and inhibitor, and includes ΔEinternal (bond, angle, and dihedral energies), ΔEElect (electrostatic), and ΔEVDW (van der Waals) energies. ΔGsolv is the change of the solvation free energy upon binding, and includes the electrostatic solvation free energy ΔGGB (polar contribution calculated using generalized Born model), and the nonelectrostatic solvation component ΔGSA (nonpolar contribution estimated by solvent accessible surface area). TΔS is the change of the conformational entropy upon binding, which was calculated using normal-mode analysis Rigid Rotor Harmonic Oscillator (RRHO) contained in MacroModel module25. Synthesis of bisabolangelone oxime derivatives. The starting bisabolangelone reduction deratives (1a and 1b) was prepared a ccording to the procedures described in our previous work9. As t he sesquiterpene-type bisabolangelone exhibited remarkably pre ventive and therapeutic action on gastric ulcer, the structure mo dification on this compound was undertaken in this work with th e aim of searching the better P-CABs. The bisabolangelone oxi mes (2, 8) were synthesized through the condensation reaction o f hydroxylamine hydrochloride in high yields, which was alkyla ted with various haloalkanes in pyridine to produce the target ox ime ethers 3~6 in moderate yield (Scheme 1). In order to study t he sturucture-activity relationship, the oxime 2 was further trans fer into corresponding carbamoyl oxime 7. All of these compou

nds were confirmed by NMR, IR and ESI-MS. The =C-H in 3methylbutylidene for 2~7 overlapped with C(8)-H at 4.40~4.73 ppm as multiple peaks in 1H NMR. The strong carbonyl absorpti on peak at 1588-1598 cm-1 could be clearly observed in the IR s pectra. In the positive ion ESI-MS spectrum, the sodium ion add uct was usually observed as the base peak ion for all target com pounds. H+/K+-ATPase inhibition activity measurement of compounds. According to the measurement method of Saccomani et al.26 and Yoon et al.27, ion-leaky membrane vesicle enriched in gastric H+/K+-ATPase was derived from pig stomach. The inhibition activities of H+/K+-ATPase was calculated based on the difference between the activity of H+/K+-ATPase with and without K+ ion. The lyophilized vesicle in HEPES/Tris buffer was incubated in the presence of various concentrations of compounds. Final assay concentrations: 5 mM HEPES/Tris buffer (pH 7.4), 0.25 M sucrose, 10 mM KCl, 5 mM MgCl2, 10 μM valinomycin. The enzymatic reaction was started by addition of 3 mM ATP. The assay was incubated for 30 min at 37ºC. Enzymatic activity was stopped by adding colorimetric reagent and the amount of mono phosphate (Pi) in the reaction was measured at 620 nm using the microplate reader. The difference between Pi production with and without K+ was taken as K+ stimulated H+/K+-ATPase activity. The inhibitory rate (%) was determined from the activity value of the control and the activity values of various concentrations of the test compound, and the 50% inhibitory concentration (IC50) of the H+/K+-ATPase activity was determined. Calculation and experimental results of ligands with H+,K+-ATPase. The molecular docking and QM/MM optimization results (Glide Gscores, IFD scores and QM/MM energies (the best pose) of compounds) were listed in Table 1. By MM/GBSA calculations, the ΔGbind values (Table 1) show that the order of favorable binding interaction is compound 5 > 3 > 7 > 4 > 6 > 2 > 8. H+,K+-ATPase inhibition activity of compounds were measured (positive drug revaprazan IC50: 0.95 μM at pH 7.4). The order of pIC50 (negative logarithms of 50 % inhibition concentration, Table1) of ligands is compound 5 > 3 > 4 > 7 > 6 > 2 > 8. The correlation coefficient R between ΔGbind and pIC50 of ligands is -0.9104 (Fig. 1). The binding free energy calculations are in good agreement with the experiment results. Table 1. Glide docking Gscores, IFD scores, QM/MM energies, binding free energies (kcal/mol) and pIC50 of bisabolangelone oxime derivatives with H+,K+ -ATPase Compd. Gscore IFD QM/MM ΔGbind pIC50 score energy -5.76 -1720.42 -936.47 -33.08 5.54 2 -9.39 -1724.68 -1207.02 -47.09 6.25 3 -7.55 -1724.36 -1242.84 -40.40 6.07 4 -6.35 -1720.35 -1052.29 -47.67 6.36 5 -5.57 -1722.99 -1093.62 -37.17 5.94 6 -7.93 -1722.45 -1336.56 -45.83 6.00 7 -6.17 -1718.80 -937.88 -32.49 5.30 8

embedded in Maestro 9.328. To provide quantitative information of the key residues, the binding free energy between ligands and H +,K+ATPase was decomposed into the contribution of each residue. The energy comparisons of residues in binding sites were shown in Fig. 3. The bisabolangelone oxime derivatives all interact with residues Gly812-Ile814, especially with the key residue Cys813. The ΔG bind between Cys813 and the highest activity compound 5 reached the highest value (-16.02 kcal/mol, Fig. 3D) by hydrophobic interaction (Fig. 2D), and ΔGbind of the second high activity compound 3 with Cys813 is -12.43 kcal/mol (Fig. 3B) through hydrophobic and hydrogen bond interactions (Fig. 2B, Table 2), while the contribution of Cys813 to the low activity compound 2 is only -2.99 kcal/mol (Fig. 3A). So Cys813 is the very important binding site, and the hydrophobic substituted group of bisabolangelone oxime derivatives such as acetylene and phenyl group could increase the interaction with Cys813. The information of hydrogen bonds between compounds and H+,K+-ATPase are listed in Table 2. Compound 7 and 4 have strong H-bond interactions with Ile814 (ΔGbind = -11.43 and -10.60 kcal/mol, Fig. 3F and 3C, Fig. 2F and 2C, respectively). Leu811 interacts with compound 3 also by H-bond (-6.97 kcal/mol, Fig. 2B and Fig. 3B). Because of the H-bond interaction (Fig. 2D), the binding free energy between Tyr928 and compound 5 (-1.69 kcal/mol) is higher than that of other compound (Fig. 3). Due to the polar molecular structure, compound 2 and 8 has weak hydrophobic interactions and low activity. But compound 2 has strong interactions with Asp137 (-4.96 kcal/mol) and Asn138 (-5.33 kcal/mol, Fig. 3A) by electrostatic and H-bond interactions (Fig. 2A, Table 2). Compared to compound 2, the lowest activity compound 8 has the strong interactions with Cys813 (-8.43 kcal/mol, Fig. 3G) and Tyr925 (-5.31 kcal/mol) by H-bonds (Fig. 2G, Table 2). Through negative charged interaction, ΔGbind between Asp137 and compound 3 is the highest (-5.25 kcal/mol) among the ligands, because the carbon atom in benzylic of bisabolangelone oxime 3 (near Asp137, Fig. 2B) has partial positive charge. The isopentene chain of bisabolangelone oxime derivatives insert into Val331-Ile336 and Tyr799-Leu809 binding pocket (Fig. 2). The contribution of Val331 to compound 6 and 7 is high (-7.23 and -7.50 kcal/mol, Fig. 3E and 3F, respectively) because of hydrophobic interaction. The binding free energies of compound 4 with Ala335 (-3.24 kcal/mol) and Tyr799 (-4.92 kcal/mol) are more favorable than those of other ligands. Therefore, besides Cys813, interacting with Asp137, Asn138, Val331, Ala335, Tyr799, Leu811, Gly812, Ile814 and Tyr928 is also important to bisabolangelone oxime derivatives.

(A)

(C)

(B)

(D)

Fig. 1. The relationship between binding free energy and pIC 50 of compounds.

(E) After the molecular docking and QM/MM optimization, the interaction modes of bisabolangelone oxime derivatives with H +,K+ATPase were compared in Fig. 2 by Ligand Interactions module

(F)

Development Foundation and Scientific Foundation from graduate school (2015CX131) of China Three Gorges University.

References and notes 1.

(G) Fig.2 Interaction modes of ligands with H+,K+ -ATPase, (A)-(G): compound 2-8 Table 2 The hydrogen bonds between compounds and H +,K+ -ATPase. Compound Group Residue Distance/ Å —OH Asp137 1.488 2 =N— Asn138 2.153 —OH Leu811 2.308 3 —OH Cys813 2.229 =O Ile814 2.144 4 —O— Tyr928 2.191 5 —NH Asp137 1.804 7 =O Ile814 2.088 —O— Cys813 2.412 8 —OH Tyr925 1.845

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

(A)

(B)

14. 15. 16. 17.

(C)

(D)

18. 19. 20.

(E)

(F)

21. 22. 23. 24.

(G) Fig.3 The energy comparisons of residues in binding sites (A)-(G) for compound 2-8.

In summary, seven bisabolangelone oxime derivatives as P-CABs were designed and synthesized by computer aided drug design methods including homology modeling, induced-fit docking, QM/MM optimization and MM/GBSA binding free energy calculations. After evaluating H+,K+-ATPase inhibition activity of these derivatives, the binding free energy calculations were in good agreement with the experiment results. The correlation coefficient R between ΔGbind and pIC50 of ligands was -0.9104. With substituted propinyl group, Compound 5 (pIC50 = 6.36) exhibited the higher activity and more favorable binding free energy (ΔGbind = -47.67 kcal/mol) than other derivatives. By the decomposed binding free energy analysis of each residue, Cys813 was found to be the most important binding site by strong interactions with the hydrophobic substituted group of bisabolangelone oxime derivatives. These calculation results could promote the rational design of novel PCABs.

Acknowledgements: The authors thank the finance supported by Natural Science Foundation of China (No. 21272136), Natural Science Foundation of Hubei Province in China (No. 2014CFB684), Youth Talent

25. 26. 27. 28.

Shin, J.M.; Munson, K.; Vagin, O.; Sachs, G. Pflugers Arch. – Eur. J. Physiol. 2009, 457, 609. Li, H.; Meng, L.; Liu, F.; Wei, J.F.; Wang, Y.Q. Expert Opin. Ther. Patents, 2013, 23, 99. Shin, J.M.; Sachs, G. Dig. Dis. Sci. 2006, 51, 823. Sachs, G.; Shin, J.M.; Vagin, O.; Lambrecht, N.; Yakubov, I.; et al. J. Clin. Gastroenterol. 2007, 41, S226. Sachs, G.; Shin, J.M.; Howden, C.W. Aliment Pharmacol. Ther. 2006, 23, S2. Andersson, K.; Carlsson, E. Pharmacol. Ther. 2005, 108, 294. Yoon, Y.A.; Park, C.S.; Cha, M.H.; Choi, H.; Sim, J.Y.; et al. Bioorg. Med. Chem. Lett. 2010, 20, 5735. Wang, J.; Zhu, L.; Zou, K.; Cheng, F.; Dan, F.J.; et al. J. Ethnopharm. 2009, 123, 343. Huang, N.Y.; Chen, L.; Liao, Z.J.; Fang, H.B.; Wang, J.Z.; et al. Chin. J. Chem. 2012, 30, 71. Abe, K.; Tani, K.; Nishizawa, T.; Fujiyoshi, Y. Embo. J. 2009, 28, 1637. Abe, K.; Tani, K.; Fujiyoshi, Y. Nat. Commun. 2011, 2, 155. Luo, H.J.; Wang, J.Z.; Huang, N.Y.; Deng, W.Q.; Zou, K. J. Comput. Aided Mol. Des. 2016, 30, 27. Sherman, W.; Day, T.; Jacobson, M.P.; Friesner, R.A.; Farid, R. J. Med. Chem. 2006, 49, 534. Schrödinger, LLC, New York, 2010, www.schrodinger.com Zhong, H.; Tran, L.M.; Stang, J.L. J. Mol. Graph. Model, 2009, 28, 336. Wanga, H.; Aslanian, R.; Madison, V.S. J. Mol. Graph. Model, 2008, 27, 512. Luo, H.J.; Wang, J.Z.; Deng, W.Q.; Zou, K. Med. Chem. Res. 2013, 22, 4970. Friesner, R.A.; Murphy, R.B.; Repasky, M.P.; Frye, L.L.; Greenwood, J.R.; et al. J. Med. Chem. 2006, 49, 6177. Vagin, O.; Denevich, S.; Munson, K.; Sachs, G. Biochemistry, 2002, 41, 12755. Vagin, O.; Munson, K.; Lambrecht, N.; Karlish, S.J.D.; Sachs, G. Biochemistry, 2000, 40, 7480. Jacobson, M.P.; Pincus, D.L.; Rapp, C.S.; Day, T.J.F.; Honig, B.; et al. Proteins, 2004, 55, 351. Murphy, R.B.; Philipp, D.M.; Friesner, R.A. J. Comp. Chem. 2000, 21, 1442. Kollman, P.A.; Massova, I.; Reyes, C.; Kuhn, B.; Huo. S.; et al. Acc. Chem. Res. 2011, 33, 889. Massova, I.; Kollman, P.A. Perspect Drug. Discov. Des. 2000, 18, 113. MacroModel, version 9.8, Schrödinger, LLC, New York, NY, 2010, www.schrodinger.com Saccomani, G.; Stewart, H.B.; Show, D.; Lewin, M.; Sachs, G. Biochem. Biophy. Acta, 1977, 465, 311. Yoon, Y.A.; Park, C.S.; Cha, M.H.; Choi, H.; Sim, J.Y.; et al. Bioorg. Med. Chem. Lett. 2010, 20, 5735. Maestro, version 9.3. Schrödinger, LLC, New York, 2012, www.schrodinger.com