Design, synthesis and antiviral efficacy of a series of potent chloropyridyl ester-derived SARS-CoV 3CLpro inhibitors

Design, synthesis and antiviral efficacy of a series of potent chloropyridyl ester-derived SARS-CoV 3CLpro inhibitors
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  Design, Synthesis and Antiviral Efficacy of a Series of PotentChloropyridyl Ester-derived SARS-CoV 3CLpro Inhibitors  Arun K. Ghosh a, Gangli Gong a, Valerie Grum-Tokars b, Debbie C. Mulhearn b, Susan C.Baker  c, Melissa Coughlin d, Bellur S. Prabhakar  d, Katrina Sleeman c, Michael E. Johnson b,and  Andrew D. Mesecar  b a Departments of Chemistry and Medicinal Chemistry, Purdue University,West Lafayette, IN 47907 b Center for Pharmaceutical Biotechnology, Department of Medicinal Chemistry andPharmacognosy, University of Illinois at Chicago, 900 S. Ashland, IL 6060 c Department of Microbiology and Immunology, Loyola University of Chicago, Stritch School of Medicine, Maywood, IL d Department of Microbiology and Immunology, University of Illinois at Chicago, IL 60607.  Abstract Design, synthesis and biological evaluation of a series of 5-chloropyridine ester-derived severe acuterespiratory syndrome-coronavirus chymotrypsin-like protease inhibitors is described. Position of thecarboxylate functionality is critical to potency. Inhibitor 10 with a 5-chloropyridinyl ester at position4 of the indole ring is the most potent inhibitor with a SARS 3Clpro IC 50  value of 30 nM and antiviralEC 50  value of 6.9 μ M. Molecular Docking studies have provided possible binding modes of theseinhibitors. © 2008 Elsevier Ltd. All rights reserved. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.  NIH Public Access Author Manuscript  Bioorg Med Chem Lett  . Author manuscript; available in PMC 2009 October 15. Published in final edited form as:  Bioorg Med Chem Lett  . 2008 October 15; 18(20): 5684–5688. doi:10.1016/j.bmcl.2008.08.082. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    Keywords synthesis; SARS 3CLpro; ester; antiviral; inhibitor Since its first appearance in southern China in late 2002, severe acute respiratory syndrome(SARS) has been recognized as a global threat. 1  It has affected more than 8000 individuals in32 countries and caused nearly 800 fatalities worldwide within a few months. 2  Its causative pathogen is a novel coronavirus and termed as SARS-CoV. 3 , 4  While SARS is contained in theworld and no more cases have been reported since April 2004, there is expectation that thisepidemic will strike again in an even more severe form. Furthermore, the nature of itsunpredictable outbreak is a potential threat to the global economy and public heath. To date,no effective therapy exists for this viral illness.The SARS coronavirus is a positive-strand RNA virus. The 5 ′  two-thirds of the genome encodestwo overlapping polyproteins, pp1a and pp1ab, which are processed to generate the viralreplication complex. During viral replication, the replicase polyprotein undergoes extensive processing by two viral proteases namely, chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro). 5 , 6  Because of their essential roles in viral replication, both proteases arerecognized as attractive targets for development of anti-SARS therapeutics. 7  The structure and activity of active sites of both SARS-CoV 3CLpro and SARS-CoV PLpro have been elucidated.Thus far, inhibitor design efforts are mostly limited to SARS-CoV 3CLpro and numerouscovalent and noncovalent inhibitors have been reported. 7  In our continuing interest in thedesign and development of SARS-CoV 3CLpro inhibitors, we recently reported structure- based design of a number of potent peptidomimetic SARS-CoV 3CLpro inhibitors ( 1  and 2 ). Ghosh et al.Page 2  Bioorg Med Chem Lett  . Author manuscript; available in PMC 2009 October 15. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    8  The SARS-CoV 3CLpro active site contains a catalytic dyad where a cysteine residue actsas a nucleophile and a histidine residue acts as the general acid base. 9  The inhibitors bind toSARS-CoV-3CLpro through covalent bonding with the active site cysteine 145 residue. Theseinhibitors contain peptidomimetic scaffolds and lacked adequate potency, particularly antiviralactivity suitable for drug-development. Recently, Wong and co-workers reported a new classof potent small molecule benzotriazole ester-based 3CLpro inhibitors. Compound 3  is the most potent inhibitor among the benzotriazole esters. 10  The mode of action involved acylation of the active site Cys-145 assisted by the catalytic dyad. This irreversible enzyme acylation wasverified by electrospray ionization mass spectrometry of the inhibited enzyme. While theseinhibitors have shown very impressive SARS-CoV 3CLpro enzyme inhibitory activity, their antiviral activity required improvement. 11  It seems the indole-5-carboxylate moiety plays animportant role in binding with the enzyme active site. Another class of hetereoaromatic ester inhibitors was also identified and studied. 12 , 13  The 5-chloropyridine moiety in 4  proved to bethe key unit for the activity against 3CLpro. The report however lacked antiviral data. Wereport herein the development of 3-chloropyridyl ester-based SARS-CoV 3CLpro inhibitorsthat exhibit potent enzyme inhibitory activity as well as very good SARS-CoV antiviral activityin cell culture assays. We have also carried out molecular docking studies to obtain the potential binding mode of these inhibitors.The general synthetic method for 5-chloropyridinyl ester inhibitors is outlined in Scheme 1.Various chloro-3-pyridinyl esters 5 , 9 , 10 , 12 - 14  (Table 1) were synthesized by esterificationof 5-chloro-3-pyridinol and the corresponding carboxylic acids 14  mediated by DCC and DMAP at 23 °C in CH 2 Cl 2 . The synthesis of 1-acetylindolecarboxlate inhibitors were carried out by acetylation of indole 5  and 10  with acetic anhydride and pyridine under reflux to provideamide 6  and 11  respectively in excellent yields.The synthesis of 1-sulfonylindolecarboxlate inhibitors is outlined in Scheme 2. Directsulfonamidation of the indole under regular TsCl/DMAP condition at 23 °C or higher temperatures could not provide the desired product. To increase its reactivity, the indole 15 was reduced to indoline 16  by sodium cyanoborohydride in excellent yield. 15  The resultingindoline readily reacted with tosyl chloride or 3-nitrobenzenesulfonyl chloride to givesulfonamides 17  or 18  in good yield. Oxidation of indolines 17  and 18  to their correspondingindoles 19  and 20 , respecively was achieved by manganese dioxide at high temperature. 16 Hydrolysis of the methyl esters to the corresponding acids 21  or 22  using sodium hydroxidefollowed by the general esterification method described in Scheme 1 afforded the targetcompounds 7  or 8 .The structure and activity of inhibitors are shown in Table 1. The enzyme inhibitory activityof the active esters against SARS-CoV-3CLpro was determined using the full-length, authenticversion of the enzyme in a FRET-based, microplate assay described by Grum-Tokars and co-workers. 8 , 17  The assays were performed in 96-well microplates using a reaction volume of 100 μ L which contained 50 mM HEPES, pH 7.5, 100 nM authentic SARS-CoV-3CLpro enzyme,1 mM DTT, 0.01 mg/mL BSA and varying concentrations of inhibitors. The reactioncomponents, with the exception of substrate, were incubated for 20 minutes and the reactionwas initiated by the addition of FRET-substrate HiLyte Fluor™ 488-Glu-Ser-Ala-Thr-Leu-Gln-Ser-Gly-Leu-Arg-Lys-Ala-Lys(QXL520™)-NH 2 , giving a final substrate concentrationof 2 μ M as described. 8 , 17  The IC 50  values for inhibitors were determined by measuring therates of reaction with increasing inhibitor concentrations.As shown in Table 1, the known 10  benzotriazole ester inhibitor 3  was evaluated in our assayas a control. In inhibitor 5 , the benzotriazole unit was replaced by 5-chloropyridine unit.Inhibitor 5  has shown comparable enzymatic inhibitory potency (IC 50  0.31 μ M) as that of 3 .However, inhibitor 3  did not exhibit any antiviral activity while 5-chloropyridine ester 5  has Ghosh et al.Page 3  Bioorg Med Chem Lett  . Author manuscript; available in PMC 2009 October 15. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    antiviral activity with an EC 50  value of 24 μ M. 18  When the indole nitrogen was acetylated, theresulting compound 6  remained quite potent (IC 50  of 0.40 μ M) as did the tosylated indole 7 (IC 50  of 0.37 μ M). Interestingly, its nitrobenzenesulfonamide analog 8  shows an improvedIC 50  value (2 μ M). We then investigated the importance of the carboxylic position on the benzene ring of indole. Accordingly, carboxylate substitution on indole rings at 5, 6, 4 and 7 positions resulted in chloropyridinyl esters 5 , 9 , 10  and 12 , respectively. These inhibitors wereevaluated and as it turned out, inhibitor 10 , with a carboxylate at the 4-position, was the most potent inhibitor with an IC 50  of 30 nM, a 10-fold potency enhancement over 5  containing acarboxylate at the 5-position. Compound 10  also shows the best SARS-CoV antiviral activitywith an EC 50  value of 6.9 μ M. Acylation of 10  to 11  resulted in a 30-fold loss of potency.Penicillin-derived chloropyridine 13  did not show any appreciable activity.Tetrahydroisoquinoline derivative 14 , however, exhibited an enzyme IC 50  value of 0.14 μ M.In general, indole with free nitrogen is more potent than its corresponding protected analogue.To confirm that 3CLpro is covalently modified by 10 , we determined enzyme modificationusing MALDI-TOF. 19  Authentic SARS-CoV 3CLpro was incubated with compound 10  for 20 minutes and then analyzed in comparison with untreated enzyme. A shift of approximately217 Da was observed after treatment of SARS-CoV 3CLpro with the inhibitor confirmingcovalent modification. Covalent modification by similar reactive esters has also been reported 13a , 20 .To obtain molecular insight into the binding properties of these active ester-based inhibitors,we conducted docking studies in the 3CLpro active site. GOLD3.2 21  was used to dock our most active compound, 10  (GRL-0496), into the active site of the authentic SARS-CoV 3Clprostructure (PDBID: 2HOB). 22  In search of obtaining a model of the associated complex betweenthe unreacted ester and protein, (i.e. prior to nucleophilic attack by CYS145), the distance between the carbonyl carbon atom of 10  and the sulfur atom of CYS145 was constrained to bein the range of 2.5-3.5Å. This pre-reaction or ‘collision complex’ is shown in Figure 2, andresulted in a distance of 2.8Å between the carbonyl carbon of 10  and the sulfur of CYS145.This orientation of the ligand has the pyridinyl chloride group situated in the S1 pocket, withthe chloro group pointing towards the surface of the protein. The nitrogen of the chloropyridinylleaving group is in close proximity (2.4Å) to the imidazole nitrogen of HIS163. The carbonyloxygen is situated between three backbone nitrogens, forming three hydrogen bonds. As shownin Figure 2: the first hydrogen bond is from CYS145(NH) (2.32Å), the second is from SER144(NH) (2.38Å), and the third is from GLY143(NH) (2.78Å). This suggests that a fairly stronghydrogen bonding network is present within the active site which likely aids in positioning andstabilizing the carbonyl group of the ester for nucleophilic attack by the CYS145. The indolegroup of 10  is positioned near the more hydrophobic S2 pocket, with the indole nitrogen likelyinteracting with the imidazole group of HIS41, see Figure 2. Next, we analyzed the interaction between 10  and 3CLpro in the ‘post reaction’ or covalentlymodified state. The product of the reaction of 10  with 3CLpro was docked using a more recentlyreleased 3CLpro crystal structure, (PDBID: 2V6N). 20  This crystal structure contains a benzotriazole ester molecule which has reacted with the thiol of CYS145, forming a covalently bound ligand similar to the compounds presented in this paper. We have used this crystalstructure for the post-reaction complex due to the notable movement of the HIS41 in 2V6N,which flips and is able to pi stack with the aromatic moiety of the smaller covalently boundligand. 20  GOLD3.2 21  was chosen again for generating this model, as it has the capability for docking covalently bound ligands.Figure 3 is a docked model of 10  covalently attached to the CYS145 sulfur atom. The modelsuggests that the indole group of 10  shifts and positions itself where the leaving group was inthe complex, more towards the S1 pocket. This positioning of inhibitor 10  in the complex is Ghosh et al.Page 4  Bioorg Med Chem Lett  . Author manuscript; available in PMC 2009 October 15. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t    not surprising as a similar orientation has been proposed before by James’ group for similar esters. 23 , 24  More importantly though is the obvious pi-pi stacking of the indolyl of 10  with theimidazole ring of HIS41, which is also seen in the benzotriazole group of the referenced crystalstructure (2V6N). 20  There is approximately 4Å between the aromatic rings of the indolyl andthe imidazole of HIS41. This interaction clearly determines the position of the indolyl groupof our compounds. Other residues shown in Figure 3, such as ASP187 and GLN189 are morethan 5Å away from the indolyl, but might come into play with larger substitutents, such as incompound 11 .The results of the docking studies presented here suggest that the indole group of 10  can potentially occupy two different binding pockets during the course of the reaction. Dynamicenzymatic rearrangement in the vicinity of CYS145 have recently been suggested from the X-ray structure of SARS-CoV 3CLpro that had been reacted with 1-(4-dimethylaminobenzoyloxyl)-benzotriazole, 20  and suggests that after covalently linking toCYS145, the indolyl of 10 shifts towards the S1 pocket and stacks with the shifted imidazolering of HIS41, locking the orientation, with implications of where substitutions on the indolylring would be most beneficial. By having both of these models now available, structuralmodifications of the indole group can be tailored to enhance interactions with the S2 pocketfor the complex formation to readily occur, but at the same time, consider that the ligand must be mobile enough to then occupy the S1 pocket post reaction.In conclusion, our design strategies by combining the key parts of two mechanism-basedinhibitors led to a series of 5-chloropyridinyl indolecarboxylate inhibitors with enzymatic potency at submicromolar levels. The position of the carboxylic acid ester is critical to its potency. Indolecarboxylate 10  with carboxylate functionality at 4-position is the most potentinhibitor with enzyme inhibitory activity against SARS-CoV 3CLpro at IC 50  of 30 nM andantiviral potency with an EC 50  value of 6.9 μ M. Further design and synthesis of more effectiveinhibitors are in progress in our laboratories.  Acknowledgements The financial support of this work is provided by the National Institute of Health (NIAID, P01 A1060915). References and Notes 1. World Health Organization. Communicable Disease Surveillance & Response. website: and He J-F, Peng G-W, Min J, Yu D-W, Liang W-L, Zhang S-Y, Xu R-H, Zheng H-Y, Wu X-W, Xu J,Wang Z-H, Fang L, Zhang X, Li H, Yan X-G, Lu J-H, Hu Z-H, Huang J-C, Wan Z-Y, Hou J-L, LinJ-Y, Song H-D, Wang S-Y, Zhou X-J, Zhang G-W, Gu B-W, Zheng H-J, Zhang X-L, He M, ZhengK, Wang B-F, Fu G, Wang X-N, Chen S-J, Chen Z, Hao P, Tang H, Ren S-X, Zhong Y, Guo Z-M,Liu Q, Miao Y-G, Kong X-Y, He W-Z, Li Y-X, Wu C-I, Zhao G-P, Chiu RWK, Chim SSC, Tong Y-K, Chan PKS, Tam JS, Lo YMD. Science 2004;303:1666. [PubMed: 14752165]3. Drosten C, Gunther S, Preiser W, van der Werf S, Brodt HR, Becker S, Rabanau H, Panning M,Kolesnikova L, Fouchier RA, Berger A, Burguiere AM, Cinatl J, Eickmann M, Escriou N, GrywnaK, Kramme S, Manuguerra JC, Muller S, Rickerts V, Sturmer M, Vieth S, Klenk HD, Osterhaus AD,Schmitz H, Doerr HW. N. Engl. J. Med 2003;348:1967. [PubMed: 12690091]4. Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, Tong S, Urbani C, Comer JA, LimW, Rollin PE, Dowell SF, Ling AE, Humphrey CD, Shieh WJ, Guarner J, Paddock CD, Rota P, FieldsB, DeRisi J, Yang JY, Cox N, Hughes JM, LeDuc JW, Bellini WJ, Anderson LJ. N. Engl. J. Med2003;348:1953. [PubMed: 12690092] Ghosh et al.Page 5  Bioorg Med Chem Lett  . Author manuscript; available in PMC 2009 October 15. NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  NI  H-P A A  u t  h  or M an u s  c r i   p t  

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