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Accepted Manuscript Structure guided design of potent and permeable inhibitors of MERS coronavirus 3CL protease that utilize a piperidine moiety as a novel design element Anushka C Galasiti Kankanamalage Yunjeong Kim Vishnu C Damalanka Athri D Rathnayake Anthony R Fehr Nurjahan Mehzabeen Kevin P Battaile Scott Lovell Gerald H Lushington Stanley Perlman Kyeong Ok Chang William C Groutas PII S0223 5234 18 30239 3 DOI 10 1016 j ejmech 2018 03 004 Reference EJMECH 10269 To appear in European Journal of Medicinal Chemistry Received Date 7 October 2017 Revised Date 28 February 2018 Accepted Date 1 March 2018 Please cite this article as A C Galasiti Kankanamalage Y Kim V C Damalanka A D Rathnayake A R Fehr N Mehzabeen K P Battaile S Lovell G H Lushington S Perlman K O Chang W C Groutas Structure guided design of potent and permeable inhibitors of MERS coronavirus 3CL protease that utilize a piperidine moiety as a novel design element European Journal of Medicinal Chemistry 2018 doi 10 1016 j ejmech 2018 03 004 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we 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 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 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 1 Structure Guided Design of Potent and Permeable Inhibitors of MERS Coronavirus 3CL Protease that Utilize a Piperidine Moiety as a Novel Design Element Anushka C Galasiti Kankanamalage Yunjeong Kim Vishnu C Damalanka Athri D Rathnayake Anthony R Fehr Nurjahan Mehzabeen Kevin P Battaile Fax 316 978 3431 e mail bill groutas wichita edu Department of Diagnostic Medicine Fax 785 532 4039 e mail kchang vet ksu edu M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 2 Abstract There are currently no approved vaccines or small molecule therapeutics available for the prophylaxis or treatment of Middle East Respiratory Syndrome coronavirus MERS CoV infections MERS CoV 3CL protease is essential for viral replication consequently it is an attractive target that provides a potentially effective means of developing small molecule therapeutics for combatting MERS CoV We describe herein the structure guided design and evaluation of a novel class of inhibitors of MERS CoV 3CL protease that embody a piperidine moiety as a design element that is well suited to exploiting favorable subsite binding interactions to attain optimal pharmacological activity and PK properties The mechanism of action of the compounds and the structural determinants associated with binding were illuminated using X ray crystallography M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 3 1 Introduction Coronaviruses are enveloped positive sense single stranded RNA viruses of the family Coronaviridae 1 Human and animal coronaviruses are classified into at least 25 species in four genera to coronaviruses 1 Mouse hepatitis virus MHV is the most prominently studied coronavirus both in vitro and in vivo serving as the prototype coronavirus Human coronaviruses including 229E NL63 OC43 and HKU1 strains have been implicated in respiratory infections otitis media exacerbations of asthma diarrhea myocarditis and neurological disease 1 4 In contrast severe acute respiratory distress syndrome coronavirus SARS CoV and Middle East respiratory syndrome coronavirus MERS CoV pose a significant threat to public health worldwide due to their ability to cause serious human disease with high mortality rates 2 4 MERS CoV has become a global threat due to continuous outbreaks in countries on the Arabian peninsula and the potential of spread to other countries with a high mortality rate 5 8 Coronaviruses are also important pathogens in animals and are associated with respiratory and enteric illnesses including transmissible gastroenteritis virus TGEV porcine epidemic diarrhea virus PEDV feline infectious peritonitis virus FIPV and bovine coronavirus 1 There are currently no effective vaccines or small molecule therapeutics for the treatment of MERS or SARS coronavirus infection 9 11 The highly conserved genome of coronaviruses contains two open reading frames ORF1a and ORF1b on the 5 end that encode nonstructural proteins Genes encoding M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 4 the coronavirus structural proteins S spike glycoprotein E envelope protein M membrane glycoprotein and N nucleocapsid protein which play a critical role in virion cell receptor binding replication and virion assembly are located at the 3 end of the genome 1 12 Coronavirus entry is initiated by the binding of the spike protein S to cell receptors specifically dipeptidyl peptidase 4 DDP4 and angiotensin converting enzyme 2 ACE2 for MERS CoV and SARS CoV respectively 1 5 Entry into cells requires host proteases for cleavage at two sites in the S protein in the case of most CoV 13 14 Translation of the genomic mRNA of ORF1a yields polyprotein pp1a while a second polyprotein pp1b is the product of a ribosomal frame shift that joins ORF1a together with ORF1b ORF1a encodes a papain like cysteine protease PLpro and a 3C like cysteine protease 3CLpro Polyproteins pp1a and pp1b are processed by 3CLpro 11 cleavage sites and PLpro 3 cleavage sites resulting in sixteen mature nonstructural proteins including RNA dependent RNA polymerase RdRp and helicase which play important roles in the transcription and replication of coronaviruses Figure 1 Both proteases are essential for viral replication making them attractive targets for drug development 9 10 15 17 MERS CoV 3CLpro is a chymotrypsin like cysteine protease having a catalytic Cys148 His41 dyad and an extended binding site 18 21 The protease displays a stringent primary substrate specificity for a P1 Gln residue 22 and has a strong preference for a P2 Leu residue The P3 residue side chain is oriented toward the solvent while the S4 subsite is shallow preferring a small hydrophobic P4 residue Ala Functional and structural studies have delineated the similarities between the 3CLpro of coronaviruses that can be exploited in the design of broad spectrum inhibitors 23 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 5 We have recently reported the first demonstration of clinical efficacy of a coronavirus protease inhibitor a dipeptidyl aldehyde bisulfite adduct inhibitor designated GC376 24 25 Specifically administration of GC376 to cats infected with FIPV a coronavirus that is 100 fatal in cats reversed the progression of fatal FIP and resulted in clinical remission in a majority of animals 90 Since FIP disease progression is quite rapid and its pathogenesis primarily immune mediated features shared by MERS CoV we hypothesized that a viral protease inhibitor could reverse the pathogenesis of MERS CoV in affected hosts Interrogation of this hypothesis entailed as a first step the design of a new and versatile class of peptidomimetic inhibitors of MERS CoV 3CL protease We describe herein the structure guided design of inhibitors of MERS CoV 3CLpro that embody a piperidine moiety as a novel design element as well as pertinent structural and biochemical studies These inhibitors were also examined against other coronaviruses including SARS CoV FIPV and MHV to evaluate the spectrum of activity against multiple coronaviruses 2 Results and Discussion 2 1 Inhibitor design rationale The structure guided design of inhibitor I encompassed the following steps a we first determined a high resolution X ray crystal structure of MERS CoV 3CLpro in complex with GC376 Figure 2 Panel A Examination of the active site of the complex revealed that the aldehyde bisulfite adduct had reverted to the precursor peptidyl aldehyde which subsequently formed a tetrahedral hemi thioacetal upon reaction with the active site Cys148 Notably the electron density at this stereocenter was consistent with the formation of both R and S enantiomers at the covalent binding site also observed for M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 6 the other structures described in the following sections The structure reveals a network of backbone hydrogen bonds which ensure correct positioning of the inhibitor to the active site as well as two critical hydrogen bonds with the P1 Gln surrogate 26 side chain The inhibitor P2 Leu side chain is ensconced in the hydrophobic S2 subsite of the enzyme Importantly the structure shows a hydrophobic driven interaction between the benzyl group of the inhibitor and the lactam ring of the Gln surrogate side chain b based on the forgoing we reasoned that extending the cap would allow the inhibitor to assume an extended conformation and orient the phenyl ring toward the hydrophobic S4 pocket of the enzyme Validation of this idea was obtained by synthesizing extended inhibitor GC813 and determining a high resolution X ray crystal structure of the MERS CoV 3CLpro GC813 complex Figure 2 Panel B The m Cl phenethyl side chain is clearly shown to occupy the hydrophobic S4 subsite In addition to an array of H bonds with Gln192 Glu169 and Gln167 and the backbone of the inhibitor which serve to correctly position the inhibitor at the active site the inhibitor interacts with the S1 S2 and S4 subsites but not the S3 subsite c we hypothesized that the attachment of a piperidine ring to the peptidyl component would yield a structurally novel peptidomimetic I capable of 1 orienting recognition elements R3 and R4 in a correct vector relationship for optimal interactions with the S3 and S4 subsites 2 rendering a dipeptidyl inhibitor equivalent to a tetrapeptidyl inhibitor with potentially diminished PK liabilities and 3 providing a flexible means for the structure guided parallel optimization of ADMET PK and physicochemical properties using diversity sites R3 and R4 in inhibitor I Figure 3 In summary the piperidine based design strategy is a hitherto unrecognized effective means of rendering a dipeptidyl inhibitor equivalent to a M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 7 tetrapeptidyl inhibitor capable of engaging in optimal binding interactions with all four S1 S4 subsites but which however is anticipated to display diminished PK liabilities due to its reduced peptidyl character Furthermore the aforementioned piperidine based design strategy has wide applicability and can be extended to any protease with an extended binding site Preliminary evidence in support of this approach is provided by the results of enzyme and cell based screening of derivatives of I Tables 1 and 2 as well as the results of structural studies vide infra 2 2 Chemistry The synthesis of final compounds 9 a f and 10 a f is outlined in Scheme 1 1 Boc 4 piperidinone was reacted with different Grignard reagents to yield the corresponding 1 Boc 4 piperidinol derivatives 1c and 1e Refluxing L leucine methyl ester hydrochloride with trichloromethyl chloroformate yielded the isocyanate which was reacted with 1c and 1e or commercially available N substituted 4 piperidinol 1a to form the corresponding carbamate adducts 4a 4c and 4e that were hydrolyzed to the corresponding acids 5a 5c and 5e with lithium hydroxide in aqueous THF Subsequent coupling with glutamine surrogate methyl ester hydrochloride 11 afforded the desired dipeptidyl esters 6a 6c and 6e which were either treated with lithium borohydride directly or were first treated with dry HCl in dioxane followed by reaction with an alkyl sulfonyl chloride or alkyl chloroformate to yield esters 7b 7d and 7f prior to reduction with lithium borohydride to yield alcohols 8 a f Dess Martin oxidation followed by flash chromatography yielded pure aldehydes 9 a f The enantiomeric purity of the aldehydes was consistently high with the amount of epimerized aldehyde ranging between 0 10 The corresponding bisulfite adducts 10 a f were readily M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 8 obtained as white solids by stirring the aldehydes with sodium bisulfite in an ethyl acetate water mixture The synthesized compounds are listed in Table 1 2 3 Biochemical Studies The inhibitory activity of the synthesized compounds against 3CLpro of MERS CoV SARS CoV or FIPV and the antiviral activity of two representative compounds compounds 10a and 10c in a cell based system including MERS CoV FIPV and MHV were evaluated as described in the experimental section The IC50 EC50 and CC50 values are listed in Tables 1 and 2 These are the average of at least two determinations It is evident that derivatives of I function as highly potent inhibitors of all tested coronaviruses in enzyme Table 1 and cell based assays Table 2 and Figure 4 More importantly representative aldehyde bisulfite adduct compounds 10a and 10c display potent inhibition toward MERS CoV in both enzyme and cell based systems with low cytotoxicity CC50 100 M Table 2 and Figure 4 For example compound 10a has a selectivity index SI CC50 EC50 of 250 With the exception of compounds 9e 10e the aldehyde and aldehyde bisulfite adducts were found to have comparable in vitro potency toward MERS CoV 3CLpro Furthermore pharmacological activity was found to be dependent on the nature of the R3 group compounds 9e 10e are 10 fold less active toward MERS CoV 3CLpro than compounds 9a d 10a d and 9f 10f In order to establish the mechanism of action of I as well as obtain structural information that can be used to guide the optimization of pharmacological activity the high resolution X ray crystal structures of several derivatives of I bound to MERS CoV M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 9 3CLpro were determined including the cocrystal structure of the MERS CoV 3CLpro inhibitor 10c complex Figure 5A The formation of a tetrahedral adduct via the reaction of the aldehyde generated from aldehyde bisulfite adduct 10c under the crystallization conditions used 27 28 with the active site cysteine Cys148 is clearly evident confirming the mechanism of action of I Inspection of the structure reveals the presence of prominent electron density consistent with the structure of inhibitor 10c however the N Boc piperidinyl moiety was disordered The position and orientation of the benzyl group suggest that the piperidine ring is likely projecting toward the S4 subsite Inhibitor 10c is bound to the active site of the enzyme via a network of backbone H bonds with Gln192 Gln167 and Glu169 Figure 5B Additionally a H bond with His41 serves to stabilize the hemi thioacetal tetrahedral adduct Also clearly evident are three critical H bonds involving the P1 Gln surrogate ring oxygen and nitrogen with Glu169 His166 and Phe143 The H bonding interactions are near identical to those of inhibitor GC813 Figure 2 Panel B The structural complementarity of inhibitors 10c and GC813 is also evident in the electrostatic surface representation of the enzyme with the two inhibitors nestled in the active site Figure 6 The cocrystal structure of the MERS CoV 3CLpro aldehyde bisulfite adduct 10e complex also showed that under the crystallization conditions used the aldehyde bisulfite adduct reverted to the precursor aldehyde which subsequently formed a tetrahedral adduct with the active site cysteine Cys148 Figure 7A The piperidinyl moiety was disordered and consequently its precise location could not be discerned However inhibitor 10e is engaged in the same H bonding interactions as inhibitor 10c Figure 7B M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 10 3 Conclusion MERS CoV constitutes a global public health concern There are currently no licensed vaccines or antiviral drugs for the prevention and treatment of coronavirus infections We disclose herein for the first time the design and utilization of a general class of piperidine based peptidomimetic inhibitors of coronavirus 3CL proteases Attachment of the piperidine moiety to a dipeptidyl component permits the resultant hybrid inhibitor to engage in favorable binding interactions with the S3 and S4 subsites of the enzyme More importantly the approach disclosed herein can be extended to other proteases of medical relevance Finally the disclosed compounds potently inhibit MERS CoV and their mechanism of action and mode of binding to MERS CoV 3CL protease have been illuminated using X ray crystallography M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 11 4 Experimental section 4 1 General Reagents and dry solvents were purchased from various chemical suppliers Aldrich Acros Organics Chem Impex TCI America and Bachem and were used as obtained Silica gel 230 450 mesh used for flash chromatography was purchased from Sorbent Technologies Atlanta GA Thin layer chromatography was performed using Analtech silica gel plates Visualization was accomplished using UV light and or iodine NMR spectra were recorded in CDCl3 or DMSO d6 using a Varian XL 400 spectrometer Melting points were recorded on a Mel Temp apparatus and are uncorrected High resolution mass spectrometry HRMS was performed at the University of Kansas Mass Spectrometry lab using an LCT Premier mass spectrometer Waters Milford MA equipped with a time of flight mass analyzer and an electrospray ion source The purity of the compounds was determined by high performance liquid chromatography HPLC using a Waters Alliance HPLC system with a reverse phase column Symmetry C18 3 5 m 4 6 x 75 mm and a Varian Pro star HPLC system with a normal phase column Kinetex 2 6u HILIC 100A 75 x 4 6mm at 254 nm Analysis was conducted using two different methods Method A Revered phase system with gradient elution Gradient was M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 12 started with water 98 methanol 2 to water 60 methanol 40 over a period of 15 minutes mobile phase flow rate 1 0 mL min Method B Normal phase system with isocratic with 20 acetonitrile and 80 dichloromethane mobile phase flow rate 1 0 mL min All final compounds had a purity of 95 by both methods 4 1 1 Synthesis of Boc piperidinol derivatives 1c and 1e General procedure An appropriate Grignard reagent 11 mmol 1 1 eq was added dropwise under a N2 atmosphere to a solution of 1 Boc 4 piperidinone 10 mmol in dry THF 15 mL in an ice bath kept at 0oC The reaction mixture was stirred for 3 h at room temperature under a N2 atmosphere while monitoring completion of the reaction by TLC The reaction mixture was diluted with water 25 mL and the solution was acidified to pH 3 using 5 hydrochloric acid The solvent was removed on the rotary evaporator and the residue was extracted with ethyl acetate 75 mL and the layers separated The organic layer then washed with brine 40 mL and dried over anhydrous sodium sulfate filtered and concentrated to yield a colorless oily product which was purified by flash chromatography to yield 1c and 1e 4 1 2 Synthesis of L leucine methyl ester isocyanate 3 L Leucine methyl ester hydrochloride 2 100 mmol was placed in a dry 500 mL RB flask and then dried overnight on the vacuum pump The flask was flushed with nitrogen and dry dioxane 200 mL was added followed by trichloromethyl chloroformate 29 67 g 150 mmol and the reaction mixture was refluxed for 10 h The solvent was M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 13 removed on the rotary evaporator and the residue was vacuum distilled to yield pure isocyanate 3 as a colorless oil 27 28 4 1 3 Synthesis of substituted piperidine derived carbamates 4a 4c and 4e General procedure A solution of substituted or unsubstituted 1 Boc 4 piperidinol 1c 1e or 1a 20 mmol in dry acetonitrile 15 mL was treated with triethylamine 4 05 g 40 mmol followed by the amino acid methyl ester isocyanate 3 20 mmol The resulting solution was refluxed for 2 h and then allowed to cool to room temperature The solution was concentrated a