key: cord-0851765-42vqmndl authors: Stanley, Mathew; Mayr, Juliane; Huber, Wolfgang; Vlasak, Reinhard; Streicher, Hansjörg title: Synthesis and inhibitory activity of sialic acid derivatives targeted at viral sialate-O-acetylesterases date: 2011-04-08 journal: Eur J Med Chem DOI: 10.1016/j.ejmech.2011.04.008 sha: db10d33c71cc8ecb074914269e4575b5b53daf77 doc_id: 851765 cord_uid: 42vqmndl A series of sialosides modified at the 4- and 9-hydroxy group were synthesised and tested for inhibition of the viral haemagglutinin-esterase activity from various Orthomyxoviruses and Coronaviruses. While no inhibition of the sialate-4-O-acetylesterases from mouse hepatitis virus strain S or sialodacryoadenitis virus was found, a 9-O-methyl derivative displayed inhibitory activity against recombinant sialate-9-O-acetylesterase from influenza C virus. Sialic acids are a group of biologically important 9-carbon sugars which decorate, ketosidically linked to cell surface glycoconjugates, the glycocalix and thus the cells of higher organisms. At this exposed position, they serve as receptors for sialic acid-recognising proteins and are consequently involved in wide variety of biological events, both pathogenic and non-pathogenic [1e3]. One of the most common modifications of sialic acids in mammals is O-acetylation. It occurs either at C-4 or at any position within the glycerol side chain of sialic acid and multiple acetylations are possible ( Fig. 1) [4, 5] . Sialate-O-acetylation has attracted increased interest in recent years due its abundance and involvement in many, including pathological, biological processes. For instance, acetylation may promote or hinder recognition of sialic acid by proteins, cells or pathogens. O-acetylation may also slow down the activity of degradative enzymes such as sialate lyases or sialidases [3] . Related biological events include cell differentiation, tumor growth, immunity, apoptosis, microbial infections and in particular cancer where they are considered markers for certain skin tumors and a form of leukaemia [2e7] . Another important function of O-acetylation is masking of siglec binding sites. CD22, a siglec regulating the activity of the B-cell receptor, binds to a2-6 linked sialic acids, and binding can be masked by 9-O-acetylation [8] . This masking is regulated by the cellular sialate-O-acetylesterase, which thereby also regulates B-cell receptor signal strength [9] . Loss of function of the esterase activity results in autoimmune disease [10, 11] . Besides these functions, sialic acids are also used as docking platforms for viral pathogens. Several RNA viruses which infect the respiratory and gastrointestinal tract utilize sialic acids as a receptor determinant. To facilitate release of progeny virus from infected cells, a number of viruses express "receptor-destroying enzymes" (RDE), which are targets for antiviral drugs. The best known are the sialidase-inhibitors Zanamivir (Relenza) and Oseltamivir (Tamiflu). Besides sialidases, the haemagglutinin-esterases (HE) of influenza C virus, isavirus, betacoronaviruses and toroviruses represent another class of RDEs. They are sialate-O-acetylesterases (SOAE) hydrolysing O-acetyl esters of O-acetylated sialic acid derivatives (Fig. 1) . Two main subtypes of HEs are known: sialate-4-O-acetylesterases (4-SOAE) and sialate-9-O-acetylesterases (9-SOAE). HE expressing viruses include human pathogens like influenza C viruses, the respiratory human coronavirus OC43 (HCoV OC43) and HKU1 (HCoV HKU1), several important animal betacorona-and toroviruses and infectious salmon anaemia virus (ISAV), a piscine orthomyxovirus. HCoVs account for 10e30% of respiratory infections, in particular, the common cold, but they can also cause gastroenteritis and neurological disorders [12, 13] . The infections are usually mild and subclinical, but strains related to OC43 and HKU1 were associated with severe human disease [14, 15] . Betacoronaviruses have attracted attention after the outbreak of SARS CoV in 2002/2003. This genus consists of human and animal viruses. Coronaviruses are able to cross the animal-to-human species barrier: bat-to-human in case of SARS CoV [16, 17] and bovine-to-human in case of BCoV leading to HCoV OC43 [18] . Toroviruses (ToV) are evolutionary related to coronaviruses [19] . ToV are associated with asymptomatic enteric infections in pigs [20] . Studies suggest that they are highly prevalent in swine populations [21, 22] . The closely related bovine toroviruses (BToV) are implicated with serious or even fatal infections [23e25]; they are found worldwide [26e30] . ToV are also associated with human gastroenteritis. In fecal samples from children and adults with diarrhoea, torovirus antigens were detected by ELISA [31, 32] , immunoelectron microscopy [33e35] and by reverse transcription polymerase chain reaction with primers covering a highly conserved region of the ToV genomes [35] . Regarding the potential for transmission of betacoronaviruses from animal to human and the danger of the emergence of further epidemics, efficient treatment(s) would be of great interest (Table 1) . Interestingly, the comparison of the crystal structures of influenza C virus HEF [36] , BCoV HE [37] and two ToV HEs [38] shows highly conserved sites of the SOAE domains. Therefore, the SOAE active site is probably an excellent target for broad-spectrum antivirals against sialate-O-acetylesterases of both orthomyxoand coronaviruses. The successful development and introduction of the antiinfluenza drugs Tamiflu and Relenza which are inhibitors of the 'receptor-destroying' sialidase from influenza virus has demonstrated that such an approach is promising. Earlier we postulated two essential pharmacophoric groups of Neu5,9Ac 2 in correct spatial arrangement required for strong substrateeenzyme interaction with sialate-9-O-acetylesterases: the 9-O-acetyl and the alpha-C2 carboxylate group [39] . Although no investigations about the substrateeenzyme interactions of sialate-4-O-acetylesterases are available, a similar mechanism as for 9-Oacetylesterases is suggested. In light of these, we embarked on a study which aims to find competitive inhibitors of 9-and 4-SOAE, which are serine esterases. Enzymes from four different viruses, influenza C virus (INF-C) [36,40e42] , bovine coronavirus (BCoV) [43, 44] , mouse hepatitis virus strain S (MHV-S) [45, 46] and sialodacryoadenitis virus (SDAV) [47] were investigated. The allyl group was chosen as an aglycon mimetic in target allyl sialosides 1e4 and control sialoside 5 because it offers a range of selective chemical methods, such as e. g. olefin metathesis, for further functionalisation or immobilisation of the inhibitors. To probe the active sites of 4-SOAE and 9-SOAE, two types of modifications of the positions 4 and 9 of the sialosides were introduced (Fig. 2) . Firstly, methylation resulting in target structures 1 and 3 should yield information whether additional hydrophobic interactions could contribute to more efficient binding and about the role of the respective hydroxyl group as hydrogen bond donor. Secondly, methylphosphonate groups were introduced as mimetics of the suspected tetrahedral intermediate of acetate hydrolysis. In theory, compounds 2 and 4 could interact with active site amino acids stabilising this polar transition state, including the 'oxyanion hole' common in serine esterases [48] . a-Allyl sialoside 6, which serves as intermediate for all syntheses, was conveniently synthesised in high yield in 4 steps from commercially available N-acetylneuraminic acid using a wellestablished Koenigs-Knorr methodology (Scheme 1) [49, 50] . Control compound 5 was obtained from 6 through saponification. Selective methylation of the 4-OH was then made possible by blocking positions 8 and 9 as the isopropylidene ketal through acid catalysed reaction of 6 with 2,2-dimethoxypropane to give 7 in 85% yield. Alkylation of 7 under Williamson-conditions followed by acid-mediated ketal hydrolysis and basic saponification of the methyl ester gave crude inhibitor 1 which was purified by gel permeation chromatography. For the introduction of a phosphonate group at position 4, hydroxyl groups 8 and 9 were protected as the cyclic carbonate using diphosgene and dimethylaminopyridine to give 8. Introduction of the phosphonate was achieved with methyl methylphosphonyl chloride and Hünig's base to give phosphonate diester 9. The position of the phosphonylation was confirmed by acetylation of the remaining hydroxyl group at position 7 and analysis of the product (not shown). Selective cleavage of the phosphonic acid methyl ester with thiophenol and triethylamine followed by basic hydrolysis of all other ester groups and purification furnished compound 2 in good yield. (Scheme 2). For methylation at position 9, compound 6 was converted into the 8,9-epoxide by subsequent treatment with toluenesulfonyl chloride in pyridine and sodium methoxide in methanol to give 10. Acid-mediated opening of the epoxide in methanol gave the 9methyl ether and, after saponification of the methyl ester and purification, target inhibitor 3. For the 9-phosphonate, direct reaction of 5 with methyl methylphosphonylchloride and Hünig's base followed by per-O-acetylation to yield 11 proved to be the best route. Deprotection of 10 was carried out as described for 9 and thus inhibitor 4 was obtained in 20% overall yield (4 steps) (Scheme 3). Inhibition of the SOAE activity of three viruses, influenza C virus (INF-C), bovine coronavirus (BCoV) and mouse hepatitis virus strain S (MHV-S) and of two chimeric recombinant viral haemagglutinin esterases, from influenza C/Cal/78 virus (HE12-GFP) and sialodacryoadenitis virus (SDAV-HE) was investigated. The inhibitory effect of compounds 3 and 4 toward the 9-SOAE activities of HE12-GFP, INF-C virus and BCoV and of compounds 1 and 2 toward the 4-SOAE activity of SDAV-HE and MHV-S were determined by pNPA assay and fluorimetric HPLC. Compound 5 was used as a negative control. No sialidase activity against any of the inhibitors was detected in the esterase preparations. The production, isolation and purification of the viruses and enzymes as well as the assays are described in the experimental section. Sialate-O-acetylesterases were incubated in the presence of different sialoside concentrations. Table 2 summarises the inhibitory effect of 9-modified sialosides 3 and 4 toward the 9-SOAE enzymes determined by pNPA assay. We identified the 9-O-methyl sialoside 3 as a potential inhibitor of HE12-GFP. At a concentration of 5 mM compound 3 the inhibition is approximately 86%. Analysis by fluorimetric HPLC revealed a 10e15% inhibition by 3 of Neu5,9Ac 2 -hydrolysis by HE12-GFP at a concentration of 1 mM, thus confirming the results of the pNPA assay (data not shown). Less inhibitory effect of 3 was detected with the whole influenza virus particles. We suggest that the different conformation of the haemagglutinin protein (recombinant HE12-GFP ¼ monomeric protein; whole virus ¼ trimeric protein) may contribute to the differences in the inhibitory effect of sialoside 3. The 9-SOAE of BCoV was also less effected by 3. According to [39] , it seems that the BCoV is more dependent on the aglycon moiety than the influenza C virus esterase. It may be that the allyl group of the sialosides has an effect on the enzymatic reaction and consequently less inhibition was detected. Comparison of the inhibitory activities of 9-modified sialosides 3 and 4 suggests that the negatively charged methylphosphonate does not induce detectable inhibition when compared to the unmodified sialoside 5. There is however, a significant effect of the small, hydrophobic, methyl group at the same position indicating that inhibition can be improved by further modifications at this position. This effect has so far been seen only with the recombinant esterase from the influenza C virus, not with whole virus. It can be speculated that this is a result of it being a monomer rather than the native trimer, but that will require further studies with optimised inhibitors of higher affinity. For influenza C virus esterase, these results are in line with those from an earlier study where a K i of 4.2 mM was determined for a 9-acetamido-9-deoxy-sialic acid derivative [51] . Table 3 summarises the inhibitory behaviour of compounds 1 and 2 toward MHV-S and SDAV-HE determined by the pNPA assay. No significant inhibition was observed even at concentrations of 5 mM, indicating that there is less scope for modification of the 4- hydroxy group. It should be kept in mind as well that the mode of action of 4-SOAE is may be different to that of 9-SOAE. Unmodified control sialoside 5 (negative control) did not show detectable inhibition of any of the enzymes. 3,4-Dichloroisocoumarin (positive control) was highly reactive towards all esterase activities at 0.1 mM concentration. We have synthesised a set of modified sialosides useful for probing the active sites of 4-and 9-sialate-O-acetylesterase enzymes. We have screened the compounds for inhibition of a set of viral SOAE's and while no inhibition of 4-SOAE could be detected, a 9-O-methyl derivative showed inhibition of the recombinant SOAE from influenza C virus. Further studies on how his can be exploited to develop high-affinity inhibitors of the enzyme as potential lead compounds for drug development are under way. Where anhydrous solvents were required for reactions, these were purchased (anhydrous) and used as received. DCM was doubly distilled (over CaH 2 ) before use. Fine chemicals were purchased from Aldrich-, Sigma-or Acros-Chemicals and were of the highest purity available. Reactions were monitored via thin layer chromatography (TLC) using pre-coated silica sheets with fluorescent indicator UV 254 . Compound detection was achieved by UV absorption and by developing plates by staining with a molybdenum phosphate reagent (20 g ammonium molybdate and 0.4 g cerium (IV) sulphate in 400 mL of 10% aqueous sulphuric acid) with subsequent heating. Chromatographic purification was performed using silica gel 60A Compound 7 (55.6 mg, 0.138 mmol) was dissolved in dry CH 3 CN (0.7 mL), under N 2 (g) and was cooled to 0 C (ice bath). DMSO 4 (24.5 mL, 0.259 mmol) was added to the solution, followed by NaH (s) (6.6 mg, 0.276 mmol). The resulting suspension was allowed to stir for 4 h at 0 C, more DMSO 4 (24.5 mL, 0.259 mmol) was added and the suspension was left to stir overnight. When TLC indicated completion of the reaction, the reaction was quenched with excess NH 4 Cl (s) and the solution was diluted with DCM (3 mL), filtered over Celite and evaporated to dryness. After flash chromatography (Tol:Acetone 5 mL) , the mixture was stirred for 2 h, the solvent was evaporated and the oily product was then co-evaporated with toluene and dried in vacuo. Aqueous NaOH (1.5 mL, 0.1 M) was added to the oil and the resulting solution was left to stir for 2 h. After completion, the solution was neutralised using Amberlite IR-120 (H þ ) ion-exchange resin, the resin was removed and the solvent removed in vacuo yielding 1 (17.9 mg, wqu). A sample of 1 (9.9 mg) was purified by gel permeation chromatography which was then lyophilised to give a white powder. A stirred solution of epoxide 10 (50 mg, 0.145 mmol) in methanol (1.2 mL) was acidified (to pH 2) with Amberlite IR-120 (H þ ) ion-exchange resin, the resin was filtered off and the solvent evaporated from the solution to leave an oily residue. The crude product was purified by flash chromatography (EA / EA:MeOH; 5:1) to yield the 9-O-methyl ether ( The ether (5 mg, 0.0132 mmol) was dissolved in dioxane (0.5 mL) and NaOH (0.05M, 0.5 mL) was added with stirring. The mixture was stirred for 2 h at room temperature, neutralised with Amberlite IR-120 (H þ ) ion-exchange resin, filtered and the solvent was evaporated in vacuo. The residue was purified by gel permeation chromatography to afford compound 3 (5 mg, qu.) as a powder. HR-ESI-MS (m/z) [M þ Na] þ calculated for: C 15 Compound 6 (10 mg, 0.0275 mmol) was dissolved in dioxane (0.5 mL) with stirring. Aqueous NaOH (0.1 M, 0.5 mL) was added and the solution was stirred for 2 h. The solution was then neutralised with Amberlite IR-120 (H þ ) ion-exchange resin, filtered and the solvent was removed in vacuo. The crude residue was then purified by gel permeation chromatography and lyophilised to give a white powder ( 6.2.6. Methyl (allyl 5-acetamido-3,5-dideoxy-d-glycero-a-dgalacto-2-nonulopyranosid)onate (6) Compound 6 was synthesised as described in the literature [20] . Compound 6 (56 mg, 0.154 mmol) was dissolved in dry acetone (1.85 mL) under a atmosphere of N 2 (g). 2,2-Dimethoxypropane (200 mL) was added to the solution, followed by p-TsOH$H 2 O (7 mg). The solution was left to stir at room temperature, under N 2 , for 2 h. Once complete, NEt 3 (few drops) was added to the solution to neutralise the reaction and the solvent was evaporated to dryness. After flash chromatography (Tol:Acetone 1:1), a clear oil was obtained, which after further drying in vacuo yielded 7 as a white foam (53 mg, 85%). the solution via microsyringe. The reaction was allowed to warm to room temperature and was stirred overnight. The reaction was again then cooled to À15 C, where a further portion of N,N-diisopropylethylamine (67 mL, 0.386 mmol) and Methyl methylphosphonyl chloride (27 mL, 0.270 mmol) was added. Once TLC indicated completion, the reaction was quenched by the addition of MeOH (1 mL) and a small spatula of NaHCO 3 (s). After stirring for 10 min, the solvent was removed in vacuo and the product was purified by flash chromatography (EA:MeOH; 7:1 / 5:1) to give 9 (57 mg, 60% 6.2.10. Methyl (allyl 5-acetamido-8,9-anhydro-3,5-dideoxy-dglycero-a-d-galacto-2-nonulopyranosid)onate (10) Compound 6 (560 mg, 1.54 mmol) was co-evaporated with dry pyridine and was then suspended in dry pyridine (12 mL) under N 2 (g). The resulting suspension was cooled to to 0 C (ice bath) and toluenesulfonyl chloride (293 mg, 1.54 mmol) was quickly added to the stirred solution. After 20 min, the solution was allowed to warm up to room temperature and was subsequently stirred for a further 5 h after which another portion of toluenesulfonyl chloride (293 mg, 1.54 mmol) was added. The solution was left to stir overnight. Once completed, solvent was removed in vacuo and the crude product was purified by flash chromatography (EA / EA:MeOH; 5:1) to yield the 9-O-tosylate as a white foam (470 mg, 59% Further 0.1 mL portions of NaOMe (0.5 M) were added as required (a total of 0.7 mL NaOMe was used). The reaction was monitored by TLC. On completion, the solution was neutralised with Amberlite IR-120 (H þ ) ion-exchange resin. The resin was removed by filtration and the solvent was removed in vacuo. Flash chromatography of the crude material (EA:MeOH; 15:1 þ 0.5% NEt 3 ) gave epoxide 10 as a white foam (87 mg, 68%). R f : 0.34 (EA:methanol; 10:1). HR-ESI-MS (m/z) [M þ Na] þ calculated for C 15 (11) Under an N 2 (g) atmosphere, compound 6 (77 mg, 0.212 mmol) was suspended in dry DCM (2 mL) and the mixture was ultrasonicated. Diisopropylethylamine (81 mL, 0.466 mmol) was added to the suspension followed by further ultrasonication. With continuous stirring, the suspension was cooled to approximately À15 C. Once acclimatised to temperature, methyl methylphosphonyl chloride (29 mL, 0.297 mmol) was added dropwise to the solution via microsyringe. After 5 h the reaction was again cooled to À15 C, where a further portion of N,Ndiisopropylethylamine (81 mL, 0.466 mmol) and methyl methylphosphonyl chloride (29 mL, 0.297 mmol) was added. The reaction was left to stir overnight. Once TLC indicated completion, the reaction was quenched by the addition of MeOH (1 mL) and a small spatula of NaHCO 3 (s). After stirring for 10 min, the solvent was removed in vacuo and the product was purified by flash chromatography (EA:MeOH; 10:1 / 5:1) to give the 9-O-phosphonate ( eCH 2 eCH ¼ CH 2 ) CDCl 3 ) d P : 34.24 (s), 34.34 (s) HR-ESI-MS (m/z) [M þ Na] þ calculated for : C 23 H 36 NO 14 PNa : 604.1771. Found: 604.1766. 1 H NMR (500 MHz, CDCl 3 ) d H : 1.45 (dd, 1H, J ¼ 3.4, 17.6 Hz, P-CH 3 ), 1.85, 2.12e2.14 (s,m, 9H, 3 Â eOCOCH 3 ), 2.00 (s, 3H, eNHCOCH 3 ), 1.95 (dd, 1H, J ¼ 12.4, 12.6 Hz, H 3ax 17 (C3), 49.69, 49.73 (C5), w52.31 (m, PeOCH 3 ), 52.83 (eCO 2 CH 3 ), 63.88 (m, C9), 66.02 (eCH 2 eCH ¼ CH 2 ) 73 (s). . Abbreviations INF-C: influenza C; HEF: haemagglutinin esterase fusion; BCoV: bovine coronavirus; SDAV-HE: sialodacryoadenitis virus haemagglutinin esterase Bovine coronavirus (BCoV) and mouse hepatitis virus strain S (MHV-S) were grown in MadineDarby bovine kidney (MDBK) cells and mouse L cells, respectively. The INF-C virus HEF and SDAV-HE were expressed as chimeric recombinant influenza C/Cal/78 virus and sialodacryoadenitis virus haemagglutinin esterase, respectively, fused in frame to enhanced green fluorescent protein (HE12-GFP and SDAV-HE). Enzymes were expressed in insect Sf9 cells in serum-free media by recombinant baculovirus Enzymes and inhibitors The three different viruses (INF-C virus, BCoV and MHV-S) were The five sialosides were dissolved in ddH 2 O to a final concentration of 10 mM. Stock solutions of p-nitrophenyl acetate (100 mM) were prepared in acetonitrile, and 3,4-dichlor-HPLC analysis Reverse-phase high-pressure liquid chromatography (HPLC) analysis of sialic acids was performed as described previously Chemistry, biochemistry and biology of sialic acids Identification and characterization of a porcine torovirus The receptor-destroying enzyme of influenza C virus is neuraminate-O-acetylesterase Supplementary data related to this article can be found online at doi:10.1016/j.ejmech.2011.04.008.