Synthesis of acylguanidine zanamivir derivatives as neuraminidase inhibitors and the evaluation of their bio-activities

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Biomolecular Chemistry

COMMUNICATION

Cite this: Org. Biomol. Chem., 2013, 11, 3943

Received 28th March 2013, Accepted 9th May 2013 DOI: 10.1039/c3ob40624e www.rsc.org/obc

Synthesis of acylguanidine zanamivir derivatives as

neuraminidase inhibitors and the evaluation of their

bio-activities

†

Chien-Hung Lin,

a

Tsung-Che Chang,

a

Anindya Das,

a

Ming-Yu Fang,

b

Hui-Chen Hung,

b

Kai-Cheng Hsu,

c

Jinn-Moon Yang,

c

Mark von Itzstein,

d

Kwok Kong T. Mong,

c

Tsu-An Hsu*

b

and Chun-Cheng Lin*

a

A series of acylguanidine-modiﬁed zanamivir analogs were syn-thesized and their inhibitory activities against the NAs of avian inﬂuenza viruses (H1N1 and H3N2) were evaluated. In particular, zanamivir derivative 3j, with a hydrophobic naphthalene substitu-ent, exhibits the best inhibitory activity against group-1 NA with an IC50of 20 nM.

Due to its rapid global spread, influenza is one of the most widespread pandemic human diseases. Despite advances in the understanding of the molecular and cellular aspects of influenza, this disease aﬀects millions of people, causing serious public health and economic problems. The influenza virus is an RNA virus of the Orthomyxoviridae family, and the subtypes of influenza virus are classified based on the distinct antigenic properties of two viral surface membrane glyco-proteins, hemagglutinin (HA) and neuraminidase (NA). Both glycoproteins are essential for virus infection, and there are currently 17 HA (H1–H17) and 9 NA (N1–N9) serotypes of influ-enza A virus circulating in the avian population.1a,bThe NAs are phylogenetically categorized into two groups: group 1 tains the N1, N4, N5, and N8 subtypes, whereas group 2 con-tains N2, N3, N6, N7, and N9.2 Furthermore, NA has been targeted in the development of structure-based drug design programs. As a result, two drugs, oseltamivir (1, Tamiflu),3an orally active NA inhibitor, and zanamivir (2, Relenza),4

administered via nasal inhalation, have been developed and are commercially available. However, the serious threat of Tamiflu-resistant viruses5increases the urgency of developing a new generation of anti-influenza NA inhibitors.6a–c

The analyses of the X-ray crystal structures of NAs have revealed conformational diﬀerences among the subgroups with respect to the 150-loop adjacent to the active site of NA:7 the group-1 NAs adopt an open 150-loop conformation with a 150-cavity near the active site, whereas the group-2 NAs show a closed 150-loop. Moreover, molecular dynamics simulations have suggested that the 150-loop and adjacent binding site loops may be more flexible than observed in the crystal struc-tures.8In addition, recent evidence suggests that the 150-cavity exists not only in group-1 NAs but also in group-2 NAs.9These findings clearly provide insights that may aid in the design of new NA inhibitors that target the 150-cavity.

Previously, NA inhibitors were designed based on the tran-sition state mimic of a hydrolyzed-sialic acid residue and the structural information of the group-2 NAs.3,4Since the discov-ery of the 150-cavity near the active site of group-1 NAs,7many eﬀorts have been made to target the newly found cavity by extending the structures of the existing inhibitors by attaching additional groups with a suitable shape, size, and hydrophobi-city.10a–iIn general, the amine or guanidine functional groups of oseltamivir10a–d and zanamivir10e provide the chemical foundation for the generation of compounds either by click reaction or amide bond formation. Although several derivatives have been proposed to access the 150-cavity based on compu-tational methods,10a,c,d,i the synthetic derivatives seemed not to maintain the same level of aﬃnity for group-1 NAs as the parent compounds did. For example, Mohan et al.10bemployed a click reaction to construct triazole derivatives at the C-3 posi-tion of the similar structure of oseltamivir. However, the modi-fication resulted in a decrease in the inhibition potency. In addition, Wen et al.10e synthesized C-4 modified zanamivir derivatives by employing N-alkylation, and the resulting deriva-tives only showed inhibitory activities at the micromolar level. Recently, von Itzstein et al.10f modified the well-known †Electronic supplementary information (ESI) available: Experimental details,

characterization data and NMR spectra for new compounds. See DOI: 10.1039/c3ob40624e

a_{Department of Chemistry, National Tsing Hua University, 101, Section 2,} Kuang-Fu Road, Hsinchu 30013, Taiwan

b_{Division of Biotechnology and Pharmaceutical Research, National Health Research} Institutes, 35, Keyan Road, Zhunan 35053, Taiwan

c

Department of Biological Science and Technology and Applied Chemistry Department, National Chiao Tung University, University Road, Hsinchu 30010, Taiwan

d_{Institute for Glycomics, Griﬃth University (Gold Coast Campus), Southport,} Queensland 4222, Australia. E-mail: cclin66@mx.nthu.edu.tw;

Fax: +886 3-571-1082

Published on 10 May 2013. Downloaded by National Chiao Tung University on 28/04/2014 02:00:34.

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neuraminidase inhibitor

5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enonic acid (Neu5Ac2en) at its C-3

position with a ( p-tolyl)allyl group and demonstrated by X-ray crystallography that the inhibitor locks the flexible 150-loop of group-1 NAs. Furthermore, these two synthesized derivatives exhibited the selective inhibition of group-1 NAs, including the NAs of A/Hong Kong/156/97 (H5N1), A/California/07/2009 (H1N1), and A/Paris/0497/2007 (H1N1), over the group-2 NA A/Paris/908/97 (H3N2). However, due to the lack of a guanidine group at the C-4 position of Neu5Ac2en, the activities of the synthesized compounds are far lower (Ki= 15.3 × 10−5M) than that of zanamivir (Ki = 0.13 × 10−9 M) against the pandemic 2009 A/H1N1 influenza virus.

However, a close examination of the crystal structure of the zanamivir-bound group-1 NA revealed that the C-4 guanidino group of zanamivir is also located near the 150-cavity and can serve as a potential modification site in the design of group-1 NA-specific inhibitors. Inspired by these findings and the identification of viruses less resistant to zanamivir, we modi-fied the C-4 guanidino group of zanamivir and evaluated the activities of the derivatives against NAs. In contrast to the pre-vious findings for zanamivir with an N-alkylated guanidino group, the N-acylated zanamivir analogs (3 in Fig. 1) main-tained potent inhibitory eﬀects.

To synthesize the C-4 guanidino-modified zanamivir deriva-tives 3, zanamivir 2 (Scheme 1) was prepared from sialic acid by following the reported procedures.11The initial attempt to synthesize acylguanidines by the direct coupling of the guani-dino group in zanamivir with acids using coupling reagents,

such as EDC with HOBt or HBTU, HBTU with HOBt, and ByBop, resulted in low yields. To circumvent this problem, as illustrated in Scheme 1, the synthetic route was modified by first forming the acylguanidine derivative 5, followed by react-ing this derivative with the correspondreact-ing amine 612 to give the N-acylated guanidine 7. Initially, the N-terminal region of 1H-pyrazole-1-carboximidamide (4),13 a guanidinylation reagent, was protected with Boc2O,14followed by deprotona-tion using NaH and then acyladeprotona-tion with N-hydroxysuccinimide-activated acid to give 5 as a mixture of two stereoisomers. Notably, the yields of the amide bond formation reaction were moderate (40–60%) due to the decomposition of the activated acid by NaH. In addition, the direct coupling of 4 with acid failed. The guanidinylation of 6 with 5 under slightly basic conditions in THF gave acylguanidine 7 (75–87%), which was deprotected under acidic conditions (TFA in DCM) to aﬀord compound 8 as a single isomer with an excellent yield. Finally, the hydrolysis of 8 with K2CO3gave the zanamivir derivatives 3a–3ac (Table 1). However, when the acid was protected as the methyl ester, in some cases (such as 3r), the hydrolysis of the methyl ester resulted in a low yield due to the instability of the acylguanidine group under strong basic conditions, and further attempts to improve the yield were unsuccessful (see Table S1†). Considering the potential instability of the acyl-guanidine group,15the methyl ester-protecting group in 6 was replaced with an ethyl ester, which has been demonstrated to be much more labile than the former under basic deprotection conditions.16In addition, the release of ethanol is less toxic in vivo, benefiting future prodrug development.17 Thus, full deprotection was achieved by using molar equivalents of K2CO3in H2O–methanol (v/v = 1/4) at 0 °C (Table S1, entry 7†) to aﬀord the final products in 70–90% yields.

However, the amide bond formed at the guanidino group may aﬀect the ability of the guanidine to form hydrogen bonds with the interacting amino acid residues of NA due to the elec-tron-withdrawing property of the amide group. Thus, the N-alkylated guanidino groups of zanamivir derivatives 14ad–14af were synthesized to investigate the electronic eﬀect of the gua-nidine on H-bonding interactions. As shown in Scheme 2, the synthesis of 14 was similar to that described in Scheme 1 except that Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-protected thiourea (11) was used as a guanidine

precursor. Compound 11 was obtained by reacting

tetrabutylammonium thiocyanate [(n-Bu)4NSCN] with PbfCl, followed by the addition of an amine.18Finally, the guanidine was formed by coupling thiourea 11 with 6 in the presence of EDC and DIEPA19to aﬀord 12. The removal of the Pbf group under acidic conditions (TFA–DCM) was followed by the hydro-lysis of the ethyl ester under basic conditions (K2CO3–EtOH) to yield the N-alkylated guanidino group on zanamivir, derivative 14, in 73–88% yield.

The inhibitory activities of synthetic compounds 3a–3ac and 14ad–14af against the H1N1 and H3N2 NAs were evalu-ated, and the results are shown in Table 1. In our assay, zana-mivir, with an IC50of 0.74 ± 0.03 nM, was used as a positive control. The results indicate that the amide derivatives Fig. 1 NA inhibitors of transition state and zanamivir derivatives.

Scheme 1 Synthesis of acylated zanamivir derivatives by modifying the guani-dino group of zanamivir. Reagents and conditions: (a) Boc2O, DIPEA, DCM, DMF, rt, 95%; (b) NaH, RCOOSuc, DCM, rt, 40–60%; (c) 6, Et3N, THF, rt, 75%–87%; (d) TFA, DCM, rt, 95%; (e) K2CO3, EtOH, H2O, rt, 70–90%.

Communication Organic & Biomolecular Chemistry

3944 | Org. Biomol. Chem., 2013, 11, 3943–3948 This journal is © The Royal Society of Chemistry 2013

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Table 1 Inhibitory activities of novel zanamivir derivatives against group-1 (H1N1) and group-2 (H3N2) neuraminidases Compd R H1N1 H3N2 Compd R H1N1 H3N2 Compd R H1N1 H3N2 IC50(nM) IC50(nM) IC50(nM) IC50(nM) IC50(nM) IC50(nM) Relenza 0.74 ± 0.03 3k 275 ± 1 334 ± 63 3v 421 ± 9 399 ± 1 3a >1000 >1000 3l 203 ± 10 234 ± 7 3w 180 ± 9 173 ± 2 3b >1000 >1000 3m 189 ± 5 211 ± 6 3x 397 ± 1 472 ± 4 3c 152 ± 3 147 ± 9 3n 462 ± 60 606 ± 17 3y 260 ± 21 421 ± 72 3d >4000 >4000 3o 196 ± 2 205 ± 27 3z 293 ± 23 360 ± 3 3e 46.1 ± 0.4 44.9 ± 2.7 3p 134 ± 17 134 ± 16 3aa 775 ± 16 868 ± 38 3f 54.9 ± 0.5 58.3 ± 0.6 3q 332 ± 22 472 ± 31 3ab 541 ± 24 615 ± 20 3g 98.3 ± 2.9 84.4 ± 8.9 3r 176 ± 10 209 ± 8 3ac 203 ± 1 196 ± 1 3h 209 ± 18 243 ± 13 3s 1200 ± 10 1400 ± 14 14ad 39 400 ± 1400 29 600 ± 1100 3i 74.9 ± 0.8 66.9 ± 0.5 3t 160 ± 13 182 ± 1 14ae 34 644 ± 2786 38 303 ± 859 3j 20.1 ± 0.7 25.5 ± 1.0 3u 239 ± 16 337 ± 20 14af >40 000 (43%) >40 000 (35%) Organic & Biomolecular Chemis try C ommunica This journal is © The R o ya l Society of Chemis try 2013 Org. Biomol .Chem ., 2013, 11 , 3943 – 3948

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(3a–3ac) are, in general, more potent inhibitors than the N-alkylated derivatives (14ad–14af) but are slightly less active than the parent compound, zanamivir. Although the inhibitory activity gradually decreased with increasing alkyl chain length in the arylguanidine groups (3e, 3l, 3y, and 3z), compound 3ac, with the longest alkyl chain tested, still maintained inhibi-tory activity, with an IC50of 203 nM. This result may be due to the increased flexibility of the benzene ring, allowing it to fit into the 150-cavity, when the linker is longer. Notably, the activity seems to be reduced in the absence of a linker between the aromatic ring and the acylguanidine (3a, 3b, and 3d). However, the presence of a strong electron-withdrawing group (NO2) on the aromatic ring of 3c shows an intricate eﬀect on the enhancement of the inhibitory activity (3c vs. 3a and 3d). Moreover, the inhibitory activity is significantly enhanced to 46.1 nM by introducing a methylene unit between the acyl-guanidine and aromatic ring (3e vs. 3a). Although the potency of inhibition decreases slightly to 54.9 nM with the incorporation of a more hydrophobic naphthalene group (3f ), compound 3j, with an internal S atom, is the most potent NA inhibitor (IC50 = 20.1 nM) among those tested in this study. The results show that the heteroatom, S, in the internal chain and the hydro-phobic naphthalene group are important factors to increase the inhibitory activity (3i vs. 3j, 3l vs. 3m, and 3i vs. 3l). Although the heteroatom located at the ortho position of an aromatic ring can increase the inhibitory activity (3p), the steric hindrance imposed would likely decrease the inhibitory activity, as for 3e vs. 3h and 3l vs. 3n–3q. Substituents at the other positions (3t and 3u) of the benzene ring or the replace-ment of benzene with acyclic substituents (3aa–3ab) or other carbocyclic analogs (3s and 3x) only produce moderate inhibi-tory activities. In addition, the replacement of the benzene ring (3y) with cyclohexane (3x) results in a decrease in the inhi-bition potency.

To further investigate whether the synthesized inhibitors exhibit selectivity among the diﬀerent NA subgroups, the activities of the inhibitors against the H3N2 NA were evaluated. The results indicate that the zanamivir analogs show inhibi-tory activities against N2 that are similar to those against N1 (Table 1), although the activity against N2 is lower. Therefore, the acylguanidium moiety of the zanamivir analogs may also

participate in an interaction that contributes to the observed inhibitory activities against the neuraminidase of group-2 influenza viruses. Although the 150-loop of the N2 neuramini-dase has the closed form, the inhibitor may induce a confor-mational change in the 150-loop, yielding the open form, when it interacts with NA.7

In addition, the N-alkylated zanamivir analogs, such as 14ad, 14ae, and 14af, show very low inhibitory activities for N1 (IC50> 30μM), similar to a previous observation.10e Interest-ingly, the N-alkylated zanamivir analog 14ad exhibits more than 200-fold lower potency relative to acyl zanamivir analogs, such as 3m (Fig. 2). These results clearly indicate that the change in the electronic properties of the guanidium moiety aﬀects the binding aﬃnity and that the carbonyl function of the acylguanidino group has a prominent role in the NA inhibitory activity.

To better understand the eﬀect of a carbonyl group at the acylguanidino group on activity, computer docking studies of the inhibitors with NA were performed (Fig. 3). GEMDOCK20 was employed to predict the docked conformations of the inhibitors in the binding cavity of H1N1 neuraminidase based on calculated binding energies. The 3D structures of the inhibitors were generated using ACD/ChemSketch, and the open-form crystal structure of H1N1 neuraminidase (PDB code 3BEQ) was selected for the docking procedure. The binding site of NA was defined to include the residues within a 10 Å radius sphere centered around the 150-loop (residues 147–152) of the H1N1 neuraminidase. The simulation for compound 3j clearly shows that the hydrophobic substituent of the guani-dine interacts with the 150-cavity, as shown in Fig. 3A. In addition, to avoid the steric hindrance involving the Asp151 residue of NA generated by the modification of the guanidine, the docking conformation of compound 3j is slightly diﬀerent from the docking mode of zanamivir, as shown in Fig. 3B and 3D. The carboxylic acid of zanamivir forms an H-bond with residue Arg118 of NA, but in 3j, Arg118 interacts with the car-bonyl group of the amide of the guanidine moiety. In addition, the guanidine of zanamivir forms six hydrogen bonds with the residues Glu119 and Asp151, and the carboxylic acid of zana-mivir forms five hydrogen bonds with the residues Arg118, Arg292, and Arg371. However, the total number of hydrogen bonds generated by the same groups as above is reduced to seven in the interaction between 3j with NA. The acylguanidine of 3j forms three hydrogen bonds with the Arg118 and Asp151 Fig. 2 Structures and inhibition activities of zanamivir and its derivatives. Scheme 2 Synthesis of N-alkylated zanamivir derivatives by modifying the

guanidino group of zanamivir. Reagents and conditions: (a) (n-Bu)4NSCN, DCM, rt, 60%; (b) R-NH2, DCM, rt 78%; (c) 6, EDC, DIPEA, DCM, rt, 65%–83%; (d) TFA, DCM, rt, 70%–83%; (e) K2CO3, EtOH, H2O, rt, 73–88%.

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residues, whereas the carboxylic acid forms four hydrogen bonds with Arg292 and Arg371 (Fig. 3B and 3D). In contrast, the N-alkylated guanidino group of compound 14ad has only one hydrogen bond with NA (Fig. 3C). In addition, the aro-matic or heterocyclic group of compound 3 fit into the 150-cavity and interacted with residues Q136, G147, V149, and T439, which form a hydrophobic pocket. Overall, although the modification of the guanidino group of zanamivir results in the loss of four H-bonds, the hydrophobic interaction between the 150-cavity and the hydrophobic substituent compensates for this loss in binding energy, resulting in only a minimal reduction in the binding energy. These results suggest that the addition of a hydrophobic group to zanamivir that fits into the 150-cavity could be a good strategy to enhance the binding aﬃnity. In addition, the modification at the C-3 position of zanamivir seems to be another approach to obtain a more potent inhibitor because the hydrogen bonding between the guanidine group and NA may be retained, and thus the extended group can target the 150-cavity.

Conclusions

In summary, the results show that acylguanidine derivatives of zanamivir are more potent inhibitors than the corresponding N-alkylated derivatives and that the carbonyl group plays a dominant role in binding by generating three additional H-bonds with R118 and D151 of NA. Although the modifications at the guanidino group of zanamivir interrupt the H-bonding interactions with NA, the addition of a carbonyl group and a hydrophobic moiety can compensate for the binding energy loss. Among the compounds tested, 3j displayed the highest potency against both N1 and N2, with IC50values of 20 nM and 25 nM, respectively. Although there is no apparent selecti-vity among the NAs when targeting the 150-loop, our results

may provide an overall advantage of a better oral bioavailability due to the improved hydrophobicity of zanamivir resulting from the addition of hydrophobic groups at its guanidine site. Finally, we anticipate that these findings may provide new insight that will aid in the development of a new generation of anti-influenza drugs.

The research was supported by the National Tsing Hua Uni-versity, the National Health Research Institutes, the National Chiao Tung University and the National Science Council in Taiwan.

Notes and references

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H1N1 neuraminidase. Molecular surface of N1 neuraminidase with bound compound 3j (A) and the docking results of compounds 3j (B), 14ad (C), and zanamivir (D) with neuraminidase in the open form with carbon atoms of inhibitor in cyancolor and protein carbons in light blue color. Oxygen and nitrogen atoms are red and blue, respectively. Hydrogen bonds between the compounds and the protein are represented by green dotted lines.

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