Oseltamivir Analogues Bearing N-Substituted Guanidines as Potent

The Gilead team involved in the original development of oseltamivir found that analogue 6 was a more potent neuraminidase inhibitor with an IC50 of 0...
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Oseltamivir Analogues Bearing N‑Substituted Guanidines as Potent Neuraminidase Inhibitors Caitlin A. Mooney,†,§ Stuart A. Johnson,†,§ Peter ’t Hart,† Linda Quarles van Ufford,† Cornelis A. M. de Haan,‡ Ed E. Moret,† and Nathaniel I. Martin*,† †

Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands ‡ Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands S Supporting Information *

ABSTRACT: A series of oseltamivir analogues bearing an Nsubstituted guanidine unit were prepared and evaluated as inhibitors of neuraminidases from four strains of influenza. The two most potent analogues identified contain relatively small N-guanidine substituents (N-methyl and N-hydroxyl) and display enhanced inhibition with IC50 values in the low nanomolar range against neuraminidases from wild-type and oseltamivir-resistant strains. Potential advantages of including the N-hydroxyguanidine moiety in neuraminidase inhibitors are also discussed.

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as effective treatment of influenza A and B infections.2b,c The most successful NAIs are illustrated in Figure 1 and include oseltamivir (1),8 a prodrug of the corresponding oseltamivircarboxylate, zanamivir (2),9 laninamivir (3),10 and peramivir (4)11 which is currently under development. Among the clinically used NAIs, oseltamivir has risen to prominence largely because of its convenient oral administration compared with zanamivir and laninamivir, both of which are delivered via inhalation, and peramivir which is administered by injection.2a Despite its success in recent years, resistance to oseltamivir is a growing concern.12 Oseltamivir resistance is generally conferred via a single amino acid mutation (H274Y) in strains possessing the N1 subtype of the neuraminidase enzyme.13 By comparison, this mutation has little effect on the antiviral activity of the other NAIs used to treat influenza infection.14 As shown in Figure 1, oseltamivir (1) differs from the other NAIs in that it lacks an exocyclic guanidine moiety. Upon the basis of available cocrystal structures, the guanidine unit present in zanamivir (2), laninamivir (3), and peramivir (4) provides active site hydrogen-bonding interactions that are not present in the oseltamivir−neuraminidase complex.14,15 These additional interactions may offer a partial explanation for the observation that oseltamivir resistant strains of influenza are generally sensitive to NAIs that contain an exocyclic guanidine group.16

INTRODUCTION Influenza viruses infect millions of people annually and can lead to life threatening respiratory illness. The pandemic nature of the virus and its ability to rapidly mutate present a serious public health concern and underscore the importance of developing effective anti-influenza agents.1 Strategies for developing new compounds to combat influenza have generally targeted mechanisms essential for viral replication.2 The earliest such agents to be used clinically were the adamantanes including amantadine3 and rimantadine,4 which operate by blocking the M2 ion channel required for disassembly of the viral particle after entry into the host cell. Adamantanes were found to be effective against influenza A virus infection but not against influenza viruses of other genera. Despite early success, the vast majority of influenza A virus strains now exhibit resistance to these drugs and their use has largely been discontinued.5 In response, the search for alternative antiinfluenza strategies led to the identification of the viral neuraminidase enzyme as a promising alternative target.2 The neuraminidase enzyme (NA) is essential to the influenza virion and functions by cleaving terminal sialic acid residues from glycoconjugates that are otherwise bound by the hemagglutinin receptor-binding protein. In doing so, neuraminidases play an essential role in the release of newly formed virions from infected cells and facilitate propagation of the virus.6 As early as the 1970s,7 neuraminidase inhibitors were proposed as a means of combating influenza, and to date, a number of clinically relevant neuraminidase inhibitors (NAIs) have been developed © 2014 American Chemical Society

Received: January 7, 2014 Published: March 20, 2014 3154

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fold lower than the value measured for 5 in the same inhibition assay.17 In the present study we describe a series of new oseltamivir analogues that incorporate various NG-substituted guanidines in place of the amino group at the C-5 position and examine the effect that such modifications have on neuraminidase inhibition. In recent years a number of groups have reported various approaches to modifying the structures of oseltamivir and zanamivir at the C-5 and C-6 positions (oseltamivir numbering; see Figure 1). In most cases the rationale for doing so was based on an attempt to identify analogues bearing exocyclic substituents capable of occupying the so-called “150-cavity” adjacent to the active site of certain neuraminidase subtypes (N1, N4, and N8).18 Recently, the groups of Pinto and Cairo employed click chemistry based strategies to generate oseltamivir analogues bearing substituted triazoles at the C-5 and C-6 positions.19 While some of the analogues prepared in this manner did show neuraminidase inhibition, they were generally less potent than oseltamivir and zanamivir.19,20 In another approach, von Itzstein and co-workers developed novel sialic acid based compounds also bearing extended exocylic substituents that were shown by crystallography to occupy the 150-cavity.21 These compound were found to effectively inhibit the influenza virus including drug-resistant strains.21 In an alternative approach, the groups of Lin and Fang independently described the incorporation of various substituents at the guanidine moiety of zanamivir (2) to produce analogues with a range of activities.22,23 Most notable among the compounds described by Lin and co-workers are a subset of Nacylguanidine analogues that exhibited IC50 as low as 20 nM against the H1N1 neuraminidase.22

Figure 1. Oseltamivir (1) with atom numbering indicated and structures of the other known NAIs used clinically or under development.

Further support for the inhibition-enhancing effect of the guanidine moiety is provided by the known oseltamivir analogue 6 wherein the C-5 amine is transformed into a guanidine group (Figure 2). The Gilead team involved in the

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RESULTS AND DISCUSSION Our rationale for pursuing oseltamivir analogues with NGsubstituted guanidines at the C-5 position was twofold: first, introduction of the guanidine moiety at the C-5 position of oseltamivir (6) is known to enhance the neuraminidase inhibitory activity17 and, second, the NG-substituents introduced might lead to beneficial binding interactions should they be able to access the 150-cavity adjacent to the neuraminidase site. In designing the series of oseltamivir analogues to be prepared, we chose to incorporate a range of small NG-

Figure 2. Structures of oseltamivir carboxylate (5) and the guanidine analogue of oseltamivir carboxylate (6).

original development of oseltamivir found that analogue 6 was a more potent neuraminidase inhibitor with an IC50 of 0.2 nM, 5-

Scheme 1. Synthetic Route Employed in Preparing Oseltamivir Analogues with NG-Substituted Guanidine Moiety at C-5 Position

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substituents containing three carbon atoms or less. The synthetic approach employed in preparing these analogues is outlined in Scheme 1. Briefly, starting from shikimic acid, oseltamivir (1) was prepared on multigram scale according to the routes independently described by Karpf and Trussardi at Roche24 and the Shi group.25 Oseltamivir was then treated with CbzNCS26 to yield thiourea 7 which served as a common intermediate in the preparation of the various NG-modified oseltamivir analogues prepared. Treatment of thiourea 7 with EDCI, NEt3, and the amine of choice led to clean formation of the protected guanidine species 8−15 as previously described for the synthesis of other NG-substituted guanidines.26−28 A two-step deprotection strategy29 was then utilized to yield 16− 23 which were subsequently purified by RP-HPLC. Oseltamivir carboxylate (5) and the unsubstituted guanidine analogue (6) were also prepared as shown in Scheme 2.

Table 1. Inhibitory Activities against Wild-Type and Mutant Influenza Neuraminidases neuraminidase inhibition, IC50 (nM)a compd

H1N1 wild-typeb

H1N1 mutantc

H5N1 wild-typed

H5N1 mutante

5 6 16 23

2.1 ± 0.1 0.87 ± 0.04 1.73 ± 0.09 3.7 ± 0.5

250 ± 20 1.7 ± 0.2 4.2 ± 0.5 43 ± 4

250 ± 30 1.32 ± 0.09 3.6 ± 0.3 33 ± 2

330 ± 20 1.9 ± 0.4 4±1 38 ± 3

a

IC50 values reported based on triplicate measurement. bNeuraminidase from influenza virus A/California/04/2009 (H1N1). cNeuraminidase (H274Y) from influenza virus A/California/04/2009 (H1N1). dNeuraminidase from influenza virus A/Anhui/1/2005 (H5N1). eNeuraminidase (H274Y) from influenza virus A/Anhui/1/ 2005 (H5N1).

The results obtained with NG-substituted analogues 16−23 indicate that the size of the substituent that can be appended to the guanidine moiety while maintaining activity is rather limited. Incorporation of NG-substituents larger than N-methyl or N-hydroxyl resulted in dramatically reduced neuraminidase inhibition, suggesting that the larger substituents here evaluated are not able to enhance binding by accessing the 150-cavity. By comparison, 16 and 23 showed strong inhibition of all neuraminidases tested, including those from oseltamivirresistant strains. The activity of N-methyl analogue 16 was found to be very similar to that of known unsubstituted analogue 6 with IC50 values in the low nanomolar range against all four neuraminidases tested. The N-hydroxy analogue (23) was slightly less potent with IC50 values approximately 10-fold higher than those measured for 16 (Table 1). Docking studies were next performed to compare the binding modes of 16 and 23 with that of oseltamivir in the neuraminidase active site (Figure 3). Figure 3A shows oseltamivir docked into the H274Y mutant H1N1 neuraminidase, according to the crystal structure published by Collins and co-workers14 (PDB code 3CL0 with oseltamivir and PDB code 3CKZ with zanamivir, the sequence of this neuraminidase is 100% identical to the sequence of the H1N1 mutant enzyme used in our inhibition assays). Figure 3B illustrates the hydrogen-bonding interactions found between oseltamivir’s exocyclic amine and the carboxylate side chains of Glu119 and Asp 151 of the neuraminidase active site. Shown in Figure 3C and Figure 3D, are the possible new hydrogen bonding interactions resulting from introduction of the guanidine moiety in analogues 16 and 23. In addition, the N-methyl substituent of 16 appears to introduce beneficial nonpolar interactions, while the oxygen of the N-hydroxy moiety in 23 functions as a hydrogen bond acceptor. Taken together, the results of these docking experiments indicate that addition of an exocyclic guanidine moiety to the oseltamivir core can lead to beneficial hydrogen bonding interactions and restore affinity for the H274Y mutant. The enhanced binding suggested by the docking analysis and the potent inhibitory activity measured for NG-hydroxy analogue 23 with oseltamivir-sensitive and -resistant neuraminidases is of particular note. The pKa for a typical NGhydroxyguanidine moiety is generally between 8.0 and 8.5, significantly lower than that of the corresponding guanidine (pKa ≳ 12.5).31 Guanidines are very strong bases and are completely ionized in serum, limiting their bioavailability and restricting their modes of action to peripheral tissues.31a One

Scheme 2. Preparation of Oseltamivir Carboxylate (5) and Unsubstituted Guanidine Analogue (6)

Compounds 16−23 were first evaluated as inhibitors of the neuraminidase from the H1N1 wild-type influenza virus using an established activity assay employing the fluorescent substrate 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA). In addition, oseltamivir carboxylate (5) and the unsubstituted guanidine analogue (6)17,30 were included as positive controls. As expected, oseltamivir carboxylate (5) effectively inhibited the wild-type H1N1 neuraminidase with an IC50 of 2.06 ± 0.14 nM while against the H5N1 neuraminidase and the H274Y mutants of the H1N1 and H5N1 enzymes, the IC50 values measured were more than 100-fold higher. By comparison, oseltamivir analogue 6, containing the unsubstituted guanidine group, was found to be a potent inhibitor of all four neuraminidases tested with IC50 values in the low nanomolar range. An initial evaluation of analogues 16−23 was performed by measuring each compound’s ability to inhibit the wild-type H1N1 neuraminidase at 10, 100, and 1000 nM. This revealed 16 and 23 to be potent inhibitors while 17−22 were found to display little or no inhibition at the highest concentration tested (1000 nM). Compounds 16 and 23 where therefore carried forward and more extensively evaluated as inhibitors of the neuraminidases from wild-type and oseltamivir-resistant H1N1 and H5N1 influenza strains (Table 1). 3156

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Figure 3. Oseltamivir carboxylate (1) and guanidine-modified analogues 16 and 23 docked into the active site of H274Y mutant H1N1 neuraminidase. (A) MSMS molecular surface representation32 of the neuraminidase active site with oseltamivir carboxylate (1) bound and the 150cavity indicated. Entry to the cavity appears to be via a relatively small channel, and the cavity itself is quite polar. (B) Hydrogen-bonding interactions between oseltamivir’s exocyclic amine and the carboxylate side chains of Glu119 and Asp 151. (C) Binding of NG-methylguanidine containing analogue 16 is predicted to be facilitated by hydrogen-bonding with Trp178, Glu227, and Glu 277. (D) NG-Hydroxyguanidine moiety in analogue 23 is predicted to function as a hydrogen-bond donor with Glu119, Asp151, and Glu227 and as a hydrogen-bond acceptor with Arg156.

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approach to overcoming these limitations is to “tailor the basicity” of guanidine compounds by instead using the corresponding NG-hydroxy analogue. The reduced basicity of the NG-hydroxyguanidine moiety has been exploited to generate analogues that are less ionized at physiological pH and show altered physiological distributions relative to the corresponding guanidine.31a As described above, oseltamivir’s oral bioavailability is largely responsible for its popularity compared with the other NAIs. A significant contribution to oseltamivir’s bioavailability as a prodrug is attributable to the ethyl ester that is hydrolyzed in vivo. In addition, the fact that oseltamivir contains an exocyclic primary amine rather than a guanidine moiety may further enhance its oral bioavailability. We here show that introduction of a guanidine moiety into the oseltamivir carboxylate structure as in 6, 16, and 23 leads to an enhanced inhibitory activity against oseltamivir-resistant neuraminidases in vitro. In a recent report, Schade and Clement described the in vivo activity of the ethyl ester prodrugs of structurally similar compounds.33 Most notable is their finding that an oseltamivir analogue bearing an exocyclic amidine moiety at C-5 maintains the bioavailability of the parent compound and displays enhanced antiviral activity against an oseltamivir resistant H1N1 influenza strain. Taken together with our own results, these findings indicate that the inclusion of modified amidine and guanidine groups at the C-5 position provides a means by which oseltamivir resistance can be overcome.

CONCLUSION The oseltamivir analogues described in this study provide new insights into the opportunities and limitations for enhancing neuraminidase inhibition via introduction of N-substituted guanidine groups into the parent compound. Among the analogues here prepared, those bearing NG-substituents larger than methyl and hydroxyl groups displayed dramatically reduced activity. These findings suggest that access to the 150-cavity is not possible for substituents appended to the terminal nitrogen atoms of the exocyclic guanidine moiety. By comparison, N-methyl and N-hydroxy analogues 16 and 23 exhibit potent inhibition of influenza neuraminidases, including those from oseltamivir-resistant strains. The activity of the NGhydroxy analogue 23 is particularly noteworthy given that NGhydroxy guanidines can show improved bioavailability relative to the corresponding unsubstituted guanidine species. These findings also point toward a possible approach for altering the bioavailability of the other major NAIs (Figure 1). In contrast to oseltamivir, the poor oral bioavailability observed with zanamivir (2) and the other major NAIs is largely attributable to the presence of a free carboxylate and an exocyclic guanidine. While the carboxylate moiety in these compounds can be masked as an ester prodrug (as in oseltamivir), attenuating the highly basic guanidine group provides an additional challenge. On the basis of the findings here presented, it may be of interest to prepare the corresponding NG-hydroxyguanidine analogues of zanamivir (2), laninamivir (3), and peramivir (4) and evaluate their inhibitory activity and (oral) bioavailability. Such investigations are ongoing in our laboratories, the results of which will be presented in due course. 3157

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to 2:1). Yields of 8−15 ranged from 70% to quantitative. As an example, analytical data for 8 are given below; data for all compounds are provided in the Supporting Information. (3R,4R,5S)-Ethyl 4-Acetamido-5-(2-(benzyloxycarbonyl)-3methylguanidino)-3-(pentan-3-yloxy)cyclohex-1-enecarboxylate (8). Yield: 227 mg, 96%. 1H NMR (300 MHz, CDCl3) δ 8.97 (br s, 1H), 7.41−7.23 (m, 5H), 6.79 (s, 1H), 5.85−5.64 (m, 2H), 5.10 (s, 2H), 4.38−4.25 (m, 1H), 4.20 (q, 2H, J = 7.2 Hz), 4.14−3.94 (m, 2H), 3.36 (app quin, 1H, J = 5.6 Hz), 2.86 (dd, 1H, J = 23.2, 5.3 Hz), 2.76 (d, 3H, J = 5.1 Hz), 2.34 (ddt, 1H, J = 18.0, 9.0, 2.2 Hz), 1.92 (s, 3H), 1.58−1.42 (m, 4H), 1.29 (t, 3H, J = 7.3 Hz), 0.95−0.83 (m, 6H); 13 C NMR (75 MHz, CDCl3) δ 171.8, 166.0, 164.0, 160.8, 137.8, 136.5, 129.7, 128.3, 127.8, 127.6, 82.1, 75.2, 66.5, 61.0, 54.0, 49.7, 31.0, 27.5, 26.3, 25.8, 23.2, 14.2, 9.5, 9.3. HRMS (ESI, M + H) calcd for C26H39N4O6, 503.2864; found, 503.2875. Synthesis of N-Substituted Guanidine Compounds 16−23. General Procedure. The Cbz-protected ethyl ester intermediate (0.4 mmol) was dissolved in 1.0 mL of MeOH (or dioxane if methanol does not dissolve). Once fully dissolved, an aqueous solution of 2 M KOH (0.5 mL, 1.0 mmol, 2.5 equiv of KOH) was added and the reaction mixture stirred for 3−4 h. Once TLC confirmed complete hydrolysis, Amberlite IR-120 resin (1 g) was added directly to acidify the mixture. After the mixture was stirred slowly for 5 min the resin was filtered off and rinsed with an additional 25 mL of MeOH. The MeOH and H2O were then removed under vacuum, and the resulting oil was used directly in the next step. The crude carboxylic acid species from the previous step (0.4 mmol) was treated with trifluoroacetic acid (15.0 mL, 202 mmol) and thioanisole (2.4 mL, 20 mmol) at room temperature and the reaction mixture stirred overnight. The next morning the TFA was removed under vacuum to yield the crude product as an oil (also contains residual thioanisole). The product was purified using RP-HPLC employing a preparative C18 column and an H2O/MeCN gradient moving from 28.5% to 50% MeCN (0.1% TFA) over 60 min (flow rate, 18.0 mL/min). Fractions containing the desired product were combined and lyophilized to yield the pure compounds as amorphous white powders. As an example, analytical data for compound 16 are given below; data for all compounds are provided in the Supporting Information. (3R,4R,5S)-4-Acetamido-5-((E)-2-methylguanidino)-3-(pentan-3-yloxy)cyclohex-1-enecarboxylic Acid (16). Yield: 49 mg, 36%. 1H NMR (300 MHz, D2O) δ 6.72 (s, 1H), 4.21 (d, 1H, J = 8.4 Hz,), 3.80 (q, 1H, J = 9.0 Hz), 3.72−3.64 (m, 1H), 3.43 (app quin, 1H, J = 5.1 Hz), 2.73 (d, 1H, J = 5.1 Hz), 2.70 (s, 3H), 2.29 (dd, 1H, J = 17.1, 9.9 Hz), 1.89 (s, 3H), 1.46−1.24 (m, 4H), 0.78−0.68 (m, 6H); 13 C NMR (75 MHz, D2O) δ 177.2, 171.9, 159.1, 140.8, 130.3, 86.9, 78.0, 57.4, 53.1, 32.4, 30.0, 28.1, 27.7, 24.4, 11.0; HRMS (ESI, M + H) calcd for C16H29N4O4, 341.2183; found, 341.2202. Neuraminidase Inhibition Assay. Compounds 2, 6, and 16−23 were evaluated as neuraminidase inhibitors by following an established protocol provided in a commercially available assay kit (NA-Fluor influezna neuraminidase assay kit from Life Technologies). For 5, 6, 16, and 23, IC50 values were determined from the dose−response curves by plotting percent inhibition of neuraminidase activity versus inhibitor concentration using GraphPad Prism 4. Docking Studies. The docking studies performed made use of the crystal structure of the H274Y mutant H1N1 neuraminidase, previously published by Collins and co-workers14 (PDB codes 3CL0 with oseltamivir and 3CKZ with zanamivir). Oseltamivir carboxylate (5) and 16 and 23 were docked into the neuraminidase active site using Autodock 4.235 as part of YASARA 12.4.1.36 For the docking experiments the original oseltamivir carboxylate structure was converted to those of analogues 16 and 23 in YASARA, which also allowed for cleaning of the molecules with regard to atom types and bond types. Initial minimization and simulated annealing of the ligand and the side chains of the neuraminidase residues within 7 Å were performed using the AMBER03 force field37 followed by 250 rigid docking runs with ga_pop_size set at 15 000 (the only change to the default YASARA macro36). The local search algorithm used was based on the Solis−Wets method,38 and the free energy was estimated

EXPERIMENTAL SECTION

General Procedures. Reagents, Solvents, and Solutions. Oseltamivir was prepared from shikimic acid based upon a combination of recently published synthetic routes.24,25,34 Oseltamivir carboxylate (5) and the corresponding unsubstituted guanidine analogue (6) were prepared as previously described.30 CbzNCS was prepared following literature protocols.26 All other reagents employed were of American Chemical Society (ACS) grade or finer and were used without further purification unless otherwise stated. Recombinant influenza A neuraminidase enzymes H1N1 wild-type, H1N1(H274Y), H5N1 wild-type, and H5N1(H274Y) were obtained from Sino Biological Inc. Purification Techniques. All reactions and fractions from column chromatography were monitored by thin layer chromatography (TLC) using plates with a UV fluorescent indicator (normal SiO2, Merck 60 F254). One or more of the following methods were used for visualization: UV absorption by fluorescence quenching; iodine staining; phosphomolybdic acid/ceric sulfate/sulfuric acid/H2O (10 g/1.25 g/12 mL/238 mL) spray; 50% sulfuric acid spray. Flash chromatography was performed using Merck type 60, 230−400 mesh silica gel. Product purities were confirmed to be ≥95% by RP-HPLC employing an analytical C18 column and a water/acetonitrile gradient moving from 5% to 95% MeCN (0.1% TFA) over 30 min (flow rate 1.0 mL/min). Instrumentation for Compound Characterization. 1H NMR spectra were recorded at 300.1 MHz with chemical shifts reported in parts per million (ppm) downfield relative to tetramethylsilane (TMS). 1H NMR data are reported in the following order: multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; sex, sextet; sept, septet; m, multiplet), number of protons, and coupling constant (J) in hertz (Hz). When appropriate, the multiplicity is preceded by br, indicating that the signal was broad. 13C NMR spectra were recorded at 75.5 MHz with chemical shifts reported relative to CDCl3 δ 77.0. 13 C NMR spectra were recorded using the attached proton test (APT) sequence. High resolution mass spectrometry (HRMS) analyses were performed using a TOF LC/MS instrument. All literature compounds had NMR and mass spectra consistent with the assigned structures. Synthetic Procedures and Compound Characterization. (3R,4R,5S)-Ethyl 4-Acetamido-5-(3-((benzyloxy)carbonyl)thioureido)-3-(pentan-3-yloxy)cyclohex-1-enecarboxylate (Thiourea Intermediate 7). Oseltamivir freebase (2.31 g, 7.41 mmol) was dissolved in dichloromethane (100 mL) and treated with a 0.5 M solution of CbzNCS in CH2Cl2 (15 mL, 7.5 mmol) and triethylamine (1.1 mL, 7.8 mmol). After the mixture was stirred for 1 h at room temperature, TLC analysis indicated partial conversion to the thiourea. An additional 3.0 mL of the 0.5 M CbzNCS solution (1.5 mmol) was added to the mixture. After the mixture was stirred for another 0.5 h, TLC verified that the reaction had reached completion. The CH2Cl2 was then removed under reduced pressure, and the residue applied directly to a silica column, eluting with EtOAc/hexane (1:2). The desired thiourea was obtained as a white solid (2.63g, 5.20 mmol). Yield: 70.0%. 1H NMR (300 MHz, CDCl3) δ 9.94 (d, 1H, J = 8.4 Hz), 8.02 (s, 1H), 7.46−7.31 (m, 5H), 6.86 (s, 1H), 5.82 (d, 1H, J = 9.3 Hz), 5.17 (q, 2H, J = 5.1 Hz), 4.84−4.70 (m, 1H), 4.36−4.26 (m, 1H), 4.21 (q, 2H, J = 6.9 Hz), 4.07−4.00 (m, 1H), 3.37 (app quin, 1H, J = 6.0 Hz), 2.88 (dd, 1H, J = 17.7, 5.4 Hz), 2.55 (ddt, 1H, J = 18.0, 9.3, 2.7 Hz), 1.97 (s, 3H), 1.55−1.42 (m, 4H), 1.29 (t, 3H, J = 7.2 Hz), 0.93−0.80 (m, 6H); 13C NMR (75 MHz, CDCl3) δ 175.0, 165.7, 161.0, 147.0, 132.8, 129.6, 124.2, 124.1, 124.0, 123.8, 78.0, 71.0, 63.7, 56.3, 49.1, 48.7, 24.9, 21.4, 21.0, 18.7, 18.6, 10.1, 5.4. HRMS (ESI, M + H) calcd for C25H36N3O6S, 506.2319; found, 506.2331. Synthesis of Cbz-Protected N-Substituted Guanidine Compounds 8−15. General Procedure. Thiourea precursor 7 (253 mg, 0.50 mmol) was dissolved in CH2Cl2 (8 mL) and treated with the amine of choice (2 equiv, 1.0 mmol), triethylamine (2 equiv, 0.14 mL, 1.0 mmol), and EDCI−HCl (2 equiv, 191 mg, 1.0 mmol) after which the mixture was stirred at room temperature. After TLC confirmed complete conversion to the substituted guanidine (typically 2−5 h) CH2Cl2 was removed under reduced pressure and the residue applied directly to a silica column (typical solvent system EtOAc/hexane 1:2 3158

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according to existing methods.39 The best docking result was minimized with the YASARA minimization experiment (steepest decent followed by simulated annealing with the AMBER03 force field). Explicit solvent was not included in the docking simulation or in the free energy estimate, where a charge-based desolvation term was computed.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental details and protocols for the synthesis of oseltamivir and all new compounds; 1H and 13C NMR spectra for 2, 6−24; analytical RP-HPLC traces for 2, 6, 16−23; neuraminidase inhibition assay details and IC50 curves for 2, 6, 16, and 23. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: +31-618785274. Author Contributions §

C.A.M. and S.A.J. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Javier Sastre Toraño is kindly acknowledged for providing HRMS analysis results. Financial support was provided by The Netherlands Organization for Scientific Research (VIDI grant to N.I.M.). The Utrecht Institute for Pharmaceutical Sciences (UIPS) and Utrecht University are also gratefully acknowledged for their support.

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ABBREVIATIONS USED NA, neuraminidase; NAI, neuraminidase inhibitor; RP-HPLC, reverse phase high performance liquid chromatography REFERENCES

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