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subsite of the influenza neuraminidase (NA) active site was characterized and optimized for a series of trisubstituted pyrrolidine inhibitors modified...
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J. Med. Chem. 2005, 48, 3980-3990

Structure-Based Characterization and Optimization of Novel Hydrophobic Binding Interactions in a Series of Pyrrolidine Influenza Neuraminidase Inhibitors Clarence J. Maring,* Vincent S. Stoll, Chen Zhao, Minghua Sun, Allan C. Krueger, Kent D. Stewart, Darold L. Madigan, Warren M. Kati, Yibo Xu, Robert J. Carrick, Debra A. Montgomery, Anita Kempf-Grote, Kennan C. Marsh, Akhteruzzaman Molla, Kevin R. Steffy,† Hing L. Sham, W. Graeme Laver,‡ Yu-gui Gu, Dale J. Kempf, and William E. Kohlbrenner Departments of Infectious Disease Research and Advanced Technology, Global Pharmaceutical R & D, Abbott Laboratories, 200 Abbott Park Road, Abbott Park, Illinois 60064, and John Curtin School of Medical Research, the Australian National University, Canberra 260, Australia Received September 2, 2004

The structure-activity relationship (SAR) of a novel hydrophobic binding interaction within a subsite of the influenza neuraminidase (NA) active site was characterized and optimized for a series of trisubstituted pyrrolidine inhibitors modified at the 4-position. Previously, potent inhibitors have targeted this subsite with hydrophilic substituents such as amines and guanidines. Inhibitor-bound crystal structures revealed that hydrophobic substituents with sp2 hybridization could achieve optimal interactions by virtue of a low-energy binding conformation and favorable π-stacking interactions with the residue Glu119. From a lead methyl ester, investigation of five-membered heteroaromatic substituents at C-4 produced a 3-pyrazolyl analogue that improved activity by making a targeted hydrogen bond with Trp178. The SAR of substituted vinyl substituents at C-4 produced a Z-propenyl analogue with improved activity over the lead methyl ester. The C-1 ethyl ester prodrugs of the substituted C-4 vinyl analogues gave compounds with excellent oral bioavailability (F > 60%) when dosed in rat. Introduction The control of annual epidemic outbreaks of respiratory influenza virus continues to be an important problem in public health.1,2 The effects of influenza infection on the mortality and morbidity of the most vulnerable populations, the elderly and very young, are especially serious.3-5 The threat of a worldwide epidemic, or pandemic, has warranted continuous monitoring to identify and isolate outbreaks of hypervirulent strains that could result in the catastrophic rates of mortality experienced in the pandemic of 1918.6 Because of numerous challenges to effecting protection of the general population by vaccination, there will be a continuing need for therapeutic intervention in order to control newly emerging strains of influenza virus. The discovery of inhibitors of the viral enzyme neuraminidase (NA) for the treatment of influenza infection has been an active area of research.7,8 The ready availability of crystal structures of inhibitor/ protein complexes has enabled a detailed analysis of the structural basis for potent inhibition. Several potent inhibitors (2, 3, and 5; Figure 1) have been studied extensively, with 2 and 4, the ethyl ester prodrug of 3, demonstrating clinical efficacy.9-14 To describe the key interactions of NA inhibitors, we have previously created an operative division of the active site of NA into five subsites21 (S1-S5, Figure 2). The NA active site has been characterized as highly polar by virtue of the 10 * Corresponding author. Address: Department R47D, AP52N, 200 Abbott Park Road, Abbott Park, IL 60064-6217. Phone: (847) 9375870. Fax: (847) 938-2756. E-mail: [email protected]. ‡ The Australian National University, Canberra 260, Australia. † Present address: Anadys Pharmaceuticals, San Diego, CA 92121.

Figure 1. Structures of influenza neuraminidase inhibitors.

Figure 2. NA active site designations and important interactions of oseltamivir carboxylate.

charged and only four hydrophobic amino acids that bind and cleave conjugated sialic acid substrates. Initial approaches to the design of NA inhibitors targeted substrate transition state mimics such as 4-amino-

10.1021/jm049276y CCC: $30.25 © 2005 American Chemical Society Published on Web 05/19/2005

Pyrrolidine Influenza Neuraminidase Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 3981

Scheme 1a

a Reagents and conditions: (a) NaBH , MeOH; (b) TBDMSCl, Et N, DMAP; (c) OsO , NMO; (d) ammonium formate, Pd/C, EtOH, 4 3 4 reflux; (e) Boc2O, MeOH/H2O; (f) NaIO4, THF, H2O; (g) isobutylMgBr, THF; (h) Dess-Martin or Swern oxidation; (i) NH4+OAc-, NaCNBH3, MeOH, reflux; (j) (i) Ac2O, Et3N,CH2Cl2, (ii) chromatography of diastereomers; (k) Bu4N+F-, THF.

DANA 1, an inhibitor of good potency that interacts directly with four subsites (S1, S2, S3, and S5) of the enzyme active site. Extending the primary amine of 1 as a guanidine into the S2 subsite resulted in the identification of zanamivir 2. Zanamivir is highly potent and inhibits NA primarily through electrostatic and hydrogen-bonding interactions. Because of its low lipophilicity and low oral bioavailability, it must be administered by inhalation to achieve efficacy. Two inhibitors, oseltamivir carboxylate (3)15 and peramivir (BCX-1812, 5),16 have subsequently been discovered that interact with subsites S4 and S5 of the active site with hydrophobic interactions. The increased lipophilicity of oseltamivir (4), the ethyl ester prodrug of 3, has enabled it to demonstrate good oral bioavailability. Strong interactions within S2 are important components of the potency of the inhibitors 2, 3, and 5. However, the substrates for NA do not interact substantially with this subsite.17 Therefore, S2 is a subsite wherein resistance-conferring mutations might occur without compromising viral infectivity. In vitro selection studies with both 2 and 3 have identified resistant variants with mutations at residue Glu119.18-20 This pattern has not been demonstrated consistently in vivo, and the overall clinical incidence of resistance to NA inhibitors appears to be low. The resistance profiles of S2 mutants selected by 2, 3, and 5 are substantially different, presumably due to the differences in substituents and trajectories16 directed toward S2. Previously, we have described the use of combinatorial and structure-based methods to increase the inhibitory potency of a series of pyrrolidine and cyclopentanebased NA inhibitors.21 The salient conclusion of that process was the unprecedented observation of a hydrophobic substituent (methyl ester group) projected from both core ring structures into S2. The hydrophobic interactions of the methyl ester substituents within the S2 subsite were sufficiently strong to produce a common stable binding conformation. The pyrrolidine inhibitor 6 presented itself as a novel inhibitor core amenable to further optimization. Here we report the systematic characterization of the hydrophobic character of S2 and the structure-based optimization of hydrophobic substitutents projected into S2 from the pyrrolidine core of

6. By virtue of increased hydrophobicity, these novel neuraminidase inhibitors display good oral bioavailability and the potential for a novel resistance profile. Chemistry Scheme 1 summarizes the synthetic route to two versatile C-4 primary alcohol intermediates, 14a and 14b, in differentially protected pyrrolidine cores. From these cores, essentially all of the S2 structure-activity relationship (SAR) was developed. The 2,4,5-trisubstituted pyrrolidine precursor 7 was assembled via an acid catalyzed [3 + 2]-dipolar cycloaddition22 of an azomethine ylide derived from the reaction of acrolein and tert-butyl N-benzylglycinate and a second molecule of acrolein acting as the dipolarophile. Equilibration of the crude reaction mixture with triethylamine produced 7 as an 8:1 (2,4-cis:trans) mixture favoring the depicted relative stereochemistry. Sodium borohydride reduction of 7 gave the corresponding mixture of diastereomeric alcohols 8 that were separated after protection as their tert-butyldimethylsilyl ethers (TBS) to give 9. Dihydroxylation of 9 with catalytic osmium tetroxide and N-methylmorpholine N-oxide produced a mixture of diols 10a that was carried forward without purification to the Boc-protected pyrrolidine 10c via a high-yield deprotection/reprotection sequence. The hindered nature of the pyrrolidine nitrogen of 10a necessitated the relatively vigorous transfer hydrogenolysis conditions. Subsequent reactions with benzyl- or Boc-protected pyrrolidine intermediates, 10a or 10c, proceeded under essentially the same reaction conditions with similar diastereoselectivity. Higher yields were usually observed for the Boc-protected pyrrolidine intermediates. Oxidative cleavage of the diols 10a,c with periodate produced the aldehyde 11. Addition of isobutylmagnesium bromide to the aldehydes 11 followed by Dess-Martin or Swern oxidation of the resulting alcohols gave the ketones 12. Reductive amination of 12 produced essentially a 1:1 mixture of amines that upon acylation with acetic anhydride and separation by chromatography gave the desired acetamide 13. Fluoride deprotection of the silyl ethers 13 produced 14. Scheme 2 outlines the syntheses of additional key intermediates, the aldehydes 15 and carboxylic acids 16,

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Scheme 2a

a Reagents and Conditions: (a) Swern Oxidation; (b) NaClO , NaH PO , 2-methyl-2-butene, CH CN, t-BuOH, H O; (c) CH I, AgO; (d) 2 2 4 3 2 3 EtMgBr, THF; (e) CH2N2; (f) EDCI, HOBT, EtOH; (g) (i) isobutyl chloroformate, Et3N, (ii) CH3NH2; (h) DPPA, BnOH; (i) NH4+HCO2-, Pd/C, EtOH, reflux; (j) 6N HCl; (k) TFA,CH2Cl2.

Table 1. Inhibition of B/Memphis Neuraminidase: SAR of Methyl Ester Derivatives and Primary Amine 21

a In a previous report,21 6 was described as an inhibitor with a Ki ) 0.037 µM (A/N2/Tokyo) using enzyme assay conditions previously described.24 However, these conditions appeared to result in an overestimate of potency for compounds in this series relative to that observed in cell culture.25 The results reported above use a modified protocol for both strains A and B (Supporting Information) that result in a Ki ) 0.15 µM (A/N2/Tokyo) for 6. The Ki and EC50 values for all compounds refer to their activities as racemates.

and analogues of Table 1. The aldehydes 15 were derived from a Swern oxidation of the alcohols 14 that upon subsequent oxidation with sodium chlorite afforded carboxylic acids 16. The esters 6 and 17 and the methylamide 20 proceeded from standard transformations of the carboxylic acid of 16a followed by sequential deprotection of the pyrrolidine nitrogen by transfer hydrogenolysis and acidic deprotection of C-1 tert-butyl ester. The C-4 primary amine 21 was obtained from a Curtius rearrangement of 16a followed by simultaneous

Table 2. Biochemical and Antiviral Cell Culture Activities of Five-Membered Heterocycles

hydrogenolysis of both nitrogen protecting groups and removal of the tert-butyl ester with trifluoroacetic acid. Addition of ethylmagnesium bromide to 15a followed by Swern oxidation and deprotection produced the ethyl ketone 19. Methylation of 14b with silver oxide and methyl iodide followed by deprotection with 6 N HCl provided 18. The syntheses of several of the heterocycles of Table 2 are depicted in Scheme 3. The bromo ketones 22 proceeded from carboxylic acids 16 via their respective diazo ketones and subsequent treatment with HBr. The 4-imidazole 23 and 4-thiazole 24 were obtained by reaction of 22a with formamide and ammonia or with thioformamide respectively followed by and acid deprotection. The 2-thiazole analogue 25 proceeded from a multistep transformation of the carboxylic acid 16 to a

Pyrrolidine Influenza Neuraminidase Inhibitors

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 3983

Scheme 3a

a Reagents and conditions: (a) (i) ClCO iBu, THF, (ii) CH N , (iii) HBr; (b) HC(O)NH , liquid NH ; (c) NH +HCO -, Pd/C, EtOH, reflux; 2 2 2 2 3 4 2 (d) HC(S)NH2, EtOH; (e) (i) ClCO2i-Bu, NMO, THF, (ii) NH4OH, (iii) P4S10; (f) ClCH2CHO; (g) 6 N HCl.

Scheme 4a

a Reagents: (a) HCOCHO, NH , MeOH; (b) NH +HCO -, Pd/C, EtOH, reflux; (c) 6 N HCl; (d) HCCMgBr, THF; (e) Jones oxidation; (f) 3 4 2 H2NNH2, EtOH; (g) NH2OH‚HCl, Na2CO3, EtOH; (h) TFA, CH2Cl2.

primary thioamide that upon treatment with chloroacetaldehyde and deprotection gave 25. The remaining C-4 heterocyclic analogues of Table 2 were synthesized from the aldehyde intermediates 15 as shown in Scheme 4. Reaction of 15a with glyoxal and ammonia followed by deprotection gave 26 in high yield. Reaction of 15b with ethynylmagnesium bromide followed by Jones oxidation produced 27. Treatment of 27 with hydrazine and subsequent one step deprotection gave the 3-pyrazole 28. Similarly, the 3- and 5-isoxazolyl analogues 29a,b were produced as a mixture from 27 when reacted with hydroxylamine and deprotected. All of the substituted vinyl analogues in Scheme 5 with the exception of 30 were synthesized as described from the aldehyde 15b. Wittig olefination conditions and acid deprotection provided analogues 31-33, 35, and 36. The Z-propenyl analogue 32 was formed exclusively, whereas the E and Z-chlorovinyl groups leading to 35 and 36 were formed in a 1:1 ratio. Horner-Emmons olefination with difluoromethylphosphonate gave the difluorovinyl group in 34. Formation of the ethyl ester prodrugs 37-40 proceeded smoothly in good yield from 30, 32, 34, and 36, respectively, by reaction with thionyl

chloride in ethanol at room temperature and purification by chromatography. Results and Discussion Prior to the discovery of 6 with a hydrophobic methyl ester substituent in the S2 subsite, all potent NA inhibitors utilized amines and guanidines to target the negatively charged amino acid residues, Glu119, Glu227, and Asp151, lining the S2 subsite. When compared to the amine-substituted analogue 21 of Table 1 (Ki ) 1.56 µM), the methyl ester 6 exhibited promising activity with submicromolar NA inhibition (Ki ) 0.15, 0.26 µM) against two representative strains of neuraminidase, A/N2/Tokyo and B/Memphis, respectively. The crystal structure of 6 bound to NA (Figure 3) exhibited exclusively hydrophobic contacts of the methyl ester moiety with S2. Further analysis of the crystal structure of 6 with respect to the native NA structure and the bound structures of the guanidine-substituted inhibitors 2 and 5 revealed several characteristics allowing binding by the methyl ester. First, the methyl ester occupies essentially the same plane of space as the guanidine substituents and similarly displaces two

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Scheme 5a

a

Reagents: (a) R1R2CHPPh3+X-, t-BuO-K+/toluene; (b) F2CHP(O)(OEt)2, LDA/THF; (c) 6 N HCl or TFA, CH2Cl2; (d) SOCl2, EtOH. Supporting Information.

bSee

Figure 3. Crystal structure of 6 bound in the active site of neuraminidase. Key amino acid residues forming the S2 subsite are highlighted.

water molecules from S2 for a net entropic gain on binding. Second, the methyl and oxygen groups of the ester appear to form van der Waals contacts with Leu134 and the side chain methylene of Asp152. Finally, the trajectory of the methyl ester into S2 produces a favorable π-stacking interaction with the π-face of the side chain carboxyl group of Glu119. Previously, both electrostatic and π-stacking interactions with Glu119 have been invoked to understand the positive components of the binding of the guanidine substituent of 2.23 Equipped with a new perspective into the nature of the S2 subsite, a structure-based medicinal chemistry program was initiated to discover optimized, metabolically stable replacements for the methyl ester. Although the analogues of Table 1 did not improve on the in vitro activity of the methyl ester, the SAR demonstrated by these analogues was consistent with the interpretations of the crystal structure and illustrative of the contribution to binding of the structural components of the methyl ester. The loss of activity of the ethyl ester 17 was consistent with the space available in S2, but the 60-fold loss in potency of the methyl ether 18 highlighted the importance of the π-stacking interaction and the conformational aspects of sp2 hybridization for analogue design. In order for 18 to achieve interactions similar to the methyl ester, the first carbon atom from the ring must adopt a high-energy, eclipsed conformation with the pyrrolidine ring carbons. In contrast, sp2 hybridization, allows binding in a lowenergy conformation. Although the N-methylamide 20 was sp2 hybridized and conformationally restricted,

modeling placed the polar N-H bond directly toward the hydrophobic methylene of Asp151. This mismatch in polarity and subsequent uncompensated desolvation energy could account for the >100-fold loss exhibited by 20. Replacing the polar amide nitrogen with a methylene of the ethyl ketone 19 produced a less active analogue than the methyl ester 6. This result was attributed to a combination of at least two factors: a suboptimal interaction of the sp3 carbon relative to the ester oxygen with Asp151 and a weaker π-stacking interaction with Glu119 by a less delocalized π-system of a ketone relative to that of an ester. The survey of methyl ester isosteres illustrated the strong structural preference for hydrophobic sp2 hybridized substituents that can displace water from the pocket via a low-energy conformation, while simultaneously maximizing π-stacking interactions with the π-face of Glu119. This structural criterion together with the observation of available space within S2 for one additional atom led to the design and investigation of a series of five-membered heterocycles. In general, the heterocycles of Table 2 were more consistent in retaining activity than the analogues of Table 1. The thiazoles 24 and 25 represent attempts to maximize the hydrophobic interactions with S2. While assessing the size constraints of this subsite, these analogues did not improve on the activity of 6. Similar results were also seen with the mixture of 4- and 5-isoxazolyl analogues 29a,b. On the basis of modeling and crystal structures of close analogues, the imidazole analogues 23 and 26 were targeted for synthesis for their potential to make hydrogen-bonding interactions in addition to hydrophobic contacts. The 4-imidazole 23 was designed to make a hydrogen bond with the backbone carbonyl of Trp178 that forms part of the “back” of the S2 subsite. Alternatively, the 2-imidazole 26 could interact with a tightly bound water molecule (Figure 4a) that also interacts with the acetamide-NH in S3. However, the 23 and 26 did not display improved NA inhibition, most likely due to significantly higher desolvation energies that negated any positive contributions to binding from the targeted hydrogen bonds for these more polar S2 substituents. Desolvation effects also were invoked to explain potency losses observed with the addition of a second positively charged amino group to the NA inhibitor 4-aminoDANA 1,26 even though the targeted binding interactions were demonstrated by crystal structures. There-

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Journal of Medicinal Chemistry, 2005, Vol. 48, No. 12 3985 Table 3. Biochemical and Antiviral Cell Culture Activities of Substituted Vinyl Analogues

Figure 4. (a) Model of interactions of 26 (2-imidazole) with a tightly bound water based on a crystal structure of the corresponding 2-imidazole analogue with (1′-acetylamino-3′ethylpentyl) side chain at C-5. (b) Crystal structure of interactions of 28 with a hydrogen bond from the 3-pyrazole substituent to the carbonyl of tryptophan 178.

fore, the analogue 28 containing a 3-pyrazole group was designed to achieve a hydrogen bond with Trp178 (Figure 4b) with a group with a substantially lower basicity than that of the 4-imidazole 23. Indeed, 28 was able to exhibit the net benefit of the targeted hydrogen bond with a 10-fold improvement in both IC50 and EC50 from that of 23 and 3-5-fold higher activity than the methyl ester 6. The results in Table 3 summarize a hybrid design strategy that combined the insights of the first two studies described above. The E-propenyl analogue 30 was designed to serve as a stable isostere that could mimic the key features of the bound conformation of the methyl ester 6. The vinyl analogue 31 was prepared first because it was synthetically expedient and provided a baseline for both the preferred orientation of the olefin in S2 and its contribution to binding. The crystal structure of 31 (Figure 5a) determined that the trajectory of the vinyl substituent into S2 was similar to that of the OMe component of the methyl ester 6 with the terminal methylidene atom displaced slightly from that of the ether oxygen. Importantly, 31 retained most of the NA inhibitory activity and cell culture potency of methyl ester 6. Encouraged by the activity of 31, a multistep synthesis of the E-propenyl-substituted inhibitor 30 was executed to produce a compound with NA inhibitory potency essentially equal to that of the methyl ester 6 but only 2-fold greater than 31. The above observations reinforce the hypothesis that displacement of water from the S2 pocket and the π-stacking interactions with Glu119 are major contributors to the binding of the methyl ester. Fortunately, with the more accessible Z-propenyl analogue 32, the effect

of methyl substitution proved to be substantial, and nearly 10-fold improved activity over the original methyl ester was observed. Interestingly, the E- and Z-chlorovinyl analogues 35 and 36 were significantly less active than their corresponding propenyl analogues, but the differential potency observed for the Z-configuration over the E-isomer was even more dramatic. When the terminal methylidene carbon was disubstituted with either methyl 33 or fluorine 34, the inhibitors produced were less active than either of the propenyl isomers but similar in activity to the vinyl analogue 31. Modeling and crystal structures of the above structures provided additional insights into the SAR of substituted olefins contained in Table 3. In the light of the results for the methyl ester 6 and the E-propenyl analogue 30, the increase in activity of 32 must be attributed to the excess of productive van der Waals interactions by the Z-propenyl substituent. The methyl group of 32 (Figure 5b) extends further along the side chain methylene of Asp 151 into a small hydrophobic pocket formed by the side chain and backbone atoms of this residue. The crystal structure of 32 revealed a significant deflection of the trajectory of the double bond compared to that of 31 in order to accommodate or optimize interaction of the methyl group. Conceivably, the increased size and electronegativity of the Z-chloro group of 35 relative to the Z-methyl of 32 accounts for the 4-fold reduction in its activity. Although the initial analysis of the crystal structure of the methyl ester focused on hydrophobic contacts of the methyl group with the Leu134, the pronounced potency differential seen in the E/Z-chloro analogues 35 and 36 prompted a reexamination of the potential negative effect of the other two residues, Trp178 and Glu227, that project polar groups toward S2: the backbone carbonyl of Trp178 and side chain carboxyl

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Figure 5. (a) Crystal structure of vinyl analogue 31 and (b) overlay with methyl ester 6. (c) Crystal structure of 32 and (d) overlay with 6.

of Glu227. To accommodate the dimethyl-substituted substituent, 33 must adopt a position for the E-methyl group in close enough proximity to the carboxyl of Glu227 for it to exert a negative effect that neutralizes the benefit of the Z-methyl. Similarly, the modest improvement of 30 over 31 can be attributed to an interaction of the methyl of 30 that is less directed toward Leu134 and more toward Glu227. This effect was even more accentuated in 36 with a more electronegative chlorine atom producing a more repulsive polar effect with both Trp178 and Glu227. The discovery of the Z-propenyl analogue 32 represented an important advance in the efforts to exploit hydrophobic approaches into the S2 subsite. Although the activity of 32 was promising, even as a racemate, it remained approximately 50-100-fold lower in potency than zanamivir (2) and oseltamivir carboxylate (3).27,28 Precedent from work in a number of inhibitor series has shown that optimization within the other subsites (S4 and S5) can yield up to 3-4 log increases in potency. Therefore, it was important to demonstrate for this novel series of NA inhibitors that hydrophobic S2 substituents can lead to orally bioavailable compounds. Formulation of the analogues 31, 32, 34, and 35 as their ethyl ester prodrugs 37-40 resulted in very soluble compounds that effectively delivered high plasma levels of their respective parent inhibitors after both iv and oral dosing in rat. All of the ethyl ester prodrugs were hydrolyzed very rapidly, producing undetectable prodrug levels 30 min after dosing. The pharmacokinetic (PK) results for 37-40 are summarized in Table 4. The PK parameters reported were calculated on the basis of the levels of parent drug observed after dosing their respective ethyl ester prodrugs. The relatively high plasma levels for 37-39 (∼6.0-8 µg/mL) and good exposures (AUC ∼ 5-13) compared very favorably to those of oseltamivir (4). These results confirm the high

Table 4. Single Dose Rat Pharmacokinetics of Ethyl Ester Prodrugsa

a Data shown is for parent drug. Prodrugs were dosed at 5 mg equiv/kg (iv) and 10 mg equiv/kg (oral). Cmax (µg/mL), t1/2 (h), AUC (µg h/mL), F (%).

potential of this series as a substrate for additional optimization in the S4,5 subsites. Conclusions Historically, the key to developing orally bioavailable influenza neuraminidase inhibitors has been the discovery and optimization of new hydrophobic binding interactions within what has been previously perceived to be a very polar enzyme active site. The SAR of a novel hydrophobic approach to binding within the S2 subsite of the influenza neuraminidase has been described with a variety of hydrophobic substituents projected from a

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pyrrolidine core inhibitor lead structure 6. This hydrophobic approach is a sharp contrast to previous approaches that targeted this subsite with cationic substituents such as amines and guanidines. For this approach, a structural requirement for sp2 hybidization allows substituents to displace water from the pocket while a low-energy conformation and an important hydrophobic interaction with the π-face of the Glu119 side chain carboxyl group are maintained. Similar studies of hydrophobic S2 substituents projected from a six-membered ring core related to 3 yielded similar preferences for sp2 hybridization.29 Expanding on this concept, a series of five-membered heterocycles demonstrated for the first time the ability of S2 to accommodate substitutents of this size; however, no hydrophobic interactions were optimized, as evidenced by generally equivalent activity relative to the methyl ester lead structure. Attempts to improve binding by targeting additional hydrogen-bonding interactions in S2 with relatively basic imidazole substituents were unsuccessful due to the higher desolvation energy required for binding of these inhibitors, although improved binding could be achieved by introducing an additional hydrogenbonding interaction from a neutral pyrazole substituent targeted toward a backbone carbonyl projected into S2 from Trp178. Another structure-based strategy to mimic the characteristics of the methyl ester demonstrated that a relatively simple substituent such as the E-propenyl olefin in 30 could serve as stable and equipotent binder to the lead structure 6 within S2. Further optimization of the hydrophobic character of S2 yielded the Zpropenyl analogue 32, a potent inhibitor of both A and B neuraminidases. Formulation of a set of the substituted vinyl series of inhibitors as ethyl ester prodrugs yielded highly bioavailable compounds in rat. These results demonstrate the potential of this new hydrophobic approach in S2 to produce orally bioavailable NA inhibitors. Details of the optimization of 32 in S4,5 to produce a drug candidate compound with a distinctive resistance profile30,31 will be reported shortly.

on a Silicon Graphics Inc., workstation using the program QUANTA 97/2001 (Molecular Simulations Inc.), and the small molecule ligand structures were fit to electron density. Structures were determined to a resolution of 2.0-3.0 Å and refined to Rwork between 21 and 25% and Rfree between 25 and 29%. General Procedures. Commercial reagents were utilized without further purification unless otherwise noted. THF was dried over benzophenone ketyl and was freshly distilled. All nonaqueous reactions were run under an atmosphere of dry nitrogen and stirred with a magnetic stir bar unless otherwise stated. All 1H NMR spectra were recorded at 300 or 500 MHz and are reported in ppm (δ). Chromatographic separations were carried on silica gel unless otherwise indicated. Solvent evaporation or concentration was at reduced pressure with the use of a rotary evaporator. Dried implies the use of MgSO4 to remove the water from a given solvent. Combustion analyses were performed by Robertson Microlit Laboratories, Inc., Madison, NJ, and are within (0.4% of the theoretical values for C, H, and N. (()-(2R,4R,5S)- and (()-(2R,4S,5S)-1-Benzyl-4-formyl5-vinylpyrrolidine-2-carboxylic Acid tert-Butyl Ester (7). To a solution of tert-butyl N-benzylglycinate (39 g, 177 mmol) and acetic acid (0.51 mL, 8.8 mmol) in toluene (300 mL) was added acrolein (59 mL, 882 mmol). The reaction was refluxed for 2.5 h, cooled, and concentrated. The crude product was stirred with triethylamine (5 mL, 36 mmol) in ethyl acetate (300 mL) at ambient temperature for 3 h. The reaction was concentrated and chromatographed with 5% ethyl acetate/ hexanes to afford 7 (27.8 g, 50%) as an 8:1 mixture of the title compounds as an oil. 1H NMR (CDCl3) (4R isomer only, 7): δ 9.71 (d, J ) 1.2 Hz, 1H), 7.21-7.35 (m, 5H), 5.7 (ddd, J ) 17.7, 10.2, 7.8 Hz, 1H), 5.22-5.33 (m, 2H), 3.94 (d, J ) 13.5 Hz, 1H), 3.93 (m, 1H), 3.61 (d, J ) 13.5 Hz, 1H), 3.49 (dd, J ) 7.8, 3.0 Hz, 1H), 2.69 (m, 1H), 2.26 (m, 1H), 1.45 (s, 9H). (()-(2R,4R,5S) and (()-(2R,4S,5S)-1-Benzyl-4-hydroxymethyl-5-vinylpyrrolidine-2-carboxylic Acid tert-Butyl Ester (8). A solution of 7 (17.4 g, 55.2 mmol) in methanol (175 mL) at 0 °C was reacted with sodium borohydride (3.5 g, 92.0 mmol) for 0.5 h and then warmed to ambient temperature for 1 h. The reaction was quenched with aqueous ammonium chloride (100 mL) and concentrated. The residue was partitioned between ethyl acetate (200 mL) and water (200 mL). The organic layer was dried and concentrated. The residual oil was purified by chromatography using a gradient of 2550% ethyl acetate/hexanes to give 8 (17.3 g, 99%) as a colorless oil. 1H NMR (CDCl3): δ 7.23-7.34 (m, 5H), 5.70 (m, 1H), 5.175.22 (m, 2H), 3.91 (d, J ) 13.2 Hz,1H), 3.66-3.75 (m, 2H), 3.48-3.53 (m, 2H), 2.54 (m, 1H), 2.39 (m, 1H), 2.16 (m, 1H), 1.80 (m, 1H), 1.46 (s, 9H). MS (ESI): m/z 318 (M + H)+. (()-(2R,4R,5S)-1-Benzyl-4-(tert-butyldimethylsilanyloxymethyl)-5-vinylpyrrolidine-2-carboxylic Acid tertButyl Ester (9). A solution of 8 (3.6 g, 11.4 mmol), tertbutyldimethylsilyl chloride (3.7 g, 24.5 mmol), and imidazole (2.8 g, 41.2 mmol) in DMF (80 mL) was stirred at room temperature for 1.5 h. The reaction was diluted with ethyl acetate (100 mL), washed with water (50 mL) and then brine (50 mL), dried, and concentrated. The residue was purified by chromatography using 5% ethyl acetate/hexanes to provide 9 (3.5 g, 71%) as a colorless oil. 1H NMR (CDCl3): δ 7.287.19 (m, 5H), 5.69 (ddd, J ) 17.1, 9.9, 8.4 Hz, 1H), 5.17 (dd, J ) 9.6, 1.8 Hz, 1H), 5.12 (m, 1H), 3.90 (d, J ) 13.5 Hz, 1H), 3.67 (dd, J ) 9.6, 5.7 Hz, 1H), 3.66 (d, J ) 13.5 Hz, 1H), 3.54 (dd, J ) 9.6, 7.8 Hz, 1H), 3.48 (dd, J ) 9.0, 3.0 Hz, 1H), 3.43 (dd, J ) 8.4, 7.2 Hz, 1H), 2.29 (dt, J ) 13.5, 9.7 Hz, 1H), 2.09 (m, 1H), 1.44 (s, 9H), 0.87 (s, 9H), 0.03 (s, 3H), 0.02 (s, 3H). MS (ESI): m/z 342 (M + H)+. (()-(2R,4R,5R,1′RS)-1-Benzyl-4-(tert-butyldimethylsilanyloxymethyl)-5-(1′,2′-dihydroxyethyl)pyrrolidine-2carboxylic Acid tert-Butyl Ester (10a). A solution of 9 (3.5 g, 8.12 mmol), N-methylmorpholine N-oxide (3.0 g, 25.6 mmol), and osmium tetroxide (20 mg) in acetone/water (8:1, 60 mL) was stirred at ambient temperature for 6 h. The reaction was quenched with saturated aqueous Na2S2O3 (50 mL) for 10 min and concentrated. The residue was partitioned between dichlo-

Experimental Details X-ray Crystallography. Isolation, purification, and crystallization of type A N9/tern/Australia/G70c/75 neuraminidase was performed as reported.32 To prepare protein/ligand complexes for type A neuraminidase, crystals were soaked in solutions of 0.93 M KH2PO4, 1.0 M K2HPO4, 3% dimethyl sulfoxide at pH 6.7 which contained millimolar concentrations (typically 30 mM) of compound for 3-24 h. Type A crystals exposed to compound were then serially transferred into buffers containing 0, 10, 20, and 27% glycerol for 1-2 min per step to prevent subsequent damage during the cryocooling process. Data for the type A crystals were collected frozen in a stream of 100 K nitrogen using an Oxford Cryo-stream cooling device. Type B crystals were soaked with compounds in solutions of 15% PEG 3350, 20% sodium nitrate, and 0.01 M calcium chloride containing millimolar concentrations (typically 30 mM) of compound for 3-24 h. Data for the type B crystals were collected at room temperature. Diffraction data were recorded using either a RAXIS-IIc or MAR-130 image plate system on a Rigaku RU-2000 rotating anode X-ray generator operating at 100 mA and 50 kV. Diffraction data were reduced using DENZO33 and the neuramidase N9 model (accession number 1NNA) from the Protein Data Bank was used for initial phasing. Generation of initial electron density maps and structure refinement was achieved using the CNX program package.34,35 Electron density maps were inspected

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romethane (100 mL) and water (100 mL). The organic layer was dried and concentrated to provide the crude diols 10a (3.8 g) as an oil. (()-(2R,4R,5R,1′RS)-4-(tert-Butyldimethylsilanyloxymethyl)-5-(1′,2′-dihydroxyethyl)pyrrolidine-2-carboxylic Acid tert-Butyl Ester (10b). A solution of the diols (10a) (21.5 g, 46.2 mmol), 10% Pd/C (2.0 g), and ammonium formate (29.2 g, 462 mmol in ethanol (250 mL) was degassed, heated to reflux for 40 min, and then filtered through Celite. The filtrate was concentrated to provide 10b (16 g) and used without further purification. 1H NMR (CDCl3): 3.70-3.51 (m, 4H), 3.44-3.30 (m, 2H), 3.13 (m, 1H), 2.40-2.14 (m, 2H), 1.18 (br s, 4H), 1.46 (s, 9H), 0.90 (s, 9H), 0.09 (s, 6H). MS (ESI): m/z 376 (M + H)+. (()-(2R,4R,5R,1′RS)-4-(tert-Butyldimethylsilanyloxymethyl)-5-(1′,2′-dihydroxyethyl)pyrrolidine-1,2-dicarboxylic Acid Di-tert-butyl Ester (10c). A solution of 10b (16 g, 42.6 mmol) and di-tert-butyl dicarbonate (14.0 g, 64 mmol) in methanol/water (3:1, 160 mL) was reacted at ambient temperature for 72 h. The reaction was concentrated and purified by chromatography using ethyl acetate/hexanes (1:1) to provide 10c (15.4 g, 70% for two steps) as light yellow solid. 1H NMR (CDCl3): δ 4.02-4.52 (m, 1H), 3.85 (dd, J ) 5.2, 1.9 Hz, 1H), 3.66-3.43 (m, 4H), 3.28 (d, J ) 8.1 Hz, 1H), 2.30-2.50 (m, 2H), 1.93 (d, J ) 14.2 Hz, 1H), 1.47 (s, 9H), 1.42 (s, 9H), 1.37 (s, 9H), 0.05 (s, 3H), 0.03 (s, 3H). MS (ESI): m/z 476 (M + H)+. (()-(2R,4R,5R)-1-Benzyl-4-(tert-butyldimethylsilanyloxymethyl)-5-formylpyrrolidine-2-carboxylic Acid tertButyl Ester (11a). The diols 10a (3.8 g, 9.68 mmol) and sodium periodate (3.0 g, 14.0 mmol) in THF/water (6:1, 50 mL) were reacted at ambient temperature for 1 h. The reaction was diluted with ethyl acetate (100 mL), washed with water (50 mL), dried, and concentrated. The residue was purified by chromatography eluting with 3% ethyl acetate/hexanes to provide 11a (2.1 g, 63%) as colorless oil. 1H NMR (CDCl3): δ 9.32 (d, J ) 3.3 Hz, 1H), 7.27-7.31 (m, 5H), 3.84 (q, J ) 13.5 Hz, 2H), 3.53-3.71 (m, 3H), 2.26-2.45 (m, 3H), 1.73 (m, 1H), 1.46 (s, 9H), 0.87 (s, 9H), 0.04 (s, 3H), 0.03 (s, 3H). MS (ESI): m/z 434 (M + H)+. (()-(2R,4R,5R)-4-(tert-Butyldimethylsilanyloxymethyl)5-formylpyrrolidine-1,2-dicarboxylic Acid Di-tert-butyl Ester (11b). The diols 10c (6.0 g, 12.6 mmol) were reacted with sodium periodate (4.4 g, 20.6 mmol) in THF/water (6:1, 110 mL) for 3 h at ambient temperature. The reaction was diluted with ethyl acetate (100 mL), washed with water (50 mL), dried, and concentrated. The residue was purified by chromatography using 20% ethyl acetate/hexanes to provide 11b (4.4 g, 79%) as a white waxy solid. 1H NMR (CDCl3) (mixture of two rotamers): δ 9.53 and 9.43 (2 × m, 1H), 4.024.34 (m, 2H), 3.54-3.67 (m, 2H), 2.37-2.43 (m, 2H), 1.891.99 (m, 1H), 1.47 and 1.48 (2 × s, 9H), 1.42 and 1.44 (2 × s, 9H), 0.88 and 0.90 (2 × s, 9H), 0.05 and 0.06 (2 × s, 6H). MS (ESI): m/z 444 (M + H)+. (()-(2R,4R,5R)-1-Benzyl-4-(tert-butyldimethylsilanyloxymethyl)-5-(3-methylbutyryl)pyrrolidine-2-carboxylic Acid tert-Butyl Ester (12a). To a solution of 11a (2.1 g, 4.85 mmol) in THF (40 mL) at 0 °C was added isobutylmagnesium chloride (2 M in ether, 18 mL, 36 mmol). The reaction was stirred at 0 °C for 1 h, quenched with aqueous ammonium chloride (50 mL), and concentrated to remove the THF. The reaction mixture was extracted with dichloromethane (100 mL), and the organics were dried and concentrated to give the crude alcohol products (2.3 g). The crude alcohol mixture in dichloromethane (200 mL) was reacted with Dess-Martin periodinane reagent (2.5 g, 5.82 mmol) for 30 min, quenched with aqueous Na2S2O3 (1N, 50 mL), and extracted with dichloromethane (2 × 50 mL). The organic layers were dried and concentrated. The residue was purified by chromatography using 2.5% ethyl acetate/hexanes to provide 12a (0.32 g, 15%). 1 H NMR (CDCl3) δ 7.20-7.34 (m, 5H), 3.87 (q, J ) 10.5 Hz, 2H), 3.83 (m, 1H), 3.50-3.71 (m, 3H), 2.00-2.45 (m, 5H), 1.62 (m, 1H), 1.44 (s, 9H), 0.85 (s, 9H), 0.83 (m, 6H), 0.04 (s, 3H), 0.03 (s, 3H). MS (ESI): m/z 490 (M + H)+. (()-(2R,4R,5R)-4-(tert-Butyldimethylsilanyloxymethyl)5-(3-methylbutyryl)pyrrolidine-1,2-dicarboxylic Acid Ditert-butyl Ester (12b). A solution of 11b (7.1 g, 16.03 mmol)

in diethyl ether (75 mL) was reacted with isobutylmagnesium chloride (24 mL, 2.0 M in ether, 48 mmol) at 0 °C for 2.5 h. The reaction was quenched with aqueous saturated NH4Cl (50 mL) and diluted with ethyl acetate (200 mL). The organic layer was separated and washed with water (100 mL) and then brine (100 mL), dried, and concentrated. The crude alcohol products were used without further purification. To a solution of oxalyl chloride (16 mL, 2 M in CH2Cl2) in CH2Cl2 (100 mL) at -78 °C under a nitrogen atmosphere was added DMSO (4.26 mL, 64.1 mmol) slowly. After 15 min, the crude alcohol in dichloromethane (30 mL) was added. After another 1 h triethylamine (17 mL, 128 mmol) was added slowly to the reaction mixture. The solution was allowed to warm slowly to ambient temperature, quenched with saturated aqueous sodium bicarbonate (10 mL), and diluted with dichloromethane (100 mL). The organic layer was washed with water (50 mL) and then brine (50 mL), dried, filtered, and concentrated. The residue was purified by chromatography and gradient elution 5-10% ethyl acetate/hexanes to provide 12b (6.3 g, 79%). 1H NMR (CDCl3): δ 4.28-4.46 (m, 2H), 3.47-3.69 (m, 1H), 2.15-2.45 (m, 4H), 1.72-1.82 (m, 1H), 1.46 and 1.47 (2 × s, 9H), 1.40 and 1.42 (2 × s, 9H), 0.81-0.96 (m, 15H), 0.07 (m, 6H). MS (ESI): m/z 500 (M + H)+. (()-(2R,4R,5R,1′S)-5-(1′-Acetylamino-3′-methylbutyl)1-benzyl-4-(tert-butyldimethylsilanyloxymethyl)pyrrolidine-2-carboxylic Acid tert-Butyl Ester (13a). A solution of 12a (0.32 g, 0.65 mmol), ammonium acetate (2.2 g. 28.6 mmol), and sodium cyanoborohydride (1.1 g, 18.1 mmol) in methanol (15 mL) was refluxed for 48 h. The reaction was quenched with 1 N NaOH (10 mL) and concentrated. The residue was extracted with dichloromethane (2 × 50 mL), and the organics were dried and concentrated. The crude amines (132 mg) were acylated with acetic anhydride (0.27 mL, 2.6 mmol) and triethylamine (0.373 mL) in dichloromethane (3 mL) for 1 h at ambient temperature. The reaction was concentrated and purified by chromatography using 25% ethyl acetate/hexanes to provide 13a (95 mg, 27% two steps) as a white solid. 1H NMR (CDCl3) δ 7.25 (m, 5H), 5.16 (bs, 1H), 4.38 (m, 1H), 4.03 (d, J ) 13.5 Hz, 1H), 3.88 (d, J ) 13.5 Hz, 1H), 3.65-3.40 (m, 3H), 3.00 (m, 1H), 2.10 (m, 2H), 1.98 (s, 3H), 1.75 (m, 1H), 1.43 (s, 9H), 1.40-1.20 (m, 3H), 0.88 (t, J ) 5.7 Hz, 6H), 0.87 (s, 9H), 0.03 (s, 6H). MS (ESI): m/z 533 (M + H)+. (()-(2R,4R,5R,1′S)-5-(1′-Acetylamino-3′-methylbutyl)-4(tert-butyldimethylsilanyloxymethyl)pyrrolidine-1,2-dicarboxylic Acid Di-tert-butyl Ester (13b). A solution of 12b (6.20 g, 12.4 mmol), ammonium acetate (22.0 g. 286 mmol), and sodium cyanoborohydride (11.4 g, 181 mmol) in methanol (160 mL) was refluxed for 24 h. The reaction was quenched with 1 N NaOH (100 mL) and concentrated. The residue was extracted with EtOAc (2 × 100 mL), and the organics were dried, filtered, and concentrated. The residue was purified by chromatography using 0.5% ammonium hydroxide and 5% methanol in CH2Cl2 to provide the amine (3.1 g, 50%) as a white foam. In addition, desilylated 4-alcohol (2.5 g) was also obtained and reprotected with tert-butyldimethylsilyl chloride and imidazole in DMF. Acylation of the above amines as described for 13a gave 13b (combined yield 40%) as a white solid. 1H NMR (CDCl3): δ 7.35 (bs, 1H), 4.27 (m, 1H), 4.16 (m, 1H), 3.67 (m, 1H), 3.61 (m, 1H), 3.48 (m, 1H), 2.30 (m, 1H), 2.07 (m, 1H), 1.99 (s, 3H), 1.86 (m, 1H), 1.46 (s, 9H), 1.44 (s, 9H), 1.17-1.25 (m, 2H), 1.04 (m, 1H), 0.89 and 0.95 (2 × d, 6H), 0.86 (s, 9H), 0.03 and 0.04 (2 × s, 6H). MS (ESI): m/z 543 (M + H)+. (()-(2R,4R,5R,1′S)-5-(1′-Acetylamino-3′-methylbutyl)-1benzyl-4-hydroxymethylpyrrolidine-2-carboxylic Acid tert-Butyl Ester (14a). To a solution of 13a (85 mg, 0.16 mmol) in THF (3 mL) was added tetrabutylammonium fluoride (1 M in THF, 0.23 mL) slowly at ambient temperature. After 1 h, the reaction was concentrated and purified by chromatography eluting with 75% ethyl acetate/hexanes to provide 14a (75 mg, 100%) as a white solid. 1H NMR (CDCl3) δ 7.357.20 (m, 5H), 5.25 (m, 1H), 4.32 (m, 1H), 3.96 (d, J ) 13.5 Hz, 1H), 3.90 (d, J ) 13.5 Hz, 1H), 3.75-3.58 (m, 2H), 3.48 (m, 1H), 3.25 (m, 1H), 3.18 (m, 1H), 2.28-2.10 (m, 2H), 2.00 (s,

Pyrrolidine Influenza Neuraminidase Inhibitors

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3H), 1.62 (m, 1H), 1.42 (s, 9H), 1.37-1.23 (m, 3H), 0.89 (m, 6H). MS (ESI): m/z 419 (M + H)+. (()-(2R,4R,5R,1′S)-5-(1′-Acetylamino-3′-methylbutyl)-4hydroxymethylpyrrolidine-1,2-dicarboxylic Acid Di-tertbutyl Ester (14b). The compound was prepared from 13b as described for 14a to give 14b (355 mg, 98%) as a white solid. 1 H NMR (CDCl3): δ 7.18 (bs, 1H), 4.27 (td, J ) 10.3, 3.0 Hz, 1H), 4.16 dd, J ) 9.2, 5.8 Hz, 1H), 3.67 (m, 2H), 3.18 (bs, 1H), 2.30 (m, 1H), 2.13 (m, 1H), 2.01 (s, 3H), 1.62 (m, 1H), 1.46 (s, 9H), 1.43 (s, 9H), 1.29 (m, 2H), 1.09 (m, 1H), 0.92 (d, J ) 6.8 Hz, 3H), 0.90 (d, J ) 6.8 Hz, 3H). MS (ESI): m/z 429 (M + H)+. (()-(2R,4R,5R,1′S)-5-(1′-Acetylamino-3′-methylbutyl)-1benzyl-4-formylpyrrolidine-2-carboxylic Acid tert-Butyl Ester (15a). To a solution of oxalyl chloride (2 M in dichloromethane) (6.2 mL, 12.4 mmol) in dichloromethane (30 mL) at -78 °C was added DMSO (1.7 mL, 24.9 mmol) dropwise. After 15 min, a solution of compound 14a (1.30 g, 3.1 mmol) in dichloromethane (30 mL) was added dropwise and reacted at -78 °C for 1 h, followed by the addition of triethylamine (7.0 mL, 49.8 mmol). The mixture was slowly warmed to room temperature, quenched with 10% aqueous NaHCO3 (50 mL), extracted with CH2Cl2 (3 × 50 mL), dried, filtered, and concentrated. The residue was purified by chromatography using 1:1 ethyl acetate/hexanes to provide 15a (900 mg, 70%) as a white solid. 1H NMR (CDCl3): δ 9.68 (s, 1H), 7.28 (m, 5H), 5.10 (d, J ) 9.2 Hz, 1H), 4.43 (m, 1H), 4.13 (m, 1H), 3.73 (m, 2H), 3.43 (m, 1H), 2.62 (m, 1H), 2.25 (m, 1H), 1.99 (s, 3H), 1.42 (s, 9H), 1.23-1.33 (m, 3H), 0.93 (d, J ) 6.4 Hz, 6H). MS (ESI): m/z 417 (M + H)+. (()-(2R,4R,5R,1′S)-5-(1′-Acetylamino-3′-methylbutyl)-4formylpyrrolidine-1,2-dicarboxylic Acid Di-tert-butyl Ester (15b). The title compound was prepared from 14b as described for 15a to give 15b (328 mg, 95%) as a white solid. 1 H NMR (CDCl3): δ 7.12 (m, 1H), 4.39 (m, 1H), 4.32 (m, 1H), 4.19 (m, 1H), 3.18 (m, 1H), 2.46 (m, 1H), 2.39 (m, 1H), 2.03 (s, 3H), 1.64 (m, 1H), 1.44 (s, 9H), 1.43 (s, 9H), 1.23-1.33 (m, 2H), 1.07 (m, 1H), 0.94 (d, J ) 6.8 Hz, 3H), 0.92 (d, J ) 6.8 Hz, 3H). MS (ESI): m/z 427 (M + H)+. (()-(2R,4R,5R,1′S)-5-(1′-Acetylamino-3′-methylbutyl)-1benzylpyrrolidine-2,4-dicarboxylic Acid 2-tert-Butyl Ester (16a). To a solution of 15a (400 mg, 0.96 mmol) and 2-methyl-2-butene (8.8 mL) in acetonitrile (16 mL) and 2-methyl-2-propanol (16 mL) at 0 °C was added a solution of sodium chlorite (2.4 g) and sodium phosphate monobasic (2.4 g) in water (5 mL) slowly. The reaction was stirred at 0 °C for 1 h, quenched with saturated aqueous Na2S2O3 (25 mL), and extracted with EtOAc (2 × 25 mL). The organics were washed with water (50 mL) and then brine (50 mL), dried, filtered, and concentrated to provide the crude acid 16a (520 mg) and used without additional purification. 1H NMR (CDCl3): δ 7.35-7.25 (m, 5H), 5.16 (br d, J ) 9.3 Hz, 1H), 4.44 (m, 1H), 4.10 (d, J ) 13.2 Hz, 1H), 3.88 (d, J ) 13.2 Hz, 1H), 3.55 (dd, J ) 9.0, 3.0 Hz, 1H), 3.43 (t, J ) 3.3 Hz, 1H), 2.85 (ddd, J ) 10.2, 3.9, 2.4 Hz, 1H), 2.38 (ddd, J ) 14.1, 9.6, 9.3 Hz, 1H), 2.17 (dt, J ) 13.8, 2.4 Hz, 1H), 2.02 (s, 3H), 1.56 (m, 1H), 1.46 (s, 9H), 1.36 (ddd, J ) 14.5, 10.2, 4.8 Hz, 1H), 1.20 (ddd, J ) 13.8, 9.0, 4.5 Hz, 1H), 0.88 (dd, J ) 6.9, 1.5 Hz, 6H). MS (ESI): m/z 433 (M + H)+. (()-(2R,4R,5R,1′S)-5-(1′-Acetylamino-3′-methylbutyl)pyrrolidine-1,2,4-tricarboxylic Acid 1,2-Di-tert-butyl Ester (16b). The title compound was prepared from 15b as described for 16a to give 16b (126 mg) and used without additional purification. 1H NMR (CDCl3): δ 7.64 (m, 1H), 4.38 (m, 1H), 4.23 (m, 2H), 2.81-2.92 (m, 1H), 2.44-2.37 (m, 2H), 2.05 (s, 3H), 1.62 (m, 1H), 1.45 (s, 9H), 1.43 (s, 9H), 1.31-1.19 (m, 2H), 1.09 (m, 1H), 0.96 (d, J ) 6.8 Hz, 3H), 0.93 (d, J ) 6.8 Hz, 3H). MS (ESI): m/z 443 (M + H)+. (()-(2R,4R,5S,1′S)-5-(1′-Acetylamino-3′-methylbutyl)pyrrolidine-2,4-dicarboxylic Acid 4-Methyl Ester Hydrochloride Salt (6). A solution of 16a (92 mg, 0.21 mmol) in THF (3 mL) at 0 °C was treated with diazomethane (∼1 mmol/ ether) and reacted for 30 min. The reaction was concentrated and purified by chromatography using 2.5% methanol in dichloromethane to give the protected C-4 methyl ester (75 mg, 79%). MH+ (ESI): 447. Hydrogenolysis of the methyl ester (62 mg, 0.139 mmol) as described for 10b and chromatography

with 2.5% methanol gave the free pyrrolidine diester (26 mg, 52%) as a white solid. The diester (22 mg, 0.062 mmol) was deprotected with 6 N HCl (2 mL) at ambient temperature for 30 min. The reaction was concentrated, redissolved in dioxane (0.5 mL), and concentrated to provide 6 (21 mg, 100%) as a white foam.1H NMR (DMSO-d6): δ 8.08 (d, J ) 9 Hz, 1H), 4.44 (m, 1H), 4.23 (m, 1H), 3.86 (m, 1H), 3.69 (m, 1H), 3.65 (s, 3H), 3.49 (m, 1H), 3.17 (m, 1H), 2.56 (m, 1H), 2.20 (m, 1H), 1.84 (s, 3H), 1.57 (m, 1H), 1.36 (m, 1H), 0.88 (d, J ) 7.5 Hz, 3H), 0.82 (d, J ) 7.5 Hz, 3H). MS (ESI): m/z 301 (M + H)+. Anal. (C14H24N2O5·1.5HCl‚C4H8O2) C, H, N. (()-(2R,4R,5R,1′S)-5-(1′-Acetylamino-3′-methylbutyl)-4(1H-imidazol-2-yl)pyrrolidine-2-carboxylic Acid Dihydrochloride Salt (26). Ammonia gas was bubbled slowly through a solution of 15a (30.0 mg, 0.072 mmol) and glyoxal (40%, 30 µL, 0.26 mmol) in methanol (5 mL) at 0 °C for 20 min. The reaction was sealed and reacted at ambient temperature. After 16 h, additional glyoxal (30 µL) and ammonia gas was added and reacted for another 6 h. The reaction was concentrated and purified by chromatography using 5% methanol in dichloromethane to provide the 2-imidazole product as a white solid (27.4 mg, 83%). Deprotection according to the procedure described for 6 gave 26 (17.8 mg, 100%) as white foam. 1H NMR (DMSO-d6): δ 8.03 (d, J ) 8.4 Hz, 1H), 7.65 (s, 2H), 4.55 (m,1H), 4.28 (m, 1H), 4.05 (m, 1H), 3.83 (m, 1H), 2.80 (m, 1H), 2.43-2.47 (m, 1H), 1.71 (s, 3H), 1.44 (m, 1H), 1.18 (m, 2H), 0.82 (d, J ) 6.6 Hz, 3H), 0.76 (d, J ) 6.6 Hz, 3H). MS (ESI): m/z 326 (M + H)+. Anal. (C14H25N3O4·2.0HCl) C, H, N. (()-(2R,4R,5R,1′S)-5-(1′-Acetylamino-3′-methylbutyl)-4propynoylpyrrolidine-1,2-dicarboxylic Acid Di-tert-butyl Ester (27). To a solution of ethynylmagnesium bromide (16 mL, 0.5 M in THF) in THF (20 mL) at 0 °C was added 15b (250 mg, 0.589 mmol) in THF (20 mL) dropwise. The reaction mixture was stirred at 0 °C for 5 min and then warmed to ambient temperature for 16 h. The reaction was quenched with aqueous ammonium chloride (20 mL) and partitioned between ethyl acetate (50 mL) and water (50 mL). The organic layer was dried and concentrated to provide the alkynyl alcohol that was oxidized with Jones reagent (3.0 M in acetone, 0.33 mL) in acetone (90 mL) at 0 °C to room temperature for 1 h. The reaction was diluted with ethyl acetate (50 mL), washed with water (50 mL) and brine (50 mL), dried, and concentrated. The residue was purified by column chromatography using 50% ethyl acetate/hexanes to provide 27 (143 mg, 54%) as a white solid. 1H NMR (CDCl3): δ 5.11 (m, 1H), 4.11-4.56 (m, 3H), 3.38 (m, 1H), 2.83 (m, 1H), 2.68 (d, J ) 13.6 Hz, 1H), 2.38 (m, 1H), 2.10 (s, 3H), 1.47 (s, 9H), 1.44 (s, 9H), 1.00-1.30 (m, 2H), 0.94 (m, 6H). MS (ESI): m/z 451 (M + H)+. (()-(2R,4R,5R,1′S)-5-(1′-Acetylamino-3′-methylbutyl)-4(1H-pyrazol-3-yl)pyrrolidine-2-carboxylic Acid Ditrifluoroacetic Acid Salt (28). 27 (140 mg, 0.311 mmol) was reacted with hydrazine monohydrate (0.24 mL, 4.944 mmol) in ethanol (12 mL) at ambient temperature for 4 h. The reaction was concentrated and purified by chromatography using ethyl acetate to provide the protected 4-pyrazole (131 mg, 91%) as a white solid. Deprotection with trifluoroacetic acid (1.9 mL) in dichloromethane (0.3 mL) at ambient temperature for 7 h and concentration gave 28 (148 mg, 89% overall). 1H NMR (DMSO-d6): δ 9.34 (br s, 1H), 9.07 (br s, 1H), 8.06 (d, J ) 7.6 Hz, 1H), 7.65 (d, J ) 2.2 Hz, 1H), 6.11 (d, J ) 2.2 Hz, 1H), 4.30 (m, 1H), 4.16 (m, 1H), 3.85 (m, 1H), 3.48 (m, 1H), 2.53 (m, 1H), 2.08 (m, 1H), 1.79 (s, 3H), 1.34 (m, 1H), 1.13 (m, 1H), 1.03 (m, 1H), 0.71 (d, J ) 6.6 Hz, 3H), 0.62 (d, J ) 6.6 Hz, 3H). MS (ESI): m/z 309 (M + H)+. Anal. (C15H24N4O3· 2.0H2O‚2.0C2HF3O2) C, H, N. (()-(2R,4S,5R,1′S)-5-(1′-Acetylamino-3′-methylbutyl)-4cis-propen-1-ylpyrrolidine-2-carboxylic Acid Trifluoroacetic Acid Salt (32). To a suspension of ethyl triphenylphosphonium bromide (479 mg, 1.29 mmol) in toluene (3 mL) was added potassium tert-butoxide (1.0 M in THF, 0.94 mmol) dropwise at ambient temperature. After 2.5 h, 15b (90 mg, 0.211 mmol) in toluene (5 mL) was added dropwise and reacted for 1 h. The reaction was quenched with saturated aqueous ammonium chloride (10 mL) and diluted with ethyl acetate (50 mL). The organic layer was washed with water (25 mL)

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and brine (25 mL), dried, and concentrated. The residue was purified by chromatography using 50% ethyl acetate/hexanes to provide the protected cis-propenyl analogue (71 mg, 76%) as an oil. Deprotection was done with trifluoroacetic acid (3.8 mL) and dichloromethane (0.6 mL) at ambient temperature for 7 h. The solvent was evaporated to provide 32 (64 mg, 76%). 1 H NMR (DMSO-d6): δ 9.18 (br s, 1H), 8.04 (d, J ) 7.9 Hz, 1H), 5.51 (m, 1H), 5.26 (m, 1H), 4.32 (dd, J ) 9.8, 7.3 Hz, 1H), 4.18 (m, 1H), 3.45 (m, 1H), 3.18 (m, 1H), 2.39 (m, 1H), 1.88 (s, 3H), 1.73 (m, 1H), 1.63 (dd, J ) 7.0, 1.5 Hz, 3H), 1.58 (m, 1H), 1.38 (m, 1H), 1.28 (m, 1H), 0.88 (d, J ) 6.7 Hz, 3H), 0.81 (d, J )6.7Hz,3H).MS(ESI): m/z283(M+H)+.Anal.(C15H26N2O3·1.1C2HF3O2) C, H, N. (()-(2R,4R,5S,1′S)-5-(1′-Acetylamino-3′-methylbutyl)-4cis-propen-1-ylpyrrolidine-2-carboxylic Acid Ethyl Ester (38). To a solution of thionyl chloride (2.10 mL, 28.7 mmol) and ethanol (25 mL) reacted at 0 °C for 10 min was added 32 (1.14 g, 2.87 mmol) in ethanol (75 mL) dropwise. The reaction continued for 17 h at ambient temperature. The reaction was concentrated and purified by chromatography using 90:10:0.5% dichloromethane/methanol/ammonium hydroxide to provide 38 (838 mg, 94%). 1H NMR (CDCl3): δ 5.50 (m, 1H), 5.41 (m, 1H), 5.28 (m, 1H), 4.21 (q, J ) 7.5 Hz, 2H), 4.06 (m,1H), 3.87 (t, J ) 7.5 Hz, 1H), 3.10 (m, 1H), 2.97 (m, 1H), 2.39 (m, 1H), 1.97 (s, 3H), 1.66 (m, 3H), 1.60 (m, 1H), 1.40 (m, 2H), 1.31 (t, J ) 7.5 Hz, 3H), 0.94 (d, J ) 7.5 Hz, 3H), 0.93 (d, J ) 7.5 Hz, 3H). MS (ESI): m/z 311 (M + H)+. Supporting Information Available: X-ray parameters and Protein Data Bank file names for neuraminidase/inhibitor complexes of compounds 6, a side chain analogue of 26 (1′acetylamino-3′-ethylpentyl), 28, and 32. Experimental details for biological assays, synthesis, and analytical data for intermediates and final compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

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