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Peramivir Phosphonate Derivatives as Influenza Neuraminidase Inhibitors Peng-Cheng Wang, Jim-Min Fang, Keng-Chang Tsai, Shi-Yun Wang, Wen-I Huang, Yin-Chen Tseng, Yih-Shyun E. Cheng, Ting-Jen Rachel Cheng, and Chi-Huey Wong J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00029 • Publication Date (Web): 11 May 2016 Downloaded from http://pubs.acs.org on May 14, 2016

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Journal of Medicinal Chemistry

Peramivir Phosphonate Derivatives as Influenza Neuraminidase Inhibitors

Peng-Cheng Wanga, Jim-Min Fanga,b,*, Keng-Chang Tsaic, Shi-Yun Wangb, Wen-I Huangb, Yin-Chen Tsengb, Yih-Shyun E. Chengb, Ting-Jen Rachel Chengb, and Chi-Huey Wongb

a

Department of Chemistry, National Taiwan University, Taipei, 106, Taiwan.

b

The Genomics Research Center, Academia Sinica, Taipei, 115, Taiwan.

c

National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei, 112, Taiwan.

Abstract Peramivir is a potent neuraminidase (NA) inhibitor for treatment of influenza infection by intravenous administration. By replacing the carboxylate group in peramivir with a phosphonate group, phosphono-peramivir (6a), the dehydration and deoxy derivatives (7a and 8a) as well as their corresponding monoalkyl esters are prepared from a pivotal intermediate epoxide 12. Among these phosphonate compounds, the dehydration derivative 7a that has a relatively rigid cyclopentene core structure exhibits the strongest inhibitory activity (IC50 = 0.34.1 nM) against several NAs of wild-type human and avian influenza viruses (H1N1, H3N2, H5N1 and H7N9), though the phosphonate congener 6a is unexpectedly less active than peramivir. The inferior binding affinity of 6a is attributable to the deviated orientations 1

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of its phosphonic acid and 3-pentyl groups in the NA active site as inferred from the NMR, X-ray diffraction and molecular modeling analyses. Compound 7a is active to the oseltamivir-resistant H275Y strains of H1N1 and H5N1 viruses (IC50 = 73–86 nM). The phosphonate monoalkyl esters (6b, 6c, 7b, 7c, 8b and 8c) are better anti-influenza agents (EC50 = 19–89 nM) than their corresponding phosphonic acids (EC50 = 50–343 nM) in protection of cells from the viral infection. The phosphonate monoalkyl esters are stable in buffer solutions (pH 2.0–7.4) and rabbit serum; furthermore, the alkyl group is possibly tuned to attain the desired pharmacokinetic properties.

Keywords: Influenza; Neuraminidase inhibitor; Peramivir; Bioisosteres; Phosphonate.

2


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Table of Contents (Graphic)

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INTRODUCTION Influenza continues to affect human health through seasonal and pandemic infections. Influenza viruses that belong to Orthomyxoviridae family constantly undergo gene mutations. The genome of influenza virus comprises eight single-stranded negative-sense RNA segments to produce at least 11 proteins.1, 2 Hemagglutinin (HA) and neuraminidase (NA) are two important glycoproteins which are situated on the surface of influenza virus particle. HA is responsible for the virus to bind with the sialo-receptor of host cell, and to promote the fusion of viral envelope with the cell endosomal member.3 When progeny viruses are produced, their connection with the host cell is cut off by the NA-catalyzed hydrolysis of the sialic acid residue on the cellular receptor. Inhibition of the viral NA will prevent releasing the viral particles, and thus suppress the viral infection to the neighboring cells.4 There are 11 subtypes of influenza NAs; however, the structures of their active sites are rather conserved. Thus, an effective NA inhibitor may act as a universal anti-influenza drug to all subtypes. Taking the advantage of its location on the surface of virus for easy access to drugs and the conserved structure of the active site for design of universal inhibitors,5, 6 several NA inhibitors have been successfully developed to become anti-influenza drugs for clinical use. Figure 1 shows the chemical structures of zanamivir (1), oseltamivir carboxylate (3a) and peramivir (5) that are potent inhibitors against influenza virus NAs. Zanamivir is administered by oral inhalation due to its poor bioavailability.7 Oseltamivir (3b) is an ester prodrug that is 4


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converted to the active carboxylate 3a by endogenous esterase.8,

9

Peramivir is an

intravenously administered anti-influenza drug.10, 11 Unlike zanamivir and oseltamivir that have the scaffolds of (oxa)cyclohexene rings, peramivir has a relatively flexible cyclopentane ring as the core structure. In drug design, phosphonate group is often used as a bioisostere of carboxylate. Phosphonic acid is more acidic than carboxylic acid to render a thermodynamically favored ion-pair with the basic guanidine group.12 We have previously synthesized zanaphosphor (2)13 and tamiphosphor (4)14 as the phosphonate congeners of 1 and 3a. By replacing the carboxylate group in 1 and 3a with a phosphonate group, 2 and 4 show more potent NA inhibitory activity and better protect host cells from influenza viral infection. These results are consistent with the molecular docking experiments, which indicate that the phosphonate ion in 2 and 4 exhibits more extensive electrostatic interactions with the three arginine residues (R118, R292 and R371) in the active site of NA. Our docking experiments also show that the phosphonic acid of 2 and 4 in tetrahedral configuration, compared with carboxylic acid in a planar structure, is complementary to the array of the three arginine residues in influenza NA. Along this line of research, we further synthesized a series of peramivir analogs, including phosphono-peramivir (6a), the dehydration compound (7a) and the deoxy compound (8a). The phosphonate monoalkyl esters 6b, 6c, 7b, 7c, 8b and 8c were also synthesized to evaluate their anti-influenza activities. 5



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Figure 1. Chemical structures of influenza neuraminidase inhibitors.

RESULTS AND DISCUSSION Chemical synthesis. Figure 2 shows our retrosynthetic analysis of phosphono-peramivir (6a). The guanidine moiety in 6a could be derived from the corresponding amino group, and the phosphonic acid could be obtained by solvolysis of the phosphonate diester. The phosphonate group in A would be introduced by a ring opening reaction of epoxide B with diethyl phosphite. Epoxide B would be constructed by an intramolecular SN2 reaction of β-iodo alcohol C, which could be obtained from decarboxylative iodination of carboxylic acid D. A (3+2) dipolar cycloaddition between alkene F and nitrile oxide G would be performed to give the oxazolidine adduct E, and the N–O bond would be subsequently cleaved to afford the highly functionalized cyclopentane D. 6


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Figure 2. Retrosynthetic analysis of phosphono-peramivir 6a.

Scheme 1 shows the syntheses of 6a and its monoalkyl esters. According to the previously reported method,15,

16

an optically active cyclopentene F was reacted with

2-ethylbutanenitrile oxide (G) to give the (3+2) cycloaddition product. After reductive cleavage of the N–O bond,17 the exposed amino group was subjected to acetylation to afford the multiple substitutive cyclopentane 9 in the desired stereochemical disposition. The hydroxyl group in 9 was protected as a tert-butyldimethylsilyl (TBS) ether, and the ester group was saponified to give the corresponding acid 10b. Treatment of 10b with iodobenzene diacetate and iodine under irradiation condition renders an iododecarboxylation to give the iodo product 11.18 The structure of 11 was confirmed by an X-ray diffraction analysis, which 7



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supports the iododecarboxylation occurred in a stereoselective manner with retention of the S-configuration. The TBS protecting group was removed by tetrabutylammonium fluoride, and the intermediate β-iodo alcohol readily proceeded with an intramolecular SN2 reaction to afford the epoxide 12, that is the same with the structure B delineated in Figure 2. The stereochemistry of epoxide 12 was unambiguously determined by an X-ray diffraction analysis (Figure 3).

Scheme 1. Syntheses of phosphono-peramivir 6a and its monoalkyl esters. Reagents and reaction conditions: (a) TBSCl, imidazole, DMF, rt, 16 h; 85%. (b) NaOH, THF/EtOH (1:1), rt, 3 h. (c) PhI(OAc)2, I2, CCl4, hν, 8.5 h; 45% from 10a. (d) TBAF, THF, rt, 2 h; 78%. (e) (EtO)2PH(=O) or (H13C6O)2PH(=O), n-BuLi, Et2O:BF3, THF, –78 °C (1 h) to rt (14 h); 13, 8


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45%, 13-isomer, 21%; 14, 41%. (f) 2 M HCl in Et2O, rt, 18 h; then BocN=C(SMe)NHBoc, HgCl2, Et3N, DMF, rt, 17 h; 15, 73%; 16, 60%. (g) TMSBr, CH2Cl2, rt, 18 h; 6a, 60%. (h) TFA, CH2Cl2, rt, 16 h; then LiBr, DMF, 110 °C, 19 h; 6b, 38%; 6c, 26%. Boc = tert-butoxycarbonyl; TBS = tert-butyldimethylsilyl; TBAF = tetrabutylammonium fluoride; TMSBr = bromotrimethylsilane; TFA = trifluoroacetic acid.

Figure 3. ORTEP drawing of epoxide 12 and the structures of compounds 13 and 13-isomer determined by COSY (blue) and NOESY (red) correlations.

Attempts to prepare phosphonate compound 13 by nucleophilic reaction of epoxide 12 with diethyl phosphite in the presence of sodium ethoxide or sodium hydride failed, leaving the starting material unchanged. In contrast, complicated reactions occurred when epoxide 12 was treated with diethyl trimethylsilyl phosphite in the presence of TMSOTf (CH2Cl2, rt, 8 h) or ZnCl2 (THF, reflux, 18 h). Finally, the reaction of 12 with diethyl phosphite at low temperature (–78 oC) in the presence of boron trifluoride etherate provided the desired phosphonate diester 13 in 45% yield.19 The regioisomer (13-isomer) with transposition of the hydroxyl and phosphonate substituents was also obtained as a side product in 21% yield.19 9



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Compound 13 and its regioisomer were separated by silica gel chromatography, and their structures were characterized by NMR spectral analyses (Figure 3). The COSY of compound 13 showed the correlation of H-4 (at δ 2.23–2.14) with the two C-5 protons (at δ 2.462.35 and 1.691.54) as well as the correlation between H-2 (at δ 2.06) and H-3 (at δ 4.25). Thus, compound 13 has the phosphonate group at C-4 adjacent to C-5, and the hydroxyl group at C-3 adjacent to C-2. The H-2 and H-3 in compound 13 should orient on the same face of the cyclopentane ring because the nucleophilic attack of phosphite was expected to occur from the opposite face of the oxirane ring. This deduction is supported by the nuclear Overhauser effect (nOe) between H-2 and H-3 that displayed in the NOESY of compound 13. In the COSY of 13-isomer, the C-5 proton at δ 1.791.72 was correlated to the signal at δ 4.35 (H-4) adjacent to the hydroxyl group, and H-3 (at δ 2.182.09) adjacent to the phosphonate group was correlated to H-2 (at δ 2.282.19). The H-2 and H-3 of 13-isomer were disposed on opposite faces as no nOe was observed, in agreement with the nucleophilic attack of diethyl phosphite at the C-3 position from the opposite face of the oxirane ring. Interestingly, epoxide 12 reacted with dihexyl phosphite gave exclusively the desired phosphonate compound 14 (41% yield) without formation of its regioisomer, presumably the reaction at the more steric-demanding C-2 position of epoxide 12 was disfavored because the phosphite nucleophile had two relatively bulky hexyl substituents. The Boc protecting group in 13 was removed under acidic condition (2 M HCl in Et2O), 10


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and


the

intermediate

amine

(as

the

hydrochloride

salt)

was

treated

with

1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea in the presence of HgCl2 and Et3N to provide the bis-Boc-guanidine compound 15.15 By a similar procedure, compound 14 was converted to the gaunidination compound 16. Upon treatment with bromotrimethylsilane, 6a was obtained from 15 in 60% yield by concurrent removal of the Boc groups and solvolysis of the phosphonate diethyl ester. The structure of 6a was rigorously determined by spectroscopic methods ([α], IR, ESI–MS, 1H,

13

C and

31

P NMR) and X-ray crystallography. Alternatively,

15 (or 16) was first treated with TFA to remove the Boc groups, and then treated with lithium bromide to afford the phosphonate monoester 6b (or 6c). Because the crystal structure of 5–NA complex (PDB code: 1L7F)20 has revealed that the hydroxyl group of peramivir makes no direct interaction with influenza neuraminidase, we considered that elimination of the hydroxyl group might not cause any adverse effect in binding with NA. Thus, the dehydration and deoxy derivatives 7a–8c were prepared (Schemes 2 and 3). Mesylation of alcohol 13 (or 14), followed by elimination of MsOH in the presence of a base DBU, provided the cyclopentene phosphonate compound 19 (or 20). Alternatively, the hydroxyl group of 13 (or 14) was activated as a thiocarbamate, which was subsequently reacted with tributyltin hydride in the presence of a radical initiator AIBN to afford the deoxy derivative 25 (or 26).15 By the procedure similar to that delineated in Scheme 1, the bis-Boc protected guanidine compounds 21, 22, 27 and 28 were prepared from the 11



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corresponding Boc-amino compounds 19, 20, 25 and 26. Compounds 21 and 27 were then treated with TMSBr to afford phosphonic acids 7a and 8a, respectively. Alternatively, the bis-Boc guanidine compounds 21, 22, 27 and 28 were first treated with TFA to remove the Boc groups, and then treated with LiBr for partial solvolysis of the dialkyl phosphonate to yield the corresponding phosphonate monoesters 7b, 7c, 8b and 8c.

Scheme 2. Syntheses of dehydration compound 7a and its monoalkyl esters. Reagents and reaction conditions: (a) MsCl, Et3N, DMAP, CH2Cl2, rt, 11 h; 17, 59%; 18, 57%. (b) DBU, THF, rt, 18 h; 19, 87%; 20, 84%. (c) 2 M HCl in Et2O, rt, 16–18 h; then BocN=C(SMe)NHBoc, HgCl2, Et3N, DMF, rt, 17 h; 21, 92%; 22, 55%. (d) TMSBr, CH2Cl2, rt, 16 h; 7a, 98%. (e) TFA, CH2Cl2, rt, 18 h; then LiBr, DMF, 110 °C, 19 h; 7b, 81%; 7c, 61%. DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene; DMAP = 4-dimethylaminopyridine; Im = imidazole; MsCl = methanesulfonyl chloride.

12


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Scheme 3. Syntheses of deoxy compound 8a and its monoalkyl esters. Reagents and reaction conditions: (a) Im2C=S, THF, reflux, 7 h; 23, 87%; 24, 54%. (b) Bu3SnH, AIBN, toluene, reflux, 20 min; 25, 71%; 26, 69%. (c) 2 M HCl in Et2O, rt, 16–18 h; then BocN=C(SMe)NHBoc, HgCl2, Et3N, DMF, rt, 17 h; 27, 77%; 28, 70%. (d) TMSBr, CH2Cl2, rt, 18 h; 8a, 63%. (e) TFA, CH2Cl2, rt, 20 h; then LiBr, DMF, 110 °C, 19 h; 8b, 73%; 8c, 63%. AIBN = 2,2′-azobis(2-methylpropionitrile); Im = imidazole.

Enzymatic and cell-based assays. The enzymatic assays for NA inhibition were measured using 2-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) as the substrate (Table 1). All peramivir phosphonate derivatives (6a–8c) possess potent inhibitory activity with IC50 values in low nanomolar to sub-nanomolar range against the influenza NA of A/WSN/33 H1N1 virus. The anti-influenza activities were measured by the cytopathic effect of Madin–Darby canine kidney (MDCK) cells due to the infection of influenza viruses. Compounds 6a–8c also exhibit good activity against the wild-type H1N1 virus with EC50 13



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values in sub-micromolar range. With regard to cytotoxicity, these potent anti-influenza agents were nontoxic to MDCK cells at the highest testing concentrations (>100 μM).

Table 1. Neuraminidase inhibition, anti-influenza activity, cytotoxicity and lipophilicity of peramivir phosphonate derivatives. Compound

IC50a (nM)

EC50b (nM)

CC50c (nM)

clog Pd

clog Dd

1

0.6 ± 0.1

16.3 ± 1.6

> 105

4.13

5.77

3a

0.7 ± 0.1

5.5 ± 0.4

> 105

0.43

1.84

5

0.07 ± 0.005

1.5 ± 0.7

> 105

0.48

2.12

6a

5.2 ± 1.6

260.3 ± 117.5

> 105

1.32

1.78

6b

0.9 ± 0.1

66.5 ± 25.2

> 105

0.42

0.81

6c

0.5 ± 0.1

18.6 ± 10.6

> 105

1.33

0.94

7a

0.3 ± 0.1

50.3 ± 12.7

> 105

0.88

1.31

7b

0.6 ± 0.3

19.1 ± 6.7

> 105

0.02

0.37

7c

1.5 ± 0.3

18.5 ± 6.9

> 105

1.77

1.38

8a

2.9 ± 1.9

343.2 ± 75.1

> 105

0.41

0.86

8b

1.0 ± 0.3

88.7 ± 12.3

> 105

0.49

0.10

8c

1.6 ± 0.5

43.2 ± 14.1

> 105

2.24

1.85

14


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a


A fluorescent substrate, 2-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA), was used to determine the IC50 value which indicates compound concentration causing 50% inhibition of influenza neuraminidase (A/WSN/33 human H1N1 virus). Data are shown as mean ± SD of three experiments.

b

The anti-influenza activities against A/WSN/33 human H1N1 virus were measured as EC50 value which indicates the compound concentration for 50% protection of the cytopathic effect due to the infection by influenza virus. Data are shown as mean ± SD of three experiments.

c

The 50% cytotoxic concentration on Madin–Darby canine kidney cells. The highest testing concentration was 100 μM.

d

The calculated partition coefficient (clog P) and distribution coefficient (clog D) were obtained using MarvinSketch accessed on 15 March 2016 (https://www.reaxys.com/reaxys/js/sre_5_3_1_05/child_java.jsp).

Though 6a is a good NA inhibitor, its inhibitory activity against human H1N1 virus is unexpectedly 74 fold inferior to peramivir, a result in contrast to the previous computational study21 that predicts 6a to be a stronger binder with N1 neuraminidase. Due to the flexible cyclopentane core structure, the phosphonate compound 6a may display the conformations different from that of carboxylate congener 5. Thus, the NA inhibitory activities of this series 15



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compounds are less predictable. To give insights into the conformations of 6a in solid state and aqueous solution, we meticulously inspected its X-ray crystal structure and 1H NMR spectrum in D2O solution. According to the graph of Karplus equation,22 the torsional angles in structure 6a were deduced from the coupling constants of specific protons in the NMR spectrum. For example, the coupling constant of 5.0 Hz between H-2 and H-3 was related to a torsional angle (φH2,3) of 51o, whereas the coupling constant of 7.0 Hz between H-1 and H-5α was assigned to a torsional angle (φH1,5) of 133o. By comparing the torsional angles deduced from X-ray and NMR (Table 2), compound 6a in solution and crystal states obviously exists as distinct conformations.

Table 2. Comparison of torsional angles of phosphono-peramivir in solution (by NMR), solid state (by X-ray) and molecular modeling.

solutiona

solid state

molecular modeling

H1–C–C–H5α

133o

150o

150o

H1–C–C–H5β

3336o

29o

28o

H1–C–C–H2

90o

124o

124o

16


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a


H2–C–C–H3

51o

33o

33o

H3–C–C–H4

139147o

173o

173o

H4–C–C–H5α

133139o

165o

166o

H4–C–C–H5β

3336o

44o

44o

H3–C–C–H7

145147o

175o

175o

The 1H NMR spectrum was measured in D2O solution, and the torsional angles are determined by the graph of Karplus equation.

Wulff et al. have previously found that peramivir (5) has different conformational preferences in the solid state, solution phase and NA-bound complex.23 To understand the binding modes in the active site of NA, we carried out the molecular docking experiments of 5 and the phosphonate congener 6a. Analysis of the molecular docking conformations of 5 in the NA active site (N1 subtype, PDB code: 2HU4)6 reveals that there is only little difference between modeling result and 5–NA cocrystal (Figures S1A and S1B in Supporting Information (SI)). A flexible molecular docking of 6a was performed based on its X-ray diffraction data. The molecular docking conformation of 6a in the NA active site (Figure S1C) is almost the same as that in solid state (Figure S1D) with RMSD = 0.01Å . Our molecular docking experiments also indicate that 6a provides two less hydrogen bondings (Figure 4B) than 5 (Figure 4A) with the key arginine residues (R292 and R371) in the S1 site of influenza 17



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NA. This result is in sharp contrast to the phosphonate congeners 2 and 4 that attain more extensive hydrogen bondings than zanamivir and oseltamivir carboxylate in the NA active site.13, 14 The conformations of cyclopentane ring in the NA-bound 5 and 6a, based on the molecular modeling (Figure S2 in SI), are similar to that of 5–NA complex. However, the carboxylate group of 5 can interact with the three arginine residues (R118, R292 and R371) in the S1 active site of influenza NA more firmly than the phosphonic acid of 6a. This discrepancy is attributable to the different orientations of the carboxylic acid in planar shape and the phosphonic acid in tetrahedral configuration. Furthermore, the molecular modeling also reveals that the 3-pentyl group of 6a deviates from that of 5 to exhibit less hydrophobic interactions with the side chains of I222 and R224 in the active site of influenza NA. By forming a more rigid conformation of cyclopentene ring, the dehydration compound 7a regains extensive electrostatic interactions with the key arginine residues in NA (Figure 4C), and thus displays high NA inhibitory activity (IC50 = 0.3 nM) against influenza H1N1 virus. The phosphonate monoalkyl esters 7b and 7c are also potent NA inhibitors with IC50 values of 0.6 and 1.5 nM, respectively. We further selected compounds 6a, 7a and 7b to evaluate their binding affinity to the NAs of various human and avian influenza viruses. These peramivir phosphonate compounds also showed high inhibitory activities (IC50 = 0.3–7.3 nM) against H3N2, H5N1 and H7N9 18


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viruses. The binding affinity of 6a and 7b to the H275Y and R292K mutant viruses decreased appreciably (IC50 = 177–7481 nM) presumably due to less hydrophobic interactions attained by the 3-pentyl group in the active sites of the mutant NAs.20,

24

However, the 7a still

displayed strong inhibition against the H275Y strains of H1N1 and H5N1 viruses with IC50 83000

> 83000

225 ± 30

> 83000

> 83000

> 83000

H3N2 wt

H3N2 R292K 45 ± 24 4371 ± 2650 146 ± 109 2831 ± 2292 2131 ± 1353 7481 ± 3341 H5N1 wt

0.7 ± 0.5

H5N1 H275Y 1.7 ± 1.1 H7N9 wt

6.0 ± 4.7

0.9 ± 0.4

0.3 ± 0.2

0.9 ± 0.1

0.3 ± 0.1

0.9 ± 0.5

26 ± 9

3.3 ± 0.6

705 ± 74

73 ± 2

177 ± 94

1.0 ± 0.5

0.8 ± 0.4

3.1 ± 0.1

4.1 ± 0.5

7.3 ± 3.5 19



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a

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Influenza recombinant neuraminidases were prepared from HEK293 cells, and the purified enzymes

were

used

for

the

studies.

A

fluorescent

substrate,

2-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA), was used to determine the IC50 value which indicates compound concentration causing 50% inhibition of influenza neuraminidases. Data are shown as mean ± SD of three experiments. b

The strains of wild-type (wt) and mutant influenza viruses: H1N1, A/WSN/1933; H3N2, A/Brisbane/10/2007; H5N1, A/Vietnam/1194/2004; and H7N9, A/Shanghai/01/2014.

In the cell-based assays, all the phosphonate monoalkyl esters (6b/6c, 7b/7c and 8b/8c) exhibit superior anti-influenza activities to their parental phosphonic acids (6a, 7a and 8a). Consistent with our previous reports,25,

26

the single-charged phosphonate monoester still

retains the necessary electrostatic interactions with the three arginine residues in NA, whereas the monoalkyl ester may increase lipophilicity to enhance intracellular uptake as inferred from the calculated partition and distribution coefficients (Table 1). Our molecular docking experiment further demonstrates that the ethyl substituent of 6b is located in the region of 430-loop to attain additional hydrophobic interaction with I427 residue (Figure 4D). This result is in agreement with the prediction by ensemble-based virtual screening, which indicates that the 430-cavity of NA favors to accept hydrophobic substituents.27

20


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(A)

(B)

(C)

(D)

Figure 4. Molecular modeling of 5 (A), 6a (B), 7a (C) and 6b (D) in the active site of influenza viral neuraminidase (N1 subtype, PDB code: 2HU4).6 The complex of 6a shows 4 hydrogen bonds with the key residues (R118, R292, R371 and Y347) in the neuraminidase active site, two hydrogen bonds less compared with 5. The phosphonic acid moiety of 7a exhibits 5 hydrogen bonding interactions with the key residues (R118, R292, R371 and Y347) in the NA active site. The ethyl substituent in 6b extend to the 430-loop to gain hydrophobic interaction.

So far, we do not have detailed pharmacokinetics studies for the peramivir phosphonate derivatives 6a–8c while their ADMET properties are predicted by Discovery Studio 3.5 program (Table S1 in SI). Among these compounds, phosphonate monoethyl ester 7b seems to be water soluble with modest intestinal absorption. The pharmacological characteristics of 7b, including the molecular mass (374 dalton), clog P (0.02), hydrogen bond donors (5) and 21



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Page 22 of 71

hydrogen bond acceptors (6), seem to fit Lipinski’s rule of five.28 The calculated distribution coefficient of 7b (clog D = –0.37) is comparable to that of the oral drug oseltamivir (clog D = –0.72). Compound 7b was stable in PBS (pH 7.4), acetate buffer (pH 4.0), HCl buffer (pH 2.0) and rabbit serum. According to the HPLC and ESI–HRMS analyses, no chemical degradation or bioconversion of phosphonate monoester 7b to its parental phosphonic acid 7a occurred on incubation at 37 oC for 96 h (Figure S3–S6 in SI). Thus, the phosphonate monoester 7b is an anti-influenza drug, rather than a prodrug of 7a.25 As a potent anti-influenza agent (IC50 = 0.6 nM and EC50 = 19 nM), 7b may be a candidate for further developed.

CONCLUSION Peramivir is a potent neuraminidase inhibitor approved by FDA to treat influenza infection. Although peramivir phosphonate derivatives have been mentioned in a patent,29 no detailed synthetic procedures or biological activities are available. In this study, we first developed an efficient synthetic method, involving Barton–Crich iododecarboxylation to give the iodo compound 11 and a ring-opening reaction of the key intermediate epoxide 12 by dialkyl phosphite, for preparation of the peramivir phosphonate derivatives 6a–8c,. Among these peramivir bioisosteres, the dehydration compound 7a that bears a relatively rigid cyclopentene ring shows the most potent NA inhibitory activity (IC50 = 0.3–4.1 nM) against several NAs of wild-type human and avian influenza viruses (H1N1, H3N2, H5N1 and 22


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H7N9). In comparison, 6a that has a more flexible cyclopentane ring shows somewhat less affinity to the viral NAs (IC50 = 0.9–14 nM). From the NMR, X-ray diffraction and molecular modeling analyses, the decreased binding of 6a is possibly because its phosphonic acid and 3-pentyl groups deviate from the perfect match orientations in NA active site. Compound 7a also possesses high inhibitory activities (IC50 = 73–86 nM) against the oseltamivir-resistant H275Y strains of H1N1 and H5N1 viruses. It is noted that the monoalkyl esters 7b and 7c exhibit excellent anti-influenza activity (EC50 = 19 nM) to protect MDCK cells from the infection caused by human H1N1 virus. A phosphonate monoalkyl ester still possesses a negative charge to provide the necessary electrostatic interactions with the three arginine residues (R118, R292 and R371) in the NA active site, and the alkyl substituent may provide additional hydrophobic interaction in the 430-loop near the S1-site of NA. The alkyl substituent may also improve lipophilicity for better cellular uptake. In a similar trend, the monoalkyl phosphonate esters 6b, 6c, 8b and 8c all exhibit better anti-influenza activities than their corresponding phosphonic acids 6a and 8a. The phosphonate monoesters are considered anti-influenza agents, instead of prodrugs, as no chemical degradation or bioconversion of these monoesters (e.g. 7b) to its parental phosphonic acid (e.g. 7a) is found by incubation in several buffer solutions (pH 2.0, 4.0 and 7.4) and rabbit serum at 37 oC for 96 h. In comparison with peramivir that is administered by intravenous injection, the phosphonate monoalkyl esters such as 7b and 7c would have better chance for development as orally 23



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Page 24 of 71

available anti-influenza drugs.

EXPERIMENTAL SECTION General. All the reagents were commercially available and used without further purification unless indicated otherwise. All solvents were anhydrous grade unless indicated otherwise. Reactions were magnetically stirred and monitored by thin-layer chromatography. Silica gel (40–63 μm particle size) and LiChroprep RP-18 (40–63 μm particle size) were used for flash chromatography. Yields were reported for spectroscopically pure compounds. Melting points were recorded on a Yanaco melting point apparatus. 1H,

13

C and

31

P NMR

spectra were recorded on Varian Unity Plus-400 (400 MHz) or Bruker AVIII FT-NMR (500 MHz) spectrometers. Chemical shifts were given in δ values relative to tetramethylsilane (TMS); coupling constants J were given in hertz (Hz). Internal standards were CDCl3 (δH = 7.24), CD3OD (δH = 3.31) or D2O (δH = 4.81) for 1H NMR spectra; and CDCl3 (δC = 77.0) or CD3OD (δC = 49.15) were for

13

C NMR spectra. The splitting patterns are reported as s

(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad), dd (double of doublets), ddd (double of doublets of doublets) and dddd (double of doublets of doublets of doublets). Infrared (IR) spectra were recorded on Varian 640-IR FT–IR spectrometer. Optical rotations were recorded on digital polarimeter of Japan JASCO Co. DIP-1000; [α]D values were given in units of 10–1 deg cm2 g–1. Electrospray ionization (ESI) mass spectra were recorded on a 24


Page 25 of 71

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high-resolution mass spectrometer.

Material. Influenza A/WSN/1933 (H1N1) was obtained from Dr. Shin-Ru Shih at Chang Gung University in Taiwan. All viruses were cultured in the allantoic cavities of 10-day-old embryonated chicken eggs for 72 h, and purified by sucrose gradient centrifugation. Madin– Darby canine kidney (MDCK) cells were obtained from American Type Culture Collection (Manassas, VA), and were grown in DMEM (Dulbecco’s modified Eagle medium, GibcoBRL) containing 10% fetal bovine serum (GibcoBRL) and penicillin–streptomycin (GibcoBRL) at 37 oC under 5% CO2.

Computer modeling. The model of a specific compound in complex with the NA was constructed through docking this compound to the crystallographic structure of N1 neuraminidase (PDB code: 2HU4).6 By modifying the 3D structure of peramivir (5, from PDB code: 2HU4) with SYBYL 8.0 program (Tripos Associates, St. Louis, MO), the 3D structure of compound (6a, 6b and 7a) was built up. GOLD 4.0.1 was used to dock the compound onto the protein with flexible docking option turned on.30, 31 Kollmann-all atom charges32 were assigned to the protein atoms, and Gasteiger–Hückel charges33 were assigned to ligand atoms using the SYBYL 8.0 program. Initial 1000 independent genetic algorithm cycles of computation were carried out with ligand torsion angles varying between 180 and 25



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Page 26 of 71

180o. The search efficiency was set at 200% to ensure the most exhaustive search for the docking conformational space. All other parameters were kept the same as the default settings. The docking processes were distributed to a 40-processor Linux cluster with Intel(R) Xeon(TM) CPU 3.00 GHz CPUs. The resultant ligandprotein complex structures were ranked with the GOLDSCORE scoring function to determine the top 1000 hits.

Determination of influenza virus TCID50. The TCID50 (50% tissue culture infectious dose) was determined by serial dilution of the influenza virus stock solution onto 100 μL MDCK cells at 1 × 105 cells/mL in 96-well microplates. The infected cells were incubated at 37 oC under 5.0% CO2 for 48 h and added to each well with 100 μL of CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay reagent (Promega). After the incubation at 37 oC for 15 min, absorbance at 490 nm was read on a plate reader. Influenza virus TCID50 was determined using Reed–Müench method.34, 35

Cloning, Expression, and Purification of Influenza neuraminidases (NAs). The NA genes from the influenza viruses A/WSN/1933 (H1N1), A/Brisbane/10/2007 (H3N2), A/Vietnam/1194/2004(H5N1), and A/Shanghai/01/2014(H7N9) were

cloned into a

mammalian expression vector as described by Schmidt PM.36 The drug-resistant mutants were created by using site-directed mutagenesis (Stratagene). The plasmid that encodes the soluble 26


Page 27 of 71

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NA, wild-type or the mutants, were transfected into the human embryonic kidney cell line HEK293 by using polyethyleneimine. The transfected cells were cultured in DMEM (Invitrogen) containing 10% fetal bovine serum. After 48 h, the supernatant was collected and cleared by centrifugation. NA proteins were purified by metal affinity chromatography using Ni-nitrilotriacetic acid (NTA) resin (GE Healthcare).

Determination of neuraminidase activity by a fluorescent assay. The NA activity was measured by using diluted allantoic fluid harvested from influenza virus infected embryonated eggs or by recombinant neuraminidase proteins. A fluorometric assay was used to

determine

the

NA

activity

2-(4-methylumbelliferyl)-α-D-N-acetylneuraminic

with acid

the

fluorogenic

(MUNANA;

substrate

Sigma).

The

fluorescence of the released 4-methylumbelliferone was measured in Envision plate reader (Perkin-Elmer, Wellesley, MA) by using excitation and emission wavelength of 365 and 460 nm, respectively. Neuraminidase activity was determined at 200 μM of MUNANA (for diluted allantoic fluid) or at 5 µM of MUNANA (for recombinant influenza neuraminidases). Enzyme activity was expressed as the fluorescence increase during 15 min incubation at room temperature.

Determination of IC50 of neuraminidase inhibitors. Neuraminidase inhibition was 27



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Page 28 of 71

determined by mixing inhibitor (0.02–83000 nM) and NA for 10 min at room temperature followed by the addition of substrate. The IC50 values were determined from the dose– response curves by plotting the percent inhibition of NA activity versus inhibitor concentrations using Graph Pad Prism 4.

Determination of EC50 and CC50 of neuraminidase inhibitors. The anti-influenza activities of neuraminidase inhibitors were measured by the EC50 values, which were the concentrations of NA inhibitor for 50% protection of the influenza virus infection-mediated cytopathic effects (CPE). Fifty microliters of diluted influenza virus at 100 TCID50 was mixed with equal volumes of NA inhibitors at varied concentrations. The mixtures were used to infect 100 μL of MDCK cells at 1 × 105 cells/mL in 96-wells. After 48 h of incubation at 37 o

C under 5.0% CO2, the cytopathic effects were determined with CellTiter 96 AQueous

Non-Radioactive Cell Proliferation Assay reagent as described above. The EC50 values were determined by fitting the curve of percent CPE versus the concentrations of NA inhibitor using Graph Pad Prism 4. The CC50 values (50% cytotoxic concentrations) of NA inhibitor to MDCK cells were determined by the procedures similar to the EC50 determination but without virus infection.

Chemical stability studies. Stock solution (20 mM) of phosphonate monoethyl ester 7b 28


Page 29 of 71

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in DMSO was prepared, and diluted to 1 mM with an appropriate buffer such as phosphate (pH 7.4), acetate (pH 4.0) and HCl (pH 2.0) buffered solutions. The sample solution was shaken at 1000 rpm at 37 oC. At different time intervals (24, 48, 72 and 96 h), 30 μL of reaction mixture was taken to HPLC analysis on an HC-C18 column (Merck Chromolith® , 100 × 4.6 mm i.d., 2 µm particle size) using mixed solvents A/B for elution, whereas solvent A was MeOH and solvent B was H2O containing 0.1% TFA, at a flow rate of 1 mL/min with detection at 214 nm wavelength. The reaction mixture was also analyzed by ESI–HRMS. The extent for hydrolysis of sample 7b to phosphonic acid 7a was evaluated.

Stability test in rabbit serum. Stock solution (20 mM) of 7b in DMSO was prepared, and diluted to 1 mM with rabbit serum. The sample mixture was shaken at 1000 rpm at 37 oC. At different time intervals (24, 48, 72 and 96 h), 0.25 mL of sample mixture was extracted with MeOH (0.5 mL) by vortex mixing at 4 oC for 2 h, and then subjected to centrifugation at 10,000 rpm for 20 min. The supernatant (0.5 mL) was evaporated under reduced pressure. The residue was redissolved in distilled deionized water (0.25 mL), and analyzed by HPLC and ESI–HRMS under the above-described conditions to evaluate the extent for hydrolysis of 7b to 7a.

Compound Characterization. New compounds were characterized by their physical and 29



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spectroscopic properties (mp, TLC, [α], IR, ESI−MS, 1H,

Page 30 of 71

13

C and

31

P NMR). Purity of

synthetic compounds was assessed to be ≥95% by HPLC analysis with detection at 214 or 254 nm wavelengths.

(1S,2S,3R,4R)-[3-((S)-1-Acetamido-2-ethylbutyl)-4-guanidino-2-hydroxycyclopentyl] phosphonic acid (6a). Bromotrimethylsilane (1.02 mL, 7.71 mmol) was added to a solution of bis-Boc-guanidine 15 (114 mg, 0.18 mmol) in CH2Cl2 (2 mL) at 0 oC. The mixture was stirred at room temperature for 18 h, and concentrated under reduced pressure. The residue was purified by reversed-phase RP-18 column (MeOH/H2O = 3:7) to afford the phosphonic acid 6a (59 mg, 60% yield). The purity of product 6a was 96.8% as shown by HPLC on an HC-C18 column (Agilent, 4.6 × 250 mm, 5 µm porosity), tR = 5.5 min [MeOH/(0.5% TFA aqueous solution) = 2:3] at a flow rate of 0.5 mL/min. C14H29N4O5P; white solid, mp 275 oC (decomposed); [α] D25 52.9 (c = 1.0, H2O); IR max (neat) 3375, 2966, 1634, 1544, 1156, 1070, 1005 cm1; 1H NMR (500 MHz, D2O) δ 4.45 (1 H, dd, J = 10.0, 5.0 Hz, H-2), 4.45 (1 H, d, J = 10.0 Hz, H-7), 3.95 (1 H, q, J = 8.5 Hz, H-4), 2.67 (1 H, dddd, J = 14.0, 14.0, 9.0, 9.0, H-5β), 2.35 (1 H, ddd, J = 10.5, 10.5, 5.0 Hz, H-3), 2.19 (1 H, ddd, J = 16.5, 8.5, 7.0 Hz, H-1), 2.03 (3 H, s, Ac), 1.71 (1 H, dddd, J = 21.0, 14.0, 7.0, 7.0 Hz, H-5α), 1.621.51 (3 H, m), 1.141.04 (2 H, m), 1.01 (3 H, t, J = 7.0 Hz, Me), 0.95 (3 H, t, J = 7.0 Hz, Me); 13C NMR (100 MHz, D2O) δ 173.9, 155.8, 71.5 (d, 2JC-P = 3.0 Hz), 55.5 (d, 3JC-P = 7.6 Hz), 50.4, 49.9, 30


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44.8 (d, 1JC-P = 131.2 Hz), 43.4, 32.5 (d, 2JC-P = 2.3 Hz), 22.8, 22.1, 21.1, 12.3, 11.4; 31P NMR (162 MHz, D2O) δ 23.8; ESI–HRMS calcd for C14H30N4O5P: 365.1954, found: m/z 365.1958 [M + H]+. Ethyl

hydrogen

(1S,2S,3R,4R)-[3-((S)-1-acetamido-2-ethylbutyl)-4-guanidino-2-hydroxycyclopentyl]phos phonate (6b). To a solution of bis-Boc-guanidine 15 (62 mg, 0.10 mmol) in CH2Cl2 (3 mL) was added TFA (0.15 mL, 1.95 mmol) at 0 oC. The mixture was stirred at room temperature for 16 h, and concentrated under reduced pressure to give the corresponding guanidine (as the TFA salt). Lithium bromide (29 mg, 0.33 mmol) was added to the solution of the above-prepared guanidinium salt (35 mg, 0.07 mmol) in DMF (1 mL). The mixture was stirred at 110 oC for 19 h, and concentrated under reduced pressure. The residue was purified by reversed-phase RP-18 column (MeOH/H2O = 2:3) to afford the phosphonate monoethyl ester 6b (15 mg, 38% yield from 15). The purity of product 6b was 97.0% as shown by HPLC on an HC-C18 column (Agilent, 4.6 × 250 mm, 5 µm porosity), tR = 12.2 min [MeOH/(0.5% TFA aqueous solution) = 3:7] at a flow rate of 0.5 mL/min. C16H33N4O5P; white solid, mp 293–295 oC; [α] D25 105.3 (c = 1.0, MeOH); IR max (neat) 3318, 2966, 2362, 1646, 1540, 1168, 1050 cm1; 1H NMR (400 MHz, CD3OD) δ 4.51 (1 H, d, J = 9.2 Hz), 4.33 (1 H, dd, J = 8.8, 4.8 Hz), 4.003.93 (2 H, m), 3.88 (1 H, q, J = 8.0 Hz), 2.702.58 (1 H, m), 2.47 (1 H, ddd, J = 10.4, 10.4, 4.8 Hz), 2.13 (1 H, ddd, J = 18.4, 9.6, 6.0 Hz), 1.98 (3 H, s), 1.691.55 (3 31



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Page 32 of 71

H, m), 1.501.41 (1 H, m), 1.28 (3 H, t, J = 7.2 Hz), 1.110.95 (8 H, m); 13C NMR (100 MHz, CD3OD) δ 173.1, 157.4, 74.1, 61.5, 57.2, 51.5, 50.8, 45.3 (d, 1JC-P = 130.7 Hz), 45.1, 34.3, 24.3, 23.3, 22.5, 17.5 (d, 3JC-P = 6.1 Hz), 13.4, 12.8;

31

P NMR (162 MHz, CD3OD) δ 25.3;

ESI–HRMS calcd for C16H34N4O5P: 393.2267, found: m/z 393.2262 [M + H]+. Hexyl

hydrogen

(1S,2S,3R,4R)-[3-((S)-1-acetamido-2-ethylbutyl)-4-guanidino-2-hydroxycyclopentyl]phos phonate (6c). To a solution of bis-Boc-guanidine 16 (37 mg, 0.05 mmol) in CH2Cl2 (2 mL) was added TFA (0.08 mL, 1.04 mmol) at 0 oC. The mixture was stirred at room temperature for 16 h, and concentrated under reduced pressure to give the corresponding guanidine (as the TFA salt). Lithium bromide (7 mg, 0.08 mmol) was added to the solution of the above-prepared guanidinium salt (11 mg, 0.017 mmol) in DMF (0.28 mL). The mixture was stirred at 110 oC for 19 h, and concentrated under reduced pressure. The residue was purified by reversed-phase RP–18 column (MeOH/H2O = 7:3) to afford the phosphonate monohexyl ester 6c (6 mg, 26% yield from 16). The purity of product 6c was 95.1% as shown by HPLC on an HC-C18 column (Merck, 4.6 × 100 mm, 2 µm porosity), tR = 4.7 min [MeOH/(0.1% TFA aqueous solution) = 6:4] at a flow rate of 1.0 mL/min. C20H41N4O5P; white solid, mp 279 C (decomposed); [α] D25 113.5 (c = 1.0, MeOH); IR max (neat) 3264, 2929, 1634, 1552,

o

1164, 1058 cm1; 1H NMR (400 MHz, CD3OD) δ 4.52 (1 H, dd, J = 11.2, 2.4 Hz), 4.34 (1 H, dd, J = 8.0, 4.4 Hz), 3.923.86 (3 H, m), 2.71 2.58 (1 H, m), 2.48 (1 H, ddd, J = 10.8, 10.8, 32


Page 33 of 71

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4.4 Hz), 2.14 (1 H, ddd, J = 18.4, 9.6, 4.8 Hz), 1.97 (3 H, s), 1.701.53 (5 H, m), 1.491.31 (7 H, m), 1.100.90 (11 H, m); 13C NMR (100 MHz, CD3OD) δ 173.4, 157.6, 74.3, 65.8, 57.3, 51.6, 51.2, 45.3 (d, 1JC-P = 131.3 Hz), 45.2, 34.2, 32.9, 32.4 (d, 3JC-P = 6.0 Hz), 26.8, 24.3, 23.9, 23.3, 22.5, 14.5, 13.4, 12.7; 31P NMR (162 MHz, CD3OD) δ 25.1; ESI–HRMS calcd for C20H42N4O5P: 449.2893, found: m/z 449.2849 [M + H]+. (3R,4R)-[3-((S)-1-Acetamido-2-ethylbutyl)-4-guanidinocyclopent-1-en-1-yl]phospho nic acid (7a). Bromotrimethylsilane (0.39 mL, 2.22 mmol) was added to a solution of bis-Boc-guanidine 21 (32 mg, 0.05 mmol) in CH2Cl2 (1 mL) at 0 oC. The mixture was stirred at room temperature for 16 h, and concentrated under reduced pressure. The residue was purified by reversed-phase RP-18 column (MeOH/H2O = 3:7) to afford the phosphonic acid 7a (18 mg, 98% yield). The purity of product 7a was 99.9% as shown by HPLC on an HC-C18 column (Agilent, 4.6 × 250 mm, 5 µm porosity), tR = 9.8 min [MeOH/(0.5% TFA aqueous solution= 3:7] at a flow rate of 0.5 mL/min. C14H27N4O4P; white solid, mp 268 oC (decomposed); [α] D25 137.6 (c = 1.0, H2O); IR max (neat) 3170, 2970, 2880, 1659, 1548, 1377, 1152, 1062 cm1; 1H NMR (400 MHz, D2O) δ 7.90 (1 H, d, J = 10.4 Hz), 6.17 (1 H, dd, J = 11.6, 2.0 Hz), 3.983.93 (2 H, m), 3.092.99 (2 H, m), 2.46 (1 H, dd, J = 16.8, 1.2 Hz), 2.00 (3 H, s), 1.571.42 (3 H, m), 1.221.13 (2 H, m), 0.920.85 (6 H, m); 13C NMR (100 MHz, D2O) δ 174.2, 156.0, 138.2 (d, 2JC-P = 11.4 Hz), 138.1 (d, 1JC-P = 178.9 Hz), 55.6 (d, 3

JC-P = 16.1 Hz), 55.2 (d, 2JC-P = 12.8 Hz), 53.4, 42.8, 40.2 (d, 2JC-P = 13.4 Hz), 22.2, 22.0, 33



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20.9, 11.1, 10.7;

31

Page 34 of 71

P NMR (162 MHz, D2O) δ 9.8; ESI–HRMS calcd for C14H28N4O4P:

347.1848, found: m/z 347.1845 [M + H]+. Ethyl

hydrogen

(3R,4R)-[3-((S)-1-acetamido-2-ethylbutyl)-4-guanidinocyclopent-1-en-1-yl]phosphonate (7b). To a solution of bis-Boc-guanidine 21 (100 mg, 0.17 mmol) in CH2Cl2 (4 mL) was added TFA (0.26 mL, 3.40 mmol) at 0 oC. The mixture was stirred at room temperature for 18 h, and concentrated under reduced pressure to give the corresponding guanidine (as the TFA salt). Lithium bromide (52 mg, 0.60 mmol) was added to the solution of the above-prepared guanidinium salt (60 mg, 0.12 mmol) in DMF (2 mL). The mixture was stirred at 110 oC for 19 h, and concentrated under reduced pressure. The residue was purified by reversed-phase RP-18 column (MeOH/H2O = 2:3) to afford the phosphonate monoethyl ester 7b (50 mg, 81% yield from 21). The purity of product 7b was 99.9% as shown by HPLC on an HC-C18 column (Agilent, 4.6 × 250 mm, 5 µm porosity), tR = 12.8 min [MeOH/(0.5% TFA aqueous solution ) = 3:7] at a flow rate of 0.5 mL/min. C16H31N4O4P; white solid, mp 273 oC (decomposed); [α] D25 132.3 (c = 1.0, MeOH); IR max (neat) 3264, 2970, 1654, 1561, 1189, 1042, 768 cm1; 1H NMR (400 MHz, CD3OD) δ 6.18 (1 H, dd, J = 10.8, 2.0 Hz), 4.02 (1 H, dd, J = 10.0, 3.6 Hz), 3.923.79 (3 H, m), 3.052.99 (2 H, m), 2.392.34 (1 H, m), 1.97 (3 H, s), 1.681.62 (1 H, m), 1.501.42 (1 H, m), 1.391.33 (1 H, m), 1.25 (3 H, t, J = 7.6 Hz), 1.201.10 (2 H, m), 0.98 (3 H, t, J = 7.6 Hz), 0.91 (3 H, t, J = 7.2 Hz); 13C NMR (100 MHz, 34


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CD3OD) δ 173.2, 157.7, 139.8 (d, 1JC-P = 176.4 Hz), 139.7 (d, 2JC-P = 10.6 Hz), 61.5, 57.2 (d, 3

JC-P = 12.2 Hz), 56.9 (d, 3JC-P = 15.2 Hz), 54.0, 45.5, 42.0 (d, 2JC-P = 12.2 Hz), 24.0, 22.9,

22.5, 17.3 (d, 3JC-P = 6.9 Hz), 12.8, 12.5;

31

P NMR (162 MHz, CD3OD) δ 10.1; ESI-HRMS

calcd for C16H32N4O4P: 375.2161, found: m/z 375.2164 [M + H]+. Hexyl

hydrogen

(3R,4R)-[3-((S)-1-acetamido-2-ethylbutyl)-4-guanidino

cyclopent-1-en-1-yl]phosphonate (7c). To a solution of bis-Boc-guanidine 22 (30 mg, 0.04 mmol) in CH2Cl2 (1 mL) was added TFA (0.06 mL, 0.78 mmol) at 0 oC. The mixture was stirred at room temperature for 18 h, and concentrated under reduced pressure to give the corresponding guanidine (as the TFA salt). Lithium bromide (14 mg, 0.16 mmol) was added to the solution of the above-prepared guanidinium salt (22 mg, 0.04 mmol) in DMF (0.59 mL). The mixture was stirred at 110 oC for 19 h, and concentrated under reduced pressure. The residue was purified by reversed-phase RP-18 column (MeOH/H2O = 7:3) to afford the phosphonate monohexyl ester 7c (11 mg, 61% yield from 22). The purity of product 7c was 98.4% as shown by HPLC on an HC-C18 column (Merck, 4.6 × 100 mm, 2 µm porosity), tR = 11.4 min [MeOH/(0.1% TFA aqueous solution) = 1:1] at a flow rate of 1.0 mL/min. C20H39N4O4P; white solid, mp 257 oC (decomposed); [α] D25 120.3 (c = 1.0, MeOH); IR max (neat) 3269, 2962, 1650, 1467, 1381, 1193, 1058 cm1; 1H NMR (400 MHz, CD3OD) δ 6.17 (1 H, dd, J = 10.8, 2.0 Hz), 4.02 (1 H, dd, J = 10.0, 3.2 Hz), 3.89 (1 H, ddd, J = 6.4, 3.6, 3.6 Hz), 3.833.73 (2 H, m), 3.042.98 (2 H, m), 2.37 (1 H, d, J = 16.0 Hz), 1.97 (3 H, s), 35



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1.681.58 (3 H, m), 1.511.29 (8 H, m), 1.211.07 (2 H, m), 1.000.89 (9 H, m); 13C NMR (100 MHz, CD3OD) δ 173.6, 158.0, 140.1 (d, 1JC-P = 176.8 Hz), 139.6 (d, 2JC-P = 11.4 Hz), 65.9, 57.3 (d, 3JC-P = 12.2 Hz), 57.1, 54.0, 45.5, 42.1 (d, 2JC-P = 12.2 Hz), 32.9, 32.2 (d, 3JC-P = 6.8 Hz), 26.9, 24.0, 23.9, 22.8, 22.5, 14.5, 12.8, 12.5;

31

P NMR (162 MHz, CD3OD) δ 10.2;

ESI-HRMS calcd for C20H40N4O4P: 431.2787, found: m/z 431.2759 [M + H]+. (1R,3R,4R)-[3-((S)-1-Acetamido-2-ethylbutyl)-4-guanidinocyclopentyl]

phosphonic

acid (8a). Bromotrimethylsilane (0.49 mL, 3.68 mmol) was added to a solution of bis-Boc-guanidine 27 (53 mg, 0.09 mmol) in CH2Cl2 (1 mL) at 0 oC. The mixture was stirred at room temperature for 18 h, and concentrated under reduced pressure. The residue was purified by reversed-phase RP-18 column (MeOH/H2O = 1:4) to afford the phosphonic acid 8a (19 mg, 63% yield). The purity of product 8a was 99.9% as shown by HPLC on an HC-C18 column (Agilent, 4.6 × 250 mm, 5 µm porosity), tR = 16.9 min [MeOH/(0.5% TFA aqueous solution) = 1:4] at a flow rate of 0.5 mL/min. C14H29N4O4P; white solid, mp 270 oC (decomposed); [α] D25 118.2 (c = 1.0, H2O); IR max (neat) 3367, 2933, 1650, 1458, 1381, 1062, 907 cm1; 1H NMR (400 MHz, D2O) δ 3.98 (1 H, dd, J = 10.0, 3.2 Hz), 3.63 (1 H, q, J = 8.0 Hz), 2.382.32 (1 H, m), 2.272.05 (3 H, m), 2.00 (3 H, s), 1.731.60 (2 H, m), 1.571.36 (3 H, m), 1.081.00 (2 H, m), 0.950.88 (6 H, m); 13C NMR (100 MHz, D2O) δ 174.0, 155.9, 57.5 (d, 3JC-P = 16.1 Hz), 54.9, 45.2 (d, 3JC-P = 7.4 Hz), 43.7, 34.9, 33.3 (d, 1JC-P = 140.6 Hz), 29.4, 22.6, 22.0, 20.8, 11.9, 11.3; 31P NMR (162 MHz, D2O) δ 27.3; ESI–HRMS 36


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calcd for C14H30N4O4P: 349.2005, found: m/z 349.2008 [M + H]+. Ethyl

hydrogen

(1R,3R,4R)-[3-((S)-1-acetamido-2-ethylbutyl)-4-guanidino

cyclopentyl]phosphonate (8b). To a solution of bis-Boc-guanidine 27 (30 mg, 0.05 mmol) in CH2Cl2 (2 mL) was added TFA (0.08 mL, 1.04 mmol) at 0 oC. The mixture was stirred at room temperature for 20 h, and concentrated under reduced pressure to give the corresponding guanidine (as the TFA salt). Lithium bromide (15 mg, 0.18 mmol) was added to the solution of the above-prepared guanidinium salt (18 mg, 0.04 mmol) in DMF (1 mL). The mixture was stirred at 110 oC for 19 h, and concentrated under reduced pressure. The residue was purified by reversed-phase RP-18 column (MeOH/H2O = 2:3) to afford the phosphonate monoethyl ester 8b (14 mg, 73% yield from 27). The purity of product 8b was 98.7% as shown by HPLC on an HC-C18 column (Agilent, 4.6 × 250 mm, 5 µm porosity), tR = 11.1 min [MeOH/(0.5% TFA aqueous solution) = 4:6] at a flow rate of 0.5 mL/min. C16H33N4O4P; white solid, mp 272–273 oC; [α] D25 110.1 (c = 1.0, MeOH); IR max (neat) 3330, 3166, 2933, 1663, 1548, 1291, 1050, 943 cm1; 1H NMR (400 MHz, CD3OD) δ 4.05 (1 H, d, J = 8.8 Hz), 3.93 (2 H, quin, J = 6.8 Hz), 3.61 (1 H, q, J = 8.0 Hz), 2.352.16 (4 H, m), 1.96 (3 H, s), 1.611.58 (3 H, m), 1.471.44 (1 H, m), 1.281.25 (4 H, m), 1.091.04 (2 H, m), 0.970.94 (6 H, m); 13C NMR (100 MHz, CD3OD) δ 173.6, 157.8, 61.3, 59.0 (d, 3JC-P = 14.4 Hz), 55.7, 46.5, 46.0, 36.6, 33.1, (d, 1JC-P = 139.1 Hz), 31.3, 24.0, 23.0, 22.3, 17.4, 13.2, 12.7;

31

P NMR (162 MHz, CD3OD) δ 28.3; ESI-HRMS calcd for C16H34N4O4P: 377.2318, 37



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Page 38 of 71

found: m/z 377.2319 [M + H]+. Hexyl

hydrogen

(1R,3R,4R)-[3-((S)-1-acetamido-2-ethylbutyl)-4-guanidino

cyclopentyl]phosphonate (8c). To a solution of bis-Boc-guanidine 28 (29 mg, 0.04 mmol) in CH2Cl2 (1 mL) was added TFA (0.06 mL, 0.80 mmol) at 0 oC. The mixture was stirred at room temperature for 20 h, and concentrated under reduced pressure to give the corresponding guanidine (as the TFA salt). Lithium bromide (9 mg, 0.10 mmol) was added to the solution of the above-prepared guanidinium salt (14 mg, 0.02 mmol) in DMF (0.15 mL). The mixture was stirred at 110 oC for 19 h, and concentrated under reduced pressure. The residue was purified by reversed-phase RP-18 column (MeOH/H2O = 4:1) to afford the phosphonate monohexyl ester 8c (11 mg, 63% yield from 28). The purity of product 8c was 97.6% as shown by HPLC on an HC-C18 column (Merck, 4.6 × 100 mm, 2 µm porosity), tR = 8.6 min [MeOH/(0.1% TFA aqueous solution) 11:9 to 13:7 gradients] at a flow rate of 1.0 mL/min. C20H41N4O4P; white solid, mp 253–254 oC; [α] D25 108.5 (c = 1.0, MeOH); IR max (neat) 3297, 2958, 2929, 1659, 1381, 1164, 1058 cm1; 1H NMR (400 MHz, CD3OD) δ 4.05 (1 H, dd, J = 10.4, 2.0 Hz), 3.903.83 (2 H, m), 3.60 (1 H, m), 2.362.26 (2 H, m), 2.222.11 (2 H, m), 1.96 (3 H, s), 1.661.54 (5 H, m), 1.491.31 (7 H, m), 1.261.24 (1 H, m), 1.121.01 (2 H, m), 0.980.90 (9 H, m); 13C NMR (100 MHz, CD3OD) δ 173.6, 157.8, 65.6, 59.1, 59.0, 55.8, 46.5, 46.1, 36.6, 33.0 (d, 1JC-P = 139.1 Hz), 32.9, 31.4, 26.9, 24.0, 23.9, 23.0, 22.4, 14.6, 13.3, 12.7;

31

P NMR (162 MHz, CD3OD) δ 28.3; ESI-HRMS calcd for 38


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C20H42N4O4P: 433.2944, found: m/z 433.2959 [M + H]+. Methyl (1S,2S,3S,4R)-3-((S)-1-acetamido-2-ethylbutyl)-4-((tert-butoxycarbonyl)amino)-2-((tert-b utyldimethylsilyl)oxy)cyclopentanecarboxylate (10a). Compound 9 was prepared according to the previously reported method with slight modification.15,

16

In brief, The substrate

(1R,4S)-2-azabicyclo[2.2.1]hept-5-en-3-one (5.00 g, 45.82 mmol) was heated in 1.25 M HCl methanolic solution (79.91 mL, 99.89 mmol) under reflux for 12 h, followed by treatment with di-tert-butyl dicarbonate (11.57 mL, 50.35 mmol) and Et3N (12.76 mL, 91.54 mmol) in CH2Cl2

(130

mL)

at

0–25

°C

for

3.5

h

to

give

methyl

(1S,4R)-4-[(tert-butoxycarbonyl)amino]cyclopent-2-ene-1-carboxylate (9.56 g, 87% yield). The above-prepared olefin compound (750 mg, 3.11 mmol) and Et3N (0.43 mL, 3.09 mmol) in toluene (17 mL) was added NaOCl (16.79–39.17 mmol, 17.35 mL of 6–14% aqueous solution) at 60 oC, followed by addition of (E)-2-ethylbutanal oxime (358 mg, 3.11 mmol) via syringe pump over a period of 1 h. This process was repeated by successive addition of NaOCl solution (17.35 mL) and (E)-2-ethylbutanal oxime (358 mg) over a period of 1 h for three times. After that, the mixture was stirred at 60 oC for another 24 h to afford the (3+2) dipolar cycloaddition product (650 mg, 59% yield). To the cycloaddition product (4.42 g, 12.47 mmol) in MeOH (51 mL) were added nickel (ΙΙ) chloride hexahydrate (2.97 g, 12.49 mmol) and 0.25 M NaOH in methanol (2.50 mL, 0.63 39



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Page 40 of 71

mmol) at –25 oC, followed by slow addition of sodium borohydride (1.18 g, 31.19 mmol) at – 25 oC. The mixture was stirred at 0 oC for 2 h, and concentrated by rotary evaporation under reduced pressure. The residue was dissolved in CH2Cl2/water (5:1), followed by addition of sodium nitrite (861 mg, 12.48 mmol), ammonium chloride (2.67 g, 49.92 mmol) and ammonium hydroxide (102 mL, 0.73 mol). After the aqueous layer turned purple, the mixture was extracted with CH2Cl2 (× 3 times). The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure to give the corresponding amine compound. To a solution of the above-prepared amine compound (3.91 g, 10.91 mmol) in CH2Cl2 (36 mL) were added triethylamine (1.52 mL, 10.91 mmol) and acetic anhydride (1.13 mL, 11.95 mmol) at 0 oC. The mixture was stirred at room temperature for 14 h, and extracted with saturated aqueous NaHCO3. The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, EtOAc/hexane 1:2 to 1:1 gradients) to afford the acetamide 9 (3.60 g, 72% yield). To a solution of 9 (1.54 g, 3.85 mmol) in anhydrous DMF (2 mL) were added imidazole (1.31 g, 19.23 mmol) and tert-butyldimethylsilyl chloride (1.39 g, 9.23 mmol). The mixture was stirred at room temperature for 16 h. The mixture was dissolved in CH2Cl2, washed with water and brine. The organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel column, EtOAc/hexane 1:6 to 2:5 gradients) to afford the silylation compound 10a (1.68 g, 85% yield). 40


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C26H50N2O6Si; foam; TLC (EtOAc/hexane = 1:2) Rf = 0.30; [α] D25 9.5 (c = 1.0, CHCl3); IR max (neat) 3305, 2958, 1720, 1642, 1364, 1172, 1103, 837 cm1; 1H NMR (400 MHz, CDCl3) δ 6.33 (1 H, d, J = 7.6 Hz), 4.99 (1 H, br), 4.38 (1 H, br), 4.20 (1 H, s), 4.12 (1 H, br), 3.66 (3 H, s), 2.76 (1 H, d, J = 8.0 Hz), 2.46 (1 H, ddd, J = 14.0, 8.8, 8.8 Hz), 1.89 (4 H, br), 1.69 (1 H, d, J = 13.2 Hz), 1.39 (9 H, s), 1.201.16 (4 H, m), 1.07 (1 H, br), 0.890.84 (15 H, m), 0.07 (3 H, s), 0.05 (3 H, s); 13C NMR (100 MHz, CDCl3) δ 175.3, 169.3, 155.4, 79.5, 78.0, 52.1 (2 ×), 51.9, 51.2, 48.5, 45.3, 33.6, 28.3 (3 ×), 25.7 (3 ×), 23.5, 23.4, 21.6, 17.8, 12.8, 12.0, 4.7 (2 ×); ESI–HRMS calcd for C26H51N2O6Si: 515.3516, found: m/z 515.3521 [M + H]+. Tert-butyl (1R,2S,3S,4S)-[2-((S)-1-acetamido-2-ethylbutyl)-3-((tert-butyldimethylsilyl)oxy)-4-iodocy clopentyl]carbamate (11). To a solution of methyl ester 10a (2.58 g, 5.01 mmol) in THF (15 mL) and ethanol (15 mL) was added 1 M NaOH (18 mL, 18 mmol). The mixture was stirred at room temperature for 3 h, neutralized by Dowex 50W×8 resin (acid form), and then filtered and concentrated under reduced pressure to give the corresponding carboxylic acid. Iodobenzene diacetate (884 mg, 2.74 mmol) and iodine (633 mg, 2.49 mmol) were added to the solution of the above-prepared carboxylic acid (2.50 g, 4.99 mmol) in CCl4 (160 mL) . The mixture was irradiated with 500 W tungsten filament lamp; after 1 h, iodosobenzene diacetate (884 mg, 2.74 mmol) and iodine (633 mg, 2.49 mmol) were added at 1.5 h intervals for five times. The reaction was completed by irradiation for total 8.5 h, and then quenched 41



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Page 42 of 71

with saturated aqueous Na2S2O3. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, EtOAc/hexane 1:8 to 1:4 gradients) to afford the iodo compound 11 (1.31 g, 45% yield from 10a). C24H47N2O4SiI; pale yellow solid, mp 183–184 oC; TLC (EtOAc/hexane = 1:2) Rf = 0.40; [α] D25 19.1 (c = 1.0, CHCl3); IR max (neat) 3367, 2962, 1720, 1646, 1258, 1168, 1091 cm1; 1H NMR (400 MHz, CDCl3) δ 5.98 (1 H, br), 4.81 (1 H, br), 4.474.43 (1 H, m), 4.27 (1 H, d, J = 3.6 Hz), 4.05 (1 H, d, J = 6.4 Hz), 3.16 (1 H, br), 2.71 (1 H, ddd, J = 8.0, 8.0, 3.6 Hz), 2.031.99 (1 H, m), 1.91 (3 H, s), 1.421.33 (12 H, m), 1.251.13 (3 H, s), 0.96 (3 H, t, J = 7.6 Hz), 0.91 (3 H, t, J = 7.6 Hz), 0.87 (9 H, s), 0.09 (3 H, s), 0.06 (3 H, s); 13C NMR (100 MHz, CDCl3) δ 169.5, 155.0, 82.3, 79.7, 52.7, 49.8, 49.4, 44.7, 43.0, 30.0, 28.4 (3 ×), 25.7 (3 ×), 23.8, 23.4, 21.6, 17.8, 13.0, 12.1, 4.2, 4.7; ESI– HRMS calcd for C24H48N2O4SiI: 583.2428, found: m/z 583.2427 [M + H]+. Tert-butyl (1S,2S,3R,5R)-[2-((S)-1-acetamido-2-ethylbutyl)-6-oxabicyclo[3.1.0]hexan-3-yl]carbamat e (12). To a solution of iodo compound 11 (1.60 g, 2.75 mmol) in THF (11 mL) was added tetra-n-butylammonium fluoride (5.50 mmol, 5.50 mL of 1 M solution in THF) at 0 oC. The mixture was stirred at room temperature for 2 h, concentrated under reduced pressure, and purified by flash chromatography (silica gel, EtOAc/hexane 1:1 to 4:1 gradients) to afford the epoxide 12 (729 mg, 78% yield). C18H32N2O4; white solid, mp 178–180

o

C; TLC 42


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(EtOAc/hexane = 8:1) Rf = 0.28; [α] D25 90.1 (c = 1.0, MeOH); IR max (neat) 3350, 2966, 1691, 1630, 1430, 1287, 1172 cm1; 1H NMR (400 MHz, CD3OD) δ 7.52 (1 H, d, J = 10.0 Hz), 4.384.32 (1 H, m), 3.52 (1 H, q, J = 8.0 Hz), 3.44 (1 H, s), 3.42 (1 H, s), 2.43 (1 H, dd, J = 14.4, 8.0 Hz), 2.192.15 (1 H, m), 1.94 (3 H, s), 1.601.53 (2 H, m), 1.441.42 (11 H, m), 1.311.14 (2 H, m), 0.960.91 (6 H, m); 13C NMR (100 MHz, CD3OD) δ 173.4, 157.5, 80.2, 58.9, 55.4, 52.3, 50.1, 47.8, 44.6, 36.5, 29.0 (3 ×), 23.6, 23.1, 22.6, 12.1, 12.0; ESI–HRMS calcd for C18H33N2O4: 341.2440, found: m/z 341.2440 [M + H]+. Tert-butyl (1R,2S,3S,4S)-[2-((S)-1-acetamido-2-ethylbutyl)-4-(diethoxyphosphoryl)-3-hydroxycyclo pentyl]carbamate

(13)

and

tert-butyl

(1R,2S,3R,4R)-[2-((S)-1-acetamido-2-ethylbutyl)-3-(diethoxyphosphoryl)-4-hydroxycyclo pentyl]carbamate (13-isomer). A solution of diethyl phosphite (597 mg, 4.32 mmol) in anhydrous THF (2 mL) was stirred and cooled to 78 oC. BuLi (4.32 mmol, 1.73 mL of 2.5 M solution in hexane) was added dropwise via syringe pump. The mixture was stirred for 15 min, and a solution of epoxide 12 (490 mg, 1.44 mmol) in anhydrous THF (9 mL) was added dropwise. The mixture was stirred for another 15 min, and BF3·OEt2 (0.74 mL, 2.88 mmol) was added slowly. The mixture was stirred at 78 oC for 1 h, warmed to room temperature, and stirred at room temperature for 14 h. The reaction was quenched with saturated aqueous NH4Cl. The mixture was concentrated by rotary evaporation, and the residual water layer was 43



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Page 44 of 71

extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, CH2Cl2/MeOH 50:1 to 25:1 to 10:1 gradients) to afford the phosphonate compound 13 (310 mg, 45% yield) and 13-isomer (146 mg, 21% yield). Compound 13: C22H43N2O7P; foam; TLC (EtOAc) Rf = 0.27; [α] D25 58.8 (c = 1.0, CHCl3); IR max (neat) 3330, 2996, 2933, 1711, 1548, 1368, 1058 cm1; 1H NMR (400 MHz, CDCl3) δ 7.53 (1 H, d, J = 10.0 Hz), 5.00 (1 H, d, J = 9.2 Hz), 4.25 (1 H, dd, J = 10.8, 4.4 Hz), 4.133.95 (6 H, m), 2.462.35 (1 H, m), 2.232.14 (1 H, m), 2.06 (1 H, dd, J = 10.8, 4.4 Hz), 2.02 (3 H, s), 1.691.54 (1 H, m), 1,461.38 (11 H, m), 1.341.15 (9 H, m), 0.80 (3 H, t, J = 7.2 Hz), 0.73 (3 H, t, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) δ 171.4, 156.1, 79.9, 74.8, 62.0 (d, 2JC-P = 6.8 Hz), 61.9 (d, 2JC-P = 6.1 Hz), 51.8, 50.3 (d, 3JC-P = 8.4 Hz), 48.1, 43.2, 40.0 (d, 1JC-P = 140.7 Hz), 30.9, 28.3 (3 ×), 23.2, 21.7, 21.2, 16.4 (d, 3JC-P = 5.3 Hz), 16.3 (d, 3JC-P = 4.6 Hz), 10.3, 10.0;

31

P NMR (162 MHz, CDCl3) δ 32.6; ESI–HRMS calcd for

C22H44N2O7P: 479.2886, found: m/z 479.2878 [M + H]+. 13-isomer: C22H43N2O7P; foam; TLC (EtOAc) Rf = 0.15; [α] D25 10.2 (c = 1.0, CHCl3); IR max (neat) 3297, 2970, 1687, 1544, 1172, 1025, 968 cm1; 1H NMR (400 MHz, CDCl3) δ 6.66 (1 H, d, J = 10.4 Hz), 4.96 (1 H, d, J = 9.6 Hz), 4.35 (1 H, d, J = 12.4 Hz), 4.204.03 (6 H, m), 3.70 (1 H, br), 2.282.09 (2 H, m), 2.00 (3 H, s), 1.95 (1 H, dd, J = 13.2, 6.8 Hz), 1.791.72 (1 H, m), 1,451.35 (11 H, m), 1.331.27 (8 H, m), 1.231.14 (1 H, m), 0.82 (3 H, 44


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t, J = 7.2 Hz), 0.75 (3 H, t, J = 6.8 Hz); 13C NMR (100 MHz, CDCl3) δ 170.9, 155.6, 79.6, 70.1, 62.6 (d, 2JC-P = 6.9 Hz), 61.7 (d, 2JC-P = 7.6 Hz), 51.3, 50.6 (d, 2JC-P = 12.1 Hz), 47.5, 46.9 (d, 1JC-P = 138.3 Hz), 41.7 (2 ×), 28.3 (3 ×), 23.4, 21.6, 21.3, 16.3 (2 ×), 10.1, 10.0; 31P NMR (162 MHz, CDCl3) δ 32.7; ESI–HRMS calcd for C22H44N2O7P: 479.2886, found: m/z 479.2909 [M + H]+. Tert-butyl (1R,2S,3S,4S)-[2-((S)-1-acetamido-2-ethylbutyl)-4-(dihexoxyphosphoryl)-3-hydroxycyclo pentyl]carbamate (14). A solution of dihexyl phosphite (551 mg, 2.20 mmol) in anhydrous THF (0.74 mL) was stirred and cooled to 78 oC. BuLi (2.20 mmol, 0.88 mL of 2.5 M solution in hexane) was added dropwise via syringe pump. The mixture was stirred for 15 min, and a solution of epoxide 12 (250 mg, 0.73 mmol) in anhydrous THF (4 mL) was added dropwise. The solution was stirred for another 15 min, and BF3·OEt2 (0.38 mL, 1.47 mmol) was added slowly. The mixture was stirred at 78 oC for 1 h, warmed to room temperature, and stirred at room temperature for 14 h. The reaction was quenched with saturated aqueous NH4Cl. The mixture was concentrated by rotary evaporation, and the residual water layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (silica gel, EtOAc/hexane 1:2 to 2:1 to 3:1 gradients) to afford the phosphonate compound 14 (178 mg, 41% yield). C30H59N2O7P; foam; TLC (EtOAc/hexane = 45



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Page 46 of 71

5:1) Rf = 0.27; [α] D25 52.4 (c = 1.0, CHCl3); IR max (neat) 3330, 2958, 1691, 1548, 1176, 1005 cm1; 1H NMR (400 MHz, CDCl3) δ 7.55 (1 H, d, J = 10.4 Hz), 4.86 (1 H, d, J = 9.6 Hz), 4.27 (1 H, dd, J = 10.4, 4.0 Hz), 4.10 (1 H, quin, J = 9.6 Hz), 4.043.90 (5 H, m), 2.492.38 (1 H, m), 2.262.17 (1 H, m), 2.08(1 H, dd, J = 10.4, 4.4 Hz), 2.03 (3 H, s), 1.681.55 (5 H, m), 1.40 (9 H, s), 1.361.17 (17 H, m), 0.870.72 (12 H, m); 13C NMR (100 MHz, CDCl3) δ 171.5, 156.1, 80.0, 75.0, 66.1 (d, 2JC-P = 6.8 Hz), 66.0 (d, 2JC-P = 4.6 Hz), 52.0, 50.3 (d, 3JC-P = 8.3 Hz), 48.1, 43.2, 40.0 (d, 1JC-P = 141.5 Hz), 31.3 (2 ×), 31.0, 30.5 (2 ×), 28.3 (3 ×), 25.2, 25.1, 23.3, 22.5 (2 ×), 21.8, 21.2, 13.9 (2 ×), 10.4, 9.9; 31P NMR (162 MHz, CDCl3) δ 32.5; ESI–HRMS calcd for C30H60N2O7P: 591.4138, found: m/z 591.4150 [M + H]+. Diethyl (1S,2S,3R,4R)-[3-((S)-1-acetamido-2-ethylbutyl)-4-(1,3-bis(tert-butoxycarbonyl)guanidin o-2-hydroxycyclopentyl]phosphonate (15). A solution of 2 M HCl in Et2O (0.40 mL, 0.80 mmol) was added to a solution of phosphonate compound 13 (120 mg, 0.25 mmol) in Et2O (0.53 mL). The mixture was stirred at room temperature for 18 h, and concentrated under reduced pressure to give the corresponding amine hydrochloride. The above-prepared amine hydrochloride

(95

mg,

0.23

mmol)

was

dissolved

in

DMF

(0.82

mL),

and

1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (67 mg, 0.23 mmol), mercury chloride (62 mg, 0.23 mmol) and Et3N (0.11 mL, 0.80 mmol) were added. The mixture was stirred at room temperature for 17 h, and filtered through a pad of Celite. The filtrate was 46


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concentrated under reduced pressure, and the residue was purified by flash chromatography (silica gel, CH2Cl2/MeOH 50:1 to 25:1 gradients) to afford the bis-Boc-guanidine product 15 (114 mg, 73% yield from 13). C28H53N4O9P; foam; TLC (EtOAc) Rf = 0.32; [α] D25 88.9 (c = 1.0, CHCl3); IR max (neat) 3280, 2978, 1650, 1413, 1368, 1160, 1062 cm1; 1H NMR (400 MHz, CDCl3) δ 11.38 (1 H, s), 8.65 (1 H, d, J = 10 Hz), 8.50 (1 H, d, J = 8.4 Hz), 4.42 (1 H, quin, J = 8.8 Hz), 4.344.30 (1 H, m), 4.134.03 (5 H, m), 3.94 (1 H, t, J = 10 Hz), 2.522.42 (1 H, m), 2.242.17 (1 H, m), 2.152.11 (1 H, m), 2.06 (3 H, s), 1.811.68 (1 H, m), 1.44 (9 H, s), 1.42 (9 H, s), 1.371.34 (2 H, m), 1.291.26 (6 H, m), 1.231.11 (3 H, m), 0.75 (3 H, t, J = 7.2 Hz), 0.70 (3 H, t, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) δ 171.7, 162.7, 155.8, 153.0, 83.8, 79.9, 75.3 (d, 2JC-P = 2.3 Hz) , 62.0 (d, 2JC-P = 6.8 Hz), 61.9 (d, 2JC-P = 6.8 Hz), 51.6 (d, 3JC-P = 3.1 Hz), 50.6 (d, 3JC-P = 9.2 Hz), 48.5, 43.1, 40.4 (d, 1JC-P = 142.2 Hz), 31.7 (d, 2

JC-P = 3.8 Hz), 28.2 (3 ×), 27.9 (3 ×), 23.3, 22.4, 21.7, 16.4 (d, 3JC-P = 6.0 Hz), 16.3 (d, 3JC-P

= 5.4 Hz), 10.6, 10.1;

31


C28H54N4O9P: 621.3628, found: m/z 621.3623 [M + H]+. Dihexyl

(1S,2S,3R,4R)-[3-((S)-1-acetamido-2-ethylbutyl)-4-(1,3-bis(tert-butoxy

carbonyl)guanidino-2-hydroxycyclopentyl]phosphonate (16). A solution of 2 M HCl in Et2O (0.16 mL, 0.32 mmol) was added to a solution of phosphonate compound 14 (60 mg, 0.10 mmol) in Et2O (0.25 mL). The mixture was stirred at room temperature for 18 h, and concentrated under reduced pressure to give the corresponding amine hydrochloride. The 47



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Page 48 of 71

above-prepared amine hydrochloride (52.6 mg, 0.10 mmol) was dissolved in DMF (0.50 mL), and 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (29 mg, 0.10 mmol), mercury chloride (27 mg, 0.10 mmol) and Et3N (0.05 mL, 0.36 mmol) were added. The mixture was stirred at room temperature for 17 h, and filtered through a pad of Celite. The filtrate was concentrated under reduced pressure, and the residue was purified by flash chromatography (silica gel, EtOAc/hexane 1:4 to 1:2 gradients) to afford the bis-Boc-guanidine product 16 (45 mg, 60% yield from 14). C36H69N4O9P; foam; TLC (EtOAc/hexane = 5:1) Rf = 0.25; [α] D25 77.4 (c = 1.0, CHCl3); IR max (neat) 3322, 3281, 2958, 1724, 1650, 1140 cm1; 1H NMR (400 MHz, CDCl3) δ 11.40 (1 H, s), 8.69 (1 H, d, J = 10.4 Hz), 8.52 (1 H, d, J = 8.8 Hz), 4.45 (1 H, quin, J = 9.2 Hz), 4.33 (1 H, dd, J = 11.2, 4.4 Hz), 4.083.94 (6 H, m), 2.542.44 (1 H, m), 2.272.18 (1 H, m), 2.14 (1 H, dd, J = 10.8, 4.8 Hz), 2.08 (3 H, s), 1.811.70 (1 H, m), 1.671.60 (4 H, m), 1.47 (9 H, s), 1.45 (9 H, s), 1.381.09 (17 H, m), 0.870.81 (6 H, m), 0.78 (3 H, t, J = 7.6 Hz), 0.73 (3 H, t, J = 7.6 Hz); 13C NMR (100 MHz, CDCl3) δ 171.7, 162.7, 155.8, 153.0, 83.8, 79.9, 75.3, 66.1, 66.0, 51.6 (d, 3JC-P = 3.1 Hz), 50.8 (d, 3JC-P = 9.1 Hz), 48.5, 43.0, 40.3 (d, 1JC-P = 142.1 Hz), 31.8 (d, 2JC-P = 3.8 Hz), 31.3 (2 ×), 30.5 (2 ×), 28.3 (3 ×), 28.0 (3 ×), 25.2, 25.1, 23.4, 22.5 (2 ×), 22.4, 21.6, 13.9 (2 ×), 10.6, 10.1; 31P NMR (162 MHz, CDCl3) δ 31.9; ESI–HRMS calcd for C36H70N4O9P: 733.4880, found: m/z 733.4862 [M + H]+. (1S,2S,3R,5S)-2-((S)-1-Acetamido-2-ethylbutyl)-3-((tert-butoxycarbonyl)amino)-5-(d 48


Page 49 of 71

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iethoxyphosphoryl)cyclopentyl methanesulfonate (17). To a solution of phosphonate 13 (224 mg, 0.47 mmol) in anhydrous CH2Cl2 (2 mL) at 0 oC were added Et3N (0.72 mL, 5.15 mmol) and 4-dimethylaminopyridine (6 mg, 0.05 mmol). Then, a solution of methanesulfonyl chloride (0.18 ml, 2.34 mmol) in anhydrous CH2Cl2 (0.75 mL) was added via syringe pump over a period of 20 min. The mixture was stirred at 0 oC for 1 h, warmed to room temperature, and stirred at room temperature for 11 h. The mixture was partitioned between EtOAc and 1 M HCl aqueous solution. The organic layer was washed successively with saturated aqueous NaHCO3 and brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, CH2Cl2/MeOH 50:1 to 30:1 gradients) to afford the methanesulfonate 17 (171 mg, 59% yield). C23H45N2O9PS; colorless oil; TLC (EtOAc) Rf = 0.47; [α] D25 65.1 (c = 1.0, CHCl3); IR max (neat) 3326, 2970, 1716, 1679, 1368, 1176, 1029 cm1; 1H NMR (400 MHz, CDCl3) δ 6.90 (1 H, d, J = 9.6 Hz), 5.30 (1 H, d, J = 8.8 Hz), 5.095.06 (1 H, m), 4.304.22 (2 H, m), 4.164.03 (4 H, m), 3.10 (3 H, s), 2.82 (1 H, ddd, J = 21.2, 9.6, 4.4 Hz), 2.652.52 (1 H, m), 2.392.36 (1 H, m), 2.16 (1 H, s), 1.96 (3 H, s), 1.821.72 (1 H, m), 1.40 (9 H, s), 1.361.25 (8 H, m), 1.231.10 (2 H, m), 0.910.82 (6 H, m); 13C NMR (100 MHz, CDCl3) δ 169.6, 155.7, 83.8, 80.0, 62.7 (2 ×), 51.1, 50.7, 47.3, 45.1, 40.0 (d, 1JC-P = 136.9 Hz), 38.3, 31.0, 28.3 (3 ×), 23.4, 22.3, 21.4, 16.3, 16.2, 11.7, 11.1;

31

P NMR (162 MHz, CDCl3) δ 28.9; ESI–HRMS calcd for C23H46N2O9PS:

557.2662, found: m/z 557.2664 [M + H]+. 49



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Page 50 of 71

(1S,2S,3R,5S)-2-((S)-1-Acetamido-2-ethylbutyl)-5-(dihexoxyphosphoryl)-3-((tert-but oxycarbonyl)amino)cyclopentyl methanesulfonate (18). To a solution of phosphonate 14 (69 mg, 0.12 mmol) in anhydrous CH2Cl2 (0.46 mL) at 0 oC were added Et3N (0.18 mL, 1.29 mmol) and 4-dimethylaminopyridine (1 mg, 0.01 mmol). Then, a solution of methanesulfonyl chloride (0.05 mL, 0.65 mmol) in anhydrous CH2Cl2 (0.2 mL) was added via syringe pump over a period of 20 min at 0 oC. The mixture was stirred at 0 oC for 1 h, warmed to room temperature, and stirred at room temperature for 11 h. The mixture was partitioned between EtOAc and 1 M HCl aqueous solution. The organic layer was washed successively with saturated aqueous NaHCO3 and brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, EtOAc/hexane 1:2 to 3:2 gradients) to afford the methanesulfonate 18 (45 mg, 57% yield). C31H61N2O9PS; colorless oil; TLC (EtOAc/hexane = 5:1) Rf = 0.46; [α] D25 40.8 (c = 1.0, CHCl3); IR max (neat) 3314, 2962, 1716, 1679, 1548, 1364 cm1; 1H NMR (400 MHz, CDCl3) δ 6.85 (1 H, d, J = 9.6 Hz), 5.23 (1 H, d, J = 10.0 Hz), 5.105.07 (1 H, m), 4.334.24 (2 H, m), 4.073.94 (4 H, m), 3.11 (3 H, s), 2.84 (1 H, ddd, J = 21.2, 10.0, 4.4 Hz), 2.682.54 (1 H, m), 2.41 (1 H, ddd, J = 8.8, 4.0, 4.0 Hz), 1.97 (3 H, s), 1.821.72 (1 H, m), 1.41 (9 H, s), 1.361.24 (15 H, m), 1.191.11 (2 H, m), 0.910.85 (12 H, m); 13C NMR (100 MHz, CDCl3) δ 169.6, 155.7, 84.0, 80.0, 66.8 (d, 2JC-P = 6.9 Hz), 66.7 (d, 2JC-P = 6.8 Hz), 51.0 (2 ×), 47.3, 45.3, 40.3 (d, 1JC-P = 136.1 Hz), 38.3, 31.3 (2 ×), 31.1, 30.5 (d, 3JC-P = 3.0 Hz), 30.4 (d, 3JC-P = 50


Page 51 of 71

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3.8 Hz), 28.3 (3 ×), 25.1 (2 ×), 23.5, 22.5 (2 ×), 22.4, 21.4, 14.0 (2 ×), 11.8, 11.3;

31

P NMR

(162 MHz, CDCl3) δ 28.9; ESI–HRMS calcd for C31H62N2O9PS: 669.3914, found: m/z 669.3939 [M + H]+. Tert-butyl

(1R,2S)-[2-((S)-1-acetamido-2-ethylbutyl)-4-(diethoxyphosphoryl)

cyclopent-3-en-1-yl]carbamate (19). To a solution of methanesulfonate 17 (134 mg, 0.22 mmol) in anhydrous THF (2 mL) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (0.13 mL, 0.87 mmol). The mixture was stirred at room temperature for 18 h, concentrated under reduced pressure, and purified by flash chromatography (silica gel, CH2Cl2/MeOH 50:1 to 30:1 gradients) to afford the cyclopentene phosphonate 19 (88 mg, 87% yield). C22H41N2O6P; foam; TLC (EtOAc) Rf = 0.31; [α] D25 19.8 (c = 1.0, CHCl3); IR max (neat) 3285, 2970, 1704, 1654, 1507, 1168, 1033, 964 cm1; 1H NMR (400 MHz, CDCl3) δ 6.43 (1 H, d, J = 11.6 Hz), 6.27 (1 H, d, J = 8.8 Hz), 4.81 (1 H, d, J = 6.4 Hz), 4.083.98 (5 H, m), 3.923.90 (1 H, m), 2.97 (1 H, dd, J = 16.4, 8.0 Hz), 2.88 (1 H, d, J =8.4 Hz), 2.27 (1 H, d, J = 16.8 Hz), 2.01 (3 H, s), 1.441.34 (11 H, m), 1.301.21 (8 H, m), 1.161.12 (1 H, m), 0.900.84 (6 H, m); 13C NMR (100 MHz, CDCl3) δ 170.6, 155.4, 146.3 (d, 2JC-P = 13.0 Hz), 131.7 (d, 1JC-P = 190.2 Hz), 79.7, 62.0 (d, 2JC-P = 5.3 Hz), 61.8 (d, 2JC-P = 5.4 Hz), 57.6 (d, 3JC-P = 17.5 Hz), 53.5, 52.0, 43.8, 39.3 (d, 2JC-P = 13.7 Hz), 28.3 (3 ×), 23.3, 22.9, 21.6, 16.4, 16.3, 11.9, 11.6;

31

P

NMR (162 MHz, CDCl3) δ 15.6; ESI–HRMS calcd for C22H42N2O6P: 461.2781, found: m/z 461.2782 [M + H]+. 51



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Page 52 of 71

Tert-butyl (1R,2S)-[2-((S)-1-acetamido-2-ethylbutyl)-4-(dihexoxyphosphoryl)cyclopent-3-en-1-yl]ca rbamate (20). To a solution of methanesulfonate 18 (45 mg, 0.07 mmol) in anhydrous THF (0.70 mL) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (0.03 mL, 0.20 mmol). The mixture was stirred at room temperature for 18 h, concentrated under reduced pressure, and purified by flash chromatography (silica gel, EtOAc/hexane 1:1 to 3:1 gradients) to afford the cyclopentene phosphonate 20 (32 mg, 84% yield). C30H57N2O6P; colorless oil; TLC (EtOAc/hexane = 5:1) Rf = 0.27; [α] D25 18.4 (c = 1.0, CHCl3); IR max (neat) 3289, 2962, 1704, 1654, 1540, 1254, 1180 cm1; 1H NMR (400 MHz, CDCl3) δ 6.42 (1 H, d, J = 12.0 Hz), 6.28 (1 H, d, J = 8.8 Hz), 4.78 (1 H, d, J = 6.4 Hz), 4.003.88 (6 H, m), 2.96 (1 H, ddd, J = 16.8, 7.2, 1.6 Hz), 2.87 (1 H, d, J =8.8 Hz), 2.25 (1 H, d, J = 16.8 Hz), 1.98 (3 H, s), 1.651.59 (4 H, m), 1.391.24 (24 H, m), 1.161.10 (2 H, m), 0.910.83 (12 H, m); 13C NMR (100 MHz, CDCl3) δ 170.7, 155.4, 146.2, 131.7 (d, 1JC-P = 188.5 Hz), 79.7, 66.0 (d, 2JC-P = 6.1 Hz), 65.9 (d, 2JC-P = 6.1 Hz), 57.7 (d, 3JC-P = 17.4 Hz), 53.6 (d, 3JC-P = 12.2 Hz), 52.0, 43.9, 39.3 (d, 2JC-P = 12.9 Hz), 31.3 (2 ×), 30.4 (2 ×), 28.3 (3 ×), 25.2, 25.1, 23.3, 23.0, 22.5 (2 ×), 21.6, 13.9 (2 ×), 12.0, 11.7;

31


C30H58N2O6P: 573.4033, found: m/z 573.4057 [M + H]+. Diethyl

(3R,4R)-[3-((S)-1-acetamido-2-ethylbutyl)-4-(1,3-bis(tert-butoxy

carbonyl)guanidino)cyclopent-1-en-1-yl]phosphonate (21). A solution of 2 M HCl in Et2O 52


Page 53 of 71

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(0.33 mL, 0.67 mmol) was added to a solution of cyclopentene phosphonate 19 (96 mg, 0.21 mmol) in Et2O (0.42 mL). The mixture was stirred at room temperature for 16 h, and concentrated under reduced pressure to give the corresponding amine hydrochloride. The above-prepared amine hydrochloride (83 mg, 0.21 mmol) was dissolved in DMF (0.79 mL), and 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (61 mg, 0.21 mmol), mercury chloride (57 mg, 0.21 mmol), and Et3N (0.10 mL, 0.74 mmol) were added. The mixture was stirred at room temperature for 17 h, and filtered through a pad of Celite. The filtrate was concentrated under reduced pressure, and the residue was purified by flash chromatography (silica gel, EtOAc/hexane 1:1 to 10:1 gradients) to afford the bis-Boc-guanidine 21 (115 mg, 92% yield from 19). C28H51N4O8P; foam; TLC (EtOAc) Rf = 0.36; [α] D25 20.5 (c = 1.0, CHCl3); IR max (neat) 3322, 2978, 1646, 1368, 1258, 1164, 1058, 968 cm1; 1H NMR (400 MHz, CDCl3) δ 11.21 (1 H, s), 8.39 (1 H, d, J = 4.8 Hz), 7.80 (1 H, d, J = 8.8 Hz), 6.54 (1 H, d, J = 11.2 Hz), 4.114.02 (5 H, m ), 3.863.81 (1 H, m), 3.20 (1 H, d, J = 11.6 Hz), 3.093.02 (1 H, m), 2.34 (1 H, d, J = 17.2 Hz), 2.03 (3 H, s), 1.491.44 (19 H, m), 1.371.13 (10 H, m), 0.920.87 (6 H, m); 13C NMR (100 MHz, CDCl3) δ 170.9, 162.4, 155.2, 152.9, 147.4 (d, 2JC-P = 13.0 Hz), 131.5 (d, 1JC-P = 188.7 Hz), 83.6, 79.5, 62.0 (2 ×), 56.8 (d, 3JC-P = 18.0 Hz), 54.4 (d, 3JC-P = 12.2 Hz), 52.5, 45.1, 38.7 (d, 2JC-P = 12.9 Hz), 28.3 (3 ×), 28.0 (3 ×), 23.8, 23.1, 22.0, 16.4, 16.3, 12.4, 12.2;

31

P NMR (162 MHz, CDCl3) δ 15.5; ESI–HRMS

calcd for C28H52N4O8P: 603.3523, found: m/z 603.3531 [M + H]+. 53



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Dihexyl

Page 54 of 71

(3R,4R)-[3-((S)-1-acetamido-2-ethylbutyl)-4-(1,3-bis(tert-butoxy

carbonyl)guanidino)cyclopent-1-en-1-yl]phosphonate (22). A solution of 2 M HCl in Et2O (0.14 mL, 0.28 mmol) was added to a solution of cyclopentene phosphonate 20 (49 mg, 0.09 mmol) in Et2O (0.20 mL). The mixture was stirred at room temperature for 16 h, and concentrated under reduced pressure to give the corresponding amine hydrochloride. The above-prepared amine hydrochloride (43 mg, 0.09 mmol) was dissolved in DMF (0.44 mL), and 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (25 mg, 0.09 mmol), mercury chloride (23 mg, 0.09 mmol), and Et3N (0.04 mL, 0.32 mmol) were added. The mixture was stirred at room temperature for 17 h, and filtered through a pad of Celite. The filtrate was concentrated under reduced pressure, and the residue was purified by flash chromatography (silica gel, EtOAc/hexane 1:3 to 1:1 gradients) to afford the bis-Boc-guanidine 22 (33 mg, 55% yield from 20). C36H67N4O8P; foam; TLC (EtOAc/hexane = 5:1) Rf = 0.46; [α] D25 20.4 (c = 1.0, CHCl3); IR max (neat) 3318, 3281, 2958, 1720, 1614, 1368, 1123 cm1; 1H NMR (400 MHz, CDCl3) δ 11.21 (1 H, s), 8.37 (1 H, d, J = 4.8 Hz), 7.81 (1 H, d, J = 8.8 Hz), 6.53 (1 H, d, J = 11.6 Hz), 4.10 (1 H, t, J = 5.2 Hz), 4.023.90 (4 H, m ), 3.853.80 (1 H, m), 3.18 (1 H, d, J = 11.2 Hz), 3.083.01 (1 H, m), 2.32 (1 H, d, J = 17.6 Hz), 2.03 (3 H, s), 1.681.60 (4 H, m), 1.471.43 (19 H, m), 1.371.25 (14 H, m), 1.201.12 (2 H, m), 0.910.83 (12 H, m); 13

C NMR (100 MHz, CDCl3) δ 171.0, 162.4, 155.2, 152.9, 147.3 (d, 2JC-P = 12.9 Hz), 131.3

(d, 1JC-P = 188.5 Hz), 83.5, 79.5, 66.0 (d, 2JC-P = 3.8 Hz, 2 ×), 56.8 (d, 3JC-P = 18.3 Hz), 54.4 54


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(d, 3JC-P = 12.2 Hz), 52.5, 45.0, 38.8 (d, 2JC-P = 12.9 Hz), 31.3 (2 ×), 30.4, 30.3, 28.2 (3 ×), 27.9 (3 ×), 25.2, 25.1, 23.8, 23.1, 22.5 (2 ×), 22.0, 13.9 (2 ×), 12.4, 12.2; 31P NMR (162 MHz, CDCl3) δ 15.5; ESI–HRMS calcd for C36H68N4O8P: 715.4775, found: m/z 715.4758 [M + H]+. O-(1S,2S,3R,5S)-[2-((S)-1-Acetamido-2-ethylbutyl)-3-((tert-butoxycarbonyl)amino)-5 -(diethoxyphosphoryl)cyclopentyl]-1H-imidazole-1-carbothioate (23). To a solution of phosphonate 13 (331 mg, 0.69 mmol) in anhydrous THF (4 mL) was added 1,1′-thiocarbonyldiimidazole (90% purity, 274 mg, 1.38 mmol). The mixture was stirred for 7 h at 66 oC under reflux, cooled, and then concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, CH2Cl2/MeOH 50:1 to 40:1 gradients) to afford the 1H-imidazole-1-carbothioate 23 (354 mg, 87% yield). C26H45N4O7PS; colorless oil; TLC (EtOAc) Rf = 0.26; [α] D25 45.4 (c = 1.0, CHCl3); IR max (neat) 3309, 2974, 2876, 1687, 1548, 1230, 1168, 1025, 756 cm1; 1H NMR (400 MHz, CDCl3) δ 8.32 (1 H, s), 7.63 (1 H, s), 7.00 (1 H, s), 6.33 (1 H, d, J = 10.0 Hz), 6.05 (1 H, s), 5.71 (1 H, d, J = 8.8 Hz), 4.42 (1 H, s), 4.304.29 (1 H, m), 4.214.02 (4 H, m), 2.622.48 (3 H, m), 2.42 (1 H, s), 1.89 (3 H, s), 1.861.82 (1 H, m), 1.39 (9 H, s), 1.351.23 (7 H, m), 1.131.08 (1 H, m), 1.02 (2 H, br), 0.910.82 (3 H, m), 0.70 (3 H, t, J = 6.8 Hz); 13C NMR (100 MHz, CDCl3) δ 182.8, 169.7, 155.4, 137.5, 131.0, 117.5, 85.4, 79.6, 62.8, 62.6, 52.4, 50.5, 47.3, 45.1, 39.7 (d, 1JC-P = 138.3 Hz), 31.8, 28.3 (3 ×), 23.3, 22.4, 21.5, 16.4 (d, 3JC-P = 7.6 Hz), 16.3 (d, 3JC-P = 6.9 Hz), 12.1, 11.3;

31

P NMR (162 MHz, CDCl3) δ 28.7; ESI–HRMS calcd for C26H46N4O7PS: 589.2825, 55



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found: m/z 589.2830 [M + H]+. O-(1S,2S,3R,5S)-[2-((S)-1-Acetamido-2-ethylbutyl)-5-(dihexoxyphosphoryl)-3-((tertbutoxycarbonyl)amino)cyclopentyl]-1H-imidazole-1-carbothioate (24). To a solution of phosphonate 14 (47 mg, 0.08 mmol) in anhydrous THF (0.50 mL) was added 1,1′-thiocarbonyldiimidazole (90% purity, 31 mg, 0.16 mmol). The mixture was stirred for 7 h at 66 oC under reflux, cooled, and then concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, EtOAc/hexane 1:1 to 3:1 to 5:1 gradients) to afford the 1H-imidazole-1-carbothioate 24 (30 mg, 54% yield). C34H61N4O7PS; colorless oil; TLC (EtOAc/hexane = 5:1) Rf = 0.13; [α] D25 42.6 (c = 1.0, CHCl3); IR max (neat) 3314, 2966, 2933, 1683, 1536, 1393, 1291, 1225 cm1; 1H NMR (400 MHz, CDCl3) δ 8.33 (1 H, s), 7.63 (1 H, s), 7.00 (1 H, s), 6.27 (1 H, d, J = 10.4 Hz), 6.05 (1 H, br), 5.68 (1 H, d, J = 9.6 Hz), 4.43 (1 H, br), 4.32 (1 H, br), 4.143.92 (4 H, m), 2.632.48 (3 H, m), 1.91 (3 H, s), 1.851.80 (1 H, m), 1.701.55 (4 H, m), 1.39 (9 H, s), 1.351.24 (14 H, m), 1.131.02 (3 H, m), 0.870.82 (9 H, m), 0.70 (3 H, t, J = 6.4 Hz); 13C NMR (100 MHz, CDCl3) δ 182.8, 169.9, 155.4, 137.5, 131.0, 117.5, 85.6, 79.6, 66.8 (2 ×), 52.4, 50.7, 47.4, 45.2, 39.9 (d, 1JC-P = 139.8 Hz), 31.9, 31.3, 31.2, 30.5 (d, 3JC-P = 6.0 Hz), 30.4 (d, 3JC-P = 6.0 Hz), 28.3 (3 ×), 25.2, 25.1, 23.3, 22.5 (2 ×), 22.4, 21.5, 13.9 (2 ×), 12.1, 11.4; 31P NMR (162 MHz, CDCl3) δ 28.8; ESI– HRMS calcd for C34H62N4O7PS: 701.4077, found: m/z 701.4095 [M + H]+. Tert-butyl

(1R,2S,4R)-[2-((S)-1-acetamido-2-ethylbutyl)-4-(diethoxyphosphoryl) 56


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cyclopentyl]carbamate

(25).

Tributyltin

hydride

(0.15

mL,

0.55

mmol)

and

2,2′-azobis(2-methylpropionitrile) (4 mg, 0.02 mmol) were added to a solution of 1H-imidazole-1-carbothioate 23 (249 mg, 0.42 mmol) in anhydrous toluene (6 mL) at 110 oC. The mixture was stirred at 110 oC under reflux for 20 min, cooled, and then concentrated under reduced pressure. The residue was dissolved in acetonitrile, and washed with hexanes for 3 times. The acetonitrile layer was concentrated under reduced pressure, and purified by flash chromatography (silica gel, CH2Cl2/MeOH 50:1 to 40:1 gradients) to afford the phosphonate 25 (139 mg, 71% yield). C22H43N2O6P; foam; TLC (EtOAc) Rf = 0.17; [α] D25 29.2 (c = 1.0, CHCl3); IR max (neat) 3297, 2966, 2880, 1708, 1654, 1540, 1250, 1058, 1029 cm1; 1H NMR (400 MHz, CDCl3) δ 6.44 (1 H, d, J = 10.0 Hz), 5.47 (1 H, d, J = 8.8 Hz), 4.083.96 (4 H, m), 3.873.83 (1 H, m), 3.773.73 (1 H, m), 2.242.06 (3 H, m), 2.041.97 (4 H, m), 1.711.60 (2 H, m), 1.35 (9 H, s), 1.301.17 (9 H, m), 1.121.01 (2 H, m), 0.840.76 (6 H, m); 13C NMR (100 MHz, CDCl3) δ 170.4, 155.7, 79.3, 61.8 (2 ×), 53.4 (d, 3

JC-P = 9.9 Hz), 51.0, 46.6 (d, 3JC-P = 5.3 Hz), 43.1, 32.6, 30.6 (d, 1JC-P = 145.9 Hz), 29.1, 28.3

(3 ×), 23.4, 22.4, 21.3, 16.4 (2 ×), 11.3, 11.0;

31

P NMR (162 MHz, CDCl3) δ 26.4; ESI–

HRMS calcd for C22H44N2O6P: 463.2937, found: m/z 463.2939 [M + H]+. Tert-butyl (1R,2S,4R)-[2-((S)-1-acetamido-2-ethylbutyl)-4-(dihexoxyphosphoryl)cyclopentyl]carba mate (26). Tributyltin hydride (0.04 mL, 0.13 mmol) and 2,2′-azobis(2-methylpropionitrile) 57



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Page 58 of 71

(1 mg, 0.006 mmol) were added to a solution of 1H-imidazole-1-carbothioate 24 (71 mg, 0.10 mmol) in anhydrous toluene (2 mL) at 110 oC. The mixture was stirred at 110 oC under reflux for 20 min, cooled, and then concentrated under reduced pressure. The residue was dissolved in acetonitrile, and washed with hexanes for 3 times. The acetonitrile layer was concentrated under reduced pressure, and purified by flash chromatography (silica gel, EtOAc/hexane 1:3 to 1:1) to afford the phosphonate 26 (40 mg, 69% yield). C30H59N2O6P; colorless oil; TLC (EtOAc/hexane = 5:1) Rf = 0.23; [α] D25 32.5 (c = 1.0, CHCl3); IR max (neat) 3301, 2958, 1712, 1544, 1225, 1176, 1001 cm1; 1H NMR (400 MHz, CDCl3) δ 6.39 (1 H, d, J = 10.4 Hz), 5.36 (1 H, d, J = 8.8 Hz), 4.003.89 (4 H, m), 3.883.82 (1 H, m), 3.793.75 (1 H, m), 2.212.19 (2 H, m), 2.122.09 (1 H, m), 1.98 (3 H, s), 1.721.57 (6 H, m), 1.37 (9 H, s), 1.341.24 (16 H, m), 1.101.02 (2 H, m), 0.850.77 (12 H, m); 13C NMR (100 MHz, CDCl3) δ 170.5, 155.7, 79.3, 65.9 (2 ×), 53.4 (d, 3JC-P = 8.4 Hz), 51.0, 46.8 (d, 3JC-P = 6.1 Hz), 43.2, 32.7, 31.3 (2 ×), 30.7 (d, 1JC-P = 146.1 Hz), 30.5 (2 ×), 29.2, 28.3 (3 ×), 25.1 (2 ×), 23.4, 22.5 (3 ×), 21.4, 13.9 (2 ×), 11.3, 11.1; 31P NMR (162 MHz, CDCl3) δ 35.3; ESI–HRMS calcd for C30H60N2O6P: 575.4189, found: m/z 575.4196 [M + H]+. Diethyl

(1R,3R,4R)-[3-((S)-1-acetamido-2-ethylbutyl)-4-(1,3-bis(tert-butoxy

carbonyl)guanidinocyclopentyl]phosphonate (27). A solution of 2 M HCl in Et2O (0.42 mL, 0.84 mmol) was added to a solution of cyclopentene phosphonate 25 (120 mg, 0.26 mmol) in Et2O (0.54 mL). The mixture was stirred at room temperature for 18 h, and concentrated under 58


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reduced pressure to give the corresponding amine hydrochloride. The above-prepared amine hydrochloride (103 mg, 0.26 mmol) was dissolved in DMF (0.92 mL), and 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (75 mg, 0.26 mmol), mercury chloride (70 mg, 0.26 mmol) and Et3N (0.13 mL, 0.93 mmol) were added. The mixture was stirred at room temperature for 17 h, and filtered through a pad of Celite. The filtrate was concentrated under reduced pressure, and the residue was purified by flash chromatography (silica gel, CH2Cl2/MeOH 50:1 to 40:1 gradients) to afford the bis-Boc-guanidine product 27 (121 mg, 77% yield from 25). C28H53N4O8P; foam; TLC (EtOAc) Rf = 0.11; [α] D25 76.0 (c = 1.0, CHCl3); IR max (neat) 3322, 2978, 1724, 1654, 1418, 1160, 1058, 964 cm1; 1H NMR (400 MHz, CDCl3) δ 11.28 (1 H, s), 8.38 (1 H, d, J = 7.6 Hz), 7.83 (1 H, d, J = 10.0 Hz), 4.083.97 (5 H, m), 3.823.76 (1 H, m), 2.282.12 (3 H, m), 2.01 (3 H, s), 1.991.91 (1 H, m), 1.811.63 (2 H, m), 1.42 (9 H, s), 1.40 (9 H, s), 1.351.28 (2 H, m), 1.261.21 (7 H, m), 1.121.06 (2 H, m), 0.78 (3 H, t, J = 7.2 Hz), 0.74 (3 H, t, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) δ 170.6, 162.6, 155.7, 152.9, 83.6, 79.6, 61.8 (d, 2JC-P = 6.9 Hz), 61.7 (d, 2JC-P = 6.0 Hz), 52.9, 50.9, 46.4 (d, 3JC-P = 7.6 Hz), 43.6, 33.3, 30.8 (d, 1JC-P = 148.3 Hz), 29.5, 28.2 (3 ×), 27.9 (3 ×), 23.4, 23.0, 21.9, 16.5, 16.4, 11.3, 11.0;

31

P NMR (162 MHz, CDCl3) δ 33.9;

ESI-HRMS calcd for C28H54N4O8P: 605.3679, found: m/z 605.3680 [M + H]+. Dihexyl

(1R,3R,4R)-[3-((S)-1-acetamido-2-ethylbutyl)-4-(1,3-bis(tert-butoxy

carbonyl)guanidinocyclopentyl]phosphonate (28). A solution of 2 M HCl in Et2O (0.10 mL, 59



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Page 60 of 71

0.20 mmol) was added to a solution of cyclopentene phosphonate 26 (37 mg, 0.06 mmol) in Et2O (0.17 mL). The mixture was stirred at room temperature for 18 h, and concentrated under reduced pressure to give the corresponding amine hydrochloride. The above-prepared amine hydrochloride

(32

mg,

0.06

mmol)

was

dissolved

in

DMF

(0.30

mL),

and

1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea (18 mg, 0.06 mmol), mercury chloride (17 mg, 0.06 mmol) and Et3N (0.03 mL, 0.22 mmol) were added. The mixture was stirred at room temperature for 17 h, and filtered through a pad of Celite. The filtrate was concentrated under reduced pressure, and the residue was purified by flash chromatography (silica gel, EtOAc/hexane 1:1 to 5:1 to 10:1 gradients) to afford the bis-Boc-guanidine product 28 (33 mg, 70% yield from 26). C36H69N4O8P; foam; TLC (EtOAc/hexane = 5:1) Rf = 0.28; [α] D25 84.5 (c = 1.0, CHCl3); IR max (neat) 3322, 3281, 2958, 2933, 1724, 1650, 1156 cm1; 1H NMR (400 MHz, CDCl3) δ 11.33 (1 H, s), 8.45 (1 H, d, J = 7.6 Hz), 7.88 (1 H, d, J = 10.0 Hz), 4.073.92 (5 H, m), 3.873.81 (1 H, m), 2.332.16 (3 H, m), 2.05 (3 H, s), 2.031.97 (1 H, m), 1.861.67 (2 H, m), 1.641.58 (4 H, m), 1.46 (9 H, s), 1.44 (9 H, s), 1.391.27 (15 H, m), 1.181.08 (2 H, m), 0.920.73 (12 H, m); 13C NMR (100 MHz, CDCl3) δ 170.8, 162.6, 155.7, 152.9, 83.7, 79.7, 66.0, 65.9, 53.1 (d, 3JC-P = 14.5 Hz), 51.1, 46.4 (d, 3

JC-P = 7.6 Hz), 43.8, 33.4, 31.3 (2 ×), 30.8 (d, 1JC-P = 148.4 Hz), 30.6, 30.5, 29.6, 28.2 (3 ×),

27.9 (3 ×), 25.2 (2 ×), 23.5, 23.0, 22.5 (2 ×), 21.9, 14.0 (2 ×), 11.4, 11.1; 31P NMR (162 MHz, CDCl3) δ 33.9; ESI–HRMS calcd for C36H70N4O8P: 717.4931, found: m/z 717.4923 [M + H]+. 60


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ASSOCIATED CONTENT Supporting Information Available: NMR spectra, HPLC diagrams and X-ray data. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author J.-M.F.: phone, 8862-33661663; fax, 8862-23637812; E-mail, [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We thank Ms. Hsin-Yu Liao for preparation of expression vectors for influenza NA. We thank the Ministry of Science & Technology and Academia Sinica for financial support.

ABBREVIATIONS USED Boc, tert-butoxycarbonyl; COSY, correlation spectroscopy; CPE, cytopathic effect; DMAP, 4-dimethylaminopyridine; HA, hemagglutinin; MDCK, Madin−Darby canine kidney; 61



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Page 62 of 71

MUNANA, 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid; NA, neuraminidase; NOESY; nuclear Overhauser effect spectroscopy; SD, standard deviation; TBAF, tetrabutylammonium fluoride; TBS, tert-butyldimethylsilyl; TCID50, 50% cell culture infectious dose; TFA, trifluoroacetic acid; TMSBr, bromotrimethylsilane; TMSOTf, trimethylsilyl trifluoromethanesulfonate.

62


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Legends of Figures, Schemes, and Tables. Figure 1. Chemical structures of influenza neuraminidase inhibitors. Figure 2. Retrosynthetic analysis of phosphono-peramivir 6a. Figure 3. ORTEP drawing of epoxide 12 and the structures of compounds 13 and 13-isomer determined by COSY (blue) and NOESY (red) correlations. Figure 4. Molecular modeling of 5 (A), 6a (B), 7a (C) and 6b (D) in the active site of influenza viral neuraminidase (N1 subtype, PDB code: 2HU4).6 The complex of 6a shows 4 hydrogen bonds with the key residues (R118, R292, R371 and Y347) in the neuraminidase active site, two hydrogen bonds less compared with 5. The phosphonic acid moiety of 7a exhibits 5 hydrogen bonding interactions with the key residues (R118, R292, R371 and Y347) in the NA active site. The ethyl substituent in 6b extend to the 430-loop to gain hydrophobic interaction. Scheme 1. Syntheses of phosphono-peramivir 6a and its monoalkyl esters. Scheme 2. Syntheses of dehydration compound 7a and its monoalkyl esters. Scheme 3. Syntheses of deoxy compound 8a and its monoalkyl esters. Table 1. Neuraminidase inhibition, anti-influenza activity, cytotoxicity and lipophilicity of peramivir phosphonate derivatives. Table 2. Comparison of torsional angles of phosphono-peramivir in solution (by NMR), solid state (by X-ray) and molecular modeling. 69



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Table 3. Neuraminidase inhibition (IC50, nM)a of phosphono-peramivir (6a), the dehydration compound 7a and the monoethyl ester 7b against various influenza viruses.

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