The hydrophobic side chain of oseltamivir influences type A subtype selectivity of neuraminidase inhibitors

Article abstract of DOI:10.1111/cbdd.13060

Neuraminidase, which plays a critical role in the influenza virus life cycle, is a target for new therapeutic agents. The study of structure–activity relationships revealed that the C-5 position amino group of oseltamivir was pointed to 150-cavity of the neuraminidase in group 1. This cavity is important for selectivity of inhibitors against N1 versus N2 NA. A serial of influenza neuraminidase inhibitors with the oseltamivir scaffold containing lipophilic side chains at the C-5 position have been synthesized and evaluated for their influenza neuraminidase inhibitory activity and selectivity. The results indicated that compound 13o (H5N1 IC₅₀?=?0.1?±?0.04?μm, H3N2 IC₅₀?=?0.26?±?0.18?μm) showed better inhibitory activity and selectivity against the group 1 neuraminidase. This study may provide a clue to design of better group 1 neuraminidase inhibitors.

Full text of DOI:10.1111/cbdd.13060

MS. XIONG LIN (Orcid ID : 0000-0003-1963-7813)
Article type
: Research Article
The hydrophobic side chain of oseltamivir influences type A subtype
selectivity of Neuraminidase inhibitors
Lin Xiong a,b, Qin-Hua Chenb, Peng Lib, Chun-Lei Lia,b,Guang-De Yanga*
a School of Pharmacy, Xi’an Jiaotong University, Xi’an, P. R. China
b Affiliated Dongfeng Hospital, Hubei University of Medicine, Hubei 442008, China
Abstract
Neuraminidase, which plays a critical role in the influenza virus life cycle, is a target for new
therapeutic agents. The study of structure–activity relationships revealed that the C-5 position amino
group of oseltamivir was pointed to 150-cavity of the neuraminidase in group-1. This cavity is
important for selectivity of inhibitors against N1 versus N2 NA. A serial of influenza neuraminidase
inhibitors with the oseltavimir scaffold containing lipophilic side chains at the C-5 position have been
synthesized and evaluated for their influenza neuraminidase inhibitory activity and selectivity. The
* Address correspondence to Dr. Guang-De Yang, School of Pharmacy, Xi’an Jiaotong University, 76 Yanta West Road, Xi’an
710061, Shaanxi, P. R. China, E-mail address: jmw52@mail.xjtu.edu.cn
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13060
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results indicated that compound 13o (H5N1 IC50 = 0.1 ±0.04µM，H3N2 IC50 = 0.26±0.18 µM)
showed better inhibitory activity and selectivity against the group-1 neuraminidase. This study may
provide a clue to design of better group-1 neuraminidase inhibitors.
Keywords: Neuraminidase Inhibitors; Anti-Influenza; Oseltavimir Derivatives; Selectivity
1. Introduction
Influenza, a severely viral infection of the respiratory system which can cause serious public health
and economic problems in the worldwide [1-2].Currently, anti-influenza medications consist of two
classes of drugs: M2 protein inhibitors and neuraminidase inhibitors[3-4]. M2 protein inhibitors are only
active against influenza virus A, their adverse drug reactions such as severe central nervous system side
effects and the rapid emergence of resistance limited its use[3-5]. Neuraminidase is one of the major
surface glycoproteins of the influenza virus. This viral enzyme is thought to promote virus entry and
release of virion progeny, thereby enhances infection efficiency[6-9]. Therefore neuraminidase has been
considered as a suitable target for designing anti-influenza drugs. Up to date, three NAIs have been
licensed worldwide for therapeutic and prophylactic uses: zanamivir1 (Relenza), oseltamivir2 (Tamiflu)
and peramivir 3(Fig.1). Oseltamivir is more commonly used for its good oral bioavailability. However,
due to the NA and hemagglutinin gene mutation frequency and the host immune system,
oseltamivir-resistant mutants of seasonal influenza, such as H5N1 and H1N1, present a new challenge
for therapeutic and prophylactic of virus infection. Therefore, it is urgent to develop novel
neuraminidase inhibitors to address these challenges [10-14]
.
Based on the differences of structural features near the oseltamivir active binding site,the
neuraminidase of influenza viruses A are divided into two phylogenetically distinct groups known as
group-1 (comprising N1, N4, N5, N8) and group-2 (comprising N2, N3, N6, N7, N9) neuraminidase[15]
.
Structural comparison of the X-ray solved neuraminidases revealed that their major difference between
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the two groups is that there is a large cavity, known as ‘150-cavity’ adjacent to the active site in group-1
but not presented in group-2 neuraminidase (Fig.2a)[16]. Currently most widely used neuraminidase
inhibitors(oseltamivir, zanamivir and peramivir) should be crossed out targeted against the structures of
neuraminidases in group-2, therefore the discovery of ‘150-cavity’ in group-1 neuraminidases
provides a new direction for designing the novel inhibitors for group -1 NA[17-19]
.
The previous studies showed that C-5 position amino group of oseltamivir was exposed towards to
the‘150-cavity’ (Fig.2b)[18]. From this point we intended to improve the activity and selectivity of this
serial of analogues against N1 versus N2 NA by introducing some groups to fill the ‘150-cavity’. In this
work, we have devised and synthesized a serial of compounds with lipophilic side chains at the C-5
amino group and evaluated their selectivity and inhibitory activities against N1 versus N2 NA [20-28]
.
2. Chemistry
A serial of (3R, 4R, 5S) ethyl 4-acetamido-5-amino-3-(pentan-3-yloxy) cyclohex-1- enecarboxylate
(oseltamivir) derivatives were synthesized following the reaction route outlined in scheme 1. Amine
12, prepared from shikimic acid[29-30] , was coupled with different carboxylic acids to give novel amide
derivatives 13a~13i in high yields(62~94%）.Meanwhile, amine 12 was treated with AcCl in the
presence of TEA to afford 13h in high yield.After that, compound 13j reacted with different amine, and
K2CO3 was used to neutralize hydrochloric acid, DMF as solvent. Finally, we got compound 13k~13t in
yield from 61~78% [31-36]
.
Results
All 20 target compounds were evaluated for their NA inhibitory activities and selectivity using
2'-(4-methylumbelliferyl)-α-D-acetylneuraminic acid (MUNANA) as the substrate. Then, crossed out
N1 (H5N1) from group-1 and N2 (H3N2) from group-2 were selected as representatives for testing, and
the results are summarized in Table 1.
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Discussion
Compared with oseltamivir, most compounds displayed decreased inhibitory activities against both
N1 and N2. However, almost all compounds have similar inhibitory activities against both N1 and N2.
Some of them displayed inhibitory activities against N1 better than N2 which showed better selectivity.
When we compared compounds 13a, 13b, 13c and 13i，it showed that phenyl ring as a substituent
group would have better inhibitory activities than alkyl chain, and the length of linker between phenyl
ring and scaffold of oseltamivir also affect the inhibitory activities，possibly due to the better
hydrophobic effect of the phenyl ring. Compared compounds 13c, 13d, 13e, 13f and 13g, it showed that
rigid linker between phenyl ring and mother ring can increase the inhibitory activities. It seems that
the rigid structure can lock the ‘150-cavity’ of neuraminidase enzyme and make it interact with
hydrophobic group well. Comparison among compounds 13m, 13n, 13o and 13p revealed that methyl
group at C-4 in benzene ring have better inhibitory activities of neuraminidase than other groups, which
may due to methyl group in benzene ring has much better hydrophobic effect with amino residue of
neuraminidase. Contrast to 13j, 13k ~ 13t all have better inhibitory activities as the amino group
interacts with amino residue of neuraminidase more than chlorine. However, when the chlorine was
replaced by amino group, the secondary amino groups showed better activities than the tertiary amino
group.It can be explained that secondary amine group have hydrogen atom which can form hydrogen
bond with amino acid residues of neuraminidase enzyme. Comparison of 13k~13p indicated that
aromatic amino group has better inhibitory activities than aliphatic amino group. Particularly, 13o has
the best inhibitory activities and selectivity. The chlorine in compound 13j was replaced by a series of
alicyclic amine such as piperidine，N-methyl piperazidine and morpholine to get compound 13q, 13r
and 13s. Comparison between13q, 13r and 13s showed that 13r has better inhibitory activities than 13q
and 13s. May be it is that substituent group of N-methyl piperazidine has stronger basicity than 13q and
13s which showed more ligand-binding effects.
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Data and molecular modeling
Auto-Dock was applied to study molecular docking. The crystal structure of NA was retrieved from
RCSB Protein Data Bank (PDB entry code: 2HU0). The protein structure was utilized in subsequent
docking experiments without energy minimization. All ligands and water molecules were removed
first, and then the polar hydrogen atoms were added. Automatic docking was employed. Other
parameters were established using the default values in the software. All molecules were minimized
using default parameters.
Accordingly, a substituent group exert from the amino at C-5 position of oseltamivir may interact
with the additional 150-cavity (Fig.3). Molecular modeling of oseltamivir with crystal structure of N1
neuraminidase in the open form (PDB entry code: 2HU0) showed that carboxyl group at C-1 of
compound 13o interacts with three arginine residues (Arg118, Arg292, Arg371) of NA, acetamido
group at C-4 interacts with other amino acids (Arg152, Glu119, Asp151 and Ile222). The side chain
extent from C-5 position of oseltamivir fitted into the 150-cavity. Superimposition compound 13o and
oseltamivir to bind with neuraminidase N1 in an open form, it revealed that compound 13o has a similar
to oseltamivir in an open form except amino group at C-5. However compound 13o had a lower
inhibitory activity than oseltamivir indicated that amino group at C-5 was very important to ligands to
interact with amino acid residues of neuraminidase which provide a clue to future design of novel NAIs.
Conclusion
In summary, we have designed and synthesized a serials of oseltamivir analogues with an extended
substituent at the internal N-position of the amino group as NA inhibitors. The results of inhibitory
activities showed that some of them as a possible inhibitor specifically against the group-1
neuraminidases. Particularly compound 13o exhibited strong inhibitor activity and good selectivity
against the group-1 neuraminidases (H5N1 IC50 = 0.1 µM，H3N2 IC50 = 0.26 µM). According to the
molecular modeling, the extended side chain of compound 13o provide additional interactions with the
150-cavity in the open form of N1 neuraminidase. However, this disposition also weakened the salt
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bridge or hydrogen bond interaction between the carbonyl and other group of 13o and the S1 and S3
pocket in the active site. This study may provide a clue to future design of better group-1 neuraminidase
inhibitors against influenza virus.
Experimental
All the solvents and reagents were purchased from commercial sources. All reactions were monitored
by thin-layer chromatography on 0.254 mm silica gel plates (60GF-254) and visualized with UV light,
or iodine vapor. 1H NMR and 13C NMR spectra were determined on a Varian Mercury VX400 or
Bruker Avance 400 apparatus in CDCl3 using TMS as internal standard. Mass spectra of final products
were records on a LC/MS Trap SL Plus spectrometer (Agilent technologies, Waldbronn, Germany)
equipped with electrospray ionization (ESI) interface and an ion trap mass analyzer. Melting points
were obtained on a XT-4 apparatus.
Neuraminidase inhibition assay
All the target compounds (20) were tested for their NA inhibitory activity in vitro(experimental
group). The in vitro assay is based on the method reported by Duand coworkers. The NA from influenza
virus (H5N1and H3N2) was obtained by the method described by Laver et al [20-21]. Values for the IC50
were measured by a spectrofluorometric technique and 2'-(4-methylumbelliferyl)-α-D-
acetylneuraminic acid (MUNANA) as substrate, this substrate cleavage by NA to yield a fluorescent
product, which can emit an emission wavelength of 450 nm with excitation wavelength of 360 nm. The
intensity of fluorescence can reflect the activity of NA sensitively.
The assay mixture contained inhibitors at various concentrations (6-8 points), there were 30 µL of the
enzyme in 32.5 mM MES buffer (pH 3.5), 10 µL of 4 mM CaCl2, 20 µL of 20 µM MUNANA, and 30
µL water in a 96-well microplate. After 10 min at 37 0C, 150 µL of 14 nM NaOH (pH = 10.2) was added
to 0.1 mL of the reaction mixture to terminate the reaction. A blank was run with the same substrate
solution with no enzyme(blank group) and oseltamivir as control group. Fluorescence was read using an
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Aminco-Bowman fluorescence spectrophotometer and substrate blanks were subtracted from the
sample readings. The IC50 was calculated by plotting percent inhibition versus the inhibitor
concentration, and determination of each point was performed in duplicate.
(3R, 4S, 5R)-Ethyl 3, 4, 5-trihydroxycyclohex-1-enecarboxylate 5
To a solution of (-)-Shikimic acid 4 (5 g, 28.26 mmol) in ethanol (25 ml), SOCl2 (1 ml, 13.85 mmol)
was added dropwise to mixture, the solution was refluxed for 3 hours, then the solvent was evaporated
under reduced pressure. The residue was dissolved in ethyl acetate and evaporated for three
times.Compound 5 was obtained as a white solid. 1H-NMR (400 MHz, DMSO-d6) δ6.62 (1H, s), 4.87
(1H, s), 4.85 (1H, s), 4.63 (1H, s), 4.23 (1H, s), 4.12 (2H, m), 3.86 (1H, s), 3.58 (1H, s), 2.41 (1H, d),
2.08 (1H, d), 1.22 (3H, t); ESI-MS m/z (M+H) 203.40.
(3R, 4S, 5R)-Ethyl 3, 4, 5-tris ((methylsulfonyl) oxy) cyclohex-1-enecarboxylate 6
A stirred solution of (3R,4S,5R)-3,4,5,-trihydroxy-cyclohex-1-enecarboxylic acid ethyl ester (5,
20.22 g, 100 mmol) in 600mL EtOAc was treated with CH3SO2Cl (24.0 mL, 310 mmol). The dark
brown solution was cooled to 0°C and treated crossed out with NEt3 (45.0 mL, 323 mmol) over a period
of 45 min keeping the temperature in the range of 0-5 0C. The brown suspension was stirred for 30 min
at 0-5 0C, filtered over a porous glass filter and the filter cake was washed with EtOAc. The organic
phase was washed by H2O and brine then dried over Na2SO4 and evaporated under reduced pressure to
yield the product 6 (42.24 g, 97%) as a brown resin. 1H-NMR(400 MHz,CDCl3) δ 1.32 (t, J= 7.1Hz,
3H), 2.70 (dd, J1 = 17.5 Hz; J2 = 5.9 Hz, 1H), 3.14 (s, 3H), 3.19 (s,3H), 3.20 (s, 3H), 3.22 (dd, J1=13.9
Hz; J2 = 5.9 Hz, 1H), 4.26 (q, J = 7.1 Hz, 2H), 4.99 (dd, J1 = 9.2 Hz; J2 = 4.0 Hz, 1H), 5.09-5.18 (m, 1H),
5.52 (dd, J1= J2 = 4.2 Hz, 1H), 6.80-6.86 (m, 1H); ESI-MS m/z (M+H) 436.06.
(3S, 4R, 5R)-Ethyl 3-azido-4, 5-bis[(methylsulfonyl)oxy]cyclohex-1-enecarboxylate 7
(3R, 4S, 5R)-3, 4, 5-tris-methanesulfonyloxy-cyclohex-1-enecarboxylic acid ethyl ester (6，10.58 g,
24.2 mmol) was dissolved in acetone (30 mL). To this solution NaN3 (1.73 g, 26.7 mmol) in H2O (10
mL) was added at room temperature and the suspension was stirred for 3h. The orange solution was
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diluted with EtOAc (80 mL) and toluene (80 mL) and extracted 3 times with aqueous NaHCO3. The
organic phase was separated, dried over Na2SO4, filtered and the filtrate was evaporated under reduced
pressure. The crude product was purified by column chromatography to get compound 7 (yield: 74%)
as yellow oil. ESI-MS m/z (M+H) 383.24; 1H-NMR(400 MHz,CDCl3) δ1.32 (t, J=7.1 Hz, 3H),
2.64-2.72 (m, 1H), 3.16 (s, 3H), 3.21 (dd, J1= 15.1 Hz; J2 = 5.7 Hz, 1H), 3.23 (s, 3H), 4.25 (q, J= 7.1 Hz,
2H), 4.31-4.38 (m,1H), 4.77 (dd, J1 = 9.9 Hz; J2 = 8.1 Hz, 1H), 4.86-4.94 (m, 1H), 6.74-6.79 (m,1H).
(1R, 5S, 6R)-Ethyl 5-azido-7-azabicyclo [4.1.0] hept-3-ene-3-carboxylate 8
(3R, 4S, 5R)-3-azido-4, 5-bis-methanesulfonyloxy-cyclohex-1-enecarboxylic acid ethyl ester (7, 2.0
g, 5.23 mmol) was dissolved under argon with stirring in THF (40 mL). The clear solution was treated at
room temperature with triphenylphosphine (1.5 g, 5.87 mmol) and stirred at room temperature for
1~2hours, then 10mL H2O and 6mL TEA were added to the mixture, the reaction was continued for
another 8 hours. After that the solvent was evaporated under reduced pressure, the residual liquor was
diluted by toluene (25 mL) and washed by KSCN (7%), combined water layer and adjusted pH = 9-10
by NaHCO3. The water layer was extracted by EtOAc and organic layer washed with H2O and brine
then dried over Na2SO4 and evaporated solvent under reduced pressure to yield the crude product as a
yellow oil, the crude product was purified by column chromatography to get compound 8 (yield: 52%).
ESI-MS m/z (M+H) 262.08; 1H NMR (400 MHz,CDCl3) δ1.17 (br s, 1H), 1.29 (t, J= 7.1 Hz, 3H),
2.40-2.49 (m, 1H), 2.69-2.79 (m, 1H), 2.83-3.12 (m, 2H), 3.18 (s, 3H), 4.20 (q, J= 7.1 Hz, 2H),
5.29-5.38 (m, 1H), 7.15-7.24 (m, 1H).
(1S, 5R, 6S)-Ethyl-7-acetyl-5-((methylsulfonyl)oxy)-7-azabicycl[4.1.0] hept-2-ene-3-carboxylatte
9
(1R, 5S, 6R)-Ethyl 5-azido-7-azabicyclo [4.1.0] hept-3-ene-3-carboxylate (8, 0.5 g 1.9mmol) was
dissolved in EtOAc (7 mL) and 0.8 mL TEA, 0.1g DMAP was added to mixture. Then acetic anhydride
(0.8 g) was added dropwise to mixture under the temperature at 0~5°C, the reaction was continued for
another hour. After the reaction, it was washed the mixture with K2CO3 (5% aq.) and organic layer
washed with H2O and brine then dried over Na2SO4 and evaporated under reduced pressure to yield the
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crude product as a yellow oil, which was purified by column chromatography to get compound 9 (yield:
71%). ESI-MS m/z (M+H) 304.14; 1H-NMR (400 MHz,CDCl3) δ1.30 (t, J = 7.1 Hz, 3H), 2.21 (s, 3H),
2.32-2.46 (m, 1H), 3.04-3.14 (m, 1H), 3.18 (s, 3H), 3.24 (dd, J1 = 5.9 Hz; J2 = 4.7 Hz, 1H), 3.35-3.41
(m,1H), 4.22 (q, J = 7.1 Hz, 2H), 4.96-5.06 (m, 1H), 7.10 (dd, J1 = 4.5 Hz; J2 = 3.5 Hz, 1H).
(3R, 4S, 5R)-Ethyl 4-acetamido-5-((methylsulfonyl)oxy)-3-(pentan-3-yloxy)cyclohex-
1-enecar-boxylate 10
(1S, 5R, 6S)-Ethyl 7-acetyl-5-((methylsulfonyl) oxy)-7-azabicycl [4.1.0]hept-2-ene-3- carboxylatte
(9, 0.25g, 0.8mmol) was dissolved under argon with stirring in 3-pentanol (20 mL), the clear light
yellow solution was treated with boron trifluoride ethyl etherate (0.157 mL, 1.24 mmol) keeping the
temperature in the range of 0-5 °C. The reaction mixture was stirred for 8 h at room temperature then
diluted with EtOAc and washed twice with water then dried over Na2SO4 and evaporated solvent under
reduced pressure to yield the crude product as a yellow solid, the crude product was purified by column
chromatography to get compound 10 (yield: 92%). m.p.137-139 oC；ESI-MS m/z (M+H) 392.31；1H
NMR (400 MHz,CDCl3) δ 0.91 (t, J = 7.5 Hz, 6H), 1.31 (t, J = 7.1 Hz, 3H), 1.43-1.58 (m, 4H), 2.02 (s,
3H), 2.73 (dd, J1 = 19.1 Hz; J2 = 4.4 Hz, 1H), 2.80-2.91 (m, 1H), 3.06 (s, 3H), 3.34-3.44 (m, 1H), 4.08
(d, J = 6.8 Hz, 1H), 4.23 (q, J =7.1 Hz, 1H), 4.27-4.35 (m, 1H), 5.21 (dd, J1 = 6.8 Hz; J2 = 4.3 Hz, 1H),
6.03 (d, J = 8.0 Hz, 1H), 6.88 (s, 1H).
(3R, 4R, 5S)-Ethyl 4-acetamido-5-azido-3-(pentan-3-yloxy) cyclohex-1-enecarboxylate 11
(3R, 4S, 5R)-4-acetylamino-3-(1-ethyl-propoxy)-5-methanesulfonyloxy-cyclohex-1- enecarboxylic
acid ethyl ester (10，0.5 g, 1.4 mmol) was dissolved in EtOH (20 mL) and 5 mL H2O and the mixture
was treated with NaN3. The colourless suspension was heated with stirring at 80°C for 8 h. The solvent
was evaporated under reduced pressure and then diluted with EtOAc and washed twice with water then
dried over Na2SO4 and evaporated solvent to yield the crude product as a yellow solid, the crude product
was purified by column chromatography to get compound 11 (yield:86%). m.p.135-137 0C；ESI-MS
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m/z (M+H) 355.11; 1H-NMR (400 MHz,CDCl3) δ 0.83-0.95 (m, 6H), 1.30 (t, J =7.1 Hz, 3H), 1.42-1.58
(m, 4H), 2.04 (s, 3H), 2.23 (dd, J1 = 17.5 Hz; J2 = 10.5 Hz, 1H), 2.85 (dd, J1 = 17.6 Hz; J2 = 5.4 Hz, 1H),
3.27-3.40 (m, 1H), 3.46 (dd, J1 = 18.6 Hz; J2 = 8.4 Hz, 1H), 4.08-4.27 (m, 3H), 4.50 (d, J = 8.3 Hz, 1H),
6.38 (d, J = 7.6 Hz, 1H), 6.78 (s, 1H).
(3R, 4R, 5S)-Ethyl 4-acetamido-5-amino-3-(pentan-3-yloxy)cyclohex-1-enecarboxylate
phosphate 12
To a three-necked flask which was equipped with an inlet and an outlet were added compound 11
(0.51 g, 1.51 mmol), ethanol (20 mL), and Lindlar catalyst (0.30 g). After the flask was purged with
hydrogen gas several times, the mixture was well stirred at room temperature under an atmosphere of
hydrogen gas for 16 h. The mixture was then filtered through a thin layer of Celite to remove the Lindlar
catalyst. The solvent was concentrated to dryness, and a mixed solvent of ethyl acetate (5 mL) and
ethanol (4 mL) was added. The solution was well stirred and warmed to 50°C, and a freshly prepared
solution of phosphoric acid (0.36 g, 85%, 1.80 mmol) in ethanol (1 mL) was added dropwise. After
stirring at 50°C for 0.5 h, white crystals formed, and the suspension was cooled to room temperature.
Filtration and rinsing with cooled acetone afforded compound 12 (yield: 84%). m.p.203-206 0C;
ESI-MS m/z (M+H) 314.11; 1H-NMR (400 MHz,D2O) δ 0.84 (t, J = 11 Hz, 3H), 0.89 (t, J = 7.3 Hz,
3H), 1.29 (t, J = 7.1 Hz, 3H), 1.40-1.63 (m, 4H), 2.09 (s, 3H), 2.52 (dd, J1 = 15.5 Hz; J2 = 12.2 Hz, 1H),
2.97 (dd, J1 = 17.1 Hz; J2 = 4.7 Hz, 1H), 3.48-3.66 (m, 2H), 4.06 (dd, J1 = J2 = 10.1 Hz, 1H), 4.25 (dd, J1
= 13.7 Hz; J2 = 6.7 Hz, 1H), 4.34 (d, J = 8.3 Hz, 1H), 6.86 (s, 1H).
(3R, 4R, 5S)-Ethyl 4-acetamido-5-(benzoylamide)-3-(pentan-3-yloxy) cyclohex-1- enecarboxylate
13a
(3R, 4R, 5S)-ethyl 4-acetamido-5-amino-3-(pentan-3-yloxy)cyclohex-1-enecarboxylate phosphate
(12, 0.2g, 0.64mmol) and 0.2 mL was dissolved in CH2Cl2(2 mL), then TEA 0.22g (1.92 mmol) was
added to mixture at rt. TLC showed that the reaction was complete, and washed the reaction solution
with saturation sodium bicarbonate, water for one time, dried over Na2SO4 and evaporated solvent
under reduced pressure to yield the crude product as a yellow solid, which was purified by column
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chromatography to get compound 13a.White solid, yield 74%, ESI-MS m/z (M+H) 428.11; 1H NMR
(400 MHz, CDCl3) δ 7.74 (d, J = 6.9 Hz, 2H), 7.57 – 7.47 (m, 1H), 7.45 (s, 1H), 7.34 (d, J = 6.2 Hz, 2H),
6.83 (s, 1H), 6.60 (d, J = 16.5 Hz, 1H), 4.34 (dd, J1 = 16.5, J2 =7.1 Hz, 4H), 4.27-4.16 (m, 1H), 3.44 (q,
J = 5.7 Hz, 1H), 2.91 (dd, J1 = 17.8, J2 = 4.5 Hz, 1H), 2.47 (dd, J1 = 17.8, J2 = 9.5 Hz, 1H), 1.83 (s, 3H),
1.63-1.46 (m, 4H), 1.30 (t, J = 7.1 Hz, 3H), 0.92 (q, J = 8.2 Hz, 6H). 13C NMR (100 MHz, CDCl3)
δ171.60, 167.93, 165.94, 138.08, 134.11, 131.47, 129.33, 128.31, 127.20, 82.26, 75.58, 60.79, 54.78,
49.36, 30.43, 26.31, 25.71, 22.92, 14.18, 9.63, 9.25.
(3R, 4R, 5S)-Ethyl 4-acetamido-5-(phenylacetamide)-3-(pentan-3-yloxy) cyclohex-1-enecar
-boxylate 13b
White solid, yield 77%, ESI-MS m/z (M+H) 442.02; 1H NMR (400 MHz, CDCl3) δ 7.30 (t, J = 7.3
Hz, 2H),7.26(m, 1H), 7.23 (t, J = 7.3Hz, 2H), 6.74 (s, 1H), 6.65 (t, J = 7.3 Hz, 1H), 6.08 (d, J = 7.7 Hz,
1H), 4.20 (q, J = 7.1 Hz, 2H), 4.11 – 4.04 (m, 1H), 4.00 (m, 1H), 3.48 (t, J = 8.9 Hz, 1H), 3.30 (m, J =
5.7 Hz, 1H), 2.73 (dd, J1 =18.1, J2 = 8.9 Hz, 1H), 2.25 (dd, J1 = 17.7, J2 = 9.5 Hz, 1H), 1.75 (s, 3H), 1.46
(dd, J1 = 13.1, J2= 6.0 Hz, 4H), 1.28 (t, J = 7.1 Hz, 3H), 0.90-0.72 (m, 6H).13C NMR (100 MHz, CDCl3)
δ 171.63, 170.66, 165.09, 137.16, 134.45, 130.30, 129.22, 128.20, 126.88, 81.98, 75.12, 60.59, 54.04,
48.51, 42.68, 30.51, 26.79, 25.05, 22.65, 14.07, 10.23, 8.49.
(3R, 4R, 5S)-Ethyl 4-acetamido-5-(3-phenylpropionamido)-3-(pentan-3-yloxy) cyclohex-1-
enecar-boxylate 13c
White solid, yield 79%, ESI-MS m/z (M+H) 456.31; 1H NMR (400 MHz, CDCl3) δ 7.31-7.26 (m,
2H), 7.22-7.15 (m, 3H), 6.77 (s, 1H), 6.54 (d, J = 8.0 Hz, 1H), 5.96 (d, J = 4.2 Hz, 1H), 4.20 (q, J = 7.1
Hz, 1H), 4.07-4.15 (m, 1H), 4.03 (t, J = 7.1 Hz, 2H), 3.37 (m, J = 5.7 Hz, 1H), 2.95 (t, J = 7.8 Hz, 2H),
2.71 (dd, J1 = 17.9, J2 = 4.9 Hz, 1H), 2.45 (t, J = 7.8 Hz, 2H), 2.22 (dd, J1 =18.1, J2 = 8.9 Hz, 1H), 1.92
(s, 3H), 1.55 – 1.44 (m, 4H), 1.30 (t, J = 7.1 Hz, 3H), 0.89 (q, J = 7.5 Hz, 6H). 13C NMR (100 MHz,
CDCl3) δ 173.24, 171.41, 166.08, 140.41, 137.26, 129.94, 128.78, 127.62, 126.09, 82.18, 75.45, 61.62,
54.00, 48.33, 37.83, 31.49, 30.53, 26.33, 25.67, 23.17, 14.03, 9.68, 9.02.
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(3R, 4R, 5S)-Ethyl 4-acetamido-5-[3-(4-nitrophenyl)propionamido]-3-(pentan-3-yloxy)
cyclohex-1-enecar-boxylate 13d
Yellow solid, yield 62%, ESI-MS m/z (M+H) 488.42; 1H NMR (400 MHz, CDCl3) δ 8.23 (d, J = 15.2
Hz, 1H), 7.66 (d, J = 8.1 Hz, 2H), 7.61 (d, J = 8.1 Hz, 2H), 6.99 (d, J = 6.6 Hz, 1H), 6.84 (s, 1H), 6.49 (d,
J = 15.5 Hz, 1H), 5.76 (s, 1H), 4.23 (dd, J1 = 14.1, J2 = 7.1 Hz, 2H), 4.15 (q, J = 7.0 Hz, 2H), 4.08 (m,
1H), 3.51 (m, 1H), 3.42 (m, 1H), 2.90 (dd, J1 = 17.6, J2 = 4.6 Hz, 1H), 2.42-2.34 (dd, J1 = 16.7, J2 = 9.8
Hz, 1H), 2.00 (s, 3H), 1.58-1.49 (m, 4H), 1.30 (t, J = 7.0 Hz, 3H), 0.93 (m, 6H). 13C NMR (100 MHz,
CDCl3) δ 171.63, 166.14, 165.09, 147.83, 141.30, 138.20, 137.14, 129.23, 127.83, 125.14, 123.70,
82.34, 75.12, 60.59, 54.04, 49.22, 30.94, 26.79, 25.70, 23.69, 14.38, 9.92, 9.19.
(3R, 4R, 5S)-Ethyl 4-acetamido-5-(3-(4-methylphenyl)propionamido)-3-(pentan-3-yloxy)
cyclohex-1-enecar-boxylate 13e
White solid, yield 68%, ESI-MS m/z (M+H) 458.42; 1H NMR (400 MHz, CDCl3) δ 7.47 (d, J = 15.6
Hz, 1H), 7.33 (t, J = 13.3 Hz, 1H), 7.19 (d, J = 7.8 Hz, 2H), 7.16 (m, 1H), 6.95 (d, J = 7.8 Hz, 2H), 6.72
(s, 1H), 6.38 (d, J = 13.9 Hz, 1H), 4.25 (m, 1H), 4.12 (q, J = 7.1, 1H), 4.08-4.00 (m, 1H), 3.40-3.29 (m,
1H), 2.78 (dd, J1 = 17.6, J2 = 4.6 Hz, 1H), 2.33 (dd, J1 = 16.7, J2 = 9.8 Hz, 1H), 2.22 (s, 3H), 1.82 (s, 3H),
1.44 (q, J1 = 13.3, J2 = 6.8 Hz, 4H), 1.22 (t, J = 7.1 Hz, 3H), 0.89–0.76 (m, 6H). 13C NMR (100 MHz,
CDCl3) δ 170.51, 165.67, 164.87, 139.95, 138.89, 137.32, 130.87, 128.52, 128.04, 126.70, 119.06,
81.28, 74.61, 60.00, 53.97, 47.74, 29.57, 25.27, 24.73, 22.36, 20.13, 13.04, 8.80, 8.09.
(3R, 4R, 5S)-Ethyl4-acetamido-5-(3-(4-chlorophenyl)propionamido)-3-(pentan-3-yloxy)
cyclohex-1-enecar-boxylate 13f
White solid, yield 72%, ESI-MS m/z (M+H) 478.24; 1H NMR (400 MHz, CDCl3) δ 7.53 (d, J = 15.4
Hz, 1H), 7.40 (d, J = 8.1 Hz, 2H), 7.31 (d, J = 8.1 Hz, 2H), 6.88 (d, J = 6.6 Hz, 1H), 6.82 (s, 1H), 6.35 (d,
J = 15.5 Hz, 1H), 6.01 (d, J = 7.8 Hz, 1H), 4.21 (dd, J1 = 14.1, J2 = 7.1 Hz, 2H), 4.19 (q, J = 7.0 Hz, 2H),
4.10 (m, 1H), 3.40 (s, 1H), 2.87 (dd, J1 = 17.6, J2 = 4.6 Hz, 1H), 2.44-2.30 (dd, J1 = 16.7, J2 = 9.8 Hz,
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1H), 1.96 (s, 3H), 1.58-1.47 (m, 4H), 1.29 (t, J = 7.0 Hz, 3H), 0.91 (m, 6H).13C NMR (100 MHz,
CDCl3) δ 171.50, 166.29, 165.90, 139.70, 137.84, 135.74, 133.11, 129.36, 129.02, 128.86, 121.65,
75.80, 60.58, 54.71, 49.22, 30.93, 26.79, 26.10, 22.64, 14.36, 10.22, 8.86.
(3R, 4R, 5S)-Ethyl4-acetamido-5-(3-(2-fluorophenyl)propionamido)-3-(pentan-3-yloxy)
cyclohex-1-enecar-boxylate 13g
White solid, yield 64%, ESI-MS m/z (M+H) 461.54; 1H NMR (400 MHz, CDCl3) δ7.56 (d, J = 15.5
Hz, 1H), 7.43–7.50(m, 2H), 7.04 (t, J = 8.4 Hz, 2H), 6.83 (s, 1H), 6.78 (s, 1H), 6.30 (d, J = 15.5 Hz, 1H),
5.93 (s, 1H), 4.28-4.20 (m, 1H), 4.15 (q J = 6.5 Hz, 2H), 4.09 (s, 1H), 3.37-3.44(m, 1H), 2.86 (dd, J1 =
17.2, J2 = 4.8Hz, 1H), 2.42–2.35 (dd, J1= 16.0, J2= 9.4 Hz, 1H), 1.96 (s, 3H), 1.47-1.57 (m, 4H), 1.29 (t,
J = 6.5 Hz, 1H), 0.92 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 171.65, 165.47, 162.67, 140.02, 130.79,
130.74, 129.73, 129.59, 129.41, 120.35, 116.02, 115.48, 81.97, 75.42, 60.99, 55.08, 49.22, 30.56,
26.11, 25.43, 23.70, 14.37, 9.54, 8.87.
(3R, 4R, 5S)-Ethyl 4-acetamido-5-(acetylamino)-3-(pentan-3-yloxy)cyclohex-1- enecarboxylate
13h
(3R, 4R, 5S)-Ethyl 4-acetamido-5-amino-3-(pentan-3-yloxy) cyclohex-1-enecarboxylate phosphate
(12, 0.2 g, 0.64 mmol) was dissolved in CH2Cl2(2 mL) and 0.2 mL TEA, 0.1 g DMAP was added to
mixture. After that 0.12 g (1mmol) acetic anhydride was added dropwise to reaction solution, TLC
showed that the reaction was complete, and washed the reaction solution with dilute hydrochloric acid,
saturation sodium bicarbonate, water for one time, then dried over Na2SO4 and evaporated solvent to
yield the crude product as a white solid, the crude product was purified by column chromatography to
get compound 13h (yield: 94%). ESI-MS m/z (M+H) 356.03; 1H NMR (400 MHz, CDCl3) δ 6.80 (s,
1H), 6.58 (d, J = 7.8 Hz, 1H), 5.99 (d, J = 7.3 Hz, 1H), 4.21 (q, J = 7.1 Hz, 2H), 4.16-4.10 (m, 1H),
4.10-4.06 (m, 1H), 4.04 (s, 1H), 3.39 (m, J = 5.6 Hz, 1H), 2.76 (dd, J1 = 17.8, J2 = 4.6 Hz, 1H), 2.32 (dd,
J1 = 17.5, J2 = 9.0 Hz, 1H), 1.99 (s, 1H), 1.95 (s, 1H), 1.52 (dt, J = 13.9, 7.1 Hz, 1H), 1.29 (t, J = 7.1 Hz,
1H), 0.90 (dd, J1 = 13.9, J2 = 7.1 Hz, 1H).13C NMR (100 MHz, CDCl3) δ 171.64, 170.28, 165.77,
137.83, 129.27, 82.05, 75.43, 60.87, 54.13, 48.25, 30.79, 26.38, 25.56, 23.20, 14.17, 9.76, 9.34.
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(3R, 4R, 5S)-Ethyl 4-acetamido-5-(propionamido)-3-(pentan-3-yloxy)cyclohex-1- enecarboxylate
13i
White solid, yield 86%, ESI-MS m/z (M+H) 368.43; 1H NMR (400 MHz, CDCl3) δ 6.79 (s, 1H), 6.45
(d, J = 7.7 Hz, 1H), 5.98 (d, J = 7.4 Hz, 1H), 4.21 (q, J = 7.1 Hz, 2H), 4.16-4.12 (m, 1H), 4.07 (d, J = 8.1
Hz, 2H), 4.04 (s, 1H), 3.45-3.33 (m, 1H), 2.76 (dd, J1 = 17.8, J2 = 4.6 Hz, 1H), 2.31 (dd, J1 = 17.5, J2 =
9.0 Hz, 1H), 2.18 (ddd, J1 = 15.1, J2=7.6, J3=2.6 Hz, 2H), 1.98 (s, 3H), 1.57-1.45 (m, 4H), 1.29 (t, J =
7.1 Hz, 3H), 1.12 (t, J = 7.6 Hz, 3H), 0.90 (q, J = 7.2 Hz, 6H).13C NMR (100 MHz, CDCl3) δ 174.44,
171.37, 165.91, 137.63, 129.07, 82.06, 75.47, 60.94, 54.42, 48.55, 30.53, 29.74, 26.39, 25.87, 23.32,
14.38, 10.10, 9.55, 9.17.
(3R, 4R, 5S)-Ethyl 4-acetamido-5-(2-chloroacetamido)-3-(pentan-3-yloxy)cyclohex-1-
enecar-boxylate 13j
(3R, 4R, 5S)-ethyl 4-acetamido-5-amino-3-(pentan-3-yloxy)cyclohex-1-enecarboxylate phosphate
(12, 0.2 g, 0.64mmol) and 0.2 mL was dissolved in CH2Cl2(2 mL), then chloroacteyl chloride 0.22 g
(1.92 mmol) was added dropwise to mixture under the condition of ice-bath. TLC showed that the
reaction was complete, and washed the reaction solution with saturation sodium bicarbonate, water for
one time, dried over Na2SO4 and evaporated solvent under reduced pressure to yield the crude product
as a yellow solid, the crude product was purified by column chromatography to get compound 13j. yield
71%, ESI-MS m/z (M+H) 388.18; 1H NMR (400 MHz, CDCl3) δ 7.58 (d, J = 7.3 Hz, 1H), 6.86 (s, 1H),
5.61 (d, J = 7.4 Hz, 1H), 4.32-4.15 (m, 4H), 4.03-3.95 (m, 3H), 3.48-3.31 (m, 1H), 2.72 (s, 1H),
2.55-2.43 (m, 1H), 2.04-1.96 (m, 1H), 1.59-1.49 (m, 4H), 1.32 (t, J = 7.1 Hz, 3H), 0.92 (dd, J1 = 16.2, J2
= 8.8 Hz, 6H).13C NMR (100 MHz, CDCl3) δ 171.33, 167.20, 165.77, 136.45, 129.55, 82.60, 74.80,
61.19, 52.76, 48.35, 42.46, 29.78, 26.23, 25.95, 23.35, 14.19, 9.76, 9.28.
(3R, 4R, 5S)-Ethyl4-acetamido-5-[2-(isopropylamino)acetamido]-3-(pentan-3-yloxy)cyclohex
-1-enecarboxylate 13k
To (3R, 4R, 5S)-ethyl4-acetamido-5-(2-chloroacetamido)-3-(pentan-3-yloxy)
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cyclohex-1-enecarboxylate 0.1 g (0.26 mmol) solution in DMF (2 mL) and 0.072g (0.52 mmol) K2CO3
was added to mixture then the mixture was keep in 55℃for 8h, after that the mixture was poured into a
large amount of water and extract by CH2Cl2, and the solvent was washed by water , brine water crossed
out for one time, dried over Na2SO4 and evaporated solvent under reduced pressure to yield the crude
product as a yellow solid, the crude product was purified by column chromatography to get compound
13k. Yield 75%, ESI-MS m/z (M+H) 412.18; 1H NMR (400 MHz, CDCl3) δ 7.74 (d, J = 7.9 Hz, 1H),
6.81 (s, 1H), 6.06 (d, J = 7.7 Hz, 1H), 4.21 (q, J = 7.1 Hz, 2H), 4.12 (dd, J1 = 11.6, J2 = 8.1 Hz, 2H), 4.05
(d, J = 10.3 Hz, 1H), 3.43-3.33 (m, 1H), 3.27-3.17 (m, 1H), 2.81-2.75 (m, 2H), 2.72 (d, J = 6.3 Hz, 2H),
2.36 (dd, J1 = 16.7, J2 = 8.2 Hz, 1H), 1.94 (s, 3H), 1.54-1.47 (m, 4H), 1.29 (t, J = 7.1 Hz, 3H), 1.12–1.00
(m, 6H), 0.89 (m, J1 = 10.7, J2 = 7.4 Hz, 6H).13C NMR (100 MHz, CDCl3) δ 173.10, 170.91, 165.90,
137.99, 128.87, 82.50, 75.89, 60.94, 54.38, 50.23, 49.37, 47.89, 30.57, 26.25, 25.88, 23.32, 22.94,
14.17, 9.54, 9.34.
(3R, 4R, 5S)-Ethyl4-acetamido-5-[2-(n-butylamino)acetamido]-3-(pentan-3-yloxy)cyclohex
-1-enecarboxylate 13l
White solid, yield 68%, ESI-MS m/z (M+H) 426.56; 1H NMR (400 MHz, CDCl3) δ 7.71 (d, J = 8.1
Hz, 1H), 6.81 (s, 1H), 6.13 (d, J = 6.8 Hz, 1H), 4.21 (q, J = 6.9 Hz, 2H), 4.09 (dd, J = 13.1, 4.8 Hz, 2H),
4.11–3.99 (m, 2H), 3.41-3.33 (m, 1H), 3.21 (t, J = 9.9 Hz, 2H), 2.74 (dd, J1 = 17.5, J2= 4.3 Hz, 1H),
2.64-2.50 (m, 2H), 2.35 (dd, J1 =17.6, J2= 9.4 Hz, 1H), 1.95 (s, 3H), 1.57-1.47 (m, 4H), 1.47-1.41 (m,
2H), 1.39-1.33 (m, 2H), 1.29 (t, J = 7.1 Hz, 3H), 0.95–0.89 (m, 6H), 0.87 (t, J = 5.5 Hz, 3H).13C NMR
(100 MHz, CDCl3) δ 172.81, 170.91, 165.98, 137.92, 129.15, 82.32, 75.76, 60.82, 54.51, 52.18, 49.76,
47.51, 31.91, 30.80, 26.30, 25.60, 23.28, 20.26, 14.36, 13.95, 9.74, 9.17.
(3R, 4R, 5S)-Ethyl4-acetamido-3-(pentan-3-yloxy)-5-[2-(phenylamino)acetamido]cyclohex
-1-enecarboxylate 13m
Gray solid, yield 66%, ESI-MS m/z (M+H) 446.55; 1H NMR (400 MHz, CDCl3) δ 7.46 (d, J = 7.8
Hz, 1H), 7.19 (t, J = 7.9 Hz, 2H), 6.82-6.74 (m, 2H), 6.58 (d, J = 7.7 Hz, 2H), 5.74 (d, J = 7.4 Hz, 1H),
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4.33 (s, 1H), 4.20 (q, J = 7.1 Hz, 2H), 4.08-3.97 (m, 2H), 3.75 (q, J = 17.3 Hz, 2H), 3.47-3.23 (m, 1H),
2.78 (dd, J1 = 17.7, J2 = 4.9 Hz, 1H), 2.28 (dd, J1 = 18.0, J2 = 9.2 Hz, 1H), 1.75 (s, 3H), 1.54-1.42 (m,
4H), 1.28 (t, J = 7.1 Hz, 3H), 0.88 (m, 6H).13C NMR (100 MHz, CDCl3) δ 171.51, 171.48, 165.92,
147.32, 137.83, 129.35, 129.19, 118.18, 113.37, 82.25, 75.37, 60.98, 53.78, 48.66, 48.62, 30.38, 26.28,
25.71, 22.94, 14.28, 9.82, 9.19.
(3R, 4R, 5S)-Ethyl 4-acetamido-3-(pentan-3-yloxy)-5-[2-(p-tolylamino)acetamido]cyclohex
-1-enecarboxylate 13n
Light solid, yield 61%, ESI-MS m/z (M+H) 460.45; 1H NMR (400 MHz, CDCl3) δ 7.48 (d, J = 8.1
Hz, 1H), 6.99 (d, J = 8.2 Hz, 2H), 6.77 (s, 1H), 6.49 (d, J = 8.3 Hz, 2H), 5.96 (d, J = 7.3 Hz, 1H), 4.19 (d,
J = 7.0 Hz, 2H), 4.13-4.07 (m, 1H), 4.04 (t, J = 7.1 Hz, 2H), 3.71 (q, J = 17.3 Hz, 2H), 3.34 (p, J = 5.6
Hz, 1H), 2.76 (dd, J = 17.8, 4.9 Hz, 1H), 2.36-2.25 (m, 1H), 2.23 (s, 3H), 1.94 (s, 1H), 1.80 (s, 3H), 1.46
(dt, J = 14.5, 7.2 Hz, 4H), 1.28 (t, J = 7.1 Hz, 3H), 0.96-0.75 (m, 6H). 13C NMR (100 MHz, CDCl3) δ
171.65, 171.31, 165.91, 144.88, 137.26, 129.90, 129.39, 128.10, 113.16, 82.30, 75.57, 61.16, 53.89,
48.99, 48.57, 30.53, 26.28, 25.73, 22.92, 20.38, 14.19, 9.59, 9.24.
(3R, 4R, 5S)-Ethyl 4-acetamido-5-{2-[(4-methoxyphenyl)amino]acetamido}-3-(pentan-3-
yloxy)-cyclohex-1-enecarboxylate 13o
White solid, yield 70%, ESI-MS m/z (M+H) 446.55; 1H NMR (400 MHz, CDCl3) δ 7.49 (d, J = 7.9
Hz, 1H), 6.78 (dd, J1 = 6.5, J2 = 2.4 Hz, 2H), 6.54 (d, J = 8.9 Hz, 2H), 5.63 (d, J = 8.0 Hz, 1H), 4.20 (q,
J = 6.8 Hz, 2H), 4.12-4.06 (m, 2H), 4.02 (d, J = 6.7 Hz, 1H), 3.76 (s, 3H), 3.71 (q, 2H), 3.39–3.28 (m,
1H), 2.77 (dd, J1 = 17.6, J2 = 4.7 Hz, 1H), 2.29 (dd, J1 = 17.8, J2 = 8.9 Hz, 1H), 1.81 (s, 3H), 1.56-1.39
(m, 4H), 1.28 (t, J = 7.1 Hz, 3H), 0.93-0.82 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 171.64, 170.97,
165.77, 153.03, 141.00, 137.15, 129.55, 115.78, 114.42, 82.38, 75.42, 61.04, 55.89, 53.80, 49.51,
48.56, 30.20, 26.21, 25.85, 23.11, 14.18, 9.52, 9.25.
(3R, 4R, 5S)-Ethyl 4-acetamido-5-{2-[(4-bromophenyl)amino]acetamido}-3-(pentan-3-yloxy)
-cyclohex-1-enecarboxylate 13p
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White solid, yield 71%, ESI-MS m/z (M+H) 492.85; 1H NMR (400 MHz, CDCl3) δ 7.47 (t, J = 14.6
Hz, 1H), 7.27 (d, 1H), 7.26 (d, J = 8.8 Hz, 2H), 6.76 (s, 1H), 6.46 (d, J = 8.7 Hz, 1H), 4.21 (dt, J1 = 15.9,
J2 = 4.2Hz, 3H), 4.02 (qd, J1 = 19.1, J2 = 9.4 Hz, 3H), 3.72 (q, J = 17.2 Hz, 2H), 3.36 (dq, J1 = 22.1, J2 =
5.5 Hz, 1H), 2.77 (dd, J1 = 17.7, J2 = 4.9 Hz, 1H), 2.40-2.20 (m, 1H), 1.81 (s, 2H), 1.55–1.44 (m, 4H),
1.34-1.29 (m, 2H), 1.00-0.81 (m, 7H). 13C NMR (100 MHz, CDCl3) δ 172.00, 170.96, 165.76, 145.81,
137.16, 132.34, 129.26, 115.09, 110.58, 82.34, 75.27, 61.08, 53.71, 48.93, 48.44, 30.45, 29.70, 26.34,
25.68, 22.91, 14.28, 9.68, 9.12.
(3R, 4R, 5S)-ethyl4-acetamido-3-(pentan-3-yloxy)-5-[2-(piperidin-1-yl)acetamido]cyclohex
-1-enecarboxylate 13q
Yellow solid, yield 78%; ESI-MS m/z (M+H) 438.56;1H NMR (400 MHz, CDCl3) δ 7.64 (d, J = 8.1
Hz, 1H), 6.81 (s, 1H), 5.73 (d, J = 6.8 Hz, 1H), 4.21 (q, J = 7.0 Hz, 2H), 4.13 (s, 2H), 4.02 (s, 1H), 3.35
(dd, J1 = 11.2, J2 = 5.5 Hz, 1H), 2.99–2.86 (m, 2H), 2.74 (d, J = 14.8 Hz, 1H), 2.41 (s, 3H), 2.34-2.30 (m,
1H), 1.94 (s, 3H), 1.60 (dd, J1 = 11.1, J2 = 5.6 Hz, 3H), 1.51 (dd, J1 = 11.1, J2 = 6.4 Hz, 4H), 1.43 (d, J =
5.3 Hz, 2H), 1.30 (t, J = 7.1 Hz, 4H), 0.89 (m, 6H).13C NMR (100 MHz, CDCl3) δ 171.64, 170.97,
166.13, 138.21, 128.57, 82.38, 75.89, 62.33, 60.95, 54.95, 53.91, 47.52, 30.80, 26.23, 25.88, 25.74,
23.57, 23.26, 14.20, 9.59, 9.35.
(3R, 4R, 5S)-ethyl4-acetamido-5-[2-(4-methylpiperazin-1-yl)acetamido]-3-(pentan-3-yloxy)
cyclohex-1-enecarboxylate 13r
White solid, yield 76%, ESI-MS m/z (M+H) 453.59; 1H NMR (400 MHz, CDCl3) δ 7.52 (d, J = 8.0
Hz, 1H), 6.81 (s, 1H), 5.70 (d, J = 6.4 Hz, 1H), 4.21(q, J = 7.0 Hz, 2H), 4.14-4.09 (m, 2H), 4.00 (s, 1H),
3.44-3.27 (dd, J1 = 11.4, J2 = 5.8 Hz, 1H), 3.05–2.89 (m, 2H), 2.74 (dd, J1 = 17.5, J2= 4.3 Hz, 1H), 2.52
(s, 8H), 2.37 (dd, J1 =17.6, J2= 9.4 Hz, 1H), 2.30 (s, 3H), 1.94 (s, 3H), 1.57–1.43 (m, 4H), 1.30 (t, J =
7.1 Hz, 3H), 0.95–0.84 (m, 6H).13C NMR (100 MHz, CDCl3) δ 171.09, 170.64, 165.84, 138.22, 128.94,
82.42, 75.95, 61.45, 61.10, 54.90, 54.01, 53.41, 47.54, 45.92, 30.76, 26.24, 25.76, 23.24, 14.20, 9.56,
9.35.
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(3R, 4R, 5S)-ethyl4-acetamido-5-(2-morpholinoacetamido)-3-(pentan-3-yloxy)cyclohex
-1-enecarbo-xylate 13s
Yellow solid, yield 74%; ESI-MS m/z (M+H) 440.56; 1H NMR (400 MHz, CDCl3) δ 7.56 (d, J = 8.0
Hz, 1H), 6.82 (s, 1H), 5.68 (d, J = 6.4 Hz, 1H), 4.22 (q, J = 7.0 Hz, 2H), 4.17-4.07 (m, 2H), 4.03-3.96
(m, 1H), 3.73 (d, J1 = 11.2, J2 = 4.4 Hz, 3H), 3.42-3.30 (m, 1H), 2.98 (q, J = 16.3 Hz, 2H), 2.75 (d, J =
15.7 Hz, 1H), 2.49 (s, 3H), 2.35 (dd, J = 15.5 Hz, 1H), 1.94 (s, 3H), 1.54-1.42 (m, 4H), 1.30 (t, J = 7.0
Hz, 3H), 0.98-0.85 (m, 6H).13C NMR (100 MHz, CDCl3) δ 170.97, 169.91, 166.13, 137.79, 128.58,
82.35, 75.80, 66.46, 61.94, 60.28, 53.79, 53.75, 47.87, 30.60, 26.33, 25.84, 23.26, 14.23, 9.60, 9.37.
(3R, 4R, 5S)-Ethyl 4-acetamido-5-[2-(benzylamino)acetamido]-3-(pentan-3-yloxy)cyclohex
-1-enecarboxylate 13t
White solid, yield 66%, ESI-MS m/z (M+H) 446.55; 1H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 8.0
Hz, 1H), 7.34 (d, J = 7.2 Hz, 2H), 7.31 (dd, J1 = 12.1, J2= 6.9 Hz, 1H), 7.30-7.24 (m, 2H), 6.80 (s, 1H),
6.22 (d, J = 7.6 Hz, 1H), 4.20 (q, J = 7.1 Hz, 2H), 4.12 (dd, J = 12.8, 8.7 Hz, 1H), 4.06 (s, 1H), 3.75 (q,
J = 13.3 Hz, 2H), 3.43–3.32 (m, 1H), 3.22 (t, J = 8.2 Hz, 2H), 2.78-2.62 (m, 1H), 2.32 (dd, J1 = 16.8, J2
= 8.2 Hz, 1H), 1.93 (s, 3H), 1.58-1.44 (m, 4H), 1.29 (t, J = 7.1 Hz, 3H), 0.88 (m, 6H). 13C NMR (100
MHz, CDCl3) δ 172.08, 171.14, 165.98, 139.20, 137.82, 129.22, 128.59, 128.11, 127.36, 82.27, 75.67,
60.93, 54.34, 53.66, 51.75, 47.79, 30.41, 26.42, 25.76, 23.24, 14.28, 9.62, 9.19
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Acknowledgments
We thank Guofu Qiu (School of Pharmaceutical Sciences, Wuhan University, Wuhan, China) for
1H NMR and 13C NMR spectrums. These works were supported by the Natural Science Foundation of
Hubei Province of China (Nos. 2015CFB615 and 2015CFC811),the Science Research Program
Foundation of Hubei Provincial Department of Education（Nos. B2016502 and B2016505）the School
Foundation for Hubei University of Medicine (No. FDFR201614), the Science and Technology Key
Program of Shiyan (Nos.16Y57 and 16Y58), the Natural Science Foundation of Hubei Provincial
Department of Education (Nos.Q20162113，B2015468 and B2016140) and the Key Discipline Project
of Hubei University of Medicine.
Conflict of interest statement
There is no conflict of interests in this manuscript.
Table1. The structure and in vitro inhibitory activities of compounds against NAa（n=3）.
H3N2/IC50
Compounds
R
H
H5N1/IC50 (µM)
(µM)
12
0.08±0.01
144.56±27.27
120.31±19.50
105.34±23.95
58.46±10.27
44.07±8.86
0.004±0.001
146.31±22.00
13a
13b
13c
13d
13e
13f
117.43±20.17
105.22±23.64
60.04±18.40
41.32±12.26
84.27±17.74
86.39±13.23
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13g
90.52±13.17
100.47±22.04
13h
13i
>200
>200
>200
>200
13j
150.40±23.55
6.40±2.72
8.40±2.14
4.68±2.19
111.70±16.87
7.40±2.36
5.20±2.25
3.54±2.11
13k
13l
13m
13n
13o
13p
13q
13r
13s
0.38±0.15
0. 10±0.04
0.44±0.17
20.48±5.25
15.46±3.59
44.58±6.08
0.86±0.32
0.26±0.18
0.54±0.16
17.56±3.49
11.39±3.08
35.68±10.04
13t
0.46±0.19
0.89±0.31
aMean IC50 ± SD of three independent experiments.
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Figure Captions
Fig.1. The chemical structure of three NA inhibitors (NAIs) approved by FDA: zanamivir, oseltamivir,
peramivir.
Fig.2. (a) Superposition of the active site of N1 (green) and N9 (yellow) neuraminidases demonstrating
that N9 is markedly different to N1 in the 150-loop region (b) Active site complex of the N1 subtype
with oseltamivir carboxylate showing the C-5 amino group as a potential modification site.
Fig.3. Docking result: Compound 13o in the active site of NA (PDB ID: 2HU0). (a) Compound 13m
binding with active pocket of NA in stick mode. (b) Superimposition of N1–inhibitor complexes
compound 13o and oseltamivir.
Fig.1. The chemical structure of three NA inhibitors (NAIs) approved by FDA: zanamivir, oseltamivir,
peramivir.
a
b
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Fig.2. (a) Superposition of the active site of N1 (green) and N9 (yellow) neuraminidases demonstrating
that N9 is markedly different to N1 in the 150-loop region (b) Active site complex of the N1 subtype
with oseltamivir carboxylate showing the C-5 amino group as a potential modification site.
Fig.3. Docking result: Compound 13o in the active site of NA (PDB ID: 2HU0). (a) Compound 13m
binding with active pocket of NA in stick mode. (b) Superimposition of N1–inhibitor complexes
compound 13o and oseltamivir.
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Scheme 1
Scheme 1.Synthesis of Oseltamivir phosphate1starting from (-)-shikimic acid: Reagents and reaction
conditions (a) Ethanol, SOCl2, reflux 6 h; (b) MsCl, TEA, DMAP, DCM, rt, 2 h; (c) NaN3,
CH3COCH3/H2O, ethyl acetate, rt, 8h; (d) PPh3, THF, H2O/TEA, 0-5℃, 8 h; (e) acetic anhydride, TEA,
DMAP, DCM, 0-5℃, 30min; (f) 3-pentanol, BF3.O(Et)2; (g) NaN3, CH3CH2OH/H2O, reflux; (h) PPh3,
THF, H2O, 50℃, 10 h; (i)R-COOH, EDCI, TEA, CH2Cl2. (j) ClCOCH2Cl, R-NH2
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