Novel N-Substituted oseltamivir derivatives as potent influenza neuraminidase inhibitors: Design, synthesis, biological evaluation, ADME prediction and molecular docking studies

Article abstract of DOI:10.1016/j.ejmech.2019.111635

The discovery of novel potent neuraminidase (NA) inhibitors remains an attractive approach for treating infectious diseases caused by influenza. In this study, we describe the design and synthesis of novel N-substituted oseltamivir derivatives for probing the 150-cavity which is nascent to the activity site of NA. NA inhibitory studies showed that new derivatives demonstrated the inhibitory activity with IC₅₀ values at nM level against NA of a clinical influenza virus strain. Moreover, the in silico ADME predictions showed that the selected compounds had comparable properties with oseltamivir carboxylate, which demonstrated the druggablity of these derivatives. Furthermore, molecular docking studies showed that the most potent compound 6f and 10i could adopt different modes of binding interaction with NA, which may provide novel solutions for treating oseltamivir-resistant influenza. Based on the research results, we consider that compounds 6f and 10i have the potential for further studies as novel antiviral agents.

Full text of DOI:10.1016/j.ejmech.2019.111635

Journal Pre-proof
Novel N-Substituted oseltamivir derivatives as potent influenza neuraminidase
inhibitors: Design, synthesis, biological evaluation, ADME prediction and molecular
docking studies
Jiqing Ye, Xiao Yang, Min Xu, Paul Kay-sheung Chan, Cong Ma
PII:
S0223-5234(19)30769-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.111635
EJMECH 111635
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 May 2019
Revised Date: 14 August 2019
Accepted Date: 19 August 2019
Please cite this article as: J. Ye, X. Yang, M. Xu, P.K.-s. Chan, C. Ma, Novel N-Substituted oseltamivir
derivatives as potent influenza neuraminidase inhibitors: Design, synthesis, biological evaluation, ADME
prediction and molecular docking studies, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.111635.
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Novel N Substituted Oseltamivir Derivatives as
Potent Influenza Neuraminidase Inhibitors: Design,
Synthesis, Biological Evaluation, ADME Prediction
and Molecular Docking Studies
a
Jiqing Ye a, Xiao Yang b, Min Xu c, Paul Kay-sheung Chan *b,d, Cong Ma
a State Key Laboratory of Chemical Biology and Drug Discovery, and Department of
Applied Biology and Chemical Technology, The Hong Kong Polytechnic University,
Kowloon, Hong Kong SAR
b
Department of Microbiology, The Chinese University of Hong Kong, Prince of
Wales Hospital, Shatin, Hong Kong SAR
c
School of Electrical and Data Engineering, Faculty of Engineering and IT,
University of Technology Sydney, Sydney, Australia
d
Stanley Ho Centre for Emerging Infectious Diseases, The Chinese University of
Hong Kong, Shatin, Hong Kong SAR
Abstract
The discovery of novel potent neuraminidase (NA) inhibitors remains an
attractive approach for treating infectious diseases caused by influenza. In this study,
we describe the design and synthesis of novel N-substituted oseltamivir derivatives
for probing the 150-cavity which is nascent to the activity site of NA. NA inhibitory
studies showed that new derivatives demonstrated the inhibitory activity with IC50
Corresponding author, E-mail: cong.ma@polyu.edu.hk (C.M.) and
paulkschan@cuhk.edu.hk (P.K.S.C.)

values at nM level against NA of a clinical influenza virus strain. Moreover, the in
silico ADME predictions showed that the selected compounds had comparable
properties with oseltamivir carboxylate, which demonstrated the druggablity of these
derivatives. Furthermore, molecular docking studies showed the most potent
compound 6f and 10i adopt different modes of interaction with NA, which may
provide novel solutions for treating oseltamivir-resistant influenza. Based on the
research results, we consider that compounds 6f and 10i have the potential for further
studies as novel antiviral agents.
Key words: Oseltamivir, Derivative, Neuraminidase, 150-cavity, Influenza virus

1. Introduction
Influenza, commonly known as flu virus, represents a severe viral infection
which can outbreak to become epidemic and even pandemic with elevated mortality
rate [1]. Influenza viruses are unique among the viruses with regard to their antigenic
variability, seasonality, and impact on the general population [2]. The continual
emergence of new strains of influenza virus presents an ongoing threat to the global
health and economic system. In the three types of influenza viruses (A, B and C)
according to the serological reactivity of internal protein, influenza A caused more of
the serious respiratory illness in humans [3]. While amongst influenza viruses of 18
hemagglutinin (HA) subtypes (H1 – 18) and 11 neuraminidase (NA) types (N1 – N11),
influenza virus A subtype H3N2 (A/H3N2) is the most dominant flu strain infecting
people associated with complications according to a report of the Centers for Disease
Control and Prevention (CDC) [4], which caused one million death throughout the
world in the Hong Kong Flu pandemic in 1968-1969 [5], and generates the current
seasonal flu worldwide every year since 2004 [6]. Additionally, most A/H3N2 viruses
contain genes from human, swine and avian viruses that demonstrated the possibility
of cross-species infections evidenced by the Fujian flu epidemic in 2003 – 2004 [7],
while the endemic of H3N2 in certain swine farms in China indicates the threat for
emergence of future epidemics [8].
NA inhibitors (NAIs) can prevent nascent viruses infecting intact cells by
inhibiting their release from host cells. The most successful NAIs include oseltamivir
phosphate (1, ethyl ester prodrug) for oral administration [9], zanamivir (2) for

inhalation [10], Laninamivir (3) [11], and peramivir (4) [12] (Fig. 1). However,
various NA mutants with resistance to oseltamivir have been identified in clinics. This
is because some of the amino acids of NA must undergo a conformational change to
accommodate the hydrophobic side chain of oseltamivir when the latter binds to the
NA active site. A report showed that three sets of mutations (Arg292Lys, Asn294Ser,
and His274Tyr) were able to prevent this conformational arrangement, and the
presence of any of these mutants resulted in oseltamivir resistance [13]. Thus, there is
an urgent need to discover novel oseltamivir derivatives for anti-influenza therapy.
Figure 1. Structures of the marketed NA inhibitors.
In 2006, Russell et al. determined the crystal structures of NAs from the N1, N4
and found that a loop of amino acids consisting of residues 147–152 (also known as
the 150-loop or 150-cavity) together with the active site residues Asp151 and Glu119,
adopted an open conformation [14]. A report demonstrated that the cavity opening of
N2 NA could also be induced by the binding of oseltamivir carboxylate (OC, active
drug after ester hydrolysis) to NA [15] (Fig. 3). These findings provided an
opportunity for the de novo design of oseltamivir derivatives. In literature, the
modifications on OC mostly focused on the derivatization at C-5-NH2 of OC such as

compounds (5) – (9) [16-18], and the introduction of a triazole side chain at the C-6
position as shown in compounds (10) and (11) has also been described [19]. In
addition, some OC derivatives such as compound (12) with a triazole ring
incorporating the nitrogen at C-5 position and the double bond migrated towards the
C-6 position have been reported [20] (Fig. 2). Results suggested that these derivatives
might be capable of interacting with NA additionally through the 150-cavity, despite
some of them lacking selectivity or structural data.
Figure 2. OC derivatives reported in literature for probing the 150-cavity.
In this study, we planned to modify the C-5 amino group of OC, and several
derivatives were designed, synthesized and evaluated the inhibitory activity of NA of
a clinical A/H3N2 strain isolated from Hong Kong, as which is more valid to
represent the real epidemic situation. Further in silico ADME predictions showed that
the derivatives have comparable pharmacokinetic and pharmacodynamic properties
with OC.

2. Results and discussion
2.1. Design of Oseltamivir derivatives
Our main objective in this research was to develop new NA inhibitors by
targeting the 150-cavity. According to the docking model of OC to the active center
of NA, C-5-NH2 is very close to the 150-cavity. Liu et al. reported a series of
oseltamivir derivatives bearing substituted benzyl amines at C-5 position [16-18].
While M2 channel blockers represent another class of successful anti-influenza drugs,
such as amantadine and rimantadine [21], and recently, Wang et al. have reported a
series of M2-S31 proton channel inhibitors with a general structure of
+
adamantly-1-NH2 CH2-heteroaryl compounds [22-24] (Fig. 3). This inspired us to
synthesize novel oseltamivir C-5-NH2 substituted derivatives as anti-influenza agents
(Fig. 3) by adapting the bioactive heteroaryl groups to substitute the C-5-NH2 of OC.
These modifications were used in this study for probing the 150-cavity and supposed
to maintain the inhibitory activity and pharmacokinetic properties.
Figure 3. Design of the new oseltamivir derivatives inspired by heteroaryl M2
channel blockers

2.2. Chemistry
The synthesis of compounds 6a – l bearing 1,2,4-oxadiazole are shown in Scheme 1.
2-chloro-N-hydroxyacetimidamide 2 was prepared from 2-chloroacetonitrile 1 and
hydroxylamine. Condensation of various aryl chlorides with 2 gave the
hydroxylamine esters 3a – l. These esters cyclized to form the chloromethyl
oxadiazole 4a – l, then the chloride group was substituted by the C-5 amine of OP in
the presence of N, N-diisopropylethylamine (DIPEA) to give 5a – l. The hydrolysis of
ethyl ester using sodium hydroxide (NaOH) provided acids 6a – l.
Scheme 1. Synthetic route of compounds 6a – l. Reagents and conditions: a)
NH2OH HCl, Na2CO3, H2O, r.t., 35 %; b) RC(O)Cl, K2CO3, acetone, r.t., 88 %; c)
AcOH, 120oC, 60 ~ 80 %; d) Oseltamivir phosphate, DIPEA, DMF, 70oC, 40 % ~ 70 %;
e) NaOH, MeOH/H2O, 50oC, 30 % ~ 50 %.
The synthesis of compounds 10a – j with the furan scaffold is depicted in Scheme 2.
Suzuki coupling of 5-bromofuran-2-carbaldehyde 7 with various boric acids furnished
aldehydes 8a – j with 50 % - 90 % overall yields. Reductive amination of 8a – j using
sodium triacetoxyborohydride (NaBH(OAc)3) provided amines 9a – j with the overall

yields of 40 % - 60 %. After hydrolysis, the desired compounds 10a – j were obtained.
Scheme 2. Synthesis of compounds 10a – j. Reagents and conditions: a) R2B(OH)2,
Pd(dppf)2Cl2, Na2CO3, Tol/EtOH/H2O, 110oC, 50 % ~ 90 %; b) Oseltamivir phosphate,
NaBH(OAc)3, EtOH; 40 % ~ 60 %; c) NaOH, MeOH/H2O, 50oC, 30 % ~ 50 %.
The synthesis of compounds 17a – h with the isoxazole scaffold is outlined in
Scheme 3. Condensation of dimethyl oxalate with methyl ketones 11a – h using
potassium tert-butoxide as the base to afford intermediates 12a – h, which cyclized
with hydroxylamine hydrochloride at 50°C in methanol to give the isoxazole ester
intermediates 13a – h with overall yields of 50 % - 80 %. The esters were reduced to
provide alcohols 14a – h by sodium borohydride and then converted to bromides 15a
– h by Appel-Lee reaction. The yields of these two steps ranged from 30 % - 60 %.
Finally, a substitution reaction of bromides by C-5-NH2 of oseltamivir was performed
at 70°C in DMF with DIPEA to provide 16a – h, which were hydrolyzed to afford the
desired products 17a – h.

Scheme 3. Synthetic route of compounds 17a - h. Reagents and conditions: a)
Dimethyl oxalate, t-BuOK, Tol, r.t.; b) NH2OH HCl, MeOH, 50oC, 50 % ~ 80 %; c)
NaBH4, MeOH, reflux, 60 % ~ 75 %; d) CBr4, PPh3, CH2Cl2, 50 % ~ 60 %; e)
Oseltamivir phosphate, DIPEA, DMF, 70oC, 40 % ~ 70 %; f) NaOH, MeOH/H2O, 50oC,
30 % ~ 60 %.
2.3. Neuraminidase enzyme inhibitory assay
All the newly synthesized compounds were preliminarily screened for
NA-inhibitory activity using MUNANA (4-(methylumbelliferyl)-N-acetylneuraminic
acid) as the substrate of NA [25]. As mentioned above, a clinical A/H3N2 strain from
Hong Kong was chosen and OC employed as the reference. Table 1 showed the
tested inhibitory potencies of the designed compounds at 1 nM.
Amongst the first series of OC derivatives (6a – l) with 1,2,4-oxadiazole
substituted at C-5 amine, most of the compounds displayed the NA inhibitory activity
at 1 nM, in which 6b with a 4-CF3 phenyl, 6d with a pyridine-2-yl, 6e with a 3-nitro
phenyl, and 6f with a furan-2-yl group demonstrated the significant activity with
percentages of inhibition ≥ 10 %. Interestingly, 6a with a thiophen-2-yl didn’t show
any activity, which differed from 6f with one atom (S and O). The results suggested
the size of the group at the C-5-NH-1,2,4-oxadiazole terminal position of OC was
important for binding.
For compounds 10i – j, all the substitutions at the C-5-NH-furanyl terminal

position exhibited the inhibitory activity. While the electron-donating substituents
such as 10a, 10e, 10g and 10i respectively possessing 4-methoxyphenyl,
3,4-dimethoxyphenyl, thiophen-3-yl and 3,5-dimethylisoxazol-4-yl demonstrated the
significant activity with percentages of inhibition > 10 %.
Lastly, all the C-5-NH-isoxazole derivatives 17a – j showed mild inhibitory
activity against NA at 1 nM regardless the electronic effects of substituents, compared
to the other two series of compounds (Table 1).
In summary, most of our compounds possess the inhibitory activity against a
clinically isolated of A/H3N2 NA. Particularly, 6f and 10i respectively bearing
5-(furan-2-yl)-1,2,4-oxadiazol-3-yl
and
3,5-dimethylisoxazol-4-yl)furan-2-yl
exhibited ~ 30 % of inhibition at 1 nM. Therefore, we further assessed the IC50 values
of compounds 6f and 10i (Table 2). Results showed that both compounds have very
good inhibitory activity against the A/H3N2 influenza NA of a clinical sample, with
IC50 values of 1.92 ± 0.24 nM and 1.63 ± 0.16 nM, respectively (Table 2).
Table 1. Inhibition rate of designed compounds (1 nM) against a clinically isolated
A/H3N2 influenza NA.
%
%
Derivatives Cmpd.
R
/
Cmpd.
6g
R
Inhibitiona
Inhibitiona
OC
6a
66.8 ± 0.4
0.0 ± 2.2
8.0 ± 1.7
0.0 ± 2.0
1.5 ± 1.2
0.5 ± 2.0
6h
6b
6i

6c
0.0 ± 3.5
6j
3.5 ± 3.1
4.0 ± 5.9
6d
17.1 ± 2.0
6k
6e
18.4 ± 3.6
6l
0.0 ± 2.6
6f
28.1 ± 1.7
14.7 ± 4.4
10a
10f
10g
10h
10i
5.5 ± 2.4
12.5 ± 2.3
4.8 ± 1.1
30.2 ± 1.5
10.9 ± 0.7
8.1 ± 2.3
0.4 ± 1.6
3.0 ± 0.7
0.3 ± 2.0
10b
10c
10d
10e
17a
17b
17c
17d
3.2 ± 3.7
8.4 ± 1.8
3.7 ± 3.8
12.9 ± 1.2
1.6 ± 0.4
5.2 ± 0.9
3.9 ± 3.6
5.4 ± 3.8
10j
17e
17f
17g
17h
a
The data were the mean of the three independent experiments. % Inhibition =
ꢀꢁ ꢃꢁ ꢆꢃꢀꢁ ꢃꢁ ꢆ
ꢄꢅ
ꢂ
ꢄꢅ
ꢇ
× 100%, A0 is the absorbance of the control (no drug control), Abg is
ꢁ ꢃꢁ
ꢇ
ꢄꢅ

the absorbance of the background, Ax is the absorbance of the sample or OC.
Table 2. Inhibitory activity of 6f and 10i against a clinically isolated A/H3N2 influenza
NA (IC50, nM)a.
Compound
IC50
OC
6f
10i
0.11 ± 0.01
1.92 ± 0.24
1.63 ± 0.16
a IC50 is the compound concentration producing 50 % inhibition of NA, the values are
the mean of three independent experiments.
2.4. In silico ADME evaluation
Computational calculation to predict absorption, distribution, metabolism and
excretion (ADME) was used in the evaluation the drug-like properties of synthesized
compounds [26-27]. Various pharmacokinetic and pharmacodynamic parameters of
four compounds (6d, 6e, 6f and 10i) with the greatest inhibitory activity in this
research were calculated such as octanol-water partitioning coefficient (QPlogPo/w),
aqueous solubility (QPlogS), binding to human serum albumin (QPlogKhsa),
brain/blood partition coefficient (QPlogBB), number of likely metabolic reactions
(#metab), central nervous system activity (CNS), predicted IC50 value for blockage of
HERG K+ channels (QPloghERG), apparent Caco-2 cell permeability (QPPCaco),
apparent MDCK cell permeability (QPPMDCK) , and human oral absorption (Table
3).
As shown in Table 3, compared to OC, the synthetic derivatives have higher

lipophilicity, and lower aqueous solubility. These results could be reasoned out
because of the substitutions of C-5 amine group. Nevertheless, lipophilicity and
solubility of the derivatives were still drug-like. Moreover, the synthetic derivatives
were suggested to bind slightly better to human serum albumin (OC ˂ 6f ˂ 6d ˂ 6e ˂
10i) which indicated that more derivatives are required for the first dose if used in
vivo. Furthermore, QPlogBB, CNS, #metab, QPloghERG were well within the
standard ranges. These results indicated that the derivatives should possess very low
central nervous and heart toxicity with good metabolic stability. Clinical using
anti-influenza drug oseltamivir phosphate is an ester prodrug of OC because of the
poor absorptivity of the latter. This was consistent with our predicted results that both
OC and its derivatives had quite poor permeability of Caco-2 and MDCK cells, which
can be transformed to ester prodrugs to resolve this problem.
Table 3. Compliance of OC derivatives with the standard ranges of computational
pharmacokinetic parameters (ADME).
Principal
Standard
Rangea
OC
6d
6e
6f
10i
descriptors
-1.654
-1.164
-0.873
-1.05
3
-0.472
-3.549
-0.659
-1.741
5
-0.692
-4.143
-0.538
-2.936
5
-0.555
-3.135
-0.692
-1.55
5
0.407
-3.732
-0.236
-1.465
7
-2.0 - 6.5
-6.5 - 0.5
-1.5 - 1.5
-3.0 - 1.2
1 - 8
QPlogPo/w
QPlogS
QPlogKhsa
QPlogBB
#metab
-2 (inactivity), +2
(acitivity)
-2
-1.322
7.34
-2
-2
-2
-2
CNS
-3.575
5.374
-3.690
0.749
-3.236
6.759
-2.591
6.731
˂ -5
QPloghERG
QPPCaco
˂ 25 poor,
˃ 500 great

˂ 25 poor,
˃ 500 great
6.146
32.756
Middle
3.876
37.254
Middle
0.461
7.688
Low
5.006
38.548
Middle
4.841
44.147
Middle
QPPMDCK
(%) Human Oral
Absorption
˂ 25 % is poor,
˃ 80 % is high
Human Oral
Absorption
a Statistics of 95 % of known drugs according to Qikprop (Schrödiner@ 2018).
2.4. Molecular docking model analysis of compound 6f and 10i with NA
The potential binding mode and key interactions of compounds 6f and 10i with
NA were analyzed using Glide of Schrödiner@2018. As shown in Fig. 4A, though 6f
and 10i are structurally very similar, it was demonstrated that these two compounds
bound with the enzyme in different modes. The oseltamivir core fragment of
compound 6f occupied the active site in a similar manner to the binding pattern of OC
while the 5-(furan-2-yl)-1,2,4-oxadiazole fragment stretched into the 150-cavity as
desired. In Fig. 4B, it was suggested that 6f retained the interactions of carboxyl acid
with three residues (Arg118, Arg292 and Arg371) and acetamide on the C-4 position
with Arg152. The C-5-NH formed one hydrogen bond with Asp151. Most of the
residues around the heteroaromatic fragment were hydrophobic, such as Gly147,
Trp438, Val116 (Fig. S1). In addition, 6f made a directly interaction with Gln136 by
H-bond. The docking model also explained why the inhibitory activity of 6f was
significant, while 6a contains a sulfur atom which may not be able to fit into the
150-cavity.
Nevertheless, the pattern of interaction of 10i with NA was suggested to be
distinct from OC or 6f. Indeed, 10i was docked for binding to NA by overturning the

molecule to prolong the 1-ethylpropoxy group into the inside area of enzyme, while
the 3,5-dimethylisoxazol-4-yl group took over where the 1-ethylpropoxy group of OC
occupies. As shown in Fig. 4C, the interactions would be formed between the
carboxylic acid of 10i and Arg118 and Arg371, as well as acetamide at the C-4
position and Ser246. This type of docking model demonstrated less interactions with
the enzyme when compared to 6f or OC (Fig. S1). Note that the electron-donating
groups substituted at C-5-NH-furanyl were favored for the inhibitory activity, which
may be explained by the improved H-bond interactions of acetamide with NA.
Fig. 4. (A) Binding modes of compounds 6f (green) and 10i (yellow) with NA (PDB
2HU0). Details of H-bond interactions of 6f (B) and 10i (C).

3. Conclusions
In this study, based on the crystal structure of NA and combination principle of
drug design, we have designed, synthesized and evaluated the inhibitory activity of
N-substituted oseltamivir derivatives containing C-5-NHCH2-Aryl fragments. Four of
the synthesized compounds, 6d, 6e, 6f and 10i, showed potent inhibitory activity (˃
10 %) against NA at 1.0 nM. Among which, 6f and 10i were determined to be the
most potent inhibitors against A/H3N2 NA of a clinical sample with IC50 values of
1.92 ± 0.24 and 1.63 ± 0.16 nM, respectively. Further in silico ADME predictions
showed that the pharmacokinetic and pharmacodynamic parameters of the selected
compounds were all comparable to OC which suggested the druggability of these
derivatives. Molecular docking studies of compound 6f and 10i have been performed,
and the results showed that 6f and 10i adopted different modes of interaction with the
NA, in which 6f recruited the extra 150-cavity of the OC-NA binding site as designed.
Therefore, further studies on the optimal binding with NA by employing the
150-cavity is worthwhile. While the conformation of 10i was demonstrated to turn
over to display a totally novel binding mode with NA with the 1-ethylpropoxy group
extend into the inside of NA. As 1-ethylpropoxy was the problematic group causing
the oseltamivir-resistance in influenza viruses, the structural diversification and
optimization based on 10i may provide new solutions of anti-influenza therapy.

4. Experimental section
4.1 Biology
4.1.1 NA enzyme inhibitory assay
The NA inhibition assay was performed using the commercially available
NA-FluorTM Influenza Neuraminidase Assay Kit. The substrate, MUNANA
(4-(methylumbelliferyl)-N-acetylneuraminic acid) was cleaved by NA to yield a
quantifiable fluorescent product. The tested compounds were dissolved in DMSO and
diluted by the 1 × assay buffer (66.6 mM 2-(N-morpholino)ethanesulfonic acid (MES)
buffer, 8 mM CaCl2, pH 6.5) to 4 × the desired concentrations. In a 96-well plate, 25
µL of the 4 × compounds solution, 25 µL of the diluted virus sample were added and
incubated for 20 to 30 min at 37°C. 50 µL of diluted 200 µM NA-FluroTM Substrate
working solution was added to each well and incubated for 60 min at 37oC. Finally,
the reaction was terminated by adding 100 µL of NA-FluorTM Stop Solution (0.2 M
Na2CO3) and the fluorescence was read at an excitation wavelength of 350 nm and an
emission of 460 nm.
4.2. Chemistry
4.2.1 General methods
Starting materials and regents, unless otherwise started, were of commercial
grade and were used without further purification. All reactions were monitored by
thin-layer chromatography (TLC) on glass sheets (Silica gel F254) which can be
visualized under UV light. Flash chromatography was carried out using silica gel
1
(200-300 mesh). H NMR (400 MHz) and 13C NMR (100 MHz) spectra were

measured on BRUKER AVANCE-III spectrometer. Chemical shifts are expressed in
δ (ppm) and coupling constants (J) in Hz. The solvents for NMR were DMSO-d6 (δ
1
2.5 for H). High resolution MS spectra were measured using a QTOF-2 micromass
Spectrometer by electron spray ionization.
4.2.2. 2-chloro-N-hydroxyacetimidamide (2)
A round bottom flask was charged a magnetic stirring, chloroacetonitrile (132
mmol, 10 g), hydroxylamine (9.2 g, 132 mmol) and 100 ml water in ice bath. Na2CO3
was added portion wise to the mixture, then warm to room temperature. After 2 h,
sodium chloride aqueous solution was added, extracted with Et2O, combined organic
phase, washed by NaCl solution and filtered, the filtration was evaporated, and the
residue was dried in vacuo to provide the titled compound as white solid, 5 g, 35 %
yield.
4.2.3. General procedure for the synthesis of compound 3a – l
To a solution of compound 2a (1 eq.) in acetone was added acyl chloride
dropwise at 0oC. Subsequently, the mixture was warmed to room temperature. After 4
h, the volatiles were removed in vacuo and the residue was partitioned between water
and EA, the organic phase was washed with brine and dried with anhydrous Na2SO4.
The mixture was filtered, and the filtration was evaporated to afford crude product as
pale-yellow solid.
4.2.4. General procedure for the synthesis of compound 4a – l
Compound (3a – l) and acetic acid were added to a round bottom flask and
heated to reflux. When TLC indicated that the reaction was completed, it was cooled

to room temperature and the solvents was removed in vacuum. NaHCO3 aqueous
solution was added to the residue, extracted by EA. The organic phase was combined
and washed with brine and dried by anhydrous Na2SO4 and the crude mixture was
evaporated and purified by column chromatography to provide the titled compound.
4.2.5. General procedure for the synthesis of compound 5a – l
To a round-bottom flask were added Oseltamivir phosphate (1eq), compound (4a
– l) (1.2 eq.) and DMF. DIPEA (3 eq.) was added at room temperature and then heated
to 70oC. After 12 h, water was added to the reaction mixture followed by extracting
with EA. The combined organic phase was washed with NaCl solution, dried by
Na2SO4 and evaporated to provide the titled compounds.
4.2.6. General procedure for the synthesis of compound 6a – l
Compound (5a – l) (1 eq.) was dissolved in methanol and water (v: v = 5: 1),
NaOH (5 eq.) was added. The reaction mixture was heated to 50oC overnight.
Methanol was removed in vacuo and a small amount of water was added to the
reaction mixture. The aqueous phase was washed by CH2Cl2 twice, then acidified to
precipitate the target compounds.
4.2.7.
(3R,4R,5S)-4-Acetamido-3-(pentan-3-yloxy)-5-(((5-(thiophen-2-yl)-1,2,4-oxadiazol-3-
yl)methyl)amino)cyclohex-1-ene-1-carboxylic acid (6a)
Yield 38 %; white solid. 1H NMR (400 MHz, DMSO) δ 8.08 (d, J = 4.8 Hz, 1H),
8.01 (d, J = 3.5 Hz, 1H), 7.75 (d, J = 9.0 Hz, 1H), 7.42 – 7.25 (m, 1H), 6.60 (s, 1H),
3.99 (d, J = 7.9 Hz, 1H), 3.87 (dd, J = 31.2, 15.1 Hz, 2H), 3.64 (dd, J = 18.7, 9.1 Hz,

1H), 3.35 – 3.30 (m, 2H), 2.79 – 2.65 (m, 2H), 1.85 – 2.06 (m, 1H), 1.84 (s, 3H), 1.35
– 1.45 (m, 4H), 0.83 (t, J = 7.3 Hz, 3H), 0.78 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz,
DMSO) δ 171.1, 170.2, 170.1, 168.1, 137.6, 134.2, 132.9, 129.8, 129.7, 125.2, 81.3,
75.7, 54.8, 54.6, 41.4, 30.9, 26.1, 25.6, 23.4, 9.9, 9.4. HRMS (ESI, m/z) calcd for
C21H27N4O5S, 447.1708 [M-H-]; found, 447.1699.
4.2.8.
(3R,4R,5S)-4-Acetamido-3-(pentan-3-yloxy)-5-(((5-(4-(trifluoromethyl)phenyl)-1,2,4-
oxadiazol-3-yl)methyl)amino)cyclohex-1-ene-1-carboxylic acid (6b)
Yield 46 %; white solid. 1H NMR (400 MHz, DMSO) δ 8.33 (d, J = 8.1 Hz, 2H),
8.02 (d, J = 8.2 Hz, 2H), 7.72 (d, J = 8.9 Hz, 1H), 6.38 (s, 1H), 3.93 (dd, J = 34.1,
15.3 Hz, 3H), 3.62 (d, J = 9.1 Hz, 2H), 3.30 (s, 1H), 2.72 (d, J = 18.2 Hz, 2H), 2.02 –
1.89 (m, 1H), 1.48 – 1.30 (m, 4H), 0.83 (t, J = 7.3 Hz, 3H), 0.77 (t, J = 7.3 Hz, 4H).
13C NMR (101 MHz, DMSO) δ 174.2, 170.6, 170.2, 133.2, 132.9, 129.2, 127.7, 127.0
(q, J = 3.6 Hz), 125.4, 122.7, 81.1, 76.2, 55.0, 41.4, 39.7, 39.5, 39.3, 31.6, 26.2, 25.6,
23.5, 9.9, 9.4. HRMS (ESI, m/z) calcd for C24H28F3N4O5, 509.2017 [M-H-]; found,
509.2009.
4.2.9.
(3R,4R,5S)-4-Acetamido-5-(((5-(4-fluorophenyl)-1,2,4-oxadiazol-3-yl)methyl)amino)-
3-(pentan-3-yloxy)cyclohex-1-ene-1-carboxylic acid (6c)
Yield 39 %; white solid. 1H NMR (400 MHz, DMSO) δ 8.18 (dd, J = 8.5, 5.4 Hz,
2H), 7.76 (d, J = 9.0 Hz, 1H), 7.49 (t, J = 8.8 Hz, 2H), 6.60 (s, 1H), 3.99 (d, J = 7.7
Hz, 1H), 3.90 (dd, J = 29.6, 15.2 Hz, 2H), 3.65 (dd, J = 18.2, 9.0 Hz, 1H), 2.81 – 2.65

(m, 2H), 2.01 (dd, J = 16.3, 8.7 Hz, 1H), 1.40 (dd, J = 11.6, 5.5 Hz, 4H), 0.83 (t, J =
13
7.3 Hz, 3H), 0.78 (t, J = 7.3 Hz, 3H). C NMR (101 MHz, DMSO) δ 174.5, 170.4,
170.1, 168.0, 165.4 (d, J = 253.1 Hz), 137.7, 131.1 (d, J = 9.5 Hz), 129.8, 120.8,
117.3 (d, J = 22.5 Hz), 81.3, 75.7, 54.7, 54.7, 41.5, 30.9, 26.1, 25.6, 23.5, 9.9, 9.4.
HRMS (ESI, m/z) calcd for C23H28FN4O5, 459.2049 [M-H-]; found, 459.2035.
4.2.10.
(3R,4R,5S)-4-Acetamido-3-(pentan-3-yloxy)-5-(((5-(pyridin-2-yl)-1,2,4-oxadiazol-3-yl
)methyl)amino)cyclohex-1-ene-1-carboxylic acid (6d)
Yield 56 %; white solid. 1H NMR (400 MHz, DMSO) δ 8.83 (d, J = 4.5 Hz, 1H),
8.26 (d, J = 7.8 Hz, 1H), 8.11 (td, J = 7.8, 1.5 Hz, 1H), 7.71 (dd, J = 7.1, 5.0 Hz, 1H),
7.66 (d, J = 16.5 Hz, 1H), 7.01 (s, 1H), 6.28 (s, 1H), 3.83 – 4.05 (m, 3H), 3.64 – 3.57
(m, 1H), 3.25 – 3.33 (m, 1H), 2.78 – 2.60 (m, 2H), 1.92 (dd, J = 17.5, 8.9 Hz, 1H),
1.82 (s, 3H), 1.44 – 1.33 (m, 4H), 0.83 (t, J = 7.3 Hz, 3H), 0.77 (t, J = 7.4 Hz, 3H).
13C NMR (101 MHz, DMSO) δ 174.4, 170.5, 170.1, 151.0, 143.5, 143.4, 138.6, 130.9,
127.8, 124.8, 80.9, 76.7, 55.3, 55.2, 41.5, 32.3, 26.2, 25.6, 23.5, 9.9, 9.4. HRMS (ESI,
m/z) calcd for C22H28N5O5, 442.2096 [M-H-]; found, 442.2090.
4.2.11.
(3R,4R,5S)-4-Acetamido-5-(((5-(3-nitrophenyl)-1,2,4-oxadiazol-3-yl)methyl)amino)-3
-(pentan-3-yloxy)cyclohex-1-ene-1-carboxylic acid (6e)
Yield 66 %; white solid. 1H NMR (400 MHz, DMSO) δ 8.80 (s, 1H), 8.61 – 8.47
(m, 2H), 7.97 (t, J = 7.9 Hz, 2H), 6.64 (s, 1H), 4.15 (d, J = 46.0 Hz, 3H), 3.80 (s, 1H),
3.28 – 2.91 (m, 2H), 2.81 (d, J = 15.5 Hz, 1H), 2.27 (s, 1H), 1.87 (s, 3H), 1.33 – 1.52

(m, 4H), 0.81 (dt, J = 20.5, 7.2 Hz, 6H). 13C NMR (101 MHz, DMSO) δ 174.0, 170.6,
167.7, 148.8, 137.9, 134.3, 132.1, 128.8, 128.2, 125.1, 122.9, 81.4, 75.2, 55.0, 53.3,
26.1, 25.6, 23.6, 9.9, 9.4. HRMS (ESI, m/z) calcd for C23H28N5O7, 486.1994 [M-H-];
found, 486.1985.
4.2.12.
(3R,4R,5S)-4-Acetamido-5-(((5-(furan-2-yl)-1,2,4-oxadiazol-3-yl)methyl)amino)-3-(pe
ntan-3-yloxy)cyclohex-1-ene-1-carboxylic acid (6f)
Yield 51 %; white solid. 1H NMR (400 MHz, DMSO) δ 8.13 (s, 1H), 7.76 (d, J =
8.9 Hz, 1H), 7.55 (d, J = 3.0 Hz, 1H), 7.04 (s, 1H), 6.84 (s, 1H), 6.43 (s, 1H), 3.93 (s,
1H), 3.92 – 3.79 (m, 2H), 3.61 (d, J = 9.0 Hz, 1H), 3.30 (d, J = 4.8 Hz, 1H), 2.77 –
2.62 (m, 2H), 2.02 – 1.90 (m, 1H), 1.82 (s, 3H), 1.38 (s, 5H), 0.83 (d, J = 6.9 Hz, 3H),
13
0.76 (t, J = 7.1 Hz, 3H). C NMR (101 MHz, DMSO) δ 170.2, 170.0, 169.7, 167.3,
148.6, 139.7, 133.9, 133.2, 117.7, 113.5, 81.1, 76.2, 55.1, 55.0, 41.3, 31.8, 26.2, 25.6,
23.4, 9.9, 9.4. HRMS (ESI, m/z) calcd for C21H27N4O6, 431.1936 [M-H-]; found,
431.1930.
4.2.13.
(3R,4R,5S)-4-Acetamido-3-(pentan-3-yloxy)-5-(((5-phenyl-1,2,4-oxadiazol-3-yl)methy
l)amino)cyclohex-1-ene-1-carboxylic acid (6g)
Yield 49 %; white solid. 1H NMR (400 MHz, DMSO) δ 8.12 (d, J = 7.3 Hz, 2H),
7.76 (d, J = 9.1 Hz, 1H), 7.71 (d, J = 7.3 Hz, 1H), 7.64 (t, J = 7.5 Hz, 2H), 7.04 (s,
1H), 6.54 (s, 1H), 3.91 (dd, J = 32.1, 15.1 Hz, 3H), 3.64 (d, J = 9.3 Hz, 1H), 3.33 –
3.30 (m, 1H), 2.73 (dd, J = 17.6, 7.4 Hz, 2H), 2.00 (dd, J = 18.4, 10.7 Hz, 1H), 1.83 (s,

3H), 1.47 – 1.28 (m, 4H), 0.82 (dd, J = 14.4, 7.0 Hz, 3H), 0.77 (d, J = 7.4 Hz, 3H).
13C NMR (101 MHz, DMSO) δ 175.4, 170.4, 170.1, 168.7, 136.3, 133.6, 131.1, 130.0,
128.2, 124.0, 81.2, 75.9, 54.9, 54.8, 41.5, 31.2, 26.1, 25.6, 23.5, 9.9, 9.4. HRMS (ESI,
m/z) calcd for C23H29N4O5, 441.2143 [M-H-]; found, 441.2136.
4.2.14.
(3R,4R,5S)-4-Acetamido-5-(((5-(naphthalen-2-yl)-1,2,4-oxadiazol-3-yl)methyl)amino)
-3-(pentan-3-yloxy)cyclohex-1-ene-1-carboxylic acid (6h)
Yield 70 %, white solid. 1H NMR (400 MHz, DMSO) δ 8.81 (s, 1H), 8.17 (dt, J
= 15.3, 8.2 Hz, 3H), 8.06 (d, J = 7.9 Hz, 1H), 7.78 (d, J = 9.0 Hz, 1H), 7.75 – 7.64 (m,
2H), 6.62 (s, 1H), 4.00 (s, 1H), 3.95 (q, J = 13.6 Hz, 2H), 3.67 (dd, J = 18.3, 9.2 Hz,
1H), 2.85 – 2.70 (m, 2H), 2.04 (ddd, J = 11.7, 8.1, 5.8 Hz, 1H), 1.84 (s, 3H), 1.48 –
1.30 (m, 4H), 0.83 (t, J = 7.3 Hz, 3H), 0.78 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz,
DMSO) δ 175.5, 170.5, 170.2, 168.0, 137.9, 135.2, 132.8, 129.8, 129.7, 129.6, 129.4,
129.3, 128.4, 127.9, 124.0, 121.3, 81.3, 75.7, 54.8, 54.7, 41.6, 30.9, 26.1, 25.6, 23.5,
9.9, 9.4. HRMS (ESI, m/z) calcd for C27H31N4O5, 491.23 [M-H-]; found, 491.2288.
4.2.15.
(3R,4R,5S)-4-Acetamido-3-(pentan-3-yloxy)-5-(((5-(o-tolyl)-1,2,4-oxadiazol-3-yl)met
hyl)amino)cyclohex-1-ene-1-carboxylic acid (6i)
Yield 69 %; white solid. 1H NMR (400 MHz, DMSO) δ 8.04 (d, J = 7.7 Hz, 1H),
7.78 (d, J = 9.1 Hz, 1H), 7.57 (t, J = 7.2 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.44 (t, J =
7.7 Hz, 1H), 6.59 (s, 1H), 4.01 (s, 1H), 3.99 – 3.90 (m, 2H), 3.66 (d, J = 9.4 Hz, 1H),
3.33 (s, 1H), 2.83 – 2.69 (m, 2H), 2.65 (s, 3H), 2.02 (dd, J = 17.0, 9.3 Hz, 1H), 1.83 (s,

3H), 1.50 – 1.32 (m, 4H), 0.83 (t, J = 7.4 Hz, 3H), 0.78 (t, J = 7.4 Hz, 3H). 13C NMR
(101 MHz, DMSO) δ 175.9, 170.1, 169.9, 168.2, 138.9, 137.4, 133.0, 132.4, 130.2,
130.0, 127.1, 123.2, 81.3, 75.8, 54.8, 54.7, 41.5, 30.9, 26.1, 25.6, 23.4, 21.8, 9.9, 9.4.
HRMS (ESI, m/z) calcd for C24H31N4O5, 455.23 [M-H-]; found, 455.2312.
4.2.16.
(3R,4R,5S)-4-Acetamido-3-(pentan-3-yloxy)-5-(((5-(3,4,5-trimethoxyphenyl)-1,2,4-ox
adiazol-3-yl)methyl)amino)cyclohex-1-ene-1-carboxylic acid (6j)
Yield 55 %; white solid. 1H NMR (400 MHz, DMSO) δ 7.77 (d, J = 9.0 Hz, 1H),
7.38 (s, 2H), 6.59 (s, 1H), 3.99 (d, J = 8.0 Hz, 1H), 3.93 (d, J = 15.3 Hz, 2H), 3.89 (s,
6H), 3.77 (s, 3H), 3.66 (dd, J = 18.8, 9.0 Hz, 1H), 3.36 – 3.29 (m, 1H), 2.78 – 2.66 (m,
2H), 2.02 (dd, J = 17.4, 9.3 Hz, 1H), 1.85 (s, 3H), 1.33 – 1.50 (m, 4H), 0.83 (t, J = 7.3
Hz, 3H), 0.78 (t, J = 7.4 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 175.2, 170.4, 170.1,
168.1, 153.9, 142.0, 137.5, 130.0, 119.1, 105.6, 81.3, 75.7, 60.8, 56.7, 54.94, 54.6,
41.6, 31.0, 26.1, 25.6, 23.5, 9.9, 9.4. HRMS (ESI, m/z) calcd for C26H35N4O8,
531.246 [M-H-]; found, 531.2468.
4.2.17.
(3R,4R,5S)-4-Acetamido-5-(((5-(4-chlorophenyl)-1,2,4-oxadiazol-3-yl)methyl)amino)-
3-(pentan-3-yloxy)cyclohex-1-ene-1-carboxylic acid (6k)
Yield 39 %; white solid. 1H NMR (400 MHz, DMSO) δ 8.13 (d, J = 6.7 Hz, 2H),
7.84 (s, 1H), 7.74 (d, J = 6.6 Hz, 2H), 6.63 (s, 1H), 4.04 (s, 3H), 3.73 (s, 1H), 2.92 (s,
1H), 2.76 (d, J = 17.1 Hz, 1H), 2.15 (s, 1H), 1.84 (s, 3H), 1.41 (s, 4H), 0.90 – 0.69 (m,
6H). 13C NMR (101 MHz, DMSO) δ 174.7, 170.4, 167.8, 138.7, 138.0, 136.0, 131.6,

130.3, 130.1, 129.2, 124.4, 122.7, 81.4, 75.5, 54.9, 54.0, 29.5, 26.1, 25.6, 23.6, 9.9,
9.4. HRMS (ESI, m/z) calcd for C23H28ClN4O5, 475.1754 [M-H-]; found, 475.1762.
4.2.18.
(3R,4R,5S)-4-acetamido-5-(((5-(2,6-difluorophenyl)-1,2,4-oxadiazol-3-yl)methyl)amin
o)-3-(pentan-3-yloxy)cyclohex-1-ene-1-carboxylic acid (6l)
1
Yield 43 %; white solid. H NMR (400 MHz, DMSO) δ 7.89 – 7.77 (m, 2H),
7.43 (t, J = 9.0 Hz, 2H), 6.62 (s, 1H), 4.11 – 3.93 (m, 3H), 3.69 (dd, J = 18.7, 9.1 Hz,
1H), 3.37 – 3.31 (m, 1H), 2.85 (s, 1H), 2.73 (dd, J = 17.4, 4.2 Hz, 1H), 2.08 (t, J =
12.2 Hz, 1H), 1.83 (s, 3H), 1.49 – 1.35 (m, 4H), 0.83 (t, J = 7.3 Hz, 3H), 0.78 (t, J =
7.4 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 170.2, 169.9, 168.2, 167.9, 160.5 (d, J =
259.2 Hz), 160.3 (d, J = 259.4 Hz), 138.0, 136.3 (d, J = 11.1 Hz), 129.4, 113.6 (d, J =
21.2 Hz), 113.5 (d, J = 21.2 Hz), 103.0 (d, J = 16.3 Hz), 81.3, 75.6, 54.8, 54.4, 41.3,
30.5, 26.1, 25.6, 23.4, 9.9, 9.4. HRMS (ESI, m/z) calcd for C23H27F2N4O5, 477.1955
[M-H-]; found, 477.1965.
4.2.19. General procedure for the synthesis of compound 8a – h
5-Bromofuran-2-carbaldehyde (175 mg, 1 mmol), boronic acid (1 mmol, 1
equiv), Pd(dppf)2Cl2.DCM (16 mg, 2 % equiv), Na2CO3 (318 mg, 3 mmol, 3 equiv)
were added to a Shrenk flask with 6 ml of Toluene, 1.5 ml H2O and 1.5 ml EtOH. The
reaction mixture was charged with N2 and heated at 110oC overnight. The solution
was extracted with EtOAc and the combined extracts were dried over Na2SO4.
Chromatographic purification gave the titled compounds.
4.2.20. General procedure for the synthesis of compound 9a – h

To a solution of oseltamivir phosphate (123 mg, 0.3 mmol) and aldehyde (0.36
mmol, 1.2 equiv) in 10 ml ethanol, NaBH(OAc)3 (318 mg, 1.5 mmol) was slowly
added. The mixture was stirred at room temperature overnight and then concentrated.
To the residue, 20 ml saturated NaHCO3 solution was added, and the mixture was
extracted with EtOAc. The combined extracts were dried over anhydrous Na2SO4 and
purified by chromatograph to give the products.
4.2.21. General procedure for the synthesis of compound 10a – h
Compound (9a – h) (1 eq.) was dissolved in methanol and water (v: v = 5: 1),
NaOH (5 eq.) was added. The reaction mixture was heated to 50oC overnight.
Methanol was removed in vacuum and a small amount of water was added to the
reaction mixture. The aqueous phase was washed by CH2Cl2 twice, then acidified to
precipitate the target compounds.
4.2.22.
(3R,4R,5S)-4-Acetamido-5-(((5-(4-methoxyphenyl)furan-2-yl)methyl)amino)-3-(penta
n-3-yloxy)cyclohex-1-ene-1-carboxylic acid (10a)
Yield 68 %; white solid. 1H NMR (400 MHz, DMSO) δ 7.81 (d, J = 9.0 Hz, 1H),
7.59 (d, J = 8.7 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 6.68 (d, J = 3.1 Hz, 1H), 6.61 (s,
1H), 6.29 (d, J = 3.1 Hz, 1H), 4.00 (d, J = 7.8 Hz, 1H), 3.80 (d, J = 18.1 Hz, 4H), 3.74
– 3.63 (m, 2H), 3.34 (dt, J = 10.7, 5.3 Hz, 1H), 2.84 – 2.65 (m, 2H), 2.02 (dd, J = 16.9,
13
9.2 Hz, 1H), 1.85 (s, 3H), 1.33 – 1.52 (m, 4H), 0.73 – 0.89 (m, 6H). C NMR (101
MHz, DMSO) δ 170.1, 168.1, 159.0, 154.0, 152.5, 137.8, 129.8, 125.1, 124.0, 114.7,
109.1, 105.0, 81.3, 75.8, 55.6, 54.7, 54.7, 43.2, 30.9, 26.1, 25.6, 23.5, 9.9, 9.4. HRMS

(ESI, m/z) calcd for C26H33N2O6, 469.2344 [M-H-]; found, 469.2350.
4.2.23.
(3R,4R,5S)-4-Acetamido-5-(((5-(4-fluorophenyl)furan-2-yl)methyl)amino)-3-(pentan-
3-yloxy)cyclohex-1-ene-1-carboxylic acid (10b)
Yield 59 %; white solid. 1H NMR (400 MHz, DMSO) δ 7.81 (d, J = 8.9 Hz, 1H),
7.75 – 7.52 (m, 2H), 7.25 (t, J = 8.5 Hz, 2H), 6.83 (s, 1H), 6.60 (s, 1H), 6.33 (s, 1H),
3.99 (s, 1H), 3.77 (dd, J = 36.1, 15.0 Hz, 2H), 3.67 (d, J = 9.2 Hz, 1H), 3.34 (s, 1H),
2.73 (dd, J = 23.4, 11.8 Hz, 2H), 2.01 (dd, J = 16.1, 9.2 Hz, 1H), 1.85 (s, 3H), 1.40
(dd, J = 11.0, 5.5 Hz, 4H), 0.90 – 0.66 (m, 6H). 13C NMR (101 MHz, DMSO) δ 170.1,
164.9 (d, J = 245.1 Hz), 155.0, 151.5, 137.6, 130.0, 127.7 (d, J = 2.9 Hz), 125.6 (d, J
= 8.1 Hz), 116.2 (d, J = 22.0 Hz), 109.3, 106.7, 81.3, 75.8, 54.7, 43.2, 40.6, 40.4, 40.2,
40.0, 39.8, 39.6, 39.4, 31.0, 26.1, 25.6, 23.5, 9.9, 9.4. HRMS (ESI, m/z) calcd for
C25H30FN2O5, 457.2144 [M-H-]; found, 457.2152.
4.2.24.
(3R,4R,5S)-4-Acetamido-3-(pentan-3-yloxy)-5-(((5-(o-tolyl)furan-2-yl)methyl)amino)c
yclohex-1-ene-1-carboxylic acid (10c)
Yield 70 %; white solid. 1H NMR (400 MHz, DMSO) δ 7.80 (d, J = 9.0 Hz, 1H),
7.64 (d, J = 7.4 Hz, 1H), 7.31 – 7.18 (m, 3H), 6.66 – 6.58 (m, 2H), 6.37 (d, J = 3.0 Hz,
1H), 4.01 (d, J = 7.8 Hz, 1H), 3.79 (dd, J = 39.2, 14.8 Hz, 2H), 3.68 (dd, J = 18.7, 9.3
Hz, 1H), 3.38 – 3.30 (m, 1H), 2.65 – 2.85 (m, 2H), 2.44 (s, 3H), 2.02 (dd, J = 17.2,
9.1 Hz, 1H), 1.85 (s, 3H), 1.33-1.50 (m, 4H), 0.84 (t, J = 7.5 Hz, 3H), 0.79 (t, J = 7.4
Hz, 3H). 13C NMR (101 MHz, DMSO) δ 170.0, 168.2, 154.5, 151.9, 137.7, 134.1,

131.7, 130.2, 129.9, 127.7, 126.6, 126.5, 110.1, 109.0, 81.3, 75.8, 54.8, 54.7, 43.1,
30.9, 26.1, 25.6, 23.4, 22.1, 9.9, 9.4. HRMS (ESI, m/z) calcd for C26H33N2O5,
453.2395 [M-H-]; found, 453.2404.
4.2.25.
(3R,4R,5S)-4-Acetamido-5-(((5-(3,5-difluorophenyl)furan-2-yl)methyl)amino)-3-(pent
an-3-yloxy)cyclohex-1-ene-1-carboxylic acid (10d)
Yield 61 %; white solid. 1H NMR (400 MHz, DMSO) δ 7.79 (d, J = 9.0 Hz, 1H),
7.37 (d, J = 6.9 Hz, 2H), 7.10 (dd, J = 18.8, 6.2 Hz, 2H), 6.61 (s, 1H), 6.39 (d, J = 2.9
Hz, 1H), 4.01 (d, J = 7.6 Hz, 1H), 3.79 (dd, J = 35.1, 15.0 Hz, 2H), 3.67 (dd, J = 18.3,
9.1 Hz, 1H), 3.30 – 3.40 (m, 1H), 2.83 – 2.62 (m, 2H), 2.02 (dd, J = 16.8, 9.1 Hz, 1H),
1.86 (s, 3H), 1.33 – 1.50 (m, 4H), 0.90 – 0.74 (m, 6H). 13C NMR (101 MHz, DMSO)
δ 170.0, 168.1, 163.4 (d, J = 245.9 Hz), 163.3 (d, J = 246.4 Hz) , 156.2, 150.1, 137.8,
134.2, 134.1, 134.0, 129.7, 109.7 (d, J = 8.3 Hz), 106.5 (d, J = 27.5 Hz), 102.6 (d, J =
26.2 Hz) , 81.3, 75.7, 54.7, 54.6, 43.0, 30.8, 26.1, 25.6, 23.4, 9.9, 9.4. HRMS (ESI,
m/z) calcd for C25H29F2N2O5, 475.205 [M-H-]; found, 475.2060.
4.2.26.
(3R,4R,5S)-4-Acetamido-5-(((5-(3,4-dimethoxyphenyl)furan-2-yl)methyl)amino)-3-(pe
ntan-3-yloxy)cyclohex-1-ene-1-carboxylic acid (10e)
Yield 66 %; white solid. 1H NMR (400 MHz, DMSO) δ 7.98 (d, J = 6.6 Hz, 1H),
7.25 (s, 2H), 7.04 – 6.93 (m, 1H), 6.80 (d, J = 2.7 Hz, 1H), 6.65 (s, 1H), 6.51 (s, 1H),
3.95 – 4.22 (m, 3H), 3.86 (d, J = 13.3 Hz, 1H), 3.82 (s, 3H), 3.78 (s, 3H), 3.33 – 3.42
(m, 1H), 3.10 (s, 1H), 2.88 (d, J = 16.5 Hz, 1H), 2.35 (s, 1H), 1.89 (s, 3H), 1.48 – 1.36

13
(m, 4H), 0.77 – 0.89 (m, 6H). C NMR (101 MHz, DMSO) δ 170.6, 167.6, 153.7,
149.6, 149.1, 138.0, 128.9, 123.8, 116.7, 112.7, 112.0, 108.0, 105.6, 81.5, 75.3, 56.1,
56.1, 54.5, 53.0, 41.3, 28.5, 26.1, 25.6, 23.7, 9.8, 9.3. HRMS (ESI, m/z) calcd for
C27H35N2O7, 499.245 [M-H-]; found, 499.2459.
4.2.27.
(3R,4R,5S)-4-Acetamido-5-(((5-(3-chlorophenyl)furan-2-yl)methyl)amino)-3-(pentan-
3-yloxy)cyclohex-1-ene-1-carboxylic acid (10f)
Yield 65 %; white solid. 1H NMR (400 MHz, DMSO) δ 7.75 (d, J = 8.8 Hz, 1H),
7.70 (s, 1H), 7.62 (d, J = 7.5 Hz, 1H), 7.43 (t, J = 7.8 Hz, 1H), 7.30 (d, J = 7.5 Hz,
1H), 6.99 (d, J = 2.0 Hz, 1H), 6.34 (d, J = 9.8 Hz, 2H), 3.92 (d, J = 6.4 Hz, 1H), 3.77
(dd, J = 47.8, 14.8 Hz, 2H), 3.63 (dd, J = 17.6, 8.8 Hz, 1H), 3.31 (s, 1H), 2.70 (t, J =
11.6 Hz, 2H), 1.99 – 1.89 (m, 1H), 1.85 (s, 3H), 1.33-1.45 (m, 4H), 0.87 – 0.72 (m,
6H). 13C NMR (101 MHz, DMSO) δ 170.0, 155.9, 150.7, 135.9, 134.17, 132.9, 131.2,
127.2, 123.1, 122.1, 109.4, 108.4, 80.9, 76.6, 55.3, 55.3, 43.2, 32.2, 26.2, 25.7, 23.5,
10.0, 9.5. HRMS (ESI, m/z) calcd for C25H30ClN2O5, 473.1849 [M-H-]; found,
473.1861.
4.2.28.
(3R,4R,5S)-4-Acetamido-3-(pentan-3-yloxy)-5-(((5-(thiophen-3-yl)furan-2-yl)methyl)a
mino)cyclohex-1-ene-1-carboxylic acid (10g)
Yield 55 %; white solid. 1H NMR (400 MHz, DMSO) δ 7.80 (d, J = 9.0 Hz, 1H),
7.62 (s, 2H), 7.40 (dd, J = 4.0, 1.9 Hz, 1H), 6.64 (d, J = 2.9 Hz, 1H), 6.61 (s, 1H),
6.29 (d, J = 2.8 Hz, 1H), 4.01 (d, J = 7.7 Hz, 1H), 3.76 (dd, J = 32.9, 11.8 Hz, 2H),