Discovery of acylguanidine oseltamivir carboxylate derivatives as potent neuraminidase inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.03.052

In search of novel anti-influenza agents with higher potency, a series of acylguanidine oseltamivir carboxylate analogues were synthesized and evaluated against influenza viruses (H1N1 and H3N2) in vitro. The representative compounds with strong inhibitory activities (IC₅₀ <40?nM) against neuraminidase (NA) were further tested against the NA from oseltamivir-resistant strain (H259Y). Among them, compounds 9 and 17 were potent NA inhibitors that exhibited a 5 and 11-fold increase in activity comparing with oseltamivir carboxylate (2, OC) against the H259Y mutant, respectively. Furthermore, the effect against influenza virus H259Y mutant (H1N1) replication and cytotoxicity assays indicated that compounds 9 and 17 exhibited a 20 and 6-fold increase than the parent compound 2, and had no obvious cytotoxicity in vitro. Moreover, the molecular docking studies revealed that the docking modes of compounds 9 and 17 were different from that of oseltamivir, and the new hydrogen bonds and hydrophobic interaction were formed in this case. This work provided unique insights in the discovery of potent inhibitors against NAs from wild-type and oseltamivir-resistant strains.

Full text of DOI:10.1016/j.bmc.2017.03.052

Accepted Manuscript
Discovery of acylguanidine oseltamivir carboxylate derivatives as potent neu-
raminidase inhibitors
Zhaoliang Li, Yanchun Meng, Shengtao Xu, Wang Shen, Zhaoqing Meng,
Zhenzhong Wang, Gang Ding, Wenzhe Huang, Wei Xiao, Jinyi Xu
PII:
S0968-0896(17)30143-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.03.052
BMC 13650
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 January 2017
22 March 2017
24 March 2017
Please cite this article as: Li, Z., Meng, Y., Xu, S., Shen, W., Meng, Z., Wang, Z., Ding, G., Huang, W., Xiao, W.,
Xu, J., Discovery of acylguanidine oseltamivir carboxylate derivatives as potent neuraminidase inhibitors,
Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.03.052
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Discovery of acylguanidine oseltamivir carboxylate derivatives as
potent neuraminidase inhibitors
Zhaoliang Lia,b,c, Yanchun Menga, Shengtao Xua, Wang Shenb,c, Zhaoqing Mengb,c,
Zhenzhong Wangb,c, Gang Dingb,c, Wenzhe Huangb,c, Wei Xiaob,c,*and Jinyi Xua,*
a State Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China
Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, P.R. China
b Jiangsu Kanion Pharmaceutical Co. Ltd., 58 South Haichang Road, Lianyungang, 222001 P.R.
China
c State Key Laboratory of New-tech for Chinese Medicine Pharmaceutical Process, Lianyungang,
222001, P.R. China
Abstract:
In search of novel anti-influenza agents with higher potency, a series of acylguanidine
oseltamivir carboxylate analogues were synthesized and evaluated against influenza
viruses (H1N1 and H3N2) in vitro. The representative compounds with strong
inhibitory activities (IC50<40nM) against neureminidase (NA) were further tested
against the NA from oseltamivir-resistant strain (H259Y). Among them, compounds 9
and 17 were potent NA inhibitors that exhibited a 5 and 11-fold increase in activity
comparing with oseltamivir carboxylate (2, OC) against the H259Y mutant,
respectively. Furthermore, the effect against influenza virus H259Y mutant (H1N1)
replication and cytotoxicity assays indicated that compounds 9 and 17 exhibited a 20
and 6-fold increase than the parent compound 2, and had no obvious cytotoxicity in
vitro. Moreover, the molecular docking studies revealed that the docking modes of
compounds 9 and 17 were different from that of oseltamivir, and the new hydrogen
bonds and hydrophobic interaction were formed in this case. This work provided
unique insights in the discovery of potent inhibitors against NAs from wild-type and
oseltamivir-resistant strains.

Keywords: Influenza viruses; Neuraminidase inhibitors; Acylguanidine oseltamivir
carboxylate; H1N1; H3N2; Oseltamivir-resistant strain (H259Y)
.* Corresponding authors.Tel.: +86-25-83271299; fax: +86-25-83302827;
E-mail addresses: jinyixu@china.com, kanionlunwen@163.com
1. Introduction
Influenza has been a serious threat to human health for centuries. Influenza viruses
were found around the world and infected millions of people annually, leading to life
threatening respiratory illness. Three pandemic influenza outbreaked during the 20th
century, the 1918 Spanish flu, the 1957 Asian flu and the 1968 Hong Kong flu, which
represented three different antigenic subtypes of influenza A virus: H1N1, H2N2, and
H3N2, respectively.1-3 In 2009, the swine-origin H1N1 influenza virus spread rapidly
throughout the world, bringing people into a renewed panic.4 Even worse, since the
influenza viruses evolve rapidly, H5N1, H7N9 and H10N8 influenza viruses were
identified constantly in recent years.5 Strategies for combating influenza mainly aimed
at the M2 ion channel protein and neuraminidase (NA), which were essential for viral
replication. However, the M2 ion channel protein inhibitors were no longer
recommended for the treatment of influenza infection due to their severe side effects
and drug resistance.6 The NA, a surface glycoprotein of influenza viruses, facilitates
the release of viral progeny from infected cells and accelerates viral invasion of the
upper airways.7
NA is a good therapeutic target because it’s active site is relatively well conserved
across the influenza A and B viral strains.7, 8 The NAs are phylogenetically categorized
into two groups: group 1 (N1, N4, N5, and N8 subtypes) and group 2 (N2, N3, N6,
N7, and N9 subtypes).9 To date four neuraminidase inhibitors (NAI) have been
currently approved for the treatment or prophylaxis of influenza infections in some
parts of the world: oseltamivir (1),10 zanamivir (3),11 peramivir (4)12 and laninamivir
octanoate (5).13
Oseltamivir (TamifluTM) is the first orally available anti-influenza drug, which is

readily converted to oseltamivir carboxylate (2, OC) by endogenous esterases, as the
real active form widely distributed in the body. The crystal structure of OC (2) bound
to NA was investigated by X-ray.14 The carboxylate was held strongly by Arg292,
Arg371 and Arg118. The amino group formed strong hydrogen bond with Glu119 and
Asp151. The hydrophobic interaction was formed between the 3-pentyloxy side chain
and four amino acid residues (Ile222, Arg224, Ala246 and Glu276). The oxygen atom
of the amide interacted with Arg152. However, the oseltamivir-resistant influenza
viruses have emerged in recent years.8,15 Some oseltamivir-resistant mutations have
been identified, such as H274Y, I117V, E119A, R292K and so on.16 The mutations
may lead to changes in the interaction effect between functional groups of OC (2) and
amino acid residues of NA. These emerging problems of oseltamivir increased the
demand for the development of novel anti-influenza NA inhibitors.
Figure 1. Structures of clinically used NA inhibitors
Fortunately, new characteristics have been found through the analysis of the X-ray
crystal structures of NAs. Russell’s team found that group-1 and group-2 NAs were
structurally distinct, and group-1 NAs contained a so-called “150-cavity” adjacent to

their active sites.17 In addition, 371-cavity and 430-cavity were also reported by Wong
and the Amaro team, respectively.18-20 These structure characteristics implied
additional interactions between NAs and their inhibitors, suggesting the design of new
anti-influenza drugs should take advantage of 150-, 371- and 430- cavities. For fully
exploiting 150-pocket found in group-1 NA, the groups of Pinto21, Martin22 and Xu23
designed various kinds of oseltamivir analogues bearing different substitutents at the
C-5 position of oseltamivir and the results were satisfactory. Wong pointed that
tamiphosphor derivatives exhibited potent inhibitory activities against influenza
viruses because the 371-cavity could accommodate the alkyl substituents of
tamiphosphor monoesters.20 Lin and co-workers synthesized a series of
acylguanidine-modified zanamivir analogs and evaluated their inhibitory activities
against the NAs of influenza viruses (H1N1 and H3N2).24, 25 The results showed that
acylguanidine-modified zanamivir analogs bearing hydrophobic groups exhibited
inhibitory activity against NAs at the nanomolar scale. Though these compounds
didn’t show selectivity between H1N1 and H3N2, they correlated well with Amaro’s
research findings that the 150-cavity may exist not only in group-1 NAs but also in
group-2 NAs.26
Figure 2. Design of acylguanidine oseltamivir carboxylate derivatives
Enlightened by these newly revealed sites of NA and Lin’s work24, 25, we attempted
to find novel anti-influenza agents with higher potency and displayed much more
advantages.Therefore, we supposed that more potent NA inhibtors may be obtained
by introducing acylguanidine into oseltamivir carboxylate. Making good use of the

150-cavity, 371-cavity or 430-cavity of the neuraminidase, we modified the C-5
amino group of oseltamivir by substituted acylguanidine analogues for improving
interaction with NA (Figure 2) and evaluated the activities of the derivatives against
NAs from wild-type and oseltamivir-resistant strains (H259Y). Thus a series of
acylguanidine oseltamivir carboxylate derivatives were synthesized and further
evaluated for their biological activities.
2. Results and discussion
2.1 Chemistry
The synthetic route of acylguanidine oseltamivir carboxylate derivatives was
shown in Scheme 1. Compounds 6a-33a were prepared according to the previously
reported method.27 Then, commercially available oseltamivir phosphate was used as
the starting material to install the acylguanidine to the C-5 position of oseltamivir. The
oseltamivir phosphate was subjected to react with 6a-33a in the presence of
Mercury(II) chloride (HgCl2), triethylamine (Et3N) and N,N-dimethyl formamide
(DMF) to give compounds 6b-33b, which were subsequently treated with
trifluoroacetic acid (TFA) to afford acylguanidine oseltamivir derivatives 6c-33c.
Finally, the hydrolysis of 6c-33c, using the weak base LiOH in H2O-THF (v/v=1/1) or
K2CO3 in H2O-ethanol (v/v=1/4)24, offered the target acylguanidine oseltamivir
carboxylate derivatives in total yield of 40–90% by pre-HPLC.
For the formation of acylguanidine bond, our attempts for the direct coupling
reaction of the carboxyl group with the guanidino group failed, even using promoting
agents
benzotriazol-1-yl-oxytrispyrrolidinophosphonium
hexafluorophosphate
(PyBOP) (Scheme 2, Supporting Information). In addition, the strategy that
compounds 6b-33b were directly hydrolysed followed by removal of the Boc
protecting group with TFA to afford 6-33, was not viable because the acylguanidine
group protected by Boc was unstable to the alkali (Scheme 3, Supporting
Information).

HO
R
H
BocN
N
R
BocN
NH2
HN
NH2
a
O
b
S
O
S
S
6a-33a
O
O
O
O
O
O
O
O
O
O
d
c
HN
HN
HN
H
N
H
N
6a-33a
NH2
HN
R
HN
R
O
O
O
NBocO
6b-33b
NH
6c-33c
oseltamivir phosphate
O
O
OH
e, f
HN
H
HN
N
R
O
NH
6-33
O
Scheme 1. General route for the synthesis of acylguanidine oseltamivir carboxylate
derivatives.
Reagents and conditons: (a) Boc2O, NaOH, t-BuOH, rt; (b) PyBOP, NMM, DMF, rt; (c) HgCl2,
Et3N, DMF, rt; (d) TFA/CH2Cl2, rt; (e) LiOH, THF, H2O, rt; (f) HPLC purify.
2.2 In vitro inhibitory activities on NAs
The H1N1 and H3N2 were the representatives of group-1 and group-2 NAs. The
inhibitory activities of synthetic compounds 6-33 against the H1N1, H3N2 and
H1N1(H259Y) NAs were evaluated, and the results are shown in Table 1. OC (2) was
used as the positive control with IC50 values of 0.76 nM, 0.80 nM and 169.3 nM
against H1N1, H3N2 and H1N1(H259Y), respectively. Acylguanidine oseltamivir
carboxylate derivatives were found to be less potent than parent compound OC (2)
against H1N1 and H3N2 NAs and didn’t display selectivity for H1N1 and H3N2 NAs.
The results were similar to Lin’s work24, 25 and Amaro’s research findings26. However,
to our delight, part of the synthesized compounds showed better inhibitory activities
against the H1N1(H259Y) NA than OC (2). The further structure activity relationship
(SAR) studies showed that the inhibitory activities of these compounds mainly
depended on the kinds and positions of the substituents on acylguanidine. Compounds

6-11, with alkyl substituents at the acylguanidine group, had moderate inhibitory
activities against all three NAs, and the length of alkyl chain had no obviously impact
on the activity. Interestingly, compound 9 bearing unsaturated double bonds at the
terminal of the long alkyl chain, was 5-fold (30.5 nM) more potent than OC (169.3
nM) against H1N1(H259Y) NA, and still maintained inhibitory activities against
H1N1 and H3N2 NAs with IC50 values of 8.1 nM and 5.4 nM respectively. When the
steric hindrance of alkyl substituents increased (compounds 10, 11), the inhibitory
activities gradually decreased, especially against the H1N1 (H259Y) NA.
Unfortunately, compounds 19-33, bearing various aryl substitents, exhibited
significantly decreased activities aganist NAs from wild-type strains except
compounds 25 and 31-33, and compounds 31-33 also remained activity against
H1N1(H259Y) NA. The SARs of these substituted compounds seemed intricate,
mainly depending on the types and positions of the aryl substituents. However, when
a methylene unit was introduced between the acylguanidine and aromatic ring
(compounds 12-15), the inhibitory activities against H1N1 and H3N2 NAs were
significantly improved, especially compound 14 with a halogen atom (Br) on the
benzene ring. At the same time, compound 14 also showed good inhibitory activity
against H1N1 (H259Y) with a 3-fold increase relative to OC. Although the potency of
inhibition decreased slightly with longer linkers between the acylguanidine and
aromatic ring (compounds 16, 18), compound 17 with the methoxyl substituents at the
C-3 and C-4 positions of the benzene ring, was the most potent NA inhibitor among
all these tested compounds. It showed good inhibitory activities against H1N1
(IC50=1.43 nM) and H3N2 (IC50=3.64 nM), and it also exhibited a approximately
11-fold increased activity (IC50=14.47nM) than OC (IC50=169.3 nM ) against
H1N1(H259Y) NA. Furthermore, the heteroaromatic ring substituented compounds
(e.g. pyridine compounds 32, 33) also displayed good inhibitory activity against
H1N1 (H259Y) with a 4-8 folds increase compared with OC.
In contrast to the OC (2) , most acylguanidine oseltamivir carboxylate derivatives
not merely maintained potent inhibitory effects against NAs from wild-type strains
but also enhanced activities against NA from the resistant strain (Table 1), especially

compounds 9 and 17 which exhibited a 5-fold and 11-fold increase, respectively.
Furthermore, cell-based assays further indicated that compounds 9 and 17 were safe
in vitro ( Table 2).
Table 1. Inhibitory activities of novel acylguanidine oseltamivir carboxylate
derivatives against NAs
O
O
OH
HN
H
HN
N
R
O
NH
6-33
O
Compd
R
H1N1a
H3N2b
IC50 (nM)
0.80 0.08
H1N1-H259Yc
IC50 (nM)
IC50 (nM)d
0.76 0.06
169.3 9.1
OC(2)
39.1 1.3
34.8 1.6
37.0 1.8
8.1 0.6
11.4 0.8
11.2 0.8
6.0 0.5
5.4 0.3
74.2 3.4
132.9 7.7
163.3 5.0
30.5 2.0
6
7
8
9
38.2 2.6
88.3 13.8
86.5 7.8
29.4 1.8
13.7 1.1
18.4 1.0
22.8 2.1
9.5 0.7
132.2 5.6
445.2 18.5
225.1 23.6
131.9 10.6
10
11
12
13

14.5 1.8
21.1 3.7
30.1 2.1
1.43 0.21
64.9 5.0
573.8 30.3
8.0 0.9
40.6 3.4
66.7 12.2
113.7 18.2
14.47 0.59
221.1 20.1
NDe
14
15
16
17
18
19
7.2 0.9
9.4 1.7
3.64 0.35
21.4 1.4
334.2 58.0
>1000
>1000
ND
ND
ND
ND
ND
20
21
22
23
24
CH3
CF3
OH
>1000
911.1 98.7
184.7 25.1
>1000
49.8 4.9
>1000
53.5 4.6
125.2 13.9
11.5 1.9
19.5 3.5
76.1 4.0
>1000
ND
25
26
216.5 15.5

175.3 15.2
155.3 18.1
533.2 47.6
158.9 10.8
ND
ND
27
28
>1000
>1000
ND
29
30
31
32
33
68.6 10.6
20.1 3.0
10.2 1.8
15.2 2.4
214.6 25.1
14.2 2.0
14.6 2.2
ND
57.8 4.8
21.1 3.2
36.8 4.6
16.1 2.0
a
b
c
A/PuertoRico/8/1934(H1N1). A/Hong Kong/498/97(H3N2). A/PuertoRico/8/1934
d
(H259Y, NA resisitant strain). IC50: compound concentration required to inhibit virus
e
production by 50%, as determined by NA activity. ND: not determined.
2.3 Docking analysis
To better understand the effect of acylguanidine group on activity, computer
docking studies of the inhibitors with NA were performed. The binding models of
compounds 9 and 17 with the H1N1 enzyme were analyzed using Auto Dock
Vinaprogam. As shown in Figure 3A, the acylguanidine moiety of compounds 9 and
17 interacts with the space nearby the 150- cavity and 430- cavity by hydrophobic
interaction. In addition, to avoid the steric hindrance, the docking conformations of
compounds 9 and 17 are different from the docking mode of oseltamivir, as shown in
Figure 3B and 3C. Though the structures of compounds 9 and 17 are distorted in
active pockets, compound 9 forms five hydrogen bonds with the residues Arg118,
Asp151, Ser179, Arg292 and Arg371 and compound 17 also forms five hydrogen

bonds with the residues Arg118, Glu119, Asp151, Arg371 and Tyr406. Furthermore,
comparing to compound 9, pi-pi stacking effect may be formed between the benzene
ring of compound 17 and Tyr347. The pi-pi stacking effect may illustrate the slight
better inhibitory activity of compound 17 against the H1N1(H259Y) NA. Of course,
the methoxyl moiety may also play a role in the action. Overall, although the
modification of the guanidino group of oseltamivir results in the loss of interaction
with the original active pockets, the new formed H-bonds and hydrophobic interaction,
between the acylguanidine moiety and the space nearby the 150-cavity and 430-cavity,
compensates for this loss in binding energy.
Figure 3. Computational conformations of the compounds in H1N1 neuraminidase.
Molecular surface of H1N1 neuraminidase with bound compound 9 (A) and the
docking results of compounds 9 (B), 17 (C), and oseltamivir (D) with H1N1
neuraminidase (PDB ID: 2HU4).
2.4 In vitro anti-influenza virus activity
Next, we evaluated the anti-influenza activity and cytotoxicity of representative
compounds (6, 9, 14, 15, 17, 31) by cell-based assays (Table 2). The results showed
that these compounds could inhibit influenza virus replication in vitro and had no

obvious cytotoxicity. The selective values (SI) of these compounds were all higher
than OC (2), especially compound 9. These promising results in cells correlated well
with their high inhibitory activities against NAs, suggesting these compounds
inhibited influenza virus replication by binding to NAs. All the tested compounds
showed better inhibitory activities than OC (2) in cells infected by H3N2 subtype
except compound 31. In addition, the inhibitory effects of these compounds were
significantly improved against the H259Y mutant (H1N1), as comparing with OC (2),
especially compounds 9 and 17 which exhibited a 20 and 6-fold increase, respectively.
Interestingly, compounds 9 and 15 showed better inhibitory activities against the
H259Y mutant (H1N1) than compounds 17 using cell-based assays, disagreeing with
inhibitory activities against NA. The prominent anti-infulenza activities together with
the inhibitory activities against NAs indicate that compounds 9 and 17 are promising
for further development of novel anti-influenza drugs.
Table 2. Potency against influenza virus replication and cytotoxicity of compounds by
cell-based assays
Compd
H1N1a
EC50 (nM)
8.26
H3N2b
EC50 (nM)
14.77
5.30
H1N1-H259Yc CC50(nM)
EC50 (nM)
SId
OC(2)
6
151.7
37.32
7.18
> 1000
> 1000
> 1000
> 1000
> 1000
> 1000
> 6.6
> 26.8
> 139.3
> 14.5
> 88.6
> 43.4
> 7.4
23.88
24.52
9.45
9
12.88
2.58
69.08
11.29
23.02
135.7
14
15
17
31
8.45
3.95
20.06
5.62
159.5
39.17
> 1000
a
b
c
A/PuertoRico/8/1934(H1N1). A/Hong Kong/498/97(H3N2). A/PuertoRico/8/1934
d
(H259Y, NA resisitant strain). SI (selective index) is determined by the ratio between
CC50 and EC50 ( H1N1-H259Y) values.
3. Conclusion

In summary, a series of acylguanidine oseltamivir carboxylate derivatives were
synthesized, which showed potent NAs (H1N1 and H3N2) inhibitory effect,
especially some compounds exhibited potent inhibition of influenza NAs from the
oseltamivir-resistant strain. Among which, compounds 9 and 17 showed the best
inhibitory activities against NAs, with IC50 values of 30.5 and 14.5 nM, respectively.
Though the IC50 values of 9 and 17 slightly lower than OC , they exhibited a 5-fold
and 11-fold increase in activity against the H259Y mutant. Furthermore, cell-based
assays futher illustrated that compounds (6, 9, 14, 15, 17, 31) were safe and could
inhibit influenza virus ( H1N1, H3N2 and H1N1- H259Y) replication in vitro.
Comparing with oseltamivir carboxylate, the inhibitory activities of the compounds 9
and 17 significantly improved with EC50 values of 7.18 and 23.02 nM against the
H259Y mutant (H1N1), exhibiting a 20 and 6-fold increase, respectively. All the
results suggest that compounds 9 and 17 are promising for further development of
novel anti-influenza drugs. Moreover, The docking results displayed that the new
formed H-bond and the hydrophobic interaction of compounds 9 and 17, between the
acylguanidine moiety and the space nearby the 150-cavity and 430-cavity of NAs,
maybe compensate for loss in binding energy, comparing with oseltamivir carboxylate
(2). We are interested in investigation the active compounds for further development
of novel anti-influenza drugs.
4. Experimental sections
4.1 General information
All the reagents were commercially available and used without further purification
unless otherwise stated. Reactions and fractions were monitored by thin-layer
chromatography on silica gel (GF-254). Flash chromatography was performed on
silica gel (300−400 mesh). Some products were prepared by RP-HPLC employing an
semi-preparative C18 column and a water/acetonitrile gradient moving from 5% to
95% MeCN (0.1% formic acid or trifluoroacetic acid) over 25 min (flow rate 1.0
1
13
mL/min). H-NMR and C-NMR spectra were recorded on a BRUKER-ACF-300 or
BRUKER-ACF-400 instrument (chemical shifts are expressed as d values relative to

TMS as internal standard). The splitting patterns are reported as s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), br (broad) and dd (doublet of doublets).
HRMS analysis was performed using an Agilent 6520 Q-TOF LC/MS spectrometer
(Agilent, Germany).
4.2 General procedure for the preparation of 6-33
Compounds 6a-33a were prepared according to the previously repored method. 27
A mixture of oseltamivir phosphate (164 mg, 0.4 mmol), appropriate
S-methylisothiourea derivatives (6a-33a, 0.4 mmol) and Et3N (0.166 mL, 1.2 mmol)
in anhydrous DMF (4.0 mL) was stirred for 0.5 h and then added HgCl2 (130 mg,
0.48 mmol) at 0°C. The mixture was stirred overnight at room temperature, then
diluted with ethyl acetate (40 mL), and filtered through Celite. The filtered solution
was washed with water and brine, and the organic layer was dried and concentrated.
The residue was purified by column chromatography (PE/EA mixtures) to yield
6b-33b. To a mixture of 6b-33b (0.2 mmol) in CH2Cl2 (2 mL) at room temperature
was added TFA (2 mL), and the reaction mixture was stirred for 6 h at room
temperature. After that, the solvent was removed completely under reduced pressure,
the oily residue was repeatedly dissolved in CH2Cl2 and the solvent was evaporated to
remove TFA. A slightly white solid precipitated, after the addition of diethyl ether.
Then, the white solid was filtered, washed with cold diethyl ether and dried in vacuo
to afford compounds 6c-33c. To a mixture of 6c-33c (0.15 mmol) in tetrahydrofuran
(4 mL) and water (4 mL) at room temperature was added 1 N LiOH (0.6 mL, 0.6
mmol). The reaction mixture was stirred for 2h, then, neutralized with 1 M aq HCl
and concentrated. The residues were purified by preparative HPLC to yield the
desired product 6-33 in total yield of 40–90%.
(3R,4R,5S)-4-acetamido-5-(2-propionylguanidino)-3-(pentan-3-yloxy)cyclohex-1-ene
-1-enecarboxylic acid (6).
1
White solid, 69%. H NMR (400 MHz, DMSO-d6), δ (ppm): 8.00 (1H, d, J = 8.5
Hz), 6.68(1H, s), 4.12-4.10 (1H, m), 4.04-3.94 (1H, m), 3.91-3.85 (1H, m), 3.62-3.59

(1H, m), 2.72 (1H, dd, J = 17.4 Hz, 4.7 Hz), 2.42 (2H, q, J = 7.3 Hz), 2.34-2.28(1H,
13
m), 1.83 (3H, s), 1.50-1.37 (4H, m), 1.03 (3H, t, J = 7.3 Hz), 0.87-0.79 (6H, m); C
NMR (100 MHz, DMSO-d6) δ (ppm): 176.8, 170.4, 167.9, 153.8, 136.8, 129.5, 81.8,
74.6, 52.7, 49.9, 30.2, 29.6, 26.2, 25.7, 23.1, 9.9, 9.5, 8.7; HR-MS (ESI) m/z: calcd for
C18H30N4O5 [M+H]+ 383.2294, [M+Na]+ 405.2114, found 383.2294, 405.2109.
(3R,4R,5S)-4-acetamido-5-(2-butyrylguanidino)-3-(pentan-3-yloxy)cyclohex-1-ene-1
-enecarboxylic acid (7).
White solid, 90%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.77 (1H, s), 8.98(1H,
d, J = 7.8 Hz), 8.85 (2H, s), 8.00 (1H, d, J = 8.4 Hz), 6.69(1H, s), 4.12-4.05 (1H, m),
4.04-3.95 (1H, m), 3.95-3.85 (1H, m), 3.44-3.36 (1H, m), 2.72(1H, dd, J = 17.5 Hz,
4.3 Hz), 2.39 (2H, t, J = 7.2 Hz), 2.34-2.29 (1H, m), 1.82 (3H, s), 1.61-1.52 (2H, m),
1.49-1.37 (4H, m), 0.91-0.78 (9H, m); 13C NMR (100 MHz, DMSO-d6) δ (ppm):
175.6, 170.2, 167.5, 153.4, 137.2, 129.0, 81.8, 74.6, 52.6, 50.0, 38.5, 29.4, 26.2, 25.7,
23.1, 17.8, 13.7, 9.9, 9.5; HR-MS (ESI) m/z: calcd for C19H32N4O5 [M+H]+ 397.2451,
found 397.2478.
(3R,4R,5S)-4-acetamido-5-(2-valerylguanidino)-3-(pentan-3-yloxy)cyclohex-1-ene-1-
enecarboxylic acid (8).
White solid, 70%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.77 (1H, s), 8.98 (1H,
d, J = 7.9 Hz), 8.83 (2H, s), 8.00 (1H, d, J = 8.4 Hz), 6.69 (1H, s), 4.11-4.06 (1H, m),
4.05-3.95 (1H, m), 3.95-3.85 (1H, m), 3.43-3.35 (1H, m), 2.72 (1H, dd, J = 17.4 Hz,
4.4 Hz), 2.40 (2H, t, J = 7.2 Hz), 2.36-2.29 (1H, m), 1.82 (3H, s), 1.57-1.49 (2H, m),
1.47-1.39 (2H, m), 1.34-1.24 (4H, m), 0.89-0.78 (9H, m); 13C NMR (100 MHz,
DMSO-d6) δ (ppm): 175.7, 170.3, 167.5, 153.4, 137.2, 129.0, 81.8, 74.6, 52.6, 49.9,
36.4, 29.4, 26.4, 26.1, 25.7, 23.1, 21.9, 14.0, 9.9, 9.5; HR-MS (ESI) m/z: calcd for
C20H34N4O5 [M+H]+ 411.2607, found 411.2624.
(3R,4R,5S)-4-acetamido-5-(2-(4-ene-valeryl)guanidino)-3-(pentan-3-yloxy)cyclohex-
1-ene-1-enecarboxylic acid (9).

White solid, 66%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.77 (1H, s), 8.98 (1H,
d, J = 7.7 Hz), 8.84 (2H, s), 8.01 (1H, d, J = 8.4 Hz), 6.69(1H, s), 5.86-5.76 (1H, m),
5.08-4.99 (2H, m), 4.15-4.06 (1H, m), 4.06-3.94 (1H, m), 3.94-3.85 (1H, m),
3.42-3.38 (1H, m), 2.71 (1H, dd, J = 17.5 Hz, 4.4 Hz), 2.54-2.51 (2H, m),
2.36-2.28(3H, m), 1.82 (3H, s), 1.49-1.37 (4H, m), 0.87-0.78 (6H, m); 13C NMR (100
MHz, DMSO-d6) δ (ppm): 174.9, 170.3, 167.5, 153.4, 137.2, 137.0, 129.0, 116.2, 81.8,
74.6, 52.6, 50.0, 35.8, 29.5, 28.2, 26.2, 25.7, 23.1, 9.9, 9.5; HR-MS (ESI) m/z: calcd
for C20H32N4O5 [M+H]+ 409.2451, found 409.2458.
(3R,4R,5S)-4-acetamido-5-(2-(2-methylacetyl)guanidino)-3-(pentan-3-yloxy)cyclohe
x-1-ene-1-enecarboxylic acid (10).
1
White solid, 56%. H NMR (400 MHz, DMSO-d6), δ (ppm): 12.78 (1H, s), 8.96
(1H, d, J = 8.2 Hz), 8.84 (2H, s), 8.00 (1H, d, J = 8.5 Hz), 6.70 (1H, s), 4.13-4.05 (1H,
m), 4.05-3.95 (1H, m), 3.95-3.87 (1H, m), 3.42-3.39 (1H, m), 2.72 (1H, dd, J = 17.5
Hz, 4.6 Hz), 2.65-2.58 (1H, m), 2.37-2.30 (1H, m), 1.82 (3H, s), 1.48-1.39 (4H, m),
1.10 (6H, d, J = 6.8 Hz), 0.86-0.78 (6H, m); 13C NMR (100 MHz, DMSO-d6) δ (ppm):
179.3, 170.3, 167.5, 153.7, 137.1, 129.0, 81.8, 74.5, 52.5, 49.9, 35.8, 29.4, 26.1, 25.7,
23.1, 18.9, 18.8, 9.9, 9.5; HR-MS (ESI) m/z: calcd for C19H32N4O5 [M+H]+ 397.2451,
found 397.2478.
(3R,4R,5S)-4-acetamido-5-(2-(3-methylbutyryl)guanidino)-3-(pentan-3-yloxy)cycloh
ex-1-ene-1-enecarboxylic acid (11).
1
White solid, 54%. H NMR (400 MHz, DMSO-d6), δ (ppm): 12.77 (1H, s), 9.00
(1H, d, J = 7.6 Hz), 8.83 (2H, s), 7.99 (1H, d, J = 8.4 Hz), 6.69 (1H, s), 4.12-4.05 (1H,
m), 4.05-3.96 (1H, m), 3.96-3.88 (1H, m), 3.42-3.37 (1H, m), 2.72 (1H, dd, J = 17.4
Hz, 4.3 Hz), 2.37-2.32 (1H, m), 2.28 (2H, d, J = 7.0 Hz), 2.07-1.97 (1H, m), 1.82 (3H,
13
s), 1.49-1.38 (4H, m), 0.92 (6H, d, J = 6.7 Hz), 0.87-0.78 (6H, m); C NMR (100
MHz, DMSO-d6) δ (ppm): 175.0, 170.3, 167.5, 153.4, 137.1, 129.1, 81.8, 74.6, 52.7,
49.9, 45.6, 29.4, 26.2, 25.7, 25.3, 23.1, 22.4, 9.9, 9.5; HR-MS (ESI) m/z: calcd for
C20H34N4O5 [M+H]+ 411.2607, found 411.2612.

(3R,4R,5S)-4-acetamido-5-(2-(2-(2-methylphenyl)acetyl)guanidino)-3-(pentan-3-ylox
y)cyclohex-1-ene-1-enecarboxylic acid (12).
1
White solid, 69%. H NMR (400 MHz, DMSO-d6), δ (ppm): 12.77 (1H, s), 9.00
(1H, s), 8.89 (2H, s), 7.99 (1H, d, J = 8.5 Hz), 7.22-7.13 (4H, m), 6.68 (1H, s),
4.12-4.05 (1H, m), 4.05-3.94 (1H, m), 3.92-3.88 (1H, m), 3.80 (2H, s), 3.39-3.37 (1H,
m), 2.72 (1H, dd, J = 17.4 Hz, 4.6 Hz), 2.36-2.30 (1H, m), 2.24 (3H, s), 1.81 (3H, s),
1.45-1.36 (4H, m), 0.85-0.77 (6H, m); 13C NMR (100 MHz, DMSO-d6) δ (ppm):
173.4, 170.3, 167.5, 153.5, 137.4, 137.2, 132.6, 130.8, 130.5, 129.0, 127.9, 126.4,
81.8, 74.5, 52.7, 50.1, 41.2, 29.4, 26.1, 25.7, 23.1, 19.6, 9.9, 9.5; HR-MS (ESI) m/z:
calcd for C24H34N4O5 [M+H]+ 459.2607, found 459.2610.
(3R,4R,5S)-4-acetamido-5-(2-(2-(4-methylphenyl)acetyl)guanidino)-3-(pentan-3-ylox
y)cyclohex-1-ene-1-enecarboxylic acid (13).
White solid, 56%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.76 (1H, s), 8.95 (1H,
d, J = 8.1 Hz), 8.85 (2H, s), 7.98 (1H, d, J = 8.4 Hz), 7.18-7.13 (4H, m), 6.68(1H, s),
4.12-4.05 (1H, m), 4.05-3.94 (1H, m), 3.92-3.88 (1H, m), 3.71 (2H, s), 3.39-3.37 (1H,
m), 2.71 (1H, dd, J = 17.4 Hz, 4.5 Hz), 2.35-2.30 (1H, m), 2.28 (3H, s), 1.81 (3H, s),
1.46-1.36 (4H, m), 0.86-0.76 (6H, m); 13C NMR (100 MHz, DMSO-d6) δ (ppm):
173.7, 170.3, 167.5, 153.5, 137.2, 136.8, 130.8, 129.8, 129.5, 129.0, 81.8, 74.6, 52.6,
50.0, 42.9, 29.4, 26.1, 25.7, 23.1, 21.1, 9.9, 9.5; HR-MS (ESI) m/z: calcd for
C24H34N4O5 [M+H]+ 459.2607, found 459.2650.
(3R,4R,5S)-4-acetamido-5-(2-(2-(4-Br-phenyl)acetyl)guanidino)-3-(pentan-3-yloxy)c
yclohex-1-ene-1-enecarboxylic acid (14).
White solid, 67%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.76 (1H, s), 8.93 (1H,
s), 8.85 (2H, s), 7.99 (1H, d, J = 8.5 Hz), 7.54 (2H, d, J = 8.3 Hz), 7.25 (2H, d, J = 8.3
Hz), 6.68(1H, s), 4.12-4.05 (1H, m), 4.04-3.94 (1H, m), 3.92-3.88 (1H, m), 3.77 (2H,
s), 3.39-3.37 (1H, m), 2.71 (1H, dd, J = 17.5 Hz, 4.7 Hz), 2.35-2.29 (1H, m), 1.80 (3H,
13
s), 1.46-1.35 (4H, m), 0.85-0.76 (6H, m); C NMR (100 MHz, DMSO-d6) δ (ppm):

173.1, 170.3, 167.5, 153.6, 137.2, 133.3, 132.3, 131.7, 129.0, 120.9, 81.8, 74.5, 52.6,
50.1, 42.5, 29.4, 26.1, 25.7, 23.1, 9.9, 9.5; HR-MS (ESI) m/z: calcd for C23H31BrN4O5
[M+H]+ 523.1556 found 523.1564, 525.1546.
(3R,4R,5S)-4-acetamido-5-(2-(2-naphthylacetyl)guanidino)-3-(pentan-3-yloxy)cycloh
ex-1-ene-1-enecarboxylic acid (15).
White solid, 63%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.76 (1H, s), 8.94 (1H,
d, J = 7.6 Hz), 8.86 (2H, s), 7.98 (1H, d, J = 8.5 Hz), 7.92-7.86 (3H, m), 7.82 (1H, s),
7.55-7.49 (2H, m), 7.44 (1H, d, J = 8.3 Hz), 6.68 (1H, s), 4.11-4.06 (1H, m),
4.02-3.97 (1H, m), 3.95 (2H, s), 3.92-3.86 (1H, m), 3.39-3.36 (1H, m), 2.72 (1H, dd, J
= 17.4 Hz, 4.6 Hz), 2.36-2.30 (1H, m), 1.80 (3H, s), 1.45-1.35 (4H, m), 0.84-0.75 (6H,
m); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 173.5, 170.3, 167.5, 153.4, 137.2,
133.4, 132.5, 131.5, 129.0, 128.6, 128.4, 128.1, 128.0, 127.9, 126.8, 126.5, 81.8, 74.6,
52.6, 50.1, 43.5, 29.4, 26.1, 25.7, 23.1, 9.9, 9.5; HR-MS (ESI) m/z: calcd for
C27H34N4O5 [M+H]+ 495.2607, found 495.2618.
(3R,4R,5S)-4-acetamido-5-(2-(3-phenylpropionyl)guanidino)-3-(pentan-3-yloxy)cycl
ohex-1-ene-1-enecarboxylic acid (16).
White solid, 45%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.78 (1H, s), 9.04 (1H,
d, J = 7.0 Hz), 8.78 (2H, s), 8.00 (1H, d, J = 8.4 Hz), 7.31-7.18 (5H, m), 6.69 (1H, s),
4.13-4.07 (1H, m), 4.03-3.95 (1H, m), 3.93-3.86 (1H, m), 3.42-3.36 (1H, m), 2.87 (2H,
t, J = 7.3 Hz), 2.76-2.68 (3H, m), 2.35-2.29 (1H, m), 1.81 (3H, s), 1.47-1.38 (4H, m),
0.87-0.78 (6H, m); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 174.9, 170.3, 167.5,
153.4, 140.6, 137.2, 129.0, 128.9, 128.7, 126.7, 81.9, 74.6, 52.6, 50.0, 38.2, 29.9, 29.5,
26.2, 25.7, 23.1, 9.9, 9.5; HR-MS (ESI) m/z: calcd for C24H34N4O5 [M+H]+ 459.2607,
found 459.2610.
(3R,4R,5S)-4-acetamido-5-(2-(3-(3,4-bis(methoxyl)phenyl)propionyl)guanidino)-3-(p
entan-3-yloxy)cyclohex-1-ene-1-enecarboxylic acid (17).
White solid, 43%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.74 (1H, s), 9.00 (1H,

d, J = 7.9 Hz), 8.88 (2H, s), 8.00 (1H, d, J = 8.4 Hz), 6.85 (1H, d, J = 8.2 Hz), 6.84
(1H, s), 6.72 (1H, d, J = 8.2 Hz), 6.69 (1H, s), 4.12-4.08 (1H, m), 4.03-3.95 (1H, m),
3.93-3.87 (1H, m), 3.73 (3H, s), 3.71 (3H, s), 3.42-3.36 (1H, m), 2.81 (2H, t, J = 6.9
Hz), 2.73-2.68 (3H, m), 2.35-2.29 (1H, m), 1.81 (3H, s), 1.49-1.36 (4H, m), 0.87-0.78
(6H, m); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 175.0, 170.3, 167.5, 153.4, 149.1,
147.7, 137.2, 132.9, 129.0, 120.5, 112.6, 112.3, 81.9, 74.6, 56.0, 55.8, 52.6, 50.0, 38.6,
29.7, 29.5, 26.2, 25.7, 23.1, 9.9, 9.5; HR-MS (ESI) m/z: calcd for C26H38N4O7 [M+H]+
519.2819, found 519.2830.
(3R,4R,5S)-4-acetamido-5-(2-(3-phenylbutyryl)guanidino)-3-(pentan-3-yloxy)cycloh
ex-1-ene-1-enecarboxylic acid (18).
White solid, 50%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.76 (1H, s), 8.92 (1H,
d, J = 7.4 Hz), 8.78 (2H, s), 8.01 (1H, d, J = 8.4 Hz), 7.29 (2H, t, J = 7.5 Hz),
7.21-7.17 (3H, m), 6.69 (1H, s), 4.11-4.07 (1H, m), 4.03-3.95 (1H, m), 3.93-3.88 (1H,
m), 3.41-3.38 (1H, m), 2.71 (1H, dd, J = 17.6 Hz, 4.2Hz), 2.60 (2H, t, J = 7.3 Hz),
2.42 (2H, t, J = 7.3 Hz), 2.36-2.29 (1H, m), 1.91-1.85 (2H, m), 1.82 (3H, s), 1.49-1.39
13
(4H, m), 0.87-0.78 (6H, m); C NMR (100 MHz, DMSO-d6) δ (ppm): 175.4, 170.3,
167.5, 153.4, 140.7, 137.1, 129.0, 128.8, 128.7, 126.4, 81.9, 74.6, 52.5, 50.0, 36.1,
34.6, 29.4, 26.2, 26.0, 25.7, 23.1, 9.9, 9.5; HR-MS (ESI) m/z: calcd for C25H36N4O5
[M+H]+ 473.2764, found 473.2769.
(3R,4R,5S)-4-acetamido-5-(2-benzoylguanidino)-3-(pentan-3-yloxy)cyclohex-1-ene-1
-enecarboxylic acid (19).
White solid, 63%. 1H NMR (400 MHz, DMSO-d6) δ 12.71 (1H, br), 9.45 (1H , d, J
= 8.6 Hz), 9.25 (1H, s), 8.13 (2H, d, J = 7.7 Hz), 7.74 (1H, t, J = 7.7 Hz), 7.61 (2H, t,
J = 7.7 Hz), 6.72 (1H, s), 4.24-4.22 (1H, m), 4.17-4.08 (1H, m), 4.02-3.90 (1H, m),
3.45-3.37 (1H, m), 2.79 (1H, dd, J = 17.3 Hz, 4.5 Hz), 2.44-2.37 (1H, m), 1.81 (3H, s),
1.52-1.37 (4H, m), 0.88-0.78 (6H, m); 13C NMR (100 MHz, DMSO-d6) δ (ppm):
170.2, 167.9, 167.5, 154.4, 131.5, 129.4, 128.9, 129.2, 128.8, 127.6, 81.8, 74.7, 52.8,
50.4, 29.7, 26.1, 25.7, 23.2, 9.9, 9.4; HR-MS (ESI) m/z: calcd for C22H30N4O5 [M+H]+

431.2294, found 431.2317.
(3R,4R,5S)-4-acetamido-5-(2-(2-methylbenzoyl)guanidino)-3-(pentan-3-yloxy)cycloh
ex-1-ene-1-enecarboxylic acid (20).
1
White solid, 64%. H NMR (400 MHz, DMSO-d6) δ (ppm): 12.71 (1H, br), 9.22
(1H, d, J = 8.7 Hz), 9.10 (1H, s), 8.29 (1H, d, J = 7.7 Hz), 7.69 (1H, t, J = 7.7 Hz),
7.52 (1H, t, J = 7.7 Hz), 7.36 (1H, t, J = 7.7 Hz), 6.70 (1H, s), 4.26-4.24 (1H, m),
4.19-4.09 (1H, m), 4.01-3.95 (1H, m), 3.46-3.37 (1H, m), 2.78 (1H, dd, J =17.4 Hz,
4.8 Hz), 2.45 (3H, s), 2.42-2.37 (1H, m), 1.83 (3H, s), 1.52-1.40 (4H, m), 0.88-0.78
(6H, m); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 170.2, 169.1, 167.5, 154.1, 137.8,
137.4, 132.8, 132.5, 131.9, 128.9, 128.6, 126.4, 81.9, 74.6, 52.8, 50.5, 29.6, 26.2, 25.7,
23.2, 20.2, 9.9, 9.5; HR-MS (ESI) m/z: calcd for C23H32N4O5 [M+H]+ 445.2451, found
445.2530.
(3R,4R,5S)-4-acetamido-5-(2-(2-trifluoromethylbenzoyl)guanidino)-3-(pentan-3-ylox
y)cyclohex-1-ene-1-enecarboxylic acid (21).
1
White solid, 68%. H NMR (400 MHz, DMSO-d6) δ (ppm): 9.22 (1H, d, J = 8.4
Hz), 8.24 (1H, d, J = 8.7 Hz), 7.93 (1H, d, J = 7.4 Hz), 7.87-7.81 (2H, m), 6.71 (1H,
s), 4.26-4.22 (1H, m), 4.18-4.08 (1H, m), 4.02-3.95 (1H, m), 3.43-3.36 (1H, m), 2.76
(1H, dd, J =17.4 Hz, 4.3 Hz), 2.44-2.38 (1H, m), 1.85 (3H, s), 1.53-1.35 (4H, m),
0.88-0.78 (6H, m); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 170.2, 168.3, 167.5,
153.5, 138.3, 137.4, 133.3, 132.5, 129.4, 128.9, 127.4, 126.7, 82.0, 74.5, 52.7, 50.6,
29.5, 26.2, 25.7, 23.1, 15.72, 9.9, 9.5; HR-MS (ESI) m/z: calcd for C23H9F3N4O5
[M+H]+ 499.2168, found 499.2200.
(3R,4R,5S)-4-acetamido-5-(2-(2-hydroxylbenzoyl)guanidino)-3-(pentan-3-yloxy)cycl
ohex-1-ene-1-enecarboxylic acid (22).
White solid, 70%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.51 (1H, s), 7.97 (1H,
d, J = 7.5 Hz), 7.89 (1H, d, J = 7.5 Hz), 7.70 (1H, t, J = 7.5 Hz), 7.35 (1H, t, J = 7.5
Hz), 6.66 (1H, s), 4.21-4.18 (1H, m), 4.14-4.05 (1H, m), 3.95-3.88 (1H, m), 3.46-3.39

(1H, m), 2.70-2.64 (1H, m), 2.42-2.35(1H, m), 1.69 (3H, s), 1.49-1.36 (4H, m),
0.87-0.75 (6H, m); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 170.1, 167.6, 166.1,
158.6, 153.9, 138.1, 134.7, 129.2, 127.3, 125.7, 117.5, 116.2, 81.5, 75.2, 53.9, 50.4,
29.9, 26.2, 25.7, 23.1, 9.9, 9.4; HR-MS (ESI) m/z: calcd for C22H30N4O5 [M+H]+
447.2244, found 447.2254.
(3R,4R,5S)-4-acetamido-5-(2-(2-Br-benzoyl)guanidino)-3-(pentan-3-yloxy)cyclohex-
1-ene-1-enecarboxylic acid (23).
1
White solid, 58%. H NMR (400 MHz, DMSO-d6) δ (ppm): 8.15 (1H, s, J = 8.7
Hz), 7.97 (1H, d, J = 7.6 Hz), 7.89 (1H, d, J = 7.6 Hz), 7.70 (1H, t, J = 7.6 Hz), 7.35
(1H, t, J = 7.5 Hz), 6.74 (1H, s), 4.41-4.38 (1H, m), 4.26-4.22 (1H, m), 4.15-4.09 (1H,
m), 3.38-3.32 (1H, m), 2.80-2.74 (1H, m), 2.34-2.28(1H, m), 1.84 (3H, s), 1.50-1.35
13
(4H, m), 0.84-0.74 (6H, m); C NMR (100 MHz, DMSO-d6) δ (ppm): 175.7, 172.6,
172.2, 158.5, 142.8, 139.4, 138.6, 138.1, 137.9, 134.2, 132.6, 124.4, 79.0, 65.7, 58.3,
55.3, 34.6, 30.9, 30.5, 27.9, 14.4, 14.1; HR-MS (ESI) m/z: calcd for C22H29BrN4O5
[M+H]+ 509.1400, found 509.1423.
(3R,4R,5S)-4-acetamido-5-(2-(2-methoxylbenzoyl)guanidino)-3-(pentan-3-yloxy)cycl
ohex-1-ene-1-enecarboxylic acid (24).
1
White solid, 78%. H NMR (MeOD, 400 MHz) δ (ppm): 7.99 (1H, d, J = 7.8 Hz),
7.64 (1H, t, J = 7.8 Hz), 7.24 (1H, d, J = 7.8 Hz), 7.15 (1H, d, J = 7.8 Hz), 6.80 (1H,
s), 4.24-4.18 (1H, m), 4.16-4.04 (1H, m), 4.06 (3H, s), 3.89-3.81 (1H, m), 3.49-3.41
(1H, m), 2.74 (1H, dd, J =17.4 Hz, 4.8 Hz), 2.96-2.88 (1H, m), 1.97 (3H, s), 1.58-1.50
(4H, m), 0.97-0.89 (6H, m); 13C NMR (100 MHz, MeOD) δ (ppm): 172.6, 166.7,
158.5, 154.6, 138.6, 138.3, 135.4, 133.7, 132.1, 130.1, 121.8, 112.9, 82.9, 75.9, 56.4,
55.4, 54.1, 32.1, 26.7, 26.3, 14.1, 9.5, 9.3; HR-MS (ESI) m/z: calcd for C23H32N4O6
[M+H]+ 461.2400, found 461.2418.
(3R,4R,5S)-4-acetamido-5-(2-(3-methylbenzoyl)guanidino)-3-(pentan-3-yloxy)cycloh
ex-1-ene-1-enecarboxylic acid (25).

White solid, 85%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.93 (1H, s), 7.73 (1H,
d, J = 7.3 Hz), 7.42 (1H, t, J = 7.3 Hz), 7.38 (1H, d, J = 7.9 Hz), 6.72 (1H, s),
4.12-4.08 (1H, m), 4.06-4.01 (1H, m), 4.00-3.93 (1H, m), 3.43-3.38 (1H, m), 2.80 (1H,
dd, J = 17.9 Hz, 4.9 Hz), 2.36 (3H, s), 2.30-2.25(1H, m), 1.81 (3H, s), 1.55-1.44 (4H,
13
m), 1.30 (3H, t, J = 7.1Hz), 0.87-0.77 (6H, m); C NMR (100 MHz, DMSO-d6) δ
(ppm): 170.0, 168.4, 166.1, 154.0, 138.3, 138.1, 133.8, 132.9, 129.5, 129.1, 128.8,
125.7, 81.8, 61.1, 52.5, 54.9, 48.1, 30.8, 26.1, 25.8, 21.3, 14.5, 9.9, 9.6; HR-MS (ESI)
m/z: calcd for C23H32N4O5 [M+H]+ 445.2451, found 445.2459.
(3R,4R,5S)-4-acetamido-5-(2-(3-trifluoromethylbenzoyl)guanidino)-3-(pentan-3-ylox
y)cyclohex-1-ene-1-enecarboxylic acid (26).
1
White solid, 70%. H NMR (400 MHz, DMSO-d6) δ (ppm): 9.21 (1H, s), 8.4 (1H,
s), 8.18 (1H, d, J = 8.7 Hz), 8.09 (1H, d, J = 8.7 Hz), 7.86 (1H, t, J = 8.7 Hz), 6.72
(1H, s), 4.23-4.20 (1H, m), 4.14-4.10 (1H, m), 4.00-3.94 (1H, m), 3.48-3.38 (1H, m),
2.79 (1H, dd, J = 17.2 Hz, 4.3 Hz), 2.43-2.37 (1H, m), 1.85 (3H, s), 1.50-1.33 (4H, m),
0.88-0.79 (6H, m); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 170.2, 167.3, 166.3,
154.0, 137.6, 137.4, 132.9, 130.8, 128.9, 125.5, 125.3, 122.8, 81.8, 74.5, 52.7, 50.4,
29.7, 26.2, 25.7, 23.1, 15.6, 9.9, 9.5; HR-MS (ESI) m/z: calcd for C23H9F3N4O5
[M+H]+ 499.2168, found 499.2201.
(3R,4R,5S)-4-acetamido-5-(2-(3-methoxylbenzoyl)guanidino)-3-(pentan-3-yloxy)cycl
ohex-1-ene-1-enecarboxylic acid (27).
White solid, 75%. 1H NMR 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.61 (1H, s),
8.78 (1H, s), 8.09 (1H, s), 7.54 (1H, s), 7.52 (1H, d, J = 7.9 Hz), 7.41 (1H, t, J = 7.9
Hz), 7.17 (1H, d, J = 7.9 Hz), 6.69 (1H, s), 4.14-4.10 (1H, m), 4.10-3.90 (1H, m), 3.82
(3H, s), 3.45-3.40 (1H, m), 3.40-3.36 (1H, m), 2.80 (1H, dd, J = 17.4 Hz, 4.8 Hz),
2.25-2.19 (1H, m), 1.82 (3H, s), 1.49-1.39 (4H, m), 0.87-0.78 (6H, m); 13C NMR (100
MHz, DMSO-d6) δ (ppm): 170.0, 167.7, 159.6, 153.9, 137.6, 134.3, 130.1, 129.4,
120.9, 119.5, 113.2, 112.9, 81.6, 75.0, 55.8, 52.6, 48.4, 30.6, 26.1, 25.8, 23.1, 9.9, 9.5;
HR-MS (ESI) m/z: calcd for C23H32N4O6 [M+H]+ 461.2400, found 461.2426.

(3R,4R,5S)-4-acetamido-5-(2-(3,5-bis(methyl)benzoyl)guanidino)-3-(pentan-3-yloxy)
cyclohex-1-ene-1-enecarboxylic acid (28).
White solid, 84%. 1H NMR (400 MHz, MeOD) δ (ppm): 7.83 (1H, s), 7.57 (1H, s),
7.36 (1H, s), 6.68 (1H, s), 4.35-4.31 (1H, m), 4.21-4.14 (1H, m), 3.99-3.94 (1H, m),
3.44-3.39 (1H, m), 2.85-2.77 (1H, m), 2.44-2.39(1H, m), 2.35 (6H, s), 1.83 (3H, s),
1.47-1.42 (4H, m), 0.88-0.78 (6H, m); 13C NMR (100 MHz, MeOD) δ (ppm): 170.0,
168.5, 167.7, 165.7, 154.6, 153.9, 138.8, 138.1, 126.6, 126.3, 81.7, 75.1, 61.0, 53.2,
30.5, 26.2, 23.1, 21.2, 14.5, 9.6, 9.4; HR-MS (ESI) m/z: calcd for C24H34N4O5 [M+H]+
459.2607, found 459.2642.
(3R,4R,5S)-4-acetamido-5-(2-(4-methylbenzoyl)guanidino)-3-(pentan-3-yloxy)cycloh
ex-1-ene-1-enecarboxylic acid (29).
1
White solid, 73%. H NMR (400 MHz, MeOD) δ (ppm): 7.87 (2H, d, J = 8.0 Hz),
7.36 (2H, d, J = 8.0 Hz), 6.80 (1H, s), 4.28-4.20 (1H, m), 4.19-4.09 (1H, m),
4.02-3.95 (1H, m), 3.48-3.47 (1H, m), 2.94 (1H, dd, J = 17.4 Hz, 4.8 Hz), 2.45 (3H, s),
2.43-2.36 (1H, m), 1.97 (3H, s), 1.59-1.53 (4H, m), 0.98-0.90 (6H, m); 13C NMR (100
MHz, MeOD) δ (ppm): 172.5, 169.0, 166.8, 154.9, 144.3, 138.4, 130.7, 129.8, 129.5,
128.7, 83.0, 75.9, 61.4, 54.0, 31.2, 26.7, 26.3, 22.5, 14.2, 9.6, 9.4; HR-MS (ESI) m/z:
calcd for C23H32N4O5 [M+H]+ 445.2451, found 445.2468.
(3R,4R,5S)-4-acetamido-5-(2-(4-methoxylbenzoyl)guanidino)-3-(pentan-3-yloxy)cycl
ohex-1-ene-1-enecarboxylic acid (30).
White solid, 70%. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 12.62 (1H, s), 8.75 (1H,
s), 8.07 (1H, s), 7.74 (2H, d, J = 7.3 Hz), 7.04 (2H, d, J = 7.3 Hz), 6.69 (1H, s),
4.19-4.13 (1H, m), 4.13-3.95 (1H, m), 3.87-3.80 (1H, m), 3.38 (3H, s), 3.44-3.40 (1H,
m), 2.82-2.78 (1H, m), 2.31-2.21(1H, m), 1.82 (3H, s), 1.47-1.39 (4H, m), 0.87-0.78
(6H, m); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 170.0, 168.4, 167.7, 153.9, 138.3,
132.9, 130.7, 129.1, 125.7, 114.2, 81.6, 75.0, 56.0, 52.6, 48.4, 30.6, 26.2, 25.8, 23.1,
9.9, 9.5; HR-MS (ESI) m/z: calcd for C23H32N4O6 [M+H]+ 461.2400, found 461.2422.

(3R,4R,5S)-4-acetamido-5-(2-(4-nitro-benzoyl)guanidino)-3-(pentan-3-yloxy)cyclohe
x-1-ene-1-enecarboxylic acid (31).
1
White solid, 40%. H NMR (400 MHz, DMSO-d6), δ (ppm): 12.74 (1H, s), 9.34
(1H, d, J = 7.9 Hz), 9.22 (2H, s), 8.42 (2H, d, J = 8.7 Hz), 8.30 (2H, d, J = 8.7 Hz),
8.21 (1H, d, J = 8.7 Hz), 6.72 (1H, s), 4.24-4.22 (1H, m), 4.16-4.07 (1H, m),
4.01-3.94 (1H, m), 3.44-3.41 (1H, m), 2.78 (1H, dd, J = 17.4 Hz, 4.5 Hz), 2.43-2.36
13
(1H, m), 1.85 (3H, s), 1.53-1.38 (4H, m), 0.88-0.79 (6H, m); C NMR (100 MHz,
DMSO-d6) δ (ppm): 170.3, 167.5, 150.8, 137.4, 130.4, 128.9, 124.4, 81.8, 74.6, 52.7,
50.6, 29.6, 26.2, 25.7, 23.2, 9.9, 9.5; HR-MS (ESI) m/z: calcd for C22H29N5O7 [M+H]+
476.2145 [M+Na]+ 498.1965, found 476.2190.
(3R,4R,5S)-4-acetamido-5-(2-(3-(pyridin-4-yl)propionyl)guanidino)-3-(pentan-3-ylox
y)cyclohex-1-ene-1-enecarboxylic acid (32).
White solid, 63%. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 8.45 (1H, s), 8.40 (1H,
d, J = 3.9 Hz), 8.20 (1H, s), 8.04 (1H, d, J = 8.2 Hz), 7.66 (1H, d, J = 7.9 Hz),
7.32-7.28 (1H, m), 6.63 (1H, s), 4.15-4.10 (1H, m), 3.99-3.92 (1H, m), 3.89-3.81 (1H,
m), 3.41-3.33 (1H, m), 2.86 (2H, t, J = 7.1 Hz), 2.75 (2H, t, J = 7.1 Hz), 2.70-2.67
13
(1H, m), 2.35-2.26 (1H, m), 1.80 (3H, s), 1.50-1.40 (4H, m), 0.86-0.76 (6H, m); C
NMR (75 MHz, DMSO-d6) δ (ppm): 175.8, 170.2, 168.7, 164.8, 154.5, 150.0, 147.9,
136.3, 130.2, 123.9, 115.0, 81.7, 74.7, 53.0, 50.0, 37.9, 29.9, 27.3, 26.2, 25.7, 23.1,
9.9, 9.5; HR-MS (ESI) m/z: calcd for C23H33N5O5 [M+H]+ 460.2560, found 460.2547.
(3R,4R,5S)-4-acetamido-5-(2-isonicotinoylguanidino)-3-(pentan-3-yloxy)cyclohex-1-
ene-1-enecarboxylic acid (33).
1
White solid, 42%. H NMR (300 MHz, DMSO-d6) δ 8.67 (2H , s), 8.14 (1H, s),
7.92-7.88 (3H, m), 6.69 (1H, s), 4.40-4.33 (1H, m), 4.15-4.10 (1H, m), 3.92-3.84 (1H,
m), 3.43-3.38 (1H, m), 2.84-2.71 (1H, m), 2.33-2.11 (1H, m), 1.82 (3H, s), 1.50-1.42
13
(4H, m), 0.89-0.80 (6H, m); C NMR (75 MHz, DMSO-d6) δ (ppm): 170.2, 167.7,
163.5, 161.8, 150.3, 138.0, 129.5, 122.9, 122.6, 81.6, 75.6, 53.3, 49.5, 31.4, 26.1, 25.7,

23.3, 9.9, 9.5; HR-MS (ESI) m/z: calcd for C21H29N5O5 [M+H]+ 432.2247, found
432.2242.
4.3 Determination of IC50 of NA inhibitors
A/PuertoRico/8/1934(H1N1),
A/HongKong/498/97(H3N2)
and
A/PuertoRico/8/1934(H259Y, NA resistant strain) were obtained from Wuhan
Institute Of Virology, Chinese Academy of Sciences, stored at -80°C. The substrate
used in the enzyme inhibition assay, 2’-(4-methylumbelliferyl)-α-D-acetylneuraminic
acid sodium salt hydrate (4-MU-NANA), was purchased from Sigma. Alamar-Blue®
was purchased from Invitrogen. Oseltamivir carboxylate (OC) was obtained from
Wuhan Institute Of Virology, Chinese Academy of Sciences.
The NA inhibition assay was performed according to the modified method.28 The
substrate, 2’-(4-methylumbelliferyl)-α-D-acetylneuraminic acid sodium salt hydrate
(MUNANA), was cleaved by NA to yield a quantifiable fluorescent product. The
tested compounds were dissolved in DMSO and diluted to the corresponding
concentrations in MES buffer. For the NA inhibition assay, 30 µL of the diluted virus
supernatant was first incubated with 10 µL of compounds at different concentrations
in a 96-well plate at 37°C for 10 min. Next, 20µM MUNANA dissolved in 33 mM
2-[N-morpholino]ethanesulfonic acid (pH 6.5) and 4 mM CaCl2, at 37°C for 1 h. The
reaction was terminated by adding 0.14 M NaOH in 83% ethanol. The fluorescence
intensity was measured at an excitation wavelength of 355 nm and an emission
wavelength of 485 nm using a multi-label plate reader (Wallac Envision, PerkinElmer,
MA, USA). OC was used as a positive control.
4.4 Docking studies.
Molecular docking studies with flexible ligand and rigid receptor were performed
with the AutoDockVinaprogram.29 Structures of the proteins and ligand were prepared
using AutoDockTools GUI for docking preparation. The pictures were generated

using Pymol and Discover Studio Visualizer 4.0 software. For wild type H1N1,
crystallographic structure in complex with oseltamivir (PDB ID: 2HU4) were used for
docking. The volume chosen for the grid box was 16 Å ×16 Å ×18 Å, center point (x
= 0.509, y = 83.305, z = 110.903). Exhaustiveness was increase to 20, and 9 ligand
poses were generated.
4.5 Cytotoxicity assay
Monolayers of MDCKs in 96-well culture plates were incubated with the
compounds at different concentrations for 72 h. Then, Alamar-Blue® (Invitrogen) was
added. After incubation for 2 h at 37°C, the plates were read in a spectrophotometer at
Ex= 570 nm and Em= 595 nm. The 50% cytotoxic concentration (CC50) was
calculated by linear regression analysis of the dose-response curves generated from
the data.
4.6 Determination of EC50 of NA inhibitors
Monolayers of MDCK cells in 96-well plates were infected with influenza virus for
1 h at 37°C. Cells were washed to remove residual viruses and various concentrations
of the compounds were added. After 36 h, viruses in the supernatant were harvested
and secondly infected the new monolayers of MDCK cells in 96-well plates for 1h.
Then cells were washed to remove residual viruses and the tested compounds. Then
new culture medium was added and monolayers of MDCK cells in 96-well plates
were infected with influenza (the harvested supernatant) for hours at 37°C. After
hours (A/PuertoRico/8/1934(H1N1) for 12h, A/Hong Kong/498/97(H3N2) for 24h,
and A/PuertoRico/8/1934(H259Y, NA resistant strain) for 20h), viruses in the
supernatant were harvested and determined as catalog 4.3. OC was used as a positive
control. Linear regression of the dose-response curve was performed to determine the
50% inhibitory effect on viral replication (EC50) for the tested and reference

compounds.
Acknowledgment
This work was supported by a grant from “Eleventh Five-Year” Major Innovation
Project for New Drug Candidates (2011ZX09401-014); the National Natural Science
Foundation (No. 81673306); the Project of State Key Laboratory of Natural
Medicines, China Pharmaceutical University (No. SKLNMKF201710). Dr. Hao Cai
and Zhipeng Ke are kindly acknowledged for providing results of molecular modeling.
We also thank Dr. Wei Tang at Wuhan Institute Of Virology, Chinese Academy of
Sciences for their work in the biological activity assays.
Supplementary Material
Supplementary data associated with this article can be found in the online version,
at dio:
Scheme 2. Synthesis of compound 16c.
Scheme 3. Synthesis of compound 16.
Copies of 1H and 13C NMR spectra for compounds 6-18 and 31-33.
RP-HPLC Data (C18 analytical column) for compounds 6-18 and 31-33.
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