Design, synthesis, and evaluation of a novel macrocyclic anti-EV71 agent

Article abstract of DOI:10.1016/j.bmc.2020.115551

We describe here the design, synthesis, and evaluation of a macrocyclic peptidomimetic as a potent agent targeting enterovirus A71 (EV71). The compound has a 15-membered macrocyclic ring in a defined conformation. Yamaguchi esterification reaction was used to close the 15-membered macrocycle instead of the typical Ru-catalyzed ring-closing olefin metathesis reaction. The crystallographic characterization of the complex between this compound and its target, 3C protease from EV71, validated the design and paved the way for the generation of a new series of anti-EV71 agents.

Full text of DOI:10.1016/j.bmc.2020.115551

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Design, synthesis, and evaluation of a novel macrocyclic anti-EV71 agent
Peng Li, Siqi Wu, Tianyichen Xiao, Yunlong Li, Zhiming Su, Wei Wei, Fei
Hao, Guoping Hu, Fusen Lin, Xinsheng Chen, Zhengxian Gu, Tianwei Lin,
Haiying He, Jian Li, Shuhui Chen
PII:
S0968-0896(20)30381-3
https://doi.org/10.1016/j.bmc.2020.115551
BMC 115551
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
7 January 2020
20 April 2020
5 May 2020
Please cite this article as: P. Li, S. Wu, T. Xiao, Y. Li, Z. Su, W. Wei, F. Hao, G. Hu, F. Lin, X. Chen, Z. Gu, T.
Lin, H. He, J. Li, S. Chen, Design, synthesis, and evaluation of a novel macrocyclic anti-EV71 agent, Bioorganic
& Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115551
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Design, synthesis, and evaluation of a novel macrocyclic anti-EV71 agent
Peng Li1,2,3, Siqi Wu1,2, Tianyichen Xiao1,2, Yunlong Li1,2, Zhiming Su1,2, Wei Wei3, Fei Hao3, Guoping
Hu3, Fusen Lin3, Xinsheng Chen3, Zhengxian Gu3, Tianwei Lin1,2*, Haiying He3, Jian Li3*, Shuhui Chen3
1 State Key laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, State-province Joint Engineering Laboratory of Targeted Drugs
from Natural Products, School of Life Sciences, Xiamen University, Xiamen, Fujian, China;
2 Cancer Research Center of Xiamen University, Xiamen, Fujian, P. R. China;
3 State Key Laboratory of Drug Lead Compound Research, WuXi AppTec (Shanghai) Co., Ltd., Shanghai, China.
* Corresponding authors. E-mail address: twlin@xmu.edu.cn (T. Lin), jian_li@wuxiapptec.com (J. Li).
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
We describe here the design, synthesis, and evaluation of a macrocyclic peptidomimetic as a
potent agent targeting enterovirus A71 (EV71). The compound has a 15-membered macrocyclic
ring in a defined conformation. Yamaguchi esterification reaction was used to close the 15-
membered macrocycle instead of the typical Ru-catalyzed ring-closing olefin metathesis reaction.
The crystallographic characterization of the complex between this compound and its target, 3C
protease from EV71, validated the design and paved the way for the generation of a new series of
anti-EV71 agents.
Keywords:
Enterovirus 71
3C Protease Inhibitor
Macrocycles
X-ray crystallography
Introduction
The human Enterovirus A71 (EV71) is the major causative agent in outbreaks of the hand, foot and mouth disease (HFMD) in
Asia in recent years. In particular, EV71 can cause infections in the central nervous system, leading to high mortality rates in young
children.1,2 Up to date, there is no specific antiviral therapy available to treat HFMD. 3
EV71 is an icosahedral virus with a single stranded and positive sense RNA genome. After entry to the host cell, its replication
starts with the translation of a polyprotein precursor from the monocistronic genome, which is subsequently processed into mature
proteins by virally encoded 2A protease and 3C protease (3Cpro). 3Cpro also plays crucial roles in evading and suppressing the host
defense systems.4 Because of the essential role in the life cycle of enteroviruses, 3Cpro has been widely recognized as an attractive
therapeutic target. Recent years, several examples of 3Cpro inhibitors for enteroviruses, including those for Human Rhinoviruses
(HRV), have been reported in literatures but no effective anti-enterovirus compounds have entered the market up to date.5-7 It is of
great importance to identify novel series of compounds targeting enteroviruses.
Design Strategy
EV71 3Cpro is a cysteine protease, and its enzymatic activity depends on the Cys-His-Glu catalytic triad in the active site. The
substrate-binding pocket of 3Cpro is shallow and a non-covalent chemical binder generally does not afford sufficient binding affinity
to inhibit 3Cpro. Consequently typical 3Cpro inhibitors are with warheads to form covalent bonds. AG7088 (Figure 1), also known as
Rupintrivir, is a peptidomimetic inhibitor containing an α,β-unsaturated ester as the warhead to form a covalent bond with the
nucleophilic Cys147 in the catalytic triad of 3Cpro. AG7088 is of high potency designed to target the 3Cpro of HRV and was advanced
to clinical trials for common cold. However, due to unfavorable stability, bioavailability, and efficacy, it did not pass the Phase II
trial.8

Typical Covalent inhibitors of EV71 3Cpro
H
N
O
NH
O
O
O
O
O
O
O
N
H
N
H
N
H
N
H
N
O
O
O
O
N
O
O
F
F
2
1 (AG7088)
HN
HN
O
O
O
H
N
O
O
H
N
O
H
N
N
H
N
H
N
H
O
O
F
O
O
5 (NK-1.8K)
6
Non-covalent inhibitors of EV71 3Cpro
HN
HN
O
O
O
H
N
O
NH
N
H
N
O
O
CN
OH
N
N
N
H
N
H
O
O
O
F
O
F
7
8
Cell-based
EC50
Mouse plasma
stability
Warhead type
Reference
1
2
5
6
7
8
α,β-unsaturated ester
α,β-unsaturated lactone
Aldehyde
1 ~ 9 nM
28 nM
2.7% left @ 2h
9, 10
9
88% left @ 2h
108 nM
1.12 μM
48 / 88 nM
0.1 μM
-
11, 12
13
α-keto amide
-
Cyanohydrin
t1/2 = 7 min
t1/2 > 2 h
14, 15
15
4‑Iminooxazolidin-2-one
Figure 1. Chemical structures and profile comparison of typical inhibitors of EV71 3Cpro with various warheads
We previously showed that AG7088 was a very potent anti-EV71 agent with an EC50 value of 9 nM in a cell-based assay. Other
typical EV71 3Cpro inhibitors such as compound 2, 5 and 6, shown in Figure 1, contain various types of covalent warheads, and recent
years, some novel inhibitors, like compound 7 and 8, with non-covalent warheads were also reported.9-15 Among above mentioned
inhibitors, some of them achieved a better balance between the potency and stability. But up to date, except AG7088, none of the
inhibitors advanced into the clinical trials.
Although AG7088 is the most advanced compound in development, it was unstable in the mouse plasma with only 2.7% remains
after 2 hours of incubation.9 We found however that the stability of AG7088 could be improved by increasing the size of the esters
and replacing the ethyl ester with lactone. For example, Compound 2 (Figure 1) was of enhanced mouse plasma stability and PK
profiles.9 These data suggested that the increase of steric hindrance, which restricted the conformation, might be an avenue to address
the issues of AG7088 in stability and pharmacokinetics (PK). Limiting the number of conformations would also reduce the entropy
loss and facilitate compound interaction with the target.
Macrocyclization has been adopted in medicinal chemistry to generate more rigid products with better stability and potency. 16-
18 Our design of a new series of anti-EV71 compounds starting with AG7088 for macrocyclization. The basic design principle was
for the product to maintain, if not enhance, the interaction with the target as for AG7088 with a suitable macrocyclic linkage.
P1
O
P4
P3
P2
NH
O
O
O
N
N
H
H
N
O
O
O
P1'
F
1 (AG7088)
EC50 = 9 nM

P1
H
H
O
N
O
N
P3
O
P1'
O
O
n
O
HN
N
O
HN
CO2Et
H
N
HN
O
N
O
N
O
H
O
N
O
O
n
P2
F
3
4
Figure 2. According to the structure of AG7088 and its conformation adopted in EV71 3Cpro, it can be found the orientation & distances of P3-P1
(5.1 Å) and P2-P1’ (5.3 Å) are quite suitable for macrocylic linkers. And our preliminary design of macrocyclic compounds with different type of
linkers is shown as general structures 3 and 4.
AG7088 fits snuggly in the substrate-binding pocket of 3Cpro, and, based on its complex structure with 3Cpro, the possible place
that could accommodate extra moieties in a macrocyclic compound was either in between P3 and P1 or in between P2 and P1’, judging
by the orientations to extend the newly attached atoms and the distances to the nearest atoms (5.1 Å and 5.3 Å). Synthesis feasibility
were also taken into consideration. The Ring-Closing Metathesis (RCM) reaction was our initial choice for the macrocyclic
compounds synthesis.19, 20 The unsaturated olefins in the intermediates of the RCM reaction however were kept to reduce the total
number of rotatory bonds for the further enhancement of the rigidity of the molecules.
The search for macrocycles with double bonds was first carried out with the Shape Screening module in the Schrodinger suite.
The in silico screening resulted in several macrocycles with the desired linkage types that could fit in the space either in between P3
and P1 or in between P2 and P1’ (Figure 2). Further modeling indicated that connecting the P1’ and P2 of AG7088 to form the
macrocyclic compound 4 could maintain most of the interactions. Compound 4 has four chiral isomers. Among these, isomer-4 was
the one that fit the substrate-binding pocket of 3Cpro in the similar fashion as AG7088 did (Figure 3).
Figure 3. Predicted binding modes of different isomers of compound 4. Isomer-1 was colored brown, isomer-2 was colored yellow, isomer-3 was
colored cyan, and isomer-4 was colored green.
Encouraged by the in silico screening results, compound 4 was selected for synthesis and evaluation.
Chemistry
Since we did not know which synthetic route would result in the desired isomer in advance, the two chiral centers were made as
a mixture. As shown in Figure 4, retrosynthetic analysis of compound 4 indicated that the desired macrocycle could be built in a
convergent manner using three building blocks B1, B2, and B3 (Figure 3). In the initial attempt, RCM reaction was employed to link
the terminal olefin moieties of B2 and B3 to make the macrocyclic ring.
H
O
N
O
OH
NH
O
O
O
O
H
2N
O
HN
O
O
N
N
H
O
N
HN
H
O
O
H2N
N
OH
O
O
O
O
4
B1
B2
B3
Figure 4. Retrosynthetic analysis of compound 4
Scheme 1 was the first synthetic route for compound 4 based on the retrosynthetic analysis. The synthesis of B1 was illustrated
in the supporting information. B3 was prepared according to the method described previously.21 Commercially available benzaldehyde
1-1 was converted to 1-2 via condensation with ethyl 2-nitroacetate under catalysis of TiCl4. Michael addition of but-3-en-1-ol to 1-
2 can provide 1-3 in near quantitative yield. The nitro group of 1-3 was then reduced by zinc dust to the corresponding intermediate
amine 1-4 (B2), which was coupled with intermediate B1 to give condensation product 1-5 under T3P condition. The ethyl ester

moiety in 1-5 was hydrolyzed to give acid 1-6, which was transformed to 1-7 via HOBT and EDCI mediated condensation reaction.
However, the ring-closing olefin metathesis reaction was not successful in the transformation of 1-7 to compound 4 under several
standard ruthenium-catalyzed olefin metathesis conditions including 1st, 2nd generation Grubbs' catalysts and Zhan Catalyst.
Interestingly, instead of desired conversion, olefin metathesis reaction occurred between the two double bonds on B3 moiety, and
mass of 1-8 was observed on LCMS as the likely main byproduct (data not shown).
Scheme 1 a
O
O
O
O
O
O
2N
O
2N
H2N
O
O
O
a
b
c
O
1-1
1-2
1-3
1-4 (B2)
O
O
O
OH
O
O
N
H
N
O
O
N
H
OH
H
e
N
O
B1
N
HN
O
O
N
HN
O
O
O
d
O
1-5
1-6
O
NH
H
O
O
NH
N
O
O
H
2N
O
O
O
B3
g
g
O
O
O
O
N
HN
N
HN
H
H
N
HN
O
f
N
HN
O
O
O
O
O
O
1-7
Compound 4
O
NH
O
O
N
HN
H
N
HN
O
O
O
1-8
a Synthetic Method A. Reagents and conditions: (a) ethyl 2-nitroacetate,TiCl4, THF,-10~25°C, 18h; (b) but-3-en-1-ol, LHMDS, 18-C-6, -78 °C; (c) Zn, HCl
a.q., EtOH, 0-25 °C, 12 h; (d) T3P, THF, 0-25 °C, 12h; (e) LiOH, THF/H2O, 0-20 °C, 12 h; (f) EDCI, HOBT, NMM, CH2Cl2, 0-25 °C, 12 h; (g) Grubbs,
Catalyst 2nd Generation, DCM, 25 °C, 12h.
Since the olefin metathesis reaction failed to close the large ring in the last step, an alternative route was adopted by using the
Yamaguchi esterification reaction,22 which could use the available 1-6 as the common intermediate.
Scheme 2 a
O
NH
O
NH
1-9
O
O
O
H2N
COOEt
N
H
OH
O
b
N
HN
COOEt
N
HN
O
O
H
a
N
HN
O
O
O
O
1-6
1-10
O
O
NH
NH
O
O
O
O
OEt
O
O
OH
c
d
N
H
HN
N
H
HN
N
HN
N
O
HN
O
O
O
O
OH
O
OH
1-11
1-12
H
O
N
O
O
O
O
HN
N
H
N
HN
O
O
O
4
a Synthetic Method B. (a) EDCI, HOBT, NMM, CH2Cl2, 0-25 °C, 12 h; (b) (E)-but-2-ene-1,4-diol, Grubbs Catalyst 2nd Generation, DCM, 25 °C, 12h (c)

LiOH, THF/H2O, 25°C, 12 h; (d) 2,4,6-trichlorobenzoyl chloride, Et3N, THF, 0-25°C, 2 h.
As Shown in Scheme 2, EDCI and HOBT mediated the coupling of intermediate 1-6 and 1-9 (prepared according to established
9
methods ) to afford 1-10, which underwent standard olefin metathesis reaction by the catalysis of 2nd generation Grubbs’ catalyst
with (E)-but-2-ene-1, 4-diol to give alcohol 1-11 under a very mild reaction condition with a 78% yield. Hydrolysis of α,β-unsaturated
ester moiety of 1-11 can give the corresponding acid 1-12 which was cyclized in the presence of 2, 4, 6-trichlorobenzoyl chloride and
Et3N to give the macrocyclic Compound 4 as a mixture of four diastereoisomers with moderate yield. Compound 4 was resolved into
three products after preparative HPLC purification. One of the three products was a mixture of two inseparable diastereoisomers. The
products were examined for potency and, if it was so identified, the stereochemistry of the active product would be determined.
The Properties of Compound 4
The inhibitory activities of the three isolated products from HPLC purification were separately evaluated in the anti-EV71 CPE
assay, and one of the single products exhibited a high anti-EV71 potency with an EC50 value of 4.5 μM.
To identify the chiral configuration of the active diastereoisomer in compound 4, the complex structure between the active
diastereoisomer in compound 4 and EV71 3Cpro were analyzed by macromolecular X-ray crystallography. The complex was obtained
by soaking the compound into the crystalline EV71 3Cpro for the structure determination.23 The stereochemistry of active
diastereoisomer of compound 4 was well defined in the X-ray structure, as is shown in Figure 5C. The crystal structure (PDB ID:
6LKA, Table 1) showed that isomer-4 was indeed the active isomer of compound 4 (Figure 5) in agreement with the modeling results
(Figure 3) and formed a covalent bond with Cys147 with a bond length of 1.8 Å, which was similar to AG7088. Like AG7088,
compound 4 bound at the S1-S4 and S’ sub-pocket and formed hydrogen bonds with Gly164, Thr142, and Gly145. The NH group
that replaced the methylene moiety in AG7088 formed an additional hydrogen bond with the side chain from Ser128. Although the
linker of macrocyclic ring did not form any specific interactions with the protein, it fitted well with defined electron density, probably
due to its rigid conformation (Figure 5).
H
O
N
C
O
O
O
O
HN
N
H
N
HN
O
O
O
Figure 5. Compound 4 bound at the active site of 3Cpro (A), its density (B) and the defined stereochemistry of the active diastereoisomer (C), PDB
ID: 6LKA.
Further test indicated, however, that the mouse plasma stability for isomer-4 of compound 4 was still unsatisfactory and over 90%
(5.6% left@2h) were degraded after an incubation of 2 hours.
Concluding Remarks
Macrocyclic compound 4 was synthesized by a highly convergent route from acyclic precursors via the Yamaguchi esterification
reaction instead of the standard Ru-catalyzed ring-closing olefin metathesis reaction.
The macrocyclic compound bound in the mode as was designed in the complex with EV71 3Cpro and its potency against EV71
was maintained although not to the full extent. With the complex structure in high resolution, a novel insight for 3Cpro inhibitor
development was gained from the interactions of the first macrocyclic inhibitor with the linker between P2 and P1’. It was obvious
that the macrocyclic linker was well accommodated in the substrate-binding groove of 3Cpro. However, none of additional interactions
was formed, which could be the reason that activity was dampened. Additional interactions between linker atoms and 3Cpro should be
vigorously designed and incorporated in future studies, especially for adopting this macrocycle strategy to other types of warheads.
The stability of compound 4 still needs further improvement. We further compared the stability data of AG7088, compound 2,
and other 3Cpro inhibitors with different esters. It seems that the stability could improve only if the steric hindrance group was placed
at the carbon atom adjacent to the carboxyl group, differing our current macrocyclic ester. It is an indication that the introduction of
additional steric hindrance would be of benefit to the stability of macrocycles.

In conclusion, we demonstrated the feasibility of a novel macrocyclic strategy for developing inhibitors to 3Cpro with a novel
compound 4 and its complex structure with EV71 3Cpro. With the improvements in potency and stability, new series of macrocyclic
compounds would be a way forward to develop new generations of EV71 3Cpro inhibitors.
Methods
Cell-Based Assay
Human embryonic rhabdomyosarcoma (RD) cells were seeded in a 96-well cell culture plate (8000 cells/well) in Dulbecco’s
Modified Eagle’s Medium (DMEM) with 2% fetal bovine serum (FBS). Cells were pre-incubated overnight in a 5% CO2 incubator
at 37 °C. The virus (EV71/Shenzhen/120F1/09 at 100×TCID50) and the chemical compounds in DMSO were added to the cells and
incubated at 37 °C for 72 hours. The final concentration of DMSO in each well was 1%. CCK-8 was added to each well in the end of
incubation to determine the cell viability. The optical density in each well was measured at 450 nm. The compound concentration
required to reduce the virus-induced cell death by 50% was defined as EC50.
Plasma stability assay
The compound stability was evaluated in CD-1 Mouse Plasma. The compounds were prepared in 10 mM DMSO and store at
2-8 °C. The solution was first diluted to 400 μM DMSO with 50% ACN/H2O and then to 40 μM DMSO with 20% ACN/H2O. The
plasma was thawed and warmed in water bath and centrifuged at 4,000 rpm for 5 min to remove clots. pH of the plasma was adjusted
to pH 7.4 ± 0.1 with either NaOH or phosphoric acid. Five microliters of the compound solution in 40 μM DMSO were mixed with
95 μl of the plasma and incubated for 0, 10, 30, 60, and 120 minutes in duplicates at 37 °C. The incubation was terminated with 300
μl of a stop solution (50% ACN/MeOH containing 100 ng/mL Tolbutamide and 10 ng/mL Buspirone), mixed for 20 min, centrifuged
at 3,220 RCF for 20 min, and subjected to LC / MS / MS analysis. The percentage of remaining compounds after incubation in plasma
was calculated as the ratio between PAR at a specific time point and PAR at 0 min. PAR is the peak area ratio between the test
compound and an internal standard.
Crystallization and structure determination
Crystals of EV71 3Cpro were obtained by the methods of hanging-drop vapour diffusion and the droplets were prepared with a
1:1(v:v) mixture of EV71 3Cpro (12 mg/ml-1) and reservoir solution consisting of 100 mM Tris–HCl pH 8.5, 25% PEG4000, and 0.8
M lithium chloride. Crystals suitable for data collection were obtained after one week at 289 K.
The complex was prepared by socking the EV71 3Cpro crystal with the compound solution in a 1:5 molar ratio and incubating
overnight at 277 K. The conditions used for co-crystallization were basically the same as those used for the protein alone.
The crystals were flash-cooled to 100 K under liquid nitrogen for data collection. X-ray diffraction data for the complex were
collected using an in house X-ray facility equipped with a Rigaku rotating-anode X-ray generator (λ= 1.5418 Å) and a MAR345dtb
detector and processed with the Automar package from MAR Research GmbH. The structure of EV71 3Cpro was solved by molecular
replacement using the program Phaser (McCoy et al., 2007). The coordinates of EV71 3Cpro complex (Protein Data Bank accession
code 4GHT; Wu C et al., 2013) were used as the search model. Manual model building and refinement were carried out with Coot
(Emsley et al., 2010) and PHENIX24 .The compound library file was generated using AceDRG (Fei Long et al., 2017). The figures
were produced using PyMOL 2.2.3 (Schrödinger). The statistics for data collection and refinement were shown in Table 1.
Table 1
Data-collection and refinement statistics.
Values in parentheses are for the highest resolution shell.
Data collection
Space group
Unit cell
a(Å)
C2221
64.27
b(Å)
64.64
c(Å)
75.59
α(°)
90.00
β(°)
90.00
γ(°)
90.00
6.9（42.6）
38077
Rmerge (%)
Total observations

Unique reflections
Resolution range (Å)
Completeness (%)
Average I/σ(I)
Multiplicity
8598
50.00-2.03（2.11-2.03）
95.1
6.2（1.1）
3.96（3.61）
Refinement
0.1955（0.2589）
R-factor/Rfree
no. of atoms
Protein
1364
87
Ligand/ion
Water
118
B factors (Å2)
Protein
40
Ligand/ion
55.2
43.1
Water
r.m.s deviation
Bond lengths (Å)
Bond angles (°)
Ramachandran statistics† (%)
Most favoured region
Allowed regions
Outliers
0.008
0.017
91.48
6.25
2.27
Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (31670729 and 31370724), Project 111
sponsored by the State Bureau of Foreign Experts and Ministry of Education of China (BP2018017). The crystallographic data
collections at Beamline BL17U1, BL18U1, and BL19U1 at Shanghai Synchrotron Radiation Facility are gratefully acknowledged.
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Supplementary Material
General Information.
Reagents and solvents were obtained from commercial suppliers and were used without further purification. 1H NMR measurements
were recorded at 400 MHz using a Bruker AMX400instrument. Chromatographic purifications were performed silica gel (300-400
mesh). Preparative HPLC separations were performed on a Boston Green ODS 150*30*5u column using gradient mixtures of
water/0.1% TFA and water (10%)/acetonitrile (90%)/0.1% TFA. All of the compounds were established by a variety of LC/MS, and
NMR analytical techniques, and purities were >95% for all final products.
General Procedure for preparation of B1.
O
O
O
O
H2N
COOMe
B1-3
Cl
OH
SOCl2
N
COOMe
N
H
COOH
LiOH
H
N
N
O
N
O
O
N
O
THF/H2O
B1-1
B1-2
B1-4
B1
(S)-methyl 2-amino-3-methylbutanoate (B1-3). To a solution of L-Valine (23.43 g, 0.2 mol) in methanol (140 mL) was added
SOCl2 (28.4 mL, 0.40 mol) at 25 oC. The resulting mixture was stirred at 80 oC for 12 h. The reaction mixture was concentrated to
give B1-3 as white solid (33.5 g, yield: 100%). 1H NMR (400 MHz, CDCl3) δ 8.80 (brs, 2H), 3.98 (s, 1H), 3.84 (s, 3H), 2.48-2.47
(m, 2H), 1.17-1.13 (m, 6H).
5-methylisoxazole-3-carbonyl chloride (B1-2). A solution of compound B1-1 (30 mg, 0.24 mmol) in SOCl2 (1 mL) was stirred at
80oC under N2 for 2 hrs. The mixture was concentrated to give compound B1-2 (30 mg, crude). It was used for next step without
further purification.
(S)-methyl 3-methyl-2-(5-methylisoxazole-3-carboxamido)butanoate (B1-4). To a solution of compound B1-2 (1.0 g, 6.0 mol)
o
and Et3N (1.80 g, 18.0 mol) in dry DCM (10 mL) was added compound B1-3 (1.04 g, in 5.0 mL DCM) dropwise at 25 C. The
o
resulting mixture was stirred at 25 C for 1h. The reaction mixture was concentrated and EtOAc (50 mL) and water (50 mL) was
added. The mixture was separated and the organic phase was washed with water (50 mL). The organic phase was dried over Na2SO4,
filtered and concentrated to give B1-4 as white solid (0.86 g, yield: 60%). 1H NMR (400 MHz, CDCl3) δ 7.25-7.23 (m, 1H), 6.42
(s, 1H), 4.75-4.65 (m, 1H), 3.77 (s, 3H), 2.50 (s, 3H), 2.35-2.20 (m, 1H), 1.10-0.90 (m, 6H).
(S)-3-methyl-2-(5-methylisoxazole-3-carboxamido)butanoic acid (B1). To a solution of B1-4 (4.3 g, 17.9 mol) in THF/H2O (120
mL, 1:1) was added LiOH (0.86 g, 36.0 mmol) at 25 oC. The resulting mixture was stirred at 25 oC for 3h. The reaction mixture was
concentrated and acidified to pH = 4 by citric acid, then extracted by DCM (50 mL*3). The organic phase was dried over Na2SO4,
filtered and concentrated to give B1 as yellow oil (3.5 g, yield: 88%). 1H NMR (400 MHz, CDCl3) δ 7.30-7.23 (m, 1H), 6.46 (s,
1H), 4.80-4.65 (m, 1H), 2.49 (s, 3H), 2.40-2.25 (m, 1H), 1.10-0.90 (m, 6H).
General procedure for Scheme 1
Ethyl 2-nitro-3-phenylacrylate (1-2). To a stirred solution of ethyl 2-nitroacetate (88 g, 0.66 mol) and benzaldehyde (100 g, 0.94

mol) in THF (1000 mL) was added TiCl4 (198 g, 1.06 mol) at -10 ℃. The mixture was stirred for 10 min and treated with N-methyl
morpholine (284 g, 2.82 mol) dropwise over 20 min. The mixture was allowed to warm to room temperature for 18h. The reaction
was quenched with water and extracted with EA (300 mL*3), dried with Na2SO4, filtered and concentrated. The crude product was
purified by silica gel chromatography (PE / EA = 30 / 1) to give compound 1-2 (55 g, yield: 34.8%). 1H NMR (400 MHz, CDCl3)
δ 7.39-7.60 (m, 6H), 4.35-4.51 (m, 2H), 1.33-1.43 (m, 3H).
Ethyl 3-(but-3-en-1-yloxy)-2-nitro-3-phenylpropanoate (1-3). But-3-en-1-ol (3.9 g, 54 mmol) in THF (30 mL) was added KHMDS
o
o
(54 mL, 1M, 54.0 mmol) at -78 C under N2. The resulting mixture was stirred at -78 C for 20 min. Then 18-crown-6 (14.2 g, 54
o
mmol) in dry toluene (50mL) was added. The resulting mixture was stirred at -78 C for 30 min. Then Compound 1-2 (8.0 g, 36
mmol) in dry THF (60 mL) was added dropwise. The resulting mixture was stirred at -78 oC for 3h. Then water (100 mL) was added.
The organic phase was separated and the aqueous phase was extracted by EA (50 mL*2). The combined organic phase was dried over
Na2SO4, filtered and concentrated to give 1-3 as yellow oil (10 g, yield: 94%). 1H NMR (400 MHz, CDCl3) δ 7.27-7.40 (m, 5H),
5.73-5.80 (m, 1H), 5.01-5.08 (m, 2H), 4.79 (d, J = 4.4 Hz, 1H), 4.12-4.17 (m, 2H), 3.88 (d, J = 4.4 Hz, 1H), 3.31-3.52 (m, 2H), 2.68
(brs., 1H), 2.27-3.35 (m, 2H), 1.18 (t, J = 7.2 Hz, 3H). MS (ESI+) m/z : 280 ([M＋H] +).
Ethyl 2-amino-3-(but-3-en-1-yloxy)-3-phenylpropanoate (1-4). Zn (44.3 g, 0.68 mmol) was added to a mixture of compound 1-3
(10 g, 34 mmol) and HCl (4N, 100 mL) at 0oC under N2. The resulting mixture was stirred at 25oC for 18 h. Then aq. NaHCO3 was
added until pH = 8. The organic phase was separated and the aqueous phase was extracted by EA (150 mL*3). The combined organic
phase was dried over Na2SO4, filtered and concentrated. The crude product was purified by silica gel chromatography (PE / EA = 5 /
1) to give compound 1-4. MS (ESI+) m/z : 264 ([M＋H] +).
Ethyl 3-(but-3-en-1-yloxy)-2-((S)-3-methyl-2-(5-methylisoxazole-3-carboxamido)butanamido) -3-phenylpropanoate (1-5). To
a solution of compound 1-5 (100 mg, 0.44 mmol) and compound 1-4 (140 mg, 0.53 mmol) in anhydrous THF (5 mL) was added
dropwise T3P (560 mg, 50% in EA, 0.88 mmol) at 0 oC under N2 and stirred for 12 h. After the reaction was complete, water (10
mL) was added and EA (10 mL*3) was used to extract the product, the organic layer was washed with brine, dried over with Na2SO4,
filtered and concentrated to give compound 1-5 (150 mg, yield: 72%). 1H NMR (400 MHz, CDCl3) δ 7.15-7.23 (m, 8H), 6.35-6.46
(m, 2H), 5.68-5.70 (m, 1H), 4.87-5.00 (m, 3H), 4.59-4.75 (m, 1H), 4.10-4.35 (m, 3H), 3.25-3.47 (m, 2H), 2.22-2.44 (m, 8H), 0.65-
0.91 (m, 13H). MS (ESI+) m/z : 472 ([M＋H] +).
3-(but-3-en-1-yloxy)-2-((S)-3-methyl-2-(5-methylisoxazole-3-carboxamido)butanamido)-3-phenylpropanoic acid (1-6). To a
solution of compound 1-5 (150 mg, 0.32 mmol) in THF (5 mL) and H2O (1 mL) was added LiOH.H2O (54 mg, 1.27 mmol) in portions.
o
The reaction was stirred at 25 C for 16 hours. After the reaction was complete, water (30 mL) was added, extracted with EA (20
mL*2). The aqueous phase was adjusted pH = 3 with 1N HCl, EA (20 mL*3) was used to extract the product. The organic phase was
washed with brine, dried over with Na2SO4, concentrated to give 1-6 (100 mg, yield: 71 %). 1H NMR (400 MHz, CDCl3) δ 7.16-
7.26 (m, 6H), 6.36-6.60 (m, 2H), 5.61-5.78 (m, 1H), 4.79-5.00 (m, 3H), 4.33-4.36 (m, 1H), 3.33-3.50 (m, 2H), 2.40-2.44 (m, 4H),
2.24-2.27 (m, 2H), 0.80-1.29 (m, 8H). MS (ESI+) m/z : 444 ([M＋H] +) .
(4S,E)-ethyl
4-(3-(but-3-en-1-yloxy)-2-((S)-3-methyl-2-(5-methylisoxazole-3-carboxamido)
butanamido)-3-
phenylpropanamido)-5-((S)-2-oxopyrrolidin-3-yl)pent-2-enoate (1-7). To a solution of compound 1-6 (500 mg, 1.13 mmol),
NMM (685 mg, 6.78 mmol) and compound B3 (444 mg, 1.69 mmol) in dry DCM (10 mL) was added EDCI (434 mg, 2.26 mmol)
o
and HOBT (305 mg, 2.26 mmol) in turns at 0 C under N2. The reaction was stirred at 25 oC for 12 h. The reaction mixture was
quenched with water at 0oC. extracted with EA（50 mL*3）, and the combined organic phase was washed with brine, dried over
Na2SO4 and concentrated in vacuum. The residue was purified by prep-HPLC (TFA) to give compound 1-7 (300 mg, yield: 41%).
1H NMR (400 MHz, CDCl3) δ 8.81-9.06 (m, 1H), 6.50-7.33 (m, 9H), 5.62-5.91 (m, 2H), 4.62-5.74 (m, 4H), 4.13-4.19 (m, 2H), 3.33-
3.47 (m, 4H), 2.28-2.54 (m, 7H), 1.61-1.95 (m, 3H), 0.53-0.99 (m, 9H). MS (ESI+) m/z : 652 ([M＋H] +).
General procedure for Scheme 2
(4S,E)-ethyl-4-(3-(((E)-5-hydroxypent-3-en-1-yl)oxy)-2-((S)-3-methyl-2-(5-methylisoxazole-3 -carboxamido)butanamido)-3-
phenylpropanamido)-5-((S)-2-oxopyrrolidin-3-yl)pent-2-enoate (1-11). To a solution of compound 1-10 (100 mg, 48.5 mmol，
prepared using the same procedure of 1-7) and (E)-but-2-ene-1,4-diol (244 mg, 3 mmol) in DCM (10 mL) was added Grubb’s 2nd (20
mg, cat.) at 25 oC. The reaction mixture was stirred at 25 oC for 12 h. The reaction was concentrated and the crude product was
purified by prep-HPLC (TFA) to give compound 1-11 (80 mg, yield: 78%) as a white solid. MS (ESI+) m/z : 682 ([M＋H] +).

(4S,E)-4-(3-(((E)-5-hydroxypent-3-en-1-yl)oxy)-2-((S)-3-methyl-2-(5-methylisoxazole-3-carboxamido)butanamido)-3-
phenylpropanamido)-5-((S)-2-oxopyrrolidin-3-yl)pent-2-enoic acid (1-12). A solution of compound 1-11 (58 mg, 0.1 mmol) and
o
LiOH.H2O (17 mg, 0.4 mmol) in THF (4 mL) and H2O (1 mL) was stirred at 25 C for 4 hours. The progress of the reaction was
monitored by TLC. After completion of the reaction, the mixture was concentrated and adjust pH = 3 with HCl (1M), extracted with
EA (20 mL*3) and concentrated to give compound 1-12 (50 mg, yield: 91%), which was used for the next step without further
purification. 1H NMR (400 MHz, CDCl3) δ 7.56-8.45 (m, 1H), 7.20-7.54 (m, 5H), 6.50-6.97 (m, 2H), 5.62-5.94 (m, 2H), 4.06-5.17
(m, 6H), 3.33-3.75 (m, 6H), 1.83-2.54 (m, 14H), 0.60-1.43 (m, 6H). MS (ESI+) m/z : 654 ([M＋H] +).
N-((2S)-1-(((6S,7E,12E)-4,9-dioxo-6-(((S)-2-oxopyrrolidin-3-yl)methyl)-2-phenyl-1,10-dioxa-5-azacyclopentadeca-7,12-dien-
3-yl)amino)-3-methyl-1-oxobutan-2-yl)-5-methylisoxazole-3-carboxamide (Compound 4) To a solution of compound 1-12 (70
mg, 0.11 mmol) and TEA (111 mg, 1.1 mmol) in THF (5 mL) was added 2,4,6-trichlorobenzoyl chloride (134 mg, 0.55 mmol). The
reaction mixture was stirred at 15 oC for 1 h and 25 oC for 2 h. The reaction mixture was added dropwise to a mixture of DMAP (268
mg, 2.2 mmol) in toluene (60 mL) at 25 oC under N2 and stirred at 25 oC for 2 h. 1N HCl (aq.) was added until pH = 3. EA (50 mL*2)
was used to extract the product, dried over Na2SO4, concentrated in vacuum and the residue was purified with prep-HPLC (TFA) to
give compound 4 (3 diastereoisomers, total yield: 33%) as white solid.
Diastereoisomer 1 (the active diastereoisomer): 1H NMR (400 MHz, CDCl3) δ 7.52-8.61 (m, 1H), 6.95-7.52 (m, 7H), 6.39-6.49 (m,
1H), 5.30-6.04 (m, 2H), 4.46-4.70 (m, 3H), 3.45-3.52 (m, 1H), 2.87-3.13 (m, 2H), 2.01-2.48 (m, 12H), 1.60-1.65 (m, 1H), 1.20-1.30
(m, 1H), 0.45-0.89 (m, 6H). 13C NMR (400 MHz, CD3OD) δ 171.9, 170.5, 166.7, 159.5, 158.3, 128.2, 127.9, 126.9, 125.2, 125.0,
122.8, 121.9, 110.6, 103.1, 101.9, 100.6, 81.1, 67.7, 62.2, 59.6, 58.9, 67.5, 57.3, 50.2, 40.1, 39.0, 38.2, 31.7, 17.9, 17.5, 10.6. MS
(ESI+) m/z: 636 ([M＋H] +). HRMS (ESI) m/z calcd for C33H41N5O8 [M+H]+ 636.3028, found 636.3043.
Diastereoisomer 2: 1H NMR (400 MHz, CDCl3) δ 7.71-7.73 (m, 1H), 7.16-7.35 (m, 5H), 7.18-7.19 (m, 1H), 6.54-6.57 (m, 1H), 5.35-
6.06 (m, 2H), 5.05-5.06 (m, 1H), 4.17-4.66 (m, 4H), 3.35-3.65 (m, 2H), 3.02-3.26 (m, 2H), 1.76-2.51 (m, 11H), 0.74-0.89 (m, 7H).
MS (ESI+) m/z : 636 ([M＋H] +) . HRMS (ESI) m/z calcd for C33H41N5O8 [M+H]+ 636.3028, found 636.3035.
Diastereoisomer 3: 1H NMR (400 MHz, CDCl3) δ 8.60-8.78 (m, 1H), 7.23-7.40 (m, 6H), 6.04-6.92 (m, 2H), 5.46-5.71 (m, 2H), 5.18-
5.23 (m, 1H), 4.53-4.63 (m, 3H), 4.19-4.23 (m, 1H), 3.58-3.65 (m, 2H), 3.08-3.20 (m, 2H), 2.28-2.75 (m, 7H), 1.75-1.83 (m, 2H),
0.78-0.85 (m, 4H), 0.39-0.49 (m, 4H). MS (ESI+) m/z : 636 ([M＋H] +). HRMS (ESI) m/z calcd for C33H41N5O8 [M+H]+ 636.3028,
found 636.3040.

Graphical Abstract
We describe here the design, synthesis, and evaluation of a macrocyclic peptidomimetic as a potent agent targeting enterovirus
A71 (EV71). The compound has a 15-membered macrocyclic ring in a defined conformation. Yamaguchi esterification reaction
was used to close the 15-membered macrocycle instead of the typical Ru-catalyzed ring-closing olefin metathesis reaction. The
crystallographic characterization of the complex between this compound and its target, 3C protease from EV71, validated the
design and paved the way for the generation of a new series of anti-EV71 agents.
Leave this area blank for abstract info.
Design, synthesis, and evaluation of a novel
macrocyclic anti-EV71 agent
Peng Li, Siqi Wu, Tianyichen Xiao, Yunlong Li, Zhiming Su, Fei Hao, Jiao Hu, Fusen Lin, Xinsheng
Chen, Zhengxian Gu, Tianwei Lin, Haiying He, Jian Li, Shuhui Chen
H
O
O
N
NH
O
O
O
O
O
Macrocyclation
O
HN
OEt
N
N
N
H
HN
N
H
H
O
N
O
O
O
O
O
F
Rupintrivir (AG7088)
4 (Isomer-4)
Co-crystal structure of compound 4 and EV71 3Cpro