Design, synthesis and biological evaluation of amino acids-oleanolic acid conjugates as influenza virus inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.115147

Viral entry inhibitors are of great importance in current efforts to develop a new generation of anti-influenza drugs. Inspired by the discovery of a series of pentacyclic triterpene derivatives as entry inhibitors targeting the HA protein of influenza vi

Full text of DOI:10.1016/j.bmc.2019.115147

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Design, synthesis and biological evaluation of amino acids-oleanolic acid con-
jugates as influenza virus inhibitors
Lingkuan Meng, Yangqing Su, Fan Yang, Sulong Xiao, Zhili Yin, Jiaxin Liu,
Jindong Zhong, Demin Zhou, Fei Yu
PII:
S0968-0896(19)30941-1
https://doi.org/10.1016/j.bmc.2019.115147
BMC 115147
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
29 June 2019
9 September 2019
30 September 2019
Please cite this article as: Meng, L., Su, Y., Yang, F., Xiao, S., Yin, Z., Liu, J., Zhong, J., Zhou, D., Yu, F., Design,
synthesis and biological evaluation of amino acids-oleanolic acid conjugates as influenza virus inhibitors,
Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.115147
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Design, synthesis and biological evaluation of amino acids-oleanolic
acid conjugates as influenza virus inhibitors
Lingkuan Menga, Yangqing Sua, Fan Yanga, Sulong Xiaob, Zhili Yina,
Jiaxin Liua, Jindong Zhongc, Demin Zhoub, Fei Yua,*,feiyuz8@kmust.edu.cn
aMedical School of Kunming University of Science and Technology, Kunming,
Yunnan 650500, China
bState Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical
Sciences, Peking University, Beijing 100191, China
cFaculty of Life Science and Technology of Kunming University of Science and
Technology, Kunming, Yunnan 650500, China
*Corresponding author at: Medical School, Kunming University of
Science and Technology, No.727 South Jingming Rd., Chenggong District, Kunming,
650500, China.
Graphical abstract
Abstract
Viral entry inhibitors are of great importance in current efforts to
develop a new generation of anti-influenza drugs. Inspired by the
discovery of a series of pentacyclic triterpene derivatives as entry
inhibitors targeting the HA protein of influenza virus, we designed and
synthesized 32 oleanolic acid (OA) analogues in this study by

conjugating different amino acids to the 28-COOH of OA. The antiviral
activity of these compounds was evaluated in vitro. Some of these
compounds revealed impressive anti-influenza potencies against
influenza A/WSN/33 (H1N1) virus. Among them, compound 15a
exhibited robust potency and broad antiviral spectrum with IC50 values at
the low-micromolar level against four different influenza strains.
Hemagglutination inhibition (HI) assay and docking experiment indicated
that these OA analogues may act in the same way as their parent
compound by interrupting the interaction between HA protein of
influenza virus and the host cell sialic acid receptor via binding to HA,
thus blocking viral entry.
Keywords: Influenza A virus; Entry inhibitor; Oleanolic acid; Amino
acids; Hemagglutinin
1. Introduction
Influenza A virus (IAV) can cause substantial morbidity and mortality
through routine seasonal spread and seasonal epidemics, and therefore it
can cause a serious threat to human health.1,2 IAV infects 3 million people
leading to approximately 250,000 to 500,000 deaths every year
worldwide.3,4 Subtypes of influenza A virus are classified based on the
antigenicity of two major surface glycoproteins, hemagglutinin (HA) and
neuraminidase (NA).5 HA binds to sialic acid-containing receptors on
target cells to initiate virus infection, whereas NA cleaves sialic acids
from cellular receptors and extracellular inhibitors to facilitate progeny
virus release.6 The high mutation rate of the RNA genome of IAV
promotes antigenic diversity and new subtypes, allowing the virus to
avoid vaccines and become resistant to antiviral drugs.7 Hence, the
development of novel anti-influenza virus agents with high efficiency and

broad antiviral spectrum is highly importance. Recently, antiviral
therapeutics are available targeting two of the three major influenza virus
surface proteins: the NA and the M2 ion channel.8-10 Unfortunately,
because most of the currently circulating influenza viruses are resistant to
M2 inhibitors, amantadine and rimantadine are no longer recommended
as common clinical anti-influenza treatments.11 Furthermore, mutant
viruses resistant to NA inhibitors have also been detected in humans.12,13
IAV infection begins with the binding of HA protein to its cellular
receptor, sialic acid (SA) linked glycoproteins, followed by fusion of the
viral and endosomal membranes. Because it is the first step of viral
infection, a favorable target for drug discovery is to inhibit IAV entry.14,15
Thus, effective blocking of the interaction between HA and SA could
lead to suppression of viral infection.16,17
In our previous research, we revealed that oleanolic acid (OA), a kind
of pentacyclic triterpene natural product, and its analogues, show
biological activity against A/WSN/33 (H1N1) virus invasion into MDCK
cells.18,19 Mechanistically, such pentacyclic triterpenes and their
derivatives interrupt the interaction between viral HA protein and SA
receptor by binding to HA, thus blocking the attachment of viruses to
host cells.
Herein, we designed and synthesized a series of OA derivatives, in
which different amino acids were coupled to the C-28 carboxyl group of
OA via an amide bond. Through the introduction of different functional
groups, we aimed to enhance the affinity of the compound with its target
protein HA so as to increase its anti-IAV activity. All the synthesized
compounds were tested, and we found that compound 15a showed
significantly higher anti-IAV activity than its precursor and the results are
discussed below.

2. Results and Discussion
2.1. Chemistry
Amino acids are usually considered as useful modules to synthesize
biologically active compounds. The introduction of such molecules with a
variety of different functional groups might enhance the binding affinity
of certain compounds to the target protein and thereby enhance their
biological activity. Herein, we designed and synthesized a series of OA
derivatives, in which different amino acids were coupled to the 28-COOH
of OA via an amide bond. The general synthesis for compounds 2-11
were conducted according to the following procedures (Scheme 1).
Compound 1 was obtained with over 85% yield by stirring OA with
O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate
(TBTU) and N,N-Diisopropylethylamine (DIEA) in THF for 4h.20
Treatment of intermediate 1 with the corresponding amino acid methyl
esters in the presence of Na2CO3 in DMF afforded target compounds
2-1121. After that, the methyl esters of compounds 2-11 were hydrolyzed
by NaOH in THF/CH3OH (1:1), which yielded compounds 2a-11a,
respectively.
However, it is difficult to obtain compounds 12–17 by the same
strategy, owing to the steric hindrance and decreased nucleophilicity.
Therefore, 1-ethyl-3-(3-dimethylaminepropyl) carbodiimide (EDC) was
used as the coupling reagent to conjugate amino acid methyl esters and
28-COOH of OA. As shown in Scheme 2, compounds 12-17 were
synthesized by condensation of OA 28-COOH and certain amino acid
methyl esters in the presence of EDC and Triethylamine (TEA) in dry
THF. Using the same strategy which yielded 2a-11a from 2-11,
compounds 12a-17a can be obtained in high yields. All the synthesized
compounds were characterized by 1H NMR, 13C NMR and ESI-HRMS.

2.2. Anti-influenza virus activity of OA-amino acid conjugates
The CellTiter-Glo® screening, an assay which monitors cell viability,
was utilized to evaluate the antiviral activity of the synthesized
conjugates and to exclude compounds with significant toxicity toward
MDCK cells22. As shown in Figure 1A, all the OA-amino acid conjugates
exhibited increased cytotoxicity compared to OA. Interestingly, in
addition to compounds 7 (the OA-serine methyl ester conjugate), 9 (the
OA-histidine methyl ester conjugate), 13a (the OA-aspartic acid
conjugate) and 15a (the OA-arginine conjugate), compounds with the
carboxyl group of the amino acids protected by the methyl ester, all
exhibited significant less cytotoxic than those with free carboxyl group
(2-6, 8, 10-17) at 100 μM. This result indicates that the substituted groups
of amino acid modules have a critical effect on the toxicity of the
conjugates.
In the antiviral experiment, the activities of all the compounds against
the influenza A/WSN/33 (H1N1) virus were evaluated by the CPE
reduction assay, and the inhibition rate was shown in Figure 1B.
Compared with DMSO which is used as a negative control, influenza
A/WSN/33(H1N1) virus causes a severe CPE in MDCK-infected cells.
As shown in Figure 1B, most of the compounds displayed higher
inhibition rate than OA. However, when compared with Figure 1A, this
observation might reflect cellular toxicity rather than potency
enhancement. According to the results, compound 15a, whose viability
and inhibition rate were both over 75%, was selected for anti-H1N1
dose-response assay. We also inquired whether 15a could exert broad
antiviral spectrum, and it was evaluated against three strains of influenza
A and B viruses (Table 1). Among them, one belongs to influenza A type,
and the other two viruses belong to the Yamagata and Victoria lineages of
the influenza B virus. All of them are oseltamivir (OSV) resistant strains.

The IC50 values of compound 15a, along with the positive control
(oseltamivir phosphate, OSV-p), were determined and listed in Table 1.
We found compound 15a displayed anti-influenza virus activity with IC50
values in the range of 6.64−61.58 μM. Furthermore, compound 15a was
effective for all tested strains including the OSV-resistant
A/Texas/50/2012 strain and even the influenza B virus (BX-35 and
BX-51B). Such a broad anti-influenza spectrum is significantly different
from that of oseltamivir, which is much less effective against
A/Texas/50/2012 (H3N2), BX-35 and BX-51B viruses (IC50 > 100 μM).
2.3. Hemagglutination Inhibition Assay
As we know, the transmembrane viral envelope protein HA plays an
important role at the early stage of influenza virus infection. Therefore,
hemagglutination inhibition (HAI) assay was applied to investigate
whether compound 15a targets HA protein. In this assay, 2-fold serial
dilutions of compound 15a were made from 100 μM to 1.56 μM. In
parallel, inhibition of hemagglutination by anti-HA antibody was used as
the positive control. As shown in Figure 2, 15a displayed a similar
capability as anti-HA antibody in effectively inhibiting influenza
virus-induced aggregation of chicken erythrocytes. This result suggests
that 15a and anti-HA antibody may have the same target, HA, and thus
blocks the interactions between viruses and target cells.
2.4. Docking calculation
Considering that HA may be a potential target for OA derivatives, we
investigated the binding pocket and binding free energy of compound 15a
and HA through blind docking calculations (Figure 3). Compound 15a

was docked into the influenza HA protein, which was obtained from the
RCSB Protein Data Bank, using AutoDock 4.2 program. The docked
conformations of HA-15a were determined based on the minimum free
energy analyses. The computer-aided docking data suggests that
compound 15a exhibits a similar binding mode as compound Y3, by
occupying the binding pocket for sialic acid receptor with an estimated
binding energy of -9.57 kcal/mol and an inhibition constant (Ki) of 24.11
nM. This means compound 15a can bind tightly to HA protein. The
docking result indicated that the 3-OH of the OA moiety formed two
hydrogen bonds with S193 residues within the HA1 conserved domain
associated with sialic acid binding. For the arginine moiety, the carboxyl
and the sub-amino groups formed three hydrogen bonds with residue
A137 and Q226, which are also critical residues involved in sialic acid
binding.
3. Conclusion
In summary, to find compounds with higher anti-IAV activity and
broader antiviral spectrum, we have designed and synthesized 32 OA
analogues by conjugating different amino acids to the 28-COOH of OA.
Compound 15a exhibited better inhibitory potency against influenza
A/WSN/33 (H1N1) virus, with an IC50 value of 6.64 μM. Moreover, it
also showed a broad anti-influenza spectrum, with IC50 values of 14.05
μM, 61.58 μM and 45.8 μM against A/Texas/50/2012, BX-35 and
BX-51B, better than that of OSV-p (>100 μM，>100 μM and >100 μM,
respectively). Hemagglutination inhibition assay indicated that these
compounds may target the HA protein of influenza virus, and docking
studies suggested that these compounds may occupy HA’s conserved
binding pocket for sialic acid receptor, disrupting the interaction between
HA and the SA receptor and thus blocking the attachment of viruses to

host cells.
4. Experimental protocols
4.1.Chemistry
General Procedure A for the Synthesis of OA O-methylation amino acids
conjugates (Compounds 2-11)
To Compound 1 (1 equiv.) and Na2CO3 (1.5 equiv.) stirring in 8 mL
DMF was added O-methylation amino acid (1.5 equiv.). The mixture was
stirred at room temperature for 20 min. After completion (TLC), the
solvent was removed under reduced pressure. The mixture was resolved
in AcOEt and washed with water and brine twice. The organic layer was
dried over Na2SO4, then filtered and concentrated. The crude residue was
purified by column chromatography.
General Procedure B for the Synthesis of OA O-methylation amino acids
conjugates (Compounds 12-17)
OA (1 equiv.) was added to a stirred solution of O-methylation amino
acid (1.5 equiv.) and TEA (1.5 equiv.) in THF. After 10 min, EDC (1.5
equiv.) was added to the stirred solution, and the mixture was then stirred
at room temperature overnight. The mixture was resolved in AcOEt and
washed with water and brine twice. The organic layer was dried over
Na2SO4, then filtered and concentrated. The crude residue was purified by
column chromatography.
General Procedure C for the Synthesis of OA amino acids conjugates
(Compounds 2a-17a)
To the O-methylationamino acids conjugates stirring in THF/CH3OH (1:1)
was added sodium hydroxide (cat.). The mixture was stirred at rt. After
completion (TLC), the reaction mixture was neutralized with HCl (1 M).
Water was added, and the resulting suspension was filtered.

Compound 2 Prepared from O-methylation glycine (133.5 mg, 1.5 mmol)
according to general procedure A. The residue was purified by column
chromatography (petroleum ether/ethyl acetate, 3/1 v/v) to afford 2 as a
white solid (418.9 mg, 78%). m.p.: 133.3–135.1 °C; 1H NMR (600 MHz,
(CD3)2SO) δ: 0.64-1.08 (m, 7×CH3), 1.08-2.00 (m, other aliphatic ring
protons) 2.76 (d, J=9.6 Hz, 1H) 2.97-3.00 (m, 1H) 3.59 (s, 3H) 3.64 (dd,
J=5.52, 17.04 Hz, 1H) 3.80 (dd, J=6, 16.98 Hz, 1H), 4.29 (d, J=5.16 Hz,
1H) 5.17 (t, J=3.36 Hz, 1H), 7.76 (t, J=5.7 Hz, 1H). 13C NMR (150 MHz,
(CD3)2SO) δ: 15.1, 16.1, 16.6, 18.0, 22.4, 22.9, 23.5, 25.6, 26.8, 27.0,
28.2, 30.4, 32.4, 32.5, 32.9, 33.6, 36.6, 38.1, 38.4, 38.9, 40.4, 40.9, 41.2,
45.2, 46.1, 47.1, 51.5, 54.8, 76.8, 121.4, 144.0, 170.5, 176.9. ESI-HRMS
(m/z): [M]+ calcd for C33H53NO4, 527.3975; found, 527.3965.
Compound 2a Prepared from 2 (200 mg, 0.37 mmol) according to
general procedure C. The crude product was washed with water three
times without further purification to afford 2a as a white solid (181 mg,
93%). m.p.: 173.2–175.2°C; 1H NMR (400 MHz, (CD3)2SO) δ: 0.64-1.08
(m, 7×CH3), 1.08-2.00 (m, other aliphatic ring protons) 2.76 (dd, J=3.84,
13.38 Hz, 1H) 2.97-3.00 (m, 1H) 3.56 (dd, J=3.54, 17.28 Hz, 1H) 3.73
(dd, J=6, 17.28 Hz, 1H) 4.28 (d, J=4.44 Hz, 1H) 5.19 (t, J=3.36 Hz, 1H)
7.57 (t, J=5.58 Hz, 1H) 12.37 (s, 1H). 13C NMR (100 MHz, (CD3)2SO) δ:
15.1, 16.1, 16.6, 18.0, 22.5, 23.0, 23.5, 25.7, 26.9, 27.0, 28.2, 30.4, 32.4,
32.5, 32.9, 33.6, 36.6, 38.1, 38.4, 38.9, 40.5, 40.9, 41.2, 45.1, 46.1, 47.2,
54.8, 76.8, 121.5, 144.0, 171.5, 176.7. ESI-HRMS (m/z): [M+H]+ calcd
for C32H52NO4, 514.3896; found, 514.3897.
Compound 3 Prepared from O-methylation alanine (154.5 mg, 1.5 mmol)
according to general procedure A. The residue was purified by column
chromatography (petroleum ether/ethyl acetate, 3/1 v/v) to afford 3 as a
1
white solid (443.6 mg, 82%). m.p.: 191.4–192.1°C; H NMR (400 MHz,

CDCl3) δ: 0.69-1.15 (m, 7×CH3), 1.15–2.00 (m, other aliphatic ring
protons) 1.37 (d, J = 7.0 Hz 3H), 2.63 (d, J = 10.08 Hz 1H) 3.18-3.22 (m,
1H), 3.72 (s, 3H), 4.41-4.48 (m, 1H), 5.42 (brs, 1H), 6.57 (d, J =5.56 Hz,
13
1H). C NMR (100 MHz, CDCl3) δ: 15.5, 15.7, 16.7, 18.2, 18.8, 23.7,
23.8, 23.8, 25.9, 27.3, 27.5, 28.2, 30.9, 32.7, 33.0, 33.4, 34.3, 37.1, 38.7,
38.9, 39.6, 42.1, 42.2, 46.4, 46.6, 47.7, 48.5, 52.5, 55.3, 79.1, 123.4,
144.0, 173.8, 177.6. ESI-HRMS (m/z): [M]+ calcd for C34H55NO4,
541.4131; found, 541.4136.
Compound 3a Prepared from 3 (200 mg, 0.36 mmol) according to
general procedure C. The crude product was washed with water three
times without further purification to afford 3a as a white solid (161.8 mg,
1
83%). m.p.: 170.4–171.8°C; H NMR (400 MHz, CD3OD) δ: 0.75-1.18
(m, 7×CH3), 1.18-2.15 (m, other aliphatic ring protons), 2.76 (d, J=10.48
Hz, 1H), 3.15 (dd, J=5.6, 12 Hz, 1H), 4.31 (q, J=7.08 Hz, 1H), 5.38 (s,
13
1H), 7.38 (d, J=6.12 Hz, 1H). C NMR (100 MHz, CD3OD) δ: 15.9,
16.3, 17.5, 18.2, 19.5, 24.0, 24.0, 24.6, 26.4, 27.9, 28.5, 28.7, 31.6, 33.5,
33.9, 35.1, 38.1, 39.8, 39.9, 40.7, 43.0, 47.4, 47.5, 48.9, 49.3, 49.5, 56.7,
79.7, 124.6, 144.7, 175.9, 179.8. ESI-HRMS (m/z): [M+H]+ calcd for
C33H54NO4, 528.4053; found, 528.4096.
Compound 4 Prepared from O-methylation valine (196.5 mg, 1.5 mmol)
according to general procedure A. The residue was purified by column
chromatography (petroleum ether/ethyl acetate, 3/1 v/v) to afford 4 as a
1
white solid (516.1 mg, 93%). m.p.: 203.8–205°C; H NMR (400 MHz,
(CD3)2SO) δ: 0.61-1.08 (m, 7×CH3), 1.08-2.00 (m, other aliphatic ring
protons), 2.02-2.09 (m, 1H) , 2.77 (d, J=13.64 Hz, 1H) 2.96-3.01 (m, 1H),
3.59 (s, 3H), 4.02 (t, J=7.88 Hz, 1H), 4.27 (d, J=4.36 Hz, 1H), 5.20 (s,
13
1H), 7.25 (d, J=7.88 Hz, 1H). C NMR (100 MHz, (CD3)2SO) δ: 15.6,
16.5, 17.2, 18.5, 19.5, 19.6, 22.6, 23.4, 24.0, 26.0, 27.4, 27.4, 28.7, 30.1,
30.8, 32.6, 33.0, 33.4, 34.0, 37.0, 38.6, 38.8, 39.3, 41.0, 41.7, 45.9, 46.3,

47.6, 51.9, 55.3, 58.6, 77.3, 122.0, 144.4, 172.7, 177.1. ESI-HRMS (m/z):
[M]+ calcd for C36H59NO4, 569.4444; found, 569.4452.
Compound 4a Prepared from 4 (200 mg, 0.34 mmol) according to
general procedure C. The crude product was washed with water three
times without further purification to afford 4a as a white solid (167.9 mg,
1
86%). m.p.: 211.4–214.2°C; H NMR (400 MHz, CD3OD) δ: 0.75-1.18
(m, 7×CH3), 1.18-2.11 (m, other aliphatic ring protons), 2.74 (d, J=11.08
Hz, 1H), 3.14 (dd, J=4.6, 11.76 Hz, 1H), 3.71 (s, 1H), 4.18 (d, J=4.84 Hz,
1H), 5.42 (s, 1H). 13C NMR (100 MHz, CD3OD) δ: 14.5, 14.9, 16.3, 17.6,
18.0, 18.1, 22.5, 22.8, 23.2, 24.9, 26.5, 27.2, 27.3, 30.2, 31.2, 32.1, 32.6,
33.0, 33.7, 36.7, 38.4, 38.5, 39.3, 41.6, 41.8, 46.3, 46.5, 47.7, 55.3, 58.4,
78.3, 123.6, 142.9, 178.4. ESI-HRMS (m/z): [M+H]+ calcd for
C35H58NO4, 556.4366; found, 556.4344.
Compound 5 Prepared from O-methylation leucine (218 mg, 1.5 mmol)
according to general procedure A. The residue was purified by column
chromatography (petroleum ether/ethyl acetate, 3/1 v/v) to afford 5 as a
1
white solid (455.2 mg, 80%). m.p.: 215.5–216.6°C; H NMR (400 MHz,
CDCl3) δ: 0.71-1.15 (m, 7×CH3), 1.15-2.00 (m, other aliphatic ring
protons), 2.61 (d, J=10.04 Hz, 1H), 3.18-3.22 (m, 1H), 3.70 (s, 3H), 4.52
13
(q, J=6.88 Hz, 1H), 5.41 (brs, 1H) 6.34 (d, J=6.84 Hz, 1H). C NMR
(100 MHz, CDCl3) δ: 15.5, 15.7, 16.8, 18.4, 22.6, 22.9, 23.7, 23.7, 24.0,
25.1, 25.8, 27.3, 27.5, 28.2, 30.8, 32.8, 33.0, 33.1, 34.3, 37.1, 38.7, 38.9,
39.5, 42.1, 42.2, 42.2, 46.5, 46.7, 47.7, 51.2, 52.2, 55.3, 79.1, 123.4,
144.0, 173.7, 177.8. ESI-HRMS (m/z): [M+Na]+ calcd for C37H61NO4Na,
606.4498; found, 606.4497.
Compound 5a Prepared from 5 (200 mg, 0.33 mmol) according to
general procedure C. The crude product was washed with water three
times without further purification to afford 5a as a white solid (181.6 mg,
1
93%). m.p.: >250°C; H NMR (400 MHz, CD3OD) δ: 0.76-1.17 (m,

7×CH3), 1.17-2.20 (m, other aliphatic ring protons), 2.79 (d, J=10.04 Hz,
1H), 3.14 (dd, J=4.4, 10.88 Hz, 1H), 3.49-3.65 (m, 1H), 4.38 (q, J=3.36
Hz, 1H), 5.35 (s, 1H) 5.50 (s, 1H). 13C NMR (100 MHz, CD3OD) δ: 15.9,
16.3, 17.9, 19.5, 19.6, 22.4, 23.5, 24.0, 24.01, 24.6, 26.1, 26.3, 27.9, 28.5,
28.7, 30.3, 31.6, 33.5, 34.0, 34.1, 35.2, 38.1, 39.8, 40.6, 41.9, 42.8, 43.0,
47.6, 47.6, 49.1, 52.5, 56.7, 79.7, 124.4, 144.8, 180.1. ESI-HRMS (m/z):
[M+H]+ calcd for C36H60NO4, 570.4522; found, 570.4541.
Compound 6 Prepared from O-methylation isoleucine (218 mg, 1.5
mmol) according to general procedure A. The residue was purified by
column chromatography (petroleum ether/ethyl acetate, 3/1 v/v) to afford
1
6 as a white solid (438.1 mg, 77%). m.p.: 192.5–195.1°C; H NMR (400
MHz, CDCl3) δ: 0.68 (s, 1H) 0.77 (s, 1H), 0.86-1.15 (m, 7×CH3),
1.15-2.00 (m, other aliphatic ring protons) 2.64 (d, J=11.06 Hz, 1H)
3.19-3.23 (m, 1H) 4.50 (dd, J=1.68, 6.76 Hz, 1H) 5.43 (t, J=3.52 Hz, 1H)
6.46 (d, J=7.68 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ: 15.2, 15.4, 15.6,
16.7, 18.3, 23.5, 23.6, 23.7, 25.6, 25.7, 27.2, 27.4, 28.1, 30.7, 32.7, 33.0,
33.1, 34.1, 37.0, 38.2, 38.5, 38.8, 39.4, 42.0, 42.2, 46.5, 46.6, 47.6, 51.9,
55.2, 56.3, 77.2, 79.0, 123.3, 143.7, 172.3, 177.5. ESI-HRMS (m/z):
[M+Na]+ calcd for C37H61NO4Na, 606.4498; found, 606.4494.
Compound 6a Prepared from 6 (200 mg, 0.33 mmol) according to
general procedure C. The crude product was washed with water three
times without further purification to afford 6a as a white solid (189.4 mg,
97%). m.p.: 199.3–200.9°C; 1H NMR (400 MHz, (CD3)2SO) δ: 0.64-1.08
(m, 7×CH3), 1.08-2.00 (m, other aliphatic ring protons), 2.71 (d, J=11.04
Hz, 1H), 2.96-3.00 (m, 1H), 3.94 (t, J=5.52 Hz, 1H), 5.23 (s, 1H), 7.17 (d,
J=6.64H). 13C NMR (100 MHz, (CD3)2SO) δ: 15.3, 15.5, 16.1, 16.7, 18.1,
22.8, 23.1, 23.4, 25.4, 25.6, 27.0 (2C), 28.3, 30.5, 32.7, 32.7, 32.9, 33.8
(2C), 36.6 (2C), 38.2, 38.5, 41.0, 41.5, 45.5, 46.2, 47.3, 54.9, 57.2, 76.9,

122.1, 143.4, 173.2, 175.8. ESI-HRMS (m/z): [M+H]+ calcd for
C36H60NO4, 570.4522; found, 570.4539.
Compound 7 Prepared from O-methylation serine (179 mg, 1.5 mmol)
according to general procedure A. The residue was purified by column
chromatography (petroleum ether/ethyl acetate, 3/1 v/v) to afford 7 as a
1
white solid (551.4 mg, 99%). m.p.: 139.7–140.8°C; H NMR (400 MHz,
CDCl3) δ: 0.69-1.16 (m, 7×CH3), 1.16-2.00 (m, other aliphatic ring
protons), 2.67 (d, J=11.24 Hz, 1H), 3.21 (s, J=10.64 Hz, 1H), 3.78 (s,
3H), 3.84 (d, J=11 Hz, 1H), 3.94 (brs, 1H), 4.12 (q, J=7.08 Hz, 1H), 4.54
13
(s, 1H), 5.46 (s, 1H), 6.87 (d, J=4.72 Hz, 1H). C NMR (100 MHz,
CDCl3) δ: 15.5, 15.7, 16.6, 18.4, 23.7, 23.8, 23.8, 25.9, 27.3, 27.6, 28.2,
30.8, 32.7, 33.1, 33.2, 34.2, 37.1, 38.7, 38.9, 39.6, 42.1, 42.2, 46.6, 46.7,
47.7, 52.7, 55.3, 55.9, 64.5, 79.1, 123.7, 143.6, 171.0, 179.4. ESI-HRMS
(m/z): [M+Na]+ calcd for C34H55NO5Na, 580.3978; found, 580.3972.
Compound 7a Prepared from 7 (200 mg, 0.35 mmol) according to
general procedure C. The crude product was washed with water three
times without further purification to afford 7a as a white solid (179.5 mg,
92%). m.p.: 174.4–176.2°C; 1H NMR (400 MHz, (CD3)2SO) δ: 0.65-1.09
(m, 7×CH3), 1.09-2.00 (m, other aliphatic ring protons), 2.73 (d, J=13.12
Hz, 1H), 2.99 (s, 1H), 3.55-3.72 (m, 2H), 4.16-4.24 (m, 1H) 7.19 (q,
J=6.92 Hz, 1H). 13C NMR (100 MHz, (CD3)2SO) δ: 15.2, 16.1, 16.5, 18.1,
22.6, 23.0, 23.6, 25.7, 27.0, 27.1, 28.3, 30.5, 32.5, 32.5, 33.0, 33.7, 36.6,
38.2, 38.5, 40.9, 41.4, 41.4, 45.4, 46.0, 47.2, 54.4, 54.9, 61.5, 76.9, 122.0,
143.8, 172.1, 176.3. ESI-HRMS (m/z): [M]+ calcd for C33H53NO5,
543.3924; found, 543.3917.
Compound 8 Prepared from O-methylation threonine (199 mg, 1.5 mmol)
according to general procedure A. The residue was purified by column
chromatography (petroleum ether/ethyl acetate, 3/1 v/v) to afford 8 as a
1
white solid (518 mg, 93%). m.p.: 139.8–140.5°C; H NMR (400 MHz,

CDCl3) δ: 0.70-1.16 (m, 7×CH3), 1.16-2.00 (m, other aliphatic ring
protons), 1.19 (d, J=6.16 Hz, 3H), 2.69 (d, J=9.04 Hz, 2H), 3.20 (d,
J=7.32 Hz, 1H), 3.73 (s, 3H), 4.20 (brs, 1H), 4.51 (d, J=6.92 Hz, 1H),
5.42 (s, 1H), 6.61 (d, J=7.2 Hz, 1H). 13C NMR (100 MHz, CDCl3) δ: 15.5,
15.7, 17.0, 18.4, 20.0, 23.7, 23.8, 24.0, 25.8, 27.3, 27.6, 28.3, 30.8, 32.9,
33.1, 33.4, 34.3, 37.2, 38.7, 38.9, 39.6, 42.2 (2C), 46.7, 46.9, 47.7, 52.5,
55.3, 57.7, 68.6, 79.1, 123.4, 143.7, 171.8, 179.0. ESI-HRMS (m/z):
[M+Na]+ calcd for C34H57NO5Na, 594.4134; found, 594.4135.
Compound 8a Prepared from 8 (200 mg, 0.34 mmol) according to
general procedure C. The crude product was washed with water three
times without further purification to afford 8a as a white solid (190.5 mg,
1
93%). m.p.: 178.6–179.7°C; H NMR (400 MHz, CD3OD) δ: 0.77-1.19
(m, 7×CH3), 1.19-2.20 (m, other aliphatic ring protons), 2.81 (d, J=10.6
Hz, 1H), 3.13-3.17 (m, 1H), 4.27-4.29 (m, 1H), 4.37 (s, 1H), 5.40 (s, 1H).
13C NMR (100 MHz, CD3OD) δ: 16.0, 16.3, 17.9, 19.5, 20.7, 24.0, 24.3,
24.6, 26.4, 27.9, 28.6, 28.8, 31.6, 33.5, 34.1, 34.5, 35.2, 38.1, 39.8, 39.9,
40.7, 43.0, 43.1, 47.7, 48.0, 49.1, 56.7, 59.0, 68.7, 79.7, 124.7, 144.5,
180.6. ESI-HRMS (m/z): [M]+ calcd for C34H55NO5, 557.4080; found,
557.4072.
Compound 9 Prepared from O-methylation histidine (253 mg, 1.5 mmol)
according to general procedure A. The residue was purified by column
chromatography (petroleum ether/ethyl acetate, 3/1 v/v) to afford 9 as a
1
white solid (594 mg, 98%). m.p.: 238.6–239.4°C; H NMR (400 MHz,
CDCl3) δ: 0.63-1.15 (m, 7×CH3), 1.15-2.00 (m, other aliphatic ring
protons), 2.59 (d, J=12.4 Hz, 1H), 3.00-3.06 (m, 1H), 3.13-3.20 (m, 1H),
3.35 (s, 1H), 3.72 (s, 3H), 4.56-4.61 (m, 4H), 5.35 (brs, 1H), 6.80 (s, 1H).
13C NMR (100 MHz, CDCl3) δ: 15.0, 15.2, 16.2, 18.0, 23.0, 23.1, 23.2,
25.4, 26.4, 26.9, 27.6, 28.8, 30.3, 32.3, 32.5, 32.5, 33.8, 36.6, 38.3, 38.4,
39.1, 41.4, 41.6, 46.1 (2C), 47.3, 52.0, 52.5, 55.0, 78.3, 123.1, 134.8,

143.2, 171.9, 178.4. ESI-HRMS (m/z): [M+Na]+ calcd for C37H57N3O4Na,
630.4247; found, 630.4240.
Compound 9a Prepared from 9 (200 mg, 0.32 mmol) according to
general procedure C. The crude product was washed with water three
times without further purification to afford 9a as a white solid (163.6 mg,
80%). m.p.: 247.2–248.2°C; 1H NMR (400 MHz, (CD3)2SO) δ: 0.35-1.02
(m, 7×CH3), 1.02-2.00 (m, other aliphatic ring protons), 2.71 (d, J=9.96
Hz, 1H), 2.96-3.09 (m, 3H), 4.33-4.38 (m, 1H), 5.11 (s, 1H), 7.16 (s, 1H),
7.61 (d, J=6.76 Hz, 1H), 8.43 (s, 1H). 13C NMR (100 MHz, (CD3)2SO) δ:
15.2, 16.1, 16.3, 18.1, 22.3, 23.0, 23.6, 25.6, 26.8 (2C), 27.0, 28.3, 30.4,
32.3, 32.4, 33.0, 33.6, 36.6, 38.2, 38.5, 38.7, 40.5, 41.2, 45.3, 46.0, 47.2,
52.1, 54.9, 76.9, 116.7, 121.5, 131.8, 134.0, 143.9, 172.8, 176.5.
ESI-HRMS (m/z): [M+H]+ calcd for C36H56NO4, 594.4271; found,
594.4263.
Compound 10 Prepared from O-methylation phenylalanine (268 mg, 1.5
mmol) according to general procedure A. The residue was purified by
column chromatography (petroleum ether/ethyl acetate, 3/1 v/v) to afford
1
10 as a white solid (549 mg, 89%). m.p.: 107.7–108.9°C; H NMR (400
MHz, CDCl3) δ: 0.62-1.12 (m, 7×CH3), 1.12-2.00 (m, other aliphatic ring
protons), 2.44 (d, J=9.4 Hz, 1H), 3.04 (dd, J=5.88, 13.8 Hz, 1H),
3.16-3.22 (m, 2H), 3.69 (s, 3H), 4.75 (q, J=5.92 Hz, 1H), 5.29 (s, 1H),
13
6.39 (d, J=6.12 Hz, 1H), 7.10 (d, J=7.2 Hz, 2H) 7.23-7.29 (m, 2H). C
NMR (100 MHz, CDCl3) δ: 15.5, 15.7, 16.6, 18.4, 23.6, 23.6, 23.9, 25.8,
27.3, 27.4, 28.2, 30.8, 32.7, 32.8, 33.1, 34.3, 37.1, 38.0, 38.6, 38.7, 38.9,
39.5, 42.1, 46.5, 46.7, 47.7, 52.3, 53.6, 55.3, 79.1, 123.5, 127.2, 128.6
(2C), 129.5 (2C), 136.3, 143.8, 172.2, 177.7. ESI-HRMS (m/z): [M+Na]+
calcd for C40H59NO4Na, 640.4342; found, 640.4352.
Compound 10a Prepared from 10 (200 mg, 0.31 mmol) according to
general procedure C. The crude product was washed with water three

times without further purification to afford 10a as a white solid (182 mg,
1
89%). m.p.: >250°C; H NMR (400 MHz, (CD3)2SO) δ: 0.66-0.98 (m,
7×CH3), 0.98-2.00 (m, other aliphatic ring protons), 2.62 (d, J=10.28 Hz,
1H) 2.88-2.96 (m, 2H), 3.06 (dd, J=3.92, 13.44 Hz 1H), 4.28-4.32 (m,
2H), 5.04 (s, 1H), 7.14-7.18 (m, 1H), 7.23 (d, J=4.16 Hz, 4H) 7.33 (d,
13
J=7.48 Hz, 1H) 12.67 (s, 1H). C NMR (100 MHz, (CD3)2SO) δ: 15.2,
16.0, 16.1, 17.9, 22.9, 23.5, 25.6, 26.8, 26.8, 28.3, 30.4, 31.9, 32.1, 33.0,
36.2, 36.6, 38.5, 38.7, 39.3, 40.1, 40.4, 41.2, 45.2, 46.0, 47.2, 54.1, 54.1,
54.9, 76.9, 121.3, 126.3, 128.2 (2C), 129.3 (2C), 138.2, 144.1, 173.4,
176.4. ESI-HRMS (m/z): [M+H]+ calcd for C39H58NO4, 604.4366; found,
604.4323.
Compound 11 Prepared from O-methylation tryptophan (327 mg, 1.5
mmol) according to general procedure A. The residue was purified by
column chromatography (petroleum ether/ethyl acetate, 3/1 v/v) to afford
1
11 as a white solid (632 mg, 95%). m.p.: >250°C; H NMR (400 MHz,
(CD3)2SO) δ: 0.67-0.93 (m, 7×CH3), 0.93-2.00 (m, other aliphatic ring
protons), 2.62 (d, J=10.36 Hz, 1H), 2.92-2.97 (m, 1H), 3.02-3.08 (m, 1H),
3.15-3.20 (m, 1H), 3.62 (s, 3H), 4.26 (d, J=5.04 Hz, 1H), 4.37-4.42 (m,
1H), 4.94 (s, 1H), 6.96 (t, J=7.6 Hz, 1H), 7.04 (t, J=7.36 Hz, 1H), 7.19 (s,
1H), 7.28-7.34 (m, 2H), 7.49 (d, J=7.8 Hz, 1H), 10.83 (s, 1H). 13C NMR
(100 MHz, (CD3)2SO) δ: 15.0, 15.1, 16.0, 18.0, 22.1, 22.8, 23.5, 25.5,
26.4, 26.5, 26.9, 28.3, 30.3, 31.7, 31.9, 32.9, 33.5, 36.5, 38.0, 38.3, 38.3,
40.2, 40.9, 44.9, 45.7, 47.0, 51.7, 53.3, 54.8, 76.8, 109.6, 111.5, 117.8,
118.3, 120.7, 121.0, 124.0, 127.0, 136.3, 144.0, 172.7. ESI-HRMS (m/z):
[M+Na]+ calcd for C42H60N2O4Na, 679.4451; found, 679.4441.
Compound 11a Prepared from 11 (200 mg, 0.28 mmol) according to
general procedure C. The crude product was washed with water three
times without further purification to afford 11a as a white solid (178.4 mg,
1
91%). m.p.: >250°C; H NMR (400 MHz, (CD3)2SO) δ: 0.67-0.93 (m,

7×CH3), 0.93-2.00 (m, other aliphatic ring protons), 2.6 (d, J=10.4 Hz,
1H), 2.92-2.97 (m, 1H), 3.02-3.08 (m, 1H), 3.19 (dd, J=4.04, 14.28 Hz,
1H) 4.26 (d, J=4.72 Hz, 1H), 4.34 (dd, J=4.12, 9.76 Hz, 1H), 4.95 (brs,
1H), 6.95 (t, J=7.52 Hz, 1H), 7.04 (t, J=7.32 Hz, 1H), 7.17 (s, 1H), 7.29
(d, J=8.08 Hz, 1H), 7.52 (d, J=7.84 Hz, 1H), 10.81 (s, 1H), 12.51 (s, 1H).
13C NMR (100 MHz, (CD3)2SO) δ: 15.0, 15.1, 16.0, 18.0, 22.2, 22.8, 23.4,
25.5, 26.4, 26.9, 28.3, 30.3, 31.7, 32.0, 32.9, 33.5, 36.5, 38.0, 38.3, 40.3,
40.9, 44.9, 45.8, 47.0, 53.1, 53.2, 54.8, 76.8, 109.9, 111.4, 118.0, 118.2,
120.6, 121.1, 123.9, 127.1, 136.3, 144.0, 173.8, 176.1, 176.2. ESI-HRMS
(m/z): [M+Na]+ calcd for C41H58NO4Na, 665.4294; found, 665.4287.
Compound 12 Prepared from O-methylation methionine (245 mg, 1.5
mmol) according to general procedure B. The residue was purified by
column chromatography (petroleum ether/ethyl acetate, 3/1 v/v) to afford
1
12 as a white solid (571 mg, 95%). m.p.: 196.1–198.5°C; H NMR (400
MHz, CDCl3) δ: 0.68-1.15 (m, 7×CH3), 1.15-2.00 (m, other aliphatic ring
protons), 2.41-2.55 (m, 2H), 2.64 (d, J=9.44 Hz, 1H), 3.21 (dd, J=4.04,
6.8 Hz, 1H), 3.30 (s, 3H), 3.73 (s, 3H), 4.12 (q, J=7.68 Hz, 1H), 4.59 (q,
13
J=6.04 Hz, 1H) 5.44 (t, J=3.84 Hz, 1H) 6.63 (d, J=6.08 Hz, 1H). C
NMR (100 MHz, CDCl3) δ: 15.5, 15.7 (2C), 16.7, 18.4, 23.7, 23.8, 25.9,
27.4, 27.6, 28.3, 30.1, 30.9, 32.0, 32.8, 33.1, 33.2, 34.3, 37.1, 38.7, 38.9,
39.6, 42.1, 42.2, 46.6, 46.7, 47.8, 51.9, 52.6, 55.3, 77.4, 79.1, 123.6,
143.8, 172.7, 178.0. ESI-HRMS (m/z): [M+Na]+ calcd for C36H59NO4Na,
624.4063; found, 624.4055.
Compound 12a Prepared from 12 (200 mg, 0.32 mmol) according to
general procedure C. The crude product was washed with water three
times without further purification to afford 12a as a white solid (185.7 mg,
1
95%). m.p.: 168.9–171.7°C; H NMR (400 MHz, CD3OD) δ: 0.76-1.18
(m, 7×CH3), 1.18-2.20 (m, other aliphatic ring protons), 2.06 (s, 3H), 2.49
(t, J=7.68 Hz, 2H), 2.76 (d, J=11.08 Hz, 1H), 3.04 (s, 1H), 3.12-3.06 (m,

13
1H), 4.31 (t, J=5.4 Hz, 2H), 5.41 (s, 1H). C NMR (100 MHz, CD3OD)
δ: 15.4, 15.9, 16.3, 17.7, 19.5, 24.0, 24.2, 24.5, 24.6, 26.4, 28.0, 28.7,
28.8, 31.1, 31.6, 32.7, 33.5, 34.0, 34.1, 35.1, 38.1, 39.8, 39.9, 40.7, 43.0
(2C), 47.6, 47.7, 49.1 (2C), 54.4, 56.7, 79.7, 124.9, 144.5, 179.8.
ESI-HRMS (m/z): [M+Na]+ calcd for C35H57NO4S, 587.4008; found,
587.4005.
Compound 13 Prepared from diO-methylation asparticacid (242 mg, 1.5
mmol) according to general procedure B. The residue was purified by
column chromatography (petroleum ether/ethyl acetate, 3/1 v/v) to afford
13 as a white solid (543 mg, 88%). m.p.: 87–88.7°C; 1H NMR (400 MHz,
(CD3)2SO) δ: 0.71-1.16 (m, 7×CH3), 1.16-2.00 (m, other aliphatic ring
protons), 2.67 (d, J=10.24 Hz, 1H) 2.95 (d, J=4.68 Hz, 1H), 3.21 (d,
J=7.64 Hz, 1H), 3.67 (s, 3H), 3.76 (s, 3H), 4.69 (dd, J=4.68, 5.88 Hz,
13
1H), 5.43 (s, 1H), 6.81 (d, J=6.24 Hz, 1H). C NMR (100 MHz,
(CD3)2SO) δ: 15.5, 15.7, 16.8, 18.4, 23.7, 23.7, 25.9, 27.4, 27.5, 28.2,
30.9, 32.9, 33.2, 33.2, 34.2, 36.2, 37.1, 38.7, 38.9, 39.6, 42.0, 42.1, 46.5,
46.6, 47.7, 49.2, 52.0, 52.9, 55.3, 77.4, 79.1, 123.4, 143.6, 171.5, 171.5,
177.8. ESI-HRMS (m/z): [M+Na]+ calcd for C36H57NO6Na, 622.4084;
found, 622.4092.
Compound 13a Prepared from 13 (200 mg, 0.32 mmol) according to
general procedure C. The crude product was washed with water three
times without further purification to afford 13a as a white solid (164.2 mg,
1
86%). m.p.: >250°C; H NMR (400 MHz, (CD3)2SO) δ: 0.67-1.08 (m,
7×CH3), 1.08-2.00 (m, other aliphatic ring protons) 2.50-2.56 (m, 4H)
2.72 (dd, J=6.08, 12.28 Hz, 1H), 2.95-3.03 (m, 1H), 4.27 (d, J=5.28 Hz,
1H), 4.42 (q, J=6.52 Hz, 1H), 5.19 (brs, 1H), 7.45 (d, J=7.24 Hz, 1H),
13
12.48 (s, 2H). C NMR (100 MHz, (CD3)2SO) δ: 15.2, 16.1, 16.8, 18.1,
22.4, 23.0, 23.5, 25.6, 26.9, 27.0, 28.3, 30.5, 32.4, 32.5, 32.9, 33.6, 35.8,
36.6, 38.2, 38.5, 40.1, 40.7, 41.4, 45.3, 46.0, 47.2, 48.9, 54.9, 76.9, 121.7,

143.8, 171.9, 172.6, 176.2. ESI-HRMS (m/z): [M]+ calcd for C34H53NO6,
571.3873; found, 571.3882.
Compound 14 Prepared from diO-methylation a glutamicacid (242 mg,
1.5 mmol) according to general procedure B. The residue was purified by
column chromatography (petroleum ether/ethyl acetate, 3/1 v/v) to afford
1
14 as a white solid (543 mg, 88%). m.p.: 166.5–167.5°C; H NMR (400
MHz, CDCl3) δ: 0.66-1.15 (m, 7×CH3), 1.15-2.50 (m, other aliphatic ring
protons), 2.63 (d, J=11.56 Hz, 1H), 3.20 (dd, J=4.00, 10.88 Hz, 1H),
3.67 (s, 3H), 3.72 (s, 3H), 4.50 (q, J=6.24 Hz, 1H), 5.43 (t, J=3.56 Hz,
13
1H). C NMR (100 MHz, CDCl3) δ: 15.5, 15.7, 16.7, 18.4, 23.7, 23.8,
25.9, 27.4, 27.6, 27.7, 28.2, 30.1, 30.9, 32.8, 33.2, 33.2, 34.2, 37.1, 38.7,
38.9, 39.6, 42.1, 42.2, 46.6, 46.6, 47.7, 51.9, 52.0, 52.5, 55.3, 79.1, 123.5,
143.8, 172.6, 173.4, 178.1. ESI-HRMS (m/z): [M+Na]+ calcd for
C37H59NO6Na, 636.4240; found, 636.4248.
Compound 14a Prepared from 14 (200 mg, 0.31 mmol) according to
general procedure C. The crude product was washed with water three
times without further purification to afford 14a as a white solid (166.2 mg,
87%). m.p.: 248.3–248.7°C; 1H NMR (400 MHz, (CD3)2SO) δ: 0.64-1.08
(m, 7×CH3), 1.08-2.00 (m, other aliphatic ring protons) 2.76 (d, J=9.76
Hz, 1H), 2.95-3.03 (m, 1H) 3.56 (dd, J=5.36, 17.4 Hz, 1H), 3.74 (dd,
J=5.96, 17.24 Hz, 1H), 4.28 (d, J=4.36 Hz, 1H), 5.19 (brs, 1H), 7.56 (t,
13
J=5.2 Hz, 1H), 12.37 (s, 1H). C NMR (100 MHz, (CD3)2SO) δ: 15.1,
16.0, 16.6, 18.0, 22.5, 22.9, 23.5 (2C), 25.6, 26.8, 27.0, 28.2 (2C), 30.4,
32.4, 32.5, 32.9, 33.6, 36.6, 38.1, 38.4, 39.6, 40.5, 40.9, 41.2, 45.1, 46.0,
47.1, 54.8, 76.8, 121.5, 143.9, 171.4, 176.7. ESI-HRMS (m/z): [M+Na]+
calcd for C32H51NO4Na, 536.3716; found, 536.3711.
Compound 15 Prepared from O-methylation arginine (282 mg, 1.5 mmol)
according to general procedure B. The residue was purified by column
chromatography (petroleum ether/ethyl acetate, 3/1 v/v) to afford 15 as a

1
white solid (502 mg, 78%). m.p.: 119.3–121.8°C; H NMR (400 MHz,
CDCl3) δ: 0.73-1.18 (m, 7×CH3), 1.18-2.00 (m, other aliphatic ring
protons), 2.14 (s, 1H) 2.32 (d, J=13.53 Hz, 1H), 2, 51 (d, J=13. 68 Hz,
1H), 2.84 (d, J=13.36 Hz, 1H), 3.32 (s, 1H), 3.74 (s, 3H), 4.68-4.72 (m,
13
2H), 5.29 (s, 1H) 6.58 (d, J=8.28 Hz, 1H) 7.94 (s, 1H), 8.89 (s, 1H). C
NMR (100 MHz, CDCl3) δ: 16.5, 16.6, 18.1, 18.3, 21.3, 21.6, 23.3, 23.8,
24.9, 25.6, 26.4, 28.0, 32.5, 33.1, 33.6, 34.4, 36.1, 37.1, 37.3, 38.2, 38.5,
40.3, 40.9, 41.7, 44.5, 49.0, 50.4, 52.8, 55.3, 77.4, 86.3, 113.4, 116.2,
127.1, 143.6, 172.2, 177.9. ESI-HRMS (m/z): [M+Na]+ calcd for
C37H62N4O4Na, 649.4669; found, 649.4664.
Compound 15a Prepared from 15 (200 mg, 0.31 mmol) according to
general procedure C. The crude product was washed with water three
times without further purification to afford 15a as a white solid (175.5 mg,
87%). m.p.: 222.6–224.4°C; 1 H NMR (600 MHz, (CD3)2SO) δ: 0.66-1.13
(m, 7×CH3), 1.13-2.00 (m, other aliphatic ring protons), 2.21-2.23 (m,
1H), 2.43 (d, J=13.68 Hz, 1H) 2.78 (d, J=11.7 Hz, 1H) 2.99-3.02 (m, 1H)
4.22-4.26 (m, 1H) 6.60 (d, J=7.56 Hz, 1H) 7.79 (s, 1H) 8.56 (s, 1H)
13
12.70 (s, 1H). C NMR (150 MHz, (CD3)2SO) δ: 16.3, 16.7, 17.9, 18.4,
21.4, 21.7, 24.3 (2C), 25.4, 26.4, 27.6 (2C), 28.6, 28.7, 32.5, 33.1, 33.3,
34.6, 35.7, 36.8, 37.2, 38.9, 39.0, 40.5, 41.1, 44.4, 48.5, 50.3, 52.3, 55.3,
77.3, 128.0, 141.2, 152.8, 173.7, 175.2. ESI-HRMS (m/z): [M]+ calcd for
C36H60N4O4, 612.4615; found, 612.4600.
Compound 16 Prepared from O-methylation tyrosine (293 mg, 1.5 mmol)
according to general procedure B. The residue was purified by column
chromatography (petroleum ether/ethyl acetate, 3/1 v/v) to afford 16 as a
1
white solid (436 mg, 67%). m.p.: 137.7–139.0°C; H NMR (400 MHz,
CDCl3) δ: 0.62-1.12 (m, 7×CH3), 1.12-2.00 (m, other aliphatic ring
protons), 2.45 (d, J=8.72 Hz, 1H), 2.94 (dd, J=5.68, 13.8 Hz, 1H), 3.08
(dd, J=5.8, 14.04 Hz, 1H), 3.21 (dd, J=3.84, 10.28 Hz, 1H) 3.68 (s, 3H),

4.11 (q, J=7.12 Hz, 1H), 4.71 (dd, J=5.88, 14.04 Hz, 1H), 5.30 (s, 1H),
6.45 (d, J=6.16 Hz, 1H), 6.73 (d, J=5.36 Hz, 2H), 6.94 (d, J=7.68 Hz,
13
2H). C NMR (100 MHz, CDCl3) δ: 15.5, 15.7, 16.6, 18.4, 23.6, 23.6,
23.9, 25.8, 27.3, 27.4, 28.2, 30.8, 32.7, 32.8, 33.1, 34.3, 37.1, 37.4, 38.7,
38.9, 39.6, 42.1, 42.2, 46.6, 46.7, 47.7, 52.3, 53.8, 55.3, 79.2, 115.6 (2C),
123.6, 127.6, 130.5 (2C), 143.7, 155.6, 172.2, 178.1. ESI-HRMS (m/z):
[M+Na]+ calcd for C40H59NO5Na, 656.4291; found, 656.4281.
Compound 16a Prepared from 16 (200 mg, 0.30 mmol) according to
general procedure C. The crude product was washed with water three
times without further purification to afford 16a as a white solid (142.8 mg,
1
73%). m.p.: >250°C; H NMR (400 MHz, (CD3)2SO) δ: 0.67-1.00 (m,
7×CH3), 1.00-2.00 (m, other aliphatic ring protons), 2.62 (d, J=10.4 Hz,
1H) 2.79 (dd J=10.04, 13.52 Hz, 1H), 2.92-2.98 (m, 2H) 4.22 (dd J=5.52,
7.72 Hz, 1H) 4.28 (d, J=4.88 Hz, 1H) 5.05 (s, 1H), 6.61 (d, J=8.28 Hz,
2H) 7.00 (d, J=8.28 Hz, 2H) 7.22 (d, J=7.24 Hz, 1H), 9.13 (s, 1H) 12.58
13
(s, 1H). C NMR (100 MHz, (CD3)2SO) δ: 15.1, 15.9, 16.1, 18.0, 22.9,
23.4, 25.6, 26.8, 27.0, 28.3, 30.3, 31.9, 32.0, 32.9, 33.6, 35.4, 36.5, 38.4,
38.6, 40.4, 41.2, 45.1, 45.9, 47.2, 54.2, 54.9, 76.8, 76.9, 114.8, 114.9,
121.3, 127.8, 130.0, (2C), 144.0, 156.1, 173.4, 176.2, 176.3. ESI-HRMS
(m/z): [M+H]+ calcd for C39H28NO5, 620.4315; found, 620.4306.
Compound 17 Prepared from O-methylation proline (193 mg, 1.5 mmol)
according to general procedure B. The residue was purified by column
chromatography (petroleum ether/ethyl acetate, 3/1 v/v) to afford 16 as a
1
white solid (503 mg, 86%). m.p.: >250°C; H NMR (600 MHz,
(CD3)2SO) δ: 0.63-1.08 (m, 7×CH3), 1.08-2.05 (m, other aliphatic ring
protons), 2.92 (d, J=12.06 Hz, 1H), 2.97-3.0 (m, 1H), 3.43 (dd, J=9.12
J=16.26 Hz, 1H), 3.57 (s, 3H), 3.82 (brs, 1H), 4.13 (d, J=7.38 Hz, 1H),
4.29 (d, J=5.16 Hz, 1H), 5.06 (s, 1H). 13C NMR (150 MHz, (CD3)2SO) δ:
15.6, 16.5, 16.8, 18.4, 20.8, 23.3, 24.2, 25.9, 26.2, 27.3, 27.4, 27.5, 28.7,

30.6, 32.9, 33.4, 33.7, 37.1, 38.5, 38.9, 39.2, 40.5, 42.0, 42.7, 46.0, 46.7,
47.7, 47.9, 51.9, 55.4, 61.4, 77.3, 121.6, 144.9, 173.2, 174.6. ESI-HRMS
(m/z): [M+Na]+ calcd for C36H57NO4Na, 590.4185; found, 590.4187.
Compound 17a Prepared from 17 (200 mg, 0.34 mmol) according to
general procedure C. The crude product was washed with water three
times without further purification to afford 17a as a white solid (138.6 mg,
1
71%). m.p.: 188.8–191.3°C; H NMR (400 MHz, CD3OD) δ: 0.75-1.16
(m, 7×CH3), 1.16-2.20 (m, other aliphatic ring protons) 3.03 (d, J=13.08
Hz, 1H), 3.14 (dd, J=4.8, 11.12 Hz, 1H), 3.52-3.60 (m, 1H), 3.93 (t,
13
J=7.96 Hz, 1H), 4.34 (d, J=6.24 Hz, 1H), 5.19 (s, 1H). C NMR (100
MHz, CD3OD) δ: 15.9, 16.3, 17.5, 19.5, 22.0, 24.4, 24.5, 26.6, (2C), 27.9,
28.6, 28.8, 30.1, 31.4, 33.7, 34.0, 34.8, 38.2, 39.8, 39.8, 40.5, 43.1, 44.2,
47.2, 48.5, 49.2, 49.2, 56.8, 63.3, 63.3, 79.7, 123.5, 145.5, 176.6, 177.5.
ESI-HRMS (m/z): [M+H]+ calcd for C35H56NO4, 554.4209 ; found,
554.4181.
4.2. Anti-influenza virus assay
MDCK cells at 5,000 cells/well were cultured in 96-well plates for a
day before IAV infection. The infection medium used was DMEM (High
Glucose) containing 1% FBS and 0.2% trypsin (1 µg/mL). Various
concentrations of compounds were added to the cell culture. The cells
were then infected with IAV at a multiplicity of infection (MOI) of 0.1,
unless otherwise indicated. After 45 h of incubation, Promega
CellTiter-Glo® reagent was added to each well following the protocol
provided by the supplier. The luminescence (RLU) emitted from each
well was quantified with a Molecular Device SpectraMax M2 plate reader.
IC50, the concentration required to inhibit 50% of A/WSN/33 was

calculated using the software GraphPad Prism 7.
4.3. HI assay
Compound 15a from a 2-fold serial dilution in saline was mixed with
an equal volume of influenza virus (2 HA units) in the V-bottomed
96-well microplates. Subsequently, 50 μL of freshly prepared chicken red
blood cells (cRBC) (1% v/v in saline) was added to each well. The
mixture was incubated for 30 min at room temperature before observing
cRBC aggregation on the plate.
4.4. Docking Simulation
Compound 15a were docked into the HA protein using AutoDock
4.2.4. The structural template of influenza HA (Protein Data Bank: 1RVT)
was
obtained
from
the
RCSB
Protein
Data
Bank
(http://www.rcsb.org/pdb/home/home.do). To carry out blind docking
experiments, grids of points covering the whole HA head and partial stem
were generated with ADT.46 (Box size: 100 × 100 × 100 points with a
standard space of 0.375 Å). Docking simulations of the compound were
carried out using the Lamarckian genetic algorithm and through a
protocol with a number of 50 GA runs, an initial population of 100
randomly placed individuals, a maximum number of 25 million energy
evaluations, a mutation rate of 0.02, and a crossover rate of 0.80. The
resulting conformations that differed by less than 2.0 Å in positional
root-mean-square deviation (rmsd) were clustered together. Other

parameters were set as default. All the relevant torsion angles were
treated as rotatable during the docking process, thus allowing a search of
the conformational space. After running autogrid and autodock, the
possible poses of compounds in HA were obtained.
Conflict of interest
The authors declared that they have no conflicts of interest to this work.
Acknowledgments
This work was supported by National Natural Science Foundation of
China (grant No. 81560560, 31660100), Open Fund Program of the State
Key Laboratory of Natural and Biomimetic Drugs (grant No.
K20170209).
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Figure 1. Inhibitory effects of natural amino acids-oleanolic acid conjugates
against influenza A/WSN/33 (H1N1) virus. (A) The cytotoxic effect-based screen of
natural amino acids-oleanolic acid conjugates (100 μM) using CellTiter-Glo® Assay.
DMSO acted as negative control. Error bars indicate standard deviations of triplicate
experiments. (B) The cytopathic effect-based screen of natural amino
acids-oleanolic acid conjugates (100 μM). MDCK was utilized as the host cell to test
A/WSN/33 virus infection; DMSO acted as negative control. Error bars indicate
standard deviations of triplicate experiments.
Figure 2. Comparisons of the behaviors of compound 15a vs. monoclonal antibody
against HA protein in inhibition of influenza virus-induced aggregation of chicken
erythrocytes. 15a exerted identical capability as anti-HA antibody in
hemagglutination inhibition in a dose-dependent manner.
Figure 3. Structural representative of compound 15a binding within HA protein
(Protein Data Bank: 1RVT) according to blind docking calculation. (A) Overview
of HA protein. The inhibitor pocket is highlighted in yellow square. (B) Closer

view of the inhibitor pocket. Compound 15a is shown as green sticks. (C) 2D
interaction plot green dashes and red curves indicate hydrogen bonds and
hydrophobic interactions correspondingly.
Scheme 1. Reagents and conditions: (a)TBTU, DIEA, THF, rt, overnight;(b) amino
acid methyl esters, Na2CO3, rt, 20min, DMF;(c) NaOH, rt, 3h, THF/CH3OH (1:1).
Scheme 2. Reagents and conditions: (a) amino acid methyl esters, TEA, EDC, rt,
overnight, THF; (a') proline methyl ester, TEA, EDC, rt, overnight, THF; (b) NaOH,
rt, 3h, THF/CH3OH (1:1).
Table 1. Comparisons of the anti-influenza virus activity of compound 15a with that
of OSV-P.
IC50 (μM)b
Compd
CC50
(μM)a
A/WSN/
33c
A/Texas/50
/2012
BX-35
BX-51B
15a
>100
>100
6.64
14.05
>100
61.58
>100
45.8
OSV-P
1.82
>100
aCC50, the concentration required to reduced normal, non-infected cell viability
by 50%. Values represent the mean of duplicate samples from three independent
experiments.
bIC50, the concentration required to reduced inhibition of viral infection-induced
cytopathogenicity by 50%. Values represent the mean of duplicate samples from
three independent experiments.
cA/WSN/33 (H1N1); A/Texas/50/2012 (H3N2); BX-35, B/Brisbane/60/2008
(Influenza B Victoria lineage); BX-51B, B/Massachusetts/2/2012 (Influenza B
Yamagata lineage).