Synthesis, Structure Activity Relationship and Anti-influenza A Virus Evaluation of Oleanolic Acid-Linear Amino Derivatives

Article abstract of DOI:10.1248/cpb.c19-00485

Oleanolic acid (OA) was discovered as a mild influenza hemagglutinin (HA) inhibitor in our earlier studies. In the present work, 20 compounds were prepared by structural modifications of OA, and their antiviral activities against influenza A/WSN/33 (H1N1) virus in Madin–Darby canine kidney (MDCK) cells were evaluated. Based on the biological result, structure–activity relationship (SAR) was discussed. Compound 10 with six-carbon chain and a terminal hydroxyl group showed the strongest anti-influenza activity with an IC₅₀ of 2.98μM, which is an order of magnitude more potent than OA. Hemagglutination inhibition and Surface plasmon resonance (SPR) assay indicated that compound 10 might interfere with influenza invasion by interacting with HA protein.

Full text of DOI:10.1248/cpb.c19-00485

Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/cpb.c19-00485
Advance Publication
August 22, 2019
Synthesis, structure activity relationship and anti-influenza A virus evaluation of
oleanolic acid-linear amino derivatives
Weijia Li,a, # Fan Yang,a, # Lingkuan Meng,a Jiaqi Sun,b Yangqing Su,a Liang Shao,a
Demin Zhoub and Fei Yua,*
a
Medical School of Kunming University of Science and Technology, Kunming, Yunnan
650500, China
b State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences,
Peking University, Beijing 100191, China
Address correspondence to:
Dr. Fei Yu
Medical School
Kunming University of Science and Technology
No.727 South Jingming Rd, Chenggong District, Kunming, 650500, China
Tel: +86-18087122003
Email: feiyuz8@kmust.edu.cn
# These authors contributed equally to this work.
* Correspondence e-mail: feiyuz8@kmust.edu.cn
Ⓒ 2019 The Pharmaceutical Society of Japan

Summary
Oleanolic acid (OA) was discovered as a mild influenza hemagglutinin (HA) inhibitor in
our earlier studies. In the present work, 20 compounds were prepared by structural
modifications of OA, and their antiviral activities against influenza A/WSN/33 (H1N1) virus
in MDCK cells were evaluated. Based on the biological result, structure–activity relationship
(SAR) was discussed. Compound 10 with six-carbon chain and a terminal hydroxyl group
showed the strongest anti-influenza activity with an IC50 of 2.98 μM, which is an order of
magnitude more potent than OA. Hemagglutination inhibition and Surface plasmon
resonance (SPR) assay indicated that compound 10 might interfere with influenza invasion by
interacting with HA protein.
Keywords
Oleanolic acid; Influenza virus; Entry inhibitor; Cytotoxicity; Synthesis design.
Chemical and Pharmaceutical Bulletin Advance Publication

Introduction
Influenza A viruses (IAVs) are the main cause of annual epidemics and occasional
pandemics of respiratory diseases worldwide1-3). According to estimates by the WHO, IAVs
infect 5%-15% of the world's population4), killing between 250,000 and 500,000 people each
year5). Based on the viral surface glycoproteins hemagglutinin (HA) and neuraminidase (NA),
IAVs are classified into various subtypes. There are 18 variants of HA (H1-H18) and 11
variants of NA (N1-N11) on the IAVs6), which may form 198 subtypes of IAVs,
theoretically7, 8)
.
Two classes of anti-influenza drugs, M2 ion-channel inhibitors (amantadine and
rimantadine) and NA inhibitors (oseltamivir, zanamivir and peramivir), have been used in
clinic for the interruption of specific processes in influenza infections9, 10). In 2018, baloxavir
was licensed in Japan for the treatment of both adult and pediatric patients infected with
influenza virus. It is approved for use in children older than twelve age with acute
uncomplicated influenza11). However, frequent application of the marketed anti-influenza
drugs, in combination with the high mutation rate of the RNA genome of the influenza virus,
has led to the rapid emergence of drug-resistant viral strains. Therefore, cost-effective
antiviral strategies must be developed to prevent emerging influenza pandemics.
IAVs entry represents a favorable target of drug discovery, because it is the first step of
viral infection. Inhibition of this first step should result in the virus propagation effectively
blocked12, 13). In our previous studies, we found that oleanolic acid (OA) and its derivatives,
which belongs to the family of pentacyclic triterpenes, display inhibitory activity against
IAVs entry by interrupting the interaction between HA and sialic acid receptor14).
Chemical and Pharmaceutical Bulletin Advance Publication

As a continuation of our project aimed at discovering novel entry-targeting IAVs
inhibitors, a total of 20 OA C-28 derivatives were designed, synthesized, and evaluated for
their anti-IAVs activities in vitro.
Results and discussion
Chemistry
Certain structural modifications of these triterpenoids, many of which are simple
derivatizations of the molecule’s functional groups, can have a significant impact on their
biological properties. For example, studies have shown increased biological activities of the
C-28 amino conjugates of various pentacyclic triterpenic acids as compared to their natural
precursors.
As shown in Chart 1, the C-28 of OA was first activated by EDCI and (CH3CH2)3N in
the presence of 1-Hydroxybenzotriazole (HOBt), then reacted with amine reagents, giving the
corresponding OA derivatives (compounds 1–20) with high yields. The amine reagents were
selected with different chain lengths and terminal functional groups to evaluate their
influence on the anti-IAVs activity.
Chemical and Pharmaceutical Bulletin Advance Publication

Biological evaluation
Anti-IAVs activity of OA derivatives.
All the synthesized OA derivatives were evaluated for their in vitro anti-A/WSN/33
(H1N1) virus in an MDCK cell line at a concentration of 100 μM. The inhibition rates of the
compounds were calculated and shown in Figure 1. To exclude the possibility that the
observed anti-IAVs virus activity was due to cytotoxicity, a preliminary screening was
performed to determine their cytotoxic activity in MDCK cells via the CellTiter-Glo assay
and the cytopathic effect (CPE) reduction assay14). Oseltamivir (OSV, 100 μM) and 1%
DMSO were used as positive and negative controls, respectively.
In Figure 1A, compounds 1-5, the diamine conjugates of OA, exhibited strong
cytotoxicity. This is in agreement with Parra’s observation that that triterpene derivatives with
a diamine, showed extreme cytotoxicity against cancer-cell lines (B16-F10, HT29 and Hep
G2)15). On the other hand, no obvious toxicity was observed when the terminal of the side
chain is a methylated carboxyl group at the final concentration of 100 μM (compounds 11-15).
However, the removal of the methyl group (compounds 16-20) resulted in significant
cytotoxicity at the same concentration, indicating that the terminal group of the side chain has
a significant effect on cytotoxicity. Interestingly, compounds 7 and 8 display much higher
cytotoxicity than 6, 9 and 10, which means the length of the side chain might play a critical
role when the terminal is a hydroxyl group (compounds 6-10). Figure 1B shows that most of
the compounds displayed a much higher inhibition rate than OA. However, in light of
Figure 1A, this observation might, to a large extent, reflect the cellular toxicity rather than
Chemical and Pharmaceutical Bulletin Advance Publication

anti-IAVs potency enhancement. According to the results above, six compounds, 9, 10, and
12-15, with high inhibition rates (>80%) and low cytotoxicity (cell viability >70%) in the
initial screening, were selected for the dose response assays. As shown in Table 1, compound
10 displayed the strongest inhibitory activity in this study with an IC50 value of 2.98 μM.
Compared with DMSO-treated MDCK cells, influenza A/WSN/33 virus causes a severe CPE
in MDCK-infected cells. We found that compound 10 (Figure. 2A) could significantly reduce
the CPE, which indicating these compounds were able to protect MDCK cells from influenza
virus-induced CPE.
Compound 10 inhibits virus-induced hemagglutination.
Hemagglutinin (HA) plays an important role in the early stages of IAVs infection and
mediates attachment of IAVs to host cells via the sialic acid receptor16, 17). HA can also bind
to sialic acid on the surface of red blood cells (RBCs), causing agglutination18). To investigate
whether compound 10 can block the ability of viral particles to bind to cell membrane
receptors, a hemagglutination inhibition (HI) assay was performed. We found that compound
10 inhibited the binding of influenza virus A/WSN/33 to RBCs, suggesting that compound 10
may directly act on the HA protein (Figure. 2B).
In addition, surface plasmon resonance (SPR) assay was used to characterize the a nity
between HA protein and the compounds 10, OA 19). We found that compound 10 could bind
tightly to the HA immobilized on the sensor chip (CM5) and at concentrations of 0.39−15 μM
exerted dose-dependent responses. As shown in Figure 2C, the binding curves are fitted well
with the Langmuir equation for monovalent binding, which allows the determination of the
apparent dissociation constant, KD. The calculated KD values for compound 10 and OA were
Chemical and Pharmaceutical Bulletin Advance Publication

4 μM and 24.4 μM, respectively, indicating that compound 10 bound more tightly to HA than
OA.
Conclusion
In summary, we have designed and synthesized 20 compounds by structural
modification of OA, a mild influenza HA inhibitor discovered in our previous work. Among
them, compound 10, which contains a six-carbon chain with a hydroxyl terminal group, was
less cytotoxic and much more potent than other compounds with an IC50 of 2.98 μM.
Hemagglutination inhibition and SPR assays indicated that compound 10 might interfere with
influenza invasion by interaction with the HA protein.
Experimental
Chemistry
General procedure A for the synthesis of oleanolic acid derivatives (1-15):
To OA-OBt (0.2 mmol) and Na2CO3 (0.6 mmol) stirring in DMF (20 mL) was added the
corresponding amine (0.2 mmol) or alcohol (0.4 mmol) or methyl ester (0.6 mmol). The
mixture was stirred at room temperature for 12 h. After completion (TLC) the solvent was
removed under reduced pressure. The mixture was dissolved in EtOAc and washed with
water and brine twice. The organic layer was dried over Na2SO4, then filtered and
concentrated. The crude product was purified by column chromatography.
General procedure B for the synthesis of oleanolic acid derivatives (16-20):
To compounds 11-15 (0.1 mmol) stirring in methanol (10 mL) was added 1N NaOH
(3 mL). The mixture was stirred at rt. After completion (TLC) the reaction mixture was
Chemical and Pharmaceutical Bulletin Advance Publication

neutralized with 1N HCl (3 mL). Water was added and the resulting suspension was filtered.
Crude product was purified by column chromatography.
OA-OBt This compound was prepared from OA (10 mmol), EDCI (13mmol),
(CH3CH2)3N (13mmol) and 1-Hydroxybenzotriazole (HOBt) according to the Lei’ s
studies20).
Compound 1 This compound was prepared from OA-OBt (0.2 mmol) and
ethylenediamine (2 mmol) according to the general procedure A. The residue was purified by
column chromatography (dichloromethane/methanol, 15/1 v/v). Yield: 62 mg, 62%; white
1
solid. Mp 202.5-203.3 °C. H NMR (400 MHz, CDCl3) δ: 0.76, 0.78, 0.92, 0.94, 0.98, 1.18
(7×CH3), 0.76-2.00 (m, other aliphatic ring protons), 2.66 (d, J=11.4 Hz, 1H), 2.81 (t, J=6 Hz,
2H), 3.11-3.21 (m, 2H), 3.36-3.43 (m, 2H), 5.40 (s, 1H); 13C NMR (100 MHz, CDCl3) δ:
15.00, 15.31, 16.67, 18.07, 23.15, 23.18, 23.27, 25.57, 26.48, 27.07, 27.73, 30.42, 32.26,
32.66 (2C), 33.85, 36.74, 38.35, 38.51, 39.18, 40.44, 40.80, 41.47, 41.69, 46.23, 46.31, 47.35,
55.01, 78.43, 122.79, 144.00, 179.73. ESI-HRMS (m/z) [M+H]+ calcd for C32H55N2O2,
499.4258; found, 499.4257.
Compound 2 This compound was prepared from OA-OBt (0.2 mmol) and
1,3-propanediamine (2 mmol) according to the general procedure A. The residue was purified
by column chromatography (dichloromethane/methanol, 15/1 v/v). Yield: 79 mg, 77%; white
1
solid. Mp 187.6-188.4 °C. H NMR (400 MHz, CD3OH) δ: 0.76, 0.78, 0.91, 0.98, 1.17
(7×CH3), 0.76-2.00 (m, other aliphatic ring protons), 2.56 (d, J=12.4 Hz, 1H), 2.69 (t, J =6.56
Hz, 2H), 3.04-3.10 (m, 1H), 3.17-3.21 (m, 1H), 3.37-3.44 (m, 1H), 5.33 (s, 1H), 5.38 (s, 1H),
7.38 (s, 1H); 13C NMR (100 MHz, CD3OH) δ: 15.90, 16.32, 17.93, 19.48, 23.98, 24.03, 24.56,
Chemical and Pharmaceutical Bulletin Advance Publication

26.49, 27.86, 28.54, 28.76, 31.62, 33.10, 33.56, 33.88, 34.50, 35.12, 37.91, 38.15, 39.81,
39.84, 40.68, 42.55, 42.93, 47.55, 47.67, 56.71, 79.66, 124.03, 145.27, 180.48. ESI-HRMS
(m/z) [M+H]+ calcd for C33H57N2O2, 513.4415; found, 513.4413.
Compound 3 This compound was prepared from OA-OBt (0.2 mmol) and
1,4-diaminobutane (2 mmol) according to the general procedure A. The residue was purified
by column chromatography (dichloromethane/methanol, 15/1 v/v). Yield: 80 mg, 76%; white
1
solid. Mp 179.3-180.2 °C. H NMR (400 MHz, (CD3)2SO) δ: 0.66, 0.84, 0.86, 0.87, 0.88,
1.07 (7×CH3), 0.66-2.00 (m, other aliphatic ring protons), 2.77 (d, J=13.8 Hz, 1H), 2.89-3.05
(m, 6H), 5.20 (s, 1H), 7.24 (t, J=5.4 Hz, 1H), 8.31 (s, 1H); 13C NMR (100 MHz, (CD3)2SO) δ:
15.08, 16.00, 16.81, 18.01, 22.27, 22.92, 23.56, 25.66, 26.59, 26.95 (2C), 28.22, 30.41, 30.54,
32.45, 32.74, 32.95, 33.66, 36.57, 38.08, 38.36, 38.74, 40.45, 41.23, 41.27, 45.15, 46.04,
47.12, 54.83, 76.80, 79.18, 121.34, 144.12, 176.01. ESI-HRMS (m/z) [M+H]+ calcd for
C34H59N2O2, 527.4571; found, 527.4572.
Compound 4 This compound was prepared from OA-OBt (0.2 mmol) and
1,5-pentanediamine (2 mmol) according to the general procedure A. The residue was purified
by column chromatography (dichloromethane/methanol, 15/1 v/v). Yield: 94 mg, 86%; white
1
solid. Mp 142.3-143.2 °C. H NMR (400 MHz, (CD3)2SO) δ: 0.65, 0.66, 0.83, 0.85, 0.87,
0.88, 1.07 (7×CH3), 0.65-2.00 (m, other aliphatic ring protons), 2.54-2.56 (m, 2H), 2.77 (d,
J=9.54 Hz, 1H), 2.87-3.05 (m, 4H), 5.20 (t, J=4.92 Hz, 1H), 7.24 (t, J=5.64 Hz, 1H); 13C
NMR (100 MHz, (CD3)2SO) δ: 15.15, 16.10, 16.90, 18.06, 22.30, 22.99, 23.64, 23.92, 25.72,
27.00, 28.30, 28.96, 30.49, 31.21, 32.53, 32.82, 33.02, 33.72, 36.63, 38.13, 38.44, 38.84,
38.96, 40.06, 40.50, 40.80, 41.30, 45.25, 46.11, 47.17, 54.88, 76.89, 121.40, 144.20, 176.18.
Chemical and Pharmaceutical Bulletin Advance Publication

ESI-HRMS (m/z) [M+H]+ calcd for C35H61N2O2, 541.4728; found, 541.4728.
Compound 5 This compound was prepared from OA-OBt (0.2 mmol) and
1,6-Hexylenediamime (2 mmol) according to the general procedure A. The residue was
purified by column chromatography (dichloromethane/methanol, 15/1 v/v). Yield: 86 mg,
1
77%; white solid. Mp 138.9-139.5 °C. H NMR (400 MHz, (CD3)2SO) δ: 0.67, 0.85, 0.87,
0.88, 0.89, 1.09 (7×CH3) 0.67-2.00(m, other aliphatic ring protons), 2.6 (t, J=6.8Hz, 2H),
2.79 (d, J=10.64 Hz, 1H), 2.91-3.07 (m, 4H), 5.21 (brs, 1H), 5.76 (s, 1H), 7.22 (t, J=5.52Hz,
1H); 13C NMR (100 MHz, (CD3)2SO) δ: 15.07, 16.03, 16.85, 18.00, 22.27, 22.93, 23.58,
25.66, 26.02, 26.43, 26.95, 28.23, 29.09, 30.84, 32.47, 32.77, 32.95, 33.66, 38.07, 38.76,
39.78, 40.43 (2C), 40.46, 46.06, 47.11, 54.81, 76.81, 121.32. ESI-HRMS (m/z) [M+H]+ calcd
for C36H63N2O2, 555.4884; found, 555.4883.
Compound 6 This compound was prepared from OA-OBt (0.2 mmol) and
2-aminoethanol (0.4 mmol) according to the general procedure A. The residue was purified
by column chromatography (petroleum ether/EtOAc, 1/1 v/v). Yield: 76 mg, 76%; white solid.
1
Mp 232.4-233.8 °C. H NMR (400 MHz, (CD3)2SO) δ: 0.67, 0.84, 0.86, 0.87, 0.89, 1.08
(7×CH3), 0.67-2.00 (m, other aliphatic ring protons), 2.75(d, J=9.28 Hz, 1H), 2.943.02 (m,
2H), 3.12-3.40 (m, 1H), 3.33-3.36 (m, 2H), 4.27 (d, J=5.16 Hz, 1H), 4.59 (t, J=5.32 Hz, 1H),
5.21 (brs, 1H), 7.15 (t, J=5.56 Hz,1H); 13C NMR (100 MHz, (CD3)2SO) δ: 15.08, 16.01,
16.72, 17.97, 22.34, 22.91, 23.51, 25.64, 26.93(3C), 28.21, 30.41, 32.36, 32.68, 32.89, 33.61,
36.55, 38.36, 40.54, 41.23, 41.54(2C), 45.24, 46.02, 47.09, 54.78, 59.84, 76.80, 121.48,
144.00, 176.50. ESI-HRMS (m/z) [M+Na]+ calcd for C32H53NO3Na, 522.3918; found,
522.3919.
Chemical and Pharmaceutical Bulletin Advance Publication

Compound 7 This compound was prepared from OA-OBt (0.2 mmol) and
3-aminopropanol (0.4 mmol) according to the general procedure A. The residue was purified
by column chromatography (petroleum ether/EtOAc, 1/1 v/v). Yield: 80 mg, 77%; white solid.
1
Mp 210.9-211.3 °C. H NMR (400 MHz, (CD3)2SO) δ: 0.67, 0.84, 0.86, 0.88, 0.89, 1.08
(7×CH3), 0.67-2.00 (m, other aliphatic ring protons), 2.76 (d, J=9.84 Hz, 1H), 2.96-3.04 (m,
2H), 3.07-3.16 (m, 1H), 3.38 (q, J=5.44 Hz, 2H), 4.27 (d, J=5.12 Hz, 1H), 4.41 (t, J=5.24 Hz,
1H), 5.21 (brs, 1H), 7.22 (t, J=5.56 Hz, 1H); 13C NMR (100 MHz, (CD3)2SO) δ: 15.06, 15.99,
16.79, 17.97, 22.27, 22.90, 23.51, 25.63, 26.93, 28.20, 30.40, 32.25, 32.40, 32.77, 32.89,
33.63, 36.32, 36.55, 38.05, 38.35, 40.06, 40.46(2C), 41.21, 45.17, 46.04, 47.08, 54.78, 58.84,
76.80, 121.43, 144.01, 176.21. ESI-HRMS (m/z) [M+Na]+ calcd for C33H55NO3Na, 536.4074;
found, 536.4074.
Compound 8 This compound was prepared from OA-OBt (0.2 mmol) and
4-amino-1-butanol (0.4 mmol) according to the general procedure A. The residue was
purified by column chromatography (petroleum ether/EtOAc, 1/1 v/v). Yield: 86 mg, 81%;
1
white solid. Mp 205.7-206.9 °C. H NMR (400 MHz, (CD3)2SO) δ: 0.66, 0.67, 0.84, 0.86,
0.88, 0.89, 1.08 (7×CH3), 0.66-2.00 (m, other aliphatic ring protons), 2.78 (d, J=9.68 Hz, 1H),
2.90-3.08 (m, 3H), 3.38 (q, J=6 Hz, 2H), 4.28 (d, J=5.16 Hz, 1H), 4.37 (t, J=4.96 Hz, 1H),
5.20 (brs, 1H), 7.21 (t, J=4.96 Hz, 1H); 13C NMR (100 MHz, (CD3)2SO) δ: 15.08, 16.00,
16.80, 18.00, 22.25, 22.91, 23.56, 25.66, 25.78, 26.95, 28.22, 30.07, 30.42, 32.45, 32.75,
32.94, 33.66, 36.57, 38.07, 38.36, 38.62, 38.74, 40.44, 41.23, 45.14, 45.16, 46.03, 47.11,
54.81, 60.54, 76.82, 121.34, 144.11, 176.00. ESI-HRMS (m/z) [M+Na]+ calcd for
C34H57NO3Na, 550.4231; found, 550.4231.
Chemical and Pharmaceutical Bulletin Advance Publication

Compound 9 This compound was prepared from OA-OBt (0.2 mmol) and
5-amino-1-pentanol (0.4 mmol) according to the general procedure A. The residue was
purified by column chromatography (petroleum ether/EtOAc, 1/1 v/v). Yield: 74 mg, 68%;
1
white solid. Mp 195.9-200.9 °C. H NMR (400 MHz, (CD3)2SO) δ: 0.69, 0.84, 0.86, 0.88,
0.89, 1.08 (7×CH3), 0.67-2.00 (m, other aliphatic ring protons), 2.78 (d, J=10.2 Hz, 1H),
2.93-3.03 (m, 3H), 3.17 (d, J=5.36 Hz, 1H), 3.31-3.38 (m, 3H), 4.28 (d, J=3.96 Hz, 1H), 4.33
(t, J=5.04 Hz, 1H), 5.20 (s, 1H), 7.19 (t, J=5.24 Hz, 1H); 13C NMR (100 MHz, (CD3)2SO) δ:
15.07, 16.01, 16.82, 18.01, 22.26, 22.92, 23.10, 23.56, 25.66, 26.95, 28.23, 29.01, 30.42,
32.25, 32.30, 32.47, 32.75, 32.95, 33.67, 36.57, 38.08, 38.37, 40.46, 41.24, 45.16, 46.05,
47.12, 54.83, 60.58, 60.70, 76.70, 76.82, 121.34, 144.12, 176.00. ESI-HRMS (m/z) [M+Na]+
calcd for C35H59NO3Na, 564.4387; found, 564.4385.
Compound 10 This compound was prepared from OA-OBt (0.2 mmol) and
6-amino-1-hexanol (0.4 mmol) according to the general procedure A. The residue was
purified by column chromatography (petroleum ether/EtOAc, 1/1 v/v). Yield: 90 mg, 80%;
1
white solid. Mp 183.8-184.5 °C. H NMR (400 MHz, (CD3)2SO) δ: 0.66, 0.67, 0.84, 0.86,
0.88, 0.89, 1.08 (7×CH3), 0.66-2.00 (m, other aliphatic ring protons), 2.78 (dd, J=3.96, 13.38
Hz, 1H), 2.92-3.04 (m, 3H), 3.36 (dd, J=6.6, 11.76 Hz, 2H), 4.29 (d, J=5.22 Hz, 1H), 4.33 (t,
13
J=5.16Hz,1H), 5.20 (brs, 1H), 7.20 (t, J=5.52 Hz, 1H); C NMR (100 MHz, (CD3)2SO) δ:
15.07, 16.04, 16.85, 18.00, 22.26, 22.94, 23.59, 25.38, 25.67, 26.62, 26.96, 28.24, 29.20,
30.45, 32.47, 32.63, 32.77, 32.97, 33.67, 36.58, 38.08, 38.39, 38.83, 38.91, 40.06, 40.46,
41.25, 45.19, 46.06, 47.12, 54.83, 60.69, 76.83, 121.33, 144.18, 176.02. ESI-HRMS (m/z)
[M+Na]+ calcd for C36H61NO3Na, 578.4544; found, 578.4545.
Chemical and Pharmaceutical Bulletin Advance Publication

Compound 11 This compound was prepared from OA-OBt (0.2 mmol) and Glycine
methyl ester hydrochloride (0.8 mmol) according to the general procedure A. The residue was
purified by column chromatography (petroleum ether/EtOAc, 3/1 v/v). Yield: 94 mg, 89%;
1
white solid. Mp 137.8-138.6 °C. H NMR (600 MHz, (CD3)2SO) δ: 0.64, 0.67, 0.84, 0.87,
0.88, 0.89, 1.08 (7×CH3), 0.64-2.00 (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 Hz, 17.04 Hz, 1H), 3.80 (dd, J=6Hz, 16.98
13
Hz, 1H), 4.29 (d, J=5.16 Hz, 1H), 5.17(t, J=3.36 Hz, 1H), 7.76(t, J=5.7Hz, 1H); C NMR
(150 MHz, (CD3)2SO) δ: 15.13, 16.05, 16.60, 18.02, 22.40, 22.94, 23.50, 25.64, 26.84, 26.97,
28.24, 30.42, 32.42, 32.50, 32.90, 33.61, 36.59, 38.09, 38.40, 38.86, 40.35, 40.93, 41.24,
45.19, 46.05, 47.14, 51.51, 54.83, 76.83, 121.42, 143.95, 170.54, 176.94. ESI-HRMS (m/z)
[M+Na]+ calcd for C33H53NO4Na, 550.3867; found, 550.3862.
Compound 12 This compound was prepared from OA-OBt (0.2 mmol) and methyl
3-aminopropionate hydrochloride (0.8 mmol) according to the general procedure A. The
residue was purified by column chromatography (petroleum ether/EtOAc, 3/1 v/v). Yield:
1
90 mg, 83%; white solid. Mp 218.8-219.6 °C. H NMR (600 MHz, (CD3)2SO) δ: 0.65, 0.67,
0.84, 0.86, 0.87, 0.89, 1.07 (7×CH3), 0.65-2.00 (m, other aliphatic ring protons), 2.40-2.43 (m,
2H), 2.74 (dd, J=3.96, 13.38 Hz, 1H), 2.97-3.00 (m, 1H), 3.15-3.21 (m, 1H), 3.25-3.21 (m,
1H), 3.57 (s, 3H), 4.28 (d, J=4.74 Hz, 1H), 5.19 (t, J=3.42 Hz, 1H), 7.35 (t, J=5.58 Hz, 1H);
13C NMR (150 MHz, (CD3)2SO) δ: 15.10, 16.03, 16.77, 17.99, 22.27, 22.92, 23.52, 25.65,
26.86, 26.96, 28.23, 30.42, 32.40, 32.61, 32.92, 33.57, 33.61, 35.02, 36.57, 38.08, 38.38,
38.89, 40.43, 41.22, 45.22, 46.01, 47.10, 51.26, 54.81, 76.82, 121.48, 143.93, 171.93, 176.47.
ESI-HRMS (m/z) [M+Na]+ calcd for C34H55NO4Na, 564.4023; found, 564.4025.
Chemical and Pharmaceutical Bulletin Advance Publication

Compound 13 This compound was prepared from OA-OBt (0.2 mmol) and methyl
4-aminobutyrate hydrochloride (0.8 mmol) according to the general procedure A. The residue
was purified by column chromatography (petroleum ether/EtOAc, 3/1 v/v). Yield: 87 mg,
1
78%; white solid. Mp 190.4-191.7 °C. H NMR (400 MHz (CD3)2SO) δ: 0.54, 0.64, 0.67,
0.84, 0.86, 0.89, 1.08 (7×CH3), 0.54-1.97 (m, other aliphatic ring protons), 2.27 (t, J=7.48 Hz,
2H), 2.78 (d, J=10.00 Hz, 1H), 2.97-3.04 (m, 3H), 3.58 (s, 3H), 4.28 (d, J=5.08 Hz, 1H), 5.21
(s, 1H), 7.31 (t, J=5.72 Hz, 1H); 13C NMR (100 MHz, (CD3)2SO) δ: 15.12, 16.08, 16.86,
18.08, 22.27, 22.98, 23.66(2C), 24.47, 25.77, 27.03(2C), 28.30, 30.51, 30.93, 32.53, 32.87,
33.03, 33.71(2C), 36.64, 38.16, 38.20, 38.45, 40.52, 41.30, 45.32, 46.07, 47.19, 51.28, 54.90,
76.90, 121.47, 144.16, 173.22, 176.33. ESI-HRMS (m/z) [M+Na]+ calcd for C35H57NO4Na,
578.4180; found, 578.4183.
Compound 14 This compound was prepared from OA-OBt (0.2 mmol) and methyl
5-aminopentanoate hydrochloride (0.8 mmol) according to the general procedure A. The
residue was purified by column chromatography (petroleum ether/EtOAc, 3/1 v/v). Yield: 77
mg, 67%; white solid. Mp 178.8-179.6 °C. 1H NMR (400 MHz, (CD3)2SO) δ: 0.65, 0.67, 0.84,
0.86, 0.88, 0.89, 1.08 (7×CH3), 0.65-2.00 (m, other aliphatic ring protons), 2.27 (t, J=7.24 Hz,
2H), 2.76 (d, J=9.84 Hz, 1H), 2.93-3.05 (m, 3H), 3.57 (s, 3H), 4.26 (d, J=5.16 Hz, 1H), 5.20
(s, 1H), 7.24 (t, J=5.52 Hz, 1H); 13C NMR (100 MHz, (CD3)2SO) δ: 15.01, 15.99, 16.79,
17.97, 22.01, 22.22, 22.87, 23.55, 25.64, 26.94, 28.21, 28.50, 30.42, 32.43, 32.77, 32.93,
33.02(2C), 33.63, 36.55, 38.06, 38.31, 38.35, 38.88, 40.44, 41.22, 45.19, 46.02, 47.10, 51.11,
54.79, 76.80, 121.36, 144.08, 173.21, 176.08. ESI-HRMS (m/z) [M+Na]+ calcd for
C36H59NO4Na, 592.4336; found, 592.4333.
Chemical and Pharmaceutical Bulletin Advance Publication

Compound 15 This compound was prepared from OA-OBt (0.2 mmol) and methyl
6-aminocappoate hydrochloride (0.8 mmol) according to the general procedure A. The
residue was purified by column chromatography (petroleum ether/EtOAc, 3/1 v/v). Yield: 95
mg, 81%; white solid. Mp 116.2-117.4 °C. 1H NMR (600 MHz (CD3)2SO) δ: 0.66, 0.67, 0.84,
0.86, 0.87, 0.89, 1.08 (7×CH3), 0.66-2.00 (m, other aliphatic ring protons), 2.27 (t, J=7.44 Hz,
2H), 2.77 (dd, J=4.02, 13.44 Hz, 1H), 2.91-3.04 (m, 3H), 3.57 (s, 3H), 4.27 (d, J=5.16 Hz,
13
1H), 5.20 (t, J=3.42 Hz, 2H), 7.21 (t, J=5.58 Hz, 2H). C NMR (150 MHz, (CD3)2SO) δ:
15.06, 16.03, 16.85, 18.00, 22.26, 22.93, 23.59, 24.23, 25.67, 26.06, 26.96, 28.24, 28.77,
30.45, 32.46, 32.77, 32.97, 33.30, 33.66, 36.58, 38.08, 38.39, 38.60, 38.91, 40.06, 40.46,
41.25, 45.20, 46.05, 47.12, 51.17, 54.82, 76.83, 121.34, 144.17, 173.26, 176.07. ESI-HRMS
(m/z) [M+Na]+ calcd for C37H61NO4Na, 606.4493; found, 606.4493.
Compound 16 This compound was prepared from 11 (0.2 mmol) and sodium hydroxide
(0.6 mmol) according to the general procedure B. Yield: 84 mg, 82%; white solid. Mp
1
176.0-177.8 °C. H NMR (600 MHz, (CD3)2SO) δ: 0.64, 0.67, 0.83, 0.87, 0.87, 0.89, 1.08
(7×CH3), 0.64-2.00 (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=6.0, 17.28 Hz, 1H), 4.28 (d,
13
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); C NMR
(150 MHz, (CD3)2SO) δ: 15.12, 16.05, 16.60, 18.00, 22.45, 22.95, 23.50, 25.65, 26.85, 26.97,
28.24, 30.42, 32.39, 32.48, 32.91, 33.62, 36.58, 38.10, 38.39, 38.88, 40.47, 40.90, 41.24,
45.14, 46.05, 47.15, 54.82, 76.83, 121.48, 143.97, 171.48, 176.68. ESI-HRMS (m/z)
[M+Na]+ calcd for C32H51NO4Na, 536.3710; found, 536.3713.
Compound 17 This compound was prepared from 12 (0.2 mmol) and sodium hydroxide
Chemical and Pharmaceutical Bulletin Advance Publication

(0.6 mmol) according to the general procedure B. Yield: 90 mg, 85%; white solid. Mp >
1
250 °C. H NMR (600 MHz (CD3)2SO) δ: 0.65, 0.67, 0.84, 0.86, 0.86, 0.88, 1.07 (7×CH3),
0.65-2.00 (m, other aliphatic ring protons), 2.33 (t, J=7.08 Hz, 2H), 2.72 (dd, J=3.78, 13.80
Hz, 1H), 2.97-3.00 (m, 1H), 3.123.15 (m, 1H), 3.23-3.29 (m, 1H), 3.37 (s, 1H), 5.19 (brs, 1H),
7.30 (t, J=5.58 Hz, 1H), 12.20 (s,1H); 13C NMR (150 MHz, (CD3)2SO) δ: 15.16, 16.09, 16.83,
18.05, 22.33, 22.97, 23.55, 25.69, 26.93, 27.00, 28.28, 30.47, 32.43, 32.66, 32.96, 33.64,
33.82, 35.02, 36.61, 38.13, 38.43, 38.95, 40.56, 41.28, 45.23, 46.05, 47.15, 54.84, 76.87,
121.62, 143.93, 173.21, 176.44. ESI-HRMS (m/z) [M+Na]+ calcd for C33H53NO4Na,
550.3867; found, 550.3865.
Compound 18 This compound was prepared from 13 (0.2 mmol) and sodium hydroxide
(0.6 mmol) according to the general procedure B. Yield: 94 mg, 87%; white solid. Mp
228.7-229.5 °C. 1H NMR (400 MHz, (CD3)2SO) δ: 0.65, 0.67, 0.84, 0.86, 0.89, 1.08 (7×CH3),
0.65-2.00 (m, other aliphatic ring protons), 2.18 (t, J=7.4 Hz, 2H), 2.78 (d, J=9.68 Hz, 1H),
2.96-3.03 (m, 3H), 3.32 (d, J=8.92 Hz, 2H), 4.28 (d, J=5.08 Hz, 1H), 5.21 (brs, 1H), 7.29 (t,
J=5.44 Hz, 1H), 12.0 (s, 1H); 13C NMR (100 MHz, (CD3)2SO) δ: 15.16, 16.10, 16.92, 18.08,
22.27, 22.99, 23.67, 24.53, 25.76, 27.04, 28.31, 30.52, 31.29, 32.53, 32.87, 33.03, 33.71,
36.65, 38.23, 38.43, 38.46, 40.50, 41.31, 45.29, 45.30, 46.09, 47.19, 54.89, 76.79, 76.90,
121.48, 144.17, 174.36, 176.24. ESI-HRMS (m/z) [M+Na]+ calcd for C34H55NO4Na,
564.4023; found, 564.4024.
Compound 19 This compound was prepared from 14 (0.2 mmol) and sodium hydroxide
(0.6 mmol) according to the general procedure B. Yield: 79 mg, 71%; white solid. Mp
1
139.1-140.9 °C. H NMR (600 MHz (CD3)2SO) δ: 0.65, 0.66, 0.83, 0.86, 0.87, 0.88, 1.07
Chemical and Pharmaceutical Bulletin Advance Publication

(7×CH3), 0.65-2.00 (m, other aliphatic ring protons), 2.17 (t, J=7.26 Hz, 2H), 2.78 (dd,
J=4.08, 13.56 Hz, 1H), 2.94-3.02 (m, 3H), 3.41 (s, 1H), 5.20 (brs, 1H), 7.26 (t, J=5.52 Hz,
1H), 11.98 (s, 1H); 13C NMR (150 MHz, (CD3)2SO) δ: 15.16, 16.09, 16.83, 18.05, 22.33,
22.97, 23.55, 25.69, 26.93, 27.00, 28.28, 30.47, 32.43, 32.66, 32.96(2C), 33.64, 33.82, 35.02,
36.61, 38.13, 38.43, 38.95, 40.06, 40.56, 41.28, 45.23, 46.05, 47.15, 54.84, 76.87, 121.62,
143.93, 173.21, 176.44. ESI-HRMS (m/z) [M+Na]+ calcd for C35H57NO4Na, 578.4180; found,
578.4180.
Compound 20 This compound was prepared from 15 (0.2 mmol) and sodium hydroxide
(0.6 mmol) according to the general procedure B. Yield: 82 mg, 72%; white solid. Mp
1
119.8-120.7 °C. H NMR (600 MHz, (CD3)2SO) δ: 0.66, 0.67, 0.84, 0.86, 0.87, 0.88, 1.08
(7×CH3), 0.66-2.00 (m, other aliphatic ring protons), 2.17 (t, J=7.38 Hz, 2H), 2.78 (dd, J=3.9,
13
13.32 Hz, 1H), 2.91-3.04 (m, 3H), 5.20 (brs, 1H), 7.22(t, J=5.58 Hz, 2H), 11.98 (s, 1H); C
NMR (150 MHz, (CD3)2SO) δ: 15.08, 16.05, 16.86, 18.01, 22.27, 22.94, 23.60, 24.30, 25.68,
26.17, 26.97, 28.25, 28.86, 30.46, 32.47, 32.77, 32.98, 33.67(2C), 36.59, 38.08, 38.39, 38.66,
38.91, 40.05, 40.47, 41.26, 45.20, 46.06, 47.13, 54.83, 76.84, 121.35, 144.16, 174.40, 176.06.
ESI-HRMS (m/z) [M+Na]+ calcd for C36H59NO4Na, 592.4346; found, 592.4344.
Chemical and Pharmaceutical Bulletin Advance Publication

Bioassays
Cell activity assay.
MDCK cells were seeded into 96-well plates in DMEM supplemented with 10% FBS
cultured overnight at 37 °C in 5% CO2. Then, cells were suspended in DMEM supplemented
with 1% FBS, containing test compound and 2 μg/mL TPCK-treated trypsin, and the cells
were further incubated at 37°C in 5% CO2 for 40 hours. Cell viability was assessed using the
CellTiter-Glo assay kit (Promega Corp., Madison, WI, USA) as recommended by the supplier,
and the plates were read using a plate reader (Molecular Devices SpectraMax M2). Viability
was calculated using the background corrected absorbance as follows: Viability (%) = (CA of
experiment well/DA of control well) × 100, where CA, DA represent the reading values of
test compound and DMSO, respectively.
Cytopathic Effect (CPE) Reduction Assay.
MDCK cells were seeded into 96-well plates, incubated overnight and infected with
influenza virus (MOI = 0.1) suspended in DMEM supplemented with 1% FBS, containing
test compound and 2 μg/mL TPCK-treated trypsin, with a final DMSO concentration of 1% in
each well. After 40 h of incubation, CellTiter-Glo reagent was added and the plates were read
using a plate reader (Molecular Devices SpectraMax M2).
Hemagglutination Inhibition (HI) Assay.
Compound from a 2-fold serial dilution in saline was mixed with an equal volume of
influenza virus (The HA titers of A/WSN/33 (H1N1) virus is 1:2) in the V-bottomed 96-well
microplate. Subsequently, 50 μL of freshly prepared chicken red blood cells (RBCs) (1% v/v
Chemical and Pharmaceutical Bulletin Advance Publication

in saline) were added to each well. The mixture was incubated for 30 min at RT before
observing RBCs aggregation on the plate.
Surface plasmon resonance (SPR).
Interactions between the influenza HA and the compounds were analyzed using the
Biacore T200 system (GE Healthcare, Uppsala, Sweden) at 25ꢀ°C. Recombinant influenza
HA (Sino Biological Inc., Beijing, China) was immobilized on a sensor chip (CM5) using an
amine coupling kit (GE Healthcare, Buckinghamshire, UK). Final HA-immobilized levels
were typically ∼16,000 RU. Subsequently, compounds were injected as analytes at various
concentrations, and PBS-P (10ꢀmM phosphate buffer with 2.7ꢀmM KCl and 137ꢀmM NaCl,
0.05% surfactant P20, pH 4.5) was used as running buffer. For binding studies, analytes were
applied at corresponding concentrations in running buffer at a flow rate of 30ꢀmL/min with a
contact time of 60s and a dissociation time of 60s. Chip platforms were washed with running
buffer and 50% DMSO. Data were analyzed with the Biacore evaluation software (T200
version 1.0) by curve fitting using a binding model of 1:1.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (grant no.
81560560) and the Scientific Research Foundation of Kunming University of Science and
Technology (No. KKSY20169517).
Conflict of Interest
The authors declare no conflict of interest.
Chemical and Pharmaceutical Bulletin Advance Publication

Supplementary Materials
The online version of this article contains supplementary materials.
Chemical and Pharmaceutical Bulletin Advance Publication

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Chemical and Pharmaceutical Bulletin Advance Publication

Table 1. Anti-IAV activity data of target compounds 9, 10 and 12-15.
Compound
IC50 (μM)a
CC50 (μM)b
9
10
12
13
14
15
OSV
OA
>50
2.98
5.36
39.49
>50
>50
1.82
72.27
>100
>100
>100
>100
>100
>100
>100
>100
a
All data presented are averages of at least three separate experiments. IC50: compound
concentration required to achieve 50% inhibition of replication of virus, as determined by the
b
CellTiter-Glo assay. CC50: compound concentration required to cause 50% death of
uninfected MDCK cells, as determined by the CellTiter-Glo assay.
Chemical and Pharmaceutical Bulletin Advance Publication

Fig 1. Inhibitory effects of OA derivatives against influenza A/WSN/33 (H1N1) virus. A.
Cytotoxicity screening of OA derivatives (final concentration 100 μM) using CellTiter-Glo®
assay. B. The CPE-based screening of OA derivatives by using a CellTiter-Glo® assay.
MDCK was utilized as the host cell to test A/WSN/33 (H1N1) virus infection; 1% DMSO
(final concentration) was used as negative control, and OSV (oseltamivir) was utilized as
positive control, respectively. The error bars indicate the standard deviations of triplicate
experiments.
Chemical and Pharmaceutical Bulletin Advance Publication

Fig 2. A. Validation of the protection of MDCK cells from influenza A/WSN/33 virus by
compound 10. B. Identify HA as the potential target of compound. Comparisons of the
behaviors of compound 10 vs anti-HA antibody in inhibition of influenza virus-induced
aggregation of chicken erythrocytes. Compound 10 exerted identical capability as anti-HA
antibody in inhibition of hemagglutination in a dose dependent manner. C. Characterization
of the affinity between compounds (10 and OA) and HA protein, which were immobilized on
a CM5 sensor chip, based on the SPR assay. Their KD values are labeled on the
corresponding curves.
Chemical and Pharmaceutical Bulletin Advance Publication

Chart 1. Reagents and conditions: (a) EDCI, HOBt, (CH3CH2)3N, CH2Cl2, 5-10℃, 12h. (b)
Amine compound, Na2CO3, DMF, rt, 12h. (c) Alcohol compound, Na2CO3, DMF, rt, 12h. (d)
Methyl ester compound, Na2CO3, DMF, rt, 12h. (e) NaOH, MeOH, rt, 6h.
Chemical and Pharmaceutical Bulletin Advance Publication