Total synthesis of dryocrassin ABBA and its analogues with potential inhibitory activity against drug-resistant neuraminidases

Article abstract of DOI:10.1016/j.bmc.2019.07.013

The stems of Dryopteris crassirhizoma, one of the main components of Lianhua-Qingwen Formula (LQF) was traditionally used for heat-clearing and detoxifying. Dryocrassin ABBA is a key antiviral component in the herbal medicine while the compound is hard to

Full text of DOI:10.1016/j.bmc.2019.07.013

Accepted Manuscript
Total Synthesis of Dryocrassin ABBA and its Analogues with Potential Inhib-
itory Activity against Drug-resistant Neuraminidases
Bo Hou, Ze Liu, Xiao-Bei Yang, Wen-Fei Zhu, Jin-Yu Li, Liu Yang, Fu-Cai
Reng, Yong-Feng Lv, Jiang-Miao Hu, Guo-Yang Liao, Jun Zhou
PII:
S0968-0896(19)30887-9
https://doi.org/10.1016/j.bmc.2019.07.013
BMC 14997
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 May 2019
4 July 2019
7 July 2019
Please cite this article as: Hou, B., Liu, Z., Yang, X-B., Zhu, W-F., Li, J-Y., Yang, L., Reng, F-C., Lv, Y-F., Hu, J-
M., Liao, G-Y., Zhou, J., Total Synthesis of Dryocrassin ABBA and its Analogues with Potential Inhibitory Activity
against Drug-resistant Neuraminidases, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.bmc.2019.07.013
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Total Synthesis of Dryocrassin ABBA and its Analogues with
Potential Inhibitory Activity against Drug-resistant Neuraminidases
Bo Hou, a, c Ze Liu, b Xiao-Bei Yang, a, c Wen-Fei Zhu,d Jin-Yu Li, a, b Liu Yang, a Fu-
Cai Reng, a. b Yong-Feng Lv, a, b Jiang-Miao Hu, * a Guo-Yang Liao, * b Jun Zhou * a
a
b
State Key Laboratory of Phytochemistry and Plant Resources in West China, and Yunnan Key Laboratory of
Natural Medicinal Chemistry. Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201,
People’ s Republic of China.
E-mail: hujiangmiao@mail.kib.ac.cn (J.-M. Hu) or jzhou@mail.kib.ac.cn (J. Zhou).
The Fifth Department of Biological Products, Institute of Medical Biology, Chinese Academy of Medical Science
and Peking Union Medical College, Kunming, People’s Republic of China.
E-mail: mybelieze@126.com (G.-Y. Liao).
c
d
University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China.
National Institute for Viral Disease Control and Prevention, Chinese Centre for Disease Control and Prevention,
China.
Electronic Supplementary Information (ESI) available, retrosynthetic analysis, enzyme inhibition assay, docking
method and compounds characterization.

Abstract
The stems of Dryopteris crassirhizoma, one of the main components of Lianhua-
Qingwen Formula (LQF) was traditionally used for heat-clearing and detoxifying.
Dryocrassin ABBA is a key antiviral component in the herbal medicine while the
compound is hard to get in large amounts with the features of homologous compounds,
polyphenol groups, and low contents. Therefore, the present work aims to seek
influenza H7N9 virus inhibitors from natural source by synthesis of dryocrassin ABBA
and its analogues. As a result, total synthesis of the compound was achieved in nine
steps with an over-all yield of 4.6 %. Neuraminidases (NAs) inhibitory activities of the
synthesized product and its analogues were evaluated afterward. Comparing with the
positive control, OSV (9.6 μM), it was very exciting that dryocrassin ABBA and its
analogues (b5 and e2) showed better NAs inhibitory activity against Anhui H7N9 with
IC50 values of 3.6 μM, 2.5 μM and 1.6 μM. For the highly resistant Shanghai N9, these
compounds can also show medium inhibitory activities. Docking results indicated the
direct interaction of synthesized 3 hits with the key K294 by hydrogen bonds, but no
direct interaction of OSV with the key K294 was observed in Shanghai N9. This study
suggested that dryocrassin ABBA and its analogues especially AB, which consisted of
polyphenol groups may have beneficial effects on treating avian influenza H7N9 virus.
Key words: dryocrassin ABBA; neuraminidase; total synthesis; Shanghai H7N9;
Anhui H7N9

1. Introduction
Influenza continues to be a serious health issue triggering substantial morbidity and
mortality, particularly among kids, elderly people, and persons with respiratory disease
1. The outbreak of H1N1 in Spain in 1918 caused around 500 million human infections
2
and killed 50 – 100 million people worldwide . From then on, other influenza
pandemics such as H5N1, H5N8 and H7N9 associated with morbidity and mortality in
humans were subsequently discovered and caused serious social and economic
3
problems . These new emerging influenza viruses have been identified and caused
worldwide attention 4. Especially, the increased preference of H7N9 virus for human-
type receptors may result in its higher cross-species transmissibility than H5N1 viruses
which prefer avian-type receptors 5. For these reasons, the present analysis focused on
Shanghai and Anhui H7N9.
Two glycoproteins including hemagglutinin and neuraminidase on the surface of the
influenza virus play key roles in the virus replicative cycle, which facilitate the entry
and exit of virus from the host cell. The agents which can interfere with one of
glycoproteins should have antiviral properties 6. Both types A and B influenza viruses
7
contain neuraminidase . For the neuraminidases of influenza A and B, up to 75%
sequence variation is conserved in the active site. The highly conserved site makes it
1
an attractive target for structure-based drug design . Therefore, inhibition of
neuraminidase by interaction with the active site could be designed as drugs which have
a broad application of activity against various seasonal flu 8.
Drugs on the market for anti-influenza Acontains M2 ion channel blockers (amantadine
9
and rimantadine) and neuraminidase inhibitors (zanamivir and Oseltamivir) . The
problem of known drugs is the emergence of drug-resistant strains to influenza A 10-11
.
In the past decade, the key S31N mutants of amantadine-resistant H1N1, H5N1, and
H3N2 strains as predominant in 98 % − 100 % of the transmissible have been isolated
from humans and animals 12. Oseltamivir (OSV) appears drug resistance during the flu
season in the United States and China 13-15. Even dual resistance to both adamantane

and neuraminidase have been emerged 16-17. Therefore, it is urgent to develop
alternative anti-influenza agents for resistant strains.
Fig. 1. Molecular fragments were derived from natural inhibitors of NA.
Natural products have been reported as important source of drug precursors. Thus,
potent and selective neuraminidase inhibitors which could be easily and economically
prepared were developed herein. Dryocrassin ABBA were isolated and elucidated in
1971-1973 from the rhizomes of Dryopteris crassirhizoma Nakai (Fig. 1) 18-19, which
is one of the main compositions of 11 herb medicines in LQF and was used for the
prevention and treatment of viral influenza in China. Post inoculation with highly
pathogenic avian influenza virus (H5N1) in mice, the survival rate was 87% in
20
dryocrassin ABBA treated groups at a dose of 33 mg/kg . Dryocrassin ABBA is a
potential novel leading compound which had antiviral effects on amantadine-resistant
avian influenza virus H5N1 infection. The promising experimental results inspired us
to synthesize the natural compound and its analogues. This study not only provided
total synthesis method of dryocrassin ABBA skeleton but also underlined that its
structural analogues have good efficient to inhibit influenza NAs.

2. Results and discussion
2.1. Chemistry
A retrosynthetic analysis of dryocrassin ABBA was illustrated in Scheme S1. The
simple synthesis of A cycle from phloroglucinol (a1) as shown in Scheme 1 has been
reported previously. Briefly, the treatment of a1 with acetic acid-boron trifluoride and
acetyl chloride to obtain a2, followed by a reaction with methyl iodide in the presence
of sodium methoxide and afforded 2,6-deacetyl-3,5-dihydroxy-4,4-dimethy-2,5-
cyclohexadien-1-one in 46 % yield. Deacetylation of a3 with 80% of the concentrated
sulfuric acid afforded 2-acetyl-3,5-dihydroxy-4,4-dimethy-2,5-cyclohexadien-1-one
(a4). Albaspidin AA was obtained in 70% yield by using formalin and concentrated
sulfuric acid in dichloromethane (DMC) 21.
Scheme 1. Synthesis of A cycle and albaspidin AA (a5).
Scheme 2. Synthesis of B cycle and b5.
Scheme 3. B cycle was linked to A cycle through TCRA reaction and synthesis of dryocrassin
ABBA.

Phloroglucinol on treatment with Butanoyl chloride in the presence of boron trifluoride
etherate led to the formation of desired 2-Butanoyl phloroglucinol (b1) in 49.3 % yield.
Synthesis of 3-btyryl-2,4,6-trihydroxybenzaldehyde (b2) was carried out by treatment
of b1 with Vilsmeier–Haack reagent (POCl3, DMF) in ethyl acetate in 71.6 % yield.
Treatment of b2 with excess the methyl iodide and dry potassium carbonate generated
3-butyryl-2,4,6-trimethoxybenzaldehyde (b3). Other hydroxy-protecting group
(Chlormethyl methyl ether, MOMCl) was preparation for the next reaction. The b4 was
obtained in NaH reaction conditions as shown in Scheme 2. The BB cycle (b5) was
obtained with formalin and concentrated sulfuric acid in 50 °C.
B cycle linked to A cycle was prepared in good yield using an organocatalytic three-
component reductive alkylation (TCRA) reaction. Firstly, we observed that the reaction
of a4 with b2 under proline catalysis can’t lead to adduct (Scheme S2, entry 1).
However, when we used a4 and 4-hydroxybenzaldehyde in TCRA reaction, mono-
adduct was detected and was obtained with 40 % yield under the standard TCRA
conditions (Scheme S2, entry 2). The optimum conditions involved the use of 5-mol%
proline catalyst and two equiv. of 4-hydroxybenzaldehyde to obtain c1 in 55 % yield.
This is an important model reaction for the synthesis of the linked AB cycles. The
results indicated the TCRA reaction was terminated due to more phenol hydroxyl of
reactive substances. Methyl iodide and MOMCl were used in hydroxy-protecting group
of b2. However, the reaction of a4 and b4 under proline catalysis would not lead to the
desired product (d1) formation (Scheme S2, entry 3). The steric hindrance of

methylether may be an important factor in hindering the reaction. Treatment of a4 and
b3 generated e1 which was obtained in DMC at room temperature for 7 h in very good
yield. This interesting result represents a novel methodology for the preparation
methylene linked polyphenols and a new reactivity for amino acid catalysis.
The demethylation of e1 was treated with boron tribromide at –78 °C to afford the
corresponding compounds e2. The final step in Scheme 3 seems to be possible since
the key factors in phenol–aldehyde incorporation are methylene source and acid. Using
different methylene conditions to polymerize e2 were shown in Table 1. Lastly,
dryocrassin ABBA was obtained in 49 % yield by using formalin and concentrated
sulfuric acid in the room temperature. Nevertheless, the natural product can be easily
isolated by regular chromatography.
Table 1
Phenol and aldehyde conditions and results of dryocrassin ABBA.
Entry
conditions
Results
CH3OH (M=0.3), CHONa (3 equvi), CH2O (4.3 equvi), 30 min, RT
CH3OH (M=0.3), KOH (3 equvi), CH2O (5.7 equvi), 30 min, RT
MSA (100, equvi), CH2O (0.6 equvi), 30 min, 80 oC
No product
No product
No product
No product
1
2
3
4
5
6
CH3OH (M=0.3), KOH (3 equvi), CH2O (5.7 equvi), 30 min, RT
EtOAc (M=0.3), H2SO4 (con, 15 equvi), CH2O (30 equvi), 30 min, RT 40 %
DMC (M=0.3), H2SO4 (con, 2 equvi), CH2O ( 17.5 equvi), 8 h, RT 49 %
2.2. Biological tests
2.2.1 NA Inhibition Assay of four Compounds
Four DNA sequences encoding the Influenza A virus (A/California/04/2009 (H1N1),
A/Babol/36/2005 (H3N2), A/Anhui/1/2005 (H5N1), A/Anhui/1/2013 (H7N9) and
A/Shanghai/1/2013 (H7N9)) NAs were expressed by HEK293 cells. The cell lysates
were collected and bioactivity were tested through 2'-(4-Methylumbelliferyl)-α-D-N-
acetylneuraminic acid. Shanghai N9 (K294-containing) displayed extreme resistance to
OSV (IC50 > 400 μM). For b5, e2 and dryocrassin ABBA, the R294K mutation
conferred less resistance relative to OSV (Table 2). For Shanghai N9, the IC50 values

for dryocrassin ABBA, b5 and e2 were 279.6 μM, 83.6 μM and 168.5 μM, respectively.
More potent inhibition of Anhui N9 was apparent with the IC50 values of 3.6 μM, 2.5
μM and 1.6 μM for dryocrassin ABBA, b5 and e2, respectively.

Table 2
Binding sites using the Autodock program and neuraminidase activity of test compounds.
IC50 (Mean± SD)b
Comps.
Ligands Binding sitesa
California
N1c
Babol
N2 c
Anhui
N9 c
Shanghai
N9 c
Anhui N1 c
SId
R119, E120, R153, W180, I224, R372

1

OSV
a5
0.05 (0.02)
NA
0.1 (0.01) 2.6 (0.32)
9.6 (4.1) 441.3 (212.7)
NA
NA
NA
NA
R119, E120, R153, E180, A248, E279, K294, N296, R372, Y406
R119, E120, R153, E229, E279, K294, N296, R372, Y406
3.8
6
b5
11.1 (3.7)
9.2 (3.6)
9.8 (3.8)
13.4 (4.5)
2.5 (1.1)
83.6 (12.6)
e2
5.6 (1.5) 52.8 (10.7)
1.6 (0.17) 168.5 (34.5)
3.6 (2.1) 279.6 (83.3)
Dry. ABBAR153, D152, R119, A248, E279, Y406, R372, K294, N296, N347
2.6
25.5 (10.0) 36.1 (10.7) 61.8 (18.2)
[a] All amino acid residues of Shanghai N9 located within 4 Å from the original ligand in the pymol program. [b] NA, not active. IC50: The concentration that affords 50%
inhibition of neuraminidase and alues express in terms of μM. Mean± SD: Means ± standard deviations of three independent experiments. [c] The neuraminidases were derived
from A/California/04/2009 (H1N1), A/Babol/36/2005 (H3N2), A/Anhui/1/2005 (H5N1), A/Anhui/1/2013 (H7N9) and A/Shanghai/1/2013 (H7N9), respectively. [d] The
sensitivity of Anhui N9 to hits was determined using IC50(OSV)/IC50(hit).

2.2.2 Analysis based on docking Shanghai N9 protein
To reveal the binding sites between four hits and Shanghai N9 protein (PDB entries:
4MWW), the rigid docking calculations were performed with the AutoDock 4.2
program. The structure of Shanghai N9 was retrieved from the Protein Data Bank and
used for visual docking. The water molecules were removed in the PDB file. Polar
hydrogen atoms were added, and non-polar hydrogen atoms were merged. The
Lamarckian genetic algorithm was used to acquire the conformation of the ligand in the
binding pocket. The docking results were clustered with an RMSD tolerance of 2.0 Å,
and the selected conformation was the best-ranked member of the cluster that adopted
the best binding energy. The fifty independent docking runs were carried out for every
ligand, and other parameters were set to default. OSV and 3 hits were docked to the
prepared N9 target. In the Shanghai and Anhui N9-oseltamivir carboxylate complex
structures (PDB entries: 4MWQ), E278 (RMSD = 1.1) displays different conformations
as in the crystal structures (Fig. 2). Consequently, Anhui N9 prefer well to interact with
OSV with a hydrophobic pocket formed by the farther from the residue E278. For
Shanghai N9 E278 is oriented toward the hydrophobic side chains of OSV.
Fig. 2. Overview of Anhui N9 protein and comparison of Anhui N9 (cyan) and Shanghai N9 (bright orange)
crystalline complex for active sites. In Shanghai N9 the distances ~ 5Å and toward of the key 294 residue aren’t able
to hydrogen-bond with OSV. Because of E278 forms improper orientation (RMSD = 1.1) leads to the adverse
interactions with alkoxy chains of OSV.
Fig. 3A shows the interaction between OSV and Shanghai N9. The six key residues

(R119, E120, R153, W180, I224 and R372) involve the charged salt bridge, hydrogen
bonding interactions and unfavorable hydrophobic effect. This unfavorable interaction
leads to the hydrophobic side chains of OSV away from the active site 15. As one of the
three key and conserved residues, R294 is thought to be the key residue for distorting
22
the sialic acid pyranose ring from a chair to a boat conformation . The process is a
critical step for NA to catalyze the hydrolytic cleavage of terminal sialic acid. The N9
from A/Shanghai/1/2013 H7N9 has much lower NA activity. In additional the mutants
associated with R294K substitution cause OSV with weak hydrogen bonding
interactions in active site. Compound e2 shows the stable inhibition of Shanghai N9
enzyme (Fig. 3B). Strong hydrogen bonding interactions among R372, Y406 and the
acetyl group of A cycle. It enhances binding energy of the active site and is close to the
five residues (R119, R153, E279, K294 and N296) of Shanghai N9 receptor. Butyl
chain to form hydrophobic interactions with E120 and E229 may account for suitable
the spatial occupation. 9 forms H-bond interactions with all above residues and having
no π bond was observed. Compound b5 was docked to the active site of Shanghai N9
protein. Its hydrogen-bond interactions with R119, E120, R153, A248, E279, K294,
N296, R372, and Y406 were illustrated in Fig. 3C. The 3-butyryl-2,4,6-
trihydroxybenzaldehyde unit leads to stimulate π-π stacking interactions with the
aromatic residues Y406 and reinforces the inhibition of N9 enzyme. Previously, Gao
and his coworkers reported the IC50 (18.59 μM) of dryocrassin ABBA against Anhui
H5N1 at NA 1 U/ml 23. In this work the IC50 of dryocrassin ABBA is approximate 3.5-
fold lower (61.8 μM). The result may due to NA enzyme concentration at NA 0.1 U/ml.
Dryocrassin ABBA with polyphenol cycles displayed approximate 2-fold decrease in
potency compared with e2. Although the polyphenol groups offer more hydrogen bonds
to amino acids of NA, the large steric size far away from the active site was observed
in Fig. 3D. Redundant hydrogen bonds may lead to excessively evaluate affinity
energies of Anhui N9 (-8.3 kcal/mol) and Shanghai N9 (-7.8 kcal/mol) in Table 3.

Fig. 3. Structural analysis of inhibitor binding to Shanghai N9. Inhibitors in bright orange are shown using a fatter
stick representation. Residues are shown using cartoon and thin stick representation. OSV (A), e2 (B), b5 (C) and
dryocrassin ABBA (D) binding modes in Shanghai N9.
Table 3
Properties and binding energies of phloroglucinols.using Vina program.
Comps.
LogP
NHBD
NHBA Affinity of Anhui Affinity of Shanghai
N9 (kcal/mol)
N9 (kcal/mol)
-1.33
3.17
2.38
2.87
4
3
4
3
6
- 6.5
- 6.3
- 7.3
- 7.1
- 7.8
OSV
6
- 7.0
- 7.2
- 8.3
b5
5
e2
Dry. ABBA
10
[a] log P= Log of the octanol–water partition coefficient were obtained by using the ALOGPS 2.1
program (www.vcclab.org/lab/alogps/). [b] NHBD = the number of hydrogen bond donor, NHBA
= the number of hydrogen bond acceptor.
In summary, except for dryocrassin ABBA, the key residues displayed in Table 2 by
the hydrogen bonds and electrostatic interactions revealed the encouraging agreement
between the IC50 values and affinity energies. Docking results indicated the direct

interaction between 3 hits and the K294 of Shanghai N9 by the hydrogen bonds. The
computed affinities of Shanghai N9 were -6.3 kcal/mol, -7.3 kcal/mol, -7.1 kcal/mol
and - 7.8 kcal/mol for OSV, b5, e2, and dryocrassin ABBA, respectively. The IC50
values for inhibition of Shanghai N9 by OSV, dryocrassin ABBA, b5 and e2 were >
400 μM, 279.6 μM, 83.6 μM and 168.5 μM, respectively. In contrast, the corresponding
IC50 values for the Anhui N9 were 9.6 μM (- 6.5 kcal/mol), 3.6 μM (- 7.2 kcal/mol), 2.5
μM (- 7.0 kcal/mol) and 1.6 μM (- 8.3 kcal/mol), respectively.
3. Conclusion
Natural products remain an attractive source to explore their therapeutic potential
against influenza virus. Dryocrassin ABBA and its analogues showed stable inhibitory
activities against NAs. Both b5 and e2 had higher activities against the influenza N9
protein than dryocrassin ABBA. This will give insight into the possible utilization of
dryocrassin ABBA and its synthetic analogues as the potential sources of H7N9
inhibitors and the leading compounds.
4. Experimental section
4.1. Chemistry
Solvents were purified in a conventional manner. Fractions were monitored by TLC
detection which was achieved by spraying the silica gel plates (Si gel G and GF254,
Qingdao haiyang Chemical Co., Ltd, Qingdao, China). Flash column chromatography
was performed on silica gel (200-300 mesh, Qingdao, China). NMR spectra were
performed on Bruker AV-400 or AVANCE III-600 spectrometers with tetramethylsilane
as an internal standard, and chemical shifts are recorded in ppm values. IR spectra were
recorded on a Tenor 27 spectrophotometer as KBr pellets. MS and HR-ESI-MS were
measured on a VG-Auto-spec-3000 or API-Qstar-Pulsar instruments.
The preparation of a2: To a solution of phlroglucinol (15 g, 118.94 mmol) in
BF32AcOH (75 ml) was stirred on an oil bath at 100 °C for 4 h. The reaction mixture
was cooled to room temperature (RT) and poured into an NaOAc (1.5 L, 51 g/L). After

stirring at RT for 24 h the precipitated orang prims were collected by filtration and then
extracted with ethyl acetate three time. The combine extracts were washed with water
and brine, dried over Na2SO4. The residues was purified by silica-gel column
chromatography (EtOAc-petroleum ether, 1:3) to give 17.5 g white powder. 1H NMR
(400 MHz, CDCl3, 25 °C) δ 16.26 (br s, 1H), 13.19 (br s, 2H), 5.82 (s, 1H), 2.56 (s,
6H). 13C NMR (101 MHz, CDCl3, 25 °C) δ 203.5, 171.2, 168.7, 103.5, 94.6, 40.1, 32.5.
(-)-HRESIMS m/z [M-H]-: calcd for C10H12O4, 195.0663, found m/z: 195.0683.
The preparation of a4: To a stirred solution of 2,4-Diaceetyl-1,3,5-benzentetriol (3.0
g, 14.27 mmol) in menthol (2.67 ml) was slowly added 28% sodium methoxide solution
(4.43 g, 85.64 mmol) at 0 °C. The mixture was stirred for 2h and then add dropwise
methyl iodide (6.08g, 2.67ml). The reaction mixture was stirred in the dark at RT for
4d. The residue, after the addition of 1 M hydrochloric acid, was extracted with ethyl
acetate. The ethyl acetate was washed with water, dried over anhydrous Na2SO4. The
solution is concentrated by reducing pressure and dried by vacuum, and a mixture a3
and 80% H2SO4 was heated at 80 °C for 30 min. The reaction mixture was poured onto
ice water mixture and then extracted with ethyl acetate three times. The combined
extracted were washed water and brine, dried over Na2SO4, and concentrated in vacuo.
The residues was purified by silica-gel column chromatography (EtOAc-petroleum
ether, 1:6) to give 1.3 g white powder a4 (46.4%).1H (400 MHz, (CD3)2SO, 25 °C): δ
18.36 (s, 1H), 5.41 (s, 1H), 2.44 (s, 3H), 1.25 (s, 6H); 13C (400 MHz, (CD3)2SO, 25 °C):
δ 200.2, 196.3, 188.4, 182.3, 104.9, 94.6, 48.4, 28.0, 24.3. (-)-HRESIMS m/z [M-H]-:
calcd for C10H12O4, 195.0663, found m/z: 195.0683.
General procedure for the preparation of a5, b5 and dryocrassin ABBA: To
a solution of reactant (50 mg, 0.3 mmol) in DMC (0.3 M) (THF for b5) was
added 37% formalin (0.72 ml, 9 mmol) and concentrated sulphuric acid (2.5
mmol) and the reaction mixture was stirred for 3h (50
°C and 8h for b5).
Saturated sodium bicarbonate was added to neutralize and ethyl acetate was
added to extract. Organic layers were combined and then washed with brine,
dried with anhydrous Na2SO4, and concentrated under reduced pressure. The

residue was purified by silica gel column chromatography to obtain the product.
a5 (white powder, 70%) 1H NMR (400 MHz, CDCl3, 25 °C): δ 18.44 (s, 2H),
:
12.31 (s, 2H), 3.31 (s, 2H), 2.73 (s, 6H), 1.54 (s, 6H), 1.47 (s, 6H). 13C NMR (101
MHz, CDCl3, 25 °C): δ 203.3, 199.5, 187.7, 173.6, 110.8, 108.6, 44.6, 29.4, 25.4,
24.3, 18.1; IR (KBr) 
max: 2989, 2659, 1639, 1573, 1431, 1202, 1207 cm-1; (-)-
ESIMS m/z [M-H]-: 403; (-)-HRESIMS m/z [M-H]-: calcd for C21H24O8,
1
403.1398, found m/z: 403.1402. b5 (white powder, 61%): H NMR (400 MHz,
CDCl3, 25 °C): δ 10.16 (s, 2H), 9.75 (br s,2H), 3.72 (s, 2H), 1.73 (q, J = 1.73 Hz,
4H), 1.01 (t, J = 7.4 Hz, 6H); 13C NMR (101 MHz, CDCl3, 25 °C): δ 207.9, 193.3,
169.8, 168.7, 165.1, 105.0, 104.9, 103.6, 45.6, 17.9, 14.8, 14.0; IR (KBr) max
:
3269; 2963, 1633, 1472, 1186, 1020 cm-1; (-)-ESIMS m/z [M-H]-: 459; (-)-
HRESIMS m/z [M-H]-: calcd for C23H24O10, 459.129, found m/z: 459.1302.
Dryocrassin ABBA (49 %, see supporting information, Table S1 and Scheme
S2).
The preparation of b1: To a solution of a1 (30 g, 237 mmol) in BF3Et2O (90
mL, 713 mmol) was added butyryl chloride (24.7 mL, 240 mmol) at 0 °C. The
reaction mixture was stirred for 3d at room temperature, quenched with 10%
aqueous solution of AcONa (450 mL). The mixture was stirred for overnight at
room temperature and extracted with AcOEt (400 mL x 3). The combined
organic layer was washed with a saturated aqueous solution of NaHCO3 (400 mL
x 3), dried over Na2SO4, and filtered. The filtrate was concentrated under reduced
pressure and The residue was purified by silica gel column chromatography
(EtOAc-petroleum ether, 1:6) to give compound b1 (23 g, 49.3 %) as a white
1
solid. H NMR (400 MHz, DMSO, 25 °C): δ 12.23 (br s, 2H), 10.32 (br s, 1H),
5.79 (s, 2H), 2.49 (t, J = 7.28 Hz, 2H), (q, J = 7.34 Hz, 2H), (t, J = 7.38 Hz, 3H);
13C NMR (101 MHz, DMSO, 25 °C): δ 205.2, 164.5, 164.3, 103.8, 94.7, 17.8,
13.9. (-)-HRESIMS m/z [M-H]-: calcd for C10H12O4, 195.0663, found
m/z:195.0668.
The preparation of b2: To the solution of b1 (11g, 56 mmol) in ethyl acetate
(30 mL) were added dimethylformamide (6.54 ml, 84 mmol) and phosphoryl

chloride (7.7 ml, 84 mmol) at room temperature. The reaction mixture was
further stirred for 45 min. Water was added to the reaction mixture and extracted
with ethyl acetate. Ethyl acetate layer was washed with brine solution and dried
over Na2SO4 and concentrated to afford the crude product. Column
chromatography over silica gel using petroleum ether/EtOAc (6:1) as eluent
1
provided formyl butyryl phloroglucinols b2 (9 g, 71.6 %) as a white powder. H
NMR (400 MHz, (CD3)2SO, 25 °C): δ 9.98 (s, 1H), 5.88 (s, 1H), 2.99 (t, J = 7.12
Hz 2H), 1.60 (q, J = 7.28 Hz, 2H), 0.91 (t, J = 7.36 Hz, 3H); 13C NMR (101 MHz,
(CD3)2SO, 25 °C): δ 205.7, 191.8, 170.8, 169.6, 167.4, 103.9, 103.3, 94.5, 45.2,
17.2, 13.7. (-)-HRESIMS m/z [M-H]-: calcd for C11H12O5, 223.0612, found m/z:
223.0618.
The preparation of b3: To a stirred solution of b2 (1.28 g, 5.71 mmol) in 14 mL
of DMF was slowly added oven-dried potassium carbonate (6.20 g, 35.40 mmol)
and methyl iodide (3.55 ml, 57.09 mmol). After stirring at 80 °C for 8 h, the
resulting mixture was extracted with ethyl acetate. The organic layers were
combined, dried with Na2SO4, and evaporated. The residues mixture was purified
1
over silica to obtain compound b3 as a yellow oil in 78.9 % yield (1.2 g). H
NMR (400 MHz, CDCl3, 25 °C): δ 10.10 (s, 1H), 6.18 (s, 1H), 3.78 - 3.61(3s, 3H
13
each s), 2.53 (t, J = 6.74 Hz, 2H), 1.49 0.77 (t, J = 6.93 Hz, 3H); C NMR (101
MHz, CDCl3, 25 °C): δ 203.2, 186.8, 164.6, 162.0, 159.9, 119.1, 111.8, 90.9,
63.9, 56.0, 55.8, 46.6, 16.7, 13.3. (+)-HRESIMS m/z [M+Na]+: calcd for
C14H18O5, 289.1046, found m/z: 289.1053.
The preparation of b4: NaH (4.5 equiv. ) in dry DMF was slowly added while
stirring at 0 °C to a solution of b2 (1 equiv.) in dry DMF. The reaction mixture
was stirred for 15 min. MOMCl (4.5 equiv.) was slowly added over a period of
15 min so that the reaction temperature was maintain at less than 5 °C. The
mixture was stirred at room temperature for another 45 min and quenched by the
addition of ice water and then extracted with EtOAc. The combined the organic
layer and washed with water and brine and dried over Na2SO4. The organic layer
was concentrated under vacuum and the residues was purified by silica gel

column chromatography with n-hexane/EtOAc to obtain b4 as a colorless oil (60
1
- 70% yield). H NMR (400 MHz, CDCl3, 25 °C): δ 10.31(s, 1H), 6.77 (s, 1H),
5.27, 5.20 (3s, 2H each s), 3.46 (3s, 3H each s), 2.74 (t, J = 2.76 Hz, 2H), 1.69
(q, J = 1.69 Hz, 2H), 0.96 (t, J = 6.93 Hz, 3H); 13C NMR (101 MHz, CDCl3, 25
°C): δ 203.1, 187.7, 162.3, 159.5, 156.9, 113.9, 102.4, 97.5, 95.2, 94.6, 58.0,
56.9, 47.3, 17.1, 13.8. (+)-HRESIMS m/z [M+Na]+: calcd for C14H18O5,
379.1363, found m/z: 379.1372.
The preparation of e1: To a stirred solution of the aldehyde b3 (719 mg, 2.70
mmol), a4 (265.0 mg, 1.35 mmol) and Hantzsch ester (376.3 mg, 1.49 mmol)
were added 4.5 mL of DCM, and then the catalyst L-proline (31 mg, 0.27 mmol)
was added. The reaction mixture was stirred at room temperature for 9h. The
crude reaction mixture was directly loaded to a silica gel column with n-
hexane/EtOAc (3:1) to give the product e1 as a yellow solid in 58 % yield (350
mg). 1H NMR (400 MHz, CDCl3, 25 °C): δ 19.23 (s, 1H), 6.29 (s, 1H), 3.88-3.81
(3s, 2H each s ), 3.67 (s, 2H), (t, J = 7.23 Hz, 2H), 1.65 (q, J = 7.37 Hz, 2H), ,
0.94(t, J = 7.40 Hz, 3H); 13C NMR (101 MHz, CDCl3, 25 °C): δ 204.7, 200.3,
197.2, 176.1, 159.1, 156.7, 155.3, 113.1, 106.1, 106.0, 92.2, 64.5, 56.2, 56.0,
48.7, 47.1, 28.1, 24.9, 17.5, 14.7, 13.8. (-)-HRESIMS m/z [M-H]-: calcd for
C24H30O8, 445.1867, found m/z: 445.1941.
The preparation of e2: To a solution of e1 (50 mg, 111.98 mol) in dichloromethane
(746.5 l) was added boron tribromide (190.56 ml, 2.02 mmol) at -78 °C in an dry ice
bath. Remove dry ice bath and the solution was stirred at room temperature for 18h.
The reaction mixture was quenched at -78 °C with ice water and extracted with ethyl
acetate. The combined organic layers was washed brine, and dried with Na2SO4 and
concentrated under reduced pressure. The residue was purified by silica gel column
chromatography (petroleum ether/EtOAc = 7:1) to obtain e2 (32 mg, 50 %) as a light
yellow oil. 1H NMR (600 MHz, CDCl3, 25 °C): δ 11.38 (s, 1H), 9.94 (s, 1H), 5.94 (s,
1H), 3.53 (s, , 2H), 3.07 (s, 2H), 2.72-2.60 (s, 3H), 1.73 (q, J = 7.26, 2H), 1.49-1.39 (s,
6H), 0.99 (t, J = 7.33, 3H). 13C NMR (151 MHz, CDCl3, 25 °C): δ 206.9, 203.5, 198.8,
187.4, 171.9, 163.4, 162.5, 158.8, 111.1, 108.4, 103.7, 96.9, 45.5, 44.3, 29.3, 25.7, 24.7,

18.1, 16.6, 14.0. IR (KBr) max: 3192, 2964, 1636, 1197, 1026 cm-1; (-)-ESIMS m/z [M-
H]-: 403; (-)-HRESIMS m/z [M-H]-: calcd for C24H30O8, 403.1398, found m/z:
403.1410.
Acknowledgments
The work was supported by grants from the National Key Research and Development
Program of China (2017YFD0201402) and the Yunnan Provincial Science and
Technology Department (No. 2018HA001, the Academician Free Exploration Project).
The authors are grateful to the staffs of the analytical group at State Key Laboratory of
Phytochemistry and Plant Resources in West China, Kunming Institute of Botany,
Chinese Academy of Sciences, for measuring the spectral data.
Supplementary Material
Supporting data including retrosynthetic analysis, enzyme inhibition assay, docking
method, the preparation and spectra of compounds mentioned in this paper can be found
at http://..

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Graphical abstract for
Total Synthesis of Dryocrassin ABBA and Analogue Structures with
Potential Inhibitory Activity against Drug-resistant Neuraminidases

Highlights
Total synthesis of dryocrassin ABBA was achieved in nine steps with an over-all yield
of 4.6 %
Dryocrassin ABBA and analogue structures have beneficial effects on treating avian
influenza neuraminidases
The AB cycle is high active against Anhui N9;