An Efficient Regioselective Synthesis of 8-Formylhomoisoflavonoids with Neuroprotective Activity by Enhancing Autophagy

Article abstract of DOI:10.1021/acs.jnatprod.0c00830

6-Formylisoophiopogonone B (7a) and 8-formylophiopogonone B (7b), two natural products isolated fromOphiopogonjaponicus, represent a subgroup of rare 6/8-formyl/methyl-homoisoflavonoid skeletons. Herein we report an efficient method for the synthesis of these formyl/methyl-homoisoflavonoids. The synthesized compounds were evaluated for their neuroprotective effects on the MPP⁺-induced SH-SY5Y cell injury model and showed marked activity. Exploration of the neuroprotective mechanisms of compound7bled to an increased expression of autophagy marker LC3-II and down-regulation of autophagy substrate p62/SQSTM1. Molecular docking studies showed that7bmay prevent the inhibition of the classic PI3K-AKT-mTOR signaling pathway by interfering with the human HSP90AA1.

Full text of DOI:10.1021/acs.jnatprod.0c00830

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Note
An Efficient Regioselective Synthesis of 8‑Formylhomoisoflavonoids
with Neuroprotective Activity by Enhancing Autophagy
Jie Li, Fan Yang, Lin-Wei Zeng, Fang-Min Zhang, Chang-Xin Zhou, and Li-She Gan*
Cite This: J. Nat. Prod. 2021, 84, 1385−1391
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sı Supporting Information
ABSTRACT: 6-Formylisoophiopogonone B (7a) and 8-formylophiopogonone B (7b), two natural products isolated from
Ophiopogon japonicus, represent a subgroup of rare 6/8-formyl/methyl-homoisoflavonoid skeletons. Herein we report an efficient
method for the synthesis of these formyl/methyl-homoisoflavonoids. The synthesized compounds were evaluated for their
+
neuroprotective effects on the MPP -induced SH-SY5Y cell injury model and showed marked activity. Exploration of the
neuroprotective mechanisms of compound 7b led to an increased expression of autophagy marker LC3-II and down-regulation of
autophagy substrate p62/SQSTM1. Molecular docking studies showed that 7b may prevent the inhibition of the classic PI3K-AKT-
mTOR signaling pathway by interfering with the human HSP90AA1.
omoisoflavonoids are a special subclass of flavonoids that
derivatives with different substitutions on rings A and B were
H
contain an additional carbon between the B and C rings
also synthesized through one-carbon extension annulation
1
,2
22
of the isoflavonoid core (Figure 1). To date, more than 240
using DMF/MeSO Cl. Structure−activity relationship stud-
2
ies showed that the 6/8-methyl or formyl groups are crucial for
their bioactivities, especially in their myocardial- and neuro-
protective effects. With our ongoing interest in constructing
the homoisoflavonoid-related scaffold, a series of quinazoli-
none derivatives were synthesized by a four-step sequence with
23
adoption of the scaffold hopping principle. For the C-6/C-8
methyl- or formyl-homoisoflavonoids, a concise regioselective
strategy was developed with a key step to introduce the 8-
formyl group and to form ring C simultaneously. Herein, we
report the regioselective total synthesis of formyl-containing
homoisoflavonoids and derivatives in high yield. An in vitro
Figure 1. Isoflavonoid and homoisoflavonoid carbon skeletons.
naturally occurring homoisoflavonoids have been isolated from
several plant genera, such as Polygonatum, Ophiopogon,
Muscari, Eucomis, and Caesalpinia, in which many plant species
+
assay and a mechanism of action study on the MPP -induced
3
,4
have been applied in traditional herbal medicines. From a
structure diversity point of view, a subclass of rarely occurring
C-6/C-8 methylated homoisoflavonoids were subsequently
discovered, mostly from the genus Ophiopogon. These natural
compounds have attracted growing attention due to their
SH-SY5Y cell injury model indicated that some of them
showed promising neuroprotective activity via enhancing
autophagy.
As shown in Scheme 1, synthesis of 6-formylisoophiopogo-
none B (7a) and 8-formylophiopogonone B (7b) was first
attempted starting from the commercially available 2′,4′,6′-
trihydroxyacetophenone (1), which were protected using
benzyl chloride in the presence of K CO to afford diprotected
5
various biological activities, especially antitumor, anti-
6
7,8
9
inflammation, antioxidation, antipathogen, and cardiovas-
1
0
cular protection. Accordingly, significant efforts have been
2
3
devoted to the synthetic approach of these chromone
acetophenone 2 in good yield. The remaining hydroxy group
did not react due to the hydrogen bond with the adjacent
1
1−16
derivatives.
Among the synthesized homoisoflavonoids,
some were found to be potent and selective MAO-B or
1
7−19
cholinesterase inhibitors,
indicating potential neuro-
Received: July 26, 2020
Published: March 16, 2021
protective activity of these natural-product-like compounds.
In our prior investigations, we have reported the isolation of a
2
0
series of homoisoflavonoids from O. japonicus and P.
2
1
cyrtonema with antioxidative, cytotoxic, and myocardial-
protective activities. Subsequently, using a readily prepared
chalcone as a key intermediate, 20 homoisoflavonoid
©
2021 American Chemical Society and
American Society of Pharmacognosy
https://dx.doi.org/10.1021/acs.jnatprod.0c00830
1
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Note
a
Scheme 1. Synthesis of 6-Formylisoophiopogonone B and 8-Formylophiopogonone B
a
Reagents and conditions: (a) BnCl, K CO , DMF, 70 °C, 16 h, 62%; (b) p-anisaldehyde, NaH, DMF, 0 °C, 30 min, 95%; (c) 10% Pd/C, H , rt,
2
3
2
1
2 h, 96%; (d) POCl , DMF, rt, 5 h, 80%; (e) MeI, MeOH, KOH, 70 °C, 2 h, 42%; (f) MeSO Cl, DMF, BF ·OEt , 80 °C, 2 h, 24% yield for 7a
3
2
3
2
and 41% yield for 7b.
a
Scheme 2. Selective Synthesis of 8-Formylophiopogonone B
a
Reagents and conditions: (a) K CO , acetone, MOMCl, rt 3 h, 65%; (b) p-anisaldehyde, NaH, DMF, rt, 30 min, 90%; (c) 10% Pd/C, H , rt, 3 h,
2
3
2
9
8%; (d) POCl , DMF, rt, 5 h, 61%; (e) MeI, MeOH, KOH, 70 °C, 2 h, 42%; (f) Zn/Hg, HCl, CH OH, 62%.
3 3
carbonyl group. Next, chalcone 3 was assembled by an aldol
condensation of di-O-benzylacetophenone (2) and 4-methox-
ybenzaldehyde. The resulting chalcone was reduced with 10%
palladium/carbon, affording the corresponding deprotected
Finally, the target compound 8-formylophiopogonone B (7b)
was prepared by methylation with MeI and KOH. To facilitate
the structure−activity relationship analysis, the natural product
7b was reduced by a classical Clemmensen reduction to give
the dimethyl derivative 12 in 85% yield.
dihydrochalcone 4 in 98% yield. Using POCl and DMF, the
3
formyl moiety was introduced at C-3 (A ring) of the
dihydrochalcone 4 by a Vilsmeier−Haack reaction. Sub-
sequently, methylation of product 5 with MeI and KOH
afforded compound 6 in 50% yield. With the key intermediate
To investigate the effects of different substituents on the
activity, compounds 15a and 15b with C-6 and C-8 benzyl
groups were prepared via similar procedures to those in
Scheme 1. Under more vigorous reaction conditions, the
benzylation product 13 was formed in 48% yield, in which an
additional benzyl group was introduced on ring A compared to
compound 2. Having the benzylation product 13 in hand, we
proceeded to the synthesis of dihydrochalcone 14 using the
aforementioned condensation/reduction/formylation se-
quence. Not surprisingly, a mixture of homoisoflavonoids
15a and 15b was formed in the final cyclization step using
6
in hand, the final cyclization proceeded smoothly with
MeSO Cl in the presence of catalytic BF ·OEt , to furnish the
2
3
2
natural products 6-formylisoophiopogonone B (7a) and 8-
formylophiopogonone B (7b) in 24% and 41% yields,
respectively.
Considering the promising neuroprotective activity of 8-
formylophiopogonone B (7b), the regioselective synthesis of
this compound was attempted. With the same starting material,
the modified synthetic route began with a different phenolic
protecting group by replacing the benzyl group with the
methoxymethyl (MOM) group (Scheme 2, step a). The di-
MOM-protected acetophenone 8 was subjected to the same
aldol condensation/reduction sequence to afford the dihy-
drochalcone 10 in 90% yield, over two steps. Treatment of
DMF/MeSO Cl. These two products were separated by flash
2
chromatography on silica gel.
+
Using the MPP -injured human neuroblastoma cell line SH-
SY5Y as a model system, the synthesized homoisoflavonoid
derivatives were screened for their neuroprotective capacity. As
illustrated in Table 1, most of these compounds showed
neuroprotective capacity at 5 μM, and the natural product 8-
formylophiopogonone B (7b) showed the best protective
activity. The A-ring formyl group is crucial for these structures
to maintain their neuroprotective activity. Additionally, the
colorimetric MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium bromide] assay on the SH-SY5Y cell line was
conducted to test the potential cytotoxic effects of the active
compound 10 with excess POCl in the presence of DMF gave
3
24
the homoisoflavonoid 11 in 65% yield. This conversion is
highly efficient because the annulation, deprotection of the
MOM groups, and introduction of the formyl group occurred
in one step. A plausible mechanism for regioselective formation
of the C ring is proposed in the Supporting Information.
1
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Table 1. Evaluation of Neuroprotective Efficacy and
Cytotoxicity of the Synthesized Homoisoflavonoids
neuroprotective efficacy: cell
viability
cytotoxicity: inhibitory rate
MPP+
MPP + comp @ 5 μM
+
comp @ 50 μM
compd
7
7
1
1
1
1
a
b
1
3
5a
5b
55.13
53.24
55.86
52.72
49.57
48.62
61.33 ± 1.08
62.08 ± 1.21
60.45 ± 1.17
51.68 ± 2.33
52.32 ± 1.28
53.68 ± 1.49
11.25%
16.60%
8.21%
−1.97%
5.54%
10.40%
compounds. After incubation for 24 h, the compounds showed
no significant effect on cell viability at the high-dose level (50
μM).
Based on our previous findings that the homoisoflavonoids
22
may protect nerve cells by inducing autophagy, the effect of
-formylophiopogonone B (7b) on autophagy was further
studied. As summarized in Figure 2A, monodansylcadaverine
MDC) was applied to compound 7b-treated cells, which is a
8
(
probe for autophagic vacuoles. According to the results of flow
cytometry analysis, autophagy ratios were increased after SH-
SY5Y cells were treated with compound 7b. Quantitative
analysis of autophagy was further pursued, and it was found
that MDC positive ratios were notably increased after
treatment with compound 7b at high-dose level (50 μM).
Furthermore, an enhanced expression of autophagy marker
LC3-II and down-regulation of autophagy substrate p62/
SQSTM1 were also observed after treatment with compound
7
b (Figure 2B).
The autophagy-inducing effects of the test compounds were
further investigated by docking and reverse docking studies. As
summarized in Table 2, a pool of potential targets was
constructed with the help of the reverse docking procedure,
Swiss Target Prediction. Human HSP90AA1, a classic
autophagy biomarker, appeared frequently in the predicted
targets. Subsequently, the binding conformations of the most
active homoisoflavonoid, 7b, to HSP90AA1 was predicted by
molecular docking modeling utilizing the Libdock method. As
shown in Figure 3, 7b interacted with the active site of
HSP90AA1 (PDB: 6N8X) through a hydrogen bond with
LEU103 and THR184 amino acid residues. Overall,
compound 7b may rescue the inhibition of the classic PI3K-
AKT-mTOR signaling pathway on autophagy by interfering
with HSP90AA1.
Figure 2. 8-Formylophiopogonone B (7b) induces autophagy in SH-
SY5Y cells. (A) Flow cytometry analysis of MDC staining was used to
determine autophagy ratios after 7b treatment for concentrations of 0,
1
2.5, 25, and 50 μM. (B) Western blot analysis of the expression level
of p62 and LC3 after treatment with 7b for concentrations of 0, 12.5,
5, and 50 μM.
2
without further purification. Unless otherwise stated, reagents were
commercially available and used as purchased without further
purification. Chemicals were purchased from Sigma-Aldrich (Shang-
hai, China), TCI China (Shanghai, China), Acros (Beijing, China),
In conclusion, an efficient total synthesis of the rare 6/8-formyl
homoisoflavonoids, especially regioselective synthesis of 8-
formylophiopogonone B (7b), was accomplished in good yield.
The 8-formylhomoisoflavonoid 7b showed promising neuro-
1
Alfa Aesar (Shanghai, China), or J&K (Shanghai, China). H NMR
+
protective activity by inducing autophagy on the MPP -
13
and C NMR spectra were obtained using a Bruker AVANCE III 500
induced SH-SY5Y cell injury model. Increased expression of
autophagy marker LC3-II and down-regulation of autophagy
substrate p62/SQSTM1were observed in the mechanism of
action study. Further docking experiments revealed that these
neuroprotective homoisoflavonoids may involve autophagy
regulation via the PI3K-AKT-mTOR signaling pathway. Thus,
compound 7b represents a lead structure for developing new
drug candidates against neurodegenerative disorders.
MHz spectrometer (Bruker Co., Switzerland) with TMS as the
internal standard. The chemical shifts are expressed in δ values
(ppm), and the coupling constants (J) are reported in Hz. HRMS
data were obtained on an Agilent Q-TOF 1290 LC/6224 MS system
using ESI in positive or negative mode. The progress of reactions was
monitored by TLC on silica gel and visualized by short-wave
ultraviolet light. Flash chromatography was performed with Qingdao
Haiyang flash silica gel (200−300 mesh).
Synthesis of 7a, 7b, 11, 12, 15a, and 15b. The reaction
conditions for the synthesis of the above-mentioned compounds are
shown in Schemes 1−3. The intermediates were prepared as follows.
1-[2,4-Bis(benzyloxy)-6-hydroxyphenyl]ethan-1-one (2). To a
solution of ketone 1 (3.36 g, 20 mmol, 1 equiv) in DMF (28 mL)
EXPERIMENTAL SECTION
■
General Experimental Procedures. All anhydrous solvents were
purchased from Sigma-Aldrich (Shanghai, China) and directly used
1
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Note
Table 2. Selection of the Human Protein Targets Found by Swiss Target Prediction for Compounds 7a, 7b, 11, 15a, and 15b
compd
reverse docking procedure targets
7
a
HSP90AB1, HSP90AA1, HSP90B1, GCGR, NQO1, CNR1, PDE10A, GPR55, ALPL, CYP11B1, CYP11B2, CFTR, NOS2, EGLN1, DNASE1L3, CNR2,
PDK1, SIRT1, KIT, OPRD1, ABCB1, PCSK7, CYP2C9, SHBG
7
b
HSP90AB1, HSP90AA1, HSP90B1, GCGR, NQO1, CNR1, PDE10A, GPR55, ALPL, CYP11B1, CYP11B2, CFTR, NOS2, EGLN1, DNASE1L3, CNR2,
PDK1, SIRT1, KIT, OPRD1, CDK1, CDK4, IKBKB
1
1
1
5a
NQO1, CNR1, CNR2, GPR55, CYP11B1, CYP11B2, MAOB
HSP90AB1, HSP90AA1, HSP90B1, GCGR, NQO1, CNR1, CALCA, GPR55, MELK, CYP11B1, CYP11B2, CFTR, NOS2, ABCB1, CTNNB1, CNR2,
PDK1, PRKCG, PRKCD, PRKCA, PRKCB, PRKCE, PRKCH, AGTR1, BCL2L1, PLAU, CCR5, MPI, TNF, MAPK3, CDK6, GSK3B, F10
1
5b
HSP90AB1, HSP90AA1, CXCR2, NQO1, CXCR1, CNR1, CNR2, KCNMA1, GPR55, CYP11B1, CYP11B2
1
5
4
28.6, 128.4, 128.2, 127.7, 125.2, 114.1, 106.4, 95.1, 92.5, 71.4, 70.3,
+
5.4; HRESIMS m/z [M + H] 467.1836 (calcd for C H O ,
3
0
27
5
67.1853).
-(4-Methoxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one
4). Under a nitrogen atmosphere, to a stirred solution of chalcone 3
2.33 g, 5 mmol, 1 equiv) in THF (20 mL) was added Pd/C (0.15 g,
.4%) at room temperature. The reaction mixture was stirred at room
3
(
(
6
Figure 3. Compound 7b docked in the active site of HSP90AA1.
temperature under 1 atm H for 16 h. After filtering off the solids, the
2
solvent was removed under reduced pressure. The crude material was
purified by flash chromatography on silica gel (eluted with hexanes/
were added K CO (7 g, 50 mmol, 2.5 equiv) and benzyl chloride
2
3
(
4.5 mL, 40 mmol, 2 equiv) at 0 °C. After stirring at 70 °C for 12 h,
EtOAc = 4:1) to give the product (1.38 g, 96% yield) as a white solid:
the reaction mixture was diluted with water (30 mL) and the resulting
1
H NMR (500 MHz, DMSO-d ) δ 12.23 (2H, s), 10.35 (1H, s), 7.15
6
mixture was extracted with EtOAc (3 × 15 mL). The organic layer
(
1H, d, J = 8.6 Hz), 6.84 (d, J = 8.6 Hz, 1H), 5.80 (1H, s), 3.71 (1H,
was washed with water and brine, dried over anhydrous MgSO , and
4
13
s), 3.24 (2H, t, J = 7.7 Hz), 2.81 (2H, t, J = 7.7 Hz); C NMR (125
concentrated in vacuo. The crude material was purified by flash
MHz, CDCl ) δ 204.1, 164.6, 164.2, 157.4, 133.5, 129.2, 113.7, 103.7,
3
chromatography on silica gel (eluted with hexanes/EtOAc = 4:1) to
+
1
94.6, 54.9, 45.3, 29.3; HRESIMS m/z [M + H] 289.1065 (calcd for
give the product (4.31 g, 62% yield) as a white solid: H NMR (500
C H O , 289.1071).
MHz, CDCl ) δ 14.02 (1H, s), 7.44−7.31 (10H, m), 6.17 (1H, d, J =
16 17
5
3
2
,4,6-Trihydroxy-3-[3-(4-methoxyphenyl)propanoyl]-
2
(
.3 Hz), 6.10 (1H, d, J = 2.3 Hz), 5.06 (2H, s), 5.06 (2H, s), 2.56
3H, s); 1 C NMR (125 MHz, CDCl ) δ 203.2, 167.6, 165.1, 162.0,
3
benzaldehyde (5). Under a nitrogen atmosphere, to a stirred solution
of dihydrochalcone 4 (1.44 g, 5 mmol, 1 equiv) in EtOAc (20 mL)
3
1
9
35.9, 135.6, 128.8, 128.7, 128.5, 128.4, 128.0, 127.7, 106.4, 94.8,
2.4, 71.1, 70.3, 33.3; HRESIMS m/z [M + H] 349.1438 (calcd for
+
were added DMF (0.52 mL, 5.5 mmol, 1.1 equiv) and POCl (0.73
3
mL, 5.5 mmol, 1.1 equiv) at room temperature. After stirring for 3 h,
the reaction mixture was quenched with water (30 mL) and the
resulting mixture was extracted with EtOAc (3 × 15 mL). The organic
C H O , 349.1434).
22
21
4
(
E)-1-[2,4-Bis(benzyloxy)-6-hydroxyphenyl]-3-(4-methoxy-
phenyl)prop-2-en-1-one (3). To a stirred solution of 2 (3.48 g, 10
layer was washed with water and brine, dried over anhydrous Na SO ,
mmol, 1 equiv) in DMF (20 mL) were added NaH (0.36 g, 15 mmol,
2
4
1
.5 equiv) and anisaldehyde (1.36 g, 10 mmol, 1 equiv) at 0 °C. After
and concentrated in vacuo. The crude material was purified by flash
stirring for 30 min, the reaction mixture was quenched with water (20
chromatography on silica gel (eluted with hexanes/EtOAc = 7:1) to
1
mL), and the resulting mixture was extracted with EtOAc (10 mL ×
give the product (1.26 g, 80% yield) as a yellow solid: H NMR (500
3
). The organic layer was washed with water and brine, dried over
MHz, DMSO-d
6
) δ 14.83 (1H, s), 13.65 (1H, s), 12.24 (1H, s), 9.99
anhydrous MgSO , and concentrated in vacuo. The crude material
(1H, s), 7.16 (2H, d, J = 8.6 Hz), 6.84 (2H, d, J = 8.6 Hz), 5.90 (1H,
4
1
3
was purified by flash chromatography on silica gel (eluted with
s), 3.71 (3H, s), 3.29 (2H, t, J = 7.6 Hz), 2.83 (2H, t, J = 7.6 Hz); C
hexanes/EtOAc = 5:1) to give chalcone 3 (4.43 g, 95% yield) as a
NMR (125 MHz, DMSO-d ) δ 204.8, 191.9, 170.6, 169.6, 167.4,
6
1
yellow solid: H NMR (500 MHz, CDCl ) δ 14.72 (1H, d, J = 1.1
157.5, 133.0, 129.3, 113.8, 103.9, 103.3, 94.5, 55.0, 45.4, 28.8;
3
+
Hz), 7.80 (1H, d, J = 15.5 Hz), 7.71 (1H, d, J = 15.5 Hz), 7.54−7.49
HRESIMS m/z [M + H] 317.1015 (calcd for C H O , 317.1020).
1
7
17
6
(
=
(
2H, m), 7.48−7.32 (8H, m), 7.02 (2H, d, J = 8.8 Hz), 6.71 (2H, d, J
2,4,6-Trihydroxy-3-[3-(4-methoxyphenyl)propanoyl]-5-methyl-
benzaldehyde (6). Under a nitrogen atmosphere, to a stirred solution
of compound 5 (2.1 g, 6.64 mmol, 1 equiv) in MeOH (34 mL) were
added KOH (747 mg, 13.3 mmol, 2 equiv) and MeI (1.89 g, 13.3
8.8 Hz), 6.22 (1H, d, J = 2.4 Hz), 6.17 (1H, d, J = 2.4 Hz), 5.10
13
2H, s), 5.06 (2H, s); C NMR (125 MHz, CDCl ) δ 192.6, 168.8,
3
1
65.1, 161.7, 161.2, 142.8, 135.9, 135.6, 130.2, 129.0, 128.8, 128.8,
a
Scheme 3. Synthesis of Homoisoflavonoids 15a and 15b
a
Reagents and conditions: (a) BnCl, K CO , DMF, 90 °C, 24 h, 48%; (b) p-anisaldehyde, NaH, DMF, 0 °C, 30 min, 92%; (c) 10% Pd/C, H , rt,
2
3
2
1
2 h, 95%; (d) POCl , DMF, rt, 5 h, 75%; (e) MeSO Cl, DMF, BF ·Et O, 80 °C, 2 h.
3
2
3
2
1
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Note
mmol, 2 equiv). After stirring at 70 °C for 2 h, the reaction mixture
was quenched with water (30 mL), and the resulting mixture was
extracted with EtOAc (3 × 15 mL). The organic layer was washed
with water and brine, dried over anhydrous Na SO , and concentrated
5,7-Dihydroxy-3-(4-methoxybenzyl)-4-oxo-4H-chromene-8-car-
baldehyde (11). Under a nitrogen atmosphere, to a stirred solution of
10 (3.76 g, 10 mmol, 1 equiv) in EtOAc (50 mL) were added DMF
(2.08 mL, 22 mmol, 2.2 equiv) and POCl (2.92 mL, 22 mmol, 2.2
2
4
3
in vacuo. The crude material was purified by flash chromatography on
equiv) slowly at room temperature. After stirring at room temperature
for 5 h, the reaction mixture was quenched with water (50 mL), and
the resulting mixture was extracted with EtOAc (3 × 25 mL). The
organic layer was washed with water and brine, dried over anhydrous
silica gel (eluted with hexanes/EtOAc = 9:1) to give the product (921
1
mg, 42% yield) as a yellow solid: H NMR (500 MHz, DMSO-d ) δ
6
1
0.05 (1H, s), 7.17 (2H, d, J = 8.7 Hz), 6.84 (2H, d, J = 8.7 Hz), 3.71
Na SO , and concentrated in vacuo. The crude material was purified
(
3H, s), 3.35 (2H, t, J = 7.6 Hz), 2.85 (2H, t, J = 7.6 Hz), 1.94 (3H,
2
4
+
by flash chromatography on silica gel (eluted with hexanes/EtOAc =
s); HRESIMS m/z [M + H] 331.1159 (calcd for C H O ,
1
8
19
6
1
9
:1) to give the product (1.99 g, 61% yield) as a yellow solid: H
3
31.1176).
NMR (500 MHz, CDCl ) δ 13.55 (1H, s), 12.69 (1H, s), 10.19 (1H,
6-Formylisoophiopogonone B (7a). Under a nitrogen atmosphere,
3
s), 7.56 (1H, t, J = 1.2 Hz), 7.19 (2H, d, J = 8.6 Hz), 6.88 (2H, d, J =
to a stirred solution of 6 (1.44 g, 5 mmol, 1 equiv) in DMF (20 mL)
1
3
8
.6 Hz), 6.26 (1H, s), 3.79 (3H, s), 3.72 (2H, s); C NMR (125
were added BF ·Et O (0.69 mL, 5.5 mmol, 1.1 equiv) and MeSO Cl
3
2
2
MHz, CDCl ) δ 189.8, 180.9, 169.6, 168.6, 160.1, 158.7, 152.8, 130.1,
(
0.46 mL, 6 mmol, 1.2 equiv) at 0 °C. After stirring at 80 °C for 2 h,
3
1
+
29.0, 125.5, 114.4, 105.0, 103.0, 99.9, 55.4, 30.0; HRESIMS m/z [M
the reaction was diluted with water (30 mL) and the resulting mixture
was extracted with EtOAc (3 × 15 mL). The organic layer was
washed with water and brine, dried over anhydrous Na SO , and
+
H] 327.0843 (calcd for C H O , 327.0863).
18 15 6
5
,7-Dihydroxy-3-(4-methoxybenzyl)-6,8-dimethyl-4H-chromen-
2
4
4
-one (12). To a solution of 7b (100 mg, 0.3 mmol, 1 equiv) in
concentrated in vacuo. The crude material was purified by flash
MeOH (5 mL) was added 3 N HCl (3 mL, 3 mmol, 10 equiv). After
warming the mixture to 90 °C, amalgamated Zn was added slowly.
After stirring vigorously under reflux for 12 h, the reaction mixture
was filtered. The filtrate was concentrated under reduced pressure and
the crude material was purified by flash chromatography on silica gel
chromatography on silica gel (eluted with CH Cl /MeOH = 9:1) to
2
2
1
give the product (408 mg, 24% yield) as a yellow solid: H NMR (500
MHz, CDCl ) δ 14.04 (1H, s), 12.62 (1H, s), 10.34 (1H, s), 7.53
3
(
1H, s), 7.19 (2H, d, J = 8.6 Hz), 6.87 (2H, d, J = 8.6 Hz), 3.80 (3H,
1
3
s), 3.70 (2H, s), 2.10 (3H, s); C NMR (125 MHz, CDCl ) δ 192.8,
3
(
eluted with hexanes/EtOAc = 7:1) to give the product (60 mg, 62%
1
1
2
3
82.3, 165.9, 165.2, 159.8, 158.6, 153.8, 130.1, 129.4, 123.6, 114.3,
1
yield) as a white solid: H NMR (500 MHz, CDCl ) δ 12.94 (s, 1H),
3
06.2, 104.1, 103.0, 55.4, 30.0, 6.5 (for detailed assignments, see ref
+
7.52 (1H, s), 7.19 (2H, t, J = 1.2 Hz), 6.86 (2H, dd, J = 9.1, 2.5 Hz),
0); HRESIMS m/z [M + H] 341.1013 (calcd for C H O ,
1
9
17
6
3
.79 (3H, s), 3.72 (2H, s), 2.18 (3H, s), 2.16 (3H, s). The NMR
41.1020). The 8-formylophiopogonone B (7b) isomer was
23
1
spectral data match the previously published data.
simultaneously obtained in 42% yield: H NMR (500 MHz,
1
-[3-Benzyl-4,6-bis(benzyloxy)-2-hydroxyphenyl]ethanone (13).
CDCl ) δ 13.78 (1H, s), 12.97 (1H, s), 10.21 (1H, s), 7.54 (1H,
3
To a solution of ketone 1 (1.18 g, 10 mmol, 1 equiv) in DMF (15
s), 7.19 (2H, d, J = 8.6 Hz), 6.88 (2H, d, J = 8.6 Hz), 3.80 (3H, s),
mL) were added K CO (3.5 g, 25 mmol, 2.5 equiv) and benzyl
1
3
2
3
3
1
1
2
3
.73 (2H, s), 2.09 (3H, s); C NMR (125 MHz, CDCl ) δ 189.8,
3
chloride (2.3 mL, 20 mmol, 2 equiv) at 0 °C. After stirring at 90 °C
for 24 h, the reaction mixture was diluted with water (30 mL) and the
resulting mixture was extracted with EtOAc (3 × 15 mL). The organic
layer was washed with water and brine, dried over anhydrous MgSO4,
and concentrated in vacuo. The crude material was purified by flash
chromatography on silica gel (eluted with hexanes/EtOAc = 5:1) to
81.0, 167.5, 165.9, 158.7, 158.4, 152.7, 130.2, 129.3, 125.3, 114.4,
08.5, 104.5, 102.5, 55.4, 30.1, 6.5 (for detailed assignments, see ref
0); HRESIMS m/z [M + H] 341.1013 (calcd for C H O ,
+
1
9
17
6
41.1020).
1-[2-Hydroxy-4,6-bis(methoxymethoxy)phenyl]ethan-1-one (8).
Under a nitrogen atmosphere, to a stirred solution of ketone 1 (1
1
give the product (2.1 g, 48% yield) as a white solid: H NMR (500
g, 5.95 mmol, 1 equiv) in acetone (20 mL) were added K CO (5 g,
2
3
MHz, DMSO-d ) δ 10.06 (1H, s), 7.23 (2H, t, J = 7.5 Hz), 7.15 (5H,
6
3
6 mmol, 6 equiv) and MOMCl (1.08 mL, 14.28 mmol, 2.4 mmol)
dd, J = 15.7, 7.2 Hz), 6.83 (2H, d, J = 8.5 Hz), 3.89 (2H, s), 3.70 (3H,
slowly. After stirring at room temperature for 3 h, the reaction mixture
was diluted with water (30 mL), and the resulting mixture was
extracted with EtOAc (3 × 15 mL). The organic layer was washed
with water and brine, dried over anhydrous Na SO , and concentrated
13
s), 3.34 (2H, t, J = 7.6 Hz), 2.84 (2H, t, J = 7.6 Hz); C NMR (125
MHz, DMSO-d ) δ 205.3, 192.9, 170.1, 167.8, 164.9, 157.5, 140.2,
6
1
33.0, 129.3, 128.2, 128.0, 125.7, 113.8, 106.6, 104.5, 103.5, 55.0,
+
2
4
45.7, 28.9, 26.8; HRESIMS m/z [M + H] 439.1911 (calcd for
in vacuo. The crude material was purified by flash chromatography on
C H O , 439.1904).
2
9
27
4
silica gel (eluted with hexanes/EtOAc = 10:1) to give the product
8
-Benzyl-5,7-dihydroxy-3-(4-methoxybenzyl)-4-oxo-4H-chro-
1
(
990 mg, 65% yield) as a yellow oil: H NMR (500 MHz, CDCl ) δ
3
mene-6-carbaldehyde (15a). Compound 15a was prepared accord-
1
3.70 (1H, s), 6.23−6.21 (2H, m), 5.23 (2H, s), 5.14 (2H, s), 3.50
1
ing to the procedure described for 7a in 31% yield: H NMR (500
1
3
(
3H, s), 3.44 (3H, s), 2.63 (3H, s); C NMR (125 MHz, CDCl ) δ
3
MHz, CDCl ) δ 14.18 (1H, s), 12.79 (1H, s), 10.39 (1H, s), 7.53
3
2
03.3, 166.9, 163.5, 160.4, 106.9, 97.1, 94.5, 94.0, 56.8, 56.5, 33.0;
(
1H, s), 7.30−7.26 (4H, m), 7.21 (2H, d, J = 8.6 Hz),− 7.20 (1H, m),
+
13
HRESIMS m/z [M + H] 257.1003 (calcd for C H O , 257.1020).
1
2
17
6
6.92 (2H, d, J = 8.6 Hz), 4.04 (2H, s), 3.84 (3H, s), 3.73 (2H, s); C
(
E)-1-[2-Hydroxy-4,6-bis(methoxymethoxy)phenyl]-3-(4-
NMR (125 MHz, CDCl ) δ 192.8, 182.2, 166.5, 165.2, 159.8, 158.6,
3
methoxyphenyl)prop-2-en-1-one (9). Compound 9 was prepared
1
1
53.8, 139.7, 130.2, 129.2, 128.5, 128.5, 126.3, 123.8, 114.3, 106.5,
1
according to the procedure described for 3 in 90% yield: H NMR
06.3, 104.1, 55.4, 30.0, 27.3 (for detailed assignments, see the
(
(
500 MHz, CDCl ) δ 13.93 (1H, s), 7.81 (1H, d, J = 15.6 Hz), 7.80
1H, d, J = 15.6 Hz), 7.56 (2H, d, J = 8.8 Hz), 6.93 (2H, d, J = 8.8
+
3
Supporting Information); HRESIMS m/z [M + H] 417.1316 (calcd
for C H O , 417.1333).
2
5
21
6
Hz), 6.32 (1H, d, J = 2.3 Hz), 6.24 (1H, d, J = 2.3 Hz), 5.29 (2H, s),
6
-Benzyl-5,7-dihydroxy-3-(4-methoxybenzyl)-4-oxo-4H-chro-
1
3
5
.18 (2H, s), 3.85 (3H, s), 3.54 (3H, s), 3.48 (3H, s); C NMR (125
mene-8-carbaldehyde (15b). Compound 15b was attained in the
1
MHz, CDCl ) δ 193.0, 167.4, 163.4, 161.6, 160.0, 142.8, 130.2, 128.3,
same steps as for the synthesis of 15a in 43% yield: H NMR (500
3
1
25.1, 114.6, 107.7, 97.7, 95.3, 94.9, 94.2, 57.0, 56.6, 55.5; HRESIMS
MHz, CDCl ) δ 14.02 (1H, s), 13.17 (1H, s), 10.30 (1H, s), 7.63
3
+
m/z [M + H] 375.1421 (calcd for C H O , 375.1438).
(1H, t, J = 1.1 Hz), 7.47 (2H, d, J = 7.2 Hz), 7. Twenty-seven (2H,
m), 7.21 (2H, d, J = 8.6 Hz), 7.19 (1H, m), 6.97 (2H, d, J = 8.6 Hz),
2
0
23
7
1
-[2-Hydroxy-4,6-bis(methoxymethoxy)phenyl]-3-(4-methoxy-
1
3
phenyl)propan-1-one (10). Compound 10 was prepared according to
4
.08 (2H, s), 3.90 (3H, s), 3.82 (2H, s); C NMR (125 MHz,
1
the procedure described for 4 in 98% yield: H NMR (500 MHz,
CDCl ) δ 189.9, 181.0, 167.3, 165.9, 158.7, 158.7, 152.7, 140.1, 130.1,
3
CDCl ) δ 13.71 (1H, s), 7.15 (2H, d, J = 8.6 Hz), 6.84 (2H, d, J = 8.6
129.2, 128.9, 128.4, 126.2, 125.3, 114.4, 112.1, 104.7, 102.6, 55.4,
30.1, 27.2 (for detailed assignments, see Supporting Information);
3
Hz), 6.28 (1H, d, J = 2.3 Hz), 6.25 (1H, d, J = 2.3 Hz), 5.23 (2H, s),
+
5
.17 (2H, s), 3.79 (3H, s), 3.47 (6H, d, J = 1.3 Hz), 3.35−3.32 (2H,
HRESIMS m/z [M + H] 417.1316 (calcd for C H O , 417.1333).
2
5
21
6
+
m), 2.98−2.95 (2H, m); HRESIMS m/z [M + H] 377.1599 (calcd
for C H O , 377.1595).
Experimental Procedure for Biological Screening. Cell
Culture. The human neuroblastoma cell line (SH-SY5Y) was
20
25
7
1
389
https://dx.doi.org/10.1021/acs.jnatprod.0c00830
J. Nat. Prod. 2021, 84, 1385−1391

Journal of Natural Products
pubs.acs.org/jnp
Note
purchased from American Type Culture Collection (Manassas, VA,
USA). SH-SY5Y cells were maintained in DMEM supplemented with
Authors
Jie Li − Department of Pharmacy, School of Medicine,
Zhejiang University City College, Hangzhou 310015,
1
0% heat-inactivated FBS and 1% penicillin/streptomycin at 37 °C in
+
a humidified atmosphere of 5% CO and 95% air. MPP was dissolved
2
People’s Republic of China; orcid.org/0000-0002-4726-
838
in 10 mM DMEM stock solution right before use and diluted to the
appropriate concentration in treatment medium. SH-SY5Y cells were
9
6
Fan Yang − Department of Pharmacy, School of Medicine,
Zhejiang University City College, Hangzhou 310015,
People’s Republic of China
seeded at a density of 1.0 × 10 cells/mL and incubated with each
+
compound for 16 h prior to adding MPP to the medium. Cells were
+
treated with MPP at 50 μM and cultured for the last 8 h of each
compound treatment.
Lin-Wei Zeng − College of Pharmaceutical Sciences, Zhejiang
University, Hangzhou 310058, People’s Republic of China
Fang-Min Zhang − Department of Pharmacy, School of
Medicine, Zhejiang University City College, Hangzhou
In Vitro Cytotoxicity Assay. The cytotoxic effects of all compounds
were determined using the standard MTT assay. SH-SY5Y cells were
harvested from culture flasks, resuspended in cell culture medium, and
plated at a density of 1.0 × 10 cells/mL onto a 96-well culture plate.
Cells were incubated for 72 h (5% CO ). Activation solution (20 μL)
was added to the MTT reagent. The reaction solutions were added to
each well. The plate was incubated for 2 h and shaken gently to evenly
distribute the dye in the wells. Absorbance was measured at a
wavelength of 490 nm.
Autophagy Assay. The SH-SY5Y cells were treated with or
without 5 μM of each compound for 24 h and cultured with 0.05 mM
MDC at 37 °C for 30 min. The fluorescence intensity of cells was
analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ,
USA). Cells were transfected with GFP-LC3 plasmid using the
Lipofectamine 2000 reagent (Invitrogen) according to the manu-
facturer’s instructions. The fluorescence of GFP-LC3 was observed
under a fluorescence microscope. Electron microscopy analysis was
also performed to observe the autophagic vacuoles (Hitachi 7000,
Japan).
6
310015, People’s Republic of China; School of Biotechnology
2
and Health Sciences, Wuyi University, Jiangmen 529020,
People’s Republic of China; College of Pharmaceutical
Sciences, Zhejiang University, Hangzhou 310058, People’s
Republic of China
Chang-Xin Zhou − College of Pharmaceutical Sciences,
Zhejiang University, Hangzhou 310058, People’s Republic of
China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c00830
Notes
The authors declare no competing financial interest.
Monodansylcadaverine Staining Assays. Treated cells were
stained with MDC (0.05 mmol/L) for 15 min. The cells were
collected, washed with PBS three times, and resuspended. MDC
positive ratios were measured by a Beckman CytoFLEX FCM flow
cytometer.
Western Blot. Proteins were extracted with lysis buffer solution.
The lysates were centrifuged at 12000g for 10 min at 4 °C, the
supernatants were transferred to a new tube, and the protein
concentration was determined with a BCA kit (Beyotime, Jiangsu,
China). The solubilized protein was run on 12% SDS-polyacrylamide
gels and blotted electrophoretically onto a nitrocellulose membrane.
The membrane was sequentially incubated with a 5% skimmed milk
ACKNOWLEDGMENTS
■
We are grateful to the National Natural Science Foundation of
China (31670357, 81872756) and the Zhejiang Provincial
Natural Science Foundation of China (LY20C020003,
LR17H300001) for financial support of this research.
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*
sı Supporting Information
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(
(
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Additional information (PDF)
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AUTHOR INFORMATION
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Corresponding Author
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Li-She Gan − College of Pharmaceutical Sciences, Zhejiang
University, Hangzhou 310058, People’s Republic of China;
School of Biotechnology and Health Sciences, Wuyi
University, Jiangmen 529020, People’s Republic of China;
orcid.org/0000-0002-5688-5894; Phone: +86-750-
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299323; Email: ganlishe@163.com
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