Preparation, cox-2 inhibition and anticancer activity of sclerotiorin derivatives

Article abstract of DOI:10.3390/md19010012

The latest research has indicated that anti-tumor agents with COX-2 inhibitory activity may benefit their anti-tumor efficiency. A series of sclerotiorin derivatives have been synthesized and screened for their cytotoxic activity against human lung cancer cells A549, breast cancer cells MDA-MB-435 using the MTT method. Among them, compounds 3, 7, 12, 13, 15, 17 showed good cytotoxic activity with IC₅₀ values of 6.39, 9.20, 9.76, 7.75, 9.08, and 8.18 μM, respectively. In addition, all compounds were tested in vitro the COX-2 inhibitory activity. The results disclosed compounds 7, 13, 25 and sclerotiorin showed moderate to good COX-2 inhibition with the inhibitory ratios of 58.7%, 51.1%, 66.1% and 56.1%, respectively. Notably, compound 3 displayed a comparable inhibition ratio (70.6%) to the positive control indomethacin (78.9%). Furthermore, molecular docking was used to rationalize the potential of the sclerotiorin derivatives as COX2 inhibitory agents by predicting their binding energy, binding modes and optimal orientation at the active site of the COX-2. Additionally, the structure-activity relationships (SARS) have been addressed.

Full text of DOI:10.3390/md19010012

marine drugs
Article
Preparation, COX-2 Inhibition and Anticancer Activity of
Sclerotiorin Derivatives
Tao Chen 1, Yun Huang 2,3, Junxian Hong 1, Xikang Wei 1, Fang Zeng 1, Jialin Li 1, Geting Ye 1, Jie Yuan 2,* and
Yuhua Long 1,
*
1
2
School of Chemistry, Guangzhou Key Laboratory of Analytical Chemistry for Biomedicine,
South China Normal University, Guangzhou 510006, China; ct2020@m.scnu.edu.cn (T.C.);
hjx2020@m.scnu.edu.cn (J.H.); xk20192421139@m.scnu.edu.cn (X.W.); zengfang@m.scnu.edu.cn (F.Z.);
jialinli@m.scnu.edu.cn (J.L.); yegeting@m.scnu.edu.cn (G.Y.)
Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University,
Guangzhou 510080, China; huangyun11910173@i.smu.edu.cn
3
School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China
Correspondence: yuanjie@mail.sysu.edu.cn (J.Y.); longyh@scnu.edu.cn (Y.L.)
*
Abstract: The latest research has indicated that anti-tumor agents with COX-2 inhibitory activity
may benefit their anti-tumor efficiency. A series of sclerotiorin derivatives have been synthesized
and screened for their cytotoxic activity against human lung cancer cells A549, breast cancer cells
MDA-MB-435 using the MTT method. Among them, compounds
3,
7,
12, 13, 15, 17 showed good
cytotoxic activity with IC50 values of 6.39, 9.20, 9.76, 7.75, 9.08, and 8.18
µM, respectively. In addition,
all compounds were tested in vitro the COX-2 inhibitory activity. The results disclosed compounds
13 25 and sclerotiorin showed moderate to good COX-2 inhibition with the inhibitory ratios of 58.7%,
51.1%, 66.1% and 56.1%, respectively. Notably, compound displayed a comparable inhibition ratio
7,
,
3
(70.6%) to the positive control indomethacin (78.9%). Furthermore, molecular docking was used to
rationalize the potential of the sclerotiorin derivatives as COX2 inhibitory agents by predicting their
binding energy, binding modes and optimal orientation at the active site of the COX-2. Additionally,
the structure-activity relationships (SARS) have been addressed.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Chen, T.; Huang, Y.; Hong,
J.; Wei, X.; Zeng, F.; Li, J.; Ye, G.; Yuan,
J.; Long, Y. Preparation, COX-2
Inhibition and Anticancer Activity of
Sclerotiorin Derivatives. Mar. Drugs
2021, 19, 12. https://doi.org/
10.3390/md19010012
Keywords: sclerotiorin derivatives; cytotoxic activity; COX-2 inhibition; molecular docking
1. Introduction
Cancer has become one of the most important factors affecting human life and health
in terms of incidence, mortality, and prevalence. In 2018, estimates for global statistics on
cancer rates show that there were 18.1 million new cases and 9.6 million deaths. Lung
cancer is the most frequent cancer and the leading cause of cancer death among males,
followed by prostate and colorectal cancer. Among females, breast cancer is the most
Received: 3 December 2020
Accepted: 23 December 2020
Published: 29 December 2020
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
ms in published maps and institutio-
nal affiliations.
commonly diagnosed cancer and the leading cause of cancer death [
1]. Cancer metastasis
is the major reason of treatment failure and death [ ]. As the research moves along,
2,3
authoritative studies have shown that an inflammatory environment plays an important
role in various stages of tumor development and affects the body response to chemother-
apeutic agents [
inflammatory mediators are present in the tumor microenvironment, including cytokines,
growth factors, reactive oxygen species and reactive nitrogen species [ ]. These mediators
also activate signaling molecules involved in inflammation and carcinogenesis, including
nuclear transcription factor, inducible nitric oxide synthase and cyclooxygenase-2. All
these factors together result in tumor initiation by increasing cell cycling, inhibiting tumor
suppressor pathways and activating oncogenes [8–10].
4,5]. Chronic inflammation is associated with tumor development, and
Copyright: © 2020 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
6–8
COX-2 is a member of the cyclooxygenase family, and has long been a research focus
in the treatment of inflammation. It is the key enzyme in the conversion of arachidonic
Mar. Drugs 2021, 19, 12. https://doi.org/10.3390/md19010012
https://www.mdpi.com/journal/marinedrugs

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acid to prostanoids, lipid mediators’ participation in multifarious physiological and patho-
logical processes. COX-2 is known as an important enzyme in the inflammation process
that has tumorigenesis function, and can promote cancer cells proliferation, migration
and invasion [11
closely associated with malignant tumors in recent years [13
has been detected in lung cancer, colon cancer, stomach cancer, breast cancer and other
tumors [16 18]. In lung cancer, studies show that COX-2 was highly expressed in all stages
,12]. Zoological and epidemiological studies have shown that COX-2 is
–15]. COX-2 over expression
–
of NSCLS (non-small-cell lung cancer). Meanwhile, most of invasive and non-invasive
lesions have shown elevated COX-2 expression when compared with nonmalignant tis-
sue [19]. Concerning to colorectal cancer, studies have reported that COX-2 was over
expressed in 85% and 50% of human colorectal carcinomas and adenomas, respectively.
Immunohistochemical studies showed that COX-1 and trace COX-2 is expressed in normal
smooth muscle cell and intestinal mucosal epithelial cell. However, COX-2 expression
is dramatically increased in cancerous colorectal tissue compared with adjacent normal
mucosa [20]. Studies indicated that COX-2 inhibitors aspirin and celecoxib can abrogate
the stimulatory effects on lung cancer cell proliferation and migration by inhibition of
COX-2 [21]. Moreover, celecoxib has been proved to inhibit growth and induce apoptosis
of lung cancer cells [22]. Epidemiologic data demonstrated a role for aspirin in suppressing
prostate carcinogenesis and suggested that inhibition of COX-2 via pharmacological means
or regulation of its expression can limit the development of human prostate cancer [23].
Therefore, targeting the inhibition of COX-2 and its downstream pathways could be helpful
for cancer therapy.
Marine creatures are natural treasures and have played important roles as sources of
natural medicinal products [24
derived marine natural products (MNPs) exhibited great potential for biological activities
such as antifouling, antimalarial and anticancer, etc. [26 28]. Sclerotiorine was first isolated
,25]. Scientific researches showed that marine organisms
–
and identified from the mycelium of Penicillium multicolor G.M.P. in 1952 [29]. Its wide
broad bioactivity makes it bound to get much more attention. Sclerotiorine and other
azaphilones isolated from Penicillium sclerotiorum OUCMDZ-3839 displayed significant
inhibitory activity against α-glycosidase and moderate bioactivity against H1N1 virus [30].
A series of sclerotioramine derivatives were synthesized and showed potent antifouling
activity against the larval settlement of the barnacle Balanus amphitrite [31]. It is reported
that sclerotiorin could substantially decrease the mycobacterial growth inside macrophages
and with no cytotoxicity, suggesting that it has potential to supplement antibiotic therapy
for tuberculosis (TB) [32]. Recently, evidence showed that sclerotiorin can provide clues for
further searching on safer and effective entity against Alzheimer’s disease (AD) [33].
During our continuous search for biologically active marine natural compounds, we
found an endophytic fungus SCNU-0016 from marine mangrove plant Acanthus ilicifolius L.
could produce sclerotiorin in high productivity. Our primary research indicated sclerotiorin
possesses COX-2 inhibition activity. The literature retrieval showed azaphilones can be
antitumor agents [34]. In order to obtain dual targeted pharmaceutical compounds, since
COX-2 selective inhibitors can both be anti-inflammatory candidates and benefit antitumor
activity, we did the modification of sclerotiorin to afford the cytotoxic compounds with
COX-2 inhibitory activity. Herein, we report synthesis, antitumor activity and COX-2
inhibition of a series of sclerotiorin derivatives.
2. Results and Discussion
2.1. Chemistry
The parent compound sclerotiorin was extracted from the fungal Penicillium sclero-
tiorum SCNU-0016. The synthetic route of sclerotiorin derivatives
1–27 was shown in
Schemes 1 and 2. Twenty-four amide-derivatives ( 24) have been successfully synthesized
1–
by one-step reaction of sclerotiorin with various amines in high yields (up to 90%). The
derivative 25 was obtained by hydrolysis of sclerotiorin (yields up to 85%). Followed by
esterification reaction of propionic anhydride and glutaric anhydride produced 26 and 27

Mar. Drugs 2021, 19, 12
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in 82% and 78% yields, respectively. The resulting extract was subjected to silica gel column
chromatography to obtain the pure products. The structures of target compounds 1–27
were confirmed by extensive spectroscopic methods including 1H NMR, 13C NMR, MS
and HRMS.
Cl
Cl
O
O
a
N
O
R1
AcO
AcO
O
O
sclerotiorin
1-24
2. R1=
3: R1=
1: R1=
OH
6: R1=
5: R1=
8: R1=
O
4: R1=
OH
O
9: R1=
7: R1=
N
OH
12: R1=
15: R1=
18: R1=
21: R1=
11: R1=
14: R1=
17: R1=
20: R1=
23: R1=
10: R1=
13: R1=
16: R1=
O
S
S
O
N
19: R1=
22: R1=
N
N
H
24: R1=
Br
Scheme 1. Reagents and conditions: (a) amine, DCM, r.t.
◦
◦
Scheme 2. Reagents and conditions: (b) CH3ONa, CH3OH, 40 C. (c) Pyridine, anhydride, 90 C.

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2.2. Biological Studies
2.2.1. Cytotoxic Activity of Sclerotiorin Derivatives
The cytotoxic activity of all compounds was evaluated against A549 (human lung
cancer) and MDA-MB-435 (breast cancer cells) by using the MTT method as described
previously.
As shown in Table 1, the four yellow pigments, sclerotiorin and the other three acyl
changed derivatives 25
(IC50 > 50 µM). Compared with sclerotiorin, most of the amine modified sclerotiorin
derivatives except and displayed moderate to fine cytotoxic activities against the
MDA-MB-435 and A549 cell lines. Particularly noteworthy, compounds 12 13 15
and 17 showed excellent cytotoxic activities with IC50 values of 6.39, 9.20, 9.76, 7.75,
9.08 and 8.18 M, respectively. Further SAR analysis can give the following clues: (1) a
vinylogous -pyridone formed by the corresponding nitrogen atom substituted pyran
, 26, 27, present no cytotoxic activity on both cancer cell lines
1
2
3,
7,
,
,
µ
γ
nucleus of sclerotiorin increase the cytotoxicity; (2) a certain suitable bulky structure
can obtain high cytotoxic activity, which was disclosed by comparing the IC50 values of
compounds 1, 2, 24 to those of 7, 10, 12; (3) from the observation of compounds 13, 14,
15, 16, 17, we can conclude one methylene connected aryl side chain have good effect on
cytotoxic activity. In order to illustrate the underlying mechanisms for the cytotoxicity of
these compounds, several rational deduces are summed up. Firstly, the amine modified
sclerotiorin derivatives may be more efficiently served as a basic factor to change the acidic
tumor microenvironment. In this respect, we can explain why the nitrogen substituted
structures are more cytotoxic to tumor cells. Secondly, sclerotiorin derivatives possess
α,
β-unsaturated ketone skeleton and could be Michael receptor. Cytotoxic activity of these
compounds may be attributed to the Michael addition reactions of sclerotiorin derivatives
with active nucleophilic group in the biomolecules, such as amino acids, nucleic acids and
other compounds in the tumor cells to irreversibly affect the functions of the biomolecules
or regulate the cellular signal pathway. Thirdly, the literature addressing tumor cells always
showed some high expression of cellular factors, the sclerotiorin derivatives may exhibit
cytotoxicity by inhibiting some of them, such as the COX-2. Further COX-2 inhibitory
activity for these compounds was conducted as following.
Table 1. Cytotoxic activity of sclerotiorin derivatives.
MDA-MB-435
IC50 (µM)
A549
IC50 (µM)
MDA-MB-435
IC50 (µM)
A549
IC50 (µM)
Compd.
Compd.
1
3
>50
6.39
40.29
22.75
20.27
21.47
40.35
25.46
14.08
22.70
14.15
25.29
28.18
23.75
>50
2
>50
35.49
44.27
23.52
11.76
9.76
>50
>50
4
5
20.93
9.20
6
>50
7
8
21.16
20.19
25.98
21.07
15.06
32.05
23.35
16.53
21.46
>50
9
38.74
11.13
7.75
10
11
13
15
17
19
21
23
25
27
EPI
12
14
15.66
21.29
26.21
15.71
16.26
42.92
>50
9.08
16
8.18
18
16.34
22.42
10.23
>50
20
22
24
26
>50
>50
Sclerotiorin
>50
>50
0.15
0.37

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2.2.2. In Vitro Primary Screen COX-2 Inhibitory Activity
In recent years, COX-2 inhibitors become a new target and hotspot for anticancer
drug research and get a lot of attention. For further investigations the biological activity of
semi-synthetic analogs of sclerotiorin, all compounds were primarily screened the COX-2
inhibitory activity at a concentration of 20 µM in vitro.
As shown in Figure 1, most of semi-synthetic derivatives and parent compound
sclerotiorin displayed good inhibitory activity for COX-2. Among all the derivatives,
compound
3
displayed perfect COX-2 inhibition with a ratio of 70.6%, which is comparable
13 15 17 25
to the positive control indomethacin (78.9%). Furthermore, compounds
7,
,
,
,
and sclerotiorin showed moderate COX-2 inhibitory ratio of 58.7%, 51.1%, 46.7%, 47.3%,
66.1% and 56.1%, respectively. More interestingly, compared to the parent compound
sclerotiorin, the inhibitory activity of the esterlysis derivative 25 increased from 56.1% to
66.1%. However, 26 and 27, the esterification product of 25, displayed low COX-2 inhibitory
ratio of 42.8%, 35.1%, respectively.
Figure 1. Inhibition ratio of compounds 1–27 to COX-2 in vitro.
Based on the above results, observation was that the COX-2 inhibitory activity of
sclerotiorin derivatives were associated with the cytotoxic activity. Overall, the cytotoxic
activity of the compounds were positively correlated with the inhibitory activity of COX-2.
Out of 28 tested compounds,
cancer cell lines and potential COX-2 inhibitory activity. Particularly, compound
found to be most potent derivative with high cytotoxic activity against the breast cancer
cells and superior COX-2 inhibitory activity. Otherwise, compounds 25 26 27 and parent
3
,
7,
13,
15 and 17 showed both cytotoxic activity against two
3
was
,
,
compound sclerotiorin were exhibiting good COX-2 inhibitory activity, but almost have
no cytotoxic activity. This find suggests sclerotiorin and its esterlysis product 25 deserve
intensive investigation on anti-inflammatory activity based on COX-2 inhibition selectivity.
2.2.3. Molecular Docking Studies
The COX-2 inhibitory effects of compound
3 led us to perform molecular docking
studies to insight understand the ligand-protein interactions in detail, and dock simulations
in COX-2 (PDB ID: 5GMN) [35] were carried out in AutoDock4.2.6 [36]. Docking procedure
was validated by docking of 949 (the co-crystallized ligand of COX-2 protein) in the active
site of COX-2 and root-mean-square deviation (RMSD) of 0.13 Å to the X-ray structure
(Figure 2).

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Figure 2. Comparison between the docked pose of 949 (yellow) as produced by docking experiment
and the co-crystallized ligand of this inhibitor within COX-2 (green).
Compound
8.24 kcal/mol was obtained. One hydrogen bonding interaction between the ester group
of compound with Gln92 (2.96 Å) of COX-2 active site was observed (Figure 3). It is
clearly visible in Figure 4 that arachidonic acid and compound were located deeply inside
3 was docked into the active site of COX-2 and the interaction energy of
3
3
the same pocket of COX-2. Three hydrogen bonding interactions between carboxyl of
arachidonic acid with Thr198 (2.92 Å, 2.95 Å) and Thr199 (2.85 Å) of COX-2 active site
were observed respectively (Figure 5). The hydrophilic groups of compound 3 including
carbonyl and ester group were inclined to via a hydrogen bonding interactions with the
hydrophilic amino acid of the outside of the COX-2 protein active pocket. However, alkyl
chain and cyclohexene of compound
3 is easier access to the interior of hydrophobic pocket
by means of hydrophobic action, which is blocking the binding site of arachidonic acid in
COX-2 enzyme to some extent. This may be the reason why the compound
perfect inhibitory activity to COX-2 cyclooxygenase (Figure 5).
3 showed a
Figure 3. Compound 3 (cyan) docked into the active site of COX-2.
Compound 25 was docked into the active site of COX-2 and the interaction energy of
7.66 kcal/mol was obtained. Unexpectedly, it displayed no H-bonding interaction with
the amino acid residue of COX-2 protein. Compound 26 was docked into the active site of
COX-2 and the interaction energy of 6.26 kcal/mol was obtained. Three hydrogen bonding
interactions between ester group of 26 with His4 (3.40 Å) and Trp5 (2.53 Å, 2.74 Å) of COX-2
active site was observed. One hydrogen bonding interaction between carbonyl group of
26 with Lys169 (3.15 Å) of COX-2 active site was observed. Additionally, Compound 26
showed H-bond interactions of 3.31 Å between its oxygen atom in the ring and Glu238
residues of the COX-2 active site (Figure 6A). However, we found that compound 26 did
not located deeply inside the hydrophobic pocket of COX-2 protein as compound 25 did,

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but docked on the surface of the protein by hydrogen bonding (Figure 6B). Consequently,
we deduce that the presence of ester group can resulted in lower activity of compound 26
.
Figure 4. Two-dimensional (2D) diagram of the binding pose of
3 and arachidonic acid in the active
site of COX-2.
Figure 5. Model showing the placement of 3 (cyan) and arachidonic acid (yellow) in the active site
pocket of COX-2.
Figure 6.
(A
) 3D diagram of the binding model of 25 (brown) and 26 (yellow) in the active site of
COX-2. (
COX-2.
B) Model showing the placement of 25 (brown) and 26 (yellow) in the active site pocket of

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3. Materials and Methods
3.1. Chemistry
3.1.1. General Information
Proton and carbon NMR spectra were recorded on Bruker AVANCE NEO 600 MHz
spectrometer (Bruker BioSpin, Switzerland). The spectra obtained in CDCl3 were referenced
to the residual solvent peak. Chemical shifts (d) are reported in parts per million (ppm)
relative to residual undeuterated solvent as an internal reference. HR-ESI-MS data were
measured on a Thermo Fisher Scientific Q-TOF mass spectrometer (Thermo Fisher Scientific,
Waltham, MA, USA). Silica gel (100–200 and 200–300 mesh) (Qingdao Haiyang Chemical
Co., Ltd., Qingdao, China) was used for column chromatography. TLC silica gel GF254
plates (Yantai Zi Fu Chemical Co., Ltd., Yantai, China) and TLC silica gel 60 F254 (MERCK
CHEMICALS (SHANGHAI) CO, LTD) were used for thin layer chromatography.
3.1.2. Fungal Materials.
The fungus SCNU-0016 used in the study was isolated from fresh fruit of the mangrove
plant Acanthus ilicifolius L, which was collected in October 2019 from Hailing island Man-
grove Nature Reserve in Guangdong province, China. The fungal isolation was conducted
as following. Initially, the plant fruit was washed with sterile water and surface-sterilized
in a 100 mL beaker with 75% ethanol for 1 min. This was followed by dipping the sam-
ple into 5% sodium hypochlorite for 1 min, then the plant parts were rinsed with sterile
water and cut into 3 mm sections and plated on potato dextrose agar (PDA, potatoes
300 mg/mL, dextrose 20 mg/mL, agar 15 mg/mL, chloramphenicol 1 mg/mL) with peni-
cillin (100 units/mL) and streptomycin (0.8 mg/ mL). The plates were incubated at 25
±
1 ◦C. The endophytic fungal strains were isolated by routine microbiological. The fungal
◦
isolates were numbered and stored at 4 C in triplicate on PDA slants. Fungal identification
was carried out using a molecular biological protocol by DNA amplication and sequencing
of the ITS region [37]. The sequence data of the fungal strain have been deposited at Gen
Bank with accession no. MW-309502. A BLAST search result showed that the sequence was
the most similar (100%) to the sequence of P. sclerotiorum. A voucher strain was deposited in
School of Chemistry, South China Normal University, Guangzhou, China, with the access
code SCNU-F0016.
3.1.3. Fungal Culture and Sclerotiorin Extraction
The fungal strain P. sclerotiorum SCNU-0016 was cultured on autoclaved potato liquid-
substrate media (one-hundred Erlenmeyer flasks (1000 mL); each containing 400 mL of
potato liquid medium, 8 g of glucose and 1.2 g artificial sea salts) at room temperature
under static conditions and daylight for 28 days. Following incubation, pancake thallus
grew on top of the potato liquid media. Then the air dried fungal were soaked in the solvent
MeOH/CH2Cl2 (v:v, 1:1). Extracts were filtered and concentrated under reduced pressure
to yield 820 g of residue. The residue was fractionated by column chromatography on silica
gel by eluting with a gradient of EtOAc/petroleum ether from 1:10 to 1:1 give five fractions
(Fr.1-Fr.5). sclerotiorin was extracted successfully by elution with EtOAc/petroleum ether
(v:v, 1:10). sclerotiorin appear good solubility in the EtOAc/petroleum ether solution. Then
removal of the solvent afforded 7.82 g of yellow powder.
3.1.4. General Procedure for Synthesis of Sclerotioramine Derivatives 1–24
A mixture of sclerotiorin (39 mg, 0.10 mmol) and varied amines (0.12 mmol) in
anhydrous dichloromethane (2 mL) was stirred at room temperature until the sclerotiorin
was disappeared. The resulting mixture was extracted with EtOAc (3
×
5 mL) and the
organic phase was washed with saturated brine (3 15 mL). Then the organic phase
×
was dried with MgSO4 and concentrated in vacuo. The crude product was purified by
column chromatography with eluent of EA/PE (5:1, v/v) to give the pure compound.
All the synthesized compounds were identified by MS and NMR spectra, please see
Figures S1–S79 in the supplementary materials.

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Compound 1
(R)-5-chloro-3-((S, 1E, 3E)-3,5-dimethylhepta-1, 3-dien-1-yl)-7-methyl-6, 8-dioxo-2-
propyl-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of 90%; m.p.
230.1–232.2 ◦C. [α]D25 +13.2 (c 0.02, MeOH); UV (MeOH)
λmax (log ε): 234 (3.13), 376 (3.03),
488 (1.68) nm; IR (KBr) νmax: 2956, 2922, 1743, 1708, 1590, 1491, 1368, 1251, 1190, 1092,
1
853 cm−1. H NMR (600 MHz, CDCl3) δH 7.75 (s, 1H), 7.02 (s, 1H), 6.96(d, J = 15.4 Hz, 1H),
6.12 (d, J = 15.4 Hz, 1H), 5.70 (d, J = 9.7 Hz, 1H), 3.89–3.72 (m, 2H), 2.59–2.34 (m, 1H), 2.16
(s, 3H), 1.85 (s, 3H), 1.80 (dd, J = 14.9, 7.4 Hz, 2H), 1.55 (s, 3H), 1.48–1.40 (m, 1H), 1.39–1.31
(m, 1H), 1.01 (dd, J = 15.3 7.2 Hz, 6H), 0.88 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CDCl3)
δC 193.9, 184.2, 170.1, 148.2, 147.8, 145.0, 144.6, 141.1, 131.6, 114.6, 114.5, 111.7, 102.2, 84.8,
55.8, 35.1, 30.0, 23.6, 23.2, 20.3, 20.2, 12.6, 12.0, 10.9. LRMS (EI) m/z 432 [M]+.
Compound 2
(R)-2-butyl-5-chloro-3-((S, 1E, 3E)-3,5-dimethylhepta-1, 3-dien-1-yl)-7-methyl-6, 8-
dioxo-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of 92%; m.p.
216.3–218.5 ◦C
[α]2D5 +13.6 (c 0.02, MeOH); UV (MeOH)
. λmax (log ε): 238 (3.10), 378
(3.09), 485 (1.78) nm; IR (KBr) νmax: 2953, 2921, 1743, 1708, 1590, 1493, 1368, 1252, 1190,
1
1092, 851 cm−1. H NMR (600 MHz, CDCl3) δH 7.74 (s, 1H), 7.02 (s, 1H), 6.96 (d, J = 15.4
Hz, 1H), 6.13 (d, J = 15.4 Hz, 1H), 5.70 (d, J = 9.7 Hz, 1H), 3.94–3.76 (m, 2H), 2.59–2.39 (m,
1H), 2.16 (s, 3H), 1.84 (s, 3H) 1.74 (s, 2H), 1.54 (s, 3H), 1.48–1.31 (m, 4H), 1.02 (d, J = 6.6 Hz,
3H), 0.98 (t, J = 7.4 Hz, 3H), 0.88 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δC 194.1,
184.3, 170.2, 148.3, 147.9, 145.1, 144.7, 141.2, 131.7, 114.8, 114.6, 111.8, 102.3, 84.9, 54.2, 35.2,
32.3, 30.2, 23.4, 20.4, 20.4, 19.8, 13.7, 12.7, 12.1. LRMS (EI) m/z 446 [M]+.
Compound 3
(R)-5-chloro-2-(2-(cyclohex-1-en-1-yl)ethyl)-3-((S, 1E, 3E)-3,5-dimethylhepta-1, 3-dien-
1-yl)-7-methyl-6, 8-dioxo-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield
of 92%; m.p. 180.6–182.8 ◦C. [α]2D5 +14.2 (c 0.02, MeOH); UV (MeOH)
λmax (log ε): 235
(3.11), 382 (3.28), 491 (1.66) nm; IR (KBr) νmax: 2965, 2920, 1743, 1703, 1600, 1491, 1368, 1288,
1
1248, 1180, 1089, 953 cm−1. H NMR (600 MHz, CDCl3) δH 7.64 (s, 1H), 7.01 (s, 1H), 6.96 (d,
J = 15.4 Hz, 1H), 6.16 (d, J = 15.4 Hz, 1H), 5.70 (d, J = 9.7 Hz, 1H), 5.41 (s, 1H), 4.05–3.74 (m,
2H), 2.56–2.38 (m, 1H), 2.31 (t, J = 7.1 Hz, 2H), 2.16 (s, 3H), 2.00–1.87 (m, 4H), 1.85 (s, 3H),
1.63 (dd, J = 10.1, 4.1 Hz, 2H), 1.56–1.48 (m, 5H), 1.47–1.40 (m, 1H), 1.35 (dd, J = 14.3, 6.9,
1H), 1.02 (d, J = 6.6 Hz, 3H), 0.88 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δC 194.0,
184.3, 170.1, 148.2, 147.8, 145.1, 144.7, 141.5, 131.9, 131.7, 127.0, 114.7, 114.4, 111.7, 102.1,
84.9, 53.1, 38.6, 35.2, 30.2, 28.4, 25.4, 23.4, 22.7, 22.0, 20.4, 20.4, 12.7, 12.1. HRMS (ESI) for [M
+ H]+: calcd for C29H37ClNO4: 498.23332. Found: 498.24115.
Compound 4
(7R)-5-chloro-2-(2, 3-dihydroxypropyl)-3-((S, 1E, 3E)-3,5-dimethylhepta-1, 3-dien-1-
yl)-7-methyl-6, 8-dioxo-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield
of 88%; m.p. 152.8–154.3 ◦C. [α]2D5 +14.5 (c 0.02, MeOH); UV (MeOH)
λmax (log ε): 236
(3.09), 379 (3.10), 488 (1.64) nm; IR (KBr) νmax: 3537, 2958, 2926, 1733, 1701, 1581, 1489,
1
1242, 1154, 858 cm−1. H NMR (600 MHz, CD3OD) δH 8.15 (s, 1H), 7.20 (s, 1H), 7.11 (d,
J = 15.4 Hz, 1H), 6.64 (d, J = 15.4 Hz, 1H), 5.79 (d, J = 9.7 Hz, 1H), 4.46 (dd, J = 14.4, 2.5 Hz,
1H), 3.94 (dd, J = 14.4, 9.2 Hz, 1H), 3.91–3.84 (m, 1H), 3.63 (dd, J = 11.1, 4.7 Hz, 1H), 3.53
(dd, J = 11.1, 6.7 Hz, 1H), 2.56–2.38 (m, 1H), 2.13 (d, J = 20 Hz, 3H), 1.92 (d, J = 0.9 Hz, 3H),
1.51 (d, J = 5.2 Hz, 3H), 1.50–1.46 (m, 1H), 1.41–1.34 (m, 1H), 1.05 (dd, J = 6.7, 2.1 Hz, 3H),
0.91 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CD3OD) δC 195.1, 185.5, 171.6, 151.8, 148.7,
148.3, 146.5, 144.9, 134.0, 117.4, 116.1, 112.3, 101.2, 86.2, 71.7, 64.5, 58.3, 36.2, 31.2, 23.8, 20.6,
20.2, 12.8, 12.4. HRMS (ESI) for [M
462.16899.
−
H]−: calcd for C24H29ClNO6: 462.17617. Found:
Compound 5
(R)-5-chloro-3-((S, 1E, 3E)-3,5-dimethylhepta-1, 3-dien-1-yl)-2-(furan-2-ylmethyl)-7-
methyl-6, 8-dioxo-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of 96%;
m.p. 160.0–161.5 ◦C. [α]D25 +16.6 (c 0.02, MeOH); UV (MeOH)
λmax (log
ε): 240 (3.16), 380
(3.06), 496 (1.66) nm; IR (KBr) νmax: 2960, 2928, 1735, 1703, 1595, 1497, 1371, 1256, 1144,

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1
1104, 1083, 860 cm−1. H NMR (600 MHz, CDCl3) δH 7.84 (s, 1H), 7.45–7.41 (m, 1H), 6.97 (s,
1H), 6.90 (d, J = 15.3Hz, 1H), 6.42–6.37 (m, 2H), 6.33 (d, J = 15.4 Hz, 1H), 5.67 (d, J = 9.8 Hz,
1H), 4.97 (d, J = 4.8 Hz, 2H), 2.52–2.40 (m, 1H), 2.14 (s, 3H), 1.84 (d, J = 0.9 Hz, 3H), 1.53 (s,
3H), 1.46–1.40 (m, 1H), 1.36–1.28 (m, 1H), 1.01 (d, J = 6.7 Hz, 3H), 0.87 (t, J = 7.4 Hz, 3H).
13C NMR (150 MHz, CDCl3) δC 193.8, 184.5, 170.2, 148.3, 148.1, 147.0, 145.1, 144.5, 144.2,
141.1, 131.9, 115.1, 115.0, 111.6, 111.1, 110.6, 102.7, 85.0, 50.3, 35.1, 30.1, 23.3, 20.4, 20.3, 12.7,
12.1. HRMS (ESI) for [M + H]+: calcd for C26H29ClNO5: 470.16560. Found: 470.17326.
Compound 6
(R)-2-(benzyloxy)-5-chloro-3-((S, 1E, 3E)-3,5-dimethylhepta-1, 3-dien-1-yl)-7-methyl-6,
8-dioxo-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of 96%; m.p.
166.6–168.3 ◦C. [α]D25 +14.3 (c 0.02, MeOH); UV (MeOH)
λmax (log ε): 248 (3.09), 386 (3.06),
491 (1.67) nm; IR (KBr) νmax: 3080, 2964, 2929, 1723, 1703, 1611, 1487, 1375, 1252, 1221, 1148,
1
1080, 952, 781 cm−1. H NMR (600 MHz, CDCl3) δH 7.82 (s, 1H), 7.44 (d, J = 7.1 Hz, 3H),
7.36 (dd, J = 7.6, 1.7 Hz, 2H), 7.08 (d, J = 15.9 Hz, 1H), 7.00 (s, 1H), 6.29 (d, J = 15.9 Hz, 1H),
5.74 (d, J = 9.7 Hz, 1H), 5.06 (dd, J = 25.6, 10.1 Hz, 2H), 258–2.43 (m, 1H), 2.17 (s, 3H), 1.83
(d, J = 0.7 Hz, 3H), 1.53 (s, 3H), 1.48–1.40 (m, 1H), 1.38–1.34 (m, 1H), 1.03 (d, J = 6.6 Hz,
3H), 0.90 (d, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δC 193.15, 184.41, 170.22, 149.11,
145.86, 144.88, 143.54, 137.20, 132.12, 131.62, 130.58, 130.27, 129.37, 128.98, 113.77, 112.08,
109.43, 103.14, 84.74, 81.96, 65.71, 35.28, 30.17, 29.83, 23.29, 20.35, 12.63, 12.13. HRMS (ESI)
for [M + H]+: calcd for C28H31ClNO5: 496.18125. Found: 496.18889.
Compound 7
(R)-5-chloro-3-((S, 1E, 3E)-3,5-dimethylhepta-1, 3-dien-1-yl)-7-methyl-6, 8-dioxo-2-(2-
(pyrrolidin-1-yl)ethyl)-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of
96%; m.p. 145.5–147.6 ◦C. [α]2D5 +16.1 (c 0.02, MeOH); UV (MeOH)
λmax (log ε): 246 (3.09),
379 (3.00), 488 (1.64) nm; IR (KBr) νmax: 2958, 2922, 1748, 1708, 1591, 1491, 1365, 1252, 1190,
1
1092, 860 cm−1. H NMR (600 MHz, CDCl3) δH 7.78 (s, 1H), 7.00 (s, 1H), 6.95 (d, J = 15.3 Hz,
1H), 6.23 (d, J = 15.3 Hz, 1H), 5.70 (d, J = 9.8 Hz, 1H), 3.98 (s, 2H), 2.84 (s, 2H), 2.61 (s, 4H),
2.53–2.43 (m, 1H), 2.17 (s, 3H), 1.86 (s, 3H), 1.81 (s, 4H), 1.54 (s, 3H), 1.49–1.40 (m, 1H),
1.38–1.34 (m, 1H), 1.03 (d, J = 6.6 Hz, 3H), 0.90 (d, J = 7.4 Hz, 3H). 13C NMR (150 MHz,
CDCl3) δC 194.0, 184.4, 170.2, 149.2, 147.6, 145.0, 144.5, 136.5, 131.6, 115.4, 115.3, 112.7, 102.3,
84.9, 58.8, 35.1, 30.1, 30.0, 23.3, 20.9, 20.7, 20.4, 20.3, 12.7, 12.1, 10.8, 10.8. HRMS (ESI) for [M
+ H]+: calcd for C27H36ClN2O4: 487.22854. Found: 487.23595.
Compound 8
(7R)-2-(sec-butyl)-5-chloro-3-((S, 1E, 3E)-3,5-dimethylhepta-1, 3-dien-1-yl)-7-methyl-6,
8-dioxo-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of 93%; m.p.
164.4–466.8 ◦C. [α]D25 +16.8 (c 0.02, MeOH); UV (MeOH)
λmax (log ε): 256 (3.05), 386 (2.98),
498 (1.53) nm; IR (KBr) νmax: 2964, 2920, 1736, 1701, 1599, 1497, 1370, 1252, 1200, 1141, 1084,
1
861 cm−1. H NMR (600 MHz, CDCl3) δH 7.83 (s, 1H), 6.95 (s, 1H), 6.87 (d J = 15.3 Hz, 1H),
6.13 (d, J = 15.3 Hz, 1H), 5.67 (d, J = 9.7 Hz, 1H), 4.23 (dd, J = 13.8, 6.9 Hz, 1H), 2.53–2.41 (m,
1H), 2.17 (s, 3H), 1.85 (s,3H), 1.80 (dd, J = 14.8, 7.4 Hz, 2H), 1.56 (s, 3H), 1.47–1.42 (m, 4H),
1.38–1.33 (m, 1H), 1.02 (d, J = 6.6 Hz, 3H), 0.93 (t, J = 7.3 Hz, 3H), 0.88 (t, J = 7.4 Hz, 3H). 13
C
NMR (150 MHz, CDCl3) δC 194.0, 184.5, 170.2, 148.4, 147.9, 145.1, 144.6, 142.2, 131.7, 114.6,
114.4, 111.7, 102.3, 84.9, 54.0, 53.4, 51.5, 35.2, 30.1, 29.8, 23.4, 20.4, 20.3, 12.8, 12.1. HRMS
(ESI) for [M + H]+: calcd for C25H33ClNO4: 446.20199. Found: 446.20947.
Compound 9
(R)-5-chloro-3-((S, 1E, 3E)-3, 5-dimethylhepta-1, 3-dien-1-yl)-2-(2-hydroxyethyl)-7-
methyl-6, 8-dioxo-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of 88%;
m.p. 180.2–182.8 ◦C. [α]D25 +15.8 (c 0.02, MeOH); UV (MeOH)
λmax (log ε): 233 (3.06), 389
(3.18), 498 (1.64) nm; IR (KBr) νmax: 3535, 2950, 2926, 1733, 1706, 1581, 1484, 1242, 1155,
1
866 cm−1. H NMR (600 MHz, CDCl3) δH 7.93 (s, 1H), 7.05 (s, 1H), 6.94 (d, J = 15.3 Hz,
1H), 6.28 (d, J = 15.4 Hz, 1H), 5.70 (d, J = 9.7 Hz, 1H), 4.04 (d, J = 5.4 Hz, 2H), 3.91 (d,
J = 4.6 Hz, 2H), 2.46 (d, J = 8.6 Hz, 1H), 2.14 (s, 3H), 1.84 (s, 3H), 1.53 (s, 3H), 1.46–1.42 (m,
1H), 1.36–1.30 (m, 1H), 1.01 (d, J = 6.6 Hz, 3H), 0.87 (t, J = 7.4 Hz, 3H). LRMS (EI) m/z
434 [M]+.

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Compound 10
(R)-5-chloro-3-((S, 1E, 3E)-3, 5-dimethylhepta-1, 3-dien-1-yl)-2-heptyl-7-methyl-6, 8-
dioxo-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of 93%; m.p.
95.6–97.3 ◦C
[α]2D5 +13.9 (c 0.02, MeOH); UV (MeOH)
. λmax (log ε): 246 (3.05), 389 (3.20),
488 (1.64) nm; IR (KBr) νmax: 2966, 2928, 1748, 1705, 1591, 1493, 1368, 1252, 1190, 1093,
1
862 cm−1. H NMR (600 MHz, CDCl3) δH 7.74 (s, 1H), 7.01 (s, 1H), 6.95 (d, J = 15.3 Hz, 1H),
6.12 (d, J = 15.4 Hz, 1H), 5.70 (d, J = 9.8 Hz, 1H), 3.82 (td, J = 7.2, 2.9 Hz, 2H), 2.52–2.37 (m,
1H), 2.16 (s, 3H), 1.84 (s, 3H), 1.77–1.72 (m, 2H), 1.54 (s, 3H), 1.46–1.42 (m, 1H), 1.37–1.25 (m,
9H), 1.02 (d, J = 6.7 Hz, 3H), 088 (t, J = 7.3 Hz, 6H). 13C NMR (150 MHz, CDCl3) δC 194.1,
184.4, 170.2, 148.2, 147.9, 145.0, 144.7, 141.1, 131.7, 114.8, 114.7, 111.8, 102.2, 84.9, 54.5, 35.2,
31.6, 30.3, 30.1, 28.8, 26.4, 23.4, 22.6, 20.4, 20.3, 14.1, 12.7, 12.1. HRMS (ESI) for [M + H]+:
calcd for C28H39ClNO4: 488.24894. Found: 488.25679.
Compound 11
(R)-5-chloro-3-((S, 1E, 3E)-3, 5-dimethylhepta-1, 3-dien-1-yl)-2-(2-(furan-2-yl)ethyl)-7-
methyl-6, 8-dioxo-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of 90%;
m.p. 130.3–132.8 ◦C. [α]D25 +15.9 (c 0.02, MeOH); UV (MeOH)
λmax (log ε): 256 (3.03), 379
(3.16), 493 (1.54) nm; IR (KBr) νmax: 2966, 2923, 1738, 1703, 1595, 1497, 1372, 1256, 1148,
1
1104, 1083, 869 cm−1. H NMR (600 MHz, CDCl3) δH 7.59 (s, 1H), 7.33 (d, J = 1.6 Hz, 1H),
6.96 (d, J = 15.3 Hz, 1H), 6.91 (d, J = 15.3 Hz, 1H), 6.28 (dd, J = 2.9, 2.0 Hz, 1H), 6.09 (d,
J = 3.1 Hz, 1H), 5.98 (d, J = 15.3 Hz, 1H), 5.68 (d, J = 9.7 Hz, 1H), 4.17–4.05 (m, 2H), 3.22–2.98
(m, 2H), 2.47 (dt, J = 8.0, 7.3 Hz, 1H), 2.16 (s, 3H), 1.83 (s, 3H), 1.53 (s, 3H), 1.46–1.42 (m,
1H), 1.37–1.31 (m, 1H), 1.02 (d, J = 6.6 Hz, 3H), 0.88 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz,
CDCl3) δC 193.8, 184.5, 170.2, 149.4, 148.1, 148.0, 145.2, 144.5, 142.7, 141.0, 131.8, 114.9, 114.5,
111.5, 110.9, 108.6, 102.5, 84.9, 52.8, 35.2, 30.2, 29.0, 23.3, 20.4, 20.4, 12.7, 12.1. HRMS (ESI)
for [M + H]+: calcd for C27H31ClNO5: 484.18125. Found: 484.18897.
Compound 12
(R)-5-chloro-2-cyclopropyl-3-((S, 1E, 3E)-3, 5-dimethylhepta-1, 3-dien-1-yl)-7-methyl-6,
8-dioxo-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of 91%; m.p.
172.3–174.4 ◦C. [α]D25 +15.6 (c 0.02, MeOH); UV (MeOH)
λmax (log ε): 251 (3.01), 388 (3.31),
489 (1.66) nm; IR (KBr) νmax: 2968, 2921, 1743, 1708, 1610, 1491, 1369, 1288, 1245, 1180, 1089,
1
955 cm−1. H NMR (600 MHz, CDCl3) δH 7.90 (s, 1H), 7.07 (s, 1H), 6.98 (d, J = 15.6 Hz, 1H),
6.55 (d, J = 15.6 Hz, 1H), 5.72 (d, J = 9.8 Hz, 1H), 3.27 (dd, J = 6.8, 3.3 Hz, 1H), 2.68–2.39
(m, 1H), 2.16 (s, 3H), 1.86 (s, 3H), 1.53 (s, 3H), 1.48–1.40 (m, 1H), 1.37–1.31 (m, 1H), 1.22 (d,
J = 6.8 Hz, 2H), 1.05 (dd, J = 15.1, 2.5 Hz, 2H), 1.02 (d, J = 6.7 Hz, 3H), 0.88 (t, J = 7.4 Hz,
3H). LRMS (EI) m/z 431 [M]+.
Compound 13
(R)-5-chloro-3-((S, 1E, 3E)-3, 5-dimethylhepta-1, 3-dien-1-yl)-7-methyl-6, 8-dioxo-2-
(thiophen-2-ylmethyl)-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of
89%; m.p. 106.5–108.4 ◦C. [α]2D5 +13.3 (c 0.02, MeOH); UV (MeOH)
λmax (log ε): 244 (3.00),
381 (3.10), 497 (1.68) nm; IR (KBr) νmax: 2960, 2924, 1735, 1704, 1591, 1499, 1371, 1256, 1140,
1
1080, 1008, 860 cm−1. H NMR (600 MHz, CDCl3) δH 7.86 (s, 1H), 7.35 (dd, J = 3.9, 2.4 Hz,
1H), 7.10–6.98 (m, 3H), 6.92 (d, J = 15.3 Hz, 1H), 6.22 (dd, J = 15.3 Hz, 1H), 5.68 (d, J = 9.8 Hz,
1H), 5.17 (q, J = 16.2 Hz, 2H), 2.54–2.41 (m, 1H), 2.15 (s, 3H), 1.80 (d, J = 10 Hz, 3H), 1.54 (s,
3H), 1.46–1.40 (m, 1H), 1.37–1.29 (m, 1H), 1.01 (d, J = 6.7 Hz, 3H), 0.87 (t, J = 7.4 Hz, 3H).
13C NMR (150 MHz, CDCl3) δC 193.8, 184.6, 170.3, 148.3, 148.0, 145.3, 144.4, 140.9, 136.3,
131.9, 127.8, 127.5, 127.2, 115.1, 114.9, 111.7, 103.0, 85.0, 52.6, 35.2, 30.1, 23.3, 20.4, 20.3, 12.7,
12.1. HRMS (ESI) for [M + H]+: calcd for C26H29ClNO5S: 486.14276. Found: 486.15086.
Compound 14
(R)-5-chloro-3-((S, 1E, 3E)-3,5-dimethylhepta-1, 3-dien-1-yl)-7-methyl-6, 8-dioxo-2-(2-
(thiophen-2-yl)ethyl)-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of
91%; m.p. 100.2–102.0 ◦C. [α]2D5 +16.2 (c 0.02, MeOH); UV (MeOH)
λmax (log ε): 251 (3.09),
389 (3.00), 501 (1.55) nm; IR (KBr) νmax: 2966, 2920, 1735, 1708, 1591, 1489, 1371, 1258, 1140,
1
1088, 1008, 849 cm−1. H NMR (600 MHz, CDCl3) δH 7.59 (s, 1H), 7.19 (dd, J = 5.1, 1.1 Hz,
1H), 6.97 (s, 1H), 6.95–6.88 (m, 2H), 6.78 (d, J = 3.3 Hz, 1H), 5.98 (d, J = 15.3 Hz, 1H), 5.68

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(d, J = 9.7 Hz, 1H), 4.08 (ddt, J = 78.5, 14.6, 7.2 Hz, 2H), 3.34–3.13 (m, 2H), 2.52–2.39 (m,
1H), 2.16 (s, 3H), 1.82 (d, J = 1.0 Hz, 3H), 1.52 (s, 3H), 1.49–1.39 (m, 1H), 1.38–1.31 (m,1H),
1.02 (d, J = 6.7 Hz, 3H), 0.88 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δC 193.8, 184.4,
170.2, 148.3, 147.9, 145.2, 144.4, 140.9, 137.4, 131.8, 127.8, 126.9, 125.4, 114.8, 114.5, 111.6,
102.6, 85.0, 55.5, 35.2, 30.6, 30.2, 23.4, 20.4, 20.4, 12.7, 12.1. HRMS (ESI) for [M + H]+: calcd
for C27H31ClNO5S: 500.15841. Found: 500.16563.
Compound 15
(R)-5-chloro-3-((S, 1E, 3E)-3, 5-dimethylhepta-1, 3-dien-1-yl)-7-methyl-6, 8-dioxo-2-
phenyl-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of 93%; m.p.
190.2–192.1 ◦C. [α]D25 +16.5 (c 0.02, MeOH); UV (MeOH)
λ
max (log
506 (1.53) nm; IR (KBr) νmax: 3088, 2964, 2916, 1731, 1707, 1611, 1591, 1495, 1363, 1275, 1204,
1130, 1084, 1020, 856, 773 cm−1 1H NMR (600 MHz, CDCl3) δH 7.83 (s, 3H), 7.59–7.47
ε): 241 (3.19), 379 (3.12),
.
(m, 3H), 7.32–7.27 (m, 2H), 7.15 (s, 1H), 6.96 (d, J = 15.6 Hz), 5.65 (d, J = 9.7 Hz, 1H), 5.58
(d, J = 15.6 Hz, 1H), 2.46–2.30 (m, 1H), 2.17 (s, 3H), 1.58 (s, 3H), 1.49 (d, J = 0.9 Hz, 3H),
1.45–1.36 (m, 1H), 1.33–1.28 (m, 1H), 0.97 (d, J = 6.6 Hz, 3H), 0.84 (t, 7.4 Hz, 3H). 13C NMR
(150 MHz, CDCl3) δC 193.9, 184.8, 170.3, 148.0, 147.6, 144.2, 143.3, 141.3, 140.5, 131.9, 130.3,
130.3, 130.2, 126.7, 126.7, 116.3, 114.5, 109.9, 103.4, 84.9, 35.1, 30.1, 23.3, 20.4, 20.3, 12.3, 12.1.
LRMS (EI) m/z 467 [M]+.
Compound 16
(R)-5-chloro-3-((S, 1E, 3E)-3,5-dimethylhepta-1, 3-dien-1-yl)-7-methyl-6, 8-dioxo-2-
phenethyl-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of 91%; m.p.
180.2–181.0 ◦C. [α]D25 +15.3 (c 0.02, MeOH); UV (MeOH)
λmax (log ε): 243 (3.06), 386 (3.14),
498 (1.55) nm; IR (KBr) νmax: 3080, 2962, 2916, 1731, 1715, 1611, 1588, 1495, 1366, 1275,
1
1212, 1130, 1084, 1022, 856, 780 cm−1. H NMR (600 MHz, CDCl3) δH 7.51 (s, 1H), 7.30 (dd,
J = 10.2, 4.6 Hz, 2H), 7.26–7.22 (m, 1H), 7.15- 7.01 (m, 2H), 6.94 (s, 1H), 6.88 (d, J = 15.2 Hz,
1H), 5.99 (d, J = 15.4 Hz, 1H), 5.67 (d, J = 9.7 Hz, 1H), 4.32–3.89 (m, 2H), 3.28–2.84 (m,
2H), 2.53–2.40 (m, 1H), 2.14 (s, 3H), 1.80 (d, J = 1.0 Hz, 3H), 1.50 (s, 3H), 1.48–1.40 (m, 1H),
1.38–1.31 (m, 1H), 1.02 (d, J = 6.7 Hz, 3H), 0.88 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz,
CDCl3) δC 193.8, 184.4, 170.1, 148.1, 148.0, 145.1, 144.5, 141.1, 135.9, 131.8, 129.3, 129.3, 128.9,
128.9, 127.8, 114.7, 114.7, 111.5, 102.3, 85.0, 55.6, 36.7, 35.1, 30.1, 23.4, 20.4, 20.3, 12.7, 12.1.
LRMS (EI) m/z 495 [M]+.
Compound 17
(R)-2-benzyl-5-chloro-3-((S, 1E, 3E)-3,5-dimethylhepta-1, 3-dien-1-yl)-7-methyl-6, 8-
dioxo-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of 92%; m.p.
188.8–190.2 ◦C. [α]D25 +13.9 (c 0.02, MeOH); UV (MeOH)
λmax (log ε): 259 (3.06), 399
(3.10), 499 (1.65) nm; IR (KBr) νmax: 3082, 2964, 2918, 1731, 1710, 1611, 1591, 1495, 1363,
1
1276, 1204, 1130, 1084, 1020, 858, 773 cm−1. H NMR (600 MHz, CDCl3) δH 7.86 (s, 1H),
7.45–7.33 (m, 3H), 7.18 (d, J = 7.2 Hz, 2H), 7.04 (s, 1H), 6.89 (d, J = 15.4 Hz, 1H), 6.02 (d,
J = 15.4 Hz, 1H), 5.64 (d, J = 9.8 Hz, 1H), 5.19–4.88 (m, 2H), 2.54–2.30 (m, 1H), 2.16 (s, 3H),
1.66 (d, J = 1.0 Hz, 3H), 1.57 (s, 3H), 1.46–1.40 (m, 1H), 1.34–1.28 (s, 1H), 0.98 (d, J = 6.7 Hz,
3H), 0.84 (t, J = 6.7 Hz, 3H). 13C NMR (150 MHz, CDCl3) δC 194.0, 184.6, 170.3, 148.4, 148.2,
145.0, 144.5, 141.7, 134.2, 131.8, 129.7, 129.7, 129.1, 126.6, 126.6, 115.2, 114.9, 111.7, 102.9, 84.9,
57.9, 35.1, 30.1, 23.4, 20.4, 20.3, 12.6, 12.1. HRMS (ESI) for [M + H]+: calcd for C28H31ClNO4:
480.1863. Found: 480.1935.
Compound 18
(R)-5-chloro-3-((S, 1E, 3E)-3, 5-dimethylhepta-1, 3-dien-1-yl)-7-methyl-2-(3-
morpholinopropyl) -6, 8-dioxo-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid,
the yield of 96%; m.p. 110.2–112.6 C. [α]2D5 +16.6 (c 0.02, MeOH); UV (MeOH)
λmax (log ε):
◦
245 (3.19), 376 (3.00), 505 (1.63) nm; IR (KBr) νmax: 2960, 2920, 1727, 1706, 1595, 1499, 1371,
1
1248, 1208, 1148, 1080, 864 cm−1. H NMR (600 MHz, CDCl3) δH 7.93 (s, 1H), 7.01 (s, 1H),
6.96 (d, J = 15.4 Hz, 1H), 6.18 (d, J = 15.4 Hz, 1H), 5.71 (d, J = 9.7 Hz, 1H), 4.01 (s, 2H), 3.75
(s, 4H), 2.68–2.24 (m, 7H), 2.16 (s, 3H), 1.98 (dd, J = 28.3, 21.6 Hz, 2H), 1.86 (s, 3H), 1.54 (s,
3H), 1.46–1.40 (m, 1H), 1.37–1.32 (m, 1H), 1.02 (d, J = 6.6 Hz, 3H), 0.88 (t, J = 7.4 Hz, 3H).
13C NMR (150 MHz, CDCl3) δC 194.1, 184.5, 170.2, 148.4, 147.9, 145.1, 144.6, 142.2, 131.7,

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114.6, 114.4, 111.7, 102.3, 84.9, 54.0, 53.4, 51.5, 35.2, 30.1, 29.8, 23.4, 20.4, 20.3, 12.8, 12.1. 10.9,
10.8. HRMS (ESI) for [M + H]+: calcd for C28H38ClN2O5: 517.22345. Found:517.24647.
Compound 19
(R)-5-chloro-2-(2-(dimethylamino)ethyl)-3-((S, 1E, 3E)-3,5-dimethylhepta-1, 3-dien-1-
yl)-7-methyl-6, 8-dioxo-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield
of 92%; m.p. 185.6–187.2 ◦C. [α]2D5 +14.8 (c 0.02, MeOH); UV (MeOH)
λmax (log ε): 236
(3.01), 371 (3.23), 487 (1.45) nm; IR (KBr) νmax: 2960, 2923, 1731, 1706, 1595, 1499, 1371, 1251,
1
1209, 1140, 1080, 866 cm−1. H NMR (600 MHz, CDCl3) δH 7.77 (s, 1H), 7.00 (s, 1H), 6.94
(d, J = 15.3 Hz, 1H), 6.21 (d, J = 15.4 Hz, 1H), 5.70 (d, J = 9.70 Hz, 1H), 3.93 (s, 2H), 2.64
(s, 2H), 2.53–2.41 (m, 1H), 2.31 (s, 6H), 2.16 (s, 3H), 1.85 (d, J = 0.8 Hz, 3H), 1.54 (s, 3H),
1.46–1.40 (m, 1H), 1.39–1.31 (m, 1H), 1.02 (d, J = 6.6 Hz, 3H), 0.88 (t, J = 7.4 Hz, 3H). 13
C
NMR (150 MHz, CDCl3) δC 193.9, 184.4, 170.1, 148.1, 148.0, 145.1, 144.5, 141.5, 131.7, 114.8,
114.6, 111.6, 102.3, 84.9, 58.5, 51.8, 45.6, 35.1, 30.0, 29.3, 23.3, 20.3, 20.2, 12.6, 12.0. HRMS
(ESI) for [M + H]+: calcd for C25H34ClN2O4: 461.26289. Found: 461.22027.
Compound 20
(R)-2-(2-(benzylamino)ethyl)-5-chloro-3-((S, 1E, 3E)-3, 5-dimethylhepta-1, 3-dien-1-
yl)-7-methyl-6, 8-dioxo-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield
of 91%; m.p. 160.1–161.8. [α]2D5 +14.3 (c 0.02, MeOH); UV (MeOH)
λmax (log ε): 251 (3.03),
381 (3.26), 497 (1.55) nm; IR (KBr) νmax: 3412, 2963, 2924, 1737, 1703, 1589, 1495, 1369, 1249,
1
1145, 1082, 1007, 965, 861 cm−1. H NMR (600 MHz, CDCl3) δH 7.81 (s, 1H), 7.32–7.27 (m,
2H), 7.24 (td, J = 7.6, 2.1 Hz, 3H), 6.94 (s, 1H), 6.83 (d, J = 15.3 Hz, 1H), 6.16 (d, J = 15.4 Hz,
1H), 5.66 (d, J = 9.7 Hz, 1H), 4.09–3.80 (m, 2H), 3.79 (s, 2H), 3.12–2.77 (m, 2H), 2.53–2.37 (m,
1H), 2.16 (s, 3H), 1.80 (d, J = 1.0 Hz, 3H), 1.55 (s, 3H), 1.49–1.41 (m, 1H), 1.37–1.32 (m, 1H),
1.01 (d, J = 6.7 Hz, 3H), 0.88 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δC 194.1, 184.5,
170.2, 148.1, 148.0, 145.1, 144.8, 142.1, 131.8, 128.8, 128.8, 128.8, 128.3, 128.3, 127.6, 114.9,
114.4, 111.8, 102.2, 85.0, 54.1, 53.6, 47.5, 35.13, 30.2, 23.4, 20.5, 20.4, 12.7, 12.1. HRMS (ESI)
for [M + H]+: calcd for C30H36ClN2O5: 523.22854. Found: 523.23594.
Compound 21
(R)-5-chloro-2-cyclopentyl-3-((S, 1E, 3E)-3, 5-dimethylhepta-1, 3-dien-1-yl)-7-methyl-6,
8-dioxo-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of 95%; m.p.
132.3–133.5 ◦C. [α]D25 +15.3 (c 0.02, MeOH); UV (MeOH)
λmax (log ε): 233 (3.09), 369 (3.13),
499 (1.65) nm; IR (KBr) νmax: 2971, 2925, 1743, 1700, 1600, 1491, 1368, 1288, 1246, 1180, 1089,
1
955 cm−1. H NMR (600 MHz, CDCl3) δH 7.88 (s, 1H), 6.95 (s, 1H), 6.87 (d, J = 15.3 Hz, 1H),
6.19 (d, J = 15.3 Hz, 1H), 5.67 (d, J = 9.7 Hz, 1H), 4.53 (p, J = 7.4 Hz, 1H), 2.55–2.38 (m, 1H),
2.29–2.12 (m, 5H), 1.91 (ddd, J = 16.4, 8.5, 5.0 Hz, 2H), 1.85 (d, J = 1.0 Hz, 3H), 1.84–1.70
(m, 4H), 1.54 (s, 3H), 1.46–1.39 (m,1H), 1.38–1.30 (m, 1H), 1.01 (d, J = 6.7 Hz, 3H), 0.88
(t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δC 194.1, 184.4, 170.2, 149.1, 147.7, 144.9,
144.5, 136.9, 131.7, 115.6, 115.3, 112.7, 102.2, 84.9, 62.7, 35.1, 33.3, 33.2, 30.1, 24.3, 24.3, 23.4,
20.4, 20.4, 12.8, 12.1. HRMS (ESI) for [M + H]+: calcd for C26H33ClNO4: 458.20199. Found:
458.20981.
Compound 22
(R)-5-chloro-2-cyclohexyl-3-((S, 1E, 3E)-3, 5-dimethylhepta-1, 3-dien-1-yl)-7-methyl-6,
8-dioxo-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of 95%; m.p.
136.2–138.1 ◦C. [α]D25 +16.5 (c 0.02, MeOH); UV (MeOH)
λmax (log ε): 248 (3.08), 379 (3.16),
503 (1.51) nm; IR (KBr) νmax: 2955, 2918, 1743, 1705, 1600, 1491, 1368, 1286, 1248, 1180, 1089,
1
923 cm−1. H NMR (600 MHz, CDCl3) δH 7.90 (s, 1H), 6.96 (s, 1H), 6.88 (d, J = 15.2 Hz, 1H),
6.14 (d, J = 15.3 Hz, 1H), 5.68 (d, J = 9.7 Hz, 1H), 3.97 (tt, J = 12.0, 3.2 Hz, 1H), 2.65–2.34 (m,
1H), 2.16 (s, 3H), 1.98 (dd, J = 23.3, 12.1 Hz, 5H), 1.85 (d, J = 1.0 Hz, 3H), 1.77 (d, J = 13.3 Hz
,
1H), 1.64 (tt, 12.4, 6.3 Hz, 2H), 1.47–1.31 (m, 4H), 1.29–1.16 (m, 3H), 1.02 (d, J = 6.7 Hz, 3H),
0.88 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δC 194.1, 184.4, 170.2, 148.6, 147.7,
145.1, 144.6, 136.9, 131.6, 115.1, 115.0, 112.9, 102.1, 84.9, 61.2, 35.1, 33.3, 33.1, 30.1, 25.8, 25.8,
25.0, 23.4, 20.4, 20.4, 12.7, 12.1. LRMS (EI) m/z 472 [M]+.

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Compound 23
(R)-2-(4-bromophenyl)-5-chloro-3-((S, 1E, 3E)-3, 5-dimethylhepta-1, 3-dien-1-yl)-7-
methyl-6, 8-dioxo-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of 91%;
m.p. 190.0–192.3 ◦C. [α]D25 +16.8 (c 0.02, MeOH); UV (MeOH)
λmax (log ε): 236 (3.03), 377
(3.25), 493 (1.48) nm; IR (KBr) νmax: 3101, 2959, 2919, 1735, 1707, 1601, 1508, 1487, 1365, 1268,
1
1250, 1207, 1129, 1080, 960 cm−1. H NMR (600 MHz, CDCl3) δH 7.76 (s, 1H), 7.73–7.63
(m, 2H), 7.24–7.15 (m, 2H), 7.12 (s, 1H), 6.96 (d, J = 15.5 Hz, 1H), 5.67 (d, J = 9.7 Hz, 1H),
5.55 (d, J = 15.5 Hz, 1H), 2.53–2.32 (m, 1H), 2.17 (s, 3H), 1.63–1.49 (m, 6H), 1.46–1.40 (m,
1H), 1.39–1.31 (m, 1H), 0.99 (d, J = 6.6 Hz, 3H), 0.85 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz,
CDCl3) δC 193.7, 184.9, 170.3, 148.3, 147.2, 143.8, 143.6, 140.9, 139.5, 133.6, 133.6, 131.9, 128.3,
128.3, 124.3, 116.0, 114.7, 110.1, 104.0, 84.8, 35.1, 30.1, 23.2, 20.4, 20.3, 12.5, 12.1. HRMS (ESI)
for [M + H]+: calcd for C27H28ClBrNO4: 544.08120. Found: 544.08855.
Compound 24
(R)-5-chloro-3-((S, 1E, 3E)-3, 5-dimethylhepta-1, 3-dien-1-yl)-2-ethyl-7-methyl-6, 8-
dioxo-2, 6, 7, 8-tetrahydroisoquinolin-7-yl acetate. Red solid, the yield of 91%; m.p.
250.0–251.1 ◦C
[α]2D5 +15.5 (c 0.02, MeOH); UV (MeOH)
. λmax (log ε): 238 (3.12), 385
(3.33), 499 (1.47) nm; IR (KBr) νmax: 2955, 2922, 1748, 1712, 1591, 1491, 1368, 1252, 1190,
1
1093, 853 cm−1. H NMR (600 MHz, CDCl3) δH 7.78 (s, 1H), 6.99 (s, 1H), 6.95 (d, J = 15.3 Hz,
1H), 6.14 (d, J = 15.4 Hz, 1H), 5.69 (d, J = 9.7 Hz, 1H), 3.92 (q, J = 7.3 Hz, 2H), 2.52–2.36
(m, 1H), 2.14 (s, 3H), 1.84 (d, J = 0.9 Hz, 3H), 1.52 (s, 3H), 1.46–1.36 (m, 4H), 1.36–1.27 (m,
1H), 1.00 (d, J = 6.7 Hz, 3H), 0.86 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δC 193.9,
184.3, 170.2, 148.2, 147.9, 145.1, 144.8, 140.8, 131.7, 115.0, 114.5, 111.7, 102.0, 84.9, 49.5, 35.1,
30.1, 23.2, 20.4, 20.3, 15.7, 12.7, 12.1. HRMS (ESI) for [M + H]+: calcd for C23H29ClNO4:
418.17069. Found: 418.17845.
3.1.5. General Procedure for Synthesis of 25
To a solution of sclerotiorin (39 mg, 0.10 mmol) in anhydrous dichloromethane (2 mL),
the CH3ONa (54 mg, 10 mmol) was add. The resulting reaction mixture was stirred at
◦
40 C until the sclerotiorin was disappeared. 12 mL HCl (2M) was added to the mixture to
regulate the pH to 2. The mixture was extracted with EtOAc (3
×
5 mL) and the organic
phase was washed with saturated brine (3 15 mL). Then the organic phase was dried
×
with MgSO4 and concentrated in vacuo. The crude product was purified by column
chromatography with eluent of EA/PE (2:1, v/v) to give the pure compound.
Compound 25
(R)-5-chloro-3-((S, 1E, 3E)-3, 5-dimethylhepta-1, 3-dien-1-yl)-7-hydroxy-7-methyl-6H-
isochromene-6, 8 (7H)-dione. Yellow solid, the yield of 88%; m.p. 130.5–132.1 C. [α]D25
◦
+221.7 (c 0.6, MeOH); UV (MeOH)
λ
max (log
ε
): 234 (3.01), 344 (3.13) nm; IR (KBr) νmax
:
3537, 2949, 1625, 1510, 1183, 1154, 1131, 1079, 966 cm−1. H NMR (600 MHz, CD3OD) δH
8.13 (s,1H), 7.17 (d, J = 15.7 Hz, 1H), 6.81 (s, 1H), 6.36 (d, J = 15.7 Hz, 1H), 5.74 (d, J = 9.8 Hz,
1H), 2.63–2.42 (m, 1H), 1.90 (d, J = 1.1 Hz, 3H), 1.52 (s, 3H), 1.49–1.46 (m, 1H), 1.38–1.34
(m, 1H), 1.00 (d, J = 6.7 Hz, 3H), 0.86 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CD3OD) δC
196.8, 191.8, 160.2, 153.6, 149.2, 143.8, 141.4, 133.8, 117.5, 116.4, 110.4, 107.0, 84.9, 36.3, 31.2,
27.7, 20.6, 12.5, 12.3. HRMS (ESI) for [M + H]+: calcd for C19H22ClO4: 349.1128. Found:
349.1212.
1
3.1.6. General Procedure for Synthesis of 26 and 27
A solution of 25 (39 mg, 0.11 mmol) and anhydride (1.10 mmol) in anhydrous pyridine
(3 mL) was refluxed at 90 ◦C until the 25 disappeared. After being cooled to room tem-
perature, 2 mL HCl (2M) was added to the mixture to regulate the pH to 2. The resulting
mixture was extracted with EtOAc (3
×
5 mL) and the organic phase was washed with
saturated brine (3 15 mL). Then the organic phase was dried with MgSO4 and concen-
×
trated in vacuo. The crude product was purified by column chromatography with eluent
of EA/PE (1:10, v/v) to give the pure compound.

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Compound 26
(R)-5-chloro-3-((S, 1E, 3E)-3, 5-dimethylhepta-1, 3-dien-1-yl)-7-methyl-6, 8-dioxo-7,
8-dihydro-6H-isochromen-7-yl butyrate. Yellow oil, the yield of 78%. [α]2D5 +42.8 (c 0.2,
MeOH); UV (MeOH)
λ
max (log
ε
): 241 (3.08), 348 (3.15) nm; IR (KBr) νmax: 2949, 1737,
1625, 1509, 1180, 1135, 1079, 958 cm−1. H NMR (600 MHz, CD3OD) δH 8.19 (s, 1H), 7.19 (d,
J = 15.7 Hz, 1H), 6.87 (s, 1H), 6.38 (d, J = 15.7 Hz, 1H), 5.75 (d, J = 9.8 Hz, 1H), 2.63-2.51 (m,
1H), 2.43 (t, J = 7.4 Hz, 2H), 1.90 (d, J = 1.1 Hz, 3H), 1.64 (ddd, J = 7.1, 5.0, 2.5 Hz, 2H), 1.55
(s, 3H), 1.51–1.44 (m, 1H), 1.38-1.34 (m, 5H), 1.04 (d, J = 6.7 Hz, 3H), 0.94 (t, J = 7.1HZ, 3H),
0.90 (t, J = 7.4 Hz, 3H). 13C NMR (150 MHz, CD3OD) δC 192.9, 188.1, 174.2, 160.5, 155.1,
149.4, 144.1, 141.5, 133.9, 117.4, 115.8, 110.7, 107.3, 86.0, 36.3, 34.0, 32.2, 31.2, 25.5, 23.4, 22.9,
20.6, 14.2, 12.5, 12.4. HRMS (ESI) for [M + H]+: calcd for C22H32ClO5: 447.1600. Found:
447.1936.
1
Compound 27
(R)-5-chloro-3-((S, 1E, 3E)-3, 5-dimethylhepta-1, 3-dien-1-yl)-7-methyl-6, 8-dioxo-7,
8-dihydro-6H-isochromen-7-yl propionate. yellow oil, the yield of 83%. [α]2D5 +37.2 (c 0.2,
MeOH); UV (MeOH)
λ
max (log
ε
): 238 (3.11), 340 (3.08) nm; IR (KBr) νmax: 2959, 1738,
1625, 1506, 1180, 1135, 1079, 962 cm−1. H NMR (600 MHz, CD3OD) δH 8.20 (s, 1H), 7.19
(d, J = 15.7 Hz, 1H), 6.88 (s, 1H), 6.38 (d, J = 15.7 Hz, 1H), 5.76 (d, J = 9.8 Hz, 1H), 2.59–2.51
(m, 1H), 2.49-2.45 (m, 2H), 1.90 (d, J = 1.0 Hz, 3H), 1.56 (s, 3H), 1.51–1.44 (m, 1H), 1.39–1.34
(m, 1H), 1.14 (t, J = 7.6 Hz, 3H), 1.04 (d, J = 6.6 Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H). 13C NMR
(150 MHz, CD3OD) δC 192.9, 188.1, 174.9, 160.5, 155.1, 149.5, 144.1, 141.5, 133.9, 117.4, 115.8,
110.7, 107.3, 86.1, 36.3, 31.2, 27.5, 22.9, 20.6, 12.5, 12.3, 9.1. HRMS (ESI) for [M + H]+: calcd
for C22H26ClO5: 405.1391. Found: 405.1465.
1
3.2. Cytotoxic Activity
The test compounds at a serial final concentration of 50, 25, 12.5, 6.25, and 3.125 µM
were evaluated against A549 (human lung cancer cells), MDA-MB-435 (breast cancer
cells) using the MTT method. Human breast cancer cell lines MDA-MB-435, human
lung cancer cell line A549 were obtained from Keygen Biotech (Nanjing, China) and
cultuRed in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA,
USA) supplemented with 5% fetal bovine serum (Hyclone, Logan, UT, USA), 2 mM l-
glutamine, 100 mg/mL streptomycin, and 100 units/mL penicillin (Invitrogen). The
cultures were maintained at 37 ◦C in a humidified atmosphere of 5% CO2.
3.3. COX-2 Inhibitory Activity
The in vitro inhibitory of the compounds were evaluated by using COX-2 (human)
inhibitor screening assay kit (Item NO:701080) supplied by Cayman chemicals USA. In-
domethacin was used as a positive control and the tested compounds was dissolved in
DMSO.
Each compound was tested in triplicate at 20 µM, and the percent inhibition for COX-2
was obtained for each experiment. The amount of prostaglandins produced by enzyme
in the presence of the test compounds was measured and compared with the control
experiments (also performed in triplicate) with enzyme inhibition = 1/concentration of
prostaglandin in each enzymatic reaction (The percent inhibition of test compound was
inversely proportional to the amount of prostaglandins produced by each wells). Finally,
the calculations were performed as per the kit guidelines.
3.4. Molecular Docking
The X-ray crystal structure of COX-2 cyclooxygenase (PDB ID: 5GMN) enzyme was
obtained from protein data bank in PDB format as initialing point. The crystal original
ligand was extracted from the crystal structure prior to docking. Then, all waters were
removed in the crystal structure. Hydrogens addition and Gasteiger charges were executed
in turn. The protein was regarded as rigid and the conformation of the ligand was regarded
as changeable. The parameter of grid box was set as 100
×
100
×
100 points and center on

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the protein. The Lamarckian genetic algorithm was used to dock algorithm and number of
GA runs was 100. PyMOL [38] and LigPolt [39] were used to visualize and analyze results.
4. Conclusions
In summary, a novel series of sclerotiorin derivatives were synthesized. All com-
pounds have been screened for their cytotoxic activity in vitro. Most of sclerotiorin deriva-
tives showed good to great cytotoxic activity. In addition, the COX-2 inhibitory results
disclosed that most of the derivatives displayed considerable inhibition of COX-2. Particu-
larly, compound
ratio to positive indomethacin (78.9%) in 20
3
displayed superior inhibitory ratio of 70.6%, a comparable inhibition
M. Moreover, both in vitro and in silico
µ
studies showed that some of the new compounds act as promising COX-2 inhibitors. The
results of this research will provide useful information for the design of novel series of
anticancer agents with COX-2 inhibitory activity.
Supplementary Materials: The following are available online at https://www.mdpi.com/1660-339
7/19/1/12/s1. Figure S1. MS spectrum of compound 1; Figures S2 and S3: NMR (in CDCl3) spectra of
compound 1; Figure S4: MS spectrum of compound 2; Figures S5 and S6: NMR (in CDCl3) spectra of
compound 2; Figure S7: HRMS spectrum of compound 3; Figures S8 and S9: NMR (in CDCl3) spectra
of compound 3; Figure S10. HRMS spectrum of compound 4; Figures S11 and S12: NMR (in CD3OD)
spectra of compound 4; Figure S13: HRMS spectrum of compound 5; Figures S14 and S15: NMR (in
CDCl3) spectra of compound 5; Figure S16: HRMS spectrum of compound 6; Figures S17 and S18
:
NMR (in CDCl3) spectra of compound 6; Figure S19: HRMS spectrum of compound 7; Figures S20
and S21: NMR (in CDCl3) spectra of compound 7; Figure S22: HRMS spectrum of compound 8;
Figures S23 and S24: NMR (in CDCl3) spectra compound 8; Figure S25: MS spectrum of compound 9;
Figures S26 and S27: NMR (in CDCl3) spectrum of compound 9; Figure S27: HRMS spectrum of
compound 10; Figures S28 and S29: NMR (in CDCl3) spectra of compound 10; Figure S30: HRMS
spectrum of compound 11; Figures S31 and S32: NMR (in CDCl3) spectra of compound 11; Figure S33:
1
MS spectrum of compound 12; Figure S34: H NMR (in CDCl3) spectra of compound 12; Figure S35:
HRMS spectrum of compound 13; Figures S36 and S37: NMR (in CDCl3) spectra of compound 13;
Figure S38: HRMS spectrum of compound 14; Figures S39 and S40: NMR (in CDCl3) spectra of
compound 14; Figure S41: MS spectrum of compound 15; Figures S42 and S43: NMR (in CDCl3)
spectra of compound 15; Figure S44: MS spectrum of compound 16; Figures S45 and S46: NMR (in
CDCl3) spectra of compound 16; Figure S47: HRMS spectrum of compound 17; Figures S48 and S49:
NMR (in CDCl3) spectra of compound 17; Figure S50: HRMS spectrum of compound 18; Figures S51
and S52: NMR (in CDCl3) spectra of compound 18; Figure S53: HRMS spectrum of compound 19;
Figures S54 and S55: NMR (in CDCl3) spectra of compound 19; Figure S56: HRMS spectrum of
compound 20; Figures S57 and S58: NMR (in CDCl3) spectra of compound 20; Figure S59: HRMS
spectrum of compound 21; Figures S60 and S61: NMR (in CDCl3) spectra of compound 21; Figure S62:
MS spectrum of compound 22; Figures S63 and S64: NMR (in CDCl3) spectra of compound 22;
Figure S65: HRMS spectrum of compound 23; Figures S66 and S67: NMR (in CDCl3) spectra of
compound 23; Figure S68c: HRMS spectrum of compound 24; Figures S69 and S70: NMR (in CDCl3)
spectra of compound 24; Figure S71: HRMS spectrum of compound 25; Figures S72 and S73: NMR (in
CDCl3) spectra of compound 25; Figure S74: HRMS spectrum of compound 26; Figures S75 and S76:
NMR (in CDCl3) spectra of compound 26; Figure S77: HRMS spectrum of compound 27; Figures S78
and S79: NMR (in CDCl3) spectra of compound 27.
Author Contributions: Conceptualization, Y.L.; methodology, T.C., Y.H., J.Y.; software, T.C.; formal
analysis, Y.L., J.Y.; investigation, T.C., Y.H., J.H., X.W., F.Z., J.L., G.Y.; resources, Y.L., J.Y.; data
curation, Y.L., J.Y.; writing—original draft preparation, T.C.; writing—review and editing, T.C., Y.L.;
visualization, T.C., Y.H., J.H., X.W., F.Z., J.L., G.Y., Y.L., J.Y.; supervision, Y.L.; project administration,
Y.L.; funding acquisition, Y.L. All authors have read and agreed to the published version of the
manuscript.
Funding: This research received no external funding.
Data Availability Statement: The data presented in this study are available in the Supplementary
Materials.

Mar. Drugs 2021, 19, 12
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Acknowledgments: The authors thank Idrees Sumra (South China Normal University) for her help in
language polish of this paper and gratefully acknowledge grants from the National Natural Science
Foundation of China (41876153), the Key Project of Natural Science Foundation of Guangdong
Province (2016A030311026) and the Guangdong MEPP Fund (GDOE [2019]A21).
Conflicts of Interest: The authors declare no conflict of interest
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