Design, synthesis, biological evaluation and silico prediction of novel sinomenine derivatives

Article abstract of DOI:10.3390/molecules26113466

Sinomenine is a morphinan alkaloid with a variety of biological activities. Its derivatives have shown significant cytotoxic activity against different cancer cell lines in many studies. In this study, two series of sinomenine derivatives were designed and synthesized by modifying the active positions C1 and C4 on the A ring of sinomenine. Twenty‐three compounds were synthesized and characterized by spectroscopy (IR,¹H‐NMR,¹³C‐NMR, and HRMS). They were further evaluated for their cytotoxic activity against five cancer cell lines, MCF‐7, Hela, HepG2, SW480 and A549, and a normal cell line, Hek293, using MTT and CCK8 methods. The chlorine‐containing compounds exhibited significant cytotoxic activity compared to the nucleus structure of sinomenine. Furthermore, we searched for cancer‐related core targets and verified their interaction with derivatives through molecular docking. The chlorine‐containing compounds 5g, 5i, 5j, 6a, 6d, 6e, and 6g exhibited the best against four core targets AKT1, EGFR, HARS and KARS. The molecular docking results were consistent with the cytotoxic results. Overall, results indicate that chlorine‐containing derivatives might be a promising lead for the development of new anticancer agents.

Full text of DOI:10.3390/molecules26113466

molecules
Article
Design, Synthesis, Biological Evaluation and Silico Prediction
of Novel Sinomenine Derivatives
Shoujie Li 1, Mingjie Gao 2, Xin Nian 3, Liyu Zhang 4, Jinjie Li 5 , Dongmei Cui 3,*, Chen Zhang 4,
*
and Changqi Zhao 1,
*
1
2
Gene Engineering and Biotechnology Beijing Key Laboratory, College of Life Science, Beijing Normal
University, 19 XinjiekouWai Avenue, Beijing 100875, China; 201831200027@mail.bnu.edu.cn
Research Group Pharmaceutical Bioinformatics, Institute of Pharmaceutical Sciences,
Albert-Ludwigs-Universität Freiburg, 79106 Freiburg im Breisgau, Germany;
mingjie.gao@pharmazie.uni-freiburg.de
3
4
5
College of Pharmaceutical Science, Zhejiang University of Technology, 18 Chaowang Road,
Hangzhou 310014, China; zjdqnx@126.com
College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China;
liyuzhang@zju.edu.cn
Beijing Key Laboratory of Bioactive Substances and Functional Foods, Beijing Union University,
197 Beitucheng West Road, Beijing 100191, China; lijinjie.7785004@163.com
Correspondence: cuidongmei@zjut.edu.cn (D.C.); chenzhang@zju.edu.cn (C.Z.); 04020@bnu.edu.cn (C.Z.);
Tel.: +86-010-58805046 (Changqi Zhao)
*
Abstract: Sinomenine is a morphinan alkaloid with a variety of biological activities. Its derivatives
have shown significant cytotoxic activity against different cancer cell lines in many studies. In this
study, two series of sinomenine derivatives were designed and synthesized by modifying the active
positions C1 and C4 on the A ring of sinomenine. Twenty-three compounds were synthesized and
characterized by spectroscopy (IR, 1H-NMR, 13C-NMR, and HRMS). They were further evaluated
for their cytotoxic activity against five cancer cell lines, MCF-7, Hela, HepG2, SW480 and A549, and
a normal cell line, Hek293, using MTT and CCK8 methods. The chlorine-containing compounds
exhibited significant cytotoxic activity compared to the nucleus structure of sinomenine. Furthermore,
we searched for cancer-related core targets and verified their interaction with derivatives through
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Li, S.; Gao, M.; Nian, X.;
Zhang, L.; Li, J.; Cui, D.; Zhang, C.;
Zhao, C. Design, Synthesis, Biological
Evaluation and Silico Prediction of
Novel Sinomenine Derivatives.
Molecules 2021, 26, 3466.
https://doi.org/10.3390/
molecular docking. The chlorine-containing compounds 5g, 5i, 5j, 6a, 6d, 6e, and 6g exhibited the
molecules26113466
best against four core targets AKT1, EGFR, HARS and KARS. The molecular docking results were
consistent with the cytotoxic results. Overall, results indicate that chlorine-containing derivatives
might be a promising lead for the development of new anticancer agents.
Academic Editor:
Giovanni Palmisano
Received: 9 May 2021
Accepted: 2 June 2021
Published: 7 June 2021
Keywords: sinomenine; synthesis; cancer; cytotoxic activity; docking study
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Natural products, also known as secondary metabolites, are organic compounds with
biological functions and activities synthesized by microorganisms or plants in their cells.
They play an irreplaceable role and have broad application prospects in pharmaceutical,
agricultural, chemical industries and other fields [1,2]. In fact, 6.1% of the total 1394 small
molecule drugs developed from 1981 to 2019 were natural compounds, and 27.5% were de-
veloped by structural optimization. Furthermore, small molecule cancer drugs developed
between 1940 and 2019 are directly or indirectly derived from natural products [3].
Alkaloids are nitrogen-containing organic compounds widely found in plants, ani-
mals and microorganisms. Most alkaloids have complex structures with basic nitrogen-
containing heterocycles. A few alkaloids are organic amine alkaloids in which nitrogen
atoms are absent in the ring structure. Thus far, around 12,000 alkaloids have been isolated
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
from nature [
4]. Based on their source or chemical structure, the alkaloids are divided
into about 60 categories, including tropine, isoquinoline, indole alkaloids, terpenoids and
Molecules 2021, 26, 3466. https://doi.org/10.3390/molecules26113466
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3466
2 of 25
steroidal alkaloids. Tropine alkaloids contain a tropinyl skeleton which is formed by the
combination of pyrrole and piperidine rings [
tetrandrine, berberine, sanguinarine, sinomenine and lycorine, are derived from the pheny-
lalanine and tyrosine pathways based on isoquinoline or tetrahydroisoquinoline [ 10].
5]. Isoquinoline alkaloids, such as papaverine,
6–
Indole alkaloids, such as vinblastine and vincristine have indole skeletons and complex
structures [11]. Terpenoids and steroidal alkaloids such as dendrobine, aconitine, and
peiminine have a terpenoid or steroidal structure [12,13]. Alkaloids are well known for
their high efficiency and low toxicity in antitumor activity.
The dry canes of Sinomenium acutum (Thunb.) Rehd. et Wils. and Sinomenium acutum
(Thunb.) Rehd. et Wils. var. cinereum Rehd. et Wils., also known as Qingfeng Teng in
traditional Chinese medicine, have been used in the treatment diseases [14]. In the 1920s,
sinomenine was isolated from Qing Feng Teng (Japanese sabia stem) grown in Japan [15].
Several studies reported that sinomenine (an isoquinoline alkaloid monomer) carrying a
phenanthrene nucleus and ethylamine bridge, has a structure is similar to the narcotics
morphine and codeine. Its chemical structure is shown in Figure 1.
Figure 1. The structures of sionmenine, morphine and codeine.
In previous work, structural modification of sinomenine was carried out in the active
groups of its four rings [9]. The A ring is a benzene ring where C3 and C4 are substituted
by methoxy and hydroxyl groups. Structural modification on the A ring is often present
on the active reaction sites C1 and C4, and sometimes on C2 or C3, but it is relatively
rare [16]. Different sinomenine derivatives were reported in the previous studies as having
good anti-inflammatory, immunosuppressive and neuroprotective activities compared to
sinomenine [17]. In this study, we designed and synthesized two new series of sinomine
derivatives in a quest to obtain potent compounds.
Many scholars have reported the structural modification of the sinomenine A ring.
Due to the good application effect of piperidine and tetrahydropyrrole in medicine, we
took sinomenine as the leading compound and introduced piperidine methylene or tetrahy-
dropyrrole methylene at C1 by Mannich reaction to obtain 1-piperidine methylated sinome-
nine (5a–5k) and 1-tetrahydropyrrole methylated sinomenine (6a–6l) [18]. As the phenolic
hydroxyl group of C4 is easy to oxidize and decompose and may be a factor leading to aller-
gic reactions in vivo, it is necessary to modify the structure to protect the phenolic hydroxyl
group. Esters are important intermediates of organic synthesis. The phenol hydroxyl ester
can not only increase liposolubility, enhance substance activity and change metabolic char-
acteristics, but it can also slow down the oxidation rate of compounds and greatly reduce
their allergic reactions. Therefore, a series of 1,4-bisubstituted sinomenine derivatives were
synthesized by the reaction of phenylacetyl chloride with 1-tetrahydropyrrole methylated
sinomenine. All structures of the products were determined by IR, 1H-NMR, 13C-NMR,
and HRMS.
Furthermore, the cytotoxicity of the synthesized compounds was assessed. Com-
pounds 5a
–5k were tested against three cancer cell lines, MCF-7, Hela, and HepG2, using
the MTT method. Compounds 6a
–6l were tested against four cancer cell lines, MCF-7,
Hela, SW480, and A549, and a normal cell line Hek293 with the CCK8 method. Finally, all
the synthesized compounds underwent network pharmacology and molecular-docking
studies in order to forecast the possible targets of the most active compounds.

Molecules 2021, 26, 3466
3 of 25
2. Results and Discussion
2.1. Synthesis
Two series of sinomenine derivatives were synthesized as shown in Scheme 1 and
Table 1. A total of 23 new 1,4-disubstituted sinomenine derivatives (5a 5k and 6a 6l) were
obtained by the introduction of piperidine and tetrahydropyrrole methylene (at 1- position,
step ) using the Mannich reaction, and then the substitution of the phenylacetyl group by
–
–
a
an acylation reaction was performed (at 4- OH, step b).
0
Scheme 1. Synthetic scheme of compounds
2–6
(R = Compound
4, the red gradient circle represents the ester substitution R.)
2.2. Anticancer Activity of the Synthesized Compounds 5a–5k
The in vitro growth inhibitory activity of the sinomenine derivatives (5a–5k) was
evaluated against different three cell lines, MCF-7, Hela, and HepG2, using sinomenine
as the reference compound kept under the same conditions in an MTT assay [19]. The
inhibition ratios of synthetic compounds were determined at concentrations of 2, 20 and
200
Figure 2.
The results indicated that all the compounds had significant activity against the cancer
cell lines. The synthesized compounds had a structure and dose-dependent relationship.
At a low drug concentration of 2 M, all of the tested compounds showed more toxicity
µM. The obtained data in triplicate for each concentration were plotted, as shown in
µ
towards the three cancer cells lines with inhibition ratios in the lower to medium percentage
range for Hela and HepG2 cell lines, or higher inhibition ratios for MCF-7 cell lines. At a
moderate drug concentration of 20 µM, the compounds 5i and 5j had significant effects on
MCF-7, Hela, and HepG2 cell lines with the inhibition ratios of 54.13 and 63.35%, 94.43 and
80.22%, and 90.69 and 89.57%, respectively. Moreover, the compound 5g showed better
anticancer activity against HepG2 cell lines with inhibition ratios reaching more than 53.2%.
Other tested compounds showed moderate to weak inhibition against both cancer cell lines.
At a high drug concentration of 200 µM, the inhibition ratios of all synthetic compounds,
except 5c, against three cell lines exceeded 50%. In conclusion, the presence of two chlorine
atoms in the R group was critical for the observed cytotoxicity activities.

Molecules 2021, 26, 3466
4 of 25
Table 1. Chemical structures of synthesized sinomenine derivatives (5a–5k, 6a–6l).
NO.
Compounds R1
R2
R3
Product
Yield (%)
1
5a
H
H
H
Cl
90
64
2
5b
H
H
3
5c
H
OMe
OMe
90
4
5d
Me
H
H
85
5
5e
H
H
H
55
6
5f
Cl
H
H
77

Molecules 2021, 26, 3466
5 of 25
Table 1. Cont.
NO.
Compounds R1
R2
R3
Product
Yield (%)
7
5g
H
H
Cl
H
72
81
8
5h
H
OMe
9
5i
5j
Cl
H
H
Cl
Cl
90
52
10
Cl
11
5k
H
H
F
90
12
13
6a
H
H
H
Cl
23
37
6b
OMe
OMe

Molecules 2021, 26, 3466
6 of 25
Table 1. Cont.
NO.
Compounds R1
R2
R3
Product
Yield (%)
14
6c
Cl
Cl
H
H
H
29
22
15
6d
Cl
16
17
18
6e
6f
H
Me
H
Cl
H
H
H
51
42
57
6g
Cl
Cl
19
20
6h
6i
H
H
H
H
H
27
36
OMe

Molecules 2021, 26, 3466
7 of 25
Table 1. Cont.
NO.
Compounds R1
R2
R3
Product
Yield (%)
21
6j
6k
6l
H
OMe
F
H
H
H
NO2
7
22
23
H
H
19
21
Figure 2. Cont.

Molecules 2021, 26, 3466
8 of 25
Figure 2. Anticancer activity of compounds 5a
–
5k against MCF-7, Hela, and HepG2 cell lines at 2,
20, and 200 M concentrations. (A) Human breast cancer cell lines, (B) human cervical cancer cells
µ
lines, (C) human hepatocellular carcinoma cell lines.
2.3. Anticancer Activity of the Synthesized Compounds 6a–6l
Cytotoxicity screens were performed to assess the in vitro anticancer activity of the syn-
thesized sinomenine derivatives 6a 6l on the various cancer cell lines MCF-7, Hela, SW480,
and A549, as well as on Hek293 as a normal cell line, at doses of 2.5 and 25 µM. The ob-
tained inhibition ratios data were used to construct a heat map (Figure 3, Tables S1 and S2
–
)
in order to select compounds that exert cancer cell line-specific cytotoxicity. Then, the IC50
values of the promising compound were determined. Cisplatin was used as a reference
standard. The antiproliferative activity was based on the evaluation of the percent viability
using the CCK8 assay, which is a rapid and sensitive method for determining the number
of viable cells in culture. In the presence of electronic coupling reagents, WST-8 can be
reduced by a dehydrogenase in the mitochondria to produce a highly water-soluble orange-
yellow formazan product. Its color is proportional to the proliferation of the number of
viable cells and is related to cytotoxicity. Using a microplate reader to measure the OD
value can indirectly reflect the number of living cells at 450 nm wavelength [20].
Based on the heat map, 6a and 6c–6g exhibited a minimum of 50% inhibition ratios
on at least one cancer cell line and had no or a very mild effect on normal cell lines. To
understand structure–function relationships, these selected compounds were subjected
to subsequent analyses. All six compounds (6a and 6c–6g) were further examined to
determine their IC50 concentrations on the previously mentioned cell lines, and their
efficacy was compared to the reference drug cisplatin. For this, six compounds and
cisplatin were evaluated on Hela, MCF-7, SW480, and A549, and on noncancerous Hek293
cell lines at various concentrations for 60 h. IC50 values were calculated (Table 2).
In agreement with the primary cytotoxicity screen, the obtained IC50 concentrations
clearly indicated which compounds were selectively effective on one or more cancer cell
lines with IC50 values less than 20 µM. In compounds 6a, 6d, 6e and 6g, with a chlorine
atom attached to the R group, significant anticancer activities were observed compared
to 6f. The derivative 6d, being the superior compound, displayed an IC50 of 5.73, 8.20,
and 6.08
of compound 6e, significant IC50 values were observed for MCF-7 and Hela cell lines (6e
IC50 = 14.86, 13.28, and 16.57 M for MCF-7, Hela, and Hek293, respectively), indicating 6e
had more selective toxicity toward cancer cells than normal cells.
µM against MCF-7, Hela, and SW480, respectively. Concerning the cytotoxicity
,
µ

Molecules 2021, 26, 3466
9 of 25
Figure 3. Heat map representation of the primary cytotoxic effects of novel sinomenine derivatives
on different cell lines (concentration = 2.5 and 25 µM; incubation time = 60 h).
Table 2. The anticancer activities of the synthesized compounds and standard anticancer agent
expressed in terms of IC50 (µM) a.
Compounds
MCF-7
Hela
SW480
A549
Hek293
6a
6c
6d
6e
6f
14.34 ± 0.83
11.52 ± 1.04
14.94 ± 0.06
>25
12.98 ± 0.62
6.52 ± 0.19
3.46 ± 0.02
16.57 ± 0.64
10.11 ± 0.42
4.71 ± 0.48
3.19 ± 0.60
>25
>25
>25
18.72 ± 0.70
11.57 ± 1.61
17.91 ± 0.74
25.05 ± 1.72
>25
5.73 ± 0.36
14.86 ± 0.15
>25
>25
3.45 ± 0.82
8.20 ± 0.52
13.28 ± 0.95
>25
6.08 ± 0.28
>25
>25
>25
1.98 ± 0.15
6g
11.88 ± 0.60
Cisplatin
4.57 ± 0.43
14.35 ± 0.70
a
The cytotoxic activity of compounds on the cancer cell lines determined using the CCK8 assay. The data are
expressed as the mean
inhibition of cancer cell growth (µM) after 60 h.
±
SD of three independent experiments. IC50 values are the concentrations that cause 50%
2.4. Computer-Aided Evaluation
2.4.1. Number of Gene Screens
The candidate genes of the five cell-line-related diseases viz. MCF-7 (1245 genes), Hela
(501 genes), HepG2 (1006 genes), SW480 (583 genes) and A549 (702 genes), were searched,
and duplicates were removed through a series of databases (Figure 4).

Molecules 2021, 26, 3466
10 of 25
Figure 4. The number of genes collected in each database and the total number of genes after duplicates
removed for each cancer cell.
2.4.2. PPI Interaction Network Construction
The gene was uploaded to the String database (https://string-db.org/, version 11.0b,
accessed on 17 October 2020) for the analysis of PPI; the species were limited to Homo sapiens
and the minimum interaction threshold was set to “medium confidence” 0.4. The network
construction used Cytoscape software (https://cytoscape.org/, release 3.8.0, accessed on
15 April 2020). We calculated the degree of each node through the CytoHubba plug-in
and selected the top 10 targets as potential targets for cancer diseases according to their
associated degree. A total of 16 targets playing an important role in cancer diseases were
obtained (Table 3). Subsequently, the PPI network was constructed for the top ten targets of
each cancer disease and visual analysis (Figure 5). The genes AKT1, TP53, EGFR, MYC and
PTEN were determined as high-frequency genes that may have a potential anticancer role.
Table 3. The key targets related to five cancer diseases.
No.
Genes
Uniprot ID
Name
1
2
AKT1
CCND1
CDH1
EGF
EGFR
ERBB2
GAPDH
HRAS
IL6
INS
KRAS
MYC
Q96B36
Q64HP0
P12830
P01133
P00533
P04626
P04406
P01112
P05231
P01308
P01116
P01106
Proline-rich AKT1 substrate 1
G1/S-specific cyclin-D1
3
Cadherin-1
4
Proepidermal growth factor
Epidermal growth factor receptor
Receptor tyrosine-protein kinase erbB-2
Glyceraldehyde-3-phosphate dehydrogenase
GTPase HRas
5
6
7
8
9
Interleukin-6
10
11
12
Insulin
GTPase KRas
Mycproto-oncogene protein
Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase
and dual-specificity protein phosphatase PTEN
Signal transducer and activator of transcription 3
Cellular tumor antigen p53
13
PTEN
P60484
14
15
16
STAT3
TP53
VEGFA
P40763
P04637
P15692
Vascular endothelial growth factor A
2.4.3. GO and KEGG Enrichment Analysis
The GO and KEGG enrichment analysis of the top 10 targets related to five cancer
diseases were analyzed with the DAVID gene annotation tool. GO analysis was applied
to enrich and functionally interpret differentially expressed key candidate genes (KCGs)
at the molecular and cellular levels. The result of the KCGs GO was decomposed into
three subontologies viz. Biological Process (BP), Cellular Component (CC) and Molecular
Function (MF) as shown in Figure 6. For BP, the KCGs were mainly enriched in positive
regulation of transcription from RNA polymerase II promoter, negative regulation of the
apoptotic process, positive regulation of protein phosphorylation and positive regulation of
cell proliferation. The KCGs in MF mainly participate in protein binding. In addition, most
of the KCGs were localized to regions called the cytoplasm, plasma membrane, cytosol and
the nucleus of the CC.

Molecules 2021, 26, 3466
11 of 25
Figure 5. Five cancer disease-related protein interaction networks constructed by Cytoscape. Each
network has 10 nodes with the top 10 degrees. (
related genes, ( ) hepatocellular cancer-related genes, (D) colonic cancer-related genes, and (
adenocarcinoma-related genes.
A
) Breast cancer-related genes, (
B
) cervical cancer-
C
E) lung
Figure 6. Gene ontology terms of top 10 targets of five cancer diseases (p-value < 0.05). BP, MF,
and CC represent Biological Process, Molecular Function, and Cellular Component groups of GO,
respectively. (
A) Breast cancer, (B) cervical cancer, (C) hepatocellular cancer, (D) colonic cancer, and
(E) lung adenocarcinoma.

Molecules 2021, 26, 3466
12 of 25
KEGG pathway databases contain vital information for systematic pathway enrich-
ment analysis of gene functions. Significantly enriched pathways as therapeutic targets
in cancer, including the PI3K/Akt signaling pathway, MAPK signaling pathway, ErbB
signaling pathway, p53 signaling pathway and FoxO signaling pathway, were identified.
The top 30 integrated KEGG pathways of each disease are shown in Figure 7.
Figure 7. Bubble plot of KEGG pathway enrichment analysis of the genes related to the five cancer
diseases. Bubble plot: letters on the left are KEGG names, numbers on the bottom are the proportions
of genes, sizes of the circles indicate the numbers of enriched genes, and colors reflect p-values. The
redder the colors are, the more enriched the genes, and the smaller the p values. (p is normalized
according to
cancer and (E) lung adenocarcinoma.
−
log10). (A) Breast cancer, (B) cervical cancer, (C) hepatocellular cancer, (D) colonic

Molecules 2021, 26, 3466
13 of 25
2.4.4. Molecular Docking
Molecular docking was carried out to elucidate the binding modes of the 24 com-
pounds to the 16 targets (AKT1, CCND1, CDH1, EGF, EGFR, ERBB2, GAPDH, HRAS,
IL6, INS, KRAS, MYC, PTEN, STAT3, TP53 and VEGFA) for which crystal structures were
known. Interestingly, most of the compounds showed a relatively much higher binding
affinity against targets such as AKT1 (PDB ID: 4EJN), KRAS (PDB ID: 4LYH), HRAS (PDB
ID: 121P) and EGFR (PDB ID: 1M17) than the other 10 targets (Figures 8 and 9). For exam-
ple, 6a interacted with AKT1 with a docking score of
−
155.153 kcal/mol, and 6d interacted
with HRAS with a docking score of 133.267, which should be deemed as potent binding.
−
The docking scores of the best-ranked molecules against the selected targets are shown in
Tables S3 and S4 and Figure S59.
Figure 8. The total energy of molecular docking between compounds 5a–5k and 11 potential targets.
Figure 9. The total energy of molecular docking between compounds 6a–6l and 13 potential targets.
As shown in Figures 10 and 11 and Tables S5–S8, a stable composite structure was
formed between the compound and the protein through hydrophobic interaction, hydrogen
bonding, halogen bonding, salt bridges and
π stacking, thereby affecting the structure and
function of the protein and causing cancer cell death. For example, 6e bound to the cavity
of 121P (HRAS) and interacted with eight amino acid residues. The interaction between 6e
and six amino acid residues (ALA18, PHE28, TYR32, LYS117, ALA146 and LYS147) was
hydrophobic. Moreover, 6e interacted with ASP30 and LYS117 through hydrogen bonds,
with GLY15A through halogen bonds, with LYS117 through salt bridges and with PHE28
through π stacking interaction.

Molecules 2021, 26, 3466
14 of 25
Figure 10. The binding pose of the selected ligands. (
protein AKT1; (
binding cavity of the protein HRAS; (D) 5g docked into the binding cavity of the protein HRAS.
A
)
5g docked into the binding cavity of the
B) 5j docked into the binding cavity of the protein EGFR; (C) 5i docked into the
Figure 11. The binding pose of the selected ligands. (A) 6e docked into the binding cavity of the

Molecules 2021, 26, 3466
15 of 25
protein AKT1; (
binding cavity of the protein HRAS; (
6g docked into the binding cavity of the protein HRAS; (F)
B
)
6d docked into the binding cavity of the protein EGFR; (
C
)
6a docked into the
D)
6e docked into the binding cavity of the protein HRAS;
6a docked into the binding cavity of
(E
)
the protein KRAS. The ligand is represented by orange sticks. The active site residues are shown as
blue sticks. The main atoms involving hydrogen bonds are indicated by blue lines. The main atoms
involving hydrophobic bonds are indicated by grey dashes. The main atoms involving halogen
bonds are indicated by green lines. The main atoms involving salt bridges are indicated by yellow
dotted lines. The main atoms involving π-stacking are indicated by green dotted lines. The key
residues participating in hydrogen bonds and hydrophobic interactions are labeled.
3. Materials and Methods
3.1. Chemistry
Unless specified otherwise, all the materials were obtained from commercial suppliers
and used without further purification. Thin layer chromatography (TLC) was performed
using silica gel 60 F254 and visualized using UV light. Column chromatography was
1
performed with silica gel (mesh 300–400). H NMR and 13C NMR spectra were recorded on
a Bruker Avance 500 MHz spectrometer in CDCl3 with Me4Si as an internal standard. Data
were reported as follows: chemical shift in ppm (δ), multiplicity (s = singlet, d = doublet,
t = triplet, q = quartet, br = broad and m = multiplet), coupling constant in Herts (Hz), and
integration. IR spectra were recorded on an FT-IR spectrometer, and only major peaks are
reported in cm−1. Mass data were recorded by ESI on an FT mass spectrometer.
3.1.1. General Procedure for the Synthesis of Compounds 5a–5k
To a mixture of 1-(1-pyrrolidinylmethyl)-Sinomenine or 1-(1-piperidinylmethyl)-Sinomenine
(1.0 mmol) and Et3N (2.5 mmol) in CH2Cl2 (5 mL) was added acyl chloride (2.5 mmol) at
0 ◦C. The reaction mixture was then stirred at room temperature. After the completion
of the reaction, sat. NaHCO3 was added and then extracted with CH2Cl2. The organic
layers were combined and dried with anhydrous Na2SO4 and evaporated under reduced
pressure. The mixture was evaporated under vacuum, and the residue was purified by
flash chromatography with dichloromethane and methanol as the eluents to produce the
pure product.
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(piperidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(4-chlorophenyl)acetate (5a)
White solid; Yield: 493.0 mg, 90%; m.p.:105–107 ◦C; IR (KBr, cm−1): 3426, 2931, 1761,
1693, 1633, 1469, 1384, 1120, 1016, 867; 1H NMR (500 MHz, CDCl3):
δ
7.37 (d, J = 8.0 Hz, 2H),
7.32 (d, J = 8.3 Hz, 2H), 6.70 (s, 1H), 5.45 (s, 1H), 3.92 (d, J = 15.1 Hz, 1H), 3.82 (d, J = 15.1 Hz
,
1H), 3.66 (d, J = 15.9 Hz, 1H), 3.59 (s, 3H), 3.44 (s, 3H), 3.21–3.39 (m, 3H), 3.19 (s, 1H), 2.91
(s, 1H), 2.62 (d, J = 16.3 Hz, 1H), 2.42–2.47 (m, 1H), 2.40 (s, 3H), 2.36 (d, J = 16.0 Hz, 1H),
2.21–2.39 (m, 4H), 1.98 (t, J = 11.0 Hz, 1H), 1.74 (t, J = 10.2 Hz, 1H), 1.44–1.53 (m, 5H),
1.35–1.43 (m, 2H) (Figure S1); 13C NMR (125 MHz, CDCl3):
δ 192.2, 168.9, 152.4, 148.8,
138.5, 133.4, 133.1, 131.8, 131.1, 129.8, 129.5, 128.6, 115.2, 113.3, 61.9, 56.2, 55.8, 54.7, 54.4,
50.1, 46.6, 45.7, 42.7, 40.8, 40.6, 36.9, 26.2, 24.4, 20.8 (Figure S2).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(piperidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-phenylacetate (5b)
White solid; Yield: 513.5 mg, 64%; m.p.:192–194 ◦C; IR (KBr, cm−1): 3413, 2917, 1749,
1686, 1632, 1471, 1382, 1134, 1030, 700; 1H NMR (500 MHz, CDCl3):
δ
7.44 (d, J = 7.4 Hz
2H), 7.36 (t, J = 7.6, 2H), 7.24 -7.31 (m, 1H), 6.71 (s, 1H), 5.45 (d, J = 1.9 Hz, 1H), 3.96
d, J = 14.9 Hz, 1H), 3.88 (d, J = 14.9 Hz, 1H,), 3.68 (d, J = 16.2 Hz, 1H), 3.61 (s, 3H), 3.45 (s,
,
(
3H), 3.23–3.39 (m, 3H), 3.17–3.22 (m, 1H), 2.91 (s, 1H), 2.64 (d, J = 14.9 Hz, 1H), 2.43–2.47
(m, 1H), 2.41 (s, 3H), 2.22–2.37 (m, 5H), 1.91–2.07 (m, 1H), 1.65–1.77 (m, 1H), 1.45–1.56 (m,
5H), 1.42 (d, J = 4.6 Hz, 2H) (Figure S3); 13C NMR (125 MHz, CDCl3):
δ 192.3, 169.4, 152.4,
149.0, 138.7, 133.4, 133.3, 129.9, 129.7, 129.5, 128.5, 127.1, 115.2, 113.4, 62.0, 56.3, 55.8, 54.8,
54.5, 50.0, 46.7, 45.7, 42.7, 41.5, 40.6, 36.9, 26.3, 24.5, 20.8 (Figure S4).

Molecules 2021, 26, 3466
16 of 25
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(piperidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(3,4-dimethoxyphenyl)acetate (5c)
White solid; Yield: 654.6 mg, 90%; m.p.:151–154 ◦C; IR (KBr, cm−1): 3415, 2935, 1762,
1690, 1618, 1466, 1384, 1106, 863; 1H NMR (500 MHz, CDCl3):
δ 6.99–7.05 (m, 1H), 6.97
(dd, J = 8.2, 1.8 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.73 (s, 1H), 5.44 (d, J = 1.8 Hz, 1H), 3.94
(s, 3H), 3.79–3.91 (m, 5H), 3.65 (s, 3H), 3.58 (d, J = 15.5 Hz, 1H), 3.46 (s, 3H), 3.23–3.42 (m,
3H), 3.17–3.22 (m, 1H), 2.89 (s, 1H), 2.64 (d, J = 16.3 Hz, 1H), 2.43–2.50 (m, 1H), 2.42 (s, 3H),
2.26–2.35 (m, 4H), 2.23 (d, J = 16.1 Hz, 1H), 2.00 (t, J = 13.8 Hz, 1H), 1.66 (t, J = 10.3 Hz, 1H),
1.38–1.56 (m,7H) (Figure S5); 13C NMR (125 MHz, CDCl3):
δ 192.4, 169.5, 152.5, 149.2, 149.0,
148.4, 138.8, 133.4, 130.0, 129.6, 125.8, 122.0, 115.2, 113.5, 113.3, 111.6, 62.1, 56.3, 56.0, 55.96,
54.8, 54.6, 49.8, 46.8, 45.7, 42.8, 41.2, 40.6, 36.9, 26.3, 24.5, 20.9 (Figure S6).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(piperidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(o-tolyl)acetate (5d)
White solid; Yield: 472.7 mg, 85%; m.p.:177–178 ◦C; IR (KBr, cm−1): 3417, 2938, 1745,
1686, 1633, 1472, 1384, 1198, 1145, 1107, 1068, 751; 1H NMR (500 MHz, CDCl3):
δ 7.29–7.40
(m, 1H), 7.16–7.24 (m, 3H), 6.71 (s, 1H), 5.44 (d, J = 1.4 Hz, 1H), 3.97 (d, J = 15.4 Hz, 1H), 3.91
(d, J = 15.5 Hz, 1H), 3.56–3.66 (m, 4H), 3.45 (s, 3H), 3.22–3.39 (m, 3H), 3.20 (s, 1H), 2.91 (s,
1H), 2.56–2.71 (m, 1H), 2.43–2.55 (m, 4H), 2.42 (s, 3H), 2.23–2.36 (m, 5H), 1.92–2.07 (m, 1H),
1.66–1.80 (m, 1H), 1.46–1.55 (m, 5H), 1.36–1.45 (m, 2H) (Figure S7); 13C NMR (125 MHz,
CDCl3):
δ 192.2, 169.3, 152.4, 149.0, 138.7, 137.3, 133.3, 132.0, 130.6, 130.4, 130.0, 129.5, 127.4,
126.1, 115.1, 113.4, 62.0, 56.3, 55.8, 54.7, 54.5, 49.8, 46.7, 45.7, 42.7, 40.6, 39.2, 36.9, 26.2, 24.5,
20.9, 19.6 (Figure S8).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(piperidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 3-phenylpropanoate (5e)
White solid; Yield: 306.3 mg, 55%; m.p.:181–185 ◦C; IR (KBr, cm−1): 3459, 2931, 1760,
1692, 1629, 1469, 1384, 1203, 1126, 987; 1H NMR (500 MHz, CDCl3):
δ 7.26–7.29 (m, 4H),
7.16–7.22 (m, 1H), 6.72 (s, 1H), 5.46 (d, J = 1.5 Hz, 1H), 3.76 (d, J = 16.0 Hz, 1H), 3.63 (s, 3H),
3.44 (s, 3H), 3.22–3.37 (m, 3H), 3.18–3.21 (m, 1H), 3.03–3.09 (m, 2H), 3.00 (t, J = 7.0 Hz, 1H),
2.87–2.94 (m, 2H), 2.63 (d, J = 15.2 Hz, 1H), 2.41 (s, 3H), 2.34–2.40 (m, 2H), 2.23–2.35 (m,
4H), 1.90–2.05 (m, 1H), 1.59–1.75 (m, 1H), 1.45–1.58 (m, 5H), 1.38–1.44 (m, 2H) (Figure S9);
13C NMR (125 MHz, CDCl3):
δ = 192.2, 170.7, 152.4, 148.9, 140.3, 138.5, 133.2, 129.8, 129.4,
128.5, 128.3, 126.1, 115.3, 113.2, 62.0, 56.2, 55.7, 54.7, 54.4, 50.2, 46.6, 45.7, 42.6, 40.6, 36.8,
35.7, 30.5, 26.2, 24.4, 20.8 (Figure S10).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(piperidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(2-chlorophenyl)acetate (5f)
White solid; Yield: 442.8 mg, 77%; m.p.:188–190 ◦C; IR (KBr, cm−1): 3414, 2926, 1749,
1684, 1633, 1472, 1384, 1225, 1145, 1107, 1031, 762; 1H NMR (500 MHz, CDCl3):
δ 7.46 (s,
1H), 7.40 (dd, J = 7.8, 1.4 Hz, 1H), 7.19–7.30 (m, 2H), 6.72 (s, 1H), 5.46 (d, J = 1.9 Hz, 1H),
4.12 (d, J = 16.5 Hz, 1H), 4.05 (d, J = 16.5 Hz, 1H), 3.73–3.79 (m, 1H), 3.68 (s, 3H), 3.45
(s, 3H), 3.22–3.40 (m, 3H), 3.17–3.22 (m, 1H), 2.94 (s, 1H), 2.62 (dd, J = 18.2, 3.9 Hz, 1H),
2.43–2.49 (m, 1H), 2.36–2.42 (m, 4H), 2.20–2.35 (m, 4H), 1.80 (t, J = 12.5, 1H), 1.62 (d, J = 12.4
Hz, 1H), 1.45–1.57 (m, 5H), 1.35–1.44 (m, 2H) (Figure S11); 13C NMR (125 MHz, CDCl3):
δ
192.3, 168.3, 152.4, 148.9, 138.7, 134.5, 133.4, 132.1, 131.8, 129.9, 129.5, 129.4, 128.8, 127.0,
115.3, 113.4, 62.0, 56.3, 55.9, 54.7, 54.5, 50.0, 46.7, 45.7, 42.7, 40.7, 39.1, 37.0, 26.2, 24.5, 20.9
(Figure S12).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(piperidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(3-chlorophenyl)acetate (5g)
White solid; Yield: 418.1 mg, 72%; m.p.:164–167 ◦C; IR (KBr, cm−1): 3415, 2934, 1763,
1691, 1619, 1467, 1384, 1120, 939; 1H NMR (500 MHz, CDCl3):
δ 7.43 (s, 1H), 7.24–7.35 (m,
3H), 6.72 (s, 1H), 5.46 (d, J = 1.4 Hz, 1H), 3.95 (d, J = 15.1 Hz, 1H), 3.84 (d, J = 15.1 Hz, 1H),
3.65–3.71 (m, 1H), 3.63 (s, 3H), 3.45 (s, 3H), 3.23–3.41 (m, 3H), 3.17–3.23 (m, 1H), 2.93 (s, 1H),
2.63 (d, J = 16.1 Hz, 1H), 2.47 (dd, J = 12.2, 2.7 Hz, 1H), 2.42 (s, 3H), 2.25–2.40 (m, 5H), 2.00
(t, J = 11.2 Hz, 1H), 1.70–1.85 (m, 1H), 1.44–1.56 (m, 5H), 1.37–1.45 (m, 2H) (Figure S13); 13
C
NMR (126 MHz, CDCl3):
δ 192.2, 168.8, 152.5, 148.9, 138.5, 135.2, 134.3, 133.5, 129.9, 129.8,

Molecules 2021, 26, 3466
17 of 25
129.7, 129.5, 128.1, 127.4, 115.2, 113.4, 62.0, 56.3, 55.8, 54.8, 54.5, 50.1, 46.7, 45.7, 42.7, 41.0,
40.7, 37.0, 26.2, 24.5, 20.9 (Figure S14).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(piperidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(4-methoxyphenyl)acetate (5h)
White solid; Yield: 558.3 mg, 81%; m.p.:121–122 ◦C; IR (KBr, cm−1): 3419, 2936, 1760,
1
1693, 1614, 1514, 1470, 1384, 1250, 1120, 1028, 865; H NMR (500 MHz, CDCl3):
δ 7.35
(d, J = 8.5 Hz, 2H), 6.90 (d, J = 8.7 Hz, 2H), 6.71 (s, 1H), 5.45 (d, J = 1.7 Hz, 1H), 3.89
d, J = 15.1 Hz, 1H), 3.78–3.86 (m, 4H), 3.64–3.72 (m, 1H), 3.62 (s, 3H), 3.46 (s, 3H), 3.22–3.40
(
(m, 3H), 3.16–3.22 (m, 1H), 2.90 (s, 1H), 2.57–2.72 (m, 1H), 2.42–2.48 (m, 1H), 2.37–2.48 (m,
3H), 2.23–2.36 (m, 5H), 1.90–2.06 (m, 1H), 1.59–1.76 (m, 1H), 1.44–1.54 (m, 5H), 1.38–1.44
(m, 2H) (Figure S15); 13C NMR (125 MHz, CDCl3):
δ 192.3, 169.9, 158.9, 152.5, 149.0, 138.8,
133.3, 130.8, 129.0, 129.6, 125.4, 115.3, 114.0, 113.5, 62.0, 56.3, 55.9, 55.3, 54.8, 54.5, 50.0, 46.7,
45.8, 42.7, 40.7, 40.6, 36.9, 26.3, 24.5, 20.9 (Figure S16).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(piperidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(2,4-dichlorophenyl)acetate (5i)
White solid; Yield: 667.5 mg, 90%; m.p.:135–137 ◦C; IR (KBr, cm−1): 3411, 2930, 1766,
1694, 1625, 1473, 1384, 1202, 1098, 869; 1H NMR (500 MHz, CDCl3):
δ 7.50–7.35 (m, 2H),
7.28 (dd, J = 7.8, 2.5 Hz, 1H), 6.74 (s, 1H), 5.47 (d, J = 1.7 Hz, 1H), 4.09 (d, J = 16.6 Hz, 1H),
4.03 (d, J = 16.5 Hz, 1H), 3.74 (d, J = 16.0 Hz, 1H), 3.69 (s, 3H), 3.46 (s, 3H), 3.22–3.41 (m,
3H), 3.18–3.23 (m, 1H), 2.96 (s, 1H), 2.63 (dd, J = 18.4, 4.9 Hz, 1H), 2.47 (dt, J = 9.4, 4.7 Hz,
1H), 2.39–2.45 (m, 4H), 2.24–2.37 (s, 4H), 1.93–2.09 (m, 1H), 1.82 (td, J = 12.5, 4.5 Hz, 1H),
1.61 (d, J = 12.5 Hz, 1H), 1.48–1.46 (m, 4H), 1.39–1.45 (m, 2H) (Figure S17); 13C NMR (125
MHz, CDCl3):
δ 192.2, 168.0, 152.5, 148.9, 138.6, 135.2, 134.0, 133.6, 133.0, 130.5, 129.9, 129.5,
129.2, 127.4, 115.3, 113.4, 62.0, 56.3, 56.0, 54.8, 54.5, 50.2, 46.7, 45.8, 42.8, 40.8, 38.6, 37.2, 26.3,
24.5, 20.9 (Figure S18).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(piperidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(3,4-dichlorophenyl)acetate (5j)
White solid; Yield: 262.3 mg, 52%; m.p.:143–146 ◦C; IR (KBr, cm−1): 3415, 2933, 1766,
1693, 1616, 1471, 1384, 1120, 945; 1H NMR (500 MHz, CDCl3):
δ
7.54 (d, J = 1.5 Hz, 1H), 7.44
(d, J = 8.2 Hz, 1H), 7.27–7.34 (m, 1H), 6.72 (s, 1H), 5.47 (d, J = 1.5 Hz, 1H), 3.94 (d, J = 15.4 Hz
,
1H), 3.82 (d, J = 15.4 Hz, 1H), 3.69 (d, J = 16.4 Hz, 1H), 3.63 (s, 3H), 3.46 (s, 3H), 3.24–3.41 (m,
3H), 3.17–3.22 (m, 1H), 2.93 (s, 1H), 2.62 (d, J = 16.4 Hz, 1H), 2.44–2.49 (m, 1H), 2.39–2.44
(m, 4H), 2.22–2.38 (m, 4H), 1.99 (t, J = 11.0 Hz, 1H), 1.85–1.73 (m, 1H), 1.45–1.57 (m, 5H),
1.35–1.46 (m, 2H) (Figure S19); 13C NMR (125 MHz, CDCl3):
δ = 192.2, 168.4, 152.4, 148.7,
138.3, 133.6, 133.5, 132.4, 131.5, 131.4, 130.4, 129.8, 129.6, 129.3, 115.3, 113.2, 62.0, 56.2, 55.8,
54.8, 54.5, 50.3, 46.5, 45.8, 42.7, 40.8, 40.4, 37.1, 26.2, 24.5, 20.8 (Figure S20).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(piperidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(4-fluorophenyl)acetate (5k)
White solid; Yield: 667.5 mg, 90%; m.p.:128–130 ◦C; IR (KBr, cm−1): 3416, 2932, 1761,
1693, 1627, 1510, 1468, 1221, 1119, 1025, 865; 1H NMR (500 MHz, CDCl3):
δ 7.40 (dd, J = 7.4,
5.7 Hz, 2H), 7.04 (t, J = 8.7 Hz, 2H), 6.70 (s, 1H), 5.46 (s, 1H), 3.93 (d, J = 15.1 Hz, 1H), 3.83
(d, J = 15.1 Hz, 1H), 3.67 (d, J = 16.0 Hz, 1H), 3.60 (s, 3H), 3.45 (s, 3H), 3.23–3.39 (m, 3H),
3.19 (s, 1H), 2.91 (s, 1H), 2.57–2.69 (m, 1H), 2.43–2.47 (m, 1H), 2.41 (s, 3H), 2.23–2.38 (m,
5H), 1.99 (t, J = 11.3 Hz, 1H), 1.74 (dd, J = 12.0, 8.5 Hz, 1H), 1.55–1.45 (m, 5H), 1.37–1.44 (m,
2H) (Figure S21); 13C NMR (125 MHz, CDCl3):
δ 192.2, 169.2, 162.1 (d, J = 244.3 Hz), 152.5,
148.9, 138.6, 133.4, 131.3 (d, J = 8.0 Hz), 129.9, 129.6, 129.0, 115.3 (d, J = 21.1 Hz), 115.2, 113.4,
62.0, 56.2, 55.8, 54.7, 54.5, 50.1, 46.6, 45.8, 42.7, 40.7, 40.6, 37.0, 26.2, 24.6, 20.8 (Figure S22).
3.1.2. General Procedure for the Synthesis of Compounds 6a–6l
To a mixture of 1-(1-pyrrolidinylmethyl)-Sinomenine or 1-(1-piperidinylmethyl)-Sinomenine
(1.0 mmol) and Et3N (2.5 mmol) in CH2Cl2 (5 mL) was added acyl chloride (2.5 mmol)
◦
at 0 C. The reaction mixture was then stirred at room temperature. After completion
of the reaction, sat. NaHCO3 was added and then extracted with CH2Cl2. The organic
layers were combined and dried with anhydrous Na2SO4 and evaporated under reduced

Molecules 2021, 26, 3466
18 of 25
pressure. The mixture was evaporated under vacuum, and the residue was purified
by flash chromatography with dichloromethane and methanol as the eluent to give the
pure product.
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(pyrrolidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(4-chloroph◦enyl)acetate (6a)
White solid; Yield: 239.5 mg, 23%; m.p.:162–165 C; IR (KBr, cm−1): 3465, 1634,
1
1384, 1217, 1089, 771, 534; H NMR (500 MHz, CDCl3):
δ 7.39 (d, J = 8.1 Hz, 2H), 7.34
(
d, J = 8.1 Hz, 2H), 6.77 (s, 1H), 5.47 (s, 1H), 3.94 (d, J = 15.1 Hz, 1H), 3.84 (d, J = 15.1 Hz,
1H), 3.68 (d, J = 16 Hz, 1H), 3.62 (s, 3H), 3.52 (br, 2H), 3.46 (s, 3H), 3.21 (br, 2H), 2.93 (br, 1H),
2.67 (d, J = 15.9 Hz, 1H), 2.45–2.40 (m, 5H), 2.40 (s, 3H), 2.01 (dd, J = 11.7, 10.7 Hz, 1H), 1.83
(br, 2H), 1.74 (br, 4H), 1.49 (d, J = 10.7 Hz, 1H) (Figure S24); 13C NMR (125 MHz, CDCl3):
δ
192.4, 169.1, 152.4, 148.9, 138.4, 134.4, 133.1, 131.8, 131.1, 129.8, 129.0, 128.6, 115.2, 112.6,
58.5, 56.1, 55.8, 54.7, 54.0, 50.1, 46.6, 45.7, 42.7, 40.8, 40.7, 36.9, 23.6, 20.8 (Figure S25). HRMS
(ESI) m/z [M+H]+ calcd for C32H38ClN2O5 565.24637, found 565.24548 (Figure S23).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(pyrrolidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(3,4-dimethoxyphenyl)acetate (6b)
Yellow solid; Yield: 780.3 mg, 37%; m.p.: 80–83 ◦C; IR (KBr, cm−1): 3466, 3018, 2958,
2839, 1759, 1688, 1632, 1515, 1465, 1384, 1263, 1145, 1107, 1026, 758, 666, 540; 1H NMR (500
MHz, CDCl3):
δ 7.02 (s, 1H), 6.97 (dd, J = 8.2, 1.9 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.78 (s,
1H), 5.44 (d, J = 1.8 Hz, 1H), 3.94 (s, 3H), 3.88 (s, 3H), 3.87 (d, J = 14.5 Hz, 1H), 3.82 (d, J
= 14.5 Hz, 1H), 3.65 (s, 3H), 3.57 (d, J = 15.8 Hz, 1H), 3.51 (br, 2H), 3.44 (s, 3H), 3.14–3.10
(m, 2H), 2.89–2.86 (m, 1H), 2.67 (dd, J = 18.2, 3.7 Hz, 1H), 2.45–2.40 (m, 5H), 2.38 (s, 3H),
2.23 (d, J=16.1, 1H), 2.00 (t, J = 11.2 Hz, 1H), 1.76–1.71 (m, 4H), 1.65 (td, J = 13.2, 4.0 Hz,
1H), 1.44–1.42(m, 1H) (Figure S27); 13C NMR (125 MHz, CDCl3):
δ 192.5, 169.6, 152.3, 149.1,
149.0, 148.3, 138.5, 134.3, 129.9, 128.9, 125.6, 121.9, 115.1, 113.0, 112.6, 111.3, 58.5, 56.2, 55.9,
54.7, 54.1, 49.6, 46.7, 45.6, 42.7, 41.2, 40.5, 36.7, 23.6, 20.9 (Figure S28). HRMS (ESI) m/z
[M+H]+ calcd for C34H43N2O7 591.3064781, found 591.30579 (Figure S26).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(pyrrolidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(2-chlorophenyl)acetate (6c)
White solid; Yield: 432.5 mg, 29%; m.p.: 79–83 ◦C; IR (KBr, cm−1): 3423, 3013, 2932,
2840, 2801, 1765, 1690, 1629, 1469, 1444, 1414, 1379, 1344, 1320, 1202, 1122, 1021, 990, 859,
754, 665, 558; 1H NMR (500 MHz, CDCl3):
δ 7.48 (d, J = 7.6 Hz, 1H), 7.41 (dd, J = 7.6, 1.5
Hz, 1H), 7.28 (td, J = 7.6, 1.6 Hz, 1H), 7.24 (td, J = 7.6, 1.6 Hz, 1H), 6.78 (s, 1H), 5.46 (d, J
= 1.9 Hz, 1H), 4.13 (d, J = 16.5 Hz, 1H), 4.06 (d, J = 16.5 Hz, 1H), 3.75 (d, J = 16 Hz, 1H),
3.70 (s, 3H), 3.55 (d, J = 12.5 Hz, 1H), 3.50 (d, J = 12.3 Hz, 1H), 3.45 (s, 3H), 3.25–3.20 (m,
2H), 2.98–2.95 (m, 1H), 2.69 (dd, J = 18.2, 4.4 Hz, 1H), 2.50 (ddd, J = 12.2, 4.4, 1.8 Hz, 1H),
2.46–2.37 (m, 8H), 2.03 (td, J = 12.2, 3.3 Hz, 1H), 1.82 (td, J = 12.7, 4.6 Hz, 1H), 1.76–1.69 (m,
4H), 1.64 (dt, J = 12.5, 4.6 Hz, 1H) (Figure S30); 13C NMR (125 MHz, CDCl3):
δ 192.3, 168.3,
152.3, 149.1, 138.6, 134.5,134.2, 132.1, 131.7, 129.7, 129.4, 128.8, 128.7, 127.0, 115.0, 112.7, 58.5,
56.1, 55.9, 54.7, 54.0, 49.8, 46.6, 45.4, 42.5, 40.6, 39.1, 36.8, 23.6, 20.9 (Figure S31). HRMS (ESI)
m/z [M+H]+ calcd for C32H38ClN2O5 565.24637, found 565.24530 (Figure S29).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(pyrrolidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(2,4-dichlorophenyl)acetate (6d)
White solid; Yield: 342.8 mg, 22%; m.p.: 82–86 ◦C; IR (KBr, cm−1): 3451, 2927, 1765,
1688, 1633, 1474, 1384, 1202, 1125, 1099, 990, 928, 770, 524; 1H NMR (500 MHz, CDCl3):
δ
7.47–7.39 (m, 2H), 7.27 (dd, J = 8.2, 2.1 Hz, 1H), 6.78 (s, 1H), 5.46 (d, J = 1.7 Hz, 1H), 4.09
(d, J = 16.5 Hz,1H), 4.02 (d, J = 16.5 Hz, 1H), 3.73 (d, J = 16.4 Hz, 1H), 3.69 (s, 3H), 3.56
(d, J = 11.7 Hz, 1H), 3.50 (d, J = 11.7 Hz, 1H), 3.45 (s, 3H), 3.28–3.17 (m, 2H), 2.97 (br, 1H),
2.69 (d, J = 16.2 Hz, 1H), 2.51 (dd, J = 11.8, 2.3 Hz, 1H), 2.46–2.41 (m, 8H), 2.03 (t, J = 12.2 Hz,
1H), 1.84 (td, J = 12.5, 4.3 Hz, 1H), 1.76–1.71 (m, 4H), 1.61 (d, J = 12.3 Hz, 1H) (Figure S33);
13C NMR (125 MHz, CDCl3):
δ 192.2, 168.0, 152.4, 149.0, 138.4, 135.2, 134.3, 133.9, 132.9,
130.4, 129.7, 129.2, 128.8, 127.4, 115.1, 112.7, 58.5, 56.1, 55.9, 54.7, 54.0, 50.0, 46.6, 45.5, 42.5,
40.6, 38.5, 36.9, 23.6, 20.9 (Figure S34). HRMS (ESI) m/z [M+H]+ calcd for C32H37Cl2N2O5
599.20740, found 599.20679 (Figure S32).

Molecules 2021, 26, 3466
19 of 25
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(pyrrolidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(3-chlorophenyl)acetate (6e)
White solid; Yield: 765.6 mg, 51%; m.p.: 68–72 ◦C; IR (KBr, cm−1): 3419, 2959, 1760,
1
1635, 1470, 1384, 1216, 1106, 990, 770, 524; H NMR (500 MHz, CDCl3):
δ 7.45–7.43 (m,
1H), 7.34–7.26 (m, 3H), 6.78 (s, 1H), 5.46 (d, J = 1.9 Hz, 1H), 3.95 (d, J = 15.1 Hz,1H), 3.85
d, J = 15.1 Hz, 1H), 3.67 (d, J = 16.1 Hz, 1H), 3.64 (s, 3H), 3.52 (br, 2H), 3.45 (s, 3H), 3.23–3.19
(
(m, 2H), 2.95–2.92 (m, 1H), 2.67 (d, J = 15.1 Hz, 1H), 2.49–2.40 (m, 9H), 2.02 (t, J = 13.1 Hz,
1H), 1.78 (dd, J = 12.5, 4.4 Hz, 1H), 1.76–1.70 (m, 4H), 1.50 (d, J = 10.4 Hz, 1H) (Figure S36);
13C NMR (125 MHz, CDCl3):
δ 192.3, 168.9, 152.4, 149.0, 138.4, 135.2, 134.4, 134.2, 129.8,
129.7, 129.6, 128.9, 128.1, 127.4, 115.2, 112.6, 58.5, 56.1, 55.8, 54.7, 54.0, 50.0, 46.6, 45.6, 42.6,
41.0, 40.7, 36.8, 23.6, 20.8 (Figure S37). HRMS (ESI) m/z [M+H]+ calcd for C32H38ClN2O5
565.24637, found 565.24530 (Figure S35).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(pyrrolidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(o-tolyl)acetate (6f)
White solid; Yield: 600.5 mg, 42%; m.p.: 72–75 ◦C; IR (KBr, cm−1): 3417, 2958, 1755,
1
1635, 1463, 1384, 1217, 1091, 990, 771, 530; H NMR (500 MHz, CDCl3):
δ 7.38–7.32 (m,
1H), 7.23–7.18 (m, 3H), 6.76 (s, 1H), 5.44 (d, J = 1.9 Hz, 1H), 3.96 (d, J = 15.4 Hz, 1H), 3.91
d, J = 15.4 Hz, 1H), 3.63 (s, 3H), 3.58 (d, J = 16.1 Hz, 1H), 3.51 (br, 2H), 3.44 (s, 3H), 3.26–3.10
(m, 2H), 2.92–2.88 (m, 1H), 2.68 (d, J = 16.0 Hz, 1H), 2.45–2.39 (m, 11H), 2.28 (d, J = 16.1 Hz,
1H), 2.01 (t, J = 11.6 Hz, 1H), 1.75–1.70 (m, 5H), 1.50 (d, J = 12.1 Hz, 1H) (Figure S39); 13
NMR (125 MHz, CDCl3): 192.3, 169.3, 152.3, 149.1, 138.5, 137.3, 134.2, 131.9, 130.6, 130.4,
(
C
δ
129.8, 128.9, 127.4, 126.0, 115.1, 112.6, 58.5, 56.2, 55.8, 54.7, 54.0, 49.7, 46.7, 45.6, 42.7, 40.5,
39.2, 36.8, 23.6, 20.8. 19.6 (Figure S40). HRMS (ESI) m/z [M+H]+ calcd for C33H41N2O5
545.30099, found 545.30072 (Figure S38).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(pyrrolidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(3,4-dichlorophenyl)acetate (6g)
◦
White solid; Yield: 917.1 mg, 57%; m.p.: 160–164 C; IR (KBr, cm−1): 3416, 2926,
1
1761, 1689, 1636, 1618, 1470, 1384, 1201, 1106, 1032, 753, 618, 482; H NMR (500 MHz,
CDCl3):
1H), 5.47 (d, J = 1.7 Hz, 1H), 3.94 (d, J = 15.3 Hz, 1H), 3.82 (d, J = 15.3 Hz, 1H), 3.68
d, J = 16.0 Hz, 1H), 3.64 (s, 3H), 3.52 (br, 2H), 3.45 (s, 3H), 3.21 (t, J = 4.3 Hz, 2H), 2.95–2.92
δ 7.56–7.53 (m, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H), 6.78 (s,
(
(m, 1H), 2.66 (d, J = 16.6 Hz, 1H), 2.48–2.40 (m, 9H), 2.04–2.00 (m, 1H), 1.79 (td, J = 12.5,
4.4 Hz, 1H), 1.75–1.70 (m, 4H), 1.51 (d, J = 12.1 Hz, 1H) (Figure S42); 13C NMR (125 MHz,
CDCl3):
δ 192.2, 168.4, 152.4, 148.8, 138.2, 134.5, 133.5, 132.3, 131.5, 131.3, 130.4, 129.7, 129.3,
129.0, 115.3, 112.5, 58.5, 56.1, 55.8, 54.7, 54.0, 50.2, 46.6, 45.7, 42.7, 40.7, 40.4, 37.0, 23.6,
20.8 (Figure S43). HRMS (ESI) m/z [M+H]+ calcd for C32H37Cl2N2O5 599.20740, found
599.20667 (Figure S41).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(pyrrolidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-phenylaceta◦te (6h)
White solid; Yield: 156.0 mg, 27%; m.p.: 165–168 C; IR (KBr, cm−1): 3417, 2958,
1
1760, 1687, 1636, 1616, 1464, 1413, 1384, 1215, 1148, 1107, 940, 770, 621, 481; H NMR
(500 MHz, CDCl3): δ 7.47–7.42 (m, 2H), 7.38–7.35 (m, 2H), 7.31–7.28 (m, 1H), 6.77 (s, 1H),
5.44 (d, J = 1.8 Hz, 1H), 3.96 (d, J = 14.9 Hz, 1H), 3.88 (d, J = 14.9 Hz, 1H), 3.66 (d, J = 16.2
Hz, 1H), 3.62 (s, 3H), 3.53 (br, 2H), 3.45 (s, 3H), 3.27–3.19 (m, 2H), 2.96–2.92 (m, 1H), 2.71
(dd, J = 17.9, 4.0 Hz, 1H), 2.51–2.41 (m, 8H), 2.31 (d, J = 15.9 Hz, 1H), 2.07–2.03 (m, 1H),
1.78–1.70 (m, 5H), 1.48 (d, J = 11.1 Hz, 1H) (Figure S45); 13C NMR (125 MHz, CDCl3):
δ
192.3, 169.7, 152.4, 149.2, 138.6, 134.2, 133.2, 129.8, 128.7, 128.6, 127.2, 114.9, 112.8, 58.5, 56.2,
55.9, 54.8, 54.0, 49.7, 46.7, 45.3, 42.5, 41.5, 40.4, 36.6, 23.6, 20.9 (Figure S46). HRMS (ESI) m/z
[M+H]+ calcd for C32H39N2O5 531.28534, found 531.28503 (Figure S44).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(pyrrolidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(4-methoxyphenyl)acetate (6i)
White solid; Yield: 282.1 mg, 36%; m.p.: 76–79 ◦C; IR (KBr, cm−1): 3435, 2959, 2838,
1760, 1685, 1630, 1514, 1466, 1382, 1322, 1302, 1250, 1202, 1180, 1148, 1105, 1026, 989, 537;
1H NMR (500 MHz, CDCl3):
δ 7.33 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2H), 6.74 (s,

Molecules 2021, 26, 3466
20 of 25
1H), 5.43 (d, J =1.0 Hz, 1H), 3.87 (d, J = 15.1 Hz, 1H), 3.79 (d, J = 15.1 Hz, 1H), 3.76 (s,
3H), 3.65 (d, J = 16.1 Hz, 1H), 3.60 (s, 3H), 3.49 (br, 2H), 3.41 (s, 3H), 3.21–3.10 (m, 2H),
2.87 (br, 1H), 2.65 (d, J = 15.5 Hz, 1H), 2.45–2.37 (m, 5H), 2.36 (s, 3H), 2.28 (d, J = 16.1 Hz,
1H), 1.98 (t, J = 11.3 Hz, 1H), 1.74–1.63 (m, 5H), 1.49 (d, J = 10.7 Hz, 1H) (Figure S48); 13
C
NMR (125 MHz, CDCl3):
δ 192.3, 169.6, 158.7, 152.2, 148.9, 138.4, 134.1, 130.6, 129.7, 128.8,
125.2, 115.1, 113.8, 112.5, 58.4, 56.0, 55.7, 55.1, 54.6, 53.9, 49.7, 46.6, 45.5, 42.5, 40.5, 40.4, 36.6,
23.5, 20.7 (Figure S49). HRMS (ESI) m/z [M+H]+ calcd for C33H41N2O6 561.29591, found
561.29486 (Figure S47).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(pyrrolidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(4-nitrophenyl)acetate (6j)
◦
Yellow solid; Yield: 71.6 mg, 7% yield; m.p.: 82–85 C; IR (KBr, cm−1): 3450, 2962, 1762,
1685, 1630, 1520, 1466, 1383, 1347, 1203, 1182, 1122, 1018, 989, 857, 560; 1H NMR (500 MHz,
CDCl3):
δ 8.24 (d, J = 8.7 Hz, 2H), 7.65 (d, J = 8.7 Hz, 2H), 6.78 (s, 1H), 5.48 (d, J =1.4 Hz,
1H), 4.10 (d, J = 15.4 Hz, 1H), 3.98 (d, J = 15.4 Hz, 1H), 3.67 (d, J = 15.9 Hz, 1H), 3.61 (s,
3H), 3.52 (br, 2H), 3.46 (s, 3H), 3.28–3.16 (m, 2H), 2.98–2.93 (m, 1H), 2.67 (d, 1H, J =17.0 Hz)
2.48–2.39 (m, 9H), 2.02 (t, J = 11.1 Hz, 1H), 1.81 (td, J =12.4Hz, 1H) 1.77–1.71 (m, 4H), 1.52
(d, J = 8.7 Hz, 1H) (Figure S51); 13C NMR (125 MHz, CDCl3):
δ 192.1, 169.1, 152.4, 148.8,
147.3, 140.7, 138.2, 134.7, 130.8, 129.7, 129.1, 123.7, 115.3, 112.6, 58.6, 56.1, 55.8, 54.8, 54.1,
50.3, 46.6, 45.8, 42.7, 41.2, 40.8, 37.1, 23.6, 20.8 (Figure S52). HRMS (ESI) m/z [M+H]+ calcd
for C32H38N3O7 576.27042, found 576.26965 (Figure S50).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(pyrrolidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(2-methoxyphenyl)acetate (6k)
White solid; Yield: 239.5 mg, 19%; m.p.: 180–182 ◦C; IR (KBr, cm−1): 3443, 3023, 3007,
2965, 2926, 2849, 2790, 1766, 1700, 1622, 1606, 1498, 1467, 1340, 1291, 1249, 1201, 1128, 1112,
1091, 1023, 991, 929, 766, 745, 534, 492; 1H NMR (500 MHz, CDCl3):
δ 7.33 (d, J = 7.1 Hz,
1H), 7.28 (td, J = 7.1, 1.5 Hz, 1H), 6.96 (t, J = 7.1 Hz, 1H), 6.91 (d, J = 7.1 Hz, 1H), 6.78 (s, 1H),
5.45 (d, J =1.7 Hz, 1H), 4.06 (d, J = 15.9 Hz, 1H), 3.87 (s, 3H), 3.84 (d, J = 15.9 Hz, 1H), 3.77
(d, J = 16.1 Hz, 1H), 3.71 (s, 3H), 3.57–3.48 (m, 2H), 3.45 (s, 3H), 3.25–3.13 (m, 2H), 2.95–2.89
(m, 1H), 2.70 (dd, J = 18.4, 5.2 Hz, 1H), 2.49–2.37 (m, 8H), 2.31 (d, J = 16.1 Hz, 1H), 2.03
(td, J = 11.6, 1.7 Hz, 1H), 1.79–1.70 (m, 5H), 1.63 (dt, J = 12.4, 2.5 Hz, 1H) (Figure S54); 13
C
NMR (125 MHz, CDCl3):
δ 192.5, 169.7, 157.5, 152.3, 149.2, 138.7, 134.1, 131.3, 129.9, 128.9,
128.6, 122.2, 120.4, 115.1, 112.6, 110.4, 58.5, 56.2, 56.0, 55.3, 54.7, 54.0, 49.4, 46.8, 45.7, 42.7,
40.5, 36.6, 35.8, 23.6, 20.8 (Figure S55). HRMS (ESI) m/z [M+H]+ calcd for C33H41N2O6
561.29591, found 561.29504 (Figure S53).
(4bR,8aS,9S)-3,7-dimethoxy-11-methyl-6-oxo-1-(pyrrolidin-1-ylmethyl)-6,8a,9,10-tetrahydro-
5H-9,4b-(epiminoethano)phenanthren-4-yl 2-(2-fluorophenyl)acetate (6l)
White solid; Yield: 210.3 mg, 21%; m.p.: 178–180 ◦C; IR (KBr, cm−1): 3445, 2965, 2924,
2892, 2800, 1752, 1687, 1633, 1604, 1495, 1471, 1459, 1414, 1373, 1320, 1305, 1295, 1237, 1200,
1146, 1107, 1033, 926, 899, 876, 851, 767, 665, 613, 565; 1H NMR (500 MHz, CDCl3):
δ 7.43
(t, J = 7.5 Hz, 1H), 7.31–7.24 (m, 1H), 7.15 (td, J = 7.5, 1.0 Hz, 1H), 7.09 (dd, J = 7.5, 1.0 Hz,
1H), 6.78 (s, 1H), 5.46 (d, J =1.9 Hz, 1H), 4.02 (d, J = 16.4 Hz, 1H), 3.96 (d, J = 16.4 Hz, 1H),
3.75 (d, J = 16.0 Hz, 1H), 3.69 (s, 3H), 3.52 (br, 2H), 3.45 (s, 3H), 3.27–3.14 (m, 2H), 2.95–2.91
(m, 1H), 2.67 (dd, J = 18.0, 3.9 Hz, 1H), 2.49–2.38 (m, 9H), 2.01 (t, J = 11.7, 1.7 Hz, 1H), 1.80
(td, J = 12.5, 4.6 Hz, 1H), 1.76–1.70 (m, 4H), 1.59 (d, J = 12.5 Hz, 1H) (Figure S57); 13C NMR
(125 MHz, CDCl3):
δ 192.4, 168.7, 161.1 (d, J = 245.3 Hz), 152.3, 149.0, 138.5, 134.3, 132.0
(d, J = 3.8 Hz), 129.8, 129.2 (d, J = 8.1 Hz), 129.0, 124.2 (d, J = 3.7 Hz), 120.8 (d, J = 15.7 Hz),
115.4, 115.2, 112.6, 58.6, 56.2, 56.0, 54.7, 54.0, 49.9, 46.7, 45.7, 42.7, 40.7, 36.9, 34.6, 23.6, 20.8
(Figure S58). HRMS (ESI) m/z [M+H]+ calcd for C32H38FN2O5 549.27592, found 549.27588
(Figure S56).
3.2. Biological Activity
3.2.1. Drugs and Drug Treatments
Cisplatin was purchased from Biyuntian (Shanghai, China), CAS: 15663-27-1, purity >99%
;
dimethyl sulfoxide (DMSO) was purchased from Bailingwei (Beijing, China); synthetic and

Molecules 2021, 26, 3466
21 of 25
purchased positive control compounds under investigation were dissolved in DMSO to
produce a stock solution.
3.2.2. Cell Lines
The MCF-7 (human breast cancer cell lines), Hela (human cervical cancer cells lines),
and HepG2 (human hepatocellular carcinoma cell lines) cells were used to measure the
cytotoxicity of the 5a–5k series, which were provided by the Academy of Military Medical
Sciences (Beijing, China). The cell lines were maintained on RPMI 1640 nutrient medium
supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, 100 mg/mL
streptomycin and 100 units/mL penicillin. The cells were grown at 37 ◦C in a humidified
atmosphere with 5% CO2 and were subcultured two to three times a week.
The MCF-7 (human breast cancer cell lines), Hela (human cervical cancer cell lines),
SW480 (human colon adenocarcinoma cell lines), A549 (human lung adenocarcinoma cell
lines) and nontoxic normal cells Hek293 (human embryonic kidney cell lines) cells were
used to measure the cytotoxicity of the 6a–6l. They were obtained from the Beijing Normal
University (Beijing, China). The test cell lines were cultured in DMEM, IMDM, F12 and
DMEM nutrient medium containing 10% heat-inactivated fetal bovine serum, 100 mg/mL
streptomycin and 100 units/mL penicillin in a humified, 5% (v/v) CO2 atmosphere at 37 ◦C.
DMEM culture medium (Dulbecco’s modified essential medium), PBS (Phosphate
Buffer Solution), penicillin and streptomycin were purchased from Gibco (Beijing, China).
FBS (fetal bovine serum), RPMI 1640, and F12 culture medium were purchased from
HyClone (Beijing, China). IMEM culture medium and 0.25% Trypsin-EDTA were purchased
from Macgene (Beijing, China). MTT was purchased from Sigma (Beijing, China). CCK8
(Cell Counting Kit-8) was purchased from Dojindo (Shanghai, China).
3.2.3. Anticancer Evaluation
MTT Assay
The MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) assay
with a slight modification was used to determine the inhibition effects of substrates 5a
MCF-7, Hela and HepG2 were used for testing. Briefly, cells (5.0
103 cells per well) were
seeded in 100 L of the RPMI-1640 medium in 96-well plates for 24 h, treated with drugs
and complete medium in wells up to 200 L for 24 h. RPMI-1640 samples were employed
–5k.
×
µ
µ
as negative controls, and cisplatin as a positive control. Four quadruplicates of each
concentration for all tested compounds were evaluated in three independent assays. For
this, after removing the drug containing media 100 µL, 10 µL of MTT solution was added
to wells and incubated for 4 h under similar conditions. After that, the supernatant from
each well was removed and the formazan crystals formed by viable cells were dissolved
with DMSO (100 µL/well). At the end of incubation, optical densities at 570 nm were
measured using a plate reader (BIO-TEK, USA). Cell viability was calculated based on the
measured absorbance relative to the absorbance of the cells exposed to the negative control,
which represented 100% cell viability.
CCK8 Assay
Cell viability was estimated using the CCK8 assay. Tumor cells (100 mL) viz. MCF-7,
Hela, SW480, A549 and nontoxic normal cells Hek293 were seeded in medium containing
Corning® 96-well tissue culture plates at confluences of 20, 15, 20, 15, 40 and 13%, respec-
tively, in Incucyte ZOOM. Fresh medium (100 µL) containing different concentrations of
the test sample was added after 12 h of seeding. The microtiter plates were incubated at
37 ◦C in a humidified incubator with 5% CO2 for 60 h. Triplicates from each concentration
were used. Negative control cells were incubated without sample and cisplatin as a positive
control. At the end of treatment, the numbers of viable cells were determined by the CCK8
test. In brief, the media was removed from the 96 well plates and replaced with 100 µL of
fresh culture medium with 10% CCK8 solution added to each well including the untreated
controls. The 96 well plates were then incubated at 37 ◦C and 5% CO2 for 1–4 h. Then,

Molecules 2021, 26, 3466
22 of 25
the absorbance was measured at 450 nm using a BMG LABTECH®-POLAR star Omega
microplate reader (Ortenberg, Germany) to determine the number of viable cells, and the
percentage of viability was calculated as ((ODt- ODb/ODc- ODb))
×
100%, where ODt is
the mean optical density of wells treated with the tested sample, ODc is the mean optical
density of wells treated with DMSO and ODb is the mean optical density of PBS wells
without cells. The relation between surviving cells and drug concentrations were plotted to
get the survival curve of each tumor cell line after treatment with the specified compound.
The 50% inhibitory concentration (IC50), the concentration required to cause toxic effects in
50% of intact cells, was estimated from graphic plots of the dose response curve for each
concentration [21,22].
3.3. In Silico Study
3.3.1. Collection of Related Genes
Multiple disease-related gene databases viz. Online Mendelian Inheritance in Man
(OMIM) [23], Human Gene Function and Network Analysis (CooLGeN) [24], The Com-
parative Toxicogenomics Database (CTD) [25] and GeneCards [26], were used to collect
the cancer-associated genes. Either advanced search or custom filtering criteria were used
for the gene retrieval. For Cool GeN, genes collected with all human genes were retrieved.
For CTD, genes annotated with direct evidence and labeled as “M” (marker/mechanism)
and/or “T” (therapeutic) were retrieved. For GeneCards, genes associated with “Protein
Coding” were retrieved.
3.3.2. Pharmacology Network Analysis
STRING [27] was used to construct the protein-protein interaction (PPI) networks of
the targets related to the five cancer diseases. Analysis and modularization were performed
using Cytoscape. The MCODE algorithm [28] was used to determine highly interconnected
regions in the PPI network. The degree cut off, node density cutoff, and node score cutoff
were kept to 2, 0.1 and 0.2, respectively.
3.3.3. Enrichment Analysis of Five Cancer Diseases Targets
All targets of five cancer diseases-related cell lines were mapped into Gene Ontology
(GO) and Kyoto Encyclopedia of Genes and Genomes (KEGGs) [29]. The GO functional
annotations were carried for the biological process (BP), molecular function (MF) and
cellular components (CC) terms [30].
3.3.4. Molecular Docking
Molecular docking was performed using iGEMDOCK (BioXGEM, Taiwan) [31]. iGEM-
DOCK makes use of the genetic algorithm that selects the best solution from the population
by calculating their fitness. Genetic algorithm is a general-purpose optimization and
search technique which is based on the evolutionary process of natural selection. Genetic
algorithm permits the individuals of a population to evolve under certain specific selec-
tion rules to a condition that maximizes the fitness function [32]. The PyMOL Molecular
Graphics (Version 1.8.4.0, Schrödinger, LLC) was used for the visualizations and graphics
generations [33]. To determine the interactions of all the docking complexes, the protein-
ligand interaction profiler (PLIP) was used to analyze the crystal structure of the available
structural complexes [34].
4. Conclusions
In the current work, a series of sinomenine derivatives were synthesized with good
yields. All the derivatives were tested for their cytotoxic activity against a variety of
cancer cells in vitro and applied to a molecular docking study to investigate the potential
molecular targets. The results show that compounds containing chlorine had significant
anticancer activity. Furthermore, we obtained 16 core targets and some key signal pathways
related to cancer diseases through comprehensive bioinformatics analysis. The molecular

Molecules 2021, 26, 3466
23 of 25
docking results showed that AKT1, EGFR, HARS and KARS could be considered as the
most potential anticancer targets of sinomenine derivatives with high binding affinity. We
found that these targets are important key genes in the PI3K/AKT and MAPK signaling
pathways combined with the results of KEGG enrichment. Therefore, we boldly speculate
that chlorine-containing sinomenine derivatives may cause the pathological death of cancer
cells by regulating multiple genes in these pathways.
As the current amount of the compound is insufficient for more experimental verifica-
tion, we intend to synthesize chlorine-substituted sinomenine derivatives with different
substitutions to conduct a detailed structure-activity relationship (SAR) analysis. We will
further explore the relationships between structure and activity combined with activity
experiments in vitro.
1
Supplementary Materials: The list of supplementary materials is as follows. Figure S1: H NMR
spectrum of compound 5a. Figure S2: 13C NMR spectrum of compound 5a. Figure S3: 1H NMR
1
spectrum of compound 5b. Figure S4: 13C NMR spectrum of compound 5b. Figure S5: H NMR
spectrum of compound 5c. Figure S6: 13C NMR spectrum of compound 5c. Figure S7: 1H NMR
1
spectrum of compound 5d. Figure S8: 13C NMR spectrum of compound 5d. Figure S9: H NMR
1
spectrum of compound 5e. Figure S10: 13C NMR spectrum of compound 5e. Figure S11: H NMR
1
spectrum of compound 5f. Figure S12: 13C NMR spectrum of compound 5f. Figure S13: H NMR
1
spectrum of compound 5g. Figure S1:. 13C NMR spectrum of compound 5g. Figure S15: H NMR
1
spectrum of compound 5h. Figure S16: 13C NMR spectrum of compound 5h. Figure S17: H NMR
1
spectrum of compound 5i. Figure S18: 13C NMR spectrum of compound 5i. Figure S19: H NMR
1
spectrum of compound 5j. Figure S20: 13C NMR spectrum of compound 5j. Figure S21: H NMR
spectrum of compound 5k. Figure S22: 13C NMR spectrum of compound 5k. Figure S23: HRMS
1
spectrum of compound 6a. Figure S24: H NMR spectrum of compound 6a. Figure S25: 13C NMR
spectrum of compound 6a. Figure S26: HRMS spectrum of compound 6b. Figure S27: 1H NMR
spectrum of compound 6b. Figure S28: 13C NMR spectrum of compound 6b. Figure S29: HRMS
1
spectrum of compound 6c. Figure S30: H NMR spectrum of compound 6c. Figure S31: 13C NMR
spectrum of compound 6c. Figure S32: HRMS spectrum of compound 6d. Figure S33: 1H NMR
spectrum of compound 6d. Figure S34: 13C NMR spectrum of compound 6d. Figure S35: HRMS
1
spectrum of compound 6e. Figure S36: H NMR spectrum of compound 6e. Figure S37: 13C NMR
spectrum of compound 6e. Figure S38: HRMS spectrum of compound 6f. Figure S39: 1H NMR
spectrum of compound 6f. Figure S40: 13C NMR spectrum of compound 6f. Figure S41: HRMS
1
spectrum of compound 6g. Figure S42: H NMR spectrum of compound 6g. Figure S43: 13C NMR
spectrum of compound 6g. Figure S44: HRMS spectrum of compound 6h. Figure S45: 1H NMR
spectrum of compound 6h. Figure S46: 13C NMR spectrum of compound 6h. Figure S47: HRMS
1
spectrum of compound 6i. Figure S48: H NMR spectrum of compound 6i. Figure S49: 13C NMR
spectrum of compound 6i. Figure S50: HRMS spectrum of compound 6j. Figure S51: 1H NMR
spectrum of compound 6j. Figure S52: 13C NMR spectrum of compound 6j. Figure S53: HRMS
1
spectrum of compound 6k. Figure S54: H NMR spectrum of compound 6k. Figure S55: 13C NMR
spectrum of compound 6k. Figure S56: HRMS spectrum of compound 6l. Figure S57: 1H NMR
spectrum of compound 6l. Figure S58: 13C NMR spectrum of compound 6l. Figure S59.:The total
energy of molecular docking between compounds and each target. Table S1: Mean
of growth inhibition ratios at 2.5 M drug concentration. Table S2: Mean SD values of growth
inhibition ratios at 25 M drug concentration. Table S3: The total energy for the tested compounds
5k against docked proteins binding pockets. Table S4: The total energy for the tested compounds
6l against docked proteins binding pockets. Table S5: The various interactions for the most
±
SD values
µ
±
µ
5a
6a
–
–
promising test compounds into the AKT1 binding pockets. Table S6: The various interactions for the
most promising test compounds into the EGFR binding pockets. Table S7: The various interactions
for the most promising test compounds into the HRAS binding pockets. Table S8: The various
interactions for the most promising test compounds into the KRAS binding pockets.
Author Contributions: S.L. (biology, computational calculations, writing, editing, revision, and
submitting); M.G. (computational calculations, writing, and revision); X.N. and L.Z. (synthesis
and writing); J.L. (HRMS experiment); C.Z. (Changqi Zhao), C.Z. (Chen Zhang) and D.C. (project
administration, funding acquisition and revision). All authors have read and agreed to the published
version of the manuscript.

Molecules 2021, 26, 3466
24 of 25
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: No applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: No applicible.
References
1.
2.
3.
4.
5.
Li, J.W.H.; Vederas, J.C. Drug discovery and natural products: End of an era or an endless frontier? Science 2009, 325, 161–165.
[CrossRef]
Walsh, C.T.; Yi, T. Natural Product Biosynthesis: Chemical Logic and Enzymatic Machinery; Royal Society of Chemistry Publishing:
Cambridge, UK, 2017; p. 765.
Newman, D.J.; Cragg, G.M. Natural products as sources of new drugs over the nearly four decades from 01/1981 to 09/2019. J.
Nat. Prod. 2020, 83, 770–803. [CrossRef]
Wink, M. Alkaloids: Properties and determination. In Encyclopedia of Food and Health; Caballero, B., Finglas, P.M., Toldrá, F., Eds.;
Elsevier: Amsterdam, The Netherlands, 2016; pp. 97–105.
Castilla-Fernandez, D.; Moreno-Gonzalez, D.; Garcia-Reyes, J.F.; Ballesteros, E.; Molina-Diaz, A. Determination of atropine and
scopolamine in spinach-based products contaminated with genus Datura by UHPLC-MS/MS. Food Chem. 2021, 347, 129020.
[CrossRef] [PubMed]
6.
7.
8.
9.
Ibis, E.; Hayme, S.; Baysal, E.; Gul, N.; Ozkavukcu, S. Efficacy and safety of papaverine as an in vitro motility enhancer on human
spermatozoa. J. Assist. Reprod. Genet. 2021, 2, 1–15.
Gaba, S.; Saini, A.; Singh, G.; Monga, V. An insight into the medicinal attributes of berberine derivatives: A review. Bioorg. Med.
Chem. 2021, 38, 116143. [CrossRef] [PubMed]
Liu, Z.Y.; Wang, X.L.; Ou, S.Q.; Hou, D.X.; He, J.H. Sanguinarine modulate gut microbiome and intestinal morphology to enhance
growth performance in broilers. PLoS ONE 2020, 15, e0234920. [CrossRef]
Ng, J.P.L.; Coghi, P.; Law, B.Y.K.; Liu, L.; Wong, V.K.W. The present and future synthetic strategies of structural modifications of
sinomenine. Org. Chem. Front. 2020, 7, 4089–4107. [CrossRef]
10. Zhao, S.; Guo, Y.; Wang, Q.; An, B. Antifungal effects of lycorine on Botrytis cinerea and possible mechanisms. Biotechnol. Lett.
2021. [CrossRef]
11. Sezer, E.N.S.; Uysal, T. Anti-Proliferative and apoptotic effects of vincristine and vinblastine on multiple myeloma. Fresenius
Environ. Bull. 2019, 28, 4001–4006.
12. Liu, J.; Han, X.; Wu, D.; Jia, Z. 2D NMR studies on sesquiterpene alkaloids from celastrus angulatus (in Chinese). Chin. J. Magn.
Reson. 1992, 9, 289–295.
13. Zhou, W.; Liu, H.; Qiu, L.Z.; Yue, L.X.; Zhang, G.J.; Deng, H.F.; Ni, Y.H.; Gao, Y. Cardiac efficacy and toxicity of aconitine: A new
frontier for the ancient poison. Med. Res. Rev. 2021, 41, 1798–1811. [CrossRef] [PubMed]
14. Wang, Y.; Zhou, L.; Li, R. Study progress in Sinomenium acutum (Thunb.) Rehd. et Wils. J. Chin. Med. Mater. 2002, 25, 209–211.
15. Ishiwari, N. An alkaloid of sinomeium diversifolius. J. Chem. Soc. Abstr. 1921, 1, 354.
16. Tang, J.; Raza, A.; Chen, J.; Xu, H. A systematic review on the sinomenine derivatives. Mini-Rev. Med. Chem. 2018, 18, 906–917.
[CrossRef] [PubMed]
17. Wei, C.J.; Xu, F.; Shi, M.J.; Hu, J.W.; Wang, J.J.; Zhen, B.; Wang, X.; Ji, T.F.; Wang, J.H.; Du, G.H. Synthesis and antitumor activities
of sinomenine derivatives on rings A and C. J. Asian Nat. Prod. Res. 2018, 20, 277–291. [CrossRef] [PubMed]
18. Arend, M.; Westermann, B.; Risch, N. Modern variants of the Mannich reaction. Angew. Chem. Int. Ed. 1998, 37, 1044–1070.
[CrossRef]
19. Liu, J.; Pang, Y.; Chen, J.; Huang, P.; Huang, W.; Zhu, X.; Yan, D. Hyperbranched polydiselenide as a self assembling broad
spectrum anticancer agent. Biomaterials 2012, 33, 7765–7774. [CrossRef]
20. Huang, H.Y.; Liu, J.J.; Xi, R.R.; Xing, X.M.; Yuan, J.H.; Yang, L.Q.; Tao, G.H.; Gong, C.M.; Zhuang, Z.X. An investigation of
hormesis of trichloroethylene in L-02 liver cells by differential proteomic analysis. Mol. Biol. Rep. 2009, 36, 2119–2129. [CrossRef]
[PubMed]
21. Mosmann, T. Rapid calorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J.
Immunol. Methods 1983, 65, 55–63. [CrossRef]
22. Gomha, S.M.; Riyadh, S.M.; Mahmmoud, E.A.; Elaasser, M.M. Synthesis and anticancer activities of thiazoles, 1,3-thiazines, and
thiazolidine using chitosan-graftedpoly (vinylpyridine) as basic catalyst. Heterocycles 2015, 91, 1227–1243.
23. Amberger, J.; Bocchini, C.A.; Scott, A.F.; Hamosh, A. McKusick’s Online Mendelian Inheritance in Man (OMIM). Nucleic Acids
Res. 2009, 37, D793–D796. [CrossRef] [PubMed]

Molecules 2021, 26, 3466
25 of 25
24. Wang, J.H.; Zhao, L.F.; Wang, H.F.; Wen, Y.T.; Jiang, K.K.; Mao, X.M.; Zhou, Z.Y.; Yao, K.T.; Geng, Q.S.; Guo, D.; et al. GenCLiP 3:
Mining human genes’ functions and regulatory networks from PubMed based on co-occurrences and natural language processing.
Bioinformatics 2020, 36, 1973–1975. [CrossRef]
25. Davis, A.P.; Grondin, C.J.; Johnson, R.J.; Sciaky, D.; Wiegers, J.; Wiegers, T.C.; Mattingly, C.J. Comparative Toxicogenomics
Database (CTD): Update 2021. Nucleic Acids Res. 2021, 49, D1138–D1143. [CrossRef] [PubMed]
26. Stelzer, G.; Rosen, N.; Plaschkes, I.; Zimmerman, S.; Twik, M.; Fishilevich, S.; Stein, T.I.; Nudel, R.; Lieder, I.; Mazor, Y.; et al. The
GeneCards suite: From gene data mining to disease genome sequence analyses. Curr. Protoc. Bioinf. 2016, 54, 1.30.1–1.30.33.
[CrossRef] [PubMed]
27. Szklarczyk, D.; Gable, A.L.; Lyon, D.; Junge, A.; Wyder, S.; Huerta-Cepas, J.; Simonovic, M.; Doncheva, N.T.; Morris, J.H.;
Bork, P.; et al. STRING v11: Protein-protein association networks with increased coverage, supporting functional discovery in
genome-wide experimental datasets. Nucleic Acids Res. 2019, 47, D607–D613. [CrossRef]
28. Bader, G.D.; Hogue, C.W. An automated method for finding molecular complexes in large protein interaction networks. BMC
Bioinform. 2003, 4, 2. [CrossRef]
29. Kanehisa, M.; Furumichi, M.; Tanabe, M.; Sato, Y.; Morishima, K. KEGG: New perspectives on genomes, pathways, diseases and
drugs. Nucleic Acids Res. 2017, 45, D353–D361. [CrossRef]
30. Hill, D.P.; Smith, B.; McAndrews-Hill, M.S.; Blake, J.A. Gene Ontology annotations: What they mean and where they come from.
BMC Bioinform. 2008, 9 (Suppl. 5), S2. [CrossRef] [PubMed]
31. Yang, J.M.; Chen, C.C. GEMDOCK: A generic evolutionary method for molecular docking. Proteins Struct. Funct. Bioinform. 2004
55, 288–304. [CrossRef]
,
32. Loncaric, M.; Strelec, I.; Pavic, V.; Subaric, D.; Rastija, V.; Molnar, M. Lipoxygenase inhibition activity of coumarin derivatives-
QSAR and molecular docking study. Pharmaceuticals 2020, 13, 154. [CrossRef]
33. Delano, W.L. The PyMol molecular graphics system. Proteins Struct. Funct. Bioinform. 2002, 30, 442–454.
34. Adasme, M.F.; Linnemann, K.L.; Bolz, S.N.; Kaiser, F.; Salentin, S.; Haupt, V.J.; Schroeder, M. PLIP 2021: Expanding the scope of
the protein-ligand interaction profiler to DNA and RNA. Nucleic Acids Res. 2021. [CrossRef] [PubMed]