Synthesis and Biological Evaluation of Glycyrrhetic Acid Derivatives as Potential VEGFR2 Inhibitors

Article abstract of DOI:10.1002/cmdc.201700271

Vascular endothelial growth factor receptor 2 (VEGFR2) has been proven to play a major role in the regulation of tumor angiogenesis. A series of novel glycyrrhetic acid derivatives were synthesized and evaluated for their VEGFR2 inhibitory activity as well as their antiproliferative properties against four cancer cell lines (MCF-7, HeLa, HepG2, and A549). In vitro biological evaluations against these human tumor cell lines indicate that most of the prepared compounds have antiproliferative activities; compound 3 a (3β-hydroxy-30-(4-phenyl-1-piperazinyl)olean-12-ene-11,30-dione) exhibited the best inhibitory activity against MCF-7 cells, with an IC₅₀ value of 1.08 μm. Compound 3 a also showed the most potent inhibitory activity against VEGFR2 tyrosine kinase, with an IC₅₀ value of 0.35 μm. Docking simulations were performed with the aim of discovering the binding mode of compound 3 a, and the results indicate that 3 a could bind at the VEGFR2 active site.

Full text of DOI:10.1002/cmdc.201700271

Accepted Article
Title: Synthesis and Biological Evaluation of Glycyrrhetic Acid
Derivatives as Potential VEGFR2 Inhibitors
Authors: Pengcheng Lv, Tian-Long Yan, Li-Fei Bai, Hai-Liang Zhu, and
Wei-Ming Zhang
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To be cited as: ChemMedChem 10.1002/cmdc.201700271
Link to VoR: http://dx.doi.org/10.1002/cmdc.201700271
A Journal of
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10.1002/cmdc.201700271
ChemMedChem
Synthesis and Biological Evaluation of
Glycyrrhetic Acid Derivatives as Potential VEGFR2 Inhibitors
Dr. Tian-Long Yan†1,2, Dr. Li-Fei Bai†3, Prof. Dr. Hai-Liang Zhu1,2, Prof. Dr. Wei-
Ming Zhang*1,2, Prof. Dr. Peng-Cheng Lv*1,2
1 Nanjing Institute for the Comprehensive Utilization of Wild Plant, Nanjing 210042,
P. R. China
2
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210046, P. R. China
3
Jiangsu Key Laboratory of Biofunction Molecule, College of Life Science and
Chemistry &Chemical Engineering, Jiangsu Second Normal University, Nanjing
210013, P. R. China
*Corresponding
authors.
Tel./fax:
+86
25
89682572.
E-mail
address: pclv@nju.edu.cn (P.-C. Lv).
†These authors equally contributed to this work.
1
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Abstract: Vascular endothelial growth factor receptor 2 (VEGFR2) has been proven
to play a major role in the regulation of tumor angiogenesis. A series of novel
glycyrrhetic acid derivatives have been synthesized and evaluated for their VEGFR2
inhibitory activity as well as their antiproliferative properties against four cancer cell
lines (MCF-7, Hela, HepG2 and A549). In vitro biological evaluation against four
human tumor cell lines indicated that most of the prepared compounds showed
antiproliferative activities, and compound 3a exhibited the best inhibitory activity
against MCF-7 with the IC50 value of 1.08 μM. Also, compound 3a showed the most
potent inhibitory activities against VEGFR2 tyrosine kinase with the IC50 value of
0.35 μM. A docking simulation study was performed with the aim of discovering the
binding mode of compound 3a, and the result showed that compound 3a could bind
well at the VEGFR2 active site.
Keywords: Glycyrrhetic acid derivatives; VEGFR2 inhibitors; Anticancer activity;
Molecular docking.
2
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Introduction:
Malignant tumors threaten human health so there is a pertinent and urgent
challenge for medicinal chemists to discover new types of anticancer drugs that
prevent complications such as severe side-effects and drug resistance [1]. Tumor cell
hyperplasia is quite a complex process. Angiogenesis, the formation of new blood
vessels out of pre-existing vasculature, plays a significant role in tumor growth and
metastasis [2]. This process is stimulated and regulated by a variety of factors, among
them are vascular endothelial growth factors (VEGFs), which play an important role
in tumor angiogenesis, vascular permeability, and endothelial cell activation,
proliferation, and migration [3]. The VEGFRs are transmembrane tyrosine kinases and
consist of three members (VEGFR1, VEGFR2 and VEGFR3) in humans [4]. VEGFR2,
of which blood vessels are comprised, plays a significant role in the regulation of
VEGF-driven responses in endothelial cells and has the capability of mediating
[5]
endothelial cell proliferation, differentiation, and micro-vascular permeability
.
Therefore, the inhibition of this signaling pathway should be desirable approach to
[6]
cancer therapy. In recent years, a number of compounds such as quinolinones
phthalazines [7], benzimidazoles [8], imidazopyridines [9], quinoline amides
,
,
[10]
quinazolines [11] and pyridines [12], have been reported as potent inhibitors of VEGFR2
in vitro or of possessing antiangiogenic activity.
Natural products have played a significant role in drug discovery and
[13]
development in recent years, especially with regards to antitumor agents
.
Glycyrrhiza glabra, a member of legumes family, indigenous to Europe, the Middle
East and Asia, is well known as licorice, and is widely used in herbal medicines for its
[14, 15]
emollient, antitussive, anti-inflammatory, antiviral and antitumor activities
.
These biological activities are attributable to a variety of biological components, such
as alkaloids, terpenoids, polysaccharides, saponins, polyamines and numerous others
[16, 17]. Among these components, the primary active constituent is glycyrrhizic acid (1,
(3β,20β)-[(2-O-β-D-glucopyranuronosyl-α-D-glucopyranuronosyl)-oxy]-11-oxo-12-
oleanen-29-oic acid), which is a triterpene saponin heavily present in the dried roots
of Glycyrrhiza glabra [18]. Glycyrrhetic acid (2, (3β,20β)-3-hydroxy-11-oxoolean-12-
en-29-oic acid), is an aglycone of glycyrrhizic acid from which it can be accessed in
3
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abundance, and it has been shown to have antiproliferative properties on various
tumor cell lines in continued research. However, glycyrrhetic acid demonstrated poor
inhibitory capability when applied to different tumor cell lines in most of the studies
[19-22]. Thus, the synthesis of new synthetic derivatives with elevated antitumor
activity was the focus. From the molecular structure, glycyrrhetic acid has three main
functional groups; they are located on rings A, C, and E. Therefore, chemical
modifications and skeleton transformations were also concentrated on these sites
(Figure 1).
Piperazine is a hexa-heterocyclic compound which contains two nitrogen atoms.
In comparison to traditional organic drugs, the piperazine ring has excellent property
because of its nitrogen symmetrical structure. In organic synthesis, piperazine can be
used as a reactive intermediate in order to obtain various novel compounds which
have strong pharmacological activity. Some of these compounds have been developed
[25]
into clinical drugs, such as levodropropizine [23], ciprofloxacin [24], trifluonerazine
and others.
In previous reports [26], research has shown that glycyrrhetic acid analogs could
regulate the expression of VEGFR2, which is associated with cancer cell survival,
growth, and angiogenesis. In addition, piperazine derivatives were associated with a
broad spectrum of pharmacological activities including antitumor properties. The two
combined moieties, glycyrrhetic acid and piperazine may exhibit a synergistic effect
on anticancer activities. In this study, 18 glycyrrhetic acid derivatives were designed
and synthesized, and their biological activities against the four human tumor cell lines
were evaluated in detail. These compounds proven to be potent in an antiproliferative
test against four tumor cell lines and also inhibited migration in a dose-dependent
manner in the MCF-7 cell line. Enzymatic assay and molecular docking studies
indicated that the synthesized compounds were potent inhibitors of VEGFR2.
2. Results and discussion
2.1. Chemistry
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The synthesis of compounds 3a-3r followed the general pathway outlined in
Scheme 1. Glycyrrhizic acid (1) was isolated from Glycyrrhiza glabra according to
the procedure in the literature [27]. Glycyrrhetic acid (2) is an aglycone of glycyrrhizic
acid, which was obtained by acid hydrolysis of glycyrrhizic acid. Lastly, glycyrrhetic
acid reacted with different piperazine derivatives in the presence of DCC and HOBt to
obtain the desired compounds 3a-3r. The refined compounds were finally obtained by
subsequent purification along with silica gel column chromatography using petroleum
ether and ethyl acetate as the eluent. The chemical structures of these glycyrrhetic
acid derivatives are displayed in Table 1. Additionally, all the synthetic compounds
have presented satisfactory analytical and spectroscopic data, which were in full
accordance with their depicted structures.
2.2. Biological evaluation
2.2.1. Antiproliferative activity
Compounds 3a-3r were evaluated by in vitro antiproliferation assays against four
human cancer cell lines: MCF-7 (breast cancer, with VEGFR protein overexpression),
Hela (cervical cancer cell line), HepG2 (human hepatoma cells), and A549
(carcinomic human alveolar basal epithelial cell). The test followed the MTT assay
and used vandetanib under identical conditions as a control. The results are shown in
Table 2. The results revealed that most of the synthetic compounds exhibited
significant antiproliferative activity. Among the given compounds, it was observed
that compound 3a showed the most potent biological activity (IC50 = 1.08µM for
MCF-7), and the dose-dependent inhibition assay curve of MCF-7 for 3a is shown in
Figure 2.
According to the data listed in Table 2, it can be concluded that the activity of
the tested compounds may be correlated to the variation and modification of the
structure. The activities of compounds (3a-3m) that contained a phenylpiperazine
skeleton were much better than those (3n-3r) that contained an alkyl and heterocyclic
piperazine skeleton. The indication is that a fragment of a phenylpiperazine skeleton
plays a significant role in the antiproliferative effect in vitro. Among the thirteen
glycyrrhetic acid derivatives (3a-3m) containing phenylpiperazine skeleton,
5
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compound 3a, which has smaller steric hindrance showed better inhibitory capability
than the others, and compounds 3p-3r that contain alkylpiperazine had the same trend,
with compound 3p showing better activity than compounds 3q and 3r. According to
the data (Table 1) of compounds 3b–3m, compounds with electron-withdrawing
substituents (such as F, NO2) improved inhibitory activity when compared to those
with electron-donating substituents (such as CH3, OCH3). The above results
demonstrated that the phenylpiperazine skeleton plays an important role in the
antiproliferative activity.
2.2.2. VEGFR2 inhibitory activity
In order to validate that the above antiproliferative effect was produced by
interaction between VEGFR2 protein and the synthesized compounds, the synthesized
compounds were evaluated for their abilities to inhibit the activity of four protein
kinases relevant to cancer: VEGFR2, EGFR (epidermal growth factor receptor), bFGF
(basic fibroblast growth factor) and PDGFR (platelet-derived growth factor receptor).
As the results show in Table 3, all compounds exhibited inhibitory activity for
VEGFR2. It was revealed that all compounds displayed selectivity for VEGFR2
protein over the other three protein kinases. In addition, the IC50 values of the
enzymatic activity exhibited a similar trend as that in the antiproliferative assay. A
further study comparing the anti-proliferative activity against the MCF-7 cell line
with the VEGFR2 inhibitory activity of the compounds was performed and the results
indicated that there was a moderate correlation between VEGFR2 inhibition and the
inhibition of cancer cellular proliferation, as shown in Figure 3, with an R2 value of
0.9481. Therefore, it could be concluded that the synthesized inhibitors can inhibit the
function of VEGFR2 and the antiproliferative effect was partly due to the interaction
of the VEGFR2 protein and the synthesized compounds.
2.2.3. Cytotoxicity test.
The inhibitory activity of the compounds is sometimes a result of their toxic
effects and consequently might cause an erroneous conclusion. Prior to the following
bioactivity analysis, some synthesized glycyrrhetic acid derivatives were tested in
6
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vitro for their cytotoxicity on human kidney epithelial cell 293T by the MTT assay to
evaluate the viability of 293T. The pharmacological results are summarized in Table
4. The data demonstrated that most of the glycyrrhetic acid derivatives had quite a
low toxicity.
2.2.4. Analysis of the apoptosis induced in cells.
In order to appraise the biological ability of the compounds, an apoptosis
experiment of compound 3a was determined in the MCF-7 cell line using Annexin V
and 7-AAD double staining by flow cytometry and treating the MCF-7 cells with 2.0,
4.0, and 8.0 µM of compound 3a for 24 h. The results are shown in Figure 4, and
revealed that compound 3a is very effective in the induction of apoptosis in a dose-
dependent manner. Additionally, the percentage of total apoptotic cells from 19.51 to
60.80% was markedly elevated in a dose-dependent manner, as compared to 8.88% of
apoptotic cells in the untreated control.
2.2.5. Flow cytometric determination of the mitochondrial membrane potential
Apoptosis was induced by compound 3a through both intrinsic and extrinsic
apoptotic pathways, which was confirmed by mitochondrial membrane potential
(MMP) loss. The damage of mitochondria on cells leads to the perturbation of MMP.
The change of MMP (Δψm) in MCF-7 cells caused by compound 3a was studied
using the Rhodamine123 dye, which was lessened by the healthy mitochondria into a
fluorescent probe and the fluorescence was detected by a flow cytometer in the FL-1
channel. The results are shown in Figure 5.
2.2.6. Wound healing migration assay
Angiogenesis is one of the hallmark manifestations of all cancers, and it occurs
in the beginning stages of the development of tumor growth and malignancy.
Although there are several methods of testing the anti-angiogenesis activity, in this
study, the chemotactic motility of MCF-7 cells was performed by wound healing
7
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migration assay with the aim to evaluate the anti-angiogenic properties of compound
3a in vitro. Cell migration is necessary for tumor growth and metastasis. As shown in
Figure 6, it was observed that compound 3a significantly inhibited MCF-7 migration
and decreased wound closure very effectively.
2.3. Molecular docking
A molecular docking study was performed to fit compound 3a into the active
center of the VEGFR2 protein (PDB code: 1YWN) in order to obtain a greater
understanding of the structure-activity relationships observed at VEGFR2. All
docking runs were performed by Discovery Studio 3.5. The results obtained from the
study are presented in Figure 7. Figures 7A and 7B exhibit the binding mode of
compound 3a as it interacts with the VEGFR2 protein. The docking results revealed
that two amino acids, Cys917 and Asp1044, located in the binding pocket of the
protein played vital roles in the conformation with compound 3a. In the binding mode,
compound 3a was effectively bound to VEGFR2 via three charge interactions, two
hydrogen bonds, and one π-anion interaction.
Here we also take the compound 3c as an example. From the binding mode of
compound 3c interacting with VEGFR2 protein, it is revealed that two amino acids
Lys866 and Phe1045, which are located at the binding pocket of the protein, played a
vital role in the interaction. Compound 3c binds to VEGFR2 via two charge
interactions; one hydrogen bond and one Pi-Lone Pair interaction.
The predicted binding interaction energy was also used as the criterion for
ranking. Due to steric hindrance, different binding sites and other possible reasons,
compound 3a has the best estimated binding free energy for VEGFR2. The histogram
about CDOCKER_INTERACTION_ENERGY (-kcal/mol) of selected compounds for
VEGFR2 was displayed in Figure S1.
This molecular docking result showed that compound 3a would bind compatible
when interacting with a 1YWN protein. These results provide a foundation at the
molecular level for demonstrating that compound 3a has the ability to bind well at the
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active site of a VEGFR2 kinase and therefore, it could be a potential inhibitor of
VEGFR2.
3. Conclusions
In summary, a series of novel glycyrrhetic acid containing piperazine structure
derivatives were synthesized and evaluated for their VEGFR2 inhibitory activity. In
vitro biological evaluation against MCF-7, Hela, HepG2 and A549 cell lines revealed
that most target compounds displayed significant antiproliferative activities. Among
these compounds, compound 3a exhibited the most potent activity, showing a
VEGFR2 inhibitory activity of 0.35 μM. An experiment concerning cell apoptosis and
wound healing migration was conducted to validate the relative biological activities,
and the ability of compound 3a to induce MCF-7 cell apoptosis and anti-angiogenesis
potential. This study indicated that compound 3a could be a potent VEGFR2 and
antitumor agent. Docking simulation was performed to position compound 3a into the
VEGFR2 active site and the result showed that compound 3a was stabilized by
interactions with Cys917 and Asp1044. These results provide valuable information
for further studies of VEGFR2 inhibitors as future anticancer therapeutics.
4. Experimental part
4.1 Materials and measurements
All chemicals and reagents used in the current work were of analytical grade, and
the dried Glycyrrhiza glabra roots were purchased from a commercial supplier.
Melting points were determined on an XT4 MP apparatus (Taike Corp., BeiJing,
China) and are uncorrected. The reactions were monitored by thin layer
chromatography (TLC) on Merck pre-coated silica GF254 plates and visualized in
1
UV light (254 nm). All the H NMR spectra were recorded on a Bruker DPX400
model spectrometer with DMSO-d6 as the solvent and tetramethylsilane (TMS) as the
internal standard, Chemical data for protons were reported in ppm (δ).
4.2. General procedure for the extraction of glycyrrhizic acid
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The roots of Glycyrrhiza glabra were oven-dried, sliced and crushed into a
powder before extraction. The powder was extracted with an ethanol to water ratio of
3:7 at a temperature of 50 °C. The crude products were purified by washing with
dichloromethane and acetone to remove any low molecular weight phenolics. A
constituent containing a large quantity of glycyrrhizic acid was attained by repeating
this procedure. This product was directly used in the next experiment [28]
.
4.3. General procedure for the preparation of glycyrrhetic acid
The glycyrrhizic acid was hydrolyzed with 6% aq. HCl at a temperature of
100 °C, and the progress of the reaction was monitored by thin layer chromatography
(TLC) to obtain glycyrrhetic acid, which was isolated by column chromatography on
silica gel with a EtOAc to PE ratio of 1:4. Glycyrrhetic acid was characterized by
comparing the melting points and spectral data with literature values [29]
.
4.4. General procedure for the synthesis of compounds 3a–3r
A mixture of DCC (1.2 mmol) and HOBt (1.2 mmol) was dissolved into 20 mL
of CH2Cl2, and the mixture was stirred at room temperature for 30 min. Compound 2
was added into the reaction with continued stirring for 20 min; then, the piperazine
derivatives (1.2 mmol) and triethylamine (3 mmol) were added into the reaction while
stirring continued for 24 hrs. The reaction mixture was diluted with CH2Cl2. Water
was added to the resultant mixture creating an aqueous layer, which was decanted off
until the organic layer remained. This process was repeated 3-4 times. The combined
CH2Cl2 layer was evaporated under reduced pressure and the residue obtained was
purified by silica gel column chromatography using petroleum ether and ethyl acetate
as the eluent to yield the compounds 3a–3r.
4.4.1.
3β-Hydroxy-30-(4-phenyl-1-piperazinyl)-olean-12-ene-11,30-dione (3a)
Yield: 83%. M.p. 282–284 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 7.2-7.27 (m,
2H, Ar-7’,9’), 6.94 (d, J = 7.9 Hz, 2H, Ar-6’,10’), 6.81 (t, J = 7.2 Hz, 1H, Ar-8’), 5.48
10
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(s, 1H, CH-12), 4.32 (d, J = 5.2 Hz, 1H, -OH), 3.70 (s, 4H, CH2-1’,CH2-4’), 3.10 (t, J
= 4.6 Hz, 4H, CH2-2’,CH2-3’), 2.98-3.05 (m, 1H, CH-3), 2.57 (d, J = 13.3 Hz, 1H,
CH-18), 2.33 (s, 1H, CH-9), 2.26-1.36 (m, 16H, CH2-2, CH2-6, CH2-7, CH2-15, CH2-
16, CH2-19, CH2-21, CH2-22), 1.35 (s, 3H, Me-27), 1.19 (s, 3H, Me-29), 1.02 (s, 6H,
Me-25, Me-26), 0.96 (d, J = 13.4 Hz, 2H, CH2-1), 0.90 (s, 3H, Me-23), 0.73 (s, 3H,
13
Me-24), 0.71 (s, 1H, CH-5), 0.69 (s, 3H, Me-28). C NMR (150 MHz, DMSO-d6): δ
(ppm) 199.0, 172.9, 169.8, 150.7, 128.9, 127.3, 119.2, 115.6, 76.5, 61.0, 54.0, 48.6,
47.6, 44.6, 43.2, 43.1, 42.9, 38.4, 37.3, 36.6, 32.1, 32.0, 31.3, 28.2, 28.0. HPLC purity:
98.57%.
4.4.2.
3β-Hydroxy-30-(4-(2,4-dimethylphenyl)-1-piperazinyl)-olean-12-ene-11,30-dione
(3b)
Yield: 79%. M.p. 175–177 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 6.88-7.00 (m,
3H, Ar-6’7’,9’), 5.52 (s, 1H, CH-12), 4.32 (d, J = 5.2 Hz, 1H, -OH), 3.68 (s, 4H, CH2-
1’,CH2-4’), 2.99-3.05 (m, 1H, CH-3), 2.75 (s, 4H, CH2-2’,CH2-3’), 2.57 (d, J = 13.4
Hz, 1H, CH-18), 2.33 (s, 1H, CH-9), 2.22 (d, J = 7.0 Hz, 6H, Me-8’, Me-10’), 2.17-
1.37 (m, 16H, CH2-2, CH2-6, CH2-7, CH2-15, CH2-16, CH2-19, CH2-21, CH2-22),
1.35 (s, 3H, Me-27), 1.19 (s, 3H, Me-29), 1.03 (d, J = 1.6 Hz, 6H, Me-25, Me-26),
0.97 (d, J = 14.3 Hz, 2H, CH2-1), 0.91 (s, 3H, Me-23), 0.75 (s, 3H, Me-24), 0.71 (s,
1H, CH-5), 0.69 (s, 3H, Me-28). 13C NMR (150 MHz, DMSO-d6): δ (ppm) 199.6,
173.5, 170.4, 148.8, 132.5, 132.2, 132.0, 127.9, 127.4, 119.3, 77.0, 61.6, 54.6, 52.4,
48.3, 45.2, 43.8, 43.5, 40.5, 39.0, 37.9, 37.1, 32.7, 31.9, 28.8, 28.6. HPLC purity:
95.63%.
4.4.3.
3β-Hydroxy-30-(4-(2-chlorophenyl)-1-piperazinyl)-olean-12-ene-11,30-dione (3c)
Yield: 80%. M.p. 172–174 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 7.41-7.45 (m,
H, Ar-9’), 7.29-7.34 (m, 1H, Ar-7’), 7.14-7.18 (m, 1H, Ar-8’), 7.05-7.10 (m, 1H, Ar-
6’), 5.51 (s, 1H, CH-12), 4.31 (s,1H, -OH), 3.71 (s, 4H, CH2-1’, CH2-4’), 2.99-3.05
(m, 1H, CH-3), 2.94 (d, J = 4.0 Hz, 4H, CH2-2’, CH2-3’), 2.58 (d, J = 13.3 Hz, 1H,
CH-18), 2.33 (s, 1H, CH-9), 2.25-1.37 (m, 16H, CH2-2, CH2-6, CH2-7, CH2-15, CH2-
16, CH2-19, CH2-21, CH2-22), 1.35 (s, 3H, Me-27), 1.20 (s, 3H, Me-29), 1.03 (d, J =
11
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1.24 Hz, 6H, Me-25, Me-26), 0.97 (d, J = 13.9 Hz, 2H, CH2-1), 0.91 (s, 3H, Me-23),
13
0.75 (s, 3H, Me-24), 0.71 (s, 1H, CH-5), 0.69 (s, 3H, Me-28). C NMR (150 MHz,
DMSO-d6): δ (ppm) 199.1, 173.2, 170.0, 148.6, 130.4, 128.2, 127.8, 127.5, 124.3,
121.1, 76.6, 61.1, 54.1, 51.3, 47.9, 44.8, 43.4, 43.0, 38.8, 38.6, 37.5, 36.7, 32.2, 31.5,
28.3, 28.2, 27.0, 26.3. HPLC purity: 95.96%.
4.4.4.
3β-Hydroxy-30-(4-(4-nitrophenyl)-1-piperazinyl)-olean-12-ene-11,30-dione (3d)
Yield: 74%. M.p. 252–254 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.09 (d, J =
9.44 Hz, 2H, Ar-7’,9’), 7.00 (d, J = 9.5 Hz, 2H, Ar-6’,10’), 5.50 (s, 1H, CH-12), 4.32
(d, J = 5.2 Hz, 1H, -OH), 3.72 (s, 4H, CH2-1’, CH2-4’), 3.48 (d, J = 4.6 Hz, 4H, CH2-
2’, CH2-3’), 2.99-3.05 (m, 1H, CH-3), 2.57 (d, J = 13.4 Hz, 1H, CH-18), 2.33 (s, 1H,
CH-9), 2.24-1.36 (m, 16H, CH2-2, CH2-6, CH2-7, CH2-15, CH2-16, CH2-19, CH2-21,
CH2-22), 1.35 (s, 3H, Me-27), 1.19 (s, 3H, Me-29), 1.03 (s, 6H, Me-25, Me-26), 0.97
(d, J = 13.6 Hz, 2H, CH2-1), 0.91 (s, 3H, Me-23), 0.74(s, 3H, Me-24), 0.71 (s, 1H,
13
CH-5), 0.69 (s, 3H, Me-28). C NMR (150 MHz, DMSO-d6): δ (ppm) 198.9, 173.1,
169.7, 154.3, 136.9, 127.3, 125.6, 112.3, 76.4, 60.9, 53.9, 47.5, 46.0, 44.6, 43.3, 42.9,
42.8, 38.3, 37.2, 36.5, 32.0, 31.3, 28.1, 28.0, 26.8. HPLC purity: 97.29%.
4.4.5.
3β-Hydroxy-30-(4-(2,3-dichlorophenyl)-1-piperazinyl)-olean-12-ene-11,30-dione
(3e)
Yield: 86%. M.p. 277–279 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 7.32-7.35 (m,
H, Ar-7’, 8’), 7.14-7.18 (m, 1H, Ar-6’), 5.52 (s, 1H, CH-12), 4.31 (d, J = 5.2 Hz, 1H,
-OH), 3.72 (s, 4H, CH2-1’, CH2-4’), 2.99-3.05 (m, 1H, CH-3), 2.95 (s, 4H, CH2-
2’,CH2-3’), 2.58 (d, J = 13.3 Hz, 1H, CH-18), 2.33 (s, 1H, CH-9), 2.25-1.37 (m, 16H,
CH2-2, CH2-6, CH2-7, CH2-15, CH2-16, CH2-19, CH2-21, CH2-22), 1.35 (s, 3H, Me-
27), 1.20 (s, 3H, Me-29), 1.03 (s, 6H, Me-25, Me-26), 0.97 (d, J = 13.7 Hz, 2H, CH2-
1), 0.91 (s, 3H, Me-23), 0.75 (s, 3H, Me-24), 0.71 (s, 1H, CH-5), 0.69 (s, 3H, Me-28).
13C NMR (150 MHz, DMSO-d6): δ (ppm) 198.9, 173.0, 169.8, 150.5, 132.5, 128.4,
127.3, 126.0, 124.6, 119.7, 76.4, 60.9, 53.9, 51.1, 47.6, 44.6, 43.2, 42.8, 38.3, 37.3,
36.5, 32.0, 31.3, 28.1, 28.0, 26.8, 26.0. HPLC purity: 98.12%.
4.4.6.
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3β-Hydroxy-30-(4-(3,4-dichlorophenyl)-1-piperazinyl)-olean-12-ene-11,30-dione
(3f)
Yield: 80%. M.p. 283–285 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 7.42 (d, J =
9.0 Hz, 1H, Ar-7’), 7.13 (d, J = 2.9 Hz, 1H, Ar-10’), 6.92-6.96 (m, 1H, Ar-6’), 5.48 (s,
1H, CH-12), 4.32 (d, J = 5.2 Hz, 1H, -OH), 3.69 (s, 4H, CH2-1’, CH2-4’), 3.16 (t, J =
4.7 Hz, 4H, CH2-2’, CH2-3’), 2.99-3.05 (m, 1H, CH-3), 2.57 (d, J = 13.3 Hz, 1H, CH-
18), 2.33 (s, 1H, CH-9), 2.25-1.35 (m, 16H, CH2-2, CH2-6, CH2-7, CH2-15, CH2-16,
CH2-19, CH2-21, CH2-22), 1.35 (s, 3H, Me-27), 1.19 (s, 3H, Me-29), 1.02 (s, 6H, Me-
25, Me-26), 0.96 (d, J = 13.5 Hz, 2H, CH2-1), 0.91 (s, 3H, Me-23), 0.73 (s, 3H, Me-
24), 0.71 (s, 1H, CH-5), 0.69 (s, 3H, Me-28). HPLC purity: 98.45%.
4.4.7.
3β-Hydroxy-30-(4-(4-methoxyphenyl)-1-piperazinyl)-olean-12-ene-11,30-dione
(3g)
Yield: 77%. M.p. 218–220 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 6.90 (d, J =
9.2, 2H, Ar-6’,10’), 6.89 (d, J = 9.2, 2H, Ar-7’,9’), 5.50 (s, 1H, CH-12), 4.32 (d, J =
5.2 Hz, 1H, -OH), 3.69 (s, 7H, CH2-1’, CH2-4’, -OCH3), 2.99-3.05 (m, 1H, CH-3),
2.96 (t, J = 4.4 Hz, 4H, CH2-2’, CH2-3’), 2.57 (d, J = 13.4 Hz, 1H, CH-18), 2.33 (s,
1H, CH-9), 2.25-1.35 (m, 16H, CH2-2, CH2-6, CH2-7, CH2-15, CH2-16, CH2-19, CH2-
21, CH2-22), 1.35 (s, 3H, Me-27), 1.19 (s, 3H, Me-29), 1.03 (s, 6H, Me-25, Me-26),
0.97 (d, J = 13.2 Hz, 2H, CH2-1), 0.91 (s, 3H, Me-23), 0.73 (s, 3H, Me-24), 0.71 (s,
1H, CH-5), 0.69 (s, 3H, Me-28). HPLC purity: 95.72%.
4.4.8.
3β-Hydroxy-30-(4-(2-fluorophenyl)-1-piperazinyl)-olean-12-ene-11,30-dione (3h)
Yield: 75%. M.p. 253–255 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 7.10-7.18 (m,
2H, Ar-7′,9′), 6.97-7.07 (m, 2H, Ar-8’,10’), 5.51 (s, 1H, CH-12), 4.31 (d, J = 5.0, 1H,
-OH), 3.71 (s, 4H, CH2-1’, CH2-4’), 3.00-3.05 (m, 1H, CH-3), 2.97 (d, J = 4.2, 4H,
CH2-2’, CH2-3’), 2.58 (d, J = 13.4, 1H, CH-18), 2.33 (s, 1H, CH-9), 2.27-1.36 (m,
16H, CH2-2, CH2-6, CH2-7, CH2-15, CH2-16, CH2-19, CH2-21, CH2-22), 1.35 (s, 3H,
Me-27), 1.19 (s, 3H, Me-29), 1.03 (s, 6H, Me-25, Me-26), 0.97 (d, J = 13.8, 2H, CH2-
1), 0.91 (s, 3H, Me-23), 0.74 (s, 3H, Me-24), 0.71 (s, 1H, CH-5), 0.69 (s, 3H, Me-28).
HPLC purity: 97.75%.
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4.4.9.
3β-Hydroxy-30-(4-(3-methoxyphenyl)-1-piperazinyl)-olean-12-ene-11,30-dione
(3i)
1
Yield: 70%. M.p. 196–198 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) 7.13 (t, J =
8.2, 1H, Ar-9′), 6.53 (d, J = 8.3, 2H, Ar-10’), 6.46 (s, 1H, Ar-6’), 6.40 (d, J = 8.0, 1H,
Ar-8’), 5.48 (s, 1H, CH-12), 4.31 (d, J = 5.0, 1H, -OH), 3.72 (s, 3H, -OCH3), 3.68 (s,
4H, CH2-1’, CH2-4’), 3.10 (s, 4H, CH2-2’, CH2-3’), 2.99-3.05 (m, 1H, CH-3), 2.57 (d,
J = 13.5, 1H, CH-18), 2.33 (s, 1H, CH-9), 2.24-1.55 (m, 16H, CH2-2, CH2-6, CH2-7,
CH2-15, CH2-16, CH2-19, CH2-21, CH2-22), 1.35 (s, 3H, Me-27), 1.19 (s, 3H, Me-29),
1.02 (s, 6H, Me-25, Me-26), 0.96 (d, J = 13.6, 2H, CH2-1), 0.91 (s, 3H, Me-23), 0.73
(s, 3H, Me-24), 0.71 (s, 1H, CH-5), 0.69 (s, 3H, Me-28). HPLC purity: 95.84%.
4.4.10.
3β-Hydroxy-30-(4-(3-(trifluoromethyl)phenyl)-1-piperazinyl)-olean-12-ene-11,30-
dione (3j)
Yield: 78%. M.p. 247–249 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 7.44 (t, J =
8.0, 1H, Ar-7′), 7.23 (d, J = 8.5, 1H, Ar-6’), 7.17 (s, 1H, Ar-10’), 7.10 (d, J = 7.6, 1H,
Ar-8’), 5.49 (s, 1H, CH-12), 4.30 (d, J = 5.2, 1H, -OH), 3.71 (s, 4H, CH2-1’, CH2-4’),
3.22 (d, J = 4.6, 4H, CH2-2’, CH2-3’), 2.98-3.05 (m, 1H, CH-3), 2.57 (d, J = 13.2, 1H,
CH-18), 2.33 (s, 1H, CH-9), 2.26-1.36 (m, 16H, CH2-2, CH2-6, CH2-7, CH2-15, CH2-
16, CH2-19, CH2-21, CH2-22), 1.35 (s, 3H, Me-27), 1.20 (s, 3H, Me-29), 1.03 (s, 6H,
Me-25, Me-26), 0.97 (d, J = 10.1, 2H, CH2-1), 0.91 (s, 3H, Me-23), 0.74 (s, 3H, Me-
24), 0.71 (s, 1H, CH-5), 0.69 (s, 3H, Me-28). 13C NMR (150 MHz, DMSO-d6): δ
(ppm) 198.9, 172.9, 169.8, 150.8, 129.9, 127.3, 118.7, 110.9, 76.4, 60.9, 55.8, 53.9,
47.8, 47.5, 44.6, 43.2, 43.1, 42.8, 38.3, 37.3, 36.5, 32.0, 31.2, 28.1, 27.9, 26.8. HPLC
purity: 97.59%.
4.4.11.
3β-Hydroxy-30-(4-(3-chlorophenyl)-1-piperazinyl)-olean-12-ene-11,30-dione (3k)
Yield: 78%. M.p. 284–286 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 7.23 (t, J =
8.1, 1H, Ar-9’), 6.95 (s, 1H, Ar-6′), 6.90 (d, J = 8.4, 2H, Ar-10’), 6.82 (m, 1H, Ar-8’),
5.49 (s, 1H, CH-12), 4.30 (s, 1H, -OH), 3.68 (s, 4H, CH2-1’,CH2-4’), 3.15 (t, J = 4.6,
4H, CH2-2’, CH2-3’), 2.98-3.05 (m, 1H, CH-3), 2.57 (d, J = 13.6, 1H, CH-18), 2.33 (s,
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1H, CH-9), 2.25-1.37 (m, 16H, CH2-2, CH2-6, CH2-7, CH2-15, CH2-16, CH2-19, CH2-
21, CH2-22), 1.35 (s, 3H, Me-27), 1.19 (s, 3H, Me-29), 1.02 (s, 6H, Me-25, Me-26),
0.96 (d, J = 13.4, 2H, CH2-1), 0.91 (s, 3H, Me-23), 0.74 (s, 3H, Me-24), 0.71 (s, 1H,
CH-5), 0.69 (s, 3H, Me-28). HPLC purity: 99.18%.
4.4.12.
3β-Hydroxy-30-(4-(4-fluorophenyl)-1-piperazinyl)-olean-12-ene-11,30-dione (3l)
Yield: 72%. M.p. 170–172 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 6.94-7.01 (m,
2H, Ar-7′,9′), 6.88 (d, J = 7.5, 2H, Ar-6’,10’), 5.51 (s, 1H, CH-12), 4.33 (s, 1H, -OH),
3.68 (s, 4H, CH2-1’, CH2-4’), 2.99-3.05 (m, 1H, CH-3), 2.91 (s, 4H, CH2-2’, CH2-3’),
2.58 (d, J = 13.3, 1H, CH-18), 2.33 (s, 1H, CH-9), 2.26-1.38 (m, 16H, CH2-2, CH2-6,
CH2-7, CH2-15, CH2-16, CH2-19, CH2-21, CH2-22), 1.35 (s, 3H, Me-27), 1.19 (s, 3H,
Me-29), 1.03 (s, 6H, Me-25, Me-26), 0.96 (d, J = 12.3, 2H, CH2-1), 0.91 (s, 3H, Me-
23), 0.74 (s, 3H, Me-24), 0.71 (s, 1H, CH-5), 0.69 (s, 3H, Me-28). 13C NMR (150
MHz, DMSO-d6): δ (ppm) 198.9, 172.8, 169.8, 151.8, 140.5, 127.3, 122.7, 120.6,
118.0, 111.7, 76.4, 60.9, 55.1, 53.9, 50.4, 47.6, 44.6, 43.2, 42.8, 38.3, 37.3, 36.5, 32.0,
31.2, 28.1, 28.0, 26.8, 26.1. HPLC purity: 95.68%.
4.4.13.
3β-Hydroxy-30-(4-(2-methoxyphenyl)-1-piperazinyl)-olean-12-ene-11,30-dione
(3m)
Yield: 75%. M.p. 175–177 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 6.92-7.01 (m,
2H, Ar-7′,9′), 6.88 (d, J = 3.8, 2H, Ar-6’,8’), 5.51 (s, 1H,CH-12), 4.30 (d, J = 5.1, 1H,
-OH), 3.78 (s, 3H, -OCH3), 3.68 (s, 4H, CH2-1’,CH2-4’), 2.98-3.05 (m, 1H, CH-3),
2.91 (s, 4H, CH2-2’,CH2-3’), 2.57 (d, J = 13.3, 1H, CH-18), 2.32 (s, 1H, CH-9), 2.27-
1.35 (m, 16H, CH2-2, CH2-6, CH2-7, CH2-15, CH2-16, CH2-19, CH2-21, CH2-22),
1.34 (s, 3H, Me-27), 1.19 (s, 3H, Me-29), 1.03 (s, 6H, Me-25, Me-26), 0.96 (d, J =
13.4, 2H, CH2-1), 0.91 (s, 3H, Me-23), 0.74 (s, 3H, Me-24), 0.71 (s, 1H, CH-5), 0.69
(s, 3H, Me-28). 13C NMR (150 MHz, DMSO-d6): δ (ppm) 198.9, 172.8, 169.8, 151.8,
140.5, 127.3, 122.7, 120.6, 118.0, 111.7, 76.4, 60.9, 55.2, 53.9, 50.4, 47.7, 44.6, 43.2,
43.0, 42.8, 38.3, 37.3, 36.5, 32.0, 31.3, 28.1, 28.0, 26.8. HPLC purity: 97.37%.
4.4.14.
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3β-Hydroxy-30-(4-(2-pyridinyl)-1-piperazinyl)-olean-12-ene-11,30-dione (3n)
1
Yield: 73%. M.p. 272–274 °C. H NMR (400 MHz, DMSO-d6): δ (ppm) 8.11-8.14
(m, 1H, CH-6’), 7.53-7.58 (m, 1H, CH-8’), 6.82 (d, J = 8.6, 1H, CH-7’), 6.66-6.69 (m,
1H, CH-9’), 5.50 (s, 1H, CH-12), 4.31 (d, J = 5.2, 1H, -OH), 3.67 (s, 4H, CH2-1’,
CH2-4’), 3.46 (t, J = 4.8, 4H, CH2-2’, CH2-3’), 2.99-3.05 (m, 1H, CH-3), 2.57 (d, J =
13.3, 1H, CH-18), 2.33 (s, 1H, CH-9), 2.27-1.36 (m, 16H, CH2-2, CH2-6, CH2-7,
CH2-15, CH2-16, CH2-19, CH2-21, CH2-22), 1.35 (s, 3H, Me-27), 1.20 (s, 3H, Me-29),
1.02 (s, 6H, Me-25, Me-26), 0.97 (d, J = 13.4, 2H, CH2-1), 0.91 (s, 3H, Me-23), 0.73
(s, 3H, Me-24), 0.71 (s, 1H, CH-5), 0.69 (s, 3H, Me-28). 13C NMR (150 MHz,
DMSO-d6): δ (ppm) 199.0, 173.1, 169.8, 158.7, 147.4, 137.5, 127.3, 113.3, 107.1,
76.5, 61.0, 54.0, 47.6, 44.8, 44.6, 43.3, 42.9, 38.4, 37.3, 36.5, 32.1, 31.3, 28.1, 28.0,
26.9, 26.1, 26.0. HPLC purity: 98.22%.
4.4.15.
3β-Hydroxy-30-(4-(3-pyridinyl)-1-piperazinyl)-olean-12-ene-11,30-dione. (3o)
Yield: 72%. M.p. 246–248 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.11 (d, J =
3.7, 1H, CH-6’), 7.65 (s, 1H, CH-7’), 6.92 (d, J = 8.4, 1H, CH-8’), 6.73 (t, J = 5.7, 1H,
CH-9’), 5.49 (s, 1H, CH-12), 4.03 (d, J = 5.2, 1H, -OH), 3.68 (s, 4H, CH2-1’, CH2-4’),
3.50 (s, 4H, CH2-2’, CH2-3’), 2.99-3.05 (m, 1H, CH-3), 2.57 (d, J = 4.4, 1H, CH-18),
2.33 (s, 1H, CH-9), 2.24-1.39 (m, 16H, CH2-2, CH2-6, CH2-7, CH2-15, CH2-16, CH2-
19, CH2-21, CH2-22), 1.35 (s, 3H, Me-27), 1.20 (s, 3H, Me-29), 1.02 (s, 6H, Me-25,
Me-26), 0.97 (d, J = 13.6, 2H, CH2-1), 0.91 (s, 3H, Me-23), 0.74 (s, 3H, Me-24), 0.71
(s, 1H, CH-5), 0.69 (s, 3H, Me-28). HPLC purity: 95.81%.
4.4.16.
3β-Hydroxy-30-(4-ethyl-1-piperazinyl)-olean-12-ene-11,30-dione (3p)
Yield: 82%. M.p. 173–175 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 5.48 (s, 1H,
CH-12), 4.30 (d, J = 5.1, 1H, -OH), 3.52 (s, 4H, CH2-1’,CH2-4’), 2.98-3.05 (m, 1H,
CH-3), 2.57 (d, J = 13.5, 1H, CH-18), 2.32 (s, 7H, CH-9, CH2-2’, CH2-3’, CH2-5’),
2.20-1.38 (m, 16H, CH2-2, CH2-6, CH2-7, CH2-15, CH2-16, CH2-19, CH2-21, CH2-
22), 1.34 (s, 3H, Me-27), 1.15 (s, 3H, Me-29), 1.03 (s, 6H, Me-25, Me-26), 0.99 (d, J
= 7.2, 3H, Me-6’), 0.95 (s, 2H, CH2-1), 0.91 (s, 3H, Me-23), 0.73 (s, 3H, Me-24), 0.71
(s, 1H, CH-5), 0.69 (s, 3H, Me-28). HPLC purity: 95.49%.
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4.4.17.
3β-Hydroxy-30-(4-benzhydryl-1-piperazinyl)-olean-12-ene-11,30-dione (3q)
Yield: 81%. M.p. 170–172 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 7.41-7.47 (m,
4H, Ar-2”,6”,8”,12”), 7.30 (t, J = 7.2, 4H, Ar-3”,5”,9”,11”), 7.19 (t, J = 7.3, 2H, Ar-
4”,10”), 5.49 (s, 1H, CH-12), 4.31 (d, J = 4.7, 1H, -OH), 4.27 (s, 1H, CH-5’), 3.56 (s,
4H, CH2-1’, CH2-4’), 3.33 (s, 4H, CH2-2’, CH2-3’), 2.98-3.04 (m, 1H, CH-3), 2.58 (d,
J = 13.5, 1H, CH-18), 2.31(s, 1H, CH-9), 2.19-1.57 (m, 16H, CH2-2, CH2-6, CH2-7,
CH2-15, CH2-16, CH2-19, CH2-21, CH2-22), 1.32 (s, 3H, Me-27), 1.11 (s, 3H, Me-29),
1.03 (d, J = 3.1, 6H, Me-25, Me-26), 0.95 (d, J = 13.5, 2H, CH2-1), 0.91 (s, 3H, Me-
23), 0.72 (s, 3H, Me-24), 0.71 (s, 1H, CH-5), 0.69 (s, 3H, Me-28). HPLC purity:
95.87%.
4.4.18.
3β-Hydroxy-30-(4-(bis(4-fluorophenyl)methyl)-1-piperazinyl)-olean-12-ene-
11,30-dione (3r)
Yield: 81%. M.p. 169–171 °C. 1H NMR (400 MHz, DMSO-d6): δ (ppm) 7.41-7.50 (m,
4H, Ar-2”,6”,8”,12”), 7.13 (t, J = 8.3, 4H, Ar-3”,5”,9”,11”), 5.49 (s, 1H,CH-12), 4.36
(s, 1H, CH-5’), 4.30 (s, 1H, -OH), 3.55 (s, 4H, CH2-1’, CH2-4’), 2.98-3.05 (m, 1H,
CH-3), 2.59 (d, J = 13.2, 1H, CH-18), 2.31 (s, 1H, CH-9), 2.25 (s, 4H, CH2-2’, CH2-
3’), 2.19-1.36 (m, 16H, CH2-2, CH2-6, CH2-7, CH2-15, CH2-16, CH2-19, CH2-21,
CH2-22), 1.32 (s, 3H, Me-27), 1.11(s, 3H, Me-29), 1.03 (d, J = 3.8, 6H, Me-25, Me-
26), 0.95 (d, J = 4.4, 2H, CH2-1), 0.91 (s, 3H, Me-23), 0.72 (s, 3H, Me-24), 0.71 (s,
13
1H, CH-5), 0.69 (s, 3H, Me-28). C NMR (150 MHz, DMSO-d6): δ (ppm) 199.1,
172.9, 170.0, 161.9, 160.3, 129.5, 129.4, 128.7, 127.5, 115.5, 115.4, 76.6, 73.0, 61.1,
54.1, 51.9, 47.8, 44.8, 43.3, 43.0, 40.1, 38.5, 37.5, 36.7, 32.2, 31.4, 30.0, 28.3. HPLC
purity: 96.67%.
4.5. Cell proliferation assay
The inhibitory activity of the prepared compounds 3a-3r against MCF-7, Hela,
HepG2 and A549 were measured by the MTT assay. Tumor cell lines were cultured
in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum and
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penicillin (100 units mL−1), and incubated at 37 °C in an atmosphere with 5% CO2,
20% O2. The cells were added in 96-well plates at a density of 3-5×103 cells per well
at 37 °C for 24 h. Subsequently, the cells were treated with all prepared compounds at
preset concentrations and Glycyrrhetic acid being employed as a positive reference.
After 24h, viable cells were determined by adding 10 μL of PBS containing 5 mg
mL−1of MTT each well. After waiting 4 h, the medium was discarded and add 150 μL
DMSO per well to dissolve the purple formazan crystals completely. The plates were
read on a Victor-V multilabel counter (Perkin-Elmer) using the default europium
detection protocol with the optical absorbance was measured at 570 nm. Percent
inhibition or IC50 values of compounds were calculated by comparing with DMSO
treated control wells. The results are shown in Table S2.
4.6. VEGFR2 kinase assay
The human KDR/GST chimera is composed of the human KDR (NP_002244)
(Asp807-Val1356) and the N-terminal polyhistidine tagged GST tag. The
recombinant has a molecular weight of 89.3 kDa which consists of 787 amino acids.
The VEGFR2 kinase assay was conducted according to the literature description.
96-well plates were coated at room temperature for 1–2h with 100mL per well of
25 μg mL−1 poly-(Glu4–Tyr) peptide (Sigma) in Tris-buffered saline (TBS) (25 mM
Tris, pH 7.2, 150 mM NaCl). Unbound peptide was washed three times with PBS.
The cytoplasmic domains of VEGFR2, PDGFR, EGFR, bFGF enzymes were diluted
(depending on the specific activity of the batch, from 10- to 20- fold) in 0.1% BSA/4
mM HEPES. A master mix of enzyme plus kinase buffer was prepared: (per well)
10μL of diluted enzyme, 10 μL of kinase buffer (4 mM HEPES, pH 7.4, 1.25 mM
MnCl2, 20mM Na3VO4) and compound (10 μL) prepared in 100% dimethyl sulfoxide
(DMSO) were added to wells with appropriate density. Controls were done by adding
DMSO alone, i.e., no test compound, to wells containing the master mix of enzyme
plus kinase buffer. After 15 min at room temperature, ATP–MgCl2(20μL of 25μM
ATP, 25 mM MgCl2, 10 mM HEPES, pH 7.4) was added to each well to initiate the
reaction. Final concentrations of the assay components were 10μM ATP, 10 mM
MgCl2, 1 mM MnCl2, 4 mM HEPES, pH 7.4, 20μM Na3VO4, 20μg mL−1 BSA. After
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40 min at room temperature, the liquid was removed, and plates were washed three
times with PBST (PBS with 0.05% Tween-20). The wells were then incubated for 1 h
at room temperature with 75μL of 0.1 μg mL−1 europium-conjugated anti-
phosphotyrosine antibody (PT66; Perkin-Elmer) prepared in the assay buffer (Perkin-
Elmer). Plates were washed three times with PBST and then incubated for 15 min in
the dark with 100 μL of enhancement solution (Perkin-Elmer). Percent inhibition or
IC50 values of compounds were calculated by comparing with DMSO-treated control
wells.
4.7. Analysis of the apoptosis induced in cells
Approximately 105 cells per well were plated in a 24-well plate and allowed to
adhere. After 12 h, the medium was replaced with fresh culture medium containing
compound 3a at final concentrations of 1, 2 and 4μM. Nontreated wells received an
equivalent volume of ethanol (<0.1%). To determine the optimal time course for the
induction of apoptosis, cells were harvested after 24 h. They were trypsinized, washed
with PBS and centrifuged at 1800 rpm for 5 min. The pellet was then resuspended in
500 μL of staining solution (containing 1μL Annexin V-PE and 5μL 7-AAD in
binding buffer), mixed gently and incubated for 15 min at room temperature (15–
25 °C) in the dark. The samples were then read in a FACSCalibur flow cytometer
(USA) at 488 nm excitation. A 578 nm band pass filter for fluorescein detection and
a >600 nm filter for 7-AAD detection were used. Analyses were performed using the
soft-ware supplied with the instrument.
4.8. Flow cytometric determination of the mitochondrial membrane potential
Changes in the mitochondrial transmembrane potential (Δψm) as a result of
mitochondrial perturbation were measured after staining with Rhodamine-123.
HepG2 cells were incubated with the indicated doses of compound 3a for 24 h.
Rhodamine-123 (5μM) was added 1 h before the termination of the experiment and
cells were collected and washed with PBS. The fluorescence intensity of 10000 events
was analyzed in the FL-1 channel of the FACSCalibur flow cytometer (USA). The
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10.1002/cmdc.201700271
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decrease in the fluorescence intensity caused by the loss of mitochondrial membrane
potential was analyzed in the FL-1 channel.
4.9. Wound healing migration assay
The wound-healing migration assay was performed as described previously.
Briefly, MCF-7 cells were treated with mitomycin C to inactivate cell proliferation,
wounded by a micro-tip, washed with PBS, and supplemented with fresh medium.
Capturing the images of the cells at the beginning and at regular intervals(24h) during
cell migration to close the wound, and comparing the images to quantify the migration
rate of the cells.
4.10. Molecular docking
The crystal structures of the protein complexes were retrieved from the RCSB
Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). The three-dimensional
structures of the aforementioned compounds were constructed using Chem. 3D ultra
12.0 software [Chemical Structure Drawing Standard; Cambridge Soft Corporation,
USA (2010)]; then they were energetically minimized by using MMFF94 with 5000
iterations and a minimum RMS gradient of 0.10. Molecular docking of compound 3a
into the three-dimensional VEGER2 complex structure (1YWN.pdb, downloaded
from the PDB) was carried out using the Discovery Studio (version 3.5) as
implemented through the graphical user interface DS-CDOCKER protocol. DS-
CDOCKER is an implementation of a CHARMM based molecular docking tool using
a rigid receptor, all bound water and ligands were eliminated from the protein and the
polar hydrogen was added to the proteins.
5. Acknowledgements
This work was supported by National Natural Science Foundation of China (No.
21602103) and Major Projects on Control and Rectification of Water Body Pollution
(No. 2011ZX07204-001-004), and ‘PCSIRT’ (IRT1020).
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Table 1. Chemical structures of glycyrrhetic acid derivatives.
Table 2 Antiproliferative activities in vitro of target compounds 3a-3r against four
cancer cell lines
Table 3. Inhibition of selected kinases IC50a (μM)
Table 4. The median cytotoxic concentration (CC50) data of tested compounds
Figure 1. Chemical structures of glycyrrhizic acid (1) and its aglycone glycyrrhetic
acid (2).
Figure 2. The dose-dependent inhibition assay curve of MCF-7 for 3a.
Figure 3. Correlation between the anti-proliferative activity against MCF-7 and the
VEGFR2 inhibitory activity.
Figure 4 MCF-7 Cells treated with compound 3a at 2, 4, and 8 μM concentration for
24 h were collected and processed for analysis. The percentage of early apoptotic cells
in the lower right quadrant (annexin V-FITC positive/PI negative cells), as well as late
apoptotic cells located in the upper right quadrant (annexin V-FITC positive/PI
positive cells). Images are representative of three independent experiments. Data are
mean ± S.E.M. of three independent experiments. * P < 0.05, ***P < 0.001.
Figure 5. Compound 3a induced mitochondrial membrane potential change in breast
cancer MCF-7 cells. The cells were treated with compound 3a at 2, 4, and 8 μM
concentration for a 24 h time period. The cells were stained with Rhodamine-123 (200
nM) dye for 30 min and analyzed in FL-1 channel of a flow cytometer. Data are
representative of one of three independent experiments.
Figure 6. (A) Effect of compound 3a on MCF-7 cells. Compound 3a inhibits MCF-7
migration in the wound healing assay. The MCF-7 cells were scratched by a sterile
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10.1002/cmdc.201700271
ChemMedChem
micro-tip. Capturing the images at the beginning and at 24h during cell migration to
close the wound. (B) Wound closure percentage of MCF-7 cells, Data are mean ±
S.E.M. of three independent experiments. **P < 0.01.
Figure 7. (A) 2D molecular docking model of compound 3a with 1YWN. (B) 3D
interaction map between compound 3a and 1YWN binding sites of VEGFR2 protein.
Scheme 1. General method of the preparation of compounds 3a-3r. Reagents and
conditions: (a) 6% aq. HCl, MeOH, 100 °C, 20 h; (b) CH2Cl2, HOBt, DCC,
piperazine derivatives, RT, 24 h.
Table 1. Chemical structures of glycyrrhetic acid derivatives
Compounds
R
Compounds
R
3a
3j
3b
3c
3k
3l
3d
3m
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3e
3n
3f
3o
3p
3g
3h
3i
3q
3r
Table 2 Antiproliferative activities in vitro of target compounds 3a-3r against four
cancer cell lines
IC50
IC50
IC50
IC50
(µMa±SD)
HELAb
(µMa±SD)
HEPG2b
(µMa±SD)
MCF-7b
(µMa±SD)
A549b
Compound
2.39±0.21
13.98±1.03
13.26±1.59
4.21±0.24
7.37±0.51
5.85±0.27
27.22±2.44
14.01±0.91
8.87±0.72
4.08±0.36
3.13±0.27
19.31±1.82
12.08±0.78
4.76±1.09
5.56±0.67
4.48±0.35
20.35±3.73
13.95±4.19
9.12±0.56
3.93±0.15
1.08±0.31
15.53±0.97
18.80±1.50
3.02±0.52
6.42±0.38
5.23±0.89
23.51±1.69
11.78±1.13
10.61±1.12
5.37±0.80
2.72±0.15
19.02±1.66
19.81±1.94
2.89±3.18
5.95±0.53
5.06±0.49
31.46±2.26
16.54±1.75
9.03±0.70
4.71±0.95
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
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16.83±1.77
10.19±1.55
16.74±4.68
36.13±5.1
41.62±3.27
69.76±5.65
81.85±15.88
84.24±4.05
3.23±0.86
12.77±0.93
8.07±0.67
23.65±1.64
39.44±3.15
37.53±1.20
58.16±7.86
69.72±2.09
95.16±9.36
2.07±0.73
14.19±2.56
8.95±0.91
18.39±1.77
38.20±3.59
32.45±3.56
49.07±4.34
74.91±5.95
79.89±6.55
1.40±1.05
17.62±2.12
3k
3l
9.27±0.72
20.51±2.33
41.74±4.31
38.14±2.01
63.33±7.19
88.47±6.49
92.17±11.72
2.11±0.38
3m
3n
3o
3p
3q
3r
Vandetanib
a Antiproliferation activity was measured using the MTT assay. Values are the average of three
independent experiments run in triplicate. Variation was generally 5–10%.
b Cancer cells kindly supplied by State Key Laboratory of Pharmaceutical Biotechnology, Nanjing
University.
Table 3. Inhibition of selected kinases IC50a (μM)
Compounds
VEGFR2
0.35
2.79
3.21
0.62
1.28
1.07
5.44
3.61
2.73
0.92
4.25
3.96
4.14
9.39
8.51
13.65
16.32
15.14
0.11
EGFR
10.09
18.68
20.95
9.77
bFGF
18.12
25.31
38.20
19.84
25.63
31.26
29.74
32.35
30.11
21.23
37.59
29.47
38.46
32.55
31.62
50.29
53.87
40.48
4.69
PDGFR
13.58
22.74
24.36
12.96
19.35
17.09
18.23
25.13
21.27
17.65
23.78
24.61
20.69
30.42
28.57
31.12
35.37
33.88
1.33
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
12.26
13.39
21.41
19.03
15.64
11.52
17.84
18.32
20.71
26.54
23.47
31.93
30.76
28.15
0.58
3q
3r
Vandetanib
a
IC50 values were averaged values determined by at least three independent experiments.
Variation was generally 5-10%.
Table 4. The median cytotoxic concentration (CC50) data of tested compounds
Compounds
CC50, µM a
162.28
Compounds
CC50, µM a
94.84
3a
3d
3i
3j
126.71
137.65
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105.30
114.92
98.57
184.41
3e
3f
3l
Vandetanib
a
The cytotoxicity of each compound was expressed as the concentration of compound that
reduced cell viability to 50% (CC50).
Figure 1. Chemical structures of glycyrrhizic acid (1) and its aglycone glycyrrhetic
acid (2).
Figure 2. The dose-dependent inhibition assay curve of MCF-7 for 3a.
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10.1002/cmdc.201700271
ChemMedChem
Figure 3. Correlation between the anti-proliferative activity against MCF-7 and the
VEGFR2 inhibitory activity.
Figure 4. MCF-7 Cells treated with compound 3a at 2, 4, and 8 μM concentration for
24 h were collected and processed for analysis. The percentage of early apoptotic cells
in the lower right quadrant (annexin V-FITC positive/PI negative cells), as well as late
apoptotic cells located in the upper right quadrant (annexin V-FITC positive/PI
positive cells). Images are representative of three independent experiments. Data are
mean ± S.E.M. of three independent experiments. * P < 0.05, ***P < 0.001.
28
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ChemMedChem
Figure 5. Compound 3a induced mitochondrial membrane potential change in breast
cancer MCF-7 cells. The cells were treated with compound 3a at 2, 4, and 8 μM
concentration for a 24 h time period. The cells were stained with Rhodamine-123 (200
nM) dye for 30 min and analyzed in FL-1 channel of a flow cytometer. Data are
representative of one of three independent experiments.
Figure 6. (A) Effect of compound 3a on MCF-7 cells. Compound 3a inhibits MCF-7
migration in the wound healing assay. The MCF-7 cells were scratched by a sterile
micro-tip. Capturing the images at the beginning and at 24h during cell migration to
29
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