Synthesis and antiproliferative activity of novel heterocyclic glycyrrhetinic acid derivatives

Article abstract of DOI:10.3390/molecules24040766

A new series of glycyrrhetinic acid derivatives has been synthesized via the introduction of different heterocyclic rings conjugated with an α,β-unsaturated ketone in its ring A. These new compounds were screened for their antiproliferative activity in a panel of nine human cancer cell lines. Compound 10 was the most active derivative, with an IC₅₀ of 1.1 μM on Jurkat cells, which is 96-fold more potent than that of glycyrrhetinic acid, and was 4-fold more selective toward that cancer cell line. Further biological studies performed in Jurkat cells showed that compound 10 is a potent inducer of apoptosis that activates both the intrinsic and extrinsic pathways.

Full text of DOI:10.3390/molecules24040766

molecules
Article
Synthesis and Antiproliferative Activity of Novel
Heterocyclic Glycyrrhetinic Acid Derivatives
Daniela P. S. Alho 1,2 , Jorge A. R. Salvador 1,2,*, Marta Cascante 3,4 and Silvia Marin 3,4,
*
1
Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Coimbra,
3000-548 Coimbra, Portugal; dani.alho23@gmail.com
Centre for Neuroscience and Cell Biology, 3004-504 Coimbra, Portugal
Department of Biochemistry and Molecular Biomedicine, Faculty of Biology, University of Barcelona,
Diagonal 643, 08028 Barcelona, Spain; martacascante@ub.edu
Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD),
Instituto de Salud Carlos III (ISCIII), 28029 Madrid, Spain
2
3
4
*
Correspondence: salvador@ci.uc.pt (J.A.R.S.); silviamarin@ub.edu (S.M.);
Tel.: +351-239-488-400 (J.A.R.S.); +34-934-021-593 (S.M.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 16 January 2019; Accepted: 15 February 2019; Published: 20 February 2019
Abstract: A new series of glycyrrhetinic acid derivatives has been synthesized via the introduction
of different heterocyclic rings conjugated with an -unsaturated ketone in its ring A. These new
compounds were screened for their antiproliferative activity in a panel of nine human cancer cell
lines. Compound 10 was the most active derivative, with an IC50 of 1.1 M on Jurkat cells, which is
α,β
µ
96-fold more potent than that of glycyrrhetinic acid, and was 4-fold more selective toward that cancer
cell line. Further biological studies performed in Jurkat cells showed that compound 10 is a potent
inducer of apoptosis that activates both the intrinsic and extrinsic pathways.
Keywords: pentacyclic triterpenoids; glycyrrhetinic acid; heterocyclic derivatives; antiproliferative
activity; apoptosis
1. Introduction
Triterpenoids are a large group of natural compounds that are widely found in nature and are
particularly prevalent in plants. They display a wide spectrum of biological and pharmacological
activities. Within this group, the pentacyclic triterpenoids are the most potent compounds and
their anti-inflammatory, antiviral, and anticancer effects have been extensively studied. Moreover,
there is an increasing interest in these triterpenoids as lead compounds for the development of new
anticancer agents, which is reflected in the large number of scientific papers and patents emerging in
this field [1–11].
Glycyrrhetinic acid (GA)
1 (Figure 1) is the aglycone of glycyrrhizin, a major pentacyclic
triterpenoid saponin found in the roots of licorice (Glycyrrhiza species) [12]. This compound has a
proven antiproliferative activity against several cancer cell lines and its anticancer effects were also
observed in animal models [13
the induction of apoptosis [16
–
16]. Recent reports have shown that its cytotoxicity is mediated by
–
21]. In addition, GA can be extracted from the roots of licorice in
1
high yields (up to 24%) [22]. These findings have increased its scientific interest as a scaffold for the
development of new derivatives for potential cancer treatment [23–27].
In previous work performed by our group, introduction of imidazole, methyl-imidazole, and
triazole heterocyclic rings was achieved in several positions of betulinic and ursolic acids, affording
several derivatives with better antiproliferative activity compared with the parental compounds.
The structure–activity relationship analysis revealed that the introduction of a heterocyclic ring in
Molecules 2019, 24, 766; doi:10.3390/molecules24040766
www.mdpi.com/journal/molecules

Molecules 2019, 24, 766
2 of 21
conjugation with an
derivatives [28,29].
α
,β-unsaturated ketone in ring A of the skeleton seems to provide the most potent
Cycloaddition of azides and alkynes, commonly referred to as “click chemistry”, has
acquired great interest in the development of novel heterocyclic compounds with varied biological
activities [30–33]. This efficient and simple process has been successfully used in the synthesis of
pentacyclic triterpenoid derivatives with improved anticancer activity [34–37].
The information provided above prompted us to synthesize new GA
1 derivatives via the
introduction of different heterocyclic rings conjugated with an -unsaturated ketone in its ring
α,β
A. These novel semisynthetic derivatives were tested for their antiproliferative activity against a series
of cancer cell lines. Further biological assays were performed for the most active compound 10 in the
cancer cell line that yielded the best results (Jurkat cells), to elucidate its mechanism of action.
Figure 1. Chemical structure of glycyrrhetinic acid 1.
2. Results and Discussion
2.1. Chemistry
The synthesis of three pairs of novel GA 1 heterocyclic derivatives is summarized in Scheme 1.
In addition to the intention to prepare heterocyclic derivatives with improved cytotoxicity, compounds
9–14 were synthesized to explore the effect of the keto group in position C-11 on the antiproliferative
activity. Therefore, the first step performed in this sequence of reactions was the removal of the
keto group via Clemmensen reduction with zinc dust and concentrated HCl in dioxane at room
temperature, to afford
structures, providing successive pairs of derivatives. Methyl esters
the reaction of compounds and with methyl iodide in the presence of potassium carbonate [39].
The 3 -hydroxyl group of these esters was oxidized using the Jones reagent [40], to give the 3-keto
derivatives and . The reaction of these derivatives with ethyl formate and sodium methoxide
allowed the preparation of the 2-hydroxyvinyl-3-keto compounds and 8 [40]. The heterocyclic
derivatives 14 were prepared via the reaction of the vinyl alcohol at C-2 with the appropriate
2
[
38]. The following steps were executed in both original and reduced
3
and were obtained from
4
1
2
β
5
6
7
9–
heterocyclic reagent, 1,10-carbonyldiimidazole (CDI), 1,10-carbonylbis(20-methylimidazole) (CBMI), or
1,10-carbonyl-di(1,2,4-triazole) (CDT) in reflux of THF under inert atmosphere [29], in yields ranging
from 41% to 70%.
Compounds 17 and 18 were synthesized as depicted in Scheme 2. These derivatives were prepared
to evaluate if the presence of another heterocyclic ring at C-30 would benefit the antiproliferative
activity. The steps of this sequence of reactions were performed using the same methods of oxidation,
reaction with ethyl formate, and introduction of heterocyclic rings described in Scheme 1. These novel
heterocyclic derivatives, 17 and 18, were obtained in yields of 56% and 38%, respectively.
Click chemistry was handled as shown in Scheme 3. We decided to synthesize 1,4-substituted-
triazolyl derivatives in conjugation with an
α,β-unsaturated ketone in ring A of pentacyclic
triterpenoids via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAc) with terminal alkynes. We
prepared the azido derivatives 19 (62%) and 20 (57%) by tosylation of the vinyl alcohol at C-2 and

Molecules 2019, 24, 766
3 of 21
subsequent replacement by azide. The triazolyl derivatives 21 and 22 were obtained via the “click
◦
reaction” of those compounds with methyl propiolate, catalyzed by CuI, in THF, at 65 C (which were
the best reaction conditions observed here), in yields of 42% and 44%, respectively.
Scheme 1. Reagents and conditions: (
DMF, r.t.; (
) Jones reagent, acetone, 0 ◦C; (
CBMI or CDT, dry THF, reflux, N2.
a
) zinc dust, concentrated HCl, dioxane, r.t.; (
b
) CH3I, K2CO3,
) CDI,
c
d) ethyl formate, NaOMe, benzene, r.t., N2; and (e
Full structural elucidation of the new glycyrrhetinic acid derivatives was achieved using infrared
spectroscopy (IR), nuclear magnetic resonance (NMR), and mass spectrometry (MS). The data obtained
from these analytical techniques, for the known compounds 2–8, were in agreement with those reported
in the literature [38–40].
The removal of the keto group at position C-11, via Clemmensen reduction, was confirmed by the
absence of a band around 1664 cm−1 on the IR spectrum, which is present in glycyrrhetinic acid, and
corresponds to the carbonyl group in ring C. On the 13C NMR spectrum, a
δ
signal around 200 ppm,
which corresponds to the presence of that carbonyl group, was absent, and the signals for carbons
C-12 and C-13 were present at higher field than that observed in compounds with the carbonyl group.
The preparation of compounds , and 16 was confirmed by the presence of a signal at
~8.6 ppm in the 1H NMR spectra, corresponding to the proton in the exocyclic double bond at C-2.
On the 13C NMR spectrum, the presence of this moiety was confirmed by the observation of a
signal
signal at ~106 ppm, corresponding to the
δ
7,
8
δ
δ
at ~188 ppm, corresponding to the exocyclic carbon, and a
C-2 carbon.
δ
The successful preparation of the heterocyclic derivatives 9–14, 17 and 18, and 21 and 22, was
confirmed by the presence of a δ signal at ~7.7–8.0 ppm, corresponding to the proton of the exocyclic
double bond at C-2 in the 1H NMR spectrum. On the 13C NMR spectrum the presence of this moiety
was confirmed by the presence of a δ signal at ~127–131 ppm, corresponding to the exocyclic carbon.

Molecules 2019, 24, 766
4 of 21
Scheme 2. Reagents and conditions: (
benzene, r.t., N2; and (c) CDI or CDT, dry THF, reflux, N2.
a b) ethyl formate, NaOMe,
) Jones reagent, acetone, 0 ◦C; (
Scheme 3. Reagents and conditions: (
propiolate, CuI, THF, 65 C.
a) TsCl, Et3N, CH2Cl2, r.t.; (
b) NaN3, acetone, r.t.; and (c) methyl
◦
1
The presence of heterocyclic ring(s) can be detected by the presence of extra
δ signals in H
and 13C NMR spectra. On the 1H NMR spectra, the imidazole group had three specific protons that
appeared at values higher than 7 ppm, the methyl-imidazole group had two protons ranging from 6.9

Molecules 2019, 24, 766
5 of 21
to 7.7 ppm, the triazole group had two proton signals with a
δ
higher than 8 ppm, and the triazole
group on compounds 21 and 22 had only one proton at ~8.3–8.4 ppm. In the 13C NMR spectra, the
δ
signals of the carbons of heterocyclic rings were present at values ranging from 118 to 153 ppm,
varying in accordance with the different heterocyclic rings.
2.2. Biological Activity
2.2.1. Antiproliferative Activity
Several cancer lines were cultured and used in experiments aimed at evaluating the potential
cytotoxicity of the synthesized compounds against human cancers. This evaluation was based on
the determination of IC50 values using 3-(4,5-dimethylthiozol-2-yl)-3,5-dipheryl tetrazolium bromide
(MTT) or triphenyl tetrazolium chloride (XTT) assays after 72 h of treatment with the compounds.
All compounds were screened for their antiproliferative activity on the HT-29 (colon
adenocarcinoma) cell line. As shown in Table 1, most of the novel heterocyclic derivatives (
17 18 21, and 22) showed improved cytotoxicity compared with the parental compound GA and the
intermediate compounds. The methylation of compounds and provided derivatives ( and ) that
exhibited an important increment in antiproliferative activity, but lacked selectivity towards tumor
cells [39]. The intermediates 16 19, and 20, which preceded the final heterocyclic derivatives,
9–14,
,
,
1
1
2
3
4
7
,
8,
,
were also tested on the A549 (lung adenocarcinoma) cell line (Table 1). Analysis of the IC50 values
of the heterocyclic derivatives with respect to their substrates in the two cancer cell lines showed
that the introduction of a heterocyclic ring provided more potent compounds, with the exception of
compound 13.
Table 1. Antiproliferative activities of GA, its derivatives, and cisplatin against HT-29 and A549
cell lines.
Cell line (IC50, µM) 1
Compound
HT-29
A549
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
115.7 ± 1.6
88.1 ± 1.4
19.6 ± 0.6
18.5 ± 0.9
46.3 ± 2.3
63.9 ± 1.1
14.3 ± 0.3
14.0 ± 0.2
11.5 ± 0.5
3.3 ± 0.2
110.5 ± 3.9
N.D.
N.D.
N.D.
N.D.
N.D.
18.5 ± 1.5
17.0 ± 0.6
11.1 ± 0.1
2.8 ± 0.2
10.3 ± 0.6
3.1 ± 0.1
24.7 ± 0.9
12.3 ± 0.6
N.D.
54.4 ± 2.6
23.1 ± 0.8
24.5 ± 0.6
48.5 ± 0.8
44.3 ± 3.6
10.6 ± 0.1
7.9 ± 0.4
12.6 ± 0.8 [42]
9.4 ± 0.7
3.6 ± 0.1
31.2 ± 1.5
12.1 ± 0.2
92.3 ± 1.5
60.3 ± 2.0
22.4 ± 0.5
21.8 ± 1.6
38.5 ± 0.6
36.3 ± 1.4
11.0 ± 0.8
8.9 ± 0.5
17
18
19
20
21
22
Cisplatin
6.1 [41]
1
The cell lines were treated with different concentrations of each compound for 72 h. IC50 values were determined
by 3-(4,5-dimethylthiozol-2-yl)-3,5-dipheryl tetrazolium bromide (MTT) assay and are expressed as means SD
±
(standard deviation) of three independent experiments. IC50 is the concentration that inhibits 50% of cellular growth.
N.D.: not determined.

Molecules 2019, 24, 766
6 of 21
The novel compounds
9–
14
,
17
,
18
,
21, and 22, and the parental compound GA
1
were further
tested in seven additional human cancer cell lines: MIAPaCa 2 (pancreas adenocarcinoma), HeLa
(cervix adenocarcinoma), A375 (melanoma), MCF7 (breast adenocarcinoma), HepG2 (hepatocellular
carcinoma), SH-SY5Y (neuroblastoma), and Jurkat (acute T cell leukemia) cells (Table 2). The synthesis
of compounds 17 and 18 was performed to investigate the effect of the combined introduction
of heterocyclic rings at the vinyl alcohol at the C-2 and at the C-30 positions. This combination
resulted in a loss of cytotoxicity in all tested lines compared with compounds 10 and 14, respectively.
The preparation of the four pairs of derivatives 9–14, 21, and 22 allowed us to evaluate the effect of the
keto group at position C-11 on the antiproliferative activity. The results of the assays in all tested cell
lines were consistent and revealed that the compounds from which the keto group was removed from
ring C were more potent. The type of heterocyclic ring conjugated with the α,β-unsaturated ketone in
ring A also influenced the antiproliferative activity. Within the group of heterocyclic derivatives with a
reduced ring C, compound 10, which bears an imidazole ring, was the most active derivative in all
tested cell lines. This compound was 31- to 96-fold more potent than GA 1, depending on the cancer
cell line. Moreover, with the exception of SH-SY5Y cells, compound 10 showed a similar or slightly
improved antiproliferative activity against all cancer cell lines compared to the chemotherapy agent
cisplatin [41–46] (Tables 1 and 2).
The selectivity towards cancer cells was studied for GA
with a human nontumoral cell line (BJ) (Table 2). Compound 10 and GA
1
and compound 10 by incubating them
showed IC50 values that
1
were 6.3 and 1.6 times lower on Jurkat cells than on the nontumoral BJ cells, respectively. Therefore, the
heterocyclic derivative 10 was more selective towards malignant cells than its parental compound 1.

Molecules 2019, 24, 766
7 of 21
Table 2. Antiproliferative activities of GA, its heterocyclic derivatives, and cisplatin against several cancer cell lines and the nontumoral BJ cell line.
Cell line (IC50, µM) 1
Compound
MIAPaca2
HeLa
A375
MCF7
HepG2
SH-SY5Y
Jurkat
BJ
1
9
10
11
12
13
14
17
101.6 ± 1.6
14.2 ± 0.9
3.3 ± 0.2
107.2 ± 2.5
12.4 ± 1.2
2.2 ± 0.1
112.2 ± 2.6
10.4 ± 1.0
2.0 ± 0.2
97.8 ± 3.9
6.4 ± 0.3
3.0 ± 0.2
5.6 ± 0.3
3.2 ± 0.3
N.D.
N.D.
N.D.
N.D.
125.1 ± 9.1
14.3 ± 0.3
3.1 ± 0.1
13.5 ± 0.4
3.5 ± 0.2
N.D.
N.D.
N.D.
N.D.
11.8 ± 0.4
9.0 ± 0.1
2.9 [41]
109.7 ± 2.5
6.0 ± 0.2
1.7 ± 0.15
5.6 ± 0.2
2.2 ± 0.2
N.D.
105.8 ± 5.0
3.2 ± 0.1
1.1 ± 0.06
2.4 ± 0.1
1.3 ± 0.12
N.D.
N.D.
N.D.
N.D.
1.7 ± 0.10
1.5 ± 0.12
1.9 [45]
165.0 ± 7.1
N.D.
6.9 ± 0.07
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
10.8 ± 0.2
3.3 ± 0.2
10.9 ± 1.0
2.6 ± 0.2
7.1 ± 0.3
2.3 ± 0.2
28.7 ± 2.6
13.4 ± 0.5
17.8 ± 1.1
15.2 ± 0.5
12.0 ± 1.0
6.9 ± 0.3
28.9 ± 1.7
11.8 ± 0.7
22.2 ± 0.8
17.9 ± 0.6
10.6 ± 0.1
5.4 ± 0.3
26.5 ± 0.9
10.3 ± 1.0
17.9 ± 0.2
13.4 ± 1.0
7.2 ± 0.5
N.D.
N.D.
N.D.
18
21
22
6.0 ± 0.3
5.2 ± 0.2
19.1 ± 4.5 [43]
3.7 ± 0.1
3.2 ± 0.1
0.7 ± 0.1 [44]
4.9 ± 0.1
Cisplatin
5.0 ± 1.0 [46]
2.3 ± 0.3 [43]
3.1 ± 1.0 [42]
10.1 ± 2.0 [43]
1
The cell lines were treated with different concentrations of each compound for 72 h. IC50 values were determined by XTT assay in Jurkat and SH-SY5Y cells and by MTT assay in all the
other cell lines. Results are expressed as means ± SD of three independent experiments. IC50 is the concentration that inhibits 50% of cellular growth. N.D.: not determined.

Molecules 2019, 24, 766
8 of 21
2.2.2. Cell Viability over Time
The Jurkat cell line, which exhibited the best results regarding antiproliferative activity among
the tested cancer cell lines, was used to study the mechanism of action of the most potent compound
synthesized here, the heterocyclic derivative 10. Trypan blue cell-counting assays were conducted
to assess the antiproliferative profile of this compound over time. The counting of viable cells was
performed after incubation of Jurkat cells with compound 10, at a concentration corresponding to its
IC50 value at 72 h of treatment, or with only the vehicle (control with DMSO). As shown in Figure 2,
compound 10 affected the cell growth of Jurkat cells as early as at 6h of treatment. At this incubation
time, a reduction of 18% of cell viability was observed in cells treated with compound 10 compared
with cells that were not treated with the same compound. This effect on cell viability increased during
next hours, until cell viability was 45% lower than it was in control cells at 24 h, and reached 50%
at 72 h of treatment. Based on the antiproliferative profile obtained in these Trypan Blue counting
experiments, we established the incubation times for the subsequent biological studies that were
performed in this study.
Figure 2. Effect of compound 10 on cell viability. Jurkat cells were treated with 1.1
µM compound 10 for
different time periods (( ): 3, 6, 12, and 24 h; ( ): 24, 48, and 72 h). Cell numbers were determined by
A
B
Trypan Blue counting assays. Results are presented as means
±
SD of three independent experiments.
2.2.3. Analysis of Cell Cycle Distribution and Apoptosis
Cell cycle and apoptosis assays were performed to elucidate the underlying antiproliferative
mechanisms of compound 10. To examine the effects on the cell cycle pattern, Jurkat cells were treated
with compound 10, at a concentration corresponding to its IC50 value at 72 h of treatment, for 3, 6, and
24 h. The study was performed using flow cytometry, and the calculation of the fraction of cells in
phases G0/G1, S, and G2/M was executed using the fraction of live cells. No significant changes on
the cell cycle distribution were detected after 3 and 6 h of exposure; treatment for 24 h increased the
population at the G0/G1 phase, with a concomitant decrease in the populations of cells at the S and the
G2/M phases with respect to the untreated cells (Figure 3). Hypodiploid sub-G0 peaks that correspond
to cells with DNA fragmentation were observed after 6 and 24 h of incubation. These results suggest
that compound 10 has the ability to induce cell death in Jurkat cells.

Molecules 2019, 24, 766
9 of 21
Figure 3. Effect of compound 10 on cell cycle distribution. Cell cycle analysis of Jurkat cells untreated
(Control) or treated with 1.1 M compound 10 for 3 ( ), 6 ( ), and 24h ( ). After treatment, cells
were stained with PI and DNA content analyzed by flow cytometry. A representative histogram
µ
A
B
C
is shown for each time of incubation and condition. Results are presented as means
independent experiments.
±
SD of three
Apoptosis assays were then conducted to elucidate the mechanism underlying the cytotoxic
effect of this heterocyclic derivative. The Annexin V-FITC/PI flow cytometry assay employs the
ability of Annexin V to bind to phosphatidylserine (PS) and the ability of propidium iodide (PI) to
enter cells with damaged cell membranes and to bind to DNA. Early apoptosis is characterized by
the loss of membrane asymmetry, with translocation of PS from the inner to the outer membrane,
prior to the loss of membrane integrity. Therefore, this assay allows the discrimination of live cells
(Annexin-V−/PI−) from early apoptotic (Annexin-V+/PI−), late apoptotic (Annexin-V+/PI+), or
necrotic (Annexin-V−/PI+). Considering the results obtained in the cell viability and cell cycle
experiments, we performed this assay on Jurkat cells after 6 and 24 h of treatment with compound
10 at a concentration corresponding to its IC50 value at 72 h of treatment. Flow cytometry dot plots
representing Annexin V and PI staining are shown in Figure 4. Compound 10 induced 32% of cells
to enter the early stage of apoptosis after 6 h of treatment. No significant changes were observed in
the late apoptotic and necrotic populations. Exposure to the compound for 24 h increased the early
apoptotic population by 25% and the late apoptotic population by 20%. The percentage of necrotic
cells did not change significantly. We also performed this assay after 48 and 72 h of treatment; the
Annexin V/PI profiles observed for these incubation times were similar to those obtained at 24 h (data

Molecules 2019, 24, 766
10 of 21
not shown). The results of the Annexin V-FITC/PI assays are in good agreement with the hypodiploid
sub-G0 peaks and their respective magnitudes, observed in the cell cycle experiments. Moreover, the
fact that the appearance of G0/G1 arrest occurred after the beginning of the apoptotic events indicates
that cell death was caused by a primary effect of compound 10 and not by the activation of apoptosis
as a consequence of the inability of cells to overcome cell growth arrest and proceed through the
cell cycle.
Figure 4. Induction of apoptosis by compound 10. Flow cytometric quantification of apoptosis in Jurkat
cells untreated (Control) or treated with 1.1
µM compound 10 for 6 (A) and 24 h (B). After treatment,
cells were stained with annexin V-FICT/PI and analyzed by flow cytometry. A representative dot plot
−
is shown for each time of incubation and condition; the right quadrants of each diagram (annexin+/PI
and annexin+/PI+) represent apoptotic cells. The percentage of early (dark gray bar) and late (light
gray bar) apoptotic cells in each condition is represented as a bars diagram, calculated from dot plots.
Results are presented as means ± SD of three independent experiments.
We further confirmed the induction of apoptosis by examining its characteristic morphological
changes in Jurkat cells treated with the same concentration of compound 10 for 6 h. As shown in
Figure 5, apoptotic bodies were observed in the treated cells using a phase-contrast microscope,
and Hoechst 33342 staining showed that some of those cells exhibited a highly condensed
nuclear morphology. In contrast, untreated cells presented intact plasma membranes and normal
nuclear morphology.
Taken together, the results described above reveal that compound 10 is a potent inducer
of apoptosis.
The activation of several caspases is involved in the execution phase of apoptosis. Caspase 8 is the
upstream caspase for the extrinsic (death receptor) pathway, whereas caspase 9 is that of the intrinsic
(mitochondrial) pathway. These pathways converge to caspase 3, the action of which is required for
the morphological changes that are associated with apoptosis [47]. Previous reports have shown that
GA 1 induces apoptosis involving both the extrinsic and intrinsic pathways [17,18,48–51]. To explore
the mechanism via which the GA heterocyclic derivative 10 triggers this programmed cell death, we
investigated its effects on the activation of caspases 3, 8, and 9 via Western blotting. The proteolytic
cleavage of all mentioned caspases was observed after 6 h of treatment at a dose corresponding to the
IC50 recorded at 72 h (Figure 6). These results indicate that the induced apoptosis is mediated by the
activation of the both extrinsic and intrinsic pathways.

Molecules 2019, 24, 766
11 of 21
Figure 5. Morphological changes in Jurkat cells. Morphology of Jurkat cells untreated (Control) or
treated with 1.1 M compound 10 for 6 h was examined using a phase-contrast microscope ( ) and
fluorescence microscopy after Hoechst 33342 staining (B).
µ
A
Figure 6. Effect of compound 10 on the activation of caspases 3, 8, and 9 in Jurkat cells after 6 h of
treatment; α-actin was used as a loading control.
Apoptosis is a highly regulated process that occurs in physiological conditions and plays a major
role in the pathogenesis of many diseases. In fact, acquired resistance towards apoptosis is a hallmark
of most types of cancer. Despite its involvement in the carcinogenesis, apoptosis is a widespread target
of many treatment strategies, including the development of novel chemotherapeutic molecules [47,52].
The p53 tumor suppressor protein is a critical component of the apoptotic signaling circuitry in both
pathways. The loss of its function is the most common strategy employed by cancer cells to evade
death. In fact, approximately 50% of human cancers bear P53 gene mutations and, in the majority of
the remaining cases, its function is compromised by different mechanisms [52–54]. The fact that Jurkat
cells are a cancer p53-deficient cell line [55] indicates that compound 10 can activate the apoptotic
machinery in a p53-independent manner.

Molecules 2019, 24, 766
12 of 21
Taken together, our results suggest that the induction of apoptosis involving both the intrinsic
and extrinsic pathways is the main mechanism responsible for the antiproliferative activity of the GA
heterocyclic derivative 10. Efforts are currently underway to elucidate further its mechanism of action.
3. Materials and Methods
3.1. Chemistry
Glycyrrhetinic acid and all reagents were purchased from Sigma-Aldrich Co (St. Louis, MO,
USA). The solvents used in the reactions were obtained from Merck Co (kenilworth, NJ, USA).
and were purified and dried according to the literature procedures. The solvents used in workups
were purchased from VWR Portugal (Radnor, PA, USA). Thin-layer chromatography (TLC) analysis
was performed in Kieselgel 60HF254/Kieselgel 60G. Purification of compounds by flash column
chromatography (FCC) was carried out using Kiesegel 60 (230–400 mesh, Merck) (kenilworth, NJ,
USA). Melting points were determined using a BUCHI melting point B-540 apparatus and were
1
uncorrected. IR spectra were obtained on a Fourier transform spectrometer. H and 13C NMR spectra
(see Supplementary Materials) were recorded on a Bruker Avance-400 Digital NMR spectrometer,
in CDCl3,, with Me4Si as the internal standard. Chemical shifts values (δ) are given in parts per
million (ppm) and coupling constants (J) are presented in hertz (Hz). Massa spectra were obtained
using a Quadrupole/Ion Trap Mass Spectrometer (QIT-MS) (LCQ Advantage MAX, THERMO
FINNINGAN). Elemental analysis was performed in an Analyzer Elemental Carlo Erba 1108 by
chromatographic combustion.
3
β
-hydroxy-olean-12-en-30-oic acid (
2): Compound 2 was prepared according to the literature [38], from
1 to give a white solid (90%). m.p.: 315–317 ◦C.
Methyl 3 -hydroxy-11-oxo-olean-12-en-30-oate (
literature [39], from 1 to give a colorless solid (90%). m.p.: 254–256 ◦C
Methyl 3 -hydroxy-olean-12-en-30-oate ( ): Compound was prepared from
as for the preparation of 3, with the obtention of a white solid (88%). m.p.: 239–242 ◦C.
Methyl 3,11-dioxo-olean-12-en-30-oate ( ): Compound was prepared according to the literature [40],
from 3 to give a white solid (94%). m.p.: 248–250 ◦C.
Methyl 3-oxo-olean-12-en-30-oate ( ): Compound was prepared from
the preparation of 5, with the obtention of a white solid. (92%). m.p.: 185–188 ◦C
Methyl 2-hydroxymethylene-3,11-dioxo-olean-12-en-30-oate ( ): Compound was prepared according to
the literature [40], from 5 to give a colorless solid (82%). m.p.: 231–234 ◦C.
Methyl 2-hydroxymethylene-3-oxo-olean-12-en-30-oate ( ): Compound was prepared from
same method as for the preparation of 7, with the obtention of a white solid (80%). m.p.: 136–139 ◦C.
Methyl 2-(1H-imidazol-1-yl)-methylene-3,11-dioxo-olean-12-en-30-oate ( ): To a solution of compound
β
3): Compound 3 was prepared according to the
β
4
4
2, using the same method
5
5
6
6
4, using the same method as for
7
7
8
8
6, using the
9
7
(300 mg, 0.59 mmol) in anhydrous THF (5 mL), CDI (191 mg, 1.18 mmol) was added. After 4 h under
magnetic stirring at reflux temperature and N2 atmosphere, the reaction was completed. Water (50 mL)
and ethyl acetate (50 mL) were added to the reaction mixture. The aqueous phase was further extracted
with ethyl acetate (2
×
50 mL). The combined organic extract was then washed with water (2
×
50 mL)
and brine (50 mL), dried over Na2SO4, filtered and evaporated to dryness. The resulting solid was
subjected to FCC [petroleum ether/ ethyl acetate from (1:1) to (1:2)] to afford
9
as a white solid. (67%).
max/cm−1 (KBr): 3113, 2953, 1728, 1685, 1649, 1601, 1518, 1485, 1458, 1385, 1306,
7.76 (1H,s), 7.66 (1H, s), 7.33 (1H,s), 7.10 (1H, s), 5.75 (1H, s), 4.19
m.p.: 249–251 ◦C. IR
ν
1
1028. H NMR (400MHz, CDCl3):
δ
(1H, d, J = 16.5), 3.67 (3H, s), 2.52 (1H, s), 1.39 (3H, s), 1.18 (3H, s), 1.16 (3H, s), 1.13 (6H, s), 1.12 (3H, s),
0.81 (3H, s). 13C NMR (100MHz, CDCl3):
206.2, 199.2, 176.8, 170.6, 139.1, 130.7, 130.3, 128.4, 122.1,
119.0, 59.2, 52.8, 51.7, 48.4, 45.3, 44.8, 44.0, 43.3, 43.2, 41.2, 37.6, 35.9, 31.8, 31.3, 31.0, 29.7, 28.5, 28.2, 26.5,
δ

Molecules 2019, 24, 766
13 of 21
26.3, 23.1, 22.3, 19.5, 17.9, 15.5. ESI-MS m/z: 561.71 ([M + H]+, 100%). Found C 74.45, H 8.80, N 5.05,
.
calcd for C35H48N2O4 0: C 74.96, H 8.63, N 5.00%.
Methyl 2-(1H-imidazol-1-yl)-methylene-3-oxo-olean-12-en-30-oate (10): The method followed that described
for compound 9 but using compound 8 (300 mg, 0.60 mmol) and CDI (195 mg, 1.20 mmol) in anhydrous
THF (5 mL) at reflux for 5 h. The resulting solid was purified by FCC with petroleum ether/ethyl
◦
−1
acetate (1:1) and afforded compound 10 as a white solid (60%). m.p.: 151–154 C. IR
ν
max/cm (KBr):
7.78
1
3118, 2951, 1730, 1687, 1610, 1518, 1487, 1458, 1383, 1306, 1028. H NMR (400MHz, CDCl3):
δ
(1H,s), 7.70 (1H,s), 7.22 (1H,s), 7.15 (1H,s), 5.34 (1H, t, J = 3.4), 3.68 (3H, s), 2.90 (1H, d, J = 16.0), 1.18
(6H, s), 1.13 (6H, s), 1.02 (3H, s), 0.91 (3H, s), 0.78 (3H, s). 13C NMR (100MHz, CDCl3):
206.7, 177.6,
δ
144.6, 138.7, 130.7, 130.5, 122.9, 122.0, 119.2, 52.6, 51.6, 48.4, 45.4, 45.2, 44.3, 43.2, 42.8, 41.9, 39.7, 38.3,
36.0, 32.0, 31.7, 31.3, 29.8, 28.5, 28.2, 27.0, 26.1, 25.7, 23.8, 22.5, 20.3, 16.4, 15.6. ESI-MS m/z: 547.73
([M + H]+, 100%). Found C 76.72, H 9.43, N 4.98, calcd for C35H50N2O3: C 76.88, H 9.22, N 5.12%.
Methyl 2-(20-methyl-1H-imidazol-1-yl)-methylene-3,11-dioxo-olean-12-en-30-oate (11): To a solution of
compound
After 2 h under magnetic stirring at reflux temperature and N2 atmosphere, the reaction was completed.
The workup was performed according to the same method as for . The resulting solid was submitted
7 (300 mg, 0.59 mmol) in anhydrous THF (5 mL), CBMI (231 mg, 1.18 mmol) was added.
9
to FCC [petroleum ether/ ethyl acetate from (1:1) to (1:4)] to afford 11 as a white solid (70%). m.p.:
146–149 ◦C. IR
NMR (400MHz, CDCl3):
(3H, s), 2.52 (1H, s), 2.47 (3H, s), 1.39 (3H, s), 1.19 (3H, s), 1.17 (3H, s), 1.14 (9H, s), 0.82 (3H, s). 13C
NMR (100MHz, CDCl3): 206.4, 199.3, 176.8, 170.5, 147.3, 129.9, 128.7, 128.5, 122.3, 118.1, 59.2, 53.0,
ν
max/cm−1 (KBr): 3116, 2953, 1730, 1686, 1657, 1608, 1541, 1502, 1458, 1412, 1385. H
1
δ
7.64 (1H,s), 7.29 (1H,s), 6.94 (1H,s), 5.75 (1H,s), 4.17 (1H, d, J = 16.3), 3.67
δ
51.8, 48.5, 45.4, 44.9, 44.0, 43.4, 42.6, 41.2, 37.7, 36.0, 31.8, 31.3, 31.1, 29.7, 28.6, 28.3, 26.5, 26.3, 23.2, 22.4,
19.5, 18.0, 15.4, 13.7. ESI-MS m/z: 575.86 ([M + H]+, 100%). Found C 74.91, H 9.01, N 4.78, calcd for
C36H50N2O4: C 75.22, H 8.77, N 4.87%.
0
Methyl 2-(2 -methyl-1H-imidazol-1-yl)-methylene-3-oxo-olean-12-en-30-oate (12): The method followed that
described for compound 11 but using compound 8 (300 mg, 0.60 mmol) and CBMI (235 mg, 1.20 mmol)
in anhydrous THF (5 mL) at reflux for 4 h. The resulting solid was subjected to FCC (petroleum ether/
ethyl acetate from (1:1) to (1:4)) to afford 12 as a white solid (63%). m.p.: 134–137 ◦C. IR
(KBr): 3118, 2951, 1730, 1687, 1606, 1541, 1502, 1456, 1412, 1383. H NMR (400MHz, CDCl3):
ν
max/cm−1
7.66
1
δ
(1H,s), 7.14 (1H,s), 7.00 (1H,s), 5.33 (1H, t, J = 3.4), 3.67 (3H, s), 2.87 (1H, d, J = 15.9), 2.48 (3H, s), 1.18
(6H, s), 1.15 (3H, s), 1.12 (3H, s), 1.01 (3H, s), 0.90 (3H, s), 0.78 (3H, s). 13C NMR (100MHz, CDCl3):
δ
206.7, 177.6, 147.3, 144.6, 130.2, 128.3, 123.8, 122.0, 118.2, 52.7, 51.6, 48.4, 45.4, 45.3, 44.3, 42.8, 42.5, 41.9,
39.7, 38.3, 36.1, 32.0, 31.7, 31.3, 29.7, 28.5, 28.2, 26.9, 26.1, 25.7, 23.7, 22.6, 20.3, 16.4, 15.5, 13.6. ESI-MS
m/z: 561.77 ([M + H]+, 100%). Found C 76.85, H 9.67, N 4.64, calcd for C36H52N2O3: C 77.10, H 9.35,
N 5.00%.
Methyl 2-(1H-triazol-1-yl)-methylene-3,11-dioxo-olean-12-en-30-oate (13): To a solution of compound
7
(300 mg, 0.59 mmol) in anhydrous THF (6 mL), CDT (581 mg, 3.54 mmol) was added. The workup was
performed after 24 h of reaction, under magnetic stirring at reflux temperature and N2 atmosphere,
using the same procedure as for compound 9. The resulting solid was purified by FCC with petroleum
◦
ether/ ethyl acetate (1:2) and afforded 13 as a white solid (41%). m.p.: 148–151 C. IR
ν
max/cm−1
δ 8.40 (1H,s),
1
(KBr): 3122, 2953, 1730, 1691, 1657, 1618, 1508, 1460, 1385. H NMR (400MHz, CDCl3):
8.06 (1H,s), 7.80 (1H,s), 5.76 (1H,s), 4.42 (1H, d, J = 18.6), 3.69 (3H, s), 2.57, 1.40 (3H, s), 1.22 (3H, s), 1.17
(3H, s), 1.15 (3H, s), 1.14 (6H, s), 0.82 (3H, s). 13C NMR (100MHz, CDCl3):
206.4, 199.1, 176.9, 170.1,
δ
152.7, 145.3, 128.7, 128.6, 125.1, 59.1, 53.0, 51.8, 48.5, 45.4, 44.9, 44.0, 43.7, 43.4, 41.2, 37.7, 35.7, 31.9, 31.4,
31.1, 29.8, 28.6, 28.3, 26.6, 26.4, 23.1, 22.4, 19.6, 18.0, 15.5. ESI-MS m/z: 274.48 (87%), 302.51 (15), 318.49
(100), 346.54 (22), 362.37 (37), 374.60 (11), 562.68 ([M + H]+, 93), 563.71 ([M + 2H] 2+, 26), 575.80 (24),
622.48 (15), 624.80 (21).

Molecules 2019, 24, 766
14 of 21
Methyl 2-(1H-triazol-1-yl)-methylene-3-oxo-olean-12-en-30-oate (14): The method followed that described
for compound 13 but using compound (300 mg, 0.60 mmol) and CDT (591 mg, 3.60 mmol) in
8
anhydrous THF (6 mL) at reflux for 26 h. The resulting solid was purified by FCC with petroleum
◦
ether/ ethyl acetate (3:1) and afforded 14 as a white solid (44%). m.p.: 137–140 C. IR
(KBr): 3122, 2951, 1732, 1689, 1622, 1512, 1456, 1383. H NMR (400MHz, CDCl3):
ν
max/cm−1
8.34 (1H,s), 8.07
1
δ
(1H,s), 7.78 (1H,s), 5.36 (1H, t, J = 3.2), 3.69 (3H, s), 3.41 (1H, d, J = 17.6), 1.20 (3H, s), 1.19 (3H, s), 1.14
(3H, s), 1.13 (3H, s), 1.03 (3H, s), 0.92 (3H, s), 0.79 (3H, s). 13C NMR (100MHz, CDCl3):
207.4, 177.8,
δ
153.1, 146.0, 144.5, 128.4, 125.6, 122.5, 52.9, 51.7, 48.6, 45.5, 45.3, 44.4, 43.5, 43.0, 42.0, 39.8, 38.5, 35.9,
32.2, 31.8, 31.4, 29.9, 28.7, 28.4, 27.1, 26.2, 25.8, 23.9, 22.6, 20.5, 16.5, 15.9. ESI-MS m/z: 274.53 (89%),
302.57 (23), 318.52 (100), 330.56 (14), 346.56 (27), 362.53 (25), 374.60 (16), 548.68 ([M + H]+, 93), 549.70
([M + 2H] 2+, 28), 561.81 (23), 578.36 (20).
3-Oxo-olean-12-en-30-oic acid (15): Compound 15 was prepared from
the preparation of
, with the obtention of a white solid (93%). m.p.: 245-248 ◦C. IR
3396, 2949, 1732, 1703, 1460, 1385. H NMR (400MHz, CDCl3):
m), 1.20 (3H, s), 1.14 (3H, s), 1.09 (3H, s), 1.06 (3H, s), 1.05 (3H, s), 1.01 (3H, s), 0.82 (3H, s). 13C NMR
(100MHz, CDCl3): 217.9, 183.4, 144.2, 122.5, 55.3, 48.0, 47.4, 46.8, 44.0, 42.4, 41.6, 39.7, 39.2, 38.2, 36.6,
34.1, 32.1, 31.9, 31.0, 28.6, 28.1, 26.9, 26.4, 26.0, 25.8, 23.5, 21.4, 19.6, 16.6, 15.1. ESI-MS2 (25%) m/z:
2
, using the same method as for
max/cm−1 (KBr):
5.31 (1H,t, J = 3.2), 2.33-2.59 (2H,
5
ν
1
δ
δ
274.98 ([M + H
−
C12H20O]+, 16%), 408.61 ([M + H
−
H2O
−
C2H4]+ or [M + H
−
HCOOH]+, 39); 437
([M + H − H2O]+, 7), 455.42 ([M+H]+, 100).
2-Hydroxymethylene-3-oxo-olean-12-en-30-oic acid (16): The method followed that described for
compound
7
but using compound 15 (850 mg, 1.87 mmol), ethyl formate (1.1 mL, 13.09 mmol) a◦nd
sodium methoxide (1010 mg, 18.70 mmol), with the obtention of a white solid (77%). m.p.: 154–158 C.
−1
1
IR
(1H, d, J = 3.0), 8.59 (1H, d, J = 2.7), 5.35 (1H, t, J = 3.4), 2.30 (1H, d, J = 14.4), 1.21 (3H, s), 1.20 (3H, s),
1.16 (3H, s), 1.13 (3H, s), 1.02 (3H, s), 0.93 (3H, s), 0.83 (3H, s). 13C NMR (100MHz, CDCl3):
190.8,
188.4, 183.4, 144.3, 122.6, 105.8, 52.1, 48.1, 45.6, 44.1, 42.6, 41.7, 40.2, 39.6, 39.4, 38.3, 36.2, 32.0, 31.9, 31.1,
νmax/cm (KBr): 3442, 2953, 1731, 1699, 1643, 1587, 1456, 1383. H NMR (400MHz, CDCl3): δ 14.91
δ
28.7, 28.5, 28.2, 27.0, 26.1, 25.8, 23.5, 20.9, 19.6, 16.6, 14.7. ESI-MS2 (25%) m/z: 437.35 ([M + H
−
H2O
−
C2H4]+ or [M + H − HCOOH]+, 44%), 465.36 ([M + H − H2O]+, 100), 483.32 ([M + H]+, 56).
30-(1H-Imidazol-1-yl)-3,30-dioxo-olean-12-en-2-(1H-imidazol-1-yl)-methylene (17): Compound 17 was
prepared using the same method as for the preparation of , but using compound 16 (300 mg, 0.62
9
mmol) and CDI (302 mg, 1.86 mmol) in anhydrous THF (6 mL) at reflux for 6 h. The resulting solid
was submitted to FCC (petroleum ether/ ethyl acetate from (1:2) to (1:8)) to afford 17 as a white solid
◦
−1
(56%). m.p.: 162–165 C. IR
ν
max/cm (KBr): 3124, 2937, 1713, 1687, 1610, 1522, 1489, 1456, 1383, 1308,
8.30 (1H, s), 7.78 (1H, s), 7.70 (1H, s), 7.58 (1H, s), 7.24
1
1227, 1173, 1030. H NMR (400MHz, CDCl3):
δ
(1H, s), 7.17 (1H, s), 7.08 (1H, s), 5.19 (1H, t, J = 3.4), 2.90 (1H, d, J = 16.0), 1.44 (3H, s), 1.19 (3H, s), 1.18
(3H, s), 1.13 (3H, s), 0.99 (3H, s), 0.90 (3H, s), 0.79 (3H, s). 13C NMR (100MHz, CDCl3):
206.6, 174.0,
δ
143.8, 138.7, 137.2, 130.7, 130.6, 129.8, 122.8, 122.7, 119.2, 117.4, 52.5, 48.1, 46.6, 45.4, 45.2, 44.4, 43.2, 41.9,
39.6, 37.5, 35.9, 32.9, 32.2, 31.7, 29.8, 27.9, 27.9, 27.2, 25.9, 25.5, 23.7, 22.4, 20.3, 16.4, 15.6. ESI-MS2 (25%)
m/z: 487.54 ([M + H
−
C3H3N2
−
C2H4]+ or [M + H
−
C4H3N2O]+, 100%), 555.31 ([M + H
−
C2H4]+,
2), 583.44 ([M + H]+, 16).
30-(1H-Triazol-1-yl)-3,30-dioxo-olean-12-en-2-(1H-triazol-1-yl)-methylene (18): The method followed that
described for compound 17 but using compound 16 (300 mg, 0.62 mmol) and CDT (1018 mg, 6.20 mmol)
in anhydrous THF (6 mL) at reflux for 38 h. The resulting solid was submitted to FCC (petroleum ether/
ethyl acetate from (2:1) to (1:1)) to afford 18 as a white solid (38%). m.p.: 142–145 ◦C. IR
(KBr): 3126, 2954, 1713, 1695, 1618, 1512, 1456, 1383, 1279, 1161, 1132. H NMR (400MHz, CDCl3):
ν
max/cm−1
1
δ
8.39 (1H, s), 8.25 (2H, s), 8.09 (1H, s), 7.80 (1H, s), 5.39 (1H, t, J = 3.4), 3.39 (1H, d, J = 17.5), 1.21 (3H, s),
1.20 (6H, s), 1.14 (3H, s), 1.03 (3H, s), 0.92 (3H, s), 0.82 (3H, s). 13C NMR (100MHz, CDCl3):
207.6,
182.0, 152.8, 146.9 (2), 146.0, 144.5, 128.4, 125.8, 122.6, 52.9, 48.4, 45.5, 45.3, 44.2, 43.5, 42.8, 42.0, 39.8,
δ

Molecules 2019, 24, 766
15 of 21
38.4, 35.9, 32.2, 31.8, 31.3, 29.9, 28.9, 28.4, 27.1, 26.2, 25.8, 23.9, 22.6, 20.5, 16.5, 15.9. ESI-MS2 (35%) m/z:
488.42 ([M + H C2H3N3
C2H4]+ or [M + H C3H3N3O]+, 100%), 514.64 ([M + H C4H6O]+, 5),
584.67 (M+, 4), 585.77 ([M + H]+, 3).
−
−
−
−
Methyl 2-azidomethylene-3,11-dioxo-olean-12-en-30-oate (19): To a solution of compound
7 (700 mg,
1.37 mmol) in dichloromethane (14 mL), triethylamine (Et3N) (573 L, 4.11 mmol) and tosyl chloride
µ
(TsCl) (654 mg, 3.43 mmol) were added. After 3.5 h, under magnetic stirring at room temperature, the
reaction was completed. The solvent of the reaction mixture was removed under reduced pressure and
the residue was suspended in acetone (14 mL); sodium azide (NaN3) (134 mg, 2.06 mmol) was added.
After 5 h under magnetic stirring at room temperature, the reaction was completed. The acetone
was removed under reduced pressure and water (60 mL) and ethyl acetate (60 mL) were added to
the residue. The aqueous phase was further extracted with ethyl acetate (2
×
60 mL). The combined
organic extract was then washed with water (3 50 mL), dried over Na2SO4, filtered and evaporated
×
to the dryness, to afford a solid. The solid was subjected to flash FCC with petroleum ether/-ethyl
acetate (15:1) to afford 19 as a white solid (62%). m.p.: 167–170 ◦C. IR
ν
max/cm−1 (KBr): 2951, 2127,
1
1730, 1659, 1460, 1387. H NMR (400MHz, CDCl3):
δ 7.37 (1H, d, J = 1.5), 5.72 (1H, s), 3.76 (1H, d, J =
16.5), 3.69 (3H,s), 2.46 (1H,s), 1.37 (3H,s), 1.15 (3H,s), 1.14 (3H,s), 1.11 (3H,s), 1.09 (6H,s), 0.81 (3H,s).
13C NMR (100MHz, CDCl3):
205.0, 199.1, 176.9, 169.7, 137.2, 128.6, 123.0, 59.1, 53.4, 51.8, 48.3, 45.2,
δ
44.9, 44.0, 43.3, 41.2, 40.6, 37.7, 35.7, 31.8, 31.6, 31.1, 29.3, 28.6, 28.3, 26.5, 26.4, 23.2, 22.5, 19.5, 18.0, 15.2.
ESI-MS2 (35%) m/z: 462.38 ([M + H − C3H6O2]+, 100%), 508.46 ([M + H − CO]+ or [M + H − C2H4]+,
12), 518.37 ([M + H − H2O]+, 43), 536.61 ([M + H]+, 7).
Methyl 2-azidomethylene-3-oxo-olean-12-en-30-oate (20): Compound 20 was prepared using the same
method as for the preparation of 19, but using compound 8 (700 mg, 1.41 mmol) in dichloromethane
(14 mL), Et3N (492 µL, 3.53 mmol), TsCl (538 mg, 2.82 mmol), and then NaN3 (138 mg, 2.12 mmol) in
acetone (14 mL). The resulting solid was subjected to flash FCC with petroleum ether/ ethyl acetate
◦
(20:1) to afford 20 as a white solid (57%). m.p.: 186–189 C. IR
1589, 1452, 1383. H NMR (400MHz, CDCl3):
ν
max/cm−1 (KBr): 2943, 2116, 1730, 1672,
1
δ 7.36 (1H, d, J = 1.6), 5.33 (1H, t, J = 3.4), 3.68 (3H,s),
2.66 (1H, d, J = 16.6), 1.15 (3H,s), 1.13 (3H,s), 1.09 (6H,s), 1.01 (3H,s), 0.88 (3H,s), 0.79 (3H,s). 13C NMR
(100MHz, CDCl3): 205.7, 177.6, 144.3, 137.1, 123.2, 122.4, 53.1, 51.6, 48.3, 45.2, 45.1, 44.3, 42.8, 41.8,
40.2, 39.6, 38.3, 35.7, 32.0, 31.8, 31.3, 29.2, 28.5, 28.2, 27.0, 26.0, 25.7, 23.6, 22.6, 20.3, 16.4, 15.4. ESI-MS2
(35%) m/z: 462.48 ([M + H H3COH
C2H4]+ or [M + H H3CCOOH]+, 32%), 504.31 ([M + H
H2O]+, 100), 522.38 ([M + H]+, 7).
δ
−
−
−
−
Compound 21: To a solution of compound 19 (350 mg, 0.65 mmol) in THF (7 mL), methyl propiolate
(64.4 µL, 0.72 mmol), and CuI (12.4 mg, 0.065 mmol) were added. After 4 h under magnetic stirring
at 65 ◦C, the reaction was completed. The reaction mixture was filtered and evaporated to dryness.
The resulting solid was purified by FCC (petroleum ether/ ethyl acetate from (8:1) to (2:1)) to afford 21
◦
−1
as a white solid (42%). m.p.: 184–186 C. IR
ν
max/cm (KBr): 3136, 2951, 1747, 1726, 1691, 1651, 1620,
8.42 (1H, s), 7.99 (1H, s), 5.74 (1H,s), 4.17
1
1554, 1458, 1385, 1209, 1034. H NMR (400MHz, CDCl3):
δ
(1H, d, J = 17.5), 3.96 (3H, s), 3.69 (3H,s), 2.53 (1H,s), 1.41 (3H, s), 1.22 (3H, s), 1.16 (3H,s), 1.14 (9H,s),
0.81 (3H,s). 13C NMR (100MHz, CDCl3):
205.8, 198.8, 176.9, 170.3, 160.7, 140.1, 128.8, 128.5, 128.2,
δ
127.7, 59.0, 53.0, 52.4, 51.8, 48.5, 45.6, 44.9, 44.0, 43.6, 43.4, 41.2, 37.7, 35.9, 31.9, 31.3, 31.1, 29.7, 28.6,
28.3, 26.5, 26.4, 23.1, 22.3, 19.5, 17.9, 15.6. ESI-MS m/z: 620.59 ([M + H]+, 100%). Found C 69.39, H 8.21,
N 6.66, calcd for C36H49N3O6: C 69.76, H 7.97, N 6.78 %.
Compound 22: Compound 22 was prepared using the same method as for the preparation of 21, but
using compound 20 (350 mg, 0.67 mmol), methyl propiolate (66.0 µL, 0.74 mmol), and CuI (12.8 mg,
0.067 mmol). The resulting solid was purified by FCC [petroleum ether / ethyl acetate from (8:1) to
◦
−1
(2:1)] to afford 22 as a white solid (44%). m.p.: 125–127 C. IR
ν
max/cm (KBr): 3143, 2953, 1730, 1699,
8.30 (1H, s), 7.90 (1H, s), 5.32 (1H, t,
1
1620, 1556, 1456, 1385, 1221, 1034. H NMR (400MHz, CDCl3):
δ
J = 3.4), 3.98 (3H, s), 3.68 (3H,s), 3.18 (1H, d, J = 18.5), 1.21 (3H, s), 1.18 (3H,s), 1.15 (3H, s), 1.13 (3H,

Molecules 2019, 24, 766
16 of 21
s), 1.01 (3H,s), 0.92 (3H,s), 0.78 (3H, s). 13C NMR (100MHz, CDCl3):
δ
206.6, 177.6, 160.7, 144.3, 139.8,
129.2, 128.5, 128.3, 122.2, 52.7, 52.5, 51.6, 48.4, 45.5, 45.2, 44.2, 43.6, 42.7, 41.8, 39.6, 38.3, 35.9, 32.0, 31.6,
31.3, 29.6, 28.5, 28.2, 26.9, 26.0, 25.6, 23.7, 22.4, 20.3, 16.3, 15.8. ESI-MS m/z: 606.33 ([M + H]+, 100%)
Found C 71.58, H 8.58, N 6.88, calcd for C36H51N3O5: C 71.37, H 8.49, N 6.94 %.
3.2. Biology
HT-29, A549, MIA Paca 2, HeLa, A375, MCF7, HepG2, SH-SY5Y, Jurkat, and BJ cells were
purchased from the American Type Culture Collection (ATCC, Rockville, MD). Dulbecco’s Modified
Eagle’s Medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640 medium, Phosphate
Buffered Saline (PBS), glucose 45%, human insulin 10 mg/mL, dimethyl sulfoxide (DMSO),
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) powder, Trypan Blue (TB)
0.4%, propidium iodide (PI), Hoescht 33342, and protease inhibitor cocktail were obtained from
Sigma-Aldrich Co. (St. Luis, MO, USA). Minimum Essential Medium (MEM), penicillin/streptomycin
(P/S) and L-glutamine were purchased from Gibco-BRL (Eggenstein, Germany). Sodium pyruvate,
trypsin/EDTA (0.05%/0.02%), MEM-Eagle nonessential amino acids 100x and ECL Western Blotting
Detection Kit Reagent were obtained from Biological Industries (Kibbutz Beit Haemek, Israel). Fetal
Bovine Serum (FBS) was obtained from PAA Laboratories (Pasching, Austria), the Cell Proliferation
Kit II (XTT kit) was purchased from Roche (Roche Molecular Biochemicals, Indianapolis, IN,
USA), and annexin V-FITC was obtained from Bender MedSystems (Vienna, Austria). Primary
antibodies against caspases 3 (#9662), 8 (#9746) and 9 (#9502) were purchased from Cell Signaling
Technology (Beverly, MA, USA) and primary antibody against α-actin (#69100) was obtained from
MP Biomedicals (Santa Ana, CA, USA). Secondary antibodies HRP-conjugated goat anti-rabbit
(NA934) and HRP-conjugated rabbit anti-mouse (P02060) were purchased from Amersham Biosciences
(Uppsala, Sweden) and DAKO (Copenhagen, Denmark), respectively.
Stock solutions of 20 mM in DMSO of the synthesized compounds were prepared and stored at
◦
−
20 C. Working solutions were prepared in culture medium and appropriate amounts of DMSO were
included in controls; all solutions had a final concentration of 0.5% DMSO.
3.2.1. Cell Culture
HT-29, A549, MIA Paca 2, HeLa, A375, and SH-SY5Y cells were cultured in DMEM supplemented
with 10% heat-inactivated FBS and 1% P/S. HepG2 and BJ cells were grown in DMEM supplemented
with 10% heat-inactivated FBS, 1% P/S and 1 mM sodium pyruvate. Jurkat cells were cultured in
RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 1% P/S, and 2 mM L-glutamine.
MCF7 cells were maintained in MEM supplemented with 10% heat-inactivated FBS, 0.1% P/S, 1 mM
sodium pyruvate, 2 mM L-glutamine, 1x MEM-Eagle nonessential amino acids, 0.01mg/mL insulin
human and 10 mM glucose.
All cell cultures were incubated in a 5% CO2 humidified atmosphere at 37 ◦C.
3.2.2. Antiproliferative Activity Assays
The antiproliferative activity of the synthesized compounds on HT-29, A549, MIA Paca 2, HeLa,
A375, MCF7, HepG2, and BJ cells was determined by the MTT assay. Exponentially growing cells
were plated in 96-well plates at a density of 1–8
medium was replaced with fresh medium containing either the compounds dissolved in DMSO at
different concentrations or only with DMSO, in triplicate, and the cells were continued to culture for
×
103 cells/ well. After 24 h of incubation, the growth
72 h. After incubation with the compounds, the medium was removed and 100
(0.5 mg/mL) were added to each well and the plates were incubated for 1 h. MTT was removed and
100 L of DMSO was added to dissolve the formazan crystals. The absorbance was immediately read at
µL of MTT solution
µ
550 nm on an ELISA plate reader (Tecan Sunrise MR20-301, TECAN, Austria). For SH-SY5Y and Jurkat
cells, the antiproliferative activity was determined by XTT assay. SH-SY5Y cells were plated with
1.6 × 104 cells/well in 96-well plates in 100
µL medium. The compounds at different concentrations or

Molecules 2019, 24, 766
17 of 21
only the vehicle (medium with 0.5% DMSO), in 100
µ
L, were added 24 h after seeding, in triplicate,
and incubated for 72 h. For Jurkat cells, 5.5
addition of the different concentrations of compounds or vehicle, in triplicate, and were allowed to
incubate for 72 h. In both cases, after that incubation period, 100 L of the XTT labeling mixture was
×
103 cells/well were plated simultaneously with the
µ
added to each well and the plates were incubated again for 4 h. Then, the absorbance was read at
450 nm on the ELISA plate reader.
Concentrations that inhibit cell proliferation by 50% (IC50) represent an average of a minimum of
three independent experiments and were expressed as means ± standard deviation (SD).
3.2.3. Cell Viability over Time Assays
The viability over time of Jurkat cells, treated with compound 10, was assessed by TB cell counting
experiments. For these assays, 1.6
×
105 cells were plated per well in 6-well plates simultaneously
with the addition of compound 10, in the concentration of its IC50 value at 72 h, or only vehicle, in a
total volume of 2 mL of medium. After each incubation time (3, 6, 12, 24, 48, and 72h), the suspension
of cells of each well was collected. Equal amounts of TB solution and suspension were mixed and the
mixture was placed in a Neubauer chamber in order to perform the counting. The number of cells
determined for each condition, in each incubation time, represents an average of three independent
experiments, with two replicates per experiment.
3.2.4. Cell Cycle Analysis
Cell cycle was assessed by flow cytometry using a fluorescence-activated cell sorter (FACS). Jurkat
cells were plated in 6-well plates at a density of 1.6
×
105 cells, simultaneously with the addition of
compound 10, at a concentration corresponding to its IC50 value at 72 h of treatment, or with only
the vehicle, in a total volume of 2 mL of medium. The cells were allowed to incubate for 3, 6, and
24 h. After incubation, cells were collected and centrifuged. The supernatant was removed and the
pellet was resuspended in 1 mL of TBS containing 1mg/mL PI, 10mg/mL RNase free of DNase,
◦
and 0.1% Igepal CA-630, for 1 h, at 4 C. FACS analysis was performed at 488 nm in an Epics XL
flow cytometer (Coulter Corporation, Hialeah, FL, USA). Data were collected and analyzed using the
Multicycle software (Phoenix Flow Systems, San Diego, CA, USA). Three independent experiments
were performed, with two replicates per experiment.
3.2.5. Annexin V-FITC/PI Flow Cytometry Assay
Apoptosis was assessed by flow cytometry using a FACS. The same number of Jurkat cells taken
for the cell cycle assay was treated as described above. The cells were allowed to incubate for 6 and
24 h. After incubation, cells were collected and centrifuged. The supernatant was removed and the
pellet was resuspended in 95
µL of binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl,
2.5 mM CaCl2). Annexin V-FITC conjugate (3
µL) was added and cells were incubated for 30 min, at
room temperature, in darkness. After incubation, 0.8 mL of binding buffer was added. Just before the
FACS analysis, cells were stained with 20 µL of 1mg/mL PI solution.
Three independent experiments were performed, with two replicates per experiment.
3.2.6. Observation of Morphological Changes
The morphological changes were observed by fluorescence microscopy using Hoechst staining.
Jurkat cells were plated in 6-well plates at a density of 1.6
×
105 cells, simultaneously with the addition
of compound 10, at a concentration corresponding to its IC50 value at 72 h of treatment, or with only
the vehicle, in a total volume of 2 mL of medium (six wells per condition). The cells were incubated
for 6 h. Before collecting, morphological changes were observed under a phase-contrast microscope.
Cells were collected by centrifugation, washed twice with PBS and stained with 500
33342 solution (2 g/mL in PBS) for 15 min, at room temperature, in darkness. Finally, cells were
washed and resuspended in 10 L PBS. The samples were mounted on a slide and observed with a
µL of Hoechst
µ
µ

Molecules 2019, 24, 766
18 of 21
fluorescence microscope (DMRB, Leica Microssystems, Wetzlar, Germany) with a DAPI filter. Three
independent experiments were conducted.
3.2.7. Western Blot Analysis
Jurkat cells (1.6
×
106 cells) were cultured in 25 cm2 flasks and treated for 6 h with compound
10 at different concentrations. After incubation, the cells were washed twice with ice-cold PBS and
resuspended in lysis buffer containing 20 mM Tris/ acetate, pH 7.5, 270 mM sucrose, 1 mM EDTA,
1 mM EGTA, 1% Triton X-100, and 1% protease inhibitor cocktail. The samples were sonicated,
incubated on ice for 20 min, and centrifuged at 13,200 rpm for 12 min at 4 ◦C, and the pellets were
discarded. The supernatants were assayed for protein concentration using a BCA kit (Thermo fisher
Scientific, Waltham, MA, USA). Protein extracts (50 µg) were separated on 15% sodium dodecyl sulfate
(SDS)-polyacrylamide gels and transferred to a polyvinylnitrocellulose transfer membrane (Bio-Rad
Laboratories, Hercules, CA, USA). The membranes were blocked by incubation in TBS buffer (20 mM
Tris, pH = 7.5 and 132 mM NaCl) containing 0.1% of Tween and 5% of BSA or nonfat milk, for 1 h, at
room temperature. Then, membranes were blotted with the primary antibodies anti-caspase 3, 8 and 9
(1:1000) overnight at 4 ◦C. The blots were washed three times with TBS-0.1% Tween and incubated
with the appropriate secondary antibodies, for 1 h, at room temperature. Before protein detection,
membranes were washed again five times with TBS-0.1% Tween. Immunocomplexes were visualized
using the Immobilon ECL Western Blotting Detection Kit Reagent. Three independent experiments
were performed.
4. Conclusions
In this study, we synthesized a series of novel GA derivatives via the introduction of
different heterocyclic rings conjugated with an α,β-unsaturated ketone in its ring A. Screening for
antiproliferative activity in a panel of nine human cancer cell lines showed that the most active
compound 10 was 31- to 96-fold more potent than GA. This derivative was also more selective
towards tumor cells. Further biological studies performed in Jurkat cells suggest that potent apoptosis
induction involving both the intrinsic and extrinsic pathways is the main mechanism underlying the
antiproliferative activity of compound 10. The enhanced potency and selectivity, and the biological
activity of this new GA heterocyclic derivative warrant further preclinical evaluation.
1
Supplementary Materials: H-NMR and 13C-NMR spectra of selected compounds are available online.
Author Contributions: Conceptualization, J.A.R.S. and S.M.; Synthesis and Structural Characterization,
D.P.S.A. and J.A.R.S.; Biological Experiments, D.P.S.A. and S.M.; Writing—Original Draft Preparation, D.P.S.A.;
Writing—Review and Editing, J.A.R.S., M.C., and S.M.; Supervision, J.A.R.S. and S.M; Project Administration,
J.A.R.S. and M.C.; Funding Acquisition, J.A.R.S. and M.C.
Funding: Jorge A. R. Salvador thanks PT2020 (Programa Operacional do Centro 2020), project nº 3269,
drugs2CAD, and the financial support by FEDER (European Regional Development Fund) through the
COMPETE 2020 Programme (Operational Programme for Competitiveness and Internationalisation). Jorge
A. R. Salvador also wishes to thank Universidade de Coimbra for financial support. Daniela P. S. Alho thanks
FEDER (Programa Operacional Factores de Competitividade—COMPETE 2020) and Fundação para a Ciência
e Tecnologia (FCT) through Projecto Estratégico: UID/NEU/04539/2013 and the financial support for the
PhD grant SFRH/BD/66020/2009. Marta Cascante and Silvia Marin thank MINECO-European Commission
FEDER—Una manera de hacer Europa (SAF2017-89673-R) and Agència de Gestió d’Ajuts Universitaris i de Recerca
(AGAUR)—Generalitat de Catalunya (2017SGR1033). Marta Cascante also acknowledges the support received
through the prize “ICREA Academia” for excellence in research, funded by ICREA Foundation—Generalitat de
Catalunya. The authors acknowledge UC-NMR facility, which is supported by FEDER and FCT funds through
the grants REEQ/481/QUI/2006, RECI/QEQ-FI/0168/2012, and CENTRO-07-CT62-FEDER-002012, and Rede
Nacional de Ressonância Magnética Nuclear (RNRMN), for NMR data.
Acknowledgments: The authors are grateful to Laboratório de Espectometria de Massa (LEM)-CEF/UC
integrated in the Rede Nacional de Espectrometria de Massa (RNEM) of Portugal for the MS analysis, and
Centro de Apoio Científico e Tecnolóxico á Investigación (CACTI), Universidade de Vigo, for elemental analysis.
The authors acknowledge Centres Científics I Tecnològics de la UB (CCiTUB) for flow cytometry analysis support.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2019, 24, 766
19 of 21
References
1.
Dzubak, P.; Hajduch, M.; Vydra, D.; Hustova, A.; Kvasnica, M.; Biedermann, D.; Markova, L.; Urban, M.;
Sarek, J. Pharmacological activities of natural triterpenoids and their therapeutic implications. Nat. Prod. Rep.
2006, 23, 394–411. [CrossRef] [PubMed]
2.
3.
Salvador, J.A.R.; Moreira, V.M.; Goncalves, B.M.F.; Leal, A.S.; Jing, Y.K. Ursane-type pentacyclic triterpenoids
as useful platforms to discover anticancer drugs. Nat. Prod. Rep. 2012, 29, 1463–1479. [CrossRef] [PubMed]
Salvador, J.A.R.; Leal, A.S.; Alho, D.P.S.; Goncalves, B.M.F.; Valdeira, A.S.; Mendes, V.I.S.; Jing, Y.K.
Highlights of pentacyclic triterpenoids in the cancer settings. In Studies in Natural Products Chemistry;
AttaUrRahman, F.R.S., Ed.; Elsevier Science Bv: Amsterdam, The Netherland, 2014; Volume 41, pp. 33–73.
Chudzik, M.; Korzonek-Szlacheta, I.; Krol, W. Triterpenes as potentially cytotoxic compounds. Molecules
2015, 20, 1610–1625. [CrossRef] [PubMed]
Salvador, J.A.R.; Leal, A.S.; Valdeira, A.S.; Goncalves, B.M.F.; Alho, D.P.S.; Figueiredo, S.A.C.; Silvestre, S.M.;
Mendes, V.I.S. Oleanane-, ursane-, and quinone methide friedelane-type triterpenoid derivatives: Recent
advances in cancer treatment. Eur. J. Med. Chem. 2017, 142, 95–130. [CrossRef] [PubMed]
Heller, L.; Schwarz, S.; Per, V.; Kowitsch, A.; Siewert, B.; Csuk, R. Incorporation of a michael acceptor
enhances the antitumor activity of triterpenoic acids. Eur. J. Med. Chem. 2015, 101, 391–399. [CrossRef]
[PubMed]
4.
5.
6.
7.
8.
Goncalves, B.M.F.; Salvador, J.A.R.; Marin, S.; Cascante, M. Synthesis and anticancer activity of novel
fluorinated asiatic acid derivatives. Eur. J. Med. Chem. 2016, 114, 101–117. [CrossRef] [PubMed]
Mendes, V.I.S.; Bartholomeusz, G.A.; Ayres, M.; Gandhi, V.; Salvador, J.A.R. Synthesis and cytotoxic activity
of novel a-ring cleaved ursolic acid derivatives in human non-small cell lung cancer cells. Eur. J. Med. Chem.
2016, 123, 317–331. [CrossRef] [PubMed]
9.
Sommerwerk, S.; Heller, L.; Kuhfs, J.; Csuk, R. Urea derivates of ursolic, oleanolic and maslinic acid induce
apoptosis and are selective cytotoxic for several human tumor cell lines. Eur. J. Med. Chem. 2016, 119, 1–16.
[CrossRef]
10. Sommerwerk, S.; Heller, L.; Kerzig, C.; Kramell, A.E.; Csuk, R. Rhodamine b conjugates of triterpenoic acids
are cytotoxic mitocans even at nanomolar concentrations. Eur. J. Med. Chem. 2017, 127, 1–9. [CrossRef]
11. Spivak, A.; Khalitova, R.; Nedopekina, D.; Dzhemileva, L.; Yunusbaeva, M.; Odinokov, V.; D’Yakonov, V.;
Dzhemilev, U. Synthesis and evaluation of anticancer activities of novel c-28 guanidine-functionalized
triterpene acid derivatives. Molecules 2018, 23, 22. [CrossRef]
12. Asl, M.N.; Hosseinzadeh, H. Review of pharmacological effects of glycyrrhiza sp and its bioactive
compounds. Phytother. Res. 2008, 22, 709–724. [CrossRef] [PubMed]
13. Shanmugam, M.K.; Nguyen, A.H.; Kumar, A.P.; Tan, B.K.H.; Sethi, G. Targeted inhibition of tumor
proliferation, survival, and metastasis by pentacyclic triterpenoids: Potential role in prevention and therapy
of cancer. Cancer Lett. 2012, 320, 158–170. [CrossRef] [PubMed]
14. Kuang, P.H.; Zhao, W.X.; Su, W.X.; Zhang, Z.Q.; Zhang, L.; Liu, J.M.; Ren, G.L.; Yin, Z.Y.; Wang, X.M.
18 beta-glycyrrhetinic acid inhibits hepatocellular carcinoma development by reversing hepatic stellate
cell-mediated immunosuppression in mice. Int. J. Cancer 2013, 132, 1831–1841. [CrossRef] [PubMed]
15. Hasan, S.K.; Khan, R.; Ali, N.; Khan, A.Q.; Rehman, M.U.; Tahir, M.; Lateef, A.; Nafees, S.; Mehdi, S.J.;
Rashid, S.; et al. 18-glycyrrhetinic acid alleviates 2-acetylaminofluorene-induced hepatotoxicity in wistar
rats: Role in hyperproliferation, inflammation and oxidative stress. Hum. Exp. Toxicol. 2015, 34, 628–641.
[CrossRef] [PubMed]
16. Wang, S.S.; Shen, Y.; Qiu, R.F.; Chen, Z.L.; Chen, Z.H.; Chen, W.B. 18 beta-glycyrrhetinic acid exhibits potent
antitumor effects against colorectal cancer via inhibition of cell proliferation and migration. Int. J. Oncol.
2017, 51, 615–624. [CrossRef]
17. Lee, C.S.; Kim, Y.J.; Lee, M.S.; Han, E.S.; Lee, S.J. 18 beta-glycyrrhetinic acid induces apoptotic cell death
in siha cells and exhibits a synergistic effect against antibiotic anti-cancer drug toxicity. Life Sci. 2008, 83,
481–489. [CrossRef]
18. Sharma, G.; Kar, S.; Palit, S.; Das, P.K. 18 beta-glycyrrhetinic acid (concur) induces apoptosis through
modulation of akt/foxo3a/bim pathway in human breast cancer mcf-7 cells. J. Cell. Physiol. 2012, 227,
1923–1931. [CrossRef]

Molecules 2019, 24, 766
20 of 21
19. Wang, D.; Wong, H.K.; Feng, Y.B.; Zhang, Z.J. 18beta-glycyrrhetinic acid induces apoptosis in pituitary
adenoma cells via ros/mapks-mediated pathway. J. Neuro-Oncol. 2014, 116, 221–230. [CrossRef]
20. Hasan, S.K.; Siddiqi, A.; Nafees, S.; Ali, N.; Rashid, S.; Ali, R.; Shahid, A.; Sultana, S. Chemopreventive effect
of 18 beta-glycyrrhetinic acid via modulation of inflammatory markers and induction of apoptosis in human
hepatoma cell line (hepg2). Mol. Cell. Biochem. 2016, 416, 169–177. [CrossRef]
21. Cai, Y.; Zhao, B.X.; Liang, Q.Y.; Zhang, Y.Q.; Cai, J.Y.; Li, G.F. The selective effect of glycyrrhizin and
glycyrrhetinic acid on topoisomerase ii alpha and apoptosis in combination with etoposide on triple negative
breast cancer mda-mb-231 cells. Eur. J. Pharmacol. 2017, 809, 87–97. [CrossRef]
22. Baltina, L.A. Chemical modification of glycyrrhizic acid as a route to new bioactive compounds for medicine.
Curr. Med. Chem. 2003, 10, 155–171. [CrossRef]
23. Guo, W.B.; Yan, M.M.; Xu, B.; Chu, F.H.; Wang, W.; Zhang, C.Z.; Jia, X.H.; Han, Y.T.; Xiang, H.J.;
Zhang, Y.Z.; et al. Design, synthesis, and biological evaluation of the novel glycyrrhetinic acid-cinnamoyl
hybrids as anti-tumor agents. Chem. Cent. J. 2016, 10, 11. [CrossRef]
24. Li, Y.; Feng, L.; Song, Z.F.; Li, H.B.; Huai, Q.Y. Synthesis and anticancer activities of glycyrrhetinic acid
derivatives. Molecules 2016, 21, 20. [CrossRef] [PubMed]
25. Yan, T.L.; Bai, L.F.; Zhu, H.L.; Zhang, W.M.; Lv, P.C. Synthesis and biological evaluation of glycyrrhetic acid
derivatives as potential vegfr2 inhibitors. ChemMedChem 2017, 12, 1087–1096. [CrossRef] [PubMed]
26. Xu, B.; Wu, G.R.; Zhang, X.Y.; Yan, M.M.; Zhao, R.; Xue, N.N.; Fang, K.; Wang, H.; Chen, M.; Guo, W.B.; et al.
An overview of structurally modified glycyrrhetinic acid derivatives as antitumor agents. Molecules 2017, 22,
24. [CrossRef] [PubMed]
27. Wang, R.; Li, Y.; Huai, X.D.; Zheng, Q.X.; Wang, W.; Li, H.J.; Huai, Q.Y. Design and preparation of derivatives
of oleanolic and glycyrrhetinic acids with cytotoxic properties. Drug Des. Dev. Ther. 2018, 12, 1321–1336.
[CrossRef] [PubMed]
28. Santos, R.C.; Salvador, J.A.R.; Marin, S.; Cascante, M.; Moreira, J.N.; Dinis, T.C.P. Synthesis and
structure-activity relationship study of novel cytotoxic carbamate and n-acylheterocyclic bearing derivatives
of betulin and betulinic acid. Bioorg. Med. Chem. 2010, 18, 4385–4396. [CrossRef] [PubMed]
29. Leal, A.S.; Wang, R.; Salvador, J.A.R.; Jing, Y.K. Synthesis of novel ursolic acid heterocyclic derivatives with
improved abilities of antiproliferation and induction of p53, p21(waf1) and noxa in pancreatic cancer cells.
Bioorg. Med. Chem. 2012, 20, 5774–5786. [CrossRef]
30. Thirumurugan, P.; Matosiuk, D.; Jozwiak, K. Click chemistry for drug development and diverse
chemical-biology applications. Chem. Rev. 2013, 113, 4905–4979. [CrossRef]
31. Musumeci, F.; Schenone, S.; Desogus, A.; Nieddu, E.; Deodato, D.; Botta, L. Click chemistry, a potent tool in
medicinal sciences. Curr. Med. Chem. 2015, 22, 2022–2050. [CrossRef]
32. Ma, N.; Wang, Y.; Zhao, B.X.; Ye, W.C.; Jiang, S. The application of click chemistry in the synthesis of agents
with anticancer activity. Drug Des. Dev. Ther. 2015, 9, 1585–1599. [CrossRef]
33. Meghani, N.M.; Amin, H.H.; Leel, B.J. Mechanistic applications of click chemistry for pharmaceutical drug
discovery and drug delivery. Drug Discov. Today 2017, 22, 1604–1619. [CrossRef]
34. Pertino, M.W.; Lopez, C.; Theoduloz, C.; Schmeda-Hirschmann, G. 1,2,3-triazole-substituted oleanolic acid
derivatives: Synthesis and antiproliferative activity. Molecules 2013, 18, 7661–7674. [CrossRef] [PubMed]
35. Dangroo, N.A.; Singh, J.; Rath, S.K.; Gupta, N.; Qayum, A.; Singh, S.; Sangwan, P.L. A convergent synthesis
of novel alkyne-azide cycloaddition congeners of betulinic acid as potent cytotoxic agent. Steroids 2017, 123,
1–12. [CrossRef]
36. Pokorny, J.; Borkova, L.; Urban, M. Click reactions in chemistry of triterpenes - advances towards
development of potential therapeutics. Curr. Med. Chem. 2018, 25, 636–658. [CrossRef] [PubMed]
37. Pertino, M.W.; Petrera, E.; Alche, L.E.; Schmeda-Hirschmann, G. Synthesis, antiviral and cytotoxic activity of
novel terpenyl hybrid molecules prepared by click chemistry. Molecules 2018, 23, 12. [CrossRef] [PubMed]
38. Li, X.J.; Wang, Y.T.; Gao, Y.; Li, L.; Guo, X.; Liu, D.; Jing, Y.K.; Zhao, L.X. Synthesis of methyl
2-cyano-3,12-dioxo-18 beta-olean-1,9(11)-dien-30-oate analogues to determine the active groups for inhibiting
cell growth and inducing apoptosis in leukemia cells. Org. Biomol. Chem. 2014, 12, 6706–6716. [CrossRef]
[PubMed]
39. Schwarz, S.; Csuk, R. Synthesis and antitumour activity of glycyrrhetinic acid derivatives. Bioorg. Med. Chem.
2010, 18, 7458–7474. [CrossRef]

Molecules 2019, 24, 766
21 of 21
40. Rao, G.; Kondaiah, P.; Singh, S.K.; Ravanan, P.; Sporn, M.B. Chemical modifications of natural
triterpenes-glycyrrhetinic and boswellic acids: Evaluation of their biological activity. Tetrahedron 2008
64, 11541–11548. [CrossRef]
,
41. Chu, F.H.; Xu, X.; Li, G.L.; Gu, S.; Xu, K.; Gong, Y.; Xu, B.; Wang, M.N.; Zhang, H.Z.; Zhang, Y.Z.; et al. Amino
acid derivatives of ligustrazine-oleanolic acid as new cytotoxic agents. Molecules 2014, 19, 18215–18231.
[CrossRef] [PubMed]
42. Porchia, M.; Dolmella, A.; Gandin, V.; Marzano, C.; Pellei, M.; Peruzzo, V.; Refosco, F.; Santini, C.; Tisato, F.
Neutral and charged phosphine/scorpionate copper(i) complexes: Effects of ligand assembly on their
antiproliferative activity. Eur. J. Med. Chem. 2013, 59, 218–226. [CrossRef] [PubMed]
43. Goncalves, B.M.F.; Salvador, J.A.R.; Marin, S.; Cascante, M. Synthesis and biological evaluation of novel
asiatic acid derivatives with anticancer activity. RSC Advances 2016, 6, 3967–3985. [CrossRef]
44. Chen, L.; Zhao, Y.; Halliday, G.C.; Berry, P.; Rousseau, R.F.; Middleton, S.A.; Nichols, G.L.; Del Bello, F.;
Piergentili, A.; Newell, D.R.; et al. Structurally diverse mdm2-p53 antagonists act as modulators of mdr-1
function in neuroblastoma. Br. J. Cancer 2014, 111, 716–725. [CrossRef]
45. Antunovic, M.; Kriznik, B.; Ulukaya, E.; Yilmaz, V.T.; Mihalic, K.C.; Madunic, J.; Marijanovic, I. Cytotoxic
activity of novel palladium-based compounds on leukemia cell lines. Anti-Cancer Drugs 2015, 26, 180–186.
[CrossRef] [PubMed]
46. Rajic, Z.; Zorc, B.; Raic-Malic, S.; Ester, K.; Kralj, M.; Pavelic, K.; Balzarini, J.; De Clercq, E.; Mintas, M.
Hydantoin derivatives of l- and d-amino acids: Synthesis and evaluation of their antiviral and antitumoral
activity. Molecules 2006, 11, 837–848. [CrossRef] [PubMed]
47. Wong, R.S.Y. Apoptosis in cancer: From pathogenesis to treatment. J. Exp. Clin. Cancer Res. 2011, 30, 14.
[CrossRef]
48. Hibasami, H.; Iwase, H.; Yoshioka, K.; Takahashi, H. Glycyrrhetic acid (a metabolic substance and aglycon of
glycyrrhizin) induces apoptosis in human hepatoma, promyelotic leukemia and stomach cancer cells. Int. J.
Mol. Med. 2006, 17, 215–219. [CrossRef]
49. Satomi, Y.; Nishino, H.; Shibata, S. Glycyrrhetinic acid and related compounds induce g1 arrest and apoptosis
in human hepatocellular carcinoma hepg2. Anticancer Res. 2005, 25, 4043–4047.
50. Lee, C.S.; Yang, J.C.; Kim, Y.J.; Jang, E.R.; Kim, W.; Myung, S.C. 18 beta-glycyrrhetinic acid potentiates
apoptotic effect of trichostatin a on human epithelial ovarian carcinoma cell lines. Eur. J. Pharmacol. 2010
649, 354–361. [CrossRef]
,
51. Yang, J.C.; Myung, S.C.; Kim, W.; Lee, C.S. 18 beta-glycyrrhetinic acid potentiates hsp90 inhibition-induced
apoptosis in human epithelial ovarian carcinoma cells via activation of death receptor and mitochondrial
pathway. Mol. Cell Biochem. 2012, 370, 209–219. [CrossRef]
52. Hanahan, D.; Weinberg, R.A. Hallmarks of cancer: The next generation. Cell 2011, 144, 646–674. [CrossRef]
[PubMed]
53. Vogelstein, B.; Lane, D.; Levine, A.J. Surfing the p53 network. Nature 2000, 408, 307–310. [CrossRef] [PubMed]
54. Brady, C.A.; Attardi, L.D. P53 at a glance. J. Cell Sci. 2010, 123, 2527–2532. [CrossRef] [PubMed]
55. Cheng, J.; Haas, M. Frequent mutations in the p53 tumor suppressor gene in human leukemia t-cell lines.
Mol. Cell Biol. 1990, 10, 5502–5509. [CrossRef] [PubMed]
Sample Availability: Samples of all compounds are available from the authors.
©
2019 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 (http://creativecommons.org/licenses/by/4.0/).