Synthesis and antiproliferative activity of novel A-ring cleaved glycyrrhetinic acid derivatives

Article abstract of DOI:10.3390/molecules24162938

A series of new glycyrrhetinic acid derivatives was synthesized via the opening of its ring A along with the coupling of an amino acid. The antiproliferative activity of the derivatives was evaluated against a panel of nine human cancer cell lines. Compound 17 was the most active compound, with an IC₅₀ of 6.1 μM on Jurkat cells, which is 17-fold more potent than that of glycyrrhetinic acid, and was up to 10 times more selective toward that cancer cell line. Further biological investigation in Jurkat cells showed that the antiproliferative activity of compound 17 was due to cell cycle arrest at the S phase and induction of apoptosis.

Full text of DOI:10.3390/molecules24162938

molecules
Article
Synthesis and Antiproliferative Activity of Novel
A-Ring Cleaved 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
Centre for Neuroscience and Cell Biology, 3000-504 Coimbra, Portugal
Department of Biochemistry and Molecular Biomedicine, Faculty of Biology, University of Barcelona,
Diagonal 643, 08028 Barcelona, Spain
2
3
4
Centro de Investigaci
ón Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Instituto
de Salud Carlos III (ISCIII), 28029 Madrid, Spain
*
Correspondence: salvador@ci.uc.pt (J.A.R.S.); silviamarin@ub.edu (S.M.);
Tel.: +351-239-488-400 (J.A.R.S.); +34-934-021-217 (S.M.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Carlo Siciliano
Received: 27 July 2019; Accepted: 12 August 2019; Published: 14 August 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A series of new glycyrrhetinic acid derivatives was synthesized via the opening of its
ring A along with the coupling of an amino acid. The antiproliferative activity of the derivatives
was evaluated against a panel of nine human cancer cell lines. Compound 17 was the most active
compound, with an IC50 of 6.1
µM on Jurkat cells, which is 17-fold more potent than that of
glycyrrhetinic acid, and was up to 10 times more selective toward that cancer cell line. Further
biological investigation in Jurkat cells showed that the antiproliferative activity of compound 17 was
due to cell cycle arrest at the S phase and induction of apoptosis.
Keywords: pentacyclic triterpenoids; glycyrrhetinic acid; A-ring cleaved derivatives; antiproliferative
activity; cell cycle arrest; apoptosis
1. Introduction
Cancer is a leading cause of death worldwide. Its global incidence continues to rise due to the
aging and growth of the world population and the increasing adoption of lifestyle choices associated
with cancer in developed countries [
drugs for cancer treatment and nowadays they play an important role as a source of leads for the
development of potential new agents [ ]. The plant-derived triterpenoids are proving to be interesting
leading compounds as reported in a large number of scientific papers emerging in this field [4–15].
Glycyrrhetinic acid (GA) is the hydrolyzed metabolite of glycyrrhizin, a major pentacyclic
triterpenoid saponin obtained from the roots of licorice (Glycyrrhiza species) in high yields up to
24% [16 17]. This compound has been shown to inhibit tumor initiation [18 22] and proliferation in
several cancer cell lines; its antiproliferative activity is mediated by cell cycle arrest [23 24] and induction
of apoptosis [21 25 28]. The antitumor effects of GA were also observed in animal models [20 29 30].
Nevertheless, it lacks potency and selectivity as an antitumor agent. Many derivatizations have been
performed in order to enhance the potency of GA 31 36]. However, the cleavage of its ring A is
1]. Plants have been a major source of highly effective conventional
2,3
1
,
–
,
,
–
1
,
,
1
[ –
still poorly explored [37]. On the other hand, is well known that the conjugation of an amino acid
moiety to pentacyclic triterpenoids improves their cytotoxicity and their selectivity towards tumor
cells [38–40]. These findings prompted us to synthesize new GA 1 derivatives via the opening of its
ring A along with the coupling with an amino acid. The novel semisynthetic derivatives were tested
for their antiproliferative activity against a panel of nine human cancer cell lines. Further biological
Molecules 2019, 24, 2938; doi:10.3390/molecules24162938
www.mdpi.com/journal/molecules

Molecules 2019, 24, 2938
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assays were conducted for the most potent compound 17 in the cancer cell line that yielded the best
results (Jurkat cells), to investigate its preliminary mechanism of action. The study of selectivity was
performed on human fibroblasts (BJ).
2. Results and Discussion
2.1. Chemistry
The synthesis of the glycyrrhetinic acid 1 derivatives is outlined in Schemes 1–3. Full structural
elucidation of the new glycyrrhetinic acid derivatives was achieved using nuclear magnetic resonance
(NMR), mass spectrometry (MS) and elemental analysis. The analytical data obtained for the known
compounds 1–5 and 8–10 were in agreement with those reported in the literature [39,41–43].
The synthesis of compounds
the reaction of compound , the starting material, with methyl iodide in the presence of potassium
carbonate [39]. The 3 -hydroxyl group of compound was then oxidized using the Jones reagent [41
to give the 3-keto derivative . The reaction of this derivative with m-chloroperbenzoic acid (m-CPBA)
provided lactone . The lactone ring of was opened by treatment with p-toluenesulfonic acid (p-TSA)
2–7 is summarized in Scheme 1. Methyl ester 2 was obtained from
1
β
2
]
3
4
4
in dichloromethane [42]. Reaction of compound 5 with bis(2-methoxyethyl)aminosulfur trifluoride
(Deoxo-Fluor®) [44] provided the acyl fluoride intermediate which was reacted either with glycine
methyl ester hydrochloride or with L-alanine methyl ester hydrochloride to afford compounds 6 and
7
, in yields of 69% and 61%, respectively. We found that the acyl fluoride, in this position of the
structure, decomposes on standing. For that reason, the crude compound was employed without
further purification, and immediately, in the subsequent reactions. The preparation of compounds
and
was confirmed by the presence of the proton signals of the amino acid side chains. On the 1H
NMR spectrum of compound , the signals of the glycine methyl ester side chain were observed
around 6.1 ppm (NH), 4.0 ppm (NCH2) and 3.7 ppm (CH3). Compound , with an alanine methyl
ester side chain, had δ signals around 6.1 ppm (NH), 4.6 ppm (NCH) and 3.7 ppm (CH3).
6
7
6
δ
7
Scheme 1. Reagents and conditions: (a) CH3I, K2CO3, DMF, r.t.; (b) Jones reagent, acetone, 0 ◦C; (c)
m-CPBA, CH2Cl2, r.t.; (d) p-TSA, CH2Cl2, r.t.; (e) Deoxo-Fluor , CH2Cl2, r.t.; (f) glycine methyl ester
®
hydrochloride or L-alanine methyl ester hydrochloride, Et3N, CH2Cl2, r.t.
Compounds 8–17 were synthesized as depicted in Scheme 2. Compound
1
was oxidized using the
Jones reagent [41] to afford compound 8, which was reacted with m-CPBA to give the derivative 9. The

Molecules 2019, 24, 2938
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lactone ring of 9 was cleaved by treatment with p-TSA in methanol and dichloromethane [42] to provide
compound 10. The derivative 11 and the three pairs of compounds synthesized in the following steps
were prepared to explore the influence of the keto group in position C-11 on the antiproliferative
activity. The removal of the keto group was performed by a Clemmensen reduction [45] with zinc dust
and concentrated HCl in dioxane at room temperature to afford 11 (75%). The reduction was confirmed
on the 13C NMR spectrum, by the absence of the
δ signal around 200 ppm, which corresponds to the
carbonyl group in ring C. Acyl fluorides 12 and 13 were obtained from the reaction of compounds
10 and 11 with Deoxo-Fluor, in yields of 75% and 61%, respectively. The synthesis of acyl fluorides
was detected on the 13C NMR spectra. The carbon C30 appeared as a doublet with a
δ signal around
166 ppm and a coupling constant of 375 Hz, in both compounds 12 and 13. These derivatives were
reacted either with glycine methyl ester hydrochloride or with L-alanine methyl ester hydrochloride to
afford compounds 14–17, in yields ranging from 43% to 83%. The glycine methyl ester side chain of
compounds 14 and 15 was detected on the 1H NMR spectra. Its proton signals were observed around
6.2 ppm (NH), 4.0 ppm (NCH2) and 3.8 ppm (CH3). Compounds 16 and 17, with an alanine methyl
ester side chain, had δ signals around 6.2 ppm (NH), 4.6 ppm (NCH) and 3.8 ppm (CH3).
Deprotection of the carboxyl group of the amino acid chain was performed in compounds
and 16, by alkaline hydrolysis (Scheme 3). This reaction also caused deprotection of the other carboxyl
group on compounds 14 and 16. Compounds 18 21 were obtained in yields ranging from 94% to 98%.
Deprotection of the carboxyl group of the amino acid chains was confirmed by the absence of signals
6, 7, 14
–
δ
around 3.7–3.8 ppm, on the 1H NMR spectra of compounds 18
–21. The loss the other methyl group of
compounds 14 and 16 was detected by the absence of the
spectra of compounds 20 and 21.
δ
signal around 3.6 ppm, on the 1H NMR
O
O
O
OH
OH
OH
H
H
H
O
O
X
H
a
b
c
GA
1
H3COOC
H
H
O
O
O
10
: X= C=O
9
8
d
11: X= CH2
O
O
F
R
H
H
X
X
e
f
H3COOC
H3COOC
H
H
14: X= C=O; R= NHCH2COOCH3
15: X= CH2; R= NHCH2COOCH3
12
13
: X= C=O
: X= CH2
16: X= C=O; R= NHCH(CH3)COOCH3
17: X= CH2; R= NHCH(CH3)COOCH3
◦
Scheme 2. Reagents and conditions: (a) Jones reagent, acetone, 0 C; (b) m-CPBA,CH2Cl2, r.t.; (c)
MeOH, p-TSA, CH2Cl2, r.t.; (d) zinc dust, conc. HCl, dioxane, r.t.; (e) Deoxo-Fluor , CH2Cl2, r.t.; (f)
glycine methyl ester hydrochloride or L-alanine methyl ester hydrochloride, Et3N, CH2Cl2, r.t.
®

Molecules 2019, 24, 2938
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Scheme 3. Reagents and conditions: (a) KOH 4N, THF/MeOH, r.t.
2.2. Biology
2.2.1. Antiproliferative Activity
Several cancer cell lines were used to evaluate the potential cytotoxicity of the synthesized
compounds against human cancers. This evaluation was based on the determination of the concentration
that inhibits cell proliferation at 50% (IC50), using 3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyltetrazolium
bromide (MTT) or 2,3-bis(2-methoxy-4-nitro- 5-sulphophenyl)-2H-tetrazolium-5-carboxanilide (XTT)
assays, after 72 h of treatment with the compounds.
Compounds
adenocarcinoma) and HT-29 (colon adenocarcinoma) cell lines (Table 1). Compounds
were not evaluated because they have already been tested with no improvements in potency and/or
selectivity [37 39 46 47]. Intermediates and 10, afforded by the cleavage of the ring A, were more
potent than the parental compound GA . Removal of the keto group from ring C, that provided
compound 11, resulted in an increment of cytotoxicity. Acyl fluorides 12 and 13 were less potent
compared to their substrates. Analysis of the IC50 values of the derivatives 6, 7 and 14 17 showed
that the conjugation of an amino acid methyl ester provided more potent compounds. Deprotection
of the carboxyl groups resulted in a loss of cytotoxicity. Compounds 18 21, afforded by the alkaline
1
,
5–
7
and 10
–
21 were screened for their antiproliferative activity on A549 (lung
2–
4,
8
and
9
,
,
,
5
1
–
–
hydrolysis, were further tested in Jurkat (acute T-cell leukemia) and MOLT-4 (acute lymphoblastic
leukemia) cell lines (Table 2). The results of these assays confirmed that the deprotection provided less
active compounds. Derivatives 6, 7 and 14–17 and the parental compound GA 1 were also screened for
their antiproliferative activity in seven additional human cancer cell lines: Jurkat, MOLT-4, MIAPaca
2 (pancreas adenocarcinoma), MCF7 (breast adenocarcinoma), HeLa (cervix adenocarcinoma), A375
(melanoma) and HepG2 (hepatocellular carcinoma). Comparing IC50 values of compounds 6 and 7
with those obtained for compounds 14 and 16, no significant differences were found regarding the
position in which the amino acid methyl ester was introduced. Derivatives 15 and 17 were respectively
more potent than compounds 14 and 16 in all tested cell lines, which confirmed that the removal
of the keto group from ring C enhanced the cytotoxicity. Compounds
7, 16 and 17, which have an
alanine methyl ester chain, were more active than compounds 14 and 15, with a glycine methyl ester
6
,
chain, respectively. These results suggest that the type of amino acid moiety introduced influences the
antiproliferative activity. Within the newly synthesized derivatives, compound 17, with a reduced ring
C and with an alanine methyl ester chain, was the most potent derivative. This compound was 5 to
17-fold more active than GA 1, depending on the cancer cell line.

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Table 1. Antiproliferative activities of GA 1, its derivatives 5–7 and 10–21, and cisplatin against A549
and HT-29 cell lines.
Cell line (IC50, µM) 1
A549 HT-29
Compound
1
5
6
7
10
11
12
110.5 ± 3.9
59.4 ± 2.1
31.6 ± 1.5
26.2 ± 2.4
52.2 ± 3.0
33.7 ± 2.0
> 100
115.7 ± 1.6
66.6 ± 3.2
37.4 ± 1.0
24.4 ± 1.7
61.7 ± 1.4
43.0 ± 1.5
> 100
13
> 100
> 100
14
15
16
17
33.7 ± 1.8
26.4 ± 2.2
24.4 ± 1.4
14.8 ± 0.9
> 100
35.0 ± 1.7
24.7 ± 0.9
23.8 ± 0.3
13.0 ± 0.5
> 100
18
19
20
> 100
> 100
> 100
> 100
21
> 100
> 100
Cisplatin
12.6 ± 0.8 [48]
6.1 [49]
1
The cell lines were treated with different concentrations of each compound for 72 h. IC50 values were determined
by MTT assay and are expressed as means ± SD (standard deviation) of three independent experiments.
Table 2. Antiproliferative activities of GA
1, its derivatives 6, 7 and 14–21, and cisplatin against several
cancer cell lines and the human nontumoral BJ cell line.
Cell line (IC50, µM) 1
Compound
Jurkat
MOLT-4
MIAPaca2
MCF7
HeLa
A375
HepG2
BJ
1
6
7
14
15
16
17
18
105.6 ± 5.0
11.9 ± 0.2
11.7 ± 0.6
13.3 ± 1.1
12.5 ± 0.5
9.6 ± 0.4
6.1 ± 0.2
46.4 ± 3.7
40.8 ± 2.7
> 100
95.5 ± 3.9
18.9 ± 1.6
18.5 ± 0.9
23.5 ± 0.8
18.9 ± 1.6
19.1 ± 1.3
15.3 ± 0.7
51.9 ± 2.5
49.0 ± 1.6
> 100
101.6 ± 1.6
28.2 ± 0.5
24.9 ± 1.2
32.5 ± 3.2
20.2 ± 1.2
22.6 ± 0.6
11.8 ± 1.1
N.D.
97.8 ± 3.9
32.9 ± 1.6
24.9 ± 0.9
28.8 ± 0.7
24.8 ± 1.3
23.8 ± 1.6
21.6 ± 0.6
N.D.
107.2 ± 2.5
34.5 ± 2.5
25.7 ± 0.6
34.2 ± 2.4
22.2 ± 0.3
19.1 ± 0.5
13.0 ± 0.5
N.D.
112.2 ± 2.6
30.0 ± 1.5
24.5 ± 1.0
30.0 ± 2.2
18.8 ± 1.1
17.0 ± 1.1
11.3 ± 0.4
N.D.
125.1 ± 9.1
30.6 ± 0.5
24.8 ± 0.4
34.7 ± 1.1
25.4 ± 1.3
25.7 ± 0.8
16.0 ± 0.3
N.D.
165.0 ± 7.1
N.D.
N.D.
N.D.
N.D.
N.D.
> 100
N.D.
N.D.
N.D.
19
20
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
21
> 100
> 100
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
Cisplatin
1.9 [50]
1.4 [50]
5.0 ± 1.0 [51]
19.1 ± 4.5 [52]
2.3 ± 0.3 [52]
3.1 ± 1.0 [48]
2.9 [49]
10.1 ± 2.0 [52]
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 MOLT-4 cells and by MTT assay in all the other cell lines. Results are expressed as means
± SD of three independent experiments. N.D.: not determined.
The selectivity towards cancer cells was studied for GA
1 and compound 17 by incubating them
with a human nontumoral cell line (BJ) (Table 2). GA and compound 17 showed IC50 values that
1
were 1.6 and more than 16.4 times lower on Jurkat cells than on the nontumoral BJ cells, respectively.
Therefore, the novel derivative 17 was up to 10 times more selective towards malignant cells than its
parental compound 1. This compound also showed a significant improvement in selectivity compared
to the chemotherapy agent cisplatin. Considering also the Jurkat cell line, cisplatin presented an IC50
value that was 5.3 times lower than on BJ cells (Table 2); therefore, compound 17 was up to 3 times
more selective than cisplatin towards Jurkat cells.
2.2.2. Analysis of Cell Cycle Distribution and Apoptosis
The Jurkat cell line which was the most susceptible to these derivatives was selected to investigate
the mechanism of action of compound 17. To evaluate the effects on the cell cycle distribution, Jurkat
cells were treated with compound 17, at a concentration corresponding to its IC50 value at 72 h of

Molecules 2019, 24, 2938
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treatment, for 24, 48 and 72 h and then analyzed by flow cytometry. The calculation of the fraction of
cells in G0/G1, S and G2/M phases was performed using the fraction of live cells. Treatment for 24
h induced significant increase in the population at S phase with respect to untreated cells (Figure 1);
after 48 h this effect has decreased and after 72 h it was no longer observed. DNA fragmentation was
detected after 72 h of incubation based on the appearance of a sub-G0 peak. This sequence of events
suggests that the cell cycle arrest at S phase may have led cells to undergo apoptosis.
Figure 1. Effect of compound 17 on cell cycle distribution. Cell cycle analysis of Jurkat cells untreated
(Control) or treated with 6.1
µM compound 17 for 24 h (A), 48 h (B), and 72 h (C). After treatment,
cells were stained with PI and DNA content analyzed by flow cytometry. A representative histogram
is shown for each incubation time and condition. Results are presented as means
independent experiments.
±
SD of three
Apoptosis assays were then performed to better elucidate the mechanism of cell death involved
in the cytotoxic effect of compound 17. The Annexin V-FITC/PI flow cytometric assay employs the
property of fluorescein isothiocyanate (FITC) conjugated to Annexin V (Annexin V-FITC) to bind to
phosphatidylserine (PS) and the property 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 cells (Annexin-V+/PI+). The experiments
were conducted on Jurkat cells treated with compound 17 at a concentration corresponding to its
IC50 value at 72 h of treatment (6.1
M for 72 h. Exposure to this compound for 24 and 48 h did not change significantly the apoptotic
(Figure 2A) and necrotic (data not shown) populations. Treatment for 72 h with compound 17 at
concentrations of 6.1 M and 12.2 M increased the early apoptotic population by 19% and 30%,
µM) for 24 and 48 h, and at concentrations of 6.1 µM and 12.2
µ
µ
µ

Molecules 2019, 24, 2938
7 of 17
respectively. No significant changes were observed in the late apoptotic population. These results
were in good agreement with those obtained in the cell cycle experiments.
Figure 2. Induction of apoptosis by compound 17. (A) Flow cytometry quantification of apoptosis
in Jurkat cells untreated (Control) or treated with compound 17 at specified concentrations for 24, 48
and 72 h. After treatment, cells were stained with annexin V-FITC/PI and analyzed by flow cytometry.
The percentage of early (dark gray bar) and late (light gray bar) apoptotic cells in each condition is
represented as a bar diagram, calculated from dot plots. Results are presented as means
±
SD of three
independent experiments. ( ) Upper panel: Representative dot plots of annexin V-FITC/PI assays of
B
Jurkat cells untreated (Control) or treated with compound 17 at specified concentrations for 72 h; the
right quadrants of each diagram (annexin+/PI− and annexin+/PI+) represent apoptotic cells. Lower
panel: Representative fluorescence microscopic images of Jurkat cells untreated (Control) or treated
with compound 17 at specified concentrations for 72 h; Jurkat cells were stained with Hoechst 33342
before analysis by fluorescence microscopy.
The induction of apoptosis was further confirmed by the observation of its characteristic
morphological changes. Hoechst 33342 staining showed volume reduction, chromatin condensation
and apoptotic bodies in Jurkat cells treated in the same conditions for 72 h (Figure 2B). In contrast,
untreated cells presented a normal morphological profile.
Taken together, the results described above suggest that compound 17 inhibits cell growth through
cell cycle arrest at the S phase and induction of apoptosis. Its mechanism of action needs to be
studied further.
3. Materials and Methods
3.1. Chemistry
Glycyrrhetinic acid and all reagents were purchased from Sigma-Aldrich Co. (Saint 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

Molecules 2019, 24, 2938
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purchased from VWR Portugal. 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). Melting points were determined using a BUCHI
1
melting point B-540 apparatus and were uncorrected. H and 13C NMR spectra were recorded on
a Bruker Avance-400 Digital NMR spectrometer, in CDCl3, with Me4Si as the internal standard (see
Supplementary Materials). Chemical shifts values (δ) are given in parts per million (ppm) and coupling
constants (J) are presented in hertz (Hz). Mass 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.
Methyl 3
β-hydroxy-11-oxo-olean-12-en-30-oate (2): Compound 2 was prepared according to the
literature [39], from 1 to give a colorless solid (90%). m.p.: 254–256 ◦C.
Methyl 3,11-dioxo-olean-12-en-30-oate (3): Preparation of
3 was performed according to a previously
described method [41], from 2 providing a white solid (94%). m.p.: 248–250 ◦C.
Methyl 3,11-dioxo-4-oxa-A-homo-olean-12-en-30-oate (4): Compound
literature [42], from 3 to give a white solid (77%). m.p.: 168–170 ◦C.
4 was prepared according to the
3,4-seco-30-methyloxicarbonyl-11-oxo-olean-4(23),12-dien-3-oic acid (5): Preparation of
5 was performed
according to a previously described method [42], from
4
providing a white solid (72%). m.p.: 90–92 ◦C.
Methyl 3,4-seco-3-N-methylglycinamido-11-oxo-olean-4(23),12-dien-30-oate (6): To a solution of compound
(300 mg, 0.60 mmol) in dichloromethane (8 mL), Deoxo-Fluor (50% in THF, 0.52 mL, 1.20 mmol) was
5
added and the reaction mixture was stirred, at room temperature, for 2.5 h, after which additional
Deoxo-Fluor (50% in THF, 0.26 mL, 0.60 mmol) was added. After 2 h, the reaction was completed.
The reaction mixture was quenched by addition of water (2 mL). The organic layer was diluted with
chloroform (40 mL), washed with water (2
×
30 mL), dried over Na2SO4, filtered and evaporated to the
dryness (280 mg, 93%). The residue was dissolved in dichloromethane (6 mL) and glycine methyl ester
hydrochloride (105 mg, 0.84 mmol) and triethylamine (0.15 mL, 1.12 mmol) were added. After 1 h
under magnetic stirring at room temperature, the reaction was completed. The reaction mixture was
evaporated to dryness and ethyl acetate (40 mL) and water (30 mL) were added to the residue. The
aqueous phase was further extracted with ethyl acetate (2
×
40 mL). The combined organic phase was
30 mL), water (30 mL) and
washed with 5% aqueous HCl (2 30 mL), 10% aqueo+us NaHCO3 (2
×
×
brine (30 mL), dried over Na2SO4, filtered and the solvent was removed under reduced pressure to
afford a white solid. The solid was purified by flash column chromatography (FCC) wi◦th petroleum
1
ether/ethyl acetate (2:1) to afford compound
6
as a white solid (69%). m.p.: 199–201 C. H NMR
(400 MHz, CDCl3):
3.93-4.05 (2H, m, NCH2), 3.74 (3H, s, COOCH3), 3.69 (3H, s, COOCH3), 1.76 (3H, s), 1.38 (3H, s), 1.16
(3H, s), 1.15 (6H, s), 0.81 (3H, s). 13C NMR (100 MHz, CDCl3):
200.3 (C11), 177.1, 173.7, 170.6, 170.3,
δ 6.14 (1H, m, NH), 5.69 (1H, s, H-12), 4.90 (1H, br s, H-23), 4.73 (1H, br s, H-23),
δ
146.7, 128.6 (C12), 114.5 (C23), 53.3, 52.4, 51.9, 51.3, 48.5, 45.2, 44.2, 43.8, 41.4, 41.3, 39.1, 37.9, 35.7, 32.0,
31.8, 31.5, 31.3, 28.7, 28.4, 26.7, 26.6, 24.0, 23.6, 23.5, 19.7, 18.8. ESI-MS m/z: 570.34 ([M + H] +, 100%).
Found C 70.78, H 9.39, N 2.41, calcd for C34H51NO6·0.25H2O: C 71.11, H 9.04, N 2.44%.
Methyl 3,4-seco-3-N-methyalaninamido-11-oxo-olean-4(23),12-dien-30-oate (7): The method followed
followed that described for compound 6. The resulting solid (274 mg, 0.55 mL; 91%) from the
first step was dissolved in dichloromethane (6 mL) and alanine methyl ester hydrochloride (116 mg,
0.83 mmol) and triethylamine (0.15 mL, 1.10 mmol) were added. After 1 h, the reaction was completed.
The workup was performed as described for compound
with petroleum ether/ethyl acetate (2:1) to afford compound
1H NMR (400 MHz, CDCl3):
6.09 (1H, m, NH), 5.70 (1H, s, H-12), 4.91 (1H, br s, H-23), 4.74 (1H, br
6
. The resulting solid was subjected to FCC
7
as a white solid (61%). m.p.: 193–195 ◦C.
δ

Molecules 2019, 24, 2938
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s, H-23), 4.55 (1H, m, -NCH(CH3)-), 3.73 (3H, s, COOCH3), 3.69 (3H, s, COOCH3), 1.77 (3H, s), 1.38
(3H, s), 1.37 (3H, m, -NCH(CH3)-), 1.17 (3H, s), 1.15 (3H, s), 1.15 (3H,s), 0.82 (3H, s). 13C NMR (100
MHz, CDCl3):
δ 200.3 (C11), 177.1, 173.8, 173.0, 170.2, 146.7, 128.6 (C12), 114.5 (C23), 53.3, 52.5, 51.9,
51.1, 48.5, 48.1, 45.2, 44.2, 43.9, 41.4, 39.1, 37.9, 35.8, 32.0, 31.9, 31.5, 31.3, 28.8, 28.4, 26.7, 26.6, 24.0, 23.7,
23.5, 19.7, 18.8, 18.5. ESI-MS m/z: 584.29 ([M + H] +, 100%). Found C 71.40, H 9.31, N 2.32, calcd for
C35H53NO6·0.25H2O: C 71.46, H 9.17, N 2.38 %.
3,11-Dioxo-olean-12-en-30-oic acid (8): Compound
8 was prepared according to the literature [41], from 1
to give a colorless solid (92%). m.p.: 308–310 ◦C.
3,11-Dioxo-4-oxa-A-homo-olean-12-en-30-oic acid (9): Compound
9 was prepared from 8, using the same
method as for the preparation of 4, with the obtention of a white solid. (75%). m.p.: 268–270 ◦C.
3,4-seco-3-methyloxicarbonyl-11-oxo-olean-4(23),12-dien-30-oic acid (10): Preparation of 10 was performed
according to a previously described method [42], from
130–132 ◦C.
9 providing a colorless solid (62%). m.p.:
3,4-seco-3-methyloxicarbonyl-olean-4(23),12-dien-30-oic acid (11): Preparation of 11 was done according to
a previously described method [45]. Compound 10 (900 mg, 1.80 mmol) was dissolved in dioxane (25
mL) and Zn powder (941 mg, 14.40 mmol) was added. Concentrated HCl (37%, 3.6 mL, 43.20 mmol)
was added dropwise for 30 min with stirring. After 4.5 h under magnetic stirring at room temperature,
the reaction was completed. The reaction mixture was filtered and the solvent was removed under
pressure. Diethyl ether (75 mL) and water (60 mL) were added to the residue. The aqueous phase was
further extracted with diethyl ether (2
×
70 mL). The combined organic extract was then washed with
50 mL), water (50 mL) and brine (50 mL),
5% aqueous HCl (2 50 mL), 10% aqueous NaHCO3 (2
×
×
dried over Na2SO4, filtered and evaporated to the dryness. The resulting solid was purified by (FCC)
with petroleum ether/ ethyl acetate (1:1) to afford compound 11 as a white solid (75%). m.p.: 138–140
◦C. 1H NMR (400 MHz, CDCl3):
δ
5.32 (1H, m, H-12), 4.87 (1H, br, s, H-23), 4.67 (1H, br, H-23), 3.65 (3H,
s, COOCH3), 1.75 (3H, s), 1.21 (3H, s), 1.16 (3H, s), 1.02 (3H, s), 0.94 (3H, s), 0.82 (3H, s). 13C NMR (100
MHz, CDCl3): 183.1 (C30), 174.8 (C3), 147.6, 144.3, 122.8 (C12), 113.7 (C23), 51.8, 50.6, 48.2, 44.2, 42.7,
δ
42.2, 39.7, 39.3, 38.4, 38.0, 34.1, 32.2, 31.5, 31.2, 28.8, 28.6, 28.3, 27.1, 26.2, 26.0, 24.7, 23.8, 23.6, 19.7, 17.0.
ESI-MS m/z: 118.11 (19%), 274.50 (32), 318.46 (75), 346.48 (11), 362.45 (15), 485.38 ([M + H] +, 100%).
Methyl 3,4-seco-30-fluorcarbonyl-11-oxo-olean-4(23),12-dien-3-oate (12): To a solution of compound 10
(500 mg, 1.00 mmol) in dichloromethane (10 mL), Deoxo-Fluor (50% in THF, 0.87 mL, 2.00 mmol)
was added. After 1.5 h under magnetic stirring at room temperature, the reaction was completed.
The reaction mixture was quenched by addition of water (3 mL). The organic layer was diluted with
chloroform (50 mL), washed with water (2
dryness. The resulting solid was subjected to (FCC) [petroleum ether/ethyl acetate from (6:1) to (4:1)]
to afford compound 12 as a white solid (75%). m.p.: 183–185 ◦C. 1H NMR (400 MHz, CDCl3):
5.71
(1H, s, H-12), 4.90 (1H, br s, H-23), 4.69 (1H, br s, H-23), 3.62 (3H, s, COOCH3), 1.75 (3H, s), 1.38 (3H, s),
1.30 (3H, s), 1.17 (3H, s), 1.16 (3H, s), 0.86 (3H, s). 13C NMR (100 MHz, CDCl3):
199.5 (C11), 174.5
×
40 mL), dried over Na2SO4, filtered and evaporated to the
δ
δ
(C3), 168.1, 166.3 (J = 374.6 Hz, C30), 146.7, 129.1 (C12), 114.4 (C23), 53.0, 51.7, 51.0, 48.1, 45.3, 44.4 (J =
42.0 Hz), 43.8, 40.8, 38.9, 37.6, 34.6, 32.1, 31.6, 30.9, 29.4, 28.5, 27.0, 26.6, 26.5, 23.9, 23.6, 23.5, 19.7, 18.8.
ESI-MS m/z: 501.16 ([M + H] +, 100%).
Methyl 3,4-seco-30-fluorcarbonyl-olean-4(23),12-dien-3-oate (13): The method followed that described for
compound 12 but using compound 11 (600 mg, 1.24 mmol) and Deoxo-Fluor (50% in THF, 1.08 mL,
2.48 mmol) in dichloromethane (12 mL) for 2 h. The resulting solid was purified by FCC [petroleum
ether /ethyl acetate from (10:1) to (4:1)] to afford compound 13 as a white solid (61%). m.p.: 174–176 ◦C.
1H NMR (400 MHz, CDCl3):
δ 5.32 (1H, m, H-12), 4.87 (1H, br s, H-23), 4.67 (1H, br s, H-23), 3.65 (3H, s,

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COOCH3), 1.75 (3H, s), 1.27 (3H, s), 1.15 (3H, s), 1.02 (3H, s), 0.94 (3H, s), 0.83 (3H, s). 13C NMR (100
MHz, CDCl3): 174.7 (C3), 167.0 (J = 375.0 Hz, C30), 147.5, 143.6, 123.4 (C12), 113.7 (C23), 51.7, 50.6,
δ
48.3, 44.6 (J = 41.2 Hz), 42.4, 42.2, 39.7, 39.3, 38.1, 38.0, 34.1, 32.2, 31.5, 31.1, 28.6, 28.1, 27.2, 27.0, 26.1,
26.0, 24.6, 23.8, 23.6, 19.7, 17.0. ESI-MS m/z: 439.51 (38%), 440.52 (14), 485.44 (27), 487.36 ([M + H] +,
100%).
Methyl 3,4-seco-30-N-methylglycinamido-11-oxo-olean-4(23),12-dien-3-oate (14): To a solution of compound
12 (300 mg, 0.60 mmol) and glycine methyl ester hydrochloride (226 mg, 1.80 mmol) in dichloromethane
(8 mL), triethylamine (0.33 mL, 2.40 mmol) was added. After 13 h under magnetic stirring at room
temperature, the reaction was completed. The reaction mixture was evaporated to dryness and ethyl
acetate (50 mL) and water (40 mL) were added to the residue. The aqueous phase was further extracted
with ethyl acetate (2
mL), 10% aqueous NaHCO3 (2
×
50 mL). The resulting organic phase was washed with 5% aqueous HCl (2
×
30
×
30 mL), water (30 mL) and brine (30 mL), dried over Na2SO4, filtered
and the solvent was removed under reduced pressure to afford a solid. The solid was subjected to
(FCC) [petroleum ether /ethyl acetate from (2:1) to (1:1)] to afford compound 14 as a white solid (77%).
m.p.:126–128 ◦C. 1H NMR (400 MHz, CDCl3):
δ
6.17 (1H, m, NH), 5.74 (1H, s, H-12), 4.88 (1H, br s,
H-23), 4.68 (1H, br s, H-23), 3.95-4.14 (2H, m, NCH2), 3.76 (3H, s, COOCH3), 3.61 (3H, s, COOCH3),
1.75 (3H, s), 1.39 (3H, s), 1.16 (6H, s), 1.15 (3H, s), 0.83 (3H, s). 13C NMR (100 MHz, CDCl3):
199.7
δ
(C11), 176.3, 174.5, 170.7, 169.5, 146.7, 128.6 (C12), 114.3 (C23), 52.9, 52.5, 51.7, 51.0, 47.9, 45.2, 43.8 (2),
42.1, 41.3, 38.9, 37.4, 34.6, 32.0, 31.6 (2), 29.5, 29.4, 28.6, 26.7, 26.5, 23.9, 23.6, 23.4, 19.6, 18.8. ESI-MS m/z:
570.32 ([M + H] +, 100%). Found C 70.83, H 9.27, N 2.49, calcd for C34H51NO6
0.25H2O: C 71.11, H
·
9.04, N 2.44%.
Methyl 3,4-seco-30-N-methylglycinamido-olean-4(23),12-dien-3-oate (15): The method followed that
described for compound 14 but using compound 13 (290 mg, 0.60 mmol), glycine methyl ester
hydrochloride (226 mg, 1.80 mmol) and triethylamine (0.33 mL, 2.40 mmol) in dichloromethane (8 mL).
The workup was performed after 16 h. The resulting solid was purified by FCC with petroleum ether/
ethyl acetate (3:2) to afford compound 15 as a white solid (43%). m.p.: 190–192 ◦C. 1H NMR (400 MHz,
CDCl3):
(2H, m, NCH2), 3.77 (3H, s, COOCH3), 3.65 (3H, s, COOCH3), 1.75 (3H, s), 1.17 (3H, s), 1.13 (3H, s), 1.01
(3H, s), 0.93 (3H, s), 0.79 (3H, s). 13C NMR (100 MHz, CDCl3):
177.0, 174.7, 171.0, 147.6, 144.4, 122.8
δ 6.18 (1H, m, NH), 5.34 (1H, m, H-12), 4.87 (1H, br s, H-23), 4.67 (1H, br s, H-23), 3.97-4.17
δ
(C12), 113.7 (C23), 52.5, 51.7, 50.6, 48.1, 44.1, 43.6, 42.2, 41.4, 39.7, 39.3, 38.0 (2),34.2, 32.2, 31.7, 31.5, 29.8,
28.6, 28.3, 27.0, 26.2, 25.9, 24.6, 23.9, 23.6, 19.7, 17.0. ESI-MS m/z: 274.37 (25%), 318.32 (27), 556.24 ([M +
H] +, 100%). Found C 73.18, H 9.83, N 2.55, calcd for C34H53NO5: C 73.47, H 9.61, N 2.52 %.
Methyl 3,4-seco-30-N-methylalaninamido-11-oxo-olean-4(23),12-dien-3-oate (16): To a solution of compound
12 (300 mg, 0.60 mmol) and alanine methyl ester hydrochloride (251 mg, 1.80 mmol) in dichloromethane
(8 mL), triethylamine (0.33 mL, 2.40 mmol) was added and the reaction mixture was stirred, at room
temperature, for 8 h, after which additional dichloromethane (1 mL) and triethylamine (0.17 mL, 1.20
mmol) were added. After 16 h, the reaction was completed. The workup was performed as described
for compound 14. The solid was subjected to FCC with petroleum ether/ethyl acetate (1:2) to afford
compound 16 as a white solid (83%). m.p.: 130–132 ◦C. 1H NMR (400 MHz, CDCl3):
δ 6.15 (1H, m,
NH), 5.77 (1H, s, H-12), 4.89 (1H, br s, H-23), 4.69 (1H, br s, H-23), 4.62 (1H, m, -NCH(CH3)-), 3.76 (3H,
s, COOCH3), 3.61(3H, s, COOCH3), 1.75 (3H, s), 1.39 (6H, m), 1.17 (3H, s), 1.16 (3H, s), 1.14 (3H, s), 0.82
(3H, s). 13C NMR (100 MHz, CDCl3):
δ 199.7 (C11), 175.4, 174.5, 173.7, 169.5, 146.7, 128.6 (C12), 114.3
(C23), 53.0, 52.7, 51.7, 51.0, 47.9 (2), 45.2, 43.8, 43.7, 42.1, 38.9, 37.5, 34.6, 32.0, 31.6, 31.5, 29.5, 29.4, 28.6,
26.7, 26.5, 24.0, 23.6, 23.4, 19.7, 18.8, 18.6. ESI-MS m/z: 570.36 (17%), 584.36 ([M + H] +, 100%). Found C
71.09, H 9.51, N 2.35, calcd for C35H53NO6·0.25H2O: C 71.46, H 9.17, N 2.38%.
Methyl 3,4-seco-30-N-methylalaninamido-olean-4(23),12-dien-3-oate (17): To a solution of compound 13
(290 mg, 0.60 mmol) and alanine methyl ester hydrochloride (251 mg, 1.80 mmol) in dichloromethane

Molecules 2019, 24, 2938
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(8 mL), triethylamine (0.33 mL, 2.40 mmol) was added and the reaction mixture was stirred, at room
temperature, for 10 h, after which additional dichloromethane (2 mL) and triethylamine (0.17 mL, 1.20
mmol) and alanine methyl ester hydrochloride (84 mg, 0.60 mmol) were added. After 24 h, the reaction
was completed. The work-up was performed as described for compound 15. The solid was purified
by FCC with petroleum ether/ ethyl acetate (3:2) to afford compound 17 as a white solid (48%). m.p.:
100–102 ◦C. 1H NMR (400 MHz, CDCl3):
δ 6.22 (1H, m, NH), 5.37 (1H, m, H-12), 4.87 (1H, br s, H-23),
4.67 (1H, br s, H-23), 4.63 (1H, m, -NCH(CH3)-), 3.76 (3H, s, COOCH3), 3.65 (3H, s, COOCH3), 1.75
(3H, s), 1.40 (3H, m, -NCH(CH3)-), 1.17 (3H, s), 1.11 (3H, s), 1.01 (3H, s), 0.94 (3H, s), 0.79 (3H, s). 13C
NMR (100 MHz, CDCl3):
δ 176.2, 174.7, 173.9, 147.6, 144.5, 122.7 (C12), 113.7 (C23), 52.6, 51.7, 50.6, 48.0,
47.9, 44.0, 43.6, 42.2, 39.7, 39.3, 38.1, 38.0, 34.2, 32.2, 31.6, 31.5, 29.8, 28.6, 28.3, 27.0, 26.2, 25.9, 24.6, 23.9,
23.6, 19.7, 18.7, 17.0. ESI-MS m/z: 570.33 ([M + H] +, 100%). Found C 73.04, H 9.94, N 2.45, calcd for
C35H55NO5·0.25H2O: C 73.20, H 9.74, N 2.44%.
Methyl 3,4-seco-3-N-glycinamido-11-oxo-olean-4(23),12-dien-30-oate (18): To a solution of compound
6
(120 mg, 0.21 mmol) in methanol (1 mL) and tetrahydrofuran (THF) (1.5 mL), KOH 4N (0.53 ml,
2.10 mmol) was added. After 10 min under magnetic stirring at room temperature, the reaction was
completed. The pH of the reaction mixture was neutralized with 10% aqueous HCl. Dichloromethane
(30 mL) and water (20 mL) were added to the mixture. The aqueous phase was further extracted with
dichloromethane (2
brine (30 mL), dried over Na2SO4, filtered and the solvent was removed under reduced pressure to
afford compound 18 as a white solid (98%). m.p.: 223–225 ◦C. 1H NMR (400 MHz, CDCl3):
6.76 (1H,
×
30 mL). The combined organic phase was washed with water (2
×
30 mL) and
δ
m, NH), 5.70 (1H, s, H-12), 4.93 (1H, br s, H-23), 4.78 (1H, br s, H-23), 3.88-4.25 (2H, m, NCH2), 3.70
(3H, s, COOCH3), 1.80 (3H, s), 1.36 (3H, s), 1.17 (3H, s), 1.15 (3H, s), 1.14 (3H, s), 0.81 (3H, s). 13C NMR
(100 MHz, CDCl3):
δ 202.2 (C11), 177.0, 174.5, 173.3, 172.3, 146.5, 127.7 (C12), 114.8 (C23), 53.3, 52.0,
50.8, 48.6, 45.4, 44.2, 44.1, 41.8, 41.4, 39.2, 37.8, 36.1, 32.0, 31.9, 31.5, 31.3, 28.8, 28.3, 26.7, 26.5, 23.9, 23.8,
23.4, 19.7, 18.7. ESI-MS m/z: 556.26 ([M + H] +, 100%).
Methyl 3,4-seco-3-N-alaninamido-11-oxo-olean-4(23),12-dien-30-oate (19): Compound 19 was prepared
using the same method as for the preparation of 18, but using compound
methanol (1 mL), THF (1.5 mL) and KOH 4N (0.50 mL, 2.00 mmol), at room temperature for 10 min, to
afford a white solid (94%). m.p.: 216–218 ◦C. 1H NMR (400 MHz, CDCl3):
6.89 (1H, m, NH), 5.69 (1H,
7 (116 mg, 0.20 mmol),
δ
s, H-12), 4.94 (1H, br s, H-23), 4.78 (1H, br s, H-23), 4.61 (1H, m, -NH(CH3)-), 3.69 (3H, s, COOCH3),
1.80 (3H, s), 1.42 (3H, m, -NH(CH3)-), 1.35 (3H, s), 1.16 (3H, s), 1.14 (6H, s), 0.81 (3H, s). 13C NMR (100
MHz, CDCl3):
δ 202.0 (C11), 176.8, 175.3, 174.0, 173.1, 146.3, 127.6 (C12), 114.6 (C23), 53.2, 51.9, 50.6,
48.5, 48.0, 45.3, 44.0 (2), 41.2, 39.0, 37.7, 36.1, 31.9, 31.8, 31.3, 31.1, 28.6, 28.1, 26.5, 26.4, 23.8 (2), 23.2, 19.5,
18.7, 18.6. ESI-MS m/z: 570.26 ([M + H] +, 100%).
3,4-seco-30-N-glycinamido-11-oxo-olean-4(23),12-dien-3-oic acid (20): The method followed that of
compound 18, using compound 14 (145 mg, 0.26 mmol), methanol (1 mL), THF (1.5 mL) and
KOH 4N (0.65 mL, 2.60 mmol), at room temperature for 10 min, to afford compound 20 as a white
solid (97%). m.p.: 213–215 ◦C. 1H NMR (400 MHz, CDCl3):
δ
7.58 (1H, m, NH), 5.77 (1H, s, H-12), 4.88
(1H, br s, H-23), 4.62 (1H, br s, H-23), 3.50-4.48 (2H, m, NCH2), 1.72 (3H, s), 1.42 (3H, s), 1.21 (3H, s),
1.16 (3H, s), 1.09 (3H, s), 0.83 (3H, s). 13C NMR (100 MHz, CDCl3):
202.1 (C11), 181.0, 177.3, 173.1,
δ
172.7, 146.5, 128.0 (C12), 114.5 (C23), 52.5, 50.6, 47.5, 45.7, 44.2, 44.0, 42.1, 41.1, 39.5, 37.5, 34.5, 31.8, 31.6
(2), 30.2, 29.1, 28.7, 26.8 (2), 23.7, 23.4, 22.9, 19.8, 18.8. ESI-MS m/z: 542.26 ([M + H] +, 100%).
3,4-seco-30-N-alaninamido-11-oxo-olean-4(23),12-dien-3-oic acid (21): Compound 21 was prepared using
the same method as for the preparation of 18, but using compound 16 (170 mg, 0.29 mmol), methanol
(1 mL), THF (1.5 mL) and KOH 4N (0.73 mL, 2.90 mmol), at room temperature for 10 min, to afford
a white solid (98%). m.p.: 224–226 ◦C. 1H NMR (400 MHz, CDCl3):
δ
7.37 (1H, m, NH), 5.74 (1H, s,
H-12), 4.88 (1H, br s, H-23), 4.78 (1H, m, -NH(CH3)-), 4.68 (1H, br s, H-23), 1.74 (3H, s), 1.15-1.35 (15H,

Molecules 2019, 24, 2938
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m), 0.82 (3H, s). 13C NMR (100 MHz, CDCl3):
δ
201.1 (C11), 179.8, 176.9, 176.3, 171.8, 146.6, 128.1 (C12),
114.5 (C23), 52.7, 50.7, 47.8, 47.7, 45.7, 44.0, 43.8, 41.9, 39.2, 37.6, 34.1, 31.9, 31.6 (2), 29.6, 29.1, 28.7, 26.8,
26.7, 23.8, 23.5, 23.2, 19.8, 18.8, 17.6. ESI-MS m/z: 272.01 (10%), 527.35 (12), 556.25 ([M + H] +, 100%).
3.2. Biology
A549, HT-29, Jurkat, MOLT-4, MIA Paca 2, MCF7, HeLa, A375, HepG2, and BJ cells were
obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Dulbecco´s
Modified Eagle 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) and Hoescht 33342 were purchased from Sigma Aldrich Co. (St Louis, 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%)
and MEM-Eagle Non-Essential Aminoacids 100 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).
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
A549, HT-29, MIA Paca 2, HeLa and A375 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 1mM sodium pyruvate. Jurkat and MOLT-4 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, 1mM
sodium pyruvate, 2 mM l-glutamine, 1 MEM-Eagle Non-Essential Aminoacids, 0.01 mg/mL insulin
×
human and 10 mM glucose.
All cell cultures were performed at 37 ◦C in an atmosphere of 5% CO2.
3.2.2. Antiproliferative Activity Assays
The antiproliferative activity of the synthesized compounds on A549, HT-29, MIA Paca 2, MCF7,
HeLa, A375, HepG2 and BJ adherent cells was determined by the MTT assay. Exponentially growing
cells were plated in 96-well plates at a density of 1–8
the plate, and the growth medium was replaced with fresh medium containing either the compounds
×
103 cells/ well. After 24 h, cells were attached to
dissolved in DMSO at different concentrations or only DMSO, in triplicate, and the cells were continued
to culture for 72 h. After incubation with the compounds, the medium was removed and 100
MTT solution (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
µL of
µ
immediately read at 550 nm on an ELISA read plater (Tecan Sunrise MR20-301, TECAN, Austria). For
Jurkat and MOLT-4 non-adherent cells, the antiproliferative activity was determined by XTT assay.
These cell lines were plated with 5.5
medium. The seeding was executed simultaneously with the addition of the different concentrations of
×
103 and 1
×
104 cells/well, respectively, in 96-well plates in 100
µL
compounds or vehicle, in triplicate, and cells were allowed to incubate for 72 h. After that incubation
period, 100 µL of the XTT labelling mixture were 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).

Molecules 2019, 24, 2938
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3.2.3. Cell Cycle Analysis
Cell cycle was assessed by flow cytometry using a fluorescence-activated cell sorter (FACS). Jurkat
cells were plated in six-well plates at a density of 1.6
105 cells/well, simultaneously with the addition
×
of compound 17, 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 24, 48 and
72 h. After incubation, cells were collected and centrifuged. The supernatant was removed and
the pellet was resuspended in 1 mL of◦TBS containing 1 mg/mL PI, 10 mg/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.
3.2.4. Annexin V-FITC/PI Flow Cytometry Assay
Apoptosis was assessed by flow cytometry using a FACS. Jurkat cells were plated in six-well
plates at a density of 1.6
×
105 cells/well, simultaneously with the addition of compound 17, at specified
concentrations, or with only the vehicle, in a total volume of 2 mL of medium. The cells were allowed
to incubate for 24, 48 and 72 h. After incubation, cells were collected and centrifuged. The supernatant
was removed and the pellet was resuspended in 95
140 mM NaCl, 2.5 mM CaCl2). Annexin V-FITC conjugate (3
for 30 min, at room temperature, in darkness. After incubation, 0.8 mL of binding buffer were added.
Just before the FACS analysis, cells were stained with 20 L of 1 mg/mL PI solution. Three independent
µL of binding buffer (10 mM HEPES/NaOH, pH 7.4,
µL) was added and cells were incubated
µ
experiments were performed, with two replicates per experiment.
3.2.5. Hoechst 33342 Staining
The morphological changes were observed by fluorescence microscopy using Hoechst staining.
Jurkat cells were plated in six-well plates at a density of 1.6
addition of compound 17, at specified concentrations, or with only the vehicle, in a total volume of 2 mL
×
105 cells/well, simultaneously with the
of medium. The cells were incubated for 72 h. After incubation, cells were collected by centrifugation,
washed twice with PBS and stained with 500
µL of Hoechst 33342 solution (2
µg/ml in PBS), for 15
min, at room temperature, in darkness. Finally, cells were washed and resuspended in 10
µL PBS.
The samples were mounted on a slide and observed with a fluorescence microscope (DMRB, Leica
Microssystems, Wetzlar, Germany) with a 40,6-diamidine-20-phenylindole dihydrochloride (DAPI)
filter. Three independent experiments were conducted.
4. Conclusions
In summary, we synthesized a series of new GA derivatives via the opening of its ring A along
with the coupling of an amino acid. Antiproliferative activity assays in a panel of nine human cancer
cell lines showed that the most potent compound 17 was 5 to 17-fold more active than GA 1. The study
of selectivity revealed that this new derivative was up to 10 times more selective towards malignant
cells than its parental compound. Preliminary mechanism investigation indicated that compound 17
may act through arresting cell cycle progression at the S phase and inducing apoptosis. The enhanced
potency and the high selectivity of this new GA derivative warrant further biological evaluation.
Supplementary Materials: 1H-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 n
º 3269,
drugs2CAD, and the financial support by FEDER (European Regional Development Fund) through the

Molecules 2019, 24, 2938
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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 Fundaç
Ci ncia e Tecnologia (FCT) through Projecto Estrat gico: 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 Ag ncia de Gesti d’Ajuts Universitaris i de Recerca
ão para a
ê
é
è
ó
(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 Laboratory of Mass Spectrometry (LEM) of the Node UC integrated
in the National Mass Spectrometry Network (RNEM) of Portugal, for the MS analyses, 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 conflicts of interest.
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Sample Availability: Samples of all compounds are available from the authors.
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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/).