Synthesis and antileukemic activity of an ursolic acid derivative: A potential co-drug in combination with imatinib

Article abstract of DOI:10.1016/j.cbi.2021.109535

Imatinib, a specific Bcr-Abl tyrosine kinase inhibitor, is the most commonly used drug in the treatment of chronic myeloid leukemia. However, optimal response is not achieved in up to 33% of patients. Therefore, development of novel therapeutic strategies for chronic myeloid leukemia is critical. Betulinic (1) and ursolic (2) acids are natural pentacyclic triterpenes that exhibit antileukemic activities. In this study, we evaluated the effects of pharmacomodulations at the C-3 position of the triterpene moiety of betulinic and ursolic acids on their activity against K562 leukemia cells. Six new derivatives (1a-2c) were synthesized and evaluated for pro-apoptotic and anti-proliferative effects in mammalian and leukemic cells. 2c derivative containing an amine group at the C-3 position of ursolic acid was the most active against leukemia cells with an IC₅₀ value of 5.2 μM after 48 h of treatment. 2c did not exhibit cytotoxic effects against VERO and HepG2 cells and human lymphocytes, showing a good selectivity index for cancer over normal cells. Induced cell death by apoptosis via caspases 3 and 8, and also caused cell cycle arrest as evidenced by accumulation of cells in the G1 phase and decreased cell population in the G2 phase. Furthermore, co-treatment of 2c with imatinib, the chemotherapy drug most commonly used to treat leukemia, resulted in a synergistic effect. Our findings provide a strong rationale for further investigation of combination therapy using the 2c derivative and imatinib in pre-clinical studies.

Full text of DOI:10.1016/j.cbi.2021.109535

Chemico-Biological Interactions 344 (2021) 109535
Contents lists available at ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Synthesis and antileukemic activity of an ursolic acid derivative: A
potential co-drug in combination with imatinib
a
a
b
a
Elenilson F. da Silva , Artur S. de Vargas , Julia B. Willig , Cristiane B. de Oliveira ,
a
b
b
a,*
Aline R. Zimmer , Diogo A. Pilger , Andr ´e ia Buffon , Simone C.B. Gnoatto
a
Laboratory of Phytochemistry and Organic Synthesis, Graduate Program in Pharmaceutical Sciences, Federal University of Rio Grande do Sul, Av. Ipiranga, 2752, Porto
Alegre, RS, 90610-000, Brazil
b
Laboratory of Biochemical and Cytological Analysis, Graduate Program in Pharmaceutical Sciences, Federal University of Rio Grande do Sul, Av. Ipiranga, 2752, Porto
Alegre, RS, 90610-000, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Imatinib, a specific Bcr-Abl tyrosine kinase inhibitor, is the most commonly used drug in the treatment of chronic
myeloid leukemia. However, optimal response is not achieved in up to 33% of patients. Therefore, development
of novel therapeutic strategies for chronic myeloid leukemia is critical. Betulinic (1) and ursolic (2) acids are
natural pentacyclic triterpenes that exhibit antileukemic activities. In this study, we evaluated the effects of
pharmacomodulations at the C-3 position of the triterpene moiety of betulinic and ursolic acids on their activity
against K562 leukemia cells. Six new derivatives (1a-2c) were synthesized and evaluated for pro-apoptotic and
anti-proliferative effects in mammalian and leukemic cells. 2c derivative containing an amine group at the C-3
Triterpene
Betulinic acid
Ursolic acid
Leukemia
K562 cell
Antic ˆa ncer
position of ursolic acid was the most active against leukemia cells with an IC50 value of 5.2 M after 48 h of
μ
treatment. 2c did not exhibit cytotoxic effects against VERO and HepG2 cells and human lymphocytes, showing a
good selectivity index for cancer over normal cells. Induced cell death by apoptosis via caspases 3 and 8, and also
caused cell cycle arrest as evidenced by accumulation of cells in the G1 phase and decreased cell population in
the G2 phase. Furthermore, co-treatment of 2c with imatinib, the chemotherapy drug most commonly used to
treat leukemia, resulted in a synergistic effect. Our findings provide a strong rationale for further investigation of
combination therapy using the 2c derivative and imatinib in pre-clinical studies.
1
. Introduction
Chronic myeloid leukemia (CML) is a hematopoietic stem cell dis-
drugs, to minimize drug resistance in CML is needed.
Semisynthetic molecules derived from natural products have been
important in cancer research and therapeutics [8–10]. Betulinic (1) and
ursolic (2) acids (Fig. 1) are aglycones belonging to the pentacyclic
triterpene class of molecules, which exert a variety of known therapeutic
activities [11–13]. These molecules exhibit pronounced anti-
proliferative activity against several tumor cell lines such as neuro-
blastoma, melanoma, glioma, prostate cancer, lung cancer, and
leukemia [14–17]. Recent studies showed that betulinic and ursolic
acids act via multiple mechanisms against tumor cells such as induction
of apoptosis via caspases 3, 8 and 9, halting of the cell cycle, autophagic
death, and others [18–20]. Moreover, investigations have shown that
modifications at positions C-3 and C-28 of triterpenes can result in
greater activity and selectivity toward cancer cells than the corre-
sponding precursor molecules [21–23]. In this study, we modified the
order associated with abnormal formation of the Philadelphia chromo-
some (Ph), which results from a translocation between chromosomes 9
and 2 [1,2]. CML is most prevalent in older patients, but can affect in-
dividuals of all ages [3]. In 2020, approximately about 1130 people will
die of CML (670 men and 460 women) in the United States of America
[
4].
Imatinib, a specific Bcr-Abl tyrosine kinase inhibitor, is considered
the most successful antineoplastic agent and remains the drug of choice
for the treatment of CML [5]. Although discovery of imatinib has been a
medical breakthrough, studies have shown that imatinib is subject to a
significant drug resistance response [6,7]. So, development of new
therapeutic agents that are effective alone, or in combination with other
*
Corresponding author. Graduate Program in Pharmaceutical Sciences, Federal University of Rio Grande do Sul (UFRGS), Av. Ipiranga, 2752, Porto Alegre, RS,
9
0610-000, Brazil.
E-mail address: simone.gnoatto@ufrgs.br (S.C.B. Gnoatto).
https://doi.org/10.1016/j.cbi.2021.109535
Received 22 March 2021; Received in revised form 14 May 2021; Accepted 24 May 2021
Available online 26 May 2021
0
009-2797/© 2021 Elsevier B.V. All rights reserved.

E.F. da Silva et al.
Chemico-Biological Interactions 344 (2021) 109535
C-3 position of ursolic and betulinic acids to evaluate their cytotoxic
activity against K562 CML cells. We further investigated the mechanism
of action of the most active derivative in cell death and cell cycle assays.
In addition, we evaluated possible synergistic effects in combination
with imatinib mesylate.
2.3.1. Syntheses of 3-oxo-lup-20(29)-en-28-oic acid (1a)
◦
To a solution of 1 (455 mg, 1.0 mmol) in acetone (6 mL) at 0 C was
added Jones reagent (1.5 mL). The solution was stirred for an hour at
◦
room temperature, then was cooled to 0 C and added (10 mL) of iso-
propanol and kept under stirring at room temperature for 30 min. Then,
the solution was filtered, and the filtrate was dried under reduced
2
. Methods and Materials
.1. General
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
pressure. The product was purified on silica gel column. A white powder
1
was obtained with 89% yield (1a). H NMR (400 MHz, CDCl
3
): δ 0.82 (s,
-25), 0.95 (s, 3H,
-11 and CH -12);
-6 and CH-9), 1.51–1.59 (m, 4H, CH -1 and CH
2), 1.60–1.67 (m, 2H, CH-13 and CH-5), 1.69 (s, 3H, CH -30), 1.79–1.88
(m, 4H, CH -15 and CH
-16), 1.91–2.02 (m, 1H, CH-18), 3.19–3.21 (m,
2H, CH -21 and CH -22), 4.78–4.85
-7), 3.32–3.39 (m, 5H, CH-19, CH
(m, 2H, CH
-29).13C NMR (125 MHz, CDCl
): δ 16.1, 16.5, 17.5, 20.2,
2
3H, CH
CH -23), 0.97 (s, 3H, CH
1.37–1.42 (m, 3H, CH
3
-26), 0.85 (s, 3H, CH
3 3
-27), 0.87 (s, 3H, CH
3
3
-24), 1.26–1.33 (m, 4H, CH
2
2
(
bromide)
2
2
2
-
tetrazolium (MTT), dimethyl sulfoxide (DMSO), Dulbecco’s Modified
Eagle’s Medium (DMEM), penicillin/streptomycin, and 0.5% trypsin/
EDTA solution were obtained from Sigma-Aldrich (Servylab, S a˜ o Leo-
poldo, RS, Brazil). Fetal bovine serum (FBS) was obtained from Gibco
3
2
2
2
2
2
2
3
(
Gibco BRL, Carlsbad, CA, USA). Annexin V-FITC/PI kit was obtained
21.4, 21.5, 21.8, 21.9, 24.1, 26.7, 29.6, 30.1, 30.9, 35.5, 36.2, 36.3,
38.5, 39.5, 39.9, 41.2, 41.8, 47.6, 48.5, 51.1, 55.5, 56.7, 109.1, 151.4,
from Quatro G Pesquisa e Desenvolvimento (Porto Alegre, RS, Brazil).
Propidium iodide (PI) was obtained from Sigma-Aldrich (St. Louis,
USA). Trizol and Reverse Transcriptase (MLV) were obtained from
Invitrogen (Carlsbad, California, USA). Real time PCR kit was obtained
from Promega (Madison, Wisconsin, USA). Analytical TLC was per-
formed on precoated Macherey-Nagel plates (Layer 0.20 mm, silica gel
179.8, 216.6. HRMS m/z calcd. for [M+H], C30
46 3
H O , 464.3447 found
464.3438.
2.3.2. Syntheses of 3-oxo-urs-12-en-28-oic acid (2a)
This compound was prepared form ursolic acid (2) as described for
6
0) using cyclohexane-dichloromethane (70:30 or 90:10), and com-
1a. It was obtained a white powder was obtained with 79% yield (2a).
1
pounds were visualized using sulfuric anisaldehyde. Column chroma-
H NMR (400 MHz, CDCl
3
): δ 0.87 (s, 4H, CH
3
-25 and CH-5), 0.89 (d, J
ˆ
= 6.4, 3H, CH -29), 0.91–0.98 (m, 3H, CH -11 and CH-20), 0.99 (d, J =
tography was performed using silica gel (technical grade, pore size 60 A,
3
2
7
0–230 mesh, 63–200
μm, Aldrich chemistry).
6.3, 3H, CH
CH
3
-30), 1.07 (s, 3H, CH
3 3
-26), 1.11 (s, 3H, CH -24), 1.13 (s, 6H,
3
-23 and CH
s, 1H, CH-9), 1.53–1.62 (m, 5H, CH
m, 2H, CH
-6), 1.76–2.11 (m, 4H, CH
6.6, CH-18), 5.13–5.38 (m, 1H, CH-12).13C NMR (125 MHz, CDCl
3
-27), 1.19–1.36 (m, 5H, CH
-1, CH
-16 and CH
2
-2, CH
-15 and CH
-22), 2.24 (1H, d, J
): δ
2
-7 and CH-19), 1.51
(
(
2
2
2
-21), 1.64–1.71
2
.2. Plant materials
2
2
2
=
3
Ursolic acid (1) was obtained from Malus domestica shells (apple),
1
3
5
5.6, 15.9, 17.3, 19.6, 19.9, 21.6, 21.8, 21.9, 23.8, 24.5, 26.9, 28.4,
1.0, 33.7, 34,1, 37.1, 39.4, 39.7, 39.8, 40.6, 42.7, 47.8, 48.4, 48.6,
2.9, 55.6, 125.9, 138.5, 181.1, 217.7. HRMS m/z calcd. for [M+H],
and its corresponding isomer betulinic acid (2) was obtained from the
bark of Platanus acerifolia L. (Maple) according to previously described
procedures [24].
30 46 3
C H O , 464.3447 found 464.3438.
Structural characterization of ursolic and betulinic acids and purity
analyses were performed using thin layer chromatography (TLC), nu-
clear magnetic resonance (NMR), and mass spectrometry (MS).
2
.3.3. Syntheses of (E)-3-oxime-lup-20(29)-en-28-oic acid (1b)
To a solution of (1a) (150 mg, 0.319 mmol) in ethanol and pyridine
2
2
:1 was added, under N atmosphere, hydroxylamine hydrochloride (70
mg, 1,0 mmol) previously dissolved in ethanol. The reaction mixture
was kept on stirring at room temperature for 72 h. Subsequently, it was
2
.3. Chemistry
2
poured into cold H O and the precipitate formed was filtered, dried and
All of the solvents were dried and distilled prior to use. Column
stored. The product was purified on silica gel column chromatography.
chromatography (CC) was carried out on silica gel using gradient eluent
mixtures (cyclohexane/dichloromethane). 1H and 13C NMR spectra
1
A white powder was obtained with 87% yield (1b). H NMR (400 MHz,
CDCl
5), 0.93 (s, 3H, CH
CH
-12), 1.34–1.41 (m, 3H, CH
and CH
-2), 1.61–1.72 (m, 2H, CH-13 and CH-5), 1.79 (s, 3H, CH
.81–1.89 (m, 4H, CH -15 and CH -16), 1.93–2.01 (m, 1H, CH-18), 2.82
-7), 3.33–3.39 (m, 5H, CH-19, CH
-22), 4.48–4.59 (m, 2H, CH
-29).13C NMR (125 MHz,
): δ 15.9, 16.4, 16.9, 19.7, 21.3, 21.5, 21.8, 21.9, 24.2, 26.5, 29.5,
3
): δ 0.79 (s, 3H, CH
-23), 0.97 (s, 3H, CH
-6 and CH-9), 1.53–1.60 (m, 4H, CH
-30),
3
-26), 0.82 (s, 3H, CH
3
-27); 0.84 (s, 3H, CH
-11 and
-1
3
-
were recorded with a Varian Inova 500 NMR spectrometer. Chemical
shifts are shown in parts per million (δ) with tetramethylsilane (TMS) as
a reference. Data are reported as follows: chemical shifts, multiplicity (s
2
3
3
-24), 1.26 (d, 4H. CH
2
2
2
2
2
3
=
singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling
1
2
2
constants J (Hz), relative integration value. Mass spectra were recorded
using with an Ultrot of (Bruker Daltonics, SP, Brazil) mass spectrometer.
The synthetic compounds were obtained with yields between 62%
and 96% (Fig. 2), and their structures were elucidated using full spec-
troscopic data [25].
(
s, 1H, OH), 3.17–3.23 (m, 2H, CH
2
2
-
2
1 and CH
2
2
CDCl
3
3
0.7, 30.8, 35.8, 36.8, 36.9, 38.5, 39.5, 39.9, 41.2, 41.7, 47.6, 48.5,
Fig. 1. Structure of betulinic acid - lupane skeleton (1) and ursolic acid - ursano skeleton (2).
2

E.F. da Silva et al.
Chemico-Biological Interactions 344 (2021) 109535
◦
Fig. 2. Synthetic Route of 1a-2c compounds. I Jones reagent, 0 C, 1h, acetone. II ethanol, pyridine, hydroxylamine hydrochloride, N2 (g), 72 h, reflux. III. THF, N2
g), LiAlH4, 6 h.
(
1
5
1.7, 55.5, 58.7, 110.1, 149.9, 169.8, 178.8. HRMS m/z calcd for
It was obtained a white powder was obtained with 51% yield (2c). H
NMR (400 MHz, CDCl
): δ 0.74–0.78 (m, 1H, CH-5), 0.73 (s, 3H, CH
5), 0.83 (s, 3H, CH -29), 0.93 (s, 3H,
-26), 0.92 (d, J = 6.4, 3H, CH
CH -30), 1.06 (m, 5H, CH -23 and CH
-24), 1.02 (d, J = 6.0, 3H, CH
11), 1.08–1.12 (m, 1H, CH-20), 1.14 (s, 3H, CH -27), 1.21–1.29 (m, 1H,
CH-19), 1.32–1.41 (m, 3H, CH -16, CH-7), 1.46–1.52 (m, 3H, CH-9,
CH -1, CH -15), 1.72–1.81 (m, 2H, CH
-21), 1.60–1.71 (m, 4H, CH
6), 1,84 (s, 1H, CH-3), 1.98–2.13 (m, 2H, CH -22), 2.15 (d, J = 6.9, 1H,
CH-18), 2.17–2.27 (m, 2H, CH -2), 5.48–5.56 (m, 1H, H-12). C NMR
(125 MHz, CDCl
[
M+H], C30 47NO , 479.3556 found 479.3553.
H
3
3
3
-
2
3
3
2
.3.4. Syntheses of (E)-3-oximeurs-urs-12-en-28-oic acid (2b)
3
3
3
2
-
This compound was prepared 3-oxo-urs-12-en-28-oic acid 2a as
3
described for 1b. It was obtained a white powder was obtained with 87%
2
1
yield (1b). H NMR (400 MHz, CDCl
3
): δ 0.74–0.78 (m, 1H, CH-5), 0.81
-26), 0.92 (d, J = 6.4, 3H, CH -29), 0.94
-24), 1.02 (d, J = 6.0, 3H, CH -30), 1.06–1.12 (m, 5H, CH -23
-27), 1.27–1.32
-7 and CH-9), 1.60–1.68
-21), 1.72–1.87 (m, 6H, CH -6, CH -16 and
-22), 2.07 (d, J = 6.2, 1H, CH-18), 3.42 (s, 1H, OH), 5.15–5.31 (m,
2
2
2
2
-
(
s, 3H, CH
s, 3H, CH
3
-25), 0.83 (s, 3H, CH
3
3
2
1
3
(
3
3
3
2
and CH
2
-11), 1.15–1.22 (m, 1H, CH-20), 1.25 (s, 3H, CH
m, 1H, CH-19), 1.46–1.52 (m, 5H, CH -2, CH
m, 6H, CH -1, CH -15, CH
3
3
): δ 14.3, 15.3, 17.1, 17.5, 19.4, 21.5, 23.4, 23.6, 23.8,
(
(
2
2
24.5, 27.0, 27.9, 31.0, 32.9, 34.2, 37.4, 38.7, 39.6, 39.7, 40.5, 41.5,
2
2
2
2
2
42.2, 47.8, 48.1, 53.1, 55.5, 69.6, 125.8, 138.5, 181.4. HRMS m/z calcd
CH
2
for [M+H], C30
2
H49NO , 465.3763 found 465.3751.
1
3
1
2
3
1
4
H, CH-12). C NMR (125 MHz, CDCl
3
): δ 14.3, 15.3, 17.1, 17.5, 19.4,
1.5, 23.4, 23.6, 23.8, 24.5, 27.0, 27.9, 31.0, 32.9, 34.2, 37.4, 38.7,
9.6, 39.7, 40.5, 41.5, 42.2, 47.8, 48.1, 53.1, 55.5, 125.8, 138.5, 168.0,
2
2
.4. Antileukemic activity and cytotoxic effect
81.4. HRMS m/z calcd for [M+H], C30
3
H47NO , 479.3556 found
.4.1. Maintenance of cell lines
79.3533.
K562 cells (human chronic myelogenous leukemia) were obtained
from Banco de C ´e lulas do Rio de Janeiro (BCRJ, Rio de Janeiro, RJ,
2
.3.5. Syntheses of 3-amino-lup-20(29)-en-28-oic acid (1c)
Brasil). Cells were cultured in RPMI-1640 medium supplemented with
To a solution of (1b), (200 mg, 0.4 mmol) in distilled THF (10 mL) at
◦
1
0% FBS and 0.5 U/mL penicillin/streptomycin at 37 C in a 5% CO2
◦
0
C under N2 atmosphere, LiAlH4 (351 mg, 9.24 mmol) was added. The
humidified atmosphere.
mixture was maintained at reflux for 6 h. Then, the mixture was treated
with aqueous 1 N NaOH (2 mL) and stirred for another hour. Finally, the
solution was extracted with diethylether (3 × 10 mL) and the organic
phase was separated and the product was purified on silica gel column
The cytotoxicity against lymphocytes from healthy donors was
evaluated to determine general blood toxicity. Human lymphocytes
were isolated from venous blood of volunteers by gradient centrifuga-
tion using Histopaque®. Cells were cultured in RPMI-1640 medium
1
chromatography. A white powder was obtained with 63% yield (1c). H
supplemented with 10% FBS and 0.5 U/mL penicillin/streptomycin at
NMR (400 MHz, CDCl
3
): δ 0.81 (s, 3H, CH
-25), 0.93 (s, 3H, CH -23), 0.97 (s, 3H, CH
-11 and CH -6 and CH-9),
-12), 1.39–1.48 (m, 3H, CH
.53–1.60. (m, 4H, CH -2), 1.63–1.72 (m, 2H, CH-13 and
-30), 1.99–2.01 (m, 4H, CH -15 and CH -16),
-7), 3.33–3.43 (m,
-22), 4,68–4.82 (m, 2H, CH
-29).13C NMR
): δ 15.9, 16.4, 16.9, 19.7, 21.3, 21.5, 21.8, 21.9, 24.2,
6.5, 29.5, 30.1, 30.8, 35.8, 36.5, 36.7, 38.5, 39.2, 39.8, 41.2, 41.7,
7.6, 48.5, 51.7, 55.5, 58.7, 68.4, 111.1, 150.9, 179.4. HRMS m/z calcd
49NO , 465.3763 found 465.3761.
3
-26), 0.83 (s, 3H, CH
3
-27),
◦
3
7
C, in a 5 %CO2 humidified atmosphere. All procedures were
0
4
1
.89 (s, 3H, CH
3
3
3
-24), 1,26 (d,
approved for the Federal University of Rio Grande do Sul Ethical Com-
mittee under authorization number 666.655/2014.
H, CH
2
2
2
2
-1 and CH
2
VERO (African green monkey kidney cells) and HepG2 cells (liver
hepatocellular carcinoma) cell lines were obtained from American Tis-
sue Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured
in DMEM supplemented with 10% FBS and 0.5 U/mL penicillin/strep-
CH-5), 1.79 (s, 3H, CH
3
2
2
2
.17–2.23 (m, 1H, CH–18), 2.87–3.03 (m, 2H, CH
H, CH-19, CH -21 and CH
2
5
2
2
2
(
125 MHz, CDCl
3
◦
tomycin at 37 C in a 5% CO2 humidified atmosphere.
2
4
2
.4.2. Treatments
for [M+H], C30
H
2
All compounds were dissolved in culture medium containing 0.5% of
dimethyl sulfoxide (DMSO). After reaching sub-confluence (70–80%
2
.3.6. Syntheses of (E)-3-amino-urs-12-en-28-oic acid (2c)
confluence), the cells were exposed to compounds for 24 h or 48 h at
This compound was prepared from 2b as described for 1c.
concentrations of 10–100
μM. Cells treated with 0.5% DMSO in culture
3

E.F. da Silva et al.
Chemico-Biological Interactions 344 (2021) 109535
medium were used as a negative control. Experiments were performed
in triplicate. Results were expressed as percentage relative to control and
IC50 values (concentration of compound causing 50% reduction in cell
viability) were calculated using nonlinear regression analysis with
GraphPad Prism software. Selectivity index (SI) was determinate by
comparison of the IC50 values for the tumor cell line and the non-tumor
cell lines.
2.7. Cell cycle distribution analysis
Cell cycle analysis was performed using K562 cells treated with 2c
for 48 h at concentrations of 7.5 and 12.5 M. Treated cells were labeled
μ
with a solution containing 6 mM PI, Triton X-100, and RNAse. DNA
content was analyzed by flow cytometry.
2
.8. Evaluation of synergistic effects of imatinib and 2c co-treatment
2
.4.3. Flow cytometric assay
Inhibition of cell viability was evaluated in non-adherent K562 cell
Synergistic effects of 2c and imatinib co-treatment were evaluated in
line and human lymphocytes flow cytometric assay. After 24 h of cul-
ture, cells were treated with compounds 1-2c (10–100 M) for 24 h and
c (5–50 M) and Imatinib for 48 h. Four independent experiments were
performed in triplicate.
K562 cell line by isobologram analysis as previously described [26]. The
concentrations of imatinib used were 0.1–0.6 M. Compound 2c was
added at 15% (0.8 M), 25% (1.3 M), 50% (2.5 M) and 75% (3.9 M)
M).
μ
μ
2
μ
μ
μ
μ
μ
of IC50 value (IC50 = 5.2
μ
Cell counting was performed using a FACSVerse™ flow cytometer
equipped with a 488 nm blue laser and flow sensor (BD Biosciences, San
Jose, CA, EUA) (BD FacSuiteTM Software).
The interaction between the two compounds was calculated based on
the following equation:
Interaction index = 2 cc/2ce + Imbc/Imbe, Where 2 cc and Imbc are
the concentrations of 2c and imatinib, respectively, required for 50%
inhibition of cell viability when used alone. 2ce and Imbe represent the
concentrations of 2c and imatinib, respectively, which reduce 50% of
cell viability when used in combination.
2
.4.4. MTT assay
Inhibition of cell viability was evaluated in adherent VERO and
HepG2 cell lines using the MTT assay. After 24 h of culture, cells were
treated with compounds 1-2c (10–100
μ
M) for 24 h and 2c (5–100
μ
M)
According to this method, an interaction index of less than 1.0 in-
dicates synergistic effects between two drugs. An index higher than 1.0
indicates antagonism and an index of 1.0 indicates an additive effect.
The results were presented using the software R statistic and R studio.
for 48 h.
2
.4.5. Trypan blue assay
Compound 2c was evaluated using trypan blue on K562 cell line to
determine the number of viable cells. K652 cells were treated with
compound 2c (10–100 M) for 48 h. After, cells were treated with trypan
blue and counted using a hemocytometer.
2.9. Statistical analysis
μ
Data are presented as mean ± standard deviation of at least three
individual experiments. Statistical analyses were performed by one-way
ANOVA, followed by Tukey’s test for multiple comparisons, using
GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA). P < 0.05
was considered statistically significant.
2
.5. Annexin V/PI assay
Compound 2c-induced apoptosis in K562 cells was evaluated after
4
8 h of treatment at 25, 50 and 100 M. After treatment, K562 cells were
μ
double stained with FITC-conjugated annexin-V and PI. Cells were
analyzed using a FACSVerse™ flow cytometer (BD Biosciences) equip-
ped with FACSuiteTM software. Triton X-100 (TX100) was used as a
necrosis positive control and imatinib mesylate was used as an apoptosis
positive control.
3. Results
3.1. Synthesis of compounds
Betulinic (1) and ursolic (2) acids were extracted from natural
sources at yields of 2% and 3%, respectively. Six new derivatives, 1a-2c,
with modifications at the C-3 position were synthesized (Fig. 2).
Initially, the ketone derivatives 1a and 2a were obtained by Jones
oxidation reaction with yields of 89 and 79%. Derivatives 1b and 2b
were obtained from 1a and 2a using hydroxylamine hydrochloride to
form oximes, with yields of approximately 87 and 77%. Finally, de-
rivatives 1c and 2c were synthesized from 1b and 2b by reduction of the
oxime moieties with lithium aluminum hydride, with yields of 63% and
51%, respectively.
2
.6. Caspase-3, -9, and -8 activity
Total RNA was isolated from cells using Trizol reagent (Life Tech-
nologies, Carlsbad, CA, USA) according to manufacturer’s instructions.
RNA concentration was determined using a Nano-Drop® spectropho-
tometer (Thermo Scientific, Wilmington, DE, USA). Five micrograms of
total RNA was used to produce cDNA using M-MLV reverse transcriptase
with a total reaction volume of 20 L. The real-time polymerase chain
μ
reaction was performed using GoTaq® qPCR Master Mix (Promega)
following manufacturer’s instructions, utilizing Rotor-Gene Q (Qiagen).
3.2. Triterpene derivatives 2c reduce K562 cell viability
◦
Reaction conditions were as follows: 95 C for 1 min, 40 cycles of 10 s at
◦
◦
◦
9
5
C, 15 s at 60 C, and 20 s at 72 C. Relative quantification was
All derivatives were evaluated for antitumor activity against K562
leukemia cells after 24 h of treatment. Cytotoxic effects on lymphocytes,
HepG2, and VERO cells were also evaluated. Results are expressed as
performed by the ΔΔCt method using glyceraldehyde-3-phosphate de-
hydrogenase (GAPDH) as an endogenous control. Primer sequences
were as follows:
IC50 (
tains at C-3 a hydrogen-bond donor amine group was the most active
derivative, with an IC50 of 14.9 M. Derivatives 1a and 2a, which
contained ketone groups (hydrogen-bond acceptors), were considered
inactive, with IC50 values of 74.3 and 90.2 M, respectivelly. The de-
rivatives containing oxime functional groups, 1b and 2b, had IC50
values of 55.2 and 21.9 M, respectively. In general, ursano derivatives
μM) for each derivative (Table 1). The 2c derivative, which con-
′
′
Caspase-3 Sense 5 - ACA TGG CGT GTC ATA AAA TAC C -3 Anti-
′
sense 5 - CAC AAA GCG ACT GGA TGA AC -3’ - Fragment size: 120.
μ
′
′
′
Caspase-8 Sense 5 - CTG GGG ATG GCC ACT GTG -3 Antisense 5 -
TCG CCT CGA GGA CAT CGC TCT C -3’ - Fragment size: 113.
μ
′
′
′
Caspase-9 Sense 5 - GAG TCA GGC TCT TCC TTT G -3 Antisense 5 -
CCT CAA ACT CTC AAG AGC AC -3’ - Fragment size: 241.
μ
′
′
′
GAPDH - Sense 5 - CAA AGT TGT CAT GGA TGA CC-3 Antisense 5 -
had better activities than lupane derivatives. Interesting, after 24 h, the
2c compound was about 6.7 times more active against K562 tumor cells
than human normal lymphocytes, presenting an important SI.
CCA TGG AGA AGG CTG GGG-3’ - Fragment size: 195.
Time-dependent effects of 2c were evaluated over 48 h in K562,
HepG2, VERO and lymphocytes cell lines (Table 1). The activity of 2c for
4

E.F. da Silva et al.
Chemico-Biological Interactions 344 (2021) 109535
Table 1
Cell cycle analysis (Fig. 5) showed an accumulation of cells in the G1
phase and a decreased number of cells in the G2 phase after 48 h of
Activity in leukemia K562, VERO, lymphocytes and HepG2 cell treated with 1-
2
c compounds using MTT and Flow cytometric assays for 24 and 48 h.
treatment with 2c at 7.5 and 12.5 M. These results demonstrated that
μ
a
Compound
IC50 (
μ
M) 24h
derivative 2c perturbs the cell cycle, leading to decreased proliferation
of K562 cells. This hypothesis was confirmed by cell counting using
trypan blue (Fig. 3C), which showed a decrease of approximately 50% of
viable cells count after of treatment with compound 2c at concentrations
K562
VERO
Lymphocytes
HepG2
1
2
85.5 ± 1.5
33.4 ± 0.4
74.3 ± 2.1
55.2 ± 0.8
19.5 ± 0.4
90.2 ± 0.9
21.9 ± 0.6
14.9 ± 0.7
nd
99.0 ± 1.5
86.0 ± 0.4
89.0 ± 2.1
72.0 ± 0,8
>100 ± 0.4
90.2 ± 0.9
>100 ± 0.9
>100 ± 0.7
nd
78.1 ± 1.1
91.2 ± 2.5
78.4 ± 0.9
84.2 ± 0.7
>100 ± 1.8
98.2 ± 0.5
>100 ± 1.1
>100 ± 0.9
nd
>100 ± 1.2
87.2 ± 3.3
74.3 ± 2.1
>100 ± 1.1
99.4 ± 0.9
>100 ± 1.6
98.8 ± 2.1
97.9 ± 2.7
nd
b
b
of 10, 20, and 30
μ
M. However, cell death was not observed at these
1
1
1
2
2
2
a
b
c
a
b
c
concentrations. At 50 and 100
μM of 2c cell death was observed in K562
cells corroborating with the results found in the apoptosis assay.
b
b
3.4. Derivative 2c induces synergistic effects in combination with imatinib
Imatinib
a
IC50 ( M) 48h
μ
We evaluated the synergistic effects of co-treatment between deriv-
b
b
b
2
c
5.2 ± 0.7
0.8 ± 0.4
78.2 ± 0.7*
82.1 ± 0.7
66.6 ± 0.7
b
b
ative 2c and imatinib (Fig. 6). The IC50 of imatinib and 2c alone was 0.8
Imatinib
nd
96.5 ± 0.7
nd
μ
M and 5.2
resulting from co-treatment with both drugs was 0.57, 0.46, 0.27, and
0.0125 M when 2c was added at 15%, 25%, 50%, and 75% of the 2c
μ
M, respectively (Fig. 6A and Table 1). The IC50 of imatinib
b
nd = not determined.
Data represent mean ± SD.
a
μ
Drug concentration that inhibits cell growth in 50%. Results expresses as
IC50, respectively (Fig. 6B). The synergistic effect was confirmed from
the construction of an isobologram curve, which showed an interaction
index <1 for all associations between derivative 2c and imatinib
mean ± standard deviation (n = 3).
b
p < 0.01 cells compared to the vehicle-treated control.
(
Fig. 6C). The results of isobologram analysis showed that treatment
leukemia cells was increased after 48 h of treatment, with an IC50 of 5.2
with a combination of imatinib and 2c synergistically inhibited the
μ
μ
M. The IC50 for 2c in non-tumor cell lines assayed was greater than 60
growth of K562 cells.
M, representing a promising estimative of higher selectivity against
K562 tumor cells instead the normal cells (SI > 12). Furthermore, we
4
. Discussion
evaluated the cytotoxicity of imatinib in K562 cells and lymphocytes
over 48 h, which had an IC50 of 0.8
μ
M and 96.5 M, respectivelly.
μ
CML is caused by constitutively active Bcr-Abl tyrosine kinase [27]
Table 1: Activity in leukemia K562, VERO, lymphocytes and HepG2
cell treated with 1-2c compounds using MTT and Flow cytometric assays
for 24h and 2c/imatinib for 48h.
Imatinib, a competitive inhibitor at the tyrosine kinase ATP binding site,
was approved in 2002 for treatment of CML and is currently the standard
initial treatment for patients in the chronic phase of the disease [28].
However, approximately 20%–33% of CML patients do not respond to
imatinib or experience relapse after an initial positive response [29]. In
addition to drug resistance limiting treatment, imatinib causes severe
side effects such as cerebral and cutaneous edema, which can result in
death [30]. Studies have shown that skin reactions are dose-dependent,
suggesting that skin-related side effects are not only because of the hy-
persensitivity reaction. These datas has fueled many studies searching
for new forms of treatment for CML, or additional strategies that
augment existing treatments, resulting in better patient outcomes [31].
One alternative currently employed is to increase the daily dose of
imatinib for patients in the chronic phase. Despite better outcomes,
many side effects are observed when the concentration is doubled. As-
sociations with imatinib have been evaluated in clinical phase studies,
Compound 2c was selected for further study based on its promising
effect in K562 leukemic cells and due its low cytotoxicity against the
other normal cells tested.
3
.3. Induction of apoptosis (via caspases 3 and 8), G2/M arrest, and G1
arrest by 2c
Apoptosis and cell cycle arrest were investigated as possible mech-
anisms for the selective effects of 2c on K562 cell line. Apoptosis assay
results are shown in Fig. 4A and B. 2c induced apoptosis only at higher
concentrations (50–100
μ
M), as evidenced by activation of caspases 3
and 8 (Fig. 4C). No cell death occurred at 5, 10, 15, or 25
assay.
μM in this
Fig. 3. Cell count with trypan blue to K562 cells after 48 h treatment with 2c, the closed boxes represent the number of viable cells and the open boxes represent the
number of dead. ***p < 0.01 for treatments compared with the DMSO control of viable cells and or dead cells.
5

E.F. da Silva et al.
Chemico-Biological Interactions 344 (2021) 109535
Fig. 4. Apoptosis assay (flow cytometric analysis, 10.000 events) of K562 cell line after 48 h of treatment with compounds and by staining with annexin V–FITC and
PI. A - B) treatment with imatinib (2 M) and 2c (25, 50 or 100 M). C) Caspases (3, 9 and 8) activation investigation after 48 h of treatment with derivative 2c. ***p
0.01 and **p < 0.05 for treatments compared with the DMSO control.
μ
μ
<
Fig. 5. Effects of 2c on cell cycle distribution of K562 cells. The cells were harvested after 48 h of treatment with 2c at concentrations of 7.5 e 12.5
the means ± SE of at least three independent experiments in triplicate. **p < 0.05 for treatments compared with the DMSO control.
μM. All values are
6

E.F. da Silva et al.
Chemico-Biological Interactions 344 (2021) 109535
Fig. 6. Synergistic analysis obtained by association between 2c derivative and the imatinib drug. A) Effect of imatinib drug in the decrease of cell viability of K562
cell line. B) IC50 values from the combination between imatinib mesylate drug and 2c derivative. C) Analysis by isobologram, demonstrating the level of interaction
between the two compounds.
demonstrating that the combination of a tyrosine kinase inhibitor and
additional drugs may be a promising strategy [32,33].
imatinib used in the clinical treatment of leukemias, we believe that the
use of this drug in combination with the derivative 2c could be impor-
tant to increase the potential of this drug as well as to minimize the side
effects. In summary, we present the synthesis of a pro-apoptotic and
antiproliferative ursolic acid derivative, 2c. This derivative showed
potential as a co-drug in combination with imatinib, allowing for
possible reduction in imatinib dose.
In this study we demonstrated antitumor activity of ursolic acid,
betulinic acid, and a series of semisynthetic derivatives against K562
leukemic cell line. Drug rational design were based on literature review
showing that insertion of nitrogen-containing functional groups at the C-
3
and C-28 positions of triterpenes may potentiate antitumor activities
against several types of cancer in vitro [34].
Our results demonstrated that the ursano skeleton confers higher
activity against K562 cells compared with lupane skeleton. In addition,
modifications containing an amine functional group are important for
activity toward K562 cells. Compounds 1c, 2b, and 2c showed small
cytotoxicity against VERO, HepG2 and lymphocytes cells, which sug-
gested selectivity to the tumor cell and a low potential of nephro and
hepatotoxicity. In addition, treatment of K562 cells with 2c, the most
active derivative, was clearly time-dependent whith higher results in
Author statement
The supervision and design of synthesis was conceived by Simone C B
Gnoatto and apart from providing funding. Aline R Zimmer, Diogo A
Pilger and Andreia Buffon were responsable for the design of biological
experiements. Elenilson F da Silva and Artur S. de Vargas contributed to
2c and others derivatives synthesis and their NMR characterization;
Julia B Willig and Cristiane B de Oliveira contributed to biological
experiements in vitro.
4
8
8h. 2c induced cell death by apoptosis via activation of caspases 3 and
, and also exhibited antiproliferative activity, halting the K562 cell
cycle. These results are similar to other studies showed that compounds
and 1 induce apoptosis in K562 cells, demonstrating that semi-
Declaration of competing interest
2
synthetic derivatives have similar mechanism of action, but higher ac-
tivity. Therefore, rational drug design was successful.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Moreover, we tested the hypothesis that the semisynthetic derivative
2
c and imatinib would exhibit synergistic activity, potentially reducing
imatinib-related side effects by decreasing the required imatinib dose.
Our results showed a synergistic effect between 2c and imatinib,
allowing for reduction in the effective concentration of imatinib without
a decrease in pharmacological effects.
Acknowledgments
This work was supported by grants and financial support from Pro-
grama de P o´ s-graduaç ˜a o em Ci ˆe ncias Farmac ˆe uticas UFRGS, Conselho
Nacional de Desenvolvimento Cientifico e Tecnol o´ gico (CNPQ-Brazil-
Universal/CNPq 425970/2018–3, 310282/2020–9), and Coordenaç ˜a o
de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Brazil). The
Laborat o´ rio Regional de Nanotecnologia (LRNANO) of Universidade
Federal do Rio Grande do Sul UFRGS for the NMR analysis and Central
analítica de instrumentaç ˜a o da Universidade de S a˜ o Paulo USP for the
HRMS analysis.
5
. Conclusion
In this study, eight derivatives were obtained by a low cost and easily
applied method. The 2c derivative containing an amine group at the C-3
position of ursolic acid showed potential as a new leukemia treatment as
evidenced by selectively decreasing K562 cell viability compared to
VERO, human lymphocytes, and HepG2 cells. Furthermore, this com-
pound induced cell death through early and late apoptosis via activation
of caspases 3 and 8, and exerted antiproliferative activity by halting the
cell cycle at the G0/G1 phases. Finally, this derivative showed a syn-
ergistic effect when combined with imatinib, strongly reducing its
effective concentration.
References
[
[
1] M.D. Kendra Sweet, Chronic myeloid leukemia: a review of TKI therapy, Hematol.
Oncol. 14 (1) (2019) 6–13.
2] A.M. Mendizabal, N. Younes, H.P. Levine, Geographic and income variations in age
at diagnosis and incidence of chronic myeloid leukemia, Int. J. Hematol. 103
Considering the various side effects associated with the high doses of
(
2016) 70–78.
7

E.F. da Silva et al.
Chemico-Biological Interactions 344 (2021) 109535
[
3] D.A. Howell, F. Warburton, A. Ramirez, E. Roman, A.G. Smith, L.J.L. Forbes, Risk
factors and time to symptomatic presentation in leukaemia, lymphoma and
myeloma, Br. J.Cancer. 113 (2015) 1114–1120.
[19] A. Spivak, R. Khalitova, D. Nedopekina, L. Dzhemileva, M. Yunusbaeva,
V. Odinokov, et al., Synthesis and evaluation of anticancer activities of novel C-28
guanidine-functionalized triterpene acid derivatives, Molecules 11 (2018) 16–23.
[20] G. Li, L. Zhou, L. Liu, J. Chen, Z. Zhao, Y. Peng, et al., Ezrin dephosphorylation/
downregulation contributes to ursolic acid-mediated cell death in human leukemia
cells, Blood Canc. J. 12 (2013) 1–10.
[
[
4] American Cancer Society, Key Statistics for Chronic Myeloid Leukemia, 2020. https
:
//www.cancer.org/cancer/chronic-myeloid-leukemia/about/statistics.html.
5] Experts in Chronic Myeloid Leukemia, The price of drugs for chronic myeloid
leukemia (CML) is a reflection of the unsustainable prices of cancer Drugs : from
the perspective of a large group of CML experts, Blood 121 (2013) 4439–4443.
6] D. Matthias, B. Mariano, Targeting the MAPK pathway in KRAS-driven tumors,
Cencer cell 37 (2020) 543–550.
[21] J. Xu, X. Wang, H. Zhang, J. Yue, Y. Sun, X. Zhang, Synthesis of triterpenoid
derivatives and their anti-tumor and anti-hepatic fibrosis activities, Nat. Prod. Res.
16 (2018) 1–7.
[
[
[22] L. Potze, F.D. Mullauer, S. Colak, J.H. Kessle, J.P. Medema, Betulinic acid-induced
mitochondria-dependent cell death is counterbalanced by an autophagic salvage
response, Cell Death Dis. 5 (2014) 1–8.
7] H. Sundar, R. Jerald, Optimizing patient care in chronic phase chronic
myelogenous leukemia: a multidisciplinary approach, J. Natl. Compr. Canc. Netw.
1
–6 (2016).
[23] H. Weng, Z. Tan, Y. Hu, Y. Shu, R. Bao, L. Jiang, et al., Ursolic acid induces cell
cycle arrest and apoptosis of gallbladder carcinoma cells, Canc. Cell Int. 14 (2014)
1–10.
[
[
8] S. Genovese, F. Epifano, P. Medina, N. Caron, A. Rives, M. Poirot, et al., Natural
and semisynthetic oxyprenylated aromatic compounds as stimulators or inhibitors
of melanogenesis, Bioorg. Chem. 87 (2019) 181–190.
[24] A.N. Innocente, G.N.S. Silva, L.N. Cruz, M.S. Moraes, M. Nakabashi, P. Sonnet, et
al., Synthesis and antiplasmodial activity of betulinic acid and ursolic acid
analogues, Molecules 17 (2012) 12003–12014.
9] S. Abdelfatah, E. Fleischer, A. Klinger, V.K.W. Wong, T. Efferth, Identification of
inhibitors of the polo-box domain of polo-like kinase 1 from natural and
semisynthetic compounds, Invest. N. Drugs 38 (2020) 1–9.
[25] L. Dalla-vechia, A. Dassonville-klimpt, P. Grellier, P. Sonnet, G. Gosmann, S.C.
B. Gnoatto, The beckmann rearrangement applied to ursolic acid with antimalarial
activity in medicinal chemistry studies, Lett. Org. Chem. 9 (2012) 92–95.
[26] N. Sawada, K. Kataoka, K. Kondo, H. Arimochi, H. Fujino, Y. Takahashi, et al.,
Betulinic acid augments the inhibitory effects of vincristine on growth and lung
metastasis of B16F10 melanoma cells in mice, Br.J.Cancer. 90 (2004) 1672–1678.
[27] S. Soverini, M. Mancini, L. Bavaro, M. Cavo, et al., Chronic myeloid leukemia: the
paradigm of targeting oncogenic tyrosine kinase signaling and counteracting
resistance for successful cancer therapy, Mol. Canc. 17– 49 (2018).
[28] P.K. Bhamidipati, H. Kantarjian, J. Cortes, A.M. Cornelison, Management of
imatinib-resistant patients with chronic myeloid leukemia, Ther Adv Hematol 2
(2013) 103–117. https://doi:10.1177/2040620712468289.
[
10] F. Waechter, C.B. Oliveira, B.D. De Vieira, D.B.B. Trivella, A.R. Zimmer, D.
A. Pilger, et al., Design, synthesis and biological evaluation of betulinic acid
derivatives as new antitumor agents for leukemia, Anti Canc. Agents Med. Chem.
1
7 (2017) 1777–1785.
[
[
[
[
11] H.A. Ibrahim, M.R. Elgindi, R.R. Ibrahim, D.G. El-Hosari, Antibacterial activities of
triterpenoidal compounds isolated from Calothamnus quadrifidus leaves, BMC
Complement.Altern.Med. 9 (2019) 19–102.
12] G. Li, J. Wang, M. Jin, C. Jin, C. Ye, Y. Zhou, A new pentacyclic triterpenoid from
the leaves of Rhododendron dauricum L. with inhibition of NO production in LPS-
induced RAW 264.7 cells, Nat. Prod. Res. 27 (2019) 1–7.
13] G.N. Silva, D.C. Schuck, L.N. Cruz, M.S. Moraes, M. Nakabashi, G.G. Gosmann,
Investigation of antimalarial activity, cytotoxicity and action mechanism of
piperazine derivatives of betulinic acid, Trop. Med. Int. Health 1 (2015) 29–39.
14] J. Mączy n´ ska, A. Choroma n´ ska, J. Kutkowska, M. Kotulska, M. Zalewski,
J. Zalewski, Effect of electrochemotherapy with betulinic acid or cisplatin on
regulation of heat shock proteins in metastatic human carcinoma cells in vitro,
Oncol. Rep. 41 (2019) 3444–3454.
[29] A. Hochhaus, et al., European LeukemiaNet 2020 recommendations for treating
chronic myeloid leukemia, Leukemia 34 (2020) 966–984, https://doi.org/
10.1038/s41375-020-0776-2.
[30] I. Amitay-Laish, S.M. Stemmer, M.E. Lacouture, Adverse cutaneous reactions
secondary to tyrosine kinase inhibitors including imatinib mesylate, nilotinib, and
dasatinib, Dermatol. Ther. 24 (2011) 386–395.
[
[
[
15] T. Kowalczyk, P. Sitarek, E. Skała, M. Toma, M. Wielanek, D. Pytel, et al., Induction
of apoptosis by in vitro and in vivo plant extracts derived from Menyanthes
trifoliata L. in human cancer cells, Cytotechnology 71 (2019) 165–180.
16] K. Sułkowska-Ziaja, A. Szewczyk, A. Galanty, J. Gdula-Argasi n´ ska, B. Muszy n´ ska,
Chemical composition and biological activity of extracts from fruiting bodies and
mycelial cultures of Fomitopsis betulina, Mol. Biol. Rep. 45 (2018) 2535–2544.
17] J. Wiemann, L. Heller, V. Perl, R. Kluge, D. Str o¨ hl, R. Csuk, et al., Betulinic acid
derived hydroxamates and betulin derived carbamates are interesting scaffolds for
the synthesis of novel cytotoxic compounds, Eur. J. Med. Chem. 106 (2015)
[31] P.E. Westerweel, et al., New approaches and treatment combinations for the
management of chronic myeloid leukemia, Frontiers in oncology (2011) 9–665.
[32] A. Burchert, M.C. Müller, P. Kostrewa, Sustained molecular response with
interferon alfa maintenance after induction therapy with imatinib plus interferon
alfa in patients with chronic myeloid leukemia, J. Clin. Oncol. 28 (8) (2018)
1429–1435.
[33] S.I. Yamaguchi, A. Ueki, E. Sugihara, et al., Synergistic antiproliferative effect of
imatinib and adriamycin in platelet-derived growth factor, Canc. Sci. 106 (2015)
875–882.
1
94–210.
[34] A. Hordyjewska, A. Ostapiuk, A. Horecka, et al., Betulin and betulinic acid:
triterpenoids derivatives with a powerful biological potential, Phytochemistry Rev.
18 (2019) 929–951.
[
18] D.M. Zhang, H.G. Xu, Y.J. Li, P.H. Sun, X.M. Wu, G.J. Wang, et al., Betulinic acid
and its derivatives as potential antitumor agents, Med. Res. Rev. 6 (2015)
1
127–1155.
8