Design, synthesis and biological evaluation of pentacyclic triterpene derivatives: Optimization of anti-Abl kinase activity

Article abstract of DOI:10.3390/molecules24193535

Imatinib, an Abelson (ABL) tyrosine kinase inhibitor, is a lead molecular-targeted drug against chronic myelogenous leukemia (CML). To overcome its resistance and adverse effects, new inhibitors of ABL kinase are needed. Our previous study showed that the

Full text of DOI:10.3390/molecules24193535

molecules
Article
Design, Synthesis and Biological Evaluation of
Pentacyclic Triterpene Derivatives: Optimization
of Anti-ABL Kinase Activity
Halil I. Ciftci 1,2 , Mohamed O. Radwan 1,2,3 , Safiye E. Ozturk 4, N. Gokce Ulusoy 4, Ece Sozer 4,
Doha E. Ellakwa 2,5 , Zeynep Ocak 6, Mustafa Can 2,7, Taha F.S. Ali 2,8, Howaida I. Abd-Alla 3
,
Nurettin Yayli 9, Hiroshi Tateishi 2, Masami Otsuka 1,2,* and Mikako Fujita 2,
*
1
Department of Drug Discovery, Science Farm Ltd., 1-7-30-805 Kuhonji, Chuo-ku, Kumamoto 862-0976, Japan;
hiciftci@kumamoto-u.ac.jp (H.I.C.); mohamedradwan@kumamoto-u.ac.jp (M.O.R.)
Medicinal and Biological Chemistry Science Farm Joint Research Laboratory, Faculty of Life Sciences,
Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan;
profdoha@gmail.com (D.E.E.); mustafacan80@yahoo.com (M.C.); tahafaroukali@gmail.com (T.F.S.A.);
htateishi@kumamoto-u.ac.jp (H.T.)
Chemistry of Natural Compounds Department, Pharmaceutical and Drug Industries Research Division,
National Research Centre, Dokki 12622, Cairo, Egypt; howaida_nrc@yahoo.com
Chemistry Department, Faculty of Science, Ege University, Erzene Mahallesi, Genclik Caddesi,
Bornova/Izmir 35040, Turkey; sfymrt14@gmail.com (S.E.O.); ng.ulusoy@gmail.com (N.G.U.);
ecsozer95@gmail.com (E.S.)
Department of Biochemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Nasr City 11651, Cairo, Egypt
Department of Microbiology, Kocaeli State Hospital, Cedit Mahallesi Gunes Cad, Hastane Yolu Sk,
Kocaeli 41300, Turkey; zeynepocak@yahoo.com
Department of Engineering Sciences, Faculty of Engineering and Architecture, Izmir Katip Celebi University,
Havaalani Sosesi Caddesi No:25, Cigli/Izmir 35620, Turkey
2
3
4
5
6
7
8
9
Medicinal Chemistry Department, Faculty of Pharmacy, Minia University, Minia 61519, Egypt
Faculty of Pharmacy, Karadeniz Technical University, Trabzon 61080, Turkey; yayli@ktu.edu.tr
Correspondence: motsuka@gpo.kumamoto-u.ac.jp (M.O.); mfujita@kumamoto-u.ac.jp (M.F.);
Tel.: +81-96-371-4622 (M.F.)
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Nazim Sekeroglu, Anake Kijjoa and Sevgi Gezici
Received: 26 July 2019; Accepted: 26 September 2019; Published: 30 September 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Imatinib, an Abelson (ABL) tyrosine kinase inhibitor, is a lead molecular-targeted drug
against chronic myelogenous leukemia (CML). To overcome its resistance and adverse effects, new
inhibitors of ABL kinase are needed. Our previous study showed that the benzyl ester of gypsogenin
(1c), a pentacyclic triterpene, has anti-ABL kinase and a subsequent anti-CML activity. To optimize its
activities, benzyl esters of carefully selected triterpenes (PT1–PT6), from different classes comprising
oleanane, ursane and lupane, and new substituted benzyl esters of gypsogenin (GP1–GP5) were
synthesized. All of the synthesized compounds were purified and charachterized by different
spectroscopic methods. Cytotoxicity of the parent triterpenes and the synthesized compounds
against CML cell line K562 was examined; revealing three promising compounds PT5
(IC50 5.46, 4.78 and 3.19 M, respectively). These compounds were shown to inhibit extracellular
signal-regulated kinase (ERK) downstream signaling, and induce apoptosis in K562 cells. Among
them, PT5 was identified to have in vitro activity (IC50 = 1.44 M) against ABL1 kinase, about sixfold
, GP2 and GP5
µ
µ
of 1c, which was justified by molecular docking. The in vitro activities of GP2 and GP5 are less than
PT5, hence they were supposed to possess other more mechanisms of cytotoxicity. In general, our
design and derivatizations resulted in enhancing the activity against ABL1 kinase and CML cells.
Keywords: leukemia; chronic myelogenous leukemia; ABL kinase; pentacyclic triterpenes;
gypsogenin; apoptosis
Molecules 2019, 24, 3535; doi:10.3390/molecules24193535
www.mdpi.com/journal/molecules

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1. Introduction
Leukemia, a group of blood cancer which initiates in the bone marrow resulting in an abnormal
production of white blood cells, is the leading cause of cancer-related deaths in a wide population
range, from children to aged people. In 2016, the annual number of new cases of leukemia was
467,000 worldwide, with 310,000 leukemia deaths [1]. Based on disease progression and cell types,
leukemia is classified into four main types, acute lymphocytic leukemia (ALL), chronic lymphocytic
leukemia (CLL), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) [2].
CML is a slow-growing type of leukemia characterized by a chromosome translocation between
chromosome 9 and 22 to form the Philadelphia (Ph) chromosome [
the connected breakpoint cluster region (BCR) and the Abelson (ABL) oncogene, resulting in BCR–ABL
fusing gene with constitutive tyrosine kinase activity [ ]. The tyrosine kinase protein plays an
3–6]. The Ph chromosome contains
6–9
important role in the activation of signalling pathways (Janus kinase: JAK, signal transducers and
activators of transcription: STAT, Src family kinase: SFK, phosphoinositide 3-kinase: PI3K, protein
kinase B: AKT and extracellular signal-regulated kinase: ERK) and cellular processes such as growth,
differentiation, and metabolism [10–12]. The introduction of imatinib mesylate, a representative
molecular-targeted drug and the first tyrosine kinase inhibitor (TKI) with specific activity against
ABL, controls CML in chronic phase. However, a significant percentage of CML patients treated with
imatinib developed drug resistance and adverse side effects [13–15]. Second and third generation
of TKIs such as dasatinib, nilotinib and ponatinib have been developed to overcome these major
limitations, but patients treated with these TKIs still did not achieve desired CML treatment and some
patients were left with unexpected lung and vascular problems [16
for new therapeutic agents for CML patients.
–19]. Hence, there is an urgent need
In recent years, natural products and their derivatives have attracted great deal of interest as a
source for the development of new diverse bioactive compounds due to their high safety profile and
cost effectiveness [20–26]. Among them, pentacyclic triterpenes are the subjects of numerous studies
carried out by chemists engaged in organic synthesis and pharmacology. A large number of pentacyclic
triterpenes such as glycyrrhetic acid, oleanolic acid, ursolic acid, betulinic acid and their derivatives
have been reported to exhibit various biological activities including antiinflammatory, antimicrobial,
antiviral, antioxidant, antitumor and anticancer [27–32]. However, a pentacyclic triterpene named
gypsogenin has not yet received significant attention in drug discovery. Gypsogenin extracted from
Gypsophila with sugar chains is a unique pentacyclic triterpene possessing an aldehyde group at its
C4 distinguished from other pentacyclic triterpenes. In our previous studies [33,34] and [35], it was
demonstrated that gypsogenin and its derivatives exhibit remarkable activities against diverse human
cancer cell lines. Furthermore, we recently discovered that the benzyl ester of gypsogenin compound
1c has anti-ABL kinase and anti-CML activities [36]. To the best of our knowledge, this was the first
study to show the inhibitory activity of pentacyclic triterpenoids and their derivatives on ABL kinase.
In this study, we conduct biological evaluation of some pentacyclic triterpenes and their derivatives
including newly synthesized compounds to optimize anti-ABL kinase and anti-CML activities.
2. Results and Discussion
As representative pentacyclic triterpenes, we selected six compounds, asiatic acid (AA), betulinic
acid (BA), glycyrrhetinic acid (GA), hederagenin (HE), oleanolic acid (OA) and ursolic acid (UA
(Figure 1). AA and UA represent the ursane type, and BA represents the lupane type. GA HE and
OA are of the oleanane type. On these pentacyclic triterpenes and each benzyl ester like 1c PT1 PT6),
biological activities were examined. Furthermore, five new substituted benzyl esters of gypsogenin
GP1 GP5 were also designed. GP1 has a 4-methoxy substitution as an electron-donating group
)
,
(
–
–
whereas other compounds have electron-withdrawing substations such as fluoro-, trifluoromethyl-,
and cyano- groups.

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Figure 1. Structures of pentacyclic triterpenes and its derivatives used in this study.
The synthesis of benzyl esters of different triterpenes PT1
esters of gypsogenin GP1 GP5 was performed as outlined in Scheme 1. AA
UA were reacted with benzyl bromide in the presence of potassium carbonate as a base to afford
the corresponding esters PT1 PT2 PT3 PT4 PT5, and PT6 respectively in good yields (44–81%).
–PT6 and the new substituted benzyl
–
,
BA GA HE OA and
,
,
,
,
,
,
,
In a similar way, GP was reacted with different substituted benzyl bromides producing five new
compounds GP1, GP2, GP3, GP4, and GP5 in 40–48% yield.
To evaluate the antiproliferative effects of all compounds against CML cell line K562, MTT assay
was carried out. The results were summarized in Table 1. Imatinib was used as a control. IC50 values
of free triterpenes were detected to be higher than 10 µM. Introduction of benzyl ester group into
triterpene led to an increase in anticancer activity on K562 except for PT3 and PT6, based on GA and
UA, respectively. Specially, PT4 has more than 15 times stronger cytotoxicity than its parent triterpene
HE. The IC50 values of all new gypsogenin derivatives GP1–GP5 were lower than 6 µM. In order
to investigate whether PT1 PT6 and GP1 GP5 are toxic to healthy blood cells or not, the cytotoxic
–
–
effects of these compounds on peripheral blood mononuclear cells (PBMC) were also examined. Each
compound, except HE which lacks cytotoxicity against K562, showed a considerable selectivity index
(SI). Notably, free triterpenes GP and OA have IC50 of around 13
7. We identified GP2 GP5 and PT5 as the strongest and selective anticancer agents on CML cell line.
These three compounds were also more selective than imatinib.
µM against K562 and SI of more than
,

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Scheme 1. Synthesis of PT1–PT6 and GP1–GP5. PT: pentacyclic triterpene skeleton.
Table 1. The cytotoxic effects of all compounds on K562 and peripheral blood mononuclear cells
(PBMC).
IC50 Values (µM) a
SI b
ID
K562 Cell Line
12.67 ± 2.15
29.31 ± 3.42
52.27 ± 4.79
41.03 ± 5.34
>100
PBMC
>100
>100
>100
>100
GP
AA
BA
GA
HE
>7.89
>3.41
>1.91
>2.43
>1
Free Triterpenes
>100
OA
UA
1c
13.46 ± 2.55
16.51 ± 1.13
8.94 ± 1.73
4.10 ± 0.85
8.49 ± 1.22
69.50 ± 8.43
6.48 ± 1.27
5.46 ± 1.51
21.77 ± 1.24
5.53 ± 1.78
4.78 ± 1.25
4.58 ± 1.49
5.13 ± 1.38
3.19 ± 1.21
5.49 ± 1.81
>100
>100
>100
>7.43
>6.06
>11.19
3.79
3.29
>1.44
3.56
9.19
1.99
4.53
11.04
5.07
PT1
PT2
PT3
PT4
PT5
PT6
GP1
GP2
GP3
GP4
GP5
Imatinib c
15.54 ± 1.08
27.96 ± 2.74
>100
Triterpenes benzyl esters
23.08 ± 3.11
50.21 ± 4.58
43.31 ± 1.93
25.05 ± 2.92
52.76 ± 3.82
23.22 ± 3.08
28.28 ± 2.25
25.60 ± 2.18
28.66 ± 3.16
Gypsogenin substituted-
benzyl esters
5.51
8.03
5.22
Control
Cell lines include chronic myelogenous leukemia (K562) and peripheral blood mononuclear cells (PBMC). b The
selectivity index (SI) values are calculated as the ratio of the IC50 between PBMC and K562 cells. Used as
a
c
the reference.
The cytotoxic effects of these compounds were also evaluated on different leukemic cells
(HL-60, MT-2 and Jurkat), HeLa, MCF7 and A549 cell lines as shown in Table 2. Among them,
the cyano-substituted compound GP5 were found as the most effective on all cancer cell lines. GP2

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and PT5 were inactive against A549 cell line at 100
µM concentration. PT5 was less active compared to
GP2 and GP5 on HL-60, Jurkat, HeLa and MCF7 cell lines. PT5 exhibited selectivity against leukemia
cells like imatinib.
Due to their significant anticancer potentials on CML, GP2
for their apoptotic activities in K562 cells using Hoechst 33342/annexin V/ethidium homodimer III
staining method. K562 cells were treated with GP2 GP5 and PT5 at their IC50 concentrations for 8
h, then stained and observed by a florescence microscope (Figure 2a). The apoptotic effects of GP2
GP5 PT5 and imatinib on CML cell line were determined as 24%, 9.5%, 14% and 18%, respectively,
, GP5 and PT5 were further investigated
,
,
,
while their late apoptotic/necrotic effects were determined as 4.5%, 5.1%, 11% and 5.6% respectively
(Figure 2b). According to these findings, GP2 having 3,5-ditrifluoromethyl substitutions induced more
pronounced apoptosis of K562 cells than GP5, PT5 and imatinib.
(a)
(b)
Figure 2. Alteration in K562 cells at IC50 concentrations of control, GP2
8 h. ( ) Fluorescent microscopic images. ( ) A total of approximately 100 stained cells was selected
randomly in each experiment of ( ) and were classified into 4 types “alive” (blue), “apoptosis” (green),
“necrosis or late apoptosis” (both green and red, yellow) and “necrosis” (red).
, GP5, PT5 and imatinib for
a
b
a

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Table 2. The cytotoxicity of selected compounds against other cancer cell lines than K562.
IC50 Values (µM)
HL-60
MT-2
Jurkat
HeLa
MCF7
A549
GP2
GP5
PT5
5.27 ± 0.42
4.36 ± 0.28
15.71 ± 1.36
11.75 ± 1.08
36.82 ± 4.21
7.26 ± 0.97
34.70 ± 3.85
12.96 ± 1.33
9.14 ± 0.71
4.83 ± 0.35
13.85 ± 1.58
9.69 ± 0.63
35.28 ± 2.84
5.66 ± 0.32
67.05 ± 4.19
28.68 ± 1.92
51.58 ± 4.75
15.31 ± 2.11
53.91 ± 6.46
90.56 ± 5.81
>100
24.55 ± 1.94
>100
Imatinib
95.07 ± 8.33
As a consequence of the central role of ABL kinase in cellular processes including cell replication
and apoptosis, GP2 GP5 and PT5 were selected for further investigation of the potential inhibitory
activity against ABL kinase using multipoint dose-response experiments (Table 3). Among them, PT5
was found to be the most potent ABL kinase inhibitor with the IC50 value of 1.44 M, followed by
GP5 and GP2 with IC50 values of 6.16 M and 7.19 M, respectively. IC50 of PT5 is six times stronger
than 1c. Although it is still incomparable to imatinib (IC50 0.21 M), they showed similar cellular
activity (IC50 ~5.5 M) against K562 cells (Table 1) and similar selectivity against various cancer cell
,
µ
µ
µ
µ
µ
lines (Table 2). PT5 is considered to express more efficient cellular activity, but the detailed reason
remains elusive.
Table 3. The Abelson (ABL) 1 tyrosine kinase inhibition of selected compounds and imatinib.
IC50 Values (µM)
1c a
GP2
GP5
PT5
Imatinib
ABL1
8.71 ± 1.14
7.19 ± 0.65
6.16 ± 0.63
1.44 ± 0.12
0.21 ± 0.44
a
A value in the previous paper [36].
To investigate this outstanding activity of PT5, we conducted a molecular docking study into the
ATP-binding site of ABL kinase. Herein, we used the most updated version of Molecular Operating
Environment (MOE) software that explains the slightly shifted placement of 1c from our previous
work [36]. Both compounds demonstrated a similar and superimposed binding mode typically settling
three CH-π interaction with His361, Asp381 and Phe401. Their hydrophobic fused cyclohexyl rings
lie in proximity to hydrophobic residues Val289, Phe359 and Ile360, Ala380 and Phe382 (Figure 3).
We believe that the presence of the polar aldehyde group on C4 of compound 1c (binding energy -7.11)
in proximity to the hydrophobic residues Ala399 and Phe401 is unfavorable compared to the lower
methyl group of PT5 which is at the same position (binding energy -7.31). Finally, the rigid skeleton of
PT5 limits its deep burial into the pocket as fulfilled by imatinib.
In order to explore the inhibitory potency of GP2, GP5, and PT5 on downstream signaling
of BCR-ABL kinase such as rapidly accelerated fibrosarcoma (Raf)/mitogen-activated protein
kinase/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK), ERK1/2
and their phosphorylated forms in K562-treated with GP2, GP5, or PT5 were examined at 10 µM
concentration for 6 h (Figure 4). In the presence of the titled compounds, amount of ERK1/2 moderately
decreased. In contrast, the reduction of phosphorylated ERK1/2 was prominent where the three
compounds exhibited inhibitory effect of ERK phosphorylation in a similar pattern to imatinib.
The inhibitory activity against ERK phoshorylation is not in accordance with cytotoxicity data in K562
cells (Table 1). This suggests that there is a pathway other than ERK signaling suppressed by the
titled compounds.

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Figure 3. Superimposition of PT5 (magenta) and 1c (green) into ATP binding site of ABL kinase (PDB:
1IEP) as predicted by MOE 2019.0101 followed by a zoom-in separate view for each compound. Amino
acids are depicted in blue sticks. All heteroatoms are colored by element. Ligand-protein interactions
are shown as black-dash lines. Some amino acid residues are hidden for clarity purpose.
Figure 4. The effect of GP2, GP5, PT5 and imatinib on ERK signaling. K562 cells were incubated with
the compounds at 10 µM concentration for 6 h, and then immunoblot analysis was conducted. Band
intensity was quantitated and relative values are shown below.
3. Materials and Methods
3.1. Chemistry
All reactions were performed in an efficient fume hood. Chemicals were purchased from
Sigma-Aldrich (St. Louis, MO, USA), Honeywell Fluka (Morristown, NJ, USA), Kanto Chemical
(Tokyo, Japan), Nacalai Tesque (Kyoto, Japan), Tokyo Chemical Industry (Tokyo, Japan) and FUJIFILM
Wako (Osaka, Japan). Commercially available reagent-grade chemicals were used without further

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purification. The reaction progress was monitored by a thin layer chromatography (TLC) on precoated
plates [Merck (Darmstadt, Germany) TLC sheets silica 60 F254]. The flash column chromatography
was carried out on Silica Gel 60N [40–100 mesh, Kanto Chemical (Tokyo, Japan)]. Melting points were
1
determined on a Yanaco (Kyoto, Japan) melting point apparatus and were uncorrected. H and 13
C
NMR spectra were registered on a Bruker (Billerica, MA, USA) Avance 600 (600 MHz). Chemical shits
are referenced to TMS. Mass spectra (MS) and high-resolution mass spectra (HRMS) were recorded on
a JEOL (Tokyo, Japan) JMS-DX303HF using positive fast atom bombardment (FAB) with 3-nitrobenzyl
alcohol as the matrix.
3.2. General Method of Preparation of PT1–PT5 and GP1–GP5 Compounds
A 0.5 mmol of a triterpenic compound was dissolved in 5 mL dry DMF followed by the addition
of anhydrous K2CO3 (1.5 mmol) and stirred for 30 min. The appropriate free or substituted benzyl
bromide was added and the mixture was stirred for an additional 6 h at room temperature. Upon
the consumption of the reactant, 5 mL of the brine solution was added and the mixture was extracted
with ethyl acetate. The organic phase was washed with water until pH = 7, dried over anhydrous
sodium sulfate, filtered and concentrated under vacuo. The resulted residue was applied for column
chromatography using an appropriate eluent [31].
Asiatic acid benzyl ester (PT1) [37]. Compound PT1 was obtained from AA and benzyl bromide
1
as a white solid (173 mg, 60%). H NMR (600 MHz, CDCl3)
δ: 7.36–7.30 (m, 5H, H-Ar), 5.23 (t, J = 3.6 Hz,
1H), 5.09 (d, J = 12.5 Hz, 1H, Bn-CHH’), 4.98 (d, J = 12.5 Hz, 1H, Bn-CHH’), 3.77–3.73 (m, 1H), 3.64
(d, J = 10.6 Hz, 1H), 3.51 (brs, 1H), 3.41 (d, J = 9.8 Hz, 2H), 2.64 (brs, 2H), 2.26 (d, J = 11.3 Hz, 1H),
2.01–1.86 (m, 4H), 1.80–1.67 (m, 5H), 1.63–1.55 (m, 2H), 1.50–1.45 (m, 2H), 1.36–1.25 (m, 5H), 1.07 (s,
3H), 1.05–1.02 (m, 2H), 1.00 (s, 3H), 0.93 (d, J = 6.5 Hz, 3H), 0.86 (s, 3H), 0.85 (d, J = 6.5 Hz, 3H), 0.63 (s,
3H). 13C NMR (150 MHz, CDCl3)
δ: 177.29, 138.22, 136.36, 128.42, 128.16, 127.97, 125.41, 80.56, 70.62,
68.77, 66.01, 52.87, 49.15, 48.12, 47.48, 46.29, 42.44, 42.14, 39.56, 39.11, 38.84, 38.15, 36.62, 32.68, 30.66,
27.92, 24.23, 23.64, 23.33, 21.17, 18.33, 17.16, 17.05, 17.01, 12.79. MS (FAB) m/z 601.2 (M + Na)+; HRMS
(FAB). Calcd for C37H54O5Na: 601.3869. Found: 601.3868. (Figure S1).
Betulinic acid benzyl ester (PT2) [38]. Compound PT2 was obtained from BA and benzyl bromide
as a white solid (136 mg, 50%). 1H NMR (600 MHz, CDCl3)
δ: 7.35–7.29 (m, 5H, H-Ar), 5.12 (d,
J = 12.3 Hz, 1H, Bn-CHH’), 5.07 (d, J = 12.3 Hz, 1H, Bn-CHH’), 4.70 (d, J = 2.3 Hz, 1H), 4.57 (dd, J = 2.3,
1.4 Hz, 1H), 3.17–3.13 (m, 1H), 3.02–2.98 (m, 1H), 2.27–2.24 (m, 1H), 2.18–2.14 (m, 1H), 1.89–1.83 (m,
2H), 1.63–1.56 (m, 3H), 1.54 (s, 3H), 1.52–1.16 (m, 14H), 1.08 (dt, J = 13.5, 3.1 Hz, 1H), 0.99 (td, J = 12.8,
4.6 Hz, 1H), 0.93 (s, 3H), 0.92 (s, 3H), 0.86 (td, J = 12.8, 3.9 Hz, 1H), 0.78 (s, 3H), 0.74 (s, 3H), 0.73 (s,
3H), 0.65–0.63 (m, 1H). 13C NMR (150 MHz, CDCl3)
δ 175.82, 150.59, 136.52, 128.50, 128.25, 128.06,
109.57, 79.01, 65.74, 56.58, 55.39, 50.60, 49.50, 46.96, 42.42, 40.69, 38.88, 38.75, 38.24, 37.21, 36.96, 34.34,
32.15, 30.62, 29.60, 28.00, 27.45, 25.58, 20.91, 19.39, 18.32, 16.13, 15.86, 15.36, 14.69. MS (FAB) m/z 569.5
(M + Na)+; HRMS (FAB). Calcd for C37H54O3Na: 569.3971. Found: 569.4046. (Figure S2).
Glycyrrhetic acid benzyl ester (PT3) [31
bromide as a white solid (123 mg, 44%). H NMR (600 MHz, CDCl3)
,
38]. Compound PT3 was obtained from GA and benzyl
1
δ: 7.40–7.32 (m, 5H, H-Ar), 5.55 (s,
1H), 5.20 (d, J = 12.2 Hz, 1H, Bn-CHH’), 5.09 (d, J = 12.2 Hz, 1H, Bn-CHH’), 3.24–3.21 (m, 1H), 2.79
(dt, J = 13.4, 3.5 Hz, 1H), 2.32 (s, 1H), 2.05–2.01 (m, 3H), 1.93 (ddd, J = 13.6, 4.3, 2.7 Hz, 1H), 1.81 (td,
J = 13.6, 4.3 Hz, 1H), 1.67–1.61 (m, 4H), 1.59–1.57 (m, 1H), 1.47–1.42 (m,1H), 1.39 (ddd, J = 11.0, 6.9,
2.7 Hz, 2H), 1.35 (s, 3H), 1.32–1.26 (m, 4H), 1.16 (s, 3H), 1.13 (s, 3H), 1.11 (s, 3H), 1.00 (s, 3H), 0.99–0.94
(m, 1H), 0.80 (s, 3H), 0.73 (s, 3H), 0.69 (dd, J = 11.9, 1.5 Hz, 1H). 13C NMR (150 MHz, CDCl3)
δ: 200.11,
176.21, 168.97, 136.16, 128.63, 128.56, 128.31, 128.26, 78.80, 66.24, 61.82, 54.98, 48.23, 45.37, 44.00, 43.20,
41.12, 39.18, 39.15, 37.68, 37.11, 32.80, 31.80, 31.20, 28.43, 28.30, 28.12, 27.34, 26.50, 26.44, 23.37, 18.70,
17.52, 16.38, 15.58. MS (FAB) m/z 561.5(M + H)+; HRMS (FAB). Calcd for C37H53O4: 561.3944. Found:
561.3970. (Figure S3).
Hederagenin benzyl ester (PT4) [39]. Compound PT4 was obtained from HE and benzyl bromide
1
as a white solid (160 mg, 57%). H NMR (600 MHz, CDCl3)
δ: 7.36–7.29 (m, 5H, H-Ar), 5.28 (t, J = 3.6 Hz,

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1H), 5.09 (d, J = 12.6 Hz, 1H, Bn-CHH’), 5.04 (d, J = 12.6 Hz, 1H, Bn-CHH’), 3.72 (d, J = 10.3 Hz, 1H),
3.64–3.62 (m, 1H), 3.42 (d, J = 10.3 Hz, 1H), 2.90 (dd, J = 13.7, 4.3 Hz, 1H), 2.55 (brs, 1H), 2.34 (brs, 1H),
1.97 (td, J = 13.9, 4.4 Hz, 1H), 1.85 (dd, J = 8.9, 3.6 Hz, 2H), 1.71 (td, J = 13.9, 4.4 Hz, 1H), 1.67–1.62
(m, 3H), 1.57–1.51 (m, 2H), 1.40–1.33 (m, 3H), 1.42–1.33 (m, 4H), 1.23–1.13 (m, 3H), 1.12 (s, 3H), 1.03
(dt, J = 13.5, 3.1 Hz, 1H), 0.98–0.94 (m, 1H), 0.93 (s, 3H), 0.92 (s, 3H), 0.89 (s, 3H), 0.89 (s, 3H), 0.84 (dd,
J = 10.6, 3.1 Hz, 1H), 0.60 (s, 3H). 13C NMR (150 MHz, CDCl3)
δ: 177.46, 143.69, 136.45, 128.42, 128.00,
127.92, 122.44, 76.89, 72.16, 65.95, 49.82, 47.64, 46.76, 45.90, 41.85, 41.74, 41.41, 39.31, 38.14, 36.92, 33.88,
33.10, 32.54, 32.39, 30.71, 27.64, 26.84, 25.90, 23.65, 23.39, 23.07, 18.50, 16.92, 15.67, 11.37. MS (FAB) m/z
585.2 (M + Na)+; HRMS (FAB). Calcd for C37H54O4Na: 585.3920. Found: 585.3912. (Figure S4).
Oleanolic acid benzyl ester (PT5) [31
bromide as a white solid (185 mg, 68%). H NMR (600 MHz, CDCl3)
,
38]. Compound PT5 was obtained from OA and benzyl
1
δ
: 7.35–7.29 (m, 5H, H-Ar), 5.29
(t, J = 3.6 Hz, 1H), 5.09 (d, J = 12.6 Hz, 1H, Bn-CHH’), 5.05 (d, J = 12.6 Hz, 1H, Bn-CHH’), 3.20 (dt,
J = 10.1, 4.9 Hz, 1H), 2.90 (dd, J = 13.8, 4.4 Hz, 1H), 1.98 (td, J = 13.8, 4.1 Hz, 1H), 1.85 (dd, J = 8.9, 3.7
Hz, 2H), 1.74–1.59 (m, 7H), 1.57–1.50 (m, 4H), 1.42 (td, J = 12.6, 3.7 Hz, 1H), 1.37–1.16 (m, 7H), 1.12 (s,
3H), 1.04 (dt, J = 6.6, 3.5 Hz, 1H), 0.98 (s, 3H), 0.92 (s, 3H), 0.90 (s, 3H), 0.88 (s, 3H), 0.77 (s, 3H), 0.71 (dd,
J = 11.7, 1.8 Hz, 1H), 0.61 (s, 3H). 13C NMR (150 MHz, CDCl3)
δ: 177.46, 143.72, 136.48, 128.42, 127.99,
127.91, 122.53, 79.04, 65.94, 55.25, 47.65, 46.78, 45.92, 41.73, 41.42, 39.33, 38.77, 38.48, 37.04, 33.90, 33.11,
32.76, 32.41, 31.59, 30.71, 28.12, 27.66, 27.23, 25.89, 23.66, 23.42, 23.09, 22.66, 18.34, 16.91, 15.58, 15.31,
14.11. MS (FAB) m/z 569.4 (M + Na)+; HRMS (FAB). Calcd for C37H54O3Na: 569.3967. Found: 569.4046.
(Figure S5).
Ursolic acid benzyl ester (PT6) [38]. Compound PT6 was obtained from UA and benzyl bromide
1
as a white solid (221 mg, 81%). H NMR (600 MHz, CDCl3)
δ: 7.36–7.30 (m, 5H, H-Ar), 5.23 (t, J = 3.6 Hz,
1H), 5.10 (d, J = 12.5 Hz, 1H, Bn-CHH’), 4.98 (d, J = 12.5 Hz, 1H, Bn-CHH’), 3.21 (dd, J = 11.3, 4.7 Hz,
1H), 2.26 (dd, J = 11.3, 0.9 Hz, 1H), 2.01 (td, J = 13.4, 4.5 Hz, 1H), 1.90–1.83 (m, 2H), 1.82–1.76 (m, 1H),
1.73–1.70 (m, 2H), 1.67–1.44 (m, 9H), 1.36–1.25 (m, 6H), 1.07 (s, 3H), 1.05–1.01 (m, 1H), 0.98 (s, 3H), 0.93
(d, J = 6.4 Hz, 3H), 0.89 (s, 3H), 0.85 (d, J = 6.4 Hz, 3H), 0.77 (s, 3H), 0.70 (dd, J = 11.7, 1.3 Hz, 1H), 0.64
(s, 3H). 13C NMR (150 MHz, CDCl3)
δ: 177.29, 138.13, 136.41, 128.41, 128.16, 127.95, 125.74, 79.04, 65.99,
55.26, 52.93, 48.15, 47.60, 42.07, 39.56, 39.12, 38.87, 38.77, 38.67, 36.98, 36.66, 33.07, 31.60, 30.70, 28.17,
27.99, 27.26, 24.28, 23.59, 23.29, 22.66, 21.19, 18.33, 17.04, 17.01, 15.64, 15.46, 14.13. MS (FAB) m/z 569.3
(M + Na)+; HRMS (FAB). Calcd for C37H54O3Na: 569.3971. Found: 569.3970. (Figure S6).
Gypsogenin 4-methoxybenzyl ester (GP1). Compound GP1 was obtained from GP and
4-methoxybenzyl bromide as a white solid (123 mg, 42%); mp 78–80 ◦C. 1H NMR (600 MHz, CDCl3)
δ
: 9.39 (s, 1H), 7.26 (d, J = 8.8 Hz, 2H, H-Ar), 6.86 (d, J = 8.8 Hz, 2H, H-Ar), 5.28 (t, J = 3.6 Hz, 1H),
5.02 (d, J = 12.1 Hz, 1H, Bn-CHH’), 4.97 (d, J = 12.1 Hz, 1H, Bn-CHH’), 3.80 (s, 3H), 3.76 (dd, J = 11.6,
4.3 Hz, 1H), 2.88 (dd, J = 13.9, 4.3 Hz, 1H), 1.95 (td, J = 13.6, 4.1 Hz, 1H), 1.87 (dd, J = 8.9, 3.6 Hz, 2H),
1.71–1.44 (m, 14H), 1.32 (td, J = 13.9, 4.3 Hz, 1H), 1.27–1.25 (m, 1H), 1.19–1.14 (m, 2H), 1.13 (s, 3H),
1.06 (s, 3H), 1.04–1.02 (m, 1H), 1.04–0.97 (m, 3H), 0.93 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.59 (s, 3H). 13
C
NMR (150 MHz, CDCl3) δ: 207.08, 177.41, 159.46, 143.84, 129.84, 128.59, 122.06, 113.81, 71.93, 65.75,
55.29, 55.19, 48.25, 47.56, 46.65, 45.91, 41.79, 41.42, 39.67, 38.07, 36.01, 33.87, 33.10, 32.31, 32.14, 30.71,
27.60, 26.13, 25.89, 23.65, 23.37, 22.99, 20.76, 16.91, 15.57, 8.95. MS (FAB) m/z 613.5 (M + Na)+; HRMS
(FAB). Calcd for C38H54O5Na: 613.3869. Found: 613.3865. (Figure S7).
Gypsogenin 3,5-bis(trifluromethyl)benzyl ester (GP2): Compound GP2 was obtained from GP
and 3,5-bis(trifluoromethyl)benzyl bromide as a white solid (149 mg, 43%); mp 85–87 ◦C. 1H NMR
(600 MHz, CDCl3) δ: 9.39 (s, 1H), 7.83 (s, 1H, H-Ar), 7.81 (s, 2H, H-Ar), 5.28 (t, J = 3.6 Hz, 1H), 5.20 (d,
J = 13.2 Hz, 1H, Bn-CHH’), 5.13 (d, J = 13.2 Hz, 1H, Bn-CHH’), 3.76 (dd, J = 11.5, 4.5 Hz, 1H), 2.89
(dd, J = 13.7, 4.1 Hz, 1H), 2.01 (td, J = 13.7, 4.1 Hz, 1H), 1.87–1.84 (m, 2H), 1.74–1.44 (m, 14H), 1.36 (td,
J = 13.7, 4.1 Hz, 1H), 1.26–1.17 (m, 3H), 1.15 (s, 3H), 1.06 (s, 3H), 0.98–0.95 (m, 1H), 0.93 (s, 3H), 0.91
(s, 3H), 0.89 (s, 3H), 0.48 (s, 3H). 13C NMR (150 MHz, CDCl3)
δ: 207.10, 177.18, 143.69, 138.97, 131.92
(q, J = 33.5 Hz), 128.09, 124.09, 122.32, 122.01–121.91 (m), 71.94, 64.35, 55.15, 48.20, 47.46, 46.88, 45.85,
41.77, 41.38, 39.61, 38.01, 35.97, 33.78, 33.01, 32.39, 31.98, 31.58, 30.66, 27.49, 26.10, 25.89, 23.51, 23.19,

Molecules 2019, 24, 3535
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23.11, 22.64, 20.65, 16.67, 15.36, 14.09, 8.95. MS (FAB) m/z 719.4 (M + Na)+; HRMS (FAB). Calcd for
C39H50O4F6Na: 719.3511. Found: 719.3511. (Figure S8).
Gypsogenin 4-trifluoromethyl benzyl ester (GP3). Compound GP3 was obtained from GP and
4-trifluoromethyl benzyl bromide as a white solid (128 mg, 41%); mp 175–177 ◦C. 1H NMR (600 MHz,
CDCl3) δ: 9.39 (s, 1H), 7.60 (d, J = 8.0 Hz, 2H, H-Ar), 7.46 (d, J = 8.0 Hz, 2H, H-Ar), 5.28 (t, J = 3.6 Hz,
1H), 5.15 (d, J = 12.9 Hz, 1H, Bn-CHH’), 5.07 (d, J = 12.9 Hz, 1H, Bn-CHH’), 3.76 (dd, J = 11.4, 4.4 Hz,
1H), 2.89 (dd, J = 13.5, 4.1 Hz, 1H), 1.99 (td, J = 13.5, 4.1 Hz, 1H), 1.87–1.84 (m, 2H), 1.74–1.19 (m, 7H),
1.55 (ddd, J = 18.5, 11.1, 4.1 Hz, 2H), 1.49–1.42 (m, 3H), 1.34 (td, J = 13.7, 4.1 Hz, 1H), 1.21 (ddd, J = 13.5,
4.1, 2.1 Hz, 2H), 1.14 (s, 3H), 1.06 (s, 3H), 1.04–0.96 (m, 3H), 0.93 (s, 3H), 0.91 (s, 6H), 0.89–0.87 (m, 1H),
0.86–0.83 (m, 1H), 0.50 (s, 3H). 13C NMR (150 MHz, CDCl3)
δ: 207.09, 177.22, 143.73, 140.35, 128.27,
125.42 (q, J = 3.8 Hz), 123.15, 122.22, 71.95, 65.07, 55.16, 48.24, 47.50, 46.78, 45.86, 41.78, 41.45, 39.64,
38.06, 35.99, 33.82, 33.06, 32.38, 32.10, 30.70, 29.71, 27.57, 26.12, 25.90, 23.62, 23.35, 23.06, 20.67, 16.77,
15.49, 8.95. MS (FAB) m/z 651.5 (M + Na)+; HRMS (FAB). Calcd for C38H51O4F3Na: 651.3637. Found:
651.3605. (Figure S9).
Gypsogenin 3,5-difluorobenzyl ester (GP4). Compound GP4 was obtained from GP and
1
3,5-difluorobenzyl bromide as a white solid (119 mg, 40%); mp 58–60 ◦C. H NMR (600 MHz,
CDCl3) δ: 9.40 (s, 1H), 6.86 (dd, J = 7.9, 1.8 Hz, 2H, H-Ar), 6.76–6.72 (m, 1H, H-Ar), 5.32 (t, J = 3.6 Hz,
1H), 5.09 (d, J = 13.1 Hz, 1H, Bn-CHH’), 4.96 (d, J = 13.1 Hz, 1H, Bn-CHH’), 3.77 (d, J = 10.0 Hz, 1H), 2.90
(dd, J = 13.5, 4.1 Hz, 1H), 2.00 (td, J = 13.7, 4.1 Hz, 1H), 1.89 (dd, J = 8.9, 3.6 Hz, 2H), 1.72–1.61 (m, 7H),
1.59 (s, 3H), 1.56–1.44 (m, 4H), 1.38–1.18 (m, 6H),1.15 (s, 3H), 1.06 (s, 3H), 1.05–1.04 (m, 1H), 1.00–0.97
(m, 1H), 0.93 (s, 3H), 0.91 (s, 3H), 0.88 (t, J = 7.0 Hz, 1H), 0.59 (s, 3H). 13C NMR (150 MHz, CDCl3)
δ:
207.09, 177.16, 163.86 (d, J = 12.5 Hz), 162.21 (d, J = 12.5 Hz), 143.72, 140.26, 122.35, 110.55, 110.42,
103.29, 71.94, 64.55, 55.49, 55.17, 48.26, 47.51, 46.83, 45.86, 41.81, 41.45, 39.68, 38.05, 36.02, 33.81, 33.06,
32.39, 32.14, 31.59, 30.70, 27.56, 26.12, 25.91, 23.61, 23.30, 23.07, 22.65, 20.74, 16.85, 15.53, 14.11, 8.96.
MS (FAB) m/z 619.2 (M + Na)+; HRMS (FAB). Calcd for C37H50O4F2Na: 619.3575. Found: 619.3576.
(Figure S10).
Gypsogenin 4-cyanobenzyl ester (GP5). Compound GP5 was obtained from GP and 4-cyanobenzyl
bromide as a white solid (140 mg, 48%); mp 175–177 ◦C. 1H NMR (600 MHz, CDCl3)
δ 9.39 (s, 1H), 7.65
(d, J = 8.3 Hz, 2H, H-Ar), 7.45 (d, J = 8.3 Hz, 2H, H-Ar), 5.29 (t, J = 3.5 Hz, 1H), 5.16 (d, J = 13.3 Hz, 1H,
Bn-CHH’), 5.05 (d, J = 13.3 Hz, 1H, Bn-CHH’), 3.76 (dd, J = 11.4, 4.1 Hz, 1H), 2.89 (dd, J = 13.8, 4.1 Hz,
1H), 2.00 (td, J = 13.7, 4.1 Hz, 1H), 1.90–1.77 (m, 2H), 1.73–1.45 (m, 12H), 1.35 (td, J = 13.7, 4.1 Hz, 1H),
1.27–1.17 (m, 4H), 1.15 (s, 3H), 1.06 (s, 3H), 1.05–1.04 (m, 1H), 1.00–0.97 (m, 1H), 0.93 (s, 3H), 0.92 (s, 3H),
0.91 (s, 3H), 0.88 (t, J = 7.0 Hz, 1H), 0.56 (s, 3H). 13C NMR (150 MHz, CDCl3)
δ 207.12, 177.16, 143.67,
141.70, 132.30, 128.37, 122.27, 118.56, 111.87, 71.95, 64.86, 55.17, 48.20, 47.48, 46.85, 46.84, 45.81, 41.80,
41.79, 41.47, 39.66, 38.05, 35.99, 33.79, 33.04, 32.38, 32.11, 31.58, 30.70, 30.69, 30.68, 30.68, 27.57, 26.11,
25.90, 23.61, 23.35, 23.06, 22.65, 20.71, 16.87, 15.57, 14.11, 8.98. MS (FAB) m/z 608.5 (M + Na)+; HRMS
(FAB). Calcd for C38H51NO4Na: 608.3716. Found: 608.3710. (Figure S11).
3.3. Biochemistry
3.3.1. Cell Cultures
A549 human lung carcinoma and HeLa human cervical carcinoma cell lines were cultured in
Dulbecco’s Modified Eagle Medium (DMEM). MCF7 human breast cancer and leukemic (K562, HL-60,
MT-2 and Jurkat) cells, and PBMC (Precision Bioservices, Frederic, MD, USA) were cultured in RPMI
1640. All media were purchased from FUJIFILM Wako (Osaka, Japan) and supplemented with 10%
fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA) and 89 µg/mL streptomycin (Meiji Seika
Pharma, Tokyo, Japan) at 37 ◦C in a humid atmosphere and 5% CO2. In experiments, all cancer cell
lines and PBMC were cultured in 24-well and 96-well plates (Iwaki brand Asahi Glass, Chiba, Japan)
at 2
×
104 cells/mL and 1
×
106 cells/mL concentrations, respectively for 24 h. The stock solution of
compounds and imatinib in concentrations between 0,1-10 mM were prepared in DMSO (FUJIFILM

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Wako, Osaka, Japan) and further diluted with fresh culture medium. The concentration of DMSO in
the final culture medium was 1%, which had no effect on the cell viability [40].
3.3.2. Cell Viability Assay
The effect of PTs, their derivatives and imatinib on cell viability was assessed by using MTT
(Dojindo, Kumamoto, Japan) as previously described in the literature [41
various concentrations (1–100
M) of the compounds for 24 h at 37 ◦C. The cells were then stained with
MTT solution and incubated for additional 4 h. At the end of this period, supernatants were removed
and 100 L DMSO was added to each well to solubilize the formazan crystals. The absorbance of the
,42]. The cells were exposed to
µ
µ
solution was determined on the Infinitive M1000 (Tecan, Mannedorf, Switzerland) plate reader at a
wavelength of 550 nm with background subtraction at 630 nm. All experiments were performed in
triplicate and IC50 values defined as the drug concentrations that reduced absorbance to 50% of control
values were calculated from MTT results.
3.3.3. Cell Death Detection
After the cells were incubated with the selected compounds in this series at IC50 concentration for
6 h, the apoptotic/necrotic/healthy/detection kit (PromoKine, Heidelberg, Germany) was performed
according to the manufacturer‘s instructions. The cells were then briefly washed twice with 1
buffer. A staining solution containing 50 L of 1 binding buffer and 5 L of FITC-Annexin V solution,
L of ethidium homodimer III solution, or 5 L of Hoechst 33342 solution was added, and incubated
for 15 min at room temperature in the dark. Then, the cells were washed with 1 binding buffer, and
×
binding
µ
×
µ
5
µ
µ
×
analyzed by the all-in-one fluorescence microscope Biorevo Fluorescence BZ-9000 (Keyence, Osaka,
Japan). The number of healthy cells (Hoechst 33342), apoptotic cells (Annexin V), late apoptotic or
necrotic cells (Annexin V and Ethidium homodimer III) and necrotic cells (Ethidium homodimer III)
were counted as previously described [43].
3.3.4. Determination of ABL1 Kinase Inhibition
The ABL1 kinase profiling assay protocol was applied according to the manufacturer‘s instructions
(Promega, Madison, WI, USA) with the modification proposed in [36]. In this system, the ABL1 kinase
and its substrate were diluted with 95
respectively. Then, the reaction of ABL1 kinase was performed using 2
varying concentrations (0.1–30 M), 4 L of kinase working stock, and 4 µ
µ
L 2.5x Kinase Buffer and 15
µ
L of 100
L of the compound solution at
L of ATP/substrate working
µM ATP solutions,
µ
µ
µ
stock in the 384-well plate. After 1 h of incubation at room temperature, the activity of the ABL1 kinase
was detected using the ADP-Glo Kinase Assay (Promega, Madison, WI, USA) and the inhibitory kinase
activity of compounds in dose-response mode was measured by the Infinitive M1000 luminescence
plate reader (Tecan, Groding, Austria). The IC50 values of tested compounds required to decrease the
kinase activity by 50% were calculated.
3.3.5. Immunoblot Analysis
The K562 cells were cultured with compounds GP2, GP5, PT5 and imatinib at 10 µM concentrations.
After 6 h of treatment at 37 ◦C, the cells were lysed in PBS-Laemmli sample buffer and then, immunoblot
analysis was conducted using phospho-specific-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E)
XP Rabbit mAb (1:1000) (Cell Signaling Technology, Danvers, MA, USA) or anti-
β-actin clone AC-15
(Sigma-Aldrich, St. Louis, MO, USA). For immunoreactivity detection, the chemiluminescence method
was performed as previously described [44
software (NIH, Bethesda, MD, USA).
,45]. The band intensity was measured using the ImageJ

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3.4. Molecular Docking
Crystal structure of the ABL kinase in complex with imatinib was obtained from Protein Data
Bank (PDB: 1IEP); PT5 was built by ChemDraw Professional 16. Before the docking simulations,
PT5 and 1IEP preparation included the addition of hydrogens, the assignment of bond order, and
assessment of the correct protonation as previously described [46,47]. The MOE 2019.01 software
(Chemical Computing Group, Montreal, Canada) was employed for the preparation, interactive
docking, visualization and the analysis procedures using its default parameters [48–50].
4. Conclusions
We herein designed and synthesized benzyl esters of different triterpenes (PT1
newly substituted benzyl esters of gypsogenin (GP1 GP5), based on our finding that 1c (benzyl ester
of gypsogenin) has anti-ABL kinase and anti-CML activities [36]. These derivatives and the parent
triterpenes were biologically evaluated, and three compounds with good activities, GP2 GP5 and
–PT6) and the
–
,
PT5, were found. Among them, PT5 has six times stronger in vitro IC50 against ABL kinase compared
to 1c, and stronger activity against CML cell line K562. The stronger interaction of PT5 with ABL
kinase was explained using the difference between the molecular docking by the methyl group in
PT5 on its C4 position and the aldehyde group on the same position of 1c. The PT5 was shown
to inhibit ERK signaling to induce apoptosis in K562. The other compounds GP2 and GP5 have
stronger activity against K562 than PT5, although their in vitro activity against ABL1 is weaker. It is
considered that these two compounds have different mechanisms to induce cell death from inhibition of
BCR-ABL. Actually, GP2 and GP5 have stronger activity against some cell lines other than K562. Taken
together, we succeeded in increasing the activity against ABL kinase and CML cells by derivatization.
The strongest inhibitor PT5 is based on oleanolic acid which is widely spread in many plants. This
study would lead to the basis of new anti-CML drugs based on naturally found pentacyclic triterpens.
1
Supplementary Materials: The following are available online. Figure S1: H and 13C spectra of PT1; Figure S2:
1H and 13C spectra of PT2; Figure S3: 1H and 13C spectra of PT3; Figure S4: 1H and 13C spectra of PT4; Figure S5:
1H and 13C spectra of PT5; Figure S6: 1H and 13C spectra of PT6; Figure S7: 1H and 13C spectra of GP1; Figure S8:
1H and 13C spectra of GP2; Figure S9: 1H and 13C spectra of GP3; Figure S10: 1H and 13C spectra of GP4; Figure
S11: 1H and 13C spectra of GP5.
Author Contributions: H.I.C., M.O.R. and M.F. designed the research; S.E.O., N.G.U. and E.S. performed isolation
of gypsogenin; M.O.R. synthesized and purified the compounds and H.I.A.-A. interpreted spectroscopic data.
H.I.C. was responsible for the biological work and mainly wrote the manuscript. D.E.E. and H.T. performed a
part of biological experiment. M.O.R. undertook the docking study. Z.O. took part in the analysis of biological
data and discussion of the results. M.C., T.F.S.A. and N.Y. did discussion. M.F. and M.O. were responsible for the
correspondence of the manuscript. All authors discussed, edited and approved the final version.
Funding: This study was supported by Bilateral Joint Research Project from the Japanese Society for the Promotion
of Science (the grant no: 18039111-000102).
Acknowledgments: MCF7 and A549 cells were provided by the RIKEN BRC through the National Bio Resource
Project of the MEXT/AMED, Japan.
Conflicts of Interest: The authors report no conflicts of interest.
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Sample Availability: Samples of the compounds (1c, PT1, PT2, PT3, PT4, PT5, PT6, GP1, GP2, GP3, GP4, GP5 and
GP6) are available from the authors.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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