New 30-substituted derivatives of pentacyclic triterpenes: preparation, biological activity, and molecular docking study

Article abstract of DOI:10.1016/j.molstruc.2020.129394

A series of 30-substituted derivatives of 3,28-O,O′-diacetylbetulin 4-19 were prepared and assessed for anticancer activities in vitro against five human cancer cell lines (SNB-19, C-32, SKOV-3, MCF-7, and T47D). The triterpene 14, containing the butyryloxycarbonyl group at the C-30 position, showed the highest cytotoxicity, with IC₅₀ values within the range of 1.24 to 6.03 μM towards applied cancer cells. The ADME study of the betulinines 4-19 were performed by determination of molecular weight (M), hydrogen bond acceptors (nHBA), hydrogen bond donors (nHBD), lipophilicity (cLogP), rotatable bonds (nROTB) and topological polar surface area (TPSA). Molecular docking was applied to examine the probable interaction between the new synthesized compounds and the Akt1 kinase. The analysis showed that triterpenes 13-16 formed numerous hydrophobic interactions inside the binding pocket of Akt1.

Full text of DOI:10.1016/j.molstruc.2020.129394

Journal of Molecular Structure 1226 (2021) 129394
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
New 30-substituted derivatives of pentacyclic triterpenes: preparation,
biological activity, and molecular docking study
Elwira Chrobaka, Ewa Be˛beneka,∗, Krzysztof Marcinieca, Monika Kadela-Tomaneka,
Szymon Siudakb, Małgorzata Latochac, Stanisław Boryczkaa
a Department of Organic Chemistry, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of Silesia in Katowice, 4 Jagiellon´ska Str., 41-200
Sosnowiec, Poland
b Zdrowit Pharmacy, 19a Ludowa Str., 41-300 Da˛browa Górnicza, Poland
c Department of Cell Biology, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of Silesia in Katowice, 8 Jedno´s´ci Str., 41-200 Sosnowiec,
Poland
a r t i c l e i n f o
a b s t r a c t
ꢀ
Article history:
A series of 30-substituted derivatives of 3,28-O,O -diacetylbetulin 4-19 were prepared and assessed for
anticancer activities in vitro against five human cancer cell lines (SNB-19, C-32, SKOV-3, MCF-7, and T47D).
The triterpene 14, containing the butyryloxycarbonyl group at the C-30 position, showed the highest cy-
totoxicity, with IC50 values within the range of 1.24 to 6.03 μM towards applied cancer cells. The ADME
study of the betulinines 4-19 were performed by determination of molecular weight (M), hydrogen bond
acceptors (nHBA), hydrogen bond donors (nHBD), lipophilicity (cLogP), rotatable bonds (nROTB) and topo-
logical polar surface area (TPSA). Molecular docking was applied to examine the probable interaction be-
tween the new synthesized compounds and the Akt1 kinase. The analysis showed that triterpenes 13-16
formed numerous hydrophobic interactions inside the binding pocket of Akt1.
Received 15 August 2020
Revised 30 September 2020
Accepted 1 October 2020
Available online 4 October 2020
Keywords:
Natural compounds
Triterpenes
Antiproliferative activity
Molecular docking
© 2020 Elsevier B.V. All rights reserved.
1. Introduction
at the C-30 position. Some bioactive C-30 substituted lupane-type
triterpenes, such as 30-hydroxylupeol and 30-hydroxybetulin, were
In recent years, there has been a growing interest in natural
substances, in particular triterpenes showing anticancer or antiviral
activity against human immunodeficiency virus (HIV) [1–3]. One of
the most commonly found compounds in this group is betulin. In
addition, this triterpene, isolated from birch bark in the 18th cen-
tury, is one of the first natural substances obtained from plant ma-
terial [4,5]. It is possible to obtain derivatives, which have diverse
biological effects including antitumor, antiviral, antibacterial, anti-
malarial and anti-inflammatory activity, due to the chemical mod-
ifications of betulin in positions C-3, C-17, C-19, C-28 and C-30 [6–
11]. An example of such a derivative is betulinic acid, which is pre-
pared through the oxidation of betulin [12]. The interest in the an-
titumor properties of triterpenes has increased since Pisha discov-
ered in 1995 that betulinic acid is a factor inducing the apoptosis
process of human melanoma cells (Mel-1, Mel-2), with no toxicity
to normal cells [13].
isolated from stems of Microtropis fokienensis and Perrottetia arisa-
nensis [14]. However, the main source of 30-substituted betulin and
betulinic acid derivatives is chemical synthesis. The introduction a
new moiety into C-30 position of pentacyclic triterpenes makes it
possible to obtain compounds with potential pharmacological effi-
ciency (Fig. 1) [15–21].
Sidova et al. reported synthesis of triterpenes containing a 1,2,3-
triazole ring in the C-30 position. The C-30 substituted triazole
derivatives were chosen for the cytotoxic activity MTS test against
eight human cancer cell lines (CCRF-CEM, CEM-DNR, K562, K562-
TAX, A549, HCT116, HCT116p53-/-, and U2OS). The obtained results
show that the most active compound was a derivative containing
a free benzaldehyde group connected to the position 30 through
the triazole ring (IC50 = 3.3-14.8 μM for tested cancer lines)
[15]. In 1996, Evers et al. carried out synthesis of isopropylidene-
substituted derivatives of betulinic acid with the anti-HIV-1 ac-
tivity. The introduction of sulfur and nitrogen nucleophiles at po-
sition 30 into betulinic acid was achieved via the Trost-Tsuji π-
allylpalladium method. It was observed that substituting the iso-
propylidene moiety at position C-30 by the 30-(hydroxyethyl)thio
group led to an increase in activity against human immunodefi-
ciency virus type 1 (HIV-1) strain IIIB/LAI [18].
An important group of betulin and betulinic acid derivatives
are compounds obtained by modification of the triterpene skeleton
∗
Corresponding author
E-mail address: ebebenek@sum.edu.pl (E. Be˛benek).
https://doi.org/10.1016/j.molstruc.2020.129394
0022-2860/© 2020 Elsevier B.V. All rights reserved.

E. Chrobak, E. Be˛benek, K. Marciniec et al.
Journal of Molecular Structure 1226 (2021) 129394
Fig. 1. Biologically activity of 30-substituted derivatives of pentacyclic triterpenes.
(cm−1). The progress of all reactions was monitored by thin layer
chromatography (TLC) on silica gel 60 254F plates. The spots were
visualized by spraying with a solution of 5% sulfuric (VI) acid and
then heating to 100 ˚C. The obtained compounds were purified by
column chromatography on silica gel 60, <63 μm (Merk) using a
mixture of various organic solvents as an eluent. All solvents for
reactions were dried and purified prior to use.
Studies carried out by Huang et al. have shown that in the
group of pentacyclic triterpenes there are compounds with an-
tifeedant activity. The authors reported that a simple preparation
of 30-hydroxybetulin from betulin displayed antifeedant activity
against larvae of the Colorado potato beetle (Leptinotarsa decemlin-
eata) [20]. Additionally, Spivak et al. described the antistosomal ac-
tivity of a derivative possessing a covalent linkage to the hydropho-
bic fragment of 3,28-diacetylbetulin at the C-30 position with the
triphenylphosphonium moiety via a hydrocarbon bridge. This allyl
salt (IC50 = 0.65 μg/mL) has shown the best activity towards Schis-
tosoma mansoni. A high activity of 30-substituted triphenylphos-
phonium salt towards adult S. mansoni most likely is caused by
presence of an allyl group. The allyl group delocalizes the phospho-
nium cation and therefore lowers the activation energy for mem-
brane passage [21].
2.1.1. Synthesis of derivatives 2 and 3
ꢀ
ꢀ
Synthesis of 3,28-O,O -diacetylbetulin 2 and 3,28-O,O -diacetyl-
30-hydroxybetulin 3 was performed according to the methods de-
scribed previously [25,20].
2.1.2. General procedure for the synthesis of derivatives 4-9
Inspired by the previous studies, we used the hydroxyl group
at the C-30 of 3,28-diacetylbetulin as a starting point to obtain
semisynthetic derivatives holding an ester and an ester-triazole
linker between the triterpene skeleton at the C-30 position [22–
24]. Additionally, molecular docking was performed to examine the
probable interaction between the obtained 30-substituted triter-
penes and the Akt1 kinase. The new derivatives were tested for
anticancer activity against human cancer cell lines: glioblastoma
(SNB-19), amelanotic melanoma (C-32), ovarian adenocarcinoma
(SKOV-3) and breast adenocarcinoma (T47D, MCF-7).
The solution of dicyclohexylcarbodiimide (0.08 g, 0.41
mmol) and 4-dimethylaminopyridine (0.004 g, 0.03 mmol) in
dichloromethane (0.4 mL) was added slowly to the mixture of
ꢀ
3,28-O,O -diacetyl-30-hydroxybetulin 3 (0.20 g, 0.37 mmol) and
the corresponding carboxylic acid (0.41 mmol) in dichloromethane
(2.5 mL) at -10 ˚C. Stirring was continued for 5 h at -10 ˚C under
argon atmosphere. The reaction was allowed to warm to room
temperature and stirred overnight. Subsequently, the reaction
mixture was filtered off. The solvent was removed under re-
duced pressure and the residue was purified by silica gel column
chromatography (dichloromethane/ethanol 60:1, v/v).
ꢀ
2. Experimental
3,28-O,O -diacetyl-30-propanoylbetulin 4 Yield 70%; mp 72-76˚C;
Rf 0.37 (dichloromethane/ethanol, 60:1, v/v); IR (ν
cm−1 KBr):
max
2.1. Methods and materials
3660, 2947, 1739, 1726, 1708, 1465, 1246; 1H NMR (600 MHz,
CDCl3) δ ppm: 0.76 (s, 3H, CH3), 0.77 (s, 3H, CH3), 0.78 (s, 3H,
CH3), 0.89 (s, 3H, CH3), 0.96 (s, 3H, CH3), 1.11 (t, 3H, J = 7.2 Hz,
CH2CH3), 1.97 (s, 3H, CH3C=O), 2.01 (s, 3H, CH3C=O), 2.30-2.33
(m, 3H, CH2CH3, H-19), 3.75 (d, 1H, J = 11.4 Hz, H-28), 4.17 (d,
1H, J = 11.4 Hz, H-28), 4.39 (m, 1H, H-3), 4.49 (m, 2H, CH2, C-30),
4.87 (s, 1H, H-29), 4.88 (s, 1H, H-29); 13C NMR (150 MHz, CDCl3)
δ ppm: 9.14, 14.75, 16.05, 16.17, 16.50, 18.16, 20.89, 21.06, 21.34,
23.69, 26.55, 27.01, 27.68, 27.94, 29.73, 31.11, 34.13, 34.37, 37.06,
37.50, 37.80, 38.38, 40.91, 42.66, 43.88, 46.32, 49.45, 50.23, 55.35,
62.47, 65.85, 80.89, 110.56, 148.89, 171.05, 171.64, 174.17. HR-MS
(APCI) m/z: C37H58O6 [M - H]−, Calcd. 597.4155; Found 597.3987.
All melting points were determined in open capillary tubes on
a Boetius melting point apparatus without correction. The 1HNMR
and 13CNMR spectra were measured in CDCl3 as solvent on a
Bruker Avance III 600 spectrometer. The chemical shifts values are
reported in ppm (δ) and the coupling constants (J) in Hz. The spin
multiplicities are given as singlet (s), doublet (d), triplet (t) and
multiplet (m). The high-resolution mass spectral analysis was per-
formed on a Bruker Impact II instrument using APCI method (neg-
ative mode). Infrared spectra (KBr, pellet) were recorded using the
IRAffinity-1 Shimadzu spectrometer and reported in wave number
2

E. Chrobak, E. Be˛benek, K. Marciniec et al.
Journal of Molecular Structure 1226 (2021) 129394
ꢀ
3,28-O,O -diacetyl-30-(2-propenoyl)betulin 5 Yield 47%; mp 70-
31.00, 34.12, 34.35, 37.05, 37.48, 37.81, 38.39, 40.91, 42.66, 46.31,
49.53, 50.21, 55.36, 62.48, 67.06, 72.32, 80.90, 85.97, 111.60, 147.87,
153.51, 171.06, 171.64. HR-MS (APCI) m/z: C38H56O6 [M - H]−, Calcd.
607.3998; Found 607.3834.
74˚C; Rf 0.38 (dichloromethane/ethanol, 60:1, v/v); IR (ν
cm−1
max
KBr): 3703, 2945, 1730, 1710, 1467, 1246; 1H NMR (600 MHz,
CDCl3) δ ppm: 0.76 (s, 3H, CH3), 0.77 (s, 3H, CH3), 0.78 (s, 3H,
CH3), 0.89 (s, 3H, CH3), 0.99 (s, 3H, CH3), 1.97 (s, 3H, CH3C=O),
1.99 (s, 3H, CH3C=O), 2.32 (m, 1H, H-19), 3.75 (d, 1H, J = 11.4 Hz,
H-28), 4.17 (d, 1H, J = 10.8 Hz, H-28), 4.39 (m, 1H, H-3), 4.57 (m,
2H, CH2, C-30), 4.91 (both br. s, 2H, H-29), 5.80 (m, 1H, CH=CH2),
6.10 (m, 1H, CH=CH2), 6.37 (m, 1H, CH=CH2); 13C NMR (150 MHz,
CDCl3) δ ppm: 14.77, 16.05, 16.17, 16.50, 18.16, 20.89, 21.06, 21.35,
23.69, 26.58, 27.01, 27.95, 29.74, 31.14, 34.13, 34.37, 37.06, 37.51,
37.80, 38.38, 40.91, 42.67, 46.32, 49.49, 50.23, 55.35, 62.48, 66.09,
80.89, 110.90, 128.36, 131.06, 148.66, 165.89, 171.05, 171.64. HR-MS
2.1.3. General procedure for the synthesis of derivatives 10-16
The solution of the corresponding chloroformate (1.11 mmol) in
ꢀ
benzene (2 mL) was added to the mixture of 3,28-O,O -diacetyl-
30-hydroxybetulin 3 (0.20 g, 0.37 mmol) and pyridine (0.9 mL) in
benzene (2.5 mL) at 0-5 ˚C. Stirring was continued for 4 h at 0-
5 ˚C. The reaction was allowed to warm to room temperature and
stirred overnight. The reaction mixture was diluted with 2 mL of
chloroform and washed successively with 1 N sulfuric acid and
water, then dried and concentrated under reduced pressure. The
crude product was purified by silica gel column chromatography
(dichloromethane/ethanol 60:1, v/v).
(APCI) m/z: C37H56O6 [M - H]−, Calcd. 595.3998; Found 595.3828.
ꢀ
3,28-O,O -diacetyl-30-propynoylbetulin
6
Yield 46%; mp 128-
cm−1
130˚C; Rf 0.43 (dichloromethane/ethanol, 60:1, v/v); IR (ν
max
KBr): 3228, 2951, 2112, 1732, 1721, 1452, 1247; 1H NMR (600 MHz,
CDCl3) δ ppm: 0.81 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.86 (s, 3H,
CH3), 0.99 (s, 3H, CH3), 1.07 (s, 3H, CH3), 2.07 (s, 3H, CH3C=O),
ꢀ
3,28-O,O -diacetyl-30-ethoxycarbonylbetulin 10 Yield 79%; mp 69-
72˚C; Rf 0.39 (dichloromethane/ethanol, 60:1, v/v); IR (ν
cm−1
max
KBr): 2947, 1739, 1467, 1244; 1H NMR (600 MHz, CDCl3) δ ppm:
0.76 (s, 3H, CH3), 0.77 (s, 3H, CH3), 0.78 (s, 3H, CH3), 0.89 (s, 3H,
CH3), 0.98 (s, 3H, CH3), 1.25 (t, 3H, J = 7.2 Hz, OCH2CH3), 1.98
(s, 3H, CH3C=O), 2.00 (s, 3H, CH3C=O), 2.33 (m, 1H, H-19), 3.76
(d, 1H, J = 10.8 Hz, H-28), 4.15 (m, 3H, OCH2, H-28), 4.39 (m, 1H,
H-3), 4.53 (s, 2H, CH2, C-30), 4.91 (s, 1H, H-29), 4.93 (s, 1H, H-
29); 13C NMR (150 MHz, CDCl3) δ ppm: 13.26, 13.32, 13.64, 15.01,
15.13, 15.48, 17.12, 19.85, 20.02, 20.31, 22.66, 25.52, 25.96, 26.91,
28.70, 29.98, 33.09, 33.30, 36.02, 36.43, 36.77, 37.34, 39.87, 41.62,
45.28, 48.41, 49.16, 54.31, 61.42, 62.76, 63.04, 68.09, 79.85, 110.15,
147.20, 154.06, 170.14, 170.58. HR-MS (APCI) m/z: C37H58O7 [M -
H]−, Calcd. 613.4104; Found 613.3933.
≡
2.09 (s, 3H, CH3C=O), 2.41 (m, 1H, H-19), 2.94 (s, 1H, C CH), 3.84
(d, 1H, J = 10.8 Hz, H-28), 4.26 (d, 1H, J = 10.2 Hz, H-28), 4.48 (m,
1H, H-3), 4.68 (m, 2H, CH2, C-30), 5.03 (s, 1H, H-29), 5.04 (s, 1H,
H-29); 13C NMR (150 MHz, CDCl3) δ ppm: 14.74, 16.05, 16.17, 16.50,
18.15, 20.89, 21.06, 21.34, 23.69, 26.59, 26.99, 27.94, 29.74, 34.13,
34.37, 37.05, 37.50, 37.80, 38.39, 40.91, 42.68, 46.33, 49.58, 50.22,
55.35, 62.44, 74.56, 75.02, 80.89, 152.45, 171.06, 171.63. HR-MS
(APCI) m/z: C37H54O6 [M - H]−, Calcd. 593.3842; Found 593.3673.
ꢀ
3,28-O,O -diacetyl-30-(3-cyclopropyl-2-propynoyl)betulin
7 Yield
41%; mp 130-133˚C; Rf 0.48 (dichloromethane/ethanol, 60:1, v/v);
IR (ν
cm−1 KBr): 3660, 2958, 2227, 1735, 1728, 1710, 1483,
max
1251; 1H NMR (600 MHz, CDCl3) δ ppm: 0.82 (s, 3H, CH3), 0.85
(s, 3H, CH3), 0.86 (s, 3H, CH3), 0.95 (s, 3H, CH3), 1.05 (s, 3H,
CH3), 1.22-1.30 (m, 5H, CH, CH2), 2.07 (s, 3H, CH3C=O), 2.10 (s,
3H, CH3C=O), 2.41 (m, 1H, H-19), 3.84 (d, 1H, J = 10.8 Hz, H-28),
4.25 (d, 1H, J = 10.8 Hz, H-28), 4.48 (m, 1H, H-3), 4.65 (m, 2H,
CH2, C-30), 5.01 (both br. s, 2H, H-29); 13C NMR (150 MHz, CDCl3)
δ ppm: -1.56, 8.28, 13.70, 15.01, 15.13, 15.47, 17.11, 19.84, 20.04,
20.33, 22.64, 25.44, 25.94, 26.92, 28.67, 33.08, 33.31, 36.00, 36.43,
36.75, 37.34, 39.85, 41.62, 45.25, 48.47, 49.18, 54.33, 61.44, 67.31,
79.86, 92.77, 152.53, 170.04, 170.64. HR-MS (APCI) m/z: C40H58O6
[M - H]−, Calcd. 633.4155; Found 633.3929.
ꢀ
3,28-O,O -diacetyl-30-propoxycarbonylbetulin 11 Yield 81%; mp
65-69˚C; Rf 0.45 (dichloromethane/ethanol, 60:1, v/v); IR (ν
max
cm−1 KBr): 2962, 1743, 1734, 1712, 1458, 1246; 1H NMR (600 MHz,
CDCl3) δ ppm: 0.85 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.90 (t, 3H,
J = 7.2 Hz, CH2CH3), 0.98 (s, 3H, CH3), 0.99 (s, 3H, CH3), 1.05 (s,
3H, CH3), 1.73 (m, 2H, CH2CH3), 2.06 (s, 3H, CH3C=O), 2.11 (s, 3H,
CH3C=O), 2.40 (m, 1H, H-19), 3.84 (d, 1H, J = 11.4 Hz, H-28), 4.11
(t, 2H, J = 6.6 Hz, OCH2), 4.26 (d, 1H, J = 10.8 Hz, H-28), 4.47 (m,
1H, H-3), 4.62 (s, 2H, CH2, C-30), 5.00 (s, 1H, H-29), 5.02 (s, 1H, H-
29); 13C NMR (150 MHz, CDCl3) δ ppm: 9.18, 13.10, 13.64, 15.01,
15.13, 15.47, 17.12, 20.02, 20.31, 21.01, 21.64, 22.66, 25.51, 25.97,
26.92, 28.70, 29.97, 30.57, 33.09, 33.31, 36.02, 36.44, 36.77, 37.34,
39.87, 41.62, 45.29, 48.40, 49.17, 54.32, 61.42, 68.41, 68.64, 79.86,
110.16, 147.23, 154.19, 170.00, 170.58. HR-MS (APCI) m/z: C38H60O7
[M - H]−, Calcd. 627.4260; Found 627.4083.
ꢀ
3,28-O,O -diacetyl-30-(2-butenoyl)betulin 8 Yield 76%; mp 79-
82˚C; Rf 0.37 (dichloromethane/ethanol, 60:1, v/v); IR (ν
cm−1
max
KBr): 3695, 2945, 1737, 1726, 1465, 1246; 1H NMR (600 MHz,
CDCl3) δ ppm: 0.76 (3H, s, CH3), 0.77 (3H, s, CH3), 0.78 (3H, s,
CH3), 0.89 (3H, s, CH3), 0.96 (3H, s, CH3), 1.83 (3H, m, CH=CHCH3),
1.97 (s, 3H, CH3C=O), 2.00 (s, 3H, CH3C=O), 2.30 (m, 1H, H-19),
3.75 (d, 1H, J = 10.8 Hz, H-28), 4.17 (d, 1H, J = 10.2 Hz, H-28),
4.39 (m, 1H, H-3), 4.54 (m, 2H, CH2, C-30), 4.89 (both br. s, 2H, H-
29), 5.81 (m, 1H, CH=CHCH3), 6.94 (1H, m, CH=CHCH3); 13C NMR
(150 MHz, CDCl3) δ ppm: 14.75, 16.05, 16.17, 16.51, 18.09, 18.16,
20.89, 21.07, 21.35, 23.69, 26.54, 27.02, 27.95, 29.73, 31.13, 34.12,
34.36, 37.06, 37.50, 37.81, 38.38, 40.91, 42.66, 43.85, 46.31, 49.46,
50.21, 55.34, 62.50, 65.74, 80.89, 110.46, 122.52, 145.12, 148.93,
166.21, 171.05, 171.65. HR-MS (APCI) m/z: C38H58O6 [M - H]−, Calcd.
609.4155; Found 609.3988.
ꢀ
3,28-O,O -diacetyl-30-allyloxycarbonylbetulin 12 Yield 83%; mp
72-75˚C; Rf 0.49 (dichloromethane/ethanol, 60:1, v/v); IR (ν
max
cm−1 KBr): 2949, 1735, 1458, 1251; 1H NMR (600 MHz, CDCl3)
δ ppm: 0.85 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.87 (s, 3H, CH3),
0.99 (s, 3H, CH3), 1.05 (s, 3H, CH3), 2.07 (s, 3H, CH3C=O), 2.09
(s, 3H, CH3C=O), 2.41 (m, 1H, H-19), 3.84 (d, 1H, J = 11.4 Hz, H-
28), 4.26 (d, 1H, J = 11.4 Hz, H-28), 4.49 (m, 1H, H-3), 4.63 (s, 2H,
CH2, C-30), 4.66 (m, 2H, OCH2), 5.01 (s, 1H, H-29), 5.02 (s, 1H, H-
29), 5.31 (m, 1H, CH=CH2), 5.40 (m, 1H, CH=CH2), 5.97 (m, 1H,
CH=CH2); 13C NMR (150 MHz, CDCl3) δ ppm: 13.65, 15.01, 15.13,
15.47, 17.12, 19.86, 20.02, 20.31, 22.66, 25.54, 25.96, 26.91, 28.70,
29.99, 33.09, 33.30, 36.02, 36.42, 36.77, 37.34, 39.87, 41.62, 45.29,
48.42, 49.16, 54.31, 61.41, 67.48, 68.36, 79.86, 110.23, 117.91, 130.57,
147.12, 153.89, 170.00, 170.57. HR-MS (APCI) m/z: C38H58O7 [M -
H]−, Calcd. 625.4104; Found 625.3868.
ꢀ
3,28-O,O -diacetyl-30-(2-butynoyl)betulin 9 Yield 72%; mp 83-
86˚C; Rf 0.46 (dichloromethane/ethanol, 60:1, v/v); IR (ν
KBr): 3298, 2941, 2243, 1743, 1726, 1703, 1456, 1247; 1H NMR (600
cm−1
max
MHz, CDCl3) δ ppm: 0.76 (s, 3H, CH3), 0.77 (s, 3H, CH3), 0.78 (s,
ꢀ
≡
3H, CH3), 0.89 (s, 3H, CH3), 0.96 (s, 3H, CH3), 1.94 (s, 3H, C CCH3),
3,28-O,O -diacetyl-30-propargyloxycarbonylbetulin 13 Yield 76%;
1.97 (s, 3H, CH3C=O), 2.00 (s, 3H, CH3C=O), 2.32 (m, 1H, H-19),
3.75 (d, 1H, J = 10.8 Hz, H-28), 4.17 (d, 1H, J = 10.8 Hz, H-28),
4.41 (m, 1H, H-3), 4.55 (m, 2H, CH2, C-30), 4.92 (both br. s, 2H,
H-29); 13C NMR (150 MHz, CDCl3) δ ppm: 3.90, 14.71, 16.05, 16.17,
16.52, 18.16, 20.89, 21.07, 21.35, 23.69, 26.56, 27.00, 27.95, 29.73,
mp 56-60˚C; Rf 0.47 (dichloromethane/ethanol, 60:1, v/v); IR (ν
max
cm−1 KBr): 3279, 2963, 2131, 1734, 1457, 1261; 1H NMR (600 MHz,
CDCl3) δ ppm: 0.81 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.86 (s, 3H,
CH3), 0.98 (s, 3H, CH3), 1.05 (s, 3H, CH3), 2.06 (s, 3H, CH3C=O),
2.09 (s, 3H, CH3C=O), 2.39 (m, 1H, H-19), 2.55 (t, 1H, J= 1.2 Hz,
3

E. Chrobak, E. Be˛benek, K. Marciniec et al.
Journal of Molecular Structure 1226 (2021) 129394
≡
C
CH), 3.83 (d, 1H, J = 11.4 Hz, H-28), 4.25 (d, 1H, J = 10.8 Hz, H-
1H, J = 10.8 Hz, H-28), 4.26 (d, 1H, J = 10.8 Hz, H-28), 4.48 (m,
1H, H-3), 4.83 (m, 2H, CH2, C-30), 5.01 (s, 1H, H-29), 5.04 (s, 1H,
H-29), 5.61 (2H, s, CH2), 7.32-7.33 (2H, m, HAr), 7.41-7.44 (3H, m,
HAr), 7.99 (1H, s, CH-triazole); 13C NMR (150 MHz, CDCl3) δ ppm:
14.75, 16.05, 16.17, 16.51, 18.16, 20.88, 21.04, 21.33, 23.69, 26.61,
27.01, 27.95, 29.75, 31.16, 34.13, 34.36, 37.06, 37.49, 37.81, 38.37,
40.91, 42.67, 46.33, 49.55, 50.20, 53.43, 54.55, 55.35, 62.47, 80.90,
110.72, 127.34, 128.28, 129.20, 129.36, 133.69, 148.23, 160.32,
171.02, 171.58. HR-MS (APCI) m/z: C44H60N3O6 [M - H]−, Calcd.
726.4482; Found 726.4544.
28), 4.48 (m, 1H, H-3), 4.66 (s, 2H, CH2, C-30), 4.78 (m, 2H, OCH2),
5.02 (both br. s, 2H, H-29); 13C NMR (150 MHz, CDCl3) δ ppm:
14.68, 15.24, 16.04, 16.17, 16.51, 18.16, 20.89, 21.07, 21.36, 23.68,
27.95, 29.72, 31.06, 34.12, 34.32, 37.05, 37.44, 37.80, 38.37, 40.90,
42.65, 46.32, 49.48, 50.19, 55.31, 55.34, 55.72, 62.44, 69.95, 75.72,
76.02, 80.90, 111.53, 147.89, 154.49, 171.07, 171.50. HR-MS (APCI)
m/z: C38H56O7 [M - H]−, Calcd. 623.3947; Found 623.3726.
ꢀ
3,28-O,O -diacetyl-30-butyryloxycarbonylbetulin 14 Yield 77%; mp
62-65˚C; Rf 0.51 (dichloromethane/ethanol, 60:1, v/v); IR (ν
max
cm−1 KBr): 3456, 2956, 1741, 1732, 1467, 1244; 1H NMR (600 MHz,
CDCl3) δ ppm: 0.76 (s, 3H, CH3), 0.77 (s, 3H, CH3), 0.78 (s, 3H,
CH3), 0.87 (t, 3H, J = 7.2 Hz, CH2CH2CH3), 0.89 (s, 3H, CH3), 0.96
(s, 3H, CH3), 1.34 (m, 4H, CH2CH2CH3), 1.98 (s, 3H, CH3C=O), 2.00
(s, 3H, CH3C=O), 2.31 (m, 1H, H-19), 3.75 (d, 1H, J = 10.8 Hz, H-
28), 4.09 (m, 2H, OCH2), 4.17 (d, 1H, J = 11.4 Hz, H-28), 4.39 (m,
1H, H-3), 4.53 (s, 2H, CH2, C-30), 4.91 (both br. s, 2H, H-29); 13C
NMR (150 MHz, CDCl3) δ ppm: 13.71, 14.68, 16.05, 16.16, 16.51,
18.16, 18.94, 20.89, 21.07, 21.35, 23.69, 26.55, 27.00, 27.95, 29.73,
30.71, 31.01, 34.12, 34.34, 37.05, 37.46, 37.80, 38.37, 40.91, 42.65,
43.70, 46.32, 49.43, 50.20, 55.35, 62.46, 67.99, 69.11, 80.90, 111.19,
148.26, 155.23, 171.05, 171.63. HR-MS (APCI) m/z: C39H62O7 [M -
H]−, Calcd. 641.4417; Found 641.4234.
3,28-O,O -diacetyl-30-[1-(4-fluorobenzyl-1H-1,2,3-triazol-
ꢀ
4-yl]carbonylbetulin 18 Yield 78%; mp 115-118˚C; Rf 0.43
(dichloromethane/ethanol, 40:1, v/v); IR (ν
cm−1 KBr):
max
2947, 1732, 1456, 1246; 1H NMR (600 MHz, CDCl3) δ ppm: 0.76
(3H, s, CH3), 0.77 (s, 3H, CH3), 0.78 (s, 3H, CH3), 0.88 (s, 3H, CH3),
0.91 (s, 3H, CH3), 1.99 (s, 3H, CH3C=O), 2.01 (s, 3H, CH3C=O), 2.32
(m, 1H, H-19), 3.74 (d, 1H, J = 10.8 Hz, H-28), 4.16 (d, 1H, J = 10.8
Hz, H-28), 4.39 (m, 1H, H-3), 4.75 (m, 2H, CH2, C-30), 4.92 (s,
1H, H-29), 4.95 (s, 1H, H-29), 5.50 (s, 2H, CH2), 7.01-7.04 (2H, m,
HAr), 7.29-7.38 (2H, m, HAr), 7.90 (1H, s, CH-triazole); 13C NMR
(150 MHz, CDCl3) δ ppm: 14.21, 14.74, 16.04, 16.17, 16.51, 18.15,
20.87, 21.06, 21.35, 23.68, 26.99, 27.95, 29.72, 34.11, 34.34, 37.04,
37.47, 37.80, 38.37, 40.90, 42.66, 46.32, 50.19, 53.80, 55.34, 60.42,
62.45, 80.89, 116.35, 116.50, 127.24, 130.19, 130.24, 171.05, 171.62.
HR-MS (APCI) m/z: C44H59FN3O6 [M - H]−, Calcd. 744.4388; Found
744.4432.
ꢀ
3,28-O,O -diacetyl-30-(3-butynyloxycarbonyl)betulin 15 Yield 76%;
oil; Rf 0.44 (dichloromethane/ethanol, 60:1, v/v); IR (ν
KBr): 3307, 2943, 1743, 1460, 1247; 1H NMR (600 MHz, CDCl3) δ
ppm: 0.76 (s, 3H, CH3), 0.77 (s, 3H, CH3), 0.78 (s, 3H, CH3), 0.89
cm−1
max
ꢀ
3,28-O,O -diacetyl-30-[1-(4-cyanobenzyl-1H-1,2,3-triazol-4-
≡
(s, 3H, CH3), 0.96 (s, 3H, CH3), 1.96 (t, 1H, J = 2.4 Hz, C CH), 1.97
yl]carbonylbetulin 19 Yield 52%; mp 104-107˚C; Rf 0.36
(s, 3H, CH3C=O), 2.00 (s, 3H, CH3C=O), 2.31 (m, 1H, H-19), 2.52
(m, 2H, OCH2CH2), 3.75 (d, 1H, J = 10.8 Hz, H-28), 4.17 (m, 4H,
OCH2, H-28), 4.39 (m, 1H, H-3), 4.54 (s, 2H, CH2, C-30), 4.92 (both
br. s, 2H, H-29); 13C NMR (150 MHz, CDCl3) δ ppm: 14.24, 14.69,
16.04, 16.17, 16.50, 18.16, 19.08, 20.89, 21.06, 21.34, 23.68, 26.60,
26.99, 27.95, 29.73, 31.04, 34.12, 34.33, 37.05, 37.45, 37.80, 38.37,
40.91, 42.66, 46.32, 49.46, 50.20, 55.35, 62.43, 65.44, 65.55, 70.21,
70.29, 79.33, 80.89, 111.26, 148.05, 154.59, 171.04, 171.61. HR-MS
(APCI) m/z: C39H58O7 [M - H]−, Calcd. 637.4104; Found 637.3846.
(dichloromethane/ethanol, 40:1, v/v); IR (ν
cm−1 KBr):
max
2949, 1732, 1458, 1246; 1H NMR (600 MHz, CDCl3) δ ppm: 0.85
(3H, s, CH3), 0.86 (s, 3H, CH3), 0.87 (s, 3H, CH3), 0.96 (s, 3H, CH3),
1.05 (s, 3H, CH3), 2.07 (s, 3H, CH3C=O), 2.09 (s, 3H, CH3C=O), 2.43
(m, 1H, H-19), 3.85 (d, 1H, J = 10.8 Hz, H-28), 4.24 (d, 1H, J = 10.8
Hz, H-28), 4.48 (m, 1H, H-3), 4.85 (m, 2H, CH2, C-30), 5.03 (s, 1H,
H-29), 5.06 (s, 1H, H-29), 5.69 (2H, s, CH2), 7.40-7.41 (2H, m, HAr),
7.72-7.73 (2H, m, HAr), 8.08 (1H, s, CH-triazole); 13C NMR (150
MHz, CDCl3) δ ppm: 14.75, 16.05, 16.17, 16.50, 18.15, 20.89, 21.05,
21.33, 23.68, 26.67, 27.00, 27.95, 29.73, 34.13, 34.35, 37.05, 37.49,
37.80, 38.40, 40.91, 42.67, 46.33, 49.62, 50.22, 53.71, 55.37, 62.43,
80.88, 117.93, 127.53, 128.54, 133.09, 138.84, 160.06, 171.04, 171.62.
HR-MS (APCI) m/z: C45H59N4O6 [M - H]−, Calcd. 751.4435; Found
751.4421.
ꢀ
3,28-O,O -diacetyl-30-(2-butynyloxycarbonyl)betulin 16 Yield 73%;
mp 67-70˚C; Rf 0.47 (dichloromethane/ethanol, 60:1, v/v); IR (ν
max
cm−1 KBr): 3660, 2947, 1739, 1726, 1708, 1465, 1244; 1H NMR (600
MHz, CDCl3) δ ppm: 0.85 (s, 3H, CH3), 0.86 (s, 3H, CH3), 0.87 (s,
3H, CH3), 0.97 (s, 3H, CH3), 1.05 (s, 3H, CH3), 1.88 (t, 3H, J = 2.4
≡
Hz, C CCH3), 2.06 (s, 3H, CH3C=O), 2.09 (s, 3H, CH3C=O), 2.39 (m,
1H, H-19), 3.83 (d, 1H, J = 10.8 Hz, H-28), 4.26 (d, 1H, J = 11.4
Hz, H-28), 4.47 (m, 1H, H-3), 4.64 (s, 2H, CH2, C-30), 4.73 (m, 2H,
OCH2), 5.02 (both br. s, 2H, H-29); 13C NMR (150 MHz, CDCl3)
δ ppm: 3.72, 14.69, 16.04, 16.17, 16.50, 18.16, 20.89, 21.06, 21.35,
23.69, 26.60, 26.99, 27.95, 29.73, 31.04, 34.12, 34.32, 37.05, 37.45,
37.80, 38.37, 40.91, 42.66, 46.32, 49.44, 50.19, 55.35, 56.23, 62.44,
72.62, 80.90, 84.11, 110.17, 147.99, 154.67, 171.06, 171.61. HR-MS
(APCI) m/z: C39H58O7 [M - H]−, Calcd. 637.4104; Found 637.3904.
2.2. In silico study
The ADME properties of new 30-substituted derivatives of
ꢀ
3,28-O,O -diacetylbetulin 3-19 like molecular weight (M), hydrogen
bond acceptors (nHBA), hydrogen bond donors (nHBD), lipophilic-
ity (cLogP), rotatable bonds (nROTB) and topological polar surface
area (TPSA) were predicted using the ACD/Labs software [26].
The target macromolecule for molecular docking studies were
obtained from the Protein Data Bank (https://www.rcsb.org/). In
our studies we applied 3D crystal structure of Akt1 (PDB ID:
3QKK). The three-dimensional structures of tested triterpenes re-
quired were generated in their low-energy conformation using
Gaussian 16 (revision A.03) computer code [27] at the density
functional theory (DFT, B3LYP) and 6-311 + G(d,p) basis sets. In
docking study Genetic Optimisation for Ligand Docking (GOLD)
5.6.3 was used [28]. The docking was defined for all the protein
2.1.4. General procedure for the synthesis of derivatives 17-19
The appropriate organic azide (0.18 mmola) was added to the
ꢀ
mixture of 3,28-O,O -diacetyl-30-propynoylbetulin 6 (0.1 g, 0.17
mmola) and copper(I) iodide (0.003 g, 0.001 mmola) in toluene
(3.3 mL). Stirring was continued under reflux for 72 h. The solvent
was removed under reduced pressure and the residue was puri-
fied by silica gel column chromatography using different mixtures
of organic solvents.
ꢀ
˚
3,28-O,O -diacetyl-30-(1-benzyl-1H-1,2,3-triazol-4-
residues within 10 A of the reference ligand that accompanied the
yl)carbonylbetulin 17 Yield 66%; mp 91-93˚C; Rf 0.43 (heksane/ethyl
downloaded protein complex. For all other parameters a default
values were used and the complexes were submitted to 10 genetic
algorithm runs using the CHEMPLP fitness function. The BIOVIA
Discovery Studio virtual environment was used to visualize molec-
ular docking details [29].
acetate, 3:2, v/v); IR (ν
cm−1 KBr): 2946, 1734, 1465, 1246;
max
1H NMR (600 MHz, CDCl3) δ ppm: 0.86 (3H, s, CH3), 0.87 (s, 3H,
CH3), 0.88 (s, 3H, CH3), 0.99 (s, 3H, CH3), 1.08 (s, 3H, CH3), 2.07 (s,
3H, CH3C=O), 2.09 (s, 3H, CH3C=O), 2.43 (m, 1H, H-19), 3.83 (d,
4

E. Chrobak, E. Be˛benek, K. Marciniec et al.
Journal of Molecular Structure 1226 (2021) 129394
Scheme 1. Synthesis of derivatives 2-16. Reagents and conditions: (a) AcO2, pyridine, rt, 18 h; (b) m-CPBA, CHCl3, reflux, 8 h; (c) RCOOH, CH2Cl2, DCC, DMAP, rt, 24 h or
ROC(O)Cl, benzene, pyridine, rt, 24 h.
2.3. Anticancer activity – WST-1 assay
the C-30 position of compound 2 with m-chloroperbenzoic acid
(m-CPBA) in refluxing chloroform led to the formation of 3,28-
O,O’-diacetyl-30-hydroxybetulin 3 at a 52% yield [32]. Derivatives
4-9 were prepared using the Steglich-type esterification of 3,28-
O,O’-diacetyl-30-hydroxybetulin 3 with various carboxylic acids in
dichloromethane in the presence of DMAP and DCC. The com-
pounds 10-16 were obtained by the esterification of hydroxyl group
at the C-30 position with chloroformates in benzene in the pres-
ence of pyridine. The synthesis of 30-substituted esters of 3,28-
O,O’-diacetylbetulin 4-16 is outlined in Scheme 1.
2.3.1. Cell culture
Antiproliferative activity of the tested triterpenes was as-
sessed in vitro using following cultured cell lines: human glioblas-
toma SNB-19 (DSMZ, Braunschweig, Germany), human amelanotic
melanoma C-32 (ATCC, Rockville, USA), human ovarian adenocarci-
noma SKOV-3 (ATCC, Manassas, VA, USA), human breast adenocar-
cinoma T47D (ATCC, Manassas, VA, USA) and human breast adeno-
carcinoma MCF-7 (DSMZ, Braunschweig, Germany). The cells were
inoculated into 96-well plates (Corning). Each well contained an
equal number of cells 5 × 103 per well and 100 μL DMEM sup-
plemented with 10% fetal bovine serum (FBS, Biological Industries
Cromwell, CT, US) and antibiotics (streptomycin-10 mg/mL and
penicillin-10,000 U/mL, Lonza, Basel, Switzerland). The cells were
incubated at 37˚C in a 5% CO2, 95% air humidified atmosphere for
24 hours.
The alkynyl group is an important structural moiety occurring
in the many biologically active compounds. Alkynyl-functionalized
derivatives have widespread application in the synthetic trans-
formations like cycloaddition and cross-coupling reactions
[33–36].
The next stage was to synthesis 30-substituted triazoles of 3,28-
O,O’-diacetylbetulin. The 30-propynoylated derivative 6 was trans-
formed to triazoles 17-19 via Huisgen 1,3-cycloaddition reaction.
Application of various organic azides facilitated the introduction
of a new moiety into the triazole ring, which may affect the
anticancer activity and its mechanism of action [15,37–39]. Final
1,4-disubstituted triazoles were produced from reaction of alkynyl
derivative 6 with organic azides in toluene in the presence of cop-
per(I) iodide under reflux. The preparation of 30-substituted tria-
zolyl derivatives 17-19 is depicted in Scheme 2.
2.3.2. WST-1 assay
Measurement of cell lines growth inhibition was performed in
vitro using the WST-1 test (Roche). The tested compounds were
added at four concentrations in the range of 0.1-100 μg/mL and
incubated for 72 hours. At the end of the incubation, the WST-1
assay was performed using a method described by Tominaga et al
[30]. The absorbance was measured against a back-ground control
at 450 nm using a the UVM340 microplate reader (Biogenet, Józe-
fów, Poland) with a reference wavelength of 600 nm. Each experi-
ment was performed triplicate. Cisplatin was applied as a reference
compound in the study of antiproliferative activity of the tested
triterpenes.
The obtained compounds were purified by column chromatog-
raphy using the mixture of various organic solvents. The chem-
ꢀ
ical structures of new 30-substitued derivatives of 3,28-O,O -
diacetylbetulin 4-19 were determined on the basis of their 1H-
NMR, 13C-NMR, IR and HR-MS spectra.
3. Results and discussion
3.2. ADME properties and molecular docking study
3.1. Synthesis
The ADME (absorption, distribution, metabolism, excretion)
studies are considered to be the major pharmacokinetic param-
eters of the compounds as drug candidates [40,41]. The pharma-
cokinetic properties of the triterpenes 3-19 were computed by us-
ing the ACD/Labs software [26]. The molecular weight (M), the
number of hydrogen bond acceptors (nHBA), the number of hydro-
The synthesis of derivatives 4-16 was achieved using betulin
1 as the substrate. Triterpene 1 was easily converted into 3,28-
O,O’-diacetylbetulin 2 using acetic anhydride in pyridine at room
temperature [31]. The allylic oxidation of the isopropenyl group at
5

E. Chrobak, E. Be˛benek, K. Marciniec et al.
Journal of Molecular Structure 1226 (2021) 129394
Scheme 2. Synthesis of triazoles 17-19. Reagents and conditions: (a) RN3, CuI, toluene, reflux
Table 1
Predicted the physicochemical parameters of compounds 3-19.
Compound
Molecular weighta
nHBAb
nHBDc
cLogPd
nROTBe
TPSAf
3
542.80
598.86
596.85
594.83
634.89
610.87
608.86
614.86
628.89
626.87
624.86
642.92
638.88
638.88
727.98
745.97
752.99
5
6
6
6
6
6
6
7
7
7
7
7
7
7
9
9
10
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
7.11
8.07
7.85
7.29
8.07
8.24
7.68
8.29
8.68
8.46
7.90
9.07
8.29
8.29
8.61
8.75
8.49
5
7
7
6
6
7
6
7
8
8
7
9
8
7
9
9
9
72.83
78.90
78.90
78.90
78.90
78.90
78.90
88.13
88.13
88.13
88.13
88.13
88.13
88.13
106.86
106.86
130.65
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
a
molecular weight (M)
b
c
d
e
f
number of hydrogen bond acceptors (nHBA)
number of hydrogen bond donors (nHBD)
lipophilicity (cLogP)
number of rotatable bonds (nROTB)
topological polar surface area (TPSA)
gen bond donors (nHBD), lipophilicity (cLogP), values of rotatable
bonds (nROTB), and topological polar surface area (TPSA) are tab-
ulated in Table 1. The obtained derivatives had fewer than ten hy-
drogen bond acceptors (nHBA) and less than five hydrogen bond
donors (nHBD), according to the rule of five. The compounds 3-19
violated Lipinski’s rule of five due to their high values of molec-
ular weight and cLogP. The number of rotatable bonds are less
than ten of what affects the large one conformational flexibility of
tested compounds. The high values of the rotatable bonds can lead
to good affinity of a variety of intracellular enzymes with binding
pocket of the triterpenes [42]. The number of nHBA (≤ 10) and
nHBD (≤ 5) and value of the topological polar surface area (TPSA
Following our previous article on 3-substituted derivatives of
betulin as potential serine/threonine protein kinase inhibitors,
molecular modeling was carried out to predict the possible mode
of action of the newly synthesized compounds 3-19 [45]. As a re-
sult of docking the computational models of 30-substituted betulin
derivatives to the active site of Akt1, 17 possible ligand-protein
complexes were obtained. All docked compounds were located in
similar spaces inside the active site. The superposition of stud-
ied compounds inside Akt1 (poses) was scored according to their
binding interactions with amino acids. Docking solutions with the
highest total score corresponded to the highest binding affinities
and present the most probable systems in vivo. According to the
obtained results, compounds 13-16 show the highest degree of fit
defined in the arbitrary units (a.u.) of the GOLD program (Table 2).
Comparing these results with those previously obtained for 3-
substituted betulin derivatives, it can be stated that the new com-
pounds are arranged in a similar way at the active site of the pro-
tein [45]. The chemistry of the active site cavity in enzyme shows
that the acetyl group at C-3 of the betulin moiety of compounds 3
and 13-16 has deeply penetrated the strong hydrophobic cavity of
the CAT domain of Akt1 (Fig. 2 and Fig. 3).
˚
< 140 A) determined the high oral bioavailability of 30-substituted
ꢀ
derivatives of 3,28-O,O -diacetylbetulin 4-19.
The anticancer activity of triterpenes towards tumor cells is re-
lated to their interaction with various molecular targets [43]. Zhuo
et al. obtained a new acetyl derivative of betulin, which effectively
reduced the viability of cancer cells (Huh7). The described results
of the in vitro study demonstrated that this triterpene inhibited the
PI3K/Akt signaling pathway by decreasing the expression levels of
Akt [44].
6

E. Chrobak, E. Be˛benek, K. Marciniec et al.
Journal of Molecular Structure 1226 (2021) 129394
Table 2
The top-ranked CHEMPLP fitness score function obtained with GOLD for betulin deriva-
tives 3-19.
Compound
Docking Score [a.u.]
57.58
Compound
Docking Score [a.u.]
3
4
71.63
70.03
73.62
71.88
70.76
68.92
68.02
71.80
12
13
14
15
16
17
18
19
73.06
75.63
75.16
76.21
74.03
73.88
66.71
71.61
5
6
7
8
9
10
11
Fig. 2. Visualization of interactions between reference compound 3 inside the binding site of Akt 1.
Detailed analysis of the reference compound 3 complex (Fig. 2)
19), amelanotic melanoma (C-32), ovarian adenocarcinoma (SKOV-
3) and breast adenocarcinoma (MCF-7, T47D) using the WST-1 test.
The IC50 values (half-maximum inhibitory concentrations) of the
new 30-substituted triterpenes 4-20 are presented in Table 3. Cis-
platin was applied as the reference drug.
determined hydrogen bonding of the acetyl group of ligand with
THR160 residue of protein chain. In addition, the hydrophobic in-
teractions can also be seen. These interactions enhance the binding
affinity.
Hydrophobic interactions seem to be a major responsible factor
for the potent inhibitory activity of triterpenoids 13-16 [46]. Ac-
cording to data from the literature, ligands that bind their target
with high efficiency are more hydrophobic, and hydrophobic inter-
actions are a driving factor of increased ligand efficiency and help
in stabilizing the biochemical environments of target-drug complex
[47].
In the case of parent compound 3, a low anticancer activ-
ity was observed. Among the synthetized compounds 4-6, alkynyl
derivative 6 demonstrated the highest activity against MCF-7 cells
(IC50 = 7.06 μM). Unfortunately, the presence of a propanoyl, 2-
propenoyl and 2-butynoyl substituent at the C-30 position (com-
pounds 4, 5, and 9) caused complete disappearance of the anti-
cancer activity. Introduction of a 3-cyclopropyl-2-propynoyl group
at the C-30 position in compound 7 led to an increase in the ac-
tivity against T47D cell line. The C-30 substituted esters 11-13 had
significant IC50 values in the range of 2.74-8.49 μM towards SNB-19
cells. The obtained results demonstrated that compound 12 with
30-allyloxycarbonyl moiety was 4.8-fold more potent against this
cell line than cisplatin. Replacing the hydroxy group with the bu-
tyryloxycarbonyl moiety at the C-30 position produced a triter-
pene 14 with the highest activity against all tested cancer cell lines
(IC50 = 1.24-6.03 μM). Moreover, for alkynyl derivative 16 an in-
crease in anticancer activity against SNB-19, MCF-7 and T47D was
observed.
Fig. 4A–D present the possible interaction of compounds 13–
16 inside the binding pocket of Akt1 after 2D analysis, using
the function “analyze single complex” in the Discovery Studio
Visualizer. The figure shows the complexes of compounds 13–
16 inside the binding site of Akt1. Corresponding amino acids
that are significantly involved in the interactions are as fol-
lows: THR291, THR195, ASP292, GLU191 (carbon−hydrogen bond);
VAL164, LYS179, MET281, VAL164, LYS179, LEU295 (alkyl); PHE161
(pi-alkyl); and 26 amino acids (Van der Waals). In addition, con-
ventional hydrogen bond interactions (THR160, LYS179) influence
the increased stability of the complex.
The docking results are in line with cytotoxicity binding assay
findings. The most active compound 14, forms the largest number
of interactions identified by the GOLD program (Fig. 5).
Presence in the 1,2,3-triazole scaffold of the aromatic ring
bonded to electronegative groups often improve cytotoxic activity
[37,39]. It was observed that changing the 4-fluorobenzyl moiety
(triazole 18) with 4-benzyl moiety (triazole 17) led to the loss of
anticancer activity against SNB-19, C-32, SKOV-3 and MCF-7 cells.
Derivative 18 with a 4-fluorobenzyl substituent at the triazole ring
presented the highest activity towards tested cancer cells com-
pared with others 30-substituted triazoles.
3.3. Anticancer activity
The anticancer activity of all tested triterpenes was evaluated
against a panel of human cell lines including glioblastoma (SNB-
7

E. Chrobak, E. Be˛benek, K. Marciniec et al.
Journal of Molecular Structure 1226 (2021) 129394
Fig. 3. Hydrophobic view of selected compounds locations: (A) compound 13; (B) compound 14; (C) compound 15; (D) compound 16. For clarity, non-polar hydrogens are
invisible.
Table 3
In vitro antiproliferative activity of 30-substituted derivatives of betulin 3-19 towards a panel
of anticancer cells (IC50 in μM).
Compound
SNB-19
C-32
SKOV-3
MCF-7
T47D
3
94.71 5.03
Neg
Neg
Neg
21.54 2.15
Neg
Neg
4
Neg
Neg
Neg
5
Neg
Neg
Neg
Neg
Neg
6
9.61 3.36
39.36 2.87
9.31 2.14
Neg
17.43 1.78
Neg
11.26 3.09
13.34 1.59
43.62 1.99
Neg
7.06 2.34
8.57 2.12
13.14 1.69
Neg
13.35 1.51
3.98 1.05
26.26 3.84
Neg
7
8
17.14 2.16
Neg
9
10
30.69 6.28
8.25 1.62
2.74 1.14
8.49 2.29
1.24 0.40
21.77 2.17
4.38 1.88
Neg
17.29 4.59
38.43 2.48
7.74 2.37
21.09 3.68
6.03 1.54
44.48 4.32
8.91 2.80
Neg
23.84 2.68
79.06 5.74
25.81 2.65
27.49 2.28
1.83 0.71
33.93 4.04
Neg
15.19 2.73
13.66 1.41
12.68 1.50
13.36 2.06
1.82 0.57
21.22 2.71
3.05 0.78
Neg
17.95 3.45
15.91 3.94
10.10 3.68
12.29 3.21
3.09 1.20
32.21 4.91
4.51 1.09
48.72 3.87
25.27 1.71
Neg
11
12
13
14
15
16
17
Neg
18
75.01 3.97
Neg
23.03 4.95
80.41 5.64
14.39 2.33
78.69 10.92
Neg
15.46 3.12
89.02 5.81
17.53 2.66
19
Cisplatin
13.10 4.66
36.77 6.66
31.50 9.83
Neg - negative in the concentration used
8

E. Chrobak, E. Be˛benek, K. Marciniec et al.
Journal of Molecular Structure 1226 (2021) 129394
Fig. 4. Two-dimensional analysis of ligands 13–16 in the binding site of Akt1.
Fig. 5. Frequency distribution of hydrophobic and hydrogen bond interactions in complexes of compounds 3, 13-16 and Akt1.
4. Conclusion
was used for the prediction of physicochemical properties of triter-
penes. The values of the three parameters (nHBA, nHBD and TPSA)
for 30-substituted derivatives suggest that compounds 3-19 may
have high oral bioavailability. The obtained results showed that the
derivative 14 has promising anticancer activity and fits the active
site of Akt1 kinase well.
In summary, a library of sixteen triterpenes was obtained by
ꢀ
modification of the C-30 position of the 3,28-O,O -diacetylbetulin.
All compounds were evaluated for their anticancer activity against
SNB-19, C-32, SKOV-3, MCF-7, and T47D cells. The in silico method
9

E. Chrobak, E. Be˛benek, K. Marciniec et al.
Journal of Molecular Structure 1226 (2021) 129394
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CRediT authorship contribution statement
Elwira Chrobak: Formal analysis, Visualization, Writing - orig-
inal draft. Ewa Be˛benek: Conceptualization, Writing - review &
editing, Formal analysis, Methodology. Krzysztof Marciniec: Soft-
ware, Data curation. Monika Kadela-Tomanek: Formal analysis.
Szymon Siudak: Data curation. Małgorzata Latocha: Resources, In-
vestigation. Stanisław Boryczka: Supervision.
Acknowledgements
Energy-minimization calculations have been carried out using
resources provided by Wroclaw Centre for 818 Networking and Su-
percomputing (http://wcss.pl), Grant No. 382. This work was sup-
ported by the Medical University of Silesia in Katowice (grant
KNW-1-055/K/9/O and KNW-1-133/K/7/B).
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