Enhancing effect of cystamine in its amides with betulinic acid as antimicrobial and antitumor agent in vitro

Article abstract of DOI:10.1016/j.steroids.2019.04.004

Amides of betulinic acid with cystamine were synthesized to investigate their antimicrobial and antitumor activity, and their influence on the cell cycle and cell apoptosis. The former target amide (6) displayed cytotoxicity in CEM cell line after 72 h of treatment (IC₅₀ = 3.0 ± 0.7 μM; TI = 20), and induced apoptosis by caspase-3/7 activation in CEM cells. The latter target amide (9) displayed antimicrobial activity against Streptococcus mutans (MIC 3.125 μM; MBC 3.125 μM) and Bacillus cereus (MIC 25 μM; MBC 25 μM). The achieved results demonstrate enhancing of their biological activity over that of the parent compounds. However, two intermediate compounds (2 and 7) displayed either considerable cytotoxicity (2; 7.5 ± 0.8 μM; TI = 10, against G361) or antimicrobial activity (7; both against Actinomyces odontolycus and Clostridium perfrigens with MIC 12.5 μM and MBC 12.5 μM). The experimental data were compared with the in silico calculated physico-chemical and ADME parameters of the target compounds, including successful intermediates.

Full text of DOI:10.1016/j.steroids.2019.04.004

Accepted Manuscript
Enhancing effect of cystamine in its amides with betulinic acid as antimicrobial
and antitumor agent in vitro
Uladzimir Bildziukevich, Lucie Rárová, Lucie Janovská, David Šaman,
Zdeněk Wimmer
PII:
S0039-128X(19)30069-8
https://doi.org/10.1016/j.steroids.2019.04.004
STE 8399
DOI:
Reference:
Steroids
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Revised Date:
Accepted Date:
12 December 2018
27 March 2019
17 April 2019
Please cite this article as: Bildziukevich, U., Rárová, L., Janovská, L., Šaman, D., Wimmer, Z., Enhancing effect
of cystamine in its amides with betulinic acid as antimicrobial and antitumor agent in vitro, Steroids (2019), doi:
https://doi.org/10.1016/j.steroids.2019.04.004
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Enhancing effect of cystamine in its amides with betulinic acid as antimicrobial and antitumor
agent in vitro
Uladzimir Bildziukevich,a,b Lucie Rárová,c Lucie Janovská,d,e David Šaman,f and Zdeněk
Wimmer *,a,b
a University of Chemistry and Technology, Department of Chemistry of Natural Compounds,
Technická 5, 166 28 Prague 6, Czech Republic; E-mail: wimmerz@vscht.cz;
wimmer@biomed.cas.cz
b Institute of Experimental Botany AS CR, v.v.i., Isotope Laboratory, Vídeňská 1083, 142 20
Prague 4, Czech Republic
c Palacký University, Centre of the Region Haná for Biotechnological and Agricultural
Research, Department of Chemical Biology and Genetics, Šlechtitelů 27, 783 71 Olomouc,
Czech Republic
d Palacký University, Department of Microbiology, Faculty of Medicine and Dentistry,
Hněvotínská 3, 775 15 Olomouc, Czech Republic
e Palacký University & Institute of Experimental Botany AS CR, Laboratory of Growth
Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research,
Šlechtitelů 27, 783 71 Olomouc, Czech Republic
f Institute of Organic Chemistry and Biochemistry AS CR, v.v.i., Flemingovo náměstí 2, 166
10 Prague 6, Czech Republic
1

Abstract
Amides of betulinic acid with cystamine were synthesized to investigate their antimicrobial
and antitumor activity, and their influence on the cell cycle and cell apoptosis. The former
target amide (6) displayed cytotoxicity in CEM cell line after 72 h of treatment (IC50 = 3.0 ±
0.7 M; TI = 20), and induced apoptosis by caspase-3/7 activation in CEM cells. The latter
target amide (9) displayed antimicrobial activity against Streptococcus mutans (MIC 3.125
M; MBC 3.125 M) and Bacillus cereus (MIC 25 M; MBC 25 M). The achieved results
demonstrate enhancing of their biological activity over that of the parent compounds.
However, two intermediate compounds (2 and 7) displayed either considerable cytotoxicity
(2; 7.5 ± 0.8 M; TI = 10, against G361) or antimicrobial activity (7; both against
Actinomyces odontolycus and Clostridium perfrigens with MIC 12.5 µM and MBC 12.5 µM).
The experimental data were compared with the in silico calculated physico-chemical and
ADME parameters of the target compounds, including successful intermediates.
Keywords: Betulinic acid; cystamine; cytotoxicity; apoptosis; antimicrobial activity.
1. Introduction
Chemical biology of natural products is a challenging area of investigation. Increasing
importance has been paid to the investigation of different conjugates formed on the basis of
plant products for the possibility to study enhancement of biological effects of the target
conjugates in comparison with the biological effects of their parent components [1].
Betulinic acid is a natural product, found in a number of plants [2]. Even if it is practically
insoluble in water [3], it displays activity against HIV [4], cancer [5,6] and diabetes [7,8]. To
improve the pharmacological characteristics of betulinic acid, especially bioavailability, its
novel derivatives with polyamines, simple and complex aromatic amines or amino acids and
2

peptides have always been a challenge for researchers [9-11]. The anti-tumor effect of
betulinic acid and its derivatives was found against a variety of tumor cell lines, such as
malignant brain tumor, primitive neuroectodermal tumor [12], human chronic myelogenous
leukemia, and against most of prevalent human cancer types, such as cervical, prostate, breast,
lung or colorectal cancer [9-11].
The radioprotective activity of cysteamine was described as early as in 1953, and it relied on
its effect against the formation of OH radicals [13]. Both, cysteamine and its oxidized form,
cystamine, were used in clinical trials to treat radiation sickness in patients receiving
radiotherapy [14]. Cystamine also displays potent effect against HIV [14]. Cystamine and
cysteamine show anti-cancer effects, and they have been subjects of clinical trials for
neurodegenerative diseases [15]. Both compounds have been selected as potential practical
candidate drugs for treating these diseases on the basis of experimental animal work [16,17].
Cystamine gives sulfane sulfur (S0) when metabolized by diamine oxidase. Its beneficial
effects, including prevention of carcinogen-induced cancer, dementia, diabetes, lowering of
blood cholesterol, decreasing plasma homocysteine levels or preventing atherosclerosis and
heart disease have already been reported [15]. The secondary main reason for choosing
cystamine for this investigation consisted in its structure that mimics the structure of di- and
polyamines used for the structural modification of betulinic acid earlier [10].
The objective of the current investigation was (a) to study aspects of chemical biology with
amide conjugates of betulinic acid with cystamine, (b) to find out if the target compounds are
cytotoxic and cause cell apoptosis, (c) to find out if they display antimicrobial activity, and (d)
to find out if they enhance the effects of betulinic acid. Amide bond formation is easily
possible to form either at C(28) carboxyl group or at the short chained diacid (e.g., succinic)
hemiester formed at the C(3)-OH in advance. To support the obtained experimental data, in
3

silico calculation of physico-chemical and ADME parameters has been done to find potential
coincidence between the experimental and calculated data.
2. Material and Methods
2.1. General
The 1H NMR and the 13C NMR spectra were recorded on a Bruker AVANCE 600 MHz
spectrometer at 600.13 MHz and 150.90 MHz in deuterochloroform, using tetramethylsilane
(δ = 0.0) as internal reference. 1H NMR data are presented in the following order: chemical
shift (δ) expressed in ppm, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet), coupling constants in Hertz, number of protons. For unambiguous assignment of
both 1H and 13C signals 2D NMR 1H,13C gHSQC and gHMBC spectra were measured using
standard parameters sets and pulse programs delivered by producer of the spectrometer.
Infrared spectra (IR) were measured with a Nicolet 205 FT-IR spectrometer. Mass spectra
(MS) were measured with a Waters ZMD mass spectrometer in a positive ESI mode. Optical
rotations were measured on an Autopol IV instrument (Rudolph Research Analytical, USA) at
589 nm wavelength, and the values were corrected to 20 oC. A PE 2400 Series II CHNS/O
Analyzer (Perkin Elmer, USA) was used for simultaneous determination of C, H, and N
(accuracy of CHN determination better than 0.30 % abs.). TLC was carried out on silica gel
plates (Merck 60F254) and the visualization was performed by the UV detection and by
spraying with the methanolic solution of phosphomolybdic acid (5%) followed by heating.
For column chromatography, silica gel 60 (0.063-0.200 mm) from Merck was used. All
chemicals and solvents were purchased from regular commercial sources in analytical grade
and the solvents were purified by general methods before use. Betulinic acid was purchased
from Dr. Jan Šarek – Betulinines (www.betulinines.com).
4

2.2. (3β)-3-(Acetyloxy)lup-20(29)-en-28-oic acid (2)
Acetic anhydride (0.45 mL; 4.7 mmol; 1.44 eq), DMAP (52 mg; 0.43 mmol, 0.13 eq) and
EDIPA (1 mL) was added to a solution of betulinic acid (1.5 g; 3.3 mmol) in dry THF (10
ml). The reaction mixture was heated under reflux over 3 h. After stopping the reaction, water
(10 mL) was added, and the mixture was stirred for an additional 1 h. The resulting mixture
was extracted with chloroform, and dried over sodium sulfate. Evaporation of the solvent
afforded a solid and its purification by a column chromatography yielded 1.55 g (95 %) of 2.
1H NMR: δ 0.83 (3H, s, H23), 0.84 (3H, s, H25), 0.85 (3H, bs, H24), 0.93 (3H, s, H26), 0.97
(3H, bs, H27), 1.05 (2H, dq, J1 = 4.9 Hz, J2 = 13.0 Hz, J3 = 13.0 Hz, J4 = 13.0 Hz, H12), 1.19
(2H, dt, J1 = 3.2 Hz, J2 = 3.2 Hz, J3 = 13.6 Hz, H21), 1.70 (3H, dd, J1 = 0.7 Hz, J2 = 1.4 Hz,
H29), 2.04 (3H, s, OAc), 2.13 (2H, ddd, J1 = 3.7 Hz, J2 = 11.6 Hz, J3 = 12.8 Hz, H1), 2.27
(2H, dt, J1 = 3.8 Hz, J2 = 3.8 Hz, J3 = 13.1 Hz, H16), 3.01 (1H, dt, J1 = 5.0 Hz, J2 = 10.9 Hz, J3
= 10.9 Hz, H19), 4.47 (1H, dd, J1 = 5.5 Hz, J2 = 10.9 Hz, H3), 4.61 (1H, dq, J1 = 1.4 Hz, J2 =
1.4 Hz, J3 = 1.4 Hz, J4 = 2.3 Hz, H30), 4.74 (1H, dq, J1 = 0.7 Hz, J2 = 0.7 Hz, J3 = 0.7 Hz, J4 =
2.3 Hz, H30). 13C NMR: δ 14.64 (q, C27), 16.02 (q, C24), 16.17 (q, C25), 16.46 (q, C26),
18.15 (t, C6), 19.32 (q, OAc), 19.33 (q, C29), 20.84 (t, C11), 23.68 (t, C2), 25.43 (t, C12),
27.94 (q, C23), 29.68 (t, C21), 30.54 (t, C15), 32.13 (t, C16), 34.22 (t, C22), 37.03 (d, C13),
37.11 (s, C10), 37.79 (t, C7), 38.37 (s, C4), 38.39 (t, C1), 40.68 (s, C8), 42.41 (s, C14), 46.92
(d, C19), 49.25 (d, C18), 50.38 (d, C9), 55.40 (d, C5), 56.35 (s, C17), 80.94 (d, C3), 109.74 (t,
C30), 150.36 (s, C20), 171.06 (s, OAc), 181.47 (s, C28). MS (ES-): m/z 497.1 [M-H]-. IR
(KBr; cm-1): 2945 (C–H), 2871 (–CH3), 1735 (–C=O), 1694 (–C=O). For C32H50O4 (498.74)
calculated C (77.06), H (10.10), found C (77.09), H (10.08). M.p. 263-266 oC.
2.3. (3β)-28-{[2-({2-[(tert-Butoxycarbonyl)amino]ethyl}disulfanediyl)ethyl]-amino}-28-
oxolup-20(29)-en-3-yl acetate (3)
5

A solution of oxalyl chloride in dry dichloromethane (2M; 1.6 mL; 3.2 mmol; 8 eq) was
added to a solution of 2 (200 mg, 0.4 mmol) in dry dichloromethane (4 mL), and the reaction
mixture was stirred overnight. The reaction mixture was evaporated, and the residue was
dissolved in dry dichloromethane (3 mL). EDIPA (0.182 mL; 1 mmol; 2.6 eq) and Boc
protected cystamine hydrochloride (174 mg, 0.6 mmol; 1.5 eq) were added. The reaction
mixture was stirred at r.t. overnight. After stopping the reaction, evaporation of the solvent
afforded a solid. Its purification by column chromatography yielded 273 mg (93 %) of 3.
1H NMR: δ 0.82 (3H, s, H23), 0.84 (3H, d, J = 0.6 Hz, H24), 0.84 (3H, s, H25), 0.93 (3H, s,
H26), 0.96 (3H, s, H27), 1.45 (9H, s, Boc), 1.68 (3H, dd, J1 = 0.7 Hz, J2 = 1.4 Hz, H29), 1.80
(2H, bdd, J1 = 7.7 Hz, J2 = 12.0 Hz, H1), 1.94 (2H, ddt, J1 = 8.2 Hz, J2 = 10.7 Hz, J3 = 10.7
Hz, J4 = 13.2 Hz, H21), 2.04 (3H, s, OAc), 2.46 (1H, bdt, J1 = 3.5 Hz, J2 = 12.2 Hz, J3 = 12.2
Hz, H13), 2.74-2.79 (2H, m, H3'), 2.79-2.86 (2H, m, H2'), 3.11 (1H, dt, J1 = 4.5 Hz, J2 = 11.1
Hz, J3 = 11.1 Hz, H19), 3.38-3.52 (2H, m, H4'), 3.38-3.52 (1H, m, H1'), 3.65 (1H, dq, J1 = 5.9
Hz, J2 = 5.9 Hz, J3 = 5.9 Hz, J4 = 14.0 Hz, H1'), 4.47 (1H, dd, J1 = 6.2 Hz, J2 = 10.3 Hz, H3),
4.59 (1H, dq, J1 = 1.4 Hz, J2 = 1.4 Hz, J3 = 1.4 Hz, J4 = 2.4 Hz, H30), 4.73 (1H, dq, J1 = 0.7
Hz, J2 = 0.7 Hz, J3 = 0.7 Hz, J4 = 2.4 Hz, H30), 5.02 (1H, bt, J = 5.0 Hz, NH), 6.31 (1H, bt, J
= 5.0 Hz, NH). 13C NMR: δ 14.60 (q, C27), 16.18 (q, C24), 16.21 (q, C25), 16.47 (q, C26),
18.18 (t, C6), 19.46 (q, C29), 20.96 (t, C11), 21.32 (q, OAc), 23.69 (t, C2), 25.58 (t, C12),
27.93 (q, C23), 28.37 (q, C7'), 29.48 (t, C21), 30.88 (t, C15), 33.62 (t, C16), 34.32 (t, C22),
37.11 (s, C10), 37.67 (d, C13), 37.79 (t, C7), 37.92 (t, C3'), 38.03 (t, C4'), 38.33 (s, C4), 38.39
(t, C1), 38.45 (t, C2'), 39.41 (t, C1'), 40.79 (s, C8), 42.45 (s, C14), 46.74 (d, C19), 50.13 (d,
C18), 50.55 (d, C9), 55.46 (d, C5), 55.70 (s, C17), 79.65 (s, C6'), 80.95 (d, C3), 109.36 (t,
C30), 150.95 (s, C20), 155.81 (s, C5'), 171.03 (s, OAc), 176.56 (s, C28). MS (ES+): m/z 733.5
[M+H]+, 755.4 [M+Na]+; (ES-): 731.4 [M-H]-. IR (KBr; cm-1): 3447 (N–H), 2926 (C–H),
2870 (–CH3), 1718 (–C=O), 1701 (–C=O), 1642 (–C=O).
6

2.4. tert-Butyl {2-[(2-{[(3β)-3-hydroxy-28-oxolup-20(29)-en-28-yl]amino}ethyl)-
disulfanediyl]ethyl}carbamate (4) and (3β,3'β)-N,N'-(disulfanediyldiethane-2,1-diyl)bis[3-
hydroxylup-20(29)-en-28-amide] (5)
LiOH·H2O (0.36 g; 8.6 mmol; 21 eq) was added to a solution of 3 (294 mg; 0.4 mmol) in
methanol (25 mL). The reaction mixture was stirred for 1 day. After stopping the reaction, the
mixture was diluted by water, extracted by chloroform (10 times 10-15 mL each) and dried
over sodium sulfate. Evaporation of the solvent afforded a solid that was purified by column
chromatography, yielding 155 mg (56 %) of 4 and 80 mg (39 %) of 5.
4: 1H NMR: δ 0.75 (3H, s, H23), 0.81 (3H, d, J = 0.6 Hz, H24), 0.92 (3H, s, H25), 0.96 (3H,
s, H26), 0.96 (3H, s, H27), 1.44 (9H, s, Boc), 1.66 (2H, dt, J1 = 3.5 Hz, J2 = 3.5 Hz, J3 = 13.0
Hz, H16), 1.67 (3H, dd, J1 = 0.7 Hz, J2 = 1.4 Hz, H29), 2.78 (2H, t, J = 5.8 Hz, H3'), 2.82
(2H, t, J = 5.8 Hz, H2'), 3.11 (1H, dt, J1 = 4.4 Hz, J2 = 11.1 Hz, J3 = 11.1 Hz, H19), 3.18 (1H,
dd, J1 = 4.7 Hz, J2 = 11.5 Hz, H3), 3.39-3.52 (1H, m, H4'), 3.39-3.52 (2H, m, H1'), 3.64 (1H,
dt, J1 = 5.8 Hz, J2 = 5.8 Hz, J3 = 5.8 Hz, J4 = 13.9 Hz, H4'), 4.58 (1H, dq, J1 = 1.4 Hz, J2 = 1.4
Hz, J3 = 1.4 Hz, J4 = 2.4 Hz, H30), 4.73 (1H, bd, J1 = 0.1 Hz, J2 = 0.1 Hz, J3 = 0.1 Hz, J4 =
2.4 Hz, H30), 4.97 (1H, bt, J = 4.6 Hz, NH), 6.32 (1H, bt, J = 4.6 Hz, NH). 13C NMR: δ 14.64
(q, C27), 15.34 (q, C24), 16.14 (q, C25), 16.17 (q, C26), 18.29 (t, C6), 19.49 (q, C29), 20.94
(t, C11), 25.62 (t, C12), 27.41 (t, C2), 27.97 (q, C23), 28.37 (q, C7'), 29.49 (t, C21), 30.89 (t,
C15), 33.62 (t, C16), 34.38 (t, C22), 37.19 (s, C10), 37.69 (d, C13), 37.91 (t, C3'), 38.02 (t,
C4'), 38.33 (t, C7), 38.43 (t, C2'), 38.71 (s, C4), 38.84 (t, C1), 39.41 (t, C1'), 40.77 (s, C8),
42.45 (s, C14), 46.71 (d, C19), 50.13 (d, C18), 50.64 (d, C9), 55.37 (d, C5), 55.70 (s, C17),
78.97 (d, C3), 79.63 (s, C6'), 109.30 (t, C30), 150.97 (s, C20), 155.81 (s, C5'), 176.54 (s,
C28). MS (ES+): m/z 691.3 [M+H]+, 713.3 [M+Na]+; (ES-): 689.0 [M-H]-. IR (KBr; cm-1):
2957 (C–H), 2870 (–CH3), 1695 (–C=O), 1641 (–C=O).
7

5: 1H NMR: δ 0.76 (3H, s, H23), 0.82 (3H, bs, H24), 0.93 (3H, s, H25), 0.96 (3H, s, H26),
0.96 (3H, s, H27), 1.36-1.39 (1H, m, H21), 1.42-1.45 (1H, m, H7), 1.54-1.57 (1H, m, H16),
1.68 (3H, dd, J1 = 0.6 Hz, J2 = 1.4 Hz, H29), 1.83 (1H, dd, J1 = 7.5 Hz, J2 = 12.2 Hz, H7),
1.93 (1H, ddd, J1 = 8.0 Hz, J2 = 11.2 Hz, J3 = 13.2 Hz, H21), 2.07 (1H, dt, J1 = 3.4 Hz, J2 =
3.4 Hz, J3 = 13.4 Hz, H16), 2.49 (1H, ddd, J1 = 3.7 Hz, J2 = 11.5 Hz, J3 = 13.0 Hz, H13),
2.81-2.88 (2H, m, H2'), 3.12 (1H, dt, J1 = 4.4 Hz, J2 = 11.1 Hz, J3 = 11.1 Hz, H19), 3.18 (1H,
dd, J1 = 4.8 Hz, J2 = 11.6 Hz, H3), 3.50 (1H, dq, J1 = 6.6 Hz, J2 = 6.6 Hz, J3 = 6.6 Hz, J4 =
14.0 Hz, H1'), 3.61 (1H, dq, J1 = 7.1 Hz, J2 = 7.1 Hz, J3 = 7.1 Hz, J4 = 14.0 Hz, H1'), 4.59
(1H, dq, J1 = 1.4 Hz, J2 = 1.4 Hz, J3 = 1.4 Hz, J4 = 2.4 Hz, H30), 4.72 (1H, bd, J = 2.4 Hz,
H30), 6.48 (1H, t, J = 6.9 Hz, NH). 13C NMR: δ 14.62 (q, C27), 15.34 (q, C24), 16.12 (q,
C25), 16.17 (q, C26), 18.29 (t, C6), 19.51 (q, C29), 20.96 (t, C11), 25.64 (t, C12), 27.41 (t,
C2), 27.97 (q, C23), 29.51 (t, C15), 30.91 (t, C21), 33.75 (t, C16), 34.39 (t, C22), 37.20 (s,
C10), 37.61 (d, C13), 38.17 (t, C2'), 38.45 (t, C1'), 38.45 (t, C7), 38.72 (t, C1), 38.85 (s, C4),
40.79 (s, C8), 42.45 (s, C14), 46.61 (d, C19), 50.15 (d, C18), 50.62 (d, C9), 55.38 (d, C5),
55.69 (s, C17), 79.00 (d, C3), 109.29 (t, C30), 150.96 (s, C20), 176.72 (s, C28). MS (ES+):
m/z 1029.8 [M+H]+, 1051.8 [M+Na]+; (ES-): 1027.8 [M-H]-. IR (KBr; cm-1): 2930 (C–H),
2870 (–CH3), 1640 (–C=O). [α]D20 = –6.8 (c 0.191; CH3OH).
2.5. (3β)-N-{2-[(2-Aminoethyl)disulfanediyl]ethyl}-3-hydroxylup-20(29)-en-28-amide (6)
A solution of HCl in 1,4-dioxane (4 M; 0.2 mL; 0.7 mmol; 20 eq) was added to a solution of 4
(50 mg; 0.072 mmol) in dry 1,4-dioxane (3 mL). The reaction mixture was stirred for 7 h at
30 °С. After stopping the reaction, the mixture was filtered, washed with a large amount of
diethyl ether and dried over sodium sulfate, yielding 29 mg (68 %) of 6.
1H NMR: δ 0.68 (1H, dd, J1 = 2.0 Hz, J2 = 11.5 Hz, H5), 0.75 (3H, s, H23), 0.81 (3H, d, J =
0.6 Hz, H25), 0.92 (3H, s, H26), 0.95 (3H, s, H24), 0.96 (3H, d, J = 0.7 Hz, H27), 1.63 (2H,
8

dt, J1 = 3.5 Hz, J2 = 3.5 Hz, J3 = 11.0 Hz, H16), 1.67 (3H, dd, J1 = 0.7 Hz, J2 = 1.4 Hz, H29),
2.42 (1H, ddd, J1 = 3.7 Hz, J2 = 11.5 Hz, J3 = 13.0 Hz, H13), 2.78-2.82 (2H, m, H3'), 2.79
(2H, t, J = 6.0 Hz, H1'), 3.04 (2H, t, J = 6.0 Hz, H2'), 3.11 (1H, dt, J1 = 4.5 Hz, J2 = 11.1 Hz,
J3 = 11.1 Hz, H19), 3.14 (1H, dd, J1 = 4.7 Hz, J2 = 11.5 Hz, H3), 3.48-3.54 (1H, m, H4'),
3.58-3.66 (1H, m, H4'), 4.59 (1H, dq, J1 = 1.4 Hz, J2 = 1.4 Hz, J3 = 1.4 Hz, J4 = 2.3 Hz, H30),
4.73 (1H, dq, J1 = 0.7 Hz, J2 = 0.7 Hz, J3 = 0.7 Hz, J4 = 2.3 Hz, H30), 6.03 (2H, t, J = 5.9 Hz,
NH). 13C NMR: δ 14.64 (q, C27), 15.34 (q, C24), 16.14 (q, C25), 16.18 (q, C26), 18.29 (t,
C6), 19.46 (q, C29), 20.92 (t, C11), 25.60 (t, C12), 27.40 (t, C2), 27.97 (q, C23), 29.48 (t,
C15), 30.86 (t, C21), 33.73 (t, C16), 34.38 (t, C22), 37.19 (s, C10), 37.75 (d, C13), 37.75 (t,
C4'), 37.94 (t, C2'), 38.33 (t, C7), 38.70 (t, C1), 38.86 (s, C4), 40.42 (t, C1'), 40.77 (s, C8),
41.94 (t, C3'), 42.47 (s, C14), 46.78 (d, C19), 50.08 (d, C18), 50.61 (d, C9), 55.36 (d, C5),
55.73 (s, C17), 78.97 (d, C3), 109.38 (t, C30), 150.87 (s, C20), 176.37 (s, C28). MS (APCI):
m/z 591.4 [M+H]+, 589.3 [M-H]-, 635.4 [M+HCOO]-. IR (KBr; cm-1): 2945 (C–H), 2870
(–CH3), 1640 (–C=O). [α]D20 = –9.1 (c 0.407; CH3OH).
2.6. (3β)-3-[(3-Carboxypropanoyl)oxy]lup-20(29)-en-28-oic acid (7)
Succinic anhydride (1.62 g; 16.2 mmol; 3.5 eq) and DMAP (170 mg; 1.39 mmol, 0.30 eq)
were added to a solution of betulinic acid (2.1 g; 4.6 mmol) in dry pyridine (20 mL). The
reaction mixture was stirred at r.t. for 5 days. After stopping the reaction, the resulting
mixture was poured onto ice, and hydrochloric acid was added to adjust pH = 7, extracted
with chloroform, and dried over sodium sulfate. Evaporation of the solvent afforded a solid
that was purified by a column chromatography, yielding 1.93 g (75 %) of 7.
1H NMR: δ 0.79 (3H, s, H23), 0.79 (3H, s, H24), 0.80 (3H, s, H25), 0.87 (3H, s, H26), 0.94
(3H, s, H27), 1.64 (3H, dd, J1 = 0.7 Hz, J2 = 1.4 Hz, H29), 2.09-2.13 (2H, m, H16), 2.22 (1H,
ddd, J1 = 3.6 Hz, J2 = 11.6 Hz, J3 = 12.9 Hz, H13), 2.44-2.52 (4H, m, H2'-H3'), 2.95 (1H, dt,
9

J1 = 4.8 Hz, J2 = 11.0 Hz, J3 = 11.0 Hz, H19), 4.38 (1H, dd, J1 = 4.7 Hz, J2 = 11.6 Hz, H3),
4.56 (1H, dq, J1 = 1.4 Hz, J2 = 1.4 Hz, J3 = 1.4 Hz, J4 = 2.4 Hz, H30), 4.69 (1H, dq, J1 = 0.7
Hz, J2 = 0.7 Hz, J3 = 0.7 Hz, J4 = 2.4 Hz, H30). 13C NMR: δ 14.31 (q, C27), 15.65 (q, C24),
15.81 (q, C25), 16.35 (q, C26), 17.67 (t, C6), 18.90 (q, C29), 20.42 (t, C11), 23.27 (t, C2),
25.00 (t, C12), 27.56 (q, C23), 28.77 (t, C2'), 29.15 (t, C21), 29.15 (t, C3'), 30.06 (t, C15),
31.64 (t, C16), 33.68 (t, C22), 36.28 (t, C1), 36.60 (s, C10), 37.38 (s, C4), 37.54 (d, C13),
37.67 (t, C7), 40.21 (s, C8), 41.99 (s, C14), 46.57 (d, C19), 48.50 (d, C18), 49.61 (d, C9),
54.62 (d, C5), 55.36 (s, C17), 79.98 (d, C3), 109.58 (t, C30), 150.26 (s, C20), 171.56 (s, C1'),
173.32 (s, C4'), 177.17 (s, C28). MS (ES-): m/z 555.2 [M-H]-. IR (KBr; cm-1): 2946 (C–H),
2872 (–CH3), 1734 (–C=O). For C34H52O6 (556.77) calculated C (73.34), H (9.41), found C
(73.37), H (9.40). M.p. 244-246 oC.
2.7. (3β)-3-[(2,2-dimethyl-4,13,16-trioxo-3-oxa-8,9-dithia-5,12-diazahexadecan-
-16-yl)oxy]lup-20(29)-en-28-oic acid (8)
Boc-protected cystamine hydrochloride (118 mg; 0.4 mmol; 1.1 eq) and T3P (0.64 mL; 1
mmol; 3 eq) were added to a solution of 7 (200 mg; 0.36 mmol) in dry pyridine (10 mL), and
the reaction mixture was stirred at r.t. for 1 day. The resulting mixture was washed with
water, extracted with chloroform, and dried over sodium sulfate. Evaporation of the solvent
afforded a solid that was purified by a column chromatography, yielding 215 mg (76 %) of 8.
1H NMR: δ 0.82 (1H, dd, J1 = 1.8 Hz, J2 = 11.2 Hz, H5), 0.86 (3H, s, H23), 0.87 (3H, s, H26),
0.89 (3H, d, J = 0.4 Hz, H25), 0.98 (3H, s, H24), 1.02 (3H, s, H27), 1.07 (2H, dq, J1 = 4.6 Hz,
J2 = 13.1 Hz, J3 = 13.1 Hz, J4 = 13.1 Hz, H2), 1.18 (2H, dt, J1 = 3.2 Hz, J2 = 3.2 Hz, J3 = 13.5
Hz, H21), 1.27 (2H, dq, J1 = 4.8 Hz, J2 =13.0 Hz, J3 = 13.0 Hz, J4 = 13.0 Hz, H11), 1.43 (9H,
s, Boc), 1.70 (3H, dd, J1 = 0.7 Hz, J2 = 1.4 Hz, H29), 2.24 (2H, dt, J1 = 3.4 Hz, J2 = 3.4 Hz, J3
= 12.6 Hz, H16), 2.31 (1H, ddd, J1 = 3.6 Hz, J2 = 11.7 Hz, J3 = 12.9 Hz, H13), 2.50 (2H, t, J =
10

6.6 Hz, H2'), 2.56-2.65 (2H, m, H3'), 2.78 (2H, t, J = 6.7 Hz, H6'), 2.81 (2H, t, J = 6.6 Hz,
H7'), 3.02 (1H, dt, J1 = 4.8 Hz, J2 = 10.9 Hz, J3 = 10.9 Hz, H19), 3.32-3.37 (2H, m, H8'),
3.43-3.51 (2H, m, H5'), 4.46 (1H, dd, J1 = 5.1 Hz, J2 = 11.3 Hz, H3), 4.59 (1H, dq, J1 = 1.4
Hz, J2 = 1.4 Hz, J3 = 1.4 Hz, J4 = 2.4 Hz, H30), 4.71 (1H, dq, J1 = 0.7 Hz, J2 = 0.7 Hz, J3 =
0.7 Hz, J4 = 2.4 Hz, H30). 13C NMR: δ 15.14 (q, C27), 16.64 (q, C24), 16.77 (q, C25), 17.09
(q, C26), 19.27 (t, C6), 19.55 (q, C29), 22.12 (t, C11), 24.68 (t, C12), 26.83 (t, C2), 28.54 (q,
C23), 28.79 (q, Boc), 30.75 (t, C2'), 30.83 (t, C21), 31.40 (t, C3'), 31.70 (t, C15), 33.34 (t,
C16), 35.48 (t, C22), 38.14 (t, C1), 38.30 (d, C13), 38.62 (t, C6'), 38.94 (s, C10), 39.07 (t,
C7'), 39.57 (t, C7), 39.62 (s, C4), 40.78 (t, C5'), 40.90 (t, C8'), 41.94 (s, C8), 43.61 (s, C14),
48.50 (d, C19), 50.42 (d, C18), 51.87 (d, C9), 56.88 (d, C5), 57.48 (s, C17), 80.18 (s, Boc),
82.62 (d, C3), 110.20 (t, C30), 151.97 (s, C20), 158.41 (s, Boc), 174.09 (s, C1'), 174.39 (s,
C4'), 180.07 (s, C28). MS (ES+): m/z 791.5 [M+H]+, 813.3 [M+Na]+; (ES-): 789.4 [M-H]-. IR
(KBr; cm-1): 2938 (C–H), 2874 (–CH3), 1732 (–C=O), 1695 (–C=O), 1662 (–C=O).
2.8. (3β)-3-{[4-({2-[(2-aminoethyl)disulfanediyl]ethyl}amino)-4-oxobutanoyl]-oxy}lup-
20(29)-en-28-oic acid (9)
A solution of HCl in 1,4-dioxane (4 M; 0.6 mL; 2.4 mmol; 13 eq) was added to a solution of 8
(142 mg; 0.179 mmol) in dry 1,4-dioxane (9 mL). Reaction mixture was stirred at 30 °С for
20 h. Then the reaction mixture was put to the freezer, next day it was melted, filtered,
washed with diethyl ether and chloroform and dried over sodium sulfate, yielding 101 mg (82
%) of 9.
1H NMR: δ 0.83 (1H, dd, J1 = 2.3 Hz, J2 = 3 x 11.8 Hz, H5), 0.86 (3H, s, H23), 0.87 (2H, d, J
= 0.7 Hz, H26), 0.89 (3H, s, H25), 0.97 (3H, s, H24), 1.02 (2H, d, J = 0.5 Hz, H27), 1.27 (2H,
dq, J1 = 4.7 Hz, J2 = 3 x 13.1 Hz, H2), 1.27 (2H, dq, J1 = 4.6 Hz, J2 = 3 x 12.8 Hz, H11), 1.70
(3H, dd, J1 = 0.7 Hz, J2 = 1.4 Hz, H29), 2.23 (2H, dt, J1 = 3.4 Hz, J2 = 3.4 Hz, J3 = 12.8 Hz,
11

H16), 2.43 (1H, ddd, J1 = 3.6 Hz, J2 = 11.2 Hz, J3 = 13.1 Hz, H13), 2.49 (2H, m, H2’), 2.59
(2H, m, H3’), 2.85 (2H, dt, J1 = 0.8 Hz, J2 = 6.8 Hz, H6’), 2.98 (2H, dt, J1 = 0.8 Hz, J2 = 6.8
Hz, H7’), 3.02 (1H, dt, J1 = 4.9 Hz, J2 = 11.0 Hz, J3 = 11.0 Hz, H19), 3.28 (2H, bt, J = 6.8 Hz,
H5’), 3.50 (2H, bt, J = 6.8 Hz, H8’), 4.47 (1H, dd, J1 = 5.1 Hz, J2 = 11.2 Hz, H3), 4.59 (1H,
dq, J1 = 3 x 1.4 Hz, J2 = 2.4 Hz, H30), 4.71 (1H, dq, J1 = 3 x 0.7 Hz, J2 = 2.4 Hz, H30). 13C
NMR: δ 15.11 (q, C27), 16.62 (q, C24), 16.75 (q, C25), 17.06 (q, C26), 19.26 (t, C6), 19.54
(q, C29), 22.11 (t, C11), 24.68 (t, C12), 26.82 (t, C2), 28.53 (q, C23), 30.69 (t, C21), 30.82 (t,
C3’), 31.37 (t, C2’), 31.68 (t, C15), 33.30 (t, C16), 35.46 (t, C22), 35.51 (t, C6’), 38.11 (t,
C1), 38.22 (d, C13), 38.29 (s, C10), 38.94 (t, C7’), 39.38 (t, C5’), 39.55 (s, C4), 39.57 (t, C7),
39.61 (t, C8’), 41.93 (s, C8), 43.59 (s, C14), 48.49 (d, C19), 50.40 (d, C18), 51.87 (d, C9),
56.89 (d, C5), 57.45 (s, C17), 82.63 (d, C3), 110.19 (t, C30), 151.96 (s, C20), 174.12 (s, C1’),
174.53 (s, C4’), 179.96 (s, C28). MS (ES+): m/z 691.3 [M+H]+; (ES-): 689.3 [M-H]-. IR (KBr;
cm-1): 2941 (C–H), 1728 (–C=O), 1695 (–C=O), 1662 (–C=O), 1642 (N–H). [α]D20 = +13.6 (c
0.381; CH3OH).
2.9. Cell cultures
The screening cell lines, i.e., T-lymphoblastic leukemia, CEM; cervical carcinoma, HeLa;
breast carcinoma, MCF7; human malignant melanoma, G-361; and human foreskin
fibroblasts, BJ, were obtained from the American Type Culture Collection (Manassas, VA,
USA). Cells were cultured in DMEM (Dulbecco's Modified Eagle Medium, Sigma Aldrich,
MO, USA). Media used were supplemented with 10% of fetal bovine serum, 2 mM of L-
glutamine, and 1% of penicillin-streptomycin (all from Sigma Aldrich, MO, USA). The cell
lines were maintained under standard cell culture conditions at 37 °C and 5% CO2 in humid
environment. Cells were subcultured twice or three times a week using the standard
trypsinization procedure.
12

3.10. Cytotoxicity screening tests
Description of the experimental procedure used in cytotoxicity assay was already published
[10]. IC50 values in CEM, MCF7, HeLa, G-361 and BJ measured with the compounds 2, 5, 6,
7 and 9 after 72 h of treatment are shown in Table 1.
3.11. Flow cytometry analysis
CEM cancer cells were treated with 6 and 9 for 24 h, stained with propidium iodide and their
DNA contents were measured by a flow cytometer (FACS VerseTM, Becton Dickinson, NJ,
USA). The distribution of cells in subG1 (“apoptotic cells”), G0/G1, S and G2/M peaks were
quantified by histogram analysis using BD FACSuite software (Becton Dickinson, NJ, USA).
The experiments were repeated three times. The differences between control and treated cells
were analyzed by one-way analysis of variance (ANOVA) using Microsoft Excel 2010.
Signification was marked with asterisk (p ˂ 0.05).
2.12. SDS–polyacrylamide gel electrophoresis and immunoblotting
CEM cells were treated for 24 h with tested compounds. The procedure was done as described
earlier [18]. The antibody against caspase-7 recognized the zymogen and fragment. The
experiments were repeated three times. The protein expression in treated cells was compared
with the untreated controls.
2.13. Activities of caspase 3/7
After 24 h, treated CEM cells were analyzed for activity of caspases 3/7 using a procedure
published earlier [18]. The experiments were repeated three times in triplicates. The
13

differences between control and treated cells were analyzed by one-way analysis of variance
(ANOVA) using Microsoft Excel 2010. Signification was marked with asterisk (p ˂ 0.05).
3. Results and Discussion
3.1. Chemistry
The amide 6 (Scheme 1) was prepared by a series of reactions, starting from betulinic acid
(1), protected at the C(3)-OH by acetate formation, yielding 2 [11]. Subsequently, the C(17)-
COOH group of 2 was converted to the corresponding acyl chloride [11], which was used
directly in the synthesis of 3, using mono-Boc-protected cystamine under the presence of
EDIPA as a base in dichloromethane. When the acetyl protecting group was removed from 3
to prepare the required amide 4, a side reaction was observed, resulting in obtaining a mixture
of the products 4 and 5 (Scheme 1), which were separated by column chromatography, finally
yielding 56 % of 4 and 39 % of 5. Partial substitution of the Boc-protecting group in 4 by
another molecule appeared, resulting in the dimeric by-product 5. Protecting Boc group was
removed from the purified 4 by a solution of hydrogen chloride in 1,4-dioxane [11], yielding
the amide 6.
The amide 9 (Scheme 2) was also prepared from betulinic acid (1), which was converted to its
hemisuccinate at the C(3)-OH center (7), using succinic anhydride as a reagent and DMAP as
a reaction promotor, in dry pyridine [10]. Because the C(17)-COOH in 7 is much less reactive
than the free carboxyl group in hemisuccinate group, no protection of the C(17)-COOH group
was necessary before the subsequent amide bond formation at the hemisuccinate carboxyl
group to get 8. This amide bond was made by reacting 7 with mono-Boc-protected cystamine
in dry pyridine, using T3P as reaction promotor. Finally, the Boc-protecting group in 8 was
removed by a solution of hydrogen chloride in 1,4-dioxane [11], yielding the required amide
9.
14

3.2. Biological activity
The amides 6 and 9, the dimeric product 5 and two intermediates (2 and 7) were subjected to
the cytotoxicity screening tests in vitro for 72 h. The compounds were tested in human T-
lymphoblastic leukemia cell line (CEM), breast carcinoma cell line (MCF7), cervical
carcinoma cell line (HeLa) and malignant melanoma cell line (G-361). Normal human
fibroblasts (BJ) were used for comparison of the toxicity. The dimeric product 5 displayed
cytotoxicity towards BJ but did no activity in either of the cancer cell lines. A difference was
found between treating the cells with 6 and 9 for 72 h: while the amide 6 was not toxic
towards normal human fibroblasts BJ, the amide 9 showed comparable toxicity in MCF7 and
in BJ. Neither 6 nor 9 were cytotoxic in HeLa and G-361. The amide 6 showed selectivity
against CEM cancer line (IC50 = 3.0 ± 0.7 M; TI = 20; 72 h). In turn, only the amide 9
showed antimicrobial activity against Streptococcus mutans (MIC 3.125 M; MBC 3.125
M), and Bacillus cereus (MIC 25 M; MBC 25 M). The intermediate compound 2 was
found to be cytotoxic on G361 cancer cell line (IC50 = 7.5 ± 0.8 M; TI = 10; 72 h). The
intermediate compound 7 displayed no cytotoxicity at all, however, its antimicrobial activity
against Actinomyces odontolycus and Clostridium perfrigens (MIC 12.5 µM; MBC 12.5 µM)
demonstrated its potential and selectivity. The cytotoxicity data of 2, 5, 6, 7 and 9 and the
antimicrobial activity of 7 and 9 are summarized in Table 1. The cytotoxicity and
antimicrobial screening methods were described earlier [10].
We have investigated whether 6 and 9 influence the cell cycle and cause apoptosis in human
acute lymphoblastic leukemia cells CEM. Cells were treated with 1, 5 and 15 M of 6 and 9
for 24 h. Then the cells were harvested, stained and analyzed by flow cytometry, western
blotting or caspase activation assay. Flow cytometric analysis showed an increase in the
subG1 phase of the cell cycle (apoptotic cells) in CEM cell line after treatment with the amide
15

9. In contrast, the amide 6 did not enhance the number of subG1 cells, but slightly induced the
proportion of cells in G0/G1 and G2/M phases with decrease of S-phase cells. In these cells,
treatment with the amide 9 resulted in increasing the proportion of cells in the G0/G1 phase
and decreased their proportion in the concomitant phases, S and G2/M (Fig. 1A). Histograms
with the distributions of CEM cells in the G0/G1, S, and G2/M cell cycle phases (Fig. 1A), and
the subG1 fraction of cells are shown in Fig. 1B, and were measured by flow cytometric
analysis, after 24 h treatment with the amides 6 and 9 relative to untreated control.
Apoptotic markers were detected by western blotting to examine the antiproliferative
activities of tested amides 6 and 9. Caspase cascade activation initiates apoptosis [19].
Caspase-3 and caspase-7 as executioner proteases cleave poly-(ADP-ribose) polymerase
(PARP) and cause subsequent DNA degradation and apoptotic death [20]. The western blots
showed degradation of caspase-7 into cleaved fragments after treatments with the amides 6
and 9 in CEM cells in a dose-dependent manner (Fig. 2). A final step of caspase activation,
PARP cleavage, was also observed in human lymphoblastic leukemia cells treated by the
amides 6 and 9 (Fig. 2). No changes in the expressions of anti-apoptotic proteins (Bcl-2 and
Mcl-1) and p53 were observed.
These data were supplemented by estimating the caspase-3/7 activity in CEM treated cells
using the fluorogenic substrate Ac-DEVD-AMC and/or caspase-3/7 inhibitor Ac-DEVD-
CHO. Compound 6 increased activity of caspase-3/7 after the treatment with 5 M up to
eleven fold; compound 9 induced increase in the effector caspases up to eight fold compared
to the untreated control (Fig. 3). These results show that the amides 6 and 9 can induce
apoptosis by caspase-3/7 activation in CEM cells.
3.3. In silico calculation of physico-chemical and ADME parameters
16

To support experimental data determined for the target compounds 5, 6 and 9, and for the
intermediates 2 and 7, their physico-chemical and ADME parameters have been in silico
calculated using ACD/iLabs software and databases [21]. The calculated physico-chemical
parameters were compared with the Lipinski [22] rule of five and with the Ghose [23] rule.
The rules describe molecular properties important for a small molecule drug pharmacokinetics
in the human body, including their absorption, distribution, metabolism and excretion (known
as ADME parameters). However, the rules do not predict displaying of the pharmacological
activity. We described in details those rules and their importance in designing
pharmacologically prospective structures recently [18]. The target compounds 5, 6 and 9 and
the intermediates 2 and 7 do not correspond to the whole range given for any of the Lipinski
[22] and Ghose [23] rules, except of several parameters (Table 2). Despite those facts, the
compounds 2, 6 and 9 display cytotoxicity (Table 1). Exceptions are already known, when
pharmacologically active compounds do not correspond to all rules, and we have observed
such result as well [10,18,24]. More details on this topic can be found in the Supplementary
material.
4. Conclusion
To conclude, we can state that the planned synthetic procedure corresponded well with the
technical procedure of the synthesis realized. The only unexpected product was the dimer 5,
which appeared due to a partial substitution of the Boc-protecting group in 4 by another
molecule, and which structure was proven by the analytical data. However, this compound (5)
was cytotoxic in the normal fibroblasts, but displayed no effect in the tumor cell lines. The
compound 6 showed selective cytotoxicity against CEM cell line (IC50 3.0 ± 0.7 M, TI =
20), while the compound 9 was less selective. The intermediate compound 2 was successfully
cytotoxic on the G361 cancer cell line (IC50 7.5 ± 0.8 M, TI = 10). Both compounds (6 and
17

9) can induce apoptosis by caspase-3/7 activation in CEM cells, and they enhance the effect
of betulinic acid (1) due to their better bioavailability in comparison with it. In addition, the
amide 9 showed antimicrobial activity against Streptococcus mutans (MIC 3.125 M; MBC
3.125 M), and it is also active against Bacillus cereus (MIC 25 M; MBC 25 M).
Therefore, a synthesis of the target molecules 6 and 9 resulted in compounds displaying either
cytotoxicity or antimicrobial activity with a clear selectivity of their effects, and with
enhancing the corresponding effects of their parent components. Finally, the intermediate
compound 7 displayed antimicrobial activity against Actinomyces odontolycus and
Clostridium perfrigens (MIC 12.5 µM; MBC 12.5 µM).
Supporting Information Summary
More details on physico-chemical and ADME parameters are given, including in silico
calculated activity values of the compounds 6 and 9 on central nervous system that are shown
in Figs. S1 and S2.
Acknowledgments
The authors thank for funding of this investigation through the projects FV10599, FV20666
and FV30300 (MPO; U.B., Z.W.), and NPUI LO1204 (MŠMT; L.J., L.R.). The excellent
technical assistance from Ms. Olga Hustáková is gratefully acknowledged.
References
[1] M. Ali-Seyed, I. Jantan, K. Vijayaraghavan, S. N. A. Bukhari, Betulinic acid: recent
advances in chemical modifications, effective delivery, and molecular mechanisms of a
promising anticancer therapy, Chem. Biol. Drug Des. 87 (2016) 517–536.
18

[2] R. H. Cichewicz, S. A. Kouzi, Chemistry, biological activity, and chemotherapeutic
potential of betulinic acid for the prevention and treatment of cancer and HIV infection,
Med. Res. Rev. 24 (2004) 90–114.
[3] B. G. Bag, C. Garai, R. Majumdar, M. Laguerre, Natural triterpenoids as renewable nanos,
Struct. Chem. 23 (2012) 393–398.
[4] Y. Kashiwada, J. Chiyo, Y. Ikeshiro, T. Nagao, H. Okabe, L. M. Cosentino, K. Fowke, K.
H. Lee, 3,28-Di-O-(dimethylsuccinyl)betulin isomers as anti-HIV agents, Bioorg. Med.
Chem. Lett. 11 (2001) 183–185.
[5] V. Zuco, R. Supino, S. C. Righetti, L. Cleris, E. Marchesi, C. Gambacorti-Passerini, F.
Formelli, Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal
cells, Cancer Lett. 175 (2002) 17–25.
[6] Y. J. You, Y. Kim, N. H. Nam, B. Z. Ahn, Synthesis and cytotoxic activity of A-ring
modified betulinic acid derivatives, Bioorg. Med. Chem. Lett. 13 (2003) 3137–3140.
[7] S. Fulda, Betulinic acid for cancer treatment and prevention, Int. J. Mol. Sci. 9 (2008)
1096–1107.
[8] C. Genet, A. Strehle, C. Schmidt, G. Boudjelal, A. Lobstein, K. Schoonjans, M. Souchet,
J. Auwerx, R. Saladin, A. Wagner, Structure-activity relationship study of betulinic acid, a
novel and selective TGR5 agonist, and its synthetic derivatives: potential impact in
diabetes, J. Med. Chem. 53 (2010) 178–190.
[9] J. Šarek, M. Kvasnica, M. Vlk, M. Urban, P. Džubák, M. Hajdúch, The potential of
triterpenoids in the treatment of melanoma. In: Research on melanoma – a glimpse into
current directions and future trends (Ed.: M. Murph), Chapter 7, InTech: Rijeka, Croatia
2011, pp 125–158.
19

[10] U. Bildziukevich, N. Vida, L. Rárová, M. Kolář, D. Šaman, L. Havlíček, P. Drašar, Z.
Wimmer, Polyamine derivatives of betulinic acid and β-sitosterol: a comparative
investigation, Steroids 100 (2015) 27–35.
[11] U. Bildziukevich, E. Kaletová, D. Šaman, E. Sievänen, E. T. Kolehmainen, M. Šlouf, Z.
Wimmer, Spectral and microscopic study of self-assembly of novel cationic spermine
amides of betulinic acid, Steroids 117 (2017) 90–96.
[12] Y. Iwamoto, Diagnosis and treatment of Ewing's sarcoma, Jpn. J. Clin. Oncol. 37 (2007)
79–89.
[13] Z. M. Bacq, G. Deschamps, P. Fischer, A. Herve, H. Le Bihan, J. Lecomte, M. Pirotte, P.
Rayet, Protection against X-rays and therapy of radiation sickness with β-
mercaptoethylamine, Science 117 (1953) 633–636.
[14] M. V. Vasin, I. B. Ushakov, Comparative efficacy and the window of radioprotection for
adrenergic and serotoninergic agents and aminothiols in experiments with small and large
animals, J. Rad. Res. 56 (2015) 1–10.
[15] J. I. Toohey, A. J. L. Cooper, Thiosulfoxide (sulfane) sulfur: new chemistry and new
regulatory roles in biology. Molecules 19 (2014) 12789–12813.
[16] C. Gibrat, F. Cicchetti, Potential of cystamine and cysteamine in the treatment of
neurodegenerative diseases, Prog. Neuropsychopharmacol. Biol. Psychiatry 35 (2011)
380–389.
[17] M. Besouw, R. Masereeuw, L. van den Heuvel, E. Levtchenko, Cysteamine: an old drug
with new potential, Drug Discov. Today 18 (2013) 785–792.
[18] U. Bildziukevich, L. Rárová, D. Šaman, Z. Wimmer, Picolyl amides of betulinic acid as
antitumor agents causing tumor cell apoptosis, Eur. J. Med. Chem. 145 (2018) 41–50.
[19] I. Budihardjo, H. Oliver, M. Lutter, X. Luo, X. Wang, Biochemical pathways of caspase
activation during apoptosis, Annu. Rev. Cell Dev. Biol. 15 (1999) 269–290.
20

[20] R. T. Allen, M. W. Cluck, D. K. Agrawal, Mechanisms controlling cellular suicide: role
of Bcl-2 and caspases, Cell. Mol. Life Sci. 54 (1998) 427–445.
[21] Advanced Chemistry Development, Software ACD/iLabs, Version 12.02, (2011).
[22] C. A. Lipinski, F. Lombardo, B. W. Dominy, P. J. Feeney, Experimental and
computational approaches to estimate solubility and permeability in drug discovery and
developmental settings, Adv. Drug Deliv. Rev. 46 (2001) 3–26.
[23] A. K. Ghose, V. N. Viswanadhan, J. J. Wendoloski, A knowledge-based approach in
designing combinatorial or medicinal chemistry libraries for drug discovery. 1. A
qualitative and quantitative characterization of known drug databases, J. Combin. Chem. 1
(1999) 55–68.
[24] U. Bildziukevich, L. Rárová, D. Šaman, L. Havlíček, P. Drašar, Z. Wimmer, Amides
derived from heteroaromatic amines and selected steryl hemiesters, Steroids 78 (2013)
1347–1352.
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[ Scheme and Figure Captions ]
Scheme 1
Scheme 1. Synthetic procedure for 6: i: Acetic anhydride, EDIPA, DMAP, THF; ii: oxalyl
chloride, dichloromethane, then cystamine, EDIPA, dichloromethane; iii: LiOH•H2O,
methanol; iv: HCl (g) in 1,4-dioxane, additional 1,4-dioxane.
Scheme 2
Scheme 2. Synthetic procedure for 9: i: Succinic anhydride, DMAP, pyridine; (ii) cystamine,
T3P, pyridine; (iii) HCl in 1,4-dioxane, additional 1,4-dioxane.
Figure 1
Figure 1. Histograms with the distributions of CEM cells.
Legend: Histograms with the distributions of CEM cells in the G0/G1, S, and G2/M cell cycle
phases (A), and the subG1 fraction of cells (B) measured by flow cytometric analysis, after 24
h treatment with 6 and 9 relative to untreated control. Data indicate the percentages (%) of the
number of cells in respective phases. Experiments were repeated three times in triplicates.
Error bars are omitted for clarity. (A) Analysis of variance (one-way ANOVA) between
control and treated cells failed due to high biological variability in replicates. (B) Differences
between control and treated cells with 9 among subG1 phase were significant (p ˂ 0.05) *.
Figure 2
Figure 2. Western blot analysis.
Legend: Western blot analysis of apoptotic proteins (PARP, zymogen and fragment of
caspase-7) in CEM cells treated with amides. The expression of proteins in cells treated with
22

1, 5 and 15 µM by compounds 6 and 9 for 24 h were compared with the protein expression of
untreated control cells (0+ means control with DMSO). The expression of β-actin was used as
a protein loading control.
Figure 3. Activity of caspase-3/7 in CEM cells.
Legend: Activity of caspase-3/7 in CEM cells treated with 1, 5 and 15 µM of amides 6 and 9
compared with untreated control cells for 24 h. The data indicate the relative increase in
caspase-3/7 activity. Experiments were repeated three times in triplicates. * Differences
between control and treated cells were significant (p ˂ 0.05).
23

Table 1. Cytotoxicity and antimicrobial activity of 2, 5, 6, 7 and 9
Compound
MW
Cytotoxicity (IC50 [µM]), 72 h
CEM
MCF7
HeLa
G-361
BJ
2
498.75
18.9 ± 0.8 16.9 ± 1.3 14.6 ± 1.8 7.5 ± 0.8 a > 50
5
1029.67 > 50
> 50
> 50
> 50
> 50
>50
13.4 ± 4.0
> 50
6
590.97
556.78
691.04
3.0 ± 0.7 b 46.0 ± 5.2 > 50
7 c
9 d
> 50
> 50
>50
> 50
4.0 ± 1.9
16.8 ± 0.3 > 50
> 50
28.0 ± 4.5
a therapeutic index TI = 10; b therapeutic index TI = 20; c antimicrobial activity of 7:
Actinomyces odontolycus (MIC 12.5 µM; MBC 12.5 µM), Clostridium perfrigens (MIC 12.5
µM; MBC 12.5 µM); d antimicrobial activity of 9: Streptococcus mutans (MIC 3.125 µM;
MBC 3.125 µM), Bacillus cereus (MIC 25 µM; MBC 25 µM).
24

Table 2. Physico-chemical and ADME parameters of the compounds 2, 5, 6, 7 and 9 calculated in silico using the ACD/iLabs software [21]
Compd.
or ref.
No.
MW
Physico-chemical and ADME parameters a
log P
log D
log S (pH bioav.
log PS * log PS
log PB
log BB
PPB [%] Hacc
/
(pH 7.4) 7.4)
[%]
fu, brain
Hdon /
n.m.b.
4/1/4
2
5
6
7
9
498.74
1029.65
590.97
556.77
691.04
7.45
14.32
6.66
6.67
7.10
4.96
14.32
4.80
1.80
4.60
-4.31
-10.1
-4.83
-2.52
-8.26
< 30
-5.2
-10.2
-5.0
-5.0
-5.5
-3.4
-7.9
-2.8
-3.2
-3.3
-0.55
-2.00
-0.00
-0.59
-0.60
-0.0
-2.0
-0.0
-0.0
-0.0
99.56
100.00
99.74
99.59
99.84
30-70
30-70
< 30
6/4/11
4/4/8
6/2/7
< 30
7/4/13
a log P – partition coefficient; log D – distribution coefficient; log S – predicted aqueous solubility; bioav. = bioavailability – the degree of
availability of a chemical by the target tissue; log PS * fu, brain – the brain/plasma equilibration rate, the parameter that is a mathematical modeling
parameter based on time required for reaching brain equilibrium; log PS – logarithm of the permeability-surface area coefficient; log PB – the
extent of brain penetration parameter; log BB – a hybrid parameter determined by permeability, plasma and brain tissue binding, and active
transport mechanism; PPB – plasma protein binding; Hacc / Hdon / n.m.b. = number of H-bond acceptors / number of H-bond donors / number of
25

movable bonds; limits given in literature [22,23] for several physico-chemical parameters: MW max. 500 [22,23]; log P max. 5.0 [22]; max. 5.6
[23]; n.m.b. max. 10 / max. 5 / - [22].
26

Scheme 1
H
H
i
COOH
COOH
H
H
H
H
HO
AcO
2
1
H
H
ii
O
H
H
S
S
O
NH
NH
O
H
AcO
iii
3
H
O
H
H
S
S
O
NH
NH
O
H
HO
+
4
H
H
H
OH
H
O
H
NH
O
S
S
NH
H
H
H
iv
HO
5
H
H
S
NH2
NH
S
H
4
O
H
HO
6
H
27

Scheme 2
H
H
i
COOH
COOH
H
H
O
H
H
HO
7
8
9
1
HO
O
H
H
O
ii
H
COOH
H
O
O
H
S
S
NH
O
NH
O
H
O
iii
H
COOH
H
O
H
S
S
NH
H2N
O
H
O
28

Figure 1
A
B
29