Synthesis and cytotoxicity of hybrids of 1,3,4- or 1,2,5-oxadiazoles tethered from ursane and lupane core with 1,2,3-triazole

Article abstract of DOI:10.1016/j.steroids.2020.108698

Ursane and lupane type (1-((5-aryl-1,3,4-oxadiazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl and (1-((4-methyl-2-oxido-1,2,5-oxadiazol-3-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl hybrids were prepared by 1,3-cycloaddition reactions of azole-derived azides w

Full text of DOI:10.1016/j.steroids.2020.108698

Journal Pre-proofs
Synthesis and cytotoxicity of hybrids of 1,3,4- or 1,2,5-oxadiazoles tethered
from ursane and lupane core with 1,2,3-triazole
Sergey A. Popov, Marya D. Semenova, Dmitry S. Baev, Tatiana S. Frolova,
Michael A. Shestopalov, Chengzhang Wang, Zhiwen Qi, Elvira E. Shults,
Māris Turks
PII:
S0039-128X(20)30124-0
DOI:
Reference:
https://doi.org/10.1016/j.steroids.2020.108698
STE 108698
To appear in:
Steroids
Received Date:
Revised Date:
Accepted Date:
2 April 2020
5 July 2020
11 July 2020
Please cite this article as: Popov, S.A., Semenova, M.D., Baev, D.S., Frolova, T.S., Shestopalov, M.A., Wang, C.,
Qi, Z., Shults, E.E., Turks, M., Synthesis and cytotoxicity of hybrids of 1,3,4- or 1,2,5-oxadiazoles tethered from
ursane and lupane core with 1,2,3-triazole, Steroids (2020), doi: https://doi.org/10.1016/j.steroids.2020.108698
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Synthesis and cytotoxicity of hybrids of 1,3,4- or 1,2,5-oxadiazoles tethered from ursane and
lupane core with 1,2,3-triazole.
Sergey A. Popova*, Marya D. Semenovaa, Dmitry S. Baeva , Tatiana S. Frolovab,c, Michael A.
Shestopalovd, Chengzhang Wang e, Zhiwen Qi e, Elvira E. Shultsa, Māris Turksf.
aNovosibirsk Institute of Organic Chemistry, Acad. Lavrentyev ave. 9, Novosibirsk, 630090, Russia
bThe Federal Research Center Institute of Cytology and Genetics, Acad. Lavrentyev Ave., 10, 630090,
Novosibirsk, Russia
cNovosibirsk State University, Pirogova Street, 2, 630090, Novosibirsk, Russia
dNikolaev Institute of Inorganic Chemistry SB RAS, Acad. Lavrentiev ave., 3, 630090 Novosibirsk
(Russia)
eInstitute of Chemical Industry of Forest Products, Chinese academy of forestry, Nanjing 210042,
China
fInstitute of Technology of Organic Chemistry, Faculty of Materials Science and Applied Chemistry,
Riga Technical University, P. Valdena Str. 3, Riga LV-1048, Latvia
*Corresponding author. E-mail:spopov@nioch.nsc.ru
Keywords: 1,3-cycloaddition; Triterpenoid conjugate; 1,2,3-Triazole; 1,3,4-,
1,2,5-Oxadiazole; Cytotoxicity tests; MDM-2-docking.
Abstract
Ursane and lupane type (1-((5-aryl-1,3,4-oxadiazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl and (1-
((4-methyl-2-oxido-1,2,5-oxadiazol-3-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl hybrids were prepared
by 1,3-cycloaddition reactions of azole-derived azides with alkyne esters connected to positions C-3
and C-28 of triterpene core and tested for cytotoxicity. Hybrid compounds of 1,3,4-oxadiazoles
attached at positions 3- and 28- of triterpenoid frame via triazole spacer and combinations of 1,2,5-
oxadiazole or 1,3,4-oxadiazole, tethered with succinate linker and 1,2,3-triazole at the position 3- of
the ursane backbone, were inactive in relation to all the cancer cells tested. Eventually, combinations
of furoxan fragment and 1,2,3-triazole linked to C-28 position of triterpene backbone demonstrated
marked cytotoxic activity towards MCF-7 and HepG2 cells. The most active ester of ursolic acid with
(1-((4-methyl-2-oxido-1,2,5-oxadiazol-3-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl substituent and 3-O-

acetyl group was superior in activity and selectivity over doxorubicin and ursolic acid on MCF-7 cells.
The length of the carbon spacer group may be of crucial importance for cytotoxicity. The introduction
of the additional ester linker between the C-28 of triterpenoid and triazole or changing triazole spacer
between furoxan moiety and triterpenoid core resulted in activity decrease against all the tested cells.
In accordance with molecular modeling results, the activity of new derivatives may be explained in
terms of the interaction of the new hybrid molecules and Mdm2 binding sites.
1. Introduction
Pentacyclic triterpenoids available from the natural pool possess a wide range of biological activities
that may be associated with the affinity of pentacyclic triterpenoids towards different biological targets
[1-7]. Ursane and lupane natural triterpenoids generally exhibit only moderate anti-inflammatory and
anti-proliferative activity and are regarded as promising scaffolds for the synthesis of new derivatives
with pronounced anti-inflammatory properties or selective cytotoxic effects against series of different
cancer cells [8-10].
H
OH
OH
H
H
O
O
H
H
HO
O
H
Ursolic acid
1
Betulonic acid
2
Fig.1 Structures of ursolic and betulonic acids.
Accessible and non-toxic ursolic and semisynthetic betulonic acids (Fig. 1) are widely used as starting
material for the preparation of novel bioactive derivatives. A synthetic combination of various
pharmacophores with triterpenoid molecules can lead to hybrid compounds with improved
physicochemical properties and / or new types of biological activity [11-13].
The addition of certain of heterocyclic moieties, including azoles (1,3,4-oxadiazoles, 1,2,5-
oxadiazoles, and 1,2,3-triazoles) to the triterpenoid framework opens routes to compounds exhibiting
more pronounced antiproliferative or chemopreventive activity that may be due to enhanced binding to
a series of corresponding molecular targets or bioavailability improvement. [14-16, 19-25]
Due to the availability of alkyne-derived esters and amides of triterpenoids [14-16] and development
of methods of selective Cu-catalyzed 1,3 –cycloaddition [17,18] the syntheses of lupane and ursane
conjugates including 1,2,3- triazole functionalities are among the most researched. Lupane and ursane
moieties were connected using 1,3-dipolar cycloaddition to afford hybrids with triazole linker that

exerted excellent activity against breast cancer cells [14]. Ursolic acid hybrids with AZT bearing
1,2,3- triazole linkage attached via C-28 amide or ester with moderate cytotoxic activity were obtained
[19,20]. Ursonic acid-derived compounds comprising aryl-1,2,3- triazole pendant groups at the C-28
position of triterpenoid frame demonstrated superior activity towards different malignant cells [21].
Similarly, triazole-bearing lupane-type esters with C-3 keto group, [15] and (1-aryl-1H-1,2,3-triazol-4-
yl)methyl oleanonic esters [22,23] might display better cytotoxic activity comparing corresponding
triterpenic acid hybrids with 3-OH group. 1,2,3-Triazole lupane conjugates with peptides were
synthesized through 1,3-dipolar cycloaddition of azido peptides to (3-oxolup-20(29)-en-28-oyl)-4-
ethynyl aniline [24]. The synthetic amino acid conjugates with 1,2,3-triazole and betulonic acid of
other type exerted good anti-inflammatory activity[25]. In this regard, the preparation of new C-3-keto
hybrids including 1,2,3-triazole group starting from the available betulonic acid is of interest for the
further study of anticancer activity. Combined derivatives of other oxadiazole, 1,2,5-oxadiazole-2-
oxide (furoxan) displayed activity as NO donors [26] and preparation and cytotoxicity of furoxan
hybrids with triterpenoids have been studied [27,28].
A series of derivatives with furoxan-type substituents and different linker groups attached to the
positions C-28 and C-3 of lupane and ursane backbones were characterized by significant activity
against cancer cells [27,28]. Thus, furoxan derivatives spacered from ursane core by the 3-O-succinate
group were prepared, and some of them were found potently cytotoxic [27] against HepG2 cells. The
results of work [28] showed that furoxan-holding betulonic acid hybrids were superior in antitumor
activity against HepG2 and B16 cell lines over nitrate-based lupane conjugates. Betulonic acid C-28
hybrids were found more cytotoxic than betulinic acid-furoxan derivatives attached at C-3 via
succinate linker. Fewer works focused conjugates of ursane and lupane derivatives with 1,3,4
oxadiazoles. The synthesis and the moderate cytotoxicity of lupane-type hybrids with 1,3,4-
oxadiazoles were reported [29]. Ursane derived esters with aryl oxadiazoles substituents were found
non-cytotoxic but possessing promising anti-inflammatory action [30]. Recently, we reported the
preparation of low toxic conjugates with anti-inflammatory properties including 1,2,5- and 1,3,4-
oxadiazoles linked to betulonic acid core via aminoacid functionalities [31]. Certain azoles are
bioisosteres of amides and peptides, therefore, the introduction of a sequence of different heterocycles
at the triterpene backbone may lead to hybrids with improved target affinity for cytotoxicity or anti-
inflammatory activity. In the literature, there is no information on hybrids of 1,3,4- or 1,2,5-
oxadiazoles connected to different positions of triterpenoids through 1,2,3- triazole linker. We were
interested in synthetic combination of two different types of azoles (1,3,4- oxadiazole and 1,2,3-
triazole or 1,2,5- oxadiazole and 1,2,3- triazole ) as pendant groups at different positions of the
triterpenoid frame for the subsequent study of cytotoxic activity of novel compounds. Thus, the
objective of the present work was to obtain novel ursane and lupane conjugates using 1,3-

cycloaddition of azides - derived from 1,3,4- and 1,2,5- oxadiazoles to propargyl esters of
triterpenoids, and to study the cytotoxicity of the hybrid compounds. To estimate affinity to molecular
targets, docking of the novel triterpenoid hybrid derivatives in Mdm2 binding sites was performed and
supported with flow cytometry data.
2. Experimental
General
IR spectra were recorded on a Bruker Vector 22 FTIR spectrometer in KВr pellets. The mass spectra
were recorded on a Thermo Scientific DFS high-resolution mass spectrometer (evaporator temperature
200–250°C, EI ionization at 70 eV). 1Н and 13C NMR spectra were registered on a Bruker AV-300
(300.13 MHz for 1Н, 75.48 MHz for 13C), Bruker AV-400 (400.13 MHz for 1Н, 100.62 MHz for 13C)
and DRX-500 (operating frequencies 500.13 and 125.76 MHz, respectively) at room temperature. The
chemical shifts are given in ppm relative to signals of the solvents used as internal standards: in 1Н
NMR spectra H 7.24 (CHCl3) and 13C NMR spectra C 76.90 (CDCl3). Signals in the NMR 1Н and
13C spectra of ursane series were assigned by correlation with those of ursolic acid [32] and ursolic
acid acetate [33]. Signals in the NMR 1Н and 13C spectra of lupane series were assigned by correlation
with those of betulonic acid (in CDCl3) [34] J values are given in Hertz. The reaction progress was
controlled by TLC on Sorbfil UV-254 plates using MTBE and 1:1 MTBE–hexane mixture as eluents,
with visualization by FeCl3 solution and under UV light. The reaction products were isolated by
column chromatography using silica gel from Acros (35-70 μm). In the NMR 1Н and 13C spectra the
assignments marked with the same symbols §, # are interchangeable.
2.1.1. Methods and reagents
Propargyl betulinate 13b [35], 2-ethoxy-2-oxoethyl ursolate 5 [36] were prepared as reported before.
2-(Chloromethyl)-5-aryl-1,3,4-oxadiazoles 3a-d [37], furoxane derivative 3-(bromomethyl)-4-methyl-
1,2,5-oxadiazole-2-oxide 4 [38], propargyl 4-chloro-4-oxobutanoate [39], (4-methyl-1,2,5-oxadiazole-
2-oxide-3-yl)methyl-N-(3-oxo-lup-20 (29)-en-28-oyl)-2-aminoethanoate 16a, (4-methyl-1,2,5-
oxadiazole-2-oxide-3-yl)methyl-N-(3-oxo-lup-20 (29)-en-28-oyl)-3-aminopropanoate 16b, (4-methyl-
1,2,5-oxadiazole-2-oxide-3-yl)methyl-N-(3-oxo-lup-20 (29)-en-28-oyl)-4-aminobutyrate 16c [31] were
synthesized following corresponding previously published procedures.
2.1.2. Synthesis of intermediate and hybrid compounds

General procedure for the preparation of 2-azidomethyl-5-aryl-1,3,4-oxadiazoles 3’a, b, c, d, and 3-
(azidomethyl)-4-methyl-1,2,5-oxadiazole 2-oxide 4 ’.
2-(Chloromethyl) -5-aryl-1,3,4-oxadiazole 3a, b, c, d (1 mmol) or 3-(bromomethyl)-4-methyl-1,2,5-
oxadiazole 2-oxide 4 and sodium azide (3 mmol) in DMF (10 mL ) was stirred at 50-55° C for 5-7
hours, (TLC control). The reaction mixture was poured into ice water, extracted with MTBE (3 × 30
mL). The organic layer was washed with brine, dried (Na2SO4), and concentrated in vacuum. The
product was purified by chromatography over a short silica gel column (CH2Cl2-MTBE ) to afford
azides 3’a, b, c,d or 4’.
3-(Azidomethyl)-4-methyl-1,2,5-oxadiazole 2-oxide 4’
The title compound was prepared from 4 in 98% yield as pale yellow oil.
IR (cm-1): 500, 554, 594, 658, 704, 773, 849, 883, 941, 993, 1039, 1093, 1122, 1221, 1271, 1315,
1352, 1387, 1427, 1477, 1520, 1606, 1674, 2116, 2935. NMR 1Н (400.13 MHz , CDCl3, δ, ppm, J/Hz):
2.35 (3Н, s, Ме-1), 4.31 (2Н, s, 2Н-4), NMR 13C (125.76 MHz , CDCl3, δ, ppm, J/Hz): 10.8 (C-1),
42.3 (C-4), 112.3 (C-2), 153.9 (C-3). Found, m/z: 155,0448 [M]+ . C4H5N5O2 Calculated, m/z:
155,0443.
Synthesis of propargyl ursolate 6a and propargylbetulonate 2a.
A suspension of ursolic acid 1 or betulonic acid 2 (10 mmol), K2CO3 (1 mol), KI (0.1 mmol) in
acetone (50 mL) and propargyl bromide (12 mmol) was stirred for 20-24 hours at room temperature
(TLC control ). The solvent was removed in vacuo, the residue was suspended in water. The
precipitate was separated on a filter, washed with HCl aq. (3%, 30 mL), water (2×50 mL), and air-
dried to give 2a (82%), 6a (84%). 1H, 13C NMR spectra of compounds 2а, 6a were identical to those
published [35].
3-(prop-1-yn)oxy-2-oxoethyl ursolate (6b)
A suspension of ursolic acid carboxymethyl ester 5b (1 mmol), K2CO3 (10 mmol), KI (0.1 mmol) in
DMF (15 mL), propargyl bromide (1.1 mol) was stirred at room temperature for 20-24 hours (TLC
control). The reaction mixture was poured into water, acidified with 10% HCl to pH ~ 1. The
precipitate was separated by filtration, rinsed with water (3×20 mL), air-dried, chromatographed over a
short silica gel column (CH2Cl2-MTBE ), to afford ester 6b as off-white powder, 98%.
IR (KBr, cm-1): 434, 451, 527, 602, 631, 677, 708, 756, 791, 810, 833, 866, 935, 970, 997, 1028, 1051,
1078, 1115, 1140, 1165, 1192, 1227, 1252, 1273, 1308, 1329, 1360, 1383, 1419, 1448, 1464, 1657,
1726, 1772, 2135, 2856, 2926, 2941, 2968, 3292, 3437, 3537. NMR 1Н (400.13 MHz , CDCl3, δ, ppm,
J/Hz): 0.69 (3H, s, Мe-26), 0.74 (3H, s, Мe-25), 0.82 (3H, d, J29-19 = 5.9 Hz, Мe-29), 0.87 (3H, Мe-
24), 0.91 (3H, d, J30-18 = 5.1 Hz, Мe-30), 0.94 (3H, s, Мe-23), 1.04, (3H, s, Мe-27), 2.21 (1H, d, J18-19

= 11.1 Hz, H-18), 2.46 (1H, s, H-5’), 3.12-3.21 (1H, m, H-3), 4.54 (2H, AB, JAB = 16.1 Hz, Δη =
22.01, 2H-3’), 4.69 (2H, s, 2H-1’), 5.20 (1H, br.s, H-12). NMR 13C (125.76 MHz , CDCl3, δ, ppm,
J/Hz): 15.4 (C-25, q), 15.6 (C-24, q), 16.9 (C-26, q), 17.0 (C-29, q), 18.2 (C-6, t), 21.1 (C-30, q), 23.2
(C-11, t), 23.4 (C-27, t), 24.1 (C-16, q), 27.2 (C-2, t), 27.9 (C-15, t), 28.1 (C-23, q), 30.6 (C-21, t), 32.9
(C-7, t), 36.4 (C-22, t), 36.9 (C-10, s), 38.6 (C-1, t), 38.7 (C-4, s), 38.7 (C-20, d), 39.0 (C-19, d), 39.5
(C-8, s), 42.0 (C-14, s), 47.5 (C-9, d), 48.1 (C-17, s), 52.5 (C-3', t), 52.6 (C-18, d), 55.1 (C-5, d), 60.1
(C-1', t), 75.5 (C-5', d), 76.9 (C-4', c), 78.9 (C-3, d), 125.7 (C-12, d), 137.8 (C-13, s), 167.3 (C-2', s),
176.7 (C-28, s). Found, m/z: 552.3809 [M]+ . C35H52O5 Calculated, m/z: 552.3812
3β-(4-Oxo-4-(prop-2-ynyloxy)-butanoyloxy)-urs-12-en-28-oic acid (10)
To a stirred mixture of ursolic acid 1 (0.1 mmol), 4-DMAP (0.01 mmol) in CHCl3 (25 mL) and
pyridine (8 mL) a solution of propargyl 4-chloro-4-oxobutanoate (0.4 mmol) in CHCl3 (20 mL) was
added dropwise at 0- 5ºC. The reaction mixture was stirred at room temperature for 48 h.
The solvent was distilled off in vacuo, H2SO4 aq, (5%, 50 mL) was added dropwise to the residue. The
mixture was extracted with MTBE (3 × 30 mL). The organic phase was washed with water (2 × 30
ml), dried (Na2SO4), concentrated in vacuo, chromatographed (SiO2, CCl4-MTBE), and concentrated
to afford succinate 10, 87%.
IR (KBr, cm-1): 544, 571, 663, 771, 806, 829, 860, 883, 968, 993, 1026, 1065, 1101, 1157, 1211, 1240,
1257, 1273, 1315, 1350, 1375, 1414, 1458, 1689, 1736, 2131, 2623, 2874, 2929, 2941, 2976, 3292.
NMR 1Н (400.13 MHz , CDCl3, δ, ppm, J/Hz): 0.73 (3H, c, Мe-26), 0.82 (6H, s, Мe-24, Мe-25), 0.84
(3H, s Мe-29), 0.93 (6H, c, Мe-23, Мe-30), 1.04 (3H, s, Мe-27), 2.15 (1H, d J18-19 = 10.9 Hz, H-18),
2.45 (1H, s, H-7’), 2.59-2.70 (4H, m, 2H-2’, 2H-3’), 4.46-4.53 (1H, m, H-3), 4.67 (2H, ddd, J1=J2 =
2.6 Hz, 2H-5’), 5.18-5.22 (1H, m, H-12). NMR 13C (125.76 MHz , CDCl3, δ, ppm): 15.4 (C-25), 16.6
(C-24), 16.8 (C-26), 16.9 (C-29), 18.0 (C-6), 21.1 (C-30), 23.1 (C-11), 23.4 (C-2), 23.4 (C-27), 24.0
(C-16), 27.9 (C-15), 27.9 (C-23), 29.0 (C-3'), 29.4 (C-2'), 30.5 (C-21), 32.8 (C-7), 36.7 (C-22), 36.7
(C-10), 37.6 (C-1), 38.1 (C-4), 38.7 (C-20), 38.9 (C-19), 39.3 (C-8), 41.9 (C-14), 47.3 (C-9), 47.7 (C-
17), 52.1 (C-5'), 52.6 (C-18), 55.2 (C-5), 75.0 (C-7'), 77.5 (C-6'), 81.5 (C-3), 125.3 (C-12), 138.0 (C-
13), 171.6 (C-4'), 171.9 (C-1'), 181.4 (C-28). Found, m/z: 594.3923 [M]+ . C37H54O6 Calculated, m/z:
594.3915
Synthesis of triterpenoid 5-aryl-1,3,4-oxadiazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl esters 7,
14; 4-methyl-2-oxido-1,2,5-oxadiazol-3-yl)methyl esters 8, 13 by the interaction of 2- (azidomethyl) -
5-methyl-1,3,4-oxadiazoles 3'or 3- (azidomethyl) -4-methyl-1,2,5-oxadiazole 2-oxide 4 'with
propargyl ethers 6a, 6b, 2a

A mixture of triterpenoid derived propargyl ester 6а, 6b, 2a (1 mmol), 2-azidomethyl-5-aryl-1,3,4-
oxadiazol 3’ or 3-(azidomethyl)-4-methyl-1,2,5-oxadiazole 2-oxide 4’ (1 mmol), CuSO4×5H2O (0,2
mmol) in DMF (15 mL) was stirred at ambient temperature for 30 min, followed by Na ascorbate (0,2
mmol) addition. The mixture was stirred at 50ºC for 10-15 h. (TLC control). The reaction mixture was
poured into ice water, acidified with 10% H2SO4 solution to pH ~3, the precipitate was filtered off,
washed with water to pH ~ 7, and dried on air. The product was purified by column chromatography
(SiO2, CCl4-MTBE).
(((5-Phenyl-1,3,4-oxadiazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl-3β -hydroxy-urs-12-en-28-oate
(7a)
The title compound was prepared in the reaction of ester 6a with azide 3’a as white powder in 65%
yield. IR (KBr, cm-1): 498, 517, 536, 559, 604, 663, 690, 712, 733, 756, 806, 829, 866, 912, 951, 964,
974, 995, 1030, 1045, 1072, 1092, 1107, 1140, 1167, 1180, 1198, 1228, 1269, 1284, 1306, 1360, 1379,
1387, 1450, 1483, 1552, 1578, 1591, 1610, 1657, 1722, 2247, 2872, 2926, 2945, 3149, 3435. NMR 1Н
(400.13 MHz , CDCl3, δ, ppm, J/Hz): 0.53 (3H, s, Me-26), 0.73 (3H, s, Me-25), 0.79 (3H, d, J29-19
=
6.8 Hz, Me-29), 0.83 (3H, s, Me-24), 0.89 (3H, br.s., Me-30), 0.94 (3H, s, Me-23), 1.02 (3H, s, Me-
27), 2.17 (1H, d, J18-19 = 11.4 Hz, H-18), 3.14-3.20 (1H, m, H-3), 5.08-5.20 (3H, m (d + AB), H-12,
2H-1’), 5.83 (2H, AB-sys, JAB = 16.2, Δη = 6.9, H-4’), 7.45-7.58 (3H, m, H-9’, H-10’, H-11’), 7.78
(1H, s, H-3’), 7.97-8.03 (2H, m, H-8’, H-12’). NMR 13C (125.76 MHz , CDCl3, δ, ppm): 15.4 (C-25,
q), 15.6 (C-24, q), 16.7 (C-26, q), 16.9 (C-29, q), 18.2 (C-6, t), 21.0 (C-30, q), 23.2 (C-11, t), 23.4 (C-
27, q), 24.1 (C-16, t), 27.1 (C-2, t), 27.9 (C-15, t), 28.1 (C-23, q), 30.5 (C-21, t), 32.9 (C-7, t), 36.5 (C-
22, t), 36.8 (C-10, s), 38.5 (C-1, t), 38.7 (C-4, s), 38.7 (C-20, d), 39.0 (C-19, d), 39.4 (C-8, s), 42.0 (C-
14, s), 44.1 (C-4', t), 47.4 (C-9, d), 48.1 (C-17, s), 52.7 (C-18, d), 55.1 (C-5, d), 57.2 (C-1', t), 78.9 (C-
3, d), 122.9 (C-7', s), 124.3 (C-3', d), 125.6 (C-12, d), 127.1 (C-8', d), 127.1 (C-12', d), 129.1 (C-9', d),
129.1 (C-11', d), 132.3 (C-10', d), 137.9 (C-13, s), 144.2 (C-2', s), 159.8 (C-5', s), 166.1 (C-6', s), 177.3
(C-28, s). Found, m/z: 695.4404 [M]+ . C42H57O4N5 Calculated, m/z: 695.4405
(((5-(4-Bromophenyl)-1,3,4-oxadiazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl-3β -hydroxy-urs-12-
en-28-oate (7c)
The title compound was prepared in the reaction of ester 6a with azide 3’c as white powder in 55%
yield. IR (KBr, cm-1): 461, 478, 505, 536, 559, 627, 660, 677, 694, 731, 791, 804, 833, 951, 976, 997,
1011, 1030, 1045, 1084, 1109, 1140, 1169, 1178, 1200, 1,2,3-0, 1269, 1306, 1325, 1365, 1379, 1387,
1408, 1458, 1481, 1547, 1585, 1605, 1655, 1724, 2872, 2926, 2970, 3090, 3149, 3437. NMR 1Н
(400.13 MHz , CDCl3, δ, ppm, J/Hz): 0.53 (3H, s, Me-26), 0.74 (3H, s, Me-25), 0.80 (3H, d, J29-19
=
6.3 Hz, Me-29), 0.84 (3H, s, Me-24), 0.89 (3H, br.s, Me-30), 0.95 (3H, s, Me-23), 1.02 (3H, s, Me-27),

2.17 (1H, d J18-19 = 10.8 Hz, H-18), 3.12-3.23 (1H, m, H-3), 5.08-5.21 (3H, m, H-12, 2H-1’), 5.30 (2H,
m, H-4’), 7.64 (2H, d, J = 8.2 Hz, H-9’, H-11’), 7.77 (1H, s, H-3’), 7.87 (2H, d, J = 8.2 Hz, H-8’, H-
12’). NMR 13C (125.76 MHz , CDCl3, δ, ppm): 15.4 (C-25, q), 15.6 (C-24, q), 16.7 (C-26, q), 16.9 (C-
29, q), 18.2 (C-8, t), 21.1 (C-30, q), 23.2 (C-11, t), 23.4 (C-27, q), 24.1 (C-16, t), 27.1 (C-2, t), 27.9 (C-
15, t), 28.1 (C-23, q), 30.5 (C-21, t), 32.9 (C-7, t), 36.5 (C-22, t), 36.8 (C-10, s), 38.5 (C-1, t), 38.6 (C-
4, s), 38.7 (C-20, d), 39.0 (C-19, d), 39.4 (C-8, s), 41.9 (C-14, s), 44.1 (C-4', t), 47.4 (C-9, d), 48.0 (C-
17, s), 52.7 (C-18, d), 55.1 (C-5, d), 57.1 (C-1', t), 78.9 (C-3, d), 121.7 (C-10', s), 124.4 (C-3', d), 125.6
(C-12, d), 127.3 (C-7', s), 128.4 (C-8', d), 128.4 (C-12', d), 132.5 (C-9', d), 132.5 (C-11', d), 137.8 (C-
13, s), 144.3 (C-2', s), 160.0 (C-5', s), 165.5 (C-6', s), 177.4 (C-28, s). Found, m/z: 773.3505 [M]+ C42
79
H56 O4 N5 Br1. Calculated, m/z: 773.3510.
(((5-(3,4-Dichlorophenyl)-1,3,4-oxadiazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl-3β -hydroxy-urs-
12-en-28-oate (7d)
The title compound was prepared in the reaction of ester 6a with azide 3’d as white powder in 37%
yield. IR (KBr, cm-1): 440, 461, 501, 555, 604, 660, 677, 729, 766, 812, 829, 849, 889, 912, 951, 976,
995, 1034, 1045, 1086, 1105, 1138, 1169, 1198, 1,2,3-2, 1269, 1306, 1325, 1365, 1387, 1408, 1456,
1543, 1566, 1581, 1606, 1724, 2872, 2926, 2945, 2970, 3091, 3147, 3439. NMR 1Н (400.13 MHz ,
CDCl3, δ, ppm, J/Hz): 0.53 (3H, s, Me-26), 0.73 (3H, s, Me-25), 0.80 (3H, d, J29-19 = 6.5 Hz, Me-29),
0.84 (3H, s, Me-24), 0.89 (3H, br.s, Me-30), 0.95 (3H, s, Me-23), 1.02 (3H, s, Me-27), 2.17 (1H, d J18-
19 = 11.5 Hz, H-18), 3.14-3.22 (1H, m, H-3), 5.09-5.21 (3H, m, H-12, 2H-1’), 5.83 (2H, AB-sys, JAB
16.3, Δη = 6.2, 2H-4’), 7.58 (1H, d, J = 8.4, H-9’), 7.78 (1H, s, H-3’), 7.85 (1H, dd, J1 = 8.6, J2 = 2.0
Hz, H-8’), 8.10 (1H, d, J = 2.0 Hz, H-12’). NMR 13C (125.76 MHz , CDCl3, δ, ppm): 15.4 (C-25, q),
=
15.6 (C-24, q), 16.6 (C-26, q), 16.9 (C-29, q), 18.1 (C-8, t), 21.0 (C-30, q), 23.1 (C-11, t), 23.3 (C-27,
q), 24.0 (C-16, t), 27.1 (C-2, t), 27.8 (C-15, t), 28.0 (C-23, q), 30.4 (C-21, t), 32.8 (C-7, t), 36.4 (C-22,
t), 36.8 (C-10, s), 38.5 (C-1, t), 38.6 (C-4, s), 38.6 (C-20, d), 38.9 (C-19, d), 39.3 (C-8, s), 41.9 (C-14,
s), 44.0 (C-4', t), 47.3 (C-9, d), 48.0 (C-17, s), 52.7 (C-18, d), 55.0 (C-5, d), 57.1 (C-1', t), 78.8 (C-3,
d), 122.5 (C-7', s), 124.5 (C-3', d), 125.5 (C-12, d), 126.0 (C-8', d), 128.7 (C-12', d), 131.3 (C-9', d),
133.8 (C-10', s), 137.0 (C-11', s), 137.8 (C-13, s), 144.2 (C-2', s), 160.3 (C-5', s), 164.3 (C-6', s), 177.3
35
(C-28, s). Found, m/z: 763.3616 [M]+ C42H55O4N5 Cl2 Calculated, m/z: 763.3626.
(1-((4-Methyl-2-oxido-1,2,5-oxadiazol-3-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl-3β-hydroxy-urs-12-
en-28-oate (8a)
The title compound was prepared in the reaction of ester 6a with azide 4’ as white powder in 67%
yield. IR (KBr, cm-1): 500, 517, 559, 600, 661, 679, 700, 725, 795, 829, 849, 914, 951, 976, 995, 1043,
1080, 1107, 1140, 1169, 1198, 1228, 1269, 1308, 1327, 1387, 1456, 1520, 1552, 1564, 1610, 1657,

1722, 2872, 2928, 2945, 2964, 3147, 3437. NMR 1Н (400.13 MHz , CDCl3 δ, ppm, J/Hz): 0.53 (3H, s,
Me-26), 0.75 (3H, s, Me-25), 0.81 (3H, d, J29-19 = 6.6 Hz, Me-29), 0.87 (3H, s, Me-24), 0.88-0.92 (3H,
br.s., Me-30), 0.96 (3H, s, Me-23), 1.04 (3H, s, Me-27), 2.18 (1H, d, J18-19 = 11.4 Hz, H-18), 2.42 (3H,
s, Me-7’), 3.15-3.21 (1H, m, H-3), 5.11 (2H, AB, JAB = 12.9, Δη = 13.3, 2H-1’), 5.20 (1H, ddd, J1 =
3.5, J2 = 3.8, H-12), 5.40 (2H, s, 2H-4’). 7.71 (1H, s, H-3’). NMR 13C (125.76 MHz , CDCl3, δ, ppm):
10.9 (C-7', q), 15.4 (C-25, q), 15.6 (C-24, q), 16.7 (C-26, q), 16.9 (C-29, q), 18.3 (C-6, t), 21.1 (C-30,
q), 23.2 (C-11, t), 23.4 (C-27, q), 24.2 (C-16, t), 27.2 (C-2, t), 27.9 (C-15, t), 28.1 (C-23, q), 30.6 (C-
21, t), 33.0 (C-7, t), 36.5 (C-22, t), 36.9 (C-10, s), 38.6 (C-1, t), 38.7 (C-4, s), 38.8 (C-20, d), 39.0 (C-
19, d), 39.5 (C-8, s), 41.2 (C-4', t), 42.0 (C-14, s), 47.5 (C-9, d), 48.1 (C-17, s), 52.8 (C-18, d), 55.2 (C-
5, d), 57.1 (C-1', t), 78.9 (C-3, d), 111.8 (C-6', s), 124.5 (C-3', d), 125.7 (C-12, d), 137.9 (C-13, s),
144.2 (C-2', s), 153.9 (C-5', s), 177.3 (C-28, s). Found, m/z: 649.4186 [M]+ C37H55O5N5 Calculated,
m/z: 649.4198
3-((4-((Ursoloyloxyacetoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-4-methyl-1,2,5-oxadiazole 2-oxide
(9a)
The title compound was prepared in the reaction of ester 6b with azide 4’ as white powder in 84%
yield. IR (KBr, cm-1): 500, 563, 602, 661, 700, 723, 756, 791, 829, 849, 914, 951, 976, 995, 1045,
1080, 1113, 1138, 1167, 1180, 1194, 1230, 1275, 1306, 1327, 1377, 1387, 1423, 1456, 1520, 1562,
1610, 1740, 1765, 2872, 2928, 2945, 2968, 3145, 3444.NMR 1Н (400.13 MHz , CDCl3, δ, ppm, J/Hz):
0.69 (3H, s, Me-26), 0.76 (3H, s, Me-25), 0.83 (3H, d, J29-19 = 6.3 Hz, Me-29), 0.89 (3H, s, Me-24),
0.92 (3H, br.s, Me-30), 0.96 (3H, s, Me-23), 1.06 (3H, s, Me-27), 2.21 (1H, d J18-19 = 11.2 Hz, H-18),
2.42 (3H, s, Me-9’), 3.15-3.22 (1H, m, H-3), 4.51 (2H, AB-sys, JAB = 15.9, Δη = 24.8, 2H-1’), 5.22
(1H, ddd, J1 = J2 = 3.8, H-12), 5.25 (2H, s, 2H-3’), 5.42 (2H, s, 2H-6’), 7.77 (1H, s, H-5’). NMR 13C
(125.76 MHz , CDCl3, δ, ppm): 10.9 (C-9', q), 15.2 (C-25, q), 15.6 (C-24, q), 16.9 (C-26, q), 16.9 (C-
29, q), 18.3 (C-6, t), 21.1 (C-30, q), 23.2 (C-11, t), 23.4 (C-27, q), 24.1 (C-16, t), 27.2 (C-2, t), 27.9 (C-
15, t), 28.1 (C-23, q), 30.6 (C-21, t), 33.0 (C-7, t), 36.4 (C-22, t), 36.9 (C-10, s), 38.6 (C-1, t), 38.7 (C-
4, s), 38.7 (C-20, d), 39.0 (C-19, d), 39.5 (C-8, s), 41.3 (C-6', t), 42.0 (C-14, s), 47.5 (C-9, d), 48.1 (C-
17, s), 52.7 (C-18, d), 55.2 (C-5, d), 57.8 (C-3', t), 60.2 (C-1', t), 78.9 (C-3, d), 111.8 (C-8', s), 124.5
(C-5', d), 125.7 (C-12, d), 137.8 (C-13, s), 143.3 (C-4', s), 153.9 (C-7', s), 167.8 (C-2', s), 176.8 (C-28,
s). Found, m/z: 707.4262 [M]+ C39H57O7N5 Calculated, m/z: 707.4253
Preparation of 3-O-succinyl-oxadiazoles 11 a-d, 12.
A solution of ursolic acid (4-propargyl)-succinate 10 (0,1 mmol), 2-(azidomethyl)-5-aryl-1,3,4-
oxadiazole 3’a-d or 3-(azidomethyl)-4-methyl-1,2,5-oxadiazole 2-oxide 4’ (0,1 mmol) and

diisopropylethylamine (1 mmol) in DMF (20 mL) was stirred at ambient temperature for 30 min,
followed by addition of CuBr (0.01 mmol). The mixture was stirred under N2 at 50-55ºC for 48 h
(TLC control). The reaction mixture was poured onto ice and acidified to pH~3 with H2SO4 aq (10%)
The precipitate was filtered, washed with water to pH ~7, and dried on air. The products were purified
by column chromatography (SiO2, MTBE -CCl4).
3 β-(4-Oxo-4-((1-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)-methoxy)-
butanoyloxy)-urs-12-en-28-oic acid (11a).
The title compound was prepared in the reaction of propargyl ester 10 with azide 3’a as white powder
in 37% yield. IR (KBr, cm-1): 569, 661, 690, 712, 744, 775, 806, 829, 858, 881, 908, 928, 966, 993,
1016, 1047, 1070, 1090, 1105, 1161, 1,2,3-4, 1254, 1271, 1315, 1369, 1377, 1389, 1421, 1452, 1483,
1552, 1578, 1591, 1610, 1693, 1732, 2623, 2874, 2928, 2945, 2970, 3145, 3442. NMR 1Н (400.13
MHz , CDCl3, δ, ppm, J/Hz): 0.74 (3H, s, Me-26), 0.79 (3H, s, Me-25), 0.81 (3H, s, Me-24), 0.83 (3H,
d, J29-19 = 6.4 Hz, Me-29), 0.91 (3H, s, Me-23), 0.93 (3H, s, Me-30), 1.05 (3H, s, Me-27), 2.16 (1H, d
J18-19 = 11.0 Hz, H-18), 2.61 (4H, m, 2H-2’, 2H-3’), 4.43-4.50 (1H, m, H-3), 5.18-5.22 (1H, m, H-12),
5.22-5.26 (2H, m, 2H-5’), 5.85 (2H, c, 2H-8’), 7.45-7.57 (3H, m, H-13’, H-14’, H-15’), 7.84 (1, s, H-
7’), 8.00 (2H, d, J = 7.2 Hz, H-12’, H-16’). NMR 13C (125.76 MHz , CDCl3, δ, ppm): 15.4 (C-25, q),
16.6 (C-24, q), 16.9 (C-26, q), 17.0 (C-29, q), 18.0 (C-6, t), 21.1 (C-30, q), 23.2 (C-11, t), 23.4 (C-2, t),
23.5 (C-27, q), 24.0 (C-16, t), 27.9 (C-15, t), 28.0 (C-23, q), 29.0 (C-2', t), 29.3 (C-3', t), 30.5 (C-21, t),
32.7 (C-7, t), 36.6 (C-22, t), 36.8 (C-10, s), 37.6 (C-1, t), 38.1 (C-4, s), 38.7 (C-20, d), 38.9 (C-19, d),
39.4 (C-8, s), 41.8 (C-14, s), 44.1 (C-8', t), 47.3 (C-9, d), 47.8 (C-17, s), 52.4 (C-18, d), 55.2 (C-5, d),
57.6 (C-5', t), 81.3 (C-3, d), 122.9 (C-11', s), 124.2 (C-7', d), 1,2,5-.5 (C-12, d), 127.1 (C-12', d), 127.1
(C-16', d), 129.1 (C-13', d), 129.1 (C-15', d), 132.3 (C-14', d), 137.9 (C-13, s), 143.8 (C-6', s), 159.9
(C-9', s), 166.1 (C-10', s), 171.7 (C-4', s), 172.1 (C-1', s), 183.6 (C-28, s). Found, m/z: 795.4559 [M]+
C46H61O7N5 Calculated, m/z: 795.4566
3β-(4-Oxo-4-((1-((5-(3,5-dichlorophenyl)-1,3,4-oxadiazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)-
methoxy)-butanoyloxy)-urs-12-en-28-oic acid (11b)
The title compound was prepared in the reaction of propargyl ester 10 with azide 3’b as white powder
in 27% yield. IR (KBr, cm-1): 519, 548, 571, 667, 729, 771, 806, 866, 908, 951, 968, 987, 1030, 1049,
1105, 1161, 1238, 1255, 1271, 1315, 1377, 1390, 1412, 1446, 1460, 1514, 1547, 1570, 1691, 1732,
2621, 2874, 2928, 2972, 3078, 3145, 3439. NMR 1Н (400.13 MHz , CDCl3, δ, ppm, J/Hz): 0.76 (3H, s,
Me-26), 0.80 (6H, s, Me-25, Me-24), 0.84 (3H, d, J29-19 = 6.4 Hz, Me-29), 0.91 (3H, s, Me-23), 0.93
(3H, s, Me-30), 1.06 (3H, s, Me-27), 2.16 (1H, d J18-19 = 11.7 Hz, H-18), 2.57-2.66 (4H, m, 2H-2’, 2H-
3’), 4.43-4.50 (1H, m, H-3), 5.21-5.27 (3H, m, H-12, 2H-5’), 5.86 (2H, c, 2H-8’), 7.53 (1H, br.s, H-

14’), 7.83 (1H, s, H-7’), 8.00 (2H, s, H-12’, H-16’). NMR 13C (125.76 MHz , CDCl3, δ, ppm, Hz): 15.4
(C-25, q), 16.7 (C-24, q), 17.0 (C-26, q), 17.0 (C-29, q), 18.0 (C-6, t), 21.1 (C-30, q), 23.2 (C-11, t),
23.4 (C-2, t), 23.5 (C-27, q), 24.0 (C-16, t), 27.9 (C-15, t), 28.0 (C-23, q), 29.0 (C-2', t), 29.3 (C-3', t),
30.5 (C-21, t), 32.7 (C-7, t), 36.7 (C-22, t), 36.8 (C-10, s), 37.6 (C-1, t), 38.1 (C-4, s), 38.7 (C-20, d),
38.9 (C-19, d), 39.4 (C-8, s), 41.8 (C-14, s), 44.1 (C-8', t), 47.3 (C-9, d), 47.8 (C-17, s), 52.4 (C-18, d),
55.1 (C-5, d), 57.6 (C-5', t), 81.4 (C-3, d), 124.4 (C-7', d), 125.3 (C-12', d), 1,2,5-.3 (C-16', d), 125.4
(C-11', s), 1,2,5-.5 (C-12, d), 132.2 (C-14', d), 136.1 (C-13', s), 136.1 (C-15', s), 137.9 (C-13, s), 143.9
(C-6', s), 160.5 (C-9', s), 164.0 (C-10', s), 171.8 (C-4', s), 172.1 (C-1', s), 183.4 (C-28, s). Elemental
analysis: found C 63.81%, H 6.83%, Cl 8.20% N 8.15%; calculated C 63.88%, H 6.88%, Cl 8.20%, N
8.10%.
3β-(4-Oxo-4-((1-((5-(4-bromophenyl)-1,3,4-oxadiazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-
butanoyloxy)-urs-12-en-28-oic acid (11c)
The title compound was prepared in the reaction of propargyl ester 10 with azide 3’c as white powder
in 33% yield. IR (KBr, cm-1): 461, 503, 571, 661, 679, 694, 731, 758, 781, 806, 833, 881, 908, 966,
995, 1011, 1030, 1049, 1070, 1086, 1103, 1113, 1159, 1215, 1238, 1,2,5-5, 1273, 1315, 1367, 1377,
1387, 1410, 1460, 1481, 1549, 1585, 1605, 1689, 1730, 2617, 2872, 2926, 2939, 2974, 3088, 3144,
3437. NMR 1Н (400.13 MHz , CDCl3, δ, ppm, J/Hz): 0.74 (3H, s, Me-26), 0.79 (3H, s, Me-25), 0.80
(3H, s, Me-24), 0.83 (3H, d, J29-19 = 6.2 Hz, Me-29), 0.91 (3H, s, Me-23), 0.93 (3H, s, Me-30), 1.05
(3H, s, Me-27), 2.16 (1H, d J18-19 = 11.1 Hz, H-18), 2.56-2.66 (4H, m, 2H-2’, 2H-3’), 4.42-4.49 (1H,
m, H-3), 5.19-5.22 (1H, m, H-12), 5.22-5.26 (2H, m, 2H-5’), 5.85 (2H, c, 2H-8’) 7.63 (2H, d, J = 8.6
Hz, H-13’, H-15’), 7.83 (1H, s, H-7’), 7.87 (2H, d, J = 8.4 Hz, H-12’, H-16’). NMR 13C (125.76 MHz ,
CDCl3, δ, ppm): 15.5 (C-25, q), 16.7 (C-24, q), 17.0 (C-26, q), 17.0 (C-29, q), 18.1 (C-6, t), 21.2 (C-
30, q), 23.2 (C-11, t), 23.5 (C-2, t), 23.6 (C-27, q), 24.0 (C-16, t), 27.9 (C-15, t), 28.0 (C-23, q), 29.1
(C-2', t), 29.4 (C-3', t), 30.6 (C-21, t), 32.7 (C-7, t), 36.7 (C-22, t), 36.8 (C-10, s), 37.7 (C-1, t), 38.1
(C-4, s), 38.8 (C-20, d), 39.0 (C-19, d), 39.4 (C-8, s), 41.8 (C-14, s), 44.1 (C-8', t), 47.4 (C-9, d), 47.9
(C-17, s), 52.5 (C-18, d), 55.2 (C-5, d), 57.7 (C-5', t), 81.4 (C-3, d), 121.8 (C-11', s), 124.3 (C-7', d),
125.6 (C-12, d), 127.3 (C-14', s), 128.5 (C-12', d), 128.5 (C-16', d), 132.6 (C-13', d), 132.6 (C-15', d),
137.9 (C-13, s), 143.9 (C-6', s), 160.0 (C-9', s), 165.5 (C-10', s), 171.8 (C-4', s), 172.2 (C-1', s), 183.5
(C-28, s). Elemental analysis: found C 63.16%, H 6.91%, N 8.04%; calculated C 63.15%, H 6.91%,
Br 8.13%, N 8.00%.
3β-(4-Oxo-4-((1-((5-(3,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl)methyl)-1H-1,2,3-triazol-4-
yl)methoxy)-butanoyloxy)-urs-12-en-28-oic acid (11d)

The title compound was prepared in the reaction of propargyl ester 10 with azide 3’d as white powder
in 38% yield. IR (KBr, cm-1): 440, 501, 555, 569, 661, 677, 729, 758, 771, 810, 829, 889, 908, 924,
966, 995, 1034, 1049, 1093, 1159, 1240, 1,2,5-5, 1271, 1315, 1350, 1377, 1389, 1410, 1458, 1543,
1566, 1581, 1606, 1689, 1732, 2619, 2872, 2928, 2974, 3090, 3144, 3435. NMR 1Н (400.13 MHz ,
CDCl3, δ, ppm, J/Hz): 0.74 (3H, s, Me-26), 0.79 (3H, s, Me-25), 0.80 (3H, s, Me-24), 0.83 (3H, d, J29-
19 = 6.4 Hz, Me-29), 0.91 (3H, s, Me-23), 0.93 (3H, s, Me-30), 1.05 (3H, s, Me-27), 2.16 (1H, d J18-19
11.1 Hz, H-18), 2.55-2.66 (4H, m, 2H-2’, 2H-3’), 4.42-4.49 (1H, m, H-3), 5.19-5.23 (1H, m, H-12),
5.23-5.25 (2H, d, J = 4.2 Hz, 2H-5’), 5.86 (2H, c, 2H-8’), 7.58 (1H, dd, J1 = 8.3, J2 = 1.3 Hz, H-16’),
7.83 (1H, s, H-7’), 7.86 (1H, ddd, J1 = J2 = 2.0 Hz, H-12’), 8.02 (1H, ddd, J1 = J2 = 1.8 Hz, H-15’).
NMR 13C (125.76 MHz , CDCl3, δ, ppm): 15.4 (C-25, q), 16.7 (C-24, q), 17.0 (C-26, q), 17.0 (C-29,
q), 18.0 (C-6, t), 21.1 (C-30, q), 23.2 (C-11, t), 23.4 (C-2, t), 23.5 (C-27, q), 24.0 (C-16, t), 27.9 (C-15,
t), 28.0 (C-23, q), 29.0 (C-2', t), 29.3 (C-3', t), 30.5 (C-21, t), 32.7 (C-7, t), 36.6 (C-22, t), 36.8 (C-10,
s), 37.6 (C-1, t), 38.1 (C-4, s), 38.7 (C-20, d), 38.9 (C-19, d), 39.4 (C-8, s), 41.8 (C-14, s), 44.1 (C-8',
t), 47.3 (C-9, d), 47.8 (C-17, s), 52.4 (C-18, d), 55.1 (C-5, d), 57.6 (C-5', t), 81.4 (C-3, d), 122.6 (C-11',
s), 124.3 (C-7', d), 125.5 (C-12, d), 126.1 (C-16', d), 128.8 (C-12', d), 131.4 (C-15', d), 133.8 (C-14', s),
137.0 (C-13', s), 137.9 (C-13, s), 143.9 (C-6', s), 160.3 (C-9', s), 164.4 (C-10', s), 171.8 (C-4', s), 172.1
=
35
(C-1', s), 183.4 (C-28, s). Found, m/z: 863.3800 [M]+ C46 H59 O7 N5 Cl2 Calculated, m/z: 863.3786
3β-(4-Oxo-4-((1-((4-methyl-2-oxido-1,2,5-oxadiazol-3-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-
butanoyloxy)-urs-12-en-28-oic acid (12)
The title compound was prepared in the reaction of propargyl ester 10 with azide 4’c as off-white
powder in 76% yield. IR (KBr, cm-1): 571, 600, 660, 675, 771, 781, 793, 808, 829, 849, 881, 910, 924,
968, 991, 1020, 1045, 1103, 1159, 1188, 1203, 1,2,3-6, 1,2,5-5, 1271, 1315, 1369, 1389, 1423, 1458,
1520, 1610, 1691, 1732, 2621, 2874, 2928, 2943, 2974, 3144, 3435. NMR 1Н (400.13 MHz , CDCl3,
δ, ppm, J/Hz): 0.75 (3H, s, Me-26), 0.81 (3H, s, Me-25), 0.82 (3H, s, Me-24), 0.83 (3H, d, J29-19 = 6.4
Hz, Me-29), 0.92 (6H, s, Me-23б Me-30), 1.05 (3H, s, Me-27), 2.16 (1H, d J18-19 = 11.4 Hz, H-18),
2.41 (3H, s, Me-10’), 2.58-2.66 (4H, m, 2H-2’, 2H-3’), 4.45-4.51 (1H, m, H-3), 5.18-5.24 (3H, m, H-
12, 2H-5’), 5.43 (2H, c, 2H-8’), 7.78 (1H, s, H-7’). NMR 13C (125.76 MHz , CDCl3, δ, ppm): 10.9 (C-
11', q), 15.4 (C-25, q), 16.7 (C-24, q), 17.0 (C-26, q), 17.0 (C-29, q), 18.0 (C-6, q), 21.1 (C-30, q), 23.2
(C-11, t), 23.4 (C-2, t), 23.5 (C-27, q), 23.9 (C-16, t), 27.9 (C-15, t), 29.0 (C-2', t), 29.3 (C-3', t), 30.5
(C-23, q), 32.7 (C-21, t), 36.6 (C-7, t), 36.8 (C-22, t), 37.6 (C-10, s), 38.1 (C-1, t), 38.7 (C-4, s), 38.8
(C-20, d), 38.9 (C-19, d), 39.4 (C-8, s), 41.3 (C-8', t), 41.8 (C-14, t), 47.3 (C-9, d), 47.8 (C-17, s), 52.4
(C-18, d), 55.1 (C-5, d), 57.5 (C-5', d), 81.3 (C-3, d), 111.9 (C-10', s), 124.3 (C-7', s), 125.6 (C-12, d),
137.9 (C-13, s), 143.9 (C-6', s), 154.0 (C-9', s), 171.8 (C-4', s), 172.1 (C-1', s), 183.9 (C-28, s).
Elemental analysis: found C 65.71%, H 7.87%, N 9.29%; calculated C 65.66%, H 7.93%,, N 9.34%.

(1-((4-Methyl-2-oxido-1,2,5-oxadiazol-3-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl 3-oxo-lup-20(29)-en-
28-oate (14a)
The title compound was prepared in the reaction of propargyl ester 13a with azide 4’ as white powder
in 75% yield. IR (cm-1): 444, 500, 544, 582, 600, 652, 669, 702, 789, 849, 883, 916, 939, 966, 989,
1009, 1024, 1045, 1084, 1126, 1140, 1173, 1228, 1281, 1294, 1317, 1352, 1379, 1458, 1520, 1552,
1610, 1641, 1703, 1724, 2870, 2949, 3072, 3145, 3435. NMR 1Н (400.13 MHz , CDCl3, δ, ppm, J/Hz):
0.71 (3H, s, Me-) 0.88 (3H, s), 0.92 (3H, s), 0.99 (3H, s), 1.03 (3H, s), 1.65 (3H, s, Me-30), 2.43 (3H,
s, Me-7’), 2.91-3.01 (1H, m, H-19), ), 4.58 (1H, br.s, H-30), 4.71 (1H, br.s, H-30), 5.18 (2H, AB, JAB
=
12.4 Hz, Δη = 18.9, 2H-1’), 5.42 (2H, AB, JAB = 12.1 Hz, Δη = 8.7, 2H-4’), 7.79 (1H, C, H-3’). NMR
13C (125.76 MHz , CDCl3, δ, ppm): 11.0 (C-7', q), 14.5 (C-27, q), 15.2 (C-25, q), 15.9 (C-26, q), 19.3
(C-30, q), 19.5 (C-6, t), 20.9 (C-24, q), 21.3 (C-11, t), 25.4 (C-12, t), 26.5 (C-23, q), 29.5 (C-15, t),
30.4 (C-21, t), 31.8 (C-16, t), 33.4 (C-7, t), 34.1 (C-2, t), 36.8 (C-10, s), 36.8 (C-22, t), 38.2 (C-13, d),
39.5 (C-1, t), 40.5 (C-8, s), 41.2 (C-4', t), 42.3 (C-14, c), 46.8 (C-19, d), 47.2 (C-4, c), 49.2 (C-9, d),
49.8 (C-18, d), 54.8 (C-5, d), 56.4 (C-17, c), 56.8 (C-1', t), 109.7 (C-29, t), 111.8 (C-6', s), 124.5 (C-3',
d), 144.2 (C-2', s), 150.2 (C-20, s), 153.9 (C-5', s), 175.9 (C-28, s), 218.1 (C-3, c). Found, m/z:
647.4035 [M]+ C37H53O5N5 Calculated, m/z: 647.4041.
(1-((4-Methyl-2-oxido-1,2,5-oxadiazol-3-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl-3β-hydroxy-lup-
20(29)-en-28-oate (14b)
The title compound was prepared in the reaction of propargyl ester 13b with azide 4’ as off-white
powder in 87% yield. IR (cm-1): 544, 600, 652, 669, 789, 849, 885, 914, 945, 984, 1009, 1043, 1074,
1107, 1134, 1151, 1174, 1230, 1271, 1294, 1319, 1360, 1377, 1389, 1454, 1520, 1608, 1641, 1726,
2870, 2943, 3072, 3145, 3439. NMR 1Н (400.13 MHz , CDCl3, δ, ppm, J/Hz): 0.67 (3H, s, Me-24),
0.70 (3H, s, Me-26), 0.73 (3H, s, Me-25), 0.91 (3H, s, Me-27), 0.93 (3H, s, Me-23), 1.64 (3H, C, Me-
29), 2.43 (3H, C, Me-7’), 2.90-2.99 (1H, m, H-19), ),3.11-3.18 (1H, m, H-3), 4.57 (1H, br.s, H-29),
4.70 (1H, br.s, H-29), 5.18 (2H, AB, JAB = 12.7 Hz, Δη = 20.3, 2H-1’), 5.41 (2H, AB, JAB = 11.5 Hz,
Δη = 10.0, 2H-4’), 7.79 (1H, s, H-3’). NMR 13C (125.76 MHz , CDCl3, δ, ppm): 11.0 (C-7', q), 14.6
(C-27, q), 15.3 (C-24, q), 15.4 (C-25, q), 16.1 (C-26, q), 18.1 (C-6, ), 19.3 (C-30, q), 20.7 (C-11, t),
25.4 (C-12, t), 27.3 (C-2, t), 27.9 (C-23, q), 29.5 (C-21, t), 30.4 (C-15, t), 31.9 (C-16, t), 34.1 (C-22, t),
36.8 (C-7, t), 37.1 (C-10, s), 38.2 (C-13, d), 38.6 (C-1, t), 38.8 (C-4, s), 40.6 (C-8, s), 41.2 (C-4', t),
42.2 (C-14, s), 46.8 (C-19, d), 49.3 (C-18, d), 50.4 (C-9, d), 55.2 (C-5, d), 56.4 (C-17, s), 56.7 (C-1', t),
78.8 (C-3, d), 109.6 (C-29, d), 111.8 (C-6', s), 124.6 (C-3', d), 144.2 (C-2', s), 150.2 (C-20, s), 153.9
(C-5', s), 175.9 (C-28, s). Found, m/z: 649.4193 [M]+ C37H55O5N5 Calculated, m/z: 649.4198.
(1-((5-Phenyl-1,3,4-oxadiazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)-3-oxo-lup-20(29)-en-28-oate (15a)

The title compound was prepared in the reaction of propargyl ester 13b with azide 4’ as off-white
powder in 66% yield. IR (KBr, сm-1): 434, 442, 498, 519, 544, 582, 665, 690, 710, 756, 802, 885, 918,
935, 964, 985, 1016, 1047, 1070, 1086, 1126, 1140, 1153, 1173, 1215, 1227, 1292, 1317, 1350, 1379,
1452, 1483, 1552, 1578, 1591, 1610, 1641, 1703, 1724, 1778, 2870, 2949, 3070, 3145, 3435. NMR 1Н
(400.13 MHz , CDCl3, δ, ppm, J/Hz): 0.68 (3H, s, Me-25) 0.83 (3H, s, Me-26), 0.90 (3H, s, Me-27),
0.97 (3H, s, Me-24), 1.02 (3H, s, Me-23), 1.60 (1H,t, J18-19 = 11.3 Hz, H-18), 1.63 (3H, s, Me-30),
2.89-2.98 (1H, m, H-19), 4.56 (1H, br.s, H-29), 4.69 (1H, br.s, H-29), 5.23 (2H, AB-sys, JAB = 12.0
Hz, Δη = 14.2, 2H-4’), 5.89 (2H, AB-sys, JAB = 13.8 Hz, Δη = 14.8, 2H-1’), 7.46-7.58 (3H, m, H-9’,
H-10’, H-11’), 7.93 (1H, s, H-3’), 7.97-8.03 (2H, m, H-8’, H-12’). NMR 13C (125.76 MHz , CDCl3, δ,
ppm): 14.45 (С-27, q), 15.15 (С-25, q), 15.80 (С-26, q), 19.2 (С-30, q), 19.4 (С-6, t), 20.9 (С-24, q),
21.2 (С-11, t), 25.3 (С-12, t), 26.4 (С-23, q), 29.4 (С-15, t), 30.3 (С-21, t), 31.7 (С-16, t), 33.4 (С-7, t),
34.0 (С-2, t), 36.7 (С-10, s), 36.7 (С-22, t), 38.2 (С-13, d), 39.4 (С-1, t), 40.4 (С-8, s), 42.2 (С-14, s),
44.1 (С-4', t), 46.7 (С-19, d), 47.2 (С-4, s), 49.1 (С-9, d), 49.7 (С-18, d), 54.8 (С-5, d), 56.3 (С-17, s),
56.8 (С-1', t), 109.7 (С-29, t), 122.8 (С-7', s), 124.4 (С-3', d), 127.0 (С-8', d), 127.0 (С-12', d), 129.1
(С-9', d), 129.1 (С-11', d), 132.3 (С-10', d), 144.2 (С-2', s), 150.1 (С-20, s), 159.8 (С-5', s), 166.0 (С-6',
s), 175.8 (С-28, s), 217.9 (С-3, s). Calculated m\z: 693.4259 Found m\z: 693.4252.
Synthesis of acetates 8b, 9b, 14c
To a stirred mixture of 8a or 9a or 14b (1 mmol), Et3N (2.0 g, 20 mmol) in CH2Cl2 (20 mL) acetic
anhydride (1.0 g, 10 mmol) was added at room temperature. The mixture was stirred overnight (TLC
control) after which MeOH (1.0 g, 30 mmol) was added. The resulting mixture was washed with HClaq
(3%, 2×20 mL), NaHCO3 aq (5%, 20 mL), dried (Na2SO4) and concentrated. The residue was
chromatographed (SiO2, CH2Cl2, MTBE) to afford acetates 9a or 9b or 17.
(1-((4-Methyl-2-oxido-1,2,5-oxadiazol-3-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl-3β-acetoxy-urs-12-
en-28-oate (8b)
The title compound was prepared in the acylation reaction of compound 9a as white powder in 90%
yield. IR (KBr, cm-1): 602, 652, 660, 700, 733, 756, 791, 829, 849, 906, 916, 953, 987, 1028, 1043,
1078, 1113, 1138, 1167, 1178, 1194, 1248, 1308, 1371, 1390, 1456, 1520, 1610, 1659, 1732, 1765,
2874, 2929, 2949, 2968, 3145, 3435. NMR 1Н (400.13 MHz , CDCl3, δ, ppm, J/Hz): 0.69 (3H, s, Me-
26), 0.82 (3H, s, Me-25), 0.84 (6H, s, Me-29, Me-24), 0.91 (3H, s, Me-30), 0.93 (3H, s, Me-23), 1.05
(3H, s, Me-27), 2.01 (4H, s, Me-2”), 2.20 (1H, d, J=11.2 Hz, H-18), 2.42 (3H, s, Me-9’), 4.43-4.57
(3H, m, H-3 + 2H-1’), 5.19-5.22 (1H, ddd, J1=J2 = 3.4 Hz, H-12), 5.24 (2H, s), 5.37 (2H, s, 2H-4’),
7.77 (1H, s, H-6’). NMR 13C (125.76 MHz , CDCl3, δ, ppm, J/Hz): 10.9 (C-7', q), 15.4 (C-25, q), 16.7
(C-24, q), 16.7 (C-26, q), 16.9 (C-29, q), 18.1 (C-6, t), 21.1 (C-30, q), 21.2 (C-2", q), 23.2 (C-11, t),

23.4 (C-27, q), 23.5 (C-16, t), 24.1 (C-2, t), 27.9 (C-15, t), 28 (C-23, q), 30.5 (C-21, t), 32.8 (C-7, t),
36.5 (C-22, t), 36.8 (C-10, s), 37.6 (C-1, t), 38.2 (C-4, s), 38.7 (C-20, d), 39 (C-19, d), 39.4 (C-8, s),
41.2 (C-4', t), 42 (C-14, s), 47.3 (C-9, d), 48.1 (C-17, s), 52.7 (C-18, d), 55.2 (C-5, d), 57.1 (C-1', t),
80.8 (C-3, d), 111.8 (C-6', s), 124.5 (C-3', d), 125.5 (C-12, d), 137.9 (C-13, s), 144.2 (C-2', s), 153.9
(C-5', s), 170.9 (C-1", s), 177.3 (C-28, s). Found, m/z: 691,4311 [M]+ C39H57O6N5 Calculated, m/z:
691,4302.
3-((4-((3β-Acetoxy-urs-12-en-28-oyl-oxyacetoxy)methyl)-1H-1,2,3-triazol-1-yl)methyl)-4-methyl-1,2,5-
oxadiazole 2-oxide (9b)The title compound was prepared in the acylation reaction of compound 9a as
white powder in 86% yield. IR (KBr, cm-1): 600, 661, 679, 700, 733, 795, 806, 829, 849, 903, 922,
953, 970, 987, 1007, 1028, 1078, 1109, 1140, 1167, 1180, 1198, 1248, 1308, 1371, 1389, 1456, 1520,
1610, 1659, 1730, 2874, 2928, 2949, 2966, 3147, 3435. NMR 1Н (400.13 MHz , CDCl3, δ, ppm, J/Hz):
0.55 (3H, s, Me-26), 0.81-0.85 (9H, m, Me-25, Me-29, Me-24), 0.87-0.92 (6H, m, Me-30, Me-23),
1.03 (3H, s, Me-27), 2.02 (3H, s, Me-2”), 2.18 (1H, d, О = 11.6 Hz, H-18), 2.41 (3H, s, Me-9’), 4.43-
4.50 (1H, m, H-3), 5.12 (2H, AB, JAB = 9.5 Hz, Δη = 12.7, 2H-1’), 5.17-5.21 (1H, m, H-12), 5.40 (2H,
s, 2H-4’), 7.73 (1H, s, H-3’) NMR 13C (125.76 MHz , CDCl3, δ, ppm, J/Hz): 11.0 (C-9', q), 15.5 (C-25,
q), 16.7 (C-24, q), 16.9 (C-26, q), 17.0 (C-29, q), 18.1 (C-6, t), 21.1 (C-30, q), 21.3 (C-2", q), 23.2 (C-
11, t), 23.4 (C-27, q), 23.5 (C-16, t), 24.1 (C-2, t), 27.9 (C-15, t), 28.0 (C-23, q), 30.6 (C-21, t), 32.9
(C-7, t), 36.4 (C-22, t), 36.8 (C-10, s), 37.6 (C-1, t), 38.2 (C-4, s), 38.7 (C-20, d), 39 (C-19, d), 39.5
(C-8, s), 41.3 (C-6', t), 42 (C-14, s), 47.4 (C-9, d), 48.1 (C-17, s), 52.6 (C-18, d), 55.2 (C-5, d), 57.8
(C-3', t), 60.2 (C-1', t), 80.9 (C-3, d), 111.9 (C-7', s), 124.6 (C-5', d), 125.6 (C-12, d), 137.8 (C-13, s),
143.3 (C-4', s), 154.0 (C-8', s), 167.9 (C-2', s), 171.0 (C-1", s), 176.9 (C-28, s). Found, m/z: 749.4367
[M]+ C41H59O8N5 Calculated, m/z: 749.4358.
(1-((4-Methyl-2-oxido-1,2,5-oxadiazol-3-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl 3-acetoxy-lup-
20(29)-en-28-oate (14c)
The title compound was prepared in the acylation reaction of compound 9a as white powder in 92%
yield. IR (KBr, cm-1): 490, 544, 600, 652, 667, 700, 756, 789, 849, 885, 916, 980, 1028, 1041, 1107,
1130, 1149, 1196, 1250, 1317, 1375, 1390, 1454, 1479, 1520, 1556, 1610, 1641, 1726, 2727, 2872,
2947, 3072, 3145. NMR 1Н (400.13 MHz , CDCl3, δ, ppm, J/Hz): 0.67 (3H, s, Me-25), 0.81 (9H, m,
Me-24, Me-26, Me-27), 0.90 (3H, s, Me-23), 1.60 (1H, m, H-18), 1.65 (3H, s, Me-30), 2.01 (3H, s,
Me-2”), 2.03-2.13 (1H, m, H-15), 2.43 (3H, s, Me-7’), 2.90-3.00 (1H, td, J1 = 10.8 Hz, J2 = 4.6 Hz, H-
19), 4.39-4.47 (1H, m, H-3), 4.58 (1H, br.s., H-29), 4.71 (1H, br,s., H-29), 5.18 (2H, AB-sys, JAB
=
12.9 Hz, Δη = 19.66, 2H-1’), 5.41 (2H, AB-sys, JAB = 10.6 Hz, Δη = 9.76, 2H-4’), 7.79 (1H, s, H-3’).
NMR 13C (125.76 MHz , CDCl3, δ, ppm, J/Hz): 11.0 (C-7', q), 14.6 (C-27, q), 15.4 (C-24, q), 16.1 (C-
25, q), 16.4 (C-26, q), 18.1 (C-6, t), 19.3 (C-30, q), 20.8 (C-11, t), 21.3 (C-2", q), 23.6 (C-12, t), 25.4
(C-2, t), 27.9 (C-23, q), 29.5 (C-21, t), 30.4 (C-15, t), 31.9 (C-16, t), 34.1 (C-22, t), 36.8 (C-7, t), 37.0

(C-10, s), 37.7 (C-4, s), 38.2 (C-13, d), 38.3 (C-1, t), 40.6 (C-8, s), 41.2 (C-4', t), 42.3 (C-14, s), 46.8
(C-19, d), 49.3 (C-18, d), 50.3 (C-9, d), 55.3 (C-5, d), 56.5 (C-17, s), 56.8 (C-1', t), 80.9 (C-3, d), 109.7
(C-29, d), 111.8 (C-6', s), 124.5 (C-3', d), 144.3 (C-2', s), 150.3 (C-20, s), 153.9 (C-5', s), 171.0 (C-1",
s), 175.9 (C-28, s). Found, m/z: 691.4303 [M]+ C39H57O6N5 Calculated, m/z: 691.4304.
2.1.3. Cytotoxicity of triterpenoid-azole hybrids
DMEM with 10% fetal bovine serum (Gibco, USA) was used for culturing. Optical density was
measured on a Multiskan RC spectrophotometer (ThermoFischer Scientific, USA). Cultures of breast
cancer MCF7 (ATCC number HTB-22), U-87 MG glioblastoma multiform cells (ATCC number HTB-
14), A549 lung carcinoma (ATCC number CRM-CCL-18), HepG2 hepatocarcinoma, (ATCC number
HB-8065) were obtained commercially (ATCC, USA). The noncancer control was immortalized
human fibroblasts (ATCC number CRL-4058). Cytotoxicity of the tested compounds was assessed
using the MTT assay and the standard procedure [40]. Cells were inoculated into 96-well plates (3,000
cells per well) and incubated at 37°C in 5% CO2 for attachment. Medium in wells was replaced after
24 h with fresh medium containing the tested compounds in DMSO (1 % v/v) and incubated for 72 h.
Optical density was measured in a plate spectrophotometer as usual. All compounds were tested at
concentrations of 10, 25, 50, and 100 μM using the required controls, i.e., negative, DMSO (solvent),
and positive, doxorubicin (standard cytostatic). Each experiment was performed independently in
triplicate with three tests in each. Statistical processing of the results was performed using the
Microsoft Excel-2007, STATISTICA 6.0 and GraphPad Prism 5.0 programs. Results were reported as
mean inhibitory concentration IC50 ± SEM. Reliability of differences (p) was estimated using the
Student t test. The differences with p < 0.05 were considered as reliable.
2.1.4. Flow cytometry analysis
For apoptosis and cell cycle analysis two different types of MCF7 cell line was used: wild type (p53
+/+) and p53-deficient (p53 -/-), kindly provided by P.N. Chumakov (Engelhardt Institute of
Molecular Biology RAS, Russia). Cells were cultivated on DMEM with 10% fetal bovine serum
(Gibco, USA). 48 h before experiment cells were seeded into 6-well plates (30000 cells per plate) and
incubated 24 h at 37°C in 5% CO2 for attachment. Then medium in wells was replaced with fresh
medium containing the tested compounds in GI50 concentration and incubated for 24 h followed by
flow cytometry analysis. Also induction for overexpression of p53 protein was performed using an 10
nM estrogen (Sigma) treatment.
For apoptosis detection FITC Annexin V Apoptosis Detection Kit I was used (BD Pharmingen). For
investigation of cell cycle Hoechst 33342 Ready Flow Reagent was used (Invitrogen, ThermoFisher
Scientific). Sample preparation was performed according to the protocol, doxorubicin as cytostatic and

nutlin-3 (Calbiochem) as MDM2 inhibitor were used as positive controls. Analysis performed on at
least 30000 cells. CytoFLEX flow cytometer (Beckman Coulter) was used for analysis. The results are
presented as a percentage of cell population standing in different stages of the cell cycle.
2.1.5. Molecular docking methods
Molecular modeling was carried out in the Schrodinger Maestro visualization environment using
applications from the Schrodinger Small Molecule Drug Discovery Suite 2016-1 package [41]. Three-
dimensional structures of the derivatives were obtained empirically in the LigPrep application using
the OPLS3 force field [42]. For the calculations, the XRD model of Mdm2 protein with PDB ID 5OAI
[43] (resolution 2 Å) was chosen. To model a possible mechanism of inhibition of selected target,
molecular docking of new compounds was performed at the binding site of 3-[(1R)-2-(Tert-
butylamino)-1-[formyl-[(3,4,5-trifluorophenyl)methyl]amino]-2-oxoethyl]-6-chloro-1H-indole-2-
carboxylic acid (B5K) using Glide [44]. The search area for docking was selected according to the size
of known inhibitor. Docking was performed in comparison with the known inhibitors B5K and nutlin-
3A. The three-dimensional structures of inhibitors were obtained in the PubChem database and
prepared in the LigPrep application. Non-covalent interactions of compounds in the binding site were
visualized using Schrodinger Maestro.
3. Results and discussion
3.1. Chemistry
The synthetic pathways to the intermediate compounds and new hybrid derivatives are depicted on the
schemes 1-4.
-N
N+
Na+
N
N
N-
N
N
R
R
N
Cl
N+
O
O
N-
3'a-d,
76-84%
3a-d
Cl
Cl
R:
Cl
Br
a
Cl
c
d
b
N-
N+
N
-N
N+
Na+
O
O
N-
N
N
N
N
O
Br
O
4'
, 84%
4

Scheme 1. Synthesis of azide derivatives from 2-(chloromethyl)-5-aryl-1,3,4-oxadiazoles and 3-
(bromomethyl)-4-methyl-1,2,5-oxadiazole 2-oxide.
2-(azidomethyl)-5-aryl-1,3,4-oxadiazoles 3’a-d were prepared by the reaction of corresponding
chloromethyl derivatives 3a-d with the excess of NaN3 in dimethylformamide (76-84% yield, Scheme
1). Similarly to it, 3-(azidomethyl)-4-methyl-1,2,5-oxadiazole 2-oxide 4’ was obtained from
bromomethyl derivative 4 in 84% yield.
30
20
N
6'
29
H
R
N
5'
19
18
Br
N
3'
H
3'a,c d
NaAscorbate
DMF, 50oC
, ,CuSO4,
N
2'
O
12
26
4'
N
OH
O
K2CO3,
Me2CO
O
28
25
17
1'
1
9
14
O
O
c
O
8
H
H
1
H
3
5
27
HO
HO
7 a,c d 62-70%
,
84%
6a
HO
H
H
12'
Cl
24
23
11'
10'
R:
7'
Cl
Br
8'
4
a9'
'
d
,
C
N
u
a
S
A
F
O
s
c
D
ClCH2COOEt,
K2CO3,DMF, 84%
4
,
o
M
r
b
a
,
t
5
e
o
C
0
O
O
N
N
6'
N
N
2'
5'
O
N
7'
4'
O
1'
O
H
3'
OR
H
72%
90%
8a
, R=H
O
5a
, R=H
O
H
KOH,
EtOH,
Ac2O, Et3N
H
RO
78%
HO
8b
, R=Ac
5b
, R=Ac
5b
K2CO3,DMF
Br
4'
,CuSO4,
O
2'
NaAscorbate
DMF, 50oC
O
5'
O
O
4'
3'
4'
6'
3'
H
O
O
2'
O
1'
N
O
H
7'
1'
65-72%
N
N
N
5'
H
O
O
9'
H
9a
, R=H
8'
RO
N
Ac2O, Et3N,
88%
HO
87%
6b
9b
, R=Ac
Scheme 2. Synthesis of ursolic acid C-28 ester conjugates with 5-aryl-1,3,4-oxadiazoles and 4-methyl-
1,2,5-oxadiazole 2-oxide tethered with 1H-1,2,3-triazol-4-yl)methyl- linkers.
Synthesis of propargyl esters of triterpenoids
Alkylation of ursolic acid with propargyl bromide in acetone in the presence of K2CO3 afforded
propargyl ursolate 6a in 84% yield (Scheme 2). Similarly, propargyl betulonate 2a was prepared by
alkylation of betulonic acid 2 in 82% yield (Scheme 4).
Compound 6b was synthesized in 3 steps (alkylation-hydrolysis-propargylation) in general 57% yield
starting from ursolic acid 1. (Scheme 2). Alkylation of ursolic acid 1 with ethyl chloroacetate resulted
in ursolic acid carboxymethyl ester. The terminal ester group of 5a was hydrolyzed selectively

following with the known method [45]. The alkylation of acid 5b was carried out in DMF to give
compound 6b with propargyl ester group tethered by the additional ester-type linker from ursane core
(87%). Certain ursolic acid hybrids including 3-O-succinyl linker and terminal 1,2,5- oxadiazole group
displayed notable cytotoxic activity [46]. Therefore, we were interested in the preparation of ursane
derivatives with a free C-28 carboxyl group and heterocyclic combinations attached at the 3-O-
position on the triterpene core. Ursolic acid (4-propargyl)-succinate 10 was prepared by acylation of 1
with prop-2-ynyl 4-chloro-4-oxobutanoate in the presence of pyridine and 4-DMAP in 78% yield
(Scheme 3). Propargyl betulonate 13a was prepared from betulonic acid 2a in 88% yield (Scheme 4).
Reduction of 13a with NaBH4 following the previously published method [35] resulted in propargyl
betulinate 13b in 75% yield.
Synthesis of triterpenoid-triazole-oxadiazole hybrids.
Ursane and lupane type (1-((5-aryl-1,3,4-oxadiazol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl esters
and (1-((4-Methyl-2-oxido-1,2,5-oxadiazol-3-yl)methyl)-1H-1,2,3-triazol-4-yl)methyl esters were
prepared by 1,3-cycloaddition reactions of corresponding azole-derived azides with propargyl esters
tailored to C-3 or C-28 position of triterpenoids. Propargyl esters 6a, 6b reacted with azides 3’a-d, 4’
in DMF at 50ºC in the presence of CuSO4 and sodium ascorbate (Scheme 2) to afford triple hybrids
7a-d (62-70%), 8a, 9a (65-72%). Lupane-type hybrids 14 a,b, 15a were prepared similarly to above
mentioned from the esters 13 a,b in 82-84% yield (Scheme 4). In the specified conditions, the
reactions of propargyl succinate 10 with azides were sluggish and the yields of hybrids 11, 12 were
low. Therefore, the interaction of 10 with azides 3’a-d, 4’ was carried out in DMF under N2
atmosphere at ambient temperature using a combination of CuBr-DIPEA as a catalyst. In this way,
compounds 11a-d and 12 were prepared in 35-40% and 75% yield (Scheme 3).
N
OH
OH
R
N
H
H
10'
N
7'
O
N
6'
O
1'
O
O
1
9' 8'
H
H
N
5'
2'
O
4'
3'
11 a-d
35-40%
O
HO
N
N
16'
O
,
Cl
11'
12'
A
15'
14'
E
Cl
R:
P
Cl
I
D
,
r
m
t
B
a
d Cu
F
O
N
N2
,
Br
O
13'
a
Cl
-
M
Cl
b
a
'
D
c
d
3
O
4
'
C
OH
u
B
r
,
D
H
I
P
D
M
E
A
,
F
,
N2 a
N
OH
10' 11'
9'
t
m
O
O
H
H
N
N
N
6'
O
O
O
7'
8'
H
N
O
3'
6'
1'
O
2'
4'
O
5'
4'
2'
1'
3'
76%
12
O
O
7'
10
, 94%
5'
O
O

Scheme 3. Synthesis of ursolic acid C-3 ester conjugates with 5-aryl-1,3,4-oxadiazoles and 4-methyl-
1,2,5-oxadiazole 2-oxide tethered with 4-(1H-1,2,3-triazol-4-yl)methoxy)-4-oxobutanoate- linkers.
H
H
O
Br
OH
H
H
O
O
K2CO3, DMF
H
1
2
R2
R1
13a
13b
H
, R , R =O, 82%
NaBH4,
THF
2
1
2
O
, R =OH, R =H, 75%
H
H
29
20
3a'
, CuSO4
4
'
uSO
N
N
, C
4
DM
O
30
O
21
NaAsc, DMF
N
F
N
c,
As
Na
N
19
17
N
N
6'
O
H
N
12
26
5'
22
H
N
N
7'
O
4'
28
25
O
1'
2'
H
3'
9
14
1
H
15
O
10
8
O
1
2
H
H
6
3
14a
14b
R2
R1
5
R , R =O, 75%
, R =OH, R =H, 87%
27
1
2
O
13a
15a
, 66%
Ac2O,
Et3N
H
H
1
2
23
24
14c
R =OAc, R =H, 92%
O
O
H
2
H
N
n
N
O
O
H
N
O
H
n= 1, 2, 3
16 a n=1, b n=2, c n=3
O
H
Scheme 4. Synthesis of lupane-type conjugates with 5-aryl-1,3,4-oxadiazoles and 4-methyl-1,2,5-
oxadiazole 2-oxide.
The structures of the new compounds were elucidated by NMR, IR, MS, and elemental analysis (11b,
11c, 12) data. In the 1H NMR spectra of the triazolo-oxadiazole derivatives, a triazole proton as a
singlet was observed in the region of 7.8 ppm. For hybrid triazole-1,3,4-oxadiazoles 7a,c,d, 11 a-d and
triazole-furoxan hybrids 8a,b, 9a,b, 12, 14a,b,c the signals of O-CH2-triazole were registered at 5.08-
5.25 ppm (1H spectra) and 57.1-57.8 (13C spectra). The corresponding signals of triazole-CH2-
oxadiazole were found for 1,3,4-oxadiazole compounds at 5.30-5.86 ppm (1H) and 44.0-44.1 (13C) and
for furoxan derivatives 5.40 (1H) and 41.3 (13C). The methylene protons of the linker group O-CH2-
COOCH2- for compounds 9a,b were observed at 4.50 (1H) and 60.2 ppm (13C). In the 13C spectra of
hybrids of triazoles with 1,3,4 oxadiazoles 7a,c,d, 11 a-d, and triazoles with furoxan 8a,b, 12, 14a,b,c
signals of carbon atoms pair of the 1,2,3 triazoles were observed at 124.2-124.5; 143.8-144.3 ppm,
while for furoxan derivatives 9a,b with additional ester linkage signals of carbons of triazole moiety
were 124.5; 143.3 ppm. The derivatives of 7a,c,d, 11 a-d showed characteristic signals of 1,3,4-
oxadiazole carbon atoms at 159.8-160.5 and 164.4-166.1 ppm. The corresponding pair of carbon
signals of furoxan moiety in compounds 8a,b, 9a,b, 12, 14a,b was registered at 111.8-111.9 and
153.9-154.0 ppm.

3.2. Cytotoxicity of hybrid compounds
Cytotoxicity of the novel compounds was assessed using the MTT assay towards breast cancer MCF7,
glioblastoma multiform cells U-87 MG, lung carcinoma A549, hepatocarcinoma U-87 MG, using
immortalized human fibroblasts as non-cancer control (Table 1).
Table 1. Concentrations of half-maximal inhibition (IC50 ± SEM, µM) of test compounds on
immortalized human fibroblasts, MCF-7, U-87 MG, A549, HepG2 cells.
Compound*
immortalized
human
MCF7
U-87 MG
A549
HepG2
fibroblasts
3.33±0.67
4.51±1.12
2.05±0.22
6.17±1.17 10.02±1.67
Doxorubicin
Ursolic acid 1
90.89±5.5
16.7±2.99
25.85±3.04
10.41±1.25
25.05±3.17 43.82±3.88
41.02±3.77 37.28±5.02
10.91±1.13 45.0±6.7
Betulonic acid 2
28.78±4.12
22.9±10.02
1.55±0.08
45.12±6.78
29.71±6.52
˃100
8a
8b
>100
˃100
12.89±2.63
˃100
9a
82.59±16.87 26.23±8.76
>100
>100
35.58±8.83
>100
11a
12
74.9±4.58
48.35±11.47
17.48±2.11
22.46±2.50
33.16±2.77
>100
>100
>100
40.26±7.55
>100
81.67±2.89
˃100
>100
14a
14b
14c
˃100
˃100
54.13±7.05
>100
13.38±5.69
45.21±6.18
>100
>100
36.33±5.11
41.08±7.06 88.27±9.71
* Compounds 7a, 7c, 7d, 9b, 11b, 11c, 11d, 11e, 11f, 15a, 16a, 16b, 16c were found non-active (IC50
˃100 µM for all cells tested).
For the starting compounds ursolic acid 1 and betulonic acid 2, cytotoxicity values were comparable
towards MCF-7, U-87-MG, and HepG2 cells, while betulonic acid 2 exhibited a significantly higher
cytotoxicity with respect to non-cancer cells and A-549 cells. The cytotoxicity of doxorubicin
significantly exceeded the activity of the starting acids 1,2 for all studied cells. All hybrid derivatives,
including a combination of 1,3,4-oxadiazoles and 1,2,3-triazoles, attached to the 28- position of ursane
(7a, 7c, 7d) and lupane backbone (15a) were found non-active towards all the tested cells. Also, no
cytotoxicity was revealed for the majority of double heterocyclic hybrids of 1,3,4-oxadiazoles and
triazole, connected to position 3- of the ursane backbone via succinate linker (11b, 11c, 11d). In this
series, only compound 11a, including a combination of 5-phenyl-1,3,4-oxadiazole and 1,2,3-triazole,
was slightly toxic towards to lung cancer cells line A-549, which was close to the activity of the
starting ursolic acid 1. The introduction of a furoxan fragment, attached via 1,2,3-triazole linker to the

triterpene backbone, led in certain cases to compounds with marked cytotoxic activity.
Compound 8a that was a combination of triazole and furoxan groups tailored to ursane C-28 position,
showed cytotoxicity comparable to the starting compound 1 (MCF-7 cells), whereas the superior
activity comparing 1 was obtained towards U-87-MG cells. Concerning HepG2, the activity value of
hybrid 8a (12.89±2.63) was comparable with the data obtained for doxorubicin (10.02±1.67). At the
same time, compound 8a significantly exceeded doxorubicin in terms of the selectivity index
(2.0 vs 0.33), respectively. The introduction of the additional ester spacer between the triterpene
backbone and triazole fragment (compound 9a) resulted in activity decrease against all the tested cells.
The cytotoxicity of 9a against MCF-7, HepG2 was close to the data obtained for ursolic acid 1. Hybrid
compound 12, including a combination of 1,2,3-triazole and 1,2,5-oxadiazole, connected via a
succinate linker to position C-3 of the ursane backbone, was inactive to all the cancer cells studied,
while being more toxic against non-cancerous cells than ursolic acid 1. It is known that the
introduction of a 3-O-acetyl group into a triterpenoid molecule may result in the enhancement of
cytotoxicity properties[14]. However, 3-O-acetate 9b turned out to be completely non-toxic for all
studied cells, in contrast to the initial 3-hydroxy derivative 9a. The acetate derivative 8b obtained
from 8a containing 3-hydroxyl group, was significantly less active than the parent compound against
U-87-MG, HepG2, and A-549 cells. Surprisingly, in relation to MCF7 breast cancer cells,
acetate 8b demonstrated an excellent activity (1.55±0.08) which was superior over that for doxorubicin
(4.51±1.12). The selectivity index of 8b for MCF7 (6.72) also significantly exceeded the values
obtained for ursolic acid (3.68) and doxorubicin (0.74). Hybrid of betulonic acid with furoxan and
1,2,3-triazole 14a was found non-efficient towards cancer cells MCF-7, U-87 MG, A549, while it
demonstrated toxicity similar to those of betulonic acid 2a towards HepG2 and non-cancer cells.
Therefore, ketoester 13a was reduced with NaBH4 to prepare the C-3-OH propargyl ester 13b which
was converted to lupane-type furoxan hybrid 14b. Similarly to ursane analog 8a, hydroxyl-
bearing 14b comprising a succession of 1,2,3-triazole and 1,2,5-oxadiazole attached to position C-28
of lupane frame, displayed selective activity towards MCF7 that was higher than those for betulonic
acid 2: 13.38±5.69 vs. 28.78±4.12, respectively, and besides was found less toxic to non-cancer cells.
Acylation of 14b led to acetate 14c, which, however, was poorly active to all cells tested. Unlike
ursane derivative 8b, the introduction of the 3-O-acetate group in 14c did not lead to an activity
improvement towards MCF-7 cells. In general, furoxan-triazole-containing hybrids of betulinic
acid 14b,c showed greater cytotoxicity compared to the parent betulonic derivative 14a. It should be
noted that betulonic acid-derived hybrids with furoxan and amino acid linkers 16a, b, c [31] exhibited
no cytotoxic properties. At the same time, notable anti-inflammatory activity was previously detected
for compounds 16 a, b, c [31].

The highest activity was found for triple ursane hybrids 8a, 8b which demonstrated generally higher
cytotoxicity levels than their lupane analogs 14a, 14b.
Thus, the combination of heterocyclic fragments of 1,2,3-triazole and 3-(methyl)-4-methyl-1,2,5-
oxadiazole-2-oxide attached at position 28 of ursolic acid provides conditions for the cytotoxic activity
of the hybrid derivatives.
3.3. Molecular modeling results
Taking into account the hybrid structure of the new compounds, in silico screening of their possible
interaction with both antitumor targets of lupane and ursane triterpenoids and targets of compounds
containing 1,2,3-triazole, 1,3,4- and 1,2,5- oxadiazole was performed (Table 2).
Table 2. Molecular targets used for in silico screening of possible interaction with novel hybrid
compounds
Entry
1
Target
PDB ID
6RQ4
5NGU
3OCB
4IQK
2P33
Mitogen-activated protein kinase 1 (Erk1)
Mitogen-activated protein kinase 2 (Erk2)
Akt1 kinase
2
3
4
Keap1 Kelch domain
5
c-Jun N-terminal kinase (JNK)
ABL2 kinase
6
2XYN
6GU6
4KD1
5L2T
7
Cyclin-dependent kinase 1
Cyclin-dependent kinase 2
Cyclin-dependent kinase 6
Cyclin-dependent kinase 9
E3 ubiquitin-protein ligase (Mdm2)
Serine protease hepsin
8
9
10
11
12
13
14
15
16
3TN8
5OAI
5CE1
4ALW
3GXL
6GXK
5CF0
Pim kinase
TGF-beta receptor type-1 (ALK5)
Aldo-Keto Reductase 1C3 (AKR1C3)
Heat shock protein 90-alpha (Hsp90-alpha)

17
18
19
Indoleamine 2,3-dioxygenase 1 (IDO1)
6AZV
3NAX
Phosphoinositide-dependent kinase 1 (PDK1)
Vascular endothelial growth factor receptor 1 4ZAU
(VEGFR1)
20
Vascular endothelial growth factor receptor 2 4AG8
(VEGFR2)
21
22
23
24
25
MEK1 kinase
4LMN
1SA0
6O0K
4KIK
4CXT
Tubulin alpha chain
Apoptosis regulator Bcl-2
IkB kinase beta
BTB domain of Keap1
Among these targets, only for the model of Mdm2 ligase (PDB ID 5OAI), results comparable to the
corresponding known inhibitor nutlin-3A were obtained. There is evidence of the possible ability of
some triterpenoids to inhibit the activity of the Mdm2 protein, which is a negative regulator of the
tumor suppressor p53 [47,48].
The estimated binding energy of the new compounds in comparison with the known Mdm2 inhibitors
is presented in Table 3. Interestingly, the highest theoretical affinity among the new derivatives was
observed for compound 8a, which was active towards tumor cell lines in in vitro experiment (Table 1).
Compounds with cytotoxic effect found 9a, 14a, including a combination of triazole and furoxan
linked to position C-28 of triterpenoid were also on the top of the affinity list. On the other hand, only
moderate and low affinity was found for furoxan-triazole hybrid 12 linked to the C-3 position of
ursane core and furoxan compounds 16a, 16c without triazole spacer (shown low cytotoxicity in vitro).
Therefore, modeling results are generally in agreement with the results of the in vitro test.
Table 3. Docking results of new derivatives at the Mdm2 binding site.
Ligand
B5K
Binding energy*, kcal/mol
-9.994
-7.537
-7.490
-6.835
-6.779
Nutlin-3A
8a
14a
16b

9a
11c
11d
12
-6.578
-6.418
-6.298
-6.183
-5.961
-5.949
-5.936
-5.610
-5.552
-5.487
-5.458
-5.388
-5.322
-5.276
-5.198
-5.156
-4.631
-4.628
11a
15a
11b
14c
1
16c
14b
8b
7a
9b
16a
2
7d
7c
* value is not genuine binding energy but estimated docking score.
The estimated binding energy of compound 8a is comparable to that of a nutlin-3A inhibitor. Since
only molecules including combinations of 1,2,3-triazole and furoxan connected to C-28 of triterpenoid
core demonstrated cytotoxic effects, we compared the features of non-covalent interactions of new
compounds 8a, 8b, 9a in the Mdm2 binding site with known inhibitors nutlin-3A and B5K (Figure 2).
The Mdm-2 binding site is characterized by a deep hydrophobic pocket into which the nutlin-3A
chlorophenyl substituent and the B5K 6-chloro-indole motif are immersed. The formation of stacking
interactions with the π-system of the amino acid residue HIS96 is important for the binding of these
inhibitors. Also, for the B5K molecule, a hydrogen bond is formed between the proton at the nitrogen
atom of the indole ring and the acceptor oxygen of the amino acid residue LEU54. Apparently, the
formation of hydrogen bonds between the carboxyl group and amino acid residues PHE55 and GLN59
through the bridging water molecule 312 is also an important binding feature of this inhibitor. New
compounds do not interact with this water molecule; however, molecules 8a and 8b can form stacking
interactions with the HIS96 π-system. The π system of the furoxan ring is involved in the binding of
compound 8a, and in the case of compound 8b, stacking is noted for the π-systems of the furoxan and
triazole cycles. The bonding feature of these compounds is the formation of hydrogen bonds due to the

strong acceptor nature of the 2-oxide oxygen atom of the furoxan motif. In compounds 8a and 8b, this
atom can interact with the proton of the hydroxyl group of TYR100, while in compound 9a it can form
a hydrogen bond with a proton bound to the nitrogen of the HIS96 imidazole ring. The oxygen atom
inside the furoxan ring of compound 9a can interact with the proton of the hydroxyl group of TYR100.
Such a feature of the interactions of compound 9a leads to a reversal of its hydrophobic triterpene
backbone compared to the configuration of compounds 8a and 8b. These compounds exhibit a partial
penetration of the methyl substituent of cycle E of the triterpene backbone into the hydrophobic pocket
of the binding site, which can promote successful binding by the type of known inhibitors.
A
B
D
C

Figure 2. Docking in Mdm2. Non-covalent interactions of compounds (A - B5K, B – 8a, C – 8b, D –
9a) are shown by dashed lines: green - hydrogen bonds, blue - stacking interactions.
3.3. Results of flow cytometry analysis
Mdm-2 oncogene was shown to form a complex with the tumor suppressor p53, and inhibit p53-
mediated gene transactivation [49]. Therefore, we studied the impact of nutlin and compounds 8а, 8b
on the apoptosis and cell cycle of p53-wild and -deficient MCF7 cells. Tables 4, 5 show the data on
apoptosis and the cell cycle of MCF7 under the action of a nutlin inhibitor and compounds 8a, 8b.
From the data presented in table 4 it is seen that 8a, 8b do not cause apoptosis in the studied cells
within 24 hours. At the same time, when combined with nutlin, both 8a and 8b remove the apoptotic
effect caused by nutlin. Obviously, compounds 8a, 8b are competitive in the action of nutlin against
Mdm2. This may be due to the concurrent penetration of substituents of novel hybrids into the
hydrophobic pocket of the binding site of Mdm2 (docking data). The effect of the compounds on the
cell cycle is presented in Table 5. The effect of individual compounds, as well as the combined action
with nutlin on deficient MCF7 cells, leads to an increase in the number of cells in the S phase
(replication stage). The most pronounced effect was noted for 8b and its combination with nutlin. On
deficient cells, one can see a similar character of the action of compound 8b and nutlin, significantly
different from the action of doxorubicin, for which a decrease in the number of cells in the S phase was
detected. From the data obtained, it follows that one of the possible mechanisms of the antitumor
action of ursolic acid derivatives with a furoxan-triazolo bis-heterocyclic substituent at position C-28
is the binding of compounds to the Mdm2 protein.

Table 4. The effect of nutlin inhibitor and compounds 8a, 8b on apoptosis of MCF-7 a cells a.
MCF7 p53 +/+b
MCF7 p53 -/-c
w/o estrogen
with estrogen
w/o estrogen
with estrogen
Сoncen
Compound
tration,uM
normal apoptosis necrosis normal apoptosis necrosis normal apoptosis necrosis normal
apoptosis necrosis
-
control
doxorubicin
nutlin
97.22
87.22
88.29
97.28
97.79
98.16
97.71
1.7
11.84
9.09
0.57
0.25
0.42
0.46
1.08
0.93
2.62
2.15
1.96
1.42
1.83
96.59
90.14
90.85
98.98
98.84
98.02
96.92
0.55
5.78
7.7
2.86
4.08
1.45
0.9
98.02
90.04
87.31
96.54
96.84
96.24
97.37
0.93
5.74
9.05
0.3
1.05
4.22
3.64
3.16
2.99
3.7
98.13
87.67
86.62
94.48
96.18
93.14
91.86
0.8
10.25
11.23
0.68
0.35
0.27
1.8
1.07
2.08
2.15
4.84
3.47
6.59
6.34
2.5
17.0
8a
22.9
0.12
0.11
0.24
0.54
8a + (nutlin)
8b
22.9+(17.0)
1.55
1.05
1.74
2.54
0.17
0.06
0.12
8b + nutlin
1.55+(17.0)
2.51
aData is given as a percentage of cell population; b wild type MCF-7; c deficient type MCF-7
Table 5. The effect of nutlin inhibitor and compounds 8a, 8b on cell cycle of MCF-7a.
MCF7 p53 +/+b
MCF7 p53 -/-с
w/o estrogen
with estrogen
w/o estrogen
with estrogen
Сoncen
Compound
tration,uM
G1
S
G2
G1
S
G2
G1
S
G2
G1
S
G2
65.43
14.14
20.43
61.41
14.35
24.24
57.87
20.46
21.67
66.69
20.86
12.45
42.22
12.16
-
control
doxorubicin
nutlin
56.85
56.95
5.99
37.16
26.07
59.4
6.31
34.29
25.11
50.51
56.5
4.35
45.14
19.04
49.64
65.79
8.14
2.5
16.98
56.17
18.72
24.46
22.05
17.0
22.9
8a
69.68
11.89
12.1
18.43
12.87
21.34
22.73
64.57
70.6
18.09
13.35
16.89
17.02
17.34
16.05
26.31
22.14
63.66
71.6
18.43
13.53
25.73
32.04
17.91
14.87
27.25
41.82
64.81
68.51
61.4
16.85
14.43
22.83
25
18.34
17.06
15.77
21.09
8a + (nutlin)
8b
22.9+(17.0) 75.03
1.55 66.55
1.55+(17.0) 60.45
12.11
56.8
47.02
26.14
8b + (nutlin)
16.82
60.84
53.91
aData is given as a percentage of cell population; b wild type MCF-7; c deficient type MCF-7

4. Conclusion
In conclusion, we designed novel heterocyclic ensembles of 1,3,4- or 1,2,5-oxadiazoles with
lupane and ursane triterpenoids tethered with 1,2,3-triazole. Propargyl derivatives of triterpenoids were
reacted utilizing click chemistry protocol with 2-azidomethyl-5-aryl-1,3,4-oxadiazoles, and 3-
(azidomethyl)-4-methyl-1,2,5-oxadiazole 2-oxide (furoxan derivative). The location of heterocyclic
substituents relative to the positions C-3 and C-28 of the triterpenoid frame and ester-type linkers were
varied. The obtained new triterpenoid hybrids with 1,2,3-triazoles and 1,3,4- or 1,2,5- oxadiazoles, as
well as those including lupane conjugates with aminoacids and 1,2,5-oxadiazoles, were tested for
cytotoxic efficacy towards MCF7 breast cancer, A549 lung carcinoma, U-87 MG multiform
glioblastoma, and HepG2 hepatocarcinoma cells. The succession of heterocyclic fragments of 1,2,3-
triazole and 3-(methyl)-4-methyl-1,2,5-oxadiazole-2-oxide attached at position C-28 of ursane core
provided the best cytotoxic activity and selectivity on HepG2 (compound 8a, activity comparable to
doxorubicin) and MCF-7 cells (compound 8b, activity superior over doxorubicin). The introduction of
an additional ester-type linker between triazole and triterpenoid or employing aminoacid-type spacers
between the triterpenoid frame and furoxan led to the loss of cytotoxicity. Lupane-type hybrids were
found less active compared to their corresponding ursane analogs. According to the molecular docking
data supported by flow cytometry analysis data, the most likely mechanism of the cytotoxic action is
the affinity of triterpenoid hybrid derivatives to Mdm2 binding sites. The obtained furoxan-triazole-
ursane hybrids 8a, 8b are candidates for the further mechanism and bioactivity studies.
Acknowledgements
This research was financially supported by the ERA.Net RUS Plus project # RUS_ST2017-139
“AnticancerBet” (Foundation of Basic Research Grant 18-53-76001)). Immortalized human fibroblasts
were kindly provided by A. Schilov (Institute of Cytology and Genetics of Siberian Branch of Russian
Academy of Sciences, Novosibirsk, Russia). Analytical and spectroscopic studies were performed at
the Chemical Service collective Center of SB RAS.
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