Cytotoxic conjugates of betulinic acid and substituted triazoles prepared by Huisgen Cycloaddition from 30-azidoderivatives

Article abstract of DOI:10.1371/journal.pone.0171621

In this work, we describe synthesis of conjugates of betulinic acid with substituted triazoles prepared via Huisgen 1,3-cycloaddition. All compounds contain free 28-COOH group. Allylic bromination of protected betulinic acid by NBS gave corresponding 30-bromoderivatives, their substitution with sodium azides produced 30-azidoderivatives and these azides were subjected to CuI catalysed Huisgen 1,3-cycloaddition to give the final conjugates. Reactions had moderate to high yields. All new compounds were tested for their in vitro cytotoxic activities on eight cancer and two non-cancer cell lines. The most active compounds were conjugates of 3β-O-acetylbetulinic acid and among them, conjugate with triazole substituted by benzaldehyde 9b was the best with IC50 of 3.3 μM and therapeutic index of 9.1. Five compounds in this study had IC50 below 10 μM and inhibited DNA and RNA synthesis and caused block in G0/G1 cell cycle phase which is highly similar to actinomycin D. It is unusual that here prepared 3β-O-acetates were more active than compounds with the free 3-OH group and this suggests that this set may have common mechanism of action that is different from the mechanism of action of previously known 3β-O-acetoxybetulinic acid derivatives. Benzaldehyde type conjugate 9b is the best candidate for further drug development.

Full text of DOI:10.1371/journal.pone.0171621

RESEARCH ARTICLE
Cytotoxic conjugates of betulinic acid and
substituted triazoles prepared by Huisgen
Cycloaddition from 30-azidoderivatives
Veronika Sidova1, Pavel Zoufaly1, Jan Pokorny1, Petr Dzubak2, Marian Hajduch2,
Igor Popa1,2, Milan Urban1,2
*
1 Department of Organic Chemistry, Faculty of Science, Palacky University, Olomouc, Czech Republic,
2 Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University,
Olomouc, Czech Republic
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* milan.urban@gmail.com
Abstract
In this work, we describe synthesis of conjugates of betulinic acid with substituted triazoles
prepared via Huisgen 1,3-cycloaddition. All compounds contain free 28-COOH group. Allylic
bromination of protected betulinic acid by NBS gave corresponding 30-bromoderivatives,
their substitution with sodium azides produced 30-azidoderivatives and these azides were
subjected to CuI catalysed Huisgen 1,3-cycloaddition to give the final conjugates. Reactions
had moderate to high yields. All new compounds were tested for their in vitro cytotoxic activi-
ties on eight cancer and two non-cancer cell lines. The most active compounds were conju-
gates of 3β-O-acetylbetulinic acid and among them, conjugate with triazole substituted by
benzaldehyde 9b was the best with IC50 of 3.3 μM and therapeutic index of 9.1. Five com-
pounds in this study had IC50 below 10 μM and inhibited DNA and RNA synthesis and
caused block in G0/G1 cell cycle phase which is highly similar to actinomycin D. It is unusual
that here prepared 3β-O-acetates were more active than compounds with the free 3-OH
group and this suggests that this set may have common mechanism of action that is differ-
ent from the mechanism of action of previously known 3β-O-acetoxybetulinic acid deriva-
tives. Benzaldehyde type conjugate 9b is the best candidate for further drug development.
OPEN ACCESS
Citation: Sidova V, Zoufaly P, Pokorny J, Dzubak P,
Hajduch M, Popa I, et al. (2017) Cytotoxic
conjugates of betulinic acid and substituted
triazoles prepared by Huisgen Cycloaddition from
30-azidoderivatives. PLoS ONE 12(2): e0171621.
doi:10.1371/journal.pone.0171621
Editor: Joseph J Barchi, National Cancer Institute
at Frederick, UNITED STATES
Received: November 22, 2016
Accepted: January 23, 2017
Published: February 3, 2017
Copyright: © 2017 Sidova et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Introduction
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Triterpenes are natural compounds that may be found in almost all living organisms and they
are particularly prevalent in plants [1]. These compounds are not part of the main metabolic
pathways, they are secondary metabolites. Interestingly, they have a variety of biological activi-
ties, which may be the reason why organisms produce them. Among the activities, we may
find antitumor, antibacterial, anticariogenic, antiparasitic, antifungal and many others [2–11].
In our research group we are developing new derivatives of betulinic acid (1) in order to find
compounds with higher cytotoxicity and better pharmacological properties than the parent
compound. One of the possibilities explored was annealing of a heterocycle to the main terpe-
nic skeleton, which resulted in a small library of about fifty new compounds [12–15]. Among
Funding: All material, chemicals, and small
equipment for both chemistry and biology was paid
by Czech Science Foundation (15-05620S);
stipendia to students were paid by internal grants
of Palacky University IGA_PrF_2016_020,
IGA_LF_2016_19. Starting terpenes were paid
from the Technology Agency of the Czech Republic
(TE01020028). The infrastructural part (Institute of
PLOS ONE | DOI:10.1371/journal.pone.0171621 February 3, 2017
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Cytotoxic conjugates of betulinic acid and substituted triazoles
Molecular and Translational Medicine) is supported
by the National Sustainability Programme
(LO1304). The funders had no role in study design,
data collection and analysis, decision to publish, or
preparation of the manuscript.
them, four derivatives had IC50 in low micromolar range and currently belong to our most
promising compounds in in vivo tests. All four active heterocycles are derivatives of betulinic
acid (1). Recently, a number of new triterpenoid heterocycles were prepared and a number of
them had high cytotoxic activity [16–20]. To improve pharmacological properties of triter-
penes, especially their solubility in water, various modifications of triterpenes were done and
some of them were successful, especially compounds with another (polar) molecule connected
to them. Examples include esters with sugars, glycosides, esters with dicarboxylic acids, conju-
gates with polyethylene glycol, ammonium salts etc [21–29].
Competing Interests: The authors have declared
that no competing interests exist.
Recently, a number of new articles were published on connecting a terpene with another
molecule of interest via Huisgen 1,3-cycloaddition reaction. The first approach used terpenes
substituted with alkynes in the position 3 [30–34], second approach used propargylesters or
amides prepared at 28-COOH group [35–45], and the third approach used 30-azidoderivatives
prepared via 30-bromoderivatives [46;47]. Rarely, also position 2 is modified [48] or two posi-
tion at once (3 and 30) in [49].
In this work, we decided to explore the third option and to connect betulinic acid (1) to
other molecules of interest. Introduction of a rather polar triazole ring capable of forming
hydrogen bonds was expected to improve solubility of the target molecules in water based
media and bioavailability [50;51]. Possibly, the triazole ring may also become a part of the
pharmacophore. On the other hand, introduction of a completely new moiety on the other
side of the triazole ring (another aromatic rings—both heterocyclic and carbocyclic, aldehydes,
amines etc.) could change the biological properties such as cytotoxicity or selectivity and the
new compounds could act by different mechanism of action than the parent betulinic acid (1).
Among the derivatives prepared by other research groups there are only few examples [46;47]
containing both free 28-carboxylic group and free 3-hydroxy group. In most cases, cycloaddi-
tion reactions were done with acid 1 protected as methyl ester or as acetate and the final mole-
cules were also tested with the protective group on. There are many examples in the literature
[21;52;53] that betulinic acid (1) derivatives are highly cytotoxic when unprotected while
methylesters and acetates are usually inactive. Therefore, the main aim of this work was to
explore unprotected derivatives of betulinic acid (1) modified by cycloaddition reactions in
the position 30 and to explore their cytotoxic activity and influence on cancer cells.
Results and discussion
Chemistry
Bromination of betulinic acid (1) derivatives at the allylic position (C-30) is described in the
literature [54–57] that mostly used NBS as the bromination agent, AIBN as a radical source
and CCl4 as an inert solvent. In this work, we found that the method afforded only low yields
when free acid 1 was used. All reagents have limited solubility in CCl4 and dibromoderivative
starts forming before the full conversion of the starting betulinic acid (1) to 30-bromobetulinic
acid. To increase the solubility of acid 1, we choose to protect it at the position 3 as acetate or
triphenylsilylether. We decided to leave the 28-COOH group unprotected, since this neopen-
tyl-type ester requires harsh conditions for its deprotection and the presence of free carboxylic
group should not interfere with the following reactions. For protection of 3β-OH group, ace-
tate was chosen as a stable protective group that would be cleavable in basic conditions, triphe-
nylsilyl group was chosen as more labile protective group easily removable in acidic conditions
or by the fluorine anion. This should allow for almost unlimited variability of the new substitu-
ents. Both acetate 2 and triphenylsilyl derivative 3 were synthesized by standard procedures.
Bromination of 2 and 3 afforded good yields of pure bromoderivatives 4 and 5. The reaction
PLOS ONE | DOI:10.1371/journal.pone.0171621 February 3, 2017
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Cytotoxic conjugates of betulinic acid and substituted triazoles
Fig 1. The preparation of all derivatives. Reagents and conditions: (a) Ac2O, pyridine, r.t. 16 h; (b) Ph3SiCl, DMF, imidazole, r.t. 36 h; (c) NBS, AIBN,
CCl4, 75˚C for 1 h then 50˚C for 3 h; (d) NaN3, DMSO, r.t., 36 h; (e) TBAF, THF, r.t., 18–32 h, or HCl, CH2Cl2, r.t., 5–11 h; (f) azides 6, 7 or 8, CuSO4Á5H2O,
sodium L-ascorbate, alkynes.
doi:10.1371/journal.pone.0171621.g001
of bromoderivatives 4 and 5 with sodium azide gave corresponding 30-azidoderivatives 7, 8,
and to obtain the unprotected azide 6, silylether 8 was deprotected by TBAF in THF; Fig 1.
Final derivatives containing acetate or triphenylsilylether at the position 3 (compounds 9a–
9h, 10a–10g) were prepared by Huisgen 1,3-cycloaddition from azides 6 and 7. Depending on
the reactivity, one or two equivalents of alkyne was used and the reaction was performed either
at r.t. or at 50˚C. Yields were moderate to high with the only exception—reaction of each azide
6–8 with propargylamine that gave 10% yield of acetate 9h but no sililated nor free product
was obtained. Reaction with FMOC or BOC protected propargylamine also did not yield the
desired product. In all cases, a mixture of polar unseparable compounds was obtained and we
were unable to isolate the desired product. The cycloaddion was catalyzed by CuI species
which ensured that only the proposed 1,4-isomer formed. Traces of Cu were removed by treat-
ment of the crude products with H2S before chromatography but the amount of remaining
metal was not determined. This will be performed if any compound of this set will enter fur-
ther biological tests. Free derivatives 11a–11g were obtained by the deprotection of their sily-
lated analogues using TBAF in THF (all 11a–11g were prepared this way) with reasonable
yields 58–81%. To find out if the compounds are stable during acidic deprotection procedure,
derivatives 10a, 10c, 10d, and 10f were also deprotected by HCl in CHCl3 with comparable
yields of 62–76%. Attempts to prepare compounds 11c and 11d directly from the unprotected
azide 8 were also successful with average yields of 75%. We may conclude, that all three ways
to the unprotected derivatives 11a–11g give similar results.
Biological assay
Cytotoxicity. Cytotoxic activity of all synthesized compounds was investigated in vitro
against eight human cancer cell lines and two non-tumor fibroblasts using the standard MTS
test (Table 1). The cancer cell lines were derived from T-lymphoblastic leukemia CCRF-CEM,
leukemia K562 and their multiresistant counterparts expressing P-glycoprotein, MRP1 and
LRP proteins (CEM-DNR, K562-TAX) [58], solid tumors including lung (A549) and colon
PLOS ONE | DOI:10.1371/journal.pone.0171621 February 3, 2017
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Cytotoxic conjugates of betulinic acid and substituted triazoles
Table 1. Cytotoxic activities of prepared derivatives on eight tumor (including resistant) and two normal fibroblast cell lines. All other compounds
prepared in this work were also tested but their activities on these 10 cell lines were higher than 50 μM which is considered inactive.
Comp. IC50 (μM/L)a
CCRF-CEM
45.5
5.7
CEM-DNR
45.4
15.3
13.2
19.2
25.8
19.5
4.0
K562
40.0
K562- TAX
43.1
15.8
12.2
15.5
23.9
19.1
3.9
A549
43.4
HCT116
38.0
15.9
35.6
18.6
25.5
25.1
6.4
HCT116p53-/-
>50.0
14.1
U2OS
>50.0
21.2
BJ
MRC-5
32.9
TIb
0.7
1
4
37.6
21.7
13.2
31.7
24.2
4.9
5
>50.0
7.4
>50.0
12.3
>50.0
17.6
31.1
27.9
>50.0
26.7
>50.0
20.0
-
6
19.7
20.9
3.2
8
21.7
13.4
3.3
17.2
23.0
24.0
25.0
34.0
19.1
1.2
9a
9b
9c
9d
9e
9f
15.1
23.4
31.1
24.8
>50.0
31.3
32.4
>3.1
9.1
3.6
14.8
9.5
12.8
28.9
9.0
14.4
12.0
29.6
15.0
30.8
28.3
23.8
11.5
8.5
22.1
13.8
11.2
30.0
23.4
29.1
>50.0
23.3
13.7
7.2
13.2
30.3
19.2
32.9
41.9
38.3
32.0
>50.0
26.3
>50.0
36.7
33.5
13.7
16.0
29.5
27.6
3.2
14.9
19.4
34.5
26.2
20.2
16.6
8.5
13.3
11.3
18.7
17.3
29.9
28.2
1.9
35.4
26.6
33.1
31.0
35.1
30.3
1.6
15.6
45.0
48.4
45.8
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>1.4
>1.9
>2.5
>3.0
>5.9
>3.5
-
9g
9h
11a
11b
11c
11f
11g
43.6
>50.0
30.5
47.6
44.1
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
26.9
30.6
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
14.4
>50.0
16.7
>50.0
>50.0
>50.0
13.7
38.0
16.0
23.3
>3.0
aThe lowest concentration that kills 50% of cells. The standard deviation in cytotoxicity assays is typically up to 15% of the average value. Compounds with
IC50 > 50 μM are considered inactive.
bTherapeutic index is calculated for IC50 of CCRF-CEM line vs average of both fibroblasts.
doi:10.1371/journal.pone.0171621.t001
(HCT116, HCT116p53-/-) carcinomas, osteosarcoma cell line (U2OS), and for comparison,
tests were performed on two human non-cancer fibroblast cell lines (BJ, MRC-5).
All derivatives prepared within this study have free 28-COOH group that was expected to
be essential for retaining of the biological activity. Cytotoxicity of the selected starting com-
pounds and also compounds modified at C-30 by Huisgen 1,3-cycloadditions are in Table 1
(acetylated derivatives 9a–9h, silylated analogues 10a–10g, and fully deprotected derivatives
11a–11g that were expected to be the most active).
Among the starting material, bromide 4 and azide 6 had significant activity (IC50 5.7 and
7.4 μM) on multiple cancer cell lines with therapeutic index of 4.9 or 3.2 (calculated for the ref-
erence CCRF-CEM line). To our surprise, both compounds 4 and 6 are 3β-O-acetates, which
is in contrast to our initial assumptions that acetates should be less active than compounds
with the free 3β-hydroxy group. In addition, our results indicate, that acetates 9a–9h are often
highly active (with IC50 in low micromolar ranges) on multiple cancer cell lines (parental and
mutiresistant) and in most cases, they are more active than their non-acetylated analogues
11a–11g. The most active compound of this study is derivative 9b (IC50 3.3 μM on the refer-
ence CCRF-CEM cell line) which belongs among acetates and contains benzaldehyde
connected to the position 30 through the triazole ring formed by the cycloaddition. The com-
pound has reasonable therapeutic index 9.1 and seems the most promising derivative of this
study. Its non-acetylated derivative is also active on the reference line (IC50 8.5 μM) and we see
this trend throughout all of the prepared derivatives, compounds 9a–9g are more active than
free compounds 11a–11g with only few exceptions.
PLOS ONE | DOI:10.1371/journal.pone.0171621 February 3, 2017
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Cytotoxic conjugates of betulinic acid and substituted triazoles
Table 2. Influence of compounds 4, 6, 9b, 9c, 11b on cell cycle, DNA and RNA synthesis at 1a × and 5b × IC50
.
Comp.
Used conc. (μM)
Sub G1 (%)
2.2
G0/G1 (%)
38.4
S (%)
42.4
35.9
43.0
30.4
34.8
21.9
46.2
30.9
38.5
25.3
35.4
G2/M (%)
19.3
16.8
23.2
20.3
23.8
19.5
18.1
20.3
21.0
15.7
19.1
pH3Ser10 (%)
DNA synthesis
37.5
RNA synthesis
42.1
Control
0
5.7a
28.5b
7.4a
37.0b
3.3a
16.5b
9.0a
45.0b
8.5a
2.1
4
4
9.4
47.3
1.34
38.6
56.3
39.7
10.7
70.0
10.8
51.9
7.2
33.7
2.35
5.80
24.8
6
49.3
1.80
33.0
14.1
6
41.4
2.68
1.00
1.70
9b
9b
9c
9c
11b
11b
58.6
1.19
22.6
15.8
35.7
0.37
0.55
0.08
48.9
1.47
20.3
25.4
65.6
65.6
65.6
40.5
0.89
2.90
0.40
59.0
0.88
20.0
48.3
42.5b
45.5
1.19
6.40
38.1
aThe values were obtained at 1 × IC50
bThe values were obtained at 5 × IC50
.
.
doi:10.1371/journal.pone.0171621.t002
In general, it seems that modified C-30 position, conjugated to a large triazole-aromatic
substituent became an important part of the pharmacophore and is responsible for the cyto-
toxicity. In contrast, the functional group at C-3 probably influences the bioavailability of each
molecule. Compounds with free both 28-COOH and 3β-OH groups contain two hydrophilic
functional group on the opposite sides of their molecules and this may interfere with their per-
meability through cellular membranes. Small and lipophilic acetate on one side of the molecule
can solve this problem.
Cell cycle analysis. We have observed that most cytotoxic derivatives from this study are
3O-acetylated 30-bromo, 30-azido derivatives 4 and 6, 3O-acetylated conjugates 9b and 9c,
and one 3-hydroxyderivative 11b. All of them are inhibiting DNA and RNA synthesis. The
inhibition of the cell cycle in G0/G1 was observed with highest accumulation after treatment
with acetylated derivative 9b. The high percentage of apoptotic cells (sub G1) is observed at
5 × IC50 concentration, pointing on rapid induction of apoptosis (Table 2).
Conclusions
Three sets of betulinic acid derivatives modified at C-30 were prepared by Huisgen 1,3-cyclo-
addition catalyzed by CuI species. All compounds have free 28-COOH and the first set are 3β-
O-acetates 9a–9h, the second set are 3β-silylethers 10a–10g, and the third set are compounds
with free 3β-OH group 11a–11g. All compounds were subjected to tests of cytotoxicity on 8
cancer cell lines and 2 non cancer fibroblasts. Several derivatives had IC50 in low micromolar
ranges for parental and multiresistant cell lines, the best compound was aldehyde-acetate 9b
which also had high therapeutic index and this makes the compound the most promising can-
didate for future in vivo tests and for studies of mechanism of action. In this work, unusual
trend was found between the activities of 3β-O-acetates vs. free compounds. Acetates 9a–9h,
were usually more active than free derivatives 11a–11g. This suggests that compounds pre-
pared in this study may have mechanism of action that differs from acetylated betulinic deriva-
tives known from the literature [21] where this trend is opposite. Moreover, the inhibition of
DNA and RNA was observed even at 1 × IC50 concentrations together with G1 cell cycle block
which is highly similar to actinomycin D behavior [59]. Thus, one may speculate that conjuga-
tion of the new triazole ring equipped with carbocyclic (or heterocyclic) ring forms a new type
of pharmacophore. To prove it, however, the compound will have to be further transformed
PLOS ONE | DOI:10.1371/journal.pone.0171621 February 3, 2017
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Cytotoxic conjugates of betulinic acid and substituted triazoles
into a probe suitable for pull down assays [49] or into a fluorescent probe and more biological
tests will have to be done.
Experimental
General experimental procedures
Materials and instruments. Melting points were determined using a Bu¨chi B-545 appara-
tus and are uncorrected. Optical rotations were measured on an Autopol III (Rudolph
Research, Flanders, USA) polarimeter in MeOH at 25˚C unless otherwise stated and are in
[10−1 deg cm2 g-1]. 1H and 13C NMR spectra were recorded on VarianUNITY Inova 400 (400
MHz for 1H) or VarianUNITY Inova 300 (300 MHz for 1H) or Jeol ECX-500SS (500 MHz for
1H) instruments, using CDCl3, D6-DMSO or CD3OD as solvents (25˚C). Chemical shifts
were eider referenced to the residual signal of the solvent (CDCl3, D6-DMSO) or to tetra-
methylsilane added as an internal standard. 13C NMR spectra were eider referenced to CDCl3
(77.00 ppm) or D6-DMSO (39.51 ppm) or to tetramethylsilane added as an internal standard.
EI MS spectra were recorded on an INCOS 50 (Finigan MAT) spectrometer at 70 eV and an
ion source temperature of 150˚C. The samples were introduced from a direct exposure probe
at a heating rate of 10 mA/s. Relative abundances stated are related to the most abundant ion
in the region of m/z > 180. HRMS analysis was performed using LC-MS an Orbitrap high-res-
olution mass spectrometer (Dionex Ultimate 3000, Thermo Exactive plus, MA, USA) operat-
ing at positive full scan mode in the range of 100–1000 m/z. The settings for electrospray
ionization were as follows: oven temperature of 150˚C, source voltage of 3,6 kV. The acquired
data were internally calibrated with phthalate as a contaminant in methanol (m/z 297.15909).
Samples were diluted to a final concentration of 0.1 mg/mL in methanol. The samples were
injected to mass spectrometer over autosampler after HPLC separation: precolumn phenom-
enex 2.6 μm C18. Mobile phase isokrat. CH3CN/IPA/amonium acetate 0.01M 80/10/10, flow
0,3 mL/min. IR spectra were recorded on a Nicolet Avatar 370 FTIR. DRIFT stands for Diffuse
Reflectance Infrared Fourier Transform. TLC was carried out on Kieselgel 60 F254 plates
(Merck) detected by spraying with 10% aqueous H2SO4 and heating to 150–200˚C. Starting tri-
terpenes—betulin (1), dihydrobetulonic acid (2b), and allobetulin (3a) were obtained from
company Betulinines (www.betulinines.com). All other chemicals and solvents were obtained
from Sigma-Aldrich.
Synthetic procedures
General procedure for Huisgen cycloaddition of triterpenic azides. Each azide was dis-
solved in DMF (4 mL/100 mg) and sodium L-ascorbate (0.5 equiv.) was added followed by
CuSO4Á5H2O. The reaction mixture was stirred until its color turned green which is the sign
for CuI species being formed (usually 20 min). Then, each alkyne was added (1–2 equiv.) and
the reaction mixture was stirred at room temperature (or 50˚C) for various time, conditions
are specified for each compound. The reaction was monitored using TLC in hexane/EtOAc in
ratios 3: 1–1: 2 depending on substrates. After the reaction was completed, the mixture was
poured on ice where the product precipitated. The precipitate was filtered on frit, washed with
water and dried in desiccator, then it was dissolved in EtOAc, traces of copper ions were pre-
cipitated by H2S and filtered off. Product was then purified by column chromatography on sil-
ica gel (100 × weight of the terpene) in hexane/EtOAc or cyclohexane/EtOAc in various ratio.
Analytical samples were purified on HPLC, crystallized or lyophilized. Specific conditions,
such as reaction times, temperature, and mobile phase for TLC, CC or HPLC are specified in
each experiment.
PLOS ONE | DOI:10.1371/journal.pone.0171621 February 3, 2017
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Cytotoxic conjugates of betulinic acid and substituted triazoles
General procedures for the deprotection of silylated compounds. Procedure 1: each
triazole (0.2 mmol) was dissolved in THF (5 mL), then TBAF (2 mL; 10 equiv.; 1M solution in
THF) was added. The reaction mixture was stirred at various temperature until the reaction
was completed (monitored by TLC with 5% MeOH in CHCl3 as mobile phase), the deprotec-
tion usually took 18–32 h. The reaction mixture was poured to water and the product was
extracted to EtOAc. The organic phase was washed twice with 5% NaHCO3 and with water,
dried over MgSO4 and evaporated. Crude product was chromatographed on silica gel (10–20
g) in gradient CHCl3 to 10% MeOH in CHCl3. Analytical samples and samples for biological
tests were purified on reverse phase C-18 HPLC in isocratic mobile phase: 80% CH3CN,
20% buffer (0.1% NH4OAc in water). Reaction temperature and time is specified at each
experiment.
Procedure 2: each triazole (0.2 mmol) was dissolved in CH2Cl2 (5 mL) and HCl (0.3 mL,
35% in water) was added. The reaction mixture was stirred at r.t. for 5–11 h while monitored
on TLC with 5% MeOH in CHCl3 as mobile phase. After the reaction was completed, 5%
NaHCO3 in water was added to adjust the pH to about 5. The mixture was stirred yet another
1 h, then poured to water, extracted to CHCl3, washed with water and dried over MgSO4.
Organic solvents were evaporated in vacuo and the crude product was chromatographed on
silica gel (10–20 g) in gradient CHCl3 to 10% MeOH in CHCl3. Analytical samples and sam-
ples for biological tests were purified on reverse phase C-18 HPLC in isocratic mobile phase:
80% CH3CN, 20% buffer (0.1% NH4OAc in water).
3β-Triphenylsililbetulinic acid 3. 5 g (10.9 mmol) of betulinic acid 1 was dissolved in
DMF (100 mL), then Ph3SiCl (5.9 g, 20 mmol) and imidazole (1.4 g, 21 mmol) was added. The
reaction mixture was stirred at r.t. for 36 h while being monitored on TLC (hexane/EtOAc 4:
1). The crude reaction mixture was poured on ice while the product precipitated, the precipi-
tate was filtered off on a frit, washed with water and dried in desiccator. Crude product was
chromatographed on silica gel (200 g) in gradient of hexane/EtOAc from 5: 1 to 2: 1 and crys-
tallized from hexane.
Compound 3 was obtained as white crystals, 6.6 g (85%): mp 147–148˚C. IR (DRIFT):
2400–3400, 1720, 1692, 1642 cm-1. 1H NMR (500 MHz, CDCl3): δ 0.84 (s, 3H); 0.88 (s, 3H);
0.92 (s, 3H); 0.93 (s, 3H); 0.96 (s, 3H, H-23, 24, 25, 26, 27); 1.68 (s, 3H, H-30); 2.16 (td, 1H, J1 =
12.9 Hz, J2 = 3.7 Hz); 2.27 (dt, 1H, J1 = 12.9 Hz, J2 = 3.2 Hz); 3.00 (td, 1H, J1 = 10.9 Hz, J2 = 4.6
Hz, H-19β); 3.34 (dd, 1H, J1 = 11.8 Hz, J2 = 4.6 Hz, H-3α); 4.60 (dd, 1H, J1 = 2.0 Hz, J2 = 1.5
Hz, H-29 pro-E), 4.73 (d, 1H, J = 2.0 Hz, H-29 pro-Z); 7.35–7.41 (m, 6H); 7.42–7.46 (m, 3H);
7.63–7.68 (m, 6H, 15 × H-Ph). 13C NMR (125 MHz, CDCl3): δ = 14.68; 15.99; 16.12; 16.33;
18.40; 19.35; 20.77; 25.43; 27.92; 28.45; 29.65; 30.65; 32.14; 34.26; 37.00; 38.39; 38.54; 39.57;
40.63; 42.39; 46.89; 49.24; 50.32; 55.24; 56.37; 77.20; 81.16; 109.64; 127.67; 129.69; 135.38;
135.55; 150.37; 182.06. MS (ESI-): m/z (%) = 713 (100, [M-H]-). HRMS (ESI-) m/z calcd for
C48H63O3Si [M-H]- 713.4384, found 713.4373.
Bromination of the position C-30 in derivatives 2 and 3. Each derivative 2 and 3 (2.8
mmol) was dissolved in CCl4 (30 mL). Then, NBS (0.8 g, 4.5 mmol) and AIBN (0.14 mmol,
5%) was added and the reaction mixture was stirred at 75˚C for 1h. Then, the reaction mixture
was stirred at 50˚C for 3 h and another several hours (TLC) at 5˚C to finish the reaction com-
pletion. The reaction had to be frequently monitored by TLC in hexane/EtOAc (2: 1 for prod-
uct 4 and 5: 1 for 5) because keeping the reaction mixture at elevated temperature for longer
period than necessary leads to dibrominated sideproducts. After the completion, the reaction
mixture was poured into water, extracted to EtOAc, 3 × washed with water, dried over MgSO4
and evaporated. Crude products were purified on silica gel in gradient of hexane/EtOAc 4: 1 to
1: 1 and crystallized from hexane.
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Cytotoxic conjugates of betulinic acid and substituted triazoles
Compound 4 (3β-O-Acetyl-30-bromobetulinic acid) was obtained as white crystals, 1.1 g
(65%): mp 170–172˚C. IR (DRIFT): 2600–3400, 1721, 1642 cm-1. 1H NMR (500 MHz, CDCl3):
δ 0.84 (s, 3H); 0.85 (s, 3H); 0.86 (s, 3H); 0.94 (s, 3H); 0.99 (s, 3H, H-23, 24, 25, 26, 27); 1.98 (q,
1H, J = 8.0 Hz); 2.05 (s, 3H, Ac); 2.20 (dt, 2H, J1 = 11.5 Hz, J2 = 2.9 Hz); 2.31 (dd, 1H, J1 = 12.9
Hz, J2 = 2.9 Hz); 3.04 (td, 1H, J1 = 11.2 Hz, J2 = 4.6 Hz, H-19β); 4.01 (AB-system, 2H, JGEM
=
10.3 Hz, H-30); 4.48 (dd, 1H, J1 = 10.9 Hz, J2 = 5.7 Hz, H-3α); 5.05 (s, 1H, H-29 pro-E); 5.16 (s,
1H, H-29 pro-Z). 13C NMR (125 MHz, CDCl3): δ = 14.65; 16.08; 16.17; 16.45; 18.14; 20.95;
21.30; 23.67; 26.77; 27.94; 29.68; 32.03; 33.11; 34.25; 36.74; 37.12; 37.14; 37.79; 38.38; 38.41;
40.71; 42.37; 42.99; 50.35; 50.82; 55.40; 56.43; 80.91; 113.46; 151.20; 171.06; 181.89. MS (ESI-):
m/z (%) = 575 (100, [M-H]-). HRMS (ESI-) m/z calcd for C32H49BrO4 [M-H]Ã 575.2730,
found 575.2722 and 577.2710, found 577.2701.
Compound 5 (3β-O-Triphenylsilyl-30-bromobetulinic acid) was obtained as white crystals,
1.8 g (81%): mp 208–210˚C. IR (DRIFT): 2600–3400, 1718, 1642 cm-1. 1H NMR (500 MHz,
CDCl3): δ 0.84 (s, 3H); 0.88 (s, 3H); 0.92 (s, 3H); 0.94 (s, 3H); 0.96 (s, 3H, H-23, 24, 25, 26, 27);
1.75 (t, 2H, J = 10.9 Hz); 1.97 (dd, 1H, J1 = 12.0 Hz, J2 = 4.6 Hz); 2.17 (m, 2H); 2.30 (d, 1H,
J = 9.7 Hz); 3.03 (td, 1H, J1 = 11.5 Hz, J2 = 4.6 Hz, H-19β); 3.33 (dd, 1H, J1 = 12.0 Hz, J2 = 4.6
Hz, H-3α); 3.99 (s, 2H, H-30); 5.03 (s, 1H, H-29 pro-E), 5.14 (s, 1H, H-29 pro-Z); 7.37–7.45 (m,
9H); 7.64–7.65 (d, 6H, J = 6.3 Hz, 15 × H-Ph). 13C NMR (125 MHz, CDCl3): δ = 14.68; 16.04;
16.12; 16.32; 18.39; 20.87; 23.67; 26.75; 27.90; 28.45; 29.64; 32.02; 33.04; 34.28; 36.73; 37.00;
38.38; 38.53; 39.56; 40.64; 42.35; 43.04; 50.27; 50.77; 55.22; 56.39; 81.13; 113.44; 127.67; 129.69;
135.36; 135.54; 151.18; 181.78. MS (ESI-): m/z (%) = 791 (100, [M-H]-). HRMS (ESI-) m/z
calcd for C48H61BrO3Si [M+H]+ 791.3495, found 791.3491 and 793.3475, found 793.3480.
Azides 6–8. Bromoderivative 4 (2 mmol) was dissolved in DMSO (40 mL) and NaN3 (260
mg, 2 equiv.) was added. The reaction mixture was stirred at r.t. for 36 h, the reaction was
monitored on TLC in hexane/EtOAc 10: 1. After that, the reaction mixture was worked up in
the usual manner and crude product was chromatographed on silica gel (100 g) in gradient
hexane/EtOAc 10: 1 to hexane/EtOAc 2: 1.
Compound 6 (3β-O-Acetyl-30-azidobetulinic acid) was obtained as 700 mg (65%): mp
148–150˚C (hexane). IR (DRIFT): 2600–3400, 2103, 1719, 1642 cm-1. 1H NMR (500 MHz,
CDCl3): δ 0.83 (s, 3H); 0.85 (s, 3H); 0.93 (s, 3H); 0.99 (s, 3H); 1.04 (s, 3H, H-23, 24, 25, 26, 27);
2.05 (s, 3H, Ac); 2.29 (d, 1H, J = 10.2 Hz); 2.94 (td, 1H, J1 = 10.9 Hz, J2 = 4.6 Hz, H-19β); 3.77
(AB-system, 2H, JGEM = 16.0 Hz, H-30); 4.47 (dd, 1H, J1 = 10.9 Hz, J2 = 5.2 Hz, H-3α); 4.99 (s,
1H, H-29 pro-E), 5.04 (s, 1H, H-29 pro-Z). 13C NMR (125 MHz, CDCl3): δ = 14.61; 16.03;
16.15; 16.44; 18.11; 20.92; 21.29; 23.64; 26.73; 27.92; 29.65; 31.97; 34.20; 36.72; 37.09; 37.10;
37.77; 38.31; 38.35; 40.68; 42.33; 43.41; 50.29; 55.36; 55.62; 55.63; 56.41; 80.89; 111.55; 148.85;
171.07; 182.21. MS (ESI-): m/z (%) = 538 (100, [M-H]-). HRMS (ESI-TOF) m/z calcd for
C32H49N3O4 [M-H]- 538.3639, found 538.3632.
Compound 7 (3β-O-Triphenylsilyl-30-azidobetulinic acid) was prepared by the same pro-
cedure as compound 6 except CH3CN was used as a solvent because of low solubility of 5 in
DMSO. The reaction gave 815 mg (54%): mp 171–175˚C (hexane). IR (DRIFT): 2600–3400,
2106, 1723, 1490 cm-1. 1H NMR (500 MHz, CDCl3): δ 0.84 (s, 3H); 0.88 (s, 3H); 0.91 (s, 3H);
0.94 (s, 3H); 0.96 (s, 3H, H-23, 24, 25, 26, 27); 1.97 (dd, 1H, J1 = 12.6 Hz, J2 = 8.0 Hz); 2.28 (d,
1H, J = 9.2 Hz); 2.93 (td, 1H, J1 = 11.5 Hz, J2 = 5.2 Hz, H-19β); 3.33 (dd, 1H, J1 = 12.0 Hz, J2 =
4.6 Hz, H-3α); 3.75 (AB-system, 2H, JGEM = 13.8 Hz, H-30); 4.96 (s, 1H, H-29 pro-E); 5.02 (s,
1H, H-29 pro-Z); 7.36–7.45 (m, 9H); 7.65 (d, 6H, J = 8.0 Hz, 15 × H-Ph). 13C NMR (125 MHz,
CDCl3): δ = 14.11; 14.66; 16.00; 16.12; 16.32; 18.38; 20.87; 22.64; 26.72; 27.88; 28.44; 29.63;
31.91; 31.97; 34.26; 36.72; 37.00; 38.29; 38.53; 39.56; 40.63; 42.33; 43.46; 50.27; 55.21; 55.49;
56.37; 81.11; 127.67; 129.69; 135.35; 135.54; 148.84; 181.73. MS (ESI-): m/z (%) = 754 (100,
[M-H]-). HRMS (ESI-TOF) m/z calcd for C48H61N3O3Si [M+H]+ 754.4398, found 754.4384.
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Cytotoxic conjugates of betulinic acid and substituted triazoles
1.5 g (2 mmol) of compound 7 was deprotected according to the general procedure 1 to
give colourless crystals of 30-azidobetulinic acid (8) 833 mg (84%): mp 171–175˚C (CHCl3/
MeOH). IR (DRIFT): 2600–3400, 2115, 1714, 1640 cm-1. 1H NMR (500 MHz, CDCl3): δ 0.76
(s, 3H); 0.83 (s, 3H); 0.94 (s, 3H); 0.97 (s, 3H); 0.99 (s, 3H, H-23, 24, 25, 26, 27); 1.97 (dd, 1H,
J1 = 12.6 Hz, J2 = 7.7 Hz); 2.16 (m, 2H); 2.30 (d, 1H, J = 12.6 Hz); 2.95 (td, 1H, J1 = 11.2 Hz,
J2 = 4.8 Hz, H-19β); 3.20 (dd, 1H, J1 = 11.5 Hz, J2 = 4.6 Hz, H-3α); 3.78 (AB-system, 2H JGEM
14.3 Hz, H-30); 4.98 (s, 1H, H-29 pro-E), 5.04 (s, 1H, H-29 pro-Z). 13C NMR (125 MHz,
CDCl3): δ = 14.67; 15.32; 16.04; 16.10; 18.24; 20.94; 26.78; 27.33; 27.97; 29.36; 31.94; 34.31;
36.74; 37.19; 38.32; 38.69; 38.85; 40.68; 42.37; 43.46; 50.30; 55.30; 55.54; 56.37; 78.98; 111.50;
113.98; 127.76; 148.88; 181.25. MS (ESI-): m/z (%) = 496 (100, [M-H]-). HRMS (ESI-) m/z
calcd for C30H47N3O3 [M-H]- 496.3534, found 496.3525.
=
Acetylated compounds 9a–9h. Compound 9a was obtained from 150 mg (0.28 mmol) 6
by the general procedure using 1 equiv. of alkyne, at 50˚C while reaction time was 16 h. The
yield of white crystals was 169 mg (95%): mp 177–178˚C (hexane). IR (DRIFT): 2600–3400,
1725, 1647 cm-1. 1H NMR (500 MHz, CDCl3): δ 0.83 (s, 3H); 0.85 (s, 6H); 0.92 (s, 3H); 0.97 (s,
3H, H-23, 24, 25, 26, 27); 1.77 (t, 1H, J = 11,5 Hz); 1.95 (dd, 1H, J1 = 8.3 Hz, J2 = 4.6 Hz); 2.05
(m, 3H, Ac); 2.17 (td, 1H, J1 = 12.3 Hz, J2 = 3.4 Hz); 2.29 (m, 1H); 2.99 (td, 1H, J1 = 10.9 Hz,
J2 = 4.6 Hz, H-19β); 4.48 (dd, 1H, J1 = 8.2 Hz, J2 = 6.0 Hz, H-3α); 4.75 (s, 1H, H-29 pro-E); 5.00
(AB-system, 2H, JGEM = 15.5 Hz, H-30); 5.10 (s, 1H, H-29 pro-Z); 7.34 (t, 1H, J = 7.2 Hz, H-
36); 7.43 (t, 2H, J = 7.2 Hz, H-35, 37); 7.77 (s, 1H, H-31); 7.84 (d, 2H, J = 7.2 Hz, H-34, 38). 13
C
NMR (125 MHz, CDCl3): δ = 14.63; 16.02; 16.15; 16.44; 18.10; 20.90; 21.29; 23.64; 26.84; 27.92;
29.60; 29.66; 31.92; 34.18; 36.61; 37.07; 37.76; 38.29; 38.34; 40.66; 42.35; 43.29; 50.27; 50.40;
54.75; 55.34; 56.28; 80.88; 112.03; 120.02; 125.74; 128.17; 128.81; 130.53; 147.97; 149.48; 171.06;
181.39. MS (ESI+): m/z (%) = 642 (100, [M+H]+), 664 (75, [M+Na]+). MS (ESI-): m/z (%) =
640 (100, [M-H]-). HRMS (ESI-) m/z calcd for C40H55N3O4 [M-H]- 640.4109, found 640.4098.
Compound 9b was obtained from 150 mg (0.28 mmol) of 6 by the general procedure using
2 equiv. of alkyne at r.t. while reaction time was 19 h. The yield of white crystals was 160 mg
(86%): mp 161–165˚C (hexane). IR (DRIFT): 2600–3400, 1720, 1450 cm-1. 1H NMR (500
MHz, CDCl3): δ 0.82 (s, 3H); 0.84 (s, 6H); 0.91 (s, 3H); 0.97 (s, 3H, H-23, 24, 25, 26, 27); 1.77
(t, 1H, J = 11.4 Hz); 1.96 (dd, 1H, J1 = 8.0 Hz, J2 = 4.4 Hz); 2.05 (s, 3H, Ac); 2.15 (td, 1H, J1 =
12.2 Hz, J2 = 3.1 Hz); 2.29 (d, 1H, J = 12.5 Hz); 2.99 (td, 1H, J1 = 10.6 Hz, J2 = 4.2 Hz, H-19β);
4.47 (dd, 1H, J1 = 10.4 Hz, J2 = 4.9 Hz, H-3α); 4.75 (s, 1H, H-29 pro-E); 5.05 (t, 2H, J = 4.7 Hz,
H-30); 5.12 (s, 1H, H-29 pro-Z); 7.54 (t, 1H, J = 8.0 Hz); 7.66 (t, 1H, J = 7.3 Hz); 7.72 (d, 1H,
J = 7.8 Hz); 7.86 (s, 1H, H-31); 8.03 (d, 1H, J = 7.8 Hz, H-35, 36, 37, 38); 10.38 (s, 1H, H-39).
13C NMR (125 MHz, CDCl3): δ = 14.28; 15.67; 15.81; 16.10; 17.74; 20.54; 20.98; 23.29; 26.56;
27.57; 29.24; 31.55; 33.82; 36.24; 36.72; 37.41; 37.94; 37.99; 40.29; 40.90; 41.99; 42.81; 49.90;
50.07; 53.39; 54.97; 55.99; 56.26; 80.53; 111.95; 123.42; 128.34; 128.49; 129.74; 132.69; 133.44;
144.50; 148.97; 170.78; 181.08; 192.00. MS (ESI+): m/z (%) = 670 (100, [M+H]+), 692 (21,
[M+Na]+). HRMS (ESI+) m/z calcd for C41H55N3O5 [M+H]+ 670.4214, found 670.4211.
Compound 9c was obtained from 150 mg (0.28 mmol) of 6 by the general procedure using
2 equiv. of alkyne at r.t. while reaction time was 24 h. The yield of white crystals was 131 mg
(73%): mp 193–196˚C (hexane). IR (DRIFT): 2600–3400, 1721, 1461 cm-1. 1H NMR (500
MHz, CDCl3, referenced to TMS): δ 0.76 (d, 1H, J = 10.3 Hz, H-5); 0.80 (s, 3H, H-24); 0.82 (s,
3H, H-26); 0.83 (s, 3H, H-23); 0.90 (s, 3H, H-25); 0.94 (m, 1H, H-1a); 0.95 (s, 3H, H-27); 1.07
(d, 1H, J = 12.4 Hz, H-12); 1.18 (dt, 1H, J1 = 13.4 Hz, J2 = 2.9 Hz, H-21a); 1.23 (m, 1H, H-2a);
1.24 (m, 1H, H-9); 1.34, (m, 1H, H-6a); 1.36 (m, 2H, H-7); 1.36 (m, 1H, J = 13.0 Hz, H-15a);
1.41 (m, 1H, H-2b); 1.42 (m, 1H, 12.4 Hz, H-12b); 1.44 (dd, 1H, J1 = 12.6 Hz, J2 = 2.6, H-16a);
1.48 (m, 1H, H-6b); 1.51 (q, 1H, J = 13.2 Hz, H-22a); 1.53 (m, 1H, J = 12.5 Hz, H-11a); 1.53 (q,
1H, J = 13.4 Hz, H-21b); 1.62 (m, 1H, J = 12.5 Hz, H-11b); 1.63 (dd, 1H, J1 = 13.0 Hz, J2 = 3.5
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Cytotoxic conjugates of betulinic acid and substituted triazoles
Hz, H-1b); 1.73 (t, 1H, J = 11.4 Hz, H-18); 1.93 (m, 1H, J = 13.2 Hz, H-22b); 2.02 (s, 3H, Ac);
2.03 (m, 1H, ∑J = 13.0 Hz, H-15b); 2.18 (td, 1H, J1 = 12.0 Hz, J2 = 3.0 Hz, H-13); 2.28 (td, 1H,
J1 = 12.6 Hz, J2 = 2.6, H-16b); 3.00 (td, 1H, J1 = 11.1 Hz, J2 = 4.5 Hz, H-19β); 4.45 (m, 1H, J1 =
11.0 Hz, J2 = 5.0 Hz, H-3α); 4.69 (s, 1H, H-29 pro-E); 4.96 (d, 1H, J = 15.6 Hz, H-30a); 5.04 (d,
1H, J = 15.6 Hz, H-30b); 5.07 (s, 1H, H-29 pro-Z); 7.24 (td, 1H, J1 = 6.2 Hz, J2 = 1.0 Hz, H-36);
7.79 (td, 1H, J1 = 7.8 Hz, J2 = 1.7 Hz, H-37); 8.20 (dt, 1H, J1 = 7.9 Hz, J2 = 1.0 Hz, H-38); 8.20
(s, 1H, H-31); 8.58 (dq, 1H, J1 = 4.9 Hz, J2 = 0.8 Hz, H-35). 13C NMR (125 MHz, CDCl3): δ =
14.73 (C27); 16.14 (C25); 16.25 (C26); 16.55 (C24); 18.22 (C6); 21.01 (C2); 21.39 (Ac, CH3);
23.76 (C11); 26.91 (C12); 28.02 (C23); 29.72 (C21); 32.04 (C15); 32.11 (C16); 34.32 (C7); 36.82
(C22); 37.19 (C10); 37.87 (C4); 38.39 (C13); 38.46 (C1); 40.78 (C8); 42.48 (C14); 43.66 (C19);
50.38 (C9); 50.63 (C18); 54.72 (C30); 55.45 (C5); 56.35 (C17); 80.98 (C3); 112.19 (C29); 120.59
(C38); 122.83 (C31); 123.09 (C36); 137.36 (C37); 148.23 (C32); 149.20 (C35); 149.39 (C33);
150.11 (C20); 171.17 (Ac, C = O); 180.72 (C28). MS (ESI+): m/z (%) = 643 (100, [M+H]+), 665
(15, [M+Na]+). HRMS (ESI+) m/z calcd for C39H54N4O4 [M+H]+ 643.4218, found 643.4220.
Compound 9d was obtained from 150 mg (0.28 mmol) of 6 by the general procedure using
2 equiv. of alkyne at r.t. while reaction time was 28 h. The yield of white crystals was 135 mg
(76%): mp 176–179˚C (cyclohexane). IR (DRIFT): 2500–3400, 1723, 1451 cm-1. 1H NMR (500
MHz, CDCl3, referenced to TMS): δ 0.76 (d, 1H, J = 10.3 Hz, H-5); 0.80 (s, 3H, H-24); 0.81 (s,
3H, H-25); 0.82 (s, 3H, H-23); 0.90 (s, 3H, H-26); 0.95 (m, 1H, H-1a); 0.96 (s, 3H, H-27); 1.09
(dd, 1H, J1 = 12.4 Hz, J2 = 4.6 Hz, H-12a); 1.18 (dt, 1H, J1 = 13.4 Hz, J2 = 2.9 Hz, H-21a); 1.24
(m, 1H, H-6a); 1.25 (m, 1H, H-2a); 1.25 (m, 1H, H-9); 1.26 (m, 1H, H-15a); 1.36 (m, 2H, H-7);
1.36 (m, 1H, J = 12.4 Hz, H-12b); 1.38 (m, 1H, H-2b); 1.40 (dd, 1H, J1 = 12.5 Hz, J2 = 3.6 Hz,
H-6b); 1.42 (m, 1H, H-16a); 1.49 (m, 1H, H-22a); 1.53 (m, 1H, H-21b); 1.54 (m, 1H, J = 12.5
Hz, H-11a); 1.62 (m, 1H, J = 12.5 Hz, H-11b); 1.64 (dd, 1H, J1 = 13.0 Hz, J2 = 3.5 Hz, H-1b);
1.75 (t, 1H, J = 11.4 Hz, H-18); 1.93 (m, 1H, J = 13.2 Hz, H-22b); 1.96 (m, 1H, H-15b); 2.02 (s,
3H, Ac); 2.22 (td, 1H, J1 = 12.0 Hz, J2 = 3.0 Hz, H-13); 2.30 (td, 1H, J1 = 12.6 Hz, J2 = 2.6 Hz,
H-16; 2.97 (td, 1H, J1 = 11.1 Hz, J2 = 4.5 Hz, H-19β); 4.45 (dd, 1H, J1 = 10.5 Hz, J2 = 5.2 Hz, H-
3α); 4.82 (s, 1H, H-29 pro-E); 5.00 (d, 1H, J = 15.6 Hz, H-30a); 5.04 (d, 1H, J = 15.6 Hz, H-29
pro-Z); 5.11 (s, 1H, H-29b); 7.39 (m, 1H, J = 7.8 Hz, H-37); 7.92 (s, 1H, H-31); 8.28 (dt, 1H,
J1 = 8.0 Hz, J2 = 1.8 Hz, H-38); 8.55 (dd, 1H, J1 = 4.9 Hz, J2 = 1.0 Hz, H-36); 8.98 (d, 1H, J = 1.5
Hz, H-34). 13C NMR (125 MHz, CDCl3): δ = 14.73 (C27); 16.15 (C26); 16.27 (C25); 16.56
(C24); 18.23 (C6); 21.07 (C2); 21.39 (Ac, CH3); 23.76 (C11); 27.22 (C12); 28.02 (C23); 29.76
(C21); 32.21 (C16); 32.31 (C15); 34.34 (C7); 36.79 (C22); 37.20 (C10); 37.88 (C4); 38.32 (C13);
38.49 (C1); 40.80 (C8); 42.49 (C14); 43.11 (C19); 50.44 (C9); 50.84 (C18); 55.47 (C5); 55.59
(C30); 56.33 (C17); 80.97 (C3); 112.29 (C29); 120.71 (C31); 124.14 (C37); 127.24 (C33); 133.73
(C38); 144.64 (C32); 146.37 (C34); 148.48 (C36); 149.65 (C20); 171.14 (Ac, C = O); 180.15
(C28). MS (ESI+): m/z (%) = 643 (100, [M+H]+). HRMS (ESI+) m/z calcd for C39H54N4O4
[M+H]+ 643.4218, found 643.4221.
Compound 9e was obtained from 150 mg (0.28 mmol) of 6 by the general procedure using
2 equiv. of alkyne at r.t. while reaction time was 20 h. The yield of white crystals was 144 mg
(76%): mp 196–198˚C (cyclohexane). IR (DRIFT): 2450–3400, 1731, 1651 cm-1. 1H NMR (500
MHz, CDCl3): δ 0.83 (s, 3H); 0.85 (s, 6H); 0.92 (s, 3H); 0.97 (s, 3H, H-23, 24, 25, 26, 27); 1.76
(t, 1H, J = 11,5 Hz); 1.95 (dd, 1H, J1 = 12.6 Hz, J2 = 8.0 Hz); 2.05 (m, 3H, Ac); 2.18 (m, 1H);
2.29 (m, 1H); 2.99 (s, 7H, Me2N); 2.99 (m, 1H, H-19β); 4.48 (dd, 1H, J1 = 8.2 Hz, J2 = 6.0 Hz,
H-3α); 4.74 (s, 1H, H-29 pro-E); 4.98 (AB-system, 2H, JGEM = 15.5 Hz, H-30); 5.08 (s, 1H, H-
29 pro-Z); 6.77 (s, 1H, aniline); 6.79 (s, 1H, aniline); 7.63 (s, 1H, H-31); 7.70 (s, 1H, aniline);
7.71 (s, 1H, aniline). 13C NMR (125 MHz, CDCl3): δ = 14.62; 16.03; 16.15; 16.45; 18.11; 20.91;
21.29; 23.66; 26.80; 27.93; 29.61; 31.17; 31.87; 31.93; 34.18; 36.62; 37.09; 37.77; 38.27; 38.35;
40.53; 40.66; 42.36; 43.36; 50.28; 50.34; 54.58; 55.34; 56.26; 80.89; 111.87; 112.58; 118.58;
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121.52; 126.69; 148.40; 149.60; 150.37; 171.02; 180.99. MS (ESI+): m/z (%) = 485 (100, [M+H]+).
HRMS (ESI+) m/z calcd for C42H60N4O4 [M+H]+ 685.4687, found 685.4687.
Compound 9f was obtained from 150 mg (0.28 mmol) of 6 by the general procedure using
1 equiv. of alkyne at r.t. while reaction time was 20 h. The yield of white crystals was 165 mg
(85%): mp 193–196˚C (hexane). IR (DRIFT): 2600–3500, 1724, 1655 cm-1. 1H NMR (500
MHz, CDCl3): δ 0.83 (s, 3H); 0.85 (s, 6H); 0.92 (s, 3H); 0.98 (s, 3H, H-23, 24, 25, 26, 27); 1.35
(s, 9H, H-t-Bu); 1.78 (t, 1H, J = 11.4 Hz); 2.05 (s, 3H, Ac); 2.17 (td, 1H, J1 = 12.5 Hz, J2 = 2.6
Hz); 2.30 (dt, 1H, J1 = 13.0 Hz, J2 = 3.1 Hz); 2.99 (td, 1H, J1 = 10.9 Hz, J2 4.2 Hz, H-19β); 4.48
(dd, 1H, J1 = 10.4 Hz, J2 = 4.7 Hz, H-3α); 4.74 (s, 1H, H-29 pro-E); 5.00 (AB-system, 2H, JGEM
= 16.6 Hz, H-30); 5.09 (s, 1H, H-29 pro-Z); 7.46 (d, 2H, J = 8.8 Hz, H-35, 37); 7.74 (s, 1H, H-
31); 7.78 (d, 2H, J = 8.3 Hz, H-34, 38). 13C NMR (125 MHz, CDCl3): δ = 14.62; 16.02; 16.14;
16.44; 18.10; 20.90; 21.31; 23.65; 26.83; 27.91; 29.60; 29.68; 31.26; 31.92; 34.16; 34.64; 36.59;
37.08; 37.76; 38.27; 38.34; 40.64; 42.34; 43.22; 50.25; 50.36; 54.76; 55.33; 56.27; 80.87; 111.86;
119.74; 125.47; 125.72; 127.70; 147.93; 149.53; 151.29; 171.06; 181.43. MS (ESI-): m/z (%) = 696
(100, [M-H]-). HRMS (ESI-) m/z calcd for C44H63N3O4 [M-H]- 696.4735, found 696.4723.
Compound 9g was obtained from 150 mg (0.28 mmol) of 6 by the general procedure using
1 equiv. of alkyne at 50˚C while reaction time was 18 h. The yield of white crystals was 147 mg
(83%): mp 158–159˚C (hexane). IR (DRIFT): 2600–3400, 1726, 1661 cm-1. 1H NMR (500
MHz, CDCl3): δ 0.83 (s, 3H); 0.85 (s, 6H); 0.91 (s, 3H); 0.94 (s, 3H, H-23, 24, 25, 26, 27); 1.67
(m, 8H, H-34, 35, 36, 37) 2.05 (s, 3H, Ac); 2.29 (d, 1H, J = 12.9 Hz); 2.95 (td, 1H, J1 = 10.9 Hz,
J2 = 4.3 Hz, H-19β); 3.21 (t, 1H, J = 8.3 Hz, H-33); 4.48 (dd, 1H, J1 = 10.9 Hz, J2 = 5.2 Hz, H-
3α); 4.69 (s, 1H, H-29 pro-E); 4.91 (AB-system, 2H, JGEM = 15.8 Hz, H-30); 5.05 (s, 1H, H-29
pro-Z); 7.26 (s, 1H, H-31). 13C NMR (125 MHz, CDCl3): δ = 14.59; 14.62; 15.99; 16.03; 16.12;
16.16; 16.46; 18.10; 20.90; 21.28; 23.65; 25.11; 26.68; 27.93; 29.59; 29.66; 31.90; 33.26; 34.18;
36.61; 36.68; 37.07; 37.76; 38.24; 28.36; 40.64; 42.34; 43.26; 50.20; 54.47; 55.34; 56.27; 80.87;
111.89; 120.05; 149.61; 152.93; 171.04; 180.94. MS (ESI-): m/z (%) = 632 (100, [M-H]-). HRMS
(ESI-) m/z calcd for C39H59N3O4 [M-H]- 632.4422, found 632.4414.
Compound 9h was obtained from 300 mg (0.56 mmol) of 6 by the general procedure using
2 equiv. of alkyne at r.t. while reaction time was 30 h. The yield of white crystals was 33 mg
(10%): mp 178–184˚C (cyclohexane). IR (DRIFT): 3650, 2600–3500, 1726, 1643 cm-1. 1H
NMR (500 MHz, CDCl3): δ 0.84 (s, 3H); 0.85 (s, 6H); 0.93 (s, 3H); 0.97 (s, 3H, H-23, 24, 25, 26,
27); 2.05 (s, 3H, Ac); 2.30 (bd, 1H, J = 12.9 Hz); 2.95 (m, 1H, H-19β); 4.48 (dd, 1H, J1 = 10.9
Hz, J2 = 5.2 Hz, H-3α); 4.70 (s, 1H, H-29 pro-E); 4.85–5.20 (m, 2H, CH2-NH2); 5.03 (AB-sys-
tem, 2H, JGEM = 15.8 Hz, H-30); 5.13 (s, 1H, H-29 pro-Z); 8.13 (s, 1H, H-31); 10.17 (s, NH2).
13C NMR (125 MHz, CDCl3): δ = 14.61; 16.01; 16.17; 16.46; 18.12; 20.90; 21.29; 23.65; 27.05;
27.93; 29.58; 31.97; 34.22; 36.20; 37.10; 37.79; 38.23; 38.39; 40.69; 42.37; 43.11; 50.29; 50.84;
55.34; 55.38; 56.33; 64.17; 77.20; 80.82; 112.57; 125.64; 132.51; 148.84; 171.02; 185.11. MS (ESI
+): m/z (%) = 595 (100, [M+H]+). HRMS (ESI+) m/z calcd for C35H54N4O4 [M+H]+ 595.4218,
found 595.4220.
Silylated compounds 10a–10g. Compound 10a was obtained from 150 mg (0.20 mmol)
of 7 by the general procedure using 1 equiv. of alkyne at 50˚C while reaction time was 20 h.
The yield of white crystals was 157 mg (92%): mp 164–166˚C (cyclohexane). IR (DRIFT):
2600–3400, 1734, 1652 cm-1. 1H NMR (500 MHz, CDCl3): δ 0.83 (s, 3H); 0.89 (s, 3H); 0.90 (s,
3H); 0.92 (s, 3H); 0.96 (s, 3H, H-23, 24, 25, 26, 27); 2.15 (dd, 1H, J1 = 12.5 Hz, J2 = 3.1 Hz); 2.29
(d, 1H, J = 12.5 Hz); 3.00 (td, J1 = 10.9 Hz, J2 = 4.4 Hz, 1H, H-19β); 3.34 (dd, 1H, J1 = 11.9 Hz,
J2 = 4.2 Hz, H-3α); 4.70 (s, 1H, H-29 pro-E); 4.99 (AB-system, 2H, JGEM = 15.6 Hz, H-30); 5.07
(s, 1H, H-29 pro-Z); 7.31–7.46 (m, 12H, H-35, 36, 37, 3 × Ph); 7.66 (dd, 6H, J1 = 7.8 Hz, J2 = 1.3
Hz, 3 × Ph); 7.75 (s, 1H, H-31); 7.84 (d, 2H, J = 8.3 Hz, H-34, 38). 13C NMR (125 MHz,
CDCl3): δ = 14.66; 15.98; 16.10; 16.32; 18.35; 20.82; 26.81; 27.88; 28.42; 29.56; 31.81; 31.90;
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34.21; 36.59; 36.95; 38.27; 28.52; 39.54; 40.59; 42.33; 43.38; 50.20; 50.31; 54.55; 55.16; 56.25;
81.08; 111.91; 120.01; 125.73; 127.67; 128.15; 128.80; 129.70; 130.53; 135.32; 135.53; 147.95;
149.43; 181.32. MS (ESI-): m/z (%) = 856 (100, [M-H]-). HRMS (ESI-) m/z calcd for
C56H67N3O3Si [M-H]- 856.4868, found 856.4849.
Compound 10b was obtained from 150 mg (0.20 mmol) of 7 by the general procedure
using 2 equiv. of alkyne at r.t. while reaction time was 22 h. The yield of white crystals was 148
mg (84%): mp 161–163˚C (cyclohexane). IR (DRIFT): 2600–3400, 1724, 1642 cm-1. 1H NMR
(500 MHz, CDCl3): δ 0.83 (s, 3H); 0.88 (s, 3H); 0.90 (s, 3H); 0.92 (s, 3H); 0.96 (s, 3H, H-23, 24,
25, 26, 27); 2.14 (td, 1H, J1 = 12.6 Hz, J2 = 2.6 Hz); 2.29 (d, 1H, J = 12.6 Hz); 2.99 (td, J1 = 10.9
Hz, J2 = 4.3 Hz, 1H, H-19β); 3.34 (dd, 1H, J1 = 11.7 Hz, J2 = 4.3 Hz, H-3α); 4.75 (s, 1H, H-29
pro-E); 5.04 (AB-system, 2H, JGEM = 15.8 Hz, H-30); 5.10 (s, 1H, H-29 pro-Z); 7.34–7.45 (m,
9H, 3 × Ph); 7.51 (t, 1H, J = 7.7 Hz, H-36); 7.62 (m, 1H, H-37); 7.66 (m, 6H, 3 × Ph); 7.72 (d,
1H, J = 8.6 Hz, H-38); 7.83 (s, 1H, H-31); 8.03 (d, 2H, J = 8.9 Hz, H-35); 10.38 (s, 1H, H-39).
13C NMR (125 MHz, CDCl3): δ = 14.67; 15.98; 16.11; 16.32; 18.36; 20.84; 22.63; 26.87; 27.88;
28.43; 29.56; 31.56; 31.89; 34.23; 36.58; 38.29; 38.54; 39.54; 40.60; 42.35; 43.28; 50.22; 54.37;
54.78; 55.18; 56.27; 81.08; 112.24; 119.89; 123.71; 127.67; 128.63; 128.83; 129.70; 130.07; 133.05;
133.72; 133.84; 135.34; 135.53; 144.83; 149.30; 192.27. MS (ESI+): m/z (%) = 886 (100, [M
+H]+), 908 (8, [M+Na]+). HRMS (ESI+) m/z calcd for C57H67N3O4Si [M+H]+ 886.4974, found
886.4975.
Compound 10c was obtained from 150 mg (0.20 mmol) of 7 by the general procedure
using 2 equiv. of alkyne at r.t. while reaction time was 26 h. The yield of white crystals was 130
mg (75%): mp 197–200˚C (cyclohexane). IR (DRIFT): 2600–3400, 1718, 1641 cm-1. 1H NMR
(500 MHz, CDCl3): δ 0.82 (s, 3H); 0.88 (s, 3H); 0.89 (s, 3H); 0.92 (s, 3H); 0.95 (s, 3H, H-23, 24,
25, 26, 27); 2.17 (t, 1H, J = 11.7 Hz); 2.28 (d, 1H, J = 11.7 Hz); 3.01 (td, 1H, J1 = 10.4 Hz, J2 =
6.5 Hz, H-19β); 3.33 (dd, 1H, J1 = 12.2 Hz, J2 = 3.6 Hz, H-3α); 4.66 (s, 1H, H- 29 pro-E); 5.01
(AB-system, 2H, JGEM = 15.8 Hz, H-30); 5.06 (s, 1H, H-29 pro-Z); 7.24 (m, 1H, H-36); 7.36–
7.45 (m, 9H, 3 × Ph); 7.65 (d, 6H, J = 6.5 Hz, 3 × Ph); 7.79 (t, 1H, J = 7.8 Hz, H-35); 8.19 (s, 1H,
H-31); 8.21 (d, 1H, J = 8.8 Hz, H-37); 8.59 (d, 1H, J = 4.7 Hz, H-34). 13C NMR (125 MHz,
CDCl3): δ = 14.65; 15.99; 16.10; 16.33; 18.36; 20.81; 26.79; 27.87; 28.42; 29.68; 31.56; 32.00;
34.23; 36.70; 36.95; 38.24; 38.49; 39.53; 40.59; 42.33; 43.54; 50.19; 50.43; 54.52; 55.16; 56.21;
81.09; 111.83; 119.88; 122.69; 127.50; 127.67; 129.70; 135.32; 136.30; 137.21; 142.18; 148.13;
149.09; 149.98, 181.13. MS (ESI+): m/z (%) = 859 (100, [M+H]+), 881 (12, [M+Na]+). HRMS
(ESI+) m/z calcd for C55H66N4O3Si [M+H]+ 859.4977, found 859.4977.
Compound 10d was obtained from 150 mg (0.20 mmol) of 7 by the general procedure
using 2 equiv. of alkyne at r.t. while reaction time was 36 h. The yield of white crystals was 131
mg (77%): mp 202–204˚C (cyclohexane). IR (DRIFT): 2650–3450, 1729, 1646 cm-1. 1H NMR
(500 MHz, CDCl3): δ 0.82 (s, 3H); 0.88 (s, 3H); 0.89 (s, 3H); 0.91 (s, 3H); 0.93 (s, 3H); 0.95 (s,
3H, H-23, 24, 25, 26, 27); 2.30 (d, 1H, J = 12.6 Hz); 2.98 (td, 1H, J1 = 10.6 Hz, J2 = 4.0 Hz, H-
19β); 3.33 (dd, 1H, J1 = 11.7 Hz, J2 = 4.0 Hz, H-3α); 4.79 (s, 1H, H-29 pro-E); 5.01 (AB-system,
2H, JGEM = 16.6 Hz, H-30); 5.10 (s, 1H, H-29 pro-Z); 7.36–7.45 (m, 9H, 3 × Ph); 7.65 (d, 6H,
3 × Ph); 7.89 (s, 1H, H-31); 8.28 (d, 1H, J = 7.7 Hz, H-37); 8.57 (d, 1H, J = 3.7 Hz, H-35); 8.99
(s, 1H, H-34). 13C NMR (125 MHz, CDCl3): δ = 14.67; 16.01; 16.13; 16.34; 18.39; 20.91; 27.11;
27.89; 28.44; 29.62; 32.07; 32.13; 34.27; 36.66; 36.99; 38.21; 28.56; 39.55; 40.65; 42.38; 43.02;
50.27; 50.72; 55.20; 55.40; 56.19; 81.10; 112.12; 120.54; 123.97; 127.07; 127.68; 129.70; 133.51;
135.35; 135.54; 144.57; 146.40; 148.50; 149.52; 181.13. MS (ESI+): m/z (%) = 859 (100, [M+H]+),
881 (7, [M+Na]+). HRMS (ESI+) m/z calcd for C55H66N4O3Si [M+H]+ 859.4977, found
859.4974.
Compound 10e was obtained from 150 mg (0.20 mmol) of 7 by the general procedure using
2 equiv. of alkyne at r.t. while reaction time was 24 h. The yield of white crystals was 128 mg
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Cytotoxic conjugates of betulinic acid and substituted triazoles
(72%): mp 194–196˚C (cyclohexane). IR (DRIFT): 2600–3400, 1724, 1652 cm-1. 1H NMR
(500 MHz, CDCl3, referenced to TMS): δ 0.53 (d, 1H, J = 10.3 Hz, H-5); 0.64 (td, 1H, J1 = 13.2
Hz, J2 = 3.5 Hz, H-1a); 0.82 (s, 3H, H-25); 0.86 (s, 3H, H-24); 0.88 (s, 3H, H-27); 0.92 (s, 3H, H-
26); 0.94 (s, 3H, H-23); 1.02 (qd, 1H, J1 = 12.4 Hz, J2 = 3.8 Hz, H-12a); 1.16 (t, 1H, J = 12.5 Hz,
H-9); 1.19 (dd, 1H, J1 = 13.4 Hz, J2 = 2.9 Hz, H-21a); 1.28 (m, 1H, J = 12.5 Hz, H-2a); 1.31 (m,
2H, H-7); 1.33 (m, 1H, H-6a); 1.38 (m, 1H, J = 12.4 Hz, H-12b); 1.41 (dd, 1H, J1 = 12.5 Hz, J2 =
2.2 Hz, H-2b); 1.44 (m, 1H, H-15a); 1.45 (m, 1H, J = 12.6 Hz, H-16a); 1.49 (m, 1H, J = 13.5 Hz,
H-22a); 1.50 (mm, 4H, H-11a, H-1, 6, 21b); 1.74 (t, 1H, J = 11.4 Hz, H-18); 1.74 (m, 1H, H-
11b); 1.92 (m, 1H, J = 13.5 Hz, H-22b); 1.99 (m, 1H, J = 13.0 Hz, H-15b); 2.13 (td, 1H, J1 = 12.0
Hz, J2 = 3.0 Hz, H-13); 2.26 (dt, 1H, J1 = 12.6 Hz, J2 = 2.6 Hz, H-16b); 2.95 (td, 1H, J1 = 11.1
Hz, J2 = 4.5 Hz, H-19β); 2.98 (s, 6H, H-40, 41); 3.32 (dd, 1H, J1 = 11.0 Hz, J2 = 5.0 Hz, H-3α);
4.67 (s, 1H, H-29 pro-E); 4.94 (m, 2H, H-30); 5.03 (s, 1H, H-29 pro-Z); 6.80 (br, 2H, H-35, 37);
7.36 (t, 6H, J = 7.2 Hz, H-3´,3´´a,b,c); 7.42 (tt, 3H, J1 = 7.2 Hz, J2 = 1.5 Hz, H-4´a,b,c); 7.60 (s,
1H, H-31); 7.63 (d, 6H, J = 7.2 Hz, H-2´,2´´a,b,c); 7.69 (d, 2H, J = 8.2 Hz, H-34,38). 13C NMR
(125 MHz, CDCl3): δ = 14.79 (C27); 16.11 (C26); 16.22 (C25); 16.44 (C24); 18.46 (C6); 20.97
(C2); 26.92 (C12); 28.00 (C11); 28.54 (C23); 29.70 (C21); 31.96 (C15); 32.05 (C16); 34.36 (C7);
36.71 (C22); 37.10 (C10); 38.38 (C13); 38.66 (C1); 39.67 (C4); 40.74 (C8); 40.86 (C40,C41);
42.47 (C14); 43.41 (C19); 50.34 (C18); 50.43 (C9); 54.65 (C29); 56.30 (C5); 56.32 (C17); 91.22
(C3); 111.62 (C29); 112.95 (C35, 37); 118.76 (C31); 122.23 (C33); 126.84 (C34,C38); 127.77
(Ca,b,c3´,3´´); 129.81 (Ca,b,c4´); 135.46 (Ca,b,c1´,1´´); 135.64 (Ca,b,c2´,2´´); 148.42 (C32);
149.70 (C20); 150.21 (C36); 180.72 (C28). MS (ESI+): m/z (%) = 901 (100, [M+H]+), 923 (6,
[M+Na]+). HRMS (ESI+) m/z calcd for C58H72N4O3Si [M+H]+ 901.5446, found 901.5441.
Compound 10f was obtained from 150 mg (0.20 mmol) of 7 by the general procedure using
1 equiv. of alkyne at r.t. while reaction time was 24 h. The yield of white crystals was 147 mg
(81%): mp 195–197˚C (cyclohexane). IR (DRIFT): 2600–3400, 1717, 1639 cm-1. 1H NMR (500
MHz, CDCl3): δ 0.84 (s, 3H); 0.89 (s, 3H); 0.90 (s, 3H); 0.95 (s, 3H); 0.96 (s, 3H, H-23, 24, 25,
26, 27); 1.35 (s, 9H, H-40, 41, 42); 1.76 (t, 2H, J = 1.2 Hz); 2.15 (td, 1H, J1 = 12.6 Hz, J2 = 2.9
Hz); 2.29 (d, 1H, J = 12.9 Hz); 2.98 (td, 1H, J1 = 11.2 Hz, J2 = 4.9 Hz, H-19β); 3.35 (dd, 1H, J1 =
11.7 Hz, J2 = 4.3 Hz, H-3α); 4.69 (s, 1H, H-29 pro-E); 4.98 (s, 2H, H-30); 5.05 (s, 1H, H-29 pro-
Z); 7.37–7.47 (m, 11H, H-35, 37, 3 × Ph); 7.66 (d, 6H, J = 6.6 Hz, 3 × Ph); 7.78 (d, 2H, H-34,
38). 13C NMR (125 MHz, CDCl3): δ = 14.68; 16.00; 16.11; 16.33; 18.36; 20.84; 26.82; 27.89;
28.43; 29.58; 29.67; 31.26; 31.93; 34.22; 34.63; 36.59; 36.98; 38.26; 38.52; 39.55; 40.60; 42.34;
43.22; 50.21; 50.34; 54.37; 54.67; 55.18; 56.24; 81.09; 111.63; 119.73; 125.46; 125.71; 127.67;
129.70; 135.34; 135.52; 147.91; 149.58; 151.27; 181.28. MS (ESI+): m/z (%) = 914 (100,
[M+H]+), 937 (75, [M+Na]+). HRMS (ESI+) m/z calcd for C60H75N3O3Si [M+H]+ 914.5650,
found 914.5650.
Compound 10g was obtained from 150 mg (0.20 mmol) of 7 by the general procedure
using 1 equiv. of alkyne at 50˚C while reaction time was 15 h. The yield of white crystals was
148 mg (88%): mp 146–148˚C (cyclohexane). IR (DRIFT): 2650–3400, 1730, 1452 cm-1. 1H
NMR (500 MHz, CDCl3): δ 0.84 (s, 3H); 0.89 (s, 9H); 0.96 (s, 3H, H-23, 24, 25, 26, 27); 2.29 (d,
1H, J = 11.5 Hz); 2.97 (t, 1H, J = 12.9 Hz, H-33); 3.21 (td, 1H, J1 = 15.5 Hz, J2 = 7.7 Hz, H-19β);
3.34 (d, 1H, J = 10.9 Hz, H-3α); 4.65 (s, 1H, H-29 pro-E); 4.89 AB-system, 2H, JGEM = 15.8 Hz,
H-30); 5.03 (s, 1H, H-29 pro-Z); 7.25 (s, 1H, H-31); 7.37–7.45 (m, 9H, 3 × Ph); 7.66 (d, 6H,
J = 8.0 Hz, 3 × Ph). 13C NMR (125 MHz, CDCl3): δ = 14.65; 15.99; 16.10; 16.32; 18.37; 20.82;
25.11; 26.66; 27.89; 28.42; 29.58; 29.66; 31.76; 31.93; 33.23; 34.23; 36.69; 36.97; 38.20; 38.53;
39.53; 40.58; 42.32; 43.47; 50.11; 50.22; 54.19; 55.20; 56.29; 81.11; 111.92; 120.00; 127.65;
129.68; 135.32; 135.50; 149.62; 152.89; 181.13. MS (ESI+): m/z (%) = 850 (100, [M+H]+).
HRMS (ESI+) m/z calcd for C55H71N3O3Si [M+H]+ 850.5337, found 850.5334.
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Cytotoxic conjugates of betulinic acid and substituted triazoles
Unprotected compounds 11a–11g. Compound 11a was obtained from 150 mg (0.17
mmol) of 10a by the general deprotection procedure 1 at 60˚C for 18 h. The yield of white crys-
tals was 143 mg (79%): mp 137–138˚C (CH2Cl2). IR (DRIFT): 2600–3400, 1724, 1650 cm-1. 1H
NMR (500 MHz, CDCl3): δ 0.76 (s, 3H); 0.82 (s, 3H); 0.92 (s, 3H); 0.98 (s, 6H, H-23, 24, 25, 26,
27); 2.18 (dd, 1H, J1 = 13.5 Hz, J2 = 3.4 Hz); 2.30 (d, 1H, J = 12.7 Hz); 3.00 (td, 1H, J1 = 11.2 Hz,
J2 = 4.6 Hz, H-19β); 3.21 (dd, 1H, J1 = 11.2 Hz, J2 = 4.9 Hz, H-3α); 4.72 (s, 1H, H-29 pro-E);
5.00 (AB-system, 2H, JGEM = 15.8 Hz, H-30); 5.10 (s, 1H, H-29 pro-Z); 7.34 (tt, 1H, J1 = 7.3 Hz,
J2 = 1.3 Hz, H-36); 7.43 (t, 2H, J = 7.3 Hz, H-35, 37); 7.77 (s, 1H, H-31); 7.84 (d, 2H, J = 7.0 Hz,
H-34, 38). 13C NMR (125 MHz, CDCl3): δ = 14.66; 15.32; 16.01; 16.08; 18.21; 20.89; 26.84;
27.29; 27.94; 29.60; 29.67; 31.86; 31.92; 34.25; 36.62; 37.14; 38.30; 38.66; 38.81; 40.63; 42.36;
43.39; 50.36; 54.59; 55.24; 56.25; 78.95; 111.95; 120.02; 125.74; 128.16; 128.81; 130.54; 147.97;
149.48; 180.97. MS (ESI+): m/z (%) = 600 (100, [M+H]+). HRMS (ESI+) m/z calcd for
C38H53N3O3 [M+H]+ 600.4160, found 600.4162. Note: deprotection procedure 2 at r.t. for 4 h
was also tried with the yield of 76%.
Compound 11b was obtained from 150 mg (0.17 mmol) of 10b by the general deprotection
procedure 1 at r.t. for 32 h. The yield of white crystals was 117 mg (62%): mp 127–129˚C
(CH2Cl2). IR (DRIFT): 2600–3400, 1727, 1453 cm-1. 1H NMR (500 MHz, CDCl3, referenced to
TMS): δ 0.66 (d, 1H, J = 9.0 Hz, H-5); 0.73 (s, 3H, H-25); 0.80 (s, 3H, H-24); 0.88 (m, 1H,
J = 13.2 Hz, H-1a); 0.90 (s, 3H, H-26); 0.95 (s, 3H, H-23); 0.96, (s, 3H, H-27); 1.04 (dd, 1H, J1 =
11.7 Hz, J2 = 4.2 Hz, H-12a); 1.23 (m, 1H, H-9); 1.24 (mm, 2H, H-15, 21a); 1.25 (m, 1H, H-
11a); 1.36 (mm, 2H, H-7); 1.36 (m, 1H, H-6a); 1.38 (m, 1H, H-12b); 1.44 (m, 1H, H-11b); 1.44
(m, 1H, J = 12.2 Hz, H-16a); 1.50 (m, 1H, H-15b); 1.52 (mm, 3H, H-2,6,21b); 1.52 (m, 1H,
J = 13.1 Hz, H-22a); 1.61 (m, 1H, H-2b); 1.63 (m, 1H, J = 13.2 Hz, H-1b); 1.75 (t, 1H, J = 11.4
Hz, H-18); 1.95 (m, 1H, H-22b); 2.16 (td, 1H, J1 = 12.1 Hz, J2 = 3.3 Hz, H-13); 2.28 (dt, 1H, J1 =
12.2 Hz, J2 = 3.1 Hz, H-16b); 2.98 (td, 1H, J1 = 11.1 Hz, J2 = 4.3 Hz, H-19β); 3.18 (dd, 1H, J1 =
11.3 Hz, J2 = 4.7 Hz, H-3α); 4.75 (s, 1H, H-29); 5.00 (d, 1H, J = 15.6 Hz, H-30); 5.06 (d, 1H,
J = 15.6 Hz, H-30b); 5.10 (s, 1H, H-29); 7.51 (tt, 1H, J1 = 7.5 Hz, J2 = 0.8 Hz, H-36); 7.64 (td,
1H, J1 = 7.5 Hz, J2 = 1.5 Hz, H-37); 7.71 (dd, 1H, J1 = 7.8 Hz, J2 = 1.1 Hz, H-38); 7.82 (s, 1H, H-
31); 8.02 (dd, 1H, J1 = 7.8, J2 = 1.2 Hz, H-35); 10.36 (d, 1H, J = 0.5 Hz, H-39). 13C NMR (125
MHz, CDCl3): δ = 14.78 (C27); 15.43 (C25); 16.12 (C26); 16.20 (C24); 18.34 (C6); 21.02 (C11);
27.06 (C12); 27.44 (C2); 28.06 (C23); 29.71 (C21); 29.77 (C15); 32.02 (C16); 34.38 (C7); 36.71
(C22); 37.28 (C10); 38.41 (C13); 38.80 (C1); 38.94 (C4); 40.77 (C8); 42.49 (C14); 43.39 (C19);
50.50 (C18); 50.52 (C9); 54.93 (C30); 55.38 (C5); 56.35 (C17); 79.03 (C3); 112.37 (C29); 123.84
(C31); 128.76 (C36); 128.96 (C35); 130.20 (C38); 133.18 (C33); 133.83 (C37); 133.96 (C34);
144.93 (C32); 149.45 (C20); 180.36 (C28); 192.41 (C39). MS (ESI+): m/z (%) = 628 (100,
[M+H]+). HRMS (ESI-TOF) m/z calcd for C39H53N3O4 [M+H]+ 628.4109, found 628.4111.
Compound 11c was obtained from 150 mg (0.30 mmol) of 10c by the general deprotection
procedure 1 at r.t. for 28 h. The yield of white crystals was 105 mg (58%): mp 141–142˚C
(CH2Cl2). IR (DRIFT): 2500–3450, 1735, 1654 cm-1. 1H NMR (500 MHz, CDCl3): δ 0.76 (s,
3H); 0.82 (s, 3H); 0.92 (s, 3H); 0.97 (s, 3H); 0.98 (s, 3H, H-23, 24, 25, 26, 27); 1.75 (t, 1H,
J = 11.5 Hz); 1.97 (dd, 1H, J1 = 12.6 Hz, J2 = 8.3 Hz); 2.06 (m, 1H); 2.22 (td, 1H, J1 = 13.9 Hz,
J2 = 3.5 Hz); 2.31 (dt, 1H, J1 = 12.5 Hz, J2 = 3.2 Hz); 3.04 (td, 1H, J1 = 11.2 Hz, J2 = 4.9 Hz, H-
19β); 3.21 (dd, 1H, J1 = 11.6 Hz, J2 = 4.9 Hz, H-3α); 4.67 (s, 1H, H-29 pro-E); 5.04 (AB-system,
2H, JGEM = 15.7 Hz, H-30); 5.09 (s, 1H, H-29 pro-Z); 7.26 (m, 1H, H-pyridine); 7.81 (td, 1H,
J1 = 7.7 Hz, J2 = 1.7 Hz, H-pyridine); 8.22 (s, 1H, H-31); 8.22 (m, 1H, H-pyridine); 8.61 (dq,
J1 = 4.9 Hz, J2 = 0.8 Hz, H-pyridine). 13C NMR (125 MHz, CDCl3): δ = 14.66; 15.34; 16.10;
18.23; 20.90; 26.81; 27.31; 27.96; 29.62; 31.17; 31.95; 32.02; 34.29; 36.72; 37.16; 38.28; 38.72;
38.82; 40.66; 42.38; 43.59; 50.39; 50.46; 54.47; 55.27; 56.21; 78.93; 111.81; 120.49; 122.76;
122.97; 137.24; 148.11; 149.07; 149.35; 150.01; 180.18. MS (ESI-): m/z (%) = 599 (100, [M-H]-).
PLOS ONE | DOI:10.1371/journal.pone.0171621 February 3, 2017
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Cytotoxic conjugates of betulinic acid and substituted triazoles
HRMS (ESI-) m/z calcd for C37H52N4O3 [M-H]- 599.3956, found 599.3947. Note: deprotection
procedure 2 was also tried with yield of 58% and the attempt to prepare compound 11c from
free azide 8 by the general procedure for click reaction gave the best yield of 74%.
Compound 11d was obtained from 150 mg (0.30 mmol) of 10d by the general deprotection
procedure 1 at r.t. for 28 h. The yield of white crystals was 112 mg (62%): mp 141–142˚C
(CH2Cl2). IR (DRIFT): 2600–3400, 1734, 1645 cm-1. 1H NMR (500 MHz, DMSO): δ 0.64 (s,
3H); 0.75 (s, 3H); 0.85 (s, 3H); 0.87 (s, 3H); 0.93 (s, 3H, H-23, 24, 25, 26, 27); 2.12 (m, 2H) 2.18
(m, 1H); 2.97 (td, 1H, J1 = 10.9 Hz, J2 = 4.3 Hz, H-19β); 3.21 (dd, 1H, J1 = 8.3 Hz, J2 = 3.7 Hz,
H-3α); 4.56 (s, 1H, H-29 pro-E); 5.02 (s, 2H, H-30); 5.05 (s, 1H, H-29 pro-Z); 7.48 (m, 1H, H-
37); 8.23 (td, 1H, J1 = 7.7 Hz, J2 = 2.0 Hz, H-36); 8.54 (dd, 1H, J1 = 4.9 Hz, J2 = 1.7 Hz, H-35);
8.71 (s, 1H, H-31); 9.06 (d, 1H, J = 2.0 Hz, H-34). 13C NMR (125 MHz, CDCl3): δ = 14.65;
15.35; 16.11; 18.24; 20.95; 26.13; 27.09; 27.96; 27.92; 29.05; 29.58; 32.05; 34.31; 36.68; 37.17;
38.11; 38.84; 40.63; 40.86; 42.39; 48.72; 50.43; 50.51; 51.43; 55.29; 56.12; 78.92; 110.77; 120.63;
128.42; 128.55; 132.13; 144.54; 146.36; 148.57; 149.58; 168.10; 178.88. MS (ESI-): m/z (%) =
599 (100, [M-H]-). HRMS (ESI-TOF) m/z calcd for C37H52N4O3 [M-H]- 599.3956, found
599.3945. Note: deprotection procedure 2 was also tried with yield of 60% and the attempt to
prepare compound 11c from free azide 8 by the general procedure for click reaction gave the
best yield of 76%.
Compound 11e was obtained from 150 mg (0.17 mmol) of 10e by the general deprotection
procedure 1 at r.t. for 30 h. The yield of white crystals was 124 g (64%): mp 150–151˚C
(CH2Cl2). IR (DRIFT): 2600–3400, 1731, 1651 cm-1. 1H NMR (500 MHz, CDCl3): δ 0.76 (s,
1H); 0.82 (s, 3H); 0.92 (s, 3H); 0.97 (s, 6H, H-23, 24, 25, 26, 27); 2.30 (d, 1H, J = 9.2 Hz); 2.99
(s, 6H, H-39, 40); 2.99 (m, 1H, H-19β) 3.20 (dd, 1H, J1 = 11.2 Hz, J2 = 4.9 Hz, H-3α); 4.71 (s,
1H, H-29 pro-E); 4.97 (AB-system, 2H, JGEM = 15.5 Hz, H-30); 5.07 (s, 1H, H-29 pro-Z); 6.67
(d, 2H, J = 9.1 Hz, H-34, 38); 7.62 (s, 1H, H-31); 7.70 (d, 2H, J = 8.7 Hz, H-35, 37). 13C NMR
(125 MHz, CDCl3): δ = 14.67; 15.33; 16.04; 16.10; 18.23; 20.92; 26.83; 27.31; 27.96; 29.64; 31.85;
34.28; 36.55; 36.69; 37.18; 38.32; 38.68; 38.84; 40.48; 40.49; 40.66; 42.38; 43.53; 50.39; 54.48;
55.26; 78.96; 111.79; 112.52; 118.61; 118.86; 126.71; 126.90 148.43; 149.65; 150.44; 179.98. MS
(ESI+): m/z (%) = 643 (100, [M+H]+). HRMS (ESI+) m/z calcd for C40H58N4O3 [M+H]+
643.4582, found 643.4583.
Compound 11f was obtained from 150 mg (0.16 mmol) of 10f by the general deprotection
procedure 1 at r.t. for 30 h. The yield of white crystals was 148 mg (75%): mp 141–142˚C
(EtOAc). IR (DRIFT): 2500–3450, 1729, 1647 cm-1. 1H NMR (500 MHz, CDCl3): δ 0.76 (s,
3H); 0.82 (s, 3H); 0.92 (s, 3H); 0.97 (s, 6H, H-23, 24, 25, 26, 27); 2.18 (dd, 1H, J1 = 12.5 Hz, J2 =
2.9 Hz); 2.29 (d, 1H, J = 12.8 Hz); 3.02 (td, 1H, J1 = 10.9 Hz, J2 = 4.6 Hz, H-19β); 3.20 (dd, 1H,
J1 = 11.2 Hz, J2 = 4.7 Hz, H-3α); 4.72 (s, 1H, H-29 pro-E); 5.00 (AB-system, 2H, JGEM = 15.8
Hz, H-30); 5.08 (s, 1H, H-29 pro-Z); 7.45 (d, 2H, J = 8.6 Hz, H-35, 37); 7.73 (s, 1H, H-31); 7.77
(d, 2H, J = 8.6 Hz, H-34, 38). 13C NMR (125 MHz, CDCl3): δ = 14.65; 15.33; 16.02; 16.09;
18.23; 20.92; 26.87; 27.31; 27.95; 29.63; 30.14; 31.27; 31.90; 32.00; 34.26; 34.64; 36.67; 37.15;
38.27; 38.67; 38.82; 40.63; 42.36; 43.31; 43.43; 50.37; 54.71; 55.24; 56.29; 78.95; 111.78; 119.76;
125.47; 125.72; 127.72; 147.93; 149.67; 151.29; 180.99. MS (ESI+): m/z (%) = 456 (100, [M
+H]+), 478 (4, [M+Na]+). HRMS (ESI+) m/z calcd for C42H61N3O3 [M+H]+ 456.4786, found
456.4786. Note: deprotection procedure 2 at r.t. for 4 h was also tried with the yield of 73%.
Compound 11g was obtained from 150 mg (0.18 mmol) of 10g by the general deprotection
procedure 1 at 50˚C for 20 h. The yield of white crystals was 144 mg (81%): mp 127–129˚C
(CHCl3). IR (DRIFT): 2500–3400, 1724, 1652 cm-1. 1H NMR (500 MHz, CDCl3): δ 0.76 (s,
3H); 0.82 (s, 3H); 0.92 (s, 3H); 0.95 (s, 3H); 0.97 (s, 3H, H-23, 24, 25, 26, 27); 2.12 (m, 2H) 2.13
(m, 1H); 2.29 (d, 1H, J = 3.0 Hz); 2.97 (td, 1H, J1 = 10.9 Hz, J2 = 4.3 Hz, H-19β); 3.21 (dd, 1H,
J1 = 8.3 Hz, J2 = 3.7 Hz, H-3α); 4.66 (s, 1H, H-29 pro-E); 4.90 (AB-system, 2H, JGEM = 15.5 Hz,
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Cytotoxic conjugates of betulinic acid and substituted triazoles
H-30); 5.05 (s, 1H, H-29 pro-Z); 7.24 (s, 1H, H-31). 13C NMR (125 MHz, CDCl3): δ = 14.66;
15.33; 16.01; 16.09; 18.20; 20.87; 25.12; 26.67; 27.31; 27.95; 29.61; 31.92; 33.24; 34.28; 36.64;
36.74; 37.16; 38.26; 38.73; 38.82; 40.63; 40.66; 42.36; 43.45; 50.16; 50.37; 54.22; 55.28; 56.29;
78.96; 111.87; 120.02; 149.66; 152.98; 180.95. MS (ESI+): m/z (%) = 592 (100, [M+H]+), 614
(10, [M+Na]+). HRMS (ESI+) m/z calcd for C37H57N3O3 [M+H]+ 592.4473, found 592.4474.
General information about the biological assays
Cell lines. Biological assays were performed in concordance with our previous publica-
tions [29, 58; 60; 61]. All cells (if not indicated otherwise) were purchased from the American
Tissue Culture Collection (ATCC). The CCRF-CEM line is derived from T lymphoblastic leu-
kemia, evincing high chemosenzitivity, K562 represent cells from an acute myeloid leukemia
patient sample with bcr-abl translocation, U2OS line is derived from osteosarcoma, HCT116
is colorectal tumor cell line and its p53 gene knock-down counterpart (HCT116p53-/-, Hori-
zon Discovery Ltd, UK) is a model of human cancers with p53 mutation frequently associated
with poor prognosis, A549 line is lung adenocarcinoma. The daunorubicin resistant subline of
CCRF-CEM cells (CEM-DNR bulk) and paclitaxel resistant subline K562-TAX were selected
in our laboratory by the cultivation of maternal cell lines in increasing concentrations of dau-
norubicine or paclitaxel, respectively. The CEM-DNR bulk cells overexpress MRP-1 and P-
glycoprotein protein, while K562-TAX cells overexpress P-glycoprotein only. Both proteins
belong to the family of ABC transporters and are involved in the primary and/or acquired
multidrug resistance phenomenon [58]. MRC-5 and BJ cell lines were used as a non-tumor
control and represent human fibroblasts. The cells were maintained in nunc/corning 80 cm2
plastic tissue culture flasks and cultured in cell culture medium according to ATCC or Hori-
zon recommendations (DMEM/RPMI 1640 with 5 g/L glucose, 2 mM glutamine, 100 U/mL
penicillin, 100 mg/mL streptomycin, 10% fetal calf serum, and NaHCO3).
Cytotoxic MTS assay. MTS assay was performed at Institute of Molecular and Transla-
tional Medicine by robotic platform (HighResBiosolutions). Cell suspensions were prepared
and diluted according to the particular cell type and the expected target cell density (25000–
35000 cells/mL based on cell growth characteristics). Cells were added by automatic pipetor
(30 μL) into 384 well microtiter plates. All tested compounds were dissolved in 100% DMSO
and four-fold dilutions of the intended test concentration were added in 0.15 μL aliquots at
time zero to the microtiter plate wells by the echoacustic non-contact liquid handler Echo550
(Labcyte). The experiments were performed in technical duplicates and three biological repli-
cates at least. The cells were incubated with the tested compounds for 72 h at 37˚C, in a 5%
CO2 atmosphere at 100% humidity. At the end of the incubation period, the cells were assayed
by using the MTS test. Aliquots (5 μL) of the MTS stock solution were pipetted into each well
and incubated for additional 1–4 h. After this incubation period, the optical density (OD) was
measured at 490 nm with an Envision reader (Perkin Elmer). Tumor cell survival (TCS) was
calculated by using the following equation: TCS = (ODdrug-exposed well/mean ODcontrol wells) ×
100%. The IC50 value, the drug concentration that is lethal to 50% of the tumor cells, was calcu-
lated from the appropriate dose-response curves in Dotmatics software.
Cell cycle and apoptosis analysis. Suspension of CCRF-CEM cells, seeded at a density of
1.106 cells/mL in 6-well panels, were cultivated with the 1 or 5 × IC50 of tested compound in a
humidified CO2 incubator at 37˚C in RPMI 1640 cell culture medium containing 10% fetal
calf serum, 10 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. Together
with the treated cells, control sample containing vehicle was harvested at the same time point
after 24 h. After another 24 hours, cells were then washed with cold PBS and fixed in 70% etha-
nol added dropwise and stored overnight at -20˚C. Afterwards, cells were washed in hypotonic
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Cytotoxic conjugates of betulinic acid and substituted triazoles
citrate buffer, treated with RNAse (50 μg/mL) and stained with propidium iodide. Flow cytom-
eter using a 488 nm single beam laser (Becton Dickinson) was used for measurement. Cell
cycle was analyzed in the program ModFitLT (Verity), and apoptosis was measured in loga-
rithmic model expressing percentage of the particles with propidium content lower than cells
in G0/G1 phase (<G1) of the cell cycle. Half of the sample was used for pH3Ser10 antibody
(Sigma) labeling and subsequent flow cytometry analysis of mitotic cells [61].
BrDU incorporation analysis (DNA synthesis). For this analysis, the same procedure
of cultivation as previously was used. Before harvesting, 10 μM 5-bromo-2-deoxyuridine
(BrDU), was added to the cells for puls-labeling for 30 min. Cells were fixed with ice-cold 70%
ethanol and stored overnight. Before the analysis, cellswere washed with PBS, and resuspended
in 2 M HCl for 30 min at room temperature to denature their DNA. Following neutralization
with 0.1 M Na2B4O7 (Borax), cells were washed with PBS containing 0.5% Tween-20 and 1%
BSA. Staining with primary anti-BrDU antibody (Exbio) for 30 min at room temperature in
the dark followed. Cells were than washed with PBS and stained with secondary antimouse-
FITC antibody (Sigma). Cells were then washed with PBS again and incubated with propidium
iodide (0.1 mg/mL) and RNAse A (0.5 mg/mL) for 1 h at room temperature in the dark and
afterwards analyzed by flow cytometry using a 488 nm single beam laser (FACSCalibur, Becton
Dickinson) [61].
BrU incorporation analysis (RNA synthesis). Cells were cultured and treated as above.
Before harvesting, pulse-labeling with 1 mM 5-bromouridine (BrU) for 30 min followed. The
cells were then fixed in 1% buffered paraformaldehyde with 0.05% of NP-40 in room tempera-
ture for 15 min, and then stored in 4˚C overnight. Before measurement, they werewashed in
PBS with 1% glycin, washed in PBS again, and stained by primary anti-BrDU antibody cross-
reacting to BrU (Exbio) for 30 min at room temperature in the dark. After another washing step
in PBS cells were stained by secondary antimouse-FITC antibody (Sigma). Following the stain-
ing, cells were washed with PBS and fixed with 1% PBS buffered paraformaldehyde with 0.05%
of NP-40 for 1 hour. Cells were washed by PBS, incubated with propidium iodide (0.1 mg/mL)
and RNAse A (0.5 mg/mL) for 1 h at room temperature in the dark, and finally analyzed by
flow cytometry using a 488 nm single beam laser (FACS Calibur, Becton Dickinson) [61].
Supporting information
S1 Fig. 1H NMR spectrum of 3.
(DOCX)
S2 Fig. 13C NMR spectrum of 3.
(DOCX)
S3 Fig. 1H NMR spectrum of 4.
(DOCX)
S4 Fig. 13C NMR spectrum of 4.
(DOCX)
S5 Fig. 1H NMR spectrum of 5.
(DOCX)
S6 Fig. 13C NMR spectrum of 5.
(DOCX)
S7 Fig. 1H NMR spectrum of 6.
(DOCX)
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Cytotoxic conjugates of betulinic acid and substituted triazoles
S8 Fig. 13C NMR spectrum of 6.
(DOCX)
S9 Fig. 1H NMR spectrum of 7.
(DOCX)
S10 Fig. 13C NMR spectrum of 7.
(DOCX)
S11 Fig. 1H NMR spectrum of 8.
(DOCX)
S12 Fig. 13C NMR spectrum of 8.
(DOCX)
S13 Fig. 1H NMR spectrum of 9a.
(DOCX)
S14 Fig. 13C NMR spectrum of 9a.
(DOCX)
S15 Fig. 1H NMR spectrum of 9b.
(DOCX)
S16 Fig. 13C NMR spectrum of 9b.
(DOCX)
S17 Fig. 1H NMR spectrum of 9c.
(DOCX)
S18 Fig. 13C NMR spectrum of 9c.
(DOCX)
S19 Fig. 2D COSY NMR spectrum of 9c.
(DOCX)
S20 Fig. 2D ROESY NMR spectrum of 9c.
(DOCX)
S21 Fig. 2D HMQC NMR spectrum of 9c.
(DOCX)
S22 Fig. 2D HMBC NMR spectrum of 9c.
(DOCX)
S23 Fig. 2D 15N-HMBC NMR spectrum of 9c.
(DOCX)
S24 Fig. 1H NMR spectrum of 9d.
(DOCX)
S25 Fig. 13C NMR spectrum of 9d.
(DOCX)
S26 Fig. 2D COSY NMR spectrum of 9d.
(DOCX)
S27 Fig. 2D ROESY NMR spectrum of 9d.
(DOCX)
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Cytotoxic conjugates of betulinic acid and substituted triazoles
S28 Fig. 2D HMQC NMR spectrum of 9d.
(DOCX)
S29 Fig. 2D HMBC NMR spectrum of 9d.
(DOCX)
S30 Fig. 2D 15N-HMBC NMR spectrum of 9d.
(DOCX)
S31 Fig. 1H NMR spectrum of 9e.
(DOCX)
S32 Fig. 13C NMR spectrum of 9e.
(DOCX)
S33 Fig. 1H NMR spectrum of 9f.
(DOCX)
S34 Fig. 13C NMR spectrum of 9f.
(DOCX)
S35 Fig. 1H NMR spectrum of 9g.
(DOCX)
S36 Fig. 13C NMR spectrum of 9g.
(DOCX)
S37 Fig. 1H NMR spectrum of 9h.
(DOCX)
S38 Fig. 13C NMR spectrum of 9h.
(DOCX)
S39 Fig. 1H NMR spectrum of 10a.
(DOCX)
S40 Fig. 13C NMR spectrum of 10a.
(DOCX)
S41 Fig. 1H NMR spectrum of 10b.
(DOCX)
S42 Fig. 13C NMR spectrum of 10b.
(DOCX)
S43 Fig. 1H NMR spectrum of 10c.
(DOCX)
S44 Fig. 13C NMR spectrum of 10c.
(DOCX)
S45 Fig. 1H NMR spectrum of 10d.
(DOCX)
S46 Fig. 13C NMR spectrum of 10d.
(DOCX)
S47 Fig. 1H NMR spectrum of 10e.
(DOCX)
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Cytotoxic conjugates of betulinic acid and substituted triazoles
S48 Fig. 13C NMR spectrum of 10e.
(DOCX)
S49 Fig. 2D COSY NMR spectrum of 10e.
(DOCX)
S50 Fig. 2D ROESY NMR spectrum of 10e.
(DOCX)
S51 Fig. 2D HMQC NMR spectrum of 10e.
(DOCX)
S52 Fig. 2D HMBC NMR spectrum of 10e.
(DOCX)
S53 Fig. 2D 15N-HMBC NMR spectrum of 10e.
(DOCX)
S54 Fig. 1H NMR spectrum of 10f.
(DOCX)
S55 Fig. 13C NMR spectrum of 10f.
(DOCX)
S56 Fig. 1H NMR spectrum of 10g.
(DOCX)
S57 Fig. 13C NMR spectrum of 10g.
(DOCX)
S58 Fig. 1H NMR spectrum of 11a.
(DOCX)
S59 Fig. 13C NMR spectrum of 11a.
(DOCX)
S60 Fig. 1H NMR spectrum of 11b.
(DOCX)
S61 Fig. 13C NMR spectrum of 11b.
(DOCX)
S62 Fig. 2D COSY NMR spectrum of 11b.
(DOCX)
S63 Fig. 2D ROESY NMR spectrum of 11b.
(DOCX)
S64 Fig. 2D HMQC NMR spectrum of 11b.
(DOCX)
S65 Fig. 2D HMBC NMR spectrum of 11b.
(DOCX)
S66 Fig. 2D 15N-HMBC NMR spectrum of 11b.
(DOCX)
S67 Fig. 1H NMR spectrum of 11c.
(DOCX)
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Cytotoxic conjugates of betulinic acid and substituted triazoles
S68 Fig. 13C NMR spectrum of 11c.
(DOCX)
S69 Fig. 1H NMR spectrum of 11d.
(DOCX)
S70 Fig. 13C NMR spectrum of 11d.
(DOCX)
S71 Fig. 1H NMR spectrum of 11e.
(DOCX)
S72 Fig. 13C NMR spectrum of 11e.
(DOCX)
S73 Fig. 1H NMR spectrum of 11f.
(DOCX)
S74 Fig. 13C NMR spectrum of 11f.
(DOCX)
S75 Fig. 1H NMR spectrum of 11g.
(DOCX)
S76 Fig. 13C NMR spectrum of 11g.
(DOCX)
Acknowledgments
All material, chemicals, and small equipment for both chemistry and biology was paid by
Czech Science Foundation (15-05620S); stipendia to students were paid by internal grants of
Palacky University IGA-PrF-2016-020, IGA-LF-2016-19. Starting terpenes were paid from
the Technology Agency of the Czech Republic (TE01020028). The infrastructural part (Insti-
tute of Molecular and Translational Medicine) is supported by the National Sustainability Pro-
gramme (LO1304). We are grateful to Tereza Volna for measurement of HRMS.
Author contributions
Conceptualization: VS PD MH MU.
Data curation: VS IP MU.
Formal analysis: VS PZ JP PD MU.
Funding acquisition: JP MH MU.
Investigation: VS PZ JP IP.
Methodology: VS PZ PD MU.
Project administration: MU.
Resources: MU PD MH.
Supervision: JP PD MU.
Validation: PD MU.
Visualization: JP PD MU.
PLOS ONE | DOI:10.1371/journal.pone.0171621 February 3, 2017
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Cytotoxic conjugates of betulinic acid and substituted triazoles
Writing – original draft: MU.
Writing – review & editing: JP PD.
References
1. Hill RA, Connolly JD. Triterpenoids. Nat Prod Rep. 2015; 32(2): 273–327. doi: 10.1039/c4np00101j
PMID: 25342440
2. Dzubak P, Hajduch M, Vydra D, Hustova A, Kvasnica M, Biedermann D, et al. Pharmacological activi-
ties of natural triterpenoids and their therapeutic implications. Nat Prod Rep. 2006; 23(3): 394–411. doi:
10.1039/b515312n PMID: 16741586
3. Sarek J, Kvasnica M, Vlk M, Biedermann D. Semisynthetic lupane triterpenes with cytotoxic activity. In:
Salvador Jorge A.R., editor. Pentacyclic triterpenes as promising agents in cancer. Hauppauge, N.Y.:
Nova Science Publishers; 2010. p. 159–89.
4. Dang Z, Ho P, Zhu L, Qian K, Lee KH, Huang L, et al. New Betulinic Acid Derivatives for Bevirimat-
Resistant Human Immunodeficiency Virus Type-1. J Med Chem. 2013 Mar 14; 56(5): 2029–37. doi: 10.
1021/jm3016969 PMID: 23379607
5. Zuo WJ, Dai HF, Chen J, Chen HQ, Zhao YX, Mei WL, et al. Triterpenes and Triterpenoid Saponins
from the Leaves of Ilex kudincha. Planta Med. 2011; 77(16): 1835–40. doi: 10.1055/s-0030-1271164
PMID: 21870320
6. Innocente A, Casanova BB, Klein F, Lana AD, Pereira D, Muniz MN, et al. Synthesis of isosteric triterpe-
noid derivatives and antifungal activity. Chem Biol Drug Des. 2014 Mar; 83(3): 344–9. doi: 10.1111/
cbdd.12251 PMID: 24138556
7. Chianese G, Yerbanga SR, Lucantoni L, Habluetzel A, Basilico N, Taramelli D, et al. Antiplasmodial Tri-
terpenoids from the Fruits of Neem, Azadirachta indica. J Nat Prod. 2010 Aug 27; 73(8): 1448–52. doi:
10.1021/np100325q PMID: 20669933
8. Fu Y, Zhou E, Wei Z, Liang D, Wang W, Wang T, et al. Glycyrrhizin inhibits the inflammatory response
in mouse mammary epithelial cells and a mouse mastitis model. FEBS J. 2014 Jun; 281(11): 2543–57.
doi: 10.1111/febs.12801 PMID: 24698106
9. Yano S, Harada M, Watanabe K, Nakamaru K, Hatakeyama Y, Shibata S, et al. Antiulcer Activities of
Glycyrrhetinic Acid Derivatives in Experimenta Glastric Lesion Models. Chem Pharm Bull. 1989; 37(9):
2500–4. PMID: 2605700
10. Morikawa T, Ninomiya K, Imura K, Yamaguchi T, Akagi Y, Yoshikawa M, et al. Hepatoprotective triter-
penes from traditional Tibetan medicine Potentilla anserina. Phytochemistry. 2014 Jun; 102: 169–81.
doi: 10.1016/j.phytochem.2014.03.002 PMID: 24697904
11. Sanchez-Quesada C, Lopez-Biedma A, Warleta F, Campos M, Beltran G, Gaforio JJ. Bioactive Proper-
ties of the Main Triterpenes Found in Olives, Virgin Olive Oil, and Leaves of Olea europaea. J Agric
Food Chem. 2013 Dec 18; 61(50): 12173–82. doi: 10.1021/jf403154e PMID: 24279741
12. Urban M, Sarek J, Kvasnica M, Tislerova I, Hajduch M. Triterpenoid Pyrazines and Benzopyrazines
with Cytotoxic Activity. J Nat Prod. 2007 Apr 1; 70(4): 526–32. doi: 10.1021/np060436d PMID:
17371067
13. Urban M, Vlk M, Dzubak P, Hajduch M, Sarek J. Cytotoxic heterocyclic triterpenoids derived from betu-
lin and betulinic acid. Bioorg Med Chem. 2012 Jun 1; 20(11): 3666–74. doi: 10.1016/j.bmc.2012.03.066
PMID: 22551630
14. Vlk M, Micolova P, Urban M, Kvasnica M, Saman D, Sarek J. 15N-labelled pyrazines of triterpenic
acids. J Radioanal Nucl Chem. 2016; 308(2): 733–9.
15. Urban M, Klinot J, Tislerova I, Biedermann D, Hajduch M, Cisarova I, et al. Reactions of Activated
Lupane Oxo-Compounds with Diazomethane: An Approach to New Derivatives of Cytotoxic Triter-
penes. Synthesis. 2006; 2006(23): 3979–86.
16. Kvasnica M, Urban M, Dickinson NJ, Sarek J. Pentacyclic triterpenoids with nitrogen- and sulfur-con-
taining heterocycles: synthesis and medicinal significance. Nat Prod Rep. 2015; 32(9): 1303–30. doi:
10.1039/c5np00015g PMID: 26030604
17. Xu J, Li Z, Luo J, Yang F, Liu T, Liu M, et al. Synthesis and Biological Evaluation of Heterocyclic Ring-
Fused Betulinic Acid Derivatives as Novel Inhibitors of Osteoclast Differentiation and Bone Resorption.
J Med Chem. 2012 Apr 12; 55(7): 3122–34. doi: 10.1021/jm201540h PMID: 22435650
18. Csuk R, Stark S, Nitsche C, Barthel A, Siewert B. Alkylidene branched lupane derivatives: Synthesis
and antitumor activity. Eur J Med Chem. 2012 Jul; 53: 337–45. doi: 10.1016/j.ejmech.2012.04.023
PMID: 22578785
PLOS ONE | DOI:10.1371/journal.pone.0171621 February 3, 2017
22 / 25

Cytotoxic conjugates of betulinic acid and substituted triazoles
19. Dinh Ngoc T, Moons N, Kim Y, De Borggraeve W, Mashentseva A, Andrei G, et al. Synthesis of triterpe-
noid triazine derivatives from allobetulone and betulonic acid with biological activities. Bioorg Med
Chem. 2014 Jul 1; 22(13): 3292–300. doi: 10.1016/j.bmc.2014.04.061 PMID: 24844757
20. Ghosh P, Rasul MdG, Chakraborty M, Mandal A, Saha A. Microwave assisted one-pot synthesis of pyr-
azine derivatives of pentacyclic triterpenoids and their biological activity. Indian J Chem. Sect B-Org
Chem Incl Med Chem. 2011; 50B(10): 1519–23.
21. Urban M, Kvasnica M, Dickinson NJ, Sarek J. Biologically Active Triterpenoids Usable As Prodrugs. In:
Bates AR, editor. Terpenoids and Squalene: Biosynthesis, Function and Health Implications. Haup-
pauge, N.Y.: Nova Science Publishers; 2015. p. 25–50.
22. Kommera H, Kaluderovic GN, Kalbitz J, Drager B, Paschke R. Small structural changes of pentacyclic
lupane type triterpenoid derivatives lead to significant differences in their anticancer properties. Eur J
Med Chem. 2010 Aug; 45(8): 3346–53. doi: 10.1016/j.ejmech.2010.04.018 PMID: 20472329
23. Willmann M, Wacheck V, Buckley J, Nagy K, Thalhammer J, Paschke R, et al. Characterization of
NVX-207, a novel betulinic acid-derived anti-cancer compound. Eur J Clin Invest. 2009 May; 39(5):
384–94. doi: 10.1111/j.1365-2362.2009.02105.x PMID: 19309323
24. Gauthier C, Legault J, Lavoie S, Rondeau S, Tremblay S, Pichette A. Synthesis and Cytotoxicity of
Bidesmosidic Betulin and Betulinic Acid Saponins. J Nat Prod. 2009 Jan 23; 72(1): 72–81. doi: 10.
1021/np800579x PMID: 19115839
25. Gao J, Li X, Gu G, Liu S, Cui M, Lou HX. Facile synthesis of triterpenoid saponins bearing β-Glu/Gal-
(1!3)-β-GluA methyl ester and their cytotoxic activities. Bioorg Med Chem Lett. 2012 Apr 1; 22(7):
2396–400. doi: 10.1016/j.bmcl.2012.02.032 PMID: 22406153
26. Borkova L, Jasikova L, Rehulka J, Frisonsova K, Urban M, Frydrych I, et al. Synthesis of cytotoxic 2,2-
difluoroderivatives of dihydrobetulinic acid and allobetulin and study of their impact on cancer cells. Eur
J Med Chem. 2015 May 26; 96: 482–90. doi: 10.1016/j.ejmech.2015.03.068 PMID: 25942059
27. Flekhter OB, Karachurina LT, Poroikov VV, Nigmatullina LP, Baltina LA, Zarudii FS, et al. The synthesis
and hepatoprotective activity of esters of the lupane group triterpenoids. Russ J Bioorg Chem. 2000; 26
(3): 192–200.
28. Leunis JC, Couche E, inventors; Betulonic and Betulinic Acid Derivatives. 8586569. 2013.
29. Borkova L, Gurska S, Dzubak P, Burianova R, Hajduch M, Sarek J, et al. Lupane and 18α-oleanane
derivatives substituted in the position 2, their cytotoxicity and influence on cancer cells. Eur J Med
Chem. 2016 Oct 4; 121: 120–31. doi: 10.1016/j.ejmech.2016.05.029 PMID: 27236068
30. Zhang L, Jia X, Dong J, Chen D, Liu J, Zhang L, et al. Synthesis and evaluation of novel oleanolic acid
derivatives as potential antidiabetic agents. Chem Biol Drug Des. 2014 Mar; 83(3): 297–305. doi: 10.
1111/cbdd.12241 PMID: 24119242
31. Pertino WM, Lopez C, Theoduloz C, Schmeda-Hirschmann G. 1,2,3-Triazole-Substituted Oleanolic
Acid Derivatives: Synthesis and Antiproliferative Activity. Molecules. 2013 Jul 1; 18(7): 7661–7674. doi:
10.3390/molecules18077661 PMID: 23884119
32. Chakraborty B, Dutta D, Mukherjee S, Das S, Maiti NC, Das P, et al. Synthesis and biological evaluation
of a novel betulinic acid derivative as an inducer of apoptosis in human colon carcinoma cells (HT-29).
Eur J Med Chem. 2015 Sep 18; 102: 93–105. doi: 10.1016/j.ejmech.2015.07.035 PMID: 26248310
33. Majeed R, Sangwan PL, Chinthakindi PK, Khan I, Dangroo NA, Thota N, et al. Synthesis of 3-O-propar-
gylated betulinic acid and its 1,2,3-triazoles as potential apoptotic agents. Eur J Med Chem. 2013 May;
63: 782–92. doi: 10.1016/j.ejmech.2013.03.028 PMID: 23584541
34. Govdi AI, Sokolova NV, Sorokina IV, Baev DS, Tolstikova TG, Mamatyuk VI, et al. Synthesis of new
betulinic acid-peptide conjugates and in vivo and in silico studies of the influence of peptide moieties on
the triterpenoid core activity. Med Chem Commun. 2015; 6(1): 230–8.
35. Khan I, Guru SK, Rath SK, Chinthakindi PK, Singh B, Koul S, et al. A novel triazole derivative of betulinic
acid induces extrinsic and intrinsic apoptosis in human leukemia HL-60 cells. Eur J Med Chem. 2016
Jan 27; 108: 104–16. doi: 10.1016/j.ejmech.2015.11.018 PMID: 26629862
36. Csuk R, Barthel A, Sczepek R, Siewert B, Schwarz S. Synthesis, Encapsulation and Antitumor Activity
of New Betulin Derivatives. Arch Pharm Chem Life Sci. 2011 Jan 1; 344(1): 37–49.
¨
37. Csuk R, Barthel A, Kluge R, Strohl D. Synthesis, cytotoxicity and liposome preparation of 28-acetylenic
betulin derivatives. Bioorg Med Chem. 2010 Oct 15; 18(20): 7252–9. doi: 10.1016/j.bmc.2010.08.023
PMID: 20846866
38. Vasilevsky SF, Govdi AI, Sorokina IV, Tolstikova TG, Baev DS, Tolstikov GA, et al. Rapid access to
new bioconjugates of betulonic acid via click chemistry. Bioorg Med Chem Lett. 2011 Jan 1; 21(1): 62–
5. doi: 10.1016/j.bmcl.2010.11.072 PMID: 21159508
39. Govdi AI, Vasilevsky SF, Nenajdenko VG, Sokolova NV, Tolstikov GA. 1,3-Cycloaddition synthesis of
1,2,3-triazole conjugates of betulonic acid with peptides. Russ Chem Bull. 2011; 60(11): 2401–5.
PLOS ONE | DOI:10.1371/journal.pone.0171621 February 3, 2017
23 / 25

Cytotoxic conjugates of betulinic acid and substituted triazoles
40. Dang Thi TA, Kim Tuyet NT, Pham The C, Thanh Nguyen H, Ba Thi C, Doan Duy T, et al. Synthesis
and cytotoxic evaluation of novel ester-triazole-linked triterpenoid-AZT conjugates. Bioorg Med Chem
Lett. 2014 Nov 15; 24(22): 5190–4. doi: 10.1016/j.bmcl.2014.09.079 PMID: 25442310
´
´
41. Rodrıguez-Hernandez D, Demuner AJ, Barbosa LC, Heller L, Csuk R. Novel hederagenin-triazolyl
derivatives as potential anti-cancer agents. Eur J Med Chem. 2016 Jun 10; 115: 257–67. doi: 10.1016/
j.ejmech.2016.03.018 PMID: 27017553
42. Rashid S, Dar BA, Majeed R, Hamid A, Bhat BA. Synthesis and biological evaluation of ursolic acid-tria-
zolyl derivatives as potential anti-cancer agents. Eur J Med Chem. 2013 Aug; 66: 238–45. doi: 10.
1016/j.ejmech.2013.05.029 PMID: 23811086
´
´
43. Rodrıguez-Hernandez D, Barbosa LC, Demuner AJ, de Almeida RM, Fujiwara RT, Ferreira SR.
Highly potent anti-leishmanial derivatives of hederagenin, a triperpenoid from Sapindus saponaria
L. Eur J Med Chem. 2016 Nov 29; 124: 153–159. doi: 10.1016/j.ejmech.2016.08.030 PMID:
27569196
44. Gruza M, Jatczak K, Komor K, Świerk P, Szeja W, Grynkiewicz G. Synthesis of protoescigenin
glycoconjugates with O-28 triazole linker. Acta Pol Pharm. 2014 Nov-Dec; 71(6): 959–65. PMID:
25745768
45. Wei G, Luan W, Wang S, Cui S, Li F, Liu Y, Liu Y, Cheng M. A library of 1,2,3-triazole-substituted olea-
nolic acid derivatives as anticancer agents: design, synthesis, and biological evaluation. Org Biomol
Chem. 2015 Feb 7; 13(5):1507–14. doi: 10.1039/c4ob01605j PMID: 25476168
46. Antimonova A, Petrenko N, Shakirov M, Rybalova T, Frolova T, Shul’ts E, et al. Synthesis and study of
mutagenic properties of lupane triterpenoids containing 1,2,3-triazole fragments in the C-30 position.
Chem Nat Compd. 2013 Sep; 49(4): 657–64.
47. Shi W, Tang N, Yan WD. Synthesis and cytotoxicity of triterpenoids derived from betulin and betulinic
acid via click chemistry. J Asian Nat Prod Res. 2015; 17(2): 159–69. doi: 10.1080/10286020.2014.
979164 PMID: 25379995
48. Spivak AY, Gubaidullin RR, Galimshina ZR, Nedopekina DA, Odinokov VN. Effective synthesis of novel
C(2)-propargyl derivatives of betulinic and ursolic acids and their conjugation with β-d-glucopyranoside
azides via click chemistry. Tetrahedron. 2016 Mar 3; 72(9): 1249–56.
49. Parida PK, Sau A, Ghosh T, Jana K, Biswas K, Raha S, Misra AK. Synthesis and evaluation of triazole
linked glycosylated 18β-glycyrrhetinic acid derivatives as anticancer agents. Bioorg Med Chem Lett.
2014 Aug 15; 24(16): 3865–8. doi: 10.1016/j.bmcl.2014.06.054 PMID: 25027936
50. Waring MJ. Lipophilicity in drug discovery. Expert Opin Drug Discov. 2010 Mar; 5(3): 235–48. doi: 10.
1517/17460441003605098 PMID: 22823020
51. Dalvie DK, Kalgutkar AS, Khojasteh-Bakht SC, Obach RS, O’Donnell JP. Biotransformation reactions
of five-membered aromatic heterocyclic rings. Chem Res Toxicol. 2002 Mar; 15(3): 269–99. PMID:
11896674
52. Urban M, Sarek J, Tislerova I, Dzubak P, Hajduch M. Influence of esterification and modification of A-
ring in a group of lupane acids on their cytotoxicity. Bioorg Med Chem. 2005 Oct 1; 13(19): 5527–35.
doi: 10.1016/j.bmc.2005.07.011 PMID: 16087342
53. Kim DSHL, Pezzuto JM, Pisha E. Synthesis of betulinic acid derivatives with activity against human mel-
anoma. Bioorg Med Chem Lett. 1998 Jul 7; 8(13): 1707–12. PMID: 9873420
54. Qian K, Kim SY, Hung HY, Huang L, Chen CH, Lee KH. New betulinic acid derivatives as potent protea-
some inhibitors. Bioorg Med Chem Lett. 2011 Oct 1; 21(19): 5944–7. doi: 10.1016/j.bmcl.2011.07.072
PMID: 21856154
55. Uzenkova NV, Petrenko NI, Shakirov MM, Shul’ts EE, Tolstikov GA. Synthesis of 30-Amino Derivatives
of Lupane Triterpenoids. Chem Nat Compd. 2005; 41(6): 692–700.
56. Srinivasan T, Srivastava GK, Pathak A, Batra S, Raj K, Singh K, et al. Solid-phase synthesis and bioe-
valuation of Lupeol-based libraries as antimalarial agents. Bioorg Med Chem Lett. 2002 Oct 21; 12(20):
2803–6. PMID: 12270150
57. Soural M, Hodon J, Dickinson NJ, Sidova V, Gurska S, Dzubak P, et al. Preparation of Conjugates of
Cytotoxic Lupane Triterpenes with Biotin. Bioconjugate Chem. 2015 Dec 16; 26(12): 2563–70.
58. Noskova V, Dzubak P, Kuzmina G, Ludkova A, Stehlik D, Trojanec R, et al. In vitro chemoresistance
profile and expression/function of MDR associated proteins in resistant cell lines derived from CCRF-
CEM, K562, A549 and MDA MB 231 parental cells. Neoplasma. 2002; 49(6): 418–25. PMID:
12584592
59. Kim HK, Kong MY, Jeong MJ, Han DC, Choi JD, Kim HY, et al. Investigation of cell cycle arrest effects
of actinomycin D at G1 phase using proteomic methods in B104-1-1 cells. Int J Biochem Cell Biol. 2005
Sep; 37(9): 1921–9. doi: 10.1016/j.biocel.2005.04.015 PMID: 15964235
PLOS ONE | DOI:10.1371/journal.pone.0171621 February 3, 2017
24 / 25

Cytotoxic conjugates of betulinic acid and substituted triazoles
60. Patro JN, Urban M, Kuchta RD. Interaction of Human DNA Polymerase alpha and DNA Polymerase I
from Bacillus stearothermophilus with Hypoxanthine and 8-Oxoguanine NucleotidesBiochemistry 2009
Sep 1; 48(34): 8271–8. doi: 10.1021/bi900777s PMID: 19642651
ˇ
´
´
´
61. Bourderioux A, Naus P, Perlıkova P, Pohl R, Pichova I, Votruba I, et al. Synthesis and Significant Cyto-
static Activity of 7-Hetaryl-7-deazaadenosines. J Med Chem. 2011 Aug 11; 54(15): 5498–507. doi: 10.
1021/jm2005173 PMID: 21711054
PLOS ONE | DOI:10.1371/journal.pone.0171621 February 3, 2017
25 / 25