Lupane and 18α-oleanane derivatives substituted in the position 2, their cytotoxicity and influence on cancer cells

Article abstract of DOI:10.1016/j.ejmech.2016.05.029

Lupane derivatives containing an electronegative substituent in the position 2 of the skeleton are often cytotoxic, however, the most active compounds are not selective enough. To further study the influence of a substituent in the position 2 in lupane and 18α-oleanane derivatives on their biological properties, we prepared a set of 38 triterpenoid compounds, 19 of them new, most of them substituted in the position 2. From betulin, we obtained 2-bromo dihydrobetulonic acid and 2-bromo allobetulon and their substitutions yielded derivatives with various substituents in the position 2 such as amines, amides, thiols, and thioethers. Nitration of allobetulon and dihydrobetulonic acid gave 2-nitro and 2,2-dinitro derivatives. Fifteen derivatives had IC₅₀ < 50 μM on a chemosensitive CCRF-CEM (acute lymphoblastic leukemia) cell line and were tested on another seven cancer cell lines including resistant and two non-cancer lines. 2-Amino allobetulin had IC₅₀ 4.6 μM and caused significant block of the tumor cells in S and slightly in G2/M transition and caused strong inhibition of DNA and RNA synthesis at 5 × IC₅₀. 2-Amino allobetulin is the most active derivative of 18α-oleanane skeletal type prepared in our research group to date.

Full text of DOI:10.1016/j.ejmech.2016.05.029

Accepted Manuscript
Lupane and 18α-oleanane derivatives substituted in the position 2, their cytotoxicity
and influence on cancer cells
Lucie Borkova, Sona Gurska, Petr Dzubak, Renata Burianova, Marian Hajduch, Jan
Sarek, Igor Popa, Milan Urban
PII:
S0223-5234(16)30425-1
10.1016/j.ejmech.2016.05.029
EJMECH 8621
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 March 2016
Revised Date: 11 May 2016
Accepted Date: 13 May 2016
Please cite this article as: L. Borkova, S. Gurska, P. Dzubak, R. Burianova, M. Hajduch, J. Sarek, I.
Popa, M. Urban, Lupane and 18α-oleanane derivatives substituted in the position 2, their cytotoxicity
and influence on cancer cells, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.05.029.
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ACCEPTED MANUSCRIPT
1

ACCEPTED MANUSCRIPT
Lupane and 18α-oleanane derivatives substituted in the
position 2, their cytotoxicity and influence on cancer cells
1
2
2
2
2
Lucie Borkova, Sona Gurska, Petr Dzubak, Renata Burianova, Marian Hajduch, Jan
2
1,2
1,2
Sarek, Igor Popa, Milan Urban *
1
Department of Organic Chemistry, Faculty of Science, Palacky University Olomouc, 17.
listopadu 1192/12, 771 46 Olomouc, Czech Republic.
2
Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry,
Palacky University Olomouc, Hnevotinska 5, 779 00 Olomouc, Czech Republic.
*
Milan Urban is the corresponding author. Tel.: +420 585 632 197, Fax: +420 585 632 180;
E-mail: milan.urban@gmail.com
1

ACCEPTED MANUSCRIPT
Abstract
Lupane derivatives containing an electronegative substituent in the position 2 of the skeleton
are often cytotoxic, however, the most active compounds are not selective enough. To further
study the influence of a substituent in the position 2 in lupane and 18α-oleanane derivatives
on their biological properties, we prepared a set of 38 triterpenoid compounds, 19 of them
new, most of them substituted in the position 2. From betulin, we obtained 2-bromo
dihydrobetulonic acid and 2-bromo allobetulon and their substitutions yielded derivatives
with various substituents in the position 2 such as amines, amides, thiols, and thioethers.
Nitration of allobetulon and dihydrobetulonic acid gave 2-nitro and 2,2-dinitro derivatives.
Fifteen derivatives had IC < 50 µM on a chemosensitive CCRF-CEM (acute lymphoblastic
50
leukemia) cell line and were tested on another seven cancer cell lines including resistant and
two non-cancer lines. 2-Amino allobetulin had IC 4.6 µM and caused significant block of
50
the tumor cells in S and slightly in G2/M transition and caused strong inhibition of DNA and
RNA synthesis at 5 × IC . 2-Amino allobetulin is the most active derivative of 18α-oleanane
5
0
skeletal type prepared in our research group to date.
2

ACCEPTED MANUSCRIPT
Keywords
Betulinic acid, allobetulin, cytotoxicity, cell cycle, triterpene, activity
1
. Introduction
Triterpenes are natural compounds usually occurring in plants, marine organisms, fungi, and
bacteria. There are hundreds of new triterpenes being isolated from natural sources every
1
2
year. Pentacyclic triterpenes display a wide range of biological activities; they are often
3
4
5
6
7
8
9
cytotoxic, antiviral, antimicrobial, antifungal, antimalarial, anti-inflammatory, antiulcer,
1
0
11
hepatoprotective, and cardioprotective. A large number of triterpenes are cytotoxic against
various cancer cell lines and anti-tumor activity was also observed in preclinical animal
2
models. Cytotoxicity of the isolated natural terpenes often inspires researchers to prepare
1
2
quantities of semisynthetic derivatives, such as heterocyclic compounds, derivatives with
13-15
16-18
19
modified positions C-3,
C-20,
or C-28. Recently, a number of patents emerged on
betulinic acid derivatives dealing with low solubility in water while retaining selective
2
0
cytotoxicity against cancer cells. Recent review summarizes synthesis and biological
2
1
activity of 18α-oleanane (allobetulin) derivatives. Acute toxicity of triterpenes is usually
low, which is important for their potential use as therapeutics.
2
2-26
Despite that triterpenes are biologically active via various mechanisms of action,
the
parent compounds isolated from natural resources usually have two main drawbacks. Firstly,
triterpenes are not active at sufficiently low concentration. The value of IC reaches low
5
0
micromolar ranges at best, which usually cannot compete with the already available
therapeutics. Therefore, significant improvement of the activity of triterpenoid derivatives is
one of the major goals of many research groups. Inappropriate pharmacological properties are
the second drawback. Low solubility in water is one of the main reasons why compounds with
high in vitro activities often fail during in vivo screening and why their administration is
difficult.
The main interest in our group is to modify the structure of triterpenoid derivatives in order to
improve their pharmacological properties and to increase their activity and selectivity against
tumors. We had synthesized a number of lupane, oleanane, and other terpenoid derivatives
and in some of them, we found significant anticancer effects. In addition to that, we are
2
7
developing tools to identify molecular targets of active triterpenes. Very promising are
3

ACCEPTED MANUSCRIPT
2
8
29
30,31
32
derivatives with modified A-ring and E-ring, heterocyclic structures,
various esters
3
3
and fluoroderivatives. Based on this research, we made structure-activity relationship
assumptions and discovered trends how the chemical structure may affect the anti-tumor
activity. One of the significant trends shows that introducing an electronegative substituent to
the position 2 of lupane skeleton increases cytotoxicity significantly and this works especially
well for betulinic acid derivatives. The examples are 2,2-difluoroderivatives of
2
8
34,35
dihydrobetulinic acid and diosphenols.
difluoroderivatives, selectivity was compromised.
However, among the most active 2,2-
In this work, we choose to introduce more substituent types to the position 2 of
dihydrobetulonic acid (2b) and allobetulon (3b) via nucleophilic substitution of 2-bromo
derivatives or by selective nitration of the 2-position in 3-oxocompounds. New derivatives
were designed to contain heteroatoms such as nitrogen, sulfur, and oxygen. Resulting
compounds were further reduced to get a larger set of more variable derivatives in order to
make more accurate assumption about the influence of each substituent at C-2 on cytotoxic
activity. Hemisuccinates were prepared from amines because they are soluble in water based
γ-cyclodextrine formulation and this would be useful in the intended future in vivo
experiments.
2
. Results and discussion
2
.1. Chemistry
From betulin (1), we prepared betulonic acid (2a), dihydrobetulonic acid (2b), and a mixture
3
1,36
of diastereoisomers of 2-bromo dihydrobetulonic acid (2c).
From betulin (1), we also
prepared allobetulin (3a), allobetulon (3b), and a diastereomeric mixture of 2-bromo
3
7-41
allobetulones 3c by known procedures
(Scheme 1).
4

ACCEPTED MANUSCRIPT
Reaction of bromo ketone 2c with sodium azide was performed in N-methylpyrrolidone
(NMP) in a presence of a small amount of acetic acid according to a precedent from steroid
4
2
chemistry. An attempt to purify crude azido acid 4 on a silica gel column failed due to fast
decomposition which yielded yellow enaminoketone 5 that spontaneously dimerizes to imine
4
3
6
the same way as it was described for analogous betulinic acid derivative. It is worth to
mention, that in general, compounds with an electronegative substituent at C-2 often occur in
4
4
their enolforms. Unlike azide 4, both enaminoketone 5 and dimer 6 were stable enough that
we were able to isolate and characterize them. Moreover compounds 5 and 6 were prepared
directly and faster when a reaction of bromo ketone 2c with sodium azide was stirred in a
solution of DMSO containing a drop of H SO at 70 °C. All attempts to reduce azide 4 to
2
4
aminoketone, azido alcohol or aminoalcohol led to compound 5 and 6 (Scheme 2).
Reaction of bromo ketone 3c with sodium azide in NMP in presence of a small amount of
acetic acid yielded azido ketone 7. Compound 7 was more stable than its analogue 4. We were
able to obtain sufficient amount of pure 7 by HPLC to get the physical and spectral data and
also to perform basic cytotoxicity assay on a reference CCRF-CEM cell line. It was observed,
that the compound remained stable at -18 ºC for at least 6 months and that slow
decomposition was occurring at room temperature which was fastened by heating, especially
in oxygen containing solvents such as EtOAc, THF, EtOH (especially during attempts to
crystallize the compound) giving enaminoketone 8 in all cases. Attempts to reduce the azido
ketone 7 with Ph P also gave enaminoketone 8. Heating 8 in refluxing xylene gave dimer 9
3
4
3
similarly to lit. In order to obtain enaminoketone 8 directly and faster, a reaction of bromo
ketone 3c with sodium azide was performed in DMSO and H SO as mentioned earlier.
2
4
5

ACCEPTED MANUSCRIPT
Higher stability of 7 allowed its reduction with sodium borohydride that gave a mixture of
diastereomers of azido alcohols 10a and 10b that were separable by HPLC. Reduction of
azide 7 with LAH afforded a diastereomeric mixture of aminoalcohols 11. It was impossible
to separate them directly (due to very low solubility) but a reaction of this mixture with acetic
anhydride gave less polar diacetates 12a (2α-acetamidoallobetulin 3β-acetate) and 12b (2β-
acetamidoallobetulin 3β-acetate) which were separable by HPLC and whose structures were
unambiguously determined by NMR. Similarly, reaction of aminoalcohols 11 with succinic
anhydride gave hemisuccinates 13a (2α; 3β isomer) and 13b, (2β; 3β isomer). Because their
spectral data was difficult to interpret (very wide signals caused by low solubility),
methylesters 14a and 14b were prepared and the full characterization was performed on them
(Scheme 2).
A reaction of bromo ketone 2c with sodium sulfide gave compound 15 which was unstable
while chromatographed but when transformed to its methylester before HPLC
chromatography, we were able to obtain and characterize it as methyl ester 16. Both
6

ACCEPTED MANUSCRIPT
derivatives 15 and 16 succumb to slow decomposition while being purified on silica gel
column or using HPLC. In order to obtain 18α-oleanane analogue of compound 15, a number
of reactions of bromo ketone 3c with variety of reaction conditions were investigated. The
most promising reaction gave a mixture of three compounds that was supposed to contain the
45
desired thiol derivative; however, the only isolable product was known compound 17, the
rest was repeatedly lost during the chromatography, probably due to decomposition
(Scheme 3).
Nucleophilic substitution of bromo ketone 3c with mercaptoethanol gave two compounds: 2-
hydroxyethylsulfanylderivative 18 and a heterocycle 19, formed by dehydratation of 18.
Structure of 19 was confirmed by full assignment of NMR signals using multiple 2D
techniques; diosphenol 20a was a byproduct of this reaction. Since 2´,3´-dihydro-1´,4´-
oxathiine derivative 19 is a very interesting heterocycle, we attempted to obtain its lupane
analogue from bromo ketone 2c; however, the only isolated product in all experiments was
diosphenol 20b, which is similar result as for many attempts to substitute bromine atom in 2-
3
1
bromo-3-oxoterpenes described in the lit. Both diosphenols 20a and 20b are known
4
6,47
compounds.
It is worth to point out, that it has been observed several times that a reaction
that works well at the A-ring of lupane analogues does not work in 18α-oleanane derivatives
and vice versa. We often see that a chemical modification that occurs on one side of the
terpenoid skeleton may interfere with a reactivity on its other side.
7

ACCEPTED MANUSCRIPT
HS
a
b
CO Me
2
CO H
2
HS
O
Br
O
O
15
2c
16
d or e
O
S
HO
O
HO
d
e
Br
O
HO
18
20b
3c
c
-H O
2
S
HO
+
O
O
O
19
17
20a
Scheme 3. Reagents and conditions: (a) Na S, N-methylpyrolidone, r.t.; (b) CH N Et O, CHCl r.t.;
2
2
2,
2
3,
(
c) Na S, DMSO, a drop of water, 75 °C; (d) mercaptoethanol (used as cosolvent), NaOH, EtOH
2
(
anh.), 0 °C to r.t.; (e) mercaptoethanol (1 eq.), NaOH, EtOH (anh.), 0 °C to r.t.
Nitroderivatives 21 - 24 were obtained in rather low yields by using nitration conditions
48,49
analogous to lit.
to nitrate dihydrobetulonic acid (2b) and allobetulin (3a).
Dihydrobetulonic acid 2b gave mononitroderivative 21 after 24 h treatment with nitration
mixture (Scheme 4) at 25 ºC while dinitroderivative 23 formed in 6 h using the same nitration
mixture when temperature was elevated to 35 ºC. Allobetulin (3a) gave a mixture of three
compounds 22, 24, and 25 while the same enolacetate 25 became the only isolated product
when the reaction time was extended. Once we had the nitro derivative 21, it was reduced by
zinc in acetic acid to give aminoketone 26 that was directly acetylated to get acetate 27. It was
necessary to acetylate compound 26 because the unprotected product was extremely difficult
to isolate during the work-up procedure due to its low solubility in organic solvents and its co-
precipitation with inorganic zinc salts, which together diminished its yield. In contrast,
extraction of acetates into organic phase proceeded always well. We found that it is also
possible to prepare acetylated enaminoketone 28 when heating 26 in toluene and acetic acid
under reflux without inert atmosphere (Scheme 4).
8

ACCEPTED MANUSCRIPT
2
2
.2. Biological assays
.2.1 Cytotoxicity
Cytotoxic activity of all synthesized compounds and stable intermediates was investigated in
vitro against human acute T lymphoblastic leukemia cell line CCRF-CEM using the standard
MTS test (Table 1). New derivatives with IC below 50 µM were further examined on seven
5
0
different cancer cell lines derived from leukemia (K562) and multiresistant counterparts
CEM-DNR, K562-TAX), solid tumors including lung (A549) and colon (HCT116,
(
HCT116p53-/-) carcinomas, osteosarcoma cell line (U2OS), and for comparison, on two
human non-cancer fibroblast lines (BJ, MRC-5) (Table 2). From this set of new derivatives,
containing a substituent at the position C-2 of triterpenic skeleton, only one compound had
activity lower than 10 µM; aminoalcohol 11 with IC50 4.6 µM. Other derivatives with
cytotoxicity in low micromolar range prepared within this paper (2b, 2c, 20b) are already
2
1
known from and therefore they were not further examined. Aminoalcohol 11 is up to date
the most active derivative with 18α-oleanane skeleton found by our research group, but its
cytotoxic activity is not limited to the tumor cells.
9

ACCEPTED MANUSCRIPT
2
.2.2. Cell cycle analysis
As a part of the study into the mechanism of action of compound 11 and to further
characterize the anti-tumor properties, we analysed its influence on the cell cycle regulation in
highly sensitive CCRF-CEM cells (Table 3). After the 24 hour treatment with 1 × and 5 ×
IC50 concentration, the apoptotic, sub-G1 population was just slightly increased (2.79% /
7
.1%). There was visible interference with the cell cycle regulation at 5 × IC , showing
50
accumulation of the cells in S and slightly in G2/M transition. Phosphorylation of the
Histone-3 at Ser10 was not increased in the studied cell population which is indicating that
cells were accumulating in the G2 phase and not entering the M phase of the cell cycle. It is in
the concordance with the observation that DNA / RNA synthesis was inhibited significantly.
Based on the collected data it seems that some of studied triterpenoid derivatives are showing
interesting cytotoxic activity. Unfortunately, selectivity towards cancer cell lines is low and
not sufficient to start in vivo anticancer tests.
Table 1. Cytotoxic activity of compounds 1 – 28 against the acute T lymphoblastic leukemia
CCRF-CEM cell line.
a
a
a
a
IC50 (ꢀmol/L )
IC50 (ꢀmol/L )
IC50 (ꢀmol/L )
IC50 (ꢀmol/L )
Comp.
Comp.
Comp.
Comp.
CCRF-CEM
CCRF-CEM
CCRF-CEM
>50
CCRF-CEM
b
1
>50
14
5
27
27
12a
12b
13a
13b
14a
14b
16
20a
20b
21
13
6
b
2
2
2
3
3
3
a
b
c
6
>50
4
7
8
>50
>50
>50
>50
>50
5
>50
32
2
>50
22
>50
29
a
b
c
>50
>50
>50
17
9
>50
23
10a
10b
11
>50
24
>50
32
45
27
4
19
>50
28
30
a
The lowest concentration that kills 50% of cells. The standard deviation in cytotoxicity assays is typically up to
b
1
5 % of the average value. Compounds with IC50 > 50 µM are considered inactive. Compounds were tested as
mixture.
Table 2.
Cytotoxic activities of selected compounds on eight tumor (including resistant) and two
normal fibroblast cell lines.
1
0

ACCEPTED MANUSCRIPT
a
IC50 (µM/L)
Comp.
HCT116
CCRF- CEM-
K562-
TAX
c
K562
A549 HCT116
U2OS
BJ
MRC-5
TI
-
/-
CEM
DNR
p53
4
17.5
16.0
33.9
33.9
5.9
14.2
>50.0
>50.0
4.5
10.6
20.1
20.1
3.9
25.7
>50.0
>50.0
6.2
>50.0
>50.0
>50.0
4.9
>50.0
>50.0
>50.0
4.8
48.9
>50.0
>50.0
6.2
>50.0
>50.0
>50.0
7.1
>50.0
>50.0
>50.0
6.3
>2.9
>1.9
>1.9
1.4
b
2
2
6.6
6.6
5
b
6
1
1
1
6
4.6
45.3
13.1
31.8
28.6
32.2
29.9
>50.0
>50.0
36.1
46.6
>50.0
35.3
>50.0
16.9
>50.0
45.3
20.6
29.9
>50.0
30.5
>50.0
>50.0
31.3
>50.0
41.6
>50.0
33.4
>50.0
45.8
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
25.4
>1.1
>2.9
>1.6
>1.7
>1.1
>1.7
2
0a
2
1
3
7
8
10.1
>50.0
>50.0
42.9
>50.0
>50.0
38.7
>50.0
>50.0
>50.0
>50.0
>50.0
>50.0
21.6
2
>50.0
32.4
>50.0
>50.0
>50.0
2
2
37.9
>50.0
>50.0
>50.0
a
The lowest concentration that kills 50% of cells. The standard deviation in cytotoxicity assays is typically up to
b
1
5 % of the average value. Compounds were tested as mixture. Compounds with IC > 50 µM are considered
50
c
inactive. Therapeutic index is calculated for IC50 of CCRF-CEM line vs average of both fibroblasts.
Table 3.
a
b
Influence of compound 11 on cell cycle, DNA and RNA synthesis at 1 × and 5 × IC .
5
0
Used
conc.
DNA RNA
synthesis synthesis
Ser10
sub G1 G0/G1
(%)
S
(%)
G2/M pH3
(%)
(
%)
(%)
(µM)
(%)
(%)
Control
-
1.80
2.79
7.1
36.41
40.73
28.90
50.33
47.77
54.92
13.28
11.50
16.18
1.66
56.37
53.53
1.88
43.37
42.98
2.23
a
1
1
1
1
4.60
1.44
0.49
b
23.00
3
. Conclusion
We prepared a set of 38 triterpenoid compounds (19 of them new) and studied the influence
of the substituent in the position C-2 on their biological properties. Higher electronegativity of
the substituents resulted in compounds with higher IC50 which is in agreement with our
previous hypothesis. Aminoallobetulin 11 is the most active compound among 18α-olenanane
derivatives. However, in most of the new derivatives, the cytotoxicity was not limited to
cancer cell lines exclusively and therefore their use as therapeutics is unlikely. A lot of
difficulties had to be overcome during the synthesis. First of all, most of the reactions
produced mixtures of epimers. In few cases, it was possible to separate the products or their
derivatives by HPLC, however, in some cases it was impossible and therefore the biological
testing had to be done with epimeric mixtures. Products with hydrophilic substituents at C-2
(e. g. amines) are strongly amphiphilic and they often do not dissolve well in solvents used for
1
1

ACCEPTED MANUSCRIPT
NMR and therefore their characterization had to be performed after acetylation or methylation
of carboxylic groups or amines. Despite all difficulties with synthesis and small chance to find
a new therapeutic agent among the presented set of triterpenes, these compounds gave us
important data points for our structure-activity relationships evaluations and based on those
data, structures of better anti-cancer inhibitors may be proposed in the future research.
Aminoallobetulin 11 is most cytotoxic compound of this study and it has influence on cell
cycle (cells accumulate in S and slightly in G2/M transition) and causes strong inhibition of
DNA and RNA synthesis at 5 × IC .
5
0
4
. Experimental part
4
4
.1. General experimental procedures
.1.1. Materials and instruments
Melting points were determined using a Büchi B-545 apparatus and are uncorrected. Optical
rotations were measured on an Autopol III (Rudolph Research, Flanders, USA) polarimeter in
-1
2
-1
1
13
MeOH at 25 °C unless otherwise stated and are in [10 deg cm g ]. H and C NMR spectra
UNITY
1
UNITY
were recorded on Varian
Inova 400 (400 MHz for H) or Varian
Inova 300 (300
1
1
MHz for H) or Jeol ECX-500SS (500 MHz for H) instruments, using CDCl , D -DMSO or
3
6
CD OD as solvents (25°C). Chemical shifts were eider referenced to the residual signal of the
3
1
3
solvent (CDCl , D -DMSO) or to tetramethylsilane added as an internal standard. C NMR
3
6
spectra were eider referenced to CDCl (77.00 ppm) or D -DMSO (39.51 ppm) or to
3
6
tetramethylsilane added as an internal standard. EI MS spectra were recorded on an INCOS
0 (Finigan MAT) spectrometer at 70 eV and an ion source temperature of 150 °C. The
5
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 an Orbitrap Elite high-resolution mass spectrometer (Thermo
Fischer Scientific, MA, USA) operating at positive full scan mode (120 000 FWMH) in the
range of 200–900 m/z. The settings for electrospray ionization were as follows: oven
temperature of 300 °C, sheath gas of 8 arb. units and source voltage of 1.5 kV. The acquired
data were internally calibrated with diisooctyl phthalate as a contaminant in methanol (m/z
3
91.2843). Samples were diluted to a final concentration of 20 ꢀmol/L with 0.1% formic acid
in water and methanol (50:50, v/v). The samples were injected by direct infusion into the
mass spectrometer. 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
1
2

ACCEPTED MANUSCRIPT
plates (Merck) detected by spraying with 10% aqueous H SO and heating to 150 - 200 °C.
2
4
Starting triterpenes – 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.
4
4
2
.2. Synthetic procedures
.2.1. Starting material
-Bromo dihydrobetulonic acid (2c) was obtained from dihydrobetulonic acid (2b) using a
procedure which we described earlier, all spectral and physical data was in agreement with
3
1,36
published data.
2
-Bromo allobetulone 3c was obtained from allobetulone (3b) using a procedure that we
3
7-41
described earlier, all spectral and physical data was in agreement with published data.
4
.2.2. Reaction of acid 2c with sodium azide; 2-azido-4-oxolupan-28-oic acid (4)
Sodium azide (219 mg; 3.37 mmol) was added to a solution of bromo derivative 2c (300 mg;
.56 mmol) in N-methylpyrrolidone (NMP, 15 mL). After 15 minutes, bromo derivative 2c
0
was converted completely to azide 4 according to TLC (toluene/Et O 5:1 with a drop of
2
AcOH). Azide 4 was precipitated by pouring the reaction mixture to tenfold volume of water,
filtered off, dried under vacuum and lyophilized from tert-butanol to give 183 mg (66 %), mp.
was not possible to measure due to decomposition of 4 to enaminoketon 5 before melting. All
physical and spectral data were obtained from crude mixture of 2α and 2β diastereoisomers
-1 1
(
1:1): [α] +27.8 (c 0.36). IR (DRIFT): 2500 – 3500, 2100, 1719, 1656 cm . H NMR (400
D
MHz, CDCl ): δ = 0.75, 0.76, 0.77, 0.86, 0.87, 0.92, 0.94, 0.98, 1.00, 1.08, 1.09, 1.13, 1.14,
3
1
2
6
.15 (42H, all s, 14 × CH from both diastereoisomers), 2.18 (t, 1H, H-1α from 2β isomer),
3
.22 – 2.28 (m, 6H, H-13, H-18, H-19 all from both isomers), 2.32 (dd, 1H, J = 12.6 Hz, J =
1
2
.0 Hz, H-1β from 2α isomer), 4.25 (dd, 1H, J = 13.3 Hz, J = 6.0 Hz, H-2β from 2α
1
2
1
3
isomer), 4.29 (dd, 1H, J = 11.2 Hz, J = 8.7 Hz, H-2α from 2β isomer). C NMR (100 MHz,
1
2
CDCl ): δ = 14.50, 14.63, 14.66, 15.35, 16.16, 16.31, 17.84, 18.36, 19.07, 19.46, 19.90,
3
2
3
4
5
1.14, 21.37, 22.03, 22.72, 22.94, 24.92, 26.60, 26.86, 29.25, 29.57, 29.62, 29.70, 29.73,
0.63, 31.90, 31.98, 32.84, 33.98, 37.02, 37.36, 37.84, 38.12, 38.44, 39.98, 40.56, 40.80,
2.62, 42.74, 44.07, 44.13, 46.56, 46.61, 47.66, 48.59, 48.63, 48.74, 49.48, 49.61, 49.82,
+
2.03, 56.72, 56.79, 56.95, 59.89, 60.81, 181.80, 181.82, 210.62, 213.43. MS (ESI ): m/z (%)
1
3

ACCEPTED MANUSCRIPT
+
+
+
-
=
498 (100, [M+H] ), 520 (75, [M+Na] ), 540 (40, [M+K] ). MS (ESI ): m/z (%) = 496 (100,
-
+
[
M-H] ). HRMS (ESI-TOF) m/z calcd for C H N O [M+H] 498.3696, found 498.3692.
30 47 3 3
4
.2.3. 2-amino-3-oxolup-1-en-28-oic acid (5) and its dimer 6
Bromo derivative 2c (500 mg; 0.9 mmol) was dissolved in DMSO (15 mL) and sodium azide
486 mg; 7.5 mmol) and one drop of sulfuric acid were added. The reaction mixture was
(
stirred at 70 °C, monitored on TLC in toluene/Et O 5:1. After 4 hours, the reaction mixture
2
was poured to double volume of water and extracted with chloroform. The organic phase was
collected, washed with water and the solvents were removed in vacuo. The crude
enaminoketone 5 was purified by column chromatography on a silica gel in CHCl /EtOAc
3
1
0:1 and was spontaneously decomposing. Fractions containing mixture of compounds 5 and
6
were collected and evaporated to give 134 mg (30 %); mp. 242 – 243 °C (CHCl /EtOAc);
3
-1 1
[
α] -41° (c 0.20). IR (DRIFT): 3337, 2400 – 3400, 1697, 1675, 1613 cm . H NMR (400
D
MHz, CDCl ): δ = 0.78 (d, 9H, J = 6.3 Hz, 3 × CH ), 0.93 (d, 9H, J = 6.3 Hz, 3 × CH ), 0.96
3
3
3
(
s, 9H, 3 × CH ), 1.05 (s, 9H, 3 × CH ), 1.13 (s, 9H, 3 × CH ), 1.19 (s, 9H, 3 × CH ), 1.22 (s,
3 3 3 3
9
H, 3 × CH ), 2.27 – 2.34 (m, 9H, H-13, H-18, H-19 all from both monomer and dimer), 6.14
3
13
(
s, 1H, H-1 monomer), 6.55 (s, 3H, H-1, H-1', NH dimer), 11.42 (bs, 2H, NH ). C NMR
2
(
100 MHz, CDCl ): δ = 14.60, 14.72, 16.56, 18.97, 21.12, 21.31, 21.91, 22.77, 23.07, 26.90,
3
2
4
7.72, 29.69, 29.77, 31.96, 34.10, 37.63, 38.17, 38.53, 41.64, 42.97, 44.04, 44.41, 45.74,
+
8.49, 53.14, 56.96, 132.28, 132.81, 183.45, 200.80. MS (ESI ): m/z (%) = 471 (100,
+
-
-
-
[
M+H] ). MS (ESI ): m/z (%) = 469 (100, [M-H] ), 423 (70, [M-COOH] ). HRMS (ESI-TOF)
+
m/z calcd for C H NO [M+H] 470.3629, found 470.3628.
3
0
47
3
4
.2.4. Reaction of 2-bromo allobetulone 3c with sodium azide; 2-azido allobetulon 7
Bromo derivative 3c (100 mg; 0.193 mmol) was dissolved in NMP (3 mL) with a drop of
acetic acid and then sodium azide (75 mg; 1.154 mmol) was added. The reaction mixture was
stirred at r.t. 30 min, while monitored on TLC in hexane/EtOAc 5:1. The reaction mixture was
poured to tenfold volume of water and extracted to an organic solvent. The organic phase was
collected, washed with water and the solvents were removed in vacuo. A pale yellow honey
like product was crystallized from cyclohexane and dried under the flow of N to give white
2
1
crystals of azide 7 (24 mg; 26 %), mp. 152 – 154 °C (cyklohexane). H NMR was identical to
4
3
the spectrum in lit.
4
.2.5. 2-amino-18α-olean-1-en-3-one (8)
1
4

ACCEPTED MANUSCRIPT
Sodium azide (5.1 g; 78 mmol) and 20 drops of sulfuric acid were added to the solution of 2-
bromallobetulone (3c) (5.6 g; 10.8 mmol) in DMSO (150 mL). The reaction mixture was
heated 4 hours at 70 °C under continuous stirring. The reaction mixture was poured to double
volume of water, extracted to CHCl , washed with water and solvents were evaporated. The
3
crude product was chromatographed on silica gel (300 g) in toluene/Et O 5:1 and crystallized
2
from EtOAc to give enaminoketone 8 (2.5 g; 50 %). 700 mg of enaminoketone 8 was purified
by HPLC in hexane/EtOAc 4:1 and crystallized from EtOAc to give pure compound 8 (500
1
mg; 71 %), mp. 223 – 226 °C (ethylacetate), [α] +38° (c 0.35). H NMR was identical to the
D
43
spectrum in lit.
4
.2.6. Dimer 9
Azido allobetulone 7 (1.1 g; 2.3 mmol) was added to triphenylphosphine (2.4 g; 9.2 mmol) in
anhydrous THF (22 mL) under argon atmosphere. The reaction mixture was stirred for 6
hours at r.t. Initially, formation of nitrogen in the mixture was observed as small bubbles and
originally colorless solution became yellowish. Then water (1.1 mL) was added to the
solution. The reaction mixture was stirred under argon atmosphere for 45 hours while the
solution darkened. At this time, azido allobetulone 7 was completely consumed according to
TLC (toluene/Et O 20:1). The reaction mixture was processed by evaporation of solvents
2
under vacuo; residues of water were removed by 5 × azeotropic distillation with toluene under
vacuo. After toluene evaporation the mixture was heated with xylene under reflux for 30
hours. Xylene was evaporated under vacuo, dry residue was dissolved with p-toluenesulfonic
acid (40 mg) in EtOH (55 mL) and the solution was vigorously stirred 3 days in presence of
air. The resulting precipitate was filtered over on a pad of cellite, washed with EtOH and
eluted from the column by chloroform. Chloroform was evaporated under vacuo and crude
product was purified by HPLC (hexane/EtOAc 20:3). After the crystallization from the
mixture EtOAc/MeOH compound 9 obtained as yellow crystals (200 mg; 17 %), mp. 345 –
2
9
29
1
3
48 °C (Lit. > 300 °C); [α] +60° (c 0.31) (Lit. +47° (c 0.1, CHCl )). H NMR was
D 3
2
9
identical to the spectrum in lit.
4
.2.7. Reduction of azido allobetulon 7 with sodium borohydride
Azido ketone 7 (500 mg; 1.0 mmol) was dissolved in EtOH (1 mL) and the solution was
cooled to 0 °C. NaBH (0.5 g; 13.5 mmol) was added to the mixture and the suspension was
4
stirred for 2 hours while temperature was allowed to reach r.t. The reaction mixture was
poured to double volume of diluted hydrochloric acid (1:4) and extracted with an organic
1
5

ACCEPTED MANUSCRIPT
solvent. The organic phase was washed with water and 5% solution of NaHCO . Solvents
3
were removed on vacuo. The crude product was purified by column chromatography on silica
gel (100 g) in CHCl . Pure azido alcohols 10a and 10b were obtained. 2α-azido alcohol 10a
3
(
0.14 g; 28 %), mp. 216 - 219 °C (MeOH); [α] +9° (c 0.29). IR (DRIFT): 3605, 2103, 1454
D
-1 1
cm . H NMR (400 MHz, CDCl ) δ: 0.80, 0.82, 0.91, 0.93, 0.94, 0.98, 1.03 (21H, all s, 7 ×
3
CH ), 1.67 (1H, dm, J(H-1α, H-1β) = 12.5 Hz, H-1α), 2.09 (1H, dd, J(H-1β, H-1α) = 12.5 Hz,
3
J(H-1β, H-2β) = 4.4 Hz, H-1β), 3.03 (1H, dd, J(H-3α, H-2β = 10.7 Hz, J(H-3α, H-OH) = 3.1
Hz, H-3α), 3.44 (1H, d, J = 7.8 Hz, H-28a), 3.50 (1H, dd, J(H-2β, H-3α) = 10.7 Hz, J(H-2β,
1
3
H-1β) = 4.4 Hz, H-2β), 3.53 (1H, s, H-19), 3.77 (1H, d, J = 7.8 Hz, H-28b). C NMR (100
MHz, CDCl ) δ: 13.44, 15.67, 16.33, 17.37, 18.12, 21.07, 24.50, 26.19, 26.24, 26.34, 28.33,
3
2
5
8.75, 32.65, 33.66, 34.04, 36.22, 36.68, 38.19, 39.34, 40.49, 40.65, 41.42, 43.91, 46.75,
+
0.92, 55.23, 61.53, 71.19, 81.30, 87.88. MS-EI: m/z (%) [for C H N O : M 483]: 483
3
0
49
3
2
+
(
M , 14), 455 (31), 441 (100), 424 (5), 412 (86), 382 (31), 370 (12). HRMS (ESI-TOF) m/z
+
calcd for C H N O [M+H] 484.3903, found 484.3908.
3
0
49
3
2
2
β-azido alcohol 10b (0.12 g; 24 %), mp. 260 - 262 °C (MeOH); [α] +50° (c 0.31). IR
D
-1 1
(
DRIFT): 3671, 2112, 1454 cm . H NMR (400 MHz, CDCl ) δ: 0.80, 0.85, 0.91, 0.93, 0.94,
3
1
.00, 1.12 (21H, all s, 7 × CH ), 1.67 (1H, dm, J(H-1α, H-1β) = 13.6 Hz, H-1α), 2.23 (1H, dd,
3
J(H-1β, H-1α) = 14.7 Hz, J(H-1β, H-2α) = 2.9 Hz, H-1β), 3.27 (1H, dd, J(H-3α, H-OH) = 9.8
Hz, J(H-3α, H-2α) = 4.7 Hz, H-3α), 3.44 (1H, d, J = 7.8 Hz, H-28a), 3.53 (1H, s, H-19), 3.78
(
1H, dd, J = 7.9 Hz, J = 1.8 Hz, H-28b), 4.04 (1H, ddd, J(H-2α, H-1α) = 4.7 Hz, J(H-2α, H-
1 2
1
3
3
1
3
7
4
4
α) = 4.7 Hz, J(H-2α, H-1β) = 2.9 Hz, H-2α). C NMR (100 MHz, CDCl ) δ: 13.42, 15.77,
3
6.35, 16.68, 18.05, 21.19, 24.53, 26.22, 26.27, 26.37, 28.75, 29.47, 32.70, 33.72, 34.04,
6.26, 36.73, 36.94, 38.54, 40.70, 40.87, 41.46, 41.76, 46.79, 50.71, 55.31, 62.65, 71.23,
+
+
7.58, 87.89. MS-EI: m/z (%) [for C H N O : M 483]: 483 (M , 20), 455 (24), 440 (100),
3
0
49
3
2
+
24 (35), 412 (67), 382 (57), 369 (28). HRMS (ESI-TOF) m/z calcd for C H N O [M+H]
3
0
49
3
2
84.3903, found 484.3902.
4
.2.8. Reduction of azido allobetulon 7 with LAH and further modifications of resulting
aminoalcohol 11
LAH (10 g; 0.3 mmol) was added to the solution of azido ketone 7 (10 g; 20.8 mmol) in THF
(250 mL) and the reaction mixture was headed under reflux for 2 hours. After cooling to r.t.,
1
6

ACCEPTED MANUSCRIPT
unreacted LAH was slowly decomposed by gradually added EtOAc (10 mL), EtOH (10 mL)
and 10% HCl (15 mL). The organic phase was filtered off inorganic salts and THF was
removed in vacuo. Crude aminoalcohol 11 was purified in Soxhlet extractor (Et O to remove
2
impurities) and by purification over silica gel (50 g) in CHCl /isopropanol 1:1. Derivative 11
3
was obtained (5.8 g; 61 %, a mixture of two enantiomers based on NMR analysis 85:15;
major one is 2α-amino-18α-oleanane-3α-ol), mp. 280 - 283 °C (non-crystallized); [α] +65°
D
-
1 1
(
c 0.16 in CHCl with 5 % MeOH). IR (nujol): 3648 cm . H NMR (400 MHz, CDCl ) δ:
3
3
0
.83, 0.92, 0.96, 0.98, 1.04, 1.22, (21H, all s, 7 × CH ), 3.46 (1H, ddd, J(H-2β, H-1α) = 10.5
3
Hz, J(H-2, H-1β) = 7.9 Hz, J(H-2, H-3β) = 2.4 Hz, H-2), 3.49 (1H, d, J = 7.8 Hz, H-28a), 3.53
1
3
(
1H, d, J(H-3, H-2) = 2.4 Hz, H-3), 3.56 (1H, s, H-19), 3.79 (1H, bd, J = 7.6 Hz, H-28b). C
NMR (100 MHz, CDCl ) δ: 13.82, 16.19, 19.46, 20.23, 20.68, 23.15, 24.83, 27.09, 27.51,
3
2
4
4
7.70, 29.24, 29.24, 33.75, 34.32, 35.89, 37.20, 37.57, 38.62, 38.71, 41.27, 42.01, 42.20,
+
2.69, 49.54, 51.94, 53.00, 72.21, 75.72, 80.00, 89.55. MS-EI: m/z (%) [for C H NO : M
3
0
51
2
+
57]: 457 (M , 100), 442 (15), 424 (19), 384 (35), 236 (11), 203 (10), 189 (14), 149 (67).
+
HRMS (ESI-TOF) m/z calcd for C H NO [M+H] 458.3998, found 458.3991.
3
0
51
2
Diacetates 12a and 12b. Crude aminoalcohol 11 (400 mg; 0.88 mmol) was dissolved in the
mixture of pyridine (10 mL) and Ac O (3 mL; 30 mmol) and the solution was stirred 4 days at
2
r.t. The reaction mixture was poured to double volume of diluted hydrochloric acid (1:4) and
extracted with an organic solvent. The organic phase was washed with water and 5% solution
of NaHCO . Solvents were removed on vacuo. Crude product was chromatographed on
3
HPLC in hexane/EtOAc 1:3. Fractions containing 12a and 12b were crystallized from MeOH
to give acetates 12a and 12b. 2α-acetamido-18α-oleanane-3β-yl acetate 12a (274 mg; 58 %),
mp. 208 – 211 °C (MeOH); [α] +10° (c 0.24). IR (DRIFT): 3428, 3375, 1722, 1668, 1520
D
-1 1
cm . H NMR (400 MHz, CDCl ) δ: 0.80, 0.88, 0.91, 0.92, 0.93, 0.98, 1.00 (21H, all s, 7 ×
3
CH ), 1.90 (3H, s, 2-NHAc), 2.08 (3H, s, 3-OAc), 3.44 (1H, d, J = 7.8 Hz, H-28a), 3.52 (1H,
3
s, H-19), 3.76 (1H, dd, J = 7.8 Hz, J = 1.4 H-28b), 4.25 (1H, m, H-2), 4.45 (1H, d, J(H-3, H-
1
2
1
3
2
1
3
7
) = 11.0 Hz, H-3), 5.58 (1H, d, J = 8.6 Hz, NH). C NMR (100 MHz, CDCl ) δ: 13.44,
3
5.64, 17.15, 17.20, 18.20, 21.10, 24.51, 26.21, 26.28, 26.37, 28.30, 28.76, 32.67, 33.69,
4.05, 36.23, 36.71, 38.02, 38.86, 40.64, 40.71, 41.43, 46.45, 46.77, 46.77, 50.97, 55.53,
+
1.20, 82.19, 87.85. MS-EI: m/z (%) [for C H NO : M 541]: 541 (M+, 89), 511 (6), 481
3
4
55
4
(40), 470 (23), 449 (8), 440 (31), 422 (38), 187 (62), 139 (100). HRMS (ESI-TOF) m/z calcd
+
for C H NO [M+H] 542.4209, found 542.4210.
3
4
55
4
1
7

ACCEPTED MANUSCRIPT
2β-acetamido-18α-oleanane-3β-yl acetate 12b (85 mg; 18 %), mp. 269 – 271 °C (MeOH);
-1 1
[
α] +51° (c 0.24). IR (DRIFT): 3446, 1737, 1668, 1510 cm H NMR (400 MHz, CDCl ) δ:
D . 3
0
.80, 0.86, 0.92, 0.93, 0.98, 1.04, 1.10 (21H, all s, 7 × CH ), 1.96 (3H, s, 2-NHAc), 2.11 (3H,
3
s, 3-OAc), 3.44 (1H, d, J = 7.8 Hz, H-28a), 3.52 (1H, s, H-19), 3.79 (1H, bd, J = 7.8 Hz, H-
1
3
2
8b), 4.32 (1H, bd, J = 6.6 Hz, H-2), 4.85 (1H, d, J = 2.6 Hz, H-3), 5.58 (1H, bs, NH). C
NMR (100 MHz, CDCl ) δ: 13.41, 15.70, 18.78, 19.10, 19.10, 21.12, 21.61, 23.49, 24.52,
3
2
4
6.20, 26.32, 26.45, 28.78, 29.96, 32.68, 33.27, 34.32, 36.23, 36.70, 37.49, 37.60, 40.86,
0.96, 41.47, 42.21, 45.39, 46.70, 51.36, 51.53, 71.24, 79.35, 87.93, 169.69, 170.89. MS-EI:
+
+
m/z (%) [for C H NO : M 541]: 541 (M , 85), 481 (21), 470 (9), 440 (20), 422 (17), 203
3
4
55
4
+
(
19), 187 (60), 139 (100). HRMS (ESI-TOF) m/z calcd for C H NO [M+H] 542.4209,
34 55 4
found 542.4215.
Hemisuccinates 13a and 13b were prepared by reaction of crude aminoalcohol 11 (5 g;
0.9 mmol) with Succinic anhydride (11g; 93 mmol) in the mixture of solvents THF (100
1
mL) and pyridine (20 mL). The reaction mixture was poured to double volume of diluted
hydrochloric acid (1:4) and extracted with an organic solvent. The organic phase was washed
with water and 5% solution of NaHCO . Solvents were removed on vacuo. Crude product was
3
chromatographed by HPLC with reverse phase in the mixture MeCN/H O 6:1. Two
2
compounds were obtained: 13a (1.2 g; 19 %), mp. 290 – 294 °C (MeOH); [α] +18° (c 0.27).
D
-1 1
IR (KBr): 3303, 1718, 1646 cm . H NMR (400 MHz, CDCl ) δ: 0.81, 0.93, 0.97, 1.24, 1.24,
3
1
.43, 1.46 (21H, all bs, 7 × CH ), 2.5 (3H), 3.45 (1H, m, H-28a), 3.56 (1H, s, H-19), 3.76 (1H,
3
+
+
m, H-28b). MS-EI: m/z (%) [for C H NO : M 557]: 557 (M , 3), 513 (2), 457 (100), 441
34
55
5
+
(
13). HRMS (ESI-TOF) m/z calcd for C H NO [M+H] 558.4158, found 558.4157.
34 55 5
1
3b (1.3 g; 21 %), mp. 279 – 281 °C (MeOH); [α] +58° (c 0.36). IR (KBr): 3284, 1718,
D
-1 1
1
643 cm . H NMR (400 MHz, CDCl ) δ: 0.80, 0.91, 0.93, 0.97, 1.24, 1.43 (21H, all bs, 7 ×
3
CH ), 2.5 (2H), 3.45 (1H, bd, J = 7.4 Hz, H-28a), 3.55 (1H, s, H-19), 3.77 (1H, bd, J = 7.4
3
+
+
Hz, H-28b). EI-MS: m/z (%) [all C H NO : M 557]: 557 (M , 1), 513 (6), 457 (100), 441
3
4
55
5
+
(
25). HRMS (ESI-TOF) m/z calcd for C H NO [M+H] 558.4158, found 558.4152.
34 55 5
Esters 14a and 14b. The solution of the mixture of hemisuccinates 13a and 13b (0.5 g;
.1 mmol) was alkylated by diazomethane in Et O according to the common procedure. After
1
2
1
8

ACCEPTED MANUSCRIPT
the purification by HPLC in hexane/EtOAc 10:1 and lyophilization from t-BuOH methyl-
esters 14a and 14b were obtained: 14a (0.25 g; 49 %), mp. 140 – 143 °C (MeOH); [α] +23°
D
-1 1
(
c 0.26). IR (DRIFT): 3426, 1741, 1668, 1520 cm . H NMR (400 MHz, CDCl ) δ: 0.80,
3
0
.85, 0.91, 0.93, 0.95, 0.97, 1.01 (21H, all s, 7 × CH ), 1.95 (1H, dd, J(H-1a, H-1b) = 12.4 Hz,
3
J(H-1a, H-3α) = 4.1 Hz, H-1a), 2.49 (2H, m, CH - Suc), 2.66 (2H, m, CH - Suc), 2.95 (1H,
2
2
d, J = 10.5 Hz), 3.47 (1H, d, J = 8.8 Hz, H-28a), 3.51 (1H, s, H-19), 3.70 (3H, s, O-CH ), 3.77
3
(1H, d J = 7.3 Hz, H-28b), 4.04 (1H, m, H-2β), 5.66 (1H, d, J(H-3α, H-2α) = 7.8 Hz, H-3α).
1
3
C NMR (100 MHz, CDCl ) δ: 13.47, 15.65, 16.26, 17.32, 18.19, 21.09, 24.53, 26.20, 26.30,
3
2
4
6.35, 26.35, 28.24, 28.78, 31.19, 32.67, 33.71, 34.05, 36.24, 36.70, 38.18, 39.77, 40.63,
0.73, 41.44, 45.75, 46.78, 48.94, 50.95, 51.97, 55.27, 71.21, 83.38, 87.87, 173.11, 173.67.
+
+
FAB-MS: [for C H NO : M 571]: 572 (M + H), 557, 516. HRMS (ESI-TOF) m/z calcd
3
5
57
5
+
for C H NO [M+H] 572.4315, found 572.4321.
3
5
57
5
1
4b (0.21 g; 41 %), mp. 167 – 170 °C (MeOH); [α] +70° (c 0.20). IR (DRIFT): 3424, 1734,
D
-1 1
1
670, 1522 cm . H NMR (400 MHz, CDCl ) δ: 0.80, 0.91, 0.93, 0.98, 1.10 (21H, all s, 7 ×
3
CH ), 2.38 (1H, bs), 2.47 (2H, t, J = 6.9 Hz, CH - Suc), 2.68 (2H, m, CH - Suc), 3.45 (1H,
3
2
2
m, H-28a), 3.52 (1H, s, H-19), 3.69 (1H, s, OCH ), 3.77 (1H, d J = 7.7 Hz, H-28b), 4.22 (1H,
3
qd, J(H-2α, H-3α) = 7.8 Hz, J = 3.2 Hz, H-2α), 6.02 (1H, d, J(H-3α, H-2α) = 7.8 Hz, H-3α).
2
1
3
C NMR (100 MHz, CDCl ) δ: 13.40, 15.73, 18.63, 18.85, 19.07, 21.49, 24.52, 26.21, 26.30,
3
2
4
6.41, 28.78, 29.39, 30.26, 31.32, 32.38, 33.38, 34.24, 36.23, 36.70, 37.29, 37.74, 40.84,
0.88, 41.45, 42.05, 46.71, 47.37, 51.45, 51.88, 51.93, 71.24, 77.91, 87.89, 171.28, 173.52.
+
+
FAB-MS: [for C H NO : M 571]: 572 (M + H), 557, 517. HRMS (ESI-TOF) m/z calcd
3
5
57
5
+
for C H NO [M+H] 572.4315, found 572.4318.
3
5
57
5
4
.2.9. 2-sulfanyl-3-oxolupane-28-oic acid (15)
Bromo derivative 2c (1.0 g; 1.9 mmol) was dissolved in NMP (100 mL) and sodium sulfide
800 mg; 10.3 mmol) was added. The reaction was stirred 90 minutes at r.t., monitored on
(
TLC in toluene/Et O 5:1 with a drop of AcOH. The reaction was quenched when poured to
2
volume of water and extracted to an organic solvent. The organic phase was collected, washed
with water and the solvents were removed in vacuo to give 854 mg (81 %) of crude 15. IR
-1
+
+
-
(
DRIFT): 2500 – 3300, 1704, 1683 cm . MS (ESI ): m/z (%) = 528 (15, [M+K] ). MS (ESI
)
: m/z (%) = 470 (100, [M-H O]). Compound 15 decomposed when being purified on column
2
chromatography, probably due to oxidation by atmospheric oxygen.
1
9

ACCEPTED MANUSCRIPT
4
.2.10. Methyl ester 16
1
00 mg (0.204 mmol) of crude 15 was transformed to methyl ester by the reaction with
diazomethane in a mixture of Et O and CHCl , and purified by HPLC in cyklohexane/EtOAc
2
3
1
1
3:1 to give methyl ester 16 (8 mg; 8 %), mp. 110 – 112 °C (CHCl ). H NMR (500 MHz,
3
CDCl ): δ = 0.76 (d, 3H, J = 6.9 Hz), 0.87 (d, 3H, J = 6.8 Hz), 0.94 (s, 3H), 0.95 (s, 3H), 0.98
3
(
s, 3H), 1.07 (s, 3H), 1.21 (s, 3H, 7 × CH ), 2.22 – 2.26 (m, 3H, H-13, H-18, H-19), 2.31 (dd,
3
1
H, J = 13.7 Hz, J = 7.4 Hz, H-2), 3.66 (s, 3H, CH ), 3.91 (dd, 1H, J = 10.9 Hz, J = 7.5
1 2 3 1 2
1
3
Hz, SH). C NMR (125 MHz, CDCl ): δ = 14.12, 14.50, 14.66, 15.78, 19.65, 20.08, 22.63,
3
2
4
2.74, 22.97, 26.82, 28.24, 29.60, 29.69, 29.73, 30.92, 31.57, 31.91, 31.99, 37.85, 38.07,
0.60, 42.60, 44.11, 47.72, 48.79, 51.21, 55.22, 56.96, 176.85, 201.73. HRMS (ESI-TOF) m/z
+
calcd for C H O S [M+H] 503.3559, found 503.3557.
3
1
50
3
4
.2.11. Reactions of bromo derivative 3c with sodium sulfide; olefine 17
Bromo derivative 3c (200 mg; 0.385 mmol) was dissolved in DMSO (10 mL) with a drop of
water and then sodium sulfide (1 g; 12.820 mmol) was added. According to TLC
(hexane/EtOAc 5:1), starting bromo derivative 3c was completely converted to three
compounds after 5.5 hours at 75 °C. The reaction mixture was poured to tenfold volume of
water and extracted to chloroform. The organic phase was collected, washed with water and
the solvents were removed in vacuo to give 165 mg of crude mixture of products which was
then chromatographed on silica gel (25 g) in gradient from toluene to toluene/EtOAc 10:1.
The only isolated product was olefine 17. Derivative 17 was crystallized from a mixture of
solvents dichloromethan and methanol dried under the flow of N and purified by
2
chromatography on silica gel in hexane/EtOAc 10:1. Pure olefin 17 was obtained (13 mg; 7.7
4
5
1
%
), mp. 196 – 198 °C (hexane/EtOAc) (Lit. 249 – 251 °C). H NMR was identical to the
4
5
spectrum in lit. The other two compounds were not obtained probably they decomposed
while being chromatographed.
4
.2.12. Reactions of bromo derivative 2c with mercaptoethanol; diosphenol 20b
The mixture of sodium hydroxide (19 mg; 0.468 mmol), mercaptoethanol (330 µL;
.696 mmol) and anhydrous ethanol (15 mL) was stirred at r.t. until the solution was
completed. The reaction mixture was cooled to 0 – 5 °C (an ice-bath) and then bromderivative
c (250 mg; 0.467 mmol) was slowly added. The ice-bath was removed and the reaction
mixture was stirred 3 days at r.t., while monitored on TLC in toluene/Et O 5:1 with a drop of
4
2
2
2
0

ACCEPTED MANUSCRIPT
AcOH. The reaction mixture was poured to tenfold volume of water and extracted to an
organic solvent. The organic phase was collected, washed with water and the solvents were
removed in vacuo. The mixture of products was separated by column chromatography on
silica gel in toluene/Et O 15:1 with 0.5 % AcOH with gradient of Et O to 50 % to give
2
2
diosphenol 20b (46 mg; 21 %). The other compounds decomposed during the attempts for
1
46
theit purification. H NMR was identical to the spectrum in lit.
4
.2.13. Thioderivative 18
Sodium hydroxide (3.9 mg; 0.095 mmol) in mercaptoethanol (7 mL) and anhydrous ethanol
1 mL) was stirred at r.t. until fully dissolved. The reaction mixture was cooled by ice bath
(
and then bromo derivative 3c (100 mg; 0.19 mmol) was slowly added. The ice-bath was
removed and the reaction mixture was stirred 5 days at r.t., while monitored on TLC in
hexne/EtOAc 5:1. The reaction mixture was poured to tenfold volume of water and extracted
to an organic solvent. Organic phase was collected, washed with water and the solvents were
removed in vacuo. Thioderivative 18 was crystallized from EtOAc to give 81 mg (78 %) of
-1
pure compound. IR (DRIFT): 1033, 1654, 3341 cm . HRMS (FAB) m/z calcd for C H O S
32
52
3
+
[
M+Na] 537.3457, found 537.3455. Compound was unstable and during NMR measurement
and during attempts for its purification, it dehydrated to heterocycle 19. 40 mg of crystals
were purified by chromatography on silica gel in hexane/EtOAc 30:1; however, dehydratation
to 2',3'-dihydro-1',4'-oxathiin derivative 19 (17 mg; 36 %) was observed again. Spectral data
for 19 are shown in the experiment below that describes intentional synthesis of 19.
4
.2.14. 2',3'-dihydro-1',4'-oxathiin derivative 19 and diosphenol 20a
The mixture of sodium hydroxide (79 mg; 1.927 mmol), mercaptoethanol (135 µL;
.927 mmol) and anhydrous ethanol (10 mL) was stirred at r.t. until the solution was
completed. The reaction mixture was cooled to 0 – 5 °C (an ice-bath) and then bromderivative
c (850 mg; 1.638 mmol) was slowly added. The ice-bath was removed and the reaction
1
3
mixture was stirred 3 days at r.t., while monitored on TLC in hexne/EtOAc 5:1. The reaction
mixture was poured to tenfold volume of water and extracted to an organic solvent. The
organic phase was collected, washed with water and the solvents were removed in vacuo. The
mixture of products was separated by column chromatography on silica gel in hexane/EtOAc
1
0:1 with gradient of EtOAc to 100 % to give white crystals of 2',3'-dihydro-1',4'-oxathiin
derivative 19 (89 mg; 11 %), mp. 269 – 271 °C (cyclohexane); [α] +88.2° (c 0.34). IR
D
-1
(
DRIFT): 1655 cm . The full assignment of NMR signals was performed using the 2D NMR
2
1

ACCEPTED MANUSCRIPT
1
that is part of the supplemental file. H NMR (500 MHz, CDCl ): δ = 0.79 (s, 3H, H-29), 0.90
3
(s, 3H, H-27), 0.92 (s, 3H, H-30), 0.92 (m, 1H, H-12a), 0.93 (s, 6H, H-24,26), 0.98 (s, 3H, H-
2
5), 1.04 (s, 3H, H-23), 1.27 (m, 1H, H-7a), 1.28 (m, 1H, H-11a), 1.30 (m, 1H, H-a), 1.30 (tt,
1
H, J = 12.3 Hz, J = 3.7 Hz, H-21a), 1.38 (m, 1H, H-9), 1.40 (m, 1H, H-6b), 1.42 (m, 1H,
1
2
H-7b), 1.43 (m, 1H, H-16b), 1.44 (m, 1H, H-12b), 1.45 (m, 1H, H-11b), 1.46 (m, 1H, H-22b),
.46 (mm, 2H, H-13,18), 1.52 (dd, 1H, J = 12.3, 4.7 Hz, H-21b), 1.63 (m, 1H, H-15b), 1.79
d, 1H, J = 15.2 Hz, H-1a), 1.87 (d, 1H, J = 15.2 Hz, H-1b), 2.92 (dq, 1H, J = 12.8 Hz, J =
1
(
1
2
2
3
3
2
4
.5 Hz, H-31a), 3.04 (m, 1H, H-31b), 3.36 (m, 1H, H-6a), 3.43 (d, 1H, J = 7.8 Hz, H-28a),
.52 (m, 1H, H-19), 3.77 (d, 1H, J = 7.8 Hz, H-28b), 4.06 (m, 1H, H-32a), 4.27 (m, 1H, H-
1
3
2b). C NMR (125 MHz, CDCl ): δ = 13.56, 15.53, 16.28, 19.79, 19.48, 21.55, 24.65,
3
6.33, 26.51, 26.52, 27.09, 28.19, 28.89, 32.82, 33.09, 34.37, 36.36, 36.85, 37.12, 38.77,
0.57, 40.78, 41.57, 45.79, 46.87, 49.58, 53.28, 65.27, 71.36, 88.00, 96.28, 149.23. MS
+
+
+
+
(
ESI ): m/z (%) = 499 (65, [M+H] ), 521 (100, [M+Na] ), 1019 (45, [2M+Na] ). HRMS
+
(
ESI-TOF) m/z calcd for C H O S [M+H] 499.3604, found 499.3604.
32
50
2
Diosphenol 20a was obtained as a byproduct (304 mg; 41 %), mp. 204 – 206 °C
4
7
1
(
cyklohexane), lit. 231.5 – 233 °C (CHCl /MeOH). H NMR was identical to the spectrum
3
4
7
in lit.
4
.2.15. 2-nitro-3-oxolupane-28-oic acid (21)
Dihydrobetulonic acid (2b) (200 mg; 0.44 mmol) was dissolved in acetic acid (4 mL) by
heating. The solution was gradually cooled to r.t. and nitric acid (2 mL, 67 %) was added
dropwise. The reaction mixture was vigorously stirred 25 h at r.t., monitored on TLC in
hexane/EtOAc 5:1. The reaction mixture was poured to tenfold volume of water. The
precipitate was filtered off, washed with KHCO and water and crystallized from CHCl and
3
3
cyklohexane. Crude product was chromatographed on silica gel in CH Cl :MeOH:AcOH
2
2
5
00:10:1 and purified by chromatography on silica gel in cyklohexane/EtOAc 5:1 to give
nitrocompound 21 (51 mg; 23 %), mp. 248 – 249 °C (CHCl /cyklohexane). IR (DRIFT): 2450
3
-
1 1
–
0
3200, 1692, 1607, 1570 cm . H NMR (500 MHz, CDCl ): δ = 0.78 (d, 3H, J = 6.9 Hz),
3
.87 (s, 3H), 0.88 (d, 3H, J = 7.4 Hz ), 0.98 (s, 6H), 1.19 (s, 3H), 1.27 (s, 3H, 7 × CH ), 1.98
3
(d, 1H, J = 15.5 Hz, H-1a), 2.23 – 2.29 (m, 3H, H-13, H-18, H-19), 2.84 (d, 1H, J = 15.5 Hz,
1
3
H-1b). C NMR (125 MHz, CDCl ): δ = 14.52, 14.64, 15.68, 16.07, 19.45, 20.45, 21.40,
3
2
4
2.71, 22.96, 26.68, 28.62, 29.63, 29.71, 31.91, 33.04, 36.31, 37.34, 38.29, 39.99, 40.12,
+
0.47, 42.62, 44.10, 48.56, 48.61, 51.77, 56.78, 122.88, 178.38, 181.90. MS (ESI ): m/z (%) =
2
2

ACCEPTED MANUSCRIPT
+
-
-
5
19 (40, [M+H O] ). (ESI ): m/z (%) = 500 (100, [M-H] ). HRMS (ESI-TOF) m/z calcd for
2
+
C H NO [M+H] 502.3527, found 502.3527.
30
47
5
4
.2.16. 2,2-dinitro-3-oxolupane-28-oic acid (23)
Dihydrobetulonic acid (2b) (1.0 g; 2.19 mmol) was dissolved in acetic acid (20 mL) by
heating. The solution was gradually cooled to 35 °C and nitric acid (10 mL, 67 %) was added
dropwise. The reaction mixture was vigorously stirred 6 h at 35 °C, monitored on TLC in
hexane/EtOAc 5:2. The reaction mixture was poured to tenfold volume of water. The
precipitate was filtered off, washed with KHCO and water and crystallized from CHCl and
3
3
cyklohexane. Crude product was purified by column chromatography on silica gel in
CH Cl :MeOH:AcOH 500:10:1. Fractions containing 23 were collected and evaporated to
2
2
give shiny white crystals of dinitroderivative 23 (432 mg; 36 %), mp. 226 – 228 °C
-1 1
(
CHCl /hexane); [α] +9.6° (c 0.47). IR (DRIFT): 2500 – 3300, 1734, 1692, 1573 cm . H
3 D
NMR (500 MHz, CDCl ): δ = 0.78 (d, 3H, J = 6.3 Hz), 0.88 (d, 3H, J = 6.9 Hz), 0.98 (s, 3H),
3
1
1
.00 (s, 3H), 1.01 (s, 3H), 1.23 (s, 3H), 1.26 (s, 3H, 7 × CH ), 2.25 – 2.30 (m , 3H, H-13, H-
3
8, H-19), 2.93 (d, 1H, J = 16.0 Hz, H-1a), 3.10 (d, 1H, J = 16.0 Hz, H-1b), 10.92 (bs, 1H,
1
3
COOH). C NMR (125 MHz, CDCl ): δ = 14.50, 14.63, 15.43, 17.32, 19.59, 21.81, 22.71,
3
2
4
5
5
2.94, 23.12, 26.57, 29.53, 29.73, 30.34, 31.82, 32.65, 36.73, 37.34, 38.17, 40.56, 42.83,
+
4.06, 47.49, 48.48, 48.48, 49.32, 52.26, 56.76, 119.13, 182.19, 197.20. MS (ESI ): m/z (%) =
+
+
64 (100, [M+H O] ). HRMS (ESI-TOF) m/z calcd for C H N O [M+H] 547.3383, found
2
30 46
2
7
47.3385.
.2.17. 2-nitroallobetulone 22, 2,2-dinitroallobetulone 24, and enolacetate 25
4
Allobetulin (3a) (500 mg; 1.129 mmol) was dissolved in acetic acid (10 mL) by heating at
0 °C. By gradual cooling to r.t., tiny crystals of starting material precipitated. Nitric acid
5 mL, 58%) was added dropwise to the vigorously stirred reaction mixture. The reaction
8
(
mixture was stirred for 5 hours at r.t., monitored on TLC in hexane/EtOAc. The reaction
mixture was poured to tenfold volume of water and extracted to an organic solvent. The
organic phase was collected, washed with water and the solvents were removed in vacuo. The
mixture of nitroderivative 22, dinitroderivative 24 and enolacetate 25 was separated by
chromatography on silica gel in dichlormethan. Fractions containing pure nitroderivative 22
or dinitroderivative 24 were collected and evaporated to dryness to give shiny white crystals
4
8
of 2-nitroallobetulone 22 (111 mg; 20 %), mp. 236 – 238 °C (Lit. 235.8 °C) and white
2
3

ACCEPTED MANUSCRIPT
48
1
crystals of 2,2-dinitroallobetulone 24 (109 mg; 18 %), mp. 212 – 213 °C (Lit. 202.4 °C). H
48
NMR was in both cases identical to the corresponding spectrum in lit.
By extending the reaction time to 17 hours, allobetulon-1(2)-en-3-acetate (25) was the main
product of the identical reaction (74 mg; 14 %), mp. 287 °C (evaporated from dichlormethan).
4
8
1
48
Lit. 262 – 264 °C (EtOH/CHCl ). H NMR was identical to the spectrum in lit.
3
4
.2.18. Acetate 27
Zinc dust (133 mg; 2.046 mmol) was added to the solution of 2-nitroallobetulone 22 (100 mg;
.206 mmol) in acetic acid (6 mL). The reaction mixture was heated under reflux for 10 min
0
then the heating was stopped and the mixture stirred another 15 min, while being monitored
by TLC in hexane/EtOAc 5:1. The reaction was quenched by filtering the zinc dust out of the
reaction mixture. Acetic anhydride (3 mL; 31.7 mmol) was added to the reaction mixture. The
acylation was completed after 1 hour of stirring at r.t., monitored on TLC in hexane/EtOAc
1
:1. The reaction mixture was poured to tenfold volume of water and extracted to an organic
solvent. The organic phase was collected, washed with water and the solvents were removed
in vacuo. Crude products from the two identical reactions were collected and purified by
chromatography on silica gel in hexane/EtOAc 1:1. Fractions containing derivative 27
(mixture of epimers) were collected and evaporated to give white powder of 27 (87 mg; 47
-1
%
), mp. 230 – 232 °C (cyklohexane); [α] -9.2° (c 0.33). IR (DRIFT): 3426, 1704, 1677 cm .
D
1
H NMR (500 MHz, CDCl ): δ = 0.73, 0.79, 0.81, 0.88, 0.93, 0.95, 1.03, 1.10, 1.11, 1.12, 1.28
3
(
21H, all s, 7 × CH ), 1.82 – 1.84 (m, 1H, H-1a), 2.01 (s, 3H, Ac), 2.59 – 2.64 (m, 1H, H-1b),
3
3
.45 (d, 1H, J = 8 Hz, H-28a), 3.52 (s, 1H, H-19), 3.77 (d, 1H, J = 8 Hz, H-28b), 4.86 – 4.97
1
3
(
m, 1H, H-2α,2β), 6.49 (dd, 1H, J = 75, 5.5 Hz, NH). C NMR (125 MHz, CDCl ): δ =
3
1
2
2
4
5
3.40, 13.40, 14.17, 15.00, 15.83, 16.67, 19.03, 19.14, 19.30, 19.83, 20.98, 21.35, 22.06,
3.23, 23.33, 24.49, 24.55, 24.58, 26.18, 26.18, 26.24, 26.24, 26.36, 26.36, 28.75, 28.75,
8.94, 32.34, 32.67, 32,67, 33.69, 34.03, 34.41, 36.23, 36.23, 36.68, 36.68, 36.96, 37.98,
0.37, 40.73, 40.75, 40.79, 41.40, 41.43, 46.30, 46.67, 46.75, 48.62, 49.12, 50.00, 50.68,
0.78, 51.53, 51.70, 58.49, 71.19, 71.19, 87.85, 87.91, 169.54, 169.70, 212.98, 215.55. MS
+
+
+
-
(
ESI ): m/z (%) = 498 (100, [M+H] ), 996 (18, [2M+H] ). MS (ESI ): m/z (%) = 496 (100,
-
+
[
M-H] ). HRMS (ESI-TOF) m/z calcd for C H NO [M+H] 498.3942, found 498.3940.
3
2
51
3
4
.2.19. Acetate of enaminoketone 28
2
4

ACCEPTED MANUSCRIPT
Zinc dust (100 mg; 1.538 mmol) was added to the solution of 2-nitroallobetulone 22 (75 mg;
0
.155 mmol) in acetic acid (5 mL). The reaction mixture was heated to reflux for 10 min and
stirred 15 min more, monitored on TLC in hexane/EtOAc 5:1. The reaction was quenched by
filtered zinc off the reaction mixture. Toluene (10 mL) was added to the reaction vial and the
mixture was boiled for 3 days, monitored on TLC in hexane/EtOAc 1:1. The reaction mixture
was poured to tenfold volume of water and extracted to an organic solvent. The organic phase
was collected, washed with water and the solvents were removed in vacuo. Crude product
(270 mg) from the two identical reactions was purified by chromatography on silica gel in
cyklohexane/EtOAc 5:1. Fractions containing 28 were collected and evaporated to dryness to
give yellow-orange powder of acetate 28 (46 mg; 20 %), mp. 135 – 137 °C (acetone); [α]D
-
1 1
+
58° (c 0.25). IR (DRIFT): 3383, 1693, 1659, 1632, 1510, 1035 cm . H NMR (500 MHz,
CDCl ): δ = 0.81, 0.92, 0.94, 1.05, 1.13, 1.13, 1.18 (21H, all s, 7 × CH ), 2.09 (s, 3H, Ac),
3
3
3
1
2
3
1
.45 (d, 1H, J = 7.8 Hz, H-28a), 3.55 (s, 1H, H-19), 3.77 (d, 1H, J = 7.5 Hz, H-28b), 7.77 (s,
1
3
H, H-1), 8.16 (s, 1H, NH). C NMR (125 MHz, CDCl ): δ = 13.31, 14.10, 16.14, 18.82,
3
0.52, 21.24, 21.73, 22.61, 24.47, 26.20, 26.29, 27.95, 28.72, 31.55, 32.64, 34.21, 36.21,
6.65, 38.67, 40.99, 41.40, 41.49, 44.03, 45.86, 46.64, 52.67, 71.20, 87.83, 129.13, 140.81,
+
+
68.82, 200.09. MS (ESI ): m/z (%) = 497 (100, [M+H] ), 992 (10 [2M]). HRMS (ESI-TOF)
+
m/z calcd for C H NO [M+H] 496.3785, found 496.3784.
3
2
49
3
4
.3. Cell lines
All cells (if not indicated otherwise) were purchased from the American Tissue Culture Collection
ATCC). The CCRF-CEM line is derived from T lymphoblastic leukemia, 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-/-, Horizon 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 daunorubicine 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
5
0
primary and/or acquired multidrug resistance phenomenon. MRC-5 and BJ cell lines were used as a
2
non-tumor control and represent human fibroblasts. The cells were maintained in nunc/corning 80 cm
plastic tissue culture flasks and cultured in cell culture medium according to ATCC or Horizon
2
5

ACCEPTED MANUSCRIPT
recommendations (DMEM/RPMI 1640 with 5 g/L glucose, 2 mM glutamine, 100 U/mL penicillin,
1
00 mg/mL streptomycin, 10% fetal calf serum, and NaHCO3).
4
.4. Cytotoxic MTS assay
MTS assay was performed at Institute of Molecular and Translational 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 ꢀL) 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 ꢀL 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 replicates at least. The cells were incubated with the tested
compounds for 72 h at 37 °C, in a 5% CO atmosphere at 100% humidity. At the end of the incubation
2
period, the cells were assayed by using the MTS test. Aliquots (5 ꢀL) 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
calculated from the appropriate dose-response curves in Dotmatics software.
4
.5. Cell Cycle and Apoptosis Analysis
6
Suspension of CCRF-CEM cells, seeded at a density of 1.10 cells/mL in 6-well panels, were
cultivated with the 1 or 5 × IC of tested compound in a humidified CO incubator at 37 °C in RPMI
5
0
2
1
640 cell culture medium containing 10% fetal calf serum, 10 mM glutamine, 100 U/mL penicillin,
and 100 ꢀg/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% ethanol added dropwise and stored overnight at -20 °C. Afterwards, cells were
washed in hypotonic citrate buffer, treated with RNAse (50 ꢀg/mL) and stained with propidium
iodide. Flow cytometer 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 logarithmic model expressing percentage of the particles with propidium content lower
Ser10
than cells in G0/G1 phase (<G1) of the cell cycle. Half of the sample was used for pH3
antibody
5
1
(
Sigma) labeling and subsequent flow cytometry analysis of mitotic cells.
4
.6. BrDU Incorporation Analysis (DNA synthesis)
2
6

ACCEPTED MANUSCRIPT
For this analysis, the same procedure of cultivation as previously was used. Before harvesting, 10 ꢀM
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 Na B O (Borax), cells were washed with PBS containing 0.5% Tween-20
2
4
7
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
5
1
by flow cytometry using a 488 nm single beam laser (FACSCalibur, Becton Dickinson).
4
.7. BrU Incorporation Analysis (RNA synthesis)
Cells were cultured and treated as above. Before harvesting, pulse-labeling with 1 mM 5-bromo
uridine (BrU) for 30 min followed. The cells were then fixed in 1% buffered paraformaldehyde with
0
.05 % of NP-40 in room temperature 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 crossreacting 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 staining, 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
37
Dickinson).
5
. Acknowledgment
The chemical part was supported by Czech Science Foundation (15-05620S) and internal
grants of Palacky University IGA_PrF_2015_00, IGA-PrF-2016-020, and IGA_LF_2016_19.
The biological part was paid by Technology Agency of the Czech Republic (TE01020028).
The infrastructural part (Institute of Molecular and Translational Medicine) is supported by
the National Sustainability Programme (LO1304). We are grateful to Lukas Najdekr for
measurement of HRMS.
6
. References
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