Synthesis, structure and cytotoxic activity of new acetylenic derivatives of betulin

Article abstract of DOI:10.3390/molecules18044526

A new series of betulin derivatives containing one or two pharmacophores bearing an acetylenic and carbonyl function at the C-3 and/or C-28 positions has been synthesized and characterized by ¹H- and ¹³C-NMR, IR, MS and elemental analyses. The crystal structure of 28-O-propynoylbetulin was determined by X-ray structural analysis. All new compounds, as well as betulin, were tested in vitro for their antiproliferative activity against human SW707 colorectal, CCRF/CEM leukemia, T47D breast cancer, and against murine P388 leukemia and Balb3T3 normal fibroblasts cell lines. Most of the compounds showed better cytotoxicity than betulin and cisplatin used as reference agent. 28-O-Propynoylbetulin was the most potent derivative, being over 500 times more potent than betulin and about 100 times more cytotoxic than cisplatin against the human leukemia (CCRF/CEM) cell line, with an ID₅₀ value of 0.02 μg/mL.

Full text of DOI:10.3390/molecules18044526

Molecules 2013, 18, 4526-4543; doi:10.3390/molecules18044526
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Structure and Cytotoxic Activity of New Acetylenic
Derivatives of Betulin
Stanisław Boryczka 1,*, Ewa Bębenek 1, Joanna Wietrzyk 2, Katarzyna Kempińska 2,
Maria Jastrzębska 3, Joachim Kusz 4 and Maria Nowak 3,4
1
Department of Organic Chemistry, Medical University of Silesia, 4 Jagiellonska Str.,
41-200 Sosnowiec, Poland
2
Department of Experimental Oncology, Ludwik Hirszfeld Institute of Immunology and
Experimental Therapy, Polish Academy of Sciences, 12 R. Weigla Str., 53-114 Wrocław, Poland
3
Department of Solid State Physics, Institute of Physics, University of Silesia, 4 Uniwersytecka Str.,
40-007 Katowice, Poland
4
Department of Physics of Crystals, Institute of Physics, University of Silesia, 4 Uniwersytecka Str.,
40-007 Katowice, Poland
* Author to whom correspondence should be addressed; E-Mail: boryczka@sum.edu.pl;
Tel.: +48-32-364-1605; Fax: +48-32-364-1600.
Received: 21 March 2013; in revised form: 8 April 2013 / Accepted: 11 April 2013 /
Published: 17 April 2013
Abstract: A new series of betulin derivatives containing one or two pharmacophores
bearing an acetylenic and carbonyl function at the C-3 and/or C-28 positions has been
1
13
synthesized and characterized by H- and C-NMR, IR, MS and elemental analyses. The
crystal structure of 28-O-propynoylbetulin was determined by X-ray structural analysis.
All new compounds, as well as betulin, were tested in vitro for their antiproliferative
activity against human SW707 colorectal, CCRF/CEM leukemia, T47D breast cancer, and
against murine P388 leukemia and Balb3T3 normal fibroblasts cell lines. Most of the
compounds showed better cytotoxicity than betulin and cisplatin used as reference agent.
28-O-Propynoylbetulin was the most potent derivative, being over 500 times more potent
than betulin and about 100 times more cytotoxic than cisplatin against the human leukemia
(CCRF/CEM) cell line, with an ID50 value of 0.02 μg/mL.
Keywords: acetylenic betulins; synthesis; crystal structure; cytotoxic activity

Molecules 2013, 18
4527
1. Introduction
Betulin [lup-20(29)-ene-3,28-diol, C30H50O2, 1] also known as betulinic alcohol, is a pentacyclic
triterpene of the lupane type which was one of the first natural products identified and isolated from
plants as a pure chemical substance in 1788 by Lowitz [1]. The still growing interest in betulin (1) and
its derivatives results from their wide spectrum of biological activities such as anticancer, antiviral,
anti-inflammatory, antibacterial and hepatoprotective properties [2–4]. Despite the fact that betulin (1)
has been known for over 200 years, the X-ray crystal structure of this compound was investigated for
the first time in 2010 by Drebushchak and in 2011 by Boryczka as betulin-EtOH and betulin-DMSO
solvates, respectively [5,6]. Betulin (1) has three available sites for simple chemical modification,
namely the secondary hydroxyl group at position C-3, the primary hydroxyl group at position C-28
and the isopropenyl side chain at position C-19.
Compound 1 can be easily converted in high yield to betulinic acid (2), which possesses a wide
spectrum of biological and pharmacological activities and is now a very attractive and promising agent
for clinical treatment for various types of cancer [7–9]. The chemical structures of these two
compounds differ only in one group at C-17 [betulin (1) R=CH2OH, betulinic acid (2) R=COOH], and
they are shown in Figure 1. In contrast to 2, betulin (1) has been described as inactive or less active
against cancer cells [3,10]. However, the results of recent reports suggest that betulin (1) shows a
significant cytotoxic activity in a similar way as betulinic acid (2) [11,12].
Figure 1. Structure of betulin (1) and betulinic acid (2).
20
19
17
R28
1
3
HO
Betulin (1)
R = CH2OH
Betulinic acid (2) R = COOH
The high content of betulin (1) in white birch bark (up to 30%) compared to betulinic acid (2)
(up to 2.5%), make it an important starting material for synthesis of new compounds with various
interesting medical properties. In the last few years a large number of betulin derivatives have been
reported to possess anticancer, anti-inflammatory, anti-HIV and anti-leishmanial activity [2,13–16].
It is well known that the carbon-carbon triple bond is one of the most important functional groups
in medicinal and organic chemistry [17]. Nevertheless, only a few reports have been described the
synthesis of acetylenic derivatives of betulin which possess useful biological activity such as cytotoxic
and antiviral properties [18–22]. It is interesting to note that the presence of an alkynyl group offers a
possibility for further functionalization of betulins [23,24].
As an extension of our study on the development of novel acetylenic derivatives with potential
anticancer activity [25,26], we became interested in the synthesis and evaluation of cytotoxicity of new

Molecules 2013, 18
4528
derivatives of betulin containing one or two pharmacophores including both carbonyl group and a
triple bond at the C-3 and/or C-28 positions. In order to investigate structure-activity relationships for
the anticancer effects, two types of reagents were used for modifications of the hydroxyl groups of
betulin (1), namely acetylene chloroformate and propynoic acids. All new synthesized compounds
were tested for cytotoxic activity against human: breast cancer (T47D), leukemia (CCRF/CEM),
colorectal adenocarcinoma (SW707), and murine: leukemia (P388) as well as normal fibroblasts
(Balb3T3) cell lines in order to obtain more information about the influence of triple bond and
carbonyl group on anticancer activity in this class of compounds.
2. Results and Discussion
2.1. Chemistry
Synthesis of acetylenic derivatives of betulin 3–9 was accomplished starting with betulin (1).
Compound 1 was isolated from birch bark by extraction with dichloromethane. The crude betulin (1)
was purified by column chromatography using a mixture of chloroform and ethanol as an eluent. Full
1
13
structural elucidation of the betulin (1) was performed using H- and C-NMR and IR spectroscopy,
and the assignments were done based on our analysis and related literature [6,27]. The synthesis of
acetylenic derivatives of betulin 3–9 is described in Schemes 1 and 2.
Scheme 1. Synthesis of acetylenic derivatives of betulin 3–6.
O
O
2
1
OCR
OCOR
HO
HO
O
3
5
1
R OCCl
R2COOH
+
1
+
CH2Cl2, rt, 24 h
DCC, DMAP
C6H6, Py, rt, 24 h
O
O
2
OCR
1
OCOR
O
O
2
R CO
1
R OCO
1
2
6
a. R = CH2C
CH
b. R = CH2CH2C
a. R = C
4
CH
CPh
1
2
b. R = C
2
CH
CCH3
d. R = CH2CH3
1
c. R = CH2CH3
c. R = CH2C
1
Treatment of betulin (1) with the corresponding chloroformate in benzene in the presence of
pyridine at room temperature gave a mixture of mono- 3 and diesters 4, in which the first isomers 3
were the major products due to the fact that reactivity of the hydroxyl group at C-28 is much higher
than that of the C-3 one. The mixtures were separated by column chromatography to afford pure
products 3 and 4 in 54%–69% and 23%–28% yields, respectively. The reaction of betulin (1) with

Molecules 2013, 18
4529
propynoic acid or phenylpropynoic acid or propanoic acid in dichloromethane in the presence of
catalytic amount of dicyclohexylcarbodiimide (DCC) and 4-dimetylaminopyridine (DMAP) afforded
mixtures of mono- 5 and diester 6, which were separated by column chromatography to give pure 5
and 6 in 50%–86% and 11%–27% yields, respectively.
Scheme 2. Synthesis of betulin derivatives 7 and 8.
O
OAc
OAc
R1OCCl, Py
C6H6, rt, 24 h
O
1
HO
R OCO
8
7a
Ac2O, DMAP
Py, rt, 18 h
1
1
+
a. R = CH2C
CH
b. R = CH2CH2C
1
CH
CCH3
1
c. R = CH2C
1
d. R = CH2CH3
OAc
AcO
7b
For the synthesis of compounds 8 (Scheme 2) the corresponding monoacetylated substrate 7a was
prepared, according to the method described in literature [28]. The synthesis of 28-O-acetylbetulin
(7a) was achieved by selective acetylation of the primary hydroxyl group of betulin (1) with an
equimolar amount of acetic anhydride in dichloromethane in the presence of DMAP and pyridine at
room temperature. The reaction also gave small amount of 3,28-O,O'-diacetylbetulin (7b) which was
separated by column chromatography.
The 28-O-acetylbetulin (7a) was then treated with corresponding chloroformates in benzene in the
presence of pyridine under mild conditions to afford 28-O-acetyl-3-O'-alkynyloxycarbonylbetulins 8 in
good yields (63%–89%). The structure of all new compounds 3–8 were determined on the basis of
their 1H, 13C-NMR, IR and MS spectra, together with elemental analyses. In addition, crystal structure
of 5a was determined by X-ray structural analysis. For the first time, crystal structure of an acetylenic
derivative of betulin-DMSO solvate is reported here.
2.2. Crystal Structure
Crystals of 28-O-propynoylbetulin (5a) for X-ray structural analysis were grown from a DMSO
solution as a 28-O-propynoylbetulin-DMSO solvate of 1:1 stoichiometry. The crystal structure of 5a is
shown in Figure 2. The compound crystallizes in the hexagonal system (space group P65) with
a = 2.6511 (1) nm, b = 2.6511 (1) nm, c = 8.3895 (2) nm, α = 90°, β = 90°, γ = 120°, V = 510.6 (1) nm3,
Z = 6. The six-membered rings have a chair conformation, while the cyclopentane ring adopts a twisted

Molecules 2013, 18
4530
conformation. All ring junctions in the 28-O-propynoylbetulin-DMSO solvate are trans-fused. A
similar ring conformation was also observed in betulin-DMSO solvate [6], 3,28-O,O'-diacetylbetulin [29]
and betulinic acid-DMSO solvate [30].
Figure 2. Molecular structure with atomic numbering scheme of 28-O-propynoylbetulin –
DMSO solvate 5a with displacement ellipsoids of 50% probability.
The methyl groups at carbon atoms C-24, C-25, C-26, C-27 occupy the axial positions, whereas the
methyl group at C-23 and the isopropenyl group at C-19 are equatorial. The torsion angle
C29-C20-C19-C21 describing the orientation of the isopropenyl group is equal to 105.4°. This
conformation is very similar to that observed for the corresponding angles in 3,28-O,O'-diacetylbetulin
(107.18°) [29] and in betulin-ethanol solvate (88.6°) [5] but differs from that observed for the structure
of betulin-DMSO solvate (−96.8°) [6], and for betulinic acid-DMSO solvate (−112.2°) [30].
The OH group at C-3 is an equatorial orientation, while the substituted hydroxymethyl group is
attached to the atom C-17 in an axial orientation. The oxygen atom O-4 of DMSO and the hydroxyl
group at C-3 of betulin (1) are involved in the O1 – H1…O4 bond, which is relatively strong with
short H…O distance (1.86 Å) and almost linear directionality (169.4°).
2.3. Cytotoxic Activity
Cytotoxic activities of all newly synthesized compounds including betulin (1) were tested in SRB or
MTT (in the case of leukemia cells) assay for their antiproliferative activity in vitro against three
human cancer cell lines: SW707 (colorectal adenocarcinoma), CCRF/CEM (leukemia), T47D (breast
cancer), and murine leukemia P388 as well as Balb3T3 normal fibroblasts cell lines. The results of the
cytotoxic activity in vitro were expressed as an IC50 value (μg/mL), i.e., the concentration of
compound which inhibits the proliferation of 50% of tumor cells as compared to the control untreated
cells. Cisplatin was used as a reference cytotoxic agent (positive test control). A value of less than
4 μg/mL was considered as an antiproliferative activity criterion for synthetic compounds [31]. The
results of the cytotoxicity studies are summarized in Table 1.

Molecules 2013, 18
Table 1. Cytotoxic activity of betulin (1), acetylenic betulins 3–8 and cisplatin reference
4531
compound against the cells of the tested human and murine cancer cell lines.
Cytotoxic activity IC50 [µg/mL]
Compd.
Human
Murine
P388 Balb3T3
R1
R2
T47D
32.4 ± 10.7
7.2 ± 1.7
6.9 ± 2.3
20.1 ± 5.3
6.6 ± 1.3
Neg
CCRF/CEM
10.9 ± 5.5
3.2 ± 0.4
5.2 ± 1.4
6.8 ± 2.9
4.1 ± 0.2
Neg
SW707
22.9 ± 15.4 5.5 ± 3.3
9.2 ± 7.1 2.9 ± 0.2
1
3a
47.3 ± 7.9
3.9 ± 2.8
-
-
CH2C CH
-
CH2CH2C CH
CH2C CCH3
CH2CH3
3b
22.6 ± 11.9 3.6 ± 1.3 19.5 ± 13.9
36.9 ± 6.3 5.5 ± 2.8 28.4 ± 17.6
20.8 ± 14.7 3.8 ± 1.9 19.1 ± 13.5
-
3c
-
3d
-
CH2C CH
4a
Neg
Neg
Neg
Neg
33.4 ± 1.7
Neg
Neg
Neg
-
CH2CH2C CH
CH2C CCH3
CH2CH3
4b
Neg
Neg
-
4c
Neg
Neg
50.4 ± 31.1
46.8 ± 21.6
0.4 ± 0.1
Neg
Neg
-
4d
69.1 ± 12.4
9.1 ± 1.9
Neg
52.6 ± 8.3
Neg
-
C CH
5a
0.02 ± 0.001 14.9 ± 3.3
0.3 ± 0.05
Neg
-
-
-
-
-
-
-
C CPh
CH2CH3
5b
49.0 ± 9.8
8.1 ± 0.9
9.9 ± 7.0
66.0 ± 2.8
Neg
Neg
5c
12.1 ± 4.4
16.2 ± 4.3
Neg
29.2 ± 24.4 3.3 ± 0.8 32.3 ± 23.0
C CH
6a
9.5 ± 1.9
Neg
31.9 ± 1.4 21.1 ± 2.6
C CPh
CH2CH3
6b
Neg
Neg
Neg
6c
26.6 ± 6.8
4.9 ± 0.6
Neg
42.3 ± 9.4 68.1 ± 14.8
7a
3.4 ± 0.3
34.1 ± 6.4
Neg
5.1 ± 2.5 12.5 ± 14.7 29.7 ± 35.8
-
-
-
-
-
-
7b
Neg
Neg
40.2 ± 5.0
Neg
Neg
Neg
-
CH2C CH
8a
Neg
CH2C CCH3
CH2CH2C CH
CH2CH3
8b
Neg
Neg
Neg
46.7 ± 16.2
Neg
Neg
8c
Neg
Neg
Neg
Neg
8d
Neg
Neg
Neg
Neg
Neg
Cisplatin
3.1 ± 1.0
2.0 ± 0.5
2.2 ± 0.5
0.5 ± 0.3
2.7 ± 0.3
Neg – negative in the concentration used.
As shown in Table 1, betulin (1) exhibited notably lower anticancer activity against the investigated
cancer cell lines, with IC50 values in the 10.9–47.3 μg/mL range, except for leukemia P388 cell line
(IC50 = 5.5 μg/mL). In this case, the selectivity index (SI) value, expressed as the ratio of IC50 of
normal fibroblast/ IC50 on corresponding cancer cell line, is equal to 8.6. The introduction of an
acetylenic formate group at the C-28 led to an increase of activity against the tested human and murine
cancer lines. In the series of derivatives 3, the most active compound 3a was three to four times more
cytotoxic than betulin (1) against cancer cells applied. Moreover, the degree of selectivity of this
compound was lower than that of betulin (1) and derivatives 3b–d. By comparing the cytotoxic
activities of monoesters 3a–c, it was found that the cytotoxic potency may be dependent on the length
of the alkynyl chain and position of the triple bond on the formate group at the C-28 of betulin
derivatives 3. The compound, having a shorter alkynyl chain, was found to be more cytotoxic to the
cancer cell lines. A terminal triple bond seems to be necessary for a good activity. The cytotoxic
activity of compound 3d is similar to that of 3b and it is lower than that observed for 3a. This indicates

Molecules 2013, 18
4532
that ethyl formate and 3-butynyl formate substituents at position 28 had a similar effect on the
antiproliferative activity of these compounds. The structure-activity relationships indicated that the
rank order of the cytotoxic activity observed in monoesters 3, according to the nature of the formate
substituent is as follows: propargyl > 3-butynyl = ethyl > 2-butynyl. The introduction of a second
formate group at the C-3 position in monoesters 3 led to the complete loss of the activity (compounds 4).
Replacement of the hydroxyl group at C-28 position in betulin (1) by a propynoyloxy function, i.e.,
monoester 5a, resulted in increase of activity, especially against the cells of CCRF/CEM, with IC50
value of 0.02 μg/mL. It is interesting to note that compound 5a was 545 times more cytotoxic than
betulin (1) and 100 times more cytotoxic than cisplatin against CCRF/CEM leukemia cells. It was also
observed that 5a was more selective (SI = 15) towards CCRF/CEM than betulin (1). These results
suggest that the propynoyloxy function is important for anti-leukemia activity. On the other hand,
addition of a second propynoyl group at C-3 in monoester 5a led to a decrease of activity except for
the activity against the SW707 colon cancer cell (compound 6a).
Though the exact reasons for this influence of propynoyl group are not quite well understood, we
assume that the cytotoxic activity of compound 5a may be a consequence of the higher electrophilicity
of its triple bond. We suggest that due to the strong negative effect of the carbonyl group, the
mesomeric formula describing such a delocalization of the electron density of the triple bond has a
cumulene structure 5-A and carbene structure 5-B (Figure 3) and it can interact very easily with
nucleophilic reactive groups in living systems such as amines or thiols. In order to understand the
effect of the triple bond of the propynoyl pharmacophore, derivative 5c was tested. It is interesting to
note that replacement of the ethynyl group (compound 5a) by the ethyl group (compound 5c) resulted
in a decrease of activity, especially against leukemia (CCRF/CEM and P388) cell lines. These results
suggested that the triple bond directly bonded to carbonyl group of the substituent at the C-28 position
seem to be essential for anticancer activity. This finding is in a good agreement with results from Csuk
and co-workers showing that the 28-etynylbetulin and its carbonyl derivatives exhibited a higher
cytotoxic activity compared to betulinic acid [23]. It was also reported that several acetylenic natural
products possessing fungicidal properties have a conjugation of the triple bond to an adjacent carbonyl
function [32]. However, the mono- 5b and diester 6b containing phenylproynoyl groups are inactive.
The structure-activity relationships observed in compounds 5 indicated that the rank order of the
cytotoxic activity, against all cancer cell lines applied, according to the nature of the acyloxy
substituent, is as follows: propynoyl > propanoyl > phenylpropynoyl. It indicates that the betulins
where the carbonyl group (at C-28 position) is directly bonded to triple bond of the ethynyl substituent,
show a strong cytotoxic effect especially against human leukemia (CCRF/CEM) cancer cells with SI
value equal to 15 and higher than betulin (1).
The 28-O-acetylbetulin 7a showed a higher activity than the parent compound 1. But the
introduction of the acetyl group at position C-3 in the compound 7a significantly decreased the
cytotoxicity (compound 7b) in all the tested cell lines when compared to betulin (1) and 7a. These
results are consistent with the reported data for the anticancer activity in vitro of acetyl derivatives of
betulin [33]. On the other hand, esterification of the hydroxyl group at the C-3 of 28-O-acetylbetulin
(7a) with acetylenic chloroformates led to the total inactive compounds 8. The obtained results also
suggest that the free C-3 hydroxyl function group is important for the cytotoxicity of studied compounds.

Molecules 2013, 18
4533
Figure 3. Proposed mesomeric structures of propynoyl pharmacophore of 28-O-
propynoylbetulin (5a).
-
O
O
+
Bet
CH2 O C C C H
Bet
CH2 O C C C H
5a
5-A
H
O
Bet =
HO
Bet
CH2 O C C C
5-B
3. Experimental
3.1. General Techniques
Melting points were determined in open capillary tubes on a Boetius melting point apparatus and
are uncorrected. NMR spectra (600/150 MHz) were recorded on a Bruker MSL 600 spectrometer in
CDCl3 solvents with tetramethylsilane as internal standard; chemical shifts are reported in ppm () and
J values in Hz. Multiplicity is designated as singlet (s), doublet (d), triplet (t), quartet (q), multiplet
(m). Mass spectra were recorded under EI conditions on a Finnigan MAT 95 instrument, IR spectra
(KBr, pellet) on a IRAffinity-1 Shimadzu spectrophotometer. Elemental C, H analyses were obtained
on a Carlo Erba Model 1108 analyzer. TLC was performed on silica gel 60 254F plates (Merck,
Darmstadt, Germany) using a mixture of chloroform and ethanol (20:1 or 40:1, v/v) as an eluent. Spots
were visualized by spraying with solution of 5% sulfuric acid, followed by heating. Column
chromatography was performed on silica gel 60, <63 μm (Merck) using a mixture of chloroform and
ethanol (40:1, v/v) as an eluent. Solvents were dried and purified according to usual procedures.
3.2. Isolation of Betulin (1)
External bark (100 g) of the white birch (Betula verrucosa), collected in Poland, was soaked in
dichloromethane (1 L) and refluxed for 8 hours. The solvent was removed under reduced pressure and
the crude product was purified by column chromatography (chloroform/ethanol, 20:1, v/v) to give 1 as
a white solid (19 g, 19%): m.p. 250–252 °C (lit. m.p. 252–253 °C [6]; 255–256 °C [27]); Rf 0.42
(chloroform/ethanol, 20:1, v/v). The 1H- and 13C-NMR spectral data of 1 were in agreement with those
published in the literature [6].

Molecules 2013, 18
4534
3.3. General Procedure for the Synthesis of Monoesters 3 and Diesters 4
To a mixture of betulin (1, 0.44 g, 1 mmol) and pyridine (2.5 mL) in dry benzene (6 mL) at 0–5 °C, a
solution of propargyl chloroformate or 2-butyn-1-yl chloroformate or 3-butyn-1-yl chloroformate or
ethyl chloroformate (3.0 mmol) in dry benzene (5 mL) was added. Stirring at this temperature was
continued for 4 h. Then the reaction mixture was allowed to warm to room temperature and stirred
over night. At the end of the reaction 5 mL of chloroform was added and the reaction mixture was
washed with 1 N sulfuric acid, followed by water, then dried with anhydrous magnesium sulfate and
concentrated under reduced pressure. The crude product was purified by column chromatography
(chloroform/ethanol, 40:1, v/v) to give pure corresponding monoesters 3 and diesters 4.
28-O-Propargyloxycarbonylbetulin (3a). Yield: 69%, m.p. 190–193 °C, Rf 0.49 (chloroform/ethanol,
1
40:1, v/v); H-NMR (CDCl3) : 0.76 (s, 3H, CH3), 0.82 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.98 (s, 3H,
CH3), 1.03 (s, 3H, CH3), 1.68 (s, 3H, CH3), 1.06–2.01 (m, 25H, CH, CH2), 2.42 (m, 1H, H-19), 2.53 (t,
J = 2.4 Hz, 1H, C≡CH), 3.18 (m, 1H, H-3), 3.95 (d, J = 10.8 Hz, 1H, H-28), 4.38 (d, J = 10.8 Hz, 1H,
13
H-28), 4.59 (s, 1H, H-29), 4.69 (s, 1H, H-29), 4.74 (d, J = 2.4 Hz, 2H, OCH2). C-NMR (CDCl3) :
14.9, 15.3, 15.4, 15.9, 16.2, 18.3, 19.1, 20.8, 25.2, 26.9, 27.4, 27.9, 28.0, 29.5, 34.2, 34.3, 37.1, 37.6,
38.7, 38.8. 40.8, 42.7, 46.5, 47.6, 48.8, 50.3, 55.2, 55.4, 67.3, 75.6, 78.9, 109.9, 149.9, 155.1. IR (KBr,
cm−1) ν: 3421, 3309, 2132, 1749, 1250, 885. EI MS (70 eV) m/z (rel. intensity): 524 (M+, 11), 411
(45), 207 (59), 203 (58), 189 (100), 135 (58); Elemental anal. (%), calcd. for C34H52O4: C, 77.82; H,
9.99; found: C, 77.59; H, 9.84.
28-O-(3-Butynyloxycarbonyl)betulin (3b). Yield: 64%, m.p. 86–88 °C, Rf 0.45 (chloroform/ethanol,
1
40:1, v/v); H-NMR (CDCl3) : 0.76 (s, 3H, CH3), 0.82 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.98 (s, 3H,
CH3), 1.03 (s, 3H, CH3), 1.68 (s, 3H, CH3), 1.05–2.02 (m, 25H, CH, CH2), 2.03 (t, J = 2.7 Hz, 1H, C≡CH),
2.43 (m, 1H, H-19), 2.59 (m, 2H, J = 2.7 Hz, J = 7.2 Hz, OCH2CH2), 3.19 (m, 1H, H-3), 3.93 (d,
J = 10.8 Hz, 1H, H-28), 4.25 (t, J = 7.2 Hz, 2H, OCH2), 4.36 (d, J = 10.8 Hz, 1H, H-28), 4.59 (s, 1H,
13
H-29), 4.69 (s, 1H, H-29). C-NMR (CDCl3) : 14.7, 15.3, 15.9, 16.1, 18.2, 19.0, 20.7, 25.1, 26.9,
27.3, 27.9, 29.4, 34.1, 34.3, 37.1, 37.5, 38.6, 38.8, 40.8, 42.6, 46.5, 47.6, 48.7, 50.3, 55.2, 65.3, 66.7,
70.2, 76.7, 78.9, 79.4, 109.9, 150.0, 155.4. IR (KBr, cm−1) ν: 3485, 3310, 2124, 1747, 1248, 884. EI
MS (70 eV) m/z (rel. intensity): 538 (M+, 10), 207 (47), 203 (52), 189 (100), 135 (57); Elemental anal.
(%), calcd. for C35H54O4: C, 78.02; H, 10.10; found: C, 78.32; H, 9.98.
28-O-(2-Butynyloxycarbonyl)betulin (3c). Yield: 54%, m.p. 112–114 °C, Rf 0.48 (chloroform/ethanol,
1
40:1, v/v); H-NMR (CDCl3) : 0.76 (s, 3H, CH3), 0.82 (s, 3H, CH3), 0.96 (s, 3H, CH3), 0.97 (s, 3H,
CH3), 1.03 (s, 3H, CH3), 1.68 (s, 3H, CH3), 1.06–2.01 (m, 25H, CH, CH2), 1.87 (t, J = 2.4 Hz, 3H,
C≡CCH3), 2.42 (m, 1H, H-19), 3.19 (m, 1H, H-3), 3.94 (d, J = 10.8 Hz, 1H, H-28), 4.37 (d, J = 10.8 Hz,
1H, H-28), 4.59 (s, 1H, H-29), 4.69 (s, 1H, H-29), 4.70 (q, 2H, J = 2.4 Hz, OCH2). 13C-NMR (CDCl3)
: 3.7, 14.7, 15.3, 15.9, 16.1, 18.2, 19.0, 20.7, 25.1, 26.9, 27.3, 27.9, 29.4, 29.4, 34.1, 34.3, 37.1, 37.5,
38.6, 38.8, 40.8, 42.6, 46.5, 47.6, 48.7, 50.2, 55.2, 56.1, 66.9, 72.5, 76.7, 78.9, 109.9, 150.0, 155.2. IR
(KBr, cm−1) ν: 3462, 2942, 2248, 1748, 1248, 883. EI MS (70 eV) m/z (rel. intensity): 538 (M+, 14),
207 (48), 203 (53), 189 (100), 135 (60); Elemental anal. (%), calcd. for C35H54O4: C, 78.02; H, 10.10;
found: C, 77.84; H, 10.22.

Molecules 2013, 18
4535
28-O-Ethoxycarbonylbetulin (3d). Yield: 68%, m.p. 94–96 °C, Rf 0.47 (chloroform/ethanol, 40:1, v/v);
1H-NMR (CDCl3) : 0.75 (s, 3H, CH3), 0.81 (s, 3H, CH3), 0.96 (s, 3H, CH3), 0.97 (s, 3H, CH3), 1.03
(s, 3H, CH3), 1.31 (t, J = 7.2 Hz, 3H, OCH2CH3), 1.67 (s, 3H, CH3), 1.05–2.02 (m, 25H, CH, CH2),
2.42 (m, 1H, H-19), 3.18 (m, 1H, H-3), 3.90 (d, J = 10.8 Hz, 1H, H-28), 4.20 (q, J = 7.2 Hz, 2H,
OCH2), 4.32 (d, J = 10.8 Hz, 1H, H-28), 4.58 (s, 1H, H-29), 4.68 (s, 1H, H-29). 13C-NMR (CDCl3) :
14.2, 14.7, 15.3, 15.9, 16.0, 18.2, 19.0, 20.7, 25.1, 26.9, 27.3, 27.9, 29.5, 34.1, 34.3, 37.1, 37.5, 38.6,
38.8, 40.8, 42.6, 46.4, 47.6, 48.7, 50.3, 55.2, 63.9. 66.3, 78.9, 109.8, 150.0, 155.7. IR (KBr, cm−1) ν:
3486, 2942, 1744, 1249, 1013, 882. EI MS (70 eV) m/z (rel. intensity): 514 (M+, 18), 424 (40), 203
(54), 189 (100), 135 (56); Elemental anal. (%), calcd. For C33H54O4: C, 77.00; H, 10.57; found: C,
77.28; H, 10.43.
3,28-O,O'-Di(propargyloxycarbonyl)betulin (4a). Yield: 27%, m.p. 125–127 °C, Rf 0.77
(chloroform/ethanol, 40:1, v/v); 1H-NMR (CDCl3) : 0.84 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.91 (s, 3H,
CH3), 0.96 (s, 3H, CH3), 1.02 (s, 3H, CH3), 1.67 (s, 3H, CH3), 1.07–2.02 (m, 25H, CH, CH2), 2.42 (m,
1H, H-19), 2.51 (t, J = 2.4 Hz, 1H, C≡CH), 2.53 (t, J = 2.4 Hz, 1H, C≡CH), 3.94 (d, J = 10.8 Hz, 1H,
H-28), 4.33 (m, 1H, H-3), 4.37 (d, J = 10.8 Hz, 1H, H-28), 4.59 (s, 1H, H-29), 4.68 (s, 1H, H-29), 4.73
13
(d, J = 2.4 Hz, 2H, OCH2), 4.76 (d, J = 2.4, Hz, 2H, OCH2). C-NMR (CDCl3) : 15.9, 16.0, 16.1,
16.2, 16.3, 18.0, 19.0, 20.7, 23.5, 25.1, 27.0, 27.7, 27.9, 29.4, 34.0, 34.3, 37.0, 37.6, 38.0, 38.3, 40.8,
42.7, 46.5, 47.6, 48.7, 50.2, 54.9, 55.2, 55.6, 67.2, 75.6, 75.9, 77.0, 86.2, 109.9, 149.9, 153.9, 154.5. IR
(KBr, cm−1) ν: 3305, 2941, 2133, 1730, 1442, 1253. EI MS (70 eV) m/z (rel. intensity): 606 ((M+, 7),
506 (41), 203 (52), 189 (100), 121 (42); Elemental anal. (%), calcd. for C38H54O6: C, 75.21; H, 8.97;
found: C, 75.03; H 9.12.
3,28-O,O'-Di(3-butynyloxycarbonyl)betulin (4b). Yield: 23%, m.p. 140–142 °C, Rf 0.75 (chloroform/
ethanol, 40:1, v/v); 1H-NMR (CDCl3) : 0.84 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.91 (s, 3H, CH3), 0.97
(s, 3H, CH3), 1.03 (s, 3H, CH3), 1.68 (s, 3H, CH3), 1.05–2.02 (m, 25H, CH, CH2), 2.01 (t, J = 2.4 Hz,
1H, C≡CH), 2.03 (t, J = 2.4 Hz, 1H, C≡CH), 2.43 (m, 1H, H-19), 2.59 (m, 4H, 2 × OCH2CH2), 3.94
(d, J = 10.8 Hz, 1H, H-28), 4.24 (m, 4H, 2 × OCH2), 4.31 (m, 1H, H-3), 4.36 (d, J = 10.8 Hz, 1H,
H-28), 4.59 (s, 1H, H-29), 4.69 (s, 1H, H-29). 13C-NMR (CDCl3) : 14.6, 15.9, 16.1, 16.3, 18.0, 19.0,
20.7, 23.5, 25.0, 26.9, 27.8, 29.4, 33.9, 34.3, 36.9, 37.5, 38.0, 38.2, 40.8, 42.6, 46.4, 47.6, 48.7, 50.1,
55.3, 64.9, 65.3, 66.7, 70.1, 70.2, 76.7, 79.4, 79.5, 79.4, 85.6, 109.9, 149.9, 154.9, 155.4. IR (KBr,
cm−1) ν: 3312, 2951, 2128, 1745, 1249, 884. EI MS (70 eV) m/z (rel. intensity): 634 (M+, 3), 203 (40),
189 (100), 135 (48), 121 (46); Elemental anal. (%), calcd. for C40H58O6: C, 75.67; H, 9.21; found: C,
75.89; H, 9.14.
3,28-O,O'-Di(2-butynyloxycarbonyl)betulin (4c). Yield: 28%, m.p. 70–73 °C, Rf 0.78 (chloroform/
ethanol, 40:1, v/v); 1H-NMR (CDCl3) : 0.84 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.91 (s, 3H, CH3), 0.97
(s, 3H, CH3), 1.03 (s, 3H, CH3), 1.68 (s, 3H, CH3), 1.05–2.02 (m, 25H, CH, CH2), 1.86 (t, J = 2.4 Hz,
3H, C≡CCH3), 1.87 (t, J = 2.4 Hz, 3H, C≡CCH3), 2.42 (m, 1H, H-19), 3.94 (d, J = 10.8 Hz, 1H, H-28),
4.32 (m, 1H, H-3), 4.37 (d, J = 10.8 Hz, 1H, H-28), 4.59 (s, 1H, H-29), 4.69 (s, 1H, H-29), 4.71 (m,
4H, 2 × OCH2). 13C-NMR (CDCl3) : 5.1, 5.3, 15.9, 16.1, 16.3, 18.0, 19.0, 20.7, 23.5, 25.0, 26.9, 27.8,
29.4, 33.9, 34.2, 36.9, 37.5, 37.9, 38.2, 40.8, 42.6, 46.4, 47.6, 48.7, 50.1, 55.3, 55.8, 56.1, 56.4, 66.9,

Molecules 2013, 18
4536
72.5, 72.7, 83.9, 84.1, 84.2, 85.8, 109.9, 149.9, 154.7, 155.2. IR (KBr, cm−1) ν: 2948, 2241, 1747,
1456, 1265, 884. EI MS (70 eV) m/z (rel. intensity): 634 (M+, 6), 520 (94), 203 (42), 189 (100), 135
(41); Elemental anal. (%), calcd. for C40H58O6: C, 75.67; H, 9.21; found: C, 75.42; H, 9.33.
3,28-O,O'-Di(ethoxycarbonyl)betulin (4d). Yield: 24%, m.p. 84–86 °C, Rf 0.76 (chloroform/ethanol,
1
40:1, v/v); H-NMR (CDCl3) : 0.86 (s, 3H, CH3), 0.87 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.99 (s, 3H,
CH3), 1.05 (s, 3H, CH3), 1.32 (m, 6H, 2 × OCH2CH3), 1.69 (s, 3H, CH3), 0.81–2.05 (m, 25H, CH,
CH2), 2.44 (m, 1H, H-19), 3.92 (d, J = 10.8 Hz, 1H, H-28), 4.22 (m, 4H, 2 × OCH2), 4.31 (m, 1H, H-3)
4.35 (d, J = 10.8 Hz, 1H, H-28), 4.60 (s, 1H, H-29), 4.70 (s, 1H, H-29). 13C-NMR (CDCl3) : 14.2, 14.7,
16.0, 16.1, 16.4, 18.1, 19.1, 20.8, 23.6, 25.1, 27.0, 27.8, 29.5, 34.1, 34.3, 37.0, 37.6, 38.0, 38.3, 40.9,
42.7, 46.5, 47.6, 48.8, 50.2, 55.4, 63.6, 63.9, 66.4, 85.1, 109.9, 150.0, 155.2, 155.7. IR (KBr, cm−1) ν:
2948, 1741, 1466, 1265, 1009, 885. EI MS (70 eV) m/z (rel. intensity): 586 (M+, 4), 496 (70), 203 (42),
189 (100), 135 (41). Elemental anal. (%), calcd. for C36H58O6: C, 73.68; H, 9.96; found: C, 73.56; H, 9.85.
3.4. General Procedure for the Synthesis of Monoesters 5 and Diesters 6
To a mixture of betulin (1, 0.44 g, 1 mmol) and propynoic acid (0.08 g, 1.10 mmol), phenylpropynoic
acid (0.16 g, 1.10 mmol) or propanoic acid (0.08 g, 1.10 mmol) in dry dichloromethane (5 mL) under
argon at −10 °C, a solution of DCC (0.23 g, 1.12 mmol) and DMAP (0.01 g, 0.08 mmol) in dry
dichloromethane (1 mL) was added. Stirring at this temperature was continued for 5 h. Then the
reaction mixture was allowed to warm to room temperature and stirred over night. After the reaction
was complete, the reaction mixture was filtered and the solvent was removed under reduced pressure.
The crude product was purified by column chromatography (chloroform/ethanol 40:1, v/v) to give pure
corresponding monoesters 5 and diesters 6.
28-O-Propynoylbetulin (5a). Yield: 60%, m.p. 133–135 °C, Rf 0.46 (chloroform/ethanol, 20:1, v/v);
1H-NMR (CDCl3) : 0.76 (s, 3H, CH3), 0.82 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.98 (s, 3H, CH3), 1.03
(s, 3H, CH3), 1.68 (s, 3H, CH3), 1.06–1.98 (m, 25H, CH, CH2), 2.43 (m, 1H, H-19), 2.89 (s, 1H,
C≡CH), 3.18 (m, 1H, H-3), 3.99 (d, J = 10.8 Hz, 1H, H-28), 4.38 (d, J = 10.8 Hz, 1H, H-28), 4.59 (s,
1H, H-29), 4.69 (s, 1H, H-29). 13C-NMR (CDCl3) : 14.7, 15.3, 15.9, 16.0, 18.2, 19.1, 20.7, 25.1, 26.9,
27.3, 27.9, 29.4, 29.5, 34.1, 34.4, 37.1, 37.6, 38.6, 38.8, 40.8, 42.6, 46.3, 47.6, 48.7, 50.3, 55.2, 64.8,
74.6, 74.6, 78.9, 109.9, 149.8, 153.2. IR (KBr, cm−1) ν: 3419, 3302, 2994, 2120, 1715, 1227. EI MS
(70 eV) m/z (rel. intensity): 494 (M+, 32), 207 (66), 203 (50), 189 (100), 135 (59); Elemental anal. (%),
calcd. for C33H50O3: C, 80.11; H, 10.19; found: C, 80.39; H, 10.02.
28-O-Phenylpropynoylbetulin (5b). Yield: 70%, m.p. 204–206 °C, Rf 0.47 (chloroform/ethanol, 20:1,
1
v/v); H-NMR (CDCl3) : 0.76 (s, 3H, CH3), 0.83 (s, 3H, CH3), 0.97 (s, 3H, CH3), 0.99 (s, 3H, CH3),
1.05 (s, 3H, CH3), 1.69 (s, 3H, CH3), 1.07–2.05 (m, 25H, CH, CH2), 2.46 (m, 1H, H-19), 3.18 (m, 1H,
H-3), 4.04 (d, J = 10.8 Hz, 1H, H-28), 4.42 (d, J = 10.8 Hz, 1H, H-28), 4.60 (s, 1H, H-29), 4.70 (s, 1H,
13
H-29), 7.36–7.60 (m, 5H, Ar-H). C-NMR (CDCl3) : 14.7, 15.3, 16.0, 16.1, 18.2, 19.1, 20.7, 25.1,
27.0, 27.3, 27.9, 29.5, 29.6, 34.1, 34.5, 37.1, 37.6, 38.6, 38.8, 40.8, 42.7, 46.4, 47.6, 48.8, 50.3, 55.2,
64.5, 78.9, 80.7, 86.2, 109.9, 119.6, 128.5, 128.5, 130.5, 132.9, 132.9, 149.9, 154.7. IR (KBr, cm−1) ν:
3587, 2993, 2220, 1691, 1287, 1195. EI MS (70 eV) m/z (rel. intensity): 570 (M+, 15), 189 (71), 135

Molecules 2013, 18
4537
(42), 129 (100), 95 (41); Elemental anal. (%), calcd. for C39H54O3: C, 82.06; H, 9.53; found: C, 82.31;
H, 9.48.
28-O-Propanoylbetulin (5c). Yield: 86%, m.p. 160–162 °C, Rf 0.46 (chloroform/ethanol, 20:1, v/v);
1H-NMR (CDCl3) : 0.75 (s, 3H, CH3), 0.81 (s, 3H, CH3), 0.96 (s, 3H, CH3), 0.97 (s, 3H, CH3), 1.02
(s, 3H, CH3), 1.15 (t, J = 7.2 Hz, 3H, CH2CH3), 1.67 (s, 3H, CH3), 0.88–1.93 (m, 25H, CH, CH2), 2.34
(q, J = 7.2 Hz, 2H, CH2CH3), 2.43 (m, 1H, H-19), 3.18 (m, 1H, H-3), 3.84 (d, J = 10.8 Hz, 1H, H-28),
4.25 (d, J = 10.8 Hz, 1H, H-28), 4.58 (s, 1H, H-29), 4.68 (s, 1H, H-29). 13C-NMR (CDCl3) : 9.2, 14.7,
15.3, 15.9, 16.0, 18.2, 19.1, 20.7, 25.1, 27.0, 27.3, 27.7, 27.9, 29.5, 29.7, 34.1, 34.5, 37.1, 37.5, 38.6,
38.8, 40.8, 42.6, 46.3, 47.6, 48.7, 50.3, 55.2, 62.5, 78.9, 109.8, 150.1, 174.9. IR (KBr, cm−1) ν: 3357,
2940, 1735, 1276, 1186, 882. EI MS (70 eV) m/z (rel. intensity): 498 (M+, 17), 207 (52), 203 (54), 189
(100), 135 (58); Elemental anal. (%), calcd. for C33H54O3: C, 79.46; H, 10.91; found: C, 79.34; H, 10.82.
3,28-O,O'-Dipropynoylbetulin (6a). Yield: 12%, m.p. 102–104 °C, Rf 0.73 (chloroform/ethanol, 20:1,
1
v/v); H-NMR (CDCl3) : 0.86 (s, 3H, CH3), 0.87 (s, 3H, CH3), 0.89 (s, 3H, CH3), 0.98 (s, 3H, CH3),
1.03 (s, 3H, CH3), 1.68 (s, 3H, CH3), 1.07–2.01 (m, 25H, CH, CH2), 2.43 (m, 1H, H-19), 2.85 (s, 1H,
C≡CH), 2.89 (s, 1H, C≡CH), 3.99 (d, J = 10.8 Hz, 1H, H-28), 4.39 (d, J = 10.8 Hz, 1H, H-28), 4.59
(m, 1H, H-3), 4.60 (s, 1H, H-29), 4.69 (s, 1H, H-29). 13C-NMR (CDCl3) : 14.7, 15.9, 16.1, 16.4, 18.1,
19.1, 20.7, 23.4, 25.1, 26.9, 27.8, 29.4, 29.5, 34.0, 34.4, 37.0, 37.6, 37.9, 38.3, 40.8, 42.6, 46.3, 47.6,
48.7, 50.2, 55.3, 64.8, 74.1, 74.6, 74.7, 75.1, 83.6, 110.0, 149.8, 152.7, 153.2. IR (KBr, cm−1) ν: 3301,
2945, 2119, 1714, 1234, 886. EI MS (70 eV) m/z (rel. intensity): 546 (M+, 9), 195 (46), 189 (53), 113
(41), 55 (100); Elemental anal. (%), calcd. for C36H50O4: C, 79.08; H, 9.22; found: C, 79.16; H, 9.28.
3,28-O,O'-Di(phenylpropynoyl)betulin (6b). Yield: 27%, m.p. 91–92 °C, Rf 0.79 (chloroform/ethanol,
1
20:1, v/v); H-NMR (CDCl3) : 0.88 (s, 3H, CH3), 0.92 (s, 3H, CH3), 0.93 (s, 3H, CH3), 0.99 (s, 3H,
CH3), 1.06 (s, 3H, CH3), 1.70 (s, 3H, CH3), 1.06–2.05 (m, 25H, CH, CH2), 2.46 (m, 1H, H-19), 4.04
(d, J = 10.8 Hz, 1H, H-28), 4.42 (d, J = 10.8 Hz, 1H, H-28), 4.61 (s, 1H, H-29), 4.66 (m, 1H, H-3),
13
4.71 (s, 1H, H-29), 7.36–7.60 (m, 10H, 2 × Ar-H). C-NMR (CDCl3) : 14.7, 16.0, 16.1, 16.5, 18.1,
19.1, 20.8, 23.6, 25.1, 25.8, 25.9, 27.0, 27.9, 29.4, 29.5, 29.6, 34.1, 34.5, 37.0, 37.6, 38.0, 38.4, 40.9,
42.7, 46.4, 47.6, 48.8, 50.2, 55.4, 64.5, 80.7, 81.0, 83.2, 85.7, 86.3, 109.9, 119.6, 119.8, 128.4, 128.5,
130.4, 130.5, 132.9, 132.9, 149.9, 154.2, 154.7. IR (KBr, cm−1) ν: 2945, 2223, 1706, 1490, 1285, 1187.
EI MS (70 eV) m/z (rel. intensity): 698 (M+, 6), 552 (30), 189 (33), 135 (15), 129 (100); Elemental
anal. (%), calcd. for C48H58O4: C, 82.48; H, 8.36; found: C, 82.35; H, 8.27.
3,28-O,O'-Dipropanoylbetulin (6c). Yield: 11%, m.p. 142–144 °C, Rf 0.71 (chloroform/ethanol, 20:1,
1
v/v); H-NMR (CDCl3) : 0.83 (s, 3H, CH3), 0.83 (s, 3H, CH3), 0.84 (s, 3H, CH3), 0.96 (s, 3H, CH3),
1.02 (s, 3H, CH3), 1.15 (m, 6H, 2 × CH2CH3), 1.68 (s, 3H, CH3), 1.06–1.96 (m, 25H, CH, CH2), 2.34
(m, 4H, 2 × CH2CH3), 2.43 (m, 1H, H-19), 3.83 (d, J = 10.8 Hz, 1H, H-28), 4.26 (d, J = 10.8 Hz, 1H,
H-28), 4.65 (m, 1H, H-3), 4.58 (s, 1H, H-29), 4.68 (s, 1H, H-29). 13C-NMR (CDCl3) : 9.2, 9.3, 14.7,
15.9, 16.1, 16.5, 18.1, 19.1, 20.7, 23.6, 25.1, 27.0, 27.7, 27.9, 28.0, 29.5, 29.7, 34.0, 34.5, 37.0, 37.4,
37.8, 38.3, 40.8, 42.6, 46.3, 47.6, 48.7, 50.2, 55.3, 62.5, 80.5, 109.8, 150.1, 174.3, 174.9. IR
(KBr, cm−1) ν: 2960, 1736, 1461, 1261, 1185, 891. EI MS (70 eV) m/z (rel. intensity): 554 (M+, 7), 480

Molecules 2013, 18
4538
(100), 203 (45), 189 (98), 135 (37); Elemental anal. (%), calcd. for C36H58O4: C, 77.93; H, 10.54;
found: C, 77.85; H, 10.62.
3.5. Synthesis of 28-O-Acetylbetulin (7a) and 3,28-O,O'-Diacetylbetulin (7b)
To a mixture of betulin (1, 1.0 g, 2.26 mmol), DMAP (0.01 g, 0.07 mmol) and pyridine (1.5 mL) in
dry dichloromethane (9 mL) was added dropwise acetic anhydride (0.24 g, 2.26 mmol) at 0 °C, and the
reaction mixture was stirred at room temperature. After being stirred for 18 h, the mixture was washed
with 10% aqueous hydrochloric acid (30 mL), water (15 mL), saturated aqueous NaHCO3 solution and
then brine (20 mL). After separation of phases, the organic layer was dried with anhydrous magnesium
sulfate and concentrated under reduced pressure. The crude product was purified by column
chromatography (chloroform/ethanol, 40:1, v/v) to give 28-O-acetylbetulin (7a, 0.77 g, 71%): m.p.
214–215 °C (lit. m.p. 210–212 °C [28]; 217–219 °C [34]); Rf 0.44 (chloroform/ethanol, 20:1, v/v) and
1
3,28-O,O'-diacetylbetulin (7b, 0.21 g, 18%): m.p. 220–222 °C (lit. m.p. 221–223 °C [34]). H and
13C-NMR spectral data of 7a and 7b were in agreement with those published in the literature [28,34].
3.6. Synthesis of 28-O-acetyl-3-O'-alkynyloxycarbonylbetulins (8)
To a mixture of 28-O-acetylbetulin (7a, 0.48 g, 1 mmol) and pyridine (2.5 mL) in dry benzene (6 mL)
at 0–5 °C, a solution of propargyl chloroformate or 2-butyn-1-yl chloroformate or 3-butyn-1-yl
chloroformate or ethyl chloroformate (2.0 mmol) in dry benzene (5 mL) was added. Stirring at this
temperature was continued for 4 h. Then the reaction mixture was allowed to warm to room
temperature and stirred over night. At the end of the reaction 5 mL of chloroform was added and the
reaction mixture was washed with 1 N sulfuric acid, followed by water, then dried with anhydrous
magnesium sulfate and concentrated under reduced pressure. The crude product was purified by
column chromatography using chloroform/ethanol (40:1, v/v) to give pure corresponding esters 8.
28-O-Acetyl-3-O'-propargyloxycarbonylbetulin (8a). Yield: 89%, m.p. 188–189 °C, Rf 0.73 (chloroform/
ethanol, 20:1, v/v); 1H-NMR (CDCl3) : 0.84 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.92 (s, 3H, CH3), 0.97
(s, 3H, CH3), 1.03 (s, 3H, CH3), 1.68 (s, 3H, CH3), 1.06–1.99 (m, 25H, CH, CH2), 2.07 (s, 3H,
COCH3), 2.44 (m, 1H, H-19), 2.51 (t, 1H, J = 2.4 Hz, C≡CH), 3.85 (d, J = 10.8 Hz, 1H, H-28), 4.24
(d, J = 10.8 Hz, 1H, H-28), 4.33 (m, 1H, H-3), 4.58 (s, 1H, H-29), 4.68 (s, 1H, H-29), 4.71 (d, 2H,
J = 2.4 Hz, OCH2). 13C-NMR (CDCl3) : 14.6, 16.0, 16.1, 18.1, 19.1, 20.8, 21.0, 23.5, 25.1, 27.0, 27.7,
27.8, 29.5, 29.7, 34.1, 34.5, 37.0, 37.5, 38.0, 40.8, 42.6, 46.3, 47.7, 48.7, 50.2, 54.9, 55.6, 62.7, 75.4,
75.9, 77.2, 86.2, 109.8, 150.1, 154.5, 171.6. IR (KBr, cm−1) ν: 3298, 2997, 2140, 1735, 1243, 889. EI
MS (70 eV) m/z (rel. intensity): 566 (M+, 7), 466 (76), 203 (46), 189 (100), 135 (40); Elemental anal.
(%), calcd. for C36H54O5: C, 76.28; H, 9.60; found: C, 76.49; H, 9.52.
28-O-Acetyl-3-O'-(3-butynyloxycarbonyl)betulin (8b). Yield: 63%, m.p. 118–121 °C, Rf 0.72
(chloroform/ethanol, 20:1, v/v); 1H-NMR (CDCl3) : 0.83 (s, 3H, CH3), 0.84 (s, 3H, CH3), 0.90 (s, 3H,
CH3), 0.96 (s, 3H, CH3), 1.02 (s, 3H, CH3), 1.67 (s, 3H, CH3), 1.06–1.99 (m, 25H, CH, CH2), 2.00 (t,
J = 2.7 Hz, 1H, C≡CH), 2.06 (s, 3H, COCH3), 2.44 (m, 1H, H-19), 2.57 (m, J = 2.7 Hz, J = 7.2 Hz 2H,
OCH2CH2), 3.84 (d, J = 10.8 Hz, 1H, H-28), 4.21 (t, J = 7.2 Hz, 2H, OCH2), 4.25 (d, J = 10.8 Hz, 1H,

Molecules 2013, 18
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13
H-28), 4.32 (m, 1H, H-3), 4.58 (s, 1H, H-29), 4.68 (s, 1H, H-29). C-NMR (CDCl3) : 14.7, 15.9,
16.1, 16.3, 18.0, 19.0, 20.7, 21.0, 23.5, 25.0, 26.9, 27.8, 29.5, 29.6, 34.0, 34.5, 36.9, 37.4, 38.0, 38.2,
40.8, 42.6, 46.2, 47.6, 48.7, 50.2, 55.3, 62.7, 65.0, 65.5, 70.1, 79.4, 85.6, 109.8, 150.1, 154.9, 171.6. IR
(KBr, cm−1) ν: 3284, 2944, 2212, 1741, 1249, 889. EI MS (70 eV) m/z (rel. intensity): 580 (M+, 7), 466
(66), 203 (42), 189 (100), 135 (46); Elemental anal. (%), calcd. for C37H56O5: C, 76.51; H, 9.72; found:
C, 76.79; H, 9.59.
28-O-Acetyl-3-O'-(2-butynyloxycarbonyl)betulin (8c). Yield: 67%, m.p. 126–129 °C, Rf 0.75
(chloroform/ethanol, 20:1, v/v); 1H-NMR (CDCl3) : 0.84 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.91 (s, 3H,
CH3), 0.97 (s, 3H, CH3), 1.03 (s, 3H, CH3), 1.68 (s, 3H, CH3), 1.06–1.99 (m, 25H, CH, CH2), 1.86 (t,
J = 2.4 Hz, 3H, C≡CCH3), 2.07 (s, 3H, COCH3), 2.44 (m, 1H, H-19), 3.84 (d, J = 10.8 Hz, 1H, H-28),
4.24 (d, J = 10.8 Hz, 1H, H-28), 4.32 (m, 1H, H-3), 4.59 (s, 1H, H-29), 4.68 (s, 1H, H-29), 4.71 (q,
13
J = 2.4 Hz, 2H, OCH2). C-NMR (CDCl3) : 3.6, 14.7, 16.1, 16.3, 18.0, 19.0, 20.7, 21.0, 23.5, 25.0,
26.9, 27.8, 29.5, 29.6, 34.0, 34.5, 36.9, 37.4, 38.0, 38.2, 40.8, 42.6, 46.2, 47.6, 48.7, 50.2, 55.3, 56.4,
62.7, 72.7, 83.8, 84.2, 85.9, 109.8, 150.1, 154.7, 171.6. IR (KBr, cm−1) ν: 2956, 2233, 1742, 1457,
1256, 920. EI MS (70 eV) m/z (rel. intensity): 580 (M+, 3), 466 (64), 203 (37), 189 (100), 187 (47), 135
(40); Elemental anal. (%), calcd. for C37H56O5: C, 76.51; H, 9.72; found: C, 76.63; H, 9.87.
28-O-Acetyl-3-O'-(ethoxycarbonyl)betulin (8d). Yield: 84%, m.p. 200–202 °C, Rf 0.73 (chloroform/
ethanol, 20:1, v/v); 1H-NMR (CDCl3) : 0.84 (s, 3H, CH3), 0.85 (s, 3H, CH3), 0.91 (s, 3H, CH3), 0.97
(s, 3H, CH3), 1.03 (s, 3H, CH3), 1.30 (t, J = 7.2 Hz, 3H, CH2CH3), 1.68 (s, 3H, CH3), 1.05–2.00 (m,
25H, CH, CH2), 2.07 (s, 3H, COCH3), 2.44 (m, 1H, H-19), 3.85 (d, J = 10.8 Hz, 1H, H-28), 4.18 (q,
J = 7.2 Hz, 2H, OCH2), 4.24 (d, J = 10.8 Hz, 1H, H-28), 4.30 (m, 1H, H-3), 4.59 (s, 1H, H-29), 4.69 (s,
13
1H, H-29). C-NMR (CDCl3) : 14.3, 14.7, 16.0, 16.1, 16.4, 18.1, 19.1, 20.8, 21.0, 23.6, 25.1, 27.0,
27.8, 29.6, 34.1, 34.5, 37.0, 37.5, 38.0, 38.3, 40.9, 42.7, 46.3, 47.7, 48.7, 50.2, 55.4, 62.8, 63.6, 85.1,
109.8, 150.1, 155.2, 171.5. IR (KBr, cm−1) ν: 2941, 1741, 1446, 1253, 1012, 890. EI MS (70 eV) m/z
(rel. intensity): 556 (M+, 6), 466 (73), 203 (38), 189 (100), 135 (41); Elemental anal. (%), calcd. for
C35H56O5: C, 75.50; H, 10.14; found: C, 75.68; H, 10.04.
3.7. Crystal Structure Determination
X-ray diffraction measurement was performed at 100(1) K. The colorless single crystal was
preselected under polarization microscope. The crystal was mounted on a glass capillary and cooled
down by a cold, dry nitrogen gas stream (Oxford Cryosystem Equipment). The data were collected
using SuperNova kappa diffractometer with Atlas CCD detector and CuKα radiation. Accurate cell
parameters were determined and refined with CrysAlis Pro program [35]. Also for the integration of
the collected data program CrysAlis Pro was used. The structure was solved using direct methods with
SHELXS97 program and then refined using SHELXL-97 program [36]. Nonhydrogen atoms were
refined with anisotropic displacement parameters. The hydrogen atoms were fixed geometrically at
calculated distances and allowed to ride on the parent carbon or oxygen atoms.
CCDC-873113 contains the supplementary crystallographic data for this paper. These data can be
obtained free of charge at http://www.ccdc.cam.ac.uk/conts/retrieving.html or from the Cambridge

Molecules 2013, 18
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Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44-1223-336033;
E-Mail: deposit@ccdc.cam.ac.uk.
3.8. Antiproliferative Assay in Vitro
3.8.1. Cells
The following established in vitro cell lines were applied: SW707 (human colorectal
adenocarcinoma), CCRF/CEM (human leukemia), T47D (human breast cancer), P388 (mouse
leukemia) and Balb3T3 (normal mouse fibroblasts). All lines were obtained from the American Type
Culture Collection (Rockville, MD, USA) and maintained at the Cell Culture Collection of the
Institute of Immunology and Experimental Therapy (Wroclaw, Poland).
Twenty-four hours before addition of the tested agents, the cells were plated in 96-well plates
(Sarstedt, Numbrecht, Germany) at a density of 104 cells per well in 100 μL of culture medium. The
cells were cultured in the opti-MEM medium supplemented with 2 mM glutamine (Gibco, Grand
Island, NY, USA), streptomycin (50 μg/mL), penicillin (50 U/mL) (both antibiotics from Polfa,
Tarchomin, Poland) and 5% fetal calf serum (Gibco). The cell cultures were maintained at 37 C in
humid atmosphere saturated with 5% CO2.
3.8.2. Compounds
The compounds were dissolved in DMSO and culture medium (1:9) to the concentration of 1 mg/mL,
and subsequently diluted in culture medium to reach the required concentrations (ranging from 1 to
100 mg/mL).
3.9. Antiproliferative Assay
The in vitro cytotoxic effect of all agents was examined using the SRB assay for adherent cells and
MTT assay for leukemia cells as described by Wietrzyk et al. [37]. Each compound in given
concentration was tested in triplicates in each experiment, which was repeated 3–5 times. The results
of cytotoxic activity in vitro were expressed as an IC50 – the concentration of compound (in μg/mL)
that inhibits proliferation rate of the tumor cells by 50% as compared to the control untreated cells.
4. Conclusions
New acetylenic derivatives of betulin containing one or two acetylenic formate or propynoyl
functions at the C-3 and/or C-28 positions were synthesized. The structures of the title compounds
1
were confirmed by H, 13C-NMR, IR, MS, and elemental analyses. Furthermore, the structure of
28-O-propynoylbetulin (5a) was also determined by X-ray crystal analysis. The presented studies
demonstrated that a simple modification of the parent structure of betulin (1) can produce new
potentially interesting anti-cancer agents. Among all studied compounds, 28-O-propynoylbetulin (5a)
exhibited the strongest cytotoxic activity and it was over 500 times more cytotoxic than betulin (1) and
100 times more cytotoxic than cisplatin against CCRF/CEM cancer cells with promising selectivity
index (SI = 15) under in vitro conditions. It was found that the betulins which possess a carbonyl group

Molecules 2013, 18
4541
at C-28 position directly bonded to the triple bond of an ethynyl substituent, showed strong cytotoxic
effects against human leukemia (CCRF/CEM) and murine leukemia (P388) cancer cells. It was also
found that the free C-3 hydroxyl function is a structural requirement for the cytotoxicity of the studied
compounds. The compound 5a has been selected for further studies. Noteworthy feature of the
obtained results was the observation that leukemia (CCRF/CEM and P388) cells appear to be more
sensitive to the cytotoxic effects of the compounds 3 and 5 than the other cancer cells lines applied.
Acknowledgments
This work was supported by the Medical University of Silesia in Katowice, Poland. Grant No
KNW-1-006/P/2/0.
References
1. Lowitz, J.T. Űber eine neue, fast benzoeartige substanz der briken. Crell’s Chem. Ann. 1788, 1,
312–317.
2. Tolstikov, G.A.; Flekhter, O.B.; Shultz, E.E.; Baltina, L.A.; Tolstikov, A.G. Betulin and its
derivatives. Chemistry and biological activity. Chem. Sustain. Develop. 2005, 13, 1–29.
3. Alakurtti S.; Mäkelä, T.; Koskimies, S.; Yli-Kauhaluoma, J. Pharmacological properties of the
ubiquitous natural product betulin. Eur. J. Pharm. Sci. 2006, 29, 1–13.
4. Tolstikova, T.G.; Sorokina, I.V.; Tolstikov, G.A.; Tolstikov, A.G.; Flekhter, O.B. Biological
activity and pharmacological prospects of lupane terpenoids: II. Semisynthetic lupane derivatives.
Russ. J. Bioorg. Chem. 2006, 32, 261–276.
5. Drebushchak, T.N.; Mikhailenko, M.A.; Brezgunova, M.E.; Shakhtshneider, T.P.; Kuznetsova S.A.
Crystal structure of betulin ethanol solvate. J. Struct. Chem. 2010, 51, 798–801.
6. Boryczka, S.; Michalik, E.; Jastrzębska, M.; Kusz, J.; Zubko, M.; Bębenek, E. X-ray crystal
structure of betulin-DMSO solvate. J. Chem. Crystallogr. 2012, 42, 345–351.
7. Mukherjee, R.; Kumar, V.; Srivastava, S.K.; Agarwal, S.K.; Burman, A.C. Betulinic acid
derivatives as anticancer agents: Structure activity relationship. Anticancer Agents Med. Chem.
2006, 6, 271–279.
8. Pisha, E.; Chai, H.; Lee, I.S.; Chagwedera, T.E.; Farnsworth, N.R.; Cordell, G.A.; Beecher, C.W.W.;
Fong, H.H.S.; Kinghorn, A.D.; Brown, D.M.; et al. Discovery of betulinic acid as a selective
inhibitor of human melanoma that functions by induction of apoptosis. Nat. Med. 1995, 1, 1046–1051.
9. Eiznhamer, D.A.; Xu, Z.-Q. Betulinic acid: A promising anticancer candidate. Idrugs 2004, 7,
359–373.
10. Mullauer, F.B.; Kessler, J.H.; Medema, J.P. Betulinic acid, a natural compound with potent
anticancer effects. Anticancer Drugs 2010, 21, 215–227.
11. Rzeski, W.; Stepulak, A.; Szymański, M.; Juszczak, M.; Grabarska, A.; Sifringer, M.; Kaczor, J.;
Kandefer-Szerszeń, M. Betulin elicits anti-cancer effects in tumour primary cultures and cell lines
in vitro. Basic Clin. Pharmacol. Toxicol. 2009, 105, 425–432.
12. Dehelean, C.A.; Feflea, S.; Molnar, J.; Zupko, I.; Soica, C. Betulin as an antitumor agent tested in vitro
on A431, HeLa and MCF7, and as an angiogenic inhibitor in vivo in the CAM assay. Nat. Prod.
Commun. 2012, 7, 981–985.

Molecules 2013, 18
4542
13. Alakurtti, S.; Heiska, T.; Kiriazis, A.; Sacerdoti-Sierra, N.; Jaffe, C.L.; Yli-Kauhaluoma, J.
Synthesis and anti-leishmanial activity of heterocyclic betulin derivatives. Bioorg. Med. Chem.
2010, 18, 1573–1582.
14. Urban, M.; Vlk, M.; Dzubak, P.; Hajduch, M.; Sarek, J. Cytotoxic heterocyclic triterpenoids
derived from betulin and betulinic acid. Bioorg. Med. Chem. 2012, 20, 3666–3674.
15. Pohjala, L.; Alakurtti, S.; Ahola, T.; Yli-Kauhaluoma, J.; Tammela, P. Betulin-derived
compounds as inhibitors of Alphavirus replication. J. Nat. Prod. 2009, 72, 1917–1926.
16. Kommera, H.; Kaluderović, G.N.; Dittrich, S.; Kalbitz, J.; Dräger, B.; Mueller, T.; Paschke, R.
Carbamate derivatives of betulinic acid and betulin with selective cytotoxic activity. Bioorg. Med.
Chem. Lett. 2010, 20, 3409–3412.
17. Ben-Zvi, Z.; Danon, A. Supplement C2: The Chemistry of Triple Bonded Functional Groups. In
The Chemistry of Functional Groups; Patai, S., Ed.; Wiley & Sons: New York, NY, USA, 1994;
pp. 739–788.
18. Csuk, R.; Barthel, A.; Schwarz, S.; Kommera, H.; Paschke, R. Synthesis and biological
evaluation of antitumor-active -butyrolactone substituted betulin derivatives. Bioorg. Med. Chem.
2010, 18, 2549–2558.
19. Kazakova, O.B.; Giniyatullina, G.V.; Yamansarov, E.Y.; Tolstikov, G.A. Betulin and ursolic acid
synthetic derivatives as inhibitors of Papilloma virus. Bioorg. Med. Chem. Lett. 2010, 20, 4088–4090.
20. Csuk, R.; Sczepek, R.; Siewert, B.; Nitsche, C. Cytotoxic betulin-derived hydroxypropargylamines
trigger apoptosis. Bioorg. Med. Chem. 2013, 21, 425–435.
21. Csuk, R.; Barthel, A.; Kluge, R.; Ströhl, D. Synthesis, cytotoxicity and liposome preparation of
28-acetylenic betulin derivatives. Bioorg. Med. Chem. 2010, 18, 7252–7259.
22. Kazakova, O.B.; Yamansarov, E.Yu.; Spirikhin, L.V.; Yunusov, M.S.; Baikova, I.P.;
Kukovinets, O.S.; Musin, R.Z. Effective synthesis and transformations of alkyne betulin
derivatives. Russ. J. Bioorg. Chem. 2011, 47, 456–460.
23. 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, 1, 37–49.
24. Bori, I.D.; Hung, H.-Y.; Qian, K.; Chen, C.-H.; Morris-Natschke, S.L.; Lee, H.-K. Anti-AIDS
agents 88. Anti-HIV conjugates of betulin and betulinic acid with AZT prepared via click
chemistry. Tetrahedron Lett. 2012, 53, 1987–1989.
25. Mól, W.; Matyja, M.; Filip, B.; Wietrzyk, J.; Boryczka, S. Synthesis and antiproliferative activity
in vitro of novel (2-butynyl)thioquinolines. Bioorg. Med. Chem. 2008, 16, 8136–8141.
26. Boryczka, S.; Mól, W.; Milczarek, M.; Wietrzyk, J.; Bębenek, E. Synthesis and in vitro
antiproliferative activity of novel (4-chloro- and 4-acyloxy-2-butynyl)thioquinolines Med. Chem. Res.
2011, 20, 1402–1410.
27. Lugemwa, F.L.; Huang, F.-Y.; Bentley, M.D.; Mendel, M.J.; Alford, A.R. A Heliothis zea
antifeedant from the abundant birchbark triterpene betulin. J. Agric. Food Chem. 1990, 38, 493–496.
28. Deng, Y.; Snyder, J.K. Preparation of a 24-nor-1,4-dien-3-one triterpene derivative from betulin:
A new route to 24-nortriterpene analogues. J. Org. Chem. 2002, 67, 2864–2873.
29. Mohammed, I.E.; Choudhary, M.I.; Ali, S.; Anjum, S.; Atta-ur-Rahman. 20(29)-Lupene-3,28-
diacetate. Acta Crystallogr. 2006, E62, o1352–o1354.

Molecules 2013, 18
4543
30. Boryczka, S.; Bębenek, E.; Jastrzębska, M.; Kusz, J.; Zubko, M. Crystal structure of betulinic
acid-DMSO solvate. Z. Kristallogr. 2012, 227, 379–384.
31. Geran, R.I.; Greenberg, N.H.; Macdonald, M.M.; Schumacher, A.M.; Abott, B.J. Cell culture
technical procedures. Cancer Chemother. Rep. 1972, 3, 59–72.
32. Lamberth, C. Alkyne chemistry in crop protection. Bioorg. Med. Chem. 2008, 17, 4047–4063.
33. Gauthier, C.; Legault, J.; Lebrun, M.; Dufour, P.; Pichette, A. Glycosidation of lupine-type
triterpenoids as potent in vitro cytotoxic agents. Bioorg. Med. Chem. 2006, 14, 6713–6725.
34. Bodrikov, I.V.; Borisova, N.V.; Chiyanov, A.A.; Kurskii, Y.A.; Fukin, G.K. Vinylic substitution
in the reaction of betulin diacetate with tert-butyl hypochlorite. Russ. J. Org. Chem. 2013, 49, 78–82.
35. Oxford Diffraction, CrysAllis CCD and CrysAllis RED, Versions1.171.32.29; Oxford Diffraction
Ltd.: Wrocław, Poland, 2008.
36. Sheldrick, G.M. A short history of SHELX. Acta Crystallogr. 2008, A64, 112–122.
37. Wietrzyk, J.; Chodynski, M.; Fitak, H.; Wojdat, E.; Kutner, A.; Opolski, A. Antitumor properties
of diastereomeric and geometric analogs of vitamin D3. Anticancer Drugs 2007, 18, 447–457.
Sample Availability: Samples of the compounds 3–9 are available from the authors.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
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