212
E. B e˛ benek et al. / Journal of Molecular Structure 1106 (2016) 210e219
CH OH
COOH
2
CrO , H SO
4
3
2
o
acetone, 0 C
HO
O
1
2
Scheme 1. Synthesis of betulonic acid 2.
2
.1.4. General procedure for the synthesis of derivatives 8e11
Sodium tetrahydroborate 0.4 g (1.1 mmol) was added to a
mixture of derivatives 4e7 (0.33 mmol) in dry tetrahydrofuran
10 mL) and the reaction was stirred at room temperature for 24 h.
For this measurement, colorless single crystals of good quality were
preselected under a polarized light microscope. The crystals were
mounted on a quartz capillary. The data were collected using an
Oxford Diffraction diffractometer with Sapphire 3 CCD detector.
Accurate cell parameters were determined and refined using Cry-
sAlis CCD program [24]. For the integration of the collected data,
the CrysAlis RED program [24] was used.
The crystal structures have been deposited in the Cambridge
Crystallographic Data Centre and allocated the deposition
numbers: CCDC 1050588 for 4 and 1050589 for 8A.
(
At the end of the reaction 7 mL of water was added and the reaction
mixture was extracted with dichloromethane (2 ꢂ 5 mL). The
organic layer was separated, then dried with anhydrous magne-
sium sulfate and concentrated under reduced pressure. The crude
products were purified by column chromatography (SiO , eluent:
2
chloroform/ethanol, 40:1, v/v) to give pure compounds 8e11.
Propargyl betulinate 8A. Recrystallization from DMF-acetone
ꢀ
1
solution (1:10, v/v); mp 118e120 C. H NMR (CDCl
s, CH ), 0.81 (3H, s, CH ), 0.92 (3H, s, CH ), 0.95 (3H, s, CH
3H, s, CH ), 0.86e2.30 (24H, m, CH, CH ),1.68 (3H, s, CH ), 2.43 (1H,
), 2.95 (3H, s, NCH ), 2.98e3.03
1H, m, 19-H), 3.15e3.19 (1H, m, 3-H), 4.59 (1H, s, 29-H), 4.61e4.72
3
)
d
¼ 0.75 (3H,
3.1.1. Refinement
3
3
3
3
), 0.96
The structure were solved using direct method with SHELXS-
014 [25] program and then the solutions were refined using
(
3
2
3
2
t, J ¼ 2.4 Hz, CCH), 2.88 (3H, s, NCH
3
3
SHELXL-2014 [25] program. H atoms were treated as riding atoms
in geometrically idealized positions, fixing the CeH bond lengths at
(
(
{
13
2H, m, OCH
2
), 4.73 (1H, s, 29-H), 8.01(1H, s, CH from DMF) ppm.
C
0
.95, 1.00, 0.99, 0.95 and 0.98 Å for aldehyde CH, methine CH,
methylene CH , terminal methylene CH and methyl CH atoms
respectively, and with Uiso(H) ¼ 1.5Ueq(C) for methyl H atoms or
.2Ueq(C) otherwise. Hydrogen atoms involved in H-bonding
acetylenic and hydroxyl H atoms) were introduced into the
1
H} NMR (CDCl
3
)
d
¼ 14.7, 15.4, 16.0, 16.1, 18.3, 19.4, 20.9, 25.5, 27.4,
2
2
3
2
4
8.0, 29.6, 30.5, 31.5, 32.0, 34.3, 36.4, 36.8, 37.2, 38.3, 38.7, 38.9,
0.8, 42.4, 46.9, 49.5, 50.6, 51.3, 55.4, 56.6, 74.3, 78.1, 79.0, 109.7,
1
(
150.5, 162.5, 175.2 ppm.
ꢀ
2
-Butynyl betulinate 9. Yield 68%; mp 156e158 C; R
f
0.24
¼ 0.75 (3H, s,
), 0.96 (3H, s, CH ), 0.97 (3H,
), 1.68 (3H, s, CH ), 1.84 (3H, t,
), 2.99e3.04 (1H, m, 19-H), 3.17e3.19 (1H, m, 3-H),
.60 (1H, s, 29-H), 4.65e4.69 (2H, m, OCH ), 4.74 (1H, s, 29-H) ppm.
calculated positions and then refined freely with isotropic atomic
displacement parameters.
1
(
chloroform/ethanol, 40/1, v/v). H NMR (CDCl
CH ), 0.82 (3H, s, CH ), 0.94 (3H, s, CH
s, CH ), 1.18e2.29 (24H, m, CH, CH
J ¼ 2.4 Hz, CCH
3
): d
3
3
3
3
3
2
3
3.2. Differential scanning calorimetry (DSC)
3
4
2
1
3
1
Thermal analysis was performed with the differential scanning
C { H} NMR (CDCl
3
):
d
¼ 3.6, 14.7, 15.3, 15.9, 16.1, 18.3, 19.4, 20.9,
calorimetry (DSC, Perkin Elmer, DSC-7) in the temperature range
2
4
5.5, 27.4, 27.9, 29.6, 30.5, 32.0, 34.3, 36.8, 37.2, 38.3, 38.7, 38.9, 40.7,
2.4, 46.9, 49.5, 50.6, 52.1, 55.4, 56.5, 73.7, 78.9, 82.4, 109.6, 150.6,
ꢀ
from room temperature to 220 C and heating rate 20 K/min.
1
75.4 ppm. IR (KBr)
n
¼ 3388 (OH), 2870e2942 (CH), 2238 (C^C),
1730 (C]O), 1123 (s, CeO). Mass spectrum (EI, 70eV) m/z (rel.
3.3. Antyproliferative assay in vitro
þ
ꢁ1
int.) ¼ 508 (M , 43%), 410 (76), 207 (51), 189 (100) cm
.
ꢀ
3
-Butynyl betulinate 10. Yield 69%; mp 102e105 C; R
f
0.25
¼ 0.75 (3H, s,
), 0.96 (3H, s, CH ), 0.97 (3H,
), 1.68 (3H, s, CH ), 2.20e2.25 (m,
Study of cytotoxic activity in vitro was performed using the
following cell lines: T47D (human breast cancer), CCRF/CEM (hu-
man leukemia), SW707 (human colorectal), P388 (mouse leuke-
mia) and BALB3T3 (normal mouse fibroblasts). All tested cell lines
were obtained from the American Type Culture Collection (Rock-
ville, MD, USA) and maintained at the Cell Culture Collection of the
Institute of Immunology and Experimental Therapy (Wrocław,
Poland). Twenty-four hours before addition of the obtained com-
pounds the cells were plated in 96-well plates (Sarstedt, Num-
brecht, Germany) at a density of 10 cells per well in 100
culture medium. The cells were cultured in the opti-MEM medium
supplemented with 2 mM glutamine (Gibco, Grand Island, NY,
USA), streptomycin (50 mg/mL), penicillin (50 U/mL) (both antibi-
1
(
chloroform/ethanol, 40/1, v/v). H NMR (CDCl
CH ), 0.82 (3H, s, CH ), 0.92 (3H, s, CH
s, CH ), 1.13e2.01 (24H, m, CH, CH
H, OCH CH
.17e3.19 (m,1H, 3-H), 4.12e4.20 (m, 2H, OCH
3
): d
3
3
3
3
3
2
3
2
3
2
2
), 2.54 (1H, t, J ¼ 2.4 Hz, CCH), 3.00e3.02 (1H, m,19-H),
2
CH
):
2
d
), 4.60 (1H, s, 29-
¼ 14.3,14.7,15.3,
13
1
H), 4.73 (1H, s, 29-H) ppm. C { H} NMR (CDCl
3
16.1, 18.3, 19.1, 20.9, 21.0, 25.6, 27.4, 28.0, 29.6, 30.6, 32.2, 34.3, 37.0,
3
7
8.3, 38.9, 40.7, 42.4, 47.0, 49.4, 50.6, 55.4, 56.4, 56.6, 59.8, 61.6, 69.7,
9.0, 80.3, 109.6, 150.6, 175.9 ppm. IR (KBr)
4
n
¼ 3423 (OH), 3312
mL of
(
C^CH), 2869e2944 (CH), 2123 (C^C), 1724 (C]O), 1263 (CeO).
þ
Mass spectrum (EI, 70eV) m/z (rel. int.) ¼ 508 (M , 12%), 411 (32),
ꢁ
1
2
07 (47), 189 (100) cm
.
otics from Polfa, Tarchomin, Poland) and 5% fetal calf serum (Gibco).
The cell cultures were maintained at 37 C in atmosphere saturated
ꢀ
3
. Crystal structure determination
2
with 5% CO . The alkynyl derivatives, as well as betulin 1 and
betulinic acid 3 were dissolved in DMSO and culture medium (1:9)
to the concentration of 1 mg/mL, and next diluted in culture me-
dium to reach the required concentrations (ranging from 1 to
3.1. X-ray diffraction experiment
The single-crystal X-ray experiments were performed at 100K.
100 mg/mL). The in vitro cytotoxic effect of all compounds was