G. Fontana, et al.
Bioorganic Chemistry 90 (2019) 103054
gel (Kiesegel 60/230–400 mesh) was used for flash chromatography
poored into an iced 1 M HCl solution. The resulting mixture was stirred
(
FC) columns. Optical rotations were measured by Jasco P-1010 digital
for 30′ while adjusting the pH to 2 in order to protonate the carboxylic
polarimeter. Microanalysis data (C, H, N) obtained by an Elemental
Vario EI. III apparatus were within ± 0.4% of the theoretical value. Dry
THF was obtained by distilling commercial THF onto Na/benzophenone
mixture.
function. Then the mixture was extracted with CHCl (4 X 30 mL), the
3
collected organic phase was neutralized with water and dried over
anhydrous Na
2
SO . The solvent was evaporated and the residue was
4
purified by FC (cyclohexane/acetone 9:1) giving 28 mg (10%) com-
Compounds 1–4, 7–10 and 13–16 were recognized by comparing
physical and spectral properties with literature data.
pound 6 and 96 mg (35%) of compound 5.
(
3α)-allyl-(3β)-hydroxyolean-12-en-28-oic acid (5): amorphous white
4.2. Extraction and purification of compounds 1 and 7.
solid, anal. C 79.11%, H 10.81%; calcd for C33
H
52
O
3
, C 79.79%, H
2
5
1
13
1
0.55%. [α]
D
+ 55.4 (c = 0.01, CHCl
3
); H NMR and C NMR:
1
.0 Kg of Olea europea leaves collected in the campus garden were
see Table 1.
dried on air for one week, cut in little slices and charged into the
Soxhlet apparatus and extracted with EtOAc for 6 h. Then the solvent
was removed by rotavapor distillation and the extract was purified by
column chromatography employing deactivated (with 15% V/W of
(3β)-allyl-(3α)-hydroxyolean-12-en-28-oic acid (6): amorphous white
solid, anal. C 79.89%, H 10.77%; calcd for C33
H
52
O
3
, C 79.79%, H
2
5
1
13
10.55%. [α]
see Table 1.
D
+ 31.4 (c = 0.0026, CHCl
3
); H NMR and C NMR:
H
2
O) Merck silica gel (Kiesegel 60/70–230 mesh) as stationary phase
(3α)-allyl-(3β)-hydroxyurs-12-en-28-oic acid (11): amorphous white
and Me
2
CO/n-hexane 2:8 as eluent. 1.30 g (0.13%) of pure OA 1 [22]
solid, anal. C 79.55%, H 10.80%; calcd for C33
H
52
O
3
, C 79.79%, H
2
5
1
13
were obtained.
10.55%. [α]
see Table 1.
D
+ 29.0 (c = 0.136, CHCl
3
); H NMR and C NMR:
2
.0 kg of Malus domestica purchased in a local market were peeled
and the peel was cut in little slices that were subsequently freeze-dried.
The resulting material was subjected to the same isolation procedure
described above obtaining 1.45 g (0.07%) of pure UA 7 [27].
(3β)-allyl-(3α)-hydroxyurs-12-en-28-oic acid (12): amorphous white
solid, anal. C 79.80%, H 10.60%; calcd for C33
H
52
O
3
, C 79.79%, H
2
5
1
13
10.55%. [α]
D
+ 40.0 (c = 0.3, CHCl
3
); H NMR and C NMR: see
Table 1.
4
.3. General procedure for the methylation with CH
2
N , preparation of
2
compounds 2, 4, 8, 10, 14 and 16.
4.7. Cell cultures
To a solution of 0.5 mmol of the starting compound in 5 mL of THF,
an ethereal solution of CH was added dropwise at 0 °C until a per-
sistent pale yellow colour developed. The resulting solution was stirred
for 30′. The CH excess was removed by gentle warming under hood
and the solvent was evaporated. The residue was purified by FC
CHCl /MeOH 50:1) giving in quantitative yield reaction products
identical in all respect to reported 2 [28], 4 [29], 8 [30], 10 [29], 14
31] and 16 [32].
The culture medium of human HCC cell lines is Roswell Park
Memorial Institute (RPMI) 1640, supplemented with 10% heat-in-
activated fetal calf serum, 2 mML-glutamine, 1 mM sodium pyruvate,
100U/mL penicillin, and 100 μg/mL streptomycin (all reagents were
from HyClone Europe Ltd., Cramlington, U.K.); the cells are cultured in
N
2 2
N
2 2
(
3
a humidified atmosphere at 37 °C in 5% CO and routinely tested for
2
Mycoplasma contamination. HA22T/VGH cell line was kindly provided
by Professor M. Levrero (Laboratory of Gene Expression, Fondazione
Andrea Cesalpino, University of Rome ‘La Sapienza’, Rome, Italy),
Hep3B and HepG2 cell lines were obtained from the American Type
Culture Collection (ATCC). After obtaining the cells, the first passage
carried out was assigned passage number 1 and having a narrow range
of passage numbers were used for all experiments.
[
4
.4. General procedure for the acetylation reaction; preparation of
compounds 3 and 9
3
00 mg of compounds 1 or 7 were solubilized in 3 mL of 1:2 Ac O/
2
Py and stirred at rt for 24 h. Then the volatiles were removed by
azeotropic evaporation with toluene. The residue was treated with 5 mL
4.8. Cell growth assays
of 1:1 THF/H O and stirred for 3 h in order to hydrolyze in situ the
2
mixed anhydride. The solvent was evaporated and the residue was
purified by FC (CHCl /acetone 0.3% in acetone) giving 278 mg (85%)
of a compound identical in all respect to reported 3 [33] and 9 [34].
For MTS assay we estimate cell growth inhibition as a percentage of
the absorbance measured in the control cells. The cells were seeded
onto 96-well plates at 2 × 104 cells/well and after 24 h the compounds
were added in different concentrations. After 72 h, 15 μl of a commer-
cial solution containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carbox-
ymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) and phe-
nazine ethosulfate (obtained from Promega Corporation, Madison, WI,
USA) were added and after a period variable for each cell line, the
bioreduction of the MTS dye was assessed by measuring the absorbance
of each well at 490 nm.
3
4
.5. General procedure for the Jones oxidations, preparation of compounds
1
3 and 15
To a solution of 300 mg (0.64 mmol) of compound 1 (7) in Me CO,
2
Jones reagent was added dropwise at 0 °C until a persistent pale yellow
colour developed in the solution. The greenish solid residue was filtered
off and the solvent was evaporated. The residue was purified by FC
(
light petroleum/acetone 5% in acetone) to give 195 mg (65%) of a
4.9. NF-κB activation
compound identical in all respect to reported 15 [34], from 7 and
65 mg (89%) of a compound identical in all respect to reported 13
35], from 1.
2
NF-κB activation was evaluated by the DNA-binding capacity of NF-
κB (p65 subunit) using the TransAM™ NF-κB and Nuclear Extract™ Kits
[
(
Active Motif, Carlsbad, CA, USA). The assay is based on the measure of
4
.6. Preparation of stereoisomers 5, 6 and 11, 12 by Grignard addition
binding between an oligonucleotide containing the NF-κB consensus
binding site (5′-GGGACT TTCC-3′) and the activated NF-κB contained in
the nuclear extracts of cells treated. In particular, the NF-κB bound to
the oligonucleotide is detected using an antibody directed against an
epitope on p65 that is accessible only when NF-κB is bound to its target
DNA. Addition of a secondary antibody conjugated to horseradish
peroxidase provides sensitive colorimetric readout that is quantified by
reaction on ketones 13 and 15.
To a solution of 250 mg (0.55 mmol) of compound 13 (15) in 15 mL
of dry THF, 700 mL (1.4 mmol) of 2 M allylmagnesium chloride in THF
were slowly added via syringe under an Ar atmosphere at a temperature
of 0 °C. The mixture was left to warm until rt overnight and then was
6