Cytotoxic effect of some pentacyclic triterpenes and hemisynthetic derivatives of stigmasterol

Article abstract of DOI:10.1007/s10600-011-0045-8

Two different oxidation reactions (PCC and KMnO₄) of stigmasterol gave three products, stigmasta-4,22-dien-3-one (1a), stigmasta-4,22-diene-3,6-dione (1b), and 3-O-acetyl-5β,6β- epoxystigmast-22-ene-3-ol (1c). The cytotoxic activities of hemisynthetic compounds, stigmasterol, and four pentacyclic triterpens 2-5 previously isolated from cultivated and wild Triumfetta cordifolia and identified were investigated against human fibrosarcoma cell line (HT1080). Most of the drugs showed moderate cytotoxic activity. It was also notice that the triterpene skeleton had a range of number of OH functions in which activity was observed. Spectroscopic analyses (¹H and ¹³C NMR) and mass were used to elucidate the structure of hemisynthetic compounds.

Full text of DOI:10.1007/s10600-011-0045-8

Chemistry of Natural Compounds, Vol. 47, No. 5, November, 2011 [Russian original No. 5, September–October, 2011]
CYTOTOXIC EFFECT OF SOME PENTACYCLIC TRITERPENES
AND HEMISYNTHETIC DERIVATIVES OF STIGMASTEROL
Louis P. Sandjo,1,2* Vincent Rincheval, Bonaventure T. Ngadjui,
and Gilbert Kirsch
3
1
UDC 547.918
2
Two different oxidation reactions (PCC and KMnO ) of stigmasterol gave three products, stigmasta-4,22-
4
dien-3-one (1a), stigmasta-4,22-diene-3,6-dione (1b), and 3-O-acetyl-5ꢁ,6ꢁ-epoxystigmast-22-ene-3-ol (1c).
The cytotoxic activities of hemisynthetic compounds, stigmasterol, and four pentacyclic triterpens 2–5
previously isolated from cultivated and wild Triumfetta cordifolia and identified were investigated against
human fibrosarcoma cell line (HT1080). Most of the drugs showed moderate cytotoxic activity. It was also
notice that the triterpene skeleton had a range of number of OH functions in which activity was observed.
1
13
Spectroscopic analyses ( H and C NMR) and mass were used to elucidate the structure of hemisynthetic
compounds.
Keywords: oxidation reaction, stigmasterol derivatives, pentacyclic triterpenes, cytotoxic activity.
Steroids and triterpenes are two classes of secondary metabolites widespread in vegetables possessing a wide spectrum
of biological activities. Some previous studies showed that many pentacyclic triterpenoids such as betulinic, oleanolic, and
ursolic acids have antitumor activity ꢂ1ꢃ. Near this class, we have their biosynthetic descents that show important pharmacological
properties. Thus, stigmasterol has shown anticancer activities ꢂ2ꢃ and analgesic ꢂ3ꢃ and hypoglycemic effect ꢂ4ꢃ. In continuation
of our study and from the above-mentioned information, we have found it interesting to evaluate the cytotoxicity properties of
three steroids, 1a, 1b, 1c, obtained from oxidation reactions of stigmasterol (1), and four pentacyclic triterpenes (lupeol (2),
oleanolic acid (3), maslinic acid (4), and tormentic acid) isolated from the stems of wild and cultivated Triumfetta cordifolia
(
Tiliaceae). We hereby describe the valorization of natural products and its derivatives.
Chemistry. Stigmasterol and pentacyclic triterpenes were previously isolated from Triumfetta cordifolia (Tiliaceae)
and identified on the basis of their spectroscopic data. Some of them were also compared by TLC with the authentic samples
in our laboratory. Stigmasterol (1), obtained in the largest amount, was used as starting material for oxidation reactions.
After its acetylation in pyridine and acetic anhydride, the product obtained was subjected to oxidation with KMnO4,
yielding the 5ꢁ,6ꢁ-epoxide derivative 1c (Scheme 1). This reaction, which was carried out in the heterogeneous phase, followed
the procedure described by Syamala et al. ꢂ5ꢃ. It was already noticed that this reaction was regioselective ꢂ5ꢃꢄ according to
which the trisubstituted olefinic function was more easily transformed to epoxide than di- and mono- substituted compound.
Comparison between the carbons C-5 (ꢅ 63.0) and C-6 (63.6) of compound 1c from the APT spectrum with the data of the
C
literature ꢂ6ꢃ confirmed also that this transformation is also stereoselective.
Compound 1 was also subjected to two oxidation reactions with PCC (pyridinium chlorochromate), yielding two
compounds 1a, and 1b (Scheme 1) ꢂ7ꢃ. The main factor was the time of reaction according to which after 24 h, the product was
different from those obtained after 36 h.
–
1
In the IR spectra of 1a and 1b, no band of the hydroxyl function was observed, and one band appeared at 1678 (1670) cm
1
3
corresponding to a conjugated ketone. The C NMR spectrum of 1a exhibited the signal of carbonyl at ꢅ 200.4 (C-3), while
C
those of 1b showed two carbonyls at ꢅ 202.3 (C-3) and 199.5 (C-6).
C
1
) Department of Organic Chemistry, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon, e-mail:
plsandjo@yahoo.fr; 2) Laboratoire dꢀIngenierie Moleculaire et Biochimie Pharmacologique, Institut Jean Barriol, University of
Paul-Verlaine Metz, 1Bld Arago 57070 Metz, France; 3) Laboratoire de genetique et biologie cellulaire, University of Versailles-St-
Quentin-en-Yvelines, Batiment Fermat-Maison 4; Niveau 2; 45, avenue des Etats-Unis, 78035 Versailles Cedex, France. Published
in Khimiya Prirodnykh Soedinenii, No. 5, pp. 642–644, September–October, 2011. Original article submitted May 15, 2010.
0
0
009-3130/11/4705-0731 2011 Springer Science+Business Media, Inc.
731

2
4
1
2
22
2
3
1
1
1
i
O
1
0
15
3
7
1
O
HO
5
ii
iii
iia
iib
O
O
O
O
O
O
1
a
75%
1b
92%
1c 87%
i. C H N/(H CCO) O 1:1 (1.5 eq), room temperature, 3 h; ii. PCC–Al O (10 eq)/
5
5
3
2
2 3
CH Cl , room temperature; iia) 24 h, room temperature, R 31/40 (hexane–ethyl
2
2
f
acetate 9:1); iib) 36 h, R 1/2 (hexane–ethyl acetate 9:1); iii. KMnO /CuSO 4 5H O
f
4
4
2
(
2:1), CH Cl /tert-ButOH/H O (20:0.3:1), R 20/37 (hexane–ethyl acetate 85:15),
2
2
2
f
room temperature, 2 h.
Scheme 1
Cytotoxicity Assay. The cytotoxic activities of eight compounds were investigated on the human HT1080 tumoral
cell line. These cells are able to undergo physiological cell death, a process termed apoptosis ꢂ8ꢃꢄ and are sensitive to a wide
variety of cytotoxic drugs such as etoposide, cisplatin, staurosporine, or TNF-ꢆ. In order to avoid a putative toxicity of the
solvents, the maximum used volume of the compound solution never exceeded 1/100 of the total volume of culture medium.
Controls with analogous concentrations of solvents were always carried out in parallel to check their lack of toxicity. Three
compounds induced less than 50% cell death after 48 hours of treatment at maximum concentrations, rendering impossible the
determination of an IC . This was the case of stigmasterol (1) (C 500 mM), stigmasta-4,22-diene-3,6-dione (1b) (Cmax 100 mM),
50
max
and tormentic acid (5) (Cmax 100 mM). Among the eight compounds, it was possible to determine the IC50 value of six:
stigmasta-4,22-dien-3-one (1a) (IC50 0.3 mM), stigmasta-4,22-diene-dione (1b), 3-O-acetyl-5ꢁ,6ꢁ-epoxystigmast-22-en-
3
-ol (1c) (IC50 0.54 mM), lupeol (2) (IC50 1.1 mM), oleanolic acid (3) (IC50 0.7 mM), and maslinic acid (4) (IC50 0.075 mM).
Thus, stigmasta-4,22-dien-3-one showed high specificity (IC50 ꢇ 0.3 mM) in inducing efficient cellular toxicity. The epoxide
ring and the ꢆ,ꢁ-unsaturated ketone appear to be the biologically active sites. Additionnaly, considering the compounds 1, 1c,
2
, 3, 4, and 5, it was noticed that cytotoxic activity increased with the number of hydroxyl group until a maximal number
of OH functions. These means that triterpene skeletons have a range of number of OH functions in which some could
have cytotoxic activity against these cells line. Even if the OH group is an alcohol function or in a carboxylic group, it
is one of the great parameters of lipophilicity and hydrophobicity; accordingly, the cell membrane permeability depends
on these two descriptors ꢂ9ꢃ.
HO
OH
OH
R
HO
HO
O
O
HO
3
, 4
5
3
: R = H; 4:R = OH
EXPERIMENTAL
IR spectra were recorded on a Perkin–Elmer FT-IR system spectrum BX spectrometer using KBr disks. 1D NMR
spectra were carried out on a Bruker DRX-400 MHz spectrometer. Melting points were measured in a Stuart Scientific Melting
Point apparatus SMP and are uncorrected. Silica gel GF254 and 60A (size 70–200 ꢈm) were used to perform thin layer
3
chromatography and column chromatography, respectively. The matrix used for the MALDI-TOF-MS was a 1 M solution of
732

2
,5-dihydroxybenzoic acid (2,5-DHB) in 50% acetonitrile, 50% ultrapure water, and 0.1% trifluoroacetic acid (TFA); its
measurements were possible using a Bruker Reflex IV time-of-flight mass spectrometer (TOF-MS) (Bruker-Daltonic, Bremen,
Germany) equipped with a Scout 384 probe ion source, using a nitrogen pulsed laser (337 nm, model VSD-337ND, Laser
Science Inc., Boston, MA) with energy output of 400 ꢈJ/pulse.
We dissolved 0.034 g (0.082 mmol) of stigmasterol and 0.75 g of PCC/Al O3 in DCM under magnetic stirring at
2
room temperature for 24 h. The product (0.025 g, 75%) was obtained as a brown solid after filtration under vacuum on Celite.
–
1
Stigmasta-4,22-dien-3-one (1a). White solid; mp 123ꢉC. IR (KBr, ꢊ , cm ): 1678, 1640. MALDI-TOF m/z 433.347
max
+
13
[
C H O + Na] . C NMR: 37.8 (C-1), 32.2 (C-2), 200.4 (C-3), 125.4 (C-4), 168.5 (C-5), 46.9 (C-6), 31.2 (C-7), 31.4 (C-8),
29 46
5
0.9 (C-9), 36.5 (C-10), 21.7 (C-11), 39.8 (C-12), 42.4 (C-13), 56.4 (C-14), 24.2 (C-15), 28.2 (C-16), 56.2 (C-17), 11.9
(
2
C-18), 20.1 (C-19), 40.4 (C-20), 21.4 (C-21), 138.1 (C-22), 129.3 (C-23), 51.2 (C-24), 31.8 (C-25), 21.1 (C-26), 18.3 (C-27),
5.4 (C-28), 11.9 (C-29).
We treated 0.034 g (0.082 mmol) of stigmasterol under the same conditions as the reaction mentioned above. The
difference was the reaction time, which was 36 h in this case. Compound 1b (0.032 g, 92%) was obtained by filtration under
vacuum on Celite.
–
1
Stigmasta-4,22-diene-3,6-dione (1b). Amorphous solid. IR (KBr, ꢊ , cm ): 2985, 1670, 1639. MALDI-TOF
max
+
13
m/z 447.331 [C H O + Na] . C NMR: 34.2 (C-1), 34.0 (C-2), 202.3 (C-3), 125.4 (C-4), 165.5 (C-5), 199.5 (C-6), 46.8
2
9 50 2
(
(
C-7), 34.2 (C-8), 51.0 (C-9), 39.1 (C-10), 20.9 (C-11), 39.8 (C-12), 42.5 (C-13), 56.5 (C-14), 24.0 (C-15), 28.0 (C-16), 55.8
C-17), 12.0 (C-18), 17.5 (C-19), 36.0 (C-20), 18.7 (C-21), 149.5 (C-22), 129.8 (C-23), 51.2 (C-24), 28.7 (C-25), 21.1 (C-26),
2
1.1 (C-27), 25.4 (C-28), 17.5 (C-29).
Oxydation by KMnO . We acetylated 0.153 g (0.3713 mmol) of stigmasterol in 1.0 mL of pyridine and 1.0 mL
4
acetic anhydride for 3 h under stirring at room temperature. The acetylated compound was obtained after rotary evaporation
under vacuum. This compound was dissolved in a mixture of 20 mL of DCM, 1 mL of tert-butanol, and 0.3 mL of H O. The
2
medium was stirred at room temperature. We added 1.6 g of KMnO and 0.8 of CuSO ·5H O to the medium, and the reaction
4
4
2
was carried out under N for 24 h. After the reaction stop, 25 mL of DCM was added, and the medium was filtered under
2
vacuum on Celite. The DCM solution was concentrated under vacuum with a rotary evaporator, yielding compound 1c
(
0.152 g, 87%).
–
1
3
-O-Acetyl-5ꢁ,6ꢁ-epoxystigmast-22-en-3-ol (1c). White solid; mp 140–141.3ꢉC. IR (KBr, ꢊ , cm ): 1738, 1635.
max
+
13
MALDI-TOF m/z 471.385 [C H O + H] . C NMR: 37.8 (C-1), 32.2 (C-2), 71.3 (C-3), 42.4 (C-4), 63.0 (C-5), 63.6
3
1 50 3
(
(
C-6), 31.2 (C-7), 31.4 (C-8), 50.9 (C-9), 36.5 (C-10), 21.7 (C-11), 39.8 (C-12), 42.4 (C-13), 56.4 (C-14), 24.2 (C-15), 28.2
C-16), 56.2 (C-17), 11.9 (C-18), 20.1 (C-19), 40.4 (C-20), 21.4 (C-21), 138.1 (C-22), 129.3 (C-23), 51.2 (C-24), 31.8 (C-25),
2
1.1 (C-26), 19.0 (C-27), 25.4 (C-28), 11.7 (C-29), 170.6 (C=O), 21.4 (CH3).
Cellular Viability. Human HT1080 fibrosarcoma adherent cell line was cultured at 37ꢉC in a humidified atmosphere
containing 5% CO in Dulbeccoꢀs modified Eagleꢀs medium (DMEM/F12) supplemented with 10% fetal bovine serum together
2
with penicillin (100 ꢈg/mL), streptomycin (100 U/mL), and glutamax (1% v/v) from Invitrogen. The cells were seeded in
4
1
2-well plates (5.10 cells/well). After 24 h, the medium was replaced in each well by 1 mL of complete medium with the
appropriate concentrations of the tested drugs in THF and stored at –20ꢉC. The cells were then incubated for 48 h, and cellular
viability was determined by flow cytometric analysis. It was shown that type I (apoptosis), type II (autophagy), and type III
(
necrosis) cell death triggers permeabilization of mitochondria ꢂ10ꢃ. Global cell death was then determined with the cationic
lipophilic DiOC (3) dye (Invitrogen), which specifically probes mitochondrial membrane potential (ꢋꢌm) ꢂ11ꢃ. The decrease
6
in forward light scattering (FSC) was also checked to confirm the cell death process ꢂ12ꢃ. After drug treatment, the media from
each well were kept in centrifuge tubes. The adherent cells were detached using trypsin, pooled with the corresponding media,
centrifuged, and resuspended in complete medium. The cells were then loaded with 100 nM DiOC (3) and incubated for 30 min
6
at 37ꢉC. Flow cytometric measurements were performed using an XL3C flow cytometer (Beckman-Coulter). Fluorescence
was induced by the blue line of an argon ion laser (488 nm) at 15 mW. Green fluorescence of DiOC (3) was collected with a
6
5
25 nm band pass filter. The percentage of dead cells was determined by measuring the percentage of cells harboring low
4
DiOC (3) fluorescence. Analyses were performed on 10 cells.
6
733

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