Cytotoxicity of semisynthetic acetal triterpenes from one-pot vicinal diol cleavage following by lactolization: Reaction promoted by NaIO 4/SiO2 gel in THF

Article abstract of DOI:10.1080/14786419.2012.691494

In situ C-C bond cleavage of vicinal diol following by the lactolisation resulted from separated treatment of Arjunolic acid (1), 24-hydroxytormentic acid (2) and 3-O-β-D-glucopyranosylsitosterol (3) with sodium periodate and silica gel in dried THF according to the strategic position of hydroxyl functions in the molecule. The reaction led to a lactol pentacyclic triterpenes 1A, 2A and a bicyclotriacetal of β-sitosterol 3A. These products were further acetylated and the cytotoxicity of all molecules was evaluated against human fibrosarcoma HT1080 cancer cells lines.

Full text of DOI:10.1080/14786419.2012.691494

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Cytotoxicity of semisynthetic acetal
triterpenes from one-pot vicinal diol
cleavage following by lactolization:
Reaction promoted by NaIO4/SiO2 gel in
THF
Louis Pergaud Sandjo a , Aurelie Vigee Barry Songfack Djoumessi a
, Vincent Rincheval b , Hervé Martial Poumale Poumale a , Berhanu
M. Abegaz c & Bonaventure T. Ngadjui a d
a Department of Organic Chemistry Faculty of Sciences , P.O. Box
812 University of Yaoundé 1, Yaoundé , Cameroon
b Laboratoire de Génétique et Biologie Cellulaire Bâtiment Fermat
– Maison 4 , University of Versailles St Quentin-en-Yvelines, Niveau
2; 45, Avenue des Etats-Unis , 78035 Versailles Cedex , France
c Department of Chemistry , University of Botswana , Private Bag
00704 , Gaborone , Botswana
d Department of Pharmaceutical Sciences and Traditional
Pharmacopeia, Faculty of Medicine and Biomedical Sciences ,
University of Yaoundé I , P.O. Box. 8664, Yaoundé , Cameroon
Published online: 23 May 2012.
To cite this article: Louis Pergaud Sandjo , Aurelie Vigee Barry Songfack Djoumessi , Vincent
Rincheval , Hervé Martial Poumale Poumale , Berhanu M. Abegaz & Bonaventure T. Ngadjui (2013):
Cytotoxicity of semisynthetic acetal triterpenes from one-pot vicinal diol cleavage following by
lactolization: Reaction promoted by NaIO4/SiO2 gel in THF, Natural Product Research: Formerly
Natural Product Letters, 27:8, 711-718
To link to this article: http://dx.doi.org/10.1080/14786419.2012.691494
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Natural Product Research, 2013
Vol. 27, No. 8, 711–718, http://dx.doi.org/10.1080/14786419.2012.691494
Cytotoxicity of semisynthetic acetal triterpenes from one-pot vicinal diol
cleavage following by lactolization: Reaction promoted by NaIO4/SiO2
gel in THF
a
Louis Pergaud Sandjo , Aurelie Vigee Barry Songfack Djoumessi ,
a
*
Vincent Rinchevalb, Herve Martial Poumale Poumalea, Berhanu M. Abegazc and
´
Bonaventure T. Ngadjuiad
a
´
Department of Organic Chemistry Faculty of Sciences, P.O. Box 812 University of Yaounde 1,
b
´
´ ´
ˆ
Yaounde, Cameroon; Laboratoire de Genetique et Biologie Cellulaire Batiment Fermat – Maison 4,
University of Versailles St Quentin-en-Yvelines, Niveau 2; 45, Avenue des Etats-Unis, 78035
Versailles Cedex, France; cDepartment of Chemistry, University of Botswana, Private Bag 00704,
Gaborone, Botswana; Department of Pharmaceutical Sciences and Traditional Pharmacopeia,
´ ´
Faculty of Medicine and Biomedical Sciences, University of Yaounde I, P.O. Box. 8664, Yaounde,
d
Cameroon
(Received 25 January 2012; final version received 4 April 2012)
In situ C–C bond cleavage of vicinal diol following by the lactolisation resulted
from separated treatment of Arjunolic acid (1), 24-hydroxytormentic acid (2) and
3-O-�-D-glucopyranosylsitosterol (3) with sodium periodate and silica gel
in dried THF according to the strategic position of hydroxyl functions in the
molecule. The reaction led to a lactol pentacyclic triterpenes 1A, 2A and a
bicyclotriacetal of �-sitosterol 3A. These products were further acetylated and the
cytotoxicity of all molecules was evaluated against human fibrosarcoma HT1080
cancer cells lines.
Keywords: hemisynthesis; acetal triterpenoids; cytotoxicity
1. Introduction
Arjulonic acid (1), 24-hydroxyltormentic acid (2) and �-sitosterol glucopyranoside (3) were
transformed into the acetal derivatives by using NaIO4–SiO2 in THF. Formerly, the bond
between vicinal diol in triterpenoids was cleaved with the aim of structure elucidation and
this was performed with sodium periodate in MeOH for 30 min (Bombardelli, Bonati,
Gabetta, & Mustich, 1974) or in ethyl acetate : ethanol overnight (Bhagirath & Rastogi,
1969) to yield dialdehyde product (Kumaraswamy, Nivedita, Sastry, & Ramakrishna,
2005). In contrast, a seven-member ring lactol was formed if there was an OH function
at C-23 or C-24 of this class of compounds (Bombardelli et al., 1974). The problem of
weak solubility of the starting compounds (1–3) in MeOH prompted us to select THF as
solvent for this reaction. But, the product was barely formed after 3 h and overnight, the
transformation was not effective. To get around this obstacle, dried silica gel described
as a solid phase catalyst in several kinds of reactions was added and a seven-member
ring triterpenoid was obtained within 6 h. The same reaction was repeated successfully by
*Corresponding author. Email: plsandjo@yahoo.fr
© 2013 Taylor & Francis

712
L. Pergaud Sandjo et al.
R
R
4
3
R
R
3
R
4
2
R
21
R
2
1
21
R
12
18
1
12
18
25
11
HO
2
O
1
4
COOH
28
NaIO SiO
4,
25
14
27
2
11
COOH
28
1
14
27
HO
THF, 6 h, rt
2
3
5
7
5
23
7
4
HO
HO
3
24
O
24 23
1A-2A
1-2
Acetic anhydride
C H N, 3 h, rt
5
5
R
2
R
3
4
R
R
1
AcO
COOH
O
1B-2B
O
Compound
Arjulonic acid 1
Product
R
R
R
R
4
Yield (%)
1
2
3
1A
1B
2A
2B
H
H
Me
Me
97
98
95
98
24-hydroxytormentic acid 2
Me
OH
H
Me
OAc
Figure 1. Hemisynthesis route of 1A, 2A, 1B and 2B.
dissolving starting compounds in dried THF and adding an adequate quantity of sodium
periodate and dried silica gel.
Silica gel has been used to perform cyclisation of polyketone into the phenolic
derivative and olefinic aldehyde into cyclo alcohol (Banerjee, Laya Mimo, & Vera Vegas,
´
2001); it was also used for acetal and ketal preparation (R. Kumar, D. Kumar, &
Chakraborti, 2007); silica gel and sodium periodate under certain conditions constitute
an efficient co-catalyst to cleave vicinal diol (Ravindranadh & Rodney, 2005).
We herein report the intramolecular lactolisation of Arjulonic acid (1), 24-hydro-
xyltormentic acid (2) and �-sitosterol glucopyranoside (3), catalysed by sodium periodate
and dried silica gel in absolute THF. The cytotoxicities of phytocomponents (1–3) and their
semi-synthetic products (1A–3A and 1B–3B) will also be reported.
2. Results and discussion
Arjunolic acid (1) was dissolved in dried THF and treated with sodium periodate and silica
gel as solid phase catalyst (Figure 1). The reaction was complete within 6 h and the
structure of an acetal triterpene was characterised using NMR data. The adsorbent seemed
to play a role of acid catalyst in the medium facilitating after the cleavage of the vicinal
diol into dialdehyde, the ring A closing to form an acetal function. The second step
required a free hydroxyl group at C-24 which was the �-position of the aldehyde function
(C-3) suitable for the ring closing (Figure. 2). The stereo-selectivity of the reaction was
confirmed by one of the 2D NMR spectra of the afforded compound. From NOESY
correlations between the acetalic proton at �H 5.13 (dd, 5.0, 9.5) and those of CH3 group
(C-25) at �H 1.05 (s), the absolute configuration of the acetal carbon was found to be S.

Natural Product Research
713
SiO2
OH
SiO2
HO
O
Na
O
O
HO
NaIO4 +
O
O
O
O
O
O
O
O
- NaIO
3
I
HO
O
H
H
O
O
O
SiO2
O
SiO2
SiO2
O
H
OH
O
H
HO
HO
O
O
O
O
OH
OR
O
O
O
OR
O
O
O
H
O
OR
O
H
O
OR
Figure 2. Vicinal diol cleavage and lactolization mechanism.
HO
5'
OH
O
HO
HO
NaIO , SiO
4'
4 2
7'
OH
O
O
O
THF, 6 h, rt
2'
OH
O
1'
O
3A
3
95.5 %
Acetic anhydride
C H N, 3 h, rt
5
5
AcO
O
O
OAc
O
O
3B
97.0 %
Figure 3. Hemisynthesis route of 3A and 3B.
Furthermore, same interactions were found between the aldehyde proton at �H 9.95 (s) and
one of the oxymethylene hydrogens H-23 at �H 3.73 which correlated with the angular
proton H-5 (�H 1.18) suggesting an absolute configuration S at C-4.
This reaction is close to Ferrier rearrangement of glucals (Misra, Tiwari, & Agnihotri,
2005) or to acetal preparation using acetic anhydride and silica supported perchloric acid
(Khan, Choudhury, & Ghosh, 2006). Two additional secondary metabolites were further
subjected to this couple of catalysts as 1 in order to have more bioactive compounds and to
check the efficiency of this reaction according that it was so far catalysed by sulphuric acid
(Hill, Alto, Calif, & Shipp, 1970) or zeolite coupled to a Lewis acid (Bejoy, Sreedharan, &
Sankaran 2005). Thus, 24-hydroxytormentic acid 2 was subjected to the same reaction
yielding like arjulonic acid an acetal seven-member ring A triterpene (Figure 1) with the
same absolute configurations at C-2 and C-4 as in 1A. 3-O-�-D-glucopyranosylsitosterol 3
was the best candidate to this one-pot preparation because of the presence of sugar
containing many vicinal diol systems; obviously, the reaction afforded a bicyclotriacetal
moiety with five- and six-member ring containing two additional acetal functions
(Figure 3). The NOESY spectrum of 3A (Figure 4) displayed correlation between
the proton at �H 4.88 and the oxymethine proton H-3 at �H 3.56 in the steroid scaffold.

714
L. Pergaud Sandjo et al.
H
H
RO
OR
H
O
H
RO
O
H
H
O
O
H
O
H
H
H
H
O
Figure 4. NOESY correlations of products 1A, 2A and 3A.
Table 1. Cytotoxic activities of compounds (IC50, 48 h) against HT1080.
Compound
IC50 (mM)
1
210
1A
400
1B
110
2
10
2A
135
2B
10
3
3A
350
3B
460
RD
1
4500
Note: Reference drug: etoposide.
This observation suggested the acetal proton which was attached to anomeric carbon
before, kept its configuration as in the case of �-D-glucopyranoside (Figure 4). The
resonance at �H 4.88 (H-10) further revealed correlations with those at �H 5.72 (H-20) and
4.02 (H-80). The proton H-80 at �H 4.02 correlated with the methylene protons at �H 3.62
(H-70) and oxymethine proton at �H 3.70 (H-40). Both acetal proton at �H 5.49 (H-50) and
CH2 protons at �H 3.64 (H-70a) showed NOE interactions with the protons of oxymethine
at �H 3.70 (H-40). All the correlations led to determine the absolute configurations in the
triacetal moeity to be 10S, 20R, 40S, 50S and 80S.
Compounds 1A–3A were further subjected to the acetylation reaction and the products
along with the starting materials were tested against fibrosarcoma HT1080 a cancer cell
line. The cytotoxicity was carried out by flow cytometry and most of the products showed
moderate activities with the IC50 at 10 and 460 mM (Table 1). The acetal 1A was less
cytotoxic than arjulonic acid but after its acetylation the activity of the product 1B was
improved and was better than those of 1 and 1A. Furthermore, the same feature was
observed with 2 which give IC50 at 10 mM but its acetal 2A was cytotoxic at 135 mM as IC50
and the activity became interesting with the acetylated product 2B. In the pentacyclic
triterpenes, the acetal function seems to reduce the cytotoxicity against HT1080 but its
protection improves the activity. Nevertheless, this function provides the cytotoxic
property to compound 3 although reducing the activity in some cases.
3. Experimental
3.1. General
1D and 2D NMR spectra were carried out on a Brucker DRX-400 MHz. Optical rotation
was measured by Perkin-Elmer polarimeter model 341 at 589 nm. SiO2 gel GF254 was
used to perform thin layer chromatography. Iodine–silica gel was employed to visualise the
spots on the TLC plates. HRESIMS and ESI–MS were carried out using MicroTOF-Q 98
(Bruker-Daltonics, Germany).
3.2. General procedure for the preparation of lactol and acetylation
Seven milligrams (0.0144 mmol) of arjulonic acid (1), 7 mg (0.014 mmol) of
24-hydroxytormentic acid (2) and 9 mg (0.016 mmol) of �-sitosterol glucopyranoside (3)
were separately dissolved in 20 mL of THF. Each solution was separately treated with

Natural Product Research
715
15.4 mg (0.072 mmol, 5 eq) of NaIO4 and 50 mg of SiO2. The reaction mixture
was vigorously stirred for 6 h and monitored with TLC. The mixture was dried and
poured onto water. The products were extracted with CHCl3 and concentrated under
vacuum yielding 6.76 mg (97%) of 1A, 6.63 mg (95%) of 2A and 8.77 mg (95.5%) of 3A,
respectively. Acetylation of 1A (4 mg), 2A (4 mg) and 3A (5 mg) were performed separately
in 2 mL of pyridine with 3 mL of acetic anhydride stirred at room temperature for 3 h
affording 1B (4.25 mg, 98.0%), 2B (4.57 mg, 98.0%) and 3B (5.56 mg, 97.0%).
3.2.1. Compound 1A
Colorless amorphous solid, Rf ¼ 0.6 (CH2Cl2–MeOH 37:3); [�] þ 50.5 (c 1, CH2Cl2);
IR; HR-ESI-MS m/z 509 [C30H46O5 þ Na]þ; 1H-NMR (400 MHz, CDCl3) 0.83 (s, Me-26),
0.89 (s, Me-29), 0.92 (s, Me-30), 1.00 (s, Me-24), 1.05 (s, Me-25), 1.09 (m, H-7a), 1.13
(s, Me-27), 1.15 (m, H-19a), 1.18 (m, H-5), 1.22 (m, H-15a), 1.26 (m, H-21), 1.34
(m, H-15b), 1.36 (m, H-6a), 1.45 (m, H-1a), 1.58 (m, H-22a), 1.59 (m, H-9), 1.62 (m, H-
19b), 1.62 (m, H-16a), 1.66 (m, H-7b), 1.71 (m, H-6b), 1.76 (m, H-22b), 1.93 (m, H-11a),
1.97 (m, H-16b), 2.04 (m, H-11b), 2.17 (dd, 5.1, 15.5, H-1b), 2.83 (dd, 4.2, 13.8, H-18), 3.73
(d, 13.3, H-23a),3.94 (d, 13.3, H-23b), 5.13 (dd, 5.0, 9.5, H-2), 5.32 (br t, 3.3, H-12), 9.95
(s, H-3); 13C-NMR (100 MHz, CDCl3) 14.5 (C-25), 17.8 (C-26), 20.5 (C-6), 20.7 (C-24),
23.0 (C-16), 23.6 (C-29), 24.8 (C-11), 25.6 (C-27), 27.9 (C-7), 30.8 (C-21), 32.4 (C-22), 33.2
(C-30), 33.4 (C-20), 34.0 (C-15), 40.0 (C-10), 40.3 (C-8), 41.3 (C-18), 42.3 (C-14), 43.8
(C-9), 44.8 (C-1), 45.7 (C-19), 46.8 (C-17), 53.8 (C-4), 61.2 (C-5), 65.5 (C-23), 93.9 (C-2),
122.8 (C-12), 143.6 (C-13), 183.2 (C-28), 206.2 (C-3);
3.2.2. Compound 1B
Yellow amorphous solid, Rf ¼ 0.7 (CH2Cl2); [�] þ 41.8 (c 1, CH2Cl2); HR-ESI-MS: m/z
551.3342 (Calcd 551.3349) [C32H48O6 þ Na]þ; Some characteristic 1H-NMR data
(400 MHz, CDCl3) 0.83 (s, Me-26), 0.90 (s, Me-29), 0.92 (s, Me-30), 1.00 (s, Me-24),
1.08 (s, Me-25), 9.96 (s, H-3), 1.12 (s, Me-27), 1.18 (m, H-5), 2.04 (s, MeCOO), 3.33
(d, 13.4, H-23a), 3.93 (d, 13.4, H-23b), 5.31 (br t, 3.3, H-12), 6.01 (dd, 5.0, 10.0, H-2);
13C-NMR (100 MHz, CDCl3) 14.7 (C-25), 17.8 (C-26), 20.5 (C-6), 20.6 (C-24), 23.1 (C-16),
23.7 (C-29), 24.9 (C-11), 25.6 (C-27), 27.7 (C-7), 30.8 (C-21), 32.4 (C-22), 33.2 (C-20), 33.2
(C-30), 34.0 (C-15), 40.0 (C-10), 40.5 (C-8), 41.2 (C-18), 42.3 (C-14), 42.7 (C-1), 43.9 (C-9),
45.8 (C-19), 46.7 (C-17), 53.5 (C-4), 61.4 (C-5), 67.8 (C-23), 94.5 (C-2), 122.7 (C-12), 143.8
(C-13), 183.2 (C-28), 205.4 (C-3), (CH3COO) 21.5, 170.2;
3.2.3. Compound 2A
Brown amorphous gum; Rf ¼ 0.6 (CH2Cl2–MeOH 9:1); [�] þ 16.52 (c 0.14, MeOH);
HR-ESI-MS m/z 525.3190 [C30H46O6 þ H]þ (Calcd 527.3187); ESI-MS: m/z 503.3
[C30H46O6 þ H]þ; 1H-NMR (400 MHz, CDCl3); 0.86 (s, Me-26), 0.93 (d, 6.7 Me-30),
0.98 (m, H-6a), 1.02 (m, H-15a), 1.17 (s, Me-25), 1.20 (s, Me-29), 1.20 (s, Me-24), 1.25 (m,
H-16a), 1.30 (m, H-7a), 1.37 (m, H-20), 1.38 (s, Me-27), 1.52 (m, H-6b), 1.53 (m, H-1a),
1.53 (m, H-21a),1.64 (m, H-7b), 1.64 (m, H-22a), 1.73 (m, H-5), 1.74 (m, H-22b), 1.75 (m,
H-16b), 1.80 (m, H-15b),1.83 (m, H-9), 2.05 (m, H-11a), 2.15 (m, H-1b), 2.16 (m, H-11b),
2.52 (dd, 4.2, 13.8, H-18), 2.57 (m, H-21b), 3.05 (d, 12.7, H-23a), 4.09 (d, 12.7, H-23b), 5.05
(dd, 4.9, 9.6, H-2), 5.34 (br t, 3.6, H-12), 9.40 (s, H-3); 13C-NMR (100 MHz, CDCl3) 14.6
(C-24), 15.1 (C-25), 16.5 (C-30), 18.0 (C-26), 23.1 (C-6), 24.5 (C-27), 25.6 (C-11), 26.6
(C-21), 27.3 (C-16), 27.3 (C-29), 30.1 (C-15), 33.6 (C-7), 39.3 (C-22), 41.3 (C-8), 41.3
(C-10), 43.1 (C-20), 43.2 (C-14), 45.4 (C-9), 46.4 (C-1), 49.1 (C-17), 54.9 (C-5), 55.2 (C-18),

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L. Pergaud Sandjo et al.
57.3 (C-4), 64.4 (C-23), 73.5 (C-19), 129.6 (C-12), 140.0 (C-13), 95.2 (C-2), 182.3 (C-28),
207.7 (C-3);
3.2.4. Compound 2B
Brown dark amorphous gum; Rf ¼ 0.55 (CH2Cl2); HR-ESI-MS: m/z 587.3575
[C34H50O8 þ H]þ (Calcd 587.3578); ESI-MS: m/z 587.4 [C34H50O8 þ H]þ; some character-
istic 1H-NMR data (400 MHz, CDCl3) 0.86 (s, Me-26), 0.94 (d, 6.5, Me-30), 1.18
(s, Me-25), 1.20 (s, Me-24), 1.20 (s, Me-29), 1.28 (s, Me-27), 2.54 (d, 9.8, H-18), 3.25 (d,
12.7, H-23a), 4.05 (d, 12.7, H-23b), 5.36 (br s, H-12), 6.04 (dd, 5.2, 10.0, H-2), 9.44 (s, H-3),
C-2 (CH3COO) 2.04 (s), C-19 (CH3COO) 2.20 (s); 13C-NMR (100 MHz, CDCl3) 14.2
(C-24), 14.7 (C-25), 16.2 (C-30), 17.6 (C-26), 22.8 (C-6), 24.0 (C-27), 24.7 (C-11), 26.1
(C-21), 27.5 (C-16), 27.5 (C-29), 29.5 (C-15), 32.1 (C-7), 36.3 (C-22), 40.3 (C-8), 40.4
(C-10), 41.1 (C-20), 42.5 (C-1), 42.5 (C-14), 53.1 (C-18), 44.1 (C-9), 49.3 (C-17), 54.3 (C-5),
56.0 (C-4), 65.5 (C-23), 73.1 (C-19), 94.9 (C-2), 129.7 (C-12), 137.4 (C-13), 182.2 (C-28),
206.1 (C-3), C-2 (CH3COO) 21.5, 170.4, C-19 (CH3COO) 22.7, 167.3
3.2.5. Compound 3A
Yellow bright amorphous solid, Rf ¼ 0.5 (CH2Cl2–MeOH 37 : 3); HR-ESI-MS: m/z
575.4315 [C35H58O6 þ H]þ (Calcd 575.4306), 597.4135 [C35H58O6 þ Na]þ (Calcd
597.4126); ESI-MS: m/z 575.4 [C35H58O6 þ H]þ; [�] � 6.2 (c 0.22, CH2Cl2); 1H-NMR
(400 MHz, CDCl3) 0.68 (s, CH3-18), 0.81 (d, 6.9, CH3-26), 0.83 (d, 7.2, CH3-27), 0.85 (m,
CH3-29), 0.91 (overlapped with C-21, H-9), 0.92 (overlapped, H-24), 0.92 (d, 6.5, CH3-21),
0.98 (m, H-14), 1.00 (s, CH3-19), 1.00 (m, H-22a), 1.06 (m, H-1a), 1.06 (m, H-15a), 1.10 (m,
H-17), 1.15 (H-12a), 1.25 (m, H-16a), 1.26 (m, H-23a), 1.28 (m, H-23b), 1.28 (m, H-28),
1.32 (m, H-22b), 1.35 (m, H-20), 1.42 (br s, H-8), 1.43 (m, H-11a), 1.48 (m, H-11b), 1.53
(m, H-7a), 1.57 (m, H-15b), 1.58 (m, H-2a), 1.65 (m, H-25), 1.83 (m, H-16b), 1.85 (m,
H-1b), 1.90 (m, H-2b), 1.97 (m, H-7b), 2.00 (m, H-12b), 2.23 (m, H-4a), 2.32 (ddd, 1.9, 4.7,
13.0, H-4b), 3.57 (m, H-3), 5.35 (d, 5.1, H-6); Triacetal moiety 3.64 (br d, 5.7, H-70a), 3.67
(d, 15.4, H-70b), 4.02 (br d, 1.0, H-80), 3.72 (dd, 5.7, 8.2, H-40), 4.88 (br d, 1.3, H-10), 5.72
(br s, H-20), 5.49 (br s, H-50). 13C-NMR (100 MHz, CDCl3) 12.0 (C-18), 12.1 (C-29), 18.9
(C-26), 19.2 (C-21), 19.5 (C-19), 20.0 (C-27), 21.2 (C-11), 23.1 (C-23), 24.4 (C-15), 28.3
(C-16), 29.2 (C-28), 29.3 (C-25), 29.9 (C-2), 32.0 (C-8), 32.1 (C-7), 34.0 (C-22), 36.3 (C-20),
36.2 (C-10), 37.2 (C-1), 38.9 (C-4), 39.8 (C-12), 42.4 (C-13), 46.0 (C-24), 50.3 (C-9), 56.1
(C-17), 56.8 (C-14), 78.5 (C-3), 122.4 (C-6), 140.2 (C-5); Triacetal part 61.7 (C-70), 75.9
(C-40), 80.4 (C-80), 94.4 (C-20), 94.5 (C-10), 100.6 (C-50).
3.2.6. Compound 3B
Brown amorphous solid, Rf ¼ 0.69 (CH2Cl2); [�] � 4.2 (c 0.18, CH2Cl2); HR-ESI-MS: m/z
681.4331 [C39H62O8 þ Na]þ (Calcd 681.4337); ESI-MS: m/z 659.4 [C39H62O8 þ H]þ; some
1
characteristic H-NMR signals (400 MHz, CDCl3): 0.68 (s, Me-18), 0.82 (d, 6.9, Me-26),
0.84 (d, 7.2, Me-27), 0.92 (H-24), 0.85 (m, Me-29), 0.91 (overlapped with C-21, H-9), 0.92
(d, 6.5, CH3-21), 0.98 (m, H-14), 1.00 (s, CH3-19), 1.10 (m, H-17), 1.06 (m, H-15a), 1.15
(H-12a), 1.25 (m, H-16a), 1.26 (m, H-23a), 1.28 (m, H-23b), 1.28 (m, H-28), 1.42 (br s,
H-8), 1.43 (m, H-11a), 1.48 (m, H-11b), 1.53 (m, H-7a), 1.57 (m, H-15b), 1.65 (m, H-25),
1.83 (m, H-16b), 1.97 (m, H-7b), 2.00 (m, H-12b), 5.36 (d, 5.2, H-6); Triacetal moiety 3.81
(t, 6.9, H-40), 4.02 (dd, 7.2, 11.4, H-70b), 4.13 (dd, 6.0, 11.6, H-70a), 4.19 (br d, 1.5, H-80)
4.91 (br d, 1.5, H-10), 5.55 (br s, H-50), 6.53 (br s, H-20); Acetyl part 2.06 (CH3), 2.11 (CH3)
13C-NMR (100 MHz, CDCl3) 12.0 (C-18), 12.1 (C-29), 18.9 (C-26), 19.2 (C-21), 19.5
(C-19), 20.0 (C-27), 21.2 (C-11), 23.2 (C-23), 24.4 (C-15), 28.4 (C-16), 29.3 (C-28), 29.5

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(C-25), 29.8 (C-2), 32.1 (C-8), 32.1 (C-7), 34.1 (C-22), 36.3 (C-20), 36.9 (C-10), 37.3 (C-1),
38.9 (C-4), 39.9 (C-12), 42.5 (C-13), 45.9 (C-24), 50.3 (C-9), 56.2 (C-17), 56.9 (C-14), 78.5
(C-3), 122.4 (C-6), 140.2 (C-5) Triacetal part 62.3 (C-70), 73.4 (C-40), 79.2 (C-80), 92.8
(C-20), 94.0 (C-10), 101.1 (C-50). Acetyl part 20.9, 170.2 (CH3COOH), 22.8, 170.6
(CH3COOH)
3.3. Cellular viability
The human HT1080 fibrosarcoma adherent cell line was cultured at 37�C in a humidified
atmosphere containing 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM/F12)
supplemented with 10% fetal bovine serum together with penicillin (100 mg mL�1),
streptomycin (100 U mL�1) and glutamax (1% v/v) from Invitrogen. The cells were seeded
in 12 well plates (5.104 cells well�1). 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 DMSO and stored at �20�C. Cells were then incubated for 48 h and cellular viability
was determined by flow cytometric analysis. Global cell death was then determined with
the cationic lipophilic DiOC6 (3) dye (Invitrogen) which specifically probes mitochondrial
membrane potential (D�m) (Bras, Queenan, & Susin, 2005). After drug treatment, the
media from each well were kept in centrifuge tubes. The adherent cells were detached using
trypsine, pooled with the corresponding media, centrifuged and resuspended in complete
medium. Cells were then loaded with 100 nM DiOC6 (3) and incubated for 30 min at
37�C. Flow cytometric measurements were performed using a 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 DiOC6 (3) was collected with a 525 nm band
pass filter. The percentage of dead cells was determined by measuring the percentage of
cells harboring low DiOC6 (3) fluorescence. Analyses were performed on 104 cells.
4. Conclusions
Many methods have been reported for the preparation of acetal or ketal but the solubility
of starting material always remained a difficulty to be surmounted. Thus, THF has a
facility to dissolve many natural products which are pyridine or DMSO soluble. From this
advantage, some acetal triterpenes were prepared and most of the products showed a
moderated cytotoxicity activity against HT1080 cancer cell lines. The obtained products
showed that intra-molecular acetalisation required systems of 1,2,3-triol and 1,2,4-triol
(Figure 2). Although, the aldehyde function is unsuitable as drug candidate, it could be
useful in further synthesis reactions such as Claisen–Schmidt condensation, Grignard or
Wittig reactions in order to develop more bioactive compounds. Besides, this lactol can
also be oxidised into lactone triterpene.
Acknowledgements
AVBD would like to thank NABSA (Network for Analytical and Bio-assay Services in Africa)
for the financial support of a short term visit in the University of Gaberone, Botswana.
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