2-O-(2-chlorobenzoyl) maslinic acid triggers apoptosis in A2780 human ovarian carcinoma cells

Article abstract of DOI:10.1016/j.ejmech.2019.07.049

Depending on the conditions of the reactions, maslinic acid can be converted into the corresponding 2-O-, 3-O-, or 2,3-di-O-acylated compounds in good yields. These compounds showed in SRB assays a significantly increased cytotoxicity as compared to the p

Full text of DOI:10.1016/j.ejmech.2019.07.049

Accepted Manuscript
2-O-(2-chlorobenzoyl) maslinic acid triggers apoptosis in A2780 human ovarian
carcinoma cells
Immo Serbian, Bianka Siewert, Ahmed Al-Harrasi, René Csuk
PII:
S0223-5234(19)30670-1
DOI:
https://doi.org/10.1016/j.ejmech.2019.07.049
EJMECH 11546
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 May 2019
Revised Date: 28 June 2019
Accepted Date: 15 July 2019
Please cite this article as: I. Serbian, B. Siewert, A. Al-Harrasi, René. Csuk, 2-O-(2-chlorobenzoyl)
maslinic acid triggers apoptosis in A2780 human ovarian carcinoma cells, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.07.049.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
2-O-(2-Chlorobenzoyl) maslinic acid triggers apoptosis in A2780 human ovarian
carcinoma cells
Immo Serbian 1,a, Bianka Siewert 2,a, Ahmed Al-Harrasi 3, René Csuk 1,*
1
Martin-Luther-University Halle-Wittenberg; Organic Chemistry, Kurt-Mothes-Str. 2, D-
06120 Halle (Saale), Germany; rene.csuk@chemie.uni-halle.de
2
University of Innsbruck, Institute of Pharmacy/Pharmacognosy, Center for Molecular
Biosciences Innsbruck (CMBI), Center for Chemistry and Biomedicine, Innrain 80-82, A-
6020 Innsbruck, Austria; bianka.siewert@uibk.ac.at
3
University of Nizwa, Natural and medical sciences Center, P.O. Box 33, Birkat Al Mauz,
Nizwa 616, Oman; aharrasi@unizwa.edu.om
a These authors contributed equally
* Corresponding author
Abstract
Depending on the conditions of the reactions, maslinic acid can be converted into the
corresponding 2-O-, 3-O-, or 2,3-di-O-acylated compounds in good yields. These compounds
showed in SRB assays a significantly increased cytotoxicity as compared to the parent
compound maslinic acid. For the most active compound of this series, i.e. 2-O-(2-
chlorobenzoyl) maslinic acid (5), more detailed cell biological tests (i.e. AO/PI dye exclusion
experiments, an annexin V assay, and microscopic investigations) on A2780 (human ovarian
carcinoma cells) revealed that this compound triggers apoptosis.
Keywords: Maslinic acid; triterpenoids; acylation; cytotoxicity; apoptosis
Introduction
Recently the pentacyclic triterpenoid maslinic acid [(2α, 3β) dihydroxy-12-oleanen-28-oic
acid, MA; Fig. 1) moved into the focus of phytomedical interest due to its broad and
promising activity spectrum.[1-5] As a secondary metabolite, MA was detected in and
isolated from a variety of different plants.[1, 6-9] Part of MA’s success, however, is based on
the fact that it can be easily obtained from the waste of olive oil production (available only in
the countries dealing with the production of olive oil) [9], from pickled olives (which can be
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bought in every supermarket)[10], or – even more conveniently – by a partial synthesis from
commercially available oleanolic acid.[11]
Fig. 1. Structure and numbering scheme for maslinic acid (MA)
Maslinic acid displays interesting pharmacological properties. The most promising, however,
is its anti-carcinogenic activity. Yu et al. recently showed the potent activity of MA to inhibit
the NFκΒ mediated survival of gallbladder cancer cells in vivo.[12] Furthermore, cytotoxic
effects of MA have been reported for different malignant cell lines, among them
colorectal,[13] pancreatic,[14] renal,[15] bladder,[16] melanoma,[17] and lung cancer [18].
The same holds true for a number of derivatives of MA.[10, 19-22] Interesting though, while
numerous MA derivatives were modified at position 28,[10, 19, 22-25] only a limited number
of derivatives were studied being exclusively modified in Ring A.[20, 26] Of those modified
at position 28, aromatic esters and amides proved to be active and selective against cancer
cells even in low µM concentrations, especially if compared to their aliphatic analogues.[10,
22] Of the handful A-ring modified derivatives, Parra and coworkers [20] studied a series of
aliphatic acyl moieties at positions C-2 and/or C-3. These derivatives, however, showed only
moderate anticancer activity against murine melanoma cancer cells (b16f10). Saxena and
colleagues [26], in contrast, showed for tormentic acid a significant increase in its cytotoxic
activity against liver cancer cells (WRL-68) due to the acetylation of position 2 and 3.
Furthermore, also a couple of aromatic acyl derivatives of tormentic acid proved to be highly
active on a low µM level.[26] Thus, we became interested in studying a series of aromatic
acyl derivatives in ring A in order to contribute to the understanding of the structure-activity
relation of these modifications.
Results and Discussion
As previously reported, acetylation of MA with acetyl chloride in pyridine yielded a mixture
1
of 2-O-acetyl maslinic acid (1) and the 2,3-diacetate 2 (Scheme 1).[10] The H NMR
2

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spectrum of 1 is characterized by the presence of an additional sharp singlet for the methyl
group of the acetyl moiety at δ = 1.97 ppm. For H-C(2) a shifting of the signal to lower fields
is observed (|∆δ| = 1.36 ppm) whereas the position of the signal for H-C(3) remains
practically unchanged (|∆δ| = 0.13 ppm). For the diacetate 2 H-C(2) as well as H-C(3) are
shifted to lower fields (|∆δ|H-C(2) = 1.32 and |∆δ|H-C(3) = 1.66 ppm).
Scheme 1. Synthesis of maslinic acid derivatives 1-11: compounds 2-8 were obtained from
the reaction of with RCOCl in dry pyridine in the presence of DMAP (catal.) while 9-11 were
synthesized using Steglich conditions (DCC, DMAP, DCM).
A similar de-shielding of H-C(2) and H-C(3) was observed upon acylation of MA with
substituted benzoyl chlorides. The obtained products of this reaction, however, differed
significantly. Utilizing substituted benzoyl chlorides yielded the two mono-substituted
derivatives of MA; for example, from the reaction of MA with 2-fluorobenzoyl chloride a
mixture of 2-O-(2-fluorobenzoate) 3 and 3-O-(2-fluorobenzoate) 4 but no dibenzoate was
obtained. Similarly, acylation of MA with 2-chloro-benzoyl chloride or 3,5-dinitro-benzoyl
chloride yielded the 2-O-mono-benzoates 5 and 7 as well as the 3-O-mono-benzoates 6 and 8,
respectively. These observations indicate that the second esterification step is sterically
hindered, due to the methyl groups at position C-4, the benzoyl moiety at position C-2, and
the electrophile itself. Thus, the second step is not favored under the applied reaction
conditions at room temperature.
19
Compound 3 is characterized in its F NMR spectrum by the presence of a signal at δ = -
109.7 ppm; for 4 a signal at δ = -109.4 ppm was observed (Figure 2). Because of additional
fluorine-proton and fluorine-carbon couplings, the NMR spectra are complex. Finally, a
3

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combination of several 1D and 2D NMR techniques allowed the unambiguous assignment of
all signals and the determination of all coupling constants. Fluorine-carbon coupling constants
were taken from the 13C APT spectra. Interestingly enough, although C-37 is in meta-position
to the fluorine substituent, no JC,F was found for this carbon whereas carbon C-35 – being also
in meta-position – showed a JC,F = 8.9 Hz.
1
19
Fig. 2. Expansions from the H NMR spectrum (500 MHz, CDCl3, upper trace) and the F
NMR spectrum (188 MHz, CDCl3) of 4 and the simulated spectrum (bottom trace).
4

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Di-acylated compounds, however, could be obtained by applying Steglich esterification
conditions. Thus, reaction of MA with benzoic acid in the presence of DCC gave a mixture of
the 2-O-benzoate 9 and the 2,3-di-O-benzoate 10. In a similar way, the 2,3-di-O-(2-
chloronicotinoate) 11 was obtained.
In a next step, to investigate the anticancer activity of the new compounds, the photometric
SRB assay was performed.[27] The EC50 values from these tests employing several different
human cancer cell lines are compiled in Table 1.
Table 1. Cytotoxicity (EC50 in µM) of compounds) 1-11, maslinic acid (MA) and betulinic
acid (BA, as a standard); SRB assays after 96 h of treatment; the values are averaged from
three independent experiments each performed in triplicate; cut-off of the assay 30 µM; n.d.
not detected/not determined. Human tumor cell lines: 518A2 (melanoma), 8505C (thyroid
carcinoma), A253 (submandibular carcinoma), A2780 (ovarian carcinoma), A549 (alveolar
basal epithelial adenocarcinoma), DLD1 (colorectal adenocarcinoma), HT29 (colorectal
adenocarcinoma), MCF-7 (breast adenocarcinoma), SW1736 (thyroid carcinoma) and non-
malignant mouse fibroblasts NIH 3T3.
EC50
BA
518A2
11.9
1.1
8505C
A253
A2780
12.7
1.2
A549
14.9
1.2
DLD1
HT29
18.4
2.0
MCF-7
12.0
1.7
SW1736
NIH 3T3
±
±
±
±
±
±
6.7 ± 0.4
n.d.
±
±
±
±
±
±
n.d.
±
±
±
±
±
±
16.4
1.9
±
16.1
1.8
±
MA
1
13.7
1.9
17.0
±
n.d.
19.5
1.7
23.4
0.6
n.d.
28.8
1.0
24.4
2.6
n.d.
16.6
0.5
±
±
±
±
±
±
2.0
21.0
2.0
8.8 ± 0.9
11.0
1.1
±
19.9
2.0
4.3 ± 0.4
4.2 ± 0.4
9.4 ± 0.7
9.8 ± 0.4
7.5 ± 1.2
5.8 ± 0.5
6.1 ± 1.0
n.d.
23.1
2.3
±
±
±
±
10.7
1.1
11.5
1.0
n.d.
n.d.
15.4
1.5
2
14.6
1.3
5.7 ± 0.6
7.7 ± 0.8
17.3
1.7
17.0
1.5
8.5 ± 0.9
6.3 ± 0.8
n.d.
8.5 ± 0.7
7.1 ± 1.0
6.7 ± 1.3
5.6 ± 1.3
3.3 ± 0.6
5.1 ± 0.8
4.2 ± 0.6
n.d.
10.4
1.1
3
11.6
0.8
12.6
1.4
±
12.9
±
6.0 ± 0.5
6.0 ± 0.6
4.7 ± 0.3
n.d.
10.7
0.5
16.9
0.2
±
±
±
±
±
±
±
±
16.9
0.8
0.3
4
11.8
0.2
12.6
1.4
±
6.6 ± 0.6
10.1
1.1
21.2
0.5
12.5
0.5
5
8.3 ± 0.3
6.8 ± 0.5
8.9 ± 0.8
6.9 ± 0.6
8.1 ± 0.6
7.5 ± 0.9
10.4
±
7.6 ± 0.7
5.2 ± 1.0
7.1 ± 0.6
7.2 ± 0.7
8.5 ± 0.6
n.d.
13.9
0.6
10.4
0.1
2.3
6
5.4 ± 0.6
12.9
1.4
7.1 ± 0.3
7
12.1
0.8
±
9.7 ± 1.4
8.3 ± 2.4
9.0 ± 1.2
6.2 ± 0.6
5.5 ± 0.7
6.4 ± 0.9
11.1
±
14.8
1.1
17.4
0.8
±
1.0
8
n.d.
9.4 ± 0.8
12.8
0.6
15.7
0.6
±
±
±
9
15.5
0.3
±
±
11.2
0.9
±
9.1 ± 2.0
10.3
1.2
±
12.7
0.6
±
18.2
2.0
15.0
1.1
10
11
n.d.
16.4
0.3
±
12.7
1.9
±
11.2
1.0
±
17.4
0.7
±
17.9
0.5
±
16.5
0.6
±
n.d.
19.4
1.7
23.2
2.0
17.1
> 30
>30
29.3
±
>30
>30
>30
>30
>30
>30
5

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1.2
0.5
As shown in Table 1, the effect of the classical acylation of parent MA depends on the origin
of cell line: While 1 lost its activity against cells of the melanoma cell line (518A2) compared
to MA, compound 1 is five times more active against cells of a lung cancer cell line (A549).
The substituted acyl benzoyl derivatives, in contrast, were constantly and independent of the
substitution pattern (mono or di) more potent than parent MA.
Of all synthesized compounds, the mono 2-chlorobenzoyl derivative 5 showed the most
potent activity along all tested cell lines. Therefore, we submitted this compound to more
detailed biological studies. Fig. 3 depicts the results from a dye-exclusion experiment
(acridine orange/propidium assay AO/PI). In this test, floating A2780 ovarian cancer cells
were stained with AO/PI and investigated by fluorescence microscopy after having been
incubated with MA or compound 5 for 72 h. In this assay, green colored cells indicate a
functional cell membrane, whereas complete red stained cells are characterized by a loss of
cell membrane integrity and thus indicate necrosis; necrosis may be either of primary or of
secondary nature. The latter typically occurs in vitro as a final event of apoptosis because of a
lack of phagocytic cells. The green stained dead cells were characterized by membrane blebs
(Figure 3 white arrows), which is a typical hallmark of apoptosis.[28] Furthermore, the
majority of the treated and dead cells showed red/orange dots in the cytosol. They occur
because AO readily crosses all membranes of the cell including those of lysosomes and other
acidic vesicles. However, once entered, AO cannot escape the acidic compartments anymore,
because AO is easily protonated due to its tertiary amines and thus trapped in these
compartments as a charged molecule.[29] As a consequence, the concentration of AO
increases and stacks of the dye occur, which are characterized by a red fluorescence.[30, 31]
Thus, an increased occurrence of red/orange dots in the cytosol is handled as autophagy
marker.[32]
Fig. 3. AO/PI dye exclusion assay utilizing ovarian cancer cells (A2780). In this fluorescence
spectroscopic analysis, the cells were treated with MA (30 µM) or 5 (15 µM) for 72 h,
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respectively. Green colored cells indicate a programmed cell death, red fluorescent cells were
necrotic. White arrows mark the typical hallmarks of apoptosis, the orange-colored arrows
indicate acidic compartments, hence indicating an autophagy dependent apoptosis.
To quantify the induced apoptosis an extra flow cytometry assay (Annexin V/PI test) was
performed. The results of this assay confirmed the conclusions of the AO/PI microscopic
assays and are depicted in Fig. 4. As previously shown, MA is able to induce apoptosis [29]
and so does compound 5 albeit more slowly than MA. This might be due to a retarded
transport through the cell membrane. Taking together, the conducted biological studies hint
towards an autophagy mediated apoptotic mode-of-cell death which was recently also
reported for parent MA. [33, 34]
Fig. 4. Results of the annexin V assay. Ovarian cancer cells (A2780) were treated with MA
(30 µM, red bars) or compound 5 (15 µM, green bars) for 24, 48 and 72 h, respectively. The
upper part shows the distribution (in %) of the cells (vital, apoptosis, secondary necrosis and
necrotic cells). Blue squares show the control experiment. In the lower part representative
plots are given for the treatment of 5 (after 24, 48 or 72 h of incubation).
7

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Conclusion
Depending on the reaction conditions used, maslinic acid can be converted into the
corresponding acylated compounds in good yield: reactions with (substituted) benzoyl
chlorides resulted in a mixture of 2-O-acylated and 3-O-acylated maslic acid derivatives.
Acylation under Steglich conditions gave in good yields the doubly acylated derivatives
instead. For biological screening, SRB assays were performed for all compounds and showed
a significantly increased cytotoxicity as compared to the starting material. Detailed
investigations (AO/PI dye exclusion, annexin V assay and microscopy) of the effect of 2-O-
(2-chlorobenzoyl) maslinic acid (5) on A2780 (human ovarian carcinoma cells) revealed that
this compound is capable of inducing autophagy mediated apoptosis.
Acknowledgment
We like to thank Dr. R. Kluge for the MS spectra and Dr. D Ströhl and his team for the NMR
measurements, Ms V. Simon for the UV/Vis, IR and optical rotation measurements, and Dr.
S. Ludwig for supporting the synthesis. The cell lines were kindly provided by Dr. Th. Müller
(Dep. of Haematology/Oncology, Martin-Luther-University Halle-Wittenberg). Financial
support by ScienceCampus Halle (W13004216 to R.C) and the Oman Research Council
(ORG/EBR/15/007 to A.A.-H.) are gratefully acknowledged.
Experimental
Reagents were bought from commercial suppliers without any further purification. Melting
points were measured with a LEICA hot stage microscope and were not corrected; other
equipment was used as previously described. SRB-assays were prepared according to Vichai
et al. [27] and the EC50 values were calculated from semi logarithmic dose response curves
(7x3 data points) by non-linear regression applying a two parametrical Hill-slope equation
and GraphPad Prism 5. Values are given with a confidence interval CI = 95%. AO/PI staining
as well as the annexin V/PI assay was performed as previously described.[22] The calculation
of the data for the latter assay was performed as described in the suppliers (BD Biosciences®)
manual. In short, approximately 500,000 cells (A2780) were seeded in cell culture flasks and
were allowed to grow for 1 day. After removing of the medium, the substance-loaded medium
was added, and the flasks were incubated for about 72 h. The supernatant medium was
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collected and centrifuged; the cell pellet was suspended in PBS and centrifuged again. Equal
amounts of a PI/AO solution (1:1, 100 µg/ ml) and a suspension of the cell pellet in PBS (w/o
Ca2+ and Mg2+) were softly mixed. Visual analysis was performed under a fluorescence
microscope (Axioskop II (Carl Zeiss, Jena). While green fluorescence shows apoptosis, a
deep colored nucleus indicates necrotic cells; weak orange dots mark lysosomes.
Maslinic acid (MA)
This compound was synthesized from oleanolic acid as previously reported.[11]
2-O-Acetyl-maslinic acid (1) and 2,3-di-O-acetyl-maslinic acid (2).
Acetylation of maslinic acid (475 mg, 1.0 mmol) in dry pyridine (10 mL) with acetyl chloride
(215 µL, 3 mmol) for 24 h at 5 °C followed by usual aqueous workup and chromatography
(silica gel, n-hexane/ethyl acetate, 10:1 ‰ 5:1) gave 1 (380 mg, 74%) and 2 (70 mg, 13%).
Data for 1: colorless solid; m.p. 181-183 °C; RF = 0.64 (n-hexane/ethyl acetate, 1:2); [α]D =
9.0° (c 0.3, MeOH) (lit.: [α]D = 3.8° (c 0.03, MeOH))[35]; MS (ESI, MeOH): m/z 515.3
(100%, [M+H]+).
Data for 2: [22] colorless solid; m.p. 221-223 °C (lit.: 225-229 °C [36]); RF = 0.30 (n-
hexane/ethyl acetate, 3:1); [α]D = 29.3° (c 0.3, CHCl3) (lit: [α]D = 30° (c 8.3, CHCl3)[30]);
MS (ESI, MeOH): m/z 557.3 (50%, [M+H]+, 1135.2 (100%, [2M+Na]+).
2-O-(2-Fluorobenzoyl) maslinic acid (3) and 3-O-(2-fluorobenzoyl) maslinic acid (4)
Acylation of maslinic acid (475 mg, 1 mmol) with 2-fluoro-benzoyl chloride (0.39 mL, 3
mmol) in dry pyridine (10 mL) at room temperature followed by usual aqueous workup and
chromatography (silica gel, n-hexane/ethyl acetate, 15:1 ‰ 5:1) gave 3 (330 mg, 56%) and 4
(165 mg, 28%).
Data for 3: colorless solid; m.p. = 235-238 °C; RF = 0.50 (n-hexane/ethyl acetate, 3:2); [α]D =
+ 4.7° (c 0.2, MeOH); IR (KBr): ν = 3433m, 2948s, 1696s, 1614m, 1488m, 1457s, 1304s,
1
1250m, 1128m, 757m cm-1; UV/vis (MeOH): λmax (log ε) = 275 (3.15) nm; H NMR (400
MHz, CDCl3): δ = 7.92 (ddd, J = 7.8, 7.3, 1.9 Hz, 1H, 37-H), 7.50 (dddd, J = 8.3, 7.4, 4.9, 1.9
Hz, 1H, 35-H), 7.20 (ddd, J = 7.8, 7.4, 1.1 Hz, 1H, 36-H), 7.13 (ddd, 1H, J = 10.9, 8.3, 1.1 Hz,
34-H), 5.26 (dd, J = 3.6, 3.6 Hz, 1H, 12-H), 5.18 (ddd, J = 12.3, 10.0, 4.5 Hz, 1H, 2-H), 3.38
(d, J = 10.0 Hz, 1H, 3-H), 2.78 (dd, J = 13.7, 4.0 Hz 1H, 18-H), 2.17 (m, J = 12.3, 4.5 Hz, 1H,
1-H), 1.96 (m, 1H, 16-H), 1.90 (m, 1H, 11-H), 1.88 (m, 1H, 11-H), 1.74 (m, 1H, 22-H), 1.70
(m, 1H, 15-H), 1.60 (m, 1H, 9-H), 1.59 (m, 1H, 16-H), 1.58 (m, 1H, 6-H), 1.57 (m, 1H, 19-
9

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H), 1.55 (m, 1H, 22-H), 1.46 (m, 1H, 7-H), 1.40 (m, 1H, 6-H), 1.33 (m, 1H, 21-H), 1.30 (m,
1H, 7-H), 1.21 (m, 1H, 21-H), 1.10 (m, 1H, 19-H), 1.09 (s, 3H, 27-H), 1.08 (m, 1H, 1-H),
1.07 (s, 3H, 24-H), 1.06 (s, 3H, 26-H), 1.05 (m, 1H, 15-H), 0.91 (m, 1H, 5-H), 0.90 (s, 3H,
13
29-H), 0.89 (s, 3H, 23-H), 0.88 (s, 3H, 30-H), 0.77 (s, 3H, 25-H) ppm; C NMR (100 MHz,
CDCl3): δ = 184.5 (C-28), 164.7 (C-31, JC,F =3.6 Hz), 161.6 (C-33, JC,F = 259.1 Hz), 143.4
(C-13), 134.5 (C-35, JC,F = 9.0 Hz), 132.0 (C-37), 124.1 (C-36, JC,F = 3.7 Hz), 122.6 (C-12),
119.3 (C-32, JC,F = 10.0 Hz), 116.6 (C-34, JC,F = 22.7 Hz), 80.8 (C-3), 75.1 (C-2), 55.3 (C-5),
47.8 (C-9), 46.6 (C-17), 45.6 (C-19), 43.5 (C-1), 41.7 (C-14), 40.7 (C-18), 39.6 (C-4), 39.5
(C-8), 38.4 (C-10), 33.9 (C-21), 33.2 (C-30), 32.6 (C-7), 32.5 (C-22), 30.4 (C-20), 28.7 (C-
24), 27.7 (C-15), 25.9 (C-27), 23.3 (C-11), 23.5 (C-29), 22.6 (C-16), 18.3 (C-6), 17.0 (C-25)
16.6 (C-23), 16.3 (C-26) ppm; 19F NMR (188 MHz, CDCl3): δ = -109.7 (ddd, JH,F = 10.9, 7.3,
4.9 Hz) ppm; MS (ESI, MeOH): m/z 595.3 (100%, [M+H]+); analysis calcd for C37H51FO5
(594.80): C 74.71, H 8.64; found: C 74.50, H 8.82.
Data for 4: colorless solid; m.p. = 171-174 °C; [α]D = 33.2° (c 0.41, MeOH); RF = 0.41 (n-
hexane/ethyl acetate, 3:2); IR (KBr): ν = 3445m, 2948s, 1698s, 1615m, 1490m, 1457m,
1366m, 1299s, 1250m, 1129m cm-1; UV/vis (MeOH): λmax (log ε) = 277 (3.10) nm; 1H NMR
(400 MHz, CDCl3): δ = 7.96 (ddd, J = 7.8, 7.2, 1.9 Hz, 1H, 37-H), 7.52 (dddd, J = 8.3, 7.3,
4.8, 1.9 Hz, 1H, 35-H), 7.18 (ddd, J = 7.8, 7.3, 1.1 Hz, 1H, 36-H), 7.14 (ddd, , J = 10.8, 8.3,
1.1 Hz, 1H, 34-H), 5.29 (dd, J = 3.2, 3.2 Hz, 1H, 12-H), 4.76 (d, J = 10.0 Hz, 1H, 3-H), 3.95
(ddd, J = 12.6, 10.0, 4.5 Hz, 1H, 2-H), 2.80 (dd, J = 13.6, 3.9 Hz, 1H, 18-H), 2.09 (dd, J =
12.6, 4.5 Hz 1H, 1-H), 1.98 (m, 1H, 16-H), 1.95 (m, 1H, 11-H), 1.91 (m, 1H, 11-H), 1.74 (m,
1H, 22-H, 1.71 (m, 1H, 15-H), 1.66 (m, 1H, 9-H), 1.61 (m, 1H, 19-H), 1.59 (m, 1H, 16-H),
1.55 (m, 1H, 6-H), 1.54 (m, 1H, 22-H), 1.47 (m, 1H, 7-H), 1.40 (m, 1H, 6-H), 1.33 (m, 1H,
21-H), 1.30 (m, 1H, 7-H), 1.21 (m, 1H, 21-H), 1.15 (m, 1H, 19-H), 1.15 (s, 3H, 27-H), 1.06
(m, 1H, 15-H), 1.04 (m, 1H, 1-H), 1.00 (s, 3H, 26-H), 1.00 (m, 1H, 5-H), 0.95 (s, 3H, 23-H),
0.94 (s, 3H, 24-H), 0.91 (s, 3H, 29-H), 0.90 (s, 3H, 30-H), 0.75 (s, 3H, 25-H) ppm; 13C NMR
(100 MHz, CDCl3): δ = 183.7 (C-28), 165.5 (C-31, JC,F = 3.7 Hz), 161.7 (C-33, JC,F = 259.7
Hz), 143.5 (C-13), 134.5 (C-35, JC,F = 8.9 Hz), 132.4 (C-37), 124.1 (C-36, JC,F = 3.8 Hz),
122.5 (C-12), 119.1 (C-32, JC,F = 10.0 Hz), 117.2 (C-34, JC,F = 22.6 Hz), 86.3 (C-3), 67.7 (C-
2), 55.2 (C-5), 47.4 (C-9), 47.6 (C-1), 46.5 (C-17), 45.9 (C-19), 41.5 (C-14), 40.8 (C-18), 39.6
(C-4), 39.3 (C-8), 38.3 (C-10), 33.9 (C-21), 33.1 (C-30), 32.5 (C-7), 32.4 (C-22), 30.5 (C-20),
28.4 (C-24), 27.5 (C-15), 26.1 (C-27), 23.4 (C-29), 23.3 (C-11), 22.9 (C-16), 18.4 (C-6), 17.6
19
(C-23), 17.2 (C-25) 16.7 (C-26) ppm; F NMR (188 MHz, CDCl3): δ = -109.4 (ddd, JH,F
=
10

ACCEPTED MANUSCRIPT
10.8, 7.2, 4.8 Hz); MS (ESI, MeOH): m/z 595.4 (100%, [M+H]+); analysis calcd for
C37H51FO5 (594.80): C 74.71, H 8.64; found: C 74.49, H 8.88.
2-O-(2-Chlorobenzoyl) maslinic acid (5) and 3-O-(2-chlorobenzoyl) maslinic acid (6)
Acylation of maslinic acid (475 mg 1 mmol) with 2-chloro-benzoyl chloride (0.40 mL, 3
mmol) in dry pyridine (10 mL) at room temperature followed by usual aqueous workup and
chromatography (silica gel, n-hexane/ethyl acetate, 15:1 ‰ 5:1) gave 5 (430 mg, 71%) and 6
(130 mg, 21%).
Data for 5: colorless solid; m.p. = 234-237 °C; [α]D = 5.5° (c 0.2, MeOH); RF = 0.49 (n-
hexane/ethyl acetate, 3:2); IR (KBr): ν = 3435m, 2948s, 1696s, 1593m, 1463m, 1436m,
1300s, 1250s, 1138m, 1050m, 750m cm-1; UV/Vis (MeOH): λmax log (ε) = 280 (2.75) nm; 1H
NMR (400 MHz, CDCl3): δ = 7.80 (ddd, J = 7.8, 1.7, 0.5 Hz, 1H, 37-H), 7.44 (ddd, J = 8.1,
1.5, 0.5 Hz, 1H, 34-H), 7.40 (ddd, J = 8.1, 7.1, 1.7, 1H, 35-H, Hz), 7.28 (ddd, J = 7.8, 7.1, 1.5
Hz, 1H, 36-H), 5.25 (dd, J = 3.6, 3.6 Hz, 1H, 12-H), 5.21 (ddd, J = 12.2, 10.0, 4.5 Hz, 1H, 2-
H), 3.38 (d, J = 10.0 Hz, 1H, 3-H), 2.81 (dd, J = 13.9, 4.5 Hz, 1H, 18-H), 2.17 (m, J = 12.2, J
= 4.5 Hz, 1H, 1-H), 1.93 (m, 1H, 16-H), 1.90 (m, 1H, 11-H), 1.87 (m, 1H, 11-H), 1.74 (m,
1H, 22-H), 1.71 (m, 1H, 15-H), 1.62 (m, 1H, 9-H), 1.58 (m, 1H, 16-H), 1.57 (m, 1H, 6-H),
1.56 (m, 1H, 19-H), 1.54 (m, 1H, 22-H), 1.44 (m, 1H, 7-H), 1.40 (m, 1H, 6-H), 1.32 (m, 1H,
21-H), 1.31 (m, 1H, 7-H), 1.22 (m, 1H, 21-H), 1.13 (m, 1H, 19-H), 1.12 (s, 3H, 27-H), 1.10
(m, 1H, 1-H), 1.09 (s, 3H, 26-H), 1.08 (s, 3H, 24-H), 1.06 (m, 1H, 15-H), 0.91 (s, 3H, 23-H),
0.90 (s, 3H, 29-H), 0.88 (m, 1H, 5-H), 0.86 (s, 3H, 30-H), 0.74 (s, 3H, 25-H) ppm; 13C NMR
(100 MHz, CDCl3): δ = 184.3 (C-28), 166.0 (C-31), 143.5 (C-13), 133.4 (C-33), 132.7 (C-
35), 131.6 (C-37), 131.1 (C-34), 130.6 (C-32), 126.8 (C-36), 122.4 (C-12), 80.6 (C-3), 75.2
(C-2), 55.3 (C-5), 47.7 (C-9), 46.4 (C-17), 45.7 (C-19), 43.4 (C-1), 41.7 (C-14), 40.7 (C-18),
39.6 (C-4), 39.4 (C-8), 38.6 (C-10), 33.7 (C-21), 33.1 (C-30), 32.4 (C-7), 32.3 (C-22), 30.6
(C-20), 28.5 (C-24), 27.4 (C-15), 25.8 (C-27), 23.4 (C-11), 23.3 (C-29), 22.7 (C-16), 18.2 (C-
6), 17.4 (C-25), 16.6 (C-23), 16.4 (C-26) ppm; MS (ESI, MeOH): m/z 611.5 (84%, [M+H]+);
analysis calcd for C37H51ClO5 (611.25): C 72.70, H 8.41; found: C 72.52, 8.59.
Data for 6: colorless solid; m.p. = 166-169 °C; [α]D = 24.0° (c 1.8, MeOH); RF = 0.43 (n-
hexane/ethyl acetate, 3:2); IR (KBr): ν = 3424m, 2946s, 1732s, 1697s, 1472m, 1365m, 1296s,
1251s, 1126m, 1050m, 960m, 747m cm-1; UV/Vis (MeOH): λmax (log ε) = 281 (2.78) nm; 1H
NMR (400 MHz, CDCl3): δ = 7.83 (ddd, 1H, J = 7.7, 1.7, 0.5 Hz, 37-H),), 7.43 (ddd, J = 8.1,
1.4, 0.5 Hz, 1H, 34-H), 7.39 (ddd, J = 8.1, 7.2, 1.7 Hz, 1H, 35-H), 7.30 (ddd, J = 7.7, 7.2, 1.4
11

ACCEPTED MANUSCRIPT
Hz, 1H, 36-H), 5.27 (dd, J = 3.5, 3.5 Hz, 1H, 12-H), 4.80 (d, J = 10.0 Hz, 1H, 3-H), 3.93 (ddd,
J = 12.6, 10.0, 4.5 Hz, 1H, 2-H), 2.81 (dd, J = 13.8, 4.1 Hz, 1H, 18-H), 2.08 (dd, J = 12.6, 4.5
Hz, 1H, 1-H), 1.97 (m, 1H, 16-H), 1.94 (m, 1H, 11-H), 1.92 (m, 1H, 11-H), 1.75 (m, 1H, 22-
H), 1.70 (m, 1H, 15-H), 1.65 (m, 1H, 9-H), 1.61 (m, 1H, 19-H), 1.60 (m, 1H, 16-H), 1.56 (m,
1H, 6-H), 1.55 (m, 1H, 22-H), 1.48 (m, 1H, 7-H), 1.39 (m, 1H, 6-H), 1.33 (m, 1H, 21-H), 1.31
(m, 1H, 7-H), 1.20 (m, 1H, 21-H), 1.15 (m, 1H, 19-H), 1.13 (s, 3H, 27-H), 1.06 (m, 1H, 15-
H), 1.05 (m, 1H, 1-H), 1.00 (m, 1H, 5-H), 1.00 (s, 3H, 26-H), 0.98 (s, 3H, 24-H), 0.93 (s, 3H,
13
23-H), 0.91 (s, 3H, 29-H), 0.89 (s, 3H, 30-H), 0.74 (s, 3H, 25-H) ppm; C NMR (100 MHz,
CDCl3): δ = 183.9 (C-28), 166.8 (C-31), 143.6 (C-13), 133.3 (C-33), 132.4 (C-35), 131.4 (C-
37), 131.0 (C-34), 130.7 (C-32), 126.6 (C-36), 122.3 (C-12), 86.3 (C-3), 67.4 (C-2), 55.2 (C-
5), 47.6 (C-9), 47.5 (C-1), 46.5 (C-17), 45.8 (C-19), 41.6 (C-14), 40.9 (C-18), 39.5 (C-4), 39.3
(C-8), 38.2 (C-10), 33.8 (C-21), 33.0 (C-30), 32.4 (C-7), 32.4 (C-22), 30.6 (C-20), 28.6 (C-
24), 27.6 (C-15), 25.9 (C-27), 23.5 (C-29), 23.4 (C-11), 22.8 (C-16), 18.3 (C-6), 17.8 (C-23),
17.1 (C-25) 16.6 (C-26) ppm; MS (ESI): m/z 611.5 (81%, [M+H]+); analysis calcd for
C37H51ClO5 (611.25): C 72.70, H 8.41; found: C 72.49, H 8.63.
2-O-(3, 5-Dinitrobenzoyl) maslinic acid (7) and 3-O-(3, 5-dinitrobenzoyl) maslinic acid
(8)
Acylation of maslinic acid (475 mg, 1 mmol) with 3,5-dinitro-benzoyl chloride (1.10 g, 4.5
mmol) in dry pyridine (10 mL) at room temperature followed by usual aqueous workup and
chromatography (silica gel, n-hexane/ethyl acetate, 15:1 ‰ 5:1) gave 7 (520 mg, 78%) and 8
(120 mg, 18%).
Data for 7: slightly yellowish solid; m.p. = 230-233 °C; [α]D = -14.5° (c 0.2, MeOH); RF =
0.50 (n-hexane/ethyl acetate, 3:2); IR (KBr): ν = 3439m, 3103m, 2949s, 1732s, 1697m,
1
1549s, 1344s, 1282m, 1171m, 730m cm-1; H NMR (400 MHz, CDCl3): δ = 9.25 (t, J = 2.1
Hz, 1H, 35-H), 9.14 (d, J = 2.1 Hz, 2H, 33-H + 37-H), 5.33 (ddd, J = 12.2, 10.0, 4.6 Hz, 1H,
2-H), 5.25 (dd, J = 3.5, 3.5 Hz, 1H, 12-H), 3.40 (d, J = 10.0 Hz, 1H, 3-H), 2.82 (dd, J = 13.8,
4.0 Hz, 1H, 18-H), 2.12 (m, J = 12.2, 4.6 Hz, 1H, 1-H), 1.99 (m, 1H, 16-H), 1.91 (m, 1H, 11-
Ha), 1.87 (m, 1H, 11-Hb), 1.75 (m, 1H, 22-H), 1.70 (m, 1H, 15-H), 1.68 (m, 1H, 9-H), 1.59
(m, 1H, 6-H), 1.58 (m, 1H, 16-H), 1.57 (m, 1H, 19-H), 1.56 (m, 1H, 22-H), 1.48 (
m, 1H, 7-
H), 1.42 (m, 1H, 6-H), 1.35 (m, 1H, 7-H), 1.31 (m, 1H, 21-H), 1.22 , 1H, 1-H), 1.20 (m,
(m
1H, 21-H), 1.15 (s, 3H, 27-H), 1.10 (m, 1H, 19-H), 1.09 (s, 3H, 24-H), 1.08 (s, 3H, 26-H),
1.05 (m, 1H, 15-H), 0.96 (m, 1H, 5-H), 0.90 (s, 3H, 23-H), 0.88 (s, 3H, 29-H), 0.88 (s, 3H,
30-H), 0.76 (s, 3H, 25-H) ppm; 13C NMR (100 MHz, CDCl3): δ = 183.6 (C-28), 162.9 (C-31),
12

ACCEPTED MANUSCRIPT
148.5 (C-34 + C-36), 143.6 (C-13), 134.3 (C-32), 129.5 (C-33 + C-37), 122.5 (C-12), 122.2
(C-35), 80.7 (C-3), 76.4 (C-2), 55.0 (C-5), 47.5 (C-9), 46.7 (C-17), 45.9 (C-19), 43.6 (C-1),
41.5 (C-14), 40.8 (C-18), 40.0 (C-4), 39.4 (C-8), 38.6 (C-10), 33.8 (C-21), 33.1 (C-30), 32.5
(C-7), 32.4 (C-22), 30.7 (C-20), 28.5 (C-24), 27.8 (C-15), 25.7 (C-27), 23.6 (C-29), 23.5 (C-
11), 22.9 (C-16), 18.4 (C-6), 17.0 (C-25) 16.7 (C-23), 16.5 (C-26) ppm; MS (ESI, MeOH):
m/z 667.4 (92%, [M+H]+); analysis calcd for C37H50N2O9 (666.80): C 66.65, H 7.56, N 4.20;
found : C 66.42, H 7.71, N 3.98.
Data for 8: slightly yellowish solid; m.p. = 181-183 °C; [α]D = 21.1° (c 0.3, MeOH); RF =
0.44 (n-hexane/ethyl acetate, 3:2); IR (KBr): ν = 3445m, 3105m, 2946s, 1734s, 1697m,
1550s, 1461m, 1345s, 1281s, 1171m, 730m cm-1 1H NMR (400 MHz, CDCl3): δ = 9.22 (t, J =
2.1 Hz, 1H, 35-H), 9.15 (d, J = 2.1 Hz, 2H, 33-H + 37-H), 5.29 (dd, J = 3.4, 3.4 Hz, 1H, 12-
H), 4.87 (d, J = 10.0 Hz, 1H, 3-H), 4.01 (ddd, J = 12.7, 10.0, 4.6 Hz, 1H, 2-H), 2.84 (dd, J =
13.6, 3.8 Hz, 1H, 18-H), 2.13 (dd, J = 12.7, J = 4.6 Hz, 1H, 1-H), 1.99 (m, 1H, 16-H), 1.97
(m, 1H, 11-H), 1.94 (m, 1H, 11-H), 1.77 (m, 1H, 22-H), 1.69 (m, 1H, 15-H), 1.66 (m, 1H, 9-
H), 1.62 (m, 1H, 16-H), 1.60 (m, 1H, 19-H), 1.59 (m, 1H, 6-H), 1.55 (m, 1H, 22-H), 1.49 (m,
1H, 7-H), 1.45 (m, 1H, 6-H), 1.35 (m, 1H, 7-H), 1.34 (m, 1H, 21-H), 1.20 (m, 1H, 21-H), 1.15
(m, 1H, 19-H), 1.14 (s, 3H, 27-H), 1.09 (m, 1H, 1-H), 1.07 (m, 1H, 15-H), 1.06 (s, 3H, 26-H),
1.04 (s, 3H, 23-H), 1.03 (m, 1H, 5-H), 0.98 (s, 3H, 24-H), 0.91 (s, 3H, 29-H), 0.90 (s, 3H, 30-
13
H), 0.76 (s, 3H, 25-H) ppm; C NMR (100 MHz, CDCl3): δ = 183.9 (C-28), 163.4 (C-31),
148.9 (C-34 + C-36), 143.5 (C-13), 134.0 (C-32), 129.4 (C-33 + C-3), 122.5 (C-35), 122.0
(C-12), 87.8 (C-3), 67.6 (C-2), 55.0 (C-5), 47.8 (C-1), 47.5 (C-9), 46.4 (C-17), 45.9 (C-19),
41.5 (C-14), 41.1 (C-18), 39.5 (C-4), 39.4 (C-8), 38.4 (C-10), 33.9 (C-21), 33.1 (C-30), 32.4
(C-7), 32.3 (C-22), 30.8 (C-20), 28.7 (C-24), 27.7 (C-15), 25.8 (C-27), 23.5 (C-11), 23.4 (C-
29), 22.6 (C-16), 18.3 (C-6), 17.7 (C-23), 17.7 (C-25) 16.6 (C-26) ppm; MS (ESI): m/z 667.4
(74%, [M+H]+); analysis calcd for C37H50N2O9 (666.80): C 66.65, H 7.56, N 4.20; found: C
66.40, H 7.70, N 4.03.
2-O-Benzoyl maslinic acid (9) and 2,3-di-O-benzoyl maslinic acid (10)
To a suspension of maslinic acid (475 mg, 1 mmol) in dry DCM (20 mL), benzoic acid (366
mg, 3 mmol), DCC (620 mg; 3 mmol) and DMAP (catl.) were added, and stirring at 25 °C
was continued for 2 days. Usual aqueous work-up followed by chromatography (silica gel, n-
hexane/ethyl acetate, 3:1) gave 9 (440 mg, 76%) and 10 (110 mg, 15%).
13

ACCEPTED MANUSCRIPT
Data for 9: colorless solid; m.p. = 196-199 °C; [α]D = -6.1° (c 0.4, MeOH); RF = 0.49 (n-
hexane/ethyl acetate, 2:1); IR (KBr): ν = 3448m, 2947s, 1718s, 1693s, 1452m, 1386w, 1276s,
1
1160w, 1116m, 1029w, 715m cm-1; UV/Vis: λmax (log ε) = 273 (2.82) nm; H NMR (500
MHz, CDCl3): δ = 8.00 (dd, J = 8.4, 1.3 Hz, 2H, 33-H + 37-H), 7.55 (tt, J = 7.4, 1.3 Hz, 1H,
35-H), 7.40 (dd, J = 8.4, 7.4 Hz, 2H, 34-H + 36-H), 5.25 (dd, J = 3.7, 3.7 Hz, 1H, 12-H), 5.21
(ddd, J = 11.4, 10.0, 4.5 Hz, 1H, 2-H), 3.38 (d, J = 10.0 Hz, 1H, 3-H), 2.79 (dd, J = 13.9, 4.6
Hz, 1H, 18-H), 2.10 (m, 1H, 1-H), 1.97 (m, 1H, 16-H), 1.90 (m, 1H, 11-H), 1.85 (m, 1H, 11-
H), 1.74 (m, 1H, 22-H), 1.71 (m, 1H, 15-H), 1.61 (m, 1H, 9-H), 1.60 (m, 1H, 16-H), 1.59 (m,
1H, 6-H), 1.58 (m, 1H, 19-H), 1.54 (m, 1H, 22-H), 1.46 (m, 1H, 7-H), 1.40 (m, 1H, 6-H), 1.33
(m, 1H, 21-H), 1.31 (m, 1H, 7-H), 1.18 (m, 1H, 21-H), 1.13 (s, 3H, 27-H), 1.12 (m, 1H, 1-H),
1.11 (m, 1H, 19-H), 1.09 (s, 3H, 24-H), 1.08 (s, 3H, 26-H), 1.07 (m, 1H, 15-H), 0.91 (m, 1H,
5-H), 0.90 (s, 3H, 23-H), 0.89 (s, 3H, 29-H), 0.88 (s, 3H, 30-H), 0.75 (s, 3H, 25-H) ppm; 13C
NMR (100 MHz, CDCl3): δ = 184.1 (C-28), 167.1 (C-31), 143.9 (C-13), 133.2 (C-35), 130.2
(C-32), 129.7 (C-33 + C-37), 128.5 (C-34 + C-36), 122.4 (C-12), 81.1 (C-3), 74.0 (C-2), 55.3
(C-5), 47.7 (C-9), 46.6 (C-17), 45.9 (C-19), 43.8 (C-1), 41.7 (C-14), 40.7 (C-18), 40.1 (C-4),
39.5 (C-8), 38.6 (C-10), 33.9 (C-21), 33.3 (C-30), 32.6 (C-7), 32.2 (C-22), 30.8 (C-20), 28.6
(C-24), 27.8 (C-15), 26.1 (C-27), 23.5 (C-29), 23.4 (C-11), 23.1 (C-16), 18.4 (C-6), 17.3 (C-
25) 16.8 (C-23), 16.5 (C-26); MS (ESI, MeOH): 577.4 (100%, [M+H]+); analysis calcd for
C37H52O5 (576.81): C 77.04, H 9.09; found: C 76.81, H 9.27
Data for 10: colorless solid: m.p. = 179-181 °C; [α]D = -28.4° (c 0.2, MeOH); RF = 0.50 (n-
hexane/ethyl acetate, 2:1); IR (KBr): ν = 3425m, 2947s, 1725s, 1603w, 1452m, 1395w,
1315m, 1279s, 1178m, 1118m, 1070w, 1027m, 710m cm-1; UV/Vis (MeOH): λmax (log ε) =
274 (3.0) nm; 1H NMR (500 MHz, CDCl3): δ = 7.96 + 7.85 (dd, J = 8.4, 1.3 Hz, 4H, 33-H +
37-H + 40-H + 44-H), 7.46 + 7.40 (tt, J = 7.4, 1.3 Hz, 2H, 35-H + 42-H), 7.31 + 7.29 (dd, J =
8.4, 7.4 Hz, 4H, 34-H + 36-H + 41-H + 43-H), 5.47 (ddd, J = 11.5, 10.4, 4.6 Hz, 1H, 2-H),
5.27 (dd, J = 3.6, 3.6 Hz, 1H, 12-H), 5.23 (d, J = 10.4 Hz, 1H, 3-H), 2.83 (dd, J = 13.8, 4.2
Hz, 1H, 18-H), 2.27 (m, J = 12.4, 4.6 Hz, 1H, 1-H), 1.99 (m, 1H, 16-H), 1.95 (m, 1H, 11-H),
1.89 (m, 1H, 11-H), 1.76 (m, 1H, 22-H), 1.72 (m, 1H, 15-H), 1.70 (m, 1H, 9-H), 1.63 (m, 1H,
6-H), 1.62 (m, 1H, 16-H), 1.61 (m, 1H, 19-H), 1.57 (m, 1H, 22-H), 1.50 (m, 1H, 7-H), 1.47
(m, 1H, 6-H), 1.36 (m, 1H, 7-H), 1.34 (m, 1H, 21-H), 1.24 (m, 1H, 1-H), 1.21 (m, 1H, 21-H),
1.19 (s, 3H, 26-H), 1.15 (s, 3H, 27-H), 1.13 (m, 1H, 5-H), 1.12 (m, 1H, 19-H), 1.09 (m, 1H,
15-H), 1.08 (s, 3H, 23-H), 1.01 (s, 3H, 24-H), 0.90 (s, 3H, 29-H), 0.89 (s, 3H, 30-H), 0.79 (s,
13
3H, 25-H) ppm; C NMR (100 MHz, CDCl3): δ = 184.5 (C-28), 166.4 + 166.2 (C-31 + C-
38), 143.8 (C-13), 132.9 (C-35 + C-42), 130.2 + 130.0 (C-32 + C-39), 129.7 + 129.4 (C-33 +
14

ACCEPTED MANUSCRIPT
C-37 + C-40 + C-44), 128.3 + 128.3 (C-34 + C-36 + C-41 + C-43), 122.4 (C-12), 81.0 (C-3),
71.3 (C-2), 55.1 (C-5), 47.5 (C-9), 46.4 (C-17), 45.9 (C-19), 43.7 (C-1), 41.6 (C-14), 40.7 (C-
18), 39.7 (C-4), 39.4 (C-8), 38.5 (C-10), 33.9 (C-21), 33.0 (C-30), 32.6 (C-7), 32.4 (C-22),
30.8 (C-20), 28.6 (C-24), 27.8 (C-15), 26.1 (C-27), 23.5 (C-29), 23.4 (C-11), 22.8 (C-16),
18.4 (C-6), 17.6 (C-23), 17.1 (C-25) 16.5 (C-26) ppm; MS (ESI, MeOH): m/z 681.1 (100%,
[M+H]+), 698.3 (81%, [M+NH4]+) 703.5 (68%, [M+Na]+), 1383.2 (60%, [2M+Na]+); analysis
calcd for C44H56O6 (680.91): C 77.61, H 8.29; found: C 77.48, H 8.47.
2, 3-Di-O-(2-chloronicotinoyl) maslinic acid (11)
Reaction of maslinic acid (475 mg,1 mmol) with 2-chloronicotinic acid (788 mg, 5 mmol) in
dry DCM (20 mL) in the presence of DCC (1.03 g, 5 mmol) and DMAP (catal.) for 2 days
followed by usual workup and chromatography (n-hexane/ethyl acetate, 3:1) gave 11 (465
mg, 71%) as a colorless solid; m.p. = 315-317 °C; [α]D = 14.1° (c 0.25, CHCl3); RF = 0.60 (n-
hexane/ethyl acetate, 1:2); IR (KBr): ν = 3452w, 2947s, 1794m, 1743s, 1579m, 1561m,
1405s, 1291s, 1242m, 1143s, 1066s, 1051s, 1003m, 978m, 954m cm-1; UV/Vis (CHCl3): λmax
(log ε) = 271 (3.85) nm; 1H NMR (400 MHz, CDCl3): δ = 8.45 + 8.56 (dd, 2H, 34-H + 40-H,
J = 4.8, 2.0 Hz), 8.09 + 8.03 (dd, 2H, 36-H + 42-H, J = 7.7, 2.0 Hz), 7.27 + 7.25 (dd, 2H, 35-
H + 41-H, J = 7.7, 4.8 Hz), 5.51 (ddd, 1H, 2-H, J = 11.6, 10.3, 4.7 Hz), 5.32 (dd, 1H, 12-H, J
= 3.4, 3.4 Hz), 5.21 (d, 1H, 3-H, J = 10.3 Hz), 2.83 (dd, 1H, 18-H, J = 13.9, 4.2 Hz), 2.27 (m,
1H, 1-H, J = 12.3, J = 4.7 Hz), 2.04 (m, 1H, 16-H), 1.95 (m, 1H, 11-H), 1.90 (m, 1H, 11-H),
1.80 (m, 1H, 22-H), 1.79 (m, 1H, 15-H), 1.68 (m, 1H, 9-H), 1.64 (m, 1H, 19-H), 1.60 (m, 1H,
16-H), 1.58 (m, 1H, 6-H), 1.53 (m, 1H, 22-H), 1.50 (m, 1H, 7-H), 1.48 (m, 1H, 6-H), 1.37 (m,
1H, 7-H), 1.32 (m, 1H, 21-H), 1.25 (m, 1H, 1-H), 1.24 (m, 1H, 21-H), 1.17 (s, 3H, 27-H),
1.15 (m, 1H, 19-H), 1.14 (s, 3H, 26-H), 1.09 (m, 1H, 5-H), 1.08 (m, 1H, 15-H), 1.06 (s, 3H,
23-H), 1.02 (s, 3H, 24-H), 0.93 (s, 3H, 29-H), 0.89 (s, 3H, 30-H), 0.83 (s, 3H, 25-H) ppm; 13C
NMR (100 MHz, CDCl3): δ = 173.1 (C-28), 164.5 + 163.3 (C-31 + C-37), 152.0 + 152.1 (C-
34 + C-40), 150.5 + 149.8 (C-33 + C-39), 143.5 (C-13), 140.5 + 140.1 (C-36 + C-42), 126.7 +
125.9 (C-32 + C-38), 122.4 (C-12), 122.3 (C-35 + C-41), 82.5 (C-3), 71.9 (C-2), 55.1 (C-5)
,
48.4 (C-17), 47.7 (C-9), 45.8 (C-19), 43.8 (C-1), 41.6 (C-14), 41.3 (C-18), 39.8 (C-4), 39.4
(C-8), 38.2 (C-10), 33.7 (C-21), 33.1 (C-30), 32.6 (C-7), 31.5 (C-22), 30.8 (C-20), 28.7 (C-
24), 27.5 (C-15), 25.8 (C-27), 23.8 (C-11), 23.6 (C-29), 22.8 (C-16), 18.1 (C-6), 17.8 (C-23),
17.2 (C-25) 16.4 (C-26) ppm; MS (ESI, MeOH): 751.3 (86%, [M+H]+); analysis calcd for
C42H52N2Cl2O6 (751.78): C 67.10, H 6.97, N 3.72; found: C 66.81, H 7.18, N 3.56.
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References
[1] G. Lozano-Mena, M. Sánchez-González, M.E. Juan, J.M. Planas, Maslinic Acid, a Natural
Phytoalexin-Type Triterpene from Olives — A Promising Nutraceutical?, Molecules, 19
(2014) 11538-11559.
[2] W.H. Yap, Y.M. Lim, Mechanistic Perspectives of Maslinic Acid in Targeting
Inflammation, Biochem. Res. Int. 2015 (2015) 279356.
[3] Q. Wei, B. Zhang, P. Li, X. Wen, J. Yang, Maslinic Acid Inhibits Colon Tumorigenesis by
the AMPK-mTOR Signaling Pathway, J. Agric Food. Chem. 67 (2019) 4259-4272.
[4] A. Loesche, M. Kahnt, I. Serbian, W. Brandt, R. Csuk, Triterpene-Based Carboxamides
Act as Good Inhibitors of Butyrylcholinesterase, Molecules, 24 (2019).
[5] H. Sharma, P. Kumar, R.R. Deshmukh, A. Bishayee, S. Kumar, Pentacyclic triterpenes:
New tools to fight metabolic syndrome, Phytomedicine, 50 (2018) 166-177.
[6] A. Venditti, C. Frezza, M. Riccardelli, S. Foddai, M. Nicoletti, M. Serafini, A. Bianco,
Unusual molecular pattern in Ajugoideae subfamily: the case of Ajuga genevensis L. from
Dolomites, Nat. Prod. Res.30 (2016) 1098-1102.
[7] N.K. Ban, N.T.K. Thoa, T.M. Linh, D.T. Trang, P. Van Kiem, N.X. Nhiem, B.H. Tai, C.
Van Minh, J.H. Song, H.J. Ko, S.H. Kim, Labdane-type diterpenoids from Vitex limonifolia
and their antivirus activities, J. Nat. Med. 72 (2018) 290-297.
[8] L. Bai, H. Zhang, Q. Liu, Y. Zhao, X. Cui, S. Guo, L. Zhang, C.T. Ho, N. Bai, Chemical
characterization of the main bioactive constituents from fruits of Ziziphus jujuba, Food Funct.
7 (2016) 2870-2877.
[9] A. Fernandez-Hernandez, A. Martinez, F. Rivas, J.A. Garcia-Mesa, A. Parra, Effect of the
solvent and the sample preparation on the determination of triterpene compounds in two-
phase olive-mill-waste samples, J. Agric. Food Chem. 63 (2015) 4269-4275.
[10] B. Siewert, E. Pianowski, A. Obernauer, R. Csuk, Towards cytotoxic and selective
derivatives of maslinic acid, Bioorg. Med. Chem. 22 (2014) 594-615.
[11] S. Sommerwerk, L. Heller, I. Serbian, R. Csuk, Straightforward partial synthesis of four
diastereomeric 2,3-dihydroxy-olean-12-en-28-oic acids from oleanolic acid, Tetrahedron, 71
(2015) 8528-8534.
[12] Y. Yu, J. Wang, N. Xia, B. Li, X. Jiang, Maslinic acid potentiates the antitumor activities
of gemcitabine in vitro and in vivo by inhibiting NF-kappaB-mediated survival signaling
pathways in human gallbladder cancer cells, Oncol. Rep. 33 (2015) 1683-1690.
[13] F.J. Reyes-Zurita, E.E. Rufino-Palomares, L. Garcia-Salguero, J. Peragon, P.P. Medina,
A. Parra, M. Cascante, J.A. Lupianez, Maslinic Acid, a Natural Triterpene, Induces a Death
16

ACCEPTED MANUSCRIPT
Receptor-Mediated Apoptotic Mechanism in Caco-2 p53-Deficient Colon Adenocarcinoma
Cells, PLoS One, 11 (2016) e0146178.
[14] Y. Tian, H. Xu, A.A. Farooq, B. Nie, X. Chen, S. Su, R. Yuan, G. Qiao, C. Li, X. Li, X.
Liu, X. Lin, Maslinic acid induces autophagy by down-regulating HSPA8 in pancreatic
cancer cells, Phytother. Res. 32 (2018) 1320-1331.
[15] P. Thakor, W. Song, R.B. Subramanian, V.R. Thakkar, D.A. Vesey, G.C. Gobe, Maslinic
Acid Inhibits Proliferation of Renal Cell Carcinoma Cell Lines and Suppresses Angiogenesis
of Endothelial Cells, J. Kidney Cancer VHL, 4 (2017) 16-24.
[16] S. Zhang, D. Ding, X. Zhang, L. Shan, Z. Liu, Maslinic acid induced apoptosis in bladder
cancer cells through activating p38 MAPK signaling pathway, Mol. Cell. Biochem. 392
(2014) 281-287.
[17] K. Mokhtari, E.E. Rufino-Palomares, A. Perez-Jimenez, F.J. Reyes-Zurita, C. Figuera, L.
Garcia-Salguero, P.P. Medina, J. Peragon, J.A. Lupianez, Maslinic Acid, a Triterpene from
Olive, Affects the Antioxidant and Mitochondrial Status of B16F10 Melanoma Cells Grown
under Stressful Conditions, Evid. Based Complement Alternat. Med. 2015 (2015) 272457.
[18] X. Bai, Y. Zhang, H. Jiang, P. Yang, H. Li, Y. Zhang, P. He, Effects of maslinic acid on
the proliferation and apoptosis of A549 lung cancer cells, Mol. Med. Rep.13 (2016) 117-122.
[19] M. Ortega-Muñoz, F. Rodríguez-Serrano, E. De los Reyes-Berbel, N. Mut-Salud, F.
Hernández-Mateo, A. Rodríguez-López, J.M. Garrido, F.J. López-Jaramillo, F. Santoyo-
González, Biological Evaluation and Docking Studies of Synthetic Oleanane-type
Triterpenoids, ACS Omega, 3 (2018) 11455-11468.
[20] A. Parra, S. Martin-Fonseca, F. Rivas, F.J. Reyes-Zurita, M. Medina-O'Donnell, A.
Martinez, A. Garcia-Granados, J.A. Lupiañez, F. Albericio, Semi-synthesis of acylated
triterpenes from olive-oil industry wastes for the development of anticancer and anti-HIV
agents, Eur. J. Med. Chem. 74 (2014) 278-301.
[21] Z. Liang, L. Zhang, L. Li, J. Liu, H. Li, L. Zhang, L. Chen, K. Cheng, M. Zheng, X.
Wen, P. Zhang, J. Hao, Y. Gong, X. Zhang, X. Zhu, J. Chen, H. Liu, H. Jiang, C. Luo, H.
Sun, Identification of pentacyclic triterpenes derivatives as potent inhibitors against glycogen
phosphorylase based on 3D-QSAR studies, Eur. J. Med. Chem. 46 (2011) 2011-2021.
[22] B. Siewert, E. Pianowski, R. Csuk, Esters and amides of maslinic acid trigger apoptosis
in human tumor cells and alter their mode of action with respect to the substitution pattern at
C-28, Eur. J. Med. Chem. 70 (2013) 259-272.
[23] S. Alam, F. Khan, 3D-QSAR studies on Maslinic acid analogs for Anticancer activity
against Breast Cancer cell line MCF-7, Sci. Rep. 7 (2017) 6019.
17

ACCEPTED MANUSCRIPT
[24] K. Chouaib, S. Delemasure, P. Dutartre, H.B. Jannet, Microwave-assisted synthesis, anti-
inflammatory and anti-proliferative activities of new maslinic acid derivatives bearing 1,5-
and 1,4-disubstituted triazoles, J. Enzyme Inhib. Med. Chem. 31 (2016) 130-147.
[25] M. Medina-O'Donnell, F. Rivas, F.J. Reyes-Zurita, A. Martinez, F. Galisteo-Gonzalez,
J.A. Lupianez, A. Parra, Synthesis and in vitro antiproliferative evaluation of PEGylated
triterpene acids, Fitoterapia, 120 (2017) 25-40.
[26] M. Saxena, U. Faridi, R. Mishra, M.M. Gupta, M.P. Darokar, S.K. Srivastava, D. Singh,
S. Luqman, S.P.S. Khanuja, Cytotoxic Agents from Terminalia arjuna, Planta Med. 73 (2007)
1486-1490.
[27] V. Vichai, K. Kirtikara, Sulforhodamine B colorimetric assay for cytotoxicity screening,
Nature Protocols, 1 (2006) 1112-1116.
[28] D.V. Krysko, T. Vanden Berghe, K. D'Herde, P. Vandenabeele, Apoptosis and necrosis:
detection, discrimination and phagocytosis, Methods, 44 (2008) 205-221.
[29] Z. Darzynkiewicz, J. Kapuscinski, Acridine orange: A versatile probe of nucleic acids
and other cell constituents, in “Flow cytometry and sorting; Methods in cell biology”, M.
Melamed, T. Lindmo, M. Mendelson (Editors), Wiley, New York, 1990, 291-314.
[30] A. Pierzyńska-Mach, P.A. Janowski, J.W. Dobrucki, Evaluation of acridine orange,
LysoTracker Red, and quinacrine as fluorescent probes for long-term tracking of acidic
vesicles. Cytometry Part A, 85 (2014), 729-737.
[31] Zelenin, A.V., Acridine Orange as a Probe for Cell and Molecular Biology, in
Fluorescent and Luminescent Probes for Biological Activity (Second Edition), W.T. Mason,
Editor. 1999, Academic Press: London, 117-135.
[32] M.P. Thomé, E.C. Filippi-Chiela, E.S. Villodre, C.B. Migliavaca, G.R. Onzi, K.B.
Felipe, G. Lenz, Ratiometric analysis of Acridine Orange staining in the study of acidic
organelles and autophagy, J. Cell Sci. 129 (2016) 4622-4632.
[33] X. Dong, J. Zhang, Z. Zhou, Z. Ye, J. Chen, J. Yuan, F. Cao, X. Wang, W. Liu, W. Yu,
X. Li, Maslinic acid promotes autophagy by disrupting the interaction between Bcl2 and
Beclin1 in rat pheochromocytoma PC12 cells, Oncotarget, 2017. 8(43): p. 74527-74538.
[34] Y. Tian, H. Xu, A. A. Farooq, B. Nie, X. Chen, S. Su, R. Yuan, G. Qiao, C. Li, X. Li, X.
Liu, X. Lin, Maslinic acid induces autophagy by down-regulating HDSPA8 in pancreatic
cancer cells, Phytother. Res. (2018), 32, 1320-1331.
[35] S. Siddiqui Bina, N. Kardar Muhammad, S. Khan, Two New Triterpenoids from
Lawsonia alba, Z.Naturforsch. B 60 (2005) 37-40.
18

ACCEPTED MANUSCRIPT
[36] B. Tursch, D. Daloze, E. Tursch, G. Chiurdoglu, Triterpenes. II. Sapogenins J and L
from Stryphnodendron coriaceum, Bull. Soc. Chim. Belg., 75 (1966) 26-28.
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Highlights
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•
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Triterpenoidic maslinic acid was acylated at positions C-2 and/or C-3
A small library of acylated compounds was prepared and screened for cytotoxicity
2-O-(2-Chlorobenzoyl)-maslinic acid showed good cytotoxicity for A2780 cells
This compound acts by an autophagy mediated apoptotic mode of cell death