Hederagenin as a triterpene template for the development of new antitumor compounds

Article abstract of DOI:10.1016/j.ejmech.2015.10.006

In this study, a series of novel C-28 esters and amides derivatives of hederagenin (He) were designed and synthesized in attempt to develop potent antitumor agents. Their structures were confirmed by MS, IR, ¹H NMR and ¹³C NMR spectroscopic analyses and their cytotoxic activities were screened in SRB assays using a panel of six human cancer cell lines. Although most of the compounds displayed moderate to high levels of cytotoxic activity they were all more potent than the natural product He. The most active compounds had either an ethylpyrimidinyl (27) or an ethylpyrrolidinyl (28) substituent, with EC₅₀ in the range of 1.1-6.5 μM for six human cancer cell lines. Notably, this corresponds to an approximately 30-fold times greater potency than He.

Full text of DOI:10.1016/j.ejmech.2015.10.006

Accepted Manuscript
Hederagenin as a triterpene template for the development of new antitumor
compounds
Diego Rodríguez-Hernández, Antonio J. Demuner, Luiz C.A. Barbosa, René Csuk,
Lucie Heller
PII:
S0223-5234(15)30288-9
10.1016/j.ejmech.2015.10.006
EJMECH 8141
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 July 2015
Revised Date: 2 October 2015
Accepted Date: 5 October 2015
Please cite this article as: D. Rodríguez-Hernández, A.J. Demuner, L.C.A. Barbosa, R. Csuk, L. Heller,
Hederagenin as a triterpene template for the development of new antitumor compounds, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.10.006.
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ACCEPTED MANUSCRIPT
Graphical abstract

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HEDERAGENIN AS A TRITERPENE TEMPLATE FOR THE
DEVELOPMENT OF NEW ANTITUMOR COMPOUNDS
a,b
a
a,b*
c*
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Diego Rodríguez-Hernández , Antonio J. Demuner , Luiz C. A. Barbosa , René Csuk ,
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Lucie Heller
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Department of Chemistry, Universidade Federal de Viçosa, Av. P. H. Rolfs, s/n, CEP
36570-900, Viçosa, MG, Brazil.
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Department of Chemistry, Universidade Federal de Minas Gerais, Av. Pres. Antônio
Carlos 6627, Campus Pampulha, CEP 31270-901, Belo Horizonte, MG, Brazil.
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Martin-Luther-University Halle-Wittenberg, Organic Chemistry, Kurt-Mothes-Str.2, D-
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06120 Halle (Saale) Germany
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* Corresponding author: Tel.: +55 31 34093396; fax: +55 31 34095757; E-mail addresses:
lcab@ufmg.br (L.C.A. Barbosa) or rene.csuk@chemie.uni-halle.de (R. Csuk).
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Abstract:
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In this study, a series of novel C-28 esters and amides derivatives of hederagenin (He) were
designed and synthetized in attempt to develop potent antitumor agents. Their structures
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were confirmed by MS, IR, H NMR and C NMR spectroscopic analyses and their
cytotoxic activities were screened in SRB assays using a panel of six human cancer cell
lines. Although most of the compounds displayed moderate to high levels of cytotoxic
activity they were all more potent than the natural product He. The most active compounds
had either an ethylpyrimidinyl (27) or an ethylpyrrolidinyl (28) substituent, with EC in
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the range of 1.1-6.5 µM for six human cancer cell lines. Notably, this corresponds to an
approximately 30-fold times greater potency than He.
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Keywords: Sapindus saponaria; pentacyclic triterpenes; hederagenin derivatives; SRB
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assay; folk medicinal plant.
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1. Introduction
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Natural products have been used to treat human diseases for thousands of years. The results
of these treatments have been variable and inconsistent, but advances in pharmaceutical
science have seen natural products used as models for the development of new drugs,
including certain anti-cancer compounds. Within the plethora of known natural products,
triterpenoic acids are considered promising candidates for anti-cancer drug development [1-
2].
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Among the many sources of bioactive natural products are the fruits of Sapindus saponaria
L., (Sapindaceae), popularly known in Brazil as "sabão-de-soldado" (soldier soap) and
"saboeiro" (soap-maker). This medium sized tropical tree found principally in South
America and India, produces great amounts of small fruits where a sap is accumulated [3].
In the tropics these fruits are mainly used as a substitute for soap. However, they are also
used in the folk medicine for treating skin lesions, inflammation and ulcers [3]. In their
pericarps these fruits accumulate great quantities of saponins carrying the aglycone
hederagenin. Hederagenin is a pentacyclic triterpene possessing two hydroxyl groups in
ring A, a double bond in ring C and a carboxylic group at C-28 position. In addition,
hederagenin acts as a chemotaxonomic marker for plants of the Sapindaceae family. Other
plant families like the Dipsacaceae also contain large amounts of hederagenin as aglycone
of saponins [4]. Furthermore it is known that this oleane type triterpenoic acid shows some
interesting biological properties, such as an anti-inflammatory [5], antifungal [6-7],
antimicrobial [8-9] and an anticancer activity [10].
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Our group and others have demonstrated that structural modifications of sesquiterpenoids
[11], diterpenoids [12] and triterpenoids [2, 13-15] might have a high impact onto their
biological activities.
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Compared to other triterpenoids, modification of hederagenin has had little attention and,
therefore, very little is known with respect to their antitumor properties. To the best of our
knowledge only one report [10] has been published showing hederagenin being moderately
active for A549 cancer cell line (EC = 39±6 µM) whilst being inactive for DLD-1 cells.
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Recently, it was shown that the cytotoxic activity of maslinic acid (a constitutional isomer
of the hederagenin) increases by esterification at position 28. Although, introducing a
benzylic substituent at C-28 did not result in significant improvement of the cytotoxicity it
improved selectivity for tumor cells [13].
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In addition, antitumor screening of derivatives of maslinic, oleanolic or ursolinic acids
revealed the presence of a C = O moiety to be essential for antitumor activity [16-18]. It has
also been shown that bulky ester residues seems to interact quite well with an hitherto
unknown intracellular target/receptor [13].
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During the last few years we have been investigating the use of natural products as lead
structures for the discovery of novel putative pharmaceutical drugs [19-21]. Consequently,
in line with this interest, we report here our preliminary findings involving the hederagenin
scaffold.
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2. Results and discussion
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2.1. Chemistry
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Firstly, hederagenin (He) was isolated from the pericarp of S. saponaria using a previously
reported procedure [22] as detailed in the experimental section. In general, the yield of
hederagenin from the dried pericarp of the fruits was around 0.9%. The isolated compound
was fully characterized by comparing its spectroscopic data with those previously reported
[30-32], along with its melting point. Subsequently, He was transformed into various esters
(1-23), by its reaction with alkyl bromides in the presence of finely grounded potassium
carbonate. Aqueous work-up using aqueous HCl (5%), then washing with brine facilitated
the isolation of the products [23]. Thus, following this simple general procedure, the target
alkyl esters were obtained in yields ranging between 35% and 90% (Fig. 1).
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[Insert Figure 1]
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Reaction of hederagenin with several amines in the presence of O-(benzotriazol-1-yl)-
N,N,N',N'-tetramethyluroniumtetrafluoro-boratetetrabutyl (TBTU) as a coupling catalyst
provided the desired amides (25-30) in 72% to 96% yield (Fig. 2) [24].
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[Insert Figure 2]
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The structures of the hederagenin-derived esters and amides (1-30) were confirmed by
extensive spectroscopic analysis. All compounds showed in their respective IR spectra
typical signals expected for the functional groups being present in the molecules. For
-1
example, in the IR spectra of 1-23, strong absorption bands around 1720-1735 cm due to a
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C = O stretching of esters were observed, associated with absorptions between 1250-1150
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cm due to stretching of C-CO-O. For compounds (25-30) the C=O absorptions were
-1
observed around ῡ = 1620-1650 cm being typical for amides. In the case of alkynes 17
-1
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and 29 the bands for the C≡C were observed between ῡ = 2118-2120 cm [25].
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In the H NMR spectra the signals of the aromatic hydrogens were detected between 7.20
and 8.50 ppm; for compound 18, the resonances due to aromatic hydrogen atoms were
found at 8.20 ppm and 7.50 ppm each as a doublet (J = 8.7 Hz). For the amides (25-30) the
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signals of the NH were observed between 6.20-6.30 ppm. In addition, all C NMR signals
for the triterpenoic skeleton in this compound series were similar with the exception of the
signals for C-28 and the group attached at this position. For carbon C-28 a shift to higher
field for the esters and amides was observed as compared to parent He (δ = 180.7 for He to
δ = 177.5±0.75 for the ester carbonyls, and to δ = 178±0.75 for the amide carbonyls). A
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detailed assignment of the NMR spectra ( H and C) for all compounds is given in the
supplementary material associated to this paper. The assignments were possible by using
2D NMR techniques (when required) and the data were consistent with the proposed
structures.
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2.2. Biological screening
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Natural triterpenes as glycyrrhetinic, betulinic, ursolic, oleanolic and maslinic acid, and
their derivatives, are well-known for their anti-cancer activities [2, 26]. Beside cytotoxicity,
many of these compounds have been shown to trigger apoptosis. For the parent compounds,
EC values between 10-80 µM have been determined; some of them were also shown to
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trigger apoptosis. Hederagenin derivatives 1-30 were tested for their cytotoxic activity
using a photometric sulforhodamine B assay (SRB) [27] and the EC50 values were
determined for six different human cancer cell lines [518A2 (melanoma cells), A2780
(ovarian carcinoma), HT29 (colon adenocarcinoma), MCF7 (breast adenocarcinoma), A549
(lung cancer), 8505C (thyroid carcinoma)]. The EC50 values were calculated from dose
response curves applying a non-linear regression using the two parametric Hills-slope
equation (Table 1).
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[Insert Table 1]
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Among all esters and amides, compounds 1-29, were more cytotoxic than parent He, while
the 1-ethylmorpholinyl amide (30) was the only one with a reduced activity (Fig. 3). These
results support the hypothesis that the presence of a bulky group bonded to carbonyl-28 of
the triterpene skeleton modulates their cytotoxic activity. Similar results have previously
been observed for glycyrrhetinic acid: in its natural form (free acid) a 3-fold lower
cytotoxicity was determined as compared to the corresponding esters derivatives [28-29].
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[Insert Figure 3]
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The amides carrying an ethylpyrimidinyl (27) or ethylpyrrolidinyl (28) moiety were the
most active derivatives for all cell lines tested, showing EC values ranging between 1.3-
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6.5 µM for 27 and values between 1.1-3.9 µM for 28. This corresponds to an approximately
30 times higher cytotoxicity than parent He (Fig. 4). In addition, the esters carrying a
benzyl (1) or an ortho-nitrobenzyl (18) group were the most active among all ester
derivatives for the cells lines tested, exhibiting EC50 values between 7.0-9.7 µM for 1 and
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6.1-8.4 µM for 18. These results revealed that compounds 1 and 18 are approximately 20
times more cytotoxic than hederagenin (Fig. 4).
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[Insert Figure 4]
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For all cells lines tested (Fig. 5) the activity of para substituted esters (20, 21, 22)
decreased with the atomic radius of the halogen substituent. Furthermore, amides derived
from heterocycles carrying an additional nitrogen atom (27, 28) were the most active
compounds for all cell lines evaluated in this study.
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[Insert Figure 5]
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To gain a deeper insight into the mode of action of these molecules some additional
experiments were performed. For such experiments we have chosen only the most active
compound 28. For this compound, the death of A2780 human ovarian cancer cells was
investigated using an acridine orange/propidium iodide assay (AO/PI) and fluorescence
microscopy (Fig. 6). Thus, the cells were treated for 48 h with an EC90 concentration of 28.
The results from this assay indicated that a controlled cell death has occurred as evidenced
by the presence of green fluorescent A2780 cells. Several cells showed an orange color, an
evidence that they died by secondary necrosis.
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3. Conclusion
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To sum up, a series of 30 different esters and amides of hederagenin has been designed and
synthesized. All these compounds (carrying a bulky group at C-28) were screened for their
cytotoxic activity employing a panel of six human cancer cell lines including melanoma
cells, ovarian carcinoma, colon adenocarcinoma, breast adenocarcinoma, lung cancer and
thyroid carcinoma using a photometric sulforhodamine-B assay. From these data, it was
evident that almost all compounds exhibited higher cytotoxicity activity for all tested
cancer cell lines compared to hederagenin. Amides carrying a heterocyclic group (27 and
28) were found to be the most promising derivatives showing EC50 ranging between 1.1-6.5
µM. Moreover, as shown by an additional AO/PI staining experiment, it was found that
compound 28 mainly acts by apoptosis. Further intensive modifications at C-28 and studies
concerning the mode of action of these compounds are currently performed in our
laboratory, and the results will be reported in due course.
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4. Experimental part
4.1. General procedures
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Reagents were obtained from Sigma-Aldrich (Milwaukee, Wisconsin, USA) and were used
without any purification. Solvents were purchased from Vetec (Rio de Janeiro, Brazil).
Analytical thin layer chromatography (TLC) was performed on silica gel 60 F254 0.2 mm
thick plates (supplied by Merck, Rio de Janeiro, Brazil), and the spots were visualized by
UV-B light or by spraying with phosphomolybdic acid in 10% EtOH, followed by heating.
Flash column chromatography (typical size of 20 cm length and 2 cm of diameter) was
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performed using silica gel 230-400 mesh. All compounds were fully characterized by IR,
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EI-MS, H NMR and C NMR spectroscopy. Infrared spectra were recorded on a Perkin-
Elmer Paragon 1000 FTIR spectrophotometer, preparing the samples as potassium bromide
disks (1% w/w). Mass spectra were recorded on a Shimadzu GCMS-QP5050A instrument
by direct insertion, using EI mode (70 eV). High resolution mass spectra were recorded on
a Bruker Micro TOF (resolution = 10,000 FWHM) using electro spray ionization (ESI) and
the result reported to four decimal figures. Elemental analyses were measured on a Foss-
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Heraeus Vario EL unit. The H and C NMR spectra were recorded on a Varian Mercury
300 spectrometer at 300 and 75 MHz, respectively, using CDCl as solvent and TMS as
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internal reference, unless otherwise stated. Melting points were measured on a MQAPF-
301 apparatus and were not corrected.
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The H NMR spectra for all compound were assigned for the signals that were clearly well
defined as described for each compound, following the structural numbers presented in the
Supporting Material. For all compounds a multiplet was observed in the range of δ = 0.5 to
2.5 ppm, so the hydrogen atoms in this range could not be assigned.
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4.2. Isolation of Hederagenin (He)
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Dried and ground pericarp of Sapindus saponaria L., (2 kg) was extracted with methanol.
A total of 3 extractions at room temperature was carried out, keeping the solvent in contact
with the sample for 24 hours. For each extraction 1 L of solvent was used. The combined
methanol extracts were concentrated under reduced pressure in a rotary evaporator to afford
650 g of a crude brown residue. To 300 g of this residue a solution of sulfuric acid in
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methanol (1.5 L, 5% v/v) was added, and the mixture was heated under reflux for 3 hours.
After this hydrolysis, the pH was adjusted to 6-7 by adding potassium hydroxide in
methanol (5% v/v). To the resultant mixture activated charcoal (100 g) was added, followed
by a heating under reflux for 30 min. The resulting mixture was filtered through a Celite®
pad (2 g), and the filtrate was concentrated under reduced pressure to approximately 10%
of the initial volume. To this solution 1 L of distilled hot water was added, and the residual
methanol was evaporated under reduced pressure. The residue was filtered off to afford
crude hederagenin as a pale brown solid.
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This crude material was washed twice with hot of acetonitrile (1 L) affording 8.5 g (2.8%
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of dry mass) He as a white solid, m.p. 318-320 C (lit.: 317-320 C [22]); R = 0.24
(hexane/ethyl acetate, 1:1 v/v). All spectroscopic data (IR, MS, and NMR) were in full
agreement with the literature [30-32].
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4.3. General procedure for the synthesis of ester 1-23
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A 25 mL one necked round-bottomed flask was charged with a solution of hederagenin
(100 mg, 0.23 mmol) in dry DMF (5 mL), and finely grounded potassium carbonate (1
mmol) was added. To this mixture an alkyl bromide (0.46 mmol) was added, and the
reaction was stirred for 18 h at 25 ºC. The reaction mixture was quenched by adding ethyl
acetate (20 mL), washed with an aqueous solution of HCl (5%, 50 mL), followed by brine
(50 mL) and dried over Na SO . The mixture was filtered, the solvent was removed under
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reduced pressure in a rotary evaporator, and the crude product was obtained as a yellow
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solid. This solid was purified by silica gel column chromatography eluting with
hexane/ethyl acetate (3:1 v/v) to afford the pure product as a solid.
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All yields and physical and spectroscopic data for the compounds are included in the
Supplementary Material
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4.4. Benzotriazol-1-yl-(3β)3,23-dihydroxyolean-12-en-28-oate (24)
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A 50 mL two-necked round bottomed flask was charged with hederagenin (100 mg, 0.23
mmol) TBTU (0.25 mmol), DIPEA (0.25 mmol) and THF (8 mL). The mixture was stirred
at room temperature overnight. The precipitate was filtered off, and the filtrate was diluted
with ethyl acetate (20 mL). The organic layer was washed with water (50 mL) and brine
(50 mL) and dried (Na SO ). The solvent was evaporated under reduced pressure to afford
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the crude product as a brown solid. This crude product was purified by column
chromatography (silica gel, hexane/ethyl acetate, 2:1 v/v) to afford 24 in 92% yield (105
mg).
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White solid; m.p. 232-234 C; R
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= 0.43 (hexane/ethyl acetate 1:1 v/v); IR (KBr): ῡ = 3400,
-
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1806, 1088, 1050, 998, 738 cm ; H NMR (300 MHz, CDCl
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): δ = 8.03 (dt, 1H, J = 8.1,
0.8 Hz, H-34), 7.51 (ddd, 1H, J = 9.0, 8.0, 0.9 Hz), 7.40 (m, 1H, H-35), 7.35 (m, 1H, H-
36), 5.36 (t, 1H, J = 3.5 Hz, H-12), 3.73 (d, 1H, J = 10.4 Hz, H-23 ), 3.64 (brt, 1H, J = 7.6
a
Hz, H-3), 3.42 (d, 1H, J = 10.4 Hz, H-23 ), 2.96 (dd, 1H, J = 13.4, 4.2 Hz, H-18), 1.10 (s,
b
3H, CH ), 0.98 (s, 3H, CH ), 0.96 (s, 3H, CH ), 0.94 (s, 3H, CH ), 0.88 (s, 3H, CH ), 0.84
3
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(s, 3H, CH ); C NMR (75 MHz, CDCl ): δ = 173.75 (C-28), 143.64 (C-13), 142.19 (C-
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31), 122.88 (C-32), 128.61 (C-36), 124.79 (C-35), 123.98 (C-12), 120.59 (C-34), 108.27
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(C-33), 76.79 (C-3), 72.02 (C-23), 49.90 (C-9), 47.73 (C-17), 47.67 (C-5), 45.58 (C-19),
42.06 (C-4), 41.92 (C-14), 41.72 (C-18), 39.60 (C-8), 38.34 (C-1), 37.03 (C-10), 33.80 (C-
21), 33.04 (C-29), 32.75 (C-7), 32.56 (C-22), 30.77 (C-20), 28.17 (C-15), 26.74 (C-27),
25.90 (C-2), 23.65 (C-30), 23.57 (C-11), 23.25 (C-16), 18.58 (C-6), 17.42 (C-26), 15.88
+
+
(C-25), 11.61 (C-24); HRMS (ESI TOF-MS) [M+H] calcd. for [C H N O ] : 589.8079,
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found 590.3959. CHN calcd.: C, 73.31; H, 8.71; found: C, 73.07; H, 8.95.
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4.5. General procedure for the synthesis of amides 25-30
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A 50 mL two-necked round bottomed flask was charged with hederagenin (100 mg, 0.23
mmol), the appropriate amine (0.50 mmol) and THF (8 mL). To the resultant solution, kept
under nitrogen atmosphere, were added TBTU (0.25 mmol) and DIPEA (0.45 mmol). This
reaction mixture was stirred vigorously for 2 h at room temperature, until TLC analysis
revealed a total consumption of the starting material. The mixture was filtered, and the
filtrate was diluted with ethyl acetate (20 mL), washed with water (50 mL) and brine (50
mL) and dried (Na SO ). The solvent was evaporated under reduced pressure to afford the
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crude product. This product was purified by silica gel column chromatography (silica gel,
hexane/ethyl acetate, 1:2 v/v).
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All yields and physical and spectroscopic data for compounds 25-30 are presented in the
Supplementary Material associated to this paper.
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4.6. Cytotoxicity assay
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The cytotoxicity of the compounds was evaluated using the sulforhodamine-B (SRB,
procured from Sigma-Aldrich, Wilwaukee, Wisconsin, USA) micro-culture colorimetric
assay. In short, exponentially growing cells were seeded into a 96-well plate on day 0 at the
appropriate cell densities to prevent confluence of the cells during the period of experiment.
After 24 hours, the cells were treated with serial dilutions of the compounds (0-30 µM) for
96 hours. The final concentration of DMSO never exceeded 0.5%, which was non-toxic to
the cells. The percentages of surviving cells relative to untreated controls were determined
96 h after the beginning of drug exposure. After 96 hours of treatment, the supernatant
medium was discarded from the 96-well plates, and the cells were fixed with 10% TCA.
For a thorough fixation, the plates were allowed to rest at 4 °C. After fixation, the cells
were washed in a strip washer. The washing was done four times with water using alternate
dispensing and aspiration procedures. The plates were dyed with 100 µL of 0.4% SRB for
about 20 min. After dying, the plates were washed with 1% acetic acid to remove the
excess of the dye and allowed to air-dry overnight. Tris base solution (100 µL, 10 mM) was
added to each well and absorbance was measured at λ = 570 nm (using a 96 well plate
reader, Tecan Spectra, Crailsheim, Germany). EC50 values were calculated from semi
logarithmic dose response curves by non-linear regression applying a two parametrical
Hill-slope equation. Values are given with a confidence interval CI = 95%.
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The AO/PI assay was performed as previously described [1, 18]. In short, approximately
500 000 A2780 cells were seeded in cell culture flasks and were allowed to grow for 24 h.
The medium was removed, and the loaded medium (applying an EC concentration) was
9
0
added. After 48 h, the supernatant medium was collected and centrifuged; the pellet was
1
4

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2
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83
84
85
suspended in phosphate-buffer saline (PBS) and centrifuged again. The liquid was
removed, and the pellet was suspended in PBS. After mixing the suspension with a solution
of AO/PI, analysis was performed under a fluorescence microscope. While a green
fluorescence showed apoptosis, a red colored nucleus indicated necrotic cells.
2
86
Acknowledgements
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88
89
90
91
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93
We are grateful to the following Brazilian agencies: Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) for research fellowships (AJD, LCAB).
Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG) for financial support and
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for research
fellowship (DRH). The cell lines were kindly provided by Dr. Thomas Müller (Dept. of
Haematology/Oncology, Martin-Luther University Halle-Wittenberg). Support by the
“Gründerwerkstatt – Biowissenschaften” is gratefully acknowledged.
2
94
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Table 1.
Cytotoxicity for He and analogs 1-30 (EC50 values in µM from SBR assays after 96 h of
treatment; the values are averaged from at least three independent experiments performed each in
triplicate; confidence interval CI = 95%; individual positive (upper value) and negative (lower
value) errors are given in the supplementary part); cut-off in all experiments 30 µM except for
parent He where the cut-off was 60 µM); employing human tumor cell lines.
EC50 518A2
A2780
19.9
9.7
HT29
50.0
9.5
MCF7
25.7
7.9
A549
29.0
7.0
8505C
38.0
7.6
He
1
34.9
7.8
2
3
4
5
6
7
8
9
10.3
10.9
12.9
10.0
9.5
7.5
11.7
9.0
12.9
14.3
13.1
13.4
12.9
11.6
13.6
12.9
14.4
15.0
13.8
13.7
12.1
11.9
13.5
17.0
7.8
14.7
11.4
13.0
13.2
14.6
8.8
11.4
15.9
1.8
12.8
14.3
16.1
13.2
13.0
12.0
13.5
13.2
13.3
13.4
14.8
14.4
13.4
12.0
14.0
17.7
7.8
14.8
11.3
13.5
13.7
15.3
11.2
15.1
19.3
1.3
10.2
12.7
16.5
12.5
11.5
10.2
12.7
11.3
12.7
12.7
13.0
13.5
9.0
10.9
13.2
14.8
13.2
11.4
11.7
13.6
12.6
12.9
13.1
13.0
13.5
11.4
8.1
8.8
7.8
14.1
11.2
8.0
8.7
9.5
8.4
9.4
13.1
11.9
9.7
10.3
6.6
12.8
13.2
6.1
8.8
6.7
8.1
8.6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
9.2
11.1
11.7
10.9
8.7
8.1
8.3
12.7
12.4
8.4
11.8
10.2
11.4
11.6
13.8
8.6
13.7
12.5
3.7
2.0
21.6
>30
13.4
15.4
7.8
13.7
9.2
12.7
13.5
14.2
7.7
14.6
15.2
6.5
14.0
15.9
6.5
13.7
8.5
9.8
13.5
14.2
9.5
15.7
17.2
2.7
1.9
16.8
>30
13.7
9.5
13.5
12.7
3.6
1.8
22.1
>30
1.1
14.8
>30
1.2
17.3
>30
3.7
17.0
>30

ACCEPTED MANUSCRIPT
OH
O
R
2
K CO3, R-Br
O
O
o
DMF, 25 C, 18 h
HO
HO
He
5
1-23
OH
OH
F
Me
Me
1
9
13
17
21
Cl
(
92%)
(
56%)
(57%)
F
(86%)
Cl
(53%)
N
(93%)
NO
2
O
2
3
6
10
14
18
22
2
O
(76%)
F
Cl
NO
Br
Br
(
43%)
(38%)
F
(75%)
O
(58%)
(32%)
(81%)
R =
Br
NO
2
7
11
15
19
20
23
O
F
Me
F
(
55%)
(79%)
Cl
(84%)
(47%)
(55%)
F
F
F
Cl
OMe
4
8
12
16
F
(
90%)
(76%)
(35%)
OMe
(68%)
F
(18%)
Figure 1. Synthesis of hederagenin ester derivatives 1-23.

ACCEPTED MANUSCRIPT
H
OH
N
R
TBTU, DIPEA
O
O
o
THF, 25 C,
2
h, R-NH2
HO
HO
He
26
25-30
OH
OH
N
N
N
N
R =
25
27
30
O
28
29
(96%)
(79%)
(76%)
(93%)
(90%)
(
72%)
Figure 2. Synthesis of hederagenin amide derivatives 25-30.

ACCEPTED MANUSCRIPT
Figure 3. Comparison of EC values (from SBR assays after 96 h of incubation with several
5
0
human tumor cell lines, confidence interval = 95%) for hederagenin derivatives 1-30.

ACCEPTED MANUSCRIPT
Figure 4. Cytotoxicity (EC50 values in µM with standard error (SE), from SBR assays) for He,
esters 1, 18 and amides 27, 28 for all cells lines tested.

ACCEPTED MANUSCRIPT
Figure 5. Comparison of cytotoxicity of hederagenin (He) and some of its ester (20, 21 and 22)
and amide (27 and 28) derivatives (EC in µM from SRB with a confidence interval (95%)) for
5
0
various human tumor cell lines.

ACCEPTED MANUSCRIPT
Figure 6. Fluorescence microscopy image of A2780 cells treated with an EC90 concentration of
hederagenin derivative 28 for 48 hours.

ACCEPTED MANUSCRIPT
Highlights
•
•
•
•
•
A series of ester and amide derivatives of hederagenin has been synthesized.
Some derivatives were more actives than hederagenin for six human cancer lines.
Amides with pyrimidinyl and pyrrolidinyl groups were the most active derivatives.
EC values for pyrimidinyl and pyrrolidinyl derivatives ranged from 1.1 to 6.5 µM.
50
AO/PI staining experiment showed that compound 28 mainly acts by apoptosis.

ACCEPTED MANUSCRIPT
Supporting information
HEDERAGENIN AS A TRITERPENE TEMPLATE FOR THE DEVELOPMENT OF
NEW ANTITUMOR COMPOUNDS
a,b
a
a,b*
c*
Diego Rodríguez-Hernández , Antonio J. Demuner , Luiz C. A. Barbosa , René Csuk , Lucie
c
Heller
a
Department of Chemistry, Universidade Federal de Viçosa, Av. P. H. Rolfs, s/n, CEP 36570-
9
00, Viçosa, MG, Brazil.
b
Department of Chemistry, Universidade Federal de Minas Gerais, Av. Pres Antônio Carlos
6
627, Campus Pampulha, CEP 31270-901, Belo Horizonte, MG, Brazil.
c
Martin-Luther-University Halle-Wittenberg, Organic Chemistry, Kurt-Mothes-Str.2, D-06120
Halle (Saale) Germany
*
Corresponding author: Tel.: +55 31 34093396; fax: +55 31 3899 3065; E-mail addresses:
lcab@ufmg.br (L.C.A. Barbosa) or rene.csuk@chemie.uni-halle.de (R. Csuk).
CONTENTS:
Section A:
Spectroscopic data of all compounds
2-29
Table 1
30
Section B:
1
13
H and C NMR Spectras
31-60
1