Novel hederagenin-triazolyl derivatives as potential anti-cancer agents

Article abstract of DOI:10.1016/j.ejmech.2016.03.018

A series of novel aryl-1H-1,2,3-triazol-4-yl methylester and amide derivatives of the natural product hederagenin was synthesized aiming to develop new antitumor agents, using Huisgen 1,3-dipolar cycloaddition reactions, with yields between 35% and 95%. T

Full text of DOI:10.1016/j.ejmech.2016.03.018

Accepted Manuscript
Novel hederagenin-triazolyl derivatives as potential anti-cancer agents
Diego Rodríguez-Hernández, Antonio J. Demuner, Luiz C.A. Barbosa, Lucie Heller,
René Csuk
PII:
S0223-5234(16)30192-1
10.1016/j.ejmech.2016.03.018
EJMECH 8443
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 December 2015
Revised Date: 4 March 2016
Accepted Date: 5 March 2016
Please cite this article as: D. Rodríguez-Hernández, A.J. Demuner, L.C.A. Barbosa, L. Heller, R. Csuk,
Novel hederagenin-triazolyl derivatives as potential anti-cancer agents, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.03.018.
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Graphical abstract
N
R
N
N
OH
O
Bulky groups
O
O
HO
m-Br
R = m-Cl
m-NO2
HO
OH
OH
µ
EC50 = 3-4.1 M against: 518A2 (melanoma), A2780 (ovarian),
Hederagenin
HT29 (colon), A549 (lung), MCF7 (breast), 8505C (thyroid)

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NOVEL HEDERAGENIN-TRIAZOLYL DERIVATIVES AS
POTENTIAL ANTI-CANCER AGENTS
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Diego Rodríguez-Hernándeza,b, Antonio J. Demunera, Luiz C. A. Barbosaa.b*, Lucie Hellerc,
René Csukc*.
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aDepartment of Chemistry, Universidade Federal de Viçosa, Av. P. H. Rolfs, s/n, CEP
36570-900, Viçosa, MG, Brazil.
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bDepartment 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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cMartin-Luther-University Halle-Wittenberg. Organic Chemistry. Kurt-Mothes-Str.2. D-
06120 Halle (Saale) Germany.
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* 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).
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Abstract:
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A series of novel aryl-1H-1,2,3-triazol-4-yl methylester and amide derivatives of the
natural product hederagenin was synthesized aiming to develop new antitumor agents,
using Huisgen 1,3-dipolar cycloaddition reactions, with yields between 35% to 95%. The
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structures of all derivatives (2-31) were confirmed by MS, IR, H NMR and 13CNMR
spectroscopic data. The cytotoxic activities of all compounds were screened against a panel
of six human cancer cell lines using SRB assay. It was found that most of the compounds
displayed higher levels of antitumor activities as compared to parent hederagenin.
Compounds 4, 8 and 15 were the most potent againstall human cancer cell lines.
Furthermore, compound 11 was the most cytotoxic against cell HT29 showing EC50 = 1.6
µM and a selectivity index of 5.4.
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Keywords: Sapindus saponaria; Huisgen 1,3-dipolar cycloaddition; hederagenin
derivatives; SRB assay
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1. Introduction
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Hederagenin (He) is a pentacyclic oleane-type triterpenoid that exhibits anti-inflammatory
[1], antifungal [2, 3], anti-leishmania [4] and antimicrobial [5, 6] activities as well as
cytotoxicity to different tumor cell lines [7, 8]. This triterpenoic acid can be extracted in
high quantities from the fruits of Sapindus saponaria L., (Sapindaceae), a tropical tree
being popularly known in Brazil as "sabão-de-soldado" (soldier soap) or "saboeiro" (soap-
maker) [9]. The fruits of S. saponaria accumulate in their pericarps great quantities of
saponins carrying hederagenin as the most abundant aglycone. In addition, hederagenin acts
as a chemotaxonomic marker for plants of the Sapindaceae family. Hence great interest has
been aroused with the investigation of the pharmaceutical effects attributed to this natural
product. It has also been reported that other families like Dipsacaceae also produce large
amounts of hederagenin as aglycone of their saponins [10].
During the last few years our research groups have been working on structural
modifications of natural products, in the search for more active compounds as potential
pharmaceuticals or agrochemicals [11-15]. We have a special interest in utilizing terpenes
largely abundant and available from several natural sources as sesquiterpenes, diterpenes
and triterpenes as lead structures for new drug discovery, since many of these compounds
have been reported to show important bioactivities, including anti-cancer effects [8, 13-18].
Recently, we have demonstrated that derivatives of oleanolic acid, 11-keto-β-boswellic
acid, glycyrrhetinic acid maslinic acid and 23-hydroxyoleanolic acid (He) bearing bulky
lipophilic groups attached to carbonyl-28 are amongst the most cytotoxic derivatives [8, 16-
19]. Based on our previous success with hederagenin and maslinic acid, we proposed a
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modification at position-28, by adding a triazolyl group, a well known pharmacophore.
Triazole containing compounds are able to bind to a variety of enzymes and receptors by
non-covalent interactions, hence displaying a large array of biological activities [18].
Several compounds of this class have found application as anti-infective, anticancer,
antiviral, and anti-hypertensive agents [20-22]. Recently, it has been found that triazolyl
derivatives of the natural pentacyclic triterpene ursolic, betulinic and oleanolic acid shows
cytotoxic activity against several cancer cell lines, while not affecting normal cell lines [19,
23-24]. Usually, the most used and effective methodology to prepare triazolyl derivatives
involves a [3+2] cycloaddition reaction between a terminal alkyne and an azide [25].
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Considering, our on-going projects on the use of natural products as scaffold for the
discovery of new potential pharmaceutical and agrochemical compounds [8, 26-28]
besides, the addition of a bulky group substituent at the C-28 of He can enhance its
anticancer potential, herein we report the preparation of a library of novel 1,2,3-triazolyl
derivatives of hederagenin bound to the C-28 and their cytotoxic profiles against several
cancer cell lines were evaluated.
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2. Results and discussion
2.1. Chemistry
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Hederagenin (He) has been recently reported to show low cytotoxic properties for several
human cancer cell lines with EC50 > 30 µM [8]. This compound, however, offers several
hot spots (e.g. C-3, C-12, C-23 and C-28) to provide enough space for chemical
modifications. We have undertaken a research program directed towards the structural
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modifications of hederagenin at its C-28 position, supported by previous reports that
showed derivatives holding bulky groups attached to C-28 to modulate the cytotoxic
activity for this class of compounds for several cancer cell lines [8, 16]. The preparation of
1,2,3-triazolyl derivatives of hederagenin was completed by a Huisgen 1,3-dipolar
cycloaddition. Firstly, hederagenin (He) was isolated from the pericarp of S. saponaria as
previously described [8, 29]. Subsequently, a (prop-2-yn-1-yl) hederageninester (1a) was
prepared (92% yield) by a reaction of He with propargyl bromide in the presence of finely
grounded potassium carbonate in N,N-dimethylformamide (DMF) [8]. In addition, a (prop-
2-yn-1-yl) hederagenin-amide (1b) was also obtained in excellent yields (93%) by reacting
He with 2-propynylamine in the presence of O-(benzotriazol-1-yl)-N,N,N',N'-
tetramethyluronium- tetrafluoroboratetetrabutyl (TBTU) as a coupling catalystand N,N-
diisopropylethylamine (DIPEA) as a base (Scheme1) [8].
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[Insert Scheme1]
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Benzyl azides were prepared from their respective benzylbromides with sodium
azide in DMF (Scheme 2) [30]. The azides were obtained in excellent yields (94-97%) and
were used without further purification.
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[Insert Scheme2]
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1,3-Dipolar cycloaddition reaction of 1a or 1b with benzyl azides in presence of
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CuSO4 5H2O and sodium ascorbate resulted into the formation of 1,4-substitutedtriazolyl
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derivatives 2-31.The overall chemical yields of the synthesized compounds ranged from
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35% to 95% (Scheme3) [25].
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[Insert Scheme 3]
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The structures of the triazolyl derivatives (2-31) were confirmed by extensive
spectroscopic analyses. All compounds showed in their respective IR spectra typical
absorptions expected for the major functional groups present. For example, the stretch
vibration of the ester C=O was detected as a strong absorption band located between ῡ =
1720-1735 cm-1 for 2-16 together with absorptions between ῡ =1250-1150 cm-1 due to
stretching of C-CO-O. For amides 17-31 the C=O stretch vibrational bands were observed
in the range of ῡ = 1640-1620 cm-1 [31].
In the1H NMR spectra, the formation of the heterocyclic unit was confirmed by the
signal around δ = 7.48-7.72 corresponding to H-33 of the triazole ring. In addition, the
signals of the aromatic hydrogen atoms were detected between δ = 6.80 and 8.22. For the
amides the signals for the NH group were observed at δ = 6.58-6.65 each as a triplet (J =
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5.0±0.6 Hz). All C NMR signals for the triterpenoic skeleton in this series of compounds
were similar to the parent hederagenin with 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 =177.4±0.4 for the esters
carbonyl and to δ = 178±0.4 for the amide carbonyl). A detailed assignment of the NMR
spectra (1H and 13C) for all compounds is presented in the supplementary material
associated to this paper. The assignments were possible by means of 2D NMR techniques
when required, and the data were fully consistent with the proposed structures.
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2.2. Biological screening
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The cytotoxic activity of the novel hederagenin derivatives (2-31) was evaluated
employing six human tumour cell lines (melanoma cells - 518A2; ovarian carcinoma -
A2780; colon adenocarcinoma - HT29; breast adenocarcinoma - MCF7; lung cancer -
A549; thyroid carcinoma - 8505C) and non-malignant mouse fibroblasts (NIH 3T3), using
the well-established photometric sulforhodamine B assay (SRB) [32]. The results of these
assays are summarized in Table 1.
[Insert Table 1]
In general, conversion of parent He into esters and amides bearing a 1,2,3-triazole
unit resulted in an increase of cytotoxicity. As shown forvirtually all human cancer cell
lines, ester substituted compounds bearing a 1,2,3-triazolyl group showed higher
cytotoxicity than parent hederagenin (Table 1; Fig. S1). The only exception is compound
14 that presented EC50 > 30 µM in 8505C (thyroid cell line), comparable to parent
hederagenin. In the case of amide derivatives, all compounds (17-31) were more active than
He against the cell lines A2780, HT29, MCF7. For the cell lines 518A2 and A549 most
amide derivatives were more active than He (Table 1; Fig. S2). However, for the thyroid
cell line (8505C) only compound 25-28 were more active compared to He. These results
indicate 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
hederagenin (He), when in its natural form (free acid) a 20-fold lower cytotoxicity was
observed as compared to the corresponding ester and amide derivatives [8]. 1,2,3-Triazolyl
compounds having an ester group are generally more cytotoxic than compounds carrying an
amide moiety. Hence, we assumed that the improvements may be the result either of a
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different transport mechanisms of the compounds through the membrane or of a different
integration into the cell membrane [33-34].
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For human cell line MCF-7 (breast adenocarcinoma), all derivatives (2-31) showed
cytotoxic activity at 10 µM or at lower dose, (Table 1). For the human cell line HT29
(colon adenocarcinoma), the activity of the ortho substituted compounds (3, 18, 9, 24, 11
and 26) decreased with the atomic radius of the halogen substituent (Fig. 1). In addition, the
ortho-fluorobenzyl-1,2,3-triazolyl ester 11 was the most active compound against this cell
line showing EC50 = 1.6 µM. This compound (11) also exhibited some selectivity (SI= 5.4;
SI is defined as the quotient of EC50 values according to EC50 [NIH 3T3]/EC50 [tumor cell line]) in
cytotoxicity discriminating between the cancer cell line HT29 and non-malignant mouse
fibroblasts NIH 3T3. Moreover, the 1,2,3-triazol benzyl ester 2 (EC50 = 2.8 µM) also gave a
good selectivity (SI = 4.2) between the cancer cell line HT29 and non-malignant mouse
fibroblasts NIH 3T3 (Table S2).
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[Insert Figure 1]
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The ester derivatives 4, 8 and 15 possessing a m-bromo, m-chloro and m-nitro
substituent were the most active compounds against all human cell lines tested so far,
showing EC50 values ranging between 3.2-4.0 µM for 4, between 3.1-4.0 µM for 8 and
between 3.2-4.1 µM for 15 (Fig.2). These results revealed that these compounds (4, 8 and
15) are at least ~8 times more active than parent hederagenin (Table 1, Fig. 2); however,
they were not selective between malignant and non-malignant cells (Table S2).
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[Insert Figure 2]
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3. Conclusion
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In summary, a series of novel aryl-1H-1,2,3-triazol-4-yl-methyl ester and amide
derivatives of hederagenin has been synthesized by employing Cu(I) catalysed Huisgen 1,3-
dipolar cycloaddition reactions of terminal alkyne derivatives of He with various benzyl
azides. Compared to hederagenin most compounds exhibited higher cytotoxicity against all
tested cancer cell lines. The ester derivatives were more cytotoxic than the amides.
Furthermore, esters 4, 8 and 15 were the most active derivatives towards all cell lines
tested, showing EC50in the range of 3.0-4.1 µM. On the other hand, compounds 2 and 11
were the most cytotoxic against the colon cancer cell (HT29) with EC50 = 2.8 µM and 1.6
µM, respectively. In addition, compound 11 showed some selectivity for the cancer cell
line HT29 and was less toxic for non-malignant mouse fibroblasts NIH 3T3 displaying SI =
5.4.
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4. Experimental part
4.1. General procedures.
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Reagents were procured from Sigma-Aldrich (Milwaukee, Wisconsin, USA) and were used
without any purification. Solvents were supplied by Vetec (Rio de Janeiro, Brazil).
Analytical thin layer chromatography (TLC) were performed on silica gel 60 F254 0.2 mm
thick plates (supplied by Merck, Rio de Janeiro, Brazil) and were visualized under UV-B
light or by spraying with phosphomolybdic acid in 10% ethanol, followed by heating. Flash
column chromatography (typical size of 20 cm length and 2 cm of diameter) was performed
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using silica gel 230-400 mesh. All compounds were fully characterized by IR, EI-MS, H
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NMR and 13C 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) under electro spray ionization (ESI) and
the results are reported to four decimal figures. Elemental analyses were measured on a
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Foss-Heraeus Vario EL unit. The H and 13C-NMR spectra were recorded on a Varian
Mercury 300 spectrometer at 300 and 75 MHz, respectively, using CDCl3 as solvent and
TMS as internal reference, unless otherwise stated. Melting points were measured on a
MQAPF-301 apparatus and were not corrected.
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The 1H NMR spectra for all compound were assigned for the signals that were clearly well
defined as described for each compound, For all compounds a multiplet was observed in
the range of 0.5 to 2.5 ppm, so the hydrogen atoms in such range could not be assigned.
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4.2 Hederagenin (He).
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He was isolated from pericarp of Sapindus saponaria L., as previously reported [8], and it
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was obtained as a white solid; m.p. 318-320 C (lit.: 317-320 C [29]); Rf = 0.24
(hexane/ethyl acetate, 1:1 v/v). All spectroscopic data (IR, MS and NMR) were in
agreement with the literature [35-37].
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4.3 Procedure for the preparation of compounds 1a and 1b.
Compounds 1a and 1b were synthesized using methods previously published [8].
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4.4 General procedure for synthesis of benzyl azides (a-o).
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The benzyl azides were preparedfrom the reaction of a solution of the appropriate benzyl
bromide (0.24 mmol) in DMF (10 mL) with sodium azide (25 mg; 0.38 mmol); in short, the
reaction mixture was stirred for 2 h at room temperature until TLC analyses revealed the
consumption of the starting material. The mixture was poured into water (50 mL), extracted
with diethyl ether (2x30 mL), washed with brine (3x20 mL), dried over Na2SO4, filtered,
and the filtrate was concentrated in a rotary evaporator under reduced pressure to afford the
benzyl azides (a-o). Since TLC analysis of the crude products revealed only one spot, they
were used without further purification.
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4.5 General procedure for synthesis of compounds (2-31).
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To a solution ofalkynes1a or 1b (0.15 mmol) and the appropriate azide (0.30 mmol) in
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CH2Cl2 (5 mL) and H2O (5 mL) CuSO4 5H2O (63 mg; 0.25 mmol) and Na-L-ascorbate
(100 mg; 0.5 mmol) were added. This reaction mixture was stirred vigorously for 24h at
room temperature, until TLC analysis revealed a total consumption of the starting material.
The mixture was diluted with H2O (20 mL), extracted with CH2Cl2 (3 x 20 mL). The
combined organic extracts were washed with brine (50 mL), dried over Na2SO4, filtered,
and the solvent was removed in a rotary evaporator under reduced pressure to afford the
crude product. This product was purified by column chromatography (silica gel,
hexane/ethyl acetate, 2:1 v/v) to yield the target compounds 2-31each as a solid material.
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4.5.1 (Benzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-28-oate (2).
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White solid; yield: 65mg, 60%; m.p. 101-102.8oC; Rf = 0.45 (hexane/ethylacetate 1:1 v/v);
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IR (KBr): ῡ = 3420, 3148, 3064, 1724, 1160, 1048, 1008, 722 cm-1; H NMR (300 MHz,
CDCl3): δ = 7.47 (s, 1H, H-33), 7.35 (brs, 3H), 7.26 (brs, 2H), 5.51 (d, 1H, J = 15.0 Hz, H-
34a), 5.45 (d, 1H, J = 15.0 Hz, H-34b), 5.21 (brs, 1H, H-12), 5.13 (brs, 2H, H-31), 3.71 (d,
1H, J = 10.3 Hz, H-23a), 3.62 (brt, 1H, J = 8.0 Hz, H-3), 3.40 (d, 1H, J = 9.6 Hz, H-23b),
2.78 (brdd, 1H, J = 13.3 Hz, H-18), 1.08 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.88 (s, 3H, CH3),
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0.86 (s, 6H, 2xCH3), 0.50 (s, 3H, CH3); C NMR (75 MHz, CDCl3): δ = 177.81 (C-28),
143.71 (C-13), 134.32 (C-32), 131.04 (C-35), 129.26 (C-37), 129.26 (C-37’), 128.97 (C-
38), 128.22 (C-37’), 128.22 (C-37), 123.03 (C-12), 122.44 (C-33), 76.91 (C-3), 72.18 (C-
23), 57.61 (C-31), 54.31 (C-34), 49.88 (C-9), 47.64 (C-17), 46.81 (C-5), 45.96 (C-19),
41.90 (C-4), 41.83 (C-14), 41.37 (C-18), 39.38 (C-8), 38.22 (C-1), 37.01 (C-10), 33.92 (C-
21), 33.17 (C-29), 32.55 (C-7), 32.41 (C-22), 30.76 (C-20), 27.72 (C-15), 26.82 (C-27),
25.93 (C-2), 23.71 (C-30), 23.45 (C-11), 23.06 (C-16), 18.61 (C-6), 16.84 (C-26), 15.85
(C-25), 11.57 (C-24); HRMS (ESI TOF-MS) [M+H]+calcd. for [C40H58N3O4]+: 644.4427,
found: 644.4424; CHN calcd.: C, 74.61; H, 8.92; N, 6.52; found: C, 74.48; H, 9.07; N,
6.39.
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4.5.2
(o-Bromobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-28-
oate (3).
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White solid; yield: 110mg, 91%; m.p. 94.8-96.3 oC; Rf = 0.44 (hexane/ethylacetate 1:1 v/v);
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IR (KBr): ῡ = 3408, 3154, 3068, 1722, 1160, 1046, 1030, 1008, 750, 732 cm-1; H NMR
(300 MHz, CDCl3): δ = 7.60 (d, 1H, J = 7.8 Hz, H-37), 7.57 (s, H, H-33), 7.23 (m, H-38,
H-39, H-40), 5.62 (t, 2H, J = 16.3 Hz H-34), 5.22 (brs, 1H, H-12), 5.14 (s, 2H, H-31), 3.70
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(d, 1H, J = 10.2 Hz, H-23a), 3.61 (brt, 1H, J = 8.0 Hz, H-3), 3.39 (d, 1H, J = 10.2 Hz, H-
23b), 2.79 (brdd, 1H, J = 11.2 Hz, H-18), 1.08 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.87 (s, 9H,
3xCH3), 0.52 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3): δ = 177.77 (C-28), 143.68 (C-13),
143.62 (C-32), 134.00 (C-35), 133.39 (C-37), 130.63 (C-40), 130.58 (C-39), 128.30 (C-38),
124.68 (C-12), 123.69 (C-36), 122.45 (C-33), 76.85 (C-3), 72.10 (C-23), 57.58 (C-31),
53.95 (C-34), 49.86 (C-9), 47.63 (C-17), 46.80 (C-5), 45.93 (C-19), 41.87 (C-4), 41.81 (C-
14), 41.36 (C-18), 39.37 (C-8), 38.21 (C-1), 36.99 (C-10), 33.91 (C-21), 33.15 (C-29),
32.53 (C-7), 32.41 (C-22), 30.75 (C-20), 27.71 (C-15), 26.75 (C-27), 25.92 (C-2), 23.70
(C-30), 23.46 (C-11), 23.04 (C-16), 18.60 (C-6), 16.86 (C-26), 15.83 (C-25), 11.58 (C-24);
HRMS (ESI TOF-MS) [M+H]+calcd. for [C40H57BrN3O4]+: 722.3532, found: 722.3520;
CHN calcd.: C, 66.47; H, 7.81; N, 5.81; found: C, 66.30; H, 7.98; N, 5.62.
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4.5.3
(m-Bromobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-28-
oate (4).
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White solid; yield: 78 mg, 65%; m.p. 125.4-125.6 C; Rf = 0.44 (hexane/ethylacetate 1:1
v/v); IR (KBr): ῡ = 3414, 3148, 3094, 1718, 1160, 1048,1008, 772, 734 cm-1; H NMR
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(300 MHz, CDCl3): δ = 7.51 (s, H, H-33), 7.47 (d, 1H, J = 7.7 Hz, H-38), 7.39 (s, 1H, H-
36), 7.24 (d, H, J = 7.7 Hz, H-40), 7.18 (t, H, J = 7.5 Hz, H-39), 5.45 (t, 2H, J = 17.7 Hz,
H-34), 5.21 (brs, 1H, H-12), 5.14 (s, 2H, H-31), 3.70 (d, 1H, J = 10.3 Hz, H-23a), 3.61 (brt,
1H, J = 8.0 Hz, H-3), 3.40 (d, 1H, J = 10.2 Hz, H-23b), 2.79 (brdd, 1H, J = 11.0 Hz, H-18),
1.08 (s, 3H, CH3), 0.89 (s, H, CH3), 0.87 (s, 9H, 3xCH3), 0.50 (s, 3H, CH3); 13C NMR (75
MHz, CDCl3): δ = 177.85 (C-28), 143.89 (C-13), 143.66 (C-32), 136.69 (C-35), 131.13
(C-38), 131.12 (C-36), 130.79 (C-39), 126.66 (C-40), 124.11 (C-12), 123.25 (C-37), 122.45
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271
272
273
274
275
276
(C-33), 76.89 (C-3), 72.15 (C-23), 57.56 (C-31), 53.47 (C-34), 49.87 (C-9), 47.62 (C-17),
46.82 (C-5), 45.94 (C-19), 41.88 (C-4), 41.83 (C-14), 41.38 (C-18), 39.38 (C-8), 38.21 (C-
1), 36.99 (C-10), 33.91 (C-21), 33.16 (C-29), 32.55 (C-7), 32.41 (C-22), 30.75 (C-20),
27.71 (C-15), 26.73 (C-27), 25.92 (C-2), 23.71 (C-30), 23.44 (C-11), 23.07 (C-16), 18.60
(C-6), 16.84 (C-26), 15.84 (C-25), 11.59 (C-24); HRMS (ESI TOF-MS) [M+H]+calcd. for
[C40H57BrN3O4]+: 722.3532, found: 722.3522; CHN calcd.: C, 66.47; H, 7.81; N, 5.81;
found: C, 66.32; H, 8.02; N, 5.61.
277
278
4.5.4
(p-Bromobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-28-
oate (5).
o
279
280
281
282
283
284
285
286
287
288
289
290
291
White solid; yield: 109 mg, 91%; m.p. 111.6-112.3 C; Rf = 0.44 (hexane/ethyl acetate 1:1
v/v); IR (KBr): ῡ = 3420, 3150, 3056, 1722, 1160, 1048, 1012, 732 cm-1; H NMR (300
1
MHz, CDCl3): δ = 7.48 (m, 3H, H-33, H-37, H-37’), 7.12 (d, 2H, J = 8.1 Hz, H-36, H-36’),
5.43 (t, 2H, J =13.4 Hz, H-34), 5.21 (brs, 1H, H-12), 5.13 (s, 2H, H-31), 3.70 (d, 1H, J =
10.2 Hz, H-23a), 3.61 (brt, 1H, J = 7.9 Hz, H-3), 3.40 (d, 1H, J = 10.2 Hz, H-23b), 2.79
(brdd, 1H, J = 13.0, 2.3 Hz, H-18), 1.08 (s, 3H, CH3), 0.87 (s, 12H, 4xCH3), 0.49 (s, 3H,
CH3); 13C NMR (75 MHz, CDCl3): δ = 177.84 (C-28), 143.91 (C-13), 143.68 (C-32),
133.53 (C-35), 132.43 (C-37, C-37’), 129.77 (C-36, C-36’), 123.96 (C-12), 123.16 (C-
38),122.43 (C-33), 76.87 (C-3), 72.12 (C-23), 57.57 (C-31), 53.56 (C-34), 49.86 (C-9),
47.61 (C-17), 46.82 (C-5), 45.93 (C-19), 41.88 (C-4), 41.82 (C-14), 41.36 (C-18), 39.37
(C-8), 38.20 (C-1), 36.98 (C-10), 33.90 (C-21), 33.15 (C-29), 32.54 (C-7), 32.41 (C-22),
30.75 (C-20), 27.70 (C-15), 26.78 (C-27), 25.92 (C-2), 23.69 (C-30), 23.42 (C-11), 23.07
(C-16), 18.57 (C-6), 16.85 (C-26), 15.81 (C-25), 11.59 (C-24); HRMS (ESI TOF-MS)
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292
293
[M+H]+calcd. for [C40H57BrN3O4]+: 722.3532, found: 722.3526; CHN calcd.: C, 66.47; H,
7.81; N, 5.81; found: C, 66.29; H, 7.92; N, 5.69.
294
295
4.5.5 (2,6-Dichlorobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-
28-oate (6).
o
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
White solid; yield: 100mg, 84%; m.p. 113-114.6 C; Rf = 0.45 (hexane/ethylacetate 1:1
v/v); IR (KBr): ῡ = 3420, 3154, 3086, 1718, 1160, 1046, 764, 732 cm-1; H NMR (300
1
MHz, CDCl3): δ = 7.49 (s, 1H, H-33), 7.38 (d, 2H, J = 7.5 Hz, H-37, H-39), 7.30 (d, 1H, J
= 8.3 Hz, H-38), 5.81 (s, 2H, H-31), 5.20 (brs, 1H, H-12), 5.14 (d, 1H, J = 12.7 Hz, H-34a),
5.08 (d, 1H, J = 12.7 Hz, H-34b), 3.69 (d, 1H, J = 10.2 Hz, H-23a), 3.61 (brt, 1H, J = 7.9
Hz, H-3), 3.38 (d, 1H, J = 10.2 Hz, H-23b), 2.78 (brdd, 1H, J = 11.0 Hz, H-18), 1.07 (s,
13
3H, CH3), 0.89 (s, 3H, CH3), 0.86 (s, 9H, 3xCH3), 0.48 (s, 3H, CH3); C NMR (75 MHz,
CDCl3): δ = 177.77 (C-28), 143.71 (C-13), 143.20 (C-32), 136.91 (C-35), 131.26 (C-36),
129.96 (C-40), 129.02 (C-37), 129.02 (C-39), 129.02 (C-38), 123.77 (C-12), 122.41 (C-33),
76.83 (C-3), 72.08 (C-23), 57.53 (C-31), 49.86 (C-9), 49.16 (C-34), 47.62 (C-17), 46.78
(C-5), 45.92 (C-19), 41.84 (C-4), 41.79 (C-14), 41.34 (C-18), 39.34 (C-8), 38.21 (C-1),
36.98 (C-10), 33.90 (C-21), 33.14 (C-29), 32.51 (C-7), 32.40 (C-22), 30.74 (C-20), 27.68
(C-15), 26.70 (C-27), 25.92 (C-2), 23.66 (C-30), 23.45 (C-11), 22.99 (C-16), 18.58 (C-6),
16.73 (C-26), 15.82 (C-25), 11.59 (C-24); HRMS (ESI TOF-MS) [M+H]+calcd. for
[C40H55Cl2N3O5]+: 712.3648, found: 712.3632; CHN calcd.: C, 65.92; H, 7.61; N, 5.77;
found: C, 65.77; H, 7.81; N, 5.54.
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312
313
4.5.6
(p-Chlorobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-28-
oate (7).
o
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
White solid; yield: 39 mg, 33%; m.p. 169.3-170.2 C; Rf = 0.45 (hexane/ethylacetate 1:1
v/v); IR (KBr): ῡ = 3412, 3150, 3100, 1722, 1160, 1048, 1016 cm-1; H NMR (300 MHz,
1
CDCl3): δ = 7.48 (s, 1H, H-33), 7.33 (d, 2H, J = 8.2 Hz, H-37, H-37’), 7.19 (d, 2H, J = 8.2
Hz, H-36, H-36’), 5.45 (t, 2H, J = 16.6 Hz, H-34), 5.21 (brs, 1H, H-12), 5.13 (s, 2H, H-31),
3.71 (d, 1H, J = 10.2 Hz, H-23a), 3.61 (brt, 1H, J = 8.2 Hz, H-3), 3.40 (d, 1H, J = 10.2 Hz,
H-23b), 2.78 (brdd, 1H, J = 13.0 Hz, H-18), 1.08 (s, 3H, CH3), 0.87 (s, 12H, 4xCH3), 0.49
13
(s, 3H, CH3); C NMR (75 MHz, CDCl3): δ = 177.86 (C-28), 143.92 (C-13), 143.70 (C-
32), 135.06 (C-35), 133.04 (C-38), 129.52 (C-36, C-36’), 129.49 (C-37, C-37’), 123.98 (C-
12), 122.44 (C-33), 76.91 (C-3), 72.18 (C-23), 57.60 (C-31), 53.53 (C-34), 49.87 (C-9),
47.63 (C-17), 46.83 (C-5), 45.95 (C-19), 41.91 (C-4), 41.84 (C-14), 41.38 (C-18), 39.38
(C-8), 38.21 (C-1), 36.99 (C-10), 33.92 (C-21), 33.16 (C-29), 32.56 (C-7), 32.43 (C-22),
30.77 (C-20), 27.72 (C-15), 26.84 (C-27), 25.93 (C-2), 23.69 (C-30), 23.44 (C-11), 23.08
(C-16), 18.58 (C-6), 16.85 (C-26), 15.81 (C-25), 11.52 (C-24); HRMS (ESI TOF-MS)
[M+H]+calcd. for [C40H56ClN3O4]+: 678.4038, found: 678.4029; CHN calcd.: C, 70.82; H,
8.32; N, 6.19; found: C, 70.59; H, 8.42; N, 6.07.
329
330
4.5.7 (m-Chlorobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-28-
oate (8).
o
331
332
White solid; yield: 106 mg, 90%; m.p. 111.9-113.3 C; Rf = 0.49 (hexane/ethylacetate 1:1
v/v); IR (KBr): ῡ = 3412, 3148, 3083, 1722, 1160, 1048, 1008, 772, 734 cm-1; H NMR
1
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333
334
335
336
337
338
339
340
341
342
343
344
345
(300 MHz, CDCl3): δ = 7.51 (s, 1H, H-33), 7.27 (m, 3H, H-36, H-38, H-39), 7.12 (d, 1H, J
= 6.6 Hz, H-40), 5.45 (t, 2H, J = 16.2 Hz, H-34), 5.21 (brs, 1H, H-12), 5.13 (s, 2H, H-31),
3.70 (d, 1H, J = 10.4 Hz, H-23a), 3.61 (brt, 1H, J = 7.6 Hz, H-3), 3.39 (d, 1H, J = 10.4 Hz,
H-23b), 2.79 (brdd, 1H, J = 13.7 Hz, H-18), 1.08 (s, 3H, CH3), 0.89 (s, 3H, CH3), 0.86 (s,
9H, 3xCH3), 0.50 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3): δ = 177.84 (C-28), 143.92 (C-
13), 143.65 (C-32), 136.44 (C-35), 135.17 (C-37), 130.52 (C-39), 129.18 (C-36), 128.20
(C-38), 126.18 (C-40), 124.12 (C-12), 122.44 (C-33), 76.85 (C-3), 72.09 (C-23), 57.54 (C-
31), 53.85 (C-34), 49.85 (C-9), 47.60 (C-17), 46.81 (C-5), 45.92 (C-19), 41.86 (C-4), 41.82
(C-14), 41.36 (C-18), 39.37 (C-8), 38.20 (C-1), 36.98 (C-10), 33.89 (C-21), 33.14 (C-29),
32.54 (C-7), 32.40 (C-22), 30.74 (C-20), 27.69 (C-15), 26.74 (C-27), 25.91 (C-2), 23.69
(C-30), 23.42 (C-11), 23.05 (C-16), 18.58 (C-6), 16.82 (C-26), 15.82 (C-25), 11.58 (C-24);
HRMS (ESI TOF-MS) [M+H]+calcd. for [C40H56ClN3O4]+: 678.4038, found: 678.4033;
CHN calcd.: C, 70.82; H, 8.32; N, 6.19; found: C, 70.64; H, 8.47; N, 6.03.
346
347
4.5.8
(o-Chlorobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-28-
oate (9).
o
348
349
350
351
352
353
354
White solid; yield: 64 mg, 54%; m.p. 108.6-110.2 C; Rf = 0.44 (hexane/ethylacetate 1:1
v/v); IR (KBr): ῡ = 3410, 3156, 3072, 1724, 1160, 1048, 1008, 752 cm-1; H NMR (300
1
MHz, CDCl3): δ = 7.56 (s, 1H, H-33), 7.42 (d, 1H, J = 7.5 Hz, H-37), 7.25 (m, 3H, H-38,
H-39, H-40), 5.62 (t, 2H, J = 16.0 Hz H-34), 5.22 (brs, 1H, H-12), 5.14 (s, 2H, H-31), 3.71
(d, 1H, J = 10.1 Hz, H-23a), 3.62 (brt, 1H, J = 8.1 Hz, H-3), 3.40 (d, 1H, J = 10.1 Hz, H-
23b), 2.80 (brdd, 1H, J = 10.3 Hz, H-18), 1.08 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.87 (s, 9H,
3xCH3), 0.53 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3): δ = 177.79 (C-28), 143.70 (C-13),
17

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355
356
357
358
359
360
361
362
143.64 (C-32), 133.71 (C-35), 132.33 (C-36), 130.59 (C-40), 130.48 (C-37), 130.11 (C-38),
127.72 (C-39), 124.27 (C-12), 122.46 (C-33), 76.92 (C-3), 72.19 (C-23), 57.61 (C-31),
51.56 (C-34), 49.88 (C-9), 47.65 (C-17), 46.82 (C-5), 45.96 (C-19), 41.91 (C-4), 41.83 (C-
14), 41.38 (C-18), 39.39 (C-8), 38.22 (C-1), 37.01 (C-10), 33.93 (C-21), 33.17 (C-29),
32.55 (C-7), 32.42 (C-22), 30.77 (C-20), 27.73 (C-15), 26.83 (C-27), 25.94 (C-2), 23.71
(C-30), 23.47 (C-11), 23.06 (C-16), 18.61 (C-6), 16.86 (C-26), 15.83 (C-25), 11.56 (C-24);
HRMS (ESI TOF-MS) [M+H]+calcd. for [C40H56ClN3O4]+: 678.4038, found: 678.4026;
CHN calcd.: C, 70.82; H, 8.32; N, 6.19; found: C, 70.69; H, 8.58; N, 6.09.
363
364
4.5.9 (2,4-Difluorobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-28-
oate (10).
o
365
366
367
368
369
370
371
372
373
374
375
376
White solid; yield: 101mg, 89%; m.p.104.5-106.2 C; Rf = 0.46 (hexane/ethylacetate 1:1
v/v); IR (KBr): ῡ = 3420, 3154, 3084, 1724, 1508, 1160, 1048, 972, 732 cm-1; H NMR
1
(300 MHz, CDCl3): δ = 7.56 (s, 1H, H-33), 7.28 (m, 1H, H-40), 6.87 (m, 2H, H-37, H-39),
5.49 (t, 2H, J =15.1 Hz, H-34), 5.22 (brs, 1H, H-12), 5.12 (s, 2H, H-31), 3.69 (d, 1H, J =
10.2 Hz, H-23a), 3.61 (brt, 1H, J = 7.9 Hz, H-3), 3.39 (d, 1H, J = 10.2 Hz, H-23b), 2.79
(brdd, 1H, J = 13.1 Hz, H-18), 1.08 (s, 3H, CH3), 0.89(s, 3H, CH3), 0.86 (s, 9H, 3xCH3),
0.48 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3): δ = 177.82 (C-28), 163.47 (dd, J = 250.3,
11.9 Hz, C-38), 160.85 (dd, J = 248.9, 11.7 Hz, C-36), 143.81 (C-13), 143.66 (C-32),
131.83 (dd, J = 9.8, 4.7 Hz, C-40), 124.08 (C-12), 122.46 (C-33), 117.92 (dd, J = 15.0, 4.0
Hz, C-35), 112.31 (dd, J = 21.2, 3.6Hz, C-39), 104.54 (t, J = 25.2 Hz, C-37), 76.81 (C-3),
72.04 (C-23), 57.51 (C-31), 49.84 (C-9), 47.61 (C-17), 47.23 (d, J = 3.6 Hz, C-34), 46.81
(C-5), 45.93 (C-19), 41.86 (C-4), 41.81 (C-14), 41.36 (C-18), 39.35 (C-8), 38.21 (C-1),
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ACCEPTED MANUSCRIPT
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378
379
380
381
36.97 (C-10), 33.91 (C-21), 33.14 (C-29), 32.54 (C-7), 32.41 (C-22), 30.74 (C-20), 27.69
(C-15), 26.72 (C-27), 25.92 (C-2), 23.67 (C-30), 23.43 (C-11), 23.05 (C-16), 18.55 (C-6),
16.75 (C-26), 15.78 (C-25), 11.57 (C-24);HRMS (ESI TOF-MS) [M+H]+calcd. for
[C40H55F2N3O4]+: 680.4239, found: 680.4233; CHN calcd.: C, 76.03; H, 8.77; N, 6.65;
found: C, 75.85; H, 8.91; N, 6.47.
382
383
4.5.10 (o-Fluorobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-28-
oate (11).
o
384
385
386
387
388
389
390
391
392
393
394
395
396
397
White solid; yield: 94mg, 85%; m.p. 142.3-143.6 C; Rf = 0.46 (hexane/ethylacetate 1:1
v/v); IR (KBr): ῡ = 3412, 3150, 3098, 1732, 1234, 1158, 758 cm-1; H NMR (300 MHz,
1
CDCl3): δ = 7.56 (s, 1H, H-33), 7.35 (m, 1H, H-40), 7.26 (m, 1H, H-38), 7.11 (m, 2H, H-
37, H-39), 5.54 (t, 2H, J = 15.5 Hz, H-34), 5.22 (brs, 1H, H-12), 5.13 (s, 2H, H-31), 3.70
(d, 1H, J = 10.3 Hz, H-23a), 3.61 (brt, 1H, J = 7.9 Hz, H-3), 3.39 (d, 1H, J = 10.2 Hz, H-
23b), 2.79 (brdd, 1H, J = 12.6 Hz, H-18), 1.08 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.87 (s, 9H,
3xCH3), 0.50 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3): δ = 177.79 (C-28), 160.67 (d, J =
246.6 Hz, C-36), 143.70 (C-13), 143.67 (C-32), 131.14 (d, J = 32.6 Hz, C-40), 130.74 (d, J
= 3.8 Hz, C-38), 124.69 (d, J = 3.6 Hz, C-35), 124.12 (C-12), 122.46 (C-33), 121.76 (d, J =
14.5 Hz, C-39), 115.98 (d, J = 20.9 Hz, C-37), 76.86 (C-3), 72.10 (C-23), 57.51 (C-31),
49.86 (C-9), 47.63 (C-17), 47.23 (d, J = 3.6 Hz, C-34), 46.80 (C-5), 45.93 (C-19), 41.87
(C-4), 41.87 (C-14), 41.36 (C-18), 39.36 (C-8), 38.22 (C-1), 36.98 (C-10), 33.91 (C-21),
33.15 (C-29), 32.53 (C-7), 32.39 (C-22), 30.75 (C-20), 27.69 (C-15), 26.75 (C-27), 25.92
(C-2), 23.60 (C-30), 23.43 (C-11), 23.05 (C-16), 18.58 (C-6), 16.77 (C-26), 15.80 (C-25),
19

ACCEPTED MANUSCRIPT
398
399
11.58 (C-24); HRMS (ESI TOF-MS) [M+H]+calcd. for [C40H56FN3O4]+: 662.4333, found:
662.4325; CHN calcd.: C, 72.58; H, 8.53; N, 6.35; found: C, 70.46; H, 8.60; N, 6.03.
400
401
4.5.11 (2,6-Difluorobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-
28-oate (12).
o
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
White solid; yield: 76 mg, 67%; m.p. 108.6-110.1 C; Rf = 0.44 (hexane/ethylacetate 1:1
v/v); IR (KBr): ῡ = 3418, 3150, 3074, 1724, 1628, 1160, 1036, 802, 734 cm-1; H NMR
1
(300 MHz, CDCl3): δ = 7.57 (s, 1H, H-33), 7.36 (m, 1H, H-38), 6.96 (m, 2H, H-37, H-39),
5.58 (t, 2H, J = 15.6 Hz, H-34), 5.22 (brs, 1H, H-12), 5.12 (s, 2H, H-31), 3.70 (d, 1H, J
=10.1 Hz, H-23a), 3.61 (brt, 1H, J = 7.8 Hz, H-3), 3.39 (d, 1H, J = 10.1 Hz, H-23b), 2.80
(brdd, 1H, J = 12.7 Hz, H-18), 1.08 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.87 (s, 9H, 3xCH3),
0.49 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3): δ = 177.80 (C-28), 161.48 (dd, J = 249.2,
6.8 Hz, C-36, C-40), 143.69 (C-13), 143.56 (C-32), 131.64 (t, J = 10.1 Hz, C-38), 124.01
(C-12), 122.46 (C-33), 111.96 (m, C-37, C-39), 110.67 (t, J = 18.5 Hz, C-35), 76.91 (C-3),
72.17 (C-23), 57.52 (C-31), 49.89 (C-9), 47.64 (C-17), 46.80 (C-5), 45.96 (C-19), 41.87
(C-4), 41.81 (C-14), 41.49 (t, J = 4 Hz, C-34), 41.37 (C-18), 39.36 (C-8), 38.22 (C-1),
36.99 (C-10), 33.92 (C-21), 33.16 (C-29), 32.54 (C-7), 32.41 (C-22), 30.76 (C-20), 27.69
(C-15), 26.77 (C-27), 25.93 (C-2), 23.69 (C-30), 23.44 (C-11), 23.05 (C-16), 18.58 (C-6),
16.67 (C-26), 15.79 (C-25), 11.57 (C-24); HRMS (ESI TOF-MS) [M+H]+calcd. for
[C40H55F2N3O4]+: 680.4239, found: 680.4232; CHN calcd.: C, 76.03; H, 8.77; N, 6.65;
found: C, 75.87; H, 8.91; N, 6.50.
20

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418
419
4.5.12 (p-Fluorobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-28-
oate (13).
o
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
White solid; yield: 85 mg, 77%; m.p. 111.3-112.6 C; Rf = 0.46 (hexane/ethylacetate 1:1
v/v); IR (KBr): ῡ = 3420, 3152, 3080, 1722, 1512, 1228, 1160, 1048, 732 cm-1; H NMR
1
(300 MHz, CDCl3): δ = 7.47 (s, 1H, H-33), 7.25 (m, 2H, H-36, H-36’), 7.04 (m, 2H, H-37.
H-37’), 5.48 (d, 1H, J = 15.0 Hz, H-34a), 5.42 (d, 1H, J = 15.0 Hz, H-34b), 5.21 (t, 1H, J =
2.9 Hz, H-12), 5.12 (s, 2H, H-31), 3.70 (d, 1H, J = 10.3 Hz, H-23a), 3.61 (brt, 1H, J = 8.4
Hz, H-3), 3.39 (d, 1H, J = 10.3 Hz, H-23b), 2.78 (dd, 1H, J = 13.8, 3.8 Hz, H-18), 1.07 (s,
3H, C), 0.89 (s, 3H, C), 0.87 (s, 3H, CH3), 0.86 (s, 6H, 2xCH3), 0.48 (s, 3H, CH3); 13C
NMR (75 MHz, CDCl3): δ = 177.85 (C-28), 162.99 (d, J = 246.8 Hz, C-38), 144.26 (C-13),
143.65 (C-32), 130.38 (d, J = 3.0 Hz, C-35), 130.11 (d, J = 8.1 Hz, C-36, C-36’), 124.42
(C-12), 122.47 (C-33), 116.28 (d, J = 21.7 Hz, C-37, C-37’), 76.85 (C-3), 72.12 (C-23),
57.50 (C-31), 53.20 (C-34), 49.85 (C-9), 47.61 (C-17), 46.81 (C-5), 45.93 (C-19), 41.88
(C-4), 41.82 (C-14), 41.36 (C-18), 39.36 (C-8), 38.20 (C-1), 36.98 (C-10), 33.91 (C-21),
33.14 (C-29), 32.54 (C-7), 32.41 (C-22), 30.70 (C-20), 27.70 (C-15), 26.77 (C-27), 25.91
(C-2), 23.67 (C-30), 23.43 (C-11), 23.06 (C-16), 18.56 (C-6), 16.81 (C-26), 15.81 (C-25),
11.55 (C-24); HRMS (ESI TOF-MS) [M+H]+calcd. for [C40H56FN3O4]+: 662.4333, found:
662.4327; CHN calcd.: C, 72.58; H, 8.53; N, 6.35; found: C, 72.40; H, 8.68; N, 6.09.
436
437
4.5.13
(p-Nitrobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-28-
oate (14).
21

ACCEPTED MANUSCRIPT
o
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
White solid; yield: 50 mg, 44%; m.p. 183.6-184.8 C; Rf = 0.43 (hexane/ethylacetate 1:1
v/v); IR (KBr): ῡ = 3404, 3152, 3086, 1720, 1526, 1162, 1054, 1038 802 cm-1; H NMR
1
(300 MHz, CDCl3): δ = 8.22 (d, 2H, , J = 8.4 Hz,H-37, H-37’), 7.58 (s, 1H, H-33), 7.39 (d,
2H, J = 8.3 Hz, H-36, H-36’), 5.61 (t, 2H, J = 16.6 Hz, H-34), 5.23 (brs, 1H, H-12), 5.16
(s, 2H, H-31), 3.70 (d, 1H, J = 10.3 Hz, H-23a), 3.61 (brt, 1H, J = 8.1 Hz, H-3), 3.40 (d,
1H, J = 10.3 Hz, H-23b), 2.79 (brdd, 1H, J = 13.4 Hz, H-18), 1.08 (s, 3H, CH3), 0.87 (s,
13
12H, 4xCH3), 0.50 (s, 3H, CH3); C NMR (75 MHz, CDCl3): δ = 177.92 (C-28), 148.23
(C-38), 144.26 (C-13), 143.65 (C-32), 141.59 (C-35), 128.72 (C-36, C-36’), 124.22 (C-37,
C-37’), 124.42 (C-12), 122.47 (C-33), 76.85 (C-3), 72.12 (C-23), 57.50 (C-31), 53.20 (C-
34), 49.81 (C-9), 47.61 (C-17), 46.86 (C-5), 45.93 (C-19), 41.89 (C-4), 41.85 (C-14), 41.39
(C-18), 39.39 (C-8), 38.20 (C-1), 36.98 (C-10), 33.92 (C-21), 33.16 (C-29), 32.57 (C-7),
32.46 (C-22), 30.76 (C-20), 27.72 (C-15), 26.81 (C-27), 25.91 (C-2), 23.67 (C-30), 23.45
(C-11), 23.10 (C-16), 18.56 (C-6), 16.88 (C-26), 15.82 (C-25), 11.53 (C-24); HRMS (ESI
TOF-MS) [M+H]+calcd. for [C40H56N4O6]+: 689.4278, found: 689.4269; CHN calcd.: C,
69.74; H, 8.19; N, 8.13; found: C, 69.51; H, 8.35; N, 8.00.
453
454
4.5.14
(m-Nitrobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-28-
oate (15).
o
455
456
457
458
459
White solid; yield: 60 mg, 52%; m.p. 152.7-153.9 C; Rf = 0.43 (hexane/ethylacetate 1:1
v/v); IR (KBr): ῡ = 3404, 3156, 3092, 1718, 1534, 1174, 1158, 1048, 728 cm-1; H NMR
1
(300 MHz, CDCl3): δ = 8.21 (m, 1H, H-38), 8.13 (brs, 1H, H-36), 7.59 (brs, 1H, H-33),
7.56 (m, 2H, H-39, H-40), 5.63 (d, 1H, J = 15.3 Hz, H-34a), 5.57 (d, 1H, J = 15.3 Hz, H-
34b), 5.22 (t, 1H, J = 3.0 Hz, H-12), 5.15 (s, 2H, H-31), 3.69 (d, 1H, J = 10.3 Hz, H-23a),
22

ACCEPTED MANUSCRIPT
460
461
462
463
464
465
466
467
468
469
470
3.60 (brt, 1H, J = 8.6 Hz, H-3), 3.39 (d, 1H, J = 10.3 Hz, H-23b), 2.79 (dd, 1H, J = 13.6,
3.7 Hz, H-18), 1.08 (s, 3H, CH3), 0.88 (s, 3H, CH3), 0.86 (s, 9H, 3xCH3), 0.50 (s, 3H,
CH3); 13C NMR (75 MHz, CDCl3): δ = 177.89 (C-28), 148.67 (C-37), 144.19 (C-13),
142.63 (C-32), 136.66 (C-35), 133.96 (C-40), 130.42 (C-39), 124.31 (C-12), 123.94 (C-36),
122.95 (C-38), 122.46 (C-33), 76.86 (C-3), 72.10 (C-23), 57.49 (C-31), 53.20 (C-34), 49.83
(C-9), 47.59 (C-17), 46.83 (C-5), 45.91 (C-19), 41.87 (C-4), 41.82 (C-14), 41.38 (C-18),
39.37 (C-8), 38.19 (C-1), 36.97 (C-10), 33.89 (C-21), 33.13 (C-29), 32.55 (C-7), 32.42 (C-
22), 30.74 (C-20), 27.70 (C-15), 26.77 (C-27), 25.91 (C-2), 23.66 (C-30), 23.42 (C-11),
23.07 (C-16), 18.56 (C-6), 16.85 (C-26), 15.80 (C-25), 11.56 (C-24); HRMS (ESI TOF-
MS) [M+H]+calcd. for [C40H56N4O6]+: 689.4278, found: 689.4269; CHN calcd.: C, 69.74;
H, 8.19; N, 8.13; found: C, 69.54; H, 8.33; N, 8.04.
471
472
4.5.15
(o-Nitrobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-28-
oate (16).
o
473
474
475
476
477
478
479
480
481
White solid; yield: 87 mg, 76%; m.p. 123.8-125.3 C; Rf = 0.43 (hexane/ethylacetate 1:1
v/v); IR (KBr): ῡ = 3046, 3158, 3060, 1722, 1530, 1160, 1048, 1004, 728 cm-1; H NMR
1
(300 MHz, CDCl3): δ = 8.14 (dd, 1H, J = 7.9, 1.3 Hz,H-37), 7.71 (brs, 1H, H-33), 7.60 (td,
2H, J = 7.4, 1.3 Hz, H-38), 7.53 (td, 2H, J = 7.4, 1.3 Hz, H-39);7.07 (dd, 1H, J = 7.6, 1.3
Hz, H-40), 5.90 (s, 2H, H-34), 5.24 (t, 1H, J = 3.1 Hz, H-12), 5.17 (s, 2H, H-31), 3.70 (d,
1H, J = 10.3 Hz, H-23a), 3.61 (brt, 1H, J = 8.2 Hz, H-3), 3.40 (d, 1H, J = 10.3 Hz, H-23b),
2.81 (dd, 1H, J = 13.8, 3.8 Hz, H-18), 1.09 (s, 3H, CH3), 0.90 (s, 3H, CH3), 0.87 (s, 9H, 3x
13
CH3), 0.55 (s, 3H, CH3); C NMR (75 MHz, CDCl3): δ = 177.77 (C-28), 147.59 (C-36),
143.90 (C-13), 143.64 (C-32), 134.43 (C-35), 130.53 (C-39), 130.51 (C-40), 129.88 (C-38),
23

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482
483
484
485
486
487
488
125.55 (C-37), 125.04 (C-12), 122.52 (C-33), 76.89 (C-3), 72.13 (C-23), 57.55 (C-31),
50.94 (C-34), 49.86 (C-9), 47.64 (C-17), 46.84 (C-5), 45.92 (C-19), 41.89 (C-4), 41.83 (C-
14), 41.38 (C-18), 39.40 (C-8), 38.21 (C-1), 37.00 (C-10), 33.92 (C-21), 33.16 (C-29),
32.55 (C-7), 32.44 (C-22), 30.77 (C-20), 27.73 (C-15), 26.80 (C-27), 25.94 (C-2), 23.71
(C-30), 23.45 (C-11), 23.07 (C-16), 18.59 (C-6), 16.92 (C-26), 15.81 (C-25), 11.56 (C-24);
HRMS (ESI TOF-MS) [M+H]+calcd. for [C40H56N4O6]+: 689.4278,found: 689.4270; CHN
calcd.: C, 69.74; H, 8.19; N, 8.13; found: C, 69.63; H, 8.40; N, 8.02.
489
490
4.15.16
(Benzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-28-amide
(17).
o
491
492
493
494
495
496
497
498
499
500
501
502
503
White solid; yield: 97mg, 90%; m.p. 124.8-126.3 C; Rf = 0.42 (hexane/ethylacetate 1:1
v/v); IR (KBr): ῡ = 3402, 3150, 3066,1722, 1636, 1520, 1048, 724 cm-1; H NMR (300
1
MHz, CDCl3): δ = 7.72 (s, 1H, H-33), 7.33(brs, 3H), 7.26 (brs, 2H), 6.61 (t, 1H, J = 4.7 Hz,
N-H), 5.49 (d, 1H, J = 14.8 Hz, H-34a), 5.42 (d, 1H, J = 14.8 Hz, H-34b), 5.35 (brs, 1H, H-
12), 4.45 (dd, 1H, J =15.0, 5.2 Hz, H-31a), 4.31 (dd, 1H, J =15.0, 5.2 Hz, H-31b), 3.68 (d,
1H, J = 10.3 Hz, H-23a), 3.61 (brt, 1H, J = 7.9 Hz, H-3), 3.38 (d, 1H, J = 10.3 Hz, H-23b),
2.51 (brdd, 1H, J = 11.1 Hz, H-18), 1.10 (s, 3H, CH3), 0.87 (s, 6H, 2xCH3), 0.86 (s, 6H,
13
2xCH3), 0.44 (s, H, CH3); C NMR (75 MHz, CDCl3): δ = 178.56 (C-28), 145.17 (C-13),
144.28 (C-32), 134.60 (C-35), 129.19 (C-37), 129.19 (C-37’), 128.89 (C-38), 128.19 (C-
36’), 128.19 (C-36), 123.27 (C-12), 122.55 (C-33), 76.63 (C-3), 71.84 (C-23), 54.28 (C-
34), 49.68 (C-9), 47.59 (C-17), 46.72 (C-5), 46.32 (C-19), 42.08 (C-4), 42.06 (C-14), 41.89
(C-18), 39.37 (C-8), 38.26 (C-1), 36.91 (C-10), 35.14 (C-31), 34.19 (C-21), 33.08 (C-29),
32.55 (C-7), 32.20 (C-22), 30.80 (C-20), 27.30 (C-15), 26.69 (C-27), 25.85 (C-2), 23.92
24

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504
505
506
(C-30), 23.68 (C-11), 23.52 (C-16), 18.52 (C-6), 16.61 (C-26), 15.87 (C-25), 11.62 (C-24);
HRMS (ESI TOF-MS) [M+H]+calcd. for [C40H58N4O3]+: 643.4587, found: 643.4578; CHN
calcd.: C, 74.73; H, 9.09; N, 8.71; found: C, 74.51; H, 9.23; N, 8.55.
507
508
4.5.17 (o-Bromobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-28-
amide (18).
o
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
White solid; yield: 102mg, 85%; m.p. 128.3-130.2 C; Rf = 0.42 (hexane/ethylacetate 1:1
v/v); IR (KBr): ῡ = 3386, 3150, 3070,1720, 1636, 1522, 1046, 1030, 748, 732 cm-1; H
1
NMR (300 MHz, CDCl3): δ = 7.58 (brs, 1H, H-37), 7.56 (s, 1H, H-33), 7.22 (m, H-38, H-
39, H-40), 6.62 (t, 1H, J = 6.3 Hz, N-H), 5.59 (s, 2H, H-34), 5.36 (brs, 1H, H-12), 4.47 (dd,
1H, J =14.8, 5.1 Hz, H-31a), 4.32 (dd, 1H, J =14.8, 5.1 Hz, H-31b), 3.68 (d, 1H, J = 10.0
Hz, H-23a), 3.60 (brt, 1H, J = 7.9 Hz, H-3), 3.37 (d, 1H, J = 10.0 Hz, H-23b), 2.51 (brdd,
1H, J = 11.2 Hz, H-18), 1.10 (s, 3H, CH3), 0.86 (s, 6H, 2xCH3), 0.85 (s, 6H, 2xCH3), 0.46
(s, H, CH3); 13C NMR (75 MHz, CDCl3): δ = 178.55 (C-28), 145.05 (C-13), 144.29 (C-32),
134.10 (C-35), 133.36 (C-37), 130.56 (C-40), 130.56 (C-39), 128.25 (C-38), 123.68 (C-36),
123.26 (C-12), 122.97 (C-33), 76.60 (C-3), 71.80 (C-23), 53.93 (C-34), 49.67 (C-9), 47.58
(C-17), 46.71 (C-5), 46.32 (C-19), 42.06 (C-4), 42.06 (C-14), 41.88 (C-18), 39.37 (C-8),
38.25 (C-1), 36.90 (C-10), 35.12 (C-31), 34.20 (C-21), 33.08 (C-29), 32.56 (C-7), 32.20
(C-22), 30.79 (C-20), 27.32 (C-15), 26.66 (C-27), 25.85 (C-2), 23.91 (C-30), 23.68 (C-11),
23.53 (C-16), 18.53 (C-6), 16.62 (C-26), 15.87 (C-25), 11.63 (C-24); HRMS (ESI TOF-
MS) [M+H]+calcd. for [C40H57BrN4O3]+: 721.3672, found: 721.3679; CHN calcd.: C,
66.56; H, 7.96; N, 7.76; found: C, 66.39; H, 8.11; N, 7.59.
25

ACCEPTED MANUSCRIPT
525
526
4.5.18 (m-Bromobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-28-
amide (19).
o
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
White solid; yield: 108mg, 90%; m.p. 123.6-125.2 C; Rf = 0.44 (hexane/ethylacetate 1:1
v/v); IR (KBr): ῡ = 3394, 3148, 3064, 1730, 1636, 1519, 1240, 1048, 770, 734 cm-1; H
1
NMR (300 MHz, CDCl3): δ = 7.51 (s, H, H-33), 7.46 (d, 1H, J = 7.3 Hz, H-38), 7.40 (s,
1H, H-36), 7.22 (m, 2H, H-39, H-40), 6.61 (t, 1H, J = 4.7 Hz, N-H), 5.47 (d, 1H, J = 15.7
Hz, H-34a), 5.39 (d, 1H, J = 15.7 Hz, H-34b), 5.36 (brs, 1H, H-12), 4.46 (dd, 1H, J =15, 5.1
Hz, H-31a), 4.33 (dd, 1H, J =15, 5.1 Hz, H-31b), 3.69 (d, 1H, J = 10.1 Hz, H-23a), 3.61
(brt, 1H, J = 8.0 Hz, H-3), 3.39 (d, 1H, J = 10.2 Hz, H-23b), 2.52 (brdd, 1H, J = 11.6 Hz,
H-18), 1.11 (s, 3H, CH3), 0.86 (s, 12H, 4xCH3), 0.44 (s, H, CH3); 13C NMR (75 MHz,
CDCl3): δ = 178.65 (C-28), 145.44 (C-13), 144.31 (C-32), 136.83 (C-35), 132.06 (C-38),
131.15 (C-36), 130.72 (C-39), 126.71 (C-40), 123.20 (C-37), 123.27 (C-12), 122.72 (C-33),
76.73 (C-3), 71.93 (C-23), 53.47 (C-34), 49.71 (C-9), 47.59 (C-17), 46.73 (C-5), 46.34 (C-
19), 42.12 (C-4), 42.08 (C-14), 41.90 (C-18), 39.38 (C-8), 38.25 (C-1), 36.91 (C-10), 35.16
(C-31), 34.20 (C-21), 33.09 (C-29), 32.57 (C-7), 32.24 (C-22), 30.81 (C-20), 27.32 (C-15),
26.74 (C-27), 25.86 (C-2), 23.96 (C-30), 23.69 (C-11), 23.53 (C-16), 18.56 (C-6), 16.63
(C-26), 15.87 (C-25), 11.61 (C-24); HRMS (ESI TOF-MS) [M+H]+calcd. for
[C40H57BrN4O3]+: 721.3672, found: 721.3686; CHN calcd.: C, 66.56; H, 7.96; N, 7.76;
found: C, 66.44; H, 8.14; N, 7.51.
544
545
4.5.19 (p-Bromobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-28-
amide (20).
26

ACCEPTED MANUSCRIPT
o
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
White solid; yield: 114mg, 95%; m.p. 143.6-145.2 C; Rf = 0.42 (hexane/ethylacetate 1:1
v/v); IR (KBr): ῡ = 3414, 3150, 1636, 1048, 1012 cm-1; H NMR (300 MHz, CDCl3): δ =
1
7.50 (s, 1H, H-33), 7.47 (d, 2H, J = 8.2 Hz, H-37, H-37’), 7.13 (d, 2H, J = 8.1 Hz, H-36,
H-36’), 6.60 (t, 1H, J = 5.1 Hz, N-H), 5.45 (d, 1H, J = 14.8 Hz, H-34a), 5.37 (d, 1H, J =
14.8 Hz, H-34b), 5.34 (brs, 1H, H-12), 4.44 (dd, 1H, J =15.0, 5.3 Hz, H-31a), 4.32 (dd, 1H,
J =15.0, 5.3 Hz, H-31b), 3.69 (d, 1H, J = 10.2 Hz, H-23a), 3.61 (brt, 1H, J = 7.0 Hz, H-3),
3.38 (d, 1H, J = 10.2 Hz, H-23b), 2.50 (brdd, 1H, J = 11.0 Hz, H-18), 1.10 (s, 3H, CH3),
13
0.86 (s, 12H, 4xCH3), 0.41 (s, H, CH3); C NMR (75 MHz, CDCl3): δ = 178.67 (C-28),
145.39 (C-13), 144.31 (C-32), 133.69 (C-35), 132.36 (C-37, C-37’), 129.82 (C-36, C-36’),
123.26 (C-12), 123.08 (C-38), 122.68 (C-33), 76.68 (C-3), 71.90 (C-23), 53.55 (C-34),
49.69 (C-9), 47.57 (C-17), 46.73 (C-5), 46.33 (C-19), 42.09 (C-4), 42.06 (C-14), 41.90 (C-
18), 39.35 (C-8), 38.25 (C-1), 36.90 (C-10), 35.16 (C-31), 34.19 (C-21), 33.07 (C-29),
32.56 (C-7), 32.21 (C-22), 30.80 (C-20), 27.29 (C-15), 26.72 (C-27), 25.84 (C-2), 23.95
(C-30), 23.66 (C-11), 23.52 (C-16), 18.49 (C-6), 16.62 (C-26), 15.86 (C-25), 11.63 (C-24);
HRMS (ESI TOF-MS) [M+H]+calcd. for [C40H58BrN4O3]+:721.3672, found: 721.3689;
CHN calcd.: C, 66.56; H, 7.96; N, 7.76; found: C, 66.31; H, 8.10; N, 7.61.
562
563
4.5.20 (2,6-Dichlorobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-
28-amide (21).
o
564
565
566
567
White solid; yield: 89mg, 75%; m.p. 144.8-146.3 C; Rf = 0.44 (hexane/ethylacetate 1:1
v/v); IR (KBr): ῡ = 3404, 3150, 3078, 1636, 1520, 1048, 764, 732 cm-1; H NMR (300
1
MHz, CDCl3): δ = 7.47 (s, 1H, H-33), 7.37 (d, 2H, J = 7.2 Hz, H-37, H-40), 7.29 (d, 1H, J
= 7.9 Hz, H-38), 6.59 (t, 1H, J = 4.9 Hz, N-H), 5.79 (t, 2H, J = 15.6 Hz, H-34), 5.34 (brs,
27

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568
569
570
571
572
573
574
575
576
577
578
579
1H, H-12), 4.45 (dd, 1H, J =15.0, 5.1 Hz, H-31a), 4.31 (dd, 1H, J =15.0, 5.1 Hz, H-31b),
3.67 (d, 1H, J = 10.3 Hz, H-23a), 3.60 (brt, 1H, J = 7.9 Hz, H-3), 3.37 (d, 1H, J = 10.3 Hz,
H-23b), 2.51 (brdd, 1H, J = 11.5 Hz, H-18), 1.09 (s, 3H, CH3), 0.86 (s, 6H, 2xCH3), 0.85 (s,
6H, 2xCH3), 0.42 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3): δ = 178.43 (C-28), 144.65 (C-
13), 144.23 (C-32), 136.88 (C-35), 131.20 (C-36), 130.04 (C-40), 128.99 (C-37), 128.99
(C-39), 128.99 (C-38), 123.24 (C-12), 122.23 (C-33), 76.61 (C-3), 71.81 (C-23), 49.10 (C-
34), 49.68 (C-9), 47.58 (C-17), 46.70 (C-5), 46.30 (C-19), 42.02 (C-4), 42.02 (C-14), 41.87
(C-18), 39.35 (C-8), 38.25 (C-1), 36.89 (C-10), 35.02 (C-31), 34.20 (C-21), 33.08 (C-29),
32.57 (C-7), 32.21 (C-22), 30.79 (C-20), 27.29 (C-15), 26.66 (C-27), 25.85 (C-2), 23.88
(C-30), 23.66 (C-11), 23.51 (C-16), 18.52 (C-6), 16.52 (C-26), 15.85 (C-25), 11.63 (C-24);
HRMS (ESI TOF-MS) [M+H]+calcd. for [C40H56Cl2N4O3]+: 711.3808, found: 711.3799;
CHN calcd.: C, 67.59; H, 7.80; N, 7.88; found: C, 67.41; H, 7.99; N, 7.67.
580
581
4.5.21 (p-Chlorobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3,23-dihydroxyolean-12-en-28-
amide (22).
582
583
584
585
586
587
588
589
White solid; yield: 92mg, 81%; m.p. 139.3-141 oC; Rf = 0.44 (hexane/ethylacetate 1:1 v/v);
1
IR (KBr): ῡ = 3402, 3152, 3068, 1718, 1636, 1520, 1048, 1016, 732cm-1; H NMR (300
MHz, CDCl3): δ = 7.50 (s, H, H-33), 7.31 (d, 2H, J = 8.2 Hz, H-37, H-37’), 7.19 (d, 2H, J
= 8.2 Hz, H-36, H-36’), 6.60 (t, 1H, J = 4.4 Hz, N-H), 5.47 (d, 1H, J = 14.9 Hz, H-34a),
5.38 (d, 1H, J = 14.9 Hz, H-34b), 5.35 (brs, 1H, H-12), 4.44 (dd, 1H, J =14.9, 5.2 Hz, H-
31a), 4.32 (dd, 1H, J =14.9, 5.2 Hz, H-31b), 3.68 (d, 1H, J = 10.2 Hz, H-23a), 3.60 (brt, 1H,
J = 8.0 Hz, H-3), 3.38 (d, 1H, J = 10.2 Hz, H-23b), 2.50 (brdd, 1H, J = 11.8 Hz, H-18),
1.10 (s, 3H, CH3), 0.85 (s, 12H, 4xCH3), 0.41 (s, 3H, CH3); 13C NMR (75 MHz, CDCl3): δ
28