Leishmanicidal and cytotoxic activity of hederagenin-bistriazolyl derivatives

Article abstract of DOI:10.1016/j.ejmech.2017.09.045

Aiming to obtain new potent leishmanicidal and cytotoxic compounds from natural sources, the triterpene hederagenin was converted into several new 1,2,3-triazolyl derivatives tethered at C-23 and C-28. For this work hederagenin was isolated from fruits of

Full text of DOI:10.1016/j.ejmech.2017.09.045

Accepted Manuscript
Leishmanicidal and cytotoxic activity of hederagenin-bistriazolyl derivatives
Diego Rodríguez-Hernández, Luiz C.A. Barbosa, Antonio J. Demuner, Amalyn Nain-
Perez, Sebastião R. Ferreira, Ricardo T. Fujiwara, Raquel M. de Almeida, Lucie
Heller, René Csuk
PII:
S0223-5234(17)30760-2
10.1016/j.ejmech.2017.09.045
EJMECH 9760
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 July 2017
Revised Date: 19 September 2017
Accepted Date: 21 September 2017
Please cite this article as: D. Rodríguez-Hernández, L.C.A. Barbosa, A.J. Demuner, A. Nain-Perez,
Sebastiã.R. Ferreira, R.T. Fujiwara, R.M. de Almeida, L. Heller, René. Csuk, Leishmanicidal and
cytotoxic activity of hederagenin-bistriazolyl derivatives, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.09.045.
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Graphical abstract

ACCEPTED MANUSCRIPT
Leishmanicidal and cytotoxic activity of hederagenin-bistriazolyl
derivatives
Diego Rodríguez-Hernándeza, Luiz C. A. Barbosaa,b*, Antonio J. Demunerb, Amalyn
Nain-Pereza, Sebastião R. Ferreirac,e, Ricardo T. Fujiwarac, Raquel M. de Almeidac,
Lucie Hellerd, René Csukd
a Department of Chemistry, Universidade Federal de Minas Gerais, Av. Pres. Antônio
Carlos, 6627, Campus Pampulha, CEP 31270-901, Belo Horizonte, MG, Brazil
b Department of Chemistry, Universidade Federal de Viçosa, Av. P. H. Rolfs, s/n, CEP
36570-900, Viçosa, MG, Brazil
c
Department of Parasitology, Universidade Federal de Minas Gerais, Av. Pres.
Antônio Carlos, 6627, Campus Pampulha, CEP 31270-901, Belo Horizonte, MG,
Brazil
d Martin-Luther-University Halle-Wittenberg, Organic Chemistry, Kurt-Mothes-Str.2, D
06120, Halle (Saale), Germany.
eHealth Science Center, Universidade Federal de Roraima, Av. Cap. Ene Garcez, CEP
69310-000, Boa Vista, RR, Brazil
*Corresponding authors: Tel.: +55 3899 3065 and +39 0532 455311. E-mail address:
lcab@ufmg.br (Luiz C. A. Barbosa) or rene.csuk@chemie.uni-halle.de (R. Csuk).
_____________________________
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Abstract
Aiming to obtain new potent leishmanicidal and cytotoxic compounds from natural
sources, the triterpene hederagenin was converted into several new 1,2,3-triazolyl
derivatives tethered at C-23 and C-28. For this work hederagenin was isolated from
fruits of Sapindus saponaria and reacted with propargyl bromide to afford as a major
product bis-propargylic derivative 1 in 74%. Submitting this compound to Huisgen 1,3-
dipolar cycloaddition reactions with several azides afforded the derivatives 2-19 with
yields in the range of 40-87%. All compounds have been screened for in vitro cytotoxic
activity in a panel of five human cancer cell lines by a SRB assay. The bioassays
showed that compound 19 was the most cytotoxic against all human cancer cell lines
with EC50 = 7.4-12.1 µM. Moreover, leishmanicidal activity was evaluated through the
in vitro effect in the growth of Leishmania infantum, and derivatives 1, 2, 5 and 17 were
highly effective preventing proliferation of intracellular amastigote forms of L. infantum
(IC50 = 28.8, 25.9, 5.6 and 7.4 µΜ, respectively). All these compounds showed a higher
selectivity index and low toxicity against two strains of kidney BGM and liver HepG2
cells. Compound 5 has higher selectivity (1780 times) in comparison with the
commercial antimony drug and is around 8 times more selective than the most active
compound previously reported hederagenin derivative. Such high activity associated
with low toxicities make the new bis-traiazolyl derivatives promising candidates for the
treatment of leishmaniasis. In addition, hederagenin and some derivatives (2, 5 and 17)
showed interaction in the binding site of the enzyme CYP51Li.
Keywords: Hederagenin, tryazol, Leishmania infantum, Cytotoxicity, CYP51Li
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1. Introduction
Natural pentacyclic triterpenes are widely found in several plant species like those
from the Celastraceae, Lamiaceae, Sapindaceae, Fabaceae family [1-3]. These
compounds are also found in olives, apples, figs, and cranberries [2-4], as well as in
some oriental and traditional medicine herbs widely distributed all over the world [5-6].
They often exhibit a wide spectrum of biological activities such as anti-inflammatory
[7], cytotoxic [8], antiprotozoal [9], anti-hypoglycemic [10], antibacterial [11], antiviral
[12], and larvicidal [13].
Among the numerous terpenoids found in nature, we highlight Hederagenin (Fig.
1), a pentacyclic oleane-type triterpenoid found in large quantities in the pericarps of
fruits of Sapindus saponaria (Sapindaceae) [14]. Several biological activities have been
reported for hederagenin, including anti-inflammatory [15], antifungal [16],
antimicrobial [17], anti-leishmanial [18] and antitumoral [14]. Due to such reports and
our continued interest in the search for bioactive compounds based on natural products
[19-24] we have recently started investigating the potential of hederagenin as a source
of new anti-leishmanial [18] and antitumor [14, 25] drugs.
During this endeavor, we have synthesized a set of hederagenin derivatives
modified at the carbonyl-28 via introduction of different groups such as benzylic, amine
or heterocycles triazolyl (Fig. 1) [14, 18, 25]. Some derivatives holding a triazolyl
moiety showed activity against several cancer cell lines being much higher than natural
hederagenin [25]. In addition, some hederagenin derivatives are known for the ability to
inhibit the proliferation of amastigote forms of Leishmania infantum (BH46) (Fig. 1),
being active at micromolar level, presenting good selectivity index and low toxicity
against two strains of kidney BGM and liver HepG2 cells. Thus, these results lead
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hederagenin derivatives to be more potent than a commercial positive drug control
(potassium antimonyl tartrate trihydrate; IC50 = 80 µM) [18].
[Insert Figure 1]
At the moment, the molecular target(s) or the mechanism(s) of action of
hederagenin triazolyl derivatives against amastigote form of L. infantum remains
unclear and is yet to be elucidated. Despite that, some studies report the role of triazolyl
derivatives as effective antiparasitic drugs, potentially blocking sterol biosynthesis by
inhibiting the enzymatic activity of a sterol 14α-demethylase (CYP51). This inhibition
can lead to the accumulation of toxic methylated sterols, which in turn leads to parasitic
death [26]. Sterol 14α-demethylase (CYP51) is an essential enzyme involved in the
survival and virulence of Trypanosoma and Leishmania species, acting mainly in the
biosynthesis of ergosterol. The disruption of this biosynthesis affects cytokinesis, stops
cell growth, and can lead to the collapse of the cellular membrane of these parasites [26-
27].
Crystal structures of protozoan sterol 14α-demethylases provide an opportunity
for the structure-directed development of such inhibitors [26]. Hargrove et al. [28]
determined the crystal structure of sterol 14α-demethylase (CYP51Li) from L. infantum
bound to fluconazole (PDB ID: 3L4D). In this context, the enzyme CYP51 has emerged
as a promising target for antiprotozoal chemotherapy [26]. There are reports involving
virtual screening of oleanolic acid, a pentacyclic triterpene that shows affinity for this
enzyme CYP51Li [28-29].
In continuation of our studies aiming to obtain more active compounds based on
the hederagenin scaffold, we report here a series of new bis-triazolyl derivatives
modifying the C-23 and C-28 positions simultaneously. For all new compounds, the
cytotoxic profiles against several cancer cell lines and the in vitro effect on the
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proliferation of intracellular amastigote forms of L. infantum were investigated. An
approach of the binding interactions of the most active compounds with sterol 14α-
demethylase enzyme CYP51 from L. infantum (PDB ID: 3L4D) were explored through
molecular docking.
2. Results and Discussion
2.1 Chemistry
The strategy envisaged in the present study involved the preparation of 18 new
1,2,3-triazolyl derivatives of hederagenin employing Huisgen 1,3-dipolar cycloaddition
reactions [30]. The essential hederagenin was isolated from the pericarp of S. saponaria
as previously described [14]. Subsequently, the bis-propargyl derivative 1 was prepared
in 74% by the reaction of hederagenin with propargyl bromide in the presence of NaH
(Scheme 1). A small quantity of the propargylic ester, not alkylated at the alcohols OH,
was isolated as a side product and was identical with a previously reported ester [14].
The required substituted alkylazides (a-r) were prepared via the reaction of
sodium azide and the corresponding alkylbromides (for details see Scheme S1 in the
Supplementary material). The reaction of 1 with azides (a-r), catalyzed by copper (I) in
the presence sodium ascorbate as reducing agent, afforded 18 new bis-triazolyl-
hederagenin derivatives 2-19. The overall chemical yields of the synthesized derivatives
ranged from 41-87% (Scheme 1).
[Insert Scheme 1]
The purity of the compounds was proven by HRMS and microanalysis, and their
1
identity confirmed by extensive NMR analyses. Thus, in the H NMR spectra, the
formation of the heterocyclic unit was confirmed by the signal around δ = 7.45-7.75
corresponding to the hydrogen of the triazolyl ring. The corresponding signals of the
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13
aromatic hydrogen atoms were detected between δ = 6.80 and 8.25. Concerning to C
NMR, all signals for the triterpenoic skeleton in this series of compounds showed
similar shifts to the parent hederagenin with exception of the signals for C-23 and C-28
and the groups attached at these positions. For the ester carbon C-28 a shift to low
frequency was observed when compared with parent (δ = 180.7 for hederagenin to δ =
177.4 ± 0.4 for the esters carbonyl-28). Regarding to C-23 a shift to high frequency was
observed as compared to natural triterpene (δ = 68.7 for hederagenin to δ = 75 ± 0.5 for
the carbon-23). The signals of the aromatic carbons were detected at high frequency at δ
= 163.5-104.5. For compounds, 5 and 15 the occurrence of 19F-13C couplings were
13
detected in the C NMR spectra. 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 for 13C NMR the assignments were also supported by the literature [31].
2.2 Biological assays – cytotoxicity
The cytotoxic activity of the hederagenin derivatives (2-19) was investigated in
vitro employing five human tumor cell lines (pharynx carcinoma - FaDu; ovarian
carcinoma - A2780; colon adenocarcinoma - HT29; malignant melanoma - A375;
thyroid carcinoma - SW1736); by using the well-established photometric
sulforhodamine B assay (SRB) [32]. For comparison, betulinic acid was used as a
positive standard control. The results of these assays are summarized in Table 1.
[Insert Table 1]
Amongst all new hederagenin derivatives, few were endowed with high
cytotoxicity against the human tumor cell lines tested. Most were less active that the
cut-off concentration of 30 µM. The derivatives 5, 6 and 7 showed low cytotoxicity with
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EC50 around 16.3-29.1 µM in the cell lines tested. However, compound 19 carrying in
the triazolyl core a substituted 2-(p-cyanophenyl)-2-oxoethyl substituent, was the most
cytotoxic against all human cancer cell lines showing EC50 in the range of 7.4-12.1 µM.
The colon adenocarcinoma (HT29), a class of solid tumor, proved to be the most
sensitive cell lines, even the compound 19 (EC50 = 7.4 µM) presented higher cytotoxic
activity compared to standard compound (betulinic acid; EC50 = 18.4 µΜ) (Table 1).
To gain a deeper insight into the mode of action of compound 19 onto tumor
cells HT29, an extra experiment was performed through fluorescence microscopic
studies using acridine orange and propidium iodide staining (Fig. 2).
[Insert Figure 2]
The result for this assay have shown that no changes in cell morphology and cell
membrane permeability occurred compared to the control. Hence, neither necrosis nor
apoptosis occurred. Thus, the impact of the derivative 19 is likely the result from
cytostatic effects rather than from being cytotoxic, since a significant inhibition of cell
growth was observed. Furthermore, when comparing these results with those previously
obtained by our group (Fig. 1) [25], we noticed that adding of a triazolyl core at carbon-
23 has no influence on the cytotoxicity of this class of compounds.
2.3 Biological assays – antileishmania activity
The in vitro antileishmanial activity for all hederagenin derivatives 1-19 was
evaluated in intracellular amastigote form of L. infantum (BH46) parasitized
macrophages (DH82). Initially, all compounds were tested at 1000 µM (phase I) to
select those with the most promising activity (see all results in Table S1 in the
Supplementary material). The results revealed that hederagenin and seven derivatives
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(1, 2, 5, 8, 15, 17 and 18) reduced the multiplication of amastigotes of L. infantum by at
least 50% (Table S1). Although no clear structure-activity relationship can be deduced
at this stage, a comparison of the activity of 14 (Table S1) with those of other benzyl
derivatives (2-15), shows that in general the most active compounds bear a substituent
at para or ortho position. None of the compounds with a meta-substituent had a
significant activity. On the other hand, among the four aryl-ketone derivatives (16-19),
two were highly active at this phase I test. A comparison of the activities for the pairs
8/17 (with a p-methylphenyl group) and 14/16 (phenyl group), revealed no clear
influence of the extra C=O moiety attached to the phenyl ring on the activity.
Following the initial assay, the most active compounds were then subjected to the
same assay at lower concentrations as 0.1, 1.0, 10 and 100 µM (phase II). It was noted
that although compounds 8 and 18 were able to reduce the growth of amastigotes in the
phase 1 test, there was no clear correlation between the bioactivity and concentration,
precluding the calculation of the IC50 values in the phase 2 test. Along with these tests,
the hederagenin, selected compounds (1, 2, 5, 15 and 17) and a positive drug control
(potassium antimonyl tartrate trihydrate) were tested on cellular viability, using the
canine macrophages cell line DH82, Monkey African green kidney BGM and human
hepatocytes-like HepG2 cells, by microscopically monitoring and by using the
colorimetric tetrazolium salt assay (MTT).
Hederagenin and compounds 1, 2, 5, 15 and 17 showed good results, preventing
the proliferation of intracellular amastigote forms of L. infantum (BH46), presenting
IC50 values in the range of 5.4-154.8 µM (Table 2). All these calculated IC50 values
were obtained from four replicates experiments. A representative sigmoidal dose-
response curve (log [substance] versus percentage inhibition of intracellular amastigote
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forms of L. infantum) is illustrated for compound 5 (Fig. 3). Evaluation of the curves
allowed the determination of IC50 values.
[Insert Figure 3]
Among these, the derivatives 1, 2, 5 and 17 were more active than parent
hederagenin, while compound 15 (holding a 2,4-difluorobenzyl moiety) was the only
one with a reduced activity (with IC50 = 154.8 µM) compared to the commercial drug
based on antimony (III) salt used as a positive control (IC50 = 80 µM) (Table 2). The
hederagenin derivatives substituted with o-fluorobenzyl (5) and 2-(p-methylphenyl)-2-
oxoethyl (17) moiety linked to a 1,2,3-triazolyl group were most potent showing IC50
values between 5.4 and 7.4 µM. These results represent an 11- and 8-fold higher activity
than hederagenin and 14- and 10-fold higher than the antimonyl tartrate drug used as
positive control, respectively.
Since it has been shown that interaction between some lupane derivatives and
the antileishmanial drug miltefosine result in synergistic effect [33], we could expect
that a similar interaction could also potentiate the hederagenin derivatives.
Having now selected the most active compounds, these were subjected to a MTT
assay, and the results of the cytotoxic concentration to canine macrophages (DH82) are
presented in Table 2. As the experiments revealed, hederagenin and derivatives 1, 2, 5
and 17 to exhibit a remarkably high selectivity index (ranging from 9 to >178)
compared with that of antimony commercial drug (SI = 0.1). It is important to point out
that although the most active bis-triazolyl derivative 5 had IC50 2.8 times higher than the
most active amide (Fig. 1, X = o-NO2) previously reported (IC50 = 5.6 versus 2.0 µM), it
was at least 8 times more selective (SI = >178 versus 22.5).
[Insert Table 2]
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Following this selectivity study, the most active compounds were tested for their
cytotoxicity against two strains of liver (HepG2) and kidney (BGM) cells to evaluate
their effects on different parts of the organisms; the results are compiled in Table 3.
Concerning the kidney cells, the natural hederagenin and compounds 2, 5 and 17
presented higher values of selective index (SI) than the corresponding value for the
positive control. Thereby, the antimony drug presented SI = 1.6 (BGM) confirming its
high toxicity as already reported [34]. In relation to this parameter the most selective
compounds were 2 (SI = 38), 5 (SI = >178) and 17 (SI = >135). It is generally accepted
that a drug candidate should have a selectivity index of at least 10 [35] to be regarded as
a promising candidate for further testing.
In the same way, for the liver cells, the hederagenin derivatives 2, 5 and 17
showed selective index equal to 38, >178 and >135, respectively (Table 3), much higher
in comparison with the observed for the antimony commercial drug (SI = 0.3). Such
results make these compounds 127 to 593 times more selective towards the human
hepatocytes than the reference drug.
When comparing these results with those previously obtained by our group [19],
we observed that adding a triazolyl core at carbon-23, the toxicity towards L. infantum
parasite was approximately the same, however, the selectivity is highly improved in
comparison with the most active amide derivatives previously reported (Table 1) [36].
Considering these results on the toxicity of some bis-tryazolyl derivatives
towards macrophages, monkey kidney and human hepatocytes cells, these compounds
seem to be promising candidates for further development of a new drug for the
treatment of L. infantum infection.
[Insert Table 3]
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2.4. Prediction of the binding mode of hederagenin and bis-tryazolyl-derivatives (2, 5
and 17) in the CYP51Li structure
An approach of molecular modeling was used to verify a possible leishmanicidal
mode of action via the ergosterol inhibition pathway. We employed the Autodock 4.2
program [37] that has a free energy scoring function, which uses an AMBER force field
to estimate the free energy of binding of a ligand to its target [38]. Hederagenin and the
most active derivatives (2, 5 and 17) were docked into the crystallographic structure of
CYP51 from L. infantum available in the Protein Data Bank (PDB code: 3L4D;
resolution: 2.75 Å). These compounds showed satisfactory docking with acceptable
statistics near the active site of CYP51Li. Detailed results of analysis (free energy of
binding values for each ligand along with the description of interacting residues) are
presented in Table 4.
[Insert Table 4]
The residues involved in bonding and nonbonding interaction with ligands are as
follows: Ile-167, Glu-204, Ser-205, Cys-206, Leu:-207, Pro-209, Ala-210, Phe-213,
Gln-224, Phe-289, Ile-297, His-293, Arg-346, Met-405, Phe-412, Phe-415, Gly-416,
Asn-455, His-457, Trh-458, Met-459, Gly-462, Pro-463 and Ala-465. The residues
involved hydrogen bonding with the ligands are as follows: Glu-204, Ser-205, Arg-227,
Lys-406, Glu-425, Met-459, Val-460. These residues are found near the active site
(heme cofactor). These ligands (hederagenin and derivatives 2, 5, 17) are surrounded by
the above residues; it can be concluded that these residues are highly conserved and
play important functional roles. Thus, interaction of the ligands with these residues
might be changing the active site structure, which leads to an inhibition of the enzyme.
In addition, these ligands show low free energy of binding against CYP51Li; thus, they
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bind with high affinity. For example, the most active compound (5) showed the best
binding energy followed by 17, 2 and hederagenin in that order. The interactions for this
derivative 5 with the enzyme are represented in Fig. 4A. The figure shows that the lead
molecules docks at a position near the active site of CYP51Li, where also the cofactor
heme (shown in green color) is bound.
[Insert Figure 4]
The protein contact potential was generated for the derivative 5 (Fig. 4B)
depicting the whole surface of the protein. Red and blue colors show negative and
positive charges, respectively. In this depiction, the triazolyl derivative 5 was shown to
be bound inside a cavity formed at the interface of the enzyme. Although the docked
conformation was different for each compound tested, the ligands are positioned at
almost the same place, thus showing a consensus pattern of interactions with the
residues near the active site. Figures showing interactions of hederagenin and
derivatives 2 and 17 with the enzyme CYP51Li can be found in the Supplementary
material. It can be predicted that the inhibition modes of all of these ligands could be
similar, because of their interactions with almost the same residues.
3. Conclusion
We have described the synthesis and biological activities of 18 new triterpenoid
derivatives of natural product hederagenin bearing two triazolyl-aromatic substituted
moieties. Regarding the cytotoxic activity, the majority of the bis-triazolyl-derivatives
were active in high micromolar ranges. Concerning antileishmanial activity, the most
active bis-triazolyl derivatives presented very high selectivity index in relation to BGM
and HepG2 line cells, revealing to be much less toxic in comparison with the antimonyl
commercial drug used in the treatment of leishmaniasis. Thus, considering that
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hederagenin can be obtained in large quantities from natural source, the new derivatives
herein reported can be used as a lead for the development of the novel drug against
Leishmania.
Experimental Section
4.1. General experimental procedures
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 using silica gel 230-400 mesh. All compounds were
1
fully characterized by IR, ESI-MS (or ASAP-MS), H 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). Mass spectra were recorded on an Advion express ionL CMS
instrument (ion source: ESI or ASAP, positive and negative mode). Elemental analyses
1
13
were measured on a Foss-HeraeusVario EL unit. The H and C NMR spectra were
recorded on a Bruker Avance at 400 MHz and 100 MHz, using CDCl3 as solvent
(25°C). Chemicals shifts (δ) are reported from tetramethylsilane with the solvent
resonance as the internal standard. Data are reported as follows: chemical shift (δ),
multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet),
integration, coupling constants (J = Hz) and assignment. Melting points were measured
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with on a MQAPF-301 apparatus. 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 0.5-2.5 ppm, so the hydrogen
atoms in such range could not be assigned.
4.2. Isolation of Hederagenin (He)
Fruits of Sapindus saponaria were collected in the municipality Tocantins
(21°10ʹ30ʹʹS and 43°01ʹ04ʹʹW), Minas Gerais State, Brazil. Voucher specimens were
dried and placed in the collection of the VIC Herbarium of the Plant Biology
Department of Viçosa Federal University (UFV), under the register number VIC
35.403. Hederagenin was isolated from pericarp of S. saponaria L., using the method
previously reported [36]. It was obtained as a white solid; m.p. 319-321 °C (lit.: 318-
320 °C) [14]; Rf = 0.24 (hexane/ethyl acetate, 1:1 v/v). All spectroscopic data (IR, MS
and NMR) were in agreement with the literature [31].
4.3. Procedure for the synthesis of (3β)3-hydroxyolean-12-en-23,28-dipropargyl-28-
oate (1).
Hederagenin (1.0 g, 2.1 mmol) was allowed to react with NaH (80% in oil, 0.19
g, 8 mmol) in dry THF (20 mL) under nitrogen atmosphere for 2 hours before a solution
of propargyl bromide (0.53 g; 4.5 mmol in 5 mL THF) was slowly added. The reaction
mixture was stirred for another 24 h, and quenched by adding water (75 mL). The
aqueous phase was extracted with ethyl acetate (3 x 50 mL) and the combined organic
phases were dried over sodium sulfate (Na2SO4). The filtrate was evaporated under
reduced pressure to afford a white residue that was purified by silica gel column
chromatography (gradient elution: hexane/ethyl acetate 6:1 to hexane/ethyl acetate 1:1)
to yield compound 1 as a white solid in 74% yield (767 mg; 1.40 mmol): Rf = 0.55
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(hexane/ethyl acetate 1:1 v/v); m.p: 83.5-85.1 °C. 1H NMR (400 MHz, CDCl3): δ= 5.30
(brs, 1H, H-12); 4.67 (d, 1H, J = 15.4 Hz, H-31a); 4.57 (d, 1H, J = 15.5 Hz, H-31b);
4.17 (d, 1H, J = 16.0 Hz, H-34a); 4.11 (d, 1H, J = 16.0 Hz, H-34b); 3.61 (m, 1H, H-3);
3.56 (d, 1H, J = 8.6 Hz, H-23a); 3.31 (d, 1H, J = 8.6 Hz, H-23b); 2.86 (brd, 1H, J = 12.6
Hz, H-18); 2.44 (brs, 1H, H-33); 2.42 (brs, 1H, H-36); 1.13 (s, 3H, CH3); 0.93 (s, 3H,
13
CH3); 0.92 (s, 3H, CH3); 0.89 (s, 3H, CH3); 0.86 (s, 3H, CH3); 0.74 (s, 3H, CH3); C
NMR (100 MHz, CDCl3): δ= 176.78 (C-28); 143.41 (C-13); 122.69 (C-12); 79.71 (C-
32); 79.25 (C-35); 78.20 (C-3); 75.74 (C-23); 74.67 (C-33); 74.51 (C-36); 58.73 (C-31);
51.71 (C-34); 49.88 (C-9); 47.66 (C-17); 46.84 (C-5); 45.91 (C-19); 41.87 (C-4); 41.79
(C-14); 41.35 (C-18); 39.44 (C-8); 38.13 (C-1); 36.94 (C-10); 33.92 (C-21); 33.16 (C-
29); 32.56 (C-7); 32.27 (C-22); 30.74 (C-20); 27.43 (C-15); 26.09 (C-27); 25.94 (C-2);
23.69 (C-30); 23.45 (C-11); 23.09 (C-16); 18.69 (C-6); 17.19 (C-26); 15.68 (C-25);
12.24 (C-24).
4.4. General procedure for the synthesis of azides a-r
The azides a-r were prepared as indicated in Scheme S1 (Supplementary
material) using methods previously published [25, 39].
4.5. General procedure for the synthesis of bis-triazolyl-hederagenin derivatives 2-19)
A 50 mL round-bottomed flask was charged with compound 1 (44 mg; 0.08
mmol), the appropriate azide (0.50 mmol), CuSO4.5H2O (126 mg; 0.50 mmol) and Na-
L-ascorbate (200 mg; 1 mmol). To this mixture were added CH2Cl2 (8 mL) and H2O (8
mL). The reaction mixture was stirred vigorously for 24 h at room temperature, until
TLC analysis revealed a total consumption of the starting material 1. The reaction
mixture was quenched by addition of H2O (20 mL) followed by extraction of the
product with CH2Cl2 (3 x 20 mL). The combined organic extracts were washed with
15

ACCEPTED MANUSCRIPT
brine (50 mL), dried over Na2SO4, filtered, and the solvent was removed in a rotary
evaporator under reduced pressure. The crude product was purified by column
chromatography on silica gel, using gradient elution: hexane/ethyl acetate 4:1 to
hexane/ethyl acetate 1:2 to afford the products 2-19 each as a solid material,
respectively.
4.5.1 (p-Nitrobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3-hydroxyolean-12-en-23-(p
nitrobenzyl)-1H-1,2,3-triazol-4-yl-methyloxy-28-oate (2)
White solid (43 mg; 60 %): Rf = 0.39 (hexane/ethyl acetate 1:1 v/v); m.p: 114.3-
116.1 °C; IR (KBr): ῡmax = 3408, 3154, 3152, 3086, 1720, 1528, 1162, 1058, 1034 807
1
cm-1; H NMR (400 MHz, CDCl3): δ= 8.18 (d, 4H, J = 8.4 Hz, H-37, H-37’, H-45, H-
45’); 7.59 (s, 1H, H-33); 7.55 (s, 1H, H-41); 7.38 (d, 4H, J = 8.4 Hz, H-36, H-36’, H-
44, H-44’); 5.62 (d, 4H, J = 13.6 Hz, H-34, H-42); 5.20 (brs, 1H, H-12); 5.17 (d, 1H, J
= 12.9 Hz, H-31a); 5.12 (d, 1H, J = 12.9 Hz, H-31b); 4.62 (brt, 2H, J = 13.5 Hz, H-39);
3.56 (d, 1H, J = 8.6 Hz, H-23a); 3.50 (m, 1H, H-3); 3.25 (d, 1H, J = 8.6 Hz, H-23b);
2.79 (brd, 1H, J = 8.5 Hz, H-18); 1.02 (s, 3H, CH3); 0.86 (s, 3H, CH3); 0.85 (s, 3H,
CH3); 0.82 (s, 3H, CH3); 0.79 (s, 3H, CH3); 0.45 (s, 3H, CH3); 13C NMR (100 MHz,
CDCl3): δ= 177.78 (C-28); 148.37 (C-38, C-46); 144.20 (C-13); 143.51 (C-32, C-40);
141.76 (C-43); 141.70 (C-35); 128.68 (C-36, C-36’, C-44, C-44’); 124.37 (C-45, C-
45’); 124.34 (C-37, C-37’); 122.62 (C-12); 122.48 (C-33, C-41); 80.02 (C-3); 75.89 (C-
23); 64.91 (C-39); 57.42 (C-31); 53.25 (C-34); 53.11 (C-42); 49.87 (C-9); 47.56 (C-17);
46.81 (C-5); 45.91 (C-19); 41.90 (C-4); 41.77 (C-14); 41.34 (C-18); 39.30 (C-8); 38.10
(C-1); 36.87 (C-10); 33.88 (C-21); 33.07 (C-29); 32.48 (C-7); 32.42 (C-22); 30.70 (C-
20); 27.68 (C-15); 26.03 (C-27); 25.78 (C-2); 23.61 (C-30); 23.35 (C-11); 23.05 (C-16);
18.63 (C-6); 16.82 (C-26); 15.65 (C-25); 12.16 (C-24); EIMS m/z 905.3 [M+H]+; anal.
C 66.03, H 7.27, N 12.00%, calcd for C50H64N8O8, C 66.35, H 7.13, N 12.38%.
16

ACCEPTED MANUSCRIPT
4.5.2 (p-Bromobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3-hydroxyolean-12-en-23-(p
bromobenzyl)-1H-1,2,3-triazol-4-yl-methyloxy-28-oate (3)
White solid (58 mg; 75%): Rf = 0.52 (hexane/ethyl acetate 1:1 v/v); m.p: 102.8-
1
104.3 °C; IR (KBr): ῡmax = 3411, 3153, 3150, 1635, 1041, 1014 cm-1; H NMR (400
MHz, CDCl3): δ= 7.43 (d, 4H, J = 7.9 Hz, H-37, H-37’, H-45, H-45’); 7.46 (s, 1H, H-
33); 7.44 (s, 1H, H-41); 7.11 (d, 4H, J = 8.3 Hz, H-36, H-36’, H-44, H-44’); 5.46 (brs,
2H, H-42); 5.45 (d, 2H, J = 15.0 Hz, H-34a); 5.41 (d, 2H, J = 15.0 Hz H-34b); 5.20 (brs,
1H, H-12); 5.13 (t, 1H, J = 12.9 Hz, H-31); 4.60 (brs, 2H, H-39); 3.57 (d, 1H, J = 8.7
Hz, H-23a); 3.50 (m, 1H, H-3); 3.25 (d, 1H, J = 8.7 Hz, H-23b); 2.79 (dd, 1H, J = 3.8,
13.9 Hz, H-18); 1.04 (s, 3H, CH3); 0.87 (s, 3H, CH3); 0.86 (s, 3H, CH3); 0.85 (s, 3H,
13
CH3); 0.82 (s, 3H, CH3); 0.48 (s, 3H, CH3); C NMR (100 MHz, CDCl3): δ= 177.73
(C-28); 145.87 (C-13); 143.90 (C-40); 143.61 (C-32); 133.68 (C-43); 133.59 (C-35);
132.39 (C-37, C-37’, C-45, C-45’); 129.73 (C-44, C-44’); 129.70 (C-36, C-36’); 123.10
(C-38); 123.06 (C-46); 123.89 (C-12); 122.46 (C-33); 122.23 (C-41); 80.13 (C-3); 75.93
(C-23); 64.96 (C-39); 57.57 (C-31); 53.60 (C-34); 53.50 (C-42); 49.96 (C-9); 47.59 (C-
17); 46.79 (C-5); 45.92 (C-19); 41.90 (C-4); 41.78 (C-14); 41.35 (C-18); 39.32 (C-8);
38.11 (C-1); 36.88 (C-10); 33.90 (C-21); 33.11 (C-29); 32.47 (C-7); 32.39 (C-22); 30.70
(C-20); 27.69 (C-15); 26.03 (C-27); 25.85 (C-2); 23.65 (C-30); 23.36 (C-11); 23.06 (C-
16); 18.67 (C-6); 16.82 (C-26); 15.66 (C-25); 12.21 (C-24); EIMS m/z 995.2 [M+Na]+;
anal. C 61.52, H 6.91, N 8.54%, calcd for C50H64Br2N6O4, C 61.73, H 6.63, N 8.64%.
4.5.3 (p-Chlorobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3-hydroxyolean-12-en-23-(p
chlorobenzyl)-1H-1,2,3-triazol-4-yl-methyloxy-28-oate (4)
White solid (57 mg; 82%): Rf = 0.51 (hexane/ethyl acetate 1:1 v/v); m.p: 112.6-
114.1 °C; IR (KBr): ῡmax = 3403, 3156, 3152, 3061, 1719, 1631, 1523, 1047, 1014, 731
1
cm-1; H NMR (400 MHz, CDCl3): δ= 7.47 (s, 1H, H-33); 7.44 (s, 1H, H-41); 7.31 (d,
17

ACCEPTED MANUSCRIPT
4H, J = 8.3 Hz, H-37, H-37’, H-45, H-45’); 7.17 (d, 4H, J = 8.2 Hz, H-36, H-36’, H-44,
H-44’); 5.44 (m, 4H, J = 15.0 Hz H-34, H-42); 5.20 (brs, 1H, H-12); 5.13 (s, 1H, H-31);
4.59 (brs, 2H, H-39); 3.56 (d, 1H, J = 8.7 Hz, H-23a); 3.50 (m, 1H, H-3); 3.25 (d, 1H, J
= 8.7 Hz, H-23b); 2.78 (dd, 1H, J = 3.1, 13.6 Hz, H-18); 1.04 (s, 3H, CH3); 0.86 (s, 6H,
2xCH3); 0.84 (s, 3H, CH3); 0.81 (s, 3H, CH3); 0.48 (s, 3H, CH3); 13C NMR (100 MHz,
CDCl3): δ= 177.63 (C-28); 145.78 (C-13); 143.78 (C-40); 143.51 (C-32); 134.87 (C-
46); 134.82 (C-38); 133.12 (C-35); 133.00 (C-43); 129.37 (C-45, C-45’); 129.32 (C-36,
C-36’, C-37, C-37’, C-44, C-44’); 123.79 (C-12); 122.51 (C-33); 122.10 (C-41); 79.97
(C-3); 75.80 (C-23); 64.89 (C-39); 57.48 (C-31); 53.41 (C-34); 53.34 (C-42); 49.84 (C-
9); 47.50 (C-17); 46.69 (C-5); 45.83 (C-19); 41.81 (C-4); 41.68 (C-14); 41.26 (C-18);
39.22 (C-8); 38.01 (C-1); 36.78 (C-10); 33.80 (C-21); 33.01 (C-29); 32.37 (C-7); 32.30
(C-22); 30.62 (C-20); 27.59 (C-15); 25.93 (C-27); 25.73 (C-2); 23.55 (C-30); 23.27 (C-
11); 22.96 (C-16); 18.56 (C-6); 16.71 (C-26); 15.55 (C-25); 12.11 (C-24); EIMS m/z
905.2 [M+Na]+; anal. C 67.69, H 7.58, N 9.39%, calcd for C50H64Cl2N6O4, C 67.93, H
7.30, N 9.51%.
4.5.4 (o-Fluorobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3-hydroxyolean-12-en-23-(o
fluorobenzyl)-1H-1,2,3-triazol-4-yl-methyloxy-28-oate (5)
White solid (45 mg; 66%): Rf = 0.48 (hexane/ethyl acetate 1:1 v/v); m.p: 84.6-
85.8 °C; IR (KBr): ῡmax = 3404, 3152, 3150, 3052, 1637, 1524, 1233, 1048, 1005, 756
1
cm-1; H NMR (400 MHz, CDCl3): δ= 7.54 (s, 1H, H-33); 7.52 (s, 1H, H-43); 7.32 (m,
2H, H-40, H-50); 7.23 (m 2H, H-38, H-48); 7.10 (m 4H, H-37, H-39, H-47, H-49); 5.56
(brs, 2H, H-34); 5.53 (brs, 2H, H-44); 5.20 (t, 1H, J = 3.0 Hz, H-12); 5.12 (brs, 1H, H-
31); 4.59 (brs, 2H, H-41); 3.56 (d, 1H, J = 8.7 Hz, H-23a); 3.50 (m, 1H, H-3); 3.25 (d,
1H, J = 8.7 Hz, H-23b); 2.79 (dd, 1H, J = 3.8, 13.8 Hz, H-18); 1.05 (s, 3H, CH3); 0.86
(s, 9H, 3xCH3); 0.81 (s, 3H, CH3); 0.49 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3): δ=
18

ACCEPTED MANUSCRIPT
177.69 (C-28); 160.65 (d, J = 246.7 Hz, C-36); 160.62 (d, J = 246.3 Hz, C-46); 145.59
(C-13); 143.70 (C-32); 143.62 (C-42); 131.10 (d, J = 3.5 Hz, C-40); 131.02 (d, J = 3.6
Hz, C-50); 130.70 (d, J = 3.0 Hz, C-38); 130.61 (d, J = 12.6 Hz, C-48); 124.94 (d, J =
2.0 Hz, C-35); 124.91 (d, J = 2.1 Hz, C-45); 124.02 (d, J = 1.0 Hz, C-49); 122.48 (C-
12); 122.01 (C-33); 121.88 (d, J = 1.5 Hz, C-39); 121.74 (C-43); 115.94 (d, J = 21.0
Hz, C-37, C-47); 80.16 (C-3); 75.96 (C-23); 64.92 (C-41); 57.58 (C-31); 49.99 (C-9);
47.84 (d, J = 4.2 Hz, C-34); 47.74 (d, J = 4.2 Hz, C-44); 47.60 (C-17); 46.78 (C-5);
45.94 (C-19); 41.88 (C-4); 41.79 (C-14); 41.37 (C-18); 39.33 (C-8); 38.12 (C-1); 36.90
(C-10); 33.91 (C-21); 33.13 (C-29); 32.45 (C-7); 32.39 (C-22); 30.72 (C-20); 27.68 (C-
15); 26.00 (C-27); 25.86 (C-2); 23.66 (C-30); 23.39 (C-11); 23.05 (C-16); 18.69 (C-6);
16.76 (C-26); 15.66 (C-25); 12.19 (C-24); EIMS m/z 873.6 [M+Na]+; anal. C 70.31, H
7.72, N 9.72%, calcd for C50H64F2N6O4, C 70.56, H 7.58, N 9.87%.
4.5.5 (o-Chlorobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3-hydroxyolean-12-en-23-(o
chlorobenzyl)-1H-1,2,3-triazol-4-yl-methyloxy-28-oate (6)
White solid (29 mg; 41%): Rf = 0.51 (hexane/ethyl acetate 1:1 v/v); m.p: 94.9-
96.3 °C; IR (KBr): ῡmax = 3400, 3153, 3150, 3072, 1723, 1636, 1520, 1048, 751, 735
cm-1; 1H NMR (400 MHz, CDCl3): δ= 7.57 (s, 1H, H-33); 7.55 (s, 1H, H-43); 7.43 (brd,
2H, J = 7.8 Hz, H-37, H-47); 7.32 (brt, 2H, J = 7.8 Hz, H-40, H-50); 7.26 (m, 2H, H-
38, H-48); 7.19 (brt, 2H, J = 7.5 Hz, H-39, H-49); 5.66 (brs, 2H, H-44); 5.65 (d, 1H, J
= 15.1 Hz, H-34a); 5.61 (d, 1H, J = 15.1 Hz, H-34b); 5.23 (brs, 1H, H-12); 5.18 (s, 1H,
J = 13.1 Hz, H-31a); 5.14 (s, 1H, J = 13.1 Hz, H-31b); 4.63 (brs, 2H, H-41); 3.59 (d,
1H, J = 8.7 Hz, H-23a); 3.53 (m, 1H, H-3); 3.26 (d, 1H, J = 8.7 Hz, H-23b); 2.81 (brdd,
1H, J = 3.5, 13.9 Hz, H-18); 1.08 (s, 3H, CH3); 0.89 (s, 9H, 3xCH3); 0.84 (s, 3H, CH3);
13
0.53 (s, 3H, CH3); C NMR (100 MHz, CDCl3): δ= 177.71 (C-28); 145.50 (C-13);
143.65 (C-32); 143.65 (C-42); 133.69 (C-35); 133.59 (C-45); 132.47 (C-36); 132.35 (C-
19

ACCEPTED MANUSCRIPT
46); 130.55 (C-37); 130.42 (C-40, C-47); 130.40 (C-50); 130.07 (C-38); 130.05 (C-48);
127.71 (C-39); 127.68 (C-49); 124.18 (C-12); 122.68 (C-33); 122.49 (C-43); 80.12 (C-
3); 75.98 (C-23); 64.90 (C-41); 57.61 (C-31); 51.60 (C-34); 51.60 (C-44); 50.01 (C-9);
47.62 (C-17); 46.80 (C-5); 45.94 (C-19); 41.90 (C-4); 41.80 (C-14); 41.38 (C-18); 39.34
(C-8); 38.14 (C-1); 36.91 (C-10); 33.93 (C-21); 33.14 (C-29); 32.47 (C-7); 32.40 (C-
22); 30.73 (C-20); 27.70 (C-15); 26.02 (C-27); 25.88 (C-2); 23.68 (C-30); 23.41 (C-11);
23.05 (C-16); 18.71 (C-6); 16.83 (C-26); 15.67 (C-25); 12.20 (C-24); EIMS m/z 905.3
[M+Na]+; anal. C 67.81, H 7.51, N 9.30%, calcd for C50H64Cl2N6O4, C 67.93, H 7.30, N
9.51%.
4.5.6 (p-Methylbenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3-hydroxyolean-12-en-23-(p
methylbenzyl)-1H-1,2,3-triazol-4-yl-methyloxy-28-oate (7)
White solid (27 mg; 40%): Rf = 0.51 (hexane/ethyl acetate 1:1 v/v); m.p: 126.9-
128.3 °C; IR (KBr): ῡmax = 3406, 3155, 3152, 3067, 1729, 1626, 1530, 1046, 749 cm-1;
1H NMR (400 MHz, CDCl3): δ= 7.46 (s, 1H, H-33); 7.43 (s, 1H, H-44); 7.17 (s, 8H, H-
36, H-36’, H-37, H-37’, H-45, H-45’, H-46, H-46’); 5.46 (brs, 2H, H-43); 5.45 (m, 2H,
H-34); 5.22 (brs, 1H, H-12); 5.14 (brs, 2H, J = 13.1 Hz, H-31); 4.62 (brs, 2H, H-40);
3.59 (d, 1H, J = 8.6 Hz, H-23a); 3.52 (m, 1H, H-3); 3.28 (d, 1H, J = 8.7 Hz, H-23b);
2.81 (brdd, 1H, J = 3.2, 13.6 Hz, H-18); 2.35 (s, 6H, 2xCH3); 1.08 (s, 3H, CH3); 0.89 (s,
13
9H, 3xCH3); 0.85 (s, 3H, CH3); 0.53 (s, 3H, CH3); C NMR (100 MHz, CDCl3): δ=
177.74 (C-28); 145.49 (C-13); 143.67 (C-32); 143.61 (C-41); 138.87 (C-38); 138.85 (C-
47); 131.56 (C-35); 131.46 (C-44); 129.92 (C-36, C-36’); 129.91 (C-45, C-45’); 128.26
(C-37, C-37’); 128.21 (C-46, C-46’); 123.78 (C-12); 122.48 (C-33); 122.20 (C-42);
80.39 (C-3); 76.10 (C-23); 64.97 (C-40); 57.65 (C-31); 54.20 (C-34); 54.09 (C-43);
50.09 (C-9); 47.63 (C-17); 46,80 (CH-5); 45,94 (CH-19); 41,90 (C-4); 41,80 (C-14);
41,38 (CH-18); 39,34 (C-8); 38,14 (CH2-1); 36,92 (C-10); 33,94 (CH2-21); 33,16 (CH3-
20

ACCEPTED MANUSCRIPT
29); 32,49 (CH2-7); 32,40 (CH2-22); 30,75 (C-20); 27,72 (CH2-15); 26,00 (CH3-27);
25,90 (CH2-2); 23,69 (CH3-30); 23,40 (CH2-11); 23,06 (CH2-16); 21.27 (C-38, C-48);
18,72 (CH2-6); 16,84 (CH3-26); 15,66 (CH3-25); 12,21 (CH3-24); EIMS m/z 843.4
[M+H]+; anal. C 73.88, H 8.35, N 9.65%, calcd for C52H70N6O4, C 74.07, H 8.37, N
9.97%.
4.5.7 (o-Bromobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3-hydroxyolean-12-en-23-(o
bromobenzyl)-1H-1,2,3-triazol-4-yl-methyloxy-28-oate (8)
White solid (36 mg; 47%): Rf = 0.50 (hexane/ethyl acetate 1:1 v/v); m.p: 97.8-
98.9 °C; IR (KBr): ῡmax = 3386, 3153, 3150, 3070, 1720, 1639, 1522, 1047, 1033, 743,
731 cm-1; 1H NMR (400 MHz, CDCl3): δ= 7.56 (m, 4H, H-33, H-37, H-43, H-47); 7.26
(brt, 2H, J = 6.8 Hz, H-39, H-49); 7.18 (brt, 2H, J = 7.4 Hz, H-40, H-50); 7.14 (m, 2H,
H-38, H-48); 7.26 (m, 2H, H-38, H-48); 7.19 (brt, 2H, J = 7.5 Hz, H-39, H-49); 5.66
(brs, 2H, H-44); 5.60 (m, 4H, H-34, H-44); 5.18 (brs, 1H, H-12); 5.12 (brs, 2H, H-31);
4.59 (brs, 2H, H-41); 3.52 (m, 2H, H-3, H-23a); 3.22 (d, 1H, J = 8.3 Hz, H-23b); 2.77
(brd, 1H, J = 12.6 Hz, H-18); 1.03 (s, 3H, CH3); 0.84 (s, 9H, 3xCH3); 0.78 (s, 3H, CH3);
13
0.48 (s, 3H, CH3); C NMR (100 MHz, CDCl3): δ= 177.52 (C-28); 145.34 (C-13);
143.46 (C-32); 143.41 (C-42); 134.06 (C-35); 133.88 (C-45); 133.20 (C-37); 133.16 (C-
47); 130.45 (C-40); 130.39 (C-38, C-50); 130.22 (C-48); 128.18 (C-39); 128.16 (C-49);
123.50 (C-36); 123.36 (C-46); 124.13 (C-12); 122.65 (C-33); 122.31 (C-43); 79.52 (C-
3); 75.55 (C-23); 64.70 (C-41); 57.45 (C-31); 53.79 (C-34); 53.74 (C-44); 49.67 (C-9);
47.42 (C-17); 46.60 (C-5); 45.75 (C-19); 41.73 (C-4); 41.61 (C-14); 41.19 (C-18); 39.15
(C-8); 37.96 (C-1); 36.72 (C-10); 33.74 (C-21); 33.01 (C-29); 32.28 (C-7); 32.24 (C-
22); 30.58 (C-20); 27.53 (C-15); 25.86 (C-27); 25.74 (C-2); 23.54 (C-30); 23.25 (C-11);
22.87 (C-16); 18.51 (C-6); 16.67 (C-26); 15.55 (C-25); 12.12 (C-24); EIMS m/z 995.3
21

ACCEPTED MANUSCRIPT
[M+Na]+; anal. C 61.70, H 6.74, N 8.44%, calcd for C50H64Br2N6O4, C 61.73, H 6.63, N
8.64%.
4.5.8 (o-Nitrobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3-hydroxyolean-12-en-23-(o
nitrobenzyl)-1H-1,2,3-triazol-4-yl-methyloxy-28-oate (9)
White solid (59 mg; 81%): Rf = 0.39 (hexane/ethyl acetate 1:1 v/v); m.p: 94.8-
96.4 °C; IR (KBr): ῡmax = 3046, 3158, 3155, 3064, 1718, 1528, 1162, 1048, 1006, 729
cm-1; 1H NMR (400 MHz, CDCl3): δ= 8.12 (d, 2H, J = 7.2 Hz H-37, H-47); 7.72 (s, 1H,
H-33); 7.70 (brs, 1H, H-43); 7.59 (brt, 2H, J = 7.3 Hz, H-38, H-48); 7.52 (brt, 2H, J =
7.2 Hz H-39, H-49); 7.05 (brt, 2H, J = 6.3 Hz, H-40, H-50); 5.91 (d, 2H, J = 7.4 Hz, H-
34, H-44); 5.23 (brs, 1H, H-12); 5.17 (brs, 2H, H-31); 4.64 (brs, 2H, H-41); 3.59 (d, 1H,
J = 8.3 Hz, H-23a); 3.53 (brt, 1H, J = 8.0 Hz, H-3); 3.28 (d, 1H, J = 8.2 Hz, H-23b);
2.81 (brd, 1H, J = 12.6 Hz, H-18); 1.06 (s, 3H, CH3); 0.87 (s, 9H, 3xCH3); 0.82 (s, 3H,
13
CH3); 0.53 (s, 3H, CH3); C NMR (100 MHz, CDCl3): δ= 177.50 (C-28); 147.47 (C-
36); 147.47 (C-46); 145.68 (C-13); 143.76 (C-32); 143.49 (C-42); 134.40 (C-35);
134.32 (C-45); 130.63 (C-39); 130.48 (C-49); 130.37 (C-40); 130.29 (C-50); 129.75 (C-
38); 129.71 (C-48); 125.41 (C-37); 125.38 (C-47); 124.94 (C-12); 123.46 (C-33);
122.43 (C-43); 79.71 (C-3); 75.67 (C-23); 64.75 (C-41); 57.46 (C-31); 50.88 (C-34);
50.80 (C-44); 49.75 (C-9); 47.50 (C-17); 46.70 (C-5); 45.80 (C-19); 41.83 (C-4); 41.68
(C-14); 41.27 (C-18); 39.23 (C-8); 38.01 (C-1); 36.80 (C-10); 33.81 (C-21); 33.05 (C-
29); 32.36 (C-7); 32.32 (C-22); 30.64 (C-20); 27.60 (C-15); 25.96 (C-27); 25.77 (C-2);
23.59 (C-30); 23.30 (C-11); 22.94 (C-16); 18.57 (C-6); 16.77 (C-26); 15.58 (C-25);
12.15 (C-24); EIMS m/z 905.4 [M+H]+; anal. C 66.14, H 12.60, N 12.11%, calcd for
C50H64N8O8, C 66.35, H 7.13, N 12.38%.
22

ACCEPTED MANUSCRIPT
4.5.9 (m-Chlorobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3-hydroxyolean-12-en-23-(m
chlorobenzyl)-1H-1,2,3-triazol-4-yl-methyloxy-28-oate (10)
White solid (34 mg; 48%): Rf = 0.48 (hexane/ethyl acetate 1:1 v/v); m.p: 122.4-
123.9 °C; IR (KBr): ῡmax = 3401, 3155, 3150, 3088, 1633, 1520, 1048, 774, 733 cm-1;
1H NMR (400 MHz, CDCl3): δ= 7.51 (s, 1H, H-33); 7.47 (s, 1H, H-43); 7.30 (m, 6H, H-
36, H-38, H-39, H-46, H-48, H-49); 7.11 (brd, 2H, J = 6.7 Hz, H-40, H-50); 5.50 (brs,
2H, H-34); 5.47 (d, 2H, J = 5.6 Hz, H-44); 5.22 (brs, 1H, H-12); 5.15 (s, 2H, H-31);
4.63 (brs, 2H, H-41); 3.59 (d, 1H, J = 8.6 Hz, H-23a); 3.52 (m, 1H, H-3); 3.27 (d, 1H, J
= 8.7 Hz, H-23b); 2.81 (brdd, 1H, J = 2.9, 13.6 Hz, H-18); 1.06 (s, 3H, CH3); 0.88 (s,
13
9H, 3xCH3); 0.84 (s, 3H, CH3); 0.51 (s, 3H, CH3); C NMR (100 MHz, CDCl3): δ=
177.80 (C-28); 145.98 (C-13); 143.98 (C-32); 143.65 (C-42); 136.68 (C-35); 136.53 (C-
45); 135.20 (C-37); 135.19 (C-47); 130.56 (C-39); 130.53 (C-49); 129.18 (C-36);
129.14 (C-46); 128.22 (C-40); 128.12 (C-50); 126.18 (C-38); 126.12 (C-48); 124.05 (C-
12); 122.51 (C-33); 122.31 (C-43); 80.28 (C-3); 76.12 (C-23); 65.04 (C-41); 57.61 (C-
31); 53.59 (C-34); 53.54 (C-44); 50.06 (C-9); 47.63 (C-17); 46.84 (C-5); 45.97 (C-19);
41.92 (C-4); 41.83 (C-14); 41.44 (C-18); 39.37 (C-8); 38.14 (C-1); 36.94 (C-10); 33.94
(C-21); 33.16 (C-29); 32.52 (C-7); 32.43 (C-22); 30.76 (C-20); 27.73 (C-15); 26.05 (C-
27); 25.88 (C-2); 23.70 (C-30); 23.41 (C-11); 23.09 (C-16); 18.74 (C-6); 16.84 (C-26);
15.71 (C-25); 12.22 (C-24); EIMS m/z 905.3 [M+Na]+; anal. C 67.75, H 9.74, N 9.36%,
calcd for C50H64Cl2N6O4, C 67.93, H 7.30, N 9.51%.
4.5.10 (m-Nitrobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3-hydroxyolean-12-en-23-(m
nitrobenzyl)-1H-1,2,3-triazol-4-yl-methyloxy-28-oate (11)
White solid (46 mg; 63%): Rf = 0.37 (hexane/ethyl acetate 1:1 v/v); m.p: 120.9-
122.3 °C; IR (KBr): ῡmax = 3404, 3156, 3153, 3092, 1719, 1534, 1174, 1158, 1048, 729
1
cm-1; H NMR (400 MHz, CDCl3): δ= 8.20 (d, 2H, J = 7.4 Hz, H-38, H-48); 8.14 (d,
23

ACCEPTED MANUSCRIPT
2H, J = 4.8 Hz, H-36, H-46); 7.58 (m, 6H, H-33, H-39, H-40, H-42, H-49, H-50); 5.62
(m, 4H, H-34, H-44); 5.21 (brs, 1H, H-12); 5.16 (s, 2H, H-31); 4.64 (brs, 2H, H-41);
3.58 (d, 1H, J = 8.6 Hz, H-23a); 3.52 (m, 1H, H-3); 3.26 (d, 1H, J = 8.7 Hz, H-23b);
2.80 (brdd, 1H, J = 3.0, 13.7 Hz, H-18); 1.04 (s, 3H, CH3); 0.87 (s, 3H, CH3); 0.86 (s,
3H, CH3); 0.84 (s, 3H, CH3); 0.81 (s, 3H, CH3); 0.48 (s, 3H, CH3); 13C NMR (100 MHz,
CDCl3): δ= 177.76 (C-28); 148.65 (C-37, C47); 146.17 (C-13); 144.14 (C-32); 143.53
(C-42); 136.87 (C-35); 136.73 (C-45); 133.94 (C-40); 133.90 (C-50); 130.38 (C-49);
130.35 (C-49); 124.25 (C-12); 123.84 (C-36); 123.81 (C-46); 122.91 (C-38); 122.83 (C-
48); 122.48 (C-33, C-43); 79.89 (C-3); 75.84 (C-23); 65.91 (C-41); 57.47 (C-31); 53.17
(C-34); 53.11 (C-44); 49.83 (C-9); 47.53 (C-17); 46.78 (C-5); 45.88 (C-19); 41.89 (C-
4); 41.75 (C-14); 41.34 (C-18); 39.29 (C-8); 38.07 (C-1); 36.85 (C-10); 33.85 (C-21);
33.07 (C-29); 32.45 (C-7); 32.38 (C-22); 30.63 (C-20); 27.66 (C-15); 26.02 (C-27);
25.79 (C-2); 23.61 (C-30); 23.34 (C-11); 23.01 (C-16); 18.61 (C-6); 16.78 (C-26); 15.64
(C-25); 12.15 (C-24); EIMS m/z 905.6 [M+H]+; anal. C 66.11, H 7.36, N 12.18%, calcd
for C50H64N8O8, C 66.35, H 7.13, N 12.38%.
4.5.11 (m-Bromobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3-hydroxyolean-12-en-23-(m
bromobenzyl)-1H-1,2,3-triazol-4-yl-methyloxy-28-oate (12)
White solid (37 mg; 48%): Rf = 0.52 (hexane/ethyl acetate 1:1 v/v); m.p: 166.1-
167.3 °C; IR (KBr): ῡmax = 3394, 3151, 3148, 3061, 1730, 1639, 1519, 1241, 1048, 772,
1
732 cm-1; H NMR (400 MHz, CDCl3): δ= 7.50 (s, 1H, H-33); 7.46 (brs, 2H, H-38, H-
48); 7.44 (brs, 1H, H-43); 7.38 (brs, 2H, H-36, H-46); 7.21 (brt, 2H, J = 7.8 Hz, H-40,
H-50); 7.15 (brd, 2H, J = 7.6 Hz, H-39, H-49); 5.47 (brs, 2H, H-44); 5.42 (m, 2H, J =
5.6 Hz, H-34); 5.19 (brs, 1H, H-12); 5.12 (s, 2H, H-31); 4.60 (brs, 2H, H-41); 3.56 (d,
1H, J = 8.6 Hz, H-23a); 3.50 (m, 1H, H-3); 3.24 (d, 1H, J = 8.6 Hz, H-23b); 2.78 (brdd,
1H, J = 2.2, 13.6 Hz, H-18); 1.03 (s, 3H, CH3); 0.85 (s, 9H, 3xCH3); 0.81 (s, 3H, CH3);
24

ACCEPTED MANUSCRIPT
13
0.48 (s, 3H, CH3); C NMR (100 MHz, CDCl3): δ= 177.72 (C-28); 145.92 (C-13);
143.91 (C-32); 143.58 (C-42); 136.90 (C-35); 136.74 (C-45); 132.05 (C-38); 132.00 (C-
48); 131.06 (C-36); 130.96 (C-46); 130.75 (C-39); 130.73 (C-49); 126.61 (C-40);
126.55 (C-50); 124.00 (C-12); 123.19 (C-37, C-47); 122.45 (C-33); 122.30 (C-43);
80.00 (C-3); 75.92 (C-23); 64.96 (C-41); 57.55 (C-31); 53.44 (C-34); 53.39 (C-44);
49.91 (C-9); 47.57 (C-17); 46.77 (C-5); 45.91 (C-19); 41.88 (C-4); 41.77 (C-14); 41.35
(C-18); 39.31 (C-8); 38.09 (C-1); 36.87 (C-10); 33.88 (C-21); 33.11 (C-29); 32.44 (C-
7); 32.37 (C-22); 30.70 (C-20); 27.68 (C-15); 26.02 (C-27); 25.83 (C-2); 23.67 (C-30);
23.36 (C-11); 23.04 (C-16); 18.68 (C-6); 16.78 (C-26); 15.68 (C-25); 12.20 (C-24);
EIMS m/z 995.2 [M+Na]+; anal. C 61.52, H 6.81, N 8.53%, calcd for C50H64Br2N6O4, C
61.73, H 6.63, N 8.64%.
4.5.12 (m-Methylbenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3-hydroxyolean-12-en-23-
(m-methylbenzyl)-1H-1,2,3-triazol-4-yl-methyloxy-28-oate (13)
White solid (28 mg; 41%): Rf = 0.56 (hexane/ethyl acetate 1:1 v/v); m.p: 101.7-
103.3 °C; IR (KBr): ῡmax = 3406, 3153, 3150, 3067, 1730, 1626, 1534, 1047, 750 cm-1;
1H NMR (400 MHz, CDCl3): δ= 7.48 (s, 1H, H-33); 7.44 (brs, 1H, H-44); 7.26 (m, 2H,
H-39, H-50); 7.17 (brs, 1H, H-36); 7.15 (brs, 1H, H-47); 7.06 (m, 4H, H-38, H-40, H-
49, H-51); 5.49 (d, 1H, J = 14.7 Hz, H-34a); 5.48 (brs, 2H, H-45); 5.43 (d, 1H, J = 14.7
Hz, H-34b); 5.22 (t, 1H, J = 3.2 Hz, H-12); 5.15 (s, 2H, H-31); 4.62 (brs, 2H, H-42);
3.59 (d, 1H, J = 8.7 Hz, H-23a); 3.52 (m, 1H, H-3); 3.28 (d, 1H, J = 8.6 Hz, H-23b);
2.82 (brdd, 1H, J = 3.9, 13,9 Hz, H-18); 2.34 (s, 6H, 2xCH3); 1.07 (s, 3H, CH3); 0.89 (s,
13
9H, 3xCH3); 0.85 (s, 3H, CH3); 0.52 (s, 3H, CH3); C NMR (100 MHz, CDCl3): δ=
177.74 (C-28); 145.60 (C-13); 143.66 (C-32); 143.66 (C-43); 136.08 (C-37); 136.05 (C-
48); 134.58 (C-35); 134.43 (C-46); 129.68 (C-36); 129.64 (C-47); 129.11 (C-39, C-50);
128.92 (C-38); 128.83 (C-49); 125.28 (C-40); 125.18 (C-51); 123.89 (C-12); 122.48 (C-
25

ACCEPTED MANUSCRIPT
33); 122.23 (C-44); 80.40 (C-3); 76.16 (C-23); 65.04 (C-42); 57.67 (C-31); 54.31 (C-
34); 54.26 (C-45); 50.11 (C-9); 47.63 (C-17); 46.79 (C-5); 45.96 (C-19); 41.88 (C-4);
41.81 (C-14); 41.39 (C-18); 39.35 (C-8); 38.12 (C-1); 36.92 (C-10); 33.93 (C-21); 33.15
(C-29); 32.48 (C-7); 32.39 (C-22); 30.74 (C-20); 27.71 (C-15); 25.99 (C-27); 25.88 (C-
2); 23.69 (C-30); 23.39 (C-11); 23.06 (C-16); 21.43 (C-41, C-52); 18.73 (C-6); 16.82
(C-26); 15.67 (C-25); 12.20 (C-24); EIMS m/z 843.5 [M+H]+; anal. C 73.83, H 8.43, N
9.75%, calcd for C52H70N6O4, C 74.07, H 8.37, N 9.97%.
4.5.13 (Benzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3-hydroxyolean-12-en-23-(benzyl)-
1H-1,2,3-triazol-4-yl-methyloxy-28-oate (14)
White solid (46 mg; 70%): Rf = 0.61 (hexane/ethyl acetate 1:1 v/v); m.p: 104.9-
106.1 °C; IR (KBr): ῡmax = 3404, 3150, 3147, 3062, 1725, 1636, 1523, 1048, 721 cm-1;
1H NMR (400 MHz, CDCl3): δ= 7.47 (s, 1H, H-33); 7.44 (brs, 1H, H-44); 7.35 (brs,
6H); 7.26 (brs, 4H); 5.51 (brs, 2H, H-44); 5.48 (m, 2H, H-34); 5.21 (brs, 1H, H-12);
5.13 (brs, 2H, H-31); 4.61 (brs, 2H, H-41); 3.58 (d, 1H, J = 8.3 Hz, H-23a); 3.52 (m,
1H, H-3); 3.26 (d, 1H, J = 8.3 Hz, H-23b); 2.80 (dl, 1H, J = 3.9, 13.0 Hz, H-18); 1.06
(s, 3H, CH3); 0.87 (s, 9H, 3xCH3); 0.83 (s, 3H, CH3); 0.50 (s, 3H, CH3); 13C NMR (100
MHz, CDCl3): δ= 177.73 (C-28); 145.65 (C-13); 143.70 (C-32); 143.63 (C-42); 134.68
(C-35); 134.52 (C-45); 122.29 (C-37, C-39, C-47, C-49); 128.92 (C-38); 128.89 (C-48);
128.18 (C-36, C-40); 128.10 (C-46, C-50); 123.89 (CH-12); 122.70 (C-33); 122.44 (C-
43); 80.35 (C-3); 76.11 (C-23); 65.02 (C-41); 57.62 (C-31); 54.30 (C-34); 54.25 (C-44);
50.08 (C-9); 47.61 (C-17); 46.79 (C-5); 45.96 (C-19); 41.88 (C-4); 41.81 (C-14); 41.39
(C-18); 39.35 (C-8); 38.12 (C-1); 36.91 (C-10); 33.92 (C-21); 33.14 (C-29); 32.47 (C-
7); 32.39 (C-22); 30.72 (C-20); 27.70 (C-15); 25.99 (C-27); 25.87 (C-2); 23.68 (C-30);
23.40 (C-11); 23.05 (C-16); 18.72 (C-6); 16.81 (C-26); 15.68 (C-25); 12.19 (C-24);
26

ACCEPTED MANUSCRIPT
EIMS m/z 815.4 [M+H]+; anal. C 73.55, H 10.58, N 10.01%, calcd for C50H66N6O4, C
73.68, H 8.16, N 10.31%.
4.5.14 (2,4-Difluorobenzyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3-hydroxyolean-12-en-
23-(2,4-difluorobenzyl)-1H-1,2,3-triazol-4-yl-methyloxy-28-oate (15)
White solid (32 mg; 45%): Rf = 0.47 (hexane/ethyl acetate 1:1 v/v); m.p: 100.9-
102.1 °C; IR (KBr): ῡmax = 3404, 3151, 3148, 3083, 1639, 1508, 1273, 1048, 1004, 735
1
cm-1; H NMR (400 MHz, CDCl3): δ= 7.57 (s, 1H, H-33); 7.53 (s, 1H, H-41); 7.30 (m,
2H, H-40, H-50); 6.87 (m, 4H, H-37, H-39, H-47, H-49); 5.54 (brs, 2H, H-44); 5.54
(brs, 2H, H-34); 5.23 (brs, 1H, H-12); 5.14 (s, 2H, H-31); 4.61 (brs, 2H, H-39); 3.59 (d,
1H, J = 8.6 Hz, H-23a); 3.53 (m, 1H, H-3); 3.28 (d, 1H, J = 8.6 Hz, H-23b); 2.81 (dd,
1H, J = 3.3, 13.8 Hz, H-18); 1.07 (s, 3H, CH3); 0.88 (s, 9H, 3xCH3); 0.84 (s, 3H, CH3);
13
0.50 (s, 3H, CH3); C NMR (100 MHz, CDCl3): δ= 177.74 (C-28); 163.50 (dd, J =
12.1, 249.1 Hz, C-38, C-48); 157.91 (dd, J = 13.2, 249.1 Hz, C-36, C-46); 145.76 (C-
13); 143.83 (C-32); 143.61 (C-42); 131.73 (m, C-40, C-50); 123.96 (CH-12); 122.49
(C-33); 122.33 (C-43); 118.13 (dd, J = 3.8, 12.6 Hz, C-35); 117.90 (dd, J = 3.9, 10.9
Hz, C-45); 112.26 (dd, J = 3.7, 21.5 Hz, C-39, C-49); 104.40 (t, J = 23.2 Hz, C-37, C-
47); 80.22 (C-3); 75.99 (C-23); 64.97 (C-41); 57.55 (C-31); 49.99 (C-9); 47.61 (C-17);
47.26 (d, J = 3.7 Hz, C-34); 47.17 (d, J = 3.7 Hz, C-44); 46.80 (C-5); 45.93 (C-19);
41.90 (C-4); 41.79 (C-14); 41.37 (C-18); 39.33 (C-8); 38.12 (C-1); 36.90 (C-10); 33.91
(C-21); 33.11 (C-29); 32.48 (C-7); 32.40 (C-22); 30.72 (C-20); 27.68 (C-15); 26.00 (C-
27); 25.84 (C-2); 23.64 (C-30); 23.38 (C-11); 23.05 (C-16); 18.67 (C-6); 16.74 (C-26);
15.63 (C-25); 12.17 (C-24); EIMS m/z 887.3 [M+H]+; anal. C 67.42, H 7.30, N 9.37%,
calcd for C50H62F4N6O4, C 67.70, H 7.04, N 9.47%.
27

ACCEPTED MANUSCRIPT
4.5.15 (Phenyl-oxoethyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3-hydroxyolean-12-en-23-
(phenyl-oxoethyl)-1H-1,2,3-triazol-4-yl-methyloxy-28-oate (16)
White solid (50 mg; 72%): Rf = 0.46 (hexane/ethyl acetate 1:1 v/v); m.p: 90.8-
92.1 °C; IR (KBr): ῡmax = 3404, 3156, 3150, 3062, 1702, 1699, 1694, 1630, 1518,
1
1048, 724 cm-1; H NMR (400 MHz, CDCl3): δ= 7.97 (d, 4H, J = 7.4 Hz H-37, H-41,
H-48, H-52); 7.73 (s, 1H, H-33); 7.69 (brs, 1H, H-44); 7.65 (brt, 2H, J = 7,3 Hz, H-39,
H-50); 7.52 (brt, 4H, J = 7.2 Hz H-38, H-40, H-49, H-51); 5.87 (dl, 2H, J = 18.4 Hz, H-
34a, H-45a); 5.79 (brd, 2H, J = 18.4 Hz, H-34b, H-45b); 5.24 (brs, 1H, H-12); 5.19 (brs,
2H, H-31); 4.66 (brs, 2H, H-42); 3.59 (d, 1H, J = 8.5 Hz, H-23a); 3.54 (brt, 1H, J = 6.0
Hz, H-3); 3.29 (d, 1H, J = 8.3 Hz, H-23b); 2.82 (brd, 1H, J = 12.7 Hz, H-18); 1.08 (s,
13
3H, CH3); 0.86 (s, 9H, 3xCH3); 0.82 (s, 3H, CH3); 0.57 (s, 3H, CH3); C NMR (100
MHz, CDCl3): δ= 190.34 (C-35); 190.08 (C-46); 177.69 (C-28); 145.54 (C-13); 143.63
(C-32); 143.58 (C-43); 134.70 (C-36, C-39, C-47); 133.97 (C-50); 129.25 (C-38, C-40,
C-49, C-51); 128.21 (C-37, C-41, C-48, C-52); 125.81 (C-12); 124.37 (C-33); 122.49
(C-44); 79.93 (C-3); 75.86 (C-23); 64.88 (C-42); 57.58 (C-31); 55.54 (C-34); 55.38 (C-
45); 49.29 (C-9); 47.60 (C-17); 46.76 (C-5); 45.92 (C-19); 41.89 (C-4); 41.78 (C-14);
41.35 (C-18); 39.33 (C-8); 38.06 (C-1); 36.89 (C-10); 33.88 (C-21); 33.14 (C-29); 32.44
(C-7); 32.35 (C-22); 30.71 (C-20); 27.69 (C-15); 26.00 (C-27); 25.90 (C-2); 23.69 (C-
30); 23.38 (C-11); 23.04 (C-16); 18.68 (C-6); 16.87 (C-26); 15.64 (C-25); 12.22 (C-24);
EIMS m/z 893.2 [M+Na]+; anal. C 71.51, H 7.92, N 9.51%, calcd for C52H66N6O6, C
71.70, H 7.64, N 9.65%.
4.5.16 (p-Methylphenyl-oxoethyl)-1H-1,2,3-triazol-4-yl-methyl-(3β)3-hydroxyolean-12-
en-23-(p-methylphenyl-oxoethyl)-1H-1,2,3-triazol-4-yl-methyloxy-28-oate (17)
White solid (45 mg; 62%): R = 0.48 (hexane/ethyl acetate 1:1 v/v); m.p: 99.7-
f
101.5 °C; IR (KBr): ῡmax = 3408, 3153, 3151, 3064, 1706, 1698, 1695, 1633, 1522,
28