Oxidation at C-16 enhances butyrylcholinesterase inhibition in lupane triterpenoids

Article abstract of DOI:10.1016/j.bioorg.2018.05.012

A set of triterpenoids with different grades of oxidation in the lupane skeleton were prepared and evaluated as cholinesterase inhibitors. Allylic oxidation with selenium oxide and Jones's oxidation were employed to obtain mono-, di- and tri-oxolupanes, starting from calenduladiol (1) and lupeol (3). All the derivatives showed a selective inhibition of butyrylcholinesterase over acetylcholinesterase (BChE vs. AChE). A kinetic study proved that compounds 2 and 9, the more potent inhibitors of the series, act as competitive inhibitors. Molecular modeling was used to understand their interaction with BChE, the role of carbonyl at C-16 and the selectivity towards this enzyme over AChE. These results indicate that oxidation at C-16 of the lupane skeleton is a key transformation in order to improve the cholinesterase inhibition of these compounds.

Full text of DOI:10.1016/j.bioorg.2018.05.012

Accepted Manuscript
Oxidation at C-16 enhances butyrylcholinesterase inhibition in lupane triterpe-
noids
María Julia Castro, Victoria Richmond, María Belén Faraoni, Ana Paula Murray
PII:
S0045-2068(18)30197-4
DOI:
https://doi.org/10.1016/j.bioorg.2018.05.012
YBIOO 2365
Reference:
Bioorganic Chemistry
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Accepted Date:
1 March 2018
7 May 2018
14 May 2018
Please cite this article as: M. Julia Castro, V. Richmond, M. Belén Faraoni, A. Paula Murray, Oxidation at C-16
enhances butyrylcholinesterase inhibition in lupane triterpenoids, Bioorganic Chemistry (2018), doi: https://doi.org/
10.1016/j.bioorg.2018.05.012
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Oxidation at C-16 enhances butyrylcholinesterase inhibition in lupane
triterpenoids.
María Julia Castroa, Victoria Richmondb, María Belén Faraonia, Ana Paula Murraya,*
a INQUISUR-CONICET, Departamento de Química, Universidad Nacional del Sur, Av. Alem
1253, B8000CPB Bahía Blanca, Argentina
b UMYMFOR (CONICET-UBA), Departamento de Química Orgánica, Facultad de Ciencias
Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, 1428
Buenos Aires, Argentina
Corresponding author e-mail: apmurray@uns.edu.ar
Abstract
A set of triterpenoids with different grades of oxidation in the lupane skeleton were
prepared and evaluated as cholinesterase inhibitors. Allylic oxidation with selenium oxide and
Jones’s oxidation were employed to obtain mono-, di- and tri-oxolupanes, starting from
calenduladiol (1) and lupeol (3). All the derivatives showed a selective inhibition of
butyrylcholinesterase over acetylcholinesterase (BChE vs. AChE). A kinetic study proved that
compounds 2 and 9, the more potent inhibitors of the series, act as competitive inhibitors.
Molecular modeling was used to understand their interaction with BChE, the role of carbonyl at
C-16 and the selectivity towards this enzyme over AChE. These results indicate that oxidation
at C-16 of the lupane skeleton is a key transformation in order to improve the cholinesterase
inhibition of these compounds.
Keywords: Cholinesterase inhibitors, Lupane derivatives, Triterpenoids, Molecular modeling
1. Introduction
Cholinesterase inhibition is the most accepted therapeutic strategy for the treatment of
Alzheimer’s disease (AD), a progressive neurodegenerative disorder that affects the elderly
population and causes memory impairment and cognitive deficit [1–3]. The inhibition of
acetylcholinesterase (AChE), the enzyme that catalyzes the hydrolysis of the neurotransmitter
1

acetylcholine, can alleviate AD symptoms by improving cholinergic functions in AD patients.
In the healthy brain, the enzyme butyrylcholinesterase (BChE) is also involved in the metabolic
degradation of acetylcholine, although the cholinesterase activity of AChE is much higher than
that of BChE [4, 5]. In AD patients, the AChE/BChE ratio depends on the brain region and the
stage of the disease progression. BChE can compensate AChE activity when its levels are
decreased. Since BChE activity increases as AD progresses, this enzyme may also play an
important role in cholinergic dysfunction, particularly at the later stages of AD [6].
As part of our research program focused on the discovery of new cholinesterase
inhibitors, we recently reported the synthesis of several analogs of calenduladiol (lup-20(29)-en-
3β,16β-diol, 1) and the selective BChE inhibition observed for 3,16-dioxolup-20(29)-en-30-al
(2) (Figure 1) [7]. Taking into account those results, in this work we further investigated the role
of the oxidation of the lupane skeleton in the BChE inhibition. For that purpose, we chose two
natural compounds, lupanes 1 and 3, as starting material and carried out chemical
transformations that provided a set of lupane derivatives with selective oxidation at C-3, C-16,
and/or C-30. All the derivatives were evaluated as cholinesterase inhibitors, and a kinetic study
was performed for the two more potent BChE inhibitors. Also, the key binding interactions
between these compounds and BChE was studied through docking modelization.
Figure 1. Structure of calenduladiol (1), 3,16-dioxolup-20(29)-en-30-al (2) and lupeol (3).
2. Results and discussion
2.1 Chemistry
2

Our previous findings revealed that trioxolupane (2) may provide a useful template for
the development of new lupane derivatives with improved and selective BChE inhibition [7]. A
set of 11 lupane derivatives were prepared by different oxidation strategies in order to obtain
analogs selectively oxidized at the same three positions as on compound 2, that is, positions 3,
16, and 30 of the lupane skeleton. Cholinesterase inhibition was determined for each compound
and the results were analyzed with the aid of molecular modeling.
Taking into account our previous results, we designed a strategy to obtain a set of
compounds with different grades of oxidation in the lupane skeleton, starting from two natural
triterpenes, calenduladiol (1) and lupeol (3), both isolated for this purpose from Chuquiraga
erinacea subsp erinacea (Asteraceae) following a procedure already reported [8]. Allylic
oxidation with selenium oxide and Jones’s oxidation were employed to obtain mono-, di- and
tri-oxolupanes that would allow us to determine which carbonyl group is necessary for enzyme
inhibition. In order to analyze the role of the hydroxyl/carbonyl groups at C-3, C-16, and C-30,
we have carried out the transformations shown in Scheme 1 and Scheme 2.
Initially, lupeol (3) was treated with Jones reagent to obtain lupenone (4) (lup-20(29)-
en-3-one) with excellent yield (93%). Starting also from 3, allylic oxidation at C-30 led us to
α,β-unsatured aldehyde 5 (3β-hydroxy-lup-20(29)-en-30-al). Then, oxidation of compound 5
with Jones reagent rendered 3,30-dioxolupane 6 (3-oxo-lup-20(29)-en-30-al) (Scheme 1).
The alternative route for preparing compound 6 by treating intermediate 4 with
selenium oxide was discarded on the basis of the results reported by Gutierrez-Nicolás et al. in
the allylic oxidation of lupenone, where this reaction also rendered the α,β-unsaturated ketone at
ring A together with the desired oxidation of C-30 [9].
Compounds 4, 5, and 6 were purified by column chromatography and identified by
comparison of their spectroscopic data with those reported in the literature. Ketone 4 has been
found as a natural compound in different plant species, and was also obtained by synthesis and
used in further preparation of other synthetic analogs [10–15]. Compound 6 was identified by
Mutai et al as a secondary metabolite found in Acacia mellifera [11]. Spectroscopic data of 5
were in agreement with those reported by Burns et al [16].
3

Scheme 1. Preparation of compounds 4–6. Reagents and conditions: (a) Jones reagent, acetone;
(b) SeO2, EtOH, reflux.
Following the same strategy but starting from calenduladiol (1), we were able to prepare
compounds 7 (3β,16β-dihydroxy-lup-20(29)-en-30-al) and 2 (3,16-dioxo-lup-20(29)-en-30-al)
(Scheme 2). The oxidation of 1 directly with Jones reagent led us to compound 8 (3,16-dioxo-
lup-20(29)-ene) as the major product, together with the partially oxidized analogs, 9 (3β-
hydroxy-lup-20(29)-en-16-one) and 10 (16β-hydroxy-lup-20(29)-en-3-one), in 42:24:34 ratio,
respectively (Scheme 2). Compounds 8–10 were separated by column chromatography. Then,
allylic oxidation of compound 9 rendered compound 11 (3β-hydroxy-16-oxo-lup-20(29)-en-30-
al, 47.2% yield), while compounds 12 (16β-hydroxy-3-oxo-lup-20(29)-en-30-al, 13.7% yield)
and 13 (16β-hydroxy-3-oxo-lup-1,20(29)-dien-30-al, 18.7 % yield) were obtained from 10 at the
same experimental conditions (Scheme 2).
4

Scheme 2. Preparation of compounds 2, 7–13. Reagents and conditions: (a) Jones reagent,
acetone; (b) SeO2, EtOH, reflux.
The synthesis of 11 and 12 starting from compound 7 was theoretically possible. When
we tried to do so by careful addition of Jones reagent onto a cooled solution of 7, the TLC
showed the presence of three compounds. The chromatographic separation of the reaction crude
rendered starting compound 7 as the major product, together with a mixture of 11 and 12 that
could not be separated. These results prompted us to try the alternative route described
previously (Scheme 2), even though we could expect the formation of some elimination product
in ring A [9]. Taking into account that ketones 9 and 10 were obtained as minor byproducts in
the synthesis of compound 8, reaction conditions were optimized in order to promote the
formation of those products instead of 8. After careful column chromatography we were able to
obtain ketones 9 and 10 in adequate amounts to approach the next step, the allylic oxidation of
each one separately. Thus, we were able to obtain compounds 11 and 12, both with the α,β-
unsatured aldehyde moiety in the side chain, but with selective oxidation of C-3 or C-16. As it
was expected, compound 13 was also obtained, together with compound 12, but their separation
was successfully achieved by column chromatography.
Lupanes 2, 7, and 8 were identified by comparison of their spectroscopic data with those
reported in our previous work [7]. Ketone 10 presented 1H and 13C NMR spectra matching those
5

reported for the natural triterpenoid isolated from Acacia cedilloi (Fabaceae), known as resinone
[17]. Compounds 9, 11, 12, and 13 are, on the other hand, new lupane derivatives and were
identified with the aid of NMR spectroscopy and MS spectrometry. Assignments of all carbon
and relevant proton signals were achieved with the aid of mono- and bi-dimensional NMR
experiments and comparison with NMR data of the known analogs and/or starting triterpenoids
[7, 9, 17, 18].
2.2 Cholinesterase inhibition
The AChE and BChE inhibitory activity of compounds 4–13 was evaluated and
compared to that of natural triterpenoids 1 and 3, and compound 2. AChE and BChE activities
were measured in vitro by the spectrophotometric method developed by Ellman with slight
modifications with tacrine as the reference inhibitor [19].
In a preliminary assay the inhibition percentage at a fixed concentration was determined
for all the derivatives. Compounds 1–13 acted like weak AChE inhibitors with low inhibition
percentages. On the other hand, compounds 2, 8, 9 and 11 showed better BChE inhibition than 1
under the same experimental conditions (Table 1). The concentration required for 50% BChE
inhibition (IC50) was then determined for these compounds. The results for cholinesterase
inhibition are summarized in Table 1.
Table 1. Cholinesterase inhibition of compounds 1-13
BChE
AChE
Compound
% Inhibitiona
% Inhibitiona
IC50 (μM)
1 b
2 b
3
8.1 ± 0.2
21.7 ± 1.2
21.3 ± 2.7
8.8 ± 1.2
5.7 ± 0.4
n.i. c
42.0 ± 0.8
86.5 ± 2.7
31.0 ± 2.2
10.2 ± 1.4
3.2 ± 1.0
28.9 ± 3.1
42.0 ± 4.4
61.4 ± 0.5
> 100
>200
21.5 ± 1.2
>200
4
-
5
-
6
7 b
-
43.5 ± 1.1
6.4 ± 0.3
40.2 ± 2.1
>200
8
154.6 ± 2.3
28.9 ± 2.1
9
6

12.6 ± 1.5
29.7 ± 0.8
n.i. c
43.5 ± 0.9
> 100
35.3
10
>200
11
76.8 ± 0.3
>200
12
n.i. c
44.6 ± 0.6
-
13
tacrine d
>200
-
0.004 ± 0.001
a at 200 μM, b From ref. 7, c n.i. : no inhibition detected, d reference inhibitor.
The best activity was observed for compound 2, with three carbonyl groups in positions
3, 16 and 30, followed by compound 9 (3β-hydroxy-lup-20(29)-en-16-one). All the compounds
bearing a 16-keto group were more effective inhibitors than the 16β-hydroxy analogs (9 vs. 1,
11 vs. 7, 8 vs. 10, 2 vs. 12,) or the unsubstituted lupanes (9 vs. 3, 11 vs. 5, 8 vs. 4, 2 vs. 6,)
(Figure 2).
Figure 2. BChE inhibitors grouped according their substitution at C-16.
2.3 Kinetic characterization of BChE inhibition
Carbonyl compounds 2 and 9, identified as the more effective BChE inhibitors of the
series, were chosen for the determination of the inhibitor type kinetic study. Enzyme activity
was evaluated at different fixed inhibitor concentrations and increasing substrate concentrations
and, the data obtained was used to elucidate the enzyme inhibition mechanism. The results are
illustrated in the form of Lineweaver–Burk plots (Figure 3). The double-reciprocal plots showed
that the inhibitors have not effect on Vmax, but increase Km. The pattern of straight lines with
intersecting y intercepts seen in Figure 3 is the characteristic signature of a competitive
7

inhibitor. Both 2 and 9, interfere with the substrate binding to the enzyme. The analysis of these
data with GraphPad Prism 5 showed a good fit with the competitive type inhibition. Estimated
Ki were 51.16 μM for 9 and 32.70 μM for 2. This kinetic study indicates that both 2 and 9 have
affinity for the active site of the enzyme and compete with the substrate.
A
1600
1400
1200
1000
800
600
400
200
0
-0.02
-0.01
0.00
0.01
0.02
0.03
0.04
0.05
-200
-400
1/[S] ( M-1)
1400
1200
1000
800
600
400
200
0
B
-0.02
-0.01
0.00
-200
0.01
0.02
0.03
0.04
0.05
-400
1/[S] ( M-1)
Figure 3. Lineweaver–Burk plots of the inhibition of BChE by compound 2 (A) and 9 (B) with
butyrylthiocholine (S) as substrate.
8

2.4 Molecular modeling study
In order to understand the selective BChE inhibition of compounds 2 and 9 over AChE
inhibition, molecular docking studies were performed with both cholinesterases to compare
their affinities. Owing to the large volume of the BChE active-site gorge (200 Å3 bigger than
that of the AChE gorge), one or more molecules of water can be placed there in order to interact
with the ligand, improving the interaction with the protein [20]. This difference in the
dimension of the active-site gorge led us to implement a hydrated docking technique with the
enzyme BChE [21].
When studying the binding mode and the interactions between these compounds and
BChE, the best molecular docking conformation showed that compounds 2 and 9 penetrate the
peripheral site through their A ring and bind the enzyme at the active site; both cases exhibit the
triterpenoid buried deep into the gorge next to the residue SER198 (belonging to the catalytic
triad), according to the competitive inhibition mechanism of the BChE that the in vitro
experiments revealed.
The study permitted us to identify hydrophobic interactions inside the gorge as well as
three hydrogen bonding interactions as stabilizing factors in the enzyme-inhibitor complex for
both compounds. The hydrophobic interactions are depicted in Figure 4. Due to both
compounds sharing the same position at the gorge of the enzyme, the figure only represents
these interactions between the BChE and compound 2. Regarding the hydrogen bonding, the
carbonyl group at C-16 of compound 9 interacts with the hydroxyl group of THR 120 (2.30Å)
and the carbonyl group of TRP82 (water mediated 2.05 and 2.27Å). Also, the hydroxyl group at
C-3 in compound 9 is close to the carbonyl group of LEU286 (2.22Å) (Figure 5). Similarly, the
carbonyl group at C-16 of 2 is involved in the same two hydrogen bonds as compound 9, with
the hydroxyl group of THR120 (2.22Å) and TRP82 water mediated (2.05 and 2.12Å). There is
another water-bridged H-bond network between the carbonyl at C-30 and the residue ASN289
(2.05 and 2.40 Å). These interactions are shown in Figure 6. The importance of the carbonyl
group at C-16 present in both compounds should be noted since both lone pairs of the oxygen
9

are involved in a hydrogen bond that strongly dock the compounds at the active site of the
protein.
On the other hand, the molecular docking with the AChE revealed that although both
compounds get into the enzyme in the same way they penetrate the BChE (through their ring A),
both compounds are located at the peripheral aromatic site of the enzyme, and the main
interactions that stabilized these compounds are hydrophobic. Additionally, compound 2
showed only one hydrogen bond between the carbonyl of C-30 and the NH of the amide group
of PHE288.
Figure 4. Hydrophobic interactions between compound 2 and the enzyme butyrylcholinesterase.
Figure 5. Hydrogen bonds between compound 9 and the enzyme BChE.
10

Figure 6. Hydrogen bonds between compound 2 and the enzyme BChE.
According to the results described for both compounds with the two cholinesterases, the
stronger interaction between these triterpenoids and BChE is highlighted. This result clearly
explains the molecular mechanism of the selectivity of these compounds toward the mentioned
enzyme.
The docking studies allowed us to establish the orientation of the inhibitors relative to
the BChE, as well as its conformation when bound to the enzyme. This study permitted us to
identify hydrophobic interactions inside the gorge as well as hydrogen bonding interactions as
the stabilizing factors in the enzyme-inhibitor complex. Moreover, this work allowed us to
understand the molecular basis of the selectivity of these compounds toward the enzyme BChE.
Further molecular dynamics studies using this complex as the starting point are
necessary to check the complex inhibitor-enzyme stability, to determinate if the enzyme
undergoes structural rearrangements, and to verify the distances and angles observed in the
interactions are within a suitable range.
3. Conclusions
Starting from calenduladiol (1) and lupeol (3), two natural triterpenoids, we have
obtained a set of lupane-type compounds with keto groups at C-3, C-16, and/or C-30. Four new
11

semisynthetic triterpenoids were obtained and fully characterized, together with seven known
ones, in order to understand the influence of the carbonyl group in the interaction of these
compounds with AChE and BChE. Compounds 2 and 9 were identified as the most effective
BChE inhibitors. A kinetic study indicated that 2 and 9 are able to bind to the enzyme in a
competitive manner. The experimental results were explained by means of molecular modeling,
which helps to understand the selectivity of these inhibitors towards BChE. Also, this study
allowed us to identify hydrophobic interactions inside the gorge, as well as hydrogen bonding
interactions as the stabilizing factors in the enzyme-inhibitor complex.
These results show that oxidation of C-16 is an efficient way to improve cholinesterase
inhibition in this kind of inhibitor. Since compounds 2 and 9, the most active in the series, can
be further modified in the isopropenyl attached to ring E, they appear to be good candidates to
explore the scope of this scaffold as potential lead compounds in the search of new anti-
Alzheimer’s drugs.
4. Experimental Section
4.1 General
Melting points were determined on a Fisher-Johns apparatus and are uncorrected. NMR
measurements, including COSY, HSQC, HMBC experiments, were carried out on Bruker
ARX300, Bruker Avance 400 and/or Bruker AMK 600 spectrometers. NMR spectra were
recorded in CDCl3. Chemical shifts are given in ppm (δ) with TMS as an internal standard.
Electrospray ionization mass spectra (ESI-MS) were recorded using an Esquire 3000 ion trap
mass spectrometer equipped with a standard ESI/APCI source. UV spectra were recorded on a
JASCO V-630BIO spectrophotometer. Elemental analyses (C, H) were performed with
EXETER ANALYTICAL, INC CE-440 Elemental Analyzer. IR spectra were recorded on a
Perkin-Elmer Paragon 1000 FT-IR spectrometer.
Silica gel 60 (0.2–0.63 mm, Merck) was used for column chromatography. Silica gel 60
(200–425 mesh, Aldrich) was used for flash chromatography. Analytical TLC was performed
12

on Silicagel 60 F254 sheets (0.2 mm thickness, Merck). p-Anisaldehyde-acetic acid spray
reagent and UV light (254 and 366 nm) were used for detection.
All chemicals and solvents were analytical grade and solvents were purified by general
methods before being used. AChE from electric eel (type VI-S), 5,5 -dithiobis(2-nitrobenzoic
acid) (DTNB), acetylthiocholine iodide (ATCI), butyrylthiocholine iodide (BTCI) and tacrine
were purchased from Sigma. BChE (horse serum) was purchased from MP Biomedicals.
Calenduladiol (1) and lupeol (3), used as starting materials for the preparation of compounds 2,
4–13, were extracted from aerial parts of C. erinacea subsp. erinacea as previously described
[8].
All derivatives were rigorously characterized by NMR spectroscopy and mass
spectrometry. The NMR data of derivatives 2, 4–8 and 10 were identical to those previously
reported [7, 11, 12, 17].
Compounds 9, 11–13 are described here for the first time and bidimensional NMR
spectra (COSY, HMBC, HSQC) were used for the unequivocal assignments of all carbons and
representative protons.
4.2 Preparation of 3,16-dioxo-lup-20(29)-en-30-al (2)
To a solution of 7 (50.0 mg, 0.11 mmol) in acetone (3 mL) Jones reagent was added
dropwise at 0 °C until the solution changed remained orange. The reaction was stirred for 30
min and quenched with i-PrOH (2 mL), filtered through Florisil and washed several times with
AcOEt. The solvent was removed and the residue was purified by flash chromatography on
silica gel with hexane/AcOEt (9:1) affording 12.9 mg (26%) of compound 2 as a white
amorphous solid. Spectroscopic and spectrometric data of 2 were in agreement with those
previously reported by our group [7]. 1H NMR (600 MHz, CDCl3)  0.89 (3H, s, H-27), 0.93
(3H, s, H-25), 1.02 (3H, s, H-24), 1.07 (3H, s, H-28), 1.13 (6H, s, H-23, H-26), 2.48 (1H, ddd, J
= 8.6, 15.7, 15.9 Hz, H-19), 2.74 (1H, d, J = 13.8 Hz, H-15a), 5.98 (1H, br s, H-29a), 6.29 (1H,
br s, H-29b), 9.52 (1H, s, H-30); 13C NMR (150 MHz, CDCl3)  217.9 (C-3), 215.4 (C-16),
194.8 (C-30), 156.2 (C-20), 133.3 (C-29), 56.9 (C-17), 54.8 (C-5), 49.2 (C-9, C-18), 47.7 (C-
13

14), 47.4 (C-4), 44.9 (C-15), 41.0 (C-8), 39.6 (C-1), 37.3 (C-13, C-19), 36.9 (C-10), 34.2 (C-2),
33.6 (C-7, C-22), 31.3 (C-12, C-21), 26.8 (C-23), 21.2 (C-11), 21.2 (C-24), 19.6 (C-6), 18.1 (C-
28), 16.3 (C-26), 15.9 (C-25), 15.4 (C-27).
4.3 Preparation of lup-20(29)-en-3-one (4)
Compound 4 was prepared from 3 (50.0 mg, 0.12 mmol) following the same procedure
described for the preparation of 2. The crude material was chromatographed over flash silica gel
with hexane/AcOEt (9:1) to afford 47.4 mg (95%) of compound 4 as a white amorphous solid.
Spectroscopic and spectrometric data of 4 were identical to those reported for lupenone [11, 12].
1H NMR (300 MHz, CDCl3) δ 0.83 (3H, s, H-28), 0.85 (3H, s, H-25), 0.94 (3H, s, H-27), 0.96
(3H, s, H-24), 1.03 (6H, s, H-23, H-26), 1.63-1.25 (27H, m), 2.41 (1H, m, H-19), 4.54 (1H, br s,
H-29b), 4.67 (1H, br s, H-29a); 13C NMR (75 MHz, CDCl3) δ 218.0 (C-3), 150.8 (C-20), 109.5
(C-29), 55.0 (C-5), 49.9 (C-9), 48.3 (C-18), 48.0 (C-19), 47.4 (C-4), 43.1 (C-17), 43.1 (C-14),
40.9 (C-8), 40.1 (C-22), 39.7 (C-13), 38.2 (C-1), 36.9 (C-10), 35.7 (C-16), 34.2 (C-2), 33.7 (C-
7), 29.9 (C-21), 27.5 (C-15), 26.8 (C-23), 25.2 (C-12), 21.6 (C-24), 21.1 (C- 11), 19.8 (C-30),
19.8 (C-6), 18.1 (C-28), 16.2 (C-26), 16.0 (C-25), 15.9 (C-27).
4.4 Preparation of 3β-hydroxy-lup-20(29)-en-30-al (5)
A solution of 3 (40.0 mg, 0.09 mmol) in EtOH (5 mL) was treated with SeO2 (24.2 mg,
0.22 mmol). The reaction mixture was heated under reflux until the disappearance of the starting
material was confirmed by TLC (24 h). Then, the reaction mixture was cooled and EtOH was
removed under reduced pressure. The crude was treated with water (20 mL) and extracted with
CH2Cl2 (3 x 30 mL). The combined organic extracts were dried over MgSO4, filtered, and
concentrated under reduced pressure. The residue was purified by column flash chromatography
on silica gel with hexane/AcOEt (7:3) to afford 18.0 mg (44%) of compound 5 as a white
crystalline solid. Compound 5 showed identical spectroscopic and spectrometric data to those
previously reported [16]. 1H NMR (300 MHz, CDCl3) δ 0.66 (1H, d, J = 9.0 Hz, H-5), 0.75 (3H,
s, H-24), 0.81 (3H, s, H-28), 0.82 (3H, s, H-25), 0.92 (3H, s, H-27), 0.96 (3H, s, H-26), 1.01
14

(3H, s, H-23), 1.30-1.19 (5H, m), 1.38 (4H, br s), 1.52-1.41 (5H, m), 1.55 (3H, br s), 1.70-1.60
(5H, m), 2.15 (1H, t, J = 11.1 Hz, H-21a), 2.75 (1H, ddd, J = 10.9, 10.9, 5.6 Hz H-19), 3.17
(1H, dd, J = 10.6, 5.3 Hz, H-3), 5.90 (1H, br s, H-29b), 6.28 (1H, br s, H-29a), 9.51 (1H, s, H-
30); 13C NMR (75 MHz, CDCl3) δ 195.2 (C-30), 157.4 (C-20), 133.3 (C-29), 79.1 (C-3), 55.4
(C-5), 50.4 (C-9, C-18), 43.4 (C-17), 42.8 (C-14), 40.9 (C-8), 40.1 (C-22), 39.0 (C-4), 38.9 (C-
1), 37.9 (C-13), 37.3 (C-10, C-19), 35.5 (C-16), 34.4 (C-7), 28.1 (C-21, C-23), 27.5 (C-2, C-12),
27.5 (C-15), 21.1 (C-11), 18.5 (C-6), 17.9 (C-28), 16.2 (C-25), 16.1 (C-26), 15.5 (C-24), 14.6
(C-27).
4.5 Preparation of 3-oxo-lup-20(29)-en-30-al (6)
Compound 6 was prepared from 5 (15.0 mg, 0.03 mmol) following the same procedure
described for the preparation of 2. The crude material was chromatographed over flash silica gel
with hexane/AcOEt (9.5:0.5) to afford 7.9 mg (53%) of compound 6 as a white amorphous
solid. Spectroscopic and spectrometric data of 6 were identical to those reported by Mutai et al
[11]. 1H NMR (300 MHz, CDCl3) δ 0.83 (3H, s, H-28), 0.91 (3H, s, H-25), 0.93 (3H, s, H-27),
1.02 (3H, s, H-24), 1.05 (3H, s, H-23), 1.06 (3H, s, H-26), 1.08 (2H, br s), 1.54-1.25 (15H, m),
1.56 (2H, m), 1.76-1.62 (4H, m), 1.87 (1H, ddd, J = 13.2, 7.5, 4.4 Hz, H-1a), 2.50-2.37 (2H, m),
2.76 (1H, ddd, J = 10.9, 10.7, 5.8 Hz, H-19), 5.91 (1H, br s, H-29b), 6.28 (1H, br s, H-29a),
9.51 (1H, s, H-30); 13C NMR (75 MHz, CDCl3) δ 218.2 (C-3), 195.2 (C-30), 157.3 (C-20),
133.3 (C-29), 55.1 (C-5), 49.8 (C-9, C-18), 47.5 (C-4), 43.4 (C-14), 42.9 (C-17), 40.9 (C-8),
40.1 (C-22), 39.8 (C-1), 38.0 (C-13, C-19), 37.0 (C-10), 35.5 (C-16), 34.3 (C-2), 33.7 (C-7),
27.5 (C-12, C-15), 26.7 (C-21, C-23), 21.6 (C-11), 21.2 (C-24), 19.8 (C-6), 17.9 (C-28), 16.0
(C-25), 15.9 (C-26), 14.5 (C-27).
4.7 Preparation of 3β,16β-dihydroxy-lup-20(29)-en-30-al (7)
Compound 7 was prepared from 1 (60.0 mg, 0.14 mmol) following the same procedure
described for the preparation of 5. The crude material was chromatographed over flash silica gel
with hexane/AcOEt (7:3) to afford 59.8 mg (97%) of compound 7 as a white crystalline solid.
15

Spectroscopic and spectrometric data of 7 were in agreement with those previously reported by
our group [7]. 1H NMR (300 MHz, CDCl3) δ 0.74 (3H, s, H-24), 0.79 (3H, s, H-28), 0.81 (3H, s,
H-26), 0.95 (3H, s, H-25), 0.95 (3H, s, H-27), 1.00 (3H, s, H-23), 2.85 (1H, ddd, J = 5.7, 10.5,
10.8 Hz, H-19), 3.15 (1H, dd, J = 5.1, 10.5 Hz, H-3), 3.66 (1H, dd, J = 4.8, 11.1 Hz, H-16), 5.91
(1H, br s, H-29a), 6.27 (1H, br s, H-29b), 9.5 (1H, s, H-30); 13C NMR (75 MHz, CDCl3) δ 195.0
(C-30), 156.4 (C-20), 133.5 (C-29), 79.0 (C-3), 77.0 (C-16), 55.4 (C-5), 49.9 (C-9, C-18), 48.9
(C-17), 44.0 (C-14), 41.0 (C-8), 39.0 (C-4), 38.8 (C-1), 37.8 (C-22), 37.2 (C-10), 37.1 (C-13, C-
19), 37.0 (C-15), 34.3 (C-7), 29.8 (C-21), 28.1 (C-23), 27.4 (C-2), 27.2 (C-12), 21.0 (C-11),
18.4 (C-6), 16.2 (C-26), 16.1 (C-25, C-27), 15.5 (C-24), 11.7 (C-28).
4.8 Preparation of 3,16-dioxo-lup-20(29)-ene (8)
Compound 8 was prepared from 1 (50.0 mg, 0.11 mmol) following the same procedure
described for the preparation of 2. The crude material was chromatographed over flash silica gel
with hexane/AcOEt (9:1) to afford 20.8 mg (42%) of compound 8 as a white amorphous solid.
Spectroscopic and spectrometric data of 8 were in agreement with those previously reported by
our group [7]. 1H NMR (400 MHz, CDCl3)  0.90 (3H, s, H-27), 0.93 (3H, s, H-25), 1.01 (3H, s,
H-24), 1.06 (3H, s, H-23), 1.09 (3H, s, H-28), 1.14 (3H, s, H-26), 1.65 (3H, s, H-30), 2.61 (1H,
ddd, J = 6.3, 10.8, 10.9 Hz, H-19), 2.71 (1H, d, J = 13.6 Hz, H-15a), 4.62 (1H, br s, H-29a),
4.73 (1H, br s, H-29b); 13C NMR (100 MHz, CDCl3)  217.8 (C-3), 215.8 (C16), 148.8 (C-20),
110.8 (C-29), 56.7 (C-17), 54.8 (C-5), 49.4 (C-9), 49.4 (C-18), 48.1 (C-14), 47.4 (C-4, C-19),
44.9 (C-15), 41.0 (C-8), 39.6 (C-1), 37.7 (C-13), 36.9 (C-10), 34.1 (C-2), 33.6 (C-7), 31.2 (C-
22), 28.6 (C-21), 26.8 (C-23), 24.8 (C-12), 21.3 (C-11), 21.1 (C-24), 19.7 (C-6), 19.0 (C-30),
18.1 (C-28), 16.3 (C-26), 16.0 (C-25), 15.4 (C-27).
4.9 Preparation of 3β-hydroxy-lup-20(29)-en-16-one (9) and 16β-hydroxy-lup-20(29)-en-3-one
(10)
Compounds 9 and 10 were prepared from 1 (50.0 mg, 0.11 mmol) following the
procedure described for the preparation of 2, with minor modifications. Jones reagent was added
16

dropwise at 0 °C until the appearance of the ketones 9 and 10 was confirmed by TLC, and there
was no evidence of the presence of diketone 8. The crude material was chromatographed over
flash silica gel with hexane/AcOEt (9.3:0.7) to afford 8.7 mg (17%) of compound 9 and 7.6 mg
(15%) of compound 10 as white amorphous solids.
Compound 9: 1H NMR (300 MHz, CDCl3) δ 0.68 (1H, d, J = 9.5 Hz, H-5), 0.77 (3H, s,
H-24), 0.85 (3H, s, H-25), 0.90 (3H, s, H-27), 0.97 (3H, s, H-23), 1.10 (3H, s, H-28), 1.11 (3H,
s, H-26), 1.44-1.36 (8H, m), 1.61- 1.54 (9H, m), 1.66 (3H, s, H-30), 1.83-1.86 (3H, m), 2.12
(1H, ddd, J = 12.5, 12.0, 4.1 Hz, H-13), 2.61 (1H, ddd, J = 11.3, 10.9, 6.0 Hz, H-19), 2.71 (1H,
d, J = 13.9 Hz, H-15a), 3.19 (1 H, dd, J = 10.9, 5.2 Hz, H-3), 4.63 (1H, br s, H-29b), 4.74 (1H,
br s, H-29a); 13C NMR (75 MHz, CDCl3) δ 216.1 (C-16), 148.9 (C-20), 110.8 (C-29), 79.0 (C-
3), 56.7 (C-5), 55.3 (C-17), 50.1 (C-9), 49.6 (C-18), 48.2 (C-14), 47.5 (C-19), 45.0 (C-15), 41.2
(C-8), 39.0 (C-1), 38.9 (C-4), 37.6 (C- 13), 37.3 (C-10), 34.3 (C-7), 31.3 (C-22), 28.7 (C-21),
28.1 (C-23), 27.5 (C-2), 24.8 (C-12), 20.8 (C-11), 19.1 (C-30), 18.4 (C-6), 18.1 (C-28), 16.6 (C-
26), 16.1 (C-25), 15.5 (C-24), 15.5 (C-27); MS (ESI, positive ions), m/z 441.4 [M+H]+. Anal.
Calcd. for C30H48O2: C 81.76; H 10.98. Found: C 81.81; H 11.03.
Compound 10: 1H NMR (300 MHz, CDCl3) δ 0.80 (3H, s, H-28), 0.93 (3H, s, H-26),
1.00 (3H, s, H-25), 1.02 (3H, s, H-24), 1.07 (6H, s, H-23, H-27), 1.68 (3H, s, H-30), 1.53-1.26
(17H, m), 2.04-1.89 (3H, m), 2.50-2.42 (3H, m), 3.61 (1H, dd, J = 10.6, 4.7 Hz, H-16), 4.60
(1H, br s, H-29b), 4.71 (1H, br s, H-29a); 13C NMR (75 MHz, CDCl3) δ 218.2 (C-3), 150.0 (C-
20), 110.0 (C- 29), 77.2 (C-16), 55.1 (C-5), 49.5 (C-9), 48.8 (C-17), 47.8 (C-18), 47.7 (C- 19),
47.5 (C-4), 44.3 (C-14), 41.0 (C-8), 39.8 (C-1), 37.8 (C-22), 37.5 (C-13), 37.0 (C-10), 37.0 (C-
15), 34.2 (C-2), 33.7 (C-7), 30.0 (C-21), 26.8 (C-23), 24.9 (C-12), 21.5 (C-11), 21.2 (C-24),
19.8 (C-6), 19.5 (C-30), 16.2 (C-25), 16.1 (C-27), 15.9 (C-26), 11.8 (C-28); MS (ESI, positive
ions), m/z 463.4 [M+Na]+. Anal. Calcd. for C30H48O2: C 81.76; H 10.98. Found: C 81.82; H
11.01.
4.10 Preparation of 3β-hydroxy-16-oxo-lup-20(29)-en-30-al (11)
17

Compound 11 was prepared from 9 (14.0 mg, 0.03 mmol) following the same procedure
described for the preparation of 5. The crude material was chromatographed over flash silica gel
with hexane/AcOEt (7:3) to afford 6.8 mg (47%) of compound 11 as a white amorphous solid.
1H NMR (300 MHz, CDCl3) δ 0.66 (1H, d, J = 10,2 Hz, H-5), 0.76 (3H, s, H-24), 0.83 (3H, s,
H-25), 0.88 (3H, s, H-27), 0.97 (3H, s, H-23), 1.09 (3H, s, H-28), 1.13 (3H, s, H-26), 1.26 (3H,
s), 1.44-1.34 (4H, m), 1.73-1.45 (8H, m), 1.82 (1H, d, J = 14.4 Hz, H-15b), 2.06-1.87 (2H, m),
2.17- 2.08 (2H, m), 2.73 (1H, d, J = 13.8 Hz, H-15a), 2.99 (1H, ddd, J = 10.9, 10.8, 6.0 Hz, H-
19), 3.17 (1H, dd, J = 10.6, 4.9 Hz, H-3), 5.97 (1H, br s, H-29b), 6.28 (1H, br s, H-29a), 9.52
(1H, s, H-30); 13C NMR (75 MHz, CDCl3) δ 215.5 (C-16), 194.7 (C-30), 156.5 (C-20), 132.4
(C-29), 79.0 (C-3), 56.9 (C-5), 55.3 (C-17), 49.9 (C-9, C-18), 47.8 (C-14), 45.0 (C-15), 41.1 (C-
8), 39.0 (C-1), 38.8 (C-4), 37.3 (C-13), 37.2 (C-10, C-19), 34.3 (C-7), 31.3 (C-22), 29.8 (C-21),
28.1 (C-23), 27.5 (C-2), 27.1 (C-12), 20.8 (C-11), 18.3 (C- 6), 18.0 (C-28), 16.5 (C-26), 16.1
(C-25), 15.5 (C-24), 15.4 (C-27); MS (ESI, positive ions), m/z 477.4 [M+Na]+. Anal. Calcd. for
C30H46O3: C 79.25; H 10.20. Found: C 79.28; H 10.23.
4.11 Preparation of 16β-hydroxy-3-oxo-lup-20(29)-en-30-al (12) and 16β-hydroxy-3-oxo-lup-
1,20(29)-dien-30-al (13)
Compound 12 and 13 were prepared from 10 (7.8 mg, 0.02 mmol) following the same
procedure described for the preparation of 5. The crude material was chromatographed over
flash silica gel with hexane/AcOEt (8.5:1.5/8.3:1.7) to afford 1.1 mg (14%) of compound 12
and 1.5 mg (19%) of compound 13.
Compound 12: 1H NMR (300 MHz, CDCl3) δ 0.83 (3H, s, H-28), 0.92 (3H, s, H-26),
0.98 (3H, s, H-25), 1.02 (3H, s, H-27), 1.06 (3H, s, H-24), 1.07 (3H, s, H-23), 1.74-1.26 (20H,
m), 2.47-2.40 (2H, m, H-2), 2.86 (1H, ddd, J = 10.8, 10.5, 5.7 Hz, H-19), 3.69 (1H, dd, J = 11.2,
4.8 Hz, H-16), 5.93 (1H, br s, H-29b), 6.29 (1H, br s, H-29a), 9.52 (1H, s, H-30); 13C NMR (75
MHz, CDCl3) δ 217.9 (C-3), 195.0 (C-30), 156.9 (C-20), 133.0 (C-29), 76.9 (C-16), 53.5 (C-5),
49.2 (C-9, C-17, C-18), 44.8 (C-4), 44.1 (C-14), 41.9 (C-8), 39.7 (C-1), 37.9 (C-22), 37.1 (C-13,
C-19), 37.0 (C-10), 36.9 (C-15), 34.2 (C-2), 33.6 (C-7), 31.7 (C-21), 27.1 (C-12), 26.7 (C-23),
18

21.3 (C-11), 21.1 (C-24), 19.7 (C-6), 16.0 (C-25), 15.8 (C-27), 15.8 (C-26), 11.7 (C-28); MS
(ESI, positive ions), m/z 455.4 [M+H]+. Anal. Calcd. for C30H46O3: C 79.25; H 10.20. Found: C
79.30; H 10.24.
Compound 13: 1H NMR (300 MHz, CDCl3) δ 0.85 (3H, s, H- 28), 0.96 (3H, s, H-27),
1.05 (3H, s, H-25), 1.08 (3H, s, H-24), 1.10 (3H, s, H-26), 1.13 (3H, s, H-23), 2.00-1.26 (16H,
m), 2.94-2.82 (1H, m, H-19), 3.69 (1H, dd, J = 11.2, 4.8 Hz, H-16), 5.78 (1H, d, J = 10.2 Hz, H-
2), 5.95 (1H, br s, H-29b), 6.30 (1H, br s, H-29a), 7.05 (1H, d, J = 10.2 Hz, H-1), 9.53 (1H, s,
H-30); 13C NMR (75 Hz, CDCl3) δ 205.5 (C-3), 195.0 (C-30), 159.5 (C-1), 156.2 (C-20), 133.5
(C-29), 125.4 (C-2), 76.9 (C-16), 53.6 (C-5), 49.0 (C-17, C-18), 44.8 (C-4), 44.2 (C-9), 44.1 (C-
14), 41.9 (C-8), 39.6 (C-10), 37.9 (C-22), 37.3 (C-13, C-19), 36.9 (C-15), 33.9 (C-7), 31.7 (C-
21), 27.9 (C-23), 27.1 (C-12), 21.5 (C-24), 21.3 (C-11), 19.3 (C-25), 19.1 (C-6), 16.6 (C-26),
14.3 (C-27), 11.8 (C-28); MS (ESI, positive ions), m/z 453.4 [M+H]+. Anal. Calcd. for
C30H44O3: C 79.60; H 9.80. Found: C 79.64; H 9.85.
4.12 Inhibition assay on AChE and BChE in vitro
Electric eel (Torpedo californica) AChE and horse serum BChE were used as source of
both the cholinesterases. AChE and BChE inhibiting activities were measured in vitro by the
spectrophotometric method developed by Ellman with slight modification [19]. The lyophilized
enzyme, 500U AChE/300U BChE, was prepared in buffer A (8 mM K2HPO4, 2.3 mM
NaH2PO4) to obtain 5/3 U/mL stock solution. Further enzyme dilution was carried out with
buffer B (8mM K2HPO4, 2.3 mM NaH2PO4, 0.15 M NaCl, 0.05% Tween 20, pH 7.6) to produce
0.126/0.06 U/mL enzyme solution. Samples were dissolved in buffer B with 2.5% of MeOH as
cosolvent. Enzyme solution (300 μL) and sample solution (300 μL) were mixed in a test tube
and incubated for 60/120 min at room temperature. The reaction was started by adding 600 μL
of the substrate solution (0.5 mM DTNB, 0.6 mM ATCI/BTCI, 0.1 M Na2HPO4, pH 7.5). The
absorbance was read at 405 nm for 180 s at 27ºC. Enzyme activity was calculated by comparing
reaction rates for the sample to the blank. All the reactions were performed in triplicate. IC50
19

values were determined with GraphPad Prism 5. Tacrine (99%) was used as reference
AChE/BChE inhibitor.
4.13 Kinetic characterization of BChE inhibition
The enzyme reaction was carried out at three fixed inhibitor concentrations (0, 20 and
70 μM for compound 2; 0, 10 and 75 μM for compound 9). In each case the initial velocity
measurements were obtained at varying substrate (S) (butyrylthiocholine) concentrations and
the reciprocal of the initial velocity (1/v) was plotted as a function of the reciprocal of [S]
(1/[S]). The double-reciprocal (Lineweaver–Burk) plot showed a pattern of intersecting lines
with increasing slopes, characteristic of a competitive inhibitor. The data of the enzyme activity
at different fixed substrate concentrations with increasing inhibitor concentrations were
analyzed with GraphPad Prism 5. The nonlinear regression of these data fitted with competitive
inhibition with R2= 0.9836 for 2 and R2= 0.9802 for 9. The calculated Ki were 51.16 μM for 9
and 32.70 μM for 2.
4.14 Molecular docking determinations
Human BChE crystal structure 1P0I [22] and Torpedo californica AChE crystal 2ACE
[23] were used for the docking simulations of compounds 2 and 9. Due to both compounds are
competitive inhibitors, the acetylcholine was not present at their active site. Geometry
optimization of the compounds was performed with semiempirical calculations (AM1) and the
Hartree-Fock method and the 6-31G (d, p) basis set incorporated in the Gaussian 03 program
[24-26]. Docking studies were performed with version 4.2.5.1 of the program AutoDock, using
the implemented empirical free energy function [21, 27]. The graphical user interface program
AutoDock Tools was used to prepare, run and analyze the docking simulations. The simulation
space was defined as box which included the active site and the peripheral site. Atomic
interaction energy on a 0.375 Å grid was calculated with the auxiliary program Autogrid 4 using
probes corresponding to each map type found in the inhibitor. All rotatable dihedrals in both
20

compounds were allowed to rotate freely. The starting position of the triterpene was outside the
grid on a random position.
The triterpenes were docked by the Lamarckian genetic algorithm protocol. A total of
256 independent simulations with a population size of 150 members were run for each
compound using AutoDock 4.2.5.1 with default parameters (random starting position and
conformation, translation step of 2.0 Å, mutation rate of 0.02, crossover rate of 0.8, local search
rate 0.06 and 2500000 energy evaluations). After docking, the 256 conformers generated for the
inhibitors were assigned to clusters based on a tolerance of 2.0 Å all atom root-mean-square
deviation (rmsd) in position from the lowest-energy solution. The clusters were also ranked
according to the energies of their representative conformations, which were the lowest-energy
solutions within each cluster.
Acknowledgments
This work was financially supported by Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET), Universidad Nacional del Sur and Comisión de
Investigaciones Científica (CIC) from Argentina. M.J.C. and V.R. are grateful to CONICET for
their postdoctoral fellowships. M.B.F. is a Research Member of CIC. A.P.M. and V.R. are
Research Members of CONICET.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in the online version, at ……
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Graphical Abstract
25

Highlights

A set of mono-, di- and tri-oxolupanes were prepared starting from calenduladiol and
lupeol.



Selective inhibition of butyrylcholinesterase was observed for all the derivatives.
Kinetic study and molecular modeling were carried with the most potent inhibitors.
Oxidation at C-16 of the lupane skeleton improves the cholinesterase inhibition.
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