Design and synthesis of new lupeol derivatives and their α-glucosidase inhibitory and cytotoxic activities

Article abstract of DOI:10.1080/14786419.2020.1758095

A series of lupeol derivatives 2, 2a-2f, 2a-2h, 3a-3e, and 4a-4b were designed, synthesised and evaluated for their α-glucosidase inhibitory and cytotoxic activities. Among synthetic derivatives, lupeol analogues 2b and 2e containing a benzylidene chain exhibited the best activity against α-glucosidase and superior to the positive agent with the IC₅₀ values of 29.4 ± 1.33 and 20.1 ± 0.91 μM, respectively. Lupeol analogues 2d and 3a showed weak cytotoxicity against K562 cell line with the IC₅₀ values of 76.6 ± 2.40 and 94.4 ± 1.51 μM, respectively.

Full text of DOI:10.1080/14786419.2020.1758095

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Design and synthesis of new lupeol derivatives
and their α-glucosidase inhibitory and cytotoxic
activities
Hoang-Vinh-Truong Phan, Thuc-Huy Duong, Duc-Dung Pham, Hoang-Anh
Pham, Van-Kieu Nguyen, Thi-Phuong Nguyen, Huu-Hung Nguyen, Ngoc-
Hong Nguyen, Pornpat Sam-ang, Kiettipum Phontree & Jirapast Sichaem
To cite this article: Hoang-Vinh-Truong Phan, Thuc-Huy Duong, Duc-Dung Pham, Hoang-Anh
Pham, Van-Kieu Nguyen, Thi-Phuong Nguyen, Huu-Hung Nguyen, Ngoc-Hong Nguyen, Pornpat
Sam-ang, Kiettipum Phontree & Jirapast Sichaem (2020): Design and synthesis of new lupeol
derivatives and their α-glucosidase inhibitory and cytotoxic activities, Natural Product Research,
DOI: 10.1080/14786419.2020.1758095
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1758095
Design and synthesis of new lupeol derivatives and their
a-glucosidase inhibitory and cytotoxic activities
Hoang-Vinh-Truong Phana, Thuc-Huy Duonga, Duc-Dung Phama, Hoang-Anh
Phama, Van-Kieu Nguyenb,c, Thi-Phuong Nguyend, Huu-Hung Nguyene,
Ngoc-Hong Nguyenf, Pornpat Sam-angg, Kiettipum Phontreeh and
Jirapast Sichaemi
b
aFaculty of Chemistry, University of Education, Ho Chi Minh City, Vietnam; Institute of Fundamental
c
and Applied Sciences, Duy Tan University, Ho Chi Minh City, Vietnam; Faculty of Natural Sciences,
d
Duy Tan University, Da Nang, Vietnam; Faculty of Biotechnology, Nguyen Tat Thanh University, Ho
e
Chi Minh City, Vietnam; Faculty of Environment and Biotechnology, Van Lang University, Ho Chi
f
g
Minh City, Vietnam; Industrial University of Ho Chi Minh City, Ho Chi Minh City, Vietnam; Faculty
h
of Science and Technology, Pibulsongkram Rajabhat University, Phitsanulok, Thailand; Division of
Pharmacognosy and Toxicology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon
i
Kaen, Thailand; Faculty of Science and Technology, Thammasat University Lampang Campus,
Lampang, Thailand
ABSTRACT
ARTICLE HISTORY
Received 28 February 2020
Accepted 14 April 2020
A series of lupeol derivatives 2, 2a-2f, 2a-2h, 3a-3e, and 4a-4b
were designed, synthesised and evaluated for their a-glucosidase
inhibitory and cytotoxic activities. Among synthetic derivatives,
lupeol analogues 2b and 2e containing a benzylidene chain
exhibited the best activity against a-glucosidase and superior to
the positive agent with the IC50 values of 29.4 1.33 and
20.1 0.91 lM, respectively. Lupeol analogues 2d and 3a showed
weak cytotoxicity against K562 cell line with the IC50 values of
76.6 2.40 and 94.4 1.51 lM, respectively.
KEYWORDS
Lupeol derivative;
a-glucosidase inhibition;
cytotoxicity
1. Introduction
Diabetes mellitus (DM), also known as diabetes, is a condition that impairs the body’s
ability to process blood glucose, otherwise known as blood sugar. There are three
CONTACT Ngoc-Hong Nguyen
nn.hong@hutech.edu.vn; Jirapast Sichaem
jirapast@tu.ac.th
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1758095.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
H.-V.-T. PHAN ET AL.
major types of the disease: type 1, type 2, and gestational DM. With all three, the
body can’t make or use insulin. (Tahlan and Verma 2017). Uncontrolled diabetes is
manifested by a very high rise in triglycerides and fatty acid levels (Ferrannini et al.
1991). It can lead to major complications such as heart attack, stroke, eye disease, kid-
ney disease, nerve disease, and infection if left untreated. Antidiabetic drugs comprise
a chemically and pharmacologically heterogeneous group of drugs and presently avail-
able to reduce hyperglycaemia in DM but have side effects. Therefore, searching for a
new class of compounds is essential to overcome these problems. Synthetic transform-
ation of plant metabolites is a significant area of medicinal chemistry that can create
new and potential drugs. Lupeol is a pharmacologically active pentacyclic triterpenoid
found in a variety of plants (Gallo and Sarachine 2009; Siddique and Saleem 2011;
Starks et al. 2011). Reddy and co-workers synthesised few novel lupeol derivatives and
screened for their in vivo antihyperglycemic activity. Most derivatives lowered the
blood glucose levels, in sucrose challenged streptozotocin induced diabetic rats (STZ-
S) model (Reddy et al. 2009).
Recent reports also revealed that lupeol exhibit various cancer types such as pros-
~
tate, skin, pancreatic, and breast cancer (Saleem 2009). Magalhaes and co-workers
~
(Magalhaes et al. 2017) designed lupeol derivatives with adequate carboxylic acid and
DIC/DMAP reagents. Some synthetic analogues displayed a selective cytostatic effect
with low GI50 values, representing a promising prototype for the development of new
anticancer drugs. Due to the several limitations associated with the use of existing
synthetic antidiabetic and anticancer drugs, the search for newer antidiabetic and anti-
cancer agents from natural source continues. In continuation of our ongoing search
for new antidiabetic and cytotoxic agents from derivatized secondary metabolite
(Sichaem et al. 2017; Nguyen et al. 2019), we described herein the synthesis of new
lupeol derivatives via three different routes. In addition, the evaluation of a-glucosi-
dase inhibitory and cytotoxic activities of the synthetic derivatives were also assayed.
2. Results and discussion
2.1. Chemistry
In this study, we successfully synthesised 15 lupeol derivatives 2, 2a-2f, 3a-3e, and
4a-4b, including eight new analogues 2a-2d, 2f, 2g, 3e, and 4b via three different
routes (Scheme 1). The first route is to prepare lupeol derivatives 2a-2g from 2.
Lupenone (2) was subjected to an aldol condensation with different aromatic alde-
hydes, including, 2-florobenzaldehyde, 2-chlorobenzaldehyde, 2-bromobenzaldehyde,
3-bromobenzaldehyde, 4-bromobenzaldehyde, 2-nitrobenzaldehyde, and benzaldehyde
1
to give 2a-2g. The identification of 2a was readily confirmed on the basis of its H
1
and 13C NMR spectra. Indeed, the comparison of H NMR of 2a and 2 showed that
the diasteretopic methylene at dH 2.91 (d, 1H, J ¼ 16.0 Hz, H-1a) and 2.11 (d, 1H,
J ¼ 16.4 Hz, H-1b), assignable to 1-CH2 in 2a was replaced for the methylenes, 2-CH2
1
and 1-CH2 in 2. The structures of 2b-2g were elucidated on the basis of their H and
13C NMR spectra as well as comparison with literature. In addition, other synthetic
products 2b-2g showed consistent NMR data with those of 2, which were in full

NATURAL PRODUCT RESEARCH
3
Scheme 1. General synthesis route towards analogues 2, 2a-2g, 3a-3e, and 4a-4b.
accordance with their depicted structures. The synthesis routes are outlined in
Scheme 1.
In an attempt to obtain more derivatives of the starting material (1), the double
R
V
bond at C-28-C-29 was modified through the oxidation using Oxone
(2KHSO5.KHSO4.K2SO4) of 2 (Uyanik et al. 2009). As the results, five products 3a-3e
were obtained. Synthetic analogue 3c, a natural product was obtained as a major
component of the reaction. NMR data of 3c was identical with the (19R)-isomer
which was previously reported from P. acidus (Sichaem et al. 2019). Interesting, this
compound was selectively synthesised by da Silva Martins and co-workers using
R
V
manganese porphyrins (da Silva Martins et al. 2016). In our case, Oxone is an
effective catalyst to form 3c without protection of 3-OH group. The structures of 3d
and 3e were identified using 1D and 2D NMR. HMBC correlations of 30-CHO, 29-
CH3, H-18 to C-28 and C-19; and of 29-CH3 to C-30 defined the E-ring of 3d and
3e. Corbett and co-workers (Corbett et al. 1985, 1987) indicated that lupeol deriva-
tives containing a chiral C-20 carbon and/or a 20-CHO group have presented as
(20R/S)-epimeric mixtures. Both diastereoisomers could be distinguished by NMR
data. In our scenario, analogues 3d and 3e was synthetically prepared as pure
diastereoisomers.
2.2. a-glucosidase inhibitory activity
Lupeol (1) and its derivatives 2, 2a-2g, 3a-3e, and 4a-4b were evaluated for their
a-glucosidase inhibitory activity (Supplementary material, Table S1). Among synthetic
derivatives, lupeol analogues containing 2-Cl- (2b) and 4-Br- (2e) benzylidene chains
exhibited more powerful a-glucosidase inhibitory activity and superior to the positive
control with the IC50 values of 29.4 1.33 and 20.1 0.91 lM, respectively, indicating
the role of these substituted groups in the a-glucosidase inhibition. It is noteworthy

4
H.-V.-T. PHAN ET AL.
that synthetic products, which lacked the 3-OH and 19-C(CH3)¼CH2 groups displayed
lower activities than their original (1) towards a-glucosidase (Sichaem et al. 2017).
Thus, this investigation indicates that these substituted groups might be involved in
a-glucosidase inhibition.
2.3. Cytotoxicity
The cytotoxicity of the starting material (1) and synthetic derivatives 2, 2a-2d, 2f, 3a-
3e, and 4a-4b against K562 cell line was evaluated (Supplementary material, Table
S2). Synthetic analogues 2d, and 3a showed weak cytotoxicity with the IC50 values of
76.6 2.40 and 94.4 1.51 lM, respectively. On the other hand, other modified com-
pounds displayed no cytotoxicity (IC50 >100 lM). Thus, this study suggests that the
modification of the 2-CH2, 3-OH, and 19-C(CH3)¼CH2 groups of 1 did not significantly
improve cytotoxicity towards K562 cell line.
3. Experimental
3.1. General
High resolution electrospray ionisation mass spectra (HRESIMS) was recorded with a
1
Bruker microTof spectrometer. H, 13C and 2D NMR spectra were recorded (CDCl3 as
solvent) at 400 and 100 MHz, respectively, on a Bruker (Avance) 400 NMR spectrometer
using tetramethylsilane (TMS) as an internal standard. Chemical shifts are reported in
ppm downfield from TMS. Reactions were monitored by thin layer chromatography
(TLC) was performed on aluminium sheets precoated with silica gel (Merck Kieselgel
60 PF254). Silica gel (No. 7729, 7734, and Merck) was used as stationary phase on
quick column chromatography and open column chromatography.
3.2. Isolation of starting material (1)
Lupeol (1) was isolated from Manilkara zapota (L.) P. Royen stems. The M. zapota
stems (10 kg) was ground into powder and exhaustively extracted at room tempera-
ture with MeOH (5 ꢀ 5 L). The filtered solution was evaporated under reduced pressure
to afford a residue (110 g). This crude extract was subsequently reextracted using sol-
vents of dichloromethane and ethyl acetate to yield dichloromethane (20 g) and EtOAc
(8.2 g) extracts. The DCM extract was fractionated by CC, eluted with n-hexane-EtOAc
(isocratic, 5:1, v/v) to give five fractions (C1-C5). Fraction C2 was rechromagraphed by
CC using n-hexane-CHCl3-EtOAc-acetone-water (isocratic, 12:1:2:2:0.02, v/v/v/v/v) as
eluent to afford seven subfractions C2.1-C2.7. Washing subfraction C2.2 (7.8 g) by
methanol (50 mL ꢀ 5) to afford 1 (3.5 g).
3.3. General procedure to synthesise 2 and 2a-2g by oxidation of 1
Lupeol (1, 200 mg, 0.469 mmol) in acetic acid (35.5 mL) was treated with 50 mL of
Jones reagent (0.05 M K2Cr2O7 in a 3 M solution of H2SO4) and stirred at 100 ꢁC for
3 hrs. The mixture was extracted with ethyl acetate–water (1:1) to gain organic layer.

NATURAL PRODUCT RESEARCH
5
The organic layer was washed with brine and dried over anhydrous Na2SO4. The resi-
due was subjected to silica gel column chromatography (CC) eluted with n-hexane-
chloroform (4:1) to afford lupeone (2). This lupeone (2, 70 mg, 0.165 mmol) together
with NaOH (35 mg, 0.875 mmol) in ethanol (7 mL) was stirred at 55 ꢁC for 15 minutes.
Then, the mixture was added appropriate benzaldehyde derivatives (0.33 mmol) and
ethanol (7 mL). The reaction was conducted at 55 ꢁC for 2 hrs under stirring and was
periodically monitored by TLC. The mixture was then extracted with ethyl acetate-
water (1:1) in the same procedure with the previous reaction. The residue was
absorbed onto column chromatography successively eluted with n-hexane-ethyl acet-
1
ate (100:1) to yield products 2a-2g. The H and 13C NMR and ESIMS spectra data of
2a-2g were supplied as supplementary material.
3.4. General procedure to synthesise analogues 3a-3e by oxidation of 1
R
V
Lupeol (1, 200 mg, 0.469 mmol) was oxidised with Oxone (317 mg, 0.516 mmol) in
acetic acid (40 mL) at 100 ꢁC for 3 hrs. The mixture was stirred and continuously moni-
tored by TLC. The mixture was extracted with ethyl acetate–water (1:1) in the similar
manner with the reaction of 1 and Jones reagent. The residue was then purified by sil-
1
ica gel CC to give products 3a-3e. The H and 13C NMR and ESIMS spectra data of 3a-
3e were supplied as supplementary material.
3.5. General procedure to synthesise analogues 4a and 4 b
In the same manner, compound 3c (70 mg, 0.163 mmol) father reacted with 4–chloro-
benzaldehyde (49.2 mg, 0.35 mmol). The reaction were applied to silica gel CC using
the gradient system of n-hexane–ethyl acetate (10:1) to obtain a new derivative 4b.
On the other hand, compound 3c was oxidised with Jones reagent to afford a known
1
product 4a. The H and 13C NMR and ESIMS spectra data of 4a-4b were supplied as
supplementary material.
3.6. a-glucosidase inhibition assay
The evaluation of the inhibitory activity of synthetic compounds 1-2, 2a-2g, 3a-3e,
and 4a-4b against baker’s yeast a-glucosidase was applied from a previous procedure
(Sichaem et al. 2017).
3.7. Cytotoxicity assay
Lupeol derivatives 1-2, 2a-2d, 2f, 3a-3e, and 4a-4b were subjected to cytotoxic evalu-
ation against K562 (chronic myelogenous leukemia) cell line using doxorubixin as the
positive control (Skehan et al. 1990).
4. Conclusion
A series of different derivatives 2, 2a-2f, 2a-2h, 3a-3e and 4a-4b from natural product
lupeol have been synthesised via three different routes and evaluated for their

6
H.-V.-T. PHAN ET AL.
a-glucosidase inhibitory and cytotoxic activities. Among them, compounds 2a-2d, 2f,
2g, 3e, and 4b are new lupeol analogues
Synthetic compounds 2b and 2e showed much stronger a-glucosidase inhibitory
activity (IC50 29.4 1.33 and 20.1 0.91 lM, respectively) than acarbose (IC50
105 0.12 lM). Synthetic products, which lacked the 3-OH and 19-C(CH3)¼CH2 groups
exhibited lower activity than their original (1) towards a-glucosidase. This result indi-
cates that these substituted groups might be involved in a-glucosidase inhibition.
Synthetic analogues 2d and 3a showed weak cytotoxicity (IC50 76.6 2.40 and
94.4 1.51 lM, respectively). This result suggests that the modification of 1 at C-2, C-3,
and C-20 did not significantly improve cytotoxicity towards K562 cell line.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This research was supported by Thailand Integrated Research and Innovation Grant (Contract
No. 10/2562).
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