Synthetic analogues of durantoside I from Citharexylum spinosum L. and their cytotoxic activity

Article abstract of DOI:10.1016/j.bmcl.2018.03.068

New iridoid glycoside derivatives from durantoside I, the latter from the dried flowers and leaves of Citharexylum spinosum, were synthesized by variously modifying a sugar moiety by silylation or acetylation and/or removal of cinnamate group at C-7 posit

Full text of DOI:10.1016/j.bmcl.2018.03.068

Accepted Manuscript
Synthetic analogues of durantoside I from Citharexylum spinosum L. and their
cytotoxic activity
Jutamas Apisornopas, Patamawadee Silalai, Teerapich Kasemsuk, Anan
Athipornchai, Uthaiwan Sirion, Kanoknetr Suksen, Pawinee Piyachaturawat,
Apichart Suksamrarn, Rungnapha Saeeng
PII:
S0960-894X(18)30263-4
https://doi.org/10.1016/j.bmcl.2018.03.068
BMCL 25722
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 January 2018
13 March 2018
23 March 2018
Please cite this article as: Apisornopas, J., Silalai, P., Kasemsuk, T., Athipornchai, A., Sirion, U., Suksen, K.,
Piyachaturawat, P., Suksamrarn, A., Saeeng, R., Synthetic analogues of durantoside I from Citharexylum
spinosum L. and their cytotoxic activity, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/
10.1016/j.bmcl.2018.03.068
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Synthetic analogues of durantoside I from Citharexylum spinosum L. and their cytotoxic
activity
Jutamas Apisornopasa, Patamawadee Silalaia, Teerapich Kasemsukb, Anan Athipornchaia,
Uthaiwan Siriona, Kanoknetr Suksenc, Pawinee Piyachaturawatc, Apichart Suksamrarnc and
Rungnapha Saeenga*
Affiliation
aDepartment of Chemistry and Center for Innovation in Chemistry, Faculty of Science,
Burapha University, Chonburi 20131, Thailand
bDepartment of Chemistry, Faculty of Science and Technology, Rambhai Barni Rajabhat
University, Chanthaburi 22000, Thailand
cDepartment of Physiology, Faculty of Science, Mahidol University, Bangkok 10400,
Thailand
dDepartment of Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240,
Thailand
Abstract
New iridoid glycoside derivatives from durantoside I, the latter from the dried flowers
and leaves of Citharexylum spinosum, were synthesized by variously modifying a sugar
moiety by silylation or acetylation and/or removal of cinnamate group at C-7 position and
subsequent screening for comparative cytotoxicity against several cancer cell lines. Addition
of alkylsilane to durantoside I and removal of cinnamate group were most effective in
improving cytotoxicity.
O
OCH3
O
OCH3
O
HO
HO
AcO
H3C
O
O
O
O
O
H3C
H
H
HO
O
HO
O
OTBDPS
OAc
OH
OH
AcO
HO
OAc
OH
Durantoside I
1
12

ED50 8.2-9.19
M

ED50 >50
M
MCF-7, A-549, ASK cancer cell line
MCF-7, A-549, ASK cancer cell line
Keywords Citharexylum spinosum L., durantoside I, cytotoxic activity, iridoid glycoside
1

Natural products are an important source of therapeutic medicines in large part a
function of their structural variability that allows for great diversity in synthetic compounds
including new medical drugs. Indeed, almost half of all current medicines are either natural
products or their semi-synthetic derivatives. Iridoid glycosides are a class of natural products,
present in several plant species that possess a variety of medicinal properties including anti-
inflammatory1, anticancer2, anti-insects3, antioxidant4,5 and antimicrobial.6 Their structure
contains cyclopentanoid monoterpene-derived moieties with a sugar group attached at C1
position.7,8 Several new iridoid glycosides have been isolated from plants but few with
scaffold modifications suggestive of enhanced therapeutic properties.9-11 This may reflect
difficulties in isolation requiring several separation circles as well as stability of iridoid
glycosides in chemical reactions. Durantoside I, is an iridoid glycoside present in some
common and widely distributed Thailand plants within the family Verbenaceae and genera,
Durantaerecta, Durantarepens L.12-15 and Citharexylum spinosum L16,17. Extracts of one
species, C. spinosum possess antiulcer, antihypertensive and hepatoprotective properties.18
Moreover, the alcoholic extract and essential oil from this plant exhibited
immunomodulatory19 and antifungal properties.20
Durantoside I consists of a cyclopentane ring fused to a six-membered oxygen
heterocycle with a cinnamic group at C-7 position of cyclopentane and glucose at position β
on the iridoid. The present study designed and synthesized new cytotoxic compounds from a
natural iridoid glycoside extracted from the dried flowers and leaves of Citharexylum
spinosum L.16,17 and measured their cytotoxic activity. Syntheses were designed through
modifications of sugar moiety by silylation, acetylation and removal of cinnamate group at C-
7 positions to obtain 13 analogues. Earlier, we demonstrated silicon in natural molecules
possesses significant cytotoxic activity.21,22 We discovered the addition of alkylsilane to
andrographolide improved anticancer properties over those of the natural parent compound.
Therefore, we converted the C-6 hydroxy group of sugar in Durantoside I structure to
silylether group to enhance activity of this natural compound.
Synthesis protocols of natural durantoside I derivatives are outlined in Scheme 1 and
2. Acetylation reaction of hydroxy groups with acetic anhydride using pyridine was carried
out at ambient temperature to produce 2", 3", 4", 6"-tetraacetyl-durantoside 3 in 25% along
with penta-acetylated product 2 in 12 % yields, which hydroxyl group was acetylated at C8
position after continue stirring the reaction overnight (Scheme 1). Hydrolysis of cinnamate
group at C-7 using Ba(OH)2 in MeOH was performed to give 4 followed by acetylation using
2

pyridine and Ac2O for 4 h to produce compounds 2", 3", 4", 6"-acetyl-durantoside 5 in 32%
overall yield in two steps.
O
OCH3
O
OCH3
O
O
HO
HO
a
O
O
+
O
O
O
H3C
H3C
H
H
HO
O
OCH3
O
AcO
O
O
O
O
OAc
OAc
OAc
OAc
HO
2
3
7
AcO
AcO
O
8
OAc
OAc
H3C
6'
H
HO
1'
O
O
O
OCH3
O
HO
OCH3
OH
OH
HO
2'
1
HO
AcO
H3C
HO
H3C
OH
O
O
O
c
H
H
b
HO
HO
O
O
O
OAc
OAc
OH
OH
AcO
HO
5
4
OAc
OH
Scheme 1 Reagents and conditions: (a) pyridine, 10 equiv. Ac2O, rt., overnight (b) 3.2 equiv.
Ba(OH)2, MeOH, rt. 40 min. (c) pyridine, excess Ac2O, rt., 4h.
Selective silylation of the hydroxy group at C-6” of the sugar ring to silyl ethers in
natural durantoside I was carried out at ambient temperature via reactions with tert-
butyldiphenylsilylchloride (TBDPSCl) or tert-butyldimethylsilylchloride (TBSCl) and
pyridine to produce silyl-analogues 6 and 7 in yields of 69% and 62%, respectively.
Acetylation of the three remaining hydroxy groups on sugar with acetic anhydride and
pyridine was performed at ambient temperature for 4 h to produce durantoside derivatives 8
and 9 in good yield (75 and 69% respectively). Hydrolysis of cinnamate group of compounds
6 and 7 at C-7 position using barium hydroxide was conducted in MeOH to give 10 and 11
followed by acetylation in step two without separation of 10 and 11. The process was
conducted at room temperature and the substances were stirred for 4 h producing yields of
iridoid glycoside compounds 12 and 13 of 55 and 27%, respectively (Scheme 2).
3

O
OCH3
O
OCH3
O
OCH3
O
O
O
HO
HO
HO
O
O
O
O
O
O
O
O
O
H3C
b
H3C
H
H3C
H
a
H
HO
HO
O
O
HO
O
OR
OH
OR
6
8
R= TBDPS
9 R= TBS
R= TBDPS
AcO
OAc
HO
OH
1
HO
OH
7 R= TBS
OAc
OH
OH
c
O
OCH3
O
OCH3
HO
HO
AcO
H3C
HO
O
O
O
O
d
H
H3C
HO
H
O
HO
O
OR
OR
10
R= TBDPS
12
AcO
OAc
R= TBDPS
13 R= TBS
HO
OH
11 R= TBS
OAc
OH
Scheme 2Reagents and conditions: (a) 2 equiv. TBDPSCl or TBSCl, pyridine, rt., 2h. (b)
pyridine, 8 equiv. Ac2O, rt., 4h. (c) 2 equiv. Ba(OH)2, MeOH, 60oC, 20 min. (d) pyridine, 10
equiv. Ac2O, rt., 4h.
2.2 Cytotoxic activity of Dutantoside I and synthetic analogues
Cytotoxic activities of all synthetic analogues were evaluated using in vitro screening
against selected cancer cell lines by in vitro screening: P-388 (murine leukaemia), KB
(human epidermoid carcinoma of the mouth), HT-29 (human colorectal adenocarcinoma),
MCF-7 (human breast cancer), A549 (human lung adenocarcinoma) and ASK (rat glioma
Table 1). Sulforhodamine B (SRB) assay was carried out to evaluate the cytotoxic activities
of synthesized compounds. All tested analogues were dissolved in DMSO (less than 0.05%).
Ellipticine, a potent anti-cancer agent was used as a positive control. Cells at concentration
of 104 cells/well were seeded in 96-well plates with varying concentrations of compounds
(0.1-50 µM). At the end of treatment period, cells were washed, fixed with trichloroacetic
acid (TCA) and were stained with 0.4% SRB dissolved in 1% acetic acid. The cellular
protein-bound dye was extracted for the determination at optical density (515 nm) with a 96
well microliter plate reader. The amount of bound dye can be extrapolated to measure cell
viability. The cytotoxic potency was expressed as IC50, the concentration that inhibit 50% of
cell viability.
4

Table 1 Cytotoxic activity of synthetic Durantoside I analogues
O
OCH3
HO
R1
H3C
H
R2O
O
O
OR'1
OR'2
R'4O
OR'3
IC50a SD (M)
HT-29 MCF-7
Compound
R1
R2
H
R’1
R’2 R’3 R’4
P-388
>50
KB
A-549 ASK
O
H
H
H
H
>50
>50
>50
>50
>50
>50
>50
>50
>50
1
O
O
O
Ac Ac
Ac Ac Ac >50
>50
2
O
O
H
H
H
Ac
Ac Ac Ac >50
Ac Ac Ac >50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
3
5
6
O
Ac
H3C
O
O
O
O
O
20.61
14.63
16.35
9.47
9.63
26.85
TBDPS
H
H
H
H
H
H
O
O
O
O
3.68
1.49
1.61
0.25
0.43
0.26
H
H
H
TBS
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
7
8
9
6.11
37.17
TBDPS Ac Ac Ac
2.07
1.63
3.11
43.33
TBS
Ac Ac Ac
>50
0.53
5.92
O
3.67
14.74
35.04
9.01
8.20
9.19
H
H
TBDPS Ac Ac Ac
TBS Ac Ac Ac
12
0.38
41.02
1.46
1.84
2.67
1.45
0.20
0.28
0.30
H3C
O
O
>50
>50
>50
>50
>50
13
H3C
O
2.15
2.00
1.99
1.96
2.02
Ellipticineb
0.082 0.02
0.084
0.039
0.033 0.280
aIC50 values (the concentration that inhibit 50% of cell viability) in M.
bPositive control
Cytotoxic activities of durantoside analogues indicated the importance of the
substituted group of the hydroxy function at C-6 on the sugar ring. Acetyl sugar durantoside
I analogues 2, 3, 4 and 5 showed no cytotoxic activity in all cell lines similar to the parent
compound 1. However, with the introduction of a tert-butyldiphenylsilyl (TBDPS) group on
the C-6 of sugar ring (compound 6), cytotoxic activity increased significantly in all cell lines
in the range 9-27 M. Analog 6 displayed stronger activity than the parent Durantoside I and
especially on MCF-7 and A-549 cancer cell lines with the IC50<10 M. Acetylation of the
remaining hydroxy group on sugar of 6 to 8, resulted in a dramatic reduction in potency of
5

all cancer cells except P-388. Compound 8 exhibited selective cytotoxic activity against P-
388 cells. Removal of the cinnamate group at C-7 of compound 8 led to analog 12, the most
active compound of this study. SilylTBDPS analogue 12 exhibited high cytotoxic activity
with an IC50 below 10 M on P-388, MCF-7, A-549 and ASK cancer cell lines. This
indicated the structural presence of cinnamate group was not important with respect to
enhancing cytotoxic activity of iridoid glycoside. Replacement of silyl-TBDPS group at C-6
of sugar with other silyl derivatives such as silyl-TBS (compound 7) did not enhance
cytotoxic activity compared to that of silyl-TBDPS analogues indicating the importance of
the type of silyl-substituted group. However, compounds bearing silyl-TBS (compounds 9
and 13) showed selective cytotoxicity against P-388 over other cell lines.
In conclusion, Durantoside I was isolated from dried flowers and leaves of
Citharexylum spinosum L. Molecular structure was modified by chemical reactions including
silylation, acetylation, and decinnamoylation to produce new analogues of iridoid glycoside.
Semi-synthetic compounds 6 and 12 exhibited higher cytotoxic activity than the original
natural durantoside I in all cell lines. Silyl-TBDPS analog 12 without cinnamate group
showed generally higher cytotoxicity than compound 6 which contained cinnamate at C-7.
This supports the design and synthesis of new iridoid analogs with smaller molecular units
without cinnamate as potentially important cytotoxic agents. To our knowledge, this is the
first report on structure modifications of natural durantoside I and their respective cytotoxic
activities against a series of cancer cell lines.
Conflicts of interest
We declare that we have no conflict of interest.
Acknowledgements
This work was supported by the Strategic Basic Research Grant of The Thailand Research
Fund to R.S. (DBG5680004) and the Center of Excellence for Innovation in Chemistry
(PERCH-CIC). Supports from the Research Grant of The Thailand Research Fund to A.S.
(DBG5980003) and to P.P (the International research network, IRN58W0004) are gratefully
6

acknowledged. Special thanks to Prof. Dr Frederick W. H. Beamish, Faculty of Science,
Burapha University, for his comments and grammatical suggestions.
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10

Highlights



Structure modifications of natural durantoside I from dried flowers and leaves of
Citharexylum spinosum L. were carried out.
New synthetic iridoid analogues were screened for cytotoxicity against six cancer
cells.
Silyl analog without cinnamate group showed generally higher cytotoxicity than
compound which contained cinnamate at C-7.
11