Quercitylcinnamates, a new series of antidiabetic bioconjugates possessing α-glucosidase inhibition and antioxidant

Article abstract of DOI:10.1016/j.ejmech.2013.05.047

Antidiabetic agents possessing dual functions, α-glucosidase inhibition and antioxidant, have been accepted to be more useful than currently used antidiabetic drugs because they not only suppress hyperglycemia but also prevent risk of complications. Herein, we design antidiabetic bioconjugates comprising of (+)-proto-quercitol as a glucomimic and cinnamic analogs as antioxidant moieties. Fifteen quercitylcinnamates were synthesized by direct coupling through ester bond in the presence of DCC and DMAP. Particular quercityl esters 6a, 7a and 8a selectively inhibited rat intestinal maltase and sucrose 4-6 times more potently than their parents 6, 7 and 8. Of synthesized bioconjugates, 6a was the most potent inhibitor against maltase and sucrose with IC₅₀ values of 5.31 and 43.65 μM, respectively. Of interest, its inhibitory potency toward maltase was 6 times greater than its parent, caffeic acid (6), while its radical scavenging (SC₅₀ 0.11 mM) was comparable to that of commercial antioxidant BHA. Subsequent investigation on mechanism underlying inhibitory effect of 6a indicated that it blocked maltase and sucrose functions by mixed inhibition through competitive and noncompetitive manners.

Full text of DOI:10.1016/j.ejmech.2013.05.047

European Journal of Medicinal Chemistry 66 (2013) 296e304
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Quercitylcinnamates, a new series of antidiabetic bioconjugates
possessing -glucosidase inhibition and antioxidant
a
,
b
Eakkaphon Rattanangkool a, Preecha Kittikhunnatham a, Thanakorn Damsud a
,
,
Sumrit Wacharasindhu a, Preecha Phuwapraisirisan a
*
a Natural Products Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
b Program of Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Antidiabetic agents possessing dual functions,
a-glucosidase inhibition and antioxidant, have been
Received 12 March 2013
Received in revised form
15 May 2013
Accepted 30 May 2013
Available online 10 June 2013
accepted to be more useful than currently used antidiabetic drugs because they not only suppress hy-
perglycemia but also prevent risk of complications. Herein, we design antidiabetic bioconjugates
comprising of (þ)-proto-quercitol as a glucomimic and cinnamic analogs as antioxidant moieties. Fifteen
quercitylcinnamates were synthesized by direct coupling through ester bond in the presence of DCC and
DMAP. Particular quercityl esters 6a, 7a and 8a selectively inhibited rat intestinal maltase and sucrose 4
e6 times more potently than their parents 6, 7 and 8. Of synthesized bioconjugates, 6a was the most
Keywords:
Diabetes mellitus
Glucosidase
Caffeic acid
Multifunctional drug
Quercitol
potent inhibitor against maltase and sucrose with IC50 values of 5.31 and 43.65
mM, respectively. Of
interest, its inhibitory potency toward maltase was 6 times greater than its parent, caffeic acid (6), while
its radical scavenging (SC50 0.11 mM) was comparable to that of commercial antioxidant BHA. Subse-
quent investigation on mechanism underlying inhibitory effect of 6a indicated that it blocked maltase
and sucrose functions by mixed inhibition through competitive and noncompetitive manners.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
exclusive formation of single bis-acetonide is critical to obtain the
desired product without stereogenic congeners, in few steps. In
Type 2 diabetes is characterized by chronic hyperglycemia
and the development of microangiopathic complications such as
retinopathy, nephropathy and neuropathy. Aggressive control
of blood glucose level is preliminary and effective therapy
for diabetic patients and reduces risk of complications [1].
Current evidences suggest that excess plasma glucose drives
overproduction of superoxide radicals and other reactive oxygen
species, which impair the cells via oxidative stress and account
for the pathogenesis of all diabetic complications [2e4]. There-
fore, antidiabetic drugs possessing antihyperglycemic effect and
radical scavenging would be potential for diabetic therapy.
fact, (þ)-proto-quercitol itself does not show inhibitory activity
against -glucosidase possibly due to its water soluble property
a
that enhances ready absorption by small intestine. However,
structural modification of (þ)-proto-quercitol by installing a series
of alkyl and acyl motifs [7] or eliminating hydroxyl group [6] led to
new generations of quercitol-based analogs with enhanced activ-
ity. With the success of this approach in hand, we expand our
application by connecting quercitol core with other bioactive
residues.
Inspired by chlorogenic acid (Fig. 2A), a well-recognized
natural product having both antioxidant [8] and antidiabetic
activities [9], we plan to introduce caffeic acid and other
related cinnamic analogs onto quercitol core (Fig. 2B). In the
current study, we synthesized fifteen quercitylcinnamates
by coupling of (þ)-proto-quercitol (10) and its epimer (11)
(Scheme 1) with a series of cinnamic analogs (1e8, Scheme 2). A
new series of quercitol-based bioconjugates with improved in-
hibition were obtained. Structureeactivity relationship of the
In the course of our research on new a-glucosidase inhibitors,
we recently reported the synthesis and inhibitory effects of ami-
noquercitols [5], conduritol F [6] and inositol analogs from natu-
rally available (þ)-proto-quercitol (Fig. 1). Although (þ)-proto-
quercitol has five contiguous hydroxy groups on cyclohexane ring,
synthesized compounds and mechanism underlying
a-glucosi-
dase inhibitory effect of the most potent inhibitor are herein
discussed.
* Corresponding author. Tel.: þ66 2 2187624; fax: þ66 2 2187598.
E-mail address: preecha.p@chula.ac.th (P. Phuwapraisirisan).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.05.047

E. Rattanangkool et al. / European Journal of Medicinal Chemistry 66 (2013) 296e304
297
quercitylcinnamates 6ae8b were accomplished by ester bond for-
mation of protected cinnamic derivatives with the chiral alcohol 10
or 11 followed by double deprotection of silyl and acetonide groups
with TBAF and amberlyst-15, respectively. Esters 6ae8b were then
isolated in 42e49% yield as white solid (Scheme 2).
OH
OH
NH2
NH2
OH
OH
OH
OH
OH
OH
OH
HO
HO
HO
OH
aminoquercitol
(+)-proto-quercitol
aminoquercitol
2.2.
a-Glucose inhibitory activity and DPPH radical scavenging
OH
HO
OH
All newly synthesized bioconjugates 1ae8b were subjected to
evaluate for their -glucosidase inhibitory effect and antioxidation
(Table 1). The commercial antidiabetic drug acarbose was used as
OH
OH
a
HO
OH
OH
OH
OH
HO
the reference and the parent cinnamic analogs (1e8) were also
HO
HO
validated for comparative purpose. For
activity, all bioconjugates showed no inhibition against yeast
glucosidase (type I -glucosidase) but some of which inhibited
maltase and sucrose, type II -glucosidases from rat intestine. The
a-glucosidase inhibitory
OH
OH
OH
a
-
conduritol F
(+)-epi-inositol
(+)-chiro-inositol
a
a
Fig. 1. Structures of (þ)-proto-quercitol and its synthetic analogs.
bioconjugates 6ae8b, whose structures encompassing caffeoyl,
ferulyl and isoferulyl moieties, displayed inhibitory effects in range
2. Results and discussion
of 5.31e954.08 mM, whereas 1ae5a were not active. Notably, their
cinnamoyl cores in 6ae8b different from those of 1ae5b in having
at least one phenolic group, suggesting that this moiety possibly
involved in exerting the observed inhibition. It was likely that the
more phenolic group in cinnamoyl moiety, the more potent inhi-
bition observed. This result was similar to previous report of in-
2.1. Compound design and synthesis
Crucial to the successful synthesis of all the conjugates
described in this work is the selective coupling reaction of cinnamic
derivatives with the desired hydroxy group on naturally available
(þ)-proto-quercitol (9) (Scheme 1). Bis-acetonides 10 and 11 were
prepared from 9, which was isolated from the stems of Arfeuillea
arborescens using the procedure described elsewhere [6,7]. Briefly,
(þ)-proto-quercitol was obtained in 0.3% (w/w) yield as white solid
after recrystallization by MeOH. Protection of two diols with
dimethoxypropane gave the target alcohol 10. In order to investi-
gate the effect of C-10 configuration on inhibitory effect, the epimer
11 was also synthesized from 10 through oxidation using acetic
anhydride/DMSO followed by LiAlH4 reduction, yielding the
desired product in 42% yield.
With the chiral coupling partners 10 and 11 in hands, the
quercitylcinnamates 1ae8b were prepared as depicted in Scheme
2. For the esters 1ae8a and their epimers 1be8b, the synthetic
route was straight forward involving the direct coupling reaction
between alcohol 10 or 11 with the corresponding cinnamic de-
rivatives 1e5 in the presence of N,N0-dicyclohexylcarbodiimide
(DCC) and 4-dimethylaminopyridine (DMAP). Removal of aceto-
nide group underwent smoothly upon treatment of amberlyst-15 in
methanol to give esters 1ae5a in 56e73% yields (Scheme 2).
On the other hand, esterification of caffeic acid (6), ferulic acid (7)
and isoferulic acid (8) required additional protection step because
free phenolic group(s) of those compounds could interfere with the
coupling reaction. The silylation of 6e8 with tert-butyldimethylsilyl
chloride (TBDMSCl) not only prevented phenolic group(s) possibly
being esterified but also improved solubility of the acids 6e8 in
CH2Cl2, thus facilitating esterification reaction. The syntheses of
testinal
a-glucosidase inhibition of hydroxylated cinnamic
derivatives 6, 7 and 8 [10]. This trend was obviously found in 6a,
whose inhibition against maltase was more potent than those of 7a
(10 times) and 8a (4 times); while 8a showed only two times more
potent than 7a (Table 2).
Further inspection of two quercityl residues (10 and 11),
installed in the active bioconjugates, on inhibitory effect revealed
significant difference in inhibition. Bioconjugates 6a, 7a and 8a, all
of which generated from natural quercitol 10, showed inhibitory
effect 3e94 times more potent than their corresponding C-
10epimers (6b, 7b and 8b). The difference in inhibitory potency
among epimeric analogs was strikingly observed in 6a, whose in-
hibition against maltase and sucrose were 93 and 22 times more
potent than 6b. Compared to their corresponding cinnamic pre-
cursors (6, 7 and 8), 6a, 7a and 8a showed more improved inhibi-
tory effects (4e6 times), whereas their epimeric analogs (6b, 7b
and 8b) displayed reverse trend (Fig. 3). The observed results
suggested that R configuration of C-10 in quercityl moiety was also
associated with exerting inhibitory effect, in addition to the pres-
ence of more phenolic groups in cinnamoyl residues. The pro-
nounced inhibitions raised by R configuration of C-10 were also
supported by a similar trend observed in our previous report of N-
alkyl aminoquercitols [7]. Of bioconjugates synthesized, 6a showed
most potent inhibition against both maltase and sucrase with IC50
values of 5.31 and 43.65 mM, respectively.
As for antioxidation of synthesized bioconjugates, 6a showed
radical scavenging activity toward DPPH with SC50 value of
Glucomimic
R = H or Me
(RO)n
Antioxidant
O
O
O
O
*
OH
OH
OH
OH
HO
HO
HO2C
OH
HO
OH
chlorogenic acid
quercitylcinnamate
B
A
Fig. 2. Structures of chlorogenic acid (A) and designed quercitylcinnamates (B) encompassing antioxidant and glucomimic residues.

298
E. Rattanangkool et al. / European Journal of Medicinal Chemistry 66 (2013) 296e304
OH
OH
OH
1) H2O, heat, 2 h
stems
Arfeuillea arborescens
2) Diaion HP-20 (H2O)
HO
3) recrystallization (MeOH, 0.3%w/w)
OH
9
-quercitol ( )
(+)-
proto
OH
OH
Me2C(OMe)2
O
O
1) Ac2O, DMSO
2) LiAlH4
42%
O
O
DMF, -TsOH
p
O
O
O
O
79%
10
11
Scheme 1. Isolation of (þ)-proto-quercitol and synthesis of bis-acetonides 10 and 11.
0.11 mM, which was comparable to that of standard antioxidant
BHA (SC50 0.10 mM).
cinnamic moieties proved to enhance a-glucosidase inhibition.
Prominent examples included 6a, whose inhibitory effect against
maltase was 6 times better than those of original caffeic acid (6). Of
bioconjugates synthesized, 6a was the most potent a-glucosidase
2.3. Mechanism underlying inhibitory effect of quercitylcaffeate
inhibitor, in addition to its radical scavenging activity equipotent to
standard antioxidant, BHA. In addition, mechanism underlying the
inhibitory effect of 6a against maltase and sucrase were proved to
be mixed-type inhibition. This suggests proposed guideline for the
application of 6a as single antidiabetic agent or combination with
other currently used drugs such as acarbose.
against rat intestinal a-glucosidase
Since 6a was the most potent inhibitor in hand, we therefore
further study mechanism underlying this inhibitory effect. The
LineweavereBurk plot of initial velocity versus maltose concen-
trations in the presence of different concentrations of 6a gave a
series of straight lines, all of which intersected within the second
quadrant (Fig. 4). The analysis showed that Vmax decreased with
increasing Km in the presence of increasing concentrations of 6a.
This behavior [11] indicated that 6a inhibits maltase by two
different pathways; competitively forming enzymeeinhibitor (EI)
complex and interrupting enzymeesubstrate (ES) intermediate by
forming enzymeesubstrateeinhibitor (ESI) complex in noncom-
petitive manner. To gain insightful the pathway in which 6a
preferentially proceeded, binding affinities of EI and ESI com-
plexes were determined. Secondary plot of slope against con-
centration of 6a (Fig. 5) showed EI dissociation constant (Ki) of
4. Experimental section
4.1. Chemistry
4.1.1. General procedure
All moisture-sensitive reactions were carried out under a ni-
trogen atmosphere. All solvents were distilled prior to use. HRESI-
MS spectra were obtained from a micrOTOF Bruker mass spec-
trometer. 1H and 13C NMR spectra were recorded (CDCl3 and CD3OD
as solvents) at 400 and 100 MHz, respectively, on a Varian Mer-
curyþ 400 NMR spectrometer. Chemical shifts were reported in
ppm downfield from TMS or solvent residue. Thin layer chroma-
tography (TLC) was performed on pre-coated Merck silica gel 60
23.8 m mM was also
M while ESI dissociation constant (Ki0) of 64.5
deduced from secondary plot of intercept against concentration of
6a (Fig. 6). A lower dissociation constant of Ki pointed out stronger
binding between enzyme and 6a and suggested preferred
competitive over noncompetitive manners. In addition, inhibitory
mechanism of 6a toward sucrase was also determined using
similar methodology. Apparently, 6a also inhibited sucrase
through mixed-inhibition (Fig. 7); in which competitive mode (Ki
F254 plates (0.25 mm thick layer) and visualized under 254 nm UV
followed by dipping in KMnO4 solution. Column chromatography
was conducted using Merck silica gel 60 (70e230 mesh) or
Sephadex LH-20.
22.4
47.5
m
m
M, Fig. 8) was preferred over noncompetitive manner (Ki0
M, Fig. 9).
4.1.2. Isolation of (þ)-proto-quercitol (9) from the stems of
A. arborescens
Our improved isolation of (þ)-proto-quercitol (9) was applied
[6,7]. Generally, ground stems (1.0e1.2 kg) of A. arborescens were
boiled with water (2 ꢀ 4 L) for 2 h. The combined decoction was
filtered, concentrated to a half and partitioned twice with equal
volume of CH2Cl2 to remove lipophilic matters. The aqueous layer
was diluted with water in a ratio of 2:1 and applied onto a Diaion
HP20 column (1 kg) equilibrated with water. The column was
excessively eluted with water (11 L), and the aqueous elutes were
lyophilized to yield white powder. More purified (þ)-proto-quer-
citol (9, 0.3% w/w) was obtained upon crystallization using hot
MeOH.
3. Conclusions
In summary, we first synthesized fifteen bioconjugates (1ae8b)
comprising two key moieties derived from (þ)-proto-quercitol as
glucomimic and cinnamic analogs as antioxidants. This synthetic
design was implemented in the hope that the bioconjugates would
provide dual activities, a-glucosidase inhibitory effect and antiox-
idant activity. Bioconjugates 6ae8b, whose structures encom-
passing hydroxylated cinnamic analogs, displayed inhibition
against rat maltase and sucrase in range of 5.31e954.08
mM.
Notably, 6a and 6b, the bioconjugates having more phenolic groups
in cinnamic core, were likely to provide pronounced inhibition. In
addition, the R configuration of C-10 in natural quercitol (10) also
enhanced inhibitory effects over those of bioconjugates having
unnatural quercitol (11). Therefore, coupling of quercityl and
4.1.3. Synthesis of bis-acetonides 10 and 11
Bis-acetonides were synthesized using our previous methods
[5e7], yielding 10 (79%) and 11 (42%). The 1H and 13C NMR data
were matched well the reports.

E. Rattanangkool et al. / European Journal of Medicinal Chemistry 66 (2013) 296e304
299
O
O
O
R4
R3
9
3
R4
R3
a
R4
R3
b
O
O
1
OH
1'
OH
OH
5
R1
HO
O
O
R1
R1
7
R2
5'
R2
R2
3'
O
OH
1-5
O
R1 = OMe, R2 = R3 = R4 = H; 1
R1 = R3 = R4 = H, R2 = OMe; 2
R1 = R2 = R4 = H, R3 = OMe; 3
R1 = R4 = H, R2 = R3 = OMe; 4
R1 = H, R2 = R3 = R4 = OMe; 5
R1 = OMe, R2 = R3 = R4 = H, 1'R; 1a
R1 = OMe, R2 = R3 = R4 = H, 1'S; 1b
R1 = R3 = R4 = H, R2 = OMe, 1'R; 2a
R1 = R3 = R4 = H, R2 = OMe, 1'S; 2b
R1 = R2 = R4 = H, R3 = OMe, 1'R; 3a
R1 = R2 = R4 = H, R3 = OMe, 1'S; 3b
R1 = R4 = H, R2 = R3 = OMe, 1'R; 4a
R1 = R4 = H, R2 = R3 = OMe, 1'S; 4b
R1 = H, R2 = R3 = R4 = OMe, 1'R; 5a
R1 = OMe, R2 = R3 = R4 = H, 1'R; 1R
R1 = OMe, R2 = R3 = R4 = H, 1'S; 1S
R1 = R3 = R4 = H, R2 = OMe, 1'R; 2R
R1 = R3 = R4 = H, R2 = OMe, 1'S; 2S
R1 = R2 = R4 = H, R3 = OMe, 1'R; 3R
R1 = R2 = R4 = H, R3 = OMe, 1'S; 3S
R1 = R4 = H, R2 = R3 = OMe, 1'R; 4R
R1 = R4 = H, R2 = R3 = OMe, 1'S; 4S
R1 = H, R2 = R3 = R4 = OMe, 1'R; 5R
O
O
O
O
c
a
OH
OH
O
R3
R3
R3
R2
R2
R2
O
O
O
R2 = R3 = OH; 6
R2 = R3 = O(CH3)2SiC(CH3)3, 1'R; 6R
R2 = R3 = O(CH3)2SiC(CH3)3; 6Si
R2 = OMe, R3 = OH; 7
R2 = OH, R3 = OMe; 8
R2 = R3 = O(CH3)2SiC(CH3)3, 1'S; 6S
R2 = OMe, R3 = O(CH3)2SiC(CH3)3; 7Si
R2 = O(CH3)2SiC(CH3)3, R3 = OMe; 8Si
R2 = OMe, R3 = O(CH3)2SiC(CH3)3, 1'R; 7R
R2 = OMe, R3 = O(CH3)2SiC(CH3)3, 1'S; 7S
R2 = O(CH3)2SiC(CH3)3, R3 = OMe, 1'R; 8R
R2 = O(CH3)2SiC(CH3)3, R3 = OMe, 1'S; 8S
O
O
OH
d, b
R3
R2
HO
OH
OH
R2 = R3 = OH, 1'R; 6a
R2 = R3 = OH, 1'S; 6b
R2 = OMe, R3 = OH, 1'R; 7a
R2 = OMe, R3 = OH, 1'S; 7b
R2 = OH, R3 = OMe, 1'R; 8a
R2 = OH, R3 = OMe, 1'S; 8b
Scheme 2. Synthesis of quercitylcinnamates. Reagents and conditions: (a) 10 or 11, DCC, DMAP, CH2Cl2, 0 ꢁC to rt; (b) amberlyst-15, MeOH; (c) TBDMSCl, imidazole, DMF, rt; (d)
TBAF, THF, rt.
4.1.4. General protection of phenolic groups
4.1.4.1. 3,4-Di-O-tert-butyldimetylsilylcaffeic acid (6Si). White pow-
der; 1H NMR (CDCl3, 400 MHz)
J ¼ 8.8 Hz, 1H), 6.81 (s, 1H), 6.62 (d, J ¼ 8.8 Hz, 1H), 6.03 (d,
J ¼ 15.8 Hz, 1H), 0.77 (d, J ¼ 4.5 Hz, 18H), 0.00 (d, J ¼ 3.2 Hz, 12H).
To a solution of 6, 7 or 8 (1 eq, 2.57 mmol), imidazole (10 eq: 1
OH group) and tert-butyldimethylsilyl chloride (TBDMSCl) (10 eq: 1
OH group) in N,N-dimethylformamide (DMF, 15 mL) in a 50 mL
round button flask. The mixture was stirred at room temperature
for 1 h under N2 gas. The reaction mixture was washed with water
(3 ꢀ 20 mL) and the organic portion was extracted with ethyl ac-
etate (EtOAc) (3 ꢀ 20 mL). The organic layer was washed with
saturated aqueous NaCl, followed by dried over Na2SO4. After
filtration and removal of the solvent under reduced pressure, the
crude product was purified by silica gel column to afford 6Si, 7Si
and 8Si in 74, 82 and 79% yield, respectively.
d
7.46 (d, J ¼ 15.8 Hz, 1H), 6.83 (d,
4.1.4.2. 4-O-tert-Butyldimetylsilylferulic acid (7Si). White powder;
1H NMR (CDCl3, 400 MHz)
J ¼ 8.6 Hz, 1H), 6.95 (s, 1H), 6.79 (d, J ¼ 8.6 Hz, 1H), 6.24 (d,
J ¼ 15.9 Hz, 1H), 3.78 (s, 3H), 0.93 (s, 9H), 0.11 (s, 6H).
d
7.65 (d, J ¼ 15.9 Hz, 1H), 6.98 (d,
4.1.4.3. 4-O-tert-Butyldimetylsilylisoferulic acid (8Si). White pow-
der; 1H NMR (CDCl3, 400 MHz)
d
7.66 (d, J ¼ 15.7 Hz, 1H), 6.97 (d,

300
E. Rattanangkool et al. / European Journal of Medicinal Chemistry 66 (2013) 296e304
Table 1
a
-Glucosidase inhibitory effect and radical scavenging activity of quercityl-
cinnamates.
Compounds
a
-Glucosidase inhibitory effect (IC50
,
mM)
Radical
scavenging
(SC50, mM)
Baker’s yeast
glucosidase
Maltase
Sucrase
1a
1b
NIa
NI
NI
NI
NI
NI
NSb
NS
2a
NI
NI
NI
NS
2b
NI
NI
NI
NS
3a
NI
NI
NI
NS
3b
NI
NI
NI
NS
4a
NI
NI
NI
NS
4b
NI
NI
NI
NS
5a
6
6a
6b
7a
7b
8a
8b
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NS
Fig. 3. Inhibition trends of bioconjugates 6ae8b compared to their cinnamic parents
6e8.
34.69 ꢂ 0.74
5.31 ꢂ 0.10
497.48 ꢂ 0.50
51.93 ꢂ 0.20
21.07 ꢂ 0.15
20.81 ꢂ 0.14
367.15 ꢂ 0.42
1.50 ꢂ 0.14
ND
87.45 ꢂ 0.62
43.65 ꢂ 0.30
954.08 ꢂ 0.50
67.21 ꢂ 0.20
171.43 ꢂ 0.35
53.00 ꢂ 0.32
805.48 ꢂ 0.65
2.30 ꢂ 0.02
ND
0.15
0.11
NS
NS
NS
1H), 6.90 (t, J ¼ 7.5 Hz, 1H), 6.85 (d, J ¼ 7.6 Hz, 1H), 6.46 (d,
J ¼ 16.1 Hz,1H), 5.47 (m, 1H), 4.30 (m, 1H), 4.23 (m, 1H), 3.83 (s, 3H),
3.66 (m, 1H), 3.54 (m, 1H), 2.16 (m, 1H), 2.04 (m, 1H), 1.46 (s, 3H),
1.38 (s, 6H), 1.30 (s, 3H).
NS
NS
AcarboseÒ
BHA
403.9 ꢂ 0.40
NDc
0.10
ND
a
No inhibition (inhibitory effect less than 30% at concentration of 10 mg/mL).
No scavenging (inhibitory effect less than 50% at concentration of 10 mg/mL).
Not determined.
4.1.5.2. 1S. 72% yield of colorless wax; 1H NMR (CDCl3, 400 MHz)
b
c
d
7.98 (d, J ¼ 16.1 Hz, 1H), 7.45 (d, J ¼ 7.5 Hz, 1H), 7.29 (t, J ¼ 7.6 Hz,
1H), 7.19 (s, 1H), 6.90 (t, J ¼ 7.6 Hz, 1H), 6.85 (d, J ¼ 7.5 Hz, 1H), 6.55
(d, J ¼ 16.1 Hz, 1H), 5.30 (m, 1H), 4.37 (m, 1H), 4.23 (m, 1H), 3.84 (m,
4H), 3.44 (m, 1H), 2.36 (m, 1H), 1.93 (m, 2H), 1.47 (s, 3H), 1.40 (s, 3H),
1.38 (s, 3H), 1.30 (s, 3H).
J ¼ 8.8 Hz, 1H), 6.95 (s, 1H), 6.79 (d, J ¼ 8.8 Hz, 1H), 6.20
(d, J ¼ 15.7 Hz, 1H), 3.78 (s, 3H), 0.93 (s, 9H), 0.11 (s, 6H).
4.1.5. General coupling reaction between bis-acetonides and
cinnamic or silylated cinnamate derivatives
4.1.5.3. 2R. 79% yield of colorless wax; 1H NMR (CDCl3, 400 MHz)
d
7.61 (d, J ¼ 16.0 Hz, 1H), 7.24 (t, J ¼ 7.9 Hz, 1H), 7.06 (d, J ¼ 7.5 Hz,
To a solution of 1, 2, 3, 4 and 5 (2.5 eq) in CH2Cl2 (5 mL) were
added N,N0-dicyclohexylcarbodiimide (DCC, 2.7 eq) and 4-dimethyl
aminopyridine (DMAP, catalytic amount). The reaction mixture was
stirred at 0 ꢁC for 30 min. 10 or 11 (1 eq) in CH2Cl2 was added
dropwise at room temperature under N2 gas. The product was fil-
trated and solvent was removed under reduced pressure. The crude
product was subsequently purified by silica gel column to afford
bis-acetonides 1Re5R.
To a solution of 6Si, 7Si and 8Si (11 eq) in CH2Cl2 (10 mL) were
added DCC (12 eq) and DMAP (catalytic amount). The reaction
mixture was stirred at 0 ꢁC for 30 min. 10 or 11 (1 eq) in CH2Cl2 was
added dropwise at room temperature under N2 gas. The bis-
acetonides 6Re8S were obtained after purification using silica gel
column.
1H), 6.98 (s, 1H), 6.89 (d, J ¼ 7.5 Hz, 1H), 6.36 (d, J ¼ 16.0 Hz, 1H),
5.45 (m, 1H), 4.30 (m, 1H), 4.23 (m, 1H), 3.77 (s, 3H), 3.66 (m, 1H),
3.56 (m, 1H), 2.15 (m, 1H), 2.06 (m, 1H), 1.46 (s, 3H), 1.39 (s, 6H), 1.20
(s, 3H).
4.1.5.4. 2S. 76% yield of colorless oil; 1H NMR (CDCl3, 400 MHz)
d
7.65 (d, J ¼ 16.0 Hz, 1H), 7.24 (t, J ¼ 7.9 Hz, 1H), 7.07 (d, J ¼ 7.5 Hz,
1H), 6.99 (s, 1H), 6.89 (d, J ¼ 7.5 Hz, 1H), 6.45 (d, J ¼ 16.0 Hz, 1H),
5.31 (m, 1H), 4.38 (m, 1H), 4.24 (m, 1H), 3.83 (m, 1H), 3.77 (s, 3H),
3.45 (m, 1H), 2.38 (m, 1H), 1.92 (m, 1H), 1.47 (s, 3H), 1.40 (s, 3H), 1.38
(s, 3H), 1.30 (s, 3H).
0.14
0.12
0.1
4.1.5.1. 1R. 75% yield of pale yellow oil; 1H NMR (CDCl3, 400 MHz)
d
7.94 (d, J ¼ 16.1 Hz, 1H), 7.43 (d, J ¼ 7.6 Hz, 1H), 7.30 (t, J ¼ 7.5 Hz,
Table 2
Kinetic data of a-glucosidase inhibition of 6a.
0.08
0.06
O
O
OH
OH
HO
0.04
No inhibitor
OH
HO
2.04 µM
0.02
OH
20.4 µM
0
-0.1
-0.05
0
0.05
0.1
0.15
1/[maltose] mM
0.2
0.25
0.3
a
-Glucosidase
Inhibition type
Ki (m
M)
Ki0
(
m
M)
Km (mM)
-0.02
Fig. 4. LineweavereBurk plots for inhibitory activity of 6a against maltase.
Maltase
sucrase
Mixed-inhibition
Mixed-inhibition
23.8
22.4
64.5
47.5
17.3
25.9

E. Rattanangkool et al. / European Journal of Medicinal Chemistry 66 (2013) 296e304
301
0.5
0.5
0.4
0.3
0.2
0.1
0
0.4
0.3
0.2
0.1
0
No inhibitor
2.04 µM
20.4 µM
-0.05
0
0.05
0.1
0.15
0
5
10
15
20
25
[I] µM
-0.1
1/[sucrose] mM
Fig. 5. Secondary plot of slope vs [I] for determination of Ki of 6a against maltase.
Fig. 7. LineweavereBurk plots for inhibitory activity of 6a against sucrase.
4.1.5.5. 3R. 83% yield of colorless viscous oil; 1H NMR (CDCl3,
4.1.5.10. 6R. 67% yield of colorless oil; 1H NMR (CDCl3, 400 MHz)
400 MHz)
d
7.60 (d, J ¼ 16.0 Hz, 1H), 7.42 (d, J ¼ 8.7 Hz, 2H), 7.19 (s,
d
7.37 (d, J ¼ 15.9 Hz,1H), 6.80 (d, J ¼ 8.7 Hz,1H), 6.78 (s,1H), 6.61 (d,
4H), 6.84 (d, J ¼ 8.7 Hz, 2H), 6.24 (d, J ¼ 16.0 Hz, 1H), 5.45 (m, 1H),
4.30 (m, 1H), 4.23 (m, 1H), 3.78 (s, 3H), 3.65 (m, 1H), 3.54 (m, 1H),
2.15 (m, 1H), 2.05 (m, 1H), 1.47 (s, 3H), 1.39 (s, 6H), 1.31 (s, 3H).
J ¼ 8.7 Hz, 1H), 6.00 (d, J ¼ 15.9 Hz, 1H), 5.30 (m, 1H), 4.17 (m, 1H),
4.09 (m, 1H), 3.51 (m, 1H), 3.40 (m, 1H), 2.00 (m, 1H), 1.91 (m, 1H),
1.32 (s, 3H), 1.24 (s, 6H), 1.16 (s, 3H), 0.78 (s, 18H), 0.00 (s, 12H).
4.1.5.6. 3S. 72% yield of colorless wax; 1H NMR (CDCl3, 400 MHz)
4.1.5.11. 6S. 77% yield of pale yellow oil; 1H NMR (CDCl3, 400 MHz)
d
7.69 (d, J ¼ 15.9 Hz, 1H), 7.49 (d, J ¼ 8.4 Hz, 2H), 6.90 (d, J ¼ 8.4 Hz,
d
7.56 (d, J ¼ 15.9 Hz, 1H), 6.96 (d, J ¼ 8.7 Hz, 1H), 6.94 (s, 1H), 6.75
2H), 6.38 (d, J ¼ 15.9 Hz, 1H), 5.35 (m, 1H), 4.43 (m, 1H), 4.30 (m,
1H), 3.87 (m, 1H), 3.83 (s, 3H), 3.50 (m, 1H), 2.43 (m, 1H), 1.98 (m,
1H), 1.53 (s, 3H), 1.46 (s, 3H), 1.44 (s, 3H), 1.36 (s, 3H).
(d, J ¼ 8.7 Hz, 1H), 6.25 (d, J ¼ 15.9 Hz, 1H), 5.29 (brs, 1H), 4.44 (m,
1H), 4.23 (m, 1H), 3.30 (m, 1H), 3.43 (m, 1H), 2.36 (m, 1H), 1.92 (s,
1H), 1.48 (s, 3H), 1.40 (s, 3H), 1.38 (s, 3H), 1.30 (s, 3H), 0.91 (s, 18H),
0.14 (s, 12H).
4.1.5.7. 4R. 78% yield of colorless oil; 1H NMR (CDCl3, 400 MHz)
d
4.1.5.12. 7R. 68% yield of colorless oil 1H NMR (CDCl3, 400 MHz)
d
7.64 (d, J ¼ 15.9 Hz,1H), 7.11 (d, J ¼ 8.3 Hz, 1H), 7.05 (s, 1H), 6.87 (d,
J ¼ 8.3 Hz, 1H), 6.30 (d, J ¼ 15.9 Hz, 1H), 5.52 (m, 1H), 4.36 (m, 1H),
4.30 (m, 1H), 3.92 (s, 9H), 3.72 (m, 1H), 3.61 (m, 1H), 2.21 (m, 1H),
2.12 (m, 1H), 1.53 (s, 3H), 1.45 (s, 6H), 1.37 (s, 3H).
7.47 (d, J ¼ 15.8 Hz,1H), 6.86 (d, J ¼ 7.2 Hz, 2H), 6.85 (s,1H), 6.80 (d,
J ¼ 7.2 Hz, 1H), 6.12 (d, J ¼ 15.8 Hz, 1H), 5.35 (m, 1H), 4.20 (m, 1H),
4.12 (m, 1H), 3.67 (s, 3H), 3.55 (m, 1H), 3.44 (m, 1H), 2.05 (m, 1H),
1.94 (m, 1H), 1.36 (s, 3H), 1.28 (s, 6H), 1.18 (m, 1H), 0.82 (s, 9H),
0.00 (s, 6H).
4.1.5.8. 4S. 81% yield pale yellow oil; 1H NMR (CDCl3, 400 MHz)
d
7.62 (d, J ¼ 15.8 Hz,1H), 7.05 (d, J ¼ 8.2 Hz,1H), 7.01 (s,1H), 6.81 (d,
4.1.5.13. 7S. 69% yield of colorless oil; 1H NMR (CDCl3, 400 MHz)
J ¼ 8.2 Hz, 1H), 6.33 (d, J ¼ 15.8 Hz, 1H), 5.31 (s, 1H), 4.38 (m, 1H),
4.24 (m, 1H), 3.83 (m, 7H), 3.45 (m, 2H), 2.37 (m, 1H), 1.90 (m, 1H),
1.48 (s, 3H), 1.40 (s, 3H), 1.38 (s, 3H), 1.30 (s, 3H).
d
7.50 (d, J ¼ 15.9 Hz,1H), 6.86 (d, J ¼ 7.6 Hz,1H), 6.83 (s,1H), 6.67 (d,
J ¼ 7.6 Hz, 1H), 6.21 (d, J ¼ 15.9 Hz, 1H), 5.20 (m, 1H), 4.27 (m, 1H),
4.13 (m, 1H), 3.72 (m, 1H), 3.67 (s, 3H), 3.34 (m, 1H), 2.27 (m, 1H),
1.81 (m,1H),1.37 (s, 3H),1.30 (s, 3H),1.27 (s, 3H),1.20 (s, 3H), 0.82 (s,
9H), 0.00 (s, 6H).
4.1.5.9. 5R. 85% yield of colorless viscous oil; 1H NMR (CDCl3,
400 MHz)
1H), 5.45 (m, 1H), 4.31 (m, 1H), 4.24 (m, 1H), 3.82 (s, 9H), 3.66 (m,
1H), 3.54 (m, 1H), 2.21 (m, 1H), 2.12 (m, 1H), 1.47 (s, 3H), 1.39 (s, 6H),
1.31 (s, 3H).
d
7.56 (d, J ¼ 15.9 Hz, 1H), 6.69 (s, 2H), 6.28 (d, J ¼ 15.9 Hz,
4.1.5.14. 8R. 81% yield of pale yellow oil; 1H NMR (CDCl3, 400 MHz)
d
7.44 (d, J ¼ 15.9 Hz, 1H), 6.94 (d, J ¼ 8.2 Hz, 1H), 6.89 (s, 1H), 6.68
0.02
0.015
3.5
3
2.5
2
0.01
1.5
1
0.005
0.5
0
-3.47E-17
0
5
10
15
20
25
0
5
10
15
20
25
[I] µM
[I] µM
Fig. 6. Secondary plot of intercept vs [I] for determination of Ki0 of 6a against maltase.
Fig. 8. Secondary plot of slope vs [I] for determination of Ki of 6a against sucrase.

302
E. Rattanangkool et al. / European Journal of Medicinal Chemistry 66 (2013) 296e304
4.1.6.2. 10S-Quercityl-2-methoxycinnamate (1b). 79% yield of color-
less oil; 1H NMR (CD3OD, 400 MHz)
7.49 (dd, J ¼ 8.0, 4.0 Hz, 1H, AreH), 7.28 (t, J ¼ 7.5 Hz, 1H, AreH),
6.95 (d, J ¼ 8.0 Hz, 1H, AreH), 6.88 (t, J ¼ 7.5 Hz, 1H, AreH), 6.52 (d,
J ¼ 16.2 Hz, 1H, H-2), 4.85 (m, 1H, H-10), 4.02 (s, 1H), 3.81 (s, 3H,
OMe), 3.52e3.24 (m, 3H), 1.93 (m, 2H, H-60); 13C NMR (CD3OD,
0.1
0.08
0.06
0.04
0.02
0
d
7.92 (d, J ¼ 16.2 Hz, 1H, H-3),
100 MHz) d 168.6, 160.0, 142.0, 133.1, 130.0, 124.3, 121.9, 119.2, 112.5,
76.1, 73.6, 72.2, 71.5, 70.8, 56.1, 32.9; HRESIMS m/z 347.1108
[M þ Na]þ (calcd for [C16H20NaO7]þ 347.1107).
4.1.6.3. 10R-Quercityl-3-methoxycinnamate (2a). 85% yield of
colorless oil; 1H NMR (CD3OD, 400 MHz)
d
7.65 (d, J ¼ 16.0 Hz, 1H,
H-3), 7.32 (t, J ¼ 7.8 Hz, 1H, AreH), 7.18 (d, J ¼ 7.8 Hz, 1H, AreH), 7.15
(s,1H, H-5), 6.98 (d, J ¼ 7.8 Hz,1H, AreH), 6.52 (d, J ¼ 16.0 Hz,1H, H-
2), 5.11 (brs, 1H, H-10), 3.93 (brs, 1H), 3.82 (s, 3H, OMe), 3.76e3.55
0
5
10
15
20
25
[I] µM
(m, 3H), 1.99 (m, 2H, H-60); 13C NMR (CD3OD, 100 MHz)
d 167.4,
Fig. 9. Secondary plot of intercept vs [I] for determination of Ki0 of 6a against sucrase.
161.6, 146.7, 137.0, 131.1, 121.9, 118.9, 117.6, 114.1, 75.9, 73.4, 73.0,
71.5, 70.8, 55.9, 32.9; HRESIMS m/z 347.1110 [M þ Na]þ (calcd for
[C16H16NaO7]þ 347.1107).
(d, J ¼ 8.2 Hz, 1H), 6.08 (d, J ¼ 15.9 Hz, 1H), 5.35 (m, 1H), 4.21
(m, 1H), 4.14 (m, 1H), 3.68 (s, 3H), 3.56 (m, 1H), 3.45 (m, 1H), 2.10e
1.91 (m, 2H), 1.37 (s, 3H), 1.29 (s, 6H), 1.21 (s, 3H), 0.83 (s, 9H),
0.00 (s, 6H).
4.1.6.4. 10S-Quercityl-3-methoxycinnamate (2b). 73% yield of color-
less oil; 1H NMR (CD3OD, 400 MHz)
d
7.72 (d, J ¼ 16.0 Hz, 1H, H-3),
7.31 (t, J ¼ 7.8 Hz, 1H, AreH), 7.18 (d, J ¼ 8.2 Hz, 1H, AreH), 7.15 (s,
1H, H-5), 6.97 (dd, J ¼ 8.2, 1.6 Hz, 1H, H-9), 6.54 (d, J ¼ 16.0 Hz, 1H,
H-2), 4.85 (m, 1H, H-10), 4.11 (s, 1H), 3.82 (s, 3H, OMe), 3.64e3.33
4.1.5.15. 8S. 79% yield of colorless oil; 1H NMR (CDCl3, 400 MHz)
d
7.48 (d, J ¼ 15.9 Hz, 1H), 6.95 (dd, J ¼ 8.3, 2.0 Hz, 1H), 6.90 (d,
J ¼ 2.0 Hz, 1H), 6.68 (d, J ¼ 8.3 Hz, 1H), 6.18 (d, J ¼ 15.9 Hz, 1H), 5.20
(m, 1H), 4.28 (m, 1H), 4.13 (m, 1H), 3.72 (m, 1H), 3.68 (s, 3H), 3.34
(m, 1H), 2.27 (m, 1H), 1.83 (m, 1H), 1.38 (s, 3H), 1.30 (s, 3H), 1.28 (s,
3H), 1.21 (s, 3H), 0.84 (s, 9H), 0.00 (s, 6H).
(m, 3H), 2.01 (m, 2H, H-60); 13C NMR (CD3OD, 100 MHz)
d 167.8,
161.6, 146.6, 137.2, 131.1, 121.8, 119.2, 117.4, 114.1, 76.1, 73.6, 72.2,
71.6, 70.8, 55.8, 32.9; HRESIMS m/z 347.1107 [M þ Na]þ (calcd for
[C16H20NaO7]þ 347.1107).
4.1.6. General deprotection of silyl groups and bis-acetonides
Deprotection of 6Re8S, which contained both silyl groups and
bis-acetonides, was performed as follow. To a solution of 6R (1 eq)
in tetrahydrofuran (THF) was added TBAF/THF 1.0 M (2 eq: 1
OTBDMS). The mixture was stirred at room temperature for 3 h. The
reaction mixture was washed with water (10 mL) and the organic
layer was extracted with ethyl acetate (EtOAc) (3 ꢀ 15 mL) followed
by being dried over Na2SO4. After filtration and removal of the
solvent under reduced pressure, the crude product was dissolved in
methanol (MeOH) and amberlyst-15 (0.5 g: 0.1 mmol) was added
into the solution. The reaction mixture was stirred at room tem-
perature for 5 h. After filtration and removal of the solvent under
reduced pressure the crude product was purified by Sephadex LH-
20 eluted with MeOH to afford 6a. Compounds 6Se8S were treated
using aforementioned procedures to yield corresponding products
6be8b, respectively.
For deprotection of 1Re5R, which contained only bis-
acetonides, general procedures were conducted as follow. To a
methanolic solution of 1R was added amberlyst-15 (0.5 g:
0.1 mmol), and the reaction mixture was stirred at room temper-
ature for 5 h. After filtration and removal of the solvent under
reduced pressure, the crude product was purified by Sephadex LH-
20 eluted with MeOH to afford 1a. Compounds 1Se5R were
transformed, using the above procedures, to corresponding prod-
ucts 1be5a, respectively.
4.1.6.5. 10R-Quercityl-4-methoxycinnamate (3a). 78% yield of white
solid; 1H NMR (CD3OD, 400 MHz)
d
7.64 (d, J ¼ 15.9 Hz, 1H, H-3),
7.56 (d, J ¼ 8.6 Hz, 2H, H-5 and H-9), 6.95 (d, J ¼ 8.6 Hz, 2H, H-6 and
H-8), 6.37 (d, J ¼ 15.9 Hz, 1H, H-2), 5.11 (m, 1H, H-10), 3.96e3.59 (m,
4H), 3.83 (s, 3H, OMe), 1.99 (m, 2H, H-60); 13C NMR (CD3OD,
100 MHz)
d 167.8, 163.3, 146.6, 131.1, 131.1, 128.2, 115.9, 115.5, 115.5,
76.0, 73.5, 72.8, 71.5, 70.8, 55.9, 33.0; HRESIMS m/z 347.1104
[M þ Na]þ (calcd for [C16H20NaO7]þ 347.1107).
4.1.6.6. 10S-Quercityl-4-methoxycinnamate (3b). 78% yield of
colorless oil; 1H NMR (CD3OD, 400 MHz)
d
7.61 (d, J ¼ 15.9 Hz, 1H,
H-3), 7.47 (d, J ¼ 8.5 Hz, 2H, H-5 and H-9), 6.86 (d, J ¼ 8.5 Hz, 2H,
H-6 and H-8), 6.31 (d, J ¼ 15.9 Hz, 1H, H-2), 4.85 (m, 1H, H-10), 4.01
(s, 1H), 3.74 (s, 3H, OMe), 3.56e3.31 (m, 3H), 1.98e1.83 (m, 2H, H-
60); 13C NMR (CD3OD, 100 MHz)
d 168.3, 163.3, 146.5, 131.0, 131.0,
128.4, 116.2, 115.5, 115.5, 76.1, 73.6, 72.3, 71.4, 70.8, 55.9, 32.9;
HRESIMS m/z 347.1103 [M þ Na]þ (calcd for [C16H20NaO7]þ
347.1107).
4.1.6.7. 10R-Quercityl-3,4-dimethoxycinnamate (4a). 85% yield of
colorless oil; 1H NMR (CD3OD, 400 MHz)
d
7.62 (d, J ¼ 15.9 Hz, 1H,
H-3), 7.22 (s, 1H, H-5), 7.17 (d, J ¼ 8.2 Hz, 1H, AreH), 6.97 (d,
J ¼ 8.2 Hz, 1H, AreH), 6.40 (d, J ¼ 15.9 Hz, 1H, H-2), 5.11 (m, 1H, H-
10), 3.97e3.56 (m, 4H), 3.86 (s, 6H, 2 ꢀ OMe), 2.00 (m, 2H, H-60); 13C
NMR (CD3OD, 100 MHz)
d 167.8, 153.0, 150.8, 146.9, 128.7, 124.2,
116.3, 112.7, 111.9, 76.0, 73.5, 72.9, 71.5, 70.8, 56.8, 56.5, 33.0;
HRESIMS m/z 377.1203 [M þ Na]þ (calcd for [C17H22NaO8]þ
377.1212).
4.1.6.1. 10R-Quercityl-2-methoxycinnamate (1a). 75% yield of color-
less oil; 1H NMR (CD3OD, 400 MHz)
d
7.97 (d, J ¼ 16.2 Hz, 1H, H-3),
7.56 (d, J ¼ 6.8 Hz, 1H, AreH), 7.38 (t, J ¼ 7.4 Hz, 1H, AreH), 7.03 (d,
J ¼ 6.8 Hz, 1H, AreH), 6.96 (t, J ¼ 7.4 Hz, 1H, AreH), 6.53 (d,
J ¼ 16.2 Hz, 1H, H-2), 5.10 (m, 1H, H-10), 3.94 (brs, 1H), 3.89 (s, 3H,
OMe), 3.84e3.60 (m, 3H), 1.99 (m, 2H, H-60); 13C NMR (CD3OD,
4.1.6.8. 10S-Quercityl-3,4-dimethoxycinnamate (4b). 79% yield of
colorless oil; 1H NMR (CD3OD, 400 MHz)
d
7.60 (d, J ¼ 15.9 Hz, 1H,
H-3), 7.12 (s, 1H, H-5), 7.09 (d, J ¼ 8.2 Hz, 1H, AreH), 6.88 (d,
J ¼ 8.2 Hz, 1H, AreH), 6.33 (d, J ¼ 15.9 Hz, 1H, H-2), 4.82 (m, 1H, H-
10), 4.01 (brs, 1H), 3.77 (s, 6H, 2 ꢀ OMe), 3.52e3.25 (m, 3H), 2.05e
100 MHz) d 168.0, 160.0, 142.1, 133.2, 130.0, 124.2, 121.9, 118.8, 112.5,
76.0, 73.5, 72.9, 71.5, 70.8, 56.2, 32.9; HRESIMS m/z 347.1100
[M þ Na]þ (calcd for [C16H16 Na O7]þ 347.1107).
1.85 (m, 2H, H-60); 13C NMR (CD3OD, 100 MHz)
d 168.2, 152.9, 150.8,

E. Rattanangkool et al. / European Journal of Medicinal Chemistry 66 (2013) 296e304
303
146.7, 128.9, 124.0,116.5, 112.7, 111.6, 76.1, 73.6, 72.3, 71.4, 70.8, 56.5,
1.92 (m, 2H, H-60); 13C NMR (CD3OD, 100 MHz)
146.9, 129.0, 122.8, 116.2, 114.8, 112.6, 76.1, 73.6, 72.3, 71.4, 70.9,
d
168.3, 151.6, 148.1,
56.5, 33.0; HRESIMS m/z 377.1209 [M
þ
Na]þ (calcd for
[C17H22NaO8]þ 377.1212).
56.5, 32.9; HRESIMS m/z 363.1056 [M
þ
Na]þ (calcd for
[C16H20NaO8]þ 363.1056).
4.1.6.9. 10R-Quercityl-3,4,5-trimethoxycinnamate (5a). 83% yield of
colorless oil; 1H NMR (CD3OD, 400 MHz)
d
7.62 (d, J ¼ 15.9 Hz, 1H,
4.2.
a-Glucosidase inhibitory activity
H-3), 6.93 (s, 1H, AreH), 6.91 (s, 1H, AreH), 6.48 (d, J ¼ 15.9 Hz, 1H,
H-2), 5.12 (m, 1H, H-10), 4.05e3.55 (m, 4H), 3.86 (s, 9H, 3 ꢀ OMe),
Inhibitory activity of test compound against
a
-glucosidase from
2.00 (m, 2H, H-60); 13C NMR (CD3OD, 100 MHz)
d
167.5, 154.9, 146.8,
baker’s yeast was assessed based on p-nitrophenoxide colorimetric
method [12]. Briefly, A 10 L of test compound (1 mg/mL in DMSO)
was pre-incubated with 40 L of -glucosidase (0.1 U/mL in 0.1 M
phosphate buffer, pH 6.9) at 37 ꢁC for 10 min. The mixture was
added with 50 L substrate solution (1 mM p-nitrophenyl-
glucopyranoside, PNPG) and incubated for additional 20 min. The
resulting mixture was quenched by adding 100 L of 1 M Na2CO3. p-
146.4,131.5, 118.1, 118.0, 106.9, 106.8, 76.0, 73.5, 73.0, 71.5, 70.8, 61.2,
56.8, 56.8, 33.0; HRESIMS m/z 407.1310 [M þ Na]þ (calcd for
[C18H24NaO9]þ, 407.1318).
m
m
a
m
a-D-
4.1.6.10. 10R-Quercitylcaffeate (6a). 84% yield of yellow oil; 1H NMR
(CD3OD, 400 MHz)
d
7.45 (d, J ¼ 15.9 Hz, 1H, H-3), 6.95 (d, J ¼ 1.6 Hz,
m
1H, H-5), 6.86 (dd, J ¼ 8.2, 1.6 Hz, 1H, H-9), 6.69 (d, J ¼ 8.2 Hz, 1H, H-
8), 6.16 (d, J ¼ 15.9 Hz, 1H, H-2), 5.00 (m, 1H, H-10), 3.83 (brs, 1H),
3.68e3.45 (m, 3H), 1.89 (m, 2H, H-60); 13C NMR (CD3OD, 100 MHz)
Nitrophenoxide ion liberated from the enzymatic reaction was
monitored at 405 nm by Bio-Rad 3550 microplate reader. The
percentage inhibition was calculated by [(A0 ꢃ A1)/A0] ꢀ 100, where
A1 and A0 are the absorbance with and without the sample,
respectively. The IC50 value was deduced from a plot of percentage
inhibition versus sample concentration and acarbose was used as a
positive control.
d
168.0, 149.7, 147.4, 146.9, 127.7, 123.1, 116.6, 115.3, 114.9, 76.0, 73.5,
72.8, 71.6, 70.8, 32.9; HRESIMS m/z 325.0930 [M ꢃ H]ꢃ (calcd for
[C15H17O8]ꢃ 325.0923).
4.1.6.11. 10S-Quercitylcaffeate (6b). 84% yield of yellow oil; 1H NMR
The inhibitory activity of the test compounds against a-gluco-
(CD3OD, 400 MHz)
d
7.51 (d, J ¼ 15.9 Hz, 1H, H-3), 6.95 (d, J ¼ 1.8 Hz,
sidases from rat intestine (as maltase and sucrase) was validated on
the basis of glucose oxidase colorimetric method [13]. Maltase and
sucrase was obtained from rat intestinal acetone powder (Sigma, St.
Louis). Generally, the powder (1 g) was homogenized with 0.9%
NaCl solution (30 mL). The aliquot containing both maltase and
sucrase was obtained upon centrifugation (12,000 g) for 30 min.
1H, H-5), 6.88 (dd, J ¼ 8.1, 1.8 Hz, 1H, H-9), 6.68 (d, J ¼ 8.1 Hz, 1H, H-
8), 6.20 (d, J ¼ 15.9 Hz, 1H, H-2), 4.81 (m. 1H, H-10), 4.00 (brs, 1H),
3.54e3.25 (m, 3H), 1.99e1.78 (m, 2H, H-60); 13C NMR (CD3OD,
100 MHz) d 168.5, 148.7, 147.3, 146.9, 127.8, 123.0, 116.5, 115.2, 115.2,
76.1, 73.6, 72.3, 71.3, 70.8, 32.9; HRESIMS m/z 349.0899 [M þ Na]þ
(calcd for [C15H18NaO8]þ 349.0899).
The test compound (1 mg/mL, 10
enzyme solution (as maltase, 20
m
L) was pre-incubated with crude
mL; as sucrase, 20 L, respectively)
m
4.1.6.12. 10R-Quercitylferurate (7a). 79% yield of yellow less oil; 1H
at 37 ꢁC for 10 min. The substrate solution (maltose: 0.58 mM,
NMR (CD3OD, 400 MHz)
d
7.51 (d, J ¼ 15.9 Hz, 1H, H-3), 7.11 (d,
20 mL; sucrose: 20 mM, 20 mL, respectively) in 0.1 M phosphate
J ¼ 1.6 Hz, 1H, H-5), 6.99 (dd, J ¼ 8.2, 1.6 Hz, 1H, H-9), 6.72 (d,
J ¼ 8.2 Hz, 1H, H-8), 6.27 (d, J ¼ 15.9 Hz, 1H, H-2), 5.01 (m, 1H,
H-10), 3.85e3.45 (m, 4H), 3.86 (s, 3H, OMe), 1.85 (m, 2H, H-60);
buffer (pH 6.9) was therefore added to the reaction mixture and
incubated at 37 ꢁC for additional 40 min. The mixture was heated in
oven at 80 ꢁC for 15 min to quench the reaction. The concentration
of glucose released from the reaction mixture was determined by
the glucose oxidase method using a commercial glucose assay kit
(SU-GLLQ2, Human). The percentage inhibition was calculated us-
ing the above expression.
13C NMR (CD3OD, 100 MHz)
d 168.0, 150.8, 149.4, 147.3, 127.6,
124.3, 116.5, 115.3, 111.8, 76.0, 73.4, 72.8, 71.5, 70.8, 56.5, 33.0;
HRESIMS m/z 363.1057 [M þ Na]þ (calcd for [C16H20NaO8]þ
363.1056).
4.1.6.13. 10S-Quercitylferurate (7b). 79% yield of colorless oil; 1H
4.3. Kinetic study of a-glucosidase inhibition
NMR (CD3OD, 400 MHz)
d
7.58 (d, J ¼ 15.9 Hz, 1H, H-3), 7.09 (s,
1H, H-5), 6.99 (d, J ¼ 8.2 Hz, 1H, AreH), 6.72 (d, J ¼ 8.2 Hz, 1H,
AreH), 6.29 (d, J ¼ 15.9 Hz, 1H, H-2), 4.73 (m, 1H, H-10), 4.01 (brs,
1H), 3.79 (s, 3H, OMe), 3.52e3.23 (m, 3H), 1.99e1.84 (m, 2H, H-
The type of inhibition was investigated by analyzing enzyme
kinetic data using aforementioned reactions. Maltase and sucrase
activities were maintained at 0.45 and 0.09 U/mg protein, respec-
60); 13C NMR (CD3OD, 100 MHz)
d
168.5, 150.7, 149.4, 147.2, 127.8,
tively, in the presence of inhibitor (from 0 to 20.4 mM) at various
124.1, 116.6, 115.6, 111.9, 76.1, 73.6, 72.4, 71.3, 70.9, 56.5, 32.9;
HRESIMS m/z 363.1057 [M þ Na]þ (calcd for [C16H20NaO8]þ
363.1056).
concentrations of maltose ranging from 1.0 to 20.0 mM. A series of
Vmax and Km values were obtained from Y intercepts and calculated
by slope ꢀ Vmax, respectively.
4.1.6.14. 10R-Quercitylisoferulate (8a). 76% yield of yellow oil; 1H
4.4. DPPH radical scavenging
NMR (CD3OD, 400 MHz)
d
7.56 (d, J ¼ 15.9 Hz, 1H, H-3), 7.08 (s, 1H,
H-5), 7.07 (d, J ¼ 8.2 Hz, 1H, AreH), 6.95 (d, J ¼ 8.2 Hz, 1H, AreH),
6.30 (d, J ¼ 15.9 Hz, 1H, H-2), 5.10 (m, 1H, H-10), 3.92 (s, 1H), 3.89 (s,
3H, OMe), 3.77e3.58 (m, 3H), 1.99 (m, 2H, H-60); 13C NMR (CD3OD,
Radical scavenging activity was validated using DPPH colori-
metric method [14]. Briefly, a sample solution (10
trations of 0.1, 1.0 and 10.0 mg/mL) was added to 0.1 mM
methanolic solution of DPPH (150 L). The mixture was kept dark
mL at concen-
100 MHz)
d
167.8, 151.7, 148.0, 147.0, 128.8, 123.0, 115.9, 114.8, 112.6,
m
76.0, 73.4, 72.8, 71.5, 70.8, 56.4, 32.9; HRESIMS m/z 363.1054
at room temperature in incubator shaker for 15 min. The absor-
bance of the resulting solution was measured at 517 nm with a
96-well microplate reader. The percentage scavenging was
calculated by [(A0 e A1)/A0] ꢀ 100, where A0 is the absorbance
without the sample whereas A1 is the absorbance with the sam-
ple. The SC50 value was deduced from a plot of percentage scav-
enging versus sample concentration. BHA was used as a standard
antioxidant.
[M þ Na]þ (calcd for [C16H20NaO8]þ 363.1056).
4.1.6.15. 10S-Quercitylisoferulate (8b). 64% yield of yellow oil; 1H
NMR (CD3OD, 400 MHz)
d
7.54 (d, J ¼ 15.9 Hz, 1H, H-3), 6.99 (s, 1H,
H-5), 6.97 (d, J ¼ 8.2 Hz, 1H, AreH), 6.85 (d, J ¼ 8.2 Hz, 1H, AreH),
6.25 (d, J ¼ 15.9 Hz, 1H, H-2), 4.73 (m, 1H, H-10), 4.00 (brs, 1H), 3.79
(s, 3H, OMe), 3.48 (t, J ¼ 9.3 Hz,1H), 3.41e3.32 (m, 1H), 3.26 (m, 1H),

304
E. Rattanangkool et al. / European Journal of Medicinal Chemistry 66 (2013) 296e304
[5] S. Wacharasindhu, W. Worawalai, W. Rungprom, P. Phuwapraisirisan,
Acknowledgments
(þ)-proto-Quercitol, a natural versatile chiral building block for the synthesis
of the alpha-glucosidase inhibitors, 5-amino-1,2,3,4-cyclohexanetetrols, Tet-
rahedron Lett. 50 (2009) 2189e2192.
This work (DBG5380037) was supported by Thailand Research
Fund and Chulalongkorn University. ER and TD are PhD candidates
supported by Chulalongkorn University Dutsadi Phiphat Scholar-
ship and The Commission of Higher Education, respectively. PK is
an undergraduate student supported through the Development
and Promotion of Science and Technology Talents Project (DPST).
[6] W. Worawalai, E. Rattanangkool, A. Vanitcha, P. Phuwapraisirisan,
S. Wacharasindhu, Concise synthesis of (þ)-conduritol F and inositol ana-
logues from naturally available (þ)-proto-quercitol and their glucosidase
inhibitory activity, Bioorg. Med. Chem. Lett. 22 (2012) 1538e1540.
[7] W. Worawalai, S. Wacharasindhu, P. Phuwapraisirisan, Synthesis of new N-
substituted aminoquercitols from naturally available (þ)-proto-quercitol and
their alpha-glucosidase inhibitory activity, Med. Chem. Commun. 3 (2012)
1466e1470.
[8] Y. Sato, S. Itagaki, T. Kurokawa, J. Ogura, M. Kobayashi, T. Hirano, M. Sugawara,
K. Iseki, In vitro and in vivo antioxidant properties of chlorogenic acid and
caffeic acid, Int. J. Pharm. 403 (2011) 136e138.
[9] A. Hunyadi, A. Martins, T.J. Hsieh, A. Seres, I. Zupkó, Chlorogenic acid and rutin
play a major role in the in vivo anti-diabetic activity of Morus alba leaf extract
on type II diabetic rats, PLoS One 11 (2012) e50619.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi:10.1016/j.ejmech.2013.05.047.
[10] S. Adisakwattana, P. Chantarasinlapin, H. Thammarat, S. Yibchok-Anun,
A series of cinnamic acid derivatives and their inhibitory activity on intestinal
alpha-glucosidase, J. Enzym. Inhib. Med. Chem. 24 (2009) 1194e1200.
[11] R.L. Stein, Kinetics of Enzyme Action: Essential Principles for Drug Hunters,
John Wiley & Sons, Singapore, 2011.
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