A novel competitive class of α-glucosidase inhibitors: (E)-1-phenyl-3-(4-styrylphenyl)urea derivatives

Article abstract of DOI:10.1002/cbic.201000376

Competitive glycosidase inhibitors are generally sugar mimics that are costly and tedious to obtain because they require challenging and elongated chemical synthesis, which must be stereo-and regiocontrolled. Here, we show that readily accessible achiral

Full text of DOI:10.1002/cbic.201000376

DOI: 10.1002/cbic.201000376
A Novel Competitive Class of a-Glucosidase Inhibitors:
(E)-1-Phenyl-3-(4-Styrylphenyl)Urea Derivatives
Jun Young Kim,[a] Ji Won Lee,[a] Young Soo Kim,[a] Yuno Lee,[a] Young Bae Ryu,[c]
Songmi Kim,[a] Hyung Won Ryu,[a] Marcus J. Curtis-Long,[b] Keun Woo Lee,[a] Woo Song Lee,[c]
and Ki Hun Park*[a]
Competitive glycosidase inhibitors are generally sugar mimics
that are costly and tedious to obtain because they require
challenging and elongated chemical synthesis, which must be
stereo- and regiocontrolled. Here, we show that readily accessi-
ble achiral (E)-1-phenyl-3-(4-strylphenyl)ureas are potent com-
petitive a-glucosidase inhibitors. A systematic synthesis study
shows that the 1-phenyl moiety on the urea is critical for en-
suring competitive inhibition, and substituents on both termi-
nal phenyl groups contribute to inhibition potency. The most
potent inhibitor, compound 12 (IC50 =8.4 mm, Ki =3.2 mm),
manifested a simple slow-binding inhibition profile for a-glu-
cosidase with the kinetic parameters k3 =0.005256 mmꢀ1 minꢀ1,
k4 =0.003024 minꢀ1, and Kiapp =0.5753 mm.
Introduction
Owing to the involvement of specific glycosidases in virtually
all carbohydrate cleavage processes in humans, plants and ani-
mals, selective inhibition of glycosidases enacting a particular
pathway offers high therapeutic promise.[1] Glycosidase inhibi-
tors have proven useful for reducing postprandial hyperglycae-
mia by suppressing the absorption of glucose, and are thus ef-
fective for the treatment of type II diabetes and obesity.[2–4] On
the other hand, glucosidase I and II sequentially trim N-linked
oligosaccharides at the Glc(1!2)Glc and Glc(1!3)Glc linkages
within the Glc3Man9GlcNAc2 glycoprotein, respectively, liberat-
ing the three terminal glucose residues.[5] Thus, these enzymes
are critical for ensuring correct processing and hence proper
function of glycoproteins. Glycosidases are accordingly excel-
lent therapeutic targets for a diverse range of diseases includ-
ing lysosomal storage disorders,[6] cancer,[7] and special atten-
tion has been given to the treatment of HIV.[8]
intermediate closest in energy to the transition state (oxocar-
benium ion). This information provides a detailed rational basis
for design of selective and potent inhibitors and the interpre-
tation of subtle differences in reaction mechanism.[12]
Until recently, the structural design of highly potent, com-
petitive glucosidase inhibitors was mostly based on carbohy-
drate-derived natural products possessing several stereogenic
centres. This is because of their high potency and well-under-
stood structure–activity relationships. However, these all re-
quire tedious multistep synthesis from carbohydrate and non-
carbohydrate sources.[13,14] Synthesis routes are further compli-
cated by the need to control/install numerous stereocentres.
These reactions can be highly substrate dependent and hence
not amenable to the synthesis of numerous analogues from
common starting materials, which complicates SAR. For these
reasons, compounds with no obvious structural similarity to
carbohydrates are a new class of inhibitors. Importantly these
species can be simple in structure (potentially having no ster-
eocentres) and thus their synthesis is simple and readily ame-
nable to analogue production. However, as these compounds
are not constrained to bind at the active site, their mode of
action can be complex and hard to model. Thus, nonsugar
Among the various types of glucosidase inhibitors, iminosu-
gars,[9] carbasugars[10] and thiosugars[11] have received the most
attention because of their potent inhibitory activity and well
established inhibition mechanisms, which are mainly competi-
tive. Some carbocyclic glucosidase inhibitors include potent
HIV inhibitors, such as conduritol epoxides and aminoconduri-
tols, as well as conduritol A analogues, which modulate the re-
lease of insulin. Thiosugars, either synthesized or isolated from
natural sources, have also been investigated as glucosidase in-
hibitors and have widened the structural diversity of com-
pounds available from natural sources. The great potency and
specificity of iminosugars is related to their ability to mimic the
transition state for hydrolysis of natural glucosidase substrates.
Competitive inhibition is observed with many of these inhibi-
tors, suggesting that both conformational (shape) and electro-
static (charge) influences could be important in binding the
active site. Amino sugars have structural features similar to the
target enzyme’s sugar substrate or the hypothetical reactive
[a] J. Y. Kim, J. W. Lee, Y. S. Kim, Y. Lee, S. Kim, H. W. Ryu, Prof. Dr. K. W. Lee,
Prof. Dr. K. H. Park
Division of Applied Life Science (BK21 Program, EB-NCRC)
Gyeongsang National University, Jinju, 660-701 (South Korea)
Fax: (+82)55-757-0178
E-mail: khpark@gnu.ac.kr
[b] M. J. Curtis-Long
12 New Road, Nafferton, Driffield, East Yorkshire YO2 5JP (UK)
[c] Dr. Y. B. Ryu, Dr. W. S. Lee
Eco-Friendly Biomaterial Research Center, KRIBB
Jeoungup 580-185 (South Korea)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000376.
ChemBioChem 2010, 11, 2125 – 2131
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2125

K. H. Park et al.
derived inhibitors with well-defined enzyme binding modes
are of utmost interest.
We have reported a new class of sulfonamide chalcone-de-
rived noncompetitive a-glucosidase inhibitors,[15] and shown
their antitumour activities in hepatocytes[16] and trans-sialidase
inhibitory activity in vitro.[17] We were, however, concerned by
the possibility of off-target effects, and hence were keen to
find equally chemically simple species that are competitive glu-
cosidase inhibitors as we believed that this could simplify SAR,
deconvolute binding modes and ultimately lead to higher se-
lectivity through rational design. The focus of our efforts was
on the stilbene skeleton. This is for three principal reasons:
1) stilbene polyphenols are abundant in edible plants and have
been shown to display a diverse array of pharmacological ac-
tivities including antitumour and anti-inflammatory proper-
ties;[18,19] 2) resveratrol (also a stilbene derivative) is a noncom-
petitive inhibitor of a-glucosidase with an IC50 of 18.5 mm;[20]
3) our unpublished results disclosed that aminated stilbenes
can act as a new class of highly potent competitive a-glucosi-
dase inhibitors. These are achiral compounds that can be ob-
tained with simple steps economically. We thus present the
most pertinent results of an extensive initiative aimed at devel-
oping new competitive glycosidase inhibitors.
Scheme 1. Reagents and conditions: a) diethyl (4-nitrobenzyl)phosphonate,
tBuOK, DMF, room temperature, 2 h; b) Fe powder, 5% acetic acid, 958C,
1 h; c) TBAF 1m, THF, room temperature, 3 h.
upon analysis of coupling constants (3J=16.2 Hz) in 1H NMR
spectra. Both E/Z isomers have sufficiently different Rf values to
be separated by silica gel chromatography. Subsequent reduc-
tion of the nitro group to the primary amine was achieved by
using Fe powder in 5% acetic acid in EtOAc to give the parent
compounds 26–28 in quantitative yield. Deprotection of TBS
groups by using TBAF eventually led to free alcohols 1–3.
Treatment of compound 2 with iodomethane and deprotec-
tion of TBS group with TBAF gave N-methylated compound 4.
N-Acetate 5 was easily prepared by treatment of 2 with acetyl-
chloride (Scheme 2). Stilbene urea analogues in Table 2 were
obtained by addition of the corresponding isocyanates to TBS
ethers 2 and ensuing desilylation of the TBS functions. For ex-
ample, treatment of compound 2 with 2,4-difluorophenyl iso-
cyanate in CH2Cl2 and subsequent desilylation with TBAF pro-
vided stilbene urea 12 (Scheme 3).
We screened inhibitory activity using maltase, an a-glucosi-
dase (E.C. 3.2.1.20), from Saccharomyces cerevisiae. This protein
is encoded by the MAL 12 gene and belongs to the glycoside
hydrolase (GH) 13 family, to which human pancreatic a-amy-
lase also belongs. It catalyzes the hydrolysis of nonreducing
1,4-linked a-d-glucose residues with the release of a-d-glucose
and retention of configuration at the anomeric carbon. It can
also catalyze transglycosylation for the synthesis of oligosac-
charides and alkylglycosides.[21] In our present study, we report
the first nonsugar-derived competitive a-glucosidase inhibitors
with micromolar IC50 values.
Results and Discussion
As outlined above, development of a novel series of a-glucosi-
dase inhibitors began with the stilbene skeleton because of its
structural similarity to resveratrol. To determine the structural
requirements for the inhibitory activity of a-glucosidase, our
initial studies focused on aminostilbenes. This is because
amines are easily converted to other functionalities with a
wide range of chemical properties.
A general route for the preparation of aminostilbenes was
developed as outlined in Scheme 1. Hydroxybenzaldehyde de-
rivatives were treated with TBSCl to form the silylated hydroxy-
benzaldehydes 20–22. The coupling reaction of protected al-
dehydes 20–22 with diethyl (4-nitrobenzyl)phosphate was ach-
ieved with the Wadsworth–Emmons reaction. The coupling re-
agent, diethyl (4-nitrobenzyl)phosphate was obtained through
Arbusov reaction of 4-nitrobenzylbromide and triethylphos-
phite. The stereoselectivity of the CꢀC coupling reaction fav-
oured the formation of the E isomer (E/Z>7:1) as generally ob-
served. The geometrical isomer selectivity was determined for
a number of bases: NaOH, nBuLi and tBuOK; tBuOK gave the
best selectivity. Assignment of the E configuration is based
Scheme 2. Reagents and conditions: a) methyl iodide, CH2Cl2, room temper-
ature, 2 h; b) acetyl chloride, CH2Cl2, room temperature, 2 h; c) TBAF (1m),
THF, room temperature, 3 h.
Scheme 3. Reagents and conditions: a) isocyanate, CH2Cl2, room tempera-
ture, 2 h; b) TBAF (1m), THF, room temperature, 3 h.
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A Competitive Class of a-Glucosidase Inhibitors
important. Further screening showed that enhanced a-glucosi-
dase inhibitory activity was obtained by replacing the amino
group with a urea function, an excellent H-bond donor. Sur-
prisingly, introduction of the aryl urea moiety led to competi-
tive inhibition as shown for compound 6.
This phenomenon was observed in all active ureas in
Table 2. As shown in Table 2, all stilbene ureas 6–19 exhibited
a dose dependent inhibitory effect on the hydrolysis activity of
a-glucosidase (IC50 8.4–104.7 mm). To determine the specificity
of compounds 6–19 for a-glucosidase, their inhibitory activi-
ties against the related enzymes b-glucosidase, a-galactosi-
dase, a-mannosidase and a-amylase were determined. None of
the inhibitors showed any activity against any of these hydro-
lases at concentrations up to 200 mm. The inhibition of com-
pound 12 is demonstrated in Figure 1 representatively. Once
Table 1. Inhibitory effects of stilbene derivatives 1–6 on a-glucosidase
activity.[a]
Compound
IC50
Inhibition type
[mm]
(Ki [mm])
1
2
3
4
5
6
>200
NT[b]
46.4
102.4
>200
>200
42.1
noncompetitive (35.1)
NT
NT
NT
competitive (10.6)
26.4
noncompetitive (21.5)
[a] Effect of compounds 1–6 on the a-glucosidase catalyzed hydrolysis of
p-nitrophenyl-a-d-glucopyranoside; [b] NT: not tested.
Table 2. Inhibitory effects of stilbene derivatives 6–19 on a-glucosidase
activity.[a]
Compound R1
R2
IC50 [mm]
Inhibition type (Ki [mm])
6
7
8
3-OH, 4-OH
H
42.1ꢁ1.1 competitive (10.6ꢁ0.5)
19.5ꢁ0.3 competitive (10.5ꢁ0.2)
68.5ꢁ2.2 NT[b]
3-OH, 4-OH 4-OCH3
3-OH, 4-OH 4-NH2
3-OH, 4-OH 4-CH3
3-OH, 4-OH 4-CF3
3-OH, 4-OH 4-F
3-OH, 4-OH 2-F, 4-F
3-OH, 4-OH 4-Cl
4-OH
9
63.6ꢁ1.8 NT
10
11
12
13
14
15
16
17
18
19
DNJ
28.8ꢁ0.4 competitive (12.1ꢁ0.3)
19.8ꢁ0.1 competitive (7.2ꢁ0.4)
8.4ꢁ0.4 competitive (3.2ꢁ0.3)
14.3ꢁ0.2 competitive (4.6ꢁ0.3)
29.1ꢁ0.3 competitive (17.0ꢁ1.3)
60.1ꢁ1.5 NT
Figure 1. Selectivity of inhibitor 12 with related hydrolases. Inhibitors were
tested on glycosidases (a-glucosidase, b-glucosidase, a-galactosidase, a-
mannosidase, and a-amylase). Data represent the results of three independ-
ent experiments performed with triplicates of each sample.
H
4-OH
4-OH
3-OH, 5-OH
4-OCH3
2-F, 4-F
H
18.5ꢁ1.2 competitive (9.4ꢁ0.4)
58.8ꢁ0.8 NT
3-OH, 5-OH 4-OCH3 104.7ꢁ2.4 NT
3-OH, 5-OH 2-F, 4-F
64.3ꢁ1.3 NT
39.5ꢁ0.5 competitive (18.0ꢁ1.2)
again, the activity was significantly influenced by position and
number of hydroxy groups on both the stilbene and the aryl
moieties. Direct comparison of inhibitory potencies of 12
(IC50 =8.4 mm), 16 (IC50 =18.5 mm) and 19 (IC50 =64.3 mm),
which have the same 2,4-difluoro substitution on the C ring
but differ in the substitution pattern in the stilbene moiety, im-
plies that a 4-hydroxyl group on the A phenyl ring is the most
important. Maximal activity was generally obtained with a 3,4-
dihydroxy motif in the A ring. The effects of varying substitu-
tion patterns and chemotypes on the C ring while holding sub-
stitution on the A ring invariant was also evaluated. 2,4-Difluor-
ide 12 was found to display an eightfold increase in activity,
relative to amino-bearing 8 and methyl-bearing 9. In line with
this analysis, the second and third most potent analogues in
this series bore 4-chloro (13, IC50 =14.3 mm) and 4-methoxy (7,
IC50 =19.5 mm) groups. This trend seems to follow neither
steric size (there might be a small preference for smaller
groups) nor electron density on the aromatic C ring. These
substituents might thus be interacting with H-bond donor spe-
cies on the protein otherwise there could be a polarity effect,
[a] Effect of compounds 6–19 on the a-glucosidase catalyzed hydrolysis
of p-nitrophenyl-a-d-glucopyranoside; [b] NT: not tested.
Stilbenes 1–6 in Table 1 were thus synthesized and subse-
quently tested for their inhibitory activity as documented in
the Experimental Section. Compounds showing significant in-
hibition at 42.1–46.4 mm were further investigated and their
IC50 and Ki values were calculated. Taken as an ensemble, the
following general SAR features can be deduced from the initial
compounds tested (Table 1, 1–6). The presence of two free hy-
droxyl groups in the A ring appears to be important for activi-
ty, with a 3,4-arrangement giving the best results. For example,
1 (bearing only one OH group, IC50 >200 mm) and 3 (3,5-dihy-
droxy-substituted derivative, IC50 =102.4 mm) versus 2 (3,4-di-
hydroxy-substituted derivative, IC50 =46.4 mm). Next, we inves-
tigated the effect of derivatizing the amino group. Alkylation
(4) and acetylation (5) resulted in considerable losses of poten-
cy, indicating that H-bond donor ability and number can be
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2127

K. H. Park et al.
the difluoro species being more polar than the chloro deriva-
tive and the methoxy analogue.
All inhibitors manifested a similar relationship between
enzyme activity and enzyme concentration. The inhibition of
a-glucosidase by compound 12, the most potent inhibitor
(Ki =3.2 mm), is illustrated in Figure 2, representatively. Raising
the concentrations of the inhibitors drastically lowered residual
enzyme activity. Plots of residual enzyme activity versus
enzyme concentration at different concentrations of com-
pound 12 gave a family of straight lines with a y-axis intercept
of 0; this indicates that 12 is a reversible inhibitor (see the Sup-
porting Information).
We then further characterized the inhibitory mechanism of
the synthesized stilbene derivatives. This was done by using
both Lineweaver–Burk and Dixon plots. As shown representa-
tively in Figure 2B, the kinetic plot shows that compound 12 is
a competitive inhibitor. This is because increasing concentra-
tion of 12 resulted in a family of lines with a common inter-
cept on the 1/V axis but with different gradients (Figure 2A).
The Ki value of the most potent inhibitor 12 was determined
to be 3.2 mm (Table 2) by using a Dixon plot (see the Support-
ing Information).
To further investigate the inhibition mechanism, time de-
pendence of the inhibition of the hydrolysis of p-nitrophenol-
a-d-glucopyranoside by these inhibitors was subsequently
probed. The enzyme showed no loss in activity over this time
range: this indicates that the enzyme is stable and steady state
is maintained throughout. This was achieved by measuring ini-
tial velocities of substrate hydrolysis as a function of preincu-
bation time of the enzyme with the inhibitor. As a decrease in
residual activity was seen as a function of preincubation time,
compound 12 emerged to be a slow-binding inhibitor at
12.5 mm (Figure 2B). Increasing concentrations of compound
12 led to a decrease in both the initial velocity (vi) and the
steady-state rate (vs; Figure 2B, insert). Figure 2C shows repre-
sentatively that residual enzyme activity decreases as a func-
tion enzyme/inhibitor incubation time. This relationship is typi-
cal for slow binding inhibitors: time dependence of the rate of
hydrolysis by a-glucosidase was measured in the presence of
different concentrations of 12 (0, 5, 7.5, 10, 12.5, 15, and
20 mm). As shown in Figure 2B and C, the time dependent re-
sidual activities obtained by using the differing concentrations
of the inhibitors were fitted to Equations (1) and (2) to deter-
mine kobs
:
½Pꢂt
ðviꢀvsÞ
obst
ð1ꢀeꢀk
Þ
Figure 2. A) Lineweaver–Burk plots for the inhibition of the hydrolysis activi-
ð1Þ
ð2Þ
¼ vst þ
kobs
½Eꢂ
!
~
*
*
ty of a-glucosidase by compound 12 ( : 0, : 7.5, : 10, : 15 mm). Condi-
tions were as follows: 75, 150, 225 mm p-nitrophenyl-a-d-glucopyranoside,
0.05 units of a-glucosidase, 0.05m potassium phosphate buffer (pH 6.8) at
v
¼ eꢀk
obst
v0
!
~
:
*
*
378C. B) Inhibition as a function of preincubation time ( : 0, : 5, : 15,
&
&
*
30, : 45, : 60 min) for active compound 12 at 12.5 mm [Eq. (2)] Inset: : 0,
!
*
:
: 12.5 mm. C) Time course for slow-binding inhibition of compound 12 (
~
!
&
&
^
*
0, : 5, : 7.5, : 10, : 12.5, : 15, : 20 mm). The kobs values at each inhibi-
tion concentration were determined by fitting the data to Equation (1).
D) Plot of kobs as a function of inhibitor 12 concentration for a slow-binding
inhibitor fitted by Equations (3) and (4).
where [P]t is the concentration of product formed, [E] is the
total enzyme concentration, vi is the initial velocity, vs is the
steady state velocity, t is time, kobs is the exponential rate con-
stant for equilibration.[22]
Then, we identified which of the four possible inhibition
modes (uninhibited, simple reversible slow binding, enzyme
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A Competitive Class of a-Glucosidase Inhibitors
(E)-(5-(4-Nitrostyryl)-1,3-phenylene)bis(oxy)bis(tert-butyldime-
thylsilane) (24): H NMR (300 MHz, CDCl3): d=0.24 (s, 12H), 1.02 (s,
isomerization, and mechanism-based inhibition) that could
give this kinetic profile was at play. Slow binding inhibition
mechanisms can be investigated by preincubation of the
enzyme with inhibitor followed by measurement of residual
active enzyme as a function of incubation time. The parame-
ters k3, k4 and Kiappt can be fitted to Equations (3) and (4).[22]
1
18H), 6.84 (d, J=4.2 Hz, 1H), 6.67 (d, J=2.1 Hz, 2H), 7.06 (d, J=
16.2 Hz, 1H), 7.15 (d, J=16.2 Hz, 1H), 7.63 (d, J=9.0 Hz, 2H),
8.22 ppm (d, J=9.0 Hz, 2H); 13C NMR (75 MHz, CDCl3): d=149.2,
147.3, 145.7, 144.4, 132.7, 129.5, 125.6, 124.3, 124.0, 121.1, 119.8,
118.9, 26.9, 18.5, ꢀ4.01 ppm; EIMS m/z: 485.
(E)-(4-(4-Nitrostyryl)-1,2-phenylene)bis(oxy)bis(tert-butyldime-
thylsilane) (25): H NMR (300 MHz, CDCl3): d=0.22 (s, 12H), 1.01(s,
k4½Iꢂ
1
kobs ¼ k4 þ
ð3Þ
ð4Þ
Kai ppt
18H), 6.84 (d, J=9.0 Hz, 1H), 6.92 (d, J=16.2 Hz, 1H), 7.04 (m, 2H),
7.14 (d, J=16.2 Hz, 1H), 7.59 (d, J=8.7 Hz, 2H), 8.19 ppm (d, J=
8.7 Hz, 2H); 13C NMR (75 MHz, CDCl3): d=148.1, 147.2, 146.4, 144.3,
133.2, 129.9, 126.5, 124.3, 124.1, 121.3, 120.8, 119.6, 25.7, 18.2,
ꢀ4.20 ppm; EIMS m/z: 485.
k4
Ki ¼
k3
We established that the progress curves yielded the follow-
ing: k3 =0.005256 mmꢀ1 minꢀ1, k4 =0.003024 minꢀ1, and Kiappt
=
General procedure for the reduction of the nitro group (26–28):
Fe powder (0.22 g, 4.0 mmol) in 5% acetic acid (15 mL) was stirred
at 958C for 30 min and cooled to 508C. The solution of nitrostil-
bene (0.39 g, 0.8 mmol) in EtOAc (5 mL) was added to the reaction
mixture. The mixture was stirred at 958C for an additional 1 h.
After being cooled to room temperature, the mixture was filtered
through Celite to remove Fe powder. The filtrate was quenched
with saturated NaHCO3 (30 mL) and extracted with EtOAc (3ꢁ
100 mL). The combined organic phase was washed with brine and
dried (Na2SO4), and the solvent was evaporated. The product was
purified by silica chromatography by using hexane/EtOAc (2:1) as
eluent to give 87% yields of aminostilbenes (27–29).
0.5753 mm. This is a characteristic feature of 12, which indicates
simple reversible slow binding mode (Figure 2B–D). A plot of
kobs versus inhibitor concentration displays a linear depend-
ence (Figure 2D), which is consistent with the single-step
mechanism:
k3
Enz þ I
Enz-I
G
H
k4
Conclusions
(E)-4-(4-(tert-Butyldimethylsilyloxy)styryl)benzenamine
(26):
In summary, we have been able to develop a range of highly
potent competitive inhibitors of a-glucosidase. These are some
of the first nonsugar-derived competitive a-glucosidase inhibi-
tors with very low (<10) micromolar IC50 values. SAR indicates
favourable substituent arrangements on both the A and
C rings and that the urea chemotype is required to ensure
competitive inhibition. We believe that these inhibitors are im-
portant lead compounds as their synthesis is simple and a
wide range can be made for developing a series of simple and
highly potent inhibitors of a-glucosidase.
1H NMR (300 MHz, [D6]DMSO): d=0.21 (s, 6H), 1.01 (s, 9H), 5.30
(brs, 2H), 6.58 (d, J=8.4 Hz, 2H), 6.84 (d, J=8.7 Hz, 2H), 6.95 (d,
J=16.5 Hz, 2H), 7.26 (d, J=8.4 Hz, 2H), 7.41 ppm (d, J=8.7 Hz,
2H); 13C NMR (75 MHz, [D6]DMSO): d=153.8, 148.4, 131.4, 127.3,
126.9, 124.9, 122.3, 119.98, 113.8, 25.5, 17.9, ꢀ4.5 ppm; EIMS m/z:
325.
(E)-4-(3,4-Bis(tert-butyldimethylsilyloxy)styryl)benzenamine (27):
1H NMR (300 MHz, [D6]DMSO): d=0.02 (s, 12H), 0.77 (s, 18H), 5.07
(s, 2H), 6.35 (d, J=8.4 Hz, 2H), 6.61 (m, 3H), 6.75 (m, 2H), 6.81 (dd,
J=8.4, 1.8 Hz, 1H), 7.05 ppm (d, J=8.4 Hz, 2H); 13C NMR (75 MHz,
[D6]DMSO): d=148.4, 146.1, 145.0, 131.35, 127.3, 124.8, 122.5,
120.9, 119.1, 118.3 113.8, 25.7, 18.1212, 25.7, 18.1, ꢀ4.22 ppm; EIMS
m/z: 455.
Experimental Section
(E)-4-(3,5-Bis(tert-butyldimethylsilyloxy)styryl)benzenamine (28):
1H NMR (300 MHz, [D6]DMSO): d=0.24 (s, 12H), 1.02 (s, 18H), 5.34
(s, 2H), 6.12 (d, J=3.9 Hz, 1H), 6.54 (d, J=8.4 Hz, 2H), 6.61 (d, J=
2.1 Hz, 2H), 6.79 (d, J=16.2 Hz, 1H), 6.96 (d, J=16.2 Hz, 1H),
7.27 ppm (d, J=8.4 Hz, 2H); 13C NMR (75 MHz, [D6]DMSO): d=
156.1, 148.8, 140.2, 129.5, 127.8, 124.4, 122.4, 113.73, 110.8, 109.8,
25.5, 17.96, ꢀ4.50 ppm.
General procedure for deprotection of the TBS group: Bu4NF
(1.0m in THF, 1.5 mL, 1.5 mmol) was added to a solution of TBS-
protected stilbene (0.36 g, 0.7 mmol) in THF (5 mL). After being
stirred for 4 h at room temperature, the mixture was quenched
with water (25 mL) and extracted with EtOAc (3ꢁ30 mL). The com-
bined organic phase was washed with brine and dried (Na2SO4),
and the solvent was evaporated. The product was purified by silica
chromatography by using hexane/EtOAc (1:3) as eluent to give
95% yields of aminostilbenes (1–3).
General: All the reagent-grade chemicals were purchased from
Sigma–Aldrich. All solvents used for isolation were of analytical
grade.
General procedure for the coupling reaction (23–25): tBuOK
(0.31 g, 2.76 mmol) was added to a solution of diethyl (4-nitroben-
zyl)phosphonate (0.58 g, 0.5 mmol) in DMF (10 mL), and this mix-
ture was stirred under a nitrogen atmosphere for 20 min. The solu-
tion of aldehyde 20–22 (2.33 mmol) in cosolvent of DMF/THF (2:1)
was added to the reaction mixture and stirred for 2 h. The mixture
was diluted with water (100 mL) and extracted with EtOAc (3ꢁ
100 mL). The combined organic phase was washed with water and
dried (Na2SO4), and the solvent was evaporated. The product was
purified by silica chromatography by using hexane/EtOAc (4:1) as
eluent to give 70% yields of nitrostilbenes (23–25) as E isomer.
(E)-tert-Butyldimethyl(4-(4-nitrostyryl)phenoxy)silane
(23):
1H NMR (300 MHz, CDCl3): d=0.07 (s, 6H), 0.83 (s, 9H), 6.69 (d, J=
8.4 Hz, 2H), 6.84 (d, J=16.2 Hz, 1H), 7.05 (d, J=16.2 Hz, 1H), 7.27
(d, J=8.4 Hz, 2H), 7.42 (d, J=8.7 Hz, 2H), 8.03 ppm (d, J=8.4 Hz,
2H); 13C NMR (75 MHz, CDCl3): d=161.1, 148.8, 137.5, 134.0, 132.9,
131.0, 128.8, 128.7, 125.1, 125.0, 30.2, 22.8, 0.1 ppm; EIMS m/z: 355.
(E)-4-(4-Aminostyryl)phenol (1): M.p. 119–1218C; 1H NMR
(300 MHz, [D6]DMSO): d=5.16 (s, 2H; NH2), 6.51 (d, J=8.4 Hz, 2H),
6.70 (d, J=8.7 Hz, 2H), 6.78 (d, 2H), 7.18 (d, J=8.4 Hz, 2H), 7.29 (d,
J=8.7 Hz, 2H), 9.38 ppm (s, 1H; OH); 13C NMR (75 MHz, [D6]DMSO):
ChemBioChem 2010, 11, 2125 – 2131
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chembiochem.org
2129

K. H. Park et al.
d=156.8, 148.6, 129.5, 127.6, 127.5, 126.4, 125.7, 123.4, 115.9,
114.4 ppm; HREIMS calcd for C14H13NO: 211.0997 [M]+; found
211.0999.
(E)-1-(4-(3,4-Dihydroxystyryl)phenyl)-3-(4-trifluoromethylphen-
yl)urea (10): M.p. 168–1708C; 1H NMR (300 MHz, [D6]DMSO): d=
6.71(d, J=8.1 Hz, 1H), 6.84 (m, 2H), 6.92 (d, J=16.8 Hz, 2H), 7.13
(m, 2H), 7.48 (m, 6H), 8.73 (s, 2H), 9.00 ppm (brs, 2H); 13C NMR
(75 MHz, [D6]DMSO): d=113.6, 116.2, 118.3, 118.8, 119.0, 125.1,
126.5, 126.6, 127.0, 127.5, 129.4, 132.1, 138.7, 143.9, 145.8, 145.9,
152.7 ppm; HREIMS calcd for C22H17F3N2O3: 414.1189 [M]+; found
414.1189.
(E)-5-(4-Aminostyryl)benzene-1,3-diol (2): M.p. 208–2108C;
1H NMR (300 MHz, CD3OD): d=6.12 (t, 1H), 6.41 (d, J=2.4 Hz, 2H),
6.67 (d, J=8.4 Hz, 2H), 6.73 (d, J=16.2 Hz, 1H), 6.91 (d, J=16.2 Hz,
1H), 7.26 ppm (d, J=8.4 Hz, 2H); 13C NMR (75 MHz, [D6]DMSO): d=
159.6, 148.7, 141.6, 129.9, 128.8, 128.6,125.7, 116.5, 105.7,
102.4 ppm; HREIMS calcd for C14H13NO2: 227.0946 [M]+; found
227.0946.
(E)-1-(4-(3,4-Dihydroxystyryl)phenyl)-3-(4-fluorophenyl)urea (11):
M.p. 156–1588C; 1H NMR (300 MHz, [D6]DMSO): d=6.74 (d, J=
8.1 Hz, 1H), 6.86 (m, 2H), 6.98 (d, J=16.8 Hz, 2H), 7.13 (m, 2H),
7.48 (m, 6H), 8.73 (s, 2H), 9.00 ppm (brs, 2H); 13C NMR (75 MHz,
[D6]DMSO): d=152.5, 145.3, 138.6, 136.0, 131.3, 128.9, 126.8, 126.5,
124.7, 120.0, 118.3, 118.2, 115.7, 115.4, 115.1, 113.1 ppm; HREIMS
calcd for C21H17FN2O3: 364.1223 [M]+; found 364.1219.
(E)-4-(4-Aminostyryl)benzene-1,2-diol (3): M.p. 140–1428C;
1H NMR (300 MHz, [D6]DMSO): d=5.19 (s, 2H, NH2), 6.50 (d, J=
8.1 Hz, 2H), 6.65 (m, 4H), 6.87 (s, 1H), 7.17 (d, J=8.1 Hz, 2H), 8.84
(s, 1H), 8.91 ppm (s, 1H); 13C NMR (75 MHz, [D6]DMSO): d=148.1,
145.3, 144.6, 129.6, 127.1, 125.8, 125.3, 123.3, 117.7, 115.7, 113.9,
112.7 ppm; HREIMS calcd for C14H13NO2: 227.0946 [M]+; found
227.0944.
(E)-1-(2,4-Difluorophenyl)-3-(4-(3,4-dihydroxystyryl)phenyl)urea
(12): M.p. 210–2128C; 1H NMR (300 MHz, [D6]DMSO): d=6.75(d,
J=8.1 Hz, 1H), 6.88 (m, 2H), 7.00 (d, J=16.2 Hz, 2H), 7.05 (m, 1H),
7.35 (m, 1H), 7.45 (m, 4H), 8.13 (m, 1H), 8.55 (s, 1H), 8.96 (s, 1H),
9.11 ppm (s, 2H); 13C NMR (75 MHz, [D6]DMSO): d=152.2, 145.3,
138.3, 131.5, 128.9, 126.9, 126.6, 124.7, 124.1, 123.9, 122.0, 121. 9,
118.2, 115.7, 113.1, 111.1, 110.8 ppm; HREIMS calcd for
C21H16F2N2O3: 382.1129 [M]+; found 382.1127.
General procedure for phenylureas: A solution of isocyanate
(0.12 g, 0.75 mmol) in CH2Cl2 (5 mL) was added to a solution of
aminostilbene (0.34 g, 0.75 mmol) in CH2Cl2 (20 mL) at room tem-
perature. The reaction mixture was stirred for 2 h. The product was
removed from the reaction by filtration and used crude in the next
step. Bu4NF (1m, in THF, 1.5 mL, 1.5 mmol) was added to a solution
of TBS urea in THF (10 mL) at room temperature. The mixture was
quenched with water (25 mL) and extracted with EtOAc (3ꢁ
50 mL). The combined organic phase was washed with brine and
dried (Na2SO4) and the solvent was evaporated. The product was
purified by silica chromatography by using hexane/EtOAc (1:3) as
eluent to give 85% yields of phenylureas (6–19).
(E)-1-(4-Chlorophenyl)-3-(4-(3,4-dihydroxystyryl)phenyl)urea
(13): M.p. 216–2188C; 1H NMR (300 MHz, [D6]DMSO): d=6.74 (d,
J=8.1 Hz, 1H), 6.89 (d, J=16.5 Hz, 2H), 7.00 (m, 2H), 7.33 (d, J=
8.7 Hz, 2H), 7.50 (m, 6H), 8.78 (s, 1H), 8.84 (s, 1H), 9.01 ppm (s,
2H); 13C NMR (75 MHz, [D6]DMSO): d=152.2, 145.3, 138.3, 131.5,
128.9, 126.9, 126.6, 124.7, 124.1, 123.9, 122.0, 121.9, 118.2, 115.7,
113.1, 111.1, 110.8 ppm; HREIMS calcd for C21H17ClN2O3: 380.0928
[M]+; found 380.0926.
(E)-1-(4-(3,4-Dihydroxystyryl)phenyl)-3-phenylurea (6): M.p. 139–
1418C; 1H NMR (300 MHz, [D6]DMSO): d=6.69 (d, J=8.1 Hz, 1H),
6.82 (d, J=16.2 Hz, 2H), 6.74 (m, 3H), 7.26 (t, 2H), 7.43 (m, 6H),
8.65 (s, 1H), 8.70 (s, 1H), 9.0 ppm (brs, 2H); 13C NMR (75 MHz,
[D6]DMSO): d=152.4, 145.4, 145.2, 139.6, 138.6, 131.3, 128.9, 128.7,
126.8, 126.5, 124.7, 121.8, 118.2, 118.2, 115.7, 113.1 ppm; HREIMS
calcd for C21H18N2O3: 346.1317 [M]+; found 346.1315.
(E)-1-(4-(4-Hydroxystyryl)phenyl)-3-phenylurea (14): M.p. 242–
2448C; 1H NMR (300 MHz, [D6]DMSO): d=6.74 (d, J=8.4 Hz, 1H),
6.89–6.94 (m, 3H), 7.26 (m, 2H), 7.35–7.45 (m, 8H), 8.65 (s, 1H),
8.70 (s, 1H), 9.50 ppm (s, 1H); 13C NMR (75 MHz, [D6]DMSO): d=
156.95, 152.4, 139.6, 138.7, 131.3, 128.8, 128.4, 127.5, 126.5, 126.4,
124.9, 121.8, 118.2, 118.2, 115.5 ppm; HREIMS calcd for C21H18N2O2:
330.1368 [M]+; found 330.1366.
(E)-1-(4-(3,4-Dihydroxystyryl)phenyl)-3-(4-methoxyphenyl)urea
1
(7): M.p. 143–1458C; H NMR (300 MHz, [D6]DMSO): d=3.69 (s, 3H),
6.69 (d, J=8.1 Hz, 1H), 6.86 (m, 5H), 6.94 (d, 1H), 7.33 (d, J=
8.7 Hz, 2H), 7.41 (m, 4H), 8.48 (s, 1H), 8.64 (s, 1H), 8.96 ppm (brs,
2H); 13C NMR (75 MHz, [D6]DMSO): d=154.4, 152.6, 145.4, 145.2,
138.9, 132.7, 131.0, 128.9, 126.7, 126.5, 124.8, 120.0, 118.2, 118.1,
115.7, 113.9, 113.0, 55.1 ppm; HREIMS calcd for C22H20N2O4:
376.1423 [M]+; found 376.1421.
(E)-1-(4-(4-Hydroxystyryl)phenyl)-3-(4-methoxyphenyl)urea (15):
M.p. 241–2438C; 1H NMR (300 MHz, [D6]DMSO): d=3.69 (s, 3H),
6.73 (d, J=8.4 Hz, 2H), 6.86 (d, J=8.7 Hz, 2H), 6.96 (d, J=16.5 Hz,
2H), 7.35–7.44 (m, 8H), 8.65 (s, 1H), 8.46 (s, 1H), 8.62 (s, 1H),
9.50 ppm (s, 1H); 13C NMR (75 MHz, [D6]DMSO): d=156.9, 154.5,
152.6, 138.9, 132.6, 131.1, 128.4, 127.5, 126.5, 126.3, 124.9, 120.0,
118.1, 1155, 113.96, 55.1 ppm; HREIMS calcd for C22H20N2O3:
360.1474 [M]+; found 360.1479.
(E)-1-(4-Aminophenyl)-3-(4-(3,4-dihydroxystyryl)phenyl)urea (8):
M.p. 220–2218C; 1H NMR (300 MHz, [D6]DMSO): d=4.79 (s, 2H),
6.49 (d, J=8.4 Hz, 2H), 6.69 (d, J=8.1 Hz, 1H), 6.82 (d, J=17.1 Hz,
2H), 6.93 (s, 1H), 7.05 (d, J=8.4 Hz, 2H), 7.42 (m, 4H), 8.13 (s, 1H),
8.53 (s, 1H), 8.89 (s, 1H), 9.02 ppm (s, 1H); 13C NMR (75 MHz,
[D6]DMSO): d=152.7, 145.4, 145.2, 143.9, 139.1, 130.8, 128.98,
128.5, 126.5, 124.8, 120.7, 118.2, 117.9, 115.7, 114.2, 113.0 ppm;
HREIMS calcd for C22H19N3O3: 361.1426 [M]+; found 361.1424.
(E)-1-(2,4-Difluorophenyl)-3-(4-(4-hydroxystyryl)phenyl)urea (16):
M.p. 238–2408C; 1H NMR (300 MHz, [D6]DMSO): d=6.73 (d, J=
8.1 Hz, 2H), 6.89–7.06 (m, 3H), 7.26–7.46 (m, 7H), 8.08 (m, 1H),
8.50 (s, 1H), 9.06 (s, 1H), 9.50 ppm (s, 1H); 13C NMR (75 MHz,
[D6]DMSO): d=156.98, 152.1, 138.3, 131.5, 128.3, 127.5, 126.6,
124.8, 124.1, 123.9, 121.9, 118.2, 115.5 ppm; HREIMS calcd for
C21H16F2N2O2: 366.1180 [M]+; found 366.1183.
(E)-1-(4-(3,4-Dihydroxystyryl)phenyl)-3-p-tolylurea (9): M.p. 164–
1
1668C; H NMR (300 MHz, [D6]DMSO): d=2.22 (s, 3H), 6.69 (d, J=
8.1 Hz, 1H), 6.82 (m, 2H), 6.92 (d, J=14.7 Hz, 2H), 7.06 (d, J=
8.1 Hz, 2H), 7.32 (d, J=8.1 Hz, 2H), 7.41 (m, 4H), 8.55 (s, 1H), 8.66
(s, 1H), 8.90 (s, 1H), 9.02 ppm (s, 1H); 13C NMR (75 MHz, [D6]DMSO):
d=152.4. 145.3, 145.2, 138.7, 136.98, 131.1, 130.6, 129.1, 128.9,
126.7, 126.5, 124.7, 118.2, 118.1, 115.6, 112.98, 20.2 ppm; HREIMS
calcd for C22H20N2O3: 360.1474 [M]+; found 360.1472.
(E)-1-(4-(3,5-Dihydroxystyryl)phenyl)-3-phenylurea (17): M.p.
230–2328C; H NMR (300 MHz, [D6]DMSO): d=6.12 (s, 1H), 6.40 (s,
2H), 6.87–6.94 (m, 2H), 6.98 (d, J=16.5 Hz, 1H), 7.24–7.29 (m, 2H),
7.43–7.50 (m, 6H), 8.66 (s, 1H), 8.73 (s, 1H), 9.20 ppm (s, 2H);
13C NMR (75 MHz, [D6]DMSO): d=158.5, 152.4, 139.6, 139.2, 139.0,
130.7, 128.8, 127.6, 127.0, 126.9, 121.8, 118.2, 118.2, 104.4,
1
2130
www.chembiochem.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2010, 11, 2125 – 2131

A Competitive Class of a-Glucosidase Inhibitors
101.97 ppm; HREIMS calcd for C21H18N2O3: 346.1317 [M]+; found
346.1315.
Keywords: alpha-glucosidases · drug design · inhibitors ·
structure–activity relationships · urea stilbene
(E)-1-(4-(3,5-Dihydroxystyryl)phenyl)-3-(4-methoxyphenyl)urea
(18): M.p. 223–2458C; 1H NMR (300 MHz, [D6]DMSO): d=3.70 (s,
3H), 6.11 (s, 1H), 6.39 (d, J=1.5 Hz, 2H), 6.85 (d, J=9.0 Hz, 2H),
6.92 (d, J=16.2 Hz, 2H), 7.33 (d, J=9.0 Hz, 2H), 7.40–7.48 (m, 4H),
8.46 (s, 1H), 8.65 (s, 1H), 9.20 ppm (s, 2H); 13C NMR (75 MHz,
[D6]DMSO): d=158.5, 154.5, 152.5, 139.4, 139.0, 132.6, 130.5, 127.6,
127.0, 126.7, 120.0, 118.0, 113.96, 104.4, 101.9, 55.1 ppm; HREIMS
calcd for C22H20N2O4: 376.1423 [M]+; found 376.1418.
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(19): M.p. 218–2208C; 1H NMR (300 MHz, [D6]DMSO): d=6.12 (t,
1H), 6.39 (d, J=1.8 Hz, 2H), 6.93 (d, J=16.5 Hz, 2H), 7.00–7.07 (m,
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dase (E.C. 3.2.1.22) and a-amylase (E.C. 3.2.1.1) inhibitory activities
were calculated by using standard experimental procedures with
some modifications. All enzyme activities were determined by
using the appropriate substrates (p-nitrophenyl-a-d-glucopyrano-
side, p-nitrophenyl-b-d-glucopyranoside, o-nitrophenyl-a-d-manno-
pyranoside, p-nitrophenyl-a-d-galactopyranoside, and starch, re-
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This work was supported by the National Research Foundation of
Korea (NRF) by the Korean government (Nos. 20090081751,
20090073267). All students were recipients of fellowship from the
BK21 Program of MEST.
Received: July 1, 2010
Published online on September 9, 2010
ChemBioChem 2010, 11, 2125 – 2131
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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