Phenylethyl cinnamides: A new series of α-glucosidase inhibitors from the leaves of Aegle marmelos

Article abstract of DOI:10.1016/j.bmcl.2008.08.024

A series of phenylethyl cinnamides, which included new compounds named anhydromarmeline (1), aegelinosides A (7) and B (8), were isolated from Aegle marmelos leaves as α-glucosidase inhibitors. The structures of new compounds were characterized by spectroscopic data and chemical degradation. Of compounds isolated, anhydroaegeline (2) revealed the most potent inhibitory effect against α-glucosidase with IC₅₀ value of 35.8 μM. The present result also supports ethnopharmacological use of A. marmelos as a remedy for diabetes mellitus.

Full text of DOI:10.1016/j.bmcl.2008.08.024

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4956–4958
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Phenylethyl cinnamides: A new series of
a-glucosidase inhibitors
from the leaves of Aegle marmelos
Preecha Phuwapraisirisan a, , Thanchanok Puksasook a,b, Jonkolnee Jong-aramruang c, Udom Kokpol a
*
a Natural Products Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand
b Program of Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
c Department of Chemistry, Faculty of Science, Burapha University, Chonburi 20231, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of phenylethyl cinnamides, which included new compounds named anhydromarmeline (1),
Received 26 June 2008
Revised 8 August 2008
Accepted 11 August 2008
Available online 14 August 2008
aegelinosides A (7) and B (8), were isolated from Aegle marmelos leaves as
The structures of new compounds were characterized by spectroscopic data and chemical degradation.
Of compounds isolated, anhydroaegeline (2) revealed the most potent inhibitory effect against -gluco-
sidase with IC50 value of 35.8 M. The present result also supports ethnopharmacological use of A.
marmelos as a remedy for diabetes mellitus.
a-glucosidase inhibitors.
a
l
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Phenylethyl cinnamide
Aegle marmelos
Anhydromarmeline
Aegelinosides A
Aegelinosides B
a-Glucosidase
Diabetes mellitus
Type 2 diabetes mellitus affects approximately 215 million peo-
ple worldwide. It is currently clear that aggressive control of
hyperglycemia in patients with type 2 diabetes can attenuate the
development of chronic complications such as retinopathy and
nephropathy.1 To date, therapy for type 2 diabetes relies mainly
on several approaches intended to suppress the hyperglycemia,
which include reducing gut glucose absorption.
Therefore inhibition of a-glucosidase, an enzyme catalyzing the
cleavage of glycosidic bonds in oligosaccharides or glycoconju-
gates, is a method of choice to control elevated glucose level in
blood. Although novel generations of a-glucosidase inhibitors have
cinnamides; three of which named anhydromarmeline (1), aegeli-
nosides A (7) and B (8), are newly discovered (see Fig. 1).
The air-dried leaves of A. marmelos, collected in Nakornpatom in
April 2006, were extracted with CH2Cl2 and 7:3 MeOH-H2O using
5'
4''
3''
O
O
1''
3
1'
5
3'
7'
1
5''
N
H
7
9
1
been consistently synthesized, those having multiple actions are
MeO
OH
greatly required. Recent investigations have reported the excep-
O
O
tional actions of
a
-glucosidase inhibitors from natural sources.2
N
H
Prominent examples included aegeline, a hydroxyl amide alkaloid
from Aegle marmelos leaves, which suppressed both blood glucose
and plasma triglyceride levels.3
Aegle marmelos is typically known as ‘bael’ in India. It belongs to
the family of Rutaceae, which is widely used in Indian Ayurvedic
medicine for the treatment of diabetes mellitus. Despite several
reports of its antihyperglycemic activity,4 the active principles
have not been identified. In this Letter, we describe the isolation,
characterization and glucosidase inhibitory effect of phenylethyl
5''
O
HO
HO
1'' O
HO
7
3''
OH
MeO
OH
N
H
O
O
HO
8
OH
* Corresponding author. Tel.: +662 218 7624; fax: +662 218 7598.
E-mail address: preecha.p@chula.ac.th (P. Phuwapraisirisan).
Figure 1. New metabolites isolated from A. marmelos.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.024

P. Phuwapraisirisan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4956–4958
4957
Table 1
Soxhlet extractor. The CH2Cl2 extract was dissolved in 4:1 MeOH–
1H and 13C NMR data for aegelinosides A (7, CD3OD) and B (8, acetone-d6)
H2O and partitioned with hexane. The methanolic layer was chro-
matographed on silica gel using stepwise MeOH–CH2Cl2 (0:1, 5:95,
10:90, 20:80 and 50:50), yielding five major fractions. The com-
bined fractions 1 and 2 were further purified on Sephadex LH-20
(1:9 MeOH–CH2Cl2) and silica gel (5:95 MeOH–CH2Cl2) to afford
anhydromarmeline5 (1, 14 mg), anhydroaegeline (2, 50 mg), and
(ꢀ)-tembamide (3, 8 mg). In addition, dehydromarmeline (4,
11 mg), (ꢀ)-aegeline (5, 65 mg), and (ꢀ)-O-methylether aegeline
(6, 20 mg) were also obtained from fraction 3, on purification using
silica gel (10:90 MeOH–CH2Cl2). The 7:3 MeOH–H2O extract was
loaded onto Diaion HP-20 and excessively eluted with H2O, MeOH
and acetone. The MeOH fraction was separated by VCC (stepwise
5:95, 10:90, 15:85, 50:50, 70:30 and 100:0 MeOH–CH2Cl2) The
combined fractions eluted with 15:85 and 50:50 MeOH–CH2Cl2
were subsequently purified by Sephadex LH-20 (3:7 MeOH–
CH2Cl2) followed by RPHPLC (ODS, 65:35 MeOH–H2O, UV
254 nm) to furnished aegelinosides A (7, 20 mg, tR 32.4 min) and
B (8, 10 mg, tR 27.1 min).
Position
Aegelinoside A (7)
Aegelinoside B (8)
13C
1H
13C
1H
1
2
3
4
5, 9
6, 8
7
10
166.5
120.5
140.5
135.4
127.5
128.1
129.3
46.0
166.4
124.1
136.4
135.9
129.8
128.0
129.4
45.4
6.65, d, 15.6
7.50, d, 15.6
6.05, d, 12.8
6.68, d, 12.8
7.57, m
7.35, m
7.35, m
3.57, d, 6.4
7.68, m
7.31, m
7.31, m
3.56, m
3.38, m
4.89, dd, 7.6, 4.4
20
77.0
130.0
128.0
113.7
159.6
99.5
73.7
76.3
70.4
76.6
5.00, t, 6.4
77.9
131.3
128.2
113.5
159.4
100.5
73.7
76.8
70.5
76.3
62.0
30
40, 80
50, 70
60
7.35, d, 8.8
6.92, d, 8.8
7.37, d, 8.6
6.89, d, 8.6
100
4.12, d, 7.2
3.28, m
3.23, m
3.26, m
3.09, m
3.87, dd, 11.6, 2.0
3.67, dd, 11.6. 5.6
3.78, s
4.18, d, 7.2
3.24, m
3.27, m
3.32, m
3.18, m
3.65, m
3.84, m
3.78, s
200
300
400
500
600
61.5
MeO
O
OMe
54.1
54.4
N
H
2
Anhydromarmeline (1)6 was obtained as yellow needle. The
molecular formula was established as C22H23NO2 by HRESIMS.
The 1H NMR spectrum displayed most signals in aromatic region
(6.1–7.7), in addition to the upfield resonances that are ascribable
to oxygenated prenyl moiety [dH 5.49 (m, 1H), 4.50 (d, J = 6.8 Hz),
1.80 and 1.74 (s, each 3H)].7 The 13C NMR showed 22 signals, five
of which were quarternary carbons which included resonance of
MeO
O
N
H
OH
3
O
O
N
H
MeO
O
3
N
H
J
2315.6 Hz
4
2
OH
MeO
H-2
O
H-
3
N
H
OR
5 R = H
MeO
O
J2312 .0 Hz
H-
6 R = Me
H-3
N
H
3
2
OH
2
7.50
7.00
6.50
6.00
5'
4''
3''
O
O
3
1''
Figure 4. Partial 1H NMR spectra of aglycones 5 (top) and 8a (bottom) obtained
from hydrolysis of 7 and 8, respectively.
1'
5
3'
7'
1
5''
N
H
7
9
Table 2
Figure 2. Selected HMBC correlations of 1.
a-Glucosidase inhibitory effect of isolated compounds
Compound
% Inhibition at various concentration (lg/mL)
10.0
5.0
2.5
1.25
MeO
1
2
3
4
5
6
7
8
30.1 1.32
56.5 0.64
13.5 0.68
19.7 1.59
36.0 0.34
12.6 1.05
17.6 1.07
8.8 0.39
13.2 0.25
25.2 0.91
11.4 0.60
15.9 1.05
20.9 1.40
NI
8.1 0.6
7.5 0.87
7.8 0.44
1.3 0.05
1.7 0.11
1.5 0.13
NI
5.3 0.77
NI
O
OH
N
H
NI
5''
O
HO
HO
11.2 1.11
NI
6.1 0.54
NI
1'' O
3''
OH
11.6 0.51
3.4 0.59
4.6 0.99
NI
Figure 3. Selected HMBC correlations of 7.

4958
P. Phuwapraisirisan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4956–4958
sponding hydrolysate named aegeline B (8a, Fig. 4), whose minus
140
120
100
80
26
IC50 35.8
μ
M
sign of specific rotation ([
a 20R-phenylethyl cinnamide.11
Compounds 1, 7 and 8 displayed slightly weak inhibition (30.1,
17.6 and 8.8%, respectively) against -glucosidase, even at concen-
tration of 10
g/mL (see Table 2).12 Of compounds isolated, anhyd-
roaegeline (2) turned out to be the most potent inhibitor
possessing IC50 value of 35.8 M (Fig. 5).
a
]
ꢀ20.6) was reminiscent to that of
D
a
l
60
l
40
In summary, we have isolated a variety of phenylethyl cinna-
mides, which included anhydromarmeline (1), aegelinosides A
20
(7) and
B (8), as a novel type of a-glucosidase inhibitors.
0
Although phenylethyl cinnamides have been commonly encoun-
tered in certain genus such as Aegle13 and Hibiscus,14 the pres-
ence of cis-cinnamide moiety in 8 is extraordinarily rare in
Nature. Recently Narender et al. have demonstrated that aegeline
(5), the related congener of 7 and 8, could serve as a potential
remedy for diabetes mellitus by suppressing blood glucose and
plasma triglyceride levels. Therefore they are likely to deserve
further development discovering new antidiabetes drugs having
dual functions.
0
20
40
60
80
100
Concentration (μM)
Figure 5. Inhibitory effect of anhydroaegeline (2) against
hydrolysis of p-nitrophenyl -glucopyranoside.
a
-glucosidase on
a-D
amide (dC 162.7). Interpretation of 2D NMR resulted in the con-
struction of two separated aromatic systems, which were con-
nected through amide linkage. A monosubstituted benzene [dH
7.53 (2H) and 7.38 (3H)] was connected to trans-olefinic protons
[dH 7.75 (d, J = 15.2 Hz) and 6.44 (d, J = 15.2 Hz)], which were in
turn linked to amide carbon based on HMBC correlations from H-
2 and H-3 to C-1. The other aromatic motif was assigned to a
para-substituted benzene [dH 7.28 (d, J = 8.4 Hz, 2H) and 6.86 (d,
J = 8.4 Hz, 2H)], which was accommodated by the oxygenated pre-
nyl (Me2C@CHCH2O–) and ethyleneamine (–CH@CH-NH–) moie-
ties. A large coupling constant (14.4 Hz) of olefinic protons (H-100
and H-200) pointed out that they were E-oriented. Therefore the
gross structure of anhydromarmeline (1) was depicted (see Fig. 2).
Aegelinoside A (7)8 was isolated from 7:3 MeOH–H2O extract
and displayed [M+Na]+ ion in HRESIMS at m/z 482.1781 that led
to molecular formula of C24H29NO8. The 1H NMR spectrum (Table
1) of 7 in CD3OD showed signals of aromatic and olefinic protons
in range of 6.6–7.6 (11H) and oxygenated methylenes and
methines (dH 3.0–5.2, 10H). The 13C NMR spectrum displayed 24
signals, which included resonance of amide (dC 166.5). The reso-
nances of dH 7.57 (m, 2H), 7.50 (d, J = 15.6 Hz, 1H), 7.35 (m, 3H),
and 6.65 (d, J = 15.6 Hz, 1H) were ascribable to trans-cinnamide
based on COSY and HMBC data. The signals at dH 7.35 (d,
J = 8.8 Hz, 2H) and 6.92 (d, J = 8.8 Hz, 2H) were assigned to p-disub-
stituted benzene which was accommodated by methoxy group (dH
3.78 and dC 54.1) at C-60 and oxygenated ethyl amine moiety
(–OCH-CH2-NH–) at C-30. The HMBC cross peaks (Fig. 3) observed
for H-2, H-3, and H-10 to C-1 indicated that these two separated
aromatic systems were linked through amide bond. The remaining
Acknowledgments
This work was financially supported by Thailand Research Fund
(MRG5180158) and the 90th Anniversary of Chulalongkorn Uni-
versity Fund (F-31-GS-ES13/28).
References and notes
1. Moller, D. E. Nature 2001, 414, 821.
2. For comprehensive reviews of natural a-glucosidase inhibitors, see: (a) Melo, E.
B.; Gomes, A. S.; Carvalho, I. Tetrahedron 2006, 62, 10277; (b) Watson, A. A.; Fleet,
G. W. J.; Asano, N.; Molyneux, R. J.; Nash, R. J. Phytochemistry 2001, 56, 265.
3. Narender, T.; Shweta, S.; Tiwari, P.; Reddy, K. P.; Khaliq, T.; Prathipati, P.; Puri,
A.; Srivastava, A. K.; Chander, R.; Agarwal, S. C.; Raj, K. Bioorg. Med. Chem. Lett.
2007, 17, 1808.
4. For recent reports, see (a) Kesari, A. N.; Gupta, R. K.; Singh, S. K.; Diwakar, S.;
Watal, G. J. Ethnopharmacol. 2006, 107, 374; (b) Panda, S.; Kar, A. Phytother. Res.
2006, 20, 1103; (c) Upadhya, S.; Shanbhag, K. K.; Suneetha, G.; Naidu, M. B.;
Upadhya, S. Indian J. Physiol. Pharmacol. 2004, 48, 476.
5. Although several phenylethyl cinnamides have been isolated from A. marmelos,
many of which have not been given the IUPAC or trivial names, for the sage of
communication in this Letter, we adopted terms ‘marmeline’ for phenylethyl
cinnamides having 60-phenyloxyl and ‘aegeline’ for those having 60-methoxyl.
6. Anhydromarmeline (1): UV (MeOH) kmax (loge
) 277 (4.81), 333 (4.46); 1H NMR
(400 MHz, CDCl3) d 1.74 (3H, s, H-500), 1.80 (3H, s, H-400), 4.50 (2H, d, J = 6.8 Hz,
H-100), 5.49 (1H, m, H-300), 6.14 (1H, d, J = 14.4 Hz, H-30), 6.44 (1H, d, J = 15.2 Hz,
H-2), 6.86 (2H, d, J = 8.4 Hz, H-50 and H-70), 7.28 (2H, d, J = 8.4 Hz, H-40 and H-
80), 7.38 (3H, m, H-6, H-7 and H-8), 7.53 (3H, m, H-5, H-9 and H-10), 7.75 (1H, d,
J = 15.2 Hz, H-3); 13C NMR (100 MHz, CDCl3) d 18.2 (C-500), 25.8 (C-400), 65.4 (C-
100), 113.2 (C-30), 115.0 (C-50 and C-70), 119.7 (C-2), 119.9 (C-200), 126.7 (C-40
and C-80), 128.0 (C-5 and C-9), 129.1 (C-6 and C-8), 130.1 (C-7), 132.4 (C-4),
137.5 (C-10), 138.3 (C-300), 143.8 (C-3), 158.1 (C-60), 162.7 (C-1); HRESIMS m/z
[M+Na]+ 356.1623 (calcd for C24H29NO8Na, 356.1626).
oxygenated methylenes and methines were assigned to b-D-glu-
cose residue, which was attached to C-20 based on HMBC correla-
tion from H-100 (4.12, d, J = 7.2 Hz) to C-20. Therefore overall
structure of 7 was accomplished. The absolute configuration of
C-20 was determined by chemical degradation. Hydrolysis of 7 in
7. Phuwapraisirisan, P.; Udomchotphruet, S.; Surapinit, S.; Tip-pyang, S. Nat. Prod.
Res. 2006, 20, 1332.
8. Aegelinoside A (7): [
25
a
]
ꢀ26.3 (c 0.05, MeOH); UV (MeOH) kmax (loge) 274
D
(4.73); 1H and 13C NMR (see Table 1); HRESIMS m/z [M+Na]+ 482.1781 (calcd
for C24H29NO8Na, 482.1791).
1 M HCl under reflux yielded
of which was identical in all respects, particularly optical rotation
D
-glucose and (ꢀ)-aegeline; the latter
9. Kamal, A.; Shaik, A. A.; Sandbhor, M.; Malik, M. S. Tetrahedron Asymmetry 2004,
15, 3939.
10. Aegelinoside B (8): [
25
26
25
a
]
ꢀ33.3 (c 0.05, MeOH); UV (MeOH) kmax (loge) 260
D
([a
]
ꢀ27.6), to R-aegeline (lit.[
a]
ꢀ35.9)9.
D
D
(4.43); 1H and 13C NMR (see Table 1); HRESIMS m/z [M+Na]+ 482.1786 (calcd
for C24H29NO8Na, 482.1791).
Aegelinoside B (8)10 was isomeric to 7 as evidenced by a molec-
ular formula of C24H29NO8. Although direct comparison of their 1H
and 13C NMR spectra could not be made since they were recorded
in different solvents, 8 revealed signals essentially identical to
those of 7. Significant difference observed by us was slightly up-
field olefinic protons H-2 (6.05, d, J = 12.8 Hz) and H-3 (6.68, d,
J = 12.8 Hz). A relative small coupling constant (J23 = 12.8 Hz) indi-
cated that D2 in 8 was cis-oriented instead of trans-oriented in 7.
The gross structure of 8 was subsequently confirmed by 2D NMR
data. The absolute configuration of C-20 was also deduced by chem-
11. Optically pure phenylethyl amides having only one chiral secondary alcohol
revealed different signs of specific rotation; (ꢀ) for R and (+) for S isomers. For
certain instances, see Ref. 9.
12. The inhibitory effects of isolated compounds were validated using previous
protocol with slight modification. For detailed experiment, see: Schäfer, A.;
Högger, P. Diabetes Res. Clin. Pract. 2007, 77, 41.
13. (a) Govindachari, T. R.; Premila, M. S. Phytochemistry 1983, 22, 755; (b) Sharma,
B. R.; Rattan, R. K.; Sharma, P. Phytochemistry 1981, 20, 2606; (c) Manandhar,
M. D.; Shoeb, A.; Kapil, R. S.; Popli, S. P. Phytochemistry 1978, 17, 1814; (d) Basu,
D.; Sen, R. Phytochemistry 1974, 13, 2329; (e) Shoeb, A.; Kapil, R. S.; Popli, S. P.
Phytochemistry 1973, 12, 2071.
14. Seca, A. M. L.; Silva, A. M. S.; Silvestre, A. J. D.; Cavaleiro, J. A. S.; Domingues, F.
M. J.; Pascoal-Neto, C. Phytochemistry 2001, 58, 1219.
ical degradation. Acid hydrolysis of 8 afforded D-glucose and corre-