Studies on the novel α-glucosidase inhibitory activity and structure-activity relationships for andrographolide analogues

Article abstract of DOI:10.1016/j.bmcl.2006.02.011

A series of analogues of andrographolide were synthesized and evaluated as novel α-glucosidase inhibitors. Among them compound 23, 15-p-methoxylbenzylidene 14-deoxy-11,12-didehydroandrographolide, was a potent inhibitor against α-glucosidase whose IC

Full text of DOI:10.1016/j.bmcl.2006.02.011

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2710–2713
Studies on the novel a-glucosidase inhibitory activity and
structure–activity relationships for andrographolide analogues
Gui-Fu Dai, Hai-Wei Xu, Jun-Feng Wang, Feng-Wu Liu and Hong-Min Liu*
New Drug Research & Development Center, Zhengzhou University, Zhengzhou 450052, PR China
Received 13 December 2005; revised 20 January 2006; accepted 7 February 2006
Available online 28 February 2006
Abstract—A series of analogues of andrographolide were synthesized and evaluated as novel a-glucosidase inhibitors. Among them
compound 23, 15-p-methoxylbenzylidene 14-deoxy-11,12-didehydroandrographolide, was a potent inhibitor against a-glucosidase
whose IC50 value was 16 lM. The structure–activity relationships were also discussed.
Ó 2006 Elsevier Ltd. All rights reserved.
Glycosidase inhibitors are of particular interest in the
development of potential pharmaceuticals such as antit-
umour,1–3 antiviral,4,5 antidiabetics,6–9 immunoregula-
tory agents10 and so forth. The plant, Andrographis
paniculata,11,12 is extensively used in the traditional med-
icines of Chinese, Indian and other Asian countries.13,14
Extracts of the plant and their isolated constituents are
reported possessing a wide spectrum of biological activ-
extent.31,32 However, there is not any report about activ-
ities of the extracts or constituents of A. paniculata
against glucosidase.
In this study, the a-glucosidase inhibitory activities of
various andrographolide analougues were first reported.
The structure–activity relationship was also investigat-
ed, which would be helpful to screen the bioactive con-
stituents from the plant, design and synthesize novel
stronger a-glucosidase inhibitors and explore the molec-
ular mechanisms of extracts from A. paniculata as drugs,
especially as antidiabetic agents.
ities including antibacterial,15,16 antiinflammatory,17,18
19,20
antimalarial,
immunological,21,22 hepatoprotec-
tive23 and anticancer24 properties. In recent years, be-
sides the above bioactivities, the antidiabetic effect of
the plant has attracted researchers’ attention.25–29
Compounds 2, 3, 4 and 5 were prepared from androgra-
pholide by condensation reaction with different carbonyl
compounds, respectively (Scheme 1).33,34 Bioactivity
evaluation showed that compound 5 has an inhibition
of 20.1% at 100 lM against a-glucosidase.35,36 But no
significant activities were observed among compounds
2–4, nor did the mother compound (1) (Table 1). That
means increasing the lipophilicity of andrographolide
by the protection of 3,19-hydroxyls with aliphatic alde-
hydes with suitable carbochain length is favourable to
the inhibitory activity against a-glucosidase. Com-
pounds 6–12 were synthesized from andrographolide
derivatives and aromatic aldehydes. According to the
biological activity evaluation results, all the above aro-
matic derivatives of andrographolide but 9 showed
inhibitory activities against a-glucosidase. The IC50
values of 6 and 7 are nearly equal and higher than those
of 8–12 (Scheme 1, Table 1).
The ethanolic extract of A. paniculata exhibited antidia-
betic property.26,27 Blood glucose was significantly re-
duced by 52.9% when hyperglycaemic rats were treated
with 50 mg /kg body weight aqueous extract of A. pan-
iculata.28 Therefore, it is very interesting to investigate
the inhibitory activities of constituents from A. panicula-
ta against glucosidase.
The plant extracts are known containing diterpenes, fla-
vonoids and stigmasterols.29 Diterpenes from A. panicu-
lata, like andrographolide 1, contain three hydroxyls, an
a-alkylidene c-butyrolactone moiety and two fused six-
membered rings. Both the six-membered rings adopt
the chair conformation, whereas the five-membered ring
is in an envelope conformation.30 They were structurally
similar to some known glycosidase inhibitors to some
Keywords: Andrographolide; Analogues; Inhibition; Glucosidase.
*
A natural compound from A. paniculata, 14-deoxy-
11,12-didehydroandrographolide (13)37 which was
Corresponding author. Tel.: +86 371 67767200; fax: +86 371
67767200; e-mail: liuhm@public.zz.ha.cn
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.011

G.-F. Dai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2710–2713
2711
O
O
O
15
14
HO
O
O
13
16
O
HO
O
HO
HO
O
O
O
a
b
12
11
a
18
1
HO
2
9
HO
17
10
5
H
H
8
HO
H
3
7
HO
4
HO
c
O
HO
H
R1
HO
20
6
13
d
1
14
H
19
O
R2
HO
21
R2
2-12
O
R1
O
O
1
O
Scheme 1. Reagents and conditions: (a) H2SO4(0.1 N), THF, reflux-
ing, 1–20 h, yield > 95%. 2: R1 = R2 = H; 3: R1 = R2 = CH3; 4:
R1 = H; R2 = (CH2)3CH3; 5: R1 = H; R2 = (CH2)5CH3; 6: R1 = H;
R2 = C6H5; 7: R1 = H; R2 = 3-Br-C6H4; 8: R1 = H; R2 = 4-F-C6H4; 9:
R1 = H; R2 = 4-Cl-C6H4; 10: R1 = H; R2 = 2-MeOC6H4; 11: R1 = H;
R2 = 4-MeOC6H4; 12: R1 = H; R2 =2,4,5-(MeO)3C6H2.
HO
H
HO
H
HO
HO
15
16-24
Scheme 2. Reagents and conditions: (a) pyridine, AL2O3, toluene,
refluxing, 6 h, yield 90%; (b) NiCl2, methanol, 0–5 °C, 0.5 h, yield 70%;
(c) methanol, Et3N, rt, 48 h, yield 10%; (d) methanol, Na2CO3,
refluxing, 5–24 h, yield 50–90%. 16: R1 = H; R2 = CH3; 17: R1 = CH3;
R2 = CH3; 18: R1 = H; R2 = (CH2)2CH3; 19: R1 = H; R2 = CHC
(CH3) (CH2)2CHC (CH3)2; 20: R1 = H; R2 = 4-F-C6H4; 21: R1 = H;
R2 = 4-Cl-C6H4; 22: R1 = H; R2 = 3-Br-C6H4; 23: R1 = H; R2 =
4-MeOC6H; 24: R1 = H; R2 = C6H5CHCH.
Table 1. Inhibitory activities of andrographolide analogues
Compound
a-Glucosidase
b-Glucosidase
1
2
nic
ni
ni
ni
3
ni
ni
4
ni
20.1a
ni
5
ni
6
48.9a (101b)
49.6a (100b)
26.2a
ndd
nd
nd
ni
Table 2. Inhibitory activities of analogues of andrographolide
7
Compound
a-Glucosidase
b-Glucosidase
8
13
14
15
16
17
18
19
20
21
22
23
24
16.5a
34.2a
ni
15.1a
17.1a
43.5a (110b)
ni
nic
ni
9
ni
10
11
12
5.5a
nd
nd
nd
13.2a
ni
14.5a
ni
ni
a Inhibition(%) determined at 100 lM concentration of compound.
ni
b IC50 (lM).
ndd
nd
nd
nd
nd
nd
c No inhibition at 100 lM.
d Not determined.
ni
ni
16.7a
100a (16b)
84.3a (58b)
synthesized from 1 also, inhibits a-glucosidase 16.5% at
100 lM (Scheme 2, Table 2). Reduction of C-12, 13 ole-
fin bond of andrographolide 1 afforded 14, which inhib-
its a-glucosidase 34.2%. But when the number of olefin
bonds was increased as compound 15, the a-glucosidase
inhibitory activity was lost. The results indicated that
the flexible chain between the c-butyrolactone moiety
and the two six-membered rings is critical to a-glucosi-
dase inhibitory activity. In other words, the large conju-
gated system between the c-butyrolactone moiety and
the two six-membered rings is unfavourable to the inhib-
itory activity.
a Inhibition (%)determined at 100 lM concentration of compound.
b IC50 (lM).
c No inhibition at 100 lM.
d Not determined.
phenylvinylidene derivative (24) were much stronger
than that of compound 13. Their IC50 values are
16 lM and 58 lM, respectively.
In order to examine the role of the C-8, C-17 epoxy moi-
ety of the new derivatives in exhibiting the a-glucosidase
inhibitory activity, compounds 25–28 were designed and
synthesized. Both 25 and 26 lose the activity when the
exocyclic double bond (n8,17) of 13 and 17 (another nat-
ural compound)38 was epoxidized, respectively. Lower
activities were observed in 27 and 28 derived by epoxida-
tion of 23 and 24, respectively (see Scheme 3, Table 3).
Compounds 16–24 are 15-ene-substituted derivatives of
compound 13. The biological activity results showed
that increasing the carbochain of C15-substituting
group to suitable length (16–19) could enhance the
inhibitory activity against a-glucosidase. But different
aromatic substitution derivatives gave inconsistent bio-
activity results. 4-Fluoro and 4-chlorobenzylidene sub-
stitution (20–21) makes the inhibitory activity lost,
while the activity is retained in 3-bromobenylidene
derivative (22). The a-glucosidase inhibitory activities
of the 4-methoxyphenylidene derivatives (23) and the
In summary, many derivatives of andrographolide
exhibited good a-glucosidase inhibitory activities with
inhibitory percentage ranging from 5.5% to 100% (23:
IC50 = 16 lM) at 100 lM. But all the compounds

2712
G.-F. Dai et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2710–2713
R2
O
Acknowledgment
O
21
R1
O
O
O
This work was supported by the National Science Foun-
dation of China (No. 20472075).
O
a
b
O
HO
HO
HO
O
H
HO
HO
13
H
References and notes
HO
25
26-28
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Scheme 3. Reagents and conditions: (a) m-CPBA, CHCl3, refluxing,
2 h, and yield 95%; (b) methanol, Na2CO3, aldehyde, refluxing, yield
70–85%. 26: R1 = CH3, R2 = CH3; 27: R1 = H, R2 = 4-MeOC6H4; 28:
R1 = H, R2 = CHCHC6H5.
4. Papandreou, M. J.; Barbouche, R.; Guieu, R.; Kieny, M.
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Table 3. Inhibitory activities of analogues of andrographolide
Compound
a-Glucosidase
b-Glucosidase
25
26
27
28
nic
ndd
nd
nd
nd
ni
37.7a(120b)
49.2a (101b)
a Inhibition(%) determined at 100 lM concentration of compound.
b IC50 (lM).
c No inhibition at 100 lM.
d Not determined.
9. Fujisawa, T.; Ikegami, H.; Inoue, K.; Kawabata, Y.;
Ogihara, T. Metabolism 2005, 54, 387.
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determined for b-glucosidase inhibitory activities
showed no inhibitory activities. The results for the struc-
ture–activity relationship studies showed that increasing
the lipophilicity by protection of 3,19-hydroxyls of
andrographolide endows compound 1 with a-glucosi-
dase inhibitory activity. The number and position of ole-
fin bonds between the c-butyrolactone moiety and the
six-membered rings were detrimental to the inhibition
effect. In general, 15-ene-substituted derivatives of 14-
deoxy-11,12,13,14-tetradehydroandrographolide signifi-
cantly increased the a-glucosidase inhibitory activities.
Epoxidation of the exocyclic double bond makes the
inhibitory activity reduced or lost.
15. Zhang, W. Chinese Patent, CN 1266699, 2000.;
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Bioactivity studies suggest that probably compounds 13
and 17 with a-glucosidase inhibitory activities may play
an important role in the plant extracts exerting antidia-
betic effect. Zhang’s and Yu’s results showed that etha-
nolic extract of A. paniculata or andrographolide can
lower plasma glucose.26,27,39 That means andrographo-
lide without glucosidase inhibitory activity may also ex-
ert antidiabetic effect. According to He’s result,40 13 is
one of the andrographolide metabolites isolated from
rat urine, faeces, and the contents of the small intestine.
So it can be deduced that the andrographolide may exert
antidiabetic effect through inhibiting glucosidase after
being metabolized to 13, an a-glucosidase inhibitor in vi-
vo. In other words, a-glucosidase inhibitory activity was
the reason or at least one of the reasons that the constit-
uents of A. paniculata had antidiabetic effects. The pro-
motion of the glucose metabolism was found when
treating diabetic rat with the plant extract or androgra-
pholide.27,39 So, it can be deduced that extracts of A.
paniculata and andrographolide lower plasma glucose
by inhibiting the disaccharide metabolism and/or pro-
moting the glucose metabolism.
17. Babish, J.G.; Howell, T.; Pacioretty, L. U.S. Patent,
20020068098, 2002.;
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1157.
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33. General methods. IR spectra were recorded as KBr pellets
on a Thermo Nicolet (IR200) Spectrometer. Mass spec-
trometry experiment was performed on a Waters Q-Tof
micro mass spectrometer. 1H and 13C spectra were
Chemical Company. Other chemicals were purchased
from native company.
35. General procedure for a-glucosidase inhibition assay. Inhi-
bition rate was determined at 37 °C in 0.067 M K2HPO4/
KH2PO4 buffer (pH 6.8). The reaction mixture contained
40 ll of enzyme solution, 40 ll of inhibitor and 20 ll of
substrate. The enzymatic reaction was started after incu-
bation of the enzyme (0.04 U/ml) for 30 min in the
presence of the inhibitor (0.1 mM) by the addition of
substrate(0.5 mM). The mixture was incubated at 37 °C
for 5 min, and the reaction was quenched by the addition
of 0.1 M Na2CO3 (pH 9.8). The absorption at 405 nm was
measured immediately and taken as the relative rate for
the hydrolysis of substrate. All experiments were carried
out in triplicate. The IC50 value is the concentration of
inhibitor at 50% of enzyme activity.
36. General procedure for b-glucosidase inhibition assay. Inhi-
bition rate was determined at 37 °C in 0.08 M citric acid/
Na2HPO4 buffer (pH 4.2). The enzymatic reaction was
started after incubation of the enzyme (0.02 U/ml) for
30 min in the presence of the inhibitor (0.1 mM) by the
addition of substrate (5 mM). The mixture was incubated
at 37 °C for 5 min, and the reaction was quenched by the
addition of 0.25 M borate buffer (pH 9.8). The absorption
at 405 nm was measured immediately and taken as the
relative rate for the hydrolysis of substrate.
recorded, respectively, at 400 and 100 Hz with a Bruker
¨
DPX-400 spectrometer with TMS as internal standard.
Melting points were determined on a Beijing Keyi XT5
apparatus. The absorbance at 405 nm was measured by a
PowerWaveX Microplate Scanning Spectrophotometer
(BIO-TEK INSTRUMENTS, INC).
34. Materials. Compound 1 was extracted from A. paniculata
Compounds 2–28 were developed from 1. Compounds 4–
12 and 15 are single isomers, but their configurations have
not been confirmed yet. Compound 13 is (11E) isomer and
its structure was confirmed by NMR which was in
accordance with Ref. 33. The configuration of compound
14 was confirmed by X-ray crystallographic analysis;
Compounds 16, 18–24, and 26–28 are (15Z) isomers,
which are established from the correlation between H-14
and H-21 in their NOESY spectra. The configuration of
epoxide ring in compounds 25–28 was confirmed by X-ray
crystallographic analysis. The enzymes of a-glucosidase
type I from baker’s yeast and b-glucosidase from almonds,
and the substrates of p-nitrophenyl a-D-glucopyranoside
and b-D-glucopyranoside were purchased from Sigma
37. Fujita, T.; Fujitani, R.; Takeda, Y.; Takaishi, Y.; Yam-
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M. Phytochemistry 2004, 65, 2317.
39. Yu, B. C.; Hung, C. R.; Chen, W. C.; Cheng, J. T. Planta
Med. 2003, 69, 1075.
40. He, X.; Li, J.; Gao, H.; Qiu, F.; Cui, X.; Yao, X. Chem.
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