Synthesis and biological evaluation of novel 8-aminomethylated oroxylin A analogues as α-glucosidase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.01.055

A series of 8-aminomethylated derivatives (1a-1j) were prepared by Mannich reaction of oroxylin A (1) with appropriate primary or secondary amines and para-formaldehyde. All the compounds were tested for their α-glucosidase inhibition activity against both yeast and rat intestinal α-glucosidase. Some of the compounds demonstrated significantly better α-glucosidase inhibitory activity than the parent compound (oroxylin A).

Full text of DOI:10.1016/j.bmcl.2008.01.055

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1659–1662
Synthesis and biological evaluation of novel 8-aminomethylated
oroxylin A analogues as a-glucosidase inhibitors
T. Hari Babu,a V. Rama Subba Rao,a Ashok K. Tiwari,b K. Suresh Babu,a P. V. Srinivas,a
Amtul Z. Alib and J. Madhusudana Raoa,*
aDivision of Organic Chemistry-I, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 007, India
bDivision of Pharmacology, Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500 007, India
Received 26 November 2007; revised 19 December 2007; accepted 15 January 2008
Available online 19 January 2008
Abstract—A series of 8-aminomethylated derivatives (1a–1j) were prepared by Mannich reaction of oroxylin A (1) with appropriate
primary or secondary amines and para-formaldehyde. All the compounds were tested for their a-glucosidase inhibition activity
against both yeast and rat intestinal a-glucosidase. Some of the compounds demonstrated significantly better a-glucosidase inhib-
itory activity than the parent compound (oroxylin A).
ꢀ 2008 Elsevier Ltd. All rights reserved.
a-Glucosidase inhibitors have become molecules of
immense pharmaceutical interest recently, as they offer
potential therapeutic benefits against numerous diseases
like type-II diabetes mellitus,1 cancer2 and viral infec-
tions.3 For instance, epithelial membrane bound a-gluco-
sidase in small intestine hydrolyses a-glucopyranoside
bond of carbohydrates and oligosaccharides and thereby
releases a-D-glucose. Inhibitors of intestinal a-glucosi-
dase activity therefore, slow down the postprandial
glucose excursion in type-II diabetes mellitus subjects.4
Glucosidase inhibition may also retard cancer growth
since the spread of cancer as well as the structural changes
of cell surface glycoconjugates within neoplasmic cells is
proliferated by glycosidases.5 a-Glucosidase inhibitors
have also been observed to block viral infections3 and
proliferation in HIV-infections.6
and that oroxylin A (1), baicalein (2) and chrysin (3)
(Fig. 1) were the major isolates responsible for the activ-
ity (Table 1). Among these three compounds, oroxylin A
happens to be the most potent inhibitor of a-glucosidase
(rat intestinal as well as yeast).
It has recently been reported that baicalein (5,6,7-trihy-
droxy flavone) and related 6-hydroxy flavones are the
new class of a-glucosidase inhibitors9 and that the
absence of hydroxyl group at 6th position (chrysin) or
replacement of hydroxyl with methoxy group at 6th
position (oroxylin A) drastically reduced the activity.9a
Furthermore, it was found that baicalein strongly inhib-
ited enzyme sucrase (IC50 = 52 lM) and mildly the en-
zyme maltase (IC50 = 500 lM) in the presence of
substrates sucrose (b-D-Fructofuranosyl-a-D-glucopyran-
oside; a-D-glucopyranosyl-b-D-Fructofuranoside) and malt-
ose (4-O-a-D-glucopyranoside-D-glucose), respectively.9b
The Indian herb Oroxylum indicum is extensively used in
traditional medicinal preparations7 and extracts of this
plant have been reported to possess diverse biological
activities like anti-inflammatory, antirheumatic, antimi-
crobial and anti-protozoal activities.8 In the course of
our study of identifying potential a-glucosidase inhibi-
tors from Indian medicinal plants, we observed that
hexane extract of this plant possess inhibitory activity
It is important to mention here that sucrase is not di-
rectly involved in dietary blood glucose level rise. In
fact, an inhibition of glucose production from malt-
ose or the isomaltose has greater benefit for control-
ling postprandial hyperglycemic excursion from
carbohydrates in non-insulin dependent diabetes mel-
litus subjects.10 The drug acarbose widely used for
mitigating dietary postprandial hyperglycemic excur-
sion has been observed to be 80 times more potent
in inhibiting carbohydrate hydrolyzing enzyme mal-
tase than the sucrase.10 Therefore, in order to better
represent the substrate maltose (4-O-a-D-glucopyrano-
Keywords: Oroxylum indicum; Oroxylin A; Mannich reaction; Primary
amines; Secondary amines; a-Glucosidase inhibition.
*
Corresponding author. Tel.: +91 40 27193166; fax: +91 40 27160512;
e-mail addresses: janaswamy@iict.res.in; janaswamy@ins.iictnet.com
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.055

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T. Hari Babu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1659–1662
HO
O
HO
HO
O
HO
O
MeO
OH
O
OH
O
OH
O
Chrysin(3)
Oroxylin A(1)
Baicalein(2)
Figure 1. Structures of oroxylin A, baicalein and chrysin.
Table 1. Enzyme inhibitory activity
The process is well documented by examples of various
phenols and is widely used in the synthesis. This reaction
provides a method for the introduction of basic amino
alkyl chain. Various drugs obtained from Mannich reac-
tion are proved to be more effective and less toxic than
parent antibiotic.13 The structure of our derivatives
combines the biological active groups of flavone (5,7-
dihydroxy-6-methoxy) and the aminomethyl moiety as
a new template for a-glucosidase inhibition activity.
Compound
IC50 (lM) value of rat IC50 (lM) value
intestinal a-glucosidase of yeast a-glucosidase
inhibition
inhibition
Hexane extract
1
84.16a
135.39
214.59
375.55
75.95
35.43
NI
80.74a
123.77
202.74
370.67
149.79
98.84
NI
2
3
1a
1b
1c
Our strategy for the synthesis of the targeted analogues
relied upon the electrophilic substitution at C-8. This
was achieved by the Mannich reaction of the oroxylin A
(1) with formaldehyde in the presence of the primary or
secondary amines in 2-propanol (Scheme 1). The classical
conditions of the Mannich reaction for the hydroxyl
compounds are based on the substrate, amine and formal-
dehyde ratio in alcohol with prolonged heating. In our
case, oroxylin A, formaldehyde and primary or secondary
amines in 1:1:1 ratio, respectively, were refluxed and
stirred in isopropanol for 1–2 h to afford the C-aminome-
thylated derivatives.14 The structures of the resulting
1d
1e
75.02
NI
NI
84.44
NI
NI
1f
1g
NI
NI
NI
NI
1h
1i
50.74
NI
6.38
166.78
NI
—
1j
Acarbose
Deoxynojirimycin —
49.29
NI, no inhibition.
a lg/mL.
1
Mannich bases were confirmed by H NMR and mass
1
analysis.15 The H NMR spectra of compounds 1a–1j
side-D-glucose) and/or isomaltose in our enzymatic
reaction, we select 4-nitrophenyl-a-D-glucopyranoside
(Sigma Chemicals, USA, cat. No. N-1377) as sub-
strate for inhibition of intestinal a-glucosidase.11 It
was observed that oroxylin A with methoxy group
at 6th position displayed better activity than baica-
lein.12 As part of our interest in the extension of sys-
tematic investigation of the chemical features of
oroxylin A, we prepared new derivatives derived from
the Mannich reaction of oroxylin A. Herein, we re-
port the rat intestinal and yeast a-glucosidase inhibi-
tory activity of the same using the methodology
reported earlier.11
clearly indicated the absence of the signal at d 6.80 for
H-8 proton of the flavanoid ring system. It should be
noted that in all cases the electrophilic substitution
occurred solely in ring A even at the large excess of the
reagent and under more severe conditions.16
Biological significance of the 8-aminomethylated deriva-
tives (1a–1j) of the oroxylin A was established by screen-
ing against both rat intestinal and yeast a-glucosidase
enzymes. Table 1 represents the IC50 values of the com-
pounds. As shown in Table 1, aminomethylene group
substitution at C-8 position significantly enhanced the
3'
R
1''
1'
HO
O
O
HO
9 O
CH2O, 10/20 amines
1
5'
7
5
3
10
MeO
MeO
2-propanol, reflux, 1-2 hrs
OH
O
OH
1
(1a-1j)
1f. R = Methyl benzyl amino (C8H10N)
1g. R = n-butyl amino (C4H10N)
1h. R = Di phenyl amino (C12H10N)
1i. R = Piperidinyl (C5H10N)
1a. R = Morpholinyl (C4H8NO)
1b. R = N-methyl piperzinyl (C5H11N2)
1c. R = Benzylamino (C7H8N)
1d. R = 1-Boc piperzinyl (C9H17N2O2)
1e. R = N-methyl furfuryl amino (C7H10NO)
1j. R = Pyrrolidinyl (C4H8N)
Scheme 1. Synthesis of 8-aminomethylated derivatives of oroxylin A.

T. Hari Babu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1659–1662
1661
10. Matsui, T.; Ogunwande, I. A.; Abesundara, K. J. M.;
Matsumoto, K. Mini. Rev. Med. Chem. 2006, 6, 109.
11. (a) Suresh Babu, K.; Tiwari, A. K.; Srinivas, P. V.; Ali, A.
Z.; China Raju, B.; Rao, J. M. Bioorg. Med. Chem. Lett.
2004, 14, 3841; (b) Rao, A. S.; Srinivas, P. V.; Tiwari, K.;
Vanka, U. M. S.; Rao, V. S.; Dasari, K. R.; Rao, J. M.
J. Chromatogr. B 2007, 855, 166; (c) Gowri, P. M.; Tiwari,
A. K.; Ali, A. Z.; Rao, J. M. Phytother. Res. 2007, 21, 796.
12. Rao, J. M.; Suresh Babu, K.; Tiwari, A. K.; Hari Babu,
T.; Srinivas, P. V.; Kumar, S. P.; Sastry, B. S.; Ali, A. Z.;
Yadav, J. S. Patent NF-201/05 PT-480 0477/DEC/2006.
13. (a) Tapzergyar, E. G. Austrian Pat. 1996, 246729; (b) Jain, S.
M.; Dhaneshwar, S. R.; Trivedi, P. Indian Drugs 1991, 29, 64.
14. General experimental procedure. A mixture of para-form-
aldehyde (0.352 mmol) and primary or secondary amine
(0.352 mmol) in 15 ml of 2-propanol was stirred at 60 ꢁC
untill complete homogenization. The solution obtained
was added slowly to a solution of (0.352 mmol) oroxylin A
(1) in 2-propanol and the reaction mixture was refluxed for
1–2 h. After completion, the reaction mixture was con-
centrated and the residue was purified by silica gel column
chromatography (60–120 mesh) to afford (1a–1j) yellow
solids in very good yields (70–85%).
intestinal a-glucosidase inhibitory activity (2–4 times).
Perusal of IC50 values shows that compound 1b is most
active, within the set, followed by compounds 1i, 1d and
1a. Concerning the structural features of the active
Mannich bases, our data indicate that the minimal
structural requirement for a-glucosidase inhibition
activity is 5,7-dihydroxy-6-methoxy groups on the
A-ring of the flavanoid. Alicyclic amine (piperzinyl or
morpholinyl ring) substituents significantly improve
intestinal a-glucosidase inhibitory potential of the
parent compound wherein N-methyl piperzinyl (1b)
and piperidinyl (1i) substitutions represent the best fit,
respectively. It is interesting to note that except piperzi-
nyl substitutions (1b and 1d) no other substitutions
could improve the yeast a-glucosidase inhibitory poten-
tial of oroxylin A. Molecular recognition in the target-
binding site in yeast a-glucosidase and rat intestinal
a-glucosidase may be the reason for different behavior
of these compounds.17
Glucosidase inhibitors have proved their usefulness in
reducing postprandial hyperglycemic excursion in both
type-I and type-II diabetes. Current interest in these
inhibitors has further been extended to a diverse range
of diseases including lysomal storage disorders and can-
cer. Special attention being focused to those compounds
with anti-HIV activity.18 Though none of the com-
pounds in the present study could displayed comparable
activity to those of the reference compounds, isolation
of suitable glycosidase inhibitors from natural sources
and/or their chemical synthesis provides biochemical
tools for the elucidation of enzyme mechanistic activity
through the variations in potential inhibitor structural
information.
15. NMR data:Compound (1a). 1H NMR (300 MHz, CDCl3):
d 12.42 (1H, s, OH), 7.86–7.82 (2H, m, H-20, 60), 7.59–7.52
(3H, m, H-30, 40, 50), 6.60 (1H, s, H-3), 4.08 (2H, s, H-100),
3.96 (3H, s, OMe), 3.84–3.80 (4H, m, H-300, 700), 2.76–2.71
(4H, m, H-400, 600). 13C NMR (75 MHz, CDCl3): d 182.56,
152.55, 150.05, 132.50,132.10, 131.15, 130.90, 125.95,
125.50, 104.54, 98.75, 66.55, 60.55, 54.25, 52.00. FABMS:
384 (M+ + 1).
Compound (1b). 1H NMR (300 MHz, CDCl3): d 12.10
(1H, s, OH), 7.82–7.78 (2H, m, H-20, 60), 7.55–7.49 (3H,
m, H-30, 40, 50), 6.56 (1H, s, H-3), 4.05 (2H, s, H-100), 3.96
(3H, s, OMe), 2.78–2.75 (4H, m, H-300, 700), 2.59–2.55 (4H,
m, H-400, 600), 2.30 (3H, s, N–Me). FABMS: 397 (M+ + 1).
Compound (1c). 1H NMR (300 MHz, CDCl3 + MeOH-d4):
d 7.75–7.60 (4H, m, ph), 7.55–7.42 (2H, m, ph), 7.38–7.28
(4H, m, ph), 6.58 (1H, s, H-3), 4.25 (2H, s, H-100), 4.09
(2H, s, CH2-Ph), 3.82 (3H, s, OMe). FABMS: 404
(M+ + 1).
Acknowledgment
Compound (1d): 1H NMR (300 MHz, CDCl3): d 12.49
(1H, s, OH), 7.88–7.82 (2H, m, H-20, 60), 7.52–7.48 (3H,
m, H-30, 40, 50), 6.60 (1H, s, H-3), 4.08 (2H, s, H-100), 3.96
(3H, s, OMe), 3.56–3.52 (4H, m, H-300, 700), 2.74–2.70 (4H,
m, H-400, 600), 1.48 (9H, s, 3· Me). 13C NMR (75 MHz,
CDCl3): d 183.00, 163.55, 153.25, 150.15, 150.06, 132.00,
130.15, 130.00, 129.50, 126.55, 106.50, 105.15, 99.55,
99.00, 80.55, 61.00, 54.56, 52.50, 43.50, 28.20. FABMS:
483 (M+ + 1).
The Authors thank Dr. J.S. Yadav, Director, IICT for
his constant encouragement.
References and notes
1. Hoffmann, J.; Splenger, M. Diabetes Care 1994, 17, 561.
2. Fernandes, B.; Sagman, U.; Auger, M.; Demetrio, M.;
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3. Metha, A.; Zitzmann, N.; Rudd, P. M.; Block, T. M.;
Dwek, R. A. FEBS Lett. 1998, 430, 17.
4. Maki, K. C.; Carson, M. L.; Miller, M. P.; Turowski, M.;
Bell, M.; Wilder, D. M.; Reeves, M. S. Diabetes Care
2007, 30, 1039.
5. Bernaeki, R. J.; Niedbala, M. J.; Korytryk, W. Cancer
Metastasis Rev. 1985, 4, 81.
6. Gruters, R. A.; Neefjes, J. J.; Tersmetti, M.; De Goede, R.
E. Y.; Tulp, A.; Huisman, H. G.; Miedeme, F.; Ploegh, H.
C. Nature 1987, 330, 74.
7. Paratta, J. A. Bealing Plants of Peninsular India 2001, 168.
8. (a) Ali, R.; Houghton, P. J.; Raman, A.; Hoult, J. R. S.
Phytomedicine 1998, 5, 375; (b) Houghton, P. J.; Matali,
R.; Azizol, M. Pharmacol. Lett. 1997, 7, 96.
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Biotechnol. Biochem. 2004, 68, 369; (b) Gao, H.; Kawa-
bata, J. Bioorg. Med. Chem. 2005, 13, 1661.
Compound (1e). 1H NMR (300 MHz, CDCl3): d 7.81–7.71
(3H, m, H-20, 60, 500), 7.55–7.48 (3H, m, H-30, 40, 50), 6.61
(1H, s, H-3), 6.32 (1H, d, J = 2 Hz, H-600), 6.25 (1H, d,
J = 2 Hz, H-700), 4.05 (2H, s, H-100), 3.95 (3H, s, OMe),
3.75 (2H, s, H-300), 2.42 (3H, s, N–Me). FABMS: 408
(M+ + 1).
1
Compound (1f). H NMR (300 MHz, CDCl3): d 7.25–7.51
(10H, m, Ph), 6.45 (1H, s, H-3), 4.12 (1H, s, H-300), 4.05
(2H, s, H-100), 3.83 (3H, s, OMe), 1.98 (3H, d, J = 5 Hz,
Me). FABMS: 418 (M+ + 1).
Compound (1g). 1H NMR (300 MHz, CDCl3): d 12.49
(1H, s, OH), 7.86–7.80 (2H, m, H-20, 60), 7.56–7.50 (3H,
m, H-30, 40, 50), 6.62 (1H, s, H-3), 4.10 (2H, s, H-100), 3.94
(3H, s, OMe), 2.80 (2H, t, J = 4 Hz, H-300), 1.65–1.55 (2H,
m, H-400), 1.47–1.36 (2H, m, H-500), 0.99-0.94 (3H, t,
J = 7 Hz, H-600). 13C NMR (75 MHz, CDCl3): d 183.50,
158.00, 152.25, 132.78, 131.55, 131.15, 129.50, 126.50,
105.55, 99.00, 98.50, 60.55, 51.55, 50.25, 33.50, 20.65,
14.55. FABMS: 370 (M+ + 1).

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T. Hari Babu et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1659–1662
Compound (1h). 1H NMR (200 MHz, CDCl3): d 12.98
Compound (1j). 1H NMR (200 MHz, CDCl3): d 12.35 (1H,
s, OH), 7.85–7.80 (2H, m, H-20, 60), 7.59–7.52 (3H, m, H-
30, 40, 50), 6.60 (1H, s, H-3), 4.13 (2H, s, H-100), 3.98 (3H, s,
OMe), 2.84–2.78 (4H, m, H-300, 600), 2.0–1.95 (4H, m, H-400,
500). FABMS: 368 (M+ + 1).
(1H, s, OH), 7.82–7.75 (2H, m, H-20, 60), 7.56–7.44 (3H,
m, H-30, 40, 50), 7.20–7.10 (6H, m, Ph), 6.99–6.92 (4H, m,
Ph), 6.60 (1H, s, H-3), 4.18 (2H, s, H-100), 4.10 (3H, s,
OMe). FABMS: 466 (M+ + 1).
Compound (1i). 1H NMR (200 MHz, CDCl3): d 12.08 (1H,
s, OH), 7.90–7.80 (2H, m, H-20, 60), 7.60–7.42 (3H, m, H-
30, 40, 50), 6.60 (1H, s, H-3) 4.02 (2H, s, H-100), 3.95 (3H, s,
OMe), 2.78–2.48 (4H, m, H-300, 700), 1.78–1.60 (6H, m, H-
400, 500, 600). FABMS: 382 (M+ + 1).
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