Novel 3-O-acyl mesquitol analogues as free-radical scavengers and enzyme inhibitors: Synthesis, biological evaluation and structure-activity relationship

Article abstract of DOI:10.1016/S0960-894X(03)00494-3

A new isomer of mesquitol (2,3-trans-3′,4′,7,8-tetrahydroxyflavan-3-ol) was isolated from Dichrostachys cinerea in excellent yields. It has shown free-radical scavenging property and α-glucosidase inhibitory activities but, it could not display xanthine o

Full text of DOI:10.1016/S0960-894X(03)00494-3

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2777–2780
Novel 3-O-Acyl Mesquitol Analogues as Free-Radical Scavengers
and Enzyme Inhibitors: Synthesis,Biological Evaluation and
Structure–Activity Relationship
R. Jagadeeshwar Rao,a Ashok K. Tiwari,b U. Sampath Kumar,a S. Venkat Reddy,a
Amtul Z. Alib and J. Madhusudana Raoa,*
aNatural Products Laboratory, Division of Organic Chemistry I, Indian Institute of Chemical Technology,
Hyderabad-500 007, India
bDivision of Pharmacology, Indian Institute of Chemical Technology, Hyderabad-500 007, India
Received 29 January 2003; revised 9 May 2003; accepted 9 May 2003
Abstract—A new isomer of mesquitol (2,3-trans-30,40,7,8-tetrahydroxyflavan-3-ol) was isolated from Dichrostachys cinerea in
excellent yields. It has shown free-radical scavenging property and a-glucosidase inhibitory activities but, it could not display xan-
thine oxidase inhibitory property. However, it was observed that acylation of 3-OH group significantly enhanced the a-glucosidase
inhibition and displayed xanthine oxidase inhibitory potential. The structure activity relationship revealed that the degree of lipo-
philicity played a major role in improving enzyme inhibitory activities. A positive correlation was observed between enzyme inhi-
bitory potential and acyl chain length (upto C-16) of aliphatic esters.
# 2003 Elsevier Ltd. All rights reserved.
Drugs with antioxidant mechanisms are being widely
proposed as bases for the development of new approa-
ches for pharmacological regulation of peroxidative-
antioxidative homeostatic imbalance which is respon-
sible for development of several disorders.1 Antioxidant
molecules isolated from natural products are being
reported to possess multiplicity in their activities either
directly related to their antioxidant property or by other
mechanisms.2 Studies on the free-radical scavenging
properties of flavonoids have permitted characterization
of the major phenolic components of naturally occur-
ring phytochemicals as antioxidants.3 Flavans, an
important class of natural antioxidants, have been
reviewed to possess a number of enzyme inhibitory
properties related to various disease conditions.4
atherosclerosis, ischemia-reperfusion, carcinogenesis
and aging and that free radicals generated in the enzy-
matic process are involved in oxidative damage.5 Thus,
it may be possible that the inhibition of this enzymatic
pathway for instance by compounds that have simulta-
neously antiradical and xanthine oxidase inhibitory
properties could have therapeutic interest. It has been
reported that some flavonoids and structurally related
antioxidants inhibit xanthine oxidase.6
Similarly, it is known for a long time that the levels of
glycosidase are elevated in the sera of many patients
with different tumors7 and inhibitors of glucosidases,
particularly a-glycosidase inhibitors possess potential of
broad spectrum therapeutic activities like anti viral, anti
cancer and anti diabetic.8 It has also been reported that
radical scavenging a-glucosidase inhibitory constituents
from medicinal plants possess variety of biological
activities like anti HIV, antiviral and anti-inflammatory
etc.9
Xanthine oxidase is considered to be the important
biological source of free radicals.3 Also, there is over-
whelming acceptance that xanthine oxidase serum levels
are significantly increased in various pathological states
like hepatitis, inflammation, hypercholesterolemia,
In this letter we report the isolation of a new isomer of
mesquitol 1 (2,3-trans-30,40,7,8-tetrahydroxyflavan-3-ol)
from traditional Indian medicinal plant Dichrostachys
cinerea10,11 in substantial yield12 along with minor
*Corresponding author. Tel.: +91-40-27193166; fax: +91-40-
27160512; e-mail: janaswamy@iict.ap.nic.in
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00494-3

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R. J. Rao et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2777–2780
amount of (ꢀ)-epicatechin (0.2%). The structure of (ꢀ)-
mesquitol was established using physical and spectro-
scopic methods.13 The 30,40-dihydroxyphenyl group at
C-2 was in trans position to the hydroxyl group at C-3
according to the large coupling constant of the H-2 sig-
nal (J=7.25 Hz) which was coincident with that of a
(+)-mesquitol moiety (JH-2=7.25 Hz).14 The specific
rotation of the compound has shown opposite sign to
that of (+)-mesquitol. Hence, the configuration at C-2
and C-3 is assumed as 2S, 3R.
target compounds (4a–g) in 85%. The aromatic esters
were obtained (Scheme 1) by treating the (ꢀ)-30,40,7,8-
tetra-O-benzyl mesquitol 2 in methylene chloride with
the corresponding aromatic acid chloride in the pre-
sence of triethylamine followed by debenzylation to
In our screening (ꢀ)-mesquitol showed better level of
free-radical scavenging and a-glucosidase inhibitory
activity than (ꢀ)-epicatechin (Table 1). The absence of
xanthine oxidase inhibition for (ꢀ)-mesquitol and (ꢀ)-
epicatechin may be due to the presence of free hydroxyl
group at C-3 position.15 It is well documented that
radical scavenging activity of antioxidant molecules is
primarily due to the pyrogallol/catechol moiety in the
molecules. Further the addition of alkyl groups intro-
duces enzyme inhibition properties of the molecule and
that inhibition of the enzyme increases with increase in
the alkyl chain length.16 Since, (ꢀ)-mesquitol has eli-
cited better free-radical scavenging and a-glucosidase
inhibitory activity than (ꢀ)-epicatechin and that the
presence of free hydroxyl group at C-3 position inhibit
it to elicit xanthine oxidase inhibitory activity, we aimed
to prepare 3-O-aliphatic acyl esters of (ꢀ)-mesquitol in
order to introduce xanthine oxidase inhibitory property
as well as to improve its enzyme inhibitory potential.
Simultaneously, we also prepared some 3-O-aromatic
acyl esters of (ꢀ)-mesquitol in order to make compara-
tive analysis (Fig. 1).
Figure 1.
The aliphatic esters were prepared (Scheme 1) by con-
densing the corresponding aliphatic acids with (ꢀ)-
30,40,7,8-tetra-O-benzyl mesquitol 2 in the presence of
dicyclohexyl carbodiimide in anhydrous methylene
chloride17 fallowed by debenzylation using palladium
on carbon under hydrogen atmosphere to yield the
Scheme 1. Reagents and conditions: (i) PhCH2Br, K2CO3, acetone,
reflux; (ii) corresponding aliphatic acid, DCC, DMAP, CH2Cl2; (iii)
corresponding aromatic acid chloride, Et3N, CH2Cl2; (iv) H2, 10% Pd-
C, MeOH.
Table 1. Free-radical scavenging activity (DPPH, ABTS), a-glucosidase and xanthine oxidase inhibition of (ꢀ)-mesquitol and its 3-O-acyl esters
Compd
Free-radical scavenging activity, SC50 (mM)
a-glucosidase inhibition
xanthineoxidase inhibition
DPPH
ABTS
IC50 (mM)
IC50 (mM)
1
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
6.72
7.15
8.89
6.68
6.07
7.51
5.17
9.43
11.53
7.97
6.72
8.99
13.47
21.67
15.94
—
14.59
13.04
14.09
14.41
17.57
11.29
10.05
17.75
—
—
—
—
—
82.32
46.31
46.18
49.34
13.46
12.56
9.56
54.45
77.76
43.26
67.26
32.82
177.56
—
NA
150
139
129
112
102
94.7
155.75
NA
NA
NA
NA
NA
—
(ꢀ)-Epicatechin
Probucol
Trolox
—
1.82
—
—
50.00
—
—
—
34.91
1-Deoxynoji rimycin
Allopurinol
—
—
NA, (Compounds were considered not active at concentration 50 mg/mL giving activity less than 15%), IC50 or SC50 values were determined by
linear regression analysis using at least five different concentrations in triplicate and represent mean of the experiment, SDM were within 10% in any
case, — Not tested.

R. J. Rao et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2777–2780
2779
yield the compounds (4h–k) in 80%. All the esters were
purified and characterized using spectroscopic methods18
before subjecting to biological evaluation.
was observed (Fig. 2). The influence of acyl substitution
on the xanthine oxidase inhibition is well noticed as
indicated by lack of activity for (ꢀ)-mesquitol. Surpris-
ingly, none of the aromatic analogues could show any
inhibition.
(ꢀ)-Mesquitol and its 3-O-acyl esters were screened for
free-radical scavenging (DPPH, ABTS) activities.19,20 It
is evident from the present study (Table 1) that the
antioxidant activity is due the phenolic hydroxyl groups
present on the ring A and B. Although, (ꢀ)-mesquitol
and its acyl esters regardless of their acyl substitution,
showed potent scavenging activity on the DPPH and
ABTS radical, compounds showed more potential radi-
cal scavenging activity on lipophilic free radical, DPPH
than the hydrophilic free radical, ABTS (Table 1).
(ꢀ)-Mesquitol and its 3-O-acyl derivatives were
assayed22 for a-glucosidase enzyme inhibition wherein
the IC50 values are found to be in the range 82.3 to 9.5
mM (Table 1). It is plausible that the inhibitory effect of
(ꢀ)-mesquitol may be related to the phenolic hydroxyls
located on its flavan framework. The introduction of an
acyl group on the C-3 position has enhanced the inhib-
itory potential to a greater extent. However, by increas-
ing the length of acyl group of aliphatic esters (4a–g)
a better affinity is favored. When the IC50 values
were plotted against the length of the acyl chain a linear
correlation was observed up to a length of C-16
(Fig. 3). All the aromatic esters of (ꢀ)-mesquitol (4h–k)
have shown better inhibitory potential than the parent
compound.
It is well documented that the presence of free hydroxyl
group at C-3 position in flavonoids reduces the xanthine
oxidase enzyme inhibition to a greater extent.15 All the
aliphatic and aromatic 3-O-acyl esters were tested21 for
xanthine oxidase inhibitory activity. The aliphatic 3-O-
acyl esters showed better inhibition pattern. The trend
observed in the experiments reported in Table 1 could
be explained on the basis of degree of lipophilicity of
the compounds. Consequently, by elongating the alkyl
chain in the acyl group, a better affinity was favored for
the space around the active site of the enzyme. When
the IC50 values of selected aliphatic esters were plotted
against the length of the acyl chain a linear correlation
In summary, a novel isomer of mesquitol is isolated in
excellent yield from Dichrostachys cinerea. The better
and improved a-glucosidase inhibitory potential, incor-
poration and increase in xanthine oxidase inhibitory
properties along with increase in lipophilicity of (ꢀ)-
mesquitol esters (4a–g) without affecting their free radi-
cal scavenging potential therefore may offer exciting
opportunity for their development as therapeutic agents
in disorders where increase in free radical generation,
xanthine oxidase and a-glucosidase levels play an
important role in pathogenesis.
Acknowledgements
We thank Drs. K. V. Raghavan, Director, IICT and J.
S. Yadav, Senior Deputy Director and Head, Organic
Division-1 for their invaluable suggestions. R. J. R.
thank to UGC, New Delhi for the fellowship.
Figure 2. Xanthine oxidase inhibitory potential of 3-O-acyl esters of
(ꢀ)-mesquitol (4a–f). The inhibitory potential of esters increased with
the length of acyl chain as indicated by good linear correlation
(r2=0.9951).
References and Notes
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Fig 3. a-Glucosidase inhibitory potential of (ꢀ)-mesquitol 1 and its
esters (4a–f). The 3-O-acyl esters are more potent than the parent
compound. In particular the inhibitory potential of 3-O-acyl esters
increased with the length of the acyl chain as indicated by the good
linearity (r2=0.9164).

2780
R. J. Rao et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2777–2780
11. Krishna, C. J.; Tara, S. J. Indian Chem. Soc. 1977, 54, 649.
12. Stem bark of D. cinerea was collected from Hyderabad
region, India in July 2000. The dried wood powder (2 kg) was
extracted with petroleum ether, CHCl3 and MeOH succes-
sively at room temp. The methanol extract was filtered and
concentrated under vacuum to obtain 50 g of residue which
was then subjected to column chromatography on silica gel
(60–120 mesh).The fractions eluted at 4% MeOH in CHCl3
yielded (ꢀ)-mesquitol (24 g).
Probucol and trolox were taken as reference compounds. All
the analysis was done in triplicates.
20. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang,
M.; Rice-Evans, C. Free Radic. Biol. Med. 1999, 26, 1231. In
brief, 7 mM ABTS (2,20-azinobis-(3-ethyl benz) thiazoline-6-
sulfonic acid) diammonium salt was reacted with 2.45 mM
potassium persulfate overnight in the dark at room temp. The
working solution was prepared by diluting it with phosphate
buffered saline (pH 7.4) to get absorbance around 0.70 at 734
nm. 10 mL of test sample was reacted with 1 mL of diluted
ABTS and absorbance was recorded within 1 min at 734 nm.
Appropriate blanks and controls were prepared and all the
determinations were carried out in triplicates. Percentage of
ABTS scavenging and SC50 values were obtained as in case of
DPPH.
13. (ꢀ)-Mesquitol 1. Amorphous powder; mp 252 ꢁC; [a]D
ꢀ36.05 (c 1.0, MeOH); 1H NMR [200 MHz; (CD3)2CO] d
7.95, 7.93, 7.25 and 7.55 (each s, 4ꢂAr-OH), 6.88–6.72 (3H, m,
H-20,50,60), 6.40 (2H, s, H-5,6), 4.62 (1H, d, J=7.5 Hz, H-2),
4.01 (1H, br s, OH-3), 4.01 (1H, m, H-3), 2.89 (1H, dd, J=5
and 15 Hz, H-4eq), 2.71 (1H, dd, J=8 and 15.0 Hz, H-4ax); 13
C
NMR [50 MHz; (CD3)2CO] d 32.08 (C-4), 66.49 (C-3), 81.08
(C-2), 108.07 (C-6), 112.07 (C-4a), 114.39 (C-5), 115.20 (C-50),
118.20 (C-60), 118.68 (C-20), 130.89 (C-10), 132.85 (C-8), 144.17
(C-7), 144.86 (C-8a), 144.94 (C-30, 40); HRFABMS: [M+H]+
291.0860 calcd for C15H14O6, 291.0868.
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18. All the new compounds synthesized in this report gave
satisfactory analytical and spectroscopic data in full accor-
dance with the assigned structures.
21. Noro, T.; Oda, Y.; Toxhio, M.; Ueno, A.; Fukushima, S.
Chem. Pharm. Bull. 1983, 31, 3984. The assay was modified
suitably. In brief, 50mL test sample dissolved in DMSO, 850
mL of 50 nM phosphate buffer (pH 7.5) and 100 mL of xanthin
oxidase (1 u/mL) were incubated 15 min and reaction was
initiated by adding 400 mL of 0.15 mM xanthine as substrate
and further incubated at 29 ꢁC for 30 min. The reaction was
stopped by adding 600 mL of 1 N HCl and absorbance was
read spectrophotometrically at 290 nm. Background absor-
bance of the compounds was corrected by preparing indivi-
dual blanks accordingly where HCl was added before enzyme
addition. Allopurinol was taken as reference compound. All
the samples were run in triplicate. Percent xanthine oxidase
inhibition was calculated as (1ꢀB/A)ꢂ100, where B is absor-
bance of reading in the presence of inhibitor and A is absor-
bance reading of control in the absence of inhibitor but
solvent. The IC50 values (50% xanthine oxidase inhibitory
concentration of compounds) were obtained by linear regres-
sion analysis.
22. Kim, J. S.; Kwon, C. S.; Son, K. H. Biosci. Biotechnol.
Biochem. 2000, 64, 2458. In brief, yeast a-glucosidase (0.74 u/
mL) were prepared in 100 mM phosphate suffer (pH 7.0)
containing 0.006% v/v 30% BSA and 0.20% w/V sodium
azide. 10 mL of test samples dissolved in DMSO were incu-
bated with 50 mL of enzyme in a 96-well microplate for 5
min and absorbance at 405 nm was recorded as zero time.
After incubation for 10 min with 50 mL substrate again
absorbance 405 nm was recorded at room temperature
(26–28 ꢁC). The increase in absorbance after zero time was
calculated and percent a-glucosidase inhibition and IC50
values were obtained as above. All the samples were run in
triplicates and 1-deoxy nojirimycin was taken as reference
compound.
19. Tiwari, A. K.; Srinivas, P. V.; Kumar, S. P.; Rao, J. M. J.
Agric. Food Chem. 2001, 49, 4642. Assay was modified suitable
for micro plate reading. In brief, in a 96-well micro plates, 25
mL test sample dissolved in dimethyl sulfoxide (DMSO, AR
grade), 125 mL of 0.1 M tris–HCl buffer (pH 7.4) and 125 mL
of 0.5 mM DPPH (1,1-diphenyl-2-picrylhydrazyl, sigma) dis-
solved in absolute ethyl alcohol were mixed and shaken well.
After incubating 20 min in dark, absorbance was recorded
spectrophotometrically (SPECTRA
PLUS384, Molecular
MAX
Devices, USA) at 517 nm. The free radical scavenging poten-
tial was determined as the percent decolorization of DPPH
due to the test samples and calculated as (1ꢀB/A)ꢂ100, where
A is absorbance of DPPH control with solvent and B absor-
bance of decolorized DPPH in the presence of test compound.
The SC50 (50% radical scavenging concentration) of the test
compound values were calculated by linear regression analysis.