Synthesis and antihyperglycemic activity of phenolic C-glycosides

Article abstract of DOI:10.1016/j.bmcl.2010.11.031

Various phenolic C-glycosides were evaluated for their in vitro and in vivo antihyperglycemic activity employing glucose uptake by rat muscle cell lines (L-6) and low dosed-streptozotocin-induced diabetic rats, respectively. Some of phenolic C-glycosides were isolated from Pterocarpus marsupium and Ulmus wallichiana and other were synthesized by unprotected sugar and phloroacetophenone using Sc(OTf)₃ in aqueous ethanol. Eight among tested compounds showed significant lowering of blood glucose level on low dosed-streptozotocin-induced diabetic rats. The compound 24 lowered the blood glucose levels by 34.9% and 33.6% during 0-5 h and 0-24 h, respectively, at the dose of 25 mg/kg body weight which is comparable to standard antidiabetic drug metformin.

Full text of DOI:10.1016/j.bmcl.2010.11.031

Bioorganic & Medicinal Chemistry Letters 21 (2011) 228–233
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antihyperglycemic activity of phenolic C-glycosides q
Preeti Rawat a, Manmeet Kumar a, Neha Rahuja b, Daya Shankar Lal Srivastava b,
Arvind Kumar Srivastava b, Rakesh Maurya a,
⇑
a Medicinal and Process Chemistry Division, Central Drug Research Institute, CSIR, Lucknow 226 001, India
b Biochemistry Division, Central Drug Research Institute, CSIR, Lucknow 226 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Various phenolic C-glycosides were evaluated for their in vitro and in vivo antihyperglycemic activity
employing glucose uptake by rat muscle cell lines (L-6) and low dosed-streptozotocin-induced diabetic
rats, respectively. Some of phenolic C-glycosides were isolated from Pterocarpus marsupium and Ulmus
wallichiana and other were synthesized by unprotected sugar and phloroacetophenone using Sc(OTf)3
in aqueous ethanol. Eight among tested compounds showed significant lowering of blood glucose level
on low dosed-streptozotocin-induced diabetic rats. The compound 24 lowered the blood glucose levels
by 34.9% and 33.6% during 0–5 h and 0–24 h, respectively, at the dose of 25 mg/kg body weight which
is comparable to standard antidiabetic drug metformin.
Received 2 August 2010
Revised 29 October 2010
Accepted 4 November 2010
Available online 11 November 2010
Keywords:
Phenolic C-glycosides
Antihyperglycemic activity
STZ
Ó 2010 Elsevier Ltd. All rights reserved.
Diabetes mellitus, often simply referred to as diabetes is a
metabolic disorder in which a person has high blood sugar, either
because the body does not produce enough insulin, or because cells
do not properly use insulin, a hormone that is required to convert
sugar, starches, and other food into energy. Human body has to
maintain the threshold blood glucose level which may be done
with insulin or glucagon depending on the condition. Patients with
diabetes are 4 times more likely to have coronary heart disease and
stroke. The chronic hyperglycemia of this disease is associated with
long-term dysfunction, damage, and failure of various organs,
especially the eyes, kidneys, nerves, heart, and blood vessels.1
One of the hallmarks of type II diabetes is decreased sensitivity
of muscle and adipose cells to insulin. Compounds that increase
the sensitivity of muscle and adipose to insulin may be useful in
the treatment of diabetes and its complications. Upon insulin treat-
ment, insulin receptor is phosphorylated which activates insulin
signal transduction pathway leading to increased glucose uptake
by glucose transporter 4 (GLUT4) in adipocytes (fat) or myocytes
(muscle). Therefore, measuring glucose uptake by these cells pro-
vides the most relevant end point assay for insulin sensitivity.2
Despite the fact that many oral therapeutic agents which in-
crease the insulin sensitivity, such as metformin, sulfonylureas,
thiazolidinedione and DPPIV inhibitors, have already been used
in clinical situations, it is still difficult to tightly control plasma glu-
cose and prevent diabetic complications.3–5 The discovery of new
lead structure has become an important area of research in
medicinal chemistry as new chemical entity is continue to fall
and toxicity of the present drugs becomes a dilemma. In continua-
tion to our antidiabetic drug discovery program, a series of C-
glycosides were isolated from terrestrial plants, derivatives
prepared and evaluated for their glucose uptake enhancing activity
and in vivo antihyperglycemic activity potential on low dosed-
streptozotocin-induced diabetic male albino rats of Sprague–
Dawley strain. Streptozotocin is a powerful alkylating agent that
induces multiple DNA strand breaks. A single large dose of strepto-
zotocin can produce type 1 diabetes mellitus in rodents by disrupt-
ing b-cell of pancreas.6
Naturally-occurring aryl C-glycosides7–9 exhibit interesting
biological activities especially C-glycosyl flavonoids which show a
variety of bioactivities such as antiviral,10 cytotoxic,11 DNA bind-
ing12 and hypotensive13 activities and their derivatives that
contain a trans-caffeoyl group show cytotoxic activity against
P388 lymphocytic leukemia cells. Biological importance of aryl
C-glycoside over aryl O-glycoside may be due to replacement of
theexocycliccarbon–oxygenbondat theanomericcenterwitha car-
bon–carbon bond which creates a hydrolytically stable carbohy-
drate mimetic with many possible biological applications.14 For
instance, examination of carbohydrate–protein interactions and cell
surface carbohydrate signaling is possible by conjugation of oligo-
saccharides to molecular probes through a C-glycoside tether.15
C-Glycosides 1–7 were isolated from an aqueous extract of the
heartwood of Pterocarpus marsupium Roxb. (Leguminaceae).16 It
has been used in the treatment of diarrhea, toothache, fever, uri-
nary tract and skin infections. The wooden glass made up of
heartwood of this plant is being used for drinking water to con-
trol blood sugar in Ayurvedic system of medicine.17,18 The
aqueous extract of the heartwood of P. marsupium has been
q
CDRI Comm. No. 7989.
⇑
Corresponding author. Tel.: +91 (522) 2612411 18x4235; fax: +91 (522)
2623405/2623938/2629504.
E-mail address: mauryarakesh@rediffmail.com (R. Maurya).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.11.031

P. Rawat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 228–233
229
HO
OH
O
tested clinically and found effective in non-insulin dependent dia-
betes mellitus patients.19
HO
OH
O
D-glucose
DMS,DMF
RT
OH
OCH3
Flavonoid C-glucosides 8–13 were isolated from ethanolic ex-
tract of stem bark of Ulmus wallichiana.20 To get the diversity in
the C-glycosides for SAR study, compound 13, 16, 19, 22 and 24
were synthesized using an elegant method developed by Sato et
al.21 for C-glycosylation using Sc(OTf)3 with unprotected sugars
Sc(OTf)3/
EtOH:H 2O (2:1)
reflux, 8h
OH
14
OH
15
HO
OH
OH
in aqueous media. The structure of 3-C-b-D-glucopyranoside-
2,4,6-trihydroxymethylbenzoate (13) isolated from U. wallichiana
was also verified by its synthesis.
The esterification of 2,4,6-trihydroxybenzoic acid (14) in the
presence of dimethyl sulfate and K2CO3 in DMF afforded esters
OH
HO
OH
OCH3
HO
O
O
+
OH
HO
HO
HO
OH
OCH3
O
OH
HO
HO
OH
13a
O
15. Unprotected
ence of a catalytic amount of Sc(OTf)3 in an aqueous ethanol, lead
to the formation of the 3-C-b- -glucopyranosylphloroacetophe-
D-glucose and ester 15 were reacted in the pres-
OH
OH
O
16
D
Scheme 1. Synthesis of compounds 13 and 16.
none 13 in 40% yield, along with the di-C-b-D-glucoside 16 in
39% yield (Scheme 1).
HO
OH
OH
OH
HO
OH
HO
OH
HO
O
O
D-glucose
HO
HO
+
OH
RO
OH
O
Sc(OTf)3
/
OH
OH
O
O
EtOH:H2O (2:1)
reflux, 8 h
OH
O
17
HO
HO
18
OH
19
OH
BnBr
K2CO3,
DMF,RT
OH
BnO
O
OBn
H
OH
BnO
O
OBn
HO
HO
O
HO
HO
OH
OBn O
21
OH
KOH, EtOH, RT
OBn O
20
H2, Pd/C
EtOH
OH
HO
OH
O
HO
HO
OH
OH
O
22
Scheme 2. Synthesis of compound 18, 19 and 22.
OH
HO
HO
OH
O
O
O
O
CH3I
O
O
HO
HO
HO
HO
K2CO3,
OH
OH
23
DMF, RT
OH
O
18
KOH, EtOH, RT
H
O
OH
O
O
O
O
HO
HO
OH
O
24
Scheme 3. Synthesis of compound 24.

230
P. Rawat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 228–233
Table 1
In vivo antihyperglycemic activity of phenolic C-glycosides on the blood glucose levels of streptozotocin-induced diabetic rats
Compounds
Structure
% Antihyperglycemic activity
% Increase in glucose
uptake by L-6 muscle cells
0–5 h AUC
0–24 h AUC
OH
HO
O
HO
HO
1
12.7
22.3
Nil
OH
OH
HO
OH
OH
OH
HO
O
OH
2
3
4
14.3*
9.78
1.99
16.1*
11.2
7.90
Nil
HO
O
O
HO
O
OH
OH
OH
ND
HO
HO
O
HO
O
OH
OH
OH
HO
Nil
HO
HO
OH
O
O
OH
OH
HO
HO
O
HO
5
6
20.0***
19.5***
ND
O
OH
HO
OH
O
OH
O
17.2***
29.0***
16.0
O
HO
HO
OH
OH
HO
OH
OH
OH
O
O
H
O
7
9.04
10.5
Nil
OH
H
HO
OH
OH
HO
HO
O
O
O
8
15.6
8.56
ND
HO
HO
OH
OH
OH

P. Rawat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 228–233
231
Table 1 (continued)
Compounds
Structure
% Antihyperglycemic activity
% Increase in glucose
uptake by L-6 muscle cells
0–5 h AUC
0–24 h AUC
OH
OH
HO
HO
O
O
O
9
17.6*
12.8*
10.2
HO
HO
OH
OH
OH
OH
OH
HO
HO
O
O
10
11
8.41
15.1
12.2
HO
HO
OH
O
OH
HO
HO
O
O
23.8***
23.8***
ND
HO
HO
OH
OH
OH
O
OH
HO
HO
O
O
12
13
16.7*
23.5*
12.2
ND
HO
HO
OH
OH
OH
OH
O
HO
HO
O
OH
HO
HO
7.27
2.90
O
OH
O
HO
OH
OH
HO
O
16
11.5
7.78
10.8
OH
HO
OH
O
HO
HO
O
OH
OH
O
HO
HO
OH
O
19
22
18.0**
20.9**
15.3
11.6
HO
HO
OH
OH
O
HO
HO
O
OH
22.3***
26.5***
HO
HO
OH
OH
O
O
O
HO
O
24
34.9***
33.6***
ND
O
HO
HO
OH
O
NH NH
Metformin
27.2***
24.1***
19.4
N
N
H
NH2
Values are mean SEM, n = 5.
*
p <0.05 versus vehicle treated control.
p <0.01 versus vehicle treated control.
p <0.001 versus vehicle treated control.
**
***

232
P. Rawat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 228–233
The 1H NMR of 13 showed one singlet at dH 6.00 for an aromatic
proton and one as doublet at dH 4.83 (J = 9.9 Hz) for anomeric pro-
ton. Configuration of the sugar moiety of 13 was determined to be
compound 2423 is a new compounds. All these synthesized com-
pounds were characterized with the help of 1H and 13C NMR, mass
spectroscopy and elemental analysis (Scheme 3).
0
0
b
as J1 2 = 9.9 Hz. The spectral data of synthesized 3-C-b-
D
-
These compounds were examined for their effect on glucose up-
take enhancing activity. The effect was measured using L-6 rat
muscle cells according to Hwang et al.24,25 with some minor mod-
ifications. Skeletal muscle is the main tissue involved in insulin-
induced stimulation of glucose uptake. Insulin increases glucose
uptake in skeletal muscle by increasing functional glucose trans-
port molecules in the plasma membrane. Glucose transport in skel-
etal muscle can also be stimulated by contractile activity. The
maximal effects of insulin and contractile activity on glucose trans-
port are additive. In skeletal muscle, both insulin and contractile
activity stimulate translocation of glucose transporter GLUT-4 pro-
tein from an intracellular membrane pool to the plasma mem-
brane. Resistance to this stimulatory effect of insulin is a major
pathological feature of diabetes. In the tested compounds only
compound 6, 19 and 22 enhance the glucose uptake by 16.0%,
15.3% and 11.6%, respectively, over control and results were com-
pared with metformin which were used as the standard anti dia-
betic drugs.
glucopyranoside-2,4,6-trihydroxymethylbenzoate was identical
with the natural compound 13 isolated from U. wallichiana stem
bark.19 The 1H NMR spectrum of 16, showed the absence of aro-
matic signal around at dH 6.00 and the presence of one doublet
at dH 4.67 (2H, d, J = 9.6 Hz, H-10, 100) integrating for two protons
corresponding to two anomeric protons, indicated the presence
of two glucose moiety in the molecule. The anomeric configuration
of both glucose moieties of 16 is due to the large coupling constant
(J = 9.6 Hz). The anomeric carbons were resonated at dC 74.6
showed that both sugar moieties were connected with aglycone
moiety by C–C linkage. Similarly 3-C-b-
D-glucopyranosylphloro-
acetophenone (18) and 3,5-di-C-b-
D-glucopyranosylphloroace-
tophenon21 (19) were synthesized by unprotected
phloroacetophenonere (17) (Scheme 2).
D-glucose and
3-C-b-D-Glucopyranosylphloroacetophenone (18) was subjected
to aromatic benzylation with 3.5 equiv of benzyl bromide in the
presence of potassium carbonate in DMF followed by Aldol
condensation with benzaldehyde in EtOH and then subsequent
All the seventeen compounds were evaluated for their in vivo
antihyperglycemic activity on low dosed streptozotocin-induced
diabetic rats.26,27 Metformin was taken as positive control. Among
the seventeen screened compounds, eight compounds (2, 5, 6, 11,
12, 19, 22 and 24) demonstrated moderate to excellent antihyper-
glycemic activity to the tune of 14–35% on the STZ-induced dia-
betic rats at 25 mg/kg oral dose (Table 1, Figs. 1–4).
hydrogenolysis with H2, Pd/C led to the formation of 30-C-b-
D-
glucopyranosyldihydrochalcone (22).22 3-C-b-
D-Glucopyranosyl-
phloroacetophenon (18) was subjected to methylation with methyl
iodide in the presence of K2CO3, in DMF followed by Aldol condensa-
tion with 4-isopropylbenzaldehyde led to the formation of
The structure–activity profile revealed that flavonoids with hy-
droxy group at position 3 and 40, showed good inhibitions compare
to without hydroxyl at these position. Activity data also revealed
that the absence of double bond in flavonoids showed an apprecia-
ble increase in activity and while an additional hydroxyl group at 30
position of flavonoids reduces the activity as the compounds 8 and
12 are less active than 9 and 11. Compound 11 is most active in this
series because it has hydroxyl group at 40 position and does not
contain double bond in ring C, hydroxyl group at 30 position. It is
evident from the activity data that if C-ring of flavanoids is chan-
ged into five membered ring as in compound 3 and 4 activity de-
creases whereas isoaurone 6 showed good activity. In chalcone it
seems that aromatic ring having more lipophilic groups are more
active as 24 is more active than compounds 22 and 5. Among all
these compounds, chalcones are found most active
Control
1
2
3
4
600
500
400
300
200
100
0
In conclusion, the C-glycosides exhibited moderate to excellent
in vivo antihyperglycemic activity ranging from 19.9% to 34.9% in
Time (min)
Figure 1. Effect of compounds 1–4 (at 25 mg/kg) on the blood glucose levels of the
streptozotocin-induced diabetic rats at various time intervals.
CONTROL
8
9
10
11
12
6
7
Control
5
Metformin
600
500
400
300
200
100
0
600
500
400
300
200
100
0
Time (min)
Time (min)
Figure 2. Effect of compounds 5–7 (at 25 mg/kg) on the blood glucose levels of the
Figure 3. Effect of compounds 8–12 (at 25 mg/kg) on the blood glucose levels of the
streptozotocin-induced diabetic rats at various time intervals.
streptozotocin-induced diabetic rats at various time intervals.

P. Rawat et al. / Bioorg. Med. Chem. Lett. 21 (2011) 228–233
233
19. Indian Council of Medical Research (ICMR), Collaborating Centres, New Delhi,
Flexible dose open trial of Vijayasar in case of newly-diagnosed non-insulin-
dependent diabetes mellitus. Indian J. Med. Res. 1998, 108, 24.
20. Rawat, P.; Kumar, M.; Sharan, K.; Chattopadhyay, N.; Maurya, R. Bioorg. Med.
Chem. Lett. 2009, 19, 4684.
Control
13
22
24
16
19
600
500
400
300
200
100
0
21. Sato, S.; Naito, Y.; Aoki, K. Carbohydr Res 2007, 342, 913.
22. Spectral data of 22: 1H NMR: (CD3OD, 300 MHz) dH 7.22 (4H, m, H-2, 3, 5, 6),
7.15 (1H, m, H-4), 5.94 (1H, s, H-50), 4.87 (1H, d, J = 9.0 Hz, H-100), 3.98 (1H, t,
J = 8.9 Hz, H-200), 3.39 (1H, m, H-300), 3.40 (2H, m, H-400, 500), 3.83 (1H, m, H-600a),
3.70 (1H, m, H-600b), 3.34 (2H, m, H- ), 2.94 (2H, t, J = 7.2 Hz, H-b).13C NMR:
a
(CD3OD, 75 MHz) dC 206.5 (C@O), 165.9 (C-40), 165.0 (C-20), 164.1 (C-60), 143.2
(C-1), 129.6 (C-2,6), 129.6 (C-3, 5), 126.9 (C-4), 105.5 (C-10), 104.4 (C-30), 96.0
(C-50), 82.6 (C-300), 80.0 (C-500), 76.1 (C-100), 73.1 (C-200), 71.6 (C-400), 62.6 (C-600),
47.1 (C-
23. Spectral data of 24: 1H NMR: (CD3OD, 300 MHz) dH 7.83 (1H, d, J = 16.0 Hz, H-b),
a
), 32.2 (C-b). ESIMS: m/z 421 [M+Na]+.
7.62 (2H, d, J = 7.2 Hz), 7.42 (1H, d, J = 16.0 Hz, H = a), 7.39 (2H, d, J = 7.2 Hz,
H-3, 5), 6.12 (1H, s, H-50), 4.85 (1H, d, J = 9.0 Hz, H-100), 3.97 (1H, t, J = 8.9 Hz,
H-200), 3.37 (1H, m, H-300), 3.40 (2H, m, H-400, 500), 3.83 (1H, m, H-600a), 3.81 (9H,
Time (min)
s, 3OCH3) 3.70 (1H, m, H-600b), 2.98 (1H, s, H-1000), 1.33 (6H, d, J = 6.9 Hz, H-2000
,
Figure 4. Effect of compounds 13, 16, 19, 22 and 24 (at 25 mg/kg) on the blood
3000). ESIMS: m/z 525 [M+Na]+.
24. Hwang, S. L.; Yang, B. K.; Lee, J. Y.; Kim, J. H.; Kim, B. D.; Suh, K. H.; Kim, D. Y.;
Kim, M. S.; Song, H.; Park, B. S.; Huh, T. L. Biochem. Biophys. Res. Commun. 2008,
371, 289.
glucose levels of the streptozotocin-induced diabetic rats at various time intervals.
STZ model. Compounds 11, 22 and 24 lowered the blood glucose to
around 23.8%, 26.5% and 33.6% after 24 h on STZ model, which is
comparable to standard drug metformin. Further study on lead
optimization and mechanism of action is in progress.
25. Methodology of glucose uptake
L6 cell culture: Stock culture of L6 myoblasts were maintained in DMEM
supplemented with 10% (v/v) FBS, streptomycin (200
lg/ml), and penicillin G
(100 g/ml) under an atmosphere of 5% CO2/95% humidified air at 37 °C. For
l
differentiation into myotubes, cells were reseeded in 24-well plate (approx.
35,000 cells/well) containing DMDE media (10% FBS) for overnight culture.
When cells were nearly confluent, 10% FBS containing DMDE media were
replaced with DMDE containing 2% FBS and cells were maintained for seven to
eight days for differentiation into myotubes. Medium was changed every 48 h
prior to use in experiments.
Acknowledgements
Manmeet Kumar, Preeti Rawat are thankful to the CSIR, New
Delhi, India for Senior Research Fellowship and SAIF division, CDRI,
for spectral data.
Glucose uptake assay by L6 rat muscle cells
Measurements of radio labeled 2-deoxyglucose uptake were carried out with
some modification in previously described method of Hwang et al.23
Differentiated L6 mature myotubes cultured on 24-well plates were treated
References and notes
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After that cells washed three-times with Krebs–Ringer N-(2-hydroxye
thyl)piperazine-N-2-ethanesulfonic acid (HEPES) buffer saline. For glucose
uptake measurement, cells were incubated in HEPES buffer saline (HBS)
containing 3 lCi radio-labeled 2-deoxy-[3H]-D-glucose and 20 lM unlabeled
2-deoxyglucose for 15 min. The reaction was terminated by three quick washes
with ice-cold HEPES buffer saline. Non-specific uptake was determined in the
presence of 10 lM cytochalasin B. Cells were lysed in 0.1 NaOH and cell-
associated radioactivity was determined by liquid scintillation counter and
results were expressed as cpm/well as compared to controls.
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27. Evaluation for antihyperglycemic activity in streptozotocin-induced diabetic
rats Male albino rats of Sprague–Dawley strain of blood glucose level between
60 and 80 mg/dl (8–10 weeks of age body weight 140 20 g) were selected for
this study. Streptozotocin was dissolved 0.1 M citrate buffer pH 4.5, and
calculated amount of the fresh solution was injected to overnight fasted rats
(60 mg/kg) intraperitoneally. Blood glucose was checked 48 h later by
glucometer by using glucostrips and animals showing blood glucose values
between 140 and 270 mg/dl were selected and divided into groups of five
animals each. Rats of experimental groups were administered suspension of
the desired test samples orally (made in 1.0% gum acacia) at a dose of 25 mg/kg
body weight. Animals of control group were given an equal amount of 1.0%
gum acacia. Control group was taken as 100%. A sucrose load of 2.5 g/ kg of
body weight was given after 30 min of drug administration. After 30 min of
post-sucrose load, blood glucose level was again checked at 1, 2, 3, 4, 5, 6 and at
24 h, respectively. Comparing the AUC of experimental and control groups
determined the percent antihyperglycemic activity. Statistical analysis was
made by Dunnett’s test (Prism Software).