Synthesis of heteroaryl/aryl kojic acid conjugates as stimulators of glucose uptake by GLUT4 translocation

Article abstract of DOI:10.1016/j.ejmech.2014.08.041

Insulin exerts its metabolic actions through the insulin receptor (IR) and plays an essential role in treatment of diabetes. The inconvenience of daily injections and the undesirable side-effects associated with insulin injections demand novel drugs for the disease. To search for bioactive insulin mimetic, we synthesized a chemical library of small molecules (2a-3f) based on the indolylkojic acid scaffold (B). An In vitro screening assay was performed to stimulate glucose transport in rat L6 skeletal muscle cells, post treatment of the compounds (2a-3f) for the time period incubation of 16 h. Compounds 2f, 2g, 2l, 3a, 3b, 3c and 3d have shown significant glucose uptake stimulation as compared to the controls at micromolar concentrations. In mechanistic studies, we observed that these compounds exert their biological action by enhancing GLUT4 translocation to cell surface via PI3K-dependent signalling pathway in agreement to the insulin mode of action. Hence, these promising conjugates should be useful for further drug development in diabetes treatment.

Full text of DOI:10.1016/j.ejmech.2014.08.041

Accepted Manuscript
Synthesis of heteroaryl/aryl kojic acid conjugates as stimulators of glucose uptake by
GLUT4 translocation
Deepak K. Sharma, Jyotsana Pandey, Akhilesh K. Tamrakar, Debaraj Mukherjee
PII:
S0223-5234(14)00776-4
10.1016/j.ejmech.2014.08.041
EJMECH 7274
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 June 2014
Revised Date: 9 August 2014
Accepted Date: 11 August 2014
Please cite this article as: D.K. Sharma, J. Pandey, A.K. Tamrakar, D. Mukherjee, Synthesis of
heteroaryl/aryl kojic acid conjugates as stimulators of glucose uptake by GLUT4 translocation, European
Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.08.041.
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ACCEPTED MANUSCRIPT
Graphical abstract
Insulin mimics (2a-3f) based on indolylkojic acid scaffold(B) were synthesized. In mechanistic
studies, biologically active compounds were found to enhance GLUT4 translocation to cell
surface via PI3K pathway.

ACCEPTED MANUSCRIPT
Synthesis of heteroaryl/aryl kojic acid conjugates as stimulators of
glucose uptake by GLUT4 translocation
a,b
c
a,c
a,b
Deepak K. Sharma, Jyotsana Pandey, Akhilesh K. Tamrakar, Debaraj Mukherjee *
a
Academy of Scientific and Innovative Research (AcSIR), India
b
CSIR-Indian Institute of Integrative MedicineJammu, J & K, 180001, India
c
CSIR-Central Drug Research Institute, Lucknow-226031, India
Keywords. Heteroaryl/aryl kojic acid conjugates, Insulin receptor, GLUT4 translocation, PI3K
*
Corresponding Author: Debaraj Mukherjee. Tel.:+91-191-2569000; fax: +91-191-2569111;
e-mail: dmukherjee@iiim.ac.in, IIIM/1690/2014
ABSTRACT. Insulin exerts its metabolic actions through the insulin receptor (IR) and plays an
essential role in treatment of diabetes. The inconvenience of daily injections and the undesirable
side-effects associated with insulin injections demand novel drugs for the disease. To search for
bioactive insulin mimetic, we synthesized a chemical library of small molecules (2a-3f) based on
the indolylkojic acid scaffold (B). An In vitro screening assay was performed to stimulate
glucose transport in rat L6 skeletal muscle cells, post treatment of the compounds (2a-3f) for the
time period incubation of 16h. Compounds 2f, 2g, 2l, 3a, 3b, 3c and 3d have shown significant
glucose uptake stimulation as compared to the controls at micromolar concentrations. In
mechanistic studies, we observed that these compounds exert their biological action by
enhancing GLUT4 translocation to cell surface via PI3K-dependent signaling pathway in
agreement to the insulin mode of action. Hence, these promising conjugates should be useful for
further drug development in diabetes treatment.
1
.0. Introduction
1

ACCEPTED MANUSCRIPT
Type 2 diabetes mellitus (T2DM) is a metabolic syndrome characterized by high blood glucose
levels due to either insulin resistance or relative insulin deficiency [1]. The prevalence of
diabetes is increasing worldwide and it has been predicted that the number of patient’s
worldwide suffering from T2DM will rise to over 360 million by 2030 [2,3]. Insulin resistance is
the major pathophysiological defect in T2DM which is characterized by the inability of insulin
sensitive cells to respond adequately to normal levels of insulin. Insulin resistance occurs
primarily within the skeletal muscles, liver and fat tissue leading to alteration of whole body
glucose homeostasis [4]. Skeletal muscle has major contribution in postprandial glucose disposal
which accounts for more than 80% of insulin-dependent glucose disposal in human [5]. Glucose
uptake is the rate-limiting step in glucose utilization in diabetic and non diabetic skeletal muscle
[6]. In skeletal muscle cells glucose uptake is the result of the enhanced translocation and
redistribution of glucose transporter 4 (GLUT4) from intracellular vesicles to plasma membrane,
where it facilitates the entry of the glucose inside the cells [7].
The discovery of the small molecule insulin mimetic demethylasterriquinone B1 from
microbial sources such as Aspergillus terreus, Pseudomassaria fungi represented a major
breakthrough in medicinal chemistry. This discovery demonstrated that a small, nonpeptidyl
natural molecule is capable of mimicking the biological function of a protein hormone by
interacting with the insulin receptor and causing activation of insulin receptor tyrosin kinase
(
IRTK) [8]. Despite these promising early results [9,10], molecules of this family have not yet
entered in clinical trials. A possible major concern is the safety of candidate pharmaceuticals that
contain the potentially problematic quinone substructure. Recent developments on the
replacement of the quinone in demethylasterriquinone B1 (A) with kojic acid (Fig. 1) showed
that it is possible to design products which do not present this safety issue but are equally active
biologically [11,12].
Fig. 1.
In continuation of our ongoing research program in the area of medicinal chemistry, we
decided to design small molecules based on the indolylkojic acid scaffold and thereafter its role
in GLUT4 translocation. In this current approach, we have introduced a one carbon spacer in
between indole/substituted indole/aryl/heteroaryl moiety and the naturally occurring kojic acid as
the modification site in indolylkojic acid scaffold (B). Thereafter, we have studied their glucose
uptake stimulatory effect in skeletal muscle cells as shown in figure 2. In skeletal muscle cells
2

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glucose uptake stimulation by insulin is the result of increased translocation of GLUT4 to cell
surface via activation of Phosphoinositide 3-kinase (PI3K) signaling pathway [13,14]. Hence to
confirm that the compounds under study have the insulin mimetic activity we have also
performed an inhibitor study of PI3K signaling pathway. Wortmannin (WRT) is a specific
inhibitor for PI3K. In the presence of WRT the PI3K pathway is inhibited and translocation of
GLUT4 by insulin or insulin mimetic compounds are restricted to the basal level only.
Fig. 2.
2
2
.0. Results and Discussion
.1. Chemistry.
The earlier reported synthetic routes for indolylkojic acid (B) are based on a Claisen
rearrangement [12] and Stille coupling [11] as shown in figure 3. However, these routes require
multistep sequences making the processes less attractive for quick access of a library of
molecules for SAR studies particularly with variations in the indole portion.
Fig. 3.
We have isolated kojic acid from its natural source Aspergillus flavus using literature method
[15] and utilized it as a raw material for the generation of target structure 1. Reddy et al. [16]
developed one pot multi-component method for the synthesis of this class of compounds using
InCl in solvent free condition at 120°C. However, we observed that method suffers from serious
3
drawbacks such as formation of diindolylmethane [17] as a side product, use of harsh condition
(120°C) which limit this methodology for gram scale synthesis and subsequent bio-evaluation.
Further, the earlier reported method [16] has very narrow substrate scope e.g. they have not taken
nucleophiles other than indole for this particular reaction. To overcome these above mentioned
drawbacks we developed a synthetic strategy which is scalable, simple and flexible enough to
create diversity. We performed initial screening of the catalyst for the synthesis of compound 1a
as shown in table 1. For this purpose we treated kojic acid (1 mmol) with benzaldehyde (1.2
mmol) in the presence of various catalysts in dioxane: water (1:1, 4 mL) at room temperature for
2
4h (Table 1). Compound 1a was obtained in excellent yield using DABCO (entry 3, Table 1). A
slight excess of aldehyde was found to be necessary for achieving the complete consumption of
the α,β-unsaturated cyclohexenone.
3

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Table 1.
To check the substrate scope of the reaction, various aldehydes including aromatic (1a-1h) and
aliphatic (1i, 1j) were allowed to react under the optimized condition as shown in table 2.
Table 2.
In all cases products (1a-1j, Table 2) were obtained in good to excellent yields. In general
aromatic aldehydes with electron-withdrawing substituents gave slightly better yield than those
having electron donating groups (1b vs 1e). With aliphatic aldehydes, reactions with small chain
aldehydes like valeraldehyde (1i) went to completion but long chain aldehydes like nonaldehyde
(1j) needed prolonged time period (2 days).
Our next target was to remove the secondary hydroxyl group of the adduct (1a-1j) with
incoming nucleophiles to obtain product 2a-2j (Table 3). Here we have investigated the role of
modified silica-H SO for C-alkylation of indole due its low cost and recyclability [18-20]. Thus,
2
4
o
compounds 1a-1j (1 equiv.) in CH
3
CN were stirred with indole (1.5 equiv) at 80 C for 2h using
silica-H SO (10 mol%) as catalyst to obtain kojic acid-indole conjugates 2a-2j in excellent
2
4
yields as shown in table 3.
Table 3.
To expand the scope of the substitution reaction various types of substituted indoles and other
nucleophiles such as thiophenol, phenol, pyrrole, thiophene and benzthiophene were allowed to
react with 1a under the optimized conditions as shown in table 4. Products 2k-3f were obtained
in excellent yields (Table 4). As anticipated, regioselectivity was observed in the case of phenol
giving only the para substituted product (3c); C-alkylation with 2-methylthiophene occurred at
C-5 position (3d) and with pyrrole at C-2 position (3f).
Table 4.
2
2
.2. Biological evaluation
.2.1. Glucose uptake stimulatory effect of compounds in L6 skeletal muscle cells. All the
synthesized compounds were evaluated for glucose uptake stimulatory effect in L6 skeletal
muscle cells stably expressing rat GLUT4 with a myc epitope inserted in the first exofacial loop
(
L6-GLUT4myc) and results are depicted in figure 4. From the tested compounds (2a-2j),
4

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compounds synthesized using electron donating benzaldehyde i.e. 2f and 2g [21] shows
significant stimulation of glucose uptake with respective 1.21- and 1.25- fold stimulation
(
p<0.05) over control. In case substituted indoles (2k-2p), compound
methoxyindole was showing better activity than other substituted indoles and showed 1.25 fold
p<0.05) increase in glucose uptake. Compound 1a substituted with various nucleophiles (3a-3f)
other than indole (2a) gives better activity. Among compounds 3a-3f, Compounds 3a, 3b, 3c and
d showed significant stimulation of glucose uptake with respective 1.33 (p<0.01), 1.38
p<0.01), 1.34 (p<0.01) and 1.21(p<0.05) fold increase at 10µM concentration. Compound 3b
2l having 5-
(
3
(
was found to be most potent in the series of synthesized insulin mimics. Insulin was taken as
positive control, which showed 1.65-fold (p<0.001) stimulation of glucose uptake at 100 nM
concentration. All other tested compounds showed very less or no effect on glucose uptake in L6
myotubes. The apparent inhibitory effect of some compounds (2a-2e, 2h-2k, 2o and 2p) on
glucose uptake might be possible due to many reasons like adverse effect on cell viability or
antagonist effect on cellular components of biological pathways. Analysis of statistical
significance of differences in measurements between samples was done by one-way ANOVA
with Dunnets post hoc test (Graph Pad Prism version 3). p<0.05 was considered statistically
significant.
Fig. 4.
Furthermore the dose dependent effect of the active compounds 2f, 2g, 2l and 3a-3b on
glucose uptake at doses of 5 µM, 10 µM and 25 µM is shown in figure 5, taking insulin as a
positive control for the experiment. Insulin at a concentration of 100 nM showed 1.6-fold
(p<0.001) stimulation of glucose uptake compared to basal. All the compounds caused a dose-
dependent increase in glucose uptake in L6-GLUT4myc cells. Compounds 2l and 3b was found
to be equipotent to insulin among active compounds and showed comparable activity to insulin
at the concentration of 25 µm.
Fig. 5.
2
.2.2. Effect of compounds on GLUT4 translocation in L6-GLUT4myc myotubes. In
skeletal muscle, translocation and redistribution of the GLUT4 to the plasma membrane is a
characteristic feature for increased glucose uptake [22]. Hence the effect of compounds on
surface GLUT4 level in non-permeabilized L6-GLUT4myc myotubes was measured by an
5

ACCEPTED MANUSCRIPT
antibody-coupled colorimetric assay as previously described [23]. As shown in figure 6, similar
to glucose uptake data, compounds 2f, 2g, 2l, 3a, 3b, 3c and 3d caused increase in surface level
of GLUT4myc with respect to the control in a dose dependent manner in a concentration range of
5
µM, 10µM and 25µM.
Fig. 6.
2
.2.3. Inhibitor based study for insulin mimetic pathway detection. To investigate the
signaling pathway responsible for the observed biological activity of 2f, 2g, 2l, 3a, 3b, 3c and 3d
and their effect on the PI3K-signaling pathway was observed. The PI3K-pathway is the major
signaling pathway in the insulin action leading to increased translocation and distribution of
GLUT4 at the cell surface [24]. To confirm whether 2f, 2g, 2l, 3a, 3b, 3c and 3d compounds
stimulated glucose uptake in skeletal muscle cells following a pathway similar to insulin, the
involvement of PI3K was investigated using inhibitor studies. Wortmannin (WRT) is a specific
inhibitor for PI3K that blocks the insulin-signaling pathway [25]. Similar to the effect of insulin,
treatment of cells with 2f, 2g, 2l, 3a, 3b, 3c and 3d compounds at 10 µM for 16 hours with a
final one hour in the presence of wortmannin completely abolished the glucose uptake
stimulatory effect of these compounds (Figure 7). These results indicate the involvement of
wortmannin-sensitive PI3-kinase mediated signaling pathway underlying the biological effect of
these compounds to stimulate glucose uptake in skeletal muscle cells.
Fig. 7.
3
.0. Conclusion
In conclusion, we have designed and synthesized of orally active insulin mimics based on
indolylkojic acid scaffold. The synthetic strategy used is concise, simple, high yielding and
flexible enough to create diversity. Our study describes the glucose uptake stimulatory effect of
the compounds 2f, 2g, 2l, 3a, 3b, 3c and 3d. These compounds exert their biological activity by
enhancing GLUT4 translocation to the cell surface via PI-3-Kinase-dependent signaling pathway
similar to the insulin’s mode of action. Results obtained during the study are indicative of future
application of these molecules as effective therapeutic agents in the management of insulin
resistance and type 2 diabetes. Further in vivo study is required to establish the observed in vitro
results and to explore the effect on other insulin sensitive tissues involved in glucose
metabolism.
6

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4
4
5
.0. Experimental section
.1. Chemistry. General information. H and C NMR spectra were recorded on 400 and
00 MHz spectrometers with TMS as the internal standard. Chemical shifts are expressed in
1
13
parts per million (δ ppm). Silica gel coated aluminium plates were used for TLC. Final products
were purified by column chromatography on silica gel (60-120/100-200 mesh) using petroleum
ether–ethyl acetate as the eluent to obtain the pure products. Exact Mass of all products were
analysed by using HRMS with QTOF analyser. Reagents used were purchased from Sigma
Aldrich.
4
.2. General procedure for preparation of compounds (1a-1j): A mixture of kojic acid (1
mmol) and aldehyde (1.2 mmol) was stirred in the presence of DABCO (1.2 mmol) in
dioxane:H O (1:1, 4mL) at room temperature for 24 h. After completion of the reaction, the
2
product was extracted with ethyl acetate (3×15 ml). The combined organic layer was dried with
anhydrous sodium sulphate, concentrated in vacuo, and purified by column chromatography
(methanol: dichloromethane =0.5:9.5) to afford the pure product.
1
Kojic Acid: C H O obtained as a colourless solid. M.pt: 195-205°C HNMR (400MHz,
6
6
4
1
3
CD OD): δ 4.30 (s, 2H), 6.40 (s, 1H), 7.86 (s, 1H); CNMR (125MHz, CD OD): 177.56,
3
3
1
71.13, 148.05, 141.72, 111.41, 61.84. HRMS (-ESI): calcd 142.0266 and found 142.0264.
-hydroxy-2-(hydroxy(phenyl)methyl)-6-(hydroxymethyl)-4H-pyran-4-one (1a): C H O
5
3
1
3
12
1
obtained in 94% yield as a colourless solid. M.pt: 140-150°C. HNMR (400 MHz, CD
3
OD): δ
4
.38 (dd, J = 15.7 Hz, 32.8 Hz, 2H), 6.19 (s, 1H), 6.46 (s, 1H), 7.29 (t, J = 7.3 Hz, 1H), 7.36 (t, J
1
3
=
7.4 Hz, 2H) 7.49 (d, J = 7.4 Hz, 2H); CNMR (125 MHz, DMSO-d ): 173.85, 167.54, 150.34,
6
1
41.22, 140.84, 128.24, 127.40, 125.99, 108.76, 65.88, 59.39. HRMS (-ESI): calcd 247.0606;
found 247.0610.
2-((4-fluorophenyl)(hydroxy)methyl)-3-hydroxy-6-(hydroxymethyl)-4H-pyran-4-one (1b):
1
C H FO obtained in 98% yield as an orange solid. M.pt: 195-205°C. HNMR (400 MHz,
13
11
5
CD3OD): δ 4.27 (dd, J = 15.7 Hz, 30.0Hz, 2H), 6.05 (s, 1H), 6.34 (s, 1H), 6.98 (t, J = 8.7 Hz,
1
3
2
1
H), 7.39 (dd, J = 8.4, 5.5 Hz, 2H); CNMR (125 MHz, CD
3
OD): 176.9, 169.6, 165.0, 162.6,
42.9, 137.7, 129.3, 116.3, 110.9, 68.0, 61.2. HRMS (-ESI): calcd 265.0512; found 265.0514.
2-((4-chlorophenyl)(hydroxy)methyl)-3-hydroxy-6-(hydroxymethyl)-4H-pyran-4-one (1c):
1
C H ClO obtained in 97% yield as a red solid. M.pt: 130-140°. HNMR (400 MHz, CD OD):
13
11
5
3
δ 4.27 (dd, J = 15.7 Hz, 30.0 Hz, 2H), 6.05 (s, 1H), 6.34 (s, 1H), 7.25 (d, J = 8.4 Hz, 2H), 7.35
7

ACCEPTED MANUSCRIPT
1
3
(
d, J = 8.4 Hz, 2H); CNMR (125 MHz, CD
3
OD+DMSO-d
40.95, 134.55, 129.76, 129.16, 110.09, 67.78, 61.17. HRMS (-ESI): calcd 281.0217; found
81.0219
-((4-bromophenyl)(hydroxy)methyl)-3-hydroxy-6-(hydroxymethyl)-4H-pyran-4-one
6
): 176.61, 169.81, 151.77, 142.81,
1
2
2
1
(
1d): C H BrO obtained in 94% yield as a red solid. M.pt: 95-105°C. HNMR (400 MHz,
13 11 5
3
CD OD): δ 4.33 – 4.16 (dd, J = 15.7 Hz, 27.2 Hz, 2H), 6.04 (s, 1H), 6.34 (s, 1H), 7.29 (d, J =
1
3
7
1
.5 Hz, 2H), 7.40 (d, J = 7.0 Hz, 2H); CNMR (125 MHz, CD OD): 176.73, 169.82, 151.61,
3
42.84, 141.13, 132.61, 129.20, 122.68, 109.84, 67.86, 61.10. HRMS (+ESI): calcd 328.9848;
found 328.9840.
3-hydroxy-2-(hydroxy(4-methoxyphenyl)methyl)-6-(hydroxymethyl)-4H-pyran-4-one
1
(1e): C14
H
14
O
6
obtained in 91% yield as a red solid. M.pt: 210-220°C. HNMR (400 MHz,
DMSO-d +D O): δ 3.70 (s, 3H), 4.26 (dd, J = 15.7 Hz, 26.8 Hz 2H), 5.91 (s, 1H), 6.31 (s, 1H),
6
2
1
3
6
1
2
.88 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4, 2H); CNMR (125 MHz, DMSO-d ): 173.89, 167.43,
6
58.57, 150.62, 140.52, 133.14, 127.7, 113.61, 108.72, 65.60, 59.41, 55.00. HRMS (-ESI): calcd
77.0712 ; found 277.0714.
2-((2,5-dimethoxyphenyl)(hydroxy)methyl)-3-hydroxy-6-(hydroxymethyl)-4H-pyran-4-
1
one (1f): C H O obtained in 89% yield as a red solid. M.pt: 240-250°C. HNMR (400 MHz,
1
5
16
7
CD OD): δ 3.61 (s, 3H), 3.73 (s, 3H), 4.24 (dd, J = 15.7 Hz, 32.6 Hz, 2H), 6.33 (s, 1H), 6.34 (s,
3
1
3
1
H), 6.87 (d, J = 8 Hz, 1H), 7.00 (t, J = 8 Hz, 1H), 7.13 (d, J = 7.6 Hz, 1H); CNMR (125 MHz
CD OD): 176.97, 169.54, 155.23, 151.93, 142.99, 130.47, 114.85, 114.42, 112.95, 109.63,
3
6
4.02, 61.10, 56.72, 56.13. HRMS (-ESI): calcd 307.0818; found 307.0820.
2-((2,3-dimethoxyphenyl)(hydroxy)methyl)-3-hydroxy-6-(hydroxymethyl)-4H-pyran-4-
1
one (1g): C H O obtained in 89% yield as a red solid. M.pt: 240-250°C. HNMR (400MHz,
1
5
16
7
3
CD OD): δ 3.61 (s, 3H), 3.73 (s, 3H), 4.24 (q, J = 15.7 Hz, 2H), 6.33 (s, 1H), 6.34 (s, 1H), 6.87
1
3
(
d, J = 8 Hz, 1H), 7.00 (t, J = 8 Hz, 1H), 7.13 (d, J = 7.6 Hz, 1H); CNMR (125MHz, DMSO-
d
5
6
): 173.95, 167.32, 151.90, 150.05, 145.36, 134.38, 123.60, 119.55, 111.92, 108.62, 61.02,
9.89, 59.29, 55.54. HRMS (-ESI): calcd 307.0818 and found 307.0820.
3-hydroxy-2-(hydroxy(naphthalen-2-yl)methyl)-6-(hydroxymethyl)-4H-pyran-4-one (1h):
1
C
17
H
14
O
5
obtained in 89% yield as a dark red solid. M.pt: 250-260°C. HNMR (400MHz,
CD OD): δ 4.25 (dd, J = 15.7 Hz, 35.7 Hz, 2H), 6.24 (s, 1H), 6.35 (s, 1H), 7.37 (s, 2H), 7.47 (d,
3
1
3
3
J = 7.9 Hz, 1H), 7.74 (s, 3H), 7.86 (s, 1H); CNMR (125MHz, CD OD): 176.76, 169.83,
8

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1
1
52.09, 142.87, 139.18, 134.74, 134.56, 129.27, 129.07, 128.68, 127.31, 127.15, 126.00, 125.20,
09.82, 68.64, 61.13. HRMS (+ESI): calcd 299.0919 and found 299.0916.
3-hydroxy-6-(hydroxymethyl)-2-(1-hydroxypentyl)-4H-pyran-4-one
(1i):
C H O
11 16 5
1
obtained in 78% yield as a brown semisolid. HNMR (400MHz, DMSO-d
6
): δ 0.83 (t, J = 6.8
Hz, 3H), 1.21-1.28 (m, 4H), 1.58-1.71 (m, 2H), 4.30 (d, J = 5.6 Hz, 2H), 5.77 (t, J = 5.6 Hz, 1H),
1
3
6
5
.32 (s, 1H); CNMR (125MHz, DMSO-d
6
): 173.88, 167.36, 151.12, 140.78, 108.61, 64.23,
9.46, 33.54, 27.14, 21.80, 13.83. HRMS (-ESI): calcd 227.0919 and found 227.0921.
3-hydroxy-6-(hydroxymethyl)-2-(1-hydroxynonyl)-4H-pyran-4-one
(1j):
C H O
15 24 5
1
obtained in 72% yield as a brown semisolid. HNMR (400MHz, CD
3
OD): δ 0.77-0.79 (m, 3H),
1
3
1
.18-1.19 (m, 12H), 1.67-1.73 (m, 2H), 4.33 (s, 2H), 5.39 (s, 1H), 6.36 (s, 1H); CNMR
125MHz, CD OD): 176.67, 169.59, 152.84, 142.83, 109.75, 66.89, 61.25, 35.25, 33.41, 30.60,
(
3
3
4
0.42, 30.36, 26.47, 23.74, 14.46. HRMS (+ESI): calcd 285.1702 and found 285.1698.
.3. General procedure for preparation of compounds 2a-2j: compound 1a-1j (1 equiv.),
o
silica-H
2
SO
4
(10 mol%) and indole (1.5 equiv) taken in CH
3
CN as solvent was stirred at 80 C
for 2 hr. After completion of the reaction, the product was extracted with ethyl acetate (3×15
ml). The combined organic layer was dried with anhydrous sodium sulphate, concentrated in
vacuo, and purified by column chromatography (methanol: dichloromethane =0.25:9.75) to
afford the pure product.
2-((1H-indol-3-yl)(phenyl)methyl)-3-hydroxy-6-(hydroxymethyl)-4H-pyran-4-one
(2a):
1
C H NO obtained in 96% yield as a colourless solid. M.pt: 210-220°C. HNMR (400MHz,
21
17
4
3
CD OD): δ 4.20 (s, 2H), 5.96 (s, 1H), 6.35 (s, 1H), 6.82 (t, J = 7.5 Hz, 1H), 6.95 (s, 1H), 6.98 (t,
J = 7.6 Hz, 1H), 7.14 (t, J = 7.1 Hz, 1H), 7.20 (t, J = 7.5 Hz, 3H), 7.26 (t, J = 8.2 Hz, 3H);
1
3
CNMR (125MHz, DMSO-d ): 173.63, 167.22, 151.05, 140.78, 140.34, 136.07, 128.43, 128.13,
6
1
26.85, 126.26, 123.86, 121.29, 118.77, 118.37, 112.75, 111.61, 108.94, 59.55. 41.07. HRMS
(+ESI): calcd 348.1236 and found 348.1235.
2-((4-fluorophenyl)(1H-indol-3-yl)methyl)-3-hydroxy-6-(hydroxymethyl)-4H-pyran-4-one
1
(
2b): C H FNO obtained in 98% yield as a brown solid. M.pt: 230-240°C. HNMR (400MHz,
2
1
16
4
CD OD): δ 4.30 (s, 2H), 6.11 (s, 1H), 6.49 (s, 1H), 6.93-7.00 (m, 3H), 7.06-7.07 (m, 2H), 7.30-
3
1
3
7
1
4
3
.37(m, 4H); CNMR (125MHz, CD OD): 176.62, 169.44, 164.24, 162.30, 153.94, 142.73,
38.22, 137.43, 131.42, 127.91, 125.00, 122.84, 120.12, 116.22, 114.41, 112.59, 109.98, 61.25,
1.40. HRMS (+ESI): calcd 366.1142 and found 366.1144.
9

ACCEPTED MANUSCRIPT
2-((4-chlorophenyl)(1H-indol-3-yl)methyl)-3-hydroxy-6-(hydroxymethyl)-4H-pyran-4-
1
one (2c): C H ClNO obtained in 96% yield as a brown solid. M.pt: 180-190°C. HNMR
2
1
16
4
(400MHz, CD OD): δ 4.20 (s, 2H), 5.98 (s, 1H), 6.34 (s, 1H), 6.84 (t, J = 7.5 Hz, 1H), 6.95-7.00
3
1
3
(
m, 2H), 7.17-7.27 (m, 6H); CNMR (125MHz, CD
3
OD): 173.04, 169.49, 153.51, 142.83,
40.37, 138.22, 133.80, 131.23, 129.57, 127.87, 125.02, 122.80, 120.08, 119.82, 114.01, 112.51,
09.91, 61.21, 41.46. HRMS (+ESI): calcd 382.0846 and found 382.0848.
1
1
2-((4-bromophenyl)(1H-indol-3-yl)methyl)-3-hydroxy-6-(hydroxymethyl)-4H-pyran-4-
1
one (2d): C H BrNO obtained in 94% yield as a brown solid. M.pt: 170-180°C. HNMR
2
1
16
4
(
(
(
400MHz, CD
m, 2H), 7.30 (d, J = 8.0 Hz, 3H), 7.38 (d, J = 8.1 Hz, 1H), 7.47 (d, J = 8.2 Hz, 2H); CNMR
125MHz, CD OD): 173.04, 169.49, 153.51, 142.83, 140.84, 138.22, 132.58, 131.56, 127.85,
25.02, 122.80, 121.78, 120.09, 119.78, 113.90, 112.50, 109.95, 61.23, 41.54. HRMS (+ESI):
calcd 426.0341 and found 426.0333.
3
OD): δ 4.33 (s, 2H), 6.09 (s, 1H), 6.48 (s, 1H), 6.96 (t, J = 7.4 Hz, 1H), 7.00-7.15
13
3
1
2-((1H-indol-3-yl)(4-methoxyphenyl)methyl)-3-hydroxy-6-(hydroxymethyl)-4H-pyran-4-
1
one (2e): C H NO obtained in 91% yield as a brown solid. M.pt: 230-240°C. HNMR
2
2
19
5
(400MHz, CD
3
OD): δ 3.78 (s, 3H), 4.31 (s, 2H), 6.05 (s, 1H), 6.48 (s, 1H), 6.87 (d, J = 8.3 Hz,
2
4
1
4
H), 6.94 (t, J = 7.4 Hz, 1H), 7.05 (s, 1H), 7.09 (t, J = 7.5 Hz, 1H), 7.33 (dd, J = 25.2, 8.2 Hz,
1
3
H); CNMR (125MHz, CD OD): 176.75,169.32, 160.02, 154.80, 142.51, 138.32, 138.17,
3
33.43, 130.70, 127.96, 125.03, 122.80, 119.97, 114.95,114.82, 112.57, 109.98, 61.24, 55.99,
1.33. HRMS (+ESI): calcd 378.1341 and found 378.1343.
2-((2,5-dimethoxyphenyl)(1H-indol-3-yl)methyl)-3-hydroxy-6-(hydroxymethyl)-4H-
1
pyran-4-one (2f): C H NO obtained in 89% yield as a brown solid. M.pt: 230-240°C. HNMR
23
21
6
(
400MHz, CD OD): δ 3.64 (s, 3H), 3.74 (s, 3H), 4.22 (d, J = 8.0 Hz, 2H), 6.24 (s, 1H), 6.34 (s,
3
1
8
1
1
H), 6.82-6.88 (m, 3H), 6.98 (t, J = 7.5 Hz, 1H), 7.06 (s, 1H), 7.10 – 7.19(m, 1H), 7.24 (d, J =
13
.1 Hz, 1H), 7.35 (d, J = 7.9 Hz, 1H); CNMR (125MHz, CD OD): 176.63, 169.56, 153.06,
3
44.37, 142.17, 138.08,127.72, 127.62, 126.99, 125.62, 124.49, 122.77, 120.08, 119.74, 114.68,
12.45, 109.79, 61.18, 56.42, 56.11, 37.43. HRMS (+ESI): calcd 408.1447 and found 408.1449.
2-((2,3-dimethoxyphenyl)(1H-indol-3-yl)methyl)-3-hydroxy-6-(hydroxymethyl)-4H-
6
pyran-4-one (2g): C23H21NO obtained in 89% yield as a brown solid. M.pt: 230-240°C.
1
HNMR (400MHz, CD OD): δ 3.65 (s, 3H), 3.75 (s, 3H), 4.20 (s, 2H), 6.36 (s, 1H), 6.39 (s, 1H),
3
13
6
.80-6.89 (m, 5H), 6.97 (t, J = 7.7 Hz, 1H), 7.22 (t, J = 7.7 Hz, 2H); CNMR (125MHz,
10

ACCEPTED MANUSCRIPT
CD
3
OD): 176.57, 169.31, 154.23, 154.10, 148.09, 142.64, 138.16, 134.95, 128.02, 124.96,
1
22.86, 119.93, 119.62, 114.85, 112.65, 112.40, 109.87, 61.25,56.34, 56.24, 35.54. HRMS
(+ESI): calcd408.1447 and found 408.1449.
2-((1H-indol-3-yl)(naphthalen-2-yl)methyl)-3-hydroxy-6-(hydroxymethyl)-4H-pyran-4-
1
one (2h): C H NO obtained in 91% yield as a brown solid. M.pt: 230-240°C. HNMR
2
5
19
4
3
(400MHz, CD OD): δ 4.21 (s, 2H), 6.18 (s, 1H), 6.37 (s, 1H), 6.82 (t, J = 7.5 Hz, 1H), 6.93 -7.06
(m, 2H), 7.22 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 8.2 Hz, 1H), 7.33 (dd, J = 6.1, 3.2 Hz, 2H), 7.42
1
3
(
d, J = 8.4 Hz, 1H), 7.63-7.72 (m, 4H); CNMR (125MHz, CD OD+DMSO-d ): 176.17, 169.49,
3 6
1
1
53.43, 142.85, 139.59, 138.13, 134.96, 134.03, 129.57, 129.31, 129.01, 128.39, 128.27, 128.23,
27.73, 127.40, 125.56, 123.12, 120.51, 120.17, 114.63, 113.11, 110.45, 61.37, 42.13. HRMS
(+ESI): calcd 398.1392 and found 398.1389.
2-(1-(1H-indol-3-yl)pentyl)-3-hydroxy-6-(hydroxymethyl)-4H-pyran-4-one
(2i):
1
C H NO obtained in 87% yield as a brown solid. M.pt: 130-140°C. HNMR (400MHz,
19
21
4
3
CD OD): δ 0.91 (t, J = 6.9 Hz, 3H), 1.21 – 1.53 (m, 4H), 2.03 – 2.33 (m, 2H), 3.37 (s, 1H), 4.36
(q, J = 15.5 Hz, 2H), 6.42 (s, 1H), 6.99 (t, J = 7.4 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 7.23 (s, 1H),
1
3
7
1
3
3
.33 (d, J = 8.1 Hz, 1H), 7.66 (d, J = 7.9 Hz, 1H); CNMR (125MHz, CD OD): 176.41, 169.21,
55.66, 142.53, 137.96, 128.09, 123.39, 122.51, 119.80, 119.72, 115.61, 112.29, 109.49, 61.25,
6.12, 33.14, 28.75, 23.74, 14.46. HRMS (+ESI): calcd 328.1549 and found 328.1551.
2
-(1-(1H-indol-3-yl)nonyl)-3-hydroxy-6-(hydroxymethyl)-4H-pyran-4-one
(2j):
1
C H NO obtained in 86% yield as a brown solid. M.pt: 110-120°C. HNMR (400MHz,
23
29
4
CD
3
OD): δ 0.90 (t, J = 6.6 Hz, 3H), 1.46 – 1.19 (m, 12H), 2.29 – 2.04 (m, 2H), 3.33 (s, 1H), 4.36
(q, J = 15.5 Hz, 2H), 6.41 (s, 1H), 6.99 (t, J = 7.4 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 7.23 (s, 1H),
1
3
7
1
3
.34 (d, J = 8.1 Hz, 1H), 7.65 (d, J = 7.9 Hz, 1H); CNMR (125MHz, CD OD): 176.41, 169.21,
3
55.66, 142.53, 137.96, 128.09, 123.39, 122.51, 119.80, 119.72, 115.61, 112.29, 109.52, 61.25,
6.12, 33.14, 33.01, 30.58,30.42, 30.37, 28.75, 23.74, 14.46. HRMS (+ESI): calcd 384.2175 and
found 384.2171.
.4. General procedure for preparation of compounds 2k-2p and 3a-3f: Compound 1a (1
equiv.), silica-H SO (10 mol%), substituted indoles and other nucleophiles (1.5 equiv) was
4
2
4
o
stirred in CH
3
CN at 80 C for 2 hr. After completion of the reaction, the product was extracted
with ethyl acetate (3x15 ml). The combined organic layer was dried with anhydrous sodium
11

ACCEPTED MANUSCRIPT
sulphate, concentrated in vacuo, and purified by column chromatography (methanol:
dichloromethane =0.25:9.75) to afford the pure product.
3-hydroxy-6-(hydroxymethyl)-2-(phenyl(2-phenyl-1H-indol-3-yl)methyl)-4H-pyran-4-one
1
(2k): C27
H21NO
4
obtained in 89% yield as a grey solid. M.pt: 190-200°C HNMR (400MHz,
CD OD): δ 4.11 (dd, J = 56.8, 15.8 Hz, 2H), 6.15 (s, 1H), 6.36 (s, 1H), 6.77 (t, J = 7.6 Hz, 1H),
3
6
2
1
1
.98 (dd, J = 15.5, 7.7 Hz, 3H), 7.20 – 7.03 (m, 4H), 7.27 (t, J = 7.2 Hz, 2H), 7.35 (t, J = 7.5 Hz,
13
H), 7.48 (d, J = 7.5 Hz, 2H); CNMR (125MHz, DMSO-d ): 173.73, 167.51, 150.71, 141.49,
6
40.14, 136.88, 136.32, 132.17, 128.79, 128.68, 128.36, 127.96, 127.34, 126.41, 121.38, 121.17,
18.76, 11.32, 108.98, 59.35, 41.8. HRMS (+ESI): calcd 424.1549 and found 424.1552.
3-hydroxy-6-(hydroxymethyl)-2-((5-methoxy-1H-indol-3-yl)(phenyl)methyl)-4H-pyran-4-
1
one (2l): C22
H19NO
5
obtained in 92% yield as a brown solid. M.pt: 130-140°C HNMR
(
400MHz, CD OD): δ 3.21 (s, 3H), 4.21 (s, 2H), 5.95 (s, 1H), 6.36 (s, 1H), 6.57 – 6.72 (m, 2H),
3
6
7
1
1
.91 (s, 1H), 7.15 (dd, J = 8.1, 5.6 Hz, 2H), 7.21 (t, J = 7.4 Hz, 2H), 7.21 (t, J = 7.4 Hz, 2H),
1
3
6
.29 (d, J = 7.3 Hz, 2H); CNMR (125MHz, DMSO-d ): 173.96, 167.17, 153.01, 151.51,
40.62, 139.95, 130.89, 128.49, 128.11, 126.92, 126.54, 124.37, 112.48, 112.34, 111.16, 108.94,
00.37, 59.34, 55.23, 41.8. HRMS (+ESI): calcd 378.1341 and found 378.1345.
3-((3-hydroxy-6-(hydroxymethyl)-4-oxo-4H-pyran-2-yl)(phenyl)methyl)-1H-indole-5-
carbonitrile (2m): C H N O obtained in 94% yield as a colourless solid. M.pt: 240-250°C
2
2
16
2
4
1
3
HNMR (400MHz, CD OD): δ 4.21 (d, J = 3.1 Hz, 2H), 6.02 (s, 1H), 6.36 (s, 1H), 7.16-7.25 (m,
1
3
7
1
1
3
H), 7.40 (d, J = 8.5 Hz, 1H), 7.58 (s, 1H); CNMR (125MHz, CD OD): 176.56, 169.42,
3
53.24, 142.88, 140.80, 140.05, 129.74, 129.5, 128.36, 127.88, 127.83, 125.75, 125.50, 121.75,
15.81, 113.81, 110.10, 102.65, 61.26, 41.81. HRMS (+ESI): calcd 373.1188 and found
73.1191.
5-hydroxy-2-(hydroxymethyl)-3-((1-methyl-1H-indol-3-yl)(phenyl)methyl)-4H-pyran-4-
1
one (2n): C H NO obtained in 91% yield as a dark red solid. M.pt: 180-190°C. HNMR
2
2
19
4
3
(400MHz, CD OD): δ 3.64 (s, 3H, NMe), 4.20 (s, 2H), 5.98 (s, 1H), 6.34 (s, 1H), 6.82 – 6.88 (m,
1
3
2
1
1
H), 7.04 (t, J= 7.2Hz, 1H), 7.12-7.27(m, 7H); CNMR (125MHz, DMSO-d +CD OD):
6 3
73.68, 166.76, 151.02, 139.97, 138.86, 135.95, 126.82, 125.86, 125.47, 120.22, 117.48, 111.20,
07.94, 58.53, 39.2, 30.30. HRMS (+ESI): calcd 362.1392 and found 362.1389.
5-hydroxy-2-(hydroxymethyl)-3-((1-pentyl-1H-indol-3-yl)(phenyl)methyl)-4H-pyran-4-
1
one (2o): C26
H27NO
4
obtained in 89% yield as a dark brown solid. M.pt: 150-160°C. HNMR
12

ACCEPTED MANUSCRIPT
(
400MHz, CD
3
OD): δ 0.90 (t, J = 7.1 Hz, 3H), 1.23 – 1.40 (m, 8H), 1.73 – 1.91 (m, 2H), 4.32 (s,
2
7
1
H), 6.11 (s, 1H), 6.48 (s, 1H), 6.97 (t, J = 7.4 Hz, 1H), 7.06 (s, 1H), 7.15 (t, J = 7.6 Hz, 1H),
1
3
.23-7.39 (m, 7H); CNMR (125MHz, CD OD): 137.91, 129.55, 128.52, 128.05, 122.69,
3
22.04, 120.21, 119.98, 110.68, 61.23, 47.06, 31.04, 30.73, 30.16, 23.36, 14.39. HRMS (+ESI):
calcd 418.2018 and found 418.2015.
3-((1-benzyl-1H-indol-3-yl)(phenyl)methyl)-5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-
1
one (2p): C H NO obtained in 89% yield as a colourless solid. M.pt: 160-170°C. H NMR
2
8
23
4
(
400 MHz,CD OD) δ 4.19 (s, 2H), 5.25 (s, 2H), 6.03 (s, 1H), 6.39 (s, 1H), 6.85 (t, J=6.8 Hz
3
1
3
1
1
1
6
H), 6.97-7.02 (m, 4H), 7.12-7.28 (m, 10H), CNMR (125MHz, DMSO-d ): 173.73, 167.51,
50.71, 141.49, 140.14, 136.88, 136.32, 132.17, 128.79, 128.68, 128.36, 127.96, 127.34, 126.41,
21.38, 121.17, 118.76, 111.32, 108.98, 59.35, 54.89, 50.12. HRMS (+ESI): calcd 437.1627 and
found 437.1625.
3-hydroxy-6-(hydroxymethyl)-2-(phenyl(phenylthio)methyl)-4H-pyran-4-one
(3a):
1
C
19
H
16
O
4
S obtained in 94% yield as a red solid. M.pt: 80-90°C. H NMR (400 MHz,CD
3
OD) δ
4
.32 (q, J = 15.7 Hz, 2H), 5.86 (s, 1H), 6.26 (s, 1H), 7.14 (d, J = 3.9 Hz, 3H), 7.21 (d, J = 7.0
1
3
Hz, 1H), 7.25 (t, J = 7.3 Hz, 2H), 7.28 – 7.34 (m, 2H), 7.46 (d, J = 7.3 Hz, 2H), CNMR
125MHz, DMSO-d ):173.32, 167.50, 146.85, 141.68, 136.91, 133.50, 131.30, 129.08, 128.78,
(
6
1
3
28.24, 128.09, 127.66, 108.85, 59.42, 48.56. HRMS (+ESI): calcd 341.0848 and found
41.0846.
3-hydroxy-6-(hydroxymethyl)-2-(((4-methoxyphenyl)thio)(phenyl)methyl)-4H-pyran-4-
1
one (3b): C20
H
18
O
5
S obtained in 89% yield as a light brown solid. M.pt: 110-120°C. HNMR
(
400MHz, CD OD): δ δ 3.64 (s, 3H), 4.34 (q, J = 15.6 Hz, 2H), 5.70 (s, 1H), 6.27 (s, 1H), 6.70
3
(d, J = 8.7 Hz, 2H), 6.77 (d, J = 8.8 Hz, 1H), 7.14 – 7.30 (m, 5H), 7.43 (d, J = 7.1 Hz, 2H);
1
3
3
CNMR (125MHz, CD OD): 176.08, 169.60, 161.83, 150.15, 143.16, 138.43, 137.26, 129.77,
1
29.75, 129.17, 124.90, 115.56, 109.69, 61.23, 55.65, 51.43. HRMS (+ESI): calcd 371.0953 and
found 371.0943.
3-hydroxy-6-(hydroxymethyl)-2-((4-hydroxyphenyl)(phenyl)methyl)-4H-pyran-4-one
1
(
3c): C H O obtained in 92% yield as a orange solid. M.pt: 170-180°C; HNMR (400MHz,
1
9
16
5
3
CD OD): δ 4.19-4.22 (m, 2H), 5.69 (s, 1H), 6.36 (s, 1H), 6.63 (d, J = 8.4 Hz, 2H), 6.95 (d, J =8.4
1
3
Hz, 2H), 7.10 – 7.19 (m, 5H); CNMR (125MHz, DMSO-d ): 173.55, 170.37, 167.25, 156.20,
6
13

ACCEPTED MANUSCRIPT
1
50.41, 141.55, 140.63, 130.10, 129.73, 128.46, 126.71, 115.23, 108.91, 59.77, 46.96. HRMS
(+ESI): calcd 325.1076 and found 325.1077.
3-hydroxy-6-(hydroxymethyl)-2-((5-methylthiophen-2-yl)(phenyl)methyl)-4H-pyran-4-
1
one (3d): C18
H
16
O
4
S obtained in 87% yield as a light brown solid. M.pt: 80-90°C. HNMR
(
400MHz, CD OD): δ 2.31 (s, 3H), 4.26 (s, 2H), 5.89 (s, 1H), 6.36 (s, 1H), 6.52 (d, J = 3.24 Hz,
3
1
3
1
1
5
6
H), 6.58 (d, J = 3.24 Hz, 1H) 7.14 – 7.23 (m, 5H); CNMR (125MHz, DMSO-d ): 173.59,
67.37, 149.09, 141.24, 139.97, 139.57, 138.94, 128.58, 128.01, 127.20, 126.40, 124.90, 109.01,
9.45, 43.32, 14.86. HRMS (+ESI): calcd 329.0848 and found 329.0850.
2-(benzo[b]thiophen-3-yl(phenyl)methyl)-3-hydroxy-6-(hydroxymethyl)-4H-pyran-4-one
1
(
3e): C H O S obtained in 87% yield as a colourless solid. M.pt: 140-150°C. HNMR
2
1
17
4
3
(400MHz, CD OD): δ 4.23-4.25 (m, 2H), 6.11 (s, 1H), 6.33 (s, 1H), 6.26-7.50 (m, 7H), 7.50 (s,
1
3
1
1
1
3
H) 7.65 (d, J = 8Hz 2H) 8.00 (d, J=7.2 Hz, 1H); CNMR (125MHz, DMSO-d ): 173.64,
6
67.44, 149.25, 141.42, 139.55, 138.69, 137.83, 133.87, 128.68, 128.44, 127.24, 125.12, 124.55,
24.31, 123.04, 121.64, 109.05, 59.44, 42.21. HRMS (+ESI): calcd 365.0848 and found
65.0843.
3-hydroxy-6-(hydroxymethyl)-2-(phenyl(1H-pyrrol-2-yl)methyl)-4H-pyran-4-one
(3f):
1
C H NO obtained in 85% yield as a black solid. M.pt: 120-130°C. HNMR (400MHz,
17
15
4
CD OD): δ 4.26 (q, J = 15.6 Hz, 2H), 5.77 (s, 1H), 5.89 -5.95 (m, 2H), 6.35 (s, 1H), 6.59 (s, 1H),
3
1
3
7
1
.12-7.20 (m, 5H); CNMR (125MHz, DMSO-d
6
): 173.64, 167.28, 149.93, 140.91, 140.38,
28.37, 127.89, 116.79, 117.41, 108.90, 107.43, 106.97, 59.47, 48.57, 41.37. HRMS (-ESI):
calcd 296.0923 and found 296.0924.
.5. Glucose Uptake Assay All the synthesized compounds were evaluated for glucose
4
uptake stimulatory effect in L6 skeletal muscle cells stably expressing rat GLUT4 with a myc
epitope inserted in the first exofacial loop (L6-GLUT4myc), a kind gift of Dr Amira Klip,
Program in Cell Biology, The Hospital for Sick Children, Toronto, Canada. Cells were
maintained in DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic solution
(10,000 U/ml penicillin G, 10 mg/ml streptomycin, 25 µg/ml amphotericin B) in a humidified
°
atmosphere of air and 5% CO at 37 C [26,27]. Differentiation was induced by switching
2
confluent cells to medium supplemented with 2% FBS. Experiments were performed in
differentiated myotubes 6–7 days after seeding. At differentiated myotubes stage the cells were
treated with test compounds 16 hours and glucose uptake was measured by incubating cells for 5
14

ACCEPTED MANUSCRIPT
3
min in HEPES-buffered saline containing 10 µM 2-DG (0.5 µCi/ml 2-[ H] DG) at room
temperature, followed by cell lysis and measurement of radioactivity incorporated by
scintillation counting, as described previously [25]. Nonspecific uptake was determined in the
presence of cytochalasin B (25 µM) during the assay and these values were subtracted from all
other values. Glucose uptake measured in duplicates and normalized to total protein, was
expressed as percent stimulation activity with respect to control cells.
4
.6. GLUT4 translocation assay. GLUT4 translocation to cell surface was determined in L6-
GLUT4myc myotubes by measuring the cell surface level of GLUT4myc by an antibody-coupled
colorimetric assay as previously described [23]. Essentially, cells grown in 24-well plates and
treated as indicated were fixed in 3% paraformaldehyde for 30 min and quenched in 100 mM
glycine for 10 min. Following blocking with 5% skimmed milk for 10 min, cells were incubated
with anti-myc antibody solution (1.0 µg/ml in PBS with 3% skimmed milk) for 1 h. After
labeling, excess antibodies were removed by extensive washing in ice-cold PBS. Cell surface
GLUT4-bound antibodies were probed by HRP-conjugated secondary antibodies followed by
detection of bound HRP by O-phenylenediamine dihydrochloride reagent. The reaction was
stopped by addition of 3N HCl and the supernatant was collected to read optical density at 492
nm. The fraction of GLUT4myc at the cell surface, measured in triplicate, was expressed as fold
induction with respect to unstimulated cells.
4
.7. Inhibitor study for PI3K. Experiments were performed in differentiated myotubes 6–7
days after seeding. At differentiated myotubes stage the cells were treated with test compounds
6 hours with final one hour in presence of wortmannin (1µm) and glucose uptake was measured
1
by incubating cells for 5 min in HEPES-buffered saline containing 10 µM 2-DG (0.5 µCi/ml 2-
3
[
H] DG) at room temperature, followed by cell lysis and measurement of radioactivity
incorporated by scintillation counting, as described previously [25]. Nonspecific uptake was
determined in the presence of cytochalasin B (25 µM) during the assay and these values were
subtracted from all other values. Glucose uptake measured in duplicates and normalized to total
protein, was expressed as percent stimulation activity with respect to control cells.
Acknowledgments
Authors are thankful to Director IIIM Jammu for encouragement and DST (GAP-1145) for
funding.
15

ACCEPTED MANUSCRIPT
Supplementary data
1
13
H, C NMR and experimental details are provides in SI.
References
[1] V. Kumar, N. Fausto, A. K. Abbas, R. S. Cotran, S. L. Robbins, Robbins and Cotran
Pathologic Basis of Disease (7th ed.). Philadelphia, Pa., Saunders. (2005) 1194–
1
195. ISBN 0-7216-0187-1.
[
2] P. Hossain, B. Kawar, M. El Nahas, Obesity and diabetes in the developing world-a growing
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Table 1. Reaction of kojic acid with benzaldehyde in the presence of various catalysts in dioxane/H O at room
2
temperature
b
Entry
Catalyst
Imidazole
DMAP
Time (d)
yield (%) of 1a
1
2
3
4
5
6
1
1
1
1
1
1
72%
60%
94%
47%
54%
30%
DABCO
DBU
Pyridine
3
Et N
a
kojic acid (1 mmol) with benzaldehyde (1.2 mmol) in the presence of various catalysts (entry 1-6, 1.5 mmol) in
b
dioxane:H O (1:1, 4 mL) at room temperature for 24 h. Isolated yield after column chromatography.
2

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Table 2. Products of reaction between kojic acid with aryl/alkyl aldehydes.
1
c,97%
1
b,98%
1a, 94%
1d, 94%
1g, 89%
1e, 91%
1f, 89%
1
h, 89%
1
i, n= 3, 85% and
b
1j, n=7, 83%
a
2
Reagents and conditions: (a) Kojic acid (1 mmol), RCHO (1.2 mmol), DABCO (1.2mmol), dioxane:H O (1:1),
b
room temp., 24 h. Reaction took 2 days for complete conversion.

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Table 3. Nucleophilic substituion of indole on various aldol adducts.
2
b, 98%
2c, 96%
2
2
2
a, 98%
d, 94%
g, 89%
2e, 91%
2h, 91%
2f, 89%
NH
OH
O
O
(
)n
OH
2i,n=3, 87% and
j, n=7 86%
Reagents and conditions: (a) 1a -1j (1equiv.), indole (1.5 equiv), silica-H SO (10 mol %), CH CN, 80°C, 2h.
2
a
2
4
3

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Table 4. Nucleophilic substitution of 1a by variously substituted indoles and different nucleophiles.
2
l, 92%
2m, 94%
3a, 94%
2
3
n, 91%
b, 89%
2
k, 89%
N
OH
O
OH
O
2
o, 89%
2p,89%
3
e, 87%
3f, 85%
3
d, 87%
3
c, 92%
a
2 4
Reagents and conditions: (a) 1a (1equiv.), Nu (substituted indoles and other nucleophiles 1.5 equiv), silica-H SO
(
10 mol %), CH CN, 80°C, 2h.
3

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Fig. 1. Orally active insulin mimic natural product demethyasterriquinone B1(A) and its kojic acid derived analogue (B) which
retains insulin mimic activity.

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IR activation
PI3K activation
O
OH
a)
Aryl/alkyl moeities
HO
O
(
(b)
indoles,
Aryl and heteroaryl
moeities
O
OH
HO
GLUT4 translocation
N
Insulin Mimic
activity
H
O
Enhance glucose uptake
by L6 skeletal muscle cells
kojic acid-derived analogue (B)
Target Structure (1)
Fig. 2. Objective of current work (a) synthesis of target molecule based on indolylkojic acid scaffold with one carbon spacer (b)
Glucose uptake stimulatory effect in L6 skeletal muscle cells via insulin like mode of action of target structure.

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Fig. 3. Literature methods for synthesis of indolylkojic acid (B): (a) Sonogashira coupling and heteroaromatic Claisen
rearrangement,of a protected iodoaniline with propargyl kojate (b) Stille coupling between stannyl derivative of indole and halide
derivative of kojic acid.

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Fig. 4. Glucose uptake stimulatory effect of compounds after overnight treatment in L6-GLUT4myc cells. (*P <0.05), (**P
<0.001) relative to controls.

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Fig. 5. Concentration-dependent effects of compounds 2f, 2g, 2l, 3a, 3b, 3c and 3d on 2-deoxyglucose uptake in L6-GLUT4myc
myotubes. Results shown are mean ± S.E.M. of three independent experiments. (*P <0.05), (**P <0.001) relative to controls.
Insulin is taken as positive control.

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Fig. 6. Effect of test compounds 2f, 2g, 2l, 3a, 3b, 3c and 3d on GLUT4 translocation in L6 GLUT-4 myc myotubes. Cells were
incubated with vehicle (DMSO), Insulin (Ins, 100nM) and test compounds at 5µM, 10µM, 25µM for 3 hours and surface density
of GLUT4 myc was determined as described. Results are expressed as fold stimulation over control. Results shown are mean±SE
of three independent experiments, each performed in triplicate. (*p<0.05), (**p<0.01) and (***p<0.001) relative to controls.