Synthesis of novel l-rhamnose derived acyclic C-nucleosides with substituted 1,2,3-triazole core as potent sodium-glucose co-transporter (SGLT) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.01.077

Sodium-glucose co-transporter (SGLT) inhibitors are a novel class of therapeutic agents for the treatment of type 2 diabetes by preventing renal glucose reabsorption. In our efforts to identify novel inhibitors of SGLT, we synthesized a series of l-rhamnose derived acyclic C-nucleosides with 1,2,3-triazole core. The key β-ketoester building block 4 prepared from l-rhamnose in five steps, was reacted with various aryl azides to produce the respective 1,2,3-triazole derivatives in excellent yields. Deprotection of acetonide group gave the desired acyclic C-nucleosides 7a-o. All the new compounds were screened for their sodium-glucose co-transporters (SGLT1 and SGLT2) inhibition activity using recently developed cell-based nonradioactive fluorescence glucose uptake assay. Among them, 7m with IC₅₀: 125.9 nM emerged as the most potent SGLT2 inhibitor. On the other hand compound 7d exhibited best selectivity for inhibition of SGLT2 (IC₅₀: 149.1 nM) over SGLT1 (IC₅₀: 693.2 nM). The results presented here demonstrated the utility of acyclic C-nucleosides as novel SGLT inhibitors for future investigations.

Full text of DOI:10.1016/j.bmcl.2014.01.077

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1528–1531
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of novel L-rhamnose derived acyclic C-nucleosides with
substituted 1,2,3-triazole core as potent sodium-glucose
co-transporter (SGLT) inhibitors
a,c,
Siddamal Reddy Putapatri a, Abhinav Kanwal b, Sanjay K. Banerjee b,c, , Srinivas Kantevari
⇑
⇑
a Organic Chemistry Division-II (CPC Division), CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
b Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
c Academy of Scientific and Innovative Research, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Sodium-glucose co-transporter (SGLT) inhibitors are a novel class of therapeutic agents for the treatment
of type 2 diabetes by preventing renal glucose reabsorption. In our efforts to identify novel inhibitors of
Received 4 November 2013
Revised 12 January 2014
Accepted 30 January 2014
Available online 6 February 2014
SGLT, we synthesized a series of
L-rhamnose derived acyclic C-nucleosides with 1,2,3-triazole core. The
key b-ketoester building block 4 prepared from
L-rhamnose in five steps, was reacted with various aryl
azides to produce the respective 1,2,3-triazole derivatives in excellent yields. Deprotection of acetonide
group gave the desired acyclic C-nucleosides 7a–o. All the new compounds were screened for their
sodium-glucose co-transporters (SGLT1 and SGLT2) inhibition activity using recently developed cell-
based nonradioactive fluorescence glucose uptake assay. Among them, 7m with IC50: 125.9 nM emerged
as the most potent SGLT2 inhibitor. On the other hand compound 7d exhibited best selectivity for inhi-
bition of SGLT2 (IC50: 149.1 nM) over SGLT1 (IC50: 693.2 nM). The results presented here demonstrated
the utility of acyclic C-nucleosides as novel SGLT inhibitors for future investigations.
Keywords:
Triazole
Rhamnose
Diabetes
Click chemistry
SGLT2 inhibitors
Ó 2014 Elsevier Ltd. All rights reserved.
In recent years, inhibition of sodium-glucose co-transporters
(SGLTs) was considered as one of the therapeutic options to reduce
blood glucose level independent of insulin.1 Of several subtypes
identified in the SGLT family, sodium-glucose co-transporters 2
(SGLT2) expressed in the early convoluted segment (S1) of the prox-
imal tubule is responsible for 90% of the renal glucose reabsorption.2
Remaining 10% of renal glucose reabsorption occurs through SGLT1
is expressed in the proximal tubule (S3). Other tissues like intestine,
heart and trachea also express SGLT1. Thus, enhancing glucose
excretion into urine by selectively inhibiting SGLT2 should lead to
effective control of type 2 diabetes mellitus (T2DM).3 Over the past
10 years, a series of O-glucosides and C-glucosides has been re-
ported as SGLT2 inhibitors (Fig. 1).4 T-10955 is the first structural
derivative of Phlorizin,6 a natural non selective SGLT inhibitor.
Sergliflozin7a and remogliflozin7b,c are other representatives of the
O-glucoside class of SGLT2 inhibitors. However, these two synthetic
aryl O-glucosides were abandoned during clinical trials due to their
poor selectivity/ efficacy and inherent metabolic instability to gas-
trointestinal b-glucosidases.8 Meanwhile, dapagliflozin,9 followed
by canagliflozin10 and empagliflozin11 have emerged as C-aryl glu-
coside class of SGLT2 inhibitors. These three were found to exhibit
more potent hSGLT2 inhibition in vitro and gastrointestinal stability
in vivo than O-glucosides.9–11 However, recent clinical results indi-
cated that none of these SGLT2 inhibitors could exert more than 50%
renal glucose reabsorption in humans.12 Thus the quest for develop-
ing novel small molecule carbohydrate mimics as inhibitors of
SGLT2 is higher than ever before.13 Besides this, C-glucosides with
triazole, indole, benzisothiazole and thiophene aglycon were also
investigated as inhibitors of SGLT2 (Fig. 2).14
From all these reports it is evident that development of SGLT
inhibitors is focused mainly at modifying aglycon unit of C-aryl glu-
coside leaving the sugar moiety untouched. Departing from this
ongoing trend, we asked ourselves whether acyclic C-nucleosides
can inhibit SGLT activity. In our efforts to find new and potent acy-
clic C-nucleosides15 as novel SGLT inhibitors, we herein present our
results on the synthesis and evaluation of the first examples of L-
rhamnose derived acyclic C-nucleosides with substituted 1,2,3-tri-
azole core as potential sodium-glucose co-transporter inhibitors.
Here,
sugar having no known role in mammalian metabolism and toxic-
ity,16a was considered as carbohydrate mimic replacing
-glucose
for the development of new antidiabetic agents. The choice of
L-rhamnose, a naturally occurring, easily available 6-deoxy-
⇑
Corresponding authors. Tel.: +91 4027191438; fax: +91 4027198933.
E-mail addresses: skbanerjee@iict.res.in (S.K. Banerjee), kantevari@yahoo.com,
D
kantevari@gmail.com (S. Kantevari).
http://dx.doi.org/10.1016/j.bmcl.2014.01.077
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

S. R. Putapatri et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1528–1531
1529
HO OH
(a)
HO
O
OH
O
OH
O
O
O
O
O
H3C
O
OH
O
O
OH
OH
(ii)
(i)
(iii)
OH
OH
HO OH
HO
O
OH
L-rhamnose
O
O
HO
HO
L-rhamnitol
1
O
O
OH
HO
OH
O
OH
Phlorizin
O
O
O
O
O
O
OH
T-1095
(iv)
O
(v)
OH
O
H
OH
O
O
OH
HO OH
O
N
N
4
2
3
O
O
Scheme 1. Synthesis of L-rhamnose derived b-ketoester building block 4. Reaction
O
O
O
O
O
O
conditions: (i) NaBH4, methanol, rt; (ii) dimethoxypropane, acetone, pTSA, rt; (iii)
35% aq AcOH; (iv) NaIO4, methanol/water (1:1); (v) N2CHCOOEt, dichloromethane,
SnCl2ꢀ2H2O.
O
O
O
HO
OH
HO
OH
OH
OH
Remogliflozin
Sergliflozin
(b)
as such without any further purification and characterization. Fi-
nally, the required key b-ketoester building block 4 was obtained
in 80% yields after reacting 3 with ethyl diazoacetate at room tem-
perature in dichloromethane. Compounds 1, 2 and 4 were fully
characterized by their IR, NMR and mass spectral data.17 On the
other hand, the required azides 5a–n were also prepared from
the respective amines by following the reported procedures,18
and were characterized by their spectral data correlating with
literature.
Click chemistry originally described by Huisgen and later devel-
oped by Sharpless, has become a ubiquitous chemical tool with
applications in nearly all areas of modern chemistry, including
drug discovery.19 Besides original Cu(I)-catalyzed azide–alkyne
cycloaddition, the variants like inverse electron demand Diels–Al-
der reaction, and other types of bio-orthogonal click ligations have
gained importance for the synthesis of bioactive molecules and
radiopharmaceuticals.20 One such variant of click reaction is organ-
ocatalytic 3+2 cycloaddition of b-ketoesters with aryl azides to
produce multi-substituted 1,2,3-triazoles.21
F
Cl
O
O
HO
HO
Empagliflozin
OH
OH
S
Cl
OEt
O
O
HO
HO
HO
HO
OH
Canagliflozin
OH
OH
OH
Dapagliflozin
Figure 1. Examples of some (a) O-glucoside and (b) C-glucoside SGLT2 inhibitors.
CN
S
N
N
N
N
O
HO
HO
O
HO
HO
Having L-rhamnose derived b-ketoester 4 in hand, we employed
OH
OH
OH
3+2 cycloaddition reaction with aryl azides in the presence of an
organocatalyst. After series of experiments with varying reaction
parameters and organocatalysts (proline, pyrrolidine), cycloaddi-
tion of b-ketoester 4 with aryl azides 5a–n (Scheme 2) were best
achieved using pyrrolidine as organocatalyst.21 For example, 3+2
cycloaddition of b-ketoester 4 with phenyl azide 5a in the presence
of pyrrolidine (10 mol %) in DMSO at 60 °C for 14 h produced ethyl
5-((4S,5S)-5-((S)-1-hydroxyethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)
-1-phenyl-1H-1,2,3-triazole-4-carboxylate (6a) in 81% yield. Use of
proline as catalyst resulted 6a in lower yield (45%). All the azides
5a–o reacted well with b-ketoester 4 to give the respective 1,4,5-tri-
substituted 1,2,3-triazole derivatives 6a–o in excellent yields
(Scheme 2). Compound 6a–o was fully characterized by IR, 1H
NMR, 13C NMR, mass spectral (HRMS) analysis.22 Finally deprotec-
tion of 1,4,5-trisubstituted 1,2,3-triazole derivatives 6a–o in dichlo-
romethane using trifluoroacetic acid/water (1:1) gave the desired
acyclic C-nucleosides 7a–o in very good (71–85%) yields. All the fi-
nal products 7a–o were purified over silica gel column chromatog-
raphy and were fully characterized by IR, 1H NMR, 13C NMR, electron
spray ionization (ESI), and high-resolution mass spectral (HRMS)
analysis.22
The in vitro inhibitory potential of all fifteen new acyclic
C-nucleosides 7a–o with aryl substituent’s appended to nitrogen on
1,2,3-triazole core was assessed using recently developed23 cell-
based nonradioactive fluorescent glucose uptake assay for functional
screening of sodium-glucose co-transporters SGLT1 and SGLT2 deter-
mined at excitation/emission maxima of 465/540 nm. SGLT1 & SGLT2
transfected Human Embryonic Kidney (HEK293) cells were propa-
gated at 37 °C in 5% CO2 in Dulbecco’s minimal essential medium
(DMEM) supplemented with 1.0% of penicillin-streptomycin and
OH
I
III
OH
H
N
Cl
S
O
F
O
O
HO
HO
HO
HO
N
OH
II
OH
IV
OH
Figure 2. Representative C-glucosides with triazole(I), indole(II), benzisothiaz-
ole(III), and thiophene(IV) aglycons as SGLT2 inhibitors.
6-deoxy L-mannose (L-rhamnose) is also due to recent discovery of
6-deoxydapagliflozin as a more active SGLT2 inhibitor (IC50 = 0.67 -
nM against human SGLT2 (hSGLT2) vs 1.16 nM for dapagliflozin.16b
Biological evaluation of fifteen new synthetic analogs revealed two
compounds 7m with IC50: 125.9 nM emerged as the most potent
SGLT2 inhibitor and 7d exhibited best selectivity for inhibition of
SGLT2 (IC50: 149.1 nM) over SGLT1(IC50: 693.2 nM).
Synthetic approach for the preparation of L-rhamnose derived
acyclic C-nucleosides with substituted 1,2,3-triazole core 7a–o
are presented in schemes 1 and 2. The key b-ketoester building
block [ethyl 3-((4R,5S)-5-((S)-1-hydroxyethyl)-2,2-dimethyl-1,3-
dioxolan-4-yl)-3-oxopropanoate] 4 required for library generation
is synthesized from commercially available
L-rhamnose (Scheme 1).
L
-Rhamnitol obtained after NaBH4 reduction,15 was protected by
reacting with 2,2-dimethoxy propane. Selective deprotection of
acetonide in 35% aqueous acetic acid gave triol 2 in 85% yield.
Oxidative cleavage of 2 with NaIO4 in methanol: water produced
reactive aldehyde 3 in excellent yield (95%). Aldehyde 3 was used

1530
S. R. Putapatri et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1528–1531
N
N
N
N
N
N
O
COOEt
OH
Pyrrolidine
DMSO
COOEt
OH
O
TFA-water
0°C-rt, 2h
R
O
R
N3
+
R
60°C, 14h
O
O
O
OH
HO
O
HO
5a-o
4
6a-o
7a-o
R=
OCH3
Br
CF3
H3C
F3CO
Ph
Br
OCH3
6h
(67%)
6a
6g
(81%)
6b
7b
6d
7d
6f
7f
(70%)
(83%)
(92%)
(85%)
(85%)
(77%)
(71%)
6c (79%)
7c
6e (75%)
7e
7h (82%)
7a (90%)
7g (77%)
(89%)
(86%)
OCH3
O2N
O2N
F
Cl
Cl
O
O2N
O
6o (69%)
7o
6k
6n
(76%)
7n (88%)
6i
6j
(71%)
6m
(73%)
(70%)
(79%)
6l (77%)
7l
(83%)
7k (80%)
7i (76%)
7j (79%)
7m (75%)
(73%)
Scheme 2. Synthesis of acyclic C-nucleosides 7a–o via organocatalytic variants of click reaction.
Table 1
10% heat inactivated fetal bovine serum (FBS). The cells were cultured
in a 90 mm dish in DMEM with 10% FBS until 70–80% confluences ob-
tained for further use for screening SGLT1 & SGLT2 inhibition activity.
To measure SGLT1 and SGLT2-mediated glucose uptake,23b transfec-
ted stable HEK cells were plated separately at 1 ꢁ 104/well in 96-well
plate; all culture medium was removed from each well and replaced
Evaluation of SGLT1 and SGLT 2 activity for 7a–o
a
a
Entry Product
SGLT2 IC50
(nM)
SGLT1 IC50
(nM)
SGLT1/SGLT2
selectivity
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
134.7
163.2
214.2
149.1
146.9
311
185.8
150.0
151.8
190.3
367
175
125.9
458
227
58.6
314.6
467.4
523.5
693.2
315.4
628.5
621.0
435.2
466.8
527.5
256.7
392.2
220.0
434.1
626.2
60.7
2.34
2.86
2.44
4.65
2.15
2.02
3.34
2.90
3.08
2.77
0.70
2.24
1.75
0.95
2.76
1.04
with 100 ll of culture medium with newly synthesized acyclic C-
nucleosides 7a–o at five different concentrations ranging from 50 to
1000 nM (i.e. 50, 100, 250, 500 and 1000 nM). After half an hour, a
fluorescently-labeled 2-deoxyglucose analog, 2-NBDG (2-deoxy-2-
[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose) was added to
the plates and incubated for 30 min. After washing three times, cells
werethenlysedandfluorescenceof aliquotsfromthelysatewasmea-
sured at excitation/emission maxima of ꢂ465/540nm. Phlorizin, a
natural non-specific SGLT1 and SGLT2 inhibitor was used as a refer-
ence compound in this fluorescent based in vitro activity evaluation
system. The IC50 values (concentration to inhibit 50% D-glucose up-
Phlorizin
take in cells) of new acyclic C-nucleosides 7a–o were determined
from the glucose uptake inhibition curves with reference to phlorizin.
IC50 values obtained for synthetic compounds 7a–o are depicted in
Table 1. All fifteen new compounds 7a–o showed SGLT1 and SGLT2
inhibition activity with IC50 ranging from 125.9 to 693.2 nM(Table 1).
Thirteen new analogs, except 7k and 7n exhibited SGLT2 inhibition at
lower concentrations (IC50 ranging from 125 to 227 nM) than SGLT1.
Among all the derivatives, p-nitrophenyl analogue7misthemostpo-
a
Average of three experiments.
identified first examples of acyclic C-nucleosides 7a–o as inhibitors
for SGLT1 and SGLT2.
In conclusion, the present study led to the identification of the
first examples of a series of new L-rhamnose derived acyclic C-
tent SGLT2 inhibitor with IC50: 125.9 nM. Other analogs 7a
nucleosides 7a–o bearing substituted 1,2,3-triazole core as potent
sodium-glucose co-transporter (SGLT) inhibitors. The key b-keto-
ester building block 4 was reacted with various aryl azides to give
6a–o. Deprotection of acetonide group in 6a–o produced the
respective 1,2,3-triazole derivatives 7a–o in excellent yields. All
the new compounds 7a–o were evaluated for sodium-glucose co-
transporters SGLT1 and SGLT2inhibitory activity using recently
developed cell-based nonradioactive fluorescence glucose uptake
(134.7 nM), 7b (163.2 nM), 7d (149.1 nM), 7e (146.9 nM), 7h
(150.0 nM), and 7i (151.8 nM) also exhibited potency for SGLT2 inhi-
bition at concentrations in comparable range. Compound 7n is non-
specific analogue similar to reference drug and inhibited both SGLT1
and SGLT2 at concentration of 434 and 458 nM respectively. Com-
pound 7k is the only compound in this series inhibited SGLT1 (IC50
:
256 nM) at slightly lower concentration than SGLT2 (IC50: 367 nM).
We next correlated selectivity index of all the compounds 7a–o for
SGLT2 over SGLT1 inhibitory activities (Table 1). Unfortunately, most
of the acyclic C-nucleoside SGLT2 inhibitors 7a–o tested showed
moderate selectivity index. Among all analogs 7a–o, one compound
assay. Among all, two compounds 7m with IC50
: 125.9 nM
emerged as the most potent SGLT2 inhibitor and 7d exhibited best
selectivity for inhibition of SGLT2 (IC50: 149.1 nM) over SGLT1
(IC50: 693.2 nM). It is worth mentioning that recently developed
cell-based nonradioactive fluorescence glucose uptake assay24 is
used for the first time in evaluating compounds as SGLT1/ SGLT2
inhibitors. Over all the results presented here demonstrated the
ethyl
1-(2,5-dimethoxyphenyl)-5-((1S,2S,3S)-1,2,3-trihydroxybu-
149 nM) inhibited
tyl)-1H-1,2,3-triazole-4-carboxylate 7d (IC50
:
SGLT2 five times more selectively over SGLT1. Although the selectiv-
ity of new compounds for SGLT2 over SGLT1 is not high, these results

S. R. Putapatri et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1528–1531
1531
17. Ethyl 3-((4R,5S)-5-((S)-1-hydroxyethyl)-2,2-dimethyl-1,3-dioxolan-4-yl)-3-
oxopropanoate (4): Compound (4 g, 22.9 mmol) in dichloromethane
utility of acyclic C-nucleosides as novel SGLT inhibitors for future
investigations in development of drugs to treat T2DM.
3
(50 mL), ethyl diazoacetate (3.1 g, 27.5 mmol) and SnCl2ꢀ2H2O (100 mg) was
added at 0 °C and stirred at rt for 7 h. The reaction was quenched with 1 N HCl
(20 mL) and extracted with dichloromethane (2 ꢁ 30 mL). The combined
organic extracts were washed with brine, dried over anhydrous Na2SO4,
concentrated under reduced pressure. The crude residue was purified by
column chromatography over silica gel eluted with ethyl acetate/hexane
Acknowledgments
Authors are thankful to Dr. M. Lakshmikantam, Director and Dr.
V.J. Rao, Head, CPC Division, IICT, Hyderabad for their continuous
support, encouragement and assistance through CSIR 12th FYP
Network (ORIGIN, CSC0108; DENOVA, CSC0205; SmiLE, CSC
0110) & MLP 0002 projects. S.K.B. is thankful to Department of
Biotechnology (DBT) for providing Ramalingaswami Fellowship.
S.K., (BT/Indo-Aus/07/06/2013) and S.K.B. (BT/PR13768/MED/30/
300/2010) thanks DBT for grants. S.R.P. & A.K. (SRF) are thankful
to CSIR for financial assistance.
(2.5:7.5) to afford 4 (4.70 g, 80%) as syrupy liquid. Yield: 80%; ½a D29
ꢃ
32.5 (c 1.0,
CHCl3). 1H NMR (500 MHz, CDCl3) d 4.43(d, J = 6.7 Hz, 1H), 4.21 (q, J = 7.1 Hz,
2H), 4.10–3.96 (m, 2H), 3.71 (d, J = 4.1 Hz, 1H), 2.39 (br s, 1H), 1.48 (s, 3H), 1.37
(s, 3H), 1.28 (t, J = 7.1 Hz, 3H), 1.22 (d, J = 6.4 Hz, 3H). 13C NMR (125 MHz,
CDCl3) d 204.3, 166.8, 110.6, 81.1, 81.0, 67.4, 61.4, 45.9, 26.6, 25.6, 18.5, 14.0. IR
(neat) 3490, 2985, 2936, 1744, 1375, 1216, 1164, 1073, 878 cmꢄ1. MS (ESI) m/z
261[M+H]+; HR-MS (ESI) calcd for C12H20O6 [M+Na]+: 283.11521, found:
283.11452.
18. Karine, B.; Adam, D. M.; John, E. M. Org. Lett. 2007, 9, 1807.
19. For recent reviews, see: (a) Thirumurugan, P.; Matosiuk, D.; Jozwiak, K. Chem.
Rev. 2013, 113, 4905; (b) Grammel, M.; Hang, H. C. Nat. Chem. Biol. 2013, 9, 475.
20. (a) Zeng, D.; Zeglis, B. M.; Lewis, J. S.; Anderson, C. J. J. Nucl. Med. 2013, 54, 829;
(b) Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8, 1128.
21. Wang, L.; Peng, S.; Danence, L. J. T.; Gao, Y.; Wang, J. Chem. Eur. J. 2012, 18,
6088.
Supplementary data
Supplementary data (Experimental methods, 1H, 13C NMR and
HRMS spectral data of all new compounds 2, 4, 6a–o and 7a–o and
copies of 1H, 13C NMR and HRMS spectra for all the new compounds
2, 4, 6a–o and 7a–o.) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmcl.2014.01.077.
22. General procedure for amine-catalyzed Huisgen [3+2] cycloaddition 6(a–o): To a
solution of aryl azide 5 (1 equiv) and b-ketoester 4 (1.2 equiv) in DMSO (1 mL)
pyrrolidine was added (0.1 equiv) and stirred at 60 °C for 14 h. The reaction
mixture was diluted with ethyl acetate (20 mL), washed with water, dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The crude
residue thus obtained was purified over silica gel column eluted with
gradient mixture of ethyl acetate/hexane to give triazoles 6a–o as pale
yellow syrupy liquids. Ethyl 5-((4S,5S)-5-((S)-1-hydroxyethyl)-2,2-dimethyl-
1,3-dioxolan-4-yl)-1-phenyl-1H-1,2,3-triazole-4-carboxylate(6a). Yield: 81%;
References and notes
½ ꢃ
a 2D9 33.5 (c 1.0, CHCl3). 1H NMR (300 MHz, CDCl3) d 7.59–7.49 (m, 5H), 5.71 (d,
1. (a) Kanwal, A.; Banerjee, S. K. Pharm. Pat. Anal. 2013, 2, 77; (b) Hinnen, D. J.
Cardiovas. Nurs. 2013, 28, 157; (c) Mather, A.; Pollock, C. Nat. Rev. Nephrol. 2010,
6, 307; (d) Wright, E. M.; Loo, D. D. F.; Hirayama, B. A. Physiol. Rev. 2011, 91,
733.
2. (a) Deshmukh, A. B.; Patel, M. C.; Mishra, B. Ren. Fail. 2013, 35, 566; (b) Raskin,
P. Diabetes Metab. Res. Rev. 2013, 5, 347.
J = 7.9 Hz, 1H), 4.52 (q, J = 7.1 Hz, 2H), 3.93–3.88 (m,2H), 3.10 (br s, 1H), 1.48 (t,
J = 7.1 Hz, 3H), 1.32 (s, 3H), 1.15 (d, J = 6.0 Hz, 3H), 0.87 (s, 3H). IR (neat) 3447,
2983, 2933, 1725, 1377, 1219, 1066, 767 cmꢄ1. MS (ESI) m/z 362[M+H]+; HR-
MS (ESI) calcd for C18H24N3O5 [M+H]+: 362.0817, found: 362.0803. A typical
procedure for the preparation of 7a–o: To
a solution of 6 (0.5 mmol) in
dichloromethane (1 mL) trifluroacetic acid: water (1:1, 4 mL) was added at
0 °C. After 2 h of stirring at rt, the reaction mixture was neutralized with
saturated NaHCO3 and extracted with ethyl acetate (2 ꢁ 15 mL). The combined
organic extracts were washed with brine, dried over anhydrous Na2SO4,
concentrated under reduced pressure. Crude residue thus obtained was
purified over silica gel column eluted with ethyl acetate/hexane (1:1)
afforded 7a–o. (4S,5R,6S,7R)-7-Methyl-3-phenyl-4,5,6,7-tetrahydro-[1,2,3]
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triazole [1,5-a]pyridine-4,5,6-triol (7a). Yield: 90%; ½a D25
ꢃ
73.7 (c 1.0, CHCl3).
Mp: 94 °C; 1H NMR (300 MHz, CDCl3) d 7.62–7.44 (m, 5H), 5.88 (d, J = 10.5 Hz,
1H), 5.05 (dd, J = 3.7, 6.0 Hz, 1H), 4.51 (q, J = 7.5 Hz, 2H), 3.68 (br s, 1H), 3.36 (br
d, J = 3.7 Hz, 1H), 2.22 (br s, 1H), 1.77 (br s, 1H), 1.48 (t, J = 7.5 Hz, 3H), 1.04 (d,
J = 6.0 Hz, 3H). 13C NMR (75 MHz, CDCl3) d 164.1, 143.1, 135.1, 130.8, 129.7,
126.2, 76.5, 68.8, 66.4, 62.7, 19.3, 14.1. IR (neat) 3399, 2969, 2926, 1731, 1500,
1384, 1217, 1081, 1057, 1019, 763, 693 cmꢄ1. MS (ESI) m/z 322[M+H]+; HR-MS
(ESI) calcd for C15H20N3O5 [M+H]+: 322.13975, found: 322.13893.
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24. Cell culture: Human embryonic kidney (HEK293) cells were purchased from
ATCC, USA and made two stable cell lines after expressing SGLT1 & SGLT2,
respectively. Previously, we searched the selectivity of these cell lines for
SGLT1 and SGLT2 inhibition study. Glucose uptake by these cell lines was only
inhibited by SGLT inhibitors but not by any other GLUTs inhibitors. SGLT1 &
SGLT2 transfected HEK cell lines were propagated at 37 °C in 5% CO2 in
Dulbecco’s minimal essential medium (DMEM) supplemented with 1.0% of
penicillin-streptomycin and 10% heat inactivated fetal bovine serum (FBS). The
cells were cultured in a 90 mm dish in DMEM with 10% FBS until 70–80%
confluency was obtained for further use for SGLT1 and SGLT2 inhibition
activity. SGLT1 inhibition assay: SGLT1 transfected stable HEK cells were plated
at 1 ꢁ 104/well in 96-well plate and used at sub confluence after 24 h pre-
incubation. For measuring SGLT1-mediated glucose uptake, all culture media
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was removed from each well and replaced with 100 ll of culture medium with
newly synthesized molecules at different concentrations (50, 100, 250, 500 and
1000 nm). After half an hour, fluorescent 2-deoxy-glucose (2-NBDG) was
added to the plates and incubated at 37 °C with 5% CO2 for a period of 30 min.
23b Cells were lysed with 50
ll of 0.1 N NaOH and fluorescence of aliquots from
the lysate was measured at excitation/emission maxima of ꢂ465/540 nm.
Phlorizin (non-Specific SGLT1 inhibitor) was used as a standard for this study.
SGLT2 inhibition assay: SGLT2 transfected stable HEK cells were plated at
1 ꢁ 104/well in 96-well plate and used at sub confluence after 24 h pre-
incubation. For SGLT2-mediated glucose uptake, all culture medium was
removed from each well and replaced with 100
ll of culture medium with
newly synthesized molecules at different concentrations (50, 100, 250, 500 and
1000 nm). After half an hour, florescent glucose (2-NBDG) was added to the
plates and incubated at 37 °C with 5% CO2 for a period of 30 min. Cells were
15. Kantevari, S.; Putapatri, S. R. Synlett 2010, 2251.
then lysed with 50 ll of 0.1 N NaOH and fluorescence of aliquots from the
16. (a) Ghoneim, A. A. Chem. Cent. J. 2011, 5, 1; (b) Zhang, L.; Wang, Y.; Xu, H.; Shi,
Y.; Liu, B.; Wei, Q.; Xu, W.; Tang, L.; Wang, J.; Zhao, G. Med. Chem. 2013. http://
dx.doi.org/10.2174/15734064113096660051.
lysate was measured at excitation/emission maxima of ꢂ465/540 nm.
Phlorizin (non-specific SGLT2 inhibitor) was used as a standard for the study.