Synthesis and SAR of benzisothiazole- and indolizine-β-D- glucopyranoside inhibitors of SGLT2

Article abstract of DOI:10.1021/ml900010b

A series of benzisothiazole- and indolizine-β-d-glucopyranoside inhibitors of human SGLT2 are described. The synthesis of the C-linked heterocyclic glucosides took advantage of a palladium-catalyzed cross-coupling reaction between a glucal boronate and the corresponding bromo heterocycle. The compounds have been evaluated for their human SGLT2 inhibition potential using cell-based functional transporter assays, and their structure-activity relationships have been described. Benzisothiazole-C-glucoside 16d was found to be an inhibitor of SGLT2 with an IC₅₀ of 10 nM.

Full text of DOI:10.1021/ml900010b

pubs.acs.org/acsmedchemlett
Synthesis and SAR of Benzisothiazole- and
Indolizine-β-D-glucopyranoside Inhibitors of SGLT2
Huiqiang Zhou, Dana P. Danger, Steven T. Dock, Lora Hawley, Shane G. Roller, Chari D. Smith,
and Anthony L. Handlon*
Metabolic Pathways Center of Excellence for Drug Discovery, GlaxoSmithKline, P.O. Box 13398, Research Triangle Park,
North Carolina 27709-3398
ABSTRACT A series of benzisothiazole- and indolizine-β-D-glucopyranoside in-
hibitors of human SGLT2 are described. The synthesis of the C-linked heterocyclic
glucosides took advantage of a palladium-catalyzed cross-coupling reaction be-
tween a glucal boronate and the corresponding bromo heterocycle. The com-
pounds have been evaluated for their human SGLT2 inhibition potential using cell-
based functional transporter assays, and their structure-activity relationships
have been described. Benzisothiazole-C-glucoside 16d was found to be an inhibitor
of SGLT2 with an IC50 of 10 nM.
KEYWORDS Benzisothiazole- and indolizine-β-D-glucopyranoside inhibitors, SAR,
SGLT2, benzisothiazole-C-glucoside
nhibitors of sodium glucose cotransporter 2 (SGLT2) have
received considerable attention lately for the treatment of
type 2 diabetes.1,2 SGLT2 is a 14 trans-membrane protein
the heart,8 we desired selectivity to avoid the potential
disruption of glucose availability in cardiac muscle cells.
All known inhibitors of SGLT2 are carbohydrate-derived
glycosides that trace their origins to the natural product
phlorizin (1, Figure 1), which is a nonselective inhibitor of
SGLT1 and SGLT2.9 Phlorizin is an O-glucoside and, as such,
is subject to hydrolysis by O-glucosidases. As a result of the
lability of glycosyl bonds, O-glucosides tend to have poor oral
bioavailability unless the glucose moiety is masked as a
prodrug, as is found in the 60-O-ethyl carbonate derivatives,
serglifozin etabonate (2) and remogliflozin etabonate (3). In
diabetic rodent models, chronic treatment with 2 and 3
lowered fasting plasma glucose concentrations and im-
proved glucose clearance following oral glucose treat-
ment.10,11 N-Glucosides and C-glucosides are more
metabolically stable and tend to have higher oral bioavail-
ability and plasma exposure without needing to be con-
verted to a prodrug.2,12
Scientists at Bristol-Myers Squibb have recently reported
the discovery of a potent inhibitor of SGLT2, dapaglifozin (4),
a C-glucoside with a long half-life and sustained duration of
action.13 Johnson & Johnson in collaboration with Mitsubishi
Tanabe have advanced canagliflozin (5), another C-aryl
glucoside, to phase III clinical development. Most recently,
scientists at Lexicon Pharmaceuticals have reported the
discovery of metabolically stable O-xyloside inhibitors of
SGLT2, their most potent analogue being 6, which possesses
the same aglycone moiety as dapagliflozin.14 Beyond the
I
located in the proximal tubule of the kidney and is respon-
sible for the reuptake of glucose from the glomerular filtrate
to the plasma. Inhibitors of SGLT2 prevent the reabsorption
of glucose leading to the rapid elimination of glucose in the
urine. In the clinic, such agents have proven to reduce blood
glucose levels and cause modest weight loss in diabetic
patients through a mechanism that does not depend on
insulin release from β cells.3,4 It has also been demonstrated
that obese animals treated with SGLT2 inhibitors undergo
weight loss in proportion to the amount of glucose that is
excreted.5 At the present time, several companies are con-
ducting clinical evaluations of SGLT2 inhibitors for the treat-
ment of metabolic disorders including diabetes and obesity.
Another closely related member of the sodium glucose
cotransporter family, SGLT1, is located on enterocytes and
transports dietary carbohydrates from the lumen of the
small intestine to the systemic circulation. Inhibitors of
SGLT1 delay glucose absorption and may also be useful in
treating diabetes. Evidence from animal studies suggests
that the profound improvements in glucose metabolism that
result from Roux-en-Y gastric bypass surgery are directly
related to altering SGLT1 expression and slowing glucose
influx.6 We were interested in identifying compounds that
would be selective for SGLT2 and that would be rapidly
absorbed from the gastrointestinal (GI) tract with high oral
bioavailability to act in the kidney. SGLT2 selectivity over
SGLT1 may be important to avoid GI side effects that could
result from carbohydrate malabsorption in the small intes-
tine. In addition, because SGLT1 is highly expressed in
cardiac myocytes7 and serves as a glucose transporter in
Received Date: December 5, 2009
Accepted Date: January 24, 2010
Published on Web Date: January 29, 2010
r
2010 American Chemical Society
19
DOI: 10.1021/ml900010b ACS Med. Chem. Lett. 2010, 1, 19–23
|

pubs.acs.org/acsmedchemlett
Figure 1
monocyclic O- and C-glucosides, Zhang and co-workers at
Johnson & Johnson have investigated O- and C-glucosides in
which the aglycone is indole (7 and 8). They found that the
C-glucosides had reduced potency as compared to the
O-glucosides and proposed that the anomeric oxygen is im-
portant for full inhibition.15 In our program to discover
novel and selective SGLT2 inhibitors, we set out to explore
C-glucosides in which the aglycone is a benzo-fused hetero-
cycle. Herein, we report our investigations into the synthesis
and structure-activity relationships (SARs) of heterobicyclic
β-D-glucopyranoside inhibitors of SGLT2.
Scheme 1a
In our initial attempts to construct heterocyclic C-aryl
glucosides, we took advantage of the attractive strategy
outlined by Meng et al.,13 namely, lithium-halogen ex-
change of a bromoaromatic ring and addition of the resulting
aryllithium to a gluconolactone followed by reduction of the
resulting gluconolactol to give the aromatic C-aryl glucoside.
Although this approach worked well when the aromatic
system was substituted phenyl or benzothiophene, when
the aromatic group was an aza heterocycle such as benzi-
sothiazole 12a or 12p, we failed to achieve addition of the
lithio species to the gluconolactone. Instead, we used an
alternative approach involving the Pd-catalyzed cross-cou-
pling of the bromoaromatic with a glucal boronate, a new
glycosylation method that has been described in a patent
application by scientists at Tanabe.16,17
The synthesis of the benzisothiazole intermediates 12a-j
is outlined in Scheme 1. Benzisothiazole 9 was readily
prepared in three steps from thiophenol.18 Bromination of
9 under acidic conditions followed by condensation with
N-methyl-O-methylhydroxylamine hydrochloride gave Wein-
reb amide 10, which was treated with various arylmagne-
sium bromides to give compounds 11a-j. The biaryl ketones
a Reagents and conditions: (a) Br2, H2SO4, HNO3, AcOH, 70 °C. (b) N-
€
Methyl-O-methylhydroxylamine hydrochloride, HATU, Hunig's base,
DMF. (c) Grignard reagent, THF, 0 °C to room temperature. (d) Et3SiH,
TFA.
were reduced with triethylsilane in TFA to provide com-
pounds 12a-j.
Heterocyclic C-aryl glucosides 16a-p were prepared from
bromoheterocycles 12a-p as outlined in Scheme 2. Palla-
dium-catalyzed cross-coupling of bromides 12a-p with
pinacol boronate ester 1316,17 provided compounds
14a-p. Hydroboration with borane-THF followed by oxida-
tion with hydrogen peroxide afforded β-glucosides 15a-p as
confirmed by the J coupling constant (∼9.5 Hz) between H10
1
and H20 in the H NMR spectrum. None of the R anomers
were isolated. Finally, deprotection of 15a-p with HF/TEAor
with TBAF gave target compounds 16a-p.
As shown in Scheme 3, the indolizine intermediates 19k-o
were prepared in two steps from 3-bromopicoline 17.Compound
r
2010 American Chemical Society
20
DOI: 10.1021/ml900010b ACS Med. Chem. Lett. 2010, 1, 19–23
|

pubs.acs.org/acsmedchemlett
Scheme 2a
a Reagents and conditions: (a) 13, Pd(PPh3)2Cl2, DME, Na2CO3, 85 °C. (b) BH3, THF, 0 °C to room temperature and then H2O2, NaOH, THF, 0 °C to
room temperature. (c) HF TEA, THF, 60 °C or TBAF, THF, room temperature.
3
Scheme 3a
Scheme 4a
a Reagents and conditions: (a) 4-Methoxyphenyl-magnesium bromide,
THF. (b) Sodium borohydride, TFA.
a Reagents and conditions: (a) MeCN, room temperature. (b) Me2SO4,
DMF, 60-80 °C. (c) NaBH4, AlCl3.
17 was treated with commercially available bromoacetophe-
nones to afford picolinium salts 18k-o, which were treated with
dimethylsulfate-DMF to provide ketones 19k-o.19,20 The ke-
tones 19k-o were reduced to diarylmethanes 12k-o using
sodium borohydride with aluminum chloride.
The imidazo[1,2-a]pyridine 12p was prepared in two steps
from commercially available aldehyde 20 (Scheme 4). Treat-
ment of 20 with 4-methoxyphenylmagnesium bromide
followed by reduction of the resulting alcohol 21 with TFA/
NaBH4 gave 12p.
The methodology that we have used to construct C-aryl
glucosides, namely, the Pd-catalyzed cross-coupling of glucal
boronates, will be a useful addition to the array of techniques
that are available to carbohydrate chemists. The method
allowed the construction of benzo-fused C-glucosides, which
were not accessible using the conventional method.
The SGLT1 and SGLT2 inhibitory activity (IC50) of the
heterocyclic C-glucosides was determined by monitoring
the inhibition of uptake of 14C-labeled R-methyl-D-glucopyr-
anoside by COS-7 cells, which transiently expressed human
SGLT2 or SGLT1 using BacMam technology.21 The results are
summarized in Table 1. Focusing initially on the benzisothia-
zole series, we found that the 4-methyl benzyl- (16a) and the
4-ethyl benzyl- (16b) substituted derivatives were potent
inhibitors with pIC50 values of 80 and 50 nM, respectively.
Increasing the size of the 4-substituent appeared to increase
the inhibition potential. Following this trend, we synthesized
the 4-isopropyl (16c) and the 4-tert-butyl (16d) analogues
and found that 16d achieved inhibition with an IC50 of
10 nM. Exploring the size limits of the 4-substituent, we
found that the 4-phenyl benzyl analogue (16e) lost potency.
Likewise, the 4-chlorobenzyl analogue (16f) displayed less
r
2010 American Chemical Society
21
DOI: 10.1021/ml900010b ACS Med. Chem. Lett. 2010, 1, 19–23
|

pubs.acs.org/acsmedchemlett
Table 1. SGLT2 and SGLT1 Inhibition of Compounds 16a-p and 4a
Table 2. Pharmacokinetic Profiles of Compounds 16b,c in Male
CD Rats Following Intravenous and Oral Dosing
dose
16b
16c
IV 2 mg/kg
1.2
424
4.3
79.1
PO 10 mg/kg
422
0.5
t
1/2 (h)
AUCinf (h ng/mL)
ss (L/kg)
CL (mL/min/kg)
1.7
393
5.3
85.5
V
pIC50
C
max (ng/mL)
321
0.5
1483
74
X
Y
Z
R1
Me
R2
SGLT2
SGLT1
t
max (h)
16a
16b
16c
16d
16e
16f
C
C
C
C
C
C
C
C
C
C
N
N
N
N
N
N
S
S
S
S
S
S
S
S
S
S
C
C
C
C
C
N
N
N
N
N
N
N
N
N
N
N
C
C
C
C
C
C
H
7.09 ( 0.21
7.30 ( 0.07
7.29 ( 0.23
8.00 ( 0.18
6.41 ( 0.15
6.85 ( 0.04
5.27 ( 0.01
6.21 ( 0.04
6.83 ( 0.08
6.43 ( 0.18
7.21 ( 0.13
6.87 ( 0.02
6.03 ( 0.21
6.81 ( 0.20
6.95 ( 0.06
<5
<5
<5
<5
<5
ND
ND
ND
ND
<5
ND
<5
ND
ND
ND
<5
ND
<5
AUCinf (h ng/mL)
F (%)
1642
77
Et
H
i-Pr
t-Bu
Ph
H
H
were rapidly absorbed after oral dosing. Although the plas-
ma clearance is high for these molecules, the bioavailability
(% F, ratio of plasma drug concentrations after oral dosing
versus IV dosing) is high, indicating that the compounds
were stable enough in the GI tract to be absorbed well.
C-Glucosides 16b and 16c did not require conversion to
prodrug to achieve oral absorption. Although the prodrug
approach has proven viable for SGLT2 inhibitors such as 2
and 3, which have clear antidiabetic effects in rodent models
of diabetes,10,11 it would be preferable to avoid the extra
chemical step of appending the prodrug moiety. A prodrug
approach also introduces the additional development risk
that the conversion of prodrug to parent may not translate
from animals to humans. Furthermore, there can be large
intrapatient variability in the pharmacokinetics of prodrug to
parent conversion.
H
Cl
H
16 g
16 h
16i
H
Me
Me
H
Me
OMe
OEt
Me
OMe
F
16j
H
16k
16l
H
H
16m
16n
16o
16p
4
H
Cl
H
CF3
OMe
H
H
9.84 ( 0.14
a Data represent -log (IC50) ( standard error.
In summary, we have taken advantage of an underutilized
Pd-catalyzed cross-coupling reaction to synthesize a series of
benzisothiazole and indolizine C-glucosides, which have
extended our understanding of SGLT2 inhibitor SARs. We
have established that potent SGLT2 inhibitors can be found in
C-glucosides in which the aglycone is a benzo-fused hetero-
cycle. We have also seen that the weakly basic imidazo
pyridines, although possessing the same overall shape as
the benzisothiazoles and indolizines, have no SGLT2 inhibi-
tion at all. Further work concerning the optimization of
SGLT2 inhibitors suitable for clinical development will be
reported in subsequent publications.
inhibition potential than the 4-alkyl analogues. We observed
that the 4-methoxy and 4-ethoxy derivatives (16i and 16j)
were less potent than the corresponding alkyl analogues.
The 3-methylbenzyl and the 3,4-dimethylbenzyl analogues
16g and 16h were significantly less potent than 16a, indicat-
ing that a methyl group in the meta position was not
tolerated.
Similar SAR trends were observed in the indolizine series.
The 4-methylbenzyl (16k) and the 4-trifluoromethylbenzyl
(16o) analogues had high SGLT2 inhibition (IC50 = 60 and
110 nM, respectively). The 4-methoxybenzyl (16l) and the
4-halobenzyl analogues (16m and 16n) were less potent than
the 4-alkyl derivatives. Probing alternative aza heterocycle
isosteres, we found that imidazo pyridine 16p, although
having a similar overall shape and size as the benzisothiazole
16i (IC50 = 155 nM) and indazole 16l (IC50 = 135 nM), was
devoid of SGLT2 inhibition activity. The most potent SGLT2
inhibitors were tested for SGLT1 inhibition and were found to
be inactive against SGLT1 in all cases. Compound 4 was
tested as a comparator and was found to be a highly selective
inhibitor of SGLT2 and more potent than the benzisothia-
zoles.
SUPPORTING INFORMATION AVAILABLE Description of
the inhibition assay, pharmacokinetic study methods, and the
synthesis of compounds 10-21. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *To whom correspondence should be
addressed. Tel: 919-483-9769. Fax: 919-315-6923. E-mail: tony.l.
handlon@gsk.com.
The pharmacokinetic profiles of two of the benzisothia-
zole analogues, 16b and 16c, were determined in male CD
rats. The results are summarized in Table 2. Both compounds
ACKNOWLEDGMENT We thank William Clay for generating
the SGLT2 and SGLT1 BacMam viruses and Sandy Kerner for the
BacMam constructs.
r
2010 American Chemical Society
22
DOI: 10.1021/ml900010b ACS Med. Chem. Lett. 2010, 1, 19–23
|

pubs.acs.org/acsmedchemlett
REFERENCES
(15) Zhang, X.; Urbanski, M.; Patel, M.; Cox, G. G.; Zeck, R. E.;
Bian, H.; Conway, B. R.; Beavers, M. P.; Rybczynski, P. J.;
Demarest, K. T. Indole-glucosides as novel sodium glucose
co-transporter 2 (SGLT2) inhibitors. Part 2. Bioorg. Med. Chem.
Lett. 2006, 16, 1696–1701.
(16) Nomura, S.; Kawanishi, E.; Ueta, K. U.S. Patent 5,233,988.
Chem. Abstr. 2005, 143, 387313.
(17) Nomura, S.; Kawanishi, E.; Ueta, K. WO 2005/012326. Chem.
Abstr. 2005, 142, 219491.
(18) Xie, W.; Herbert, B.; Ma, J.; Nguyen, T. M.; Schumacher, R.
WO2005/063767. Chem. Abstr. 2005, 143, 133366.
(19) Li, H.; Xia, Z.; Chen, S.; Koya, K.; Ono, M.; Sun, L. Develop-
ment of a Practical Synthesis of STA-5312, a Novel Indolizine
Oxalylamide Microtubule Inhibitor. Org. Process Res. Dev.
2007, 11, 246–250.
(20) Przewloka, T.; Chen, S.; Xia, Z.; Li, H.; Zhang, S.; Chimma-
namada, D.; Kostik, E.; James, D.; Koya, K.; Sun, L. Applica-
tion of DMF-methyl sulfate adduct in the regioselective
synthesis of 3-acylated indolizines. Tetrahedron Lett. 2007,
48, 5739–5742.
(1)
(2)
(3)
(4)
Bays, H. From victim to ally: The kidneyas an emerging target
for the treatment of diabetes mellitus. Curr. Med. Res. Opin.
2009, 25, 671–681.
Washburn, W. Evolution of sodium glucose co-transporter 2
inhibitors as anti-diabetic agents. Expert Opin. Ther. Pat.
2009, 19, 1485–1499.
List, J. F.; Woo, V.; Morales, E.; Tang, W.; Fiedorek, F. T.
Sodium-glucose cotransport inhibition with dapagliflozin in
type 2 diabetes. Diabetes Care 2009, 32, 650–657.
Wilding, J. P. H.; Norwood, P.; T'joen, C.; Bastien, A.; List, J. F.;
Fiedorek, F. T. A study of dapagliflozin in patients with type 2
diabetes receiving high doses of insulin plus insulin sensiti-
zers: Applicability of a novel insulin-independent treatment.
Diabetes Care 2009, 32, 1656–1662.
(5)
Devenny, J.; Harvey, S.; Rooney, S.; Godonis, H.; Washburn,
W.; Whaley, J.; Taylor, S.; Pelleymounter, M. A. The Effect of
Dapagliflozin, a Highly Selective SGLT2 Inhibitor, on Body
Weight in Diet-induced Obese Rats. Annual Scientific Meeting
of the North American Association for the Study of Obesity
(NAASO), New Orleans, LA, October 20-24, 2007.
Stearns, A. T.; Balakrishnan, A.; Tavakkolizadeh, A. Impact of
Roux-en-Y gastric bypass surgery on rat intestinal glucose
transport. Am. J. Physiol. Gastrointest. Liver Physiol. 2009,
297, G950–G957.
(21) Condreay, J. P.; Witherspoon, S. M.; Clay, W. C.; Kost, T. A.
Transient and stable gene expression in mammalian cells
transduced with a recombinant baculovirus vector. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 127–132.
(6)
(7)
Zhou, L.; Cryan, E. V.; D'Andrea, M. R.; Belkowski, S.; Con-
way, B. R.; Demarest, K. T. Human cardiomyocytes express
high level of Naþ/glucose cotransporter 1 (SGLT1). J. Cell.
Biochem. 2003, 90, 339–346.
(8)
(9)
Banerjee, S. K.; McGaffin, K. R.; Pastor-Soler, N. M.; Ahmad, F.
SGLT1 is a novel glucose transporter that is perturbed in
disease states. Cardiovasc. Res. 2009, 84, 111 –118.
Ehrenkranz, J. R. L.; Lewis, N. G.; Kahn, C. R.; Roth, J. Phlorizin:
A review. Diabetes/Metab. Res. Rev. 2005, 21, 31–38.
(10) Fujimori, Y.; Katsuno, K.; Nakashima, I.; Ishikawa-Takemura,
Y.; Fujikura, H.; Isaji, M. Remogliflozin etabonate, in a novel
category of selective low-affinity sodium glucose cotranspor-
ter (SGLT2) inhibitors, exhibits antidiabetic efficacy in rodent
models. J. Pharmacol. Exp. Ther. 2008, 327, 268–276.
(11) Fujimori, Y.; Katsuno, K.; Ojima, K.; Nakashima, I.; Nakano,
S.; Ishikawa-Takemura, Y.; Kusama, H.; Isaji, M. Sergliflozin
etabonate, a selective SGLT2 inhibitor, improves glycemic
control in streptozotocin-induced diabetic rats and Zucker
fatty rats. Eur. J. Pharmacol. 2009, 609, 148–154.
(12) Isaji, M. Sodium-glucose cotransporter inhibitors for dia-
betes. Curr. Opin. Invest. Drugs (Thomson Sci.) 2007, 8,
285–292.
(13) Meng, W.; Ellsworth, B. A.; Nirschl, A. A.; McCann, P. J.; Patel,
M.; Girotra, R. N.; Wu, G.; Sher, P. M.; Morrison, E. P.; Biller, S.
A.; Zahler, R.; Deshpande, P. P.; Pullockaran, A.; Hagan, D. L.;
Morgan, N.; Taylor, J. R.; Obermeier, M. T.; Humphreys, W. G.;
Khanna, A.; Discenza, L.; Robertson, J. G.; Wang, A.; Han, S.;
Wetterau, J. R.; Janovitz, E. B.; Flint, O. P.; Whaley, J. M.;
Washburn, W. N. Discovery of Dapagliflozin: A Potent, Selec-
tive Renal Sodium-Dependent Glucose Cotransporter 2
(SGLT2) Inhibitor for the Treatment of Type 2 Diabetes. J.
Med. Chem. 2008, 51, 1145–1149.
(14) Goodwin, N. C.; Mabon, R.; Harrison, B. A.; Shadoan, M. K.;
Almstead, Z. Y.; Xie, Y.; Healy, J.; Buhring, L. M.; DaCosta, C.
M.; Bardenhagen, J.; Mseeh, F.; Liu, Q.; Nouraldeen, A.;
Wilson, A. G. E.; Kimball, S. D.; Powell, D. R.; Rawlins, D. B.
Novel L-Xylose Derivatives as Selective Sodium-Dependent
Glucose Cotransporter 2 (SGLT2) Inhibitors for the Treatment
of Type 2 Diabetes. J. Med. Chem. 2009, 52, 6201–6204.
r
2010 American Chemical Society
23
DOI: 10.1021/ml900010b ACS Med. Chem. Lett. 2010, 1, 19–23
|