Exploration of O-spiroketal C-arylglucosides as novel and selective renal sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2009.10.088

A series of novel O-spiroketal C-arylglucosides have been prepared and evaluated in cell-based functional assays for activity against human sodium-dependent glucose co-transporters 1 and 2 (SGLT1 and 2). The core spiro[isobenzofuran-1,2′-pyran] structure proved to be an effective scaffold for diversification and a number of compounds with single digit nanomolar potency and high selectivity have been synthesized. Compound 5a promoted glucosuria when administered in vivo in rats and produced a significant blood glucose reduction effect.

Full text of DOI:10.1016/j.bmcl.2009.10.088

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6877–6881
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Exploration of O-spiroketal C-arylglucosides as novel and selective renal
sodium-dependent glucose co-transporter 2 (SGLT2) inhibitors
Binhua Lv a,b,c, , Baihua Xu a,b,c, Yan Feng c, Kun Peng c, Ge Xu c, Jiyan Du c, Lili Zhang c, Wenbin Zhang c,
*
Ting Zhang c, Liangcheng Zhu c, Haifeng Ding c, Zelin Sheng c, Ajith Welihinda d,
Brian Seed d, Yuanwei Chen a,b,c,
*
a Chengdu Institute of Organic Chemistry, Chinese Academy of Sciences, Chengdu 610041, PR China
b Graduate School of Chinese Academy of Science, Beijing 100049, PR China
c Egret Pharma (Shanghai) Company, Ltd, 4F, 1118 Halei Road, Zhangjiang Hi-Tech Park, Pudong New Area, Shanghai 201203, PR China
d Theracos Inc., 550 Del Rey Avenue, Sunnyvale, CA 94805-3528, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel O-spiroketal C-arylglucosides have been prepared and evaluated in cell-based functional
assays for activity against human sodium-dependent glucose co-transporters 1 and 2 (SGLT1 and 2). The
core spiro[isobenzofuran-1,20-pyran] structure proved to be an effective scaffold for diversification and a
number of compounds with single digit nanomolar potency and high selectivity have been synthesized.
Compound 5a promoted glucosuria when administered in vivo in rats and produced a significant blood
glucose reduction effect.
Received 3 August 2009
Revised 18 October 2009
Accepted 20 October 2009
Available online 23 October 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
O-Spiroketal C-arylglucosides
SGLT2
Spiro[isobenzofuran-1, 20-pyran]
Glucosuria
Blood glucose
Type II diabetes mellitus (DM2) is an acquired metabolic dis-
ease in which the glycemic load resulting from excess nutrient
consumption and dysregulated gluconeogenesis overwhelms the
ability of pancreatic islet beta cells to produce sufficient insulin
to maintain euglycemia. DM2 often appears in the setting of
comorbidities associated with hyperglycemia and a sedentary life-
style, including the constellation with obesity, dyslipidemia and
hypertension known as metabolic syndrome. The renal sodium
glucose linked transporter 2, SGLT2, has emerged as an attractive
target for the management of DM2 in the context of metabolic syn-
drome.1 As its name suggests, SGLT2 acts by cotransport of sodium
ion and glucose to recover glucose from urine, thereby conserving
a valuable metabolic resource under nutrient-limited conditions.2
Blockade of this activity should release glucose into urine, thereby
reducing circulating glucose levels. Individuals affected by the
genetic syndrome Familial Renal Glucosuria (FRG) have mutations
in SGLT2, sometimes resulting in a predicted complete loss of
transporter activity, but have no obvious morbidities.3 In extreme
cases of FRG a syndrome of ‘salt wasting’ is observed, with hypo-
tension and a slight elevation in aldosterone consistent with a
homeostatic mechanism that has been engaged to retain sodium.4
Selectivity for the inhibition of SGLT2 compared to the closely re-
lated SGLT1 is thought to be highly attractive because deficiency
in SGLT1 leads to the human genetic disease Glucose Galactose
Malabsorption syndrome, characterized by a severe diarrhea and
failure to thrive.5
Following the initial disclosure by Tanabe Seiyaku of SGLT2-
selective inhibitors patterned on the naturally occurring inhibitor
phlorizin,6,7 a large number of candidate inhibitors have been syn-
thesized.8 Members of of the O-glycoside class of SGLT2 inhibitor,
including T-1095A,6,7 sergliflozin-A (1),9 remogliflozin10 or their
pro-drugs have been evaluated for the treatment of DM2 but have
been found to require frequent dosing because of metabolic insta-
bility. Dapagliflozin (2), a C-aryl glucoside, has been identified as a
potent and selective SGLT2 inhibitor with a favorable pharmacody-
namic and pharmacokinetic profile in preclinical studies.11
The program described here was directed at the creation of
metabolically robust compounds with high selectivity towards
SGLT2. Modeling studies and analysis of the published X-ray struc-
ture of different classes of potential SGLT2 inhibitors,12 led to the
realization that the aryl ring (B ring) of the aglycone moiety was
almost perpendicular to carbohydrate moiety (A ring) in the lowest
energy conformation. A series of novel compounds (Fig. 1) that
have a different core structure than reported inhibitors was
* Corresponding authors. Tel.: +86 28 85232730; fax: +86 28 85259387 (Y.C.).
E-mail addresses: 51popo51@163.com, binhualv@egretpharma.com (B. Lv),
chenyw@ci.ac.cn (Y. Chen).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.088

6878
B. Lv et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6877–6881
R1
4
OMe
X
HO
HO
HO
C
B
2'
O
A
R2
2
O
O
O
OH
HO
HO
I
OH
Sergliflozin
OH
R1
4
6
X
O
O
A
Cl
O
B
C
HO
2'
R2
O
OH
HO
HO
II
HO
OH
OH
Dapagliflozin
OH
X = H, F, Cl
R1,R2 = alkyl, alkoxyl,
halogen, hydrogen
Figure 1. Origin and design of O-spiroketal C-arylglucosides SGLT2 inhibitors.
accordingly synthesized.13 The central modifications were the
formation of a O-spiro linkage between the glucose ring and the
aglycone proximal phenyl ring, wherein the spiro structure ex-
pected to combine both the character of C-glucosides and O-gluco-
sides, and the retention of a chlorine at the 4-position on the B ring,
a substitution that had been found to be critical for high in vivo po-
tency in preliminary studies. Retention of the 4-chloro substituent
and the attainment of higher potency distinguishes the program
discussed below from that disclosed by Kobayashi et al.14 albeit
at the price of a significant increase in synthetic difficulty attribut-
able to the electron deficiency of the resulting B ring.
be drawn as to which substituent would result in a favorable orien-
tation with respect to the target.
The synthesis of 3a–3p is shown in Scheme 1. Persilylated gluc-
onolactone 7 was prepared in 93% yield by a slow addition of tri-
methylsilyl chloride to commercially available gluconolactone 6
in N-methylmorpholine and tetrahydrofuran.16,17 Benzoates 11
were constructed through Sandmeyer reaction or Balz–Schiemann
reaction conditions from aniline 10,18 which was prepared by bro-
mination of commercially available 3-amino-4-methylbenzoic acid
8 followed by esterification of acid 9. Oxidation of 11 with potas-
sium permanganate in tert-butanol provided the key electron-defi-
cient tetrasubstituted benzene 12. Friedel–Crafts acylation of
substituted benzenes with the benzoyl chloride prepared from acid
12 by treatment with oxalyl chloride generated the benzophenone
13. Selective reduction of the ketone with triethylsilane and cata-
lytic trifluoromethanesulfonic acid in trifluoroacetic acid provided
As part of our ongoing SAR studies,15 O-spiroketal C-arylgluco-
sides with varied substituents on the aglycone aryl rings were sys-
tematically prepared to search for possible favorable ligand–
protein interactions. Through modulation of the electron density
and the substitution pattern of the distal ring, inferences could
O
O
O
O
HO
HO
TMSO
TMSO
a
OH
OTMS
OH
OTMS
6
7
MeO2C
Br
X
HO2C
NH2 HO2C
b
NH2
MeO2C
Br
X
MeO2C
NH2
e
d
c
CO2H
Br
Br
8
9
10
11 X=F or Cl
12 X=F or Cl
R1
R1
R1
R2
X
X
MeO2C
Br
X
HO
MOMO
Br
j
f, g
h,i
Br
R2
14 X=F or Cl
R2
O
15 X=F or Cl
13 X=F or Cl
R1
X
O
O
k,l
HO
R2
OH
3a-3p
HO
OH
Scheme 1. Construction of the aglycones and synthesis of 3a–3p. Reagents and conditions: (a) TMSCl, NMM, THF, 35 °C (93%); (b) NBS, DMF, 5 °C (87%); (c) SOCl2, MeOH,
reflux (99%); (d) NaNO2, concd HCl, CuCl, 1,4-dioxane, H2O, 0 °C (93%); or HBF4, isoamyl nitrite, anhydrous EtOH, ꢀ10 °C, then xylene, reflux (50%); (e) KMnO4, t-BuOH, 18-
crown-6, H2O, reflux (73%); (f) (COCl)2, DMF, DCM; (g) AlCl3, R1 and R2 substituted phenyl, DCM; (h) Et3SiH, CF3SO3H, TFA; (i) NaBH4, MeOH, THF; (j) MOMCl, DIPEA, DCM,
0 °C–rt; (k) n-BuLi, THF, toluene, ꢀ78 °C, lactone 7; (l) CH3SO3H, MeOH, ꢀ78 °C to rt (40–63%, two steps).

B. Lv et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6877–6881
6879
Scheme 2. Reagents and conditions: (a) 1,2-bis(trimethylsilyloxy)ethane, ethylene glycol, TsOHꢁH2O, toluene, reflux (34%); (b) NaBH4, MeOH, THF, 65 °C; (c) MOMCl, DIPEA,
DCM, 0 °C–rt (86%); (d) n-BuLi, THF, toluene, ꢀ78 °C, lactone 7; (e) CH3SO3H, MeOH, ꢀ78 °C to rt (68%, two steps); (f) Ac2O, pyridine, DMAP, DCM, rt; (g) NH2NH2ꢁH2O, TsOH,
EtOH, reflux; (h) DBH, HF/pyridine, DCM, ꢀ78 °C (36%, two steps).
the corresponding diarymethane which was further reduced with
sodium borohydride to give the benzyl alcohol 14. Protection of
the primary alcohol with chloromethyl methyl ether gave the agly-
cone 15. Lithium–halogen exchange of 15 followed by its addition
The synthesis of the cyclopropyl glucoside 29 is described in
Scheme 3. Standard Wittig reaction conditions applied to ketone
17 gave the alkene 25, which was subjected to modified
Simmons–Smith reaction conditions to give the cyclopropyl com-
pound 26.22 Reduction and protection of 26 provided the aglycone
28. Condensation and cyclization as described above gave analogue
29 in moderate yield.20
The compounds were tested using cell-based SGLT functional
assays.23 The role of substituent groups on the 2-, 3- and 4-posi-
tions of the distal aryl ring was examined via compounds that
encompassed alkoxyl, alkyl and halogen groups (Table 1). At the
4-position, methoxyl-substituted compound 3c showed an IC50 va-
lue of 2.5 nM for human SGLT2 (hSGLT2) and only 80-fold selectiv-
ity vs. human SGLT1 (hSGLT1), whereas a 2.5-fold improvement in
selectivity was found for ethoxyl-substituted compound 3b.
Replacing the ethyl substituent with isopropyl or tert-butyl gave
compounds 3d–f, with reduced binding affinity but increased affin-
ity for SGLT2 over SGLT1. In particular, the high selectivity of 3g
can be expected to favorably impact the predicted gastrointestinal
side effect profile.24 All mono-, para-substituted R1 analogues
3a–h, whether the 4-position on the proximal ring was chlorine
to 2,3,4,6-tetra-O-trimethylsilyl-b-D-gluconolactone 7 gave a mix-
ture of lactols which were converted in situ to the desired O-spi-
roketal C-arylglucosides 3a–p by treatment with methanesulfonic
acid in methanol.19,20
The suitability of substituents on the one atom bridge between
rings B and C was also evaluated. Scheme 2 details the route to the
difluoromethylene bridge, which was attained by conversion of ke-
tone 17 to the ketal 18, followed by reduction with sodium borohy-
dride to give alcohol 19 and subsequent protection with
chloromethyl methyl ether to yield the intermediate 1,3-dioxolane
20. In a one-pot reaction, compound 21 was prepared from 20
using a synthetic approach similar to that outlined for the forma-
tion of compound 3a–p in Scheme 1. An initial attempt to synthe-
size the target diaryldifluoromethane 24 by direct fluorination of
tetra acetyl glucoside 22 with DAST failed, but preparation of the
hydrazone 23 followed by fluorination with HF/pyridine complex
with catalytic DBH afforded the desired compound.20,21
Scheme 3. Reagents and conditions: (a) (C6H5)3PCH3I, t-BuOK, toluene, rt (60%); (b) Et2Zn, TFA, CH2I2, DCM, ꢀ15 °C (58%); (c) NaBH4, MeOH, THF, 65 °C (98%); (d) MOMCl,
DIPEA, DCM, 0 °C–rt; (e) n-BuLi, THF, toluene, ꢀ78 °C, lactone 7; (f) CH3SO3H, MeOH, ꢀ78 °C to rt (34%, two steps).

6880
B. Lv et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6877–6881
Table 1
O-Spiroketal C-arylglucosides (3a–p): exploration of the aglycone proximal and distal ring substituents (X, R1 and R2)23
R1
4
X
HO
HO
O
O
O
3
R2
HO
1
2
OH
OH
O
OH
HO
HO
3a-3p
415
Compound
X
R1
R2
hSGLT2 IC50 (nM)
hSGLT1 IC50
(lM)
Selectivity (hSGLT2/hSGLT1)
2
NA
H
NA
NA
H
H
H
H
H
H
H
H
3-Cl
2-Cl
2-Me
2-Me
2-Me
2-OEt
2-F
2-OMe
NA
6.7
71
6.5
2.5
6.6
7.1
13
0.3
0.89
10–100
1.3
0.2
0.6
2.5
9.7
3.1
5.3
10.5
10–100
1–10
10–100
1–10
10–100
1.5
132
>140
200
80
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
4
4-OEt
4-OEt
4-OMe
4-Et
4-iPr
4-tBu
4-OCF3
4-Et
4-OEt
4-OEt
4-OEt
4-OMe
4-F
Cl
Cl
Cl
Cl
Cl
Cl
F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
NA
91
352
742
10,333
616
154
ND
ND
ND
ND
ND
8.6
68
>1000
>1000
>1000
ꢂ100
1000
0.8
4-F
3-Me
3-Me
NA
1875
ND
ND
ꢂ10
1–10
ND
>>1000
or fluorine, had equivalent or increased potency and selectivity for
SGLT2 inhibition relative to dapagliflozin 2, possibly because of the
greater conformational constraints imposed by the spiro scaffold.
Di-substitution at the 2- and 4-positions of the distal aryl ring,
(Table 1, analogue 3j–n) resulted in products having significantly
lower SGLT2 inhibitory activities, perhaps due to the increased ste-
ric bulk at the 2-position. Analogs 3o–p exhibited a binding affinity
to SGLT2 that was unexpected from the SAR profile of reported C-
glucosides or O-glucosides in which the meta position of the distal
aryl ring was substituted with a methyl group.25 Compound 3o, in
which the meta position is methyl and ortho position is fluorine,
had ꢂ8-fold higher potency and more than 14-fold higher selectiv-
ity for inhibition of SGLT2 compared with dapagliflozin 2.
compared to 3d, perhaps due to a more favorable orientation of the
distal ring induced by the conformational effects of the cyclopropyl
group.
The SAR of replacements for the glucose primary hydroxyl
group was investigated as outlined in Scheme 4, leading to 5a–f
(Table 2). Direct and selective fluorination of 3d with DAST in
dichloromethane provided compound 5a. Compounds 5b–e could
be obtained by SN2 substitution of tosylate 16 which was formed
from 3d by reaction with p-toluenesulfonyl chloride and 2,6-luti-
dine. Reduction of azide 5e with triphenylphosphine generated
the amine 5f in moderate yield.20
Replacement of the 60 hydroxyl group with fluorine gave a sim-
ilar in vitro profile to compound 3d. The primary amine compound
5f had greatly reduced SGLT2 inhibitory activity. Replacement of
the hydroxyl by a methoxyl group, compound 5b appeared to yield
comparable SGLT2 potency and more selectivity, but the more
bulky trifluoroethoxyl group of compound 5c reduced potency at
Modification of the spacer moiety between the proximal and
distal rings of 3d afforded the high potency compounds 21, 24
and 29 (Table 2). Interestingly, compound 29 exhibited a fivefold
higher potency and a 50-fold more selectivity for SGLT2 inhibition
Table 2
O-Spiroketal C-arylglucosides (III) SAR exploration of the aglycone spacing element (L) and 60-position substituent (R)23
Cl
O
O
R
L
6'
OH
OH
HO
III
Compound
R
L
hSGLT2 IC50 (nM)
hSGLT1 IC50
(l
M)
Selectivity (hSGLT2/hSGLT1)
5a
5b
5c
5d
5e
5f
F
CH2
CH2
CH2
CH2
CH2
CH2
CO
3.8
5.8
70
43
10–100
100–1000
22
7
0.7
2.7
14
3.5
1–10
>100
43
184
466
200
82
ND
ND
CH3O
CF3CH2O
CH3COO
N3
NH2
OH
21
24
1955
229
OH
CF2
1.6
29
OH
1.3
6.0
4615

B. Lv et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6877–6881
6881
Cl
Cl
Cl
O
O
O
R
O
TsO
O
R
O
b
c
OH
OH
OH
OH
HO
HO
OH
16
HO
OH
5b-d
5e R = N3
5f R = NH2
3d R = OH
5a R = F
a
d
Scheme 4. Reagents and conditions: (a) DAST, DCM, ꢀ70 °C (61%); (b) p-toluenesulfonyl chloride, 2,6-lutidine, rt (66%); (c) CH3ONa; or CF3CH2ONa; or NaOAc; or NaN3, DMF
(78%); (d) PPh3, THF/H2O(4:1), rt (60%).
Chem. Lett. 2003, 13, 2269; (b) Nishimura, T.; Fujikura, H.; Fushimi, N.;
Tatani, K.; Katsuno, K.; Isaji, M. WO03/000712; Chem. Abstr. 2003, 138,
49945.; (c) Nishimura, T.; Fushimi, N.; Fujikura, H.; Katsuno, K.; Komatsu,
Y.; Isaji, M. WO02/068439; Chem. Abstr. 2002, 137, 232854.; (d) Washburn,
W. N.; Ellsworth, B.; Meng, W.; Wu, G.; Sher, P. M. US03/0114390; Chem.
Abstr. 2003, 139, 36736.
least 10-fold less potent compared to the parent compound 3d.
Acetylation led to compound 5d with both reduced potency and
selectivity.
Oral administration of a single dose of 25 mg/kg of 3d to CD1
mice produced severe diarrhea after 6 h. Oral administration of a
single dose of 1.0 mg/kg of 5a to normal Sprague-Dawley rats in-
duced loss of 1220 mg of glucose per 200 g body weight over
24 h, which appeared to be a ꢂ120-fold elevation in glucose dis-
posal relative to vehicle controls. In a separate experiment, a 44%
AUC reduction in blood glucose level versus controls was observed
in 2 h after a single 1 mg/kg oral dose of 5a was administrated to
normal C57BL/6 J mice with starting blood glucose levels of 68–
86 mg/dL. The above correlation of SGLT2 inhibition, glucosuria
and blood glucose-lowering effects of compound 5a suggest the
series may be merit further exploration.
14. Kobayashi, T.; Sato, T.; Nishimoto, M. WO 2006/080421; Chem. Abstr. 2006, 145,
189115.
15. Xu, B.; Lv, B.; Feng, Y.; Xu, G.; Du, J.; Welihinda, A.; Sheng, Z.; Seed, B.; Chen, Y.,
unpublished results.
16. Horton, D.; Priebe, W. Carbohydr. Res. 1981, 94, 27.
17. Deshpande, P. P.; Ellsworth, B. A.; Singh, J.; Denzel, T. W.; Lai, C.; Crispino, G.;
Randazzo, M. E.; Gougoutas, J. Z. WO2004/063209; Chem. Abstr. 2004, 141,
89317.
18. Argentini, M.; Wiese, C.; Weinreich, R. J. Fluorine Chem. 1994, 68, 141.
19. Czernecki, S.; Ville, G. J. Org. Chem. 1989, 54, 610.
20. All compounds provided satisfactory spectral data (1 HNMR, LC–MS). The
detailed synthetic procedure for each compound can be found in Chen, Y.;
Feng, Y.; Xu, B.; Lv, B.; Dong, J.; Seed, B.; Hadd, M. J. US 2007/0275907; Chem.
Abstr. 2007, 147, 542063.
In summary, we have developed a novel series of SGLT2 inhib-
itors, in which the core scaffold is spiro[isobenzofuran-1,20-pyran].
An SAR exploration shows good inhibitory activity and high selec-
tivity for SGLT2 that we attribute, in part, to a greater conforma-
tional constraint imposed by the spiro system. Further
investigation of the in vitro and in vivo SAR of O-spiroketal C-ary-
glucosides will be reported in due course.
21. Mark, L. R.; Charles, J. M.; Chyall, L. J.; Paul, K. WO 2000020364; Chem. Abstr.
2002, 132, 265874.
22. Lorenz, J. C.; Long, J.; Yang, Z.; Xue, S.; Xie, Y.; Shi, Y. J. Org. Chem. 2004, 69, 327.
23.
A plasmid bearing the human full-length SGLT1 coding sequence in the
pDream 2.1 mammalian expression vector was purchased from GenScript
Corporation. A full-length human SGLT2 cDNA (GenScript Corporation) was
cloned into the pEAK15 mammalian expression vector. Human SGLT1
expression plasmid DNA was transiently transfected into COS-7 cells
(American Type Culture Collection) using Lipofectamine 2000 (Invitrogen
Corporation). Transfected cells were evaluated for SGLT1 activity by methyl-
D
a-
-[U-14C]glucopyranoside (AMG) uptake assay and cryopreserved until use.
Acknowledgements
Plasmid containing human SGLT2 was linearized and stably transfected into
HEK293.ETN cells. SGLT2-expressing clones were selected based on resistance
to puromycin (Invitrogen Corporation) and activity in AMG uptake assay. Cells
expressing SGLT1 or SGLT2 were seeded on 96-well ScintiPlates (PerkinElmer,
We thank Dr. Jacques Y. Roberge for thoughtful comments, Ying
Chen for performing in vitro assays and the analytical group of
Egret Pharma (Shanghai) Limited for analytical service.
Inc.) in DMEM containing 10% FBS (1 ꢃ 105 cells per well in 100
lL medium)
incubated at 37 °C under 5% CO2 for 48 h prior to the assay. Cells were washed
twice with 150 lL of either sodium buffer (137 mM NaCl, 5.4 mM KCl, 2.8 mM
CaCl2, 1.2 mM MgCl2, 10 mM tris(hydroxymeth-yl) aminomethane/N-2-
hydroxyethylpiperazine-N0-ethane sulfonic acid [Tris/Hepes], pH 7.2) or
sodium-free buffer (137 mM N-methyl-glucamine, 5.4 mM KCl, 2.8 mM
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-[U-14C]glucopyranoside uptake was quantitated using a TopCount
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a-D
5. Wright, E. M. Am. J. Physiol. 1998, 275, G879.
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a reference
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