Novel C-aryl glucoside SGLT2 inhibitors as potential antidiabetic agents: Pyridazinylmethylphenyl glucoside congeners

Article abstract of DOI:10.1016/j.bmcl.2010.04.006

Novel C-aryl glucoside SGLT2 inhibitors containing pyridazine motif were designed and synthesized for biological evaluation. Among the compounds tested, pyridazine containing methylthio moiety 22l or thiadiazole ring 22ah showed the best in vitro inhibitory activities in this series (IC₅₀ = 13.4, 11.4 nM, respectively) against SGLT2 to date. Subsequently, compound 22l exhibited reasonable urinary glucose excretion and glucosuria in normal SD rats, thereby demonstrating that this pyridazine series possesses both in vitro SGLT2 inhibition and in vivo efficacy, albeit to a lower degree.

Full text of DOI:10.1016/j.bmcl.2010.04.006

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3420–3425
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel C-aryl glucoside SGLT2 inhibitors as potential antidiabetic agents:
Pyridazinylmethylphenyl glucoside congeners
Min Ju Kim, Junwon Lee, Suk Youn Kang, Sung-Han Lee, Eun-Jung Son, Myung Eun Jung, Suk Ho Lee,
*
Kwang-Seop Song, MinWoo Lee, Ho-Kyun Han, Jeongmin Kim, Jinhwa Lee
Central Research Laboratories, Green Cross Corporation, 303 Bojeong-Dong, Giheung-Gu, Yongin, Gyeonggi-Do 446-770, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel C-aryl glucoside SGLT2 inhibitors containing pyridazine motif were designed and synthesized for
biological evaluation. Among the compounds tested, pyridazine containing methylthio moiety 22l or
thiadiazole ring 22ah showed the best in vitro inhibitory activities in this series (IC50 = 13.4, 11.4 nM,
respectively) against SGLT2 to date. Subsequently, compound 22l exhibited reasonable urinary glucose
excretion and glucosuria in normal SD rats, thereby demonstrating that this pyridazine series possesses
both in vitro SGLT2 inhibition and in vivo efficacy, albeit to a lower degree.
Received 21 December 2009
Revised 3 March 2010
Accepted 3 April 2010
Available online 9 April 2010
Keywords:
SGLT
Ó 2010 Elsevier Ltd. All rights reserved.
Dapagliflozin
Pyridazine
Glucoside
Diabetes
Diabetes has become an increasing concern to the world’s popu-
lation. In 2007, approximately 246 million people were considered
diabetic, with an additional 7 million people diagnosed with the dis-
ease every year.1 Diabetes is a chronic metabolic disorder that is de-
fined by the body’s inability to generate insulin or the inability of the
body to respond adequately to circulating insulin. There are two
identified forms of diabetes: type 1 diabetes is distinguished as an
autoimmune disease involving pancreatic b-cells, while type 2 dia-
betes is defined by b-cell dysfunction and insulin resistance.2 Type
2 diabetes is the most common disorder of glucose homeostasis,
accounting for nearly 90–95% of all cases of diabetes.
Sodium-dependent glucose cotransporters (SGLTs) couple the
transport of glucose against a concentration gradient with the
simultaneous transport of Na+ down a concentration gradient.3
Two important SGLT isoforms have been cloned and identified,
SGLT1 and SGLT2.4 SGLT1 is located in the kidney and the heart,
where its expression regulates cardiac glucose transport.5 SGLT1
is a high-affinity, low-capacity transporter and therefore accounts
for only a small fraction of renal glucose reabsorption.6 In contrast,
SGLT2 is a low-affinity, high-capacity transporter located exclu-
sively at the apical domain of the epithelial cells in the early prox-
imal convoluted tubule. It is estimated that 90% of renal glucose
reabsorption is facilitated by SGLT2; the remaining 10% is likely
mediated by SGLT1 in the late proximal straight tubule.7 Since
SGLT2 appears to account for the majority of renal glucose reab-
sorption based on human mutation studies,8 it has attracted ther-
apeutic interest.
Extensive SAR studies by Bristol–Myers Squibb identified dapa-
gliflozin 1 (Fig. 1), a potent, selective SGLT2 inhibitor for the treat-
ment of type 2 diabetes.9–11 At present, dapagliflozin is the most
advanced SGLT2 inhibitors in clinical trials and is believed to be
the first SGLT2 inhibitor to market.14 On the other hand, Mitsubishi
Tanabe Pharma, in collaboration with Johnson & Johnson, is devel-
oping canagliflozin 2 (Fig. 1), another novel C-glucoside-derived
SGLT2 inhibitor.12 In August 2009, a phase 3 study was reportedly
initiated to evaluate the safety and efficacy of 2 in patients with
type 2 diabetes.13
In the present study, metabolically more stable C-glucosides
bearing a heteroaromatic ring were exploited in order to develop
novel SGLT2 targeting antidiabetic agents. We envisioned that
replacement of the distal ring of dapagliflozin 1 with a heterocyclic
ring was a worthy approach for the improvement of the partition
coefficient (log P) value, to potentially decrease plasma protein
binding. For this purpose, the structure of dapagliflozin 1 was mod-
ified into compounds bearing a heterocyclic ring. Among a variety
of heterocycles, we decided to screen pyridazine as our current ef-
forts. Herein, we report the design, synthesis and biological evalu-
ation of pyridazinylmethylphenyl glucoside congeners.
As shown in Scheme 1, reduction of commercially available
5-bromo-2-chlorobenzoic acid (4) with a borane-dimethyl sulfide
complex, and subsequent silylation of the corresponding alcohol
with TIPSCl (triisopropylsilyl chloride) in the presence of imidazole
and DMAP (4-(dimethylamino)pyridine) generated bromide 5 in
* Corresponding author. Tel.: +82 31 260 9892; fax: +82 31 260 9020.
E-mail address: jinhwalee@greencross.com (J. Lee).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.006

M. J. Kim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3420–3425
Cl OEt
3421
Me
O
O
HO
HO
F
S
HO
HO
OH
OH
OH
OH
2, canagliflozin
1, dapagliflozin
Figure 1. Structures of C-aryl glucoside SGLT2 inhibitors.
Cl
Br
CO2H
4
.
1. BH3 SMe2 / THF, 0 oC to rt
2. TIPSCl, Imidazole, DMAP / DMF, rt
(95%, 2 steps)
Cl
Cl
OTIPS
O
O
O
OH
Br
BnO
BnO
1. PBr3, pyridine / ether, rt
1.
, BuLi / THF, -78 oC
BnO
BnO
5
OBn
OBn
2. KCN / EtOH, H2O, reflux
(80%, 2 steps)
2. Et3SiH, BF3.OEt2 / CH2Cl2, -50 to -10 oC
3. Bu4NF / THF, rt
OBn
OBn
6
3
3. EtOH, (resolution)
(98%, 3 steps)
(40%, 3 steps)
Cl
Cl
NaH
NaOH
SOCl2
N
Cl
O
CO2Me
O
CN
DMF
N
BnO
BnO
BnO
BnO
EtOH, H2O
reflux
(98%)
MeOH
reflux
(89%)
+
(93%)
Cl
OBn
OBn
9
OBn
OBn
7
8
Cl
N
Cl
Cl
N
N
Cl
N
LiOH
O
O
BnO
BnO
H2O, THF, MeOH
(86%)
CO2Me
10
BnO
OBn
BnO
OBn
OBn
OBn
11
Scheme 1. Preparation of key C-glycoside dichloride 11.
95% yield over two steps. Lithium-halogen exchange, followed by
the addition of the nascent lithiated aromatic compound to perb-
enzylated gluconolactone 3,15 produced a mixture of the corre-
sponding lactols. The lactols were reduced using triethylsilane
and BF3 etherate,14 desilylated and afforded alcohol 6 in 98% yield
over the three steps. Thus, alcohol 6 was converted to bromide
using PBr3 in the presence of pyridine, which was treated with
KCN in refluxed aqueous EtOH to generate cyanide in 80% yield
for the two steps. A mixture of two isomers was resolved through
recrystallization from ethanol to produce the required beta-isomer
7 in about 40% yield. Hydrolysis of 7 with sodium hydroxide in
aqueous ethanol generated the corresponding carboxylic acid in
quantitative yield. Treatment of the carboxylic acid with thionyl
chloride in refluxed methanol produced the corresponding methyl
ester 8 in 89% yield. Thus, coupling of ester 8 and 3,6-dichloropyr-
idazine (9) using NaH in the presence of DMF yielded the corre-
sponding ester 10, which was hydrolyzed and decarboxylated
using lithium hydroxide in an aqueous solution of THF and meth-
anol to generate the key dichloride 11 in 80% yields over two steps.
Utilization of the key intermediate dichloride 11 was illustrated
in Scheme 2. Replacement of chloropyridazine with sodium alkox-
ide provided the corresponding 12. Likewise, treatment of 11 with
NaSMe yielded methylthio-pyridazine 13 in 74% yields unevent-
fully. Fe(III)-mediated alkylation21 was conducted on 11 using
Grignard reagent such as ethylmagnesium bromide to afford ethyl-
pyridazine 14 in 67% yields. Sonogashira reaction22 was conducted
on 11 with ethynylbenzene 15 to provide the compound 16.
Palladium(0)-catalyzed cyanation23 proceeded smoothly under
microwave irradiation to provide the cyanopyridazine 17. Suzu-
ki–Miyaura coupling24 of 11 with boronic acid such as phenylbo-
ronic acid 18 to generate phenylpyridazine 19 in 73% yields.
Amino group was also introduced smoothly under microwave con-
ditions in approximately 80% yields to produce the compounds.
At last, procedures for deprotection of benzyl groups which were
utilized in this article are illustrated in Scheme 3. Thus, the first re-
sort for deprotection is using TMSI (trimethyliodosilane)16 in aceto-
nitrile as in Case 1 (13 to 22l). If the first method fails, use of TMSOTf
(trimethylsilyl trifluoromethanesulfonate) in combination with

3422
M. J. Kim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3420–3425
Cl
N
O
N
R
Cl
N
S
N
O
BnO
BnO
O
BnO
BnO
Cl
N
Et
OBn
N
OBn
OBn
O
12
OBn
BnO
BnO
13
OBn
14
OBn
NaOR
NaSMe
Fe(acac)3
EtMgBr
Cl
N
NR1R2
N
Cl
N
Cl
N
O
HNR1R2
BnO
BnO
O
BnO
BnO
OBn
OBn
OBn
20
OBn
11
OH
B
OH
15
Ph
Pd(PPh3)2Cl2
CuI
Pd(PPh3)2Cl2
18
Zn(CN)2
Pd(0)
Cl
N
N
Ph
O
Cl
N
CN
BnO
BnO
N
Cl
N
N
O
OBn
BnO
O
BnO
OBn
19
BnO
OBn
BnO
OBn
17
OBn
16
OBn
Scheme 2. Preparation of various pyridazines using key C-glycoside dichloride 11.
Case 1
Cl
N
S
Cl
N
S
N
N
TMSI
O
O
HO
HO
BnO
BnO
CH3CN
OH
OBn
OH
OBn
22l
13
Case 2
Cl
N
Cl
Cl
N
Cl
N
N
O
O
BnO
TMSOTf
Ac2O
AcO
AcO
BnO
OBn
OAc
OBn
21
OAc
11
Cl
N
Cl
N
O
NaOMe
MeOH
HO
HO
OH
22a
OH
Scheme 3. Debenzylation conditions for the preparation of the target compounds.

M. J. Kim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3420–3425
3423
Table 1
In vitro inhibitory activity against hSGLT2
Cl
N
R3
R2
N
O
HO
HO
R1
OH
unit: nM
OH
Ref. Dapagliflozin (0.49 0.04)a
22
R1
H
R2
H
R3
Compound
hSGLT2 IC50
R1
H
R2
H
R3
Compound
hSGLT2 IC50
b
b
Ph
Cl
22a
202
22v
176
O
H
H
H
H
H
H
H
H
H
H
H
H
22b
22c
22d
22e
22f
43
H
H
H
H
H
H
H
H
H
H
H
H
22w
22x
64.3
O
S
S
O
O
70.8
110
109
435
17.0
61.3
71.4
75.4
168
222
22y
O
O
S
22z
O
O
Me
F
22aa
22ab
22g
O
O
H
H
H
H
H
H
H
H
22h
22i
17.3
50.5
111
H
H
H
H
H
H
H
H
22ac
22ad
22ae
22af
272
351
64.9
82.7
Me
O
O
N
22j
O
O
22k
77.6
O
O
O
S
Me
H
H
H
H
22l
13.4
71.3
H
H
H
H
22ag
22ah
103
O
N
S
S
S
N
22m
11.4
N
Me
H
H
H
H
22n
22o
1870
293
H
H
H
H
22ai
22aj
1710
700
O
Me
O
N
H
H
H
H
22p
22q
131
237
H
H
H
H
22ak
22al
1140
1040
HN
N
Me
H
H
H
H
22r
22s
259
356
H
Me
Me
22am
22an
1520
S
S
Me
Me
Me
10,000
H
H
H
H
22t
811
134
22ao
10,000
22u
a
This data was obtained by multiple determinations.
These data were obtained by single determinations.
b
acetic anhydride is another option for routine deprotection of benzyl
groups as shown in Case 2. For example, 11 was treated with TMSOTf
and acetic anhydride17 to provide the corresponding tetraacetate 21,
then hydrolyzed to yield the target compound 22a.

3424
M. J. Kim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3420–3425
including benzodioxole 22af, dihydrobenzodioxine 22ag exhibited
*
2000
1800
1600
1400
1200
1000
800
modest inhibitory activities against hSGLT2 (22af, IC50 = 82.7 nM;
22ag, IC50 = 103 nM). It is noteworthy that any substitution at the
aryl or heteroaryl ring connected with the distal pyridazine ring re-
sulted in products with significantly lower hSGLT2 inhibitory activ-
ities
(22ac,
IC50 = 272 nM;
22ad,
IC50 = 351 nM;
22ai,
IC50 = 1.71
lM). Among the biaryl-type compounds tested, thiadia-
zole 22ah demonstrated the best in vitro inhibitory activity
(IC50 = 11.4 nM). However, amine-substituted pyridazines showed
weak activities, showing IC50 = 700–1140 nM for 22aj, 22ak, and
22al. Moreover, mono- or di-substitution at the pyridazine moiety
further deteriorated hSGLT2 inhibitory activities, as shown for com-
pounds 22am, 22n, and 22ao (IC50 = 1.52 lM, >10 lM, >10 lM,
respectively), likely suggesting that increased steric hindrance is
not tolerated in the region.
600
*
400
200
0
Vehicle
Dapa. @ 1mg/kg
22l @ 10mg/kg
Figure 2. Urinary glucose excretion test of vehicle, compound 1 (1 mg/kg) and 22l
(10 mg/kg) in normal SD rats. All results are expressed as means S.E.M. The
statistical analysis was performed using
Dunnett’s post hoc test. *P <0.05 versus vehicle.
a one-way ANOVA followed by the
In order to further assess this series, the pharmacokinetic prop-
erties of a selected compound, 22l, were measured in male SD rats.
After oral administration of 5 mg/kg of 22l to rats, a Cmax of
30
0.31 lg/mL was obtained at 0.33 h. The elimination half-life of
*
22l following oral administration was 1.94 h in rats.28 Compound
22l showed decent oral bioavailability (F = 26.1%) in rats. Subse-
quently, compound 22l was tested in animal models for in vivo
efficacy.18–20,27 The urine glucose and urine volume data were nor-
malized per 200 g of body weight. As shown in Figure 2, a single
oral dose of pyridazine 22l increased urinary glucose excretion in
normal SD rats, resulting in a 180-fold elevation in glucose disposal
relative to vehicle controls. Urinary glucose excretion of com-
pounds 1 and 22l were 1648 228 mg/200 g body weight and
344 116 mg/200 g body weight, respectively (1.90 228 mg/
200 g for vehicle). Urine volume excreted in normal SD rats are also
shown in Figure 3. Dapagliflozin 1 caused increased urine volume
over vehicle in 5.7-fold, while pyridazine 22l increased urine vol-
ume in merely 1.3-fold (vehicle: 3.9 0.23, 1: 22.36 4.18, 22l:
5.07 1.09 ml/200 mg body weight, respectively).
25
20
15
10
5
0
Vehicle
Dapa. @ 1mg/kg
22l @ 10mg/kg
Figure 3. Urine volume excreted of vehicle, compound 1 (1 mg/kg) and 22l (10 mg/
kg) in normal SD rats. All results are expressed as means S.E.M. The statistical
analysis was performed using a one-way ANOVA followed by the Dunnett’s post
hoc test. *P <0.05 versus vehicle.
Obviously, the decreased in vivo efficacy of the current SGLT2
inhibitor 22l compared with dapagliflozin 1 could be attributed
to the difference in inherent in vitro potencies (22l: IC50 = 13.4 nM
vs 1: IC50 = 0.49 nM) as well as in pharmacokinetic properties.
Thus, replacement of the distal ring of dapagliflozin 1 with a pyrid-
azine ring as in compound 22l appeared to diminish the in vitro
activity and oral absorption, thereby resulting in the diminished
in vivo efficacy in animal models.
In summary, metabolically more stable C-glucosides bearing
pyridazine ring as a potential antidiabetic agent were exploited.
Among the compounds tested, pyridazine containing methylthio
moiety 22l or thiadiazole ring 22ah showed the best in vitro inhib-
itory activities against hSGLT2 in this series to date (IC50 = 13.4,
11.4 nM, respectively). Subsequently, compound 22l demonstrated
reasonable urinary glucose excretion and glucosuria in normal SD
rats, thereby demonstrating that this pyridazine series possesses
both in vitro SGLT2 inhibition and in vivo efficacy, albeit to a rela-
tively lower degree. The information acquired from this series of
compounds can be utilized as a quick reference to achieve more
advanced series in this area.
The cell-based SGLT2 AMG (methyl-a-D-glucopyranoside) inhi-
bition assay was performed to evaluate the inhibitory effects of all
prepared compounds on hSGLT2 activities.18,19,25,26 Exploration of
the SAR began by replacing the phenyl moiety at the distal ring posi-
tion of dapagliflozin 1 with pyridazine moiety. Table 1 shows the
structure–activity relationship upon alteration of the substituent
at the distal pyridazine ring employing only the b-anomer. Initially,
ethoxide on the pyridazine ring showed reasonable activity (22b,
IC50 = 43 nM). As the size of the carbon chain of alkoxy moiety on
pyridazine increases, decrease in the inhibitory activity against
hSGLT2 is observed up to C-4 chain (22e, IC50 = 109 nM), but not at
C-5 (22g, IC50 = 17.0 nM) or C-6 (22h, IC50 = 17.3 nM) before further
elongation results in decreased activity as shown in 22i
(IC50 = 50.5 nM). Branched alkoxy chains displayed moderate inhib-
itory activity against hSGLT2, showing IC50 = 110 nM for 22d or
435 nM for 22f, respectively. This pattern is also observed for cyclo-
hexyloxy 22j (IC50 = 109 nM) or pyranyloxy 22k (IC50 = 77.6 nM),
suggesting that branched aliphatic chains or slightly increased steric
hindrance is not optimal at this position. Replacement for this moi-
ety with methylthio group 22l improved the inhibitory activity
against hSGLT2 (IC50 = 13.4 nM). However, increase of lipophilicity
appeared to deteriorate the activity, showing IC50 = 71.3 nM for eth-
ylthio, 22m, or 1870 nM for phenylthio, 22n, respectively. Aliphatic
chains on the pyridazine ring displayed moderate inhibitory activ-
ity, showing IC50 = 131–356 nM for 22o, 22p, 22q, 22s, and 22u.
Again, branched aliphatic chain 22t showed reduced inhibitory
activity against hSGLT2. Alkyne 22v on the pyridazine ring also
showed moderate activity against hSGLT2 (IC50 = 176 nM). Mean-
while, diaryl-types, especially furans, thiophenes or pyridine exhib-
ited more favorable activity (22w–22z, 22ae: IC50 = 61.3–75.4 nM)
than phenyl (22ab, IC50 = 222 nM). Substitution with bicyclic groups
Acknowledgments
We appreciate Dr. Chong-Hwan Chang for his leadership and
dedication during his tenure as Head of R&D, Green Cross Corpora-
tion (GCC).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.04.006.

M. J. Kim et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3420–3425
3425
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Vazquez, E.; Springfield, S. A.; Zhou, G.; Liu, P.; Kieczykowski, G. R.; Chen, A. M.;
Phenix, B. D.; Singh, U.; Strine, J.; Izzo, B.; Krska, S. W. Org. Process Res. Dev.
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(hSGLT2) gene was amplified by PCR from cDNA-Human Adult Normal
Tissue Kidney (Invitrogen, Carlsbad, CA). The hSGLT2 sequence was cloned
into pcDNA3.1(+) for mammalian expression and were stably transfected into
chinese hamster ovary (CHO) cells. SGLT2-expressing clones were selected
based on resistance to G418 antibiotic (GeneticinÒ, Invitrogen, Carlsbad, CA)
and activity in the 14C- -glucopyranoside (14C-AMG) uptake assay.
a-methyl-D
26. For sodium-dependent glucose transport assay, cells expressing hSGLT2 were
seeded into a 96-well culture plate at a density of 5 Â 104 cells/well in RPMI
medium 1640 containing 10% fetal bovine serum. The cells were used 1 day
after plating. They were incubated in pretreatment buffer (10 mM HEPES,
5 mM Tris, 140 mM choline chloride, 2 mM KCl, 1 mM CaCl2, and 1 mM MgCl2,
pH 7.4) at 37 °C for 10 min. They were then incubated in uptake buffer (10 mM
HEPES, 5 mM Tris, 140 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and
1 mM 14C-nonlabeled AMG pH 7.4) containing 14C-labeled (8
lM) and inhibitor
or dimethyl sulfoxide (DMSO) vehicle at 37 °C for 2 h. Cells were washed twice
with washing buffer (pretreatment buffer containing 10 mM AMG at room
temperature) and then the radioactivity was measured using
a liquid
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27. For urinary glucose excretion in normal animal: urinary glucose excretion was
evaluated on normal Sprague-Dawley (SD) rats.20–22 Male SD rats weighing
250–300 g (7–8 weeks old) were purchased from Orient-Bio Laboratory Animal
Research Center Co. (Gyeonggi-do, Korea). They were housed in a temperature
(25 2 °C), and moisture (55 10%) controlled room, exposed to a controlled
12 h cycle of light and darkness, and allowed free access to food and water. All
animals were acclimated for one week prior to the experiment. For glucosuria
assessment, overnight-fasted SD rats were placed into metabolism cages for
baseline urine collection over 24 h. Rats were weighed, randomized into three
groups in plane (n = 3), dosed orally with single doses of vehicle or drug (1
@1 mg/kg, 32l @10 mg/kg), and subsequently dosed orally with 50% aqueous
glucose solution (2 g/kg). Immediately after dosing, rats were returned to the
metabolism cages for 24 h urine collection and re-fed at 1 h after the glucose
challenge.
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&
Johnson
Pharmaceutical,
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28. For comparison, after oral administration of 5 mg/kg of 1 to rats, a Cmax of
2.50 lg/mL was obtained at 0.67 h. The elimination half-life of 1 following oral
administration was 4.01 h in rats. Compound
1 showed desirable oral
bioavailability (F = 88.42%) in rats.