Pyrimidinylmethylphenyl glucoside as novel C-aryl glucoside SGLT2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.09.103

Novel C-aryl glucoside SGLT2 inhibitors containing pyrimidine motif were designed and synthesized for biological evaluation. Among the compounds assayed, pyrimidine containing methylthio moiety 11g demonstrated the best in vitro inhibitory activity against SGLT2 in this series to date (IC₅₀ = 10.7 nM).

Full text of DOI:10.1016/j.bmcl.2010.09.103

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7046–7049
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrimidinylmethylphenyl glucoside as novel C-aryl glucoside SGLT2 inhibitors
⇑
Junwon Lee, Jong Yup Kim, Jungsub Choi, Sung-Han Lee, Jeongmin Kim, Jinhwa Lee
Research Center, 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 pyrimidine motif were designed and synthesized for
biological evaluation. Among the compounds assayed, pyrimidine containing methylthio moiety 11g
demonstrated the best in vitro inhibitory activity against SGLT2 in this series to date (IC50 = 10.7 nM).
Ó 2010 Elsevier Ltd. All rights reserved.
Received 31 August 2010
Revised 16 September 2010
Accepted 21 September 2010
Available online 29 September 2010
Keywords:
SGLT
Dapagliflozin
Pyrimidine
Glucoside
Diabetes
Diabetes has become an increasing concern to the world’s
population. In 2010, approximately 285 million people around
the world will have diabetes, corresponding to 6.4% of the world’s
adult population, with a prediction that by 2030 the number of
people with diabetes will have grown to 438 million.1 Type 2 dia-
betes is the most common disorder of glucose homeostasis,
accounting for nearly 90–95% of all cases of diabetes.2
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 It
is estimated that 90% of renal glucose reabsorption is facilitated
by SGLT2.4
Bristol-Myers Squibb has identified dapagliflozin, a potent,
selective SGLT2 inhibitor for the treatment of type 2 diabetes.5–7
At present, dapagliflozin is the most advanced SGLT2 inhibitor in
clinical trials.8 On the other hand, Mitsubishi Tanabe, in collabora-
tion with Johnson and Johnson, is developing canagliflozin, another
novel C-glucoside-derived SGLT2 inhibitor.9 In addition, Boehrin-
ger Ingelheim, Lexicon, Astellas, and Pfizer are reported to be in
various phases of clinical trials (Fig. 1).10
In the present study, C-glucosides bearing a heteroaromatic ring
were exploited in order to develop novel SGLT2 targeting antidia-
betic agents with better biological activities and pharmacological
properties than dapagliflozin.11 Along this line, the structure of
dapagliflozin was modified into A or B, bearing a pyrimidine ring
as shown in Figure 2. Herein, we report the design, synthesis and
biological evaluation of pyrimidinylmethylphenyl glucoside
congeners.
Preparation of the pyrimidine compound is described in Scheme
1. Thus, a mixture of alcohols 111a,b was converted to the required
beta-isomer 2 using three steps involving benzoylation, separation
through recrystallization from isopropyl alcohol, and subsequent
hydrolysis with lithium hydroxide in aqueous mixture of metha-
nol–THF in about 60% yield. The alcohol 2 thus obtained was con-
verted to bromide 3 using PBr3 in the presence of a catalytic
amount of pyridine in quantitative yield. Suzuki–Miyaura cou-
pling12 of the key intermediate bromide 3 with boronic acid such
as 2-(methylthio)pyrimidin-5-ylboronic acid (4)13 generated pyri-
midinylmethylphenyl compound 5 in quantitative yield. Subse-
quent deprotection of the four benzyl groups using BBr3 in
methylene chloride produced the target compound 6k in approxi-
mately 40% yield.
We next extended a method previously adopted by us11b for
preparation of novel pyrimidinyl analogs as shown in Scheme 2.
Thus, coupling of ester 711a,b with 5-bromo-2-chloropyrimidine
(8) using NaH in the presence of DMF produced the corresponding
ester, which was hydrolyzed and decarboxylated using LiOH in an
aqueous solution of THF and methanol to generate the key inter-
mediate 9 in 66% yield over two steps. Methylthio group was then
introduced smoothly under microwave conditions in approxi-
mately 50% yields to produce the compound of structure 10. At
last, deprotection of benzyl groups was accomplished in two steps.
For example, 10 was treated with TMSOTf and acetic anhydride to
provide the corresponding tetraacetate, which was subsequently
hydrolyzed to yield the target compound 11 in 58% yields over
two steps.
The cell-based SGLT2 AMG (methyl-a-D-glucopyranoside)
inhibition assay was performed to evaluate the inhibitory effects
⇑
Corresponding author. Tel.: +82 31 260 9892; fax: +82 31 260 9020.
E-mail address: jinhwalee@greencross.com (J. Lee).
of all prepared compounds on hSGLT2 activities.14,15 Exploration
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.103

J. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7046–7049
7047
Cl
OEt
Me
O
O
HO
HO
F
S
HO
HO
OH
OH
OH
OH
canagliflozin
dapagliflozin
Cl
OEt
Cl
OEt
O
O
MeO
HO
O
HO
HO
OH
OH
OH
OH
Pfizer's
Lexicon's
Figure 1. Structures of C-aryl glucoside SGLT2 inhibitors.
R1
N
R2
O
HO
N
Cl
OEt
HO
OH
OH
A
O
HO
HO
R1
N
OH
R2
O
N
OH
HO
HO
dapagliflozin
OH
OH
B
Figure 2. Exploration of C-glucoside bearing pyrimidine at the distal ring.
Cl
Cl
O
OH
O
OH
d
a, b, c
BnO
BnO
BnO
BnO
99%
64%
3 steps
OBn
OBn
OBn
OBn
1
2
Cl
Cl
N
SMe
O
Br
O
N
N
SMe
e
BnO
BnO
BnO
+
N
99%
OBn
BnO
OBn
(HO)2B
OBn
OBn
3
4
5
Cl
N
SMe
O
N
f
HO
HO
41%
OH
OH
6k
Scheme 1. Reagents and conditions: (a) benzoyl chloride, pyridine, rt; (b) IPA (selective crystallization); (c) LiOHÁH2O, THF, MeOH, H2O, rt; (d) PBr3, cat. pyridine, ether, rt; (e)
Pd(PPh3)4, Cs2CO3, toluene, EtOH, 120 °C; (f) BBr3, CH2Cl2, 0 °C.
of the SAR began by replacing the phenyl moiety at the distal
ring position of dapagliflozin with pyrimidine moiety.19 Table 1
shows the structure–activity relationship upon alteration of the
substituent at the distal pyrimidine ring employing only the b-ano-
mer. Initially, n-butyl on the pyrimidine ring showed relatively
poor activity (6a, IC50 = 8870 nM). However, replacement for this
moiety with phenyl group 6b improved the inhibitory activity
against hSGLT2 (IC50 = 124 nM). Fluorophenyl on the pyrimidine
ring displayed comparable inhibitory activity against hSGLT2
(IC50 = 119 nM). Meanwhile, substitution of phenyl with thiophene
exhibited slightly better activity against hSGLT2 (6e: IC50
=
113 nM) than phenyl (6b, IC50 = 124 nM). On the other hand, furan
6d and thiadiazole 6g demonstrated twofold lower in vitro inhibi-
tory activity (IC50 = 246 nM for 6d, IC50 = 223 nM for 6g) than

7048
J. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7046–7049
Cl
Cl
Br
N
O
CO2Et
Br
O
a, b
BnO
BnO
N
BnO
BnO
N
+
66%
2 steps
OBn
Cl
Cl
N
OBn
OBn
OBn
8
7
9
SMe
Cl
SMe
N
N
O
O
c
d, e
BnO
BnO
N
HO
HO
N
53%
58%
2 steps
OBn
OH
OBn
OH
10
11g
Scheme 2. Reagents and conditions: (a) NaH, DMF, 0 °C to rt; (b) LiOHÁH2O, THF, MeOH, H2O, rt; (c) NaSMe, NMP, MW (170 °C); (d) TMSOTf, Ac2O, À30 °C to rt; (e) NaOMe,
MeOH, rt.
Table 1
In vitro inhibitory activity against hSGLT2
Cl
O
R
HO
HO
OH
OH
Compound
R
hSGL2 IC50 (nM)a
Compound
R
hSGL2 IC50 (nM)a
N
N
O(CH2)6CH3
SMe
Dapagliflozin
—
0.49 0.04b
6j
130
N
N
N
N
n-Bu
6a
6b
8870
124
6k
76.0
73.6
N
N
11a
N
N
F
F
N
N
6c
119
11b
107
N
N
N
N
N
N
N
N
N
N
O
S
O
6d
6e
6f
246
113
189
223
11c
11d
11e
11f
36.8
34.5
17.1
64.4
O
N
N
N
N
CONH2
S
N
N
S
N
N
N
N
N
6g
S
N
N
N
N
N
OEt
SMe
SEt
6h
6i
46.9
447
11g
11h
10.7
39.4
N
On-Bu
N
a
These data were obtained by single determinations.
The IC50 value was obtained by in-house multiple determinations.
b

J. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7046–7049
7049
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. J. Med.
Chem. 2008, 51, 1145.
phenyl, likely suggesting that increased polarity is not that favor-
able in the region. Alkoxy groups on the pyrimidine were also
explored. As a matter of fact, ethoxy group showed favorable
inhibitory activity against hSGLT2 (6h: IC50 = 46.9 nM), which is
particularly reminiscent of dapagliflozin. As the number of carbons
increase, the inhibitory activity against hSGLT2 becomes fluctu-
ated. For example, the tenfold drop in activity is observed in vary-
ing the length of the alkoxy chain from ethyl (6h: IC50 = 46.9 nM)
to n-butyl (6i: IC50 = 447 nM), whereas the threefold increase in
activity is observed from n-butyl (6i: IC50 = 447 nM) to n-heptyl
(6j: IC50 = 130 nM). But none improved inhibitory activity relative
to the simple ethoxy analog 6h. Methylthio group for this region
also appears to be tolerant (IC50 = 76 nM for 6k), primarily imply-
ing that small lipophilic groups are favored at this location.
Next, isomeric pyrimidine series of compounds such as 4-chloro-
3-(5-(methylthio)pyrimidin-2-yl)methylphenyl was explored
(11a–11h, Table 1). This series exhibited increase in inhibitory
activities against hSGLT2 compared with the previous pyrimidine
series such as 4-chloro-3-(2-(methylthio)pyrimidin-5-yl)methyl-
phenyl. For instance, phenylpyrimidine 11a (IC50 = 73.6 nM)
7. Washburn, W. N. J. Med. Chem. 2009, 52, 1785.
8. According to a report from Business Wire on June 26, 2010, dapagliflozin as add
on therapy to insulin demonstrated improved glycemic control in patients with
type 2 diabetes inadequately controlled with insulin.
9. Nomura, S.; Sakamaki, S.; Hongu, M.; Kawanshi, E.; Koga, Y.; Sakamoto, T.;
Yamamoto, Y.; Ueta, K.; Kimata, H.; Nakayama, K.; Tsuda-Tsukimoto, M. J. Med.
Chem. 2010. doi:10.1021/jm100332n.
10. (a) Boehringer Ingelheim Pharmaceuticals, www.clinicaltrials.gov, 2010,
NCT01164501. (b) Lexicon Pharmaceuticals, www.clinicaltrials.gov, 2009,
NCT00962065. (c) Astellas Pharma Inc., www.clinicaltrials.gov, 2010,
NCT01135433. (d) Mascitti, V. Presented at the 240th American Chemical
Society National Meeting and Exposition, Boston, MA, August 22–26, 2010;
Medi-151.
11. (a) Lee, J.; Lee, S.-H.; Seo, H. J.; Son, E.-J.; Lee, S. H.; Jung, M. E.; Lee, M.;
Han, H.-K.; Kim, J.; Kang, J.; Lee, J. Bioorg. Med. Chem. 2010, 18, 2178; (b) Kim,
M. J.; Lee, J.; Kang, S. Y.; Lee, S.-H.; Son, E.-J.; Jung, M. E.; Lee, S. H.; Song, K.-S.;
Lee, M.; Han, H.-K.; Kim, J.; Lee, J. Bioorg. Med. Chem. Lett. 2010, 20, 3420; (c)
Kang, S. Y.; Song, K.-S.; Lee, J.; Lee, S.-H.; Lee, J. Bioorg. Med. Chem. 2010, 18,
6069.
12. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
13. To the solution of 5-bromo-2-chloropyrimidine (1.00 g, 5.17 mmol) in THF
(20 mL) was added sodium methanethiolate (544 mg, 7.75 mmol). The reaction
mixture was stirred at 60 °C overnight and evaporated in vacuo. The residue
was purified by flash column chromatography (Biotage SP1™) to provide
5-bromo-2-(methylthio)pyrimidine (1.01 g, 95%) as a white solid (MH+ 205).
To the solution of 5-bromo-2-methylthiopyrimidine (1.01 g, 4.92 mmol) in THF
(20 mL) was added trimethylborate (0.84 mL, 7.39 mmol) at À78 °C. To the
reaction mixture was added n-BuLi (2.5 M in hexane, 3.0 mL, 7.5 mmol) at
À78 °C for 1 h and warmed-up to À20 °C. The mixture was quenched by 1 N
aqueous HCl solution at À20 °C and evaporated in vacuo. The residue was
poured into water (50 mL) and extracted with EtOAc (50 mL Â 2). The organic
layer was dried over MgSO4 and evaporated in vacuo to provide
2-(methylthio)pyrimidin-5-ylboronic acid (538 mg, 64%) as a yellow solid:
1H NMR (400 MHz, CD3OD) d 8.76 (s, 2H), 2.57 (s, 3H).
showed twofold increase in activity than the counterpart 6b (IC50
124 nM). As more clear demonstration, two pairs of examples
are presented, including furans 6d (IC50 = 246 nM), 11d (IC50
=
=
34.5 nM) as well as methylthiopyrimidines 6k (IC50 = 76 nM), 11g
(IC50 = 10.7 nM), demonstrating sevenfold increase of activity,
respectively. Among the substituents tested up to date, the best re-
sult was obtained when thiomethyl is installed at C-5 at pyrimidine
as shown in 11g (IC50 = 10.7 nM).18 However, increasing the number
of carbons from methylthio 11g to ethylthio 11h exhibited threefold
drop in inhibitory activity against hSGLT2 (11g: IC50 = 10.7 nM; 11h:
IC50 = 39.4 nM).
Thus, replacement of the distal ring of dapagliflozin with a
pyrimidine ring appears to weaken in vitro inhibitory activity
against hSGLT2 perhaps for unfavorable electronic environments
at the distal ring position. However, the information obtained from
this pyrimidine series should help to design more effective SGLT2
inhibitors that are structurally related.
14. For cloning and cell line construction for human SGLT2, human SGLT2
(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
15. 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
In summary, metabolically more stable C-glucosides bearing
pyrimidine ring as a potential antidiabetic agent was exploited.
Among the compounds tested, pyrimidine containing methylthio
moiety 11g showed the best in vitro inhibitory activity against
hSGLT2 in this series to date (IC50 = 10.7 nM).
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
scintillation counter. IC50 was determined by nonlinear regression analysis
Acknowledgment
using GraphPad PRISM.16,17
16. Han, S.; Hagan, D. L.; Taylor, J. R.; Xin, L.; Meng, W.; Biller, S. A.; Wetterau, J. R.;
Washburn, W. N.; Whaley, J. M. Diabetes 2008, 57, 1723.
17. Katsuno, K.; Fujimori, Y.; Takemura, Y.; Hiratochi, M.; Itoh, F.; Komatsu, Y.;
Fujikura, H.; Isaji, M. J. Pharmacol. Exp. Ther. 2007, 320, 323.
We appreciate Dr. Eun Chul Huh for his leadership as Head of
R&D, Green Cross Corporation (GCC).
18. (2S,3R,4R,5S,6R)-2-(4-Chloro-3-((5-(methylthio)pyrimidin-2-yl)methyl)phenyl)-
6-(hydroxymethyl)-tetrahydro-2H-pyran-3,4,5-triol (11g): white solid; mp
References and notes
107 °C (MeOH); 1H NMR (400 MHz, DMSO-d6)
d 8.62 (s, 2H), 7.35 (d,
J = 2.0 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.21 (dd, J = 8.4, 2.0 Hz, 1H), 4.98 (br,
1H), 4.95 (d, J = 5.2 Hz, 1H), 4.83 (d, J = 5.6 Hz, 1H), 4.45 (t, J = 5.6 Hz, 1H), 4.32
(d, J = 15.6 Hz, 1H), 4.26 (d, J = 15.6 Hz, 1H), 3.97 (d, J = 9.2 Hz, 1H), 3.68–3.64
(m, 1H), 3.40 (quint, J = 6.0 Hz, 1H), 3.25–3.17 (m, 2H), 3.15–3.06 (m, 2H), 2.50
(s, 3H); 13C NMR (100 MHz, DMSO-d6) d 164.61, 154.64, 139.34, 135.10, 132.38,
131.34, 131.00, 128.30, 127.66, 81.15, 80.58, 78.29, 74.55, 70.27, 61.31, 48.51,
42.13, 14.47; MS (ESI) m/z 413 (M+H)+, 435 (M+Na)+.
1. International Diabetes Federation. Diabetes Atlas, 4th ed.; International
Diabetes Federation: Montreal, Canada, 2009.
2. Dwarakanathan, A. J. Insur. Med. 2006, 38, 20.
3. Mackenzie, B.; Loo, D. D. J. Biol. Chem. 1996, 20, 32678.
4. Moe, O. W.; Berry, C. A.; Rector, F. C. In The Kidney; Brenner, B. M., Rector, F. C.,
Eds., 5th ed.; WB Saunders: Philadelphia, 2000; pp 375–415.
5. Ellsworth, B. A.; Meng, W.; Patel, M.; Girotra, R. N.; Wu, G.; Sher, P.; Hagan, D.;
Obermeier, M.; Humphreys, W. G.; Robertson, J. G.; Wang, A.; Han, S.; Waldron,
T.; Morgan, N. N.; Whaley, J. M.; Washburn, W. N. Bioorg. Med. Chem. Lett. 2008,
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19. In this Letter, SGLT2 inhibition data were obtained by single determinations.
Whenever we use dapagliflozin as our reference in our in-house assay, the
SGLT2 inhibition for dapagliflozin has showed a certain number in the close
range (IC50 = 0.49 0.04 nM) in each different assay. Thus, we trust that all SAR
discussions in the manuscript are scientifically valid.
6. 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, A. A.; Zahler, R.; Deshpande, P. P.;