Design and synthesis of fluorescent SGLT2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.08.036

The design and synthesis of the first fluorophore-conjugated SGLT2 inhibitors is described. The mode of linking the fluorophore to the SGLT2 pharmacophore was found to be crucial in achieving optimum potency. Superior potency to phlorizin was provided by

Full text of DOI:10.1016/j.bmcl.2008.08.036

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4944–4947
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of fluorescent SGLT2 inhibitors
a
a
a
Mark I. Lansdell a, , Denise J. Burring , David Hepworth , Matthew Strawbridge ,
*
Emily Graham a, Thierry Guyot b, Mark S. Betson b, James D. Hart b
a Pfizer Global Research and Development, Sandwich Laboratories, Ramsgate Road, Kent CT13 9NJ, UK
b Peakdale Molecular Limited, Peakdale Science Park, Sheffield Road, Chapel-en-le-Frith, High Peak SK23 0PG, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The design and synthesis of the first fluorophore-conjugated SGLT2 inhibitors is described. The mode of
linking the fluorophore to the SGLT2 pharmacophore was found to be crucial in achieving optimum
potency. Superior potency to phlorizin was provided by examples containing TAMRA, BODIPY, Cy3B
and NBD fluorophores.
Received 14 July 2008
Revised 11 August 2008
Accepted 12 August 2008
Available online 14 August 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
SGLT2
Inhibitor
Fluorophore
Conjugation
After filtration by the kidney glomerulus, renal glucose is subse-
quently reabsorbed by two sodium-dependent glucose cotrans-
intensive focus on such a limited range of chemotypes has led to
highly congested chemical space in terms of intellectual property
considerations, and limited opportunity for significant differentia-
tion within the existing structural classes. We therefore saw value
in seeking an entirely unrelated class of SGLT2 inhibitor.
porters (SGLTs).1 SGLT1 is
a low capacity, high affinity
transporter that is expressed in the small intestine and heart in
addition to the kidney,2 whereas SGLT2 is a high capacity, low
affinity transporter expressed predominantly in the proximal con-
voluted tubule of the nephron,3 where it is responsible for 90% of
renal glucose reabsorption. Inhibition of SGLT2 is thus expected
to result in elevated urinary glucose excretion, and indeed human
SGLT2 gene mutations lead to renal glucosuria in individuals who
are otherwise asymptomatic.4 The consequent lowering of blood
glucose levels is anticipated to be achieved without risk of hypo-
glycemia by virtue of a lack of impact on insulin secretion. In com-
bination with a negative energy balance, such a potential profile is
highly attractive for the treatment of diabetes mellitus and obesity.
As a result, the development of small-molecule inhibitors of SGLT2
has received widespread attention across the pharmaceutical
industry.5
A major component of our strategy to identify a novel SGLT2-
inhibiting chemotype was to perform a high-throughput screen
(HTS) of our company compound file. However, the opportunity
for undertaking such an HTS was limited by the available assay for-
mats. The most widely used in vitro SGLT2 assays are based on
functional inhibition of the uptake of [14C]-
a-methyl-D-glucopy-
ranoside in various cell lines expressing SGLT2. Although an auto-
mated 96-well format of this assay has been described,9 we
considered this still too cumbersome for the purposes of a full-file
HTS.
OMe
HO
O
OH
OH
HO
Despite the very large number of SGLT2 inhibitors that have been
described, all are structurally related to the prototypical inhibitor
O
O
O
O
phlorizin6
1 (Fig. 1), sharing the common features of a glucose-
HO
HO
2
1
based (or glucose-mimicking) ring bearing a lipophilic side-chain
at C1. The side chains contain two cyclic systems (generally both
aromatic) separated by one to three carbons. In general, known
SGLT2 inhibitors are either O-glucosides, such as phlorizin 1 and
sergliflozin-A7 2, or C-aryl glucosides such as dapagliflozin8 3. This
HO
OH
OH
OH
OH
Cl
R
O
HO
HO
3 R = OEt
4 R = Et
OH
OH
* Corresponding author. Tel.: +44 0 1304 645037; fax: +44 0 1304 651819.
E-mail address: mark.lansdell@pfizer.com (M.I. Lansdell).
Figure 1. Representative SGLT2 inhibitors.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.036

M. I. Lansdell et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4944–4947
4945
Table 1
We report here the results of our efforts to design a fluorescent
SGLT2 inhibition data for compounds 1, 4,16 and 5, and 9–12
inhibitor of SGLT2 with the aim of enabling development of a fluo-
rescence polarisation high-throughput binding assay. Fluorometric
assays offer several advantages over their radiometric counter-
parts, especially with regard to safety risks, waste disposal, and
ease of miniaturisation. Where fluorometric assays tend to present
a greater challenge is in the design of an appropriate fluorescent li-
gand, due to the difficulty in maintaining potency whilst accom-
modating the steric demands of the fluorophore, particularly
when this is conjugated to a small molecule rather than a pep-
tide.10 Nonetheless, there have been several reports of successful
fluorophore-conjugation with small molecule ligands in recent
years,10,11 and in-house experience with a high-throughput fluoro-
metric hERG assay12 has encouraged pursuit of this approach.
Our first task was to select the location on the typical SGLT-
inhibitor framework for conjugation of a fluorophore.13 Despite
the wealth of disclosed SGLT2 inhibitors, the vast majority reside
solely within the patent literature, often lacking potency data,
and there is a general dearth of well-described structure–activity
relationships (SAR) for this transporter. However, we noticed some
general flexibility in the nature and size of the substituent on the
terminal aromatic ring, and thus chose to target this location, ini-
tially employing the nitrogen of amines 8a/b (Scheme 1) as the
conjugation handle. Known phenol 514 was converted to the corre-
sponding triflate,14 which was then carbonylated to provide novel
methyl ester 6. Reaction of 6 with ammonia or methylamine pro-
vided amides 7a and 7b, respectively, and these were reduced to
benzylic amines 8a/b.
Capping of the amine as a simple benzamide to give 9a/b
(Table 1) suggested that the presence of an amide function close
to the lipophilic pharmacophore can be tolerated (albeit with some
loss of potency), especially if N-methylated. Due to the high cost of
many commercially available fluorophores, we selected the rela-
tively inexpensive fluorescein to begin our survey of fluorophore
conjugation.15 We were encouraged to achieve at least measurable
inhibition of SGLT2 with our first compounds (10a/b) although the
demands of the fluorophore were clearly detrimental to potency,
even when separated from the SGLT2 pharmacophore by a C5
tether (11a/b). Use of NBD chloride provided an alternative non-
amide mode of conjugation (12a/b), and demonstrated that sub-
micromolar potency was attainable.
a
Compound
R1
R2
SGLT2 IC50 (nM)
1
4
5
—
—
—
—
—
—
752 (545–1040)b
12 (11–14)b
33 (28, 39)c
O
O
9a
9b
H
Me
495 (480, 510)c
45 (36, 53)c
HO
O
O
10a
10b
H
Me
11000 (9600, 12400)c
350 (3100–3600)c
3
O
OH
HO
O
O
O
HN
11a
11b
H
Me
8000 (7500, 8500)c
15050 (14600, 15500)c
O
O
OH
NO2
12a
12b
H
Me
2400 (2300, 2500)c
286 (241, 330)c
N
O
N
Inhibition of uptake of [14C]-
a-methyl-D-glucopyranoside in CHO cells stably
a
transfected with human SGLT2.17
b
1 n = 13, 4 n = 50 (95% confidence interval in parentheses).
c
Values are means of n = 2 (individual measurements in parentheses).
the fluorophore. To this end, known tetra-acetylated phenol 1314
(an immediate precursor in the preparation of 5) was alkylated
with protected amino-bromoalkanes containing chains ranging
from ethyl to octyl (Scheme 2). After global deprotection of
14a–e, the resulting amines 15a, 16a, 17a, 18 and 19 were either
directly conjugated with the fluorophore of choice, or were first
alkylated before fluorophore-conjugation, providing targets
20–23.
Before examining a wider range of fluorophores, we decided to
incorporate a linker which would allow greater separation of the
pharmacophore from any polarity associated with attachment of
The sub-micromolar potency of 20 (Table 2), the first compound
made in this series, immediately suggested that this would be a
superior system for attaching the fluorophore compared to utilis-
ing the benzylic nitrogen of compound 8a/b. We therefore ex-
panded the combination of the alkoxy linker with other
fluorophores, which we anticipated would have fluorescent prop-
erties more suitable for our needs. Potency enhancement was ob-
served on moving from fluorescein-tagged 20, to TAMRA-tagged
21 to BODIPY-tagged 22e/f, all containing a 4-carbon linker. At this
point we fully explored the impact of linker length and amide sub-
stituent R1. The SAR regarding linker length was found to be rela-
tively flat (compare compounds 22a–k), although there was an
indication of a slight preference for increased distance of the fluo-
rophore from the SGLT2 pharmacophore. In contrast, the size of the
amide R1 had a much more profound impact on potency, with pro-
pyl and butyl substituents (22g–i) leading to a marked decrease in
potency. We also prepared several examples tagged with Cy3B
(23a–d) and found this fluorophore to provide marginally im-
proved potencies when compared to BODIPY with the same linker.
Finally, we investigated the impact of introducing a phenyl ring
into the linker (Scheme 3). Phenol 1314 was reacted under Mitsun-
obu conditions with benzyl alcohol 2418 to generate phenoxy ether
25, which was then globally deprotected. The resulting amine 26a
Cl
R
O
HO
HO
5 R = OH
a,b
d
6 R = CO2Me
c
OH
7a/b R = CONHR1
OH
8a/b R = CH2NHR1
e
Cl
R2
N
R1
O
HO
HO
9-12
OH
OH
Scheme 1. Reagents and conditions: (a) PhN(Tf)2, Et3N, DMAP, CH2Cl2, 63%; (b)
Pd(OAc)2, dppp, CO, MeOH, 100 °C, 95%; (c) 7a: NH3/MeOH, 100 °C, 77% 7b: MeNH2,
EtOH, 100 °C, 90%; (d) BH3-THF, THF, reflux, then MeOH/BuOH quench, 8a R1 = H
52%, 8b R1 = Me 68%; (e) benzoic acid succinimidyl ester, or 5-carboxyfluorescein
succinimidyl ester (5-FAM, SE), or 6-(fluorescein-5-carboxamido)hexanoic acid
succinimidyl ester (5-SFX), or 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD chlo-
ride), Et3N, DMF, rt.

4946
M. I. Lansdell et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4944–4947
H
14a n = 2, R1 = Boc
14b n = 3, R1 = Boc
14c n = 4, R1 = Boc
14d n = 6, R1 = Boc
14e n = 8, R1 = Phth
Cl
O
N
Cl
OH
( )
R1
n
a
O
O
AcO
AcO
AcO
AcO
OAc
OAc
13
OAc
OAc
b
H
N
17a n = 4, R1 = H
Cl
O
( )
R1
n
R2
c
O
Cl
O
N
HO
( )n
17b n = 4, R1 = Me
17c n = 4, R1 = Et
17d n = 4, R1 = n-Pr
17e n = 4, R1 = i-Pr
17f n = 4, R1 = i-Bu
R1
15a n = 2, R1 = H
15b n = 2, R1 = Me
O
HO
OH
c
d
HO
HO
OH
20-23
OH
16a n = 3, R1 = H
16b n = 3, R1 = Me
18 n=6, R1 = H
19 n=8, R1 = H
OH
c
Scheme 2. Reagents and conditions: (a) 14a–d: Br(CH2)nNHBoc, Cs2CO3, DMF, a 36%, b 71%, c 75%, d 91%; 14e: N-(8-bromooctyl)phthalimide, Cs2CO3, DMF; (b) 15a/16a/17a/
18/19: i—NH3(aq)/MeOH, ii—HCl, MeOH, 15a 95%, 16a 100%, 17a 91%, 18 60%; 19: i—K2CO3, MeOH, 25% (over two steps from 13), ii—NH2NH2, MeOH, reflux, 62%; (c) 15b/16b/
17b: i—ethyl formate, EtOH, reflux, ii—BH3.THF, THF, reflux, 15b 50%, 16b 75%, 17b 43%; 17c: i—acetyl chloride, NaOAc, THF, H2O, 75%, ii—BH3.THF, THF, reflux, 25%; 17d: i—
propionyl chloride, NaOAc, THF, H2O, 89%, ii—BH3.THF, THF, reflux, 39%; 17e: i—acetone, MeOH, ii—NaBH4, MeOH, 80%; 17d: i—isobutyraldehyde, MeOH, ii—NaBH4, MeOH,
84%; (d) 5-carboxyfluorescein succinimidyl ester (5-FAM, SE), or 5-carboxytetramethylrhodamine succinimidyl ester (5-TAMRA, SE), or 4,4-difluoro-5,7-dimethyl-4-bora-
3a,4a-diaza-s-indacene-3-propionic acid succinimidyl ester (BODIPY FL, SE), or Cy3B NHS ester, Et3N, DMF, rt.
Table 2
SGLT2 inhibition data for compounds 20–23
a,b
Compound
Linker
R1
R2
SGLT2 IC50 (nM)
HO
O
O
O
O
20
n = 4
H
835 (790, 880)
O
O
OH
O
-
N+
O
21
n = 4
H
370 (200, 540)
N
22a
22b
22c
22d
22e
22f
22g
22h
22i
n = 2
n = 2
n = 3
n = 3
n = 4
n = 4
n = 4
n = 4
n = 4
n = 6
n = 8
H
Me
H
Me
H2
Me
n-Pr
i-Pr
i-Bu
H
420 (370, 470)
315 (310, 320)
203 (166, 240)
430c
267 (254, 280)
155 (110, 200)
850c
O
N
F
F
B
N
1570 (1440, 1700)
2000c
22j
22k
148 (119, 177)
205 (120, 290)
H
O
N
O
+
N
23a
23b
23c
23d
n = 4
n = 4
n = 4
n = 6
H
145 (138, 151)
122 (79, 164)
131 (100, 162)
102 (100, 104)
Me
Et
H
-
SO3
a
Inhibition of uptake of [14C]- -glucopyranoside in CHO cells stably transfected with human SGLT2.17
a-methyl-D
Values are means of n = 2 (individual measurements in parentheses) unless otherwise stated.
n = 1.
b
c

M. I. Lansdell et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4944–4947
4947
2. (a) Wright, E. M.; Turk, E.; Hager, K.; Lescale-Matys, L.; Hirayama, B.;
Supplisson, S.; Loo, D. D. F. Acta Physiol. Scand. Suppl. 1992, 607, 201; (b)
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Skorecki, K. Kidney Int. 2005, 67, 34.
NHBoc
NHBoc
24
Cl
O
O
AcO
HO
a
25
13
AcO
OAc
Cl
OAc
b
R2
N
R1
O
O
5. (a) Handlon, A. L. Expert Opin. Ther. Patents 2005, 15, 1531; (b) Isaji, M. Curr.
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2005, 21, 31.
7. (a) Katsuno, K.; Fujimori, Y.; Takemura, Y.; Hiratochi, M.; Itoh, F.; Komatsu, Y.;
Fujikura, H.; Isaji, M. J. Pharmacol. Exp. Ther. 2007, 320, 323; (b) Pajor, A. M.;
Randolph, K. M.; Kerner, S. A.; Smith, C. D. J. Pharmacol. Exp. Ther. 2008, 324, 985.
8. 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. J. Med.
Chem. 2008, 51, 1145.
HO
HO
OH
d
OH
26a R1 = H, R2 = H
26b R1 = Me, R2 =H
27, 28
c
Scheme 3. Reagents and conditions: (a) PPh3, DIAD, THF, 65%; (b) i—NH3/MeOH,
ii—HCl/MeOH, 84% (over 2 steps); (c) i—ethylformate, EtOH, reflux, ii—BH3.THF,
THF, reflux, 79% (over 2 steps); (d) 5-carboxytetramethylrhodamine succinimidyl
ester (5-TAMRA, SE), or 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-
3-propionic acid succinimidyl ester (BODIPY FL, SE), Et3N, DMF, rt.
9. Castaneda, F.; Kinne, R. K.-H. Mol. Cell. Biochem. 2005, 280, 91.
10. Middleton, R. J.; Kellam, B. Curr. Opin. Chem. Biol. 2005, 9, 517.
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Schramm, H. W. Bioconjugate Chem. 2004, 15, 359; (b) Xie, S.-X.; Petrache, G.;
Schneider, E.; Ye, Q.-Z.; Bernhardt, G.; Seifert, R.; Buschauer, A. Bioorg. Med.
Chem. Lett. 2006, 16, 3886; (c) Cowart, M.; Gfesser, G. A.; Bhatia, K.; Esser, R.;
Sun, M.; Miller, T. R.; Krueger, K.; Witte, D.; Esbenshade, T. A.; Hancock, A. A.
Inflamm. Res. 2006, 55, S47; (d) Middleton, R. J.; Briddon, S. J.; Cordeaux, Y.;
Yates, A. S.; Dale, C. L.; George, M. W.; Baker, J. G.; Hill, S. J.; Kellam, B. J. Med.
Chem. 2007, 50, 782; (e) Amon, M.; Ligneau, X.; Camelin, J.-C.; Berrebi-
Bertrand, I.; Schwartz, J.-C.; Stark, H. ChemMedChem 2007, 2, 708; (f) Schneider,
E.; Keller, M.; Brennauer, A.; Hoefelschweiger, B. K.; Gross, D.; Wolfbeis, O. S.;
Bernhardt, G.; Buschauer, A. ChemBioChem 2007, 8, 1981; (g) Singleton, D. H.;
Boyd, H.; Steidl-Nichols, J. V.; Deacon, M.; de Groot, M. J.; Price, D.; Nettleton, D.
O.; Wallace, N. K.; Troutman, M. D.; Williams, C.; Boyd, J. G. J. Med. Chem. 2007,
50, 2931; (h) Jones, L. H.; Randall, A.; Napier, C.; Trevethick, M.; Sreckovic, S.;
Watson, J. Bioorg. Med. Chem. Lett. 2008, 18, 825.
Table 3
SGLT2 inhibition data for compounds 27-28
R1
R2
SGLT2 IC50 (nM)
a,b
Compound
O
O
-
N+
O
27a
27b
H
Me
55 (54, 55)
154 (107, 200)
O
N
12. Deacon, M.; Singleton, D.; Szalkai, N.; Pasieczny, R.; Peacock, C.; Price, D.; Boyd, J.;
Boyd, H.; Steidl-Nichols, J.; Williams, C. J. Pharmacol. Toxicol. Methods 2007, 55,
255.
13. The more ambitious approach of incorporating the fluorophore within the
minimum pharmacophore to actively contribute to SGLT2-inhibition was
briefly explored, but was found to be insufficiently promising.
F
F
O
B N
28a
28b
H
Me
123 (120, 126)
95 (91, 98)
N
14. Eckhardt, M.; Eickelmann, P.; Himmelsbach, F.; Barsoumian, E. L.; Leo, T.
WO2005092877.
15. All fluorophores were purchased from Invitrogen (Molecular Probes), with the
exception of Cy3B NHS ester and NBD chloride, which were purchased from GE
Healthcare and Sigma–Aldrich, respectively.
a
Inhibition of uptake of [14C]-
a-methyl-D-glucopyranoside in CHO cells stably
transfected with human SGLT2.17
b
Values are means of n = 2 (individual measurements in parentheses).
16. The structure of dapagliflozin 3 had not been disclosed by the time this work
was completed. We had employed close-analogue 4 (Washburn W.; Meng W.
US20060063722), alongside phlorizin 1, to benchmark our SGLT2 assay. In a
later, slightly modified, format of the assay, we have since established that 3
and 4 are equipotent.
17. Methodology analogous to that described for SGLT1 by: Lin, J.-T.; Kormanec, J.;
Wehner, F.; Wielert-Badt, S.; Kinne, R. K.-H. Biochim. Biophys. Acta 1998, 1373, 309.
18. Adlington, R. M.; Baldwin, J. E.; Becker, G. W.; Chen, B.; Cheng, L.; Cooper, S. L.;
Hermann, R. B.; Howe, T. J.; McCoull, W.; McNulty, A. M.; Neubauer, B. L.;
Pritchard, G. J. J. Med. Chem. 2001, 44, 1491.
19. Preparation of 27a: 5-TAMRA-SE (5 mg, 0.095 mmol) and amine 26a (5.7 mg,
0.011 mmol) were dissolved in anhydrous DMF (0.5 mL) and triethylamine
(0.019 mL, 0.11 mmol) was then added. The solution was stirred at room
temperature for 16 hours in the dark. Solvents were removed in vacuo and the
residue was stored under N2 (g) in the freezer until purification, which was
was either directly conjugated with a fluorophore, or was first meth-
ylated before conjugation. TAMRA-labelled 27a19 (Table 3) was
found to be considerably more potent than its alkyl-chain-linked
analogue 21, notably over 10-fold more potent than phlorizin 1
and reaching within 5-fold of the potency achieved by 4, despite
the burden of the fluorophore. In contrast, combination of the phe-
nyl-based linker (28a/b) with a BODIPY fluorophore provided more
modest improvements versus the alkyl chain analogues 22e/f.
In summary, we have described the first examples of fluoro-
phore-labelled SGLT2 inhibitors. The mode of linking the fluoro-
phore to the SGLT2 pharmacophore was found to be crucial in
achieving good potency. A variety of fluorophores are broadly
acceptable, which offers the potential for fine-tuning of the fluores-
cent properties,20 and sufficient potency has been attained that we
anticipate such compounds could find use as pharmacological tools
in assay development or other investigations of SGLT2.
performed by preparative HPLC (Column: Waters XBridge C18,
5 lm,
19 Â 150 mm, 20 mL/min; Eluent: gradient 20–55% MeCN in water over
15 minutes, then 85% for 5 min, Rt 11.2 min). The product was obtained as a
dark purple solid by freeze drying (8.3 mg, 96%). 1H NMR (CD3OD, 300 MHz,)
rotamers d 3.26–3.58 (m, 16H), 3.75 (dd, 2H), 4.02 (d, 2H), 4.08 (d, 1H), 4.63 (s,
2H), 5.04 (s, 2H), 6.87 (d, 2H), 6.90 (s, 2H), 7.00 (d, 2H), 7.11 (d, 2H), 7.22–7.40 (m,
10H), 8.04 (d, 1H), 8.51 (d, 1H). MS (ESI) m/z 912.34 (MH)+. Chemical purity by
HPLC: Waters XBridge C18, 3 Â 150 mm, 5
lm, 0.5 mL/min, gradient 5-95%
MeCN in 2% aq. HCO2 H over 15 min, then held for 10 minutes, Rt 10.61 min,
95.93% (223 nm).
References and notes
20. The excitation-emission profiles of compounds 23d, 27a, and 28b were
measured and found to be essentially unchanged from the corresponding
commercially available fluorophore precursors.
1. (a) Wright, E. M. Am. J. Physiol. Renal Physiol. 2001, 280, F10; (b) Wright, E. M.;
Turk, E. Pflugers Arch. Eur. J. Physiol. 2004, 447, 510.