Identification of novel GLUT inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.02.050

The compound class of 1H-pyrazolo[3,4-d]pyrimidines was identified using HTS as very potent inhibitors of facilitated glucose transporter 1 (GLUT1). Extensive structure–activity relationship studies (SAR) of each ring system of the molecular framework was established revealing essential structural motives (i.e., ortho-methoxy substituted benzene, piperazine and pyrimidine). The selectivity against GLUT2 was excellent and initial in vitro and in vivo pharmacokinetic (PK) studies are encouraging.

Full text of DOI:10.1016/j.bmcl.2016.02.050

Accepted Manuscript
Identification of novel GLUT inhibitors
Holger Siebeneicher, Marcus Bauser, Bernd Buchmann, Iring Heisler, Thomas
Müller, Roland Neuhaus, Hartmut Rehwinkel, Joachim Telser, Ludwig Zorn
PII:
S0960-894X(16)30162-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.02.050
BMCL 23604
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
8 December 2015
15 February 2016
18 February 2016
Please cite this article as: Siebeneicher, H., Bauser, M., Buchmann, B., Heisler, I., Müller, T., Neuhaus, R.,
Rehwinkel, H., Telser, J., Zorn, L., Identification of novel GLUT inhibitors, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.02.050
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Bioorganic & Medicinal Chemistry Letters
Identification of novel GLUT inhibitors
Holger Siebeneichera, , Marcus Bausera, Bernd Buchmanna , Iring Heislerb, Thomas Müllerb, Roland
Neuhausa, Hartmut Rehwinkela, Joachim Telserb and Ludwig Zorna
a Bayer Pharma AG, Drug Discovery, 13353 Berlin, Germany
b Bayer Pharma AG, Drug Discovery, 42096 Wuppertal, Germany
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The compound class of 1H-pyrazolo[3,4-d]pyrimidines was identified using HTS as very potent
inhibitors of facilitated glucose transporter 1 (GLUT1). Extensive structure-activity relationship
studies (SAR) of each ring system of the molecular framework was established revealing
essential structural motives (i.e. ortho-methoxy substituted benzene, piperazine and pyrimidine).
The selectivity against GLUT2 was excellent and initial in vitro and in vivo pharmacokinetic
(PK) studies are encouraging.
Keywords:
GLUT1 inhibitor
2009 Elsevier Ltd. All rights reserved.
Pyrazolopyrimidine
Warburg effect
Structure-activity relationships (SAR)
In vitro pharmacokinetics (PK)
———
 Corresponding author at present address: Bayer Pharma AG, Drug Discovery, Medicinal Chemistry 3, Muellerstraße 178, 13353 Berlin,
Germany, Tel.: +49 30 468 18486; fax: +49 30 468 15911; e-mail: holger.siebeneicher@bayer.com

Already in 1930 Otto Warburg observed a phenomenon in
which cancer cells often metabolically switch from oxidative
phosphorylation to glycolysis even under normal oxygen
supply.1-7 At first sight, the contradictory behavior of cancer cells
with regard to their increased demand on energy supply is of
crucial importance to feed the essential biochemical anabolic
pathways with higher amounts of central intermediates.8 To
fulfill the increased demand on building blocks the glycolytic
rate is often up-regulated in tumor entities.9 As the cellular
uptake of glucose is the first rate-limiting step in the glycolytic
process it is not surprising that the transporters responsible for
this uptake were found to be up-regulated in both solid and
hematological malignancies.10
inhibiting the GLUT1 transporter while being selective against
GLUT2 (CHO-hGLUT2 cells) and comparable active on GLUT3
(DLD1Glut1-/- cells, Horizon discovery).41 From the originally
found 6-(piperazin-1-yl)pyrimidin-4-aniline hits a conjunctive
approach at the central pyrimidine quickly led to a 1H-
pyrazolo[3,4-d]pyrimidine core.42 This compound class was
shown to be reversible to the glucose transporter by glucose
competition experiments.43 The SAR of each of the four elements
of the molecular framework, named as ring systems A to D, was
explored independently. Starting with the aryl ring connected to
the piperazine (ring A, Table 1) it was found that an ortho-
methoxy group (3) at the aryl ring resulted in very potent GLUT1
inhibition and excellent selectivity against GLUT2 and a
selectivity factor of around 10 against GLUT3. Omission of the
methyl group of the methoxy group in 3 in comparison to 2
markedly deteriorated the excellent GLUT2 selectivity, while
substitution by the metabolically more stable OCF3 group (4) led
to a completely inactive compound which was also observed for
the corresponding ortho-CF3 moiety (6) did. A methoxy
monosubstitution in meta- (8) and para-position (10) at the aryl
ring A decreased the GLUT1 potency by a factor of
approximately 7 and 70, respectively, in comparison to 3.
Of the two different classes of hexose transporters (SGLT:
sodium-dependent glucose transporter, GLUT: facilitative
glucose transporter) the GLUTs were found to be overexpressed
in many tumors.11 In particular, GLUT1 overexpression has been
reported in many types of human cancers, including those of
brain,12 breast,13,14 colon,15,16 kidney,17 lung,18 ovary19 and
prostate,20,21 and is correlated with advanced cancer stages and
poor clinical outcomes. It was demonstrated that the activation of
certain oncogenes like c-myc,22 KRAS,23 BRAF24 and p53,25 and
transcription factors like hypoxia inducible factor-1a26,27 can
induce the GLUT1 overexpression. Additionally, there is a
widely clinically applied diagnostic modality PET imaging,
which makes use of increased glucose uptake in some types of
cancer with [18F]fluoro-2-deoxyglucose (FDG).28,29 All these
factors demonstrate the importance of GLUT1 function for
cancer cell viability and lead to the therapeutic options that an
inhibition of this transporter might be beneficial in the treatment
of tumors with high glucose turnover.
Keeping the ortho-methoxy substitution fixed while exploring
the introduction of a second functional group in the ring revealed
that only a substituent in para-position towards the methoxy
group was beneficial regarding the GLUT1 potency. In this
position not only a small fluorine atom (compound 15) can be
incorporated but also a primary carboxamide (compound 17)
with clear reduction of GLUT2 selectivity, though. The same
observation could be made when a pyridine nitrogen was
introduced in the ortho-methoxy substituted compounds 19-22.
Insertion of a pyridine nitrogen again gave a very potent GLUT1
inhibitor when it was located at para-position towards the
methoxy group in the aryl ring. Introduction of other
heteroaromatic systems at the piperazine like pyrimidine only led
to inhibitors in the higher three-digit nM range. 23, having a non
aromatic ring A system, revealed to be a completely inactive
compound.
Whereas GLUT1 is nearly ubiquitously expressed in all
normal tissues to maintain the basal glucose supply30 the
expression of some members of the GLUT family is more
specific. Those GLUTs can be involved in central processes like
insulin secretion of pancreas (GLUT2)31 and neuronal glucose
uptake (GLUT3).32 To enable a therapeutic window with a
potential GLUT1 inhibitor selectivity within the GLUT family is
decisive for any possible cancer treatment with this approach.
Several synthetic approaches were employed to build the
desired 1H-pyrazolo[3,4-d]pyrimidines. One way started with
conversion of appropriately substituted hydrazines 24 with
(ethoxymethylene)malononitrile to obtain the 5-amino-pyrazole-
4-carbonitriles 25.44,45 These were heated with formic acid to
obtain pyrazolo[3,4-d]pyrimidin-4-ones 26.46 After activation of
Several small-molecule GLUT1 inhibitors have already been
described in literature including resveratrol,33 naringenin,34
phloretin,35 WZB117,36 thiazolidinedione,37 and STF-31.38 With
their thiazolidinediones Wang et al. demonstrated the inhibition
of [3H]-2-deoxy-D-glucose ([3H]-2-DG) uptake in LNCaP cells
and the suppressive effects on the viability of LNCaP cells in
MTT assays.37 Chan et al used the principle of chemical synthetic
lethality to demonstrate the sensitivity of VHL deficient renal
cancer cells to glucose uptake inhibition by STF-31.38 The
compound WZB117 was able to inhibit the glucose uptake in
A549 cancer cells and their cell proliferation in a dose-dependent
manner.36 All these results underline the feasibility of GLUT1
inhibition as cancer treatment.
46
the pyrimidin-4-one with POCl3 the required piperazine aryl
head 30 can be added in a one pot procedure with DBU as base.47
Alternatively, a mono-Boc-protected piperazine 28 can be added
to the 4-chloro-1H-pyrazolo[3,4-d]pyrimidines 2748 followed by
deprotection49 and cross-coupling with the desired aryl bromides
in a Buchwald-Hartwig reaction50-53 using Pd(OAc)2, XPhos and
NaOtBu. Furthermore, the piperazine aryl head 30 could be
synthesized first via Buchwald-Hartwig and then coupled to 4-
chloro-1H-pyrazolo[3,4-d]pyrimidine 32 to receive intermediates
33. A final Goldberg reaction54 using K3PO4 and trans-N,N'-
dimethylcylcohexane-1,2-diamine as ligand was employed to
couple the aryl moiety to the pyrazole.
Therefore, an HTS screen was performed using a cell-based
assay with CellTiter-GloTM,39 readout for ATP production. In a
pairwise chemical screen, DLD1 cells were co-incubated with the
potential GLUT1 inhibiting test compounds and with rotenone as
inhibitor for the oxidative phosphorylation.40 With the rotenone
co-incubation, the cells could only produce ATP via glycolysis
and the amount of produced ATP could be linked to the amount
of glucose taken up. Subsequent assays regarding cell uptake of
radioactive [3H]-2-DG, consumption of glucose and generation of
lactate in the presence of the test compounds confirmed their
competitive GLUT1 inhibitory behavior. In total 120,303
compounds were tested in this HTS and 435 hits were identified
Within the lead structure it was found that the central piperazine
played a very decisive part for the excellent GLUT1 potency
(ring B, Table 2). Trials to omit one of the piperazine nitrogens
(35 and 36) resulted in less potent test compounds. Substitution
adjacent to the ring A attachment point (37) was tolerated
whereas the corresponding regioisomer (38) led to a decrease in
GLUT1 potency by a factor of ~10. Methylene bridges across the
piperazine
ring
were
not
tolerated
(39
and

Table 1 SAR at piperazine aryl head41,a
A
GLUT1b
GLUT2c
GLUT3d
GLUT1b
GLUT2c
GLUT3d
Cpd
A
R1
Cpd
A
R1
F
IC50 [µM] IC50 [µM] IC50 [µM]
IC50 [µM] IC50 [µM] IC50 [µM]
F
OH
OMe
OCF3
CN
0.27
0.007
0.025
>10
1.81
>10
1.12
0.06
>10
>10
5.99
>10
0.68
0.06
0.25
>10
2.05
>10
1
2
3
4
5
6
0.11
1.2
1.6
18
-
-
0.18
0.23
1.36
ND
0.68
1.51
19
20
CF3
F
OMe
CN
0.29
0.18
0.039
1.21
1.48
0.22
0.54
0.32
0.069
7
8
9
-
0.009
0.51
0.06
21
OMe
1.77
8.7
1.77
10
F
CN
2.4
1.2
>10
>10
10
0.54
11
12
-
-
0.06
>10
0.95
>10
0.07
>10
22
23
F
CN
0.01
0.15
>10
1.7
0.04
0.17
13
14
15
16
17
F
CN
CONH2
0.001
0.02
0.005
>10
>10
0.10
0.01
0.05
0.037
a
d
All mean data values reported are derived from at least two
experiments.
Cell-based assay using hGLUT1 knock-out DLD1 cell line
(DLD1GLUT1-/-) mainly expressing hGLUT3 (coincubation of
test compound with 300 µM glucose and 1 µM rotenone for 15
min).
b
Cell-based assay using DLD1 cell line mainly expressing
hGLUT1 (coincubation of test compound with 100 µM glucose
and 1 µM rotenone for 15 min).
ND = not determined
c Cell-based assay using CHO cell line transfected with hGLUT2
(coincubation of test compound with 0.03 µM fructose and 1 µM
rotenone for 15 min).
28
i)
ii)
iii)
iv)
24
25
26
27
29
v)
vi)
31
30
vii)
viii)
ix)
31
28
30
32
33
1-23, 37-42, 49-68
34
Scheme 1. Reagents and conditions: (i) EtOH, Et3N, rt; (ii) HCOOH, 110 °C; (iii) POCl3, 130 °C; (iv) a) Et3N, THF, rt – 60 °C,
b) 4M HCl, dioxane, rt; (v) DBU, DMF, 120 °C or THF, Et3N, rt – 60 °C; (vi) Pd(OAc)2, XPhos, NaOtBu, toluene, 90 °C; (vii)

a) Pd(OAc)2, XPhos, NaOtBu, toluene, 90 °C, b) 4M HCl, dioxane, rt; (viii) Et3N, THF, 60 °C or DBU, DMF, 120 °C; (ix) CuI,
K3PO4, trans-N,N'-dimethylcylcohexane-1,2-diamine, NMP, microwave irradiation, 180°C.
Table 2
Table 3
Substitution of central piperazine moiety41,a
Variation of pyrazolopyrimidine core41,a
B
C
GLUT1b
GLUT2b
GLUT3b
GLUT1b
GLUT2b
GLUT3b
Cpd
C
Cpd
B
IC50 [µM] IC50 [µM] IC50 [µM]
IC50 [µM] IC50 [µM] IC50 [µM]
3
>10
>10
ND
1.4
0.25
>10
1.20
0.12
0.48
3
0.025
0.3
>10
0.6
5.0
0.25
4.0
0.025
1.9
35
36
37
38
43
44
45
0.47
0.014
0.12
4.0
2.0
>10
>10
>10
2.5
0.049
0.027
3.3
3.5
0.43
46
47
2.3
>10
4.1
39
0.064
>10
4.4
>10
ND
>10
2.2
40
41
48
0.053
>10
0.63
a
All mean data values reported are derived from at least two
experiments.
42
0.004
0.021
0.008
b For details of the biological assays, see Ref. 41
a
All mean data values reported are derived from at least two
experiments.
ND = not determined
Removing a nitrogen from 5- or 7-position (43 and 44) or
adding a nitrogen at the 6-position (45) gave only compounds of
low activity or no activity at all.
b For details of the biological assays, see Ref. 41
ND = not determined
The aryl ring connected to the pyrazole (ring D, Table 4) can
be substituted with small functional groups in the ortho-, meta-
and para-position without significant loss of GLUT1 potency.
Regarding the ortho-position a fluorine atom (49) has even
beneficial effect in comparison to compound 3. In case of a para-
substituent an electron-withdrawing group like fluorine (56) or
cyanide (57) revealed to be superior to an electron-donating
function such as methoxy (58) or methyl (59). Double
substitution at the aryl ring was also feasible but the electronic
behavior and the relative positioning of the substituents was
important to keep the excellent GLUT1 potency (60 and 61).
Introduction of a pyridine nitrogen to the benzene ring D with
(66 and 67) or without further substitution (63, 64, and 65)
40). Switching to a piperazine mimic, i.e. the spiro-compound 41,
was not successful. Ring enlargement to the corresponding 1,4-
diazepane (42) resulted in a markedly improved potency however
the selectivity over the other GLUT members was deteriorated.
Regarding GLUT1 potency some regions of the 1H-
pyrazolo[3,4-d]pyrimidine core (ring C, Table 3) allowed for
more flexibility than the piperazine moiety. For example, the
pyrazole ring could be exchanged for a triazole (46), imidazole
(47) or imidazole-2-one (48) without a loss in activity. However,
in the case of the imidazole 47, the GLUT3 selectivity
deteriorated. On the other hand, the presence of the pyrimidine
moiety revealed to be more crucial for a good GLUT1 inhibition.

Table 4
SAR at pyrazoloaryl tail41,a
D
GLUT1b
GLUT2b
IC50 [µM] IC50 [µM]
GLUT3b
GLUT1b
IC50 [µM] IC50 [µM]
GLUT2b
GLUT3b
IC50 [µM]
Cpd
D
R1
Cpd
D
IC50 [µM]
0.025
0.007
0.05
H
F
OMe
Me
3
>10
1.1
1.2
1.0
0.25
0.04
0.11
0.25
49
50
51
0.51
0.75
8.0
6.0
0.94
1.5
63
0.01
F
52
53
54
55
0.035
0.033
0.2
>10
nd
0.9
10
0.12
0.35
0.17
CN
OMe
Me
64
0.05
0.096
F
0.017
0.017
0.23
1.5
>10
>10
2.6
10
0.046
0.2
0.17
56
57
58
59
CN
OMe
Me
1.2
4.4
7.0
1.6
65
66
3.0
-
0.008
>10
0.09
0.08
NDe
60
61
-
-
0.03
0.41
0.88
2.1
0.32
0.95
0.1
ND
2.0
0.34
0.12
67
68
0.04
62
a
All mean data values reported are derived from at least two
experiments.
ND = not determined
b For details of the biological assays, see Ref. 41
Table 5
In vitro pharmacokinetic profiling of pyrazolopyrimidines
LMa stability
Hepd stability
Caco-2 permeability
Cpd
Clb,LM (L/h/kg)b; Fmax (%)c Clb,Hep (L/h/kg); Fmax (%)
Papp A-Be (ERf)
0.14; 89 (h)
0.84; 36 (h)
2.2; 47 (r)
2.7; 36 (r)
2.3; 57 (m)
59 (0.29)
3
2.6; 39 (r)
ND
15
49
0.37; 72 (h)
0.79; 40 (h)
0.5; 62 (h)
175 (0.42)
2.2; 48 (r)
1.7; 68 (m)
0.55; 58 (h)
1.9; 65 (m)
ND
ND
52
68
3.7; 11 (r)
153 (0.33)
0.83; 37 (h)
a Liver microsomes (LM) (h: human, r: rat, m: mouse)
b
Blood clearance (Clb): Clb = (QH·Clint)/(QH+Clint), QH: liver blood flow (human: 1.3 L/h/kg, rat: 4.2 L/h/kg, mouse: 5.4 L/h/kg),
Clint: intrinsic clearance (well-stirred model of hepatic clearance)
c Maximal predicted bioavailability after peroral administration (Fmax): Fmax=(1-Clb/QH)∙100%
d Hepatocytes (Hep)

e
The apparent permeability (Papp) values (nm/s) are derived from the transport of the compounds (2 µM) over a 2 h period from the
apical (A) to the basolateral (B) compartments
f The letters ER refer to the efflux ratios and are calculated by dividing the Papp B–A values by the Papp A–B values.
ND = not determined
Table 6
In vivo pharmacokinetic profile of compound 49 in male Wistar rats (0.3 mg/kg i.v.a, 5 mg/kg p.o.b)
Clb
(L/h/kg)
Vss
(L/kg)
terminal
t1/2 (h)
Cmax,po
(µM)
F[%]
67
1.2
1.9
2.7
1.7
a Formulation: PEG400 50% + Water 40% + EtOH 10%
b Formulation: PEG300 90% + NMP 10%
decreased the GLUT1 activity significantly. The same effect was observed when a substituted methylene was inserted between rings
C and D (compound 62). A complete switch to a saturated cyclopentyl replacing ring D (compound 68) was also feasible without
losing potency and selectivity, but this substituent proved to be metabolically too labile (Table 5) already in in vitro PK assays with
human liver microsomes (Fmax: 37% h) and rat hepatocytes (Fmax: 11% h). Consistent with prediction, for aromatic ring D analogs
the in vitro PK data looked more promising than for compound 68.
The in vitro PK assays with human liver microsomes and rat hepatocytes (Table 5) revealed a moderate to good metabolic
stability of the GLUT1 inhibitor compounds with exception to compound 68 containing the liable cyclopentyl moiety. Comparing
the human liver microsome data of compounds 3 and 15 the additional substitution with fluorine at ring A did not further increase
metabolic stability, neither did the introduction of a fluorine at ring D (49 and 52). The Caco-2 permeability was generally high in
this compound class, only compound 3 showed moderate apical to basolateral transport. All compounds show indication for uptake
transport in Caco-2 cells, as the efflux-ratio was found to be less than 1.
In vivo PK results showed compound 49 to have a low blood clearance (29% of liver blood flow) in Wistar rat (Table 6). The
volume of distribution was high and the terminal half-life of the compound was intermediate. Oral bioavailability was 67% which is
in line with the total blood clearance indicating complete absorption.
In summary, optimization at each ring of the molecular framework of the 1H-pyrazolo[3,4-d]pyrimidines revealed that with an
appropriate substitution pattern (e.g. 49) a single-digit nanomolar inhibition at GLUT1 is feasible. Furthermore, excellent selectivity
towards GLUT2 can be obtained within this compound class. The preliminary in vitro profile looked promising and first in vivo PK
studies showed that good oral bioavailability is attainable. Further efforts are undertaken to increase GLUT3 selectivity.
Acknowledgments
The authors thank the following for their valuable assistance and input in the experimental procedures: Dr. Marcus Koppitz, Dr.
Knut Eis, Dr. Niels Lindner, Dr. Mélanie Héroult, Dr. Heike Petrul, Dr. Maria Quanz, Dr. Sylvia Gruenewald, Dr. Andrea
Haegebarth and Dr. Holger Hess-Stumpp. The authors also thank Dr. Arwed Cleve for his fruitful suggestions and precise
proofreading during the preparation of this manuscript.
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Identification of novel GLUT inhibitors
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Holger Siebeneicher, Marcus Bauser, Bernd Buchmann , Iring Heisler, Thomas Müller, Roland Neuhaus,
Hartmut Rehwinkel, Joachim Telser, Ludwig Zorn
49
IC50
:
7 nM (GLUT1)
1.1 µM (GLUT2)
40 nM (GLUT3)