Novel compounds reducing IRS-1 serine phosphorylation for treatment of diabetes

Article abstract of DOI:10.1016/j.bmcl.2015.11.099

Activation of various interacting stress kinases, particularly the c-Jun N-terminal kinases (JNK), and a concomitant phosphorylation of insulin receptor substrate 1 (IRS-1) at serine 307 play a central role both in insulin resistance and in β-cell dysfunction. IRS-1 phosphorylation is stimulated by elevated free fatty acid levels through different pathways in obesity. A series of novel pyrido[2,3-d]pyrimidin-7-one derivatives were synthesized as potential antidiabetic agents, preventing IRS-1 phosphorylation at serine 307 in a cellular model of lipotoxicity and type 2 diabetes.

Full text of DOI:10.1016/j.bmcl.2015.11.099

Accepted Manuscript
Novel compounds reducing IRS-1 serine phosphorylation for treatment of dia-
betes
Laura Simon-Szabó, Márton Kokas, Zoltán Greff, Sándor Boros, Péter
Bánhegyi, Lilián Zsákai, Csaba Szántai-Kis, Tibor Vantus, József Mandl,
Gábor Bánhegyi, István Vályi-Nagy, László Őrfi, Axel Ullrich, Miklós Csala,
György Kéri
PII:
S0960-894X(15)30307-3
DOI:
Reference:
http://dx.doi.org/10.1016/j.bmcl.2015.11.099
BMCL 23355
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 October 2015
26 November 2015
27 November 2015
Please cite this article as: Simon-Szabó, L., Kokas, M., Greff, Z., Boros, S., Bánhegyi, P., Zsákai, L., Szántai-Kis,
C., Vantus, T., Mandl, J., Bánhegyi, G., Vályi-Nagy, I., Őrfi, L., Ullrich, A., Csala, M., Kéri, G., Novel compounds
reducing IRS-1 serine phosphorylation for treatment of diabetes, Bioorganic & Medicinal Chemistry Letters (2015),
doi: http://dx.doi.org/10.1016/j.bmcl.2015.11.099
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Novel compounds reducing IRS-1 serine
phosphorylation for treatment of diabetes
Leave this area blank for abstract info.
Ar
O
Ar
O
Ar
N
N
O
O
O
O
O
N
R
S
N
O
N
O
N
H
N
F
N
F
O
Br
O
Ar
S
N
N
O
O
F
O
F
F
12-51
F
O
S
O
O
O
O
O
O
N
N
N
N
O
O
S
Lipotoxicity-induced IRS-1 Ser 307
phosphorylation in RINm5F insulinoma cells
(Inhibitor code, Relative P-IRS-1 density)
N
N
N
N
N
N
N
N
H
N
N
H
H
H
21, 4%
18, 7%
20, 13%

Bioorganic & Medicinal Chemistry Letters
Novel compounds reducing IRS-1 serine phosphorylation for treatment of
diabetes
Laura Simon-Szabóa , Márton Kokasa , Zoltán Greffb† , Sándor Borosb , Péter Bánhegyib , Lilián Zsákaib ,
Csaba Szántai-Kisb , Tibor Vantus a, c , József Mandla, c , Gábor Bánhegyic , István Vályi-Nagyd, László
Őrfib, e , Axel Ullrichf , Miklós Csalac and György Kéri a, b
a MTA-SE Pathobiochemistry Research Group, Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, 1444
Budapest, Hungary
b Vichem Chemie Research Ltd., 1022, Budapest, Hungary
c Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, 1444, Budapest, Hungary
d United St. Istvan and St. Laszlo Hospital 1097, Budapest, Hungary
e Department of Pharmaceutical Chemistry, Semmelweis University, 1092, Budapest, Hungary
f Department of Molecular Biology, Max-Planck-Institute of Biochemistry, 82152, Martinsried, Germany
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Activation of various interacting stress kinases, particularly the c-Jun N-terminal kinases (JNK),
and a concomitant phosphorylation of insulin receptor substrate 1 (IRS-1) at serine 307 play a
central role both in insulin resistance and in β-cell dysfunction. IRS-1 phosphorylation is
stimulated by elevated free fatty acid levels through different pathways in obesity. A series of
novel pyrido[2,3-d]pyrimidin-7-one derivatives were synthesized as potential antidiabetic
agents, preventing IRS-1 phosphorylation at serine 307 in a cellular model of lipotoxicity and
type 2 diabetes.
Keywords:
Type 2 diabetes
Lipotoxicity
c-Jun N-terminal kinase
IRS-1 phosphorylation
Pyrido[2,3-d]pyrimidin
2015 Elsevier Ltd. All rights reserved.
———
 Corresponding author. Tel.: +36 1 266 2615; fax: +36 1 266 2615; e-mail: keri.gyorgy@med.semmelweis-univ.hu
† We regret to write that our co-author Zoltán Greff passed away on 25th July 2014. We dedicate this paper to his memory.

literature
JNK1
IC50
(µM)
[28]
IRS-1-P % c-jun-P %
Type 2 diabetes is a metabolic disorder based on insulin
resistance and the inability of pancreatic β-cells to adapt by a
compensatory increase in insulin secretion.
in-house
JNK1
IC50
of
of
anisomycin anisomycin
Cpd
Structure
control at
10µM
control at
10µM
(µM)
Activation of various interacting stress kinases, and a
concomitant phosphorylation of insulin receptor substrate 1 (IRS-
1) at serine 307 play a central role both in insulin resistance and
in β-cell dysfunction.1 Obesity-induced elevation in plasma free
fatty acid (FFA) levels is deleterious to most cells, and this
lipotoxicity2 is known to enhance IRS-1 serine 307
phosphorylation, too.3
N
1
2
3
4
5
6
0.009
0.028
0.025
0.17
0.05
0.04
2.75
5.42
4.22
97
69
161
0
104
46
88
42
94
81
HO
N
N
N
H
H
O
NH2
N
F
N
N
N
H
H
O
OH
Multiple interaction with inflammatory signaling, oxidative
and endoplasmic reticulum (ER) stress as well as accumulation
of diacylglycerols and ceramide, constitute the major
mechanisms of FFA-induced serine kinase activation and IRS-1
serine 307 phosphorylation.4
N
HO
N
N
N
H
H
O
OH
F
Insulin acts as an autocrine survival signal in pancreatic β-
cells. Therefore, insulin resistance is pro-apoptotic in these cells,
and creates a vicious circle by interfering with compensatory
insulin secretion. Certain stress-activated protein kinases, such as
c-Jun N-terminal kinases (JNKs) and p38 mitogen-activated
protein kinase (p38 MAPK) play a prominent role in IRS-1 serine
phosphorylation. Their inhibition proved to be β-cell protective
in isolated islets of Langerhans.5 Attenuation of IRS-1 Ser307
phosphorylation as well as reduced JNK phosphorylation and
activity have been demonstrated to contribute to β-cell protection
by the widely used insulin sensitizer metformin in ER stress6 and
in lipotoxicity.7 Accordingly, JNK inhibitors have been shown to
protect human β-cells from the deleterious effects of high glucose
and leptin8 and to reduce FFA-induced AP-1 activation and
apoptosis in INS-1 insulinoma cells.9 Moreover, they improved
functional β-cell mass in transplanted human pancreatic islets
both in vitro and in vivo in the early post-transplant period.10In
addition, several JNK inhibitors have been shown to improve
insulin sensitivity and glucose tolerance in db/db diabetic mice11-
13 and in mice fed a high-fat diet.14 Consequently, JNK inhibitors
are generally considered as potential antidiabetic agents.15,16
O
F
O
O
O
N
N
N
H
N
N
F
OH
F
O
O
14
15
N
HO
N
N
N
H
F
F
O
O
N
N
H
N
N
Table 1 Reference compounds synthesized and used in our experiments
The
2,6-disubstituted
7-oxo-pyrido[2,3-d]pyrimidines
(WO02064594) are p38 MAPK inhibitors of nanomolar IC50
values with JNK1 inhibitory effect in the micromolar range. The
most prominent representative of the pyrido[2,3-d]pyrimidin-7-
one core family is palbociclib (Pfizer) (PD-0332991), which is
known as a selective inhibitor of CDK4 and CDK6 and has been
recently approved by FDA in combination with letrozole for the
treatment of postmenopausal women with estrogen receptor
JNK as a member of MAPK family also controls cell
proliferation, survival and DNA repair through phosphorylating a
wide array of transcription factors (e.g. c-Jun, p53). Therefore,
specific JNK inhibitors are relevant for multiple medical
aspects.17 However, the molecules that efficiently inhibit IRS-1
serine 307 phosphorylation are of special importance for anti-
diabetic treatment. The aim of the present study was to apply
cellular models of lipotoxicity-related and non-related IRS-1
Ser307 phosphorylation to select inhibitors with the potential to
prevent or reduce insulin resistance and β-cell dysfunction.
(ER)-positive, human epidermal growth factor receptor
2
(HER2)-negative advanced breast cancer.20 SciFinder search on
the pyrido[2,3-d]pyrimidin-7-one core revealed that 9123 such
derivatives have been published in 681 publications, including 27
review articles. Based on the literature search, we have found a
white spot in the chemical space and designed a novel compound
group containing the pyrido[2,3-d]pyrimidin-7-one core. These
novel derivatives (listed in Table 2) were synthesized according
to the scheme shown in Fig 1. Based on Vichem's in-house data
on JNK pathway inhibitors, 46 analogues were selected for our
experiments. Instant JChem was used for structure database and
biological data management.21
The compounds tested in this work belong to the NCL of
Vichem Chemie Research Ltd (Budapest).18 We have screened
Vichem’s EVL library for JNK inhibition. Two compound
families were identified to be druglike and suitable for
optimization. The published hit compounds listed in Table 1 were
used as reference compounds in our experiments.
Bisphenylamino pyrimidines, the more active JNK inhibitors,
have nanomolar IC50 values, and the general method of their
synthesis is well established.19 The best known representative of
this compound family is pazopanib, a multitarget receptor
tyrosine kinase inhibitor, which acts against renal cell carcinoma
and soft tissue sarcoma via inhibition of angiogenesis.16 Around
32 000 compounds can be found in SciFinder having
bisphenylamino pyrimidine core structure, and 610 biological
activity values were published in 1776 publications, including
353 review articles. These data show that the chemical space is
well covered for this core structure.
Ar
b
O
c
O
O
a
O
O
N
N
O
Br
+
S
O
Ar
S
N
N
O
7
N
NH2
8,9
Ar
Ar
O
O
d
N
N
R
(e)
N
N
N
O
S
N
N
O
H
O
O
10,11
12-51
Figure 1.a: substituted phenol, N-methyl-pirrolidone, K2CO3, 6 h, RT; b: N-
methyl-pirrolidone, K2CO3, 12 h, 120oC; c: 3-chloro-perbenzoicacid, CHCl3;
d: R-amine; e: removing protecting group, salt formation

c-jun-P
% of
aniso-
mycin
control
in-
IRS-1-P % JNK-P %
of of
anisomycin anisomycin
house
JNK1
IC50
2,6-Cl2-
Ph
Cpd
12
13
14
15
16
17
18
19
20
21
22
Ar
R
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Cyclopentyl 19.32
4-(4-
8.18
9.09
108.40
58.93
75.80
45.05
81.60
96.10
96.14
96.27
129.39
131.36
68.20
138.96
74.50
21.12
81.98
114.87
74.00
control at
10µM
control at
10µM
(µM)
1-Methane-
sulfonyl-
piperidin-4-
yl
2,4-F2-
Ph
2,6-Cl2-
Ph
Hydroxy-
piperidin-1-
0.65
10.93
5.93
3.79
>23
2.72
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
2.08
2.15
2.61
8.05
77.17
80.95
40.03
29.64
94.08
152.84
37.58
84.01
52.85
30.50
133.12
35.29
173.60
55.20
87.07
118.95
66.47
>23
yl)-phenyl
2-Methane-
sulfonamido 18.94
-ethyl
2,4-F2-
Ph
Piperidin-4-
yl
2,4-F2-
Ph
12.21
13.20
14.31
14.66
26.71
40.48
46.31
51.06
68.20
71.26
74.50
79.38
81.98
67.53
4-(4-Methyl-
2,4-F2-
Ph
2,6-Cl2-
Ph
Cyclopentyl
piperazin-1-
yl)-phenyl
>23
>23
60.88
4-(4-
2,4-F2-
Ph
Hydroxy-
piperidin-1-
yl)-phenyl
2,6-Cl2- 2-(Pyridine-
57.48
115.66
127.74
102.06
99.10
Ph
2-yl)-ethyl
3-Trifluoro-
methyl-
benzyl
2,4-F2-
Ph
2,6-Cl2-
Ph
100.89
137.28
32.37
Cyclopropyl 14.92
3-(4-Methyl-
piperazin-1- 19.10
yl)-propyl
2-
2,4-F2-
Ph
2,4-F2-
Ph
Morpholin-
4-yl-ethyl
>23
>23
>23
>23
>23
4-Hydroxy-
piperidin-1-
yl
2,4-F2-
Ph
2,6-Cl2-
Ph
Cyclohexyl 16.14
2,4-F2-
Ph
2,6-Cl2- 4-Piperidin-
Cycloheptyl
6.73
2.77
0.91
3.34
>23
7.17
2.48
118.90
73.54
107.95
111.23
86.15
Ph
1-yl-phenyl
3-Methane-
sulfonamido
-propyl
3-Hydroxy-
pyrrolodon-
1-yl
2,4-F2-
Ph
2,4-F2-
Ph
1-Aminos-
ulfonyl-
piperidin-4-
yl
2,4-F2-
Ph
2,4-F2-
Ph
Pyridin-2-
methyl
89.38
4-(4-Methyl-
piperazin-1-
yl)-phenyl
2-
2,4-F2-
Ph
2,6-Cl2-
Ph
76.93
Morpholin- 27.07
4-yl-ethyl
114.48
82.55
3-Hydroxy-
2,6-Cl2- Pyridin-2-yl-
Ph
2,6-Cl2-
Ph
114.89
142.31
68.02
pyrrolodin-
1-yl
>23
>23
6.75
methyl
23
24
3-Hydroxy-
methyl-
piperidin-1-
yl
2,4-F2-
Ph
2,4-F2-
Ph
Cyclopropyl
103.36
96.42
2,6-Cl2- Tetrahydro-
Ph pyran-4-yl
2,4-F2- 2-Pyridin-2-
Ph yl-ethyl
25

2,4-F2-
Ph
4-Piperidin-
1-yl-phenyl
41
42
43
44
45
46
47
48
49
50
51
>23
>23
>23
>23
>23
>23
>23
>23
>23
>23
>23
84.01
88.71
148.70
38.07
130.80
103.90
133.35
107.86
102.90
120.51
110.16
117.82
131.10
148.70
103.97
3-Hydroxy-
piperidin-1-
yl
2,4-F2-
Ph
Figure 2 Inhibition of JNK, c-Jun and IRS-1 Ser 307 phosphorylation in
anisomycin treated HEK293 cells
3-Hydroxy-
piperidin-1-
yl
2,6-Cl2-
Ph
93.59
36.24
The relative P-JNK, P-c-Jun and Ser307-P-IRS-1 densities
were quantified by densitometry and compared to those of the
anisomycin-only-treated samples, which were regarded as 100%
of activation. As expected, there was a correlation between the
phosphorylation of JNK and c-Jun proteins, and some tested
molecules exerted remarkable inhibitory effect on these
parameters (Table 1 and 2). The observed changes in IRS-1
Ser307 phosphorylation are less tightly correlated with the extent
of JNK phosphorylation, in accordance with the multiplicity of
IRS-1 Ser307 kinases. Further investigation in the present study
was narrowed down to 12 selected inhibitors (highlighted in the
tables) based on two inclusion criteria of reducing JNK
phosphorylation below 65% and also lowering IRS-1 Ser307
phosphorylation to at least 15% (Table 1 and 2). These
2,4-F2- 4-Pyrrolidin-
Ph
98.90
98.90
1-yl-phenyl
3-Hydroxy-
methyl-
piperidin-1-
yl
2,6-Cl2-
Ph
99.30
99.30
4-Hydroxy-
methyl-
piperidin-1-
yl
2,4-F2-
Ph
102.35
104.31
110.36
123.96
127.11
165.10
91.58
compounds
share
2,6-disubstituted
7-oxo-pyrido[2,3-
d]pyrimidine core structure.
2,4-F2- 2-Dimethyl-
Ph
104.31
110.36
121.82
119.26
135.70
amino-ethyl
2-Hydroxy-
methyl-
piperidin-1-
yl
2,6-Cl2-
Ph
4-Hydroxy-
piperidin-1-
yl
2,4-F2-
Ph
4-Hydroxy-
methyl-
piperidin-1-
yl
2,6-Cl2-
Ph
2,6-Cl2- 4-Pyrrolidin-
Ph 1-yl-phenyl
Figure 3 Lipotoxicity-induced JNK phosphorylation in RINm5F insulinoma
cells
Table 2 The synthesized novel 7-oxo-pyrido[2,3-d]pyrimidines
The 12 kinase inhibitors selected on the basis of their
effectiveness in anisomycin-treated HEK293 cells were further
Effect of 46 potential inhibitors on JNK activation and JNK-
related protein phosphorylation was tested on HEK293 cells
treated with the direct (ER-stress-independent) JNK-activator,
anisomycin. The cellular model was first validated by using BI-
78D3, a known inhibitor of JNK activation. Cells were treated
with BI-78D3 (various concentrations between 0.1 and 100 µM)
and anisomycin (50 ng/ml) simultaneously and incubated for 30
min. Phosphorylated JNK, phosphorylated c-Jun and Ser307-
phosphorylated IRS-1 protein levels were assessed in the cell
lysates by Western blot. Anisomycin-induced JNK, c-Jun and
IRS-1Ser307 phosphorylations were counteracted by BI-78D3 in
a concentration dependent manner (Fig 2). It diminished all these
three parameters to the limit of detection at 50 µM, and hence it
was applied at this concentration as a positive control in further
experiments. The tested molecules were used uniformly at 10 µM
final concentration among the same experimental conditions.
examined in
a cellular model of lipotoxicity. A JNK
phosphorylation level below 61% and an IRS-1 Ser307
phosphorylation level below 15% in comparison with
anisomycin-only-treated samples comprised the selection criteria.
The stress response of RINm5F rat insulinoma cells, including
JNK, c-Jun and IRS-1 Ser307 phosphorylations, to short term
saturated fatty acid treatment, has been studied earlier in our
laboratory.18 In this study, the levels of P-JNK and P-IRS-1 were
assessed in the cells after incubation in the presence of 500 µM
albumin-conjugated palmitate for 8 hr. JNK phosphorylation was
inhibited to less than 50% of palmitate only treatment by two of
the investigated kinase inhibitors, 27 and 18 (54% and 59%
inhibition, respectively) (Fig 3). These two compounds also
exerted a remarkable albeit different inhibitory effect on IRS-1
Ser307 phosphorylation (58% and 93%, respectively) (Fig 4).

potentially applicable for the treatment of type 2 diabetes. We
have mapped the structural features taking into account novelty,
druglikeness, and IRS-1 serine phosphorylation inhibitory
activity. The development process yielded a narrow group of
novel structures with excellent inhibitory effect on IRS-1 Ser307
phosphorylation, opening the opportunity for further drug
development.
Acknowledgments
We would like to thank Mrs. Valéria Mile and Mr. Bálint
Hegymegi-Barakonyi for their skillful technical assistance, and
Dr. Eszter Illyés for the LCMS data. This work was supported by
the Hungarian Scientific Research Fund (OTKA 104113 and
106060) and by the Hungarian Government and received funding
from the National Research, Development and Innovation Fund
of Hungarian Ministry for National Economy under grant
agreement number KMR_12-1-2012-0074 (Development of
kinase inhibitors for the treatment of renal fibrosis and diabetic
nephropathy).
Figure 4 Lipotoxicity-induced IRS-1 Ser307 phosphorylation in RINm5F
insulinoma cells
Supplementary Material
Supplementary data (detailed synthetic methods, analytical
data, kinase selectivity and cytotoxicity data) associated with this
article can be found, in the online version.
Interestingly, IRS-1 phosphorylation was also markedly
reduced by another three inhibitors, namely 14 (61%), 20 (87%)
and 21 (96%) (Fig 4), which in turn did not decrease the level of
phosphorylated JNK at similar extent (Fig 3).
References and notes
Our most effective inhibitors of IRS-1 phosphorylation are
containing disubstituted phenoxy substituent in position 6 of the
pyrido-pyrimidine core. 2,4-Difluorophenoxy derivatives showed
close to 100% inhibition in case of various substituents on
position 2 amino group e.g. cyclopentylamino, cyclohexylamino,
methanesulfonamido-propylamino, 1-aminosulfonyl-piperidine-
4-yl substituents gave excellent results. 2,6-Dichlorophenoxy
1.
2.
3.
4.
5.
Ragheb, R.; Shanab, G.M.; Medhat, A.M.; Seoudi, D.M.; Adeli,
K.; Fantus, I. G. Biochem Biophys Res Commun, 2009, 389, 211.
Zambo, V.; Simon-Szabo, L.; Szelenyi, P.; Kereszturi, E.;
Banhegyi, G. Csala, M. World J Hepatol, 2013, 5, 550.
Le Marchand-Brustel, Y.; Gual, P.; Gremeaux, T.; Gonzalez, T.;
Barres, R. Tanti, J. F. Biochem Soc Trans, 2003, 31, 1152.
Gao, Z.; Zhang, X.; Zuberi, A.; Hwang, D.; Quon, M.J. Lefevre,
M.; Ye, J. Mol Endocrinol, 2004, 18, 2024.
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T.J. Agapitos, D.; Wang, R.; Rosenberg, L. Ann Surg, 2001, 233,
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substituent in position
6
still resulted <10% IRS-1
phosphorylation e.g. with tetrahydropyran-4-yl substituent in
position 2, but several other substituents in position 2 caused
similar effect also (Table 2). Small scale kinase selectivity of 10
compounds having IRS-1 Ser307 phosphorylation level below
15% were determined by Proteros Biostructures at 10 µM or 1
µM compound concentration (see supplementary material).
Based on these results the following kinases were hit remarkably:
Abl, c-kit, DDR1, JNK1, PDGFRβ, Ret, c-Src, VEGFR2. We
determined IC50 of the reported compounds at 24h on HEK293 as
a measure of citotoxicity. We found that every compound has
IC50 over 10 µM and most of them have over 30 µM (see
supplementary material).
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Pastori, R. L. Diabetologia, 2008, 51, 298.
11. Kaneto, H.; Nakatani, Y.; Miyatsuka, T.; Kawamori, D.;
Matsuoka, T.A. Matsuhisa, M.; Kajimoto, Y.; Ichijo, H.;
Yamasaki, Y.; Hori, M. Nat Med, 2004, 10, 1128.
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C.; Fornoni, A. Kidney Int, 2009, 75, 381.
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A.; Solinas, G.; Karin, M.; Pellecchia, M. Proc Natl Acad Sci
USA, 2008, 105, 16809.
14. Cho, H.; Black, S.C.; Looper, D.; Shi, M.; Kelly-Sullivan, D.
Timofeevski, S.; Siegel, K.; Yu, X. H.; McDonnell, S. R.; Chen,
P.; Yie, J.; Ogilvie, K. M.; Fraser, J.; Briscoe, C. P Am J Physiol
Endocrinol Metab, 2008, 295, 1142.
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We aimed to identify and develop new compounds with the
potential to reduce IRS-1 Ser307 phosphorylation. The selected
46 compounds were first tested in anisomycin-treated HEK293
cells and 12 of them were further studied in a cellular model of
palmitate induced lipotoxicity. Although the signaling
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in case of 5 compounds, 4 of which also decreased JNK
phosphorylation at statistically significant, yet smaller extent. It
seems likely that these molecules act by inhibiting other protein
kinases involved in IRS-1 phosphorylation beside JNK.
We have successfully achieved our triple aim: we have started
a medicinal chemistry optimization starting from druglike JNK
inhibitor structures in order to obtain novel compounds

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